Inter-rater reliability of output measures for a posture matching assessment approach: a pilot study with food service workers.

Despite the ongoing health problem of repetitive strain injuries, there are few tools currently available for ergonomic applications evaluating cumulative loading that have well-documented evidence of reliability and validity. The purpose of this study was to determine the inter-rater reliability of a posture matching based analysis tool (3DMatch, University of Waterloo) for predicting cumulative and peak spinal loads. A total of 30 food service workers were each videotaped for a 1-h period while performing typical work activities and a single work task was randomly selected from each for analysis by two raters. Inter-rater reliability was determined using intraclass correlation coefficients (ICC) model 2,1 and standard errors of measurement for cumulative and peak spinal and shoulder loading variables across all subjects. Overall, 85.5% of variables had moderate to excellent inter-rater reliability, with ICCs ranging from 0.30-0.99 for all cumulative and peak loading variables. 3DMatch was found to be a reliable ergonomic tool when more than one rater is involved.

Growth arrest-specific gene 6 expression in human breast cancer.

Growth arrest-specific gene 6 (Gas6), identified in 1995, acts as the ligand to the Axl/Tyro3 family of tyrosine kinase receptors and exerts mitogenic activity when bound to these receptors. Overexpression of the Axl/Tyro3 receptor family has been found in breast, ovarian and lung tumours. Gas6 is upregulated 23-fold by progesterone acting through the progesterone receptor B (PRB). Recently, Gas6 has been shown to be a target for overexpression and amplification in breast cancer. Quantitative real-time PCR analysis was used to determine the levels of Gas6 mRNA expression in 49 primary breast carcinomas. Expression of PRB protein was evaluated immunohistochemically with a commercially available PRB antibody. The results showed a positive association between PRB protein and Gas6 mRNA levels (P=0.04). Gas6 correlated positively with a number of favourable prognostic variables including lymph node negativity (P=0.0002), younger age at diagnosis (P=0.04), smaller size of tumours (P=0.02), low Nottingham prognostic index scores (P=0.03) and low nuclear morphology (P=0.03). This study verifies for the first time the association between PRB and Gas6 in breast cancer tissue.

Gain of imprinting of SLC22A18 sense and antisense transcripts in human breast cancer.

The 11p15.5 region harbors three imprinted sense/antisense transcript pairs, SLC22A18/SLC22A18AS, IGF2/IGF2AS (PEG8), and KCNQ1/KCNQ1OT1 (LIT1). SLC22A18 (solute carrier family 22 (organic cation transporter) member 18) and its antisense transcript SLC22A18AS are paternally suppressed in fetal samples. In adult tissue, SLC22A18 displays polymorphic imprinting, but the imprinting status of SLC22A18AS remains elusive. SLC22AI8 DNA-PCR-RFLP analysis using NlaIII restriction digestion identified SLC22A18 heterozygotes within this breast tissue cohort (n = 89). Commercial sequencing identified informative SLC22A18AS samples. Random hexamer-primed cDNA synthesis, SLC22A18/SLC22A18AS-specific PCR, and imprinting evaluation by commercial sequencing demonstrated that SLC22A18AS displays a nonimprinted profile in reduction mastectomies (n = 6). However, SLC22A18 showed a gain of imprinting (GOI) in 1/4 of these normal cases. In the malignant cohort, GOI was also demonstrated in 18% for SLC22A18 and 14% for SLC22A18AS, occurring concomitantly in one case. This study reports the imprinting status of SLC22A18AS in adult tissue, and shows that GOI affects both the sense, and antisense transcripts at this locus in human breast tissue.

A correlational test of the relationship between posttraumatic growth, religion, and cognitive processing.

The present study examined the degree to which event related rumination, a quest orientation to religion, and religious involvement is related to posttraumatic growth. Fifty-four young adults, selected based on prescreening for experience of a traumatic event, completed a measure of event related ruminations, the Quest Scale, an index of religious participation, and the Posttraumatic Growth Inventory. The three subscales of the Quest Scale, the two groups of rumination items (soon after event/within past two weeks), and the index of religious participation were entered in a standard multiple regression with the total score of the Posttraumatic Growth Inventory as the dependent variable. The degree of rumination soon after the event and the degree of openness to religious change were significantly related to Posttraumatic Growth. Congruent with theoretical predictions, more rumination soon after the event, and greater openness to religious change were related to more posttraumatic growth. Present findings offer some confirmation of theoretical predictions, and also offer clear direction for further research on the relationships of religion, rumination, and posttraumatic growth.

