Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish

The zebrafish heart has the capacity to regenerate after ventricular resection. Although this regeneration model has proved useful for the elucidation of certain regeneration mechanisms, it is based on the removal of heart tissue rather than its damage. Here, we characterize the cellular response and regenerative capacity of the zebrafish heart after cryoinjury, an alternative procedure that more closely models the pathophysiological process undergone by the human heart after myocardial infarction (MI). Localized damage was induced in 25% of the ventricle by cryocauterization (CC). During the first 24 hours post-injury, CC leads to cardiomyocyte death within the injured area and the near coronary vasculature. Cell death is followed by a rapid proliferative response in endocardium, epicardium and myocardium. During the first 3 weeks post-injury cell debris was cleared and the injured area replaced by a massive scar. The fibrotic tissue was subsequently degraded and replaced by cardiac tissue. Although animals survived CC, their hearts showed nonhomogeneous ventricular contraction and had a thickened ventricular wall, suggesting that regeneration is associated with processes resembling mammalian ventricular remodeling after acute MI. Our results provide the first evidence that, like mammalian hearts, teleost hearts undergo massive fibrosis after cardiac damage. Unlike mammals, however, the fish heart can progressively eliminate the scar and regenerate the lost myocardium, indicating that scar formation is compatible with myocardial regeneration and the existence of endogenous mechanisms of scar regression. This finding suggests that CC-induced damage in zebrafish could provide a valuable model for the study of the mechanisms of scar removal post-MI.     

Quantitative analysis of N-terminal valine peptide adducts specific for 1,2-epoxy-3-butene

Butadiene (BD) metabolism shows gender, species and concentration dependency, making the extrapolation of animal results to humans complex. BD is metabolized mainly by cytochrome P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2;3,4-diepoxybutane (DEB) and 1,2-epoxy-butanediol (EB-diol). For accurate risk assessment it is important to elucidate species differences in the internal formation of the individual epoxides in order to assign the relative risks associated with their different mutagenic potencies. Analysis of N-terminal globin adducts is a common approach for monitoring the internal formation of BD derived epoxides. Our long term strategy is to develop an LC-MS/MS method for simultaneous detection of all three BD hemoglobin adducts. This approach is modeled after the recently reported immunoaffinity LC-MS/MS method for the cyclic N,N-(2,3-dihydroxy-1,4-butadyil)-valine (pyr-Val, derived from DEB). We report herein the analysis of the EB-derived 2-hydroxyl-3-butenyl-valine peptide (HB-Val). The procedure utilizes trypsin hydrolysis of globin and immunoaffinity (IA) purification of alkylated heptapeptides. Quantitation is based on LC-MS/MS monitoring of the transition from the singly charged molecular ion of HB-Val (1-7) to the a(1) fragment. Human HB-Val (1-11) was synthesized and used for antibody production. As internal standard, the labeled rat-[(13)C(5)(15)N]-Val (1-11) was prepared through direct alkylation of the corresponding peptide with EB. Standards were characterized and quantified by LC-MS/MS and LC-UV. The method was validated with different amounts of human HB-Val standard. The recovery was >75% and coefficient of variation <25%. The LOQ was set to 100 fmol/injection. For a proof of principal experiment, globin samples from male and female rats exposed to 1000 ppm BD for 90 days were analyzed. The amounts of HB-Val present were 268.2+/-56 and 350+/-70 pmol/g (mean+/-S.D.) for males and females, respectively. No HB-Val was detected in controls. These data are much lower compared to previously reported values measured by GC-MS/MS. The difference may be due higher specificity of the LC-MS/MS method to the N-terminal peptide from the alpha-chain versus derivatization of both alpha- and beta-chain by Edman degradation, and possible instability of HB-Val adducts during long term storage (about 10 years) between the analyses. These differences will be resolved by examining recently collected samples, using the same internal standard for parallel analysis by GC-MS/MS and LC-MS/MS. Based on our experience with pyr-Val adduct assay we anticipate that this assay will be suitable for evaluation of HB-Val in multiple species.     

