Interferon γ and glycemic status in diabetes associated with chronic pancreatitis.

BACKGROUND/AIMS: Although the role of cytokines in the etiopathology of chronic pancreatitis (CP) is well recognized, information on pancreatic tissue cytokines in CP with/without associated diabetes is unavailable. The aim of the present study was to identify the differences in pancreatic cytokines and observe their correlations with the glycemic status in CP. METHODS: Pancreata were obtained from CP patients (n = 44), with/without associated diabetes and non-diabetic control subjects (n= 20). Patients with CP were classified into two groups after ascertaining their diabetic status. Pancreatic cytokines (IL 1ß, IL 6, IL 8, IL 10, IL 12P70, TNF α, IFN γ) were analyzed by flow cytometer. The influence of individual and cocktail of cytokines on glucose stimulated insulin release (GSIR) was examined by challenging the islets from control subjects. RESULTS: The pancreatic IFN γ levels in diabetic and non diabetic CP patients were significantly higher in comparison to controls. The glucose stimulated insulin release (GSIR) in response to high glucose concentration in control islets, islets from non-diabetic and diabetic CP patients was 8.2, 5.67 and 3.15 µU × 10(-3)/min/islet equivalent respectively. IFN γ resulted in 82.35% decrease in GSIR from the control islet cells at a concentration of >20 pg/ml which was reversed by epigallocatechin-3-gallate (EGCG). CONCLUSION: These results suggest that IFN γ among other cytokines, play a major role in ß-cell dysfunction associated with CP.

ß-Cell dysfunction in chronic pancreatitis.

Chronic pancreatitis (CP) is a progressive inflammatory disease characterized by irreversible destruction of pancreatic secretory parenchyma, fibrosis, exocrine atrophy, and endocrine insufficiency leading to diabetes. Secondary diabetes occurring in CP subsequent to destruction of pancreatic ß-cells is distinct, since it involves ß-cell dysfunction amidst an inflammatory milieu. Even though considerable knowledge is available on the pathophysiology and clinical management of CP, relatively much less is known about the molecular events leading to ß-cell dysfunction. Investigators have demonstrated that altered morphology, reduced ß-cell mass, and ß-cell numbers result in endocrine insufficiency. However, recent reports and our observations suggest that ß-cell dysfunction develops in the early stages of CP while clinical diabetes manifests later, when there is profound fibrosis. In the early stages, altered internal milieu and physiology arising due to inflammation and release of cytokines might lead to deranged signaling pathways and islet dysfunction. Subsequently, development of fibrosis causes islet destruction. This suggests that endocrine deficiency in CP is multifactorial. Although the role of transcription factors (Pdx-1, MafA, NeuroD) on ß-cell functions is understood, alterations in internal milieu of pancreatic tissue that affects ß-cell functions in CP has not been elucidated. In this review, we summarize the factors that have an effect on islet functions. Understanding molecular events of ß-cell dysfunction in CP can lead to the development of targeted preventive and therapeutic modalities.

Sugar coated ceramic nanocarriers for the oral delivery of hydrophobic drugs: formulation, optimization and evaluation.

In order to enhance the delivery of poorly-soluble drugs, we have explored aquasomes (three-layered, ceramic core based, oligosaccharide coated nanoparticles) as potential carriers for the delivery of model hydrophobic drug piroxicam (log P = 3.1). Ceramic nanoparticles were prepared using two techniques; namely, co-precipitation by refluxing and co-precipitation by sonication. Core preparation was finally done using sonication approach; based on the higher % yield (42.4 ± 0.4%) and shorter duration (1 day) compared to the reflux method (27.4 ± 2.05%, 6 days). Lactose loading onto ceramic core was achieved using adsorption. Colorimetric analysis of lactose coating was done using Anthrone method. Optimization of process variables namely, incubation time and core to coat ratio (for sugar loading) was carried out. Optimum time of incubation was 3 h and the core to coat ratio was 4:1. The drug loading was achieved by incubating the sugar loaded cores in different concentrations of piroxicam solution and it was found that 1.5% w/v piroxicam was optimal. Structural characterization using Fourier-Transform Infra Red Spectroscopy (FTIR) confirmed the presence of sugar coating onto the core. Morphological evaluation using transmission electron microscopy (TEM) revealed spherical nanoparticles (size 56.56 ± 5.93 nm for lactose coated core and 184.75 ± 13.78 nm for piroxicam loaded aquasomes) confirming the nanometric dimensions.

