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Biomedical subjects

J H Elder

Publications and source records attributed to J H Elder.

At least 19 recordsLinked to original sources
PubMed1998

Analysis of the S3 and S3' subsite specificities of feline immunodeficiency virus (FIV) protease: development of a broad-based protease inhibitor efficacious against FIV, SIV, and HIV in vitro and ex vivo.

The S3 and S3' subsite binding specificities of HIV and feline immunodeficiency virus proteases (FIV) proteases (PRs) have been explored by using C2-symmetric competitive inhibitors. The inhibitors evaluated contained (1S, 2R, 3R, 4S)-1,4-diamino-1, 4-dibenzyl-2,3-diol as P1 and P1' units, Val as P2 and P2' residues, and a variety of amino acids at the P3 and P3' positions. All inhibitors showed very high potency against HIV PR in vitro, and their Ki values ranged between 1.1 and 2.6 nM. In contrast to the low restriction of P3 and P3' residues observed in HIV PR, FIV PR exhibited strong preference for small hydrophobic groups at the S3 and S3' subsites. Within this series, the most effective inhibitor against FIV PR contained Ala at P3 and P3'. Its Ki of 41 nM was 415- and 170-fold lower than those of the inhibitors without the P3 and P3' moieties or with the Phe at these positions, respectively. In addition, these compounds were tested against mutant FIV PRs, which contain amino acid substitutions corresponding to those in native HIV PR at homologous sites, and their efficacy of inhibition progressively increased up to 5-fold. The most potent FIV PR inhibitor was selected for examination of its effectiveness in tissue culture, and it was able to block nearly 100% of virus production in an acute infection at 1 microg/ml (1.1 microM) against HIV, FIV, and simian immunodeficiency virus. Furthermore, it was not toxic to cells, and even after 2 months of culture there was no sign of resistance development by virus. The findings suggest that inhibitors with small P3 residue may be efficacious against a broad range of HIV variants as well as interspecies PRs.

Animals↗
PubMed1998

Evidence for boundary-specific grouping.

A prerequisite for higher-level visual tasks such as object recognition is a segmentation of the image into distinct two-dimensional regions. While it has long been assumed that the human visual system jointly exploits region and boundary cues for image segmentation, we report the results of psychophysical experiments which suggest that the visual system relies on geometric properties of bounding contours such as closure and not on the texture of the two-dimensional regions they partition. These findings suggest that the visual system may code and links contours into coherent shapes before surface properties are conjoined.

Discrimination, Psychological↗
PubMed1997

A continuous fluorometric assay for the feline immunodeficiency virus protease.

A novel fluorogenic substrate for continuous feline immunodeficiency virus (FIV) protease (PR) assay was developed in which 2-aminobenzoic acid (Abz) and p-nitrophenylalanine (F(NO2)) were used as the fluorescent donor and acceptor, respectively. The 14-amino-acid fluorogenic substrate of sequence RALTK(Abz) VQ approximately F(NO2)VQSKGR (approximately indicates cleavage site) was modeled after a naturally occurring FIV PR capsid/nucleocapsid cleavage site in the gag polyprotein. The 2-aminobenzoyl group was attached to the epsilon amino group of a lysine (K(Abz)) in position P3 and the F(NO2) is in position P1' in order to promote efficient intramolecular quenching prior to cleavage by FIV PR. We measured a K(m) of 33 +/- 6 microM and a kcat of 0.29 +/- 0.02 s-1 for the enzymatic hydrolysis of this fluorogenic substrate by FIV PR under the conditions of our assay (0.05 M sodium citrate/0.1 M sodium phosphate buffer, pH 5.25, 0.2 M NaCl, 0.1 mM EDTA, and 1 mM dithiothreitol). This assay affords a rapid and convenient means for quantitating FIV PR activities and promises to be useful for judging the relative strength of inhibitors.

DNA, Viral↗
PubMed1997

Crystal structures of the inactive D30N mutant of feline immunodeficiency virus protease complexed with a substrate and an inhibitor.

Crystal structures of complexes of a D30N mutant of feline immunodeficiency virus protease (FIV PR) complexed with a statine-based inhibitor (LP-149), as well as with a substrate based on a modification of this inhibitor (LP-149S), have been solved and refined at resolutions of 2.0 and 1.85 A, respectively. Both the inhibitor and the substrate are bound in the active site of the mutant protease in a similar mode, which also resembles the mode of binding of LP-149 to the native protease. The carbonyl oxygen of the scissile bond in the substrate is not hydrated and is located within the distance of a hydrogen bond to an amido nitrogen atom from one of the two asparagines in the active site of the enzyme. The nitrogen atom of the scissile bond is 3.25 A from the conserved water molecule (Wat301). A model of a tetrahedral intermediate bound to the active site of the native enzyme was built by considering the interactions observed in all three crystal structures of FIV PR. Molecular dynamics simulations of this model bound to native wild-type FIV PR were carried out, to investigate the final stages of the catalytic mechanism of aspartic proteases.

