Sub-aponeurotic haemorrhage: a rare but life-threatening neonatal complication associated with ventouse delivery.
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Adult↗
Biomedical subjects
R M Thomas
Publications and source records attributed to R M Thomas.
Salt effects on hydrophobic interaction and charge screening in the folding of a negatively charged peptide to a coiled coil (leucine zipper).
The stability of a coiled coil or leucine zipper is controlled by hydrophobic interactions and electrostatic forces between the constituent helices. We have designed a 30-residue peptide with the repeating seven-residue pattern of a coiled coil, (abcdefg)n, and with Glu in positions e and g of each heptad. The glutamate side chains prevented folding at pH values above 6 because of electrostatic repulsion across the helix dimer interface as well as within the individual helices. Protonation of the carboxylates changed the conformation from a random coil monomer to a coiled coil dimer. Folding at alkaline pH where the peptide had a net charge of -7e was promoted by the addition of salts. The nature of the charge screening cation was less important than that of the anion. The high salt concentrations (>1 M) necessary to induce folding indicated that the salt-induced folding resulted from alterations in the protein-water interaction. Folding was promoted by the kosmotropic anions sulfate and fluoride and to a lesser extent by the weak kosmotrope formate, whereas chloride and the strong chaotrope perchlorate were ineffective. Kosmotropes are excluded from the protein surface, which is preferentially hydrated, and this promotes folding by strengthening hydrophobic interactions at the coiled coil interface. Although charge neutralization also contributed to folding, it was effective only when the screening cation was partnered by a good kosmotropic anion. Folding conformed to a two-state transition from random coil monomer to coiled coil dimer and was enthalpy driven and characterized by a change in the heat capacity of unfolding of 3.9 +/- 1.2 kJ mol-1 K-1. The rate of folding was analyzed by fluorescence stopped-flow measurements. Folding occurred in a biphasic reaction in which the rapid formation of an initial dimer (kf = 2 x 10(7) M-1 s-1) was followed by an equally rapid concentration-independent rearrangement to the folded dimer (k > 100 s-1).
Amino Acid Sequence↗
DNA-mediated folding and assembly of MyoD-E47 heterodimers.
Basic region helix-loop-helix (bHLH) transcription factors regulate key steps in early development by binding to regulatory DNA sites as heterodimers consisting of a tissue-specific factor and a widely expressed factor. We have examined the folding, dimerization, and DNA binding properties of the muscle-specific bHLH protein MyoD and its partner E47, to understand why these proteins preferentially associate in heterodimeric complexes with DNA. In the absence of DNA, the E47 bHLH domain forms a very stable homodimer, whereas MyoD is unfolded and monomeric. Fluorescence quenching experiments show that MyoD does not dimerize with E47 under dilute conditions in the absence of DNA. Residues in and around the loop of the E47 bHLH domain contribute to its markedly greater stability. An altered MyoD bHLH substituted with the loop segment from E47 folds in the absence of DNA, and it readily dimerizes with E47. In the presence of a specific DNA binding site, MyoD and E47 both form homodimeric complexes with DNA that have similar dissociation constants, despite the very different stabilities of these protein dimers off DNA. A 1:1 mixture of these bHLH domains forms almost exclusively heterodimeric complexes on DNA. Assembly of these bHLH-DNA complexes is apparently governed by the strength of each subunit's interaction with the DNA and not by the strength of protein-protein interactions at the dimer interface. These findings suggest that preferential association of MyoD with E47 in DNA complexes results from more favorable DNA contacts made by one or both subunits of the heterodimer in comparison with either homodimeric complex.
Amino Acid Sequence↗
Synthesis, physicochemical characterization, and crystallization of a putative retro-coiled coil.
