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Biomedical subjects

R M Thomas

Publications and source records attributed to R M Thomas.

At least 19 recordsLinked to original sources
PubMed2001

Folding and self-assembly of herpes simplex virus type 1 thymidine kinase.

Thymidine kinase from herpes simplex virus type 1 (HSV1 TK) has been postulated to be a homodimer throughout the X-ray crystallography literature. Our study shows that HSV1 TK exists as a monomer-dimer equilibrium mixture in dilute aqueous solutions. In the presence of 150 mM NaCl, the equilibrium is characterized by a dissociation constant of 2.4 microm; this constant was determined by analytical ultracentrifugation and gel filtration experiments. Dimerization seems to be unfavorable for enzymatic activity: dimers show inferior catalytic efficiency compared to the monomers. Moreover, soluble oligomers formed by self-assembly of TK in the absence of physiological salt concentrations are even enzymatically inactive. This study investigates enzymatic and structural relevance of the TK dimer in vitro. Dissociation of the dimers into monomers is not accompanied by large overall changes in secondary or tertiary structure as shown by thermal and urea-induced unfolding studies monitored by circular dichroism and fluorescence spectroscopy. A disulfide-bridge mutant TK (V119C) was designed bearing two cysteine residues at the dimer interface in order to crosslink the two subunits covalently. Under reducing conditions, the properties of V119C and wild-type HSV1 TK (wt HSV1 TK) were identical in terms of expression yield, denaturing SDS PAGE gel electrophoresis, enzyme kinetics, CD spectra and thermal stability. Crosslinked V119C (V119Cox) was found to have an increased thermal stability with a t(m) value of 59.1(+/-0.5) degrees C which is 16 deg. C higher than for the wild type protein. This is thought to be a consequence of the conformational restriction of the dimer interface. Furthermore, enzyme kinetic studies on V119Cox revealed a K(m) for thymidine of 0.2 microm corresponding to wt HSV1 TK, but a significantly higher K(m) for ATP. The present findings raise the question whether the monomer, not the dimer, might be the active species in vivo.

Adenosine Triphosphate↗
PubMed2001

Disulfide bridge conformers of mature BMP are inhibitors for heterotopic ossification.

Heterotopic ossification is a frequent complication in patients who have suffered head and neck traumas or undergone total hip replacement. Heterotopic ossification occurs when osteogenic precursor cells present at the ectopic site receive the necessary signal(s) to differentiate into osteoblasts. At the protein level, the key factors in differentiation of cells to the osteogenic lineage are BMPs. Stable BMP variants derived from the identical amino acid sequence but with different disulfide bridge configurations have been investigated and found to be capable of inhibiting ossification in vitro and in vivo in rodents. These findings provide a concept for the straightforward development of a novel class of BMP antagonists that could lead to new treatments for traumatically and genetically induced heterotopic ossification and also, possibly, for disorders in which other members of the TGF-beta superfamily are involved.

Animals↗
PubMed2001

META-controlled env-initiated transcripts encoding superantigens of murine Mtv29 and Mtv7 and their possible role in B cell lymphomagenesis.

