Lippincott-Raven Publishers Research Articles

Development and Functional Characterization of Insulin-releasing Human Pancreatic Beta Cell Lines Produced by Electrofusion

Three novel human insulin-releasing cell lines designated 1.1B4, 1.4E7, and 1.1E7 were generated by electrofusion of freshly isolated of human pancreatic beta cells and the immortal human PANC-1 epithelial cell line. Functional studies demonstrated glucose sensitivity and responsiveness to known modulators of insulin secretion. Western blot, RT-PCR, and immunohistochemistry showed expression of the major genes involved in proinsulin processing and the pancreatic beta cell stimulus-secretion pathway including PC1/3, PC2, GLUT-1, glucokinase, and K-ATP channel complex (Sur1 and Kir6.2) and the voltage-dependent L-type Ca(2+) channel. The cells stained positively for insulin, and 1.1B4 cells were used to demonstrate specific staining for insulin, C-peptide, and proinsulin together with insulin secretory granules by electron microscopy. Analysis of metabolic function indicated intact mechanisms for glucose uptake, oxidation/utilization, and phosphorylation by glucokinase. Glucose, alanine, and depolarizing concentrations of K(+) were all able to increase [Ca(2+)](i) in at least two of the cell lines tested. Insulin secretion was also modulated by other nutrients, hormones, and drugs acting as stimulators or inhibitors in normal beta cells. Subscapular implantation of the 1.1B4 cell line improved hyperglycemia and resulted in glucose lowering in streptozotocin-diabetic SCID mice. These novel human electrofusion-derived beta cell lines therefore exhibit stable characteristics reminiscent of normal pancreatic beta cells, thereby providing an unlimited source of human insulin-producing cells for basic biochemical studies and pharmacological drug testing plus proof of concept for cellular insulin replacement therapy.

NMR Structure and Ion Channel Activity of the p7 Protein from Hepatitis C Virus

The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly α-helical, irrespective of the membrane mimetic medium (e.g. lysolipids, detergents, or organic solvent/water mixtures). The secondary structure elements of the monomeric form of p7 were determined by (1)H and (13)C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated with the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal α-helix to the first transmembrane helix, TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion channel gating mechanism.

The effect of joint exposures: examining the presence of interaction

Clinical epidemiological studies investigate whether an exposure, or risk factor, is causally related to the development or progression of a disease or mortality. It might be of interest to study whether this relation is different in different types of patients. To address such research questions, the presence of interaction among risk factors can be examined. Causal interaction between two risk factors is considered most clinically relevant in epidemiology. Causal interaction occurs when two risk factors act together in causing disease and is explicitly defined as a deviation from additivity on a risk difference scale. Statistical interaction can be evaluated on both an additive (absolute risk) and multiplicative (relative risk) scale, depending on the model that is used. When using logistic regression models, which are multiplicative models, several measures of additive interaction are presented to evaluate whether the magnitude of an association differs across subgroups: the relative excess risk due to interaction (RERI), the attributable proportion due to interaction (AP), or the synergy index (S). For a transparent presentation of interaction effects the recent Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement advises reporting the separate effect of each exposure as well as the joint effect compared with the unexposed group as a joint reference category to permit evaluation of both additive and multiplicative interaction.

Primary orbital endodermal sinus tumor (yolk sac tumor)

Endodermal sinus tumor, or yolk sac tumor, is the most common malignant neoplasm of germ cell origin and usually occurs in infant testes or ovaries. On rare occasions, the tumor may arise from extragonadal sites, including sacrococcygeal region, uterus, vagina, prostate, retroperitoneum, liver, mediastinum, pineal gland, and third ventricle. The orbit is an unusual location for the primary development of this neoplasm. We report the case of a girl with primary orbital endodermal sinus tumor who was managed with exenteration and chemotherapy with the result of a disease-free survival of 9 years.

