The myelin proteolipid protein gene modulates apoptosis in neural and non-neural tissues.

Mutations of the myelin proteolipid protein gene (Plp) are associated with excessive programmed cell death (PCD) of oligodendrocytes. We show for the first time that PLP is a molecule ubiquitously expressed in non-neural tissues during normal development, and that the level of native PLP modulates the level of PCD. We analyze three non-neural tissues, and show that native PLP is expressed in trophoblasts, spermatogonia, and cells of interdigital webbing. The non-neural cells that express high levels of native PLP also undergo PCD. The level of PLP expression modulates the level of PCD because mice that overexpress native PLP have increased PCD and mice deficient in PLP have decreased PCD. We show that overexpression of native PLP causes a dramatic acidification of extracellular fluid that, in turn, causes increased PCD. These studies show that the level of native PLP modulates the amount of PCD during normal development via a pH-dependent mechanism.

Altered expressions of VEGF mRNA splice variants during progression of uterine-peritoneal adhesions in the rat.

PROBLEM: Postoperative pelvic adhesions contribute to infertility, pelvic pain, bowel obstruction, and difficult reoperative procedures. METHOD OF STUDY: In the present study, a rat uterine-peritoneal adhesion model was developed to study the progression of adhesion formation during a course of 7 days following pelvic surgery. The distal 1 cm of each uterine horn and its adjacent peritoneum was abraded by six scratches with a scalpel blade, producing punctate bleeding. The scratched portion of uterine horn and the peritoneum was then held with Vicryl 3-0 to promote adhesion. The uterine tissue and the portion of peritoneum, held with suture, were then excised from a group of four rats, each at 6, 12, 24, 48, 72 hr and 5 and 7 days following surgery. Total RNA was isolated from these tissues and the expression pattern of different splice variants of vascular endothelial growth factors (VEGF) was examined using relative abundance reverse transcriptase polymerase chain reaction (RA-RT-PCR) method. RESULTS: Three known splice variants of VEGF mRNA (VEGF120, VEGF164 and VEGF188), as well as an additional band (approximately 510 bp), were amplified from these tissues. The relative abundance of known VEGF isoforms demonstrated altered expression during adhesion progression. When compared with noninjured uterine tissues, VEGF120 and VEGF188 demonstrated up-regulation during early stages of adhesion formation, whereas VEGF164 rather demonstrated down-regulation 24 and 48 hr following surgery. CONCLUSIONS: The up-regulation of VEGF isoforms during the progression of uterine-peritoneal adhesion may be a compensatory mechanism regulating angiogenesis in order to provide nutrients and oxygen to the injured tissues.

Ethanol elevates c-Myc levels in cultured mouse preimplantation embryos.

A brief exposure to ethanol accelerates the rate of early mouse embryonic development in vitro, increasing blastocyst formation, trophoblast outgrowth, and implantation rates after embryo transfer. The physiological effects of ethanol during preimplantation development are associated with rapid changes in gene expression and apparently arise from the ability of ethanol to elevate cytoplasmic free Ca2+ and alter cellular signaling pathways. The purpose of this study was to examine whether the abundance of c-Myc, a transcription factor that promotes cell proliferation and is required for blastocyst development, is upregulated in mouse blastocysts challenged with ethanol. After exposure of mouse blastocysts to 0.1% (17.5 mM) ethanol, wc determined the levels of: 1) c-Myc mRNA, using reverse transcription and the polymerase chain reaction; and 2) c-Myc protein levels, using specific monoclonal antibodies. Within 10 min of exposure to ethanol, the relative abundance of c-Myc mRNA increased 6-fold, then rapidly returned to baseline levels within 1 hr. As expected, elevation of c-Myc mRNA by ethanol was attenuated in embryos that were first treated with the intracellular Ca2+ chelator, BAPTA-AM. Western blot analysis of solubilized embryos revealed that c-Myc mRNA was translated into a single 62-kD protein that increased in intensity 30 min after treatment with ethanol. Immunocytochemical staining demonstrated that c-Myc was localized exclusively in nuclei and that staining intensity increased significantly after 10 min. Peak levels of c-Myc protein were found 30 min after ethanol exposure and persisted for at least 2 hr. The c-myc proto-oncogene seems to be an immediate early response gene for ethanol that may regulate the transcription of other genes that influence early embryogenesis and growth.

Expression of calcitonin receptors in mouse preimplantation embryos and their function in the regulation of blastocyst differentiation by calcitonin.

