A rapid screening test for HIV-1 antibodies: application to biological studies on human tissue

Human umbilical cord vessels are commonly used as a source of human vascular tissue for physiological studies and as a source of endothelial and smooth muscle cells. Blood samples from 236 umbilical cords were tested for the presence of HIV-1 antibodies to access the prevalence of HIV-1 infection and to evaluate possible methods for screening umbilical cords. Ten of the 236 samples were HIV-1 antibody positive by ELISA whereas 3 were positive by Western blot and a new method, the Quick-Western blot. Two of the 3 positive samples contained antibody bands against gp160, gp120, gp41, and p24 HIV-1 proteins, and one sample had antibodies against only gp160, gp120 and gp41. The Quick-Western blot required only 45 minutes for the analysis while the ELISA and Western blot took 3 hours and 18 hours, respectively. These data indicate that HIV-1 infection in mothers may present a hazard to researchers using human umbilical cords as a source of vascular tissue. The Quick-Western blot method is a simple, portable, rapid and accurate method that may be used to screen blood. The short analysis time of the Quick-Western blot allows the identification of infected blood before the tissue deteriorates as a source of cells or vascular tissue for experimental studies.     

Immunochemical characterisation of parathyroid hormone-related protein from tumor and non-tumor cells

The molecular forms of parathyroid hormone-related protein (PTHRP) in conditioned media from the BEN human lung cancer cell line, rat parathyroid cells (PT-r) and human keratinocytes were studied by gel-filtraton chromatography with assay of PTHRP by immunoassays and bioassay. Immunoreactivity (1–86 and 1–34) and bioactivity (1–34) in conditioned media eluted as a coincident major peak (approx. molecular mass 19–22 kDa) and there was evidence of amino-terminal species in the molecular mass range 10–16 kDa in BEN and keratinocyte media. Western blotting of PTHRP affinity purified by monoclonal antibodies directed at regions 1–34 or 37–67, identified a major species in all cell cytosols and media with an apparent molecular mass of 24–25 kDa, consistently slighty larger than recombinant PTHRP(1–141) (mobility of 21 kDa) which may represent an intact or native form of PTHRP. Additional amino-terminal species were identified in medium from keratinocytes (16 and 7 kDa), BEN cells (18 and 14 kDa) and PT-R cells (17 kDa), suggesting that processing occurs at the C-terminus and within the mid-region to form a range of amino-terminal fragments.     

Estimation of oxygen-diffusing capacity in the gills of different sizes of a tilapia, Oreochromis niloticus

Measurements on gills of features that affect gas exchange have been studied in relation to body weight in specimens (0.0112–812.3 g) of a tilapia, Oreochromis niloticus . The data were analysed with respect to body weight by means of logarithmic transformations (log Y = log a+b log W ). The slopes ( b ) of the log/log regression lines for the gill area, harmonic mean diffusion distance and oxygen diffusing capacity were 0.777, 0.077 and 0.700, respectively. The gill respiratory area of O. niloticus (Trewavas) increases as the fish develops because the number and bilateral area of secondary lamellae increase. The scaling value for oxygen-diffusing capacity is less than the value for gill area because of the slight increase in harmonic mean diffusion distance with development.     

Alcohol consumption and its relation to cardiovascular risk factors in British women.

OBJECTIVE--To examine the relation between alcohol consumption and risk factors for coronary heart disease in women. DESIGN--Cross sectional study of a stratified random sample of the population grouped into five categories of habitual alcohol consumption. SETTING--People registered with general practitioners at two large health centres in east Bristol, England. SUBJECTS--1048 women aged 25-69 years. MAIN OUTCOME MEASURES--Fasting plasma concentrations of insulin, total cholesterol, total triglycerides, and high density lipoprotein cholesterol, including its subfractions HDL2 and HDL3, and body mass index. RESULTS--Compared with non-drinkers women consuming a moderate amount of alcohol (1-20 g/day) had lower plasma concentrations of triglycerides, by 0.19 mmol/l (95% confidence interval 0.07 to 0.35); cholesterol, by 0.4 mmol/l (0.19 to 0.61); and insulin, by 1.4 mU/l (0.43 to 1.97) and a lower body mass index, by 1.2 kg/m2 (0.43 to 1.97). They also had higher concentrations of high density lipoprotein cholesterol, by 0.09 mmol/l (0.03 to 0.15); HDL2 cholesterol by 0.05 mmol/l (-0.02 to 0.10) and HDL3 cholesterol, by 0.06 mmol/l (0.06 to 0.11). All these were independent of body mass index, smoking habits, and taking oral contraceptives. CONCLUSIONS--Moderate alcohol consumption is associated with lower levels of cardiovascular risk factors in women. Insulin may have a central role.     

Depletion of U3 small nucleolar RNA inhibits cleavage in the 5'' external transcribed spacer of yeast pre-ribosomal RNA and impairs formation of 18S ribosomal RNA.

Multiple processing events are required to convert a single eukaryotic pre-ribosomal RNA (pre-rRNA) into mature 18S (small subunit), 5.8S and 25-28S (large subunit) rRNAs. We have asked whether U3 small nucleolar RNA is required for pre-rRNA processing in vivo by depleting Saccharomyces cerevisiae of U3 by conditional repression of U3 synthesis. The resulting pattern of accumulation and depletion of specific pre-rRNAs indicates that U3 is required for multiple events leading to the maturation of 18S rRNA. These include an initial cleavage within the 5' external transcribed spacer, resembling the U3 dependent initial processing event of mammalian pre-rRNA. Formation of large subunit rRNAs is unaffected by U3 depletion. The similarity between the effects of U3 depletion and depletion of U14 small nucleolar RNA and the nucleolar protein fibrillarin (NOP1) suggests that these could be components of a single highly conserved processing complex.     

