Apomictic parthenogenesis in a parasitoid wasp Meteorus pulchricornis, uncommon in the haplodiploid order Hymenoptera.

Although apomixis is the most common form of parthenogenesis in diplodiploid arthropods, it is uncommon in the haplodiploid insect order Hymenoptera. We found a new type of spontaneous apomixis in the Hymenoptera, completely lacking meiosis and the expulsion of polar bodies in egg maturation division, on the thelytokous strain of a parasitoid wasp Meteorus pulchricornis (Wesmael) (Braconidae, Euphorinae) on pest lepidopteran larvae Spodoptera litura (Fabricius) (Noctuidae). The absence of the meiotic process was consistent with a non-segregation pattern in the offspring of heterozygous females, and no positive evidence was obtained for the induction of thelytoky by any bacterial symbionts. We discuss the conditions that enable the occurrence of such rare cases of apomictic thelytoky in the Hymenoptera, suggesting the significance of fixed heterosis caused by hybridization or polyploidization, symbiosis with bacterial agents, and occasional sex. Our finding will encourage further genetic studies on parasitoid wasps to use asexual lines more wisely for biological control.

An X-ray outburst from the rapidly accreting young star that illuminates McNeil's nebula.

Young, low-mass stars are luminous X-ray sources whose powerful X-ray flares may exert a profound influence over the process of planet formation. The origin of the X-ray emission is uncertain. Although many (or perhaps most) recently formed, low-mass stars emit X-rays as a consequence of solar-like coronal activity, it has also been suggested that X-ray emission may be a direct result of mass accretion onto the forming star. Here we report X-ray imaging spectroscopy observations which reveal a factor approximately 50 increase in the X-ray flux from a young star that is at present undergoing a spectacular optical/infrared outburst (this star illuminates McNeil's nebula). The outburst seems to be due to the sudden onset of a phase of rapid accretion. The coincidence of a surge in X-ray brightness with the optical/infrared eruption demonstrates that strongly enhanced high-energy emission from young stars can occur as a consequence of high accretion rates. We suggest that such accretion-enhanced X-ray emission from erupting young stars may be short-lived, because intense star-disk magnetospheric interactions are quenched rapidly by the subsequent flood of new material onto the star.

Beta cell expression of endogenous xenotropic retrovirus distinguishes diabetes-susceptible NOD/Lt from resistant NON/Lt mice.

Endogeneous retroviral expression in beta cells is a feature of prediabetes in nonobese diabetic (NOD) mice. The purpose of this study was to characterize the class-specific pattern of retroviral gene expression in NOD/Lt beta cells versus a related, but diabetes-resistant strain, NON/Lt. Electron microscopic comparison of beta cells from both strains indicated low constitutive expression of the intracisternal type A (IAP) retroviral class. However, NOD beta cells, in contrast to NON beta cells, expressed an additional intracisternal retroviral form resembling a type C particle. Antibodies against both IAP and type C were detected in NOD, with the humoral response to type C, but not IAP, preceding decline in beta cell function. RNA was extracted from freshly isolated islets from NOD and NON males. Comparative Northern blot analysis of total type C retroviral gene expression using a gag-pol DNA probe corroborated expression of endogenous type C proviruses in both NOD and NON islet cells and thymus. Use of class-specific retroviral probes identified the class of expressed endogenous retrovirus distinguishing the two inbred strains. The single ecotropic provirus present in both the NOD and NON genome (Emv-30) was not expressed in islets or thymus of either strain. Comparison of endogenous xenotropic provirus content by Southern blot analysis revealed two unique xenotropic loci (Xmv-65, -66) in NOD; 8.4 and 3.0 kb xenotropic envelope (env) RNA transcripts were detected in NOD, but not NON islets and thymus. NON contained three xenotropic loci common to other inbred strains (Xmv-21, -25, and -28). Both strains were partially characterized for content of recombinant (polytropic and modified polytropic) proviruses. IAP RNA expression was common to both NOD and NON islets and hence could not be specifically associated with the unique intracisternal type C particle found in NOD, but not NON beta cells. In conclusion, this study shows that expression of xenotropic type C but not IAP distinguishes retroviral activity in NOD/Lt versus NON/Lt beta cells. The potential pathogenic role of retroviral gene expression in NOD beta cells is discussed.

