Synthesis and characterization of polyanhydride for local BCNU delivery carriers

p-Carboxyphenoxy propane (CPP) prepolymer consisting of 4 units and sebacic acid (SA) prepolymer consisting of about 10 units were synthesized by reacting CPP and SA in the presence of excess acetic anhydride, respectively. Polyanhydride, poly(CPP-SA) copolymers were copolymerized by a melt polycondensation process with a mixture of CPP and SA prepolymer. Copolymers of average molecular weight up to 110,000 g/mol were achieved. The crystallinity of poly(CPP-SA) copolymers was decreased by the addition of the CPP homopolymer segment to SA homopolymer. Poly(CPP-SA) copolymers gradually degraded for period of 10 days. No large difference of weight loss observed according to molecular weight variation of poly(CPP-SA) copolymers. BCNU release from wafers fabricated by poly(CPP-SA) showed a sustained release pattern with no initial burst and delay of drug release.     

Serial cloning of pigs by somatic cell nuclear transfer: restoration of phenotypic normality during serial cloning.

Somatic cell nuclear transfer (scNT) is a useful way to create cloned animals. However, scNT clones exhibit high levels of phenotypic instability. This instability may be due to epigenetic reprogramming and/or genomic damage in the donor cells. To test this, we produced transgenic pig fibroblasts harboring the truncated human thrombopoietin (hTPO) gene and used them as donor cells in scNT to produce first-generation (G1) cloned piglets. In this study, 2,818 scNT embryos were transferred to 11 recipients and five G1 piglets were obtained. Among them, a clone had a dimorphic facial appearance with severe hypertelorism and a broad prominent nasal bridge. The other clones looked normal. Second-generation (G2) scNT piglets were then produced using ear cells from a G1 piglet that had an abnormal nose phenotype. We reasoned that, if the phenotypic abnormality of the G1 clone was not present in the G2 and third-generation (G3) clones, or was absent in the G2 clones but reappeared in the G3 clones, the phenotypic instability of the G1 clone could be attributed to faulty epigenetic reprogramming rather than to inherent/accidental genomic damage to the donor cells. Blastocyst rates, cell numbers in blastocyst, pregnancy rates, term placenta weight and ponderal index, and birth weight between G1 and G2 clones did not differ, but were significantly (P < 0.05) lower than control age- and sex-matched piglets. Next, we analyzed global methylation changes during development of the preimplantation embryos reconstructed by donor cells used for the production of G1 and G2 clones and could not find any significant differences in the methylation patterns between G1 and G2 clones. Indeed, we failed to detect the phenotypic abnormality in the G2 and G3 clones. Thus, the phenotypic abnormality of the G1 clone is likely to be due to epigenetic dysregulation. Additional observations then suggested that expression of the hTPO gene in the transgenic clones did not appear to be the cause of the phenotypic abnormality in the G1 clones and that the abnormality was acquired by only a few of the G1 clone's cells during its gestational development.     

Clonorchis sinensis: molecular cloning and localization of myosin regulatory light chain

One cDNA clone was purified from an adult Clonorchis sinensis cDNA library, and its deduced polypeptide sequence was found to be homologous with myosin regulatory light chain (MRLC) of invertebrates and vertebrates. Two amino-acid residues, Thr and Ser, were conserved at the phosphorylation sites that regulate the function of MRLCs. Recombinant C. sinensis MRLC (rCsMRLC) protein was produced and purified from Escherichia coli, and mouse anti-CsMRLC immune sera recognized a protein of molecular weight 24 kDa from a soluble protein preparation of C. sinensis. The CsMRLC protein was immunohistochemically localized to the muscle fibers of the subtegumental muscle layer and to the muscles of oral and ventral suckers. However, the rCsMRLC protein proved to be less useful antigen for the serodiagnosis of human clonorchiasis.The nucleotide sequence reported herein was submitted to GenBank and assigned accession number AY519356.     

Serological specificity of phenolic glycolipid I from Mycobacterium leprae and use in serodiagnosis of leprosy

The serological activities of the specific phenolic glycolipid I from Mycobacterium leprae, its dissected parts, and related glycolipids from other mycobacteria were examined by enzyme-linked immunosorbent assay against hyperimmune anti-M. leprae rabbit antiserum and sera from patients with leprosy and other mycobacterial diseases. High anti-phenolic glycolipid I immunoglobulin M antibodies were found in 23 of 24 (96%) of lepromatous leprosy patients on short term chemotherapy and in 8 of 13 tuberculoid leprosy patients (62%). Sera from patients with tuberculosis or atypical mycobacterial infections were devoid of anti-phenolic glycolipid I activity. The structurally related phenolic glycolipids from Mycobacterium kansasii and Mycobacterium bovis and the aglycone segments of the M. leprae product showed no significant activity. Thus, the trisaccharide determinant of phenolic glycolipid I is specific in its structure, serological activity, and, to a lesser extent, the antibody class it evokes.     

