In Vitro Induction of Altered Response of Rat Atria to Acetylcholine with Histamine-Sensitizing Factor of Bordetella pertussis

Purified histamine-sensitizing factor (HSF) of Bordetella pertussis induced in vitro an alteration in the pharmacologic response of rat atria to acetylcholine. Spontaneously beating atrial preparations isolated from rats were exposed to HSF at a concentration of 50 ng/ml at 37 C for 30 min to 4 hr and washed with Krebs-Ringer solution, and then tested at 1-hr intervals up to 28 hr during incubation for their responses to epinephrine and acetylcholine. At 13 hr after exposure to HSF, irrespective of the exposure period, the HSF-treated atria, in which positive-inotropic action of epinephrine was manifested, depressed the negative-inotropic response to acetylcholine. The activity of HSF was neutralized by anti-HSF serum only in the first 3 hr of exposure. Only 1.8% of the added ¹²⁵I-labeled HSF bound “specifically” to one pair of atria for the manifestation of the altered response.     

The Effect of Gentian Violet on Virulent Properties of <Emphasis Type="Italic">Candida albicans</Emphasis>

The aim of this study was to evaluate the effect of gentian violet (GV) on phospholipase activity, proteinase activity and germ tube formation rate of Candida albicans. Both 12 phospholipase-positive and 12 proteinase-positive C. albicans isolates with Pz values ≤0.89 were obtained. A yeast suspension (1–3 × 10⁷ cfu/ml) of each isolate was prepared. After a brief exposure (60 min) to sub-therapeutic concentrations (0.5 or 2 μg/ml) of GV, Pz value of phospholipase, Pz value of proteinase and germ tube formation rate were determined. Phospholipase activity, proteinase activity and germ tube formation rate in two groups exposed to GV were significantly lower than those in the group unexposed (P < 0.05). The results of this study indicated that sub-therapeutic concentrations of GV may lead to reduction in phospholipase activity, proteinase activity and germ tube formation, and then may suppress virulence and pathogenicity of C. albicans.     

FLORIDOSIDE AS A CARBON PRECURSOR FOR THE SYNTHESIS OF CELL‐WALL POLYSACCHARIDE IN THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA)1

Although red algae are known to be obligatory photoautotrophs, the red microalga Porphyridium sp. was shown to assimilate and metabolize floridoside. A pulse‐chase experiment with [¹⁴C]floridoside showed that at the end of a 240‐min pulse, 70% of total ¹⁴C‐uptake by the cells remained in the floridoside fraction. To evaluate the assimilation of floridoside by Porphyridium sp. cells, we exposed Porphyridium sp. not only to [¹⁴C]floridoside but also to its constituents, [¹⁴C]glycerol and [¹⁴C]galactose, as compared with [¹⁴C]bicarbonate. The extent of incorporation of [¹⁴C] galactose by the Porphyridium sp. cells was insignificant (50–80 dpm·mL^?1), whereas uptake of ¹⁴C from [¹⁴C]glycerol into the algal cells was evident (2.4 × 10³ dpm·mL^?1) after 60 min of the pulse. The pattern of ¹⁴C distribution among the major constituent sugars, xylose, glucose and galactose, of the labeled soluble polysaccharide was dependent on the ¹⁴C source. The relative content of [¹⁴C]galactose in the soluble polysaccharide was highest (28.8%) for [¹⁴C]floridoside‐labeled culture and lowest (19.8%) for the [¹⁴C]glycerol‐labeled culture. Upon incubation of [¹⁴C]floridoside with a crude extract of a cell‐free system prepared from nonlabeled cells of Porphyridium sp., the label was indeed found to be incorporated into the sulfated polysaccharide. Our results suggested that the carbon metabolic pathway in Porphyridium sp. passes through the low molecular weight photoassimilatory product—floridoside—toward sulfated cell‐wall polysaccharide production.     

