Norcantharidin, a clinical used chemotherapeutic agent, acts as a powerful inhibitor by interfering with fibrinogen-integrin αIIb ß3 binding in human platelets.

During platelet activation, fibrinogen binds to its specific platelet receptor, integrin αIIb ß3 , thus completing the final common pathway for platelet aggregation. Norcantharidin (NCTD) is a promising anticancer agent in China from medicinal insect blister beetle. In this study, we provided the evidence to demonstrate NCTD (0.1-1.0 µM) possesses very powerful antiplatelet activity in human platelets; nevertheless, it had no effects on surface P-selectin expression and only slight inhibition on ATP-release reaction in activated platelets. Moreover, NCTD markedly hindered integrin αIIb ß3 activation by interfering with the binding of FITC-labelled PAC-1. It also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as clot retraction. Additionally, NCTD attenuated phosphorylation of proteins such as integrin ß3 , Src and FAK in platelets spreading on immobilized fibrinogen. These results indicate that NCTD restricts integrin αIIb ß3 -mediated outside-in signalling in human platelets. Besides, NCTD substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation and prolonged the bleeding time in mice. In conclusion, NCTD has dual activities, it can be a chemotherapeutic agent for cancer treatment, and the other side it possesses powerful antiplatelet activity for treating thromboembolic disorders.

Inflammation and oxidative stress in corneal tissue in experimental keratitis due to Fusarium solani: Amelioration following topical therapy with voriconazole and epigallocatechin gallate.

Combined antifungal and antioxidant therapy may help to reduce oxidative stress in fungal keratitis. Experimental Fusarium solani keratitis was induced by application of F. solani conidia to scarified cornea (right eye) of 16 rabbits (another four rabbits were negative controls [Group I]). Five days later, F. solani-infected animals began receiving hourly topical saline alone (Group II), voriconazole (10 mg/mL) alone (Group III), epigallocatechin gallate (EGCG, 10 mg/mL) alone (Group IV) or voriconazole and EGCG (Group V). Twenty days post-inoculation, corneal lesions were graded. After animal sacrifice, excised corneas underwent histopathological and microbiological investigations. Corneal tissue levels/activities of interleukin 1 beta (IL-1ß) and tumour necrosis factor alpha (TNF-α) gene mRNA transcripts, matrix metalloproteinase (MMP) 2 and 9 proteins, malondialdehyde (MDA) and reduced glutathione (GSH), and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), were also measured. Clinical and histopathological scores (severity of corneal lesions; [P < .05]) and mean levels (P < .05) of IL-1ß and TNF-α mRNA transcripts, MMP 2, MMP 9 and MDA were Group II > Groups IV and III > Groups V and I. Mean SOD, CAT, GPx and GSH levels (P < .05) were Group II < Groups IV and III < Groups V and I. Topical voriconazole with EGCG apparently reduces inflammation in experimental F. solani keratitis, as manifested by improved clinical, histological, microbiological and molecular parameters.

Regulatory effect of chrysin on expression of lenticular calcium transporters, calpains, and apoptotic-cascade components in selenite-induced cataract.

