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Biomedical subjects

J Lenard

Publications and source records attributed to J Lenard.

At least 19 recordsLinked to original sources
PubMed2001

Male and female car drivers--differences in collision and injury risks.

Crash data from two UK resources were examined for differences between male and female passenger car drivers in collision circumstances and injury outcomes. The proportion of female car license holders is growing, women are more likely to be the driver in a collision and are more vulnerable to injury particularly neck strain. Women drive smaller, lighter cars compared to men and are more often the driver of the smaller vehicle in a multivehicle collision. Vehicle design, crash testing programmes and regulation, currently based heavily on the average male, should give more balanced consideration to female characteristics in future activities.

Abbreviated Injury Scale↗
PubMed1999

Host cell protein kinases in nonsegmented negative-strand virus (mononegavirales) infection.

Phosphorylation of one or more viral proteins is probably an essential step in the life cycle of every member of the nonsegmented negative-strand RNA virus (mononegavirales [MNV]) group. Since no virally encoded protein kinases have been discovered in this group, phosphorylation is effected entirely by host cell kinases. The virally encoded P proteins of the MNV are the only ones consistently phosphorylated with a stoichiometry > or =1. The P protein of vesicular stomatitis virus (VSV), and perhaps also of respiratory syncytial virus, are the only ones for which a function of phosphorylation has been established. Phosphorylation by casein kinase 2 at one or more identified sites in the VSV P protein activates transcriptional activity by promoting formation of a homotrimer, which is then capable of binding the RNA polymerase and attaching it to the N protein-RNA template. A second phosphorylation of VSV P protein by a different kinase also occurs, dependent upon prior modification by casein kinase 2, but its function is not definitely known. Phosphorylation of the other MNV P proteins may serve a different purpose. No evidence has been obtained yet for any function for phosphorylation of any other MNV protein.

Casein Kinases↗
PubMed1998

The transmembrane domain in viral fusion: essential role for a conserved glycine residue in vesicular stomatitis virus G protein.

The transmembrane (TM) domains of viral fusion proteins are required for fusion, but their precise role is unknown. G protein, the fusion protein of vesicular stomatitis virus, was previously shown to lose syncytia-forming ability if six residues (GLIIGL) were deleted from its TM domain. The 20-residue TM domain of wild-type (TM20) G protein was thus changed into a TM domain of 14 residues (TM14). To assess possible sequence specificity for this loss of function, the two Gly residues in TM20 were replaced with either Ala or Leu. Both mutations resulted in complete loss of fusion activity, as measured by fusion-dependent reporter gene transfer. Single substitutions decreased activity by about half. TM14 was weakly active (15%) but reintroduction of a Gly residue into TM14 by a single Ile --> Gly substitution increased activity to 80%. All mutants retained normal hemifusion activity, i.e., lipid mixing between the outer leaflets of the reacting membranes. Thus, at least one TM Gly residue is required for a late step in fusion mediated by G protein. Gly residues were significantly (2.6-fold; P = 0.004) more abundant in the TM domains of viral fusion proteins than in those of nonfusion proteins and were distributed differently within the TM domain. Thus, Gly residues in the TM domain of other viral fusion proteins may also prove to be important for fusion activity.

Amino Acid Sequence↗
PubMed1997

Characterization of cholesterol-free insect cells infectible by baculoviruses: effects of cholesterol on VSV fusion and infectivity and on cytotoxicity induced by influenza M2 protein.

The patented cell line from the cabbage looper Trichoplusia ni (High Five from Invitrogen) was found to grow readily under cholesterol-free (CF) culture conditions. Cellular cholesterol became undetectable by CF passage 4, while growth rate and overall cell morphology remained unaffected for at least 59 CF passages. The Golgi apparatus in CF cells was significantly smaller than in control cells, and the CF cells also concentrated a ceramide-based fluorescent Golgi marker to a greater extent, but endoplasmic reticulum morphology appeared unaffected. Two proteins were expressed in High Five cells from recombinant baculoviruses under CF and control conditions: the vesicular stomatitis virus (VSV) fusion glycoprotein G and the influenza virus ion channel M2. Both proteins were expressed in comparable amounts in CF and control cells. Both were properly assembled and transported to the plasma membrane in CF cells, indicating the presence of functional Golgi. Wild-type G protein expression resulted in extensive syncytia formation in both CF and control cells, showing that cholesterol is not required for VSV fusion. However, a mutant G protein lacking six transmembrane domain residues was inactive in both CF and control cells. Influenza M2 protein was functional in control cells, as indicated by its amantadine-inhibitable cytotoxicity, but cytotoxicity was absent in CF cells expressing this protein, indicating a cholesterol-dependence for the cytotoxic action of this protein. CF and control cells were both infectible with VSV. However, infected cell centers were modestly decreased (ca. 3.5-fold) in CF cells. CF cells offer a convenient and novel approach to the study of specific cholesterol functions.

