Published Science

The versatility and resolving capacity of polyacrylamide gel electrophoresis has resulted in this group of methods becoming the most popular for the analysis of patterns of protein expression in a wide variety of complex systems. These techniques are often used to characterise protein purity and to monitor the various steps in a protein purification process. Moreover, two-dimensional poly-acrylamide gel electrophoresis (2-DE) remains the core technology of choice for separating complex protein mixtures in the majority of proteome projects (1). This is due to its ability to separate simultaneously thousands of proteins and to indicate post-translational modifications that result in alterations in protein pI or Mr. Additional advantages are the high-sensitivity visualization of the resulting 2-DE separations, compatibility with quantitative computer analysis to detect differentially regulated proteins (2), and the relative ease with which proteins from 2-DE gels can be identified and characterised by mass spectrom-etry (MS) (3).

The proteome is a dynamic entity, constantly changing both in levels of protein expression as well as in posttranslational modification and localization, all of which are hidden in the static DNA code. The repertoire of techniques and associated instrumentation which is now being applied to proteomics experiments is expanding exponentially, and although a complete visualization of the proteome is still beyond reach of any single technique, several technology platforms exist that can provide complementary datasets. Proteomics in the clinical setting is also developing, providing a major impact on the way in which diseases will be diagnosed, treated and monitored (1).In particular, approaches involving two-dimensional (2-D) gel electro-phoresis (2-DE) separations and subsequent protein identification using mass spectrometry (MS) have been useful for imaging thousands of intact proteins (including modified/proteolyzed forms) in a single run, but quantification has been challenging, particularly over a large sample set. Difference gel electro-phoresis (DIGE) has proven to be a powerful quantitative technology for differential-expression proteomics on a global level, where the individual abundance changes for thousands of intact proteins can be simultaneously monitored in replicate samples over multiple variables with statistical confidence (see Note 1). This includes quantitative information on protein isoforms that arise due to a charged post translational modification that results in a change in the isoelectric point of the protein (such as acetylation or phosphorylation), and splice variants or protein processing events, all of which can be resolved for individual quantification and subsequent analysis by mass spectrometry.

2D-PAGE remains a widely employed proteomics method because it provides the highest separating resolution of intact polypeptide isoforms that are amenable to subsequent characterization (1, 2). ProteoTope is a differential radioactive detection method that we developed based upon temporally separated exposures of iodinated proteins on 2D-PAGE gels (3, 4). We have also developed systems for extremely high resolution 2D-PAGE over 54 cm or longer, which were designed for optimal quantification of radioactively labeled proteins (5, 6). These methods provide synergistic improvement in data quality for obtaining quality results from limited samples, such as typical valuable clinical tissue samples (7–9).Here we provide a comprehensive technical description of differential 2D-radioactive ProteoTope quantification using highly standardized samples from the ABRF PRG2006 study (10). Our method is a novel application of the widespread Imaging Plate radioactive detection method (11, 12). The twofold study aims for multiple ABRF study participants were to blindly identify the eight unknown standard proteins in the two sample mixtures, and to determine a best estimate of their relative amounts in each sample by multiple measurements of mixtures of the two samples at a ratio of 1:1 (10). 2D-PAGE and Proteo-Tope differential radioactive imaging provided excellent relative quantification data of even subtle differences between the two samples. However our inverse symmetrical replicate experimental design and precise relative quantifications revealed that the quantities of proteins in both samples were apparently altered during sample resolubilization in our laboratory. Thus this case study demonstrates both the excellent quantification of limited protein samples achievable by differential radioactive imaging, and the requirement for stringently optimized sample-specific handling protocols which were not developed for this one-off time-restricted experiment.

I would like to begin by saying that the manuscript in this volume by my dear friend and mentor, Professor Phillip V. Tobias, is particularly enjoyable to me because it allows me to refl ect, and remember some of the events that he describes. I think my father probably thought that he had successfully browbeaten Phillip to accept the designation of Homo habilis as a new species of our genus. It is, I am told, a trait that I have inherited from Louis: we think we have achieved something, or persuaded someone and proceed on that basis when, in fact, we have made nowhere near the point we were trying to make. So I think that Phillip and my father were both correct in their beliefs: Philip was not browbeaten, but Louis certainly would have tried, although he would have denied all along that he was trying! I particularly like Phillip's reference to a characteristic that Louis had (and which I am told I also have). He would pick something up and say, “Isn't that obvious?” to which people would respond “No!” This is exactly the response I get, and the response that my father got from Phillip. In this case, at least, Phillip has had the good grace to say that Louis had been right, and that it had been obvious — Phillip just hadn't noticed the obviousness. So, for the family history, let me just give my Dad a little support, which I was loathe to give when he was alive, because I now fi nd myself more inclined to appreciate his qualities.I totally agree with the validity of the taxon Homo habilis, and I think that Phillip Tobias, along with John Napier and Louis Leakey, was perfectly correct in launching that species (Leakey et al., 1964). The type specimen, OH 7, which Phillip has alluded to as “Jonny's child,” and whose mandible and parietals formed the basis of the new designation, is Homo habilis. And, I think it is correctly a species of the genus Homo (cf. Wood and Collard, 1999; Collard and Wood, 2007; Wood, 2009). By the same token, however, I think some of the specimens from Olduvai that Phillip (Tobias, 1991) and others (Johanson et al., 1987) have included in H. habilis still have a question mark next to that allocation.

Understanding the origin of the genus Homo in Africa remains one of the central problems in paleoanthropology. Consi derable empirical evidence relevant to this issue derives from the Omo—Turkana Basin, which includes areas surrounding Lake Turkana in northern Kenya and the lower Omo River Valley in southern Ethiopia (Fig. 15.1). This basin is well known for it rich record of Late Cenozoic vertebrates (Coppens et al., 1976; Harris, 1983, 1991; Harris et al., 1988; Harris and Leakey, 2003; Leakey and Harris, 2003; Leakey and Leakey, 1978). It also includes some of the earliest specimens attributed to the genus Homo (Prat et al., 2005; Suwa et al., 1996), as well as some of the best specimens of the genus from near the Pliocene/Pleistocene boundary (Leakey, 1973, 1976; Walker and Leakey, 1993; Wood, 1985, 1991).

