Spontaneous loss of a conditionally dispensable chromosome from the Alternaria alternata apple pathotype leads to loss of toxin production and pathogenicity.

The Alternaria alternata apple pathotype causes Alternaria blotch of susceptible apple cultivars through the production of a cyclic peptide, host-specific toxin, AM-toxin. We recently cloned a cyclic peptide synthetase gene, AMT, whose product catalyzes the production of AM-toxin and showed that it resides on chromosomes of 1.8 Mb or less, depending on the A. alternata apple pathotype strain. Reverse transcriptase (RT)-PCR, using primers specific to AMT, on laboratory sub-cultured strains previously shown to produce AM-toxin, identified one isolate that did not express the gene. A leaf necrosis bioassay confirmed an AM-toxin-minus phenotype. However, an original isolate of this strain which had not undergone sub-culture gave a positive result by both RTPCR and bioassay. Contour-clamped homogeneous electric field electrophoresis and Southern hybridization demonstrated the loss of a 1.1-Mb chromosome in the non-toxin-producing isolate. Since this chromosome can be entirely lost without affecting growth, but is necessary for pathogenicity, we propose it is a conditionally dispensable chromosome.

Regulation of glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) gene in the mouse mammary gland differs from that of casein genes.

Mouse glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), also known as mC26 and homologous to bovine PP3, is a milk protein synthesized in the mammary gland. Several studies have investigated the regulation of casein, the major milk protein, gene in the mammary gland, but little is known about GlyCAM-1. Here we examined GlyCAM-1 gene expression in mouse mammary epithelial cells. First, we detected GlyCAM-1 expression in mammary epithelial cells in situ by immunohistochemistry; almost all mammary epithelial cells of the lactating mouse expressed GlyCAM-1. Second, mammary epithelial cells were digested with collagenase and cultured with insulin, prolactin and/or glucocorticoid. alpha-Casein and beta-casein genes were expressed following treatment with insulin, prolactin and glucocorticoid. In contrast, GlyCAM-1 expression could not be detected with any combination of these three hormones. We also analyzed changes in the levels of GlyCAM-1 and caseins mRNAs in cultured cells. The addition of hormones to the culture medium increased casein mRNAs, but surprisingly reduced GlyCAM-1 mRNA. Our results suggest that the mechanisms that regulate GlyCAM-1 gene in mammary cells of lactating mice are different from those involved in the regulation of casein genes.

Cloning and characterization of a cyclic peptide synthetase gene from Alternaria alternata apple pathotype whose product is involved in AM-toxin synthesis and pathogenicity.

Afternaria afternata apple pathotype causes Alternaria blotch of susceptible apple cultivars through the production of a cyclic peptide host-specific toxin, AM-toxin. PCR (polymerase chain reaction), with primers designed to conserved domains of peptide synthetase genes, amplified several products from A. alternata apple pathotype that showed high similarity to other fungal peptide synthetases and were specific to the apple pathotype. Screening of a Lambda Zap genomic library with these PCR-generated probes identified overlapping clones containing a complete cyclic peptide synthetase gene of 13.1 kb in length with no introns. Disruption of this gene, designated AM-toxin synthetase (AMT), by transformation of wild-type A. afternata apple pathotype with disruption vectors resulted in toxin-minus mutants, which were also unable to cause disease symptoms on susceptible apple cultivars. AM-toxin synthetase is therefore a primary determinant of virulence and specificity in the A. alternata apple pathotype/apple interaction.

Expression of cell cycle regulators and growth factor/receptor systems in gastric carcinoma in young adults: association with Helicobacter pylori infection.

