Role of intracellular pH in muscle fatigue

Intracellular pH of in vitro diaphragm preparations was determined following low- (5 Hz, 1.5 min) and high- (75 Hz, 1 min) frequency stimulation, using glass microelectrodes of the liquid membrane type (pHm). Results were compared with values obtained by the standard homogenate technique (pHh). High- and low-frequency stimulation reduced peak tetanic tension to 21 +/- 1 (SE) and 71 +/- 2% of initial values, respectively. Peak tetanic tension returned to resting values after 10- to 15-min recovery from high- or low-frequency stimulation. Resting pHm was 7.063 +/- 0.011 (n = 72), and after fatiguing stimulation declined to values as low as 6.33. During recovery pHm significantly increased and by 10 min had returned to prefatigue values. No difference was observed in the recovery of pHm between the low- and high-frequency stimulation groups (analysis of variance test, ANOVA), and in both groups pHm recovery was highly correlated to the recovery of peak tetanic tension (r = 0.94, P less than 0.001). Resting pHh was 7.219 +/- 0.023 (n = 13), which was significantly higher than the pHm value. In contrast to pHm, intracellular pHh was significantly higher during recovery from 75- vs. 5-Hz stimulation (P less than 0.05). For both groups pHh increased significantly with time and by 10 min returned to prestimulation values. The ANOVA test demonstrated that pHh values were significantly higher than pHm values during recovery from fatigue. The results from this study support our hypothesis that fatigue from both high- and low-frequency stimulation is at least partially due to the deleterious effects of intracellular acidosis on excitation-contraction coupling.     

Denaturation of covalently closed circular DNA. Kinetics, comparison of several DNAs, mechanism and ionic effects

The rates of the alkaline denaturation of the covalently closed, circular DNAs (form I) of the replicative forms (RF) of phages G4, phi X174, and fd, and of plasmid pBR322 and phage PM2 have been measured at 0 degrees C and some at higher temperatures. These rates are orders of magnitude slower than the denaturation of linear DNA because of the increased stability of the helix to deprotonation that results from the accumulating positive superhelicity during denaturation. Denaturation reactions were initiated by rapid, infrasonic mixing (Camien, M.N., and Warner, R.C. (1984) Anal. Biochem. 138, 329-334), and their progress was measured by analytical ultracentrifugal analysis for the amounts of form I and denatured (Id) DNA after neutralization of the alkaline reaction. The comparative rates of the five DNAs varied over a wide range; the fastest, G4-RF, denatured at 500-fold the rate of the slowest, fd-RF. The differences are accounted for by the interaction of positive superhelicity with the sequence-dependent regions of relative helix stability in the various DNAs. Renaturation rates of Id DNAs varied similarly for Ids prepared at 0 degrees C, but only a few-fold for Ids prepared at 50 degrees C. The rate of denaturation of G4-RF was determined over a wide range of NaOH and NaCl concentrations at 0 degrees C, and the pHm was determined as a function of ionic strength and temperature. The effects of ionic strength have been analyzed in an application of the Manning ion condensation-screening theory (Manning, G.S. (1978) Q. Rev. Biophys. 11, 179-246) which is shown to account for the large destablizing effect of salts on the helix. The pH region of transition at 50 degrees C from renaturation to denaturation was examined, and it was found that the maximum rate of renaturation occurred at a pH about 0.05 units below the pHm.     

Application of high-field 1H-NMR spectroscopy for the study of perifused amphibian and excised mammalian muscles

Frog sartorius and gastrocnemius muscles were perifused at 20 degrees C, the intracellular pH (pHi) and the concentration of phosphocreatine were determined in the resting muscle by 1H-NMR spectroscopy at 470 MHz; values of pHi = 7.31 +/- 0.05 (n = 7) and concentration of phosphocreatine = 20.4 +/- 1.1 mumol/g wet wt. (n = 6) were found. The hydrolysis of phosphocreatine and the simultaneous increase in lactate upon perifusion with 10 mM caffeine (in Ringer's solution) was followed with a time resolution of 1 min. Lactate increased at a rate of 1.0 mumol/g per min, but no pHi change was recorded during the time monitored. The lower limit for the buffering capacity of the muscle cytosol was estimated to be 16.7 mumol/g muscle per pH unit from the uncertainty in pHi determination (+/- 0.03 pH units) and from the amount of lactate produced and phosphocreatine hydrolyzed. Changes in pHi, lactate concentration and fatty acyl chain intensity were monitored by 1H-NMR spectroscopy at 361 MHz in ischemic rat skeletal muscle, excised and stored at 20 degrees C. The resonances in the 1H-NMR spectrum of a human skeletal muscle perchloric acid extract are reported and tentatively assigned.     