Neurochemical mechanisms responsible for depotentiation of synaptic transmission

It was established in experiments on murine hippocampal slices that low-frequency (1 sec⁻¹, 15 min) stimulation of the Schaffer collaterals applied 45 to 60 min after their high-frequency repetitive stimulation (60 sec⁻¹, 0.5 sec) results, in 2/3 of the slices, in reduction of the amplitude of population EPSP recorded from pyramidal neurons of theCA1 area, almost to its level before high-frequency stimulation. Depotentiation was practically completely prevented by application of a non-competitive blocker of NMDA glutamate receptors (GR), ketamine (100 μM), was weakened by a blocker of voltage-dependent L-type Ca²⁺ channels, nifedipine (10 μM), and remained significant after a competitive blocker of the AMPA/kainate receptors, CNQX (10 μM), had been applied to the slices. Depotentiation was significantly reduced by 10 μM of a calmodulin inhibitor, trifluoroperazine, by an increase in the intracellular cAMP concentration caused by activation of A2-adenosine receptors and D5-dopamine receptors, but was resistant to the action of 50 μM of a protein kinase C (PKC) inhibitor, polymixin B. Nootropic compounds possessing anti-amnestic activity enhanced the depotentiation. It is suggested that depotentiation is due to an increase in the intracellular Ca²⁺ concentration, activation of protein phosphatases, and dephosphorylation of pre- and post-synaptic substrates involved in the expression of long-term post-tetanic potentiation of synaptic transmission, which result from cooperative activation of NMDA GR and metabotropic GR.     

Glycerol Dehydrogenase from Cellulomonas sp. NT3060: Purification and Characterization

NAD⁺-dependent glycerol dehydrogenase from Cellulomonas sp. NT3060 was purified by a procedure of 10 steps involving crystallization. Dihydroxyacetone was identified as the oxidation product of glycerol with the enzyme. The purified enzyme did not lose activity on heating below 60°C. The enzyme oxidized other alcohols such as 1,2-propanediol, 2,3-butanediol and glycerol-α-monochlorohydrin, beside glycerol. The enzyme activity was inhibited by p-chloromercuribenzoate, Zn²⁺, Cu²⁺ and Cd²⁺. Oxidation of glyberol was activated by Na⁺ and reduction of dihydroxyacetone was activated by K⁺ at pH 7.5.     

Cryopreservation of in vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn.

In vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn. were successfully cryopreserved by vitrification. Shoot tips (0.5–1 mm) excised from 6-week-old shoots were precultured in hormone-free Woody Plant Medium (WPM) supplemented with 0.2 M sucrose, for 2 days at 4 °C in the dark, and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose, for 20 min at 25 °C. Osmoprotected shoot tips were first dehydrated with 50 % vitrification solution (PVS2), for 30 min at 0 °C, and then placed in 100 % PVS2, for 30 min at 0 °C. The solution was replaced with fresh 100 % PVS2, and the shoot tips were plunged directly into liquid nitrogen. The shoot tips were rewarmed in a water bath at 40 °C for 2 min, and then washed twice, for 10 min at 25 °C, with 1.2 M sucrose solution, before being transferred onto WPM supplemented with 0.5 mg l^?1 N ⁶-benzyladenine, 0.5 mg l^?1 indole-3-acetic acid, 0.2 mg l^?1 zeatin, 20 g l^?1 glucose and 6 g l^?1 Difco Bacto agar. The shoot tips were kept in darkness for 1 week and under dim lighting for another week, before being exposed to standard culture conditions (16 h photoperiod). This protocol was successfully applied to three alder genotypes, with recovery rates higher than 50 %.     

