Exploring the potent inhibitors and binding modes of phospholipase A2 through in silico investigation.

Snake venom of Naja naja comprises of several types of enzymes, and among them, water-soluble proteolytic enzyme, phospholipase A2 (PLA2), is noteworthy for its numerous adverse effects, such as cytotoxicity, cardiotoxicity, hemolytic, anti-coagulant, and hypotensive effects, including being highly potent as a neurotoxin. Limited anti-venom therapy (with their lower efficacy) has attracted considerable pharmacological interest to develop potent inhibitors of PLA2. Thus, 34 experimentally proven and diverse synthetic inhibitors of PLA2 were screened primarily on the basis of Glide extra precision docking and MM-GBSA rescoring function. Then, ten potential hits were subjected to induced fit docking, in which top three potential inhibitors were considered, and those were found to interact with Ca2+, disulfide binding site, and phosphatidylcholine activation sites, thereby, possibly disrupting the catalytic activity of Ca2+ as well as the inflammatory functions of PLA2. These compounds showed positive remarks on various physiochemical properties and pharmacologically relevant descriptors. Gap energy and thermodynamic properties were investigated by employing density functional theory for all compounds to understand their chemical reactivity and thermodynamic stability. Molecular dynamics simulation was performed for 100 ns in order to evaluate the stability and binding modes of docked complexes, and the energy of binding was calculated through MM-PBSA analysis. On the whole, the proposed compounds could be used for targeted inhibition. Communicated by Ramaswamy H. Sarma.

A regulatory approach on serum bilirubin level in subjects with impaired glucose tolerance.

The present study was undertaken to find the role of dietary intervention and physical exercise on serum bilirubin level in IGT subjects. Thirty three newly detected otherwise healthy subjects with IGT, aged 35-63 years, were randomly selected to participate in a 12 weeks diet and exercise program. Nine participants were within 35-40 years while majority fifteen participants aged 41-50 years and rest six participants were above 50 (51-63) years. A male preponderance was observed among the study participants where 53.3% of the total participants were male (n=16) and 46.7% were female (n=14). Mean bilirubin (mg/dl) level was recorded 0.68 ± 0.29 at base line and with follow-up, the value was 0.66 ± 0.26 mg/dl. For men (n=16), serum bilirubin were 0.77 ± 0.39 and 0.75 ± 0.36 mg/dl at base line and follow-up while for women (n=14), the values were 0.67 ± 0.33 and 0.59 ± 0.28 mg respectively. The 35-40 years group (n=9) showed bilirubin from 0.66 ± 0.23 at base line to 0.73 ± 0.19 mg/dl at follow-up while 41-50 years group (n=15) had 0.70 ± 0.34 and 0.58 ± 0.26 mg/dl and for 51-63 years group (n=6), the values were 0.65 ± 0.29 and 0.73 ± 0.33 mg/dl respectively. Participants with BMI 20-25 had bilirubin 0.62 ± 0.29 mg/dl at base line and 0.71 ± 0.21 mg/dl at follow-up while with BMI >25 (n=20) had 0.71 ± 0.30 and 0.63 ± 0.2 8 mg/dl respectively. No significant changes in serum bilirubin were observed among the groups and therefore, the dietary intervention and physical exercise during the period did not have a significant role in this respect.

New Report of Black Leaf Spot Mold (Pseudocercospora fuligena) on Withania somnifera from India.