Transient States of Adenylate Cyclase in Brain Membranes

Basal activity of adenylate cyclase from the amygdala of sheep brain and the neostriatum of turkey brain decays in two phases at 37 degrees C. The first phase is rapid (t1/2 = 2.3 +/- 0.3 min) and results in the loss of 60-70% of basal activity. The second phase is slow (t1/2 approximately 100 min) during which time the catalytic units denature irreversibly. The GTP analogue guanosine-5' (beta-gamma imino) triphosphate (p[NH]ppG) prevents the rapid decay by stabilizing the enzyme at its initial level of activity and also reactivates the enzyme to initial levels during or immediately following the early phase, indicating that denaturation of neither the guanylnucleotide units nor the catalytic units causes the rapid decline in basal activity. Activation by p[NH]ppG is rapid at 37 degrees C, but the binding of p[NH]ppG to the guanylnucleotide subunit also occurs at nonactivatory temperatures. This is determined by the protection of catalytic units from thermal or N-ethylmaleimide inactivation after extensive washing. Thus, at 25 degrees C all of the catalytic units can be stabilized by saturating p[NH]ppG concentrations. At 0 degree C, 35% of the catalytic units can be stabilized by saturating p[NH]ppG concentrations within 30 s. The half-saturation constant for the binding of p[NH]ppG at 0 degree C is identical to that derived in an assay at 37 degrees C, or after an incubation of the membranes for 10 min at 45 degrees C, when the process of thermal denaturation is 80% complete (K1/2 approximately 3 +/- 2 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Endocytosis and shedding of the decay accelerating factor on human polymorphonuclear cells

The decay-accelerating factor (DAF) is a cell membrane glycoprotein that functions in the control of C activation. We studied the modulation of membrane DAF on polymorphonuclear cells (PMN) by using anti-DAF antibodies. Fluorescence-activated cell sorter analysis showed that DAF expression was reduced by 43 +/- 7% on resting or stimulated cells that were held at 37 degrees C for 30 min when compared with those kept on ice. Most of this reduction occurred within the first 15 min, and was followed by a gradual further decrease in surface DAF. PMN that were held at 37 degrees C for varying periods of time before DAF measurement had a gradual decrease suggestive of release of DAF from the PMN membrane or endocytosis. To examine the latter, PMN were reacted with anti-DAF at 0 degree C, followed by 125I-Fab'2 secondary antibodies at either 0 degree C or 37 degrees C, and subsequently treated with pronase. Thirty +/- 11% of the 125I remained bound to cells kept at 37 degrees C compared to 2% in those held at 0 degrees C. Internalization was further confirmed by electron microscopy. In PMN that were not exposed to pronase, 26 +/- 2% of the surface-associated 125I was released at 37 degrees C compared with 7% at 0 degrees C. Immunoprecipitation and SDS-PAGE of surface-labeled PMN showed that the temperature-dependent released DAF had a lower m.w. than membrane DAF. Immunofluorescent studies revealed that 37 degrees C mediated the redistribution of DAF from a homogeneous pattern into caps. These results show that under the conditions studied DAF is partially internalized and partially released from the PMN membrane to the fluid phase; the latter may contribute to the presence of DAF in body fluids.     

