A model of Plasmodium falciparum lactate dehydrogenase and its implications for the design of improved antimalarials and the enhanced detection of parasitaemia.
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Amino Acid Sequence↗
Biomedical subjects
R B Sessions
Publications and source records attributed to R B Sessions.
Correlation of the enzyme activities of Bacillus stearothermophilus lactate dehydrogenase on three substrates with the results of molecular dynamics/energy minimization conformational searching.
Current methods for reengineering enzyme substrate specificities rely heavily on the use of static x-ray crystallographic models. In this article we detail the use of a molecular mechanics approach for suggesting regions of Bacillus stearothermophilus L-lactate dehydrogenase (EC 1.1.1.27) involved in substrate specificity, and hence areas of interest for protein engineers. The approach combines molecular dynamics with energy minimization (MD/EM) to search the conformational space available to a 15-A sphere of the ternary complex centered on the catalytic histidine. The search is carried out by calculating a 30-ps dynamics trajectory at 300 K and minimizing structures at 1-ps intervals. The protocol has been performed on 14 systems containing different combinations of substrate and mutant/wt LDH. In order to discover which interactions are important in defining substrate specificity, eight conformational parameters representing substrate-active site interactions were measured in each of the 420 minimized structures. These parameters were then compared to the measured catalytic activity of the protein-substrate combinations. These comparisons show that arginine 109 orientation is a major determining factor in LDH specificity. Using this methodology it is possible to estimate the catalytic activity of proteins of varied sequence by computer simulation before synthesis.
Catalysis↗
Removal of substrate inhibition in a lactate dehydrogenase from human muscle by a single residue change.
High concentrations of ketoacid substrates inhibit most natural hydroxyacid dehydrogenases due to the formation of an abortive enzyme-NAD+-ketoacid complex. It was postulated that this substrate inhibition could be eliminated from lactate dehydrogenases if the rate of NAD+ dissociation could be increased. An analysis of the crystal structure of mammalian LDHs showed that the amide of the nicotinamide cofactor formed a water-bridged hydrogen bond to S163. The LDH of Plasmodium falciparum is not inhibited by its substrate and, uniquely, in this enzyme the serine is replaced by a leucine. In the S163L mutant of human LDH-M4 pyruvate inhibition is, indeed, abolished and the enzyme retains high activity. However, the major contribution to this effect comes from a weakening of the interaction of pyruvate with the enzyme-coenzyme complex.
Humans↗
Guided evolution of enzymes with new substrate specificities.
A gene library was constructed coding for all possible variants of two amino acids (101, 102) in a solvent-exposed surface return loop (alpha E-beta D) of Bacillus stearothermophilus L-lactate dehydrogenase (bsLDH). All but one of 38 enzyme variants examined were thermally stable and had native-like hydrodynamic properties. In this sample, there was no bias detected in either the DNA or amino acid sequences encoded. We argue that the alpha E-beta D surface loop sequence is unimportant for protein folding or stability and can be fully varied to select enzymes with new substrate specificities. The selection of NAD-dependent dehydrogenases with specificity for: malate, phenyllactate, hydroxyisocaproate and 4-phenyl-2-hydroxy-butanoate from two bsLDH libraries is described. This required a highly discriminatory screen for 2-hydroxy acid dehydrogenase activity to select enzymes which, in the absence of the natural allosteric activator fructose-1,6-bisphosphate (FBP), maintained high temperature stability and catalytic activity without substrate inhibition. In general the amino acid residues at positions 101 and 102 which determined substrate specificity were as expected from hydrophobic and ionic complementarity to the substrate. For example, a bsLDH variant with Asn101Va1102 is as efficient with phenylpyruvate as is the wild-type enzyme (Asn101Gln102) with pyruvate. Using molecular modelling, the valine at position 102 can be fitted into the active site without significant structural distortion caused by the aromatic side-chain of the substrate. Similarly, nine out of ten malate dehydrogenases (MDHs) selected had an arginine residue at position 102 to complement the negatively charged carboxyl group in oxaloacetate. One, Arg101Arg102 (Kcat/Kmoxaloacetate = 1.6 x 10(6) M-1 S-1) is 25% more active than the previous best synthetic MDH. There were surprises: present understanding would not have predicted the oxaloacetate transforming activity of Ser101Leu102 or the phenylpyruvate activity of Pro101Lys102. The former is about one-third as efficient as the best malate dehydrogenase selected, whilst the latter had about one-seventh of the best phenylpyruvate dehydrogenase activity.
