Large-scale isolation of ESTs from medaka embryos and its application to medaka developmental genetics.

The medaka is becoming an attractive model organism for the study of vertebrate early development and organogenesis and large-scale mutagenesis projects that are aimed at creating developmentally defective mutants are now being conducted by several groups in Japan. To strengthen the study of medaka developmental genetics, we have conducted a large-scale isolation of ESTs from medaka embryos and developed tools that facilitate mutant analysis. In this study, we have characterized a total of 132,082 sequences from both ends of cloned insert cDNAs from libraries generated at different stages of medaka embryo development. Clustering analysis with 3-prime sequences finally identified a total of 12,429 clusters. As a pilot analysis, 924 clusters were subjected to in situ hybridization to determine the spatial localization of their transcripts. Using EST sequence data generated in the present study, a 60-mer oligonucleotide microarray with 8,091 unigenes (Medaka Microarray 8K) was constructed and tested for its usefulness in expression profiling. Furthermore, we have developed a rapid and reliable mutant mapping system using a set of mapped EST markers (M-marker 2003) that covers the entire medaka genome. These resources will accelerate medaka mutant analyses and make an important contribution to the medaka genome project.

Local injection of hepatocyte growth factor/scatter factor (HGF/SF) alters cyclic growth of murine hair follicles.

Hepatocyte growth factor/scatter factor (HGF/SF) has recently been shown to stimulate the hair follicle growth of mouse vibrissae in vitro. In this study, we analyzed the effect of cutaneous injections of recombinant human HGF/SF on hair follicle growth using mice in different hair cycle stages. Five male newborn mice, five male mice in second anagen, and five male mice in second telogen were administered a dorsal intradermal injection of 1 microg HGF/SF dissolved in 0.1% albumin-phosphate-buffered saline once daily for five or seven consecutive days, and then sacrificed on days 7 or 10. Hair follicle growth was evaluated photometrically and histologically using three parameters: the skin color of the reverse side of the resected skin, the skin thickness, and the area occupied by hair follicle tissue. The HGF/SF injected skin of newborn mice had hair follicles that were histologically longer and larger than those of the 0.1% albumin-phosphate-buffered saline injected skin. Mice that had received HGF/SF injection in second anagen, retained anagen hair follicles after 10 d only at the injection site, suggesting that HGF/SF delayed the transition from anagen to telogen. The HGF/SF injected skin of telogen mice had a significant increase in hair follicle tissue in the dermis, suggesting a mild anagen inducible activity by HGF/SF. Furthermore, precise measurements of the 20 hairs plucked from the HGF/SF injection sites revealed mild hair elongation in all the aforementioned experiments. These results imply that HGF/SF acts as a paracrine factor that alters cyclic hair growth of mice.

Hepatocyte growth factor/scatter factor stimulates hair growth of mouse vibrissae in organ culture.

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional polypeptide that acts as a mitogen, motogen, or morphogen, depending on the biologic context. In this study, we examined the effect of HGF/SF on hair growth using a serum-free organ culture system. Vibrissal hair follicles isolated from newborn mice were cultured at 31 degrees C in 95% O2/5% CO2 for 72 h in the presence of various cytokines or growth factors, and elongation of hair shaft, DNA, and protein synthesis in hair follicles were measured. Among the agents tested, only HGF/SF significantly increased hair follicle length (p < 0.001), 3H-thymidine (p < 0.001), and 35S-cysteine (p < 0.05) incorporation. The effect of HGF/SF was dose dependent, with maximal stimulation obtained at 10 ng/ml. The increase in hair follicle length and thymidine incorporation were specifically inhibited by a neutralizing antibody against HGF/SF. These results indicate that HGF/SF can promote hair growth and may have clinical utility in this regard.

The effect of various cytokines on hair growth of mouse vibrissae in organ culture.

Hepatocyte growth factor/scatter factor (HGF) is a multifunctional polypeptide which acts as a mitogen, motogen or morphogen depending on the biological context. In this study, we examined the effect of HGF on hair growth using a serum-free organ culture system. Vibrissal hair follicles isolated from newborn mice were cultured at 31 degrees C in 95% O2-5%CO2 for 72 h in the presence of various cytokines or growth factors. DNA, protein synthesis and elongation of the hair shaft in the hair follicles were measured. Among the agents tested, only HGF significantly increased hair follicle length (P < 0.001) and 3H-thymidine (P < 0.001) incorporation. The effect of HGF was dose-dependent, with maximal stimulation obtained at 10 ng/ml. The increase in hair follicle length and thymidine incorporation were specifically inhibited by a neutralizing antibody against HGF. These results indicate that HGF is able to promote hair growth and may have clinical utility in this regard.

