Biotransformation of cholesterol by Micrococcus species mediated by plasmid DNA

A steroid-biotransforming strain RJ6 was identified as Micrococcus roseus. This bacterium has a 10kb plasmid pMQV10. Curing mediated through cultivation of the culture with a low concentration (200ng/ml) of mitomycin C is described. Loss of cholesterol degradation (chol⁺) and streptomycin resistance (Sm^r) phenotypes as a consequence of the loss of plasmid indicate the extrachromosomal location of these two genes in this strain. An electroporation procedure was developed for transformation of cured strain of Micrococcus (RJC6) by plasmids. Frequency of greater than 10⁵ transformants/g DNA was achieved, which is 100-fold higher than the standard transformation procedure that yielded 5.3×10³ transformants/g DNA in the same strain.     

Optimization of cryopreservation of Artemisia annua L. callus

Cryopreservation of callus tissue of Artimisia annua L. was optimized. Two lines of calli were precultured on MS medium with 5% (v/v) dimethyl sulfoxide, and protected by a cryoprotectant containing 15% (v/v) ethylene glycol, 15% (v/v) dimethyl sulfoxide, 30% (v/v) glycerol and 13.6% (w/v) sucrose. The highest survival rate of callus A201 reached 87% after it was pretreated at 25°C, cryopreserved by liquid nitrogen, recovered in water bath at 25°C and reloaded at 25°C with 34% (w/v) sucrose solution, and that of callus A202 reached 78% after it was treated as callus A201, except pretreated at 35°C, recovered at 35°C and reloaded with 47.8% (w/v) sucrose solution.     

Purification and characterization of aspartate aminotransferase from developing embryos of the camel tick Hyalomma dromedarii

Aspartate transaminase (AST) activity in the camel tick Hyalomma dromedarii was followed throughout embryogenesis. During purification of AST to homogeneity, ion exchange chromatography lead to four separate forms (termed I, II, III and IV). AST II with the highest specific activity was pure after chromatography on Sephacryl S-300. The molecular mass of AST II was 52KDa for the native enzyme, composed of one subunit of 50KDa. AST II had a Km value of 0.67mM for -ketoglutarate and 15.1mM for aspartate. AST II had a pH optimum of 7.5 with heat stability up to 50°C for 15min. The enzyme was activated by MnCl₂, and inhibited by CaCl₂, MgCl₂, NiCl₂, and ZnCl₂.     

Recombinant Components of the EcoRII Restriction–Modification System: Restriction Endonuclease Can Interact with DNA · RNA Duplexes

To obtain recombinant restriction endonuclease (R) and methylase (M) of the EcoRII restriction–modification system, bacterial strains overproducing their functional hexahistidine derivatives were constructed. Active full-length R·EcoRII was produced only in cells that also expressed M·EcoRII from a multicopy plasmid. Recombinant R·EcoRII bound with hybrid DNA·RNA duplexes.     

Effects of constant and fluctuating temperatures on sporulation and infection by the aphid-pathogenic fungus Pandora neoaphidis

Laboratory studies were performed to assess the importance of temperature on sporulation and infection by the aphid-pathogenic fungus Pandora neoaphidis (Remaudière and Hennebert) Humber. Numbers of primary conidia discharged from mycelium formulated as alginate granules and unformulated mycelial mats were assessed, as well as infection of the potato aphid, Macrosiphum euphorbiae (Thomas) (Homoptera, Hemiptera, Aphididae), using culture plugs as inoculum sources. Sporulation from experiments at constant temperatures indicated the optimum temperature range was 10–20°C for both mycelial preparations and there was no or very little sporulation at 30°C. Infection of aphids kept at 15°C was 34–50%, while infection at 25°C was 11–44%. At 20°C, 77–79% of aphids were infected. Under fluctuating temperature cycles, conidia numbers did not differ when mycelial preparations were maintained at 18–25°C compared with 18–20°C, but fewer conidia were recorded when preparations were exposed continuously to 18–30°C. Infections of inoculated aphids kept for varying numbers of days at 18–25°C varied between 24–47%, but only 3–32% of aphids were infected when exposed to a cycle of 18–30°C for various times. Unformulated mycelial mats of P. neoaphidis appear to be superior to forumlated alginate granules for use in experimental greenhouse and field trials, since temperature stability is similar for both materials but mycelial mats are much easier to produce.     

