Atypical Ca<Superscript>2+</Superscript>-independent,raw-starch hydrolysing α-amylase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. GRE1: characterization and gene isolation

Bacillus sp. GRE1 isolated from an Ethiopian hyperthermal spring produced raw-starch digesting, Ca²⁺-independent thermostable α-amylase. Enzyme production in shake flask experiments using optimum nutrient supplements and environmental conditions was 2,360 U l⁻¹. Gel filtration chromatography yielded a purification factor of 33.6-fold and a recovery of 46.5%. The apparent molecular weight of the enzyme was 55 kDa as determined by SDS-PAGE. Presence or absence of Ca²⁺ produced similar temperature optima of 65–70°C. The optimum pH was in the range of 5.5–6.0. The enzyme maintained 50% of its original activity after 45 min of incubation at 80°C and was stable at a pH range of 5.0–9.0. The V _max and K _m values for soluble starch were 42 mg reducing sugar min⁻¹ and 4.98 mg starch ml⁻¹, respectively. Strong inhibitors of enzyme activity included Cu²⁺, Zn²⁺ and Fe²⁺. The enzyme coding gene and the deduced protein translation revealed a characteristic but markedly atypical homology to Bacillus species α-amylase sequences. The enzyme hydrolyzed wheat, corn and tapioca starch granules efficiently below their gelatinization temperatures. Rather than the higher oligosaccharides normally produced by Bacillus α-amylases operating at high temperatures, maltose was the major hydrolysis product with the present enzyme.     

Disturbance calls of five migratory Characiformes species and advertisement choruses in Amazon spawning sites

Species-specific disturbance calls of five commercially-important characiform species are described, the Curimatidae commonly called branquinhas: Potamorhina latior, Potamorhina altamazonica and Psectrogaster amazonica; Prochilodontidae: jaraquí Semaprochilodus insignis and curimatã Prochilodus nigricans. All species have a two-chambered swimbladder and the sonic mechanism, present exclusively in males, utilises hypertrophied red muscles between ribs that adhere to the anterior chamber. The number of muscles is unusually plastic across species and varies from 1 to 4 pairs suggesting considerable evolution in an otherwise conservative system. Advertisement calls are produced in river confluences in the Madeira Basin during the high-water mating season (January–February). Disturbance calls and sampling allowed recognition of underwater advertisement choruses from P. latior, S. insignis and P. nigricans. The advertisement calls of the first two species have largely similar characteristics and they mate in partially overlapping areas in the Guaporé River. However, P. latior sounds have a lower dominant frequency and it prefers to call from river confluences whereas S. insignis shoals occur mostly in the main river channel adjacent to the confluence. These results help identify and differentiate underwater sounds and evaluate breeding areas during the courtship of commercially important characids likely to be affected by two hydroelectric dams.     

Separating Instability from Aggregation Propensity in γS-Crystallin Variants

Molecular dynamics (MD) simulations, circular dichroism (CD), and dynamic light scattering (DLS) measurements were used to investigate the aggregation propensity of the eye-lens protein γS-crystallin. The wild-type protein was investigated along with the cataract-related G18V variant and the symmetry-related G106V variant. The MD simulations suggest that local sequence differences result in dramatic differences in dynamics and hydration between these two apparently similar point mutations. This finding is supported by the experimental measurements, which show that although both variants appear to be mostly folded at room temperature, both display increased aggregation propensity. Although the disease-related G18V variant is not the most strongly destabilized, it aggregates more readily than either the wild-type or the G106V variant. These results indicate that γS-crystallin provides an excellent model system for investigating the role of dynamics and hydration in aggregation by locally unfolded proteins.     

Pivotal Role of the RB Family Proteins in in Vitro Thyroid Cell Transformation

Rat thyroid differentiated cells (PC Cl 3) are an excellent model system with which to study the interaction between differentiation and cell transformation. We previously demonstrated that PC Cl 3 cells expressing the adenovirus E1A gene no longer depend on thyrotropin for growth and do not express thyroid differentiation markers. Here we show that an E1A mutant unable to bind the RB protein failed to transform the PC Cl 3 cells. Conversely, mutations in the E1A p300 interacting region did not affect its transforming ability. The pivotal role of RB family proteins in the thyroid cell transformation is supported by the thyrotropin independence induced by the E7 gene of human papilloma virus type 16, but not by a mutated form in the RB-binding region.     

In vivo DNA gene electro-transfer: a systematic analysis of different electrical parameters

BACKGROUND: Intramuscular plasmid injection followed by electroporation is an efficient method for gene therapy or vaccination. Several protocols have been described that give good transduction levels with several reporter genes. METHODS: In this work we have explored the efficiency of gene delivery upon variation of the different electrical parameters such as pulse length frequency and voltage monitoring both on short- and long-term protein production. RESULTS: Having defined the best performing parameters, we have designed a short electric treatment that gives good levels of plasmid-encoded protein in different species such as mice, rabbits and monkeys.     

Reproductive status is associated with the severity of fibrosis in women with hepatitis C

Introduction

Chronic hepatitis C is the main cause of death in patients with end-stage liver disease. Prognosis depends on the increase of fibrosis, whose progression is twice as rapid in men as in women. Aim of the study was to evaluate the effects of reproductive stage on fibrosis severity in women and to compare these findings with age-matched men.

Materials and Methods

A retrospective study of 710 consecutive patients with biopsy-proven chronic hepatitis C was conducted, using data from a clinical database of two tertiary Italian care centers. Four age-matched groups of men served as controls. Data about demographics, biochemistry, liver biopsy and ultrasonography were analyzed. Contributing factors were assessed by multivariate logistic regression analysis.

