Production of extracellular lactase fromFusarium moniliforme

Summary A strain ofFusarium moniliforme, previously used for microbial protein production, excreted lactase (-D-galactosidase, EC.3.2.1 23) when cultivated either in a whey liquid medium or on a wheat bran solid medium. The enzyme produced in both media had pH and temperature optima of 4–5 and 50–60°C respectively and was particularly suitable for processing acid whey.In the whey culture, maximum lactase yield was observed after 95 h of growth at 30°C and whey lactose concentration of 9%. The addition of ammonium, potassium and sodium ions to the growth medium considerably enhanced lactase production. A maximum enzyme yield corresponding to hydrolysis of 3 nmoles o-nitrophenyl--D-galactopyranoside sec^–1 ml^–1 of growth medium, at pH 5 and 60°C, was obtained.In the wheat bran culture, the maximum enzyme yield was obtained after 140 h of growth at 28–30°C. A marked increase in the enzyme production was observed when nitrate or phosphate was added to the growth medium. Also, the addition of certain agricultural by-products (molasses, whey) enhanced lactase production. The observed maximum yield corresponding to the hydrolysis of 182 nmoles of ONPG sec^–1 g^–1 of wheat bran, at pH 5 and 60°C, is comparable to that reported for certain microorganisms used commercially for lactase production.     

SIRT1 Activation by Small Molecules: KINETIC AND BIOPHYSICAL EVIDENCE FOR DIRECT INTERACTION OF ENZYME AND ACTIVATOR?

SIRT1 is a protein deacetylase that has emerged as a therapeutic target for the development of activators to treat diseases of aging. SIRT1-activating compounds (STACs) have been developed that produce biological effects consistent with direct SIRT1 activation. At the molecular level, the mechanism by which STACs activate SIRT1 remains elusive. In the studies reported herein, the mechanism of SIRT1 activation is examined using representative compounds chosen from a collection of STACs. These studies reveal that activation of SIRT1 by STACs is strongly dependent on structural features of the peptide substrate. Significantly, and in contrast to studies reporting that peptides must bear a fluorophore for their deacetylation to be accelerated, we find that some STACs can accelerate the SIRT1-catalyzed deacetylation of specific unlabeled peptides composed only of natural amino acids. These results, together with others of this study, are at odds with a recent claim that complex formation between STACs and fluorophore-labeled peptides plays a role in the activation of SIRT1 (Pacholec, M., Chrunyk, B., Cunningham, D., Flynn, D., Griffith, D., Griffor, M., Loulakis, P., Pabst, B., Qiu, X., Stockman, B., Thanabal, V., Varghese, A., Ward, J., Withka, J., and Ahn, K. (2010) J. Biol. Chem. 285, 8340–8351). Rather, the data suggest that STACs interact directly with SIRT1 and activate SIRT1-catalyzed deacetylation through an allosteric mechanism.     

Identification and Characterisation of the RalA-ERp57 Interaction: Evidence for GDI Activity of ERp57

RalA is a membrane-associated small GTPase that regulates vesicle trafficking. Here we identify a specific interaction between RalA and ERp57, an oxidoreductase and signalling protein. ERp57 bound specifically to the GDP-bound form of RalA, but not the GTP-bound form, and inhibited the dissociation of GDP from RalA in vitro. These activities were inhibited by reducing agents, but no disulphide bonds were detected between RalA and ERp57. Mutation of all four of ERp57’s active site cysteine residues blocked sensitivity to reducing agents, suggesting that redox-dependent conformational changes in ERp57 affect binding to RalA. Mutations in the switch II region of the GTPase domain of RalA specifically reduced or abolished binding to ERp57, but did not block GTP-specific binding to known RalA effectors, the exocyst and RalBP1. Oxidative treatment of A431 cells with H₂O₂ inhibited cellular RalA activity, and the effect was exacerbated by expression of recombinant ERp57. The oxidative treatment significantly increased the amount of RalA localised to the cytosol. These findings suggest that ERp57 regulates RalA signalling by acting as a redox-sensitive guanine-nucleotide dissociation inhibitor (RalGDI).     

