Defective interfering particles of parvovirus H-1.

Defective interfering particles of the parvovirus H-1 were produced by serial propagation at high multiplicities of infection. Such particles interfere with the synthesis of capsid proteins and infectious virus of standard H-1. The interference is sensitive to UV irradiation, dependent on the multiplicity of the challenge virus, and is active in heterotypic infections against parvovirus H-3 or LuIII. Defective interfering particle genomes have alterations characterized by integral numbers (1 to 10 or more) of a 60-base-pair addition in the neighborhood of the origin of replicative-form DNA replication and deletions that are located primarily within two regions, 32 to 44 or 80 to 90 on the genome map. Some of the implications of these findings are discussed.     

Plasma zinc concentrations of mothers and the risk of nonsyndromic oral clefts in their children: a case-control study in the Philippines

BACKGROUND: Findings from animal experiments suggest a link between poor maternal zinc status and increased risk of oral clefts in offspring; however, there are few human studies on this issue. METHODS: A case-control study was conducted using 74 case mothers of children with nonsyndromic cleft lip with or without cleft palate (CL/P, n=57) or cleft palate alone (CP, n=17), and 283 control mothers of unaffected children recruited in the Philippines in early 2003. Maternal zinc status was assessed by determining plasma zinc concentrations a mean of 5 years after delivery of the index child. Odds ratios (ORs) of estimates of the relative risk of oral clefts were calculated for quartiles of maternal plasma zinc concentrations. RESULTS: The mean plasma zinc concentration of CL/P case mothers (9.6+/-1.2, SD micromol/l) was significantly lower than that in control mothers (10.1+/-1.6 micromol/l; P<0.05). Low plasma zinc concentrations (<11.0 micromol/l) were found in 88% and 94% of CL/P and CP case mothers, respectively, and in 72% of controls (P<0.05). The ORs for CL/P and CP combined, adjusted for potential confounding factors, decreased with increasing quartile of plasma zinc as follows: 1.0 (lowest quartile reference), 0.83 (95% confidence interval 0.37-1.89), 0.70 (0.31-1.68), and 0.26 (0.10-0.70) (P trend=0.008). CONCLUSIONS: Low plasma zinc concentrations were common in Filipino women of reproductive age, and higher plasma zinc concentrations were associated with a lower risk for oral clefts in their children.     

Construction of a genetic switch for inducible trans-activation of gene expression in eucaryotic cells.

The cotransfection of selectable marker genes and the gene for the nonstructural proteins NS1 and NS2 of the autonomous parvovirus H-1 failed to produce cell lines that constitutively expressed NS1. A plasmid, pP38NS1cat, was constructed that expressed the NS1-NS2 gene from the H-1 P38 coat protein promoter in place of the natural P4 promoter. The P38 promoter is constitutively weak and is trans-activated by NS1. Stable cell lines were isolated that contained pP38NS1cat that was constitutively silent, but inducible with exogenous NS1 by superinfection or by treatment with sodium butyrate. The cells that were induced for this self-stimulatory genetic circuit did not remain in the culture, suggesting that expression of NS1-NS2 is cytotoxic or that the expression is not sustained. The properties of these cell lines and an example of the construction of a cell line inducible for expression of the viral coat protein gene and the bacterial gene for chloramphenicol acetyltransferase (cat) are described.     

Differential inhibition of human placental prostaglandin release in vitro by a GnRH antagonist

Previously, we have demonstrated that the production of prostaglandins by human placental tissue varied with gestational age. In addition, we have shown that placental prostaglandin release was affected by GnRH, and that its response was also dependent on the gestational age of the placenta. Thus, we have studied the effect of a GnRH antagonist ([N-Ac-Pro1,D-p-Cl-Phe2,D-Nal(2)3,6-LHRH, Syntex Research, Palo Alto, CA) on basal prostaglandin release from placentas of 6 to 15 weeks' gestation and found that this antagonist (1 microgram/ml) effects an inhibition of the release of prostaglandin E, prostaglandin F, and 13,14-dihydro-15-keto-prostaglandin from placentas of 13 and 15 weeks of gestation. This effect was not overridden by GnRH at 10 times the antagonist concentration in the 13-week placental cultures, but was totally reversed by GnRH (10 micrograms/ml) in the 15-week placental cultures. These data demonstrate that this GnRH antagonist can affect human placental prostaglandin production at 13 to 15 weeks of gestation and indicate that endogenous placental GnRH-like activity may exert a control over placental prostaglandin release at this gestational stage.     

