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T Benjamin

Publications and source records attributed to T Benjamin.

At least 19 recordsLinked to original sources
PubMed2000

Identification of gel-separated tumor marker proteins by mass spectrometry.

Two-dimensional gel electrophoresis with subsequent analysis by mass spectrometry was applied to study differences in protein expression between benign and malignant solid tumors from human beast, lung and ovary cells. Cells from freshly resected clinical material were lysed and the extracts were subjected to isoelectric focusing with immobilized pH gradients followed by second-dimensional separation on 10-13% sodium dodecyl sulfate (SDS)/polyacrylamide gels. Polypeptides were identified using matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry after in-gel protein digestion. Some of the upregulated polypeptides in malignant cells are of potential importance as markers of tumor proliferation. Twenty such proteins were identified, ten constituting novel identifications and ten sequence verifications of previously gel-matched proteins. The proteins identified span a wide range of functions, but several cases of protein truncation were found. Truncated forms of cytokeratins 6D and 8, and of cathepsin D were identified. Truncated froms of these over-expressed proteins support the presence of proteolytic processing steps in tumor material. The protein processing and the difference between protein and mRNA abundancies in tumors of different malignancy and origin suggest that studies at the protein level are important for an understanding of tumor phenotypes.

Biomarkers, Tumor↗
PubMed1998

Molecular characterization and sequencing of antifreeze proteins from larvae of the beetle Dendroides canadensis.

The deduced amino acid sequences of antifreeze proteins (AFPs) from larvae of the beetle Dendroides canadensis were determined from both complementary DNAs (cDNAs) and from peptide sequencing. These consisted of proteins with a 25-residue signal peptide and mature proteins 83 (Dendroides antifreeze protein; DAFP-1) or 84 (DAFP-2) amino acids in length which differed at only two positions. Peptide sequencing yielded sequences which overlapped exactly with those of the deduced cDNA sequences of DAFP-1 and DAFP-2, while the partial sequence of another AFP (DAFP-3) matched 21 of 28 residues. Seven 12- or 13-mer repeating units are present in these antifreeze proteins with a consensus sequence consisting of: Cys-Thr-X3-Ser-X5-X6-Cys-X8-X9-Ala-X11-Thr-X1 3, where X3 and X11 tend toward charged residues, X5 tends toward threonine or serine, X6 toward asparagine or aspartate, X9 toward asparagine or lysine, and X13 toward alanine in the 13-mers. The most interesting feature of these proteins is that throughout the length of the mature antifreeze proteins every sixth residue is a cysteine. These sequences are not similar to any of the known fish AFPs, but they are similar to AFPs from the beetle Tenebrio molitor.

Amino Acid Sequence↗
PubMed1997

Tumor induction by a transformation-defective polyoma virus mutant blocked in signaling through Shc.

Transformation of cells in culture by polyoma virus requires integration of signals downstream of middle T-Shc and middle T-phosphatidylinositol 3-kinase interactions, but the same is not true for induction of tumors in the mouse. Thus, a middle T mutant defective in transformation and blocked in binding Shc is able to induce a broad spectrum of tumors after inoculation into newborn mice. The "tumor profile" induced by the mutant shows enhancement of tumors at some sites and reductions at others but otherwise resembles that induced by the wild-type virus. A nontransforming double-mutant blocked in binding phosphatidylinositol 3-kinase as well as Shc is severely affected but still induces some tumors. These results show that pathways that must cooperate to induce full transformation of cells in vitro can act independently and are to a large extent redundant in tumor induction.

3T3 Cells↗
PubMed1997

Roles of N-glycans with alpha2,6 as well as alpha2,3 linked sialic acid in infection by polyoma virus.

Specific glycosylation inhibitors were used to show that N-glycans, and not O-glycans or glycolipids, constitute the major class of receptors on 3T3 cells for polyoma virus. Sialic acid (SA)-specific lectins were used in attempts to confirm the expected SA linkage on N-linked glycans. A lectin specific for alpha2,6 linked SA, a non-receptor type, was slightly more effective in blocking infection than a lectin specific for alpha2,3 linked SA which corresponds to the known receptor specificity. Possible explanations are suggested for this unexpected result.

