Academic medical center libraries on the Web.

Academic medical center libraries are moving towards publishing electronically, utilizing networked technologies, and creating digital libraries. The catalyst for this movement has been the Web. An analysis of academic medical center library Web pages was undertaken to assess the information created and communicated in early 1997. A summary of present uses and suggestions for future applications is provided. A method for evaluating and describing the content of library Web sites was designed. The evaluation included categorizing basic information such as description and access to library services, access to commercial databases, and use of interactive forms. The main goal of the evaluation was to assess original resources produced by these libraries.

A comparison of four current awareness services.

A study was undertaken to compare four current awareness services: CurrentContents/OVID, Reference Update/OVID, CARL Uncover, and FirstSearch (ContentsFirst and ArticlesFirst). The features and performances of each are evaluated. Three factors determined to be important are ease of use, health sciences coverage, and journal currency. This article is a summary of the results of this evaluation.

The synapse-specific phosphoprotein F1-20 is identical to the clathrin assembly protein AP-3.

F1-20 and AP-3 are independently described, synapse-associated, developmentally regulated phosphoproteins with similar apparent molecular masses on SDS-polyacrylamide gel electrophoresis (PAGE). F1-20 was cloned and characterized because of its synapse specificity. AP-3 was purified and studied biochemically because of its function as a clathrin assembly protein. Here we present evidence that establishes the identity of F1-20 and AP-3. Monoclonal antibodies against F1-20 and AP-3 both specifically recognize a single protein from mouse brain with an apparent molecular mass of 190 kDa on SDS-PAGE. These monoclonal antibodies also specifically recognize the cloned F1-20 protein expressed in Escherichia coli. The anti-F1-20 monoclonal antibody (mAb) stains a bovine protein with an apparent molecular mass on SDS-PAGE of 190 kDa that copurifies with brain clathrin-coated vesicles (CCVs) and that can be extracted from the brain CCVs under conditions that extract AP-3. The anti-F1-20 and anti-AP-3 mAbs specifically recognize the same spot on a two-dimensional gel run on a bovine brain clathrin-coated vesicle extract. AP-3 purified from bovine brain CCVs is recognized by both the anti-F1-20 and anti-AP-3 mAbs. Purified preparations of bovine AP-3 and bacterially expressed mouse F1-20 give identical patterns of protease digestion with bromelain and subtilisin. Sequence analyses reveal that F1-20 has an essentially neutral 30-kDa NH2-terminal domain with an amino acid composition typical of a globular structure and an acidic COOH-terminal domain rich in proline, serine, threonine, and alanine. This is consistent with proteolysis experiments that suggested that AP-3 could be divided into a 30-kDa globular uncharged clathrin-binding domain and an acidic, anomalously migrating domain.

Characterization of a novel synapse-specific protein. I. Developmental expression and cellular localization of the F1-20 protein and mRNA.

A molecular description of the nerve terminal will be required to understand synaptic function fully. The goals of this study were to contribute toward such a description by characterizing a novel synapse-specific protein. A monoclonal antibody library was screened for antibodies to synaptic proteins. The antibodies were then used to isolate cDNA clones by expression screening. Here we report a detailed characterization of the protein reactive with monoclonal antibody F1-20. Immunohistochemical and biochemical analyses revealed that the F1-20 protein is synapse associated. Western blot analyses revealed that the F1-20 protein is a brain-specific polypeptide with an apparent molecular weight on SDS-PAGE of 190,000 Da. Northern blot analyses indicated that probes generated from an F1-20 cDNA clone hybridize to a single brain-specific mRNA of approximately 4.8 kilobases. In situ hybridization experiments demonstrated that F1-20 mRNA expression is neuronal specific. Northern and Western blot analyses indicated that F1-20 mRNA levels increase abruptly at postnatal day 4 and protein levels increase abruptly at postnatal day 7. This corresponds to a period of active synaptogenesis and synaptic maturation in the mouse CNS. We characterized the neuroanatomical distribution of the F1-20 protein by immunohistochemistry, and of the F1-20 mRNA by in situ hybridization. We found that the F1-20 mRNA and protein are expressed nonuniformly in brain. Variation in the expression of the F1-20 protein is complex and reveals patterns also exhibited by probes directed against other synapse-associated molecules. The highest levels of F1-20 protein are found in the cortically organized regions of the brain. The highest levels of F1-20 mRNA are found in long-distance projection neurons. There is also variation in the expression of F1-20 mRNA between different classes of large output neuron, as well as extensive variation in the expression of F1-20 mRNA between different nuclear groups.

