Polyandry increases reproductive performance but does not decrease survival in female Brontispa longissima.

The costs and benefits of polyandry are still not well understood. We studied the effects of multiple mating on the reproductive performance of female Brontispa longissima (Coleoptera: Chrysomelidae), one of the most serious pests of the coconut palm, by using three experimental treatments: (1) singly-mated females (single treatment); (2) females that mated 10 times with the same male (repetition treatment); and (3) females that mated once with each of 10 different males (polyandry treatment). Both multiple mating treatments resulted in significantly greater total egg production and the proportion of eggs that successfully hatched (hatching success) than with the single mating treatment. Furthermore, the polyandry treatment resulted in greater total egg production and hatching success than with the repetition treatment. Thus, mate diversity may affect the direct and indirect benefits of multiple mating. Female longevity, the length of the preoviposition period, the length of the period from emergence to termination of oviposition, and the length of the ovipositing period did not differ among treatments. The pronounced fecundity and fertility benefits that females gain from multiple mating, coupled with a lack of longevity costs, apparently explain the extreme polyandry in B. longissima.

Disposition and metabolism of a novel prostanoid antiglaucoma medication, tafluprost, following ocular administration to rats.

The disposition and metabolism of tafluprost, an ester prodrug of the 15,15-difluoro-prostaglandin F(2alpha) antiglaucoma agent, have been studied in rats after ocular administration. Radioactivity was absorbed very rapidly into the eye and systemic circulation after a single ocular dose of 0.005% [(3)H]tafluprost ophthalmic solution, with maximum levels in plasma and most eye tissues occurring within 15 min. The absorption ratio of radioactivity was approximately 75%, suggesting the high availability of ocular administration of tafluprost. Approximately 10% of the dose was present in cornea at this time, and radioactivity concentrations in this tissue exceeded those in aqueous humor and iris/ciliary body throughout the 24-h study period. After repeated daily ocular doses, radioactivity levels remained greatest in cornea, followed by iris/ciliary body that replaced aqueous humor as the eye tissue containing the second highest radioactivity concentration. In female rats, radioactivity was excreted equally between urine and feces after a single ocular dose, whereas in male rats more was excreted in feces, reflecting the greater biliary excretion in males rats (50% dose) compared with females rats (33% dose). Tafluprost was extensively metabolized in the rat, such that intact prodrug was not detected in plasma, tissues, or excreta by radio-high-performance liquid chromatography. On the other hand, the active moiety, tafluprost acid, was the only noteworthy radioactive component in cornea, aqueous humor, and iris/ciliary body for at least 8 h after the ocular dose, and it was also a major plasma metabolite in early time points. The gender differences in conjugation reactions resulted in the differences in the excretion.

Characterization of cyclosporin A transport in cultured rabbit corneal epithelial cells: P-glycoprotein transport activity and binding to cyclophilin.

PURPOSE: The purpose of this study was to characterize cyclosporin A (CsA) uptake and transport in cultured rabbit corneal epithelial cells (RCECs). METHODS: CsA uptake was evaluated by measuring time-dependent 3H-CsA accumulation in confluent RCECs. Bidirectional 3H-CsA fluxes were measured across the RCEC layers grown on Transwell-COL culture plate inserts. The anti-P-gp monoclonal antibody C219 was used in western blot analysis to probe for the presence of P-gp in these cells. RESULTS: The accumulation of 3H-CsA was time and temperature dependent. Steady state was reached by 60 minutes. The initial uptake was saturable and was suppressed as a function of increases in preloading with unlabeled CsA. This uptake process was enhanced by metabolic inhibition with either 3-O-methylglucose, MG, or 10 mM NaN3 and 3-O-MG. The largest increase was obtained with 10 mM NaN3 in combination with 3-O-MG. In their presence, uptake increased by 40%. A multidrug-resistance (MDR)-reversing agent (i.e., 500 microM verapamil, 100 microM vincristine, 100 microM progesterone, 100 microM testosterone, 500 microM quinidine, or 100 microM chlorpromazine) significantly increased 3H-CsA accumulation. The largest increase was obtained with 500 microM quinidine (i.e., 36%). Conversely, verapamil and vincristine produced the largest inhibition of 3H-CsA efflux (i.e., 19% and 28%, respectively). However, in the presence of 10 microM unlabeled CsA, 3H-CsA efflux increased. 3H-CsA flux across RCEC layers showed marked directional asymmetry. The stromal (S) to tear (T) side transcellular 3H-CsA permeability coefficient (Ptrans) was approximately seven times higher than that in the T-to-S direction. The S-to-T Ptrans was reduced by an MDR-reversing agent by up to 40%. Western blot analysis of lysates revealed a 170-kDa membrane protein band. CONCLUSIONS: These results suggest that in RCEC the tear-side-facing membrane has a P-gp-mediated drug efflux pump. In addition, there is suggestive evidence for the presence of the cytosolic protein, cyclophilin. The presence of P-gp in these cells could help protect them from being damaged by the uptake of toxic substances.