Spontaneous germinal center (GC)-derived B cell lymphomas of SJL mice (RCS) transcribe a 1.8-kb Mtv-29 mRNA under control of the META-env promoter. The encoded vSAg29 stimulates syngeneic Vbeta16(+) CD4(+) T cells, thereby acquiring T cell help necessary for RCS growth. Other strains of B cell lymphoma-prone mice include Mtv29(+) C57L and MA/MyJ, and the Mtv29(-) Mtv7(+)-recombinant inbred strain, SW x J-1. The lymphomas of these mice produce similar mouse mtv-vSAg-encoding mRNA, as characterized by Northern blotting, PCR, and RNase protection. A 1.8-kb mRNA in C57L/J and MA/MyJ lymphomas hybridized with an Mtv29-specific oligonucleotide, whereas SW x J-1 lymphomas produced 1.8-kb transcripts hybridizing with an Mtv7-specific oligonucleotide. Similar META-env-initiated transcripts were absent from LPS-activated B cells from any strain examined but were detected in Peyer's patch RNA from SJL mice. Like typical SJL-derived RCS, all these lymphomas stimulated syngeneic CD4(+) T cells and Vbeta16(+) T hybridoma cells. Immunohistochemical staining of primary tumors showed the presence of peanut agglutinin binding (PNA(+)) highly mitotic lymphoblasts, suggesting their GC derivation. The findings indicate that this novel mRNA for Mtv29 is present in B cell lymphomas from several Mtv29(+) mouse strains. Additionally, this is the first description of the ability of Mtv7 to produce transcripts that are controlled and spliced identically to those of Mtv29 and that are expressed in SW x J-1, I-A(s+), lymphomas that also stimulate Vbeta16(+) T cells. Our results suggest an important role for mouse mtv-vSAgs and Vbeta16 T cell stimulation in the development of GC-derived murine B cell lymphomas.

Animals↗
PubMed2001

Chorismate synthase from the hyperthermophile Thermotoga maritima combines thermostability and increased rigidity with catalytic and spectral properties similar to mesophilic counterparts.

Chorismate synthase, the last enzyme in the shikimate pathway, catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate to chorismate, a biochemically unique reaction in that it requires reduced FMN as a cofactor. Here we report on the cloning, expression, and characterization of the protein for the first time from an extremophilic organism Thermotoga maritima which is also one of the oldest and most slowly evolving eubacteria. The protein is monofunctional in that it does not have an intrinsic ability to reduce the FMN cofactor and thereby reflecting the nature of the ancestral enzyme. Circular dichroism studies indicate that the melting temperature of the T. maritima protein is above 92 degrees C compared with 54 degrees C for the homologous Escherichia coli protein while analytical ultracentrifugation showed that both proteins have the same quaternary structure. Interestingly, UV-visible spectral studies revealed that the dissociation constants for both oxidized FMN and 5-enolpyruvylshikimate 3-phosphate decrease 46- and 10-fold, respectively, upon heat treatment of the T. maritima protein. The heat treatment also results in the trapping of the flavin cofactor in an apolar environment, a feature which is enhanced by the presence of the substrate 5-enolpyruvylshikimate 3-phosphate. Nevertheless, stopped-flow spectrophotometric evidence suggests that the mechanism of the T. maritima protein is similar to that of the E. coli protein. In essence, the study shows that T. maritima chorismate synthase exhibits considerably higher rigidity and thermostability while it has conserved features relevant to its catalytic function.

Bacterial Proteins↗
PubMed2001

Effect of indomethacin on gallbladder inflammation and contractility during acute cholecystitis.

OBJECTIVE: The aim of this study was to determine whether the prostaglandin synthase inhibitor indomethacin reverses the inflammation and abnormal gallbladder contractility that occur after common bile duct ligation (CBDL), a model of acute cholecystitis. METHODS: Gallbladder muscle contractility was studied in vitro in normal, CBDL, and sham-operated guinea pigs. Animals were treated with saline or indomethacin in vivo. Acetylcholine (ACh) was used to directly contract the muscle and electric field stimulation (EFS) to activate intrinsic nerves. Hematoxylin and eosin-stained slides of muscle strips were scored for inflammation. RESULTS: CBDL in saline-treated animals increased the inflammation score and decreased gallbladder muscle contractility to ACh and EFS. Indomethacin decreased the inflammation score and partly reversed the smooth muscle contractile response to ACh 6 and 24 h after CBDL, but not at 48 h. Indomethacin did not reverse the CBDL-induced decrease in nerve-evoked contractions. CONCLUSION: Gallbladder inflammation and contractile dysfunction after CBDL are partly reversed with indomethacin at 6 and 24 h, but not at 48 h. This suggests that, early in the course of CBDL, the inflammation and contractile dysfunction are, in part, prostaglandin-mediated.

