Effect of Copper on l-Cysteine/l-Cystine Influx in Normal Human Erythrocytes and Erythrocytes of Wilson's Disease.

Wilson's disease is a disease of abnormal copper metabolism in which free serum copper level is raised. The objective of the study was to determine, whether in Wilson disease, l-cysteine/l-cystine influx into RBC was decreased or not and the specific amino acid transporter affected by copper in normal human RBC. For l-cysteine/l-cystine influx, ten untreated cases, ten treated cases and ten age and sex matched healthy controls were recruited. To study the effect of copper on l-cysteine/l-cystine influx in RBC, 15 healthy subjects were selected. RBC GSH and l-cysteine/l-cystine influx were estimated by Beautler's and Yildiz's method respectively. In untreated cases, l-cysteine/l-cystine influx and erythrocyte GSH level were decreased showing that elevated level of free copper in serum or media decreased l-cysteine/l-cystine influx in human RBC. Copper treatment inhibited L amino acid transporter in normal RBC specifically.

Association of obesity and leptin with insulin resistance in type 2 diabetes mellitus in Indian population.

Obesity and diabetes mellitus are two modern epidemics. But their interrelationship is debated. Here we explored the probable association among obesity, leptin and insulin resistance in type 2 diabetes mellitus. 60 recent onset (< 5 years) diabetics and age-sex matched 33 non diabetic controls were assessed for physical and chemical parameters like Body Mass Index, abdominal circumference, waist/hip ratio, fasting blood glucose, insulin and leptin. Degree of insulin resistance was calculated by HOMA-IR method (Homeostatic Model Assessment). All the physical parameters showed positive correlation with leptin and the HOMA-IR score, strength of association being highest between insulin resistance and abdominal circumference. Leptin and insulin resistance showed no correlation. Findings were lower in controls. Study concluded that, obesity mainly central type might be responsible for insulin resistance in type 2 diabetes mellitus where as leptin, a potential marker for obesity, may not. This perhaps points towards the multifactorial causation of insulin resistance in type 2 diabetes mellitus.

The appearance of dermcidin isoform 2, a novel platelet aggregating agent in the circulation in acute myocardial infarction that inhibits insulin synthesis and the restoration by acetyl salicylic acid of its effects.

Hyperglycemia with severe reduction of plasma insulin level is frequently associated with acute ischemic heart disease. Since insulin is reported to be an anti thrombotic humoral factor, the mechanism of the impaired insulin synthesis was investigated. The plasma from the patients with acute myocardial infarction (AMI) was analyzed by SDS-polyacrylamide gel electrophoresis. Dermcidin isoform 2 (dermcidin) was determined by enzyme linked immunosorbent assay. Insulin synthesis was determined by in vitro translation of glucose induced insulin mRNA synthesis in the pancreatic ß cells. Nitric oxide (NO) was determined by methemoglobin method. SDS-polyacrylamide gel electrophoresis of AMI plasma demonstrated the presence of a novel protein band of Mr 11 kDa that was determined to be dermcidin. Addition of 0.1 µM dermcidin inhibited insulin synthesis by >65 fold compared to control through the inhibition of NO synthesis in the pancreatic cells. The oral administration of 150 mg acetyl salicylic acid (aspirin) to the AMI patients increased the plasma insulin level from 13 (median) to 143 µunits/dl (median) with concomitant decrease of plasma dermcidin level from 112 to 9 nM in these patients within 12 h. It was also found that while the injection of 3.0 ± 0.05 (n = 10) nmol dermcidin with 0.25 ± 0.03 µmol ADP/g body weight caused coronary thrombus in mice, ADP itself at this concentration failed to produce thrombus. These results indicated that dermcidin was a novel platelet aggregating agent, and potentiated the ADP induced thrombosis in the animal model as well as acutely inhibited glucose induced insulin synthesis.

Exploring the role of Azadirachta indica (neem) and its active compounds in the regulation of biological pathways: an update on molecular approach.

In ethnomedicine, plant parts and compounds are used traditionally to treat different diseases. Neem (Azadirachta indica A. Juss) is the most versatile and useful medicinal plant ever found. Its every part is rich in bioactive compounds, which have traditionally been used to treat different ailments including infectious diseases. Bioactive compounds such as nimbolide, azarirachtin, and gedunin of neem are reported to have a tremendous ability to regulate numerous biological processes in vitro and in vivo. The present review article aims to explore the importance of neem extracts and bioactive compounds in the regulation of different biological pathways. We have reviewed research articles up to March 2020 on the role of neem in antioxidant, anti-inflammatory, antiangiogenic, immunomodulatory, and apoptotic activities. Studies on the concerned fields demonstrate that the bioactive compounds and extracts of neem have a regulatory effect on several biological mechanisms. It has been unveiled that extensive research is carried out on limonoids such as nimbolide and azarirachtin. It is evidenced by different studies that neem extracts are the potential to scavenge free radicals and reduce ROS-mediated damage to cells. Neem can be used to normalize lipid peroxidation and minimize ROS-mediated cell death. Besides, neem extracts can significantly reduce the release of proinflammatory cytokines and elevate the count of CD4 + and CD8 + T-cells. This review indicates the pivotal roles of A. indica in the regulation of different biological pathways. However, future investigations on other bioactive compounds of neem may reveal different therapeutic potentials.

