Loss of DNA repair mechanisms in cardiac myocytes induce dilated cardiomyopathy

Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1^−/D mice). Ckmm-Cre^+/−;Ercc1^−/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre^+/−;Ercc1^−/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre^+/-;Ercc1^−/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre^+/−;Ercc1^−/fl and Ercc1^−/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death.     

A "Point of No Return" in the Cell Cycle of Chlorella

In a Chlorella culture growing synchronously at pH 6.3 undera 12 hr light/12 hr dark regime, DNA replication occurs betweenthe 8th and the 12th hour of the cycle, the main period of proteinand chlorophyll synthesis occurring between the 4th and 12thhour of the cycle. When the culture is transferred to alkalinepH at any time up to the 8 hr of the cycle, autospore releaseis prevented, and the pattern of synthesis of DNA, protein andchlorophyll is altered. However, when the culture is transferredto alkaline conditions after the 8th hour of the cycle, thepattern follows that of a culture growing at pH 6.3 with respectto cell number and volume, as well as protein, chlorophyll andDNA contents. Thus, a transition point seems to occur afterthe 8 hr of the cycle. The existence of such a point was alsodemonstrated by reciprocal experiments in which Chlorella wascultured at an alkaline pH and transferred to pH 6.3 at varioustimes in the cell cycle. ¹ Present address: Applied Research Institute, Ben-Gurion Universityof the Negev, P.O. Box 1025, Beer-Sheva 84110, Israel. (Received October 2, 1981; Accepted January 20, 1982)     

High resolution microchemical analysis using soft X-ray lithographic techniques

High resolution x-ray lithographic studies of cells from chick embryo hearts dried by the CO2 critical point method have been made with soft x-ray radiation of different wavelengths. A marked difference in the relief replica in polymethyl methacrylate (PMMA) resulting from the differential absorption by the dried cells of carbon K alpha radiation at 4.48 nm and broad band synchrotron radiation (SR) with lambda is greater than 1.5 nm demonstrates the potential usefulness of the technique in making high resolution (approximately or equal to 10 nm) chemical identification of the constitutents which make up the various parts of the cell.     

CD4+ T cell clones specific for the human p97 melanoma-associated antigen can eradicate pulmonary metastases from a murine tumor expressing the p97 antigen.

p97 is a human tumor-associated Ag present on most melanoma cells that represents a possible target for immunologic attack. To evaluate the capacity of T cells reactive with this protein to promote elimination of melanoma cells expressing p97, a murine model was developed by transfecting a C3H/HeN melanoma with the p97 cDNA, generating p97-specific CD4+ T cells by in vivo immunization of C3H/HeN mice with a vaccinia/p97 recombinant virus followed by in vitro cloning with soluble p97 protein, and determining whether these CD4+ T cells could mediate rejection of pulmonary metastases. Characterization of the T cell clones demonstrated the presence of both I-Ak and I-Ek-restricted clones, although the majority of clones recognized p97 in the context of I-Ek. Analysis of clonal specificity using truncated p97 proteins revealed that at least three epitopes were immunogenic, and further studies with overlapping 15-amino acid peptides from a region of the p97 molecule defined by these truncated proteins identified an immunodominant epitope responsible for the majority of the I-Ek response. The T cell clones were not capable of directly recognizing the p97-expressing melanoma cells but responded to the tumor if syngeneic APC were present to process the tumor-derived p97 Ag. The therapeutic efficacy of these CD4+ T cell clones was evaluated in an adoptive therapy model in which mice bearing metastatic pulmonary lesions were treated by i.v. administration of the p97-specific cells. Despite the inability of the CD4+ clones to directly respond to or lyse the tumor cells, the clones were effective in promoting tumor eradication. In vitro studies demonstrated that this may have reflected secretion of lymphokines that activated macrophages to lyse the tumor. The results suggest that noncytolytic p97-specific CD4+ T cell clones can be effective in therapy of pulmonary melanoma metastases. Moreover, if human T cells reactive with the p97 protein could be generated, the expression of this tumor-associated Ag in melanoma cells might be adequate for such T cells to mediate a therapeutic antitumor response.     

Genetic variation at the immunoglobulin allotype loci in Creoles of Trinidad

The sera of a sample of 204 Creoles from Trinidad were tested for the presence of polymorphic gene complexes occurring on immunoglobulin light- and heavy-chain molecules including the allotypic markers IGKC 1, IGHA2 1 and 2, IGHG1 A, X, F, and Z, and IGHG3 G, G5, B0, B1, B3, B4, B5, C3, C5, S, and T. Nine IGHG (GM) haplotypes occur in polymorphic frequencies (greater than .01) in this population, including known African, Asian, Caucasian, and Amerindian marker haplotypes. Significant differences (P less than .01) were found in the frequency distributions of three IGHG (GM) haplotypes and the frequency of IGKC*1 in these data and data from Creole populations of Belize and St. Vincent. The Creoles of Trinidad and St. Vincent are more similar in IGHG (GM) haplotype distributions than are Trinidad and Belize populations. Previous testing has revealed no significant differences between St. Vincent and Belize Creoles at the Ig allotypic loci. Analysis of migration patterns in the Caribbean suggests that different rates of Asian migration have maintained regional diversity at these loci, while continuous gene flow from the eastern Caribbean to Trinidad has had a relative homogenizing effect on the gene pools of this area.     

Quantitative Phosphoproteome Analysis Unveils LAT as a Modulator of CD3ζ and ZAP-70 Tyrosine Phosphorylation

Signaling through the T cell receptor (TCR) initiates adaptive immunity and its perturbation may results in autoimmunity. The plasma membrane scaffolding protein LAT acts as a central organizer of the TCR signaling machinery to activate many functional pathways. LAT-deficient mice develop an autoimmune syndrome but the mechanism of this pathology is unknown. In this work we have compared global dynamics of TCR signaling by MS-based quantitative phosphoproteomics in LAT-sufficient and LAT-defective Jurkat T cells. Surprisingly, we found that many TCR-induced phosphorylation events persist in the absence of LAT, despite ERK and PLCγ1 phosphorylation being repressed. Most importantly, the absence of LAT resulted in augmented and persistent tyrosine phosphorylation of CD3ζ and ZAP70. This indicates that LAT signaling hub is also implicated in negative feedback signals to modulate upstream phosphorylation events. Phosphorylation kinetics data resulting from this investigation is documented in a database (phosphoTCR) accessible online. The MS data have been deposited to the ProteomeXchange with identifier PXD000341.     

Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis

With the recent development of retinal prostheses, it is important to develop reliable techniques for assessing the safety of these devices in preclinical studies. However, the standard fixation, preparation, and automated histology procedures are not ideal. Here we describe new procedures for evaluating the health of the retina directly adjacent to an implant. Retinal prostheses feature electrode arrays in contact with eye tissue. Previous methods have not been able to spatially localize the ocular tissue adjacent to individual electrodes within the array. In addition, standard histological processing often results in gross artifactual detachment of the retinal layers when assessing implanted eyes. Consequently, it has been difficult to assess localized damage, if present, caused by implantation and stimulation of an implanted electrode array. Therefore, we developed a method for identifying and localizing the ocular tissue adjacent to implanted electrodes using a (color-coded) dye marking scheme, and we modified an eye fixation technique to minimize artifactual retinal detachment. This method also rendered the sclera translucent, enabling localization of individual electrodes and specific parts of an implant. Finally, we used a matched control to increase the power of the histopathological assessments. In summary, this method enables reliable and efficient discrimination and assessment of the retinal cytoarchitecture in an implanted eye.