An Irreversible and Kinetically Controlled Process: Thermal Induced Denaturation of <Emphasis Type="SmallCaps">L</Emphasis>-2-Hydroxyisocaproate Dehydrogenase from <Emphasis Type="Italic">Lactobacillus confusus</Emphasis>

The thermal denaturation of Lactobacillus confusus l-2-Hydroxyisocaproate Dehydrogenase (l-HicDH) has been studied by Differential Scanning Calorimetry (DSC). The stability of this enzyme has been investigated at different pH conditions. The results of this study indicate that the thermal denaturation of this enzyme is irreversible and the T _m is dependent on the scan-rate, which suggests that the denaturation process of l-HicDH is kinetically determined. The heat capacity function of l-HicDH shows a single peak with the T _m values between 52.14°C and 55.89°C at pH 7.0 at different scan rates. These results indicate that the whole l-HicDH could unfold as a single cooperative unit, and intersubunit interactions of this homotetrameric enzyme must play a significant role in the stabilization of the whole enzyme. The rate constant of the unfolding is analyzed as a first order kinetic constant with the Arrhenius equation, and the activation energy has been calculated. The variation of the activation energy values obtained with different methods does not support the validity of the one-step irreversible model. The denaturation pathway was described by a three-state model, N → U → F, in which the dissociation of the tetramer takes place as an irreversible step before the irreversible unfolding of the monomers. The calorimetric enthalpy associated with the irreversible dissociation and the calorimetric enthalpy associated with the unfolding of the monomer were obtained from the best fitting procedure. Thermal unfolding of l-HicDH was also studied using Circular Dichroism (CD) spectroscopy. Both methods yielded comparable values.     

Water-soluble titanocene complexes with sulfur-containing aminoacids: synthesis, spectroscopic, electrochemical and Ti(IV)–transferrin interaction studies

Three new water soluble titanocene–aminoacid complexes have been synthesized via the reaction of Cp₂TiCl₂ and two equivalents of aminoacid (L) in methanol, affording [Cp₂TiL₂]Cl₂, L=L-cysteine (2), D-penicillamine (3) and L-methionine (4). These complexes have been characterized by ¹H, IR and UV-Vis spectroscopies, elemental analysis and cyclic voltammetry. Kinetic studies of ligand hydrolysis have been monitored at low pH using UV-Vis and ¹H NMR spectroscopies to assess their stability in aqueous solution. At low pH, aminoacid ligands are lost one order of magnitude faster than cyclopentadienyl. However, at physiological pH, in Tris buffer solution, the complexes decompose rapidly to form an insoluble titanium compound. The affinity of these complexes to apo-transferrin was also investigated to elucidate how the ancillary aminoacid ligands affect the titanium intake by apo-transferrin.Electronic Supplementary Material Supplementary material is available for this article at     

Release of Calcium from Mitochondrial and Nonmitochondrial Intracellular Stores in Mouse Pancreatic Acinar Cells by Hydrogen Peroxide

In the present study we have studied how [Ca²⁺] _i is influenced by H₂O₂ in collagenase-dispersed mouse pancreatic acinar cells and the mechanism underlying this effect by using a digital microspectrofluorimetric system. In the presence of normal extracellular calcium concentration, perfusion of pancreatic acinar cells with 1 mm H₂O₂ caused a slow sustained [Ca²⁺] _i increase, reaching a stable plateau after 10–15 min of perfusion. This increase induced by H₂O₂ was also observed in a nominally calcium-free medium, reflecting the release of calcium from intracellular store(s). Application of 1 mm H₂O₂ to acinar cells, in which nonmitochondrial agonist-releasable calcium pools had been previously depleted by a maximal concentration of CCK-8 (1 nm) or thapsigargin (0.5 μm) was still able to induce calcium release. Similar results were observed when thapsigargin was substituted for the mitochondrial uncoupler FCCP (0.5 μm). By contrast, simultaneous addition of thapsigargin and FCCP clearly abolished the H₂O₂-induced calcium increase. Interestingly, co-incubation of intact pancreatic acinar cells with CCK-8 plus thapsigargin and FCCP in the presence of H₂O₂ did not significantly affect the transient calcium spike induced by the depletion of nonmitochondrial and mitochondrial agonist-releasable calcium pools, but was followed by a sustained increase of [Ca²⁺] _i . In addition, H₂O₂ was able to block calcium efflux evoked by CCK and thapsigargin. Finally, the transient increase in [Ca²⁺] _i induced by H₂O₂ was abolished by an addition of 2 mm dithiothreitol (DTT), a sulfhydryl reducing agent. Our results show that H₂O₂ releases calcium from CCK-8- and thapsigargin-sensitive intracellular stores and from mitochondria. The action of H₂O₂ is likely mediated by oxidation of sulfhydryl groups of calcium-ATPases. Received: 15 May 2000/Revised: 4 October 2000     

