Interstitial deletion of chromosome 2q32-34 associated with multiple congenital anomalies and a urea cycle defect (CPS I deficiency).

A de novo deletion of the long arm of chromosome 2 at 2q31-33 was observed in the fetal amniocyte G-banded karyotype performed because of possible multiple malformations identified by ultrasound at 23 weeks gestation. Two days after the uneventful term delivery of a 2.45 kg male, the neonate experienced cardiopulmonary decompensation and biochemical changes compatible with carbamoyl phosphate synthetase I (CPS I) deficiency (elevated ammonia with a peak of 948 micromol/L, deficiency of citrulline, and no increase in orotic acid). The child died on day 3 of life. Physical anomalies confirmed at autopsy included double superior vena cava, ectopic adrenal tissue, and metatarsus adductus. The autopsy also revealed histologic evidence consistent with CPS deficiency, most notably microvesicular steatosis of the liver and Alzheimer's Type II changes with hypertrophic astrocytes in the basal ganglia. A postnatal lymphocyte karyotype confirmed the chromosome 2q31-33 deletion. Enzyme analysis on postmortem liver tissue confirmed the diagnosis of CPS deficiency. CPS I is reported to be mapped to 2q35 by NCBI (http://www.ncbi.nlm.nih.gov/mapview/) and 2q34 by ENSEMBL (http://www.ensembl.org/). The UCSC Human Genome Browser July 2003 assembly also places the gene at 2q34 (http://genome.UCSC.edu/). Fluorescence in situ hybridization (FISH) analysis with a BAC clone (RP11-349G4) of CPS I demonstrated that one copy of the gene was deleted in this infant. Using additional probes corresponding to the bands in the region of deletion, we identified the deleted region as 2q32-2q34. Our observations support the CPS I map position (ENSEMBL, UCSC) at 2q34. Additionally, potential conditions associated with deletions narrowly defined by standard cytogenetic techniques merit consideration in prenatal counseling. As demonstrated here, deletions may not only result in malformations and mental retardation but also increase the likelihood of revealing mutated genes located in the undeleted region of the homologous chromosome.

Acidification and glucocorticoids independently regulate branched-chain alpha-ketoacid dehydrogenase subunit genes.

Acidification or glucocorticoids increase the maximal activity and subunit mRNA levels of branched chain alpha-ketoacid dehydrogenase (BCKAD) in various cell types. We examined whether these stimuli increase transcription of BCKAD subunit genes by transfecting BCKAD subunit promoter-luciferase plasmids containing the mouse E2 or human E1alpha-subunit promoter into LLC-PK(1) cells, which do not express glucocorticoid receptors, or LLC-PK(1)-GR101 cells, which we have engineered to constitutively express the glucocorticoid receptor gene. Dexamethasone or acidification increased luciferase activity in LLC-PK(1)-GR101 cells transfected with the E2 or E1alpha-minigenes; acidification augmented luciferase activity in LLC-PK(1) cells transfected with these minigenes but dexamethasone did not. A pH-responsive element in the E2 subunit promoter was mapped to a region >4.0 kb upstream of the transcription start site. Dexamethasone concurrently stimulated E2 subunit promoter activity and reduced the binding of nuclear factor-kappaB (NF-kappaB) to a site in the E2 promoter. Thus acidification and glucocorticoids independently enhance BCKAD subunit gene expression, and the glucocorticoid response in the E2 subunit involves interference with NF-kappaB, which may act as a transrepressor.

Regulation of expression of branched-chain alpha-keto acid dehydrogenase subunits in permanent cell lines.

The rat hepatoma cell line H4IIEC3 has demonstrated a response to both insulin and glucocorticoids in its accumulation of BCKAD subunit RNAs. It is amenable to BCKAD promoter minigene transfection analyses, demonstrating positive (glucocorticoids) and negative (insulin) regulatory effects. These cells can therefore be used as a model to identify cis-acting sites responsible for regulation of BCKAD subunit promoter activity.

Glucocorticoid regulation of branched-chain alpha-ketoacid dehydrogenase E2 subunit gene expression.

