Comparative safety evaluation of riot control agents of synthetic and natural origin.

Riot control agents (RCA) are lachrymatory, irritating compounds which temporarily incapacitate the uncontainable crowd. Ortho-Chlorobenzylidene-malononitrile (CS), 2-chloroacetophenone (CN), dibenz[b,f]1:4-oxazepine (CR), and nonivamide (PAVA) are synthetic RCAs, while oleoresin extract of chili known as oleoresin capsicum (OC) a natural irritant has been in use by various law enforcement agencies. Though efficacy of these agents is beyond doubt, they suffer from certain drawbacks including toxicity, production cost, and ecological compatibility. Presently, we have evaluated the safety of CR, OC, and PAVA on inhalation variables along with oral lethality. Additionally, the liver function test (LFT) in serum and lungs function was evaluated in broncho-alveolar-lavage fluid (BALF), both collected on the 14th day after RCA exposure. Animals then sacrificed and histopathology of liver and lungs was carried out. Results showed OC and PAVA to be more toxic than CR with an oral LD50 of 150 and 200 mg/kg body weight, respectively, while CR was safe at >3 g/kg body weight. All three agents caused severe impairment of respiratory variables bringing down normal respiration by >80% with rise in sensory irritation. Recovery from the irritating effect of CR was more rapid than OC and PAVA. LFT and BALF variables were not significantly different from that of control. There were no remarkable histopathological changes in liver and lungs. Hence, as per results, CR is safest among all synthetic and natural origin RCAs and can be safely used for effective dispersion of disobedient mob.

Seasonal variation in nutritional composition of Kappaphycus alvarezii (Doty) Doty-an edible seaweed.

Seasonal variation in the proximate and mineral composition of Kappaphycus alvarezii were investigated in the present study, moreover, the relationship between the nutritive components of this seaweed and the environment were also established. Carbohydrates represented the major portion of the algae (i.e. average total carbohydrate content was 23.01 ± 1.64 g/100 g DW), while the lipid content was the lowest among the constituents investigated (0.39 ± 0.04 to 0.91 ± 0.51 g/100 g DW). The protein content of K. alvarezii varied from 12.69 ± 0.6 to 23.61 ± 0.02 g/100 g DW, and the fiber content varied between 9.68 ± 0.08 to 18.57 ± 0.15 g/100 g DW. Highest total mineral content (29939.61 ± 9340.38 mg/100 g DW) was observed in April 2005, while least values were recorded in January 2006 i.e. (10997.62 ± 1120.26 mg/100 g DW). The Na/K ratio during the study ranged from 0.34 to 0.87. All the samples showed remarkable semi-refined carrageenan (SRC) yield ranging from 42.70 ± 1.07 to 63.73 ± 1.73 % (average 53.90 ± 1.37 %), and, the samples collected during December 2004 and January 2006 demonstrated maximum gel strengths i.e. 743 ± 15.28 and 783.33 ± 15.28 g·cm(-2) respectively. Various environmental parameters influenced the chemical composition of K. alvarezii, and these parameters demonstrated seasonal fluctuations. Moreover, based on the nutritional composition obtained, it could be stated that this seaweed has great scope to be incorporated into several food products as an excellent nutritional supplement, or as a value additive in animal or pet food.

Development & evaluation of biotinylated DNA probe for clinical diagnosis of chikungunya infection in patients' acute phase serum & CSF samples.