In vivo evidence for ATP-dependent and P-glycoprotein-mediated transport of cyclosporin A at the blood-brain barrier.

To evaluate the significance of P-glycoprotein (P-gp)-mediated active efflux on the blood-brain barrier (BBB) permeability of cyclosporin A (CsA) in vivo, we investigated the effects of ATP depletion in the brain and of a multidrug-resistant (MDR) reversing agent on the transport of CsA across the BBB. Using transient brain ischemia obtained by 4-vessel occlusion of vertebral and common carotid arteries in rats to deplete ATP content in the brain, the estimated permeability surface area product (PS) value of [3H]CsA was increased 2.7-fold compared with that in normal rats, whereas the PS value of [14C]sucrose was not altered. Additionally, when quinidine hydrochloride (QND) was infused into the brain through a microdialysis probe implanted in the rat hippocampus, the extravascular extraction of CsA was increased to approximately 2.5-fold of the control, whereas no difference in the extravascular extraction between control and normal rats having no implanted dialysis probe was observed. Furthermore, the efflux rate from brain to blood of CsA was decreased remarkably to 5% of control at steady-state by co-administration of CsA with QND directly into the brain through the dialysis probe. The ATP-dependent and QND-sensitive efflux of CsA from the brain strongly indicates that P-gp in the brain capillary endothelial cells functions as an efflux pump under the physiological state, and that P-gp-mediated efflux of CsA is a major mechanism of the restricted transfer from blood into the brain.

Antimicrobial constituents of Angelica dahurica roots.

One novel coumarin from Angelica dahurica roots was elucidated to be 5,8-di(2,3-dihydroxy-3-methylbutoxy)-psoralen. It occurs together with six other known coumarins and ferulic acid. The antimicrobial activity of the coumarins and ferulic acid were compared.

Chemical conversion of vibsanin C to vibsanin E and structure of 3-hydroxyvibsanin E from viburnum awabuki

Vibsanin E (4), a tricyclic vibsane-type diterpene, has been prepared in 50% yield from vibsanin C (2), a seven-membered ring vibsane-type diterpene by reaction with BF3.OEt2 at -78 degrees C. This chemical correlation not only established structure, including absolute configurations, but also has demonstrated a possible biosynthetic route to 4 via 2 derived from vibsanin B (1). The structure of 3-hydroxyvibsanin E (5), another example of a tricyclic seven-membered ring vibsane, isolated from the leaves of Viburnumawabuki, has been established by extensive analyses of 2D NMR data and comparison of its spectral data with those of 4.

A novel method for chemo-enzymatic synthesis of elicitor-active chitosan oligomers and partially N-deacetylated chitin oligomers using N-acylated chitotrioses as substrates in a lysozyme-catalyzed transglycosylation reaction system.

