Effects of Cudrania tricuspidata and Sargassum fusiforme extracts on hair growth in C57BL/6 mice.

BACKGROUND: Cudrania tricuspidata is a perennial plant, and Sargassum fusiforme is a brown seaweed with numerous potential benefits, including anticancer, anti-inflammatory, and antioxidant activities. However, the efficacies of C. tricuspidata and S. fusiforme on hair growth have not yet been elucidated. Therefore, the present study examined the effects of C. tricuspidata and S. fusiforme extracts on hair growth in C57BL/6 mice. RESULTS: ImageJ demonstrated that drinking and skin application of C. tricuspidata and/or S. fusiforme extracts significantly increased the hair growth rate in the dorsal skin of C57BL/6 mice compared to the control group. Histological analysis confirmed that drinking and skin application of C. tricuspidata and/or S. fusiforme extracts for 21 days significantly increased the length of hair follicles on the dorsal skin of treated C57BL/6 mice compared to that in the control mice. RNA sequencing analysis revealed that hair growth cycle-related factors (anagen factors) such as Catenin Beta 1 (Ctnnb1) and platelet-derived growth factor (Pdgf) were upregulated (> twofold) only by C. tricuspidate extracts, whereas vascular endothelial growth factor (Vegf) and Wnts were upregulated by both C. tricuspidata or S. fusiforme applications in treated mice (compared to the control mice). In addition, oncostatin M (Osm, a catagen-telogen factor) was downregulated (< 0.5 fold) by C. tricuspidata when administered via both skin and drinking mode in treated mice compared to that in control mice. CONCLUSIONS: Our results suggest that C. tricuspidata and/or S. fusiforme extracts show potential hair growth efficacy by upregulating anagen factor genes, including ß-catenin, Pdgf, Vegf, and Wnts, and downregulating catagen-telogen factor genes, including Osm, in C57BL/6 mice. The findings suggest that C. tricuspidata and/or S. fusiforme extracts are potential drug candidates to treat alopecia.

Anti-diabetic effect of hesperidin on palmitate (PA)-treated HepG2 cells and high fat diet-induced obese mice.

The present study examined the relationship between the anti-diabetic effect of hesperidin (HES) and the differential gene expression in HES treated high fat diet (HFD)-induced obese mice. Based on the glucose uptake assay, the treatment of HES restored the glucose uptake to control level in an insulin-independent manner in PA-treated HepG2 cells. Western blot analysis confirmed that the treatment of HES increased the insulin-stimulated phosphorylation of Akt and GSK3ß in insulin-resistant PA-treated HepG2 cells. HFD-induced obese mice treated with HES significantly reduced serum insulin, blood glucose, and homeostatic model assessment for insulin resistance (HOMA-IR) values. In addition, both glucose tolerance and insulin tolerance were significantly improved to normal level by HES in HFD-induced obese mice. RNA sequencing analysis disclosed that the expression levels of up-regulated 12 genes and down-regulated 6 genes related to insulin signaling and glucose metabolism were restored to normal level by HES in the liver of HFD-induced obese mice. A protein-protein interaction (PPI) network was constructed via search tool for the retrieval of interacting genes/proteins (STRING) analysis, and Eno1, Pik3cd, Hk2, Trib3, Myc, Nos3, Ppargc1a, and Igf2 were located in the functional hubs of the PPI network of glucose metabolism. Furthermore, Western blot analysis confirmed that HES improved insulin sensitivity and glucose homeostasis by normalizing the expression levels of hexokinase-II, enolase-1, and PI3 kinase p110δ to normal level. The overall results suggest that HES possess a potential anti-diabetic effect by normalizing the expression levels of the insulin signaling and glucose metabolism related genes which were perturbed in the liver of HFD-induced obese mice.

Broccoli Leaves Attenuate Influenza A Virus Infection by Interfering With Hemagglutinin and Inhibiting Viral Attachment.

Broccoli (Brassica oleracea L. var. Italica) leaves are a byproduct of broccoli and could be used as a food source. The study aimed to evaluate the effect of broccoli leaves on influenza A virus (IAV) infection. We investigated the effect of ethanol extract of Broccoli leaves (EBL) on IAV infection using green fluorescent protein (GFP)-tagged Influenza A/PR/8/34 virus (PR8-GFP IAV). When EBL and PR8-GFP IAV were cotreated to RAW 264.7 cells, the fluorescence microscopy and fluorescence-activated cell sorting (FACS) analysis showed that EBL significantly reduced the levels of GFP expression by influenza viral infection dose-dependently. Immunofluorescence (IF) analysis confirmed that EBL decreased the expression of IAV proteins. EBL exhibited a strong inhibitory effect of IAV binding on the cells and moderate virucidal impact. Consistently, EBL potently suppressed the hemagglutination by IAV infection. These results indicate that EBL prevents IAV attachment via the inhibition of HA upon viral infection. Finally, EBL as an HA inhibitor of IAV could be used as the natural antiviral source to protect against influenza viral infection.