PURPOSE: Selenite-induced cataract is associated with oxidative stress, loss of calcium homeostasis, activation of calpain enzymes, and apoptotic cell death in the lens. An evaluation of naturally occurring antioxidants that also restrict calcium influx into the lens and calpain activation and thus prevent lenticular cell death may lead to the development of safe and effective anticataractogenic drugs. This study focuses on a naturally occurring flavone, chrysin, and its efficacy in preventing cataractogenic changes in in vitro cultured Wistar rat lenses. METHODS: Lenses from Wistar rats incubated for 24 h at 37 °C in Dulbecco's modified Eagle's medium (DMEM) were categorized into four main groups: Group I (control, incubated in DMEM alone); Group II (selenite-challenged and untreated, incubated in DMEM that contained 100 µM/ml of sodium selenite only); Group III (selenite-challenged and chrysin-treated, incubated in DMEM that contained sodium selenite [100 µM/ml of DMEM] and chrysin [200 µM/ml of DMEM]); and Group IV (chrysin-treated, incubated in DMEM that contained chrysin [200 µM/ml of DMEM] only). The Group III (selenite-challenged and chrysin-treated) lenses were further categorized into five sub-groups: Group IIIa (incubated for 24 h in DMEM that contained sodium selenite and chrysin added simultaneously), Group IIIb (first incubated for 2 h in DMEM that contained chrysin only and then for up to 24 h in fresh DMEM that contained sodium selenite only), Group IIIc (first incubated for 30 min in DMEM that contained sodium selenite only and subsequently for up to 24 h in DMEM that contained chrysin only), and Groups IIId and IIIe (first incubated for 1 h and 2 h, respectively, in DMEM that contained sodium selenite only and subsequently for up to 24 h in DMEM that contained chrysin only). RESULTS: Gross morphological assessment revealed dense opacification (Grade +++) in the selenite-challenged, untreated lenses (Group II); however, seven of the eight selenite-challenged and simultaneously chrysin-treated (Group IIIa) lenses showed no opacification (Grade 0) after 24 h incubation, while the remaining single lens exhibited only a slight degree of opacification (Grade +). In the Group IIIa lenses, the reduced glutathione, protein sulfhydryl, and malondialdehyde concentrations appeared to have been maintained at near-normal levels. The mean lenticular concentration of calcium was significantly lower in the Group IIIa lenses than that in the Group II lenses and approximated the values observed in the normal control (Group I) lenses. The Group IIIa lenses also exhibited significantly (p<0.05) higher mean lenticular activity of calpain, significantly higher mean mRNA transcript levels of genes that encode m-calpain and lenticular preferred calpain (Lp82), and significantly higher mean levels of the m-calpain and Lp82 proteins than the corresponding values in the Group II lenses. Casein zymography results suggested that chrysin prevented calpain activation and autolysis. Significantly (p<0.05) lower mean levels of mRNA transcripts of the genes that encode calcium transporter proteins (plasma membrane Ca(2+)-ATPase-1 and sarco/endoplasmic reticulum Ca(2+)-ATPase-2) and lenticular apoptotic-cascade proteins (early growth response protein-1, caspase-3, caspase-8, and caspase-9) and significantly (p<0.05) lower mean concentrations of the proteins themselves were seen in the Group IIIa rat lenses in comparison to the values noted in the Group II rat lenses. CONCLUSIONS: Chrysin appears to prevent selenite-induced cataractogenesis in vitro by maintaining the redox system components at near-normal levels and by preventing the abnormal expression of several lenticular calcium transporters and apoptotic-cascade proteins, thus preventing accumulation of calcium and subsequent calpain activation and lenticular cell death in cultured Wistar rat lenses.

Novel synthetic benzimidazole-derived oligosaccharide, M3BIM, prevents ex vivo platelet aggregation and in vivo thromboembolism.

BACKGROUND: Thrombus formation, a phenomenon primarily related to increased platelet activation, plays a key role in cardiovascular and cerebrovascular diseases. Although the established antiplatelet agents, such as aspirin and clopidogrel, have been shown to be beneficial in treating thromboembolic diseases, they have considerable limitations. Hence, the development of more effective and safe antithrombotic agents is necessary to satisfy a substantial unmet clinical need. In recent years, the favorable properties of imidazole-related drugs have prompted medicinal chemists to synthesize numerous novel therapeutic agents. The chemical structure of the benzimidazole backbone has proven antiplatelet properties. Moreover, synthetic oligosaccharides have exhibited antiplatelet properties. Therefore, we developed a new aldo-benzimidazole-derived oligosaccharide compound, M3BIM, for achieving a stronger antiplatelet effect than the drugs which are being used in clinical aspects. We investigated the effects of M3BIM on platelet activation ex vivo and its antithrombotic activity in vivo. RESULTS: M3BIM (10-50 µM) exhibited a more potent activity in inhibiting platelet aggregation stimulated by collagen than it did in inhibiting that stimulated by thrombin in washed human platelets. The M3BIM treatment revealed no cytotoxicity in zebrafish embryos, even at the highest concentration of 100 µM. In addition, M3BIM inhibited the phosphorylation of phospholipase Cγ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase 2 and c-Jun N-terminal kinase 1), and markedly reduced the ATP-release reaction and intracellular calcium mobilization in collagen-activated platelets. By contrast, M3BIM showed no effects on either collagen-induced p38 MAPK and Akt phosphorylation or phorbol 12, 13-dibutyrate-induced PKC activation and platelet aggregation. Moreover, the M3BIM treatment substantially prolonged the closure time in human whole blood, and increased the occlusion time in mesenteric microvessels and attenuated cerebral infarction in mice. For the study of anticoagulant activities, M3BIM showed no significant effects in the prolongation of activated partial thromboplastin time and prothrombin time in mice. CONCLUSION: The findings of our study suggest that M3BIM is a potential therapeutic agent for preventing or treating thromboembolic disorders.

Comparative decline of the protein profiles of nebulin in response to denervation in skeletal muscle.

