Is local anesthesia beneficial in preventing shoulder pain after laparoscopy?
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Anesthesia, Local↗
Biomedical subjects
M Kretschmer
Publications and source records attributed to M Kretschmer.
In vivo microscopy of murine islets of Langerhans: increased adhesion of transferred lymphocytes to islets depends on macrophage-derived cytokines in a model of organ-specific insulitis.
Environmental factors contribute to the pathogenesis of type 1 diabetes (insulin-dependent diabetes mellitus). Multiple low doses of streptozotocin (MLDS) induce hyperglycaemia and insulitis in mice. Previously we demonstrated that adhesion of lymphocytes to endothelium of islets is only increased when donor animals were diabetic and recipient mice had received 5 mg/kg streptozotocin (STZ). Therefore we used streptozotocin to evaluate the immunological relevance of such an irritation of islets. Lymphocytes, separated from diabetic mice (MLDS), were fluorescently labelled and injected to recipient mice that had received 5 mg/kg STZ. With in vivo microscopy we measured lymphocyte flow and adherence in islets. Expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) in the pancreas was assessed using immunohistochemistry. Very late antigen-4 (VLA-4) and leucocyte function-associated antigen-1 (LFA-1) expression on transferred lymphocytes was measured with flow cytometry. Pretreatment of recipients with antibodies to cytokines or silica reduced lymphocyte adherence to islet endothelium from 2.04% (goat immunoglobulin G; IgG) or 1.82% (rat IgG) to 0.47, 0.58, 0.39 or 0. 19% for monoclonal antibody (mAb) interferon-gamma (IFN-gamma), polyclonal antibody (pAb) tumour necrosis factor-alpha (TNF-alpha), pAb interleukin (IL)-1alpha or silica, respectively. Reduced adhesion was associated with a decreased expression of VCAM-1 and ICAM-1 in islets of treated recipients compared with mice treated with 5 mg/kg STZ alone. In conclusion, pretreatment of recipients with 5 mg/kg STZ leads to an increased expression of adhesion molecules in the islets and lymphocyte adhesion to islet endothelium in vivo, demonstrating an immune response of the islets. Prevention of increased expression of ICAM-1 or VCAM-1 and reduction of lymphocyte adhesion in islets by silica or antibody indicate an involvement of macrophages and macrophage derived cytokines in the generation of this immune response.
Animals↗
[Practical education of geriatric nurses in Brandennburg. The aim is completion, not competition].
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Aged↗
Mutation of monofunctional 6-phosphofructo-2-kinase in yeast to bifunctional 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase.
We have shown previously that 6-phosphofructo-2-kinase in yeast has negligible fructose-2,6-bisphosphatase activity even though resembling in part of its C-terminal sequence the phosphatase domain of the bifunctional liver enzyme. Here we show that exchanging Ser-404 to His-404 in the yeast peptide creates a bifunctional enzyme with a fructose-2,6-bisphosphatase activity involving a phosphoprotein intermediate. Like mammalian bifunctional enzymes, the His-404 mutant protein is readily phosphorylated by fructose 2,6-P2 with a half-saturation of 0.4 microM, the same Km value as for its fructose-2,6-bisphosphatase activity. Protein phosphorylation by the C-subunit of cAMP-dependent protein kinase, presumably at a C-terminal consensus site, increases the Km value to 1.5 microM. The newly created fructose-2,6-bisphosphatase is inhibited competitively by its product fructose 6-P with a K(i) of 0.6 mM. No effect of the His-404 mutation was found on 6-phosphofructo-2-kinase activity, in line with the mutant yeast enzyme having independent kinase and phosphatase domains, like its mammalian wild-type counterparts. The results would fit with the evolution of the PFK26 gene having involved fusion between kinase and phosphatase genes--as proposed for the mammalian enzyme--but with accompanying or later silencing of the fructose-2,6-bisphosphatase activity.
Animals↗
Glucose metabolism in Escherichia coli and the effect of increased amount of aldolase.
We present a comparative study of Escherichia coli with normal and increased amounts of fructose-1,6-bisphosphate aldolase. Most experiments employed a resting cell system involving a high cell density (so as to obtain the soluble pool by direct extraction) and anaerobic incubation in the presence of chloramphenicol. Glucose use is linear with time with a rate ca. half of that in growth, fermentation is almost quantitative, and metabolite concentrations reach a quasi steady state. Increased amount of aldolase had little effect on glucose flux; fructose-1,6-P2 concentration decreased by ca. one-third, and the extent of equilibration of its two halves, measured by a dismutation procedure on samples taken during metabolism of [6-14C]glucose, increased from 0.33 [(cpm in C1-3)/(cpm in C1-6)] to 0.43. Using the simplest model, that increased amount of aldolase does not perturb net flux or later metabolites, together with the steady-state rate equations for aldolase and triose-P isomerase, we show that the results with resting cells fit with the extra enzyme being fully active, and do not necessitate special assumptions concerning a glycolytic complex, metabolite compartmentation, or secondary mechanisms assuring high metabolite concentration. However, the fit does require that the measured Vmax values substantially underestimate the actual ones. Calculation also shows that the forms of the predicted curves--and hence the fit with experimental data--of fructose-1,6-P2 concentration and labeling as a function of the amount of aldolase are highly dependent on glyceraldehyde-3-P concentration but independent of the kinetic parameters of aldolase.