Expression of gloshedobin,a thrombin-like enzyme from the venom of Gloydius shedaoensis,in Escherichia coli

Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, was expressed in Escherichia coli using expression vector pET-32a(+). The gene was expressed under T7 promotor with a fusion partner of Thx.Tag and a 6xHis.Tag at its 5 terminal. After induction by IPTG for 6 h, the recombinant enzyme was expressed in the cytoplasm. Expression at 25°C gave twice the amount of recombinant gloshedobin in cytoplasm than at 37°C.     

Defective remethylation of homocysteine is related to decreased synthesis of coenzymes B2 in thyroidectomized rats

Summary. We investigated the influence of hypothyroidism on homocysteine metabolism in rats, focusing on a hypothetical deficient synthesis of FAD by riboflavin kinases. Animals were allocated in control group (n=7), thyroidectomized rats (n=6), rats with diet deficient in vitamin B2, B9, B12, choline and methionine (n=7), thyroidectomized rats with deficient diet (n=9). Homocysteine was decreased in operated rats (2.6±1.01 vs. 4.05±1.0mol/L, P=0.02) and increased in deficient diet rats (29.56±4.52 vs. 4.05±1.0mol/L, P=0.001), when compared to control group. Erythrocyte-Glutathione-Reductase-Activation-Coefficient (index of FAD deficiency) was increased in thyroidectomized or deficient diet rats (P=0.004 for both). Methylenetetrahydrofolate-reductase and methionine-synthase activities were decreased in thyroidectomized rats but not in those subjected to deficient diet. Cystathionine--synthase was increased only in operated rats. Taken together, these results showed a defective re-methylation in surgical hypothyroidism, which was due in part to a defective synthesis of vitamin B2 coenzymes. This defective pathway was overcompensated by the increased Cystathionine--synthase activity.     

Isolation and characterization of a phytase with improved properties from Citrobacter braakii

Citrobacter braakii YH-15 produced an intracellular phytase which was purified 12800 fold to homogeneity with the specific activity of 3457 units mg^–1, which is 1.9 times higher than E. coli phytase previously recorded as having the highest specific activity. Its molecular weight was 47 kDa by SDS-PAGE gel. Enzyme activity was optimal at pH 4 and at 50 °C. The K _m value for sodium phytate was 0.46 mM with a V _max 6027 U mg^–1. The phytase was resistant to proteases such as trypsin, pepsin, papain, pancreatin, and elastase.     

Cotransformation of lactococcin-producing, 2.0-mega dalton and erythromycin-resistant pGB 301 plasmids toLactococcus lactis subsp.lactis protoplast

Protoplasts of plasmid-freeLactococcus lactis subsp.lactis LM 0230 and PC₄ strains were cotransformed successfully with the plasmid pools ofL. lactis subsp.lactis 484, a lactosefermenting (Lac⁺), lactococcin-producing (Lap⁺), lactococcin-resistant (Lap^r), sucrosefermenting (Suc⁺) wild strain, its derivatives, and pGB 301 erythromycin resistance plasmid (Ery^r) at the frequencies of 10⁴ transformants/g of DNA. PC₄ protoplasts were transformed at slightly lower frequencies that LM 0230 protoplasts when the same plasmid combinations were used for transformation. Agarose gel electrophoresis of plasmids from three groups of transformants, namely, Lac^–Lap^–Ery^r, Lac⁺Suc⁺Lap⁺Lap^rEry^r, and Lac^–Suc⁺Lap⁺ Lap^rLap^r, confirmed that 2.0 and 65.0 megadalton (MDa) plasmids carried genes for Suc⁺Lap⁺Lap^r and Lac⁺ phenotypes respectively. The protoplasts could be transformed with low-molecular-weight 2.0 MDa Lap plasmid at a relatively higher frequency than those with high-molecular-weight 65.0 MDa Lac plasmid. All the transformants resembled parent culture 484 in terms of lactic acid production (0.810–0.840%), milk curdling time (6 h), and lactococcin activity (7–12 mm, zone of inhibition) againstListeria monocytogenes, Salmonella typhi, andStaphylococcus aureus. The plasmids and their respective phenotypes in PC₄ transformants were genetically more stable than those of LM 0230 protoplasts. The marker plasmid pGB 301 disappeared more frequently from the transformants when present in association with the lowmolecular-weight, high-copy-number 2.0 MDa plasmid, thereby suggesting the incompatibility of these two plasmids.     

