Influence of Storage at Freezing and Subsequent Refrigeration Temperatures on β-Galactosidase Activity of Lactobacillus acidophilus

The ability of three strains of Lactobacillus acidophilus to survive and retain β-galactosidase activity during storage in liquid nitrogen at −196°C and during subsequent storage in milk at 5°C was tested. The level of β-galactosidase activity varied among the three strains (0.048 to 0.177 U/10⁷ organisms). Freezing and storage at −196°C had much less adverse influence on viability and activity of the enzyme than did storage in milk at 5°C. The strains varied in the extent of the losses of viability and β-galactosidase activity during both types of storage. There was not a significant interaction between storage at −196°C and subsequent storage at 5°C. The strains that exhibited the greatest losses of β-galactosidase activity during storage in milk at 5°C also exhibited the greatest losses in viability at 5°C. However, the losses in viability were of much greater magnitude than were the losses of enzymatic activity. This indicates that some cells of L. acidophilus which failed to form colonies on the enumeration medium still possessed β-galactosidase activity. Cultures of L. acidophilus to be used as dietary adjuncts to improve lactose utilization in humans should be carefully selected to ensure that adequate β-galactosidase activity is provided.     

Pigment patterns in mutants affecting the biosynthesis of pteridines and xanthommatin in Drosophila melanogaster

Eye-color mutants of Drosophila melanogaster have been analyzed for their pigment content and related metabolites. Xanthommatin and dihydroxanthommatin (pigments causing brown eye color) were measured after selective extraction in acidified butanol. Pteridines (pigments causing red eye color) were quantitated after separation of 28 spots by thin-layer chromatography, most of which are pteridines and a few of which are fluorescent metabolites from the xanthommatin pathway. Pigment patterns have been studied in 45 loci. The pteridine pathway ramifies into two double branches giving rise to isoxanthopterin, drosopterins, and biopterin as final products. The regulatory relationship among the branches and the metabolic blockage of the mutants are discussed. The Hn locus is proposed to regulate pteridine synthesis in a step between pyruvoyltetrahydropterin and dihydropterin. The results also indicate that the synthesis and accumulation of xanthommatin in the eyes might be related to the synthesis of pteridines.Support for this work was provided to J.F. in part by a grant from the Ministerio de Universidades e Investigación (Spain) and to F.J.S. by a grant from the Ministerio de Educación y Ciencia (Spain).     

The melanoma proteoglycan: restricted expression on microspikes, a specific microdomain of the cell surface

A cell surface chondroitin sulfate proteoglycan associated with human melanomas and defined by mAb's F24.47 and 48.7 has been characterized biochemically and localized by indirect immunogold electron microscopy. These antibodies recognize distinct epitopes on the intact proteoglycan. In addition, mAb 48.7 also recognizes an epitope on a 250,000-D glycoprotein and is therefore similar to antibody 9.2.27 (described by Bumol, T.F., and R.A. Reisfeld, 1982, Proc. Natl. Acad. Sci. USA., 79:1245-1249). Furthermore, it was shown that the glycosaminoglycan chains released by alkaline borohydride treatment of the proteoglycan recognized by mAb 48.7 had a size of approximately 60,000 D. Since the intact proteoglycan was estimated to be 420,000 D, there are probably three chondroitin sulfate chains attached to the 250,000-D core glycoprotein. Furthermore, an oligosaccharide fraction containing 42% of the 3H activity (glucosamine as precursor) was isolated. Immunolocalization studies using whole-mount electron microscopy revealed that the chondroitin sulfate proteoglycan was present almost exclusively on microspikes, a microdomain of the melanoma cell surface. These processes were present as 1-2-micron structures on the upper cell surface and as longer (up to 20 micron) structures at the cell periphery. Peripheral microspikes were involved in the initial interactions between adjacent cells and formed complex footpads that made contact with the substratum. Immunogold-labeled cells were also thin sectioned and the specific localization of the chondroitin sulfate proteoglycan antigen was quantitated. The data confirmed the results of whole-mount microscopy and demonstrated a statistically significant association of the antigen with the microspike processes as compared with other areas of the cell surface. By using two different mAb's (48.7 and F24.47) that recognize epitopes on either the core glycoprotein or the intact proteoglycan, respectively, we have demonstrated that both molecules have the same restricted distribution at the cell surface. The specific localization of the antigen to microspikes at the cell surface suggests it may play a role in cell-cell contact and cell-substratum adhesion, which could be important in the metastatic process.     

Interactions in Syntrophic Associations of Endospore-Forming, Butyrate-Degrading Bacteria and H2-Consuming Bacteria

Butyrate is an important intermediate in the anaerobic degradation of organic matter. In sulfate-depleted environments butyrate is oxidized to acetate and hydrogen by obligate proton reducers, in syntrophic association with hydrogen-consuming methanogens. This paper describes two enrichments of endospore-forming bacteria degrading butyrate in consortia with methanogens. The isolates are readily established in coculture with H₂-consuming, sulfate-reducing bacteria by pasteurizing the culture. The two original enrichments differed in that one grew to an optically dense culture while the second grew in clumps. Examination by scanning electron microscopy showed that clumping resulted from the production of large amounts of extracellular polymer. Several H₂-consuming methanogens were identified in the enrichments. Some of them grew closely associated to the butyrate degraders. This attachment to the hydrogen producer may permit some methanogens to compete for the growth substrate against other bacteria having higher substrate affinity.     

