Expression and function of Fas antigen vary in bovine granulosa and theca cells during ovarian follicular development and atresia

Fas antigen is a receptor that triggers apoptosis when bound by Fas ligand (FasL). A role for Fas antigen in follicular atresia was studied in follicles obtained during the first wave of follicular development during the bovine estrous cycle (estrus is Day 0). Granulosa and theca cells were isolated from healthy dominant follicles and the two largest atretic subordinate follicles on Day 5, atretic dominant follicles on Days 10-12, and preovulatory follicles on Day 1. Fas antigen mRNA levels were highest in granulosa cells from subordinate as compared to other follicles, and lowest in theca cells from healthy Day 5 dominant as compared to other follicles. FasL alone had no effect on viability of granulosa or theca cells but became cytotoxic in the presence of interferon-gamma (IFN). IFN has been shown to induce responsiveness to Fas antigen-mediated apoptosis in other cell types. In the presence of IFN, killing of granulosa cells by FasL was greater in subordinate compared to healthy dominant follicles on Day 5, did not differ between healthy and atretic dominant follicles, and was similar in theca among all follicles. Granulosa cells from preovulatory follicles, which had been exposed to the LH surge in vivo, were completely resistant to FasL-induced killing. In summary, Fas antigen expression, and responsiveness to Fas antigen-mediated apoptosis, vary during follicular development.     

Expression and activity of the Fas antigen in bovine ovarian follicle cells

The Fas antigen is a cell surface receptor that triggers apoptosis when bound to Fas ligand (FasL). Studies were undertaken to determine whether the cow provides a suitable model to study the role of the Fas pathway in inducing apoptosis of ovarian cells during follicular atresia. Expression of Fas antigen mRNA and responsiveness to FasL-induced killing in vitro were measured. Effects of the cytokines tumor necrosis factor (TNF)-alpha and interferon-gamma (IFN) were studied because of previous demonstrations of their role in Fas-mediated apoptosis in other cell types. Fas antigen mRNA was detectable in cultured granulosa and theca cells, and expression was increased by treatment with IFN but not TNF. Granulosa and theca cells were resistant to FasL-induced killing unless pretreated with IFN. TNF had no effect on FasL-induced killing. Granulosa and theca cell cultures in which killing occurred in response to FasL stained positively for annexin V, an early marker for cells undergoing apoptosis. These results provide a basis for further studies using the bovine ovary to examine the role of the Fas antigen in follicular atresia.     

Symplastic Solute Transport and Avocado Fruit Development: A Decline in Cytokinin/ABA Ratio is Related to Appearance of the Hass Small Fruit Variant

Studies on the effect of fruit size on endogenous ABA and isopentenyladenine(iP) in developing avocado (Persea americana Mill. cv. Hass)fruit revealed that ABA content was negatively correlated withfruit size whilst the iP/ABA ratio showed a linear relationshipwith increasing size of fruit harvested 226 d after full bloom.The effect of this change in hormone balance on the relationshipbetween symplastic solute transport and appearance of the smallfruit variant was examined following manipulation of the endogenouscytokinin (CK)/ABA ratio. Application of ABA caused seed coatsenescence and retarded fruit growth but these effects wereabsent in fruit treated with equal amounts of ABA plus iP. Thus,the underlying physiological mechanisms associated with ABA-inducedretardation of Hass avocado fruit growth appeared to be inextricablylinked to a decline in CK content and included: diminution ofmesocarp and seed coat plasmodesmatal branching, gating of mesocarpand seed coat plasmodesmata by deposition of electron densematerial in the neck region, abolishment of the electrochemicalgradient between mesocarp and seed coat parenchyma, and arrestof cell-to-cell chemical communication. (Received February 25, 1998; Accepted July 28, 1998)     

The taxonomic treatment of agamosperms in the genusLimonium mill. (Plumbaginaceae)

Agamospermous species account for a large proportion of the species ofLimonium. Agamospermy is indicated by uneven polyploidy or aneuploidy, low pollen stainability and by the presence of monomorphic self-incompatible populations. The taxonomic treatment of agamospermous taxa varies from recognition of all of them at the same specific rank or by utilising a range of ranks in the taxonomic hierarchy. The influence of evolutionary hypotheses on taxonomic systems is considered. Molecular data provide a means of measuring the genetical relationships of taxa and establishing groups in a taxonomic hierarchy.     

