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STUDY QUESTION Are there phase-specific changes in the early secretory (ES) phase human tubal lavage proteome that can inform and potentially optimize IVF culture media? SUMMARY ANSWER The human tubal lavage proteome during the ES phase relative to the menstrual phase reveals substantial differential protein abundance in pathways such as glycolysis, redox homeostasis and activation of 14-3-3 zeta-mediated signaling. WHAT IS KNOWN ALREADY The Fallopian tube is uniquely suited to the development of the preimplantation embryo as it transits the tube during the ES phase of the menstrual cycle. Euploid cleavage-stage embryo arrest may reflect incomplete recapitulation of in-vivo conditions by current media formulations. STUDY DESIGN, SIZE, DURATION Proteome-wide analysis of distal tubal lavage specimens collected from 26 healthy women undergoing open microtubal anastomosis surgery from January 2013 to January 2018 was performed. Specimens were grouped by menstrual cycle phase in order to analyze phase-specific differences in protein abundance. For the murine embryo assay, single-cell embryos (N = 482) were collected from superovulated wild type C57BL/6 female mice and cultured in microdrops over 5 days for the assessment of blastocyst development. PARTICIPANTS/MATERIALS, SETTING, METHODS Human tubal lavage specimens were processed for label-free mass spectrometry. Reported menstrual cycle day was confirmed by measuring serum hormones. Key protein targets in the ES phase were validated via immunoblot. The ES phase-specific increase in 14-3-3 zeta protein was confirmed via ELISA of conditioned media obtained from primary human Fallopian tube epithelial cell culture. A murine embryo assay was performed to investigate the impact of graduated concentrations of 14-3-3 zeta on the blastocyst development rate. MAIN RESULTS AND THE ROLE OF CHANCE Comparison of the ES and menstrual phase human tubal lavage proteomes revealed 74 differentially expressed proteins with enrichment of pathways and biological processes involved in the regulation of carbohydrate metabolism, oxidative stress and cell survival. The adapter-regulator protein 14-3-3 zeta was among the most significantly increased in the ES phase. Supplementation of embryo culture media with 14-3-3 zeta at concentrations tested did not significantly improve the murine blastocyst development. LIMITATIONS, REASONS FOR CAUTION Although select associations were recapitulated in the conditioned media from sex steroid exposed primary human tubal epithelial cells, cell culture represents an in-vitro approximation. Changes to embryo culture media, such as protein supplementation, must undergo rigorous preclinical safety testing prior to adoption for human use. WIDER IMPLICATIONS OF THE FINDINGS This study represents the first description of the human Fallopian tube lavage proteome across the menstrual cycle, revealing a unique proteomic signature during the ES phase. Although supplementation of culture media with 14-3-3 zeta at appropriate concentrations showed no significant impact on the murine blastocyst development rate, other biologically plausible candidate proteins for individual or high throughput testing strategies are identified. STUDY FUNDING/COMPETING INTEREST(S) This work was funded in part by an Army Medical Department Advanced Medical Technology Initiative grant from the United States Army Medical Research and Materiel Command’s Telemedicine and Advanced Technology Research Center. There are no competing interests. TRIAL REGISTRATION NUMBER N/A

Erythritol helps both prevent and improve periodontal disease and is therefore widely used for dental care in humans. However, only a few studies have investigated the effects of erythritol on periodontal disease in animals. We hypothesized that erythritol could be used to prevent and improve periodontal disease also in canines and investigated the effects of erythritol on canine periodontal disease-related pathogenic bacteria using both in vitro and in vivo methods. The effect of erythritol on the proliferation of Porphyromonas gulae, which is reportedly associated with canine periodontal disease, was investigated in vitro. In addition, a 4-week intervention trial using an external gel preparation containing 5% erythritol was performed in canines with mild periodontal disease; changes in the microbiota around periodontal lesions were investigated using next-generation sequencing and bioinformatics analysis. The growth of P. gulae was significantly suppressed by erythritol in vitro. In the intervention study, the Shannon index, an indicator of the species distribution α-diversity, and the occupancy of several canine periodontal disease - related bacteria ( P. gulae, P. cangingivalis) were significantly decreased in periodontal lesions. Based on the results of in vitro and in vivo studies, we conclude that, as in humans, erythritol has bacteriostatic effects against periodontal disease - related bacteria in canines.

Aqueous and KCl-soluble polysaccharides were extracted from Laurencia dendroidea (Rhodomelaceae, Ceramiales) and their chemical profile was accessed by anion-exchange chromatography, chemical and spectroscopic analyses. The homogeneous agaran DHS-4 (181.3 × 103 g. mol−1, 21.3% of NaSO3) presents A units mostly 2-sulfated (18.9 mol%), nonsubstituted (15.3 mol%) and 6-O-methylated (10.1 mol%), while B units are l -sugars composed predominantly by galactose 6-sulfate precursor units (19.2 mol%) and 3,6-anhydrogalactose (13.8 mol%), besides non-precursor galactose 6-sulfate units bearing d -xylose substituents on C-3 (8.1 mol%). The crude KCl-soluble DHS agaran (20.5% of NaSO3) inhibited proteolysis and hemolysis induced by Lachesis muta and Bothrops jararaca venoms. DHS was able to inhibit up to 75% the L. muta venom hemorrhagic effect and to reduce the lethality displayed by B. jararaca venom, increasing the mice survival time up to 3 times. Therefore, this agaran has high potential to be used as an additional tool to treat snakebite envenomation.

Snakebite is a serious occupational hazard affecting mainly rural populations of tropical and subtropical developing countries. Lachesis muta (Bushmaster) bites are extremely serious but are rarely reported in the literature. Bushmaster envenomings are characterized by intense local pain, edema, neurotoxicity, hypotension, local hemorrhage, and dramatic systemic alterations. Antivenom treatment has regularly been used for more than a century; however, it fails to neutralize local tissue damage and hemorrhage, leading to morbidity or disabilities in victims. Thus, the production and clinical use of antivenom must be improved. The present work characterizes, for the first time, a sulfated polysaccharide from the red seaweed, Laurencia aldingensis, including its neutralizing effect on some toxic activities of L. muta venom. Chemical and spectroscopic analyses showed that L. aldingensis produces sulfated agarans with the A-units partially C-2 sulfated or 6-O-methoxylated presetting the B-units in the cyclized (3,6-anhydro-α-L-galactose) or in the non-cyclized form (α-L-galactose). The latter is significantly substituted by sulfate groups on C-6. In vitro and in vivo assays showed that this sulfated agaran inhibited hemolysis, coagulation, proteolysis, edema, and hemorrhage of L. muta venom. Neutralization of hemorrhagic activity was also observed when the agaran was administered by different routes and after or before the venom injection. Furthermore, the agaran blocked the edema caused by a phospholipase A2 isolated from the L. muta venom. Experimental evidence therefore indicates that the sulfated agaran of L. aldingensis has potential to aid antivenom therapy of accidents caused by L. muta venom and may help to develop more effective antivenom treatments of snake bites in general.

This paper reports the structural and optical investigations of the structural colour of the weevil Lamprocyphus augustus . The photonic crystal structure within the weevil's scales was investigated using sequential focused ion-beam milling and scanning electron microscopy imaging. We carefully analysed the reconstructed three-dimensional structure to determine the unit cell of the photonic crystal. It was found that the cuticle network of the cubic unit cell perfectly matches the previously reported diamond-based network. However, different results were obtained for the crystal orientations of the small crystal domains that comprise the entire photonic crystal structure in the scales: directions are highly preferred along the surface normal of the scale. This finding explains the fact that the scale is almost uniformly coloured despite the multi-domain structure. It is confirmed experimentally and theoretically that the wavelength range of the reflection band corresponds to the gap of the photonic band.

