Functional and molecular evidence for Na+-HCO3- cotransporter in porcine vas deferens epithelia

This study focused on the role ofsodium-bicarbonate cotransporter (NBC1) in cAMP-stimulated iontransport in porcine vas deferens epithelium. Ion substitutionexperiments in modified Ussing chambers revealed that cAMP-mediatedstimulation was dependent on the presence of Na⁺,HCO, and Cl for a full response.HCO-dependent current was unaffected byacetazolamide, bumetanide, or amiloride but was inhibited bybasolateral 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.Na⁺-driven, HCO-dependent,stilbene-inhibitable anion flux was observed across the basolateralmembrane of selectively permeabilized monolayers. Results ofradiotracer flux studies suggest a4,4'-dinitrostilbene-2,2'-disulfonate-sensitive stoichiometry of 2 baseequivalents per Na⁺. Antibodies raised against rat kidneyNBC epitopes (rkNBC; amino acids 338-391 and 928-1035)identified a single band of ~145 kDa. RT-PCR detected NBC1 message inporcine vas deferens epithelia. These results demonstrate that vasdeferens epithelial cells possess the proteins necessary for thevectoral transport of HCO and that these mechanismsare maintained in primary culture. Taken together, the results indicatethat vas deferens epithelia play an active role in male fertility andhave implications for our understanding of the relationship betweencystic fibrosis and congenital bilateral absence of the vas deferens.

     

Two genetic circuits repress the Caenorhabditis elegans heterochronic gene lin-28 after translation initiation

The heterochronic gene lin-28 of the nematode Caenorhabditis elegans controls the relative timing of diverse developmental events during the animal's larval stages. lin-28 is stage-specifically regulated by two genetic circuits: negatively by the 22-nt RNA lin-4 and positively by the heterochronic gene lin-14. Here, we show that lin-28 is repressed during normal development by a mechanism that acts on its mRNA after translation initiation. We provide evidence that lin-14 inhibits a negative regulation that is independent of the lin-4 RNA and involves the gene daf-12, which encodes a nuclear hormone receptor. The lin-4-independent repression does not affect the initiation of translation on the lin-28 mRNA, and like the lin-4-mediated repression, acts through the gene's 3'-untranslated region. In addition, we find that lin-4 is not sufficient to cause repression of lin-28 if the lin-4-independent circuit is inhibited. Therefore, the lin-4-independent circuit likely contributes substantially to the down-regulation of lin-28 that occurs during normal development. The role of lin-4 may be to initiate or potentiate the lin-4-independent circuit. We speculate that a parallel lin-4-independent regulatory mechanism regulates the expression of lin-14.     

The Salton Sea as critical habitat to migratory and resident waterbirds

Concern about the Salton Sea ecosystem, based on potential impacts of increasing salinity, contaminants, disease outbreaks, and large die-offs of birds, is heightened because of tremendous prior loss and degradation of wetland habitat in western North America. In 1999, we used a variety of survey methods to describe patterns of abundance of birds at the Salton Sea and in adjacent habitats. Our results further documented the great importance of the Salton Sea within the Pacific Flyway to wintering, migratory, and breeding waterbirds. Exclusive of Eared Grebes, we estimated about 187000 individual waterbirds at the Salton Sea in January, 88000 in April, 170000 in August, and 261000 in November. Additional surveys of Eared Grebes in November and December suggested the total population of all waterbirds was about 434000 to 583000 in those months, respectively. We also documented breeding by about 14000 pairs of colonial waterbirds. Waterbirds were particularly concentrated along the northern, southwestern, southern, and southeastern shorelines and river deltas. By contrast, some species of wading birds (Cattle Egret, White-faced Ibis, Sandhill Crane) and shorebirds (Mountain Plover, Whimbrel, Long-billed Curlew) were much more numerous in agricultural fields of the Imperial Valley than in wetland habitats at the Sea. Various studies indicate the Salton Sea is of regional or national importance to pelicans and cormorants, wading birds, waterfowl, shorebirds, and gulls and terns. Important taxa are the Eared Grebe, American White Pelican, Double-crested Cormorant, Cattle Egret, White-faced Ibis, Ruddy Duck, Yuma Clapper Rail, Snowy Plover, Mountain Plover, Gull-billed, Caspian, and Black terns, and Black Skimmer. Proposed restoration projects should be carefully assessed to ensure they do not have unintended impacts and are not placed where large numbers of breeding, roosting, or foraging birds concentrate. Similarly, plans to enhance opportunities for recreation or commerce at the Sea should aim to avoid or minimize disturbance to birds. Future research should focus on filling gaps in knowledge needed to effectively conserve birds at the Salton Sea.     

