Transient expression of beta-galactosidase in differentiating sporozoites of Eimeria tenella

A cocktail of seven Listeria monocytogenes isolates of food, human and environmental origin was used to assess the antilisterial activity of the bacteriocins nisin and ALTA 2341 in combination with various atmospheres: air, 100% N2, 40% CO2:60% N2, or 100% CO2. Buffered tryptone soya broth (pH 6.0) was used as the growth medium and incubation was at 4 degrees C (21 days) or 12 degrees C (7 days), or when temperature fluctuated between these values for defined periods. It was observed that atmosphere alone influenced the growth rate of L. monocytogenes, with 100% CO2 exerting the greatest inhibition. A 5 log population increase was observed in all atmospheres after 7 days at 12 degrees C. At 4 degrees C a 4-5 log population increase was observed in air, 100% N2 and 40% CO2:60% N2 within 21 days. Growth was prevented by 100% CO2. In the presence of nisin (400 IU/ml), an increase in the lag phase was observed before growth (5 log population increase after 7 days) in all atmospheres at 12 degrees C. This effect was enhanced at 4 degrees C where a maximum 2 log population increase was observed in all atmospheres except 100% CO2, in which growth was prevented. Increasing the concentration of nisin to 1250 IU/ml prevented L. monocytogenes growth in all atmosphere combinations at 4 and 12 degrees C. Two concentrations of ALTA 2341 were also tested. In the presence of 0.1% ALTA 2341 and at 12 degrees C, a 3-5 log population increase was observed in all atmospheres with the exception of 100% CO2, which prevented L. monocytogenes growth. At 4 degrees C, growth was observed in the combination of 0.1% ALTA 2341 and 100% N2 only (3 log population increase). Use of a higher concentration of ALTA 2341 (1.0%) resulted in a population decrease below the detection level within 24 h in all atmosphere/temperature combinations. Re-growth occurred in the presence of 1.0% ALTA 2341 in all atmospheres at 12 degrees C, and in combination with air or 100% N2 at 4 C. When the effectiveness of either nisin or ALTA 2341 and atmosphere was tested against L. monocytogenes as temperature fluctuated for periods between 4 and 12 degrees C, only the combination of 100% CO2 and 1.0% ALTA 2341 prevented growth. Cells surviving exposure to nisin or ALTA 2341 were recovered from 28 of the 32 combinations tested that contained bacteriocin. Nisin survivors remained sensitive to the bacteriocin. ALTA 2341 survivors had become resistant to the bacteriocin.     

The solubility of the liquid oxysulfide phase in liquid Fe-O-S alloys

The solubility of the liquid oxide phase in liquid Fe-O alloys has been measured for the temperature range of 1378 to 1740 °C. Also the solubility of the liquid oxysulfide phase in liquid Fe-O-S alloys has been determined for the composition range of 0.08 to 0.30 wt pct oxygen and 0 to 0.5 wt pct sulfur. The oxygen content of liquid iron saturated with the liquid oxide phase is log O = ?6358/T + 2.76. The standard free energy for the formation of the oxide phase is: xFe(l) + O(pct) = Fe_xO(l); ΔG° = 242.4 ? 0.0829T + 166,990/T(kJ). The equation for the standard free energy in the temperature range of 1550 to 1650 °C may be written as: ?117.5 + 0.0496T (kJ). The effect of composition on temperature of saturation of liquid Fe-O-S alloys with the oxysulfide phase is:T(K) = ?6358/(log pct O ? 2.76) - (pct S)x [554 + 135.0(log O ? 2.77)]. The relationship applies for the composition range of 0.15 to 0.30 wt pct oxygen and 0.0 to 0.5 wt pct sulfur and temperatures from 1480 to 1680 °C.     