An artificial HIV enhancer-binding polypeptide has recently been dimerized by covalently linking it to the leucine zipper motif of the yeast transcriptional activator GCN4 (Liu N et al., 1997, Eur Biophys J 25:399-403). Although it seemed that the dimerization of this peptide could be best achieved by the use of the retro sequence of the leucine zipper, this approach was not implemented in the original construct. As the first step toward the synthesis of a basic region-retro leucine zipper HIV enhancer-binding fusion protein, we have now prepared the retro version of the leucine zipper (r-LZ35) and performed initial physicochemical characterization. Circular dichroism and sedimentation equilibrium studies showed that, at concentrations < 100 microM, the retro peptide was an unstructured monomer. At higher concentrations, however, the monomer was in equilibrium with a tetramer and, at 1 mM, the retro peptide was almost fully helical. N-terminal extension of the retro peptide by the tripeptide Cys-Gly-Gly resulted in a 38-residue polypeptide that could be covalently dimerized by forming a disulfide bond between two chains to give the peptide (r-LZ38)2. Even in the low micromolar concentration range peptide (r-LZ38)2 formed a stable, noncovalent, helical dimer as revealed by circular dichroism and sedimentation equilibrium in the presence and absence of guanidinium chloride. (r-LZ38)2 has been crystallized and X-ray structural analysis is under way. The disulfide-crosslinked retro-leucine zipper may lend itself to interesting protein structural studies, including protein design. The present work also highlights the structural and functional potential of retro proteins in general.
Amino Acid Sequence↗
Molecular and functional analysis of the lipopolysaccharide biosynthesis locus wlb from Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica.
The Bordetella pertussis wlb locus (wlbpe, formerly bpl) is required for the biosynthesis of a trisaccharide that, when attached to the B. pertussis lipopolysaccharide (LPS) core (band B), generates band A LPS. The equivalent loci in Bordetella bronchiseptica (wlbbr) and Bordetella parapertussis (wlbpa) were identified and cloned. The wlbbr and wlbpa loci differ from wlbpe in that they lack the insertion sequence that defines the right-hand terminus of wlbpe. Deletion of 12 kb of DNA containing the whole wlb locus (delta wlb) by allelic exchange in each of the three bordetellae had no effect on band B biosynthesis, whereas band A biosynthesis was prevented in B. pertussis and B. bronchiseptica. In B. bronchiseptica and B. parapertussis, delta wlb mutants also lacked O-antigen. Reintroduction of the wlbpe or wlbbr loci on a shuttle vector into the three delta wlb mutants restored the wild-type LPS phenotype in the B. pertussis and B. bronchiseptica mutants. In the case of B. parapertussis, which normally does not synthesize an apparent band A structure, introduction of the wlbpe or wlbbr loci now enabled the generation of band A. This suggests that the attachment point for band A trisaccharide on the LPS core is present in B. parapertussis, and further suggests that the wild-type wlbpa locus is not fully functional. Introduction of the wlbpa locus into the delta wlbpe, delta wlbbr and delta wlbpa mutants had interesting consequences. The B. bronchiseptica and B. parapertussis recipients were now able to biosynthesize O-antigen, but no band A was generated. In the B. pertussis recipient, a truncated band A was expressed consistent with a mutation in the wlbH gene, but on Western blotting the expression of a small amount of full-length band A was also seen. Evidence that the wlbHpa protein is not fully functional was provided by the failure of the wlbpa locus to fully complement a B. pertussis wlbH (delta wlbHpe) mutant. This was supported by DNA sequence data showing that a single amino acid, conserved between homologous proteins from a range of bacteria, is altered in the B. parapertussis WlbH protein.
Amino Acid Sequence↗
Rapid progression of Barrett's esophagus to metastatic esophageal carcinoma in a patient with chronic lymphocytic leukemia.
Barrett's esophagus is a common premalignant condition that predisposes to the development of adenocarcinoma of the esophagus through a process of transformation from metaplasia to dysplasia and then carcinoma. Periodic endoscopic surveillance with multiple biopsies is adopted by most physicians to detect dysplasia or early carcinoma. We report a case of an 80-year-old white man with chronic lymphocytic leukemia (CLL) who had periodic endoscopic surveillance without any evidence of dysplasia or cancer, and who died of metastatic carcinoma of the esophagus only 18 months after his last upper endoscopic examination. We suspect that the relative immunosuppressed state resulting from his CLL was the major contributor to the rapid progression of the Barrett's esophagus to cancer. Patients with CLL have higher risk of second cancers, and several cases of aggressive carcinomas have been reported in association with CLL. This is the first case report of metastatic esophageal cancer arising in Barrett's esophagus in a patient with CLL. This case suggests that we might need a more aggressive surveillance strategy for Barrett's esophagus in patients with CLL or other immunocompromised conditions.
Adenocarcinoma↗
Is risk of neuroleptic malignant syndrome increased in the postpartum period?
