Normal mouse strains differ in the site of initiation of closure of the cranial neural tube

The scanning electron microscopic study of day 9 embryos reported here documents differences among normal mouse strains in morphology of cranial neural tube closure. The site of initiation of contact and fusion of the cranial neural folds, previously defined as Closure 2 (Macdonald et al., '89), is located in the region of the junction between the forebrain (prosencephalon) and midbrain (mesencephalon) in three normal strains: LM/Bc, AEJ/RkBc, and ICR/Bc. However in a fourth normal strain, SWV/Bc, Closure 2 is initiated much further rostral, in the prosencephalic region. In addition, the anterior neuropore, rostral to Closure 2, closes late in ICR/Bc embryos, relative to the posterior progress of development of the Closure 2 seam. Initiation of closure from the most rostral end of the neural tube (Closure 3) appears to be relatively delayed in ICR/Bc embryos. We hypothesize that the observed genetic polymorphism in location of the first site of fusion between the cranial neural folds in normal mouse embryos may be one basis for differences among normal strains in liability to exencephaly induced by teratogens.     

Temporal and spatial expression of a thiolprotease gene during pea ovary senescence, and its regulation by gibberellin

Clones encoding a thiolprotease (tpp) have been isolated from a cDNA library of unpollinated, senescent pea ovaries and its pattern of expression during both ovary senescence and parthenocarpic development have been studied. The sequence of the tpp cDNA displays a high similarity with other plant and animal thiolproteases of the papain group. The homology is highest around the Cys-His of the active centre; a 109 amino acid sequence at the carboxy terminus was found to be homologous only to thiolproteases of plant origin; this part of the mRNA is also present in another pea mRNA that exhibits similar patterns of induction. tpp mRNA shows a temporal pattern of accumulation that precedes that observed for proteolytic activity. Such accumulation did not occur when ovaries were induced to grow parthenocarpically by gibberellic acid (GA) treatment; furthermore the initial low level of expression present in ovaries decreased after GA treatment, indicating that the gene is down-regulated by gibberellins. Spatially, tpp mRNA is localized mainly within the ovule and ovary vascular elements, and transiently within the endocarp of senescent ovaries. This pattern of expression precedes the development of the cytopathogenic effects observed as unpollinated ovaries undergo senescence.     

Differentiation of U937 cells induced by 5,8,11,14-eicosatetraynoic acid, a competitive inhibitor of arachidonic acid metabolism

5,8,11,14-Eicosatetraynoic acid, a competitive inhibitor of arachidonic acid metabolism, rapidly and reversibly inhibited DNA synthesis in U937 cells. This inhibition was not due to cytotoxicity, as judged by studies with trypan blue, release of 51Cr-labeled proteins, and its reversibility. When cells were cultured in the presence of ETYA for several days, morphologic, enzymatic, and functional changes consistent with differentiation occurred. Morphologic evidence of differentiation was evident by light microscopy. The cells enlarged, the ratio of cytoplasm to nuclei increased, secretory granules and vacuoles developed, the apparent activity of nonspecific esterase rose, and ingestion of latex particles increased. A morphology consistent with that of an immature monocyte was evident by electron microscopy. These features included the development of lobulated nuclei, a reduced nuclear to cytoplasmic ratio, increased complexity and development of the cytoplasmic components, and the disappearance of fimbriated plasma membrane structures. In addition, putative polyribosomes were less evident. When cells differentiated by ETYA were cultured in media free of the inhibitor, DNA synthesis reinitiated and the cell number increased; differentiation was phenotypic and not genotypic. To examine whether ETYA-induced differentiation was obligatorily related to its suppression of DNA synthesis, cells were incubated in 50 microM hydroxyurea and DNA synthesis was inhibited for 24 to 36 h without morphologic evidence of cellular differentiation. However, addition of ETYA to cells prevented from dividing by hydroxyurea and subsequent culture for 72 h induced morphologic evidence of differentiation. The effects of ETYA on cell division and cell differentiation are closely related but can be dissociated. The molecular events underlying these results remain to be established.     

