Nampt/PBEF/Visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme

Intracellular nicotinamide phosphoribosyltransferase (iNampt) is an essential enzyme in the NAD biosynthetic pathway. An extracellular form of this protein (eNampt) has been reported to act as a cytokine named PBEF or an insulin-mimetic hormone named visfatin, but its physiological relevance remains controversial. Here we show that eNampt does not exert insulin-mimetic effects in vitro or in vivo but rather exhibits robust NAD biosynthetic activity. Haplodeficiency and chemical inhibition of Nampt cause defects in NAD biosynthesis and glucose-stimulated insulin secretion in pancreatic islets in vivo and in vitro. These defects are corrected by administration of nicotinamide mononucleotide (NMN), a product of the Nampt reaction. A high concentration of NMN is present in mouse plasma, and plasma eNampt and NMN levels are reduced in Nampt heterozygous females. Our results demonstrate that Nampt-mediated systemic NAD biosynthesis is critical for beta cell function, suggesting a vital framework for the regulation of glucose homeostasis.     

Dark-mediated dormancy release in stratified Lolium rigidum seeds is associated with higher activities of cell wall-modifying enzymes and an apparent increase in gibberellin sensitivity

Dormancy release in freshly matured, imbibed annual ryegrass (Lolium rigidum) seeds is inhibited by light and involves a decrease in seed sensitivity to abscisic acid. Other processes involved in dormancy release in the dark were investigated by measuring seed storage compound mobilisation and the activity of cell wall-degrading enzymes. Activities of endo-β-mannanase and total peroxidase were higher in dark-stratified compared to light-stratified seeds, indicating that weakening of the structures constraining the embryo was accelerated in the dark. A dramatic degradation of storage proteins in light-stratified seeds, accompanied by induction of a high molecular mass protease, suggests that maintenance of storage(-like) proteins is also important in dark-mediated dormancy release. α-Amylase activity was induced in dark-stratified seeds at least 48 h prior to radicle emergence upon transfer to conditions permitting germination, or in light-stratified seeds supplied with exogenous gibberellin A₄. This suggests that (a) α-amylase is involved in stimulation of germination of non-dormant L. rigidum seeds, and (b) dark-stratified seeds have an increased sensitivity to gibberellins which permits the rapid induction of α-amylase activity upon exposure to germination conditions. Overall, it appears that a number of processes, although possibly minor in themselves, occur in concert during dark-stratification to contribute to dormancy release.     

Oocyte CD9 is enriched on the microvillar membrane and required for normal microvillar shape and distribution

Microvilli are found on the surface of many cell types, including the mammalian oocyte, where they are thought to act in initial contact of sperm and oocyte plasma membranes. CD9 is currently the only oocyte protein known to be required for sperm-oocyte fusion. We found CD9 is localized to the oocyte microvillar membrane using transmission electron microscopy (TEM). Scanning electron microscopy (SEM) showed that CD9 null oocytes, which are unable to fuse with sperm, have an altered length, thickness and density of their microvilli. One aspect of this change in morphology was quantified using TEM by measuring the radius of curvature at the microvillar tips. A small radius of curvature is thought to promote fusibility and the radius of curvature of microvillar tips on CD9 wild-type oocytes was found to be half that of the CD9 null oocytes. We found that oocyte CD9 co-immunoprecipitates with two Ig superfamily cis partners, EWI-2 and EWI-F, which could have a role in linking CD9 to the oocyte microvillar actin core. We also examined latrunculin B-treated oocytes, which are known to have reduced fusion ability, and found altered microvillar morphology by SEM and TEM. Our data suggest that microvilli may participate in sperm-oocyte fusion. Microvilli could act as a platform to concentrate adhesion/fusion proteins and/or provide a membrane protrusion with a low radius of curvature. They may also have a dynamic interaction with the sperm that serves to capture the sperm cell and bring it into close contact with the oocyte plasma membrane.     