Natural Selection VS. Random Drift: Evidence from Temporal Variation in Allele Frequencies in Nature

We have obtained monthly samples of two species, Drosophila pseudoobscura and Drosophila persimilis, in a natural population from Napa County, California. In each species, about 300 genes have been assayed by electrophoresis for each of seven enzyme loci in each monthly sample from March 1972 to June 1975. Using statistical methods developed for the purpose, we have examined whether the allele frequencies at different loci vary in a correlated fashion. The methods used do not detect natural selection when it is deterministic (e.g., overdominance or directional selection), but only when alleles at different loci vary simultaneously in response to the same environmental variations. Moreover, only relatively large fitness differences (of the order of 15%) are detectable. We have found strong evidence of correlated allele frequency variation in 13-20% of the cases examined. We interpret this as evidence that natural selection plays a major role in the evolution of protein polymorphisms in nature.     

Effect of prolactin and glucocorticoids on P-enolpyruvate carboxykinase activity in liver and mammary gland from diabetic and lactating rats

Summary The administration of 2 bromo--ergocryptine, to reduce serum prolactin decreased the activity of cytosolic P-enolpyruvatc carboxykinase (GTP) (EC4.1.1.32) about 50% in both liver and mammary gland of lactating animals. Adrenalectomy had similar effects to those of bromo-a-ergocryptine. In contrast, there was a 50% increase in enzyme activity in the mammary gland of diabetic, lactating rats and a 10-fold increase in liver as compared with normal rats. P-enolpyruvate carboxykinase activity in mammary gland as liver is coordinately regulated by prolactin, glucocorticoids and insulin.     

Segments of chromosomal DNA from Rhynchosciara americana that undergo additional rounds of DNA replication in the salivary gland DNA puffs have only weak ARS activity in yeast

We have constructed a library of recombinant phage containing DNA from salivary gland chromosomes of Rhynchosciara americana. We have isolated phage from this library that carry sequences homologous to cDNA clones that hybridize in situ to the DNA puffs at the polytene chromosome regions C3 and C8. This has enabled us to demonstrate a 16-fold amplification of the genomic DNA sequences at these regions during DNA-puffing. At the C8 site there is a sequence element that has characteristics of 'scrambled' moderately repetitive DNA. This is located within 3 kb from the gene encoding a 1.95-kb mRNA. We have assayed restriction fragments from the two DNA puffs for Ars activity in yeast. The only strong Ars activity is associated with a part of the moderately repetitive DNA element from the C8 puff which is not present at this site in all animals.     

Immunoelectrophoretic approach to the metabolic regulation of glutamine synthetase in Rhodopseudomonas capsulata E1F1: role of glutamine

Anti-glutamine synthetase serum was raised in rabbits by injecting purified glutamine synthetase (GS) of the phototrophic bacterium Rhodopseudomonas capsulata E1F1. The antibodies were purified to monospecificity by immunoaffinity chromatography in GS-sepharose gel. These anti-GS antibodies were used to measure the antigen levels in crude extracts from bacteria, grown phototrophically with dinitrogen, nitrate, nitrite, ammonia, glutamate, glutamine or alanine as nitrogen sources. The amount of GS detected by rocket immunoelectrophoresis was proportional to Mn²⁺-dependent transferase activity measured in the crude extracts. Addition of GS inhibitor l-methionine-d,l-sulfoximine (MSX) to the actively growing cells promoted increased antigen levels, that were not found in the presence of glutamine or chloramphenicol. The ammonia-induced decrease in GS relative levels was reverted by MSX. GS levels remained constant when phototrophically growing cells were kept in the dark.Abbreviations GS glutamine synthetase - MOPS 2-(N-morpholine) propane sulfonate - MSX l-methionine-d,l-sulfoximine     

Action ofN-methyl-N′-nitro-N-nitrosoguanidine inRhizobium trifolii

Rhizobium trifolii was highly resistant to the lethal effect ofN-methyl-N-nitro-N-nitrosoguanidine (MNNG), but it was sensitive to the mutagenic action of this chemical. A concentration of 500g/ml yields a survival of between 1% and 10%, which allows us to obtain a higher number of mutants than lower concentrations that yield higher survival rates. Lethal damage produced by nitrosoguanidine was repaired, and repair is inhibited by acriflavine.     

Reactions of sugar nitro-alkenes with acetoacetic esters

3,4,5,6,7-Penta-O-acetyl-1,2-dideoxy-1-nitro-d-gluco-hept-1-enitol reacts with methyl acetoacetate in an unusual Michael reaction, giving the normal adduct (6), and a bicyclic derivative (9) that arises from quasi-dimerization of the former when a high concentration of the base is used. Acetylation of compound 9 gives the hydroxylamine O-acetate (10).From the reactions of 3,4,5,6,7-penta-O-acetyl-1,2-dideoxy-1-nitro-d-galacto-hept-1-enitol with ethyl and tert-butyl acetoacetate, the normal adducts (7 and 8) were isolated. The structures of compounds 6 to 10 were established on the basis of their spectral properties (u.v., i.r., mass, and ¹H- and ¹³C-n.m.r.)