Herpes simplex virus immediate-early promoters are responsive to virus and cell trans-acting factors.

The promoters for each of the immediate-early genes from herpes simplex virus type 1 were cloned and fused to a chloramphenicol acetyltransferase cassette. These chimeric genes were used as targets in a transient expression assay to determine how the immediate-early gene products ICP4 and ICP0 and the virion-associated stimulatory protein Vmw65 affected their expression in HeLa and Vero cells. The basal level of expression from these cassettes differed significantly depending on the extent of 5'-flanking sequence and the cell line that served as host. The promoters from IE-4 and IE-0 behaved in a qualitatively similar fashion independent of the host cell. However, the promoter for ICP27 had a unique response pattern: in Vero cells it acted as an alpha gene promoter, whereas in HeLa cells its response was more like that of a beta gene promoter. The promoter sequences for ICP22 and ICP47 behaved as the IE-4 and IE-0 promoters did in HeLa cells, but their response to the effector molecules in Vero cells was unlike that of other alpha gene promoters we have studied. Evidence is also presented for a role for ICP27 in autoregulation.     

Hemolymph concentrations of host ecdysteroids are strongly suppressed in precocious prepupae of Trichoplusia ni parasitized and pseudoparasitized by Chelonus near curvimaculatus.

Regulation of ecdysteroid production in lepidopteran prepupae was studied using a parasitic wasp (C. near curvimaculatus) which specifically suppresses host prepupal ecdysteroid production after the induction of precocious host metamorphosis. At the developmental stage at which the hemolymph of the unparasitized metamorphosing host has its maximum titer of prepupal ecdysteroids, the hemolymph of 4th instar "truly parasitized" hosts (hosts with a surviving endoparasite) had a strongly reduced ecdysteroid titer. However, during the photophase about 12 h later, just prior to emergence of the parasite larva, an ecdysteroid peak was observed in the host hemolymph. Fourth instar pseudoparasitized prepupal hosts (in which the endoparasite was not present or died early in development) exhibited a sustained suppression in the hemolymph ecdysteroid titer. Small 5th instar pseudoparasitized hosts, which normally would molt to a 6th instar prior to metamorphosis, but which precociously attained the prepupal stage, also had a strongly reduced ecdysteroid titer. The late increase observed in truly parasitized hosts could be completely prevented by surgical removal of the parasite 24 h earlier, resulting in a titer similar to that in pseudoparasitized hosts. HPLC analysis of ecdysteroids in normal, truly parasitized, and 4th or 5th instar pseudoparasitized prepupae showed that both ecdysone and 20-OH ecdysone* were suppressed in truly and pseudoparasitized prepupae, with ecdysteroid levels being lowest in pseudoparasitized hosts. These data, and those of Brown and Reed-Larsen (Biol Contr 1, 136 [1992]), showing endoparasite secretion of ecdysteroids just prior to its emergence from the host, strongly indicate that: (1) the prepupal peak in truly parasitized hosts originates from the endoparasite, and (2) the low level of ecdysteroids in pseudoparasitized hosts results from the host's intrinsic inability to express a normal level of prepupal ecdysteroid titer. While precocious 4th or 5th instar prepupae of similar size had similarly suppressed ecdysteroid titers, smaller 4th instar prepupae had a lower ecdysteroid titer than larger, precocious 5th instar prepupae. Rare 5th instar pseudoparasitized prepupae that were of nearly normal size showed a prepupal ecdysteroid titer distinctly greater than those of the usual smaller, precocious 5th instar prepupae. The data suggest that the competence of the host to express a normal hemolymph titer of prepupal ecdysteroids is more closely correlated with the size of the prepupae than with the instar attained.     

Interactions of an intact proteoglycan and its fragments with basic homopolypeptides in dilute aqueous solution

The interactions between a proteoglycan and cationic polypeptides have been investigated by the use of circular-dichroism spectroscopy. The interaction produces an induced conformational change for poly(l-arginine) and poly(l-lysine), similar to the effects previously reported for mucopolysaccharide-polypeptide mixtures. For bovine nasal septum proteoglycan, the interactions are similar to those for chondroitin 4-sulphate, which comprises approximately 63% of the total polysaccharide. The results also suggest that the interactions produce a conformational change in the protein core. Similar studies for the Smith-degradation product show that the protein core can adopt a substantial alpha-helical content and is capable of interactions with poly-(l-arginine). The interactions for chondroitin sulphate ;doublets' are significantly different from those for the separated chains, indicating that the arrangement of the polysaccharide side chains in pairs (and larger groups) along the protein backbone contributes to the interaction properties of the intact proteoglycan.     

