Plasma bikunin: Half-life and tissue uptake

Bikunin is a chondroitin sulfate-containing plasma protein synthesized in the liver. In vitro, it has been shown to inhibit proteases and to have additional activities, but its biological function is still unclear. Here we have studied the dynamics of plasma bikunin in rats and mice. A half-life of 7 ± 2 min was obtained from the time course of the decrease of the plasma level of bikunin following hepatectomy. Clearance experiments with intravenously injected radiolabeled bikunin with or without the chondroitin sulfate chain showed that the polysaccharide had little influence on the elimination rate of the protein. The uptake of bikunin by different tissues was studied using bikunin labeled with the residualizing agent ¹²⁵I-tyramine cellobiose; 60 min after intravenous injection, 49% of the radioactivity was recovered in the kidneys and 6–11% in the liver, bones, skin, intestine and skeletal muscle. The uptake in the liver was analyzed by intravenous injection of radiolabeled bikunin followed by collagenase perfusion and dispersion of the liver cells. These experiments indicated that bikunin is first trapped extracellularly within the liver before being internalized by the cells. (Mol Cell Biochem 271: 61–67, 2005)     

Regulation of cardiac malonyl-CoA content and fatty acid oxidation during increased cardiac power

Myocardial fatty acid oxidation is regulated by carnitine palmitoyltransferase I (CPT I), which is inhibited by malonyl-CoA. Increased cardiac power causes a fall in malonyl-CoA content and accelerated fatty acid oxidation; however, the mechanism for the decrease in malonyl-CoA is unclear. Malonyl-CoA is formed by acetyl-CoA carboxylase (ACC) and degraded by malonyl-CoA decarboxylase (MCD); thus a fall in malonyl-CoA could be due to activation of MCD, inhibition of ACC, or both. This study assessed the effects of increased cardiac power on malonyl-CoA content and ACC and MCD activities. Anesthetized pigs were studied under control conditions and during increased cardiac power in response to dobutamine infusion and aortic constriction alone, under hyperglycemic conditions, or with the CPT I inhibitor oxfenicine. An increase in cardiac power was accompanied by increased myocardial O(2) consumption, decreased malonyl-CoA concentration, and increased fatty acid oxidation. There were no differences among groups in activity of ACC or AMP-activated protein kinase (AMPK), which physiologically inhibits ACC. There also were no differences in V(max) or K(m) of MCD. Previous studies have demonstrated that AMPK can be inhibited by protein kinase B (PKB); however, PKB was activated by dobutamine and the elevated insulin that accompanied hyperglycemia, but there was no effect on AMPK activity. In conclusion, the fall in malonyl-CoA and increase in fatty acid oxidation that occur with increased cardiac work were not due to inhibition of ACC or activation of MCD, suggesting alternative regulatory mechanisms for the work-induced decrease in malonyl-CoA concentration.     

Ultrastructure of the contact surfaces of Passalurus ambiguus (Rudolphi, 1819) (Nematoda)

The ultrastructure of the contact surfaces (integument and intestinal wall) of the nematode Passalurus ambiguus has been studied. The integument is composed according to the scheme common for all nematodes and includes a cuticle, hypodermis and a muscular layer. The specificity is with regard to the epicuticle, the different number of the cuticular sublayers in the anterior, central and the posterior parts of the worm body and the absence of a basal cuticular membrane. The intestinal wall consists of epithelial cells with microvilli. The ultrastructural characteristics of both contact surfaces indicate their main functions--absorption, secretion, transport, protection, movement, etc.     

Astrocyte- and hepatocyte-specific expression of genes from the distal serpin subcluster at 14q32.1 associates with tissue-specific chromatin structures

The distal serpin subcluster contains genes encoding alpha1-antichymotrypsin (ACT), protein C inhibitor (PCI), kallistatin (KAL) and the KAL-like protein, which are expressed in hepatocytes, but only the act gene is expressed in astrocytes. We show here that the tissue-specific expression of these genes associates with astrocyte- and hepatocyte-specific chromatin structures. In hepatocytes, we identified 12 Dnase I-hypersensitive sites (DHSs) that were distributed throughout the entire subcluster, with the promoters of expressed genes accessible to restriction enzyme digestion. In astrocytes, only six DHSs were located exclusively in the 5' flanking region of the act gene, with its promoter also accessible to restriction enzyme digestion. The acetylation of histone H3 and H4 was found throughout the subcluster in both cell types but this acetylation did not correlate with the expression pattern of these serpin genes. Analysis of histone modifications at the promoters of the act and pci genes revealed that methylation of histone H3 on lysine 4 correlated with their expression pattern in both cell types. In addition, inhibition of methyltransferase activity resulted in suppression of ACT and PCI mRNA expression. We propose that lysine 4 methylation of histone H3 correlates with the tissue-specific expression pattern of these serpin genes.     

Reversal of the sex difference in plasma leptin levels in obese children with impaired glucose tolerance

INTRODUCTION: Basal leptin level has been demonstrated to correlate positively with many indices of obesity, as well as insulin resistance. However, to date, little is known about regulation of leptin in obese children with incipient glucose metabolic disorders. OBJECTIVE: The aim of this study was to define the precise influence of the glucose tolerance status on plasma leptin in obese boys and girls separately. MATERIAL AND METHODS: 70 obese children with impaired glucose tolerance (IGT) and well-matched 70 normal glucose-tolerant (NGT) subjects were examined. Fasting and 2-h post glucose load plasma glucose and insulin levels as well as fasting leptin levels were determined, apart from anthropometric measurements. RESULTS: Leptin levels were significantly lower in girls with IGT compared to NGT girl (17.7+/-6.5 microg/L vs. 23.1+/-7.7 microg/L; p<.001). No such difference was observed in boys. In a multiple regression analysis adjusting for age and adiposity, in the female group plasma glucose and insulin levels 2-h after glucose load were the best predictors of fasting plasma leptin (r=-0.49, p<.005 and r=0.34, p<.05; respectively). In boys, plasma insulin level 2-h after glucose load was the independent determinant of leptin (r=0.36, p<.05). CONCLUSION: The differences between regulation of leptin synthesis in girls and boys with simple obesity were found. The stimulatory effect of insulin on leptin synthesis was greater in girls with normoglycemia than in girls with impaired glucose tolerance.     

