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Biomedical subjects

J H Matthews

Publications and source records attributed to J H Matthews.

At least 19 recordsLinked to original sources
PubMed2001

Intramolecular ditryptophan crosslinks enforce two types of antiparallel beta structures.

BACKGROUND: Two types of biaryl crosslinks can be formed with natural protein sidechains: ditryptophan and dityrosine. Biaryl crosslinks have the same topology as disulfide crosslinks, yet little is known about their effect on local peptide structure. RESULTS: Three ditryptophan-linked peptide dimers based on the sequence Ac-Leu-Trp-Ala-COX were prepared. The tripeptide dimer with -CONH(2) termini was too insoluble to study, but the tripeptide dimer with -COOMe termini crystallized from methanol/chloroform as an antiparallel beta-sheet. The tripeptide dimer with a -CONMe(2) termini adopted a slipped antiparallel beta structure in methanol/chloroform. CONCLUSIONS: These results suggest that intermolecular ditryptophan crosslinks that join the middle of peptide chains can confer a preference for antiparallel beta-sheet structure. The effect is most dramatic when both the inside and outside edges of the dimer can form hydrogen bonds as in the crystal structure of dimer 3b.

Crystallization↗
PubMed2000

Crystal structure analysis of recombinant human uteroglobin and molecular modeling of ligand binding.

Uteroglobin, a steroid-inducible, cytokine-like, secreted protein with immunomodulatory properties, has been reported to bind progesterone, polychlorinated biphenyls (PCB), and retinol. Structural studies may delineate whether binding of ligands is a likely physiological function of human uteroglobin (hUG). We report a refined crystal structure of uncomplexed recombinant hUG (rhUG) at 2.5-A resolution and the results of our molecular modeling studies of ligand binding to the central hydrophobic cavity of rhUG. The crystal structure of rhUG is very similar to that of reported crystal structures of uteroglobins. Using molecular modeling techniques, the three ligands--PCB, progesterone, and retinol--were docked into the hydrophobic cavity of the dimer structure of rhUG. We undocked the progesterone ligand by pulling the ligand from the cavity into the solvent. From our modeling and undocking studies of progesterone, it is clear that these types of hydrophobic ligands could slip into the cavity between helix-3 and helix-3' of the dimer instead of between helix-1 and helix-4 of the monomer, as proposed earlier. Our results suggest that at least one of the physiological functions of UG is to bind to hydrophobic ligands, such as progesterone and retinol.

Animals↗
PubMed1999

Bound structures of novel P3-P1' beta-strand mimetic inhibitors of thrombin.

The X-ray crystal structures of four beta-strand-templated active site inhibitors of thrombin containing P1' groups have been determined and refined at about 2.1-A resolution to crystallographic R-values between 0.148 and 0.164. Two of the inhibitors have an alpha-ketoamide functionality at the scissile bond; the other two have a nonhydrolyzable electrophilic group at the P1' position. The binding of lysine is compared with that of arginine at the S1 specificity site, while that of D,L-phenylalanine enantiomorphs is compared in the S3 region of thrombin. Four different P1' moieties bind at the S1' subsite in three different ways. The binding constants vary between 2.0 microM and 70 pM. The bound structures are used to intercorrelate the various binding constants and also lead to insightful inferences concerning binding at the S1' site of thrombin.

Aza Compounds↗
PubMed1998

Set-up variation of patients treated with radiotherapy to the prostate measured with an electronic portal imaging device.

The set-up variation of 11 patients treated supine with radical radiotherapy for carcinoma of the prostate was measured with an electronic portal imaging device to determine the adequacy of set-up techniques and current margins, as well as the need for immobilization. During the treatments 172 images of the anterior fields and 159 images of the left-lateral fields were taken and the errors in treatment placement were measured by template matching. The variation in the superior-inferior direction was small, 1.4-1.6 mm (1 SD), while the medio-lateral variation was 2.8 mm (1 SD). The anterior-posterior variation was largest, 4.6 mm (1 SD) with an offset of 3.3 mm anterior. This anterior offset and large anterior-posterior variation suggests that set-up techniques were not optimal for this direction. The 1 cm margin used was adequate for set-up variation except in a small number of cases, which was mainly due to the anterior trend. Random (treatment-to-treatment) variations were small (1.1-2.3 mm; 1 SD), indicating that immobilization would result in only modest improvement in reproducibility for these supine patients.

Humans↗
PubMed1998

The cytotoxic effect of the vitamin B12 inhibitor cyanocobalamin [c-lactam], and a review of other vitamin B12 antagonists.