Skeletal muscle glycogenolysis, glycolysis, and pH during electrical stimulation in men

Glycogenolytic and glycolytic rates were estimated and muscle pH (pHm) was measured in electrically stimulated quadriceps femoris muscles of seven men. Leg blood flow was occluded and muscles were stimulated 64 times at 20 Hz, with contractions lasting 1.6 s and separated by pauses of 1.6 s. Muscle biopsies were obtained at rest and following 16, 32, 48, and 64 contractions. Glycolytic intermediates and several modulators of the glycolytic enzyme phosphofructokinase (PFK) were measured. Glycogenolytic and glycolytic rates were 1.68 and 1.26 mmol glucosyl units X kg dry muscle-1 X S-1 contraction time during the initial 16 contractions and pHm decreased from 7.00 +/- 0.01 to 6.70 +/- 0.03. During the subsequent 32 contractions both glycogenolytic and glycolytic rates were maintained at approximately 0.70 mmol X kg-1 X S-1 and pHm decreased to 6.45 +/- 0.04. In the final 16 contractions, both rates were very low and pHm was unchanged. Therefore, PFK remained active despite increasing acidity until pHm decreased to approximately 6.45. We conclude that increases in the concentrations of several positive modulators partially reverses pH-dependent ATP inhibition of PFK in vivo, permitting glycolytic activity to continue in the pHm range of 6.70-6.45.     

Isolation and characteristics of scallop sarcoplasmic reticulum with calcium transport activity.

Sarcoplasmic reticulum with calcium transport activity has been isolated from the cross-striated adductor muscle of the scallop, which lives in cold (< or = 20 degrees C) sea water, by using pH 7.0 buffer solution both to homogenize the tissue and to sediment the membrane fraction. The yield of the preparation was 60-100 mg protein from 100 g of the scallop muscle. Ca(2+)-activated ATPase protein of about 100 kDa accounted for 40-50% of the protein preparation. The maximum activities of ATP-dependent, oxalate-facilitated calcium accumulation and Ca(2+)-ATPase were observed at a pH of about 7.0 and temperature of 20-30 degrees C, and their values were about 2 mumol Ca2+/mg of protein/min and about 3 mumol ATP hydrolysis/mg of protein/min, respectively. At 0 degree C, 10-20% of these activities was maintained, while at 37 degrees C, the activities were irreversibly lost. The Ca(2+)-ATPase activity was half-maximally activated at about 0.3 microM [Ca2+]. The ATPase activity exhibited non-Michaelian behavior with respect to ATP, with two different Km values of approximately 10 microM and 0.1-0.3 mM. GTP, CTP, and ITP were also hydrolyzed by the preparation at a rate of 10-30% of that of ATP. The preparation was stored at -80 degrees C with retention of function for about a year.     

Receptor-mediated internalization of high density lipoprotein by rat sinusoidal liver cells: identification of a nonlysosomal endocytic pathway by fluorescence-labeled ligand

Rat sinusoidal liver cells possess the surface receptor for high density lipoprotein (HDL) (Murakami, M., S. Horiuchi, K. Takata, and Y. Morino. 1987. J. Biochem. (Tokyo) 101: 729-741). The present study was undertaken to determine whether cell surface-bound HDL underwent subsequent endocytic internalization by using 125I-labeled HDL and fluorescein isothiocyanate-labeled HDL (FITC-HDL). The cell-associated radioactivity obtained by a 40-min incubation with 125I-labeled HDL at 37 degrees C was released into the medium as acid-precipitable forms upon further incubation at 37 degrees C. When further incubated at 0 degree C instead of 37 degrees C, however, this release was significantly reduced. A similar phenomenon was observed after the cell-associated ligands had been treated with trypsin. The cell-associated ligands obtained after a 1-hr incubation with 125I-labeled HDL at 0 degree C were largely counted for by those bound to the outer surface of the cells, thus suggesting that HDL is internalized into cells at 37 degrees C but not at 0 degree C. Moreover, when cells were incubated with FITC-HDL at 0 degree C, the cell-associated ligands were found in a pH 7.2 +/- 0.1 compartment, whereas when incubated at 37 degrees C, its microenvironmental pH became much more acidic, exhibiting pH 6.2 +/- 0.1. Furthermore, this value returned to 7.1 +/- 0.1 upon treatment with carbonylcyanide m-chlorophenylhydrazone known to dissipate the total protonomotive force. These results suggest, therefore, that the internalization process does follow receptor-mediated binding of HDL in rat sinusoidal liver cells. This notion was also supported by fluorescence microscopic observations.     