The sliding filament model of the sarcomere was developed more than half a century ago. This model, consisting only of thin and thick filaments, has been efficacious in elucidating many, but not all, features of skeletal muscle. Work during the 1980s revealed the existence of two additional filaments: the giant filamentous proteins titin and nebulin. Nebulin, a giant myofibrillar protein, acts as a protein ruler to maintain the lattice arrays of thin filaments and plays a role in signal transduction and contractile regulation. However, the change of nebulin and its effect on thin filaments in denervation-induced atrophic muscle remains unclear. The purpose of this study is to examine the content and pattern of nebulin, myosin heavy chain (MHC), actin, and titin in innervated and denervated tibialis anterior (TA) muscles of rats using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), densitometry and electron microscopic (EM) analyses. The results revealed that denervation induced muscle atrophy is accompanied by decreased nebulin content in a time-dependent manner. For instant, the levels of nebulin in denervated muscles were markedly (P < 0.05) decreased, about 24.6% and 40.2% in comparison with innervated muscle after denervation of 28 and 56 days, respectively. The nebulin/MHC, nebulin/actin, and nebulin/titin ratios were decreased, suggesting a concomitant reduction of nebulin in denervated muscle. Moreover, a western blotting assay proved that nebulin declined faster than titin on 28 and 56 days of denervated muscle. In addition, EM study revealed that the disturbed arrangements of myofilaments and a disorganized contractile apparatus were also observed in denervated muscle. Overall, the present study provides evidence that nebulin is more sensitive to the effect of denervation than MHC, actin, and titin. Nebulin decline indeed resulted in disintegrate of thin filaments and shortening of sarcomeres.

Structure-Based Virtual Screening and Biological Evaluation of a Calpain Inhibitor for Prevention of Selenite-Induced Cataractogenesis in an in Vitro System.

Calpains belong to the family of calcium-dependent, structurally related intracellular cysteine proteases that exhibit significant functions in evolution of different types of cataracts in human as well as animal models. Application of calpain inhibitors generated through a virtual screening workflow may provide new avenues for the prevention of cataractogenesis. Hence, in the current study, compounds were first screened for potent calpain inhibitory activity by employing a structure-based approach, and the screening results were then validated through biological experiments in rat lenses. A hit compound, HTS08688, was obtained by structure-based virtual screening. A micromolar concentration of HTS08688 was found to prevent in vitro cataractogenesis in isolated Wistar rat lenses, while maintaining the antioxidant and calcium concentrations at near normal levels. Inhibition of superoxide anion generation, as observed through cytochemical localization studies, and maintenance of structural integrity, as demonstrated by histological analysis of lenticular tissue, also suggested that HTS08688 can ameliorate the cataractous condition induced by selenite in an in vitro rodent model. A cell proliferation assay was performed; the IC 50 value of the screened calpain inhibitor, HTS08688, against human lenticular epithelial cells-b3 was found to be 177 µM/mL. This combined theoretical and experimental approach has demonstrated a potent lead compound, HTS08688, that exhibits putative anticataractogenic activity by virtue of its potential to inhibit calpain.

Keratitis due to Fusarium langsethiae: clinical profile, molecular identification, and susceptibility to antifungals.

We report a case of keratitis due to Fusarium langsethiae in a 56-year-old man. The patient presented with pain and tearing of 10 days duration in the right eye, which had sustained a paddy stalk injury. On examination, a hypopyon corneal ulcer was noted in the right eye. Multiple scrapings were obtained from the affected part of the cornea. A lactophenol cotton blue wet mount and a Gram-stained smear of scrapings were made. Scrapings were also inoculated on various culture media, including Sabouraud dextrose agar (SDA). A fungal etiology was sought by conventional microbiological techniques and polymerase chain reaction. In vitro susceptibility testing was performed by an agar dilution method. Direct microscopy of corneal scrapings revealed septate hyphae, leading to initiation of intensive topical therapy with natamycin (5 %). However, the keratitis progressed, necessitating therapeutic penetrating keratoplasty. White, powdery-like colonies, with abundant aerial mycelium, were recovered on SDA from corneal scrape material. Based on macroscopic and microscopic morphological features, the isolated fungus was initially identified as a Fusarium species. Sequence analysis of the 28S rRNA region of the fungal genome led to a specific identification of F. langsethiae. Antifungal susceptibility testing results suggested that the strain isolated was susceptible to voriconazole, ketoconazole, and itraconazole. To our knowledge, this is the first reported case of keratitis due to F. langsethiae; attention is drawn to the unique characteristics of the fungal isolate, difficulties in identification and non-responsiveness to medical therapy.

Evaluation of the anti-atherogenic potential of chrysin in Wistar rats.

