Association of whole blood aggregation response with immunogold-labeled glycoproteins in adult and neonatal platelets.

A growing body of recent data has provided specifics about the hemostatic system in infants, emphasizing differences from adults. Although platelet structure in newborns and adults appears to be quite similar, precise information about platelets in the first week of life indicates functional hyporeactiveness. This study was designed with a twofold purpose: one was to determine if aggregation results corresponded to immunogold-labeled activation markers; the second was to use immunogold labeling to determine if infant platelets circulate in an activated state. The results showed significant differences in ristocetin (P = .03) and collagen (P = .003) impedance, and whole blood aggregation lag times in infants when compared to adults. Treatment of neonatal platelets with collagen yielded decreased ATP release compared with adults. Immunogold labeling of specific activation markers CD62 (P-selectin) and CD63 (GP53) revealed that neonatal platelets were not circulating in an activated state. Significant (P = .04) anti-CD41 (GPIIb) immunogold labeling differences were observed after thrombin stimulation, with adults binding more particles. These data suggest that hyporeactivity of neonatal platelets is not due to a circulating preactivated state, but instead may be a consequence of impaired intracellular signaling that affects both aggregation and membrane activation labeling. Whether this signaling is secondary to an intrinsic neonatal alteration or a maternal (in utero) environmental effect is yet to be determined.

Imparied platelet--dense granule release in neonates.

PURPOSE: The aim of this study is to examine differences in platelet dense granule (PDG) uptake and release between preterm infants, term infants, and adults. METHODS: PDG uptake and release was examined by flow cytometry using mepacrine fluorescent staining in phycoerythrin-anti-GPIIb/IIIa bound platelets taken from cord blood of eight term infants and seven preterm infants and venous blood from eight adults. RESULTS: Analysis of the baseline fluorescence in the untreated versus thrombin-treated samples revealed significant differences in the way infant PDGs responded to thrombin stimulation when compared with adults. Initial uptake of mepacrine in both term and preterm platelets was similar to that in adult platelets. Statistically significant differences between adults and both term and preterm infants, at two concentrations of mepacrine, were observed after stimulation with thrombin. CONCLUSION: Persistent mepacrine staining of infant PDGs, when compared with those of adults, after thrombin stimulation implies defective infant PDG release. This may partially explain why infants have impaired response to agonists requiring ATP release from PDGs.

Flow cytometry of neonatal platelet RNA.

PURPOSE: Serious disorders of hemostasis occur more often in the small, stressed preterm infant, partly due to immaturity of the newborn hemostatic mechanism. Information regarding the maturational development of platelets in infants has been limited by the large amounts of blood historically needed for platelet studies. The objective of this study was to determine differences between platelets in adults and infants by flow cytometric analysis of "reticulated" platelets. PATIENTS AND METHODS: Eighteen normal adults, 42 healthy term infants, and 27 preterm infants were studied. The infants were subdivided by mode of delivery: vaginal or Caesarean section. Platelet-rich plasma from adult whole blood and infant cord blood samples was divided into aliquots containing 5 X 10(6) platelets, fixed with 1% paraformaldehyde, and stained with thiazole orange for RNA content. The percentage of RNA positive "reticulated" platelets in each aliquot was then determined by flow cytometry. RESULTS: Group comparisons using ANOVA statistical analysis showed a significant difference (p<0.05) for platelet RNA content between adults and term infants, the adults having a higher percentage of reticulated platelets. Although the difference was smaller, adults also had a higher percentage of reticulated platelets than preterm infants. There was no significant difference in reticulated platelet values between infants within one gestational age group compared by type of delivery. CONCLUSION: There are demonstratable differences in adult and infant platelet RNA content that may reflect developmental differences in megakaryocytic/platelet kinetics.

Viral transduction of trkA into cultured nodose and spinal motor neurons conveys NGF responsiveness.

The trkA gene encodes the tyrosine kinase receptor that transduces the NGF signal. We have used a defective herpes simplex virus-1 (HSV-1) vector containing a full-length rat trkA sequence (pHSVtrkA) to express NGF receptors in primary cultures of neonatal nodose neurons and embryonic spinal motor neurons which are normally unresponsive to NGF. Transduction of rat neonatal nodose ganglion neurons and spinal motor neurons with pHSVtrkA resulted in expression of functional NGF receptors. Stimulation of these receptors by NGF resulted in the survival and the morphologic and biochemical differentiation of these neurons. These observations indicate that nodose and spinal motor neurons can express the downstream signal transduction apparatus necessary to mediate the survival and differentiating effects mediated by ligand binding to trkA and that transduction with pHSVtrkA can effectively convert NGF nonresponsive neurons into responsive ones.

Identification of a signal necessary for initiation of reverse transcription of the hepadnavirus genome.