Hemodynamic changes in the kidney in a pediatric rat model of sepsis-induced acute kidney injury

Sepsis is a leading cause of acute kidney injury (AKI) and mortality in children. Understanding the development of pediatric sepsis and its effects on the kidney are critical in uncovering new therapies. The goal of this study was to characterize the development of sepsis-induced AKI in the clinically relevant cecal ligation and puncture (CLP) model of peritonitis in rat pups 17-18 days old. CLP produced severe sepsis demonstrated by time-dependent increase in serum cytokines, NO, markers of multiorgan injury, and renal microcirculatory hypoperfusion. Although blood pressure and heart rate remained unchanged after CLP, renal blood flow (RBF) was decreased 61% by 6 h. Renal microcirculatory analysis showed the number of continuously flowing cortical capillaries decreased significantly from 69 to 48% by 6 h with a 66% decrease in red blood cell velocity and a 57% decline in volumetric flow. The progression of renal microcirculatory hypoperfusion was associated with peritubular capillary leakage and reactive nitrogen species generation. Sham adults had higher mean arterial pressure (118 vs. 69 mmHg), RBF (4.2 vs. 1.1 ml·min(-1)·g(-1)), and peritubular capillary velocity (78% continuous flowing capillaries vs. 69%) compared with pups. CLP produced a greater decrease in renal microcirculation in pups, supporting the notion that adult models may not be the most appropriate for studying pediatric sepsis-induced AKI. Lower RBF and reduced peritubular capillary perfusion in the pup suggest the pediatric kidney may be more susceptible to AKI than would be predicted using adults models.     

Polymorphism in the DNA repair enzyme XRCC1: utility of current database and implications for human health risk assessment

Genetic polymorphisms are increasingly recognized as sources of variability not only in toxicokinetic but also in toxicodynamic response to environmental agents. XRCC1 is involved in base excision repair (BER) of DNA; it has variant genotypes that are associated with modified repair function. This analysis focuses on four polymorphisms: three in the coding region that affect protein structure and one in an upstream regulatory sequence that affects gene expression. The Arg399Gln variant is the most widely studied with evidence supporting a quantitative effect of genotype on phenotype. The homozygous variant (Gln/Gln) can have 3-4-fold diminished capacity to remove DNA adducts and oxidized DNA damage. This variant is relatively common in Caucasians and Asians where approximately 10% are homozygous variant. In contrast, the Arg194Trp variant appears to protect against genotoxic effects although the degree to which DNA repair is enhanced by this polymorphism is uncertain. The homozygous variant is rare in Caucasians and African Americans but it is present at 7% in Asians. A third coding region polymorphism at codon 280 appears to decrease repair function but additional quantitative information is needed and the homozygous variant is rare across populations studied. A polymorphism in an upstream promoter binding sequence (-77T>C) appears to lower XRCC1 levels by decreasing gene expression. Based upon genotype effect on phenotype and allele frequency, the current analysis finds that the codon 399 and upstream (-77) polymorphisms have the greatest potential to affect the toxicodynamic response to DNA damaging agents. However, the implications for risk assessment are limited by the likelihood that polymorphisms in multiple BER genes interact to modulate DNA repair.     

Adenovirus-5-vectored P. falciparum vaccine expressing CSP and AMA1. Part B: safety, immunogenicity and protective efficacy of the CSP component

Background

A protective malaria vaccine will likely need to elicit both cell-mediated and antibody responses. As adenovirus vaccine vectors induce both these responses in humans, a Phase 1/2a clinical trial was conducted to evaluate the efficacy of an adenovirus serotype 5-vectored malaria vaccine against sporozoite challenge.

Methodology/Principal Findings

NMRC-MV-Ad-PfC is an adenovirus vector encoding the Plasmodium falciparum 3D7 circumsporozoite protein (CSP). It is one component of a two-component vaccine NMRC-M3V-Ad-PfCA consisting of one adenovector encoding CSP and one encoding apical membrane antigen-1 (AMA1) that was evaluated for safety and immunogenicity in an earlier study (see companion paper, Sedegah et al). Fourteen Ad5 seropositive or negative adults received two doses of NMRC-MV-Ad-PfC sixteen weeks apart, at particle units per dose. The vaccine was safe and well tolerated. All volunteers developed positive ELISpot responses by 28 days after the first immunization (geometric mean 272 spot forming cells/million[sfc/m]) that declined during the following 16 weeks and increased after the second dose to levels that in most cases were less than the initial peak (geometric mean 119 sfc/m). CD8+ predominated over CD4+ responses, as in the first clinical trial. Antibody responses were poor and like ELISpot responses increased after the second immunization but did not exceed the initial peak. Pre-existing neutralizing antibodies (NAb) to Ad5 did not affect the immunogenicity of the first dose, but the fold increase in NAb induced by the first dose was significantly associated with poorer antibody responses after the second dose, while ELISpot responses remained unaffected. When challenged by the bite of P. falciparum-infected mosquitoes, two of 11 volunteers showed a delay in the time to patency compared to infectivity controls, but no volunteers were sterilely protected.