Anaerobiosis↗
A pocket database for cloning.
A system of programs is presented that turns the HP 48SX calculator into a pocket database for molecular biology with a library of restriction enzymes and plasmids. The operations of the calculator are extended to handle DNA and peptide sequences much like numbers. The software provides a comprehensive system for cloning and includes functions for cutting and ligation, DNA-peptide translation and its reverse, DNA probe design, and codon usage statistics. Since the calculator comes with built-in Kermit protocol, serial port, infrared interface, and sequences are in ASCII code, users can communicate sequences bench-to-bench to other calculators or upload and download them with IBM PC compatible and Macintosh computers.
Base Sequence↗
The yeast FBP26 gene codes for a fructose-2,6-bisphosphatase.
Sequencing of an open reading frame 450 bp downstream from the yeast VPS35 gene revealed a putative peptide of 452 amino acids and 52.7 kDa. The predicted amino acid sequence has 45% identity with the 55-kDa subunit of the 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase (EC 2.7.1.105/EC 3.1.3.46) from rat liver and 42% identity with 480 amino acids in the center of the recently reported 93.5-kDa subunit of yeast 6-phosphofructo-2-kinase (EC 2.7.1.105). The product of the new yeast gene is similar to the entire sequence of the bifunctional rat liver enzyme and, unlike yeast 6-phosphofructo-2-kinase, has the histidine residue essential for fructose-2,6-bisphosphatase activity. Extracts from a chromosomal null mutant strain, fbp26::HIS3, incubated in the presence of [2-32P]fructose 2,6-P2, lacked in autoradiograms the characteristic 56-kDa labeled band observed in wild-type. The same band was intensified 3-fold over wild-type level with the FBP26 gene introduced on multicopy in the fbp26::HIS3 background. A similar increase was found for fructose-2,6-bisphosphatase activity in the same extracts. The FBP26 gene did not cause detectable increase in 6-phosphofructo-2-kinase activity when introduced on multicopy in a pfk26::LEU2 mutant, indicating that its gene product is predominantly a fructose-2,6-bisphosphatase. Growth on glucose, fructose, galactose, pyruvate, and glycerol/lactate was not impaired in strains carrying the fbp26::HIS3 allele.
Amino Acid Sequence↗
[Effects of lowest levels of drugs--a contribution to homeopathy research].
After oral administration of homoeopathically prepared low-dose-amounts of Conium and Mercury phosphate to male Wistar rats enzymatic parameters were investigated in three subcellular compartments of the liver under blind conditions. 1. After seven single application of the substance amount referring to a D8 potency a maximum effect could be detected for both agents. 2. The relation between agent and magnitude of the provoked effect is not linear. 3. The importance of these results is discussed and integrated into a general context.
Administration, Oral↗
Yeast 6-phosphofructo-2-kinase: sequence and mutant.
We have reported yeast 6-phosphofructo-2-kinase (EC 2.7.1.105) as having a ca. 96-kDa subunit size, as well as isolation of its structural gene, PFK26. Sequencing now shows an open reading frame of 827 amino acids and 93.5 kDa. The deduced amino acid sequence has 42% identity with the 55-kDa subunit of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from rat liver with extra material at both ends. Although the yeast sequence is especially similar to the liver one in its bisphosphatase domain, the essential His-258 of the liver enzyme is, in yeast, a serine, which may explain the apparent lack of bisphosphatase activity. Also, the yeast enzyme known to be activated via protein kinase A, has a putative phosphorylation site near its C-terminus and lacks the N-terminal phosphorylation sequence involved in inhibition of the liver enzyme. In a chromosomal null mutant strain, pfk26::LEU2, activity was marginal and the protein was not detectable as antigen. The mutant strain grew well on glucose and contained a near-normal level of fructose 2,6-P2. But in its growth on pyruvate, by contrast with the wild-type strain, no fructose 2,6-P2 was detectable, and it did not form after glucose addition in the presence of cycloheximide either. Such resting cells, however, metabolized glucose at the normal high rate. Glucose addition to the pfk26 mutant strain in the absence of cycloheximide, on the other hand, caused a ca. 10% normal rate of fructose 2,6-P2 accumulation, presumably employing a glucose-inducible second enzyme. Using strains also lacking 6-phosphofructo-1-kinase, affinity chromatography revealed the second enzyme as a minor peak amounting to 6% of 6-phosphofructo-2-kinase activity in a PFK26 strain and as the sole peak, in similar amount, in a pfk26 mutant strain.
