The Murine Nucleolin Protein Is an Inducible DNA and ATP Binding Protein Which Is Readily Detected in Nuclear Extracts of Lipopolysaccharide-Treated Splenocytes

A 100-kDa DNA binding protein was found to be dramatically up-regulated upon the mitogenic stimulation of murine splenocytes with bacterial lipopolysaccharide (LPS). The induced DNA binding protein was also found to exhibit moderate binding specificity for the immunoglobulin isotype switch DNA repeats. Furthermore, the induction of the 100-kDa protein by LPS was found to be mediated by both an increase in the protein's stability and an increase in the synthesis of the protein. In vitro phosphorylation experiments revealed that the 100-kDa DNA binding protein was one of the most heavily phosphorylated proteins in both lymphoid and nonlymphoid nuclear extracts. Although this in vitro phosphorylation initially appeared to be mediated by a potent nuclear kinase activity, it was later determined that a significant part of the detected labeling was due to the direct binding of ATP by the 100-kDa protein. Antibodies raised to the 100-kDa DNA binding protein were used to isolate cDNA clones from a lymphocyte cDNA λgt11 expression library. Nucleotide sequence analysis revealed that the cloned cDNAs were identical to the mouse nucleolin gene. The β-galactosidase fusion proteins (encoded by exons 3-14 of nucleolin) and a more severely truncated 45-kDa protein (encoded by exons 5-14 of nucleolin) were both found to bind strongly to DNA and ATP. Furthermore, the strength of DNA binding was found to be highly dependent on the overall dG content of the DNA probes. Our experiments also revealed that apart from binding ATP and G-rich DNA, nucleolin directly bound GTP, dATP, and dGTP, but not dCTP, dTTP, or dUTP. Computer analysis revealed that the putative ATP binding domains appear to fall within two of the phylogenetically conserved RNA binding domains of nucleolin.     

Clavispora,a new yeast genus of the Saccharomycetales

In Candida lusitaniae van Uden et do Carmo-Sousa (1959), strains of opposite sex have been found. Cells of the opposite mating types conjugate and form asci with one to four clavate spores. These are easily liberated from the ascus. The type strain of Candida obtusa (Dietrichson) van Uden et do Carmo-Sousa ex van Uden et Buckley (1970) also produces ascospores after mating with one of the strains of Candida lusitaniae. As clavate ascospores are unknow in yeasts, a new genus, Clavispora, is proposed with Clavispora lusitaniae spec. nov. as type species.     

Alpha-locus hexosaminidase genetic compound with juvenile gangliosidosis phenotype: clinical,genetic, and biochemical studies.

A 3-year-old boy developed progressive neurological deterioration in his third year, characterized by dementia, ataxia, myoclonic jerks, and bilateral macular cherry-red spots. Hexosaminidase A (HEX A) was partially decreased in the patient''s serum, leukocytes, and cultured skin fibroblasts. Hexosaminidase was studied in serum and leukocytes from family members. Four members of the paternal branch appeared to be carriers of classical infantile Tay-Sachs allele, HEX alpha 2, probably receiving the gene from one great-grandparent of Ashkenazi origin. In the maternal branch, no one was a carrier of classical infantile Tay-Sachs disease, but five individuals were carriers of a milder alpha-locus defect. The patient, therefore, was a genetic compound of two different alpha-locus hexosaminidase mutations. At least 21 families with late-infantile or juvenile GM2 gangliosidosis have been reported, 18 of them with alpha-locus mutations, and three with beta-locus mutations. Genetic compounds of hexosaminidase have been reported in at least seven families, five with alpha-locus mutations and two with beta-locus mutations. The compound had the phenotype of infantile Tay-Sachs disease in one family, infantile Sandhoff disease in another, and the normal phenotype in the rest.     

Adaptation to chronic hypobaric hypoxia and sexual hormones

The role of the gonads and their hormones on body weight was studied in rats of both sexes submitted to chronic hypoxia and their controls at sea level atmospheric pressure. Intact rats were exposed to either 4 700 or 6 000 m simulated altitude in a hypopressure chamber. Castrated rats and castrated rats daily injected with either 0.5 mg of testosterone or 20 microgram of estradiol or the vehicle, were exposed to the higher altitude. The rat weight was recorded for a period of at least eight weeks. All groups of hypoxic male animals increased their weight significantly less than the controls at sea level. Also in castrated females and in castrated injected with testosterone or the vehicle the same pattern of weight curves was observed. On the contrary, groups of intact females and castrated females injected with estradiol did not show significant differences between hypoxic and control animals. Only in a group of smaller intact females (50-80 g) the body weight increase was significantly diminished by exposure to either 4 700 or 6 000 m simulated altitude.     

Determination of the chromosomal size of three different strains of Enterococcus faecalis and one strain of Enterococcus faecium.

Pulsed-field gel electrophoresis was used to determine the chromosomal size of three different strains of Enterococcus faecalis and one strain of Enterococcus faecium. The size determinations of OG1X, a strain of E. faecalis widely used in many laboratories for genetic studies, using Sma I, Not I, and Sfi I alone or in combination, ranged from 2,750 to 2,761 kb. Using the same enzymes as with OG1X, the size of HH-67, a plasmid-free clinical isolate of E. faecalis, was determined to be 2,170-2,288 kb and the size of JH2-2, an E. faecalis recipient strain, ranged from 2,008 to 2,135 kb. The size range generated for GE-1, a plasmid-free E. faecium strain, with the use of Sma I, Not I, and Apa I was 2,045-2,155 kb. Although OG1X differed in size from the other three enterococci, each individual enterococcal strain generated reproducible results in different experiments. However, for both E. faecalis OG1X and E. faecium GE-1, one of the enzymes used generated a considerably smaller molecular size than that generated by the other two enzymes. The discrepancy was due to visually undiscernible comigrating fragments, and serves to point out a potential source of error if fewer than two enzymes are used to size a genome. The size discrepancies were resolved by digesting individual fragments with a second enzyme. The molecular sizes of these enterococcal strains are larger than that recently reported for Campylobacter, smaller than that of Escherichia coli and Pseudomonas aeruginosa, and similar (OG1X) or smaller (JH2-2, HH67, and GE-1) than the 2,819-kb reported for Streptococcus mutans.     