C-type natriuretic peptide (CNP) is a potent stimulator of long bone and vertebral development via endochondral ossification. In the present study, we investigated the effects of CNP on craniofacial skeletogenesis, which consists of both endochondral and membranous ossification. Morphometric analyses of crania from CNP knockout and transgenic mice revealed that CNP stimulates longitudinal growth along the cranial length, but does not regulate cranial width. CNP markedly increased the length of spheno-occipital synchondrosis in fetal murine organ cultures, and the thickness of cultured murine chondrocytes from the spheno-occipital synchondrosis or nasal septum, resulting in the stimulation of longitudinal cranial growth. Mandibular growth includes endochondral and membranous ossification; although CNP stimulated endochondral bone growth of condylar cartilage in cultured fetal murine mandibles, differences in the lengths of the lower jaw between CNP knockout or transgenic mice and wild-type mice were smaller than those observed for the lengths of the upper jaw. These results indicate that CNP primarily stimulates endochondral ossification in the craniofacial region and is crucial for midfacial skeletogenesis.

SUMMARY Ultrasonic hearing is widespread among moths, but very few moth species have been reported to produce ultrasounds for sexual communication. In those that do, the signals are intense and thus well matched for long distance communication. By contrast, males of the Asian corn borer moth (Crambidae) were recently shown to whisper extremely low-intensity ultrasonic courtship songs close to females. Since low sound levels will prevent eavesdropping by predators, parasites and conspecific rivals, we predicted low intensity ultrasound communication to be widespread among moths. Here we tested 13 species of moths including members of the Noctuidae, Arctiidae, Geometridae and Crambidae. Males of nine species, 70%, produced broadband ultrasound close to females. Peak frequencies ranged from 38 to above 100 kHz. All sounds were of low intensity, 43-76 dB SPL at 1 cm [64±10 dB peSPL (mean ± s.d.), N=9 species]. These quiet and/or hyper-frequency ultrasounds are audible to nearby mates, but inaudible to unintended receivers. Although largely unknown because it is so inconspicuous, acoustic communication using low intensity ultrasound appears to be widespread among hearing moths. Thus, acoustic communication may be the norm rather than the exception.

Objective: To evaluate the effects of various 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) on ectopic osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2) using different administration methods. Materials and methods: Disks containing 5 μg of rhBMP-2 and type I collagen were implanted into the calf muscles of 6-week-old male rats (n = 64). Either the lactone form of simvastatin (SV), open hydroxy-acid form of simvastatin (SVA), cerivastatin (CVA), or vehicle (control) was then administered per orally (PO group) or subcutaneously (SC group) for 20 days. The disks were removed on day 21 after implantation, and ectopic induced bone formation was evaluated by radiographic, histologic, and biochemical analysis. Results: Both the projected and radiopaque area on X-ray film, and the calcium content of the SV group in the SC group (SV–SC group) were significantly greater than those in the other SC and PO groups. Alkaline phosphatase activity and tartrate-resistant acid phosphatase activity in the SV–SC group were significantly lower than those in the other SC and PO groups. Histologic examination revealed an increase of ectopic induced bone volume in the SV–SC group. Conclusion: Subcutaneous administration of SV stimulates ectopic osteoinduction by rhBMP-2 through reduction of bone turnover.

International audience; A screening was performed using nine marine-derived fungi as biocatalysts and the natural products (-)-ambrox® (1), (-)-sclareol (2), and (+)-sclareolide (3) in order to select the microorganisms able to catalyze the biooxidation of these compounds. It was observed that only the Aspergillus sydowii CBMAI 934, Botryosphaeria sp., Eutypella sp., and Xylaria sp. presented active oxidoreductases and catalyzed the regioselective hydroxylation in the natural products. The hydroxylated metabolites obtained were 1β-hydroxy-ambrox (1a) (14 %, A. sydowii CBMAI 934); 3β-hydroxy-ambrox (1b) (17 %, Botryosphaeria sp.; 11 %, Eutypella sp.); 3β-hydroxy-sclareol (2a) (31 %, Xylaria sp.; 69 %, Botryosphaeria sp.; 55 %, Eutypella sp.); 18-hydroxy-sclareol (2b) (10 %, Xylaria sp.); and 3β-hydroxy-sclareolide (3a) (34 %, Botryosphaeria sp.; 7 %, Eutypella sp.). This is the first report of biohydroxylation of (-)-ambrox® (1), (-)-sclareol (2), and (+)-sclareolide (3) by whole mycelia of marine-derived fungi.

The giant egg (Ge) locus is a Z-linked mutation that leads to the production of large eggs. Cytological observations suggest that an unusual translocation of a large fragment of the W chromosome bearing a putative egg size-determining gene, Esd, gave rise to giant egg mutants. However, there is currently no molecular evidence confirming either a W-Z translocation or the presence of Esd on the W chromosome. To elucidate the origin of giant egg mutants, we performed positional cloning. We observed that the Bombyx mori. orthologue of the human Phytanoyl-CoA dioxygenase domain containing 1 gene (PHYHD1) is disrupted in giant egg mutants. PHYHD1 is highly conserved in eukaryotes and is predicted to be a Fe(II) and 2-oxoglutarate-dependent oxygenase. Exon skipping in one of the two available Ge mutants is probably caused by the insertion of a non-long terminal repeat transposon into intron 4 in the vicinity of the 5' splice site. Segmental duplication in Ge(2) , an independent allele, was caused by unequal recombination between short interspersed elements inserted into introns 3 and 5. Our results indicate that (1) Bombyx PHYHD1 is responsible for the Ge mutants and that (2) the Ge locus is unrelated to the W-linked putative Esd. To our knowledge, this is the first report describing the phenotypic defects caused by mutations in PHYHD1 orthologues.

Eleven sesquiterpenes (1-11) and one long chain aldehyde (12) have been isolated from the dichloromethane extract of the red alga Laurencia scoparia. Four of them are new natural products. Scopariol (1) is a new natural product with an unusual rearranged chamigrane-type structure. The other three are beta-chamigrenes: isorigidol (2), (+)-3-(Z)-bromomethylidene-10 beta-bromo-beta-chamigrene (3), and (-)-3-(E)-bromomethylidene-10 beta-bromo-beta-chamigrene (4). The in vitro activity of compounds 1-12 against the parasitant stage of Nippostrongylus brasiliensis (L4) has been studied.

SC1/DM-GRASP/BEN, a cell adhesion molecule of the immunoglobulin super family, promotes the extension of neurites from neurons that express SC1 in culture, presumably by direct homophilic interactions. SC1 is specifically and transiently expressed on motoneurons during the period of their axonal growth, suggesting that it plays an important role in this growth. To explore this possibility, we expressed SC1 ectopically on the spinal cord interneurons of quail and chick embryos by in ovo electroporation at E3, when the motoneuron axonal growth starts. The axonal growth of the interneurons expressing chick SC1 was analyzed by immunohistochemistry with chick-specific anti-SC1 monoclonal antibody in quail, and by retrograde labeling with a dye in chick and quail embryos at E5. The majority of the axons of SC1-positive interneurons passed through the motor column and extended normally along the spinal cord basement membrane, but a few appeared to grow out from the cord. However, a dye back-labeling of the spinal nerves revealed that none of the interneurons were both SC1 and dye positive. These results suggest that the expression of SC1/DM-GRASP/BEN alone is insufficient for regulating the first step of the selective axonal pathfinding of motoneurons.

