Optimization of β-mannanase production by Bacillus subtilis US191 using economical agricultural substrates

The Bacillus subtilis US191 strain producing highly thermostable β-mannanase was previously selected as potential probiotic candidate for application as feed supplement in poultry industry. Initially, the level of extracellular β-mannanase production by this strain was 1.48 U ml⁻¹. To improve this enzyme titer, the present study was undertaken to optimize the fermentation conditions through experimental designs and valorization of agro-industrial byproducts. Using the Plackett–Burman design, in submerged fermentation, a set of 14 culture variables was evaluated in terms of their effects on β-mannanase production. Locust bean gum (LBG), soymeal, temperature, and inoculum size were subsequently optimized by response surface methodology using Box–Behnken design. Under optimized conditions (1 g L⁻¹ LBG, 8 g L⁻¹ soymeal, temperature of 30°C and inoculum size of 10¹⁰ CFU ml⁻¹), a 2.59-fold enhancement in β-mannanase titer was achieved. Next, to decrease the enzyme production cost, the effect of partial substitution of LBG (1 g L⁻¹) by agro-industrial byproducts was investigated, and a Taguchi design was applied. This allowed the attaining of a β-mannanase production level of 8.75 U ml⁻¹ in presence of 0.25 g L⁻¹ LBG, 5 g L⁻¹ of coffee residue powder, 5 g L⁻¹ of date seeds powder, and 5 g L⁻¹ of prickly pear seeds powder as mannans sources. Overall, a 5.91-fold improvement in β-mannanase production by B. subtilis US191 was achieved.     

Gene interaction in DNA microarray data is decomposed by information geometric measure

MOTIVATION: Given the vast amount of gene expression data, it is essential to develop a simple and reliable method of investigating the fine structure of gene interaction. We show how an information geometric measure achieves this. RESULTS: We introduce an information geometric measure of binary random vectors and show how this measure reveals the fine structure of gene interaction. In particular, we propose an iterative procedure by using this measure (called IPIG). The procedure finds higher-order dependencies which may underlie the interaction between two genes of interest. To demonstrate the method, we investigate the interaction between the two genes of interest in the data from human acute lymphoblastic leukemia cells. The method successfully discovered biologically known findings and also selected other genes as hidden causes that constitute the interaction. AVAILABILITY: Softwares are currently not available but are possibly made available in future at http://www.mns.brain.riken.go.jp/~nakahara/DNA_pub.html where all the related information is also linked.     

PCR detection of DNAs of animal origin in feed by primers based on sequences of short and long interspersed repetitive elements

PCR primers for the detection of materials derived from ruminants, pigs, and chickens were newly designed on the basis of sequences of the Art2 short interspersed repetitive element (SINE), PRE-1 SINE, and CR1 long interspersed repetitive element (LINE), respectively. These primers amplified the SINE or LINE from total DNA extracted from the target animals and from test feed containing commercial meat and bone meal (MBM). With the primers, detection of Art2, PRE-1, or CR1 in test feed at concentrations of 0.01% MBM or less was possible. This method was suitable for the detection of microcontamination of feed by animal materials.     

Effect of Li+ upon the Mg2+-dependent activation of recombinant Gialpha1

Although lithium salts have been used in the treatment and prophylaxis of manic-depressive or bipolar patients for 50 years, the mechanism of the pharmacologic action of Li+ is unknown. Based on activity studies of inhibitory and stimulatory guanine-binding (G) proteins in rat cortical membranes, it was proposed that Li+ inhibition of G-proteins may account for its pharmacologic action. We used the purified alpha subunit of the recombinant inhibitory G-protein, rGialpha1, and found that Li+ at therapeutic levels significantly inhibited the formation of the GDP.AlF4-.rGialpha1 complex. Because our studies were conducted with a purified, metal-reconstituted G-protein rather than with cell membrane suspensions, our Li+ inhibition results lend additional support to the G-protein hypothesis for Li+ action.     

Oxidative stress-related lung dysfunction by chromium(VI): alleviation by Citrus aurantium L.

Chromium(VI), a very strong oxidant, causes high cytotoxicity through oxidative stress in tissue systems. Our study investigated the potential ability of ethanolic Citrus aurantium L., family Rutaceae extract, used as a nutritional supplement, to alleviate lung oxidative damage induced by Cr(VI). A high-performance liquid chromatography coupled with a mass spectrometer method was developed to separate and identify flavonoids in C. aurantium L. Six flavonoids were identified, as (1) poncirin, (2) naringin, (3) naringenin, (4) quercetin, (5) isosinensetin, and (6) tetramethyl-o-isoscutellarein. Adult Wistar rats, used in this study, were divided into six groups of six animals each: group I served as controls which received standard diet, group II received via drinking water K₂Cr₂O₇ alone (700 ppm), groups III and IV were pretreated for 10 days with ethanol extract of C. aurantium L. at doses of 100 and 300 mg/kg body weight/day, respectively, and then K₂Cr₂O₇ was administrated during 3 weeks, and groups V and VI received during 10 days only C. aurantium L. ethanol extract at doses of 100 and 300 mg/kg/day, respectively. Ethanol extract of C. aurantium L. was administered orally. Rats exposed to Cr(VI) showed in lung an increase in malondialdehyde and protein carbonyl levels and a decrease in sulflydryl content, glutathione, nonprotein thiol, and vitamins C and E levels. Decreases in enzyme activities such as in Na⁺K⁺ ATPase, catalase, glutathione peroxidase, and superoxide dismutase were noted. Pretreatment with C. aurantium L. of chromium-treated rats ameliorated all biochemical parameters. Lung histological studies confirmed the biochemical parameters and the beneficial role of C. aurantium L.     