Supervillin binding to myosin II and synergism with anillin are required for cytokinesis

Cytokinesis, the process by which cytoplasm is apportioned between dividing daughter cells, requires coordination of myosin II function, membrane trafficking, and central spindle organization. Most known regulators act during late cytokinesis; a few, including the myosin II–binding proteins anillin and supervillin, act earlier. Anillin''s role in scaffolding the membrane cortex with the central spindle is well established, but the mechanism of supervillin action is relatively uncharacterized. We show here that two regions within supervillin affect cell division: residues 831–1281, which bind central spindle proteins, and residues 1–170, which bind the myosin II heavy chain (MHC) and the long form of myosin light-chain kinase. MHC binding is required to rescue supervillin deficiency, and mutagenesis of this site creates a dominant-negative phenotype. Supervillin concentrates activated and total myosin II at the furrow, and simultaneous knockdown of supervillin and anillin additively increases cell division failure. Knockdown of either protein causes mislocalization of the other, and endogenous anillin increases upon supervillin knockdown. Proteomic identification of interaction partners recovered using a high-affinity green fluorescent protein nanobody suggests that supervillin and anillin regulate the myosin II and actin cortical cytoskeletons through separate pathways. We conclude that supervillin and anillin play complementary roles during vertebrate cytokinesis.     

Telomere length and risk of melanoma,squamous cell carcinoma,and basal cell carcinoma

Background: Telomeres help maintain chromosomal structure and may influence tumorigenesis. We examined the association between telomere length and skin cancer in a clinic-based case-control study of 198 melanoma cases, 136 squamous cell carcinoma (SCC) cases, 185 basal cell carcinoma (BCC) cases, and 372 healthy controls. Methods: Cases were histologically confirmed patients treated at the Moffitt Cancer Center and University of South Florida Dermatology Clinic in Tampa, FL. Controls self-reported no history of cancer and underwent a skin cancer screening exam at study enrollment to rule out the presence of skin cancer. Quantitative real time PCR was used to measure telomere length in peripheral blood samples. Results: Melanoma patients had longer telomeres than controls (odds ratio (OR) = 3.75; 95% confidence interval (CI): 2.02–6.94 for highest versus lowest tertile) (P for trend = <0.0001). In contrast, longer telomere length was significantly inversely associated with SCC (OR = 0.01; 95% CI: 0.00–0.05 for highest versus lowest tertile) (P for trend = <0.0001) and BCC (OR = 0.10; 95% CI: 0.06–0.19 for highest versus lowest tertile) (P for trend = <0.0001). Conclusion: Telomere length may be involved in the development of skin cancer, although the effect on cancer risk differs for melanoma and non-melanoma carcinomas. Our findings suggest that long telomere length is positively associated with melanoma while inversely associated with SCC and BCC.     

The Role of Oxidative Stress in the Longevity and Insecticide Resistance Phenotype of the Major Malaria Vectors Anopheles arabiensis and Anopheles funestus

Oxidative stress plays numerous biological roles, both functional and pathological. The role of oxidative stress in various epidemiologically relevant biological traits in Anopheles mosquitoes is not well established. In this study, the effects of oxidative stress on the longevity and insecticide resistance phenotype in the major malaria vector species An. arabiensis and An. funestus were examined. Responses to dietary copper sulphate and hydrogen peroxide were used as proxies for the oxidative stress phenotype by determining the effect of copper on longevity and hydrogen peroxide lethal dose. Glutathione peroxidase and catalase activities were determined colorimetrically. Oxidative burden was quantified as protein carbonyl content. Changes in insecticide resistance phenotype were monitored by WHO bioassay. Insecticide resistant individuals showed an increased capacity for coping with oxidative stress, mediated by increased glutathione peroxidase and catalase activity. This effect was observed in both species, as well as in laboratory strains and F₁ individuals derived from wild-caught An. funestus mothers. Phenotypic capacity for coping with oxidative stress was greatest in strains with elevated Cytochrome P450 activity. Synergism of oxidative stress defence enzymes by dietary supplementation with haematin, 3-Amino-1, 2, 4-triazole and Sodium diethyldithiocarbamate significantly increased pyrethroid-induced mortality in An. arabiensis and An. funestus. It is therefore concluded that defence against oxidative stress underlies the augmentation of the insecticide resistance phenotype associated with multiple blood-feeding. This is because multiple blood-feeding ultimately leads to a reduction of oxidative stress in insecticide resistant females, and also reduces the oxidative burden induced by DDT and pyrethroids, by inducing increased glutathione peroxidase activity. This study highlights the importance of oxidative stress in the longevity and insecticide resistance phenotype in malaria vectors.