The vitamin B12 antagonist cyanocobalamin [c-lactam] was cytotoxic to cultured human leukemia cells, grown in methylfolate, homocysteine, and vitamin B12, but not in the presence of methionine. Small concentrations of methionine were effective in restoring the growth rate in a dose-dependent fashion, confirming methionine deficiency as the cytotoxic principle. Cyanocobalamin [c-lactam] prevented utilization of the methyl group of methylfolate, but no evidence of folate deficiency developed in long-term culture. High concentrations of non-methylated folate were unable to reverse the cytotoxicity, excluding a methylfolate 'trap' as the cause. Low concentrations of serine in the medium induced transient biochemical megaloblastosis. Cyanocobalamin [c-lactam] caused this to occur earlier, and persist. In high concentrations of serine, the inhibitor caused only transient changes in deoxyuridine suppression. Homocysteine cannot be remethylated without vitamin B12, and condensation with serine is the only other excretory pathway for this toxic amino acid. We hypothesize that impaired DNA synthesis in vitamin B12 deficiency is the result of diverting serine away from thymidylate synthesis, into homocysteine metabolism.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1997

Cyanocobalamin [c-lactam] inhibits vitamin B12 and causes cytotoxicity in HL60 cells: methionine protects cells completely.

The [c-lactam] derivative of cobalamin antagonizes vitamin B12 in vivo. Therefore, we investigated its effects in tissue culture to develop a model in which to study vitamin B12-deficient hemopoiesis. HL60 cells were cultured in medium containing either methionine or L-homocysteine thiolactone, and various concentrations of 5-methyltetrahydrofolate or pteroylglutamic acid. In medium with L-homocysteine thiolactone, 5-methyltetrahydrofolate, and dialyzed serum, cyanocobalamin [c-lactam] caused cell death, reversible by additional vitamin B12. Pteroylglutamic acid did not prevent this cytotoxic effect. Methionine completely protected cells against cyanocobalamin [c-lactam] for periods of up to 4 months of culture, irrespective of the folate source. Cyanocobalamin [c-lactam] reversibly impaired the incorporation of 5-[14CH3]-tetrahydrofolate and [1-(14)C] propionic acid by intact cells, consistent with inhibition of methionine synthase and methylmalonyl-CoA mutase. A substantial proportion of 5-[14CH3]-tetrahydrofolate uptake could not be suppressed by methionine and may, therefore, have occurred outside of the methionine synthase pathway. These findings are the first indication that cyanocobalamin [c-lactam] antagonizes vitamin B12 in vitro and causes cell death from methionine deficiency. The model should be valuable for investigating the biochemical pathology of vitamin B12-deficient hemopoiesis. The results suggest that methylfolate is not trapped when methionine synthase is inhibited in HL60 cells, but they do not disprove the methylfolate trap hypothesis as applied to normal blood cells.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1996

Crystal structures of thrombin with thiazole-containing inhibitors: probes of the S1' binding site.

Structures of the blood clotting enzyme thrombin complexed with hirugen and two active site inhibitors, RWJ-50353 10080(N-methyl-D-phenylalanyl-N-[5-[(aminoiminomethyl)amino]-1- [[(2-benzothiazolyl)carbonyl]butyl]-L-prolinamide trifluoroacetate hydrate) and RWJ-50215 (N-[4-(aminoiminomethyl)amino-1-[2- (thiazol-2-ylcarbonylethyl)piperidin- 1-ylcarbonyl]butyl]-5-(dimethylamino)naphthalenesulfonamide trifluoroacetate hydrate), were determined by x-ray crystallography. The refinements converged at R values of 0.158 in the 7.0-2.3-A range for RWJ-50353 and 0.155 in the 7.0-1.8-A range for RWJ-50215. Interactions between the protein and the thiazole rings of the two inhibitors provide new valuable information about the S1' binding site of thrombin. The RWJ-50353 inhibitor consists of an S1'-binding benzothiazole group linked to the D-Phe-Pro-Arg chloromethyl ketone motif. Interactions with the S1-S3 sites are similar to the D-phenylalanyl-prolyl-arginyl chloromethylketone structure. In RWJ-50215, a S1'-binding 2-ketothiazole group was added to the thrombin inhibitor-like framework of dansylarginine N-(3-ethyl-1,5-pentanediyl)amide. The geometry at the S1-S3 sites here is also similar to that of the parent compound. The benzothiazole and 2-ketothiazole groups bind in a cavity surrounded by His57, Tyr60A, Trp60D, and Lys60F. This location of the S1' binding site is consistent with previous structures of thrombin complexes with hirulog-3, CVS-995, and hirutonin-2 and -6. The ring nitrogen of the RWJ-50353 benzothiazole forms a hydrogen bond with His57, and Lys60F reorients because of close contacts. The oxygen and nitrogen of the ketothiazole of RWJ-50215 hydrogen bond with the NZ atom of Lys60F.