Energetically distinct early and late stages of HlyB/HlyD-dependent secretion across both Escherichia coli membranes.

The alternative secretion pathway which exports hemolysin across both Escherichia coli membranes into the surrounding medium is directed by an uncleaved C-terminal targeting signal and the membrane translocator proteins HlyD and HlyB. In order to identify stages and intermediates in this unconventional secretion process we have examined the effect of inhibition of the total proton motive force (delta P) and its components during the in vivo HlyB/HlyD-dependent export of a 22.4 kDa secretion competent HlyA C-terminal peptide (Actp). Secretion of Actp was severely inhibited by the proton ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP), which collapses simultaneously membrane potential delta psi and the proton gradient delta pH, and also by valinomycin/K+, a potassium ionophore which disrupts delta psi. The inhibition of secretion by valinomycin/K+ was ameliorated by imposition of a pH gradient, the second component of the delta P, and selective depletion of delta pH by nigericin also blocked secretion. This indicates that, as in the secretion of beta-lactamase to the periplasm, HlyB/D-directed secretion requires delta P itself and not specifically one of its components. However, inhibition of HlyB/D-dependent secretion was only marked when CCCP, valinomycin/K+ or nigericin were present during the early stage of Actp secretion; at a later stage the secretion was not significantly inhibited. HlyB/D-dependent secretion appears therefore to share with conventional secretion across the cytoplasmic membrane an early requirement for delta P, but comprises in addition a late stage which does not require delta P, delta psi or delta pH. The translocation intermediate identified in the delta P-independent late stage of secretion was associated with the membrane fraction. Analysis of the protease accessibility of this intermediate in whole cells and spheroplasts showed that it was not in the periplasm, nor was it exposed on the cell surface or on the periplasmic faces of either the inner or outer membranes. This may reflect its close association with the inner membrane or a membrane translocation complex.     

Cytoarchitecture of the fetal murine soleus muscle

The organogenesis of the soleus muscle of the 129 ReJ mouse (a mixed muscle, which in the adult contains approximately equal numbers of slow-twitch oxidative and fast-twitch oxidative-glycolytic myofibers) was studied in spaced, serial transverse, and longitudinal sections of muscles of 14-, 16-, and 18-day in utero and 1- and 5-day postnatal mice. A discrete soleus muscle was distinguished by 14 days in utero. It consisted of groups of closely apposed primary myotubes displaying junctional complexes and a pleomorphic population of mononucleated cells. Between 14 and 16 days in utero there was little de novo myotube formation. At 16 days in utero, basal lamina surrounded groups of primary myotubes; and primitive motor endplates were found on these myotubes. At 18 days in utero, the basal-lamina-enclosed groups of primary myotubes were no longer present. At this stage, basal lamina surrounded clusters (consisting of one primary myotube and one or more secondary myotubes) or independent myotubes (single myotubes surrounded by their own basal lamina). Cluster formation and cluster dispersal occurred concurrently, beginning at 18 days in utero and extending until birth. At birth, there was still a substantial population of immature, secondary myotubes that interdigitated with larger, more mature primary myofibers. At this stage, intermuscular axons had begun to myelinate, and postsynaptic specialization of the motor endplates had begun. Cluster dispersal and myonuclear migration was completed during the first 5 days postnatally with the muscle taking on adult characteristics. Beginning at 16 days in utero and extending into the neonatal period, there was evidence of myotube death in the soleus muscle.     

Structural gene for NAD synthetase in Salmonella typhimurium.

We have identified the structural gene for NAD synthetase, which catalyzes the final metabolic step in NAD biosynthesis. This gene, designated nadE, is located between gdh and nit at 27 min on the Salmonella typhimurium chromosome. Mutants of nadE include those with a temperature-sensitive lethal phenotype; these strains accumulate large internal pools of nicotinic acid adenine dinucleotide, the substrate for NAD synthetase. Native gel electrophoresis experiments suggest that NAD synthetase is a multimeric enzyme of at least two subunits and that subunits from Escherichia coli and S. typhimurium interact to form an active heteromultimer.     

Synthesis of the cyanogenic beta-glucosidase, linamarase, in white clover

The beta-glucosidase, linamarase, which specifically hydrolyzes cyanogenic substrates, linamarin and lotaustralin, in white clover, is synthesized in the early stages of leaf and seedling development in genetically competent plants. Plants, from natural populations, possessing at least one Li allele synthesize linamarase but plants with only li alleles do not, nor do they produce inactive but antigenically related linamarase. Linamarase is known to be a mannosyl glycoprotein, which in its active form is a dimer, with a subunit size of 62,000 Mr. We demonstrate that the antibiotic tunicamycin, which prevents N-acetyl-asparagine linked glycosylation, reduces in vivo synthesis of linarmarase. In vitro translation of mRNA from a Li Li plant yields a 59,000 Mr immunoprecipitated linamarase polypeptide which is modified to a 62,000 Mr product by the addition of dog pancreas microsomes. No anti-linamarase immunoprecipitable product is obtained from the in vitro translation products of mRNA from a li li plant.