Detection and identification of FaeC as a minor component of K88 fibriilae of Escherichia coli

A tribrid gene containing ompF, faeC, and lacZ sequences was constructed by subcloning a large central segment of the K88ab gene encoding the fibrillar subunit-like protein FaeC into the open reading frame expression vector pORF2. The resulting tribrid protein was isolated and used to raise antibodies against the FaeC protein. These antibodies were then used for the detection and subcellular localization of the FaeC protein in Escherichia coli harbouring the K88ab-encoding plasmid pFM205 or mutant derivatives. Immunoblotting of subcellular fractions and of purified fibrillae, and agglutination experiments using whole cells revealed that the FaeC protein is present in the periplasm and as a minor component in the K88ab fibrillae. FaeC was also detected in purified K88ac and K88ad fibrillae. Immunoelectron microscopy confirmed the presence of FaeC in K88ab fibrillae, particularly at the tips of the longer fibrillae.     

Mechanism of L-glutamate transport in membrane vesicles from Bacillus stearothermophilus.

In the presence of electrochemical energy, several branched-chain neutral and acidic amino acids were found to accumulate in membrane vesicles of Bacillus stearothermophilus. The membrane vesicles contained a stereo-specific transport system for the acidic amino acids L-glutamate and L-aspartate, which could not translocate their respective amines, L-glutamine and L-asparagine. The transport system was thermostable (Ti = 70 degrees C) and showed highest activities at elevated temperatures (60 to 65 degrees C). The membrane potential or pH gradient could act as the driving force for L-glutamate uptake, which indicated that the transport process of L-glutamate is electrogenic and that protons are involved in the translocation process. The electrogenic character implies that the anionic L-glutamate is cotransported with at least two monovalent cations. To determine the mechanistic stoichiometry of L-glutamate transport and the nature of the cotranslocated cations, the relationship between the components of the proton motive force and the chemical gradient of L-glutamate was investigated at different external pH values in the absence and presence of ionophores. In the presence of either a membrane potential or a pH gradient, the chemical gradient of L-glutamate was equivalent to that specific gradient at different pH values. These results cannot be explained by cotransport of L-glutamate with two protons, assuming thermodynamic equilibrium between the driving force for uptake and the chemical gradient of the substrate. To determine the character of the cotranslocated cations, L-glutamate uptake was monitored with artificial gradients. It was established that either the membrane potential, pH gradient, or chemical gradient of sodium ions could act as the driving force for L-glutamate uptake, which indicated that L-glutamate most likely is cotranslocated in symport with one proton and on sodium ion.     

The Saccharomyces cerevisiae SOC8-1 gene and its relationship to a nucleotide kinase

The yeast SOC8-1 gene was originally identified by partial complementation of cdc8 mutant strains. We have carried out Bal31 deletion analysis of the SOC8-1 gene to define the minimal size which is required for the complementation of the cdc8 mutation. When the SOC8-1 gene is cloned in a multicopy plasmid, it enables temperature-resistant growth in the cdc8 mutant strain, while the SOC8-1 gene in a single copy plasmid does not. Thus, its suppression of the cdc8 mutant is dosage dependent. The high copy number vector carrying the SOC8-1 gene can complement five different cdc8 alleles, indicating that the suppression is not allele specific. Since CDC8 encodes thymidylate kinase, cells bearing a high copy number plasmid containing SOC8-1 gene were tested for the ability to phosphorylate several nucleoside monophosphates, including UMP, GMP and dTMP. Significantly increased phosphorylation activity was observed, suggesting that SOC8-1 encodes a nucleotide kinase. Both restriction enzyme analysis of the SOC8-1 gene and partial purification of the overproduced kinase in SOC8-1 overproducing strains suggest that SOC8-1 may be allelic with URA6. Consistent with these results, both SOC8-1 and URA6 are located on chromosome XI. Thus, one possible suppression mechanism is that SOC8-1 may provide a trans-acting dTMP kinase activity, bypassing the cdc8 gene defect.     

