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Biomedical subjects

D Saunders

Publications and source records attributed to D Saunders.

At least 73 records · Page 4Linked to original sources
PubMed1993

Cyclic disulfide analogues of the complement component C3a. Synthesis and conformational investigations.

The flexible C-terminal region of the anaphylatoxic peptide C3a was reported to contain the receptor binding site. To elucidate the receptor binding conformation of the C-terminus, as well as to examine a synthetic approach to potential C3a-antagonists, 26 cyclic disulfide bridged C3a analogues were synthesized. Solid phase peptide synthesis was performed on different polymeric supports by individual peptide synthesis, with Fmoc strategy, and simultaneous multiple peptide synthesis, using Boc and Fmoc strategies. Both strategies gave open-chain peptides in comparable yields. Syntheses using the Boc strategy employed the HF-labile 4(methoxy)benzyl group (Mob) for beta-thiol protection of cysteine; in contrast, the TFA-stable protecting groups, acetamidomethyl (Acm) and trityl (Trt), were chosen for syntheses employing Fmoc strategy. Ring closure reactions by iodine oxidation were carried out starting from protected (Acm/Acm, Trt/Acm) or unprotected dithiols. The resulting cyclic C3a analogues were characterized by HPLC, amino acid analysis, and FAB-MS. Conformational investigations using CD spectroscopy and theoretical structural investigations by means of molecular dynamics calculations revealed that slight variations in sequence result in pronounced conformational consequences. The potential of cyclic C3a analogues to activate or to desensitize guinea pig platelets, a standard test system for biological activities of anaphylatoxic peptides like C3a, revealed relatively low activities for cyclic peptides (< 0.1% C3a activity). N-terminal acylation with cationic, arginine-rich sequences like YRRGR- led to amplified biological effects. Three of the synthesized peptides, namely CAALCLAR (P1), YRRGRCGGLCLAR (P5) and YRRGRAhxCGGLCLAR (P8), point in the direction of C3a antagonists.

Amino Acid Sequence↗
PubMed1993

An alternative to hysterectomy? GnRH analogue combined with hormone replacement therapy.

OBJECTIVE: To assess whether GnRH analogues are effective in relieving pelvic pain and congestion and whether menopausal symptoms caused by GnRH analogues can be minimised by supplementation with low dose continuous combined hormone replacement therapy (HRT). DESIGN: Open prospective study. SETTING: Tertiary referral clinic at a teaching hospital. PATIENTS: Twenty-one women with chronic pelvic pain. INTERVENTION: Four months' therapy with goserelin 3.6 mg/month combined with continuous oestradiol valerate 1 mg daily and medroxyprogesterone acetate 5 mg daily. MAIN OUTCOME MEASURES: Visual analogue scale for pain, menopausal symptoms, bleeding patterns, uterine area, endometrial status, oestradiol concentrations and venogram scores. RESULTS: Amenorrhoea was maintained in all but two women. Endometrial atrophy was maintained despite HRT supplementation. Two women had moderate menopausal symptoms but none had severe symptoms. Significant reduction of uterine cross sectional area was maintained throughout the study. There was no significant relief of pain. CONCLUSIONS: HRT supplementation of GnRH analogues abolishes menopausal symptoms and thus may improve patient acceptability. Potentially beneficial effects such as endometrial atrophy, reduction of uterine volume and amenorrhoea were not negated by HRT. This combination is not effective in the treatment of chronic pelvic pain and congestion.

Adult↗
PubMed1993

Diagnosis and management of extrauterine pregnancies.

