Spectroscopy and a high-resolution crystal structure of Tyr263 mutants of cyanobacterial phytochrome Cph1

Phytochromes are biliprotein photoreceptors that can be photoswitched between red-light-absorbing state (Pr) and far-red-light-absorbing state (Pfr). Although three-dimensional structures of both states have been reported, the photoconversion and intramolecular signaling mechanisms are still unclear. Here, we report UV-Vis absorbance, fluorescence and CD spectroscopy along with various photochemical parameters of the wild type and Y263F, Y263H and Y263S mutants of the Cph1 photosensory module, as well as a 2.0-Å-resolution crystal structure of the Y263F mutant in its Pr ground state. Although Y263 is conserved, we show that the aromatic character but not the hydroxyl group of Y263 is important for Pfr formation. The crystal structure of the Y263F mutant (Protein Data Bank ID: 3ZQ5) reaffirms the ZZZssa chromophore configuration and provides a detailed picture of its binding pocket, particularly conformational heterogeneity around the chromophore. Comparison with other phytochrome structures reveals differences in the relative position of the PHY (phytochrome specific) domain and the interaction of the tongue with the extreme N-terminus. Our data support the notion that native phytochromes in their Pr state are structurally heterogeneous.     

Crystallographic and Electron Microscopic Analyses of a Bacterial Phytochrome Reveal Local and Global Rearrangements during Photoconversion

Phytochromes are multidomain photoswitches that drive light perception in plants and microorganisms by coupling photoreversible isomerization of their bilin chromophore to various signaling cascades. How changes in bilin conformation affect output by these photoreceptors remains poorly resolved and might include several species-specific routes. Here, we present detailed three-dimensional models of the photosensing module and a picture of an entire dimeric photoreceptor through structural analysis of the Deinococcus radiodurans phytochrome BphP assembled with biliverdin (BV). A 1.16-Å resolution crystal structure of the bilin-binding pocket in the dark-adapted red light-absorbing state illuminated the intricate network of bilin/protein/water interactions and confirmed the protonation and ZZZssa conformation of BV. Structural and spectroscopic comparisons with the photochemically compromised D207A mutant revealed that substitutions of Asp-207 allow inclusion of cyclic porphyrins in addition to BV. A crystal structure of the entire photosensing module showed a head-to-head, twisted dimeric arrangement with bowed helical spines and a hairpin protrusion connecting the cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) and phytochrome-specific (PHY) domains. A key conserved hairpin feature is its anti-parallel, two β-strand stem, which we show by mutagenesis to be critical for BphP photochemistry. Comparisons of single particle electron microscopic images of the full-length BphP dimer in the red light-absorbing state and the photoactivated far-red light-absorbing state revealed a large scale reorientation of the PHY domain relative to the GAF domain, which alters the position of the downstream histidine kinase output module. Together, our data support a toggle model whereby bilin photoisomerization alters GAF/PHY domain interactions through conformational modification of the hairpin, which regulates signaling by impacting the relationship between sister output modules.     

PHY34 inhibits autophagy through V-ATPase V0A2 subunit inhibition and CAS/CSE1L nuclear cargo trafficking in high grade serous ovarian cancer

PHY34 is a synthetic small molecule, inspired by a compound naturally occurring in tropical plants of the Phyllanthus genus. PHY34 was developed to have potent in vitro and in vivo anticancer activity against high grade serous ovarian cancer (HGSOC) cells. Mechanistically, PHY34 induced apoptosis in ovarian cancer cells by late-stage autophagy inhibition. Furthermore, PHY34 significantly reduced tumor burden in a xenograft model of ovarian cancer. In order to identify its molecular target/s, we undertook an unbiased approach utilizing mass spectrometry-based chemoproteomics. Protein targets from the nucleocytoplasmic transport pathway were identified from the pulldown assay with the cellular apoptosis susceptibility (CAS) protein, also known as CSE1L, representing a likely candidate protein. A tumor microarray confirmed data from mRNA expression data in public databases that CAS expression was elevated in HGSOC and correlated with worse clinical outcomes. Overexpression of CAS reduced PHY34 induced apoptosis in ovarian cancer cells based on PARP cleavage and Annexin V staining. Compounds with a diphyllin structure similar to PHY34 have been shown to inhibit the ATP6V0A2 subunit of V(vacuolar)-ATPase. Therefore, ATP6V0A2 wild-type and ATP6V0A2 V823 mutant cell lines were tested with PHY34, and it was able to induce cell death in the wild-type at 246 pM while the mutant cells were resistant up to 55.46 nM. Overall, our data demonstrate that PHY34 is a promising small molecule for cancer therapy that targets the ATP6V0A2 subunit to induce autophagy inhibition while interacting with CAS and altering nuclear localization of proteins.Subject terms: Drug development, Target identification     