Medallion-like dermal dendrocyte hamartoma in a 36-year-old male

Medallion-like dermal dendrocyte hamartoma in a 36-year-old male

Integrin-Mediated Transforming Growth Factor-β Activation Regulates Homeostasis of the Pulmonary Epithelial-Mesenchymal Trophic Unit

Trophic interactions between pulmonary epithelial and mesenchymal cell types, known as the epithelial-mesenchymal trophic unit (EMTU), are crucial in lung development and lung disease. Transforming growth factor (TGF)-beta is a key factor in mediating these interactions, but it is expressed in a latent form that requires activation to be functional. Using intact fetal tracheal tissue and primary cultures of fetal tracheal epithelial cells and fibroblasts, we demonstrate that a subset of integrins, alpha(v)beta(6) and alpha(v)beta(8), are responsible for almost all of the TGF-beta activation in the EMTU. Both alpha(v)beta(8) and alpha(v)beta(6) contribute to fetal tracheal epithelial activation of TGF-beta, whereas only alpha(v)beta(8) contributes to fetal tracheal fibroblast activation of TGF-beta. Interestingly, fetal tracheal epithelial alpha(v)beta(8)-mediated TGF-beta activation can be enhanced by phorbol esters, likely because of the increased activity of MT1-MMP, an essential co-factor in alpha(v)beta(8)-mediated activation of TGF-beta. Autocrine alpha(v)beta(8)-mediated TGF-beta activation by fetal tracheal fibroblasts results in suppression of both transcription and secretion of hepatocyte growth factor, which is sufficient to affect phosphorylation of the airway epithelial hepatocyte growth factor receptor, c-Met, as well as airway epithelial proliferation in a co-culture model of the EMTU. These findings elucidate the function and complex regulation of integrin-mediated activation of TGF-beta within the EMTU.

Crystal Structure of the Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein Dimerization Domain Reveals Evolutionary Linkage between Corona- and Arteriviridae

The causative agent of severe acute respiratory syndrome (SARS) is the SARS-associated coronavirus, SARS-CoV. The nucleocapsid (N) protein plays an essential role in SARS-CoV genome packaging and virion assembly. We have previously shown that SARS-CoV N protein forms a dimer in solution through its C-terminal domain. In this study, the crystal structure of the dimerization domain, consisting of residues 270–370, is determined to 1.75Å resolution. The structure shows a dimer with extensive interactions between the two subunits, suggesting that the dimeric form of the N protein is the functional unit in vivo. Although lacking significant sequence similarity, the dimerization domain of SARS-CoV N protein has a fold similar to that of the nucleocapsid protein of the porcine reproductive and respiratory syndrome virus. This finding provides structural evidence of the evolutionary link between Coronaviridae and Arteriviridae, suggesting that the N proteins of both viruses have a common origin.

Decrease in pH Strongly Enhances Binding of Native, Proteolyzed, Lipolyzed, and Oxidized Low Density Lipoprotein Particles to Human Aortic Proteoglycans

Binding of low density lipoprotein (LDL) to proteoglycans and modification of LDL are key processes in atherogenesis. Recently, it has been demonstrated that during atherogenesis the extracellular pH of atherosclerotic lesions decreases. We have examined the effect of the decreased pH on the binding of LDL to human aortic proteoglycans. The binding of native, oxidized, proteolyzed (alpha-chymotrypsin-treated), or lipolyzed (sphingomyelinase- or phospholipase A(2)-treated) LDL particles to proteoglycans were measured in microtiter well assays at pH 5.5-7.5. We found that the lower the pH, the higher the amount of binding of LDL to proteoglycans. At the lowest pH tested (pH 5.5), the amounts of proteoglycan-bound native, proteolyzed, sphingomyelinase-, and phospholipase A(2)-treated LDL were 20-, 23-, 30-, and 37-fold higher, respectively, than at pH 7.5. Interestingly, although oxidized LDL failed to bind to proteoglycans at neutral pH, there was significant binding at acidic pH. Binding of native and modified LDL to proteoglycans at pH 5.5 was blocked by 1 m NaCl, indicating that at neutral pH LDL binds to proteoglycans via ionic interactions. Inhibition of this binding by acetylation and cyclohexanedione treatment of LDL showed that the positively charged amino acids of apolipoprotein B-100, lysine, and arginine, respectively, mediated the ionic interaction. Taken together, our results suggest that in areas of atherosclerotic arterial intima where the extracellular pH decreases, retention of LDL by proteoglycans is enhanced, leading to extracellular accumulation of LDL and progression of the disease.

