Adipose tissue insulin receptor and glucose transporter 4 expression, and blood glucose and insulin responses during glucose tolerance tests in transition Holstein cows with different body condition.

Glucose uptake in tissues is mediated by insulin receptor (INSR) and glucose transporter 4 (GLUT4). The aim of this study was to examine the effect of body condition during the dry period on adipose tissue mRNA and protein expression of INSR and GLUT4, and on the dynamics of glucose and insulin following the i.v. glucose tolerance test in Holstein cows 21 d before (d -21) and after (d 21) calving. Cows were grouped as body condition score (BCS) ≤3.0 (thin, T; n = 14), BCS = 3.25 to 3.5 (optimal, O; n = 14), and BCS ≥3.75 (overconditioned, OC; n = 14). Blood was analyzed for glucose, insulin, fatty acids, and ß-hydroxybutyrate concentrations. Adipose tissue was analyzed for INSR and GLUT4 mRNA and protein concentrations. During the glucose tolerance test 0.15 g/kg of body weight glucose was infused; blood was collected at -5, 5, 10, 20, 30, 40, 50, and 60 min, and analyzed for glucose and insulin. On d -21 the area under the curve (AUC) of glucose was smallest in group T (1,512 ± 33.9 mg/dL × min) and largest in group OC (1,783 ± 33.9 mg/dL × min), and different between all groups. Basal insulin on d -21 was lowest in group T (13.9 ± 2.32 µU/mL), which was different from group OC (24.9 ± 2.32 µU/mL. On d -21 the smallest AUC 5-60 of insulin in group T (5,308 ± 1,214 µU/mL × min) differed from the largest AUC in group OC (10,867 ± 1,215 µU/mL × min). Time to reach basal concentration of insulin in group OC (113 ± 14.1 min) was longer compared with group T (45 ± 14.1). The INSR mRNA abundance on d 21 was higher compared with d -21 in groups T (d -21: 3.3 ± 0.44; d 21: 5.9 ± 0.44) and O (d -21: 3.7 ± 0.45; d 21: 4.7 ± 0.45). The extent of INSR protein expression on d -21 was highest in group T (7.3 ± 0.74 ng/mL), differing from group O (4.6 ± 0.73 ng/mL), which had the lowest expression. The amount of GLUT4 protein on d -21 was lowest in group OC (1.2 ± 0.14 ng/mL), different from group O (1.8 ± 0.14 ng/mL), which had the highest amount, and from group T (1.5 ± 0.14 ng/mL). From d -21 to 21, a decrease occurred in the GLUT4 protein levels in both groups T (d -21: 1.5 ± 0.14 ng/mL; d 21: 0.8 ± 0.14 ng/mL) and O (d -21: 1.8 ± 0.14 ng/mL; d 21: 0.8 ± 0.14 ng/mL). These results demonstrate that in obese cows adipose tissue insulin resistance develops prepartum and is related to reduced GLUT4 protein synthesis. Regarding glucose metabolism, body condition did not affect adipose tissue insulin resistance postpartum.

Gene expression study of IL10 family genes in vitiligo skin biopsies, peripheral blood mononuclear cells and sera.

BACKGROUND: Vitiligo is a pigmentation disorder, the cause of which is complex and not yet fully understood. There is a significant change of epidermal cytokines in involved skin of patients with vitiligo compared with uninvolved skin and skin of healthy controls, thus suggesting a possible involvement of cytokines in the pathogenesis of vitiligo. OBJECTIVES: To evaluate potential roles of IL10 family cytokines (IL10, IL19, IL20, IL22 and IL24) in vitiligo. Along with the selected cytokines, we investigated subunits of the receptors (IL10RA, IL10RB, IL20RA and IL22RA1) which are involved in the signalling pathway of the cytokines. METHODS: Quantitative real-time polymerase chain reaction was used to detect mRNA expression levels in samples extracted from skin biopsies and peripheral blood mononuclear cells and an enzyme-linked immunosorbent assay was used to measure protein concentrations in serum from patients with vitiligo and healthy controls. RESULTS: IL22 is significantly associated with vitiligo, especially with the active stage of vitiligo, as shown by results of mRNA expression and supported by results of protein level in sera. IL22 may provoke inflammation which leads to destruction of melanocytes. CONCLUSIONS: The actual role of IL22 during pathogenesis of vitiligo remains to be better characterized. Signal transductions of other investigated cytokines seem to be regulated on the expression level of their receptor complex subunits.

