Potential sex differences in activation of pain-related brain regions in nonhuman primates with a unilateral spinal nerve ligation.

The lack of truly robust analgesics for chronic pain is owed, in part, to the lack of an animal model that reflects the clinical pain state and of a mechanism-based, objective neurological indicator of pain. The present study examined stimulus-evoked brain activation with functional magnetic resonance imaging in male and female cynomolgus macaques following unilateral L7 spinal nerve ligation and the effects of clinical analgesics pregabalin, duloxetine, and morphine on brain activation in these macaques. A modified straight leg raise test was used to assess pain severity in awake animals and to evoke regional brain activation in anesthetized animals. The potential effects of clinical analgesics on both awake pain behavior and regional brain activation were examined. Following spinal nerve ligation, both male and female macaques showed significantly decreased ipsilateral straight leg raise thresholds, suggesting the presence of radicular-like pain. Morphine treatment increased straight leg raise thresholds in both males and females whereas duloxetine and pregabalin did not. In male macaques, the ipsilateral straight leg raise activated contralateral insular and somatosensory cortex (Ins/SII), and thalamus. In female macaques, the ipsilateral leg raise activated cingulate cortex and contralateral insular and somatosensory cortex. Straight leg raises of the contralateral, unligated leg did not evoke brain activation. Morphine reduced activation in all brain regions in both male and female macaques. In males, neither pregabalin nor duloxetine decreased brain activation compared with vehicle treatment. In females, however, pregabalin and duloxetine decreased the activation of cingulate cortex compared with vehicle treatment. The current findings suggest a differential activation of brain areas depending on sex following a peripheral nerve injury. Differential brain activation observed in this study could underlie qualitative sexual dimorphism in clinical chronic pain perception and responses to analgesics. Future pain management approaches for neuropathic pain will need to consider potential sex differences in pain mechanism and treatment efficacy.

Regional brain activation during rectal distention and attenuation with alosetron in a nonhuman primate model of irritable bowel syndrome.

Greater understanding of the mechanism that mediates visceral pain and hypersensitivity associated with irritable bowel syndrome (IBS) would facilitate the development of effective therapeutics to manage these symptoms. An objective marker associated with the underlying mechanisms of visceral pain and hypersensitivity could be used to guide therapeutic development. The current study examined brain activation evoked by rectal distention with functional magnetic resonance imaging (fMRI) in a cynomolgus macaque model of visceral hypersensitivity. Male, cynomolgus macaques underwent five four-week treatments of dextran sodium sulfate (DSS)-distilled water (DW), which induced mild-moderate colitis with remission during each treatment cycle. Balloon rectal distention (RD) was performed under anesthesia 14 weeks after the final DSS-DW treatment. Colonoscopy confirmed the absence of colitis prior to the start of RD. In naïve, untreated macaques, 10, 20 and 30 ml RD did not evoke brain activation. However, insular cortex/somatosensory II cortex and cerebellum were significantly activated in DSS-treated macaques at 20 and 30 ml rectal distention. Intra-rectal pressure after DSS treatment was not significantly different from that of naïve, untreated macaques, indicating lack of alteration of rectal functioning following DSS-treatment. Treatment with 5-HT3 receptor antagonist alosetron (p.o.) reduced distension-evoked brain activation and decreased intra-rectal pressure. The current findings demonstrated activation of brain regions to RD following DSS treatments which was not present in naïve macaques, suggesting visceral hypersensitivity. Brain activation in turn was reduced by alosetron, which could underlie the analgesic effect alosetron in IBS patients.

Predicting Total Drug Clearance and Volumes of Distribution Using the Machine Learning-Mediated Multimodal Method through the Imputation of Various Nonclinical Data.

Pharmacokinetic research plays an important role in the development of new drugs. Accurate predictions of human pharmacokinetic parameters are essential for the success of clinical trials. Clearance (CL) and volume of distribution (Vd) are important factors for evaluating pharmacokinetic properties, and many previous studies have attempted to use computational methods to extrapolate these values from nonclinical laboratory animal models to human subjects. However, it is difficult to obtain sufficient, comprehensive experimental data from these animal models, and many studies are missing critical values. This means that studies using nonclinical data as explanatory variables can only apply a small number of compounds to their model training. In this study, we perform missing-value imputation and feature selection on nonclinical data to increase the number of training compounds and nonclinical datasets available for these kinds of studies. We could obtain novel models for total body clearance (CLtot) and steady-state Vd (Vdss) (CLtot: geometric mean fold error [GMFE], 1.92; percentage within 2-fold error, 66.5%; Vdss: GMFE, 1.64; percentage within 2-fold error, 71.1%). These accuracies were comparable to the conventional animal scale-up models. Then, this method differs from animal scale-up methods because it does not require animal experiments, which continue to become more strictly regulated as time passes.