BACKGROUND & OBJECTIVES: The resurgence of chikungunya virus (CHIKV) in the Indian Ocean Islands and India has drawn worldwide attention due to its explosive nature, high morbidity and complex clinico-pathological manifestations. The early confirmatory diagnosis of CHIKV is essential for management as well as control of unprecedented epidemics. The present study describes the development and evaluation of a highly sensitive and specific E1 structural gene specific biotinylated DNA probe for detection of chikungunya virus in clinical samples using a dot blot format. METHODS: The complementary DNA (cDNA) of CHIKV was spotted on to nylon membrane. The membrane was subjected to prehybridization and hybridization and developed using a colour development solution containing DAB chromogen. RESULTS: The CHIKV E1 specific DNA probe was highly sensitive detecting picogram levels of target nucleic acid. The comparative evaluation with SYBR Green I based real-time RT-PCR revealed 99 per cent accordance with a sensitivity and specificity of 99 and 98 per cent, respectively. The specificity of this assay was further confirmed through cross-reaction studies with confirmed dengue and Japanese encephalitis (JE) patient serum samples along with infected culture supernatant of Ross River and Saint Louis encephalitis and plasmid DNA of O'Nyong Nyong, Semlinki forest and Sindbis viruses. INTERPRETATION & CONCLUSION: The DNA probe reported in this study may be useful for specific, sensitive and confirmatory clinical diagnosis of chikungunya infection in acute phase human patient serum and CSF samples. This assay can also be used in the laboratory for quantification of viral antigen in cell culture supernatant for research purpose.

Association of lipid peroxidation with endothelial dysfunction in patients with overt hypothyroidism.

Hypothyroidism is associated with increased oxidative stress. The mechanism underlying the endothelial dysfunction in thyroid disease is not yet clear. This study aims to investigate lipid peroxidation and its association with endothelial dysfunction in overt hypothyroidism (OHT).Plasma malondialdehyde (MDA) as a marker of oxidative stress and plasma nitrates and asymmetric dimethyl arginine levels (ADMA) as markers of endothelial dysfunction were estimated in 25 OHT patients in comparison to 25 euthyroid controls. Plasma MDA, ADMA levels were significantly increased, whereas plasma nitrates were significantly decreased in the patient group compared to control group (p<0.01). Moreover, a significant positive association between plasma MDA and ADMA was found in the patient group (ρ=0.472, p=0.036). Our results reveal the presence of endothelial dysfunction in OHT patients as evidenced by decreased plasma nitrates and increased ADMA levels. Increased levels of MDA represent an increased generation of reactive oxygen species in these patients. A finding of significant direct relation of plasma MDA with ADMA indicates that oxidative stress has a strong impact on endothelial dysfunction in overt hypothyroidism. Further studies focusing on the role of oxidative stress in endothelial dysfunction and the effects of antioxidant supplementation on endothelial function in OHT patients are required.

Evaluation of protective efficacy of CC-2 formulation against topical lethal dose of T-2 toxin in mice.

T-2 toxin is the type-A trichothecene and a common contaminant of food and cereals, produced by Fusarium species. T-2 toxin easily penetrates skin due to its lipophilic nature and causes skin irritation and blisters in humans. Physical protection of the skin and airway is the only proven effective method of protection. To date, no chemical antidotes are available to prevent T-2 induced lethality. In the present study, we evaluated the protective efficacy of 20% N,N'-dichloro-bis(2,4,6-trichlorophenyl) urea (CC-2) formulation against lethal topical exposure dose of T-2 toxin in mice. None of the animals exposed to only T-2 toxin at lethal dose of 2 and 4 LD50 (11.8 and 23.76 mg/kg body weight) survived beyond 36 and 16 h, respectively. CC-2 application at 5 and 15 min post-exposure protected mice 100% from lethality at 2 LD50. Survival rate was 100% and 50% at 4LD50 dose if CC-2 was applied dermally within 5 and 15 min post-exposure. Recovery profile of surviving animals after 2LD50 T-2 toxin exposure at 1, 3, 7, and 14 days was assessed in terms of hepatic GSH, lipid peroxidation, serum ALP, ALT and AST. Hepatic lipid peroxidation significantly increased in all groups exposed to T-2 toxin by 3 day but normalized by day 7. A delayed GSH depletion was noted in surviving animals on day 7 but recovered by day 14. ALT and AST levels were elevated in all CC-2 protected mice on day 1 and normalized by day 3. ALP level decreased till day 7 in all protected groups. The biochemical variables recovered to control values by 14th day. GC-MS analysis after in vitro interaction of CC-2 formulation with T-2 toxin had shown that nearly 86% of T-2 toxin is decontaminated in 5 min but 8-10% of T-2 toxin was still present even after 60 min of interaction. Results of our study suggest that CC-2 may be an effective dermal decontaminant against lethal topical exposure of T-2 toxin.