Angiotensin converting enzyme: substrate inhibition

Phosphate, borate, and Tris inhibit angiotensin converting enzyme (ACE), but HEPES buffer is inert. Measurements of substrate inhibition were made in HEPES buffer at pH 7.0 and 25 degrees C and 37 degrees C. Substrate inhibition was marked and goes to completion. A new equation for substrate inhibitions enables one, under favorable circumstances, to determine whether there is cooperativity in the binding of substrate to the inhibitory and active sites. Cooperativity does occur with ACE using Hipp-His-Leu as substrate. The kinetic parameters were measured (Km = 0.21 mM, K* = 0.65 mM at 37 degrees C). The enzyme concentration (1.94 X 10(-8) M) was determined by titration with lisinopril so that kcat (5 X 10(3) at 37 degrees C) could be determined. Using this value and the molecular weight the specific activity of ACE was calculated for different common buffers. The specific activity in HEPES calculated from Vmax was 33.7 units/mg at 37 degrees C.     

Bioconversion of hemicellulose: aspects of hemicellulase production by Trichoderma reesei QM 9414 and enzymic saccharification of hemicellulose

The growth of Trichoderma reesei QM9414 in shake flasks at 28 degrees C on hemicellulose substrates and bagasse resulted in rather low yields of hemicellulolytic enzymes (1.0-1.5 units/mL xylanase and 0.05-0.08 units/mL beta-xylosidase). The influence of pH on the synthesis of beta-xylosidase was greater than on the synthesis of xylanase. Both xylanase and beta-xylosidase showed optimal activity at pH 4-5 and 55-60 degrees C. Xylanase was stable at pH 2-10 but was heat labile and totally inactivated after 1 h at 65 degrees C. Enzyme stability towards heat could be increased in the presence of bovine serum albumin. The beta-xylosidase was more tolerant to heat, but stable over a pH range 2.5-6.0. The D-xylose inhibited both enzymes in a competitive manner. Hemicellulose (heteroxylan) was degraded to the extent of 30-40%within 24 h. The degree of hydrolysis decreased as the substrate concentration increased and increased with increased amounts of enzyme. Multiple enzyme doses resulted in increased saccharification in reduced times. The degree of hydrolysis was influenced by the amount of beta-xylosidase present in the hemicellulolytic enzyme preparation. The -;xylosidase was demonstrated to play an important role in the overall conversion of heteroxylan into xylose that is analogous to the role of beta-glucosidase in the saccharification of cellulose by cellulases.     

Temperature and pH dependence of energy balance by (31)P- and (1)H-MRS in anaerobic frog muscle

The temperature (T)-dependence of energy consumption of resting anaerobic frog gastrocnemii exposed to different, changing electrochemical gradients was assessed. To this aim, the rate of ATP resynthesis (delta approximately P/deltat) was determined by (31)P- and (1)H-MRS as the sum of the rates of PCr hydrolysis (delta[PCr]/deltat) and of anaerobic glycolysis (delta[La]/ deltat, based on a approximately P/La ratio of 1.5). The investigated T levels were 15, 20 and 25 degrees C, whereas initial extracellular pH (pHe) values were 7.9, 7.3 and 7.0, i.e. higher, equal or lower, respectively, than intracellular pH (pHi). The latter was changing with T according to the neutrality point (dpH/dT=-0.0165 pH units/ degrees C). Both rates of PCr hydrolysis and of lactate accumulation and that of their sum, expressed as delta approximately P/deltat, were highly T-dependent. By contrast, the pHe-dependence of the muscle energy balance was nil or extremely limited at 15 and 20 degrees C, respectively, but remarkable at 25 degrees C (with a depression of the ATP resynthesis rate up to 25% with a decrease of pHe from 7.9 to 7.0). The pHe-dependent reduction of metabolic rate was associated with a down-regulation of anaerobic glycolysis due to reduced activity of ion-transporters controlling acid-base balance and/or to a shift from Na(+)/H(+) to a more efficient Na(+)-dependent Cl(-)/HCO(3)(-) exchanger. Uncoupling of glycogenolysis from P-metabolite concentrations, both as function of T (>or=20 degrees C) and of pHe (相似文献   

