Inhibition of coated pit formation in Hep2 cells blocks the cytotoxicity of diphtheria toxin but not that of ricin toxin

It has been recently shown (Larkin, J. M., M. S. Brown, J. L. Goldstein, and R. G. W. Anderson, 1983, Cell, 33:273-285) that after a hypotonic shock followed by incubation in a K+-free medium, human fibroblasts arrest their coated pit formation and therefore arrest receptor-mediated endocytosis of low density lipoprotein. We have used this technique to study the endocytosis of transferrin, diphtheria toxin, and ricin toxin by three cell lines (Vero, Wi38/SV40, and Hep2 cells). Only Hep2 cells totally arrested internalization of [125I]transferrin, a ligand transported by coated pits and coated vesicles, after intracellular K+ depletion. Immunofluorescence studies using anti-clathrin antibodies showed that clathrin associated with the plasma membrane disappeared in Hep2 cells when the level of intracellular K+ was low. In the absence of functional coated pits, diphtheria toxin was unable to intoxicate Hep2 cells but the activity of ricin toxin was unaffected by this treatment. By measuring the rate of internalization of [125I]ricin toxin by Hep2 cells, with and without functional coated pits, we have shown that this labeled ligand was transported in both cases inside the cells. Hep2 cells with active coated pits internalized twice as much [125I]ricin toxin as Hep2 cells without coated pits. Entry of ricin toxin inside the cells was a slow process (8% of the bound toxin per 10 min at 37 degrees C) when compared to transferrin internalization (50% of the bound transferrin per 10 min at 37 degrees C). Using the indirect immunofluorescence technique on permeabilized cells, we have shown that Hep2 cells depleted in intracellular K+ accumulated ricin toxin in compartments that were predominantly localized around the cell nucleus. Our study indicates that in addition to the pathway of coated pits and coated vesicles used by diphtheria toxin and transferrin, another system of endocytosis for receptor-bound molecules takes place at the level of the cell membrane and is used by ricin toxin to enter the cytosol.     

Large-population passages of vesicular stomatitis virus in interferon-treated cells select variants of only limited resistance.

Vesicular stomatitis virus (VSV) populations were repeatedly passaged in L-929 cells treated with alpha interferon (IFN-alpha) at levels of 25 U/ml. This IFN-alpha concentration induced a 99.9% inhibition of viral yield in standard infections. Analysis of viral fitness (overall replicative ability measured in direct competition with a reference wild-type VSV) after 21 passages in IFN-treated cells showed only a limited increase or no increase in fitness, compared with the greater increase upon parallel passage in cells not treated with IFN-alpha. However, this limited increase in fitness was more pronounced when competition assays were carried out with IFN-alpha-treated cells, suggesting the selection of VSV populations with a low level of resistance to IFN-alpha. Thus, despite the extensively documented capacity of VSV to adapt to changing environments, the antiviral state induced by IFN-alpha imposes adaptive constraints on VSV which are not readily overcome.     

Mycoleptodiscus terrestris from black pepper roots in the Dominican Republic

Mycoleptodiscus terrestris from black pepper roots in the Dominican Republic is described together with some notes and photomicrographs.     

Integration of Social,Cultural, and Biomedical Strategies into an Existing Couple-Based Behavioral HIV/STI Prevention Intervention: Voices of Latino Male Couples

Introduction

Successful HIV prevention and treatment requires evidence-based approaches that combine biomedical strategies with behavioral interventions that are socially and culturally appropriate for the population or community being prioritized. Although there has been a push for a combination approach, how best to integrate different strategies into existing behavioral HIV prevention interventions remains unclear. The need to develop effective combination approaches is of particular importance for men who have sex with men (MSM), who face a disproportionately high risk of HIV acquisition.

Materials and Methods

We collaborated with Latino male couples and providers to adapt Connect ‘n Unite, an evidence-based intervention for Black male couples, for Latino male couples. We conducted a series of three focus groups, each with two cohorts of couples, and one focus group with providers. A purposive stratified sample of 20 couples (N = 40, divided into two cohorts) and 10 providers provided insights into how to adapt and integrate social, cultural, and biomedical approaches in a couples-based HIV/AIDS behavioral intervention.

Results

The majority (N = 37) of the couple participants had no prior knowledge of the following new biomedical strategies: non-occupational post-exposure prophylaxis (nPEP); pre-exposure prophylaxis (PrEP); and HIV self-testing kits. After they were introduced to these biomedical interventions, all participants expressed a need for information and empowerment through knowledge and awareness of these interventions. In particular, participants suggested that we provide PrEP and HIV self-testing kits by the middle or end of the intervention. Providers suggested a need to address behavioral, social and structural issues, such as language barriers; and the promotion of client-centered approaches to increase access to, adaptation of, and adherence to biomedical strategies. Corroborating what couple participants suggested, providers agreed that biomedical strategies should be offered after providing information about these tools. Regarding culturally sensitive and responsive approaches, participants identified stigma and discrimination associated with HIV and sexual identity as barriers to care, language barriers and documentation status as further barriers to care, the couple-based approach as ideal to health promotion, and the need to include family topics in the intervention.

Discussion

We successfully adapted an evidence-based behavioral HIV prevention intervention for Latino male couples. The adapted intervention, called Conectando Latinos en Pareja, integrates social, cultural, behavioral and biomedical strategies to address the HIV epidemic among Latino MSM. The study highlights the promise regarding the feasibility of implementing a combination approach to HIV prevention in this population.     

