Production of a thermostable alkali-tolerant xylanase from Bacillus circulans AB 16 grown on wheat straw

Bacillus circulans AB 16 was able to produce 50 IU/ml of xylanase, with negligible cellulase activity when grown on untreated wheat straw. The pH optimum of the crude enzyme was 6–7 with a temperature optimum of 80 C. The enzyme showed high pH and thermal stability retaining 100% activity at 60 C, pH 8 and 9 after 2.5 h of incubation. The residual activity at 70 C after 2.5 h was 62% and 45% at pH 8 and 9, respectively. At 75 C only 22.2% activity remained at pH 8 after 1 h incubation. Since Kraft pulp is alkaline this enzyme could be used for prebleaching of pulp at temperatures up to 70 C without pH adjustment.     

The combined effect of high hydrostatic pressure,heat and bacteriocins on inactivation of foodborne pathogens in milk and orange juice

The objective of this study was to combine pressure (345 MPa) with heat (50 C), and bacteriocins (5000 AU/ml sample) for a short time (5 min) for the inactivation of relatively pressure-resistant strains of four foodborne pathogens: Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella in pasteurized milk and orange juice. Without bacteriocin addition, 5.5 log-cycle reduction was obtained for S. aureus 485 in milk whereas more than 8 log-cycle reduction was achieved for all the other strains studied. After storage of samples for 24 h at 4 C, S. aureus 765 also gave positive results on selective media, where no growth was observed for all the other micro-organisms assayed. Incubation of the same pressurized samples at 37 C for 48 h showed growth of L. monocytogenes strains in addition to S. aureus strains, where still no growth was observed for E. coli O157:H7 and Salmonella strains in their respective selective media. For orange juice samples, more than 8 log-cycle reduction was achieved for all the bacterial species studied. No growth was seen for these species on their respective selective media agar plates after storage at 4 C for 24 h and at 37 C for 48 h. When a bacteriocin-based biopreservative (BP₁) was combined with pressurization, more than 8 log-cycle reduction in cell population of the resistant strains of S. aureus and L. monocytogenes were achieved in milk after pressurization. Milk samples were stored at 25 C up to 30 days to test the effect of treatment and samples showed no growth whereas all the controls were positive.     

Hainan Black-crested Gibbon Is Headed For Extinction

Although Hainan black-crested gibbons have been on the list of the most endangered primate species in the world for many years, their environment is still deteriorating, especially on Hainan Island. Our findings indicate that the species is unlikely to survive the next decades unless efficient conservation policies and strategies are put in place immediately. Census data show that populations of the species used to occur across the whole island, but in 2003 only 13 individuals could be found, confined to a small region, the Bawangling Natural Reserve (19 02¹–19 08¹N and 109 02¹–10913¹ E), in the western part of the island, covering only 14–16 km². In other words, ca. 99% of the habitat has vanished in the past 300 years. Such dramatic change has pushed the species to the edge of extinction; only 2 groups and 2 solitary adult males, remained in 2003. Two adult females, 2 juveniles and one infant comprise Group A, in Donger, the core area of the western part of the reserve; and 1 adult male, 2 adult females, 1 juvenile and 1 infant formed another group (B), confined to another core area (Nanchahe) in the northern part of the reserve. The dramatic decline in the gibbon population has occurred due to vegetation reduction, ecological deterioration and extensive human impact. The forest cover was reduced from 95.5% 2000 years ago to just 4% in 1999; and the human population in 2003 was 330% larger than in 1950.     

Enhanced acetyl esterase production by Fusarium oxysporum

Production of acetyl esterase (EC 3.1.1.6) by Fusarium oxysporum strain F3 was enhanced by optimization of growth conditions. Under optimal conditions, activities as high as 0.89U/ml of culture medium were obtained. The culture filtrate was equally active on p-nitrophenyl acetate and acetylxylan. The enzyme produced 71% deacetylation of acetylxylan in 2h at 40C. Activity was optimized at pH6.5 and at 55^C. The respective K_m values for p-nitrophenyl acetate and acetylxylan were 0.25mM and 1.05% (w/v) and the V_m values were 0.65 and 0.43mol acetate/min/mg protein.     

Gibberellin translocation in Pisum sativum L.