The effect of L-carnitine supplementation on lipid parameters, inflammatory and nutritional markers in maintenance hemodialysis patients.

Protein energy malnutrition and inflammation are common and usually concurrent in maintenance hemodialysis (MHD) patients. Carnitine, a small molecule involved in fatty acid metabolism, is significantly decreased in long-term HD patients. L-Carnitine supplementation may have potential benefits in improving dialysis-related disorders. However, there are conflicting reports with regard to the beneficial effects of L-Carnitine supplementation. Hence, the present study was carried out to evaluate the effect of L-Carnitine supplementation on lipid parameters, apoproteins and inflammatory and nutritional markers in HD patients. A total of 35 patients with end-stage renal disease, on MHD for a period of 2 to 5 years were recruited into the study. The study group consisted of 20 patients who received Carnitine supplementation intravenously three times a week after each HD session, at 1 g/dose, while the control group consisted of 15 patients without supplementation with L-Carnitine. Highly sensitive C-reactive protein (hsCRP), total protein, albumin, lipid profile and apoprotein AI and B were determined at baseline and at the end of the study. A significant decrease in the hsCRP levels was observed in the Carnitine-supplemented group (P < 0.05). However, no significant change was observed in the lipid parameters and nutritional markers in the Carnitine-supplemented group. In conclusion, the present study demonstrates the significant benefit of L-Carnitine supplementation on inflammatory status in MHD patients as noted by marked decrease in hsCRP levels in comparison with the control group.

Association of serum antioxidants and risk of coronary heart disease in South Indian population.

BACKGROUND AND AIM: Higher prevalence of coronary heart disease (CHD) has been reported in south Indian population, which cannot be accounted for by the traditional risk factors like hyperlipidemia. Identification of new risk factors may help in treatment and prevention of CHD in this part of the world. In an attempt to investigate the causes of increased incidence of CHD in this part of the world, we intended to look for oxidative stress in our patients as a possible risk factor. As an initial step in this perspective, a case- control study was conducted to find out the serum antioxidant levels and their association with CHD in south Indian population. SETTINGS AND DESIGN: A tertiary care hospital; Case--control study. MATERIALS AND METHODS: One hundred thirty nine angiographically proven CHD patients (aged 29-75 years) were studied against 59 population based healthy controls (aged 29-72 years) free of CHD. Fasting serum cholesterol, triglycerides, HDL cholesterol, erythrocyte and plasma glutathione peroxidase and superoxide dismutase were estimated on automated clinical chemistry analyzer. LDL cholesterol and VLDL cholesterol were calculated. Vitamins A and E were estimated using high performance liquid chromatography (HPLC). STATISTICAL ANALYSIS: Unpaired t test was used to compare means. Binary logistic regression was done to find out the association between dependent and independent variables. RESULTS: Significantly higher levels of Total Cholesterol/HDL cholesterol and LDL cholesterol/HDL cholesterol ratio and lower HDL cholesterol levels were observed in patients when compared to controls. No significant difference of plasma and erythrocyte glutathione peroxidase and superoxide dismutase activity was observed between patients and controls. Significantly lower levels of vitamin E in patients than in controls was observed (P<0.001). Serum vitamin E was inversely associated with coronary heart disease even after controlling for age and other coronary risk factors (Odds ratio 0.898, 95% CI 0.826-0.976 P=0.01). CONCLUSIONS: The results of present study suggest that deficiency of vitamin E may be an independent risk factor of CHD. This study brings out the need for long- term monitoring of vitamin E supplementation as a preventive measure for CHD in the population studied.

Therapeutic potential of monoisoamyl and monomethyl esters of meso 2,3-dimercaptosuccinic acid in gallium arsenide intoxicated rats.

