Protein kinase A-mediated phosphorylation of connexin36 in mouse retina results in decreased gap junctional communication between AII amacrine cells

Gap junctions in AII amacrine cells of mammalian retina participate in the coordination of the rod and cone signaling pathway involved in visual adaptation. Upon stimulation by light, released dopamine binds to D(1) receptors on AII amacrine cells leading to increased intracellular cAMP (cyclic adenosine monophosphate) levels. AII amacrine cells express the gap junctional protein connexin36 (Cx36). Phosphorylation of Cx36 has been hypothesized to regulate gap junctional activity of AII amacrine cells. However, until now in vivo phosphorylation of Cx36 has not been reported. Indeed, it had been concluded that Cx36 in bovine retina is not phosphorylated, but in vitro phosphorylation for Cx35, the bass ortholog of Cx36, had been shown. To clarify this experimental discrepancy, we examined protein kinase A (PKA)-induced phosphorylation of Cx36 in mouse retina as a possible mechanism to modulate the extent of gap junctional coupling. The cytoplasmic domains of Cx36 and the total Cx36 protein were phosphorylated in vitro by PKA. Mass spectroscopy revealed that all four possible PKA consensus motifs were phosphorylated; however, domains point mutated at the sites in question showed a prevalent usage of Ser-110 and Ser-293. Additionally, we demonstrated that Cx36 was phosphorylated in cultured mouse retina. Furthermore, activation of PKA increased the level of phosphorylation of Cx36. cAMP-stimulated, PKA-mediated phosphorylation of Cx36 protein was accompanied by a decrease of tracer coupling between AII amacrine cells. Our results link increased phosphorylation of Cx36 to down-regulation of permeability through gap junction channels mediating light adaptation in the retina.     

N‐terminal residues of the yeast pheromone receptor,Ste2p,mediate mating events independently of G1‐arrest signaling

In Saccharomyces cerevisiae, mechanisms modulating the mating steps following cell cycle arrest are not well characterized. However, the N‐terminal domain of Ste2p, a G protein‐coupled pheromone receptor, was recently proposed to mediate events at this level. Toward deciphering receptor mechanisms associated with this mating functionality, scanning mutagenesis of targeted regions of the N‐terminal domain has been completed. Characterization of ste2 yeast overexpressing Ste2p variants indicated that residues Ile 24 and Ile 29 as well as Pro 15 are critical in mediating mating efficiency. This activity was shown to be independent of Ste2p mediated G1 arrest signaling. Further analysis of Ile 24 and Ile 29 highlight the residues' solvent accessibility, as well as the importance of the hydrophobic nature of the sites, and in the case of Ile 24 the specific size and shape of the side chain. Mutation of these Ile's led to arrest of mating after cell contact, but before completion of cell wall degradation. We speculate that these extracellular residues mediate novel receptor interactions with ligand or proteins, leading to stimulation of alternate signaling effector pathways. J. Cell. Biochem. 107: 630–638, 2009. © 2009 Crown in the right of Canada.     

Developing the sterile insect technique for Cryptophlebia leucotreta (Lepidoptera: Tortricidae): influence of radiation dose and release ratio on fruit damage and population growth in field cages

The effect of radiation dose and different release ratios of treated (T) to untreated (U) Cryptophlebia leucotreta (Meyrick) (Lepidoptera: Tortricidae), on the incidence of fruit damage, the competitiveness of the treated males, and population growth was examined inside field cages. Navel orange trees were individually enclosed in large nylon mesh cages. Newly emerged adult moths treated with either 150 or 200 Gy of gamma radiation were released into the cages at ratios of 5T:1U or 10T:1U. The fruit was collected after 4 wk, and the number of damaged fruit and larval entries per cage were recorded for each treatment. Infested fruit was maintained in the laboratory until all emerging F1 progeny were collected and outcrossed to untreated moths of the opposite sex. Treatment had a significant effect on the mean number of larval entries and on the number of undamaged fruit per cage. The number of larval entries as well as the number of F1 progeny per cage decreased as the overflooding ratio increased. A significant reduction in egg hatch was observed in the progeny of crosses between F1 females or F1 males originating from the treatment cages compared with crosses of F1 moths originating from the control cages. The lowest mean number of fertile F1 adult females and males was obtained from the 150 Gy and 10T:1U ratio treatment. This treatment also showed the lowest per generation rate of increase (< 1 from the parental [P1] to the F1 generation), suggesting that growth in the fertile population would have been prevented if releases of treated moths at this dose and ratio were maintained in the field.     

Cost-effectiveness of pooled nucleic acid amplification testing for acute HIV infection after third-generation HIV antibody screening and rapid testing in the United States: a comparison of three public health settings

Background

Detection of acute HIV infection (AHI) with pooled nucleic acid amplification testing (NAAT) following HIV testing is feasible. However, cost-effectiveness analyses to guide policy around AHI screening are lacking; particularly after more sensitive third-generation antibody screening and rapid testing.