Withania somnifera (family solanaceae) commonly known as ashwagandha and Indian ginseng, originated in India is one of the most powerful medicinal plants for more than 3,000 years (1). It is commercially cultivated for its roots, a natural rich source of glycowithanolides, tannins, potassium nitrate, etc., which are an anti-inflammatory, anti-tumor, anti-oxidant, anti-ulcer, and regulator of the nervous system and sleep (2). During the monsoon of July 2011, black spots on the leaves of infected plants were observed in the ashwagandha growing Lucknow, Raibareilly, and adjoining areas of Uttar Pradesh province with 10 to 20% disease incidence. Early stage of disease were characterized by the presence of light chlorotic spots on both sides of old leaves that later turned into dark black spots resulting in early defoliation. About 27 samples were collected from different locations of the fields for isolation of the causal organism and microscopic studies. Infected leaves were cut into small pieces, surface sterilized with 1% sodium hypochlorite for 1 min, rinsed thrice with sterilized distilled water, and placed onto potato dextrose agar (PDA) plates. After 21 days of dark incubation at 25°C, 8- to 10-mm grayish-brown colonies were observed. Microscopic studies at early and mature stages of infection showed production of conidia in conidiophores. Conidiophores were mostly 5 to 9, few dense pale brown, simple unbranched, septate, geniculate and 14 to 55 × 3 to 5.5 µm. Conidia were subhyline, obclavate to cylindrical, some were straight to slightly curved, multiseptate, base long obconic to long obconically truncate, and 12 to 85 × 3.5 to 5 µm. On the basis of cultural and morphological studies, the pathogen was identified as Pseudocercospora fuligena (3). The pathogen identity was further confirmed at molecular level using universal primers ITS1/ITS4 through PCR (4). An amplification of the expected size (~550 bp) was generated, eluted from agarose gel by QIAquick gel extraction kit (Qiagen), cloned into pGEM-T Easy vector (Promega), sequenced, and deposited in GenBank (Accession No. KF881898). NCBI BLASTn showed 99% identity with P. fuligena (GU214675) strain CPC 12296, isolated from Lycopersicon sp. Pathogenicity test was carried out on 10 plants of W. somnifera cv. Poshita through two approaches, one using mycelia from culture and another using spore suspension from naturally infected leaves. In the first approach, fungal mycelia were applied onto the healthy ashwagandha leaves, whereas in the second approach, infected leaves were washed with distilled water and spore suspension of 106 spores/ml was sprayed on healthy plants. Plants sprayed with sterilized distilled water served as controls. Inoculated plants were placed in a growth chamber at 28°C under 90% humidity for 3 days. After, pots were placed in the glasshouse at 27 ± 2°C with 70 to 80% humidity for 21 days. Initial symptoms appeared on the 7th day while typical symptoms appeared on all the inoculated plants after 12 to 17 days. Control plants remained free of infection. Re-isolation of the pathogen on PDA fulfilled Koch's postulates. Black leaf mold caused by P. fuligena has been reported on tomato (5). This is the first report of black leaf mould caused by P. fuligena on W. somnifera from India. P. fuligena has the potential to reduce yield of W. somnifera. References: (1) Anonymous. Alt. Med Rev. 9:211, 2004. (2) B. D. Basu and K. R. Kirtikar. Indian Medicinal Plants: Plates, vol. 1-4. Bishen Singh Mahendra Pal Singh, Dehradun, India, 1991. (3) T. C. Wang et al. Plant Dis. 79:661, 1995. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (5) S. Yamada. Ann. Phytopathol. Soc. Jpn. 15:13, 1951.

A Bioinformatics Approach for Homology Modeling and Binding Site Identification of Triosephosphate Isomerase from Plasmodium falciparum 3D7.

Malaria is a major public health concern, and malarial parasites have developed resistance against the commonly available drugs. So now a days it is a major concern to find out a new target for drug therapy. Plasmodium falciparum 3D7, one of the strains of plasmodium species also lacks in a functional tricarboxylic acid cycle and solely dependent on glycolysis for its energy supply like other plasmodium species. Although enzymes of malarial parasite have been considered as potential antimalarial drug targets, a little is known about their structural biology. The tertiary structure of triose phosphate isomerase of P. falciparum 3D7 was determined by means of homology modeling through multiple alignment followed by intensive optimization and validation. The modeling was done by Swiss-Model Workspace. The obtained model was verified with the structure validation programs such as, PROCHECK, Verify3D, and QMEAN for reliability. The verify3D value of 0.69 indicates that the environment profile of the model is good. A self-optimized prediction method with alignment or SOPMA is employed for calculation of the secondary structural features of triose phosphate isomerase. The secondary structure indicates that the predicted 3D structure of triosephosphate isomerase of P. falciparum 3D7 contains 48.37% α-helix, 29.27% random coil, and 16.67% extended strand. Active site determination through CASTp suggests that this protein can be utilized as a potential drug target. However, these will further be tested by wet lab studies for a targeted vaccine design against P. falciparum 3D7.