Transformation of rat ovarian epithelial and Rat-1 fibroblast cell lines by RAST24 does not influence cisplatin sensitivity.

Recent reports suggest that expression of an activated c-Ha-ras oncogene is associated with cisplatin resistance in NIH-3T3 fibroblasts. To investigate the generality of these observations, cisplatin cytotoxicity was determined in a series of clonal Rat-1 fibroblast and rat ovarian surface epithelial (ROSE) cell lines carrying a zinc-inducible metallothionein-RAST24 fusion gene, MTRAST24. Cisplatin sensitivity in RAS-transformed fibroblast sublines did not differ from parental controls. Induction of mutant RAST24 expression by zinc sulfate did not affect the cisplatin sensitivity of individual cell lines. Expression of mutant p21Ha-RAS varied more than 40-fold in these fibroblast sublines. Similarly, there was no difference in cisplatin sensitivity between parental ROSE controls, neomycin phosphotransferase transfected controls, or MTRAST24 transfectants. Finally, the cisplatin sensitivity of RAS-transformed ROSE cells was similar to that of spontaneously transformed ROSE cells. Overall, these observations suggest that there is little relationship between mutant ras expression and cisplatin sensitivity in rat epithelial and fibroblast cell lines.

Cross-resistance to diverse drugs is associated with primary cisplatin resistance in ovarian cancer cell lines.

We have previously obtained, by exposure to near continuous increasing concentrations of cisplatin, a panel of human ovarian cancer cell lines that exhibit a wide range of primary resistance to the drug (9- to > 400-fold). These cells had strikingly increased (4- to 50-fold) levels of glutathione (GSH) as compared with the drug-sensitive cells of origin (A. K. Godwin et al., Proc. Natl. Acad. Sci. USA, 89: 3070-3074, 1992). Utilizing this panel of resistant cell lines, we evaluated cross-resistance to classical alkylating agents, natural product drugs, and irradiation. We observed that cross-resistance to carboplatin paralleled that of cisplatin, culminating in approximately 250-fold resistance. Similarly, melphalan cross-resistance continued to increase to > 400-fold and again paralleled the primary cisplatin resistance. Cell lines with low to very high levels of resistance to cisplatin are 8- to 850-fold resistant to the epipodophyllotoxin derivative etoposide. Cross-resistance is also observed for other natural product drugs, including Adriamycin (approximately 80-fold), mitoxantrone (approximately 440-fold), and taxol (approximately 40-fold). Cross-resistance to irradiation is, however, modest (< 2-fold). The cells with the greatest primary resistance to cisplatin most commonly had the highest cross-resistance to the other drugs examined. The cross-resistance to the natural product category drugs was found not to be mediated by the products of either the multidrug resistance 1 (MDR1) or multidrug resistance-associated protein (MRP) genes based on lack of coordinate increased expression or amplification of these genes as assessed by Northern and Southern blot analyses. Furthermore, verapamil failed to markedly increase drug sensitivity. Although there was no indication that these natural product drug efflux pumps were operative, we observed decreased doxorubicin accumulation in these cell lines cross-resistant to natural products. In addition, alternations in DNA topoisomerase II mRNA levels, which have been observed in a variety of human tumor cell lines selected in vitro for resistance to etoposide or teniposide, were not detected. Only intracellular levels of GSH correlated with cross-resistance to these diverse anticancer agents and partial loss of resistance was associated with a marked decrease in glutathione levels. In the absence of alternative mechanisms, we speculate that the very broad clinically relevant cross-resistance seen in this model system may, at least in part, be the direct result of GSH-mediated drug inactivation or may be due to a combination of GSH conjugation to drug and conjugate efflux mediated by the putative ATP-dependent glutathione S-conjugate export pump.

Comparison of cytokine effects on mouse pancreatic alpha-cell and beta-cell lines. Viability, secretory function, and MHC antigen expression.