Regulation of the mammalian branched-chain alpha-ketoacid dehydrogenase complex (BCKAD) occurs under a variety of stressful conditions associated with changes in circulating glucocorticoids. Multiple levels of regulation in hepatocytes, including alteration of the levels of the structural subunits available for assembly (E1, alpha-ketoacid decarboxylase; E2, dihydrolipoamide acyltransferase; and E3, dihydrolipoamide dehydrogenase), as well as BCKAD kinase, which serves to phosphorylate the E1alpha subunit and inactivate complex activity, have been proposed. The direct role of glucocorticoids in regulating the expression of the murine gene encoding the major BCKAD subunit E2, upon which the other BCKAD subunits assemble, was therefore examined. Deletion analysis of the 5' proximal 7.0 kb of the murine E2 promoter sequence, using E2 promoter/luciferase expression minigene plasmids introduced into the hepatic H4IIEC3 cell line, suggested a promoter proximal region responsive to glucocorticoid regulation. Linker-scanning mutagenesis combined with deletion analysis established this functional glucocorticoid-responsive unit (GRU) to be located near the murine E2 proximal promoter site at -140 to -70 bp upstream from the transcription initiation site. The presence of this region in plasmid minigenes, containing varying amounts of the murine genomic sequence 5' upstream from proximal E2 promoter sequences, conferred 2-10 fold increases in luciferase reporter gene expression in H4IIEC3 cells, whether introduced by transient transfection or following co-selection for stable transfectants. The GRU region itself appeared to contain multiple interacting elements that combine to regulate overall E2 promoter activity in response to changing physiological conditions associated with varying concentrations of glucocorticoids and likely other hormonal effectors.

Molecular characterization of a unique patient with epimerase-deficiency galactosaemia.

Inherited deficiencies of UDP-galactose 4-epimerase (GALE) have been associated with two distinct phenotypes. The vast majority of North American patients are clinically asymptomatic, are identified through newborn screening programmes for classical galactosaemia, and are of African-American descent. At least two symptomatic patients have been reported, one Pakistani and the other Asian Muslim, both with severe complications in the neonatal period and subsequent mental retardation. Through newborn screening, we have identified a GALE-deficient patient who is of mixed Pakistani/caucasian ancestry. He was clinically well in the neonatal period on a lactose-containing diet, and biochemical studies, including urine reducing sugars and galactitol, were consistent with a diagnosis of peripheral GALE deficiency. Although early developmental milestones were met normally, he now shows significant developmental delays in both motor and language skills. Mutational analysis revealed this patient to be a compound heterozygote at the GALE locus, with mutations N34S and L183P identified in the patient and confirmed in the parents. This report represents the first characterization of specific mutations in a GALE-deficient patient in conjunction with biochemical and clinical phenotype, and facilitates further studies of the GALE enzyme and its role in the different clinical forms of epimerase-deficiency galactosaemia.

Isolation of the murine branched-chain alpha-ketoacid dehydrogenase E2 subunit promoter region.

The promoter region of the murine branched-chain alpha-ketoacid dehydrogenase E2 subunit (dihydrolipoyl transacylase) gene was isolated and characterized. Sequence analysis of the promoter-regulatory region showed the presence of two inverted 'CAAT box' sequences, the most proximal being -42 to -48 bp upstream from the determined transcription initiation site, but no TATA-box sequences, similar to the human BCKAD E2 gene. The boundary of the minimum promoter sequence appeared to reside just inclusive of this first inverted CAAT sequence, but minigene transfer analysis demonstrated that the promoter proximal between -65 and -140 bp is likely to be extremely important for controlling regulated changes in E2 RNA expression. The regulatory effect of this region may be modulated by a number of other upstream regions which were identified within the 7.0 kb sequence examined.

Effects of insulin on the regulation of branched-chain alpha-keto acid dehydrogenase E1 alpha subunit gene expression.

Alterations in dietary intake, especially of protein, may produce changes in the hepatic levels of the branched-chain alpha-keto acid dehydrogenase (BCKAD) complex. The possible role of insulin in the regulation of these observed changes in hepatic capacity for BCKAD expression was therefore examined. Steady-state RNA levels encoding three of the subunits, E1 alpha, E1 beta and E2, increased by 2-4-fold in the livers of mice starved for 3 days, a known hypoinsulinaemic state. In contrast, the levels of E1 beta and E2, but not E1 alpha, RNA were decreased when mice were fed 0% protein diets compared with the levels observed in mice fed standard (23%) or higher protein isocaloric diets. BCKAD subunit protein levels under these conditions changed co-ordinately even though the changes in RNA were not co-ordinate. The effects of hormonal changes that might be associated with these dietary changes were examined, using the rodent hepatoma cell line H4IIEC3. In these cells, the levels of E1 alpha protein and mRNA were significantly depressed in the presence of insulin. In contrast, the levels of E1 beta and E2 RNAs were not decreased by insulin. The half-lives of the E1 alpha and E2 RNAs were determined to be quite long, from 13 to 18 h, with insulin having no dramatic overall effect on the half-lives determined over 24 h. Therefore, it is likely that insulin directly affects the transcription of the E1 alpha gene rather than RNA stability in exerting its negative regulatory effect. This effect is specific to the E1 alpha subunit. The differences in BCKAD subunit RNA levels observed under various nutritional and developmental conditions may therefore be the result of the differential effects of insulin and other hormones on the multiple regulatory mechanisms modulating BCKAD subunit expression.