Phototransformation of the Red Light Sensor Cyanobacterial Phytochrome 2 from Synechocystis Species Depends on Its Tongue Motifs

Phytochromes are photoreceptors using a bilin tetrapyrrole as chromophore, which switch in canonical phytochromes between red (P_r) and far red (P_fr) light-absorbing states. Cph2 from Synechocystis sp., a noncanonical phytochrome, harbors besides a cyanobacteriochrome domain a second photosensory module, a P_r/P_fr-interconverting GAF-GAF bidomain (SynCph2(1-2)). As in the canonical phytochromes, a unique motif of the second GAF domain, the tongue region, seals the bilin-binding site in the GAF1 domain from solvent access. Time-resolved spectroscopy of the SynCph2(1-2) module shows four intermediates during P_r → P_fr phototransformation and three intermediates during P_fr → P_r back-conversion. A mutation in the tongue''s conserved PRXSF motif, S385A, affects the formation of late intermediate R3 and of a P_fr-like state but not the back-conversion to P_r via a lumi-F-like state. In contrast, a mutation in the likewise conserved WXE motif, W389A, changes the photocycle at intermediate R2 and causes an alternative red light-adapted state. Here, back-conversion to P_r proceeds via intermediates differing from SynCph2(1-2). Replacement of this tryptophan that is ∼15 Å distant from the chromophore by another aromatic amino acid, W389F, restores native P_r → P_fr phototransformation. These results indicate large scale conformational changes within the tongue region of GAF2 during the final processes of phototransformation. We propose that in early intermediates only the chromophore and its nearest surroundings are altered, whereas late changes during R2 formation depend on the distant WXE motifs of the tongue region. Ser-385 within the PRXSF motif affects only late intermediate R3, when refolding of the tongue and docking to the GAF1 domain are almost completed.     

Enhanced modulation of gut microbial dynamics affecting body weight in birds triggered by natural growth promoters administered in conventional feed

This study explored the effects of natural growth promoters (phytogenic feed additives and organic acids) on animal performance, carcass characteristics, blood parameters, gut microflora composition, and microbe–host interactions in broiler chickens over a 42-day feeding period. Two-hundred-fifty-day-old chicks were randomly assigned to one of five treatments: (i) control diets (CON); (ii) control diets + 40 g/tons antibiotic growth promoter (AB); (iii) control diets + 3 kg/tons organic acids (ORG); (iv) control diets + 3 kg/tons phytogenic feed additives (PHY); (v) control diets + 3 kg/tons organic acids + phytogenic feed additive combination (COM). A non-significant differences (p > 0.05) were observed in broiler performance among treatments at 21 days of age; however, a gradually increasing body weight gain and reduced feed conversion ratio were observed at 42 days in treatments versus control group. Biochemical indices were non-significant (p > 0.05) except for decreased cholesterol (p < 0.05) and increased A/G ratio (p < 0.05) recorded in the treatment groups. The addition of PHY and ORG improved total counts of Enterococcus spp. and Lactobacillus spp. (p < 0.05) as well as reduced caecal and ileal Campylobacter spp. and Escherichia coli (p < 0.05). Correlation analysis elucidated beneficial bacteria (Enterococcus spp. and Lactobacillus spp.) were positively and pathogenic bacteria (Campylobacter spp. and E. coli) were negatively correlated (p < 0.05) with host weight gain. The findings indicated that dietary supplementation of PHY and ORG sustained balanced gut microflora, which in turn improved body weight. This study broadens the significance of using PHY and ORG as safe alternatives to antibiotic growth promoters for achieving healthier and economical broiler production.     

