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The herpes simplex virus-1 (HSV-1) utilizes cell-surface glycosaminoglycan, heparan sulfate, to gain entry into cells and cause infection. In a search for synthetic mimics of heparan sulfate to prevent HSV infection, we discovered potent inhibitory activity arising from sulfation of a monomeric flavonoid. Yet, detailed screening indicated that the sulfated flavonoid was completely inactive and the potent inhibitory activity arose from a macromolecular substance present in the parent flavonoid. The active principle was identified through a battery of biophysical and chemical analyses as a sulfated form of lignin, a three-dimensional network polymer composed of substituted phenylpropanoid monomers. Mass spectral analysis of the parent lignin and its sulfated derivative indicates the presence of p-coumaryl monomers interconnected through uncondensed β-O-4-linkages. Elemental analysis of lignin sulfate correlates primarily with a polymer of p-coumaryl alcohol containing one sulfate group. High-performance size exclusion chromatography shows a wide molecular weight distribution from 1.5 to 40 kDa suggesting significant polydispersity. Polyacrylamide gel electrophoresis (PAGE) analysis indicates a highly networked polymer that differs significantly from linear charged polymers with respect to its electrophoretic mobility. Overall, macromolecular lignin sulfate presents a multitude of substructures that can interact with biomolecules, including viral glycoproteins, using hydrophobic, hydrogen-bonding, and ionic forces. Thus, lignin sulfate represents a large number of interesting structures with potential medicinal benefits.

The epoxy resins diglycidyl ether of bisphenol A and triglycidyl p-amino phenol were reacted with acrylic acid to afford the corresponding acrylated resins. These vinyl ester resins were then reacted with toluene di-isocyanate to procure their urethanized derivatives. All these resins were characterized by their viscosity, number average molecular weight and infrared spectrophotometry. Curingconditions for these resins were established by differential scanning calorimetry. The results indicated that the curing reaction follows first-order kinetics, with activation energy in the range 53-84 kJ mol-. Styrene monomer was observed to lower the curing temperature of all resin systems when incorporated prior to curing.

Using AMP deaminase (AMP aminohydrolase; EC 3.5.4.6) purified from rabbit left-ventricular heart tissue, we report direct investigation of the potential for cardiac AMP deaminase activity to be regulated by kinase-mediated phosphorylation. Rabbit heart AMP deaminase served as a substrate for Ca2+/phospholipid-dependent protein kinase (protein kinase C; PKC) exclusively; no other mammalian protein kinase phosphorylated the enzyme. PKC-dependent AMP deaminase phosphorylation was rapid, linear with respect to time and the concentrations of PKC and AMP deaminase in the reaction, and inhibitable by staurosporine. Upon phosphorylation, the apparent Km of cardiac AMP deaminase decreased from 5.6 mM to 1.2 mM, without effect on the Vmax. Whether phosphorylated or not, rabbit heart AMP deaminase was inhibited by 1.0 mM GTP, which decreased the Vmax. by approximately 50% in each case. PKC-dependent phosphorylation of cardiac AMP deaminase did not alter the enzyme's allosterism toward millimolar ATP or ADP: both nucleotides at 1.0 mM concentration decreased the apparent Km to approximately 0.5 mM. Treatment of cardiac phospho-AMP deaminase with either the protein phosphatase calcineurin or alkaline phosphatase generated a dephosphorylated form which displayed molecular and kinetic properties identical with those of the originally isolated enzyme. These data raise the possibility that a phosphorylation-dephosphorylation mechanism may regulate flux through AMP deaminase in the heart under pathological conditions, such as myocardial ischaemia, characterized by PKC activation and adenylate depletion.

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) is a ubiquitous enzyme in eukaryotes, which may play a role in ATP catabolism during myocardial ischaemia. We report isolation of AMP deaminase from rabbit myocardium with a 19% recovery and a 650-fold enrichment, using a newly devised protocol involving sequential cation-exchange, gel-permeation and affinity chromatographies. The cardiac AMP deaminase preparation described was electrophoretically and chromatographically homogeneous and contained one unique N-terminal residue (leucine). The isolated enzyme was sensitive to various cations (K+, Mg2+, Ca2+). The pH optimum of purified cardiac AMP deaminase was 6.8, its pI was 6.5, and it displayed substrate-specificity toward 5′-AMP. The subunit molecular mass of rabbit heart AMP deaminase on SDS/PAGE (81 kDa) and the holoenzyme molecular mass as estimated by non-denaturing size-exclusion h.p.l.c. (330 kDa) indicated that the native enzyme was a tetramer. Cardiac AMP deaminase displayed a sigmoidal substrate-saturation curve in the presence of 100 mM KCl. Apparent Michaelis constants were a Km of 5.8 mM AMP and a Vmax. of 11.1 mumol/min per mg of protein. ATP and ADP were positive allosteric effectors of cardiac AMP deaminase: the apparent Km was decreased to 1.7 mM by 1.0 mM ATP. The enzyme was inhibited by GTP, coformycin, coformycin 5′-phosphate, palmitoyl-CoA, inorganic phosphate compounds, and the metal chelator o-phenanthroline. No inhibition either by product nucleotide (IMP) or by nicotinamide nucleotides was detected when these agents were examined at concentrations up to 2.5 mM. We conclude that this enzyme preparation offers a means by which the kinetic mechanism and regulation of mammalian cardiac AMP deaminase may be directly investigated.

