Search PubMed⌕ Search

Biomedical subjects

R Mitchell

Publications and source records attributed to R Mitchell.

At least 55 records · Page 3Linked to original sources
PubMed1996

Albendazole resistance in Giardia is correlated with cytoskeletal changes but not with a mutation at amino acid 200 in beta-tubulin.

Albendazole resistance was induced in three different Giardia cultures following growth in successively increasing amounts of drug. One of the lines was previously resistant to high levels of metronidazole and was able to grow in 2 microM albendazole. The other two survived exposure to 0.8 microM, while normally lethal levels of albendazole against Giardia in vitro were around 0.1-0.2 microM. Albendazole-resistant Giardia were cross-resistant to parbendazole. Major chromosome rearrangements were evident in the line resistant to 2 microM albendazole and IFA with antitubulin antibody indicated differences in the cytoskeleton, particularly the median body, between sensitive and resistant lines. This implicates the cytoskeleton in the mechanism of resistance. Substitution of Tyr for Phe is a consistent beta-tubulin amino acid change in the benzimidazole-resistant helminths and fungi so far analyzed. PCR primers were designed from the published Giardia beta-tubulin gene sequence and spanned the region encoding Phe at position 200. Sequence data from albendazole-resistant Giardia demonstrated that the beta-tubulin gene did not carry a mutation in the codon for amino acid 200. These data suggest that Phe at position 200 in beta-tubulin is not necessary for benzimidazole resistance.

Albendazole↗
PubMed1996

Recombinant human luteinizing hormone: a partial physicochemical, biological and immunological characterization.

The aim of this study was to partially characterize the glycoform composition of a recombinant human luteinizing hormone preparation (rhLH; Serono), an early version of the material (LHadi) which is currently being assessed for clinical application. Specifically, the charge (pl) and internal carbohydrate complexity of this rhLH was examined and compared with that of an alternative commercially available form of recombinant LH (Crystal Chem) and a pituitary International Reference Preparation (IRP). All preparations were separated by charge by chromatofocusing them on a pH gradient (7-4) using a 4 ml mono-P column is conjunction with a fast performance liquid chromatography system and by complexity of the oligosaccharide structures using concanavalin A (con-A) lectin affinity chromatography. LH in both the unfractionated and fractionated material was assessed by immunoradiometric assay (IRMA, I-LH) and by the in-vitro Leydig cell bioassay (B-LH). Both assays were calibrated against IRP 80/552. The in-vitro biopotency of the preparations was 18187 (Serono rhLH), 12063 (Crystal Chem rhLH) and 6658 (80/552) IU/mg; biological:immunological ratios were 1.14 (80/552), 1.90 (Crystal Chem rhLH) and 1.99 (Serono rhLH). However, similar qualitative data were obtained by both bioassay and immunoradiometric assay following fractionation, with the median pl of the bioactive LH in the preparations being 5.5 (24% > pH 6), 5.52 (18% > pH 6) and 4.97 (0% > pH 6) for the Serono, Crystal Chem and pituitary preparations respectively. Further all three contain < 1% of the complex carbohydrate structures and between 36-44% and 56-63% of the intermediate and simple forms of bioactive LH. In conclusion, the Serono recombinant LH preparation has a higher in-vitro bioactivity and is more basic than the other two preparations although the complexity of its carbohydrate moities appears to be similar.

Animals↗
PubMed1996

Internal carbohydrate complexity of the oligosaccharide chains of recombinant human follicle stimulating hormone (Puregon, Org 32489): a comparison with Metrodin and Metrodin-HP.

Glycoforms of recombinant human follicle stimulating hormone (rhFSH) (Org 32489, Puregon) were characterized using concanavalin A lectin affinity chromatography to reveal information about the internal carbohydrate complexity (extent of carbohydrate side-chain branching) of the preparations. The rhFSH glycoforms were measured by radioimmunoassay and a two-site immunoradiometric assay and compared with those in two urinary preparations (Metrodin and Metrodin-HP) used in assisted reproduction programmes and a urinary FSH international standard 70/45 (uFSH IS 70/45). Similar data were obtained with both assays; rhFSH had 6% complex internal carbohydrate structures compared with 22-27% for Metrodin, Metrodin-HP and uFSH. The proportion of simple carbohydrate structures was also different, with rhFSH having 18.5 compared with 4.5-9.3% for Metrodin, Metrodin-HP and uFSH. A linear relationship was observed between the percentage glycoforms with an isoelectric point (pl) < 4 and the log percentage simple forms (logarithmic regression; r = 0.93) indicating a direct relationship between carbohydrate complexity and charge heterogeneity. In summary, rhFSH contains fewer complex forms and an increased proportion of simple carbohydrate structures in comparison with Metrodin, Metrodin-HP and IS 70/45.