Expression of vascular endothelial growth factor gene and its receptor (flt-1) gene in urinary bladder cancer

Factors affecting the ability of Escherichia coli O157:H7 to survive in foods with a(w) less than required for growth have not been fully defined. This study was undertaken to determine the ability of E. coli O157:H7 to survive in a commercial dry infant rice cereal as affected by a(w) (0.35+/-0.04, 0.52+/-0.03 and 0.73+/-0.03), pH (4.0 and 6.8), and temperature (5, 25, 35 and 45 degrees C), and in nine other reduced-a(w) foods. Death of E. coli O157:H7 in cereal was enhanced with increased temperature and decreased pH during a 16- to 24-week storage period. Survival was enhanced at pH 6.8 compared to pH 4.0 in cereal at a(w) 0.34+/-0.04 during initial storage at 5 and 25 degrees C. The effect of temperature (8, 15, 21 and 30 degrees C) on survival and growth of acid-adapted cells of E. coli O157:H7 inoculated into cereal reconstituted with milk or apple juice at two inoculum levels (8.2-12.3 cfu/ml and 82-123 cfu/ml of slurry) was also studied. Growth occurred in cereal reconstituted with milk at all test temperatures and in cereal reconstituted with apple juice at 15, 21 and 30 degrees C. Populations increased by >1 log10 cfu/ml within 3-6 h at 21 and 30 degrees C. Acid-adapted and unadapted cells had similar growth patterns. The effects of temperature and acid adaptation on survival of E. coli O157:H7 in nine commercial foods and food ingredients with pH 4.07-6.49 and a(w) 0.17-0.82 were determined. The pathogen survived in these foods for various lengths of time, depending the storage temperature, with an order of survival of 5 degrees C >21 degrees C >37 degrees C. Survival appeared to be enhanced in foods with highest pH, and acid-adapted cells retained higher viability than unadapted cells in only two of the nine test foods. Of particular importance is the ability of E. coli O157:H7 to survive well in dry foods with a wide range in a(w) and pH, particularly at refrigeration temperature.     

Effectiveness of salt, pH, and diacetyl as inhibitors for Escherichia coli O157:H7 in dairy foods stored at refrigeration temperatures

The behavior of Escherichia coli O157:H7 inoculated in 10% rehydrated nonfat dry milk adjusted to pH levels between 3.8 and 5.4 with lactic acid, salt levels of 0 to 6%, and diacetyl levels of 0, 5, and 10 micrograms/g was determined at 4 and 12 degrees C. Cell populations were determined by surface plating on tryptic soy agar after 7 and 35 days of incubation. Survival was also determined using retail cultured diary products. E. coli O157:H7 did not survive in skim milk at pH 3.8 and was reduced by 3 log cycles at pH 4.1, regardless of salt, diacetyl, and temperature levels. At pH levels above 4.4, survival was observed at lower salt concentrations for up to 35 days at both 12 and 4 degrees C. The organism grew (up to a 2.2-log increase) at pH 5.0 at 2% salt levels after 35 days of storage at 12 degrees C. Diacetyl at a concentration of 10 ppm had no effect on survival and growth. In all but one case, E. coli O157:H7 was inactivated in yogurt, sour cream, and buttermilk at a rate similar to or greater than what was consistent with the acidified skim milk data. Also consistent with the skim milk data, growth occurred in two of the three cottage cheese samples at 12 degrees C after 7 days but not after 35 days or at 4 degrees C, when a 1- to 2-log decline was observed.     

Long-term bacterial profile of refrigerated ground beef made from carcass tissue, experimentally contaminated with pathogens and spoilage bacteria after hot water, alkaline, or organic acid washes

The effects of 2% (vol/vol) lactic acid (LA), 2% (vol/vol) acetic acid (AA), 12% (wt/vol) trisodium phosphate (TSP), 72 degrees C water (HW), and 32 degrees C water (W) washes on bacterial populations which were introduced onto beef carcass surfaces after wash treatments were determined up to 21 days of storage at 4 degrees C of packaged ground beef prepared from the treated and inoculated carcasses. Beef carcass necks were collected from cattle immediately after harvest and subjected to the above treatments or left untreated (control). Neck meat was then inoculated with low levels (ca. <2 log10) of Listeria innocua, Salmonella typhimurium, Escherichia coli O157:H7, and Clostridium sporogenes contained in a bovine fecal cocktail. In general, growth of these four bacteria, aerobic bacteria, lactic acid bacteria, and pseudomonads was suppressed or not observed in the ground beef when LA, AA, or TSP treatments were used as compared to the untreated control. HW or W washes offered little suppression of growth of pathogens during subsequent storage of ground beef when these bacteria were introduced onto beef tissue posttreatment. Of the treatments used, a final LA or AA wash during the processing of beef carcasses offers the best residual efficacy for suppression of pathogen proliferation in ground beef during long-term refrigerated storage or short-term abusive temperature storage if these bacteria contaminate the carcass immediately after carcass processing.     