Prediction of Total Drug Clearance in Humans Using Animal Data: Proposal of a Multimodal Learning Method Based on Deep Learning.

Research into pharmacokinetics plays an important role in the development process of new drugs. Accurately predicting human pharmacokinetic parameters from preclinical data can increase the success rate of clinical trials. Since clearance (CL) which indicates the capacity of the entire body to process a drug is one of the most important parameters, many methods have been developed. However, there are still rooms to be improved for practical use in drug discovery research; "improving CL prediction accuracy" and "understanding the chemical structure of compounds in terms of pharmacokinetics". To improve those, this research proposes a multimodal learning method based on deep learning that takes not only the chemical structure of a drug but also rat CL as inputs. Good results were obtained compared with the conventional animal scale-up method; the geometric mean fold error was 2.68 and the proportion of compounds with prediction errors of 2-fold or less was 48.5%. Furthermore, it was found to be possible to infer the partial structure useful for CL prediction by a structure contributing factor inference method. The validity of these results of structural interpretation of metabolic stability was confirmed by chemists.

Characterization of Aspergillus oryzae glycoside hydrolase family 43 beta-xylosidase expressed in Escherichia coli.

This is the first report of glycoside hydrolase family 43 beta-xylosidase from Aspergillus oryzae. To characterize this enzyme, the recombinant enzyme was expressed in Escherichia coli. Unlike known beta-xylosidases from fungal origins, the enzyme did not show substrate ambiguity and was stable at alkaline pH.

Deletion analysis of the promoter of Aspergillus oryzae gene encoding heat shock protein 30.

In order to find a promoter that could be influenced by temperature shift, we explored and isolated an Aspergillus oryzae gene expressed at high temperatures (37-42 degrees C) by the cDNA subtraction method. Of the 96 cDNA clones isolated from the subtraction library, one cDNA clone showed 73% identity with Aspergillus nidulans heat shock protein 30 (hsp30). Based on this, we designated the isolated gene hsp30 of A. oryzae. A. oryzae hsp30 was weakly expressed at 30 degrees C, but strongly at 40 degrees C. We showed that the promoter of this hsp30 induced heterologous gene expression at high temperatures using beta-glucuronidase (GUS) gene as a reporter. Regarding elucidation of the region essential for heat shock response, we showed that the minimum length of the promoter region that was essential for heat shock response was located between -388 and -272 (+1 indicated the first position of the translation initiation codon) of the hsp30 promoter. This promoter region harbors several putative transcription factor binding sites, including heat shock elements (HSEs), a CCAAT box, and a TATA box. Furthermore, site-directed mutagenesis of this promoter revealed that HSE1 (aTTCgtcGAAacgcccaGAAa) and HSE2 (cGAAagTTCtcGACg), located between -342 and -272 of the hsp30 promoter, were its cis-acting elements for heat shock response.

A novel transformation system using a bleomycin resistance marker with chemosensitizers for Aspergillus oryzae.

Aspergillus oryzae is resistant to many kinds of antibiotics, which hampers their use to select transformants. In fact, the fungus is resistant to over 200microg/ml of bleomycin (Bm). By enhancing the susceptibility of A. oryzae to Bm using Triton X-100 as a detergent and an ATP-binding cassette (ABC) pump inhibitor, chlorpromazine, to the growing medium, we established a novel transformation system by Bm selection for A. oryzae. In a medium containing these reagents, A. oryzae showed little growth even in the presence of 30microg Bm/ml. Based on these findings, we constructed a Bm-resistance expression cassette (BmR), in which blmB encoding Bm N-acetyltransferase from Bm-producing Streptomyces verticillus was expressed under the control of a fungal promoter. We obtained a gene knockout mutant efficiently by Bm selection, i.e., the chromosomal ligD coding region was successfully replaced by BmR using ligD disruption cassette consisted of ligD flanking sequence and BmR through homologous recombination.

Role of BK channels in testosterone-induced relaxation of the aorta in spontaneously hypertensive rats.

