Isolation and characterization of a C-type lectin cDNA specifically expressed in the tip of mouthparts of the flesh fly Sarcophaga peregrina.

We have isolated a novel gene, CLEM 36, of the flesh fly Sarcophaga peregrina, which shows significant homology to the C-type lectin family. CLEM 36 mRNA was transcribed excessively from the second day after eclosion only in the tip of mouthparts. Whole mount in situ hybridization showed that CLEM 36 mRNA was expressed in the C-type lectin-producing tissue (CLPT) located at the entrance of the food canal and between the labellum and haustellum. Immunoblot analysis showed that the mature form of CLEM 36 protein was synthesized in the CLPT, then secreted into saliva. Our results indicate that CLEM 36 protein may play an important role in biological defence against pathogens during the food intake of this insect.

Improved preparation of sulfur substituted 3-vinylpyrrole and its application to the syntheses of chuangxinmycin derivatives.

Sulfur substituted 3-vinylpyrrole 10 was prepared from 3-thio-acetylpyrrole 9 by alkylation with alkyl halide in the presence of propylene oxide. Functionalized 4-alkylthioindoles were made by Diels-Alder reaction of the 3-vinylpyrrole 10 with dienophiles. Chuangxinmycin analogues were synthesized by using some of the functionalized 4-alkylthioindoles as key intermediates.

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured luteal cells from rat ovary.

We examined the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured luteal cells from rat ovary. Luteal cells from immature rats treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were cultured in the absence or presence of ovine luteinizing hormone (LH) (100 ng/ml) and PACAP-38 (10, 100 and 1000 ng/ml). Following 48 hours of incubation, the levels of progesterone, 20 alpha-hydroxy-4-pregnene-3-one (20 alpha-OH-P) and adenosine 3',5'-monophosphate (cAMP) were measured in the culture media. PACAP alone significantly stimulated the production of progesterone and 20 alpha-OH-P in a dose-dependent manner (p < 0.01 and 0.05, respectively, ANOVA). LH-induced production of progesterone and accumulation of cAMP were significantly decreased by increasing concentrations of PACAP (p < 0.05 for each, ANOVA). Conversely, LH-stimulated 20 alpha-OH-P production was enhanced by PACAP in a dose-dependent manner (p < 0.05). Since PACAP decreased the ratio of progesterone to 20 alpha-OH-P production in LH-stimulated cells, PACAP-mediated inhibition of the stimulatory action of LH on progesterone production may be involved in the initiation of luteolysis. PACAP-38 also suppressed increases in LH receptor content in cultured luteal cells. These results suggest that PACAP regulates the effects of LH on luteal cell function and that PACAP might be closely linked to reproduction.

Ehlers-Danlos syndrome type IV with a unique point mutation in COL3A1 and familial phenotype of myocardial infarction without organic coronary stenosis.

We report on a 43-year-old male patient with Ehlers-Danlos syndrome (EDS) type IV with acute myocardial infarction (MI) without organic coronary stenosis. The disease was complicated with pneumothorax, subcutaneous and mediastinal emphysema, and splenic artery rupture. Three of the patient's family members suffered sudden cardiac death or MI. A diagnosis of EDS type IV was confirmed by decreased production of type III collagen by 86%. Mutation analysis revealed a point mutation in the COL3A1 gene that substituted glycine for aspartate at amino acid position 877. This mutation had not been reported as pathogenic for EDS type IV. These findings suggest close linkage between the mutation and the phenotype with familial MI.

Embryonic development in the eggs of the silkworm, Bombyx mori, exposed to the space environment.

To investigate the effects of cosmic radiation and microgravity on embryogenesis and organogenesis in Bombyx eggs, two different stages of eggs, the early stage after oviposition and the diapause-terminated eggs, were loaded on the US Space Shuttle/Atlantis (STS-84) for a 9 day flight. More than 85% of the early stage eggs hatched in the flight sample and the ground control. In the diapause-terminated eggs, the percentage of unhatched eggs were 43% in the ground control and 56% in the flight sample. In these eggs, uncompleted embryonic reversal was observed two-fold higher percentage in the flight sample than in the ground control. The incidence of abnormality such as the larvae with segmental fusion and the appearance of abnormal crescent marking in the flight sample was significantly higher than that in the ground control. This was also observed in the 1st and 2nd filial generation of the flight sample. From these results, unsuccessful blastokinesis and the abnormal appearance was discussed in relation to cosmic radiation and microgravity.

Iron(III)picolinate-induced oxygenation and subsequent rearrangement of triterpenoid derivatives with hydrogen peroxide.