Extracellular α-amylase from Thermus filiformis Ork A2: purification and biochemical characterization

An extracellular α-amylase produced by the thermophilic bacterium Thermus filiformis Ork A2 was purified from cell-free culture supernatant by ion exchange chromatography. The molecular mass was estimated to be 60 000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was rich in both basic and hydrophobic amino acids, presenting the following NH₂-terminal amino acid sequence: Thr-Ala-Asp-Leu-Ile-Val-Lys-Ile-Asn-Phe. Amylolytic activity on soluble starch was optimal at pH 5.5–6.0 and 95°C, and the enzyme was stable in the pH range of 4.0–8.0. Calcium enhanced thermostability at temperatures above 80°C, increasing the half-life of activity to more than 8 h at 85°C, 80 min at 90°C, and 19 min at 95°C. Ethylenediaminetetraacetic acid (EDTA) inhibited amylase activity, the inhibition being reversed by the addition of calcium or strontium ions. The α-amylase was also inhibited by copper and mercuric ions, and p-chloromercuribenzoic acid, the latter being reversed in the presence of dithiothreitol. Dithiothreitol and β-mercaptoethanol activated the enzyme. The α-amylase exhibited Michaelis-Menten kinetics for starch, with a K _m of 5.0 mg·ml⁻¹ and k _cat/K _m of 5.2 × 10⁵ ml·mg⁻¹ s⁻¹. Similar values were obtained for amylose, amylopectin, and glycogen. The hydrolysis pattern was similar for maltooligosaccharides and polysaccharides, with maltose being the major hydrolysis product. Glucose and maltotriose were generated as secondary products, although glucose was produced in high levels after a 6-h digestion. To our knowledge this is the first report of the characterization of an α-amylase from a strain of the genus Thermus. Received: June 2, 1997 / Accepted: September 16, 1997     

Evaluating the Impact of Zimbabwe’s Prevention of Mother-to-Child HIV Transmission Program: Population-Level Estimates of HIV-Free Infant Survival Pre-Option A

Objective

We estimated HIV-free infant survival and mother-to-child HIV transmission (MTCT) rates in Zimbabwe, some of the first community-based estimates from a UNAIDS priority country.

Methods

In 2012 we surveyed mother-infant pairs residing in the catchment areas of 157 health facilities randomly selected from 5 of 10 provinces in Zimbabwe. Enrolled infants were born 9–18 months before the survey. We collected questionnaires, blood samples for HIV testing, and verbal autopsies for deceased mothers/infants. Estimates were assessed among i) all HIV-exposed infants, as part of an impact evaluation of Option A of the 2010 WHO guidelines (rolled out in Zimbabwe in 2011), and ii) the subgroup of infants unexposed to Option A. We compared province-level MTCT rates measured among women in the community with MTCT rates measured using program monitoring data from facilities serving those communities.

Findings

Among 8568 women with known HIV serostatus, 1107 (12.9%) were HIV-infected. Among all HIV-exposed infants, HIV-free infant survival was 90.9% (95% confidence interval (CI): 88.7–92.7) and MTCT was 8.8% (95% CI: 6.9–11.1). Sixty-six percent of HIV-exposed infants were still breastfeeding. Among the 762 infants born before Option A was implemented, 90.5% (95% CI: 88.1–92.5) were alive and HIV-uninfected at 9–18 months of age, and 9.1% (95%CI: 7.1–11.7) were HIV-infected. In four provinces, the community-based MTCT rate was higher than the facility-based MTCT rate. In Harare, the community and facility-based rates were 6.0% and 9.1%, respectively.

Conclusion

By 2012 Zimbabwe had made substantial progress towards the elimination of MTCT. Our HIV-free infant survival and MTCT estimates capture HIV transmissions during pregnancy, delivery and breastfeeding regardless of whether or not mothers accessed health services. These estimates also provide a baseline against which to measure the impact of Option A guidelines (and subsequently Option B+).     