Polarity of amber mutations and suppressed amber mutations in the galactose operon of E. coli

Summary Amber mutants in the t gene of the galactose operon have been examined for polarity in the presence and absence of the suppressors su _I and su _yMel .In the absence of suppressors there is a gradient of polarity with the more polar mutations nearer the epimerase gene. This polarity is cis-dominant. Amber t mutants have raised epimerase levels but this effect is recessive. The operon is normally inducible in the presence of amber mutations. A double amber mutant had the polarity of the mutation nearest the epimerase end of the gene.In the presence of suppressors there is practically no gradient of polarity. This is in disagreement with the model proposed by Martin et al. (1966) and Yanofsky and Ito (1966). Modifications of this model to fit the present data are proposed.     

Classification of staphylococci based on chemical and biochemical properties

Summary In the present work the chemical cell wall composition and some other biochemical characteristics were studied in staphylococci with the intention of utilizing the data obtained in their classification.According to the cell wall peptidoglycans and teichoic acids, the 130 strains of staphylococci studied are divided into 10 major groups. This division of staphylococci into groups is in good agreement with their present classification only in some cases. All of the 47Staphylococcus aureus strains contain a cell wall peptidoglycan of thel-Lys-Gly_5–6 type and ribitol teichoic acid. Coagulase-negative staphylococci are more heterogeneous and are divided according to their cell wall composition into 9 major groups. 21 strains of them are classified asS. epidermidis sensu stricto. They form a natural group and are distinguished by the occurrence of thel-Lys-Gly_4–5,l-Ser_0.5–1.8 peptidoglycan type, glycerol teichoic acid and anl-lactate dehydrogenase which is activated by fructose-1,6-diphosphate. 8 strains with peptidoglycan of thel-Lys-Gly_4–5,l-Ser_0.5–1.8 type and ribitol teichoic acid are labeled asS. saprophyticus. The remaining groups have not been given species names and require further extensive comparative study.     

The effect of carbon dioxide on reduction of methylene blue by microorganisms

Summary and Conclusions The experiments ofHes which indicate that CO₂ is essential for the reduction of methylene blue by microorganisms presumably because it is required for the functioning of the cellular hydrogen transport mechanisms, have been repeated and results have been obtained that seemingly differ from those ofHes. We could not demonstrate any effect of CO₂ on the rate of methylene blue reduction by various bacteria in the presence of utilizable compounds such as glucose, sucrose, lactate and pyruvate. UsingE. coli and other bacteria we found, however, that the time required for the reduction of methylene blue was greatly increased by CO₂ removal when no substrates were added. This effect of CO₂ on the endogenous metabolism was observed only when the cells were depleted of endogenous reserves and of CO₂ by aeration or dilution.     

Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate

Summary An L1210 cell line (JT-1), which can grow in medium supplemented with 1nm folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37°C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23±0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [³H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37°C remained as unmetabolized folic acid. Binding was also rapid at 0°C but uptake at the plateau was only one-half the value obtained at 37°C. Half-maximal saturation of the binding component (K _D) occurred at a folate concentration of 0.065nm at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (K _D=2.0nm). 5-Methyltetrahydrofolate was also bound by this component (K _i=13nm at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (K _i=45nm) and methotrexate (K _i=325nm). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500nm caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein. An additional low-affinity, high-capacity transport system for folate that had been proposed previously was not observed under a variety of experimental conditions in either the adapted or parental cells.     

Clotting of citrated plasma by a proteolytic enzyme associated with an antibacterial agent from serum

Two to 4 mg/ml of an antibacterial agent occurring in the serum of humans and animals caused the clotting of citrated rabbit, human, calf, ox, sheep, goat, guinea pig, rat, mouse, horse, chicken, pigeon and swine plasma. Heparinized plasmas of the same species were found resistant to the clotting action of the antibacterial agent. Citrated plasma previously heated at 56 C for 30 min, treated with 640 units/ml tyrosinase for 60 min, or absorbed with 0.2M Ca₃(PO₄)₂ and 0.2M Mg(OH)₂ was also found resistant to the clotting action of the antibacterial agent. The clotting action of the antibacterial agent was not affected by heating at 66 C for 60 min, nor by multiple passage through Seitz filters. The coagulation of citrated plasma proceeded most rapidly with 0.2M Tris(hydroxymethyl)-amino-methane buffer, pH 7 to 9, or distilled water as the antibacterial agent solvent; 0.2M phosphate buffer, pH 6 to 8, reduced the clotting action of the antibacterial agent, while 0.2M citrate-phosphate buffer, pH 4.0, or 0.2M carbonate-bicarbonate buffer, pH 10.0, inhibited entirely the clotting activity of this agent.     