Regulation of gene expression in preimplantation mouse embryos: effects of the zygotic clock and the first mitosis on promoter and enhancer activities

Previous studies have reported that promoters requiring enhancers for full activity in mammalian somatic cells also require enhancers when injected into mouse two-cell embryos, whereas the same promoters can be expressed just as efficiently in the absence of an enhancer when injected into arrested one-cell embryos. Experiments were designed to determine whether this phenomenon reflected normal developmental changes at the beginning of mammalian development, or simply differences in the physiological states of these cells under the experimental conditions employed. The activity of three different promoters that function in a wide variety of mammalian cells was measured both in embryos whose morphological development was arrested and in embryos that continued development in vitro. Expression of the injected gene was related to the onset of zygotic gene expression ("zygotic clock"), the phase of the cell proliferation cycle, the use of aphidicolin to arrest cell proliferation, and formation of two-cell embryos in vitro and in vivo. The results demonstrated that promoter activity was tightly linked to zygotic gene expression, while the need for enhancers to stimulate promoter activity depended only on formation of a two-cell embryo. These results further support the hypothesis that the first mitosis induces a general repression of promoters prior to initiation of zygotic gene expression that is relieved specifically by enhancers.     

Effect of methabenzthiazuron on growth and nitrogenase activity in Vicia faba

The effects of the herbicide methabenzthiazuron (175 and 220 g ha^-1) on vegetative and reproductive growth, nodulation and nitrogenase activity of Vicia faba were studied in the field under Mediterranean conditions. Nitrogenase activity of excised nodules was estimated using the acetylene reduction assay four times during the developmental period. Leaf area index, dry weight and nitrogen content of the different parts of the plants were measured. Methabenzthiazuron-treated plants showed an increase in nodulation, nitrogenase activity and vegetative growth at early pod fill. Methabenzthiazuron also caused an increase in leaf N content and fruits. These were transient effects found during early and mid pot fill. Nevertheless, plants treated with these sublethal doses of herbicide improved seed production and nitrogen content of seeds at harvest time. The stimulatory effect of methabenzthiazuron on N₂ fixation and vegetative growth seems not be related with the transient stimulatory effect on photosynthetic capacity, also caused by the herbicide, since the stimulatory effect on N₂ fixation was apparent during pod fill, when photosynthetic capacity declined and was not modified by methabenzthiazuron.     

Flavin-containing monooxygenase: a major detoxifying enzyme for the pyrrolizidine alkaloid senecionine in guinea pig tissues

Evidence based on optimal pH, thermal stability, and enzyme inhibition data suggests that the NADPH-dependent microsomal N-oxidation of the pyrrolizidine alkaloid senecionine is carried out largely by flavin-containing monooxygenase in guinea pig liver, lung, and kidney. In contrast, the hepatic microsomal conversion of senecionine to the pyrrole metabolite (+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) is catalyzed largely by cytochrome P450. However, the rate of senecionine N-oxide formation (detoxication) far exceeded the rate of DHP formation (activation) in guinea pig liver microsomes over a range of pHs (pH 6.8 to 9.8). In guinea pig lung and kidney microsomes, N-oxide was the major metabolite formed from senecionine with little or no production of DHP. The high rate of detoxication coupled with the low level of activation of senecionine in liver, lung, and kidney may help explain the apparent resistance of the guinea pig to intoxication by senecionine and other pyrrolizidine alkaloids.     

Comparison of rainbow trout and mammalian cytochrome P450 enzymes: evidence for structural similarity between trout P450 LMC5 and human P450IIIA4

Studies were undertaken to determine the immunochemical relationship between constitutive trout cytochrome P450s and mammalian cytochrome P450IIIA enzymes. Polyclonal antibodies (IgG) generated against trout P450 LMC5 reacted strongly with P450IIIA1 in dexamethasone-induced rat liver microsomes and with P450IIIA4 in human liver microsomes in immunoblots. In contrast, rabbit anti-P450 LMC1 IgG did not recognize these proteins in rat and human liver microsomes. Reciprocal immunoblots using anti-rat P450IIIA1 showed that this antibody does not recognize trout P450 LMC1 or LMC5. However, anti-human P450IIIA4 IgG was found to cross react strongly with P450 LMC1 and LMC5. Progesterone 6 beta-hydroxylase activity of trout liver microsomes, a reaction catalyzed by P450 LMC5, was markedly inhibited by anti-P450IIIA4 and by gestodene, a mechanism-based inactivator of P450IIIA4. These results provide evidence for a close structural similarity between trout P450 LMC5 and human P450IIIA4.     

A study on melanogenesis and catecholamine biosynthesis during Bufo bufo development

Tyrosinase and L-DOPA decarboxylase activities have been investigated during Bufo bufo development since catecholamines and melanin are formed from common substrates in homologous cells. Catecholamines first appear at stage 13 (neural plate), but tyrosinase, at a very low level, and L-DOPA decarboxylase are present throughout all of prior development. Hence, L-DOPA decarboxylase activity is not likely to be correlated with the control of catecholamine synthesis, although at stage 17 it is mainly localized in the nonneural part of the embryo. The distribution of young melanosomes and L-DOPA decarboxylase suggest a separation between melanogenesis and catecholamine synthesis.