Biomedical subjects

N E Sherman

Publications and source records attributed to N E Sherman.

At least 19 recordsLinked to original sources

PubMed2006

Purification and characterization of an esterase isozyme involved in hydrolysis of organophosphorus compounds from an insecticide resistant pest, Helicoverpa armigera (Lepidoptera: Noctüidae).

An esterase isozyme was purified from the insecticide resistant pest, Helicoverpa armigera collected from field crops. Purification involved ammonium sulfate precipitation, hydrophobic interaction and ion exchange chromatography followed by gel filtration chromatography. The purification was 212-fold with 1% yield of the enzyme. The optimum pH of the isozyme was found to be 10.5 and 8.5 for p-nitrophenyl phosphate and paraoxon, respectively. The enzyme was unstable at temperature >50 degrees C. The molecular mass determined by SDS-PAGE was 66 kDa. Cations such as Hg(+2), Ag(+2), Cd(+2) inhibited the activity while Zn(+2) stimulated it. Kinetic studies indicated that the enzyme had low K(m) values of 0.238 and 0.348 mM for p-nitrophenyl phosphate and paraoxon, respectively. The enzyme had broad substrate specificity with high K(m) values for ATP, ADP and beta-glycerophosphate. This enzyme was partially sequenced and identified as an alkaline phosphatase.

Alkaline Phosphatase↗

PubMed2001

High-molecular-mass receptors for ammodytoxin in pig are tissue-specific isoforms of M-type phospholipase A(2) receptor.

Studying the molecular basis of presynaptic neurotoxicity of ammodytoxin C, a secretory phospholipase A(2) from the venom of Vipera a. ammodytes snake, we demonstrated the existence of two high-molecular-mass ammodytoxin C-binding proteins in porcine tissues, one in cerebral cortex and the other in liver. These proteins differ considerably in stability and Western blotting properties. However, as shown by immunological analysis and tandem mass spectrometry sequencing of several internal peptides derived from the purified receptors, both belong to secretory phospholipase A(2) receptors of the M type, which are Ca(2+)-dependent multilectins homologous to the macrophage mannose receptor. Based on Southern blot analysis of genomic DNA and deglycosylation of the receptors, the difference between the two proteins most likely stems from the different posttranscriptional and posttranslational modifications of a single gene product. Our findings raise the possibility that the M-type receptors for secretory phospholipases A(2) may display different physiological properties in different tissues.

Amino Acid Sequence↗

PubMed2001

Proteolytic processing of the human T-cell lymphotropic virus 1 reverse transcriptase: identification of the N-terminal cleavage site by mass spectrometry.

Human T-cell lymphotropic virus 1 (HTLV-1) is a type C human retrovirus, which is the causative agent of Adult T-cell Leukemia and other diseases. The reverse transcriptase of HTLV-1 (E.C. 2.7.7.49) is synthesized as part of a Gag--Pro--Pol precursor protein, and the mature Gag, Pro, and Pol proteins, including the reverse transcriptase, are created by proteolytic processing catalyzed by the viral protease. The location of the proteolytic cleavage site, which creates the N-terminus of mature HTLV-1 reverse transcriptase, has not been previously identified. By using sequence comparisons of several retroviral polymerases, as well as information about the location of the ribosomal frameshift, we tentatively identified this N-terminal processing site. PCR amplification was used to construct a clone, which spans a region of the pro--pol junction of HTLV-1, to produce a recombinant Pro--Pol protein spanning the locations of those cleavage sites proposed by others as well as the one identified by our sequence alignment. Cleavage of the recombinant Pro--Pol protein by HTLV-1 protease generated a 5.5-kDa fragment. Analysis of this fragment by capillary LC-MS and MS/MS revealed the N-terminal cleavage site to be between Leu(147)--Pro(148) of the pro ORF. This is the first physical identification of the authentic amino acid sequence of the reverse transcriptase of HTLV-1. The data reported here provides a basis for further investigation of the function and structural aspects of protein-nucleic interaction.