This study was based on 16 women provisionally diagnosed as having extrauterine pregnancies. Of these, 13 (81.3%) were confirmed as positive at operation. Patients were managed according to 1 of 3 regimens; 1) methotrexate (n = 4), 2) methotrexate followed by surgery (n = 3) and 3) surgery (n = 6). Serial blood samples, collected before and after treatment, were analyzed for ovarian (oestradiol, E2; progesterone, P4) uterine (placental protein 14, PP14) and placental markers (chorionic gonadotrophin, HCG; pregnancy-associated plasma protein-A (PAPP-A). Of the pretreatment samples, only 30.4% and 41.7% were depressed for PP14 and HCG, respectively. By contrast, the diagnostic value of PAPP-A (77.8%) and P4 (87.5%) was greater. Biochemical monitoring of treatment was best achieved with trophoblastic derived antigens (HCG), whereas antigens of maternal origin demonstrated widely varied responses. This study established the effectiveness of chemotherapy for treatment of tubal pregnancies as an alternative to surgery, but if a biochemical marker is required, the marker of choice is HCG.

Abortion, Therapeutic↗
PubMed1992

Synthetic peptides as antagonists of the anaphylatoxin C3a.

Peptide compounds resembling the receptor-binding C-terminal domain of the anaphylatoxic peptide C3a were synthesized to examine two kinds of C3a antagonism: (a) specific desensitization of C3a-sensitive cells and (b) competitive binding to the C3a receptor. We used guinea-pig platelets, which express a C3a receptor and specifically release ATP upon stimulation, to evaluate the actions of the C3a analogues. The ATP liberation can be inhibited by pretreatment (i.e. desensitization) of the guinea-pig platelets with substimulatory concentrations of C3a or its analogues. Compared to C3a, several peptides were found with at least a tenfold greater difference between the required concentrations for C3a-specific half-maximal desensitization (DD50) and half-maximal platelet activation (ED50). The most potent compounds were YAAALKLAR and Fmoc-EAALKLAR (Fmoc: 9-fluorenylmethoxycarbonyl) with an ED50/DD50 of 140 +/- 28 and 80 +/- 17, respectively (mean +/- standard deviation). The ED50/DD50 of human C3a was found to be only 6 +/- 2. Some C3a derivatives were also tested in competitive binding studies for their ability to compete with C3a for receptor sites on guinea-pig platelets. Three of them were considered partial antagonists [YRRGRCGGLCLAR, YRRGRXCGGLCLAR and YRRGRXCGALCLAR (X = 6-aminohexanoyl)] because their Ki were smaller than their ED50 (Ki/ED50 = 0.6 +/- 0.3, 0.5 +/- 0.1 and 0.4 +/- 0.2, respectively). Interestingly, the last two compounds also had ED50/DD50 values greater than 60. Common to all three peptides are N-terminal arginine-rich sequences and intramolecular disulfide bridges which introduce conformational constraint.

Amino Acid Sequence↗
PubMed1992

A comparison of the in vivo biochemical responses to exogenous parathyroid hormone-(1-34) [PTH-(1-34)] and PTH-related peptide-(1-34) in man.

PTH-related peptide (PTHrP) is one of the etiological factors associated with hypercalcemia of malignancy in humans and rodents. In both in vivo and in vitro animal systems its actions mimic those of PTH; however, its bioactivity in humans has not previously been assessed. Therefore, we compared the actions of the synthetic human (h) analogs hPTHrP-(1-34) and hPTH-(1-34) when given by iv infusion to 15 healthy subjects, aged 25 +/- 3 yr. Three 12-h test infusions were given to each subject in the order: hPTH-(1-34) at a dose of 8 pmol/kg.h, an equimolar dose (8 pmol/kg.h) of PTHrP-(1-34) (low dose), and a 10-fold higher dose (80 pmol/kg.h) of hPTHrP-(1-34) (high dose). PTH infusion resulted in significant increases from basal values in serum total ionized calcium, urinary phosphate and cAMP, and serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2d3]. No significant increases from basal values in any of these variables were observed during low dose PTHrP infusion. However, a 10-fold higher dose of PTHrP significantly increased serum calcium from 2.36 +/- 0.07 to 2.63 +/- 0.16 mmol/L (P less than 0.003), ionized calcium from 1.22 +/- 0.03 to 1.39 +/- 0.09 mmol/L (P less than 0.003), urinary phosphate from 0.21 +/- 0.19 to 0.31 +/- 0.16 mmol/L glomerular filtrate (P less than 0.05), urinary cAMP from 37 +/- 18 to 53 +/- 28 nmol/L glomerular filtrate (P less than 0.01), and serum 1,25-(OH)2D3 from 29.8 +/- 12.1 to 46.0 +/- 20.3 pmol/L (P less than 0.01). For each variable these changes were statistically equivalent to the increases observed during PTH infusion. The molar concentrations of circulating immunoreactive PTH-(1-34) and PTHrP-(1-34) (at the higher dose) achieved during infusion were at a ratio of 1:3. These results suggest that the in vivo actions of synthetic hPTHrP-(1-34) are comparable to those of hPTH-(1-34), but its biological activity after infusion may be less than that of hPTH-(1-34). Moreover, the increased concentrations of serum 1,25-(OH)2D3 observed with administration of hPTHrP-(1-34) are unlike the changes seen in hypercalcemia of malignancy in which levels of this vitamin D metabolite are frequently depressed.

