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Biomedical subjects

T C Evans

Publications and source records attributed to T C Evans.

At least 19 recordsLinked to original sources
PubMed1998

Semisynthesis of cytotoxic proteins using a modified protein splicing element.

Two cytotoxic proteins, bovine pancreatic ribonuclease A (RNase A), and a restriction endonuclease from Haemophilus parainfluenzae (HpaI), were produced using a novel semisynthetic approach that utilizes a protein splicing element, an intein, to generate a reactive thioester at the C-terminus of a recombinant protein. Nucleophilic attack on this thioester by the N-terminal cysteine of a synthetic peptide ultimately leads to the ligation of the two reactants through a native peptide bond. This strategy was used to produce RNase A and HpaI by isolating inactive truncated forms of these proteins, the first 109 and 223 amino acids of RNase A and HpaI, respectively, as fusion proteins consisting of the target protein, an intein, and a chitin binding domain. Thiol-induced cleavage of the precursor led to the liberation of the target protein with a C-terminal thioester-tag. Addition of synthetic peptides representing the amino acids missing from the truncated forms led to the generation of full-length products that displayed catalytic activity indicative of the wild-type enzymes. The turnover numbers and Km for ligated and renatured RNase A were 8.2 s(-1) and 1.5 mM, in good agreement with reported values of 8.3 s(-1) and 1.2 mM (Hodges & Merrifield, 1975). Ligated HpaI had a specific activity of 0.5-1.5 x 10(6) U/mg, which compared favorably with the expected value of 1-2 x 10(6) U/mg (J. Benner, unpubl. obs.). Besides assisting in the production of cytotoxic proteins, this technique could allow the easy insertion of unnatural amino acids into a protein sequence.

Amino Acid Sequence↗
PubMed1997

Ionic properties of membrane association by vitamin K-dependent proteins: the case for univalency.

Ionic properties of membrane interaction by prothrombin, protein Z, and other vitamin K-dependent proteins were studied to determine the relevance of a monovalent membrane contact mechanism between one phospholipid headgroup and a calcium-lined pore in the protein [McDonald, J. F., Shah, A. M., Schwalbe, R. A., Kisiel, W., Dahlback, B., and Nelsestuen, G. L. (1997) Biochemistry 36, 5120-5127]. For comparison, multivalent ionic interaction was illustrated by peptides of +3 to +5 net charge and by blood clotting factor V. As expected, the peptides were easily dissociated by salt and gave nominal charge-charge interactions (zazb values) of -13 to -17. Factor V showed much higher binding affinity despite nominal zazb values of about 9. Membrane-bound prothrombin and protein Z showed very low sensitivity to salt as long as calcium was at saturating levels (zazb values of approximately -1.3 to -1.4), appropriate for univalent ionic attraction. Prothrombin contains +3 charge groups (Lys-2, Lys-11, Arg-10) that are absent from the GLA domain (residues 1-35) of protein Z, while protein Z contains -4 charge groups (Gla-11, Asp-34, Asp-35) that are absent in prothrombin. Thus, similar zazb relationships indicated little role for these surface charges in direct membrane contact. Calcium-saturated protein Z bound to phosphatidylcholine (PC) in a manner which indicated the addition of one calcium ion, bringing the total calcium stoichiometry in the protein-membrane complex to at least 8. Protein Z bound to phosphatidic acid (PA) in a manner suggesting the need for a fully ionized phosphate headgroup, a property expected by ion pairing in an isolated environment. Electrostatic calculations showed that the proposed protein site for phosphate interaction was electropositive. The cluster of hydrophobic amino acids (Phe-5, Leu-6, and Val-9) on the surface of prothrombin was electronegative, suggesting a role in the electrostatic architecture of the GLA domain. Overall, membrane binding by vitamin K-dependent proteins appeared consistent with the formation of an ion pair in an isolated environment.

Amino Acid Sequence↗
PubMed1997

Genes required for GLP-1 asymmetry in the early Caenorhabditis elegans embryo.