The previous data indicated that the testosterone (Tes)-induced relaxation of thoracic aorta is greater in spontaneously hypertensive rats (SHR) than in normotensive rats (Wistar-Kyoto rats; WKY) and that there were differences between SHR and WKY in the functions of K(ATP), K(v), and K(Ca) channels. The present study was carried out to ascertain the mechanisms of the Tes-induced relaxation. Indomethacin (30 muM) pretreatment suppressed the Tes-induced relaxation. Following noradrenalin (NA)-induced vasoconstriction, the relaxation induced by Tes was significantly attenuated by endothelium removal in SHR (not in WKY), but the dilatory effect of Tes following KCl-induced vasoconstriction was not attenuated by endothelium removal. After tetraethylammonium (K(Ca) channel inhibitor) or iberiotoxin (large conductance, Ca(2+) activated BK channel inhibitor) pretreatment, the Tes-induced relaxation was attenuated in SHR, but not in WKY. This attenuation in SHR was not observed after endothelium removal. The above results suggest that the relaxation induced by Tes following NA-induced vasoconstriction in SHR results from hyperpolarization due to BK channel opening.

Effect of insulin-like growth factor-binding protein 7 on steroidogenesis in granulosa cells derived from equine chorionic gonadotropin-primed immature rat ovaries.

Insulin-like growth factor (IGF)-binding protein (IGFBP) 7 is a secreted protein that regulates cellular proliferation, adhesion, and angiogenesis, and has low affinity for IGF compared with that of IGFBP1-IGFBP6. We sought to determine whether IGFBP7 is present in follicular fluid and to elucidate whether IGFBP7 participates in the steroidogenesis of rat mature follicles. Follicular fluid and granulosa cells (GCs) were collected from immature rats 2 days after their treatment with equine chorionic gonadotropin (eCG). IGFBP7 protein was detected in the follicular fluid and the conditioned medium of cultured ovarian GCs by immunoblot analysis. When subconfluent GCs were cultured and treated with FSH and activin, coincubation with FSH and activin markedly increased GC expression of Cyp19a1 (aromatase) mRNA and 17beta-estradiol (E(2)) secretion. The addition of recombinant murine IGFBP7 to these cultures decreased in the activin-enhanced, FSH-stimulated Cyp19a1 mRNA levels in the cells and suppressed the 17beta-E(2) levels in the culture medium. Treatment of GCs with Igfbp7-specific small interfering RNA (siRNA), which knocked down Igfbp7 expression, increased the FSH-stimulated levels of Cyp19a1 but not Cyp11a1 expression. Basal and FSH-stimulated 17beta-E(2) secretion into the culture medium was also enhanced by Igfbp7 siRNA. These results suggest that IGFBP7 suppresses estrogen production in GCs. These observations support the notion that this protein, which is secreted into the follicular fluid, may serve as an intraovarian factor that negatively regulates GC differentiation.

Prevention of aortic calcification by etidronate in the renal failure rat model.

Our recent clinical study indicated that etidronate may inhibit the progression of aortic calcification in hemodialysis patients. To determine whether etidronate inhibits aortic calcification in renal failure rats, renal failure was induced by subtotal nephrectomy, in which 5/6 of the kidneys were removed. Significant increases in serum creatinine levels were observed 2 weeks after the operation, at which point treatment with etidronate and calcitriol was initiated. Etidronate at 5 or 10 mg/kg significantly reduced the thoracic and abdominal aortic calcification induced by calcitriol. It also reduced the dysfunction in aortic contraction. The elevation of bone metabolism and reduction of bone mineral density observed in the nephrectomized rats were not affected by treatment with 5 mg/kg etidronate. No changes in serum Ca and the product of Ca and P levels were observed between the non etidronate-treated group and the 5 mg/kg etidronate-treated group. Moreover, the reduction in the aortic expression of matrix Gla protein mRNA observed in nephrectomized rats was reversed by 5 mg/kg etidronate. These results show that etidronate at concentrations that do not affect the bone mineral density inhibits aortic calcification and recovers vascular dysfunction in renal failure rats.

Studies on the mechanisms underlying beta-adrenoceptor-mediated relaxation of rat abdominal aorta.

Mechanisms underlying beta-adrenoceptor (beta-AR)-mediated vascular relaxation were studied in the isolated rat abdominal aorta. In the endothelium-denuded helical preparations, a non-selective beta-AR agonist isoprenaline elicited a concentration-dependent relaxation. In the absence of beta-AR antagonists, isoprenaline-induced relaxation was not practically affected by an adenylyl cyclase inhibitor SQ 22,536 (300 microM), but was strongly diminished by high-KCl (80 mM). Isoprenaline-induced relaxation in the presence of SQ 22,536 was significantly diminished by iberiotoxin (IbTx, 0.1 microM), but was not affected by 4-aminopyridine (4-AP, 3 mM). Isoprenaline-induced relaxation was not also affected by SQ 22,536 (300 microM) even in the presence of CGP20712A (a beta(1)-selective antagonist) and ICI-118,551 (a beta(2)-selective antagonist) (0.1 microM for each), but was strongly diminished by high-KCl. By contrast, SQ 22,536-resistant, isoprenaline-induced relaxation in the presence of CGP20712A plus ICI-118,551 was not affected by IbTx (0.1 microM), but was inhibited significantly by 4-AP (3 mM). These results suggest that in rat abdominal aortic smooth muscle: 1) both beta(1)-/beta(2)-AR- and beta(3)-AR-mediated relaxations substantially involve cAMP-independent mechanisms; 2) beta(1)-/beta(2)-AR-mediated, cAMP-independent relaxant mechanisms are partly attributed to the large-conductance, Ca (2+)-sensitive K(+) (MaxiK, BK) channel whereas beta(3)-AR-mediated relaxant mechanisms are attributed to K(v) channel.

