Activation and inhibition of rubber transferases by metal cofactors and pyrophosphate substrates

Metal cofactors are necessary for the activity of alkylation by prenyl transfer in enzyme-catalyzed reactions. Rubber transferase (RuT, a cis-prenyl transferase) associated with purified rubber particles from Hevea brasiliensis, Parthenium argentatum and Ficus elastica can use magnesium and manganese interchangably to achieve maximum velocity. We define the concentration of activator required for maximum velocity as [A](max). The [A](max)(Mg2+) in F. elastica (100 mM) is 10 times the [A](max)(Mg2+) for either H. brasiliensis (10 mM) or P. argentatum (8 mM). The [A](max)(Mn2+) in F. elastica (11 mM), H. brasiliensis (3.8 mM) and P. argentatum (6.8 mM) and the [A](max)(Mg2+) in H. brasiliensis (10 mM) and P. argentatum (8 mM) are similar. The differences in [A](max)(Mg2+) correlate with the actual endogenous Mg(2+) concentrations in the latex of living plants. Extremely low Mn(2+) levels in vivo indicate that Mg(2+) is the RuT cofactor in living H. brasiliensis and F. elastica trees. Kinetic analyses demonstrate that FPP-Mg(2+) and FPP-Mn(2+) are active substrates for rubber molecule initiation, although free FPP and metal cations, Mg(2+) and Mn(2+), can interact independently at the active site with the following relative dissociation constants K(d)(FPP) 相似文献   

Extracellular HCO(3)(-) dependence of electrogenic Na/HCO(3) cotransporters cloned from salamander and rat kidney

We studied the extracellular [HCOabstract (3) (-)] dependence of two renal clones of the electrogenic Na/HCO(3) cotransporter (NBC) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (DeltaV(m)) elicited by the NBC cloned from the kidney of the salamander Ambystoma tigrinum (akNBC) and by the NBC cloned from the kidney of rat (rkNBC). We used a two-electrode voltage clamp to measure the change in current (DeltaI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCOabstract (3 )(-)/CO(2) (0.33-99 mM HCOabstract (3)(-), pH(o) 7.5) elicited an immediate, DIDS (4, 4-diisothiocyanatostilbene-2,2-disulfonic acid)-sensitive and Na(+)-dependent hyperpolarization (or outward current). In DeltaV(m) experiments, the apparent K(m ) for HCOabstract (3)(-) of akNBC (10. 6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent K(m) for HCOabstract (3)(-) of rkNBC was less (6.5 mM). Because it has been reported that SOabstract (3)(=)/HSO abstract (3)(-) stimulates Na/HCO(3 ) cotransport in renal membrane vesicles (a result that supports the existence of a COabstract (3)(=) binding site with which SOabstract (3)(=) interacts), we examined the effect of SOabstract (3)(=)/HSO abstract (3)(-) on rkNBC. In voltage-clamp studies, we found that neither 33 mM SOabstract (4)(=) nor 33 mM SOabstract (3) (=)/HSOabstract (3)(-) substantially affects the apparent K(m) for HCO abstract (3)(-). We also used microelectrodes to monitor intracellular pH (pH(i)) while exposing rkNBC-expressing oocytes to 3.3 mM HCOabstract (3 )(-)/0.5% CO(2). We found that SO abstract (3)(=)/HSOabstract (3 )(-) did not significantly affect the DIDS-sensitive component of the pH(i) recovery from the initial CO(2 )-induced acidification. We also monitored the rkNBC current while simultaneously varying [CO(2)](o), pH(o), and [COabstract (3)(=)](o) at a fixed [HCOabstract (3)(-)](o) of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [COabstract (3)(=)](o ). However, a pH titration curve nicely fitted the data expressed as current versus pH(o). Thus, rkNBC expressed in Xenopus oocytes does not appear to interact with SOabstract (3 )(=), HSOabstract (3)(-), or COabstract (3)(=).     

Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy

A simple and direct assay method for glucose oxidase (EC 1.1.3.4) from Aspergillus niger and Penicillium amagasakiense was investigated by Fourier transform infrared spectroscopy. This enzyme catalyzed the oxidation of d-glucose at carbon 1 into d-glucono-1,5-lactone and hydrogen peroxide in phosphate buffer in deuterium oxide ((2)H(2)O). The intensity of the d-glucono-1,5-lactone band maximum at 1212 cm(-1) due to CO stretching vibration was measured as a function of time to study the kinetics of d-glucose oxidation. The extinction coefficient epsilon of d-glucono-1,5-lactone was determined to be 1.28 mM(-1)cm(-1). The initial velocity is proportional to the enzyme concentration by using glucose oxidase from both A. niger and P. amagasakiense either as cell-free extracts or as purified enzyme preparations. The kinetic constants (V(max), K(m), k(cat), and k(cat)/K(m)) determined by Lineweaver-Burk plot were 433.78+/-59.87U mg(-1) protein, 10.07+/-1.75 mM, 1095.07+/-151.19s(-1), and 108.74 s(-1)mM(-1), respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on horseradish peroxidase in aqueous media: 470.36+/-42.83U mg(-1) protein, 6.47+/-0.85 mM, 1187.77+/-108.16s(-1), and 183.58 s(-1)mM(-1) for V(max), K(m), k(cat), and k(cat)/K(m), respectively. Therefore, this spectroscopic method is highly suited to assay for glucose oxidase activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of glucose oxidase activity.     

Salt-induced lipid changes in Catharanthus roseus cultured cell suspensions

Salt treatment strongly affected cell growth by decreasing dry weight. Exposure of Catharanthus roseus cell suspensions to increasing salinity significantly enhanced total lipid (TL) content. The observed increase is mainly due to high level of phospholipids (PL). Hundred mM NaCl treatment increased phospholipid species phosphatidylcholine (PC) and phosphatidylethanolamine (PE), whereas it reduced glycolipid ones monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) but not sulfoquinovosyldiacylglycerol (SQDG). Moreover, fatty acid composition was clearly modified when cells were cultured in the presence of 100 mM NaCl, whereas only few changes occurred at 50 mM. Salt treatment decreased palmitic acid (16:0) level and increased that of linolenic acid (18:2). Such effect was observed in phospholipid species PC and PE and in glycolipid DGDG. Double bond index (DBI) was enhanced more than 2-fold in fatty acids of either glycolipids or phospholipids from cells submitted to 100 mM NaCl. Free sterol content was also significantly enhanced, especially at 100 mM NaCl, whereas free sterols/phospholipids (St/PL) ratio was slightly decreased. All these salt-induced changes in membrane lipids suggest an increase in membrane fluidity of C. roseus cells.     

Isolation and characterisation of a Salvia bogotensis seed lectin specific for the Tn antigen

A lectin was isolated and characterised from Salvia bogotensis seeds. Removal of the abundant pigments and polysaccharides, which are present in seeds, was an essential step in its purification. Several procedures were assayed and the best suited, including Pectinex treatment, DEAE-cellulose and affinity chromatography, led to a protein being obtained amounting to 18-20mg/100g seeds having high specific agglutination activity (SAA). The lectin specifically agglutinated human Tn erythrocytes and was inhibited by 37mM GalNAc, 0.019mM ovine submaxillary mucin (OSM) or 0.008mM asialo bovine submaxillary mucin (aBSM). Enzyme-linked lectinosorbent assay (ELLSA) revealed strong binding to aOSM and aBSM, corroborating Tn specificity, whereas no binding to fetuin or asialo fetuin was observed. The lectin's monomer MW (38,702Da), amino acid composition, pI, carbohydrate content, deglycosylated form MW, thermal stability and Ca(2+) and Mn(2+) requirements were determined. Evidence of the existence of two glycoforms was obtained. The lectin's specificity and high affinity for the Tn antigen, commonly found in tumour cells, makes this protein a useful tool for immunohistochemical and cellular studies.     

