bluuebunny/crossref_metadata_2025 huggingface dataset:
Catheter-Related
Microbacterium
Bacteremia Identified by 16S rRNA Gene Sequencing
Authors: Lau Susanna K. P. , Woo Patrick C. Y. , Woo Gibson K. S. , Yuen Kwok-Yung
Published: July 2002

We describe the application of 16S rRNA gene sequencing in defining two cases of catheter-related
Microbacterium
bacteremia. In the first case, a gram-positive bacillus was isolated from both the blood culture and central catheter tip of a 39-year-old woman with chronic myeloid leukemia. The API Coryne system identified the isolate as 98.9%
Aureobacterium
or
Corynebacterium aquaticum
. In the second case, a gram-positive bacillus was recovered from five sets of blood cultures from both central catheter and percutaneous venipuncture of a 5-year-old girl with acute myeloid leukemia. The isolate was identified by the API Coryne system as 99.7%
Cellulomonas
or
Microbacterium
species. Further phenotypic tests failed to identify the two isolates. 16S rRNA gene sequencing showed 99.4% similarity between the first isolate and
Microbacterium oxydans
and 98.7% similarity between the second isolate and
Microbacterium trichotecenolyticum
, indicating that both isolates were
Microbacterium
species.
Microbacterium
infections are rarely reported in the literature. Although the central venous catheter was previously proposed to be a source of bacteremia, the first case in this report represents the first culture-documented case of catheter-related
Microbacterium
bacteremia.
Kinetic Analysis of the Role of Intersubunit Interactions in Human Immunodeficiency Virus Type 1 Capsid Protein Assembly In Vitro
Authors: Lanman Jason , Sexton Jennifer , Sakalian Michael , Prevelige, Peter E.
Published: July 2002

The human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) plays a crucial role in both assembly and maturation of the virion. Numerous recent studies have focused on either the soluble form of CA or the polymer end product of in vitro CA assembly. The CA polymer, in particular, has been used to study CA-CA interactions because it is a good model for the CA interactions within the virion core. However, analysis of the process of in vitro CA assembly can yield valuable insights into CA-CA interactions and the mechanism of core assembly. We describe here a method for the analysis of CA assembly kinetics wherein the progress of assembly is monitored by using turbidity. At pH 7.0 the addition of either of the isolated CA domains (i.e., the N or the C domain) to an assembly reaction caused a decrease in the assembly rate by competing for binding to the full-length CA protein. At pH 8.0 the addition of the isolated C domain had a similar inhibitory affect on CA assembly. However, at pH 8.0 the isolated N domain had no affect on the rate of CA assembly but, when mixed with the C domain, it alleviated the C-domain inhibition. These data provide biochemical evidence for a pH-sensitive homotypic N-domain interaction, as well as for an N- and C-domain interaction.
Novel Endogenous Retrovirus in Rabbits Previously Reported as Human Retrovirus 5
Authors: Griffiths David J. , Voisset Cécile , Venables Patrick J. W. , Weiss Robin A.
Published: July 2002

Human retrovirus 5 (HRV-5) represented a fragment of a novel retrovirus sequence identified in human RNA and DNA preparations. In this study, the genome of HRV-5 was cloned and sequenced and integration sites were analyzed. Using PCR and Southern hybridization, we showed that HRV-5 is not integrated into human DNA. A survey of other species revealed that HRV-5 is present in the genomic DNA of the European rabbit (
Oryctolagus cuniculus
) and belongs to an endogenous retrovirus family found in rabbits. The presence of rabbit sequences flanking HRV-5 proviruses in human DNA extracts suggested that rabbit DNA was present in our human extracts, and this was confirmed by PCR analysis that revealed the presence of rabbit mitochondrial DNA sequences in four of five human DNA preparations tested. The origin of the rabbit DNA and HRV-5 in human DNA preparations remains unclear, but laboratory contamination cannot explain the preferential detection of HRV-5 in inflammatory diseases and lymphomas reported previously. This is the first description of a retrovirus genome in rabbits, and sequence analysis shows that it is related to but distinct from A-type retroelements of mice and other rodents. The species distribution of HRV-5 is restricted to rabbits; other species, including other members of the order Lagomorpha, do not contain this sequence. Analysis of HRV-5 expression by Northern hybridization and reverse transcriptase PCR indicates that the virus is transcribed at a low level in many rabbit tissues. In light of these findings we propose that the sequence previously designated HRV-5 should now be denoted RERV-H (for rabbit endogenous retrovirus H).
The Amino-Terminal Half of Sendai Virus C Protein Is Not Responsible for either Counteracting the Antiviral Action of Interferons or Down-Regulating Viral RNA Synthesis
Authors: Kato Atsushi , Ohnishi Yukano , Hishiyama Michiko , Kohase Masayoshi , Saito Sakura , Tashiro Masato , Nagai Yoshiyuki
Published: July 2002