Isolation and characterization of free glycans of the oligomannoside type from the extracellular medium of a plant cell suspension

The oligosaccharides Man₅GlcNAc and Man₃(Xyl)GlcNAc(Fuc)GlcNAc presumed to originate fromN-glycosyl proteins have been purified from an extracellular medium (concentration: 2–5 mg/l of 14 day cultures) of white campion (Silene alba) suspension culture. Their primary structures have been determined by¹H-400-MHz NMR spectroscopy and FAB-MS spectrometry. They are probably the result of an autophagic process including protein catabolism due to sucrose starvation. Additional identification of digalactosylglycerol (galactolipid breakdown) argues for this hypothesis.Abbreviations Fuc l-fucose - Man d-mannose - Xyl d-xylose - GlcNAc N-acetyl-d-glucosamine - Gal d-galactose - Glc d-glucose - FAB-MS fast atom bombardment mass spectrometry - NMR nuclear magnetic resonance     

The properties of l-fucose binding agglutinin associated with the cell wall of Rhizoctonia solani

The adherence of Escherichia coli B cells to cell wall associated-agglutinin of the soil borne plant pathogen Rhizoctonia solani, was inhibited by l-fucose, l-galactose, trypsin, SDS, cycloheximide and Na₂-EDTA. The coiling of the biocontrol agent Trichoderma harzianum around Rhizoctonia hyphae was prevented by SDS, cycloheximide, Na₂-EDTA and methyl--l-fucoside — an inhibitor of Rhizoctonia agglutinin not metabolized by both fungi. The possible role of the agglutinin in Trichoderma-Rhizoctonia interaction is discussed.     

Induction of G- and R-banding in human chromosomes by the demethylating agent S-adenosyl-L-homocysteine.

In this report we describe the procedure of growing human lymphocytes with the demethylating agent S-adenosyl-L-homocysteine (SAH). After this treatment, which is not toxic for cell survival, both R- and G-banding were obtained by new experimental procedures: R-bands have been directly demonstrated with the GC-specific fluorochrome chromomycin A3 without the necessity of any AT-specific counterstaining agent; simultaneous G-banding and active nucleolar organizer regions have been obtained by silver impregnation of chromosomes and subsequent Giemsa staining. These results suggest a possible relationship between local differences in DNA methylation and the determination of the banded chromosome structure.     

Characterization of quisqualate recognition sites in rat brain tissue usingDl-[3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and a filtration assay

The binding of [³H]AMPA (Dl--amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a ligand for the putative quisqualate excitatory amino acid receptor subtype, was evaluated using centrifugation and filtration receptor binding techniques in rat brain crude synaptosomal membrane preparations. Maximal specific binding of [³H]AMPA occurred in Triton X-100 treated membranes in the presence of the chaotropic agent potassium thiocyanate (KSCN). The effects of KSCN on binding were reversible and optimal at 100 mM. Supernatant obtained from detergent-treated membranes inhibited specific [³H]AMPA and [³H]kainic acid binding, suggesting the presence of an inhibitory agent which was tentatively identified as glutamate. Using centrifugation, saturation analysis revealed two distinct binding sites in both the absence and presence of KSCN. The chaotrope was most effective in increasing binding at the low affinity binding site, enhancing the affinity (K _d) without a concommitant change in the total number of binding sites. Using filtration, a single binding site was detected in Triton-treated membranes. Like the data obtained by centrifugation, KSCN enhanced the affinity of the receptor (K _d value=10 nM) without altering the number of binding sites (B _max=1.2 pmol/mg protein). The rank order of potency of various glutamate analogs in the [³H]AMPA binding assay was quisqualate > AMPA > l-glutamate > kainate > d-glutamate, consistent with the labeling of a quisqualate-type excitatory amino acid receptor subtype.l-glutamic acid diethylester, and 2-amino-7-phosphonoheptanoic acid (AP₇) were inactive. The present technique provides a rapid, reliable assay for the evaluation of quisqualate-type excitatory amino acid agonists and/or antagonists that may be used to discover more potent and selective agents.     