CITRIC ACID PRODUCTION BY Candida SPECIES GROWN ON A SOY-BASED CRUDE GLYCEROL

Citric acid was produced by five species of the yeast Candida after growth on a medium containing soy biodiesel-based crude glycerol. After growth on a medium containing 10 g L^?1 or 60 g L^?1 crude glycerol for 168 hr at 30°C, Candida parapsilosis ATCC 7330 and C. guilliermondii ATCC 9058 produced the highest citric acid levels. On 10 g L^?1 or 60 g L^?1 crude glycerol for 168 hr at 30°C, the citric acid level produced by C. parapsilosis ATCC 7330 was 1.8 g L^?1 or 11.3 g L^?1, respectively, while C. guilliermondii ATCC 9058 produced citric acid concentrations of 3.0 g L^?1 or 10.4 g L^?1, respectively. Biomass production by C. guilliermondii ATCC 9058 on 10 g L^?1 or 60 g L^?1 crude glycerol for 168 hr at 30°C was highest at 1.2 g L^?1 or 6.9 g L^?1, respectively. The citric acid yields observed for C. guilliermondii ATCC 9058 after growth on 10 g L^?1 or 60 g L^?1 crude glycerol (0.35 g g^?1 or 0.21 g g^?1, respectively) were generally higher than for the other Candida species tested. When similar crude glycerol concentrations were present in the culture medium, citric acid yields observed for some of the Candida species utilized in this study were about the same or higher compared to citric acid yields by Yarrowia lipolytica strains. Based on the findings, it appeared that C. guilliermondii ATCC 9058 was the most effective species utilized, with its citric acid production being similar to what has been observed when citric acid-producing strains of Y. lipolytica were grown on crude glycerol under batch conditions that could be of significance to biobased citric acid production.     

Effects of inoculum concentration,temperature, and carbon sources on tannase production during solid state fermentation of cashew apple bagasse

In this study, the optimization of tannase production by solid state fermentation was investigated using cashew apple bagasse (CAB), an inexpensive residue produced by the cashew apple agroindustry, as a substrate. To accomplish this, CAB was enriched with 2.5% (w/w) tannic acid and 2.5% (w/w) ammonium sulphate and then moistened with water (60 mL/100 g of dry CAB). The influence of inoculum concentration (10⁴ to 10⁷ spores/g), temperature (20, 25, 30, and 35°C) and several additional carbon sources (glucose, starch, sucrose, maltose, analytical grade glycerol, and glycerol produced during biodiesel production) on enzyme production by Aspergillus oryzae was then evaluated. Supplementation with maltose and glycerol inhibited tannase synthesis, which resulted in lower enzyme activity. Starch and sucrose supplementation increased enzyme production, but decreased the enzyme productivity. The maximum tannase activity (4.63 units/g of dry substrate) was obtained at 30°C, using 10⁷ spores/g and 1.0% (w/v) sucrose as an additional carbon source.     

Characterization of a novel alkaline esterase from Altererythrobacter epoxidivorans CGMCC 1.7731T

A novel esterase gene (e25) was identified from Altererythrobacter epoxidivorans CGMCC 1.7731^T by genome sequence screening. The e25 gene is 948 nucleotides in length and encodes a 315?amino acid protein (E25) with a predicted molecular mass of 33,683 Da. A phylogenetic tree revealed that E25 belongs to the hormone-sensitive lipase (HSL) family of lipolytic enzymes. An activity assay of E25 showed that it exhibited the highest catalytic efficiency when using p-nitrophenyl caproate (C6) as a substrate. The optimum pH and temperature were determined to be approximately pH 9 and 45°C, and the K_m and V_max values were 0.12?mM and 1,772?µmol/min/mg, respectively. After an incubation at 40°C for 80?min, E25 retained 75% of its basal activity. The enzyme exhibited good tolerance to metal cations, such as Ba²⁺, Ca²⁺, and Cu²⁺ (10?mM), but its activity was strongly inhibited by Co²⁺, Ni²⁺, Mn²⁺, and Zn²⁺. The E25 enzyme was stimulated by glycerol and retained over 60% of its basal activity in the presence of 1% Tween-80 and Triton X-100. Overall, the activity of E25 under alkaline conditions and its organic solvent and detergent tolerance indicate that E25 could be useful as a novel industrial catalyst in biotechnological applications.     