Novel Autoregulatory Function of Hepatitis B Virus M Protein on Surface Gene Expression

The hepatitis B virus surface gene consists of a single open reading frame divided into three coding regions: pre-S1, pre-S2, and S. By alternate translation at each of the three initiation codons, L, M, and S proteins can be synthesized. Studies have shown that M protein is not essential for viral replication, virion morphogenesis, or in vitro infectivity. In this study, we show that native M protein can regulate surface gene expression at the transcriptional level. The regulatory effect of M protein is mediated through the CCAAT box within the S promoter. Deletion mapping analysis indicated that the transactivating effect of M protein is mediated through amino acids 1-57 of M protein (the MHBs(au) domain), although its maximal transactivation activity coincides with that of the pre-S2 domain. This conclusion is supported by the fact that disruption of the putative V8 protease site at the pre-S2/S domain junction not only rendered M protein incapable of transactivating the S promoter but also inactivated its nuclear translocation potential. Immunoprecipitation and immunoblot experiments demonstrated that pre-S2 interacts with the three subunits of the CCAAT box-binding factor/nuclear factor Y, the cognate binding protein of the CCAAT box. These results demonstrate and define a novel regulatory role of M protein, which, under natural conditions, may undergo a proteolytic process to generate an MHBs(au) species that will be translocated inside the nucleus, where it will interact with the CCAAT box-binding factor to regulate surface gene expression. Because the CCAAT box is located at a fixed position within numerous promoters, these observations might provide a plausible explanation for hepatitis B virus-associated hepatocarcinogenesis.

Does Consumption of Cola Beverages Cause Bone Fractures in Children?

Does Consumption of Cola Beverages Cause Bone Fractures in Children?

16-Year-Old Boy With Gross Hematuria

16-Year-Old Boy With Gross Hematuria

Cardiovascular Drugs in the Perioperative Period

Cardiovascular Drugs in the Perioperative Period

A Specific Isozyme of 2′-5′ Oligoadenylate Synthetase Is a Dual Function Proapoptotic Protein of the Bcl-2 Family

2-5(A) synthetases are a family of interferon-induced enzymes that polymerize ATP into 2'-5' linked oligoadenylates that activate RNase L and cause mRNA degradation. Because they all can synthesize 2-5(A), the reason for the existence of so many synthetase isozymes is unclear. Here we report that the 9-2 isozyme of 2-5(A) synthetase has an additional activity: it promotes apoptosis in mammalian cells. The proapoptotic activity of 9-2 was isozyme-specific and enzyme activity-independent. The 9-2-expressing cells exhibited many properties of cells undergoing apoptosis, such as DNA fragmentation, caspase activation, and poly ADP-ribose polymerase and lamin B cleavage. The isozyme-specific carboxyl-terminal tail of the 9-2 protein was shown, by molecular modeling, to contain a Bcl-2 homology 3 (BH3) domain, suggesting that it may be able to interact with members of the Bcl-2 family that contain BH1 and BH2 domains. Co-immunoprecipitate assays and confocal microscopy showed that 9-2 can indeed interact with the anti-apoptotic proteins Bcl-2 and Bclx(L) in vivo and in vitro. Mutations in the BH3 domain that eliminated the 9-2-Bcl-2 amd 9-2-Bclx(L) interactions also eliminated the apoptotic activity of 9-2. Thus, we have identified an interferon-induced dual function protein of the Bcl-2 family that can synthesize 2-5(A) and promote cellular apoptosis independently. Moreover, the cellular abundance of this protein is regulated by alternative splicing; the other isozymes encoded by the same gene are not proapoptotic.

