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Quantitation of the amount of protein in a solution is possible in a simple spectrometer. Absorption of radiation in the near UV by proteins depends on the Tyr and Trp content (and to a very small extent on the amount of Phe and disulfide bonds). Therefore the A 280 varies greatly between different proteins (for a 1 mg/mL solution, from 0 up to 4 [for some tyrosine-rich wool proteins], although most values are in the range 0.5–1.5 [1]). The advantages of this method are that it is simple, and the sample is recoverable. The method has some disadvantages, including interference from other chromophores, and the specific absorption value for a given protein must be determined. The extinction of nucleic acid in the 280-nm region may be as much as 10 times that of protein at their same wavelength, and hence, a few percent of nucleic acid can greatly influence the absorption.

Identifying protein structure, function, and expression level under varying environmental conditions in cells is an important activity in most life science research and pharmaceutical discovery. Determining the molecular weight and purity of protein samples is routinely done in laboratories using sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE) in the last few decades (1, 2). This process is highly manual and takes many hours to prepare the gel, run the samples, stain and destain the protein fractions, capture the gel image, and analyze the image to determine the protein sizes against a ladder. With commercially available precast gel, the first step of the process is becoming simpler and less messy, but overall it is still a relatively slow and tedious procedure. In large-scale protein processing operations and proteomics research laboratories, protein analysis often is a bottleneck in the workflow. Over the last 10 years capillary gel electrophoresis-SDS (SDS-CGE) is being increasingly used for analysis of therapeutic proteins. Compared to SDS-PAGE, SDS-CGE offers advantages of on-column detection, improved quantitation and resolution, and automation. However, SDS-CGE is limited by low sensitivity, throughput, and reliability. In the past several years, microfluidic-based assay for sizing, quantitiation, and purity assessment of proteins are also finding rapid adoption. The benefits in integration, fast separation, automation, and ease of use offered by microfluidics address the limitations of SDS-PAGE and SDS-CGE, and have proven to be a faster and easier alternative to the traditional SDS-PAGE or SDS-CGE.

The solubilization process for two-dimensional (2-D) electrophoresis has to achieve four parallel goals: Breaking macromolecular interactions in order to yield separate polypeptide chains. This includes denaturing the proteins to break noncovalent interactions, breaking disulfide bonds, and disrupting noncovalent interactions between proteins and nonproteinaceous compounds such as lipids or nucleic acids. Preventing any artefactual modification of the polypeptides in the solubilization medium. Ideally, the perfect solubilization medium should freeze all the extracted polypeptides in their exact state prior to solubilization, both in terms of amino acid composition and in terms of posttranslational modifications. This means that all the enzymes able to modify the proteins must be quickly and irreversibly inactivated. Such enzymes include of course proteases, which are the most difficult to inactivate, but also phosphatases, glycosidases, and so forth. In parallel, the solubilization protocol should not expose the polypeptides to conditions in which chemical modifications (e.g., deamidation of Asn and Gln, cleavage of Asp-Pro bonds) may occur.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has become a highly reliable separation technique for protein characterization. Broad application of electrophoresis techniques has required the development of detection methods that can be used to visualize the proteins separated on polyacrylamide gels. A few of these methods are Coomassie blue (CB) staining (1), silver staining (2), fluorescent staining (3–5), specific enzyme visualization (6), and radioactive detection (7).CB staining is the most commonly used method owing to its proven reliability, simpliCity>, and economy (8, 9), but it lacks sensitivity compared with the silver-staining method. In addition, the staining/destaining process is time consuming (10). Silver staining is the most sensitive nonradioactive protein detection method currently available, and can detect as little as 10 fg of protein (11, 12). However, it has several drawbacks, such as high-background staining, multiple steps, high cost of reagent, and toxicity of formaldehyde (10).

Quantification of proteins is a common challenge. There are various methods described for estimation of the amount of protein present in a sample, for example, amino acid analysis and the bicinchoninic acid (BCA) method (see Chapters 83 and 3). Proteins may be quantified in sample solvent prior to elec-trophoresis and can provide an estimate of total amount of protein in a sample loaded onto a gel (1,2). These methods quantify total protein present but not of one protein in a mixture of several. For this, the mixture must be resolved. Liquid chromatography achieves this, and the various proteins may be quantified by their absorbancy at 220 nm or 280 nm. Microgram to submicrogram amounts of protein can be analyzed in this way, using microbore high-performance liquid chromatography (HPLC). A method has been published whereby theoretical extinction coefficients may be calculated for peptides, based on their sequence, though it is probably less suited to intact proteins because of the influence on UV aborption of longer sequence and structure. (3). Problems may occur if particular species chromatograph poorly (such as hydrophobic polypeptides on reverse-phase chromatography).An alternative is polyacrylamide gel electrophoresis (as the mixture-resolving step) followed by protein staining and densitometry. Exceptions are small peptides that are not successfully resolved and stained in gels, and which are more suited to chromatography (3). Microgram to submicrogram amounts of protein (of more than a few kilodaltons in size) may be quantified in this way. Not every protein stain is best suited to quantification in gels, however. The popular noncolloidal Coomassie gel stains such as PAGE blue ‘83 are not suitable. As discussed by Neuhoff et al. (4), staining by Coomassie dye is difficult to control—it may not fully penetrate and stain dense bands of protein, it may demonstrate a metachromatic effect (whereby the protein—dye complex may show any of a range of colors from blue through purple to red), and it may be variably or even completely decolorized by excessive destaining procedures (because the dye does not bind covalently). As a consequence, stoichiometric binding of Coomassie dye to protein is commonly not achieved. Sensitive silver stains are commonly used to detect rare proteins at levels at levels of the order of 0.05 ng/mm2 in two-dimensional (2-D) gels, for example, but they, too, are not suitable for quantification. For various reasons, proteins vary widely in their stainability by silver—some do not stain at all, others may show a metachro-matic effect. Use of silver staining for quantification is complicated by other factors, too—such as the difficulty in obtaining staining throughout the gel, not just at the surface. This subject is reviewed at greater length in ref. 5, but in summary it may be said that while silver stain methods have great sensitivity, they are problematical for quantification purposes.

