Search PubMed⌕ Search

Biomedical subjects

A M Lever

Publications and source records attributed to A M Lever.

At least 37 records · Page 2Linked to original sources
PubMed1999

Comparative antimicrobial efficacy of multi-purpose lens care solutions using the FDA's revised guidance document for industry: stand-alone primary criteria.

PURPOSE: We evaluated six single-bottle, multi-purpose lens care solutions and a two component lens care system for disinfection efficacy according to the stand-alone primary criteria within the recently published U.S. Food and Drug Administration (FDA) Guidelines. METHODS: One-tenth mL of 1 x 10(8) colony forming units (CFU)/mL of bacterial and fungal challenge organisms was added to each test solution. Following a specified period (e.g., each manufacturer's labeled minimum disinfection time), aliquots of inoculated test solution were neutralized and plated on validated recovery media. After incubation the number of viable microorganisms were enumerated and mean log reductions determined. RESULTS: ReNu and ReNu MultiPlus met the FDA's acceptance criteria for stand-alone disinfectants against all challenge organisms: Staphylococcus aureus, Serratia marcescens, Pseudomonas aeruginosa, Candida albicans, and Fusarium solani. Opti-Free Express failed to meet the FDA's stand-alone disinfectant acceptance criteria for S. aureus, S. marcescens and C. albicans and Opti-Free Express with Opti-Free Supraclens failed to meet the acceptance criteria for either S. aureus and C. albicans. Opti-One failed to meet the FDA's stand-alone disinfectant acceptance criteria for C. albicans and F. solani. Both Complete and Solo-Care failed to meet the FDA's acceptance criteria for C. albicans. CONCLUSIONS: This evaluation provides a direct comparison of antimicrobial activity (based on stand-alone criteria) for commercially available multi-purpose lens care solutions at their labeled minimum disinfection times. The results of this study should be considered when selecting appropriate lens care systems for patients.

Bacteria↗
PubMed1998

Pausing of reverse transcriptase on retroviral RNA templates is influenced by secondary structures both 5' and 3' of the catalytic site.

In the most extensive examination to date of the relationship between the pausing of reverse transcrip-tase (RT) and RNA secondary structures, pause events were found to be correlated to inverted repeats both ahead of, and behind the catalytic site in vitro. In addition pausing events were strongly associated with polyadenosine sequences and to a lesser degree diadenosines and monoadenosine residues. Pausing was also inversely proportional to the potential bond strength between the nascent strand and the template at the point of termination, for both mono and dinucleotides. A run of five adenosine and four uridine residues caused most pausing on the HIV-1 template, a region which is the site of much sequence heterogeneity in HIV-1. We propose that homopolyadenosine tracts can act as termination signals for RT in the context of inverted repeats as they do for certain RNA polymerases.

Avian Myeloblastosis Virus↗
PubMed1998

Development of cell lines stably expressing human immunodeficiency virus type 1 proteins for studies in encapsidation and gene transfer.

Experiments were done to test cell lines for their capacity to express human immunodeficiency virus type 1 (HIV-1) proteins in a stable manner. Marked differences were seen in the ability to stably express and export viral Gag and Pol proteins. Two cell lines, one suspension (MDS) and one monolayer (SW480), were established which exported these proteins at high level. Two other cell lines, HeLa and THP-1, showed poorer expression and very limited particle release. Single cell cloning was used to select the optimal producing clones from the lines. These produced large quantities of viral core particles pelletable from the supernatants. Cell lines were constructed from these clones which stably expressed in addition either the HIV-1 Envelope or a packageable HIV-based vector. The vector was shown to be packaged within the viral core particles. Transient transfection of envelope expressing constructs into a gag-pol plus vector cell line, or the vector into a gag-pol plus envelope expressing cell line resulted in gene transfer to CD4+ target cells. These cell lines provide useful tools with which to study the assembly and export of viral proteins and RNA, for assay of alternative envelope proteins to pseudotype HIV cores, for assessment of antiviral drugs and as a source of correctly processed proteins for immunological studies.