Acute Disease↗
PubMed2001

Solving a singular DAE model of unconfined detonation.

We consider a simplified model of an unconfined one-dimensional detonation problem, giving a brief survey of the history of the problem and of its numerical solution. This problem with its mathematical features is typical of those solved commercially by ICI plc, and the specific values used for the chemical constants in the example are typical of those of interest. Unfortunately, not all obvious methods work well, because of the singular nature of the problem at the Chapman-Jouguet shock front. We concentrate on shooting methods for the detonation problem based on backward differentiation formula integrators, and present a new analysis which explains how these methods work. Finally, we outline some possibilities for further work, including discussing a more general detonation problem, previous solutions and potential future solution methods.

Journal Article↗
PubMed2001

Results of steroid-based therapy for the hepatitis C-autoimmune hepatitis overlap syndrome.

OBJECTIVE: Overlap syndromes in which persons manifest clinical, histological, or immunological features of both hepatitis C infection and autoimmune hepatitis are well described. The discordant forms of treatment for hepatitis C and autoimmune hepatitis have made medical management of these patients difficult. We report our experience in using corticosteroids as first line therapy for the hepatitis C-autoimmune hepatitis overlap syndrome. METHODS: Seven patients with this overlap syndrome (diagnosis based on the presence of serum hepatitis C antibody by RIBA and serum hepatitis C RNA by polymerase chain reaction, and serum hypergammaglobulinemia, elevated ANA or ASMA titers, or histological findings consistent with autoimmune hepatitis) were treated with prednisone with or without azathioprine or cyclosporine, and followed for a median duration of 44.5 months. RESULTS: Five patients (71%) showed improvement of median serum ALT level from 162 U/L to 38 U/L (p = 0.04) and median serum gamma-globulin from 2.1 g/dl to 1.4 g/dl (p = 0.04) by 6 months of therapy. The mean modified histological activity index score also decreased from 11.4 +/- 2.5 to 6.6 +/- 2.6 (p = 0.04) by at least 1 yr of therapy. One patient discontinued prednisone while taking azathioprine and experienced a rebound elevation of serum ALT that did not respond to retreatment with prednisone. Antiviral therapy was subsequently administered and resulted in biochemical and virologic response. Hepatitis C virus RNA remained detectable in all other patients. CONCLUSION: Corticosteroids are beneficial as a first line therapy for some patients with the hepatitis C-autoimmune overlap syndrome, resulting in appreciable biochemical and histological response but without viral eradication.

Adult↗
PubMed2000

Analysis of antibody A6 binding to the extracellular interferon gamma receptor alpha-chain by alanine-scanning mutagenesis and random mutagenesis with phage display.

The monoclonal antibody A6 binds a conformational epitope comprising mainly the CC' surface loop on the N-terminal fibronectin type-III domain of the extracellular interferon gamma receptor (IFNgammaR). The crystal structure of an A6 Fab-IFNgammaR complex revealed an interface rich in the aromatic side chains of Trp, Tyr, and His residues. These aromatic side chains appear to interact with both polar and hydrophobic groups at the interface, a property which, in general, may be advantageous for ligand binding. To analyze these interactions in more detail, the affinities of 19 A6 alanine-scanning mutants for the IFNgammaR have been measured, using engineered A6 single chain variable region fragments, and a surface plasmon resonance biosensor. Energetically important side chains (DeltaG(mutant) - DeltaG(wt) > 2.4 kcal/mol), that form distinct hot spots in the binding interface, have been identified on both proteins. These include V(L)W92 in A6, whose benzenoid ring appears well situated for a pi-cation (or pi-amine) interaction with the side chain of receptor residue K47 and simultaneously for T-stacking onto the indole ring of W82 in the receptor. At another site, energetically important residues V(H)W52 and V(H)W53, as well as V(H)D54 and V(H)D56, surround the aliphatic side chain of the hot receptor residue K52. Taken together, the results show that side chains distributed across the interface, including many aromatic ones, make key energetic contributions to binding. In addition, the receptor CC' loop has been subjected to random mutagenesis, and receptor mutants with high affinity for A6 have been selected by phage display. Residues previously identified as important for receptor binding to A6 were conserved in the clones isolated. Some mutants, however, showed a much improved affinity for A6, due to changes at Glu55, a residue that appeared to be energetically unimportant for binding the antibody by alanine-scanning mutagenesis. An E55P receptor mutant bound A6 with a 600-fold increase in affinity (K(D) approximately 20 pM), which is one of the largest improvements in affinity from a single point mutation reported so far at any protein-protein interface.