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Adult↗
Orally active trifluoromethyl ketone inhibitors of human leukocyte elastase.
This paper describes the development a series of peptidyl trifluoromethyl ketone inhibitors of human leukocyte elastase which are found to have excellent pharmacological profiles. Methods have been developed that allow for the synthesis of these inhibitors in stereochemically pure form. Two of these compounds, 1k and 1l, have high levels of oral bioavailability in several species. Compound 1l has entered development as ZD8321 and is presently undergoing clinical evaluation. These compounds demonstrate that peptidyl trifluoromethyl ketone inhibitors can achieve high levels of oral activity and bioavailability, and therefore they may prove useful as therapeutic agents in the treatment of diseases in which elastase is implicated.
Administration, Oral↗
Molecular cloning of a peroxisomal Ca2+-dependent member of the mitochondrial carrier superfamily.
A cDNA from a novel Ca2+-dependent member of the mitochondrial solute carrier superfamily was isolated from a rabbit small intestinal cDNA library. The full-length cDNA clone was 3,298 nt long and coded for a protein of 475 amino acids, with four elongation factor-hand motifs located in the N-terminal half of the molecule. The 25-kDa N-terminal polypeptide was expressed in Escherichia coli, and it was demonstrated that it bound Ca2+, undergoing a reversible and specific conformational change as a result. The conformation of the polypeptide was sensitive to Ca2+ which was bound with high affinity (Kd approximately 0.37 microM), the apparent Hill coefficient for Ca2+-induced changes being about 2.0. The deduced amino acid sequence of the C-terminal half of the molecule revealed 78% homology to Grave disease carrier protein and 67% homology to human ADP/ATP translocase; this sequence homology identified the protein as a new member of the mitochondrial transporter superfamily. Northern blot analysis revealed the presence of a single transcript of about 3,500 bases, and low expression of the transporter could be detected in the kidney but none in the liver. The main site of expression was the colon with smaller amounts found in the small intestine proximal to the ileum. Immunoelectron microscopy localized the transporter in the peroxisome, although a minor fraction was found in the mitochondria. The Ca2+ binding N-terminal half of the transporter faces the cytosol.
Animals↗
Carcinoids of the jejunum and ileum: an immunohistochemical and clinicopathologic study of 167 cases.
BACKGROUND: Carcinoid tumors of the gastrointestinal tract differ in their clinical and histopathologic features, depending on the site of origin. There are few clinicopathologic studies that specifically describe jejunoileal carcinoid tumors. METHODS: One hundred sixty-seven ileal and jejunal carcinoids were retrospectively studied with emphasis on clinical, pathologic, immunohistochemical, and prognostic features. RESULTS: The mean age of patients at the time of presentation was 62 +/- 12 years (range, 13-93 years). Eight patients had carcinoid syndrome (5%) and 1 had Zollinger-Ellison syndrome. Twenty-six percent of tumors were multiple, and 77% were transmurally invasive; 31% had regional lymph node metastases only, and 32% had liver or mesenteric metastases. Ninety-three percent of tumors had an insular growth pattern. Serotonin was expressed in 86% of tumors (86 of 102), chromogranin in 92%, and neuron specific enolase in 95%. Twenty percent of tumors (10 of 51) expressed prostatic acid phosphatase; 96% were argyrophil, and 98% argentaffin. Of 80 cases with follow-up data (mean follow-up, 52 +/- 5 months), 21% were dead of disease, 16% were dead of other causes, 19% were alive with disease, and 44% had no evidence of disease at last follow-up. The 5-year Kaplan-Meier survival estimate for all cases was 58%. By univariate analysis, survival was negatively correlated with distant metastases at the time of surgery (P = 0.002), mitotic rate (P = 0.01), tumor multiplicity (P = 0.01), the presence of carcinoid syndrome (P = 0.02), depth of invasion (P = 0.03), and female gender (P = 0.05); by multivariate analysis, survival was negatively associated with distant metastasis (P = 0.002), carcinoid syndrome (P = 0.01), and female gender (P = 0.03). CONCLUSIONS: Jejunoileal carcinoid tumors have a relatively high rate of transmural invasion and aggressive clinical behavior. They are usually insular and largely argentaffin, with a high rate of chromogranin and serotonin positivity. These features differentiate jejunoileal carcinoids from other gastrointestinal carcinoids.