Autoradiography is often used to detect and quantify radiolabeled proteins present after separation by polyacrylamide gel electrophoresis (PAGE) (see Chapter 56). The method, however, requires relatively high levels of radioactivity when weak β-emitters, such as tritium, are to be detected. In addition, lengthy exposures requiring the use of fluorescent enhancers are often required. Recent developments in detection of proteins using silver staining (1, 2) have added to the problem because of the fact that tritium emissions are quenched by the silver (2). Since for many metabolic labeling studies, tritium labeled precursors are often the only ones available, it seemed useful to develop a method that would overcome these drawbacks. The method the author developed involves the use of a cleavable crosslinking agent in the polyacrylamide gels that allows the solubilization of the protein for quantification by scintillation counting. Although developed for tritium (3), the method works well with any covalently bound label, as demonstrated here with 35S. Resolution is as good as or better than autoradiography (3 and Fig. 1), turnaround time can be greatly reduced, and quantification is more easily accomplished.

Quantitation of specific proteins in complex mixtures is simplified by the use of antibodies directed against the protein of interest. If the specific protein is differentially expressed within a population of cells, quantitation of the protein in cell lysates by immunoblotting will provide an average quantity of the protein per cell. As a result, when lysates of different cell populations are compared, large changes in the amount of a protein in a small percentage of cells cannot be distinguished from small changes in the amount of a protein in a large percentage of cells. Flow cytometric analysis solves this problem by providing a means to measure the amount of a specific protein within each individual cell in a population. A fluorescently labeled probe, usually an antibody, is required for detection of the protein. A single cell suspension is reacted with an antibody and passed through a flow cytometer, which focuses the cell suspension into a stream that intersects a laser beam. As each cell passes through the beam, the fluorescent probe in each cell is excited and photomultiplier tubes register the degree of fluorescence (1).Multivariate analysis, the simultaneous measurement of multiple parameters, is a powerful feature of flow cytometry. When determining the quantity of a specific protein in a cell, the quantity of other molecules in the same cell can also be measured by using additional fluors specifically targeted to other molecules. Examples of second molecules measured simultaneously include DNA (2–4) or additional proteins (5). In addition, measurement of light scatter can provide information about cell size and granularity. Most flow cytometers are capable of measuring six colors plus light scatter, but specialized flow cytometers that are capable of measuring as many as eighteen colors are now available.

Flow Cytometry (FC) is a popular method for the simultaneous quantitation of multiple specific proteins and other molecules in each cell of a single cell suspension (1). Laser Scanning Cytometry (LSC) is a closely related technology that provides many of the benefits of FC and offers additional features that allow morphological and biochemical analysis of a cell population (2, 3). In both methods, quantitation of endogenous cellular proteins generally requires antibodies against the protein of interest. These antibodies must be either directly labeled with a fluorochrome or detected by a fluorescently-labeled secondary antibody. Using fluorescently-labeled antibodies in combination with fluorescent dyes that quantitatively bind to DNA, both FC and LSC have been valuable in measuring the quantities of specific proteins in relation to cell cycle position (4). In contrast to FC, where a single cell suspension is passed through a fixed-position laser beam, LSC analyzes cells fixed to a surface such as a microscope slide or cover slip. Since the position of the cell on the slide is part of the recorded data, it is possible to visualize the cell after the initial recording event or to subsequently stain the cell with another fluorescent probe and make a second measurement. LSC also allows analysis of cells following biochemical treatments that are incompatible with analysis by FC. In this chapter we describe the value of LSC in analyzing chromatin-associated proteins. Specifically, we describe an in situ fractionation procedure that allows measurement of total and chromatin-associated MCM2, a DNA helicase subunit, as a function of cell cycle position.

Accurate measurement of the amount of tryptophan in a sample is problematic, as it is completely destroyed under normal conditions employed for the complete hydrolysis of proteins. Strong acid is ordinarily the method of choice, and constant boiling 6 M hydrochloric acid is most frequently used. The reaction is usually carried out in evacuated sealed tubes or under nitrogen at 110°C for 18–96 h. Under these conditions, peptide bonds are quantitatively hydrolyzed (although relatively long periods are required for the complete hydrolysis of valine, leucine, and isoleucine). As well as complete destruction of tryptophan, small losses of serine and threonine occur, for which corrections are made. The advantages of amino acid analysis include the measurement of absolute amounts of protein, provided that the sample is not contaminated by other proteins. However, it may be a disadvantage if an automated amino acid analyzer is not readily available.

Two dimensional polyacrylamide gel electrophoresis (2-DE) provides not only the ability to resolve and quantify thousands of proteins, but it also gives those in research and industry the ability to monitor in-process protein purification, quickly and easily (1). It can become a valuable tool in process analytical technology (PAT) in which the goal is to understand and control a process in order to build in quality. 2-DE is also used to identify variability in protein expression in a variety of cell lines (2, 3).It is known that many parameters and laboratory conditions can influence the resolution of proteins on 2-DE, such as pH range of carrier ampholytes used, the quality of reagents and equipment used, temperature, voltage, and the skill of the researcher or technician. Even though there are a multitude of vendors that supply premade reagents, there is pride bestowed to the technician or researcher performing their work.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is one of the most powerful methods for protein analysis (1,2). However, the typical procedures for the detection of protein bands after SDS-PAGE, using the visible dye coomassie blue and silver staining, have several time-consuming steps and require the fixation of proteins in the gel. This chapter describes a rapid and very simple method for protein staining in SDS gels developed in our laboratory (3–5). The method is based on the fluorescent properties of the hydrophobic dye Nile red (9-diethylamino-5H-benzo[α]phenoxazine-5-one; see Fig. 1), and allows the detection of

Little attention has been paid to the function of lipoproteins as part of a nonspecific immune defense system that binds and inactivates microbes and their toxins effectively by complex formation. Because of high extra-capillary tissue pressure, aggregates of such complexes may be trapped in vasa vasorum of the major arteries. This complex formation and aggregation may be enhanced by hyperhomocysteinemia, because homocysteine thiolactone reacts with the free amino groups of apo-B to form homocysteinylated low-density lipoprotein (LDL), which is subject to spontaneous precipitation in vitro. Obstruction of the circulation in vasa vasorum, caused by the aggregated complexes, may result in local ischemia in the arterial wall, intramural cell death, bursting of the capillary, and escape of microorganisms into the intima, all of which lead to inflammation and creation of vulnerable plaques. The presence of homocysteinylated LDL and oxidized LDL stimulates production of LDL autoantibodies, which may start a vicious circle by increasing the complex formation and aggregation of lipoproteins. The content of necrotic debris and leukocytes and the higher temperature than its surroundings give the vulnerable plaque some characteristics of a micro-abscess that by rupturing may initiate an occluding thrombosis. This suggested chain of events explains why many of the clinical symptoms and laboratory findings in acute myocardial infarction are similar to those seen in infectious diseases. It explains the presence of microorganisms in atherosclerotic plaques and why bacteriemia and sepsis are often seen in myocardial infarction complicated with cardiogenic shock. It explains the many associations between infections and cardiovascular disease. And it explains why cholesterol accumulates in the arterial wall. Some risk factors may not cause vascular disease directly, but they may impair the immune system, promote microbial growth, or cause hyperhomocysteinemia, leading to vulnerable plaques.