Amino Acid Sequence↗

PubMed2001

A high affinity acceptor for phospholipase A2 with neurotoxic activity is a calmodulin.

One of the high affinity binding proteins for ammodytoxin C, a snake venom presynaptically neurotoxic phospholipase A(2), has been purified from porcine cerebral cortex and characterized. After extraction from the membranes, the toxin-binding protein was isolated in a homogenous form using wheat germ lectin-Sepharose, Q-Sepharose, and ammodytoxin-CH-Sepharose chromatography. The specific binding of (125)I-ammodytoxin C to the isolated acceptor was inhibited to different extents by some neurotoxic phospholipases A(2), ammodytoxins, bee venom phospholipase A(2), agkistrodotoxin, and crotoxin; but not by nontoxic phospholipases A(2), ammodytin I(2), porcine pancreatic phospholipase A(2), and human type IIA phospholipase A(2); suggesting the significance of the acceptor in the mechanism of phospholipase A(2) neurotoxicity. The isolated acceptor was identified as calmodulin by tandem mass spectrometry. Since calmodulin is generally considered as an intracellular protein, the identity of this acceptor supports the view that secretory phospholipase A(2) neurotoxins have to be internalized to exert their toxic effect. Moreover, since ammodytoxin is known to block synaptic transmission, its interaction with calmodulin as an acceptor may constitute a valuable probe for further investigation of the role of the latter in this Ca(2+)-regulated process.

Amino Acid Sequence↗

PubMed2001

Differential extraction and enrichment of human sperm surface proteins in a proteome: identification of immunocontraceptive candidates.

The objective of this study was to discover previously unknown human sperm surface proteins that may be candidate contraceptive vaccinogens. To this end, methods of concentrating human sperm proteins for microsequencing by mass spectrometry were used, which increased the likelihood of identifying surface proteins. Vectorial labeling, differential extraction and two-dimensional (2-D) gel electrophoresis were employed to identify and isolate proteins accessible at the cell surface. Percoll harvested or swim-up sperm were either solubilized directly or solubilized after surface labeling with sulfo-succinimidyl-6-(biotinamido)hexanoate (sulfo-NHS-LC-biotin). Comparisons were made of proteins extracted with four lysis buffers: (i) Celis buffer containing 9.8 M urea and 2% Igepal CA-630; (ii) 1% Triton X (TX)-100; (iii) 1.7% TX-114 followed by phase partitioning; or (iv) 1 M NaCl. Blots of proteins separated by high-resolution 2-D electrophoresis were probed with avidin and antibodies to known proteins specific for three domains: the sperm surface (SAGA-1), the acrosome (SP-10), and the cytoskeleton (alpha-tubulin). Celis buffer (45 min) extracted proteins from all three major compartments. However, a 20-s extraction in Celis buffer enriched for several proteins and enabled the identification of several novel peptides by mass spectrometry. Mild extraction with TX-100 or 1 M NaCl solubilized mainly membrane and acrosomal proteins, but not cytoskeletal proteins. Comparison of biotinylated proteins extracted by each method showed that the major vectorially labeled proteins solubilized by Celis buffer were also solubilized by TX-100, TX-114, and 1 M NaCl. Extraction with TX-114 followed by phase-partitioning significantly enriched hydrophobic surface proteins and aided resolution and isolation. Eight protein spots microsequenced following all these extraction methods proved to be novel sperm molecules.

Acrosome↗

PubMed2001

Co-localization of the inositol 1,4,5-trisphosphate receptor and calreticulin in the equatorial segment and in membrane bounded vesicles in the cytoplasmic droplet of human spermatozoa.

