Pharmacology and intracellular signaling mechanisms of the native human orphan receptor BRS-3 in lung cancer cells

Neither the native ligand nor the cell biology of the bombesin (Bn)-related orphan receptor subtype 3 (BRS-3) is known. In this study, we used RT-PCR to identify two human lung cancer lines that contain sufficient numbers of native hBRS-3 to allow study: NCI-N417 and NCI-H720. In both cell lines, [DPhe6,betaAla11,Phe13, Nle14]Bn(6-14) stimulates [3H]inositol phosphate. In NCI-N417 cells, binding of 125I-[DTyr6,betaAla11,Phe13,Nle14]Bn(6-14) was saturable and high-affinity. [DPhe6,betaAla11,Phe13,Nle14]Bn(6-14) stimulated phospholipase D activity and a concentration-dependent release of [3H]inositol phosphate (EC50 = 25 nM) and intracellular calcium (EC50 = 14 nM); the increases in intracellular calcium were primarily from intracellular stores. hBRS-3 activation was not coupled to changes in adenylate cyclase activity, [3H]-thymidine incorporation or cell proliferation. No naturally occurring Bn-related peptides bound or activated the hBRS-3 with high affinity. Four different bombesin receptor antagonists inhibited increases in [3H]inositol phosphate. Using cytosensor microphysiometry, we found that [DPhe6,betaAla11,Phe13, Nle14]Bn(6-14) caused concentration-dependent acidification. The results show that native hBRS-3 receptors couple to phospholipases C and D but not to adenylate cyclase and that they stimulate mobilization of intracellular calcium and increase metabolism but not growth. The discovery of human cell lines with native, functional BRS-3 receptors, of new leads for a more hBRS-3-specific antagonist and of the validity of microphysiometry as an assay has yielded important tools that can be used for the identification of a native ligand for hBRS-3 and for the characterization of BRS-3-mediated biological responses.     

Evaluation of clothing systems to determine heat strain

This article describes the basic evaluation process and test methodology employed when temperature extremes for clothing systems must be considered as part of the U.S. Army's Health Hazard Assessment for material in the development and acquisition process. The goals of the evaluation are to select clothing systems that minimize the hazards of heat strain and to predict the heat strain for persons wearing such clothing. Clothing evaluations begin with biophysical assessments that determine the thermal characteristics (vapor permeability and insulation) for textiles via guarded hot plate tests and for clothing systems via thermal manikin tests. The results from biophysical tests can be used to select the textile and/or clothing with the best thermal characteristics. The data from manikin evaluations also can be used in prediction modeling. Human physiological testing is best done in a controlled laboratory environment, although for realism and user acceptability field trials may also be conducted. Proven test and measurement methods must be employed, and tests must control for confounding variables; subjects serve as their own controls, and test environment and procedures are consistent between trials. The process and test methodology described can be applied to the evaluation of civilian clothing systems as well as to the military systems for which they were developed.     

Transformation of Rat-1 fibroblasts with the v-src oncogene increases the tyrosine phosphorylation state and activity of the alpha subunit of Gq/G11

Two major intermediaries in signal transduction pathways are pp60v-sre family tyrosine kinases and heterotrimeric guanine nucleotide-binding proteins. In Rat-1 fibroblasts transformed by the v-src oncogene, endothelin-1 (ET-1)-induced inositol 1,4,5-trisphosphate accumulation is increased 6-fold, without any increases in the numbers of ET-1 receptors or in the response to another agonist, thrombin. This ET-1 hyperresponse can be inhibited by an antibody directed against the carboxyl terminus of the Gq/G11 alpha subunit, suggesting that the Gq/G11 protein couples ET-1 receptors to phospholipase C (PLC). While v-src transformation did not increase the expression of the Gq/G11 alpha subunit, immunoblotting with anti-phosphotyrosine antibodies and phosphoamino acid analysis demonstrated that the Gq/G11 alpha subunit becomes phosphorylated on tyrosine residues in v-src-transformed cells. Moreover, when the Gq/G11 protein was extracted from control and transformed cell lines and reconstituted with exogenous PLC, AIF*4-stimulated Gq/G11 activity was markedly increased in extracts from v-src-transformed cells. Our results demonstrate that the process of v-src transformation can increase the tyrosine phosphorylation state of the Gq/G11 alpha-subunit in intact cells and that the process causes an increase in the Gq/G11 alpha-subunit's ability to stimulate PLC following activation with AIF-4.     