Racemic and optically active 2-methoxy-4-oxatetradecanoic acids: novel synthetic fatty acids with selective antifungal properties

The unprecedented (+/-)-2-methoxy-4-oxatetradecanoic acid and the optically pure (S)-2-methoxy-4-oxatetradecanoic acid were synthesized in six steps and in 11-14% overall yields starting with either 1,2-O-isopropylidene-rac-glycerol or 1,2-O-isopropylidene-(S)-glycerol. The key step in the synthesis was the selective monosilylation of a dibutylstannylene intermediate. The title compounds displayed selective fungitoxicity in the range of 0.08-0.22 mM against Cryptococcus neoformans ATCC 66031 and Aspergillus niger ATCC 16404, but no significant activity against C. albicans ATCC 14053 and ATCC 60193 (>2.6 mM). Albeit being good substrates for N-myristoyltransferases (NMTs), the racemic and the S-enantiomer of the oxygenated 2-methoxylated compounds showed no significant difference in antifungal activity. This finding suggests an alternative mechanism of fungitoxicity other than NMT inhibition.     

Accelerated CuZn-SOD-mediated oxidation and reduction in the presence of hydrogen peroxide

Copper, zinc-superoxide dismutase (CuZn-SOD) is a cytosolic, antioxidant enzyme that scavenges potentially damaging superoxide radical (()O(2)(-)). Under the proper conditions, CuZn-SOD also catalyzes the oxidation and reduction of certain small molecules. Here, we demonstrate that increased exposure to hydrogen peroxide (H(2)O(2)), a by-product of the ()O(2)(-) scavenging reaction, dramatically increases the ability of CuZn-SOD to oxidize melatonin and reduce S-nitrosoglutathione (GSNO). After a 15min in vitro incubation with CuZn-SOD and 1mM H(2)O(2), 76% of the melatonin was oxidized, compared to 52% with 0.25mM H(2)O(2), and just 9% without H(2)O(2). Pre-incubation with 1mM H(2)O(2) resulted in a 100% increase in the rate of GSNO breakdown by CuZn-SOD in the presence of glutathione (GSH) compared to untreated CuZn-SOD. Collectively, these data suggest that even small increases in intracellular H(2)O(2) levels may result in the oxidation and/or reduction of small molecules critical for proper cellular function.     

Protection from acute cellular injury using Sleeping Beauty mediated telomerase gene transfer

We developed a Sleeping Beauty (SB) transposon mediated hTERT gene delivery system for in vitro use. We have constructed telomerase or luciferase gene expressing SB-transposons with a SV40 enhancer (pT3.hTERT.Con and pT3.Con, respectively) or without an enhancer (pT3.Pro). Using the SB transposon system in vitro hTERT gene overexpression has protective effects from acute cellular injury by tert-butyl hydroperoxide (t-BH), carbon tetrachloride (CCl(4)), and d-galactosamine (d-GalN) in normal human cells IMR-90. pT3.hTERT.Con vector and helper plasmid co-transfection resulted in a approximately 3-fold increase in telomerase activity which was maintained for 14 days. Trypan blue and Cell Death Detection Assays showed the protective effects of the telomerase gene against toxic agents. Fourteen days after co-transfection with pT3.hTERT.Con vector and helper plasmid, IMR-90 cells were incubated with 1.2mM t-BH for 50 min, 5mM CCl(4) for 1.5h or 30 mM d-GalN for 24h. Cell viability of SB-mediated telomerase overexpressing cells significantly increased by 48% (t-BH), 43% (CCl(4)), and 25% (d-GalN) in comparison to mock treated cells. Cell Death Detection ELISA showed a decrease in the rate of apoptosis by 47%. In summary, SB transposon mediated telomerase gene transfer may have a protective effect against t-BH, CCl(4), or d-GalN induced acute cellular injury, and this results suggested SB-mediated telomerase therapy for tissue engineering.     

