Insulin regulation of pyruvate kinase activity in cultured rat hepatocytes, in the presence of vasopressin, ionophore A23187 or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate.

The short-term interactions of insulin and vasopressin on pyruvate kinase (PK) activity were studied in primary cultures of rat hepatocytes. (1) Vasopressin inhibited PK activity by approx. 30% within 15 s, but activity returned to control values by 5 min. The transient inhibition by vasopressin was mimicked by either 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) or ionophore A23187. (2) Insulin alone transiently inhibited PK activity at 1 min, but stimulated PK activity at 5 and 15 min. (3) Insulin completely antagonized the early inhibition by vasopressin, PMA or A23187 of PK activity at 15 s. (4) Insulin inhibited PK activity in the presence of vasopressin, PMA or A23187 at 5 min. (5) 8-Bromo cyclic AMP inhibited PK activity within 15 s, and this inhibition was maintained for at least 5 min. Insulin did not antagonized the inhibition by the cyclic AMP analogue. These results show that insulin under appropriate conditions can act as an inhibitor or activator of PK.     

Tandem coagulase/thermonuclease agar method for the detection of Staphylococcus aureus.

In optimizing previously reported coagulase agar media to obtain a rapid, reliable, and inexpensive coagulase test agar, variations in plasmas, pH, buffer system, fibrinogen, and fibrinolytic inhibitor were investigated. The agar with the following composition was determined best for the demonstration of coagulase production by Staphylococcus aureus: 25 ml of 15% bovine fibrinogen (fraction I, type I, citrated, Sigma Chemical Co.), 25 ml of rehydrated rabbit plasma (coagulase plasma ethylenediaminetetraacetic acid, Difco), 10.0 mg of soybean trypsin inhibitor (Schwarz/Mann), and 450 ml of brain heart infusion agar (Difco). In additional studies involving 7 different temperatures and 11 heating times, the thermal destruction of microbial nucleases on plate count agar and coagulase test agar was investigated. Heating the plates for 2.5 h at 65 degrees C destroyed all heatlabile nucleases, but not thermonucleases of S. aureus. A tandem agar plate method for the identification of S. aureus was developed. Coagulase and thermonuclease activity of 50 colonies can be detected on a single agar plate. Suspect S. aureus colonies isolated on various selective media are transferred to coagulase test agar, the plates are incubated at 37 degrees C for 18 h, and the coagulase reaction is recorded. The plates are then heated at 65 degrees C for 2.5 h, overlaid with toluidine blue-metachromatic diffusion agar, and reincubated at 37 degrees C for 3 h, and the thermonuclease reaction is recorded. Studies based on 88 enterotoxigenic S. aureus strains and 133 and 48 suspect S. aureus strains isolated from fresh salami mixtures on mannitol salt and tellurite-polymyxin-egg yolk agars, respectively, demonstrated 100% agreement between the tandem agar plate method and standard coagulase and thermonuclease tests. Overall, the tandem agar plate method is a rapid and convenient approach contributing to the identification of S. aureus from foods.     

Hydrolysis of synthetic esters by the antibacterial agent in serum

Yotis, William W. (Loyola University, Chicago, Ill.). Hydrolysis of synthetic esters by the antibacterial agent in serum. J. Bacteriol. 91:488-493. 1966.-An antistaphylococcal serum agent was assayed colorimetrically, manometrically, and titrimetrically for esterase activity. p-Nitrophenol acetate, triacetin, l-lysine methyl and ethyl ester, and norleucine methyl ester were hydrolyzed by the antistaphylococcal agent. Acetylcholine and benzoylcholine esters, triolein, tristearin, and p-tosylarginine methyl ester were not attacked by this agent. With p-nitrophenol acetate as substrate, optimal activity occurred at pH 7.4. Incubation at 60 C for 30 min reduced drastically the esterase activity of the antistaphylococcal agent, and incubation at 75 C for 30 min abolished the esterase activity of this agent. Almost complete inhibition of esterase activity was observed with 0.001 m HgCl(2), ZnSO(4), and ethylenediaminetetraacetic acid (EDTA). EDTA inhibition could be reversed by the addition of CaCl(2), but not MgCl(2). Cysteine reversed the inhibition of HgCl(2). NaF, atoxyl, diisopropyl fluorophosphate, quinine, and physostigmine did not influence the esterase activity of the antibacterial agent. The demonstration of esterase activity of both the antistaphylococcal agent and coagulase may shed further light on the reported ability of coagulase to neutralize the antistaphylococcal activity of this agent, or the prevention of absorption of the agent on the staphylococcal cell surface. In addition, the colorimetric procedure described in this report may be a convenient tool in assaying the potency of the antistaphylococcal agent.     

