Dalton Protein Research Articles

Characterization of Monoclonal Antibody 436 Recognizing the ARG-PRO-ALA-PRO Sequence of the Polymorphic Epithelial Mucin (PEM) Protein Core in Breast Carcinoma Cells

Epithelial mucins have obtained increasing clinical relevance since they were found in the serum of cancer patients and were shown to be elevated in metastatic disease. We report here the characterization of the monoclonal antibody (MAb) 436 which recognises the protein core of the polymorphic epithelial mucin (PEM) of the human breast. MAb 436 was generated by immunizing Balb/c mice with membrane-enriched fractions prepared from metastatic lesions in the axillary lymph nodes. The antigenic determinant recognized by the MAb 436 is expressed on the surface of breast cancer cells and was measured by ELISA on all of 50 cytosol preparations of primary breast tumors. Immunohistochemistry showed 98% of primary and 100% of metastatic breast cancer lesions to be positive with the 436 antigenic determinant expressed both in the cytoplasm and at the plasma membrane level of the tumor cells. Moreover, the antigen was expressed in a homogeneous fashion (80-100% of the total number of tumor cells) in more than 60% of the tumors. Reactivity with normal tissues was rare and scattered and restricted to glandular structures particularly at the luminal border level except for the distal and collecting tubules of adult and fetal kidney, where a cytoplasmic 436 antigen distribution was observed. Other cancers proved positive but the reactivity was always variable and heterogeneous. The antigen recognized by MAb 436 appears in Western Blotting as a M(r) of more than 200,000 daltons protein resolved in two bands. Epitope mapping experiments using overlapping octapeptides in the repeat unit of the PEM identified in the RPAP (Arg-Pro-Ala-Pro) sequence the binding site of the 436 antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Heat Shock and Growth Atmosphere on the Heat Resistance of Escherichia coli 0157:H7

Log phase cells of Escherichia coli 0157:H7 were grown aerobically at 30°C and heat shocked at various time/temperature combinations to determine the optimum conditions that would result in the highest number of survivors to a subsequent 55°C heat treatment. Heat shocking at 42°C for 5 min resulted in the largest increase in D55 value over nonheat-shocked controls of all heat-shock time/temperature combinations tested. Growth atmosphere significantly contributed to the heat resistance of both heat-shocked and nonheat-shocked cells, with anaerobically grown cells having D55 values higher than those of cells grown aerobically. Therefore, both heat shocking and anaerobic growth contributed to an increase in the number of survivors of E. coli 0157:H7 cells when compared with nonheat-shocked cells grown aerobically. Aerobically grown, heat-shocked cells, and anaerobically grown cells, both heat-shocked and nonheat-shocked, contained a 71,000 dalton protein not present in aerobically grown, nonheat-shocked controls. This protein was found to be immunologically similar to a sigma32 subunit of RNA polymerase, as evidenced by Western Blot using monoclonal antibodies specific for the sigma subunit. In addition to heat, anaerobic growth appeared to be a form of stress, since it resulted in the synthesis of heat-shock proteins and in an increased survival of cells to a heat treatment.

Isolation and characterization of the chicken bcl-2 gene: expression in a variety of tissues including lymphoid and neuronal organs in adult and embryo.

The expression of human bcl-2 gene is de-regulated by t(14;18) translocation in most of follicular lymphoma. Recent studies indicated that the bcl-2 gene product has an ability to block apoptosis of hematopoietic cells. To facilitate the analysis of the role of this gene in normal development using an animal model, we have isolated and partially characterized the chicken homologue of human bcl-2 gene. The analysis of nucleotide sequence showed that the organization of the chicken bcl-2 gene is very similar to that of human bcl-2 gene. The primary transcript is spliced to encode a 25,687 dalton (233 a.a.) protein. The chicken Bcl-2 protein has two regions highly homologous to human Bcl-2 protein surrounding a totally non-homologous region. The expression of the chicken bcl-2 gene was analyzed in various chicken tissues. In the adult chicken, bcl-2 transcripts were detected in thymus, spleen, kidney, heart, ovary and brain, with the highest levels being detected in the thymus. However, the bursa of Fabricius, which is the site of early B cell development, expressed much less amounts of bcl-2 RNA. On the other hand, in embryo, the gene is extensively expressed in the bursa, as well as in muscle and the above tissues. Our findings indicate that a homologue of the human bcl-2 gene does exist in the chicken and that its expression is developmentally regulated in some tissues.