Significance

The NMRC-MV-Ad-PfC vaccine expressing CSP was safe and well tolerated given as two doses, but did not provide sterile protection.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00392015","term_id":"NCT00392015"}}NCT00392015     

A phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer

BackgroundNatural killer (NK) cells derived from patients with cancer exhibit diminished cytotoxicity compared with NK cells from healthy individuals. We evaluated the tumor response and in vivo expansion of allogeneic NK cells in recurrent ovarian and breast cancerMethodsPatients underwent a lymphodepleting preparative regimen: fludarabine 25 mg/m² × 5 doses, cyclophosphamide 60 mg/kg × 2 doses, and, in seven patients, 200 cGy total body irradiation (TBI) to increase host immune suppression. An NK cell product, from a haplo-identical related donor, was incubated overnight in 1000 U/mL interleukin (IL)-2 prior to infusion. Subcutaneous IL-2 (10 MU) was given three times/week × 6 doses after NK cell infusion to promote expansion, defined as detection of ≥100 donor-derived NK cells/μL blood 14 days after infusion, based on molecular chimerism and flow cytometryResultsTwenty (14 ovarian, 6 breast) patients were enrolled. The median age was 52 (range 30–65) years. Mean NK cell dose was 2.16 × 10⁷cells/kg. Donor DNA was detected 7 days after NK cell infusion in 9/13 (69%) patients without TBI and 6/7 (85%) with TBI. T-regulatory cells (Treg) were elevated at day +14 compared with pre-chemotherapy (P = 0.03). Serum IL-15 levels increased after the preparative regimen (P = < 0.001). Patients receiving TBI had delayed hematologic recovery (P = 0.014). One patient who was not evaluable had successful in vivo NK cell expansionConclusionsAdoptive transfer of haplo-identical NK cells after lymphodepleting chemotherapy is associated with transient donor chimerism and may be limited by reconstituting recipient Treg cells. Strategies to augment in vivo NK cell persistence and expansion are needed.     

Experimental test of nest-site limitation in mature mixed forests of central British Columbia,Canada

Nest-site availability limits cavity-using populations in many harvested forests; however, little is known about the extent of nest-site limitation in mature forests with a full complement of excavator species and intact processes of cavity creation and loss. To examine the role of nest-site availability in limiting cavity-using populations in mature mixed conifer forests in central British Columbia, Canada, we conducted an 11-year before-after control-impact experiment in which we increased nest-site availability via nest box addition. Our 7 sites (3 treatments, 4 controls) had low cavity densities (<2/ha) prior to treatment and cavity occupation rates were also low (<10%/yr), which is a relationship often cited in the literature as evidence of non-limitation in cavity-nesting populations. Following nest box addition at our treatment sites, which tripled the availability of cavities, total density of bird and mammal nests more than tripled. Density of mountain chickadee (Poecile gambeli) nests increased 9-fold on treatment sites and returned to pre-treatment levels following box removal, suggesting that chickadee populations were limited by cavity availability at our study sites. Density of red squirrel (Tamiasciurus hudsonicus) and northern flying squirrel (Glaucomys sabrinus) nests and roosts also increased significantly at treatment sites following box addition and declined following box removal. We noted little change in chickadee or squirrel nest density at control sites monitored concurrently. Squirrels preferred large-sized over small-sized boxes, and significantly enlarged the entrance areas of small boxes by chewing, suggesting that there may have been a shortage of suitable nest and roost sites for them in our study area. We contend that low cavity occupancy rates may not accurately reflect nest-site availability for cavity nesters in mature forests, and that cavity size may influence the true availability of cavities on the landscape. © 2011 The Wildlife Society.