Asparagine↗
PubMed1997

Single subject experimentation for psychiatric nursing.

Mental health nurses encounter countless opportunities to assess medication and intervention efficacy but often find themselves relying solely on descriptive case study accounts and familiar group comparison methodology. Although case studies are useful, they lack scientific rigor and group comparison designs may not be suited to certain client populations or research questions that address individual client characteristics. The purpose of this article is to describe single subject experimentation, contrast this approach with case study and group comparison designs, and discuss how this type of scientific inquiry can be incorporated in psychiatric nursing practice.

Bias↗
PubMed1997

Molecular analysis of the feline immunodeficiency virus protease: generation of a novel form of the protease by autoproteolysis and construction of cleavage-resistant proteases.

The feline immunodeficiency virus (FIV) protease is essential for virion maturation and subsequent viral replication in that it cleaves the Gag and Gag/Pol polyproteins at eight sites to release the respective structural proteins and enzymes. During purification of a recombinant FIV protease (PR), we noted that it underwent autoproteolysis (autolysis) to give discrete cleavage products. These additional PR cleavage sites were defined using N-terminal amino acid sequence analysis and mass spectrometry. Protease breakdown products were also found in FIV virions and were of the same apparent molecular weights as the in vitro autolysis products. Four primary PR autolysis sites were blocked via substitution of either the P1 amino acid with a beta-branched amino acid or the P1' amino acid with lysine. Cleavage-resistant PRs which had Km and k(cat) values similar to those of FIV PR were constructed. An autolysis time course determined that blocking all four primary autolysis sites yielded a cleavage-resistant PR which was enzymatically stable. Concomitant with autolysis is the generation of an N-terminally truncated form of the PR (Thr6/PR) which has enhanced stability with respect to that of FIV PR. A structural basis for the Thr6/PR activity is presented, as are the possible roles of autolysis in the viral replication cycle.

Animals↗
PubMed1997

Blocking of feline immunodeficiency virus infection by a monoclonal antibody to CD9 is via inhibition of virus release rather than interference with receptor binding.

A monoclonal antibody, MAb vpg15, inhibits feline immunodeficiency virus (FIV) infection in tissue culture. The antibody is directed to a determinant of the feline cell surface marker, CD9, implying that CD9 may serve as a viral receptor or coreceptor in this system. In cells expressing CD9, MAb vpg15 markedly delayed acute virus infection in terms of reverse transcriptase activity detected in cell culture supernatants. This effect was evident if the antibody was added before, immediately after, or 24 h after virus infection. Binding experiments showed that MAb vpg15 did not block virus binding to the cells. PCR analyses at various intervals postinfection also indicated that MAb vpg15 did not block virus uptake, reverse transcription of viral RNA, or integration into host cell DNA. Multiply spliced mRNAs were detected up to 24 h postinfection in both control and MAb vpg15-treated cells. However, viral mRNAs were markedly diminished in MAb vpg15-treated cells after this time, consistent with a failure of the FIV infection to spread in the cell culture. Treatment of chronically infected cells with MAb vpg15 also caused a sharp diminution in viral particle production, while viral mRNA levels were the same in both untreated and MAb-treated infected cells. Analyses of intracellular and extracellular levels of virus-associated antigens showed an enhanced accumulation of intracellular p24. These findings are consistent with the interpretation that MAb vpg15 acts at a posttranscriptional stage by interfering with the assembly and/or release of virus from the cell.

Animals↗
PubMed1997

Cortical neuronal cytoskeletal changes associated with FIV infection.

HIV-1 infection is often complicated by central nervous system (CNS) dysfunction. Degenerative neuronal changes as well as neuronal loss have been documented in individuals with AIDS. Feline immunodeficiency virus (FIV) infection of cats provides a model for both the immune and the central nervous system manifestations of HIV infection of humans. In this study we have examined neurons in the frontal cortex of feline immunodeficiency virus-infected cats and controls for immunoreactivity with SMI 32, an antibody recognizing a non-phosphorylated epitope on neurofilaments. We noted a significant increase in the number of immunoreactive pyramidal cells in infected animals compared to controls. The changes seen in the neuronal cytoskeleton as a consequence of the inoculation with FIV were similar to those seen in humans undergoing the normal aging process as well as those suffering from neurological diseases, including Alzheimer's and dementia pugilistica. The changes we noted in the feline brain were also similar to that reported in animals with traumatic injuries or with spontaneously occurring or induced motor neuron diseases, suggesting that the increase in reactivity represents a deleterious effect of FIV on the central nervous system.