We studied the expression of cell cycle regulators and growth factor-receptor systems in gastric carcinoma in young adults and tried to clarify the specific alterations associated with H. pylori. We studied 33 young patients (18-29 years old, mean age 26.4) with gastric carcinoma. The patients were classified into two groups according to the degree of atrophic gastritis. Then we examined the expression of p53, cripto, cyclin-E, c-met, c-erbB2 and TGF-alpha immunohistochemically and compared the results between the two groups. The results were compared with 66 sex-, tumor histology-, and depth-matched elder controls (36-86 years old, mean age 64.0). H. pylori was judged by Giemsa staining. Seventeen patients had atrophic changes in the corpus (Group A), while 16 showed superficial gastritis or normal mucosa (Group S). All 17 patients of Group A showed H. pylori infection, while the 3 of the 16 members of Group S did not have H. pylori. p53 overexpression was observed more frequently in Group S (88%) than in Group A (41%, p<0.05). In the 3 patients without H. pylori infection, all carcinoma specimens showed p53 overexpression. Overexpression of cyclin-E was detected in 4 patients from Group S. On the other hand, cripto was observed more frequently in Group A than in Group S. No obvious differences were observed in c-erbB2, TGF-alpha and c-met expression. Overall, p53 overexpression was detected more frequently in younger than in older patients, whereas cripto expression was less detected. These results suggest that p53 and cyclin-E may act in an H. pylori-independent or -adjunctive manner for gastric carcinogenesis. Cripto expression might be correlated tightly with H. pylori infection.

A Polymerase Chain Reaction-Based Method to Specifically Detect Alternaria alternata Apple Pathotype (A. mali), the Causal Agent of Alternaria Blotch of Apple.

ABSTRACT Alternaria alternata apple pathotype (previously A. mali) causes Alternaria blotch on susceptible apple cultivars through the production of a host-specific toxin, AM-toxin. Identification of some Alternaria species, especially those that produce host-specific toxins, has been extremely difficult due to a high level of variability which extends even to nonpathogenic isolates. We have recently cloned and characterized a gene (AMT) that plays a crucial role in AM-toxin biosynthesis and demonstrated that it is only present in isolates of A. alternata apple pathotype. Using primers designed for the AMT gene, we developed a polymerase chainreaction-based method to specifically detect AM-toxin producing isolates of A. alternata apple pathotype.

(+)-catechin acts as an infection-inhibiting factor in strawberry leaf.

ABSTRACT An infection-inhibiting factor (IIF) was isolated from strawberry leaves and identified as (+)-catechin. This compound inhibited the formation of infection hyphae from appressoria of Alternaria alternata, but allowed both spore germination and appressorial formation. It is a normal component of strawberry leaves, but further accumulates as the major IIF in response to inoculation with nonpathogenic spores of A. alternata. The accumulation of (+)-catechin on a susceptible host was not induced, however, by inoculation with pathogenic spores of the strawberry pathotype or by inoculation with nonpathogenic spores supplemented with host-specific toxin (AF-toxin I). These results imply that (+)-catechin acts as a protective agent during induced resistance and that AF-toxin I acts as a fungal suppressor of induced resistance.

Molecular karyotypes for Alternaria plant pathogens known to produce host-specific toxins.

There are at least ten plant diseases caused by Alternaria species in which host-specific toxins (HSTs) are responsible for fungal pathogenicity. Of these HST-producers, seven are considered distinct pathotypes of the species Alternaria alternata, and the remaining three are among other species of pathogenic Alternaria. Inter- and intra-specific variation among Alternaria taxa, including HST-producers, was determined by electrophoretic karyotyping using pulsed-field gel electrophoresis. A. alternata including seven pathotypes of A. alternata and eight non-pathogenic strains had 9-11 chromosomal bands with estimated sizes ranging from 0.4 to 5.7 Mb. In contrast, Alternaria species that are morphologically distinct from A. alternata had 8-10 bands with sizes between 0.9 and 5.7 Mb. Estimated genome sizes of A. alternata and other Alternaria species ranged from 28.8 to 33.6 Mb and 25.1 to 30.7 Mb, respectively. Other species of pathogenic Alternaria were difficult to differentiate from A. alternata on the basis of chromosome-size polymorphisms alone, but Southern analysis using rDNA as a probe could, in some cases, differentiate between them. These results were cytologically confirmed by 4',6-diamidino-2-phenylindole (DAPI) staining and fluorescence in situ hybridization with a rDNA probe for mitotic metaphase chromosomes prepared by the germ-tube burst method.