Binding of oligonucleotides to cell membranes at acidic pH

Antisense oligodeoxynucleotides [oligo(dN)] have the ability to enter living cells and block the expression of specific genes. However, little is known about the mechanism of cellular uptake of oligo(dN). We have found that oligo(dN) can bind to the cell membranes of eukaryotic cells with much greater efficiency under acidic conditions (pH 4.0–4.5) than at neutral pH. The binding appears to be specific to poly nucleic acids since various sizes of oligo(dN), DNA and RNA, but not mononucleotides, compete for the binding. We have identified a 34 kDa membrane protein from T-cells, which binds to oligo(dT) cellulose at pH 4.5 and can be eluted at pH 7.5. This protein fraction blocked the binding of oligo(dN) to living T-cells in a competitive fashion. Our results suggest that eukaryotic cells have a receptor for oligo(dN) at acidic pH and that the 34 kDa dalton protein on the cell membrane may mediate such binding.

Yeast mutant affected for viability upon nutrient starvation: Characterization and cloning of the RVS161 gene

In yeast, nutrient starvation leads to entry into stationary phase. Mutants that do not respond properly to starvation conditions have been isolated in Saccharomyces cerevisiae. Among them the rvs161 mutant (RVS for Reduced Viability upon Starvation) is sensitive to carbon, nitrogen and sulphur starvation. When these nutrients are depleted in the medium, mutant cells show cellular viability loss with morphological changes. The mutation rvs161-1 is very pleiotropic, and besides the defects in stationary phase entry, the mutant strain presents other alterations: sensitivity to high salt concentrations, hypersensitivity to amino acid analogs, no growth on lactate or acetate medium. The addition of salts or amino acid analogs leads to the same morphological defects observed in starved cells, suggesting that the gene could be implicated mainly in the control of cellular viability. The gene RVS161 was cloned; it codes for a 30,252 daltons protein. No homology was detected with the proteins contained in the databases. Moreover, Southern analysis revealed the presence of other sequences homologous to the RVS161 gene in the yeast genome.

The Viviparous-1 developmental gene of maize encodes a novel transcriptional activator

The Viviparous-1 (Vp1) gene of maize is specifically required for expression of the maturation program in seed development. We show that Vp1 encodes a 73,335 dalton protein with no detectable homology to known proteins. An acidic transcriptional activation sequence was identified by fusion to the GAL4 DNA-binding domain. Expression of VP1 in maize protoplasts resulted in strong activation (130-fold) of a reporter gene fused to the promoter of a presumptive target gene. The acidic domain in VP1 was essential for transactivation and could be functionally replaced by the activator sequence of the herpes simplex virus VP 16 protein. Our results indicate that VP1 is a novel transcription factor possibly involved in potentiation of a seed-specific hormone response.

Nascent synthesis and secretion of cellobiase inNeocallimastix frontalis EB188

De novo synthesis and the secretion of cellobiase fromNeocallimastix frontalis EB188 were studied. Cellobiase was secreted rapidly in cellulose switch cultures. Chemical inhibition of protein synthesis and radiotracer studies suggested secretion was dependent on nascent protein synthesis. Inhibitors of protein glycosylation also inhibited secretion. An 85,000 dalton protein (and several others) represented the principal differences in de novo synthesis between cellulose- and glucose-switched cultures. Concentrations of the 85,000 daltons protein increased with culture incubation time and ultimately accounted for 7% of the total protein secreted. This protein was purified by gel electrophoresis separations and was determined to be a cellobiase. Secretion of this cellobiase was correlated with its radiolabeling. The possibility of cellobiase induction representing a specialized gene control system inNeocallimastix frontalis EB188 is discussed.