The relationship between carbonyl reductase, which is localized in the theca and interstitial cells, and ovarian follicular development was evaluated by measuring the enzyme activity and concentration of carbonyl reductase, and examining its immunohistochemical localization in immature rats treated with equine chorionic gonadotrophin (eCG), ovine(o) FSH and hCG. eCG and oFSH were administered s.c. to 26-day-old rats, and the ovaries were isolated 48 and 72 h after treatment. hCG was administered s.c. 48 h (at 28 days of age) after eCG or oFSH, and the ovaries were isolated 6 or 9 h after treatment. Both eCG and oFSH significantly increased the ovarian carbonyl reductase concentration 48 h after treatment compared with the saline-treated group, but the increase with eCG was approximately twice that with oFSH. Seventy-two hours after treatment, eCG further increased both carbonyl reductase activity and its concentration. Ovulation was also induced. oFSH stimulated only ovarian follicular growth without further increase in either activity or concentration. In addition, eCG increased the immunoreactivity to anti-carbonyl reductase antibody in the theca and interstitial cells, but oFSH did not. Treatment with hCG in saline-pretreated rats increased carbonyl reductase activity by 2.8-fold and carbonyl reductase concentration by 4.1-fold after 9 h compared with the 0 h concentration, without producing the large follicles observed with eCG and oFSH. However, the stimulatory effect of hCG on ovarian carbonyl reductase in eCG- or oFSH-pretreated rats was weaker than that in saline-pretreated rats. These results suggest that ovarian carbonyl reductase is induced by LH/hCG but not by FSH and that the enzyme induced by hCG is unrelated to preovulatory ovarian follicular growth.

In the previous study, we have demonstrated that nicotine hardly induces catecholamine release from adrenal medulla of 21-day-old rats. The present study examined the responsiveness of the adrenal chromaffin cells to acetylcholine in vitro and neostigmine and oxotremorine in vivo in 21-day-old and 8-week-old rats. As assessed by electron microscopy, the number of the chromaffin granules was markedly decreased and the content of adrenaline in adrenals was diminished significantly by oxotremorine treatment in 8-week-old rats, whereas these changes did not occur in 21-day-old rats. Morphological changes of the adrenal chromaffin cells, with respect to exocytosis, were not observed in neostigmine-treated 21-day-old rats and acetylcholine-treated adrenal slices prepared from 21-day-old rats. Catecholamine release was hardly evoked by acetylcholine in these slices as judged by measuring the catecholamine content in the medium. These results indicate that the sensitivity of the chromaffin cells to these secretagogues in 21-day-old rats is very low when compared to that in young adult rats.

We have cloned a novel matrix metalloproteinase (MMP) from cultured chicken embryo fibroblasts. The cDNA-derived protein sequence contains 608 amino acids including a C-terminal hydrophobic transmembrane domain of 24 amino acids and a cytoplasmic domain of 20 amino acids. This chicken MMP is 72% similar to a recently described membrane-type MMP (MT-MMP) from human placenta (Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994) Nature 370, 61-65). Accordingly, we name this novel MMP chicken MT-MMP. As shown by Northern blotting, two MT-MMP mRNAs of 6 and 10 kilobases are constitutively expressed but only modestly regulated by growth factors and cytokines in cultured chicken embryo fibroblasts. Both mRNAs are abundant in the head and body of 8- and 9-day-old chicken embryos. As shown by in situ mRNA hybridization, MT-MMP is expressed in embryonic neural tube, spinal ganglia, and respiratory epithelium, as well as in developing cartilage and muscle. Using reverse transcription-polymerase chain reaction, we have found MT-MMP mRNA in 2-day-old chicken embryos and extraembryonic membranes. In addition, a strong correlation was observed between the mRNA expression of MT-MMP and 72-kDa type IV collagenase. Collectively, the early MT-MMP mRNA expression and its co-localization in several tissues with 72-kDa type IV collagenase mRNA suggest that the MT-MMP plays an important role in early development.

Age-related changes and effects of gonadotrophins on ovarian carbonyl reductase (CR) of virgin female Wistar-Imamichi rats were investigated. In mature rats ovarian CR is an LH-dependent oxidoreductase and is closely involved in the periovulatory process. After 8 months of age, all of the rats exhibited persistent oestrus and the ovarian mass was significantly decreased, whereas 4- and 5-month-old rats showed the regular 4 day oestrous cycles and ovulation occurred. The ovarian CR activity towards four substrates declined steeply consistent with persistent oestrus and the enzyme activity for each substrate at 15 months of age was 1.4-53.7% lower than at 4 months of age. The ovarian CR concentration measured by western blot analysis was also lower at 8 months of age. Equine chorionic gonadotrophin (eCG) and hCG were administered to 10- and 15-month-old rats at persistent oestrus to examine whether the ovarian CR in rats at persistent oestrus is influenced by gonadotrophins. Treatment with hCG not only increased ovarian CR activity and its concentration but also caused ovulation at both ages. The immunoreactivity to anti-ovarian CR antibody in the theca cells of the hCG-treated ovary was greater than that of the control ovary. Equine(e) CG did not stimulate ovarian CR activity in rats at persistent oestrus, although ovarian mass of 10-month-old rats was increased after administration of 50 iu of eCG. These results indicate that changes in ovarian CR with ageing are related to the transition of regular oestrous cyclicity to acyclicity and that the expression of ovarian CR markedly decreased in aged rats is stimulated by hCG.

1. Binding and inhibition of antimicrobial fluoroquinolones towards liver CYP1A2 purified from 3-methylcholanthrene-treated rats were investigated using proton nuclear magnetic resonance (nmr) and phenacetin metabolism. 2. The proton nmr longitudinal relaxation rate study indicated that the paramagnetic effects of the haem iron of CYP1A2 were observed in protons of enoxacin with a 1,8-naphthyridine skeleton and its 4'-nitrogen atom on the 7-piperazine ring probably participated in specific binding to the haem iron. These data suggest a facile accessibility and strong binding of enoxacin to the active site of the enzyme. On the contrary, the binding region of norfloxacin with a quinoline skeleton could not be specified, and an 8-fluorinated derivative (AT-3970) had much lower paramagnetic effects and no specific binding region. 3. In a reconstituted CYP1A2 system, enoxacin exhibited the most potent inhibition of phenacetin O-deethylation. The metabolism was less inhibited by norfloxacin, and AT-3970 had a weak inhibitory activity. 4. The binding ability of the fluoroquinolones to the CYP1A2 active site is likely to determine their inhibitory activity against phenacetin metabolism.

We investigated the optimal conditions for the application of the MTT assay to determine the viability of cell in primary cultures of rat hepatocytes. The optimal conditions were found to be: inoculation at cell density of 1.31 x 10(5) cells/cm2; concentration of 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 0.12 mM; and MTT incorporation time, 2 h or more. We also found that insulin and glucocorticoid, which are usually added to the medium to maintain cell function, had no effect on the viability of the primary cultures of rat hepatocytes.

The identification of rat faecal metabolites of a new antihistaminic agent, ebastine, 4'-tert-butyl-4-[4-(diphenylmethoxy)piperidino]butyrophenone, is presented. After oral administration of (14C)ebastine (20 mg kg-1) to rats, 84% of the radioactive dose was excreted in the 24 h faeces. Unchanged drug and five metabolites were isolated from the faeces by thin-layer chromatography and solid-phase extraction, and their structures were identified by liquid secondary ion mass spectrometry using the B/E linked scanning technique. The main metabolic pathways were oxidation of a terminal methyl group to give the hydroxymethyl and carboxyl derivatives, and hydroxylation of a phenyl ring in the diphenylmethoxy moiety. In addition to the oxidative mechanism, metabolism of ebastine involved sulphate conjugation. It is noteworthy that M-4, having both phenolic and alcoholic hydroxyl groups, was sulphated selectively in the latter position.

