Isolation of amylolytic strains of Thermoactinomyces vulgaris and production of thermophilic actinomycete amylases

Kuo, M. J. (Iowa State University, Ames), and P. A. Hartman. Isolation of amylolytic strains of Thermoactinomyces vulgaris and production of thermophilic actinomycete amylases. J. Bacteriol. 92:723-726. 1966.-Of 759 isolates obtained from dung, compost, and soil samples, a culture of Thermoactinomyces vulgaris (strain 5) was selected for further study on the basis of quantities of amylase produced in synthetic and nonsynthetic media, rapid growth and sporulation, culture stability upon prolonged storage at 5 C, and growth temperature range. Inoculum preparation, temperature optimum for amylase formation, and the effects of various kinds and levels of carbon and nitrogen sources on amylase production were studied with T. vulgaris strain 5. An optimal procedure for production of T. vulgaris amylases is proposed.     

The Saccharomyces cerevisiae SOC8-1 gene and its relationship to a nucleotide kinase

The yeast SOC8-1 gene was originally identified by partial complementation of cdc8 mutant strains. We have carried out Bal31 deletion analysis of the SOC8-1 gene to define the minimal size which is required for the complementation of the cdc8 mutation. When the SOC8-1 gene is cloned in a multicopy plasmid, it enables temperature-resistant growth in the cdc8 mutant strain, while the SOC8-1 gene in a single copy plasmid does not. Thus, its suppression of the cdc8 mutant is dosage dependent. The high copy number vector carrying the SOC8-1 gene can complement five different cdc8 alleles, indicating that the suppression is not allele specific. Since CDC8 encodes thymidylate kinase, cells bearing a high copy number plasmid containing SOC8-1 gene were tested for the ability to phosphorylate several nucleoside monophosphates, including UMP, GMP and dTMP. Significantly increased phosphorylation activity was observed, suggesting that SOC8-1 encodes a nucleotide kinase. Both restriction enzyme analysis of the SOC8-1 gene and partial purification of the overproduced kinase in SOC8-1 overproducing strains suggest that SOC8-1 may be allelic with URA6. Consistent with these results, both SOC8-1 and URA6 are located on chromosome XI. Thus, one possible suppression mechanism is that SOC8-1 may provide a trans-acting dTMP kinase activity, bypassing the cdc8 gene defect.     

X-ray diffraction analysis on single crystals of recombinant Escherichia coli ornithine transcarbamoylase

Single crystals of recombinant Escherichia coli ornithine transcarbamoylase suitable for x-ray analysis have been grown from polyethylene glycol and 2-methyl-2,4-pentanediol. The space group has been determined as P3(1) or P3(2), with one protein trimer of three identical 36.8-kDa subunits in the asymmetric unit. The unit cell dimensions are a = b = 105.1 A and c = 87.8 A. The crystals diffract well to 3-A resolution and are quite resistant to radiation damage. Single crystals have also been grown of a genetically engineered site-specific mutant for which the replacement of an arginine (Arg-57) to a glycine has been shown to not only drastically affect the enzyme activity but also its kinetic mechanism (Kuo, L. C., Miller, A. W., Lee, S., and Kozuma, C. (1988) Biochemistry 27, 8823-8832). The crystals of the Arg-57----Gly mutant protein are isomorphous to those of the wild type. Crystal soaking experiments using both wild-type and Arg-57----Gly crystals in the presence of various ligands have provided evidence of specific conformational changes upon substrate binding which supports our previous kinetic and spectroscopic observations.     

Ganglioside Composition of Normal and Mutant Mouse Embryos

The enrichment of gangliosides in neuronal membranes suggests that they play an important role in CNS development. We recently found a marked tetrasialoganglioside deficiency in twl/twl mutant mouse embryos at embryonic day (E)-11. The recessive twl/twl mutants die at embryonic ages E-9 to E-18 from failed neural differentiation in the ventral portion of the neural tube. In the present study, we examined the composition and distribution of gangliosides in twl/twl mutant mouse embryos at E-12. The total ganglioside sialic acid concentration was significantly lower in the mutants than in normal (+/-) embryos. The mutants also expressed significant deficiencies of gangliosides in the "b" metabolic pathway (GD3, GD1b, GT1b, and GQ1b) and elevations in levels of gangliosides in the "a" metabolic pathway (GM3, GM2, GM1, and GD1a). These findings suggest that the mutants have a partial deficiency in the activity of a specific sialyltransferase in the b pathway. Regional ganglioside distribution was also studied in E-12 normal mouse embryos. The ganglioside composition in heads and bodies was similar to each other and to whole embryos. Total ganglioside concentration and the distribution of b pathway gangliosides were significantly higher in neural tube regions than in nonneural tube regions. These findings suggest that b pathway gangliosides accumulate in differentiating neural cells and that the deficiency of these gangliosides in the twl/twl mutants is closely associated with failed neural differentiation.     

