Thyroid hormone regulation of the NADH shuttles in liver and cardiac mitochondria.

Thyroid hormone can potentially regulate the malate/aspartate and alpha-glycerophosphate shuttle pathways in cardiac mitochondria either directly, by altering gene expression, or indirectly, by increasing myocardial workload. The goal of the current study was to determine the influence of thyroid hormone on the NADH shuttles in cardiac and liver mitochondria. Malate/aspartate and alpha-glycerophosphate shuttle capacities were significantly increased in cardiac mitochondria from adult rats treated for 9 days with T3 compared to saline-treated controls. Liver mitochondria demonstrated a significant increase in alpha-glycerophosphate and no change in malate/aspartate shuttle capacity. T3 increased steady-state mRNA levels and activity of mitochondrial alpha-glycerophosphate dehydrogenase in both myocardium and liver. Quantitative immunoblot studies demonstrated a significant increase in aspartate-glutamate carrier levels in T3-treated myocardium suggesting a regulatory role of the aspartate/glutamate carrier in T3-treated hearts. Thyroid hormone effects on the NADH shuttles are tissue-specific. Changes in the NADH shuttles in the presence of thyroid hormone excess occur both directly at the gene level and indirectly as an adaptive response.

Ontogeny of malate-aspartate shuttle capacity and gene expression in cardiac mitochondria.

Developmental downregulation of the malate-aspartate shuttle has been observed in cardiac mitochondria. The goals of this study were to determine the time course of the postnatal decline and to identify potential regulatory sites by measuring steady-state myocardial mRNA and protein levels of the mitochondrial proteins involved in the shuttle. By use of isolated porcine cardiac mitochondria incubated with saturating concentrations of the cytosolic components of the malate-aspartate shuttle, shuttle capacity was found to decline by approximately 50% during the first 5 wk of life (from 921 +/- 48 to 531 +/- 53 nmol.min-1.mg protein-1). Mitochondrial aspartate aminotransferase mRNA levels were greater in adult than in newborn myocardium. mRNA levels of mitochondrial malate dehydrogenase in adult cardiac tissue were 224% of levels in newborn tissue, whereas protein levels were 54% greater in adult myocardium. Aspartate/glutamate carrier protein levels were also greater in adult than in newborn tissue. mRNA and protein levels of the oxoglutarate/malate carrier were increased in newborn myocardium. It was concluded that 1) myocardial malate-aspartate shuttle capacity declines rapidly after birth, 2) divergence of mitochondrial malate dehydrogenase mRNA and protein levels during development suggests posttranscriptional regulation of this protein, and 3) the developmental decline in malate-aspartate shuttle capacity is regulated by decreased oxoglutarate/malate carrier gene expression.

Developmental regulation of the alpha-glycerophosphate shuttle in porcine myocardium.

The alpha-glycerophosphate (alpha-GP) shuttle has been shown to play a role in reducing equivalent transfer in neonatal cardiac mitochondria. In adult heart mitochondria, alpha-GP shuttle activity is not detectable. The goals of the current study were to define the time course of the age-dependent decline in alpha-GP shuttle capacity and to identify the enzymatic step(s) of the alpha-GP shuttle which are regulated during development. Intact mitochondria were isolated from porcine hearts of various ages and assayed for alpha-GP shuttle capacity. By 5 weeks of age, alpha-GP shuttle capacity had decreased by nearly 39%. The cytosolic step of the shuttle, catalysed by cytosolic alpha-glycerophosphate dehydrogenase (c alpha-GPDH), demonstrated a significant increase between 0-2-day-old animals and adults. Partial cDNA clones of porcine c alpha-GPDH and mitochondrial alpha-glycerophosphate dehydrogenase (m alpha-GPDH) were prepared and used to quantitate expression of these genes. Using mRNA isolated from neonatal and adult porcine myocardium, expression of the c alpha-GPDH was unchanged, while expression of the m alpha-GPDH gene was present in neonatal but absent in adult myocardium. These results demonstrate a rapid postnatal decline in myocardial alpha-GP shuttle capacity which appears to be regulated by a decline in m alpha-GPDH gene expression.

DNA extraction by sonication: a comparison of fresh, frozen, and paraffin-embedded tissues extracted for use in polymerase chain reaction assays.

