Effect of medium components and time of cultivation on chitin production by Mucor circinelloides (Mucor javanicus IFO 4570) -- a factorial study

Chitin production of mycelia from Mucor circinelloides (Mucor javanicus) was studied with a two-level factorial design in all combinations of the following factors in the culture medium: time of cultivation, concentration of D-glucose, L-asparagine and thiamine. Chitin was characterised by infrared and nuclear magnetic resonance spectroscopy. The four factors showed statistically significant (95%) positive main effects on chitin production, without interactions between them. The highest chitin yield (23.9%) was obtained with the medium containing 60 g/l of glucose, 3 g/l of asparagine and 0.008 mg/l of thiamine and is comparable to the highest in the literature.     

Distribution of quinic acid derivatives and other phenolic compounds in Brazilian propolis

The quinic acid derivatives (including 4-feruoyl quinic and 5-ferruoyl quinic acids characterized for first time in propolis samples) and other phenolic compounds were quantified in thirteen Brazilian propolis samples by HPLC analysis. For chemometrical analysis, the distribution of quinic acid derivatives and other phenolic compounds were considered. The results suggest that the Brazilian propolis with floral origin from Citrus sp. have the highest concentration of the quinic acid derivatives (between 11.0 to 58.4 mg/mg of the dried crude hydroalcoholic extract) and therefore would probably show a more effective hepatoprotective activity.     

Direct organogenesis from hypocotyl-derived explants of annatto (Bixa orellana)

The relative importance of explant, cytokinin type, carbon source and gelling agent for annatto organogenesis was studied. The best organogenic response, including adventitious shoot number and elongation, was obtained when hypocotyl segments and rooted hypocotyls were cultured onto MS medium supplemented with 4.56 M zeatin, 87.6 mM sucrose, and 2.8 g l^–1 Phytagel®. Adventitious shoots derived from hypocotyl segments were less frequent and more difficult to elongate than those derived from rooted hypocotyls. Thidiazuron (TDZ) promoted a higher organogenic response in rooted hypocotyls, resembling a rosette-like structure, but impaired shoot elongation. Histological investigation showed that zeatin-induced meristemoids originated mainly from wounding tissues, and that TDZ induced a high level of mitotic division resulting in several proliferation zones nearby the epidermis and outer cortical tissues. Rhizogenesis efficiency (rooting frequency and root number) was greater at the highest indole-3-butyric acid (IBA) concentration (5.0 M) employed, although calli occurred at the basal end of shoots. Eighty percent of rooted plantlets survived after acclimatization. This optimized regeneration protocol may enable further development of an efficient genetic transformation protocol for this species.     

Crystal structure of alpha-galactosidase from Trichoderma reesei and its complex with galactose: implications for catalytic mechanism

The crystal structures of alpha-galactosidase from the mesophilic fungus Trichoderma reesei and its complex with the competitive inhibitor, beta-d-galactose, have been determined at 1.54 A and 2.0 A resolution, respectively. The alpha-galactosidase structure was solved by the quick cryo-soaking method using a single Cs derivative. The refined crystallographic model of the alpha-galactosidase consists of two domains, an N-terminal catalytic domain of the (beta/alpha)8 barrel topology and a C-terminal domain which is formed by an antiparallel beta-structure. The protein contains four N-glycosylation sites located in the catalytic domain. Some of the oligosaccharides were found to participate in inter-domain contacts. The galactose molecule binds to the active site pocket located in the center of the barrel of the catalytic domain. Analysis of the alpha-galactosidase- galactose complex reveals the residues of the active site and offers a structural basis for identification of the putative mechanism of the enzymatic reaction. The structure of the alpha-galactosidase closely resembles those of the glycoside hydrolase family 27. The conservation of two catalytic Asp residues, identified for this family, is consistent with a double-displacement reaction mechanism for the alpha-galactosidase. Modeling of possible substrates into the active site reveals specific hydrogen bonds and hydrophobic interactions that could explain peculiarities of the enzyme kinetics.     

Ovarian cysts and their consequences on the reproductive performance of swine herds

The aim of this work was to determine the incidence of ovarian cysts in the breeding herd and their consequences in the reproductive performance of the herd. Data from 1990 cyclic sows from two farms, with 0-12 parities, lactation length between 6 and 47 days and weaning-estrus interval between 0 and 32 days were evaluated by ultrasound examination for cyst incidence. As cyst was considered an anaechoic structure with smooth and thin walls with a diameter larger than 2 cm that remained visible for at least 5 days after estrus onset. Cyst incidence was found to be 2.4%. Sows with ovarian cysts have a greater return to estrus rate (34.0 x 7.7%, P<0.01), and cysts were associated with around 10% of regular and irregular return to estrus patterns on both farms. The adjusted farrowing rate (52.2 x 90.0%, P<0.01) and anestrual sows that were not pregnant (10.6 x 0.6%, P<0.01) were also influenced by the appearance of ovarian cysts, but they did not influence litter size (P>0.05). The incidence of cysts was not influenced by parity (P>0.05). Sows with shorter lactation had a greater incidence of cysts (P<0.05). Sows with a weaning-estrus interval shorter than 3 days had a greater incidence of ovarian cysts (P<0.05). The time of the year had no influence on the incidence of ovarian cysts (P<0.05).     