Gross anatomical features of the tongue, lingual skeleton and laryngeal mound of Rhea americana (Palaeognathae, Aves): morpho-functional considerations

The tongue body of Rhea americana is triangular and partially pigmented with each caudo-lateral margin displaying a round, sub-divided lingual papilla. The tongue root is a smooth, non-pigmented tract of mucosa. The tongue body is supported by the paraglossum and distal half of the rostral projection of the basihyal (RPB), and the tongue root by the proximal half of the RPB, body of the basihyal and proximal ceratobranchials. An urohyal is absent; however, peculiar to R. americana, the caudal margin of the cricoid body displays a median projection, which may represent the remnant of the urohyal incorporated into the cricoid. The laryngeal mound is less elevated, the arytenoid cartilages are smaller than in other ratites, and the caudal margin displays pharyngeal papillae that vary in shape and number. The unique morphology of the lingual skeleton and its positioning within the tongue of R. americana, the rostral insertion of the M. ceratoglossus, the absence of the urohyal (enhanced ventroflexion) and the caudal positioning and mobile attachment of the ensheathed basihyal to the paraglossum would appear to allow independent movement of the tongue body relative to the hyobranchial apparatus. Additionally, the deeply indented base and rostral oval opening in the paraglossum limits the length of cartilage present in the midline of the tongue body. This may allow the tongue the necessary flexibility for the lingual papillae to clean the choana. The cleaning action of the tongue would occur simultaneously with the previously described role of this organ and associated structures during feeding. Thus, the so-called reduced, ancestral tongue of R. americana may be structurally and functionally more complex than previously believed.     

Medically Unexplained and Explained Physical Symptoms in the General Population: Association with Prevalent and Incident Mental Disorders

Background

Clinical studies have shown that Medically Unexplained Symptoms (MUS) are related to common mental disorders. It is unknown how often common mental disorders occur in subjects who have explained physical symptoms (PHY), MUS or both, in the general population, what the incidence rates are, and whether there is a difference between PHY and MUS in this respect.

Aim

To study the prevalence and incidence rates of mood, anxiety and substance use disorders in groups with PHY, MUS and combined MUS and PHY compared to a no-symptoms reference group in the general population.

Method

Data were derived from the Netherlands Mental Health Survey and Incidence Study-2 (NEMESIS-2), a nationally representative face-to-face survey of the general population aged 18-64 years. We selected subjects with explained physical symptoms only (n=1952), with MUS only (n=177), with both MUS and PHY (n=209), and a reference group with no physical symptoms (n=4168). The assessment of common mental disorders was through the Composite International Diagnostic Interview 3.0. Multivariate logistic regression analyses were used to examine the association between group membership and the prevalence and first-incidence rates of comorbid mental disorders, adjusted for socio-demographic characteristics.

Results

MUS were associated with the highest prevalence rates of mood and anxiety disorders, and combined MUS and PHY with the highest prevalence rates of substance disorder. Combined MUS and PHY were associated with a higher incidence rate of mood disorder only (OR 2.9 (95%CI:1.27,6.74)).

Conclusion

In the general population, PHY, MUS and the combination of both are related to mood and anxiety disorder, but odds are highest for combined MUS and PHY in relation to substance use disorder. Combined MUS and PHY are related to a greater incidence of mood disorder. These findings warrant further research into possibilities to improve recognition and early intervention in subjects with combined MUS and PHY.     

