Biomedical subjects
Publications and source records attributed to J E Fischer.
It should be clear that the authors of this article are not enamored of practice guidelines, thinking them of much more potential harm than good. On the other hand, if practice guidelines are deemed essential, then they must be written with the goal of quality patient care advocacy and with an eye toward the possible damage they may do.
It should be clear that the authors of this article are not enamored of practice guidelines, thinking them of much more potential harm than good. On the other hand, if practice guidelines are deemed essential, then they must be written with the goal of quality patient care advocacy and with an eye toward the possible damage they may do.
Although a linkage between aerobic glycolysis and sodium-potassium transport has been demonstrated in diaphragm, vascular smooth muscle, and other cells, it is not known whether this linkage occurs in skeletal muscle generally. Metabolism of intact hind-leg muscles from young rats was studied in vitro under aerobic incubation conditions. When sodium influx into rat extensor digitorum longus (EDL) and soleus muscles was facilitated by the sodium ionophore monensin, muscle weight gain and production of lactate and alanine were markedly stimulated in a dose-dependent manner. Although lactate production rose in both muscles, it was more pronounced in EDL than in soleus. Monensin-induced lactate production was inhibited by ouabain or by incubation in sodium-free medium. Preincubation in potassium-free medium followed by potassium re-addition also stimulated ouabain-inhibitable lactate release. Replacement of glucose in the incubation medium with pyruvate abolished monensin-induced lactate production but exacerbated monensin-induced weight gain. Muscles from septic or endotoxin-treated rats exhibited an increased rate of lactate production in vitro that was partially inhibited by ouabain. Increases muscle lactate production in sepsis may reflect linked increases in activity of the Na+, K+-ATPase, consumption of ATP and stimulation of aerobic glycolysis.
Different aspects of muscle metabolism are altered during sepsis and there is evidence that some of these changes may be regulated at the gene level. Differential display is a recently described technique to identify genes whose expression has changed during a biological process. This technique utilizes reverse transcriptase-polymerase chain reaction (RT-PCR) to compare mRNA signals in tissues during two different conditions. We used differential display to test the hypothesis that gene expression is altered in skeletal muscle during sepsis. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham-operated. Sixteen hours after CLP or sham operation, extensor digitorum longus muscles were harvested and RNA was extracted. Following differential display, 30 fragments (F1-F30) were identified that appeared to be uniquely expressed in muscles from sham-operated or septic rats. These fragments were reamplified by PCR and used as probes in Northern blot analysis. Messenger RNA levels corresponding to 2 of the 30 fragments (F5 and F24) were confirmed to be increased by Northern blot analysis in septic muscle. Following cloning and sequencing, F5 was found to display significant homology to the gene sequence of the guanine nucleotide releasing protein MSS4. The sequence of F24 did not match any reported gene sequence and may therefore represent a previously unidentified gene. The results support the hypothesis that gene expression is altered in skeletal muscle during sepsis.
We have previously shown that [D-Trp(32)]NPY can competitively antagonize NPY-induced feeding in rats (Balasubramaniam et al. J. Med. Chem. 1994, 37, 811-815). This peptide, however, did not bind to SK-N-MC cells with Y-1 receptors. Since centrally truncated NPY analogs have been shown to bind Y-1 receptors, we synthesized similar analogs of [D-Trp(32)]NPY and investigated their Y-1 (SK-N-MC) and Y-2 (SK-N-BE2) receptor affinities and their properties in human erythroleukemia (HEL) cells. None of the analogs with D-Trp(32) mobilized intracellular calcium, [Ca2]i, in HEL cells. Although Des-AA(6-24)[Aoc(6)]NPY and the corresponding D-Trp(32) analog exhibited no affinity to Y-1 receptors, Des-AA(7-24)[Aoc(6),D-Trp(32)] NPY(6) exhibited weak binding. Replacing Pro(5) in 6 with D-Ala to stabilize the central chain reversal, and hence the antiparallel alignment of the N- and C-terminal regions known to be important for Y-1 binding, resulted in an analog, Des-AA(7-24)[D-Ala(5),Aoc(6),D-Trp(32)]NPY (7), which exhibited moderate antagonist potency in attenuating NPY effects on cAMP and [Ca2+]i, in SK-N-MC and HEL cells, respectively. This analog also shifted the dose-response curve of NPY on blood pressure in anesthetized rats. Deletion of only the 7-17 and/or the incorporation of N-Me-Ala(5), superior beta-turn stabilizer, in 7 did not improve the Y-1 receptor affinity. Des-AA(7-24)[D-Ala(5), Gly(6),D-Trp(32)]NPY exhibited an affinity similar to that of 7, suggesting that a long spacer arm is not necessary for efficient Y-1 receptor interaction. Locking the antiparallel alignment via a 2/26 or 2/27 lactam bridge did not improve the binding. Finally, replacement of D-Ala(5) in 7 with D-Trp dramatically increased both the binding and the antagonistic potencies. Modeling based on the avian pancreatic polypeptide X-ray structure suggested that analogs which have the N- and C-terminal regions in close proximity might exhibit good binding, and that the D-Trp(32) substitution may induce a beta-turn that could be important for exhibiting antagonism. A systematic investigation has resulted in the development of relatively potent Y-1 receptor antagonists. Further structure-activity studies with these compounds and those previously reported by us and other investigators should result in the development of long-acting and receptor selective antagonists.