Potassium-induced insulin release and voltage noise measurements in single mouse islets of Langerhans

Summary Insulin release and membrane potential fluctuations in response to increased extracellular potassium [K⁺] _o have been measured in single perifused islets of Langerhans from normal mice. An increase in [K⁺] _o from 5mm to 50mm induced a transient insulin release with a peak at about 1 min. The peak value was [K⁺] _o -dependent but the half-timet _1/2 for the decline was constant at nearly 1 min. 2.5mm cobalt completely inhibited the potassium-induced stimulation of insulin release. The insulin release elicited by 28 and 50mm [K⁺] _o was similar in terms of peak, total release and half-time from maximum release. Stepwise increase in [K⁺] _o from 10 to 28 to 50mm resulted in a normal response to 28mm but no peak of release after the 28 to 50mm increase. The results indicate good correlation between excess voltage noise, thought to reflect calcium channel activity, and insulin release evoked by changing extracellular potassium.     

Kinetic analysis of carrier-mediated ion transport by the charge-pulse technique

Summary The charge-pulse technique has been used previously for the study of quasistationary processes in membranes which required only a moderate time resolution. It is shown here that a time resolution of about 400 nsec may be achieved with this technique and that it may be applied to the kinetic analysis of carrier-mediated ion transport. By this method we have studied the transport of alkali ions through optically black monoolein membranes in the presence of the ion carrier valinomycin. All three relaxation processes that are predicted by theory have been resolved. From the relaxation times and the relaxation amplitudes the rate constants for the association (k _R ) and the dissociation (k _D ) of the ioncarrier complex, as well as the translocation rate constants of the complex (k _MS ) and the free carrier (k _S ) could be obtained. For 1m Rb⁺ at 25° C the values arek _R =3×10⁵ m ^–1 sec^–1,k _D =2×10⁵ sec^–1,k _MS =3×10⁵ sec^–1,k _S =4×10⁴ sec^–1. The activation energies of the single rate constants which have been estimated from experiments at two different temperatures range between 50 and 90 kJ/mol.     

Comparison of the biological activity of fusicoccin in higher plants with its binding to plasma membranes

The concentration dependences of the binding of fusicoccins (FCs) A, B, C, D, J and H to plasma membranes isolated from maize (Zea mays L.) roots have been studied in parallel with the effects of these compounds on elongation and ⁸⁶Rb transport in detached maize roots. The dissociation constants obtained showed a good correlation between the affinity of the FCs for the plasmalemma and their biological activity. However, the range of physiologically active FC concentrations proved to be about two orders of magnitude higher than that calculated from the dissociation constants. It was also shown that Vicia faba L. mesophyll protoplasts, unlike isolated plasma membranes, have two FC-binding sites, one with a K _D similar to that of the isolated plasmalemma while the other has a substantially higher K _D , apparently corresponding to the physiologically active state of the FC-binding proteins.Abbreviation FC fusicoccin     

Muramyl dipeptide bound to poly-l-lysine substituted with mannose and gluconoyl residues as macrophage activators

Poly-l-lysine modified with mannose derivatives, the residual cationic charges of which being neutralized byN-acylation, were synthesized and used as carriers of a macrophage activator (N-acetylmuramyl dipeptide, MDP). The influence of the acylating agent on the targeting efficiency was investigated: a hydrosolubilizing group such as a gluconoyl moiety led to very efficient carrier conjugates, while an acetyl group did not. The effect of sugar and acyl content of the polymers was assessed using these compounds as inhibitors of red blood cell agglutination by Concanavalin A. The binding and specific endocytosis of poly-l-lysine substituted with several mannose derivatives and gluconoyl residues (GlcA_x-, Man_y-PLK) have been determined by a quantitative flow cytometry analysis. MDP bound to these conjugates was much more efficientin vitro than free MDP in macrophage cytostasis assays.Abbreviations MDP N-acetylmuramyl dipeptide - PLK poly-l-lysine - BSA bovine serum albumin - BOC t-butyloxycarbonyl - DMF dimethylformamide - DCHU N, N-dicyclohexylurea - DCCl N, N-dicyclohexylcarbodiimide - TEA triethylamine - Su succinimidyl - DMSO dimethylsulfoxide - FITC fluoresceinyl isothiocyanate - RPMI Roswell Park Memorial Institute - PBS phosphate buffered saline - Fl fluoresceinyl - GG glycyl-glycyl     

Glucose oxidase: natural occurrence, function, properties and industrial applications