Effect of troglitazone on the excess testosterone and LH secretion in thyroidectomized, insulin-resistant, type 2 diabetic Goto-Kakizaki rats.

Our previous study suggested that hypothyroidism in Goto-Kakizaki (GK) rats with insulin resistance and type 2 diabetes elevates their serum testosterone and luteinizing hormone (LH) levels and ovarian LH receptor messenger RNA (mRNA) expression. The present study assessed the effects of troglitazone (Tro), an insulin-sensitizing agent, on these hypothyroidism-induced hormonal changes in GK rats. GK and normal (Wistar strain) female rats were thyroidectomized (Tx) and then injected with 5 IU of equine chorionic gonadotropin (eCG) for 5 d starting 1 wk after thyroidectomy (the control groups). In the test groups, Tx GK and Wistar rats were injected with both eCG and Tro (100 mg kg-1) po for 5 d. Tro treatment had no effect on the elevated LH serum levels in eCG-treated Tx GK rats but suppressed their enhanced serum testosterone levels as well as significantly decreasing their LH receptor mRNA expression. Tro lowered testosterone and LH receptor mRNA levels in cultured theca cells. These results indicate that Tro lowers the elevated testosterone secretion and ovarian LH receptor mRNA expression that is induced in GK rats by Tx and gonadotropin treatment, which suggests that insulin resistance may be involved in enhancing testosterone production and LH receptor expression in the ovary.

Changes in ovarian steroidogenesis in insulin-resistant, type 2 diabetic Goto-Kakizaki rats after thyroidectomy and gonadotropin treatment.

The present study used thyroidectomized insulin-resistant, type 2 diabetic Goto-Kakizaki (GK) rats to assess whether insulin resistance and hypothyroidism modulate ovarian physiology. Animals were treated with daily injections of 5 IU equine chorionic gonadotropin for 5 days starting 1 week after thyroidectomy. Control groups included rats of GK and control (Wistar) strains treated only with equine chorionic gonadotropin or thyroidectomy, or with no treatment (intact). In Wistar rats, equine chorionic gonadotropin injections tended to increase the serum concentrations of luteinizing hormone (LH) and testosterone more in the thyroidectomy group than in intact rats. Similar changes in LH and testosterone were observed in the thyroidectomy + equine chorionic gonadotropin and equine chorionic gonadotropin groups of GK rats, but the LH and testosterone levels in the thyroidectomy + equine chorionic gonadotropin group were significantly higher in GK rats. Expression of ovarian LH receptor messenger RNA (mRNA) was enhanced by thyroidectomy. The LH receptor mRNA levels were significantly higher in the thyroidectomy+equine chorionic gonadotropin group of GK rats than in the corresponding group of control rats. These results indicate that hypothyroidism in animals with insulin resistance and type 2 diabetes promotes LH and testosterone secretions, and suggests that the enhanced-testosterone levels is partially mediated by the enhancement of LH receptor expression and an increase in the serum level of LH.

Characterization of beta 3-adrenoceptor-mediated relaxation in rat abdominal aorta smooth muscle.

The present study was carried out to characterize beta-adrenoceptor subtypes mediating relaxation of rat abdominal aorta smooth muscle. (-)-Isoprenaline and a nonconventional beta(3)-adrenoceptor agonist, (+/-)-[4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride ((+/-)-CGP12177A), induced concentration-dependent relaxation of (-)-phenylephrine (0.3 microM) preconstricted spiral preparations. Pretreatment with a combination of (+/-)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate (CGP20712A, a selective beta(1)-adrenoceptor antagonist) and (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride (ICI-118,5511, a selective beta(2)-adrenoceptor antagonist) (0.1 microM for each) produced a 14-fold rightward shift of the concentration-response curve for (-)-isoprenaline; however, the relaxation in response to (+/-)-CGP12177A was unaffected by the blockade of beta(1)- and beta(2)-adrenoceptors. In the presence of CGP20712A and ICI-118,551 (0.1 microM for each), the concentration-response curves for (-)-isoprenaline and (+/-)-CGP12177A were shifted to the right by a nonselective beta(1)-, beta(2)- and beta(3)-adrenoceptor antagonist, (+/-)-bupranolol (3 and 10 microM). These results clearly suggest that beta(3)-adrenoceptors are involved in beta-adrenoceptor-mediated relaxation of rat abdominal aorta smooth muscle.

