Identification of EcoRV fragments spanning the N alpha-tubulin gene of Physarum

The N alpha-tubulin gene of Physarum polycephalum has an EcoRV site at codons 252/253. EcoRV digestion of physarum DNA generated two EcoRV fragments per gene copy comprising both coding and flanking sequences. Hybridisation probes which included coding sequences upstream from the central EcoRV site cross-hybridised with another alpha-tubulin gene. Probes derived from either 5'- or 3'-flanking regions were gene-specific. These probes identified two EcoRV fragments in the haploid strain CLdAXE viz 5.4 kb (5'-fragment) and 6.2 kb (3'-fragment). The same two fragments were identified in EcoRV digests of DNA of the diploid strain M3CVIII, and a second form of the gene was also identified comprising two fragments viz 5.0 kb (5'-end) and 5.5 kb (3'-end). Both forms gave rise to an identical 4.65 kb HindIII fragment as judged by restriction mapping.     

An autocrine factor from Reuber hepatoma cells that stimulates DNA synthesis and acetyl-CoA carboxylase. Characterization of biologic activity and evidence for a glycan structure

Conditioned medium from Reuber H-35 or Fao hepatoma cells contains autocrine factors that both stimulate DNA synthesis and activate acetyl-coenzyme A (CoA) carboxylase in serum-deprived Fao cells. The factor(s), which appears within 4 h of serum-free culture, also increases the cell number and the mitotic index. The effects of the conditioned medium are insulinomimetic, both with respect to stimulation of DNA synthesis and acetyl-CoA carboxylase activity. However, no induction of tyrosine aminotransferase activity or stimulation of aminoisobutyric acid uptake is seen in response to the conditioned medium. Insulin over a 4-h period does not increase the concentration of DNA synthesis stimulating activity that is observed in the medium. This activity is dialyzable and is resistant to acid treatment or to heating to 60-100 degrees C and to trypsin digestion; it is not extracted with chloroform/methanol nor adsorbed by charcoal or by a C18 reverse-phase column. Fractionation of the conditioned medium derived from Reuber H-35 hepatoma cells by gel filtration chromatography reveals two low molecular weight (less than 1000) compounds that both stimulate DNA synthesis in Fao hepatoma cells. The larger compound (peak I) also stimulates the activity of acetyl-CoA carboxylase. The stimulatory effects of the peak I compound are destroyed by nitrous acid deamination, periodate oxidation, and methanolysis. Biosynthetic labeling studies indicate the probable presence of glucosamine, galactose, and perhaps phosphate in the peak I-activating material. No significant incorporation of either myoinositol or mannose into the active material has been observed. These data, taken together, are consistent with a glycan structure for this autocrine factor, which bears strong resemblance to similar insulinomimetic factors generated in BC3H1 myocytes and H-35 hepatoma cells in response to insulin and on digestion of membranes with a phosphatidylinositol-specific phospholipase C. Further characterization of this factor may provide insight into different pathways of insulin action and could provide a strategy to check autocrine-stimulated tumor growth.     

Biochemical differentiation among karyotypic forms of Australian Rattus

Isozyme electrophoresis of up to 55 loci, and microcomplement fixation of albumin were used to assess the extent of structural gene divergence among karyotypic forms of Australian Rattus. The results show that the Australian Rattus are monophyletic with respect to R. rattus or R. norvegicus. Within the Australian Rattus, rates of chromosomal evolution have varied enormously, the highest rates being found among members of the R. sordidus group, where extensive chromosomal repatterning has occurred with little or no structural gene divergence.     

Rapid endocytosis of the transferrin receptor in the absence of bound transferrin

The rate of endocytosis of transferrin receptors, occupied or unoccupied with transferrin, was measured on the cell line K562. At 37 degrees C, receptors, radioiodinated on the cell surface at 4 degrees C, were internalized equally rapidly in the presence or absence of transferrin. In both cases, 50% of the labeled receptors became resistant to externally added trypsin in 5 min. An antitransferrin antibody was used to show directly that the receptors had entered the cells without bound transferrin. The distribution of the receptors on the cell surface was revealed by antibody and protein A-gold staining after prolonged incubation in the presence or absence of transferrin. The receptors were concentrated in coated pits under both conditions. The data suggest that endocytosis of transferrin receptors is not "triggered" by ligand binding and raise the possibility that ligand-induced down-regulation of surface receptors may not occur by this mechanism. Instead receptors may be recognized as being ligand-occupied, not at the cell surface, but at some other site in the recycling pathway such as the endosome.     