Long-term studies of macrozoobenthos in intertidal and shallow subtidal habitats near the island of Norderney (East Frisian coast,Germany)

Near the East Frisian island of Norderney two sites are investigated permanently in order to study long-term fluctuations of macrozoobenthos: one transect (since 1977) at the northern side in shallow subtidal waters and another one (since 1976) at the sheltered southern side in the intertidal area of the Wadden Sea. Since 1980 the investigations have been continued in the frame of COST-47, sedimentary intertidal programme (including the shallow subtidal habitats colonized by the Macoma balthica community).The results up to 1984, respectively to 1985, are presented and the changes of abundance of the dominant species are discussed. Discussed are also the influences of water temperatures and sediment disturbances caused by wave action. There is evidence that the intertidal variety of the Macoma balthica community shows a greater stability than the subtidal variety.     

Mutation of lysine 405 to serine in the parvovirus H-1 NS1 abolishes its functions for viral DNA replication, late promoter trans activation, and cytotoxicity.

A consensus sequence in parvovirus nonstructural protein NS1 has been predicted to be an ATP-binding domain associated with an ATPase and a DNA helicase activity. To investigate the function of NS1 in viral gene expression, a site-directed mutagenesis converting NS1 lysine 405 to serine in parvovirus H-1 was carried out by the polymerase chain reaction. As shown previously, a parvovirus genome containing a deleted NS1 gene was excised from a bacterial plasmid and replicated when a wild-type NS1 gene was provided in trans but failed to be excised and replicate when the mutant NS1 gene was supplied. Interestingly, the serine 405 mutation totally lost the activity of trans activation on the virus late promoter (P38) in a chloramphenicol acetyltransferase (CAT) assay and it lost evidence for cytotoxicity in two tumor cell lines (HeLa Gey and NB324K). The serine 405 NS1 protein was translocated normally to the nucleus. These results suggest that the NS1 lysine 405 of H-1 in its putative purine nucleotide-binding site is essential for viral DNA replication and that this domain may be involved in the regulation of the P38 promoter by an unknown mechanism. The loss of NS1 cytotoxicity on tumor cells suggests that NS1 expression is the major cause of cell killing by parvoviruses, which may facilitate further study of the mechanism of oncosuppression by parvoviruses.     

Mechanisms in the development of retinotectal projections in the chick embryo studied by surgical deflection of the retinal pathway

Retinotopic analysis of the pathways of normal and aberrant retinal axons within the tectum of developing chick embryos was performed by selective labeling of retinal axons with a fluorescent dye, rhodamine-B isothiocyanate. To produce aberrant retinal axons, the presumptive optic chiasma was surgically disorganized at the 3rd day of incubation. At the 11th and 13th days of incubation, more than half of the operated embryos exhibited several aberrant retinal axons which reached ectopic parts of the tectum. The pathways of these aberrant axons within the tectum depended on the position of their initial invasion into the tectum at the diencephalotectal junction, and not on their position of origin within the retina. The aberrant retinal axons did not show any sign of correction of their pathways toward their normal sites of innervation within the tectum. As development proceeded, elimination of the aberrant retinal axons occurred. By the 16th day of incubation, almost all operated embryos lacked aberrant retinal axons and although the total number of axons often appeared reduced, a nearly normal topography of retinotectal projections was established. These findings indicate that the initial invasion of the retinal axons into the tectum is conducted predominantly by nonspecific mechanisms and, thereafter, a selective maintenance of appropriate retinal axons occurs.