Optimization of an electroporation procedure for Kluyveromyces lactis transformation

Summary An electroporation method using a Bio-Rad Gene Pulser has been optimized for introducing heterologous DNA into Kluyveromyces lactis yeasts. The plasmid pCR1, derived from a native Kluyveromyces plasmid, was used to transform K. lactis. This plasmid produces a wheat -amylase and contains both the biosynthetic marker URAA and G418 resistance genes. Transformation was optimal at 4500 V/cm, 25 F, and with 0.2 g plasmid DNA. Transformation efficiencies in the range 10⁴–10⁵ transformants/10⁷ cells/g DNA were obtained.     

Immobilization of alkaline protease from Conidiobolus macrosporus for reuse and improved thermal stability

Alkaline protease from Conidiobolus macrosporus was immobilized on polyamide using glutaraldehyde as a bifunctional agent. The immobilized enzyme was optimally active at a higher temperature of 50°C than the free enzyme (40°C ) and showed a ten-fold increased thermostability at 60°C compared to that of the free enzyme. The efficiency of immobilization was 58% under the optimal conditions of pH and temperature. There was a 14-fold decrease in the K _m of immobilized enzyme compared to the free enzyme. The immobilized enzyme was fully active even after twenty-two cycles of repeated use. It retained 80% activity at 50°C in presence of 8 M urea exhibiting its stability to the denaturant and was compatible with several commercial detergents.     

Relation between trace element levels in plasma and myocardium during coxsackievirus B3 myocarditis in the mouse

During most infections the plasma levels of trace elements change, but it is not clear if this reflects changes in the infected tissues. Coxsackievirus B3 (CB3) infection may result in viral replication, subsequent inflammation and changed trace element levels in the myocardium. In the present study, the trace element levels in the plasma and heart of adult male A/J mice were determined during the pre-inflammatory stage (day 4) of CB3 myocarditis for the following trace elements: aluminium (Al), arsenic (As), calcium (Ca), cobalt (Co), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), selenium (Se), silver (Ag), vanadium (V) and zinc (Zn). The severity of the infection was assessed through clinical signs of disease and trace element levels were measured through inductively-coupled plasma mass-spectrometry (ICP-MS). In the heart, the levels decreased for V (59%; p<0.01), Co (38%; p<0.01), Al (81%; p< 0.01), As (66%; p<0.01) and Se (16%; p<0.01). Increased levels were detected for Mn (13%; p<0.05), Fe (48%; p<0.01), Cu (34%; p<0.01) and Ag (46%; p< 0.01). In the plasma, decreases were detected in the level of Zn (32%; p<0.05), whereas increases were seen in Mn (362%; p<0.05), Fe (272%; p<0.01), Co (71%; p<0.05), Cu (25%; n.s.) and Mg (43%; p<0.01) levels. A correlation was found between the levels in plasma and myocardium for Co (r _s=–0.636; p<0.05), Fe (r _s=0.764; p<0.05), Mn (r _s=0.682; p<0.05) and Mg (r _s=–0.791; p<0.05). Thus, determination of some of these trace elements in the plasma may be useful to indicate target tissue involvement in the early pre- inflammatory stage of an infectious disease. Some of these elements are important nutrients for the immune system, while others may be associated with the development of disease complications, such as cardiac arrhythmias.     

Cloning of a gene encoding a thermo-stable endo-beta-1,4-glucanase from Thermoascus aurantiacus and its expression in yeast

A gene encoding a thermo-stable endo--1,4-glucanase was isolated from the thermophilic fungus, Thermoascus aurantiacusIFO9748, and designated as eg1. Induction of this gene expression at 50°C was stronger than at 30°C. The deduced amino acid sequence encoded by eg1 showed that it belongs to the glycoside hydrolase family 5. The cloned gene was expressed in Saccharomyces cerevisiae and the gene product was purified and characterized. No significant activity loss was detected over 2 h at 70°C and the product was stable from pH 3–10. The enzyme was optimally active at 70°C over 20 min and the optimal pH was 6.     