Cytokine effects on permanent cell lines of transformed mouse pancreatic alpha- and beta-cells were compared. The beta-tumor cell 1 (beta TC1) line (from an adenoma created in transgenic mice expressing the SV40 large T-antigen oncogene under control of the rat insulin II promoter) produced insulin predominantly, although small quantities of intracellular glucagon (100:1 insulin to glucagon) were detectable by radioimmunoassay. The alpha TC1 line (from an adenoma created in transgenic mice expressing the SV40 large T-antigen oncogene under control of the rat preproglucagon promoter) produced not only glucagon but also considerable quantities of insulin (4:1 glucagon to insulin) and preproinsulin mRNA. We therefore cloned alpha TC1 cells and obtained 12 glucagon-producing clonal cell lines that did not produce levels of insulin detectable by radioimmunoassay. Analysis by Northern blotting of total RNA from two lines, alpha TC1 clones 6 and 9, confirmed the absence of preproinsulin mRNA. No somatostatin or pancreatic polypeptide was detected by immunohistochemical staining in alpha TC1 clones 6 or 9 or beta TC1 cells. Rat recombinant gamma-interferon (IFN-gamma; 5-250 U/ml) or mouse recombinant interleukin 1 (IL-1; 1-25 U/ml) individually inhibited DNA synthesis in beta TC1 cells after 3 days of treatment. The two cytokines in combination acted synergistically to further depress DNA synthesis and increase cytotoxicity. In contrast, alpha TC1 clone 9 cells were not sensitive to inhibition of DNA synthesis by each cytokine individually, although glucagon synthesis was inhibited. The combination of these cytokines caused marked inhibition of DNA and glucagon syntheses in alpha TC1 clone 9 cells. alpha TC1 clone 9 cells were somewhat more resistant to the cytotoxic action of the combined cytokines than were beta TC1 cells. Incubation with 50 U/ml IFN-gamma induced class II MHC molecules (I-Ab, I-Ad, and I-Ed) and enhanced the constitutive expression of class I molecules (H-2Kb and H-2Kd) on the cell surfaces of beta TC1, uncloned alpha TC1, and alpha TC1 clones 6 and 9. Thus, these cell lines are heterozygous for MHC alleles derived from both parental strains used in the construction of the transgenic mice [C57BL/6J (H-2b) and DBA/2J (H-2d)]. Class II gene transcription induced by IFN-gamma was confirmed in beta TC1 and alpha TC1 clone 9 cells by Northern blot analysis with A alpha-, A beta-, E alpha, and E beta-DNA probes.(ABSTRACT TRUNCATED AT 400 WORDS)

NIT-1, a pancreatic beta-cell line established from a transgenic NOD/Lt mouse.

NOD/Lt mice harboring a hybrid rat insulin-promoter/SV40 large T-antigen gene spontaneously develop beta-cell adenomas. NIT-1 is a pancreatic beta-cell line established from one of these transgenic mice. Immunocytochemical staining of passage 18 cells showed most contained insulin, with less than 5% containing glucagon, and none containing pancreatic polypeptide or somatostatin. Glucagon content radioimmunoassayed in cell extracts was only 0.27% of the insulin content. Two-hour insulin secretion at 16.5 mM glucose was 638 ng/10(6) cells (41% of intracellular content) compared to only 1.3 ng glucagon (32% of intracellular content). Stimulated insulin secretion was consistently observed in response to 11 and 16.5 mM glucose between passages 11 and 19. At passage 19, both theophylline and tolbutamide stimulated insulin secretion at 5.5 mM glucose. Northern-blot analysis confirmed high levels of insulin mRNA but only trace glucagon mRNA and undetectable somatostatin mRNA. Interferon-gamma (IFN-gamma)-induced MHC class I RNA expression was correlated with markedly increased antigen expression at the cell surface. Similarly, a MHC-linked "occult" class I-like antigen detected by Cr release assay only after exposure of standard NOD/Lt islet cells to IFN-gamma was strongly induced by IFN-gamma in NIT-1 cells. Cell surface MHC class II antigen was not constitutively expressed on NIT-1 cells and could not be detected after IFN-gamma incubation, despite demonstration of IFN-gamma-induced Aa, Ab, and Li invariant-chain RNA transcripts. Similarly IFN-gamma induction of intercellular adhesion molecule 1 (Icam-1) transcripts was not accompanied by demonstrable cell surface expression of ICAM-1 antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+-dependent exocytosis of L-glutamate by alphaTC6, clonal mouse pancreatic alpha-cells.