In this chapter, from the engineering point of view, we introduce the results from our group and related research on three typical configurations of engineered liver tissues; cell sheet-based tissues, sheet-like macroporous scaffold-based tissues, and tissues based on special scaffolds that comprise a flow channel network. The former two do not necessitate in vitro prevascularization and are thus promising in actual human clinical trials for liver diseases that can be recovered by relatively smaller tissue mass. The third approach can implant a much larger mass but is still not yet feasible. In all cases, oxygen supply is the key engineering factor. For the first configuration, direct oxygen supply using an oxygen-permeable polydimethylsiloxane membrane enables various liver cells to exhibit distinct behaviors, complete double layers of mature hepatocytes and fibroblasts, spontaneous thick tissue formation of hepatocarcinoma cells and fetal hepatocytes. Actual oxygen concentration at the cell level can be strictly controlled in this culture system. Using this property, we found that initially low then subsequently high oxygen concentrations were favorable to growth and maturation of fetal cells. For the second configuration, combination of poly-L: -lactic acid 3D scaffolds and appropriate growth factor cocktails provides a suitable microenvironment for the maturation of cells in vitro but the cell growth is limited to a certain distance from the inner surfaces of the macropores. However, implantation to the mesentery leaves of animals allows the cells again to proliferate and pack the remaining spaces of the macroporous structure, suggesting the high feasibility of 3D culture of hepatocyte progenitors for liver tissue-based therapies. For the third configuration, we proposed a design criterion concerning the dimensions of flow channels based on oxygen diffusion and consumption around the channel. Due to the current limitation in the resolution of 3D microfabrication processes, final cell densities were less than one-tenth of those of in vivo liver tissues; cells preferentially grew along the surfaces of the channels and this fact suggested the necessity of improved 3D fabrication technologies with higher resolution. In any case, suitable oxygen supply, meeting the cellular demand at physiological concentrations, was the most important factor that should be considered in engineering liver tissues. This enables cells to utilize aerobic respiration that produces almost 20 times more ATP from the same glucose consumption than anaerobic respiration (glycolysis). This also allows the cells to exhibit their maximum reorganization capability that cannot be observed in conventional anaerobic conditions.

BACKGROUND: Algae species have been used as an important source of food because they are highly nutritive considering their vitamin, protein, mineral, fiber, essential fatty acid and carbohydrate contents. However, a large number of seaweeds have been poorly studied, especially Brazilian species. Two red macroalgae species from the Brazilian coast (Plocamium brasiliense and Ochtodes secundiramea) were assessed with respect to their total lipid, fatty acid, total nitrogen, protein, amino acid and total carbohydrate contents. RESULTS: The total lipid contents (dry weight) were 36.3 and 35.4 g kg−1; fatty acid contents were 9.3 and 12.1 g kg−1; total nitrogen contents were 37.4 and 24.9 g kg−1; protein contents were 157.2 and 101.0 g kg−1; amino acid contents were 127.5 and 91.4 g kg−1; and total carbohydrate contents were 520.3 and 450.7 g kg−1 for P. brasiliense and O. secundiramea, respectively. CONCLUSION: Considering these compositions, both algae species were determined to have sources of protein, essential amino acids and carbohydrates similar to the edible seaweeds Laminaria japonica and Palmaria palmata. Copyright © 2011 Society of Chemical Industry

The left atrial (LA) function during coronary artery occlusion followed by reperfusion using densitometric analysis of digital atrioventriculographic images was evaluated. Eight anesthetized dogs underwent atrioventriculography at baseline, 10 and 60 min after left circumflex coronary artery (LCX) occlusion and 5, 30, 60, and 120 min of reperfusion. Time-density curves were obtained for LA and left ventricle (LV). The ratios of passive atrial video-densitometric change (VC) to total VC (Passive Ratio), and active VC to total VC (Active Ratio) were calculated. Left ventricular ejection fraction (LVEF), peak ejection rate (PER), and peak filling rate (PFR) were derived. Active Ratio, an index of atrial contraction, increased to 144%, and Passive Ratio decreased to 75% of baseline at 60 min of LCX occlusion. Two hours after reperfusion, both Active and Passive Ratios returned to control level. While LVEF reduced to 70%, PER to 67%, LV peak positive dP/dt to 88% of baseline at 60 min after occlusion, and remained depressed at 2 h after reperfusion. However, PFR, LV peak negative dP/dt and LV isovolumic pressure decay rate showed recovery at 2 h after reperfusion. There were significant correlations between PFR and Passive Ratio (r = 0.41), and between Active and Passive Ratios (r = 0.55). Thus, time-course of recovery of LV post-ischemic systolic and diastolic function was different. Return of LA function to control level during 2 h after reperfusion may be depend on recovery of LV diastolic function.

It has been suggested that the calcium-binding proteins, parvalbumin and calbindin-D28K, are involved in the control of intracellular calcium levels but their exact functions are unknown. Immunoreactivity for parvalbumin has been associated with rapidly firing cells, while calbindin has been implicated in protecting neurons from excitotoxicity. Since the chick Edinger Westphal nucleus contains two populations of neurons with different firing patterns5, parvalbumin and calbindin immunoreactivities were examined in the Edinger Westphal nuclei of posthatch chicks to determine wheter a particular subpopulation of neurons is associated with either protein. Moderate levels of parvalbumin immunoreactivity were found consistently associated with repetitively firing neurons in the lateral Edinger Westphal nucleus. In contrast, medial neurons expressed much lower levels of parvalbumin immunoreactivity. Many medial neurons were negative for parvalbumin although occasionally a few medial neurons stained as strongly as lateral neurons. Definitive calbindin immunoreactivity was absent from Edinger Westphal nuclei despite robust staining of cells in other parts of the brainstem and in control sections of cerebellum.

A new cancer-associated antigen CA130, recognised by two monoclonal antibodies (moABs) 130-22 and 145-9, was often found to be present at high levels in the sera of patients with ovarian cancer. There was a strong correlation between CA130 and CA125 values. The epitopes recognised by moABs 130-22 and 145-9 were proved to differ from the CA125 epitope, but to exist on the molecule bearing CA125. Unlike OC125, the majority of 130-22/145-9 activity was associated with a much lower molecular mass (less than 200 kDa), indicating that a lower molecular mass immunoreactive determination may be a unique CA130 antigenic determinant within CA125 molecule. Clinical data demonstrate that, (1) elevated levels of CA130 determinant were found in the sera of 91.3% of women with epithelial ovarian cancer, (2) falling or rising levels of CA130 correlated with regression or progression of ovarian cancer in > 95% of cases, (3) normalisation of serum CA130 levels at response does not imply no microscopic residual disease, but CA130 changes during follow-up support the evaluation of recurrence and can be used as a monitoring marker in an individual patient.

Investigation of the bioactive crude extracts from two populations of the red alga Laurencia dendroidea from the southeastern Brazilian coast led to the identification of five sesquiterpenes: (+)-obtusane (1), a triquinane derivative (2), (-)-elatol (3), obtusol (4), and cartilagineol (5). An antileishmanial bioassay against Leishmania amazonensis was conducted for crude lipophilic extracts and for sesquiterpenes 2, 3, and 4. Compounds 3 and 4 displayed in vitro and in vivo leishmanicidal activity and very low cytotoxicity.