Adult↗
PubMed1991

Oxidation-resistant variants of recombinant antileucoprotease are better inhibitors of human-neutrophil-elastase-induced emphysema in hamsters than natural recombinant antileucoprotease.

1. Antileucoprotease, being sensitive to oxidative inactivation, can be produced by recombinant techniques. Via site-directed mutagenesis, two mutants of recombinant antileucoprotease were produced in which one or more of the oxidation-sensitive methionine residues were replaced by leucine: in rALP242, methionine-73 was replaced by leucine, and in rALP231, leucine was substituted for four methionine residues. In vitro, native antileucoprotease and the recombinant antileucoprotease preparations have similar inhibitory characteristics towards human neutrophil elastase. We hypothesized that replacement of methionine residues in the antileucoprotease molecule would result in a reduced oxidation sensitivity of the mutants. 2. After incubation of recombinant antileucoprotease and its mutants with increasing dosages of cis-platinum(II)diammine dichloride, we observed that native antileucoprotease and recombinant antileucoprotease were inactivated by this reagent to the same extent. Compared with this, rALP242 was less inactivated, whereas the inhibitory capacity of rALP231 was not influenced by cis-platinum(II)diammine dichloride at all. 3. After incubation of recombinant antileucoprotease, rALP242 and rALP231 with triggered polymorphonuclear leucocytes, which are thought to produce an excess of oxidants, we measured residual inhibitory activities towards human neutrophil elastase of 10%, 55% and 87%, respectively. 4. In vivo, the inhibitory effects of intratracheally administered rALP242 and rALP231 towards human-neutrophil-elastase-induced emphysema were significantly greater than that of recombinant antileucoprotease. There were no significant differences between the mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

The role of hysteroscopy in patients having failed IVF/GIFT transfer cycles.

Fifty patients who had undergone 2 or more failed in vitro fertilization (IVF) embryo transfer (ET) cycles or failed GIFT cycles where fertilization had been demonstrated, underwent hysteroscopy; 28% were found to have intrauterine abnormalities which may have been responsible for the failure of the IVF-ET or GIFT. Patients with an abnormality found at hysteroscopy had undergone a significantly higher mean number of transfer cycles. These results suggest that intrauterine abnormalities may be a cause for failure of IVF-ET or GIFT and therefore hysteroscopy should be part of the infertility work-up of all patients prior to undergoing IVF treatment.

Abortion, Spontaneous↗
PubMed1990

Reevaluation of the C3a active site using short synthetic C3a analogues.