Anti-adipogenic effect of the flavonoids through the activation of AMPK in palmitate (PA)-treated HepG2 cells.

BACKGROUND: Flavonoids are natural polyphenols found widely in citrus fruit and peel that possess anti-adipogenic effects. On the other hand, the detailed mechanisms for the anti-adipogenic effects of flavonoids are unclear. OBJECTIVES: The present study observed the anti-adipogenic effects of five major citrus flavonoids, including hesperidin (HES), narirutin (NAR), nobiletin (NOB), sinensetin (SIN), and tangeretin (TAN), on AMP-activated protein kinase (AMPK) activation in palmitate (PA)-treated HepG2 cells. METHODS: The intracellular lipid accumulation and triglyceride (TG) contents were quantified by Oil-red O staining and TG assay, respectively. The glucose uptake was assessed using 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) assay. The levels of AMPK, acetyl-CoA carboxylase (ACC), and glycogen synthase kinase 3 beta (GSK3ß) phosphorylation, and levels of sterol regulatory element-binding protein 2 (SREBP-2) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) expression were analyzed by Western blot analysis. The potential interaction between the flavonoids and the γ-subunit of AMPK was investigated by molecular docking analysis. RESULTS: The flavonoid treatment reduced both intracellular lipid accumulation and TG content in PA-treated HepG2 cells significantly. In addition, the flavonoids showed increased 2-NBDG uptake in an insulin-independent manner in PA-treated HepG2 cells. The flavonoids increased the AMPK, ACC, and GSK3ß phosphorylation levels and decreased the SREBP-2 and HMGCR expression levels in PA-treated HepG2 cells. Molecular docking analysis showed that the flavonoids bind to the CBS domains in the regulatory γ-subunit of AMPK with high binding affinities and could serve as potential AMPK activators. CONCLUSION: The overall results suggest that the anti-adipogenic effect of flavonoids on PA-treated HepG2 cells results from the activation of AMPK by flavonoids.

Anti-obesity effect of sulforaphane in broccoli leaf extract on 3T3-L1 adipocytes and ob/ob mice.

The present study evaluated the anti-obesity effect of sulforaphane (SFN) and glucoraphanin (GRN) in broccoli leaf extract (BLE) on 3T3-L1 adipocytes and ob/ob mice. Based on Oil Red O staining and triglyceride (TG) assay, SFN and BLE significantly reduced (P<.05) both lipid accumulation and TG content in the differentiated 3T3-L1 adipocytes. SFN and BLE increased 2-NBDG uptake by 3T3-L1 adipocytes in a dose-dependent manner. Western blot analysis confirmed that SFN and BLE increased the phosphorylation levels of both AMPK (Thr172) and ACC (Ser79), and reduced the expression of HMGCR in liver and white adipose tissues of ob/ob mice. Histological analysis revealed that SFN and BLE ameliorated hepatic steatosis, and reduced the size of adipocyte in ob/ob mice. Treatment with SFN and BLE significantly reduced (P<.05) TG content, low-density lipoprotein (LDL) cholesterol, total cholesterol (TC), and glucose in the serum of ob/ob mice. RNA sequencing analysis showed that up- or down-regulation of 32 genes related to lipid metabolism was restored to control level in both SFN and BLE-treated ob/ob mice groups. A protein-protein interaction (PPI) network was constructed via STRING analysis, and Srebf2, Pla2g2c, Elovl5, Plb1, Ctp1a, Lipin1, Fgfr1, and Plcg1 were located in the functional hubs of the PPI network of lipid metabolism. Overall results suggest that the SFN content in BLE exerts a potential anti-obesity effect by normalizing the expression of genes related to lipid metabolism, which are up- or down-regulated in ob/ob mice.

The effects of naringenin and naringin on the glucose uptake and AMPK phosphorylation in high glucose treated HepG2 cells.