Internally controlled quantitative assays for PKR mRNA and protein from liver biopsies and other finite clinical samples.

A method to quantify double-stranded RNA-dependent protein kinase (PKR) mRNA and protein from human cells is described. A competitive RT-PCR assay has been developed by synthesis of an internal standard control (ISC) species of RNA. A competitive immunoblot assay was used to quantify full-length PKR (FL-PKR) protein in a sample of total cellular proteins, using truncated PKR protein as an internal standard against which FL-PKR protein could be quantified. The method can be used for simultaneous analysis of transcriptional and postranscriptional regulation of PKR gene expression from very small clinical specimens such as liver biopsies, e.g., 2-3 mg (wet weight) and containing only 2x10(5) cells. To the best of our knowledge, this is the first report of a sensitive simultaneous assay system for this important immunoeffector molecule.

Partial activation of the insulin receptor kinase domain by juxtamembrane autophosphorylation.

Increased enzymatic activity of receptor tyrosine kinases occurs after trans-phosphorylation of one or two tyrosines in the activation loop, located near the catalytic cleft. Partial activation of the insulin receptor's kinase domain was observed at dilute concentrations of kinase, suggesting that cis-autophosphorylation was occurring. Autophosphorylation during partial activation mapped to the juxtamembrane (JM) tyrosines and not to activation loop tyrosines. Furthermore, a double JM Tyr-to-Phe mutant kinase (JMY2F) did not undergo partial activation but catalyzed substrate phosphorylation at a very low rate. Steady-state kinetics of peptide phosphorylation were determined with and without JM autophosphorylation. The JMY2F mutant was used to prevent concurrent cis-autophosphorylation and therefore to approximate the basal state apoenzyme in the kinetic analysis. Partial activation was dominated by a decreased Michaelis constant for peptide substrate, from KM,PEP >/= 2.5 mM in the basal state to 0.2 mM in the partially activated state; the KM,ATP remained virtually unchanged at approximately 1 mM, and kcat increased from 180 to 600 min-1. The high KM,PEP suggests weak binding of peptide substrates to the apoenzyme. This was confirmed by Ki > 1 mM for peptide substrates used as inhibitors of JM autophosphorylation. The absence of comparably large changes in kcat and KM,ATP suggests that the JM region is primarily a strong barrier to the peptide entry step of trans-phosphorylation reactions. The JM region therefore functions as an intrasteric inhibitor in the basal state of the insulin receptor's kinase domain.

The mechanism of trans-activation of the MDR1 gene by human T-cell leukemia virus.

Overexpression of P-glycoprotein (P-gp), the protein product of the multidrug resistance gene (MDR1), confers a drug resistant phenotype on cells. We have recently demonstrated that the MDR1 promoter is transcriptionally activated by the HTLV-I tax protein, providing an explanation for the development of drug resistance in HTLV-I infections. Here we report that HTLV-I mediated MDR1 activation is dependent on the presence of an NF-IL6-binding site located between base pairs -148 and -141 relative to the transcription start site. This finding opens up the possibility of moderating P-gp expression through interference with NF-IL6 binding to its trans recognition element and subsequent repression of MDR1 transcription.

Enhanced MDR1 gene expression in human T-cell leukemia virus-I-infected patients offers new prospects for therapy.

Overexpression of P-glycoprotein (P-gp), the protein product of the multidrug resistance gene (MDR1), confers a drug resistant phenotype on cells. This phenotype is reminiscent of human T-cell leukemia virus (HTLV)-transformed leukemic cells, for which no consistently effective chemotherapeutic regime has been found. The presence of an active multiple drug resistance (MDR) phenotype in freshly isolated peripheral blood mononuclear cells (PBMC) from HTLV-I-infected subjects was investigated. Significant P-gp-mediated efflux activity and enhanced MDR1 mRNA expression was observed in nine of 10 HTLV-infected subjects. The development of MDR phenotypes was found to be independent of disease type or status with significant MDR activities being observed in adult T-cell leukemia (ATL), HTLV-associated myelopathy (HAM)/tropical spastic paraparesis (TSP), and asymptomatic HTLV-infected individuals. P-gp-mediated drug efflux was also found to be restricted to CD3+ T-cell populations. Furthermore, we show the novel finding that the MDR1 gene promoter is transcriptionally activated by the HTLV-I tax protein, suggesting a molecular basis for the development of drug resistance in HTLV-I infections. These observations open up the possibility of new chemotherapeutic approaches to HTLV-associated diseases through the use of P-gp inhibitors.