Protein kinase C activity and reactivity to phorbol ester in vascular smooth muscle from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY)

Protein kinase C (PKC) activity in aortic and renal arterial smooth muscle from SHR (20-23 wk male; mean arterial pressure = 178 mm Hg) and WKY (age/sex matched; mean arterial pressure = 126 mm Hg) was quantitated. Activity was greatest in the particulate fractions relative to the soluble fractions in all sources. The only difference between SHR and WKY was in the soluble fraction from SHR renal arteries, which had 2 fold more activity (255 pmol/mg/min) when compared with WKY (136 pmol/mg/min). This difference was not apparently related to force modulation, since the magnitude of isometric force development in renal arteries in response to phorbol 12,13-dibutyrate was not different between SHR and WKY. The magnitude of force developed in response to phorbol 12,13-dibutyrate and PKC activity in the particulate fraction was greatest in aorta vs. renal arteries in both WKY and SHR. These results suggest that regional vascular differences in the amount of PKC activity may exist which are not apparently related to a disease state (i.e., hypertension). These differences may be related to differential sensitivity to phorbol ester-mediated contractions in isolated smooth muscle.     

Inactivation of the ribonucleoside triphosphate reductase from Lactobacillus leichmannii by 2'-chloro-2'-deoxyuridine 5'-triphosphate: a 3'-2' hydrogen transfer during the formation of 3'-keto-2'-deoxyuridine 5'-triphosphate

The ribonucleoside triphosphate reductase of Lactobacillus leichmannii converts the substrate analogue 2'-chloro-2'-deoxyuridine 5'-triphosphate (ClUTP) into a mixture of 2'-deoxyuridine triphosphate (dUTP) and the unstable product 3'-keto-2'-deoxyuridine triphosphate (3'-keto-dUTP). This ketone can be trapped by reduction with NaBH4, producing a 4:1 mixture of xylo-dUTP and dUTP. When [3'-3H]ClUTP is treated with enzyme in the presence of NaBH4, the isomeric deoxyuridines isolated after alkaline phosphatase treatment retained 15% of the 3H in ClUTP. Degradation of these isomeric nucleosides has established the location of the 3H in 3'-keto-dUTP as predominantly 2'(S). The xylo-dU had 98.6% of its label at the 2'(S) position and 1.5% at 2'(R). The isolated dU had 89.6% of its label at 2'(S) and 1.4% at 2'(R), with the remaining 9% label inferred to be at the 3'-carbon, this resulting from the direct enzymic production of dUTP. These results are consistent with enzymic production of a 1:1000 mixture of dUTP and 3'-keto-dUTP, where the 3'-hydrogen of ClUTP is retained at 3' during production of dUTP and is transferred to 2'(S) during production of 3'-keto-dUTP. The implications of these results and the unique role of the cofactor adenosylcobalamin (Ashley et al., 1986) are discussed in terms of reductase being a model for the B12-dependent rearrangement reactions.     

Protonation mechanism and location of rate-determining steps for the Ascaris suum nicotinamide adenine dinucleotide-malic enzyme reaction from isotope effects and pH studies

The pH dependence of the kinetic parameters and the primary deuterium isotope effects with nicotinamide adenine dinucleotide (NAD) and also thionicotinamide adenine dinucleotide (thio-NAD) as the nucleotide substrates were determined in order to obtain information about the chemical mechanism and location of rate-determining steps for the Ascaris suum NAD-malic enzyme reaction. The maximum velocity with thio-NAD as the nucleotide is pH-independent from pH 4.2 to 9.6, while with NAD, V decreases below a pK of 4.8. V/K for both nucleotides decreases below a pK of 5.6 and above a pK of 8.9. Both the tartronate pKi and V/Kmalate decrease below a pK of 4.8 and above a pK of 8.9. Oxalate is competitive vs. malate above pH 7 and noncompetitive below pH 7 with NAD as the nucleotide. The oxalate Kis increases from a constant value above a pK of 4.9 to another constant value above a pK of 6.7. The oxalate Kii also increases above a pK of 4.9, and this inhibition is enhanced by NADH. In the presence of thio-NAD the inhibition by oxalate is competitive vs. malate below pH 7. For thio-NAD, both DV and D(V/K) are pH-independent and equal to 1.7. With NAD as the nucleotide, DV decreases to 1.0 below a pK of 4.9, while D(V/KNAD) and D(V/Kmalate) are pH-independent. Above pH 7 the isotope effects on V and the V/K values for NAD and malate are equal to 1.45, the pH-independent value of DV above pH 7. From the above data, the following conclusions can be made concerning the mechanism for this enzyme. Substrates bind to only the correctly protonated form of the enzyme. Two enzyme groups are necessary for binding of substrates and catalysis. Both NAD and malate are released from the Michaelis complex at equal rates which are equal to the rate of NADH release from E-NADH above pH 7. Below pH 7 NADH release becomes more rate-determining as the pH decreases until at pH 4.0 it completely limits the overall rate of the reaction.     

