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Biomedical subjects

Peter Kuhn

Publications and source records attributed to Peter Kuhn.

At least 19 recordsLinked to original sources
PubMed2007

Ribonucleocapsid formation of severe acute respiratory syndrome coronavirus through molecular action of the N-terminal domain of N protein.

Conserved among all coronaviruses are four structural proteins: the matrix (M), small envelope (E), and spike (S) proteins that are embedded in the viral membrane and the nucleocapsid phosphoprotein (N), which exists in a ribonucleoprotein complex in the lumen. The N-terminal domain of coronaviral N proteins (N-NTD) provides a scaffold for RNA binding, while the C-terminal domain (N-CTD) mainly acts as oligomerization modules during assembly. The C terminus of the N protein anchors it to the viral membrane by associating with M protein. We characterized the structures of N-NTD from severe acute respiratory syndrome coronavirus (SARS-CoV) in two crystal forms, at 1.17 A (monoclinic) and at 1.85 A (cubic), respectively, resolved by molecular replacement using the homologous avian infectious bronchitis virus (IBV) structure. Flexible loops in the solution structure of SARS-CoV N-NTD are now shown to be well ordered around the beta-sheet core. The functionally important positively charged beta-hairpin protrudes out of the core, is oriented similarly to that in the IBV N-NTD, and is involved in crystal packing in the monoclinic form. In the cubic form, the monomers form trimeric units that stack in a helical array. Comparison of crystal packing of SARS-CoV and IBV N-NTDs suggests a common mode of RNA recognition, but they probably associate differently in vivo during the formation of the ribonucleoprotein complex. Electrostatic potential distribution on the surface of homology models of related coronaviral N-NTDs suggests that they use different modes of both RNA recognition and oligomeric assembly, perhaps explaining why their nucleocapsids have different morphologies.

Amino Acid Sequence↗
PubMed2006

Case study of the morphologic variation of circulating tumor cells.

We report a detailed cytomorphologic evaluation of the circulating component of widely metastatic breast carcinoma. A previously healthy 38-year-old woman was diagnosed with breast cancer. Wide local excision revealed a 1.7-cm infiltrating ductal adenocarcinoma, BSR score 7/9 with angiolymphatic invasion, and 4/20 lymph nodes positive for carcinoma. Five years later, a bone marrow biopsy revealed involvement of bone marrow by metastatic breast carcinoma, and shortly thereafter, metastases were identified in the liver and lung hilum. She enrolled in a clinical investigation for the detection of circulating tumor cells (CTCs) in breast carcinoma. A total of 659 CTCs were identified in a 10-mL blood sample using an immunofluorescent protocol targeting cytokeratins and detected using fiber-optic array scanning technology. The detected CTCs were subsequently stained with a Wright-Giemsa stain, and representative cells were evaluated in detail by light microscopy for morphologic evaluation. We find that the patient's CTCs exhibit a high degree of pleomorphism including CTCs with high and low nuclear-to-cytoplasmic ratios along with CTCs exhibiting early and late apoptotic changes. In addition, in comparison with her tumor cells in other sites, the full morphologic spectrum of cancer cells present in primary and metastatic tumor is also present in peripheral blood circulation.

Adult↗
PubMed2006

Comparative structural analysis of a novel glutathioneS-transferase (ATU5508) from Agrobacterium tumefaciens at 2.0 A resolution.

Glutathione S-transferases (GSTs) comprise a diverse superfamily of enzymes found in organisms from all kingdoms of life. GSTs are involved in diverse processes, notably small-molecule biosynthesis or detoxification, and are frequently also used in protein engineering studies or as biotechnology tools. Here, we report the high-resolution X-ray structure of Atu5508 from the pathogenic soil bacterium Agrobacterium tumefaciens (atGST1). Through use of comparative sequence and structural analysis of the GST superfamily, we identified local sequence and structural signatures, which allowed us to distinguish between different GST classes. This approach enables GST classification based on structure, without requiring additional biochemical or immunological data. Consequently, analysis of the atGST1 crystal structure suggests a new GST class, distinct from previously characterized GSTs, which would make it an attractive target for further biochemical studies.

Agrobacterium tumefaciens↗
PubMed2006

Multisite promiscuity in the processing of endogenous substrates by human carboxylesterase 1.