The dose dependent effects of monoisoamyl and monomethyl esters of meso 2,3-dimercaptosuccinic acid (DMSA) (0.1, 0.3 and 0.5 mmol kg(-1), intraperitoneally (i.p.) once daily for 5 days) to offset the characteristic biochemical, immunological, oxidative stress consequences and DNA damage (based on DNA fragmentation and comet assay) following sub-chronic administration of gallium arsenide and the mobilization of gallium and arsenic were examined. The effects of these chelators alone in normal animals too were examined on above-mentioned variables. Male Wistar rats were exposed to 10 mg kg(-1), GaAs, orally once daily for 12 weeks and were administered DMSA or two of its monoesters (monoisoamyl or monomethyl) for 5 consecutive days. DMSA was used as a positive control. DMSA and its derivatives, when given alone, generally have no adverse effects on various parameters. After 5 days of chelation therapy in GaAs pre-exposed rats, MiADMSA was most effective in the reduction of inhibited blood delta-aminolevulinic acid dehydratase (ALAD) activity and zinc protoporphyrin level while, all three chelators effectively reduced urinary ALA excretion, compared to GaAs alone exposed rats. MiADMSA was also effective, particularly at a dose of 0.3 mmol kg(-1), in enhancing the inhibited hepatic transaminase activities. Parameters indicative of oxidative stress responded less favorably to the chelation therapy, however, three chelators significantly restored the altered immunological variables. MiADMSA was relatively more effective than the other two chelators. GaAs produced significant DNA damage in the liver and kidneys and the chelation treatment had moderate but significant influence in reducing DNA damage. All three chelators significantly reduced arsenic concentration and, however, MiADMSA was more effective than the other two chelators in depleting arsenic concentration from blood and other soft tissues. A dose of 0.3 mmol kg(-1) was found to be relatively better than the other two doses examined. Gallium contents of blood and soft tissues remained uninfluenced by the chelation therapy. Significant loss of copper after MiADMSA administration, however, is of concern and requires further exploration. Additionally, further studies are required for the choice of appropriate dose, duration of treatment and possible toxic/side effects. Keeping in view the promising role of MiADMSA in the treatment of GaAs poisoning, these data will be needed for the registration of this chelating agent as licensed drug for the treatment of gallium arsenide intoxication.

Design, synthesis and biological evaluation of benzimidazole/benzothiazole and benzoxazole derivatives as cyclooxygenase inhibitors.

We have synthesised a series of 2-[[2-alkoxy-6-pentadecylphenyl)methyl]thio]-1H-benzimidazoles/benzothiazoles and benzoxazoles from anacardic acid and investigated their ability to inhibit human cyclooxygenase-2 enzyme (COX-2). The active compounds were screened for cyclooxygenase-1 (COX-1) inhibition. Compound 13 is 384-fold and 19 is more than 470-fold selective towards COX-2 compared to COX-1. Thus, this class of compounds may serve as excellent candidates for selective COX-2 inhibition.

Lessons in integration--operations research in an Indian leprosy NGO.

Since the Alma Ata Declaration in 1978, health systems supporting the treatment and control of infectious diseases like leprosy and tuberculosis have been encouraged to 'integrate' into the primary health care structure within countries. Now, more than 20 years later, countries are still grappling with the concept of integration and looking for ways to achieve it. This study reports findings from a leprosy/Tuberculosis/AIDS awareness pilot project conducted by LEPRA India, a leprosy non-governmental organization (NGO), between 1996 and 2000 in Koraput district, Orissa. The project addressed the issue of integration on two levels. On the one hand LEPRA used the context of the project to explore ways in which to integrate TB services into their existing leprosy control structure. On the other hand, lessons from the pilot study were intended to help the organization find ways of linking with the government health care structure. Following a 'qualitative approach', this operations research project assessed the perceptions of communities and providers about leprosy and tuberculosis services. Providers across the spectrum of this plural healthcare system were asked to provide comment on developing stronger networks with each other, with NGOs and with government, while patients and communities were asked to describe the resources available to them and the constraints they face in accessing health care in general, and for leprosy and TB in particular. LEPRA staff from top management to the outreach workers were also approached for their views. Patients and communities noted that physical access to treatment was a major constraint, while the existence of local providers and family support structures facilitated health and health care. Providers expressed a willingness to collaborate (with LEPRA and the government), but lacked training, adequate staff support and the appropriate equipment/technical resources. Also lacking were adequate information campaigns to inform the public about these diseases and their treatment. This information has provided LEPRA with an understanding of how they might best fill gaps in the existing system and therefore assist in the process of integrating services in their own organization and through the primary health care structure. To achieve this aim, LEPRA will increasingly become involved in developing relationships and partnerships with government in the delivery of training and services and in infrastructure development.

