Aldehyde oxidase and alcohol dehydrogenase genetics in the mouse. New alleles for the Aox-2 and Adh-3 loci

The genetic variability of one of the liver isozymes of aldehyde oxidase (AOX-B₂ or AOX-2) and the stomach isozyme of alcohol dehydrogenase (ADH-C₂) has been examined among strains of mice. Evidence is presented for a fourth allele of Aox-2 and a third allele of Adh-3 . The hybrid allozyme pattern for mouse liver AOX was consistent with a dimeric subunit structure for this enzyme.     

Sorbitol dehydrogenase genetics in the mouse: A ';null' mutant in a ';European' C57BL strain

A ';null' activity variant phenotype for sorbitol dehydrogenase (SDH) was observed in C57BL/LiA mice and used to examine the genetics of this enzyme. Linkage studies of the locus ( Sdh-1 ) with non-agouti (a) and a biochemical Iocus encoding liver L-α-hydroxyacid oxidase ( Hao-1 ) demonstrated that it is coincident with or closely linked to the structural locus, previously localized on chromosome 2. Alcohol dehydrogenase (ADH) isozymes were also examined, since the liver A₂ isozyme exhibited some activity as a sorbitol dehydrogenase on cellulose acetate zymograms. It is apparent that SDH activity is not ';essential' in this mouse strain.     

Alcohol dehydrogenase isozymes in the mouse: Genetic regulation, allelic variation among inbred strains and sex differences of liver and kidney A2 isozyme activity

Genetic analysis of a proposed cis-acting temporal locus ( Adh-3t ), which regulates alcohol dehydrogenase C₂ (ADH-C₂) acitivity in mouse epididymis extracts, among F₁ (ddN × BALB/c) × ddN male backcross progeny provided evidence for genetic distinctness between the structural ( Adh-3 ) and temporal ( Adh-3t ) loci on chromosome 3. Genetic analysis also confirmed the close, linkage of Adh-1 (encoding liver and kidney ADH-A₂) and Adh-3 (encoding stomach ADH-C₂) to within 0.3 centimorgans on the mouse genome. Evidence is presented for a proposed closely linked cis-acting temporal locus (designated Adh-1t ) for the A₂ isozyme (encoded by Adh-1 ) controlling the activity of this enzyme in mouse kidney extracts, but having no apparent affect on liver and intestine ADH-A₂ activities. An extensive survey of the distribution of Adh-1, Adh-3 and Adh-3t alleles among 65 strains of mice is reported — with the exception of two Japanese strains (ddN and KF), linkage disequilibrium between Adh-3 and Adh-3t was observed. Sex differences in mouse liver and kidney ADH-A₂ activities were observed, with male/female ratios of approximately 0.6 and 3 respectively for these tissue extracts.     

Gene markers for alcohol-metabolizing enzymes among recombinant inbred strains of mice with differential behavioural responses towards alcohol

The genetic variability of alcohol dehydrogenase (C2 isozyme), aldehyde dehydrogenase (A2 isozyme) and aldehyde oxidase (A2 isozyme) has been examined among recombinant inbred strains of mice which have been previously studied concerning their differential behavioural responses towards alcohol. The results showed no correlation between biochemical phenotype for these loci and behavioural response.     

Physiological Effects on Periplaneta americana of Bacillus sp. Exotoxin

ABSTRACT This study was performed to evaluate the physiological effects of Bacillus sp. on Periplaneta americana. The insecticidal exotoxin was fractionated by ion-exchange chromatography and gel filtration from Bacillus sp. cultivates. Lp₂ among 4 lipoproteins showed markable increase after 24 hrs, but there was no change in glycoprotein. The enzyme activities of P. americana showed mostly decreasing pattern by treatment of exotoxin fractions. Protease activities decreased at 12 hrs after treatment in comparison with normal subjects. Amylase also showed decreasing pattern and maximal decline was observed after 12 hrs. Trehalase activities decreased at 6 hrs and 24 hrs. Invertase activities decreased from 6 hrs to 24 hrs, but recovered after 48 hrs. Amylase isozyme A₁ and A₂ disappeared and new isozyme appeared after 12 hrs with exotoxin treatment. Trehalase isozyme T₁ and invertase I₄ increased markedly after 12hrs. However, non-specific esterase E1 disappeared after 24hrs.     