DNA extraction from fixed tissues can be the most laborious and complex step in amplifying DNA by the polymerase chain reaction (PCR). We have previously reported a rapid and efficient method for extracting DNA by the use of sonication and glass beads. We have extended our experiences with this technique using fresh, frozen, and formalin-fixed paraffin-embedded tissues with and without the use of glass beads and report their results. Multiple tissue types were obtained at autopsy or as part of a surgical specimen. DNA was extracted from identical tissue when the sample was fresh, frozen, or formalin-fixed paraffin-embedded. Our results indicate that in most instances the sonication technique, which takes only 30 min from start to finish, can rapidly extract fresh, frozen, or formalin-fixed paraffin-embedded tissue and is superior to other rapid extraction techniques in terms of quality and quantity of DNA. It is much more rapid than those techniques that use long digestion periods. This technique will be of great value to those investigators extracting DNA for polymerase chain reaction assays.

Cytomegalovirus detection in bone marrow transplant patients with idiopathic pneumonitis. A clinicopathologic study of the clinical utility of the polymerase chain reaction on open lung biopsy specimen tissue.

The morbidity and mortality rates of cytomegalovirus (CMV) infections, including pneumonitis in bone marrow transplant patients, are well documented and yet no rapid, sensitive diagnostic tool is available. To test the polymerase chain reaction as such a diagnostic tool, formalin-fixed, paraffin-embedded open lung biopsy material was selected from post-bone marrow transplant patients in whom pulmonary parenchymal changes were evident but viral inclusions were not seen. As a control, other immunosuppressed (non-bone marrow transplant) patients and low-risk nonimmunosuppressed patients were studied in a similar manner. Viral culture results and clinical data were available on all the high-risk patients. Two of 15 high-risk patients were found to have amplifiable CMV DNA despite the lack of histologic viral inclusions. One of these patients had been treated recently for CMV pneumonia, and the other had a positive culture of the lung specimens for CMV 13 days after biopsy. None of 12 control patients had amplifiable CMV DNA. These data indicate that the polymerase chain reaction is more sensitive than histologic examination and at least as sensitive as viral culture without evidence of false-positive results.

An efficient method for the extraction of DNA from formalin-fixed, paraffin-embedded tissue by sonication.

A method was developed for fast and efficient isolation of DNA from formalin-fixed, paraffin-embedded tissue sections for subsequent use in PCRs and DNA hybridization assays. The method relies on the use of a sonicating water bath to disrupt tissue samples to which a small amount of micro-sized glass beads have been added. The sonicating glass beads provide fast and efficient physical shearing of fixed tissue sections, allowing for quick release and solubilization of the DNA. The extraction process from paraffin section to amplifiable target DNA takes 30 minutes. The method eliminates the need for repetitive solvent extractions and exhaustive proteinase K digestion. PCR amplification of human genomic and viral target sequences was successfully carried out on DNA isolated from a number of different types of normal and infected tissues.

Growth control in cerebral microvessel-derived endothelial cells.

Endothelial (En) cells derived from the cerebral microvasculature were examined for their growth control properties. These cells were shown to be growth responsive to fetal bovine serum and arrested growth if serum was removed. They lend themselves to studies in chemically defined media since they survive well in serum-free medium with little or no proliferation. These cerebral microvessel En cells also respond to epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), unlike some other endothelial cells. Similar to other endothelial cells, the En cells responded to fibroblast growth factor (FGF). The response to EGF and PDGF occurred in serum-free media, but only if both agents were present. The cells further responded mitogenically to conditioned media obtained from transformed endothelial cells. The En cells were found to bind EGF and displayed two orders of affinity as determined by Scatchard analysis, depending upon whether the cells were in confluent or subconfluent conditions. The data indicate that some endothelial cells respond to EGF and PDGF and that one mechanism for growth control of endothelial cells may be an ability to regulate high-affinity growth factor receptors for epidermal growth factor.

Establishment and preliminary growth characteristics of a transformed mouse cerebral microvessel endothelial cell line.

A clone of spontaneously transformed mouse cerebral microvessel endothelial cells has been characterized and its growth characteristics have been investigated. The clone has been designated TEn and has been derived from parent nontransformed cerebral microvessel endothelial cells after multiple passages in culture. These transformed endothelial cells maintained characteristics of other transformed cells: anchorage-independent growth in soft agar, nude mouse tumorigenicity, and growth to high saturation density in culture with lack of contact inhibition. Although transformed, these cells still maintained endothelial functions and markers. These cells respond to both epidermal growth factor and platelet-derived growth factor. Although the TEn cells utilize serum factors for growth, they have maintained in completely serum-free media for up to 3 months. Additionally, serum-free media conditioned by these cells contained a growth factor-like activity that was mitogenic for the nontransformed parent endothelial cells. The TEn cells' maintenance of endothelial functions, growth in serum-free media, production of growth factor-like activity, and the response to well-characterized purified mitogens (epidermal growth factor and platelet-derived growth factor) suggest that these cells may be useful in studying growth control of cerebral microvessel-derived endothelial cells as well as in studies of growth factor production by transformed cells.