Initial experiences of implementation of functional integration (FI) in LEPRA India projects in Orissa.

In 2000, the Government of the State of Orissa (population 37 million) in India decided to introduce functional integration for the control of leprosy, in place of the long-established vertical programme, using the general health services and the primary health care system. This paper describes the initial (9 months) experience of implementing this strategy in two projects run by LEPRA India. One of these, in the district of Koraput, was established in 1991 and covers a population of 1.5 million people. The other, in Kalahandi district, started in 1997 and covers a population of 600,000. Both projects operate under difficult conditions with regard to terrain, the use of numerous tribal languages, illiteracy, water shortage, poor roads and communications. The preparatory phase included intensive health education of the public on leprosy, using a wide range of educational media and techniques. At the same time, LEPRA India supported the Government in the training and orientation of trainers, medical officers, primary health care staff and female health workers at village level. In all, over 2000 were trained. This paper describes all aspects of the implementation of functional integration in these two areas. In the 9-month period, 4207 suspect cases were referred to medical officers by health workers, but only 256 (6%) were confirmed as having leprosy. There were 169 confirmed self-reporting cases. Despite the clearly understood intention to involve primary health staff in case detection, 67% of all cases were in fact detected by LEPRA India, possibly due to overlapping attendance at clinics by vertical and general staff. There is obviously a need for further training of the general staff since only 6% of cases referred by them were confirmed as having leprosy. Steps must also be taken to ensure that the emphasis on case detection, confirmation and treatment shifts from the vertical to the general health staff. The supply of anti-leprosy drugs and steroids to primary health centers needs improvement. Appropriate teaching and learning material is urgently needed for both field staff and medical officers.

Lovastatin-induced cytoskeletal reorganization in lens epithelial cells: role of Rho GTPases.

PURPOSE: To understand the involvement of isoprenylated small guanosine triphosphatases (GTPases) in lovastatin-induced cataractogenesis, Rho- and Rac-mediated cell adhesion and actin cytoskeletal reorganization were investigated in lovastatin-treated lens epithelial cells. METHODS: The effects of lovastatin on F-actin reorganization (phalloidin staining), focal adhesion formation (paxillin or vinculin), cell-cell adhesions (cadherin and beta-catenin), and protein tyrosine phosphorylation were evaluated in human and porcine lens epithelial cells by immunocytochemical staining with specific antibodies. To explore the involvement of the Rho and Rac GTPases in lovastatin-mediated effects, changes in distribution of Rho and Rac GTPases were analyzed by Western blot analysis, and the effects of C3-exoenzyme on lovastatin-induced cytoskeletal changes were evaluated by immunocytochemical analysis. RESULTS: Lovastatin induced drastic changes in cell shape in both human and porcine lens epithelial cells, including a profound loss of actin stress fibers, focal adhesions, protein phosphotyrosine, and cell-cell adhesions. Lovastatin treatment also led to the accumulation of nonisoprenylated Rho and Rac GTPases in cytosolic fraction. Supplementation of culture media with geranylgeranyl pyrophosphate dramatically reversed the lovastatin-induced morphologic and cytoskeletal changes, whereas farnesyl pyrophosphate was ineffective. Treatment of cells with C3-exoenzyme (a Rho GTPase-specific inhibitor), however, abolished the geranylgeranyl-supplementation-induced recovery from the morphologic and cytoskeletal effects of lovastatin. CONCLUSIONS: This study demonstrates that inhibition of protein prenylation by lovastatin leads to disruption of actin cytoskeletal organization, and to loss of integrin-mediated focal adhesions and cadherin-mediated cell-cell adhesions in lens epithelial cells. Based on isoprenoid supplementation studies, it could be concluded that impairment of geranylgeranylated Rho and Rac GTPase function is most likely responsible for lovastatin-induced cytoskeletal changes in lens epithelial cells.

