Production and partial characterization of high molecular weight extracellular alpha-amylase from Thermoactinomyces vulgaris isolated from Egyptian soil

Optimizing production of alpha-amylase production by Thermoactinomyces vulgaris isolated from Egyptian soil was studied. The optimum incubation period, temperature and initial pH of medium for organism growth and enzyme yield were around 24 h, 55 degrees C and 7.0, respectively. Maximum alpha-amylase activity was observed in a medium containing starch as carbon source. The other tested carbohydrates (cellulose, glucose, galactose, xylose, arabinose, lactose and maltose) inhibited the enzyme production. Adding tryptone as a nitrogen source exhibited a maximum activity of alpha-amylase. Bactopeptone and yeast extract gave also high activity comparing to the other nitrogen sources (NH4CI, NH4NO3, NaNO3, KNO3, CH3CO2NH4). Electrophoresis profile of the produced two alpha-amylase isozymes indicated that the same pattern at about 135-145 kDa under different conditions. The optimum pH and temperature of the enzyme activity were 8.0 and 60 degrees C, respectively and enzyme was stable at 50 degrees C over 6 hours. The enzyme was significantly inhibited by the addition of metal ions (Na+, Co2+ and Ca2+) whereas CI- seemed to act as activator. The enzyme was not affected by 0.1 mM EDTA while higher concentration (10 mM EDTA) totally inactivated the enzyme.     

Rapid Hybridization Probe Assay and PCR for Detection of Chlamydia trachomatis in Urinary Tract Infections: A Prospective Study

Chlamydia trachomatis is a widespread bacterium that causes trachoma and genital tract infections in humans. The fact that the growth of this pathogen does not normally occur outside living cells poses a challenge in its diagnosis. The present study aimed to compare the efficacies of different molecular and cultural methods in the detection of C. trachomatis in urine samples collected from patients with urinary tract infections. Examined detection methods involved the Gen-Probe C. trachomatis (GP-CT) assay, direct antigen detection by enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) method. The efficacies of these methods were compared to that of the cell culture technique depending on sensitivity, specificity, and accuracy. C. trachomatis was detected in 25 out of 50 (50%) of examined urine samples using the cell culture method. Compared with this standard technique, the GP-CT assay was the most sensitive procedure, being able to detect the pathogen in all positive samples, followed by PCR and ELISA, which showed 60% and 40% sensitivities, respectively. PCR and ELISA displayed the highest level of specificity (100%) compared to the cell culture method with the GP-CT assay showing 40% specificity. The rate of accuracy was comparable between the GP-CT, PCR, and ELISA methods ranging from 70–80% of the accuracy of the cell culture method. The above results suggest that C. trachomatis is a frequent pathogen associated with upper and lower urinary tract infections. Both the GP-CT assay and PCR method can be recommended as reliable detection methods for C. trachomatis, and the GP-CT can be used as a screening tool.     

Where to from here? The mechanisms enabling the movement of first instar caterpillars on whole plants using Helicoverpa armigera (Hübner)

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) is an economically-important, polyphagous herbivore in Old World countries. The distribution of larvae within various host plants has been described, but few studies have tried to determine the behavioural mechanisms by which the given distributions arose. Our aim was to determine the mechanisms which enable larval movement on pea plants, starting with first instars. Observations and bioassays determined larval movement in response to light and angled surfaces, as well as the effect of feeding and plant volatiles on these responses. The majority (68–72%) of 1st instars were positively phototactic towards blue, green and white light and 42% towards UV light. In the dark, larvae showed negative geotaxis. The angle of their substrate also had a kinetic effect on larvae; the steeper the angle from horizontal the more larvae moved under all conditions. Phenylacetaldehyde (a flower volatile) suppressed larval movement except at 90°. (Z)-3-Hexenyl acetate (a green leaf volatile) reversed the direction of movement at the flattest angle. Feeding lessened the probability of moving. We suggest that phototaxis and geotaxis are behaviours common to larval lepidopterans (caterpillars), and that these basic behaviours are modulated by environmental, larval, and plant factors to give observed distributions. Using a multinomial model approach, we created a flow chart to qualitatively and quantitatively represent the decision-making process of first instar H. armigera in response to the factors influencing movement.     