Animals↗
PubMed1996

Comparative properties of feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (HIV-1) proteinases prepared by total chemical synthesis.

The aspartyl proteinase (PR) encoded by the feline immunodeficiency virus (FIV) was prepared by total chemical synthesis. The 116-amino-acid polypeptide chain was assembled in a stepwise fashion using a Boc chemistry solid-phase peptide synthesis approach and subsequently folded into the biologically active dimeric proteinase. The synthetic enzyme showed proteolytic activity against a variety of different peptide substrates corresponding to putative cleavage sites of the Gag and Gag-Pol polyproteins of FIV. A comparative study with the proteinase of human immunodeficiency virus type 1 (HIV-1) showed that the FIV and HIV-1 enzymes have related but distinct substrate specificities. In particular, HIV-1 PR and FIV PR each show a strong preference for their own MA/CA substrates, despite identical amino acid residues at four of seven positions from P3-P4' of the substrate including an identical MA/CA cleavage site (between Tyr approximately Pro residues). FIV PR also showed a requirement for a longer peptide substrate than HIV-1 PR. Defining the similarities and the differences in the properties of these two retroviral enzymes will have a significant impact on structure-based drug design.

Amino Acid Sequence↗
PubMed1996

Influence of ORF2 on host cell tropism of feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) is a lentivirus associated with an immunodeficiency syndrome of the domestic cat. A short open reading frame (ORF2), of unknown function, is present in all FIV isolates. We have investigated the role of ORF2 in determining the cell tropism of two infectious molecular clones of FIV. FIV-PPR is able to productively infect feline peripheral blood leukocytes (PBLs) and a T lymphocyte cell line (MCH5-4), but not a feline astrocyte cell line (G355-5) or Crandell feline kidney cells (CrFK). In contrast, FIV-34TF10 is able to productively infect G355-5 and CrFK cells, but not PBLs or MCH5-4 cells. The major difference in these FIV clones is that ORF2 in FIV-PPR is capable of encoding a 79-amino-acid peptide, whereas there is a stop codon in ORF2 after 43 amino acids in FIV-34TF10. We performed site-directed mutagenesis to change the stop codon (TGA) in FIV-34TF10 to a tryptophan (TGG), the amino acid present at this location in FIV-PPR. FIV-34TF10 with ORF2 repaired (FIV-ORF2rep) productively infected PBLs, MCH5-4 cells, and primary macrophages, as well as CrFK and G355-5 cells, indicating that a protein encoded by ORF2 plays a role in determining the host cell tropism of FIV. ORF2 contains hydrophobic, acidic, and leucine-rich domains similar to those shown to be important for transactivating proteins of other lentiviruses. Coexpression of a plasmid expressing the ORF2 gene product with another construct expressing the chloramphenicol acetyl transferase (CAT) gene driven by the FIV LTR, resulted in transactivation of CAT expression in both feline and human cells.

Amino Acid Sequence↗
PubMed1996

Crystal structure of dUTP pyrophosphatase from feline immunodeficiency virus.

We have determined the crystal structure of dUTP pyrophosphatase (dUTPase) from feline immunodeficiency virus (FIV) at 1.9 A resolution. The structure has been solved by the multiple isomorphous replacement (MIR) method using a P6(3) crystal form. The results show that the enzyme is a trimer of 14.3 kDa subunits with marked structural similarity to E. coli dUTPase. In both enzymes the C-terminal strand of an anti-parallel beta-barrel participates in the beta-sheet of an adjacent subunit to form an interdigitated, biologically functional trimer. In the P6(3) crystal form one trimer packs on the 6(3) screw-axis and another on the threefold axis so that there are two independent monomers per asymmetric unit. A Mg2+ ion is coordinated by three asparate residues on the threefold axis of each trimer. Alignment of 17 viral, prokaryotic, and eukaryotic dUTPase sequences reveals five conserved motifs. Four of these map onto the interface between pairs of subunits, defining a putative active site region; the fifth resides in the C-terminal 16 residues, which is disordered in the crystals. Conserved motifs from all three subunits are required to create a given active site. With respect to viral protein expression, it is particularly interesting that the gene for dUTPase (DU) resides in the middle of the Pol gene, the enzyme cassette of the retroviral genome. Other enzymes encoded in the Pol polyprotein, including protease (PR), reverse transcriptase (RT), and most likely integrase (IN), are dimeric enzymes, which implies that the stoichiometry of expression of active trimeric dUTPase is distinct from the other Pol-encoded enzymes. Additionally, due to structural constraints, it is unlikely that dUTPase can attain an active form prior to cleavage from the polyprotein.