Methanol dehydrogenase from Hyphomicrobium MS 223

Abstract The monomethyl sulfate-degrading bacterium, Hyphomicrobium MS 223 , contains a NAD(P)-independent methanol dehydrogenase (EC 1.1.99.8) which was isolated and characterized. The enzyme was activated by ammonium ions, had an M _r of 118000 and was composed of two subunits of identical M _r. It showed a broad substrate specificity for primary alcohols and was able to oxidize secondary alcohols and several aliphatic aldehydes. The new competitive inhibitor acetaldehyde oxime inhibited aldehyde oxidation more strongly than alcohol oxidation.     

Kinetic and structural evidence for the identification of 11-hydroxythromboxane B2 dehydrogenase as cytosolic aldehyde dehydrogenase

We have recently purified 11-hydroxythromboxane B₂ dehydrogenase from porcine kidney and identified it as cytosolic aldehyde dehydrogenase (EC 1.2.1.3) based on amino acid analysis and other protein characteristics. In the present paper we have studied the catalytic interaction of thromboxane B₂ (TXB₂) with different aldehyde substrates and a potent aldehyde dehydrogenase inhibitor, disulfiram. TXB₂ was a competitive inhibitor of the aldehyde dehydrogenase reaction in assays with 3,4-dihydroxyphenylacetaldehyde, a high affinity substrate. The conversion of TXB₂ to 11-dehydro-TXB₂ was also inhibited by propanal and disulfiram.

The protein characteristics of the enzyme have also been further studied. The native enzyme is a tetramer and has an isoelectric point of 7.0 which is comparable with that of cytosolic aldehyde dehydrogenases from other species. Taken together the present data further indicate that 11-hydroxythromboxane B₂ dehydrogenase is identical with cytosolic aldehyde dehydrogenase and that substrates and inhibitors of aldehyde dehydrogenase interact with thromboxane metabolism in vitro.     

Subcellular distribution of pyruvate-degrading enzymes in Chlamydomonas reinhardtii studied by an improved protoplast fractionation procedure

The subcellular distribution of pyruvate-degrading enzymes has been determined in Chlamydomonas reinhardtii (Dangeard) by protoplast induction with autolysine, dig-itonin lysis and further fractionation by differential centrifugation using a Percoll cushion. Mitochondrial and plastidic fractions contained intact and physiologically competent organelles - RC 1.7, ADP/O 2.7 and rate of malate oxidation 76 nmol O, (mg protein)^-1min^-1 for mitochondria, CO_2; fixation 46.8 μmol (mg Chi)^-1 h^-1 for chloroplasts.
Results from protoplast fractionation were further confirmed by the determination of enzyme activities within trypsin-treated organelles. Mitochondria (formate fermentation) and chloroplasts (chlorofermentation) were shown to possess the capacity for anaerobic pyruvate degradation. Pyruvate dehydrogenase (NAD⁺, EC 1.2.4.1), pyruvate formate-lyase (EC 2.3.1.54) and lactate dehydrogenase (NADH, EC 1.1.1.27) showed equal distribution between mitochondria and chloroplasts, whereas activities of phosphotransacetylase (EC 2.3.1.8) and acetate kinase (EC 2.7.2.1) were only detectable in the mitochondrial fraction. NADH- and NADPH-dependent activities of both alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase (acylating, EC 1.2.1.10) were localized in the mitochondrial and cytoplasmic or the plastidic and cytoplasmic fractions, respectively, whereas pyruvate decarboxylase (EC 4.1.1.1) was only detected in the cytoplasmic fraction.     