Tandem Coagulase/Thermonuclease Agar Method for the Detection of Staphylococcus aureus

In optimizing previously reported coagulase agar media to obtain a rapid, reliable, and inexpensive coagulase test agar, variations in plasmas, pH, buffer system, fibrinogen, and fibrinolytic inhibitor were investigated. The agar with the following composition was determined best for the demonstration of coagulase production by Staphylococcus aureus: 25 ml of 15% bovine fibrinogen (fraction I, type I, citrated, Sigma Chemical Co.), 25 ml of rehydrated rabbit plasma (coagulase plasma ethylenediaminetetraacetic acid, Difco), 10.0 mg of soybean trypsin inhibitor (Schwarz/Mann), and 450 ml of brain heart infusion agar (Difco). In additional studies involving 7 different temperatures and 11 heating times, the thermal destruction of microbial nucleases on plate count agar and coagulase test agar was investigated. Heating the plates for 2.5 h at 65°C destroyed all heatlabile nucleases, but not thermonucleases of S. aureus. A tandem agar plate method for the identification of S. aureus was developed. Coagulase and thermonuclease activity of 50 colonies can be detected on a single agar plate. Suspect S. aureus colonies isolated on various selective media are transferred to coagulase test agar, the plates are incubated at 37°C for 18 h, and the coagulase reaction is recorded. The plates are then heated at 65°C for 2.5 h, overlaid with toluidine blue-metachromatic diffusion agar, and reincubated at 37°C for 3 h, and the thermonuclease reaction is recorded. Studies based on 88 enterotoxigenic S. aureus strains and 133 and 48 suspect S. aureus strains isolated from fresh salami mixtures on mannitol salt and tellurite-polymyxin-egg yolk agars, respectively, demonstrated 100% agreement between the tandem agar plate method and standard coagulase and thermonuclease tests. Overall, the tandem agar plate method is a rapid and convenient approach contributing to the identification of S. aureus from foods.     

Activation of Coagulase Clotting by Trypsin Inhibitor

Egg white trypsin inhibitor activated coagulase clotting when added to a final concentration between 2 and 60 mg/ml. The greatest increase in clotting rate was observed in reaction mixtures containing the lowest concentrations of serum and plasma. Maximal activation was reached with 40 mg of trypsin inhibitor per ml when either serum or plasma was used as the source of coagulase-reacting factor (CRF). The increased rate of clotting is partly due to inhibition of plasmin. Freezing and thawing reduced plasma clotting inhibition. Soybean trypsin inhibitor also activated the coagulase reaction. The increased rate of clotting was observed with a coagulase preparation from organisms which produced plasminogen activators and with the culture supernatant fraction from organisms which did not activate plasminogen to plasmin. The tube test for coagulase could be made more sensitive for some strains of staphylococci by increasing the concentration of CRF (added as plasma or serum) by adding trypsin inhibitor, or both.     

