Male white grub beetles prefer the pheromone composition of young females in the field.

Females of the white grub beetle, Dasylepida ishigakiensis, release both (R)- and (S)-2-butanol as sex pheromones, but the males are only attracted to (R)-2-butanol. In laboratory-reared females, the proportion of the (R)-isomer decreased significantly as their calling opportunities increased and as they aged. We examined whether such qualitative changes also occur in field populations. We collected virgin females from the field and then trapped and analysed the volatiles emitted during their first and second callings. The ratio of (R)- to (S)-2-butanol (R/S) was 78:22 at the first calling, but shifted to 39:61 at the second calling. While investigating the composition of the female pheromones, the question arose as to whether the male preferences change in response to the shift in female pheromone composition. To answer this question, we observed the behaviour of young and old males in response to various R/S ratios as lures in the laboratory and in the field. In the flight tunnel assay of laboratory-reared individuals, young males touched female models with a 9:1 R/S ratio lure less than those with pure (R)-2-butanol; however, older males touched the two groups with equivalent frequency. In the field trap test, older males were much more attracted to (R)-2-butanol-scented lures. When we tested using lures with the same amount of (R)-2-butanol but added different amounts of the (S)-isomer, we found that increased levels of (S)-2-butanol resulted in lower attractiveness to males. (S)-2-butanol was confirmed to have an inhibitive activity in the attractiveness of (R)-2-butanol.

Age-dependent changes in the ratio of (R)- and (S)-2-butanol released by virgin females of Dasylepida ishigakiensis (Coleoptera: Scarabaeidae).

The females of the white grub beetle, Dasylepida ishigakiensis, release two enantiomers of 2-butanol, (R)-2-butanol and (S)-2-butanol. The ratio describing the relative proportions of these two enantiomers (R/S ratio) has not yet been investigated. (R)-2-Butanol has been shown to attract males in laboratory and field experiments, whereas (S)-2-butanol tends to inhibit them. To determine the R/S ratio of the 2-butanol emitted by virgin females, we collected 2-butanol from young (53 days old), mature (63 days old) and old females (73 days old) using water, extracted with an SPME fibre and subsequently injected into GC-MS. The major component of the 2-butanol emitted by the young females was (R)-2-butanol, but as the females aged, the component ratio favoured (S)-2-butanol. Young females released an 80:20 mixture of (R)- and (S)-2-butanol, whereas old females released a 45:55 mixture. The EAG response of male antennae to a 50:50 ratio (racemic mixture) showed a similar dose-response curve to that of (R)-2-butanol. The male orientation responses to (R)-2-butanol decreased when the relative proportion of (S)-2-butanol increased. An inhibitory and/or masking effect of (S)-2-butanol on male orientation behaviour was also observed in the flight tunnel assay. These results suggest that males are more strongly attracted to young females than to old females. We also discuss the possibility of using 2-butanol isomers as a control or monitoring agent for this insect.

Mating disruption by a synthetic sex pheromone in the white grub beetle Dasylepida ishigakiensis (Coleoptera: Scarabaeidae) in the laboratory and sugarcane fields.

A serious sugarcane pest, Dasylepida ishigakiensis, remains in the soil during most of its life cycle except for a short period for mating. Mating disruption by an artificial release of the sex pheromone (R)-2-butanol (R2B), therefore, may be a feasible method to control this pest. We examined the effects of artificial release of R2B and its related compounds, (S)-2-butanol (S2B) and the racemic 2-butanol (rac-2B), on the mating success of this beetle both in the laboratory and in the field. In flight tunnel experiments, almost all males orientated towards a R2B-releasing source and 40% of them landed on the source. When the atmosphere was permeated with R2B, the frequency of males landing on the model was significantly reduced. Both rac-2B and S2B were less effective, but substantial reduction in landing success by males was achieved at higher rac-2B concentrations. R2B released from polyethylene dispensers in sugarcane plots greatly reduced not only the proportion of females mated with males but also the number of males caught by R2B-baited traps, indicating that male mate-searching behaviour was strongly affected by the released R2B. Similar inhibitory effects on male behaviour were also observed when tube- or rope-type dispensers released high rac-2B concentrations in the field. These results indicate that it would be highly possible to control D. ishigakiensis through the disruption of the sexual communication by releasing either synthetic R2B or rac-2B.

