A male and hermaphrodite specific RAPD marker for papaya ( Carica papayaL.)

The random amplified polymorphic DNA (RAPD) technique was used to determine the sex of a dioecious species, Carica papaya L., with three sex types, male, female and hermaphrodite. A 450 bp marker fragment, named PSDM(Papaya Sex Determination Marker), exists in all male and hermaphrodite plants but not in the female plants so far analyzed. The DNA sequence of PSDM exhibited no significant similarity to previously reported sequences. A sequence-characterized amplified region (SCAR) marker, SCARps, was developed from PSDM to determine the sex of papaya. Southern hybridization, using PSDM as a probe, showed that PSDM exists in the male and hermaphrodite genomes, but not in the female genome. This result strongly suggests that PSDM is located on the chromosome region that is specific to the male and the hermaphrodite. SCARps is a suitable marker for the precise and rapid diagnosis of sex in papaya. Received: 1 February 2001 / Accepted: 22 May 2001     

Rapid Changes in Polyphosphoinositide Metabolism in Pea in Response to Fungal Signals

Effects of the elicitor and/or suppressor from Mycosphaerellapinodes on polyphosphoinositide metabolism (PI metabolism) inpea were examined both in vivo and in vitro. The elicitor induceda rapid and biphasic increase in levels of phosphatidylinositol-4,5-bisphosphate(PtdInsP₂) and inositol 1,4,5-trisphosphate (IP₃) in epicotyltissues that was apparent within 15 min. A transient increasein levels of PtdInsP₂ and IP₃ was detected immediately in elicitor-treatedplasma membranes. However, the concomitant presence of suppressorwith elicitor resulted in inhibition of these increases bothin vivo and in vitro. These findings suggest that the elicitorrapidly activates phosphatidylinositol kinase, phosphatidylinositol-4-monophosphatekinase and phospholipase C, which are involved in PI metabolism,whereas the suppressor markedly inhibits these enzymes. Neomycin,a known inhibitor of phospholipase C, blocked the elicitor-inducedaccumulation both of IP₃ and pisatin and it also induced localsusceptibility in pea tissues that resembled that of the fungalsuppressor. From these results, it appears that rapid changesin PI metabolism are indispensable in the signal transductionrelated to defense responses of pea plants. (Received January 18, 1993; Accepted May 13, 1993)     

Loss of cytoplasmic retention ability of mutant LKB1 found in Peutz-Jeghers syndrome patients.

LKB1 Serine/Threonine (ST) kinase (also called STK11) originally identified in our novel protein kinase search project has recently been recognized as a susceptibility gene of Peutz-Jeghers Syndrome (PJS; MIM 175200). PJS is a dominantly inherited human disorder which is characterized by gastrointestinal hamartomatous polyposis and mucocutaneous melanin pigmentation. Since PJS patients also show a predisposition to a wide spectrum of cancers, it is speculated that LKB1 has a tumor suppressor function. In the present study we have characterized the basic biochemical property of LKB1. In the analysis of mutant LKB1 identified in PJS patients, it was found that one of the mutants, SL26, does not lose its kinase function, but alters its subcellular distribution to accumulate in the nucleus only, whereas wild type LKB1 shows both nuclear and cytoplasmic localization. Domain mapping of the nuclear targeting signal of LKB1 assigned it to its amino terminal side. Furthermore, it was shown that LKB1 also has a cytoplasmic retention ability which is considered defective and pathogenic in the SL26 mutant. It is speculated that subcellular distribution of LKB1 is regulated in the balance of these two forces, importation into the nucleus and retention within the cytoplasm; and the cytoplasmic retention ability is necessary for LKB1 to fulfil its normal function.     