SDS-PAGE is the most widely used method for qualitatively analyzing protein mixtures. It is particularly useful for monitoring protein purification, and because the method is based on the separation of proteins according to size, the method can also be used to determine the relative molecular mass of proteins (see Note 14).

BACKGROUND Hormonal therapy is effective for advanced prostate cancer (PC) but the disease often recurs and becomes hormone-refractory. It is hypothesized that a subpopulation of cancer cells, that is, cancer stem cells (CSCs), survives hormonal therapy and leads to tumor recurrence. CD44 expression was shown to identify tumor cells with CSC features. PC contains secretory type epithelial cells and a minor population of neuroendocrine cells. Neuroendocrine cells do not express androgen receptor and are quiescent, features associated with CSCs. The purpose of the study was to determine the expression of CD44 in human PC and its relationship to neuroendocrine tumor cells. METHODS Immunohistochemistry and immunofluorescence were performed to study CD44 expression in PC cell lines, single cells from fresh PC tissue and archival tissue sections of PC. We then determined if CD44+ cells represent neuroendocrine tumor cells. RESULTS In human PC cell lines, expression of CD44 is associated with cells of NE phenotype. In human PC tissues, NE tumor cells are virtually all positive for CD44 and CD44+ cells, excluding lymphocytes, are all NE tumor cells. CONCLUSIONS Selective expression of the stem cell-associated marker CD44 in NE tumor cells of PC, in combination with their other known features, further supports the significance of such cells in therapy resistance and tumor recurrence. Prostate 69: 787–798, 2009. © 2009 Wiley-Liss, Inc.

Proteins often work together in protein complexes. For the analysis of the function as well as the structure of proteins it is often of interest to analyze the assembly of such complexes. While protein complexes normally dissociate into their subunits when analyzed by SDS-PAGE (Fig. 1B), nondenaturing electrophoresis (so called native electrophoresis) methods leave the complexes intact. Blue native polyacrylamide gel electrophoresis (BN-PAGE, Fig. 1C), a method developed by Schägger and Jagow (1), has proven to be a valuable tool to analyze the respiratory chain complexes of different organisms (2, 3) to investigate the complexes of the import machinery of mitochondria (4–6) or chloroplasts (7), or to analyze the proteome of isolated organelles or whole cellular lysates (8, 9).

It has long been apparent that the separation capacity of conventional 18 cm IPGs as the first separation step in two dimensional polyacrylamide gel electro-phoresis (2D-PAGE) was theoretically incapable of separating the complexity that is to be expected by the protein species of a mammalian cell line or tissue (1). We first attempted to improve the resolution of 2D-PAGE by developing IPGs of 54 cm in length (2). While this strategy was successful, it required the laborintensive preparation and batchwise validation of own-caste IPGs for each experiment. Subsequently we found that qualitatively similar excellent results could be obtained by placing commercially available IPGs in series (end to end) during IEF. We submitted this method as a patent application on September 12, 2003 (WO2005026715, but since allowed to lapse in all countries but Germany) and published it in the scientific literature in August 2005 (3). The main advantage of this analytical strategy is that less sample is consumed per high resolution protein separation as compared to the alternative use of conventional combinations of individual narrow pH range IPGs for IEF. Thus, the system is well suited to the optimal analysis of samples that are limited in amount, as we have since demonstrated in a number of analytical investigations, including cases where samples from small human clinical biopsies were employed (4–6). Farhoud and colleagues independently published an essentially identical serial IEF method and device (7), also emphasizing the maximal resolution and yield of minimally consumed scarce samples. That paper was submitted for publication in July 2005, four months after the publication of our patent application.

A central issue in proteomics is the generation of qualitative and quantitative protein inventories for a given tissue, organ, or organism. In many eukaryotes, this amounts to a formidable task. Mammals may express more than 200,000 different transcripts with a quarter of them encoding proteins (1). In addition, there exist several hundred different (2) posttranslational modifications, increasing the number of different protein species by an estimated factor of 10, compared to the number of different mRNAs (3, 4). Consequently, higher organism will display more than 1,000,000 different proteins (5). Considering as a lower, very conservative limit, the expression of 5–10% of the transcriptome in an organ, about 50,000–100,000 different protein species will be present in such a sample. Even in a single cell, > 10,000 different protein species likely exist. This number easily exceeds the potential of any known proteomic approach. Another important issue in protein analysis is the high range of protein abundance. Protein concentrations likely vary between 106–1010, depending on the tissue analyzed (6, 7). This renders the identification of low abundant proteins impossible in the bulk of total protein extracts.

Objective. The purpose of this study was to investigate prognostic impact of cholesterol and its subfractions among 75-year-old people from the general population. Methods and Results. The study comprised a random sample (222 women and 210 men) from the general population (participation rate 70%). During 10-year follow-up, 19% of women and 35% of men experienced a major cardiovascular event (MCVE). The all-cause mortality was 29% for women and 47% for men. After adjustment for cardiovascular risk factors, a low level of high-density lipoprotein cholesterol (HDL-C) was significantly associated with MCVE (P = .006) and mortality (P = .011) in men but not in women. The prognostic sex disparity was nearly significant (P = .051 for MCVE and .067 for mortality). The associations of adjusted HDL-C to MCVE and mortality were unchanged after excluding individuals with prevalent stroke or MI. Total cholesterol and low-density lipoprotein cholesterol (LDL-C) were not significantly related to prognosis in either sex. Main Conclusions. HDL-C was associated with dismal prognosis in men but not in women. Elderly men with HDL-C

The bicinchoninic acid (BCA) assay, first described by Smith et al. (1) is similar to the Lowry assay, since it also depends on the conversion of Cu2+ to Cu+ under alkaline conditions (see Chapter 2). The Cu+ is then detected by reaction with BCA. The two assays are of similar sensitivity, but since BCA is stable under alkali conditions, this assay has the advantage that it can be carried out as a one-step process compared to the two steps needed in the Lowry assay. The reaction results in the development of an intense purple color with an absorbance maximum at 562 nm. Since the production of Cu+ in this assay is a function of protein concentration and incubation time, the protein content of unknown samples may be determined spectrophotometrically by comparison with known protein standards. A further advantage of the BCA assay is that it is generally more tolerant to the presence of compounds that interfere with the Lowry assay. In particular it is not affected by a range of detergents and denaturing agents such as urea and guanidinium chloride, although it is more sensitive to the presence of reducing sugars. Both a standard assay (0.1–1.0 mg protein/mL) and a microassay (0.5–10 μg protein/mL) are described.