cAMP-dependent protein kinase from brown adipose tissue: temperature effects on kinetic properties and enzyme role in hibernating ground squirrels

Arousal from hibernation requires thermogenesis in brown adipose tissue, a process that is stimulated by beta-adrenergic signals, leading to a rise in intracellular 3',5'-cyclic adenosine monophosphate AMP (cAMP) and activating cAMP-dependent protein kinase A (PKA) to phosphorylate a suite of target proteins and activate lipolysis and uncoupled respiration. To determine whether specific adaptations (perhaps temperature-dependent) facilitate PKA kinetic properties or protein-phosphorylating ability, the catalytic subunit of PKA (PKAc) from interscapular brown adipose of the ground squirrel Spermophilus richardsonii, was purified (final specific activity = 279 nmol phosphate transferred per min per mg protein) and characterized. Physical properties of PKAc included a molecular weight of 41 kDa and an isoelectric point of 7.8 +/- 0.08. A change in assay temperature from a euthermic value (37 degrees C) to one typical of hibernating body temperature (5 degrees C) had numerous significant effects on ground squirrel PKAc including: (a) pH optimum rose from 6.8 at 37 degrees C to 8.7 at 5 degrees C, (b) K(m) values at 37 degrees C for Mg.ATP (49.2 +/- 3.4 microM) and for two phosphate acceptors, Kemptide (50.0 +/- 5.5 microM) and Histone IIA (0.41 +/- 0.05 mg/ml) decreased by 53%, 80% and 51%, respectively, at 5 degrees C, and (c) inhibition by KCl, NaCl and NH4Cl was reduced. However, temperature change had little or no effect on K(m) values of rabbit PKAc, suggesting a specific positive thermal modulation of the hibernator enzyme. Arrhenius plots also differed for the two enzymes; ground squirrel PKAc showed a break in the Arrhenius relationship at 9 degrees C and activation energies that were 29.1 +/- 1.0 kJ/mol for temperatures > 9 degrees C and 2.3-fold higher at 68.1 +/- 2.1 kJ/mol for temperatures < 9 degrees C, whereas the rabbit enzyme showed a breakpoint at 17 degrees C with a 13-fold higher activation energy over the lower temperature range. However, fluorescence analysis of PKAc in the absence of substrates, showed a linear change in fluorescence intensity and wavelength of maximal fluorescence over the entire temperature range; this suggested that the protein conformational change indicated by the break in the Arrhenius plot was substrate-related. Temperature change also affected the Hill coefficient for cAMP dissociation of the ground squirrel PKA holoenzyme which rose from 1.12 +/- 0.18 at 37 degrees C to 2.19 +/- 0.07 at 5 degrees C, making the release of catalytic subunits at low temperature much more responsive to small changes in cAMP levels. Analysis of PKAc function via in vitro incubations of extracts of ground squirrel brown adipose with 32P-ATP + cAMP in the presence versus absence of a PKA inhibitor, also revealed major differences in the patterns of phosphoproteins, both between euthermic and hibernating animals as well as between 37 and 5 degrees C incubation temperatures; this suggests that there are both different targets of PKAc phosphorylation in the hibernating animal and that temperature affects the capacity of PKAc to phosphorylate different targets. Both of these observations, plus the species-specific and temperature-dependent changes in ground squirrel PKAc kinetic properties, suggest differential control of the enzyme in vivo at euthermic versus hibernating body temperatures in a manner that would facilitate a rapid and large activation of the enzyme during arousal from torpor.     

