Large complex formation of the inhibitor of caspase-activated DNase.

Inhibitor of caspase-activated DNase (ICAD) is required for correctly folding of CAD and inhibits nuclease activity of CAD in non-apoptotic cells. From proteomic analysis of the ICAD binding proteins, we revealed that over-expressed flag-ICAD bound other ICAD molecules. Purified recombinant ICAD protein showed three bands, 66 KDa, 132 KDa and 450 KDa, by native-PAGE. ICAD fused with glutathione-S-transferase (GST) was immunoprecipitated with anti-flag antibody from Jurkat cell lysates cotransfected with ICAD fused with either GST or flag expression vectors. When purified recombinant ICAD protein was separated by gel chromatography, the molecular weight of ICAD was detected at approximately 440 and 45 K. ICAD in extracts of wild type Jurkat cells also existed at approximately 440 and 45 K as measured by gel chromatography; so that fractions of CAD coincided with fractions of approximately 440 K of ICAD. These results indicate that ICAD and/or CAD appeared to form large complexes in Jurkat cells.

Transcriptional suppression of the estrogen receptor by truncated estrogen receptor-alpha.

The estrogen receptor (ER) is composed of six major functional domains - the A/B domain as the activation function 1 domain, domain C as the DNA-binding domain, domain D as a hinge domain, and domain E/F as the ligand-dependent transcriptional domains. A novel protein (designated as SRB-RGS) that interacted with domains C and D of ER alpha (ER alpha C/D) repressed the transcriptional activity of ER alpha. In this study, we have examined whether ER alpha C/D releases transcriptional suppression of ER alpha by intrinsic SRB-RGS. The expression vector of ER alpha C/D was transfected to the human cancer cell, KPL-1, which expressed the intrinsic ER. Unexpectedly, transcriptional suppression of ER by ER alpha C/D was observed. COS-7 cells, which have no intrinsic ER, showed a similar suppression of ER alpha by co-transfection of ER alpha and ER alpha C/D. The DNA-binding and the estrogen-binding activities of ER alpha decreased on co-transfection of ER alpha C/D, suggesting a decrease in the receptor protein itself. It is likely that the degradation of ER by co-transfection caused the transcriptional suppression of the ER.

Molecular cloning and characterization of a steroid receptor-binding regulator of G-protein signaling protein cDNA.

Steroid hormone receptors are composed of six major functional domains, i.e. the A/B domains as the activation function 1 domain (AF-1), domain C as the DNA-binding domain, domain D as a hinge domain and domain E/F as the ligand-dependent transcriptional domain (AF-2). They regulate gene transcription through interactions with various nuclear factors of their domains, such as AF-1 and AF-2. We have insufficient knowledge of the function of the DNA-binding domain (domain C) except for its DNA-binding function or the hinge domain (domain D). Therefore, we attempted to identify factors interacting with the domains by using a yeast two-hybrid system. Domains C and D of estrogen receptor alpha were used as a bait to isolate cDNA clones from a rat ovary cDNA library. We isolated the cDNA clone of a novel steroid receptor-binding protein bearing the regulator of G-protein signaling (RGS) designated as SRB-RGS. The protein repressed the transcriptional activity of estrogen receptor alpha, suggesting cross-talk of steroid hormones and peptide hormones (or growth factors) for signal transductions mediated by SRB-RGS.

Peroxisomes exist in growth cones and move anterogradely and retrogradely in neurites of PC12D cells.

Localization and movement of peroxisomes have been investigated in neurites of a subline of PC12 pheochromocytoma cells (PC12D cells). The cells were transfected with a construct encoding the green fluorescent protein and bearing the C-terminal peroxisomal targeting signal 1 SKL motif (-Ser-Lys-Leu-COOH). Peroxisomes were detected as green punctate fluorescent signals. Many peroxisomes were observed in neurites of PC12D cells, especially in neural terminal-like structures, growth cones, varicosities, and branch points. Growth cones containing many peroxisomes were active, since they extended several long filopodias. Existence of peroxisomes in growth cones and neuronal terminal-like structures suggests that peroxisomes might have some role in neuronal extension and nerve terminal functioning. Peroxisomal motility was analyzed by time-lapse imaging using a fluorescence microscope at 25 degrees C. Peroxisomes were transported bidirectionally in neurites, i.e., through anterograde and retrograde transport. This result suggests that peroxisomes move to growth cones and neural terminals from the PC12D cell body, play some role in these parts, and go back to cell body.

Expression of indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase in early concepti.

Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan degradation in the placenta has been implicated in the prevention of the allogeneic fetus rejection [Munn, Zhou, Attwood, Bondarev, Conway, Marshall, Brown, and Mellor (1998) Science 281, 1191-1193]. To determine how IDO is associated with the development of the fetus and placenta, the time course of IDO expression (tryptophan-degrading activity, IDO protein and IDO mRNA) in the embryonic and extra-embryonic tissues as well as maternal tissues of mice was examined. A high tryptophan-degrading activity was detected in early concepti on days 6.5 and 7.5, whereas IDO protein and its mRNA were not expressed during early gestation, but appeared 2-3 days later, lasted for about 3 days and declined rapidly thereafter. The expression of IDO basically coincided with the formation of the placenta. On the contrary, the early tryptophan-degrading activity was due to gene expression of tryptophan 2,3-dioxygenase (TDO), as shown by Northern and Western analysis. These findings indicate that IDO is transiently expressed in the placenta but that the expression does not last until birth, and that the IDO expression is preceded by expression of another tryptophan-degrading enzyme, TDO, in the maternal and/or embryonic tissues in early concepti.

Gene analysis of genomic DNA from stored serum by polymerase chain reaction: identification of three missense mutations in patients with cholinesterasemia and ABO genotyping.

We established a method to determine the butyrylcholinesterase genotype associated with a BCHE deficiency directly using multiple PCR from stored serum, which was stored at -70 degrees C for more than 30 years. PCR products from sera of six propositi were used for DNA sequence analysis. All of these BChE variants were characterized by a single nucleotide substitution. Four of them were homozygotes and demonstrated a C-->T single nucleotide point mutation at codon 100 from CCA (Pro) to TCA (Ser). The fifth case was a heterozygote of this mutation. The remaining one was a compound heterozygote showing a T-->C transition mutation at codon 203 from TCA (Ser) to CCA (Pro) and a G-->C transversion mutation at codon 365 from GGA (Gly) to CGA (Arg). Furthermore we developed a method to determine the ABO genotype from the same serum. These results indicated that serum is useful as a starting material for amplification of genomic DNA when fresh blood samples are not available.

Essential role of trehalose in the synthesis and subsequent metabolism of corynomycolic acid in Corynebacterium matruchotii.

A previous paper indicated that corynomycolates synthesized by the fluffy layer fraction prepared from Corynebacterium matruchotii cells appeared exclusively as alpha-trehalose 6-monocorynomycolate (TMM) (T. Shimakata, K. Tsubokura, T. Kusaka, and K. Shizukuishi, 1985, Arch. Biochem. Biophys. 238, 497-508). In the present communication, the role of trehalose in the synthesis and subsequent metabolism of corynomycolic acids was reexamined. Consequently the following facts were clarified: (i) trehalose 6-phosphate (T-6-P), but not trehalose, stimulated corynomycolate synthesis from palmitate in the presence of ATP; the immediate product was TMM, which showed a rapid turnover. Since the turnover was blocked by addition of alpha-trehalose, only TMM accumulated among corynomycolate-containing substances. These results strongly suggested that T-6-P is an essential component as the acceptor in corynomycolate-synthetic system; (ii) TMM was the precursor not only to alpha-trehalose 6,6'-dicorynomycolate (TDM) and free corynomycolic acids but also to cell wall corynomycolate; (iii) addition of alpha-trehalose blocked the transfer of the corynomycolate moiety from TMM to cell wall corynomycolate, TDM, and free corynomycolic acids to a similar extent. These results clearly indicate that trehalose plays an essential role in the metabolism of corynomycolate after Claisen condensation and subsequent reduction in C. matruchotii.

[Missense mutations of the butyrylcholinesterase gene in six Japanese patients with low cholinesterasemia: genetic analysis using sera stored in a freezer].

Six serum samples with no detectable butyrylcholinesterase (BCHE) activity had been stored at -70 degrees C for more than 10 years. These sera were used for amplification of BCHE gene using polymerase chain reaction (PCR) and for nucleotide sequence analysis. Five of them demonstrated a C-->T transition at codon 100 (CCA-->TCA), resulting in a Pro-->Ser substitution. The other one was a compound heterozygote as revealed a T-->C transition mutation at codon 203 from TCA (Ser) to CCA (Pro) and G-->C transversion at codon 365 from GGA (Gly) to CGA (Arg). These results showed sera stored in a freezer could be used as a starting material for amplification of genomic DNA when it is not possible to obtain fresh blood samples.

First case of missense mutation (LDH-H:R171P) in exon 4 of the lactate dehydrogenase gene detected in a Japanese patient.