Explore the source record for details and available documents.
Recent studies suggest that sepsis-induced increase in muscle proteolysis mainly reflects energy-ubiquitin-dependent protein breakdown. We tested the hypothesis that glucocorticoids activate the energy-ubiquitin-dependent proteolytic pathway in skeletal muscle during sepsis. Rats underwent induction of sepsis by cecal ligation and puncture or were sham-operated and muscle protein breakdown rates were measured 16 h later. The glucocorticoid receptor antagonist RU 38486 or vehicle was administered to groups of septic and sham-operated rats. In other experiments, dexamethasone (2.5 or 10 mg/kg) was injected subcutaneously in normal rats. Total and myofibrillar proteolysis was determined in incubated extensor digitorum longus muscles as release of tyrosine and 3-methylhistidine, respectively. Energy-dependent proteolysis was determined in incubated muscles depleted of energy with 2-deoxyglucose and 2,4-dinitrophenol. Levels of muscle ubiquitin mRNA and free and conjugated ubiquitin were determined by Northern and Western blot, respectively. RU 38486 inhibited the sepsis-induced increase in total and myofibrillar energy-dependent protein breakdown rates and blunted the increase in ubiquitin mRNA levels and free ubiquitin. Some, but not all, sepsis-induced changes in ubiquitin protein conjugates were inhibited by RU 38486. Injection of dexamethasone in normal rats increased energy-dependent proteolysis and ubiquitin mRNA levels. The results suggest that glucocorticoids regulate the energy-ubiquitin-dependent proteolytic pathway in skeletal muscle during sepsis.
BACKGROUND: Muscle wasting and negative nitrogen balance are common findings in septic patients. It is not clear what signals this loss of body protein. Proteins modified by reactive oxygen species have been shown to be rapidly degraded. OBJECTIVE: To test the hypothesis that sepsis results in an increased amount of oxidatively damaged proteins in skeletal muscle. METHODS: Exposure of proteins to reactive oxygen species results in the incorporation of carbonyl groups into amino acids with metal binding sites. The formation of carbonyl group derivatives in sarcoplasmic and myofibrillar proteins was measured in the extensor digitorum longus and soleus muscles of septic rats 4 to 48 hours after cecal ligation and puncture and in control rats that underwent sham operation. RESULTS: Protein carbonyl content was increased 8 and 16 hours after cecal ligation and puncture in the extensor digitorum longus and soleus muscles, respectively. When muscles were incubated in vitro, the carbonyl content in protein decreased in muscles from septic rats but not in muscles from rats that had the sham operation. The loss of carbonyl groups in incubated septic muscles occurred also in energy-depleted muscles. CONCLUSIONS: Muscle proteins are oxidatively damaged during sepsis and an energy-independent proteolytic pathway participates in the degradation of these proteins. Damage to muscle proteins by reactive oxygen species may signal the selective removal of postsynthetically modified proteins, contributing to accelerated muscle protein degradation in sepsis.
Maintaining rats on TPN for 7 days was associated with a 50% reduction in gut mass and protein content. Co-infusing PYY with total parenteral nutrition (TPN) resulted in significant savings in jejunal wet mass and elevated protein content of jejunum, ileum and colon as compared with rats maintained on TPN alone. No significant effects of PYY on plasma amino acid profile were noted. Although minor alterations in mucosal polyamines were observed in rats maintained on TPN, co-infusion of PYY had no significant effect on gut polyamine concentrations. These results suggest that PYY has trophic effects upon the gut during otherwise catabolic conditions. Therefore, co-infusion of PYY with TPN may suggest methods whereby loss of intestinal mucosa and atrophy-associated complications of TPN may be modulated.