Production and Characterization of Monoclonal Antibodies to Ha-ras and N-ras p21

The mammalian ras family consists of the Ha, Ki and N-ras genes that encode a series of 21,000 dalton proteins (p21). The three ras proteins participate in normal cell physiology and have been implicated in cellular transformation by either overexpression of the normal p21 or by mutation at positions 12, 13, or 61. To help understand the biological roles of the different ras proteins, we have generated monoclonal antibodies (Mabs) to the Ha-ras and N-ras p21. Mab Ha-770, raised to a Ha-specific synthetic peptide, reacts with Ha-ras recombinant p21 (r-p21) as well as cellular Ha-ras p21 by immunoprecipitation. Western blot and sandwich ELISA assays. Mab N-838, raised to an N-ras specific synthetic peptide, reacts with the N-ras recombinant p21 by immunoprecipitation, Western blot and sandwich ELISA assays. Mabs to the Ha-ras and N-ras p21 should be valuable reagents in assessing the individual roles of ras proteins in normal and neoplastic cells.

HLA-DR is a procoagulant

Numerous incidents of thromboembolic complications have been documented in cancer patients and in recipients of mismatched organ transplants. Tumor procoagulants have also been implicated in the process of metastasis. Two protein bands of 35,000 and 28,000 daltons isolated from human ovarian carcinoma possessed procoagulant activity. The 35,000 dalton protein had an amino terminal sequence identical to that of the major histocompatibility antigen HLA-DR. Further, isolation of the protein using immunoaffinity column chromatography with monoclonal antibody to HLA-DR resulted in the isolation of procoagulant activity. The immunoaffinity purified protein enhanced thrombin generation in recalcified normal plasma approximately 20- fold. HLA-DR procoagulant activity was completely inhibited by Staphylococcal enterotoxin A. We propose that the procoagulant nature of HLA-DR may contribute to thrombotic disorders in several cancers and in association with graft rejection. The ability of enterotoxin A to inhibit this procoagulant may lead to development of future therapeutic strategies.

Concurrent Inactivation of Calcium Dependent Phosphorylation and Neurotransmitter Release in Cultured Rat Brain Neurons

Depolarization of cultured rat brain neurons preloaded with 3H-dopamine provokes a transient (t 1/2 = 9.6 sec), Ca(2+)-dependent release of the labeled neurotransmitter from cells. In parallel, the amount of 32Pi incorporated into a protein of apparent molecular weight of 43,000 increased whereas the phosphorylation of a protein with an apparent molecular weight of 55,000 daltons decreased. The time course of the change in phosphorylation pattern was examined. The depolarization-induced phosphorylation of the 45,000 protein and dephosphorylation of the 55,000 dalton protein consisted of an initial, rapidly terminating phase (t 1/2 = 5 sec), and of a slow, Ca(2+)-independent phosphorylation of both proteins which persisted during maintained depolarization. The depolarization-evoked changes in the neuronal protein phosphorylation were dependent on the extracellular Ca2+ concentration (half saturation at 0.4-0.5 mM Ca2+). These data indicate that the entry of Ca2+ into the depolarized cells induces rapid phosphorylation-dephosphorylation activities. These processes terminate within 10 sec, concurrently with the depression of neurotransmitter release.

Dephosphin, a 96 000 Da substrate of protein kinase C in synaptosomal cytosol, is phosphorylated in intact synaptosomes

A 96 000 dalton phosphoprotein, called dephosphin, is phosphorylated in intact synaptosomes from rat brain and is rapidly dephosphorylated upon depolarisation-dependent calcium entry. A 96 000 dalton phosphoprotein is also a substrate of protein kinase C synaptosomal cytosol, and the aim of the study was to determine whether the two proteins may be the same. Dephosphin in intact synaptosomes and the 96 000 dalton protein kinase C substrate comigrated on polyacrylamide gels. Both phosphoproteins had identical phosphopeptide maps after digestion with V8 protease. Both phosphoproteins ran on isoelectric focussing gels with a pf of 6.3–6.7 and focussed as a series of 5–6 spots. Both proteins were phosphorylated exclusively on serine. Both proteins could be resolved into a doublet on longer polyacrylamide gels. The two subunits were of 96 and 93 kDa in both phosphorylation conditions and had dissimilar phosphopeptide maps. However, phosphopeptide maps of either the 96 or 93 kDa subunits were identical in intact synaptosomes compared with synaptosomal cytosol. These results show that a phosphoprotein phosphorylated in intact synaptosomes and a 96 000 dalton protein kinase C substrate from rat brain synaptosomal cytosol are the same, and raise the possibility that protein kinase C is the protein kinase responsible for dephosphin phosphorylation in intact synaptosomes.