Hypercholesterolemia is one of the major risk factors that precipitate coronary heart disease and atherosclerosis. Oxidative stress is believed to contribute to the pathogenesis of hypercholesterolemic atherosclerosis; hence, various antioxidant compounds are being evaluated for potential anti-atherogenic effects. In the present study, the putative anti-atherogenic and antioxidant efficacy of a flavonoid, chrysin, was evaluated in an experimental model of atherosclerosis. In male, albino Wistar rats fed an atherogenic diet for 45 days and treated with saline, significantly higher mean levels of serum lipid profile parameters (total cholesterol, triglycerides, low-density, and very low-density lipoprotein cholesterol), lower mean levels of high-density lipoprotein cholesterol and higher mean serum levels of hepatic marker enzymes (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and lactate dehydrogenase) were observed when compared with the levels in rats fed a control diet. In addition, significantly lower mean hepatic levels of lipoprotein lipase, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase) and non-enzymatic antioxidants (reduced glutathione, and vitamins C and E), and a significantly higher mean level of hepatic malondialdehyde (MDA) were noted in comparison to the values in control rats. In atherogenic diet-fed rats that received chrysin orally (200 mg/kg b.wt) for 15 days, starting 30 days after the start of the atherogenic diet, significantly lower mean serum levels of lipid profile parameters (except for HDL-cholesterol which was elevated), hepatic marker enzymes, and significantly higher mean hepatic levels of LPL, HMG-CoA reductase, enzymatic, and non-enzymatic antioxidants and significantly lower mean levels of hepatic MDA were noted, compared to the values in atherogenic diet-fed, saline-treated rats. Histopathological studies appeared to suggest the protective effect of chrysin on the hepatic tissue and aorta of atherosclerotic rats. These results suggest that chrysin has anti-atherogenic potential in an experimental setting.

Extract of Antrodia camphorata exerts neuroprotection against embolic stroke in rats without causing the risk of hemorrhagic incidence.

In this study, the neuroprotective effect of an extract of Antrodia camphorata (A. camphorata), a fungus commonly used in Chinese folk medicine for treatment of viral hepatitis and cancer, alone or in combination with aspirin was investigated in a rat embolic stroke model. An ischemic stroke was induced in rats by a selective occlusion of the middle cerebral artery (MCA) with whole blood clots and then orally treated with A. camphorata (0.25 and 0.75 g/kg/day) alone and combined with aspirin (5 mg/kg/day). Sixty days later, the brains were removed, sectioned, and stained with triphenyltetrazolium chloride and analysed by a commercial image processing software program. Brain infarct volume, neurobehavioral score, cerebral blood perfusion, and subarachnoid and intracerebral hemorrhage incidence were perceived. In addition, potential bleeding side effect of the combinative therapy was assessed by measuring hemoglobin (Hb) content during intracerebral hemorrhage and gastric bleeding, prothrombin time (PT), and occlusion time (OT) after oral administration. Posttreatment with high dose A. camphorata significantly reduced infarct volume and improved neurobehavioral score (P < 0.05). Since A. camphorata alone or with aspirin did not alter the Hb level, this treatment is safe and does not cause hemorrhagic incident. Remarkably, the combination of A. camphorata and aspirin did not show a significant effect on the bleeding time, PT and OT increase suggesting that A. camphorata may have the neuroprotective effect without the prolongation of bleeding time or coagulation time. From these observations, we suggest that combinative therapy of A. camphorata and aspirin might offer enhanced neuroprotective efficacies without increasing side effects.

Pleurotus ostreatus, an edible mushroom, enhances glucose 6-phosphate dehydrogenase, ascorbate peroxidase and reduces xanthine dehydrogenase in major organs of aged rats.