Amino Acid Sequence↗
Minimizing pectoralis myocutaneous flap loss with the delay principle.
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Aged↗
The development of tertiary interactions during the folding of a large protein.
BACKGROUND: We have used protein engineering and relaxation kinetics to examine the order in which secondary structure elements assemble during folding. Aliphatic contacts in the core of a large domain within the monomeric protein phosphoglycerate kinase (PGK) were disrupted in order to map the development of interactions between beta-strand and alpha-helix residues, both near and distant in the sequence. RESULTS: Mutations which break sequence-local alpha-beta contacts destabilize the first identifiable intermediate in folding, showing that these contacts develop early in the folding pathway. In contrast, the removal of sequence-distant alpha-beta interactions has little effect at this stage, but reduces the rate at which the intermediate converts to the native state. Thus, contacts between these remote segments of secondary structure start to form later on in the process, during the rate-limiting transition. CONCLUSIONS: In the case of this large protein domain, our results support the hypothesis that folding proceeds by a hierarchic pathway. Interactions form rapidly between sequence-local groups to produce microdomains before the establishment of the long-range contacts necessary to define the global fold, which proceeds through a highly hydrated transition state.
Circular Dichroism↗
Comparison of Tc-99m MIBI and TI-201/Tc-99m pertechnetate for diagnosis of primary hyperparathyroidism.
The purpose of this study was to directly compare the efficacy of Tc-99m MIBI with TI-201/Tc-99m pertechnetate scintigraphy for localizing parathyroid adenomas in patients with primary hyperparathyroidism. Of 21 patients suspected of having primary hyperparathyroidism who had undergone both studies, 14 had subsequent surgery and pathologic correlation. Based on the surgical and pathologic results, Tc-99m MIBI yielded 13 true-positive and 1 false-negative result. Thallium-201/ Tc-99m pertechnetate imaging yielded eight true-positive, four false-negative, and two nondiagnostic studies because of lack of Tc-99m pertechnetate uptake from thyroid hormone suppression. In conclusion, Tc-99m MIBI scintigraphy is an accurate method for localizing parathyroid adenomas in patients with primary hyperparathyroidism and the results suggest that it is superior to using TI-201/Tc-99m pertechnetate.
Adenoma↗
Helical (spiral) CT of the upper airway with three-dimensional imaging: technique and clinical assessment.
OBJECTIVE: The purpose of this study was to evaluate the application of helical CT-generated three-dimensional images of the upper airway. MATERIALS AND METHODS: Thirty patients, 10 healthy and 20 with upper-airway disease, were studied with helical CT (5-mm collimation). Overlapping images at 2-mm intervals were retrospectively generated. In the group of healthy patients, two radiologists in independently compared overlapping with nonoverlapping images, ranked confidence in identifying small airway structures on a scale of 1-5, and tabulated the number of images demonstrating these structures. In the 20 patients with disease, three-dimensional (3D) surface models were rendered on an independent workstation and were reviewed by two radiologists and one otolaryngologist for image quality, appreciation of lesion morphology, and ability to judge lesion extent, using a similar scale. A phantom was used to optimize parameters for the 3D reconstructions. RESULTS: Viewing of the retrospectively generated overlapping images increased by 122% the number of images in which laryngeal and hypopharyngeal structures could be identified (p < .01). Image confidence scores for the radiologists averaged 3.3 for nonoverlapping and 4.0 for overlapping (p < .05). Radiologists and otolaryngologist rated the quality of the 3D images equally. The otolaryngologist's assessment of the value of the models for understanding the lesion morphology was 3.5 compared with the radiologists assessment of 2.5; and for judging the lesion extent, the otolaryngologist's assessment was 3.8 compared with 2.7 for the radiologist, a statistical significance of p < .01. CONCLUSION: Helical CT with the application of overlapping images and 3D reconstructions significantly assists the understanding of upper-airway disease.
