Autoradiographic detection of HPRT variants of human lymphocytes resistant to RNA synthesis inhibition

The feasibility of using RNA synthesis in freshly isolated, human peripheral blood lymphocytes to detect 6-thioguanine (TG)- and 8-azaguanine (AG)-resistant variants in an autoradiographic assay similar to that of Strauss and Albertini (1979) has been evaluated. In phytohemagglutinin (PHA)-stimulated cultures RNA synthesis and HPRT activity began well in advance of DNA synthesis and increased in parallel during the first 44 h of culture. Introduction of TG or AG with PHA at the beginning of culture completely inhibited DNA synthesis during the first 44 h and reduced RNA synthesis to low levels within 24 h. When TG or AG was added after cells had been in culture for 38 h, DNA synthesis was reduced quickly while RNA synthesis was inhibited more slowly. An autoradiographic assay is described in which freshly isolated lymphocytes are cultured with PHA for 24 h, with or without TG or AG, then labeled with [3H]uridine for 1 h. TG-resistant and AG-resistant variant frequencies for 2 normal individuals and a Lesch-Nyhan individual were determined with this assay. The variant frequencies for the normal individuals ranged from 0.46 to 10.6 X 10(-5) depending upon the selective conditions used. All the Lesch-Nyhan cells were resistant to 0.2 microM-2 mM AG; some were sensitive to 0.2 mM TG and most were sensitive to 2.0 mM TG.     

Amino acid sequence homologies between rabbit, rat, and human serum retinol-binding proteins

The main transporting protein for vitamin A in rabbit serum, the retinol-binding protein (RBP), was isolated and its amino acid sequence determined. Rabbit RBP was found to be highly homologous to human RBP, whose amino acid sequence was elucidated earlier, and to rat RBP. The rat RBP sequence was obtained by combining information deduced from the nucleotide sequences of two overlapping cDNA clones with the NH2-terminal sequence of the isolated protein determined by automated Edman degradation. The identity between the three proteins is approximately 90%. The high degree of homology between RBP molecules from different species is probably explained by the fact that RBP participates in at least three types of molecular interactions: in the binding of prealbumin, in the interaction with retinol, and in the recognition of a specific cell surface receptor. All these interactions should lead to a conservation of RBP structure. The amino acid differences between rabbit, rat, and human RBP are discussed in light of the recent elucidation of the three-dimensional structure of human RBP. Hybridization of a probe isolated from a rat RBP cDNA clone to restriction enzyme-digested genomic DNA from rat and mouse suggests that RBP is encoded by a single gene.     

Production of (S)-3-chlorolactaldehyde from (S)-alpha-chlorohydrin by boar spermatozoa and the inhibition of glyceraldehyde 3-phosphate dehydrogenase in vitro

The (S)-isomer of the male antifertility agent alpha-chlorohydrin was metabolized by mature boar spermatozoa in vitro to (S)-3-chlorolactaldehyde. This oxidative process, which did not occur when (R)-alpha-chlorohydrin was offered as a substrate, was catalysed by an NADP+-dependent dehydrogenase that converts glycerol to glyceraldehyde. (S)-3-chlorolactaldehyde, produced by this metabolic reaction or when added to suspensions of boar spermatozoa, was a specific inhibitor of glyceraldehyde 3-phosphate dehydrogenase as assessed by the accumulation of fructose 1,6-bisphosphate and the triosephosphates. When glycerol and (S)-alpha-chlorohydrin were added concomitantly to boar spermatozoa in vitro, the presence of glycerol decreased the degree of inhibition of glyceraldehyde 3-phosphate dehydrogenase. Extracts of glyceraldehyde 3-phosphate dehydrogenase that were obtained from boar spermatozoa incubated with (S)-alpha-chlorohydrin or (R,S)-3-chlorolactaldehyde showed significant reductions in their enzymic activity.     