Amino Acid Sequence↗
PubMed1996

Neurologic dysfunctions caused by a molecular clone of feline immunodeficiency virus, FIV-PPR.

FIV is a lentivirus of domestic cats that causes a spectrum of diseases that is remarkably similar to the clinical syndrome produced by HIV infection in people. Both HIV and FIV has been shown to cause neurologic dysfunction. Specific Pathogen-Free (SPF) cats were placed into one of three groups: FIV-PPR infected; DU-FIV-PPR (a dUTPase mutant of the FIV-PPR clone) infected; or an age-matched control group. In both infected groups, the general clinical signs of infection included lymphadenopathy, oral ulcerations, rough hair coat, and conjuntivitis. Specific neurological changes in the FIV-PPR infected cats included hind limb paresis; delayed righting and pupillary reflexes; behavioral changes; delayed visual and auditory evoked potentials; decreased spinal and peripheral nerve conduction velocities; and marked alterations in sleep patterns. Most of these changes were also observed in the DU-FIV-PPR infected cats. However, these cats tended to have a slightly less severe disease. In this study, we have demonstrated that an infectious molecular clone of FIV closely parallels the disease course of wild type FIV-infected cats. By using a knockout gene mutant of this clone, we were able to demonstrate that the dUTPase gene is not essential for neuropathogenesis. Further use of the FIV-PPR clone should prove useful in determining the essential viral elements that are important in the neuropathogenesis of lentiviral infections.

Animals↗
PubMed1995

Increased mutation frequency of feline immunodeficiency virus lacking functional deoxyuridine-triphosphatase.

Feline immunodeficiency virus (FIV) encodes the enzyme deoxyuridine-triphosphatase (DU; EC 3.6.1.23) between the coding regions for reverse transcriptase and integrase in the pol gene. Here, we report the in vivo infection of cats with a DU- variant of the PPR strain of FIV and compare its growth properties and tissue distribution with those of wild-type FIV-PPR. The results reveal several important points: (i) DU- FIV is able to infect the cat, with kinetics similar to that observed with wild-type FIV; (ii) both wild-type and DU- FIV-infected specific-pathogen free cats mount a strong humoral antibody response which is able to limit the virus burden in both groups of animals; (iii) the virus burden is reduced in the DU- FIV-infected cats, particularly in tissues such as spleen and salivary gland; and (iv) the mutation frequency in DU- FIVs integrated in the DNA of primary macrophages after 9 months of infection is approximately 5-fold greater than the frequency observed in DU- FIV DNA integrated in T lymphocytes. Mutation rate with wild-type FIV remains the same in both cell types in vivo. The dominant mutations seen in macrophages with DU- FIV are G-->A base changes, consistent with an increased misincorporation of deoxyuridine into viral DNA of DU- FIVs during reverse transcription. Because this enzyme is absent from human immunodeficiency virus type 1 and other primate lentiviruses, virus replication in cell environments with low DU activity may lead to increased mutation and contribute to the rapid expansion of the viral repertoire.

Amino Acid Sequence↗
PubMed1995

Feline immunodeficiency virus (FIV)-specific cytotoxic T lymphocytes from chronically infected cats are induced in vitro by retroviral vector-transduced feline T cells expressing the FIV capsid protein.

We have previously reported the presence of feline immunodeficiency virus (FIV)-specific, major histocompatibility complex (MHC)-restricted cytolytic T lymphocytes (CTL) in experimentally FIV-infected cats. However, the fine specificity of the CTL and the role of individual FIV proteins in inducing FIV-specific CTL responses remain unknown. In this study, we examined the in vitro induction and activity of FIV p24 capsid-specific CTL obtained from cats that had been experimentally infected with FIV Petaluma for 30 to 56 months. An amphotropic murine retroviral vector was used to generate transgenic primary feline T lymphoblasts that expressed the FIV capsid protein. When the autologous capsid-transduced T cells were used in vitro to stimulate CTL responses from peripheral blood mononuclear cells of chronically infected cats, MHC-restricted lysis of virus-infected target cells was observed. The majority of the CTL expressed CD8, and depletion of this population, but not CD4+ cells, effectively diminished the CTL activity. When the autologous capsid-transduced T cells were used as target cells, lysis by capsid-induced effectors was not observed. Analysis of capsid-transduced T cell clones revealed a variable and low level of capsid expression among the clones. This study demonstrates the potential for using retroviral vectors as a means of inducing CTL effector cells that will specifically kill lentivirus-infected cells during lentiviral infection.