Reverse transcription of the hepadnavirus genome initiates near the 3' end of the RNA template and has previously been shown to depend on sequences flanking the initiation site for DNA synthesis (C. Seeger and J. Maragos, J. Virol. 64:16-23, 1990). DNA synthesis leads to the covalent attachment of a protein to the 5' end of minus-strand DNA, and it is generally believed that this protein serves as the primer for reverse transcription. To examine priming in more detail, we have carried out a detailed genetic analysis of the nucleotide sequences at the origin of minus-strand DNA synthesis characterized in our earlier study. This mutational analysis has led to the identification of a short, four-nucleotide-long sequence as the signal for initiation of reverse transcription. This signal is a UUUC sequence motif flanking the position of the 5' end of minus-strand DNA, which alone is not sufficient for DNA synthesis, indicating that positional effects are also important to specify the origin of DNA synthesis.

The trk proto-oncogene rescues NGF responsiveness in mutant NGF-nonresponsive PC12 cell lines.

The trk tyrosine kinase proto-oncogene product gp140prototrk binds nerve growth factor (NGF) and is rapidly and selectively activated by this neurotrophic factor. To determine whether gp140prototrk is involved in transducing a functional NGF signal, PC12 cell mutants (PC12nnr) deficient in high affinity NGF binding and unresponsive to NGF were used. Northern analysis revealed that these mutant cells have greatly reduced levels of trk expression. PC12nnr cultures were transiently transfected with expression vectors encoding the full-length rat trk cDNA and assessed for responsiveness to NGF. Expression of exogenous trk rescued the capacity for NGF-promoted neurite outgrowth, cellular hypertrophy, and serum-free survival by these cells. These results indicate that gp140prototrk is necessary for functional NGF signal transduction.

The neurotrophic factors brain-derived neurotrophic factor and neurotrophin-3 are ligands for the trkB tyrosine kinase receptor.

Neurotrophic factors are essential for neuronal survival and function. Recent data have demonstrated that the product of the tyrosine kinase trk proto-oncogene binds NGF and is a component of the high affinity NGF receptor. Analysis of the trkB gene product, gp145trkB, in NIH 3T3 cells indicates that this tyrosine kinase receptor is rapidly phosphorylated on tyrosine residues upon exposure to the NGF-related neurotrophic factors BDNF and NT-3. Furthermore, gp145trkB specifically binds BDNF and NT-3 in NIH 3T3 cells and in hippocampal cells, but does not bind NGF. Thus, the trk family of receptors are likely to be important signal transducers of NGF-related trophic signals in the formation and maintenance of neuronal circuits.

Identification and characterization of the woodchuck hepatitis virus origin of DNA replication.

Replication of the woodchuck hepatitis virus (WHV) genome requires the synthesis of minus-strand DNA from an RNA template, the pregenome, by reverse transcription. During this reaction, the 5' end of minus-strand DNA becomes covalently linked to a protein. The position of the 5' end of minus-strand DNA was identified previously, but the initiation site for DNA synthesis on pregenomic RNA remained ambiguous because of a sequence repetition at the termini of the RNA template for reverse transcription. Employing a recently designed expression vector for the production of infectious WHV, we localized the origin of minus-strand DNA synthesis to the 3' end of pregenomic RNA. In addition, we identified the nucleotide sequences on pregenomic RNA that provide the signal for the initiation of reverse transcription. Removal of this signal sequence from pregenomic RNA abolished minus-strand DNA synthesis. Insertion of a DNA oligomer bearing this signal sequence at the 3' end of pregenomic RNA restored the production of minus-strand DNA joined to protein. Our results support a model in which protein is the primer for reverse transcription of minus-strand DNA of WHV.

Molecular analysis of the function of direct repeats and a polypurine tract for plus-strand DNA priming in woodchuck hepatitis virus.

The replication of the hepadnavirus DNA genome is initiated by reverse transcription of pregenome RNA into minus-strand DNA followed by plus-strand DNA synthesis. The priming of plus-strand DNA requires the transfer of an RNA primer from pregenome RNA to the primer-binding site on minus-strand DNA. Annealing of the primer to the primer-binding site is facilitated by short direct repeats, DR1 and DR2. To investigate the mechanism of plus-strand primer formation, we have introduced specific mutations into DR1 and DR2 and measured the effect of these mutants on initiation of plus-strand DNA synthesis. To facilitate such an analysis, we have constructed a vector for the efficient expression of woodchuck hepatitis virus in cultured cells. Our results suggest that the 3' end of the RNA primer is determined prior to its transfer to the primer-binding site and that the determination of the 3' end of the primer does not depend on a specific sequence motif at the cleavage site. In addition, we have identified an alternative initiation site for plus-strand DNA synthesis at a purine-rich sequence between DR1 and DR2. Initiation at this site occurs by a mechanism that is independent of the direct repeats and does not require the transfer of an RNA primer to the primer-binding site.

Tropical cyclone bebe creates a new land formation on funafuti atoll.

A huge rubble rampart 18 kilometers long was formed at Funafuti Atoll during tropical cyclone Bebe on 21 October 1972. The material forming the rampart was derived from deeper water offshore. The formation appears to be permanent and indicates that tropical storms may play a significant role in the formation of atoll islets.