Cryopreservation of <Emphasis Type="Italic">Dendrobium candidum</Emphasis> Wall. ex Lindl. protocorm-like bodies by encapsulation-vitrification

Protocorm-like bodies (PLBs) of Dendrobium candidum Wall. ex Lindl., orchid, were successfully cryopreserved using an encapsulation vitrification method. PLBs were precultured in liquid Murashige and Skoog (MS) medium containing 0.2 mg l⁻¹ α-naphthalene acetic acid and 0.5 mg l⁻¹ 6-benzyladenine enriched with 0.75 M sucrose, and grown under continuous light (36 μmol m⁻² s⁻¹) at 25 ± 1°C for 5 days. PLBs were osmoprotected with a mixture of 2 M glycerol and 1 M sucrose for 80 min at 25°C and dripped in a 0.5 M CaCl₂ solution containing 0.5 M sucrose at 25 ± 1°C and left for 15 min to form Ca-alginate beads (about 4 mm in diameter). Then, these were dehydrated with a plant vitrification solution 2 (PVS₂) consisting of 30% (w/v) glycerol, 15% (w/v) ethylene glycol, and 15% (w/v) dimethyl sulfoxide in 0.5 M sucrose, pH 5.8, for 150 min at 0°C. Encapsulated and dehydrated PLBs were plunged directly into liquid nitrogen for 1 h. Cryopreserved PLBs were then rapidly re-warmed in a water bath at 40°C for 3 min and then washed with MS medium containing 1.2 M sucrose for three times at 10 min intervals. Within 60 days, plantlets with the cryopreserved PLBs developed normal shoots and roots, and without any observed morphological abnormalities, were obtained. The survival rate of encapsulated-vitrified PLBs was above 85%. Thus, this encapsulation-vitrification method was deemed promising for cryopreservation of PLBs of D. candidum.     

A thermostable lipolytic enzyme from a thermophilic Bacillus sp.: Purification and characterization

A thermophilic Bacillus sp. was isolated that secreted an extracellular, thermostable lipolytic enzyme. The enzyme was purified to 58 folds with a specific activity of 9730 units/mg of protein and yield of 10% activity by ammonium sulphate precipitation, Phenyl Sepharose chromatography, gel-permeation followed by Q Sepharose chromatography. The relative molecular mass of the protein was determined to be 61 kDa by SDS-PAGE and approximately 60 kDa by gel permeation chromatography. The enzyme showed optimal activity at 60–65 ^∘C and retained 100% activity after incubation at 60 ^∘C and pH 8.0 for 1 h. The optimum pH was determined to be 8.5. It exhibited 50% of its original activity after 65 min incubation at 70 ^∘C and 23 min incubation at 80 ^∘C. Catalytic function of lipase was activated by Mg⁺⁺ (10 mM), while mercury (10 mM) inactivated the enzyme completely. No effect on enzyme activity was observed with trypsin and chymotrypsin treatment, while 50% inhibition was observed with thermolysin. It was demonstrated that PMSF, SDS, DTT, EDTA, DEPC, βME (100 mM each) and eserine (10 mM) inhibited the activity of the lipolytic enzyme. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K _m and V _max of 0.5 mM and 0.139 μM/min/ml. The enzyme showed preference for short chain triacylglycerol and hydrolyzes triolein at all positions. In contrast to other thermostable Bacillus lipases, this enzyme has very low content of hydrophobic amino acids (22.58 %). Immunological studies showed that the active site and antigen-binding site of enzyme do not overlap.     

Short- and Long-term Effects of High Temperatures (47–49°C) on Tobacco Leaves. II. O2 Uptake and Amylolytic Activity

Detached tobacco (Nicotiana rustiest L.) leaves were exposed for 2 min to a temperature range of 24° to 47.5°C. The heat treatment enbanced O₂ uptake by 30 Vs. Likewise, amylolytic activity in chloroplast extracts was intensified; this activity being observed also in situ. The enhancements can be reversed either by short-term cold treatment (7°C for 10 min), or by application of kinetin.     