Regional Specificity in Alterations of Rat Brain Copper and Catecholamines Following Perinatal Copper Deficiency

Abstract: Perinatal copper deficiency was studied in 1-month-old female and male Sprague-Dawley rat offspring to investigate regional changes in brain copper and catecholamine levels. Offspring of dams given the low copper treatment beginning at day 7 of gestation exhibited signs characteristic of deficiency such as impaired growth and 10-fold lower liver copper levels compared with copper-adequate controls. Regional analysis of brain copper by graphite furnace atomic absorption spectroscopy revealed uniform and severe reduction of copper to levels 20 ± 3% of controls in all regions, except the hypothalamus, where reductions to 56 and 28% of those in copper-adequate females and males, respectively, were measured. HPLC analysis revealed significant reductions in norepinephrine levels in cerebrum, midbrain, corpus striatum, cerebellum, and medulla-pons of copper-deficient offspring ranging between 39 and 67% of control values. There were no significant differences in norepinephrine concentration in the hypothalamus. There was a significant, one-third reduction of dopamine in the corpus striatum of copper-deficient male rats. Consistent with altered in vivo dopamine β-monooxygenase activity, there were five-, three-, and twofold elevations of dopamine in cerebellum, medulla-pons, and hypothalamus of copper-deficient rats. Spectrophotometric measurement of in vitro dopamine β-monooxygenase activity of brain and adrenal homogenates was higher in copper-deficient rats, confirming prior work. An explanation for the in vitro data is unclear. Changes in copper and catecholamine levels were influenced by diet and were regionally selective, especially in the hypothalamus.     

The Regional Distribution of N-Acetylaspartylglutamate (NAAG) and Peptidase Activity Against NAAG in the Rat Nervous System

Abstract: N -Acetylaspartylglutamate (NAAG), a prevalent peptide in the vertebrate nervous system, may be hydrolyzed by extracellular peptidase activity to produce glutamate and N -acetylaspartate. Hydrolysis can be viewed as both inactivating the peptide after synaptic release and increasing synaptic levels of ambient glutamate. To test the hypothesis that NAAG and the peptidase activity that hydrolyzes it coexist as a unique, two-stage system of chemical neurotransmission, 50 discrete regions of the rat CNS were microdissected for assay. In each microregion, the concentration of NAAG was determined by radioimmunoassay and the peptidase activity was assayed using tritiated peptide as substrate. The NAAG concentration ranged from 2.4 nmol/mg of soluble protein in median eminence to 64 in thoracic spinal cord. Peptidase activity against NAAG ranged from 54 pmol of glutamate produced per milligram of membrane protein per minute in median eminence to 148 in superior colliculus. A linear relationship was observed between NAAG peptidase and NAAG concentration in 46 of the 50 areas, with a slope of 2.26 and a correlation coefficient of 0.45. These data support the hypothesis that hydrolysis of NAAG to glutamate and N -acetylaspartate is a consistent aspect of the physiology and metabolism of this peptide after synaptic release. The ratio of peptide concentration to peptidase activity was >0.3 in the following four areas: ventrolateral medulla and reticular formation where the peptide is concentrated in axons of passage, thoracic spinal cord, where NAAG is concentrated in ascending sensory tracts as well as motoneuron cell bodies, and ventroposterior thalamic nucleus.     

Identification of a developmentally regulated sialidase inEimeria tenella that is immunologically related to theTrypanosoma cruzi enzyme

Sporozoites and merozoites of three species ofEimeria, E. tenella, E. maxima, andE. necatrix, that cause diarrhea in chickens worldwide, were examined for their expression of sialidase (SA) activity. The enzyme was found in three species, and the activity of merozoites was 10–20 times higher than that of sporozoites. The enzyme was resistant to degradation by proteases that are normally present in the intestine, a site inhabited by theEimeria parasites, and it was relatively resistant to heat, with optimum activity being at 40°C, which is within the range of temperature in the chicken intestine (40–43°C).E. tenella SA was immuniprecipitated by monoclonal and polyclonal antibodies raised against theTrypanosoma cruzi SA (TCSA), and enzyme activity was neutralized by these antibodies.E. tenella SA was identified by immunoblots as a doublet of molecular weight 190 000 and 180 000 using, as a probe, anti-TCSA antibodies and antibodies against a synthetic peptide (TR) derived from the long tandem repeat domain of TCSA. Binding of the monoclonal and polyclonal antibodies toE. tenella was completely blocked by TR, but not by an irrelevant peptide (BR). Therefore,E. tenella expresses a developmentally regulated SA that is structurally related to theT. cruzi counterpart. Because of the high SA activity in merozoites, and by analogy with other SA-producing microbes that inhabit mucin-rich epithelia, we suggest that theEimeria SA plays a role in desialylating intestinal mucins to reduce viscosity of the local environment and thereby facilitate parasite migration. The enzyme could also play a role in host cell-parasite interaction.Abbreviations SA sialidase (neuraminidase) - Neu5Ac N-acetylneuraminic acid - 4-MU-Neu5Ac 2-(4-methylumbelliferyl)--N-acetyl-d-neuraminic acid - BSA bovine serum albumin - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride - PNA peanut agglutinin - Ab antibody - TCN-2 monoclonal antibody toT. cruzi sialidase, anti-Ars, monoclonal antibody top-azophenylarsonate - TCSA Trypanosoma cruzi sialidase