Characterization of five human cDNAs with homology to the yeast SIR2 gene: Sir2-like proteins (sirtuins) metabolize NAD and may have protein ADP-ribosyltransferase activity.

The yeast Sir2 protein regulates epigenetic gene silencing and as a possible antiaging effect it suppresses recombination of rDNA. Studies involving cobB, a bacterial SIR2-like gene, have suggested it could encode a pyridine nucleotide transferase. Here five human sirtuin cDNAs are characterized. The SIRT1 sequence has the closest homology to the S. cerevisiae Sir2p. The SIRT4 and SIRT5 sirtuins more closely resemble prokaryotic sirtuin sequences. The five human sirtuins are widely expressed in fetal and adult tissues. Recombinant E. coli cobT and cobB proteins each showed a weak NAD-dependent mono-ADP-ribosyltransferase activity using 5, 6-dimethylbenzimidazole as a substrate. Recombinant E. coli cobB and human SIRT2 sirtuin proteins were able to cause radioactivity to be transferred from [32P]NAD to bovine serum albumin (BSA). When a conserved histidine within the human SIRT2 sirtuin was converted to a tyrosine, the mutant recombinant protein was unable to transfer radioactivity from [32P]NAD to BSA. These results suggest that the sirtuins may function via mono-ADP-ribosylation of proteins.     

In vitro long-term performance study of a near-infrared fluorescence affinity sensor for glucose monitoring

The long-term in vitro performance of a fluorescence affinity sensor for transdermal blood glucose monitoring was investigated. Affinity binding of fluorescently labeled concanavalin A (ConA) was used in this application, as previously described by Ballerstadt and Schultz [Anal. Chem. 17 (2000) 4185-4192). In this paper, the fluorescence emission of the sensor was extended to the near infrared (670 nm) using Alexa647 as the fluorochrome conjugated to concanavalin A. Sensors were alternately exposed to glucose solutions having concentrations of 2.5 and 20 mM with a dwell time of 3 h. The optical output of the sensors was monitored over a 4-month period. The sensors showed an initial increase in fluorescence over the first 3-4 weeks before gradually decreasing, with an approximately linear drop of 25% per month. In order to understand the reasons for the decrease in fluorescence output, further experiments were conducted, including time-dependent membrane leakage tests, solubility tests of ConA, temperature-dependent activity tests of ConA, and fluorescence photo-bleaching tests. From these results, it became evident that the decrease in fluorescence was not due to denaturation of the ConA. The most likely cause was leakage of the fluorescently labeled ConA through the interface between the outer sealant and the membrane. This problem is considered to be solvable and future publications will address this issue. Extrapolation of the experimental data suggests that a leak-proof sensor would be remarkably stable with a fluorescence decrease of only 15% over a 1-year period.     

Closing the loop between neurobiology and flight behavior in Drosophila

Fruit flies alter flight direction by generating rapid stereotyped turns called saccades. Using a combination of tethered and free-flight methods, both the aerodynamic mechanisms and the sensory triggers for saccades have been investigated. The results indicate that saccades are elicited by visual expansion, and are brought about by remarkably subtle changes in wing motion. Mechanosensory feedback from the fly's 'gyroscope' complements visual cues to terminate saccades, as well as to stabilize forward flight. Olfactory stimuli elicit tonic increases in wingbeat amplitude and frequency but do not alter the time course or magnitude of visual reflexes.     

Selecting representative model micro-organisms

Background

The sexually transmitted disease, gonorrhea, is a serious health problem in developed as well as in developing countries, for which treatment continues to be a challenge. The recent completion of the genome sequence of the causative agent, Neisseria gonorrhoeae, opens up an entirely new set of approaches for studying this organism and the diseases it causes. Here, we describe the initial phases of the construction of an expression-capable clone set representing the protein-coding ORFs of the gonococcal genome using a recombination-based cloning system.

Results

The clone set thus far includes 1672 of the 2250 predicted ORFs of the N. gonorrhoeae genome, of which 1393 (83%) are sequence-validated. Included in this set are 48 of the 61 ORFs of the gonococcal genetic island of strain MS11, not present in the sequenced genome of strain FA1090. L-arabinose-inducible glutathione-S-transferase (GST)-fusions were constructed from random clones and each was shown to express a fusion protein of the predicted size following induction, demonstrating the use of the recombination cloning system. PCR amplicons of each ORF used in the cloning reactions were spotted onto glass slides to produce DNA microarrays representing 2035 genes of the gonococcal genome. Pilot experiments indicate that these arrays are suitable for the analysis of global gene expression in gonococci.

Conclusion

This archived set of Gateway^® entry clones will facilitate high-throughput genomic and proteomic studies of gonococcal genes using a variety of expression and analysis systems. In addition, the DNA arrays produced will allow us to generate gene expression profiles of gonococci grown in a wide variety of conditions. Together, the resources produced in this work will facilitate experiments to dissect the molecular mechanisms of gonococcal pathogenesis on a global scale, and ultimately lead to the determination of the functions of unknown genes in the genome.     

