Biomedical subjects
Publications and source records attributed to S L Briggs.
Fibronectin is known to stimulate cell growth and migration, but research does not provide a complete picture of all the mechanisms involved. Further studies are needed before practitioners can apply in vitro results to in vivo environments.
The Relativistic Heavy Ion Collider (RHIC) is a high energy hadron accelerator built to study basic nuclear physics. It consists of two counter-rotating beams of fully stripped gold ions that are accelerated in two rings to an energy of 100 GeV/nucleon or protons at 250 GeV/c. The beams can be stored for a period of five to ten hours and brought into collision for experiments during that time. The first major physics objective is to recreate a state of matter, the quark-gluon plasma, that has been predicted to have existed at a short time after the creation of the universe. Because there are only a few other high energy particle accelerators like RHIC in the world, the rules promulgated in the US Code of Federal Regulations under the Atomic Energy Act, State regulations, or international guidance documents do not cover prompt radiation from accelerators to govern directly the design and operation of a superconducting collider. Special design criteria for prompt radiation were developed to provide guidance tor the design of radiation shielding. Environmental Management at RHIC is accomplished through the ISO 14001 Environmental Management System. The applicability, benefits, and implementation of ISO 14001 within the framework of a large research accelerator complex are discussed in the paper.
Acyl carrier protein synthase (AcpS) catalyzes the formation of holo-ACP, which mediates the essential transfer of acyl fatty acid intermediates during the biosynthesis of fatty acids and lipids in the cell. Thus, AcpS plays an important role in bacterial fatty acid and lipid biosynthesis, making it an attractive target for therapeutic intervention. We have determined, for the first time, the crystal structure of the Streptococcus pneumoniae AcpS and AcpS complexed with 3'5'-ADP, a product of AcpS, at 2.0 and 1.9 A resolution, respectively. The crystal structure reveals an alpha/beta fold and shows that AcpS assembles as a tightly packed functional trimer, with a non-crystallographic pseudo-symmetric 3-fold axis, which contains three active sites at the interface between protomers. Only two active sites are occupied by the ligand molecules. Although there is virtually no sequence similarity between the S.pneumoniae AcpS and the Bacillus subtilis Sfp transferase, a striking structural similarity between both enzymes was observed. These data provide a starting point for structure-based drug design efforts towards the identification of AcpS inhibitors with potent antibacterial activity.
The N-terminal fragment 1-34 of parathyroid hormone (PTH), administered intermittently, results in increased bone formation in patients with osteoporosis. PTH and a related molecule, parathyroid hormone-related peptide (PTHrP), act on cells via a common PTH/PTHrP receptor. To define more precisely the ligand-receptor interactions, we have crystallized human PTH (hPTH)-(1-34) and determined the structure to 0.9-A resolution. hPTH-(1-34) crystallizes as a slightly bent, long helical dimer. Analysis reveals that the extended helical conformation of hPTH-(1-34) is the likely bioactive conformation. We have developed molecular models for the interaction of hPTH-(1-34) and hPTHrP-(1-34) with the PTH/PTHrP receptor. A receptor binding pocket for the N terminus of hPTH-(1-34) and a hydrophobic interface with the receptor for the C terminus of hPTH-(1-34) are proposed.
The crystal structures of four active site-directed thrombin inhibitors, 1-4, in a complex with human alpha-thrombin have been determined and refined at up to 2.0 A resolution using X-ray crystallography. These compounds belong to a structurally novel family of inhibitors based on a 2,3-disubstituted benzo[b]thiophene structure. Compared to traditional active-site directed inhibitors, the X-ray crystal structures of these complexes reveal a novel binding mode. Unexpectedly, the lipophilic benzo[b]thiophene nucleus of the inhibitor appears to bind in the S1 specificity pocket. At the same time, the basic amine of the C-3 side chain of the inhibitor interacts with the mostly hydrophobic proximal, S2, and distal, S3, binding sites. The second, basic amine side chain at C-2 was found to point away from the active site, occupying a location between the S1 and S1' sites. Together, the aromatic rings of the C-2 and C-3 side chains sandwich the indole ring of Trp60D contained in the thrombin S2 insertion loop defined by the sequence "Tyr-Pro-Pro-Trp." [The thrombin residue numbering used in this study is equivalent to that reported for chymotrypsinogen (Hartley BS, Shotton DM, 1971, The enzymes, vol. 3. New York: Academic Press. pp 323-373).] In contrast to the binding mode of more classical thrombin inhibitors (D-Phe-Pro-Arg-H, NAPAP, Argatroban), this novel class of benzo[b]thiophene derivatives does not engage in hydrogen bond formation with Gly216 of the thrombin active site. A detailed analysis of the three-dimensional structures not only provides a clearer understanding of the interaction of these agents with thrombin, but forms a foundation for rational structure-based drug design. The use of the data from this study has led to the design of derivatives that are up to 2,900-fold more potent than the screening hit 1.