Light-induced Changes in the Dimerization Interface of Bacteriophytochromes

Phytochromes are dimeric photoreceptor proteins that sense red light levels in plants, fungi, and bacteria. The proteins are structurally divided into a light-sensing photosensory module consisting of PAS, GAF, and PHY domains and a signaling output module, which in bacteriophytochromes typically is a histidine kinase (HK) domain. Existing structural data suggest that two dimerization interfaces exist between the GAF and HK domains, but their functional roles remain unclear. Using mutational, biochemical, and computational analyses of the Deinococcus radiodurans phytochrome, we demonstrate that two dimerization interfaces between sister GAF and HK domains stabilize the dimer with approximately equal contributions. The existence of both dimerization interfaces is critical for thermal reversion back to the resting state. We also find that a mutant in which the interactions between the GAF domains were removed monomerizes under red light. This implies that the interactions between the HK domains are significantly altered by photoconversion. The results suggest functional importance of the dimerization interfaces in bacteriophytochromes.     

Tongue Volume Influences Lowest Oxygen Saturation but Not Apnea-Hypopnea Index in Obstructive Sleep Apnea

Objectives

The aim of this study was to identify correlations between sleep apnea severity and tongue volume or posterior airway space measured via three-dimensional reconstruction of volumetric computerized tomography (CT) images in patients with obstructive sleep apnea (OSA) for use in predicting OSA severity and in surgical treatment. We also assessed associations between tongue volume and Mallampati score.

Methods

Snoring/OSA male patients (n = 64) who underwent polysomnography, cephalometry, and CT scans were enrolled in this retrospective study. OSA was diagnosed when the apnea-hypopnea index (AHI) was greater than 5 (mild 5–14; moderate 15–29; severe>30). The patients were also categorized into the normal-mild group (n = 22) and the moderate-severe group (n = 42). Using volumetric CT images with the three-dimensional reconstruction technique, the volume of the tongue, posterior airway space volume, and intra-mandibular space were measured. The volumes, polysomnographic parameters, and physical examination findings were compared, and independent factors that are related to OSA were analysed.

Results

No associations between tongue volume or posterior airway space and the AHI were observed. However, multivariate linear analyses showed that tongue volume had significantly negative association with lowest O₂ saturation (r = 0.365, p = 0.027). High BMI was related to an increase in tongue volume. Modified Mallampati scores showed borderline significant positive correlations with absolute tongue volume (r = 0.251, p = 0.046) and standardized tongue volume (absolute tongue volume / intramandibular area; r = 0.266, p = 0.034). Between the normal-mild and moderate-severe groups, absolute tongue volumes were not different, although the standardized tongue volume in the moderate-severe group was significantly higher.

Conclusion

Absolute tongue volume showed stronger associations with lowest O₂ saturation during sleep than with the severity of AHI. We also found that high BMI was a relevant factor for an increase in absolute tongue volume and modified Mallampati grading was a useful physical examination to predict tongue size.     

Performance and total-tract digestibility responses to exogenous xylanase and phytase in diets for growing pigs

Sixty individually housed male pigs (Large White X Landrace, average weight 19.7±0.56 kg) were used in a completely randomised block design having five wheat-based dietary treatments (n=12 pigs per treatment) to study the interactive effects between added xylanase (XYL) and phytase (PHY), either alone or in combination, and days on trial on coefficient of total-tract digestibility (CTTAD) and performance. The diets used were: (i) positive control (PC); (ii) negative control with reduced energy and mineral content (NC); (iii) NC with added XYL (NCX); (iv) NC with added PHY (NCP); and (v) NC with added XYL plus PHY (NCXP). The performance response and CTTAD of dietary components were measured 3 and 7 weeks after commencement of feeding, with the experiment finishing when pigs reached approximately 65 kg live weight.Pigs fed the NC diet decreased average daily gain (ADG) by 13% (P<0.01) and increased feed conversion ratio (FCR) by 11% (P<0.01) compared to pigs fed the PC diet. Addition of PHY in the NC diet increased ADG by 9% (P<0.05) and decreased FCR by 5% (P=0.065), whilst addition of XYL in the NC diet had no effect on the performance indices. Combined addition of XYL and PHY in the NC diet did not improve performance of pigs for the first 21 days but improved FCR by 8% (P=0.008) during the days 22–49. Digestibility measurements showed that combined use of XYL and PHY in the NC diet improved CTTAD of dry matter (DM, P<0.05), gross energy (GE, P<0.05) and crude protein (CP, P<0.01) only during the days 22–49. Independent supplementation of PHY in the NC diet tended to improve CTTAD of GE (P=0.09) only during the days 22–49. In contrast, supplementation of PHY immediately improved CTTAD of P and maintained it to similar levels at 49 days. Results suggested that supplementation of PHY plus XYL in combination in the NC diet improved FCR and CTTAD of DM, GE, CP and P but the beneficial effects other than CTTAD of P were derived mainly during days 22–49.     