<Emphasis Type="Italic">In vitro</Emphasis> screening of sugarcane to evaluate smut susceptibility

Tissue-cultured plantlets of three sugarcane (Saccharum spp.) cultivars having a known field smut reaction were screened for susceptibility to Ustilago scitaminea H&P Sydow. Plantlets were inoculated with 0.5 l of a suspension of equally mixed quantities of plus and minus mating type sporidia of U. scitaminea at concentrations ranging from 1×10¹ to 1×10⁶ cells. Fungal sori (whips) were produced in cultivar N12 (intermediate) 6weeks following inoculation with 1×10⁵ mixed sporidia and thereafter in cultivar NCo310 (susceptible) but not in cultivar N19 (resistant). Sori bearing teliospores were produced up to 3months following inoculation and incubation at 26°C. No sori were produced at mixed sporidial concentrations lower than 1×10⁵cells. The in vitro soral production in cultivars N19, N12 and NCo310 was 0, 27.5 and 47.5% respectively. Plantlets inoculated with 1×105sporidia of only one mating-type did not produce sori in any of the three cultivars tested. Blind scoring of an unknown sugarcane cultivar by this method corresponded exactly with its field smut rating.     

Elevated temperature and chemical modification selectively abolishes levan forming activity of levansucrase of Zymomonas mobilis

A levansucrase (SacB) of Zymomonas mobilis was purified to electrophoretic homogeneity from a recombinant Escherichia coli. The 55 kDa enzyme hydrolysed -fructosides but not -glucosides and catalysed levan formation from sucrose as well as raffinose. The optimum temperature for polymerase activity (30°C ) was lower than that for hydrolase activity (50°C ). In contrast to other levansucrases, polymerase activity of levansucrase was inhibited by para- chloromercuribenzoate (1 mM) but with little or no effect on hydrolase activity. Selective modulation of polymerase activity by this inhibitor will be useful in revealing the mechanism of levansucrase catalysis.     

Light utilization and competition between Echinacea purpurea, Panicum virgatum and Ratibida pinnata under greenhouse and field conditions

Competitive interactions were compared under field and greenhouse conditions for three representative tallgrass prairie species, Echinacea purpurea, Panicum virgatum and Ratibida pinnata. These were planted in monoculture and in mixtures of two species using a replacement series design with groups of four or eight plants. Competition was determined from shoot dry weight data collected during 120days in the greenhouse and after 415days in the field. Yields declined with increased density of a single species in the greenhouse from 40days onward and in the field. Relative yields were up to 100% higher in mixtures than in monocultures for all species early on in the greenhouse experiment. Later in the experiment and in the field relative yields of E.purpurea decreased in the presence of P.virgatum and R.pinnata, whereas relative yields of these two species increased in the presence of E.purpurea. There were correlations in relative yield between the field and the greenhouse experiment at 80 and 120days. In the greenhouse P.virgatum maintained higher net assimilation rates than the other species. Relative growth rates of all species were higher in monoculture and in mixtures up to 40days, after which they declined, especially for E.purpurea in mixtures. In the field, higher light intensities occurred in pure stands of E.purpurea than in mixed stands with other species. The order of competitive ability that was apparent from these field and greenhouse studies, P.virgatum=R.pinnata>E.purpurea, could be partially explained by photosynthetic rates in relation to canopy light interception.     

Composition of Endemic Fish Assemblages in Relation to Exotic Species and River Regulation in a Temperate Stream

Spatial variation in the fish community of the regulated Raia stream (Portugal) was examined during the summer of 1995. Variation in the native fish community (abundance of species-size combinations) was explicitly related to both the abundance of exotic species and habitat variables. The fish community changed in space from assemblages characterised by the high relative abundance of Leuciscus pyrenaicus, Leuciscus alburnoides and the smaller size classes of Barbus bocagei (all Iberian endemics with total length, TL, < 100mm to assemblages characterised by the high relative abundance of Chondrostoma polylepis (Iberian endemic) and B. bocagei with TL > 200mm and of exotics Micropterus salmoides and Lepomis gibbosus (> 100mm TL). The former assemblages used shallow sites far from downstream dams with some current whereas the latter assemblages used deeper sites closer to downstream dams without current velocity and with abundant floating macrophytes. Both exotic species and habitat variables were significant correlates of endemic assemblage composition in the Raia stream and the abundance of M. salmoides > 150mm in TL was the best biotic predictor of endemic assemblage composition. The total variation in the community of endemic fish was partitioned into four components: (i) associated uniquely with exotic species - 12.6%, (ii) associated uniquely with habitat variables - 27.6%, (iii) associated both with exotic species and habitat variables - 14.5%, and (iv) that unexplained - 45.3%. A significant association of exotic species with the endemic fish community remained after accounting for the selected environmental variables and a strong (habitat) x (exotic species) interaction was indicated.     