N,N',N"-Tri(monochloro)acetylchitotriose prepared by N-monochloroacetylation of chitotriose trihydrochloride was successfully polymerized into higher-molecular-weight oligomers by a lysozyme-catalyzed transglycosylation reaction, and a following base-catalyzed N-demonochloroacetylation gave a chitosan oligomer mixture mainly composed of oligomers with dp > 6. Partially N-deacetylated chitin oligomers (DAC oligomers) with dp 4-12 were synthesized by the enzyme reaction using N,N',N"-tri(monochloro)acetylchitotriose and N,N',N"-triacetylchitotriose (chitin trimer) as initial substrates followed by N-demonochloroacetylation. The structures of synthetic oligomers were analyzed by 1H NMR spectroscopy, enzymatic hydrolysis and nitrous acid deamination-NaBH4 reduction treatment. The dp of synthetic oligomers was measured by MALDI TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) using per-N-acetylated derivatives. The synthetic chitosan and DAC oligomers were strong elicitors for phytoalexin induction in Pisum sativum and Phaseolus vulgaris. This chemo-enzymatic method utilizing N-acylated chitotrioses as substrates is a novel approach to the synthesis of high-molecular-weight chitosan oligomers and DAC oligomers of biological importance.

Effective production of dehydro cyclic dipeptide albonoursin exhibiting pronuclear fusion inhibitory activity. II. Biosynthetic and bioconversion studies.

Albonoursin production was greatly enhanced when cyclo (L-Leu-L-Phe) (CFL), a tetrahydro derivative of albonoursin, was added to the 2-day culture of an albonoursin-producing actinomycete, Streptomyces albulus KO-23. The increase in albonoursin production paralleled the amount of CFL added. Furthermore, the resting cells of the strain catalyzed the bioconversion of CFL to albonoursin. The optimum pH and temperature for the conversion were found to be pH 10.0 and 50 degrees C. The feeding experiments and the resting-cell reactions revealed that albonoursin is biosynthesized by dehydrogenation of CFL in the actinomycete. This is the first report for a dehydrogenation of amino acid residues at the alpha,beta-positions in cyclic dipeptides.

Effective production of dehydro cyclic dipeptide albonoursin exhibiting pronuclear fusion inhibitory activity. I. Taxonomy and fermentation.

Strain KO-23, an actinomycete producing albonoursin as well as streptopyrone, was identified as Streptomyces albulus by morphological and biochemical studies. Fermentation conditions for albonoursin, a dehydro cyclic dipeptide exhibiting a pronounced inhibitory activity toward pronuclear fusion of sea urchin eggs, were optimized. Under the optimum conditions, the actinomycete produced 16 mg/liter of albonoursin, 30 times higher than that in the original culture. The cells cultivated under these conditions highly express biosynthetic enzymes for albonoursin, and thus are available for biosynthetic studies of dehydro cyclic peptides.

Beta adrenergic antagonist permeation across cultured rabbit corneal epithelial cells grown on permeable supports.

PURPOSE: To determine whether cultured rabbit corneal epithelial cells (RCEC), grown on permeable supports, provide a suitable in vivo model for characterizing transcellular drug permeation and metabolism. METHODS: Primary rabbit corneal epithelial cells grown in DMEM-F12 were seeded on Transwell-COL inserts coated with fibronectin. The epithelial barrier integrity was evaluated, based on measurements of 14C-mannitol and 3H-PEG900, and their transepithelial electrical resistance (TEER). Ultrastructure evaluation was based on scanning electron microscopy and transmission electron microscopy, which were performed 8 days after seeding. Measurements of beta adrenergic antagonist permeability were performed to assess transcellular permeability. RESULTS: Eight days after seeding, the TEER reached a peak of 144 omega.cm2 and the 14C-mannitol and 3H-PEG900 permeabilities were 6.8 x 10(-6) and 2.9 x 10(-6) cm/sec, respectively. Ultrastructural analysis revealed a multilayered structure with numerous microplicae and typical cytoplasmic organelles along with desmosomes. The relationship between permeation of beta-blockers and lipophilicity resembled the intact isolated cornea. CONCLUSIONS: This is the first description of cultured RCEC grown on permeable support. Many of its properties mimic those described in the intact corneal epithelium. Even though its electrical tightness is less than that of the intact cornea, the transcellular permeability to lipophilic beta-antagonists is comparable to the isolated preparation. Therefore, this model will facilitate characterization of ocular permeation mechanisms of hydrophobic drugs whose route of permeation is transcellular.

Cultured rabbit corneal epithelium elicits levofloxacin absorption and secretion.