Effect of dietary fiber of “Rihane” barley grains and azoxymethane on serum and liver lipid variables in Wistar rats

The aim of the present study is to evaluate the effects of diet enriched with dietary fiber of barley variety “Rihane” and azoxymethane on serum and liver lipid variables in male rats. Forty male rats were divided into four groups and fed on control diet or experimental diet that contained control enriched with dietary fiber of barley variety “Rihane”. Animals were injected with saline (controls) or azoxymethane (20 mg/kg body weight s.c.) at 7 and 8 weeks of age. The experimental diet significantly decreased cholesterol level compared with the control diet. Rats fed with BR diet significantly increased the serum high-density lipoprotein (HDL) cholesterol and significantly decreased low-density lipoprotein (LDL) cholesterol concentrations. The experimental diet decreased the atherogenic index (p < 0.05) compared with the control diet. Whereas the azoxymethane induced a significant increase of liver lipid, serum LDL and triglyceride concentrations, but it caused a significant reduction of HDL. Consequently, the ratio of HDL/TC decreased significantly compared with the control (p < 0.05). Accordingly, these results indicated that the diet enriched with dietary fiber of barley variety “Rihane” could be effective in decreasing the atherogenic risk factors in rats whereas the use of the azoxymethane as colon-specific carcinogen substance altered the lipid metabolism.     

Differential tissue distribution and specificity of phenoloxidases from the Pacific oyster Crassostrea gigas

Phenoloxidases (POs) play a key role in melanin production, are involved in invertebrate immune mechanisms, and have been detected in different bivalves. Recently, we identified catecholase- and laccase-like PO activities in plasma and haemocyte lysate supernatant (HLS) of the Pacific oyster Crassostrea gigas. To go further in our investigations, the aims of this study were (i) to determine the tissue distribution of PO activities in C. gigas, and (ii) to identify and characterise the different sub-classes of POs (i.e. tyrosinase, catecholase and/or laccase) involved in these oxido-reductase activities. With dopamine and p-phenylenediamine (PPD) but not with l-tyrosine used as substrates, PO-activities were detected by spectrophotometry in the gills, digestive gland, mantle, and muscle. These results suggest the presence of catecholase and laccase but not of tyrosinase activities in oyster tissues. The highest activity was recovered in the digestive gland. PO-like activities were all inhibited by 1-phenyl-2-thiourea (PTU) and by the specific laccase inhibitor, cethyltrimethylammonium bromide (CTAB). With dopamine as substrate, the catecholase inhibitor 4-hexylresorcinol (4-HR) only inhibited PO in the muscle. SDS-PAGE zymographic assays with dopamine and PPD elicited a unique ~40kDa protein band in the muscle. In the other tissues, laccase-like activities could be related to ~10kDa and/or ~200kDa protein bands. The ~10kDa protein band was also detected in plasma and HLS, confirming the presence of a laccase in the later compartments, and probably in most of the tissues of C. gigas. This is the first time to our knowledge that a ~10kDa protein band is associated to a laccase-like activity in a mollusc species, contributing to the characterisation of phenoloxidase activities in marine bivalves.     

Dwindling genetic diversity in European ground squirrels?

The European ground squirrel (Spermophilus citellus) is endangered and in decline. Populations are increasingly fragmented, and only a coordinated conservation effort at the European level may guarantee its long-term survival. To obtain a general population genetic picture on a larger geographic scale, we screened 117 individuals from seven local populations in Hungary, Romania, and Austria for allelic variation at eleven microsatellite loci. We found a high (23.4%) proportion of private alleles, and a moderate to somewhat elevated level (15.27%) of partitioning of genetic diversity among populations, compared to that found in many other terrestrial mammals. Genetic variability was significantly higher than in earlier studied Czech populations that are considered genetically depleted, but significantly lower than in undisturbed populations of S. suslicus and S. brunneus, that are similar to the European ground squirrel in their ecological requirements, reproductive biology, and social organization. Genetic diversity was also lower than in most presumably “undisturbed” populations of other Sciurid species. This, together with the observed level and pattern of genetic differentiation among populations, such as no significant increase of genetic differentiation with geographic distance and similar variance of genetic differentiation between populations independent of geographic distance, indicated the prevalence of relatively strong drift effects for all populations. A Bayesian STRUCTURE analysis and a factorial correspondence analysis concordantly revealed a fairly complex genetic composition of local populations, but no major geographic trend in the pattern of the genetic composition. Overall, the results suggest disintegration of local colonies that might earlier have been more connected genetically. The STRUCTURE analysis also suggested anthropogenic translocations among single Hungarian populations. Our data on genetic diversity and its distribution do not object to such conservation measures. Translocation of individuals particularly from nearby populations may increase the chances of survival of small and isolated populations and counteract inbreeding at low densities.