Reversal of the sex difference in plasma leptin levels in obese children with impaired glucose tolerance

INTRODUCTION: Basal leptin level has been demonstrated to correlate positively with many indices of obesity, as well as insulin resistance. However, to date, little is known about regulation of leptin in obese children with incipient glucose metabolic disorders. OBJECTIVE: The aim of this study was to define the precise influence of the glucose tolerance status on plasma leptin in obese boys and girls separately. MATERIAL AND METHODS: 70 obese children with impaired glucose tolerance (IGT) and well-matched 70 normal glucose-tolerant (NGT) subjects were examined. Fasting and 2-h post glucose load plasma glucose and insulin levels as well as fasting leptin levels were determined, apart from anthropometric measurements. RESULTS: Leptin levels were significantly lower in girls with IGT compared to NGT girl (17.7+/-6.5 microg/L vs. 23.1+/-7.7 microg/L; p<.001). No such difference was observed in boys. In a multiple regression analysis adjusting for age and adiposity, in the female group plasma glucose and insulin levels 2-h after glucose load were the best predictors of fasting plasma leptin (r=-0.49, p<.005 and r=0.34, p<.05; respectively). In boys, plasma insulin level 2-h after glucose load was the independent determinant of leptin (r=0.36, p<.05). CONCLUSION: The differences between regulation of leptin synthesis in girls and boys with simple obesity were found. The stimulatory effect of insulin on leptin synthesis was greater in girls with normoglycemia than in girls with impaired glucose tolerance.     

Influence of hormonal therapy on growth rate and bone age progression in patients with Turner syndrome

The efficacy of growth promoting hormonal therapy is assessed on the basis of growth rate as well as bone age progression until the patients reach their final height. The aim of our study was to investigate which hormonal therapy influences in most appropriate way height velocity and bone age progression in patients with Turner syndrome (TS) and to establish the optimal age to initiate treatment. Patients were divided into five groups according to the type of hormonal therapy: 1) 11 patients treated with growth hormone (GH); 2) 18 patients treated with GH and oxandrolone (Ox); 3) 7 patients treated with GH, Ox and estrogens (E); 4) 6 patients treated with OX and E; and the control group (Group 0) of 62 untreated patients. The patients height was expressed in hSDS calculated on the basis of growth chart for patients with TS (hSDST). Bone age (BA) was assessed according to Greulich-Pyle method. Results: The mean values of deltahSDST in the first and second year of therapy in individual groups were significantly different. The difference resulted from significantly higher value of deltahSDST in group treated with GH+Ox. Analysis of regression between deltaCA and deltaBA revealed regression coefficients alpha of equation deltaBA= alpha x deltaCA: in group 0: 0.817; group GH: 1.233; group GH+Ox: 0.861; group GH+Ox+E: 0.997; group Ox+E: 1.141. There was significant difference between regression coefficients in studied groups. It resulted from significantly higher value of alpha in group treated with GH than in a group 0 and treated with GH+Ox. Only group treated with GH+Ox showed a significant negative correlation between baseline CA and deltaBA during the therapy. We can conclude that all regimens of hormonal therapy improved height in our patients but the highest increase of height during the therapy and the smallest progression of the bone age in the same time were observed in patients treated with GH+Ox.     

On the specificity of 4-amino-5-methylamino-2',7'-difluorofluorescein as a probe for nitric oxide

The specificity of 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) for nitric oxide was evaluated in in vitro systems. The probe was found fairly specific for nitric oxide. Potential sources of artifacts include the autoxidation of DAF-FM, potentiated by light, and its oxidation by sources of superoxide and peroxyl radicals, leading to fluorescence spectra indistinguishable from those of the nitric oxide adduct. Although DAF-FM reacts with peroxynitrite, this reaction seems to be of secondary importance under quasi-physiological conditions. On the other hand, a simultaneous presence of a nitric oxide source and a superoxide or hydrogen peroxide decreases or increases the fluorescence of DAF-FM, respectively, resulting in biased estimates of nitric oxide production.     

Position-specific incorporation of a highly photodurable and blue-laser excitable fluorescent amino acid into proteins for fluorescence sensing

A new fluorescent amino acid, L-2-acridonylalanine, was incorporated into proteins at specific positions using 4-base codon/anticodon strategy. The efficiency of the incorporation was high enough to obtain enough quantities of the mutants. The acridonyl group was highly fluorescent when it was excited at the wavelengths of blue-lasers and was highly photodurable compared with conventional fluorophores often used for biological analyses. The fluorescence intensity was sensitive to small changes in the polarity of the environment. When the nonnatural amino acid was incorporated into specific positions of streptavidin, the mutant protein worked as a fluorescent sensor to biotin. Similarly, when the amino acid was incorporated into camel single-chain antibody, the mutant protein sensitively responded to the antigen molecule. The high incorporation efficiency, the high photodurability, the excitability with blue-lasers, and high sensitivity to the environment make the acridonylalanine as the promising fluorescent amino acid for sensing small molecules when incorporated into specific positions of various antibodies, receptors, and enzymes.