Proteolytic anaerobic bacteria from lake sediments of Antarctica

Amongst twenty five proteolytic bacteria isolated from lake sediment samples of Antarctica, six isolates were selected based on SDS PAGE protein profile and zone of hydrolysis on casein agar at 10 degrees C. Most of the cultures were rod shaped and motile with two showing terminal bulging spores. Isolates grew between 5 degrees C to 37 degrees C and protease was induced in the late log, stationary or death phase. Isolate SPA-3 grew maximally at 10 degrees C and SPA-6 at 37 degrees C while others preferred 20 degrees C-30 degrees C for growth. The growth and protease production on casein, skimmed milk, bovine serum albumin and gelatin varied with the isolates. Acetate was the dominant volatile fatty acid (24-66% of total VFA) produced during hydrolysis of protein substrate.     

Calcium sparks in mouse ventricular myocytes at physiological temperature

In cardiac muscle, Ca2+ is released from the sarcoplasmic reticulum (SR) in units called Ca2+ sparks. Ca2+ spark characteristics have been studied almost entirely at room temperature. This study compares characteristics of spontaneous sparks detected with fluo 3 in resting mouse ventricular myocytes at 22 and 37 degrees C. The incidence and frequency of Ca2+ sparks decreased dramatically at 37 degrees C compared with 22 degrees C. Also, spark amplitudes and times to peak were significantly reduced at 37 degrees C. In contrast, spatial width and decay times were unchanged. During field stimulation, peak spatially averaged transients were similar at 22 and 37 degrees C, and experiments with fura 2 demonstrated that diastolic and systolic Ca2+ concentrations were unchanged. However, SR Ca2+ content decreased significantly at 37 degrees C. Restoration of SR Ca2+ by superfusion with 5 mM Ca2+ increased spark frequency but did not reverse the effects of temperature on spark parameters. Thus effects of temperature on spark frequency may reflect changes in SR stores, whereas changes in spark amplitude and rise time may reflect known effects of temperature on ryanodine receptor function.     

Spectrin plus band 4.1 cross-link actin. Regulation by micromolar calcium

A low-salt extract prepared from human erythrocyte membranes forms a solid gel when purified rabbit muscle G- or F-actin is added to it to give a concentration of approximately 1 mg/ml. This extract contains spectrin, actin, band 4.1, band 4.9, hemoglobin, and several minor components. Pellets obtained by centrifugation of the gelled material at 43,000 g for 10 min contain spectrin, actin, band 4.1, and band 4.9. Although extracts that are diluted severalfold do not gel when actin is added to them, the viscosity of the mixtures increases dramatically over that of G-actin alone, extract alone, or F-actin alone at equivalent concentrations. Heat-denatured extract is completely inactive. Under conditions of physiological ionic strength and pH, information of this supramolecular structure is inhibited by raising the free calcium ion concentration to micromolar levels. Low-salt extracts prepared by initial extraction at 37 degrees C (and stored at 0 degree C) gel after actin is added to them only when warmed, whereas extracts prepared by extraction at 0 degree C are active on ice as well as after warming. Preincubation of the 37 degrees C low-salt extract under conditions that favor conversion of spectrin dimer to tetramer greatly enhances gelation activity at 0 degree C. Conversely, preincubation of the 0 degree C low-salt extract under conditions that favor conversion of spectrin tetramer to dimer greatly diminishes gelation activity at 0 degree C. Spectrin dimers or tetramers are purified from the 37 dgrees or 0 degree C low-salt extract by gel filtration at 4 degrees C over Sepharose 4B. The addition of actin to either purified spectrin dimer (at 32 degrees C) or tetramer (at 0 degree C or 32 degrees C) results in relatively small increases in viscosity, whereas the addition of actin to a high-molecular-weight complex (HMW complex) containing spectrin, actin, band 4.1, and band 4.9 results in dramatic, calcium-sensitive increases in viscosity. These viscosities are comparable to those obtained with the 37 degrees or 0 degree C low-salt extracts. The addition of purified band 4.1 to either purified spectrin dimer (at 32 degrees C) or purified spectrin tetramer (at 0 degree C) plus actin results in large increases in viscosity similar to those observed for the HMW complex and the crude extract, which is in agreement with a recent report by E. Ungewickell, P. M. Bennett, R. Calvert, V. Ohanian, and W. B. Gratzer. 1979 Nature (Lond.) 280:811-814. We suggest that this spectrin-actin-band 4.1 gel represents a major structural component of the erythrocyte cytoskeleton.     