The importance of sleep to hormones and glucose metabolism was first documented more than four decades ago. Since then, sleep curtailment has become an endemic behavior in modern society. In addition, the prevalence of sleep disorders, particularly obstructive sleep apnea (OSA), has increased. OSA is very common in endocrine and metabolic disorders, but often remains undiagnosed. This Review summarizes the laboratory and epidemiologic evidence that suggests how sleep loss, either behavioral or disease-related, and poor quality of sleep might promote the development of obesity and diabetes mellitus, and exacerbate existing endocrine conditions. Treatment of sleep disorders has the potential to improve glucose metabolism and energy balance. Screening for habitual sleep patterns and OSA might be critically important for patients with endocrine and metabolic disorders.

While improvement in agricultural technology had enabled the production of abundant food, it has thus far failed to eliminate hunger. Malnutrition is expected to reach an all time high. Evidences have suggested that animal based diet has put immense pressure on the already fragile food system, contributing to problems in terms of global food security, health security, and environmental sustainability. Plant based dietary approaches may therefore, target some of these problems from the roots, and may be a solution to improving ethical issues and equity in the current food system. This paper examines how meat production and consumption contributed to the current crises in the food system through the lens of ethics--the moral compass--to find directions on how the present generation should eat, and how the food system could be maintained for a better future.

Evidence on the effects of exercise in the treatment of patients with chronic diseases should be based on well designed randomised controlled trials. The most consistent finding of the meta-analyses summarised in the present work is that aerobic/functional capacity and muscle strength can be improved by exercise training among patients with different diseases without having detrimental effects on disease progression. This is important, as with population aging exercise therapy may be an important means of reducing disability and increasing the number of older people living independently. Additionally, there is accumulating evidence that in patients with chronic disease exercise therapy is effective in improving the prognostic risk factor profile and, in certain diseases, in delaying mortality. In some diseases, such as osteoarthritis, pain symptoms may also be reduced. Severe complications during the exercise therapy programs were rare.

The morphology of the human shoulder seems well-suited to serving its role of providing a wide range of mobility for the upper limb. The dorsal position of a highly mobile scapula and the lateral orientation of the small, shallow glenoid fossa articulating with a medially directed, large round humeral head open up nearly a complete sphere of motion allowing the hand to be positioned almost anywhere around the body. For a creature that depends a great deal on its hands to manipulate objects in order to survive, such an ideal pairing of form and function would seem to be a wonderful example of the elegance of design in nature. Of course the human shoulder did not arrive at its present confi guration purely through natural selection for its current use. Humans share many features of upper limb morphology with extant apes suggesting that at least some aspects of human shoulder anatomy refl ect our phylogenetic heritage. In order to understand which features of the human shoulder have been inherited from a common hominin ancestor, including primitive retentions that continue to serve their original purpose and those that have been co-opted to serve a new function, and which are uniquely derived in humans, it is necessary to determine the course of evolutionary change in the hominin pectoral girdle and shoulder. This paper attempts to trace those changes, beginning with early hominins, but focusing on the characteristics of the shoulder region in early Homo erectus (see Larson, 2007, for a more detailed overview of hominin shoulder evolution).

What we eat, how we eat, where we eat, and when we eat are deeply embedded cultural practices. Eating is also related to how we medicate. The multimillion-dollar diet industry offers advice on how to eat for a better body and longer life, and avoiding harmful foods (or choosing healthy ones) is considered separate from consuming medicine & mdash;another multimillion-dollar industry. In contrast, most traditional medical systems view food as inseparable from medicine and regard medicinal foods as the front line of healing. Drawing on medical texts and food therapy practices from ar.

In political theory citizens are defined as being willing to serve the ‘common good’ while consumers are supposed to seek ‘pleasure’. The two terms pull in different directions, so that adding a hyphen is not enough to craft a figure capable of acting in ways that are generous as well as gratifying. This, or so I will argue, is linked with the understanding of the body that lurks in the background. The idea is that the body is naturally greedy. It only acts ‘properly’ if norms are imposed on it from the outside. To interfere with this understanding, I seek help from advertisements for ‘good food’ as well as from ethnographic research into the way bodily pleasure (rather than being innate) is being shaped in socio-material practices. It might as well be shaped in wise, sensitive and responsive ways. This leads me to suggest that, in theory, we experiment with a ‘consumer-citizen’ whose normativity is literally incorporated. I propose that, despite the caveats, we might call this normativity good taste.

Deltamethrin, an α-cyano class of pyrethroid insecticide is used in insect pest control and antimalaria programs in several countries including India. Although various toxic manifestations of deltamethrin are reported in mammals, its ecotoxicologic dimensions are not adequately researched in ecologically and commercially important fishes. In this study, we report genotoxic effect of deltamethrin in a biomarker fish Channa punctata (Bloch). Adult fish were exposed to three concentrations of technical grade deltamethrin (0.4, 0.8, and 1.2 μg/L) for 48 and 72 h. Ethyl methane sulfonate was used as a positive control. Fish were analyzed for induction of micronucleus (MN), nuclear abnormalities (NAs), and oxidative stress biomarkers in erythrocytes. Deltamethrin significantly induced MN and NAs accompanied by increased lipid peroxidation. Activity of antioxidant enzyme superoxide dismutase was significantly decreased but an increase was observed in reduced glutathione level after 72 h of exposure. The NAs in exposed fish included blebbed, lobed and notched nuclei, and binucleated erythrocytes. Our findings suggest that oxidative stress may, in part, be contributing to deltamethrin-induced genotoxic damage to erythrocytes. Although MN induction is a nonspecific biomarker, it may provide an indication of pollution load of deltamethrin in the affected fish population when used as part of suite of other biomarkers. © 2008 Wiley Periodicals, Inc.

Objective: LDL atherogenicity is frequently attributed to oxidative modification, but glycated LDL, which can participate in many of the cellular processes leading to atherosclerosis, generally circulates at higher concentration even in non-diabetic people. We tested the hypothesis that small-dense LDL, known to be most closely associated with coronary heart disease, undergoes more glycation than other LDL sub-fractions. Methods and results: The concentration of glycated apolipoprotein B (apo B) was measured in serum, LDL and its sub-fractions from 44 non-diabetic subjects. By ELISA serum glycated apoB concentration was 3.0 ± 1.1 mg/dl (mean ± S.D.) of which 84.6 ± 13.6% was in LDL. Of the glycated apo B in LDL 67.8 ± 21.9% was in small dense LDL (LDL3; D1.044-1.063 g/ml) whereas only 32.2 ± 21.9% was in more buoyant LDL subfractions (LDL1 and 2; D1.019-1.044 g/ml). The percentage of apo B present in LDL1 and 2 which was glycated was 1.8 ± 1.8% whereas in LDL3 it was 17.4 ± 18.5% (P \textless 0.001). Furthermore when LDL sub-fractions from non-diabetics (n = 29) were incubated with glucose (30-80 mmol/l) glycation of apo B in the denser LDL3 subfraction was significantly more pronounced than in less dense LDL subfractions. Conclusion: Small-dense LDL is more susceptible to glycation and this may contribute to the atherogenicity of small-dense LDL, even in non-diabetic people. © 2008 Elsevier Ireland Ltd. All rights reserved.