Evidence that the phytochrome gene family in black cottonwood has one PHYA locus and two PHYB loci but lacks members of the PHYC/F and PHYE subfamilies

The phytochrome photoreceptors play important roles in the photoperiodic control of vegetative bud set, growth cessation, dormancy induction, and cold-hardiness in trees. Interestingly, ecotypic differences in photoperiodic responses are observed in many temperate- zone tree species. Northern and southern ecotypes of black cottonwood (Populus trichocarpa Torr. & Gray), for example, exhibit marked differences in the timing of short-day-induced bud set and growth cessation, and these responses are controlled by phytochrome. Therefore, as a first step toward determining the molecular genetic basis of photoperiodic ecotypes in trees, we characterized the phytochrome gene (PHY) family in black cottonwood. We recovered fragments of one PHYA and two PHYB using PCR-based cloning and by screening a genomic library. Results from Southern analyses confirmed that black cottonwood has one PHYA locus and two PHYB loci, which we arbitrarily designated PHYB1 and PHYB2. Phylogenetic analyses which included PHY from black cottonwood, Arabidopsis thaliana and tomato (Solanum lycopersicum) suggest that the PHYB/D duplications in these species occurred independently. When Southern blots were probed with PHYC, PHYE, and PHYE heterologous probes, the strongest bands that we detected were those of black cottonwood PHYA and/or PHYB. These results suggest that black cottonwood lacks members of the PHYC/F and PHYE subfamilies. Although black cottonwood could contain additional PHY that are distantly related to known angiosperm PHY, our results imply that the PHY family of black cottonwood is less complex than that of other well-characterized dicot species such as Arabidopsis and tomato. Based on Southern analyses of five black cottonwood genotypes representing three photoperiodic ecotypes, substantial polymorphism was detected for at least one of the PHYB loci but not for the PHYA locus. The novel character of the PHY family in black cottonwood, as well as the differences in polymorphism we observed between the PHYA and PHYB subfamilies, indicates that a number of fundamental macro- and microevolutionary questions remain to be answered about the PHY family in dicots.      

The PHY domain is required for conformational stability and spectral integrity of the bacteriophytochrome from Deinococcus radiodurans

Bacteriophytochrome from Deinococcus radiodurans (DrBphP) is a plant phytochrome homolog. To investigate the interaction of chromophore and protein structure, we purified recombinant DrBphP and performed biochemical analyses. Differences of apo- and holo-protein in electrophoretic properties in native gels and their susceptibility to trypsin indicate changes in both the conformation and surface topography of this protein as a result of chromophore assembly. Furthermore, proteolysis to Pr and Pfr conformers displayed distinctive cleavage patterns with a noticeable Pr-specific tryptic fragment. Of interest, a prolonged tryptic digestion showed a more severe impact upon the Pfr form. Most importantly, when we assessed the extent of dark reversion to evaluate the role of the cleaved part, a rapidly accelerated reversion was observed upon cleavage at residues 329-505 corresponding to the PHY domain. Our data thus show that the PHY domain is necessary for the Pfr stabilization and spectral integrity of DrBphP.     