A rapid and accurate method for the estimation of protein concentration is essential in various areas of biology and biochemistry. An assay originally described by Bradford (1) has become the preferred method for quantifying protein in many laboratories. This technique is simpler, faster, and more sensitive than the Lowry method. Moreover, when compared with the Lowry method, it is subject to less interference by common reagents and non-protein components of biological samples (see Note 1). Despite the introduction of alternative protein assays, the Bradford method remains a popular technique, with the original article (1) being cited over 3,500 times in primary research papers in 2006, thirty years after its initial publication.The Bradford assay relies on the binding of the dye Coomassie Blue G250 to protein. Detailed studies indicate that the free dye can exist in four different ionic forms for which the pKa values are 1.15, 1.82 and 12.4 (2). Of the three charged forms of the dye that predominate in the acidic assay reagent solution, the more cationic red and green forms have absorbance maxima at 470 nm and 650 nm, respectively. In contrast, the more anionic blue form of the dye, which binds to protein, has an absorbance maximum at 590 nm. Thus, the quantity of protein can be estimated by determining the amount of dye in the blue ionic form. This is usually achieved by measuring the absorbance of the solution at 595 nm (see Note 2).

Origin, adaptation and diversity are eternal themes in human evolution. These issues are equally timeless with respect to our own lineage. Human paleontologists continue to grapple with questions surrounding the origin and early evolution of our own genus. How do we identify the earliest members the genus Homo? How many species of Homo were there in the Pliocene and Pleistocene, and how do they relate to one another? Where and when did they evolve? Other burning issues relate to questions about body size, proportions and the functional adaptations of the locomotor skeleton. When did the human postcranial “Bauplan” evolve, and for what reasons? What behaviors (and what behavioral limitations) can be inferred from the postcranial bones that have been attributed to Homo habilis and Homo erectus?Other issues of signifi cance relate to growth, development and life history strategies, and the biological and archeo-logical evidence for diet and behavior in early Homo. Additional issues of importance pertain to the environmental and climatic context in which the genus Homo evolved. Were there global or pan-African climatic events that relate to the appearance and/or extinction of Homo species, and if so, can they be tied to the appearance or disappearance of these species in any meaningful way? Did Homo species live in environments that differed from those inhabited by earlier hominins, and can any general trends through time be inferred from paleontological and isotopic evidence?

The most accurate method of determining protein concentration is probably acid hydrolysis followed by amino acid analysis. Most other methods are sensitive to the amino acid composition of the protein, and absolute concentrations cannot be obtained (1). The procedure of Lowry et al. (2) is no exception, but its sensitivity is moderately constant from protein to protein, and it has been so widely used that Lowry protein estimations are a completely acceptable alternative to a rigorous absolute determination in almost all circumstances in which protein mixtures or crude extracts are involved.The method is based on both the Biuret reaction, in which the peptide bonds of proteins react with copper under alkaline conditions to produce Cu+, which reacts with the Folin reagent, and the Folin—Ciocalteau reaction, which is poorly understood but in essence phosphomolybdotungstate is reduced to heteropoly-bdenum blue by the copper-catalyzed oxidation of aromatic amino acids. The reactions result in a strong blue color, which depends partly on the tyrosine and tryptophan content. The method is sensitive down to about 0.01 mg of protein/ mL, and is best used on solutions with concentrations in the range 0.01–1.0 mg/mL of protein.

The quantitation of protein in biological samples is of great importance and utility in many research laboratories. Protein measurements are widely utilized to ensure equal loading of samples on sodium dodecyl sulfide (SDS)-polyacry-lamide gels and to provide a basis for comparison of enzyme activities and other analytes.

Despite a steady increase in the number and diversity of African Middle Pliocene hominin fossils, paleoanthropolo-gists are not now substantially closer to understanding the temporal, geographical or ecological contexts of the origin of the Homo clade than was the case in 1964, when Louis Leakey, Phillip Tobias and John Napier introduced Homo habilis as the earliest species of the human genus. The reasons for this circumstance are several. The fi rst is a still data-poor record in the relevant time periods. Although the half-million year interval between 2.5 and 3 Ma witnessed heightened morphological, taxonomic and diet-driven adaptive diversity in extinct hominins, all of the known taxa from this interval are of australopith grade (i.e., Australopithecus africanus, A. garhi, A. aethiopicus, late A. afarensis) and none shares derived morphological characteristics exclusively with geologically younger, securely attributed representatives of the Homo lineage. A few specimens that at one time or another have been attributed to Homo fall in the time period 2.5–2.0 Ma but most of these are fragmentary and/or of questionable (or at least debated) diagnostic value (see discussion below). The earliest well-sampled record of fossil hominins that bear characters found only in the genus Homo date to ca. 1.7–1.8 Ma. The fact that as many as three species with unique ties to Homo (H. habilis, H. rudolfensis, H. erectus) have their apparent FADs at about this time (Kimbel, 1991; Wood, 1991; Spoor et al., 2007) speaks to an earlier differentiation of the lineage, but other than the Hadar maxilla A.L. 666-1, with an age of ca. 2.3 Ma (Kimbel et al., 1997), the record of specimens bearing on the earliest phases of the lineage's evolution is mostly uninformative.The second reason for our poor comprehension of Homo origins is conceptual. Historically, the search for the origins of Homo has been confl ated with defi ning the morphological and behavioral boundaries between ape and human. In 1871, Darwin postulated an evolutionary scenario linking terrestrial bipedality, canine reduction, tool manufacture, and brain enlargement at the base of the human lineage. When growing knowledge of Australopithecus (including Paranthropus) made it clear by the 1940s that bipedality and canine reduction evolved well before hominins became signifi cantly encephalized and began to leave a record of their lithic technology, large brains and stone tools were elevated to a foun-dational role in the divergence of the Homo lineage from some generalized australopith form. Thus, Oakley's (1959) “Man the Tool-maker” joined Leakey et al.'s (1964) recognition of the “handy man” Homo habilis in framing thinking about the emergence of Homo for the latter half of the twentieth century (Tattersall, 1998).