Subacute (90 days) oral toxicity studies of Kombucha tea.

Kombucha tea (KT) is a popular health beverage and is used as an alternative therapy. KT is prepared by placing the kombucha culture in solution of tea and sugar and allowing to ferment. The inoculum is a fungus consisting of symbiotic colony of yeast and bacteria. KT is consumed in several countries and is believed to have prophylactic and therapeutic benefits in a wide variety of ailments, viz., intestinal disorders, arthritis, ageing and stimulation of immunological system. Though KT is used in several parts of the world its beneficial effects and adverse effects have not been scientifically evaluated. Since there are no animal toxicological data on KT, subacute oral toxicity study was carried out. Five groups of rats were maintained: (a) control group given tap water orally, (b) KT given 2 ml/kg orally, (c) plain tea (PT) given 2 ml/kg orally, (d) KT given in drinking water, 1% (v/v) and (e) PT given in drinking water, 1% (v/v). The rats were given this treatment daily for a period of 90 days. Weekly records of weight, feed intake, water intake and general behaviour were monitored. There was no significant difference in the growth of the animals as evidenced by the progressive body weight change. The organ to body weight ratio and histological evaluation did not show any toxic signs. The haematological and biochemical variables were within the clinical limits. The study indicates that rats fed KT for 90 days showed no toxic effects.

Heteromorpha trifoliata (Dombwe) accelerates acetic acid-induced peptic ulcers: a preliminary study in the rat.

OBJECTIVES: To investigate the effect of H. trifoliata on: (a) acetic acid-induced ulcers, (b) food intake, (c) water intake, (d) weight gain, (e) gastric acid secretion in rats. DESIGN: Comparative study. SETTING: Laboratory. SUBJECTS: 20 female Sprague Dawley rats (220 to 250 g) with acetic acid-induced peptic ulcers randomly assigned to test and control groups (n = 10). The test rats were allowed water and normal rat diet comprising 20% H. trifoliata ('Dombwe) and 'Imfe nkulu' in Shona and Ndebele respectively) for 17 days after ulceration while control rats were allowed water and normal rat diet for 17 days after ulceration. Thirty six other rats were prepared to study the effect of H. trifoliata on gastric and acid secretion stimulated by histamine, gastrin and carbachol. MAIN OUTCOME MEASURES: Photographs of the gross anatomy and histology of test and control rat stomachs were taken. Daily food and water intake, weekly weight gain and gastric acid secretion were measured in the test and control rats. RESULTS: 17 days following the consumption of the H. trifoliata containing diet, macroscopically, no ulcers were found on the outer surface of the stomach walls of test rats. However, histological examination revealed traces of ulcer at the sites where ulcers were induced previously. In contrast, 70% of the control rats still had ulcers on the surface of their stomach walls. Histological examination showed massive denuded mucosa and submucosa at the ulcer sites which are signs of severe ulceration. Food intake in both groups was not significantly different except during the first three days when test rats consumed significantly less food (p < 0.01) than control rats. Daily water intake and weekly weight gain were also not significantly different in the test and control groups. H. trifoliata had no significant effect on gastric acid secretion stimulated by histamine, gastrin and carbachol. CONCLUSIONS: H. trifoliata does not affect daily food and water intake and weekly weight gain in rats. It also does not affect histamine, gastrin and carbachol-stimulated acid secretion in rats. However, H. trifoliata accelerates the healing of acetic acid-induced peptic ulcer in rats. This may validate the use of H. trifoliata in the treatment of peptic ulcer in humans.