The Sendai virus C proteins, C′, C, Y1, and Y2, are a nested set of independently initiated carboxy-coterminal proteins translated from a reading frame overlapping the P frame on the P mRNA. The C proteins are extremely versatile and have been shown to counteract the antiviral action of interferons (IFNs), to down-regulate viral RNA synthesis, and to promote virus assembly. Using the stable cell lines expressing the C, Y1, Y2, or truncated C protein, we investigated the region responsible for anti-IFN action and for down-regulating viral RNA synthesis. Truncation from the amino terminus to the middle of the C protein maintained the inhibition of the signal transduction of IFNs, the formation of IFN-stimulated gene factor 3 (ISGF3) complex, the generation of the anti-vesicular stomatitis virus state, and the synthesis of viral RNA, but further truncation resulted in the simultaneous loss of all of these inhibitory activities. A relatively small truncation from the carboxy terminus also abolished all of these inhibitory activities. These data indicated that the activities of the C protein to counteract the antiviral action of IFNs and to down-regulate viral RNA synthesis were not encoded within a region of at least 98 amino acids in its amino-terminal half.
Further Characterization of Equine Foamy Virus Reveals Unusual Features among the Foamy Viruses
Authors: Lecellier Charles-Henri , Neves Manuel , Giron Marie-Lou , Tobaly-Tapiero Joelle , Saïb Ali
Published: July 2002

Foamy viruses (FVs) are nonpathogenic, widely spread complex retroviruses which have been isolated in nonhuman primates, cattle, cats, and more recently in horses. The equine foamy virus (EFV) was isolated from healthy horses and was characterized by molecular cloning and nucleotide sequence analysis. Here, to further characterize this new FV isolate, the location of the transcriptional cap and poly(A) addition sites as well as the main splice donor and acceptor sites were determined, demonstrating the existence of the specific subgenomic
pol
mRNA, one specific feature of FVs. Moreover, similar to what has been described for the human foamy virus (HFV), the prototype of FVs, a replication-defective EFV genome was identified during persistent infection. At the protein level, the use of specific antibodies allowed us to determine the size and the subcellular localization of EFV Gag, Env, and Tas, the viral transactivators. While EFV Gag was detected in both the cytoplasm and the nucleus, EFV Env mainly localized in the Golgi complex, in contrast to HFV Env, which is sequestered in the endoplasmic reticulum. In addition, electron microscopy analysis demonstrated that EFV budding occurs at the plasma membrane and not intracellularly, as is the case for primate FVs. Interestingly, EFV Tas was detected both in the nucleus and the cytoplasm of Tas-transfected cells, in contrast to the strict nuclear localization of other FV Tas but similar to the equine infectious anemia virus Tat gene product. Taken together, our results reveal that this new FV isolate exhibits remarkable features among FVs, bringing new insights into the biology of these unconventional retroviruses.
Efficiency of Measles Virus Entry and Dissemination through Different Receptors
Authors: Schneider Urs , von Messling Veronika , Devaux Patricia , Cattaneo Roberto
Published: July 2002

The efficiency with which different measles virus (MV) strains enter cells through the immune cell-specific protein SLAM (CD150) or other receptors, including the ubiquitous protein CD46, may influence their pathogenicity. We compared the cell entry efficiency of recombinant MV differing only in their attachment protein hemagglutinin (H). We constructed these viruses with an additional gene expressing an autofluorescent reporter protein to allow direct detection of every infected cell. A virus with a wild-type H protein entered cells through SLAM two to three times more efficiently than a virus with the H protein of the attenuated strain Edmonston, whereas cell entry efficiency through CD46 was lower. However, these subtle differences were amplified at the cell fusion stage because the wild-type H protein failed to fuse CD46-expressing cells. We also proved formally that a mutation in H protein residue 481 (asparagine to tyrosine) results in improved CD46-specific entry. To define the selective pressure exerted on that codon, we monitored its evolution in different H protein backgrounds and found that several passages in CD46-expressing Vero cells were necessary to shift it in the majority of the MV RNA. To verify the importance of these observations for human infections, we examined MV entry into peripheral blood mononuclear cells and observed that viruses with asparagine 481 H proteins infect these cells more efficiently.
Ubiquitin-Fused and/or Multiple Early Genes from Cottontail Rabbit Papillomavirus as DNA Vaccines
Authors: Leachman Sancy A. , Shylankevich Mark , Slade Martin D. , Levine Dana , K. Sundaram Ranjini , Xiao Wei , Bryan Marianne , Zelterman Daniel , Tiegelaar Robert E. , Brandsma Janet L.
Published: July 2002

Human papillomavirus (HPV) vaccines have the potential to prevent cervical cancer by preventing HPV infection or treating premalignant disease. We previously showed that DNA vaccination with the cottontail rabbit papillomavirus (CRPV) E6 gene induced partial protection against CRPV challenge and that the vaccine's effects were greatly enhanced by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF). In the present study, two additional strategies for augmenting the clinical efficacy of CRPV E6 vaccination were evaluated. The first was to fuse a ubiquitin monomer to the CRPV E6 protein to enhance antigen processing and presentation through the major histocompatibility complex class I pathway. Rabbits vaccinated with the wild-type E6 gene plus GM-CSF or with the ubiquitin-fused E6 gene formed significantly fewer papillomas than the controls. The papillomas also required a longer time to appear and grew more slowly. Finally, a significant proportion of the papillomas subsequently regressed. The ubiquitin-fused E6 vaccine was significantly more effective than the wild-type E6 vaccine plus GM-CSF priming. The second strategy was to vaccinate with multiple CRPV early genes to increase the breadth of the CRPV-specific response. DNA vaccines encoding the wild-type CRPV E1-E2, E6, or E7 protein were tested alone and in all possible combinations. All vaccines and combinations suppressed papilloma formation, slowed papilloma growth, and stimulated subsequent papilloma regression. Finally, the two strategies were merged and a combination DNA vaccine containing ubiquitin-fused versions of the CRPV E1, E2, and E7 genes was tested. This last vaccine prevented papilloma formation at all challenge sites in all rabbits, demonstrating complete protection.
Different Patterns of Immune Responses but Similar Control of a Simian-Human Immunodeficiency Virus 89.6P Mucosal Challenge by Modified Vaccinia Virus Ankara (MVA) and DNA/MVA Vaccines
Authors: Amara Rama Rao , Villinger Francois , Staprans Silvija I. , Altman John D. , Montefiori David C. , Kozyr Natalia L. , Xu Yan , Wyatt Linda S. , Earl Patricia L. , Herndon James G. , McClure Harold M. , Moss Bernard , Robinson Harriet L.
Published: July 2002

