52K Protein Research Articles

Presence of neurotensin and neuromedin-N within a common precursor from a human pancreatic neuroendocrine tumor.

An acid extract of a human neuroendocrine pancreatic adenoma was found to contain very high concentrations of immunoreactive neurotensin (iNT; approximately 130 mumol/L) as well as immunoreactive neuromedin-N (iNMN; approximately 40 mumol/L), portions of which (iNT, 0.2%; iNMN, 30%) were found in large molecular forms. Processing of the large forms could be mimicked by treatment with pepsin, which increased their immunoreactivity 15- to 20-fold (iNT) and 1- to 2-fold (iNMN), liberating a peptide similar to NMN and 2 fragments of NT [primary product, NT-(4-13)]. Biochemical characterizations using gel electrophoresis, isoelectric focusing, and high pressure liquid chromatography indicated that the large forms were highly basic (pI 8.5-9.5) proteins with a mol wt of about 20K (78% of the total), 45K (8%), and 60K (4%). The 20K protein contained iNT and iNMN in a 1:1 ratio, while a slightly smaller species contained only NMN. These results are in agreement with cDNA studies of canine intestinal mRNA, indicating the presence of a 170-amino acid precursor containing 1 copy each of NT and NMN. They further indicate that within this tumor differential processing of precursor occurred, resulting in a NT to NMN ratio of about 3:1, with additional NMN stored in large molecular forms.

Analysis of human vaginal secretions by SDS-polyacrylamide gel electrophoresis

Proteins in 68 vaginal secretions from 50 women were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Although there were considerable numbers of intermediate types, the secretions could be classified into the following groups based on the densitometric patterns. A relatively low concentration of 67K protein (albumin) and a high concentration of 52K protein (IgG) were the patterns common to pre- and post-menstrual phases and pregnant stadium. A relatively high concentration of the 67K protein and a moderate concentration of the 52K protein characterized the midcycle. A considerably high concentration of 67K protein compared with the 79K (transferrin), 58K (IgA) and 52K bands was the pattern for menstrual phase and postpartum stage.

The P protein and the nonstructural 38K and 29K proteins of newcastle disease virus are derived from the same open reading frame

The nucleotide sequence of cloned cDNA copies of the mRNA encoding the Newcastle disease virus (NDV), strain AV, phosphoprotein (P) was determined. The sequence of 1443 nucleotides contains one long open reading frame which could encode a protein with a molecular weight of 42,126, and two smaller open reading frames which could encode proteins with molecular weights of 11,178 and 13,935. Full-length cDNA clones were constructed in an SP6 vector, mRNA was transcribed in a cell-free system using the SP6 polymerase, and the mRNA was translated in a wheat germ cell-free extract. The P mRNA directed the synthesis of, primarily, four products. One, with a molecular weight of 53,000 Da, comigrated with authentic P protein made in infected cells and was precipitable with antisera with specificity for the NDV P protein. The other products of the cell-free reaction had molecular weights of 38,000, 29,000 and 12,000. The 29,000- and the 38,000-Da polypeptides were also precipitable with anti-P protein antibody. Using truncated cDNA clones, evidence is presented that the 38,000- and 29,000-Da proteins are derived from initiation at AUG triplets in the same reading frame as the P protein. Infected cells also contain these polypeptides which may be analogous to C proteins of other paramyxoviruses. Thus the NDV P protein mRNA is different than most other paramyxovirus P protein mRNAs which are translated in two different reading frames to yield the P and C proteins.

Immunological studies on teleocalcin and salmon corpuscles of Stannius.