Complete deficiency of lactate dehydrogenase (LDH) subunit H was identified in a 41-year-old woman with paralysis of her left lower limb. The propositus had extremely low LDH activity and five of her family members had levels of LDH activity that ranged from lower than normal to normal level. A transversion mutation at codon 171 (CGC-->CCC), resulting in an Arg-->Pro substitution was identified in her DNA sequence. A new NruI restriction site was introduced into the polymerase chain reaction (PCR) product by PCR-primer introduced restriction analysis (PCR-PIRA) using a specific mismatched primer. Digestion with NruI revealed that the propositus and her mother were, respectively, homozygous and heterozygous for this mutation.

DNA-binding properties of the overexpressed recombinant estrogen receptor alpha.

Estrogen receptor alpha was overexpressed in COS-7 cells by transformation of an expression vector with the full length open reading frame of the receptor alpha. The nuclear estradiol-receptor complex and the soluble receptor from the COS-7 cells were cross-linked with an estrogen response element (ERE), which was substituted with 5-bromo-2'-deoxyuridine (BrdUVRE), as the receptor dimers by UV irradiation. In gel retardation analysis, the specific bindings of both nuclear and soluble receptors to ERE were decreased with increasing of KCl concentration compared with 0.1 M KCl. The ionic interactions of both receptors with ERE are thought to be similar.

Nitric oxide and aneurysm formation in Kawasaki disease.

The objective of this study is to show that nitric oxide plays a role in the development of coronary artery abnormalities in Kawasaki disease. We examined nitrite+nitrate, biopterin and neopterin in 316 urine samples of 34 patients with Kawasaki disease, those of 24 patients with other diseases, and those of 25 healthy children acting as a control group, because urinary nitrite + nitrate are reportedly useful as markers of nitric oxide generation in vivo, and pathways for neopterin-biopterin synthesis and nitric oxide generation are tightly coupled. In our study, the children with Kawasaki disease excreted more urinary nitrite + nitrate and neopterin than did the healthy control group children and excreted abnormally high quantities more often than did the patients with other diseases. Good relationships were found between the urinary nitrite + nitrate levels and the urinary biopterin levels, and between these biopterin levels and the urinary neopterin levels. Nitric oxide is therefore thought to be generated in abnormally high quantities, and to be closely related to the pathology of Kawasaki disease and to the development of coronary artery abnormalities. The role of nitric oxide in Kawasaki disease should be further studied to elucidate the pathophysiology of the disease and to aid in the development of new treatments.

Expression of the JNK2-alpha1 gene in the developing chick brain.

We have isolated chicken JNK2-alpha1 encoding a c-Jun N-terminal kinase, and examined the expression during embryogenesis. The kinase domain sequence is well conserved between chicken and mammals, but carboxy-terminal sequence of JNK is divergent from subtype 1 and 2, possibly derived from alternative splicing. The JNK2-alpha1 gene is preferentially expressed in the neuroepithelium of developing brain at stages 16-26, and transcripts are not detectable in the other region including spinal cord. These results suggest that JNK2-alpha1 is involved in development of the central nervous system as a mediator of stress-activated protein kinase pathway conferring competence to the external stimuli such as growth factors.

Nitric oxide may trigger lactation in humans.

OBJECTIVE: To determine whether nitric oxide is synthesized in the breast and plays a role in lactation. DESIGN: Concentrations of biopterin, neopterin, and the total concentration of nitrite plus nitrate, a marker for nitric oxide generation were measured in 242 samples of breast milk obtained from 39 women during postpartum days 1 to 30. The total concentration of nitrite plus nitrate was measured in 17 sets of breast milk and serum obtained from 17 women on postpartum day 4 or 5. RESULTS: (1) The total concentration of nitrite plus nitrate rose and peaked just before an increase in the volume of milk secreted was observed. (2) The total concentration of nitrite plus nitrate in breast milk was not correlated with that in the serum. (3) High levels of neopterin and biopterin were found in breast milk. (4) The volume of breast milk on day 5 was correlated with the total concentration of nitrite plus nitrate observed in breast milk on days 1 to 3. (5) The total concentration of nitrite plus nitrate in the breast milk of the high secretors significantly exceeded that seen in the low secretors. CONCLUSIONS: We suggest that nitric oxide is synthesized in the breast and may trigger lactation in humans.

Genetic analysis of a Japanese patient with butyrylcholinesterase deficiency.

A patient (64-year-old, male) with familial cholinesterasemia caused by BChE deficiency was studied. DNA sequence analysis of all exons identified a point mutation, an A-->G transition at codon 128, resulting in a Tyr-->Cys substitution. The propositus showed extremely low BChE activity, but his other family members (three individuals) showed from intermediate to normal BChE activity. An immunological method revealed the absence of BChE protein in serum of the propositus. Both PCR primer introduced restriction analysis (PCR-PIRA) and sequence analysis revealed all three family members to be heterozygotes for this mutation.

Liver specific kynurenine(alanine):glyoxylate aminotransferase was expressed in kidney cell line.