Human carboxylesterase 1 (hCE1) is a drug and endobiotic-processing serine hydrolase that exhibits relatively broad substrate specificity. It has been implicated in a variety of endogenous cholesterol metabolism pathways including the following apparently disparate reactions: cholesterol ester hydrolysis (CEH), fatty acyl Coenzyme A hydrolysis (FACoAH), acyl-Coenzyme A:cholesterol acyltransfer (ACAT), and fatty acyl ethyl ester synthesis (FAEES). The structural basis for the ability of hCE1 to perform these catalytic actions involving large substrates and products has remained unclear. Here we present four crystal structures of the hCE1 glycoprotein in complexes with the following endogenous substrates or substrate analogues: Coenzyme A, the fatty acid palmitate, and the bile acids cholate and taurocholate. While the active site of hCE1 was known to be promiscuous and capable of interacting with a variety of chemically distinct ligands, these structures reveal that the enzyme contains two additional ligand-binding sites and that each site also exhibits relatively non-specific ligand-binding properties. Using this multisite promiscuity, hCE1 appears structurally capable of assembling several catalytic events depending, apparently, on the physiological state of the cellular environment. These results expand our understanding of enzyme promiscuity and indicate that, in the case of hCE1, multiple non-specific sites are employed to perform distinct catalytic actions.

Binding Sites↗
PubMed2006

Structural and biophysical characterization of the EphB4*ephrinB2 protein-protein interaction and receptor specificity.

Increasing evidence implicates the interaction of the EphB4 receptor with its preferred ligand, ephrinB2, in pathological forms of angiogenesis and in tumorigenesis. To identify the molecular determinants of the unique specificity of EphB4 for ephrinB2, we determined the crystal structure of the ligand binding domain of EphB4 in complex with the extracellular domain of ephrinB2. This structural analysis suggested that one amino acid, Leu-95, plays a particularly important role in defining the structural features that confer the ligand selectivity of EphB4. Indeed, all other Eph receptors, which promiscuously bind many ephrins, have a conserved arginine at the position corresponding to Leu-95 of EphB4. We have also found that amino acid changes in the EphB4 ligand binding cavity, designed based on comparison with the crystal structure of the more promiscuous EphB2 receptor, yield EphB4 variants with altered binding affinity for ephrinB2 and an antagonistic peptide. Isothermal titration calorimetry experiments with an EphB4 Leu-95 to arginine mutant confirmed the importance of this amino acid in conferring high affinity binding to both ephrinB2 and the antagonistic peptide ligand. Isothermal titration calorimetry measurements also revealed an interesting thermodynamic discrepancy between ephrinB2 binding, which is an entropically driven process, and peptide binding, which is an enthalpically driven process. These results provide critical information on the EphB4*ephrinB2 protein interfaces and their mode of interaction, which will facilitate development of small molecule compounds inhibiting the EphB4*ephrinB2 interaction as novel cancer therapeutics.

Binding Sites↗
PubMed2006

Gynaecological operations: do they improve sexual life?

Aim of the study was to determine if gynaecological operations have an effect on sexual function using the current medlined literature. We performed a Medline search using the terms "sexual life/function after operative gynaecological treatment", "sexual life/function after operations for gynaecological problems", "sexual life/function after hysterectomy", "sexual life/function, incontinence" and "sexual life/function, pelvic organ prolapse". Reviews were excluded. We divided the operations into four groups of (1) combined prolapse and incontinence operations, (2) prolapse operations only, (3) incontinence operations only and (4) hysterectomy and compared pre-to postoperative sexual outcome. Thirty-six articles including 4534 patients were identified. Only 13 studies used a validated questionnaire. The other authors used self-designed and non-validated questionnaires or orally posed questions by the examiner to determine sexual function. Prolapse operations particularly posterior repair using levator plication seem to deteriorate sexual function, incontinence procedure have some worsening effect on sexual function and hysterectomy seems to improve sexual function with no differences between subtotal or total hysterectomy. Gynaecological operations do influence sexual function. However, little validated data are available to come to this conclusion.

Female↗
PubMed2006

High speed detection of circulating tumor cells.

Epithelial tumor cells circulate in peripheral blood at ultra-low concentrations in cancer patients. We have developed an instrument capable of rapid and accurate detection of rare cells in circulation utilizing fiber-optic array scanning technology (FAST). The FAST cytometer can locate immunofluorescently labeled rare cells on glass substrates at scan rates 500 times faster than conventional automated digital microscopy. These high scan rates are achieved by collecting fluorescent emissions using a fiber bundle with a large (50 mm) field of view. Very high scan rates make possible the ability to detect rare events without the requirement for an enrichment step. The FAST cytometer was used to detect, image and re-image circulating tumor cells in peripheral blood of breast cancer patients. This technology has the potential to serve as a clinically useful point-of-care diagnostic and a prognostic tool for cancer clinicians. The use of a fixed substrate permits the re-identification and re-staining of cells allowing for additional morphologic and biologic information to be obtained from previously collected and identified cells.

Breast Neoplasms↗