Selective precipitation of 32Pi onto filter papers. Application to ATPase and cyclic AMP phosphodiesterase determination.

A simple and selective method is described for the isolation of 32Pi based on the precipitation of the phosphomolybdate complex with triethylamine. Precipitation takes place on filter paper, which are then washed with a solution containing ammonium molybdate and triethylamine to remove other radioactive phosphates. Very large numbers of samples and very small sample volumes can be accommodated easily. The use of this method to measure ATPase and cyclic AMP phosphodiesterase activity is demonstrated.

Phosphorylation of membranes from the rat gastric mucosa.

Gastric mucosal membranes derived primarily from parietal cells were found to contain endogenous protein kinase systems as well as several phosphate-accepting substrates. One specific membrane protein with a molecular weight of 88 000 was phosphorylated only in the presence of calcium, while the degree of phosphorylation of three other membrane proteins was similarly increased. The activity of the calcium-dependent protein kinase was found to be totally inhibited in the presence of trifluoperazine, a phenothiazine known to specifically inactivate calmodulin. These results suggest that a calmodulin- and calcium-dependent phosphorylation system may be a component of the parietal cell membrane. Phosphorylation of the membrane proteins was not affected by either cyclic AMP or cyclic GMP. The heat-stable inhibitor protein of cyclic AMP-dependent protein kinase did not inhibit the endogenous protein kinase activity suggesting that the membrane enzyme is not similar to the cytosolic protein kinase. However, the catalytic subunit of the soluble enzyme was capable of phosphorylating a number of membrane proteins indicating that after maximal autophosphorylation of the gastric membranes, phosphate-acceptor sites are still available to the cytosolic cyclic AMP-dependent protein kinase.

Differences between glycogen synthases from rat and rabbit skeletal muscle as indicated by phosphopeptide maps.

Glycogen synthase I was purified from rat skeletal muscle. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme migrated as a major band with a subunit Mr of 85,000. The specific activity (24 units/mg protein), activity ratio (the activity in the absence of glucose-6-P divided by the activity in the presence of glucose-6-P X 100) (92 +/- 2) and phosphate content (0.6 mol/mol subunit) were similar to the enzyme from rabbit skeletal muscle. Phosphorylation and inactivation of rat muscle glycogen synthase by casein kinase I, casein kinase II (glycogen synthase kinase 5), glycogen synthase kinase 3 (kinase FA), glycogen synthase kinase 4, phosphorylase b kinase, and the catalytic subunit of cAMP-dependent protein kinase were similar to those reported for rabbit muscle synthase. The greatest decrease in rat muscle glycogen synthase activity was seen after phosphorylation of the synthase by casein kinase I. Phosphopeptide maps of glycogen synthase were obtained by digesting the different 32P-labeled forms of glycogen synthase by CNBr, trypsin, or chymotrypsin. The CNBr peptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the tryptic and chymotryptic peptides were separated by reversed-phase HPLC. Although the rat and rabbit forms of synthase gave similar peptide maps, there were significant differences between the phosphopeptides derived from the N-terminal region of rabbit glycogen synthase and the corresponding peptides presumably derived from the N-terminal region of rat glycogen synthase. For CNBr peptides, the apparent Mr was 12,500 for rat and 12,000 for the rabbit. The tryptic peptides obtained from the two species had different retention times. A single chymotryptic peptide was produced from rat skeletal muscle glycogen synthase after phosphorylation by phosphorylase kinase whereas two peptides were obtained with the rabbit enzyme. These results indicate that the N-terminus of rabbit glycogen synthase, which contains four phosphorylatable residues (Kuret et al. (1985) Eur. J. Biochem. 151, 39-48), is different from the N-terminus of rat glycogen synthase.

Inhibitory effect of polycations on phosphorylation of glycogen synthase by glycogen synthase kinase 3.

