High-affinity Ca2+,Mg(2+)-ATPase in plasma membrane-rich preparations from olfactory epithelium of Atlantic salmon.

High-affinity Ca2+,Mg(2+)-ATPase was identified in a plasma membrane-rich fraction of olfactory epithelium from Atlantic salmon (Salmo salar). The enzyme required both Ca2+ and Mg2+ for activation. The apparent Km for Ca2+ was 9.5 nM and Vmax was 0.85 mumol Pi/mg of protein per min. Stimulation by Ca2+ was optimal at 5-100 microM MgCl2. Bovine brain calmodulin had no effect on Ca2+,Mg(2+)-ATPase, even after multiple washes of the membrane preparation with EDTA or EGTA. Endogenous calmodulin was somewhat resistant to removal and could be detected with immunoblotting after multiple washes of the membrane preparation with EDTA or EGTA. This endogenous calmodulin may regulate Ca2+,Mg(2+)-ATPase activity because the activity was inhibited by calmidazolium. Vanadate inhibited Ca2+,Mg(2)-ATPase activity and thapsigargin, a specific inhibitor for Ca2+,Mg(2+)-ATPase of endoplasmic reticulum, had no effect on the enzyme activity. High affinity Ca2+,Mg(2+)-ATPase exists in both ciliary and nonciliary membranes with a similar Km for Ca2+. Ca2+,Mg(2+)-ATPase activity is greater in cilia preparations than in membranes from the deciliated olfactory epithelium. As a putative plasma membrane Ca2+ pump, this high-affinity Ca2+,Mg(2+)-ATPase may play an important role in the regulation of intracellular Ca2+ in olfactory epithelia. In particular, the ciliary membrane may play a prominent role in the removal of Ca2+ from ciliated olfactory receptor cells after odorant stimulation.

Factors influencing postoperative urinary retention in patients undergoing surgery for benign anorectal disease.

One hundred eleven patients who had undergone surgery for benign anorectal disease under spinal anesthesia were studied retrospectively to determine the incidence of postoperative urinary retention requiring catheterization and to assess possible influences on that incidence. The age group and sex of the patients did not affect the rate of retention. However, the use of a long-acting anesthetic agent and the administration of at least 1,000 mL of intravenous fluid perioperatively each produced a significant increase in postoperative urinary retention.

The influence of chlordecone and oestrogen on the secretion of proteinaceous molecules of the mouse uterus.

Ovariectomised mice were treated with sesame seed oil, oestradiol, or one of three different levels of the insecticide chlordecone (1, 1a, 3, 3a, 4, 5, 5a, 6-decachloro-octahydro-1, 3, 4-metheno-2H-cyclobuta[cd] pentalen-2-one). After 9 days of treatment, the uteri were injected with (35)S-methionine and radiolabelled proteins were flushed from uteri 4 h later. Reproductive tract weights and the total amount of radioactivity in uterine flushings were measured. Reproductive tract weights increased significantly only in animals treated with the highest dose of chlordecone tested and oestradiol-treated animals. Trichloroacetic acid (TCA) precipitated radioactivity was increased by all doses of insecticide chlordecone tested but increases were significant only at one level. The greatest stimulation of (35)S-methionine incorporation occurred in ovariectomised animals treated with oestradiol. Radioactive proteins were analysed by fluorography of two-dimensional gels. The fluorograms showed that the proteinaceous uterine secretions, as influenced by chlordecone, were greatly reduced in number as compared with those secreted under the influence of oestradiol. Chlordecone appears to cause the secretion of proteinaceous material from the uteri of mice in a qualitatively different manner than does oestradiol.

Reconstruction of a sagittal band and extensor tendon centralization using a palmaris longus tendon graft.

A technique of delayed extensor tendon reconstruction and centralization using a palmaris longus tendon graft is presented. The tendon graft is woven through the base of the proximal phalanx and up and over the reconstructed extensor tendon to simulate the natural sagittal bands. As described, the technique allows gliding of the extensor tendon while maintaining its vitally important central location.

Overwhelming postsplenectomy sepsis in a patient with burns: a case report and a rational approach to treatment.