Amino Acid Sequence↗
Identification and cloning of yeast phosphofructokinase 2.
Fructose-6-phosphate 2-kinase ('phosphofructokinase 2') was purified from a strain of Saccharomyces cerevisiae lacking fructose-6-phosphate 1-kinase. After chromatography on DEAE-Sephacel, Sephacryl blue, CM-Sephadex and rechromatography on CM-Sephadex with fructose-6-phosphate elution, the specific activity was 1.6 U/mg protein. Although the latter value is high for fructose-6-phosphate 2-kinase, as was the purification factor of 3 x 10(4), staining with Coomassie blue showed the fraction to still contain many proteins. Incubation with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase gave a further increase in specific activity and labeling of, only, 96-kDa and 93-kDa polypeptides. Antiserum raised against these polypeptides recognized them in an immunoblot and could be used to remove the enzyme activity from crude extracts. Tryptic peptide profiles were obtained from about 10 pmol of the 96-kDa and 93-kDa polypeptides. The profiles were similar and sequencing allowed construction of mixed probes and identification of a putative single structural gene. Returned to yeast on a multicopy plasmid, phosphofructokinase 2 activity was considerably above the wild-type level, as was polypeptide revealed by immunoblotting.
Amino Acid Sequence↗
Biochemical and histochemical observations on effects of low-level heavy metal load (lead, cadmium) in different organ systems of the freshwater crayfish, Astacus astacus L. (Crustacea: Decapoda).
The effects of low-level lead (20 micrograms/liter) and/or cadmium (2 micrograms/liter) exposure on the structure and function of different organ systems of the freshwater crayfish. Astacus astacus L. (Crustacea: Decapoda) were estimated by several biochemical and histochemical methods. The animals were incubated during 10 weeks (max.) at a temperature of 10 degrees C and a normal diurnal rhythm. Lead accumulated in high amounts especially in the digestive gland, carapax, and gills, whereas the hindgut and musculature exhibited very low lead levels. Cadmium accumulated particularly in the digestive gland and gills. Lead and cadmium levels were definitely lower in the digestive gland, gills, and carapax of animals incubated in water containing a double, i.e., lead and cadmium load, than in animals kept in water containing only one of these heavy metals. Histochemically both metals could be visualized in a typical distribution within the tissues, such as the carapax, digestive gland, or gills. After several weeks of poisoning, all organs, but especially the digestive gland, showed severe structural impairment. The activities of oxidative enzymes in the digestive gland and gills were significantly lowered after 2 weeks of incubation. Enzyme histochemical evaluation demonstrated changes of reaction intensities within the organs as compared to the controls. GSH S-transferase activities and GSH contents were also distinctly decreased following lead and/or cadmium intoxification. The histochemical demonstration of SH and S-S groups exhibited a stronger staining reaction after 10 weeks of exposure, especially in digestive gland and gills. The results obtained are discussed in view of the specific impairment of function of the organ systems studied, as related to the typical biology of the animal species tested.
Animals↗
[Multi-disciplinary cooperation: about identity and various stakes].
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Health Occupations↗
6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase from Saccharomyces cerevisiae.
In permeabilized yeast cells 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase are studied during growth. It is shown that in yeast at least two fructose 2,6-bisphosphate degrading enzyme activities occur, differing in pH profile and in their substrate affinities. The activities of 6-phosphofructo-2-kinase and of fructose-2,6-bisphosphatases drop in the exponential and the transition phase while the activity of the alkaline phosphatases steadily increases. In the stationary phase the activities of 6-phosphofructo-2-kinase and of the low Km fructose-2,6-bisphosphatase increase again. Yeast 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase were purified and separated from each other. The purified 6-phosphofructo-2-kinase was found to exhibit a very high specific activity (1.3 U/mg). The enzyme is efficiently inhibited by ATP. The ATP inhibition is most pronounced at low concentrations of magnesium and fructose-6-phosphate. Phosphoenolpyruvate and sn-glycerol 3-phosphate are inhibitors of the enzyme. The high-affinity yeast fructose-2,6-bisphosphatase releases inorganic phosphate from the 2-position of fructose 2,6-bisphosphate. It displays hyperbolic kinetics towards fructose 2,6-bisphosphate (Km = 0.3 microM) and is strongly inhibited by fructose 6-phosphate. The inhibition is counteracted by sn-glycerol 3-phosphate. The enzyme is shown to be inactivated by cAMP-dependent phosphorylation and reactivated by the action of protein phosphatase 2A.