1-Deoxynojirimycin↗
PubMed1995

Micropreparative immobilized pH gradient two-dimensional electrophoresis in combination with protein microsequencing for the analysis of human liver proteins.

Simplified methodology has been developed for the direct N-terminal amino acid microsequencing of human liver and hepatoma derived polypeptides, following micropreparative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Utilization of immobilized pH gradient (IPG) gel strips in the first dimension permitted protein loading of 0.5-2.0 mg with negligible diminution of polypeptide resolution. Following 2-D separation and electrotransfer to polyvinylidene difluoride (PVDF) membranes nearly 100 well resolved Ponceau S stained polypeptides were readily visualized, from which, 32 adult liver S-9 and 72 HepG2 nuclear cytosolic polypeptides were subjected to N-terminal microsequencing. Twenty normal adult liver and 54 HepG2 polypeptides yielded N-terminal sequence information, of which 17 and 19 polypeptides, respectively, exhibited high sequence homology to previously identified proteins. The initial yields of the proteins sequenced ranged from 2-14 pmols and yielded sequences of 14-26 amino acid residues. Many of the adult liver and HepG2 proteins contained inferred leader sequences since the first sequenced residue was several (20-30) residues from the methionine initiation site predicted by the cDNA of the adult liver. Quantitative comparison of 60 well characterized hepatic proteins between normal adult liver and two nontransformed, Chang and WRL-68, and four human hepatoma derived cell lines, HepG2, Huh-7, FOCUS, and SK-Hep, revealed a high homogeneity of protein expression both qualitatively and quantitatively in both whole cell lysate and purified nuclear preparations. Most notable differences include the previously characterized polypeptides: carbamoyl phosphate synthase, MER5 homologous protein, cytidylate kinase, phosphatidylethanolamine-binding protein and mitochondrial enoyl-CoA hydratase as well as three N-terminally blocked polypeptides: 11 (63 kDa/pI 7.00), 56 (26/6.45) and 59 (22/6.00) all of which were expressed at similar levels in normal adult liver tissue and each of the nontransformed, Chang and WRL-68, cell lines but not expressed or expressed at greatly decreased levels in each of tumor derived liver cell lines. Pyruvate carboxylase, superoxide dismutase, serotransferrin, liver fatty acid binding protein, 1-hydroxyprostaglandin dehydrogenase, NADH dehydrogenase (ubiquinone) as well as three N-terminally blocked polypeptides: 9 (57/6.00), 53 (24/4.90) and 63 (16/4.70) were detected only in whole adult liver tissue and not in any of the cultured cell lines. Two additional polypeptides: U35, (27/6.05) and 58 (22/5.70) yielded N-terminal partial amino acid sequences but were not identified in established protein databases. We have shown that micropreparative IPG 2-D PAGE In combination with protein microsequencing provides a convenient one step procedure to rapidly obtain partial amino acid sequence information for nearly 100 individual polypeptides directly from a single 2-D PAGE gel with numerous applications to a wide variety of biological model systems.

Adult↗
PubMed1994

Noninterfering synthetic peptides as internal controls for amino acid sequencing of sample unknowns.

Noninterfering synthetic peptides have been designed that may be used as internal sequencing standards (ISS-1 and ISS-2) by placing them in an amino acid sequencer with the sample. The peptides are composed of four unnatural amino acids which all yield phenylthiohydantoin derivatives having unique retention times compared with those obtained from the commonly observed natural residues. These internal standards indicate how the entire sequencing system was functioning during the actual analysis of an unknown by providing an initial yield and numerous repetitive yields. Verifying proper operation is extremely important when cycles appear blank due to the presence of modified amino acids or a blocked N-terminus. In addition, the ISS peptides can detect and identify different types of sequencing errors. This sometimes eliminates the need to rerun a sample due to blank cycles caused by mechanical malfunctions which result in failure to cleave the N-terminal residues. ISS-1 or ISS-2 may also be utilized during method development to compare different sample supports and to normalize sequencing data from proteins or peptides that have been treated differently.

Amino Acid Sequence↗
PubMed1994

Plasma proteins as early biomarkers of exposure to carcinogenic aromatic amines.