Cis-autophosphorylation of juxtamembrane tyrosines in the insulin receptor kinase domain.

Receptor tyrosine kinases undergo ligand-induced dimerization that promotes kinase domain trans-autophosphorylation. However, the kinase domains of the insulin receptor are effectively dimerized because of the covalent alpha2beta2 holomeric structure. This fact has made it difficult to determine the molecular mechanism of intraholomeric autophosphorylation, but there is evidence for both cis- and trans-autophosphorylation in the absence and presence of insulin. Here, using the cytoplasmic kinase domain (CKD) of the human insulin receptor, we demonstrate that autophosphorylation in the juxtamembrane (JM) subdomain follows a cis-reaction pathway. JM autophosphorylation was independent of CKD concentration over the range 6 nM-3 microM and was characterized kinetically: Half-saturation (K(ATP)) was observed at 75 microM ATP [5 mM Mn(CH3CO2)2] with a maximal rate of 0.24 mol of PO4 (mol of CKD)(-1) min(-1). Pairwise substitutions of Phe for Tyr in the other two autophosphorylation subdomains, generated by site-directed mutagenesis, altered the kinetics of JM autophosphorylation but did not change the pathway from a cis-reaction. Tyr(1328,1334) to Phe (in the carboxy-terminal subdomain) yielded <2-fold increase in the efficiency of JM autophosphorylation, whereas Tyr(1162,1163) to Phe (in the activation loop subdomain) yielded approximately 38-fold increased efficiency of JM autophosphorylation, due predominantly to a 23-fold decreased K(ATP). These findings demonstrate basal state binding of ATP to the CKD leading to cis-autophosphorylation and novel basal state regulatory interactions among the subdomains of the insulin receptor kinase. On the basis of these results and the crystal structure of the conserved catalytic core of this kinase [Hubbard, S. R., et al. (1994) Nature 372, 746], a model is proposed which reconciles the JM cis-reaction and the activation loop cis-inhibition/trans-reaction with the complex kinetics of insulin receptor autophosphorylation [Kohanski, R. A. (1993) Biochemistry 32, 5766].

A tyrosine kinase assay using reverse-phase high-performance liquid chromatography.

Reverse-phase HPLC can be used as a very precise and accurate routine assay for peptide phosphorylation by protein kinases that has advantages over other methods. In particular, peptides with native amino acid sequences can be used without the need for radioisotopes. However, reaction conditions that are employed can often present difficulties in recovery and quantitation of phospho- and apo-peptides. Two general problems were encountered; First, variation in the retention times of peptides and an increasing width of the injection front which can interfere with quantitation both resulted from repeated sample injections. These were caused mostly by the presence of carrier bovine serum albumin used to reduce loss of peptides during the reaction and by high concentrations of ATP used to study the kinetics of enzyme catalyzed reactions. These problems were solved by regular washing of the reverse-phase column, thus allowing a broad range of peptide and ATP concentrations to be used. Second, the stability of peptides used in the assay was affected by dithiothreitol in combination with manganese. The former is a common reagent of kinase purifications and the latter is often the metal cofactor used in kinase reactions. Minimizing the concentration of dithiothreitol or using magnesium resolved these difficulties. Consideration of these factors is therefore important when using reverse-phase HPLC to monitor peptide phosphorylation in protein tyrosine kinase assays.

Ethnic differences in women's body satisfaction: an experimental investigation.

This investigation was conducted so that a clearer picture of the complex relationship between ethnicity and body satisfaction could be obtained. Although body satisfaction has recently been shown to be influenced by several factors, such as mood, no studies investigating the stability of body satisfaction (to date) have examined whether there are ethnic differences in how such factors influence body satisfaction. Eighty-four White women and 33 Black women (U.S. undergraduates) were given bogus positive or negative social feedback so that the effect of the feedback on their body satisfaction could be determined. Results indicated positive feedback increased and negative feedback decreased the body satisfaction of White women in the expected directions, but there was no such effect for the Black women. The relevance of these findings in the understanding of bulimia nervosa and eating disorders is discussed, as is the need to differentiate between ethnic groups.

Coping with crises: an examination of the impact of traumatic events on religious beliefs.