An immunochemical analysis of the novel liver-restricted LDH activity from cichlid fishes (Cichlidae: Teleostei) confirms its non-orthology with piscine LDH-C

In an electrophoretic survey of lactate dehydrogenase (LDH) isozymes in neotropical cichlid fishes (Perciformes: Cichlidae) several species were discovered in which a cathodal liver-restricted isozyme is expressed along with the highly anodal eye-restricted isozyme (LDH-C₄) typically encountered in perciform fishes. Biochemical characterization of these two isozymes from the basketmouth cichlid, Acaronia nassa (Heckel), strongly suggested that they were non-orthologous and challenged the accepted view that eye- and liver-restricted LDH isozymes are alternative expressions of the same (LDH-C *) gene. In this study, antiserum raised against cypriniform (goldfish) liver-restricted LDH-C₄ failed to cross-react with the basketmouth liver-restricted analogue while effectively titrating the eye-restricted, anodal isozyme and, at higher titres, the LDH-B₄, heart-restricted isozyme from all cichlid species. Anti-serum raised against basketmouth muscle-restricted LDH-A₄ failed to titrate any of the eye- and liver-restricted isozymes. These data confirm the orthology of eye- and liver-restricted LDH isozymes in Cypriniform and Perciform fishes as originally proposed, suggest that the liver-restricted isozyme of cichlid fishes is non-orthologous and further raise the question of identity and evolutionary origin of this anomalous LDH activity.     

Chronic Exposure to Adenosine Receptor Agonists and Antagonists Reciprocally Regulates the A1 Adenosine Receptor-Adenylyl Cyclase System in Cerebellar Granule Cells

Abstract: Chronic treatment with the adenosine receptor antagonist caffeine evokes an up-regulation of A₁ adenosine receptors and increased coupling of the receptor to G proteins in rat brain membranes. However, chronic agonist exposure has not been explored. Primary cultures of cerebellar granule cells were exposed chronically to A₁ adenosine receptor agonists and antagonists. Exposure to the A₁ adenosine receptor agonist N ⁶-cyclopentyladenosine resulted in (1) a time- and concentration-dependent reduction in the density of receptors labeled by 1,3-[³H]dipropyl-8-cyclopentylxanthine, (2) an enhanced ability of guanyl nucleotides to decrease the fraction of A₁ adenosine receptor sites displaying high affinity for 2-chloroadenosine, and (3) a functional uncoupling of receptors from adenylyl cyclase (EC 4.6.1.1). The adenosine antagonists caffeine and 8- p -sulfophenyltheophylline produced alterations in A₁ adenosine receptor homeostasis that were antipodal to those associated with agonist treatment. Antagonist exposure (1) increased the density of A₁ adenosine receptors in cerebellar granule cell membranes, (2) blunted the effect of guanyl nucleotides on receptor coupling to G proteins, and (3) increased the functional coupling of receptors to adenylyl cyclase inhibition. Forskolin treatment of cerebellar granule cells did not affect receptor density, suggesting that cyclic AMP is not involved in the regulation of A₁ adenosine receptor expression.     

Desensitisation of the Adenosine A1 Receptor by the A2A Receptor in the Rat Striatum

Abstract: The influence of the adenosine A_2A receptor on the A₁ receptor was examined in rat striatal nerve terminals, a model for other cells in which these receptors are coexpressed. Incubation of striatal synaptosomes with the A_2A receptor agonist 2- p -(2-carboxyethyl)phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS 21680) caused the appearance of a low-affinity binding site for the A₁ receptor agonist 2-chloro- N ⁶-cyclopentyladenosine (CCPA). This effect was blocked by the A_2A receptor antagonist ZM241385 and by the protein kinase C inhibitor chelerythrine, but not by the protein kinase A inhibitor N -(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004). The effect was not seen with striatal membranes or with hypotonically lysed synaptosomes. These results demonstrate a protein kinase C-mediated heterologous desensitisation of the A₁ receptor by the A_2A receptor.     