Viral Infections of the Nervous System

2nd ed, by Richard T. Johnson, 527 pp, with illus, Philadelphia, Pa, Lippincott-Raven Publishers, 1998.Viral Infections of the Nervous System (2nd edition) represents an excellent resource and leading reference in neurovirology. Professor Richard T. Johnson has updated this important textbook (published in 1998) and assimilated new findings and information following a 16-year interval since the 1st edition. As a single-author textbook, this work is easily readable, with continuity and a unified structure difficult to realize with multiauthored works. Professor Johnson's personal insights are interspersed throughout the book, insights that stem from his lifetime of investigations with multiple viral pathogens and provide valuable perspectives regarding the historical background of important discoveries. This reviewer strongly recommends this work as a highly readable and compact textbook.Viral Infections of the Nervous System is organized into 4 main sections entitled “General Principles,” “Acute Neurological Diseases,” “Chronic Neurological Diseases,” and “Other Perspectives.” In “General Principles,” basic principles of viral pathogenesis and host responses with special emphasis on the nervous system are described. In “Acute Neurological Diseases,” the pathogenesis of meningitis and encephalitis caused by multiple viruses, human herpesviruses, rabies, postinfectious demyelinating diseases, and postviral syndromes is discussed. New material abounds in the “Chronic Neurological Diseases” section regarding human and animal retroviruses, retroviral infections of the developing nervous system, and prion diseases. A thorough examination of chronic inflammatory and demyelinating diseases has been enhanced. The final section, entitled “Other Perspectives,” provides information difficult to find in other textbooks, such as discussions of oncogenesis in the nervous system and the nature of emerging viral infections. Recent concerns about West Nile Virus in the United States came after the publication of this book, and hence this encephalitis virus is only briefly described.The rapid development of neurovirology since the publication of the 1st edition 16 years ago has necessitated the addition of new chapters describing human retroviruses and postviral syndromes. New material regarding human herpesviruses, prion diseases, and viral infections of the developing nervous system has been incorporated in this updated edition. Sixteen years represents a lengthy period between editions, but few, if any, current books describe neurovirology in such a focused and thorough manner. General virology textbooks such as Fields Virology (Lippincott Williams & Wilkins) and Principles of Virology by Flint et al (ASM Press) provide alternative textbook sources for neurovirologists and students of infectious diseases. These alternatives represent excellent references in general virology, but they lack the uniquely focused approach in neurovirology. Viral Infections of the Nervous System provides a cohesive approach to neuropathogenesis by describing neuropathologic similarities of diseases caused by different viruses while emphasizing key differences in viral pathogenesis caused by related viruses. Johnson authoritatively describes the historical context of important discoveries. Importantly, this book is not a complete reference for the diagnosis and therapy of viral infections, although these subjects are addressed briefly. Other resources, such as the Manual of Clinical Microbiology (ASM Press) and medical textbooks of infectious diseases, represent more comprehensive references for the diagnosis and treatment of diseases in medical virology.Viral Infections of the Nervous System deserves an emphatic recommendation to medical and graduate students, physicians, and virologists. This book accomplishes its goal of providing a stand-alone reference for neurovirology and viral pathogenesis of the human nervous system. All major areas of neurovirology and prion diseases are included in this textbook. The cohesive presentation of a single-author work and the personal insights of a highly respected figure in the field lend additional value to this reference. Although neurovirology is rapidly expanding with applications of molecular biology, basic principles of pathogenesis are elegantly described and provide a durable framework for further studies. I hope that a 3rd edition will be published after a shorter time interval, and I congratulate Professor Johnson on an outstanding contribution to the virologic literature.

Palliative Care and Hospice Programs

Palliative Care and Hospice Programs

Cytosine Arabinoside Lesions Are Position-specific Topoisomerase II Poisons and Stimulate DNA Cleavage Mediated by the Human Type II Enzymes

Cytosine arabinoside (araC) is an important drug used for the treatment of human leukemias. In order to exert its cytotoxic effects, araC must be incorporated into chromosomal DNA. Although specific DNA lesions that involve base loss or modification stimulate nucleic acid cleavage mediated by type II topoisomerases, the effects of deoxyribose sugar ring modification on enzyme activity have not been examined. Therefore, the effects of incorporated araC residues on the DNA cleavage/religation equilibrium of human topoisomerase IIalpha and beta were characterized. AraC lesions were position-specific topoisomerase II poisons and stimulated DNA scission mediated by both human type II enzymes. However, the positional specificity of araC residues differed from that previously reported for other cleavage-enhancing DNA lesions. Finally, additive or synergistic increases in DNA cleavage were observed in the presence of araC lesions and etoposide. These findings broaden the range of DNA lesions known to alter topoisomerase II function and raise the possibility that this enzyme may mediate some of the cellular effects of araC.