Clinical and imaging features distinguishing Susac syndrome from primary angiitis of the central nervous system.

INTRODUCTION: To assess clinical and/or imaging features useful to distinguish between Susac syndrome (SuS) and primary angiitis of central nervous system (PACNS). METHODS: Multicenter retrospective analysis of two cohorts of Argentine patients diagnosed with SuS and PACNS. RESULTS: 13 patients diagnosed with SuS (6 women and 7 men, mean age 35 ±â€¯10 years) and 15 with PACNS (10 women and 5 men, mean age 44 ±â€¯18 years) were analyzed. Cognitive impairment (11 out of 13 patients vs. 5 out of 15, p = .006), ataxia (7 out of 13 vs. 2 out of 15, p = .042) and auditory disturbances (7 out of 13 vs. 0 out of 15, p = .003) were more frequent in SuS patients; whereas seizures were more frequent in PACNS patients (8 out of 15 vs. 1 out of 13, p = .035). On MRI, corpus callosum (CC) involvement was observed more often in SuS, with abnormalities in CC genu, in 13 out of 13 SuS patients vs. only 2 out of 15 PACNS patients (p < .001); in CC body these were present in 13 out of 13 SuS patients vs. 1 out of 15 PACNS patients, (p < .001); and in CC splenium in 12 out of 13 Sus patients vs. 1 of 15 PACNS, p < .001). Cortical lesions were more frequent in PACNS patients (10 out of 15 vs. 3 out of 13 SuS patients, p = .02), as were hemorrhages (5 out of 15 vs. 0 out of 13 SuS, p = .04) and multiple basal ganglia infarcts (7 out of 15 vs. 1 out of 13 Sus, p = .037). CONCLUSION: Specific clinical and/or MRI findings may help distinguish SuS from PACNS with potential therapeutic implications.

Native and recombinant human hepatocyte growth factors are highly potent promoters of DNA synthesis in both human and rat hepatocytes.

Human hepatocyte growth factor (hHGF) has recently been expressed as a recombinant polypeptide from Chinese hampster ovary cell transfectants. Using a primary rat hepatocyte bioassay, we have tested the biological activity of recombinant hHGF and compared it with native hHGF. Dose-response curves were almost identical, with half-maximal stimulation of DNA synthesis at 1-2 ng/ml (equivalent to approximately 10 pM). S-phase labeling index was similarly enhanced and numerous mitotic cells were observed. Recombinant and native hHGF also stimulated DNA synthesis and S-phase labeling index in primary adult human hepatocytes. Human cells were more responsive than rat hepatocytes, with recombinant hHGF slightly more potent than native hHGF (half-maximal stimulation 0.3 and 0.6 ng/ml, respectively). Since HGF levels rise in patients with fulminant hepatic failure and in animals after partial hepatectomy or administration of hepatotoxins, situations where liver regeneration occurs, HGF is suggested to play a key role in regulation of hepatic growth. The high potency of the factor on human hepatocytes reinforces its candidacy as a critical mitogen in human liver growth. The availability of a recombinant hHGF opens the way for in vivo experimental studies and to the possibility of using hHGF as a clinical therapeutic agent, either alone or in combination with other factors.

Localized energization of the mitochondrial inner membrane by ATP.

Studies were made to determine whether the energy-dependent binding of ethidium to the mitochondrial inner membrane reflects the membrane potential or the energization of localized regions of the membrane. The number of binding sites of ethidium in mitochondria energized with ATP was 72 nmol/mg protein and decreased with increase in the amount of the ATPase system (F1 . F0) inactivated by oligomycin. These findings clearly show that the energy-dependent binding of ethidium to the mitochondrial inner membrane energized with ATP does not reflect the membrane potential, in good accord with the previous conclusion (Higuti, T., Yokota, M., Arakaki, N., Hattori, A. and Tani, I. (1978) Biochim. Biophys. Acta 503, 211-222), but that ethidium binds to localized regions of the energized membrane that are directly affected by ATPase (F1), reflecting the localized energization of the membrane by ATP.

Sidedness of inhibition of energy transduction in oxidative phosphorylation in rat liver mitochondria by ethidium bromide.