Interaction between chondroitin-6-sulfate and poly-L-arginine in aqueous solution

The interactions between chondroitin-6-sulfate and poly-L -arginine in aqueous salt solution have been investigated by circular dichroism techniques. In the presence of chondroitin-6-sulfate, at neutral pH, poly-L -arginine adopts the α-helical conformation rather than “charged coil” form observed in the absence of mucopolysaccharide. This interaction is at a maximum when the ratio of arginine to disaccharide residues is 2:1. Elevation of the temperature leads to a sharp melting transition at 76.0 ± 1.0°C. This behavior is in marked contrast to that for poly-L -lysine-chondroitin-6-sulfate interactions, which are at a maximum at a 1:1 residue ratio and have a melting transition at 47.0 ± 1.0°C. These results indicate a stronger interaction for poly-L -arginine than for poly-L -lysine. The positive arginine side chains appear to interact with both the negative sulfate and carboxyl residues, while those of the lysines are involved only with the sulfates. Poly-L -ornithine at neutral pH shows no conformation directing interaction with chondroitin-6-sulfate, although a small proportion of α-helix is formed on dilution of the mixture with methanol. The extent of the interaction of cationic polypeptides with chondroitin-6-sulfate increases in the order poly-L -ornithine, poly-L -lysine, poly-L -arginine, i.e., in the order of increasing side-chain length.     

Isolation and analysis of free fatty aldehydes from rat, dog, and bovine heart muscle

Procedures are described for the isolation of free fatty aldehydes from total lipid extracts of rat, dog, and bovine heart muscle. These aldehydes did not arise from hydrolysis of the naturally occurring alkenyl ethers in either the extraction or isolation procedures. The free fatty aldehydes were present in heart muscle in amounts between 0.27 and 0.56 micromole per 100 mg of lipid. Hexadecanal and octadecanal are the principal free fatty aldehydes present, except in dog heart muscle where an unidentified, unusual aldehyde constitutes 20% of the mixture.     

Depolarization and Neurotransmitters Increase Neuronal Protein Tyrosine Phosphorylation

Abstract: In rat hippocampal slices and in neurons in primary culture, K⁺-induced depolarization increased markedly and rapidly tyrosine phosphorylation of a 110-kDa protein (pp110) and, to a lesser degree, of a 120-kDa protein (pp120), in a calcium-dependent fashion. Qlutamate, 1-aminocyclopentane- trans -1,3-dicarboxylic acid (an agonist of metabotropic glutamate receptors), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (an agonist of ionotropic glutamate receptors) stimulated also tyrosine phosphorylation of pp110 and pp120. These effects were not observed in astrocytes in primary culture. In hippocampal slices tyrosine phosphorylation of pp110 and pp120 was stimulated by Ca²⁺-ionophores and by phorbol esters and antagonized by a chelator of intracellular Ca²⁺and by drugs that inhibit protein kinase C. Stimulation of muscarinic and α₁,-adrenergic receptors increased also tyrosine phosphorylation of pp110 and pp120. These results demonstrate that membrane depolarization and stimulation of neurotransmitter receptors activate a tyrosine phosphorylation pathway in neurons. This pathway involves an increase in intracellular Ca²⁺ concentrations and the activation of protein kinase C. It may provide a biochemical basis for some neurotrophic effects of electrical activity and neurotransmitters and may contribute to the role of tyrosine phosphorylation in long-term potentiation.     

Safety,Immunogenicity and Duration of Immunity Elicited by an Inactivated Bovine Ephemeral Fever Vaccine

Bovine ephemeral fever (BEF) is an economically important viral vector-borne cattle disease. Several live-attenuated, inactivated and recombinant vaccines have been tested, demonstrating varying efficacy. However, to the best of our knowledge, duration of immunity conferred by an inactivated vaccine has never been reported. In the last decade, Israel has faced an increasing number of BEF outbreaks. The need for an effective vaccine compatible with strains circulating in the Middle East region led to the development of a MONTANIDE™ ISA 206 VG (water-in-oil-in-water), inactivated vaccine based on a local strain. We tested the safety, immunogenicity and duration of immunity conferred by this vaccine. The induced neutralizing antibody (NA) response was followed for 493 days in 40 cows vaccinated by different protocols. The vaccine did not cause adverse reactions or a decrease in milk production. All cows [except 2 (6.7%) which did not respond to vaccination] showed a significant rise in NA titer of up to 1:256 following the second, third or fourth booster vaccination. Neutralizing antibody levels declined gradually to 1:16 up to 120 days post vaccination. This decline continued in cows vaccinated only twice, whereas cows vaccinated 3 or 4 times showed stable titers of approximately 1:16 for up to 267 days post vaccination. At least three vaccinations with the inactivated BEF vaccine were needed to confer long-lasting immunity. These results may have significant implications for the choice of vaccination protocol with inactivated BEF vaccines. Complementary challenge data should however be added to the above results in order to determine what is the minimal NA response conferring protection from clinical disease.