Amylin dose-dependently stimulated the secretion of amylase and cholesterol esterase from rat pancreatic acinar AR42J cells. The biochemical basis of this action was investigated using fura-2-loaded AR42J cells. Amylin increased intracellular free calcium. [Ca2+]i, in a dose-dependent manner. The Ca2+ signal persisted even in Ca(2+)-free medium, suggesting mobilization from intracellular stores rather than influx. Consistently, thapsigargin abolished amylin-induced responses, suggesting that Ca2+ is released from an inositol 1,4,5-triphosphate (IP3)-sensitive pool. This was confirmed by the finding that amylin elevated IP3 levels. AR42J cells pretreated with amylin did not respond to amylin, suggesting that the receptors mediating this response undergo homologous desensitization. However, pretreatment with related peptides, calcitonin gene-related peptide (CGRP) and salmon calcitonin, did not diminish [Ca2+]i mobilization by amylin. CGRP and calcitonin also failed to mobilize [Ca2+]i even at 10 microM. These results suggests that the stimulatory effects of amylin on pancreatic digestive enzyme secretion from AR42J cells are mediated by a G-protein-linked membrane receptor coupled to IP3-dependent calcium pools.
Tumor-bearing rats exhibited significant decreases in 1- to 4-h intake of rat chow following the intrahypothalamic injection of 2 micrograms neuropeptide Y (NPY). This refractory feeding response was present prior to the onset of anorexia and became more severe as anorexia worsened. The constant infusion of NPY (125 ng/h) into the perifornical hypothalamus of TB and control rats elicited increased feeding for only 2 days. Because chromatography revealed minipump NPY to be intact after 10 infusion days, downregulation of NPY receptors may have occurred. Daily injection of increasing doses of NPY stimulated ad lib feeding in non-TB rats, while having no effect on TB rats. Desensitization to NPY-induced feeding following daily injections of the peptide was suggested by the loss of feeding response to a dose (500 ng) of NPY that increased food intake prior to the daily NPY treatments. These results suggest that hypothalamic NPY feeding systems are refractory in TB rats, even before they exhibit anorexia. In addition, a rapid loss of the feeding response occurred in rats with constant infusion of NPY into hypothalamic tissue or with daily intrahypothalamic injections of the peptide, suggesting possible NPY receptor-mediated alterations. Therefore, control of obesity or anorexia through NPY feeding mechanisms may prove difficult due to rapid compensatory receptor changes.
We examined the influence of sepsis and endotoxemia in rats on the biosynthesis of polyamines in small-intestinal mucosa. Sepsis was induced by cecal ligation and puncture (CLP); control rats were sham-operated. In other experiments, rats were treated with two subcutaneous injections of endotoxin (1 mg/kg) or corresponding injections of sterile saline. Ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) activities and concentrations of putrescine, spermidine, and spermine were measured in jejunal mucosa at intervals during 16 hours. Sepsis stimulated ODC and SAMDC activities and increased putrescine and spermidine concentrations in jejunal mucosa. Injection of endotoxin resulted in metabolic changes similar to those observed following CLP. The results suggest that sepsis and endotoxemia stimulate polyamine biosynthesis in mucosa of small intestine. The role of polyamines in the regulation of cell proliferation and metabolic changes in the intestinal mucosa during sepsis remains to be determined.
Enterocutaneous and aortoenteric fistulas arise from a diverse array of pathophysiologic states. Classification by anatomic, physiologic, and etiologic systems is critical to both nonoperative and operative treatment planning.