Adolescent↗
An artificial HIV enhancer-binding peptide is dimerized by the addition of a leucine zipper.
A 42 residue artificial peptide that binds to the HIV-1 enhancers has been described previously. The specificity of interaction of the peptide with its target DNA sequence has been demonstrated by a variety of techniques. Naturally occurring regulatory proteins frequently bind to DNA as dimers, thereby increasing the strength and specificity of the interaction, the dimer interface often being provided by a leucine zipper type coiled coil. As a suitable binding site for this kind of system is located to the 5' end of the HIV enhancer region, it was decided to design and synthesize a fusion peptide that not only contained the DNA binding sequence of the original 42 residue peptide but also incorporated a leucine zipper based on that of the GCN4 transcriptional activator, that should, therefore, be capable of dimerizing. The resultant peptide, LZ66, has now been shown to be fully active in band shift and in vitro transcription assays and to exhibit about double the inhibitory activity of the parent 42 residue peptide. Preliminary CD measurements revealed that the peptide has a high alpha-helical content and that it adopts a stable conformation down to the low micromolar peptide concentration range. Sedimentation equilibrium studies confirmed that the principles involved in the design of the peptide are valid and that the peptide is indeed dimeric in solution.
Amino Acid Sequence↗
A trimeric, alpha-helical, coiled coil peptide: association stoichiometry and interaction strength by analytical ultracentrifugation.
Alpha-helical coiled coils are proving to be almost ideal systems for the modelling of peptide and protein self-association processes. Stable oligomeric systems, in which the stoichiometry is well defined, can be produced by the careful selection of the appropriate amino acid sequence, although the principles behind this are still not fully understood. Here we report on a 35 residue peptide, FZ, synthesized by the solid phase method, which was originally designed to form a dimer, but which, in fact, associates to the trimeric state. A detailed characterization of the associative properties of the peptide has been performed by circular dichroism spectroscopy and, in particular, by sedimentation equilibrium in the analytical ultracentrifuge. The presence of the trimeric state, which is stable even at low peptide concentrations, has been confirmed by various independent methods of analysis for molar mass. The effects of both temperature and of guanidinium chloride on the peptide have been investigated and both found to be peptide-concentration dependent. The unfolding induced by the denaturant cannot be adequately described by a simple, two state monomer-trimer equilibrium.
Amino Acid Sequence↗
Effects of oxygen radicals and radical scavenging on opossum lower esophageal sphincter.
Nitric oxide mediates esophageal peristalsis and lower esophageal sphincter (LES) relaxation. Superoxide produced with inflammation inactivates nitric oxide. Superoxide is cleared in biological systems by superoxide dismutase. We tested the hypothesis that superoxide and the superoxide scavenging system modulate LES function. Transverse strips of muscle from the opossum LES relaxed when stimulated by an electrical field. Diethyldithiocarbamite was used to inhibit copper/zinc superoxide dismutase. Xanthine and xanthine oxidase were used to generate superoxide. Xanthine with xanthine oxidase or diethyldithiocarbamite alone had no effect on the LES. However, xanthine/xanthine oxidase and diethyldithiocarbamite reduced LES relaxation 34.1% and increased its resting tone 71.2%. Superoxide dismutase did not affect LES function, but protected the tissue from the effects of diethyldithiocarbamite and xanthine/xanthine oxidase. These studies are consistent with the hypothesis that superoxide acts by inactivating nitric oxide and suggest that these antioxidant enzyme systems may play a role in the maintenance of LES function.
Animals↗
The role of antioxidant enzymes in the control of opossum sphincter of Oddi motility.
Superoxide rapidly oxidizes nitric oxide (NO) to form peroxynitrite, thus terminating the biological activity of NO. The aims of our study were to determine if superoxide alters the motor function of the sphincter of Oddi and to localize the antioxidant enzymes in the sphincter of Oddi. Immunostaining was performed and enzyme activities were measured in the sphincter of Oddi. In physiological experiments, force-displacement transducers recorded tension in the spontaneously contracting sphincter of Oddi and after electrical field stimulation (EFS) of precontracted sphincter of Oddi. Superoxide was generated by the addition of xanthine with xanthine oxidase, superoxide radicals were scavenged by the addition of superoxide dismutase (SOD), and catalase or SOD was inhibited by diethyldithiocarbamic acid. Immunostaining demonstrated SOD and catalase immunoreactivity in ganglia situated at the serosal surface of the circular muscle. Total SOD activity was 202 +/- 12 U/mg. Generation of superoxide or inhibition of SOD increased the contractile frequency and decreased relaxation after EFS. We conclude that superoxide alters sphincter of Oddi motor function, and the presence of superoxide scavenging enzymes in enteric plexuses suggests that they may regulate sphincter of Oddi neuromuscular function by clearing endogenous superoxide.
