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).     

Markedly altered membrane transport and intracellular binding of vincristine in multidrug-resistant Chinese hamster cells selected for resistance to vinca alkaloids

Studies of a multidrug-resistant variant (DC-3F/VCRd-5L) of Chinese hamster lung cells selected for resistance to vinca alkaloids revealed marked alterations in transport and intracellular binding of [3H]vincristine compared to parental DC-3F cells. Influx of [3H]vincristine in DC-3F cells appears to be an equilibrating, but mediated, process. Although saturation kinetics for [3H]vincristine influx were not demonstrated, an extremely high temperature-dependence (Q10 27-37 degrees C = 5-6) and trans-inhibition of influx following preloading of cells with nonradioactive vincristine argue in favor of a carrier-mediated process. Efflux of [3H]vincristine from parental cells conformed to first-order kinetics (t1/2 37 degrees = 3.6 +/- 0.4) and exhibited a lower temperature-dependence (Q10 27-37 degrees C = 3-3.5) than influx. In variant vs. parental cells, influx of [3H]vincristine was reduced 24-fold and efflux was increased two-fold, accounting for the large (approximately 48-fold) reduction in steady-state level of exchangeable drug accumulating in variant cells. Otherwise, transport of [3H]vincristine in these cells showed characteristics similar to parental DC-3F cells. Also, the rate and amount of intracellular binding of [3H]vincristine in variant cells was almost 40-fold lower than in parental cells. These alterations in influx and efflux of [3H]vincristine and its intracellular binding appear to account, at least to a major extent, for the high level of resistance (2,750-fold) of this variant to vinca alkaloids. In contrast, cross-resistance of this variant to daunomycin (178-fold) could be explained only minimally by a transport alteration. Only a two-fold increase in efflux of [3H]daunomycin was demonstrated in variant vs. parental cells along with some decrease in intracellular binding. Influx of [3H]daunomycin was unaltered. In view of these results, we conclude that these two agents most likely do not share the same route for entry in these cells but might share the same efflux route.     

Glycogen and trehalose accumulation during colony development in Streptomyces antibioticus

Streptomyces antibioticus accumulated glycogen and trehalose in a characteristic way during growth on solid medium. Glycogen storage in the substrate mycelium took place during development of the aerial mycelium. The concentration of nitrogen source in the culture medium influenced the time at which accumulation started as well as the maximum levels of polysaccharide stored. Degradation of these glycogen reserves was observed near the beginning of sporulation. The onset of sporogenesis was always accompanied by a new accumulation of glycogen in sporulating hyphae. During spore maturation the accumulated polysaccharide was degraded. No glycogen was observed in aerial non-sporulating hyphae or in mature spores. Trehalose was detected during all phases of colony development. A preferential accumulation was found in aerial hyphae and spores, where it reached levels up to 12% of the cell dry weight. The possible roles of both carbohydrates in the developmental cycle of Streptomyces are discussed.     

Properties of free and bound Citrobacter freundii lipopolysaccharides

Culture medium content of free lipopolysaccharide (LPS) components spontaneously released from a Citrobacter freundii culture grown in minimum synthetic medium was determined during early (8-hr culture) and late (24-hr culture) phases of growth. As judged by Limulus-lysate test, free LPS occurred in the medium as early as after 8 hrs of incubation, i.e. at the beginning of log growth phase. As the culture continued to grow the LPS amount released into culture medium kept rising, reaching 30% of endotoxin present in 24-hr Citrobacter culture. The released LPS complex was isolated by separation and its physicochemical, immunochemical and biological properties were determined and compared with those of cell-bound endotoxin recovered from cells by phenol extraction. Comparisons revealed distinct differences in the chemical composition and the degree of heterogeneity; free LPS was less heterogeneous. Immunologically, free LPS differed from bound LPS in the structure of macromolecules, but was identical with it in some antigenic determinants. The biological activity of free LPS preparation was greater than that of cell-bound LPS.     

Arbovirological survey in Silica plateau area, Roznava District, Czechoslovakia

The serosurveys conducted in the Silica plateau area of the Slovak karst region revealed the presence of specific neutralizing antibody against tick-borne encephalitis (TBE) virus in 18% of local inhabitants (33 examined, mostly goats and sheep farmers), 54% of goats (26 examined), 18% of sheep (120 examined) and 13% of cattle (60 examined), against Lipovník (LIP) virus in 30% of inhabitants, 88% of goats, 55% of sheep and 45% of cattle, and against Bhanja (BHA) virus in 27% of inhabitants, 46% of goats, 29% of sheep and 23% of cattle. The results of hemagglutination-inhibition tests with TBE and BHA antigens were analogous. A detailed analysis of these serologic data points to a recent enhancement of the circulation of LIP and BHA viruses and to a very low TBE virus activity in this natural focus of arboviral infections. The immunological surveys of the 32 former "Roznava disease" patients, conducted 25 years after an extensive epidemic of a TBE virus infection that originated in Roznava in 1951, revealed the presence of neutralizing (and also hemagglutination-inhibiting) antibodies against TBE virus in as many as 78% of cases. Antibodies against LIP and BHA viruses were also detectable in the sera of 16% and 9%, respectively, of these individuals. Populations of the ectoparasites examined for the presence of arbovirus comprised 231 Ixodes ricinus, 806 Dermacentor marginatus and 204 Haemaphysalis punctata ticks and 117 specimens of the louse-flies Melophagus ovinus. Two strains of arbivirus that were antigenically related to Lipovník and Tribec viruses belonging to a group of Kemerovo viruses were isolated from male and female I. ricinus ticks collected from cattle.     