Calcitonin secretion in the pregnant uterus is tightly regulated by the ovarian hormones, estrogen and progesterone, which limit its expression to a brief period preceding blastocyst implantation. The binding of calcitonin to a G protein-coupled receptor activates adenylate cyclase and elevates cytosolic Ca2+ levels. The acceleration of preimplantation embryonic development that is known to occur upon elevation of intracellular Ca2+ prompted an investigation into calcitonin regulation of blastocyst differentiation. Using reverse transcription and the polymerase chain reaction to estimate the relative abundance of calcitonin receptor mRNA, a 25-fold accumulation of the splice variant, CR-1a, was observed in embryos between the 1-cell and 8-cell stages. Cytosolic free Ca2+ levels were rapidly elevated in embryos at the 4-cell to blastocyst stages after exposure to 10 nM calcitonin. Blastocysts treated for 30 minutes with 10 nM calcitonin differentiated in vitro at an accelerated rate, as assessed by the translocation of alpha5beta1 integrin to the apical surface of trophoblast cells, the corresponding elevation of fibronectin-binding activity and the timing of trophoblast cell migration. Chelation of cytosolic free Ca2+ with BAPTA-AM, but not inhibition of protein kinase A activity by H-89, attenuated the effects of calcitonin on blastocyst development. These findings support the concept that calcitonin secretion within the progesterone-primed uterus and the coordinate expression of CR-1a by preimplantation embryos regulates blastocyst differentiation through receptor-mediated Ca2+ signaling.

Integrin trafficking regulates adhesion to fibronectin during differentiation of mouse peri-implantation blastocysts.

Trophoblast cells of the peri-implantation blastocyst differentiate from a polarized epithelium, the trophectoderm, into invasive cells having an apical surface occupied by integrins that mediate adhesion to the extracellular matrix. Blastocyst differentiation was assessed during serum-free culture using a fibronectin binding assay with intact mouse blastocysts. Fibronectin binding activity became elevated during a 24-h "window" after approximately 72 h of culture. Blastocyst differentiation was unaffected by transcriptional inhibition with alpha-amanitin, however, exposure of cavitating morulae to the drug significantly delayed the onset of maximal fibronectin-binding activity. Inhibition of de novo protein synthesis with cycloheximide delayed development only when added during the first 24 h of blastocyst culture, indicating that proteins required for adhesion to fibronectin were synthesized at least 24 h before blastocyst differentiation was completed. Since blastocyst differentiation did not appear to be regulated temporally by gene expression, the possible role of protein trafficking was investigated using the inhibitor, brefeldin A. Brefeldin A caused a reversible, dose-dependent decrease in fibronectin-binding activity when added to the culture medium between 48 and 72 h of culture. During the period of brefeldin A sensitivity, alpha 5 beta 1 integrin, a major fibronectin receptor, translocated to the apical surface of trophoblast cells, as determined by immunohistochemistry and confocal microscopy. Mouse blastocysts expressed other integrins that recognize the central cell-binding domain of fibronectin, including the alpha v integrins and alpha llb beta 3, but not alpha4 which recognizes the lllCS site. Trafficking of alpha 5 beta 1, and possibly other integrins, to the apical surface of trophoblast cells appears to be a critical step in the differentiation of the mouse blastocyst to an invasive phenotype.

Ethanol-induced intracellular calcium mobilization rapidly alters gene expression in the mouse blastocyst.

The induction of intracellular Ca2+ release in pre-implantation mouse embryos accelerates their subsequent rate of development in vitro through a calmodulin-dependent mechanism [Stachecki J.J., Armant D.R. Transient release of calcium from inositol 1,4,5-trisphosphate-specific stores regulates mouse pre-implantation development. Development 1996; 122: 2485-2496]. To examine the hypothesis that intracellular Ca2+ signaling alters embryonic gene expression, individual transcript levels were compared by mRNA differential display before and 1 h after intracellular Ca2+ mobilization with ethanol in mouse blastocysts. Ten up-regulated and four down-regulated genes were observed, representing 3.5% of approximately 400 transcripts that were resolved. After sequencing, most of the DNA fragments appeared to be novel; however, two amplicons that increased after Ca2+ mobilization were identified as arginase and ubiquitin conjugating enzyme (E2). The up-regulation of arginase mRNA (3.5-fold after 2 h) was confirmed by reverse transcription and the polymerase chain reaction using specific oligonucleotide primers derived from the deduced mouse embryo sequence. A corresponding 2.5-fold increase in arginase enzymatic activity peaked 9 h after ethanol exposure. Increased expression of arginase and other genes may mediate the onset of rapid cell proliferation and differentiation that is induced by Ca2+ signaling during pre-implantation development.

Alcohol dehydrogenases and aldehyde dehydrogenases among inbred strains of mice: multiplicity, development, genetic studies and metabolic roles.