Alterations of the myocardial skeletal framework in acute myocardial infarction with and without ventricular rupture. A preliminary report

Thinning and dilatation (expansion) of the infarct region and complete rupture of the ventricular wall are significant complications of acute transmural myocardial infarction associated with increased morbidity and mortality. The pathogenesis of these related events is unknown. Recent studies of myocardial connective tissue have delineated an extensive array of intercellular and pericellular structures which serve as a skeletal framework and which may modulate contractile activity. We have employed a modified silver impregnation method to visualize the connective tissue components by light microscopy. To explore whether the skeletal framework is altered in acute myocardial infarction with and without ventricular rupture, we studied 9 human hearts at autopsy, and 4 canine infarcts of known duration. The human infarctions included 4 nonruptured cases with infarcts 1-5 days old, and 5 ruptured cases with infarcts 3-10 days old. Sections from normal, lateral, and central infarct or ventricular rupture sites were stained with silver. The normal tissue from each heart served as a control. Silver staining was moderately decreased in the lateral infarct zones, and markedly decreased in the central non-ruptured infarct zones. In the 5 ventricular rupture cases, the rupture site had no silver staining. A similar pattern was observed in the 4 canine infarcts. Thus, we conclude that the skeletal framework is markedly altered in the central zone of acute myocardial infarction. The acute changes of silver stained connective tissue may contribute significantly to the development of infarct expansion or ventricular wall rupture.     

Effect of Carbohydrate Source on Kanagawa-Type Hemolysis of Vibrio parahaemolyticus

Almost all strains of Vibrio parahaemolyticus produced Kanagawa-type hemolysis on media of high salt content in the presence of fermentable carbohydrates.     

Metabolic changes after revascularization in a patient with innominate artery occlusion by localized in vivo proton magnetic resonance spectroscopy

Localized in vivo proton magnetic resonance spectroscopy ((1)H-MRS) has been used to measure the metabolic status of the human brain in a non-invasive manner; thus, it is often called "a non-invasive biochemical assay". MRS is more sensitive than magnetic resonance imaging (MRI) in detecting ischemic damage by measuring the metabolic changes that occur prior to the anatomic changes. We report a patient who presented with innominate artery occlusion and symptoms of posterior circulation insufficiency and showed favorable metabolic changes by (1)H-MRS after revascularization. He showed no visible lesion in brain MRI, but in (1)H-MRS, decreased N-acetylaspartate (NAA) signal was noted in a resting state.After revascularization, both symptomatic improvement and recovery of NAA signal were observed. (1)H-MRS may provide valuable clinical information in diagnosis and management of cerebral hypoperfusion at a much earlier stage prior to the anatomic changes.     

Laser capture microdissection and real-time PCR for analysis of glomerular endothelin-1 gene expression in mesangiolysis of rat anti-Thy 1.1 and murine Habu Snake Venom glomerulonephritis.

Molecular analysis of pathologic changes in glomeruli requires methods allowing rapid and exact detection of alterations in gene expression. Here, we analyzed endothelin-1 (ET-1) mRNA expression in mesangiolytic glomeruli during the course of a rat and murine model of mesangioproliferative glomerulonephritis (GN). A novel method combining laser capture microdissection (LCM), which permits the precise removal of selected mesangiolytic glomeruli, with a highly sensitive real-time RT-PCR technique was used. Anti-Thy 1.1. GN was introduced in male Sprague-Dawley rats (1.0 mg/kg body weight of OX-7 IV) and Habu Snake Venom GN was introduced in C57BL6 mice (habu snake venom toxin 6 mg/kg body weight IV). The degree of mesangiolysis during both GNs was analyzed using a semiquantitative scoring system. Mesangiolytic glomeruli were microdissected at different days of the diseases (day 2, 6, and 12 in anti-Thy 1.1 GN and days 1, 3, 7, and 14 in Habu Snake Venom GN) and from normal control animals. After RNA extraction and cDNA synthesis, ET-1 gene expression was measured by real-time RT-PCR. In parallel, in anti-Thy 1.1. GN ET-1 mRNA expression was analyzed using semiquantitative nonradioactive in situ hybridization; ET-1 protein expression was investigated by immunohistochemistry. Mesangiolysis peaked at day 6 in anti-Thy1.1 GN and at day 1 in Habu Snake Venom GN. Mesangiolytic glomeruli were easily microdissected on cryostat sections in both models; quantification of mRNA with RT-PCR was reliable and reproducible. Glomerular ET-1 mRNA expression increased during the course of anti-Thy 1.1 GN and Habu Snake Venom GN peaked when mesangiolysis was most pronounced. This was seen by RT-PCR after glomerular LCM and by in situ hybridization; in parallel, glomerular ET-1 protein expression was increased. Combination of LCM and RT-PCR is a reliable method for quantification of localized gene expression in isolated renal structures. The above data argue for an important role of ET-1 in pathogenesis and/or repair of mesangiolysis in experimental mesangioproliferative GN.