Purification, characterization and toxic profile of two toxins isolated from puffer fish Tetraodon patoca, available in Bangladesh.

Among the marine toxins related to human intoxication, tetrodotoxin has been known as one of the most prejudicial. Two tetrodotoxins, namely PFT-1 and PFT-2 were isolated and purified from liver of puffer fish by thin layer chromatography. The structure of both the toxins was elucidated by means of IR, 1H-NMR and 13C-NMR and mass spectroscopy. Sub acute toxicity study showed that both the toxins had pronounced effects on total RBC, WBC, platelet and ESR. Further serum levels of SGPT, SGOT, SALP, bilirubin, creatinine and urea are also affected by the toxins. The histopathological examinations showed that all the tissues such as liver, lung, heart and kidney of rat were severely changed after treatment with the toxins. The toxicity of the purified compounds, PFT-1 and PFT-2 were also performed by brine shrimp lethality bioassay.

Purification and characterization of three galactose specific lectins from mulberry seeds (Morus sp.).

Three lectins were extracted and purified from mulberry seeds by gel filtration of 100% ammonium sulfate saturated crude protein extract followed by ion-exchange chromatography on DEAE and CM-cellulose. The lectins were found to be homogeneous as judged by polyacrylamide disc gel electrophoresis. The molecular masses of the lectins as determined by gel filtration were 175 000 for MSL-1, 120 000 for MSL-2 and 89 500 for MSL-3. MSL-1 is dimer in nature, with the two monomers held together by disulfide bond(s), while MSL-2 and MSL-3 contain four nonidentical subunits that are held together by nonionic hydrophobic interactions. The lectins agglutinated rat red blood cells and this agglutination was inhibited specifically by galactose, methyl-alpha-d-galactopyranoside, methyl-beta-d-galactopyranoside, lactose and raffinose. The lectins MSL-1, MSL-2 and MSL-3 contained 5.7, 5.4 and 4.5% neutral sugars, respectively, and the sugar composition of the lectins was glucose and mannose for MSL-1 and galactose for both MSL-2 and MSL-3. The lectins exhibited strong cytotoxic effect in brine shrimp lethality bioassay.

Prevention of recurrent herpes simplex virus (HSV) infections in HIV-infected persons.

The purpose of this study was to determine the frequency of HSV infections and recurrences among HIV-infected patients and to examine different regimens for suppression of HSV recurrence. A randomized retrospective chart review of HIV-infected patients at a public hospital in Los Angeles County was conducted. We reviewed 224 patients' charts; 26 percent had AIDS based on the 1987 CDC definition. HSV infection was documented as a clinical event in 51 records (23 percent). Patients with an AIDS diagnosis had a greater incidence (53 percent) of HSV infections than did those with a diagnosis of symptomatic or asymptomatic HIV infection (p < 0.001, Fisher's exact test). Recurrences of HSV occurred in 26 (51 percent) of the 51 HSV-infected persons during a period of 1042 patient months. Eighteen patients who had received acyclovir suppression at 600 mg/day had three HSV recurrences in 382 patient months, whereas 14 who received 400 mg/day had eight recurrences in 282 patient months (p = 0.02). HSV infections occur in 23 percent of HIV-infected patients, increasing to 53 percent in AIDS patients. Acyclovir suppression prevents recurrent HSV, and a dosage of 600 mg/day is more effective than 400 mg/day.

Desensitization to trimethoprim/sulfamethoxazole in HIV-infected patients.

