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.

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.

Involvement of the carboxyl terminus of the third intracellular loop of the cannabinoid CB1 receptor in constitutive activation of Gs.

The human cannabinoid receptor CB1 functionally couples primarily to Gi-, but also to Gs-mediated pathways to modulate intracellular cyclic AMP (cAMP) levels. To probe the features of the receptor that may be involved in promoting interactions with one G protein type over another, we generated the L341A/A342L mutant CB1 receptor. The double mutation involved the swap in position of two adjacent residues in the carboxyl-terminal segment of the third intracellular loop of CB1. This resulted in partial constitutive activation of the receptor and an agonist-independent enhancement in cAMP levels. Characterization following treatment with either pertussis or cholera toxin indicated that the constitutive activity is selective for a Gs- and not a Gi-mediated pathway. Treatment with the CB1-specific inverse agonist SR141716A inhibited the basal accumulation of cAMP in the presence of pertussis toxin, establishing that the effect is CB1 mediated. The binding of the agonist CP-55,940 to the L341A/A342L receptor was not markedly different from that for the wild-type receptor despite the constitutive Gs activity. This may reflect a preference of this ligand for an activated receptor state associated with the Gi coupling form and underscores the potential for developing therapeutics that selectively activate one pathway over another.

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.

Ligand binding and modulation of cyclic AMP levels depend on the chemical nature of residue 192 of the human cannabinoid receptor 1.

The human cannabinoid receptor associated with the CNS (CB1) binds delta9-tetrahydrocannabinol, the psychoactive component of marijuana, and other cannabimimetic compounds. This receptor is a member of the seven transmembrane domain G protein-coupled receptor family and mediates its effects through inhibition of adenylyl cyclase. An understanding of the molecular mechanisms involved in ligand binding and receptor activation requires identification of the active site residues and their role. Lys192 of the third transmembrane domain of the receptor is noteworthy because it is the only nonconserved, charged residue in the transmembrane region. To investigate the properties of this residue, which are important for both ligand binding and receptor activation, we generated mutant receptors in which this amino acid was changed to either Arg (K192R), Gln (K192Q), or Glu (K192E). Wild-type and mutant receptors were stably expressed in Chinese hamster ovary cells and were evaluated in binding assays with the bicyclic cannabinoid CP-55,940 and the aminoalkylindole WIN 55,212-2. We found that only the most conservative change of Lys to Arg allowed retention of binding affinity to CP-55,940, whereas WIN 55,212-2 bound to all of the mutant receptors in the same range as it bound the wild type. Analysis of the ligand-induced inhibition of cyclic AMP production in cells expressing each of the receptors gave an EC50 value for each agonist that was comparable to its binding affinity, with one exception. Although the mutant K192E receptor displayed similar binding affinity as the wild type with WIN 55,212-2, an order of magnitude difference was observed for the EC50 for cyclic AMP inhibition with this compound. The results of this study indicate that binding of CP-55,940 is highly sensitive to the chemical nature of residue 192. In contrast, although this residue is not critical for WIN 55,212-2 binding, the data suggest a role for Lys192 in WIN 55,212-2-induced receptor activation.

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.

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.

The vesicular stomatitis virus matrix protein inhibits transcription from the human beta interferon promoter.

In cells infected by wild-type (wt) vesicular stomatitis virus (VSV) Indiana, host transcription is severely inhibited. DNA cotransfection studies have implicated the VSV matrix (M) protein in this process (B. L. Black and D. S. Lyles, J. Virol. 66:4058-4064, 1992). The M protein inhibited transcription not only from viral promoters in plasmids but also from the chromosomally integrated human immunodeficiency virus type 1 (HIV-1) provirus promoter (S.-Y. Paik, A. C. Banerjea, G. G. Harmison, C.-J. Chen, and M. Schubert, J. Virol. 69:3529-3537, 1995). In this study, we investigated the effect of wt VSV M protein on expression of a reporter gene under control of a cellular promoter (beta-interferon [IFN-beta] promoter), using double transient transfections in BHK and COS-1 cells. The cellular IFN-beta promoter was as susceptible to the inhibitory effect of the M protein as the viral promoters used previously. Viral proteins N, P, and G had no significant effect on reporter gene expression. The M protein gene from VSV mutant T1026R1, which is defective in host transcription inhibition, was cloned and sequenced, and its effect on reporter gene expression was tested. The mutant M protein had a methionine-to-arginine change at position 51 in the protein sequence and did not inhibit transcription from either the IFN-beta promoter or viral promoters. This VSV mutant is a good inducer of IFN, as opposed to the wt virus, which suppresses IFN induction. These results show that the M protein inhibits transcription from cellular as well as viral promoters and that the M protein does not regulate the IFN promoter any differently from viral promoters. While the M protein may play a role in IFN gene regulation, other viral or cellular factors that provide specificity to the induction process must also be involved.

