Biomedical subjects

S A MacFarlane

Publications and source records attributed to S A MacFarlane.

25 records · Page 2Linked to original sources

PubMed1991

Interleukin 1 beta induces rapid phosphorylation and redistribution of talin: a possible mechanism for modulation of fibroblast focal adhesion.

The majority of interleukin 1 (IL-1) receptors in human fibroblasts has been shown to be localized at focal adhesions. This study describes rapid alterations caused by IL-1 beta/IL-1-receptor interaction at these sites. Fibroblast monolayers, incubated with IL-1 beta and prepared for electron microscopy, showed successive loss of cell-substratum contact and fewer and less-pronounced processes. Immunocytochemistry revealed loss and redistribution of the talin staining initially observed after 5-15 min of IL-1 beta incubation. Similarly, the cytoskeleton showed a decrease in staining and a disorganization starting from 15 to 30 min after IL-1 addition, whereas extracellular fibronectin appeared largely unaffected. Prelabeling with [32P]phosphate showed a 2- to 3-fold increase in the level of talin phosphorylation, peaking at 15 min. Phospho amino acid analyses revealed a higher level of serine and threonine phosphorylation. The data suggest that the action of IL-1 beta on fibroblasts may be partially mediated by direct phosphorylation of talin via activation of a protein serine/threonine kinase, leading to changes in transmembrane linkage proteins and the cytoskeleton. Such alterations at focal adhesions may provide a mechanism by which IL-1 can rapidly modulate cell-matrix interactions during inflammation and wound healing.

Cell Adhesion↗

PubMed1991

Analysis of the nucleotide sequence of bean pod mottle virus middle component RNA.

The complete nucleotide sequence of the middle component RNA (M RNA) of the comovirus bean pod mottle virus (BPMV) has been determined. The sequence consists of 3662 nucleotides and contains a single long open reading frame sufficient to code for a protein of 113,353 Da. The proteolytic processing sites within this protein have been identified by comparison with the known three-dimensional structure of the virion and cleavage at these sites would lead to a range of products consistent with those observed during processing of the M RNA-encoded polyproteins in vitro. We have performed computer-aided searches for reiterated sequences within BPMV M RNA which might explain why ordered RNA is visible in the electron density map of BPMV middle component particles (Chen, Z., Stauffacher, C. V., Li, Y., Schmidt, T., Bomu, W., Kamer, G., Shanks, M., Lomonossoff, G., and Johnson, J. E., 1989, Science 245, 154-159). These searches revealed both the presence of overrepresented pentameric sequences and a consensus sequence which was repeated 15 times within the RNA sequence.

Amino Acid Sequence↗

PubMed1991

Construction and analysis of infectious transcripts synthesized from full-length cDNA clones of both genomic RNAs of pea early browning virus.

Full-length cDNA clones of both RNAs of pea early browning virus have been constructed. Synthetic transcripts derived in vitro from these clones are infectious when inoculated onto plants. Electron microscopy revealed the presence of virions in transcript-inoculated plants, and both purified RNA and virions isolated from such plants could be used to infect other plants. Transcripts of RNA1 alone were able to replicate and spread systemically which is a characteristic of members of the tobravirus group of plant viruses.

Base Sequence↗

PubMed1989

Pea early browning virus RNA1 encodes four polypeptides including a putative zinc-finger protein.

We have determined the complete nucleotide sequence of RNA1 of the tobravirus pea early browning virus [PEBV] from an overlapping series of cDNA clones. The 7073 nucleotide sequence contains four open reading frames [ORFs]. The 5' proximal ORF encodes a 141K polypeptide, and readthrough of the opal [UGA] termination codon of this ORF would lead to the synthesis of a second, 201K polypeptide. Both of these polypeptides have extensive amino acid homology with the putative replicase proteins of tobacco rattle virus [TRV] and tobacco mosaic virus [TMV]. The third ORF encodes a 30K polypeptide which has homology with the TRV 29K and TMV 30K putative cell-to-cell spread proteins. The fourth, 3' proximal ORF encodes a 12K polypeptide which has extensive homology with the TRV 16K protein whose function is unknown. Examination of the amino acid sequences of the 12K and 16K gene products reveals in each the presence of two multiple-cysteine/histidine motifs, a finding which suggests that these proteins might have zinc and/or nucleic acid-binding properties.