This report describes the characterization of an antiserum to salmon teleocalcin, a glycoprotein hormone that has recently been isolated from sockeye salmon, Oncorhynchus nerka, corpuscles of Stannius (CS). Immunodiffusion studies showed that teleocalcin was immunologically identical in four species of Pacific salmon. Histological staining of adjacent sections of sockeye salmon CS by periodic acid-Schiff reagent and immunocytochemistry revealed that the teleocalcin-immunoreactive cells were also periodic acid-Schiff positive. In bioassays using rainbow trout (Salmo gairdneri), the biological effects of teleocalcin were completely abolished by pretreatment with the antiserum before injection. The antiserum was ineffective by itself, however, in neutralizing endogenous teleocalcin. Immunoprecipitation studies of in vitro translation products from salmon CS poly(A)+ RNA identified a 26K protein, which corresponds to the size of the teleocalcin monomer as predicted by cDNA sequencing studies. Similar banding patterns were obtained with purified teleocalcin and crude CS extracts by Western blot analysis. However, there was one extra band (32K) in the CS extracts that was not present in the purified hormone preparations. This may represent the pro-form of the hormone in salmon.

Activation-dependent phosphorylation of endogeneous protein kinase c substrates in quiescent human T lymphocytes

Activation of quiescent human T lymphocytes with phorbol ester, synthetic diacylglycerol, or an antibody specific for the antigen receptor associated CD3 antigen resulted in a rapid phosphorylation of a Mr 80,000 (termed 80K) and a Mr 19,000 (termed 19K) cellular protein. The 80K (pI 4.4-5.1) protein was evidently analogous to a previously described 80K protein, which is a putative in vivo substrate for the Ca 2+-activated phospholipid-dependent protein kinase (PK.C), while the identity of the 19K protein is unknown. We present evidences that the 19K protein is variably phosphorylated on serine residues and that phosphorylation involves activation of the PK.C pathway. The biological significance of these phosphorylation events was suggested both by the ligand specificity and the correlation with subsequent induction of cellular proliferation.

Parallel inhibition of platelet-activating factor-induced protein phosphorylation and serotonin release by K-252a, a new inhibitor of protein kinases, in rabbit platelets

K-252a, (8 R,9 S,11 S)-(−)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1 H,8 H,11 H-2,7b,11a-triazadi benzo[ a, g]cycloocta[ c, d, e]trinden-1-one, an indole carbazol compound isolated from microbial origin, potently inhibits protein kinase C in partially purified enzyme and intact platelets. We examined the effects of this compound on platelet-activating factor [1- O-alkyl- α-acetyl-sn-glycero-phosphocholine (AGEPC)] induced protein phosphorylation, serotonin release and a rise in intracellular free calcium using washed rabbit platelets. In Ca 2+-containing medium (1 mM CaCl 2), AGEPC at 10 −10 and 10 −9 M markedly phosphorylated two proteins having molecular weights of 40,000 dallons (40 K protein) and 20,000 dallons (20 K protein) and evoked a marked rise in cytosolic free calcium. K-252a at 3 and 10 μM caused a concentration-dependent inhibition in the 20 K protein phosphorylation but caused only slight inhibition in the 40 K protein phosphorylation. K-252a inhibited the basal phosphorylation of 20 K protein obtained in non-stimulated platelets, and caused no significant alteration in the rise of intracellular free calcium evoked by AGEPC. It can be considered, from this evidence, that K-252a may act directly on myosin light chain kinase, resulting in the inhibition of 20 K protein phosphorylation. In Ca 2+-free medium [1 mM ethylene glycol-bis( β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid (EGTA)], AGEPC at 10 −8 M predominantly phosphorylated 40K protein, although phosphorylation of 20K protein and cytosolic free calcium were increased slightly. K-252a at 1–10 μM caused a concentration-dependent inhibition in the 40K protein phosphorylation. These results indicate that K-252a functions as an inhibitor of both protein kinase C and myosin light chain kinase in rabbit platelets. In AGEPC-stimuIated platelets, the inhibition of 20K protein phosphorylation in Ca 2+-containing medium and of 40K protein phosphorylation in Ca 2+-free medium was closely correlated with the inhibition of serotonin release by K-252a. These results strongly suggest that the phosphorylation of these two proteins may be a prerequisite for serotonin release in AGEPC-stimulated platelets.

Dihydropyridine agonist Bay K 8644 inhibits platelet activation by competitive antagonism of thromboxane A2-prostaglandin H2 receptor.