Abstract Evidence for carrier-mediated transport of levofloxacin in the isolated rabbit cornea has been found. However, it is not known whether this mechanism is located in the epithelium or the endothelium. To resolve this question, we have measured the kinetics of levofloxacin uptake in primary cultures of rabbit corneal epithelial cells. The results indicate that levofloxacin accumulation was time dependent and a steady state was reached after 30 min. Maximal uptake occurred from a solution whose pH was 6.5. The uptake process was stereoselective and concentration dependent. In addition to the uptake, secretion of levofloxacin also occurred. These results indicate that the corneal epithelium is the site of levofloxacin transport mechanisms, mediating both absorption and secretion.

Characterization of the carrier-mediated transport of levofloxacin, a fluoroquinolone antimicrobial agent, in rabbit cornea.

The cornea presents a formidable barrier to drug penetration. The fluoroquinolone levofloxacin, which is an effective antimicrobial agent, has the potential to be used in the topical treatment of ocular disease. Thus, we sought to characterize how levofloxacin penetrates the cornea. To perform this characterization, we measured the time dependent permeation of levofloxacin across the isolated rabbit cornea using a diffusion chamber, and compared it with antipyrine fluxes. Levofloxacin permeation into the receiver epithelial-side bathing solution (pH = 6.5) from the donor endothelial-side (pH = 7.4) reached 3.00 nmolcm(-2) cornea after 2h, whereas in the opposite direction permeation was 1.89 nmolcm(-2) cornea. Based on the temperature-dependent effects on permeation, the calculated energy of activation for permeation, Ea, was 31.3 kcal mol(-1), whereas Ea for antipyrine, a marker of diffusion, was 11.0 kcalmol(-1). The transport of levofloxacin from epithelium to endothelium was concentration-dependent and had both a linear and saturable component. Evaluation of the kinetic parameters, Jmax, apparent Km and k(d) showed that they were 38.78 pmol min(-1) cm(-2), 3.83 mM and 0.0135 microL min(-1) cm(-2), respectively. These results, coupled with the fact that levofloxacin permeation reached a maximum value at pH 6.5, suggest that levofloxacin transport across the cornea is carrier mediated. However, at present, it cannot be ascertained whether such a system is localized in either the corneal epithelial or the endothelial layer.

Novel diketopiperazine metabolism in a microorganism: two-step hydrolysis of cyclo(Gly-Leu) to amino acids and preliminary characterization of cyclo(Gly-Leu) hydrolase and dipeptidase.

A bacterium, strain NM 5-3, isolated from soil exhibited the highest cyclo(Gly-Leu) (CGL)-hydrolyzing activity and was identified as Agrobacterium radiobacter. The reaction products from CGL were dipeptides (Leu-Gly and Gly-Leu) and amino acids (Leu and Gly). Inhibitors for the dipeptidase of this strain did not inhibit the hydrolysis of CGL to dipeptides, indicating that two distinct enzymes, CGLase and a dipeptidase, were involved in its hydrolysis. The activities of these two enzymes were separated by anion-exchange column chromatography. The results indicated that strain NM5-3 hydrolyzed CGL via the dipeptides to the corresponding amino acids. The CGLase fraction was found to catalyze the hydrolysis of cyclo(Gly-D-Leu), cyclo(Gly-Gly), cyclo(L-Ala-Gly), and cyclo(D-Ala-Gly). On the other hand, the dipeptidase fraction exhibited L-specific substrate specificity.

Enzymatic conversion of cyclic dipeptides to dehydro derivatives that inhibit cell division.

The cell-free extract of an albonoursin-producing strain, Streptomyces albulus KO-23, was found to catalyze the conversion of several cyclic dipeptides having Phe and aliphatic side chain-containing amino acid residues to the corresponding dehydro derivatives. 3Z-Benzylidene-6S-methyl-2,5-piperazinedione, 3Z-benzylidene-2,5-piperazinedione, and 3Z, 6Z-dibenzylidene-2,5-piperazinedione were prepared by this conversion system. Among the dehydro cyclic dipeptides prepared, tetradehydro derivatives exhibited inhibitory activity toward the first cleavage of sea urchin embryo, while didehydro derivatives did not. We previously found that cyclo(Leu-Phe) and its didehydro derivatives did not show any inhibitory activity, in contrast to high activity in the case of albonoursin. Taken together, these findings indicate that dehydrogenation at the alpha,beta-positions of both amino acid residues in this type of cyclic dipeptide is required for the inhibitory activity.