Physical properties of ceramides: effect of fatty acid hydroxylation

The structural and thermotropic properties of alpha-hydroxy fatty acid (HFA) and non-hydroxy fatty acid (NFA) ceramides (CER) have been studied using differential scanning calorimetry (DSC) and X-ray diffraction techniques. The DSC of anhydrous HFA-CER shows a single, sharp reversible transition at 95.6 degrees C (delta H = 15.3 kcal/mol). At intermediate hydrations HFA-CER exhibited more complex behavior but at maximum hydration only a single reversible transition is observed at 80.0 degrees C (delta H = 8.5 kcal/mol). X-ray diffraction of hydrated (74% water) HFA-CER at 20 degrees C shows a lamellar structure with a bilayer periodicity d = 60.7 Angstrum; a single wide angle reflection at 4.2 Angstrum is characteristic of hexagonal chain packing. Above the main transition temperature at 91 degrees C, a hexagonal (HII) phase is observed. In contrast, DSC of anhydrous NFA-CER demonstrates two thermal transitions at 81.3 degrees C (delta H = 6.8 kcal/mol) and 85.9 degrees C (delta H = 3.5 kcal/mol). With increasing hydration, both transitions shift towards lower temperatures; at maximum hydration, on heating, the endothermic transitions occur at 72.7 degrees C (delta H = 9.8 kcal/mol) and 81.1 degrees C (delta H = 4.0 kcal/mol). On cooling, there is hysteresis of both transitions. X-ray diffraction of NFA-CER (80% water) at 20 degrees C shows a well-ordered lamellar structure with a bilayer periodicity d = 58.6 Angstrum and three wide-angle reflections at 4.6 Angstrum, 4.2 Angstrum, and 3.8 Angstrum. At 77 degrees C (between the two transitions), again a lamellar structure exists with reduced bilayer periodicity d = 53.1 Angstrum and four wide-angle reflections at 4.6 Angstrum, 4.2 Angstrum, and 3.8 Angstrum are observed. Above the second transition, only a single low angle reflection at 30.0 Angstrum is observed; a diffuse reflection at 4.6 Angstrum is indicative of a melted chain phase. Thus, HFA-CER exhibits a simple phase behavior involving the reversible conversion of a gel phase to a hexagonal phase (L beta-->HII). However, NFA-CER shows a more complex polymorphic phase behavior involving two gel phases.     

Phase transitions of rat stratum corneum lipids by an electron paramagnetic resonance study and relationship of phase states to drug penetration

In order to relate barrier function to stratum corneum structure and the thermal transitions of corneum lipids, samples from hairless rat skin were investigated by using ESR and drug penetration techniques. The phase transition of stratum corneum lipids was estimated using a deeper probe (16-doxyl-stearic acid) inserted in the lipid bilayers and measuring the rotational correlation time, tau(c). Results of ESR study showed that stratum corneum lipids underwent thermal transitions at 39.3 +/- 1.6 degrees C and 63.6 +/- 2.6 degrees C roughly similar to the data obtained by differential scanning calorimetry measurements. Cholesterol oxidase treatment decreased the fluidity of the lipids at lower temperatures. The treatment of stratum corneum with laurocapram (1%) and isopropyl myristate (IPM, 2%) little changed both phase transition temperatures, although the treatment highly increased the molecular motion of the lipids. The flux (J(s)) of lipophilic drugs (beta-estradiol, indomethacin and betahistine) through the skin was enhanced with increasing temperatures, with an increase in the diffusion constant within skin and a decrease in the lag time. There was a good relationship between log J(s) or log permeability coefficient (K(p)) and 1/tau(c) in the temperature range of 45 to 64 degrees C. The calculated activation energy (delta E) for diffusion of these drugs across skin was 17-40 kcal/mol. Judging from our data, stratum corneum lipids of rat probably exist as the gel, crystalline state below 39 degrees C, the mesomorphic state between 39 and 64 degrees C and the fluid, liquid-crystalline state at temperatures of 64 degrees C or above. These results are in line with the permeability of these lipophilic drugs through the intercellular lipids disordered is highly increased.     

A novel parameterization scheme for energy equations and its use to calculate the structure of protein molecules

A generalized, weighted, nonlinear least squares procedure is developed, based on pH titration data, for the refinement of octanol-water partition coefficients (log P) and ionization constants (pKa) of multiprotic substances. Ion-pair partition reactions, self-association reactions forming oligomers, and formations of mixed-substance complexes can be treated with this procedure. The procedure allows for CO2 corrections in instances where the base titrant may have CO2 as an impurity. Optionally, the substance purity and the titrant strength may be treated as adjustable parameters. The partial differentiation in the Gauss-Newton refinement procedure is based on newly derived analytical expressions. The new procedure was experimentally demonstrated with benzoic acid, 1-benzylimidazole, (+/-)-propranolol, and mellitic acid (benzenehexacarboxylic acid, AH6). Ionic strength (l) was adjusted with KNO3. Benzoic acid (20 degrees C; l 0.1 M): pKa = 3.99 +/- 0.02, log P = 1.96 +/- 0.02, log P (anion) = -1.2; 1-benzylimidazole (25 degrees C; l 0.1 M): pKa = 6.70 +/- 0.03, log P = 1.60 +/- 0.04; propranolol (25 degrees C; l 0.1 M): pKa = 9.53 +/- 0.06, log P = 3.35 +/- 0.03, log P (cation) = 0.62 +/- 0.08; mellitic acid (26 degrees C; l 0.2 M): pKas 1.10 +/- 0.46, 1.69 +/- 0.03, 2.75 +/- 0.02, 4.00 +/- 0.02, 5.05 +/- 0.01, and 6.04 +/- 0.02; in the presence of 0.01 M n-Bu4NBr, log P (AH6) = 1.5, log P (AH5-) = 1.1, log P (AH4(2-)) = 0.8, log P (AH3(3-)) = 0.3, log P (AH2(4-)) = -0.1, and log P (AH5-) = -0.5 (all +/- 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)     