Characterization of a novel synapse-specific protein. II. cDNA cloning and sequence analysis of the F1-20 protein.

The F1-20 protein is a novel neuronal-specific, synapse-associated protein that is expressed nonuniformly in mouse brain. Expression of the F1-20 protein is developmentally regulated in a pattern coincident with active synaptogenesis and synaptic maturation. Here we report the cloning of the cDNA sequence for the F1-20 protein. We found two distinct isoforms of F1-20 cDNA that differed by the presence of 15 additional nucleotides, which does not interrupt the open reading frame. RNase protection analysis and PCR amplification of mouse brain RNA revealed that both isoforms are present in cellular RNA. It is likely that the two F1-20 mRNA isoforms are derived from RNA splicing events utilizing alternative 3' acceptor sites. Analysis of the deduced amino acid sequence for the complete open reading frame revealed that the predominant F1-20 mRNA encodes an 896 amino acid polypeptide with a molecular weight of 91,319 Da. The deduced amino acid sequence does not contain a signal sequence, or any extensive hydrophobic regions. The deduced amino acid sequence does contain a number of consensus sequences for protein kinases. Searches of the protein and nucleic acid sequence data bases revealed that the F1-20 protein has not been previously characterized at the primary structure level, although a weak similarity was found between rabbit calpastatin and the C-terminal portion of the F1-20 protein. We then determined biochemically that the F1-20 protein is a substrate for Ca(2+)-dependent proteolysis, which is specifically inhibited by calpain inhibitors in vitro. This indicates that the F1-20 protein is a substrate for neuronal calpain. We observed that treatment of a synaptosomal lysate with alkaline phosphatase led to an increase in the electrophoretic mobility of the F1-20 protein, as well as to an increase in the sharpness of the electrophoretic band. This indicates that the F1-20 protein is phosphorylated in vivo.

In situ hybridization mapping of glucocorticoid receptor messenger ribonucleic acid in rat brain.

We have mapped the distribution of glucocorticoid receptor (GR) mRNA in the male adult rat brain using T7 RNA polymerase transcripts of a 1155 base pair rat GR cDNA clone comprising the coding region for amino acids 140-525. Strong expression of GR mRNA was found in the neurons of the CA1 and CA2 fields of the hippocampus and in the paraventricular and periventricular hypothalamic nuclei. Moderate to strong hybridization was found in the dorsal thalamic nuclei, layers II and VI of the cerebral cortex, the anterior olfactory nucleus and primary olfactory cortex, the hypothalamic mammillary nuclei, the subthalamus, and the granule and mitral cells of the olfactory bulb. Weak to moderate hybridization was found in many other regions of the tel- and diencephalon. Mes- and rhomboencephalic neurons displayed very low levels of GR mRNA relative to the levels observed in the tel- and diencephalon. In the cerebellum, moderate to strong levels of mRNA were detected in the granule and Purkinje cell layers with very low levels elsewhere. Nonneuronal brain elements, such as glial cells, the pia mater, and the choroid plexus, were found to express low to moderate amounts of GR mRNA. These results confirm and extend mapping studies of steroid receptors in the brain using radiolabeled steroids or monoclonal antibodies against rat liver GR and demonstrate that the relative distribution of GR protein in different brain nuclei reflects differences in GR mRNA levels. The rat GR cDNA clone is also shown to provide suitable probes for mapping GR gene expression in the mouse brain.

The neuroectodermal tumor of bone.

Four small round cell malignant tumors of bone occurring in children are described. There was no catecholamine secretion and the clinical, radiologic, and biopsy diagnosis in each was Ewing's sarcoma. Glycogen was sparse both on imprints and in tissue sections. The tumors, when extensively sampled, had areas of a lobular growth pattern and Homer Wright rosettes. The rosettes were always focal and varied in complexity from case to case; they were rudimentary in one instance and markedly fibrillar in the most obvious instance. Neuron-specific enolase was demonstrated in tissue sections and in longterm cell cultures from three of the tumors. The cultured cells put out moderately long beaded processes in serum-free medium but had no catecholamine fluorescence. Electron microscopy of the tumor rosettes and the cultured cells showed processes containing aggregates of microtubules and only one case had rare neurosecretory granules. This study suggests that some small round cell tumors of bone and soft tissue in children, which present as Ewing's sarcoma, are neuroectodermal in nature.