Gel electrophoretic procedures potentially applicable to the isolation of human growth hormone from a transformed bacterial source at the gram-preparative scale

A crude bacterial extract containing approximately 4 mg/ml protein, 25% of which was human growth hormone (hGH), was subjected to two alternative gel electrophoretic isolation procedures, designated I and II. Procedure I exploits the high electrophoretic net mobility (RM larger than 0.127) at pH 7.6, 0 degrees C, of the bacterial contaminants relative to hGH. This allows one to stack the contaminants at a protein load of 31.5 mg/cm2 of gel, using a "non-restrictive" gel concentration. Unstacked hGH is collected from the gel section between 0.3 and 0.6 of relative gel length and extracted electrophoretically as described previously. Alternatively, the unstacked hGH was concentrated on the gel by dispatching a second moving boundary behind the original stack ("re-stacking") and a gel section (relative gel length 0.45 to 0.6) between the two moving boundaries was excised and subjected to electrophoretic extraction. The yield of hGH ranged from 70 to 82%, and its purity (weight/Lowry) ranged from 86 to 115%. Procedure II exploits the high electrophoretic net mobility (RM larger than 0.064) at pH 10.5, 0 degrees C, of hGH relative to its bacterial contaminants at a gel concentration of 9 %T, 2 %CBis, at a protein load of 2.5 mg/cm2 of gel. The selectively stacked hGH is collected by preparative elution-PAGE, using an apparatus with 17.6 cm2 gel surface area. The yield of hGH was 90% and its purity ranged from 84-92%.     

Variation in natural killer activity in peripheral blood during the menstrual cycle.

Natural killer activity was measured sequentially in normal female volunteers through their menstrual cycle. During the periovulatory period there was a significant fall in natural killer activity compared with in normal male volunteers. This variation was not apparent in women taking oral contraception. Cytotoxic activity was not related to oestradiol concentrations in individual women. The data support an interaction between immunological activity and sex hormones over the normal physiological range and would account for the described reduction in natural killer activity in pooled blood from female blood donors.     

Comparison of fatty acid content and DNA homology of the filamentous gliding bacteriaVitreoscilla,Flexibacter, Filibacter

DNA hybridization experiments showed that there was a high degree of homology amongVitreoscilla strains but not with DNA fromFilibacter limicola. Flexibacter spp were much more heterogeneous indicating a low genetic similarity. These results were also reflected in the membrane fatty acids of the bacteria. TheVitreoscilla strains were very similar with the 16:17c fatty acid being dominant. The membrane fatty acids ofF. limicola were dominated by a15:0 and a17:0 components which provided additional support for its relatedness to the genusBacillus. There was much greater diversity in the fatty acid patterns of theFlexibacter spp.F. aurantiacus, F. ruber andF. elegans shared the common dominant fatty acids 16:17c with theVitreoscilla strains, but this was replaced by the 16:16c acid inF. flexilis. F. ruber was distinguished by the absence of branched odd-chain monounsaturated fatty acids andF. elegans by the dominance of the -OH i15:0 acid. Precise determination of fatty acid double bond positions and geometry are essential for correct interpretation of increasingly complex ecological and taxonomic data sets.     

Bkm sequences are polymorphic in humans and are clustered in pericentric regions of various acrocentric chromosomes including the Y

Summary Probes of uncloned Bkm satellite DNA and a Drosophila clone 2(8), consisting mainly of GATA repeasts related to a major sequence component in Bkm, have been used to probe Southern blots of human male and female DNAs obtained from a Caucasian and an Australian aboriginal population and to human chromosomes in situ. Hybridization was observed to a distinct and an indistint series of bands against a smeared background. The same distinct bands are identified in the DNA samples with both probes, but are most readily detected using the uncloned Bkm probe. Most restriction bands are common to both populations and some are polymorphic. However, certain bands appear to be characteristic of the Australian aboriginal samples. There are no distinct sex-linked patterns. However all of the small acrocentric human chromosomes, including the Y chromosome show hybridization to uncloned Bkm in situ.