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D Chapman

Publications and source records attributed to D Chapman.

At least 73 records · Page 4Linked to original sources
PubMed1994

The conformational analysis of a synthetic S4 peptide corresponding to a voltage-gated potassium ion channel protein.

The S4 region of the Drosophila Shaker voltage-gated K+ channel has been proposed to function as a voltage-sensor. We have synthesised a peptide corresponding to this S4 region. Structural studies on the S4 peptide were conducted using Fourier transform infrared (FTIR) spectroscopy. Spectra were obtained for the peptide dissolved in aqueous solution, in trifluoroethanol solvent and also after reconstitution into lipid bilayers and micelles. The peptide in trifluoroethanol adopts an alpha-helical conformation which is in good agreement with the results of a recent 2D NMR study on the structure of a S4 peptide corresponding to the rat brain sodium channel [(1989) FEBS Lett. 257, 113-117]. A predominantly alpha-helical structure is also observed when the S4 peptide is present in aqueous lysophosphatidylcholine micelles, in dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol lipid bilayers. In contrast to this, the S4 peptide in aqueous solution is in a random coil conformation. The coil-to-helix transition observed for the S4 peptide upon its transfer from aqueous solution to lipid membrane indicates that it has a high degree of conformational flexibility and can undergo large changes in its structure in response to its environment. This may have important implications for its role in the voltage activation process during which the S4 peptide has been postulated to, at least partially, move from a lipid bilayer to an aqueous extracellular media [(1992) Biophys J. 62, 238-250]. The results of our study lend support to such a model.

Amino Acid Sequence↗
PubMed1994

Compass: predicting biological activities from molecular surface properties. Performance comparisons on a steroid benchmark.

We describe a new method, Compass, for predicting the biological activities of molecules based on the activities and three-dimensional structures of other molecules. The method improves on previous techniques by representing only the surface of molecules, by incorporating a nonlinear statistical method, and by automatically choosing conformations and alignments of molecules. We use a benchmark problem of steroid binding affinity prediction to compare the performance of the method with that of two previous systems: CoMFA and a molecular similarity method. Compass predicts steroid affinities substantially more accurately than the others, which represent the state of the art. We present experiments showing that the improved performance depends on each of the technical innovations.

Algorithms↗
PubMed1994

Beta-sheet secondary structure of the trimeric globular domain of C1q of complement and collagen types VIII and X by Fourier-transform infrared spectroscopy and averaged structure predictions.

C1q plays a key role in the recognition of immune complexes, thereby initiating the classical pathway of complement activation. Although the triple-helix conformation of its N-terminal segment is well established, the secondary structure of the trimeric globular C-terminal domain is as yet unknown. The secondary structures of human C1q and C1q stalks and pepsin-extracted human collagen types I, III and IV (with no significant non-collagen-like structure) were studied by Fourier-transform i.r. spectroscopy in 2H2O buffers. After second-derivative calculation to resolve the fine structure of the broad amide I band, the Fourier-transform i.r. spectrum of C1q showed two major bands, one at 1637 cm-1, which is a characteristic frequency for beta-sheets, and one at 1661 cm-1. Both major bands were also detected for Clq in H2O buffers. Only the second major band was observed at 1655 cm-1 in pepsin-digested C1q which contains primarily the N-terminal triple-helix region. The Fourier-transform i.r. spectra of collagen in 2H2O also showed a major band at 1659 cm-1 (and minor bands at 1632 cm-1 and 1682 cm-1). It is concluded that the C1q globular heads contain primarily beta-sheet structure. The C-terminal domains of C1q show approximately 25% sequence identity with the non-collagen-like C-terminal regions of the short-chain collagen types VIII and X. To complement the Fourier-transform-i.r. spectroscopic data, averaged Robson and Chou-Fasman structure predictions on 15 similar sequences for the globular domains of C1q and collagen types VIII and X were performed. These showed a clear pattern of ten beta-strands interspersed by beta-turns and /or loops. Residues thought to be important for C1q-immune complex interactions with IgG and IgM were predicted to be at a surface-exposed loop. Sequence insertions and deletions, glycosylation sites, the free cysteine residue and RGD recognition sequences were also predicted to be at surface-exposed positions.