Recently we demonstrated the control of a mucosal challenge with a pathogenic chimera of simian and human immunodeficiency virus (SHIV-89.6P) by priming with a Gag-Pol-Env-expressing DNA and boosting with a Gag-Pol-Env-expressing recombinant modified vaccinia virus Ankara (DNA/MVA) vaccine. Here we evaluate the ability of the MVA component of this vaccine to serve as both a prime and a boost for an AIDS vaccine. The same immunization schedule, MVA dose, and challenge conditions were used as in the prior DNA/MVA vaccine trial. Compared to the DNA/MVA vaccine, the MVA-only vaccine raised less than 1/10 the number of vaccine-specific T cells but 10-fold-higher titers of binding antibody for Env. Postchallenge, the animals vaccinated with MVA alone increased their CD8 cell numbers to levels that were similar to those seen in DNA/MVA-vaccinated animals. However, they underwent a slower emergence and contraction of antiviral CD8 T cells and were slower to generate neutralizing antibodies than the DNA/MVA-vaccinated animals. Despite this, by 5 weeks postchallenge, the MVA-only-vaccinated animals had achieved as good control of the viral infection as the DNA/MVA group, a situation that has held up to the present time in the trial (48 weeks postchallenge). Thus, MVA vaccines, as well as DNA/MVA vaccines, merit further evaluation for their ability to control the current AIDS pandemic.
Explant-Induced Reactivation of Herpes Simplex Virus Occurs in Neurons Expressing Nuclear cdk2 and cdk4
Authors: Schang Luis M. , Bantly Andrew , Schaffer Priscilla A.
Published: July 2002

Herpes simplex virus (HSV) establishes productive (lytic) infections in nonneuronal cells and nonproductive (latent) infections in neurons. It has been proposed that HSV establishes latency because quiescent neurons lack cellular factors required for productive infection. It has been further proposed that these putative factors are induced following neuronal stress, as a requirement for HSV reactivation. To date, the identity of these putative cellular factors remains unknown. We have demonstrated that cyclin-dependent kinase (cdk) 1, 2, or 7 is required for HSV replication in nonneuronal cells. Interestingly, cdks 1 and 2 are not expressed in quiescent neurons but can be induced in stressed neurons. Thus, cdks may be among the cellular proteins required for HSV reactivation whose neuronal expression is differentially regulated during stress. Herein, we determined that neuronal expression of nuclear cdk2, cdk4, and cyclins E and D2 (which activate cdks 2 and 4, respectively) was induced following explant cultivation, a stressful stimulus that induces HSV reactivation. In contrast, neuronal expression of cdk7 and cytoplasmic cdk4 decreased during explant cultivation, whereas cdk3 was detected in the same small percentage of neurons before and after explant cultivation and cdks 1, 5, and 6 were not detected in neuronal cell bodies. HSV-1 reactivated specifically in neurons expressing nuclear cdk2 and cdk4, and an inhibitor specific for cdk2 inhibited HSV-1 reactivation. We conclude that neuronal levels of cdk2 are among the factors that determine the outcome of HSV infections of neurons.
Successful Boosting of a DNA Measles Immunization with an Oral Plant-Derived Measles Virus Vaccine
Authors: Webster Diane E. , Cooney Michelle L. , Huang Zhongjun , Drew Damien R. , Ramshaw Ian A. , Dry Ian B. , Strugnell Richard A. , Martin Jenny L. , Wesselingh Steve L.
Published: July 2002

Despite eradication attempts, measles remains a global health concern. Here we report results that demonstrate that a single-dose DNA immunization followed by multiple boosters, delivered orally as a plant-derived vaccine, can induce significantly greater quantities of measles virus-neutralizing antibodies than immunization with either DNA or plant-derived vaccines alone. This represents the first demonstration of an enhanced immune response to a prime-boost vaccination strategy combining a DNA vaccine with edible plant technology.
Given the Opportunity, the Sendai Virus RNA-Dependent RNA Polymerase Could as Well Enter Its Template Internally
Authors: Vulliémoz Diane , Roux Laurent
Published: July 2002