A novel intracellular mutualistic bacterium in the invasive ant Cardiocondyla obscurior

The evolution of eukaryotic organisms is often strongly influenced by microbial symbionts that confer novel traits to their hosts. Here we describe the intracellular Enterobacteriaceae symbiont of the invasive ant Cardiocondyla obscurior, ‘Candidatus Westeberhardia cardiocondylae''. Upon metamorphosis, Westeberhardia is found in gut-associated bacteriomes that deteriorate following eclosion. Only queens maintain Westeberhardia in the ovarian nurse cells from where the symbionts are transmitted to late-stage oocytes during nurse cell depletion. Functional analyses of the streamlined genome of Westeberhardia (533 kb, 23.41% GC content) indicate that neither vitamins nor essential amino acids are provided for the host. However, the genome encodes for an almost complete shikimate pathway leading to 4-hydroxyphenylpyruvate, which could be converted into tyrosine by the host. Taken together with increasing titers of Westeberhardia during pupal stage, this suggests a contribution of Westeberhardia to cuticle formation. Despite a widespread occurrence of Westeberhardia across host populations, one ant lineage was found to be naturally symbiont-free, pointing to the loss of an otherwise prevalent endosymbiont. This study yields insights into a novel intracellular mutualist that could play a role in the invasive success of C. obscurior.     

Primary productivity of natural grass ecosystems of the tropics: A reappraisal

Studies of net primary production in four contrasting tropical grasslands show that when full account is taken of losses of plant organs above- and below-ground these ecosystems are far more productive than earlier suggested. Previous values have mainly been provided by the International Biological Programme (IBP), where estimates of production were based on a change in vegetation mass alone and would not necessarily have taken full account of organ losses and turnover. Calculation at three of our sites based on estblished methodology using changes in plant mass alone (i.e. that used by the International Biological Programme, IBP) proved to be serious underestimates of when acount was taken of losses simultaneously with measurement of change in plant mass. Accounting for the turnover of material at these three sites resulted in productivities up to five times higher than were obtained using the standard IBP procedure. An emergent C₄ grass stand at a fourth site in the Amazon achieved a productivity which approached the maximum recorded for agricultural crops. In this case, productivity values, when organ losses were taken into account, only slightly exceeded that obtained with IBP methods. The findings reported here have wider implications, in prediction of global carbon cycling, remote sensing of plant productivity and impact assessment of conversion to arable cropping systems.     

Calcitonin biosynthesis: evidence for a precursor.

Calcitonin biosynthesis has been studied in chicken ultimobranchial glands incubated in vitro in the presence of radioactive amino acids. The results obtained suggest the existence of a biosynthetic precursor of higher molecular weight or procalcitonin. This precursor has been identified by pulse-chase experiments, molecular weight determinations, biological activity measurements and analysis of tryptic peptides. Its molecular weight is about 13000 (calcitonin, about 3500) as determined by polyacrylamide gel electrophoresis. Procalcitonin is present in small amounts in chicken ultimobranchial glands and it is biologically active in rats.     

The ultrastructure of odontogenesis in larval and adult urodeles; differentiation of the dental epithelial cells

Summary Sections of undemineralized tooth germs ofAmbystoma andTriturus were examined. The ultrastructure of early germs, both larval and adult, and of dentinogenesis, resembled that of mammals. In adult bicuspid teeth, once the dentine of the cusps was mineralized, mineral crystals of a similar size to early mammalian enamel crystals, appeared between the dentine and the inner dental epithelium (i.d.e). Concomitantly, the i.d.e showed features of mammalian secreting ameloblasts. This new layer, regarded as true enamel, lacked collagen, possessed an ordered arrangement of crystals and reached a maximum thickness of 6 m.In larval monocuspid teeth, once dentine mineralization had reached the plasma membranes of the i.d.e at the tip of the cusp, the i.d.e developed a ruffled border. At this stage the dentine of the tip, regarded as enameloid, was very hard and difficult to section. The ruffled border, characteristic of other cells which transport materials, was regarded as indicating that the i.d.e was removing organic matter from the enameloid. The differences in development between larval and adult teeth support the concept that there is a change in cellular activity of the i.d.e which occurs during metamorphosis from the larval to the adult urodele.     

Two Modes of Balancing Selection in Drosophila Melanogaster: Overcompensation and Overdominance

Overdominance is often invoked to account for the extensive polymorphisms found in natural populations of organisms; overcompensation, however, may be equally or more important. Overcompensation occurs when limiting resources are better exploited by a genetically mixed than by a uniform population, and is often causally related to frequency-dependent selection. We have designed experiments to test whether overcompensation occurs in Drosophila melanogaster, using the Sod locus as a marker. Tests are made at each of two densities and two temperatures for cultures with desired genetic compositions. Both temperature and density have statistically significant effects on the per-female productivity of the cultures. More important, there are strong effects due to overcompensation. Cultures that are more polymorphic are also more productive than less polymorphic ones even when the level of individual heterozygosity is the same in all. There is also overdominance for the Sod locus: the heterozygotes are more productive than either homozygote at every temperature and density, and the differences are statistically significant in several cases. These results corroborate previous studies showing that overdominance may contribute to the maintenance of the Sod polymorphisms. Moreover, our results indicate that the significance of overcompensation as a mechanism to account for polymorphism in natural populations deserves further investigation.