Several polycations were tested for their abilities to inhibit the activity of glycogen synthase kinase 3 (GSK-3). L-Polylysine was the most powerful inhibitor of GSK-3 with half-maximal inhibition of glycogen synthase phosphorylation occurring at approx. 100 nM. D-Polylysine and histone H1 were also inhibitory, but the concentration dependence was complex, and DL-polylysine was the least effective inhibitor. Spermine caused about 50% inhibition of GSK-3 at 0.7 mM and 70% inhibition at 4 mM. Inhibition of GSK-3 by L-polylysine could be blocked or reversed by heparin. A heat-stable polycation antagonist isolated from swine kidney cortex also blocked the inhibitory effect of L-polylysine on GSK-3 and blocked histone H1 stimulation of protein phosphatase 2A activity. Under the conditions tested, L-polylysine also inhibited GSK-3 catalyzed phosphorylation of type II regulatory subunit of cAMP-dependent protein kinase and a 63 kDa brain protein, but only slightly inhibited phosphorylation of inhibitor 2 or proteolytic fragments of glycogen synthase that contain site 3 (a + b + c). L-Polylysine at a concentration (200 nM) that caused nearly complete inhibition of GSK-3 stimulated casein kinase I and casein kinase II, but had virtually no effect on the catalytic subunit of cAMP-dependent protein kinase. These results suggest that polycations can be useful in controlling GSK-3 activity. Polycations have the potential to decrease the phosphorylation state of glycogen synthase at site 3, both by inhibiting GKS-3 as shown in this study and by stimulating the phosphatase reaction as shown previously (Pelech, S. and Cohen, P. (1985) Eur. J. Biochem. 148, 245-251).

Isolation and characterization of cyclic AMP-independent glycogen synthase kinase from rat skeletal muscle.

Glycogen synthase kinase was isolated from rat skeletal muscle. This kinase, which is cyclic nucleotide-independent and calcium-independent, was separated from phosphorylase kinase, cyclic AMP-dependent protein kinase and phosvitin kinase by phosphocellulose chromatography. Gel filtration on Sephadex G-100 resolved the glycogen synthase kinase into two fractions with apparent molecular weights of 68 000 (peak I) and 52 000 (peak II). This step also separated glycogen synthase kinase from the catalytic subunit of the cyclic AMP-dependent protein kinase, which had an apparent molecular weight of 39 000. Peak II glycogen synthase kinase activity was not affected by the addition of calcium, EGTA or a number of cyclic nucleotides. In addition to ATP, dATP would serve as the phosphate donor. Other trinucleotides tested were either poor or ineffective substrates. Activity was about 5-fold greater with Mg2+ than with Mn2+. Glycogen stimulated activity about 25%. Modifications of the methods of Soderling et al. ((1970) J. Biol. Chem. 245, 6317--6328) and Nimmo et al. ((1976) Eur. J. Biochem. 68, 21--30) were developed for purification of glycogen synthease (UDPglucose:glycogen 4-alpha D-glucosyltransferase, EC 2.4.1.11) to specific activity of 35 units/mg of protein. Using this preparation of glycogen synthase as substrate, the phosphorylation and inactivation catalyzed by glycogen synthase kinase was compared to that catalyzed by cyclic AMP-dependent protein kinase or phosphorylase kinase. Each of the kinases had different specificities for phosphorylation sites on glycogen synthase.

Exposure to sixty minutes of hyperoxia upregulates myocardial humanins in patients with coronary artery disease - a pilot study.

In experimental setting the concept of myocardial preconditioning by hyperoxia has been introduced and different intracellular protective mechanisms and their effects have been described. To study whether similar protective phenotype can be induced by hyperoxia also in humans, gene expression profile after hyperoxic exposure was analyzed. Adult patients were randomized to be ventilated with either FiO2 0.4 (n = 14) or 1.0 (n = 10) for 60 minutes before coronary artery bypass grafting. A tissue sample from the right atrial appendage was taken for gene analysis and expression profile analysis on genome wide level by RNA-seq analysis was applied. Exposure to > 96% oxygen for 60 minutes significantly changed the expression of 20 different genes, including upregulation of two different humanins - MTRNR2L2 and MTRNR2L8, and activated a "cell survival" network as detected by Ingenuity Pathway Analyses. We concluded that administration of > 96% oxygen for 1 hour changes gene expression in the myocardium of the patients with coronary artery disease and may enhance cell survival capability.