The product of the KIN1 locus in Saccharomyces cerevisiae is a serine/threonine-specific protein kinase.

The catalytic domain (30 kDa) of all protein kinases can be aligned for maximum homology, thereby revealing both invariant and highly conserved residues. The KIN1 locus from Saccharomyces cerevisiae was isolated by hybridization to a degenerate oligonucleotide encoding the conserved protein kinase domain, DVWSFG. The predicted amino acid sequence revealed significant homology to the catalytic domain of protein kinases. Using antibodies raised against a bacterial LacZ/KIN1 fusion protein, we have identified by immunoprecipitation the yeast KIN1 gene product as a 145,000 dalton protein (p145KIN1). In exponentially growing yeast cells, the KIN1 protein is phosphorylated primarily on serine residues. The gene product of KIN1 was shown to be a serine/threonine-specific protein kinase in immune complexes, as determined by the transfer of label from [gamma-32P]ATP to either pp145KIN1 or to an exogenously added substrate, alpha-casein. The optimal metal ion concentration in this assay was 20 mM-MnCl2. Subsequent phosphoamino acid analysis of the radiolabelled product, pp145KIN1, demonstrated that this autophosphorylation was specific for serine/threonine residues. There is no apparent difference between wild-type cells and cells containing a disrupted KIN1 gene. The biochemical characterization of protein kinases in simple eukaryotes such as yeast will aid us in determining the role of phosphorylation in cellular growth and physiology.

Comparison of Isolates of Citrus Ringspot, Psorosis, and Other Viruslike Agents of Citrus

A collection of graft-transmissibnle psorosis and psorosislike diseases of citrus were compared with a partially characterized isolate of citrus ringspot virus (CRSV-4) from Florida. Fourteen isolates of citrus ringspot and psorosis from Florida, Spain, and Argentina were mechanically transmissible to Chenopodium quinoa and induced local lesions. The infectivity associated with several isolates was seaprated into top and bottom components by sucrose gradient centrifugation, which is characteristic of CRSV-4. A 48-kilo dalton (kDa) protein, which is assumed to be the CRSV capsid protein, was detected in preparations of top and bottom components from 13 of the isolates (...)

Isolation ofEnterobacter aerogenes susceptible to beta-lactam antibiotics despite high level beta-lactamase production

This report describes a patient with nosocomial meningitis from whom four distinct isolates of Enterobacter aerogenes were recovered over a complicated course of chemotherapy. The initial isolate was susceptible to expanded spectrum beta-lactams despite constitutive production of high levels of beta-lactamase. Resistant isolates recovered during antibiotic therapy had lost a 42,000 outer membrane protein. These data suggest that b-lactam susceptibility in the original isolate was due to "hyperpermeability" mediated by the 42,000 Dalton protein.

Nitrogen-15 and carbon-13 NMR studies of ligands bound to the 280 000-dalton protein porphobilinogen synthase elucidate the structures of enzyme-bound product and a Schiff base intermediate