The role of leucocytes in the acetyl salicylic acid (aspirin) induced nitric oxide synthesis in the production of interferon-alpha, a potent inhibitor of platelet aggregation and a thrombolytic agent.

The role of aspirin-induced NO synthesis in the production of interferon-alpha (IFN-alpha) in leucocytes and the effect of IFN-alpha on platelet aggregation was studied. Treatment of Platelet Rich Plasma (PRP) with the dialyzed supernatant from the leucocyte suspension incubated with 80 microM aspirin resulted in parallel syntheses of NO and IFN-alpha as determined by methemoglobin assay and enzyme linked immunosorbent assay respectively. Incubation of PRP with 10 nM purified IFN-alpha for 40 min resulted in the maximal inhibition of platelet aggregation through the synthesis of NO due to the activation of nitric oxide synthase in platelets by IFN-alpha. The treatment of clotted PRP with IFN-alpha resulted in the lysis of the clot due to the fibrinolysis. Injection of IFN-alpha was found to protect mice from death due to the lysis of ADP-induced coronary thrombus. Interferon-alpha was found to be a potent inhibitor of platelet aggregation and a thromboprotective agent.

The Control of Stress Induced Type I Diabetes Mellitus in Humans through the Hepatic Synthesis of Insulin by the Stimulation of Nitric Oxide Production.

The role of stress induced development of Type-1 diabetes mellitus (T1DM) as opposed to autoimmunity remains obscure. It has been reported that a stress induced protein, identified to be dermcidin isoform 2 (dermcidin) inhibited insulin synthesis in both the pancreatic ß cells and the hepatic cells. As dermcidin effect could be neutralized by the increased production of systemic nitric oxide (NO), investigations were carried out to determine the feasibility of controlling stress induced T1DM through the neutralization of dermcidin by systemic increase of NO. To determine the role of plasma dermcidin level in T1DM subjects (n=45), if any, when the plasma dermcidin level were determined, it was found that the protein level was increased in 65% of the participating volunteers. Efforts were made to normalize the plasma glucose level (median=175 mg/dL) in these T1DM subjects by systemic increase of NO by applying a cotton pad containing 0.28mmol sodium nitroprusside on the abdominal skin. It was found that the systemic increase of NO level decreased the blood glucose level of 275 mg/dL (median) to 115 mg/dL (median) in these volunteers within 24 h with concomitant increase of plasma insulin level from 7.5 µunits/dL to 101 µunits/dL at the same time. The increase of plasma insulin level was accompanied by the decrease of dermcidin level of 124.5 nM to 18 nM with increase of NO from 0.43 ± 0.19 nM to 4.1 ± 1.56 nM. The results suggested that the stress induced T1DM by dermcidin could be controlled by the systemic increase of NO which in consequence led to increased synthesis of insulin.

Development of a point of care testing tool to classify peritoneal effusion as exudate and transudate.

BACKGROUND: Recently we developed a bedside test to classify pleural effusion into exudate and transudate but point of care classification of peritoneal effusion is yet not published. METHODS: We analyzed the Boyer's criteria parameters from bloodless peritoneal fluid and classified the biofluid as exudate or transudate and also estimated some parameters of oxidative stress in the biofluid by established spectrophotometric procedure. Two hundred microliters of sample was used and 10 µl of 30% hydrogen peroxide was added, followed by inspection of the sample for the appearance of bubbles. RESULTS: All exudative ascitic fluids (n=50) have shown the appearance of profuse bubbles within 1 min addition of hydrogen peroxide along with significantly more catalase activity compared to transudate. All transudative ascitic fluids (n=50) have not shown bubble formation within 1 min after the addition of hydrogen peroxide. The exudate does not show bubble formation if added with catalase inhibitors prior to the addition of hydrogen peroxide. Blood mixed transudate have shown profuse bubble formation after the addition of hydrogen peroxide. CONCLUSION: The hydrogen peroxide bubbling reaction has the potential to be developed as a point of care test to classify peritoneal fluid as exudate or transudate.

Identification of orcinol reactive substance in pleural fluid cell lysate--a new parameter for classification of pleural effusion.