The larval integument of the silkworm, Bombyx mori, is opaque because urate granules accumulate in the epidermis. Although the biosynthetic pathway of uric acid is well studied, little is known about how uric acid accumulates as urate granules in epidermal cells. In the distinct oily (od) mutant silkworm, the larval integument is translucent because of the inability to construct urate granules. Recently, we have found that the od mutant has a genomic deletion in the B. mori homologue of the human biogenesis of lysosome-related organelles complex1, subunit 2 (BLOS2) gene (BmBLOS2). Here, we performed a molecular and functional characterization of BmBLOS2. Northern blot analysis showed that BmBLOS2 was ubiquitously expressed in various tissues. We analysed the structure of a newly isolated mutant (od(B) ) allelic to od and found a premature stop codon in the coding sequence of BmBLOS2 in this new mutation. Moreover, the translucent phenotype was rescued by the germ-line transformation of the wild-type BmBLOS2 allele into the od mutant. Our results suggest that BmBLOS2 is responsible for the od mutant phenotype and plays a crucial role in biogenesis of urate granules in the larval epidermis of the silkworm. The relationships amongst Hermansky-Pudlak syndrome (HPS) genes in mammals, granule group genes in Drosophila and translucent mutant genes in B. mori are discussed.

We examined effects of hypocapnia on burst activity in the piriform-amygdala complex and C(4) inspiratory activity in limbic-brainstem-spinal cord preparations from 0- to 1-day-old rats. Hypocapnia (2% CO(2)) increased the burst rate in the piriform-amygdala complex but decreased the C(4) inspiratory burst rate. Since hyperventilation induces hypocapnia, and enhanced amygdala activity may be involved in induction of a sense of anxiety, our findings might explain the neuronal mechanism of a vicious circle between hyperventilation and an increased sense of anxiety.

Sphingosine (a potent inhibitor of protein kinase C) at 5-10 microM, which are concentrations lower than those that inhibit this enzyme activity, enhanced the aggregation of rabbit platelets induced by low concentrations of U46619, platelet-activating factor, thrombin and arachidonic acid, whereas H-7 and staurosporine, other protein kinase C inhibitors, failed to do so. Of the sphingosine analogues which also inhibit protein kinase C, psychosine and lyso-GM3 did not show such an enhancing effect. Sphingosine promoted both Ins(1,4,5)P3 formation and an increase in the cytoplasmic free Ca2+ concentration in response to all the agonists used. Furthermore, the hydrolytic action of exogenously added phospholipase C (from Clostridium perfringens) on platelet membrane phospholipids was dose-dependently enhanced by pretreatment of the platelets with sphingosine. These results imply that sphingosine, at relatively low concentrations, brings about hyperaggregability of the platelets by the agonists employed, probably owing to enhancement of the phospholipase C activity. Such an effect appears to be induced by a mechanism independent of protein kinase C inhibition. We suggest that sphingosine might act as a positive modulator for the stimulus-response coupling in the platelets.

The potential toxicity and carcinogenicity of thiabendazole (TBZ) was investigated in F344 rats. TBZ was given in pelleted diets at different dietary levels (0, 0.05, 0.1, 0.2 or 0.4%) to groups of 30 rats of each sex for 104 wk. Throughout the study, a marked decrease in body-weight gain was observed in males and females fed 0.4% TBZ. Transitional cell hyperplasia of the renal pelvis and/or papilla was observed in dosed groups of each sex with significant positive trends. Low incidence of transitional cell papilloma or carcinoma of the urinary system was recorded in the dosed groups. Preputial gland adenoma occurred in males treated with 0.4% TBZ, and the incidence was significantly higher than in controls. There was no clear-cut evidence of the carcinogenic activity of TBZ in the urinary system of male and female rats.

Three novel halogenated beta-bisabolene sesquiterpenoids (1-3), together with two know triquinane alcohol sesquiterpenes (6 and 7), were isolated from the red alga Laurencia scoparia and their structures elucidated by spectroscopic methods. Single-crystal X-ray crystallography allowed us to confirm the structure of 1 as well as to determine the absolute configuration of all stereocenters. To the best of our knowledge, the isolation of beta-bisabolenes from the genus Laurencia has no precedent in the literature. Compound 1 showed weak in vitro anthelmintic activity against parasitant stage (L4) Nippostrongilus brasiliensis.

Monoclonal antibodies (mAbs) were produced against the proteins of advanced third-stage larvae (AdL3) of Gnathostoma nipponicum. Six mAbs (Gn2C3, Gn2H3, Gn4C3, Gn4E9, GnSH1, and Gn10B7) were obtained as determined by enzyme-linked immunosorbent assay (ELISA). Gn4E9 and GnSH1 seemed to be genus-specific, as they did not cross-react with Anisakis sp., Dirofilaria immitis, Gongylonema pulchrum, Toxocara canis, Trichinella sp., Trichuris vulpis, Metagonimus sp., or Spirometra erinaceieuropaei by ELISA. Immunohistochemistry showed that Gn2C3, Gn4E9, and Gn5H1 reacted strongly with the central esophagus; Gn2H3 reacted with cuticle,muscle, intestine, and the cervical sac; and Gn4C3 and Gn10B7 reacted with cuticle, muscle, esophagus, intestine, and the cervical sac of AdL3. In Western blotting analysis, Gn2C3, Gn4E9, and Gn5H1 reacted to 60-, 53-, 46-, and 41-kDa proteins; Gn4C3 reacted to the AdL3 protein of G. nipponicum (42 kDa). Moreover, proteins purified using a mAb Gn4E9 immunoprecipitation method (sizes 60-, 53-, 46-, and 41-kDa) were used as antigens in ELISAs. A significant difference (P0.01) was shown between mouse sera infected with G. nipponicum and sera infected with Trichnella sp. or not infected. These results provide a rationale for evaluating esophageal proteins for the development of diagnostic methods for detecting G. nipponicum or Gnathostoma sp. infections.

p21/WAF1 (p21) inhibits the activity of the cyclin/cdk complex and controls the G1 to S cell phase transition. In the present study, we used a recombinant adenoviral approach and gene gun technology to introduce p21 into esophageal cancer cells in order to assess the effect of p21 on cell growth. Infection with the p21 adenovirus (AdV) using gene gun technology resulted in inhibition of TE9 and KE3 cell growth. The levels of involucrin, which is a marker of squamous epithelium differentiation, markedly increased at 48 h and 72 h after p21 AdV infection in TE9 cells. These results indicate that p21 plays an important role in esophageal cancer cell proliferation. Overexpression of the p21 gene can inhibit cell growth and induce differentiation in esophageal cancer cells. p21 gene therapy may prove beneficial in the treatment of esophageal cancer.

The enzyme glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) in an NADPH-dependent manner. A specific antibody raised against recombinant rat GR was used to localize the protein in the female reproductive organs during the estrous cycle in the rat. In the ovary, the strongest reactivity to the antibody was observed in oocytes, followed by granulosa cells, corpus luteum, and interstitial cells. A strongly positive reaction was also observed mainly in the oviduct epithelia, uterine epithelia, and endometrial gland in the reproductive tract. Oviducts contained the highest GR activity. The GR activity of uterus during metestrus was about twice as high as that for other stages of the cycle. The levels of GR proteins in the tissues roughly matched the activities. The expression of the GR mRNA was highest during metestrus. Because GSH is known to increase gamete viability and the efficiency of fertility, GR, which is expressed in these tissues, is predicted to play a pivotal role in the reproduction process as a source of GSH.