Methods and Findings

We conducted a cost-effectiveness analysis of pooled NAAT screening that assessed the prevention benefits of identification and notification of persons with AHI and cases averted compared with repeat antibody testing at different intervals. Effectiveness data were derived from a Centers for Disease Control and Prevention AHI study conducted in three settings: municipal sexually transmitted disease (STD) clinics, a community clinic serving a population of men who have sex with men, and HIV counseling and testing sites. Our analysis included a micro-costing study of NAAT and a mathematical model of HIV transmission. Cost-effectiveness ratios are reported as costs per quality-adjusted life year (QALY) gained in US dollars from the societal perspective. Sensitivity analyses were conducted on key variables, including AHI positivity rates, antibody testing frequency, symptomatic detection of AHI, and costs. Pooled NAAT for AHI screening following annual antibody testing had cost-effectiveness ratios exceeding US$200,000 per QALY gained for the municipal STD clinics and HIV counseling and testing sites and was cost saving for the community clinic. Cost-effectiveness ratios increased substantially if the antibody testing interval decreased to every 6 months and decreased to cost-saving if the testing interval increased to every 5 years. NAAT was cost saving in the community clinic in all situations. Results were particularly sensitive to AHI screening yield.

Conclusions

Pooled NAAT screening for AHI following negative third-generation antibody or rapid tests is not cost-effective at recommended antibody testing intervals for high-risk persons except in very high-incidence settings. Please see later in the article for the Editors'' Summary     

Ectopic activation of Dpp signalling in the male Drosophila germline inhibits germ cell differentiation

The mechanisms that control differentiation of stem cells to specialised cell types probably include factors intrinsic to stem cells as well as extrinsic factors produced by the microenvironment of the stem cell niche. The Drosophila male germline is renewed from a population of stem cells located in the apical tip of the adult testis. The morphological relationship between germline stem cells and their surrounding somatic cells is well understood but the factors that regulate stem cell proliferation and differentiation are still being uncovered. This study examined the effect of stimulating Dpp signalling directly in male germ cells. Ectopic Dpp or Activin signalling resulted in overproliferation of both stem cell-like and spermatogonial-like cells in the apical region of the testis. A third cell population that expressed stem cell markers was seen to proliferate in the distal testis when Dpp signalling was either stimulated or repressed in germline stem cells.     

The latex agglutination test versus counterimmunoelectrophoresis for rapid diagnosis of bacterial meningitis

A modified latex agglutination (LA) test was compared with Gram-staining and counterimmunoelectrophoresis (CIE) for the rapid detection in the cerebrospinal fluid (CSF) of antigen to Haemophilus influenzae type b, Neisseria meningitidis groups A, B and C, Escherichia coli K1, Streptococcus pneumoniae and group B streptococci, seven frequent causes of bacterial meningitis in children. Of 50 CSF samples from patients with culture-proven bacterial meningitis 90% were correctly shown by the LA test to contain antigen of the responsible organism. Gram-staining revealed organisms in 80% of 45 of these samples. In 75% of the 40 samples that were of sufficient volume for CIE, positive results for the appropriate antigen were obtained. The concentration of antigen detected in the CSF by the LA test varied from undetectable to 800 000 ng/ml. Patients with a high concentration (more than 2000 ng/ml or a positive result at dilutions of CSF over 1/8) were significantly more likely to have a poor response to therapy (two died and two had persistent pleocytosis or bacteria in the CSF) than patients with a lower concentration (4/16 v. 0/18, P < 0.05). After appropriate therapy was begun the concentration of antigen fell dramatically, but measurable amounts of antigen persisted in the CSF for up to 6 days. The LA test detected bacterial antigen at concentrations 2 to 70 times below the lower limit detected by CIE. In seven additional patients who had received antibiotics before lumbar puncture was performed the LA test detected antigen from meningitis-causing bacteria even though cultures of the CSF were sterile. In another 145 patients who did not have meningitis the results of the LA test were negative. The LA test, done as described in this article, is easier to perform than CIE and should be a useful addition to the diagnostic tests carried out on the CSF of any patient suspected of having meningitis.     

Molecular characterisation of inter and intra-specific somatic hybrids of potato using randomly amplified polymorphic DNA (RAPD) markers

Summary Protoplast fusion allows the transfer of both mono- and polygenic traits between species that are sexually incompatible. This approach has particular relevance for potato, and somatic hybridisation has been used to introduce a range of disease resistance genes from sexually incompatible wild species into the cultivated potato gene pool. In addition, protoplast fusion allows the resynthesis of tetraploid genotypes from pre-selected diploid or dihaploid donor parents. A limiting factor for the efficient exploitation of this technology in potato breeding is the difficulty of unequivocally identifying nuclear hybrids (heterokaryons). In order to facilitate the identification of hybrids at an early stage following fusion, Randomly Amplified Polymorphic DNA markers (RAPDs) have been used to characterise molecularly both inter- and intra-specific somatic hybrids of potato. RAPD markers detect naturally occurring polymorphism in the donor genotypes and utilise short oligonucleotide primers of arbitrary nucleotide sequence in combination with the polymerase chain reaction (PCR). The exploitation of RAPDs in the characterisation of both somatic and sexual hybrids is discussed.     