CONTEXT: Aging is now considered to be associated with an elevation in oxidative damage to macromolecules and enhanced levels of inflammation. Therefore, inhibition of age-related oxidative stress by natural supplement is an important study. OBJECTIVES: To investigate whether the treatment with Pleurotus ostreatus (Jacq.: Fr) Kumm, (Pleurotaceae) can ameliorate oxidative damage in aged rats. MATERIALS AND METHODS: Male Wistar rats were divided into three groups of six each: group 1, normal young rats; group 2, normal aged untreated rats; group 3, normal aged rats treated with P. ostreatus (200 mg/kg body wt administered intraperitoneally for 21 days). On the 22nd day, rats were sacrificed by decapitation; the liver, kidneys, heart and brain were removed from each rat for the biochemical and isozyme analyses of the antioxidant enzymes glucose 6-phosphate dehydrogenase (G6PDH), ascorbate peroxidase (Apx) and xanthine dehydrogenase (XDH). RESULTS: An elevated activity of XDH was observed in the liver (G2:13.72 ± 4.1 versus G1: 7.57 ± 1.15; p < 0.05), kidneys (G2:101.48 ± 12.3 versus G1: 31.15 ± 1.71; p < 0.01), heart (G2: 63.21 ± 3.96 versus G1: 37.3 ± 2.70; p < 0.01) and brain (G2: 39.02 ± 3.96 versus G1: 19.84 ± 1.22; p < 0.001). The activities of G6PDH and Apx were lowered in major organs of aged untreated rats. However, treatment of P. ostreatus to aged rats resulted in decreased XDH and increased G6PDH and Apx activities in liver, kidneys, heart and brain. Interestingly, analyses of isozyme pattern of these enzymes are support the results obtained from the spectrophotometric determinations. CONCLUSION: These results suggest that an extract of P. ostreatus can protect the age-related oxidative damage in major organs of Wistar rats by enhancing the antioxidant enzymes G6PDH and Apx and by reducing XDH.

Putative free radical-scavenging activity of an extract of Cineraria maritima in preventing selenite-induced cataractogenesis in Wistar rat pups.

PURPOSE: To investigate the possible free radical-scavenging activity of an extract of Cineraria maritima on selenite-induced cataractous lenses in Wistar rat pups. METHODS: In the present study, Wistar rat pups were divided into three experimental groups. On P10, Group I (control) rat pups received an intraperitoneal injection of 0.89% saline. Rats in groups II (selenite-challenged, untreated) and III (selenite-challenged, C. maritima treated) received a subcutaneous injection of sodium selenite (19 µmol/kg bodyweight); Group III rat pups also received an intraperitoneal injection of the extract of C. maritima (350 mg/kg bodyweight) once daily P9-14. Both eyes of each pup were examined from P16 until P30. Cytochemical localization of nitroblue tetrazolium salts and generation of superoxide, hydroxyl, and nitric oxide levels were measured. The expression of the inducible nitric oxide synthase gene was evaluated with reverse transcription-PCR. Immunoblot analysis was also performed to confirm the differential expression of the inducible nitric oxide synthase protein. RESULTS: Subcutaneous injection of sodium selenite led to severe oxidative damage in the lenticular tissues, shown by increased formation of formazan crystals, elevated generation of superoxide, hydroxyl, and nitric oxide radicals, and elevated inducible nitric oxide synthase gene and protein expression that possibly contributed to the opacification of the lens and thus cataract formation. When rat pups were treated with intraperitoneal administration of the extract of C. maritima, the generation of free radicals as well as the messenger ribonucleic acid and protein expression of inducible nitric oxide synthase were maintained at near normal levels. CONCLUSIONS: The data generated by this study suggest that an ethanolic extract of C. maritima possibly prevents cataractogenesis in a rat model by minimizing free radical generation.

Keratitis due to the wood saprobic ascomycete, Auerswaldia lignicola (Family Botryosphaeriaceae), in a carpenter in India.

Keratitis due to Auerswaldia lignicola in a 32-year-old Indian male carpenter is described. At presentation, the patient reported persistent pain and tearing (left eye) in spite of topical antimicrobial therapy for more than 3 weeks. Clinically, mycotic keratitis was suspected, and direct microscopy of corneal scrapings stained by lactophenol cotton blue and Gram stains revealed broad septate hyphae. Intensive topical antifungal therapy was then given for 15 days. The keratitis continued to progress, necessitating therapeutic penetrating keratoplasty. Following the keratoplasty, there was rapid reduction in inflammation and gradual quietening of the eye. Brown-black fungal colonies resembling Lasiodiplodia theobromae were isolated from corneal scrape and corneal button (post-surgery) material on Sabouraud glucose-neopeptone agar; however, sporulation did not occur, so the morphological identification could not be confirmed. Sequence analysis of the 18S rRNA region of extracted fungal genomic DNA yielded an identification of A. lignicola Ariyawansa, J.K. Liu & K.D. Hyde; the sequence data have been deposited in GenBank (A. lignicola strain DK/V4, accession number KC866317.1). Medical management of keratitis due to such rarely reported fungal species may be difficult, necessitating surgical procedures.

Deciphering the potential efficacy of acetyl-L-carnitine (ALCAR) in maintaining connexin-mediated lenticular homeostasis.