Carbohydrates↗
PubMed1996

Oestradiol and immunoreactive inhibin-like secretory patterns following controlled ovarian hyperstimulation with urinary (Metrodin) or recombinant follicle stimulating hormone (Puregon).

Inhibin (and its alpha-subunit) may be of particular value as a marker for follicular development in in-vitro fertilization (IVF) in comparison with the classic follicle stimulating hormone (FSH)-dependent marker oestradiol in patients following pituitary desensitization and treatment with recombinant FSH (rFSH). This preparation lacks luteinizing hormone (LH), which is essential for thecal cell androgen secretion and thus oestradiol production. Our study has assessed oestradiol and immunoreactive inhibin-like secretion following ovarian stimulation with rFSH or a purified urinary FSH preparation (Metrodin) (uFSH). A randomized, assessor-blind study was initiated using patients receiving a single treatment cycle of IVF (using fresh embryos) following pituitary desensitization with intranasal buserelin (500 microg daily) and the i.m. injection of either rFSH (n = 38) or uFSH (n = 17). Ovarian ultrasound examinations were performed and bloods (10 ml) collected prior to FSH treatment and every 1-2 days until ovulation induction with human chorionic gonadotrophin. LH and FSH concentrations were measured by an immunoradiometric assay, and inhibin-like immunoreactivity by a radioimmunoassay and an enzyme-linked immunosorbent assay, both with alpha-subunit specificity. Oestradiol concentration was measured with a coated tube radioimmunoassay. Following desensitization, basal LH, FSH and oestradiol concentrations were measured, as was that of immunoreactive inhibin. Following treatment with either rFSH or uFSH, LH concentrations remained low while FSH concentrations rose to a plateau of 5.6-6.7 IU/l in both groups. In contrast, the concentration of oestradiol was higher (P < 0.05) with rFSH than with uFSH in the last four days of treatment, a pattern that was repeated for inhibin-like immunoreactivity. The change in oestradiol and inhibin concentrations during treatment was approximately 2-fold higher with rFSH. The total number of follicles obtained with rFSH was similar to that with uFSH. However, the number of follicles with a diameter of >/= 15 mm was higher the rFSH group, and there was a concomitant increase in the number of oocytes recovered. Oestradiol concentration and inhibin-like immunoreactivity (determined by either method) were associated with total follicle number and number of follicles >/= 15 mm in diameter, as well as with each other (P < 0.001). When ovarian hormone output was normalized per follicle produced, oestradiol output was higher for rFSH than for uFSH P = 0.04). Inhibin output was clearly higher using rFSH than uFSH. There were seven pregnancies (one miscarriage) with rFSH and two with uFSH. Despite similar concentrations od FSH in patients, rFSH (Puregon) appears to be more potent in vitro in terms of follicular number, ovarian hormone secretion (both concentration and output/follicle) and oocyte recovery. In both groups, LH concentrations of approximately 1.3 IU/l were sufficient to support oestradiol secretion similar to that normally found in IVF programmes using human menopausal gonadotrophin preparations containing large amounts of LH. Despite known problems of specificity with the assays od inhibin, its measurement was of similar value to oestradiol as a marker of follicular development.

Adult↗
PubMed1996

Methods, indications, and results of percutaneous choledochoscopy. A series of 161 procedures.

