Dynamics of T cells and TCR excision circles differ after treatment of acute and chronic HIV infection

We quantified T cell proliferation and thymic function in primary HIV infection (PHI; n = 19) and chronic HIV infection (CHI; n = 14) by measuring Ki67 staining and TCR excision circle (TREC) number. After antiretroviral therapy of PHI there is a profound decrease in the number and percentage of Ki67(+) T cells (<6% Ki67(+)) with no significant increase in TREC per million cells and a transient increase in TREC per milliliter. In contrast, after antiretroviral therapy of CHI there is a reduction in the percentage but little change in the total number of Ki67(+)CD4(+) T cells associated with increases in both TREC per million cells and TREC per milliliter. Using a mathematical model that accounts for proliferation, death, and redistribution of T cells, we find that redistribution is consistent with the TREC changes observed during treatment of PHI and that an increase in thymic output is needed to explain the increase in TREC during treatment of CHI. Consideration of TREC per milliliter shows that changes in proliferation alone cannot explain the changes in TREC. In addition, although increased proliferation of memory cells in HIV infection has been established, we find no difference in TREC per million CD45RA(-) "memory" T cells between healthy and infected individuals (p = 0.154 for CD4(+); p = 0.383 for CD8(+)). Finally, although the number of TREC per million cells is always much lower in memory T cells than in naive T cells, in the setting of HIV infection, given that memory cells make up a larger proportion of total T cells, we find that 50% of TREC per milliliter in CD4(+) T cells is harbored in the CD45RA(-) "memory" subset of our infected subjects.     

Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice

A wide range of protein acyl modifications has been identified on enzymes across various metabolic processes; however, the impact of these modifications remains poorly understood. Protein glutarylation is a recently identified modification that can be nonenzymatically driven by glutaryl-CoA. In mammalian systems, this unique metabolite is only produced in the lysine and tryptophan oxidative pathways. To better understand the biology of protein glutarylation, we studied the relationship between enzymes within the lysine/tryptophan catabolic pathways, protein glutarylation, and regulation by the deglutarylating enzyme sirtuin 5 (SIRT5). Here, we identify glutarylation on the lysine oxidation pathway enzyme glutaryl-CoA dehydrogenase (GCDH) and show increased GCDH glutarylation when glutaryl-CoA production is stimulated by lysine catabolism. Our data reveal that glutarylation of GCDH impacts its function, ultimately decreasing lysine oxidation. We also demonstrate the ability of SIRT5 to deglutarylate GCDH, restoring its enzymatic activity. Finally, metabolomic and bioinformatic analyses indicate an expanded role for SIRT5 in regulating amino acid metabolism. Together, these data support a feedback loop model within the lysine/tryptophan oxidation pathway in which glutaryl-CoA is produced, in turn inhibiting GCDH function via glutaryl modification of GCDH lysine residues and can be relieved by SIRT5 deacylation activity.     

Genetic selection for protein solubility enabled by the folding quality control feature of the twin-arginine translocation pathway

One of the most vexing problems facing structural genomics efforts and the biotechnology enterprise in general is the inability to efficiently produce functional proteins due to poor folding and insolubility. Additionally, protein misfolding and aggregation has been linked to a number of human diseases, such as Alzheimer's. Thus, a robust cellular assay that allows for direct monitoring, manipulation, and improvement of protein folding could have a profound impact. We report the development and characterization of a genetic selection for protein folding and solubility in living bacterial cells. The basis for this assay is the observation that protein transport through the bacterial twin-arginine translocation (Tat) pathway depends on correct folding of the protein prior to transport. In this system, a test protein is expressed as a tripartite fusion between an N-terminal Tat signal peptide and a C-terminal TEM1 beta-lactamase reporter protein. We demonstrate that survival of Escherichia coli cells on selective medium expressing a Tat-targeted test protein/beta-lactamase fusion correlates with the solubility of the test protein. Using this assay, we isolated solubility-enhanced variants of the Alzheimer's Abeta42 peptide from a large combinatorial library of Abeta42 sequences, thereby confirming that our assay is a highly effective selection tool for soluble proteins. By allowing the bacterial Tat pathway to exert folding quality control on expressed target protein sequences, we have generated a powerful tool for monitoring protein folding and solubility in living cells, for molecular engineering of solubility-enhanced proteins or for the isolation of factors and/or cellular conditions that stabilize aggregation-prone proteins.     

Sarcopenic obesity and inflammation in the InCHIANTI study.

The aging process is often paralleled by decreases in muscle and increases in fat mass. At the extreme these two processes lead to a condition known as "sarcopenic obesity" (Roubenoff R. Ann NY Acad Sci 904: 553-557, 2000). Research suggests that inflammatory cytokines produced by adipose tissue, especially visceral fat, accelerate muscle catabolism and thus contribute to the vicious cycle that initiates and sustains sarcopenic obesity. We tested the hypothesis that obesity and poor muscle strength, hallmarks of sarcopenic obesity, are associated with high circulating levels of proinflammatory cytokines in a random sample of the residents of two municipalities in the Chianti geographic area (Tuscany, Italy). The study sample consisted of 378 men and 493 women 65 yr and older with complete data on anthropometrics, handgrip strength, and inflammatory markers. Participants were cross-classified according to sex-specific tertiles of waist circumference and grip strength and according to a cut point for obesity of body mass index > or =30 kg/m(2). After adjusting for age, sex, education, smoking history, physical activity, and history of comorbid diseases, components of sarcopenic obesity were associated with elevated levels of IL-6, C-reactive protein, IL-1 receptor antagonist, and soluble IL-6 receptor (P < 0.05). Our findings suggest that global obesity and, to a greater extent, central obesity directly affect inflammation, which in turn negatively affects muscle strength, contributing to the development and progression of sarcopenic obesity. These results suggest that proinflammatory cytokines may be critical in both the development and progression of sarcopenic obesity.     