Molecular cloning of the murine branched chain alpha-ketoacid dehydrogenase E2 subunit: presence of 3' B1 repeat elements.

The cDNA sequence encoding the murine E2 subunit (dihydrolipoyl transacylase) of the branched-chain alpha-ketoacid dehydrogenase (BCKAD) complex was determined. In the region encoding the mature E2 subunit protein, both the nucleotide composition and predicted amino acid sequence are highly conserved between murine, human, and bovine species. In contrast, the 5' sequence encoding the amino-terminal preprotein sequence and 3' untranslated region are less well conserved. The 3'-noncoding region contains sequences highly homologous to the rodent B1 repeat elements, which are related to human Alu repeat sequences. This finding is similar to the presence of three Alu repeat sequences in the 3'-noncoding region of human E2 cDNA.

Diagnostic and referral reliability in an alcoholism treatment network.

Diagnostic and referral reliability were studied throughout an integrated alcoholism referral and treatment network. Diagnosticians included volunteers from an inpatient alcohol program staff, an outpatient referral network, a hospital medical staff, and a hospital administrative staff. Participant category had no influence on diagnostic reliability and only minimal influence on referral reliability. Cases involving only physical addiction to alcohol, particularly withdrawal symptoms, were more reliably diagnosed and referred than were cases involving psychosocial problems alone. Diagnostic practices included consideration of multiple problem areas for each case. Results are related to prior research findings and current directions in the diagnosis and treatment of alcohol disorders.

Mediation of abusive childhood experiences: depression, dissociation, and negative life outcomes.

A study of 301 college undergraduates is employed to test a causal model which proposes that dissociation and depression act as mediator variables that develop from child abuse and lead to various negative outcomes. As predicted, child maltreatment was found to be related to negative life experiences, with depression and dissociation differentially mediating the various outcomes.

Molecular and biochemical basis of intermediate maple syrup urine disease. Occurrence of homozygous G245R and F364C mutations at the E1 alpha locus of Hispanic-Mexican patients.

Maple syrup urine disease (MSUD) is caused by a deficiency of the mitochondrial branched-chain alpha-keta acid dehydrogenase (BCKAD) complex. The multienzyme complex comprises five enzyme components, including the E1 decarboxylase with a heterotetrameric (alpha 2 beta 2) structure. Four unrelated Hispanic-Mexican MSUD patients with the intermediate clinical phenotype were diagnosed 7 to 22 mo after birth during evaluation for developmental delay. Three of the four patients were found homozygous for G to A transition at base 895 (exon 7) of the E1 alpha locus, which changes Gly-245 to Arg (G245R) in that subunit. The remaining patient was homozygous for T to G transversion at base 1,253 in the E1 alpha gene, which converts Phe-364 to Cys (F364C) in the gene product. Transfection studies in E1 alpha-deficient lymphoblasts indicate that both G245R and F364C mutant E1 alpha subunits were unable to significantly reconstitute BCKAD activity. Western blotting showed that both mutant E1 alpha subunits in transfected cells failed to efficiently rescue the normal E1 beta through assembly. The putative assembly defect was confirmed by pulse-chase labeling of E1 subunits in a chaperone-augmented bacterial overexpression system. The kinetics of initial assembly of the G245R E1 alpha subunit with the normal E1 beta was shown to be slower than the normal E1 alpha. No detectable assembly of the F364C E1 alpha with normal E1 beta was observed during the 2 h chase. Small amounts of recombinant mutant E1 proteins were produced after 15 h induction with isopropyl thiogalactoside and exhibited very low or no E1 activity. Our study establishes that G245R and F364C mutations in the E1 alpha subunit disrupt both the E1 heterotetrameric assembly and function of the BCKAD complex. Moreover, the results suggest that the G245R mutant E1 alpha allele may be important in the Hispanic-Mexican population.

Noncoordinated responses of branched-chain alpha-ketoacid dehydrogenase subunit genes to dietary protein.