The negative-stranded RNA viral genome is an RNA-protein complex of helicoidal symmetry, resistant to nonionic detergent and high salt, in which the RNA is protected from RNase digestion. The 15,384 nucleotides of the Sendai virus genome are bound to 2,564 subunits of the N protein, each interacting with six nucleotides so tightly that the bases are poorly accessible to soluble reagents. With such a uniform structure, the question of template recognition by the viral RNA polymerase has been raised. In a previous study, the N-phase context has been proposed to be crucial for this recognition, a notion referring to the importance of the position in which the nucleotides interact with the N protein. The N-phase context ruled out the role of the template 3′-OH congruence, a feature resulting from the obedience to the rule of six that implies the precise interaction of the last six 3′-OH nucleotides with the last N protein. The N-phase context then allows prediction of the recognition by the RNA polymerase of a replication promoter sequence even if internally positioned, a promoter which normally lies at the template extremity. In this study, with template minireplicons bearing tandem replication promoters separated by intervening sequences, we present data that indeed show that initiation of RNA synthesis takes place at the internal promoter. This internal initiation can best be interpreted as the result of the polymerase entering the template at the internal promoter. In this way, the data are consistent with the importance of the N-phase context in template recognition. Moreover, by introducing between the two promoters a stretch of 10 A residues which represent a barrier for RNA synthesis, we found that the ability of the RNA polymerase to cross this barrier depends on the type of replication promoter, strong or weak, that the RNA polymerase starts on, a sign that the RNA polymerase may be somehow imprinted in its activity by the nature of the promoter on which it starts synthesis.
The Carboxy-Terminal Region of the Human Immunodeficiency Virus Type 1 Protein Rev Has Multiple Roles in Mediating CRM1-Related Rev Functions
Authors: Hakata Yoshiyuki , Yamada Masami , Mabuchi Naoto , Shida Hisatoshi
Published: July 2002

The human immunodeficiency virus type 1 (HIV-1) regulatory protein, Rev, mediates the nuclear export of unspliced and singly spliced viral mRNAs by bridging viral RNA and export receptor human CRM1 (hCRM1). Ribonucleoprotein complex formation, including the oligomerization of Rev proteins on viral RNA, must occur to allow export. We show here that Rev-Rev interactions, which are a basis of complex formation, can be initiated without cellular factors and are subsequently enhanced by hCRM1-Ran-GTP. Furthermore, we reveal functions for the Rev carboxy-terminal (C-terminal) region, which is well conserved among many HIV-1 strains, and for which no function has been reported. This region is required for the efficient binding of Rev to hCRM1 and consequently for nuclear export, Rev-Rev dimerization, and full Rev transactivator activity. Consistent with these results, a HIV-1 proviral plasmid that expresses a C-terminally truncated Rev mutant protein produces smaller amounts of the p24 antigen than does a plasmid that possesses an intact
rev
gene. These results indicate the functional importance of the C-terminal region for full Rev activity, which leads to efficient HIV-1 replication.
The Herpes Simplex Virus Type 1 US11 Protein Binds the Coterminal UL12, UL13, and UL14 RNAs and Regulates UL13 Expression In Vivo
Authors: Attrill Helen L. , Cumming Sarah A. , Clements J. Barklie , Graham Sheila V.
Published: July 2002

The US11 protein of herpes simplex virus type 1 (HSV-1) is a small, highly basic phosphoprotein expressed at late times during infection. US11 localizes to the nucleolus in infected cells, can associate with ribosomes, and has been shown to bind RNA. The RNA substrates of US11 identified thus far have no apparent role in the virus lytic cycle, so we set out to identify a novel, biologically relevant RNA substrate(s) for this protein in HSV-1-infected cells. We designed a reverse transcriptase PCR-based protocol that allowed specific selection of a 600-bp RNA binding partner for US11. This RNA sequence, designated 12/14, is present in the coterminal HSV-1 mRNAs UL12, UL13, and UL14. We show that the binding of US11 to 12/14 is sequence-specific and mediated by the C-terminal domain of the protein. To elucidate the role of US11 in the virus life cycle, we infected cells with wild-type virus, a cosmid-reconstructed US11 HSV-1 null mutant, and a cosmid-reconstructed wild-type virus and analyzed expression of UL12, -13, and -14 during a time course of infection. These experiments revealed that this interaction has biological activity; at early times of infection, US11 down-regulates UL13 protein kinase mRNA and protein.
Identification of SRPK1 and SRPK2 as the Major Cellular Protein Kinases Phosphorylating Hepatitis B Virus Core Protein
Authors: Daub Henrik , Blencke Stephanie , Habenberger Peter , Kurtenbach Alexander , Dennenmoser Julia , Wissing Josef , Ullrich Axel , Cotten Matt
Published: July 2002

Phosphorylation of hepatitis B virus (HBV) core protein has recently been shown to be a prerequisite for pregenomic RNA encapsidation into viral capsids, but the host cell kinases mediating this essential step of the HBV replication cycle have not been identified. We detected two kinases of 95 and 115 kDa in HuH-7 total cell lysates which interacted specifically with the HBV core protein and phosphorylated its arginine-rich C-terminal domain. The 95-kDa kinase was purified and characterized as SR protein-specific kinase 1 (SRPK1) by mass spectrometry. Based on this finding, the 115-kDa kinase could be identified as the related kinase SRPK2 by immunoblot analysis. In vitro, both SRPKs phosphorylated HBV core protein on the same serine residues which are found to be phosphorylated in vivo. Moreover, the major cellular HBV core kinase activity detected in the total cell lysate showed biochemical properties identical to those of SRPK1 and SRPK2, as examined by measuring binding to a panel of chromatography media. We also clearly demonstrate that neither the cyclin-dependent kinases Cdc2 and Cdk2 nor protein kinase C, previously implicated in HBV core protein phosphorylation, can account for the HBV core protein kinase activity. We conclude that both SRPK1 and SRPK2 are most likely the cellular protein kinases mediating HBV core protein phosphorylation during viral infection and therefore represent important host cell targets for therapeutic intervention in HBV infection.
Syntheses and Structures of Donor-Acceptor Complexes of Selenium Dioxide with Pyridine and Trimethylamine
Authors: Toužín Jiří , Neplechová Kamila , Žák Zdirad , Černík Miloš
Published: July 2002