Influence of extremely low frequency magnetic fields on Ca2+ signaling and NMDA receptor functions in rat hippocampus.

Extremely low frequency (ELF<300Hz) electromagnetic fields affect several neuronal activities including memory. Because ELF magnetic fields cause altered Ca(2+) homeostasis in neural tissues, we examined their influence on Ca(2+) signaling enzymes in hippocampus and related them with NMDA receptor functions. Hippocampal regions were obtained from brains of 21-day-old rats that were exposed for 90 days to 50Hz magnetic fields at 50 and 100 microT intensities. In comparison to controls, ELF exposure caused increased intracellular Ca(2+) levels concomitant with increased activities of Ca(2+)-dependent protein kinase C (PKC), cAMP-dependent protein kinase and calcineurin as well as decreased activity of Ca(2+)-calmodulin-dependent protein kinase in hippocampal regions. Simultaneous ligand-binding studies revealed decreased binding to N-methyl-D-aspartic acid (NMDA) receptors. The combined results suggest that perturbed neuronal functions caused by ELF exposure may involve altered Ca(2+) signaling events contributing to aberrant NMDA receptor activities.

Predictors of mortality in rhinocerebral mycosis.

INTRODUCTION: Rhinocerebral mycosis is a rapidly progressive fatal opportunistic infection, predominantly affecting people in an immunocompromised state. Aggressive surgical therapy, with repeated debridement in combination with intravenous amphotericin B can lead to a high rate of cure. AIM: To determine the predictors of mortality in rhinocerebral mycosis. MATERIALS AND METHODS: The demographic data, clinical features, radiological (MRI/CT) findings, treatment details of patients with a diagnosis of rhinocerebral mycosis confirmed on histopathology were analyzed retrospectively. The outcome was assessed as alive and dead. Univariate analysis with odds ratio (OR) was employed in data analysis. Chi-square test was used for P value. RESULTS: There were 38 patients. The age range was 7-82 (mean 48.68) years; 30 (79%) were males. Craniofacial pain was the most common initial presenting symptom, noted in 29 (76.3%). Rhino-orbital involvement was noted in 24 (63.2%) and 12 (31.6%) had associated focal neurological deficits. Immunocompromised state was noted in 24 (63.2%). Eighteen (47.4%) patients died. The predictors for mortality: odds ratio (95% CI) were 2.45 (1.01-3.89) for elderly age, 5.67 (4.13-7.21) for intracranial extension, 2.6 (1.26-3.94) for immunocompromised state, 2.62 (1.25-3.99) for infection with zygomycosis and 2.33 (1.01-3.65) for anemia. CONCLUSION: Rhinocerebral mycosis is associated with high mortality in spite of aggressive therapy. Intracranial extension with focal neurological deficits is a major predictor of mortality in rhinocerebral mycosis.

Prevention of isoproterenol-induced cardiac hypertrophy by eugenol, an antioxidant.

Recent reports on the involvement of calcineurin in cardiac hypertrophy and its susceptibility to free radicals, prompted us to examine possible beneficial effects of dietary antioxidants in this regard. In continuation of initialin vitro studies revealing eugenol to be a potent calcineurin inhibitor, we investigated its ability to reverse isoproterenol-induced cardiac hypertrophy in rats. Intraperitoneal administration of isoproterenol (1 mg/kg body wt/day for 10 days) induced cardiac hypertrophy with increased heart weight and enhanced apoptosis of myocytes concomitant with accumulation of reactive oxygen species, decreased glutathione contents, increased activities of calcineurin and protein kinase C in ventricular tissue. Administering eugenol for 3 days (1 mg/kg body wt/twice a day), followed by combined administration of isoproterenol and eugenol resulted in significant reversal of cardiac hypertrophy and restoration of above changes. These results suggest that eugenol, a natural antioxidant of dietary origin, may offer potential benefits in the management of cardiac hypertrophy.

Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa.

Two metal response elements, flanking an antioxidant response element, were identified in regions upstream (-3730 bp) to copper metallothionein (CuMT) gene of Neurospora crassa. Presence of copper in culture media, but not of pro-oxidants like H2O2 or menadione, induced CuMT gene expression that could not be completely abolished by antioxidants such as N-acetyl cysteine and ascorbic acid. Gel shift assays revealed the ability of nuclear extracts from copper induced cultures to bind PCR-amplified metal response or antioxidant response elements. Similar observations could not be made with cultures exposed either to pro-oxidants or antioxidants. These results differentiate between CuMT gene induction by copper from antioxidant functions associated with the identified upstream elements.

Serum calcineurin activity in relation to oxidative stress and glycemic control in type II diabetes mellitus.

OBJECTIVES: In view of the well-recognized prevalence of oxidative stress in diabetes mellitus and the susceptibility of calcineurin (Ca(2+)-calmodulin dependent protein phosphatase 2 B) to free radicals, calcineurin was assayed in the sera of type II diabetic patients. DESIGN AND METHODS: Serum contents of thiobarbituric acid reactive substances, calcineurin and calmodulin, as well as activities of calcineurin and superoxide dismutase were measured in 81 diabetic patients and compared with age-matched controls. RESULTS: Oxidative stress in diabetic subjects was evidenced by increased thiobarbituric acid reactive substances, decreased superoxide dismutase activity concomitant with decreased calcineurin activity in sera. The observed decrease in calcineurin activity had a reciprocal correlation with fasting blood sugar, thiobarbituric acid reactive substances, and glycosylated hemoglobin. CONCLUSION: The inverse correlation observed between serum calcineurin activity and glycosylated hemoglobin levels suggests that an assay of serum calcineurin activity may be useful in simultaneous assessment of oxidative stress and glycemic control in type II diabetes mellitus.

Oxidative stress as a prerequisite for aflatoxin production by Aspergillus parasiticus.

The relevance of free radical generation and oxidative stress with regard to aflatoxin production was examined by comparing the oxygen requirement and antioxidant status of a toxigenic strain of Aspergillus parasiticus with that of a nontoxigenic strain at early (trophophase) and late logarithmic (idiophase) growth phases. In comparison to the nontoxigenic strain, wherein the oxygen requirements were relatively unaltered at various growth phases, the toxigenic strain exhibited greater oxygen requirements at trophophase coinciding with onset of aflatoxin production. The activities of antioxidant enzymes such as xanthine oxidase, superoxide dismutase, and glutathione peroxidase and the mycelial contents of thiobarbituric acid-reactive substances as well as of reduced glutathione were all enhanced during the progression of toxigenic strain from trophophase to idiophase. The combined results suggest that aflatoxin production by the toxigenic strain may be a consequence of increased oxidative stress leading to enhanced lipid peroxidation and free radical generation.

Synthesis and evaluation of 6,11-ethanohexahydrobenzo[b]quinolizidines: a new class of noncompetitive N-methyl-D-aspartate antagonists.