The survival and growth of acid-adapted mesophilic pathogens that contaminate meat after lactic acid decontamination

Lactic acid decontamination (LAD) may adapt pathogens to lactic acid. Such organisms may have an increased resistance to acid and can contaminate meat after LAD. The survival and growth of acid adapted Campylobacter jejuni, Salmonella typhimurium. Escherichia coli O157:H7 and Staphylococcus aureus inoculated on skin surface of still warm pork belly cuts 2 h after LAD was examined during chilled (4 degrees C) storage and refrigeration abuse equivalent to 12.5 degrees C. Lactic acid decontamination included dipping in 1, 2 or 5% lactic acid solutions at 55 degrees C for 120 s. Lactic acid decontamination brought sharp reductions in meat surface pH, but these recovered with time after LAD at approximately 1-1.5 pH units below that of water-treated controls. A sharp decrease in the number of cfu of pathogens occurred on chilled 2-5% lactic acid treated pork belly cuts when the skin surface was less than pH 4.8-5.2. The reductions ranged from 0.1-0.3 log10 cfu cm-2 for E. coli O157:H7 to over 1.7-2.4 log10 cfu cm-2 for Camp. jejuni, respectively. Increase in storage temperature from 4 to 12.5 degrees C reduced delayed decrease in numbers of all pathogens except Camp. jejuni by a factor of two. Deaths in Camp. jejuni at 12.5 degrees C slightly exceeded those at 4 degrees C. After the initial sharp decline, the number of cfu of mesophilic pathogens decreased gradually at a rate similar to that on water-treated controls. Growth of all mesophilic pathogens except Camp. jejuni on 2-5% LAD meat occurred during storage at 12.5 degrees C when the meat surface pH exceeded 4.8-5.2, and was slower than on water-treated controls. Low temperature and acid-adapted E. coli O157:H7, Salm. typhimurium and Staph. aureus, and acid adapted Camp. jejuni that contaminate skin surface after hot 2-5% LAD, did not cause an increased health hazard, although microbiota and intrinsic parameters (lactic acid content, pH) were created that could advantage their survival and growth.     

Effects of GABAB receptor antagonism on the development of pentylenetetrazol-induced kindling in mice

In our previous studies, the yeast Endomyces fibuliger LU677 was found to degrade amygdalin in bitter apricot seeds. The present investigation shows that E. fibuliger LU677 produces extracellular beta-glycosidase activity when grown in malt extract broth (MEB). Growth was very good at 25 degrees C and 30 degrees C and slightly less at 35 degrees C. When grown in MEB of pH 5 and pH 6 with addition of 0, 10 or 100 ppm amygdalin, E. fibuliger produced only slightly more biomass at pH 5, and was only slightly inhibited in the presence of amygdalin. Approximately, 60% of the added amygdalin was degraded (fastest at 35 degrees C) during an incubation period of 5 days. Supernatants of cultures grown at 25 degrees C and pH 6 for 5 days were tested for the effects of pH and temperature on activity (using amygdalin, linamarin and prunasin as substrates). Prunase activity had two pH optima (pH 4 and pH 6), amygdalase and linamarase only one each at pH 6 and pH 4-5 respectively. The linamarase activity evolved earlier than amygdalase (2 days and 4 days respectively). The data thus indicate the presence of at least two different glycosidases having different pH optima and kinetics of excretion. In the presence of amygdalin, lower glycosidase activities were generally produced. However, the amygdalin was degraded from the start of the growth, strongly indicating an uptake of amygdalin by the cells. The temperature optimum for all activities was at 40 degrees C. Activities of amygdalase (assayed at pH 4) and linamarase (at pH 6) evolving during the growth of E. fibuliger were generally higher in cultures grown at 25 degrees C and 30 degrees C. TLC analysis of amygdalin degradation products show a two-stage sequential mechanism as follows: (1) amygdalin to prunasin and (2) prunasin to cyanohydrin.     