Amino Acid Sequence↗
PubMed1994

The secondary structure of the von Willebrand factor type A domain in factor B of human complement by Fourier transform infrared spectroscopy. Its occurrence in collagen types VI, VII, XII and XIV, the integrins and other proteins by averaged structure predictions.

The type A domain of the von Willebrand Factor is found also in the complement proteins factor B (FB), C2, CR3 and CR4, the integrins, collagen types VI, VII, XII and XIV, and other proteins. FB is a component of the alternative pathway of the complement system of immune defence, and is cleaved into the fragments Bb and Ba during complement activation. Bb contains a von Willebrand Factor type A (vWF) domain of unknown secondary structure and a serine proteinase (SP) domain, whereas Ba contains three short consensus repeat/complement control protein (SCR/CCP) domains. Fourier transform infrared (FT-IR) spectroscopy on a recombinant vWF domain and on FB and its Bb and Ba fragments shows a broad amide I band. In H2O buffer, second derivative spectra of the amide I band show subcomponents at 1654 to 1657 cm-1, which is typical of alpha-helix, and at 1676 to 1685 cm-1 and 1636 to 1637 cm-1, which are typical of beta-strand. alpha-Helix was detected in the vWF domain, the Bb fragment and FB, and the proportion of alpha-helix present decreased in that order. This shows that the vWF domain contains appreciable amounts of alpha-helix, while the SP and SCR/CCP domains are almost entirely beta-sheet in their secondary structures. Quantitative integration of the vWF FT-IR spectrum showed that this contained 31% alpha-helix and 36% beta-sheet. In 2H2O buffer, the alpha-helix content in the vWF domain is sensitive to the solvent, while the beta-sheet content is less so. An alignment of 75 vWF type A sequences from 25 proteins was used for averaged secondary structure predictions of the total length of 206 residues by the Robson and Chou-Fasman methods. In support of the FT-IR analysis, a total of at least five well-predicted alpha-helices (35% of residues) and at least five well-predicted beta-strands (21% of residues) were identified by both predictive methods, all of which were interspersed by regions of coil or turn conformations. Eight of the ten predicted alpha-helices and beta-strands form an alternating arrangement with each other. Since the predicted alpha-helices are mostly amphipathic, and since the alpha-helix FT-IR band is sensitive to solvent, the alpha-helices are inferred to be on the protein surface.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Fourier transform infrared spectroscopy and differential scanning calorimetry of transferrins: human serum transferrin, rabbit serum transferrin and human lactoferrin.

Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) have been used to investigate the solution structure and thermal stability of human serum transferrin (HST), human lactoferrin (HLF) and rabbit serum transferrin (RST) in their diferric and apo forms. Our study shows that: (A) The secondary structure of all the proteins studied (estimated in H2O) was in the range 43-53% alpha-helix and 23-28% beta-sheet. These values differ markedly from previously reported circular dichroism (CD) data. This is attributed to the fact that FTIR and CD measure different aspects of secondary structure (hydrogen bonding and dihedral angles, respectively). (B) The secondary structural content of the proteins is not altered by iron binding or release. However, the iron-free proteins undergo a greater extent of 1H-2H exchange than the diferric proteins indicating that significant structural changes do occur upon iron binding/release. (C) The removal of iron leads to thermal destabilization of HST, HLF and RST. Structural variation in the apo transferrins is indicated by the observation of a single irreversible DSC transition for apo human lactoferrin, a double DSC transition for apo human serum transferrin (one reversible) and a broad irreversible asymmetric DSC transition for apo rabbit serum transferrin. FTIR spectroscopy shows that a distinct loss of protein secondary structure occurs at the transition temperatures shown by DSC.

Animals↗
PubMed1994

Hepatic arterial floxuridine and leucovorin for unresectable liver metastases from colorectal carcinoma. New dose schedules and survival update.