Two-dimensional gel electrophoresis (2DG) has been used to study the changes induced in dog plasma polypeptides by the known urinary bladder carcinogens, 4-aminobiphenyl (4-ABP) and 2-naphthylamine (2-NA). Treatment with 3-aminobiphenyl (3-ABP) and 1-naphthylamine (1-NA), both considered to be non-carcinogenic, were used as controls. The purpose of this study was: (1) to determine whether or not changes that occurred in the plasma protein patterns were specific to 4-ABP and/or other related carcinogenic arylamines; (2) to measure the time course in the changes of the major polypeptides during dosing and their resynthesis during a recovery period; and (3) to determine, by microsequencing, the biochemical identity of the affected proteins. The results indicate that only the most potent carcinogen, 4-ABP, had the effect of suppressing the expression of some proteins, while the other aromatic amines caused no discernible change in the 2DG patterns during a 12-week dosing period. The 4-ABP caused dramatic suppression of two sets of proteins. One set of three spots had an apparent molecular weight of 32.5 kDa, and a pI of 5.8-6.0. The major component in this group was identified as the beta-chain of haptoglobin. Expression of this protein decreased markedly during the first 2 weeks of treatment and recovered slowly after dosing stopped. Since haptoglobin functions to bind with free hemoglobin and facilitates its elimination from the blood stream, these results can be rationalized as a consequence of 4-ABP binding to hemoglobin in the erythrocyte, resulting in cell death and hemolysis. The 4-ABP modified hemoglobin then binds to haptoglobin and this tertiary complex is purged from the blood stream, resulting in the disappearance of free haptoglobin. A second set of spots (mol. wt., 65 kDa; pI, 6.5-6.6) disappeared much faster than the haptoglobin, and recovered more quickly. The major protein is about one-fifth the intensity of haptoglobin and appeared to be N-terminally blocked. Internal microsequencing of four fragments obtained from tryptic cleavage of the major spot of this group showed significant similarity to the serum albumin sequence of several species. This spot group is not the major serum albumin spot, however, since the latter is readily identified as the most abundant spot on the plasma map. During the course of this study, several other polypeptides in the 2DG map of dog plasma were identified and are presented here.

2-Naphthylamine↗
PubMed1993

The rat liver epithelial (RLE) cell nuclear protein database.

The master two-dimensional computer database of rat liver epithelial (RLE) cellular proteins (Wirth et al., Electrophoresis 1991, 12, 931-954) has been expanded to include detailed information concerning 1100 nucleoplasmic (cytosolic) and 850 particulate associated [35S]methionine labeled as well as 215 nucleoplasmic and 269 particulate associated [32P]orthophosphate labeled RLE nuclear polypeptides, respectively. The RLE nuclear protein database developed using the Elsie 5 gel analysis system contains both qualitative and quantitative annotations including polypeptide identification number, protein name (if known), molecular weight and pI information, quantitation and polypeptide spot shape, subcellular location, as well as specific information regarding transformation (chemical and spontaneous) and growth-related characteristics. Microsequencing of polypeptides directly from two-dimensional (2-D) blotted membranes has recently been established in our laboratory and provides a highly efficient and rapid means of polypeptide identification in the absence of specific antibodies. At present the RLE protein database is still in the developmental stage and is continually being updated as additional information is obtained. Nonetheless, it is anticipated that knowledge obtained concerning the identification and characterization of specific transformation and/or growth regulatory proteins in the RLE in vitro cell system will not only have direct application to other rodent and human 2-D protein databases currently under development but will also complement them.

Amino Acid Sequence↗
PubMed1993

Defect in entry and altered pathogenicity of a polyoma virus mutant blocked in VP2 myristylation.

