A 3.5 genome equivalent multi access YAC library: construction, characterisation, screening and storage.

The construction of a yeast artificial chromosome (YAC) primary gridded library of 35,000 clones from human lymphoblastoid (48,XXXX) cell line DNA is described. The average YAC size is approximately 350kb representing a greater than 3.5 times coverage of the genome. The library is stored at -70 degrees C as gridded clones on nylon filters impregnated with 20% glycerol and as glycerol suspensions of individual clones in microtitre plates providing a prolonged multi-user potential. To date we have used 14 single copy probes to screen this library by colony hybridisation as well as PCR and have isolated between 1 and 5 YAC clones for every probe.     

Differential effects of suramin on the coupling of receptors to individual species of pertussis-toxin-sensitive guanine-nucleotide-binding proteins.

The anti-helminthic drug suramin inhibited the basal high-affinity GTPase activity of both C6 BU1 glioma and NG 108-15 neuroblastoma x glioma hybrid-cell membranes with an IC50 (concentration causing half-maximal inhibition) value close to 30 micrograms/ml. This effect was shown to occur via a non-competitive mechanism in which the binding affinity of the G-proteins for GTP was not altered, but the maximal velocity of the subsequent hydrolysis was reduced. In NG 108-15 membranes, both opioid peptides and foetal-calf serum stimulated high-affinity GTPase activity in a pertussis-toxin-sensitive manner. These effects have previously been shown to be mediated by different G-proteins [McKenzie, Kelly, Unson, Spiegel & Milligan (1988) Biochem. J. 249, 653-659]. Suramin completely prevented the opioid-peptide-stimulated increase in GTP hydrolysis, but did not prevent the opioid peptide from binding to its receptor. Suramin, however, did not block the foetal-calf-serum-stimulated GTPase response. This selective action of suramin provides further evidence for distinct roles for two separate pertussis-toxin-sensitive G-proteins in signal transduction in NG 108-15 membranes and provides the first evidence for a selective effect of a drug on the functions of different G-proteins.     

Developmental expression of 44-kDa bone phosphoprotein (osteopontin) and bone γ-carboxyglutamic acid (Gla)-containing protein (osteocalcin) in calcifying tissues of rat

New aspects of the distribution and developmental appearance of the 44-kDa bone phosphoprotein (44K BPP, also called sialoprotein I or osteopontin) and bone gamma-carboxyglutamic acid (Gla)-containing protein (BGP, also called osteocalcin) during osteogenesis and dentinogenesis were investigated with immunocytochemical techniques using monospecific, affinity-purified polyclonal antibodies. Sections from newborn rat incisors and from various bone anlagen of newborn animals and fetuses were processed for detection of 44K BPP or BGP antigenicity. In addition, histochemical reactions for detection of alkaline phosphatase or calcium salts were performed on a number of the sections. The 44K BPP appears to be synthesized and secreted by chondrocytes only in the areas of cartilage-to-bone transition; these cells could participate indirectly in the process of bone formation by providing a suitable scaffold onto which primary marrow osteoblasts attach and spread. During osteogenesis, 44K BPP is found in bone-forming cells almost concomitantly with the appearance of alkaline phosphatase and before osteoid deposition, whereas BGP is still absent during early stages of mineralization. We hypothesize that this dramatic difference between the developmental appearance of 44K BPP and BGP reflects the delayed expression of the BGP gene relative to that of 44K BPP. In long-term cultures of bone marrow from adult mice, some fibroblastic cells expressed the 44K BPP phenotype; these cells could represent early osteogenic progenitor cells. Some experiments also suggested that, as with BGP, 44K BPP or an immunologically related protein is synthesized by some odontoblasts and secreted into predentin, prior to the onset of mineralization.     

Recognition of a genus-wide antigen ofLegionella by a monoclonal antibody

A monoclonal antibody was produced against a cytoplasmic membrane protein that appears to be common to all species of the genusLegionella. The antibody was positive in polyacrylamide gel electrophoresis and Western blotting with extracts of all of 22 species type strains ofLegionella that were tested. The apparent molecular mass of the protein varied from 57.2 to 62.1 kilodaltons for the 23 species type strains ofLegionella. An enzyme-linked immunosorbent assay (ELISA) was developed with the monoclonal antibody to enable rapid screening of clinical and environmental isolates forLegionella. All of 23 species type strains ofLegionella that were tested were strongly positive with the monoclonal antibody in the ELISA. Among 27 other bacterial species and 84 strains that were tested, onlyBordetella ssp. andAcinetobacter lwoffii were cross-reactive in the ELISA. These two cross-reactive species are readily distinguishable fromLegionella by culture characteristics. The monoclonal antibody may also be useful in tests to detect the genus-wide antigen in body fluids of patients with legionellosis.     

