Synthesis and evaluation of stimulatory properties of Sphingomonadaceae glycolipids

Glycosphingolipids (GSLs) from the Sphingomonadaceae family of bacteria have been reported to be potent stimulators of natural killer T cells. These glycolipids include mono-, tri- and tetraglycosylceramides. Here we have prepared the GSL-1 to GSL-4 series of glycolipids and tested their abilities to stimulate natural killer T cells. Among these glycolipids, only GSL-1 (1) is a potent stimulator. Using a series of synthetic diglycosylceramides, we show that oligoglycosylceramides from Sphingomonadaceae are not effectively truncated to GSL-1 in lysosomes in antigen-presenting cells, possibly because the higher-order GSLs are poor substrates for lysosomal acyltransfer enzymes.     

Capturing and amplifying impurities from purified recombinant monoclonal antibodies via peptide library beads: a proteomic study

Capture and amplification of low-level contaminants in purified preparations of recombinant DNA products is described here in the case of mAb meant for human consumption. Such a process is based on treatment with a vastly heterogeneous ligand library composed of hexapeptides bound to a polyhydroxymethacrylate resin. Upon this treatment, a protein solution is recovered with "normalized" relative concentration ratios, in which high-abundance proteins are strongly reduced and rare proteins are highly concentrated. Upon 2-D map analysis, the relatively few spots present in control monoclonals were seen to increase in number, reaching >100 visible polypeptide chains in the pI/M(r) plane. Most of these newly emerged spots were subjected to MS analysis and were found to be composed mainly of three classes of proteins: those derived from proteins present in the culture broth (notably albumin and transferrin), fragments of the desired final product, covering M(r) ranges from as low as 5 up to 45 kDa and some aggregates of light and heavy chains of Igs (mostly dimers and trimers). This ligand library thus appears to be a formidable tool for exploring and bringing to the limelight the "hidden proteome".     

The cytoplasmic phosphoproteome of the Gram-negative bacterium Campylobacter jejuni: evidence for modification by unidentified protein kinases

We have undertaken a comprehensive analysis of cytoplasmic protein phosphorylation in Campylobacter jejuni by mass spectrometric identification of phosphoproteins and localization of the sites of modification by phosphopeptide analyses. Cell extracts, enriched for phosphoproteins using Fe(III) IMAC or commercial phosphoprotein purification kits, were analyzed by 1-D and 2-D SDS-PAGE and subjected to mass fingerprinting by in-gel tryptic digestion and MALDI-TOF MS. Fifty-eight phosphopeptides were identified from 1-D gel bands by nano-LC-MS/MS and automated searching in a C. jejuni ORF database resulting in the unequivocal identification of 36 phosphoproteins of diverse function. In addition to elongation factors and chaperonins, which have been reported to be phosphorylated in other bacteria, the major phosphoproteins included bacterioferritin and superoxide dismutase. The sequences around the phosphorylated Ser and Thr residues are indicative of specific kinases being responsible for some of the modifications. However, many of the other identified proteins are enzymes that have phosphorylated substrates, including ATP, hence other modifications may arise from autophosphorylation. Comparative analyses of IMAC extracts from the Escherichia coli strain AD202 and Helicobacter pylori resulted in the identification of homologs of six of the C. jejuni phosphoproteins, though their overall phosphoproteome maps were distinctly different.     

Morphological variation of the spermatheca in the garden snail Cantareus aspersus

Spermathecal morphology is known to play an important role in postcopulatory sexual selection of many invertebrates. In helicid land snails, the spermatheca is subdivided into tubules, whose number is sometimes subject to a strong inter-individual variation. Significance of this variation for postcopulatory sexual selection is unknown, but it might be related to cryptic female choice. In the present work, we have investigated the fine multi-tubular structure of the sperm storage organ in Cantareus aspersus. We found between 3 and 13 tubules per individual in a single population, which represents a degree of variation rarely observed in helicid land snails.     

The complex p25/Cdk5 kinase in neurofibrillary degeneration and neuronal death: the missing link to cell cycle

Emergence of the cell cycle hypothesis in neurodegenerative disease comes from the numerous lines of evidence showing a tight link between "cell cycle-like reactivation" and neuronal death. Terminally differentiated neurons remain in G0 phase and display, compared to proliferating cells, an opposite regulation pattern of cell cycle markers in that most of the key activators and inhibitors are respectively down- and up-regulated. It has been clearly established that any experimental attempt to force terminally differentiated neurons to divide ultimately leads to their death. Conversely, cell cycle blockade in experimental models of neuronal death is able to rescue neurons. Hence, cell cycle deregulation is certainly among mechanisms governing neuronal death. However, many questions remain unresolved, especially those related to which molecular mechanisms trigger cell cycle deregulation and how this deregulation leads to cell death. In the present review, we focus on neurodegeneration in Alzheimer's disease and discuss the cell cycle deregulation related to this neurodegenerative pathology. Finally, we emphasize the role of p25/Cdk5 kinase complex in this pathological process through retinoblastoma protein phosphorylation and derepression of E2F-responsive genes and other actors such as cdc2, cyclins, and MCM proteins.     