Porphobilinogen synthase (PBGS) catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). Despite the 280,000-dalton size of PBGS, much can be learned about the reaction mechanism through 13C and 15N NMR. To our knowledge, these studies represent the largest protein complex for which individual nuclei have been characterized by 13C or 15N NMR. Here we extend our 13C NMR studies to PBGS complexes with [3,3-2H2,3-13C]ALA and report 15N NMR studies of [15N]ALA bound to PBGS. As in our previous 13C NMR studies, observation of enzyme-bound 15N-labeled species was facilitated by deuteration at nitrogens that are attached to slowly exchanging hydrogens. For holo-PBGS at neutral pH, the NMR spectra reflect the structure of the enzyme-bound product porphobilinogen (PBG), whose chemical shifts are uniformly consistent with deprotonation of the amino group whose solution pKa is 11. Despite this local environment, the protons of the amino group are in rapid exchange with solvent (kexchange greater than 10(2) s-1). For methyl methanethiosulfonate (MMTS) modified PBGS, the NMR spectra reflect the chemistry of an enzyme-bound Schiff base intermediate that is formed between C4 of ALA and an active-site lysine. The 13C chemical shift of [3,3-2H2,3-13C]ALA confirms that the Schiff base is an imine of E stereochemistry. By comparison to model imines formed between [15N]ALA and hydrazine or hydroxylamine, the 15N chemical shift of the enzyme-bound Schiff base suggests that the free amino group is an environment resembling partial deprotonation; again the protons are in rapid exchange with solvent. Deprotonation of the amino group would facilitate formation of a Schiff base between the amino group of the enzyme-bound Schiff base and C4 of the second ALA substrate. This is the first evidence supporting carbon-nitrogen bond formation as the initial site of interaction between the two substrate molecules.

Characterization of an immuno-dominant variable surface antigen from pathogenic and nonpathogenic Entamoeba histolytica.

A 125-kD surface antigen of Entamoeba histolytica is recognized by 73% of immune sera from patients with amoebic liver abscesses. Using pooled human immune sera a cDNA clone (lambda cM17) encoding this antigen (M17) has been isolated from a lambda gt11 expression library of the virulent stain E. histolytica HM1:IMSS. Monospecific antibodies, purified by binding to phage lysate of lambda cM17, and mAb FA7 reacted exclusively with the 125-kD antigen by Western blot analysis. Surface binding and cap formation are observed with patient sera, purified monospecific antiserum, and mAb FA7. Corresponding genomic clones (pBSgM17-1/2/3) were isolated by hybridization with the cDNA clone. These contained an open-reading frame of 3345 bp, which is in good agreement with the mRNA size of approximately 3.0 kb as revealed by Northern hybridization with lambda cM17. The inferred amino acid sequence predicts a 125,513 dalton protein that contains 17 potential N-linked glycosylation sites and is unusually rich in tyrosine and asparagine residues. A distinctly hydrophobic NH2-terminal region may serve as membrane anchor or signal sequence. In contrast to conservation of an immunodominant epitope recognized in pathogenic and nonpathogenic strains by monoclonal FA7 and human immune sera, amplification and sequence analysis of a 1,4000-bp fragment of this gene from a fresh nonpathogenic isolate by use of the PCR demonstrate regions of significant sequence divergence in this antigen. A 1% sequence variability among different isolates of the pathogenic strain HM1:IMSS and a 12-13% variability between pathogenic and nonpathogenic strains are revealed by comparison to published partial amino acid sequences (Tannich, E., R.D. Horstmann, J. Knobloch, and H.H. Arnold. 1989. Proc. Natl. Acad. Sci. USA. 86:5118). Some restriction enzymes were found that allowed PCR diagnosis of nonpathogenic and pathogenic isolates with the exclusion of E. histolytica-like Laredo, suggesting that a detailed study of nonpathogenic and pathogenic isolates in relation to the M17 antigen sequence will provide a basis of differentiating isolates.

The multifunctional peptide synthetase performing the first step of penicillin biosynthesis in Penicillium chrysogenum is a 421,073 dalton protein similar to Bacillus brevis peptide antibiotic synthetases.

The nucleotide sequence of the Penicillium chrysogenum Oli13 acvA gene encoding delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase, which performs the first step in penicillin biosynthesis, has been determined. The acvA gene contains an open reading frame of 11,238 bp encoding a protein of 3746 amino acids with a predicted mol. wt of 421,073 dalton. Three domains within the protein of approximately 570 amino acids have between 38% and 43% identity with each other and share similarity with two antibiotic peptide synthetases from Bacillus brevis as well as two other enzymes capable of performing ATP-pyrophosphate exchange reactions. The acvA gene is located close to the pcbC gene encoding isopenicillin N synthetase, the enzyme for the second step of beta-lactam biosynthesis, and is transcribed in the opposite orientation to it. The intergenic region of 1107 bp from which the acvA and pcbC genes are divergently transcribed has also been sequenced.