Antimicrobial, antioxidant, antitumour-promoting and cytotoxic activities of different plant part extracts of Garcinia atroviridis griff. ex T. anders.

Crude extracts (methanol) of various parts, viz. the leaves, fruits, roots, stem and trunk bark, of Garcinia atroviridis were screened for antimicrobial, cytotoxic, brine shrimp toxic, antitumour-promoting and antioxidant activities. The crude extracts exhibited predominantly antibacterial activity with the root extract showing the strongest inhibition against the test bacteria at a minimum inhibitory dose (MID) of 15.6 microg/disc. Although all the extracts failed to inhibit the growth of most of the test fungi, significant antifungal activity against Cladosporium herbarum was exhibited by most notably the fruit (MID: 100 microg), and the leaf (MID: 400 microg) extracts. None of the extracts were significantly cytotoxic, and lethal towards brine shrimps. The root, leaf, trunk and stem bark extracts (except for the fruits) showed strong antioxidant activity exceeding that of the standard antioxidant, alpha-tocopherol. Antitumour-promoting activity (>95% inhibition) was shown by the fruit, leaf, stem and trunk bark extracts.

[Single-dose toxicity studies of tazobactam/piperacillin and tazobactam].

Tazobactam (TAZ) is a newly developed beta-lactamase inhibitor. Tazobactam/Piperacillin (TAZ/PIPC) is a formulation consisting of TAZ and PIPC in a ratio of 1:4. Singe-dose toxicity studies in TAZ/PIPC and TAZ were carried out using mice and rats of both sexes and male dogs. The results were as follows. 1. A common clinical sign in mice and rats administered TAZ/PIPC or TAZ by all routes was soft stool. Other signs in mice and rats included a decrease in spontaneous motor activity and/or a decreased respiratory rate for the intraperitoneal (i.p.), subcutaneous (s.c.) or intravenous (i.v.) route. The animals administered by the i.v. route showed tremor for mice and clonic convulsion for rats before death. Hyperemia, hemorrhage or edema of the lung, and hemorrhage of the digestive tract were observed in these animals at necropsy. An enlargement of the spleen was seen in some of the surviving animals treated with TAZ/PIPC. 2. In dogs, TAZ/PIPC caused vomiting, and TAZ caused vomiting, respiratory abnormality, soft stool and diarrhea by the intravenous (i.v.) administration. 3. TAZ/PIPC or TAZ caused clinical signs such as the loss of hair at the injection site for the s.c. route, and necrosis of the tail for the i.v. route in mice and rats, also caused limping of the injected anterior limb in dogs. Necrosis and hemorrhage at the injection site, and peritonitis by the i.p. injection were observed at necropsy. These findings were due to the irritation of TAZ/PIPC or TAZ.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects on growth of rat offspring born from dams treated subcutaneously with a surfactant, polyoxyethylene(10)nonylphenyl ether(NP-10), during lactational period.

A surfactant NP-10 was administered subcutaneously to Jcl:Wistar female rats at dose levels of 5, 20 and 80 mg/kg/day from date of birth to day 21 after birth of F1 offspring to assess its effects on the growth, behavior and functions of the offspring. For F0 dams, scab formation and loss of hair at the test substance administration site were observed in all treatment groups and induration of the skin at the test substance administration site in the 20 and 80 mg/kg groups in general condition and necropsy findings at the end of the dosing period. In necropsy findings, in addition to these changes, hemorrhage and whitish change of the subcutis at the test substance administration site were seen in all treatment groups, adhesion to the somatic muscles and granulation of the subcutis at the test substance administration site in the 20 and 80 mg/kg groups, and swelling of the spleen and adrenals in the 80 mg/kg group. Reduction or a tendency for reduction in food consumption was also detected from the initial day of dosing (day 0) to day 17 after birth F1 offspring in the 80 mg/kg group. Body weight or the findings on the day after birth and day of weaning failed to reveal any evidence of an effect that could be ascribed to the test substance. In F1 born offspring, a decrease or tendency for decrease in body weight was observed from day 7 after birth in both sexes and for females during the gestation period in the 80 mg/kg group. However, body weight gains based on the weights at 4 weeks after birth or on day 0 of gestation in the 80 mg/kg group failed to reveal any difference from that in the control group. The observation on the day of birth and during the period of lactation, physical development test, reflex test, general condition, open-field test, water-maze test, reproductive ability test, observations at cesarean section, necropsy findings, organ weights, histopathological findings of females or males that did not achieve successful gestation, skeletal examination, and the observations at cesarean section and external examination of F2 fetuses failed to reveal any evidence that could be ascribed to the test substance. These results indicate that NP-10 had no effect on the behavior or functions of the offspring, although it affected the growth of the offspring born, under the conditions of this study. The non-effective dose level is considered to be 20 mg/kg for general toxicity of the dams and for their offspring.

