Opportunities on the Web: a role for information professionals, using the development of the BMA Library Online Service as a case study.

The paper considers how attributes and skills fundamental to the information profession may be applied to the development and management of Web-based services and resources. It also looks at the need for the acquisition of new skills as part of a continuing professional development programme. The report considers the changing role of the Webmaster and the implications this might have in terms of the role of information professionals. To offer a practical example, from within the healthcare sector, the article goes on to describe the development of BMA Library Online. This is the BMA Library's own suite of Web-based information services and resources for personal members, institutional members and staff. The library's own Web team has been responsible for the development and maintenance of BMA Library Online. To conclude, the paper looks forward to future possibilities for information professionals as Web developers and at how this might be influenced by changes in the Webmaster role. It is crucial that skills and expertise are shared as the roles that go to make up a Webmaster or Webmaster team continue to merge and evolve, and with the possible wider distribution of provision of Web content within organizations.

User support for a library-managed online database search service: the BMA Library free MEDLINE service.

This paper discusses user support in the context of a library-managed online database search service. Experience is drawn from the British Medical Association (BMA) Library's Free MEDLINE Service. More than 9,600 BMA members, who are largely unfamiliar with computer communications and database searching, have registered as users of the service. User support has played a significant role in the development of the service and has comprised four main aspects: an information pack, a help desk, online help, and MEDLINE courses. The paper includes an analysis of help desk usage statistics collected from January 1996 through June 1996, and highlights other relevant research. Plans for further service enhancements and their implications in terms of future user support are discussed.

AML1 is expressed in skeletal muscle and is regulated by innervation.

Although most skeletal muscle genes are expressed at similar levels in electrically active, innervated muscle and in electrically inactive, denervated muscle, a small number of genes, including those encoding the acetylcholine receptor, N-CAM, and myogenin, are expressed at significantly higher levels in denervated than in innervated muscle. The mechanisms that mediate electrical activity-dependent gene regulation are not understood, but these mechanisms are likely to be responsible, at least in part, for the changes in muscle structure and function that accompany a decrease in myofiber electrical activity. To understand how muscle activity regulates muscle structure and function, we used a subtractive-hybridization and cloning strategy to identify and isolate genes that are expressed preferentially in innervated or denervated muscle. One of the genes which we found to be regulated by electrical activity is the recently discovered acute myeloid leukemia 1 (AML1) gene. Disruption and translocation of the human AML1 gene are responsible for a form of acute myeloid leukemia. AML1 is a DNA-binding protein, but its normal function is not known and its expression and regulation in skeletal muscle were not previously appreciated. Because of its potential role as a transcriptional mediator of electrical activity, we characterized expression of the AML1 gene in innervated, denervated, and developing skeletal muscle. We show that AML1 is expressed at low levels in innervated skeletal muscle and at 50- to 100-fold-higher levels in denervated muscle. Four AML1 transcripts are expressed in denervated muscle, and the abundance of each transcript increases after denervation. We transfected C2 muscle cells with an expression vector encoding AML1, tagged with an epitope from hemagglutinin, and we show that AML1 is a nuclear protein in muscle. AML1 dimerizes with core-binding factor beta (CBF beta), and we show that CGF beta is expressed at high levels in both innervated and denervated skeletal muscle. PEBP2 alpha, which is structurally related to AML1 and which also dimerizes with CBF beta, is expressed at low levels in skeletal muscle and is up-regulated only weakly by denervation. These results are consistent with the idea that AML1 may have a role in regulating gene expression in skeletal muscle.

Dystrophin is a component of the subsynaptic membrane.

A subsynaptic protein of Mr approximately 300 kD is a major component of Torpedo electric organ postsynaptic membranes and copurifies with the AChR and the 43-kD subsynaptic protein. mAbs against this protein react with neuromuscular synapses in higher vertebrates, but not at synapses in dystrophic muscle. The Torpedo 300-kD protein comigrates in SDS-PAGE with murine dystrophin and reacts with antibodies against murine dystrophin. The sequence of a partial cDNA isolated by screening an expression library with mAbs against the Torpedo 300-kD protein shows striking homology to mammalian dystrophin, and in particular to the b isoform of dystrophin. These results indicate that dystrophin is a component of the postsynaptic membrane at neuromuscular synapses and raise the possibility that loss of dystrophin from synapses in dystrophic muscle may have consequences that contribute to muscular dystrophy.

Construction of plasmid vectors with a non-antibiotic selection system based on the Escherichia coli thyA+ gene: application to cholera vaccine development.

The construction of live oral carriers based on attenuated Salmonella strains as vectors offers a new approach to vaccine development. We have constructed a set of plasmid vectors which have the thyA gene of Escherichia coli (encoding thymidylate synthetase) as the marker for selection and maintenance of plasmid clones. The thyA system offers an alternative to antibiotic-resistance selection markers. It can be easily adapted to a particular host-vector combination since thyA chromosomal mutations can be readily introduced by trimethoprim selection. We also describe the construction of thyA-based plasmids with the Vibrio cholerae rfb genes (encoding O-antigen biosynthesis of the Inaba serotype). These have been found to be useful in the construction of candidate bivalent cholera-typhoid vaccines.

Effect of lipopolysaccharide core synthesis mutations on the production of Vibrio cholerae O-antigen in Escherichia coli K-12.