Characterization of Porphyrobacter sanguineus sp. nov., an aerobic bacteriochlorophyll-containing bacterium capable of degrading biphenyl and dibenzofuran.

Three strains of " Agrobacterium sanguineum", an aerobic marine bacterial species described previously, were re-characterized from phylogenetic and taxonomic viewpoints. 16S rDNA sequence comparisons showed that the " A. sanguineum" strains belong to the alpha-4 subgroup of alpha-Proteobacteria, with members of the genera Erythromicrobium and Porphyrobacter as their closest relatives. DNA-DNA hybridization studies indicated that the " A. sanguineum" strains were distinguishable from any previously known species of these genera. Bacteriochlorophyll a, monosaccharide-type glycosphingolipids, 2-OH fatty acids of C14:0, C15:0, C16:0, and C16:1, and ubiquinone-10 were detected in the " A. sanguineum" strains. The G+C of the DNA was 63.8-64.0 mol%. Two of the " A. sanguineum" strains, IAM 12620 (=ATCC 25659) and ATCC 25661, were able to grow with biphenyl and dibenzofuran as sole carbon source in the presence of 0.05% yeast extract. The medium in these cultures turned yellowish-orange at the exponential phase of growth due to the release of soluble chromogenic metabolites. The remaining " A. sanguineum" strain, ATCC 25660, and all test strains of Erythromicrobium and Porphyrobacter neither grew nor produced yellow-orange pigment with biphenyl or dibenzofuran. In PCR experiments, bphA1 gene, coding for the large subunit protein of biphenyl dioxygenase, was detected in " A. sanguineum" IAM 12620 and ATCC 25661. Based on these results, we propose classifying " A. sanguineum" IAM 12620 and ATCC 25661 as a new species of the genus Porphyrobacter with the name Porphyrobacter sanguineus sp. nov.

Nitric oxide (NO) primarily accounts for endothelium-dependent component of beta-adrenoceptor-activated smooth muscle relaxation of mouse aorta in response to isoprenaline.

Isoprenaline is known to produce vascular relaxation through activation of beta-adrenoceptors. In recent years, beta-adrenoceptor-activated vascular relaxation has been the focus of pharmacological study in terms of both the receptor subtypes and the intracellular signaling mechanisms which trigger smooth muscle mechanical functions. In addition, the possible contribution of the endothelium to beta-adrenoceptor-activated relaxation of vascular beds has provoked considerable discussion, with consensus still to be established. In the present study, we examined the effects of isoprenaline on isolated mouse aortic smooth muscles to determine whether the presence of the endothelium plays a substantial role in the relaxation it produces. A possible role for nitric oxide (NO) as a primary endothelium-derived factor released in response to isoprenaline was also elucidated pharmaco-mechanically. In isolated thoracic and abdominal aortae pre-contracted with phenylephrine (3 x 10(-7)-10(-6) M), isoprenaline elicited relaxation in a concentration-dependent fashion (10(-9)-10(-5) M). In endothelium-denuded preparations, isoprenaline-elicited relaxation was reduced to 40-50% of the response obtained in endothelium-intact preparations. In the preparations treated with N(G)-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-4) M; an NO synthase inhibitor) or 1H-[1,2,4]-oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ, 10(-5) M; a soluble guanylyl cyclase inhibitor), isoprenaline-elicited relaxation was attenuated almost to the same degree as the response in endothelium-denuded preparations. The degree of endothelium-dependency in isoprenaline-elicited relaxation was largely diminished when treated with propranolol (3 x 10(-6) M). The present findings indicate that isoprenaline substantially relaxes the mouse aorta with both endothelium-dependent and -independent mechanisms. The endothelium-dependent component seems to correspond to about 50% of the isoprenaline-elicited relaxation, and is almost entirely due to endothelium-derived NO. Activation of propranolol (3 x 10(-6) M)-inhibitable beta-adrenoceptors seems to be primarily responsible for the NO-mediated endothelium-dependent pathway in isoprenaline-elicited relaxant response of mouse aorta.