Kynurenine (Alanine); glyoxylate aminotransferase (AGT) expression plasmid in the COS-7 was constructed. pGV-C was used as a expression vector which contains SV-40 promoter and enhancer. pGV-C and human AGT clone, H1-2, were digested by Hind III/Sma I separately. The AGT fragment was inserted into the digested pGV-C large fragment, The constructed plasmid was named as pGV-AGT. The constructed plasmid was transfected to COS-7 cultured cell by electroporation. The best electroporation condition was checked.

The role of vasopressin, somatostatin and GABA in febrile convulsion in rat pups.

In order to further elucidate a possible role of neuropeptides and GABA in the pathogenesis of febrile convulsions, we studied changes of immunoreactive-arginine vasopressin (IR-AVP), IR-somatostatin (IR-SRIF) and gamma-aminobutyric acid (GABA) in the rat brain after febrile convulsions induced by ultra-red light (UR). Male Wistar rats at 16 days of age irradiated with UR developed generalized convulsions after 4.9 +/- 0.5 min irradiation. Six rats were killed by microwave irradiation 3 min after UR irradiation prior to convulsion development, and 29 rats were killed either 0 min, 2 h, 6 h, 24 h or 48 h after febrile convulsions. Non-irradiated rats served as controls. The rat brain was dissected into 4 regions; amygdala, hypothalamus, cortex and hippocampus, and subjected to radioimmunoassays. IR-AVP levels in hypothalamus were increased 3 min after UR and decreased at 2 h and 6 h after the convulsions. IR-SRIF levels were increased in cortex and hippocampus at 3 min after UR and 0 min after the convulsions. The GABA content increased in all regions tested at 2 h and 6 h after the convulsions. These results suggest that AVP, SRIF and GABA may be involved in the pathogenesis of febrile convulsions in different ways.

Metabolic inter-organ relations by exercise of fed rat: carbohydrates, ketone body, and nitrogen compounds in splanchnic vessels.

Fed rats were exercised until exhaustion by almost 65% VO2max on a treadmill. In 2.5 min after the exercise, blood was collected from various vessels of the splanchnic bed. Metabolites, glucose, lactate, ketone body, and nitrogencompounds in the plasma, were measured. Glucose excretion from the liver was increased by exercise, but was not significant. The absorption by the kidney decreased to 30% by exercise. Lactate was highly absorbed by the kidney, lower limbs, and digestive tract by exercise. Exercise caused a 200-300% increase of the plasma beta-hydroxybutyrate, but the absorption by the kidney and the lower limbs was decreased. These data suggest that glucose is a good carbon source for the recovery, and that lactate is more useful than glucose, but ketone body is less effective at a very early recovery phase under fed condition. Amino acid balances in each organ except digestive tract were positive showing anabolic conditions of these organs even after exhaustive exercise at fed condition. Most amino acid concentrations in the plasma tended to decrease to 60-90% by exercise. Amino acids were excreted from the digestive tract, and were eventually absorbed by the liver in both rested and exercised rat. The digestive tract, therefore, seems to be a primary amino acids pool to supply them to the liver during the inter meal. Urea excretion from the liver was more than the absorbed ammonia showing that active deamination from amino acids was carrying on. The resulted carbon skeletons of the amino acids might be used for the gluconeogenesis in the liver.

Spin-labelling study of biomembranes in spontaneously hypertensive rats: calcium- and calmodulin-dependent regulation.

1. The present study was performed to investigate alterations in membrane characteristics of spontaneously hypertensive rats (SHR) by using an electron paramagnetic resonance (EPR) and spin-labelling methods. 2. Washed erythrocytes from SHR were examined and compared with erythrocytes from age-matched normotensive Wistar-Kyoto (WKY) rats. 3. The values of outer hyperfine splitting (2T' 11) and that of the order parameter (S) obtained from EPR spectra for a spin label agent (5-nitroxide stearate) were significantly higher in the erythrocytes of SHR than in those of WKY rats. 4. When calcium (Ca2+) was loaded to erythrocytes with a Ca2+ ionophore (A 23187), the order parameter (S) of the EPR spectra showed a greater increase in SHR than in WKY rats. Furthermore, the Ca2+ -induced change in the order parameter (S) of SHR was significantly antagonized by pretreatment of the Ca2+ antagonists (verapamil, diltiazem) and a calmodulin antagonist (W-7). 5. The results show that the erythrocyte membranes of SHR tolerated different spin motions from those of normotensive WKY rats in the EPR study, which might be associated with the idea that the membrane fluidity might be lower in SHR. Furthermore, the data suggest that Ca2+ -calmodulin antagonists may ameliorate the Ca2+ -induced changes in membrane functions in hypertension.