Thymosin ß4 Reduces H2O2 Induced Oxidative Stress in MC3T3-E1 Cells on Titanium Surface.

Thymosin ß4 (Tß4) is known to inhibit an inflammatory response and to increase the survival of osteoblasts on titanium (Ti) surfaces. Ti is the most widely used graft material in dentistry; however, an inflammatory response induced following implant placement results in the generation of reactive oxygen species (ROS). The oxidative stress from the production of ROS such as nitric oxide (NO) and hydrogen peroxide (H2O2) can damage surrounding cells, resulting in implant failure by decreasing cell viability. Thus, the aim of this study was to determine the biological effects of Tß4 on the oxidative stress induced to MC3T3-E1 preosteoblasts on the Ti surface. Based on an MTT assay and bromodeoxyuridine immunofluorescence staining, Tß4 was found to increase the proliferation of the H2O2-exposed MC3T3-E1 cells on Ti discs. Reverse transcription-polymerase chain reaction and western blot analyses showed that Tß4 decreased the mRNA and protein expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in H2O2-exposed MC3T3-E1 cells on the Ti discs. Tß4 inhibited the synthesis of intracellular ROS and the secretion of NO and prostaglandin E2 (PGE2) from H2O2-exposed MC3T3-E1 cells on the Ti discs. In conclusion, Tß4 inhibits H2O2-induced iNOS and COX-2 expression with a decrease in ROS, NO, and PGE2 synthesis, which leads to improved cell survival with low cytotoxicity under an oxidative stress condition in MC3T3-E1 cells on the Ti surface. This suggests that Tß4 may be a crucial molecule to reduce oxidative stress-induced cell damage or hypoxia, leading to promoted osseointegration on the Ti surface during implant placement.

Effect of Thymosin beta4 on the Differentiation and Mineralization of MC3T3-E1 Cell on a Titanium Surface.

Osteoblasts are responsible for the synthesis of bone matrix through the secretion of collagenous and non-collagenous proteins with mineralization. Thymosin beta4 (Tbeta4) is an actin-sequestering peptide that is involved in the regulation of cell proliferation, differentiation and motility. A recent study reported that the inhibition of Tbeta4 mRNA synthesis strongly decreases the level of gene expression of bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP), osteocalcin (OCN), osteonectin (ON) and collagen type I (Col I) with mineralization during differentiation in odontoblasts. Titanium (Ti) is used commonly as an implant material for dental implants, which have strong mechanical potential and good biocompatibility with bone. This study examined whether Tbeta4 can be a potential molecule for promoting the differentiation and mineralization of MC3T3-E1 cells on a Ti surface. Tbeta4 increased the viability of MC3T3-E1 cells during differentiation on Ti discs compared to that of the control. The expression of Tbeta4 mRNA and protein in the Tbeta4-treated MC3T3-E1 cells was higher than the control during differentiation on the Ti discs. In addition, Tbeta4 increased the formation of mineralization nodules and the mRNA expression of alkaline phosphatase (ALP), DSPP, dentin matrix protein1 (DMP1), BSP and Col I compared to that of the control in MC3T3-E1 cells during differentiation on Ti discs. From the results, Tbeta4 increased the viability and promoted the differentiation and mineralization of MC3T3-E1 cells on Ti discs. This highlights the potential use of Tbeta4 for increasing osseointegration through osteoblast differentiation and mineralization on Ti discs.

Sanguinarine Induces Apoptosis of Human Oral Squamous Cell Carcinoma KB Cells via Inactivation of the PI3K/Akt Signaling Pathway.

Preclinical Research Sanguinarine, an alkaloid isolated from the root of Sanguinaria canadensis and other plants of the Papaveraceae family, selectively induces apoptotic cell death in a variety of human cancer cells, but its mechanism of action requires further elaboration. The present study investigated the pro-apoptotic effects of sanguinarine in human oral squamous cell carcinoma KB cells. Sanguinarine treatment increased DR5/TRAILR2 (death receptor 5/TRAIL receptor 2) expression and enhanced the activation of caspase-8 and cleavage of its substrate, Bid. Sanguinarine also induced the mitochondrial translocation of pro-apoptotic Bax, mitochondrial dysfunction, cytochrome c release to the cytosol, and activation of caspase-9 and -3. However, a pan-caspase inhibitor, z-VAD-fmk, reversed the growth inhibition and apoptosis induced by sanguinarine. Sanguinarine also suppressed the phosphorylation of phosphoinositide 3-kinase (PI3K) and Akt in KB cells, while co-treatment of cells with sanguinarine and a PI3K inhibitor revealed synergistic apoptotic effects. However, pharmacological inhibition of AMP-activated protein kinase and mitogen-activated protein kinases did not reduce or enhance sanguinarine-induced growth inhibition and apoptosis. Collectively, these findings indicate that the pro-apoptotic effects of sanguinarine in KB cells may be regulated by a caspase-dependent cascade via activation of both intrinsic and extrinsic signaling pathways and inactivation of PI3K/Akt signaling. Drug Dev Res 77 : 227-240, 2016. © 2016 Wiley Periodicals, Inc.