Enhanced utilization of glycerol for glyceride synthesis in isolated adipocytes from early pregnant rats

Adipose tissue normally has low glycerol kinase activity, but its expression is enhanced under conditions of augmented insulin sensitivity and/or obesity. Since these conditions occur during early pregnancy, the comparative utilization of glucose or glycerol by isolated adipocytes from rats at 0, 7, 14, or 20 days of pregnancy was studied. Incubations were carried out in the presence of [U¹⁴C]-glucose or -glycerol in medium supplemented or not with 5 mM glucose and 100 nM insulin. The conversion of glucose into esterified fatty acids and glyceride glycerol was greatest in adipocytes from 7-day pregnant rats, the effect being further enhanced by insulin. Both the amount of aquoporin 7 and the in vitro conversion of glycerol into glyceride glycerol were greatest in adipocytes of 7-day pregnant rats, the later being unaltered by insulin. In the presence of glucose, the overall glycerol utilization was lower than in its absence and glycerol conversion into glyceride glycerol was further decreased by insulin, the effect only being significant in adipocytes from 7-day pregnant rats. It is proposed that the enhanced utilization of glycerol for glyceride glycerol synthesis in adipose tissue contributes to the net accumulation of fat depots that normally takes place in early pregnancy.     

Biochemical properties of a serine protease from Aspergillus flavus and application in dehairing

The proteases are enzymes produced by several filamentous fungi with important biotechnological applications. In this work, a protease from Aspergillus flavus was characterized. The culture filtrate of A. flavus was purified to homogeneity by Sephacryl S-200 column chromatography followed by CM–cellulose. The molecular weight of the purified enzyme was estimated to be approximately 32?kDa by SDS–PAGE. The enzyme hydrolysed BTpNA (N-α-benzoyl-dl-tyrosyl-p-nitroanilide), azo-casein and casein as substrates. Optimal temperature and pH were 55?°C and 6.5, respectively. The enzyme was stimulated by Mg²⁺, Ca²⁺, Zn²⁺ and inhibited by Hg²⁺ and Ag²⁺ and Cu²⁺. The protease showed increased activity with detergents, such as Tween 80 and Triton X, and was stable to the reducing agents, such as β-mercaptoethanol. The protease activity was strongly inhibited in the presence of phenylmethylsulfonyl fluoride, indicating it is a serine protease. The enzyme entrapped in calcium alginate beads retained its activity for longer time and could be reused up to 10 times. The thermostability was increased after the immobilization and the enzyme retained 100% of activity at 45?°C after 60?min of incubation, and 90% of residual activity at 50?°C after 30?min. In contrast, the free enzyme only retained 10% of its residual activity after 60?min at 50?°C. The enzymatic preparation was demonstrated to be efficient in the capability of dehairing without destruction of the hide. The remarkable properties such as temperature, pH and immobilization stability found with this enzyme assure that it could be a potential candidate for industrial applications.     

Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) by vitrification

The seeds of a Japanese terrestrial orchid (Bletilla striata Rchb.f.) were germinated and cultured on solidified new Dogashima (ND) medium for 10 days. These embryos were then precultured on ND medium supplemented with 0.3 m sucrose for 3 days at 25°C in continuous dark. The embryos were then overlaid with a mixture of 2 m glycerol and 0.4 m sucrose for 15 min at 25°C and finally dehydrated with highly concentrated vitrification solution (PVS2) for 3 h at 0°C prior to immersion into liquid nitrogen for 30 min. After rapid warming, the embryos were washed with liquid ND medium supplemented with 1.2 m sucrose for 20 min and then plated on ND medium. Successfully vitrified and warmed embryos developed into normal plantlets. The rate of plant regeneration amounted to about 60%. This vitrification method appears to be a promising technique for cryopreservation of orchids. Received: 19 September 1996 / Revision received: 3 January 1997 / Accepted: 24 February 1997     

Purification and characterization of polygalacturonase produced by thermophilic Thermoascus aurantiacus CBMAI-756 in submerged fermentation

