Biomedical subjects

Anne Clark

Publications and source records attributed to Anne Clark.

6 recordsLinked to original sources

PubMed2004

Evidence that an isoform of calpain-10 is a regulator of exocytosis in pancreatic beta-cells.

Calpain-10 (CAPN10) is the first type 2 diabetes susceptibility gene to be identified through a genome scan, with polymorphisms being associated with altered CAPN10 expression. Functional data have been hitherto elusive, but we report here a corresponding increase between CAPN10 expression level and regulated insulin secretion. Pancreatic beta-cell secretory granule exocytosis is mediated by the soluble N-ethylmaleimide-sensitive fusion protein attachment receptor protein complex of synaptosomal-associated protein of 25 kDa (SNAP-25), syntaxin 1, and vesicle-associated membrane protein 2. We report, for the first time, direct binding of a calpain-10 isoform with members of this complex. Furthermore, SNAP-25 undergoes a Ca2+-dependent partial proteolysis during exocytosis, with calpain protease inhibitor similarly suppressing both insulin secretion and SNAP-25 proteolysis. Based upon these findings, we postulate that an isoform of calpain-10 is a Ca2+-sensor that functions to trigger exocytosis in pancreatic beta-cells.

Antigens, Surface↗

PubMed2004

Pancreatic beta-cell granule peptides form heteromolecular complexes which inhibit islet amyloid polypeptide fibril formation.

Islet amyloid polypeptide (IAPP), or 'amylin', is co-stored with insulin in secretory granules of pancreatic islet beta-cells. In Type 2 diabetes, IAPP converts into a beta-sheet conformation and oligomerizes to form amyloid fibrils and islet deposits. Granule components, including insulin, inhibit spontaneous IAPP fibril formation in vitro. To determine the mechanism of this inhibition, molecular interactions of insulin with human IAPP (hIAPP), rat IAPP (rIAPP) and other peptides were examined using surface plasmon resonance (BIAcore), CD and transmission electron microscopy (EM). hIAPP and rIAPP complexed with insulin, and this reaction was concentration-dependent. rIAPP and insulin, but not pro-insulin, bound to hIAPP. Insulin with a truncated B-chain, to prevent dimerization, also bound hIAPP. In the presence of insulin, hIAPP did not spontaneously develop beta-sheet secondary structure or form fibrils. Insulin interacted with pre-formed IAPP fibrils in a regular repeating pattern, as demonstrated by immunoEM, suggesting that the binding sites for insulin remain exposed in hIAPP fibrils. Since rIAPP and hIAPP form complexes with insulin (and each other), this could explain the lack of amyloid fibrils in transgenic mice expressing hIAPP. It is likely that IAPP fibrillogenesis is inhibited in secretory granules (where the hIAPP concentration is in the millimolar range) by heteromolecular complex formation with insulin. Alterations in the proportions of insulin and IAPP in granules could disrupt the stability of the peptide. The increase in the proportion of unprocessed pro-insulin produced in Type 2 diabetes could be a major factor in destabilization of hIAPP and induction of fibril formation.

Amino Acid Sequence↗

PubMed2003

Improvements in immunization compliance using a computerized tracking system for inner city clinics.

Vaccination compliance rates were calculated for 1995 to 2001 for enrolled patients, based on the Centers for Disease Control and Prevention guidelines and age-appropriate vaccine schedules. The results reported here indicate computerized tracking with the Doctor's Pediatric Immunization Program (Dr. PIP) maintained vaccine compliance rates (> 90%) in healthy and immunocompromised children at 2 months and 12 months of age. Instituting the computerized system has yielded nearly optimal results in both indigenous inner-city clinics. Despite the efficient progress made by automated tracking, the results for specific vaccine strategies (Varicella) and target groups (human immunodeficiency virus, high-risk indigent populations) may require on-going and intensive educational efforts to achieve optimization levels.

Adolescent↗

PubMed2002

Topological investigation of amyloid fibrils obtained from beta2-microglobulin.

Amyloid fibrils of patients treated with regular hemodialysis essentially consists of beta2-microglobulin (beta2-m) and its truncated species DeltaN6beta2-m lacking six residues at the amino terminus. The truncated fragment has a more flexible three-dimensional structure and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology of synthetic fibrils obtained from intact beta2-m and truncated DeltaN6beta2-m was investigated by the limited proteolysis/mass spectrometry approach that appeared particularly suited to gain insights into the structure of beta2-m within the fibrillar polymer. The distribution of prefential proteolytic sites observed in both fibrils revealed that the central region of the protein, which had been easily cleaved in the full-length globular beta2-m, was fully protected in the fibrillar form. In addition, the amino- and carboxy-terminal regions of beta2-m became exposed to the solvent in the fibrils, whereas they were masked completely in the native protein. These data indicate that beta2-m molecules in the fibrils consist of an unaccessible core comprising residues 20-87 with the strands I and VIII being not constrained in the fibrillar polymer and exposed to the proteases. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to occur in vivo to generate the truncated forms of beta2-m occurring in natural fibrils. On the basis of these data, a possible mechanism for fibril formation from native beta2-m is discussed and an explanation for the occurrence of truncated protein species in natural fibrils is given.

Amyloid↗

PubMed2002

Endoscopic microscopy.

In vivo endoscopic optical microscopy provides a tool to assess tissue architecture and morphology with contrast and resolution similar to that provided by standard histopathology--without need for physical tissue removal. In this article, we focus on optical imaging technologies that have the potential to dramatically improve the detection, prevention, and therapy of epithelial cancers. Epithelial pre-cancers and cancers are associated with a variety of morphologic, architectural, and molecular changes, which currently can be assessed only through invasive, painful biopsy. Optical imaging is ideally suited to detecting cancer-related alterations because it can detect biochemical and morphologic alterations with sub-cellular resolution throughout the entire epithelial thickness. Optical techniques can be implemented non-invasively, in real time, and at low cost to survey the tissue surface at risk. Our manuscript focuses primarily on modalities that currently are the most developed: reflectance confocal microscopy (RCM) and optical coherence tomography (OCT). However, recent advances in fluorescence-based endoscopic microscopy also are reviewed briefly. We discuss the basic principles of these emerging technologies and their current and potential applications in early cancer detection. We also present research activities focused on development of exogenous contrast agents that can enhance the morphological features important for cancer detection and that have the potential to allow vital molecular imaging of cancer-related biomarkers. In conclusion, we discuss future improvements to the technology needed to develop robust clinical devices.

Cell Line, Tumor↗

PubMed2002

Detection of dysplasia with near real time confocal microscopy.

The use of high resolution, in vivo confocal imaging may offer a clinical tool to detect early neoplasia and reduce the incidence and mortality of cancer. Our laboratory is currently examining the feasibility of using confocal microscopy for non-invasive diagnosis of dysplasia and early carcinoma in epithelial tissue. We are performing a series of ex vivo studies investigating the optical properties of normal and abnormal biopsies to quantify the diagnostic capability of this technology to discriminate between normal and pre-cancerous tissue. These studies use a near real time reflectance confocal microscope to acquire images at various depths throughout the epithelium. To date, we have completed a twenty-five patient study of cervical biopsies and have acquired images from six patients of an approved twenty-two patient study in the oral cavity. The cervical study has shown a distinct difference between normal and dysplastic tissue which can be used diagnostically, while initial results from the oral cavity are promising even with increased keratin scattering. In conclusion, our examination of normal and precancerous biopsies has demonstrated the confocal microscope's ability to image sub-cellular morphology at a resolution making accurate diagnosis possible and supporting this technique's potential for in vivo assessment of dysplasia.

Biopsy↗