We have examined effects of mutations of the minor capsid proteins VP2 and VP3 of polyoma virus. Infection of cells by mutants blocked in expression of either VP2 or VP3 by alterations of their initiator methionine codons led to the rapid selection of wild-type revertants. This reversion suggests a strong, if not absolute, requirement for both proteins in virus growth. A mutant virus, VP2*, in which alanine was substituted for glycine-2, expressed a nonmyristylated form of VP2 that was incorporated into virions. Studies of VP2* revealed a 15- to 20-fold lower specific infectivity and a delay in growth in both primary and established mouse cells compared to wild-type virus. Analysis of the growth delay indicated a defect in an early step of infection, at or prior to uncoating. Synthesis of viral DNA and VP1 was delayed by about 9 hr in mutant compared to that in wild-type infected cells. No evidence was obtained for an effect on either virus assembly (encapsidation) or stability of virions. Infection of mice by the VP2* mutant virus resulted in attenuated virus replication and tumor induction which were much more severely affected following intranasal inoculation than intraperitoneal inoculation.

3T3 Cells↗
PubMed1992

Polyoma tumorigenesis in mice: evidence for dominant resistance and dominant susceptibility genes of the host.

We have determined the tumor responses of nine inbred mouse strains and of two F1 hybrids following inoculation with polyoma virus. The results showed wide variations in the frequencies of mice developing tumors. Correlation with H-2 haplotype were evident. C3H/BiDa mice of H-2k type were fully susceptible, while C57BL/6 mice of H-2b type were completely resistant. DBA/2 and BALB/c mice, both H-2d, were of low susceptibility. Contributions of the major histocompatibility complex were demonstrated using pairs of H-2 congenic mice. Substitution of H-2k on either a BALB/c or a C57BL/10 background resulted in an increase in the frequencies and kinds of tumors induced. Susceptibilities of BALB and C57BL mice bearing the H-2k haplotype were still well below those of C3H/BiDa mice, however. Crosses of C3H/BiDa with BALB/c or BALB.K (H-2k) produced F1s that were of moderate and full susceptibility, respectively. These results indicate two distinct classes of host genes, one at the H-2 locus where different alleles confer varying degrees of resistance and the other a dominant susceptibility gene(s) carried in C3H/BiDa mice.

Animals↗
PubMed1992

Infection by polyomavirus of murine cells deficient in class I major histocompatibility complex expression.

Embryonic fibroblasts and kidney epithelial cells from beta 2-microglobulin-deficient mice were as infectible by polyomavirus as cells from normal littermates were, as judged by expression of nuclear viral capsid antigen, development of cytopathic effects, and yields of infectious virus. We conclude that expression of intact class I major histocompatibility complex molecules is not essential for polyomavirus infection.

Animals↗
PubMed1989

Direct N-terminal sequence analysis of rat liver plasma membrane glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis.

Nine previously uncharacterized membrane glycoproteins from normal rat liver have been analyzed by amino acid sequencing from two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after transblotting to Immobilon-P membranes. Three of these components show altered levels of expression in liver tumors. A single electroblotted polyacrylamide gel yielded sufficient quantities of these glycoproteins for amino acid sequencing and the N-terminal structure could be determined for four of them. The remaining five glycoproteins of interest were not sequenceable in this manner, presumably because they had blocked N-termini. Prior to electrophoresis, two enrichment methods were applied to the crude liver membrane preparations: affinity chromatography with concanavalin A to isolate the plasma membrane glycoproteins and then fast protein liquid chromatography on Superose 12 to obtain components having a specific range of molecular weights. These materials were next subjected to 2-D PAGE using pH 4-6 carrier ampholytes in the first dimension and 7.5% sodium dodecyl sulfate gels in the second. The proteins were then electroblotted to Immobilon-P membranes and located by staining with Coomassie Brilliant Blue R-250. Our results demonstrate that N-terminal sequencing (gas-phase) can be achieved on polypeptides obtained from approximately 250 micrograms of total glycoproteins applied to a single 2-D gel.

Amino Acid Sequence↗
PubMed1989

Familial transmission of 16p trisomy in an infant.

Based on four reported cases including the present case, 16p trisomic infants have remarkably similar features. These are severe developmental delay, psychomotor retardation, typical facies, and anomalies of extremities.

Chromosome Banding↗
PubMed1986

Effect of microsomal enzyme inducers on the urinary excretion pattern of mutagenic metabolites of the carcinogen 2,4-toluenediamine.