Predictive models of the effect of temperature, pH and acetic and lactic acids on the growth of Listeria monocytogenes

The combined effect of temperature (1-20 degrees C), pH (4.5-7.2) and acetic acid (0-10,000 mg/l; model 1) or lactic acid (0-20,000 mg/l; model 2) on growth of Listeria monocytogenes in laboratory media was studied. Growth curves at various combinations of temperature, pH and acid concentration were fitted by the model of Baranyi and Roberts (1994), and specific growth rates derived from the curve fit were modelled. Predictions of growth from the models were compared with data in the literature, and this showed the models to be suitable for use in predicting growth of L. monocytogenes in a range of foods including meat, poultry, fish, egg and milk and dairy products. The two models are compatible, i.e. they give similar predictions for cases when no acid is present.     

Influence of acetic acid on the growth of Escherichia coli K12 during high-cell-density cultivation in a dialysis reactor

High-cell-density cultivations of Escherichia coli K12 in a dialysis reactor with controlled levels of dissolved oxygen were carried out with different carbon sources: glucose and glycerol. Extremely high cell concentrations of 190 g/l and 180 g/l dry cell weight were obtained in glucose medium and in glycerol medium respectively. Different behaviour was observed in the formation of acetic acid in these cultivations. In glucose medium, acetic acid was formed during the earlier phase of cultivation. However, in glycerol medium, acetic acid formation started later and was particularly rapid at the end of the cultivation. In order to estimate the influence of acetic acid during these high-cell-density cultivations, the inhibitory effect of acetic acid on cell growth was investigated under different culture conditions. It was found that the inhibition of cell growth by acetic acid in the fermentor was much less than that in a shaker culture. On the basis of the results obtained in these investigations of the inhibitory effect of acetic acid, and the mathematical predictions of cell growth in a dialysis reactor, the influence of acetic acid on high-cell-density cultivation is discussed.     

Heterogeneity of plasma glucagon. Circulating components in normal subjects and patients with chronic renal failure

Plasma immunoreactive glucagon (IRG) concentrations were measured in 36 patients with chronic renal failure (CRF) and 32 normal subjects. In addition, the components of circulating IRG were analyzed by gel filtration in the fasting state and after physiological stimuli. Fasting IRG was elevated (P less than 0.001) in CRF patients (534 +/- 32 pg/ml) compared with the levels found in healthy subjects (113 +/- 9 pg/ml). Oral glucose suppressed plasma IRG in CRF patients from a basal level of 568 +/- 52 to a nadir of 354 +/- 57 pg/ml (120 min). This degree of suppression (38%) was comparable to that found in normal subjects (basal = 154 +/- 20 to 100 +/- 23 pg/ml) at 120 min (35%). Intravenous infusion of arginine (250 mg/kg) resulted in a 71% rise in IRG in CRF patients and a 166% increase in normal subjects. Gel filtration of fasting plasma from CRF patients showed three major peaks. The earliest (A) was found in the void volume (mol wt greater than 40,000) and constituted 16.5 +/- 4.7% of the elution profile. The middle peak (B) eluted just beyond the proinsulin marker (approximately 9,000 mol wt) and constituted the largest proportion of the elution profile (56.5 +/- 3.4%). The third peak (C) coincided with the standard glucagon and [125I]glucagon markers (3,485 mol wt) and comprised 27.0 +/- 4% of the IRG profile. In contrast, only peaks A and C were found in fasting plasma of normal subjects (53.6 +/- 10.4% in A and 46.4 +/- 10.4 in C). After oral glucose, glucagon immunoreactivity in the 3,500 mol wt peak (C) was markedly suppressed, while the B peak in patients with CRF declined to a lesser extent. The A peak in both groups was unchanged. After an arginine infusion only the C peak increased in both groups of subjects. Gel filtration of plasma in 3 M acetic acid gave similar profiles to those obtained in glycine albumin buffer. Exposure of serum to trypsin indicated that the B and C peaks were digestible, while the A peak was resistant to the action of the enzyme. In one sample, peak C increased after a 2-h exposure of serum to trypsin. We conclude that circulating IRG in normal subjects and patients with CRF is heterogenous. The hyperglucagonemia of renal failure is largely due to an increase in IRG material of approximately 9,000 mol wt, consistent with proglucagon, although the 3,500 mol wt component is also considerably elevated (threefold). The significance of circulating IRG levels should be interpreted with caution until the relative biological activity of the three components is established.     