BACKGROUND: Naringin and its aglycone naringenin are citrus-derived flavonoids with several pharmacological effects. On the other hand, the mechanism for the anti-diabetic effects of naringenin and naringin are controversial and remain to be clarified further. OBJECTIVE: This study examined the relationship between glucose uptake and AMP-activated protein kinase (AMPK) phosphorylation by naringenin and naringin in high glucose-treated HepG2 cells. METHODS: Glucose uptake was measured using the 2-NBDG fluorescent D-glucose analog. The phosphorylation levels of AMPK and GSK3ß (Glycogen synthase kinase 3 beta) were observed by Western blotting. Molecular docking analysis was performed to evaluate the binding affinity of naringenin and naringin to the γ-subunit of AMPK. RESULTS: The treatment with naringenin and naringin stimulated glucose uptake regardless of insulin stimulation in high glucose-treated HepG2 cells. Both flavonoids increased glucose uptake by promoting the phosphorylation of AMPK at Thr172 and increased the phosphorylation of GSK3ß. Molecular docking analysis showed that both naringenin and naringin bind to the γ-subunit of AMPK with high binding affinities. In particular, naringin showed higher binding affinity than the true modulator, AMP with all three CBS domains (CBS1, 3, and 4) in the γ-subunit of AMPK. Therefore, both naringenin and naringin could be positive modulators of AMPK activation, which enhance glucose uptake regardless of insulin stimulation in high glucose-treated HepG2 cells. CONCLUSIONS: The increased phosphorylation of AMPK at Thr172 by naringenin and naringin might enhance glucose uptake regardless of insulin stimulation in high glucose treated HepG2 cells.

Effect of Screw Insertion Torque on Mechanical Properties of a Hybrid Polyaxial Locking System.

OBJECTIVE: The aim of this study was to determine the effect of insertion torque and angulation on the push-out strength of screws in Atraumatic Rigid Fixation (ARIX) system. MATERIALS AND METHODS: In vitro mechanical tests of the ARIX system were conducted. Screw plate constructs (n = 120) were tested using five different insertion torques at four different angles relative to the perpendicular axis of the plate. Before the push-out test, screws were locked into the plates, and the push-out force of the screw was measured by applying a load parallel to the screw axis. RESULTS: Implant failure was observed at 0.8 Nm at an insertion angle of 15 degrees, and at 1 Nm at 0 degree, 5 degrees and 10 degrees. Two types of failures were observed: cold welding and plate deformation. An insertion torque of 0.8 Nm produced a significantly higher push-out force compared with 0.2 Nm. Non-angled specimens with 0.8 Nm insertion torque exhibited significantly higher screw push-out forces compared with other tested specimens and insertion angles. Insertion angle did not affect screw push-out force at insertion torques of 0, 0.2, 0.4 and 0.6 Nm. CONCLUSION: The ARIX locking system is much more sensitive to insertion torque than angle. An effect of insertion angle was observed only at an insertion torque of 0.8 Nm, under which all angles significantly decreased push-out force relative to zero degrees of angulation. In addition, low insertion torques can result in screws loosening over time, while greater insertion torques than 1 Nm can result in screw head stripping and plate hole deformation.

Correction to: Monitoring Dose Response of Cyanide Antidote Dimethyl Trisulfide in Rabbits Using Diffuse Optical Spectroscopy.

The online version of the original article can be found at.

Monitoring Dose Response of Cyanide Antidote Dimethyl Trisulfide in Rabbits Using Diffuse Optical Spectroscopy.

INTRODUCTION: Cyanide (CN) poisoning is a serious chemical threat from accidental or intentional exposures. Current CN exposure treatments, including direct binding agents, methemoglobin donors, and sulfur donors, have several limitations. Dimethyl trisulfide (DMTS) is capable of reacting with CN to form the less toxic thiocyanate with high efficiency, even without the sulfurtransferase rhodanese. We investigated a soluble DMTS formulation with the potential to provide a continuous supply of substrate for CN detoxification which could be delivered via intramuscular (IM) injection in a mass casualty situation. We also used non-invasive technology, diffuse optical spectroscopy (DOS), to monitor physiologic changes associated with CN exposure and reversal. METHODS: Thirty-six New Zealand white rabbits were infused with a lethal dose of sodium cyanide solution (20 mg/60 ml normal saline). Animals were divided into three groups and treated with saline, low dose (20 mg), or high dose (150 mg) of DMTS intramuscularly. DOS continuously assessed changes in tissue hemoglobin concentrations and cytochrome c oxidase redox state status throughout the experiment. RESULTS: IM injection of DMTS increased the survival in lethal CN poisoning. DOS demonstrated that high-dose DMTS (150 mg) reversed the effects of CN exposure on cytochrome c oxidase, while low dose (20 mg) did not fully reverse effects, even in surviving animals. CONCLUSIONS: This study demonstrated potential efficacy for the novel approach of supplying substrate for non-rhodanese mediated sulfur transferase pathways for CN detoxification via intramuscular injection in a moderate size animal model and showed that DOS was useful for optimizing the DMTS treatment.