Regulation of flagellar bending by cAMP and Ca2+ in hamster sperm.

Hamster sperm were immotile in the medium at free Ca2+ concentrations ([Ca2+]) below 1 x 10(-4) M. The flagellum was acutely bent in the opposite direction to the curve of the hook-shaped heads. This phenomenon seemed to be caused by the decrease in the intracellular cAMP concentration, since the cAMP concentration was low at [Ca2+] below 1 x 10(-4) M and increased abruptly at 1 x 10(-3) M, at which sperm were swimming actively. In addition, sperm became motile due to treatment with 8-bromo-cAMP, a membrane permeable analogue of cAMP, in a medium without Ca2+. These results suggested that extracellular Ca2+ is involved in the regulation of flagellar movement via increasing intracellular cAMP concentration. By the treatment with W-13, a calmodulin inhibitor, sperm also became motile, although cAMP concentration remained at a low level. These results suggested that cAMP is not always required for the flagellar movement when the function of calmodulin is depressed.

Effects of 1alpha,25-dihydroxycholecalciferol and cortisol on the growth and differentiation of primary cultures of mouse mammary epithelial cells in collagen gel.

We examined the effects of 1alpha,25-dihydroxycholecalciferol (1,25-DHCC) and the glucocorticoid, cortisol, on primary mouse mammary epithelial cells in collagen gel cell culture systems. Physiological low concentrations (10(-11)-10(-9) M) of 1,25-DHCC stimulated growth of the cells in a collagen gel matrix culture in serum-free DMEM+Ham's F12 (1:1) medium containing BSA, EGF and cholera toxin, and the cell number reached 1.8-fold the control after 6 d in culture. In contrast, supraphysiological concentrations (10(-8)-10(-7) M) of 1,25-DHCC suppressed cell growth. Cortisol produced similar, but smaller, dose-dependent effects. The addition of serum to the culture medium masked the stimulatory effect of 1,25-DHCC and both the stimulatory and inhibitory effects of cortisol. 1,25-DHCC also affected casein synthesis by cells cultured in a serum-free floating collagen gel culture containing prolactin, insulin and cortisol, enhancing synthesis at low concentrations (10(-11)-10(-9) M) and inhibiting it above 10(-8) M. In the absence of cortisol, no detectable change in casein synthesis was induced by 1,25-DHCC. These results suggest a physiological role for 1,25-DHCC in stimulating both growth and differentiation of mouse mammary epithelial cells, though 1,25-DHCC does not substitute for glucocorticoids in the differentiation of the cells.

Purification and Biological Characterization of Host-Specific SV-Toxins from Stemphylium vesicarium Causing Brown Spot of European Pear.

ABSTRACT Culture filtrates of a pathogenic isolate (IT37) of Stemphylium vesicarium, causing brown spot of European pear, induced veinal necrosis only on pear leaves susceptible to the pathogen. Two host-specific toxins, SV-toxins I and II, were purified from culture filtrates of IT37 by successively using Amberlite XAD-2 resin adsorption, cellulose thin-layer chromatography, and high-performance liquid chromatography under three different sets of conditions. Susceptible cultivars showed veinal necrosis at a SV-toxin I concentration of 0.01 to 0.1 mug/ml, whereas resistant cultivars were insensitive to the toxin at 1,000 mug/ml. SV-toxins I and II caused a dose-dependent increase in electrolyte loss from susceptible leaf tissues. No increase in electrolyte loss was detected in leaf tissues from resistant cultivars. The results of physiological studies indicated that SV-toxins appear to have an early effect on plasma membranes of susceptible leaves. Spores of a nonpathogenic isolate induced necrotic lesions on susceptible leaves in the presence of a small amount of toxin. SV-toxins were detected in intercellular fluids obtained from diseased leaves after inoculation with the pathogen. The results indicate that SV-toxins I and II produced by S. vesicarium can be characterized as host-specific toxins.

