Effects of gossypol acetic acid on the absorptive and digestive functions of rat intestine

Oral administration of Gossypol acetic acid (10 mg/kg body wt./day, daily for 15 days), an experimental antifertility agent to male rats, caused significant reduction in the uptake of glucose, alanine, leucine and calcium in the small intestinal segments. Gossypol also caused significant decrease in the intestinal brush border membrane--associated enzymes, sucrase, lactase, maltase and alkaline phosphatase. Kinetic analysis indicated that Gossypol decreased the apparent velocity of the disaccharidases while the Km was not altered. It also caused a shift in the transition temperature in these enzymes and predictably changed the energy of activation both below and above the transition temperature, although the Arrhenius expressions of the temperature dependence still showed proximity and were parallel to the control group.     

The Unusual Fold of Herpes Simplex Virus 1 UL21, a Multifunctional Tegument Protein

UL21 is a conserved protein in the tegument of alphaherpesviruses and has multiple important albeit poorly understood functions in viral replication and pathogenesis. To provide a roadmap for exploration of the multiple roles of UL21, we determined the crystal structure of its conserved N-terminal domain from herpes simplex virus 1 to 2.0-Å resolution, which revealed a novel sail-like protein fold. Evolutionarily conserved surface patches highlight residues of potential importance for future targeting by mutagenesis.     

Response surface optimization for enhanced production of cellulases with improved functional characteristics by newly isolated Aspergillus niger HN-2

Fungi isolated from partially decayed wood log samples showing characteristic diversity for spore colour, colony morphology and arrangement of spores were assessed for cellulolytic enzyme production. Isolates showing a cellulolytic index of ≥2.0 were assayed for filter paper (FP) cellulase and β-glucosidase (BGL) production. Molecular characterization confirmed the identity of the selected cellulolytic isolate as a strain of Aspergillus niger (A. niger HN-2). Addition of 2 % (w/v) urea enhanced FP and BGL activity by about 20 and 60 %, respectively. Validation studies conducted at parameters (29 °C, pH 5.4, moisture content 72 % and 66 h) optimized through response surface methodology in a solid-state static tray fermentation resulted in FP, BGL, cellobiohydrolase I (CBHI), endoglucanase (EG), xylanase activity and protein content of 25.3 FPU/g ds, 750 IU/g ds, 13.2 IU/g ds, 190 IU/g ds, 2890 IU/g ds and 0.9 mg/ml, respectively. In comparison, A. niger N402 which is a model organism for growth and development studies, produced significantly lower FP, BGL, CBHI, EG, xylanase activity and protein content of 10.0 FPU/g ds, 100 IU/g ds, 2.3 IU/g ds, 50 IU/g ds, 500 IU/g ds and 0.75 mg/ml, respectively under the same process conditions as were used for A. niger HN-2. Process optimization led to nearly 1.8- and 2.2-fold increase in FP and BGL activity, respectively showing promise for cellulase production by A. niger HN-2 at a higher scale of operation. Zymogram analysis revealed two isoforms each for EG and cellobiohydrolase and three isoforms for BGL. Crude cellulase complex produced by A. niger HN-2 exhibited thermostability under acidic conditions showing potential for use in biofuel industry.     

Thermostable pullulanase from a mesophilic Bacillus cereus isolate and its mutant UV7.4

Summary A mesophilicBacillus cereus strain was isolated from soil. Since it produced fairly good amounts of extracellular pullulanase, mutations with ultraviolet light and nitrosoguanidine were done to improve the productivity. One of the second generation mutants, UV7.4, produced larger amounts of pullulanase at 37°C than most (mesophilic or thermophilic) organisms reported. The pullulanase was highly active and stable for up to one hour at 70°C. If developed further, UV7.4 can be used commercially for the saccharification of starch.     

Continuous production of lactic acid from molasses by free and immobilized Sporolactobacillus cellulosolvens

Both free and immobilized cells of Sporolactobacillus cellulosolvens, in continuous culture on molasses (50 g sugar 1^-1) at 40°C, had maximum lactic acid productivities of 0.03 and 0.06 mol l^-1 h, at dilution rates of 0.27 and 0.25 h^-1, respectively.S.S. Kanwar is with the Department of Biotechnology, Guru Nanak Dev University, Amritsar-143 005, India; B.S. Chadha is with the Department of Microbiology, Guru Nanak Dev University, Amritsar-143 005, India. H.K. Tewari and V.K. Sharma are with the Department of Microbiology, Punjab Agricultural University, Ludhiana-141 004, India.     

Repurposing phytochemicals as anti-virulent agents to attenuate quorum sensing-regulated virulence factors and biofilm formation in Pseudomonas aeruginosa

Unregulated consumption and overexploitation of antibiotics have paved the way for emergence of antibiotic-resistant strains and ‘superbugs’. Pseudomonas aeruginosa is among the opportunistic nosocomial pathogens causing devastating infections in clinical set-ups globally. Its artillery equipped with diversified virulence elements, extensive antibiotic resistance and biofilms has made it a ‘hard-to-treat’ pathogen. The pathogenicity of P. aeruginosa is modulated by an intricate cell density-dependent mechanism called quorum sensing (QS). The virulence artillery of P. aeruginosa is firmly controlled by QS genes, and their expression drives the aggressiveness of the infection. Attempts to identify and develop novel antimicrobials have seen a sharp rise in the past decade. Among different proposed mechanisms, a novel anti-virulence approach to target pseudomonal infections by virtue of anti-QS and anti-biofilm drugs appears to occupy the centre stage. In this respect, bioactive phytochemicals have gained prominence among the scientific community owing to their significant quorum quenching (QQ) properties. Recent studies have shed light on the QQ activities of various phytochemicals and other drugs in perturbing the QS-dependent virulence in P. aeruginosa. This review highlights the recent evidences that reinforce the application of plant bioactives for combating pseudomonal infections, their advantages and shortcomings in anti-virulence therapy.     