Chemical synthesis of polysaccharides. 7. Enzymatic hydrolysis of (1 → 6)-α-DL-glucopyranan (DL-dextran)

Hydrolysis of (1 → 6)-α-DL -glucopyranan (synthetic DL -dextran) by an endo-dextranase from a Penicillium species was examined in an acetate buffer solution (pH 5.3) at 37°C. Three samples of different tacticities (isotactic dyad content, 55, 63, and 72%) were employed with a clinical dextran for comparison. Colorimetric determination of the reducing end units of the saccharides produced during hydrolysis showed that the maximum degrees of hydrolysis based on the D-glucose units, (D.H.)_D, for the DL -dextrans were 21.4, 27.8, and 33.0% in the order of increasing isotacitic dyad content, whereas the (D.H.)_D value for the clinical dextran was 51.9%. A statistical treatment of the enzymatic hydrolysis is proposed to interpret the experimental results.     

Kinetics and time-temperature equivalence of polymer degradation

We studied the hydrolysis kinetics of amorphous polylactide. It was found the hydrolysis rate had a slow-to-fast transition at a certain molecular weight (Mn). This transition was not correlated with the mass loss and water uptake of samples, nor the pH values of testing media. We speculated that this transition was due to the slow diffusion of polymer chain ends. The chain ends did not significantly promote the hydrolysis of samples until their concentrations (approximately 1/Mn) reached a critical value. The degradation tests were also conducted over a temperature range from 37 to 90 degrees C. A time-temperature equivalent relationship of degradation processes was established and a master curve spanning a time range equivalent to 3-5 years at 37 degrees C was constructed. This master curve can be used to predict polymer degradation processes based on accelerated tests. The functional time and disappearance time of degradable polymers were also discussed.     

Kinetics of hyaluronan hydrolysis in acidic solution at various pH values

Hyaluronic acid (HA) was hydrolyzed using varying temperatures (40, 60, and 80 degrees C) and acid concentrations (0.0010, 0.010, 0.10, 0.50, 1.0, and 2.0 M HCl). The degradation process was monitored by determination of weight average molecular weight ( M w) by size-exclusion chromatography with online multiangle laser light scattering, refractive index, and intrinsic viscosity detectors (SEC-MALLS-RI-visc) on samples taken out continuously during the hydrolysis. SEC-MALLS-RI-visc showed that the degradation gave narrow molecular weight distributions with polydispersity indexes ( M w/ M n) of 1.3-1.7. Kinetic plots of 1/ M w versus time gave linear plots showing that acid hydrolysis of HA is a random process and that it follows a first order kinetics. For hydrolysis in HCl at 60 and 80 degrees C, it was shown that the kinetic rate constant ( k h) for the degradation depended linearly on the acid concentration. Further, the dependence of temperature on the hydrolysis in 0.1 M HCl was found to give a linear Arrhenius plot (ln k h vs 1/ T), with an activation energy ( E a) of 137 kJ/mol and Arrhenius constant ( A) of 7.86 x 10 (15) h (-1). (1)H NMR spectroscopy was used to characterize the product of extensive hydrolysis (48 h at 60 degrees C in 0.1 M HCl). No indication of de- N-acetylation of the N-acetyl glucosamine (GlcNAc) units or other byproducts were seen. Additionally, a low molecular weight HA was hydrolyzed in 0.1 M DCl for 4 h at 80 degrees C. It was shown that it was primarily the beta-(1-->4)-linkage between GlcNAc and glucuronic acid (GlcA) that was cleaved during hydrolysis at pH < p K a,GlcA. The dependence of the hydrolysis rate constant was further studied as a function of pH between -0.3 and 5. The degradation was found to be random (linear kinetic plots) over the entire pH range studied. Further, the kinetic rate constant was found to depend linearly on pH in the region -0.3 to 3. Above this pH (around the p K a of HA), the kinetic constant decreased more slowly, probably due to either a change in polymer conformation or due to an increased affinity for protons due to the polymer becoming charged as the GlcA units dissociated.     