Phosphorylation and inactivation of rat heart glycogen synthase by cAMP-dependent and cAMP-independent protein kinases.

The regulation of cardiac muscle glycogen metabolism is not well understood. Previous studies have indicated that heart glycogen synthase is heavily phosphorylated in vivo on multiple sites. Using purified enzymes, we have investigated the effect of phosphorylation of different sites on the activity of rat heart glycogen synthase. A convenient procedure was developed for the purification of rat heart glycogen synthase. The enzyme was phosphorylated by selected kinases, and glycogen synthase activity, extent of phosphorylation, and phosphopeptide maps were analyzed. Rat heart glycogen synthase, purified to apparent homogeneity (M(r) 87,000 on SDS-PAGE), had a specific activity of 18 U/mg protein and had an activity ratio of 0.74 (activity in the absence divided by the activity in the presence of glucose 6-P). cAMP-dependent protein kinase, glycogen synthase kinase 3, Ca2+/calmodulin-dependent protein kinase II, protein kinase C, and phosphorylase kinase phosphorylated the enzyme with a concomitant decrease in the activity ratio to values ranging from 0.1 to 0.4. Casein kinase II phosphorylated but did not inactivate glycogen synthase. Six tryptic phosphopeptides, obtained from heart glycogen synthase phosphorylated by the various kinases, were separated by reverse-phase chromatography. The phosphopeptide(s) obtained with each kinase eluted at the same position(s) as corresponding phosphopeptides obtained from rat skeletal muscle glycogen synthase. The study shows that the pattern of phosphorylation and effects on activity are very similar for cardiac and skeletal muscle glycogen synthase. It is suggested that the well known differences in heart and glycogen metabolism may be due to the interplay of kinases and phosphatases which could lead to different phosphorylation and activity states of glycogen synthase.

Effects of dibutyryl adenosine 3',5'-monophosphate, angiotensin II, and atrial natriuretic factor on phosphorylation of a 17,600-dalton protein in adrenal glomerulosa cells.

Rat adrenal glomerulosa cells were incubated with [32P]phosphate and (Bu)2AMP (dbcAMP), angiotensin II, and atrial natriuretic factor (ANF). Incorporation of [32P]phosphate into cellular proteins was analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. dbAMP stimulated phosphorylation of a 17.6K protein, while angiotensin II did not stimulate it. ANF did not affect the protein phosphorylation, whether the cells were in the basal state or stimulated by dbcAMP or angiotensin II. On the other hand, ANF markedly inhibited angiotensin II-stimulated aldosterone production, but only slightly inhibited dbcAMP-stimulated aldosterone. These results suggest that in rat adrenal glomerulosa cells phosphorylation of the 17.6K protein may have a relationship with the stimulatory effect of cAMP on aldosterone production; however, neither angiotensin II nor ANF affected the phosphorylation of this protein, and phosphorylation of the 17.6K protein is not an obligatory step in the regulation of aldosterone production.

Hypertension in the transgenic rat TGR(mRen-2)27 may be due to enhanced kinetics of the reaction between mouse renin and rat angiotensinogen.

The transgenic rat TGR(mRen-2)27, in which the Ren-2 mouse renin gene is transfected into the genome of the rat, develops severe hypertension with high adrenal renin and low kidney renin. These animals express both mouse and rat renin. To investigate the cause of hypertension in the TGR rat, we compared the kinetics of mouse renin acting on mouse and rat angiotensinogens. The optimum pH of the renin reaction in the Sprague-Dawley rat was 6.5, whereas the optimum pH of the reaction in the TGR rat was approximately 8.5. The optimum pH of the renin reaction in the DBA mouse was 6.0. Purified mouse Ren-2 renin acting on rat angiotensinogen showed a pH profile similar to that for the renin reaction in the TGR rat. The angiotensinogen concentration in pooled plasma from eight DBA mice was 104.5 ng angiotensin I/mL and was clearly lower than that in Sprague-Dawley rats (772.4 +/- 37.3 ng angiotensin I/mL, n = 4). The reaction of purified mouse Ren-2 renin with rat angiotensinogen was 10 times faster than with mouse angiotensinogen. Plasma renin activity in DBA mice increased dramatically on addition of rat angiotensinogen (from 253.4 +/- 66.7 to 225,000 +/- 48,000 ng angiotensin I/mL per hour). Intravenous injection of 2 or 10 microL of DBA mouse plasma into the nephrectomized Sprague-Dawley rat increased the mean arterial pressure of the rat by 27.7 +/- 4.7 and 61.8 +/- 2.7 mmHg, respectively, whereas injection of 200 microL of Sprague-Dawley rat plasma did not change the mean arterial pressure of the rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-elimination of phosphate and subsequent addition of pyridoxamine as a method for identifying and sequencing peptides containing phosphoseryl residues.