The physiological and biochemical consequences of treating Le (tall) and le (dwarf) pea seedlings with varying quantities of the gibberellins [³H]GA₂₀ and GA₁ have been investigated. Although the percentage uptake of these compounds from the site of application on the 3 stipules was low and most of the applied GA remained unmetabolised in situ, the quantitative relationship between GA translocation and GA dosage was found to be linear for GA₁ but saturating for GA₂₀. The movement of the GAs and their subsequently produced metabolites was mainly acropetal. They accumulated in greatest quantity in the apical extremities of the shoot. Overall, the extent to which GA₂₀ was metabolished in le seedlings was considerably less than in Le pea seedlings. Although all le tissues contained significantly less [³H]GA₁ than their Le counterparts, phenotypic effects of the le mutation were apparent only on internode and tendril development. Increased tissue growth, consequent upon GA treatment, was also apparent only in the internodes and tendrils of le plants. For internodes, GA₁ content determined the mid-logarithmic-phase growth rate and, consequently, final length. For tendrils, GA₂₀ rather than GA₁ may be the primary stimulatory agent.Abbreviations GA gibberellin - HPLC high-performance liquid chromatography - 1–6 consecutive developmental numbering system for plant tissues/organs as shown in Fig. 1 The author gratefully acknowledges financial support from Imperial Chemical Industries, Plant Protection, Jealott's Hill, Bracknell, Berks., UK and the Science and Engineering Research Council.     

The diverse Michaelis constants and maximum velocities of lactate dehydrogenase in situ in various types of cell

Summary The kinetics of lactate dehydrogenase in mouse cardiac muscle fibres, skeletal muscle fibres, gastric parietal cells, parotid gland ductal and acinar cells, oocytes and mouse and human hepatocytes were studied as a function of substrate concentration in sections of unfixed mouse and human tissues incubated at 37°C on lactate agarose gel films. The absorbances of the final reaction products deposited in single cells of various types were measured continuously as a function of incubation time using an image analysis system. The initial velocities (v _i) of the dehydrogenase were calculated from two equations deduced previously by us, v _i = a₁A (equation 1) and v _i = v + a ₂A (equation 2), where v and A are, respectively, the gradient (steady-state velocity) and intercept of the linear regression line of absorbance on time for incubation times between 1 and 3 min, and a ₁ and a ₂ are constants characteristic for each cell type.Hanes plots using v _i, calculated from equation 2 gave more consistent estimates of the Michaelis constant (K _m) and the maximum reaction velocity (V _max ) than those employing either steady-state velocity measurements or v _i calculated from equation 1. The K _m thus found for mouse skeletal muscle fibres (10.4–12.5 mM) and hepatocytes (14.3–16.7 mM) agreed well with values determined previously in biochemical assays. However, the K _m for cardiac muscle fibres (13.4 mM) was higher. The K _m of the enzyme in gastric parietal cells, parotid gland cells and oocytes was in the range 7.6–9.7 mM. The V_max were more diverse, ranging from 29 moles hydrogen equivalents/cm³ cytoplasm/min units in mouse parotid gland acinar cells, 59–68 units in skeletal and cardiac muscle fibres, 62–65 units in gastric parietal cells and oocytes, and 102–110 units in hepatocytes. The diversity found for K _m and V _max in different cell types confirms the value of the quantitative histochemical approach in revealing the heterogeneity of cellular metabolism in situ.     

Opposing effects of two osmolytes – trehalose and glycerol – on thermal inactivation of rabbit muscle 6-phosphofructo-1-kinase

Trehalose and glycerol are known as good stabilizers of function and structure of several macromolecules against stress conditions. We previously reported that they have comparable effectiveness on protecting two yeast cytosolic enzymes against thermal inactivation. However, enzyme protection has always been associated to a decrease in catalytic activity at the stabilizing conditions i.e., the presence of the protective molecule. In the present study we tested trehalose and glycerol on thermal protection of the mammalian cytosolic enzyme phosphofructokinase. Here we found that trehalose was able to protect phosphofructokinase against thermal inactivation as well as to promote an activation of its catalytic activity. The enzyme incubated in the presence of 1 M trehalose did not present any significant inactivation within 2 h of incubation at 50 C, contrasting to control experiments where the enzyme was fully inactivated during the same period exhibiting a t_0.5 for thermal inactivation of 56± 5 min. On the other hand, enzyme incubated in the presence of 37.5% (v/v) glycerol was not protected against incubation at 50 C. Indeed, when phosphofructokinase was incubated for 45 min at 50 C in the presence of lower concentrations of glycerol (7.5–25%, v/v), the remaining activity was 2–4 times lower than control. These data show that the compatibility of effects previously shown for trehalose and glycerol with some yeast cytosolic enzymes can not be extended to all globular enzyme system. In the case of phosphofructokinase, we believe that its property of shifting between several different complex oligomers configurations can be influenced by the physicochemical properties of the stabilizing molecules. (Mol Cell Biochem 269: 203)     