Effects of low temperature on contraction in papillary muscles from rabbit, rat, and hedgehog

During hibernation the body temperature may fall to only a few degrees above 0 degree C. The heart of the hedgehog continues to function whereas the hearts of nonhibernating mammals stop beating. The present study was performed to investigate and compare the mechanical responses to hypothermia in rabbits, rats, and hedgehogs. Isometric force was recorded from papillary muscles mounted in an organ bath and effects of hypothermia on the mechanical restitution curve were also compared. A reduction of bath temperature from 35 degrees C caused an increase in peak developed force. Maximum force was seen at 20 degrees C in the rabbit, 15 degrees C in the rat, and 10 degrees C in the hedgehog preparations. In all the species there was a similar prolongation of time to peak force and of time from peak to half-relaxation as temperature was lowered. An increase in resting force and after-contractions were recorded in the rabbit and rat muscles at temperatures below 15 and 10 degrees C, respectively. The rabbit and rat preparations became inexcitable at temperatures below 10 and 5 degrees C, respectively. The hedgehog papillary muscle, on the other hand, still contracted at 0 degree C and did not show increased resting force nor after-contractions. The results are consistent with the hypothesis that there is a calcium overload in cardiac cells from rabbit and rat at low temperatures but there is no calcium overload in the hedgehog muscle during hypothermia.     

Human beta-interferon incubated with muscle homogenate is protected by albumin but not by proteinase inhibitors.

The scarce bioavailability of beta-interferon (IFN-beta) after intramuscular administration is probably due either to the binding of IFN-beta to interstitial matrix, or to lymphatic absorption and/or to local breakdown by lysosomal proteinases from muscle. In this work, we first showed that after intramuscular injection, the apparent bioavailability of natural human IFN-beta is about 10% of that of recombinant IFN-alpha 2 and then we evaluated the effects of proteinase inhibitors and albumin on IFN-beta incubated at 37 degrees C with muscle homogenate. IFN biological activity decreased spontaneously by about 20% after incubation for 6 hr at 37 degrees C in Hanks' solution, but it was almost completely lost after incubation with muscle homogenate. Proteinase inhibitors (alpha 1-antitrypsin, alpha 2-macroglobulin, aprotinin, soybean trypsin inhibitor, leupeptin, EP-459, and EP-475) failed to block the inactivation of IFN-beta by muscle proteinases, whereas albumin exerted a partial but consistent protection.     

Changes in enterococcal population during storage of reconstituted infant food

A six-fold increase in the enterococcal population was observed in reconstituted infant food samples after storage for 2 h at 37 degrees C. The increase in enterococcal counts at 40 degrees C and 45 degrees C was approximately five-fold during the same period. However, the corresponding total viable counts increased by twelve fold at these temperatures after 2 h. After 12 h, the enterococcal and total viable counts increased to 39 x 10(4) and 36 X 10(7) colony forming units per ml, at 37 degrees C respectively. A similar pattern in enterococcal and total bacterial count was observed at 40 degrees and 45 degrees C. TNase was detected in reconstituted infant food samples held at 37 degrees, 40 degrees and 45 degrees C, after 12 h, while pH values declined to 5.0, 5.1 and 5.2, respectively at the above temperatures. From TNase positive samples, an isolate S. faecium IF-100 capable to produce TNase was recovered. Storage of reconstituted infant food samples in the refrigerator (5 degrees C) resulted in a gradual increase in enterococcal population which reached 39 X 10(3) c.f.u. per ml after 12 days. However, TNase was not detected in any of these samples.