The dihydropyridine calcium channel antagonists, such as nifedipine, inhibit platelet aggregation in vitro and ex vivo, but the mechanism by which this occurs is uncertain. Bay K 8644 (BAY) is a substituted dihydropyridine that has effects on voltage-dependent calcium channels in cardiac and smooth muscle that are opposite the effects of nifedipine. To evaluate the mechanism responsible for dihydropyridine-induced inhibition of platelet function, we studied the in vitro effects of BAY on human platelet aggregation and secretion plus several related biochemical parameters, including cytoplasmic ionized calcium ([Ca2+]i). BAY exerted concentration-dependent effects on platelet aggregation and secretion of [14C]serotonin. BAY (1-10 microns) inhibited the second wave of platelet aggregation and secretion stimulated by adenosine diphosphate or epinephrine and blocked shape change, aggregation, and secretion induced by the thromboxane A2 (TXA2) mimic, U46619. BAY also inhibited U46619-induced phosphorylation of the approximately 40,000-dalton cytoplasmic protein substrate of protein kinase C (40K protein), formation of TXA2, and rise in [Ca2+]i, all biochemical consequences of platelet activation. The (+)-(R) enantiomer of BAY [BAY(+)] was predominantly responsible for the inhibitory effects of racemic BAY. Nifedipine had the same inhibitory effects on platelet function and biochemistry, except it was approximately 10 times less potent than BAY. Since these results suggested inhibition of the TXA2-prostaglandin H2 (PGH2) receptor, we measured binding of [3H]U46619 to intact platelets. BAY, BAY(+), and nifedipine all functioned as competitive antagonists of [3H]U46619 binding (BAY Ki = 1.47 microM). They did not inhibit binding of [3H]yohimbine to platelet alpha 2-adrenergic receptors. At 1-10 nM BAY, BAY(+) and the (-)-(S) enantiomer of BAY [BAY(-)] all resulted in slight stimulation of platelet function and biochemical events. No significant increase in [3H]U46619 binding was demonstrable, however. Therefore, dihydropyridines that function as either calcium channel agonists or antagonists in cardiac or smooth muscle exert concentration-dependent effects on platelet function. In nanomolar concentrations, they augment, and in micromolar concentrations, they inhibit platelet activation induced by TXA2 or U46619. These data indicate that dihydropyridines do not inhibit TXA2-induced platelet activation by an effect on voltage-dependent calcium channels; they define the mechanism of inhibition as competitive antagonism of the TXA2-PGH2 receptor. The mechanism responsible for augmentation of platelet activation is uncertain.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning and sequencing of the 52K cathepsin D complementary deoxyribonucleic acid of MCF7 breast cancer cells and mapping on chromosome 11.

Two lambda gt11 libraries containing complementary DNAs from human breast cancer MCF7 cells were screened by expression with monoclonal antibodies to the secreted 52K protein and with a 36-mer oligonucleotide derived from the N-terminal amino acid sequence of the secreted 52K protein. Four overlapping clones were sequenced, and found to be extensively homologous to the cathepsin D of normal human kidney, except for 5-point mutations resulting in one amino acid change (Ala to Val) in the profragment of cathepsin D. Northern blot analysis showed the 2.2 kilobase (kb) cathepsin D mRNA to be induced by estradiol in MCF7 cells and produced constitutively at high levels in the estrogen-receptor-negative BT20 cell line. A simple restriction pattern consistent with the restriction map of cathepsin D cDNA was obtained in Southern blot analysis of MCF7 cell DNA. In situ hybridization of the 52K-9 cDNA probe on normal lymphocytes assigned the 52K cathepsin D gene at the extremity of the short arm of chromosome 11, in the p15 band, close to the H-ras gene and in the region whose deletion increases the risk of invasive breast cancer. We conclude that the estrogen induced 52K protein has the same sequence as normal pro-cathepsin D and we propose that the 52K protein correspond to the only pro-cathepsin D expressed in MCF7 cells.

Surface protein composition of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein.