Purification and characterization of extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus. Purine nucleoside phosphorylase activity and evidence for intersubunit disulfide bonds

5'-Methylthioadenosine phosphorylase from Sulfolobus solfataricus, a thermoacidophilic archaeon optimally growing at 87 degrees C, has been purified to homogeneity. Reducing agents are not required for catalytic activity. The enzyme has a molecular mass of 160 kDa and is composed of six apparently identical subunits of 27 kDa. The NH2-terminal sequence shows high homology (50%) with the NH2-terminal sequence of Escherichia coli purine nucleoside phosphorylase. Physicochemical and kinetic features are reported. 5'-Methylthioadenosine phosphorylase is highly thermophilic, with an optimum temperature of 120 degrees C. The enzyme is characterized by extreme thermal stability, remaining completely active after 2 h at 100 degrees C and showing half-inactivation times of 15 and 5 min when incubated at 130 and 140 degrees C, respectively. An apparent melting temperature of 132 degrees C has been calculated. After 24 h of incubation at room temperature no loss of activity is detected in the presence of 9 M urea, 4 M guanidine hydrochloride, 0.075% SDS, 50% methanol, 50% ethanol, 50% dimethylformamide, 1 M NaCl, and 1% Triton X-100. Data are also reported on the enzyme's resistance to proteolysis and on the effect of salts, detergents, solvents, and reducing agents on enzyme thermostability. Labeling experiments with iodo[2-14C]acetic acid resulted in the incorporation of approximately 12 mol of labeled iodoacetate/mol of protein, indicating the presence of six disulfide bonds that, on the basis of SDS-polyacrylamide gel electrophoresis, are probably positioned intersubunits, resulting in the organization of the enzyme into two trimers. 5'-Methylthioadenosine (MTA) phosphorylase is endowed with a broad substrate specificity, being able to phosphorolytically cleave inosine, guanosine, and adenosine with a better efficiency than MTA, allowing us to hypothesize that in S. solfataricus the same enzyme is responsible for the catabolism of MTA and of these purine nucleosides.     

Small bowel rejection in isolated small bowel transplantation and in multivisceral transplantation: a comparative study in a large animal model

31P NMR lineshapes of multilamellar liposomes composed mostly of a bilayer-spanning tetraether lipid are consistent with rapid axially symmetric motion about the bilayer normal. The residual chemical shift anisotropy of 36 ppm is comparable to that seen for diacylphosphatidylglycerol systems and suggests comparable headgroup motion. The lateral diffusion rates for Thermoplasma acidophilum total polar lipids in mutilamellar liposomes was measured by two dimensional exchange NMR as a function of temperature. At 55 degrees C, near the growth temperature, the rate of lateral diffusion, DL, is comparable to that of diester phospholipids in the Lalpha liquid crystalline phase, having a value of 2 x 10(-8) cm2/s. DL decreases with temperature reaching a value of 8-6 x 10(-9) cm2/s at 30 degrees C. The activation energy Ea for lateral diffusion is estimated to be 10 kcal/mol (approximately 42 kJ/mol). The lateral diffusion rates indicate that the tetraether liposomes have a membrane viscosity at 30 degrees C which is considerably higher than that of diester phospholipids in the liquid crystalline phase.     

Cloned ligand-gated channels activated by extracellular ATP (P2X receptors)