CELL CYCLE PROPERTIES OF DIFFERENTIATING SPERMATOGONIA IN ADULT SPRAGUE-DAWLEY RATS

The duration of the mitotic cycle and of its components was analysed for each of the six successive generations of differentiating spermatogonia (A₁, A₂, A₃, A4, intermediate and B), using radioautographed whole mounts of seminiferous tubules from testes of adult Sprague-Dawley rats. Cell cycles were determined from two successive waves of per cent labeled metaphases obtained during the period of 81 hr after a single dose of ³H-thymidine. Except for the A₁ spermatogonia, all spermatogonial types (A₂ to B) had similar cell cycle durations of 41-42.5 hr and comparable pre-DNA synthesis phases (G₁) of 11-13 hr. Although the combined duration of DNA synthesis (S) and the post-synthesis phase (G₂) remained identical for all the cell types including A₁, there was a progressive lengthening of the S period at the expense of G₂ during the process of spermatogonial maturation. This change was most marked during the transition from A₁ to A₃ spermatogonia when the S period increased from 14 hr to 21 hr, and the G₂ phase shortened from 13 hr to 7.5 hr. This feature seems to be unique to germ cells and may be associated with an increasing amount of heterochromatin in the nucleus. Excluding the development of type A₁ cells, the entire process of spermatogonial maturation lasted for 208 hr. Combined data on cell cycle times indicated that every 313 hr or 13 days, a new sequence of spermatogonial differentiation was initiated by the A₁ cells. This was equivalent to the duration of one 'cycle' of the seminiferous epithelium as measured by other techniques.     

Adenosine A2b Receptors Mediate an Increase in Interleukin (IL)-6 mRNA and IL-6 Protein Synthesis in Human Astroglioma Cells

Abstract: The cytokine interleukin (IL)-6 has recently been demonstrated to play a role in the pathology of Alzheimer's disease (AD). The mechanisms leading to increased IL-6 levels in brains of AD patients are still unknown. Because in experimental animals ischemia increases both the level of cytokines and the extracellular concentrations of adenosine in the brain, we hypothesized that these two phenomena may be functionally connected and that adenosine might increase IL-6 gene expression in the brain. Here we show that the mixed A₁ and A₂ agonist 5'-( N -ethylcarboxamido)adenosine (NECA) induces an increase in IL-6 mRNA levels and protein synthesis in the human astrocytoma cell line U373 MG. The A₁-specific agonists R -phenylisopropyladenosine and cyclopentyladenosine are much less potent, and the A_2a-specific agonist CGS-21680 shows only marginal effects. Increased levels of mRNA are already found within 30 min after NECA treatment. The A_2a-selective antagonists 8-(3-chlorostyryl)caffeine and KF17837 [( E )-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine], which have also some antagonistic properties at A_2b receptors, and the nonspecific adenosine antagonist 8-phenyltheophylline were equipotent at inhibiting the NECA-induced increase in IL-6 protein synthesis, whereas the specific A₁ antagonist 8-cyclopentyl-1,3-dipropylxanthine is much less potent. The results indicate that adenosine A_2b receptors participate in the regulation of the IL-6 gene in astrocytoma cells.     

Aldehyde Dehydrogenases in Rat Brain. Subcellular Distribution and Properties

Abstract: Kinetic studies suggested the presence of several forms of NAD-dependent aldehyde dehydrogenase (ALDH) in rat brain. A subcellular distribution study showed that low- and high- K _m activities with acetaldehyde as well as the substrate-specific enzyme succinate semialdehyde dehydrogenase were located mainly in the mitochondrial compartment. The low- K _m activity was also present in the cytosol (<20%). The low- K _m activity in the homogenate was only 10–15% of the total activity with acetaldehyde as the substrate. Two K _m values were obtained with both acetaldehyde (0.2 and 2000 μ m ) and 3,4-dihydroxyphenylacetaldehyde (DOPAL) (0.3 and 31 μ m ), and one K _m value with succinate semialdehyde (5 μ m ). The main part of the aldehyde dehydrogenase activities with acetaldehyde, DOPAL, and succinate semialdehyde, but only little activity of the marker enzyme for the outer membrane (monoamine oxidase, MAO), was released from a purified mitochondrial fraction subjected to sonication. Only small amounts of the ALDH activities were released from mitochondria subjected to swelling in a hypotonic buffer, whereas the main part of the marker enzyme for the intermembrane space (adenylate kinase) was released. These results indicate that the ALDH activities with acetaldehyde, DOPAL and succinate semialdehyde are located in the matrix compartment. The low- K _m activity with acetaldehyde and DOPAL, but not the high- K _m activities and succinate semialdehyde dehydrogenase, was markedly stimulated by Mg²⁺ and Ca²⁺ in phosphate buffer. The low- and high- K _m activities with acetaldehyde showed different pH optima in pyrophosphate buffer.     

