Interfering mechanism of sodium bicarbonate on spore germination of Bacillus stearothermophilus

Spore germination of Bacillus stearothermophilus was progressively inhibited as the concentrations of sodium bicarbonate (NaHCO₃) in the germination media increased from 0% to 1·0% (w/v). The inhibitory effect of NaHCO₃ was attributed to the release of HCO⁻₃ and its alkaline properties, each of which played a different role. At low concentrations (< 0·3%), the inhibitory effect of NaHCO₃ was mainly due to bicarbonate. As NaHCO₃ increased from 0·3% to higher concentrations, the effect of HCO⁻₃ reached a plateau while the alkalinating effect became the more dominant inhibitory factor. Fourier transform infrared (FTIR) analysis reveals that sodium bicarbonate reacted with the carboxyl group (1570 cm⁻¹) of some acidic amino-acid residues of protein in the spore, leading to a less orientated structure. A shift of two units towards the longer frequency for carboxyl groups indicates that a stronger interaction was formed between the carboxyl group and the Na⁺ ion. The largest ratio of peak height between the absorbance of carboxylate (1570 cm⁻¹) and of amide II (1546 cm⁻¹) of spores after pretreatment with 0·3% sodium bicarbonate reflects the biggest structural alterations of keratin-like proteins in the spore. The role of NaHCO₃ in enhancing the sporicidal effect of glutaraldehyde is discussed.     

Sprint training attenuates myocyte hypertrophy and improves Ca2+ homeostasis in postinfarction myocytes

Zhang, Xue-Qian, Yuk-Chow Ng, Timothy I. Musch, Russell L. Moore, R. Zelis, and Joseph Y. Cheung. Sprint training attenuates myocyte hypertrophy and improvesCa²⁺ homeostasis in postinfarctionmyocytes. J. Appl. Physiol. 84(2): 544-552, 1998.Myocytes isolated from rat hearts 3 wk aftermyocardial infarction (MI) had decreasedNa⁺/Ca²⁺exchange currents(I_Na/Ca; 3 Na⁺ out:1Ca²⁺ in) and sarcoplasmicreticulum (SR)-releasable Ca²⁺contents. These defects in Ca²⁺regulation may contribute to abnormal contractility in MI myocytes. Because exercise training elicits positive adaptations in cardiac contractile function and myocardialCa²⁺ regulation, thepresent study examined whether 6-8 wk ofhigh-intensity sprint training (HIST) would ameliorate some of thecellular maladaptations observed in post-MI rats with limited exerciseactivity (Sed). In MI rats, HIST did not affect citrate synthaseactivities of plantaris muscles but significantly increased thepercentage of cardiac -myosin heavy chain (MHC) isoforms (57.2 ± 1.9 vs. 49.3 ± 3.5 in MI-HIST vs. MI-Sed, respectively;P  0.05). At the single myocytelevel, HIST attenuated cellular hypertrophy observed post-MI, asevidenced by reductions in cell lengths (112 ± 4 vs. 130 ± 5 µm in MI-HIST vs. MI-Sed, respectively;P  0.005) and cell capacitances (212 ± 8 vs. 242 ± 9 pF in MI-HIST vs. MI-Sed, respectively; P  0.015). ReverseI_Na/Ca wassignificantly lower (P  0.0001) inmyocytes from MI-Sed rats compared with those from rats that were shamoperated and sedentary. HIST significantly increased reverseI_Na/Ca(P  0.05) without affecting theamount ofNa⁺/Ca²⁺exchangers (detected by immunoblotting) in MI myocytes. SR-releasable Ca²⁺ content, as estimated byintegrating forwardI_Na/Ca duringcaffeine-induced SR Ca²⁺ release,was also significantly increased (P  0.02) by HIST in MI myocytes. We conclude that the enhanced cardiacoutput and stroke volume in post-MI rats subjected to HIST aremediated, at least in part, by reversal of cellular maladaptationspost-MI.