Junctional adhesion molecule-C promotes metastatic potential of HT1080 human fibrosarcoma

The junctional adhesion molecule (JAM) family is a key molecule in a process called transendothelial migration or diapedesis. Here, we report implications of JAM-C in cancer metastasis. We first determined the mRNA expression of JAMs in 19 kinds of cancer cell lines. JAM-C was expressed in most of tumors having potent metastatic properties. Especially in murine K-1735 melanoma cell lines, the highly metastatic sublines (M2 and X21) strongly expressed JAM-C when compared with the poorly metastatic ones (C-10 and C23). Next, we investigated the role of JAM-C in cancer metastasis by using human JAM-C (hJAM-C) gene-transfected HT1080 fibrosarcoma cells. In comparison with mock-transfected HT1080 cells, these cells showed a significant increase in the adhesion to various extracellular substrates and the invasion across a Matrigel-coated membrane. The knockdown of hJAM-C using small interfering RNA resulted in the suppression of both the adhesion and the invasion of HT1080 cells, suggesting that endogenous hJAM-C might be involved in tumor metastasis. Finally, we studied the role of hJAM-C in an in vivo experimental metastatic model. The results showed that the overexpression of hJAM-C in HT1080 cells significantly decreased the life spans of the tumorbearing mice. In contrast, the knockdown of hJAM-C in HT1080 cells suppressed the weight gain of the lungs with metastatic colonies. We conclude that the expression of JAM-C promotes metastasis by enhancing both the adhesion of cancer cells to extracellular matrices and the subsequent invasion.     

Hyperglycemia impairs the insulin signaling step between PI 3-kinase and Akt/PKB activations in ZDF rat liver

Akt/PKB activation is reportedly essential for insulin-induced glucose metabolism in the liver. During the hypoinsulinemic and hyperglycemic phase in the Zucker diabetic fatty (ZDF) rat liver, insulin-induced phosphorylations of the insulin receptor (IR) and insulin receptor substrate (IRS)-1/2 were significantly enhanced. Similarly, phosphatidylinositol (PI) 3-kinase activities associated with IRS-1/2 were markedly increased in ZDF rat liver compared with those in the control lean rat liver. However, interestingly, insulin-induced phosphorylation and kinase activation of Akt/PKB were severely suppressed. The restoration of normoglycemia by sodium-dependent glucose transporter (SGLT) inhibitor to ZDF rats normalized elevated PI 3-kinase activation and phosphorylation of IR and IRS-1/2 to lean control rat levels. In addition, impaired insulin-induced Akt/PKB activation was also normalized. These results suggest that chronic hyperglycemia reduces the efficiency of the activation step from PI 3-kinase to Akt/PKB kinase and that this impairment is the molecular mechanism underlying hyperglycemia-induced insulin resistance in the liver.     

Primary alveolar echinococcosis: Course of larval development and antibody responses in intermediate host rodents with different genetic backgrounds after oral infection with eggs of Echinococcus multilocularis

We investigated parasite establishment, subsequent larval development and antibody responses in gerbils, cotton rats and 4 inbred mouse strains until 16 weeks post inoculation (p.i.) with 200 eggs of Echinococcus multilocularis. The rate of parasite establishment in the liver determined at 4 weeks p.i. was highest in DBA/2, followed by AKR/N, C57BL/10 and C57BL/6 mice, whereas gerbils harboured few parasite foci. The accurate number of liver lesions in cotton rats could not be determined due to rapid growth and advanced multivesiculation of the parasite observed at 2 weeks p.i. The course of larval development was most advanced in DBA/2 mice with mature protoscolex formation at 16 weeks p.i., followed by AKR/N harbouring metacestodes with sparsely distributed immature protoscoleces. On the other hand, C57BL/6 and C57BL/10 mice had infertile metacestodes without any protoscolex formation. The parasite growth in mice was totally slower than those in gerbils and cotton rats. Specific IgG and IgM responses against 3 types of native crude antigens of larval E. multilocularis were evaluated using somatic extracts of and vesicle fluid of metacestode, and somatic extracts from purified protoscoleces. The 4 mouse strains demonstrated basically similar kinetics with apparent IgG and IgM increases at 9 weeks p.i. and thereafter, except C57BL/10, exhibited higher levels of IgM against crude antigens at some time point of infection. On the other hand, a follow-up determination of specific IgG and IgM levels against recombinant antigens from larval E. multilocularis revealed that each mouse strain showed different antibody-level kinetics. The findings in the present study demonstrate that the course of host–parasite interactions in primary alveolar echinococcosis, caused by larval E. multilocularis, clearly varies among intermediate host rodents with different genetic backgrounds.     