Ethidium bromide, a new type of inhibitor of energy transduction in oxidative phosphorylation, inhibited ATP synthesis in intact mitochondria but not in submitochondrial particles, the latter being inside-out relative to the membranes of intact mitochondria. Ethidium bromide incorporated inside the submitochondrial particles inhibited ATP synthesis in the particles. The decrease of the membrane potential by valinomycin (plus KCl) inhibited only slightly the energy-dependent binding of ethidium bromide to the mitochondria. The present results show clearly that ethidium bromide inhibited energy transduction in oxidative phosphorylation by acting on the outer side (C-side) of the inner mitochondrial membrane, perhaps by neutralizing negative charges created on the surface of the C-side, and that it had no inhibitory activity on the inner side (M-side) of the membrane. Th present results show also that the energy-dependent binding of ethidium is not due to electrophoretic transport down the membrane potential; ethidium may bind to negative charges on the surface of the C-side. The present study suggest that an anisotropic distribution of electric charge in the inner mitochondrial membrane is an intermediary high energy state of oxidatvie phosphorylation.

Stoichiometry of subunit e in rat liver mitochondrial H(+)-ATP synthase and membrane topology of its putative Ca(2+)-dependent regulatory region.

Previous studies have revealed that residues 34-65 of subunit e of mitochondrial H(+)-ATP synthase are homologous with the Ca(2+)-dependent tropomysin-binding region for troponin T and have suggested that subunit e could be involved in the Ca(2+)-dependent regulation of H(+)-ATP synthase activity. In this study, we determined the content of subunit e in H(+)-ATP synthase purified from rat liver mitochondria, and we also investigated the membrane topology of a putative Ca(2+)-dependent regulatory region of subunit e using an antibody against peptide corresponding to residues 34-65 of subunit e. Quantitative immunoblot analysis of subunit e in the purified H(+)-ATP synthase revealed that 1 mol of H(+)-ATP synthase contained 2 mol of subunit e. The ATPase activity of mitoplasts, in which the C-side of F(0) is present on the outer surface of the inner membrane, was significantly stimulated by the addition of the antibody, while the ATPase activity of submitochondrial particles and purified H(+)-ATP synthase was not stimulated. The antibody bound to mitoplasts but not to submitochondrial particles. These results suggest that the putative Ca(2+)-dependent regulatory region of subunit e is exposed on the surface of the C-side of F(0) and that subunit e is involved in the regulation of mitochondrial H(+)-ATP synthase activity probably via its putative Ca(2+)-dependent regulatory region.

Molecular cloning and sequence analysis of cDNA for a 59 kD bone sialoprotein of the rat: demonstration that it is a counterpart of human alpha 2-HS glycoprotein and bovine fetuin.

A complementary DNA (cDNA) for the 59 kD bone sialoprotein, which is supposed to be the rat counterpart of human alpha 2-HS glycoprotein (alpha 2-HSG) and is synthesized by both hepatocytes and osteoblasts, has been cloned from a rat liver cDNA library. Polyclonal rabbit antibodies to rat 59 kD bone sialoprotein were used to identify and isolate the cDNA. The amino acid sequence of 59 kD bone sialoprotein deduced from the cDNA revealed that the entire protein consisted of 352 amino acid residues, including a signal peptide of 18 amino acid residues, and contained three possible N-glycosylation sites. On Northern blot analysis of rat liver, an mRNA of about 1.5 kilobases was detected. An mRNA of 59 kD bone sialoprotein was also detectable in rat bone but not in other tissues, such as kidney, brain, and lung. A computer search of protein and nucleic acid data bases revealed that 68.2, 63.2, and 97.4% amino acid residues of 59 kD bone sialoprotein were identical with those of human alpha 2-HSG, bovine fetuin, and rat phosphorylated N-glycoprotein (pp63), respectively. The positions of cysteine residues in 59 kD bone sialoprotein also completely matched those in human alpha 2-HSG and bovine fetuin, indicating that the sialoprotein is the rat counterpart of human alpha 2-HSG and bovine fetuin. In addition, comparison of the nucleotide sequence of cDNA for rat fetuin/alpha 2-HSG with that for pp63 recently corrected showed only two differences in nucleotides in the entire protein coding regions of the two proteins, and immunoreactive rat fetuin/alpha 2-HSG in the conditioned medium of adult rat hepatocytes in primary culture was found to be phosphorylated. Thus, because rat fetuin/alpha 2-HSG isolated from bone and synthesized by osteoblasts in culture does not contain phosphorus, it seems to be pp63 dephosphorylated during circulation or in the bone matrix.