BACKGROUND: We studied three new dose schedules of hepatic arterial infusion of floxuridine (FUDR) and leucovorin and update survival analysis of a previously reported trial using these drugs by hepatic arterial infusion for patients with hepatic metastases from colorectal carcinoma. METHODS: Untreated patients with hepatic metastases from colorectal cancer were treated with three dose schedules: Group D, FUDR (0.3 mg/kg/day) and leucovorin (30 mg/m2/day) as a 14-day continuous infusion through an implantable hepatic arterial pump alternating with a 4-week rest period; Group E, a lower dose of FUDR (0.25 mg/kg/day) and leucovorin (30 mg/m2/day) as a 14-day infusion alternating with 2 weeks of saline; and Group F, FUDR (0.3 mg/kg/day) with a lower leucovorin dose (15 mg/m2/day) for 2 weeks followed by a 2-week rest. RESULTS: In 42 patients with unresectable hepatic metastases, the complete-plus-partial response rate was 56%, with a median survival of 24.2 months. Complete-plus-partial response rates for groups D, E, and F were 30%, 54%, and 75%, respectively. Twelve percent of the 42 patients developed biliary sclerosis; the percentages of patients per group were 17%, 15%, and 6%, respectively. Updated median survival of the original 24 patients treated with FUDR and leucovorin by hepatic arterial infusion and these 42 new patients (66 total) was 28.8 months. One-, two-, three-, four-, and five-year survival rates were 86%, 62%, 31%, 15%, and 7%, respectively. CONCLUSIONS: Hepatic arterial chemotherapy with FUDR and leucovorin for patients with hepatic metastases from colorectal carcinoma yields a high response rate and 1- and 2-year survivals of 86% and 62%, respectively. Although a lower dose of leucovorin (15 mg/m2) with FUDR produces a high response rate with less toxicity, before larger scale trials are initiated, further investigation is needed to reduce toxicity. A study of hepatic arterial dexamethasone with FUDR and leucovorin has been initiated for this purpose.

Adult↗
PubMed1994

A shape-based machine learning tool for drug design.

Building predictive models for iterative drug design in the absence of a known target protein structure is an important challenge. We present a novel technique, Compass, that removes a major obstacle to accurate prediction by automatically selecting conformations and alignments of molecules without the benefit of a characterized active site. The technique combines explicit representation of molecular shape with neural network learning methods to produce highly predictive models, even across chemically distinct classes of molecules. We apply the method to predicting human perception of musk odor and show how the resulting models can provide graphical guidance for chemical modifications.

Algorithms↗
PubMed1994

The conformational equilibria of a renin inhibitor peptide in solution.

The conformational equilibrium of a decapeptide renin inhibitor (Renin Inhibitory Peptide (RIP), NH-P-H-P-F-H-F-F-V-Y-K-CO2H) in water, methanol and trifluoroethanol has been investigated. The value of a combined spectroscopic approach was apparent, with the need to define conformational states that were mixtures of conformational forms. Similarities between this study and that of the Melanin Concentrating Hormone (MCH) core peptide (5-14) are notable [1]. In water, two beta-turn conformations and an extended form were found to be in equilibrium, with cis/trans isomerism at Pro-3. Extended conformations associated with the P(II) helix and irregular forms were more favoured in aqueous environments. In MeOH and TFE, two beta-turn conformations associated with overlapping sequences and cis/trans isomerism at Pro-3 amide bond were seen to be in equilibrium. 2D ROESY and chemical-exchange cross-peaks were detected by 1H NMR and used to build up detailed models of the interconverting beta-turn conformations of RIP.

Journal Article↗
PubMed1994

Studies of the pore-forming domain of a voltage-gated potassium channel protein.