Preconditioning with millimolar concentrations of vitamin C or N-acetylcysteine protects L6 muscle cells insulin-stimulated viability and DNA synthesis under oxidative stress

The effect of reactive oxygen/nitrogen species (ROS/RNS)(hydrogen peroxide -- H(2)O(2), superoxide anion radical O(2)*- and hydroxyl radical *OH -- the reaction products of hypoxanthine/xanthine oxidase system), nitric oxide (NO* from sodium nitroprusside -- SNP), and peroxynitrite (ONOO(-) from 3-morpholinosydnonimine -- SIN-1) on insulin mitogenic effect was studied in L6 muscle cells after one day pretreatment with/or without antioxidants. ROS/RNS inhibited insulin-induced mitogenicity (DNA synthesis). Insulin (0.1 microM), however, markedly improved mitogenicity in the muscle cells treated with increased concentrations (0.1, 0.5, 1 mM) of donors of H(2)O(2), O(2)*-, *OH, ONOO(-) and NO*. Cell viability assessed by morphological criteria was also monitored. Massive apoptosis was induced by 1 mM of donors of H(2)O(2) and ONOO(-), while NO* additionally induced necrotic cell death. Taken together, these results have shown that ROS/RNS provide a good explanation for the developing resistance to the growth promoting activity of insulin in myoblasts under conditions of oxidative or nitrosative stress. Cell viability showed that neither donor induced cell death when given below 0.5 mM. In order to confirm the deleterious effects of ROS/RNS prior to the subsequent treatment with ROS/RNS plus insulin one day pretreatment with selected antioxidants (sodium ascorbate - ASC (0.01, 0.1, 1 mM), or N-acetylcysteine - NAC (0.1, 1, 10 mM) was carried out. Surprisingly, at a low dose (micromolar) antioxidants did not abrogate and even worsened the concentration-dependent effects of ROS/RNS. In contrast, pretreatment with millimolar dose of ASC or NAC maintained an elevated mitogenicity in response to insulin irrespective of the ROS/RNS donor type used.     

Furostanol glycoside 26-O-beta-glucosidase from the leaves of Solanum torvum

A beta-glucosidase (torvosidase) was purified to homogeneity from the young leaves of Solanum torvum. The enzyme was highly specific for cleavage of the glucose unit attached to the C-26 hydroxyl of furostanol glycosides from the same plant, namely torvosides A and H. Purified torvosidase is a monomeric glycoprotein, with a native molecular weight of 87 kDa by gel filtration and a pI of 8.8 by native agarose IEF. Optimum pH of the enzyme for p-nitrophenyl-beta-glucoside and torvoside H was 5.0. Kinetic studies showed that Km values for torvoside A (0.06 3mM) and torvoside H (0.068 mM) were much lower than those for synthetic substrates, pNP-beta-glucoside (1.03 mM) and 4-methylumbelliferyl-beta-glucoside (0.78 mM). The enzyme showed strict specificity for the beta-d-glucosyl bond when tested for glycone specificity. Torvosidase hydrolyses only torvosides and dalcochinin-8'-beta-glucoside, which is the natural substrate of Thai rosewood beta-glucosidase, but does not hydrolyse other natural substrates of the GH1 beta-glucosidases or of the GH3 beta-glucosidase families. Torvosidase also hydrolyses C5-C10 alkyl-beta-glucosides, with a rate of hydrolysis increasing with longer alkyl chain length. The internal peptide sequence of Solanum beta-glucosidase shows high similarity to the sequences of family GH3 glycosyl hydrolases.     