Structure-guided approach on the role of substitution on amide-linked bipyrazoles and its effect on their anti-inflammatory activity

A structure-guided modelling approach using COX-2 as a template was used to investigate the effect of replacing the chloro atom located at the chlorophenyl ring of amide-linked bipyrazole moieties, aiming at attaining better anti-inflammatory effect with a good safety profile. Bromo, fluoro, nitro, and methyl groups were revealed to be ideal candidates. Consequently, new bipyrazole derivatives were synthesised. The in vitro inhibitory COX-1/COX-2 activity of the synthesised compounds exhibited promising selectivity. The fluoro and methyl derivatives were the most active candidates. The in vivo formalin-induced paw edoema model confirmed the anti-inflammatory activity of the synthesised compounds. All the tested derivatives had a good ulcerogenic safety profile except for the methyl substituted compound. In silico molecular dynamics simulations of the fluoro and methyl poses complexed with COX-2 for 50 ns indicated stable binding to COX-2. Generally, our approach delivers a fruitful matrix for the development of further amide-linked bipyrazole anti-inflammatory candidates.     

Gene-Gene Interaction Study Between Genetic Polymorphisms of Folate Metabolism and MTR SNPs on Prognostic Features Impact for Breast Cancer

Background:Breast Cancer (BC), the second leading cause of cancer mortality after lung cancer and varied across the world due to genetic and environmental factors. In this study, we evaluated the interaction between the polymorphisms in genes encoding enzymes of folate metabolism: methylenetetrahydrofolate reductase (MTHFR), methionine synthesis reductase (MTR) with the BC prognostic factors.Methods:This study was conducted on 160 Egyptian subjects, 60 controls and 100 cases. Sequencing, RFLP analysis in addition to statistical analysis including Chi‐squared test, haplotype analysis was used to evaluate associations with BC risk and its clinicopathological parameters. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression.Results:Strong significant association with breast cancer risk was observed for the haplotype (T-C-G) of MTHFR C677T/ MTHFR A1289C and MTRA2576G and hormonal receptor expression (ER-/PR-/HER2+), bigger and advanced tumor and metastatic lymph nodes. However, no significant difference was observed for age.Conclusion:The combination of SNPs from MTHFR and MTR genes has a more synergistically genetic effect on BC disease progression. These SNPs could be used as tumor aggressiveness markers among Egyptian females with BC and could help in saving money and time.Key Words: Breast cancer, Methionine synthesis reductase, MTHFR, PCR-RFLP, SNPs     

Inflammatory, hemostatic, and clinical changes in a baboon experimental model for heatstroke.

The mortality and neurological morbidity in heatstroke have been attributed to the host's inflammatory and hemostatic responses to heat stress, suggesting that immunomodulation may improve outcome. We postulated that an experimental baboon model of heatstroke will reproduce human responses and clinical outcome to allow testing of new therapeutic strategies. Eight anesthetized juvenile baboons (Papio hamadryas) were subjected to heat stress in an incubator maintained at 44-47 degrees C until rectal temperature attained 42.5 degrees C (moderate heatstroke; n = 4) or systolic arterial pressure fell to <90 mmHg (severe heatstroke; n = 4) and were allowed to recover at room temperature. Four sham-heated animals served as a control group. Rectal temperature at the end of heat stress was 42.5 +/- 0.0 and 43.3 +/- 0.1 degrees C, respectively. All heat-stressed animals had systemic inflammation and activated coagulation, indicated by increased plasma IL-6, prothrombin time, activated partial thromboplastin time, and D-dimer levels, and decreased platelet count. Biochemical markers and/or histology evidenced cellular injury/dysfunction: plasma levels of thrombomodulin, creatinine, creatine kinase, lactic dehydrogenase, and alanine aminotransferase were increased, and varying degrees of tissue damage were present in liver, brain, and gut. No baboon with severe heatstroke survived. Neurological morbidity but no mortality was observed in baboons with moderate heatstroke. Nonsurvivors displayed significantly greater coagulopathy, inflammatory activity, and tissue injury than survivors. Sham-heated animals had an uneventful course. Heat stress elicited distinct patterns of inflammatory and hemostatic responses associated with outcome. The baboon model of heatstroke appears suitable for testing whether immunomodulation of the host's responses can improve outcome.     

Phosphorylation of tyrosine in cultured human placenta

The [³²P]phosphoamino acids in proteins of first trimester and term-cultured human placentas have been separated and their relative amounts were measured. A significant phosphorylation of tyrosine residues could be detected in the cultured placental tissue at different stages of gestation. The phosphotyrosine accounts for 2–4% of the total acid-stable phosphate in the phosphoamino acids after partial acid hydrolysis. The difference in the extent of [³²P]tyrosine in various placentas seems to be a function of biological variation of the individual placentas, rather than a function of placental age and stage of gestation. In contrast, a significant difference in the phosphorylation ratio of serine and threonine could be measured between first trimester and term placentas. As more evidence is accumulating that protein phosphorylation of tyrosine is involved in the processes of cellular growth and proliferation, our findings of the relatively high tyrosine phosphorylation in human placenta strongly suggest that this type of protein phosphorylation may play an important role in the placental growth and development. Furthermore, these findings may correlate with the existence of the endogenous RNA virus-like particles found in normal human placenta.     