In 1980 the C-terminal pentapeptide LGLAR (C3a 73-77) was described (Caporale, L. H. et al. J. Biol. Chem. 1980, 255: 10758) as the minimal sequence inducing a C3a-specific activity. We have synthesized C3a-analogue peptides connected to non-peptidic acyl residues known to potentiate biological activity. Starting from the acylated hexapeptide fluorenylmethoxycarbonyl(Fmoc)-aminohexanoyl(Ahx)-ALGLAR+ ++, a related series of shorter peptides was synthesized. C3a-specific activity was measured as ATP release from guinea pig platelets. Even the tripeptide LAR, acylated with Fmoc-Ahx, exhibited C3a-specific activity. With 0.34% C3a activity, it was even more potent than the native LGLAR sequence which has 0.01% activity. N-terminal extension of the acylated tripeptide LAR by adding one to three alanines increased activity tenfold up to 3.26% (Fmoc-Ahx-AAALAR), while N-terminal addition of three glycine residues (Fmoc-Ahx-GGGLAR) only increased activity to 0.83% of native C3a. Furthermore, a stimulus-specific desensitization could be observed. Fmoc-Ahx-R and Fmoc-Ahx-AR exhibited neither activity nor desensitizing capacity, but the addition of four alanines to the dipeptide AR led to a sequence (Fmoc-Ahx-AAAAAR) with a C3a-specific activity of 0.14%. Even arginine prolonged N-terminally with five glycines (Fmoc-Ahx-GGGGGR) exhibited some C3a-specific activity so that for biological activity only the appropriate presentation of arginine seems to be essential.

Adenosine Triphosphate↗
PubMed1990

Analysis of the local and systemic immune responses induced in BALB/c mice by experimental respiratory syncytial virus infection.

Pulmonary A2 strain respiratory syncytial virus infection of BALB/c laboratory mice persisted for up to 7 days after initial infection with peak virus titres being recovered on day 4. Virus antigen within the lungs was found to be restricted essentially to the alveolar regions. Similarly, pulmonary histopathological changes remained confined to the peri-alveolar regions being consistent with mild pneumonia. Infection was found to elicit a pulmonary major histocompatibility complex-restricted cytotoxic T lymphocyte (CTL) response which was first detectable 6 days after infection and optimal 7 to 9 days after infection. This local CTL response was preceded by a rapid transient virus-specific lymphocyte transformation response which was detectable only 3 days after intranasal infection. In addition, infection induced rapid interferon production within the lungs which was accompanied by an equally rapid rise in pulmonary natural killer (NK) cell cytotoxic activity. Enhanced NK cell cytotoxicity could be detected after only 1 day post-infection and continued to rise to maximum levels on day 3. This response like the acute CTL response was found to be restricted to the lower respiratory tract. IgG was the first class of virus-specific immunoglobulin to be detected in the lungs of infected animals after experimental infection. However, IgG was not detected until day 10 post-infection, 5 days after the initial decline of virus shedding. Virus-specific IgA although detectable did not appear in the lung until day 24.

Animals↗
PubMed1989

Inactivation of dopamine beta-hydroxylase by beta-ethynyltyramine: kinetic characterization and covalent modification of an active site peptide.

beta-Ethynyltyramine has been shown to be a potent, mechanism-based inhibitor of dopamine beta-hydroxylase (DBH). This is evidenced by pseudo-first-order, time-dependent inactivation of enzyme, a dependence of inactivation on the presence of ascorbate and oxygen cosubstrates, the ability of tyramine (substrate) and 1-(3,5-difluoro-4-hydroxybenzyl)imidazole-2-thione (competitive multisubstrate inhibitor) to protect against inactivation, and a high affinity of beta-ethynyltyramine for enzyme. Inactivation of DBH by beta-ethynyltyramine is accompanied by stoichiometric, covalent modification of the enzyme. Analysis of the tryptic map following inactivation by [3H]-beta-ethynyltyramine reveals that the radiolabel is associated with a single, 25 amino acid peptide. The sequence of the modified peptide is shown to be Cys-Thr-Gln-Leu-Ala-Leu-Pro-Ala-Ser-Gly-Ile-His-Ile-Phe-Ala-Ser-Gln-Leu- His*- Thr-His-Leu-Thr-Gly-Arg, where His* corresponds to a covalently modified histidine residue. In studies using the separated enantiomers of beta-ethynyltyramine, we have found the R enantiomer to be a reversible, competitive inhibitor versus tyramine substrate with a Ki of 7.9 +/- 0.3 microM. The S enantiomer, while also being a competitive inhibitor (Ki = 33.9 +/- 1.4 microM), is hydroxylated by DBH to give the expected beta-ethynyloctopamine product and also efficiently inactivates the enzyme [kinact(app) = 0.18 +/- 0.02 min-1; KI(app) = 57 +/- 8 microM]. The partition ratio for this process is very low and has been estimated to be about 2.5. This establishes an approximate value for kcat of 0.45 min(-1) and reveals that (S)-beta-ethynyltyramine undergoes a slow turnover relative to that of tyramine (kcat approximately 50 s(-1), despite the nearly 100-fold higher affinity of the inactivator for enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Photoaffinity labeling of the lumenal K+ site of the gastric (H+ + K+)-ATPase.