Molecular screening and fetal diagnosis of β-thalassemia in the Italian population

Summary This paper reports our experience of molecular screening and fetal diagnosis of -thalassemia in 457 at risk couples of Italian descent. Molecular screening was carried out by dot blot analysis on amplified DNA with oligonucleotide probes complementary to the eight most common mutations in Italians [39 (CT); 6 (-A); ⁺-87 (CG); ⁺ IVSI nt 110 (GA); IVSI nt 1 (GA); ⁺ IVSI nt 6 (TC); IVSII nt 1 (GA); ⁺ IVSII nt 745 (CG)]. By using this approach, we have been able to define the mutation in 92.8% of cases. The rest (all but four) were defined by direct sequencing and this led to the detection of nine rare mutations [76 (-C); ⁺ IVSI nt 5 (GA); ⁺ IVSI nt 5 (GC); ⁺ IVSI -1 (cod 30) (GC); ⁺-87 (CT), -290 bp del.; ⁺-101 (CT)], and to the characterization of a novel mutation consisting of the deletion of the G at the invariant AG of the IVSII splice acceptor site of the -globin gene ( IVSII nt 850-1 bp). In the remaining four cases, the -globin gene showed entirely normal sequences and the -globin gene cluster was intact, as indicated by Southern blot analysis. Fetal diagnosis was carried out by dot blot analysis with the oligonucleotide probes defined in the parents. The procedure is simple and reliable, and the results can be obtained within 1 week of sampling. No misdiagnosis has so far occurred. The results indicate that fetal diagnosis of -thalassemia by DNA analysis may be obtained in practically all cases (even in a population showing marked heterogeneity of -thalassemia) by the combination of dot blot analysis for detecting common mutations, and direct sequencing for defining those that are uncommon.     

The electrical response ofAvena coleoptile cortex to auxins

Solute mobilities of 28 compounds in isolated cuticular membranes (CM) from Capsicum annuum L. fruit, Citrus aurantium L. and Pyrus communis L. leaves were studied using unilateral desorption from the outer surface. First-order rate constants of desorption (k^*), which are directly proportional to the diffusion coefficient in the waxy outer limiting skins of cuticles were measured. When log k^* was plotted vs. molar volumes of test compounds linear graphs were obtained. The y-intercepts of these graphs (k^*) represent the mobility of a hypothetical molecule having zero molar volume and the slopes of the graphs () represent the size selectivity of the barrier and are related to the free volume available for diffusion. Thus, solute mobilities in cuticles are composed of two independent terms which are subtractive. If k^* and are known, k^* can be estimated for any solute from its molar volume (V_x) using the equation log k^*=log k^* –V_x. These parameters were used to analyse the effects of plant species, extraction of cuticular waxes and molecular structure of solutes on solute mobilities in plant cuticles. For aliphatic solutes, k^* was a factor of 10 smaller than for cyclic compounds, while was 0.011 and 0.012, respectively. The k^*-values for CM of the three species were very similar, but was higher for bitter-orange CM (0.012) than for those of pepper fruits and pear leaves (0.009). This has the consequence that differences in solute mobilities (k^*) among cuticles from different plan species increase with increasing molar volumes of solutes. Our data and our analysis provide evidence that constituents of cuticular waxes are mobile, at least in the solid amorphous wax fraction, but mobility decreases rapidly with increasing molar volume. For instance, if amounts to 0.01, mobilities of wax monomers decrease by a factor of 10 for every increase in molar volume of 100 cm³ · mol^–1. Thus, hexadecanoic acid is quite mobile in the amorphous wax fraction of Citrus (k^*=1.5×10^–6·s^–1), but for dotriacontane having twice the molar volume, k^* was only 2.5×10^–9·s^–1, which is almost three orders of magnitude smaller. Wax esters have even higher molar volumes and their mobilities will be even smaller (about 4×10^–12·s^–1 for a C₄₈-ester). Since low chain mobilities are a prerequisite for low mobilities and permeabilities, the selective advantage of high-molecular-weight wax monomers in plant cuticular waxes becomes obvious. Extracting cuticular waxes from pear leaf CM increased solute mobilities by a factor of 182, but it had no effect on size selectivity. We interpret this result as evidence to the effect that cuticular waxes reduce mobility by increasing tortuosity of the diffusion path, rather than by decreasing the mean free path of diffusional jumps and jump frequencies of diffusants.Abbreviations CM cuticular membrane(s) - 2,4-D 2,4-dichloro-phenoxyacetic acid - LAB lactic acid buffer - MX polymer matrix membranes - UDOS unilateral desorption from the outer surface     