The preparation of fixed-concentration polyacrylamide gels has been described in Chapters 20 and 21. However, the use of polyacrylamide gels that have a gradient of increasing acrylamide concentration (and hence decreasing pore size) can sometimes have advantages over fixed-concentration acrylamide gels. During electrophoresis in gradient gels, proteins migrate until the decreasing pore size impedes further progress. Once the “pore limit” is reached, the protein banding pattern does not change appreciably with time, although migration does not cease completely (1). There are two main advantages of gradient gels over linear gels.

A substantial number of the large, slow-growing fauna on several continents were famously lost in the Pleistocene extinctions. At present, many slow-growing, long-lived mammals are threatened with extinction all over the world. Rhinos, elephants, chimpanzees, orangutans, and gorillas all make an enormous investment in their single offspring, taking years to raise one to independence. Recovery from population downturns is always diffi cult and sometimes impossible. Large investments in a single offspring, in theory, produce high quality offspring that may be larger, smarter, or more social, but slow maturers risk dying before reproducing. Of all these species, humans are the slowest to begin reproducing.In recent years it has become clear that human growth and development, like human life history in general, was not present in australopiths (see Kuykendall, 2003 for a recent review). Evidence suggests, on the contrary, that much of what makes our life history unique took shape during the evolution of the genus Homo. Much of our evidence comes from the study of incremental records of daily growth preserved in teeth (see Dean, 2006), and some from correlative studies of life history in living primates (Smith, 1989, 1991). Given time and judicious use of some partially destructive analytical techniques, it seems likely that we will eventually be able to retrieve a time scale of dental maturation for each species within the Homininae and thus be able to reconstruct the general pace of maturation for each. In this effort, even single broken or sectioned teeth can make a contribution to knowledge. Even so, the occasional rare fossil of a more complete juvenile offers the chance to attempt a more complete reconstruction of maturation, or to ask questions that go beyond time scale alone. The juvenile male Homo erectus skeleton of Nariokotome (KNM-WT 15000) is one of these.

A high protein or meat intake might be a risk factor for metabolic disorders. Stable isotopic abundances (SIA) of hair can be used as biomarkers for animal protein intake due to characteristic isotopic patterns of food proteins. We investigated if an additional meat intake (M, 200 g pork fillet/day) or an omission of meat and meat products (NOM) can influence the natural 15N and 13C SIA within 4 weeks in hair and plasma of young women. The daily protein intake (means ± SD) was 1.40 ± 0.29, 2.25 ± 0.35, and 1.15 ± 0.26 g/kg at baseline, during M, and during NOM, respectively. At baseline the animal protein intake correlated with bulk SIA of hair (15N: R2 = 0.416; 13C: R 2 = 0.664; n = 14). However, isotope ratio mass spectrometry (IRMS) analyses have not shown that hair and plasma SIA were changed significantly after M or NOM. Possible reasons were discussed. Urinary SIA were significantly lower after M than after NOM (15N: p = 0.039; 13C: p = 0.006) and close to those of pork fillet. Characteristic patterns of SIA were measured in individual amino acids (AA) by gas chromatography/combustion isotope ratio mass spectrometry (GC/C/IRMS). The results confirmed considerable differences in SIA between AA (δ15N, up to 22‰; δ13C, up to 31‰). Plots of 15N versus 13C abundances in hair revealed characteristic differences between indispensable and dispensable AA. The intervention-dependent changes of AA-specific SIA were not as clear as expected. Although the AA-specific SIA may reveal more detailed characteristics of physiological conditions, further methodological research is required. We suggest that the SIA of leucine can be potential markers of protein intake. The reliability of SIA as biomarkers of protein intake still have to be tested in longer lasting intervention studies in humans. The results may have implications in the assessment for possible benefits and risks of protein consumption. Copyright © 2009 John Wiley & Sons, Ltd.

A cluster of risk factors for cardiovascular disease and type 2 diabetes mellitus, which occur together more often than by chance alone, have become known as the metabolic syndrome. The risk factors include raised blood pressure, dyslipidemia (raised triglycerides and lowered high-density lipoprotein cholesterol), raised fasting glucose, and central obesity. Various diagnostic criteria have been proposed by different organizations over the past decade. Most recently, these have come from the International Diabetes Federation and the American Heart Association/National Heart, Lung, and Blood Institute. The main difference concerns the measure for central obesity, with this being an obligatory component in the International Diabetes Federation definition, lower than in the American Heart Association/National Heart, Lung, and Blood Institute criteria, and ethnic specific. The present article represents the outcome of a meeting between several major organizations in an attempt to unify criteria. It was agreed that there should not be an obligatory component, but that waist measurement would continue to be a useful preliminary screening tool. Three abnormal findings out of 5 would qualify a person for the metabolic syndrome. A single set of cut points would be used for all components except waist circumference, for which further work is required. In the interim, national or regional cut points for waist circumference can be used.

The publication of Richard Manning's Against the Grain has particular importance for anthropologists interested in agriculture and its modern transformations. Comments of a panel of anthropologists and Manning's response are included in this set of articles. Recent developments in the production of corn for fuel ethanol in the midwestern United States is examined.