Short- and long-term A3 adenosine receptor activation inhibits the Na+/H+ exchanger NHE3 activity and expression in opossum kidney cells

The renal function of the A(3) adenosine receptor (A3AR) is poorly characterized. In this study, we report that the A3AR-selective agonist, 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purine-9-yl]-1-deoxy-N-methyl-b-D-ribofuranuronamide (2-Cl-IBMECA) regulates the Na+/H+ exchanger-3 (NHE3) in a dose- and time-dependent fashion. In opossum kidney (OK) cells, 2-Cl-IBMECA at high (10(-6) M) and low (10(-8) M) dose inhibits NHE3 by a multiphasic time course with an acute phase of NHE3 inhibition from 15 min to 1 h, followed by a chronic phase of NHE3 inhibition from 24 to 48 h. Pre-incubation with either the selective A3AR-antagonist MRS1523 (10(-7) M) or the protein kinase C inhibitor, Calphostin C (10(-8) M) completely blocked 10(-6) M 2-Cl-IBMECA-induced acute (15 min) and chronic (24 h) phases of NHE3 inhibition. In contrast, the acute inhibitory phase (15 min) of 10(-8) M 2-Cl-IBMECA was completely prevented only when Calphostin C (10(-8) M) was added in conjunction with the protein kinase A inhibitor, H89 (10(-7) M). Acute (15 or 30 min depending on the A3AR-agonist concentration) A3AR-dependent inhibition of NHE3 activity was accompanied by decrease in cell surface NHE3 protein with no change in total NHE3 antigen. Chronic (24 h) A3AR-mediated down-regulation of NHE3 was associated with reduction of surface NHE3, decreased total NHE3 protein (70%) and a paradoxical rise of NHE3 RNA (40%). In summary, these results indicate that A3AR directly regulates NHE3 at multiple levels in a complex pattern. A3AR-dependent short- and long-term inhibition of NHE3 may be a fundamental mechanism of net sodium and fluid balance.     

Coagulase Production by Injured Staphylococcus aureus MF-31 During Recovery

Suspensions of Staphylococcus aureus MF-31 injured by heat treatment at 54 C for 15 min produced coagulase during recovery in Trypticase Soy Broth. Coagulase also was produced by injured cells during recovery in a medium that did not support growth. Coagulase synthesis during recovery was independent of the molar strength of the buffer in which the cells were injured, the age of the cells, and the degree of injury. Return of salt tolerance and coagulase production required glucose, amino acids, and phosphate in the recovery medium. Vitamins stimulated coagulase production, but did not affect recovery. Although coagulase production was not necessary for repair of thermal injury to S. aureus MF-31, its detection was interpreted as an indicator of protein synthesis.     

Application of a fluorogenic substrate in the assay of proteolytic activity and in the discovery of a potent inhibitor of Candida albicans aspartic proteinase.

A fluorescent method for monitoring the activity of the secreted Candida carboxyl (aspartic) proteinase (EC 3.4.23.6) was developed using a fluorogenic substrate based on resonance energy transfer. The fluorescent assay was used to monitor proteinase production, purification, and inhibition. The Km for the fluorogenic substrate, 4-(4-dimethylaminophenylazo)benzoyl-gamma-aminobutyryl-Ile-His-Pro - Phe-His-Leu-Val-Ile-His-Thr- [5-(2-aminoethyl)amino]naphthalene-1-sulfonic acid, was found to be 4.3 microM at the optimum pH of 4.5. Reaction products were separated by reverse-phase high-performance liquid chromatography and identified by amino acid analysis or by 252Cf plasma desorption mass spectrometry. Cleavage of the fluorogenic substrate was between the histidine-threonine residues, releasing the fluorescent product, threonine-[5-(2-aminoethyl)amino]naphthalene-1-sulfonic acid. Proteolytic activity was expressed as nanomoles of fluorescent product released at 22 degrees C/60 min, pH 4.5, and the release of 0.9 nmol product was equivalent to one hemoglobin proteolytic unit (O.D.A700 increase of 0.100) produced at 37 degrees C/60 min, pH 3.5. The aspartic proteinase inhibitor pepstatin had an IC50 of 27 nM when tested in a dose-response study with the purified enzyme. The apparent Ki for pepstatis was 2.9 nM. Several synthetic inhibitors of the enzymes were identified with IC50's in the nanomolar range. The most potent compound, A70450, was characterized as a fast, tight-binding inhibitor having an IC50 of 1.3 nM and apparent Ki of 0.17 nM.     