Capsid↗
PubMed1998

Nonreciprocal packaging of human immunodeficiency virus type 1 and type 2 RNA: a possible role for the p2 domain of Gag in RNA encapsidation.

The ability of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) to cross-package each other's RNA was investigated by cotransfecting helper virus constructs with vectors derived from both viruses from which the gag and pol sequences had been removed. HIV-1 was able to package both HIV-1 and HIV-2 vector RNA. The unspliced HIV-1 vector RNA was packaged preferentially over spliced RNA; however, unspliced and spliced HIV-2 vector RNA were packaged in proportion to their cytoplasmic concentrations. The HIV-2 helper virus was unable to package the HIV-1 vector RNA, indicating a nonreciprocal RNA packaging relationship between these two lentiviruses. Chimeric proviruses based on HIV-2 were constructed to identify the regions of the HIV-1 Gag protein conferring RNA-packaging specificity for the HIV-1 packaging signal. Two chimeric viruses were constructed in which domains within the HIV-2 gag gene were replaced by the corresponding domains in HIV-1, and the ability of the chimeric proviruses to encapsidate an HIV-1-based vector was studied. Wild-type HIV-2 was unable to package the HIV-1-based vector; however, replacement of the HIV-2 nucleocapsid by that of HIV-1 generated a virus with normal protein processing which could package the HIV-1-based vector. The chimeric viruses retained the ability to package HIV-2 genomic RNA, providing further evidence for a lack of reciprocity in RNA-packaging ability between the HIV-1 and HIV-2 nucleocapsid proteins. Inclusion of the p2 domain of HIV-1 Gag in the chimera significantly enhanced packaging.

Amino Acid Sequence↗
PubMed1998

Functional analysis of the core human immunodeficiency virus type 1 packaging signal in a permissive cell line.

Packaging of type C retrovirus genomic RNAs into budding virions requires a highly specific interaction between the viral Gag precursor and unique cis-acting packaging signals on the full-length RNA genome, allowing the selection of this RNA species from among a pool of spliced viral RNAs and similar cellular RNAs. This process is thought to involve RNA secondary and tertiary structural motifs since there is little conservation of the primary sequence of this region between retroviruses. To confirm RNA secondary structures, which we and others have predicted for this region, disruptive, compensatory, and deletion mutations were introduced into proviral constructs, which were then assayed in a permissive cell line. Disruption of either of two predicted stem-loops was found to greatly reduce RNA encapsidation and replication, whereas compensatory mutations restoring base pairing to these stem-loops had a wild-type phenotype. A GGNGR motif was identified in the loops of three hairpins in this region. Results were consistent with the hypothesis that the process of efficient RNA encapsidation is linked to dimerization. Replication and encapsidation were shown to occur at a reduced rate in the absence of the previously described kissing hairpin motif.

Base Sequence↗
PubMed1997

Variable immortalizing potential and frequent virus latency in blood-derived T-cell clones infected with human T-cell leukemia virus type I.

Human T-cell leukemia virus type I (HTLV-I)-infected T cells expanded in vitro by single-cell cloning provide a unique system for investigating virus-cell interactions in nonimmortalized T cells. By analysis of clones generated randomly from the blood of virus carriers, we confirm that CD4 T cells are the major reservoir of HTLV-I in vivo and show that most infected cells contain a single integrated provirus. Contrary to the situation in HTLV-I immortalized cell lines, the HTLV-I provirus was found to be transcriptionally silent in a high proportion of randomly generated T-cell clones and could not be reactivated by mitogenic stimulation. The spontaneous proliferation previously documented in HTLV-I-infected T-cell clones was not observed in silently infected cells, and therefore correlates directly with the expression of tax and other viral genes. The only cytokine mRNA found to be significantly elevated in the virus-producing clones was interleukin-6; however, receptor-blocking experiments argue against a role for IL-6 in the virus-induced cell proliferation. We observed a striking variation in the ability of individual HTLV-I-producing clones to immortalize fresh peripheral blood lymphocytes. This ability did not correlate with the levels of viral mRNA expression, gag p24 production, spontaneous proliferation, or tax-transactivation, possibly suggesting a role for host cell factors as determinants of viral infectivity or immortalization. Studies to elucidate the basis of this phenotypic heterogeneity should enhance our understanding of viral spread and pathogenesis.