     

The glucose transport system of the hyperthermophilic anaerobic bacterium Thermotoga neapolitana

The glucose transport system of the extremely thermophilic anaerobic bacterium Thermotoga neapolitana was studied with the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DOG). T. neapolitana accumulated 2-DOG against a concentration gradient in an intracellular free sugar pool that was exchangeable with external source of energy, such as pyruvate, and was inhibited by arsenate and gramicidin D. There was no phosphoenolpyruvate-dependent phosphorylation of glucose, 2-DOG, or fructose by cell extracts or toluene-treated cells, indicating the absence of a phosphoenolpyruvate:sugar phosphotransferase system. These data indicate that D-glucose is taken up by T. neapolitana via an active transport system that is energized by an ion gradient generated by ATP, derived from substrate-level phosphorylation.     

Ustilago maydis Mating Hyphae Orient Their Growth toward Pheromone Sources

Snetselaar, K. M., Bolker, M., and Kahmann, R. 1996. Ustilago maydis mating hyphae orient their growth toward pheromone sources. Fungal Genetics and Biology 20, 299-312. When small drops of Ustilago maydis sporidia were placed 100-200 μm apart on agar surfaces and covered with paraffin oil, sporidia from one drop formed thin hyphae that grew in a zig-zag fashion toward the other drop if it contained sporidia making the appropriate pheromone. For example, a2b2 mating hyphae grew toward a1b1 and a1b2 mating hyphae, and the filaments eventually fused tip to tip. Time-lapse photography indicated that the mating hyphae can rapidly change orientation in response to nearby compatible sporidia. When exposed to pheromone produced by cells in an adjacent drop, haploid sporidia with the a2 allele began elongating before sporidia with the a1 allele. Sporidia without functional pheromone genes responded to pheromone although they did not induce a response, and sporidia without pheromone receptors induced formation of mating hyphae although they did not form mating hyphae. Diploid sporidia heterozygous at b but not at a formed straight, rigid, aerial filaments when exposed to pheromone produced by the appropriate haploid sporidia. Again, the a2a2b1b2 strain formed filaments more quickly than the a1a1b1b2 strain. Taken together, these results suggest that the a2 pheromone diffuses less readily or is degraded more quickly than the a1 pheromone.     

Characterization of lipoprotein particles isolated by immunoaffinity chromatography. Particles containing A-I and A-II and particles containing A-I but no A-II

Two populations of A-I-containing lipoprotein particles: A-I-containing lipoprotein with A-II (Lp (A-I with A-II], and A-I-containing lipoprotein without A-II (Lp (A-I without A-II] have been isolated from plasma of 10 normolipidemic subjects by immunoaffinity chromatography and characterized. Both types of particles possess alpha-electrophoretic mobility and hydrated density in the range of plasma high-density lipoproteins (HDL). Lp (A-I without A-II) and Lp (A-I with A-II) are heterogeneous in size. Lp (A-I without A-II) comprised two distinct particle sizes with mean apparent molecular weight and Stokes diameter of 3.01 X 10(5), and 10.8 nm for Lp (A-I without A-II)1, and 1.64 X 10(5), and 8.5 nm for Lp (A-I without A-II)2. Lp (A-I with A-II) usually contained particles of at least three distinct molecular sizes with mean apparent molecular weight and Stokes diameter of 2.28 X 10(5) and 9.6 nm for Lp (A-I with A-II)1, 1.80 X 10(5) and 8.9 nm for Lp (A-I with A-II)2, and 1.25 X 10(5) and 8.0 nm for Lp (A-I with A-II)3. Apoproteins C, D, and E, and lecithin:cholesterol acyltransferase (LCAT) were detected in both Lp (A-I without A-II) and Lp (A-I with A-II) with most of the apoprotein D, and E, and LCAT (EC 2.3.1.43) in Lp (A-I with A-II) particles. Lp (A-I without A-II) had a slightly higher lipid/protein ratio than Lp (A-I with A-II). Lp (A-I with A-II) had an A-I/A-II molar ratio of approximately 2:1. The percentage of plasma A-I associated with Lp (A-I without A-II) was highly correlated with the A-I/A-II ratio of plasma (r = 0.96, n = 10). The variation in A-I/A-II ratio of HDL density subfractions therefore reflects different proportions of two discrete types of particles: particles containing A-I and A-II in a nearly constant ratio and particles containing A-II but no A-II. Each type of particle is heterogeneous in size and in apoprotein composition.     