A novel method for immobilization of invertase from the thermophilic fungus, Thermomyces lanuginosus

An invertase from the thermophilic fungus, Thermomyces lanuginosus was immobilized on phenyl-Sepharose and its properties were studied. Between the soluble and immobilized forms of the invertase, there were not much difference in their optimum pH, K _M and V _max for sucrose. In contrast, the K _M and V _max for raffinose changed significantly. The optimum temperature for the immobilized invertase was lower by 10 C. The immobilized invertase showed remarkable stability at 50 C and was less sensitive to inhibition by metal ions. There was no leaching of the enzyme for at least a month when stored in the refrigerator. The method is novel and specific for the thermophilic invertase as a mesophilic invertase (from yeast) did not bind to phenyl-Sepharose.     

Mechanisms of glycolytic control during hibernation in the ground squirrel Spermophilus lateralis

Summary The mechanisms of glycolytic rate control during hibernation in the ground squirrel Spermophilus lateralis were investigated in four tissues: heart, liver, kidney, and leg muscle. Overall glycogen phosphorylase activity decreased significantly in liver and kidney to give 50% or 75% of the activity found in the corresponding euthermic organs, respectively. The concentration of fructose-2,6-bisphosphate (F-2,6-P₂) decreased significantly in heart and leg muscle during hibernation to 50% and 80% of euthermic tissue concentrations, respectively, but remained constant in liver and kidney. The overall activity of pyruvate dehydrogenase (PDH) in heart and kidney from hibernators was only 4% of the corresponding euthermic values. Measurements of phosphofructokinase (PFK) and pyruvate kinase (PK) kinetic parameters in euthermic and hibernating animals showed that heart and skeletal muscle had typical rabbit skeletal M-type PFK and M₁-type PK. Liver and kidney PFK were similar to the L-type enzyme from rabbit liver, whereas liver and kidney PK were similar to the M₂ isozyme found primarily in rabbit kidney. The kinetic parameters of PFK and PK from euthermic vs hibernating animals were not statistically different. These data indicate that tissue-specific phosphorylation of glycogen phosphorylase and PDH, as well as changes in the concentration of F-2,6-P₂ may be part of a general mechanism to coordinate glycolytic rate reduction in hibernating S. lateralis.Abbreviations ADP adenosine diphosphate - AMP adenosine monophosphate - ATP adenonine triphoshate - EDTA ethylenediaminetetra-acetic acid - EGTA ethylene glycol tetra-acetic acid - F-6-P fructose 6-phosphate - F-1,6-P₂ fructose 1,6-bisphosphate - F-2,6-P₂ fructose-2,6-bisphosphate - K _a activation coefficient - I₅₀ concentration of inhibitor which reduces control activity by 50% - PDH pyruvate dehydrogenase - PEP phosphoenolpyruvate - PFK 6-phosphofructo-1-kinase - PK pyruvate kinase     

Requirement for nonprotonated drug molecules in the direct lethal action of miconazole against Candida albicans

At 10^–5 M, miconazole (MCZ) can exert a direct physicochemical cell-damaging lethal action against logarithmic phase yeasts of Candida albicans. The imidazole moiety of MCZ has a pKa 6.5. Thus, in media of pH >6.5 most drug molecules are nonprotonated (MCZ). Conversely, at pH < 6.5 the majority are protonated and carry a positive charge (MCZH⁺). Our earlier work suggesting that MCZ is required for direct lethal action was tested further. In support of such a requirement, we established a minimal lethal concentration of MCZ (i.e. 5×10^–6 M) that was relatively independent of pH, MCZ concentration, and MCZMCZH⁺ ratio.     