Papillomavirus-like particles induce acute activation of dendritic cells

The role of viral structural proteins in the initiation of adaptive immune responses is poorly understood. To address this issue, we focused on the effect of noninfectious papillomavirus-like particles (VLPs) on dendritic cell (DC) activation. We found that murine bone marrow-derived dendritic cells (BMDCs) effectively bound and rapidly internalized bovine papillomavirus VLPS: Exposure to fully assembled VLPs of bovine papillomavirus, human papillomavirus (HPV)16 or HPV18, but not to predominately disordered HPV16 capsomers, induced acute phenotypic maturation of BMDCS: Structurally similar polyomavirus VLPs bound to the DC surface and were internalized, but failed to induce maturation. DCs that had incorporated HPV16 VLPs produced proinflammatory cytokines IL-6 and TNF-alpha; however, the release of these cytokines was delayed relative to LPS activation. Production of IL-12p70 by VLP-exposed DCs required the addition of syngeneic T cells or rIFN-gamma. Finally, BMDCs pulsed with HPV16 VLPs induced Th1-dominated primary T cell responses in vitro. Our data provide evidence that DCs respond to intact papillomavirus capsids and that they play a central role in VLP-induced immunity. These results offer a mechanistic explanation for the striking ability of papillomavirus VLP-based vaccines to induce potent T and B cell responses even in the absence of adjuvant.     

Simplified method for determination of rosiglitazone in human plasma

Rosiglitazone is a thiazolidinedione antihyperglycemic drug used in the treatment of type 2 diabetes mellitus. Rosiglitazone is extensively metabolized by cytochrome P450 2C8 and so may have some utility as an in vivo probe for this enzyme. A liquid chromatographic method using sensitive fluorescence detection and simplified sample processing involving protein precipitation with acetonitrile was developed. The isocratic mobile phase consisted of 10 mM sodium acetate-acetonitrile (pH 5; 60:40, v/v) and was delivered at a flow rate of 1 ml/min to an Alltima phenyl column (250 mm x 4.6 mm, 5 microm). Detection was by fluorescence at (EX/EM) 247/367 for rosiglitazone and 235/310 for the internal standard betaxolol. Intra- and inter-day precision ranged from 3.1 to 8.5% and 2.3 to 5.7%, respectively. No endogenous interference was observed with either rosiglitazone or the internal standard. The assay is simple, economical, precise, and is directly applicable to human pharmacokinetic studies involving single dose rosiglitazone administration.     

Methods to Measure the Impact of Home,Social, and Sexual Neighborhoods of Urban Gay,Bisexual, and Other Men Who Have Sex with Men

Men who have sex with men (MSM) accounted for 61% of new HIV diagnoses in the United States in 2010. Recent analyses indicate that socio-structural factors are important correlates of HIV infection. NYCM2M was a cross-sectional study designed to identify neighborhood-level characteristics within the urban environment that influence sexual risk behaviors, substance use and depression among MSM living in New York City. The sample was recruited using a modified venue-based time-space sampling methodology and through select websites and mobile applications. This paper describes novel methodological approaches used to improve the quality of data collected for analysis of the impact of neighborhoods on MSM health. Previous research has focused predominately on residential neighborhoods and used pre-determined administrative boundaries (e.g., census tracts) that often do not reflect authentic and meaningful neighborhoods. This study included the definition and assessment of multiple neighborhoods of influence including where men live (home neighborhood), socialize (social neighborhood) and have sex (sexual neighborhood). Furthermore, making use of technological advances in mapping, we collected geo-points of reference for each type of neighborhood and identified and constructed self-identified neighborhood boundary definitions. Finally, this study collected both perceived neighborhood characteristics and objective neighborhood conditions to create a comprehensive, flexible and rich neighborhood-level set of covariates. This research revealed that men perceived their home, social and sexual neighborhoods in different ways. Few men (15%) had the same home, social and sexual neighborhoods; for 31%, none of the neighborhoods was the same. Of the three types of neighborhoods, the number of unique social neighborhoods was the lowest; the size of sexual neighborhoods was the smallest. The resultant dataset offers the opportunity to conduct analyses that will yield context-specific and nuanced understandings of the relations among neighborhood space, and the well-being and health of urban MSM.     

EVL regulates VEGF receptor‐2 internalization and signaling in developmental angiogenesis

Endothelial tip cells are essential for VEGF‐induced angiogenesis, but underlying mechanisms are elusive. The Ena/VASP protein family, consisting of EVL, VASP, and Mena, plays a pivotal role in axon guidance. Given that axonal growth cones and endothelial tip cells share many common features, from the morphological to the molecular level, we investigated the role of Ena/VASP proteins in angiogenesis. EVL and VASP, but not Mena, are expressed in endothelial cells of the postnatal mouse retina. Global deletion of EVL (but not VASP) compromises the radial sprouting of the vascular plexus in mice. Similarly, endothelial‐specific EVL deletion compromises the radial sprouting of the vascular plexus and reduces the endothelial tip cell density and filopodia formation. Gene sets involved in blood vessel development and angiogenesis are down‐regulated in EVL‐deficient P5‐retinal endothelial cells. Consistently, EVL deletion impairs VEGF‐induced endothelial cell proliferation and sprouting, and reduces the internalization and phosphorylation of VEGF receptor 2 and its downstream signaling via the MAPK/ERK pathway. Together, we show that endothelial EVL regulates sprouting angiogenesis via VEGF receptor‐2 internalization and signaling.