Modulation of the intracellular calcium concentration within mammalian spermatozoa is important in several pre-fertilization events including hyperactivated motility and the acrosome reaction. To identify calcium binding proteins (CBP) potentially regulating these processes, a (45)Ca overlay technique was employed on 2-D blots of human sperm extracts. Microsequencing by Edman degradation and CAD mass spectrometry identified a relatively abundant 60.5 kDa CBP with a pI of 4.2 as calreticulin (CRT). Immunofluorescent labelling with anti-CRT antibodies localized CRT to the acrosome, with highest fluorescence in the equatorial segment, and in the cytoplasmic droplets of 94 and 48% of human spermatozoa respectively. Double immunolabelling experiments demonstrated co-localization of CRT and the inositol 1,4,5-trisphosphate receptor (IP(3)R) in the acrosome, in the equatorial segment, and vesicular structures in the cytoplasmic droplets of the neck region. Electron microscopic immunogold labelling localized CRT to the equatorial segment of acrosome-reacted spermatozoa and to membrane-enclosed vesicles within the cytoplasmic droplet of both acrosome-intact and acrosome-reacted spermatozoa. Localization of the IP(3) receptor to the CRT-containing vesicles, in the sperm neck and to the acrosome, suggests that capacitative calcium entry in human spermatozoa may be regulated from these putative calcium storage sites.

Acrosome↗

PubMed2001

Outer dense fiber proteins are dominant postobstruction autoantigens in adult Lewis rats.

Obstruction of the male reproductive tract commonly results in generation of antisperm autoantibodies. However, only a few of the sperm autoantigens recognized by these antibodies have been characterized. To identify postobstruction rat sperm autoantigens, sperm proteins were separated by two-dimensional(2-D) gel electrophoresis. Spots corresponding to proteins that were stained by at least 50% of postvasectomy rat sera on 2-D Western blots were removed from polyacrylamide gels and microsequenced by tandem mass spectrometry. From a total of 21 spots, 12 contained peptides that matched solely to either of two outer dense fiber proteins, odf1 or odf2. Six additional spots contained peptides comprising odf1 or odf2 and were accompanied by peptides representing other proteins. Only three spots lacked outer dense fiber peptides but did contain sequences of other known proteins. The results indicate that the outer dense fiber proteins odf1 and odf2 are dominant postobstruction autoantigens because they were detected in the majority of the immunoreactive protein spots examined. Possible explanations for this observation include the abundance of outer dense fiber proteins in spermatozoa, slow solubility, which may provide a sustained supply of antigen, and testis-specific expression during spermiogenesis.

Amino Acid Sequence↗

PubMed2001

Pseudomonas aeruginosa and a proteomic approach to bacterial pathogenesis.

Pseudomonas aeruginosa is a Gram-negative bacterium that is ubiquitous in the environment and can cause a variety of diseases in compromised patients. The genome of P. aeruginosa strain PAO1 has been reported to contain 5570 potential proteins. The value of this genomic database is that new proteins can be recognized to use as diagnostic markers, novel drug targets, and to better understand the physiology of this organism. However, similar to what has been observed in other sequenced bacterial genomes, approximately one third of the potential proteins have no known function. This is somewhat surprising given the long-standing interest in P. aeruginosa as an opportunistic pathogen. Obviously new tools, in addition to sequence similarity analysis, are needed to determine the role of these proteins. Proteomics using two-dimensional gel electrophoresis followed by mass spectrometry to detect and identify P. aeruginosa proteins represents a novel approach to address this gap.

Animals↗

PubMed2001

Fingerprinting Trichoderma reesei hydrolases in a commercial cellulase preparation.

Polysaccharide degrading enzymes from commercial T. reesei broth have been subjected to "fingerprint" analysis by high-resolution 2-D gel electrophoresis. Forty-five spots from 11 x 25 cm Pharmacia gels have been analyzed by LC-MS/MS and the resulting peptide sequences were compared to existing databases. Understanding the roles and relationships of component enzymes from the T. reesei cellulase system acting on complex substrates is key to the development of efficient artificial cellulase systems for the conversion of lignocellulosic biomass to sugars. These studies suggest follow-on work comparing induced and noninduced T. reesei cells at the proteome level, which may elucidate substrate-specific gene regulation and response.

Biotechnology↗

PubMed2000

T cell tolerance based on avidity thresholds rather than complete deletion allows maintenance of maximal repertoire diversity.