We have previously reported that [D-Trp32]NPY and its centrally truncated analogues such as des-AA7-24[D-Trp5,32,Aoc6]NPY can competitively antagonize NPY effects on rat hypothalamus and Y1 (SK-N-MC AND HEL) cells, respectively. In continuation of this work, we performed structure-activity studies with C-terminal decapeptide sequence keeping D-Trp at position 32 to develop lower molecular weight Y1-selective antagonists. This study led to the development of des-Asn29[D-Trp28,32]NPY(27-36), which bound to both Y1 (SK-N-MC, Ki > or = 10 microM) and Y2 (SK-N-BE2, Ki = 1.01 +/- 0.03 microM) receptors. This peptide did not exhibit any agonist activity at Y1 receptors, and exhibited comparable potencies in antagonizing the effects of NPY on the synthesis of cAMP and mobilization of [Ca2+]i in HEL cells. However, in SK-N-MC cells, it was more potent in antagonizing the mobilization of [Ca2+]i than inhibition of cAMP synthesis. Substitution of Nva for Gln34 to increase the hydrophobicity without altering the carbon skeleton substantially increased Y1 affinity (Ki = 0.33 +/- 0.15 microM) and imparted Y1 selectivity (Ki for Y2 affinity = 3.16 +/- 0.50). Moreover, this peptide exhibited good antagonistic potency in HEL cells. 2D NMR studies of des-Asn29[D-Trp28,32]NPY(27-36) revealed the existence of a fairly stable loop-like structure between residues 27 and 32 and a less stable one between residues 32 and 36. The increased Y1 affinity of des-Asn29[D-Trp28,32,Nva34]NPY(27-36) may be due to the stabilization of the 32-36 loop by Nva34. It appears therefore that stabilization of the loop structures in these peptides should result in the development of more potent Y1 receptor antagonists. Our investigations also suggest that HEL cells express a homogeneous population of NPY Y1 receptors whereas SK-N-MC cells express high- and low-affinity Y1 receptors coupled to Ca2+ and cAMP, respectively.
Tumor-bearing (TB) and control rats were maintained for four to seven days on total parenteral nutrition (TPN) in which the lipid component (11.1% of total calories) was Intralipid or fish oil. Although no TB rats maintained on standard Intralipid-based TPN died prematurely in these experiments, one-third of the TB rats maintained on fish oil-based TPN died of bleeding complications by Day 4 of TPN infusion. The bleeding appeared to be due to the particle size in the fish oil emulsion, because a subsequent study employing a better emulsified TPN with smaller lipid particles exhibited fewer bleeding problems. Tumor growth, skeletal muscle atrophy, and gut hypoplasia were not reduced in the TB group maintained on fish oil-based TPN. However, immunosuppression observed in the Intralipid-based TPN group, as measured by mitogen response of cultured splenocytes, was significantly improved in the rats maintained on fish oil-based TPN. Levels of eicosapentaenoic acid were reduced in all rats receiving TPN. Although the hypothesized antitumor and anticachexia effects of fish oil administration were not realized, significant improvement in immunosuppression was noted. These results suggest that intravenously administered fish oil may have potential positive effects for the host.
Recent studies suggest that sepsis stimulates mucosal polyamine and protein synthesis. It is not known in which cell type polyamine biosynthesis is increased during sepsis and if polyamines regulate mucosal protein synthesis. We examined the effect of sepsis in rats on polyamine biosynthesis in isolated jejunal enterocytes and measured mucosal protein synthesis following inhibition of ornithine decarboxylase (ODC) activity with difluoromethylornithine. ODC and S-adenosylmethionine decarboxylase (SAMDC) activities and putrescine concentrations were increased in isolated jejunal enterocytes 16 h after induction of sepsis by cecal ligation and puncture. Enterocyte spermidine and spermine levels were not influenced by sepsis. Mucosal ODC and SAMDC activities and polyamine levels were increased following treatment of rats with interleukin-1 but not tumor necrosis factor. Treatment of rats with difluoromethylornithine prevented the sepsis-induced increase in mucosal ODC activity, putrescine concentration, and protein synthesis rate. The results suggest that sepsis increases ODC and SAMDC activities and putrescine concentrations in enterocytes of the small intestine. This metabolic response to sepsis may be regulated by interleukin-1 although other mechanisms may also be involved. Increased mucosal protein synthesis during sepsis may at least in part be regulated by increased putrescine levels.
Explore the source record for details and available documents.
Total parenteral nutrition remains a vital medical intervention, and in our institution it is considered as basic as intravenous fluids, antibiotics, and blood transfusions. As is true of most treatments, there are specific indications as well as associated risks and costs which mandate justification of its use. It is clear that the indications for TPN are diminishing as basic science and clinical studies continue to find increased benefits associated with enteral feeding, and as techniques for initiating enteral nutrition improve. Because of unproven benefits and/or increased complication rates, TPN has lost favor in the treatment of conditions that were previously thought to require parenteral alimentation, such as acute pancreatitis, pediatric and adult burns, critical care, and preoperative use in patients with mild or moderate malnutrition. Despite diminishing indications, TPN continues to generate excitement in some areas as its immunological effects become better defined. The use of TPN in patients with cancer before certain therapies, as well as in the transplant population, remains hopeful. New uses of TPN will result from a better understanding of the cellular and molecular effects of parenteral feeding. In the future, TPN may well be used as a pharmacologic agent rather than as nutritional intervention.