Early changes in mitochondrial monoamine oxidase activity of rat liver during 2-acetylaminofluorene feeding

The activities of mitochondrial type A and B monoamine oxidase were determined in the liver of rats fed a diet containing 2-acetylaminofluorene (AAF). Three days after the initiation of AAF-feeding, there was a significant decrease of type B monoamine oxidase activity without affect on type A enzyme. The decreased activity of type B monoamine oxidase, which reached a minimum after three weeks, was sustained for as long as AAF-feeding was continued. Sex-related difference in response to AAF was seen in the rat with respect to the onset and the intensity of the decreased type B monoamine oxidase activity, male rats being more sensitive to the carcinogen than female rats. In contrast to the in vivo effect, AAF showed a potent inhibitory effect on type A monoamine oxidase, rather than on type B enzyme, when added in vitro. The pI₅₀ values were estimated to be 7.5 against type A monoamine oxidase and 4.1 against type B enzyme, respectively. The in vitro inhibition of both types of monoamine oxidase by AAF was competitive. The K_i values for AAF were calculated to be 9.51 · 10^?9 M for type A monoamine oxidase and 1.30 · 10^?5 M for type B enzyme, respectively. In accordance with the potent inhibitory effect of AAF on type A monoamine oxidase in vitro, a single administration of the carcinogen, at a dose of 50 mg/kg, resulted in a marked and temporal decrease of the enzyme activity in the mitochondria of male rat liver. Recovery of the decreased type B monoamine oxidase activity was slow, and the enzyme activity did not return to control levels, even if rats were fed the basal diet for 2 or 4 weeks after the cessation of AAF-feeding.     

Relationship between general or specific immunoreactivity and prognosis in postoperative patients with hepatocellular carcinoma

Summary The levels of a variety of immunological parameters were examined in 203 preoperative patients with hepatocellular carcinoma (HCC) at various stages (I–IV). The changes in the peripheral blood lymphocyte (PBL) count, the serum level of immunosuppressive acidic protein and the degree of the skin reaction to purified protein derivative were associated significantly with the stage of HCC progression. However, the percentages of lymphocyte subsets, mitogenic responsiveness of PBL and serum immunoglobulin concentration remained at the levels of stage I. Further study demonstrated that in patients undergoing hepatic artery ligation, there were statistically significant correlations between the PBL count, immunosuppressive acidic protein concentration and intensity of the skin reaction to purified protein derivative, assayed 1 month after surgery, and the prognosis. HCC-specific immunity was examined in 34 patients treated by hepatic resection or hepatic artery ligation using in vitro responses of PBL to HCC extracts (ATS test). This test was performed using culture medium containing added arginine. None of the PBL from the patients showed a positive response to allogeneic HCC extracts, but the PBL from 12 patients (9 hepatic resections, 3 hepatic artery ligations) were stimulated significantly (SI 2.5) with autologous HCC extracts. In 7 of 9 hepatic resection patients who were positive in the ATS test, tumor recurrence was identified. Statistical analysis indicated that the ATS test result was significantly correlated with tumor recurrence in hepatic resection patients. Autologous-PBL-stimulating activities were isolated in a fraction at pH 8.3 and in fractions at pH 6.7–7.0 by chromatofocusing of the crude extract. Although identification of the HCC-specific antigen remains to be done, use of the above fractions may simplify the ATS test procedure and improve its sensitivity.     

Cloning and sequence analyses of cDNAs encoding vasotocin and isotocin precursors of chum salmon,Oncorhynchus keta: evolutionary relationships of neurohypophysial hormone precursors

Summary The nucleotide sequences of cloned cDNAs were used to determine the primary structures of the precursors of vasotocin (sVT) and isotocin (sIT) from the hypothalamus of the chum salmon,Oncorhynchus keta. Two different cDNAs were obtained for each of sVT and sIT precursors (sVT-I and sVT-II; sIT-I and sIT-II). Both sVT and sIT precursors were found to contain a signal peptide and hormone that is connected to a neurophysin by a Gly-Lys-Arg sequence. Northern and Southern blot analyses showed that the sVT and sIT genes are expressed by the same chum salmon hypothalamus, but not by the liver and kidney. Microheterogeneity was found in the nucleotide and amino acid sequences of sVT precursors between our results and the previously reported data (Heierhorst et al. 1990). The conspicuous difference is the occurrence of a stop codon in the middle of sVT-II cDNA. The carboxyl termini of both sVT and sIT neurophysins are about 30 amino acids longer than neurophysins of toad and mammalian neurohypophysial hormone precursors. Although these extended regions do not contain a glycosylation site, they show striking similarity with the glycopeptide moiety (copeptin) of toad vasotocin and mammalian vasopressin precursors. The central portion of the neurophysins shows highest homology among corresponding regions of sVT and sIT precursors. Moreover, calculation of nucleotide substitution rates suggests that a recent gene conversion may have occurred which encompasses the exon that encodes the central segment of the sVT and sIT precursors. A possible pathway for the evolution of precursor molecules of neurohypophysial hormones is discussed.Abbreviations AVP vasopressin - C carboxyl - h human - IT isotocin - MT mesotocin - N amino - OXT oxytocin - S chum salmon - SDS sodium dodecyl sulfate - t toad - VT vasotocin     