The translation of maternal glp-1 mRNA is regulated both temporally and spatially in the early Caenorhabditis elegans embryo (T. C. Evans, S. L. Crittenden, V. Kodoyianni, and J. Kimble, Cell 77, 183-194, 1994). To investigate the control of embryonic glp-1 expression, we have examined the distribution of GLP-1 protein in selected maternal effect mutants that affect pattern or fate in the early embryo. We find that mutants that disrupt anterior-posterior asymmetry in the early embryo (par-1-par-6, emb-8, Par(q537)) disrupt the spatial but not temporal control of GLP-1 expression: GLP-1 is observed at the normal stage of embryogenesis in par-like mutants; however, it is uniformly distributed. In contrast, mutants that alter blastomere identity (skn-1, pie-1, mex-1, apx-1) do not affect the normal GLP-1 pattern. We conclude that genes controlling the asymmetry of cellular components, including P granules, also control GLP-1 asymmetry in the early embryo. The finding that mutants that disrupt anterior-posterior asymmetry translate GLP-1 in all blastomeres suggests that loss of embryonic asymmetry causes translational activation of GLP-1 in the posterior.

Animals↗
PubMed1996

Importance of cis-proline 22 in the membrane-binding conformation of bovine prothrombin.

Upon addition of calcium to the metal-free protein, bovine prothrombin displays a conformational change with behavior of a classic trans- to cis-proline isomerization. The change is accompanied by a decrease of the intrinsic protein fluorescence and is essential to creating the membrane-binding conformation of prothrombin. This study showed that an identical conformational change was displayed by a peptide corresponding to residues 1-45 of prothrombin. This peptide contains a single tryptophan that underwent extensive quenching upon calcium addition. The kinetics were slow (t1/2 = 2.7 min at 24 degrees C) and displayed an activation energy of 24 kcal/mol. These properties overlapped precisely with the behavior of bovine prothrombin fragment 1 (residues 1-156). Consistent with studies on prothrombin and other vitamin K-dependent proteins that have been modified or truncated, the 1-45 peptide required about 10-fold higher calcium to elicit these behaviors than did fragment 1. The conformational change was necessary for membrane binding by the 1-45 peptide. The only proline in this sequence is at position 22. This proline is of the trans configuration in a crystallized form of calcium-bovine prothrombin fragment 1 [Soriano-Garcia, M., et al. (1992) Biochemistry 31, 2554]. Unless the protein conformational change is based on another behavior, this study showed that biochemical properties of the protein are inconsistent with structure solutions. Further studies are needed to reconcile structure/function in membrane association. Proline 22 in bovine prothrombin may constitute a useful biochemical marker for the membrane-binding conformation of a vitamin K-dependent protein.

Amino Acid Sequence↗
PubMed1996

Epirubicin, cisplatin, and protracted venous infusion of 5-fluorouracil for esophagogastric adenocarcinoma: response, toxicity, quality of life, and survival.

BACKGROUND: The results of chemotherapy for patients with esophagogastric carcinoma have generally been modest but regimens developed more recently have produced higher response rates, and rekindled interest in neoadjuvant chemotherapy. One such regimen is epirubicin, cisplatin, and 5-fluorouracil (ECF). This study evaluates its efficacy, toxicity, impact on quality of life (QL), and impact on survival in a large consecutive series of patients with metastatic and locally advanced disease (LAD). METHODS: Patients with histologically confirmed esophagogastric carcinoma were treated with ECF (epirubicin 50 mg/m2 and cisplatin 60 mg/m2 every 3 weeks with continuous infusion of 5-fluorouracil (5-FU) 200 mg/m2/d). Responses were evaluated with computed tomography (CT) scan and endoscopy. QL was assessed using the European Organization for Research and Treatment of Cancer QLQ-C30 questionnaire. RESULTS: A total of 235 patients were treated, 173 with metastatic disease and 62 with LAD. The mean number of cycles delivered was 6 (range: 1-11) and patients were followed-up for a median of 8 months. Response was observed in 135 of 220 (61%) evaluable patients, with a complete response (C(R)), 11% of the patients and a partial response in 50% of the patients. Patients with moderately differentiated adenocarcinomas and LAD responded most favorably. Symptomatic improvement was achieved in the majority of cases (63-78% depending on the symptom). Toxicity was generally only mild to moderate, with severe non hematologic toxicity in less than 12% of the patients and only 6 (2.5%) treatment related deaths. QL assessment showed no significant negative impact on emotional functioning and good symptomatic control. Surgery following response to ECF was performed in 29 of the LAD patients, and in 19 cases (66%) a potentially curative resection was possible, with histologic CR in 32% of the patients. CONCLUSIONS: ECF is a highly active regimen with acceptable toxicity in patients with esophagogastric adenocarcinoma. In a proportion of patients with LAD, chemotherapy enabled potentially curative surgery to be performed. The results justify further investigation of this regimen in a neoadjuvant setting.

