Biomedical subjects

K Govindaraju

Publications and source records attributed to K Govindaraju.

13 recordsLinked to original sources

PubMed2006

Utilization of meat industry by products: protein hydrolysate from sheep visceral mass.

Protein hydrolysate was prepared from pre-treated sheep visceral mass (including stomach, large and small intestines) by enzymatic treatment at 43+/-1 degrees C (at the in situ pH 7.1+/-0.2 of the visceral mass) using fungal protease. The enzyme readily solubilized the proteins of the visceral mass as indicated by the degree of hydrolysis (34%) and nitrogen recovery (>64%). Hydrolysis with an enzyme level of 1% (w/w of total solids) at 43+/-1 degrees C with a pH around 7.0 for 45 min was found to be the optimum condition. The yield of protein hydrolysate was about 6% (w/w). The amino acid composition of the protein hydrolysate that was very hygroscopic, was comparable to that of casein.

Animals↗

PubMed2003

Analysis of glutathione in rat airway surface liquid by capillary zone electrophoresis with conductivity detection.

Glutathione (GSH) is an important component of antioxidant defenses in airway surface liquid (ASL), a thin layer (10-30 microm) of liquid covering the epithelial cells lining the airways of the lung. Decreased levels of ASL GSH have been reported in cystic fibrosis (CF), potentially contributing to the severe oxidative stress seen in this disease. To help investigate the role of GSH in ASL, we developed a technique suitable for analysis of GSH and its oxidized form (GSSG) in microliter samples using capillary sampling followed by capillary zone electrophoresis (CZE) analysis with conductivity detection. CZE was carried out in 100 mM CHES and 40 mM lithium hydroxide with 5 mM spermine at pH 9.1 under an applied electric field of -416 V cm(-1). To prevent any autooxidation of GSH during sample manipulations, the samples were treated with N-ethylmaleimide (50 mM) to alkylate free thiol (-SH). Under these conditions, GSH and GSSG were cleanly separated without interference from common anions (e.g. Cl(-), PO(4)(3-), HCO(3)(-), etc.) and the limit of detection for ASL analysis was 11 microM for GSH and 8 microM for GSSG (S/N=3). GSH and GSSG were also measured in rat plasma. Baseline values of 897+/-210 microM (GSH) and 215+/-61 microM (GSSG) were obtained for rat ASL (n=8), whereas 12.4+/-2.7 microM (GSH) and 14.8+/-6.7 microM (GSSG) were obtained for rat plasma (n=5).

Animals↗

PubMed2001

Capillary electrophoresis analysis of nitrite and nitrate in sub-microliter quantities of airway surface liquid.

We developed a simple capillary electrophoresis (CE) method to measure nitrite and nitrate concentrations in submicroliter samples of rat airway surface liquid (ASL), a thin (10-30 microm) layer of liquid covering the epithelial cells lining the airways of the lung. The composition of ASL has been poorly defined, in large part because of the small sample volume (approximately 1-3 microl per cm2 of epithelium) and difficulty of harvesting ASL. We have used capillary tubes for ASL sample collection, with microanalysis by CE using a 50 mM phosphate buffer (pH 3), with 0.5 mM spermine as a dynamic flow modifier, and direct UV detection at 214 nm. The limit of detections (LODs), under conditions used, for ASL analysis were 10 microM for nitrate and 30 microM for nitrite (SIN= 3). Nitrate and nitrite were also measured in rat plasma. The concentration of nitrate was 102+/-12 microM in rat ASL and 70+/-1.0 microM in rat plasma, whereas nitrite was 83+/-28 microM in rat ASL and below the LOD in rat plasma. After instilling lipopolysaccharide intratracheally to induce increased NO production, the nitrate concentration in ASL increased to 387+/-16 microM, and to 377+/-88 microM in plasma. The concentration of nitrite increased to 103+/-7.0 microM for ASL and 138+/-17 microM for plasma.

Animals↗

PubMed2000

Analysis of small-scale biological compartments by capillary electrophoresis.

Two characteristics of capillary electrophoresis make the technique attractive for the separation of the components of microscale compartments within living organisms: small sample volume requirements and direct compatibility with biofluids. Indeed, capillary electrophoresis has been used for analysis down to a sub-cellular level. There are also potentially many applications of capillary electrophoresis to biological compartments on a super-cellular scale, which are nevertheless so small that they make analysis by conventional separations techniques difficult or impractical. The analytical challenges in small-scale bioanalysis are first to develop a suitable method for collection of sample and its introduction into the separation capillary, and secondly, to achieve the required separation. Examples reviewed here will primarily focus on the analysis of tear fluid or airway surface liquid, cases in which the amount of sample that can be collected range from around 10 microl to around 100 nl.

Body Fluids↗

PubMed2000

Downregulation of endothelial nitric oxide synthase in rat aorta after prolonged hypoxia in vivo.