Donor-acceptor (DA) complexes of pyridine (py)·SeO2 and trimethylamine (TMA)·SeO2 were prepared by direct reactions of solid selenium dioxide with the amines. Both are white solids decomposing slowly at room temperature. The former complex decomposes into its components, while the latter one undergoes a redox reaction. The single-crystal structure determination of py·SeO2 has revealed that the chain-like structure of (SeO2)n is destroyed during the formation of the DA bond and the py·SeO2 molecule is bound to the nearest neighbouring molecule through two weak Se···O interactions. More labile TMA·SeO2 complex could not be prepared as single crystals. Analogous molecular structure of both complexes was proved by their Raman spectra. A modified general valence force field was used for the interpretation of Raman spectra of TMA·SeO2 and TMA·SO2.
Putting Small Metallacarboranes to Work: A Review
Authors: Grimes Russell N.
Published: July 2002

The prospects for developing useful applications based on the chemistry of transition metal complexes of nido-2,3-RR'C2B4H42- and related small carborane ligands are reviewed, with primary emphasis on areas where their utility has been demonstrated in practice. Special attention is directed to the properties of small 6- and 7-vertex MC2B3 and MC2B4 clusters that distinguish them from their icosahedral metallacarborane congeners, and from structurally related metallocene sandwiches. A review with 43 references.
Some Halogenation Reactions of nido-7,8,9,11-P2C2B7H9
Authors: Holub Josef , Bakardjiev Mario , Štíbr Bohumil
Published: July 2002

Halogenations of the diphosphadicarbaborane nido-7,8,9,11-P2C2B7H9 using the AlCl3/CCl4, and I2/AlCl3/C6H6 halogenation systems resulted in the formation of a mixture of mono- and disubstituted derivatives 10-X-nido-7,8,9,11-P2C2B7H8 (for X = Cl and I, yields 6 and 21%, respectively) and 5,10-X2-nido-7,8,9,11-P2C2B7H7 (for X = Cl and I, yields 57 and 46%, respectively). These results show that the halogenation under electrophilic conditions takes place at positions most distant from the P atoms, but at sites adjacent to the CH units. In contrast, the bromination with N-bromosuccinimide in CH2Cl2 is probably of radical character, proceeding in positions adjacent to the P centres to give 3-Br-nido-7,8,9,11-P2C2B7H8 (yield 41%) and 2,3-Br2-nido-7,8,9,11-P2C2B7H7 (yield 5%). The mixtures of the halo derivatives were separated by column chromatography on silica gel. Individual compounds were characterized by mass spectrometry and multinuclear (11B, 1H, and 31P) spectroscopy combined with two-dimensional [11B-11B]-COSY NMR techniques. Various NMR effects of halogen substitutions are discussed.
Synthesis of Novel o-Carborane-Based Ketonitriles and Their Keto-Enol Tautomerism
Authors: Semioshkin Andrey A. , Inyushin Sviatoslav G. , Petrovskii Pavel V. , Bregadze Vladimir I.
Published: July 2002

Novel o-carborane-based ketonitriles were obtained in enol form via acylation of malononitrile with 2-methylcarborane-1-carbonyl chloride as well as by acylation of o-carboranylacetonitrile with benzoyl chloride. These ketonitriles appear to exist in solution mostly in enol form.
Clinical validation of methods of diagnosis of neuropathy in a field study of United Kingdom sheep dippers
Authors: Buchanan D , Jamal G A , Pilkington A , Hansen S
Published: July 2002

Objectives:To investigate the reproducibility of measured indices of chronic peripheral neuropathy from a field study of sheep dippers when compared with similar measurements carried out in a clinical setting.

Methods:A stratified random sample of field study subjects was invited to attend a clinic. Neuropathy was measured both in the field and at the clinic with a modified version of a standard symptoms questionnaire and quantitative sensory thresholds for hot, cold, and vibration. These were combined into a classification of the likelihood of neuropathy with a neuropathy scoring system. Indicators of sensory abnormality were based on comparison of sensory thresholds to age dependent reference values derived from an external reference group.

Results:Only 51% of subjects were assigned similar classifications in the field and clinic based on the neuropathy scoring system. Of the component indices, grouped symptom scores, with 65% of subjects showing exact agreement, proved to be more reproducible than quantitative sensory test indicators. There were biases in the comparison of field and clinic measurements of hot and vibration sensations, but no evidence of greater variation between individual people in sensory thresholds in the field relative to at the clinic.

Conclusions:The neuropathy scoring system proved to be of limited reproducibility, due in a large part to the lack of reproducibility of the indicators of sensory test abnormality caused by inadequate temperature control. However, the symptoms score and measured sensory thresholds could be used separately as indices of neuropathy in exposure-response analyses.
Active epidemiological surveillance of musculoskeletal disorders in a shoe factory
Authors: Roquelaure Y , Mariel J , Fanello S , Boissière J-C , Chiron H , Dano C , Bureau D , Penneau-Fontbonne D
Published: July 2002

Aims:(1) To evaluate an active method of surveillance of musculoskeletal disorders (MSDs). (2) To compare different criteria for deciding whether or not a work situation could be considered at high risk of MSDs in a large, modern shoe factory.

Methods:A total of 253 blue collar workers were interviewed and examined by the same physician in 1996; 191 of them were re-examined in 1997. Risk factors of MSDs were assessed for each worker by standardised job site work analysis. Prevalence and incidence rates of carpal tunnel syndrome, rotator cuff syndrome, and tension neck syndrome were calculated for each of the nine main types of work situation. Different criteria used to assess situations with high risk of MSDs were compared.