Organ culture conditions of human hair follicles.

Experimental results revealed that although [3H]thymidine uptake in the hair bulb increased time dependently for 12 days under normal culture conditions (95% air-5% CO2 at 37 degrees C), striking morphological changes occurred in the hair bulb cells as demonstrated by histological findings. As such, organ culture conditions applicable to human hair follicles were studied utilizing observations from both histology and DNA synthesis. We found that culture conditions of 95% O2-5% CO2 at 31 degrees C were superior when compared to normal culture conditions for cultures of human hair follicles when attempting to maintain the normal morphology of hair germinative cells. The hair bulb and the germinative cells successfully maintained their normal morphology throughout the 96 and 48 h culture period, respectively. Autoradiographs of [3H]thymidine-labeled follicles showed localization in the germinative cells below Auber's critical line. Hair bulb DNA synthesis increased time dependently for 96 h after culture initiation. Under conditions of 95% O2-5% CO2 at 31 degrees C, the synthesis of DNA in hair germinative cells was observed. Such an organ culture method may prove useful for studies on the human hair growth mechanism.

Effects of cytokines, anti-cancer agents and cocarcinogen on DNA synthesis in hair bulb cells.

We analysed the effects of cytokines, anti-cancer agents and cocarcinogen on DNA synthesis in human hair germinative cells cultured in serum-free media. Epidermal growth factor and gamma interferon were found to inhibit DNA synthesis slightly, while strong inhibition was demonstrated by doxorubicin, cytosine arabinoside and tetradecanoyl-phorbolacetate. Basic fibroblast growth factor had very little influence on DNA synthesis. This organ culture model in serum-free media is a useful method by which to examine the effects of various cytokines and drugs on DNA synthesis in hair germinative cells and/or to study the pathogenesis of various alopecia diseases.

The effect of hepatocyte growth factor/scatter factor on human hair follicle growth.

The effect of hepatocyte growth factor/scatter factor (HGF/SF) on human hair follicle growth was examined using a serum-free organ culture system. The DNA synthesis in human hair follicles and elongation of the hair shaft were measured subsequent to the follicle isolation and culture at 31 degrees C in 95% O2-5% CO2 for 72 h. Results showed that HGF/SF significantly increased 3H-thymidine (P < 0.001) incorporation and hair follicle length (P < 0.05). The effect of HGF/SF was dose-dependent with a maximal stimulation at 10 ng/ml.

Organ culture of human hair follicles in serum-free medium.

Human hair follicles were cultured in serum-free media at 31 degrees C in an atmosphere containing 95% O2 and 5% CO2. Results showed that the length of the cultured hair increased time dependently for 96 h. Histological findings revealed that the hair germinative cells maintained their normal morphology throughout the 96 h culture period. DNA synthesis in the hair bulb also increased time dependently for 96 h. Autoradiographs of 3H-thymidine-labelled follicles indicated that they were localized in the germinative cells below Auber's critical line. The effects of minoxidil sulphate on DNA synthesis in this culture system were concentration dependent. Minoxidil sulphate at concentrations of 10(-10), 10(-9) and 10(-8) M significantly increased DNA synthesis compared with DNA synthesis in the control medium. Autoradiographs of the follicles cultured in 10(-10) M minoxidil sulphate showed that 3H-thymidine localized primarily in the germinative cells below Auber's critical line. These results suggest that this organ culture system may be useful for studying DNA synthesis by hair germinative cells in serum-free media.

Thirteen-week repeated oral dose toxicity study of ecabapide, a gastroprokinetic drug, in dogs and rats.

Thirteen-week oral repeated dose toxicity of ecabapide, a gastroprokinetic drug, was investigated in dogs at dosage levels of 50, 175 or 600 mg/kg, and in rats at dosage levels of 25, 100, 400 or 1600 mg/kg. In dogs, vomiting, aqueous salivation, body weight gain inhibition, and hemolytic anemia, together with an increase in Heinz body formation, were observed at 175 and/or 600 mg/kg. Histological examination revealed enhanced hemosiderin deposition in the liver and spleen, retention of erythrocytes in the splenic sinus and enhanced erythropoiesis in bone marrow at 175 and/or 600 mg/kg. In the rat study, although increases in serum total protein, albumin and calcium, as well as increased liver and kidney weights, were observed at 400 and/or 1600 mg/kg, no obvious morphological changes were seen. The hemolytic anemia and an increased Heinz body formation were not observed in rats, indicating a species difference. On the basis of these results, the non-toxic dose of ecabapide was considered to be 50 mg/kg in dogs and 100 mg/kg in rats.