Exploring the history of obesity in Europe, this text sets out the chronology of change from the uncertainties before and during World War II. It also identifies international attempts to create an ideal body weight model to assist food aid programmes.

The association of diet with chronic disease has been well documented, and in recent years, research interest has focused on the investigation of whole dietary patterns, instead of single nutrients, for the prevention, and/or treatment of several diseases. The Mediterranean diet is recommended to the Western world as a dietary pattern that is both palatable and healthy, and that can be easily incorporated within a modern lifestyle. Although it is difficult to establish a definition of the ‘typical’ traditional Mediterranean diet, Mediterranean dietary patterns share eight characteristics that differentiate them from American and northern European food cultures. In particular: a high ratio of monounsaturated to saturated fat (MUFA:SFA); high intake of vegetables; fruits, nuts and seeds; legumes/pulses; (mainly unrefined) cereals; a low-to-moderate intake of dairy products; low intake of meat, meat products and poultry; and moderate alcohol consumption. In 1995, the use of an 8-unit ‘a priori’ dietary score to assess adherence to the Mediterranean diet was proposed, based on the above characteristics of this dietary pattern. This score was later revised to account for fish consumption, the intake of which in the Mediterranean diet was moderate-to-high. Since then, several studies have used adaptations of the original Mediterranean Diet Score, and found significant inverse associations between adherence and overall mortality, disease risk, and biomarkers of health, as well as positive associations with survival. Further, the score has been utilised to detect dietary improvements in nutrition intervention studies. The purpose of this chapter is to describe and investigate the use of the original score and its adaptations in research studies, present the findings of studies utilising such indexes, and discuss validity and reliability issues for dietary assessment purposes. Suggestions for researchers wishing to employ Mediterranean diet indexes to investigate associations with chronic disease and assess adherence to the Mediterranean diet in the future will also be provided. © 2009 by Nova Science Publishers, Inc. All rights reserved.

Recent advances in neurobiology have aided our understanding of attention-deficit hyperactivity disorder (ADHD). The higher-order association cortices in the temporal and parietal lobes and prefrontal cortex (PFC) interconnect to mediate aspects of attention. The parietal association cortices are important for orienting attentional resources in time/space, while the temporal association cortices analyse visual features critical for identifying objects/places. These posterior cortices are engaged by the salience of a stimulus (its physical characteristics such as movement and colour). Conversely, the PFC is critical for regulating attention based on relevance (i.e. its meaning). The PFC is important for screening distractions, sustaining attention and shifting/dividing attention in a task-appropriate manner. The PFC is critical for regulating behaviour/emotion, especially for inhibiting inappropriate emotions, impulses and habits. The PFC is needed for allocating/planning to achieve goals and organizing behaviour/thought. These regulatory abilities are often referred to as executive functions. In humans, the right hemisphere of the PFC is important for regulating distractions, inappropriate behaviour and emotional responses. Imaging studies of patients with ADHD indicate that these regions are underactive with weakened connections to other parts of the brain. The PFC regulates attention and behaviour through networks of interconnected pyramidal cells. These networks excite each other to store goals/rules to guide actions and are highly dependent on their neurochemical environment, as small changes in the catecholamines noradrenaline (NA) or dopamine (DA) can have marked effects on PFC function. NA and DA are released in the PFC according to our arousal state; too little (during fatigue or boredom) or too much (during stress) impairs PFC function. Optimal amounts are released when we are alert/interested. The beneficial effects of NA occur at postsynaptic alpha(2A)-receptors on the dendritic spines of PFC pyramidal cells. Stimulation of these receptors initiates a series of chemical events inside the cell. These chemical signals lead to the closing of special ion channels, thus strengthening the connectivity of network inputs to the cell. Conversely, the beneficial effects of moderate amounts of DA occur at D(1) receptors, which act by weakening irrelevant inputs to the cells on another set of spines. Genetic linkage studies of ADHD suggest that these catecholamine pathways may be altered in some families with ADHD, e.g. alterations in the enzyme that synthesizes NA (DA beta-hydroxylase) are associated with weakened PFC abilities. Pharmacological studies in animals indicate catecholamine actions in the PFC are highly relevant to ADHD. Blocking NA alpha(2A)-receptors in the PFC with yohimbine produces a profile similar to ADHD: locomotor hyperactivity, impulsivity and poor working memory. Conversely, drugs that enhance alpha(2)-receptor stimulation improve PFC function. Guanfacine directly stimulates postsynaptic alpha(2A)-receptors in the PFC and improves functioning, while methylphenidate and atomoxetine increase endogenous NA and DA levels and indirectly improve PFC function via alpha(2A)- and D(1) receptor actions. Methylphenidate and atomoxetine have more potent actions in the PFC than in subcortical structures, which may explain why proper administration of stimulant medications does not lead to abuse. Further understanding of the neurobiology of attention and impulse control will allow us to better tailor treatments for specific patient needs.