The goal of this study was to determine whether hypoxia alters expression of endothelial nitric oxide synthase (eNOS) in the systemic circulation. Rats breathed either air or 10% oxygen for 12 hours, 48 hours, or 7 days. Thoracic aortas were excised and either mounted in organ bath myographs or frozen in liquid nitrogen for later extraction of protein and RNA. eNOS protein (Western blotting) was decreased (20% of normoxic control) after 12 hours, 48 hours, and 7 days of hypoxia. eNOS mRNA (ribonuclease protection assay) was similarly reduced. Acetylcholine (10(-4) mol/L) reversed phenylephrine (10(-5) mol/L) preconstriction by 53.3+/-5.6% in aortic rings from normoxic rats and 26.1+/-4.8% in rings from rats exposed to hypoxia for 48 hours (P<0.05), with comparable impairment of relaxation by the calcium ionophore A23187 (10(-5) mol/L). Responses to diethylamine nitric oxide and 8-bromo-cGMP were unaffected. Aortic cGMP levels after incubation with acetylcholine (10(-6) mol/L) averaged 14.0+/-1.8 fmol/mg in rings from normoxic rats compared with 8.7+/-1.0 fmol/mg in rings from hypoxic rats (P<0. 05). Similarly, nitrate concentration (by capillary electrophoresis) in the media in which the rings were incubated was reduced in the hypoxic group (5.6+/-0.23 micromol/L for hypoxic rats and 7.8+/-0.7 micromol/L for normoxic rats). Impaired endothelial NO release may handicap the vascular responses that defend vital organ function during hypoxia.

Animals↗

PubMed2000

Airway surface liquid composition in mice.

Airway surface liquid (ASL) lines the conducting airways of the respiratory tract. We collected small samples of this liquid from the lower tracheae of anesthetized C57BL/6 mice and determined its ionic composition (in mM: 87.2 Na(+), 4.7 K(+), and 57.0 Cl(-)). Intravenous methacholine produced significant increases in the concentrations of Na(+), K(+), and Cl(-) within ASL. A limited analysis of liquid from cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice revealed no significant differences compared with littermate controls; however, Pseudomonas aeruginosa infection led to an increase in the salt concentration of ASL in cftr(+/+) mice. Morphometric measurements of tracheal submucosal gland volume revealed significant differences between inbred mouse strains, corresponding to ease of ASL collection. We conclude that although submucosal glands may be responsible for the production of some ASL, the ionic composition of this liquid is actively regulated by the underlying epithelial cells.

Animals↗

PubMed1998

Analysis of proteins in microsamples of rat airway surface fluid by capillary electrophoresis.

A thin layer of airway surface fluid (ASF) lining the pulmonary airways plays an important role in the primary defense mechanisms of the lung against bacterial infection. However, little is known about the composition of ASF due to the thinness (typically 5-30 microm in healthy animals) of the fluid layer and its relative inaccessibility, which causes considerable difficulties in sample collection and subsequent analysis. We have used a novel technique of capillary sampling coupled with capillary electrophoresis (CE) to analyze the protein composition of rat ASF. CE analyses were performed under two different conditions: a borate buffer, pH 9.1, or a phosphate buffer, pH 2.5, with 0.5 mM spermine. The different selectivities afforded by the two methods aid in peak identification, and quantitation of most of the major species was possible using both separation conditions. Albumin, transferrin and globulins are observed to be the major protein components in rat ASF, at concentrations of 28 mg ml(-1), 4.0 mg ml(-1) and 34 mg ml(-1) respectively, in comparison to 31 mg ml(-1), 3.1 mg ml(-1) and 40 mg ml(-1), respectively, in rat plasma.

Albumins↗

PubMed1997

Microanalysis of lung airway surface fluid by capillary electrophoresis with conductivity detection.

The thin layer of fluid that covers the surface of the epithelia lining the conducting airways plays an important role in primary pulmonary defense, and its composition may be a critical factor in the pathogenesis of several lung diseases including cystic fibrosis. Despite its physiological importance, the composition of airway surface fluid (ASF) is poorly understood due to considerable difficulties in sample collection from the 5-30 microns thick layer and subsequent analysis. We have used a novel technique for sample collection and microanalysis of ASF (nanoliter sample required) by capillary electrophoresis with conductivity detection. Limitations on the diameters of capillary required for the sample injection process and for the conductivity detector require the use of coupled separation capillaries with different external diameters. Two different methods were used to construct a butt-joint coupling for capillaries of different outer diameters. Reasonable efficiency is observed with the coupled capillaries (N = 100000 plates m-1) compared to an unbroken single capillary (N = 180000 plates m-1). The use of conductivity detection allows greater flexibility in method development and the possibility of determining a greater variety of ions than with a previous indirect-UV method. In the present study, we describe the analysis of cations (Na+, K+, Ca2+, Mg2+) and anions (Cl-, NO2-, NO3-, SO4(2-), PO4(2-), HCO3-) in rat ASF. Particular attention was paid to developing washing procedures which limited fouling of the conductivity sensor. In healthy rats, ASF was found to be hypotonic compared to plasma levels, consistent with some observations made in human airways.