Removal of the N-linked glycan structure from the peanut peroxidase prxPNC2: influence on protein stability and activity

Lines of transgenic tobacco have been generated that are transformed with either the wild-type peanut peroxidase prxPNC2 cDNA, driven by the CaMV35S promoter (designated 35S::prxPNC2-WT) or a mutated PNC2 cDNA in which the asparagine residue (Asn189) associated with the point of glycan attachment (Asn189) has been replaced with alanine (designated 35S::prxPNC2-M). PCR, using genomic DNA as template, has confirmed the integration of the 35S::prxPNC2-WT and 35S:prxPNC2-M constructs into the tobacco genome, and western analysis using anti-PNC2 antibodies has revealed that the prxPNC2-WT protein product (PNC2-WT) accumulates with a molecular mass of 34,670 Da, while the prxPNC2-M protein product (PNC2-M) accumulates with a molecular mass of 32,600 Da. Activity assays have shown that both PNC2-WT and PNC2-M proteins accumulate preferentially in the ionically-bound cell wall fraction, with a significantly higher relative accumulation of the PNC2-WT isoenzyme in the ionically-bound fraction when compared with the PNC2-M isoform. Kinetic analysis of the partially purified PNC2-WT isozyme revealed an affinity constant (apparent Km) of 11.2 mM for the reductor substrate guaiacol and 1.29 mM for H2O2, while values of 11.9 mM and 1.12 mM were determined for the PNC2-M isozyme. A higher Arrenhius activation energy (Ea) was determined for the PNC2-M isozyme (22.9 kJ mol(-1)), when compared with the PNC2-WT isozyme (17.6 kJ mol(-1)), and enzyme assays have determined that the absence of the glycan influences the thermostability of the PNC2-M isozyme. These results are discussed with respect to the proposed roles of N-linked glycans attached to plant peroxidases.     

4-benzyl-3-phenyl-5H-furan-2-one, a vasodilator isolated from Malbranchea filamentosa IFM 41300

Screening of Malbranchea filamentosa IFM 41300 for bioactive compounds led to the identification of 4-benzyl-3-phenyl-5H-furan-2-one (1) as a vasodilator and erythroglaucin (2). The structure of 1 was established on the basis of spectroscopic and chemical investigations. Compound 1 inhibited Ca2+-induced vasocintraction in aortic rings pretreated with high K+ (60mM) or norepinephrine. Finally, compound 1 did not exhibit activity against human pathogenic microorganisms.     

Low-affinity Ca and Ba binding sites in the pore of α7 nicotinic acetylcholine receptors

α7 nicotinic receptors are highly permeable to Ca²⁺ as well as monovalent cations. We extended the characterization of the Ca²⁺ permeation of non-desensitizing chick α7 receptors (S240T/L247T α7 nAChRs) expressed in Xenopus oocytes by (1) measuring the concentration dependence of conductance under conditions in which Ca²⁺ or Ba²⁺ were the only permeant cations in the extracellular solution, and (2) measuring the concentration dependence of Ca²⁺ block of K⁺ currents through the receptors. The first set of experiments yielded an apparent affinity of 0.96 mM Ca²⁺ activity (2.4 mM concentration) for Ca²⁺ permeation and an apparent affinity of 0.65 mM Ba²⁺ activity (1.7 mM concentration) for Ba²⁺ permeation. The apparent affinity of Ca²⁺ inhibition of K⁺ currents was 0.49 mM activity (1.5 mM concentration). The similarity of these apparent affinities in the millimolar range suggests that the pore of α7 receptors has one or more low-affinity Ca²⁺ binding sites and no high-affinity sites.     

Synthesis and glycosidase inhibitory activity of 1-amino-3,6-anhydro-1-deoxy-d-sorbitol derivatives

3,6-Anhydro-1-(aryl or alkylamino)-1-deoxy-d-sorbitol derivatives have been prepared in four steps from isosorbide, a by-product from the starch industry. The inhibitory activities of these new compounds have been evaluated towards 13 glycosidases. A first lead-compound was identified, which inhibited β-N-acetylglucosaminidase from bovine kidney (82% inhibition at 1 mM).     