AAL-Toxin-Deficient Mutants of Alternaria alternata Tomato Pathotype by Restriction Enzyme-Mediated Integration.

ABSTRACT Host-specific toxins are produced by three pathotypes of Alternaria alternata: AM-toxin, which affects apple; AK-toxin, which affects Japanese pear; and AAL-toxin, which affects tomato. Each toxin has a role in pathogenesis. To facilitate molecular genetic analysis of toxin production, isolation of toxin-deficient mutants utilizing ectopic integration of plasmid DNA has been attempted. However, the transformation frequency was low, and integration events in most transformants were complicated. Addition of a restriction enzyme during transformation has been reported to increase transformation frequencies significantly and results in simple plasmid integration events. We have, therefore, optimized this technique, known as restriction enzyme-mediated integration (REMI), for A. alternata pathotypes. Plasmid pAN7-1, conferring resistance to hygromycin B, with no detectable homology to the fungal genome was used as the transforming DNA. Among the three restriction enzymes examined, HindIII was most effective, as it increased transformation frequency two-to 10-fold depending on the pathotype, facilitating generation of several hundred transformants with a 1-day protocol. BamHI and XbaI had no significant effect on transformation frequencies in A. alternata pathotypes. Furthermore, the transforming plasmid tended to integrate as a single copy at single sites in the genome, compared with trials without addition of enzyme. Libraries of plasmid-tagged transformants obtained with and without addition of restriction enzyme were constructed for the tomato pathotype of A. alternata and were screened for toxin production. Three AAL-toxin-deficient mutants were isolated from a library of transformants obtained with addition of enzyme. These mutants did not cause symptoms on susceptible tomato, indicating that the toxin is required for pathogenicity of the fungus. Characterization of the plasmid integration sites and rescue of flanking sequences are in progress.

Association of p34cdc2 and cyclin B1 during meiotic maturation in porcine oocytes.

The relative levels and association of p34cdc2 and cyclin B1 have been determined in pig oocytes during meiotic progression from G2 to metaphase II (MII). Fully grown G2-arrested porcine oocytes contained large amounts of free p34cdc2 and extremely small amounts of p34cdc2-cyclin B1 complex which did not increase in amount during the GV stage. Cyclin B1 is not synthesized in measurable quantities until 23 hr after the start of maturation. During the first metaphase (MI) the amount of both cyclin B1 and the p34cdc2-cyclin B1 complex increased sharply until 35 hr. Thereafter the amount of the p34cdc2-cyclin B1 complex increased again to 45 hr, resulting in higher levels of the complex in MII than in MI. Moreover, three distinct migration forms of p34cdc2 were detected in pig oocytes by immunoblotting with anti-PSTAIRE antibody, and most of the cyclin B1-associated p34cdc2 was detected in the lower band in MI oocytes but migrated in the second band during MII. These results suggest that cyclin B1 levels in porcine oocytes differ from those in starfish, clam, and Xenopus oocytes in which considerably higher concentrations of cyclin B1 appear to be present during the GV stage and that the nature of the association between p34cdc2 and cyclin B1 changes between the first and the second metaphase.

Helicobacter pylori infection, serum gastrin, and gastric acid secretion in teen-age subjects with duodenal ulcer, gastritis, or normal mucosa.