OBJECTIVE: Nonsurgical methods for evaluation and treatment of the biliary tree are usually done under fluoroscopic guidance. Direct visualization of the bile ducts, that is, choledochoscopy, could provide a more precise method with which to perform these maneuvers. The methods, indications, and results of percutaneous choledochoscopy are discussed from a series of 161 procedures performed in 123 consecutive patients. METHODS AND RESULTS: Ninety-six patients had transhepatic drains and 27 had T-tubes implanted. Biopsy of bile duct stenosis was conducted in 52 cases. The sensitivity for the diagnosis of malignancy was 78%, and the authors recommended this method in case of nonsurgical treatment of stenosis. Gallstone extraction was performed in 75 cases (35 intrahepatic). Lithotripsy was necessary for 64 patients and complete gallstone clearance was obtained for 69 patients (92%). The rate of stone recurrence was 18.1% (median follow-up, 32 months), prompting the authors to reconsider surgery for the treatment of underlying disease. Laser photocoagulation was used successfully for two of three patients to ablate an intraductal adenoma. In eight cases of cholangiocarcinoma, the laser effect was too short to satisfactorily relieve obstruction of the intrahepatic bile ducts. Morbidity dramatically decreased from 53.7% to 5% when progressive dilation of the tract, aseptic conditions, and general anesthesia were applied systematically. CONCLUSION: Percutaneous choledochoscopy can be used routinely, provided that strict techniques are adhered to. This procedure should be reserved for only a small subset of biliary diseases.

Adolescent↗
PubMed1996

Differences in serum luteinizing hormone measurements by immunoradiometric assay induced by kinetic manipulation of assay conditions are dependent on the endocrine milieu of serum.

Divergent estimates for luteinizing hormone (LH) in individual serum samples may be given by different immunoassays. In order to investigate this phenomenom further, we have studied the effect of differences in assay kinetics within the same immunoradiometric assay (IRMA) configuration on LH measurement in sera from different endocrine states. Three pairs of monoclonal/polyclonal two-site IRMA systems for LH were developed from three LH monoclonal antibodies and a common polyclonal anti-human chorionic gonadotrophin. For IRMA systems a short and long assay, which were different only with respect to the incubation time (1/2 h and overnight respectively), of the labelled monoclonal first antibody were performed. The IRMAs were all standardized against the LH international reference preparation 68/40. LH concentrations were measured by all the IRMAs in sera obtained from normal men (n = 11) and from women with polycystic ovarian syndrome (PCO; n = 13). In normal men, there were no differences in LH estimates between the short and the long assays of the three IRMA systems, and the ratios of long to short assays were similar for all the systems. However, in PCO there were significant differences between short and long assays and the ratios of long to short assays were different for the IRMA systems. These results indicate that kinetic differences between IRMAs of the same antibody configuration can be associated with differences in measured LH concentrations, depending on the endocrine status of the sera studied. As LH glycoform patterns are known to differ between normal men and PCO, the observed changes in LH estimates may be due to the different glycoform composition.

Adult↗
PubMed1996

Bone SPECT to assess mandibular invasion by intraoral squamous-cell carcinomas.

UNLABELLED: It is important for head and neck surgeons planning their operative approach to assess tumor infiltration to the mandible by intraoral squamous-cell carcinomas. Two- to 3-hr planar bone scintigraphy is sensitive but not specific in detecting mandibular invasion by tumor. We evaluated 3-hr SPECT bone scintigraphy, which provides better anatomical detail, to determine if it more accurately assesses tumor invasion. METHODS: Thirty consecutive patients with proven intraoral squamous-cell carcinomas were studied. Semiquantitative assessment of the mandible was performed with a three-level graded scoring system, based on comparisons of tracer uptake in the mandible to that in the upper cervical vertebrae and in an unaffected part of mandible. Lesion-to-cervical spine (L/S) count ratios and lesion-to-nonlesion (L/N) count ratios in the mandible on the 3-hr SPECT images were calculated. RESULTS: All patients with proven tumor invasion showed higher tracer uptake than those with normal mandible or other dental diseases. The L/S and L/N count ratios on the SPECT images were significantly higher in patients with tumor invasion than in those without (L/S, p < 0.001; L/N, p < 0.01). L/S ratios provided better differentiation than L/N ratios. CONCLUSIONS: Bone SPECT provides a reliable means of assessing tumor invasion to the mandible by intraoral squamous-cell carcinomas.

Adult↗
PubMed1996

Prevalence of HBsAg among the non cauterized groups of blood donors.