Molecular cloning of a cysteine proteinase cDNA from the cotton boll weevil Anthonomus grandis (Coleoptera: Curculionidae)

The cotton boll weevil (Anthonomus grandis) causes severe cotton crop losses in North and South America. This report describes the presence of cysteine proteinase activity in the cotton boll weevil. Cysteine proteinase inhibitors from different sources were assayed against total A. grandis proteinases but, unexpectedly, no inhibitor tested was particularly effective. In order to screen for active inhibitors against the boll weevil, a cysteine proteinase cDNA (Agcys1) was isolated from A. grandis larvae using degenerate primers and rapid amplification of cDNA ends (RACE) techniques. Sequence analysis showed significant homologies with other insect cysteine proteinases. Northern blot analysis indicated that the mRNA encoding the proteinase was transcribed mainly in the gut of larvae. No mRNA was detected in neonatal larvae, pupae, or in the gut of the adult insect, suggesting that Agcys1 is an important cysteine proteinase for larvae digestion. The isolated gene will facilitate the search for highly active inhibitors towards boll weevil larvae that may provide a new opportunity to control this important insect pest.     

Drug resistance in Chromobacterium violaceum

Chromobacterium violaceum is a free-living bacterium commonly found in aquatic habitats of tropical and subtropical regions of the world. This bacterium is able to produce a large variety of products of biotechnological and pharmacological use. Although C. violaceum is considered to be non-pathogenic, some cases of severe infections in humans and other animals have been reported. Genomic data on the type strain ATCC 12472(T) has provided a comprehensive basis for detailed studies of pathogenicity, virulence and drug resistance genes. A large number of open reading frames associated with various mechanisms of drug resistance were found, comprising a remarkable feature of this organism. Amongst these, beta-lactam (penicillin and cephalosporin) and multidrug resistance genes (drug efflux pumps) were the most numerous. In addition, genes associated with bacitracin, bicyclomycin, chloramphenicol, kasugamycin, and methylenomycin were also found. It is postulated that these genes contribute to the ability of C. violaceum to compete with other bacteria in the environment, and also may help to explain the common drug resistance phenotypes observed in infections caused by this bacterium.     

Extractive cultivation of xylanase by Penicillium janthinellum in a poly(ethylene glycol)/cashew-nut tree gum aqueous two-phase system

Cultivation of the fungus Penicillium janthinellum for xylanase production was studied in a poly(ethylene glycol)/cashew-nut tree gum aqueous two-phase system, using a two-level fractional factorial design. The parameters studied were initial pH, cultivation time, type of agro-industrial residue (oat husk or corn cob), agitation, temperature, and phase-forming polymers. The xylanase produced during fermentation partitioned into the top phase. The agitation and temperature (negative), cultivation time and initial pH (positive) effects proved statistically significant for xylanase production. The highest percentage yield of the xylanase in the top and its production in the top phase, about 97% and 160.7 U/mL, were obtained in cultures of 120 h, 40 rpm, 25 degrees C, and pH 5.0.     

Thyroid hormone induces cerebellar astrocytes and C6 glioma cells to secrete mitogenic growth factors

In this study, the effect of thyroid hormone (triiodothyronine, T(3)) on the secretion of mitogenic growth factors in astrocytes and C6 glioma cells was examined. The proliferating activity of T(3) could be due, at least in part, to the astrocyte secretion of acidic and basic fibroblast growth factor (aFGF and bFGF), tumor necrosis factor-beta, and transforming growth factor-beta. In contrast, the conditioned medium (CM) of T(3)-treated C6 cells was mitogenic to this cell line only after hyaluronidase digestion, suggesting the impairment of growth factor mitogenic activity by hyaluronic acid. Furthermore, the presence of bFGF was significantly greater in the CM of both T(3)-treated astrocytes and T(3)-treated C6 cells than in the corresponding control CM. These data show that T(3) induces cerebellar astrocytes to secrete mitogenic growth factors, predominantly bFGF, that could influence astrocyte and neuronal proliferation via autocrine and paracrine pathways.     

Quantitation of aliphatic suberin in Quercus suber L. cork by FTIR spectroscopy and solid-state (13)C-NMR spectroscopy

This work determined that the percentage of suberin in cork may be found by solid-state (13)C cross polarization/magic angle spinning (CP/MAS) NMR spectroscopy and by FTIR with photoacoustic detection (FTIR-PAS) spectroscopy. A linear relationship is found between the suberin content measured through CP/MAS spectral areas and that measured gravimetrically. Furthermore, application of a partial least squares (PLS1) regression model to the NMR and gravimetric data sets clearly correlates the two sets, enabling suberin quantification with 90% precision. Suberin quantitation by FTIR-PAS spectroscopy is also achieved by a PLS1 regression model, giving 90% accurate estimates of the percentage of suberin in cork. Therefore, (13)C-CP/MAS NMR and FTIR-PAS proved to be useful and accurate noninvasive techniques to quantify suberin in cork, thus avoiding the traditional time consuming and destructive chemical methods.