The reaction of oleanane triterpenoid 1b with a FeIII(PA; picolinate)3/H2O2/MeCN system (reagent system A), a simple model system for mono-oxygenase, gave the 11 alpha-hydroxyl derivative 3 as major product, along with 11-oxo derivative 4 and 12-oxo derivative 6. The reaction of lupane triterpenoid 2b with reagent system A gave only oxidative rearrangement compounds, (20R)-aldehyde 8 and (20S)-aldehyde 9 were epimeric isomers. Then, we have found that iron(III) picolinate complex, FeIII(PA)3 is efficient in effecting the rearrangement of triterpenoid epoxides 5 and 7 into the corresponding carbonyl compounds, 6, 8 and 9 with 1,2-shift of the hydride.

Effectiveness of transcoronary chemoembolization for metastatic right ventricular tumor derived from hepatocellular carcinoma.

A right ventricular outflow tract was partially obstructed by a metastatic tumor from a hepatocellular carcinoma. This tumor was treated via chemoembolization of the right coronary artery, which resulted in tumor regression and improvement of the patient's symptoms.

Cloning and sequence of a cDNA for a highly basic protease from the digestive juice of the silkworm, Bombyx mori.

A serine protease of the silkworm, Bombyx mori, with an isoelectric point of pH 10-11 and a pH optimum for succinyl-Leu-Leu-Val-Tyr-MCA degrading activity of about 10, was found in a 0.33 M NaCl-eluted fraction obtained from cation-exchange chromatography of digestive juice. The activity of the enzyme was strongly inhibited by chymostatin and PMSF, indicating that the protease is a chymotrypsin-like serine protease. The N-terminal amino acid sequence of the protease was determined, and a full-length cDNA clone (0.92 kbp) which was isolated from a midgut cDNA library was sequenced. The cDNA encodes a pre-proenzyme of 284 amino acids with a pro-segment of 50 amino acids and mature protein of 234 amino acids. From its primary structure, the predicted molecular mass of the mature protein is 24.5 kDa. A sequence comparison of the Bombyx highly basic protease with other serine proteases revealed that this enzyme is a mammalian-type serine protease with a catalytic triad consisting of His45, Asp92 and Ser186. A large number of Arg residues are encoded by the cDNA which may be responsible for its stability and/or function in the alkaline condition, by remaining charged at high pH.

Biological roles of angiotensin II via its type 2 receptor during rat follicle atresia.

Type 1 angiotensin II (ANG II) receptors play crucial roles in the regulation of blood pressure and fluid osmolarity, whereas the physiological roles of type 2 ANG II receptors (AT2) remain unclear. Because AT2 is expressed in atretic follicles where granulosa cells undergo apoptosis, we examined the space and time relationship between AT2 expression and follicle atresia in vivo and the effect of AT2 on follicle-stimulating hormone (FSH) actions in vitro. Binding studies, autoradiography, and RT-PCR of AT2 revealed that the AT2 content in granulosa cells was time dependently increased at both protein and mRNA levels in equine chorionic gonadotropin-treated immature female rats. This increase paralleled the progression of atresia. ANG II suppressed FSH-caused prevention of DNA fragmentation, increases in luteinizing hormone receptor content, and estrogen production through AT2 in cultured granulosa cells. Moreover, FSH-induced stimulation of extracellular signal-regulated kinase activity, critical for cell survival, was inhibited by AT2 stimulation. These results suggest that AT2 mediates the progression of follicle atresia through granulosa cell apoptosis by inhibiting FSH actions.

Effect of growth hormone releasing hormone on luteinizing hormone stimulated progestin biosynthesis in cultured rat ovarian granulosa cells.

In the present study, we examined the effects of growth hormone releasing hormone (GHRH) on luteinizing hormone (LH)-stimulated progestin biosynthesis. Granulosa cells from pregnant mare serum gonadotropin (PMSG)-treated immature rats were cultured in vitro in the absence or presence of ovine. National Institute of Health LH (100 ng/ml) with various doses of GHRH (10(-9), 10(-8) and 10(-7) mol/l) for 24 h. At the end of the incubation period, the incubation media were collected and levels of progesterone, 20 alpha-hydroxypregn-4-en-3-one and cyclic adenosine 3',5'-monophosphate (cAMP) were measured. GHRH significantly stimulated progesterone production and cAMP accumulation in media in a dose-dependent manner in the basal state (p < 0.01 and p < 0.001, respectively). In the presence of LH, GHRH significantly inhibited LH-stimulated progesterone production in a dose-dependent manner (p < 0.01), whereas increasing concentrations of GHRH produced progressive increases in cAMP accumulation (p < 0.05). Since increasing concentrations of GHRH produced progressive decreases in the ratio of progesterone to 20 alpha-hydroxypregn-4-en-3-one production in the LH-stimulated state (p < 0.01), GHRH may be involved in modulation of key steroidogenic steps concerned with progesterone degradation rather than formation in LH-stimulated rat granulosa cells. These results suggest that GHRH may regulate the effects of LH in granulosa cells and play an important role in the reproductive process.

