Linkage mapping of fifty-eight new rat microsatellite markers

Fifty-eight new anonymous simple sequence repeats (SSR) were generated and mapped to various rat chromosomes. Among them two genes (rat homologs for human cadherin-14 and mouse fibroblast growth factor-related protein) were mapped on Chromosomes (Chrs) 2 and 11 respectively. The majority of markers were generated from a small insert genomic library specific to Chr 11, 13, 14, and 15. Twenty new markers were mapped to Chr 13, which is known to contain a blood pressure quantitative trait locus (QTL). Several approaches to obtain microsatellite markers are described. The protocols and newly generated markers should be useful for ongoing rat genome project.     

Pharmacology and subcellular distribution of [3H]rilmenidine binding sites in rat brain

We have previously reported that in rat brain membranes, [3H]rilmenidine, in addition to labelling alpha2-adrenoceptors and the I2B-subtype of imidazoline receptor binding site (I2B-RBS), may label an additional I-RBS population, distinct from previously classified I1-RBS and I2-RBS. In this study, using crude or fractionated rat brain membranes we examined the possible association of [3H]rilmenidine-labelled I-RBS with the A- and B-isoforms of monoamine oxidase (MAO) by studying the inhibition of [3H]rilmenidine binding by a number of MAO inhibitors; and comparing the maximal binding density (Bmax) and subcellular distribution of [3H]rilmenidine binding sites with that of MAO-A and MAO-B catalytic sites labelled by [3H]RO41-1049 and [3H]RO19-6327 and 12-RBS labelled by [3H]2-BFI. Inhibition of [3H]rilmenidine binding by all MAO inhibitors tested produced very shallow curves (slope 0.29-0.56). Clorgyline and moclobemide (selective MAO-A inhibitors) displayed moderate affinities (60-140 nM), while pargyline (non-selective MAO-inhibitor), RO41-1049 (selective MAO-A inhibitor) and RO19-6327 (selective MAO-B inhibitor) exhibited very low affinities (> 2 microM) for 50-75% of [3H]rilmenidine-labelled I-RBS in crude brain membranes and even lower affinity for the remaining binding. Under identical buffer conditions, the Bmax of [3H]rilmenidine-labelled I-RBS (1.45+/-0.14 pmol/mg protein) was considerably lower than those of MAO-A (13.10+/-0.15 pmol/mg) and MAO-B (10.35+/-0.50 pmol/mg) sites. These results suggest that [3H]rilmenidine does not interact directly with the active catalytic site of either MAO enzyme and could at best only associate with a subpopulation of MAO molecules. Binding studies on five fractions of rat cortex homogenates-nuclear (N), heavy (M) and light (L) mitochondrial, microsomal non-mitochondrial (P), and soluble cytosolic (S) fractions-revealed that 45% of total [3H]rilmenidine binding was present in the P fraction cf. 20 and 23% in the M and L fractions, in contrast to [3H]RO19-6327 and [3H]2-BFI which bound 11-13% in the P fraction and 36-38% and 35-44% in the M and L fractions, respectively. Binding of all ligands in the N fraction was 6-15% of total. These studies reveal that [3H]rilmenidine-labelled I-RBS, unlike the I2-RBS, are not predominantly associated with mitochondrial fractions containing the MAO enzymes (and cytochrome oxidase activity), but appear to be distributed in both the mitochondrial and plasma membrane fractions in rat cerebral cortex.     

Sugar-induced molten-globule model

Proteins denature at low pH because of intramolecular electrostatic repulsions. The addition of salt partially overcomes this repulsion for some proteins, yielding a collapsed conformation called the A-state. A-states have characteristics expected for the molten globule, a notional kinetic protein folding intermediate. Here we show that the addition of neutral sugars to solutions of acid-denatured equine ferricytochrome c induces formation of the A-state in the absence of added salt. We characterized the structure and stability of the sugar-induced A-state with circular dichroism spectropolarimetry (CD) and NMR-monitored hydrogen-deuterium exchange experiments. We also examined the stability of the sugar-induced A-state as a function of sugar size and concentration. The results are interpreted using several models and we conclude that the stabilizing effect is consistent with increased steric repulsion between the protein and the sugar solutions.     

Respiratory syncytial virus (RSV) disease and prospects for its control

Respiratory syncytial virus (RSV) is a major virus pathogen of infants and young children, an important cause of disease in adults and is responsible for a significant amount of excess morbidity and mortality in the elderly. It also can be devastating in immunosuppressed populations. Vaccines are being developed, but none are currently licensed. Moreover, even if one or more are approved, they may not be suitable for some populations vulnerable to RSV (e.g. very young infants and the immunosuppressed). Ribavirin and immunoglobulin preparations with high titers of RSV-specific neutralizing antibodies are currently approved for use to treat and prevent RSV infection. However, neither of these is cost-effective or simple to administer. New agents are needed to reduce the impact of RSV. This review is concerned with the means currently available for controlling RSV, the search for new agents effective against this virus, and future prospects for preventing and treating RSV infections.     