Modeling of neutral or mildly basic functional groups in the S1 site of thrombin led to the targeting of imidazole as a S1 binding element and correctly predicted the optimal chain length for connecting this group with the S2 and S3 binding elements. Derivatives of 4-(3-aminopropyl)-imidazole can be selective inhibitors of thrombin demonstrating potent anticoagulant activity.
Potent, subnanomolar thrombin inhibitors 4, 5, and 6 are developed through side chain optimization of novel, benzo[b]thiophene-based small organic entities 2 and 3 and through SAR additivity studies of the new structural elements identified. X-ray crystallographic studies of 4b-thrombin complex revealed a hydrophobic and an electrostatic interaction of these new elements with thrombin at the S2 and S3 binding sites. In vitro and in vivo pharmacological studies showed that 4, 5, and 6 are potent anticoagulants in human plasma with demonstrated antithrombotic efficacy in a rat model of thrombosis.
BACKGROUND: Because heterotopic pregnancy is rare, the presence of an intrauterine pregnancy tends to impede early diagnosis and definitive intervention for the ectopic component. Delay in diagnosing the condition and failure to proceed quickly with the requisite anesthesia and surgery can jeopardize both maternal well-being and survival of the intrauterine fetus. CASE: A patient with heterotopic pregnancy carried the intrauterine pregnancy to term following first-trimester rupture of the tubal pregnancy, with hypovolemic shock. CONCLUSION: Prompt diagnosis, rapid fluid and blood resuscitation, heart-sparing anesthesia and gentle, expeditious surgery collectively contributed to the favorable outcome for the mother and surviving infant.
In an effort to increase the thrombin inhibitory activity of a novel series of inhibitors (i.e., 1a), substituents were incorporated at the C-3" position of the C-3 aryl ring (2). Consistent with the X-ray crystallography studies, small hydrophobic groups at the C-3" site (Br and Me) enhanced thrombin inhibitory activity by 8-fold. However, a few more hydrophilic substituents (NO2 and OMe) also enhanced the potency of the series. The biological results are discussed in terms of molecular modeling studies.
BACKGROUND: Maternal and fetal morbidity and mortality associated with varicella pneumonia in the peripartum period are increased further with multiple complications. CASE: The patient had varicella pneumonitis acquired at 32 weeks' gestation and complicated by bacterial superinfection, adult respiratory distress syndrome, endotoxin shock, and bronchiolitis obliterans organizing pneumonia. CONCLUSION: Aggressive, timely management of respiratory failure, control of infection, correction of endotoxin shock, treatment of biopsy-proven bronchiolitis obliterans organizing pneumonia, and obstetric intervention for fetal compromise produced a good outcome for mother and infant.
Spiradoline (U62066E) a racemic mixture of the two enantiomers U63639(+) and U63640(-), appears to have kappa opioid receptor activity, but the contribution of each enantiomer toward this activity is still in question. To determine the activity of each enantiomer in comparison to the racemic mixture, the three forms were tested in the cold-water tail-flick (CWTF) assay in male Sprague-Dawley rats. Antinociception by spiradoline was completely antagonized by naloxone 0.50 mg/kg, a dose five times that required to antagonize antinociception by fentanyl in this same assay. In a second series of tests, fentanyl-induced antinociception was markedly reduced, while spiradoline-induced antinociception was essentially unchanged. in methadone-tolerant animals. Of the enantiomers, only U63640 produced antinociception, whereas U63639 failed to affect the nociceptive response. Additionally, spiradoline failed to produce antinociception in animals pretreated with norbinaltorphimine (kappa receptor specific), but antinociception was not affected in animals pretreated with beta-funaltrexamine (mu receptor specific). These results show that spiradoline is a full antinociceptive agonist in the CWTF assay and that the effects of the drug are mediated through kappa opioid receptors.