The acquisition of increased insulin-responsive hexose transport in 3T3-L1 adipocytes correlates with expression of a novel transporter gene

The expression of two genes encoding facilitated glucose transporter proteins was studied during the differentiation of the 3T3-L1 fibroblastic cell line into adipocytes. The mRNA encoding the widely expressed HepG2/brain glucose transporter (GTI) is detectable in fibroblasts and its abundance remains unchanged during differentiation. On the other hand, the mRNA encoding a glucose transporter protein (GTIII) localized exclusively to muscle and adipose tissue is undetectable in fibroblasts but present in adipocytes. GTIII mRNA is first expressed three days after differentiation of 3T3-L1 cells has begun. Similarly, it is not until 3 days following the initiation of differentiation that GTIII protein can be detected, as assayed either by Western immunoblot or indirect immunofluorescence. The latter technique localizes GTIII predominantly to the perinuclear region of the adipocyte. The appearance of GTIII in developing fat cells correlates temporally with the acquisition of an increased stimulation of hexose uptake by maximal concentrations of insulin. These data support the concept that the marked increase in hexose transport in adipocytes in response to insulin is dependent on the expression in these cells of a specific, hormone-regulatable transport protein.     

Decreased thermal denaturation temperature of osteogenesis imperfecta mutant collagen is independent of post-translational overmodifications of lysine and hydroxylysine

Fibroblasts from many patients with osteogenesis imperfecta (OI) synthesize and secrete Type I collagen which is both overmodified and exhibits a decreased thermal denaturation temperature. We have examined the relationship between overmodification and decreased melting temperature in several favorable OI mutants by selectively inhibiting lysyl hydroxylase activity with the drug Minoxidil and comparing the melting profiles of the resultant undermodified collagen with untreated control. Minoxidil treatment causes an appreciable decrease in hydroxylysine with compensatory increases in lysine content, and the delayed sodium dodecyl sulfate-polyacrylamide gel electrophoretic mobility of the overmodified collagen chains becomes normal. However, the decreased melting temperature was unchanged from untreated OI control. When unhydroxylated collagen produced by normal control and OI fibroblasts incubated with alpha,alpha'-dipyridyl was examined, mutant OI molecules melted at a lower temperature than control. These data indicate that the decreased thermal denaturation temperature of OI mutant collagen is independent of post-translational overmodification of lysine or hydroxylysine. Presumably, substitutions for glycine in the Gly-X-Y structural motif distort the helix and produce lower melting temperatures by presently unknown mechanisms.     

The effect of poly(ADP-ribosyl)ation on native and H1-depleted chromatin. A role of poly(ADP-ribosyl)ation on core nucleosome structure

The effect of poly(ADP-ribosyl)ation on native and H1-depleted chromatin was analyzed by gel electrophoresis, electron microscopy, and velocity sedimentation. In parallel, the interaction of automodified poly(ADP-ribose) polymerase with native and H1-depleted chromatin was analyzed. In H1-depleted chromatin histone H2B becomes the major poly(ADP-ribose) histone acceptor protein, whereas in native chromatin histone H1 was the major histone acceptor. Poly(ADP-ribosyl)ation of H1-depleted chromatin prevented the recondensation of polynucleosomes reconstituted with exogenous histone H1. This is probably due to the presence of modified poly(ADP-ribose) polymerase and hyper(ADP-ribosyl)ated histone H2B. Indeed, about 40% of the modified enzyme remained associated with H1-depleted chromatin, while less than 1% of the modified enzyme was bound to native chromatin. The influence of poly(ADP-ribosyl)ation on the chromatin conformation was also studied at the level of nucleosome in using monoclonal and polyclonal antibodies specific for individual histones and synthetic peptides of histones. In native chromatin incubated in the presence of Mg2+ there was a drop in the accessibility of histone epitopes to monoclonal and polyclonal antibodies whereas upon poly(ADP-ribosyl)ation their accessibility was found to remain even in the presence of Mg2+. In poly(ADP-ribosyl)ated H1-depleted chromatin an increased accessibility of some histone tails to antibodies was observed.     