Distinct physiological roles of the three [NiFe]-hydrogenase orthologs in the hyperthermophilic archaeon Thermococcus kodakarensis

Hydrogenases catalyze the reversible oxidation of molecular hydrogen (H₂) and play a key role in the energy metabolism of microorganisms in anaerobic environments. The hyperthermophilic archaeon Thermococcus kodakarensis KOD1, which assimilates organic carbon coupled with the reduction of elemental sulfur (S⁰) or H₂ generation, harbors three gene operons encoding [NiFe]-hydrogenase orthologs, namely, Hyh, Mbh, and Mbx. In order to elucidate their functions in vivo, a gene disruption mutant for each [NiFe]-hydrogenase ortholog was constructed. The Hyh-deficient mutant (PHY1) grew well under both H₂S- and H₂-evolving conditions. H₂S generation in PHY1 was equivalent to that of the host strain, and H₂ generation was higher in PHY1, suggesting that Hyh functions in the direction of H₂ uptake in T. kodakarensis under these conditions. Analyses of culture metabolites suggested that significant amounts of NADPH produced by Hyh are used for alanine production through glutamate dehydrogenase and alanine aminotransferase. On the other hand, the Mbh-deficient mutant (MHD1) showed no growth under H₂-evolving conditions. This fact, as well as the impaired H₂ generation activity in MHD1, indicated that Mbh is mainly responsible for H₂ evolution. The copresence of Hyh and Mbh raised the possibility of intraspecies H₂ transfer (i.e., H₂ evolved by Mbh is reoxidized by Hyh) in this archaeon. In contrast, the Mbx-deficient mutant (MXD1) showed a decreased growth rate only under H₂S-evolving conditions and exhibited a lower H₂S generation activity, indicating the involvement of Mbx in the S⁰ reduction process. This study provides important genetic evidence for understanding the physiological roles of hydrogenase orthologs in the Thermococcales.     

Tongue worm (subclass: Pentastomida) infection and treatment in two domesticated reptiles – A case report

Tongue worms (Subclass: Pentastomida) are endoparasites found in carnivorous reptiles, fish, amphibians, birds, and mammals. Several pentastomids cause pentastomiasis, a zoonotic disease. We encountered tongue worm infection in two reptiles imported into Japan: a Wahlberg's velvet gecko (Homopholis wahlbergii) bred in Germany and a wild-caught green tree python (Morelia viridis) from the Republic of Indonesia. The infecting tongue worms were identified as Raillietiella sp. and Armillifer sp. in the gecko and python, respectively, based on morphological and molecular analyses. Gecko is a newly recorded definitive host harboring Raillietiella tongue worm. Live tongue worms were first detected in domestic reptiles in Japan, and this indicates a risk that these tongue worms could be spread to other animals, as well as humans. Therefore, the diagnosis and treatment of infected pets are of importance. In the case of the two infected reptiles, the availability of fecal egg examination for diagnosis was shown. In addition, the effectiveness of treatment with 200 μg/kg/day ivermectin orally once weekly was demonstrated, without no side-effects and no re-detection of eggs after the cessation of treatment.     

The phytochrome gene family in tomato and the rapid differential evolution of this family in angiosperms

A reexamination of the genome of the tomato (renamed Solanum lycopersicum L.) indicates that it contains five, or at most perhaps six, phytochrome genes (PHY), each encoding a different apoprotein (PHY). Five previously identified tomato PHY genes have been designated PHYA, PHYB1, PHYB2, PHYE, and PHYF. A molecular phylogenetic analysis is consistent with the hypothesis that the angiosperm PHY family is composed of four subfamilies (A, B, C/F, and E). Southern analyses indicate that the tomato genome does not contain both a PHYC and a PHYF. Molecular phylogenetic analyses presented here, which utilize for the first time full-length PHY sequences from two completely characterized angiosperm gene families, indicate that tomato PHYF is probably an ortholog of Arabidopsis PHYC. They also confirm that the angiosperm PHY family is undergoing relatively rapid differential evolution. Assuming PHYF is an ortholog of PHYC, PHY genes in eudicots are evolving (Ka/site) at 1.52-2.79 times the rate calculated as average for other plant nuclear genes. Again assuming PHYF is an ortholog of PHYC, the rate of evolution of the C and E subfamilies is at least 1.33 times the rate of the A and B subfamilies. PHYA and PHYB in eudicots are evolving at least 1.45 times as fast as their counterparts in the Poaceae. PHY functional domains also exhibit different evolutionary rates. The C-terminal region of angiosperm PHY (codons 800-1105) is evolving at least 2.11 times as fast as the photosensory domain (codons 200-500). The central region of a domain essential for phytochrome signal transduction (codons 652-712) is also evolving rapidly. Nonsynonymous substitutions occur in this region at 2.03-3.75 times the average rate for plant nuclear genes. It is not known if this rapid evolution results from selective pressure or from the absence of evolutionary constraint.     