Enhancing phytoremediative ability of Pisum sativum by EDTA application

The aim of our research was to demonstrate how the presence of EDTA affects resistance of pea plants to Pb and Pb-EDTA presence, and to show the effectivity of lead ions accumulation and translocation. It was determined that EDTA not only increased the amount of Pb taken up by plants but also Pb ion transport through the xylem and metal translocation from roots to stems and leaves. It can be seen in the presented research results that addition of the chelator with Pb limited metal phytotoxicity. We also demonstrated a significant effect of EDTA not only on Pb accumulation and metal transport to the aboveground parts but also on the profile and amount of thiol compounds: glutathione (GSH), homoglutathione (hGSH) or phytochelatins (PCs), synthesized by the plants. We observed a significant effect of the synthetic chelator on increasing the level of Pb accumulation in roots of plants treated with Pb including EDTA (0.5 and 1 mM). Pisum sativum plants treated only with 1 mM Pb(NO3)2 accumulated over 50 mg Pb x g(-1) dry wt during 4 days of cultivation. Whereas in roots of pea plants exposed to Pb+0.5 mM EDTA 35% more Pb was observed. When 1 mM EDTA was applied roots of pea accumulated over 67% more metal. The presence of EDTA also increased metal uptake and transport to the aboveground parts. In pea plants treated only with 1 mM lead nitrate less than 3 mg Pb x g(-1) dry wt was transported, whereas in P. sativum treated with Pb-EDTA doubled amount of Pb was observed in stems and leaves.     

High molecular mass kininogen inhibits metalloproteinases of Bothrops jararaca snake venom

High molecular mass kininogen (HK) purified from Bothrops jararaca (Bj) plasma was tested on activities of the Bj venom in vivo and in vitro. Results showed that, when incubated with BjHK, the Bj venom presented inhibition on hemorrhagic, edema forming, myotoxic, and coagulant activities. It is well known that metalloproteinases are directly or indirectly involved in these activities. Similarly, human HK inhibits the hemorrhagic effect of the Bj venom as well as hemorrhagic and enzymatic effects of jararhagin, a hemorrhagic metalloproteinase isolated from Bj venom. Complex between HK and jararhagin was not detected by gel filtration. Nevertheless, the inhibitory effect of the hemorrhagic activity of the venom was only partial when HK was pre-incubated with 0.4mM ZnCl(2) or with 0.45mM CaCl(2). These data suggest that the inhibitory effect depends, at least partially, on the competition for ions between kininogen and metalloproteinases of the venom.     

Endothelium-independent vasorelaxation effects of 16-O-acetyldihydroisosteviol on isolated rat thoracic aorta

Aims

The aim of this study is to investigate the vasorelaxant effect of 16-O-acetyldihydroisosteviol (ADIS) and its underlying mechanisms in isolated rat aorta.

Main methods

Rat aortic rings were isolated, suspended in organ baths containing Kreb's solution, maintained at 37 °C, and mounted on tungsten wire and continuously bubbled with a mixture of 95% O₂ and 5% CO₂ under a resting tension of 1 g. The vasorelaxant effects of ADIS were investigated by means of isometric tension recording experiment.

Key findings

ADIS (0.1 μM–3 mM) induced relaxation of aortic rings pre-contracted by phenylephrine (PE, 10 μM) and KCl (80 mM) with intact-endothelium (E_max = 79.26 ± 3.74 and 79.88 ± 3.79, respectively) or denuded-endothelium (E_max = 88.05 ± 3.69 and 78.22 ± 6.86, respectively). In depolarization Ca²⁺-free solution, ADIS inhibits calcium chloride (CaCl₂)-induced contraction in endothelium-denuded rings in a concentration-dependent manner. In addition, ADIS attenuates transient contractions in Ca²⁺-free medium containing EGTA (1 mM) induced by PE (10 μM) and caffeine (20 mM). By contrast, relaxation was not affected by tetraethylammonium (TEA, 5 mM), 4-aminopyridine (4-AP, 1 mM), glibenclamide (10 μM), barium chloride (BaCl₂, 1 mM), and 1H-[1,2,3]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, 1 μM).