The surface protein composition of members of a serogroup of Aeromonas hydrophila which exhibit high virulence for fish was examined. Treatment of whole cells of representative strain A. hydrophila TF7 with 0.2 M glycine buffer (pH 4.0) resulted in the release of sheets of a tetragonal surface protein array. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis showed that this sheet material was composed primarily of a protein of apparent molecular weight 52,000 (52K protein). A 52K protein was also the predominant protein in glycine extracts of other members of the high-virulence serogroup. Immunoblotting with antiserum raised against formalinized whole cells of A. hydrophila TF7 showed the 52K S-layer protein to be the major surface protein antigen, and impermeant Sulfo-NHS-Biotin cell surface labeling showed that the 52K S-layer protein was the only protein accessible to the Sulfo-NHS-Biotin label and effectively masked underlying outer membrane (OM) proteins. In its native surface conformation the 52K S-layer protein was only weakly reactive with a lactoperoxidase 125I surface iodination procedure. A UV-induced rough lipopolysaccharide (LPS) mutant of TF7 was found to produce an intact S layer, but a deep rough LPS mutant was unable to maintain an array on the cell surface and excreted the S-layer protein into the growth medium, indicating that a minimum LPS oligosaccharide size was required for A. hydrophila S-layer anchoring. The 52K S-layer protein exhibited hear-dependent SDS-solubilization behavior when associated with OM, but was fully solubilized at all temperatures after removal from the OM, indicating a strong interaction of the S layer with the underlying OM. The native S layer was permeable to 125I in the lactoperoxidase radiolabeling procedure, and two major OM proteins of molecular weights 30,000 and 48,000 were iodinated. The 48K species was a peptidoglycan-associated, transmembrane protein which exhibited heat-modifiable SDS solubilization behaviour characteristic of a porin protein. A 50K major peptidoglycan-associated OM protein which was not radiolabeled exhibited similar SDS heat modification characteristics and possibly represents a second porin protein.

Interferons-α/β- and -γ-Resistant Friend Cell Variants Exhibiting Receptor Sites for Interferons but No Induction of 2-5A Synthetase and 67K Protein Kinase

A number of Friend leukemia cell variants with a interferon-gamma (IFN-gamma)-resistant phenotype have been isolated. They appear resistant to the antiproliferative action of IFN-gamma and to the induction of the antiviral state assessed by Friend leukemia virus release and vesicular stomatitis virus yield. Selection was performed via a prolonged exposure to increasing amounts of highly purified recombinant IFN-gamma of wild-type Friend cells or of variant clones thereof already resistant to IFN-alpha/beta (Affabris et al., 1982, Virology 120, 441-452). Only the clones derived from IFN-alpha/beta-resistant variants showed a phenotype fully resistant to IFN-gamma treatment while keeping their previously acquired resistance to IFN-alpha/beta. These cells are not deficient in high-affinity receptors for IFN-gamma so that their resistant phenotype appears to be mediated by events distal to binding of IFN-gamma to its receptors. Furthermore, analysis of IFN-induced dsRNA-dependent 2-5A synthetase and 67K protein kinase enzymatic activities, biochemical markers for cellular responses to IFN, showed that both these activities were not induced in IFN-alpha/beta and IFN-gamma-resistant clones when treated with either type of IFN. Accordingly, no increased expression of 2-5A synthetase mRNA(s) could be detected by probing poly(A)+-enriched RNA from cells exposed to IFN-alpha/beta or IFN-gamma treatment with murine or human specific cDNAs. On the other hand, no major changes in restriction patterns of 2-5A synthetase gene(s) were observed in these variant cells by restriction endonuclease digestion and Southern blotting. In addition, analysis of 2-5A synthetase mRNA induction, performed on wild-type cells, showed that the kinetic of induction due to IFN-gamma treatment is slower than that obtained with IFN-alpha/beta.

Protein myristoylation as an intermediate step during signal transduction in macrophages: its role in arachidonic acid metabolism and in responses to interferon gamma.

