Evidence for kinetic and immunologic heterogeneity of dihydrofolate reductase in L1210 leukemia cells.

Two species of DHFR were identified in wild-type L1210 murine leukemia cells by analysis of the kinetics of the binding of MTX and dissociation of the MTX-enzyme complex at pH 5.0 and pH 7.2. The two forms of DHFR were also distinguished by immunoinhibition of the binding of MTX and the catalytic reduction of FH2 to FH4 using an antiserum raised to the purified high affinity form of DHFR. The Ka for the binding of MTX by the low affinity form of the enzyme is 4.5 x 10(7) M-1, substantially lower than the reported Ka for the binding of this drug by the high affinity enzyme. The low affinity form of the enzyme catalyzed the reduction of FH2 to FH4 at a rate slower than the high affinity form of DHFR.     

The switch from larval to adult globin gene expression in Xenopus laevis is mediated by erythroid cells from distinct compartments

The transition of hemoglobins during metamorphosis of Xenopus laevis involves replacement of the larval erythrocytes by adult ones, suggesting that the developmental control of this event depends upon the growth characteristics of the precursor cells. To identify the erythroid precursor cells and to investigate their developmental fate, we analyzed the distribution of stage-specific globin mRNAs by northern blotting in dorsal and ventral fragments of stage 32 embryos after in vitro culture as well as presumptive erythropoietic tissues of tadpoles during metamorphosis. The histological analysis shows that erythrocytes differentiate only in ventral fragments, suggesting that the ventral blood islands and most likely also the dorsolateral mesoderm are the primary sites of erythropoiesis. We also demonstrate that the first generations of erythrocytes, already express the predominating larval-specific alpha-globin mRNAs. The globin mRNA patterns obtained from presumptive erythropoietic tissues suggest an important role of circulating precursor cells in larval erythropoiesis, whereas the liver appears to be the main site of formation and maturation of the adult erythrocytes. Tentatively we propose that anuran erythropoiesis is dependent upon a self-perpetuating stem-cell line and that the larval and the adult erythrocytes are derived from successive generations of erythroid precursors, whose commitment may be imposed by the erythropoietic sites.     

Synergistic effects in theα 1- andβ 1-adrenergic regulations of intracellular calcium levels in striatal astrocytes

1. Using indo-1 as a calcium fluorescent probe, we have observed the following in striatal astrocytes in primary culture. 2. The stimulation of alpha-adrenoceptors induces a rapid rise in cytosolic calcium resulting from an internal calcium mobilization followed by an external calcium influx (4-min duration). 3. The stimulation of beta 1-adrenoceptors evokes only a slight internal calcium mobilization (90-sec duration). 4. The simultaneous stimulation of beta 1- and alpha 1-adrenoceptors induces a more prolonged calcium influx (10 min). The latter phenomenon could explain the calcium-dependent synergistic effects of alpha 1 and beta stimulation on cAMP production already described in the brain.     

Growth and timing of puberty: reciprocal effects.

Based on the analysis of the pubertal growth spurt and final height in different pathological conditions, this paper provides evidence that variations in age at onset of puberty have a major influence on the subsequent acceleration of the growth rate but a minor impact on final height. A reciprocal effect of the growth rate on the timing of onset of puberty is also suggested by a number of clinical observations.     

Essential roles of core starvation-stress response loci in carbon-starvation-inducible cross-resistance and hydrogen peroxide-inducible adaptive resistance to oxidative challenge in Salmonella typhimurium

The starvation-stress response (SSR) of Salmonella typhimurium encompasses the physiological changes that occur upon starvation for an essential nutrient, e.g. C-source. A subset of SSR genes, known as core SSR genes, are required for the long-term starvation survival of the bacteria. Four core SSR loci have been identified in S. typhimuriumrpoSstiAstiB, and stiC. Here we report that in S. typhimurium C-starvation induced a greater and more sustainable cross-resistance to oxidative challenge (15 mM hydrogen peroxide (H₂O₂) for 40 min) than either N- or P-starvation. Of the four core SSR loci, only rpoS and stiC mutants exhibited a defective C-starvation-inducible cross-resistance to H₂O₂ challenge. Interestingly, (unadapted) log-phase S. typhimurium rpoS and stiA mutants were very sensitive to oxidative challenge. Based on this, we determined if these core SSR loci were important for H₂O₂ resistance developed during a 60 min adaptive exposure to 60 μM H₂O₂ (adapted cells). Both unadapted and adapted rpoS and stiA mutants were hypersensitive to a H₂O₂ challenge. In addition, a stiB mutant exhibited normal adaptive resistance for the first 20 mins of H₂O₂ challenge but then rapidly lost viability, declining to a level of about 1.5% of the wild-type strain. The results of these experiments indicate that: (i) the rpoS and stiC loci are essential for the development of C-starvation-inducible cross-resistance to oxidative challenge, and (ii) the rpoSstiA, and, in a delayed effect, stiB loci are needed for H₂O₂-inducible adaptive resistance to oxidative challenge. Moreover, we found that both stiA and stiB are induced by a 60 μM H₂O₂ exposure, but only stiA was regulated (repressed) by (reduced form) OxyR.     

Effect of lactic starter cultures on the organic acid composition of milk and cheese during ripening—analysis by HPLC

The major function of lactic starter cultures in cheese making is to produce lactic and other organic acids from the carbohydrates present in milk. The activity of six starter cultures consisting of two Lactococcus lactis ssp. lactis , two Lactococcus lactis ssp. lactis biovar. diacetylactis and two Leuconostoc strains, was tested by monitoring the evolution of the organic acid composition of milk by a modified HPLC method. In addition, their performance as cheese starters was also tested. The HPLC method developed proved to be a precise tool to monitor the organic acid content. Thus, it can be used to follow the fermentation ability of starter cultures, providing information about the type of fermentation. The use of any of the six starters assayed is suggested for manufacturing Afuega'l Pitu cheese.