4-Azasteroidal 5 alpha-reductase inhibitors without affinity for the androgen receptor

In efforts to develop potent 5 alpha-reductase inhibitors without affinity for the androgen receptor, synthetic 3-oxo-5 alpha-steroids were tested for their ability to inhibit 5 alpha-reductase, using [14C]testosterone as the substrate, and for their ability to inhibit the binding of [3H]5 alpha-dihydrotestosterone to the androgen receptor of rat prostate cytosol. 2',3' alpha-Tetrahydrofuran-2'-spiro-17-(5 alpha-androstan-3-one) is not an inhibitor of 5 alpha-reductase and has a high affinity for the androgen receptor; substitution of the -CH2- at the 4-position with N-H resulted in a good inhibitor of 5 alpha-reductase. The 4-N-CH3 derivative is even more active, whereas the N-CH2-CH3 derivative is inactive. These 4-aza derivatives have much lower affinity for the androgen receptor than the parent compound. The 4-N-H derivatives of several 3-oxo-5 alpha-steroids were found to be 20-100% as potent as their corresponding 4-N-CH3 analogs as inhibitors of 5 alpha-reductase, whereas their androgen receptor affinities were at least 40-fold lower than their 4-N-CH3 analogs. Their 5 beta-isomers did not inhibit either 5 alpha-reductase or the androgen receptor binding of [3H]5 alpha-dihydrotestosterone. Two of these 4-N-H steroids, 17 beta-N,N-diethylcarbamoyl-4-aza-5 alpha-androstan-3-one and 17 beta-N, N-diisopropylcarbamoyl-4-aza-5 alpha-androstan-3-one, are potent 5 alpha-reductase inhibitors with Ki values equal to 29.2 +/- 1.7 and 12.6 +/- 0.8 nM, respectively, but have little affinity for the androgen receptor. The inhibition of 5 alpha-reductase by both compounds is competitive with testosterone. When [3H]testosterone was incubated with minced rat prostate in the presence of either of these two 4-azasteroids, the nuclear concentration of 5 alpha-dihydrotestosterone decreased and that of testosterone increased. The total nuclear uptake of testosterone plus 5 alpha-dihydrotestosterone was not significantly affected. These 4-azasteroids should be useful for investigating the importance of 5 alpha-reductase in androgen action in vivo.     

Role of magnesium binding to myosin in controlling the state of cross-bridges in skeletal rabbit muscle

The effect of Mg2+ on the disposition of myosin cross-bridges was studied on myofibrils and synthetic myosin and rod filaments by employing chymotryptic digestion and chemical cross-linking methods. In the presence of low Mg2+ concentrations (0.1 mM), the proteolytic susceptibility at the heavy meromyosin/light meromyosin (HMM/LMM) junction in these three systems sharply increases over the pH range from 7.0 to 8.2. Such a change has been previously associated with the release of myosin cross-bridges from the filament surface [Ueno, H., & Harrington, W.F. (1981) J. Mol. Biol. 149, 619-640]. Millimolar concentrations of Mg2+ block or reverse this charge-dependent transition. Rod filaments show the same behavior as myosin filaments, indicating that the low-affinity binding sites for Mg2+ are located on the rod portion of myosin. The interpretation of these results in terms of Mg2+-mediated binding of cross-bridges to the filament backbone is supported by cross-linking experiments. The normalized rate of S-2 cross-linking in rod filaments at pH 8.0, kS-2/kLMM, increases upon addition of Mg2+ from 0.30 to 0.65 and approaches the cross-linking rate measured at pH 7.0 (0.75), when the cross-bridges are close to the filament surface. In rod filaments prepared from oxidized rod particles, chymotryptic digestion proceeds both at the S-2/LMM junction and at a new cleavage site located in the N-terminal portion of the molecule. Kinetic analysis of digestion rates at these two sites reveals that binding of Mg2+ to oxidized myosin rods has a similar effect at both sites over the pH range from 7.0 to 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bilirubin inhibits the activation of superoxide-producing NADPH oxidase in a neutrophil cell-free system.