Animals↗

PubMed1997

Airway surface fluid composition in the rat determined by capillary electrophoresis.

The apical surface of respiratory epithelial cells is covered by a thin layer of low-viscosity fluid termed airway surface fluid (ASF), about which relatively little is known. We collected samples of ASF from anesthetized rats, which were then analyzed using capillary electrophoresis, a method that enables extremely small quantities of fluid to be analyzed. We found values for Na+ (40.57 +/- 3.08 mM), K+ (1.74 +/- 0.36 mM), and Cl- (45.16 +/- 1.81 mM), indicating that this fluid is hypotonic compared with rat plasma. In contrast, the concentrations of nitrite and nitrate within ASF were higher than reported plasma values. Additionally, intravenous administration of the cholinergic agonist methacholine (MCh) resulted in a dose-dependent increase in the concentration of Na+ and Cl- within the ASF. This increase is approximately 50% in these ions after a dose of 100 ng MCh/g body wt. This animal model, together with this microanalytical technique, may be useful for investigating the in vivo regulation of ASF composition.

Animals↗

PubMed1997

Prevention of falls in a geriatric ward.

Falls occurring in the hospital are significant events because of the injuries sustained, extended hospital stay and potential medicolegal implications. We conducted a 3-year study to determine the characteristics of fallers in a geriatric ward and assess the effectiveness of lectures and nursing assessments in the prevention of falls. In the first year of the study we found that the rate of falls was 9% of all patients admitted. About 85.7% of falls occurred at the bedside and 41.4% happened while getting in and out of bed. Most falls occurred between 4 pm and 8 pm and half of the fallers had no preceding symptoms. In the second year, a series of lectures and educational material were given to the same nursing staff and the prevalence of falls was reduced to 7% (P > 0.05). However, the implementation of a nursing assessment protocol in the third year of the study achieved a marked reduction in prevalence of falls to 5.9% from the initial 9% (Chi-square chi 2 = 4.19, P = 0.04). Thus, the vigorous administration of a nursing assessment protocol can reduce falls in a geriatric ward.

Accidental Falls↗

PubMed1989

Chymotrypsin-catalyzed hydrolysis of chromium (III) derivatives of insulin: evidence for stabilization of the protein through interactions with metal ions.

We investigated the chymotrypsin-promoted hydrolysis of a series of chromium(III)-insulin complexes containing chelating or macrocyclic ligands. It has been shown that Cr(III) stabilizes insulin against the chymotrypsin-promoted hydrolysis of the protein. The molecular weights of Cr(III) containing peptides have been estimated to be of the order of 2,700-3,700 daltons. The Cr(III) containing peptides are richer in glutamic acid than the intact insulin and are devoid of any isoleucine. High molecular weights and the observed glutamic acid/histidine ratios in Cr(III) containing peptides have been rationalized in terms of Cr(III) being associated with insulin aggregates rather than the monomer of the protein. The chymotrypsin hydrolysis of Cr(III) insulin derivatives is influenced markedly by the nature, charge, and type of Cr(III) complex with which the protein has been reacted. Arguments have been advanced that chymotrypsin-promoted hydrolysis of insulin Cr(III) derivatives does not lead to cleavages at or near every tyrosine residue.

Chromium↗

PubMed1989

Chromium(III)-insulin derivatives and their implication in glucose metabolism.

Insulin has been reacted with five chromium(III) complexes that are capable of relatively facile substitution of aquo ligands. The new Cr(III) insulin derivatives have been characterized by means of electronic and infrared spectra, and evidence for major changes in the protein structure, including the state of aggregation, has been presented. Supporting evidence for the arguments favoring the beneficiary role of chromium(III) in glucose metabolism has been obtained using in vivo studies, and it has been shown that insulin derived with Cr(salen) (H2O)2+ is capable of reversing the blood sugar, serum cholesterol, and phospholipids levels to those of normal rats. The results emphasize the dependence of biopotency on the structure of Cr(III) complexes used for derivation of insulin and discount the postulates that Cr(III) serves to assemble insulin and receptor units through metal-sulphur bonding. The influence of Cr(III) on the structural stability and state of aggregation of insulin and their possible role in glucose metabolism is discussed.

Animals↗

PubMed1987

Active site chemistry of lysyl oxidase.

The bimolecular reduction of the Cu(II)-based enzyme lysyl oxidase with two inorganic reductants, tris bipyridylchromium(II) and (1,3,6,8,10,13,16,19)-octaazabicyclo (6,6,6)eicosanecobalt(II) has been examined at various ionic strength and [H+] conditions. The electrochemical properties of the enzyme have also been examined. The results show that Cu(II) is the redox site in the enzyme and has E 1/2 = 0.05 +/- 0.005 V against SCE. The observed rate constants, kobs, for the reduction of the enzyme by either Cr(bpy)32+ or Co(sep)2+ at any concentration of the reductant increased with the ionic strength of the medium. The ionic strength dependence of kobs has been analyzed in terms of the charge of the active site being 1 +.

2,2'-Dipyridyl↗