Overwhelming postsplenectomy sepsis is a dreaded sequel of splenectomy. The rate of overwhelming sepsis in children after splenectomy for trauma is reported to be 10 to 30 times that of the general population. Episodes of pneumonia, septicemia, and meningitis in adults after a splenectomy are 166 times more common than in the general population. The care of a patient with burns and asplenia presents many unique management challenges to the burn physician. Awareness of the development of overwhelming postsplenectomy sepsis and its most common infecting organisms is crucial. The specific immunologic deficiencies of reduced immunoglobulin production and cell-mediated immunity that exist in patients after a splenectomy may be compounded by burn injury. Specific treatment recommendations for patients with burns and asplenia are lacking. We report a fatal case of overwhelming sepsis in a patient with asplenia and with an 8% total body surface area partial-thickness burn, and we review the pathogenesis of overwhelming postsplenectomy sepsis. We focus on treatment recommendations regarding the use of prophylactic antimicrobials, intravenous immunoglobulin replacement therapy, and pneumococcal polyvalent vaccine to standardize the care of the patient with burns and asplenia and reduce infectious morbidity and deaths.

L-carnitine protects fish against acute ammonia toxicity.

1. Juvenile chinook salmon (Oncorhynchus tshawytscha) were injected intraperitoneally (i.p.) with 0.25 M mannitol followed 1 hr later by an i.p. challenge of ammonium acetate. 2. At 10.75 mmol ammonium acetate/kg body weight, 98% of the fish showed signs of ammonium toxicity and 69% died. 3. Substitution of L-carnitine (10-16 mmol/kg) for mannitol afforded striking protection from the subsequent challenge with ammonium acetate; 67% showed no signs of ammonia toxicity and only 4% died. 4. Of other quaternary amines tested, trimethylamine oxide also afforded protection, but betaine and choline did not.

Smoltification in steelhead trout (Salmo gairdneri): Developmental aspects of plasma constituents.

Selected biochemical parameters were measured in the plasma of both underyearling anadromous steelhead trout (Salmo gairdneri) and underyearling residentS. gairdneri. The analyses were conducted in an effort to determine whether or not there might be changes which could be associated with the parr-smolt transformation of anadromous strains. Plasma NH(+) 4 and plasma Na(+) were assayed and plasma proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE).Ammonia was the only plasma ion to show changes with time that were different between the two strains of fish. Proteins prepared by 2D PAGE exhibited developmental changes in both migratory and nonmigratory fish. Each strain exhibited changes with time and the anadromous fish displayed patterns of proteins that were not observed in the nonanadromous strain. It is possible that certain changes in the protein constituents found in anadromous fish are associated with the processes of smoltification. The data are consistent with the notion that this developmental event occurs over an extended period of time and is not restricted to the spring.The data suggest that there may be some changes that occur in certain plasma constituents of migratory fish beginning in the fall and continuing into the spring. The data also indicate that certain ontogenetic events that are not associated with smoltification can be ascertained by analyses of plasma.

Stimulation of Ca(2+)-regulated olfactory phospholipase C by amino acids.

L-Amino acids are potent olfactory stimuli for Atlantic salmon. A plasma membrane fraction, previously shown to be rich in amino acid binding sites, was prepared from olfactory rosettes of Atlantic salmon (Salmo salar) and utilized to investigate the role of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis in olfactory signal transduction. A cocktail of L-amino acids (Ser, Glu, Lys, and Gly) stimulated PIP2 hydrolysis by phospholipase C (PLC) in a dose-dependent manner with half-maximal stimulation when all amino acids were present at approximately 1 microM. Stimulation of PIP2 hydrolysis by amino acids required GTP gamma S, which alone had no effect on PLC activity. Unlike GTP gamma S, AlF4- and Ca2+ stimulated PIP2 breakdown. Preincubation with 1 mM GDP beta S eliminated the effect of amino acids and AlF4- on PIP2 hydrolysis, suggesting the involvement of G protein regulation. The lack of stimulation by GTP gamma S alone suggested that there was negligible exchange of GTP gamma S for GDP in the absence of odorant. There were no significant effects of amino acids on either adenylate cyclase or guanylate cyclase activities in the membrane preparation under these conditions. The effect of the amino acid cocktail was maximal at 1-10 nM free Ca2+. At or above 100 nM free Ca2+, no effect of amino acids on PIP2 hydrolysis was found. However, between 100 nM and 100 microM, Ca2+ directly stimulated PLC activity in a dose-dependent manner. This stimulation by Ca2+ appeared to be G protein independent because it did not require GTP gamma S and was not inhibited by GDP beta S.(ABSTRACT TRUNCATED AT 250 WORDS)

Atlantic salmon (Salmo salar) fed L-carnitine exhibit altered intermediary metabolism and reduced tissue lipid, but no change in growth rate.