Adenocarcinoma↗
PubMed1995

Dissociation of serum amyloid P from C4b-binding protein and other sites by lactic acid: potential role of lactic acid in the regulation of pentraxin function.

Serum amyloid P (SAP) and C-reactive protein (CRP) are two members of the pentraxin family of proteins. These proteins associate with a variety of other materials that are found in serum under normal or pathological circumstances. This study showed that carboxylated compounds, especially lactic acid, were capable of dissociating pentraxins from several macromolecular binding sites. When measured by sucrose density gradient ultracentrifugation, complete dissociation of the complex of hSAP (human SAP) with C4b-binding protein (C4BP) occurred at > or = 5 mM lactate. Lactate dissociated the hSAP-membrane complex and prevented hSAP self-association. The only interaction that was not dissociated by 10 mM lactate was the hSAP-heparin complex. The relative efficacies of several dissociating agents were O-phosphorylethanolamine > lactate > succinate > carbonate > epsilon-amino-n-caproic acid. This suggested that the carboxyl group plus a hydrogen-bonding site on the hydrocarbon chain was important, but a charged amino group was not a contributor to function when the anion was provided by a carboxyl group. The concentration of lactic acid needed to dissociate hSAP from C4BP was dependent on protein concentration in a manner suggesting the cooperative binding of lactate (coefficient = 2) to hSAP. Pure proteins, at concentrations found in normal serum, required about 12 mM lactate for half-dissociation of the hSAP-C4BP complex. Other pentraxins also interacted with lactic acid, but with lower affinities. An important observation was that lactic acid was capable of dissociating rat CRP from lipoproteins in rat serum. Human CRP bound very weakly to lactate, so that lactate probably is not a significant regulator of this pentraxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1994

Calcium and membrane-binding properties of monomeric and multimeric annexin II.

The calcium-dependent interaction of several annexins with membranes was studied. A novel technique was developed that allowed estimation of calcium binding to aggregating systems. This consisted of immobilized phospholipids on phenyl-Sepharose. Proteins associated with this affinity gel in the presence of radioactive calcium and were eluted with the same buffer containing excess EGTA. This produced an elution profile with a peak of excess calcium. Protein extinction coefficients were estimated in order to quantitate the protein more accurately. Association of annexin II with the membrane was of very high affinity and involved a calcium stoichiometry of 11 +/- 1 at 12.5 microM free Ca2+ and 10 +/- 2 at 50 microM free Ca2+. (AII)2(p11)2, a heterotetramer of two annexin II and two p11 subunits, bound 12 +/- 1 Ca2+ at 12.5 microM Ca2+ and 15 +/- 1 Ca2+ at 50 microM Ca2+. These stoichiometries showed a pattern of "all or none" calcium binding where the number of calcium ions bound to a protein-membrane complex was virtually independent of the free calcium concentration or the density of protein on the membrane. (AII)2(p11)2 contains two annexin II subunits so that calcium stoichiometry was not directly related to the number of potential sites. (AII)2(p11)2 required less calcium to support membrane binding than did annexin II. Thus, dimerization of the membrane binding unit may be needed for annexins to function at intracellular calcium levels. Annexin VI contains twice as many putative calcium binding units as annexin V and the same pattern of behavior occurred for this pair of proteins. At 25 microM free calcium, (AII)2(p11)2 alone bound no detectable calcium (< 0.1 mol of calcium/mol of protein) and annexin II bound only 0.3-0.6 calcium ions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Translational control of maternal glp-1 mRNA establishes an asymmetry in the C. elegans embryo.