Endogenous gibberellins in young seedlings of wheat (Triticum aestivum) cultivars

Gibberellins A₁, A₃, A₄ and A₇ were identified by combined gas chromatography mass spectrometry (GC-MS) in leaf and stem tissues of 17-day-old seedlings of wheat ( Triticum aestivum L. ), cvs Siete Cerros (semi-dwarf, Rht1) and Møystad (tall), of F₁, hybrids from the cross Møystad × Siete Cerros and of 2 selected lines from the cross Møystad x Sonora 64 (Rht1 and Rht2). GA, and GA, were identified by full scan mass spectra separately in all 5 extracts, GA₄ and GA₇, were identified by selected ion monitoring in a bulked fraction. About 90% of the biological activity (Tan-ginbozu dwarf rice bioassay) in all 5 extracts was due to the GA₁/GA₃-fraction.     

Modulation of adenosine A1 and A2A receptors in C6 glioma cells during hypoxia: involvement of endogenous adenosine

During hypoxia, extracellular adenosine levels are increased to prevent cell damage, playing a neuroprotective role mainly through adenosine A₁ receptors. The aim of the present study was to analyze the effect of hypoxia in both adenosine A₁ and A_2A receptors endogenously expressed in C6 glioma cells. Two hours of hypoxia (5% O₂) caused a significant decrease in adenosine A₁ receptors. The same effect was observed at 6 h and 24 h of hypoxia. However, adenosine A_2A receptors were significantly increased at the same times. These effects were not due to hypoxia-induced alterations in cells number or viability. Changes in receptor density were not associated with variations in the rate of gene expression. Furthermore, hypoxia did not alter HIF-1α expression in C6 cells. However, HIF-3α, CREB and CREM were decreased. Adenosine A₁ and A_2A receptor density in normoxic C6 cells treated with adenosine for 2, 6 and 24 h was similar to that observed in cells after oxygen deprivation. When C6 cells were subjected to hypoxia in the presence of adenosine deaminase, the density of receptors was not significantly modulated. Moreover, DPCPX, an A₁ receptor antagonist, blocked the effects of hypoxia on these receptors, while ZM241385, an A_2A receptor antagonist, was unable to prevent these changes. These results suggest that moderate hypoxia modulates adenosine receptors and cAMP response elements in glial cells, through a mechanism in which endogenous adenosine and tonic A₁ receptor activation is involved.     

Adenosine A1 Receptors in Cultured Cerebellar Granule Cells: Role of Endogenous Adenosine

Abstract: Adenosine A₁ receptors as well as other components of the adenylate cyclase system have been studied in cultured cerebellar granule cells. No significant changes in adenosine A₁ receptor number, assayed by radioligand binding in intact cells, were detected from 2 days in vitro (DIV) until 7 DIV. Nevertheless, a decline in this parameter was detected at 9 DIV. The steady-state levels of α-G_s and α-G_i, detected by immunoblotting, showed similar profiles, increasing from 2 to 5 DIV and decreasing afterward. Forskolin-stimulated adenylate cyclase levels also showed an increase until 5 DIV, decreasing at 7 and 9 DIV. The adenosine A₁ receptor analogue cyclopentyladenosine (CPA) was able to inhibit cyclic AMP accumulation at 2, 5, and 7 DIV but failed to do so at 9 DIV. This inhibition was prevented by the specific adenosine A₁ receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. The presence of adenosine deaminase in the culture increased adenosine A₁ receptor number during the period studied and induced recovery of the inhibitory effect of CPA, lost after 7 DIV. These data suggest that functional expression of adenosine A₁ receptors and the other components of the adenylate cyclase system is subjected to regulation during the maturation of cultured cerebellar granule cells and demonstrates a key role for endogenous adenosine in the process.     