Insulin-stimulating protein from human plasma.

An insulin-stimulating protein was isolated from human plasma by a procedure involving Sephadex G-100 chromatography and reverse-phase HPLC. The isolated material gave a single band on SDS-polyacrylamide gel electrophoresis. This protein itself had no insulin-like activity, but enhanced fatty acid synthesis from glucose in rat adipose tissue explants in the presence of suboptimal concentrations of insulin. It also stimulated the effect of insulin on CO2 liberation from glucose by isolated rat adipocytes and increased the maximal response to insulin.

Wolbachia-mediated parthenogenesis in the predatory thrips Franklinothrips vespiformis (Thysanoptera: Insecta).

Wolbachia are bacterial endosymbionts in arthropods and filarial nematodes. They cause thelytoky, which is a form of parthenogenesis in which females produce females without males, in hymenopteran insects. Infection of this parthenogenesis-inducing Wolbachia has been restricted to the order Hymenoptera, but was found in another insect order, Thysanoptera. A parthenogenetic colony of a predatory thrips Franklinothrips vespiformis (Aeolothripidae) possessed B-group Wolbachia. Male progeny were produced from this thrips by heat and tetracycline treatments. Males produced motile sperm, which were transferred to the female spermatheca by mating. However, the mating did not affect the sex ratios of the next generation, suggesting that the sperm do not fertilize the eggs.

The role of E-cadherin and scatter factor in tumor invasion and cell motility.

The acquisition of invasive properties by transformed epithelial cells constitutes an essential step in the progression of carcinomas. We have defined 2 types of interferences leading to enhanced motility and invasiveness of epithelial cells: (i) disturbances of intercellular adhesion, and (ii) treatment with "scatter factor", a secretory protein of mesenchymal cells. Invasive properties (invasion of collagen gels or embryonal heart tissue) are acquired by epithelial cells in vitro when intercellular adhesion is inhibited by antibodies that are specific for the cell-cell adhesion molecule E-cadherin. Furthermore, we found that differentiated human carcinoma cell lines are noninvasive and express E-cadherin, whereas dedifferentiated carcinoma lines are invasive and have lost E-cadherin expression. Invasiveness of these latter cells could be prevented by transfection with E-cadherin cDNA and was again induced by treatment of the transfected cells with anti-E-cadherin antibodies. A correlation between the degree of tumor differentiation and the amount of E-cadherin expression was also visualized on frozen sections of ovarian carcinomas, lobular breast carcinomas, and squamous carcinomas of head and neck. Thus, loss of E-cadherin appears to be a critical step in the establishment of an invasive, i.e. fully malignant phenotype. Scatter factor, which is also capable of dissociating epithelial cell colonies in vitro, was isolated from conditional medium of human fibroblasts; it is a 92,000 mol.wt glycoprotein, which is proteolytically cleaved into 62,000 and 34/32,000 mol.wt subunits. The purified glycoprotein induces invasion of MDCK cells into collagen matrices, and induces or enhances the invasive properties of various human carcinoma cell lines. Sequencing of tryptic peptides of scatter factor revealed strong similarity with hepatocyte growth factor. Furthermore, both factors exhibit identical activities, i.e. scatter factor stimulates DNA synthesis of primary hepatocytes and hepatocyte growth factor dissociates and increases the motility of various epithelial cells. Thus scatter factor and hepatocyte growth factor represent identical or closely similar proteins.

The potentiations by insulin-stimulating peptide from bovine serum albumin of the effects of insulin mimickers and insulin in stimulating glucose utilization by rat adipocytes.