An endogenous inhibitor of Ca++-ATPase from human placenta

Intracellular free calcium is regulated by Ca(++)-ATPase, one form present on the plasma membrane (PM Ca(++)-ATPase) and the other on sarcoplasmic (endoplasmic) reticulum (SR/ER Ca(++)-ATPase). An endogenous inhibitor of SR Ca(++)-ATPase from human placenta was shown to be present in normal placenta and the activity was not detectable in placenta from preeclamptic patients. The inhibitor was distributed in cytosol and microsomes. The inhibition of Ca(++)-ATPase by this inhibitor was concentration- and time-dependent. The inhibitor neither bound to DEAE- nor CM-sepharose resins at pH 7.5 and 8.5. Furthermore, it was heat stable for 15 min up to 55 degrees C and completely destroyed at 80 degrees C in a few minutes. It was also observed to be stable at room temperature for at least 3 months. The purification and characterization of this inhibitor would be valuable in achieving an understanding of the normal regulation of Ca(++)-ATPase in the placenta during pregnancy.     

Compound Inhibitory to Clostridium botulinum Type E Produced by a Moraxella Species

A Moraxella strain, A-43, produced a compound inhibitory to the outgrowth of Clostridium botulinum type E spores. The inhibitor could be produced in various laboratory media, and the outgrowth of germinated spores was inhibited by a 1/10th dilution of the A-43 spent medium. Germination was not affected. Molecular weight of the inhibitor was estimated at 800 to 1,000. The inhibitor was dialyzable and could be concentrated by lyophilization. It was stable at 37, 25, and 5 C, but was 70% inactivated when heated at 65 C for 10 min. The inhibitor was not volatile and could not be vacuum-distilled at 40 C. Solutions of acids with pH values below 2.0 destroyed the activity. The A-43 inhibitor appears to be similar, in molecular weight and inhibition characteristics, to tylosin.     

Mechanism of protein synthesis inhibition in CNS during postischaemic reperfusion.

We have used brain (dog, rat) and spinal cord (dog, rabbit) cell-free systems to study early postischaemic inhibition of protein synthesis. Ischaemia alone produced a relatively small decrease in activity of all subcellular systems used. When 15 min of normoxic reperfusion was used, more than 30% decrease (p less than 0.01) in [14C]-leucine incorporation was detected. A translational inhibitor that appeared in the postribosomal supernatant fraction at the early stage of reperfusion reduced translational capacity of an initiating cell-free system. It also phosphorylated the small (38 kDa) subunit of eukaryotic initiation factor 2 (eIF-2) in vitro. Effect of the inhibitor can be reversed by addition of partially purified intact eIF-2 and/or high concentration (2 mmol/l) of GTP. A prevention of postischaemic free oxygen radical formation by the reoxygenation with hypoxaemic blood, containing 37.5 mm Hg O2 at 0-5 min and 56 mm Hg O2 at 6-10 min of recirculation, that was followed by 5 min of normoxic reperfusion, resulted in a significant increase (p less than 0.02) of polypeptide chain synthesis in vitro when compared with normoxic reperfusion.     

Action of Lipases of Staphylococcus aureus on Milk Fat

The activity of the lipase(s) of two strains of coagulase-positive Staphylococcus aureus was determined in milk fat incubated at 15, 22, and 30 C for 8 days. Total fat hydrolysis was measured by acid degree values (ADV). Neutral lipids were separated into component groups on a Florisil column. Free fatty acids were determined by temperature-programmed gas-liquid chromatography. The ADV were 25 to 50% greater at 22 than at 15 C and 4 to 7 times greater at 30 than at 22 C. The lipases liberated as much as 0.48 g of fatty acids per gram of fat during 8 days at 30 C. The enzyme showed a predilection for the palmitic acid-glycerol bond. Addition of fatty acids C₁₄ to C₁₈ inclusive to inoculated sterile skim milk caused inhibition of S. aureus as follows: (i) complete at 0.05 and 0.10% concentration of C₁₀ and (ii) partial at 0.05 and complete at 0.10% concentration of C₈. The samples showing inhibition were negative for peptonization, coagulase, and change in pH. Addition of oleic and stearic acid to sterile skim milk inoculated with S. aureus resulted in an increase in nonprotein nitrogen, and the C₄ to C₁₂ acids caused a decrease in protease activity.     