It has been confirmed that the neurotransmitter acetylcholine (ACh) is present in blood; it is synthesized in T-lymphocytes by choline acetyltransferase (ChAT) and released upon T-lymphocyte activation. Both muscarinic and nicotinic ACh receptors have been identified on lymphocytes isolated from thymus, lymph node, spleen and blood, and their stimulation by muscarinic and nicotinic agonists elicits a variety of functional and biochemical effects, providing a strong argument that ACh synthesized and released from T-lymphocytes acts as an autocrine and/or paracrine factor regulating immune function. In the present study, we compared ACh levels in the blood, circulating mononuclear leukocytes (MNLs), thymus and spleen of spontaneously hypertensive rats (SHRs), which exhibit immune deficiencies related to the emergence of natural thymocytotoxic autoantibody, age-related decline of T-cell function and morphological changes in immune organs, with ACh levels in age-matched, normotensive Wistar Kyoto rats. In each case, ACh levels in 5-, 10- and 20-week-old SHRs were significantly lower than in WKYs. ChAT mRNA expression in MNLs was also significantly depressed in the SHRs. These results suggest that diminished synthesis and release of ACh from MNLs into blood and lymphoid organs likely reflects an immune deficiency related to T-cell dysfunction.

Glutathione, which is found in high levels in eye tissues, is involved in multiple functions, including serving as an antioxidant and as an electron donor for peroxidases. Although the activities of enzymes related to glutathione metabolism have been reported in the eye, the issue of which cells produce these proteins, where they are produced and at what levels is an important one. Glutathione reductase, an enzyme which recycles oxidized glutathione by transferring electrons from NADPH, was localized immunohistochemically in adult rat eye in this study. The reductase was distributed in the corneal and conjunctival epithelia, corneal keratocytes and endothelium, iridial and ciliary epithelia, neural retina, and retinal pigment epithelium. In addition, it was highly expressed in ganglion cells, which are responsible for transmitting photophysiological signals from the retina to the higher visual centres. To clarify the correlation of glutathione reductase expression and oxidative stress, the enzymatic activity and the level of protein expression at the pre- and postnatal stages was examined. Expression of the enzyme was detected first in the ganglion cell layer of a late prenatal stage, and appeared in the inner plexyform layer after birth. Along with an increasing differentiation between the inner nuclear and outer nuclear layers, glutathione reductase expression became detectable in the outer plexyform layer. Pigment epithelial cells were positively stained only after birth. Expression was also detected in the lens epithelium from the prenatal to early postnatal stages although its level was low in the adult lens. Collectively, these data, except for lens epithelia, suggest the pivotal role of glutathione reductase in recycling oxidized glutathione for the protection of the tissues against oxidative stress, which is caused by eye opening accompanied by the initiation of various ocular processes, such as accession of light and transduction of the photochemical signal.

A 2.4-kb truncated L-plastin promoter was inserted either 5' to the LacZ gene (Ad-Lp-LacZ) or 5' to the cytosine deaminase (CD) gene (Ad-Lp-CD) in a replication-incompetent adenoviral vector backbone. Infectivity and cytotoxicity experiments with the LacZ and CD vectors suggested that the L-plastin promoter-driven transcriptional units were expressed at much higher levels in explants of ovarian cancer cells from patients and in established ovarian or bladder cancer cell lines than they were in normal peritoneal mesothelial cells from surgical specimens, in organ cultures of normal ovarian cells, or in the established CCD minimal deviation fibroblast cell line. Control experiments showed that this difference was not attributable to the lack of infectivity of the normal peritoneal cells, the normal ovarian cells, or the minimal deviation CCD fibroblast cell line, because these cells showed expression of the LacZ reporter gene when exposed to the replication-incompetent adenoviral vector carrying the cytomegalovirus (CMV)-driven LacZ gene (Ad-CMV-LacZ). The Ovcar-5 and Skov-3 ovarian cancer cell lines exposed to the Ad-Lp-CD adenoviral vector were much more sensitive to the prodrug 5-fluorocytosine (5FC), which is converted from the 5FC prodrug into the toxic chemical 5-fluorouracil, than was the CCD minimal deviation fibroblast cell line after exposure to the same vector. A mouse xenograft model was used to show that the Ad-Lp-CD vector/5FC system could prevent engraftment of ovarian cancer cells in nude mice. Finally, injection of the Ad-Lp-CD vector into s.c. tumor nodules generated a greater reduction of the size of the tumor nodules than did injection of the Ad-CMV-LacZ vectors into tumor nodules. The Ad-Lp-CD vectors were as suppressive to tumor growth as the Ad-CMV-CD vectors. These results suggest that an adenoviral vector carrying the CD gene controlled by the L-plastin promoter (Ad-Lp-CD) may be of potential value for the i.p. therapy of ovarian cancer.

Small intestinal microsomes of cynomolgus monkeys were found to catalyze hydroxylation and dealkylation of an H(1)-antihistamine prodrug, ebastine. To identify the main enzyme responsible for ebastine hydroxylation, which has been hitherto unknown, we purified two cytochrome P450 isoforms, named P450 MI-2 and P450 MI-3, from the intestinal microsomes on the basis of the hydroxylation activity. P450 MI-2 and P450 MI-3 showed the respective apparent molecular weights of 56,000 and 53,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The internal amino acid sequence of P450 MI-2 had high similarity with those of human CYP4F2, CYP4F3, and CYP4F8. The first 27 amino acid residues of P450 MI-3 were highly homologous with those of monkey CYP3A8 and human CYP3A4/5/7. Furthermore, P450 MI-2 and P450 MI-3 were recognized by anti-CYP4F and anti-CYP3A antibodies, respectively, in immunoblot analysis and catalyzed leukotriene B(4) omega-hydroxylation and testosterone 6beta-hydroxylation, which are known to be mediated by CYP4F and CYP3A, respectively. Although both enzymes had ebastine hydroxylation activity, the V(max) value of P450 MI-2 was much higher than that of P450 MI-3 (37.0 versus 0.406 nmol/min/nmol of P450), and the former K(M) (5.1 microM) was smaller than the latter K(M) (10 microM). Anti-CYP4F antibody inhibited the hydroxylation in small intestinal microsomes strongly (70%), but anti-CYP3A antibody did not. These results indicate that P450 MI-2 belongs to the CYP4F subfamily and is mainly responsible for hydroxylation of ebastine in monkey small intestinal microsomes. This suggests that the small intestinal CYP4F enzyme, P450 MI-2, can play an important role in the metabolism of drugs given orally.

Human amniotic epithelial cells (HAEC) are formed from epiblasts on the 8th day after fertilization. Because they lack major histocompatibility complex (MHC) antigen, human amniotic tissue transplantation has been used for allotranplantation to treat patients with lysosomal diseases. We have provided evidence that HAEC have multiple functions such as synthesis and release of acetylcholine (ACh) and catecholamine (CA) as well as expressing mRNA coding for dopamine receptors and dopamine (DA) transporter (DAT). On the other hand, we showed that monkey amniotic epithelial cells (MAEC) synthesize and release CA and posses DA receptors and DAT. Detection of muscarinic actylcholine receptors indicates the presence of an autocrine mechanism in HAEC. Recently, we found that HAEC have neurotrophic function in conditioned medium from HAEC, indicating the presence of a novel neurotrohpic factor that is synthesized and released from HAEC. The amniotic membrane may have a significant role in supplying neurotrophic factors as well as neurotransmitters to the amniotic fluid, suggesting an important function in the early stages of neural development of the embryo. This review will focus on the neuropharmacological aspects of HAEC and MAEC in relation to the physiology of amniotic membrane.