The synthesis and in vitro and in vivo evaluation of 12,13-cycloalkyl-6,11-ethanobenzo[b]-quinolizidines, a new class of noncompetitive N-methyl-D-aspartate (NMDA) antagonists acting at the PCP site on the NMDA receptor complex, is reported. Structure-activity relationship studies led to the identification of 10-hydroxy-(6 alpha,11 alpha,11 alpha beta,12R*,13S*)-1,3,4,6,11,11a,13,14,15,16-decahydro-12H- 6,11[1',2']-endo-cyclopenta-2H-pyrido[1,2-b]isoquinoline hydrobromide (5h) and 9-hydroxy-(6 alpha,11 alpha,11a beta,12R*,13S*)- 1,3,4,6,11,11a,13,14,15,16-decahydro-12H-6,11-[1',2']-endo-cycl ope nta-2H- pyrido[1,2-b]isoquinoline hydrobromide (5i), the most potent members of this series with Ki values of 2.3 +/- 0.2 and 2.3 +/- 0.5 nM, respectively. Molecular modeling studies revealed that this series of compounds occupies both lipophilic sites of the Andrews PCP receptor model and shares structural features which are common to other classes of known noncompetitive NMDA antagonists such as MK-801.

Identification, synthesis, and characterization of a unique class of N-methyl-D-aspartate antagonists. The 6,11-ethanobenzo[b]quinolizinium cation.

A series of novel N-methyl-D-aspartate antagonists acting at the phencyclidine site has been identified. Compound 2 has a Ki = 8 +/- 1 nM (vs [3H]thienylcyclidine, [3H]TCP) as a mixture of enantiomers. Resolution and further testing indicate that (-)-2, Ki = 4 +/- 0.7 nM, is a potent and selective TCP site ligand with neuroprotective activity in cultured neurons in the presence of excitotoxic concentrations of NMDA (IC50 = 26 nM). Compound (-)-2 is > 1000-fold selective for the TCP site vs a panel of receptor types including opiate, adrenergic, serotonergic, dopamine, adenosine, dihydropyridine, and benzodiazepine and displays increased selectivity for the activated (open) NMDA receptor-ion channel complex vs PCP and MK801 as measured by patch recordings in cultured, voltage-clamped neurons. Highly enhanced "open-channel" selectivity leads to tentative classification of these ligands as uncompetitive vs NMDA. Ligands with these characteristics may enable deconvolution of the pharmacologic effects associated with typical noncompetitive NMDA antagonists. We report here on the identification, synthesis, and activity of compounds of this structural class.

Novel benzo[b]quinolizinium cations as uncompetitive N-methyl-D-aspartic acid (NMDA) antagonists: the relationship between log D and agonist independent (closed) NMDA channel block.

A series of permanently charged benzo[b]quinolizinium cations having lower lipophilicity than MK-801 or phencyclidine (PCP) were synthesized. Data relating agonist independent block of N-methyl-D-aspartic acid (NMDA) ion channels to log D are described. Closed channel access is predicted to result in a more noncompetitive profile of antagonism compared to selective open channel blockers, which are uncompetitive inhibitors. Reduced closed channel block may underlie the absence of PCP or MK-801-like behavioral side effects observed for benzo[b]-quinolizinium cations.

Discovery of 6,11-ethano-12,12-diaryl-6,11-dihydrobenzo[b]quinolizinium cations, a novel class of N-methyl-D-aspartate antagonists.

6,11-Ethano-12,12-diaryl-6,11-dihydrobenzo[b]quinolizinium cations 8, a novel class of N-methyl-D-aspartate (NMDA) antagonists acting at the phencyclidine site, have been identified. Structure-activity relationship studies around the lead compound 8a led to the identification of 12g (WIN 67870-2), one of the most potent compounds in this series. Compound 12g has a Ki = 1.8 +/- 0.2 nM vs [3H]TCP binding, has 700-fold selectivity for binding to the open state of the NMDA receptor-ionophore, and was devoid of MK-801- and PCP-like behavioral effects in rats. Compound 12g was neuroprotective in cultured mouse cortical neurons and exhibited antiischemic activity in a rat middle cerebral artery occlusion/reperfusion model of focal ischemia.

Orally bioavailable benzisothiazolone inhibitors of human leukocyte elastase.