One of the most dramatic contributions to human evolutionary studies made by the Leakey family was their discovery from 1959 onwards of the hominin fossils to which Louis Leakey, Phillip Tobias and John Napier in 1964 gave the name Homo habilis. The fi rst specimens were found in the lower part of Bed I of the superlative Olduvai Gorge sequence in Tanzania. The then new dating method, the potassium-argon technique, shocked everyone by revealing an age of 1.75 million years for Olduvai Bed I (Leakey et al., 1961). This high antiquity was one of the factors conducive to the shocked rejection of Homo habilis by almost all our peers.The delicate parietal bones and modest teeth, which seemed to differ from those of South Africa's Australopithecus africanus, were thus of high antiquity. Moreover, they were contemporaneous with an excessively large-toothed austral-opithecine whose remains were found in the same Bed I (Leakey, 1959a, b, c; Tobias, 1959, 1967). The history of early Homo contemporary with robust australopithecines has been recounted elsewhere (Tobias, 1978). The Olduvai discoveries of 1959–1961 were not the fi rst evidence of an apparent Homo contemporary and sympatric with an aus-tralopithecine to emerge from Africa's soil. A decade earlier, fossils showing every justifi cation to be included in the genus Homo had been found in the Swartkrans cave, in South Africa. Swartkrans had already proved to be an immensely rich source of fossils of Australopithecus (Paranthropus) robustus. In September 1949, a jaw fragment, SK 45, came to light in Member 1 of the Swartkrans Formation. This and other Swartkrans specimens were originally named by Robert Broom and John Robinson Telanthropus capensis. However, in 1957, Simonetta proposed to re-designate it Homo erec-tus, and Robinson (1961) agreed with this. By 1959, when Olduvai began to yield early Homo fossils, the existence of a Homo contemporary and sympatric with large-toothed robust australopithecines had been well-established in the paleo-anthropological record for 10 years. When the Olduvai hom-inins were being studied from 1961 to 1963, the prevailing view was that H. erectus was contemporary with the robust australopithecines.

We propose that excessive fructose intake (\textgreater50 g/d) may be one of the underlying etiologies of metabolic syndrome and type 2 diabetes. The primary sources of fructose are sugar (sucrose) and high fructose corn syrup. First, fructose intake correlates closely with the rate of diabetes worldwide. Second, unlike other sugars, the ingestion of excessive fructose induces features of metabolic syndrome in both laboratory animals and humans. Third, fructose appears to mediate the metabolic syndrome in part by raising uric acid, and there are now extensive experimental and clinical data supporting uric acid in the pathogenesis of metabolic syndrome. Fourth, environmental and genetic considerations provide a potential explanation of why certain groups might be more susceptible to developing diabetes. Finally, we discuss the counterarguments associated with the hypothesis and a potential explanation for these findings. If diabetes might result from excessive intake of fructose, then simple public health measures could have a major impact on improving the overall health of our populace.

Analysis of human serum and plasma has been a major focus of many recent proteomics studies because blood is a promising, routinely collected biological fluid that is likely to contain multiple novel biomarkers for many human diseases, such as cancers and cardiovascular disease (1). Most cells in the body are thought to secrete or shed proteins into the blood, and therefore plasma is very likely to contain information concerning the physiological status of all tissues and organs in the body (2–4). While serum and plasma hold great potential as sources of new disease biomarkers, systematic discovery of novel biomarkers in patient cohorts is greatly complicated by the presence of a small number of highly abundant (mg/ml range) proteins in blood (2–6). In particular, serum albumin constitutes about 65% of total serum protein, while immunoglobulins contribute an additional 15%. Most of the remaining 20% of total protein content is comprised of a modest number of additional high- and medium-abundance (μg/ml range) proteins, which tend to obstruct discovery of novel, specific disease biomarkers present at ng/ml to pg/ml levels.

Background/Aims Previous studies examining ethnic differences in non-alcoholic fatty liver disease (NAFLD) are limited by small sample sizes and the lack of liver biopsy as a diagnostic modality. Methods We retrospectively examined the influence of ethnicity on the biochemical and liver histological differences in NAFLD patients. Results The proportion of African Americans (AA) in the NAFLD sample (total 238 patients; 15.1% AA) was lower than in the base population (68.2%). Median ALT (47 IU/L; p=0.05) and triglyceride (134mg/dL, p=0.002) levels were lower in AA than other ethnicities. AA showed lower degrees of steatosis [odds ratio (OR) 0.44, 95% confidence interval (CI) 0.22–0.89; p=0.02] than Whites. In contrast, Asians showed higher grades of ballooning than Whites (OR 2.67, 95% CI 1.03–6.93; p=0.04) and other ethnicities combined (OR 2.71, 95% CI 1.06–6.92; p=0.04). Hispanics showed a higher rate of Mallory bodies than Whites (OR 2.38, 95% CI 1.05–5.39; p=0.04) and other ethnicities combined (OR 2.41, 95% CI 1.09–5.34; p=0.03). Conclusions African Americans showed a lower degree of steatosis than Whites. In contrast, Asians and Hispanics showed higher grades of ballooning and Mallory bodies, respectively, than Whites and other ethnicities combined. These findings highlight the need for prospective studies to evaluate ethnic differences in NAFLD.

Our understanding of the evolutionary trajectory of hominin limb proportions and overall body shape is severely hampered by the paucity of fossil individuals with associated elements from early in the human career (Walker, 1973; McHenry and Coffi ng, 2000; Richmond et al., 2002; Reno et al., 2005). It is now apparent that hind-limb elongation and modern human interlimb proportions emerged at least by the time of early Homo erectus (=ergaster) as represented by the fossils from Dmanisi at almost 1.8 million years ago (Lordkipanidze et al., 2007) and the “Nariokotome boy”, KNM-WT 15000, at approximately 1.6 million years ago (Ruff, 1993; Ruff and Walker, 1993). Interlimb proportions based on complete bone lengths in australopithecines are poorly known, and “Lucy” (A.L. 288-1, Australopithecus afarensis) still represents our best example of the “primitive” hominin condition (Johanson and Edey, 1981). With a humer-ofemoral index (100 × humerus length/femur length) near 85, A. afarensis is “intermediate” between African apes and modern humans (Jungers, 1982, 1991; Jungers and Stern, 1983; Richmond et al., 2002). This difference from humans is driven by a relatively short femur (but not as short as in apes), not by long arms (Jungers, 1994). It is also important to note that interlimb proportionality based on lengths need not correspond to estimates of interlimb size and shape based on dia-physeal and articular dimensions (McHenry, 1978; McHenry and Berger, 1998; Green et al., 2007). Long bone lengths, especially of the hind-limb elements, are most relevant to questions about the evolution of locomotor effi ciency and the kinematics of bipedalism (e.g., Jungers, 1982; Bramble and Lieberman, 2004; Pontzer, 2005; Steudel-Numbers, 2006).BOU-VP-12/1 is another skeleton of a later australopith (possibly A. garhi), but its humerus and femur are both quite damaged and incomplete (Asfaw et al., 1999). Estimated and reconstructed lengths of these long bones are controversial and exhibit large confi dence intervals, whether based on regression or by eye (Richmond et al., 2002; Haeusler and McHenry, 2004; Reno et al., 2005). Although possible, it seems premature to conclude with any confi dence that its humerofemoral proportions were already “human-like” (Reno et al., 2005). Similarly, Homo habilis as represented by OH 62 has essentially indeterminate interlimb proportions, probably lying somewhere between those of gorillas and modern humans (Korey, 1990; Reno et al., 2005; but see Haeusler and McHenry, 2004). It appears risky at this time to develop complex evolutionary scenarios about limb length proportions predicated on BOU-VP-12/1 and/or OH 62.