Phosphorylation sites and inactivation of branched-chain alpha-ketoacid dehydrogenase isolated from rat heart, bovine kidney, and rabbit liver, kidney, heart, brain, and skeletal muscle

Branched-chain alpha-ketoacid dehydrogenase complex was isolated from rat heart, bovine kidney, and rabbit liver, heart, kidney, brain, and skeletal muscle. Phosphorylation to approximately 1 mol Pi/mol alpha-subunit of the alpha-ketoacid decarboxylase component was linearly associated with 90-95% inactivation. The complex from some tissues (i.e., from rabbit kidney and heart, and rat heart) showed 30-40% more phosphate incorporation for an additional 5-10% inactivation. Reverse-phase HPLC analysis of tryptic digests of 32P-labeled complexes from all of the above tissues revealed two major (peaks 1 and 2) and one minor (peak 3) phosphopeptide which represent phosphorylation sites 1, 2, and a combination of 1 and 2, respectively. These phosphopeptides, numbered according to the order of elution from reverse-phase HPLC, had the same elution time regardless of the tissue or animal source of the complex. The amino acid sequence of site 1 from rabbit heart branched-chain alpha-ketoacid dehydrogenase was Ile-Gly-His-His-Ser(P)-Thr-Ser-Asp-Asp-Ser-Ser-Ala-Tyr-Arg. Regardless of the source of the complex, both sites were almost equally phosphorylated until total phosphorylation was approximately 1 mol Pi/mol of alpha-subunit and the rate of inactivation was correlated with the rate of total, site 1, or site 2 phosphorylation. Phosphorylation beyond this amount was associated with greater site 2 than site 1 phosphorylation. alpha-Chloroisocaproate, a potent inhibitor of branched-chain alpha-ketoacid dehydrogenase kinase activity, greatly reduced total phosphorylation and inactivation; however, phosphorylation of site 2 was almost abolished and inactivation was directly correlated with phosphorylation of site 1. Thus, the complex isolated from different tissues and mammals had an apparent conservation of amino acid sequence adjacent to the phosphorylation sites. Both sites were phosphorylated to a similar extent temporally although site 1 phosphorylation was directly responsible for inactivation.     

Characterization of human platelet basic protein, a precursor form of low-affinity platelet factor 4 and beta-thromboglobulin

Platelet basic protein (PBP) was purified from the supernatant of thrombin-stimulated, washed human platelets by ion-exchange, affinity, molecular sieve, and high-performance liquid chromatography (HPLC). The NH2-terminal amino acid sequence was determined by automated Edman degradation, revealing 9 unique residues followed by 10 residues of the established low-affinity platelet factor 4/beta-thromboglobulin (LA-PF4/beta TG) sequence. Among the nine were three basic residues, accounting for the high isoelectric point of PBP. Additional evidence for precursor status includes the immunological cross-reactivity of all three species and the ability of plasmin and trypsin to produce from PBP a species resembling beta TG in charge, hydrophobicity, and size. Tryptic peptide maps of PBP and LA-PF4 obtained by reverse-phase HPLC were very similar, and from each protein, a peptide was isolated which showed the amino acid composition predicted for the COOH-terminal tryptic peptide of beta TG. Normal platelets contained predominantly LA-PF4, with PBP ranging from 10% to 30% of total beta TG antigen. This was true even when fresh platelets were lysed with trichloroacetic acid in order to provide the most complete and rapid inhibition of proteolytic activity. beta TG itself was never detected in this situation or in the release supernatant of stimulated platelets, and only rarely in unprotected lysates. In agreement with earlier results, crude preparations of PBP were mitogenic for 3T3 cells, but highly purified preparations of PBP and LA-PF4 were free of this activity.     

Sensitivity to X-irradiation of peripheral blood lymphocytes from ageing donors

The proliferation of human blood lymphocytes from ageing donors, responding to concanavalin A, showed greater sensitivity to inhibition by X-rays than similar cells from younger donors. This increased sensitivity was associated with deficiency in repair of X-ray-induced damage to nuclear material, as measured by density in sucrose gradients, and with increased incidence of chromosomal damage following exposure of freshly isolated lymphocytes. There was also an increased frequency of spontaneous chromosomal aberrations in ageing subjects whose lymphocytes were deficient in repair of DNA damage.