Organ culture of mouse vibrissal hair follicles in serum-free medium.

We developed a method for organ culture of mouse vibrissal hair follicles in a serum-free medium. Cultures conducted at 31 degrees C in 95% O2-5% CO2 were found to be suitable for the follicles, with several findings of considerable interest pertaining to hair growth. During the 96 h culture period, the length of the isolated follicles significantly increased; the hair bulb cells maintained their normal morphology; and DNA and protein synthesis within the bulb increased time-dependently. Furthermore, autoradiography showed that 3H-thymidine-labeling was localized in the matrix cells below Auber's critical line in the hair bulb; 3H-leucine-labeling was found in the epithelial region; and 35S-cysteine-labeling was detected in the cortex of hair, particularly in the keratogenous zone. These results indicate that the culture system using mouse vibrissal hair would be potentially useful as an effective model for examination of hair growth.

The expression of endothelin-1 and its binding sites in mouse skin increased after ultraviolet B irradiation or local injection of tumor necrosis factor alpha.

Endothelin (ET)-1 is a 21-amino acid peptide which has vasoconstrictor and growth regulatory activity. Recently, cultured keratinocytes have been reported to express ET-1 and its receptor when irradiated by ultraviolet (UV) B. In order to further understand the role of ET-1 in vivo during UVB-induced inflammation, we examined the localization, intensity and time course of the expression levels of ET-1 and its binding sites in UVB-exposed BALB/c mouse skin. Frozen and paraffin sections prepared from mouse skin 48 h after treatment with UVB irradiation (0.36 or 0.72 J/cm2) or after injection with tumor necrosis factor (TNF)-alpha (1.0 microgram) or interleukin (IL)-1 alpha (0.05 microgram) were incubated with monoclonal anti-ET-1 IgG and then visualized by peroxidase staining. In normal skin, faint ET-1 immunoreactivity was observed in the epidermis, pilosebaceous structures and blood vessels. Upon exposure to UVB irradiation or administration of TNF-alpha injection or IL-1 alpha injection, such immunoreactivity was found to be significantly enhanced. Subsequently, the frozen sections were incubated with 125I ET-1 for 30 min, and visualized by autoradiographic technique. In normal skin, ET-1 weakly bound to the skin, while UVB irradiation and TNF-alpha injection significantly enhanced ET-1 binding in the epidermis, pilosebaceous structures and blood vessels. Time course experiments (1, 2, 4 and 7 days) indicated that ET-1 immunoreactivity and ET-1 binding peaked 1 or 2 days after UVB irradiation or TNF-alpha injection. These results suggest that the up-regulated expression of ET-1 and its binding sites in the epidermis and pilosebaceous structures may act as an autocrine/paracrine factor during UVB-induced inflammation.

Local tolerance of muroctasin injection in rabbits.

The local tolerance of N2-[(N-acetylmuramoyl)- N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine (MDP-Lys (L18), muroctasin), a synthetic muramyl dipeptide derivative, was investigated in rabbits. One ml of MDP-Lys (L18) injection was administered into the dorsal subcutis in single dose or multiple doses. Pathological examination revealed moderate infiltration of inflammatory cells at the injection sites 2 days after the last injection, but there was a tendency for recovery from this change to occur by the 7th day after injection. In conclusion, the tissue lesion caused by MDP-Lys (L18) injection administered subcutaneously is slight and transient.

Thirteen-week oral toxicity study of the new cognition-enhancing agent nefiracetam in rats.

Thirteen-week toxicity of nefiracetam (N-(2,6-dimethylphenyl)-2-(2-oxo-1- pyrrolidinyl) acetamide, DM-9384, CAS 77191-36-7) was examined in rats by oral administration of 30, 120, or 480 mg/kg. Rats receiving 480 mg/kg showed salivation, prone position, increased water consumption, increased levels of serum total cholesterol, total protein, albumin and total bilirubin, increased liver weight and hypertrophy of liver cells. This hypertrophy of hepatocytes with increased liver weight was also observed in males at 120 mg/kg. The non-toxic dose of nefiracetam under the present experimental conditions was therefore determined as 30 mg/kg.

Protein kinase activity is central to rat germ cell apoptosis induced by methoxyacetic acid.

