Analysis of antigenicity and topology of E2 glycoprotein present on recombinant hepatitis C virus-like particles

Purification of hepatitis C virus (HCV) from sera of infected patients has proven elusive, hampering efforts to perform structure-function analysis of the viral components. Recombinant forms of the viral glycoproteins have been used instead for functional studies, but uncertainty exists as to whether they closely mimic the virion proteins. Here, we used HCV virus-like particles (VLPs) generated in insect cells infected with a recombinant baculovirus expressing viral structural proteins. Electron microscopic analysis revealed a population of pleomorphic VLPs that were at least partially enveloped with bilayer membranes and had viral glycoprotein spikes protruding from the surface. Immunogold labeling using specific monoclonal antibodies (MAbs) demonstrated these protrusions to be the E1 and E2 glycoproteins. A panel of anti-E2 MAbs was used to probe the surface topology of E2 on the VLPs and to compare the antigenicity of the VLPs with that of truncated E2 (E2(660)) or the full-length (FL) E1E2 complex expressed in mammalian cells. While most MAbs bound to all forms of antigen, a number of others showed striking differences in their abilities to recognize the various E2 forms. All MAbs directed against hypervariable region 1 (HVR-1) recognized both native and denatured E2(660) with comparable affinities, but most bound either weakly or not at all to the FL E1E2 complex or to VLPs. HVR-1 on VLPs was accessible to these MAbs only after denaturation. Importantly, a subset of MAbs specific for amino acids 464 to 475 and 524 to 535 recognized E2(660) but not VLPs or FL E1E2 complex. The antigenic differences between E2(660,) FL E1E2, and VLPs strongly point to the existence of structural differences, which may have functional relevance. Trypsin treatment of VLPs removed the N-terminal part of E2, resulting in a 42-kDa fragment. In the presence of detergent, this was further reduced to a trypsin-resistant 25-kDa fragment, which could be useful for structural studies.     

Sensitive liquid chromatographic method using fluorescence detection for the determination of estradiol 3- and 17-glucuronides in rat and human liver microsomal incubations: formation kinetics

We have developed a sensitive and specific HPLC-fluorescence assay for the determination of estradiol-3-glucuronide and estradiol-17-glucuronide in human and rat liver microsomal incubations. The method utilizes a mobile phase comprised of acetonitrile and 50 mM ammonium phosphate buffer (35:65, v/v) that is pumped though a phenyl column at 1 ml/min; the run time is less than 15 min. Calibration curves for both metabolites were linear over the range 20-4000 pmol. The intra- and inter-day coefficients of variation were <6%. In both rat and human liver microsomes, the formation of estradiol-3-glucuronide displayed atypical kinetics (consistent with activation), while estradiol-17-glucuronide formation was consistent with classical Michaelis-Menten kinetics. Overall, the assay described is a sensitive and reproducible method for the determination of estradiol glucuronides in liver microsomal preparations.     

Development of Hydrotaea aenescens (Diptera: Muscidae) in manure of unweaned dairy calves and lactating cows

In laboratory studies performed in the United States and Hungary, the dump fly Hydrotaea aenescens (Wiedemann) was reared successfully in manure of 1- to 8-wk-old dairy calves, and in manure from adult lactating dairy cows. Survival in manure collected from 1-wk-old calves was poor (7.2%), better in manure collected from 2- and 3-wk-old calves (53.5%), and best in manure collected from 4- to 8-wk-old calves (71.4%). Survival in cow manure was slightly lower (47.4%) than that in calf manure. Reasons for different rates of development in the United States and in Hungary, and by calf age are discussed as are implications for biological control.     

Exogenous reference RNA for normalization of real-time quantitative PCR

We have utilized an in vitro transcribed 3' mRNA fragment of the plant gene ribulose bisphosphate carboxylase (RuBisCO) as an exogenous standard for normalization of quantitative PCR data. Both K562 cells and primary erythroid CD34+ progenitor cells were treated with sodium butyrate and changes in gamma-globin mRNA levels were assayed using a previously published TaqMan probe and primer set, while RuBisCO levels were assayed by a SYBR Green detection assay. The data presented show that a correction to measured gamma-globin induction was necessary with both cell types. The correction for the CD34+ progenitor cells was a striking 95% increase, while that for the K562 cells was 44%. The use of an exogenous reference such as in vitro transcribed mRNA for the RuBisCO plant gene provides a robust and sample-independent method for the normalization of quantitative PCR data in bacterial and animal cells.     