ELISA for the detection of venoms from four medically important snakes of India.

A double antibody sandwich enzyme linked immunosorbent assay (ELISA) was developed to detect Echis carinatus venom in various organs (brain, heart, lungs, liver, spleen and kidneys) as well as tissue at the site of injection of mice, at various time intervals (1, 6, 12, 18, 24 h and 12 h intervals up to 72 h) after death. The assay could detect E. carinatus venom levels up to 2.5 ng/ml of tissue homogenate and the venom was detected up to 72 h after death. A highly sensitive and species-specific avidin-biotin microtitre ELISA was also developed to detect venoms of four medically important Indian snakes (Bungarus caeruleus, Naja naja, E. carinatus and Daboia russelli russelli) in autopsy specimens of human victims of snake bite. The assay could detect venom levels as low as 100 pg/ml of tissue homogenate. Venoms were detected in brain, heart, lungs, liver, spleen, kidneys, tissue at the bite area and postmortem blood. In all 12 human victim cadavers tested the culprit species were identified. As observed in mice, tissue at the site of bite area showed the highest concentration of venom and the brain showed the least. Moderate amounts of venoms were found in liver, spleen, kidneys, heart and lungs. Development of a simple, rapid and species-specific diagnostic kit based on this ELISA technique useful to clinicians is discussed.

Identification of a novel hydroxyproline-rich glycoprotein as the major allergen in Parthenium pollen.

BACKGROUND: The airborne pollen of the Compositae weed, Parthenium hysterophorus, is a major cause of allergic rhinitis in the Indian subcontinent and in certain parts of the southern United States and western Australia. Earlier studies have identified a 31 kd protein as the major allergen in Parthenium pollen. OBJECTIVE: This study was undertaken to carry out the purification, immunochemical characterization, sequencing, and epitope analysis of this major allergen, designated as Par h I. METHODS: The IgE-binding activity of the allergen was evaluated by immunoblot and inhibition ELISAs. Pronase digestion, periodate oxidation, and chemical deglycosylation were performed to determine the role of peptide and carbohydrate components of the allergen in IgE binding. RESULTS: The data provide evidence for the involvement of carbohydrate moieties on Par h 1 in its IgE-binding ability. The N-terminal 91 amino acid sequence of Par h 1 shows 81% identity with a protein from sunflower anther, and the hydroxyproline-rich region of Par h 1 is 30% to 40% identical to similar stretches in extensins, a class of hydroxyproline-rich cell wall glycoproteins from different plant species. IgE antibodies in the sera of individuals allergic to Parthenium cross-reacted with a 50 kd hydroxyproline-arabinose-rich extensin precursor from potato tuber, and this binding was periodate-sensitive. CONCLUSIONS: It appears that a group of soluble plant glycoproteins, which are related to the ubiquitous extensins, have certain carbohydrate-containing IgE-binding epitopes that may contribute to allergenic cross-reactivity among specific pollens and foods.

Investigations into the loss of glutathione from lenses in organ culture.

PURPOSE: To investigate possible causes and implications of the decrease in glutathione concentration in rat lenses during organ culture. METHODS: Freshly excised lenses were incubated in modified TC-199 medium. Ellman's Reagent or the GSH-400 assay were used to assay glutathione levels in lenses cultured for different times and under a variety of altered culture conditions. RESULTS: In lenses from young rats the glutathione decrease was not ameliorated by reduction of oxygen tension in the incubator, nor by supplementation of the culture medium with various antioxidants or sulfhydryl compounds, nor with the amino acid precursors of glutathione. Addition of 2-mercaptoethanol stimulated cysteine transport into the lens but had only a modest effect in maintaining the level of glutathione. The decrease in glutathione concentration was less in cultured lenses from older rats. Lenses from rhesus monkeys exhibited no decrease in glutathione levels when maintained in organ culture for up to 48 h. CONCLUSIONS: The basis for the decreased glutathione in cultured young rat lenses is still uncertain. The data from the present study indicate a definite relationship between glutathione loss and age for cultured rat lenses, with young lenses being much more susceptible. The resistance of cultured monkey lenses to loss of glutathione demonstrates species differences in this property which may be relevant to previously reported differences in susceptibility to oxidative damage.