Taste is a chemical sense that aids in the detection of nutrients and guides food choice. A limited number of primary qualities comprise taste. Accumulating evidence has raised a question about whether fat should be among them. Most evidence indicates triacylglycerol is not an effective taste stimulus, though it clearly contributes sensory properties to foods by carrying flavor compounds and altering texture. However, there is increasing anatomical, electrophysiological, animal behavior, imaging, metabolic, and psychophysical evidence that free fatty acids are detectable when non-taste cues are minimized. Free fatty acids varying in saturation and chain length are detectable, suggesting the presence of multiple transduction mechanisms and/or a nonspecific mechanism in the oral cavity. However, confirmation of “fatty” as a taste primary will require additional studies that verify these observations are taste specific. Oral exposure to free fatty acids likely serves as a warning signal to discourage intake and influences lipid metabolism.

Isoelectric focusing (IEF) is an electrophoretic method for the separation of proteins, according to their isoelectric points (pI), in a stabilized pH gradient. The method involves casting a layer of support media (usually a polyacrylamide gel but agarose can also be used) containing a mixture of carrier ampholytes (low-mol-wt synthetic polyamino-polycarboxylic acids). When using a poly-acrylamide gel, a low percentage gel (4%) is used since this has a large pore size, which thus allows proteins to move freely under the applied electrical field without hindrance. When an electric field is applied across such a gel, the carrier ampholytes arrange themselves in order of increasing pI from the anode to the cathode. Each carrier ampholyte maintains a local pH corresponding to its pI and thus a uniform pH gradient is created across the gel. If a protein sample is applied to the surface of the gel, where it will diffuse into the gel, it will also migrate under the influence of the electric field until it reaches the region of the gradient where the pH corresponds to its isoelectric point. At this pH, the protein will have no net charge and will therefore become stationary at this point. Should the protein diffuse slightly toward the anode from this point, it will gain a weak positive charge and migrate back towards the cathode, to its position of zero charge. Similarly diffusion toward the cathode results in a weak negative charge that will direct the protein back to the same position. The protein is therefore trapped or “focused” at the pH value where it has zero overall charge. Proteins are therefore separated according to their charge, and not size as with SDS gel electrophoresis. In practice the protein samples are loaded onto the gel before the pH gradient is formed. When a voltage difference is applied, protein migration and pH gradient formation occur simultaneously.

The brain is dependent on glucose as a primary energy substrate, but is capable of utilizing ketones such as β-hydroxybutyrate (βHB) and acetoacetate (AcAc), as occurs with fasting, prolonged starvation or chronic feeding of a high fat/low carbohydrate diet (ketogenic diet). In this study, the local cerebral metabolic rate of glucose consumption (CMRglu; μM/min/100g) was calculated in the cortex and cerebellum of control and ketotic rats using Patlak analysis. Rats were imaged on a rodent PET scanner and MRI was performed on a 7-Tesla Bruker scanner for registration with the PET images. Plasma glucose and βHB concentrations were measured and 90-minute dynamic PET scans were started simultaneously with bolus injection of 2-Deoxy-2[18F]Fluoro-D-Glucose (FDG). The blood radioactivity concentration was automatically sampled from the tail vein for 3 min following injection and manual periodic blood samples were taken. The calculated local CMRGlu decreased with increasing plasma BHB concentration in the cerebellum (CMRGlu = −4.07*[BHB] + 61.4, r² = 0.3) and in the frontal cortex (CMRGlu = −3.93*[BHB] + 42.7, r² = 0.5). These data indicate that, under conditions of ketosis, glucose consumption is decreased in the cortex and cerebellum by about 10% per each mM of plasma ketone bodies.

Silver staining methods have long been know to provide highly sensitive detection of proteins and nucleic acids following electrophoresis in agarose and polyacrylamide gels (1, 2). Silver staining technologies can be extended to other media such as blots, thin-layer chromatography (TLC), and microtiter plates. The quantification of proteins adsorbed to microtiter plate wells provides quantitative information for enzyme-linked immunosorbent assay (ELISA) and protein interaction assays. One nonradioactive procedure, copper iodide staining, is described in Chapter 72 (3). The kinetic silver staining method for measuring the amount of adsorbed protein in a microtiter plate has been developed recently. The microtiter plate assay has a sensitivity similar to copper iodide staining (5–150 ng/well) but higher precision (

Background: Lipid levels among contemporary patients hospitalized with coronary artery disease (CAD) have not been well studied. This study aimed to analyze admission lipid levels in a broad contemporary population of patients hospitalized with CAD. Methods: The Get With The Guidelines database was analyzed for CAD hospitalizations from 2000 to 2006 with documented lipid levels in the first 24 hours of admission. Patients were divided into low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), and triglyceride categories. Factors associated with LDL and HDL levels were assessed along with temporal trends. Results: Of 231,986 hospitalizations from 541 hospitals, admission lipid levels were documented in 136,905 (59.0%). Mean lipid levels were LDL 104.9 ± 39.8, HDL 39.7 ± 13.2, and triglyceride 161 ± 128 mg/dL. Low-density lipoprotein cholesterol \textless70 mg/dL was observed in 17.6% and ideal levels (LDL \textless70 with HDL ≥60 mg/dL) in only 1.4%. High-density lipoprotein cholesterol was \textless40 mg/dL in 54.6% of patients. Before admission, only 28,944 (21.1%) patients were receiving lipid-lowering medications. Predictors for higher LDL included female gender, no diabetes, history of hyperlipidemia, no prior lipid-lowering medications, and presenting with acute coronary syndrome. Both LDL and HDL levels declined over time (P \textless .0001). Conclusions: In a large cohort of patients hospitalized with CAD, almost half have admission LDL levels \textless100 mg/dL. More than half the patients have admission HDL levels \textless40 mg/dL, whereas \textless10% have HDL ≥60 mg/dL. These findings may provide further support for recent guideline revisions with even lower LDL goals and for developing effective treatments to raise HDL. © 2009 Mosby, Inc. All rights reserved.