Function of Secretory Leukocyte Protease Inhibitor (SLPI) in Odontoblast During Mouse Tooth Development.

Secretory leuckocyte protease inhibitor (SLPI) is thought as a regulating protein on the synthesis and degradation of matrix proteins. But there was no report of expression and function of SLPI on the tooth development, especially on the odontoblasts. As observed by in-situ hybridization and immunohistochemical analysis, SLPI was expressed in odontoblasts and predentin on post-natal day 4 (PN4). On PN10, SLPI was observed under the dentin and apical region including odontoblasts processes. Further, on PN15, expression of SLPI was the same pattern compared to PN10. SLPI was expressed under layer of the odontoblasts and in odontoblasts on PN20. Matrix metalloproteinase-2 (MMP-2) and -9 levels in SLPI/MDPC-23 cells were higher than that of the MDPC-23 cells. The gene expression of SLPI, bone sialoprotein (BSP), osteocalcin (OCN), osteonectin (ON), and collagen type I (Col I) was higher in SLPI/MDPC-23 than that of MDPC-23 cells and the expression of dentin sialophosphoprotein (DSPP) was lower in SLPI/MDPC-23. Taken together, our results suggest that SLPI may be a MMP-2 and -9 regulating molecule in odontoblasts during dentin matrix formation and acts as a signaling molecule for dentin matrix related proteins during odontoblasts differentiation and mineralization.

Secretory Leukocyte Protease Inhibitor (SLPI) Increases Focal Adhesion in MC3T3 Osteoblast on Titanium Surface.

An appropriate interaction between implanted materials and the surrounding tissue is essential for successful implantation. Titanium (Ti) and some of its alloys have been used in dentistry and orthopedics as a substitutive material for hard tissue, such as teeth or natural bone. Nevertheless, metal ions released from titanium and alloy implants have adverse biological effects on biological tissues or cells. Secretory leukocyte protease inhibitor (SLPI) promotes cell migration, proliferation and wound healing. FAK and ERK1/2 signaling regulate cell adhesion and proliferation for cell survival. This study evaluated the potential of SLPI as a molecule to increase the cell adhesion on the Ti surface. Compared with the untreated cells, SLPI increased the adhesion of MC3T3-E1 cells to Ti discs, formation of actin stress fibers, paxillin expression and the phosphorylation of FAK. Moreover, SLPI enhanced the level of Grb2 and Ras expression and ERK1/2 phosphorylation in the MC3T3-E1 cells on Ti discs. These results suggest that SLPI can increase the interaction between the implanted Ti material and surrounding bone in orthodontic and dental surgery, making an effective nanomolecule for successful implantation.

The Effect of Thymosin ß4 for Osteoblast Adhesion on Titanium Surface.

Titanium (Ti) is the most widely used implant material in dentistry and orthopedics but the release of metal ions from Ti implants results in increased bone resorption by enhancing the production of inflammatory cytokines from the macrophages and facilitating osteoclast differentiation. Thymosin ß4 (Tß4) has several biological activities, such as promoting wound healing, angiogenesis, cell proliferation and migration in mammalian cells. This study examined the role of Tß4 in osteoblasts via focal adhesions (FAs) and ERK1/2 signaling related to cell adhesion and proliferation for cell survival on the Ti surface. As a result, cell adhesion and proliferation increased in the Tß4 treated cells (Tß4/MC3T3-E1) but was significantly lower in the Tß4 knock-down cells by Tß4-siRNA (si-Tß4/MC3T3-E1) than that of the untreated cells. The levels of FAK phosphorylation, paxillin expression, and paxillin localization were higher in the Tß4/IMC3T3-E1 cells than that of the untreated cells but lower in the si-Tß4/MC3T3-E1 cells. In addition, the levels of cell proliferation, Grb2 and Ras protein expression and phosphorylation of ERK1/2 were higher in the Tß4/MC3T3-E1 cells than in the untreated cells but lower in the si-Tß4/IMC3T3-E1 cells. These results suggest that Tß4 might be a good nanomolecule that promotes osteoblast survival by facilitating adhesion and proliferation on the Ti surface.

660 nm Red LED Induces Secretory Leukocyte Protease Inhibitor (SLPI) in Lipopolysaccharide-Stimulated RAW264.7 Cell.

SLPI acts as a modulator of the innate immune responses of macrophages, neutrophils and odontoblasts, and LPS-inducible anti-inflammatory cytokine to suppress the production of pro-inflammatory products by macrophages. Many studies have revealed the effects of light emitting diodes (LEDs) on the tissue repair and inflammatory responses. Although the anti-inflammatory mechanisms of irradiation with LEDs in gingival fibroblasts are known, the effects of 660 nm red LEDs on the inflammation remain unclear. Moreover, there is no report regarding the molecular mechanism for the relationship between SLPI and biological effects of LEDs. The effects of 660 nm red LEDs on inflammation with SLPI were investigated by examining the effects of 660 nm LED on the SLPI expression of RAW264.7 cells after LPS stimulation. This paper reports that the 660 nm red LED induced SLPI expression or reduced the LPS response, and inhibited NF-κB activation directly, leading to the suppression of pro-inflammatory cytokines, such as TNF-α and IL-1ß, suggesting that it might be a useful wavelength LED for inflammation therapy.