Glucose oxidase (GOX) from Aspergillus niger is a well-characterised glycoprotein consisting of two identical 80-kDa subunits with two FAD co-enzymes bound. Both the DNA sequence and protein structure at 1.9 Ǻ have been determined and reported previously. GOX catalyses the oxidation of d-glucose (C₆H₁₂O₆) to d-gluconolactone (C₆H₁₀O₆) and hydrogen peroxide. GOX is produced naturally in some fungi and insects where its catalytic product, hydrogen peroxide, acts as an anti-bacterial and anti-fungal agent. GOX is Generally Regarded As Safe, and GOX from A. niger is the basis of many industrial applications. GOX-catalysed reaction removes oxygen and generates hydrogen peroxide, a trait utilised in food preservation. GOX has also been used in baking, dry egg powder production, wine production, gluconic acid production, etc. Its electrochemical activity makes it an important component in glucose sensors and potentially in fuel cell applications. This paper will give a brief background on the natural occurrence, functions as well as the properties of glucose oxidase. A good coverage on the diverse uses of glucose oxidase in the industry is presented with a brief outline on the working principles in the various settings. Furthermore, food grade GOX preparations are relatively affordable and widely available; the readers may be encouraged to explore other potential uses of GOX. One example is that GOX-catalysed reaction generates significant amount of heat (∼200 kJ/mol), and this property has been mostly neglected in the various applications described so far.     

Purine nucleotide synthesis in lymphoblasts cultured from normal subjects and a patient with Lesch-Nyhan syndrome

Human lymphoblasts derived from normal and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient individuals have been maintained in permanent tissue culture, and comparative studies of their purine metabolism have been undertaken. In agreement with previous observations in fibroblasts, the HGPRT-deficient lymphoblasts (less than 2% normal HGPRT activity) demonstrate threefold increases in the production of purines by the de novo pathway and four- to eightfold increases in intracellular concentrations of 5-phosphoribosyl 1-pyrophosphate (PRPP). The activities of the enzymes of purine metabolism responsible for production and utilization of PRPP were measured under optimal conditions in each cell line. The activities of adenine phosphoribosyltransferase (APRT), PRPP synthetase, and PRPP amidotransferase were independent of cell density and were not significantly different in the two cell lines. The K _m values of the common substrate, PRPP, were determined in normal lymphoblast extracts for APRT (K _m of 0.033 mM), HGPRT (K _m of 0.074 mM), and PRPP amidotransferase (K _m of 0.3 m M). The relatively low affinity of PRPP amidotransferase for PRPP suggests that deficiency of the HGPRT enzyme with its attendant increase in PRPP concentration should be accompanied by increased in vivo activity of PRPP amidotransferase, the first and presumed rate-limiting enzyme of de novo purine biosynthesis.This work was supported in part by National Institutes of Health Grants AM-05646, AM-13622, and GM-17702.     

Predicting the summer chlorophylla concentration in a reservoir based on the environmental conditions of the preceding spring

From a monthly survey over a ten-year period (1983–1992) of the Ishitegawa Reservoir, Japan, two multiple regression equations describing the mean chlorophylla (Chl-a) concentration at 0.5 m depth during July, August and September (C _S ) and the maximum Chl-a concentration in the photic zone, including its subsurface maximum during this period (C _M ), were obtained. The conductivity at 0.5 m depth in May and the mean air temperature during May or during May and June were used as independent variables. These equations were calculated using seven years of data (1983–1988 and 1992). From 1989 to 1991 two impermeable plastic-coated sheet fences were installed across the upper end of the reservoir along the top 5 m of water column. The equations were used to determine the effectiveness of this flow regulation on the summer Chl-a concentration. In 1989 and 1991, when the fences were in place from June to August, no effects were found on the observed C _S and C _M values. In 1990, when the fences had been in place since October 1989, the observed values were lower than the predicted values.     

Aluminium Chloride Enhances Colchicine Production in Root Cultures of Gloriosa superba

Root cultures of Gloriosa superba were treated with 5 mm methyl jasmonate and 125 μm AlCl₃ which enhanced the intracellular colchicine content of the roots by 50-fold and 63-fold, respectively. Ten millimolar of CaCl₂ and 1 mm CdCl₂ enhanced biomass significantly (7- to 8.6-fold, respectively) while maximum release of colchicine into the medium was obtained with 10 mm CdCl₂. Casein hydrolysate, yeast extract and silver nitrate had no significant effect on growth and colchicine accumulation in root cultures. Revisions requested 2 November 2005; Revisions received 9 January 2006