Sexual maturation of ewes raised without ram exposure in a controlled lighting environment

Finn x Dorset ewe lambs (n = 70) born in the spring (March 28 to April 6) from two successive lambing seasons were evaluated for age at first ovulation in the absence of mature rams. Ewe lambs were born in a controlled, short light (8L:16D) photoperiod or in ambient light (13L:11D). At about 10 to 11 wk of age, ewe lambs were allocated to a short (8L:16D) or long (16L:8D) light environment. Plasma progesterone (P(4)) concentrations were measured as an index of first ovulation. First exposure of ewes to sexually mature rams was in November. Most ewe lambs (77%) ovulated before ram exposure. More lambs (P < 0.025) born in ambient light and raised in short light reached puberty with typical cycles of plasma progesterone compared to other treatments. Long days tended to retard the onset of puberty. Although pregnancy rate did not differ across light treatments, more ewes became pregnant from the ambient-light born and short-light raised treatment. Photoperiod is an important factor affecting the onset of sexual maturation and genesis of normal luteal progesterone secretion in the ewe lamb.     

Ig isotypes produced by EBV-transformed B cells as a function of age and tissue distribution

EBV can transform human B cells giving rise to lymphoblastoid cell lines that produce and secrete Ig. Herein B cells from various tissues of newborns and adults were transformed by EBV and their Ig products were analyzed with isotype-specific mAb. Although IgG- and IgA-bearing B cells were present in the newborn, EBV transformed IgM-producing cells almost exclusively in both newborn blood and breast milk. IgM-secreting cells were derived from IgM+ B cells and IgM- pre-B cells present in neonatal blood, but only from IgM+ cells in adult blood. Whereas in adults most EBV-transformed cells produced IgM, producers of IgG and of IgA were present in frequencies that varied according to the tissue source. Precursors of IgG-producing cells were relatively abundant in blood, spleen, and tonsil, and relatively infrequent in bone marrow and appendix. EBV-inducible IgA producers were relatively concentrated in the appendix and to a lesser extent in tonsils and blood. Differences in the subclass composition of EBV-transformed populations of IgG- and IgA-producers were also observed for the various adult lymphoid tissues. IgG1-producing cells predominated in most tissues, and precursors of IgG2 were largely confined to the circulation. Whereas IgA1-producing cells were predominant in all tissues, a marked enrichment in IgA2-producers was observed in the appendix. These results indicate a remarkable heterogeneity in the isotype distribution pattern of EBV-transformable B cells that is determined both by developmental age and tissue localization. We propose that EBV selectively transforms primed B cells, the isotype commitment of which varies according to tissue origin and age.     

Local production of fibronectin by ectopic human retinal cells

Summary The distribution of fibronectin mRNA and fibronectin in adult human retina and epiretinal membranes was investigated by in situ hybridisation and immunohistochemical techniques. The cells in normal adult retina contained little or no fibronectin mRNA and the retina only showed fibronectin immunoreactivity in retinal vessels. The cells in detached neuroretina did not contain fibronectin message but the vitreoretinal interface of the detached retina exhibited variable fibronectin immunoreactivity. Retinal glia, retinal pigment epithelium and fibroblast-like cells in membranes at the vitreoretinal juncture (epiretinal membranes) showed variable labelling with the fibronectin mRNA probe and all the membranes immunostained for fibronectin. No difference could be detected between membrane cell types in the intensity of labelling with the mRNA probe or for fibronectin immunoreactivity. The results indicate that cells in situ in attached and detached adult human retina do not produce fibronectin. Although fibronectin at the vitreoretinal juncture in retinal detachment is probably partly derived from plasma fibronectin resulting from breakdown of the blood-retinal barrier, ectopic retinal cells produce fibronectin and contribute to the glycoprotein in epiretinal membranes.     

Redox cycling of potential antitumor aziridinyl quinones

The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.     

Reduced orthostatic tolerance following 4 h head-down tilt

The cardiovascular responses to a 10-min 1.22 rad (70 degrees) head-up tilt orthostatic tolerance test (OST) was observed in eight healthy men following each of a 5-min supine baseline (control), 4 h of 0.1 rad (6 degrees) head-down tilt (HDT), or 4 h 0.52 rad (30 degrees) head-up tilt (HUT). An important clinical observation was presyncopal symptoms in six of eight subjects following 4 h HDT, but in no subjects following 4 h HUT. Immediately prior to the OST, there were no differences in heart rate, stroke volume, cardiac output, mean arterial pressure and total peripheral resistance for HDT and HUT. However, stroke volume and cardiac output were greater for the control group. Mean arterial pressure for the control group was less than HDT but not HUT. Over the full 10-min period of OST, the mean arterial pressure was not different between groups. Heart rate increased to the same level for all three treatments. Stroke volume decreased across the full time period for control and HDT, but only at 3 and 9 min for HUT. There was a higher total peripheral resistance in the HDT group than control or HUT. The pre-ejection period to left ventricular ejection time ratio was less in HDT than for control or HUT groups. These data indicate a rapid adaptation of the cardiovascular system to 4 h HDT that appears to be inappropriate on reapplication of a head to foot gravity vector. We speculate that the cause of the impaired orthostatic tolerance is decreased tone in venous capacitance vessels so that venous return is inadequate.