An insulin-stimulating peptide derived from bovine serum albumin by digestion with trypsin was shown to inhibit insulin degradation. Addition of this peptide (1.2 microM) to the medium of isolated rat adipocytes markedly inhibited the degradation of insulin in the medium, but had a little effect on degradation of cell-associated insulin. Moreover, this peptide did not prevent dissociation of cell-associated insulin, suggesting that it is a bacitracin-type, not a chloroquine-type inhibitor of insulin degradation. The peptide also potentiated the stimulation by insulin mimickers of glucose oxidation by rat adipocytes, strongly indicating that it has some other effects besides inhibition of insulin degradation. Therefore, the effect of the peptide on activation of pyruvate dehydrogenase (PDH), one of the postbinding actions of insulin, was studied. Addition of the peptide (4 microM) to adipocytes was found to activate PDH in the absence or presence of insulin. This stimulatory effect of the peptide on PDH was dose-dependent and was observed in both whole cells and subcellular fractions of rat adipocytes. The peptide also stimulated PDH in a subcellular system of either plasma membranes and mitochondria or mitochondria only. Sodium fluoride, an inhibitor of phosphatase, blocked the action of the peptide almost completely, suggesting that the stimulatory effect of the peptide on PDH activity is at least partly due to its activation of PDH phosphatase. The mechanisms of action of the peptide are discussed. The peptide should be useful in studies on modulation of the action of insulin.

Effect of hepatocyte growth factor/scatter factor on lipogenesis in adult rat hepatocytes in primary culture.

Hepatocyte growth factor (HGF)/scatter factor is known to be the most potent mitogen for hepatocytes. In this paper, we report that lipogenesis in primary cultured rat hepatocytes treated with 10 ng/ml of recombinant human HGF (rhHGF) for 24 h was stimulated, as measured by the incorporation of 3H2O into long-chain fatty acids, to more than twice as much as the control. Insulin (0.1 microM) was more effective than rhHGF but rhHGF did not show an additive or synergistic effect when added to insulin. We also showed that treatment with rhHGF increased the activities of glucose-6-phosphate dehydrogenase (G6PDH) and malic enzyme, key enzymes which supply NADPH for lipogenesis, and acetyl-CoA carboxylase, the rate-limiting enzyme of lipogenesis. The increase in G6PDH and acetyl-CoA carboxylase activities was accompanied by increases in the levels of mRNA for the enzymes. These results suggest that HGF is involved in liver regeneration not only by stimulation of cell proliferation but also by acceleration of differentiation of hepatocytes.

Insulin-stimulating peptide from a tryptic digest of bovine serum albumin: purification and characterization.

A procedure was established for isolation of a low molecular weight polypeptide with insulin-stimulating activity in apparent homogeneity from a tryptic digest of bovine serum albumin on a semipreparative scale. Purification of this insulin-stimulating peptide (ISP) was monitored by an adipose-explant assay in which stimulation of fatty acid synthesis from glucose by insulin was measured. The polypeptide was purified by a combination of DEAE-cellulose column chromatography, gel filtration on Bio-Gel P-10, hydrophobic chromatography on a semipreparative C18 reversed-phase HPLC column, and ion exchange chromatography on an SP-5PW HPLC column. The primary structure of ISP was deduced. ISP is a two-chain polypeptide consisting of 71 amino acid residues, and corresponds essentially to residues 115-143 and 144-184 (185) of bovine serum albumin connected to each other by a disulfide bridge. But comparison of the sequence of ISP with that of the relevant regions of bovine serum albumin determined by Brown indicated the presence of one tyrosine insertion between residues 155 and 156 of albumin. Therefore, the molecular weight of ISP was calculated to be 8,496.

Changes with age of the rat fetuin concentration in serum and its mRNA expression.

Rat fetuin, a counterpart of human alpha2-HS glycoprotein and bovine fetuin, shows strong intermolecular binding and association with other serum proteins. Therefore, to measure its concentration in rat serum, we pretreated serum samples with 1% SDS plus 5% (ca. 0.7 M) 2-mercaptoethanol at 100 degrees C for 3 min, and then subjected them to SDS-PAGE under reducing conditions followed by Western blotting. We found that the fetuin concentrations in normal rat serum determined by Western blotting were 2.5-4.5 mg/ml. These concentrations were three orders of magnitude higher than the previously reported concentrations. We also tried to measure the fetuin concentration in rat serum by means of an enzyme-linked immunosorbent assay after treatment of the samples with 0.1% sodium dodecyl sulfate (SDS) plus 10 mM 2-mercaptoethylamine at 100 degrees C for 3 min, but it gave a value of about 1/4 of that on Western blotting. Rat fetuin is expressed mainly in the liver, with a peak 2-4 weeks after birth, as determined by Northern blot analysis. The fetuin mRNA level in the liver changes almost in parallel with its serum concentration. The tibia also expresses fetuin, but much less than the liver.