SPERMATOGONIAL STEM CELL RENEWAL IN THE MOUSE: II AFTER CELL LOSS

The repair of the mouse seminiferous epithelium after cell loss has been studied in seminiferous tubules mounted in toto . Cell loss was inflicted by injection of Myleran in a dose of 10 mg/kg body weight. In stages 7–8, in which we mainly counted, the numbers of A_isolated (A_is), A_paired (A_pr), A_aligned (A_al) and A₁ spermatogonia and resting primary spermatocytes decreased after injection. After about 24 days normal numbers of A₁ spermatogonia were found again. Thereafter a substantial overshoot in the number of A₁ spermatogonia was found.
While normally most of the A_pr and A_al cells differentiate into A₁ spermatogonia in stages 3 and 4 and do not divide until stage 9, during repair they pass through one more division during stages 6 and 7. Normally, during these stages divisions of these spermatogonia are rare. Owing to this extra division the transformation of A_pr and A_al into A₁ spermatogonia is delayed from stage 3 or 4 to stage 8, i.e. still before stage 9, in which A₁ spermatogonia divide. From 16 days after the injection onwards the extra division takes place less generally and more and more cells transform into A₁ spermatogonia at the normal time.     

Chronic Ethanol Reduces Immunologically Detectable Gqα/11α in NG108–15 Cells

Abstract: The amyloid protein (βA₄) is found in the CNS of patients with Alzheimer's disease; however, the pathogenic role of this protein is not known. In the present study, a peptide fragment of βA₄βA₄ 25–35; Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-NH₂), which contains the conserved C-terminal sequence of substance P (X-Gly-Leu-Met-NH₂), and the neuropeptide substance P (SP) were examined for their ability to modulate nicotine-evoked secretion from cultured bovine adrenal chromaffin cells. Secretion of the released endogenous catecholamines was monitored by electrochemical detection after separation by HPLC. Secretion induced by 10⁻⁵ M nicotine was inhibited by SP and βA₄ 25–35. The IC₅₀ of SP and βA₄ 25–35 was 3 × 10⁻⁶ and 3 × 10⁻⁵ M , respectively. SP and βA₄ 25–35 both protected against nicotinic receptor desensitization. However, βA₄ 25–35 was ∼ 10-fold less effective than SP in its protective effect. The present work shows that βA₄ 25–35 can mimic the modulatory actions of SP on the nicotinic response of cultured bovine chromaffin cells, i.e., inhibition of the nicotinic response and protection against nicotinic desensitization. These modulatory actions may be associated with changes in nicotinic receptor levels reported to occur in Alzheimer's disease.     

EXPRESSION OF A BEE-VENOM PHOSPHOLIPASE A2 FROM APIS CERANA CERANA IN ESCHERICHIA COLI

Abstract  The venomous phospholipase A₂ (AcPLA₂) coding reading region of the Chinese honeybee ( Apis cerana cerana ), which is composed of 405 bp encoding a mature glycosylated peptide with 134 amino residues was transformed into the expression vector pETblue-1. Then the recombinant vector was introduced into Escherichia coli Tuner (DE3) plac I for expression. Analysis result of SDS-PAGE showed that the expression products had a protein band of about 15kD. Detection of western blot using ant-European honeybee ( Apis mellifera ) phospholipase A₂ (AmPLA₂) polyclonal serum as the first antibody showed that the expression products appeared a special blot same as the native AmPLA₂.The result demonstrated that the AcPLA₂ peptide had been expressed in E. coli and the AcPLA₂ has the similar antigenicity as the AmPLA₂.