AIM: The objective of the present study was to evaluate the influence of short-term dietary intake of farmed fish on biomarkers related to the atherosclerotic process. METHODS AND RESULTS: Lipid, inflammatory, and haemorheological variables before (T0) and after a dietary intervention with about 800 g Orbetello farmed sea bass per week for 10 weeks (T1) were evaluated in nine dyslipidemic subjects. Fish intake significantly decreased triacylglycerols (T1, 140.2+/-20.3 mg/dl versus T0, 183.3+/-29.2 mg/dl; P =0.04), whereas no significant changes for the other lipid variables have been observed. Moreover, dietary intervention significantly (P

Lipoedema is an infrequently recognized disorder in women. Lipoedema is characterized by bilateral enlargement of the legs due to abnormal depositions of subcutaneous fat associated with often mild oedema. There is substantial variability in disease severity. The diagnosis should be made as early as possible to prevent complications of the disorder, which is associated with increasing functional and cosmetic morbidity. This review describes clinical manifestations, pathogenesis, technical investigations, management and therapies of lipoedema, with the aim of optimizing management and care of patients with lipoedema.

There has been increasing interest in investigating the role of glycation as an atherogenic modification of lipoproteins; especially after the failure of antioxidant therapy to suppress the incidence of atherogenic cardiovascular diseases in high-risk individuals. Glycation of lipoproteins impairs their function. In the case of LDL, it also leads to rapid scavenger-receptor uptake by also diminishes the antiatherogenic properties of HDL. The concentration of circulating glycated LDL, even in nondiabetics, is higher than that of oxidatively modified LDL. Glycated small dense,LDL is significantly higher in nondiabetic individuals as well as in Type 2 diabetic patients. Lowering the level of small dense LDL by using statins lowers glycated LDL in Type 2 diabetic patients. This may be one of the reasons for their-effect in decreasing cardiovascular risk in these patients. Treatments that prevent glycation of lipoproteins may prevent atherosclerosis and other diabetic complications.

Objective: To compare the effects of an energy reduced, very low carbohydrate, high saturated fat diet (LC) and an isocaloric conventional high carbohydrate, low fat diet (HC) on lipids and endothelial function after 12-months. Methods: 49 overweight and obese patients (age 50.0+/-7.7 yrs, body mass index 33.7+/-4.1 kg/m2) were randomly assigned to either an energy restricted (∼6-7MJ, 30% energy deficit), isocaloric LC or HC for 52 weeks. Body weight, lipids, flow mediated dilatation (FMD), augmentation index (AIx) and pulse wave velocity (PWV) were assessed pre- and post intervention. Results: Weight loss was similar in both groups (LC -14.9+/-10.5 kg, HC -11.5+/-7.4 kg; P=0.20). There was a significant time x diet effect for FMD (P= 0.045); FMD decreased on the LC diet (5.70+/-3.59% to 3.65+/-2.65%, P=0.001) but remained unchanged on the HC diet (5.86+/-2.54% to 5.51+/-3.52%; P=0.60). PWV improved in both groups but AIx did not change. LDL cholesterol increased by 0.6 mmol/L in the LC group (19%) and did not change in the HC group. Despite a fall of triglyceride in the LC group of 0.58 mmol/L, apoB increased by 3% in the LC group and fell by 6% in the HC group. HDL cholesterol increased by 21% in the LC group and 5% in the HC group. Conclusions: Both diets achieved similar reductions in body weight and improvements in PWV. However LC impaired FMD, suggesting that consumption of LC may have adverse effects on cardiovascular risk via higher LDL cholesterol.

Low-sodium Dietary Approaches to Stop Hypertension (DASH) diets are base producing but restrict red meat without clear justification. We hypothesized that a vitality diet (VD), a low-sodium DASH-type diet with a low dietary acid load containing 6 servings of 100 g cooked lean red meat per week, would be more effective in reducing blood pressure (BP) compared with a higher acid load reference healthy diet (RHD) based on general dietary guidelines to reduce fat intake and increase intake of breads and cereals. A randomized, parallel dietary intervention study was conducted to compare the BP-lowering effect of these 2 diets in postmenopausal women with high/normal BP. Women were randomly assigned to follow either VD or RHD for 14 weeks. Home BP was measured daily with an automated BP monitor under standard conditions. Of 111 women commencing the study, 95 completed (46 VD, 49 RHD). Systolic BP (SBP) throughout the intervention was lower in the VD group compared to the RHD group (repeated-measures analysis of variance time by diet, P = .04), such that at the end of the study, the VD had a fall of SBP by 5.6 ± 1.3 mm Hg (mean ± SEM) compared with a fall of 2.7 ± 1.0 mm Hg in the RHD (group difference, P = .08). When only those taking antihypertensive medications were assessed, the VD (n = 17) had a significant fall of 6.5 ± 2.5 mm Hg SBP (P = .02) and 4.6 ± 1.4 mm Hg diastolic BP (P = .005) after 14 weeks, and their BP was lower than that of the RHD group (n = 18) throughout the study (P

2009 May;89(5):1425-32.. Epub 2009 Feb 11.

Provides an alternative history of nutrition in the U.S. that focuses on the power of scientific language. As nutritional studies proliferate, producing more and more knowledge about connection between diet and health, Americans seem increasingly confused about what to eat to stay healthy. In Measured Meals, Jessica J. Mudry looks at language used in the United States to communicate about health and nutrition, and reveals its effects on reframing, reshaping, and controlling what and how Americans eat. Analyzing the USDA and American federal food guidelines over the past one hundred years, Mudry shows how the language of nutrition has evolved over time. She critiques trend of discussing food in terms of quantification : calories, vitamins, and serving sizes. She also examines how organizations such as the USDA attempt to legislate a healthy diet by mandating quantities of food based on measurable nutrients, revealing the power of language to make meaning and influence social action--Publisher's description.