Assessing the reliability and responsiveness of 5 shoulder questionnaires

Inspired by the high response rates achieved with the DBCT regimen (dacarbazine [DTIC], carmustine [BCNU], cisplatin and tamoxifen [TAM]), we administered the nitrosourea compound fotemustine, cisplatin and TAM (FCT regimen) to 69 patients with metastatic melanoma. Fotemustine (100 mg/m2) and cisplatin (100 mg/m2) were administered every 4 weeks, preceded by TAM 160 mg daily for 7 days from the second course onwards. Pharmacokinetic blood sampling was performed in 14 patients during the initial two cycles to compare the pharmacokinetic behaviour of fotemustine with or without TAM. Previous chemo- or radiotherapy was allowed, and patients with brain metastases or concomitant other malignancies were included. Four complete and 11 partial responders were observed among 66 evaluable patients, yielding a response rate of 22.7% (95% confidence interval 12.9 32.5%). The median survival time was 6.4 months (range 0.1-52+ months). The main toxicities were thrombocytopenia, protracted nausea/vomiting and ototoxicity. Renal toxicity was generally mild, but possibly contributed to two deaths. Seven patients experienced deep venous thrombosis during the study. TAM had no influence on the pharmacokinetics of fotemustine. The activity of the FCT regimen was clearly inferior to that initially reported with DBCT treatment. However, a recent publication concludes that the latter achieves a considerably lower response rate when administered to a larger patient group. We believe our results reflect the true activity of FCT and similar regimens when administered routinely to unselected patients. Considering the number of potentially serious side effects, we cannot recommend the moderately active FCT regimen as a palliative treatment option for melanoma patients.     

Epidemiological studies in children of a low-endemic region, a high-endemic region, and dwellers of a leprosy colony: evaluation of anti-ND-BSA antibodies and lepromin response

Children residing in a low-endemic region (LER), a high-endemic region (HER), and a leprosy colony contact population (CP) were evaluated for lepromin response as well as reactivity to the Mycobacterium leprae-specific synthetic antigen, ND-BSA. The mean reactivity to ND-BSA in the LER group (OD 0.03 +/- 0.03, N = 71) was significantly lower (p < 0.001) than that in the contact population (OD 0.14 +/- 0.09, N = 140) as well as the population residing in the HER (OD 0.09 +/- 0.08, N = 1340). ELISA-positive results were the highest (21.4%) with the CP group and lowest (0.0%) in the LER group, suggesting that it was a measure of the extent of exposure of M. leprae. In the contact population, females showed a preponderance for ELISA positivity over males (p < 0.005), a finding not observed with the HER population. The Mitsuda responses showed a Gaussian-type distribution in all of the three populations examined with the mean response being highest in the LER (6.0 mm +/- 2.9) and lowest in the HER (4.5 mm +/- 2.0) groups. The percent positivity for the Mitsuda reaction was found to be highest in the LER (93.0%) and lowest in the HER (88.3%) groups. The Mitsuda response thus appears to be independent of M. leprae exposure, and its interpretation in a given population needs consideration of several factors, such as nutritional, environmental, etc.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitochondrial DNA variable number tandem repeats (VNTRs): utility and problems in molecular ecology

Analysis of mitochondrial (mt)DNA size polymorphism in the form of variable number tandem repeats (mtVNTRs) has become an increasingly popular methodology for addressing questions in molecular ecology. When detected by PCR, mtVNTR analysis can provide a sensitive, rapid, and cost-effective measure of genetic variability that may be exploited in studies of population differentiation and biogeography. Despite the emergence of this approach, there has been little critical evaluation of its success or utility as a practical tool. In this review, we identify problematic methodological, theoretical and interpretive factors that can influence the utility of mtVNTR analysis. The reliability of the procedure is considered in terms of both detection of alleles and scoring of intra-individual allele frequencies. While many of the potential technical problems of the technique do not raise serious practical concerns, this rapid and sensitive methodology is seriously compromised by the difficulty of reliably assessing allele frequencies, of assaying only germline tissue, and in our ignorance of the mechanisms generating mtVNTR diversity. Thus, although there is a considerable potential for mtVNTR pilot studies to assess genetic diversity, the utility of the technique to resolve broader questions in molecular ecology should be treated cautiously until such a time as the system is better understood.     

Flauoprotein structure and mechanism 2. Monoamine oxidases: old friends hold many surprises

The two forms of monoamine oxidase (MAO), A and B, continue to be of major interest to biochemists, pharmacologists, neurologists, and gerontologists. Despite intensive study for more than half a century, unexpected and unique properties of these enzymes continue to come to light. Recent studies have centered on their kinetic mechanism, their unique predilection for substrates related to the neurotoxic tertiary amine MPTP, and their putative role in aging and in the etiology of neurodegenerative diseases. New and potent inhibitors of MAO A and MAO B continue to be developed because of their potential use in clinical medicine. Some are effective in the picomolar range but MAO B from different mammalian species shows remarkable differences in sensitivity to these agents.     

Closed rupture of the anterior tibial tendon. A case report

Closed subcutaneous rupture of the anterior tibial tendon is a relatively uncommon injury that requires a thorough clinical examination to diagnose correctly. The authors report a case of this disorder and provide a review of the relevant literature. A method of surgical repair not previously described in the literature is also presented.     

Overshadowing and latent inhibition counteract each other: support for the comparator hypothesis

In 4 conditioned lick suppression experiments with rats, the combined effects of latent inhibition treatment followed by overshadowing treatment were assessed as a test of the comparator hypothesis's (R.R. Miller & L.D. Matzel, 1988) explanations of overshadowing and latent inhibition. Experiments 1 and 2 confirmed the prediction of the comparator hypothesis that combined latent inhibition and overshadowing treatments attenuate the response deficit produced by either treatment alone. Furthermore, consistent with the comparator hypothesis, posttraining changes in the associative status of the putative comparator stimulus altered responding to the target conditioned stimulus (Experiment 3), and switching contexts between latent inhibition and overshadowing treatments (Experiment 4) eliminated the interaction between the latent inhibition and overshadowing treatments.