One thousand two hundred and seventeen (1217) blood specimens from non-cauterized blood donors were tested for HBsAg by counterimmunoelectrophoresis technique: 48 specimens were positive giving an incidence of 3.95%; specimens from 99 blood donors with cautery marks were tested side by side using the same technique, 3 specimens were positive giving an incidence of 3.05%. These results show that Sudan can be considered among the countries with very high prevalence of HBsAg. This emphasizes the importance of testing every unit of blood or blood product before transfusion. This high prevalence is mainly attributable to the use of common improperly sterilized water-boiled syringes, needles and surgical instruments. The results also show that the incidence of HBsAg among cauterized donors is not higher than among non-cauterized; thus routine rejection of cauterized citizens from giving blood at the reception stage is unjustified as it adds to the problem of shortage of blood in Sudan.

Adolescent↗
PubMed1996

Pyruvate: ferredoxin oxidoreductase from Entamoeba histolytica recognized by a monoclonal antibody.

A mouse monoclonal antibody, Eh208C2-2 MAb, raised against whole cell antigens of Entamoeba histolytica trophozoites of the pathogenic strain HM-1: IMSS and polyclonal antisera (PAb) against membrane antigens of E. histolytica trophozoites of strain HTH-56: MUTM were screened against a cDNA library of the pathogenic strain, SFL3. The monoconal antibody detected many phage plaques expressing an E. histolytica protein. The DNA sequence encoding the protein was approximately 55% identical, over 1,100bp, to Trichomonas vaginalis pyruvate: ferredoxin oxidoreductase (PFOR) and pyruvate: flavodoxin oxidoreductase from Klebsiella pneumoniae, Anabaena variabilis and Enterobacter agglomerans. Two of seven clones detected by mouse polyclonal antisera also encoded this protein. Two others encoded Entamoeba Hsp70, another encoded Entamoeba alkyl-hydroperoxide reductase and the remaining two were unidentified sequences. Entamoeba PFOR is an abundant, antigenic protein which may be a useful target for the development of protective host immune responses against invasive amebiasis.

Amino Acid Sequence↗
PubMed1995

Activation of MAP kinase by the LHRH receptor through a dual mechanism involving protein kinase C and a pertussis toxin-sensitive G protein.

The LHRH receptor in alpha T3-1 gonadotrope cells was shown to bring about a marked and sustained activation of MAP kinase. This response was prevented by protein kinase C inhibition or down-regulation and could be partially mimicked by phorbol ester. Additional evidence for inhibition of this response by pertussis toxin and partial mimicry by mastoparan (in a pertussis toxin-sensitive manner) provides the first evidence for Gi/Go-mediated signal transduction by the LHRH receptor. This dual mechanism of MAP kinase activation appears to be exceptional amongst the G protein-linked receptors that have been investigated.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1995

Cyanide and carbon monoxide binding to the reduced form of cytochrome bo from Escherichia coli.

Cyanide binds to fully reduced cytochrome bo and induces a blue shift of the Soret absorption band of the high-spin heme o and a change in the visible region spectrum consistent with the expected conversion to a low-spin state. The dissociation constant, determined by titration of the extent of the binding spectrum, is 7.0 +/- 0.6 mM at pH 7.0. In contrast, cyanide does not bind significantly in this concentration range to the reduced form of cytochrome bd. The reduced cyanide compound of cytochrome bo can be laser photolyzed. Typically, less than 20% photolysis was attained with conditions that give essentially full photolysis of the carbon monoxide compound. The subsequent monophasic kinetics of recombination of cyanide at varying cyanide concentrations were used to determine kon, koff, and dissociation constant values at pH 7.0 of 572 +/- 43 M-1 s-1, 4.2 +/- 0.7 s-1, and 7.3 +/- 1.3 mM, respectively. The dissociation constant changes very little in the pH range 6-8, indicating that a proton is bound together with the cyanide anion, as predicted by our recent proposal of a requirement for electroneutrality in the binuclear center [Mitchell, R., & Rich, P. R. (1994) Biochim. Biophys. Acta 1186, 19-26]. Competition studies confirm that cyanide and carbon monoxide cannot bind simultaneously, so that their binding sites must overlap. A small fraction of the reduced unliganded enzyme appears to have a distinct photolysis spectrum in the absence of added ligands, and this is transformed into a typical ferrous cyanide compound only at very high cyanide concentrations. Cyanide binding and photolysis were also examined in a number of mutant forms of cytochrome bo, and in a wild-type form which was partially depleted in CuB. Dramatic changes in rate constants and binding constants were found in several cases. Data from several mutants were compared with analogous data on the binding and photolysis of carbon monoxide, and the effects of mutation were quite different with the two ligands. A model is developed to explain the observed effects of point mutations on the recombination kinetics of both carbon monoxide and cyanide. Overall, the results indicate that the CuB site is required for binding of cyanide, but not carbon monoxide, to the reduced enzyme, possibly by providing the site for binding of the associated proton.