Immunoaffinity purification, partial sequence, and subcellular localization of rat liver phospholipase A2

Monoclonal antibodies against rat liver mitochondrial phospholipase A2 were used to develop a rapid immunoaffinity chromatography for enzyme purification. The purified enzyme showed a single band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the N-terminal 24 amino acids was determined. This part of the sequence showed only 25% homology with that of rat pancreatic phospholipase A2 but was 96% identical to that of rat platelet and rat spleen membrane-associated phospholipase A2. These enzymes are distinguished from pancreatic phospholipases A2 by the absence of Cys-11. In rat liver phospholipase A2 activity has been reported in various subcellular fractions. All of these require Ca2+ and have a pH optimum in the alkaline region, but little is known about the structural relationship and quantitative distribution of these enzymes. We have investigated these points after solubilization of the phospholipase A2 activity from total homogenates and crude subcellular fractions by extraction with 1 M potassium chloride. Essentially all of the homogenate activity could be solubilized by this procedure indicating that the enzymes occurred in soluble or peripherally membrane-associated form. Gel filtration and immunological cross-reactivity studies indicated that phospholipases A2 solubilized from membrane fractions shared a common epitope with the mitochondrial enzyme. The quantitative distribution of the immunopurified enzyme activity among subcellular fractions followed closely that of the mitochondrial marker cytochrome c oxidase. Rat liver cytosol contained additional Ca2+-dependent and -independent phospholipase activities.     

Human serum amyloid A protein. The assignment of the six major isoforms to three published gene sequences and evidence for two genetic loci

Serum amyloid A protein (apo-SAA) is an acute-phase reactant and an apolipoprotein of high density lipoproteins (HDL). Six major isoforms of apo-SAA occur in humans (pI 6.0, 6.4, 7.0, 7.4, 7.5, 8.0). In this report we have rationalized the phenotypic expression of apo-SAA isoforms with published apo-SAA structures predicted from apo-SAA cDNA's pA1 and pSAA82 and the genomic DNA SAAg9. The six apo-SAA isoforms fall into three pairs, pI 6.0/6.4, 7.0/7.5, and 7.4/8.0, which are products of cDNA pA1, cDNA pSAA82, and genomic DNA SAAg9, respectively. The second of each isoform pair (i.e. pI 6.4, 7.5, and 8.0) is the "primary" product: a 104-residue peptide with the NH2-terminal sequence Arg-Ser-Phe-Phe. Each primary product is processed either to a major 103-residue peptide with the NH2-terminal sequence Ser-Phe-Phe or processed to a minor 102-residue product which results from the loss of both an Arg and a Ser residue from the NH2 termini. These "secondary" products have the lower pI values of 6.0, 7.0, and 7.4, respectively. The isoelectric points of the SAAg9 products were confirmed by expression of SAAg9 in transfected mouse L-cells. Both the pI 8.0 and 7.4 isoforms were present in cellular extracts, suggesting that post-translational modification of apo-SAA may occur intracellularly. However, the greater relative abundance of the pI 7.4 isoform extracellularly suggests that the major conversion may occur after secretion. Whereas the gene corresponding to the pA1 cDNA sequence does not show allelic variation, the segregation characteristics of the pI 7.0/7.5 and 7.4/8.0 isoform pairs amongst individuals suggests that these isoforms are the products of genes (with sequences corresponding to pSAA82 and SAAg9, respectively) which are allelic variants at a single locus distinct from that for the pI 6.0/6.4 isoform pair.     

Involvement of LFA-1 in lymphoma invasion and metastasis demonstrated with LFA-1-deficient mutants

Lymphocyte function-associated antigen-1 (LFA-1) is a leukocyte and lymphoma cell surface protein that promotes intercellular adhesion. We have previously shown that the invasion of hepatocyte cultures by lymphoma cells is inhibited by anti-LFA-1 antibodies (Roos, E., and F. F. Roossien. 1987. J. Cell Biol. 105:553-559). In addition, we now report that LFA-1 is also involved in invasion of lymphoma cells into fibroblast monolayers. To investigate the role of LFA-1 in metastasis of these lymphoma cells, we have generated mutants that are deficient in LFA-1 cell surface expression because of impaired synthesis of either the alpha or beta subunit precursor of LFA-1. We identified at least three distinct mutant clones. The invasive potential of the mutant cells in vitro, in both hepatocyte and fibroblast cultures, was considerably lower than that of parental cells. The metastatic potential of the mutants was much reduced, indicating that LFA-1 expression is required for efficient metastasis formation by certain lymphoma cells.