Effects of resveratrol on the insulin signaling pathway of obese mice.

The present study was conducted to investigate the effects of resveratrol on the insulin signaling pathway in the liver of obese mice. To accomplish this, we administered resveratrol to high fat diet-induced obese mice and examined the levels of protein phosphorylation in the liver using an antibody array. The phosphorylation levels of 10 proteins were decreased in the high fat diet and resveratrol (HFR) fed group relative to the levels in the high fat diet (HF) fed group. In contrast, the phosphorylation levels of more than 20 proteins were increased in the HFR group when compared with the levels of proteins in the HF group. Specifically, the phosphorylation levels of Akt (The308, Tyr326, Ser473) were restored to normal by resveratrol when compared with the levels in the HF group. In addition, the phosphorylation levels of IRS-1 (Ser636/Ser639), PI-3K p85-subunit α/γ (Tyr467/Tyr199), PDK1 (Ser241), GSK-3α (S21) and GSK-3 (Ser9), which are involved in the insulin signaling pathway, were decreased in the HF group, whereas the levels were restored to normal in the HFR group. Overall, the results show that resveratrol restores the phosphorylation levels of proteins involved in the insulin signaling pathway, which were decreased by a high fat diet.

Nitric oxide-mediated vasorelaxation effects of anti-angiotensin I-converting enzyme (ACE) peptide from Styela clava flesh tissue and its anti-hypertensive effect in spontaneously hypertensive rats.

In our previous study, an anti-angiotensin I converting enzyme (ACE) peptide (Ala-His-Ile-Ile-Ile, MW: 565.3Da) was isolated from Styela clava flesh tissue. In this study the fractions obtained during the isolation process and the finally purified peptide were examined to see if they had vasorelaxation effects in isolated rat aortas, and then the peptide was investigated for anti-hypertensive effect in spontaneously hypertensive rats (SHRs). The induction of vasorelaxation in the rat aortas was observed with the isolated fractions and the peptide from the enzymatic hydrolysate of S. clava flesh tissue and could be markedly blocked by pretreatment with the nitric oxide synthase (NOS) inhibitor, N(G)-nitro-l-arginine methyl ester (l-NAME). In human endothelial cells, NO synthesis was found to be increased and eNOS phosphorylation was upregulated when the cells were cultured with the purified peptide. Furthermore, systolic blood pressure was reduced by administration of the potent vasorelaxation peptide in SHRs.

A short note on a euphausiid, Euphausia pacifica, an important food source to demersal fishes in Uljin area, the eastern coast of Korea.

Gut contents of two species of demersal fishes, Pleurogrammus azonus and Dasycottus steiger were examined to understand the importance of a euphausiid, Euphausia pacifica as prey of demersal fishes at coastal waters off Uljin, the eastem Korea where aggregations of large amounts of the euphausiids were found. Euphausiids were significantly important food source to the species of fishes examined. All of the stomachs of P. azonus examined contained euphausiids (% of a food item frequency occurred, F = 100%) in March and June 2003. Individual numbers of euphusiids in the guts ranged from 10 to 540 individuals per gut. Percents of individual number of food item were 99.60 and 99.96 for euphausiids in March and June 2003, respectively The guts of D. steiger also frequently contained euphausiids (F = 86.67 and 79.20% in October 2002 and June 2003, respectively), while amphipods (F = 36.67% and 16.17%,) and other fishes (F = 33.33% and 45.80%) were also often observed. Pleurogrammus azonus preferred larger euphausiids with size range 16.9-28.4 mm, while the range of euphausiids distributed in the ambient water column was much broader (from eggs to adults).

Development of immunoassays for the detection of kanamycin in veterinary fields.