BACKGROUND: Cell-free DNA is observed to be more in exudative pleural effusions. Based on this fact development of a clinical chemistry test for classification of pleural effusion will require DNA extraction followed by PCR amplification and electrophoresis. These procedures may not be cost effective for the purpose for classification of pleural effusion as already established parameters are popular for the purpose which can be estimated by comparatively low cost colorimetric procedures. Therefore development of a simple colorimetric test for the classification of pleural fluid based on nucleic acid identification test can be attempted. The aim of this work is to develop such colorimetric test for classification of pleural effusion using only pleural fluid sample. METHODS: Cell pellet is obtained from 5 ml pleural fluid which is lysed and subjected to DNA extraction, followed by identification under UV-transilluminator after electrophoresis and orcinol and diphenylamine reaction. RESULT: Exudates show extractable DNA from 5 ml biofluid (n=52) which are not observed from transudate (n=32). Orcinol reaction is significantly positive in exudates (n=52) compared to the transudates (n=32). Diphenylamine test cannot differentiate exudate from transudate. CONCLUSION: Orcinol reaction of cell lysate obtained from pleural fluid can classify pleural fluid sample into exudate or transudate.

The glucose-induced synthesis of insulin in liver.

Pancreatic ß cells, stimulated by glucose, are known to synthesize and secrete insulin. As liver diseases are reported to cause diabetes mellitus, studies were conducted to determine the possibility of glucose-induced insulin synthesis in the liver cells. The glucose-induced insulin synthesis was determined by in vitro translation of mRNA from the hepatocytes. The cDNA from mRNA was prepared and sequence analysis was performed. Incubation of hepatocytes from the liver of adult mice (n=10) with glucose (0.02 M) resulted in the insulin synthesis [0.03 (mean)±0.006 (S.D.) µunits/mg/h] compared to the pancreatic ß cells [0.04±0.004 µunits/mg/h]. Immunohistochemical study also demonstrated the glucose-induced synthesis of insulin in liver cells. Incubation of the mice hepatocytes with glucose resulted in the synthesis of insulin mRNA. The purified mRNA which was used to prepare cDNA resulted in the formation of proinsulin I and proinsulin II genes corresponding to 182 and 188 base pairs, respectively. Sequence analysis of the cDNA indicated that proinsulin I as well as proinsulin II gene could be involved in the synthesis of insulin by hepatocytes. These results suggested that insulin synthesis in both hepatic and pancreatic cells could be involved in the control of diabetes mellitus.

A drop of hydrogen peroxide can differentiate exudative pleural effusion from transudate--development of a bedside screening test.

BACKGROUND: There is no bedside test to classify pleural fluid as exudate or transudate. The aim of the present study is to develop such a test. METHODS: We analyzed the Light's criteria parameters from bloodless pleural fluid and classified the biofluid as exudate or transudate and also estimated some parameters of oxidative stress in the biofluid by established spectrophotometric procedure. Two hundred microliters of sample was taken and added with 10 microl of 30% hydrogen peroxide followed by inspection of the sample for appearance of bubbles. RESULT: All exudative fluids (n=52) have shown appearance of profuse bubbles within 1 min of addition of hydrogen peroxide along with significantly more catalase activity compared to transudate. All transudative fluids (n=32) have not shown bubble formation within 1 min after addition of hydrogen peroxide. The exudate does not show bubble formation if supplemented with catalase inhibitors. Blood mixed transudate have shown profuse bubble formation after addition of hydrogen peroxide. CONCLUSION: In the case of blood uncontaminated pleural fluid, this newly developed protocol's sensitivity and specificity will be equivalent to Light's criteria probably with more advantage as by this procedure transport of the sample to the clinical laboratory is not required due to its inherent simplicity.

Purification and mechanism of action of "cortexin," a novel antihypertensive protein hormone from kidney and its role in essential hypertension in men.

Because kidney tissue damage is associated with both hypertension and impaired nitric oxide (NO) production, we investigated the possibility whether the kidney tissue contains any activator of endothelial NO synthase (eNOS) that could be important in essential hypertension. An activator protein of M(r) 43000 Da for eNOS from the goat kidney cortex homogenate was purified to homogeneity by chromatographic techniques. This activator trivially, called "cortexin," was determined by enzyme-linked immunosorbent assay using anticortexin antibody. NO was determined by the formation of methemoglobin. Injection of 0.5 nmol cortexin/kg body weight to rabbit pretreated with l-epinephrine that increased the systolic and diastolic pressures to 195 +/- 3.40 mm Hg and 98.14 +/- 6.64 mm Hg, respectively, reduced and kept the elevated pressures at normal ranges of 133.57 +/- 12.14 (systolic) and 51.03 +/- 3.21 (diastolic) for 45 hours with simultaneous increase of plasma NO level. The inhibition of cortexin-induced NO synthesis nullified the antihypertensive effect of cortexin. The plasma cortexin level in newly diagnosed persons with essential hypertension was 0 pmol/mL (median), which contrasted with 218.94 pmol cortexin/mL (median), in normotensive persons (P < .0005; n = 25). We concluded that the impaired production of cortexin in the cortex of kidney might lead to essential hypertension.

Familial hypercholesterolaemia--a case report.

A case report of a 9-year-old female presenting with cutaneous xanthoma and other findings suggestive of homozygous familial hypercholesterolaemia along with abnormalities in serum lipid profile in other siblings is presented in view of its rarity.