We studied the effect of bilirubin on the NADPH-dependent superoxide production induced by sodium dodecyl sulfate in a cell-free system consisting of the membrane and cytosolic fractions of pig neutrophils. Preincubation of the cytosolic fraction with bilirubin before the addition of sodium dodecyl sulfate resulted in the time- and dose-dependent inhibition of the superoxide production while the preincubation of the membrane fraction with the tetrapyrrole did not result in the inhibition. When the pigment was added after the initiation of the reaction, the ongoing production was not affected by the addition. Other tetrapyrroles, such as hemin, protoporphyrin and biliverdin, also inhibited the production. The results indicate that bilirubin inhibits the activation process of the superoxide producing NADPH oxidase by decreasing the potency of the cytosolic fraction and its inhibitory effect seems to be due to the hydrophobic nature of the tetrapyrrole.     

Affinity labeling of the membrane protein-binding component of human polymorphonuclear leukocyte receptors for leukotriene B4.

A radiolabeled N-(3-aminopropyl)-leukotriene B4 amide ([3H]LTB4-APA) analog of the potent leukocyte chemotactic factor leukotriene B4 (LTB4) binds to receptors for LTB4 in plasma membrane-enriched preparations from human blood polymorphonuclear leukocytes (PMNL) and intact PMNL with respective mean dissociation constants of 2.3 nM and 69 nM at 4 degrees C. The [3H]LTB4-APA bound to plasma membrane-enriched preparations from PMNL was covalently cross-linked to membrane proteins with disuccinimidyl suberate. Solubilization and resolution by SDS-PAGE of proteins from [3H]LTB4-APA-labeled PMNL membranes revealed predominant labeling of a 60-kDa protein. Labeling of the PMNL membrane protein was inhibited by LTB4 and its analogs at concentrations similar to those inhibiting the binding of [3H]LTB4 to its receptor, with an identical rank order of potency of LTB4 greater than 20-hydroxy-LTB4 greater than LTB4-APA = 5(S),12(R)-dihydroxy-eicosa-14-cis-6,8,10-trans-tetraenoic acid much greater than LTD4 = LTC4. GTP suppressed the labeling of the 60-kDa PMNL membrane protein to an extent consistent with the decrease in receptor affinity for LTB4 induced by GTP. The stereospecificity of the affinity cross-linking reaction and the regulation by GTP support the identification of an approximately 60-kDa protein as the binding component of the PMNL receptor for LTB4.     

Long-range physical mapping of the alpha-amylase-1 (alpha-Amy-1) loci on homoeologous group 6 chromosomes of wheat

Summary Long-range physical maps of the small multigene family of the malt -amylase genes (-Amy-1) located on the long arms of wheat chromosomes 6A (the -Amy-A1 locus) and 6B (-Amy-B1) were generated by pulsed-field gel electrophoresis analysis. By using three methylation-sensitive rare-cutter restriction endonucleases, NotI, NruI and MluI, and an -Amy-1 cDNA probe and four gene-specific genomic probes from the -Amy-B1 locus, the size of the -Amy-B1 locus was estimated to be about 700 kb and of the -Amy-B1 locus to be about approximately 4300 kb. These two maps indicate clustering of GC-rich and C-methylation-sensitive restriction enzyme recognition sites. At least five regions reminiscent of CpG islands are apparent in -Amy-B1, and three in -Amy-A1. Correlation between recombination frequency and physical distance within the -Amy-B1 locus suggests that 1 cM approximates to 1 Mb in physical distance.