The emergence of species included in the genus Homo at ~2.4 Ma, as well as appearances of other hominin taxa between 3.0 and 1.8 Ma have often been attributed to global climate change (deMenocal, 1995; deMenocal and Bloemendal, 1995; Vrba, 1995), but the precise details of these climatic transitions, including causal factors, are still debated. In addition, the Plio-Pleistocene boundary (~1.8 Ma; Pasini and Colalongo, 1997) is an important benchmark in human evolution as many hominin species (e.g., Homo erec-tus/ergaster, Paranthropus robustus) have fi rst appearance data (FADs) close to this date, while other taxa (e.g., Homo rudolfensis, Paranthropus aethiopicus) appear to have last appearance data (LADs) prior to this time. There are also some species that are found on both sides of the boundary (e.g., Paranthropus boisei, Homo habilis) (Kimbel, 1995; Wood and Richmond, 2000; Cameron, 2003; Spoor et al., 2007).Perhaps the most frequently cited relationship between climate and faunal change is Vrba's turnover pulse hypothesis (Vrba, 1995; Potts, 1998a; Behrensmeyer, 2006) which refers to an intensity of turnover (speciation and extinction events) during a brief period of time as a result of environmental change. More specifi cally, synchronous change in multiple groups was said to have occurred at 2.5 Ma due to a shift from a moist, warm habitat to drier, cooler and more open conditions. More recently, this hypothesis has been countered by one positing a prolonged and gradual period of turnover between 2.5 and 1.8 Ma (Behrensmeyer et al., 1997), although this study is specifi c to the Turkana Basin. Examinations of individual site patterns in East Africa have shown that turnovers in some mammalian lineages occur during the 3.0–2.0 Ma period, but that the exact timing appears to fl uctuate depending upon site and type of analysis (Bobe and Eck, 2001; Alemseged, 2003). It has become apparent that the picture of hominin evolution is thus contex-tually diverse. Strategic analyses in different basins across both time and space are necessary to further our understanding of evolution during this time period (Behrensmeyer, 2006).

Many high-molecular-mass proteins (MW>100 kDa) are known to be involved in cytoskeleton, defense and immunity, transcription and translation in higher eukaryotic organisms. Though a variety of protein separation techniques have been described, at the moment purification of a high-molecular-mass protein remains a difficult task. O’Farrell (1) was the first to devise a two-dimensional gel electrophoresis (2-DE) technique which could detect more than 1000 spots in a gel. Though it is evident that this method was quite powerful in his days, the method is not particularly suitable in analyzing high-molecular-mass proteins larger than 200 kDa. When skeletal muscle, for example, is simultaneously analyzed by sodium dodecyl sulfite (SDS)-polyacrylamide gel electrophoresis (PAGE) and O’Farrell’s 2-DE method, myosin heavy chain (approx 200 kDa) is clearly seen in the former gel, but not in the latter. Hirabayashi (2) was the first to develop a 2-DE method which could analyze high-molecular-mass proteins, including myosin heavy chain and dystrophin, as large as 500 kDa (3). His trick was the use of agarose gel instead of polyacrylamide gel for the first-dimensional isoelectric focusing (IEF). Agarose gel, when used for IEF, can analyze much larger proteins than the polyacrylamide gel can. Oh-Ishi and Hirabayashi (4) further improved the method by adding 1 M thiourea and 5 M urea in an agarose IEF medium. Thiourea is a potent protein solubilizing reagent especially effective for high-molecular-mass proteins that could enter the first-dimensional agarose IEF gel. Here, we describe the 2-DE method with agarose gels in the first dimension (agarose 2-DE) that is compatible with analyzing high-molecular-mass proteins.

In this chapter, we describe modifications of existing protein assays that take advantage of microwave irradiation to reduce assay incubation times from the standard 15–60 min down to just seconds (1).

A variety of techniques are available for the quantification of proteins, their degradation products and other impurities. Densitometry is particularly useful due to its sensitivity, accuracy and versatility, and it can be applied to proteins in gels or on membranes and used with numerous detection methods. In addition to being accurate, sensitive and reproducible, the technique is cost-effective, simple, and does not require a high degree of specialized training, yet provides technical advantages over other available tools. For example, Vuletich and Osawa showed that densitometry following SDS-PAGE and electroblotting was 20-fold more sensitive than HPLC at detecting oxidatively modified myoglobin (1). In addition, Morçöl and Subramanian reported the development of a sensitive Ponseau-S-stained dot blot/densitometric protein assay that was less subject to interference by pH, detergent and other reagents than the commonly used Bradford protein assay (2). More recently, Zhu et al. and Miles et al. reported a quantitative densitometric method that measures host cell derived impurity levels on immunoblots of recombinant proteins expressed in E. coli (3, 4). This method was shown to be at least 20 times more sensitive than a commercially available ELISA kit designed to measure host cell impurity levels (3). Other groups have reported on the utility of gel or fluorograph densitometry, using multiple detection methods, in carbohydrate analysis (5), taxonomic and forensic applications (6), as a clinical diagnostic tool in Balkan nephropathy (7) and in the analysis of high density lipoproteins (8), as a means of quantifying relative levels of excretory-secretory polypeptides synthesized in vitro by Schistosoma mansoni daughter sporocysts (9), and for analyzing adsorption of proteins and an oligodeoxynucle-otide in Alhydrogel®-based malaria vaccine candidates (10).

To determine utility of luteinizing hormone (LH) estimation, post intramuscular (IM) depot leuprolide in comparison with subcutaneous leuprolide stimulation test. Test for monitoring therapy in patients with gonadotropin dependent precocious puberty (GDPP). In seven patients of GDPP, who were treated with 11.25 mg depot leuprolide, the LH peak after subcutaneous (sc) test was compared with LH at hourly interval for 4 h after IM depot leuprolide for 13 tests and 3rd hour value for next ten tests. These two values were compared both before and after therapy. Before therapy, the mean +/- SD LH peak after subcutaneous leuprolide stimulation test was 20.6 +/- 7.85 IU/l (range 9.64-30.4 IU/l), and it was 27.3 +/- 12.21 IU/l 3 h after the first depot leuprolide injection (range 10.5-45.4 IU/l). During therapy, the mean +/- SD of LH peak after sc stimulation test was 1.96 +/- 0.75 IU/l (range 1.1-3.1 IU/l), and it was 2.58 +/- 0.54 IU/l (range 1.4-3.4 IU/l) 3 h after depot leuprolide injection. Three-hour LH value following IM depot leuprolide injection (11.25 mg) can be used for monitoring therapy in patients with GDPP because of its convenience and cost effectiveness.