The impact of traumatic events on empirical and metaphysical assumptions was examined, by comparing assumptions of a group of 25 persons who had recently experienced a major stressor with assumptions of a group of 25 persons who had not had such an experience. Each group was composed of 22 women and 3 men, with a mean age of 20 years. Participants completed written measures assessing levels of adjustment, empirical world assumptions, religious motivation, and religious and spiritual experiences. Naturalistic interviews were conducted with the trauma group. The trauma group obtained significantly higher scores on symptoms of psychological distress but did not differ in evaluations of the empirical world as predictable, safe, or controllable. Interviews suggested that the metaphysical assumptions were not challenged by trauma; rather, they provided a framework for understanding and coping with trauma.

Production of non-infectious human immunodeficiency virus-like particles which package specifically viral RNA.

A recombinant vector that rapidly produces large amounts of human immunodeficiency virus (HIV) virus-like particles (VLPs) was constructed. This vector lacks LTR sequences and a functional nef gene. The VLPs produced are non-infectious but similar in structure to mature, infectious HIV virions. They package specifically HIV RNAs containing appropriate signals and do not package abundant cellular mRNAs (e.g. actin). In the system described here, efficient particle production and release is decoupled from infection. Use of this VLP system offers many advantages over the study of infectious virions, permitting the expression of mutant phenotypes which interfere with virus infectivity.

Evidence that a kissing loop structure facilitates genomic RNA dimerisation in HIV-1.

Genomic RNA isolated from retroviral particles is a dimer composed of two identical strands. A region called the dimer linkage signal close to the 5' end of the RNA may be involved in forming the dimer. Several models for the formation of the HIV-1 RNA dimer have been proposed. In the kissing loop model, dimerisation results from base-pairing between homologous sequences in an RNA stem-loop. In the guanine tetrad model interstrand guanine contacts from the dimer. We have made mutations preventing the dimerisation of subgenomic RNAs in vitro by these mechanisms. To prevent the kissing loop dimer forming we changed the complementary loop sequence from 711GCGCGC716 to 711AAACGC716. To prevent the guanine tetrad dimer forming we changed G819 to U. These mutations were introduced into a clone of HIV-1NL4-3 separately and collectively. All three clones produced infectious virions. Dimeric RNA with similar thermal stabilities was isolated from viruses containing either the single or the double mutations. The results suggest that sequences involved in forming a guanine tetrad are not important for HIV-1 RNA dimerisation. In contrast sequences involved in forming a kissing loop complex are not absolutely required, but are important in forming a stable HIV-1 RNA dimer.

Molecular determinants of the V3 loop of human immunodeficiency virus type 1 glycoprotein gp120 responsible for controlling cell tropism.

We and others have identified the major determinant of cell tropism in human immunodeficiency virus type 1 (HIV-1) as the V3 loop of glycoprotein gp120. We have conducted a detailed study of two molecularly cloned isolates of HIV-1, HIVJR-CSF and HIVNL4-3, that differ in their tropism for immortalized CD4+ cell lines, by constructing a series of site-directed mutations within the V3 loop of HIVJR-CSF based on the sequence of HIVNL4-3. The phenotypes of these mutants fall into two classes, those which are viable and those which are not. A spontaneous mutant with significantly altered growth properties was also recovered and found to have an additional single amino acid change in the V3 loop sequence. The carboxy-terminal beta-strand part of the V3 loop is the major determinant of cell tropism. However, the results presented here indicate that the functional role of the V3 loop sequences can only be interpreted properly in the context of the original gp120 backbone from which they were derived. These findings show that over-simplistic interpretation of sequence data derived from unknown mixtures of HIV variants in infected persons may be highly misleading.

A single amino acid substitution in the V1 loop of human immunodeficiency virus type 1 gp120 alters cellular tropism.

Specific point mutations which affect viral tropism have been identified in both the V3 loop and in the CD4-binding region of the human immunodeficiency virus type 1 surface glycoprotein gp120. Here we report that a single point mutation in the first variable region (V1) of human immunodeficiency virus type 1 strain JRCSF is responsible for a change in viral tropism.

L-arginine: a therapeutic option for AIDS/HIV infection?

Numerous studies implicate cellular immunological effector systems in the partial containment of virus replication during the early stages of HIV infection. Immunostimulatory therapeutic regimes designed to enhance virus clearance are therefore theoretically attractive, but are accompanied by the risk of concomitant activation of HIV replication. Supra-normal levels of L-arginine have been shown to induce broad immune stimulation in vitro and in vivo, but do not increase HIV gene expression in vitro. These observations, together with the lack of toxicity of this agent, suggest a novel therapeutic approach to HIV disease.