NTR1 encodes a high affinity oligopeptide transporter in Arabidopsis

Heterologous complementation of yeast mutants has enabled the isolation of genes encoding several families of amino acid transporters. Among them, NTR1 codes for a membrane protein with weak histidine transport activity. However at the sequence level, NTR1 is related to rather non-specific oligopeptide transporters from a variety of species including Arabidopsis and to the Arabidopsis nitrate transporter CHL1. A yeast mutant deficient in oligopeptide transport was constructed allowing to show that NTR1 functions as a high affinity, low specificity peptide transporter. In siliques NTR1-expression is restricted to the embryo, implicating a role in the nourishment of the developing seed.     

Influence of nutrient excess on intracellular proteins composition in relation to spiramycin production,in Streptomyces ambofaciens

The protein profile on media favouring or reducing spiramycin biosynthesis in S. ambofaciens, was examined using SDS-polyacrylamide gradient gel electrophoresis over a time course of 144 h. Changes in the pattern of protein synthesis were observed which varied according to the nutrient medium conditions and appeared to suggest the presence of protein subsets specific to the production phase, which were probably involved in the switch to secondary metabolism and the onset of antibiotic synthesis. Excess of ammonium, glycerol or phosphate provoked the persistence of a protein band during the whole culture period, which was detected only during growth phase in the control producer culture. In addition, the nutrient excess caused the suppression of spiramycin production and the absence of two protein bands which appeared only in production phase in the control producer culture.     

S-adenosylhomocysteine metabolism in different cell lines: effect of hypoxia and cell density.

BACKGROUND/AIMS: The methylation potential (MP) is defined as the ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). It was shown recently that hypoxia increases AdoMet/AdoHcy ratio in HepG2 cells (Hermes et al., Exp Cell Res 294: 325-334, 2004). In the present study, we compared AdoMet/AdoHcy ratio and energy metabolism in HepG2, HEK-293, HeLa, MCF-7 and SK-HEP-1 cell lines under normoxia and hypoxia. METHODS: Metabolite concentrations were measured by HPLC. In addition, AdoHcy hydrolase (AdoHcyase) activity was determined photometrically. RESULTS: Under normoxia HepG2 cells show the highest AdoMet/AdoHcy ratio of 53.4 +/- 3.3 followed by MCF-7 and SK-HEP-1 cells with a AdoMet/AdoHcy ratio of 14.4 +/- 1.1 and 21.1 +/- 1.3, respectively. The lowest AdoMet/AdoHcy ratios are exhibited by HeLa and HEK-293 cells (6.6 +/- 0.7 and 7.1 +/- 0.3). Hypoxia does not significantly change the MP in MCF-7 and HeLa cells, but alters the MP in HepG2, HEK-293 and SK-HEP-1 cells. These alterations are dependent on the cell density. Under normoxia HepG2 cells exhibit AdoHcyase activity of 2.5 +/- 0.2 nmol min(-1) mg(-1) protein. All other cell lines show 3-5 times lower enzyme activity. Interestingly, hypoxia affects AdoHcyase activity only in HepG2 cells. CONCLUSIONS: Our data clearly show that the cell lines are characterized by different MP and different behavior under hypoxia. That implies that a lower MP is not necessarily associated with impaired transmethylation activity and cellular function.     

Effect of Low Temperatures on Growth,Structure, and Metabolism of Campylobacter coli SP10

The effect of low temperatures on the survival, structure, and metabolism of Campylobacter coli SP10, a virulent strain, was investigated. C. coli became nonculturable rapidly at 20 and 10°C and slightly later at 4°C. Incubation in a microaerobic atmosphere improved survival, but after day 8, campylobacters were detectable by direct-count procedures only. The increase in the number of coccoid cells was most pronounced at 37°C but also was noticeable at 20 and 10°C. Two forms of coccoid cells were seen electron microscopically, but only one (20 and 10°C) seemed to be a degenerative form. The flagella were shorter at 20 and 10°C, a result which correlates well with the observed slight changes in the 62-kDa protein band. The fatty acid composition of bacterial cells was influenced significantly by low temperatures. An increase in the short-chain and unsaturated acids was noted; above all, a drastic increase in C_19:0 cyc at 20°C with a concomitant decrease in C_18:1 trans9,cis11 was seen. The concentrations of excreted metabolites were analyzed to obtain information on metabolic activity. Depending on the magnitude of the temperature downshift, the production of organic acids decreased, but it was always observable after a temperature-specific lag phase and regardless of ability to be cultured. Under optimal conditions, succinate, lactate, and acetate were the main metabolites, other acids being of less importance. The pattern changed significantly at lower temperatures. Succinate was never detected at 20°C and was only occasionally detected at 10 and 4°C. At the same time, fumarate concentrations, which are normally not detectable at 37°C, were highest at 20°C and reduced at 10 and 4°C. Inactivation of fumarate reductase was considered to be a possible explanation.