Regulation of the α-ketoglutarate dehydrogenasecomplex during hibernation in a small mammal,the Richardson's ground squirrel (Urocitellus richardsonii)

The citric acid cycle (CAC) is a central metabolic pathway that links carbohydrate, lipid, and amino acid metabolism in the mitochondria and, hence, is a crucial target for metabolic regulation. The α-ketoglutarate dehydrogenase complex (KGDC) is the rate-limiting step of the CAC, the three enzymes of the complex catalyzing the transformation of α-ketoglutarate to succinyl-CoA with the release of CO₂ and reduction of NAD to NADH. During hibernation, the metabolic rate of small mammals is suppressed, in part due to reduced body temperature but also active controls that suppress aerobic metabolism. The present study examined KGDC regulation during hibernation in skeletal muscle of the Richardson's ground squirrel (Urocitellus richardsonii). The KGDC was partially purified from skeletal muscle of euthermic and hibernating ground squirrels and kinetic properties were evaluated at 5°, 22°, and 37 °C. KGDC from hibernator muscle at all temperatures compared with euthermic controls exhibited a decreased affinity for CoA as well as reduced activation by Ca²⁺ ions at 5 °C from both euthermic and hibernating conditions. Co-immunoprecipitation was employed to isolate the E1, E2 and E3 enzymes of the complex (OGDH, DLST, DLD) to allow immunoblot analysis of post-translational modifications (PTMs) of each enzyme. The results showed elevated phospho-tyrosine content on all three enzymes during hibernation as well as increased ADP-ribosylation and succinylation of hibernator OGDH. Taken together these results show that the KGDC is regulated by posttranslational modifications and temperature effects to reorganize enzyme activity and mitochondrial function to aid suppression of mitochondrial activity during hibernation.     

The mechanism of calcium-dependent activation of respiration of liver mitochondria from hibernating ground squirrels,Citellus undulatus

• 1.1. It is shown that Ca²⁺-dependent activation of respiration of liver mitochondria from hibernating ground squirrels is accompanied by mitochondrial swelling.
• 2.2. The swelling of mitochondria from hibernating ground squirrels, as well as the activation of mitochondrial respiration, is precluded by cyclosporin A, p-bromphenacylbromide and oligomycin. Carboxyatractiloside, on the contrary, under these conditions favors the swelling and the acceleration of respiration.
• 3.3. It was concluded that Ca²⁺-dependent activation of hibernating ground squirrel liver mitochondrial respiration resulted from the appearance of a non-specific permeability pathway and from swelling of mitochondria.

     

Cytosolic phospholipase A2 regulation in the hibernating thirteen-lined ground squirrel

Cytosolic calcium-dependent phospholipase A₂ (cPLA₂) has multiple roles including production of arachidonic acid (a key player in cellular signaling pathways) and membrane remodeling. Additionally, since catabolism of arachidonic acid generates free radicals, the enzyme is also implicated in ischemic injury to mammalian organs. Regulation of cPLA₂ could be important in the suppression and prioritization of cellular pathways in animals that undergo reversible transitions into hypometabolic states. The present study examines the responses and regulation of cPLA₂ in skeletal muscle and liver of hibernating thirteen-lined ground squirrels, Spermophilus tridecemlineatus. cPLA₂ activity decreased significantly by 43% in liver during hibernation, compared with euthermic controls, and K_m values for arachidonoyl thio-PC substrate fell in both organs during hibernation to 61% in liver and 28% in muscle of the corresponding euthermic value. To determine whether these responses were due to a change in the phosphorylation state of the enzyme, Western blotting was employed using antibodies recognizing phospho-Ser⁵⁰⁵ on α-cPLA₂. The amount of phosphorylated α-cPLA₂ in hibernator liver was just 38% of the value in euthermic liver. Furthermore, incubation of liver extracts under conditions that enhanced protein phosphatase action caused a greater reduction in the detectable amount of phospho-Ser⁵⁰⁵ enzyme content in euthermic, versus hibernator, extracts. The data are consistent with a suppression of cPLA₂ function during torpor via enzyme dephosphorylation, an action that may contribute to the well-developed ischemia tolerance and lack of oxidative damage found in hibernating species over cycles of torpor and arousal.     