49-Year-Old Woman With Chest Pain

49-Year-Old Woman With Chest Pain

Drugs, Diseases & Anesthesia

Vice Chairman; Department of Anesthesiology; Associate Medical Director for Quality; Texas A&M University HSC; Scott & White Memorial Hospital; Temple, Texas 76508James C. Eisenach, M.D., EditorDrugs, Diseases & Anesthesia. Ronald J. Stern, M.D. Philadelphia, Lippincott-Raven Publishers, 1996. ISBN: 0–397–58758–9 (softcover); 0–397–58764–3 (network version). Prices: softcover, $99.00; network version, $395.00.Drugs, Diseases & Anesthesia is one of a growing number of texts available on interactive software. This critique will focus exclusively on the Lippincott-Raven interactive software package and its usefulness for practicing anesthesiologists and residents. The information also comes in a softcover textbook. I probably represent most practicing anesthesiologists who rely on multiple software packages to accomplish daily administrative duties but have neither the time nor the interest to become a computer expert. Computers to me are useful if they make my task "easier to accomplish" or make me more "efficient."Following the user-guided instructions, I found the installation process to be straightforward and easy. The software came on four computer disks. For window-based personal computers, the minimum system configuration needed to use the software is an IBM (or compatible) 486 or a Pentium (Intel) processor, 4 MB RAM, 8 MB free hard disk space, Microsoft 3.1 (Redmond, WA), or higher. No Macintosh (Apple, Cupertino, CA) version was described.After the program is loaded, the program opens to a screen that lists Patient Drugs (drugs that a patient may be taking), Diseases (coexisting diseases that a patient may have), and Anesthetics (drugs or anesthetic agents that the anesthesiologist may administer to a patient during the course of an anesthetic). To use the program, one would select an item from one of these three categories. After selection, a description of the item will appear in a text window. To see interactions among the three items, one would click on the "see interaction" button and a window will appear over the main screen with a description of the specific interaction.Although only 63 disease states are listed, the interaction of the disease state, drug, and anesthetic can be very helpful. Confronted with patients prescribed various new drugs, it is often difficult to keep up with all the possible interactions. This software is an example of "computer decision support." This program enables the anesthesiologist to quickly select anesthetics best suited for patients and their diseases, while lessening the chance for an adverse drug interaction, therefore making a significant contribution to patient safety.Negatives: The copyright on the most recent version is 1996, hence, some of the material is out of date. For example, there were no descriptions of more recent drugs, such as cisatracurium, remifentanyl, and milrinone. The text reads like the Physicians Desk Reference, [1] there are no literature citations, most of the information is derived from textbook information: Anesthesia, [2] Anesthesia and Coexisting Disease, [3] and The Pharmacological Basis of Therapeutics. [4] The diseases discussed are not extensive; for example, no information regarding mitral regurgitation, anemia, amyloidosis, or epiglottis was found. There was no "search" or "find" mode, which would make it easier to find information. The installation required four computer disks; I hope future editions will come out on CD-ROM. Customer service to this product was lacking: calls placed on multiple occasions provided only an answering machine. This is clearly not the prompt customer service that I have experienced with other software vendors.The software version provides several advantages over the textbook version. First, the software version provides easy point and click access to listed information rather than going through the Table ofcontents or the index in the back of the text. However, the softcover version is very portable and does not require a computer to access.This program represents exciting change in the practice of anesthesiology. It is a tool for facilitating and improving the anesthesiology practitioner's performance. Our challenge as users is to use this tool to the advantage of our patients. It is an "operational" teacher, one that could be used in the operating room, at the bedside, or in the preoperative clinic when one is formulating an anesthetic plan or is confronted with an unfamiliar drug or unusual disease.Is it worth purchasing? This product is one of several interactive software products that are intended to assist the clinician to provide a safer anesthetic. It's aim is to get the right information in the hands of the clinician when needed. However, because of the rapid advance in our specialty and the dated information, I would consider waiting for the next edition before purchasing this product. Also, poor customer service limits it's potential appeal, especially to the computer novice.Frank J. Villamaria, M.D.Vice Chairman; Department of Anesthesiology; Associate Medical Director for Quality; Texas A&M University HSC; Scott & White Memorial Hospital; Temple, Texas 76508(Accepted for publication January 7, 1999.)

Abstracts of literature

Abstracts of literature

ERCP and its Applications

ERCP and its Applications . Edited by Jacobson IM. (Pp 288; illustrated; $110.00.) Philadelphia: Lippincott-Raven Publishers, 1997. ISBN 0-397-51664-9. In 1998 at least three new books entirely devoted to ERCP were published, and this is the most comprehensive of them. It is a multi-author textbook from the United States and in fact from the Eastern part of that country with only one author hailing from Canada and one group from Amsterdam. British readers will recognise a few well known exiles, however. The book is largely a very practical and up to date (many chapters contain references from 1997) review of ERCP in all or nearly all of its manifestations at the end of the 20th Century. The book is accompanied by x ray films, colour plates and line …