Selective inhibitors of germination of legume seeds in activated sludge compost

Water extracts of the compost produced from activated sludge and coffee residue were found to be selectively inhibitory to seed germination of some legumes. Germination rate of white clover (Trifolium repens L.), red clover (Trifolium pratense L.) and alfalfa (Medicago sativa L.) seeds were reduced to 2, 29 and 73% of the control, respectively, by water extracts of the compost (20 g l^–1). However, the extracts did not show any inhibition to seed germination of sorghum (Sorghum bicolor Moench), African millet (Eleusine coracana Gaertn.), and Komatsuna (Brassica rapa L.) at the same concentration. The inhibitors in the compost extracts were separated by ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC) and the inhibitory activities of seed germination were tested with white clover seeds. Five inhibitors were isolated and identified as 3,4-dichlorophenylacetic acid (3,4-DCP), 3,4-dichlorobenzoic acid (3,4-DCB), 3,4,5-trichlorophenylacetic acid, 3,4,5-trichlorobenzoic acid and mono-2-ethylhexylphthalate by ¹H-, ¹³C-NMR spectroscopy and mass spectrometry. The inhibitory activities of some authentic chemicals of the inhibitors and the related compounds were compared. The results indicated that the main inhibitor in the compost could be 3,4-DCB, which was contained at the concentration of 6.58 mg kg^–1 compost and showed the strongest inhibitory effect on seed germination of white clover among the tested compounds.     

Androgen Regulates Gene Expression of Cytoskeletal Proteins in Adult Rat Motoneurons

Expression of β-actin and β-tubulin mRNA was examined in androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats by in situ hybridization histochemistry using complementary DNAs encoding chick β-actin and mouse β-tubulin, respectively. Both hybridizable β-actin and βtubulin mRNAs were localized in the somata and proximal dendrites of SNB motoneurons. Removal of androgen by castration significantly reduced the expression levels of both β-actin and β-tubulin mRNAs in the SNB motoneurons, whereas the changes were prevented by testosterone treatment. In contrast, castration or testosterone treatment induced little or no change in the expression levels of these mRNAs in the much less androgen-sensitive motoneurons of the retrodorsolateral nucleus (RDLN). These results suggest that androgen regulates the expression of β-actin and β-tubulin genes in the SNB motoneurons and may provide evidence for the molecular mechanisms of hormonally induced neuronal plasticity in the SNB motoneurons.     

Identification and Biological Activity of Germination-Inhibiting Long-Chain Fatty Acids in Animal-Waste Composts

Long-chain fatty acids in germination-inhibiting animal-wastecomposts were identified by gas chromatography-mass spectrometryas myristic, palmitic, stearic, oleic, linoleic, and linolenicacids. These acids were found at concentrations greater than0.25 mg (g dry compost)^–1. The identified acids, togetherwith lauric acid, and five kinds of short- and medium-chainfatty acid, were tested for their effects on the germinationprocess of sorghum seeds. The authentic long-chain fatty acids, which were dissolved ina 1:9 (v/v) mixture of methanol and distilled water at 40 mgliter^–1, significantly reduced the -amylase activity,physiological water uptake, and ATP content of the germinatingseeds during the first 24h of imbibition, as well as the rateof germination of seeds. Among the tested fatty acids, myristicand palmitic acids were the most potent inhibitors of germination.The inhibitory effects of long-chain fatty acids were strongerthan those of the phenolic acids. The short- and medium-chainfatty acids did not have any significant germination-inhibitoryeffects at 40 mg liter^–1. The results indicate that thelong-chain fatty acids are the dominant inhibitors of germinationin animal-waste composts, and that the inhibition of the -amylaseactivity in germinating sorghum seeds is one aspect of the modeof action of these long-chain fatty acids. ¹On leave from the Department of Crop Science, Faculty of Agriculture,University of Peradeniya, Sri Lanka.     

Successful expression in pollen of various plant species of in vitro synthesized mRNA introduced by particle bombardment

Gold particles coated with -glucuronidase (GUS) mRNA with a 5 cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.     