Campylobacters are playing an increasingly important role in gastrointestinal disease all over the world, and in some countries they are even more frequent than salmonellae. In industrialized countries, the annual incidence of enteritis caused by campylobacters is estimated to be 1%. In underdeveloped countries it is much higher, affects mainly small children and, in contrast to the situation in developed countries, is caused more often by Campylobacter coli than by C. jejuni (12, 43, 44). The natural reservoir of campylobacters is the intestine of warm-blooded animals; 24 to 82% of examined chicken populations, 58 to 95% of Scandinavian pigs, and 23% of slaughter cattle were campylobacter positive, to name only a few animal species (1, 9, 19, 41). Campylobacters, like other intestinal pathogens, are released into the environment via the feces of infected animals or humans. On average, raw sewage contains log 2 to 4 CFU of campylobacters per 100 ml, but concentrations can rise significantly if abattoirs or chicken farms are connected to the sewer system (18). Treated sewage may contain campylobacters at log 1 to 2 CFU/100 ml, and fresh, undisinfected sewage sludge, which is used as a fertilizer in some countries, may contain campylobacters at up to log 6 CFU/100 ml (2, 17). Sewage discharge into surface water and agricultural runoff can thus lead to the contamination of bathing water areas and drinking water reservoirs and can pose a significant health risk for the population. Not surprisingly, large waterborne outbreaks of campylobacter-induced enteritis have been reported (31, 45, 48).Various surface waters have been examined in the past, and 25 to 95% were shown to be campylobacter positive, with campylobacter concentrations ranging from log 0.04 to log 2 CFU/100 ml. Detection rates were higher during the winter months, suggesting enhanced survival at low temperatures, although, apart from starvation, temperature downshift is one of the greatest stress factors that bacteria experience when they are released into the environment. While psychrophilic and psychrotrophic bacteria have adapted evolutionarily to life at low temperatures, human pathogens, being mesophilic bacteria, can be severely inhibited. In nutrient-rich media, reactions such as intracellular (p)ppGpp (guanosine 5′-triphosphate-3′-diphosphate and guanosine 5′-diphosphate-3′-diphosphate) synthesis or production of cold shock proteins have been shown to be dependent on the temperature shift (22). Changes in the composition of the cell membrane and inhibition of enzyme activities and membrane transport systems are further conceivable reactions of mesophilic bacteria to temperature downshift (29). To study the effect of low temperatures on campylobacters, a virulent C. coli strain was subjected to temperatures normally encountered in Central European aquatic systems. The influence on growth, morphology, membrane fluidity, proteins, and metabolism was examined.     

Variability in allelic DNA methylation in spermatozoa

In certain segments of human DNA, the methylation of deoxycytidine residues has been found to be highly specific and interindividually conserved. Imprinted DNA sequences in diploid primary cells show allele-specific differences in DNA methylation, usually with the active chromosomal regions being unmethylated and the inactive regions being methylated. We show here that DNA from spermatozoa exhibits variations in allelic methylation patterns. Since germ cells are haploid, individual spermatozoa can differ in DNA methylation patterns not only in the maternally or paternally derived allele, but also within each allele.     

Seasonal Changes in the Cambium of Trees. I. Sucrose Content in Thuja occidentalis

Cambium samples of Thuja occidentalis L. were collected at five different times, covering spring reactivation and early and late resting period, and used for sucrose determinations. Fragments of the different cell types - xylem ray, cambial initials, sieve-elements including phloem parenchyma cells, phloem ray - were dissected from freeze-dried radial sections and analyzed individually. Results show large differences in sucrose concentrations in the different cell types of the cambial layer. In addition, each cell type also shows seasonal fluctuations in sucrose content, whose amplitudes and patterns of variation appear specific for the particular cell type.     

THE EFFECT OF MORPHINE ON THE TRANSPORT AND METABOLISM OF INTRACISTERNALLY-INJECTED LEUCINE IN THE RAT

—Intracisternally injected l or d-[¹⁴C]leucine was retained longer in the brains of morphine-treated rats than in saline-injected control animals. This resulted in higher levels of the labelled leucine and of labelled metabolites of the l-isomer in free pools of brain tissue. However, the absolute levels of brain amino acids and the relative distribution of radioactivity among l-leucine metabolites in brain were unaffected by treatment with morphine, indicating that no disturbance of leucine oxidation through the citric acid cycle was produced by the drug. The inhibition of protein synthesis caused by acute administration of morphine was calculated to be greater than previously reported since morphine treatment increased the specific radioactivity of the free pool of leucine in brain following the intracisternal injection of the labelled amino acid. Possible mechanisms responsible for these morphine effects are discussed.     

Basic Fuchsin as A Nuclear Stain

Five distinct nuclear stains and staining procedures which utilize basic fuchsin as the dye have been studied, compared and tested on a Feulgen-weak fungus, Blastomyces dermatitidis, and other fungi.

Aqueous basic fuchsin has been shown to be an excellent, though impermanent, stain with which to study the nuclei of this and other fungi. The conditions under which formaldehyde acts as a mordant for basic fuchsin and produces a permanent nuclear stain have been established.

Comparison of crystal violet and basic fuchsin suggests that the mordanting action of the aldehyde operates through the para-amino groups of the dye. Certain other basic dyes were not mordanted by formaldehyde.

Gentle acid hydrolysis of the tissues has been found to be essential both to the specificity of the dye as a nuclear stain and to the mordanting effect of the aldehyde.

The possible relationship of these observations to the Feulgen reaction is discussed. A protocol for the method developed is presented.