Animals↗
Acute experimental colitis decreases colonic circular smooth muscle contractility in rats.
Distal colitis decreases the contractility of the underlying circular smooth muscle. We examined how time after injury and lesion severity contribute to the decreased contractility and how colitis alters the calcium-handling properties of the affected muscle. Distal colitis was induced in rats by intrarectal administration of 4% acetic acid. Contractile responses to acetylcholine, increased extracellular potassium, and the G protein activator NaF were determined for circular muscle strips from sham control and colitic rats at days 1, 2, 3, 7, and 14 postenemas. Acetylcholine stimulation of tissues from day 3 colitic rats was performed in a zero calcium buffer, in the presence of nifedipine, and after depletion of intracellular stores of calcium. The colitis was graded macroscopically as mild, moderate, or severe. Regardless of agonist, maximal decrease in force developed 2 to 3 days posttreatment, followed by a gradual return to control by day 14. The inhibitory effect of colitis on contractility increased with increasing severity of inflammation. Limiting extracellular calcium influx had a greater inhibitory effect on tissues from colitic rats; intracellular calcium depletion had a greater inhibitory effect on tissues from control animals. The data suggest that both lesion severity and time after injury affect the contractile response of circular smooth muscle from the inflamed distal colon. Impaired utilization of intracellular calcium may contribute to the decreased contractility.
Acetic Acid↗
Subfulminant liver failure and severe hepatotoxicity caused by loratadine use.
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Adult↗
The role of cavitation in the induction of cellular DNA damage by ultrasound and lithotripter shock waves in vitro.
The induction of DNA strand breaks in Chinese hamster ovary cells was measured with the comet assay after continuous wave ultrasound or lithotripter shock wave exposure. Cell lysis and hydrogen peroxide production were measured to gauge the level of inertial cavitation activity. Significant DNA damage was found after 2.17-MHz ultrasound exposure at 37 degrees C to 0.82 MPa for 2 min or 4 min, and to 0.58 MPs for 4 min. A significant portion of the damage induced at the 0.82-MPa level was repaired by the cells when warmed. Neither exposure to 500 or 1000 shock waves at 37 degrees C in a thin-walled tube, nor exposure to 1000, 1500 or 2000 shock waves at 25 degrees C in a polyethylene pipette bulb produced a significant effect, when the flash of light from the spark-gap discharge was blocked. This finding was consistent with the generally lower lysis and hydrogen peroxide production by the shock wave exposure.
Animals↗
Contrast-agent gas bodies enhance hemolysis induced by lithotripter shock waves and high-intensity focused ultrasound in whole blood.
Fresh canine whole blood was exposed in 1.3-mL disposable pipette bulbs with or without added Albunex contrast agent to lithotripter shock waves (rate: 2 Hz; mean pressure amplitude: 14.8 MPa) or 1.28-MHz high-intensity focused ultrasound up to a mean pressure amplitude of 17.8 MPa. Significant shock-wave-induced hemolysis up to about 10% was detected after 500 or more shock waves without added agent, after 200 with 1% added agent and after 100 with 10% added contrast agent. Hemolysis was increased somewhat with added contrast agent, but this enhancement was not statistically significant. For ultrasound exposure, significant hemolysis approaching 100% was detected for 100-ms continuous exposure at 17.8 MPa without added gas bodies. Addition of 1% contrast agent enhanced the hemolysis by reducing the apparent threshold to 10 MPa. Burst mode exposure with a 100-ms total on-time (20 microseconds, 100 microseconds and 1-ms bursts) also yielded hemolysis enhancement with added contrast agent, with results similar to the 100-ms continuous exposure. The added gas bodies appear to lower cavitation thresholds by serving as cavitation nuclei in the blood.
Albumins↗