Topoisomerase II-mediated DNA cleavage by amonafide and its structural analogs

Treatment of SV40-infected monkey cells with amonafide (benzisoquinolinedione), an intercalative antitumor drug, resulted in rapid accumulation of linearized intracellular SV40 DNA molecules that were protein linked. Studies using purified mammalian DNA topoisomerase II have shown that amonafide and its structural analogs interfere with the breakage-rejoining reaction of the enzyme by stabilizing a reversible enzyme-DNA "cleavable complex." Denaturation of the cleavable complex with sodium dodecyl sulfate resulted in DNA cleavage and the covalent association of topoisomerase II polypeptides with the cleaved DNA. Unwinding measurements indicate that amonafide is a DNA intercalator. These results suggest that amonafide and its structural analogs (e.g., mitonafide) represent a new class of intercalative topoisomerase II-active antitumor drugs. Different from other topoisomerase II-active antitumor drugs, amonafide and mitonafide induce specific DNA cleavage at a single major site on pBR322 DNA. The strong site specificity of amonafide may allow detailed characterization of the intercalator-stabilized, topoisomerase II-DNA cleavable complex.     

抑菌防霉剂—噻苯哒唑的合成

本文介绍了噻苯哒唑的新用途和采用“一锅煮”工艺制备噻唑酸进而制得该防霉剂的合成方法。     

一种新认识的胃癌癌前病变：人胃粘膜球样异型增生的组织病理学特点

Histopathologic features of globoid dysplasia of human gastric epithelium were described by means of observation of serial paraffin sections of 53 cases of globoid dysplasia. It was divided into three grades according to the architecture and cellular atypia. Penetration of outer layer globoid dysplastic cells through the basement membrane of "double layers structure" appeared in typical globoid dysplasia Grade III and infiltration of globoid dysplastic cells into stroma as well as the formation of incipient focus of signet ring cell carcinoma were described. The twinkling scene of infiltration of the globoid dysplastic cells into lamina propria through the basement membrane and the damage of basement membrane by globoid dysplastic cells were shown by Gordon Sweet's stain. Through the analysis of background lesions of the globoid dysplasia, a conclusion can be made that the globoid dysplasia might be an important precancerous lesion of the signet ring cell carcinoma of the stomach.     

NMDA receptors regulate developmental gap junction uncoupling via CREB signaling

Signaling through gap junctions (electrical synapses) is important in the development of the mammalian central nervous system. Abundant between neurons during postnatal development, gap junction coupling subsequently decreases and remains low in the adult, confined to specific subsets of neurons. Here we report that developmental uncoupling of gap junctions in the rat hypothalamus in vivo and in vitro is associated with a decrease in connexin 36 (Cx36) protein expression. Both developmental gap junction uncoupling and Cx36 downregulation are prevented by the blockade of NMDA glutamate receptors, action potentials and the calcium-cyclic AMP response element binding protein (CREB), and are accelerated by CREB overexpression. Developmental gap junction uncoupling and Cx36 downregulation are not affected by blockade of non-NMDA glutamate receptors, and do not occur in hypothalamic neurons from NMDA receptor subunit 1 (NMDAR1) knockout mice. These results demonstrate that NMDA receptor activity contributes to the developmental uncoupling of gap junctions via CREB-dependent downregulation of Cx36.     

A two decade survey of respiratory adenovirus in Taiwan: the reemergence of adenovirus types 7 and 4

From November 1999 to December 2001, three outbreaks of adenovirus (Ad) respiratory infection occurred in southern Taiwan. To determine the circulating serotypes and molecular epidemiology, a total of 524 virus strains were randomly selected from 1,064 strains isolated from 1981 to 2001, and were studied using restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR)-RFLP. The major subgenus found was subgenus B (45%), followed by subgenus E (29%) and subgenus C (25%). Ad3 and Ad7 were the major types found during the 1st outbreak, which occurred from November 1999 to March 2000, while Ad4 was found mainly during the 2nd and 3rd outbreaks in October 2000 and September 2001, respectively. Both Ad7 and Ad4 were reemerged serotypes, whereas Ad3 was consistently isolated during the survey, although it declined drastically from 36 to 2% in 2001. Genotype analysis in this study showed that the only strain of Ad7 found in 1983 was Ad7a, but all randomly selected strains of Ad7 isolated during 1999-2000 were Ad7b. The clinical features of 217 patients were analyzed during the 1999-2000 outbreaks. About 79% of the total cases were less than 7 years old. The ratio of male to female was 2:1. Severe infections, such as pneumonia and acute bronchitis, accounted for nearly half of the cases (43%). These results show the reemergence and changing of serotypes, the clinical association of respiratory adenovirus infections, and the molecular epidemiology of Ad7 genotypes in Taiwan during the past two decades.     