Metabolic evidence was sought to explain the reduced body fat and increased body protein observed in Atlantic salmon fed diets supplemented with L-carnitine. By stimulating fatty acid oxidation, dietary carnitine might increase flux through pyruvate carboxylase and decrease flux through the branched-chain alpha-keto acid dehydrogenase complex, by increasing regulatory ratios of acetyl CoA:free enzyme A (CoA-SH) and ATP:ADP. Such changes could conserve nitrogen by providing more carbon for amino acid biosynthesis and by blocking oxidative loss of the branched-chain amino acids. Consistent with this hypothesis, salmon fed carnitine (23 mmol/kg diet) for 9 wk exhibited greater metabolic rates than cohorts fed a carnitine-free diet (P < 0.05) for the following: 1) 1-[14C] palmitate oxidation by liver cubes (48%) and by isolated hepatocytes (151%), 2) pyruvate-dependent [14 CO2]-fixation by isolated mitochondria (81%), 3) incorporation of 1-[14C] lactate into glucose by liver cubes (120%) and by isolated hepatocytes (210%), and 4) incorporation of [35S]-methionine into the acid-insoluble fraction of liver cubes (59%) and isolated hepatocytes (89%). Hepatic concentrations of seven amino acids, including the branched-chain amino acids, were greater (7-112%), as were the plasma concentrations of three of these (45-130%). However, 230% more enzyme in the mitochondria of carnitine-fed fish, and not a difference in the ratios of acetyl CoA:CoA-SH or ATP:ADP, appeared to account for accelerated flux through pyruvate carboxylase; flux through the dehydrogenase complex was unchanged. These results implicate induction of pyruvate carboxylase (or a reduction in turnover) and enhanced protein synthesis in the mechanism for carnitine-induced changes in gluconeogenesis and nitrogen metabolism.

Hsp70 and a 54 kDa protein (Osp54) are induced in salmon (Salmo salar) in response to hyperosmotic stress.

Hsp70 and a 54 kDa osmotic stress protein (osp54) were induced in isolated tissues of anadromous Atlantic salmon (Salmo salar) upon exposure to hyperosmotic conditions. Incubation of branchial lamellae, hepatic tissue, and erythrocytes in medium supplemented with 200-600 mM NaCl dramatically reduced protein synthesis. Although general protein synthesis remained depressed following transfer of tissues from 450 mM supplemental NaCl to iso-osmotic medium, hsp70 was prominently induced in branchial lamellae and hepatic tissue. Accumulation of hsp70 mRNA and a decrease in actin mRNA suggest preferential upregulation of the hsp70 gene. Induction of osp54 was observed in branchial lamellae and erythrocytes, but not in hepatic tissue, during exposure to 75-125 mM supplemental NaCl. Use of glycerol in place of NaCl to create hyperosmotic conditions stimulated induction of hsp70 in branchial lamellae. Substitution with mannitol resulted in induction of osp54 in both branchial lamellae and erythrocytes. The solute-specific and temporal patterns of response suggest that hsp70 and osp54 might function in concert to restore osmotic homeostasis and renature proteins destabilized or denatured during the early stages of osmotic shock.

L-alanine binding sites and Na+, K(+)-ATPase in cilia and other membrane fractions from olfactory rosettes of Atlantic salmon.