An extracellular polygalacturonase was isolated from 5-day culture filtrates of Thermoascus aurantiacus CBMAI-756 and purified by gel filtration and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60–65°C. The apparent K _m with citrus pectin was 1.46 mg/ml and the V _max was 2433.3 μmol/min/mg. The apparent molecular weight of the enzyme was 30 kDa. The enzyme was 100% stable at 50°C for 1 h and showed a half-life of 10 min at 60°C. Polygalacturonase was stable at pH 5.0–5.5 and maintained 33% of initial activity at pH 9.0. Metal ions, such as Zn⁺², Mn⁺², and Hg⁺², inhibited 50, 75 and 100% of enzyme activity. The purified polygalacturonase was shown to be an endo/exo-enzyme, releasing mono, di and tri-galacturonic acids within 10 min of hydrolysis.     

Detergent-compatible,organic solvent-tolerant alkaline protease from Bacillus circulans MTCC 7942: Purification and characterization

Proteases are now recognized as the most indispensable industrial biocatalyst owing to their diverse microbial sources and innovative applications. In the present investigation, a thermostable, organic solvent-tolerant, alkaline serine protease from Bacillus circulans MTCC 7942, was purified and characterized. The protease was purified to 37-fold by a three-step purification scheme with 39% recovery. The optimum pH and temperature for protease was 10 and 60°C, respectively. The apparent molecular mass of the purified enzyme was 43 kD as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The K_m and V_max values using casein-substrate were 3.1 mg/mL and 1.8 µmol/min, respectively. The protease remained stable in the presence of organic solvents with higher (>3.2) log P value (cyclohexane, n-octane, n-hexadecane, n-decane, and n-dodecane), as compared to organic solvents with lower (<3.2) log P value (acetone, butanol, benzene, chloroform, toluene). Remarkably, the protease showed profound stability even in the presence of organic solvents with less log P values (glycerol, dimethyl sulfate [DMSO], p-xylene), indicating the possibility of nonaqueous enzymatic applications. Also, protease activity was improved in the presence of metal ions (Ca²⁺, Mg²⁺, Mn²⁺); enhanced by biosurfactants; hardly affected by bleaching agents, oxidizing agents, and chemical surfactants; and stable in commercial detergents. In addition, a protease–detergent formulation effectively washed out egg and blood stains as compared to detergent alone. The protease was suitable for various commercial applications like processing of gelatinous film and as a compatible additive to detergent formulation with its operative utility in hard water.     

Cytolytic Activity of Naegleria fowleri Cell-free Extract1

ABSTRACT. The cytotoxic activity of a cell-free extract of Naegleria fowleri amebae on B103 rat nerve cells in culture was investigated. The cell-free extract was prepared by subjecting lysed amebae to centrifugation at 100,000 g for 1 h, precipitation of the supernatant fluid with 30–60% saturated ammonium sulfate, and desalting by group exclusion chromatography utilizing Sephadex G-25. The supernatant fluid recovered from this procedure was termed the soluble fraction. The Naegleria cytotoxic activity present in the soluble fraction was assayed by ⁵¹Cr released from labeled B103 cells. The Naegleria soluble fraction, when added to nerve cells, elicited blebs on the B103 target cell surface within 5 min after exposure to the fraction. Later, holes were observed in the B103 cell plasma membrane. These alterations were never observed on untreated B103 cells. Phospholipase A, phospholipase C, and protease activities were associated with the desalted ammonium sulfate-precipitable cytotoxic activity of N. fowleri cell-free lysate. The cytotoxic activity was impaired by ethylenediamine-tetraacetate (EDTA), phospholipase A inhibitor (Rosenthal's reagent), heating at 50°C for 15 min, or incubation at pH 10 for 60 min. Repeated freeze-thawing and inhibitors of proteolytic enzymes had no effect on the cytotoxic activity. Small amounts of ethanol (5% v/v) enhanced cytotoxic activity of the fraction. Phospholipases A and C, as well as other as yet unidentified cytolytic factors may be responsible for producing ⁵¹Cr release from target cells by the soluble fraction of N. fowleri extracts.