Uterine relaxation responses to calcitonin gene-related peptide and calcitonin gene-related peptide receptors decreased during labor in rats

OBJECTIVES: Our purpose was to investigate (1) whether uterine relaxation responses to calcitonin gene-related peptide are differentially regulated during pregnancy and labor, (2) the involvement of nitric oxide in smooth muscle relaxant action of calcitonin gene-related peptide in the rat uterus, (3) whether receptors for calcitonin gene-related peptide are expressed in rat uterus, and if so (4) whether the concentrations of these receptors are differently regulated during pregnancy and labor. STUDY DESIGN: Rats were killed on day 18 of gestation, at the time of spontaneous labor, or postpartum day 2. The uteri were removed for in vitro contractility measurements, nitric oxide production, and calcitonin gene-related peptide receptor binding assay. RESULTS: (1) Calcitonin gene-related peptide induced a dose-dependent relaxation in spontaneously contracting uterine strips from pregnant rats on day 18 of gestation; (2) the relaxation effects of calcitonin gene-related peptide on the uterus were decreased during spontaneous delivery at term and post partum compared with that during pregnancy; (3) calcitonin gene-related peptide-induced relaxation was inhibited by pretreatment of the uterine tissue with a calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide(8-37); (4) nitric oxide synthesis inhibitor (N(G)-nitro-L-arginine methyl ester) and soluble guanylate cyclase inhibitor (LY83583) significantly decreased calcitonin gene-related peptide-induced relaxation of the rat uterus during pregnancy; (5) calcitonin gene-related peptide increased the uterine nitric oxide production in pregnant rats, and this increase was obliterated in the presence of calcitonin gene-related peptide(8-37); and (6) calcitonin gene-related peptide receptors are present in rat uterus, and the concentration of these receptors dramatically increases during pregnancy and decreases during labor at term. CONCLUSIONS: Calcitonin gene-related peptide inhibits uterine spontaneous contractions in rats during pregnancy but not during labor and post partum. The inhibitory effects of calcitonin gene-related peptide on uterine contractility appear to be modulated, at least in part, by the activation of nitric oxide generation in the rat uterus. Changes in calcitonin gene-related peptide receptors could contribute to the changes in calcitonin gene-related peptide-mediated uterine relaxation during pregnancy and labor.     

Trends in mortality of childhood-onset insulin-dependent diabetes mellitus in Leicestershire: 1940-1991

The relative risk of death by calendar date of diagnosis was investigated in a population-based incident cohort of 845 (463 males:382 females) IDDM diagnosed in Leicestershire before the age of 17 years between 1940 and 1989. The mortality status of 844 (99.9%) patients was determined as of the 31 December 1991, representing 14,346 person-years of risk. Trends in relative risk of death were investigated using Cox proportional hazards modelling for within cohort comparisons and age/sex and calendar time adjusted standardized mortality ratios (SMR) using generalized linear modelling for external comparisons. Median age at diagnosis was 10 years (range 3 months to 16 years); median duration of diabetes 15 years (range 1-51 years). Forty-four patients had died (5.2%; median age at death 31 years, range 11-51 years). A further four patients died at presentation (within 24 h) from ketoacidosis and are excluded from all analyses. Calendar date of diagnosis was found to be an important predictor of mortality. Adjusting for attained age there was evidence of a decline in relative risk of death with calendar date of diagnosis of 3.4% (95% CI, 0.005-6.9%) per annum, equivalent to a 32% fall per decade (95% CI, 5-51%), or 84% (95% CI, 21-97) from 1940 to 1989. The data are consistent with a large fall in mortality between the 1940s and 1950s representing over 50% of the total reduction in mortality between 1940 and 1991. Neither sex nor age at diagnosis were significant predictors of mortality. Over the study period 1940-89 the SMR (male and female combined) fell from 981 (541-1556) to 238 (60-953) relative to the general population. This population-based study shows that the prognosis for Type 1 (insulin-dependent) diabetes mellitus has improved markedly over the period 1940-1991.     

Sesquiterpenoids of the drimane class from a sponge of the genus Dysidea

Ten sesquiterpenoids, including seven new ones, have been isolated from an undescribed sponge of the genus Dysidea. Compounds 1-8 are sesquiterpenoids of the drimane class, while 9 and 10 are 12-norsesquiterpenoids of the same structural class. The structures of novel compounds have been determined by combined spectroscopic methods. These compounds exhibited moderate antimicrobial and enzyme inhibitory (Na+/K(+)-ATPase and PLA2) activities.     

Simultaneous chromizing-aluminizing coating of austenitic stainless steels

Chromium and aluminum were simultaneously co-deposited by diffusion into austenitic stainless steel substrates, by a single-step, pack-cementation process. The mechanism for the formation of diffusion-coated products on 304 and 316 stainless steels and on Incoloy 800 is discussed. The morphologies of the phases formed at the surface, i.e., an external beta layer and an underlying multiphase interdiffusion zone, are presented. The formation of the brittle, , outer layer was minimized by variations in the pack composition and activator. The coated 304 and 316 steels exhibited excellent scaling resistance upon oxidation in air at 1000°C.     

Simultaneous Aluminizing and Chromizing of Steels to Form (Fe,Cr)3Al Coatings

A cementation pack involving halide activatorsand elemental Al and Cr powders has been used to achievethe codeposition and diffusion of aluminum and chromiuminto low-alloy steels. A two-step treatment at 925°C and 1150°C yields dense anduniform ferrite coatings of about 400-m thickness,with surface compositions of approximatelyFe₃Al plus several percent Cr. The two stepheating schedule prevents the formation of a blocking chromium carbide atthe substrate surface. An attempt was made to add atrace of Ce to the Al + Cr content of the coating byintroducing Ce oxide into the pack, but there is no evidence that this doping was achieved. Uponcyclic oxidation in air at 700°C, the coated steelexhibits a negligible 0.085 mg/cm² weightgain for 1900 one-hour cycles. Virtually no attack wasobserved on coated steels tested in a simulated boileratmosphere at 500°C for 500 hr. But coatings with asurface composition of only 8 wt.% Al and 6 wt.% Crsuffered limited sulfidation attack in the simulated boiler atmosphere at temperatures higher than500°C for 1000 hr.