Results:On the basis of prevalence data, three types of work situation were detected to be at high risk of MSDs: cutting, sewing, and assembly preparation. The three types of work situations identified on the basis of incidence data (sewing preparation, mechanised assembling, and finishing) were different from those identified by prevalence data. At least one recognised risk factor for MSDs was identified for all groups of work situations. The ergonomic risk could be considered as serious for the four types of work situation having the highest ergonomic scores (sewing, assembly preparation, pasting, and cutting).

Conclusion:The results of the health surveillance method depend largely on the definition of the criteria used to define the risk of MSDs. The criteria based on incidence data are more valid than those based on prevalence data. Health and risk factor surveillance must be combined to predict the risk of MSDs in the company. However, exposure assessment plays a greater role in determining the priorities for ergonomic intervention.
Step-pool and cascade morphology, Mosquito Creek, British Columbia: a test of four analytical techniques
Authors: Wooldridge C L , Hickin E J
Published: July 2002

The identification and geometric definition of individual cascade and step-pool bedforms are investigated in a steep, coarse-grained, mountain stream, Mosquito Creek, by testing four analytical techniques: visual identification, zero-crossing, bedform differencing, and power spectral analysis. The test is the first use of these techniques in a headwater stream, and the analysis of two bed profiles showed that visual identification was able to (i) identify, (ii) determine the geometry of, and (iii) classify the type of individual bedforms better than the other methods. The other techniques were not able to differentiate step-pools from cascades, and the large range of grain sizes and bedform heights hampered their ability to consistently identify stepped bedforms. The step-pool (pronounced, channel-spanning steps that alternate with channel-spanning pools) and cascade (multi-tiered, partially channel-spanning structures) morphology in Mosquito Creek has formed in the last 20 years as fluvial action has restructured its previously engineered, revetment-lined, planar bed. The channel bed exhibits a morphologic regularity that power spectral analysis captured as periodic fluctuations in the bed profiles, with mean wavelengths slightly greater than those identified by the other methods. Further, the active reorganization of revetment has formed stepped structures with geometries similar (i.e., height to wavelength ratios) to stepped features found in natural mountain streams. Channel slope partially controlled bedform geometry (wavelength and height), and bedform height weakly controlled individual step spacing, but there was no relation between wavelength and grain size (D90).
Arctic North American seasonal temperatures from the latest Miocene to the Early Pleistocene, based on mutual climatic range analysis of fossil beetle assemblages
Authors: Elias Scott A , Matthews Jr. John V
Published: July 2002

Late Tertiary and early Quaternary fossil beds in the arctic regions of North America have yielded abundant, well-preserved remains of plants and arthropods, documenting the existence of coniferous forests in the high arctic latitudes. Nearly all of the beetle (Coleoptera) specimens in these fossil assemblages represent extant species. We have applied the mutual climatic range (MCR) method of paleotemperature analysis to fossil beetle assemblages from 11 sites to estimate mean summer (Tmax) and winter (Tmin) temperatures. We found that arctic Tmin values during the latest Miocene and Pliocene were substantially warmer than they are today. The MCR estimates therefore support the scenario derived from the paleobotanical data, namely that arctic Pliocene climates were far less continental. Several Pliocene-age assemblages from the high Arctic yielded Tmax estimates 9-10°C warmer than modern values at the sites. This is the same degree of warming required to allow coniferous forests to grow in the high Arctic. By 3 million years BP, a cooling trend is marked in the paleobotanical and fossil beetle evidence from Alaska. All assemblages dating between 5.7 and 2 million years BP yielded calibrated Tmax values between 12.4 and 13.8°C, regardless of location. Thus the insect fossil data support the theory that there was far less latitudinal gradation in temperatures during the Late Pliocene than there is today. Our reconstructions suggest regional climatic cooling (especially winter temperatures) began by at least 2 million years BP.
Early Eocene Leptictida, Pantolesta, Creodonta, Carnivora, and Mesonychidae (Mammalia) from the Eureka Sound Group, Ellesmere Island, Nunavut
Authors: Eberle Jaelyn J , McKenna Malcolm C
Published: July 2002

We describe the leptictid Prodiacodon; the pantolestids Palaeosinopa sp.nov., cf. Palaeosinopa, and Pantolestidae, gen. et sp. indet.; the creodonts Palaeonictis and Prolimnocyon; the carnivorans Viverravus, cf. Vulpavus, and Miacis; and the mesonychid Pachyaena from early Eocene (i.e., Wasatchian) strata of the Eureka Sound Group on central Ellesmere Island, Nunavut. Palaeosinopa and Palaeonictis may have originated in mid-latitude North America and subsequently migrated to Europe via a north Atlantic land bridge, while Prolimnocyon and Pachyaena probably originated in Asia. Additionally, the occurrence of Pachyaena in the Early Eocene of Europe probably is best explained by dispersal from high-latitude North America to Europe via a north Atlantic land bridge. We update the Eureka Sound Group mammalian faunal list.
New insights into the structure of the Sudbury Igneous Complex from downhole seismic studies
Authors: Snyder David , Perron Gervais , Pflug Karen , Stevens Kevin
Published: July 2002