Animals↗
PubMed1997

Lower limb injuries to passenger car occupants.

A detailed examination was undertaken of hospitalized car occupants who sustained a lower limb injury in a frontal crash. The assessment included an analysis of the type, severity and causes of these injuries and mechanisms involved in lower limb fractures. The findings showed that fractures and dislocations occurred in 88% of lower limb injury cases, that more than half were from crashes < 48 km hour-1 and that the number of fractures was directly proportional with delta V. Ankle dislocations and foot fractures from the floor and toe pand were the most common injury-source combination overall. The most frequent mechanisms of lower limb fracture were compression, perpendicular loading of the knee and crushing or twisting of the foot. The study points to the need for further regulation to reduce lower limb fractures in frontal crashes and highlights a number of possible countermeasures.

Accidents, Traffic↗
PubMed1996

The transcriptional form of the phosphoprotein of vesicular stomatitis virus is a trimer: structure and stability.

The phosphoprotein (P) of vesicular stomatitis virus was previously shown to assemble into a homomultimer upon phosphorylation by casein kinase II. It thus acquired transcriptional activity, including the ability to bind to the other two transcriptional components, the polymerase L and the N-RNA template. This multimer has now been found to be a trimer using a His-tag dilution method. Trimer stability was assessed using a variation of this method, by measuring the rate of exchange of monomers between preformed tagged and untagged trimers at different values of pH and ionic strength. Exchange rates increased with increasing ionic strength and were similar at pH 6, 8, and 10, but the trimer was completely dissociated at pH 4. This suggests that the trimer is stabilized by electrostatic interactions, probably involving carboxylate and guanidino groups. Addition of viral L protein stabilized the P trimers, completely preventing subunit exchange under transcription conditions. The association constants (Kass) for trimerization of partially active D and A substitution mutants were also determined by His-tag dilution and found to correlate well with transcriptional activity, further confirming that the active species is the trimer. Circular dichroism spectra were identical for phosphorylated and unphosphorylated wild-type P protein and for D and A mutants known to be predominantly trimeric and monomeric, respectively.

Alanine↗
PubMed1995

Multimerization and transcriptional activation of the phosphoprotein (P) of vesicular stomatitis virus by casein kinase-II.

Casein kinase-II (CK-II) is a widely distributed protein kinase, which plays numerous roles in the regulation of transcription through modification of transacting transcription factors. Phosphorylation of vesicular stomatitis virus (VSV) P protein by CK-II was found to be both necessary and sufficient for transcriptional activation. Upon treatment of P by CK-II, activity was acquired faster (t1/2 = 3.7 min) than were total phosphates (t1/2 = 7.4 min). Stoichiometry was 2 mol phosphate/mol P, indicating activation by phosphorylation at either one or both of two independent sites. The sites were identified by substituting aspartate (D) residues at either S60 or T62, producing proteins that were partly active without phosphorylation, but were fully active at higher concentrations; CK-II added only a single phosphate group to each of these, and conferred full activity. P protein doubly substituted with D at S60 and T62 was fully active without phosphorylation, and was not a substrate for CK-II. Active P protein, whether CK-II treated or doubly substituted, was shown by gel filtration and crosslinking to exist as a discretely multimeric, probably tetrameric, structure. The singly substituted mutants were partly multimeric, becoming fully so after CK-II treatment. Phosphorylation by CK-II thus mediates the self-association of P into the multimeric, transcriptionally active form.

Amino Acid Sequence↗
PubMed1995

Cooperative binding of multimeric phosphoprotein (P) of vesicular stomatitis virus to polymerase (L) and template: pathways of assembly.