The initial reaction velocities of lactate dehydrogenase in various cell types

Summary The initial reaction velocities (v _v ) of lactate dehydrogenase in hepatocytes, cardiac muscle fibres, skeletal (gastrocnemius) muscle fibres, gastric parietal cells, ductal epithelial and acinar cells of the parotid gland, and oocytes were determined, by computer-assisted image analysis, in unfixed sections of these tissues incubated at 37°C on substrate-containing agarose gel films. They were found to fit the equations v _i = a₁A (equation 1) and v _i – v = a₂A (equation 2) reported previously for mouse hepatocytes (Nakae & Stoward, 1993a, b), where v and A are, respectively, the gradients (or steady-state velocities) and the intercepts on the absorbance axis of the linear regression lines of the absorbance (A) of the finalreaction product on incubation times between 1 and 3 min, and a ₁ and a ₂ are constants. Both equations 1 and 2 fitted the observed v _i closely for mouse (a ₁ = 2.7, a ₂ = 2.2) and human (a ₁ = 3.0, a ₂ = 1.9) hepatocytes. However, equation 2 fitted the observed v _i better than equation 1 for mouse cardiac muscle fibres (a ₂ = 1.5), skeletal muscle fibres (a ₂ = 1.2), gastric parietal cells (a ₂ = 1.7), acinar (a ₂ = 1.4) and striated ductal (a ₂ = 2.2) epithelial cells of the parotid gland, and oocytes (a ₂ = 1.6). The values of v _i calculated from the two equations agreed with the observed v _i to within about 11%. They ranged from 105 mole hydrogen equivalents/cm³ cell/min units in hepatocytes to 24 units in parotid acinar cells, but for other cell types they were between 46 and 61 units. These are all considerably higher than values reported previously.     

Der Redoxzustand des NAD(P) und der Cytochrome b und c2 in Abhängigkeit vom pO2 bei einigen Athiorhodaceae

Zusammenfassung Der Gehalt an reduzierten Nicotinamid-adenin-dinucleotiden bei Rhodospirillum rubrum, Rhodopseudomonas spheroides und Rps. capsulata wird beim Absenken des pO₂ erst zwischen 0,2 und 0,5 mm Hg deutlich erhöht.Beim schnellen Wechsel (dichte Bakteriensuspension) zur Anaerobiose tritt bei R. rubrum eine deutliche Überreduktion der Nicotinamid-adenin-dinucleotide ein und bei erneuter Belüftung eine Überoxydation.Der Reduktionszustand der Cytochrome b+c₂ und c₂ (Differenzspektren 422 m bzw. 426 m oder 428 m minus 405 m und 550 m minus 545 m bzw. 550 m minus 560 m) erhöht sich etwa zur gleichen Zeit wie die Konzentration von NAD(P)H. Bei erneuter Belüftung tritt immer eine Überoxydation ein, die über 1 min andauern kann.Die änderung des Redoxgleichgewichts von NAD(P)⁺/NAD(P)H und der Cytochrome ist korreliert mit dem Zusammenbruch des Energiestoffwechels (oxydative Phosphorylierung), zeigt aber keine Beziehung zum Beginn der Bakteriochlorophyll-synthese unter semiaeroben Wachstumsbedingungen, die schon bei höherem pO₂ erfolgt.Damit konnte gezeigt werden, daß die Induktion der BChl.-Synthese in Dunkelkulturen durch Änderung des O₂-Partialdruckes in keinem direkten Zusammenhang zur Änderung des Redoxgleichgewichtes von NAD(P)⁺/NAD(P)H und der Cytochrome b und c₂ steht.

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The influence of the pO₂ on the redox balance of NAD(P) and of cytochrome b and c₂ in some Athiorhodaceae

Summary If the oxygen partial pressure in growing cultures of Rhodospirillum rubrum, Rhodopseudomonas spheroides, and Rhodopseudomonas capsulata is slowly lowered the level of reduced nicotinamide-adenine dinucleotide is strongly increased but not before the pO₂ is dropped beneath 0.2–0.5 mm Hg.A very quick change from aerobic to strict anaerobic conditions in a dense suspension of R. rubrum causes a significant overreduction of the nicotinamide-adenine dinucleotides. After switching on the aeration an overoxidation is observable.The reduction of cytochromes b and c₂ obeys the same kinetics as the reduction of NAD(P)⁺. A sudden aeration is followed by an overoxidation, which continues for more than 1 min.The changes in the ratio of the reduced to the oxidised states of the nicotinamide-adenine dinucleotide and the cytochromes are correlated to the breakdown of the metabolism (oxidative phosphorylation). But the synthesis of bacteriochlorophyll is induced by a decrease of oxygen partial pressure down to 50- mm Hg (depended from the organism). It has been shown in these experiments that the induction of the synthesis of bacteriochlorophyll in the dark by changing the pO₂ is not directly correlated to the change in the redox state of NAD(P)⁺/NAD(P)H and of the cytochromes b and c₂.