A family of proteins with thioredoxin (TRx)-dependent peroxidase activity, referred to as peroxiredoxins (PRx), has been identified in many species. The sixth member of this family, PRxVI, contains only one conserved cysteine residue, while other members contain additional cysteines. We have isolated a cDNA for rat PRxVI and constructed a large scale baculovirus system to produce the recombinant protein. The protein was purified by a simple two-step procedure utilizing ion-exchange and gel-filtration chromatography. The purified PRxVI exhibited a low level of glutathione-dependent peroxidase but not TRx-dependent activity. PRxVI expression was the highest in lung, followed by brain, kidney, heart, testis, etc. as judged by Northern and Western blot analyses using a rabbit antibody to the purified PRxVI. Immunohistochemical analyses showed strong staining in the epithelium of the bronchus and bronchioles in lung and in the epithelial cells of kidney tubules. In addition, Sertoli cells in testis and islet of Langerhans cells in pancreas were strongly stained. The developmental changes of PRxVI expression in lung and kidney were low in the prenatal stage but induced postnatally. Moreover, intraperitoneal administration of chloroform induced PRxVI mRNA in kidney. When the distribution and the induced expression of PRxVI under conditions of oxidative stress are considered, a physiological role of it as an antioxidative enzyme is indicated.

Leukemia inhibitory factor (LIF) is a widely expressed cytokine involved in both local and systemic immune response. Furthermore, it has been implicated in various immunological processes including thymic T cell maturation and embryo implantation. We investigated implication of various modalities in the application of prolonged and viable allograft to the wound, using cytokines and growth factors.BALB/c and B6D2F1 strains of mice were used either as a skin graft donor or host. LIF cDNA inserted in plasmid vector or the vector alone was injected intradermally in graft skin and observed up to 21 days. LIF, LIF-receptor, gp130, as well as type 1 and 2 T helper cytokine expressions were investigated by reverse transcription polymerasse chain reaction, in situ hybridization, and histological studies.LIF cDNA-treated groups showed significantly improved graft survival compared to the vector-treated control in 21 days postoperatively for grafting from B6D2F1 to BALB/c and BALB/c to B6D2F1. LIF and LIF receptor mRNA expressions were observed 24 hr and 21 days posttransplantation. The gp130 expression was only observed in LIF-treated B6D2F1 to BALB/c allografting on day 21 posttransplantation. LIF transcripts were strongly present in the epidermal, dermal, and subdermal tissues as determined by an in situ hybridization of LIF-treated grafting.These results suggest that LIF cDNA treatment is an effective and beneficial adjuvant for the skin allograft survival. Improved skin allograft modulation by cytokine gene transfer is a potentially promising therapy for temporary large skin coverage.

Localization of glycosaminoglycans (GAG) in Bruch's membrane of postnatal rat eyeballs was examined histochemically. Fixed eyeballs from postnatal rats (ages 5 days and 8 weeks) were routinely processed and embedded in paraffin wax or Quetol 651 resin. Paraffin-embedded tissue sections were stained with hematoxylin and eosin or sensitized high iron diamine procedure in combination with selective methods such as GAG-degrading enzyme digestions and/or a chemical modification, and examined by light microscopy. Quetol 651-embedded ultrathin sections were stained with heavy metals and examined by electron microscopy. In rats at postnatal day 5, Bruch's membrane contained mainly chondroitin sulfate (CS) and heparan sulfate (HS). In contrast, at 8 weeks after birth the membrane included a large amount of dermatan sulfate (DS) and HS. According to electron microscopic findings, Bruch's membrane on day 5 consisted of only 3 layers without a central elastic layer. However, at 8 weeks after birth the membrane was constructed of 5 layers. These findings suggested that the difference in GAG molecular species in the membranes at 5 days and at 8 weeks after birth could be correlated with the development and maturation of the collagenous layer in Bruch's membrane. Moreover, maturation of Bruch's membrane may contributes to the architectural stabilization of the outer portions of the photoreceptor cells.

The phorbol ester tumor promoters and related analogs are widely used as potent activators of protein kinase C (PKC). The phorbol esters mimic the action of the lipid second messenger diacylglycerol (DAG). The aim of this commentary is to highlight a series of important and controversial concepts in the pharmacology and regulation of phorbol ester receptors. First, phorbol ester analogs have marked differences in their biological properties. This may be related to a differential regulation of PKC isozymes by distinct analogs. Moreover, it seems that marked differences exist in the ligand recognition properties of the C1 domains, the phorbol ester/DAG binding sites in PKC isozymes. Second, an emerging theme that we discuss here is that phorbol esters also target receptors unrelated to PKC isozymes, a concept that has been largely ignored. These novel receptors lacking kinase activity include chimaerins (a family of Rac-GTPase-activating proteins), RasGRP (a Ras exchange factor), and Unc-13/Munc-13 (a family of proteins involved in exocytosis). Unlike the classical and novel PKCs, these "non-kinase" phorbol ester receptors possess a single copy of the C1 domain. Interestingly, each receptor class has unique pharmacological properties and biochemical regulation. Lastly, it is well established that phorbol esters and related analogs can translocate each receptor to different intracellular compartments. The differential pharmacological properties of the phorbol ester receptors can be exploited to generate specific agonists and antagonists that will be helpful tools to dissect their cellular function.

Hippocampal cholinergic neuronal activity is reported to be regulated, at least partly, through serotonin1A (5-HT1A) receptors. Chronic lithium treatment has been shown to alter both behavioral and neurochemical responses mediated by postsynaptic 5-HT1A receptors. We investigated whether long-term lithium treatment affects central cholinergic neurotransmission through 5-HT1A receptor-mediated pathways. Changes in acetylcholine (ACh) release induced by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT1A receptor agonist, in the rat hippocampus were measured using a microdialysis technique and a radioimmunoassay for ACh. Administration of lithium for 21 days resulted in a serum lithium concentration of 1.03 mM and caused little change in density or affinity of [3H]8-OH-DPAT binding sites in the hippocampus. The local application of 8-OH-DPAT into the hippocampus of lithium treated rats increased the ACh efflux in both the absence and the presence of physostigmine, a cholinesterase (ChE) inhibitor, in the perfusion fluid. The basal ACh efflux of lithium treated rats was not different from that of the control rats under normal conditions, but was significantly higher than that of the controls when ChE was inhibited. These results demonstrate that chronic lithium treatment increases spontaneous ACh release in the hippocampus under conditions of ChE inhibition, but not under normal conditions, and enhances cholinergic neurotransmission through 5-HT1A receptor-mediated pathways, and suggest that activation of 5-HT1A receptor function by lithium is related to the enhancement of hippocampal cholinergic neurotransmission.

Nitric oxide synthase activity is increased in the stomach in association with Helicobacter pylori infection and portal hypertension, but the mechanism by which nitric oxide contributes to mucosal damage remains unclear.To examine whether nitric oxide injures gastric mucosal cells and whether cellular glutathione affects nitric oxide-induced cytotoxicity.A confluent monolayer of RGM-1 gastric mucosal cells was exposed to nitric oxide donors (NOC5 or NOC12). Cell viability was determined by trypan blue dye exclusion, lactate dehydrogenase release and supravital staining with Hoechst 33342 and propidium iodide. The kinetics of the reduced/oxidized forms of glutathione were also measured, as well as the effect of glutathione-depletion or glutathione-precursor treatment on nitric oxide-induced cytotoxicity.Excess exogenous nitric oxide produced by NOC5 or NOC12 induced necrosis in RGM-1 cells in a time- and concentration-dependent manner. The level of reduced glutathione drastically decreased prior to the loss of cell viability and remained low, but oxidized glutathione was not affected. Glutathione depletion increased necrosis of both NOCs in an NOC-concentration-related fashion, while pre-treatment with gamma-glutamylcysteine ethyl ester reduced their necrotic susceptibility.Exogenous nitric oxide induced necrosis in gastric mucosal cells, and intracellular reduced glutathione protects gastric mucosal cells from damage by nitric oxide.