Membrane proteins serve many fundamental functions for cells such as cell-cell interactions, signal transduction, and molecular transport both within and between cells. Bioinformatic analysis has estimated that transmembrane domain containing proteins represent about 20–30% of all open reading frames in sequenced genomes (1). In addition, many proteins are anchored to the membranes via lipidic posttranslational modifications such as glycosylphos-phatidyl inositol anchors or via interaction with other membrane proteins. In total, embrane anchored proteins represent a considerable portion of all cellular proteins. They also represent 70% of all known drug targets, which prioritize them in biomedical research (2). Consequently, profiling membrane proteomes will aid in both understanding basic biological processes and discovering novel targets for therapeutic agents. Membrane proteomics, however, is rendered difficult on several levels.

Peptides are a large class of molecules that differ in size, charge, conformation, hydrophobiCity>, and the ability to form biospecific complexes. Peptides play an important role in many physiological processes, including the regulation of pain, blood pressure, and immune response, and can act as antibiotics, coenzymes/enzyme inhibitors, drugs, growth stimulators, hormones, neurotransmitters, and toxins (1).Since the identification of leu-enkephalin in the 1970s, the role of neuropeptides in the regulation of the central nervous system and signal transduction has been extensively investigated (1). Identification of specific peptides and their functions may permit the design of pharmacologically active synthetic analogs that could be used for the treatment of neurological diseases such as Parkinson’s and Alzheimer’s. Peptide analysis is also important for proteomics research. Digestion of a protein yields distinct peptide mixtures, which are separated and mapped in order to characterize the parent molecule. In some cases, multidimensional separations are needed to achieve resolution and identification of all peptide components.

Nonalcoholic fatty liver disease (NAFLD) is associated with insulin resistance, obesity, and other features of metabolic syndrome and is known to be the most common cause for abnormal liver enzymes. The recent surge in the number of patients with NAFLD has been accompanied by an increase in research on potential treatment options, particularly weight loss and dietary interventions. Given the growing interest on the role of carbohydrates in the prevention and treatment of NAFLD, this review discusses the relationship between the amount of carbohydrates in the diet and effects on NAFLD, with special emphasis on a low-carbohydrate diet. We discuss the role of insulin resistance in the pathophysiology of NAFLD and provide an overview of various popular diets and their role as a treatment option for NAFLD. Additional large, longer-duration trials studying the efficacy of a low-carbohydrate diet in the treatment and prevention of NAFLD are eagerly awaited.

SDS-PAGE (Chapter 21) is probably the most commonly used gel electro-phoretic system for analyzing proteins. However, it should be stressed that this method separates denatured protein. Sometimes one needs to analyze native, nondenatured proteins, particularly if wanting to identify a protein in the gel by its biological activity (for example, enzyme activity, receptor binding, antibody binding, and so on). On such occasions it is necessary to use a nondenatur-ing system such as described in this chapter. For example, when purifying an enzyme, a single major band on a gel would suggest a pure enzyme. However this band could still be a contaminant; the enzyme could be present as a weaker (even nonstaining) band on the same gel. Only by showing that the major band had enzyme activity would you be convinced that this band corresponded to your enzyme. The method described here is based on the gel system first described by Davis (1). To enhance resolution a stacking gel can be included (see Chapter 21 for the theory behind the stacking gel system).

Background: Mucin deposition on the shins is considered as an indicator of pretibial myxoedema, which is typically seen in patients with Graves’ disease. Objective: The purpose of this study was to report the clinical and histopathological features of a group of patients with pretibial mucinosis in the absence of thyroid disease. Methods: Five patients are included in this series and studied both clinically and histologically and compared with similar cases in the literature. Results: All patients were middle aged or elderly. Four patients were women. They were characterized clinically by morbid obesity and bilateral lower extremity pitting oedema sparing the feet. Semitranslucent papules and/or nodules and sometimes vesicles were found on the shins. Characteristic histological features include (i) hyperorthokeratosis with epidermal atrophy and effacement of the rete ridge pattern, (ii) oedema in the papillary and upper part of the reticular dermis with mucin deposition stained positively with alcian blue and colloidal iron, (iii) angioplasia in the upper part of dermis with upward-running, increased and thickened capillary vessels and (iv) variable fibrosis in the reticular dermis with separation of collagen bundles and increased stellate or linear fibroblasts. A hypocaloric diet was given in two cases, and an important weight loss was observed, which was accompanied by a marked improvement of the pretibial mucinosis. Conclusions: Pretibial mucinosis is a histological feature associated with morbid obesity and lymphoedematous features of the legs that should be distinguished from true pretibial myxoedema. The term of ‘obesity-associated lymphoedematous mucinosis’ seems to be appropriate for this condition.

Brain enlargement, reduction in molar tooth size, increased stature and other features of early Homo did not evolve in a vacuum. These evolutionary changes refl ect shifts in a complex web of relationships among their populations, between early Homo and other hominin species, and between their biotic community and abiotic forces (i.e., climate change). Archeological evidence complements and balances inferences from hominin fossil remains, non-hominin vertebrate paleontology, geology, and other component fi elds of paleoan-thropology. This paper represents an attempt to pull together the various strands of its authors' expertise to shed light on the origins and adaptations of early Homo. It is not intended to be a comprehensive review of Oldowan sites, their chronology, lithic typology, paleontological associations, and interpretive issues. For recent overviews of these subjects, see Plummer (2004), Schick and Toth (2006) as well as papers in Toth and Schick (2006), Ungar (2007) and Hovers and Braun (2009). The coincidence of knapped stone tools, butchery-marked bones and fossil remains of early Homo is usually linked to increased hominin carnivory. This paper reviews evidence for this hypothesis, and considers alternative hypotheses as well.