Monoclonal antibody against kanamycin was prepared, and competitive direct ELISA and immunochromatographic assay were developed using the antibody to detect kanamycin in animal plasma and milk. The monoclonal antibody produced was identified to be IgG1, which has a kappa light chain. No cross-reactivity of the antibody was detected with other aminoglycosides, indicating that the monoclonal antibody was highly specific for kanamycin. Based on competitive direct ELISA, the detection limits of kanamycin were determined to be 1.1 ng/ml in PBS, 1.4 ng/ml in plasma, and 1.0 ng/ml in milk. The concentration of intramuscularly injected kanamycin was successfully monitored in rabbit plasma with competitive direct ELISA. Based on the colloidal gold-based immunochromatographic assay, the detection limits of kanamycin were estimated to be about 6-8 ng/ml in PBS, plasma, and milk. The immunochromatographic assay would be suitable for rapid and simple screening of kanamycin residues in veterinary medicine. Screened positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA. Therefore, the assays developed in this study could be used to complement each other as well as other laboratory findings. Moreover, instead of slaughtering the animals to obtain test samples, these methods could be applied to determine kanamycin concentration in the plasma of live animals.

Development of ELISA and immunochromatographic assay for the detection of neomycin.

BACKGROUND: Reliable analytical methods are required to monitor neomycin residue levels in the livestock products. In particular, a more simple and rapid detection method is required in the veterinary fields. METHODS: Competitive direct ELISA and immunochromatographic assay were developed using monoclonal antibody to detect neomycin in the animal plasma and milk. RESULTS: No cross-reactivity of the antibody was observed with other aminoglycosides based on competitive direct ELISA methods, indicating that the antibody is highly specific for neomycin. Based on the standard curves, the detection limits were determined to be 6.85 ng/ml in PBS, 3.61 ng/ml in plasma, and 2.73 ng/ml in milk, respectively. Recoveries of neomycin from spiked plasma and milk at levels of 50-200 ng/ml ranged from 87% to 108%. Concentration of intramuscularly injected neomycin was successfully monitored in the rabbit plasma through competitive direct ELISA. Immunochromatographic method was also developed using colloidal gold-conjugated monoclonal antibody. Through this method, the detection limits were estimated to be about 10 ng/ml of neomycin in PBS, plasma, and milk. CONCLUSIONS: Immunochromatographic assay developed in this study is suitable for the simple screening of neomycin residues in the veterinary field. Observed positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA.

Development of ELISA and immunochromatographic assay for the detection of gentamicin.

Competitive direct enzyme-linked immunosorbent assay (ELISA) and the immunochromatographic assay were developed using a monoclonal antibody to detect gentamicin in the animal plasma and milk. No cross-reactivity of the antibody was observed with other aminoglycosides based on competitive direct ELISA, indicating that the antibody is highly specific for gentamicin. On the basis of the standard curves, the detection limits were determined to be 0.9 ng/mL in phosphate-buffered saline (PBS), 1.0 ng/mL in plasma, and 0.5 ng/mL in milk, respectively. Recoveries of gentamicin from spiked plasma and milk at levels of 25-100 ng/mL ranged from 85 to 112%. The concentration of intramuscularly injected gentamicin was successfully monitored in the rabbit plasma through competitive direct ELISA. The detection limits were estimated to be about 6 ng/mL of gentamicin in PBS, plasma, and milk using the colloidal gold-based immunochromatographic assay, which is suitable for the simple screening of gentamicin residues in the veterinary field. Observed positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA. Therefore, the assays developed in this study could complement each other as well as veterinary field and laboratory findings.

Genotoxicity and toxicological effects of acrylamide on reproductive system in male rats.

The toxicity of acrylamide was evaluated through mutagenicity of Salmonella, chromosome aberration of Chinese hamster lung fibroblasts, micronucleus formation in mice and reproductive toxicity in rats. Based on Ames test, acrylamide showed mutagenic potency for strains TA98 and TA100. Moreover, both chromosomal aberration assay and micronucleus assay indicated that acrylamide might have genotoxic potency; the chromosomal aberration frequencies were observed to be proportional to acrylamide concentrations of 5-50 mM, and acrylamide significantly increased micronuclei in peripheral blood cells of mice at doses of higher than 72.5 mg/kg. Male rats were treated with acrylamide at doses of 0, 5, 15, 30, 45, or 60 mg/kg/day for 5 consecutive days, and the toxicity of acrylamide was observed. In the group treated with the highest dose of acrylamide (60 mg/kg/day), the loss of body weight and reduced testis weight were observed. Also the epididymides weights were reduced significantly in all the groups treated with acrylamide. The number of sperms in cauda epididymidis decreased significantly in an acrylamide dose-dependent manner. Rats treated with 60 mg/kg/day of acrylamide showed several histopathological lesions in the seminiferous tubules. There were thickening and multiple layering of the tubular endothelium, and the formation of many multinucleated giant cells in seminiferous tubules. Taken together, acrylamide not only causes the genotoxicity of eukaryotic cells and mice but also shows the toxicological effects on reproductive system in male rats.