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Mutations in the obese gene (OB) or in the gene encoding the OB receptor(OB-R) result in obesity, infertility and diabetes in a variety of mouse phenotypes. The demonstration that OB protein (also known as leptin) can normalize body weight in ob/ob mice has generated enormous interest. Most human obesity does not appear to result from a mutant form of leptin: rather, serum leptin concentrations are increased and there is an apparent inability to transport it to the central nervous system (CNS). Injection of leptin into the CNS of overfed rodents resistant to peripheral administration was found to induce biological activity. Consequently, for the leptin to act as a weight-lowering hormone in human obesity, it appears that appropriate concentrations must be present in the CNS. This places a premium on understanding the structure of the hormone in order to design more potent and selective agonists. Here we report the crystal structure at 2.4A resolution of a human mutant OB protein (leptin-E100) that has comparable biological activity to wild type but which crystallizes more readily. The structure reveals a four-helix bundle similar to that of the long-chain helical cytokine family.
The crystal structure of human alpha-thrombin in complex with LY178550, a nonpeptidyl, active site-directed inhibitor, has been solved to 2.07 A resolution by the method of X-ray crystallography. The final model of the complex has a crystallographic R-value of 21.5% (Rfree = 23.1%) with 0.014 A and 2.4 degrees standard deviation from ideal bond lengths and angles, respectively. Well-defined electron density was observed for the inhibitor in the active site. The inhibitor binds to the active site in an L-shaped manner, mimicking the bound conformation of the tripeptide arginal series of thrombin inhibitors (Chirgadze NY et al., 1992, American Crystallographic Association Meeting 20: 116 [Abstr. PB311]). The basic amidine of LY178550 forms a salt bridge with Asp 189 within the specificity pocket, while the 4-benzylpiperidine side chain engages in a number of hydrophobic interactions at the S2 and S3 binding sites. The inhibitor does not interact in any fashion with the active site sequence Ser 214-Gly 216, as occurs with many of the inhibitors studied previously. The indole N-H of the inhibitor forms a hydrogen bond to the gamma-oxygen of the catalytic serine (Ser 195).
Changes in colonic motility in rats following intravenous (IV) oxymorphone (0.1 mg/kg), atropine (0.1 mg/kg), or saline were monitored to determine whether opioid-induced changes in colonic motility affect antinociceptive measurements when using colorectal distension (CRD) as a nociceptive assay. Polygraph recordings of colonic pressures, contraction frequencies, and the pressure-volume relationship of the stimulus showed that oxymorphone produced a transient increase in contraction frequencies when compared to atropine- and saline-treated rats. The transient increase in contraction frequency caused by oxymorphone declined to baseline levels at 30 min after administration, the time at which the nociceptive threshold for CRD was tested. Neither oxymorphone nor atropine changed baseline pressures or the pressure-volume curve for the balloon stimulus. Antinociceptive results from CRD at 30 min posttreatment showed that only oxymorphone produced significant antinociception. We conclude that oxymorphone does not produce changes in colonic motility that complicate antinociceptive measurements in CRD and that CRD is an effective means of testing opioid-induced visceral antinociception.