Methoxyacetic acid (MAA) is a major metabolite of ethylene glycol monomethyl ether (EGME). Previous investigations of the testicular lesion induced by EGME have found that dividing meiotic cells are the most sensitive, although several stages of spermatocytes are also vulnerable. Preliminary data from this lab suggested the involvement of protein kinase activity in the development of this lesion, a hypothesis explored in the present studies. We used cultured seminiferous tubules (STs) from juvenile rats (25-day-old), exposed in vitro to MAA and several inhibitors of protein kinases. Nineteen h following a 5-h exposure to 5 mM MAA (the plasma level in vivo after a toxic dose of EGME), apoptotic spermatocytes were seen in early- and late-stage STs. Cell death was prevented by cotreatment with broad-spectrum inhibitors of protein kinases such as H-7, H-8, K-252a, W-7, and genistein. In corroboration, immunocytochemistry with antibodies to various kinases (PKCmu, zeta, and gamma, AKAP220, CaMKII, MLCK, and Src) showed increased staining around dying spermatocytes following EGME treatment in vivo. 2D-PAGE, autoradiography, and nanospray mass spectrometry was used to separate and identify proteins whose phosphorylation status was most greatly changed following exposure to MAA. One protein was identified by sequence analysis as being glucose-regulated protein 94 (grp94). Westem blotting and immunocytochemistry confirmed this finding. The data we present implicate kinase activities in the pathogenesis of this lesion and suggest the involvement of Sertoli cells.

Hepatocyte growth factor/scatter factor expressed in follicular papilla cells stimulates human hair growth in vitro.

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional polypeptide which acts as mitogen, motogen, or morphogen. In this study, we examined the effect of HGF/SF on human hair growth using organ and cell culture systems. HGF/SF was found to stimulate hair length and DNA synthesis in hair follicles at increasing concentrations up to 10 ng/ml (P < 0.05 and P < 0.01, respectively). HGF/SF stimulated [3H]thymidine incorporation by hair bulb-derived keratinocytes with the strongest response at 30 ng/ml of HGF/SF (P < 0.05). Cultured follicular papilla cells secreted HGF/SF, measured by an enzyme-linked immunoassay, in response to interleukin 1-alpha (IL1-alpha, 10 ng/ml), tumor necrosis factor-alpha (TNF-alpha, 10 ng/ml), or tetradecanoylphorbolacetate (100 nM) at levels ranging from 0.2 to 0.3 ng/mg protein/48 h. HGF/SF mRNA expressions, measured by the reverse transcription-polymerase chain reaction, were detected in follicular papilla cells, and were also stimulated by the three reagents. Transforming growth factor-beta (10 ng/ml) suppressed both protein and mRNA levels. These results suggest that hair follicle elongation induced by HGF/SF in organ culture occurs partly due to the mitogenic activity of HGF/SF expressed in follicular papilla cells on hair bulb-derived keratinocytes.

Early pathophysiologic feature of arthropathy in juvenile dogs induced by ofloxacin, a quinolone antimicrobial agent.

Arthropathy in dogs induced by ofloxacin, a quinolone antimicrobial agent, was pathophysiologically investigated. In the in vivo studies, ofloxacin was administered orally once or twice at 20 mg/kg/day to male juvenile (3-month-old, n=3) or adult (36-month-old, n=2) dogs, and the humeral and femoral heads were examined pathologically. Unlike adult dogs, fluid-filled vesicles were macroscopically observed on the articular surfaces of one juvenile dog 24 hours after a single treatment with ofloxacin. These lesions were seen in all juvenile dogs by twice dosing. Microscopically, fissures or cavity formations in the middle zone of the articular cartilage were noted only in juvenile dogs. Furthermore, the cartilage matrix from the abnormal area to the articular surface showed a decreased safranin-O staining intensity, suggesting proteoglycan depletion. Ultrastructurally, chondrocytes in the middle zone of juvenile dogs displayed dilatation of the cisternae in the rough endoplasmic reticulum as an initial hallmark. In the in vitro studies, chondrocytes isolated from the articular cartilage of naive juvenile dogs were exposed to ofloxacin at 6.3-100 microg/ml for 24 hours. Although no changes were noted in the deoxyribonucleic acid synthesis, protein synthesis, or proteoglycan release at concentrations of up to 100 microg/ml, the proteoglycan synthesis was evidently decreased in a dose-dependent manner from 12.5 microg/ml. The results obtained suggest that the inhibitory action of ofloxacin on proteoglycan syntheses in the chondrocytes may largely contribute to the early morphologic features in the articular cartilage of the juvenile dog.