New vertical seismic profiles from the northwest margin of the Sudbury impact structure provide details of structural geometries within the lower impact melt sheet (usually called the Sudbury Igneous Complex) and the sublayer norite layer. Vertical seismic profile sections and common depth point transformation images display several continuous reflections that correlate with faults and stratigraphic boundaries logged from drill cores. Of four possible mechanisms that explain repeated rock units, late-stage flow or normal faulting that occurred within the last layers to cool and crystallize might best explain the observations, especially the most prominent reflectors observed in the seismic data. These results reaffirm previously proposed two-stage cooling and deformation models for the impact melt sheet.
Estimating abundance from gillnet samples with application to red drum (Sciaenops ocellatus) in Texas bays
Authors: Porch Clay E , Fisher Mark R , McEachron Lawrence W
Published: July 2002

A model of gillnet selection is developed to accommodate the possibility that some catch observations will be known more precisely than others and allow for nonlinear relationships between the selection parameters and mesh size. The model is used to show that gillnet selection for red drum (Sciaenops ocellatus) in Texas bays may be explained as a unimodal process approximating a skewed Laplace distribution, where the optimal length varies in proportion to mesh size and the variance in proportion to the optimal length. It is also suggested that the number of encounters with the net ought to depend on swimming speed of the quarry, which in turn varies predictably with length. This information, along with the estimates of selection, is used to develop indices of abundance for each length-class. The results indicate that the recruitment of year-old red drum to Texas bays has fluctuated markedly since 1975, but without any persistent trends. However, the survival of these and older fish has increased dramatically owing to various regulations promulgated since 1981.
Second-generation MS-based high-throughput screening system for enantioselective catalysts and biocatalysts
Authors: Schrader Wolfgang , Eipper Andreas , Pugh D Jonathan , Reetz Manfred T
Published: July 2002

A high-throughput method is described, where the enantioselectivity of approximately 10 000 catalysts or biocatalysts can be determined per day. The method is based on electrospray mass spectrometric techniques using an eight-channel multiplexed (MUX) sprayer system connected to a time-of-flight mass spectrometer. The inlet of the ion source is controlled by a stepping rotor that is continuously moving from one sprayer to the next with a recording time of 100 ms for each channel and a delay time of 50 ms, thus allowing a spectrum to be obtained from each channel every 1.2 s. One cycle, where eight samples are being sprayed in parallel, requires around 70 s, which allows a 96-well microtiter plate to be screened in 14 min. Integration of two pseudo-enantiomers (S)-glycidyl phenyl ether and (R)-D5-glycidyl phenyl ether is necessary to quantify the enantiomeric excess (ee-value), where one enantiomer is isotopically labeled to allow easy identification of the mass spectrometric signals. Errors of ~2% for the ee-values indicate that in addition to the significant improvement in sample throughput this is also a precise method for high-throughput screening. This second-generation assay is useful for combinatorial enantioselective transition-metal catalysis and in the directed evolution of enantioselective enzymes.
Tailoring the substrate specificity of secondary alcohol dehydrogenase
Authors: Phillips Robert S
Published: July 2002

Our research with the thermophilic secondary-alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus has provided novel information regarding the physical basis of enzyme substrate specificity and stereospecificity. We demonstrated that oxidation of secondary alcohols catalyzed by T. ethanolicus SADH exhibits temperature-dependent enantiospecificity. In other studies, we found that the structure of co-factor analogs also significantly affects the stereochemistry of the SADH reaction. More recently, we demonstrated that pH can also have a modest effect on SADH enantiospecificity. Organic solvents have also been shown by others to affect the stereochemistry of SADH reactions. We designed and prepared S39T and C295A mutant forms of SADH by site-directed mutagenesis, and we evaluated the effects of the mutations by analysis of the temperature dependence of the enantiomeric ratio (E) for simple chiral alcohols such as 2-butanol. This procedure allows for the determination of the differential Eyring parameters (ΔΔHand ΔΔS) for the reaction. We demonstrated that this technique is a sensitive method for analysis of the effects of mutation on enzyme stereospecificity. S39T and C295A SADH exhibit significant changes in substrate specificity and stereospecificity consistent with the changes in the volume of the alkyl-binding pockets. Thus, it is possible to alter the substrate specificity and stereospecificity of alcohol dehydrogenase by changing either the reaction medium or the protein structure.Key words: alcohol dehydrogenase, substrate specificity, stereospecificity, temperature dependence, site-directed mutagenesis.
Dipeptide sulfonamides as endothelin ETA/ETB receptor antagonists
Authors: Ksander Gary M , Shetty Suraj S , DelGrande Dominick , Balwierczak Joseph L , Bruseo Charles W , Savage Paula , deJesus Reynalda , Yuan Andrew , Webb Randy L , Jeng Arco Y
Published: July 2002