1. Membrane fractions were obtained from homogenates of olfactory rosettes from Atlantic salmon (Salmo salar) or from isolated olfactory cilia and homogenates of deciliated olfactory rosettes. 2. Specific binding of L-[3H]alanine was saturable, high-affinity, and effectively inhibited by L-threonine, L-serine and L-alanine but not by L-lysine or L-glutamic acid. Comparable results were obtained with L-[3H]serine except for the presence of a second, lower affinity, binding site for L-alanine but not L-serine. 3. Specific binding of L-[3H]alanine was inhibited by low concentrations of mercury ion, acidic pH, and high concentrations of cadmium, copper or zinc ions. Aluminum had no effect. 4. Specific binding sites for L-alanine were present in membranes from isolated cilia at a level 2-fold that of membranes prepared from the deciliated rosette. 5. Ouabain sensitive Na+, K(+)-ATPase activity was also determined in cilia preparations. This enzyme was present in cilia at a level approximately 3-fold that of membranes prepared from the deciliated rosette. 6. The results are consistent with the presence of an olfactory alanine receptor in S. salar with binding characteristics similar to those of a variety of other fish species and with a localization on olfactory cilia as well as non-ciliated receptor cell membranes.

Cloning and characterization of salmon hsp90 cDNA: upregulation by thermal and hyperosmotic stress.

Accumulating evidence suggests that glucocorticoids are essential for development of hypoosmoregulatory capacity in salmon during adaptation to seawater. Heat shock protein (hsp)90 has been reported to function in signal transduction and the maturation and affinity of glucocorticoid receptors. We sought to determine whether this hsp might be upregulated by thermal and hyperosmotic stress in salmon, a species that migrates between the freshwater and marine environments. A 2625-bp cDNA cloned from a salmon cDNA library was found to code for a protein of 722 amino acids exhibiting a high degree of identity with zebra fish (92%) and human (89%) hsp90beta. Accumulation of hsp90 mRNA was observed in isolated branchial lamellae incubated under hyperosmotic conditions and in branchial lamellae of salmon exposed to hyperosmotic stress in vivo. In contrast, exposure of kidney to hyperosmotic stress in vitro and in vivo failed to elicit an increase in the quantity of hsp90 mRNA. By way of comparison, accumulation of hsp90 mRNA was observed in both branchial lamellae and kidney tissue subjected to thermal stress in vitro and in vivo. Western blot analyses of proteins isolated from tissues under identical conditions in vitro revealed that the pool of hsp90 increased with thermal stress but not with osmotic stress. The results suggest that accumulation of hsp90 mRNA in response to osmotic stress is unrelated to cellular protein denaturation and that synthesis of hsp90 may be regulated at both the level of transcription and translation.

Binuclear [2Fe-2S] clusters in the Escherichia coli SoxR protein and role of the metal centers in transcription.

SoxR protein of Escherichia coli is activated by superoxide-generating agents or nitric oxide as a powerful transcription activator of the soxS gene, whose product activates approximately 10 other promoters. SoxR contains non-heme iron essential for abortive initiation of transcription in vitro. Here we show that this metal dependence extends to full-length transcription in vitro. In the presence of E. coli sigma 70 RNA polymerase, iron-containing SoxR mediates open complex formation at the soxS promoter, as determined using footprinting with Cu-5-phenyl-1,10-phenanthroline. We investigated the nature of the SoxR iron center by chemical analyses and electron paramagnetic resonance spectroscopy. Dithionite-reduced Fe-SoxR exhibited an almost axial paramagnetic signature with g values of 2.01 and 1.93 observable up to 100 K. These features, together with quantitation of spin, iron, and S2-, and hydrodynamic evidence that SoxR is a homodimer in solution, indicate that (SoxR)2 contains two [2Fe-2S] clusters. Treatment of Fe-SoxR with high concentrations of dithiothreitol caused subtle changes in the visible absorption spectrum and blocked transcriptional activity without generating reduced [2Fe-2S] centers, but was also associated with the loss of iron from the protein. However, lowering the thiol concentration by dilution allowed spontaneous regeneration of active Fe-SoxR.

Cysteine-to-alanine replacements in the Escherichia coli SoxR protein and the role of the [2Fe-2S] centers in transcriptional activation.