BACKGROUND: Trimethoprim-sulfamethoxazole (T/S) is an effective and important prophylactic medication for HIV-infected patients that must frequently be discontinued because of allergic reactions. OBJECTIVE: Our objective was to assess the safety, the frequency of success, and the duration of desensitization to T/S in HIV-infected patients. METHOD: We studied oral desensitization with T/S of patients with a history of allergy to the medication and longitudinal follow-up. Twenty-eight men with a history of T/S-induced skin rashes were studied. Mean age was 35 years (range, 26 to 50 years). Mean CD4 count was 89 cells/mm3 (range, 0/mm3 to 210/mm3). Patients were seen every 4 to 6 weeks. Mean follow-up was 19.07 weeks (range 2 to 81 weeks). RESULTS: After 32 weeks, 23 of 28 (82%) patients were successfully desensitized (four had rashes develop, and one could not continue for personal reasons). Of the 23 patients who were successfully desensitized, six were known to have subsequently discontinued T/S (four had rashes; two discontinued on the advice of their personal primary physicians). Six patients were lost to follow-up. One patient died of pulmonary Kaposi's sarcoma. Ten patients are taking the medication regularly without any problems. CONCLUSION: T/S desensitization is a simple, safe and effective means to provide it for most patients with a history of "allergic" rashes.

The interaction of Abrus precatorius agglutinin with saccharides as analyzed by fluorescence spectroscopy.

The binding of saccharides to Abrus precatorius agglutinin (APA) was analyzed by fluorescence spectroscopy. Upon binding of specific saccharides, the fluorescence emission maximum of APA (338 nm) shifted to shorter wavelength by 5 nm, owing to the change in the environment of tryptophan. By analyzing the change in the fluorescence intensity at 338 nm as a function of concentration of saccharides, the association constants for binding of saccharides to APA were determined. The results suggest that in the saccharide binding site on each B-chain of APA, there may be a site which interacts with the saccharide residue linked to galactopyranoside at the non-reducing end, in addition to the site which recognizes the galactopyranosyl residue. Fluorescence quenching data indicate that 8 out of 24 tryptophans in APA are located at or near the surface of the protein molecule and are available for quenching with both KI and acrylamide, and 10 tryptophans are involved in the environment to which acrylamide has access but KI does not. Binding of lactose to APA reduced by 4 the number of tryptophan residues accessible to quenchers. Based on the results, it is suggested that the tryptophan residues at the saccharide binding site on each B-chain of APA are present on the surface of the APA molecule, and they are shielded from quenching by KI and acrylamide upon binding with specific saccharides.

Fluorescence spectroscopic studies on tryptophan at the saccharide-binding site of castor bean hemagglutinin.

The environment of tryptophan in castor bean hemagglutinin (CBH) was analyzed by fluorescence spectroscopy with regard to saccharide binding. Upon binding of specific saccharides, the fluorescence maximum of 333 nm of CBH shifted to a wavelength 2 nm shorter, owing to the change in the environment of tryptophan at the saccharide-binding site. By analyzing the change in the fluorescence intensity at 320 nm as a function of concentration of saccharides, the association constants for binding of saccharides to CBH were determined. The results suggest that the saccharide-binding site on each B-chain is actually composed of a subsite with which the saccharide residue linked to galactopyranoside at the non-reducing end can interact, and another site which recognizes the galactopyranoside moiety. Quenching data indicated that five out of 22 tryptophans in CBH are surface-localized and are available for quenching with both KI and acrylamide, and three other tryptophans are buried and are available only to acrylamide. Binding of raffinose to CBH decreased by 2 the number of tryptophan residues accessible to quenchers in the CBH molecule. We speculate that raffinose binds to CBH in such a manner as to shield the tryptophan located at the subsite from quenching by KI and acrylamide. The results also suggest that the tryptophan residue at the saccharide-binding site on each B-chain is localized near the surface, and present in the positively charged environment.

States of tryptophan residues in castor bean hemagglutinin: the perturbing effects of solvents on tryptophans in hemagglutinin and its complexes as analyzed by UV-difference spectroscopy.