Phytic acid, in vitro protein digestibility, dietary fiber, and minerals of pulses as influenced by processing methods.

The objective of this project was to determine the effect of various types of processing on selected nutrition related parameters of commonly consumed Indian pulses and soybean. Germination reduced the phytic acid content of chickpea and pigeonpea seeds by over 60%, and that of mung bean, urd bean, and soybean by about 40%. Fermentation reduced phytic acid contents by 26-39% in all these legumes with the exception of pigeonpea in which it was reduced by more than 50%. Autoclaving and roasting were more effective in reducing phytic acid in chickpea and pigeonpea than in urd bean, mung bean, and soybean. Germination and fermentation greatly increased the in vitro protein digestibility (IVPD). IVPD was only slightly increased by roasting and autoclaving of all legumes. Germination and fermentation also remarkably decreased the total dietary fiber (TDF) in all legumes. Autoclaving and roasting resulted in slight increases in TDF values. All the processing treatments had little effect on calcium, magnesium and iron contents.

Recombinant antigen-based avidin-biotin microtiter enzyme-linked immunosorbent assay for serodiagnosis of invasive amebiasis.

An immunoscreening approach was used to isolate a strongly positive cDNA clone from an Entamoeba histolytica HK-9 cDNA expression library in the phage vector lambda ZAP-II. The 1.85-kb cDNA insert was found to be truncated and encoded the cysteine-rich, immunodominant domain of the antigenic 170-kDa subunit of the amebal galactose-N-acetylgalactosamine binding lectin. This domain was expressed as a glutathione S-transferase fusion protein in Escherichia coli. Inclusion bodies of the recombinant protein were solubilized with Sarkosyl, and the protein was enriched from the crude bacterial extract by thiol-affinity chromatography. The recombinant protein was used to develop a rapid, sensitive, and specific avidin-biotin microtiter enzyme-linked immunosorbent assay (ELISA) for invasive amebiasis. Sera from 38 individuals suffering from invasive amebiasis, 12 individuals with noninvasive amebiasis, 44 individuals with other infections, and 27 healthy subjects were screened by the recombinant antigen-based ELISA. The sensitivity and specificity of the assay were 90.4 and 94.3%, respectively, which correlated well with those of an ELISA developed with crude amebal antigen (r = 0.94; P < 0.0001), as well as with those of a commercially available serodiagnostic ELISA (r = 0.92; P < 0.0001). Thus, the bacterially expressed recombinant lectin can replace the crude amebal extract as an antigen in the serodiagnosis of invasive amebiasis by using avidin-biotin microtiter ELISA.

Insulin decreases the glycogen synthase kinase-3 alpha mRNA levels by altering its stability in streptozotocin-induced diabetic rat liver.

The chronic effect of insulin on the expression of the glycogen synthase kinase-3 alpha gene in streptozotocin-induced diabetic rat liver is examined. The mRNA levels of glycogen synthase kinase-3 alpha were increased (143% of normal levels) in diabetic livers and these were normalized by insulin supplementation to the diabetic animals. Neither diabetes nor insulin supplementation to diabetic rats altered the transcription rate of glycogen synthase kinase-3 alpha. However, diabetes caused an increase in the half-life of glycogen synthase kinase-3 alpha mRNA from 5 h in normal hepatocytes to 8 h in diabetic ones. Insulin supplementation to the incubation medium of diabetic hepatocytes decreased the half-life of glycogen synthase kinase-3 alpha mRNA to a level comparable with normal values. This study suggests that the chronic effect of insulin decreases the levels of glycogen synthase kinase-3 alpha mRNA by altering its stability.