Alcohol consumption and type 2 diabetes among older adults: the Cardiovascular Health Study

Objective: The objective was to examine the role of total and beverage‐specific alcohol consumption on the incidence of type 2 diabetes mellitus (DM) among elderly men and women. Research Methods and Procedures: We studied prospectively 4655 participants of the Cardiovascular Health Study who were free of DM at baseline. Alcohol consumption was obtained at baseline and during follow‐up examinations. DM was defined using fasting glucose and/or use of hypoglycemic medications. We used Cox proportional hazard models to estimate adjusted relative risks of diabetes across alcohol categories. Results: During a mean follow‐up of 6.3 years, 234 incident cases of DM were documented. Compared with never drinkers, hazard ratios [95% confidence interval (CI)] for DM were 0.7 (0.3 to 1.4), 0.5 (0.3 to 0.9), 0.6 (0.4 to 1.1), and 0.8 (0.4 to 1.3) for former drinkers and current drinkers of <1, 1 to 6, and 7+ drinks per week, respectively, for men after adjustment for age, BMI, education, and smoking. Corresponding values for women were 1.2 (0.6 to 2.3), 0.7 (0.4 to 1.1), 0.6 (0.3 to 1.1), and 0.4 (0.2 to 1.0), respectively. A reduced risk of DM was observed with all types of beverage consumed. Similar findings were observed when we repeated the above analyses using simple or weighted cumulative alcohol update and covariates over time. Discussion: Light to moderate alcohol consumption was associated with a lower incidence of DM among elderly people, irrespective of the type of beverage consumed.     

Overall and central obesity and risk of type 2 diabetes in U.S. black women

Objective: Obesity has risen to epidemic proportions in the United States, leading to an emerging epidemic of type 2 diabetes. African‐American women are disproportionately affected by both conditions. While an association of overall obesity with increasing risk of diabetes has been documented in black women, the effect of fat distribution, specifically abdominal obesity, has not been studied. We examined the association of BMI, abdominal obesity, and weight gain with risk of type 2 diabetes. Research Methods and Procedures: During eight years of follow‐up of 49,766 women from the Black Women's Health Study, 2472 incident cases of diabetes occurred. Cox proportional hazard models were used to estimate incidence rate ratios (IRRs), with control for age, physical activity, family history of diabetes, cigarette smoking, years of education, and time period of data collection. Results: Sixty‐one percent of participants had a BMI ≥25 kg/m² (WHO definition of overweight). Compared with a BMI of <23 kg/m², the IRR for a BMI of >45 kg/m² was 23 (95% confidence interval, 17.0 to 31.0). The IRR for the highest quintile of waist‐to‐hip ratio relative to the lowest was 2.3 (95% confidence interval, 2.0 to 2.7) after control for BMI. Furthermore, at every level of BMI, an increased risk was observed for high waist‐to‐hip ratio relative to low. Discussion: Central obesity, as well as overall obesity, is a strong risk factor for diabetes in African‐American women. Efforts to reduce the prevalence of obesity in African‐American women are of paramount importance.     

Bim, Bad and Bmf: intrinsically unstructured BH3-only proteins that undergo a localized conformational change upon binding to prosurvival Bcl-2 targets

All BH3-only proteins, key initiators of programmed cell death, interact tightly with multiple binding partners and have sequences of low complexity, properties that are the hallmark of intrinsically unstructured proteins (IUPs). We show, using spectroscopic methods, that the BH3-only proteins Bim, Bad and Bmf are unstructured in the absence of binding partners. Detailed sequence analyses are consistent with this observation and suggest that most BH3-only proteins are unstructured. When Bim binds and inactivates prosurvival proteins, most residues remain disordered, only the BH3 element becomes structured, and the short alpha-helical molecular recognition element can be considered to behave as a 'bead on a string'. Coupled folding and binding is typical of many IUPs that have important signaling roles, such as BH3-only proteins, as the inherent structural plasticity favors interaction with multiple targets. This understanding offers promise for the development of BH3 mimetics, as multiple modes of binding are tolerated.