The genetic origin of mouse annexin VIII

Mouse annexin VIII cDNA was characterized by DNA sequencing of expressed sequence tag clones, molecular systematic analysis, and genetic linkage mapping to investigate its evolutionary origin. Its subfamily identity, divergence pattern, and nucleotide substitution rate were established by comparison with other annexin cDNA and deduced protein sequences. The known phylogenetic association of annexin VIII in an evolutionary clade with annexins XI, IV, V, and VIa identified these close homologs as potential progenitors or duplication products. Cladistic analysis confirmed the base position of annexin XI and its relationship to annexin IV as a direct duplication product. Although annexin VIII also derived from annexin XI, the evolutionary branching order, gene separation times, and mapping results indicated that it was probably a subsequent duplication product of annexin IV about 300 million years ago. Dates were calibrated against the assumed separation time of 75 Mya for rodents from other mammals, divergence rates were based on comparisons of all available annexin species, and relative rate tests implied individually stable gene clocks for most annexins. Linkage mapping of mouse Anx8 to the centromeric region of Chromosome (Chr) 14 placed it in a more distal homology group from previously mapped Anx7 and Anx11. Despite their synteny, the combined proximity and segregation of these three annexins diminished the likelihood that they were mutual gene duplication products. Received: 25 May 1997 / Accepted: 13 September 1997     

Annexin A8 Identifies a Subpopulation of Transiently Quiescent c-Kit Positive Luminal Progenitor Cells of the Ductal Mammary Epithelium

We have previously shown that Annexin A8 (ANXA8) is strongly associated with the basal-like subgroup of breast cancers, including BRCA1-associated breast cancers, and poor prognosis; while in the mouse mammary gland AnxA8 mRNA is expressed in low-proliferative isolated pubertal mouse mammary ductal epithelium and after enforced involution, but not in isolated highly proliferative terminal end buds (TEB) or during pregnancy. To better understand ANXA8’s association with this breast cancer subgroup we established ANXA8’s cellular distribution in the mammary gland and ANXA8’s effect on cell proliferation. We show that ANXA8 expression in the mouse mammary gland was strong during pre-puberty before the expansion of the rudimentary ductal network and was limited to a distinct subpopulation of ductal luminal epithelial cells but was not detected in TEB or in alveoli during pregnancy. Similarly, during late involution its expression was found in the surviving ductal epithelium, but not in the apoptotic alveoli. Double-immunofluorescence (IF) showed that ANXA8 positive (+ve) cells were ER-alpha negative (−ve) and mostly quiescent, as defined by lack of Ki67 expression during puberty and mid-pregnancy, but not terminally differentiated with ∼15% of ANXA8 +ve cells re-entering the cell cycle at the start of pregnancy (day 4.5). RT-PCR on RNA from FACS-sorted cells and double-IF showed that ANXA8+ve cells were a subpopulation of c-kit +ve luminal progenitor cells, which have recently been identified as the cells of origin of basal-like breast cancers. Over expression of ANXA8 in the mammary epithelial cell line Kim-2 led to a G₀/G₁ arrest and suppressed Ki67 expression, indicating cell cycle exit. Our data therefore identify ANXA8 as a potential mediator of quiescence in the normal mouse mammary ductal epithelium, while its expression in basal-like breast cancers may be linked to ANXA8’s association with their specific cells of origin.     

“It’s My Secret”: Fear of Disclosure among Sub-Saharan African Migrant Women Living with HIV/AIDS in Belgium

Patients with HIV not only have to deal with the challenges of living with an incurable disease but also with the dilemma of whether or not to disclose their status to their partners, families and friends. This study explores the extent to which sub-Saharan African (SSA) migrant women in Belgium disclose their HIV positive status, reasons for disclosure/non-disclosure and how they deal with HIV disclosure. A qualitative study consisting of interviews with twenty-eight SSA women with HIV/AIDS was conducted. Thematic content analysis was employed to identify themes as they emerged. Our study reveals that these women usually only disclose their status to healthcare professionals because of the treatment and care they need. This selective disclosure is mainly due to the taboo of HIV disease in SSA culture. Stigma, notably self-stigma, greatly impedes HIV disclosure. Techniques to systematically incorporate HIV disclosure into post-test counseling and primary care services are highly recommended.