Studies on the substrate specificity of neutral alpha-mannosidase purified from Japanese quail oviduct by using sugar chains from glycoproteins.

The substrate specificity of neutral alpha-mannosidase purified from Japanese quail oviduct [Oku, H., Hase, S., & Ikenaka, T. (1991) J. Biochem. 110, 29-34] was analyzed by using 21 oligomannose-type sugar chains. The enzyme activated with Co2+ hydrolyzed the Man alpha 1-3 and Man alpha 1-6 bonds from the non-reducing termini of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc (M5A), but hardly hydrolyzed the Man alpha 1-2 bonds of Man9GlcNAc2. The hydrolysis rate decreased as the reducing end of substrates became more bulky: the hydrolysis rate for the pyridylamino (PA) derivative of M5A as to that of M5A was 0.8; the values for M5A-Asn and Taka-amylase A having a M5A sugar chain being 0.5 and 0.04, respectively. The end product was Man beta 1-4GlcNAc2. For the substrates with the GlcNAc structure at their reducing ends (Man5GlcNAc, Man6GlcNAc and Man9GlcNAc), the hydrolysis rate was remarkably increased: Man5GlcNAc was hydrolyzed 16 times faster than M5A, and Man2GlcNAc 40 times faster than Man9GlcNAc2. The enzyme did not hydrolyze Man alpha 1-2 residue(s) linked to Man alpha 1-3Man beta 1-4GlcNAc. The end products were as follows: [formula; see text] These results suggest that oligomannose-type sugar chains with the GlcNAc structure at their reducing ends seem to be native substrates for neutral alpha-mannosidase and the enzyme seems to hydrolyze endo-beta-N-acetylgucosaminidase digests of oligomannose-type sugar chains in the cytosol.     

Studies on the probing stimulants for the white-backed planthopper,Sogatella furcifera (Homoptera: Delphacidae) in rice plant (Oryza sativa L.)

To elucidate the probing stimulants in rice plants for the white-backed planthopper, Sogatella furcifera, bioassay-guided separations were conducted, which led to the isolation of four active compounds. Using NMR and LC-MS spectra, their structures were determined as isoorientin 2″-O-(6?-(E)-feruloyl)glucoside, isoorientin 2″-O-(6?-(E)-p-coumaroyl)glucoside, tricin 5-O-glucoside, and isoscoparin 2″-O-(6?-(E)-feruloyl)glucoside.     

Adaptive significance of death feigning posture as a specialized inducible defence against gape-limited predators

Death feigning is fairly common in a number of taxa, but the adaptive significance of this behaviour is still unclear and has seldom been tested. To date, all proposed hypotheses have assumed that prey manage to escape predation by sending a death-mimicking signal, although death-feigning postures are markedly different from those of dead animals. Moreover, the efficacy of this technique may largely depend on the foraging mode of the predator; death feigning seldom works with sit-and-wait predators that make the decision to attack and consume prey within a very brief time. We examined whether death feigning in the pygmy grasshopper Criotettix japonicus Haan was an inducible defence behaviour against the frog Rana nigromaculata, a sit-and-wait, gape-limited predator. The characteristic posture assumed by the grasshopper during death feigning enlarges its functional body size by stretching each of three body parts (pronotum, hind legs and lateral spines) in three different directions, thereby making it difficult for the predator to swallow the prey. Our result is the first consistent explanation for why death-mimicking animals do not always mimic the posture of dead animals.