Isolated colostomy site recurrence in rectal cancer-two cases with review of literature

Background

Colostomy site carcinomas are rare with only eight cases reported in the world literature. Various etiological factors like adenoma-cancer sequence, bile acids, recurrent and persistent physical damage at the colostomy site by faecal matter due to associated stomal stenosis have been considered responsible. Two such cases are being reported and in both cases there was no evidence of any local recurrence in the pelvis or liver and distant metastasis. Both patients had received adjuvant chemotherapy following surgery.

Case presentation

First case was a 30-year-old male that had reported with large bowel obstruction due to an obstructing ulcero-proliferative growth (poorly differentiated adenocarcinoma) at the colostomy site after abdomino-perineal resection, performed for low rectal cancer six years previously. Wide local excision with microscopically free margins was performed with a satisfactory outcome. Four years later he presented with massive malignant ascites, cachexia and multiple liver metastasis and succumbed to his disease.Second case was a 47-year-old male that presented with acute large bowel obstruction due to an annular growth (well differentiated adenocarcinoma) in the upper rectum. He was managed by Hartmann's operation and the sigmoid colostomy was closed six months later. Five years following closure of colostomy, he presented with two parietal masses at the previous colostomy site scar, which, on fine needle aspiration cytology were found to be well-differentiated adenocarcinomas of colorectal type. Surgery in the form of wide local resection with free margins was performed. He presented again after five years with recurrence along the previous surgery scar and an incisional hernia and was managed by wide local excision along with hernioplasty. Follow-up of nine years following first surgery is satisfactory.

Conclusion

Colostomy site/scar recurrence of rectal carcinoma is rare and could be due to various etiological factors, although the exact causative mechanism is not known. Surgery with microscopically free margins is recommended in the absence of metastatic disease. Stenosis of the stoma is considered as one of the most important contributory factors and should be followed carefully.

     

HLA Class-II Associated HIV Polymorphisms Predict Escape from CD4+ T Cell Responses

Antiretroviral therapy, antibody and CD8⁺ T cell-mediated responses targeting human immunodeficiency virus-1 (HIV-1) exert selection pressure on the virus necessitating escape; however, the ability of CD4⁺ T cells to exert selective pressure remains unclear. Using a computational approach on HIV gag/pol/nef sequences and HLA-II allelic data, we identified 29 HLA-II associated HIV sequence polymorphisms or adaptations (HLA-AP) in an African cohort of chronically HIV-infected individuals. Epitopes encompassing the predicted adaptation (AE) or its non-adapted (NAE) version were evaluated for immunogenicity. Using a CD8-depleted IFN-γ ELISpot assay, we determined that the magnitude of CD4⁺ T cell responses to the predicted epitopes in controllers was higher compared to non-controllers (p<0.0001). However, regardless of the group, the magnitude of responses to AE was lower as compared to NAE (p<0.0001). CD4⁺ T cell responses in patients with acute HIV infection (AHI) demonstrated poor immunogenicity towards AE as compared to NAE encoded by their transmitted founder virus. Longitudinal data in AHI off antiretroviral therapy demonstrated sequence changes that were biologically confirmed to represent CD4⁺ escape mutations. These data demonstrate an innovative application of HLA-associated polymorphisms to identify biologically relevant CD4⁺ epitopes and suggests CD4⁺ T cells are active participants in driving HIV evolution.     

Regulated Interaction of Tegument Proteins UL16 and UL11 from Herpes Simplex Virus

It is well known that proteins in the tegument (located between the viral capsid and envelope proteins) play critical roles in the assembly and budding of herpesviruses. Tegument proteins UL16 and UL11 of herpes simplex virus (HSV) are conserved among all the Herpesviridae. Although these proteins directly interact in vitro, UL16 was found to colocalize poorly with UL11 in cotransfected cells. To explain this discrepancy, we hypothesized that UL16 is initially made in an inactive form and is artificially transformed to the binding-competent state when cells are disrupted. Consistent with a regulated interaction, UL16 was able to fully colocalize with UL11 when a large C-terminal segment of UL16 was removed, creating mutant UL16(1-155). Moreover, membrane flotation assays revealed a massive movement of this mutant to the top of sucrose gradients in the presence of UL11, whereas both the full-length UL16 and the C-terminal fragment (residues 156 to 373) remained at the bottom. Further evidence for the presence of a C-terminal regulatory domain was provided by single-amino-acid substitutions at conserved cysteines (C269S, C271S, and C357S), which enabled the efficient interaction of full-length UL16 with UL11. Lastly, the binding site for UL11 was further mapped to residues 81 to 155, and to our surprise, the 5 Cys residues within UL16(1-155) are not required, even though the modification of free cysteines in UL16 with N-ethylmaleimide does in fact prevent binding. Collectively, these results reveal a regulatory function within the C-terminal region of UL16 that controls an N-terminal UL11-binding activity.