Mucopolysaccharidosis IVA: structural gene alterations identified by Southern blot analysis and identification of racial differences

Ninety-six alleles (36 alleles of Japanese and 60 of Caucasian origin) from forty-eight patients with mucopolysaccharidosis IVA were investigated for structural gene alterations using Southern blot analysis. All patients had a previously demonstrated deficiency of N-acetylgalactosamine-6-sulfate-sulfatase and exhibited a wide spectrum of clinical severity. Initially, using the fulllength cDNA as a probe, five of 36 chromosomes from the Japanese patients revealed similar rearrangements with respect to DNA digested with BamHI, SacI, and XhoI. Subsequent analysis using seven genomic fragments, covering the entire gene, enhanced the detection of aberrant fragments produced by the above restriction enzymes. Conversely, the 60 chromosomes of Caucasian origin revealed no evidence of large structural rearrangements when analyzed by these methods. There was a statistically significant difference between the two populations (P < 0.01). A severely affected Japanese patient showed structural rearrangements on both chromosomes by means of BamHI blots. An 8.0-kb fragment and a highly polymorphic 7.0-kb to 11.0-kb fragment present in normal individuals disappeared and two aberrant fragments of 11.5 kb and 12.0 kb were observed. Three other Japanese patients also showed these two aberrant fragments, in addition to the normal fragment pattern, and were thus heterozygous for this rearrangement. Interpretation of Southern blots was difficult because of the complexity of polymorphic bands resulting from variable number of tandem repeat elements. However, by utilizing these aberrant fragments or polymorphic bands, carrier detection was effective, even in families with poorly characterized mutations. Hybridization with probe MG-A (5end genomic probe in intron 1) showed a 8.4-kb fragment in BamHI blots of one Japanese and one Caucasian patient; XhoI, SacI, and EcoRI blots were normal. Since this BamHI alteration was also observed in one normal control, it appears to be a rare nonpathological polymorphism.     

Mucopolysaccharidosis IVA: polymorphic haplotypes and informative RFLPs in the Japanese population

Seven different restriction fragment length polymorphisms (RFLPs) at the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) locus were analyzed using Southern blotting and polymerase chain reaction based techniques to search for the frequency of each RFLP produced by StyI, SphI, HaeIII, StuI, HapII, XhoI, and BamHI restriction endonucleases, respectively, in 36 mutant alleles, including two sibling cases and 100 normal alleles. Calculation of heterozygosity indexes showed that these RFLPs were polymorphic, ranging from 0.31 to 0.69 in mucopolysaccharidosis IVA (MPS IVA) patients compared with 0.21 to 0.65 in normal individuals. There was some significant difference in several RFLPs and in the combination with four kinds of RFLPs (SphI, StuI, HapII, XhoI polymorphisms). The normal alleles were composed of 13 different RFLPs haplotypes; the most common among the Japanese population carrying normal alleles was haplotype 8 (bDEF1) (31.3%), the others being dispersed. The same haplotype 8 was the most frequent in the mutant alleles (44.4%), with seven further haplotypes. These findings revealed the striking variety of polymorphic haplotypes in the MPS IVA gene. By using these five kinds of RFLPs, we examined the theoretical informativity of haplotype analysis in heterozygote detection in nine unrelated MPS IVA families and ten unrelated normal families. All the members of the MPS IVA families studied were diagnosed as a patient, carrier, or noncarrier. We propose that prenatal diagnosis or family analysis in cases in which mutations have not been characterized is now feasible.     

Induction of phenylalanine ammonia-lyase and increase in phenolics in lettuce leaves in relation to the development of russet spotting caused by ethylene

Russet spotting (RS), consisting of numerous small brown spots on the midrib of head lettuce (Lactuca sativa), is a physiological disorder induced by exposure to ethylene. In leaves suffering RS, the increase in spotting was accompanied by a parallel increase in the amount of phenolic compounds. Of these, chlorogenic acid and isochlorogenic acid were identified. Ethylene induced high phenylalanine ammonia-lyase (PAL) activity and RS formation in the susceptible cultivar Salinas, but not in the resistant cultivar Calmar. In the absence of ethylene neither significant PAL induction nor RS occurred. No correlation was found between the increase in polyphenol oxidase or peroxidase and the development of RS. The increase in PAL activity, however, was closely correlated with the development of RS. The increase in PAL activity preceded the development of RS, and the extent of RS was directly related to the level of PAL. Three temperatures (0.5, 5.5, and 12.5 C) were compared on the basis of their influence on both RS and PAL induction. At the lowest temperature (0.5 C) neither PAL induction nor RS occurred to a significant extent. At the highest temperature (12.5 C) an initial rapid increase in PAL activity and an earlier development of spotting were observed, but subsequently there was a decrease in both PAL activity and the rate of development of RS. At the medium temperature (5.5 C) both PAL activity and RS increased progresively with time. The decline of PAL activity at a higher temperature might be attributed to inactivation of the enzyme. Thus, a temperature favorable for induction of PAL activity by ethylene was also favorable for RS. These observations indicate a close interrelationship between the induction of PAL activity and the development of RS in response to ethylene, and suggest a causal relationship between the two events. PAL serves as a useful biochemical marker for the RS reaction.