Case-Control Studies↗
Immunohistochemical staining for glutathione S-transferase predicts response to platinum-based chemotherapy in head and neck cancer.
The glutathione S-transferases (GSTs) play an important role in the cell's defense against toxic substances. The GSTs are a family of enzymes produced by several genes that interact with distinct but overlapping substrates and that may play a role in resistance of tumor cells to several chemotherapeutic agents. We examined the correlation between expression of GSTs determined by immunohistochemistry and clinical response to platinum-based chemotherapy in 51 patients with head and neck cancer, who received a total of 56 courses of chemotherapy. The overall response rate for the 56 chemotherapy treatment courses was 48%. The overall response rate (complete response + partial response) for patients with low GST scores was 88% (21 of 24), whereas among the patients with high GST scores, the overall response rate was 19% (6 of 32; P = 0.001). Patients with a low GST score were 4.7 times more likely to respond to chemotherapy than patients with high GST scores. GST scores corresponded to response in 84% of cases. Among 23 patients treated with neoadjuvant chemotherapy, the overall response rate for patients with low GST scores was 100% (14 of 14), whereas among the patients with high GST scores, the overall response rate was 44% (4 of 9; P = 0.002). Among 33 patients treated with chemotherapy for relapsed disease, the overall response rate for patients with low GST scores was 70% (7 of 10), whereas among the patients with high GST scores, the overall response rate was 8.6% (2 of 23; P < 0.001). We conclude that GST expression correlates well with response to platinum-based chemotherapy in head and neck cancer.
Antineoplastic Agents↗
Increase in suprabasilar integrin adhesion molecule expression in human epidermal neoplasms accompanies increased proliferation occurring with immortalization and tumor progression.
In a previous prospective study of 80 patients with squamous cell carcinoma of the upper aerodigestive tract, a progressive increase in expression of the integrin cell adhesion molecule alpha 6 beta 4 in suprabasilar cell layers of the tumor parenchyma was associated with an increase in early recurrence after therapy. In this study, we determined the relationship of the altered expression pattern of the integrin to changes occurring during benign, invasive, or metastatic stages of tumor development. Suprabasilar expression of integrin alpha 6 beta 4 appeared with neoplastic transformation in benign squamous papillomas, but homogeneous expression occurred more frequently in the parenchyma of primary and metastatic squamous cell carcinomas. The variation in the extent of suprabasilar integrin expression among the tumors corresponded to the variation in the population undergoing proliferation as determined by two independent markers of proliferation. Integrin expression was quantified in primary, HPV 16 DNA-immortalized, and v-ki-ras oncogene-transformed keratinocytes, and the pattern of expression was compared with cell cycle progression. Primary keratinocyte lines showed a bimodal distribution of integrin expression, with one population showing decreased integrin expression, cell size, and a block of cell cycle progression consistent with differentiation, whereas another population exhibited high integrin expression and full progression through the cell cycle, consistent with proliferation. HPV-immortalized and v-ki-ras-transformed cell lines undergoing continuous proliferation exhibited uniformly strong integrin expression, which was similar in intensity to that observed in the proliferating population of normal keratinocytes. Similar increases in expression of two additional integrins, alpha 2 beta 1 and alpha 3 beta 1, occurred along with integrin alpha 6 beta 4 in tissue specimens and cell lines derived from neoplasms. Thus, epidermal neoplasms display an increase in a population of cells exhibiting constitutive expression of a repertoire of integrins, which is similar to that found transiently in the acute phase of epidermal wound healing, a physiological response in which hyperproliferation, retention of multiple layers of proliferating cells, and migration occur. The association of a progressive increase in suprabasilar expression of these integrins with early tumor recurrence and advanced neoplasia suggests that constitutive expression and function of the same repertoire of integrins may be advantageous, rather than sufficient, for tumor progression.