Pancreatic islet cells express receptors and transporters for L-glutamate and are thus believed to use L-glutamate as an intercellular signaling molecule. However, the mechanism by which L-glutamate appears in the islets is unknown. In the present study, we investigated whether L-glutamate is secreted through exocytosis by alphaTC6 cells (clonal mouse pancreatic alpha-cells). An appreciable amount of L-glutamate was released from cultured cells after the addition of KCl or A23187 in the presence of Ca2+ and 10 mmol/l glucose in the medium. The KCl-induced glutamate release was significantly reduced when assayed in the absence of Ca2+ or when the cells were pretreated with EGTA-AM. The KCl-induced Ca2+-dependent glutamate release was inhibited approximately 40% by voltage-gated Ca2+ channel blockers, such as nifedipine at 20 micromol/l. The degree of KCl-induced Ca2+-dependent glutamate release was correlated with an increase in intracellular [Ca2+], as monitored by fura-2 fluorescence. Botulinum neurotoxin type E inhibited 55% of the KCl-induced Ca2+-dependent glutamate release, followed by specific cleavage of 25 kDa synaptosomal-associated protein. Furthermore, bafilomycin A1, a specific inhibitor of vacuolar H+-ATPase, inhibited 40% of the KCl-induced Ca2+-dependent glutamate release. Immunoelectronmicroscopy with antibodies against synaptophysin, a marker for neuronal synaptic vesicles and endocrine synaptic-like microvesicles, revealed a large number of synaptophysin-positive clear vesicles in cells. Digitonin-permeabilized cells took up L-glutamate only in the presence of MgATP, which is sensitive to bafilomycin A1 or 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile (a proton conductor) but insensitive to either oligomycin or vanadate. From these results, it was concluded that alphaTC6 cells accumulate L-glutamate in the synaptophysin-containing vesicles in an ATP-dependent manner and secrete it through a Ca2+-dependent exocytic mechanism. The Ca2+-dependent glutamate release was also triggered when cells were transferred in the medium containing 1 mmol/l glucose, suggesting that low glucose treatment stimulates the release of glutamate. Our results are consistent with the idea that L-glutamate is secreted by alpha-cells through Ca2+-dependent regulated exocytosis.

PDX-1 induces insulin and glucokinase gene expressions in alphaTC1 clone 6 cells in the presence of betacellulin.

The pancreatic beta- and alpha-cells are developmentally related to each other but reveal diverse gene expression patterns. Among the two important transcription factors for insulin gene expression, IEF1 is present both in alpha- and beta-cells, but PDX-1/IPF1/STF-1/IDX-1, a homeodomain-containing transcription factor, is present in beta-cells but not in alpha-cells. To elucidate the function of PDX-1 in the expression of beta-cell-specific genes, we established stable alphaTC1 clone 6 (alphaTC1.6)-derived transfectants expressing PDX-1 and examined the changes in the gene expression patterns in them. The exogenous expression of PDX-1 in alphaTC1.6 cells alone could induce islet amyloid polypeptide (IAPP) mRNA expression in the cells but not the expression of insulin, glucokinase, or GLUT2 gene. However, when betacellulin was added to the medium, the PDX-1-expressing alphaTC1.6 cells, but not the control alphaTC1.6 cells, came to express insulin and glucokinase mRNAs. This did not occur with other growth factors such as epidermal growth factor, transforming growth factor alpha, and insulin-like growth factor I. GLUT2 mRNA remained undetectable in the PDX-1--expressing alphaTC1.6 cells. These observations demonstrate the potency of PDX-1 for the expression of the insulin, glucokinase, and IAPP genes and suggest that certain regulatory factors, which can partially be modified by betacellulin, also contribute to the beta-cell specificity of gene expression.

Complications after proximal gastrectomy with jejunal pouch interposition: report of a case.

We report a rare case of proximal gastrectomy complication as a result of a severe dilatation of a jejunal pouch interposed for reconstruction. A 44-year-old man who had early gastric cancer underwent proximal gastrectomy with a jejunal pouch interposition at our department. Fourteen months after the procedure, he began to complain of left hypochondrial fullness and reflux symptoms. He had difficulty eating and his quality of life (QOL) was markedly impaired. Barium meal revealed severe dilatation of the jejunal pouch. Decompression using a stomach tube and other measures only achieved temporary improvement. 4.5 years later, the dilated jejunal pouch was resected together with apyloroplasty and double tract reconstruction. Six months after this secondary surgery, the patient recorded no further complications. Food intake increased and QOL improved.