Growth differentiation factor 9 regulates expression of the bone morphogenetic protein antagonist gremlin

Growth differentiation factor 9 (GDF9) is an oocyte-expressed member of the transforming growth factor beta (TGF-beta) superfamily and is required for normal ovarian follicle development and female fertility. GDF9 acts as a paracrine factor and affects granulosa cell physiology. Only a few genes regulated by GDF9 are known. Our microarray analysis has identified gremlin as one of the genes up-regulated by GDF9 in cultures of granulosa cells. Gremlin is a known member of the DAN family of bone morphogenetic protein (BMP) antagonists, but its expression and function in the ovary are unknown. We have investigated the regulation of gremlin in mouse granulosa cells by GDF9 as well as other members of the TGF-beta superfamily. GDF9 and BMP4 induce gremlin, but TGF-beta does not. In addition, in cultures of granulosa cells, gremlin negatively regulates BMP4 signaling but not GDF9 activity. The expression of gremlin in the ovary was also examined by in situ hybridization. A distinct change in gremlin mRNA compartmentalization occurs during follicle development and ovulation, indicating a highly regulated expression pattern during folliculogenesis. We propose that gremlin modulates the cross-talk between GDF9 and BMP signaling that is necessary during follicle development because both ligands use components of the same signaling pathway.     

An investigation of dye reduction by food-borne bacteria

S.A. LEAROYD, R.G. KROLL AND C.F. THURSTON. 1992. The rates of reduction of seven redox dyes by 13 bacterial strains were measured and found to vary greatly between different bacterium/dye combinations. Phenazine ethosulphate and toluidine blue were the most rapidly reduced dyes by the majority of bacteria and resorufin and 2-hydroxy-1,4-naphthoquinone were reduced slowly, if at all. There was also considerable variation in the rates of reduction with any single dye/organism combination. Glucose stimulated the rates of endogenous dye reduction in about half of the organisms. For Bacillus cereus, Pseudomonas fluorescens and Escherichia coli , dye reduction was stimulated by a range of exogenous substrates but lactose, the primary available carbon and energy source in milk, had little effect. In Lactococcus lactis , dye reduction was stimulated by sugars but not by organic acids. Oxygen successfully competed with dye reduction in organisms containing respiratory chains, but with membrane fractions, dye reduction was more rapid than oxygen consumption. All the organisms showed little cytosolic dye reduction, except L. lactis which showed substantial rates of reduction of some dyes by this fraction. With the membrane fraction of E. coli and Ps. fluorescens , cyanide inhibited NADH and succinate-dependent dye reduction, Antimycin A inhibited lactate and succinate and rotenone had no significant effect, but inhibition was not always observed with membrane from both organisms.     

Cost-effectiveness of HIV screening in STD clinics, emergency departments, and inpatient units: a model-based analysis

Background

Identifying and treating persons with human immunodeficiency virus (HIV) infection early in their disease stage is considered an effective means of reducing the impact of the disease. We compared the cost-effectiveness of HIV screening in three settings, sexually transmitted disease (STD) clinics serving men who have sex with men, hospital emergency departments (EDs), settings where patients are likely to be diagnosed early, and inpatient diagnosis based on clinical manifestations.

Methods and Findings

We developed the Progression and Transmission of HIV/AIDS model, a health state transition model that tracks index patients and their infected partners from HIV infection to death. We used program characteristics for each setting to compare the incremental cost per quality-adjusted life year gained from early versus late diagnosis and treatment. We ran the model for 10,000 index patients for each setting, examining alternative scenarios, excluding and including transmission to partners, and assuming HAART was initiated at a CD4 count of either 350 or 500 cells/µL. Screening in STD clinics and EDs was cost-effective compared with diagnosing inpatients, even when including only the benefits to the index patients. Screening patients in STD clinics, who have less-advanced disease, was cost-effective compared with ED screening when treatment with HAART was initiated at a CD4 count of 500 cells/µL. When the benefits of reduced transmission to partners from early diagnosis were included, screening in settings with less-advanced disease stages was cost-saving compared with screening later in the course of infection. The study was limited by a small number of observations on CD4 count at diagnosis and by including transmission only to first generation partners of the index patients.

Conclusions

HIV prevention efforts can be advanced by screening in settings where patients present with less-advanced stages of HIV infection and by initiating treatment with HAART earlier in the course of infection.