Duty or dream? Edwin G. Conklin's critique of eugenics and support for American individualism

This paper assesses ideas about moral andreproductive duty in American eugenics duringthe early twentieth century. While extremeeugenicists, including Charles Davenport andPaul Popenoe, argued that social leaders andbiologists must work to prevent individuals whowere ``unfit' from reproducing, moderates,especially Edwin G. Conklin, presented adifferent view. Although he was sympathetic toeugenic goals and participated in eugenicorganizations throughout his life, Conklinrealized that eugenic ideas rarely could meetstrict scientific standards of proof. Withthis in mind, he did not restrict his eugenicvision to hereditary measures. Relying onhis experience as an embryologist, Conklininstead attempted to balance more extremeeugenic claims – that emphasized the absolutelimits posed by heredity – with his own view of``the possibilities of development.' Throughhis critique he argued that most human beingsnever even begin to approach their hereditarypotential; he moderated his own eugenicrhetoric so that it preserved individualopportunity and responsibility, or what hasoften been labeled the American Dream.     

Development and applications of surface plasmon resonance-based von Willebrand factor-collagen binding assay

Von Willebrand factor (vWf) functions both as a carrier of factor VIII (fVIII) in plasma and as an adhesive protein providing the primary link between collagen of the extracellular matrix and platelets sequestered from blood flow. The functional activity of vWf correlates with the level of its binding to collagen, which is commonly measured in the enzyme-linked immunosorbent assay (ELISA). We developed an automated collagen-binding assay employing the surface plasmon resonance (SPR) phenomenon, which allows one to quantitatively measure the binding of purified vWf and vWf-containing therapeutic fVIII concentrates to collagen type III immobilized on a biosensor chip. The results of the SPR-based assay highly correlated (r = 0.987) with collagen-binding ELISA. The advantages of the SPR-based assay are its higher accuracy and reproducibility in comparison with ELISA. We applied the developed assay for monitoring structural changes in the vWf component of plasma-derived fVIII/vWf concentrates during a virus inactivation procedure performed by heat treatment. We determined the critical residual moisture content of 2% that can be present in lyophilized concentrates during heat-treatment procedures without causing deteriorative changes in vWf properties. Our data suggest that the SPR-based assay is a useful tool in the development of industrial virus-inactivation procedures, allowing one to preserve vWf activity and achieve the maximal therapeutic efficacy of fVIII/vWf concentrates.     

Low sequence variation among isolates of infectious hypodermal and hematopoietic necrosis virus (IHHNV) originating from Hawaii and the Americas

A 2.9 kb fragment of the infectious hypodermal and hematopoietic necrosis virus (IHHNV) genome, which contains the coding sequence of putative non-structural and capsid proteins, was amplified and sequenced from each of 14 IHHNV isolates collected from cultured penaeid shrimp stocks in Hawaii and various sites in the Americas between 1982 and 1997. The sequence comparison indicates that the IHHNV genome is very stable, with 99.6 to 100% similarity among these 14 isolates. Only nucleotide substitutions were found. The percentage of substitution was higher in the putative capsid proteins region (1.3%) than in the putative non-structural proteins region (0.6%). Out of 25 substitutions found, 14 resulted in amino acid changes. There is no apparent association between clinical outcomes and particular amino acid substitutions. Based on genetic distances, the isolates were clustered into 3 groups that generally correspond with their geographic origins.     

Unusual deep intronic mutations in the COL4A5 gene cause X linked Alport syndrome

The X-linked form of Alport syndrome is caused by mutations in the COL4A5 gene in Xq22. This large multiexonic gene has, in the past, been difficult to screen, with several studies detecting only about 50% of mutations. We report three novel intronic mutations that may, in part, explain this poor success rate and demonstrate that single base changes deep within introns can, and do, cause disease: one mutation creates a new donor splice site within an intron resulting in the inclusion of a novel in-frame cryptic exon; a second mutation results in a new exon splice enhancer sequence (ESE) that promotes splicing of a cryptic exon containing a stop codon; a third patient exhibits exon skipping as a result of a base substitution within the polypyrimidine tract that precedes the acceptor splice site. All three cases would have been missed using an exon-by-exon DNA screening approach.     