2,4-Toluenediamine [(TDA) CAS: 95-80-7] was administered to rats pretreated with the microsomal enzyme inducers phenobarbital (PB), beta-naphthoflavone (beta NF), or 3-methylcholanthrene (MCA). The 24-hour urines of male F344 rats were examined for their mutagenic potency by means of the Salmonella assay, with the Aroclor 1254-pretreated rat liver S-9 fraction as an activating system. No revertants were found with TDA or its urinary metabolites in the absence of the S-9 fraction. In the presence of S-9, the number of revertants increased as the concentration of TDA or its urinary metabolites increased. The urinary metabolites, generated after the microsomal enzyme inducers (PB, beta NF, MCA), had increased mutagenic activity as compared with the controls (saline, corn oil). In the presence of beta-glucuronidase (beta G), increased numbers of TA98 revertants were noted in the urine of rats pretreated with PB, saline, or corn oil. Addition of sulfatase did not alter the number of TA98 revertants. Conversely, beta G treatment of urine from rats pretreated with MCA or beta NF led to a decrease in the number of TA98 revertants as compared to levels in urine without beta G. Addition of known urinary metabolites of TDA, such as 4-acetylamino-2-aminobenzoic acid or 2,4-diacetylaminobenzoic acid, to beta NF-pretreated rat urine had no inhibitory effect on the mutagenicity in the absence of beta G. However, in the presence of beta G, the inhibitory effect was similar to that noted with beta NF-pretreated rat urine. Upon separation of urinary metabolites (beta NF-pretreated rat urine) into free, conjugated, and water-soluble forms, the maximum number of TA98 revertants was associated with the free ethyl acetate-extractable fraction, which accounted for the total mutagenic activity associated with the original volume of urine. Conjugated metabolites showed much less mutagenic activity, and an inhibitory principle was associated with the water-soluble fraction.

Animals↗
PubMed1986

Sequential analysis of chemically induced hepatoma development in rats by two dimensional electrophoresis.

Using the Solt-Farber hepatocarcinogenesis model, a large population of preneoplastic and neoplastic nodules were induced in male Fischer 344 rats. Total cellular polypeptides from normal liver and individual preneoplastic and neoplastic nodules were analyzed for both qualitative and quantitative changes using computer assisted high resolution two-dimensional electrophoresis. Approximately 800-1000 cytosolic and 1200-1400 membrane associated polypeptides were readily separated and detected using an ultrasensitive silver stain. The polypeptide patterns were remarkably similar for each tissue and only four qualitative polypeptide differences were noted. One cytosolic polypeptide, 6.8/57 (designated pl/Mr X 10(-3), and three membrane associated polypeptides, 6.25/41, 6.75/24, and 6.05/21, were expressed in both preneoplastic and neoplastic nodules but not in normal liver. No qualitative polypeptide differences were detected among the individual preneoplastic or individual neoplastic nodules or between preneoplastic and neoplastic nodules. Numerous quantitative changes in both known markers for hepatocarcinogenesis and in as yet unidentified polypeptides were noted. In particular, the Ya subunit of glutathione S-transferase B, the Yb subunit of glutathione S-transferase A, as well as the three isoelectric point variants of the Yp subunit of glutathione S-transferase P were increased 2-, 4-, and 7-fold, respectively, in preneoplastic and neoplastic nodules. Whereas DT-diaphorase was increased 2-3-fold in hyperplastic nodules as compared to normal liver, no differences in the expression of albumin were noted. Although no differences were observed in the expression of aldehyde dehydrogenase in preneoplastic and neoplastic nodules, polypeptide b (6.9/54) was shifted slightly toward the basic region in normal liver. alpha-Fetoprotein was not detected in either preneoplastic or neoplastic nodules. In addition to these changes in known markers, comparison of 500-800 cytosolic and 750-1000 membrane associated polypeptides showed that roughly 4-10% of the polypeptides were undergoing quantitative changes of at least 4-fold during these stages of hepatocarcinogenesis. Thirty (10 cytosolic and 20 membrane) polypeptides were significantly down-regulated while 22 (7 cytosolic and 15 membrane) polypeptides were up-regulated in both preneoplastic and neoplastic nodules. In all cases the direction and magnitude of change were the same in both preneoplastic and neoplastic nodules with the exception of three polypeptides.(ABSTRACT TRUNCATED AT 400 WORDS)

Albumins↗