The response of the murine genes encoding the subunits of branched-chain alpha-ketoacid dehydrogenase complex (BCKAD) to changes in dietary protein was determined. Steady-state RNA levels for two of the subunits, E1 beta and E2, decreased by two- to four-fold in the livers of mice fed 0% protein isocaloric diets compared to the levels observed in mice fed standard (23%) or high (50%) protein isocaloric diets. In contrast, the levels of RNA encoding the E1 alpha subunit did not change significantly in response to these dietary protein changes. The hepatic decreases in E1 beta and E2 RNA associated with 0% protein isocaloric diets were reversible, with prompt return to baseline levels following 48 hours of 50% protein isocaloric diets ad libitum. In kidney, no significant changes in the RNAs encoding any of the three BCKAD subunits were observed in response to changes in dietary protein. Studies of RNA variations associated with growth and development in several murine tissues, including liver and kidney, demonstrated coordinated changes between all subunits. Similar coordinated changes were observed during 3T3-L1 adipocyte differentiation. These studies suggest that the responses of the BCKAD subunit genes to alterations in dietary protein are noncoordinated and tissue-specific, in contrast to the coordinated changes observed during growth and/or differentiation. The differences in BCKAD subunit RNA levels observed under varying nutritional and developmental conditions suggest that multiple regulatory mechanisms modulate BCKAD subunit expression.

Molecular cloning and analysis of the expression of the E1 beta subunit of branched chain alpha-ketoacid dehydrogenase in mice.

The cDNA sequence encoding the murine liver E1 beta subunit of the branched-chain alpha-ketoacid dehydrogenase complex (BCKAD) was determined. In the region encoding the mature E1 beta subunit protein, both the nucleotide composition and predicted amino acid sequence are highly conserved between murine, rat, human, and bovine species. In contrast, they are less well conserved in the 5' sequence encoding the amino terminal preprotein sequence and 3' untranslated region. The pattern of tissue-specific expression of three BCKAD subunit RNAs was determined to be similar in both rat and murine tissues except for that observed in murine liver, where a higher than expected level of E1 beta subunit RNA was observed. Variation in the levels of this subunit might play a major role in the regulation of the overall ability of the murine liver to modulate BCKAD content in response to changing physiologic needs.

Ontogeny of adenosine deaminase in the mouse decidua and placenta: immunolocalization and embryo transfer studies.

This study has determined the cellular site of adenosine deaminase (ADA) expression in the mouse during development from Days 5 through 13 (day vaginal plug was found = Day 0) of gestation. Developmental expression of ADA progressed in two overlapping phases defined genetically (maternal vs. embryonal) and according to region (decidual vs. placental). In the first phase, ADA enzyme activity increased almost 200-fold in the antimesometrial region (decidua capsularis + giant trophoblast cells) from Days 6 through 9 of gestation but remained low in the mesometrial region. Immunohistochemical staining revealed a major localization of ADA to the secondary decidua. In the second phase, ADA activity increased several-fold in the placenta (labyrinth + basal zones) from Days 9 through 13 of gestation but remained low in the embryo proper. Immunohistochemical staining revealed a major localization of ADA to secondary giant cells, spongiotrophoblast, and labyrinthine trophoblast. Regression of decidua capsularis and growth of the spongiotrophoblast population accounted for an antimesometrial to placental shift in both ADA enzyme activity and a 40-kDa immunoreactive protein band. To verify a shift from maternal to fetal expression, studies were performed with two strains of mice (ICR, Eday) homozygous for a different ADA isozyme (ADA-A, ADA-B). Blastocysts homozygous for Adab were transferred to the uterus of pseudopregnant female recipients homozygous for Adaa. The isozymic pattern in chimeric embryo-decidual units analyzed at Days 7, 9, 11, and 13 revealed a predominance of maternal-encoded enzyme at Days 7 through 11 of gestation and a shift to fetal-encoded enzyme by Day 13. Thus, maternal expression of ADA in the antimesometrial decidua may play a role during establishment of the embryo in the uterine environment, whereas fetal expression of ADA in the trophoblast might be important to placentation.

Developmental expression of adenosine deaminase during decidualization in the rat uterus.

Adenosine deaminase (ADA) is expressed in high concentrations at the fetal-maternal interface during postimplantation stages of gestation in the mouse. The experiments reported here were designed to identify the specific uterine cells that express ADA subsequent to implantation in the rat and to determine if embryonic cells contribute to ADA expression. The results of biochemical analysis demonstrate that ADA-specific activity increases to very high levels in implantation sites, beginning approximately 72 h after blastocyst attachment. Immunocytochemical analysis localized this ADA expression to the decidualized stromal cells in the antimesometrial region of the pregnant uterus. In experimentally induced deciduoma, these cells were capable of synthesizing high levels of both ADA and mRNA for ADA in the absence of embryos. The enzyme first appeared in decidual cell cytoplasm, approximately 72 h after induction of decidualization, and later was localized in the decidual cell nuclei. Since the expression of ADA and its mRNA in decidual cells follows the appearance of desmin, a protein marker for decidualization, by at least 48 h, ADA appears to be involved in the functioning of mature decidual cells rather than in stromal cell differentiation. The expression of ADA, but not desmin, was restricted to the antimesometrial decidual cells and decreased when these cells regressed. At mid-gestation ADA activity increased and was localized principally in the fetal placenta. The results presented here demonstrate that ADA is localized to the antimesometrial decidual cell and that its expression is consequent to differentiation of the uterine stromal cell and independent of any embryonic stimulus.