Binding Sites↗
PubMed1996

Relationships between the academic community and the pharmaceutical industry: the legislative background and its effect on spending on medical research and development.

Patent legislation governing drugs has evolved through a series of amendments to the Patent Act. From 1923 until 1993, Canada operated a system of "compulsory licensing," allowing generic copies of patented medicines to be manufactured within Canada and, by 1969, to be imported. In 1987, the act was amended (Bill C-22) to provide patented medicines with a fixed period of market protection before a compulsory license could be issued and to create a price review board to monitor and control prices charged. In return for patent protection, brand-name drug companies promised to invest a growing percentage of sales revenue in research and development in Canada. A 1993 amendment to the Patent Act (Bill C-91) brought a fundamental change to the legislation by abolishing the system of compulsory licensing and applying general patent regulations to medicines, thereby bringing Canadian law into line with that of its trading partners. It is now illegal to sell a copy of a drug until the patent expires (20 years after the patent is filed). This means that marketed drugs are protected for 8 to 13 years, since drug development takes a large proportion of the life of the patent. Since this amendment was passed, the brand-name drug companies have made major contributions to research and development in Canada, increasing from 6.5% of sales revenue in 1987 to 11.6% in 1994. Major irritants in the legislation remain. Generic drug companies have complained about "linkage regulations" that allow brand-name drug companies to legally challenge generic drug production on the basis of alleged infringements of linked patents, delaying the marketing of the generic drug. The act also prohibits Canadian manufacturers from exporting a generic drug to a country where it is not protected if it still protected in Canada. Brand-name manufacturers want some means of patent term restoration if regulatory authorities prolong the time taken before marketing a drug. This legislation is being reviewed by parliament beginning in 1997.

Canada↗
PubMed1995

The effects of radiotherapy and surgery on the sexual function of women treated for cervical cancer.

PURPOSE: This study investigated the short- and medium-term effects of pelvic radiotherapy and surgery on the sexual function of women treated for cervical cancer. METHODS AND MATERIALS: Sixteen women with Stages I, II, or III disease referred for radiotherapy treatment were assessed. Six had undergone prior hysterectomy. The women were assessed with questionnaires prior to radiotherapy, at completion of radiotherapy, and at 6 weeks and 14 weeks after radiotherapy treatment. The clinical findings at routine follow-up were noted. RESULTS: The study showed significant changes in sexual activity and satisfaction as a result of treatment. This was due to a number of physical and psychological factors. The level of sexual activity was lowest at completion of radiotherapy treatment. A feeling of vaginal shortening was the most frequent reason and was more common in women who were treated with surgery and radiotherapy. Dyspareunia, bleeding, and concern of bleeding and/or recurrence were all significant factors. CONCLUSIONS: The questionnaires were an effective way of assessing women's sexual function. Radiotherapy caused sexual dysfunction in one-half of women. Combined treatment with radiotherapy and surgery results in a higher risk than radiotherapy alone. Women with cervical cancer and undergoing radiotherapy treatment require considerable counseling and support.

Coitus↗
PubMed1995

Resistance mechanisms determining the in vitro sensitivity to paclitaxel of tumour cells cultured from patients with ovarian cancer.

Paclitaxel, a drug which stabilises microtubules, demonstrates marked activity against ovarian cancer. We investigated the sensitivity to paclitaxel of tumour cells from disaggregated solid tumours or tumour-bearing ascites from 7 ovarian cancer patients, and 21 established tumour cell lines (ovarian, melanoma and lung). Response was quantitated by [3H]-thymidine incorporation in 96-well plates or by colony growth. Dose-response curves to paclitaxel were biphasic with a dose-dependent phase providing an IC50 value (50% reduction in incorporation) and dose-dependent "plateau" phase where the effect was independent of paclitaxel concentration. IC50 values ranged from 2.5 to 110 nM with evidence of multidrug resistance in the two most resistant cell lines. The "plateau" killing values varied from 0.1 log10 to > 3.4 log10 units reduction, and were found to be significantly correlated (r = 0.86; P < 0.0001) with logarithmic culture doubling times of the cell lines. Cellular glutathione levels were measured and found not to be significantly associated with response to paclitaxel. The results suggest that the ratio of paclitaxel exposure time to the culture doubling time is a major factor in paclitaxel cytotoxicity. The relationship between tumour cell cytokinetics and paclitaxel pharmacokinetics in vivo may therefore be crucial in determining clinical paclitaxel response.