Endothelin-1 (ET-1) is a potent mitogen and modulator of vascular tone. It is synthesized and released from endothelial cells and acts upon two receptor subtypes designated as ETA and ETB. In this study, a series of potent dipeptide sulfonamide dual-endothelin ETA/ETB receptor antagonists were prepared to investigate their potential benefit in vascular diseases. CGS 31398 inhibited [125I]ET-1 binding to human ETA and ETB receptors expressed in Chinese hamster ovary (CHO) cells (ETA/CHO, ETB/CHO) with respective IC50 values of 0.26 and 0.12 nM. However, in anesthetized rats, this compound markedly potentiated ET-1-induced renal vascular resistance, a response normally observed with selective ETB receptor antagonists. To determine whether species differences account for these results, a direct comparison was made between binding to rat and rabbit aortic membranes versus functional antagonism in isolated rat aortic rings. It was found that CGS 31398 had potent affinity for the ETA receptor in rat and rabbit aorta with IC50 values of 0.87 and 0.79 nM, respectively. Inhibition of ET-1-induced contractions of rat aorta by the compound was considerably weaker than expected (pKB = 6.4), while that of sarafotoxin S6c induced contraction of dog saphenous vein (100% inhibition at 100 nM) was consistent with corresponding binding data. These results suggest that although CGS 31398 is a potent dual inhibitor of ETA/ETB receptor binding, it surprisingly displays potent ETB and weak ETA receptor antagonism in functional assays.Key words: ETA receptor, ETB receptor, CGS 31398, SB 209670, receptor antagonists.
Regulation of nociceptive neurons by nerve growth factor and glial cell line derived neurotrophic factor
Authors: Priestley J V , Michael G J , Averill S , Liu M , Willmott N
Published: July 2002

Nociceptive dorsal root ganglion (DRG) cells can be divided into three main populations, namely (1) small diameter non-peptide-expressing cells, (2) small-diameter peptide-expressing (calcitonin gene related peptide (CGRP), substance P) cells, and (3) medium-diameter peptide-expressing (CGRP) cells. The properties of these cell populations will be reviewed, with a special emphasis on the expression of the vanilloid (capsaicin) receptor VR1 and its regulation by growth factors. Cells in populations 1 and 2 express VR1, a nonselective channel that transduces certain nociceptive stimuli and that is crucial to the functioning of polymodal nociceptors. Cells in population 1 can be regulated by glial cell line derived neurotrophic factor (GDNF) and those in populations 2 and 3 by nerve growth factor (NGF). In vivo, DRG cells express a range of levels of VR1 expression and VR1 is downregulated after axotomy. However, treatment with NGF or GDNF can prevent this downregulation. In vitro, DRG cells also show a range of VR1 expression levels that is NGF and (or) GDNF dependent. Functional studies indicate that freshly dissociated cells also show differences in sensitivity to capsaicin. The significance of this is not known but may indicate a difference in the physiological role of cells in populations 1 and 2.Key words: nociceptors, CGRP, IB4, vanilloid, dorsal root ganglion.
Peptide signals regulating food intake and energy homeostasis
Authors: Blevins James E , Schwartz Michael W , Baskin Denis G
Published: July 2002

The adiposity hormone leptin has been shown to decrease food intake and body weight by acting on neuropeptide circuits in the hypothalamus. However, it is not clear how this primary hypothalamic action of leptin is translated into a change in food intake. We hypothesize that the behavioral effect of leptin ultimately involves the integration of neuronal responses in the forebrain with those in the nucleus tractus solitarius in the caudal brainstem, where ingestive behavior signals are received from the gastrointestinal system and the blood. One example is the peptide cholecystokinin, which is released from the gut following ingestion of a meal and acts via vagal afferent nerve fibers to activate medial nucleus tractus solitarius neurons and thereby decrease meal size. While it is established that leptin acts in the arcuate nucleus in the hypothalamus to stimulate anorexigenic neurons that inhibit food intake while simulataneously inhibiting orexigenic neurons that increase food intake, the mechanisms linking these effects with regions of the caudal brainstem that integrate cues related to meal termination are unclear. Based on an increasing body of supportive data, we hypothesize that this integration involves a pathway comprising descending projections from neurons from the paraventricular nucleus to neurons within the nucleus tractus solitarius that are activated by meal-related satiety factors. Leptin's anorexic effect comprises primarily decreased meal size, and at subthreshold doses for eliciting an effect on food intake, leptin intensifies the satiety response to circulating cholecystokinin. The location of neurons subserving the effects of intracerebroventricular administration of leptin and intraperitoneal injection of cholecystokinin on food intake has been identified by analysis of Fos expression. These studies reveal a distribution that includes the paraventricular nucleus and regions within the caudal brainstem, with the medial nucleus tractus solitarius having the most pronounced Fos expression in response to leptin and cholecystokinin, and support the hypothesis that the long-term adiposity signal leptin and the short-term satiety signal cholecystokinin act in concert to maintain body weight homeostasis.Key words: brainstem, adiposity, satiety.
New insights into the antidepressant actions of substance P (NK1 receptor) antagonists
Authors: Rupniak Nadia M.J
Published: July 2002

Considerable progress has been made in understanding the neural circuits involved the antidepressant and anxiolytic efficacy of substance P (NK1 receptor) antagonists (SPAs). Progress has been hampered by species differences in the pharmacology of the NK1 receptor, and the availability of NK1R/ mice has been a particularly useful resource in overcoming this difficulty. Using neuroanatomical, behavioural, and electrophysiological techniques, studies have now established that pharmacological blockade or deletion of the NK1 receptor produces an antidepressant and anxiolytic-like profile in a range of behavioural assays that is distinct from that of established drugs. There is evidence from focal injection studies that some of these effects may be mediated directly by blockade of NK1 receptors in the amygdala and its projections to the hypothalamus, periaqueductal gray, and reticulopontine nucleus. Substance P and NK1 receptors are also intimately associated with ascending 5-HT and norepinephrine projections to the forebrain, and alterations in the function of these systems are also likely to be related to the antidepressant efficacy of SPAs. Unlike some established drugs, SPAs are generally well tolerated and do not induce sedation or motor impairment in preclinical species. These findings are consistent with a novel antidepressant mechanism of action of SPAs.Key words: NK1 receptor, depression, anxiety.