In C. elegans, the glp-1 gene encodes a membrane receptor that is required for anterior cell fates in the early embryo. We report that GLP-1 protein is localized to anterior blastomeres in 2- to 28-cell embryos. By contrast, glp-1 mRNA is present in all blastomeres until the 8-cell stage. Furthermore, the glp-1 3' untranslated region can restrict translation of a reporter mRNA to anterior blastomeres. Therefore, the translation of maternal glp-1 mRNA is temporally and spatially regulated in the C. elegans embryo. The regulation of maternal glp-1 mRNA has striking parallels to the regulation of maternal hunchback mRNA in the Drosophila embryo. Thus, the establishment of embryonic asymmetry in diverse organisms may involve conserved mechanisms of maternal mRNA regulation.

Animals↗
PubMed1994

GLP-1 is localized to the mitotic region of the C. elegans germ line.

In C. elegans, germline mitosis depends on induction by the somatic distal tip cell (DTC) and on activity of the glp-1 gene. Using antibodies to GLP-1 protein, we have examined GLP-1 on western blots and by immunocytochemistry. GLP-1 is tightly associated with membranes of mitotic germline cells, supporting its identification as an integral membrane protein. Furthermore, GLP-1 is localized within the germ line to the mitotic region, consistent with the model that GLP-1 acts as a membrane receptor for the distal tip cell signal. Unexpectedly, GLP-1 and the zone of mitosis extend further than the DTC processes. We present three models by which the DTC may influence GLP-1 activity and thereby determine the zone of mitosis. The spatial restriction of GLP-1 appears to be controlled at the translational level in hermaphrodites. We suggest that down-regulation of GLP-1 may be required to effect the transition from mitosis into meiosis.

Animals↗
PubMed1993

Translational regulation of tra-2 by its 3' untranslated region controls sexual identity in C. elegans.

C. elegans hermaphrodites make sperm and then oocytes in an otherwise female animal. Gain-of-function mutations in the sex-determining gene tra-2 (tra-2(gf)) transform hermaphrodites into females (spermless hermaphrodites). The tra-2(gf) mutations map to a perfect direct repeat in the 3' untranslated region; each repeat is called a direct repeat element (DRE). Three experiments demonstrate that DREs repress tra-2 at the translational level. First, tra-2(gf) mRNAs are associated with larger polysomes than are their wild-type counterparts. Second, translation of a reporter RNA is inhibited by DREs. Third, disruption of DREs does not increase tra-2 mRNA levels. An RNA binding activity specifically associates with the DREs. We propose that tra-2 translation is inhibited by association of an RNA binding-factor with the DREs and that this translational control is essential for development of C. elegans as a hermaphrodite/male species.

Animals↗
PubMed1993

Social work technician program.

The increasing use of US emergency departments has resulted in increased patient delays and resultant dissatisfaction on the part of the patients and their families. Furthermore, recent studies have indicated that these delays may jeopardize the health of the ED patient. We describe a program designed to address these concerns. The Social Work Technician Program employs social work students nearing graduation as liaisons between medical personnel and ED patients and their families. This program has been found to promote patient satisfaction and welfare while providing the students with a valuable educational experience.

Clinical Clerkship↗
PubMed1993

Clinical study of bendazac lysine for in vivo contact lens cleaning.

Bendazac Lysine (BZL), a nonsteroidal anti-inflammatory drug (NSAID), prevents denaturation of proteins secondary to chemical and physical activity. The major protein deposited on soft contact lenses is lysozyme, derived from the preocular tear film. We evaluated the effectiveness of eye drops formulated with BZL for in vivo prevention of proteinaceous deposits on contact lenses and found them to be useful in inhibiting protein deposition during a 6-month clinical trial.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1993

Acute pulmonary disease in the aged.

Due to the changes within the respiratory system that occur physiologically with aging as well as preexisting medical conditions, elderly persons are less tolerant of the insults discussed in this article. For this reason, clinicians must be particularly vigilant in their approach to the diagnosis and management of pulmonary complaints in this population.

Acute Disease↗
PubMed1991

The red blood cell distribution width.

The availability of automated blood cell analyzers that provide an index of red blood cell distribution width (RDW) has lead to new approaches to patients with anemia. While the emergency physician is primarily responsible for the detection of patients with anemia, the inclusion of the RDW in the complete blood count has made diagnosing certain anemias easier, especially those that are microcytic. The derivation of the RDW and its clinical application to emergency physicians is discussed and a categorization of anemias based on the mean corpuscular volume (MCV) and RDW is included.

Adult↗