Development of a water-soluble preparation of emamectin benzoate and its preventative effect against the wilting of pot-grown pine trees inoculated with the pine wood nematode, Bursaphelenchus xylophilus.

Water-soluble preparations have been investigated to develop a trunk injection agent based on the poorly water-soluble anti-nematode emamectin benzoate. Following tests on the phytotoxicity of some solvents and solubilizers and demonstration of the ability of some solubilizers to dissolve emamectin benzoate in water, acetone + methanol was selected as the solvent and Polysorbate 80 as the solubilizer. This water-soluble preparation of emamectin benzoate prevented the wilting of pot-grown 4-year-old trees of the Japanese black pine, Pinus thunbergii, artificially inoculated with the pine wood nematode, Bursaphelenchus xylophilus, at a dose of 20 g emamectin benzoate per cubic metre of pine tree.

Intracameral and lenticular penetration of locally applied stable isotope-labeled vitamin E.

PURPOSE: To confirm the aqueous humor and lens dynamics of 1% deuterium-labeled alpha-tocopherol acetate (D(3)-VEA) solution. METHODS: The concentrations of D(3)-VEA and D(3)-alpha-tocopherol (D(3)-VE), derived from D(3)-VEA, in the aqueous humor and lens were measured after continuous instillation of 1% D(3)-VEA into the cul-de-sac of rat eyes for 1 or 3 weeks. D(3)-VEA and D(3)-VE concentrations were determined by gas chromatography/mass selective detector. RESULTS: In the aqueous humor, the concentrations of D(3)-VEA and D(3)-VE were, respectively, 93.1 and 9.4 ng/mL after 1 week, and, respectively, 498.9 and 21.5 ng/mL after 3 weeks. In the lens, they were, respectively, 15.0 and 9.8 ng/g after 1 week, and 6.1 and 4.8 ng/g after 3 weeks. CONCLUSION: The penetration levels of alpha-tocopherol acetate by eyedrop application were confirmed in the aqueous humor and lens.

Induction of mucosal disease in cattle persistently infected with noncytopathic bovine viral diarrhea-mucosal disease virus by superinfection with cytopathic bovine viral diarrhea-mucosal disease virus.

Three head of cattle persistently infected with noncytopathic bovine viral diarrhea-mucosal disease virus (ncBVD-MDV) were superinfected naturally or experimentally with cytopathic bovine viral diarrhea-mucosal disease virus (cBVD-MDV). In the naturally superinfected case, one animal manifested pyrexia and severe diarrhea, and died without developing antibodies to cBVD-MDV. However, another animal survived with only continual slight anorexia and pyrexia, and developed strong resistance to the superinfected strain. In the experimental cases, induction of MD was unsuccessful in two persistently infected cattle when superinfected with cBVD-MDV antigenically heterologous for persistently infected ncBVD-MDV. They also developed antibodies to the cBVD-MDV strain with which they had been infected. After 6 months, these cattle were infected again with a cBVD-MDV strain different from that used in the previous experiment. One animal infected with this strain, which was antigenically homologous to the persistently infected strain, died after developing MD symptoms without developing antibodies to the infecting strain. It is suggested that the antigenic relationship between the persistent ncBVD-MDV and the superinfected cBVD-MDV was an important factor in developing MD.