S-1 is a novel oral anticancer drug, composed of tegafur (FT), gimestat (CDHP) and otastat potassium (Oxo) in a molar ratio of 1:0.4:1, based on the biochemical modulation of 5-fluorouracil (5-FU). In this study the combined effect of S-1 and low-dose CDDP as a modulator for colon 26 liver metastasis in mice was evaluated. In an experiment with S-1 (5 mg/kg/day: po) and CDDP (0.25 mg/kg/day: i.p.) for 14 days, the combined effects for both liver metastasis and tumor of spleen were not superior to those with S-1 or CDDP alone group. Body weight loss was not greater in the S-1 + CDDP group than in the control group. In an experiment with S-1 (5 mg x 2/kg/day: po) and CDDP (0.25 mg/kg/day: i.p.) for 7 days, the inhibitory effects of S-1 + CDDP of liver metastasis and tumor of the spleen were remarkable compared with the S-1 alone group. However a greater loss of body weight was seen in the S-1 + CDDP group than in other groups. This study suggests that low-dose CDDP might be a modulator of S-1 for colon 26 liver metastasis. Further study is needed to determine the optimum dose and duration of treatment.

An important role of C-terminal amino acid residues of bovine pancreatic ribonuclease A (RNase A) in the formation of the three-dimensional structure was previously implied. In this study, we replaced the C-terminal amino acid, Val124, with amino acid residues with different properties by site-directed mutagenesis. The recombinant mutant enzymes were purified and subjected to a refolding study after being converted to a fully reduced and denatured state. There was a significant difference among the mutant enzymes in the rate of recovery of the activity when air oxidation was performed: the rate decreased in the order of V124E, V124L, V124G, V124K, V124A, and V124W. On the other hand, the recovery rates for all the mutant RNase A in the presence of GSH and GSSG were almost the same. The recovered activity of V124E after 24 h incubation reached approximately 90% of that of the wild type enzyme, followed by V124L 80%, V124A and V124W 65%, and V124K and V124G 50%. The duration of the initial lag phase became shorter in the order of V124W, V124A, V124K or V124G, V124E, or V124L. The results imply that the C-terminal amino acid significantly influences the formation of correct disulfide bonds during the refolding process and that the hydrophobic interaction of Val124 is important for efficient packing of the RNase A molecule.

Objective and Design: Recently, Ogawa et al. [17] reported that the peritoneal mast cells (PMCs) of rats can release histamine by substance P (SP) in a receptor-dependent manner. In the present study, we confirmed and extended their findings.¶Material: PMCs were isolated from six strains of rats. In some experiments, peritoneal cells in the non-MC fraction were used.¶Methods: PMCs were incubated with SP, neurokinin (NK) receptor agonists or antagonists, and histamine content in the supernatant was measured. In the binding assay, PMCs were incubated with [125I]BH-SP together with SP or NK receptor antagonists. NK-1 receptor mRNA was detected using a reverse transcription-polymerase chain reaction (RT-PCR) assay.¶Results: PMCs from Slc:Wistar and F344/NSlc were highly sensitive to SP, leading to histamine release, whereas those from Slc:SD and three other strains were not. PMCs from Slc:Wistar and F344/NSlc also released histamine in the presence of an NK-1 agonist. The histamine release induced by SP and the NK-1 agonist was inhibited by the NK-1 receptor antagonists, FK888 and CP-99,994. [125I]BH-SP binding experiments revealed that PMCs from Slc:Wistar rats possessed a single high affinity binding site for SP and that the binding was blocked by NK-1 receptor antagonists. Peritoneal cells in the non-MC fraction exhibited no appreciable binding. In the RT-PCR assay, expression of NK-1 receptor mRNA was evident in Slc:Wistar PMCs, but not in the non-MC fraction from Slc:Wistar or Slc:SD PMCs.¶Conclusion: These data demonstrate the existence of functional NK-1 receptors on freshly isolated PMCs in at least some strains of rats.

Selenocysteine lyase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the exclusive decomposition of L-selenocysteine to L-alanine and elemental selenium. An open reading frame, named csdB, from Escherichia coli encodes a putative protein that is similar to selenocysteine lyase of pig liver and cysteine desulfurase (NifS) of Azotobacter vinelandii. In this study, the csdB gene was cloned and expressed in E. coli cells. The gene product was a homodimer with the subunit Mr of 44,439, contained 1 mol of PLP as a cofactor per mol of subunit, and catalyzed the release of Se, SO2, and S from L-selenocysteine, L-cysteine sulfinic acid, and L-cysteine, respectively, to yield L-alanine; the reactivity of the substrates decreased in this order. Although the enzyme was not specific for L-selenocysteine, the high specific activity for L-selenocysteine (5.5 units/mg compared with 0.019 units/mg for L-cysteine) supports the view that the enzyme can be regarded as an E. coli counterpart of mammalian selenocysteine lyase. We crystallized CsdB, the csdB gene product, by the hanging drop vapor diffusion method. The crystals were of suitable quality for x-ray crystallography and belonged to the tetragonal space group P43212 with unit cell dimensions of a = b = 128.1 A and c = 137.0 A. Consideration of the Matthews parameter Vm (3.19 A3/Da) accounts for the presence of a single dimer in the crystallographic asymmetric unit. A native diffraction dataset up to 2.8 A resolution was collected. This is the first crystallographic analysis of a protein of NifS/selenocysteine lyase family.

Calponin is a basic smooth muscle protein capable of binding to actin, calmodulin, tropomyosin, and phospholipids. We have found that the basic calponin interacted with brain tubulin under polymerized and unpolymerized conditions in vitro [Fujii, T., Hiromori, T., Hamamoto, M., and Suzuki, T. (1997) J. Biochem. 122, 344-351]. We examined the calponin-binding site on the tubulin molecule by sedimentation, limited digestion, chemical-cross linking, immunoblotting, and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometric (DE MALDI-TOF) analyses. Calponin interacts with both the alpha and beta tubulins and only slightly with the tyrosinated and acetylated form of alpha tubulin. The binding of calponin to microtubules was blocked by adding poly(L-aspartic acid) (PLAA) or MAP2. After digestion of microtubule proteins with subtilisin, the amount of calponin binding to alphabetas microtubules was reduced compared to native microtubules, but no further reduction was observed in the case of alphasbetas microtubules. The chemical cross-linked products of calponin and synthesized peptides (KDYEEVGVDSVEGE; alpha-KE) derived from the C-terminal region of alpha tubulin and (YQQYQDATADEQG; beta-YG) and (GEFEEEGEEDEA; beta-GA) from that of beta tubulin were detected by mass spectrometry. One kind of calponin-peptide complex was formed in the presence of alpha-KE or beta-YG, while five complexes (calponin:peptide = 1:1-5) were generated in the presence of beta-GA. Peptides alpha-KE and beta-GA inhibited the binding of calponin to tubulin produced by EDC in a concentration-dependent manner. These findings suggest that basic calponin interacts with both tubulin subunits and that their C-terminal regions, which also contain the binding sites of MAP2, tau, and kinesin, may be involved in calponin-binding.