Animals↗
PubMed1995

Feline immunodeficiency virus as a model for study of lentivirus infection of the central nervous system.

Feline immunodeficiency virus infects the CNS and results in predictable pathophysiology strikingly similar to that seen with HIV-1 infection of humans. The observed pathophysiology is mimicked in several physiologically assessed modalities, further supporting the validity of the feline model. Peripheral and control evoked potential findings and the occurrence of the sleep architecture changes in both cat and human disease provide an intriguing focus for further investigation. Although structurally diverse in an absolute sense, FIV and HIV-1 share basic structural features and commonalities of their life cycle. It is likely that by understanding the common mechanisms by which these lentiviruses influence CNS function, a more complete understanding of the neurological deficits seen in HIV-1 infected patients will be obtained. The cat model is particularly valuable for study of CNS disease, since it allows detailed analyses of events during the acute phase of infection, under circumstances in which the nature and timing of the infection are carefully controlled. The availability of molecular clones for mutational analysis will facilitate mapping of genomic regions critical to the perturbation of CNS function. It is suggested that development of intervention strategies in the cat model will yield treatment modalities directly applicable to HIV-1 infection of humans.

AIDS Dementia Complex↗
PubMed1995

Retroviral vector-transduced cells expressing the core polyprotein induce feline immunodeficiency virus-specific cytotoxic T-lymphocytes from infected cats.

The core polyprotein of feline immunodeficiency virus (FIV) was expressed in primary feline T-lymphocytes using a retroviral vector. These cells were used as antigen-presenting stimulator cells (APSC) for the in vitro induction of cytotoxic T-lymphocytes (CTL) from feline peripheral blood mononuclear cells (PBMC). CTL from 4 cats chronically infected with the Petaluma strain of FIV specifically lysed autologous FIV-infected targets in an MHC-restricted manner. The CD8 phenotype of more than 70% of the induced effector cells (97% for cells from one cat) was consistent with MHC class I-restricted cytotoxicity. In addition, it was possible to detect low levels of core polyprotein-specific lysis from effector cells of two of the FIV-infected cats. When observed, the level of lysis, measured as a percentage of specific 111In release, was lower for the transgenic gag-expressing targets than for FIV-infected targets. The difference in killing may reflect the low level of core CTL were not detected in either PBMC stimulated with cells transduced by a retroviral vector without the FIV gag sequence or PBMC from an uninfected cat stimulated with autologous transgenic APSC. The detection of FIV-specific CTL from infected cats following stimulation with transgenic APSC suggests a role for retroviral vectors in determining CTL specific for individual lentiviral proteins in protective immunity.

3T3 Cells↗
PubMed1995

In-home communication intervention training for parents of multiply handicapped children.

Two major questions derived from social-interactional theory were addressed: What are the effects of an in-home communication training program for parents on (1) the acquisition of child-training skills (imitating/animating and expectant waiting) by parents of developmentally delayed children who have severe language impairments and autistic features and (2) the acquisition of communication behaviors in the children themselves? Questions regarding the social value of the intervention and effects on parent-child interactions were also addressed. Four mothers were taught two parent-training skills (imitating/animating and expectant waiting) during two in-home training sessions. Following the parent training, the mothers conducted training sessions in their homes with their children for 10 minutes, three times per week for 8-12 weeks. All sessions were videotaped and frequency counts taken of the target parent skills and child target behaviors. A single subject experimental research design was used incorporating a multiple baseline across two parental child-training skills (i.e., imitating/animating, expectant waiting). Data were analyzed visually as is customary in single subject experimentation, and direct behavioral counts showed that all four mothers demonstrated increases in the frequency with which they used the child-training skills following treatment. Increases in four child behavior frequencies (i.e., vocal utterances, social responding, social initiating, and intelligible words spoken) were also noted. The ECO Scales Interaction Profile results supported direct behavioral count data, showing significantly improved parent-child interactions for the subject dyads over three conditions (p < .034). Additionally, Parental Consumer Satisfaction Questionnaire results indicated that the intervention was positively perceived by the parents. Furthermore, the utility of single subject experimentation procedures in clinical nursing practice was clearly demonstrated in this research, and the findings support nurse-conducted, in-home parent training as a practical and socially valid means of addressing the complex needs of chronically disabled children and their families.

Child↗