The role of macrophages in the regulation of inflammation and immunity is, in part, due to their secretory repertoire. Among the important mediators released by macrophages are the products of both the cyclooxygenase and lipoxygenase pathways of arachidonic acid (20:4) metabolism. The principal focus of this paper is the mechanism by which bacterial lipopolysaccharides (LPS) regulate 20:4 metabolism in macrophages. LPS has the capacity to prime macrophages for greatly enhanced 20:4 metabolism when the cells are subsequently challenged with a spectrum of triggers. Concomitant with priming, LPS also promotes the covalent attachment of myristic acid to a set of macrophage proteins. The time and concentration dependence of LPS-induced protein myristoylation is consistent with a role for myristoylation in LPS priming of the 20:4 cascade. One of the myristoylated proteins is a 68K (K = 10(3) Mr) protein kinase C substrate which associates with membranes upon myristoylation. LPS-primed macrophages show greatly increased phosphorylation of the 68K protein when the cells are subsequently treated with protein kinase C activating phorbol esters. It is proposed that the myristoylation of the 68K protein promotes its attachment to the membrane where it is more closely associated with activated protein kinase C (PKC), an association which would ensure more efficient catalysis during the mobilization and oxygenation of 20:4. This paper also examines protein myristoylation during T-cell-mediated activation of macrophages. Immune-activated macrophages have an enhanced capacity to kill several infectious agents by oxidative mechanisms. The lymphokine gamma-interferon (IFN gamma) rapidly induces the myristoylation of a 48K protein. This 48K protein is also myristoylated in murine macrophages that have been activated in vivo by intraperitoneal injection of Corynebacterium parvum, suggesting that it may be an important intermediate in the activation of macrophages for enhanced microbicidal capacity.

人血小板アラキドン酸凝集に対するポリミキシンＢの影響

Polymyxin B (PMB), a cyclic polycationic peptide antibiotic, inhibited dose-dependently human platelet aggregation induced by a high level (45μg/ml) of arachidonate (AA) or eicosadienoic acid (C20: 2), whereas this drug had no effect on a low level (0.45μg/ml) of AA-induced platelet responses. Colistin (polymyxin E), a derivative of PMB, had no inhibitory effect on platelet aggregation induced by a high level of AA or C20: 2. When human platelets were stimulated with AA, phosphorylation of 40K and 20K proteins preceded the aggregation response and platelet response by AA correlated closely with the phosphorylation of these proteins. PMB (100μM) inhibited the phosphorylation of 40K and 20K proteins induced by 45μg/ml AA, but not the phosphorylation event induced by 0.45μg/ml AA. PMB also inhibited dose-dependently [Ca2+]i elevation in response to a high level (20μg/ml) of AA, whereas PMB had no effect on a transient increase in [Ca2+]i induced by a low level (0.45μg/ml) of AA. The present results indicate that PMB has no effect on protein phosphorylation systems in human platelets although the drug has been shown to inhibit protein kinase C in vitro, and suggest that PMB selectively inhibit platelet aggregation induced by a high level of AA, possibly by interacting the lipid matrix of platelet membranes.

The 52K cathepsin-D of breast cancer: structure, regulation, function and clinical value.

A major question in cancer research is: why do cancer cells continuously proliferate and invade adjacent tissue. Breast cancer cells are particularly suitable for studying this question since when they are differentiated, they contain estrogen receptors, and estrogen, specifically, triggers their growth. One approach to understanding the estrogen-induced growth stimulation in this model is to identify the estrogen-induced factors involved in the control of cell proliferation [1–6]. Here, we illustrate this approach in a study of growth regulation of MCF7 cells by estrogen, in which we found a secreted 52K protein with mitogenic activity. It was identified as a protease that is also possibly involved in the process of tumor cell metastasis.KeywordsBreast CancerBreast Cancer CellMCF7 CellProgesterone ReceptorHuman Breast Cancer CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Estrogens and growth factors induce the mRNA of the 52K-pro-cathepsin-D secreted by breast cancer cells.

The estrogen-induced 52K protein secreted by human breast cancer cells is a lysosomal protease recently identified as a pro-cathepsin D by sequencing several cDNA clones isolated from MCF7 cells (Augereau et al., Mol. Endocr.). Using one of these clones, we detected, in MCF7 cells, a 2.2 kb mRNA whose level was rapidly increased 4- to 10-fold by estradiol, but not by other classes of steroids. Other mitogens, such as epidermal growth factor and insulin, also induced the 2.2 kb mRNA in a dose-dependent manner. Induction with epidermal growth factor was as rapid but was 2- to 3-fold lower than with estradiol. Antiestrogens had no effect on the 52K-cathepsin-D mRNA in MCF7 cells, but became estrogen agonists in two antiestrogen-resistant sublines R27 and LY2. The use of transcription and translation inhibitors and nuclear run-on experiments indicate that estradiol enhances transcription of the 52K-cathepsin-D gene in MCF7 cells.