PURPOSE: To determine the putative role of acetyl-L-carnitine (ALCAR) in maintaining normal intercellular communication in the lens through connexin. METHODS: In the present study, Wistar rat pups were divided into 3 groups of eight each. On postpartum day ten, Group I rat pups received an intraperitoneal injection (50 µl) of 0.89% saline. Rats in Groups II and III received a subcutaneous injection (50 µl) of sodium selenite (19 µmol/kg bodyweight); Group III rat pups also received an intraperitoneal injection of ALCAR (200 mg/kg bodyweight) once daily on postpartum days 9-14. Both eyes of each pup were examined from day 16 up to postpartum day 30. Alterations in the mean activity of the channel pumps, calcium-ATPase and sodium/potassium-ATPase, were determined. The expression of genes encoding key lenticular gap junctions (connexin 46 and connexin 50) and a channel pump (plasma membrane Ca(2+)-ATPase [PMCA1]) was evaluated by reverse transcription-PCR. Immunoblot analysis was also performed to confirm the differential expression of key lenticular connexin proteins. In addition, bioinformatics analysis was performed to determine the interacting residues of the connexin proteins with ALCAR. RESULTS: Significantly lower mean activities of Ca(2+)-ATPase and Na(+)/K(+) -ATPase were observed in the lenses of Group II rats than those in Group I rat lenses. However, the observed mean activities of Ca(2+)-ATPase and Na(+)/K(+)-ATPase in Group III rat lenses were significantly higher than those in Group II rat lenses. The mean mRNA transcript levels of the connexin 46 and connexin 50 genes were significantly lower, while the mean levels of PMCA1 gene transcripts were significantly higher, in Group II rat lenses than in Group I rat lenses. Immunoblot analysis also confirmed the altered expression of connexin proteins in lysates of whole lenses of Group II rats. However, the expression of connexin 46 and connexin 50 proteins in lenses from group III rats was essentially similar to that noted in lenses from normal (Group I) rats. Hydrogen bond-interaction between ALCAR and amino acid residues at the functional domain regions of connexin 46 and connexin 50 proteins was also demonstrated through bioinformatics tools. CONCLUSIONS: The results suggest that ALCAR plays a key role in maintaining lenticular homeostasis by promoting gap junctional intercellular communication.

Expression of genes of the aflatoxin biosynthetic pathway in Aspergillus flavus isolates from keratitis.

PURPOSE: To document transcriptional activation (expression) of key aflatoxin biosynthetic pathway genes in corneal isolates of Aspergillus flavus. METHODS: The expression of certain regulatory (aflatoxin regulatory [aflR] and aflatoxin J [aflJ]) and structural (polyketide synthase acetate [pksA] and norsolonic acid-1 [nor-1]) genes in four corneal A. flavus isolates was evaluated by reverse transcription PCR. The aflatoxin-producing potential of each strain was determined by thin-layer chromatography and quantified by spectrophotometry. Four environmental isolates were used for comparison. The mean expression levels of these genes were compared in the corneal and environmental A. flavus isolates. In addition, the mean expression levels were also correlated with the aflatoxin production levels. RESULTS: All isolates expressed aflJ, nor-1, and pksA, while all but one expressed aflR. Overall, significantly higher mean expression levels occurred in aflatoxigenic than in non-aflatoxigenic corneal isolates. A significant positive correlation was noted between the mean expression level of aflR and the quantum of aflatoxin production by the corneal isolates. Essentially similar patterns of expression of these genes were noted in four environmental A. flavus isolates used for comparison. CONCLUSIONS: For the first time, isolates of A. flavus from human keratitis patients have been shown to express regulatory and structural aflatoxin biosynthetic pathway genes. Further studies are needed to clarify the precise influence of the corneal microenvironment on expression of these genes and aflatoxin production by A. flavus infecting the cornea.

Alterations in the lenticular protein profile in experimental selenite-induced cataractogenesis and prevention by ellagic acid.

BACKGROUND: Accumulating evidence suggests that oxidative stress underlies age-related formation of cataract, and that antioxidants retard cataractogenesis. This study aimed to evaluate whether ellagic acid, a natural polyphenol with antioxidant properties, prevents alterations in the lenticular protein profile in an experimental model of selenite cataract. METHODS: Alterations in lenticular protein were determined by two-dimensional electrophoresis (2DE) and image analysis. Eluted αA-crystallin spots were analyzed by mass spectrometry. Western blot analysis was also performed to confirm the differential expression of certain crystallins and cytoskeletal proteins. RESULTS: In cataractous lenses, 2DE and image analysis revealed approximately 45 and 60 prominent spots in soluble and insoluble protein fractions respectively. Analysis of the pI and molecular weight of protein spots revealed differences in the expression of crystallin proteins in soluble and insoluble fractions. Western blot analysis confirmed changes in the expression of αA- and ßB1- crystallins in both soluble and insoluble protein fractions, while mass spectrometry confirmed the degradation of αA-crystallin in selenite cataractous lenses. Western blot analysis also confirmed the occurrence of altered expression of certain cytoskeletal proteins in insoluble fractions. However, the lenticular protein profile in lenses from selenite-challenged, ellagic acid-treated rats was essentially similar to that noted in lenses from normal rats. CONCLUSIONS: The present study confirms the importance of structural and cytoskeletal proteins in the maintenance of lenticular transparency; the results also suggest that ellagic acid prevents lenticular protein alterations induced by selenite in an experimental setting.

