β‐glucuronidase of family‐2 glycosyl hydrolase: A missing member in plants

Glycosyl hydrolases hydrolyze the glycosidic bond in carbohydrates or between a carbohydrate and a non‐carbohydrate moiety. β‐glucuronidase (GUS) is classified under two glycosyl hydrolase families (2 and 79) and the family‐2 β‐glucuronidase is reported in a wide range of organisms, but not in plants. The family‐79 endo-β-glucuronidase (heparanase) is reported in microorganisms, vertebrates and plants. The E. coli family‐2 β‐glucuronidase (uidA) had been successfully devised as a reporter gene in plant transformation on the basis that plants do not have homologous GUS activity. On the contrary, histochemical staining with X‐Gluc was reported in wild type (non-transgenic) plants. Data shows that, family‐2 β‐glucuronidase homologous sequence is not found in plants. Further, β‐glucuronidases of family‐2 and 79 lack appreciable sequence similarity. However, the catalytic site residues, glutamic acid and tyrosine of the family‐2 β‐glucuronidase are found to be conserved in family‐79 β‐glucuronidase of plants. This led to propose that the GUS staining reported in wild type plants is largely because of the broad substrate specificity of family‐79 β-glucuronidase on X‐Gluc and not due to the family‐2 β‐glucuronidase, as the latter has been found to be missing in plants.     

A cluster randomized controlled trial of the effectiveness and cost-effectiveness of Intermediate Care Clinics for Diabetes (ICCD): study protocol for a randomized controlled trial

ABSTRACT: BACKGROUND: World-wide healthcare systems are faced with an epidemic of type 2 diabetes. In the United Kingdom, clinical care is primarily provided by general practitioners (GPs) rather than hospital specialists. Intermediate care clinics for diabetes (ICCD) potentially provide a model for supporting GPs in their care of people with poorly controlled type 2 diabetes and in their management of cardiovascular risk factors. This study aims to (1) compare patients with type 2 diabetes registered with practices that have access to an ICCD service with those that have access only to usual hospital care; (2) assess the cost-effectiveness of the intervention; and (3) explore the views and experiences of patients, health professionals and other stakeholders. METHODS: This two-arm cluster randomized controlled trial (with integral economic evaluation and qualitative study) is set in general practices in three UK Primary Care Trusts. Practices are randomized to one of two groups with patients referred to either an ICCD (intervention) or to hospital care (control).Intervention group: GP practices in the intervention arm have the opportunity to refer patients to an ICCD - a multidisciplinary team led by a specialist nurse and a diabetologist. Patients are reviewed and managed in the ICCD for a short period with a goal of improving diabetes and cardiovascular risk factor control and are then referred back to practice.orControl group: Standard GP care, with referral to secondary care as required, but no access to ICCD.Participants are adults aged 18 years or older who have type 2 diabetes that is difficult for their GPs to control. The primary outcome is the proportion of participants reaching three risk factor targets: HbA1c (<=7.0%); blood pressure (<140/80); and cholesterol (<4 mmol/l), at the end of the 18-month intervention period. The main secondary outcomes are the proportion of participants reaching individual risk factor targets and the overall 10-year risks for coronary heart disease(CHD) and stroke assessed by the United Kingdom Prospective Diabetes Study (UKPDS) risk engine. Other secondary outcomes include body mass index and waist circumference, use of medication, reported smoking, emotional adjustment, patient satisfaction and views on continuity, costs and health related quality of life. We aimed to randomize 50 practices and recruit 2,555 patients. DISCUSSION: Forty-nine practices have been randomized, 1,997 patients have been recruited to the trial, and 20 patients have been recruited to the qualitative study. Results will be available late 2012.Trial registration[ClinicalTrials.gov: Identifier NCT00945204].     

Efficacy of Cyclin Dependent Kinase 4 Inhibitors as Potent Neuroprotective Agents against Insults Relevant to Alzheimer’s Disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disease with no cure till today. Aberrant activation of cell cycle regulatory proteins is implicated in neurodegenerative diseases including AD. We and others have shown that Cyclin dependent kinase 4 (Cdk4) is activated in AD brain and is required for neuron death. In this study, we tested the efficiency of commercially available Cdk4 specific inhibitors as well as a small library of synthetic molecule inhibitors targeting Cdk4 as neuroprotective agents in cellular models of neuron death. We found that several of these inhibitors significantly protected neuronal cells against death induced by nerve growth factor (NGF) deprivation and oligomeric beta amyloid (Aβ) that are implicated in AD. These neuroprotective agents inhibit specifically Cdk4 kinase activity, loss of mitochondrial integrity, induction of pro-apoptotic protein Bim and caspase3 activation in response to NGF deprivation. The efficacies of commercial and synthesized inhibitors are comparable. The synthesized molecules are either phenanthrene based or naphthalene based and they are synthesized by using Pschorr reaction and Buchwald coupling respectively as one of the key steps. A number of molecules of both kinds block neurodegeneration effectively. Therefore, we propose that Cdk4 inhibition would be a therapeutic choice for ameliorating neurodegeneration in AD and these synthetic Cdk4 inhibitors could lead to development of effective drugs for AD.     