Risk factors for elevated HIV incidence rates among female injection drug users in Vancouver

Background

In 1997, we found a higher prevalence of HIV among female than among male injection drug users in Vancouver. Factors associated with HIV incidence among women in this setting were unknown. In the present study, we sought to compare HIV incidence rates among male and female injection drug users in Vancouver and to compare factors associated with HIV seroconversion.

Methods

This analysis was based on 939 participants recruited between May 1996 and December 2000 who were seronegative at enrolment with at least one follow-up visit completed, and who were studied prospectively until March 2001. Incidence rates were calculated using the Kaplan–Meier method. The Cox proportional hazards regression model was used to identify independent predictors of time to HIV seroconversion.

Results

As of March 2001, seroconversion had occurred in 110 of 939 participants (64 men, 46 women), yielding a cumulative incidence rate of HIV at 48 months of 13.4% (95% confidence interval [CI] 11.0%–15.8%). Incidence was higher among women than among men (16.6% v. 11.7%, p = 0.074). Multivariate analysis of the female participants'' practices revealed injecting cocaine once or more per day compared with injecting less than once per day (adjusted relative risk [RR] 2.6, 95% CI 1.4–4.8), requiring help injecting compared with not requiring such assistance (adjusted RR 2.1, 95% CI 1.1–3.8), having unsafe sex with a regular partner compared with not having unsafe sex with a regular partner (adjusted RR 2.9, 95% CI 0.9–9.5) and having an HIV-positive sex partner compared with not having an HIV-positive sex partner (adjusted RR 2.7, 95% CI 1.0–7.7) to be independent predictors of time to HIV seroconversion. Among male participants, injecting cocaine once or more per day compared with injecting less than once per day (adjusted RR 3.3, 95% CI 1.9–5.6), self-reporting identification as an Aboriginal compared with not self-reporting identification as an Aboriginal (adjusted RR 2.5, 95% CI 1.4–4.2) and borrowing needles compared with not borrowing needles (adjusted RR 2.0, 95% CI 1.1–3.4) were independent predictors of HIV infection.

Interpretation

HIV incidence rates among female injection drug users in Vancouver are about 40% higher than those of male injection drug users. Different risk factors for seroconversion for women as opposed to men suggest that sex-specific prevention initiatives are urgently required.Recent reports in Canada and numerous other countries indicate that HIV is increasingly affecting women.¹ Before 1995, adult women in Canada had 9.6% of all positive HIV tests for which the age and sex of the person being tested were known. By 1995, this proportion had increased to 18.5% and reached 23.9% in 2000. In addition, 39% of all new HIV infections among women in 2000 were attributed to injection drug use.² These data are consistent with findings in the United States where, in 1999, women accounted for 23% of all reported AIDS cases in adults, of which 42% were attributed to injection drug use.³These data clearly indicate that the face of the epidemic is changing. Whereas some factors unique to the transmission of HIV to women are known, basic and behavioural research efforts addressing sex-related and drug-related vulnerabilities among female injection drug users (IDUs) are lacking.⁴ At a time when women''s vulnerability to HIV infection is becoming increasingly apparent worldwide,^5,6 a better understanding of the processes and factors that cause drug-related harm among women in industrialized countries is urgently required.Since the mid-1990s, the Downtown Eastside of Vancouver, British Columbia, has experienced an explosive and ongoing HIV epidemic among IDUs with annual HIV incidence rates reaching as high as 19% in 1997.^7,8 When subjects were enrolled in the Vancouver Injection Drug User Study (VIDUS), it was found that the baseline HIV prevalence was higher among women than men (35.2% v. 25.8%).⁷ Follow-up of this cohort now allows an investigation aimed at identifying the predictors of HIV seroconversion among female and male IDUs. Therefore, we sought to compare HIV incidence rates among male and female IDUs in Vancouver and to compare risk factors associated with HIV seroconversion.