Recent mutagenesis studies have identified a stretch of amino acid residues which form the ion-selective pore of the voltage-gated potassium channel. It has been suggested that this sequence of amino acids forms a beta-barrel structure making up the structure of the ion-selective pore [Hartman, H.A., Kirsch, G.E., Drewe, J.A., Taglialatela, M., Joho, R.H. and Brown, A.M. (1991) Science, 251, 942-944; Yellen, G., Jurman, M.E., Abramson, T. and MacKinnon, R. (1991) Science, 251, 939-942; Yool, A.J. and Schwarz, T.L. (1991) Nature, 349, 700-704]. We have synthesized a polypeptide corresponding to this amino acid sequence (residues 431-449 of the ShA potassium channel from Drosophila). A tetrameric version of this sequence was also synthesized by linking together four of these peptides onto a branching lysine core. Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy have been used to investigate the structure of these peptides after their reconstitution into lyso phosphatidylcholine micelles and lipid bilayers composed of dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol. The spectroscopic studies show that these peptides are predominantly alpha-helical in these lipid environments. When incorporated into planar lipid bilayers both peptides induce ion channel activity. Molecular modelling studies based upon the propensity of these peptides to form an alpha-helical secondary structure in a hydrophobic environment are described. These results are discussed in the light of recent mutagenesis and binding studies of the Drosophila Shaker potassium ion channel protein.

Amino Acid Sequence↗
PubMed1994

A molecular technique for identification of bacteria using small subunit ribosomal RNA sequences.

We have recently developed a novel molecular technique for identification of specific bacterial species within a complex mixture. The technique uses PCR to amplify small subunit ribosomal RNA (SSU rRNA) genes from a mixture of bacteria. One of the PCR primers is labeled with a fluorescent dye to allow detection of the amplified product. The PCR product is then digested with restriction enzymes and a capillary electrophoresis unit equipped with a laser-induced fluorescence detector is employed to analyze the restriction fragments. Only restriction fragments that contain the fluorescent-labeled primer are detected. Generally, the nucleotide sequence of the SSU rRNA genes is unique for each bacterial species. Consequently, the fluorescent-labeled restriction fragments from different bacterial species often have characteristic lengths. Thus, the different fluorescent peaks that appear in a capillary electropherogram correspond to labeled restriction fragments from different bacterial species. This protocol allows us to identify a number of different bacterial species in a complex mixture. Only a minute sample of bacterial DNA and a minimal amount of time (8-10 h) are required for this analysis. The protocol is sensitive, rapid and capable of identifying a broad spectrum of bacterial species.

Bacteria↗
PubMed1993

Secondary structure of M13 coat protein in phospholipids studied by circular dichroism, Raman, and Fourier transform infrared spectroscopy.

There is considerable uncertainty about the precise secondary structure adopted by the M13 coat protein when embedded in a phospholipid bilayer. Circular dichroism (CD) spectroscopy suggests that a major change in the structure of the coat protein occurs upon membrane insertion. It is reported that the structure of the protein in the membrane has only about 50% alpha-helix, the rest being mainly in a beta-sheet conformation, whereas the protein is almost completely alpha-helical when intact in the phage. In this study we have undertaken a spectroscopic analysis using Fourier transform infrared, Raman, and CD spectroscopy to characterize the secondary structure of M13 coat protein when present in membranes consisting of dioleoylphosphatidylglycerol and dimyristoylphosphatidylglycerol. In sharp contrast to earlier CD studies, our results indicate that the coat protein in its membrane-embedded state has a very high alpha-helical content with virtually no beta-sheet structures present. This result indicates that the structures of the coat protein when intact in the phage or when embedded in the membrane are similar. Although our results differ from earlier CD studies, they are consistent with a recent NMR study, which showed that the M13 coat protein in sodium dodecyl sulfate micelles is primarily alpha-helical with no evidence for beta-sheet structure [Henry, G. D., & Sykes, B.D. (1992) Biochemistry 31, 5284-5297]. These results lead to the conclusion that the M13 coat protein can insert from the membrane-bound state into a virus particle with a similar secondary structure, without large energy implications.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage M13↗
PubMed1993

Protein secondary structure from Fourier transform infrared and/or circular dichroism spectra.