Structural and functional analysis of the murine adenosine deaminase gene.

We describe the structural and functional analysis of cosmid clones that span the entire murine adenosine deaminase gene. Functional analysis indicated that these clones are capable of encoding murine adenosine deaminase activity when introduced into human cell lines. Structural analysis revealed that the gene consists of 12 exons distributed over approximately 25 kb. The exact size of each exon and the sequence of each exon/intron junction were determined. The results show that the 1056-nucleotide open reading frame for adenosine deaminase extends from exon 1 to exon 11, and that exon 12 contains untranslated sequences only. During the course of these investigations, we discovered that a gene encoding an abundant 1.3-kb polyadenylated transcript overlaps the 3' end of the murine adenosine deaminase gene and is transcribed from the opposite strand.

Identification of transcription stop sites at the 5' and 3' ends of the murine adenosine deaminase gene.

We report here the identification and nucleotide sequence of two transcription termination regions associated with the murine adenosine deaminase gene. One region is situated within or very near exon one and in mouse fibroblasts accounts for more than a 50-fold drop in the abundance of nascent transcripts. This termination region is believed to be involved in the regulation of adenosine deaminase gene transcription. The other termination region is located approximately 3.5 kilobases beyond the major polyadenylation site and defines the 3' boundary of the adenosine deaminase transcription unit.

Characteristics of participants in a large group awareness training.

A study was conducted to assess the psychosocial characteristics of individuals who become involved in large group awareness training (LGAT) programs. Prospective participants in The Forum, which has been classified as an LGAT, were compared with nonparticipating peers and with available normative samples on measures of well-being, negative life events, social support, and philosophical orientation. Results revealed that prospective participants were significantly more distressed than peer and normative samples of community residents and had a higher level of impact of recent negative life events compared with peer (but not normative) samples. Prospective participants also held preparticipation values more similar to those espoused by the LGAT than peer or normative samples, and the three groups failed to be distinguished by their levels of social support. The implications of the findings are considered for understanding participation in LGATs and other self-change promoting activities.

Developmental expression of adenosine deaminase in the upper alimentary tract of mice.

The distribution and localization of adenosine deaminase (ADA) was studied during postnatal development of the alimentary tract in mice. There was detectable enzyme activity in all organs examined, but a range of more than 10,000 fold in the relative levels of specific activity was observed among adult tissues. A comprehensive survey of multiple adult tissues revealed that the highest levels of ADA occur in the upper alimentary tract (tongue, esophagus, forestomach, proximal small intestine). Immunohistochemical analysis revealed that ADA was predominantly localized to the epithelial lining of the alimentary mucosa: the keratinized squamous epithelium that lines the forestomach, esophagus, and surface of the tongue; and the simple columnar epithelium of the proximal small intestine (duodenum, proximal jejunum). Biochemical analysis revealed that ADA was one of the most abundant proteins of these mucosal tissue layers, accounting for 5%-20% of the total soluble protein. Tissue-specific differences in ADA activity correlated both with levels of immunoreactive protein and RNA abundance. The level of ADA activity in the upper alimentary tissues was subject to pronounced developmental control, being low at birth and achieving very high levels within the first few weeks of postnatal life. The appearance in development of ADA-immunoreactivity coincided with maturation of the mucosal epithelium. These results suggest that ADA is subject to strong cell-specific developmental regulation during functional differentiation of certain foregut derivatives in mice.

Adenosine deaminase gene expression. Tissue-dependent regulation of transcriptional elongation.

The mechanism by which the expression of adenosine deaminase (ADA, EC 3.5.4.4) is regulated in murine tissues was analyzed. Elevated levels of the specific activity of this enzyme appeared to correlate directly with steady state levels of ADA mRNA. Transcriptional studies using nuclear run-on experiments revealed a pronounced asymmetric distribution of nascent transcripts across the ADA genome. The ability to detect nascent transcripts downstream of the promoter proximal region varied among tissues and correlated with tissue levels of ADA mRNA and enzymatic activity. These results indicate that processes affecting the elongation of nascent transcripts into completed primary transcripts play a role in tissue-specific expression of ADA.