Nurse practitioner in a rural setting.

The author details her experiences as a nurse practitioner in rural southeast Missouri. The passage of Missouri House Bill 564 encouraged greater utilization of nurse practitioners to provide primary care. This article shares some of the frustrations, joys, and trials by fire of the author. Among the issues presented are practice issues, prescriptive authority, professional liability, evaluation of practice, and changes for the future.

Molecular cloning and analysis of functional envelope genes from human immunodeficiency virus type 1 sequence subtypes A through G. The WHO and NIAID Networks for HIV Isolation and Characterization.

Present knowledge of human immunodeficiency virus type 1 (HIV-1) envelope immunobiology has been derived almost exclusively from analyses of subtype B viruses, yet such viruses represent only a minority of strains currently spreading worldwide. To generate a more representative panel of genetically diverse envelope genes, we PCR amplified, cloned, and sequenced complete gp160 coding regions of 35 primary (peripheral blood mononuclear cell-propagated) HIV-1 isolates collected at major epicenters of the current AIDS pandemic. Analysis of their deduced amino acid sequences revealed several important differences from prototypic subtype B strains, including changes in the number and distribution of cysteine residues, substantial length differences in hypervariable regions, and premature truncations in the gp41 domain. Moreover, transiently expressed glycoprotein precursor molecules varied considerably in both size and carbohydrate content. Phylogenetic analyses of full-length env sequences indicated that the panel included members of all major sequence subtypes of HIV-1 group M (clades A to G), as well as an intersubtype recombinant (F/B) from an infected individual in Brazil. In addition, all subtype E and three subtype G viruses initially classified on the basis of partial env sequences were found to cluster in subtype A in the 3' half of their gp41 coding region, suggesting that they are also recombinant. The biological activity of PCR-derived env genes was examined in a single-round virus infectivity assay. This analysis identified 20 clones, including 1 from each subtype (or recombinant), which expressed fully functional envelope glycoproteins. One of these, derived from a patient with rapid CD4 cell decline, contained an amino acid substitution in a highly conserved endocytosis signal (Y721C), as mediated virus entry with very poor efficiency, although they did not contain sequence changes predicted to alter protein function. These results indicate that the env genes of primary HIV-1 isolates collected worldwide can vary considerably in their genetic, phylogenetic, and biological properties. The panel of env constructs described here should prove valuable for future structure-function studies of naturally occurring envelope glycoproteins as well as AIDS vaccine development efforts targeted against a broader spectrum of viruses.

Genetic variation of HIV type 1 in four World Health Organization-sponsored vaccine evaluation sites: generation of functional envelope (glycoprotein 160) clones representative of sequence subtypes A, B, C, and E. WHO Network for HIV Isolation and Characterization.

As part of the WHO Network for HIV Isolation and Characterization, we PCR amplified, cloned, and sequenced gp120 and gp160 genes from 12 HIV-1 isolates collected in four WHO-sponsored vaccine evaluation sites (Brazil, Rwanda, Thailand, Uganda). Envelope clones were derived from PBMC-grown isolates obtained from asymptomatic individuals within 2 years of seroconversion. Analysis of their deduced amino acid sequences identified all but one to contain an uninterrupted open reading frame. Transient expression and biological characterization of selected gp160 constructs identified six clones to encode full length and functional envelope glycoproteins. Phylogenetic analysis of their nucleotide sequences revealed that they represent HIV-1 subtypes A, B, C, and E. Since current knowledge of HIV-1 envelope immunobiology is almost exclusively derived from subtype B viruses, these reagents should facilitate future envelope structure, function and antigenicity studies on a broader spectrum of viruses. This should assist in the design and evaluation of effective vaccines against HIV-1.

Vectorial release of poliovirus from polarized human intestinal epithelial cells.

Polarized epithelial cells represent the primary barrier to virus infection of the host, which must also be traversed prior to virus dissemination from the infected organism. Although there is considerable information available concerning the release of enveloped viruses from such cells, relatively little is known about the processes involved in the dissemination of nonenveloped viruses. We have used two polarized epithelial cell lines, Vero C1008 (African green monkey kidney epithelial cells) and Caco-2 (human intestinal epithelial cells), infected with poliovirus and investigated the process of virus release. Release of poliovirus was observed to occur almost exclusively from the apical cell surface in Caco-2 cells, whereas infected Vero C1008 cells exhibited nondirectional release. Structures consistent with the vectorial transport of virus contained within vesicles or viral aggregates were observed by electron microscopy. Treatment with monensin or ammonium chloride partially inhibited virus release from Caco-2 cells. No significant cell lysis was observed at the times postinfection when extracellular virus was initially detected, and transepithelial resistance and vital dye uptake measurements showed only a moderate decrease. Brefeldin A was found to significantly and specifically inhibit poliovirus biosynthetic processes by an as yet uncharacterized mechanism. The vectorial release of poliovirus from the apical (or luminal) surface of human intestinal epithelial cells has significant implications for viral pathogenesis in the human gut.

Bidirectional entry of poliovirus into polarized epithelial cells.

The interactions of viruses with polarized epithelial cells are of some significance to the pathogenesis of disease because these cell types comprise the primary barrier to many virus infections and also serve as the sites for virus release from the host. Poliovirus-epithelial cell interactions are of particular interest since this virus is an important enteric pathogen and the host cell receptor has been identified. In this study, poliovirus was observed to adsorb to both the apical and basolateral surfaces of polarized monkey kidney (Vero C1008) and human intestinal (Caco-2) epithelial cells but exhibited preferential binding to the basolateral surfaces of both cell types. Localization of the poliovirus receptor by a receptor-specific monoclonal antibody (D171) revealed a similar distribution predominantly on basolateral membranes, and treatment of cells with antibody D171 inhibited virus adsorption to both membrane surfaces. Poliovirus was able to initiate infection with similar efficiency following adsorption to either surface, and infection was blocked at both surfaces by D171, indicating that functional receptor molecules are expressed on both surfaces at sufficient density to mediate efficient infection at the apical and basolateral plasma membranes. Poliovirus infection resulted in a decrease in transepithelial resistance which was inhibited by prior treatment with monoclonal antibody D171 and occurred prior to other visible cytopathic effects. These results have interesting implications for viral pathogenesis in the human gut.

High-performance liquid chromatographic monitoring of metabolic products resulting from the treatment of mouse hepatoma cells with N6-cycloalkylated nucleosides.

High-performance liquid chromatography was used to analyze cell lysates and growth medium of mouse hepatoma cells, separately treated with N6-cyclopropyl-, N6-cyclobutyl-, and N6-cyclopentyladenosines, in an effort to gain insight into the mechanism by which these modified nucleosides exert their cytotoxic effect(s). The corresponding 5'-monophosphate of the respective modified nucleoside was detected in the separate cell lysate samples. Both the modified nucleoside and its corresponding 5'-monophosphate were detected in the separate growth medium samples and their relative concentrations therein were determined. These results indicate that the cytotoxicity of these N6-cycloalkylated nucleosides may be attributed to their 5'-monophosphates within the cells.