Prostaglandin E2 predominantly induces production of hepatocyte growth factor/scatter factor in human dental pulp in acute inflammation.

Hepatocyte growth factor (HGF), which is also known as the scatter factor, is a broad-spectrum and multifunctional cytokine, mediates epithelial-mesenchyme interaction, and is shown to be involved in the development and regeneration of various tissues, including tooth. Here, we report that HGF was present in adult human dental pulps, and its levels increased during acute inflammation of the tissue. Levels of HGF mRNA in dental pulps also increased with inflammation, as determined by reverse-transcription/polymerase chain-reaction. The production of HGF in fibroblasts from dental pulps in culture was dose-dependently stimulated by inflammatory cytokines such as interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha, and by prostaglandin (PG) E2, as determined by an enzyme-linked immunosorbent assay. We also showed that indomethacin did not affect the increase in HGF production by the cells with IL-1alpha, TNF-alpha, and PGE2. The levels of HGF mRNA in the cells were simultaneously increased by these stimulants, as determined by Northern blotting. Since the production of PGs is known to increase at the beginning of inflammation, PGE2 may be involved in the regeneration of dental pulps by the induction of HGF expression after inflammation.

Expression of hepatocyte growth factor/scatter factor and c-Met in human dental papilla and fibroblasts from dental papilla.

Hepatocyte growth factor/scatter factor (HGF/SF), a broad-spectrum and multifunctional cytokine, is essential for the development of tissues including tooth. Here it was found that the HGF/SF content of human dental papillae obtained from 8 to 16-year-old individuals decreased significantly with age. Cultured fibroblasts prepared from the dental papillae of individuals of different ages produced HGF/SF at almost the same rate, but the sensitivities of the cells to interleukin-1alpha and tumour necrosis factor-alpha for the production of HGF/SF increased with age. Generally, mesenchymal cells such as fibroblasts produce HGF/SF but do not express c-Met, a receptor for HGF/SF, yet fibroblasts in dental papilla and cultured fibroblasts prepared from dental papilla did express c-Met, as determined by immunohistochemistry, in situ hybridization and reverse transcription-polymerase chain reaction. Recombinant human [125I]iodo-HGF/SF specifically bound to cell-surface macromolecules with a mol. wt of 146,000, which is the same as that of the beta-subunit of c-Met. The physiological role of c-Met on fibroblasts in dental papilla is unknown, but the addition of 2 ng of HGF/SF per ml to the culture medium significantly stimulated DNA synthesis in the cells, as determined by pulse labelling with [3H]thymidine. Exogenous HGF/SF also stimulated secretion by the cells of vascular endothelial growth factor, a cytokine that induces blood vessel-formation. These results suggest that HGF/SF may be involved in tooth development via autocrine mechanisms.

Concurrent infection with Legionella pneumophila and Pneumocystis carinii in a patient with adult T cell leukemia.

A 48-year-old woman was admitted to our hospital with high fever, chills, cough, and exertional dyspnea. On admission, the chest roentgenogram and computed tomography scan showed bilateral alveolar infiltration in the middle and lower lung fields. Microscopic examination of the bronchial lavage fluid showed flower cells typical for adult T-cell leukemia (ATL) and cysts of Pneumocystis carinii, and Legionella pneumophila serogroup 1 grew on buffered charcoal yeast extract (BCYE)-alpha agar. The patient was successfully treated with antibiotics including trimethoprim/sulfamethoxazole, erythromycin, and sparfloxacin. Remission of ATL was achieved after three courses of antileukemic chemotherapy. Mixed infection of opportunistic pathogens should be considered in patients with ATL.

[A case of loiasis].

Loiasis is quite common in the endemic regions of Central and West Africa. But only three cases were reported in Japan. This is a report of a 28 year old male from Gabon infected with Loa loa with eye symptoms as the chief complaint. For the first time in Japan he was treated with Ivermectin (IVM) which is recently attracting attention as the drug for filariasis world wide. IVM therapy was effective, and decreased the counts of microfilarias in the patient's blood. No adverse effect was seen in this patient. This case suggested that IVM is an useful drug for loiasis, and further study is warranted.