As aptly observed by Aiello et al. (2000), any attempt to reconstruct human evolutionary history depends upon reliable hypotheses pertaining to the species groups that are represented in the hominin fossil record. There has been a notable lack of consensus regarding the species of Homo that are represented in the Plio-Pleistocene karst cave deposits of South Africa (Howell, 1978; Clarke, 1977a, b, 1985a, b, 1994; Chamberlain, 1987; Rightmire, 1990; Wood, 1991; Tobias, 1991; Kimbel and Rak, 1993; Grine et al., 1993, 1996; Ahern, 1998; Kuman and Clarke, 2000; Grine, 2001, 2005; Curnoe, 2001, 2002, 2008; Prat, 2002; Dunsworth and Walker, 2002; Schwartz and Tattersall, 2003; Curnoe and Tobias, 2006; Smith and Grine, 2008). To some degree, this is because these discussions have been restricted to a few incomplete mandibles and crania from the sites of Sterkfontein and Swartkrans. However, there are larger collections of teeth from these and other sites that have been attributed to this genus and which might shed light on this issue. We here attempt to assess the phenetic affi nities of some of these dental remains by comparison with penecontemporaneous East African fossils that have been referred to H. rudolfensis, H. habilis and H. erectus (=H. ergaster).Overall crown size and shape may sometimes serve to distinguish the molar teeth Homo from those of Australopithecus and Paranthropus, but it is not clear that these attributes are necessarily useful in differentiating among species of Homo. At the same time, however, the utility of molar cusp sizes and proportions in species-level distinctions has been explored by a number of workers (Corruccini, 1977; Lavelle, 1978; Hills et al., 1983; Hartman, 1989; Uchida, 1991, 1992, 1998a, b; Wood and Xu, 1991; Macho and Moggi-Cecchi, 1992; Matsumura et al., 1992; Smith, 1999; Bailey, 2004). Such data have been shown to be of some use in taxonomic evaluations of the hominin fossils from East Africa (Wood et al., 1983; Wood and Uytterschaut, 1987; Wood and Engleman, 1988; Suwa, 1988, 1990; Suwa et al., 1994, 1996).

Most plant tissues are not a ready source for protein extraction and need specific precautions. The cell wall and the vacuole make up the majority of the cell mass, with the cytosol representing only 1 to 2 % of the total cell volume. Subsequently, plant tissues have a relatively low protein content compared to bacterial or animal tissues. The cell wall and the vacuole are associated with numerous substances responsible for irreproducible results such as proteolytic breakdown, streaking and charge heterogeneity. Most common interfering substances are phenolic compounds, proteolytic and oxidative enzymes, terpenes, pigments, organic acids, inhibitory ions, and carbohydrates.

Long-term climate change seems to be modulated primarily by tectonic changes at both the global and local scale (Maslin et al., 2001). Late Cenozoic global cooling has been ascribed to both the uplift of Tibet (Ruddiman and Raymo, 1988), and the closure of the Panama Isthmus (Haug and Tiedemann, 1998), although the exact role of atmospheric carbon dioxide is still unclear (Sundquist and Visser, 2004). In East Africa, long-term climate change is also controlled by local tectonics, especially the dynamic development of the branching East African Rift System (Sepulchre et al., 2006). Early hominin evolution in East Africa thus occurs at the same time as both long-term global cooling and extensive local tectonic changes. There is a compelling need to understand how these two environmental factors interact at the local scale and affect fl ora and fauna living in the East African Rift. The geologic record of the last 5 million years demonstrates that both local and global infl uences can lead to extremely rapid environmental change (Maslin and Christensen, 2007).

Sample preparation is a very crucial step in two-dimensional (2-D) gel electro-phoresis in which the proteins of the sample are brought into a state where they can be separated by isoelectric focusing in the first dimension. The proteins must be denatured, reduced, and solubilized, and they must be kept so during electrophoresis without changing their pI. The buffer for this purpose is traditionally called the lysis buffer.Most bacterial studies aim to solubilize as many proteins as possible to obtain the best possible representation of the total protein content or protein expression under the investigated biological circumstances. However, prefractionation, or the successive application of different chemical reagents, can be used to investigate proteins with certain characteristics. Note that the gels will reflect the proteome of the bacteria at the time the proteins are solubilized and that preceding centrifugations or other manipulations may stress the bacteria, thereby influencing the protein profile.

The importance of bodily fluids as a source of clinically relevant biomarkers/ surrogate markers of human disease has increased significantly over the last decade (1, 2), and modern proteomic methods have evolved and been adapted to meet the demand. The specific challenges facing serum analysis include the wide dynamic range in the concentration of individual components and the tremendous number of potential variants of glycosylated proteins (3). The most dominant plasma proteins, albumin and immunoglobulin (Ig)G, typically comprise up to 70% of the plasma proteome in abundance. To enable the majority of the remaining, far less abundant proteins to be better visualized by two-dimensional gel electrophoresis (2-DE), these two proteins must first be removed or, at least, depleted in relative concentration. There are a number of currently available commercial products from a range of suppliers that enable albumin depletion by chemical affinity, exploiting the remarkable albumin-binding ability of structures closely related to the reactive dye molecule Cibacron blue 3GA (4), and the IgG binding properties of protein G (5). The blue dye has been shown to have a special affinity for proteins containing the dinucleotide fold, a structural feature that is common to several classes of proteins (4). Albumin can be separated from other plasma proteins using lectin affinity, as it is not normally glycosylated, while the majority of classical plasma proteins are. This approach allows both enrichment of lower-abundance proteins, and the study of differences in glyco-protein profiles (6). Highly effective depletion of albumin using monoclonal antibody selection has also been demonstrated, and coupled with protein G/IgG depletion (7).Recent research has identified 325 distinct proteins from 1800 2-D gel protein features following multi-component immunoaffinity extraction and further comprehensive chromatographic fractionation (8). Depletion of IgG by a protein G resin can also be coupled with NaCl/Ethanol precipitation to deplete albumin (9). However, depletion can cause problems in itself. The concentration of the high abundant proteins varies considerably within bodily fluids. Therefore once the samples are depleted, there is a significant change in the relative concentration of the protein constituents from the original sample. As an example in our own studies the percentage protein remaining after depletion in cerebrospinal fluid has an average of 17.9% with a standard deviation of 6.6%. This could be overcome by loading according to sample volumes rather than protein loading, which is often the norm in proteomic experiments (10). A major drawback is that the depletion not only adds further steps in sample processing, but undoubtedly alters the protein constituents within the sample as high abundant proteins bind other proteins. Consequently, the depletion may complicate quantitative comparisons and result in the loss of potential biomarkers (11).

Yeasts are the focus of much research, both in their role as pathogens and as biotechnically important organisms, and not the least in their role as model systems for eukaryotic cells. In particular, Saccharomyces cerevisiae has also been the object of several proteomics-related efforts. However, the preparation of protein extract from yeast is complicated by the presence of a cell wall of mainly chitin and glucans, which needs to be disrupted in the extraction process. Several methods presenting solutions to this problem, in connection with sample preparation for two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), have been described (1, 2), in which the cell wall is broken either by vortexing in the presence of glass beads or by sonication.We have found the method described below to be robust and to yield reproducible results in several studies (3–6). It is furthermore easy to perform and does not require any specialized equipment.