Alanine↗
PubMed2000

The retro-GCN4 leucine zipper sequence forms a stable three-dimensional structure.

The question of whether a protein whose natural sequence is inverted adopts a stable fold is still under debate. We have determined the 2. 1-A crystal structure of the retro-GCN4 leucine zipper. In contrast to the two-stranded helical coiled-coil GCN4 leucine zipper, the retro-leucine zipper formed a very stable, parallel four-helix bundle, which now lends itself to further structural and functional studies.

Amino Acid Sequence↗
PubMed2000

Effect of acalculous cholecystitis on gallbladder neuromuscular transmission and contractility.

BACKGROUND: Impaired smooth muscle contractility is important in the pathophysiology of acalculous cholecystitis. Common bile duct ligation (CBDL) is a model of acalculous cholecystitis, producing acute inflammatory changes and decrease in gallbladder smooth muscle contractility. The aim of this study was to determine whether there is coexistent dysfunction of neural efferent motor pathways of the gallbladder after CBDL. MATERIALS AND METHODS: Gallbladder muscle contractility was studied in vitro in normal, CBDL, and sham-operated guinea pigs. Electric field stimulation (EFS; 2-16 Hz) was used to activate intrinsic nerves and exogenous acetylcholine (ACh) was used to directly stimulate the muscle. H&E-stained slides of muscle strips were scored for inflammatory changes. RESULTS: After CBDL, there was a progressive increase in the inflammation score and decrease in gallbladder muscle contractility to ACh. There was also a progressive decline in EFS-induced contractility when expressed as absolute force or normalized to the maximal muscle contractile response to ACh. The nitric oxide synthase inhibitor l-NNA (10 microM) increased EFS-induced contractions by 50 +/- 25% (P = 0.05) in CBDL animals but had no effect in sham surgical controls. CONCLUSIONS: CBDL with its acute gallbladder inflammation affects gallbladder contractility by two mechanisms: (1) decreased smooth muscle contractility, and (2) decreased neurally mediated contractions. The neurally mediated alterations result from dysfunction of cholinergic excitatory nerves and upregulation of nitric-oxide-mediated inhibition of smooth muscle contractility.

Acetylcholine↗
PubMed2000

Alpha1-antichymotrypsin globules within hepatocytes in patients with chronic hepatitis C and cirrhosis.

Alpha1-antichymotrypsin (A1AC) is an acute phase serine protease inhibitor, similar to alpha1-antitrypsin (A1AT) in amino acid sequence. A1AT deficiency is known to be associated with emphysema and cirrhosis; deficiency of serum A1AC has been reported to be associated with emphysema, childhood asthma, and cryptogenic cirrhosis. The hepatocyte globules associated with A1AT deficiency have been well described; A1AC deficiency also has been reported to be associated with hepatocyte globules. The aim of this study was to describe the globules of A1AC and to compare them with A1AT globules. Immunohistochemistry for A1AC and A1AT was performed on liver biopsy specimens from 15 hepatitis C virus (HCV)-positive cirrhotic patients, 14 non-HCV cirrhotic patients, and 12 other patients with chronic hepatitis C but no cirrhosis, all of whom had known serum levels of A1AC; most had known serum levels of A1AT. Five of 15 HCV-positive cirrhotic patients, 1 of 14 non-HCV cirrhotic patients, and 1 of 12 noncirrhotic chronic hepatitis C patients had A1AC globules. Two of 15 HCV-positive cirrhotic patients and 2 of 14 non-HCV cirrhotic patients had A1AT globules. Histologically, the globules of A1AC were similar to those of A1AT but were smaller and fewer; the PAS/D stain was not as helpful for A1AC as it was for A1AT; immunohistochemistry was most useful. There was not a good correlation between serum levels of A1AC and its globules in hepatocytes. A1AC globules should be included in the differential diagnosis of hepatocyte inclusions.