Class B scavenger receptor-mediated intestinal absorption of dietary beta-carotene and cholesterol.

There is now a general consensus that the intestinal absorption of water-insoluble, dietary lipids is protein-mediated, but the assignment of protein(s) to this function is still a matter of debate. To address this issue, we measured beta-carotene and cholesterol absorption in wild-type and SR-BI knockout mice and the uptake of these lipids in vitro using brush border membrane (BBM) vesicles. From the comparison of the in vivo and in vitro results we conclude that both BBM-resident class B scavenger receptors, SR-BI and CD36, can facilitate the absorption of beta-carotene and cholesterol. SR-BI is essential for beta-carotene absorption, at least in mice on a high fat diet. This is due to the fact that the absorption of beta-carotene is restricted to the duodenum and SR-BI is the predominant receptor in the mouse duodenum. In contrast, SR-BI may be involved but is not essential for cholesterol absorption in the small intestine. The question of whether SR-BI contributes to cholesterol absorption in vivo is still unresolved. Transfection of COS-7 cells with SR-BI or CD36 confers on these cells lipid uptake properties closely resembling those of enterocytes and BBM vesicles. Both scavenger receptors facilitate the uptake of dietary lipids such as beta-carotene, free and esterified cholesterol, phospholipids, and fatty acids into COS-7 cells. This lipid uptake is effected from three different lipid donor particles: mixed bile salt micelles, phospholipid small unilamellar vesicles, and trioleoylglycerol emulsions which are all likely to be present in the small intestine. Ezetimibe, a representative of a new class of drugs that inhibit intestinal cholesterol absorption, blocks SR-BI- and CD36-facilitated uptake of cholesterol into COS-7 cells.

Toxicological effects of acrylamide on rat testicular gene expression profile.

Toxicological effects of acrylamide on differential gene expression profile of rat testis were evaluated. Acrylamide induced morphological sperm defects, and decreased sperm concentration in cauda epididymis. Serum testosterone level and Leydig cell viability were also decreased dose-dependently, which resulted in decreased spermatogenesis. Acrylamide-induced histopathological lesions, such as formation of multinucleated giant cells and vacuolation, and numerous apoptotic cells were observed in seminiferous tubules. cDNA microarray analysis revealed that genes related to testicular-functions, apoptosis, cellular redox, cell growth, cell cycle, and nucleic acid-binding were up/down-regulated in testes isolated from acrylamide-treated group (60 mg/kg/day). Acrylamide toxicity appears to increase Leydig cell death and perturb gene expression levels, contributing to sperm defects and various abnormal histopathological lesions including apoptosis in rat testis.

Molecular phylogenetic status of the Korean goral and Japanese serow based on partial sequences of the mitochondrial cytochrome b gene.

To investigate the molecular phylogenetic status of the Korean goral, Nemorhaedus caudatus raddeanus, and Japanese serow, Capricornis crispus, we determined partial sequences of the mitochondrial cytochrome b gene of twelve Korean gorals and sixteen Japanese serows, and compared them with those of the major lineages of Rupicaprini species including two other Nemorhaedus species and two other Capricornis species. The Korean gorals examined possessed two haplotypes with only one nucleotide difference between them, while the Japanese serows showed slightly higher sequence diversity with five haplotypes. Genetic distances and molecular phylogenetic trees indicated that there is considerable genetic divergence between the Korean goral and N. caudatus (the Chinese goral) [Groves and Shields (1996)], but virtually none between Korean and Russian gorals. The Korean and Russian gorals may therefore be distinct from the Chinese goral. The data highlight the importance of conservation of the goral populations of these regions, and the need to reconsider the taxonomic status of Korean and Russian gorals. Our study also clearly demonstrated sufficient genetic distance between serows and gorals to justify their assignment to separate genera. Of the three species of Capricornis, the Formosan serow, C. swinhoei is more closely related to C. sumatraensis than to the Japanese serow, suggesting that the Formosan serow is a distinct species. Preliminary data on intraspecific genetic variation in the Japanese serow are also presented.