Very large molecular weight proteins are difficult to separate by electrophoresis because of their poor penetration into gels using the Laemmli SDS polyacryla-mide system (1). Protein migration in SDS gels has been found to be linear with the log of the molecular weight (2), so the larger the protein, the more poorly it is resolved from other big proteins. Many workers have attempted to solve this problem by using very low concentration acrylamide gels (3), acrylamide mixed with agarose (4), or acrylamide gradients (5) to better separate large proteins. Low concentration acrylamide gels are difficult to use because of their mechanical fragility and distortion during handling; these problems are magnified when blotting is attempted. An additional difficulty in blotting very large proteins is their poor transfer to the membrane. Inclusion of 2-mercaptoethanol in the transfer buffer improves transfer efficiency, but acrylamide gels stained after transfer typically still contain most of the giant muscle protein titin (6).A new electrophoresis system using SDS agarose for protein electrophoresis and blotting has been described (7). An example showing the resolution for several muscle samples containing large proteins is shown in Fig. 1. Migration distance shows a linear relationship with the log of the molecular weight (7). This system allows quantitative transfer of proteins from the gel with much higher reproduc-ibility than can be achieved with methods using low percentage acrylamide.

Background: We developed a Waon therapy (soothing warm therapy) and have previously reported that repeated Waon therapy improves hemodynamics, peripheral vascular function, arrhythmias, and clinical symptoms in patients with chronic heart failure (CHF). The aim of this study was to investigate the effect of Waon therapy on the prognosis of CHF patients. Patients and methods: We studied 129 patients with CHF in NYHA functional class III or IV who were admitted to our hospital between January 1999 and March 2001. In the Waon therapy group, 64 patients were treated with a far infrared-ray dry sauna at 60 °C for 15 min and then kept on bed rest with a blanket for 30 min. The patients were treated daily for 5 days during admission, and then at least twice a week after discharge. In the control group, 65 patients, matched for age, gender, and NYHA functional class, were treated with traditional CHF therapy. The follow-up time was scheduled for 5 years. Results: Recent, complete follow-up data on each patient were obtained. The overall survival rate was 84.5% (Kaplan-Meier estimate). Twelve patients died in the control group and 8 patients died in the Waon therapy group at 60 months of follow-up. Cardiac events due to heart failure or cardiac death occurred in 68.7% of the control group but only 31.3% of the Waon therapy group (P \textless 0.01) at 60 months of follow-up. Conclusion: Waon therapy reduced cardiac events in patients with CHF. This therapy is a promising non-pharmacological treatment for CHF. © 2008 Japanese College of Cardiology.

Background Trials comparing the effectiveness and safety of weight-loss diets are frequently limited by short follow-up times and high dropout rates. Methods In this 2-year trial, we randomly assig...

Genus defi nitions use four criteria, two lines of evidence (phenotypic and genetic) and fall into two categories. The four criteria are the relationships among taxa, information about their adaptive grade, estimates of the genetic distance that separates them, and the estimated time of divergence. At least one researcher (Dubois, 1988) has suggested that a fi fth criterion, evidence of hybridization between species, should be the primary criterion for grouping species into genera. However, even if this suggestion had any merit, it is not clear how evidence of hybridization could be obtained from the fossil record of extinct taxa.One, or more, of the criteria has been used, or combined, to generate genus defi nitions that belong to two categories. The fi rst category draws upon phenotypic evidence to make inferences about adaptive grade, and uses both phenotypic and molecular evidence (if the latter is available) to make inferences about relationships. Defi nitions in the second category rely solely on genetic evidence for either estimates of genetic distance, or for generating estimates about the timing of evolutionary events, which are then converted into criteria for recognizing genera.

With the advent of “proteomics,” many previously unidentified proteins will be isolated in gels for subsequent structural and functional characterization. It is, therefore, important that methods are available for detecting these proteins with minimal risk of modification. This chapter describes a “reverse” staining technique that facilitates the sensitive detection of unmodified proteins (1–9).

Evolutionary medicine has gained increasing attention in recent years by implying that a food selection similar to that of the Paleolithic may prevent diseases. This article is an attempt to characterize the food selection during hominid evolution based on current paleontologic research. Hominid evolution can be divided into multiple phases; and the nutrition ecology of the plio-pleistocene hominids can be tentatively characterized. According to new results of isotope analysis, the Australopithecines did ingest small amounts of animal food already 4.5-2.5 million years ago, while consuming a mainly plant based abrasive diet, which was similar to that of recent chimpanzees. Compared to the Australopithecines, the first representatives of Homo such as H. erectus and H. habilis (2.5-1.5 million years before today) were likely to consume a diet providing more energy and nutrients, which might also have been related to the more gracile dentition. Like H. sapiens the members of this species also consumed an omnivore diet. Assumptions about the nutrition ecology of the archaic and the modern H. sapiens are often concluded by analogies based on the living of historic and recent foragers (hunter-gatherers). As the few detailed ethnographic data show, the diet composition of the individual hunter-gatherer groups varied considerably and ranged from a nearly pure animal-based diet to a diet dominated by plants. All in all the eating behaviour of prehistoric humans was, like that of their pleistocene ancestors, very flexible. Except for focussing on an energy and nutrient-rich diet there was neither specialization in certain foods, nor a typical plant-animal ratio nor a defined macronutrient distribution. Correspondingly, it is impossible to justify details given by representatives of evolutionary medicine on "the Paleolithic diet" empirically.

The vascular endothelium may develop a proinflammatory profile with aging, but evidence is limited in humans. Expression of inflammatory proteins was determined in vascular endothelial cells (EC) obtained from peripheral veins of 24 young (23 +/- 1 years, mean +/- SE) and 36 older (63 +/- 1) healthy men and women using quantitative immunofluorescence. The older subjects had lower vascular endothelium-dependent dilation (forearm blood flow responses to acetylcholine, p < 0.05), and higher plasma concentrations of C-reactive protein, interleukin-6 (IL-6), and oxidized low-density lipoprotein (all p < 0.05), but not tumor necrosis factor-alpha (TNF-alpha). Total (O: 0.52 +/- 0.04 vs. Y: 0.33 +/- 0.05 NFkappaB/HUVEC intensity, p < 0.05) and nuclear (O: 0.59 +/- 0.04 vs. Y: 0.41 +/- 0.04) expression of nuclear factor kappa B p65 (NFkappaB), a proinflammatory gene transcription factor, was greater in EC from the older subjects (p < 0.05). EC expression of the inhibitor (of nuclear translocation) of NFkappaB (IkappaBalpha) was lower in the older subjects (O: 0.16 +/- 0.02 vs. Y: 0.24 +/- 0.03, p < 0.05), whereas IkappaB kinase (IkappaK) was not different. EC expression of the proinflammatory proteins IL-6 (O: 0.42 +/- 0.06 vs. Y: 0.29 +/- 0.03, p < 0.05), TNF-alpha (O: 0.52 +/- 0.06 vs. Y: 0.33 +/- 0.05, p < 0.05) and monocyte chemoattractant protein 1 (MCP-1) (O: 0.59 +/- 0.06 vs. Y: 0.38 +/- 0.02, p