It was previously shown that the phosphoprotein (P) of vesicular stomatitis virus must undergo phosphorylation-dependent multimerization to become transcriptionally active. Phosphorylation at S-60 and/or T-62 by casein kinase II or substitution of these residues by D is required for multimer formation. We now find that substitution of either one of these residues by A prevents phosphorylation by casein kinase II and multimer formation. The binding of multimeric P to the other two transcriptional components of vesicular stomatitis virus (L protein and the N-RNA template) has been characterized by using P immobilized on beads through its poly(His) tag to facilitate recovery of bound complexes. Multimerization of P was absolutely required for binding to both L and template. Multimeric P combined with the polymerase enzyme (L) in a stoichiometric 1:1 complex, which bound to the N-RNA template much more strongly than multimeric P alone. Substitution of S-227 and S-233 by A residues had no effect on multimerization or binding of L to P but prevented binding of both P and L to template and abolished transcriptional activity. In contrast, substitution of these residues with D residues had no effect on template binding or activity. However, substitution at these sites by either D or A largely abolished phosphorylation by L-associated kinases, thus identifying S-227 and S-233 as the major sites targeted by these kinases and confirming that phosphorylation of P protein by L-associated kinases is without transcriptional effect.

Animals↗
PubMed1994

Seripauperins of Saccharomyces cerevisiae: a new multigene family encoding serine-poor relatives of serine-rich proteins.

A gene, PAU1, has been cloned from Saccharomyces cerevisiae and sequenced. It is located in a telomeric region, probably on chromosome IV, and contains an open reading frame encoding a protein of 120 amino acids (aa) (approx. 13 kDa). The deduced sequence is nearly identical to two other genes found in GenBank (named PAU2 and PAU3 by us), which are located close to the ends of chromosomes V and III, respectively. Blotting of separated chromosomes with a PAU1 probe at high stringency revealed that at least six chromosomes in addition to III, IV and V possessed related sequences, suggesting a large gene family. Probing of an ordered array of phage lambda clones containing yeast genomic DNA inserts ('Olson filters') revealed ten additional hybridizing sequences, located close to the ends of the left and/or right arms of chromosomes I, II, VII, VIII, X, XII, XIV and XV. Transcription of these sequences could not be demonstrated, however, under a wide variety of growth and culture conditions. The deduced PAU1, PAU2 and PAU3 aa sequences are all highly homologous with the SRP1 aa sequences, which contains eight serine-rich tandem repeats of 12 aa each, at its C terminus. This homology is limited, however, to the N-terminal half of SRP1, and does not include the repeats. In fact, PAU1 is quite serine-poor (5.8%), leading to the suggested name of seripauperins for this family of genes.

Amino Acid Sequence↗
PubMed1993

Photodynamic inactivation of infectivity of human immunodeficiency virus and other enveloped viruses using hypericin and rose bengal: inhibition of fusion and syncytia formation.

The mechanism of the antiviral activity of hypericin was characterized and compared with that of rose bengal. Both compounds inactivate enveloped (but not unenveloped) viruses upon illumination by visible light. Human immunodeficiency and vesicular stomatitis viruses were photodynamically inactivated by both dyes at nanomolar concentrations. Photodynamic inactivation of fusion (hemolysis) by vesicular stomatitis, influenza, and Sendai viruses was induced by both dyes under similar conditions (e.g., I50 = 20-50 nM for vesicular stomatitis virus), suggesting that loss of infectivity resulted from inactivation of fusion. Syncytium formation, between cells activated to express human immunodeficiency virus gp120 on their surfaces and CD4+ cells, was inhibited by illumination in the presence of 1 microM hypericin. Hypericin and rose bengal thus exert similar virucidal effects. Both presumably act by the same mechanism--namely, the inactivation of the viral fusion function by singlet oxygen produced upon illumination. The implications of this photodynamic antiviral action for the potential therapeutic usefulness of both hypericin and rose bengal are discussed.

Animals↗
PubMed1993

Effects of staurosporine on transcription by vesicular stomatitis virus.

The protein kinase inhibitor staurosporine has been found to inhibit vesicular stomatitis virus transcription in infected BHK cells. Both primary and secondary transcription were virtually abolished by 10 microM staurosporine. In contrast, transcription by purified virions, or by nucleocapsids isolated from them, was unaffected by staurosporine. Staurosporine inhibition did not occur at or prior to the uncoating step, since: (i) the drug was equally effective if added at t = 0 or at t = 1 hr after infection; (ii) immunofluorescence microscopy of infected cells in the presence of staurosporine showed that M protein was diffusely present in the cytoplasm, indicative of normal uncoating. Staurosporine caused a modest (< 25%) reduction in virus internalization from the cell surfaces. This was not sufficient to account for the transcription inhibition, however. Inhibition of transcription by staurosporine was not observed in extracts from infected cells, nor could it be induced by addition of uninfected cell cytosol to purified nucleocapsids. This suggests that inhibition of transcription in infected cells is secondary to substrate depletion or other cytotoxic effects of the drug. The addition of staurosporine to purified nucleocapsids, while not affecting cell-free transcription, inhibited phosphorylation of L protein completely and of P (NS) protein partially, providing addition evidence that at least two different kinases are present in nucleocapsids, and that the staurosporine inhibited ones are unnecessary for cell-free viral transcription.