Conformation-sensitive antibodies against alzheimer amyloid-beta by immunization with a thioredoxin-constrained B-cell epitope peptide

Immunotherapy against the amyloid-beta (Abeta) peptide is a valuable potential treatment for Alzheimer disease (AD). An ideal antigen should be soluble and nontoxic, avoid the C-terminally located T-cell epitope of Abeta, and yet be capable of eliciting antibodies that recognize Abeta fibrils and neurotoxic Abeta oligomers but not the physiological monomeric species of Abeta. We have described here the construction and immunological characterization of a recombinant antigen with these features obtained by tandem multimerization of the immunodominant B-cell epitope peptide Abeta1-15 (Abeta15) within the active site loop of bacterial thioredoxin (Trx). Chimeric Trx(Abeta15)n polypeptides bearing one, four, or eight copies of Abeta15 were constructed and injected into mice in combination with alum, an adjuvant approved for human use. All three polypeptides were found to be immunogenic, yet eliciting antibodies with distinct recognition specificities. The anti-Trx(Abeta15)4 antibody, in particular, recognized Abeta42 fibrils and oligomers but not monomers and exhibited the same kind of conformational selectivity against transthyretin, an amyloidogenic protein unrelated in sequence to Abeta. We have also demonstrated that anti-Trx(Abeta15)4, which binds to human AD plaques, markedly reduces Abeta pathology in transgenic AD mice. The data indicate that a conformational epitope shared by oligomers and fibrils can be mimicked by a thioredoxin-constrained Abeta fragment repeat and identify Trx(Abeta15)4 as a promising new tool for AD immunotherapy.     

Cycloartane triterpene glycosides from Astragalus trigonus

Three new cycloartane glycosides, trigonoside I, II and III, and the known astragalosides I and II were isolated from the roots of Astragalus trigonus. The structures of the new glycosides were totally elucidated by high field (600 MHz) NMR analyses as cycloastragenol-6-O-β-xylopyranoside, cycloastragenol-3-O-[-l-arabinopyranosyl(1 → 2)-β-d-xylopyranosyl]-6-O-β- d-xylopyranoside and cycloastragenol-3-O-[-l-arabinopyranosyl(1 → 2)-β-d-(3-O-acetyl)-xylopyranosyl]-6-O-β-d-xylopyranoside.     

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia.Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer.DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia.We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.     

A whole mitochondrial genome screening in a MELAS patient: a novel mitochondrial tRNA(Val) mutation

Mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome is a mitochondrial disorder characterized by a wide variety of clinical presentations and a multisystemic organ involvement. In this study, we report a Tunisian girl with clinical features of MELAS syndrome who was negative for the common m.3243A>G mutation, but also for the reported mitochondrial DNA (mtDNA) mutations and deletions. Screening of the entire mtDNA genome showed several known mitochondrial variants besides to a novel transition m.1640A>G affecting a wobble adenine in the anticodon stem region of the tRNA(Val). This nucleotide was conserved and it was absent in 150 controls suggesting its pathogenicity. In addition, no mutations were found in the nuclear polymerase gamma-1 gene (POLG1). These results suggest further investigation nuclear genes encoding proteins responsible for stability and structural components of the mtDNA or to the oxidative phosphorylation machinery to explain the phenotypic variability in the studied family.     

Multiple testing in fetal gender determination from maternal blood by polymerase chain reaction

Fetal male DNA can be identified in maternal blood by polymerase chain reaction (PCR) amplification of Y-specific sequences. This technology has not reached a satisfactory accuracy and reproducibility in fetal gender determination because of the very low concentration of fetal cells. Our purpose was to evaluate the possibility of improving the reliability of this test by setting up a repeated amplification system. We amplified, by nested PCR of the Y-specific sequence DYS14, 137 DNA samples extracted from maternal peripheral blood (93 from male-bearing and 44 from female-bearing pregnancies ranging from the 6th to the 36th gestational week). Each maternal DNA sample was tested doubly, in two different PCR sessions, with a total of four amplifications. We obtained discordant results in the four amplifications in 82/137 (60%) samples. The best interpretation of these discordant results was obtained by applying a positivity cutoff of at least two positive amplifications for considering a DNA sample as belonging to a male-bearing pregnancy. We obtained a sensitivity of 83%, a specificity of 93%, a positive predictive value of 96% and a negative predictive value of 72% in fetal male gender diagnosis. By applying this quadruple testing system, we significantly improved PCR accuracy and predictive values compared with single and double testing of the same samples. We conclude that, for future investigations of fetal DNA retrieved from maternal blood, the application of a quadruple testing system is better than the single PCR test. Received: 18 August 1997 / Accepted: 12 January 1998