The rfb genes of Vibrio cholerae O1 (Ogawa serotype) were subcloned into a derivative of pBR322. This plasmid was transformed into several Escherichia coli K-12 mutant strains which produce an incomplete lipopolysaccharide (LPS)-core-oligosaccharide region. The data indicate that the V. cholerae O-antigen is assembled onto the E. coli LPS and that at least two glucoses are needed in the core in order to achieve a high level of production. These data are consistent with the reported presence of glucose in the V. cholerae LPS-core-oligosaccharide region.

Multiple dystrophin isoforms are associated with the postsynaptic membrane of Torpedo electric organ.

We have shown previously that dystrophin is a component of postsynaptic membranes in Torpedo electric organ and is localized at mammalian neuromuscular synapses. In skeletal muscle, dystrophin is also detectable at the non-synaptic membrane of the myofiber, whereas in the electric organ, dystrophin is strictly localized to the postsynaptic membrane, and is not detectable in non-synaptic membranes. Multiple isoforms of dystrophin are present in skeletal muscle, and different isoforms could potentially be targetted to synaptic and non-synaptic membranes. We sought to determine whether the electric organ contains a single, or multiple isoforms of dystrophin, and we show here that the electric organ contains both a and b isoforms of dystrophin. Because dystrophin is found only at the postsynaptic membrane of the electric organ, we conclude that the two isoforms coexist in the postsynaptic membrane.

Cloning and sequence analysis of a rat liver cDNA encoding acyl-peptide hydrolase.

Acyl-peptide hydrolase catalyzes the removal of an N alpha-acetylated amino acid residue from an N alpha-acetylated peptide. Two overlapping degenerate oligonucleotide probes based on the sequence of a CNBr tryptic peptide, derived from purified rat acyl-peptide hydrolase, were synthesized and used to screen a rat liver lambda gt11 cDNA library. A 2.5-kilobase cDNA was cloned and sequenced. This clone contained 2364 base pairs of rat acyl-peptide hydrolase sequence but lacked a translational initiation codon. Using a 220-base pair probe derived from near the 5'-end of this almost full-length cDNA to rescreen the library, full-length clones were isolated, which contained an in-frame ATG codon at nucleotides 6-8 and encoded the NH2-terminal sequence, Met-Glu-Arg-Gln.... The DNA sequence encoded a protein of 732 amino acid residues, 40% of which were confirmed by protein sequence data from 19 CNBr or CNBr tryptic peptides. The isolated enzyme is NH2-terminally blocked (Kobayashi, K., and Smith, J. A. (1987) J. Biol. Chem. 262, 11435-11445), and based on the NH2-terminal protein sequence deduced from the DNA sequence and the sequence of the most NH2-terminal CNBr peptide, it is likely that the NH2-terminal residue is an acetylated methionine residue, since such residues are frequently juxtaposed to glutamyl residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527). The RNA blot analysis revealed a single message of 2.7 kilobases in various rat tissues examined. Although this enzyme is known to be inhibited by diisopropyl fluorophosphate and acetylalanine chloromethyl ketone (Kobayashi, K., and Smith, J. A. (1987) J. Biol. Chem. 262, 11435-11445), no strong similarity in protein sequence has been found with other serine proteases. This result suggests that acyl-peptide hydrolase may be a unique serine protease.

A physical map of the chromosomal region determining O-antigen biosynthesis in Vibrio cholerae O1.

We have previously described the cosmid cloning of the genes determining the biosynthesis of the Inaba and Ogawa O-antigens of the lipopolysaccharides of Vibrio cholerae O1 (Manning et al., 1986). By Southern hybridization analysis of chromosomal and cosmid DNA, and heteroduplex analysis between the clones we have been able to precisely define the region of contiguous chromosomal DNA in the vicinity of the O-antigen-encoding region. These data and comparison of end points of clones and of deletion derivatives demonstrate that at least 16 kb of a 19-kb SstI fragment is required to encode O-antigen biosynthesis. Expression of O-antigen is independent of the orientation of this SstI fragment with respect to cloning vectors suggesting that its regulatory region has been cloned intact. No detectable differences were observed in the restriction patterns of the Inaba and Ogawa coding regions implying that only minor changes are involved when serotype conversion (Inaba to Ogawa or vice versa) occurs. Bhaskaran [Ind. J. Med. Res. 47 (1959) 253-260] originally defined this region associated with O-antigen biosynthesis oag; however, to be consistent with other organisms [Hitchcock et al., J. Bacteriol. 166 (1986) 699-705], it is suggested this be changed to rfb.

Molecular cloning and expression in Escherichia coli K-12 of the O antigens of the Inaba and Ogawa serotypes of the Vibrio cholerae O1 lipopolysaccharides and their potential for vaccine development.

The gene clusters that determine the biosynthesis of both the Inaba and Ogawa serotypes of the O antigen of the lipopolysaccharide of Vibrio cholerae were cloned and expressed in Escherichia coli K-12. Restriction analysis of the clones demonstrated that about 15 kilobases were common to all clones and a further 5 kilobases were common to the Ogawa clones. The O antigens expressed by E. coli K-12 had the specificity of V. cholerae. Antibodies raised against E. coli K-12 that harbor one of these clones, pPM1001 (Inaba), were as highly protective in the infant mouse model system as were antibodies to V. cholerae itself. Introduction of such clones into suitable carrier strains could be expected to produce a good oral immunogen against cholera.