Adult↗
PubMed2000

Intraventricular haemorrhage and stage 3 retinopathy of prematurity.

BACKGROUND/AIMS: A recent report has highlighted the decreasing prevalence in recent years of severe intraventricular haemorrhage (IVH) in very low birthweight (VLBW) infants (<1500 g). This study attempted to identify the severity of the grade of IVH in infants with stage 3 retinopathy of prematurity (ROP), and to re-examine the association between threshold ROP and IVH. METHODS: This was a retrospective study carried out over 3 years, between December 1995 and December 1998 of neonates admitted to a single neonatal intensive care unit. 28 infants with stage 3 ROP were identified from the ROP screening database. Cranial ultrasound scans were available on 24 of these infants. The scans were reviewed and the severity of IVH was graded from grade 1 to grade 4. The birth weight, sex, ethnic origin, and gestational age of the babies were recorded. The number of infants progressing to threshold disease and the treatment provided was documented. RESULTS: The 24 infants had a median gestational age of 26 weeks (range 24-28 weeks) and a median birth weight of 762.5 g (range 540-1010 g). 17 infants were treated for threshold disease. 13 infants (54.2%) had IVH, of these eight (61.5%) had grade 1, two (15.4%) had grade 2, one (7.7%) had grade 3, and two (15.4%) had grade 4. 12 of the 13 infants (92.3%) with IVH had treatment with laser or cryotherapy for ROP compared with five of the remaining 11 infants (p = 0.023, Fisher's exact test). These data provide little evidence of any association between IVH and each of ethnic origin (p = 0.856), sex (p = 1), birth weight, or gestational age (p = 0.56 and p = 0.06 respectively) in infants with stage 3 ROP. CONCLUSIONS: These data provide strong evidence (p = 0. 023) of an association between the presence of IVH and treatment of threshold ROP. Although the numbers in this study are small the majority of infants with stage 3 ROP had grade 1 IVH, which heralds a more favourable neurological outcome. An association between the severity of ROP and severity of IVH was not demonstrated. With improvements in neonatal care and a reduction in the prevalence of severe IVH, there appears to be a weakening of the previously reported association between severe IVH and severe ROP. However, the presence of even a minor grade of IVH may be a significant risk factor for threshold ROP once stage 3 disease is encountered.

Birth Weight↗
PubMed1999

Purification and characterization of recombinant pyruvate phosphate dikinase from Giardia.