Protective effect of chrysin on carbon tetrachloride (CCl4)-induced tissue injury in male Wistar rats.

Chrysin, a natural flavonoid has been reported to possess potent anti-inflammatory, anti-cancer and antioxidation properties. In the present study, we aimed to evaluate the putative protective effect of chrysin, an isoflavone, on carbon tetrachloride (CCl(4))-induced toxicity in male Wistar rats. Intraperitoneal administration of CCl(4) (2 ml/kg) to rats for 4 days resulted in significantly elevated (p < 0.05) serum levels of glutamic oxaloacetic transaminase (SGOT), glutamic pyruvate transaminase (SGPT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH), when compared to normal rats. In addition, the tissues (liver, kidney and brain) and haemolysate samples showed considerable increase in levels (p < 0.05) of malondialdehyde (MDA) and lowered levels (p < 0.05) of reduced glutathione (GSH), vitamin C and E when compared to values in normal rats. Quantitative analysis of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (Gpx) exhibited lower activities of these antioxidant enzymes in the tissues and haemolysate of CCl(4)-administered rats. The protective action of chrysin on CCl(4)-induced rat was demonstrated with SGPT, SGOT, ALP and LDH resuming to near normal levels, while the mean levels of GSH and of vitamin C and E were elevated, the mean activities of CAT, SOD and Gpx were enhanced and the mean level of MDA was lowered in the tissue and haemolysate samples when compared to the CCl(4)-exposed untreated rats. The expression of the iNOS gene appeared to be up-regulated in the liver and kidney samples of CCl(4)-exposed untreated rats, whereas in CCl(4)-exposed chrysin-treated rats, the mRNA transcript levels of iNOS approximated normal levels. These results strongly suggest that chrysin is able to prevent the oxidative damage induced by CCl(4) in the liver, brain, kidney and haemolysate of male Wistar rats.

Keratitis due to Aspergillus flavus: clinical profile, molecular identification of fungal strains and detection of aflatoxin production.

PURPOSE: To document the clinical profile of patients with keratitis due to Aspergillus flavus and to elaborate on differences in the aflatoxin-producing potential of keratitis strains versus environmental strains of A. flavus. METHODS: Over a 6-month period, strains of Aspergillus flavus were isolated in culture from corneal scrape or biopsy material of patients who presented with suppurative keratitis (clinical isolates). The strains were confirmed to be A. flavus by molecular methods (amplification of the internal transcribed spacer 2 [ITS 2] region and direct sequencing followed by comparative GenBank analysis). The aflatoxin-producing potential of each strain was determined by thin-layer chromatography. The ability of each strain to form sclerotia in Czapek-Dox agar (CDA) after 7 days incubation at 30 degrees C in the dark and to produce a beige ring in yeast extract sucrose agar supplemented with methyl beta-cyclodextrin and sodium desoxycholate (YESD medium) after 3 days incubation at 30 degrees C was also assessed. For comparison, the tests were also run on 10 strains of A. flavus (identity confirmed by molecular methods) collected from local farming areas (environmental isolates). RESULTS: Aflatoxin B1 was detected in 16 (80%) of 20 culture filtrate or mycelial homogenate samples of the clinical isolates (mean concentration: 366.7+/-125.4 parts per billion [ppb]) but in only eight (40%) of 20 samples of environmental isolates (mean concentration: 306.6+/-125.4 ppb). Seven of the eight aflatoxin-producing clinical isolates and two of the four aflatoxin-producing environmental isolates formed sclerotia (>400 microm) and a beige ring in culture. CONCLUSIONS: Aflatoxin B1 was detected in a significantly higher percentage of growth samples of clinical isolates (80%) than growth samples of environmental isolates (40%) (chi(2)=6.667; p=0.0098); the therapeutic implications of this finding require further study. The production of sclerotia and a beige ring in culture appear to be useful markers of aflatoxin-producing potential in strains of A. flavus isolated from keratitis.

Alterations in lenticular proteins during ageing and selenite-induced cataractogenesis in Wistar rats.