Bacterial Proteins↗
PubMed1995

Phorbol ester and diacylglycerol activation of native protein kinase C species from various tissues.

The characteristics of PKC activation induced by a number of compounds were investigated using PKCs, partially-purified from sources with a naturally high abundance of certain Ca2+ dependent PKC isoforms. Native isoforms were used rather than PKC isoforms expressed from a baculovirus system to assess the effect of tissue specific factors on activity. However, some data using recombinant PKC alpha were included for comparison. The presence of specific PKC isoforms in different tissues was determined using Western blot analysis. Protein kinase C alpha, beta 1, delta, epsilon, and zeta/iota were all present in rat midbrain cytosolic extract, PKC alpha, beta 1, delta, and zeta/iota were present in spleen cytosol, and PKC alpha and zeta/iota were present in COS 7 cell cytosol. The predominance of alpha and beta activities in COS 7 and spleen extracts respectively was confirmed by enzymic assay. The PKC activity assay was configured such that the Ca2+ dependence of the PKC activity induced by different PKC activators could be determined. Phorbol 12,13-dibutyrate (PDBu) was virtually equipotent on the Ca(2+)-dependent PKC activity from midbrain and spleen and slightly less potent on that from COS 7 cells. In the absence of Ca2+, PDBu was considerably less potent overall (as, indeed, were the other PKC activators) and was less potent on COS 7 cell PKC than on those from midbrain or spleen. Mezerein was more potent than PDBu at inducing PKC activity in COS 7 cell extracts in either the absence or presence of Ca2+ whereas in the presence of Ca2+, mezerein was slightly less potent on midbrain and spleen than PDBu and equipotent in the absence of Ca2+. Maximum values for Ca(2+)-independent activation by mezerein indicated that this activator was particularly effective in recruiting Ca(2+)-dependent PKC isoform activity in a Ca2+ free environment. The greater potency of mezerein on PKC alpha was confirmed using PKC alpha and beta further purified from rat spleen by hydroxylapatite (HAP) chromatography. The effects of both PDBu and mezerein were investigated using anterior pituitary tissue where a particularly high potency of mezerein in the absence of Ca2+ was noted. The diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DOG), appeared to cause little or no activation of native Ca(2+)-dependent isoforms in Ca2+ free conditions unlike another longer chain diacylglycerol, 1,2-dioleoyl-sn-glycerol. Also DOG activated midbrain PKCs more potently than PKCs from spleen or COS 7 cells (or lung and pituitary tissue) in the presence of Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Identification of the major site of apolipoprotein B modification by advanced glycosylation end products blocking uptake by the low density lipoprotein receptor.

Advanced glycosylation end products (AGEs) arise from glucose-derived Amadori products and have been implicated in the pathogenesis of diabetic vascular disease. We recently reported the presence of an AGE-modified form of low density lipoprotein (LDL) that circulates in high amounts in patients with diabetes or renal insufficiency and that exhibits impaired plasma clearance kinetics. We utilized AGE-specific antibodies to identify the major sites of AGE modification within protease-digested preparations of apolipoprotein B that impair the binding of the AGE-modified form of LDL by human fibroblast LDL receptors. The predominant site of AGE immunoreactivity was found to lie within a single, 67-amino acid region located 1791 residues NH2-terminal of the putative LDL receptor binding domain. These data point to the high reactivity and specificity of this site for AGE formation and provide further evidence for important structural interactions between the LDL receptor binding domain and remote regions of the apolipoprotein B polypeptide.