Production of somatic hybrids of moss by electrofusion

Summary Mixtures of protoplasts of two auxotrophic mutants of Physcomitrella patens, one requiring thiamine (aneurine), the other p-aminobenzoic acid, have been subjected to electrofusion. The protoplasts were aligned in an alternating electric field (500 KHz, 20 V RMS/cm) and induced to fuse by a brief DC pulse (800 V/cm, time constant lms). After culture, first on complete medium and then on selective medium, hybrid plants were obtained at a frequency of 3%. One hybrid with a morphology typical of polyploidy was also observed.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Gastrocnemius Muscle Lipids in Relation to Diet in Two Mouse Mutants, 129Re-dy and A2G-adr, with Abnormal Muscle Function

The lipids of gastrocnemius muscle from normal and dystrophic (dy) mice of the Bar Harbor, 129Re strain were studied. Animals were fed diets containing either 3.1% or 1.1% of total calories as linoleic acid. Lipid analyses were also done on muscle from a new mouse mutant, A2G-adr, which has abnormal muscle function, characterised by an arrested development of the righting response. These animals were fed the "high" linoleic acid diet only. Total lipid, triacylglycerol, and cholesterol were elevated in the 129Re-dy irrespective of the diet, whereas A2G-adr possessed significantly higher levels of cholesterol. Total phosphorus (micrograms P/g muscle) and cholesterol/phospholipid ratios were elevated in the dy strains only. Cardiolipin was raised in the dy ("low" linoleic diet) and adr muscle, whereas phosphatidylcholine was lower in the adr strain only. Linoleic acid esterified to phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine was elevated whereas arachidonic acid in phosphatidylserine was decreased in both mutants. Docosahexanoic acid (22:6) in all three dy phospholipids was decreased, independent of dietary treatment. The adr strain possessed normal levels of this fatty acid. The results specifically point to an abnormality in long-chain polyunsaturated fatty acid metabolism in gastrocnemius muscle in the 129Re-dy mutant; in the adr mutant they could reflect an abnormal increase in the number of muscle mitochondria.     

Response and fertility of dairy heifers following injection with prostaglandin F(2alpha) during early, middle or late diestrus

After an observed estrus, 250 dairy heifers were injected once with 25 mg of PGF(2alpha) either on cycle days 5 through 7 (E), 8 through 11 (M) or 12 through 15 (L). For five days after the PGF(2alpha) injection, heifers were inseminated at about 12 h after estrus was first observed. Observed estrual response rates were 43.0%, 83.6% and 100% for E, M and L, respectively. Average time from PGF(2alpha) to observation of estrus for E, M and L was 59, 70 and 72 h. Conception rates for heifers responding to PGF(2alpha) were 56.8%, 62.1% and 78.3% for E, M and L, respectively. Based on blood samples drawn at the time of PGF(2alpha) injection, progesterone concentration was significantly correlated with response rate but not with conception rate. When compared with M and L, E had a significantly lower response rate and conception rate as well as a shorter period between injection of PGF(2alpha) and observation of estrus.     

Biochemical phenotype of 5-fluorouracil-resistant murine T-lymphoblasts with genetically altered CTP synthetase activity

From wild type mouse lymphoma cells, a clone, (FURT-1A), was isolated by virtue of its resistance to 1 microM 5-fluorouracil and 10 microM thymidine. In comparative growth rate experiments, FURT-1A cells were also less sensitive than parental cells to the growth inhibitory effects of thymidine, deoxyguanosine, 5-fluorouridine, and arabinosylcytosine. The altered growth sensitivity of FURT-1A cells to cytotoxic nucleosides was directly related to their decreased ability to accumulate the corresponding triphosphate from exogenous nucleoside. FURT-1A cells contained elevated cytidylate nucleotide pools which prevented normal growth sensitivity and interfered with the salvage of nucleosides by inhibiting nucleoside kinase activities, by stimulating nucleotide dephosphorylating activities, and by overcoming certain allosteric inhibitions imposed on ribonucleotide reductase. Metabolic flux experiments with [3H]uridine in situ indicated that FURT-1A cells had a 2-fold enhanced rate of conversion of UTP to CTP. Kinetic analyses indicated that the CTP synthetase activity in extracts of FURT-1A cells was refractory to inhibition by CTP. The genetic loss of normal allosteric inhibition of the CTP synthetase activity in FURT-1A cells could account for the unusual phenotypic properties of these cells and conferred a high spontaneous mutator phenotype to cells possessing this mutation.     

Purine phosphoribosyltransferase (EC 2.4.2.7 and 2.4.2.8) and purine de novo synthesis activity in rat testicular tissue at different stages of development, and their correlation with the circulating levels of gonadotrophins and testosterone, and with structural changes

The overall activity of the purine de novo synthesis pathway and the activities of purine phosphoribosyltransferase in the rat testis were measured at different ages and were correlated with histological observations. Similar studies of the concentration of circulating gonadotrophins and testosterone were performed. The purine phosphoribosyltransferase activities were between two and three orders of magnitude greater than purine de novo synthesis. The peak activity of the purine de novo synthesis pathway coincided with the first appearance of meiosis in the spermatocytes immediately before the luteinising hormone (LH) level rose to its peak. The highest activity of the hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) - catalysed purine salvage pathway coincided with the first appearance of mature spermatozoa in the tubules just after the occurrence of peak levels of follicle-stimulating hormone (FSH). These findings are linked to the development of testicular atrophy in cases of severe HPRT deficiency in man.