The Influence of Predator Sex on Chemically Mediated Antipredator Response in the Wolf Spider Pardosa milvina (Araneae: Lycosidae)

The wolf spider, Pardosa milvina, reduces activity in the presence of chemical cues (silk and excreta) from a larger predatory wolf spider, Hogna helluo. Hogna is sexually dimorphic in body size and males and females differ in their propensity to attack prey. Consequently, each sex may present different levels of risk to Pardosa. We measured predation risk of Pardosa in the presence of male or female Hogna. We also assessed Pardosa antipredator responses and survival in the presence or absence of previously deposited chemical cues from male or female Hogna. In the absence of predator chemical cues, Pardosa survived significantly longer in the presence of male Hogna compared with female Hogna. We then assessed Pardosa survival in the presence of chemical cues from each Hogna sex by placing Pardosa in containers previously occupied by a female Hogna, a male Hogna, or no Hogna (control). We then introduced a female Hogna into each container and measured predation latency. Pardosa survived significantly longer in the presence of female and male cues compared with the control treatment. Median survival time of Pardosa was over four times longer on substrates with female Hogna cues compared with male cues, but this difference was not statistically significant. We tested Pardosa activity levels in the presence of chemical cues from male or female Hogna. Both Hogna sexes were maintained in separate containers after which we placed an adult female Pardosa in one of the containers or a blank control container. Pardosa significantly decreased activity in the presence of chemical cues from either sex relative to the control. Activity was lowest on substrates with female Hogna cues, but not significantly lower than on substrates with male Hogna cues. Results suggest that chemical information from male or female Hogna significantly reduces Pardosa activity which results in increased survival.     

Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli

We have developed a protocol to produce large quantities of high purity myristoylated and non-myristoylated neuronal calcium sensor 1 (NCS-1) protein. NCS-1 is a member of the neuronal calcium sensor (NCS) family and plays an important role in modulating G-protein signaling and exocytosis pathways in cells. Many of these functions are calcium-dependent and require NCS-1 to be modified with an N-terminal myristoyl moiety. In our system, a C-terminally 6x His-tagged variant of NCS-1 was co-expressed with yeast N-myristoyltransferase (NMT) in ZYP-5052 auto-induction media supplemented with sodium myristate (100-200 microM). With optimized growth conditions and a high capacity metal affinity purification scheme, >50mg of homogenous myristoylated NCS-1 is obtained from 1L of culture in a single step. The properties of the C-terminally tagged NCS-1 variants are indistinguishable from those reported for untagged NCS-1. Using this system, we have also isolated and characterized mutant NCS-1 proteins that have attenuated (NCS-1 E120Q) and abrogated (NCS-1 DeltaEF) ability to bind calcium. The large quantities of NCS-1 proteins isolated from small culture volumes of auto-inducible media will provide the necessary reagents for further biochemical and structural characterization. The affinity tag at the C-terminus of the protein provides a suitable reagent for easily identifying binding partners of the various NCS-1 constructs. Additionally, this method could be used to produce other recombinant proteins of the NCS family, and may be extended to express and isolate myristoylated variants of other proteins.     

Nurse specialist in family planning.

In an experimental clinic, run by nurse specialists in family planning, a total of 768 patients were seen in the first year. Oral contraception was dispensed for 377 patients and 187 intrauterine devices (IUCDs) were inserted; a further 204 IUCD patients attended only for follow-up visits. All side effects were adequately diagnosed by the nurse specialist.     

Arylphthalazines as potent,and orally bioavailable inhibitors of VEGFR-2

A series of arylphthalazine derivatives were synthesized and evaluated as antagonists of VEGF receptor II (VEGFR-2). IM-094482 57, which was prepared in two steps from commercially available starting materials, was found to be a potent inhibitor of VEGFR-2 in enzymatic, cellular and mitogenic assays (comparable activity to ZD-6474). Additionally, 57 inhibited the related receptor, VEGF receptor I (VEGFR-1), and showed excellent exposure when dosed orally to female CD-1 mice.     

The Evolution of RecD Outside of the RecBCD Complex

The common understanding of the function of RecD, as derived predominantly from studies in Escherichia coli, is that RecD is one of three enzymes in the RecBCD double-stranded break repair DNA recombination complex. However, comparative genomics has revealed that many organisms possess a recD gene even though the other members of the complex, recB and recC, are not present. Further, bioinformatic analyses have shown that there is substantial sequence dissimilarity between recD genes associated with recB and recC (recD1), and those that are not associated with recBC (recD2). Deinococcus radiodurans, known for its extraordinary DNA repair capability, is one such organism that does not possess either recB or recC, and yet does possess a recD gene. The recD of D. radiodurans was deleted and this mutant was shown to have a capacity to repair double-stranded DNA breaks equivalent to wild-type. The phylogenetic history of recD was studied using a dataset of 120 recD genes from 91 fully sequenced species. The analysis focused upon the role of gene duplication and functional genomic context in the evolution of recD2, which appears to have undergone numerous independent events resulting in duplicate recD2 genes. The role of RecD as part of the RecBCD complex appears to have a divergence from an earlier ancestral RecD function still preserved in many species including D. radiodurans.