Stability of ribonuclease T2 from Aspergillus oryzae.

The stability of ribonuclease T2 (RNase T2) from Aspergillus oryzae against guanidine hydrochloride and heat was studied by using CD and fluorescence. RNase T2 unfolded and refolded reversibly concomitant with activity, but the unfolding and refolding rates were very slow (order of hours). The free energy change for unfolding of RNase T2 in water was estimated to be 5.3 kcal.mol-1 at 25 degrees C by linear extrapolation method. From the thermal unfolding experiment in 20 mM sodium phosphate buffer at pH 7.5, the Tm and the enthalpy change of RNase T2 were found to be 55.3 degrees C and 119.1 kcal.mol-1, respectively. From these equilibrium and kinetic studies, it was found that the stability of RNAse T2 in the native state is predominantly due to the slow rate of unfolding.

Structure of the active 27-residue fragment of human calpastatin.

A synthetic 27-residue peptide corresponding to exon 1B of the endogenous inhibitor calpastatin contains a well-conserved region and has an ability to inhibit the cysteine endopeptidase calpain specifically. We examined the solution structure of this peptide in DMSO-d6 by two-dimensional 1H NMR spectroscopy. Although regular secondary structures such as alpha-helix and beta-sheet were not found, the region from Ile18 to Arg23 formed a well-defined structure with a type I beta-turn. This region coincided well with the highly conserved region of calpastatin. The result strongly suggests that this turn structure is essential for the inhibitory activity of calpastatin.

Cell-mediated immunity to bovine P2 protein and neuritogenic synthetic peptide in experimental allergic neuritis.

Cellular reactivity to bovine P2 protein (P2) and its two synthetic peptides, SP66-78 and SP70-78, was serially examined by the lymphocyte proliferation test in animals with experimental allergic neuritis (EAN). SP66-78 and SP70-78 correspond to residues 66-78 and 70-78 of bovine P2. Proliferative response to SP66-78 as well as P2 appeared at day 7 before the onset of EAN and was clearly manifested at day 14 in the active stage, thereafter disappearing in the stable stage, whereas no response to SP70-78 was detected during the course of the disease. These results suggest that cell-mediated immune response to P2 and the specific part residues 66-78 of P2 play an important role in the pathogenesis of EAN.

Activated T lymphocyte subsets in experimental allergic neuritis.

Changes in activated T cell subsets in peripheral blood were examined during the course of experimental allergic neuritis (EAN), using two-color immunofluorescence flow cytometry. Both CD4+ and CD8+ activated T cells decreased transiently before the onset of clinical signs, and increased just around the time of onset of the disease. In contrast, during the recovery phase, the numbers of CD4+ activated T cells returned to the normal range, whereas CD8+ activated T cells continued to increase. These findings imply that activation of CD4+ helper/inducer cells contributes mainly to the evolution of EAN, and that of CD8+ suppressor cells are necessary for recovery.

Effects of combination treatment with FK506 and cyclosporine on survival time and vascular changes in renal-allograft-recipient dogs.

In our previous experiments studying the effects of FK506 on renal allografting in the dog, we encountered two major problems. One problem was anorexia and the other problem was vascular changes mainly in the recipient heart. Anorexia was generally dose dependent, but the vascular changes were seen to be more prominent at lower doses rather than at higher immunosuppressive doses. The present study was undertaken to study these two problems. A nonanorexic, vascular change-related, nonimmunosuppressive dose of FK506 was combined with a low dose of cyclosporine or prednisolone in beagle dogs after renal allografting. Treatment with either FK506 alone at a dose of 0.32 mg/kg or cyclosporine alone at 2.5 mg/kg was not effective in prolonging renal recipient survival. The recipient dogs died of rejection, and a variety of vascular changes were observed in the hearts of both groups. Combined treatment with FK506 and cyclosporine at these same doses resulted in statistically significant prolongation of the survival time of the renal recipient (P less than 0.01), and histologic studies showed that the frequency and severity of the vascular changes were suppressed in the recipient receiving the combined treatment. The combination of FK506 and prednisolone at 0.5 mg/kg was not effective in prolonging survival. Furthermore, the extent of vascular changes was similar to those found in recipients receiving FK506 alone. The data suggest that combined treatment with low doses of both FK506 and cyclosporine acted synergistically in prolonging canine renal allografts and that the vascular changes frequently seen at low doses of FK506 were reduced by additional immunosuppression with a low dose of cyclosporine.