Amino Acid Sequence↗

PubMed1989

Effects of interleukin-1 on fibroblast extracellular matrix, using a 3-dimensional culture system.

This study describes the alterations induced by Interleukin-1 alpha and -beta (IL-1 alpha and IL-1 beta) on fibroblast-synthesized extracellular matrix. Fibroblasts were grown between pieces of dentin or in collagen-coated Terasaki wells for 3 or 6-9 weeks to create 3-dimensional cell-containing matrices constituted primarily of proteoglycans and collagens, respectively. Following incubation with IL-1 alpha or IL-1 beta (10(-9) M) at 37 degrees C for 24 or 72 hr, samples were prepared for light and electron microscopy. Both IL-1 alpha and IL-1 beta induced collapse of the extracellular matrix by 72 hr, as manifested by a decrease of the cross-sectional area and an increased density of the matrices. Three-week matrices were reduced 26% and 45% by using IL-1 alpha and IL-1 beta, respectively. Comparable values obtained by using 6-week matrices were 14% and 30%. Cells within the matrix, normally stellate in shape with numerous extended processes, attained a more rounded or spindle shape with few and reduced processes and showed apparent alterations at cell matrix attachment sites and rearrangement of the cytoskeleton. Elongated cells at the top of the matrix appeared more compressed. The alterations were more pronounced in cultures incubated with IL-beta than with IL-1 alpha. Immunocytochemistry of extracellular matrix components revealed a decrease in staining intensity of chondroitin and dermatan sulfate in the 3-week matrix following IL-1 beta incubation. There was also a decrease in collagen type 1 staining of 9-week matrices treated with IL-1 alpha or IL-1 beta. These studies show that IL-1 has an effect on fibroblast-synthesized extracellular matrix and indicate that the effects of IL-1 alpha and IL-1 beta may differ. The resulting collapse of the matrix appears at least in part to be due to changes in proteoglycans and collagens.

Actin Cytoskeleton↗

PubMed1987

Analysis of the Klebsiella pneumoniae ntrB gene by site-directed in vitro mutagenesis.

A number of in-frame insertion and deletion mutations have been constructed in vitro in the Klebsiella pneumoniae ntrB gene and the effects of each mutant NtrB protein on NtrC activity have been assessed after reintroduction of the ntrB mutation into the glnA ntrBC operon. These experiments suggest that the phosphorylation of NtrC catalysed by NtrB not only makes NtrC competent as a transcriptional activator but also improves the DNA-binding properties and hence the negative control functions of NtrC. The variety of NtrB phenotypes obtained suggest a structure/function model for the protein.

Amino Acid Sequence↗

PubMed1985

The nucleotide sequence of the nitrogen regulation gene ntrB and the glnA-ntrBC intergenic region of Klebsiella pneumoniae.

The nucleotide sequence of the Klebsiella pneumoniae ntrB gene and the glnA-ntrBC intergenic region has been determined. NtrB encodes a 38,409 Dalton polypeptide with a potential DNA-binding domain between residues 67 and 86. This N-terminal domain may play a role in the co-operative control of ntr-regulated promoters by the ntrB and ntrC products. Mapping of in vivo transcripts with S1 nuclease identified three transcripts in the glnA-ntrBC intergenic region. Two transcripts originate upstream of glnA; one reading through into ntrBC and one terminating at a sequence resembling a rho-independent terminator between glnA and ntrBC. A third transcript originates from the ntrBC promoter which has a consensus binding site for the ntrC product in the -10 region. Comparison of the glnA-ntrBC intergenic sequences from K. pneumoniae, Escherichia coli and Salmonella typhimurium has identified a number of conserved features and some significant differences.

Cloning, Molecular↗