Cell Membrane Permeability↗
Biochemical parameters in various sections of bovine corpora lutea graviditatis during the course of pregnancy.
The enzyme activities of glutathione peroxidase (GPO) with cumenehydroperoxide (cumene-OOH) and H2O2 as substrates, glutathione-S-transferase (GSH-S-T) with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, phosphofructokinase (PFK) and succinate dehydrogenase (SuDH) were determined for months 1 through 9 of pregnancy in the basal and peripheral sections of the corpora lutea graviditatis of Holstein-Frisean cows. The concentration of reduced glutathione (GSH) was simultaneously measured in these tissue sections. Substantial topographical differences were apparent in the enzyme activities. GPO and GSH-S-T showed activity differences during the course of pregnancy. During the 2nd month of pregnancy, minimal values for the activity of cytoplasmic GPO were observed in the basal areas. The cytoplasmic GPO in the peripheral areas displayed a contrasting dynamic with maximal values during the 6th month. GSH-S-T activities in basal and peripheral tissues appeared similar. GPO activities with H2O2 as substrate, likewise, displayed similar courses of activity in both tissue localizations. SuDH was more active in the peripheral than in the basal area. The activity of PFK displayed just the reverse course. The concentration of GSH in the peripheral area was not higher than in basal area.
Animals↗
Investigations on the influence of copper succinate on the production of superoxide anion radicals by bovine small intestinal mucosa cells.
The effect of an experimental ischemia lasting for 45 minutes and a subsequent period of reperfusion of equal length on the activity of xanthine oxidase (XO) and microsomal NADPH-cytochrome P450 reductase (NADPH-CR) were investigated in the small intestinal mucosa of male neonatal calves of the breed German "Schwarzbunte". The activity of the NADPH-CR was determined by chemiluminescence. The activity of XO decreased during ischemia, but rose to values above the control level following reperfusion. 5 mg of Cu2(succinate)2 (CuSu) administered either intraarterially or intraluminally during reperfusion prevented the rise in XO. Formation of malondialdehyde decreased in the presence of CuSu. The NADPH-CR likewise showed subnormal activity values during ischemia, but also remained at a low level during reperfusion. The activity of this enzyme was further lowered by local intraarterial or intraluminal administration of 5 mg of CuSu and by 120 mg of CuSu administered intravenously during the reperfusion. These results are discussed with regard to the superoxide anion radical induced tissue lesions observed during reperfusion.
Animals↗
Kinetics of 6-phosphofructo-2-kinase from Saccharomyces cerevisiae: inhibition of the enzyme by ATP.
6-Phosphofructo-2-kinase (PFK-2) was purified from yeast and separated from fructose-2,6-biphosphatase (FBPase-2). The purification procedure involved polyethylene glycol fractionation followed by chromatography on DEAE-Sephacel. PFK-2 and FBPase-2 were copurified in these steps. Separation of the two enzymes resulted from Sephacryl S-300 Blue chromatography. Then, PFK-2 was chromatographed on CM-Sephadex and eluted with a gradient of KCl. Finally, PFK-2 was rechromatographed at CM-Sephadex and specifically eluted with fructose 6-phosphate. PFK-2 (specific activity 1.3 U/mg) was purified about 25,000-fold. The enzyme is inhibited by ATP which is particularly pronounced at low concentrations of magnesium and fructose 6-phosphate. Phosphoenolpyruvate and sn-glycerol 3-phosphate are inhibitors of the enzyme.
Adenosine Diphosphate↗
Regional histochemical aspects of xanthine oxidase activity in ischemic and reperfused small intestine of the rat.
The study describes regional changes of xanthine oxidase and succinate dehydrogenase activities as shown by the ischemic and reperfused small intestine of the rat. The results are obtained with enzyme histochemical methods, including densitometrical verifications, and are substantiated with biochemical enzyme determinations. The decrease of xanthine oxidase activity was best visible in the anoxic duodenum and jejunum, where the findings of histochemical enzyme determinations agreed with those achieved biochemically. The activities of succinate dehydrogenase as measured densitometrically may serve as a further control, considering also the typical intracellular distribution of the reaction products.
Animals↗
Smallest zinc quantities affect the histamine release from peritoneal mast cells of the rat.
Seven individual 0.025-mg doses of zinc administered as lactose tablets on consecutive days, significantly increase histamine release from peritoneal mast cells of the rat. Seven individual doses of 0.25 microgram caused a somewhat smaller, though still very pronounced increase in the release in comparison with zero control.
Animals↗