Involvement of platelet-derived growth factor and histocompatibility of DRB 1 in chronic renal allograft nephropathy.

BACKGROUND: The role of activated T cells in graft arteriosclerosis, which is observed in chronic renal allograft nephropathy, and the involvement of major histocompatibility complex (MHC) incompatibility remain to be determined. We examined the effect of T lymphocytes that were obtained from renal transplant patients undergoing chronic rejection treated with cyclosporine (CsA) on platelet-derived growth factor (PDGF)-induced proliferation of cultured human vascular smooth muscle cells (SMC) and compared the proliferation activity of T lymphocytes with MHC incompatibility, especially DRB 1 mismatch. METHODS: Renal transplant patients with continued allograft function, who survived more than 1 year after transplantation, were recruited. Chronic rejection was documented by graft-biopsy findings together with increasing serum creatinine levels (10-20% per year). After the incubation of supernatant (conditioning medium) of cultured T cells from CsA-treated renal transplant patients with chronic rejection (n=18) and with normal renal function (n=14) as control, normal subjects (n=11) and chronic hemodialysis (HD) patients (n=5) with cultured SMC in the presence or absence of PDGF, DNA synthesis (3H-thymidine uptake) of SMC was examined. The in vitro effects of CsA on DNA synthesis of cultured SMC were also evaluated. RESULTS: The supernatant of cultured T cells from renal transplant patients with chronic rejection stimulated PDGF-induced DNA synthesis of SMC in a dose-dependent manner, showing significant enhancement as compared with control transplant patients, normal subjects, and chronic HD patients. The supernatant itself did not significantly stimulate DNA synthesis of SMC. No significant in vitro stimulation of CsA on DNA synthesis was observed. The supernatant of T cells obtained from recipients undergoing chronic rejection with two DRB 1 mismatches showed significantly higher enhanced activity of PDGF-induced DNA synthesis than the supernatant from those recipients without mismatch of DRB 1. On the other hand, no significant correlation of the enhanced activity by T cell supernatant to HLA A and B mismatch numbers was observed. CONCLUSIONS: Growth factor-promoting factors(s) derived from activated T cells associated with MHC class II DR expression, which promotes PDGF-induced proliferation of SMC, exists in renal transplant patients with chronic renal allograft nephropathy, and is probably involved in arteriosclerosis of the graft kidney.

Expression of arachidonate 12-lipoxygenase in rat tissues: a possible role in glucagon secretion.

There are three isoforms of arachidonate 12-lipoxygenase in mammals: platelet, leukocyte, and epidermal types. We found in this study that the leukocyte-type enzyme was present in rat pineal gland, lung, spleen, aorta, adrenal gland, spinal cord, and pancreas, as assessed by RT-PCR. Immunohistochemical analysis showed that the enzyme was localized in macrophages in lung and spleen, alpha-cells of pancreatic islet, zona glomerulosa cells of adrenal cortex, and neuronal cells of spinal cord and superior cervical ganglion. The presence of the 12-lipoxygenase in pancreatic alpha-cells was confirmed by glucagon staining in a consecutive section. We overexpressed the leukocyte-type 12-lipoxygenase cDNA in a glucagon-secreting alphaTC clone 6 cell line that had been established from a transgenic mouse. Glucagon secretion was stimulated by approximately twofold in the 12-lipoxygenase-expressing cells compared to the mock-transfected and original cells. The results suggest that the 12-lipoxygenase of the leukocyte type augments glucagon secretion from pancreatic islets.

Suppression of inflammatory responses to 12-O-tetradecanoyl-phorbol-13-acetate and carrageenin by YM-26734, a selective inhibitor of extracellular group II phospholipase A2.