Adult↗
PubMed1995

Cobalamin and folate deficiency in the elderly.

Elderly persons are more likely to have low values for serum and erythrocyte folate, and for serum cobalamin. Many of those with low vitamin levels have biochemical abnormalities consistent with true deficiency, including increased formiminoglutamic acid excretion, abnormal marrow deoxyuridine suppression, and raised serum levels of methylmalonic acid and homocysteine. Therapy with the appropriate vitamin reverses the biochemical defect. Despite this, the clinical consequences for most elderly persons are remarkably few. True megaloblastic anaemia is rare, and the small number of therapeutic trials to date have not improved the levels of haemoglobin in the treated subjects, although the mean corpuscular volume has decreased significantly. There has been recent concern that these low blood vitamin levels might be important causes of nervous system damage, but studies specifically of the elderly have not demonstrated overall improvements in neurological function following therapy. Vascular damage from high blood homocysteine levels secondary to cobalamin or folate deficiency remains a potential hazard. Dietary insufficiency, malabsorption of protein-bound vitamin B12 secondary to atrophic gastritis, and defective absorption of folyl polyglutamates seem the likeliest possible causes. Pernicious anaemia, although a common cause of severe megaloblastic anaemia in the elderly, is an infrequent cause for the low cobalamin levels in population studies. Although the benefits are uncertain, the balance of the evidence suggests that one should treat elderly persons with low values of cobalamin or folate. Crystalline vitamin B12 and folic acid are absorbed normally and are therefore suitable for replacement therapy, provided that pernicious anaemia is excluded.

Aged↗
PubMed1994

Crystallization and characterization of the recombinant human Clara cell 10-kDa protein.

Crystals of recombinant human Clara cell 10-kDa protein were grown both from ammonium sulfate and polyethylene glycol (PEG) solutions. Crystals grown from ammonium sulfate solution have been characterized by X-ray diffraction studies as monoclinic with the space group C2 and lattice constants a = 69.2 A, b = 83.0 A, c = 58.3 A, and beta = 99.7 degrees. The monoclinic crystals diffract to beyond 2.5 A. Some of the crystals grown from PEG were of a similar habit to those grown from ammonium sulfate, but others were triclinic with the space group P1 and cell constants a = 40.3 A, b = 46.3 A, c = 51.3 A, alpha = 117.7 degrees, beta = 102.3 degrees, and gamma = 71.4 degrees. These crystals diffract to beyond 3.2 A.

Crystallography, X-Ray↗
PubMed1994

Radiosensitivity of new and established human melanoma cell lines: comparison of [3H]thymidine incorporation and soft agar clonogenic assays.

Seven new low-passage melanoma lines were developed in this laboratory from clinical melanoma specimens and characterised for chromosome complement, DNA ploidy and S-phase content. The radiosensitivity of these lines was compared with that of eight established melanoma cell lines, FME, MM-96, SK-MEL-5, SK-MEL-28, SK-MEL-2, MALME-3M, M19-MEL and LOX-IMVI, using a 96-well microculture assay technique. Dose-response curves were determined using a 5-day incubation period and 6-h terminal [3H]thymidine-labelling period. Radiation (60Co source) was carried out under a lead wedge to provide a radiation dose range of 0-10 Gy, or by irradiating part of the plate (radiation dose 0 or 2 Gy). Data for a range of cell densities in a single 96-well plate were combined into a single regression equation incorporating linear quadratic terms for radiation dose and cell density. SF2 values were defined as the amount of thymidine incorporated following a radiation dose of 2 Gy, expressed as a fraction of that of unirradiated cells, and varied from 0.36 to 0.93. The reproducibility in repeat assays, as defined by the standard error of determinations at different passage numbers, was +/- 0.04. The newly developed lines exhibited a similar range of radiosensitivity to that of the established lines, and melanin content did not correlate with resistance. For nine of the lines, radiation parameters were also determined using a modified Courtenay clonogenic soft agar assay technique, and the results compared with the thymidine incorporation results, and a significant linear correlation was found between SF2 and SF2' (r = 0.89). The linear (alpha) and quadratic (beta) terms of the best-fit linear quadratic dose-response curves, were significantly correlated between the two assays. It is concluded for this series of human melanoma lines that proliferation assays in 96-well plates provide radiosensitivity parameters comparable to those using clonogenic assays.

Adult↗