Human leukocyte elastase (HLE) has been proposed as a primary mediator of pulmonary emphysema and other inflammatory airway diseases. HLE is capable of cleaving many proteins, including elastin, other components of connective tissue, certain complement proteins, and receptors. Under normal conditions an appropriate balance exists in the lung between HLE and endogenous inhibitors, which scavenge the released enzyme before it exerts deleterious effects in the lung. Emphysema is thought to result from an imbalance in the lung between HLE and endogenous inhibitor (elevated elastase or insufficient inhibitor) that leads to the destruction of alveoli. We have identified WIN 64733 (2) and WIN 63759 (3) as potent (Ki* = 14 and 13 pM, respectively), selective, mechanism-based inhibitors of HLE which are orally bioavailable in the dog (absolute bioavailability 46% and 21%, respectively). In this series the in vitro stabilities of the inhibitors in blood, jejunal homogenates, and liver S9 homogenates are useful predictors of oral bioavailability. After being administered orally (30 mg/kg) to dogs, compounds 2 and 3 are found in the lung, being detected in the epithelial lining fluid obtained by bronchoalveolar lavage (Cmax of 2.5 and 0.47 microgram/mL, respectively).

Novel NMDA antagonists: replacement of the pyridinium ring of 6,11-ethanobenzo[b]quinolizinium cations with heteroisoquinolinium cations.

Replacement of the pyridinium ring of 6,11-ethanobenzo[b]quinolizinium cations with thiazolium (4a and 4b) and N-methylimidazolium (4c and 4d) resulted in equipotent compounds in the [3H]TCP binding assay. The corresponding N-methyl-1,2,4-triazolium analogs were less potent in this assay. The thiazolium derivative 4b, with a Ki = 2.9 nM, is being evaluated as a possible neuroprotective N-methyl-D-aspartic acid (NMDA) antagonist.

A putative role for calmodulin in the activation of Neurospora crassa chitin synthase.

The possible role of calmodulin in cell wall formation and chitin synthesis was studied in Neurospora crassa by examining the effects of anti-calmodulin agents on protoplast regeneration and possible associations between chitin synthase and calmodulin related proteins in microsomal isolates. Protoplast regeneration was inhibited by trifluoperazine (> 20 microM), an anticalmodulin agent. Chitin synthase activity in microsomes was associated with that of calmodulin-dependent protein kinase and inhibited by trifluoperazine (100 microM). In vitro activity of chitin synthase was enhanced upon inclusion of calmodulin (300 ng) in the assay mix, 63% over and above the stimulation brought about by trypsin, an activator of the enzyme. Autoradiography studies on microsomal proteins revealed calmodulin-dependent phosphorylation of two microsomal calmodulin-binding proteins (106 and 89 kDa). The results indicate that calmodulin-mediated phosphorylation of specific microsomal proteins may be important in the in vivo activation of chitin synthase.

Regulation of aflatoxin production by Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation.

To elucidate Ca(2+)-mediated regulation of aflatoxin production, the status of Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation was investigated employing toxigenic and non-toxigenic strains of Aspergillus parasiticus. Incubation of cytoplasmic extracts with [gamma-(32)P]ATP followed by SDS-PAGE and autoradiography revealed total absence of protein phosphorylation during periods corresponding to aflatoxin production in the toxigenic strain (NRRL 2999). In contrast, protein phosphorylation was unaffected in the non-toxigenic strain (SRRC 255). Aflatoxin production in the toxigenic strain was also accompanied by enhanced (26-fold) activity of calcineurin (calmodulin-dependent protein phosphatase 2B) concomitant with a lowered (6-fold) activity of calmodulin-dependent protein kinase. In addition, the in vitro activity of Ca(2+)/calmodulin-dependent protein kinase was susceptible to dose-dependent inhibition by aflatoxin. Since calcineurin remains active in the absence of phosphorylation by calmodulin-dependent protein kinase, it is suggested that calcineurin-mediated dephosphorylation of regulatory enzymes ensures continued production of aflatoxins.

Clinical significance of serum calcineurin in acute leukemia.