The N-terminal domain of PMTV TGB1 movement protein is required for nucleolar localization, microtubule association, and long-distance movement

The triple-gene-block (TGB)1 protein of Potato mop-top virus (PMTV) was fused to fluorescent proteins and expressed in epidermal cells of Nicotiana benthamiana under the control of the 35S promoter. TGB1 fluorescence was observed in the cytoplasm, nucleus, and nucleolus and occasionally associated with microtubules. When expressed from a modified virus (PMTV.YFP-TGB1) which formed local lesions but was not competent for systemic movement, yellow fluorescent protein (YFP)-TGB1 labeled plasmodesmata in cells at the leading edge of the lesion and plasmodesmata, microtubules, nuclei, and nucleoli in cells immediately behind the leading edge. Deletion of 84 amino acids from the N-terminus of unlabeled TGB1 within the PMTV genome abolished movement of viral RNA to noninoculated leaves. When the same deletion was introduced into PMTV.YFP-TGB1, labeling of microtubules and nucleoli was abolished. The N-terminal 84 amino acids of TGB1 were fused to green fluorescent protein (GFP) and expressed in epidermal cells where GFP localized strongly to the nucleolus (not seen with unfused GFP), indicating that these amino acids contain a nucleolar localization signal; the fusion protein did not label microtubules. This is the first report of nucleolar and microtubule association of a TGB movement protein. The results suggest that PMTV TGB1 requires interaction with nuclear components and, possibly, microtubules for long-distance movement of viral RNA.     

Hypolithic Microbial Community of Quartz Pavement in the High-Altitude Tundra of Central Tibet

The hypolithic microbial community associated with quartz pavement at a high-altitude tundra location in central Tibet is described. A small-scale ecological survey indicated that 36% of quartz rocks were colonized. Community profiling using terminal restriction fragment length polymorphism revealed no significant difference in community structure among a number of colonized rocks. Real-time quantitative PCR and phylogenetic analysis of environmental phylotypes obtained from clone libraries were used to elucidate community structure across all domains. The hypolithon was dominated by cyanobacterial phylotypes (73%) with relatively low frequencies of other bacterial phylotypes, largely represented by the chloroflexi, actinobacteria, and bacteriodetes. Unidentified crenarchaeal phylotypes accounted for 4% of recoverable phylotypes, while algae, fungi, and mosses were indicated by a small fraction of recoverable phylotypes.     

The functional geometry of local and horizontal connections in a model of V1.

A mathematical model of interacting hypercolumns in primary visual cortex (V1) is presented that incorporates details concerning the geometry of local and long-range horizontal connections. Each hypercolumn is modeled as a network of interacting excitatory and inhibitory neural populations with orientation and spatial frequency preferences organized around a pair of pinwheels. The pinwheels are arranged on a planar lattice, reflecting the crystalline-like structure of cortex. Local interactions within a hypercolumn generate orientation and spatial frequency tuning curves, which are modulated by horizontal connections between different hypercolumns on the lattice. The symmetry properties of the local and long-range connections play an important role in determining the types of spontaneous activity patterns that can arise in cortex.     

A surface-focused biotinylation procedure identifies the Yersinia pestis catalase KatY as a membrane-associated but non-surface-located protein

This study identified major surface proteins of the plague bacterium Yersinia pestis. We applied a novel surface biotinylation method, followed by NeutrAvidin (NA) bead capture, on-bead digestion, and identification by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The use of stachyose during biotinylation focused the reaction to the surface. Coupled with NA pulldown and immunoblot analysis, this method determined whether a protein was accessible to the surface. We applied the method to test the hypothesis that the catalase KatY is a surface protein of the plague bacterium Y. pestis. A rabbit serum recognized the catalase KatY as a major putative outer membrane-associated antigen expressed by Y. pestis cells grown at 37 degrees C. Similar findings by other groups had led to speculations that this protein might be exposed to the surface and might be a candidate for evaluation as a protective antigen for an improved plague vaccine. KatY was obtained only in the total membrane fraction, and stachyose greatly reduced its biotinylation as well as that of the periplasmic maltose binding protein, indicating that KatY is not on the bacterial surface. LC-MS-MS analysis of on-bead digests representing ca. 10(9) cells identified highly abundant species, including KatY, Pal, and OmpA, as well as the lipoprotein Pcp, all of which bound in a biotin-specific manner. Pla, Lpp, and OmpX (Ail) bound to the NA beads in a non-biotin-specific manner. There was no contamination from abundant cytoplasmic proteins. We hypothesize that OmpX and Pcp are highly abundant and likely to be important for the Y. pestis pathogenic process. We speculate that a portion of KatY associates with the outer membrane in intact cells but that it is located on the periplasmic side. Consistent with this idea, it did not protect C57BL/6 mice against bubonic plague.