Characterization of Yersinia pestis fibrinolysin using monoclonal antibody]

Immunochemical identity of Y. pestis fibrinolysin and coagulase is demonstrated using monoclonal antibodies. These substances are proven to exist as complex proteins with two independent activities. Possible causes of this phenomenon are discussed. Coagulase antigenic determinants are involved in specific fluorescence of Y. pestis cells grown at 28 degrees C. A new original method for screening the hybridomas producing monoclonal antibodies is proposed, based on inhibition of the functional activity of antigen.     

Association of a heat-stable inhibitor protein with cyclic-3',5'-AMP-dependent protein kinase from the nematode Ascaris suum: purification and characterization of the inhibitor.

An inhibitor protein of the catalytic subunit of the cyclic 3',5'-AMP-dependent protein kinase from the nematode Ascaris suum was isolated and characterized. The molecular weight of the inhibitor was estimated as 28,000 by electrophoresis under denaturing conditions and as 30,000 by gel permeation chromatography on Superose 12. The Trypsin-labile inhibitor was resistant to short incubations (less than or equal to 5 min) at temperatures up to 95 degrees C and at pH 3. It affected the protein kinase from Ascaris and bovine heart with almost the same affinity, and inhibition was not relieved by the presence of cAMP and cGMP. However, the inhibition was antagonized by low concentrations of heparin. Unlike in mammalian tissues, the concentration of the inhibitor was sufficiently high to exert at least 90% inhibition of the protein kinase activity in Ascaris muscle. Therefore, the inhibitor may play a role in cellular regulation in the nematode.     

The requirement of both extracellular regulated kinase and p38 mitogen-activated protein kinase for stimulation of cytosolic phospholipase A(2) activity by either FcgammaRIIA or FcgammaRIIIB in human neutrophils. A possible role for Pyk2 but not for the Grb2-Sos-Shc complex

The signal transduction pathways initiated by opsonized zymosan (OZ) leading to activation of cytosolic phospholipase A(2) (cPLA(2)) in human neutrophils remain obscure. In a previous study, we showed that the activation of cPLA(2) by OZ is tyrosine kinase-dependent. The present study demonstrates that the signals initiated by OZ involve activation of tyrosine kinase Pyk2 but not the formation of the adhesion protein complex, Shc-Grb2-Sos. Stimulation of cPLA(2) activity by OZ is mediated by Fc gamma receptors (FcgammaRs) and not by complement receptors for the C3b protein. Cross-linking of FcgammaRIIA or FcgammaRIIIB induces p38 mitogen-activated protein (MAP) kinase and extracellular regulated kinase (ERK) phosphorylation. The kinetics of cPLA(2) activity stimulated by either of the FcgammaRs or by both is similar to that of p38 MAP kinase and was detected as early as 15 s after stimulation, maintained a plateau for 10 min, and decreased thereafter. ERK activation was detected also within 15 s but decreased significantly 5 min after stimulation. The MEK inhibitor, PD-098059, or the p38 MAP kinase inhibitor, SB-203580, caused a partial inhibition during the time course of cPLA(2) activity, whereas their combination caused a total inhibition. Thus, although ERK activation is significantly shorter than that of p38 MAP kinase, it is equally required for activation and maintenance of cPLA(2) activity by occupancy of a single receptor, FcgammaRIIA or FcgammaRIIIB.     