Attachment and spreading of fibroblasts on an RGD peptide-modified injectable hyaluronan hydrogel

Hyaluronan (HA) hydrogels resist attachment and spreading of fibroblasts and most other mammalian cell types. A thiol-modified HA (3,3'-dithiobis(propanoic dihydrazide) [HA-DTPH]) was modified with peptides containing the Arg-Gly-Asp (RGD) sequence and then crosslinked with polyethylene glycol (PEG) diacrylate (PEGDA) to create a biomaterial that supported cell attachment, spreading, and proliferation. The hydrogels were evaluated in vitro and in vivo in three assay systems. First, the behavior of human and murine fibroblasts on the surface of the hydrogels was evaluated. The concentration and structure of the RGD peptides and the length of the PEG spacer influenced cell attachment and spreading. Second, murine fibroblasts were seeded into HA-DTPH solutions and encapsulated via in situ crosslinking with or without bound RGD peptides. Cells remained viable and proliferated within the hydrogel for 15 days in vitro. Although the RGD peptides significantly enhanced cell proliferation on the hydrogel surface, the cell proliferation inside the hydrogel in vitro was increased only modestly. Third, HA-DTPH/PEGDA/peptide hydrogels were evaluated as injectable tissue engineering materials in vivo. A suspension of murine fibroblasts in HA-DTPH was crosslinked using PEGDA plus PEGDA peptide, and the viscous, gelling mixture was injected subcutaneously into the flanks of nude mice; gels formed in vivo following injection. After 4 weeks, growth of new fibrous tissue had been accelerated by the sense RGD peptides. Thus, attachment, spreading, and proliferation of cells is dramatically enhanced on RGD-modified surfaces but only modestly accelerated in vivo tissue formation.     

DNA mediated immunization with encoding the nucleoprotein gene of porcine transmissible gastroenteritis virus

The immune response to a naked plasmid DNA encoding the nucleoprotein (N protein) of porcine transmissible gastroenteritis virus (TGEV) was investigated in this study. A complementary DNA of the entire N gene was amplified by RT-PCR, and inserted into a mammalian expression vector (pcDNA3.1) to construct a recombinant plasmid (pcDNA/N). To evaluate the immunogenicity of the construct, BALB/c mice were intramuscularly immunized with different doses (50, 100 and 200 μg/mouse) of pcDNA/N twice at a 5-week interval. An optimal antibody response was achieved with 100 μg of pcDNA/N. The response lasted at least 11 weeks after primary immunization. By western blotting analysis, the antibodies specifically recognized a 47 kDa protein corresponding to the viral N protein, but they did not reveal neutralizing activity against infectious TGEV in vitro. Immunoglobulin G2a was predominant among these antibodies, which was indicative of Th1 type cell activation in pcDNA/N immunized mice. Moreover, spleen cells from these mice showed stronger immune responses than those from live vaccine or parental vector immunized mice. These results suggest that the construct can elicit both humoral and cell-mediated immune (CMI) responses against TGEV N protein in mice.     

Platelet satellitism: experimental studies

Platelet satellitism (PS), the in vitro phenomenon of platelets rosetting about nonlymphocytic leukocytes, is an uncommon and poorly understood finding reported in the ethylenediaminetetra-acetic acid (EDTA)-anticoagulated blood of patients with a wide variety of clinical conditions. This report presents experimental studies investigating the nature of this phenomenon by utilizing the blood of patients with platelet satellitism. Wet preparation studies and electron microscopy (transmission and scanning) demonstrated the morphologic sequences involved in the phenomenon, including eventual phagocytosis of platelets by neutrophils. The results of varying conditions such as time, temperature, and anticoagulant are described. All of five patients tested were found to have cryofibrinogenemia. Certain blood components from all of three patients tested were capable of inducing PS in normal whole blood, whereas components from normal subjects usually were not. In one patient (A), the PS-inducing capability appeared to be present in both plasma and platelets. In another patient (B) the PS-inducing capability was present in platelets (in both 1966 and 1975) and also in the cryosupernate of serum and plasma; among various antisera, antifibrinogen had the greatest ability to reduce the degree of PS in patient blood; addition of moderate amounts of CaCl2 and/or MgCl2 did not diminish the phenomenon; and two sisters and two daughters demonstrated no PS. In a third patient (C) the PS-inducing capability appeared to be largely concentrated in the cryoprecipitable fraction of plasma. These studies suggest that there are different factors in the patients' blood resulting in PS. Further studies showed PS could be induced in normal blood by adding certain nonprimate antihuman antisera (anti-IgM, antialbumin or antifibrinogen) and also by adding some preparations of normal washed platelets to the same individuals's normal whole blood. This indicates that the phenomenon of PS can be produced by factors other than those specifically present in patients with PS. Antigen-antibody complexes, either formed in vivo (mixed cryoglobulinemia) or in vitro, did not result in PS when mixed with normal blood. These and other studies suggest that PS can result from the presence of several different factors, usually proteins (in conjunction with EDTA), which probably attach to the surface of platelets apparently resulting in some alteration (such as change in surface charge) causing the platelets to be attracted to and phagocytosed by neutrophils.