Mutation in a DNA-binding protein reveals an association between DNA-methyltransferase activity and a 26,000-Da polypeptide in frog virus 3-infected cells

The DNA of frog virus 3 (FV3), an iridovirus, is highly methylated; more than 20% of the cytosine bases are methylated at the 5-carbon position by an FV3-induced DNA methyltransferase (DNA-mt). To determine the role of this enzyme in virus replication and regulation of gene expression, we have analyzed an FV3 mutant that lacks DNA-mt activity and is resistant to 5-azacytidine (an inhibitor of DNA-mt). Comparative polypeptide analysis, using cytoplasmic extracts from the wild-type FV3 and mutant-infected cells, revealed that a single protein of 26,000 (26K) molecular weight was altered in the mutant-infected cells. The altered polypeptide migrated faster in SDS-polyacrylamide gel as compared to the wild-type FV3 26K protein. Five spontaneous revenants derived from the mutant regained the migrational characteristic of the wild-type 26K protein, DNA-mt activity, and methylation of their DNA. We further show that the 26K polypeptide is a DNA-binding protein and that 80% of the enzyme activity can be eluted from an ssDNA affinity column. Taken together, these data support the conclusion that the 26K polypeptide is associated with DNA-mt activity.

Binding properties of 9K protein to F1-ATPase: a counterpart ligand to the ATPase inhibitor.

A regulatory subunit of yeast mitochondrial ATP synthase, 9K protein, formed an equimolar complex with F1-ATPase in the presence of ATP and Mg2+, indicating that the binding of the protein to the enzyme took place in a similar manner to that of ATPase inhibitor. The ATP-hydrolyzing activity of F1-ATPase decreased 40% on binding of the 9K protein, and the remaining activity was resistant to external ATPase inhibitor. The apparent dissociation constant of the F1-ATPase-9K complex was determined by gel permeation chromatography to be 3.7 X 10(-6) M, which was in the same order of magnitude as that of enzyme-ATPase inhibitor complex (4.2 x 10(-6) M). When added simultaneously the binding of the inhibitor and 9K protein to F1-ATPase were competitive and the sum of their bindings did not exceed 1 mol per mol of enzyme. However, the binding of each protein ligand to F1-ATPase took more than 1 min for completion, and when one of these two proteins was added 10 min after the other, it did not replace the other. These observations strongly suggest that membrane-bound F1-ATPase always binds to either the 9K protein or ATPase inhibitor in intact mitochondria and that the complexes with the two ligands are active and inactive counterparts, respectively.

Difference between R5020 and the antiprogestin RU486 in antiproliferative effects on human breast cancer cells.

We have compared the effects of the progestin R5020 and the antiprogestin RU486 on the growth of the MCF-7 and T47D breast cancer cell lines. Differences between the two compounds were demonstrated in several parameters. 1. Estradiol was required for the efficient inhibition of cell growth of both lines by R5020 but not by RU486. Therefore in the total absence of estrogen (phenol-red free medium), the effects of the two drugs on cell growth were dissociated, RU486 remaining inhibitory while R5020 was inactive. 2. The proteins secreted by cells were differently affected, since R5020 induced a 48K protein and decreased the production of the estrogen-regulated 52K protein, while RU486 had no effect on these two parameters. 3. The morphology of cells treated by R5020 was more altered in the presence of estradiol than in its absence, while that of cells treated by RU486 was not affected whether or not estradiol was present. 4. There was a greater reduction of estrogen receptor sites in MCF-7 cells produced by R5020 than by RU486. Even though the two drugs appear to act through the same progesterone receptor and to inhibit total protein secretion, it is likely that they exert their antiproliferative effects on cultured breast cancer cells by different mechanisms. R5020 antagonizes the stimulation produced by estradiol. RU486 by contrast exerts a more direct progesterone receptor mediated inhibitory effect requiring no synergism by estradiol and therefore does not act through a partial progestin activity.