BACKGROUND: Calcineurin is involved in T-lymphocyte activation as well as in the maturation of hematopoietic cells. Identification of this predominantly intracellular phosphatase and of calmodulin (CaM) in human sera warranted their assessment in different types of acute leukemias. METHODS: Phosphatase activity of calcineurin (CaN) was assayed, involving the measurement of trifluoperazine-sensitive neutral phosphatase, in sera of leukemic patients before and after treatment. Calcineurin and calmodulin contents were also determined by ELISA employing monoclonal antibodies specific to the proteins. RESULTS: The activity of calcineurin was decreased by 75% and 85% in sera of patients diagnosed either for acute lymphoid leukemia and acute myeloid leukemia, respectively, without apparent changes in calmodulin or calcineurin contents under both these conditions. In addition, the decreased calcineurin activity in acute myeloid leukemia was restored to levels comparable to non-leukemic individuals upon treatment. This was not observed in cases of acute lymphoid leukemia. CONCLUSIONS: These results suggest diagnostic utility in the measurement of serum calcineurin activity in acute leukemia. Restoration of normal calcineurin activity in patients undergoing treatment for acute myeloid leukemia may provide a means to monitor patient response to the prescribed therapeutic regimen.

Proliferative stimulation of lymphocytes by calmodulin binding proteins isolated from amniotic fluid.

In view of the role of calmodulin and calmodulin binding proteins in modulating the second messenger functions of Ca2+, we studied the presence of such proteins in amniotic fluid, which may be considered as a dynamic medium for promoting foetal growth. Affinity chromatography of amniotic fluid proteins revealed the presence of calmodulin binding proteins in samples obtained either at 28 or 36 wk of pregnancy. The relative content of these proteins increased in amniotic fluid from 1.5 mg/g total protein at 28 wk to 3.6 mg/g at full term of pregnancy. Culturing murine splenocytes in presence of the isolated calmodulin binding proteins (10 micrograms protein/10(6) cells) resulted in nearly 4-fold enhancement of 3H-thymidine incorporation into them as compared to controls. In comparison, similar incorporation of the radiolabel into lymphocytes obtained from cord blood was enhanced only by 2 fold in presence of calmodulin binding proteins, though at a much lower protein concentration (50 ng/10(6) cells). SDS-PAGE on 12.5 per cent gels of eluates obtained from calmodulin-agarose columns, followed by overlay of corresponding western blots with biotinylated calmodulin revealed the presence of a 68 kDa calmodulin binding protein in samples collected either at 28 wk or at full-term of pregnancy. In addition, the amniotic fluid also contained 83 kDa calmodulin binding protein at 28 wk. This first-time demonstration of mitogenic, calmodulin binding proteins in amniotic fluid suggests that such mitogens may participate in promoting foetal growth.

Isolation and characterization of a copper containing protein from blue cell walls of Neurospora crassa.

Culturing Neurospora crassa in presence of toxic amounts of copper (0.63 mM) resulted in blue coloured mycelia and cell walls. Significant amounts (approximately 45%) of total mycelial copper were associated with cell wall isolates under conditions of copper toxicity. Hence, such blue cell walls were analysed to identify specific ligands involved in copper binding. While decuprification of the blue cell walls with 8-hydroxy quinoline (8 HQ) did not alter their copper binding abilities, similar treatment with EDTA (10 mM) decreased such abilities indicating that EDTA treatment lead to loss of copper binding ligands from cell walls. Treatment of blue cell walls with 8 HQ followed by EDTA resulted in the solubilization of a copper binding protein (relative MW approximately 14 kDa) which was associated with phosphate and carbohydrate moieties. On amino acid analysis, this protein was found to be devoid of free thiol groupings but enriched in acidic and basic amino acids, distinguishing it from classical intracellular metal binding proteins such as metallo-thioneins and phytochelatins that are inducively synthesized under conditions of metal toxicity. The biological significance of the isolated wall-bound copper binding protein, which appears to be a normal constituent of cell walls, is discussed in relation to cytoplasmic metal binding proteins and mechanism(s) adapted by fungi in countering metal toxicity.