Effects of follicle-stimulating hormone on endothelin receptors in cultured rat ovarian granulosa cells.

Two subtypes of the endothelin (ET) receptor (ETA and ETB) were studied in cultured ovarian granulosa cells. Immature 21-day-old female Wistar-Imamichi rats were implanted with diethylstilbestrol (DES) pellets for 5 days and granulosa cells were collected by repeated puncturing. Viable cells (2.5 or 5 x 10(5)) were cultured with 50--400 ng/ml of ovine NIH follicle-stimulating hormone (FSH) in the presence or absence of [125I-Tyr13]ET-1 (50 pM) in 1 ml McCoy's 5a medium for 72 h. FSH gradually increased the [125I-Tyr13]ET-1 binding to granulosa cells, whereas FSH-untreated granulosa cells had no significant changes. The dose of 200 ng/ml of FSH was most effective for [125I-Tyr13]ET-1 binding for 48-h culture, thereafter revealing a plateau. After 48 h of culture with 200 ng/ml of FSH, granulosa cells were further incubated with [125I-Tyr13]ET-1 (10 pM-1 nM) and/or [125I]IRL1620, the selective ETB receptor agonist (10 pM-1 nM) for 2 h for equilibrium study, and then the dissociation constant and the maximal binding capacity between receptors and ligands were determined by saturation curve and Scatchard plot analysis. ETA + ETB, ETB, and ETA (sites/cells) showed a 4.4-, 2.6-, and 7.5-fold increase, respectively. As for steroidogenesis, ET-1 (100 nM) or ET-3 (100 nM) suppressed FSH-induced progesterone and 17 beta-estradiol production. These results indicate that FSH upregulates both ETA and ETB receptors in DES-treated immature rat granulosa cells, with no significant differences between ET-1 and ET-3, and that ET-1 or ET-3 suppresses FSH-induced steroidogenesis. ETs may affect the granulosa cell function through the ETA and ETB receptors, and the increase in amount of ET binding does not reflect ET effects on granulosa cell function. The ET receptor plays an important role in the development of the ovary.

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured granulosa cells from rat ovary and expression of mRNA encoding PACAP type IA receptor.

The purpose of this study was to detect the presence of mRNA encoding pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor in granulosa cells from rat ovary and to examine the effect of PACAP on progestin biosynthesis. mRNA was isolated from granulosa cells from the ovaries of immature rats treated with pregnant mares' serum gonadotrophin. The technique of reverse transcription and polymerase chain reaction with primers specific to PACAP type I receptor were used to demonstrate the expression of mRNA encoding PACAP type IA receptor in these cells. Granulosa cells were also cultured in the absence or presence of 100 ng LH ml-1 with various doses of PACAP-38 (10, 100 and 1000 ng ml-1). At the end of the incubation period, the incubation media were collected and concentrations of progesterone, 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) and cAMP were measured. Increasing concentrations of PACAP-38 significantly stimulated the production of progestins (progesterone and 20 alpha-OH-P) and cAMP accumulation in a dose-dependent manner (P < 0.01; ANOVA). This effect was observed in media cultured for 24 and 48 h in both basal and LH-stimulated states. PACAP-38 did not significantly affect the ratio of progesterone: 20 alpha-OH-P produced by granulosa cells cultured for 24 h in the LH-stimulated state. However, at 1000 ng ml-1, PACAP-38 significantly decreased the ratio of progesterone to 20 alpha-OH-P production in granulosa cells cultured for 48 h (P < 0.01). These results suggest that granulosa cells from rat ovary express mRNA encoding PACAP type IA receptor and that PACAP may regulate granulosa cell differentiation and play an important role in the reproductive process.

Oxygenation reaction of methyl valproate with a mono-oxygenase model reagent: implication for stereochemical identification of the mammalian metabolites of valproic acid.