A photoaffinity label for the lumenal K+ site of the gastric (H+ + K+)-ATPase has been identified. Seven azido derivatives based upon the reversible K+ site inhibitor SCH 28080 were studied, one of which, m-ATIP (8-(3-azidophenylmethoxy)-1,2,3-trimethylimidazo[1,2-a] pyridinium iodide), was subsequently synthesized in radiolabeled form. In the absence of UV irradiation, m-ATIP inhibited K+ -stimulated ATPase activity in lyophilized gastric vesicles competitively with respect to K+, with a Ki value of 2.4 microM at pH 7.0. Irradiation of lyophilized gastric vesicles at pH 7.0 with [14C]m-ATIP in the presence of 0.2 mM ATP resulted in a time-dependent inactivation of ATPase activity that was associated with an incorporation of radioactivity into a 100-kDa polypeptide representing the catalytic subunit of the (H+ + K+)-ATPase. Both inactivation and incorporation were blocked in the presence of 10 mM KCl but not with 10 mM NaCl, consistent with interaction at the K+ site. The level of incorporation required to produce complete inhibition of ATPase activity was 1.9 +/- 0.2 times the number of catalytic phosphorylation sites in the same preparation. Tryptic digestion of gastric vesicle membranes, labeled with [14C]m-ATIP, failed to release the radioactivity from the membranes suggesting that the site of interaction was close to or within the membrane-spanning sections of this ion pump.

Affinity Labels↗
PubMed1989

Characterization of C3a receptor-proteins on guinea pig platelets and human polymorphonuclear leukocytes.

The expression of specific membrane receptors for C3a was determined on guinea pig C3a-sensitive (gp R+) platelets and human polymorphonuclear leukocytes (hu PMNL). Binding studies with 125I-labeled C3a from gp or hu sources and Scatchard analysis applied to the binding data revealed the existence of two receptor classes on gp R+ platelets; a high-affinity class with about 200 binding sites/cell and Kd = 1.7 x 10(-9) M, and a relatively low-affinity class with Kd = 10(-8) M and about 500 sites/cell. Hu PMNL express a homogeneous receptor class with Kd = 3 x 10(-8) M and 40,000 sites/cell. Molecular characterization of the C3a receptor on gp R+ platelets was achieved by (a) cross-linking photoaffinity-labeled receptors to bound 125I-labeled C3a; (b) photoaffinity labeling receptors with a 13-amino acid residue C3a analogue 125I-Nap-Ahx-13; and (c) use of chemical cross-linkers like disuccinimidylsuberate to cross-link receptors with 125I-C3a. All three techniques gave rise to very similar labeling patterns. With the photoaffinity labeling methods, a diffuse band pattern was observed with an apparent molecular mass of 95-123 kDa with 125I-C3a as label, and 85-105 kDa with 125I-Nap-Ahx-13 as label. Chemical cross-linking of 125I-C3a revealed three distinct bands with molecular masses of approximately 123, 108 and 95 kDa. Subtracting the contribution of the cross-linked ligands, the C3a receptor on gp R+ platelets appears to be a protein complex, consisting of one to three components with estimated molecular masses between 83-114 kDa.

Adenosine Triphosphate↗