Blotting, Southern↗
PubMed1997

Effects of cation substitutions on reverse transcriptase and on human immunodeficiency virus production.

Reverse transcription is a key aspect of the retroviral life cycle. The enzyme reverse transcriptase requires divalent cations, manganese or magnesium, for function. In some cation-dependent systems substitution of a physiological metal by a nonphysiological metal has been shown to work. We investigated the effect of different cations on HIV reverse transcriptase activity. The studies established reaction conditions for assaying different cations. A variety of transition metals were used in in vitro assays with HIV recombinant RT homodimer and some were delivered to HIV-infected cells in vitro to study effects on virus production. Most metals substituted adequately for magnesium. However, palladium showed a marked nonreversible inhibition of RT activity in vitro that correlated with reduced HIV virus production in tissue culture. A more extensive range of transition metals and divalent cations was tested for their effects on detection of HIV RT from infected cell supernatants. In these complex phenotypes were seen. In some cases the RT activity appeared to be more easily detectable. This may relate to calcium-dependent nucleases in cell supernatants being inhibited, leading to an apparent enhancement of RT activity, or may be due to direct effects on RT processivity.

Cations↗
PubMed1997

Location of cis-acting signals important for RNA encapsidation in the leader sequence of human immunodeficiency virus type 2.

We used a series of deletion mutations in the untranslated leader region of human immunodeficiency virus type 2 (HIV-2) to seek cis-acting packaging signals. Sequences between the 5' major splice donor and the gag initiation codon, where such signals have been identified in HIV-1, appear to make a measurable but very minor contribution to genomic RNA packaging, and deletions here had little effect on viral replication in vitro. Immediately 5' to the splice donor, two regions were identified which, when deleted, caused a significant replication defect. The most proximal of these to the splice donor demonstrated a phenotype consistent with its being a major cis-acting packaging signal in HIV-2.

Animals↗
PubMed1996

Sequences involved in the dimerisation of human T cell leukaemia virus type-1 RNA.

The formation of a genomic RNA dimer appears to be a critical step in the life cycle of all retroviruses. To investigate the site and nucleotide interactions involved in this process, a 531 bp DNA fragment encompassing sequences up- and downstream of the splice donor in human T cell leukaemia virus type 1 (HTLV-1) was inserted into a plasmid vector under the control of the SP6 promoter. RNA transcripts generated in vitro from this template formed dimers which could be dissociated by heating at 60-80 degrees C for 3 min. The physical properties of the dimeric RNA were not consistent with either Watson-Crick base pairing or guanine tetrad formation as being solely responsible for the interaction. Deletion mutagenesis identified a 32 nt sequence required for dimerisation. Computer modelling was carried out in order to identify putative RNA secondary structures within this essential region. A stem-loop structure was identified, the stem of which was conserved among different sequenced isolates of HTLV-1. This sequence also contains a 15 nt palindrome. We sought by disruptive and compensatory mutagenesis to define the possible roles of these two structures in dimer linkage.

Base Sequence↗
PubMed1996

Reasons for delay in administration of antibiotics to patients with meningitis and meningococcaemia.

To determine the extent of, and reasons for, delay in treatment of patients with bacterial meningitis or meningococcal septicaemia, we reviewed the case notes of all adults admitted to Addenbrooke's Hospital, Cambridge with these diagnoses over a 3 year period. Thirty-three patients were identified. Some 70% (21/30) patients admitted via their GPs were not treated before admission. In 12 of these cases, the diagnosis was not considered. Nine patients were not treated despite the diagnosis of meningitis being considered possible or likely; in two cases this was due to suspected penicillin allergy, but no reason was given for the remaining seven. Of 24 patients untreated prior to hospital admission, only nine were given antibiotics before lumbar puncture had been performed. Failure to treat meningitis and meningococcaemia most often resulted from failure to consider the diagnosis, but treatment was delayed in a significant number of cases for no obvious reason. General practitioners and hospital doctors need to have a low threshold for administering antibiotics as soon as the diagnosis of bacterial meningitis or meningococcaemia is considered possible.