Buspirone attenuated methotrexate-induced hippocampal toxicity in rats by regulating Nrf2/HO-1, PPAR-γ, NF-κB/nNOS,and ROS/NLRP3/caspase-1 signaling pathways

Methotrexate (MTX) is a chemotherapeutic agent widely used to treat a variety of tumors. Nonetheless, MTX-induced hippocampal neurotoxicity is a well-defined dose-limiting adverse effect that limits clinical utility. Proinflammatory cytokine production and oxidative stress are possible mechanisms for MTX-induced neurotoxicity. Buspirone (BSP), a partial agonist of the 5-HT1a receptor (5-HT1aR), has emerged as an anxiolytic drug. BSP has been shown to possess antioxidant and anti-inflammatory effects. The current study investigated BSP's potential anti-inflammatory and antioxidant effects in attenuating MTX-induced hippocampal toxicity. Rats received either BSP (1.5 mg/kg) orally for 10 days and MTX (20 mg/kg) i.p. on Day 5. BSP administration markedly protected hippocampal neurons from drastic degenerated neuronal changes induced by MTX. BSP significantly attenuated oxidative injury by downregulating Kelch-like ECH-associated protein 1 expression while potently elevating hippocampal Nrf2, heme oxygenase-1, and peroxisome proliferator-activated receptor expression. BSP dampened inflammation by reducing NO₂⁻, tumor necrosis factor-alpha, IL-6, and interleukin 1 beta levels mediated by downregulating NF-κB and neuronal nitric oxides synthase expression. Moreover, BSP potently counteracted hippocampal pyroptosis by downregulating NLRP3, ASC, and cleaved-caspase-1 proteins. Therefore, BSP may represent a promising approach to attenuate neurotoxicity in patients receiving MTX.     

DF++ : an adaptive buffer-aware probabilistic delegation forwarding protocol for Delay Tolerant Network

The delegated forwarding (DF) curb transmissions by forwarding the message to a node that holds high quality value seen by the message. However, DF assumes infinite buffer space that is not possible in real time applications. In addition, quality value computation considers the encountering history and does not account for additional network parameters such as aging and transitive connectivity. In this paper, we have proposed a routing protocol called as DF++ that compute quality value based on probabilistic model used in PRoPHET protocol and forwards the message to current node by adaptive computation of available buffer space. We have compared performance of DF++ with DF, Epidemic and PRoPHET routing protocols. The proposed DF++ has higher delivery probability and fewer message drop and transmissions.     

Fine structural changes in the ileum of mice fed on delta-endotoxin-treated potatoes and transgenic potatoes.

The present work has been designed to study the effect of feeding on transgenic potatoes, which carry the CryI gene of Bacillus thuringiensis var. kurstaki strain HD1, on the light and electron microscopic structure of the mice ileum, in comparison with feeding on potatoes treated with the 'delta-endotoxin' isolated from the same bacterial strain. The microscopic architecture of the enterocytes of the ileum of both groups of mice revealed certain common features such as the appearance of mitochondria with signs of degeneration and disrupted short microvilli at the luminal surface. However, in the group of mice fed on the 'delta-endotoxin', several villi appeared with an abnormally large number of enterocytes (151.8 in control group versus 197 and 155.8 in endotoxin and transgenic-treated groups, respectively). Fifty percent of these cells were hypertrophied and multinucleated. The mean area of enterocyte was significantly increased (105.3 microm(2) in control group versus 165.4 microm(2) and 116.5 microm(2) in endotoxin and transgenic-treated groups, respectively). Several forms of secondary lysosomes or auotophagic vacuoles were recognized in these cells. These changes were confirmed with the scanning electron microscope which revealed a remarkable increase in the topographic contour of enterocytes (23 microm in control group versus 44 microm and 28 microm in endotoxin and transgenic-treated groups, respectively) at the divulged surface of the villi. The basal lamina along the base of the enterocytes was damaged at several foci. Several disrupted microvilli appeared in association with variable-shaped cytoplasmic fragments. Some of these fragments contained endoplasmic reticulum, as well as ring-shaped annulate lamellae. In addition, the Paneth cells were highly activated and contained a large number of secretory granules. These changes may suggest that delta-endotoxin-treated potatoes resulted in the development of hyperplastic cells in the mice ileum. Although mild changes are reported in the structural configuration of the ileum of mice fed on transgenic potatoes, nevertheless, thorough tests of these new types of genetically engineered crops must be made to avoid the risks before marketing.