Significance

These findings reveal the vasorelaxant effect of ADIS, through endothelium-independent pathway. It acts as a Ca^2 + channel blocker through both intracellular and extracellular Ca^2 + release.     

Screening of reducing agents for anaerobic growth of Candida albicans SC5314

This study aimed to evaluate the influence of different redox potentials (Eh) on cell growth, whole-cell protein profile and cell surface hydrophobicity (CSH) of Candida albicans SC5314. The yeast was grown in YNB broth enriched with reducing (158 mM sodium sulfite, 4 mM sodium sulfite, 2.5 mM sodium metabisulfite, 1.3 mM 2-mercaptoethanol, 5.5 mM thioglycolic acid, and 3.2 mM l-cysteine hydrochloride) and oxidizing agents (15 mM ammonium persulfate and 80 mM potassium ferricyanide) and incubated in normoxic and anoxic atmospheres at 37 °C, for 48 h. Pre- and post-incubation Eh values were determined and cytoplasm proteins were extracted. Proteins were parted by SDS-PAGE and their profiles were compared. 3.2 mM l-cysteine and 1.3 mM 2-mercaptoethanol promoted and maintained negative Eh values during incubation. No differences were detected among SDS-PAGE profiles. CSH differences only were observed with 4 mM sodium sulfite and 3.2 mM l-cysteine. Results showed that 3.2 mM l-cysteine is a reducing agent that allows maintenance of negative Eh in both anoxic and normoxic conditions and it seems not to interfere in the global expression of plasmatic proteins.     

A novel lectin from the wild mushroom Polyporus adusta

A lectin with antiproliferative activity toward tumor cell lines and mitogenic activity toward splenocytes was isolated from the mushroom Polyporus adusta. The lectin was composed of two identical subunits each with a molecular weight of 12 kDa. It was adsorbed on both DEAE-cellulose and Q-Sepharose and unadsorbed on CM-Sepharose. The hemagglutinating activity of the lectin was inhibited by turanose and by a large variety of other carbohydrates. It was adversely affected in the presence of NaOH or HCl at a concentration of 7.5mM and above, and when the ambient temperature was raised above 70 degrees C. All divalent and trivalent metallic chlorides tested at 1.25-10mM including CaCl(2), MgCl(2), ZnCl(2), MnCl(2), and AlCl(3), did not alter the hemagglutinating activity of the lectin. FeCl(3) at 10mM caused the hemagglutinating activity to increase by 100%, but it did not change the lectin activity when tested at lower concentrations up to 5mM.     

The influence of reduced glutathione in fertilization medium on the fertility of in vitro–matured C57BL/6 mouse oocytes

It is well known that IVM oocytes show a decreased potential for fertility and development compared with in vivo–matured oocytes. In this study, we added reduced glutathione (GSH) to the fertilization medium during IVF to investigate its effect on the fertility and early embryo development of IVM oocytes. The fertilization rate for IVM oocytes and fresh sperm increased with the addition of GSH (0, 1.0, and 2.0 mM: 51%, 76%, and 70%). Moreover, the addition of GSH to the fertilization medium also improved the developmental potential compared with the control sample (0 mM). In addition, we performed IVF using IVM oocytes and frozen/thawed sperm that had been cryopreserved in a mouse bank. Results indicated a marked increase in the fertilization rate when 1.0 mM GSH was added to the fertilization medium compared with when no GSM was used (0.0 mM GSH: 2% (3/195); 1.0 mM GSH: 33% (156/468)). Furthermore, the fertilization rate improved dramatically via zona drilling using laser equipment (52%: 267/516), whereas normal offspring were obtainsed after transferring embryos created via IVF using IVM oocytes and frozen/thawed sperm. This is the first report in which offspring have been obtained via IVF using IVM oocytes and frozen/thawed sperm.