Amino Acid Sequence↗
PubMed1995

Cloning and characterization of the gene for mouse macrophage migration inhibitory factor (MIF).

An emerging body of data indicates that the protein mediator described originally as macrophage migration inhibitory factor (MIF) exerts a central and wide ranging role in host inflammatory responses. MIF is a major constituent of corticotrophic cells within the anterior pituitary gland and is secreted into the circulation in a hormone-like fashion. MIF also exists performed in monocytes/macrophages and is a pivotal mediator in the host response to endotoxic shock. To gain further insight into the biologic expression of this protein that encompasses components of both the immune and the endocrine systems, we have cloned the mouse MIF gene and identified potential regulatory sequences present within the 5'-proximal promoter region. The gene for mouse MIF is located on chromosome 10, spans approximately 1 kb, and shares a high degree of structural homology with its human counterpart. Of note, the consensus enhancer/promoter motifs identified include both inflammatory/growth factor-related elements and sites associated with the genes for certain peptide hormones. We also report the structures of two MIF pseudogenes that account for early observations suggesting that mouse MIF is encoded by a highly homologous multigene family.

Amino Acid Sequence↗
PubMed1995

The involvement of metabotropic glutamate receptors and their intracellular signalling pathways in sustained nociceptive transmission in rat dorsal horn neurons.

The excitatory responses of individual dorsal horn neurons to cutaneous brush, repeated application of the C-fibre-selective chemical algogen, mustard oil, or to ionophoretic (1S,3R)-ACPD [a metabotropic glutamate receptor (mGluR) agonist] were monitored by extracellular recording. We have previously shown that the responses of dorsal horn neurons to mustard oil are inhibited by several selective antagonists of mGluRs. Effects of ionophoresis of the mGluR antagonists (R,S)-CHPG and L-AP3 and a range of selective inhibitors of intracellular signalling pathways were examined on evoked responses here. The results suggest that protein kinase C, phospholipase A2 and perhaps Ca2+/calmodulin kinase II play a role in mediating the sustained elevated activity of dorsal horn neurons that is incrementally elicited by repeated application of mustard oil, but probably make little contribution to sustained brush-evoked activity. Concurrence in the sensitivity of mustard oil- and (1S,3R)-ACPD-evoked activity to (R,S)-CHPG, L-AP3 and to inhibitors of intracellular signalling pathways, suggests that mGluRs are an important origin of these intracellular signals required for sustained nociception.

Animals↗
PubMed1995

Effect of tyrosine kinase inhibitors on luteinizing hormone-releasing hormone (LHRH)-induced gonadotropin release from the anterior pituitary.

A range of selective tyrosine kinase inhibitors, piceatannol, methyl-2,5-dihydroxycinnamate (MDC), genistein, psi-tectorigenin and lavendustin A, all reduced luteinizing hormone-releasing hormone (LHRH)-induced luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release from pro-oestrous rat hemipituitaries incubated in vitro. In general, both 'initial' release and the augmented release resulting from LHRH self-priming, were reduced in parallel in a concentration-dependent fashion. The effects of piceatannol were independent of the steroidal status of the pituitary tissue. Both piceatannol and MDC greatly reduced LH release by ionomycin and a protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), suggesting that the tyrosine kinase(s)-dependent step is in the later stages of the stimulus-secretion pathway activated by the LHRH receptor. These data were supported by immunoblots for phosphotyrosine showing that in the gonadotrope-derived alpha T3-1 cell line, treatment with LHRH caused piceatannol-sensitive increases in specific tyrosine phosphorylation of several proteins (major bands at 65-75 and 120-130 kDa). Treatment of cells with PDBu mimicked the tyrosine phosphorylations evoked by LHRH whereas the PKC inhibitor, GF109203X, partially reduced both LHRH- and PDBu-induced tyrosine phosphorylations. Direct effects of MDC and piceatannol on PKC were assessed in an in vitro PKC assay; piceatannol, but not MDC, inhibited PKC activity but at considerably higher concentrations than required for inhibition of LHRH-induced gonadotropin secretion. These data support a role for tyrosine kinase activation in LHRH-induced secretion.

Animals↗