Purification and Characterization of Glutamyl-tRNA Synthetase

Chlorophyll biosynthesis starts with the synthesis of glutamyl-tRNA (glu-tRNA) by a glutamyl-tRNA synthetase (Glu RS). The glu-tRNA is subsequently transformed to delta-aminolevulinic acid (ALA), which is a committed and regulated precursor in the chlorophyll biosynthetic pathway. The Glu RS from a green alga, Chlamydomonas reinhardtii, was purified and shown to be able to synthesize glu-tRNA and to participate in ALA synthesis in a coupled enzyme assay. Physical and chemical characterization of the purified Glu RS indicated that the enzyme had been purified to homogeneity. The purified enzyme has a native molecular weight of 60,000, an isoelectric point of 4.6, and it formed a single band of 32,500 daltons when analyzed by a silver stained denaturing gel. The N-terminal amino acid sequence of the 32,500 dalton protein was determined to be Asn-Lys-Val-Ala-Leu-Leu-Gly-Ala-Ala-Gly. The molecular weight analyses together with the unambiguous N-terminal amino acid sequence obtained from the purified enzyme suggested that the native enzyme was composed of two identical subunits. Polyclonal antibodies raised against the purified and denatured enzyme were able to inhibit the activity of the native enzyme and to interact specifically with the 32,500 dalton band on Western blots. Thus, the antibodies provided an additional linkage for the structural and functional identities of the enzyme. In vitro experiments showed that over 90% of the glu RS activity was inhibited by 5 micromolar heme, which suggested that Glu RS may be a regulated enzyme in the chlorophyll biosynthetic pathway.

Characterization of fourteen strains of Neisseria gonorrhoeae: structural analyses and serum reactivities

Resistance to normal human serum (NHS) killing in Neisseria gonorrhoeae has been associated with particular types of Protein I (PI) and lipopolysaccharide (LPS), but many exceptions exist, and the role of these structures in determining serum reactivities remains controversial. In reality, the response of the gonococcus to NHS is probably governed by several parameters involving a number of outer-membrane (OM) components. We surveyed the serum reactivities of 14 strains of N. gonorrhoeae and characterized each of their major OM components. The strains presented a spectrum of sensitivity to pooled NHS. As assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, immunoblotting, and peptide mapping, the strains were also quite heterogeneous in terms of PI, H.8 antigen, and LPS type, and the presence of the 2-1-L8 epitope. Five of the strains had identical PIAs in varying LPS and H.8 backgrounds, and four had identical PIBs in varying LPS and H.8 backgrounds. As assessed by electrophoretic migration and monoclonal antibody binding, Protein III and the 44,000 Dalton protein were identical in these strains. We found no association between PI subclass and serum sensitivity, while H.8 and LPS variation appeared to be related to bactericidal responses. The diversity and close interaction of gonococcal components in the OM are undoubtedly involved in differential abilities to survive NHS killing.

Ubiquitin immunoreactivity shows several proteins varying with development and sporulation in the basidiomycete Coprinus cinereus

Crude extracts of mycelia and basidiocarp primordia in the basidiomycete Coprinus cinereus were resolved on sodium dodecyl sulfate--polyacrylamide gels, and ubiquitin and several proteins were detected by immunoblotting with anti-ubiquitin antibody. The molecular masses of the proteins detected were 30,900, 28,600, 27,800, 26,300, 22,500, and 15,400 daltons, respectively. Relative levels of ubiquitin and most of the ubiquitin-immunoreactive proteins in basidiocarp primordium formation increased and in basidiocarp maturation decreased in cap and upper stipe, while in lower stipe became high except for the 27,800 dalton protein and ubiquitin. During sporulation, ubiquitin and all the ubiquitin-immunoreactive proteins tended to decrease in the cap of the young wild-type basidiocarp. The levels of 30,900 and 15,400 dalton proteins increased transiently at 6-10 h after the beginning of the last light period, while ubiquitin decreased markedly. No correlation was observed between changes in levels of the ubiquitin-immunoreactive proteins and the blocked stages in sporulation-deficient mutants.