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the major enzymes responsible for the metabolism of alcohols and aldehydes in the body. Both exist as a family of isozymes in mammals, and have been extensively studied in animal models, particularly among inbred strains of mice. Mouse ADH exists as at least three major classes, which are predominantly localized in liver (classes I and III), and in stomach/cornea (class IV). Mouse ALDH exhibits extensive multiplicity, several forms of which have been characterized, including ALDH1 (liver cytoplasmic/class 1 isozyme); ALDH2 (liver mitochondrial/class 2.); ALDH3 (stomach cytosolic/class 3); ALDH4 (liver microsomal/class 3); and ALDH5 (testis cytosolic/class 3). Biochemical, genetic and molecular genetic analyses have been performed on several of these enzymes, including studies on variant forms of ADH and ALDH. Distinct metabolic roles are proposed, based upon their tissue and subcellular distribution characteristics and the biochemical properties for these enzymes.

Involvement of serine 74 in the enzyme-coenzyme interaction of rat liver mitochondrial aldehyde dehydrogenase.

It has been suggested that the active site nucleophile in sheep liver aldehyde dehydrogenase was not a cysteine residue but was a serine located at position 74 [Loomes, K. M., Midwinter, G. G., Blackwell, L. F., & Buckley, P. D. (1990) Biochemistry 29, 2070-2080]. This enzyme form has not yet been cloned and expressed, but since the rat liver mitochondrial enzyme has been and shares 70% sequence homology with other cytosolic aldehyde dehydrogenases, the residue in the rat enzyme was converted into an alanine to test for the necessity of a hydroxyl group at that position. The recombinantly expressed mutant enzyme possessed 10% catalytic activity, but the Km for NAD increased from 10 to 1900 microM while the Kms for various aldehydes were unchanged. Kinetic analysis revealed that the dissociation constant for NAD also increased in the mutant as did k1, the on velocity for NAD binding. The mutant enzyme bound poorly to an AMP-Sepharose column and did not interact as well with NADH, as determined by fluorescence enhancement binding studies, or with ADP-ribose, a competitive inhibitor. Pulse-chase analysis showed that the mutant was as stable as was the recombinantly expressed native enzyme. It was less stable to heat denaturation at 50 degrees C (half-life of 1 min compared to 4). Converting the alanine to a cysteine or a threonine did not restore native-like properties of the enzyme. These mutants had kinetic properties very similar to those of the alanine mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional distribution of low-Km mitochondrial aldehyde dehydrogenase in the rat central nervous system.

To clarify the regional capacity of the brain to oxidize biogenic aldehydes and ethanol-derived acetaldehyde, a quantitative immunohistochemical study of the microregional and cellular expression of low Km mitochondrial aldehyde dehydrogenase (mALDH; EC 1.2.1.3) in the rat central nervous system was undertaken, using antiserum raised in rabbit against low-Km aldehyde dehydrogenase purified from rat liver mitochondria. mALDH-specific immunoreactivity (IR) was observed to various extent in the majority of structures in all brain and spinal cord areas. Staining was strong in the extranuclear cytoplasm of neuronal and glial cell bodies but less pronounced in their processes and terminals, the conducting tracts, white matter and neuropile and in blood vessels. Immunostaining density was 2 to 3 times higher in neuronal perikarya as compared with neuropile. mALDH-positive neurons were found in all brain regions, being strongest in the inferior olive and hippocampus stratum pyramidale and weakest in substantia nigra. The percentage of morphologically identifiable ALDH-positive neurons ranged from 40% in the arcuate hypothalamic nucleus to 88% in the cerebellar Purkinje cells. A comparison of the heterogeneous expression of mALDH in various rat CNS regions and cells, as observed in the present study, with the corresponding previously published distributions of the potential acetaldehyde-producing enzymes ADH and cytochrome P450 2E1 indicates major differences, which may help in understanding potential acetaldehyde-mediated CNS effects of ethanol. Knowledge of the regional distribution of high-affinity aldehyde dehydrogenase should also throw light on the neurophysiological role of local regulation of the metabolism of biogenic aldehydes in the brain.

Alcohol dehydrogenases in the brain of mice.

Alcohol dehydrogenase (ADH) phenotypes were investigated in the brain of 15 different inbred mice by isoelectric focusing followed by staining of enzyme activities. The Class III ADH activity was detected in all the strains studied, whereas the Class II ADH activity was found only in few strains (including the alcohol-preferring strain--C57BL/6J) having the "a" allele (ADH-C2(2)) for this isozyme in stomach. The inbred strains having the "b" allele (ADH-C2(1)) for the Class II ADH in stomach (including the alcohol avoiding strains--BALB/c, CBA/H, C3H/He, DBA/2J, and SJL/J) demonstrated null variant for this phenotype in their brain. The Class I ADH activity was very low or absent in the brain extracts of all the strains studied. The ADH activities were confined to the cytosolic fractions of brain and were higher in the extracts of cerebral hemispheres than in cerebellum. The genetic linkage studies showed that the locus for the brain Class II ADH is closely linked to the "Adh gene complex" on chromosome 3 of mice.