The Escherichia coli soxRS regulon activates oxidative stress and antibiotic resistance genes in two transcriptional stages. SoxR protein becomes activated in cells exposed to excess superoxide or nitric oxide and then stimulates transcription of the soxS gene, whose product in turn activates>/=10 regulon promoters. Purified SoxR protein is a homodimer containing a pair of [2Fe-2S] centers essential for soxS transcription in vitro . The [2Fe-2S] centers are thought to be anchored by a C-terminal cluster of four cysteine residues in SoxR. Here we analyze mutant SoxR derivatives with individual cysteines replaced by alanine residues (Cys-->Ala). The mutant proteins in cell-free extracts bound the soxS promoter with wild-type affinity, but upon purification lacked Fe or detectable transcriptional activity for soxS in vitro . Electron paramagnetic resonance measurements in vivo indicated that the Cys-->Ala proteins lacked the [2Fe-2S] centers seen for wild-type SoxR. The Cys-->Ala mutant proteins failed to activate soxS expression in vivo in response to paraquat, a superoxide- generating agent. However, when expressed to approximately 5% of the cell protein, the Cys-->Ala derivatives increased basal soxS transcription 2-4-fold. Overexpression of the Cys119-->Ala mutant protein strongly interfered with soxS activation by wild-type SoxR in response to paraquat. These studies demonstrate the essential role of the [2Fe-2S] centers for SoxR activation in vivo ; the data may also indicate oxidant-independent mechanisms of transcriptional activation by SoxR.

Signal transduction for taurocholic acid in the olfactory system of Atlantic salmon.

Conjugated bile acids such as taurocholic acid (TChA) are potent olfactory stimuli for Atlantic salmon (Salmo salar). A plasma membrane rich fraction was derived from salmon olfactory rosettes and used to investigate TChA signal transduction and receptor binding. In the presence of GTP gamma S, TChA caused dose-dependent stimulation of phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown, half maximal at less than 10(-7) M TChA. Stimulation of PIP2 breakdown by TChA required GTP gamma S, was blocked by GDP beta S, and was mimicked by A1F4-, consistent with a G protein requirement. A1F4- and Ca2+ stimulated breakdown of PIP2, but not phosphatidylcholine, arguing against a non-specific lipase activation. Stimulation of PIP2 breakdown by TChA was maximal at low Ca2+ concentration, < or = 10 nM. Conventional binding analysis with 3H-TChA was inconclusive due to a high degree of non-specific binding and to lack of tissue specificity expected for an olfactory receptor. Analysis of odorant amino acid binding indicated possible interaction of TChA with a putative acidic amino acid receptor but no interaction of TChA with a putative neutral amino acid receptor. We conclude that olfactory discrimination between amino acids and bile acids occurs in part at the receptor level while both classes of odors appear to use the same signal transduction mechanism, G protein mediated activation of phosphoinositide specific phospholipase C (PLC).

Measurement of lung tissue mass, thoracic blood and interstitial volumes by transmission/emission scanning using [99mTc]pertechnetate.

1. We have developed a method for non-invasive measurement of lung tissue mass, thoracic blood and interstitial volumes by a combination of transmission and emission scanning with technetium isotope (99mTc). 2. In a lung model we demonstrated that emission counts could be successfully corrected for attenuation with data obtained by transmission scanning, despite an uneven distribution of radioactivity and attenuation in the model. 3. In dogs we compared regional transthoracic tissue thickness, measured by transmission scanning, and regional 'thickness' of blood measured by transmission/emission scanning with direct gravimetric measurements made post mortem. Scanning and direct measurements correlated significantly. 4. In man we used a [99mTc]pertechnetate (99mTcO4) flood source to obtain antero-posterior transmission scans with a gamma-camera. The thickness of attenuating tissue was estimated in each pixel. Scans were obtained of thoracic blood (by labelling erythrocytes with 99mTcO4) and of interstitium (with 99mTc-labelled diethylenetriaminepenta-acetic acid and subtraction of the blood image). We used a computer program to correct the emission scans for attenuation using the transmission scan derived tissue thickness, pixel by pixel. Finally we took a lateral chest radiograph to estimate chest wall thickness. 5. In normal man lung tissue thickness at hilar level was 3.1 +/- 0.5 cm (n = 8). Thoracic blood thickness increased from the apex downwards in the upright lung, being 1.2 +/- 0.1 cm at the hilar level and 2.0 +/- 0.3 cm at the lung base. Interstitial thickness was 0.8 +/- 0.3 cm at the hilum and 0.85 +/- 0.2 at the base. These values compare well with data in the literature.(ABSTRACT TRUNCATED AT 250 WORDS)