Restoration of Dioxin-Induced Damage to Fetal Steroidogenesis and Gonadotropin Formation by Maternal Co-Treatment with α-Lipoic Acid

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an endocrine disruptor, causes reproductive and developmental toxic effects in pups following maternal exposure in a number of animal models. Our previous studies have demonstrated that TCDD imprints sexual immaturity by suppressing the expression of fetal pituitary gonadotropins, the regulators of gonadal steroidogenesis. In the present study, we discovered that all TCDD-produced damage to fetal production of pituitary gonadotropins as well as testicular steroidogenesis can be repaired by co-treating pregnant rats with α-lipoic acid (LA), an obligate co-factor for intermediary metabolism including energy production. While LA also acts as an anti-oxidant, other anti-oxidants; i.e., ascorbic acid, butylated hydroxyanisole and edaravone, failed to exhibit any beneficial effects. Neither wasting syndrome nor CYP1A1 induction in the fetal brain caused through the activation of aryl hydrocarbon receptor (AhR) could be attenuated by LA. These lines of evidence suggest that oxidative stress makes only a minor contribution to the TCDD-induced disorder of fetal steroidogenesis, and LA has a restorative effect by targeting on mechanism(s) other than AhR activation. Following a metabolomic analysis, it was found that TCDD caused a more marked change in the hypothalamus, a pituitary regulator, than in the pituitary itself. Although the components of the tricarboxylic acid cycle and the ATP content of the fetal hypothalamus were significantly changed by TCDD, all these changes were again rectified by exogenous LA. We also provided evidence that the fetal hypothalamic content of endogenous LA is significantly reduced following maternal exposure to TCDD. Thus, the data obtained strongly suggest that TCDD reduces the expression of fetal pituitary gonadotropins to imprint sexual immaturity or disturb development by suppressing the level of LA, one of the key players serving energy production.     

Histochemical demonstration of O-glycosidically linked,type 3 based ABH antigens in human pancreas using lectin staining and glycosidase digestion procedures

Summary Histochemical analyses of the chemical structures of sugar sequences with or without blood group specificity were carried out by combined stepwise digestion of tissue sections with exo-and endoglycosidases and subsequent lectin stainings in formalin-fixed, paraffin-embedded human pancreas. In acinar cells from blood group A or AB secretor individuals, sequential digestion with -N-acetylgalactosaminidase and -L-fucosidase imparted reactivity with peanut agglutinin (PNA) in cells reactive with Dolichos biflorus agglutinin as well as those with Ulex europaeus agglutinin I(UEA-I). Simple fucosidase digestion imparted the PNA reactivity only in UEA-I reactive cells. Sequential digestion with -galactosidase and fucosidase likewise liberated the PNA binding sites in Griffonia simplicifolia agglutinin I-B₄ reactive cells from blood group B and AB secretors. Sialidase digestion liberated the PNA binding sites not only in acinar cells but also intercalated duct cells, islet cells of Langerhans and endothelial cells. The PNA reactivity obtained by these enzyme digestions was eliminted by endo--N-acetylgalactosaminidase (endo-GalNAcdase) digestion. Preexisting PNA affinity in acinar cells from nonsecretors was also susceptible to endo-GalNAcdase treatment. Following the endo-GalNAcdase digestion, fucosidase or sialidase digestion recovered the PNA reactivity in acinar cells from nonsecretors. These results show that ABH determinants carried on O-glycosidically linked type 3 chain (D-galactose-(1-3)-N-acetyl-D-galactosamine1-serine or threonine) are secreted in pancreatic acinar cells and suggest that product coded by the secretor gene is required for the complete conversion of type 3 precursor chains into H determinants.     

N-terminal amino acid sequence of human urinary prokallikrein

The N-terminal amino acid sequences of human urinary prokallikrein and kallikrein have been determined. Their amino acid sequences are as follows. (Formula; see text) The results showed that prokallikrein comprises an additional seven amino acids at the amino terminus of the kallikrein, of which the sequence is (H2N)Ala-Pro-Pro-Ile-Gln-Ser-Arg(COOH). Comparison of the structure of this peptide with those of other proteins revealed extensive sequence identity with the propeptide portions of rat and mouse tissue kallikreins, that were predicted from the preproenzyme-encoded nucleotide sequences. The amino acid sequence of the peptide was also highly homologous to that of the propeptide portion of EGF-binding protein, that was predicted from the nucleotide sequence, and that of the alpha-subunit of NGF. The N-terminal amino acid sequence of kallikrein was completely identical to the reported one (Lottspeich, F., et al. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1947-1950) and shows considerable amino acid sequence homology with the porcine and rat pancreatic kallikreins. As far as the present results are concerned, it is strongly indicated that the inactive kallikrein in human urine is a tissue type prokallikrein which is activated on the release of the N-terminal peptide consisting of seven amino acids.     