Adenylate deaminase (EC 3.5.4.6) may help to regulate the adenine nucleotide catabolism characteristic of such disease states as myocardial ischaemia. We report analysis of the molecular, kinetic and allosteric properties of rabbit heart adenylate deaminase when extracted and purified under phosphate-free conditions (i.e., with Hepes/KOH). The enzyme's subunit molecular mass (approximately 81 kDa), pI (6.5), substrate specificity for 5′-AMP, and activation by K+ were identical in the absence or presence of phosphate. At each chromatographic step during isolation without phosphate, cardiac adenylate deaminase showed a lower apparent activity as compared with the enzyme prepared with phosphate present. Kinetic constants for the phosphate-free rabbit heart adenylate deaminase preparation (Km 0.54 mM AMP; Vmax. 1.4 mumol/min per mg of protein) were approximately 10-fold lower than those of the enzyme isolated with phosphate. The same irreversible decrease in kinetic constants could be achieved by dialysing phosphate from the phosphate-containing enzyme preparation. The relationship between enzyme activity and substrate concentration was sigmoidal in the presence of phosphate, but hyperbolic in its absence. Cardiac adenylate deaminase under phosphate-free conditions was no longer allosterically activated by ATP and ADP, yet remained inhibitable by GTP. Enzyme inhibition by the transition-state mimic coformycin was not influenced by phosphate status. The phosphate-free preparation of rabbit heart adenylate deaminase was markedly labile and extremely susceptible to proteolysis by trypsin or chymotrypsin. The inactivation kinetics and fragmentation pattern in response to controlled proteolysis depended on whether the enzyme had been isolated with or without phosphate present, suggesting a conformational difference between the two enzyme preparations. These data constitute direct evidence that the absence of phosphate irreversibly converts cardiac adenylate deaminase into a pseudo-isoenzyme with distinct kinetic, regulatory and stability properties.

The curing behaviour of vinyl ester resin, the condensation product of acrylic acid with diglycidyl ether of bisphenol-A, was studied by differential scanning calorimetry. The glass-fibre-reinforced composites of the vinyl ester resin were prepared and evaluated for their chemical, electrical and mechanical properties. The incorporation of styrene during the curing of the vinyl ester resin showed significant improvements in tensile strength and dielectric properties.

Cardiac sarcolemmae from guinea pig ventricles were purified and incubated with cGMP-dependent protein kinase. In the presence of the purified kinase plus 10-5M cGMP or 8-Br-cGMP, a protein of approximately 50 kD, (Kilodalton) was phosphorylated. This membrane-associated cGMP-dependent protein kinase substrate is similar in MW to the regulatory subunit of the cAMP-dependent protein kinase, which is known to be a substrate for the cGMP-dependent protein kinase. Thus, this substrate, the identity of which remains to be proven, may be a possible mediator of cGMP-mediated control of cardiac function.

The curing behaviour of epoxy resin-m-amino benzoic acid, the condensation product of epoxy resin namely; diglycidyl ether of bisphenol-A (DGEBA) and m-amino benzoic acid (m-ABA) was studied by differential scanning calaorimetry (DSC). The resultant neat products of DGEBA-m-ABA were characterised by infrared (IR) spectral studies and thermogravimetric analysis (TGA). The glass fiber reinforced composites were prepared and evaluated for their chemical and mechanical properties.

Dynamics tests were preformed at the Oconee pressurized water reactor to obtain information for checking a theoretical plant model. Low level, periodic reactivity perturbations were introduced and several system responses (reactor power, temperatures, pressures) were monitored. The data were processed off-line to give frequency responses. A linear state-variable model for the plant was formulated and used to compute theoretical frequency responses. A computerized, model-reference identification procedure was used to identify the fuel temperature coefficient of reactivity and the overall fuel-to-coolant heat transfer coefficient. The study showed that dynamic tests can be performed in operating nuclear power plants with insignificant interference to normal operation. Also, the use of automatic parameter identification procedures was demonstrated.

Phospholipase A2activity in sonicates and acid extracts of ejaculated, washed human sperm was measured using [1-14C] oleate-labeled autoclaved E. coliand 1-[1-14C] stearoyl-2-acyl-3-sn- glycerophosphorylethanolamine as substrates. Phospholipase A was optimally active at pH 7.5, was calcium-dependent, and exclusively catalyzed the release of fatty acid from the 2-position of phospholipids. The activity was membrane-associated, and was solubilized by extraction with 0.18 N H2SO4. Acid extracts of human sperm had the highest specific activity (1709 nmols /h per mg), followed by mouse, rabbit and bull, which were 105, 36 and 1.7 nmols /h per mg, respectively. para-bromophenacyl bromide inhibited human sperm phospholipase A2activity, but mepacrine was without effect. In the presence of 1.0 mM added CaCl2, phospholipase A2activity was inhibited by Zn2+and Mn2+; whereas Cu2+, Cd2+, Mg2+, or Sr2+had no effect. Zn2+stimulated activity at low concentrations (10−6to 10−8M), and inhibited activity in a dose-dependent manner at concentrations of 10−5M. The extent of stimulation by low concentrations of Zn2+was dependent on CA2+concentration; at 10−7M, Zn2+activity was stimulated 160% with 0.5 mM CaCl2, and only 120% with 1.0 mM CaCl2. At low concentrations (10−5to 10−7M), methoxyverapamil (D600) and trifluoperazine stimulated human sperm phospholipase A2activity, and trifluoperazine but not D600 produced almost complete inhibition between 10−5and 10−4M of the drug. The significance of human sperm phospholipase A2activity and its modulation by CA2+, Zn2+and Mn2+in the sperm acrosome reaction is discussed.