Peptides containing phosphoseryl residues can be modified by removal of the phosphate groups via beta-elimination followed by addition of pyridoxamine to the resulting dehydroalanyl residue. Peptides containing the modified residues can be detected at nanomole levels by monitoring absorbance at 328 nm or at picomole levels by monitoring fluorescence. Photolysis of the modified peptide converts the pyridoxamino adduct to a form which can be readily identified after Edman degradation.

Identification of the phosphoserine residue in histone H1 phosphorylated by protein kinase C.

The site-specific phosphorylation of bovine histone H1 by protein kinase C was investigated in order to further elucidate the substrate specificity of protein kinase C. Protein kinase C was found to phosphorylate histone H1 to 1 mol per mol. Using N-bromosuccinimide and thrombin digestions, the phosphorylation site was localized to the globular region of the protein, containing residues 71-122. A tryptic peptide containing the phosphorylation site was purified. Modification of the phosphoserine followed by amino acid sequence analysis demonstrated that protein kinase C phosphorylated histone H1 on serine 103. This sequence, Gly97-Thr-Gly-Ala-Ser-Gly-Ser(PO4)-Phe-Lys105, supports the contention that basic amino acid residues C-terminal to the phosphorylation site are sufficient determinants for phosphorylation by protein kinase C.

The grid-blot: a procedure for screening large numbers of monoclonal antibodies for specificity to native and denatured proteins.

This report describes a procedure referred to as a grid-blot for simultaneously testing up to 30 monoclonal antibodies for specificity with an equivalent number of different proteins on a single sheet of nitrocellulose paper. Only 150 microliters of hybridoma culture supernatant is required for the screening and the entire procedure can be completed in less than five hours. This assay was developed to quickly identify those hybridoma cultures producing antibodies that preferentially recognize the native form of a protein and those that also recognize the SDS denatured form and were optimal for use in Western blots. Monoclonal antibodies raised against two distinct proteins, myofibril C-protein (120 antibodies) and the catalytic subunit of cyclic-AMP dependent protein kinase (240 antibodies) were tested. The grid-blot results indicated that 85 of the C-protein antibodies and 55 of the catalytic subunit antibodies were monospecific. Only 4 of the C-protein and 9 catalytic subunit antibodies showed a preferential staining for the appropriate native protein. The antibodies that stained the denatured protein most intensely in the grid-blot corresponded with those that produced the best immunostain in the Western blot. Finally, a version of the grid-blot was found to be an efficient means of determining antibody isotypes.

[Intramolecular alkylaton of aromatic compounds. 34. Synthesis of pyridinylmethylindolines as potential precursors of ergolines] . / Intramolekulare Aromatenalkylierungen. 34. Mitt.: Synthese von Pyridinylmethylindolinen als potenzielle Edukte für Ergoline.

The carbinoles 3 prepared from the N-protected indolaldehydes 2 and bromomethoxypyridine 1 can smoothly be hydrogenolized to the lutidinylindoles 4 which in turn give the corresponding indolines 5 by NaCNBH3-reduction. Treatment of 3a by acid the trihetarylmethane 9 and 5-methoxypyridine-2-carboxaldehyde 10 are generated. The acetylpyridine 7 is found as a by-product of 3c. As by-product of the reduction the borane adduct 8 is detected.