Purification and characterization of the active serine: pyruvate aminotransferase of rat liver mitochondria expressed in Escherichia coli

In the previous study (Oda, T., et al. (1985) Eur. J. Biochem. 150, 415-421), we isolated a cDNA clone which expressed in Escherichia coli a specific size of product having the activity of rat liver serine:pyruvate aminotransferase (SPTm). This specific product (SPT10) was purified to homogeneity through three different column chromatographies. The amino acid composition and N-terminal amino acid sequence of the purified enzyme agreed with those predicted from the nucleotide sequence of cDNA and showed that SPT10 consists of the whole amino acid sequence of mature SPTm and several extra amino acid residues at the N-terminus. The catalytic and physical properties of SPT10, such as substrate specificity, Km for alpha-keto acids, electric charge, and quaternary structure, were all very similar to those of SPTm. Using several cDNA clones which lack a 5'-terminal sequence corresponding to a portion of the N-terminal amino acid sequence of SPTm, we examined the expression profile of the specific product in bacteria transformed with each cDNA clone. The products encoded by these cDNAs were segregated into inclusion bodies and were neither catalytically active nor easily solubilized by sonication. In contrast, the inclusion bodies were not formed in the bacteria transformed with the cDNA clone for SPT10.     

Protective role of potentially lethal damage repair in the neoplastic transformation of Balb/c 3T3 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine

To determine the role of repair of potentially lethal damage (PLD) in the initiation process of neoplastic transformation, Balb/c 3T3 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were temporarily exposed to conditioned medium obtained from density-inhibited Chinese hamster cell cultures, as a post-treatment for the induction of PLD repair. With or without this exposure, cell survival and transformation frequencies were simultaneously determined by colony-formation and focus-formation assays, respectively. Temporary exposure to conditioned medium resulted in a 20-30% increase in cell survival compared with no exposure. Post-treatment with conditioned medium resulted in a 60-65% reduction in transformation frequencies. At the molecular level, the repair of MNNG-induced single-strand breaks of DNA occurred much more rapidly in conditioned medium. These data suggest that PLD repair reduces the in vitro neoplastic transformation through excision repair operative during the cessation of DNA replication. Thus, PLD repair appears to be preventive against neoplastic fixation in initiation of neoplastic development.     

Escherichia coli engineered to produce eicosapentaenoic acid becomes resistant against oxidative damages

The colony-forming ability of Escherichia coli genetically engineered to produce eicosapentaenoic acid (EPA) grown in 3mM hydrogen peroxide (H(2)O(2)) was similar to that of untreated cells. It was rapidly lost in the absence of EPA. H(2)O(2)-induced protein carbonylation was enhanced in cells lacking EPA. The fatty acid composition of the transformants was unaffected by H(2)O(2) treatment, but the amount of fatty acids decreased in cultures of cells lacking EPA and increased in cultures of cells producing EPA, suggesting that cellular EPA is stable in the presence of H(2)O(2) in vivo and may protect cells directly against oxidative damage. We discuss the possible role of EPA in partially blocking the penetration of H(2)O(2) into cells through membranes containing EPA.     

Analysis of gender difference of cardiac risk biomarkers using hGH-transgenic mice.

We investigated gender difference in the effects of chronic exposure to human growth hormone (hGH) on cardiac risk biomarkers using transgenic mice with non-pulsatile circulating hGH. Blood plasma was obtained from transgenic and control mice at 8, 12, and 16 weeks of age, and was used for the measurement of hGH and the following cardiac risk biomarkers: total cholesterol (CHO), triglyceride (TG), HDL cholesterol (HDL), LDL cholesterol (LDL), non esterified free fatty acids (NEFA), and lipid peroxides (LPO). The hearts and the livers of transgenic mice were weighed and histopathologically examined, and the results were compared with those of control mice. Transgenic males exhibited higher levels of LDL at 8 and 12 weeks of age and higher levels of LPO at every week of age examined, as compared to those of the control males, while transgenic females exhibited somewhat lower levels of LDL and LPO from 8 to 16 weeks of age, as compared to the control females. The relative heart weight in males increased with aging and was significantly higher in the 16-week-old transgenic males compared to those of the control mice. The present results demonstrate that transgenic males had cardiac risk potential caused by chronic-exposure to hGH as compared to females. The results also show that the present transgenic mouse line is a useful model for the study of gender difference in cardiac disorders caused by hGH.     