Expression of the Middle Component RNA of Cowpea Mosaic Virus in vivo

SUMMARY Upon infection of cowpea mesophyll protoplasts with cowpea mosaic virus (CPM V), the only M RNA-encoded proteins detected so far have been the two capsid proteins VP37 and VP23. We now report the detection of a M r 60000 (60K) precursor to both capsid proteins in infected protoplasts cultured in the presence of zinc ions. Furthermore a M RNA-encoded 48K protein was detected in the membrane fraction of infected cells using antisera raised against synthetic peptides. The results obtained indicate that, just like the bottom component RNA, the M RNA of CPMV is translated in vivo into a polyprotein from which proteins are derived by proteolytic cleavage.

Contribution of beta-lactamase hydrolysis and outer membrane permeability to ceftriaxone resistance in Enterobacter cloacae.

Mechanisms of ceftriaxone resistance were examined in Enterobacter cloacae. Clones were selected from four strains: susceptible (S), resistant (R1), selected by plating on ceftriaxone-containing agar, and highly resistant (R2), selected in ceftriaxone-treated mice infected with S clones. According to 14C-labeled beta-lactam binding assays, ceftriaxone resistance was not associated with altered target proteins. R1 and R2 clones stably produced 50 to 1,500 times more beta-lactamase than S clones; this production increased after cefoxitin induction in all S and some R1 clones. Experiments conducted with strain 218 suggested that ceftriaxone resistance involved beta-lactamase hydrolysis. Half-lives for the beta-lactamase-beta-lactam complexes at 37 degrees C were 0.4 and 2.2 min for ceftriaxone and Sch 34343, a drug not affected by the resistance, respectively; in chromatography experiments, 218 intact R1 cells (2 x 10(9) to 3 x 10(9) CFU) suspended in ceftriaxone-containing buffer (2 micrograms/ml) hydrolyzed 80% of the antibiotic in 30 min. Three observations also suggested decreased permeability in some clones, (i) Most of the R1 and R2 clones showed decreased expression of outer membrane proteins of 37,000 to 38,000 molecular weight (37K to 38K proteins) by electrophoresis, often associated with increased amounts of 42K protein. (ii) [14C]Sch 34343 labeling of intact cells proceeded more slowly in 218 R2 (with altered 37K to 38K proteins) than in 218 R1 (without this alteration), a difference persisting after competition with unlabeled cloxacillin. Delays in binding were not caused by different enzymatic activities, since 218 R1 and 218 R2 produce, in similar amounts, beta-lactamases undistinguishable in isoelectric point and Km of cephaloridine. (iii) Intact cells from 218 R2 hydrolyzed ceftriaxone more slowly (20% in 30 min) than did those from 218 R1. In 218 R1, beta-lactamase overproduction was responsible for a 15- to 200-fold increase in the MIC's of ceftriaxone, ceftazidime, carbenicillin, piperacillin, moxalactam, aztreonam, carumonam, and BMY 28142. Imipenem and Sch 34343 were not affected; an additional three- to fivefold increase in the MIC's of these antibiotics (with the exception of piperacillin, imipenem, Sch 34343), seen with 218 R2, was associated with decreased permeability.

Two homologous protein components of hepatic gap junctions.

Gap junctions consist of closely packed pairs of transmembrane channels, the connexons, through which materials of low relative molecular mass diffuse from the cell to neighbouring cells. In liver, connexons consist of six protein subunits which, until now, were believed to be identical. However, besides the major polypeptide of relative molecular mass (Mr) 28,000 (and see refs 4 and 6), a component of Mr 21,000 (21K) has been repeatedly observed in liver. The amino-terminal sequence (18 residues) of this less abundant protein shows that it is related to, but distinct from, the Mr 28K protein. Immuno-staining and immuno-precipitation show both proteins to be in the same gap junctional plaques. Thus, it seems that hepatic gap junction channels (and by extension possibly others) are composed of two (or more) homologous proteins.