A multivariate linear model (Gauss-Markoff model) with noise is used to analyze the estimation of protein secondary structure from spectra of 21 reference proteins whose structures are known from X-ray studies. Fourier transform infrared (FTIR) spectra from 1700 to 1500 cm-1 and circular dichroism (CD) spectra from 178 to 260 nm have been used. The secondary structure categories of interest are alpha-helix, antiparallel beta-sheets, parallel beta-sheets, beta-turns, and "other". The secondary structures are predicted from separate spectra as well as from combined FTIR and CD spectra. The characteristic spectra belonging to the secondary structures and the prediction errors are also estimated. Attention has been paid to the criteria for the choice of rank of matrices of reference spectra, which corresponds to the number of independent pieces of spectral information. Criteria used are: magnitudes of singular values, root mean square error of model fit, relative error of estimable parameters and errors in predicted secondary structure. The ranks of the spectral matrices are found to be between three and six. The model accuracy is determined by removing each protein from the sample and comparing predicted and X-ray values of secondary structure. It is concluded that the linear model is more adequate for the protein FTIR spectra than for the CD spectra. Secondary structure predictions using the FTIR amide I band (1700-1600 cm-1) and the FTIR amide II band (1600-1500 cm-1), or a combination of the two, are of comparable accuracy. In particular, antiparallel beta-sheets and "other" are more reliably estimated from FTIR spectra. However, alpha-helix is more reliably estimated from CD spectra. Combining the spectra yields the best results of both techniques for each class.

Circular Dichroism↗
PubMed1993

Biophysical studies of the Pf1 coat protein in the filamentous phage, in detergent micelles, and in a membrane environment.

During the assembly of the Pf1 phage, the membrane-bound coat proteins convert into subunits of the filamentous phage. Fourier-transform infrared (FT-IR) transmission spectroscopy has been applied to a study of the secondary structure of these coat proteins when present (a) in the phage, (b) in detergent micelles, and (c) in a phospholipid membrane aqueous system. Suspensions of the Pf1 phage in H2O and 2H2O show an amide I band at 1652 cm-1, indicative of a high content of the alpha-helical structure present. Oriented films of the Pf1 phage studied by polarized FT-IR transmission spectroscopy indicate that the alpha-helical structures as well as the tyrosine residues (band at 1515 cm-1) are both aligned along the axis of the phage. When the Pf1 coat protein is present in sodium dodecyl sulfate detergent micelles and in lipid membrane systems, the FT-IR spectra show an amide I band at 1657-1658 cm-1, also indicative of a predominantly alpha-helical secondary structure. H/2H amide proton exchange studies show that when present in a phospholipid membrane system some 50-60% of the Pf1 protein exchanges rapidly, while the rest undergoes slow exchange. This is consistent with a proportion of the protein being exposed to the solvent and the other being embedded in the lipid bilayer. The presence of a band at 1630-1640 cm-1 is indicative of the presence of random structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗
PubMed1993

A pilot study of hepatic artery floxuridine combined with systemic 5-fluorouracil and leucovorin. A potential adjuvant program after resection of colorectal hepatic metastases.