The gene encoding pyruvate phosphate dikinase (PPDK) from Giardia duodenalis was expressed using a baculovirus system. The recombinant enzyme was purified to homogeneity and its enzymological and solution structure properties characterized. The catalytic constant for the pyruvate-producing reaction was about twice as high (1560 min(-1) at 30 degrees C) as that for the reverse reaction (700 min(-1)) and the k(cat)/Km for PPi was about two orders of magnitude higher than k(cat)/Km for Pi, indicating that the pyruvate-forming reaction is much more efficient than the reverse, phosphoenolpyruvate (PEP)-forming process. The endogenous substrate levels found for PEP (0.5 mM) and pyruvate (< 80 microM) support the assumption that, under physiological conditions, the enzyme primarily performs a catabolic function. The molecular mass of the purified recombinant PPDK was analyzed by analytical ultracentrifugation and size exclusion chromatography using different assay conditions that have been reported to affect the quaternary structure of PPDKs in other organisms. Both methods clearly indicated a dimeric structure for giardial PPDK with a molecular mass of about 197 kDa (monomer mass 97.6 kDa). Several compounds, primarily structural analogs of PPi, were tested for their ability to inhibit PPDK activity. Most of the bisphosphonates examined showed either no, or only a moderate, inhibitory effect on the enzyme. Imidodiphosphate was the only competitive inhibitor with respect to PPi (Kic = 0.55 mM), whereas the bisphosphonates produced a mixed type of inhibition. The most active compound in inhibiting PPDK activity was oxalate, with a Kic value of less than 1 microM with respect to PEP.

Animals↗
PubMed1999

Facilitation of the cellular uptake of a triplex-forming oligonucleotide by novel polyamine analogues: structure-activity relationships.

The inefficient uptake of oligodeoxynucleotides, including that of TFO, through the cell membrane is a limiting factor in developing gene therapy approaches for cancer and other diseases. To develop a new strategy for oligonucleotide delivery into the nucleus, we synthesized a series of novel polyamine analogues and examined their effects on the uptake of a 37-mer [32P]-labeled TFO, targeted to the promoter region of c-myc oncogene. We used MCF-7 breast cancer cells to investigate the efficacy of polyamines on the internalization of the TFO. The uptake of TFO was enhanced by complexing it with several unsubstituted polyamine analogues at 0. 1-5 microM concentrations, with up to 6-fold increase in TFO uptake in the presence of a hexamine, 1,21-diamino-4,9,13, 18-tetraazahenicosane (H2N(CH2)(3)NH(CH2)(4)NH(CH2)(3)NH(CH2)(4)NH(CH2)(3)NH(2) or 3-4-3-4-3). TFO uptake increased with the cationicity of the polyamines; however, bis(ethyl) substitution and structural features of the methylene bridging region had significant effects on TFO uptake. The majority of labeled TFO was recovered from the nuclear fraction containing genomic DNA. Electrophoretic mobility shift assay revealed enhanced binding of TFO to a target duplex containing promoter region sequence of c-myc oncogene. Treatment of MCF-7 cells with the TFO complexed with 0.5 microM 3-4-3-4-3 suppressed c-myc mRNA level by 65%, as determined by Northern blot analysis. These data indicate a novel approach to deliver oligodeoxynucleotides to the cell nucleus, and suppress the expression of target genes, and provide new insights into the mechanism of oligonucleotide transport in living cells.

Breast Neoplasms↗
PubMed1999

Effect of experimental acalculous cholecystitis on gallbladder smooth muscle contractility.

Gallbladder motility is impaired in chronic cholelithiasis but has not been studied in acute acalculous cholecystitis. The aim of this study was to determine the effects of acute acalculous inflammation on gallbladder contractility using the common bile duct ligation (CBDL) model in guinea pigs. Three groups of guinea pigs were studied: CBDL, normal, and sham surgical controls. Gallbladder dimensions were measured, and muscle strips were used for histology and in vitro contractility studies. CBDL resulted in progressive gallbladder distension, increased serum bilirubin, and gallbladder inflammation. There was a progressive decline in muscle contractility in the CBDL group as evidenced by a decrease in the contractile response to potassium and bethanechol with the duration of CBDL. In conclusion, CBDL in the guinea pig produces acute gallbladder inflammation and decreased gallbladder muscle contractility. Direct inhibition of muscle function is indicated by impaired contractile responses to potassium depolarization and bethanechol stimulation. Although the mechanism of the decrease in contractility with CBDL is unknown, we speculate that impaired muscle contractility is secondary to inflammation and may play a role in the clinicopathology of acute acalculous cholecystitis.

Animals↗
PubMed1998

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↗