Biomethanation of low pH petrochemical wastewater using up-flow fixed-film anaerobic bioreactors

Anaerobic digestion of wastewater from a petrochemical plant, manufacturing Nylon-6 was studied in continuously fed up-flow fixed-film column reactors using different biomass support materials such as bonechar, charcoal, bricks, plastic beads and polyurethane foam under varying hydraulic and organic loading rates. Experimental results showed bonechar as the best support material with high biomass-retaining capacity because of its high specific surface area (53.35m²g^–1 of bedding material) and pore specific volume (0.244cm³g^–1 of bedding material). This system could treat waste water at hydraulic retention times (HRT) as low as 2.5 days with organic loading rates as high as 21.76kg COD m^–3 day^–1 using acidic feed of pH 2.5 resulting in a 95% COD reduction with biogas production of 11.76m³ m^–3 of reactor volume day^–1. Total alkalinity of 1700mg CaCO₃ l^–1 and pH of 7.5 of the treated wastewater were observed at 2.5 days HRT, indicating that methanogenesis appear to be alkalizing step and wastewater with pH as low as 2.5 can be treated as such without neutralization with retention of methanogenic biomass on bonechar.     

Growth, Natural Mortality, Length–weight Relationship, Maximum Length and Length-at-first-maturity of the Coelacanth Latimeria chalumnae

Based on the re-interpretation of published data, the von Bertalanffy growth function parameters of the coelacanth, Latimeria chalumnae, are estimated as L=218cm total length, s.e. 23; K=0.059 (year^–1), s.e. 0.012; t₀=–3.3 (year), s.e. 0.5, corresponding to a life span of 48 years. The length–weight relationship of the form W=a*TLb, with wet weight (W) in g and total length (TL) in cm, is estimated as a=0.0278, b=2.89, r²=0.893, n=87, range=42.5–183cm TL. Using extreme value theory, the maximum length for female coelacanths is estimated as 199cm TL (95% confidence interval=175–223cm TL) and for males as 168cm TL (95% confidence interval 155–180cm TL). Based on data from seven females with embryos or mature eggs, the length-at-first-maturity for females is estimated to be about 150cm TL, corresponding to an age of about 16 years. Based on the value of t₀=–3.3 years and on the presence of three scale rings found in a newborn coelacanth, the period of embryogenesis lasts for about three years, the longest known in vertebrates. The natural mortality rate is estimated at M=0.12. Population food consumption is found to be 1.4 times the existing biomass per year, and gross food conversion efficiency indicates that only 10% of the consumed food is utilized for somatic growth.     

Age and Growth of the Whiskery Shark, Furgaleus macki, from Southwestern Australia

Age and growth of the whiskery shark, Furgaleus macki, from southwestern Australia were examined using vertebral ageing and tag-recapture data. The readability of bands on the vertebral centra varied markedly between individuals. Four readers were used to make band counts, with the most experienced reader having the lowest index of average percent error and the highest level of agreement with final counts. Marginal increment analysis indicated that opaque bands form in January. With parturition occurring from August to October, size data suggests that the first band is probably formed 15–17 months after birth. The age at maturity was estimated to be 4.5 years for males, and 6.5 years for females. The oldest male was 10.5 years, and oldest female was 11.5 years. Von Bertalanffy growth parameters for males were L =121.5cm fork length, K=0.423 year^–1, t ₀=–0.472 years, were L =120.7cm fork length, K=0.369 year^–1, t ₀=–0.544 years for females, and were L =118.1cm fork length, K=0.420 year^–1, t ₀=–0.491 years for combined sexes. Data from a tag recapture study were analysed using a maximum likelihood method to verify the estimates of growth parameters from vertebral ageing. Von Bertalanffy growth parameters from the tag recapture study were L =128.2cm fork length, K=0.288 year^–1, t ₀=–0.654 years. The two methods of estimating growth parameters produced similar results, with rapid growth until approximately 5 years of age, after which there was little increase in length.