Antibody-directed catalysis (ADC) is a two-step method for the delivery of chemotherapeutic agents in which enzyme-antibody conjugate, prelocalized to antigen-bearing tumor cells, catalyzes the site-specific conversion of prodrug to drug. An ADC system consisting of F(ab')-beta-lactamase conjugates and a cephalosporin derivative of the oncolytic agent 4-desacetylvinblastine-3-carboxhydrazide was investigated. The ability of the system to mediate antitumor activity was compared with that of free drug given alone and with covalent drug-antibody conjugates in LS174T and T380 colon carcinoma xenografts in nude mice. Efficacy increased from moderate tumor growth inhibition by using free 4-desacetylvinblastine-3-carboxhydrazide to tumor regression and long-term stabilization with the ADC system. Labile covalent drug-antibody conjugates prepared from the same antibodies were less effective than ADC and required much higher antibody doses. The antigens KS1/4, carcinoembryonic antigen, and tumor-associated glycoprotein-72, TAG-72, present on the model cell lines, were chosen to investigate the effect of differences in subcellular location and expression heterogeneity on the efficacy of ADC delivery. Response was equivalent with the three tumor antigens. Hence, heterogeneous expression and membrane shedding of carcinoembryonic antigen and TAG-72, did not diminish the suitability of these antigens as targets for ADC therapy. In contrast, drug-antibody conjugate efficacy was more sensitive to subcellular location and heterogeneity. Thus, ADC is a highly effective form of immunochemotherapy in preclinical models, with applicability toward a variety of antigen targets.
A conjugate of 4-desacetylvinblastine-3-carboxyhydrazide (DAVLBHY) and the glioma-reactive monoclonal antibody (mAb) 9.2.27 induced long-term suppression of tumor growth in athymic nude mice engrafted with U87MG human glioma cells. In vitro, DAVLBHY had the strongest antiproliferative activity (inhibitory concentration at which incorporation of [3H]thymidine is at 50% of untreated control is 2.0 x 10(-9) M) of seven cytotoxic drugs tested and so was chosen for conjugation to mAb 9.2.27, which reacts specifically with the core protein of chondroitin sulfate proteoglycans found in human glioblastomas. After conjugation of DAVLBHY to the carbohydrate residues of mAb 9.2.27 it retained its full binding capacity. For in vivo studies, DAVLBHY and several conjugate derivatives were evaluated by using two dosages of i.v. injections, each starting 2 days after s.c. tumor inoculation. The control tumors reached a volume of nearly 3000 mm3 within 30 days. Tumor growth was delayed by about 20 days with four i.v. injections of 0.5 mg/kg 9.2.27-DAVLBHY, which was slightly superior to the unconjugated drug. Moreover, 9.2.27-DAVLBHY produced a highly significant suppression of growth so that the average tumor volume was only 3% of that observed in untreated controls after 28 days. Four injections of this conjugate at a larger dose, 2.0 mg/kg, prevented recurrence of the tumors for 130 days in all animals tested, thus demonstrating a significant increase in the therapeutic index, since the unconjugated drug provided limited inhibition of tumor growth for only 40 days. The specificity of the antitumor effect was demonstrated in a comparison with the control conjugate, KS1/4-DAVLBHY, which despite partial tumor suppression had only a transient effect. The specific antitumor effect of 9.2.27-DAVLBHY was unexpected, since the target antigen is expressed at a relatively low density (40,000 sites/cell) on U87MG glioma cells.
Antibodies reactive with human epidermal growth factor receptor (EGFr), such as 225 IgG1 (Masui et al., Cancer Res., 44: 1002-1007, 1984), are effective tumor-suppressive agents in xenograft models. In the present study an additional antibody reactive with EGFr was made and compared to 225 IgG1 for antitumor activity as an unmodified antibody or as a drug immunoconjugate. This IgG1 clone, designated EGFrL11, competed with EGF and immunoprecipitated a Mr 178,000 protein identical to that immunoprecipitated with 225 IgG1. Cross-competition and immunodepletion studies indicated that the two antibodies bound to distinct epitopes on the same molecule. Immunofluorescence studies confirmed that the EGFrL11 epitope was expressed on the surface of viable human squamous cell carcinoma lines including T222. Unmodified EGFrL11 and 225 IgG1 were tested for antitumor activity in T222 xenografts. At a dose of 81 mg/kg given twice weekly for 3 weeks, tumor suppression, but not regression, occurred with EGFrL11. A similar result was obtained with 225 IgG1. To gauge the potential of these antibodies as immunoconjugates, both were tested for antitumor activity in the T222 model after conjugation to the Vinca derivative 4-desacetylvinblastine-3-carboxhydrazide. Both immunoconjugates completely regressed established tumors. These data suggest that Vinca conjugates with EGFr-reactive monoclonal antibodies warrant further investigation as possible clinical candidates.