Pathobiological implications of MUC16 expression in pancreatic cancer

MUC16 (CA125) belongs to a family of high-molecular weight O-glycosylated proteins known as mucins. While MUC16 is well known as a biomarker in ovarian cancer, its expression pattern in pancreatic cancer (PC), the fourth leading cause of cancer related deaths in the United States, remains unknown. The aim of our study was to analyze the expression of MUC16 during the initiation, progression and metastasis of PC for possible implication in PC diagnosis, prognosis and therapy. In this study, a microarray containing tissues from healthy and PC patients was used to investigate the differential protein expression of MUC16 in PC. MUC16 mRNA levels were also measured by RT-PCR in the normal human pancreatic, pancreatitis, and PC tissues. To investigate its expression pattern during PC metastasis, tissue samples from the primary pancreatic tumor and metastases (from the same patient) in the lymph nodes, liver, lung and omentum from Stage IV PC patients were analyzed. To determine its association in the initiation of PC, tissues from PC patients containing pre-neoplastic lesions of varying grades were stained for MUC16. Finally, MUC16 expression was analyzed in 18 human PC cell lines. MUC16 is not expressed in the normal pancreatic ducts and is strongly upregulated in PC and detected in pancreatitis tissue. It is first detected in the high-grade pre-neoplastic lesions preceding invasive adenocarcinoma, suggesting that its upregulation is a late event during the initiation of this disease. MUC16 expression appears to be stronger in metastatic lesions when compared to the primary tumor, suggesting a role in PC metastasis. We have also identified PC cell lines that express MUC16, which can be used in future studies to elucidate its functional role in PC. Altogether, our results reveal that MUC16 expression is significantly increased in PC and could play a potential role in the progression of this disease.     

Fluorescent-tagged heparan sulfate precursor oligosaccharides to probe the enzymatic action of heparitinase I

Heparitinase I, a key lyase enzyme essential for structural analysis of heparan sulfate (HS), degrades HS domains that are undersulfated at glucuronyl residues through an elimination mechanism. Earlier studies employed viscosimetric measurements and electrophoresis to deduce the mechanism of action of heparitinase I and two other related lyases, heparitinase II and heparitinase III. However, these findings lack molecular evidence for the intermediates formed and could not distinguish whether the cleavage occurred from the reducing end or the nonreducing end. In the current study, 2-aminoacridone (2-AMAC)-labeled HS precursor oligosaccharides of various sizes were prepared to investigate the mechanism of heparitinase I-mediated depolymerization using sensitive and quantitative methodologies. Furthermore, fluorescent (2-AMAC) tagging of HS precursor oligosaccharides allowed us to distinguish fragments that result from cleavage of the substrates at various time intervals and sites farther away from the reducing and nonreducing ends of oligosaccharide substrates. This study provides the first direct molecular evidence for a predominantly random endolytic mechanism of cleavage of HS precursor oligosaccharides by heparitinase I. This robust strategy can be adapted to deduce the mechanism of action of other heparitinases and also to deduce structural information of complex HS oligosaccharides of biological importance.     

Physio-morphological traits and osmoregulation strategies of hybrid maize (Zea mays) at the seedling stage in response to water-deficit stress

Drought has been identified as a major factor restricting maize productivity worldwide, especially in the rainfed areas. The objective of the present study was to investigate the physiological adaptation strategies and sugar-related gene expression levels in three maize (Zea mays L.) genotypes with different drought tolerance abilities (Suwan4452, drought tolerant as a positive check; S7328, drought susceptible as a negative check; Pac339, drought susceptible) at the seedling stage. Ten-day old seedlings of maize genotypes were subjected to (i) well-watered (WW) or control and (ii) water-deficit (WD) conditions. Leaf osmotic potential of cv. S7328 under WD was significantly decreased by 1.35–1.45 folds compared with cv. Pac339 under WW, whereas it was retained in cv. Suwan4452, which utilized total soluble sugars as the major osmolytes for maintaining leaf greenness, F_v/F_m, Φ_PSII, and stomatal function (P_n, net photosynthetic rate; g_s, stomatal conductance; and E, transpiration rate). Interestingly, sucrose degradation (65% over the control) in cv. Pac339 under WD was evident in relation to the downregulation of the ZmSPS1 level, whereas glucose enrichment (1.65 folds over the control) was observed in relation to the upregulation of ZmSPS1 and ZmSUS1. Moreover, CWSI (crop water stress index), calculated from leaf temperature of stressed plants, was negatively correlated with E, g_s, and P_n. Overall, growth characteristics, aboveground and belowground parts, in the drought-susceptible cv. Pac339 and cv. S7328, were significantly decreased (> 25% over the control), whereas these parameters in the drought-tolerant cv. Suwan4452 were unaffected. The study validates the use of leaf temperature, CWSI, P_n, g_s, and E as sensitive parameters and overall growth characters as effective indices for drought tolerance screening in maize genotypes at the seedling stage. However, further experiments are required to validate the results observed in this study under field conditions.

     

Differential expression of defense‐related proteins of rice and sheath blight symptoms in response to active and inactive Rhizoctonia solani toxin

The effects of the phyotoxin from the fungal pathogen Rhizoctonia solani, causing sheath blight on the expression of defense‐related proteins of rice were investigated. The toxin inactivated by chemical treatment and by the toxin‐inactivating enzyme α‐glucosidase produced by Trichoderma viride was used in the study along with the active toxin. Toxin inactivated by T. viride α‐glucosidase and sodium periodate caused significantly less damage and electrolyte leakage to test plants. The active toxin and the pathogen induced chitinase and ß‐1,3‐glucanase synthesis in rice plants, while the inactivated toxin did not have any effect on the expression of these pathogenesis‐related proteins. The toxin was found to suppress the peroxidase activity 72 h after inoculation and the inactivated toxin restored the activity as that of untreated plants. There was no remarkable change in phenylalanine ammonia lyase activity in rice sheath treated with both the forms of the toxin.