1. YM-26734 [4-(3,5-didodecanoyl-2,4,6-trihydroxyphenyl)-7-hydroxy-2-(4-hydroxyph eny l) chroman] dose-dependently inhibited the activities of extracellular phospholipase A2 (PLA2): rabbit platelet-derived group II and porcine pancreas-derived group I PLA2, with IC50 values of 0.085 (0.056-0.129, n = 5) and 6.8 (5.0-9.6, n = 5) microM, respectively. 2. In contrast, YM-26734 did not reduce the activity of intracellular PLA2 prepared from mouse macrophages, which preferentially hydrolyzed arachidonoyl phospholipids at concentrations up to 50 microM. YM-26734 also showed no effect against either sheep seminal vesicle cyclo-oxygenase or rat leukocyte 5-lipoxygenase. 3. Linewater-Burk analysis showed that YM-26567-1 behaved as a competitive inhibitor of group II PLA2 derived from rabbit platelets, with a Ki value of 48 nM. 4. In mice, YM-26734 inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA, 1 microgram/ear)-induced ear oedema in a dose-dependent manner, with ED50 values of 45 (30-67) micrograms/ear (n = 5) and 11 (4-32) mg kg-1, i.v. (n = 5), but did not decrease arachidonic acid (4 mg/ear)-induced ear oedema at 1 mg/ear and 30 mg kg-1, i.v. 5. In rats, the accumulation of exudate fluids and leukocytes in the pleural cavity in response to carrageenin injection (2 mg) was significantly less in a group treated with YM-26734 (20 mg kg-1, i.v.) than in the control group (0.43 +/- 0.02 vs 0.59 +/- 0.03 g per cavity and 3.8 +/- 0.2 vs 4.9 +/- 0.3 x 10(7) cells per cavity, respectively; n = 5). 6. These results suggest that YM-26734 is a potent and competitive inhibitor of extracellular PLA2 with selectivity for group II PLA2, and that the inhibition of group II enzymes activity may cause the suppression of inflammatory responses to TPA and carrageenin.

Novel mutation of the DAX1 gene in a patient with X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism.

X-linked adrenal hypoplasia congenita (AHC) is characterized by primary adrenal insufficiency and is frequently associated with hypogonadotropic hypogonadism (HHG). Mutations of the DAX1 gene have been reported in patients with AHC and HHG. We found a novel DAX1 mutation in our patient. Sequence analysis of the patient's DAX1 demonstrated a 1-bp (G) deletion at codon 49 in exon 1. The mutation shifts the reading frame, resulting in completely different amino acid sequences from codon 49 to the premature stop codon at 84. The G was present at this position in the sequences of the father and 2 younger brothers. Direct sequence and single-strand conformation polymorphism analyses of polymerase chain reaction fragments revealed that the mutation at codon 49 was heterozygously present in the mother's DAX1 gene. The codon 84 is located in the first half of the DNA binding domain, and the mutation site is closer to the N-terminus than those in previously reported cases. The onset of adrenal insufficiency in the neonatal period as seen in our patient has also been reported in other patients with different DAX1 mutations, especially in a patient with DAX1 protein lacking 11 amino acids at the C-terminus. Therefore, it is less likely that position of termination codons correlate to clinical manifestations.

Binding of substrate analogues to subsites D, E, and F of hen egg-white lysozyme.

The pH dependence of the binding of dye, Beibrich Scarlet, to hen egg-white lysozyme[EC 3.2.1.17] was studied at ionic strength 0.3 and 25 degrees by following circular dichroic (CD)bands originating from the bound dye. This binding involved one of the catalytic groups, Glu 35. The effect of the binding of N-acetylglucosamine (GlcNAc), its dimer or trimer on the binding of this dye was also studied at pH 7.5 by measuring changes in the CD bands of the dye bound to lysozyme. It was shown that there are two sites for simultaneous binding of these saccharides in the lysozyme molecule. The stronger binding of the saccharide was noncompetitive and the weaker binding was competitive with dye binding. The binding constants for the stronger binding site (the upper portion of lysozyme cleft) were in good agreement with those previously determined by following changes in the tryptophyl CD bands of lysozyme. The binding constants to the weaker site were about 1.1 x 10(-4), 5 x 10(2), and 5M(-1) for the trimer, dimer, and monomer of GlcNAc, respectively. Assuming that the trimer, dimer, and monomer occupy subsites D, E, and F; E and F; and E, respectively, the unitary free energies of saccharide binding were estimated to be about --1.9, --3.3, and --2.7 kcal/mole for D, E, and F, respectively.