Crystallization and preliminary X-ray diffraction study of an endoglucanase from Clostridium thermocellum

Endoglucanase D, a cellulose degradation enzyme from Clostridium thermocellum has been cloned in Escherichia coli, purified and crystallized. The crystals are trigonal, space group P3(1)12 (or P3(2)12) with a = 57.7 (+/- 0.1) A, c = 192.1 (+/- 0.2) A, and diffract X-rays to a resolution of 2.8 A. They are suitable for a high-resolution X-ray diffraction analysis.     

Crystal structure determination, refinement and the molecular model of the alpha-amylase inhibitor Hoe-467A

The crystal and molecular structure of the alpha-amylase inhibitor Hoe-467A has been determined and refined at high resolution. The polypeptide chain is folded in two triple-stranded sheets, which form a barrel. The topology of folding is as found in the immunoglobulin domains. The amino acid triplet Trp18-Arg19-Tyr20 has an exceptional conformation and position in the molecule and is possibly involved in inhibitory activity.     

Contact areas of the turnip yellow mosaic virus tRNA-like structure interacting with yeast valyl-tRNA synthetase

The tRNA-like structure of turnip yellow mosaic virus is known to be efficiently recognized and aminoacylated by valyl-tRNA synthetase. The present work reports domains in the isolated tRNA-like fragment (159 terminal nucleotides at the 3'-end of the two viral RNAs) in contact with purified yeast valyl-tRNA synthetase. These domains were determined in protection experiments using chemical and enzymatic structural probes. In addition, new data, re-enforcing the validity of the tertiary folding model for the native RNA, are given. In particular, at the level of the amino acid accepting arm it was found that the two phosphate groups flanking the three guanine residues of loop I are inaccessible to ethylnitrosourea. This is in agreement with a higher-order structure of this loop involving "pseudo knotting", as proposed by Rietveld et al. (1982). Valyl-tRNA synthetase efficiently protects the viral RNA against digestion by single-strand-specific S1 nuclease at the level of the anticodon loop. With cobra venom ribonuclease, specific for double-stranded regions of RNA, protection was detected on both sides of the anticodon arm and at the 5'-ends of loop I, a region that is involved in the building up of the acceptor arm. Loop II, which is topologically homologous to the T-loop of canonical tRNA was likewise protected. Weak protection was observed between arms I and II, and at the 3'-side of arm V. This arm, located at the 5'-side of arm IV (homologous to the D-arm of tRNA), does not participate in the pseudo-knotted model of the valine acceptor arm. Ethylnitrosourea was used to determine the phosphates of the tRNA-like structure in close contact with the synthetase. These are grouped in several stretches scattered over the RNA molecule. In agreement with the nuclease digestion results, protected phosphates are located in arms I, II, and III. Additionally, this chemical probe permits detection of other protected phosphates on the 3'-side of arm IV and on both sides of arm V. When displayed in the three-dimensional model of the tRNA-like structure, protected areas are localized on both limbs of the L-shaped RNA. It appears that valyl-tRNA synthetase embraces the entire tRNA-like structure. This is reminiscent of the interaction model of canonical yeast tRNAVal with its cognate synthetase.     

Mini-F E protein: the carboxy-terminal end is essential for E gene repression and mini-F copy number control

Mini-F is a segment of the conjugative plasmid F consisting of two origins of replication flanked by regulatory regions, which ensure a normal control of replication and partitioning. Adjacent to the ori-2 origin is a complex coding region that consists of the E gene overlapped by three open reading frames with the coding potential for 9000 Mr polypeptides here designated 9 kd-1, 9 kd-2 and 9 kd-3. In this paper, we show that open reading frame 9 kd-3 is preceded by active promoter and Shine-Dalgarno sequences. The E coding region specifies: an initiator of replication, which acts at the ori-2 site; a function that negatively regulates the expression of the E gene; and a function involved in mini-F copy number control. To assign one of these functions to one of the overlapping coding sequence, we have isolated, characterized and sequenced mutations mapping in the E coding region. In this paper, we analyse two mutations (cop5 and pla25) that abolish the repression of the E gene. As these mutations affect the primary structure of protein E itself but not the 9 kd polypeptides, we conclude that protein E takes part in the negative regulation of its own synthesis. In addition, the localization of the cop5 and pla25 mutations indicates that the carboxy-terminal end of the E protein is involved in the autorepression function. The cop5 mutation causes an eightfold increase of the mini-F copy number. The pla25 mutation leads to the inability of the derived mini-F plasmid to give rise to plasmid-harbouring bacteria. The ways in which the cop5 and pla25 mutations may lead to such phenotypes are discussed in relation to the different functions mapping in the E coding sequence.     

What is the correct value for Na, K-ATPase activity in homogenates of cerebral cortex?

Activity of Na, K-ATPase in homogenates of fresh cerebral cortex of rats was compared with that of cortex frozen under different conditions. Activity yields after rapid in situ freezing of the exposed cerebral cortex were twice, higher (26.1 U) than in homogenates of the fresh cortex (13.3 U). Fresh brain kept on ice for 60 and 300 s and subsequently frozen in liquid nitrogen yielded activities comparable to those of the tissue frozen in situ (24.1 U and 24.9 U for 60 s and 300 s periods, respectively). Inhibition of Na, K-ATPase by 10(-7) M vanadate was significantly stronger (38%) in homogenates of the fresh brain then in those of the cortex frozen in situ (28%). High Na, K-ATPase activity (47.6 U) in suspensions of synaptosomal membranes (SM) prepared from fresh cortical homogenates was only slightly inhibited by 10(-7) M vanadate (12%). Various treatments of homogenates or SM suspensions, like increase of piston rotation speed, repeated freezing and thawing procedure or vigorous shaking did not significantly affect the enzyme activity. Mg-ATPase activity and its sensitivity to vanadate was also modified by tissue treatment but the effect was much less pronounced.