Alkaloids↗
PubMed1993

Antiretroviral activities of hypericin and rose bengal: photodynamic effects on Friend leukemia virus infection of mice.

The ability of hypericin to protect mice from splenomegaly resulting from infection with Friend leukemia virus (FLV) was re-examined in light of recent evidence showing that light is absolutely required for this drug's antiviral activity. FLV-induced splenomegaly was not prevented or ameliorated in mice injected with 100 micrograms hypericin, either mixed with the FLV inoculum or administered 1 day p.i., either under normal laboratory light or in the dark. These results contradict previous findings. Both hypericin and rose bengal, however, inactivated the FLV inoculum at low doses (< 11 micrograms), provided that the mixture was illuminated for 1 h under a normal fluorescent desk lamp. This procedure protected mice completely from FLV-induced splenomegaly, and provided a possible explanation for the discrepancy between our results and those reported previously. We conclude that for FLV, as for other enveloped viruses studied previously, illumination of hypericin with the virus is absolutely required for hypericin's antiviral (virucidal) effects, thus limiting its potential usefulness as an antiretroviral agent.

Animals↗
PubMed1993

Photoinactivation of influenza virus fusion and infectivity by rose bengal.

Rose bengal inactivated influenza virus upon exposure to light. Infectivity and fusion were inactivated with the same dose dependence, supporting the suggestion that the virucidal activity of photodynamic agents against enveloped viruses may be generally due to inactivation of their fusion protein(s). Concentrations required for inactivation were found to depend upon the ratio of rose bengal to virus, rather than on the nominal aqueous concentration. Fusion-competent virosomes were inactivated similarly to intact virus particles. The HA2 portion of the influenza fusion protein HA underwent two different, apparently mutually exclusive modifications upon illumination with rose bengal: cross-linking, and conversion to a form that moved slightly more slowly on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Inactivation of viral fusion was inhibited by oxygen removal or addition of azide or beta-carotene, and was enhanced by D2O, consistent with partial involvement of singlet oxygen. The possibility of a second mechanism of viral photoinactivation, by direct interaction between the viral fusion protein and the photoactivated dye, is also discussed.

Animals↗
PubMed1992

Occupant safety in modern passenger cars.

A study was undertaken recently for the Federal Office of Road Safety in Australia of 150 modern vehicle crashes where at least one of the vehicle occupants was admitted to hospital. The types of injuries sustained by occupants of modern Australian passenger cars involved in road crashes (including points of contact within the vehicle) were assessed to provide direction for future improvements in occupant protection. Seat belt performance in all seating positions was of particular interest. While the limited number of cases did not permit a full and detailed statistical analysis of these data, the findings nevertheless show there is scope for improving occupant protection for drivers and passengers of modern passenger cars.

Accidents, Traffic↗
PubMed1992

Characterization and partial purification of an insulinase from Neurospora crassa.

An insulin-binding metal- and thiol-dependent proteinase has been purified 1491-fold from high speed cytosolic fractions of the fungus Neurospora crassa. This enzyme resembles insulin-degrading enzymes (insulinases) present in mammalian cells and in Drosophila melanogaster in the following ways: (i) it degrades radiolabeled insulin with a specificity similar to that of rat muscle insulinase, as demonstrated by HPLC analysis of the degradation products; (ii) it is inhibited by bacitracin, EDTA, 1,10-phenanthroline, and the sulfhydryl-reactive compounds N-ethylmaleimide and p-chloromercuribenzoate, but not by inhibitors of serine proteases or by lysosomal protease inhibitors. Cross-linking with 125I-insulin labels a band of ca. 120 kDa, and several smaller bands which may represent degradation products. The N. crassa insulinase is stimulated by Mn2+ and strongly inhibited by Zn2+; Mn2+ can also reactivate the enzyme after inhibition by EDTA, but Zn2+ is ineffective. The N. crassa protein differs in this regard from mammalian and insect insulinases which are generally activated by both Mn2+ and Zn2+. This finding extends the apparent evolutionary conservation of these metal- and thiol-dependent proteases into the microbial realm.

Cations, Divalent↗