Given the flexible nature of TCR specificity, deletion or permanent disabling of all T cells with the capacity to recognize self peptides would severely limit the diversity of the repertoire and the capacity to recognize foreign Ags. To address this, we have investigated the patterns of CD8+ CTL reactivity to a naturally H-2Kb-presented self peptide derived from the elongation factor 1alpha (EF1alpha). EF1alpha occurs as two differentially expressed isoforms differing at one position of the relevant peptide. Low avidity CTLs could be raised against both variants of the EF1alpha peptide. These CTLs required 100-fold more peptide-H-2Kb complexes on the target cell compared with CTLs against a viral peptide, and did not recognize the naturally expressed levels of EF1alpha peptides. Thus, low avidity T cells specific for these self peptides escape tolerance by deletion, despite expression of both EF1alpha isoforms in dendritic cells known to mediate negative selection in the thymus. The low avidity in CTL recognition of these peptides correlated with low TCR affinity. However, self peptide-specific CTLs expressed elevated levels of CD8. Furthermore, CTLs generated against altered self peptide variants displayed intermediate avidity, indicating cross-reactivity in induction of tolerance. We interpret these data, together with results previously published by others, in an avidity pit model based on avidity thresholds for maintenance of both maximal diversity and optimal self tolerance in the CD8+ T cell repertoire.

Animals↗

PubMed2000

Molecular cloning of apobec-1 complementation factor, a novel RNA-binding protein involved in the editing of apolipoprotein B mRNA.

The C-to-U editing of apolipoprotein B (apo-B) mRNA is catalyzed by a multiprotein complex that recognizes an 11-nucleotide mooring sequence downstream of the editing site. The catalytic subunit of the editing enzyme, apobec-1, has cytidine deaminase activity but requires additional unidentified proteins to edit apo-B mRNA. We purified a 65-kDa protein that functionally complements apobec-1 and obtained peptide sequence information which was used in molecular cloning experiments. The apobec-1 complementation factor (ACF) cDNA encodes a novel 64.3-kDa protein that contains three nonidentical RNA recognition motifs. ACF and apobec-1 comprise the minimal protein requirements for apo-B mRNA editing in vitro. By UV cross-linking and immunoprecipitation, we show that ACF binds to apo-B mRNA in vitro and in vivo. Cross-linking of ACF is not competed by RNAs with mutations in the mooring sequence. Coimmunoprecipitation experiments identified an ACF-apobec-1 complex in transfected cells. Immunodepletion of ACF from rat liver extracts abolished editing activity. The immunoprecipitated complexes contained a functional holoenzyme. Our results support a model of the editing enzyme in which ACF binds to the mooring sequence in apo-B mRNA and docks apobec-1 to deaminate its target cytidine. The fact that ACF is widely expressed in human tissues that lack apobec-1 and apo-B mRNA suggests that ACF may be involved in other RNA editing or RNA processing events.

APOBEC-1 Deaminase↗

PubMed1998

Arginine to glutamine substitutions in the fourth module of Xenopus interphotoreceptor retinoid-binding protein.