Non-enzymic oxygenation reaction of methyl valproate (2) utilizing a simple model system for mono-oxygenases, Fe(MeCN)2(6+)-H2O2-Ac2O in MeCN, was investigated in connection with stereochemical analyses of the mammalian metabolites of 1. This oxygenation reaction of methyl valproate (2) gave a 92:8 mixture of the anti-isomer 4a and the syn-isomer 4b, together with 5a, and 5b corresponding to the mammalian metabolites of 1. The stereochemistry of 4a, 5a, and 5b was elucidated by spectral analyses of the corresponding beta-lactone 6a, gamma-lactone 7a and 7b prepared from the oxygenation products. The asymmetric synthesis of (+)-7a was also achieved.

Behavioral and electroencephalographic studies of beagles with an Eck's fistula: suitability as a model of hepatic encephalopathy.

Behavioral manifestations, electroencephalograms (EEGs) and visually evoked potentials (VEPs) were studied in beagles with Eck's fistula (portacaval shunt [PCS]), an established model of hyperammonemia, to determine whether they developed CNS disorders characteristic of hepatic encephalopathy. After PCS, behavioral changes occurred in the form of listlessness, sluggishness (altered gait, snapping and transient catatonia-like symptoms) and apparent blindness, which appeared in that order and progressed to coma and death in some animals. The EEGs from the frontal cortex showed a gradual decrease in voltage and frequency. Development of snapping and catatonia-like symptoms coincided with the occurrence of high voltage fast waves in the EEGs from the occipital cortex. In comatose Eck's fistula dogs. flattening of the EEGs was recorded from the frontal cortex and a lowered voltage was noted in the EEGs from the occipital cortex. After PCS, the latencies and amplitudes of the components of VEP were increased. The snapping and catatonia-like symptoms were markedly ameliorated by carbamazepine and the coma by flumazenil and thyrotropin-releasing hormone. These findings indicate that Eck's fistula dogs provide a useful model of hepatic encephalopathy.

Rat corpus luteum expresses both PACAP and PACAP type IA receptor mRNAs.

Both pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP type I receptor gene expressions were detected in the corpus luteum of pregnant mare's serum gonadotropin (PMSG)-human chorionic gonadotropin (hCG)-treated immature rats using reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR products of the poly(A)+ RNA extracted from rat corpora lutea yielded dominant DNA bands that corresponded to segments of PACAP mRNA (453 bp) and PACAP type IA receptor mRNA (290 bp) spanned by the PCR primers. The identities of the PACAP cDNA and the PACAP receptor cDNA fragments were confirmed by Southern blot hybridization analyses. Our results showed that PACAP mRNA and PACAP type IA receptor mRNA are synthesized within luteinized cells of rat ovary, and suggest that PACAP is closely linked to the reproductive process.

Structure of two cecropin B-encoding genes and bacteria-inducible DNA-binding proteins which bind to the 5'-upstream regulatory region in the silkworm, Bombyx mori.

Two genomic DNAs encoding cecropin B (CecB), an antibacterial protein from Bombyx mori, were cloned and sequenced. The number of CecB genes was estimated to be more than four copies per haploid by genomic Southern blotting. Two genes, CecB1 and CecB2, were located tandemly within 12 kb in the same orientation. These two genes encoded identical amino acids, though 15 nucleotides (nt) were different in the coding region and the intron size varied. About 90% of the nt spanning 800 bp in the 5'-untranslated region (UTR) were identical between the two genes. This 5'-flanking region contained characteristic sequences such as a repetitive element of B. mori (Bm1), an interleukin-6 response element (IL-6 RE), and two putative lipopolysaccharide (LPS) response elements (LPS RE). An electrophoretic mobility shift assay (EMSA) showed that the fat body contains at least three different nuclear proteins inducible by bacteria which bind to the 5'-UTR, suggesting that these proteins may be involved in CecB expression triggered by bacteria.

Induction of hepatic microsomal P450 by 4-phenylalkylpyridines in rat: chain length-dependent and sex-related differential induction of P450s.

The effects of 4-phenylalkylpyridines (chain length of 0-5, 7, 9 and 11 carbon atoms) on the induction of hepatic microsomal P450 and its multiple forms (1A1/2 (1A1, 1A2), 3A2, 2B1/2, 2C6 and 2E1) in the male and female rat have been investigated. P450 induction gradually declined with increasing chain length of the 4-phenylalkylpyridines. Immunoblot analysis revealed that three pyridine compounds having methylene units of 0, 1 and 2 only induced P4501A2, whereas those having 4, 5 or 7 methylene units only induced 1A1 in the male rat. In the female rat, however, we could not observe such a chain length-dependent differential induction of the P4501A subfamily. Induced levels of P4503A2 and 2E1 were dependent on total P450 specific content, but 2C6 was increased in a chain length-dependent manner in both sexes. These results provide new information on the differential effects of pyridine-containing compounds on P450 induction in the rat.