The temperature ranges for growth of Streptococcus mutans GS-5 and S. sobrinus 6715 were found to be very narrow, from about 30 to 47 degrees C, with optimal growth around 37 degrees C. Thus, the organisms showed little potential to grow in the environment outside of the animal host. In contrast wider ranges were found for Enterococcus hirae, S. rattus and S. sanguis. Detailed study of S. mutans GS-5 showed that energetic coupling, reflected in yields of biomass per mol of glucose utilized, were not greatly affected by changes in temperature within the growth range. However, since glycolysis occurred over a wider temperature range (about 10 to 52 degrees C) than growth, yield values dropped to zero at temperatures above or below the growth range. The temperature range for glycolysis could be related to temperature sensitivity of the phosphoenolypyruvate: sugar phosphotransferase system for sugar uptake. F-ATPases were active over a similar range of temperatures, but with a broad optimal range from about 30 to 50 degrees C. Proton permeability of S. mutans increased steadily with temperature in a manner similar to that of other mesophilic bacteria, such as Escherichia coli. Growth of the bacteria in media supplemented with various fatty acids had major effects on proton permeabilities but the effects were not well reflected by changes in growth or glycolysis of the bacteria. The overall conclusions were that S. mutans is a typical mesophile in relation to membrane and catabolic functions but its narrow temperature range for growth is related to temperature sensitivities of anabolic systems.     

Fractal analysis of photolysis of nitrosyl haemoglobin at low temperatures

In Mycobacterium phlei, fatty acid unsaturation increased with decreasing temperature. The 10-hexadecenoic acid content increased as the temperature was reduced from 35 degrees C to 26-20 degrees C. At lower temperatures tuberculostearic acid decreased while oleic and linoleic acids increased, the latter being found in M. phlei for the first time. Concomitantly palmitic acid content decreased, and the 6- and 9-hexadecenoic acids increased slightly on reducing the temperature from 35 to 10 degrees C. Thus, down to 26-20 degrees C palmitic acid was mainly replaced by 10-hexadecenoic acid. From this range down to 10 degrees C, palmitic and tuberculostearic acids were replaced by oleic and linoleic acids. Consequently, fatty acid branching decreased and mean chain length increased, as the temperature was reduced. These observations support the view that regulation of membrane fatty acid composition is part of microbial temperature adaptation, and that the mechanism behind the responses might be more complex than generally believed.     

Modification of venous stasis thrombosis in the rat by platelet-active drugs and by heparin

Incubation of adenovirus type 2 infected cells at 42 degrees C resulted in an inhibition of assembly of virus particles although all the major viral structural polypeptides and virus-induced cellular polypeptides so far identified were detected by electrophoretic analysis. Selective high salt-acid-urea extraction of low mol. wt. polypeptides revealed the absence of protein VII at 42 degrees C whereas precursor polypeptide P-VII and core protein V were found. Pulse-chase and temperature shift experiments indicated that cleavage of P-VII into VII was a reversible thermosensitive process, requiring de novo protein synthesis after shift-down to 37 degrees C. Virus particles assembled at 37 degrees C after transfer from 42 to 37 degrees C contained both viral DNA and polypeptides pre-labelled during the eclipse phase at 42 degrees C, including core protein VII.     

Neonatal exposure to bFGF exerts NGF-like effects on mouse behavioral development

Metabolic products secreted by the fungal mycelia of Hirsutella thompsonii var. thompsonii (CBS 556.77D) in a defined culture broth in shake culture were tested for toxicity to Galleria mellonella larvae and Drosophila melanogaster adults via injection and per os application, respectively. In addition, the toxic effect of broth filtrate was observed in vitro in a cell line of Bombyx mori. Czapek-Dox broth fortified with 1% yeast extract stimulated more rapid mycelial growth and correspondingly more toxin production in time. At 25-30 degrees C, metabolic toxin(s) was detected in broth via bioassay at about 4-5 days postinoculation when mycelial biomass reached 5 mg/ml (dry wt). At these temperatures, biological activity of the filtrate peaked at about 8-10 days when mycelial growth reached a maximum (10 mg/ml, dry wt). This suggests a positive relationship between toxic metabolite and mycelial production. After 10 days, the toxicity of the filtrate appeared to decline gradually. Pathogenicity symptoms of the metabolites developed slowly in both G. mellonella and D. melanogaster. Early signs of lethargy appeared at 4 days postinjection and cumulative mortality of G. mellonella larvae was low after 1 week; however, the percentage of mortality reached 98-100% after 14 days. At death, G. mellonella larvae displayed small dark spots on a brownish cuticle. Histopathological effects were observed in the larval midgut, malpighian tubules, hypodermis, fat body, hemocytes, muscle, and silk glands. Cellular change consisted of pycnosis of the nucleus and a reduction in cytoplasm density. Highest mortality (78.8%) to adult D. melanogaster occurred after 10 days post-treatment.(ABSTRACT TRUNCATED AT 250 WORDS)