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP) is unusual for a lipid-binding protein in that its gene is expressed uniquely by cells of photoreceptor origin and consists of four homologous repeats, each coding for a module of approximately 300 amino acid residues. All-trans retinol binding domains, which appear to be present in each module, are composed of conserved hydrophobic regions [Baer et al, Exp Eye Res 1998; 66:249-262]. Here we investigate the role of highly conserved arginines contained in these regions. METHODS: To study the arginines in an individual module without the interference of ligand-binding activity elsewhere in the protein, we expressed in E. coli the fourth module of Xenopus IRBP by itself as a soluble thioredoxin fusion protein (X4IRBP). Arginines 1005, 1041, 1073 and 1122 were separately replaced by glutamine using PCR overlap extension mutagenesis. The glutamine substitutions were confirmed by liquid chromatography-tandem mass spectrometry. The binding of all-trans retinol and 9-(9-anthroyloxy)stearic acid (9-AS) to X4IRBP and each of the mutants was evaluated by fluorescence spectroscopy. Binding was followed by monitoring the enhancement of ligand fluorescence and the quenching of protein endogenous fluorescence. The ability of the recombinant proteins to protect all-trans retinol from oxidative degradation was evaluated by monitoring absorbance at 325 nm over time. RESULTS: The substitution of Gln for Arg1005 about doubled the amount of ligand necessary to attain saturation and about doubled the level of fluorescence enhancement obtained at saturation for both all-trans retinol and 9-AS. Although there was not a significant change in the Kd, the substitution increased the calculated number of binding sites (N) from approximately 2 to approximately 4 per polypeptide. The other Arg->Gln mutants did not significantly change the Kd or N. None of the mutations compromised the ability of the module to protect all-trans retinol from degradation. CONCLUSIONS: Our data suggest that the function of the conserved arginines in IRBP is fundamentally different from that of other retinoid-binding proteins. These residues do not appear to play a role in defining the specificity of the ligand-binding domain. Rather, Arg1005 appears to play a role in defining the capacity of the domain. Our data suggest that the binding site consists of a single hydrophobic cavity promiscuous for fatty acids and all-trans retinol.

Amino Acid Substitution↗

PubMed1996

G protein gamma subunits with altered prenylation sequences are properly modified when expressed in Sf9 cells.

The gamma subunits of heterotrimeric G proteins undergo post-translational prenylation and carboxylmethylation after formation of the betagamma dimer, modifications that are essential for alpha-betagamma, betagamma-receptor, and betagamma-effector interactions. We have determined the specific prenyl group present on the beta1gamma1, beta1gamma2, and beta1gamma3 dimers purified from baculovirus-infected Sf9 cells by specific binding to G protein alpha subunits immobilized on agarose. These recombinant dimers undergo the same post-translational modifications determined for gamma1 and gamma2 isolated from mammalian tissues. Furthermore, infection of Sf9 cells with a recombinant baculovirus encoding an alteration of the gamma1 CaaX sequence (gamma1-S74L) resulted in geranylgeranylation of the resulting gamma1 subunit, and alteration of the gamma2 CaaX sequence to CAIS (gamma2-L71S) resulted in farnesylation. Both of these altered gamma subunits were able to associate stably with beta1, and the resulting betagamma dimer bound tightly to alpha-agarose and eluted specifically with aluminum fluoride. These results indicate that Sf9 insect cells properly process the CaaX motif in G protein gamma subunits and are a useful model system to study the role of prenylation in the protein-protein interactions in which the betagamma subunits participate.

Amino Acid Sequence↗

PubMed1996

A Listeria monocytogenes pentapeptide is presented to cytolytic T lymphocytes by the H2-M3 MHC class Ib molecule.

Polymorphism of MHC class Ia molecules severely constrains vaccine development against intracellular pathogens. Antigen presentation by MHC class Ib molecules, which are generally conserved between different individuals, may circumvent this obstacle. Herein, we use tandem mass spectrometry to identify a Listeria monocytogenes pentapeptide antigen that is presented to T lymphocytes by the H2-M3 MHC class Ib molecule. The peptide contains N-formyl methionine at the N terminus and exclusively hydrophobic amino acids. Mice of the H-2 d, H-2 b,and H-2 k haplotypes respond to this peptide upon infection with Listeria monocytogenes. Identification of antigens presented by MHC class Ib molecules is feasible and may provide opportunities for relatively unrestricted vaccine development.

Alleles↗

PubMed1995

Computer-aided design and fabrication of an electron bolus for treatment of the paraspinal muscles.