BACKGROUND: Many studies have confirmed the close association of Helicobacter pylori with duodenal ulcer (DU) in adults. However, in the subtype of DU known as 'childhood' or 'early onset DU' genetic factors seem to play a prominent role in the pathogenesis. The aim of this study was to investigate the prevalence of H. pylori in teen-age subjects with DU, gastritis, and normal mucosa and to examine the relationship of H. pylori to serum gastrin levels and gastric acid secretion. METHODS: Sixty-one teen-age subjects (24 with DU, 14 with gastritis, and 23 normal subjects) were investigated for the presence of H. pylori, antral histology, gastrin levels, basal acid output (BAO), and maximal acid output (MAO). RESULTS: All 24 patients with DU and 8 of 14 with gastritis were infected with H. pylori; none of the normal subjects were infected. Mean gastritis scores and fasting serum gastrin levels were significantly higher in patients with DU or H. pylori-positive gastritis than in subjects with H. pylori-negative gastritis or normal mucosa (p < 0.05). The difference in serum gastrin levels was also significant when patients with DU were compared with those with H. pylori-positive gastritis (p < 0.05). BAO and MAO were significantly higher in patients with DU than in subjects with H. pylori-positive gastritis or normal mucosa (p < 0.05), but there was no difference between subjects with H. pylori-positive gastritis and those with normal mucosa. CONCLUSION: H. pylori infection is associated closely with teen-age DU and gastritis and with hypergastrinemia but does not affect BAO and MAO in most infected teen-age subjects.

Significance of rapid urease test for identification of Helicobacter pylori in comparison with histological and culture studies.

Helicobacter pylori in the stomach is an etiological factor of gastritis and peptic ulcer. It is now considered that gastric cancer can be, at least in some cases, a late complication of H. pylori infection. In 123 consecutive endoscopic antral biopsies obtained from patients with the Okamoto Hospital, the specimens were subjected to the rapid urease test (RUT), histology (H&E stain), and culture, for the identification of H. pylori. The results of these methods were compared semi-quantitatively in order to evaluate these detection methods for identifying H. pylori. The results of these methods were found to agree well, with the Spearman's rank correlation coefficient between RUT and culture being 0.90 (P < 0.01) and that between histology and culture being 0.80 (P < 0.01). RUT is considered to be a very simple, sensitive, and highly specific test which enables the endoscopist to diagnose H. pylori infection.

Analysis of flagellar bending in hyperactivated hamster and mouse spermatozoa.

Flagellar bending was analysed using photographs of hyperactivated hamster and mouse spermatozoa. The flagellar waveform consists of several bends; the centre of each bend was therefore located on the flagellum, and the angle of the bend measured. The direction of the bend was determined by using the asymmetry of hook-shaped head to assess the asymmetry of flagellar waveform. The bend that occurred in the same direction as the curve of head was defined as the reverse bend and the bend in the opposite direction as the principal bend. In hamster spermatozoa, flagellar bending was asymmetric to the direction of the reverse bend after incubation for 5 min. After incubation for 4 h the asymmetry had increased, as the angles of the reverse bends had increased in all regions of the flagellum but the principal bend had not. In mouse spermatozoa incubated for 5 min, flagellar bending was relatively symmetric. In the hyperactivated mouse spermatozoa incubated for 3 h, the angle of the principal bend increased in the distal region and those of the reverse bends increased in almost all regions of the flagellum. Since the increase in the reverse bend was relatively high, flagellar bending became asymmetric to the direction of the reverse bend as in hamster spermatozoa. These increases in asymmetry were also evident in the measurement of the total changes in angular direction between the proximal and distal end of flagella in both species. The increase in asymmetry could provide an explanation for the changes in the motility patterns seen in spermatozoa after the onset of hyperactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the mC26 gene encoding GlyCAM-1 in the lactating mouse mammary gland.

We previously reported the partial sequence of a cloned genomic DNA, mC26, which codes for a protein highly expressed in the lactating mouse mammary gland [mC26: Satow et al. (1986) J. Biochem. 99, 1639-1643; partial sequence: Kawamura et al. (1987) J. Biochem. 101, 103-110]. In this study, we sequenced the EcoRI-HindIII fragment (5,394 bp) of this gene and found that this gene contains a sequence completely (100%) homologous to the cDNA sequence currently reported to code for GlyCAM-1, a putative ligand for L-selectin. We show by means of an RNA protection assay that the mRNA of this gene is expressed in the mammary glands of lactating mice as well as in the lymph nodes. Semi-quantitative analysis of expression of the mC26 gene in the mammary glands revealed that the amount of mRNA was not detectable in the early stage of pregnancy, increased in the late stage, and remained quite abundant during lactation. The potential role of this gene highly expressed in the mammary gland in a stage-specific and tissue-specific manner is discussed.