The states of tryptophan residues in castor bean hemagglutinin (CBH) were analyzed by solvent perturbation studies employing ultraviolet difference spectroscopy. Eight out of 22 tryptophan residues in CBH were exposed to ethylene glycol and glycerol, suggesting that the remaining 14 tryptophan residues are buried in the interior of the CBH molecule. The fraction of tryptophan residues accessible to the perturbant decreased with increase in the molecular size of the perturbant, and only 2 tryptophan residues were exposed to polyethylene glycol 600. Upon binding with raffinose, 2 tryptophan residues were shielded from the perturbing effect of the solvent, and binding of lactose reduced the number of tryptophan residues accessible to the perturbant by 1 mol per mol of protein. Binding of galactose, however, did not change the accessibility of tryptophan to the perturbant. On the other hand, the accessibility of tyrosine to the perturbant remained unchanged after binding with raffinose and lactose, suggesting that tyrosine is not directly involved in the saccharide binding of CBH. Based on these results, it is proposed that one tryptophan residue at the saccharide-binding site on each B-chain of CBH lies on the surface of the protein molecule and is located at a subsite which is accessible to a glucopyranoside moiety in the lactose molecule or a glycopyranosyl-fructofuranosyl moiety in the raffinose molecule, whereas such a residue is not present at the galactopyranoside-recognition site.

Ultraviolet difference spectroscopic studies on the saccharide-binding properties of Abrus precatorius agglutinin.

The nature of the binding of specific saccharides to Abrus precatorius agglutinin (APA) was studied by ultraviolet difference spectroscopy. Upon binding of saccharides, APA displayed difference spectra with maxima at 291-292 nm and 284-285 nm. Such spectra suggest that the state of the tryptophan residue closely associated with the saccharide-binding activity of APA is perturbed by the binding of a saccharide. The difference spectra value (delta epsilon) increased with increasing saccharide concentration. From the increase in delta epsilon at 291-292 nm, the association constant (Ka) was obtained for the binding of individual saccharides to APA. Lactose bound to APA with the highest affinity among the saccharides examined and its Ka value (8.3 X 10(3) M-1 at pH 7.0 and 25 degrees C) was approximately four times as large as that of galactose (2.2 X 10(3) M-1). Raffinose and methyl beta-galactopyranoside showed larger association constants than galactose. Galactosamine, N-acetylgalactosamine and 2-deoxy galactose were found to bind with APA with fairly low affinity. The shape of the lactose-induced difference spectrum changed with pH and the spectrum in the acidic region showed characteristic broadening of the difference maximum peaks. The affinity of lactose to APA was nearly equal in the range of pH 6-8, but decreased outside this pH region and with increasing temperature.

Ultraviolet difference spectroscopic analysis of the saccharide-binding properties of Ricinus communis agglutinin.

The nature of the binding of saccharides to Ricinus communis agglutinin was studied by ultraviolet difference spectroscopy. Upon binding of galactose and galactose-containing saccharides, R. communis agglutinin displayed difference spectra with an extreme maximum at 291-293 nm and a smaller maximum at 284-285 nm. Such difference spectra suggest that the environment of a tryptophan residue located at or near the saccharide-binding site of R. communis agglutinin is being changed by an interaction between a tryptophan residue and the bound saccharides. The value of the difference spectra (delta epsilon) increased upon progressive addition of saccharide until the saccharide binding site was saturated with ligand. From the increase in delta epsilon at 291-293 nm, the association constants were obtained for the R. communis agglutinin-saccharide interaction over the temperature range 5-35 degrees C and various pH values. The results clearly demonstrate that the association constants are nearly equal in the range of pH 5-8, but decrease beyond the above pH range and with elevation of temperature. From the thermodynamic parameters for the binding of various saccharides to R. communis agglutinin, we suggest that there exists a subsite structure in the saccharide-binding site of the R. communis agglutinin molecule.