Nurses and physicians evaluate their intensive care experiences.

OBJECTIVE: Analysis of the evaluation of intensive care experiences of nurses and physicians in relation to their educational background and degree of professionalization. DESIGN: Cross-cultural, qualitative study design: German nurses and physicians and American nurses. SETTING: German medical intensive care unit (nine beds), German surgical intensive care unit (six beds), and an American medical-surgical intensive care unit (16 beds). SUBJECTS: Thirteen German nurses (eight women, five men) of a medical intensive care unit, six German nurses (five women, one man) of a surgical intensive care unit, nine German physicians of a medical intensive care unit (two women, seven men), and 13 American nurses of a medical-surgical intensive care unit (10 women, three men). METHODS: The interviewing technique by the method of Role Repertory Grid Test by G.A. Kelly. We focused on important job experiences in intensive care and the remembered evaluation of these experiences. RESULTS: The reported experiences of intensive care unit nurses and physicians show negative as well as positive appraisals. No correlation can be shown between negative appraisals and the degree of professionalization or training of German nurses and physicians, and American nurses, respectively. The qualitative-content analysis of positively appraised experiences does indicate, for all four studied groups, that even primarily so-called "positive" experiences are closely connected with negative aspects and connotations. CONCLUSIONS: The results indicate that nurses and physicians experience intensive care work with extreme ambivalence. To be content with and successful on the job it appears necessary to develop a high level of ambivalence. From a methodologic point of view, the advantages of the interviewing technique (Role Repertory Grid) are discussed.

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.

NF-kappaB activation Is delayed in mouse L929 cells infected with interferon suppressing, but not inducing, vesicular stomatitis virus strains.

Vesicular stomatitis virus (VSV) mutant T1026R1 of the Indiana (IN) serotype is a good inducer of interferon (IFN). This mutant was used to study the activation of NF-kappaB, a transcription factor necessary for IFN induction, in mouse L929 cells that were stably transfected with a chimeric gene containing the human IFN-beta gene promoter attached to the chloramphenicol acetyltransferase (CAT) coding sequence. NF-kappaB DNA binding activity was detected as early as 30 min after virus adsorption in nuclear extracts, increased up to 4 hr, and then remained constant for at least 6 additional hr. The kinetics of CAT expression correlated with the kinetics of NF-kappaB nuclear DNA binding activity. Virus entry and delivery of viral components into the cytoplasm were required for NF-kappaB activation. Exposure of T1026R1 to one hit of UV irradiation nearly completely reduced NF-kappaB activation. In cells infected with wild-type (wt) VSV (IN), a noninducer of IFN, NF-kappaB DNA binding activity in the nucleus was delayed for several hours after virus adsorption. Coinfection of wt VSV and T1026R1 resulted in the reduction of T1026R1-promoted NF-kappaB activation. This inhibitory activity of wt VSV was abolished by one hit of UV irradiation. Under similar conditions expression of the CAT gene was more UV resistant, suggesting that IFN gene expression is regulated at multiple levels.

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.

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.

A novel bacteristatic action of bovine and porcine serum that is reversed by norepinephrine.

A previously unreported anti-bacterial activity against Escherichia coli has been found in porcine and bovine sera, but not in human or chicken sera. The activity is not affected by heat inactivation of the complement system, and is bacteriostatic rather than bactericidal. Growth inhibition is reversible by addition of norepinephrine.

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.

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.

Preparation of a cell-free translation system with minimal loss of initiation factor eIF-2/eIF-2B activity.

Cell-free translation systems prepared from suspension-cultured HeLa S3 cells or mouse L cells by hypotonic shock followed by Dounce homogenization poorly initiated the translation of exogenous mRNA. In contrast, cell extracts prepared from cells exposed to the detergent lysolecithin translated exogenous mRNA readily. The block in initiation in the former lysates was localized to the ribosome fraction. During in vitro translation polysomes from homogenized cells disaggregated but the run-off ribosomes were unable to reinitiate translation. The block resulted from a decrease in eukaryotic initiation factor 2 (eIF-2) or the guanine nucleotide exchange factor (eIF-2B) activity, since the addition of eIF-2 or eIF-2B to these latter extracts substantially improved the capacity of the extract to initiate translation of exogenous mRNA. Extracts from homogenized cells, but not from detergent-treated cells, showed enhanced ability to phosphorylate the alpha subunit of exogenous eIF-2. We show that the method of cell extract preparation greatly influences the state of eIF-2/eIF-2B activity in the resulting extract and that extracts in which this activity is maintained can readily initiate translation on exogenous mRNA and reinitiate on endogenous mRNA.

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.