狐蝠科3种蝙蝠舌长度及结构比较

为探讨旧大陆食果和食蜜蝙蝠的食性类型不同是否造成其取食器官舌长度及结构的差异,本研究以2种食果蝙蝠犬蝠(Cynopterus sphinx)和棕果蝠(Rousettus leschenaultii)以及1种食蜜蝙蝠长舌果蝠(Eonycteris spelaea)为研究对象,比较了这3个物种间舌的差异.犬蝠、棕果蝠和长舌果蝠伸入直径为2 cm试管的最大舌长度L1(包括伸入试管的吻部和吻部以外的舌长)分别为(29.19±0.52)mm、(35.05±0.82) mm、(49.34±1.64) mm;伸出吻端外部的舌长L3分别为(16.25±0.53)mm、(19.25±0.79) mm、(31.88 ± 1.56) mm;与体重转换后的最大舌长度,即转换L1分别为(8.57±0.17) mm/g1/3、(7.90 ±0.27) mm/g1/3、(12.41 ±0.40) mm/g1/3;与体重转换后的伸出吻端外部的舌长,即转换L3分别为(4.77±0.16) mm/g1/3、(4.34±0.22) mm/g1/3、(8.01 ±0.38) mm/g1/3;与体重转换后的解剖舌长分别为(5.56 ±0.16) mm/g1/3、(5.35 ±0.14) mm/g1/3、(6.65±0.38)mm/g1/3.此5个参数种间比较均差异显著,食蜜类的长舌果蝠的5个参数均显著长于食果类犬蝠和棕果蝠的相应参数.通过比较3种蝙蝠的舌结构发现,长舌果蝠的舌尖尖细且具有毛刷状丝状乳头结构,舌面及两侧凹槽较多;犬蝠和棕果蝠的舌尖钝圆,舌面乳头和凹槽较少而平缓.本文结果表明,旧大陆食蜜蝙蝠与食果蝙蝠在舌长度和舌结构上存在明显差异,可能与捕食行为的差异有关.     

Human constitutive androstane receptor mediates induction of CYP2B6 gene expression by phenytoin

Compared with its rodent orthologs, little is known about the chemical specificity of human constitutive androstane receptor (hCAR) and its regulation of hepatic enzyme expression. Phenytoin (PHY), a widely used antiepileptic drug, is a potent inducer of CYP2B6 in primary human hepatocytes, but does not activate human pregnane X receptor (PXR) significantly in cell-based transfection assays at the same concentrations associated with potent induction of CYP2B6. Based on this observation, we hypothesized that PHY may be a selective activator of hCAR. In primary human hepatocytes, expression of CYP2B6 reporter genes containing phenobarbital-responsive enhancer module (PBREM) or PBREM/xenobiotic-responsive enhancer module (XREM) response elements were activated up to 14- and 28-fold, respectively, by 50 microm PHY. By contrast, parallel experiments in HepG2 cell lines co-transfected with an hPXR expression vector did not show increased reporter activity. These results indicated that a PXR-independent pathway, which is retained in primary hepatocytes, is responsible for PHY induction of CYP2B6. Further experiments revealed that PHY effectively translocates hCAR from the cytoplasm into the nucleus in both primary human hepatocytes and CAR(-/-) mice. Compared with vehicle controls, PHY administration significantly increased CYP2B6 reporter gene expression, when this reporter construct was delivered together with hCAR expression vector into CAR(-/-) mice. However, PHY did not increase reporter gene expression in CAR(-/-) mice in the absence of hCAR vector, implying that CAR is essential for mediating PHY induction of CYP2B6 gene expression. Taken together, these observations demonstrate that, in contrast to most of the known CYP2B6 inducers, PHY is a selective activator of CAR in humans.