Antigens, Surface↗
Structure and dynamics of ion channel polypeptides from amide exchange analysis and dynamics simulations.
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Alamethicin↗
Three-dimensional imaging of the hypopharynx and larynx by means of helical (spiral) computed tomography. Comparison of radiological and otolaryngological evaluation.
A new computed tomography (CT) technology, helical (spiral) CT, allows the entire neck to be imaged in only 30 seconds. Although multiplanar and three-dimensional (3-D) imaging could be performed with conventional CT, the volumetric acquisition provided by helical (spiral) CT allows significantly improved quality and easier reconstruction for more applications. These 3-D models show an airway appearance similar to that obtained with laryngography. Independent review of the 3-D images in 12 patients with lesions by two radiologists and one otolaryngologist was performed to assess 1) image quality, 2) ability to judge lesion extent, and 3) assistance in understanding the lesion compared to that provided by routine axial scans. Rating scores of 1 to 5 were assigned, with 5 representing the best quality or greatest value. The results showed that both groups scored image quality equally: 4.7. Lesion extent for the radiologists was 2.6, while the otolaryngologist's ranking was 3.7 (p < .01). In assisting understanding of lesions versus axial scans, radiologists ranked 3-D images 2.1, while the otolaryngologist ranked them 3.1 (p < .01). In summary, 3-D models provide a complementary imaging technique in understanding upper airway disease.
Artifacts↗
Helical CT of the upper airway: normal and abnormal findings on three-dimensional reconstructed images.
Imaging of the hypopharynx, larynx, and upper airway are effectively achieved with CT and MR imaging. These techniques have proved their diagnostic usefulness in assessing the deep soft tissues not visible with laryngoscopy [1]. However, with axial imaging, large numbers of images often need to be mentally stacked to envision the appearance of the airway. With helical CT, we can create high-quality three-dimensional (3D) reconstructions [2, 3]. Advantages of helical technology include rapid scanning, decreased motion artifact, and minimization of misregistration artifacts. Recent work has suggested a role for multiplanar and 3D reconstructions of helical data for assessing the tracheobronchial tree [3]. The helically derived 3D models illustrate the normal and abnormal findings affecting the airway.
Humans↗
Use of dental implants for rehabilitation of mandibulectomy patients prior to radiation therapy.
The use of dental implants can aid in prosthetic rehabilitation of patients after ablative cancer surgery of the oral cavity. Biological effects of radiation therapy and the enhancement of radiation by metal implants have limited the use of dental implants. This article presents a case of dental implants used prior to radiation therapy for rehabilitation of a mandibulectomy patient and discusses the current understanding of radiation effects on bone and metal implants.
Alveolar Process↗
Performance status after treatment for squamous cell cancer of the base of tongue--a comparison of primary radiation therapy versus primary surgery.