[Intramolecular alkylation of aromatic compounds. 35. Di- and tetrahydropyridinemethylindolines as potential precursors of ergolines] . / Intramolekulare Aromatenalkylierungen. 35. Mitt.: Di- und Tetrahydropyridinylmethylindoline als potenzielle Vorstufen zu Ergolinen.

In the presence of methyl chloroformate, compound 6a is reduced by NaCNBH3 yielding the 1,6-dihydropyridine derivative 7. Under the same conditions the indoline 10 accessible from 6a via 9a gives a mixture of the 1,4- and 1,6-dihydropyridine derivatives 11 and 12. As by-product of the reduction the borane adduct 13 is detected. In contrast the methoiodide 1 is reduced by NaBH4 or DIBAH giving a separable mixture of the diastereomers of the tetrahydropyridines 2 and 3; on catalytic hydrogenation the piperidine derivative 4 is formed. Cleavage of the enolether moiety in 3a and 7 provides the corresponding piperidones 5 and 14, respectively. Using prolonged reaction time 7 is hydrolized quantitatively furnishing the 1,4-diketone 15.

[The photostability of antimycotics. 3. Photostability of locally acting antimycotics]. / Untersuchung der Photostabilität von Antimykotika. 3. Mittellung: Photostabilität lokal wirksamer Antimykotika.

The photochemical stability of a number of topical antimycotic drugs was tested. The light sensitivity decreases in the order naftifine, sulbentine, cloxiquin, tolnaftate and chlorphenesin. A liquid chromatography-mass spectrometry system was used to identify a number of photodegradation products. Light exposure of sulbentine leads to the formation of benzylisothiocyanate. Chlorphenesine solutions undergo photodehalogenation with the formation of varying photodegradation products depending on the solvent used. The photochemical reactions of naftifine are a cis-trans-isomerization and the formation of a dimer product. Drug preparations are also degraded under light exposure in a simulated topical application. Excipients in the drug preparations strongly influence the photodegradation kinetics and the chemical structure of photodegradation products.

[Conformationally restricted pethidine analogs. 5. Synthesis and pharmacologic testing of cis-B-10b-acyl-1,2,3,4,4a,5,6,10b- octahydrobenzo(f)isoquinolines]. / Pethidin-Analoga mit eingeschränkter Konformationsbeweglichkeit. 5. Mitteilung: Synthese und pharmacokologische Prüfung von cis-B-10b-Acyl-1,2,3,4,4a,5,6,10b-octahydrobenzo[f]ischinolinen.

Cyanophenylcyclobuten (1a) gave the corresponding cis-B-configurated 10b-acyloctahydrobenzo[f] isoquinolines 3a-f by Grignard reaction. Cis-B-3b was demethylated to yield the secondary base 3g, which in turn was realkylated to the homologous allyl- and isopentenyl derivatives 3h and 3i. In the writing test the desoxyketobemidon analogon cis-B-3b exhibits a remarkable activity, whereas 3a, c-e are somewhat weaker and 3h and 3i are inactive.

Whole Transcriptome Analysis (RNA Sequencing) of Peripheral Blood Mononuclear Cells of Vitiligo Patients.

Vitiligo is an idiopathic disorder characterized by depigmented patches on the skin due to a loss of melanocytes. The cause of melanocyte destruction is not fully understood. The aim of this study was to detect the potential pathways involved in the vitiligo pathogenesis to further understand the causes and entity of vitiligo. For that the transcriptome of peripheral blood mononuclear cells of 4 vitiligo patients and 4 control subjects was analyzed using the SOLiD System platform and whole transcriptome RNA sequencing application. Altogether 2,470 genes were expressed differently and GRID2IP showed the highest deviation in patients compared to controls. Using functional analysis, altogether 993 associations between the gene groups and diseases were found. The analysis revealed associations between vitiligo and diseases such as lichen planus, limb-girdle muscular dystrophy type 2B, and facioscapulohumeral muscular dystrophy. Additionally, the gene groups with an altered expression pattern are participating in processes such as cell death, survival and signaling, inflammation, and oxidative stress. In conclusion, vitiligo is rather a systemic than a local skin disease; the findings from an enormous amount of RNA sequencing data support the previous findings about vitiligo and should be further analyzed.