Adult↗
PubMed1996

Neisserial disease.

Explore the source record for details and available documents.

Complement Activation↗
PubMed1996

trans-acting proteins involved in RNA encapsidation and viral assembly in human immunodeficiency virus type 1.

The human immunodeficiency virus type 1 gag gene product Pr55gag self-assembles when expressed on its own in a variety of eukaryotic systems. Assembly in T lymphocytes has not previously been studied, nor is it clear whether Pr55gag particles can package genomic RNA or if the Gag-Pol polyprotein is required. We have used a series of constructs that express Gag or Gag-Pol proteins with or without the viral protease in transient transfections in COS-1 cells and also expressed stably in CD4+ T cells to study this. Deletion of the p6 domain at the C terminus of protease-negative Pr55gag did not abolish particle release, while truncation of the nucleocapsid protein reduced it significantly, particularly in lymphocytes. Gag-Pol polyprotein was released from T cells in the absence of Pr55gag but did not encapsidate RNA. Pr55gag encapsidated human immunodeficiency virus type 1 RNA whether expressed in a protease-positive or protease-negative context. p6 was dispensable for RNA encapsidation. Marked differences in the level of RNA export were noted between the different cell lines.

Amino Acid Sequence↗
PubMed1996

The human immunodeficiency virus type 1 5' packaging signal structure affects translation but does not function as an internal ribosome entry site structure.

The role of the RNA secondary structure in the 5' packaging signal region of human immunodeficiency virus type 1 (HIV-1) in initiating translation of gag mRNA has been investigated both in vitro and in the presence of cellular cofactors in vivo. Heat denaturation of the structure and mutagenic deletion both lead to an increase in levels of translated products, indicating that the structure is a significant inhibitor of translation. The proximity of the gag AUG to the packaging signal structure suggested that it might function as an internal ribosome entry site. However, in both a cell-free system and eukaryotic cells, translation will initiate at a novel upstream initiation codon introduced within the 5' noncoding region. This codon is utilized exclusively, resulting in gag protein products with an extra 11 amino acids at the amino terminus, which, when expressed in T lymphocytes, are confined intracellularly, probably because of the lack of an N-terminal glycine myristoylation signal. Deletion of the secondary structure abolishes gag production even in the presence of tat and rev in trans. Using dicistronic constructs containing the HIV-1 5' leader cloned between two heterologous open reading frames, we were unable to detect any significant expression of the second open reading frame that would have been supportive of an internal ribosome entry site mechanism. Using mutant proviruses either lacking the entire packaging signal structure region or containing the introduced upstream initiation codon in long-term replication studies, we were unable to detect reverse transcriptase activity in culture supernatants. The 5' packaging signal structure of HIV-1 does not serve as an internal ribosome entry site. The translation of gag is consistent with ribosomal scanning. However, the packaging signal structure causes significant translational inhibition.

Amino Acid Sequence↗
PubMed1996

Expression of mutant and wild-type gag proteins for gene therapy in HIV-1 infection.

The effect of expression of defective HIV-based retroviral constructs in CD4-positive lymphocytes on subsequent infection of the cell by HIV-1 was studied. A vector containing a N terminally elongated gag protein which was noncleavable and myristoylation negative was not effective at inhibiting HIV assembly or viral replication in the culture. Expression of a wild-type HIV gag in trans led to an increase in cytopathicity within the culture such that all the cells died. A control LTR containing vector had no effect. A myristoylation negative gag would not appear to be a useful dominant negative inhibitor of HIV replication, but might be usable as a post-exposure immunogen. Post-infective immunisation with wild-type HIV-1 gag would appear to risk increasing virus-related cytopathicity.

Cell Line↗