Results

Liver fibrosis was more advanced in the early menopausal than in the fully reproductive (P<0.0001) or premenopausal (P = 0.042) group. Late menopausal women had higher liver fibrosis compared with the other groups (fully reproductive, P<0.0001; premenopausal, P = <0.0001; early menopausal, P = 0.052). Multivariate analyses showed that male sex was independently associated with more severe fibrosis in the groups corresponding to premenopausal (P = 0.048) and early menopausal (P = 0.004) but not late menopausal pairs. In women, estradiol/testosterone ratio decreased markedly in early (vs. reproductive age: P = 0.002 and vs. premenopausal: P<0.0001) and late menopause (vs. reproductive age: P = 0.001; vs. premenopausal: P<0.0001). In men age-matched with menopausal women, estradiol/testosterone ratio instead increased (reproductive age group vs. early: P = 0.002 and vs. late M: P = 0.001).

Conclusions

The severity of fibrosis in women worsens in parallel with increasing estrogen deprivation and estradiol/testosterone ratio decrease. Our data provide evidence why fibrosis progression is discontinuous in women and more linear and severe in men, in whom aging-associated estradiol/testosterone ratio increase occurs too late to noticeably influence the inflammatory process leading to fibrosis.     

Flooding tolerance of Tabebuia cassinoides: Metabolic, morphological and growth responses

Tabebuia cassinoides (Lam.) DC (Bignoniaceae) is a tree species that occurs in swampy areas of the coastal “restinga” in SE Brazil (a coastal sandy plains scrub and forest formation). To elucidate possible adaptive strategies that enable this species to occupy areas subjected to seasonal or perennial waterlogging, metabolic, morphological and growth responses of plants under flooding conditions were studied. The root system of T. cassinoides plants presented elevated amounts of ethanol (10.6 μmol g⁻¹ fresh wt) only in the first 5 d of soil water saturation. The two-fold increase in ethanol production under flooding was corroborated by an increase in ADH activity in the same period. Lactic acid concentrations did not change significantly during four months of flooding treatment. The decrease of alcoholic fermentation under hypoxia was associated with the appearing of new roots. The induction of aerenchyma formation in roots developed under flooding conditions, allowed oxygen transport from the shoot to these organs, thus maintaining an aerobic respiration. We conclude that this characteristic and the capacity to oxidize the rhizosphere are probably responsible for the survival and growth of plants while flooded and for their success in an environment, which restricts the presence of the majority of competing tree species.     

Validation of a modified fluorimetric assay for the screening of trichomonacidal drugs

A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis has been optimised to provide a more sensitive method to evaluate potential trichomonacidal compounds. Resazurin has been used in recent years to test drugs against different parasites, including trichomonadid protozoa; however, the reproducibility of these resazurin-based methods in our laboratory has been limited because the flagellate culture medium spontaneously reduces the resazurin. The objective of this work was to refine the fluorimetric microassay method previously developed by other research groups to reduce the fluorescence background generated by the media and increase the sensitivity of the screening assay. The experimental conditions, time of incubation, resazurin concentration and media used in the microtitre plates were adjusted. Different drug sensitivity studies against T. vaginalis were developed using the 5-nitroimidazole reference drugs, new 5-nitroindazolinones and 5-nitroindazole synthetic derivatives. Haemocytometer count results were compared with the resazurin assay using a 10% solution of 3 mM resazurin dissolved in phosphate buffered saline with glucose (1 mg/mL). The fluorimetric assay and the haemocytometer counts resulted in similar percentages of trichomonacidal activity in all the experiments, demonstrating that the fluorimetric microtitre assay has the necessary accuracy for high-throughput screening of new drugs against T. vaginalis.     

Echinoids (Echinodermata: Echinoidea) from the Gulf of Mexico

The echinoid fauna of the Gulf of Mexico collected during three research cruises (20-1260 m depth) was surveyed from samples were taken at 43 stations. A total of 190 individuals were identified (eight orders, 11 families, 15 genera and 18 species). Six species are new records for the Gulf of Mexico: Stylocidaris lineata, Phormosoma placenta placenta, Plesiodiadema antillarum, Plethotaenia spatangoides, Brissopsis atlantica and Hypselaster limicolus. This adds to the little information available on the echinoid fauna of Tamaulipas, Veracruz, Tabasco, Campeche and Yucatan states in Mexico.     

Intersexual cooperation during male clasping of external female genitalia in the spider Physocyclus dugesi (Araneae, Pholcidae)

Sexual conflict may influence the shape and evolution of body structures that males use to grasp females during mating. Not only sexual coercion but also intersexual cooperation may be involved during clasping behavior. Among pholcid spiders, secondary sexual modifications of the male chelicerae, such as apophyses with spines or tooth-like processes, function to grasp the female by specific parts of her external genitalia such as grooves or apophyses of the epigynum. We analyzed how the female and the male respond when their structures for clasping are experimentally modified in the pholcid Physocyclus dugesi. We used three treatment groups for virgin females that differed in the manipulation of the epigynum apophyses (uncovered, partially covered, and fully covered by a plaster) and two groups of males (uncovered and fully covered cheliceral apophyses). We found that females are mainly cooperative to courting males not only when the female genital apophyses were experimentally covered but also when the male cheliceral apophyses were covered. The current data also indicate behavioral flexibility in males during courtship, especially when they had difficulty in genital intromission. Our experimental results, together with previous observational studies, support a modulated-cooperative scenario between the sexes for cheliceral clasping and genital intromission in pholcid spiders.