PURPOSE: To compare the quality of life and functional outcome in patients with squamous cell cancer of the base of tongue treated with primary radiation vs. primary surgery. METHODS AND MATERIALS: At our institution, patients with base of tongue cancer are primarily treated either by radiation or surgery depending upon the philosophy of their primary physician. Primary radiation consists of 45-54 Gy external beam radiation followed by an 192Ir implant delivering an additional 20-30 Gy over 2-3 days. A neck dissection is done at the same time as the implant for those with involved nodes. Primary surgery consists of resection of the base of tongue lesion, neck dissection and postoperative radiation therapy. Because both groups have similar local control in our experience (80-90%), we used a subjective performance status scale for head and neck cancer patients to assess the quality of life in these patients (0-100, 0 = worst function, 100 = normal function). This scale measures ability to eat in public, understandability of speech, and normalcy of diet. There were 30 radiation patients (21: T1-T2; nine: T3-T4) and ten surgery patients (five: T1-T2; five: T3-T4) available for long-term quality of life assessment. RESULTS: Patients treated with radiation had consistently better performance status scores and quality of life according to our study. This was true for those with early (T1-2) as well as more advanced (T3-4) disease. For eating in public, T1-2 patients had scores of 85 vs. 75 (p = .31) and T3-4 patients had scores of 82 vs. 35 (p < .0001) for radiation vs. surgery, respectively. For understandability of speech, T1-2 patients had scores of 92 vs. 65 (p = .0021), and T3-4 patients had scores of 95 vs. 35 (p < .0001) for radiation vs. surgery, respectively. For normalcy of diet, T1-2 patients had scores of 74 vs. 50 (p = .047), and T3-4 patients had scores of 78 vs. 32 (p = .0012) for radiation vs. surgery, respectively. In addition, we compared scores for early vs. advanced disease treated by the same modality. For radiation, there was no difference in all three functional categories for T1-2 vs. T3-4 (p = .84), showing that quality of life scores remain high for all stages. For surgery, functional status deteriorated significantly when comparing T1-2 vs. T3-4 (p = .0014), consistent with the fact that larger tumors require more extensive operations. CONCLUSION: Radiation therapy provides a better performance status than surgery for base of tongue cancer. This is true for both early and advanced disease. Because radiation also provides similar local control and survival, our data suggests that radiation may be the preferred strategy. Functional scores remain high for all T stages treated with radiation, but deteriorate with more advanced T stages for patients treated with surgery. Similar studies using objective criteria are needed to further compare these treatments.
Adult↗
Engineering surface loops of proteins--a preferred strategy for obtaining new enzyme function.
A prerequisite for the rational redesign of enzymes is that altering amino acids in an attempt to obtain new biological function does not unexpectedly alter the globular, natural framework of the native protein on which the design is being executed. The results of combinatorial-mutagenesis strategies suggest that random variation of amino acid sequence is most easily tolerated at the solvent-exposed surfaces of a protein. This review analyses effective redesigns of Bacillus stearothermophilus lactate dehydrogenase (bsLDH), in which all residue variations are at solvent-exposed surfaces. The majority of these variations were located within surface loops, which interconnect stable secondary structures traversing the globular core of the protein.
Geobacillus stearothermophilus↗
Growth factor mRNA expression in human head and neck malignant tumors.
OBJECTIVE: Growth factor overexpression has been shown to be an integral part of the growth promotion of a number of tumors, including breast carcinoma, colon carcinoma, and sarcomas. The role of peptide growth factors in the regulation of growth of head and neck cancers has not been extensively investigated, however. In this study, we present our preliminary results of the pattern of expression of four growth factors: insulin-like growth factor II, transforming growth factor-alpha, basic fibroblast growth factor, and platelet-derived growth factor A chain in human head and neck tumors. DESIGN: We examined eight benign and 27 malignant human head and neck surgical specimens for the expression of growth factor messenger RNAs using the polymerase chain reaction technique. RESULTS: Our preliminary data demonstrate that growth factor messenger RNA is expressed by most of our malignant head and neck cancer specimens and by all eight benign head and neck tissue specimens. However, we were not able to show any distinction in the pattern of growth factor RNA expression among the malignant head and neck surgical specimens for the four growth factors studied to date. CONCLUSIONS: These findings demonstrate that the expression of specific biologically active genes can be studied in routine surgical specimens using the polymerase chain reaction. The clinical prognostic significance of the overexpression of growth factors in head and neck malignancy as well as future therapeutic implications are discussed.
Base Sequence↗
Thyroid cancer.
Management of thyroid cancer varies somewhat between communities and institutions depending on tumor type and individual treatment philosophy. The differentiated thyroid cancers have a significantly better outlook than the medullary and anaplastic. This article provides an overview of the literature that describes pathogenesis, diagnosis, and treatment currently recommended for these thyroid cancers.
Humans↗