Involvement of Ca2+-dependent proteasome in the degradation of both cyclin B1 and Mos during spontaneous activation of matured rat oocytes

In matured rat oocytes, spontaneous activation from the metaphase-II (MII) stage occurred after collection from the oviducts. It is well known that the mitogen-activated protein kinase (MAPK) pathway and p34(cdc2) kinase play an important role in the arrest at MII in other species. However, there is no information about the difference in these factors among strains of rats. In the present study, in spontaneously activated oocytes from the Wistar rat, the Mos protein level and the activity of MAPK kinase (MEK)/MAPK were decreased at 120 min (13.8, 25.7, and 19.3, respectively, P<0.05), whereas Sprague-Dawley (SD) oocytes, which were not spontaneously activated, had a high level of Mos protein and MEK/MAPK activity (75.9, 76.2, and 87.9, respectively, P<0.05). Phosphorylation of MAPK in the SD oocytes was significantly suppressed by MEK inhibitor, U0126 at 60 min; this treatment decreased p34(cdc2) kinase activity via cyclin B1 degradation in a time-dependent manner. The treatment with proteasome inhibitor, MG132 or Ca2+-chelator, BAPTA-AM, overcame the spontaneous degradation of both Mos and cyclin B1 in a dose-dependent manner in Wistar oocytes. More than 90% of Wistar oocytes treated with BAPTA-AM were arrested at MII until 120 min. In conclusion, SD oocytes carrying Mos/MEK/MAPK, maintained a high activity of p34(cdc2) kinase by stabilizing cyclin B1, thus involved in their meiotic arrest. In contrast, Wistar oocytes had a relatively low cytostatic factor activity; rapid decrease of Mos/MEK/MAPK failed to stabilize both cyclin B1 and Mos, and these oocytes were likely to spontaneously activate.     

Receptor-like protein kinase 2 (RPK 2) is a novel factor controlling anther development in Arabidopsis thaliana

Receptor-like kinases (RLK) comprise a large gene family within the Arabidopsis genome and play important roles in plant growth and development as well as in hormone and stress responses. Here we report that a leucine-rich repeat receptor-like kinase (LRR-RLK), RECEPTOR-LIKE PROTEIN KINASE2 (RPK2), is a key regulator of anther development in Arabidopsis. Two RPK2 T-DNA insertional mutants (rpk2-1 and rpk2-2) displayed enhanced shoot growth and male sterility due to defects in anther dehiscence and pollen maturation. The rpk2 anthers only developed three cell layers surrounding the male gametophyte: the middle layer was not differentiated from inner secondary parietal cells. Pollen mother cells in rpk2 anthers could undergo meiosis, but subsequent differentiation of microspores was inhibited by tapetum hypertrophy, with most resulting pollen grains exhibiting highly aggregated morphologies. The presence of tetrads and microspores in individual anthers was observed during microspore formation, indicating that the developmental homeostasis of rpk2 anther locules was disrupted. Anther locules were finally crushed without stomium breakage, a phenomenon that was possibly caused by inadequate thickening and lignification of the endothecium. Microarray analyses revealed that many genes encoding metabolic enzymes, including those involved in cell wall metabolism and lignin biosynthesis, were downregulated throughout anther development in rpk2 mutants. RPK2 mRNA was abundant in the tapetum of wild-type anthers during microspore maturation. These results suggest that RPK2 controls tapetal cell fate by triggering subsequent tapetum degradation, and that mutating RPK2 impairs normal pollen maturation and anther dehiscence due to disruption of key metabolic pathways.     

Identification of a novel bifunctional delta12/delta15 fatty acid desaturase from a basidiomycete, Coprinus cinereus TD#822-2

A new gene encoding a delta12 fatty acid desaturase-related protein was cloned from a multicellular basidiomycete Coprinus cinereus TD#822-2. The 1326 bp full-length gene, designated as Cop-odeA, codes for a putative protein of 442 amino acids with a MW of 49224. The Cop-odeA yeast transformant accumulated four new fatty acids identified as 9,12-hexadecadienoic acid, 9,12,15-hexadecatrienoic acid, linoleic acid, and alpha-linolenic acid, which comprised 8.8%, 1.0%, 29.0%, and 0.6% of the total fatty acids, respectively. The Cop-odeA protein was confirmed to be a novel bifunctional fatty acid desaturase with both high delta12 desaturase activity and unusual delta15 desaturase activity.