BACKGROUND: Most patients with colorectal carcinoma metastatic to the liver have relapses after surgical resection of hepatic metastases with failures divided equally between hepatic and extrahepatic sites. A pilot study was begun using a regimen combining intrahepatic floxuridine (FUDR) and systemic 5-fluorouracil (5-FU) and leucovorin (LV) to determine its safety and efficacy. METHODS: Because this was a pilot study, 21 patients with unresectable hepatic metastases from colorectal carcinoma were treated to assess the regimen's toxicity. Eight patients had liver metastases that were resected completely; then they received treatment. FUDR was given by hepatic arterial pump through a 14-day continuous infusion at 0.25 mg/kg/day. Systemic therapy consisted of LV 200 mg/m2 and 5-FU 280 mg/m2 using a bolus dose of 5-FU for 5 days with escalation of the 5-FU dose in separate patient cohorts. The maximally tolerated 5-FU dose was 325 mg/m2. RESULTS: The median survival in the 21 unresectable patients was 16 months with a partial response rate of 56% (10 of 18 evaluable patients; 95% confidence interval, 38-79%). The major systemic toxicity was diarrhea, Grade 3 or 4, in 54% of patients being treated in the 4-week regimen and 19%, in the 5-week regimen. The level of hepatic toxicity was similar to that in previous studies using intrahepatic chemotherapy alone, i.e., 48% of patients had a 200% increase in alkaline phosphatase levels and 10% had bilirubin elevations of more than 3.0 mg/dl (one patient had documented biliary sclerosis). All eight patients treated with adjuvant therapy were alive without disease after a median follow-up of 23 months. CONCLUSIONS: Systemic 5-FU and LV can be combined safely with intraarterial FUDR without loss of efficacy or increased biliary toxicity. Eight patients treated with this regimen as adjuvant therapy after liver metastasis resection were alive and disease-free after a median follow-up of 23 months.

Adult↗
PubMed1993

Intraportal injection of monoclonal antibody in nude mice bearing hepatic metastases.

Using a model for hepatic human colorectal carcinoma metastases in athymic mice, we compared the selective [intraportal (ip)] and systemic [intravenous (iv)] injection of radiolabeled monoclonal antibody (mAb) strongly reactive against the cell line. Percent injected dose of radiolabeled antibody per gram (%id/g) of tumor or normal tissues was measured at selected time points (up to 5 days postinjection) within 3 dose levels: 0.1, 1.0, and 2.0 micrograms (micrograms). At each dose level, 3-9 animals were studied in each of 3 groups: animals receiving ip injection (group HT-29-15 ip), those receiving intravenous injection (group HT-29-15 iv), and those receiving isotype-matched control antibody via the intraportal route (group BL-3 ip). Significantly greater (P < 0.005) %id/g in tumor was seen in group HT-29-15 ip at all time points and dose levels compared to those in groups HT-29-15 iv or BL-3 ip. However, immediately after injection of mAb, there was no difference in tumor %id/g between groups HT-29-15 ip and HT-29-15 iv at the highest dose level. There was no increase in %id/g of mAb in normal liver and blood after ip injection compared to iv injection beyond day 1. Therefore ip injection resulted in higher tumor to liver and tumor to blood ratios compared to iv (P < 0.005). We conclude that delivery of mAb to hepatic metastases can be enhanced by selective injection; this has important implications in the design of future clinical trials utilizing radiolabeled mAb in the diagnosis and treatment of hepatic metastases.

Analysis of Variance↗
PubMed1993

Limited-field radiotherapy as salvage treatment of localized persistent or recurrent epithelial ovarian cancer.

Thirty-five patients with persistent or recurrent epithelial ovarian cancer were treated with salvage pelvic or para-aortic radiotherapy for disease limited to the pelvis (29), para-aortic retroperitoneum (5), or vaginal cuff (1). Prior therapy included a median of 3 chemotherapeutic drugs (range, 1-7) over a median of 12 cycles (range, 5-39); 12 patients had received intraperitoneal chemotherapy. The median number of prior laparotomies was 3 (range, 2-5). Including pelvic boosts, the median dose delivered to the treatment field was 4600 cGy (range 4000-7000); 2 patients received additional treatment with a permanent 125I implant. All patients completed therapy, although 5 (14%) with grade 3 toxicity required a treatment break. Late bowel complications unrelated to recurrence occurred in 3 patients (9%). Median actuarial and progression-free survivals for all patients from start of radiotherapy were 40 and 14 months, respectively. At least 16 of 26 (62%) recurrences involved the treatment field. Multiple prognostic factors were evaluated in terms of recurrence, survival, and acute and chronic complications, but no significant prognosticators were detected. These findings indicate that limited-field salvage radiotherapy has an acceptable complication rate and may prolong the symptom-free survival interval in selected patients.

Adult↗