PURPOSE: Demonstrate the technology for the design, fabrication, and verification of an electron bolus used in the preoperative irradiation of a mesenchymal chondrosarcoma in the paraspinal muscle region (T8-T12), in which the target volume overlay a portion of the spinal cord, both lungs, and the right kidney. METHODS AND MATERIALS: An electron-bolus design algorithm implemented on a three dimensional (3D) radiotherapy treatment planning system designed the bolus to yield a dose distribution that met physician-specified clinical criteria. Electron doses were calculated using a 3D electron pencil-beam dose algorithm. A computer-driven milling machine fabricated the bolus from modeling wax, machining both the patient surface and the beam surface of the bolus. Verification of the bolus fabrication was achieved by repeating the patient's computed tomography (CT) scan with the fabricated bolus in place (directly on the posterior surface of the prone patient) and then recalculating the patient's dose distribution using the 3D radiotherapy treatment planning system. RESULTS: A treatment plan using a 17-MeV posterior electron field with a bolus delivered a superior dose distribution to the patient than did the same plan without a bolus. The bolus plan delivered a slightly increased dose to the target volume as a result of a slightly broader range of doses. There were significant reductions in dose to critical structures (cord, lungs, and kidney) in the bolus plan, as evidenced by dose-volume histograms (DVHs). The patient dose distribution, calculated using CT scan data with the fabricated bolus, showed no significant differences from the planned dose distribution. CONCLUSIONS: A bolus can provide considerable sparing of normal tissues when using a posterior electron beam to irradiate the paraspinal muscles. Bolus design and fabrication using the tools described in this paper are adequate for patient treatment. CT imaging of the patient with the bolus in place followed by calculation of the patient's dose distribution demonstrated a useful method for verification of the bolus design and fabrication process.

Adolescent↗

PubMed1995

Human H-Y: a male-specific histocompatibility antigen derived from the SMCY protein.

H-Y is a transplantation antigen that can lead to rejection of male organ and bone marrow grafts by female recipients, even if the donor and recipient match at the major histocompatibility locus of humans, the HLA (human leukocyte antigen) locus. However, the origin and function of H-Y antigens has eluded researchers for 40 years. One human H-Y antigen presented by HLA-B7 was identified as an 11-residue peptide derived from SMCY, an evolutionarily conserved protein encoded on the Y chromosome. The protein from the homologous gene on the X chromosome, SMCX, differs by two amino acid residues in the same region. The identification of H-Y may aid in transplantation prognosis, prenatal diagnosis, and fertilization strategies.

Amino Acid Sequence↗

PubMed1995

Identification of a graft versus host disease-associated human minor histocompatibility antigen.

Minor histocompatibility antigen disparities between human leukocyte antigen (HLA)-matched bone marrow donors and recipients are a major risk factor for graft versus host disease (GVHD). An HLA-A2.1-restricted cytotoxic T cell clone that recognized the minor histocompatibility antigen HA-2 was previously isolated from a patient with severe GVHD after HLA-identical bone marrow transplantation. The HLA-A2.1-bound peptide representing HA-2 has now been identified. This peptide appears to originate from a member of the non-filament-forming class I myosin family. Because HA-2 has a phenotype frequency of 95 percent in the HLA-A2.1-positive population, it is a candidate for immunotherapeutic intervention in bone marrow transplantation.

Amino Acid Sequence↗

PubMed1991

The influence of local control on metastatic dissemination of prostate cancer treated by external beam megavoltage radiation therapy.

The influence of local control on metastatic dissemination was analyzed in 601 patients with clinically staged A2 to C prostate cancer treated by high-energy external beam radiation therapy who did not undergo hormonal manipulation before disease progression. Median follow-up for surviving patients was 7.7 years. Ninety-three patients had locally recurrent disease. The actuarial incidence of metastases in these patients (70% at 13 years) was significantly higher than in the 508 patients without local failure (40% at 13 years, P less than 0.001). High stage, high grade, prior transurethral resection, elevated acid phosphatase, disease fixation to the pelvic sidewall, and failure to perform a baseline bone scan correlated positively with the occurrence of metastases. However, except for a slight excess of Stage C, none of the metastatic predictors were more common in patients who failed locally than in those who did not. The Stage C preponderance does not account for the difference in incidence of metastases between the two groups, in as much as metastases were significantly more common in Stage C when disease recurred locally than when it did not. Thus, local control of prostate cancer does decrease the likelihood of metastatic disease. Moreover, patients with local control experienced a significantly better disease-specific survival than patients who failed locally.

Actuarial Analysis↗