NONEXISTENCE OF A TYPE C MONOAMINE OXIDASE IN RAT BRAIN

Abstract— The possible existence of type C MAO, distinct from type A and type B, in circumventricular structures of rat brain was examined by histological studies on the inhibitory effects of clorgyline. a preferential type A MAO inhibitor and deprenyl, a preferential type B inhibitor, on enzyme. Brain slices were preincubated with the inhibitors and then incubated with 5-HT, the substrate for type A MAO, and stained for MAO activity. Deposits of the product formazan were detected in circumventricular structures of slices of brain preincubated with clorgyline and deprenyl at concentrations of 10^-7–10^-4m at room temperature for 5 min. When the slices were preincubated with either of these inhibitors at room temperature for 60 min, strong activity was observed in this region, whereas when they were preincubated with either 10^-5m -clorgyline or 10^-5m -deprenyl for 20 and 30 min at 37°C, no MAO activity was seen in any region of the brain. Thus, at the higher preincubation temperature, lower concentrations of each inhibitor and a shorter preincubation period were required for inhibition of the enzyme. Preincubation for 60 min at 37°C with a combination of 10^-7m -clorgyline and 10^-8m -deprenyl did not inhibit the enzyme in the circumventricular region completely, but at the same temperature, concentrations of 10^-7m of both inhibitors inhibited the enzyme completely in 10min, Thus the effects of the inhibitors are synergistic. These results indicate that the inhibitory effects of the two inhibitors on the enzyme in circumventricular structures of the brain is time- and temperature-dependent. Moreover, the activity seems to be sensitive to deprenyl even when 5-HT is used as substrate. The results do not support the idea of the existence of type C MAO, distinct from type A and type B MAO.     

Trypsin inhibitors in xoconostle seeds (Opuntia joconostle Weber.)

Seed proteins recovered after heating a seed extract from Opuntia joconostle Weber (xoconostle, an acid cactus pear) were screened for different biochemical activities, detecting only trypsin-like inhibitory activity. Two trypsin-like inhibitor forms from seeds were separated by RP-HPLC and partially sequenced and characterized as an enriched mixture. They were evaluated for inhibition on several serine proteinases, but only trypsin-like inhibition was detected by the inhibitor extract. The two isolated forms, OjTI 1 and OjTI 2 showed low molecular weights of 4.26 and 4.17 kDa as determined by mass spectrometry. An enriched inhibitory fraction showed a high thermal stability by retaining the activity after heating the sample for 1 h at 90 °C, as well as after heating for at 120 °C under 1 kg/cm² for 15 min at different pH values. Partial sequence of the two forms was determined by mass spectrometry indicating that they were similar and after alignments analysis they showed the highest similarity with the trypsin inhibitor from O. streptacantha and to a lesser extent to other trypsin inhibitors of the MEROPS database families. The inhibitory spectrum was evaluated against several digestive enzymes from pests and beneficial insects from several taxonomic orders.     

A functionally inactive, cold-stabilized form of the Escherichia coli F1Fo ATP synthase

An unusual effect of temperature on the ATPase activity of E. coli F1Fo ATP synthase has been investigated. The rate of ATP hydrolysis by the isolated enzyme, previously kept on ice, showed a lag phase when measured at 15 degrees C, but not at 37 degrees C. A pre-incubation of the enzyme at room temperature for 5 min completely eliminated the lag phase, and resulted in a higher steady-state rate. Similar results were obtained using the isolated enzyme after incorporation into liposomes. The initial rates of ATP-dependent proton translocation, as measured by 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching, at 15 degrees C also varied according to the pre-incubation temperature. The relationship between this temperature-dependent pattern of enzyme activity, termed thermohysteresis, and pre-incubation with other agents was examined. Pre-incubation of membrane vesicles with azide and Mg2+, without exogenous ADP, resulted in almost complete inhibition of the initial rate of ATPase when assayed at 10 degrees C, but had little effect at 37 degrees C. Rates of ATP synthesis following this pre-incubation were not affected at any temperature. Azide inhibition of ATP hydrolysis by the isolated enzyme was reduced when an ATP-regenerating system was used. A gradual reactivation of azide-blocked enzyme was slowed down by the presence of phosphate in the reaction medium. The well-known Mg2+ inhibition of ATP hydrolysis was shown to be greatly enhanced at 15 degrees C relative to at 37 degrees C. The results suggest that thermohysteresis is a consequence of an inactive form of the enzyme that is stabilized by the binding of inhibitory Mg-ADP.