Activation of mitogen-activated protein kinase during meiotic maturation in mouse oocytes.

Mitogen-activated protein kinase (MAP kinase) plays a role in the cascade of protein kinase activation in cultured cells. To investigate the involvement of MAP kinase in meiotic maturation, we measured MAP kinase activity, using myelin basic protein as a substrate, with histone H1 kinase activity, in mouse oocytes. MAP kinase activity was low 1 h after isolation from follicles (when oocytes lost their germinal vesicle), increased abruptly at 2 h, and remained high until the second metaphase (13 h after isolation from follicles). Histone H1 kinase activity increased gradually from 2 to 7 h after isolation. When immature oocytes were treated with puromycin, MAP kinase activity did not increase after isolation from follicles. In the presence of 3-isobutyl-1-methylxanthine, the treatment of immature oocytes with okadaic acid, a specific inhibitor of protein phosphatase 1 and 2A, induced germinal vesicle breakdown and activation of MAP kinase. These results suggest that MAP kinase is involved in the regulation of meiotic maturation, and that the activation of MAP kinase requires protein synthesis and is inhibited by the protein phosphatase during meiotic maturation in mouse oocytes.

A deficiency in the mechanism for p34cdc2 protein kinase activation in mouse embryos arrested at 2-cell stage.

Mouse embryos of the ddY strain fertilized in vitro undergo the first cleavage to the 2-cell stage but not the second cleavage even 45 hr after insemination (2-cell block). We examined the phosphorylation state of p34cdc2 and histone H1 kinase activity in mouse 2-cell embryos to investigate the relationship of p34cdc2 with 2-cell block. In the first mitotic cell cycle, the amount of phosphorylated forms of p34cdc2, which were detected as the bands of retarded mobility on SDS-PAGE followed by immunoblotting with anti-p34cdc2 antibody, increased during interphase and abruptly decreased at M phase. Concomitant with this dephosphorylation, histone H1 kinase activity was increased. After the embryos cleaved to the 2-cell stage, the amounts of phosphorylated forms of p34cdc2 increased up to 33 hr after insemination. However, the activation of histone H1 kinase did not occur and the states of phosphorylation of p34cdc2 did not show any significant changes until 45 hr. In contrast, 2-cell embryos of B6C3F1 mice, which do not show a 2-cell block and develop normally to blastocysts in vitro, exhibit the dephosphorylation of p34cdc2 and an increase in histone H1 kinase activity between 31 and 45 hr after insemination. When the ddY mouse embryos arrested at the 2-cell stage were treated with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, the dephosphorylation of p34cdc2 occurred and histone H1 kinase activity increased. The chromosomes of these embryos stained with 4',6'-diamidino-2-phenylindole revealed the initiation of condensation. These results suggest that 2-cell-blocked embryos contain enough p34cdc2 to induce mitotic events but the protein remains in a latent form.

[The use of a Swan-Ganz catheter with a fast-response thermistor for the measurement of right ventricular performance during anesthetic management of pheochromocytoma].

We used a Swan-Ganz catheter with a fast-response thermistor to measure the right ventricular ejection fraction (RVEF) during the anesthetic management of two patients with epinephrine-dominant pheochromocytomas. Pre-operatively, one patient received alpha adrenergic blocking agents (prazocine, doxazocine) to control the blood pressure but the other patient did not receive any agents. In the latter patient who did not receive alpha adrenergic blocking agents, right ventricular function was depressed post-operatively in the recovery room. The importance of preoperative preparation with alpha adrenergic blocking agents was confirmed by the reductions in RVEF and RVEDVI (right ventricular end-diastolic volume index) after resection of the tumor. Not only left heart monitoring but also right heart monitoring with RVEF and RVEDVI are recommended for the proper management of a patient with pheochromocytoma.