PURPOSE: To determine putative alterations in the major lenticular proteins in Wistar rats of different ages and to compare these alterations with those occurring in rats with selenite-induced cataract. METHODS: Lenticular transparency was determined by morphological examination using slit-lamp biomicroscopy. Alterations in lenticular protein were determined by sodium dodecyl sulfate-PAGE (SDS-PAGE) and confirmed immunologically by western blot. RESULTS: Morphological examination did not reveal observable opacities in the lenses of the rats of different age groups; however, dense nuclear opacities were noted in lenses of rats in the selenite-cataract group. Western blot assays revealed age-related changes in soluble and urea-soluble lenticular proteins. Decreased alphaA- and betaB1-crystallins in the soluble fraction and aggregation of alphaA-crystallin, in addition to the degraded fragment of betaB1-crystallin, in the urea-soluble fraction appeared to occur in relation to increasing age of the rats from which the lenses were taken; similarly, cytoskeletal proteins appeared to decline with increasing age. The lenses from rats in the selenite-cataract group exhibited similar changes, except that there was also high molecular weight aggregation of alphaA-crystallin. CONCLUSIONS: The results of this study suggest that there is loss, as well as aggregation, of alphaA-crystallin in the aging rat lens, although there is no accompanying loss of lenticular transparency.

An extract of the oyster mushroom, Pleurotus ostreatus, increases catalase gene expression and reduces protein oxidation during aging in rats.

OBJECTIVE: The objective of the present study was to address the effect of mushroom Pleurotus ostreatus on the catalase (CAT) gene expression and the protein carbonyls in liver and kidney of aged (24 months old) rats. METHODS: Eighteen acclimated rats were divided into 3 groups of 6 each: group I, normal young (4 months old) rats; group II, normal aged (24 months old) untreated rats; group III, normal aged rats treated with mushroom P. ostreatus extract (200 mg/kg body weight administered intraperitoneally for 30 days). On the 31st day, rats were sacrificed by decapitation; the livers and kidneys were removed, washed free of blood, blotted dry and processed immediately. Reverse transcriptase-polymerase chain reaction (RT-PCR) and spectrophotometry were utilized for the analyses of CAT gene expression and protein carbonyl content in the tissues of livers and kidneys. RESULTS: In aged rats that had been treated with the extract of P. ostreatus (group III), the level of the transcript of CAT gene was found to be higher than that in liver (P<0.01) and kidney (P<0.05) of aged untreated (group II) rats, respectively. Treatment of aged rats with P. ostreatus extract (group III) resulted in protein carbonyl levels being significantly lower in liver (P<0.05) and kidney (P<0.01) than those observed in aged untreated (group II) rats. CONCLUSION: These results suggest that an extract of P. ostreatus can enhance the antioxidant enzyme (CAT) gene expression and could decrease the incidence of free radical-induced protein oxidation in aged rats, thereby protecting the occurrence of age-associated disorders that involve free radicals.

Evaluation of the Putative Efficacy of a Methanolic Extract of Ocimum Basilicum in Preventing Disruption of Structural Proteins in an in Vitro System of Selenite-induced Cataractogenesis.

Purpose: To evaluate whether a methanolic extract of Ocimum basilicum (OB) leaves prevented lenticular protein alterations in an in-vitro model of selenite-induced cataractogenesis.Materials and Methods: Transparent lenses extirpated from Wistar rats were divided into three groups: control; selenite only; treated. Control lenses were cultured in Dulbecco's modified Eagle's medium (DMEM) alone, selenite only lenses were cultured in DMEM containing sodium selenite only (100 µM selenite/ml DMEM) and treated lenses were cultured in DMEM containing sodium selenite and the methanolic extract of OB leaves (200 µg of extract/ml DMEM); all lenses were cultured for 24 h and then processed. The parameters assessed in lenticular homogenates were lenticular protein sulfhydryl and carbonyl content, calcium level, insoluble to soluble protein ratio, sodium dodecyl sulphate-polyacrylamide gel electrophoretic (SDS-PAGE) patterns of lenticular proteins, and mRNA transcript and protein levels of αA-crystallin and ßB1-crystallins.Results: Selenite only lenses exhibited alterations in all parameters assessed. Treated lenses exhibited values for these parameters that were comparable to those noted in normal control lenses.Conclusions: The methanolic extract of OB leaves prevented alterations in lenticular protein sulfhydryl and carbonyl content, calcium level, insoluble to soluble protein ratio, SDS-PAGE patterns of lenticular proteins, and expression of αA-crystallin and ßB1-crystallin gene and proteins in cultured selenite-challenged lenses. OB may be further evaluated as a promising agent for the prevention of cataract.