Humans adapt well to short- or a longer-term starvation, using their reserve stores of carbohydrates, fat and protein. Reduction of energy expenditure and conservation of body protein are further reaction to starvation. Energy stores are replenished during feeding period. Long-term partial or total cessation of energy intake leads to marasmic wasting. With the addition of the stress response, catabolism and wasting are accelerated and the normal adaptive responses to simple starvation are over-ridden. Weight loss in either situation results in impaired mental and physical function, as well as poorer clinical outcome. Previously malnourished subjects have less reserves with which to face an acute illness. They are unable to release sufficient amounts of endogenous nitrogen in response to trauma and infection with subsequent higher mortality, more complications and prolonged recovery. If surgery is planned in these patients nutritional support improves physiological functions and lessen surgical risk. © 2008 European Society for Clinical Nutrition and Metabolism.

BACKGROUND: In chronic heart failure (HF), lower total cholesterol (TC) levels have been associated with increased mortality. However, the relationship between lipid levels and outcomes in acute HF has not been studied. This study investigates the relationship between cholesterol levels and in-hospital mortality in patients hospitalized with acute HF. METHODS: The Get With the Guidelines-Heart Failure registry prospectively collects data on patients hospitalized with HF. We analyzed data on 17,791 patients admitted between January 2005 and June 2007 at 236 participating hospitals who had TC levels recorded. Baseline patient characteristics, treatment regimens, and in-hospital mortality were examined by TC level (mg/dL) quartiles (Q) as follows: Q1 (TC or =180). RESULTS: Mean TC level was 150 +/- 47 mg/dL. Patients with lower TC were older and had higher prevalence of ischemic heart disease. Of the patients, 46% were on a lipid-lowering drug, including 58%, 50%, 43%, and 34% of patients in TC Q1 to Q4, respectively. In-hospital mortality in TC Q1 to Q4 was 3.3%, 2.5%, 2.0%, and 1.3%, respectively (P

A population genetic analysis of the long-wavelength opsin (OPN1LW, "red") color vision gene in a global sample of 236 human nucleotide sequences had previously discovered nine amino acid replacement single nucleotide polymorphisms, which were found at high frequencies in both African and non-African populations and associated with an unusual haplotype diversity. Although this pattern of nucleotide diversity is consistent with balancing selection, it has been argued that a recombination "hot spot" or gene conversion within and between X-linked color vision genes alone may explain these patterns. The current analysis investigates a closely related primate with trichromatism to determine whether color vision gene amino acid polymorphism and signatures of adaptive evolution are characteristic of humans alone. Our population sample of 56 chimpanzee (Pan troglodytes) OPN1LW sequences shows three singleton amino acid polymorphisms and no unusual recombination or linkage disequilibrium patterns across the approximately 5.5-kb region analyzed. Our comparative population genetic approach shows that the patterns of OPN1LW variation in humans and chimpanzees are consistent with positive and purifying selection within the two lineages, respectively. Although the complex role of color vision has been greatly documented in primate evolution in general, it is surprising that trichromatism has followed very different selective trajectories even between humans and our closest relatives.

Changes in diet are likely to reduce chronic disorders, but after decades of active research and heated discussion, the question still remains: what is the optimal diet to achieve this elusive goal? Is it a low-fat diet, as traditionally recommended by multiple medical societies? Or a high monounsaturated fat (MUFA) diet as predicated by the Mediterranean diet? Perhaps a high polyunsaturated fat (PUFA) diet based on the cholesterol-lowering effects? The right answer may be all of the above but not for everybody. A well-known phenomenon in nutrition research and practice is the dramatic variability in interindividual response to any type of dietary intervention. There are many other factors influencing response, and they include, among many others, age, sex, physical activity, alcohol, and smoking as well as genetic factors that will help to identify vulnerable populations/individuals that will benefit from a variety of more personalized and mechanistic-based dietary recommendations. This potential could and needs to be developed within the context of nutritional genomics that in conjunction with systems biology may provide the tools to achieve the holy grail of dietary prevention and therapy of chronic diseases and cancer. This approach will break with the traditional public health approach of "one size fits all." The current evidence based on nutrigenetics has begun to identify subgroups of individuals who benefit more from a low-fat diet, whereas others appear to benefit more from high MUFA or PUFA diets. The continuous progress in nutrigenomics will allow some time in the future to provide targeted gene-based dietary advice.

BACKGROUND/OBJECTIVE: We previously reported that chronic hyperglycemia, but not hypoglycemia, was associated with the reduction of neuronal size in the rat brain. We hypothesized that hyperglycemia-induced changes in neuronal structure would have negative consequences, such as impaired learning and memory. We therefore assessed the effects of hyperglycemia and hypoglycemia on neuronal dendritic structure and cognitive functioning in young rats. DESIGN/METHODS: Experimental manipulations were conducted on male Wistar rats for 8 wk, beginning at 4 wk of age. At the completion of the treatments, all rats were trained in the radial-arm water maze, a spatial (hippocampus-dependent) learning and memory task. Three groups of rats were tested: an untreated control group, a streptozotocin-induced diabetic (STZ-D) group, and an intermittent hypoglycemic group. Following behavioral training, the brains of all animals were examined with histologic and biochemical measurements. RESULTS: Peripheral hyperglycemia was associated with significant increases in brain sorbitol (7.5 +/- 1.6 vs. 5.84 +/- 1.0 microM/mg) and inositol (9.6 +/- 1.4 vs. 7.1 +/- 1.1 microM/mg) and reduced taurine (0.65 +/- 0.1 vs. 1.3 +/- 0.1 mg/mg). Histologic evaluation revealed neurons with reduced dendritic branching and spine density in STZ-D rats but not in control or hypoglycemic animals. In addition, the STZ-D group exhibited impaired performance on the water maze memory test. CONCLUSIONS: Hyperglycemia, but not hypoglycemia, was associated with adverse effects on the brain polyol pathway activity, neuronal structural changes, and impaired long-term spatial memory. This finding suggests that the hyperglycemic component of diabetes mellitus has a greater adverse effect on brain functioning than does intermittent hypoglycemia.