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Abkürzungen NAD(P) Nicotinamid-adenin-dinucleotid + Nicotinamid-adenin-dinucleotid-phosphat - pO₂ Sauerstoffpartialdruck - BChl. Bacteriochlorphyll     

Production,purification and characterization of pectinase from a Bacillus sp. DT7

A soil isolate, Bacillus sp. DT7 has been found to produce significant amounts of an extracellular pectinase subsequently characterized as pectin lyase (EC 4.2.2.10). By optimizing growth conditions, Bacillus sp. DT7 produced higher amount of pectin lyase (53 units/ml) than that has been reported in the literature. Using gel filtration and ion exchange chromatography, this enzyme was purified and found to have a molecular mass of 106 kDa. The purified enzyme exhibited maximal activity at a temperature of 60 C and pH 8.0. The presence of 100 mM concentrations of CaCl₂ and mercaptoethanol significantly enhanced pectinase activity of the purified enzyme. This pectinase has tremendous applications in textile industry, plant tissue maceration and fruit juice wastewater treatments.     

Slow growth phenotype - A possible approach to improved plasmid maintenance in Saccharomyces cerevisiae

Summary Transformation-induced slow growth phenotype (SGP) in yeast is repressed in the presence of 2m plasmids. A full 2m-sequence-based recombinant plasmid (pJB502) was found to be more stable in a 2m-free- [cir] strain of Saccharomyces cerevisiae than in a cir⁺ strain. This could not be attributed to differences in growth rate calculated from kinetic analysis of plasmid loss, but transformed [cir] isolates, which had lost the recombinant plasmid, exhibited varying degrees of SGP in batch culture. One of these isolates was outcompeted in chemostat culture by the recombinant-plasmid-containing strain, suggesting that improved plasmid maintenance can result from SGP in cir hosts.     

Root and stem xylem embolism,stomatal conductance,and leaf turgor in Acer grandidentatum populations along a soil moisture gradient

The objective of this study was to determine how adjustment in stomatal conductance (g ^s) and turgor loss point (_tlp) between riparian (wet) and neighboring slope (dry) populations of Acer grandidentum Nutt. was associated with the susceptibility of root versus stem xylem to embolism. Over two summers of study (1993–1994), the slope site had substantially lower xylem pressures (_px) and g ^s than the riparian site, particularly during the drought year of 1994. The _tlp was also lower at the slope (-2.9±0.1 MPa; all errors 95% confidence limits) than at riparian sites (-1.9±0.2 MPa); but it did not drop in response to the 1994 drought. Stem xylem did not differ in vulnerability to embolism between sites. Although slope-site stems lost a greater percentage of hydraulic conductance to embolism than riparian stems during the 1994 drought (46±11% versus 27±3%), they still maintained a safety margin of at least 1.7 MPa between midday _px and the critical pressure triggering catastrophic xylem embolism (_pxCT). Root xylem was more susceptible to embolism than stem xylem, and there were significant differences between sites: riparian roots were completely cavitated at -1.75 MPa, compared with -2.75 MPa for slope roots. Vulnerability to embolism was related to pore sizes in intervessel pit membranes and bore no simple relationship to vessel diameter. Safety margins from _pxCT averaged less than 0.6 MPa in roots at both the riparian and slope sites. Minimal safety margins at the slope site during the drought of 1994 may have led to the almost complete closure of stomata (g _s=9±2 versus 79±15 mmol m^-2 s^-1 at riparian site) and made any further osmotic adjustment of _tlp non-adaptive. Embolism in roots was at least partially reversed after fall rains. Although catastrophic embolism in roots may limit the minimum for gas exchange, partial (and reversible) root embolism may be adaptive in limiting water use as soil water is exhausted.