Postnatal development of mouse alcohol dehydrogenases: agarose isoelectric focusing analyses of the liver, kidney, stomach and ocular isozymes.

The postnatal development of alcohol dehydrogenase (ADH) isozymes was studied in liver, kidney, stomach and eye tissues of C57BL/6J inbred male mice using agarose isoelectric focusing and histochemical methods. Development profiles were tissue specific, with adult patterns being attained by 30 days in the liver and stomach, and by 42 days in the kidneys. Ocular ADH-C2 increased in activity at the end of the first week, corresponding to the time of eye opening in the neonate.

Sister chromatid exchange in human chromosomes from normal individuals and epileptic patients on combinations of anticonvulsants.

Sister chromatid exchange (SCE) frequency, a sensitive indicator in mutagenicity testing, and mitotic index (MI) have been studied to observe genotoxic effects in epileptic patients on routine combinations of anticonvulsant therapy. All patients, both male and female and from various age groups, revealed an increased frequency of SCE per metaphase and a low MI (P less than 0.001) with respect to controls. A nonsignificant decrease in SCE frequency has been observed with an increase in the age of onset of epilepsy. Although the SCE frequency increased and the MI decreased in some groups with respect to the duration of epilepsy, there was no difference observed in SCE frequency with the duration of therapy.

Genetics and development of ocular oxidases in the mouse: evidence for a new locus (Eox-1) closely linked with the aldehyde oxidase loci on chromosome 1.

Isoelectric focusing (IEF) and histochemical techniques were used to examine the genetics, postnatal development and biochemical properties of ocular oxidases (EOXs) among inbred strains of mice. The designation as EOX was made on a provisional basis, since the 'natural' substrate(s) for this enzyme have not been identified. Five major forms were resolved from adult animals, which exhibited high activity in murine lens and low activity in the cornea. An additional ocular oxidase was observed in neonatal animals. Genetic analyses demonstrated that one of these enzymes (EOX-1) is encoded by a locus (Eox-1) which is closely linked with, but distinct from, the aldehyde oxidase (Aox) gene complex on chromosome one of the mouse. These results support the proposal that ocular oxidases are distinct from the major liver AOXs in this organism.

Developmental changes in aldehyde dehydrogenases from mouse tissues.

The postnatal development of aldehyde dehydrogenase (AHD) isozymes from C57BL/6J mouse tissues was examined using agarose-IEF zymogram methods. Mitochondrial isozymes (AHD-1 and AHD-5) were present throughout, increasing to high levels in liver, kidney and stomach by weaning (3 weeks). These activities remained high subsequently, except for kidney AHD-5, which decreased significantly after week 4. The appearance of the cytosolic isozymes was tissue specific and time dependent: liver AHD-2 was undetected until day 21, and increased subsequently; stomach AHD-4 was first observed at day 5, increasing to adult levels by day 21; AHD-6 was active in neonatal kidney and stomach extracts, but was undetected after day 8; and AHD-7 was observed in liver and kidney extracts from day 16. These results supported previous proposals for multiple genes encoding aldehyde dehydrogenases in the mouse, based upon the distinct developmental profiles for the liver, kidney and stomach isozymes.

Isoelectric focusing studies of aldehyde dehydrogenases from mouse tissues: variant phenotypes of liver, stomach and testis isozymes.

Isoelectric focusing techniques (IEF) were used to examine the tissue distribution and genetic variability of aldehyde dehydrogenases (AHDs) from inbred strains of mice. Twelve zones of AHD activity were resolved which were differentially distributed between tissues. Liver extracts exhibited highest activity for most enzymes, with the exception of isozymes found in stomach (AHD-4) and testis (AHD-4 and AHD-6). Genetic variants for AHD-1 (liver mitochondrial isozyme) and AHD-4 (stomach isozyme) were examined from inbred strains and F1 hybrid animals. The results were consistent with dimeric subunit structures (designated as A2 and D2 isozymes respectively). IEF patterns for activity variants of testis-specific AHD-6 were identical, with 3-banded phenotypes being observed. pI values for the AHD forms as well as for aldehyde oxidase and xanthine oxidase isozymes, which stain in the absence of coenzyme, were reported.