Tumor necrosis factor-alpha (TNF-α) is a therapeutic target for impaired cutaneous wound healing.

Impaired wound healing states lead to substantial morbidity and cost with treatment resulting in an expenditure of billions of dollars per annum in the U.S. alone. Both chronic wounds and impaired acute wounds are characterized by excessive inflammation, enhanced proteolysis, and reduced matrix deposition. These confounding factors are exacerbated in the elderly, in part, as we report here, related to increased local and systemic tumor necrosis factor-alpha (TNF-α) levels. Moreover, we have used a secretory leukocyte protease inhibitor (SLPI) null mouse model of severely impaired wound healing and excessive inflammation, comparable to age-related delayed human healing, to demonstrate that topical application of anti-TNF-α neutralizing antibodies blunts leukocyte recruitment and NFκB activation, alters the balance between M1 and M2 macrophages, and accelerates wound healing. Following antagonism of TNF-α, matrix synthesis is enhanced, associated with suppression of both inflammatory parameters and NFκB binding activity. Our data suggest that inhibiting TNF-α is a critical event in reversing the severely impaired healing response associated with the absence of SLPI, and may be applicable to prophylaxis and/or treatment of impaired wound healing states in humans.

Dynamin II involves in cell migration and actin formation of NIH3T3 cells.

It was previously reported that in Ras transformed NIH3T3 cells, dynamin II acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration. However, these results do not provide sufficient evidence of a relationship between dynamin II and the Ras signal transduction pathway leading to membrane ruffling and cell migration. The results showed that a dynamin II association with myosin II as a signaling molecule is involved in NIH3T3 cell migration through the Ras/PI3K signaling pathway, and is associated with the p85 subunit of PI3K. Confocal microscopy also revealed co-localization between dynamin II and paxillin after PDGF stimulation. In addition, immunofluorescence results showed that dynamin II was colocalized with the actin filament. After stimulating the NIH3T3 cells with PDGF and treating them with an actin inhibitor, such as Cytochalasin D, it was observed that dynamin II with the myosin II complex inhibited binding to the actin. Therefore, dynamin II is localized in focal adhesion when cell migration is triggered and binds to the actin filament component, suggesting that it is a good candidate nanomolecule to regulate the cell attachment and migration to the materials such as implants etc.

Hyaluronic acid immobilization on the poly-allylamine coated nano-network TiO2 surface.

Recently, biocompatibility report revealed that the TiO2 nano-network (TiO2 NT) structure has much higher cells colonization than the native TiO2 on Ti surface. In this study, we prepared the hyaluronic acid (HA) immobilized TiO2 NT layer by plasma surface modification and then evaluated biological behavior of MC3T3-E1 on the Ti, TiO2 NT and TiO2 NT/NH2/HA surface. The cell viability tests revealed slightly enhanced viability on the TiO2 NT/NH2/HA surfaces than on the untreated Ti surfaces.

Expression of dynamin II in odontoblast during mouse tooth development.

Odontoblasts secrete a collagen-based matrix and release numerous membrane-bound matrix vesicles, which are involved in dentin formation during tooth development. Dynamin II is a GTPase protein that contributes a variety of vesicular budding events, such as endocytotic membrane fission, caveolae internalization and protein trafficking in the Golgi apparatus. However, the expression and function of dynamin II in odontoblasts has not been reported. Therefore, this study examined the expression and possible role of dynamin II in odontoblasts during tooth development and mineralization. The levels of mRNA and protein expression in MDPC23 cells were significantly high at the early stages of differentiation and then decreased gradually thereafter. Immunohistochemistry showed that dynamin II was not expressed near the region of the odontoblasts at embryonic day 17 (E17) and E21. However, dynamin II was expressed strongly in the odontoblast layer at postnatal day 1 (PN1) and decreased gradually at PN3 and PN5. In addition, at PN15 in the functional stage, the dynamin II protein was also expressed in the odontoblast process as well as adjacent to the nuclear region. In conclusion, dynamin II may be involved in the transport of vesicles containing collageneous and non-collageneous proteins for dentin formation in odontoblast, suggesting that it is a good nanomolecule as a candidate to regulate the secretion of collagen on the bone and other nano material.

Effect of rosmarinic acid on differentiation and mineralization of MC3T3-E1 osteoblastic cells on titanium surface.

Titanium (Ti) is a widely used biomaterial for dental implants because of its outstanding biocompatibility for hard tissues. Osseointegration, the interaction between implanted biomaterials and living cells in bone, is essential for successful implantation. Rosmarinic acid (RA) is a plant-derived phytochemical with low toxicity and side effects and has various effects that can be applied as a therapeutic substance. The MC3T3-E1 osteoblastic cells on the Ti surface in medium with or without 14 µg/ml RA were used to test RA effects on osteoblast differentiation, cell viability and mineralization during differentiation. RA treatment increased osteoblast differentiation, cell viability and mineralization in MC3T3-E1 osteoblastic cells on Ti surface during differentiation, upregulating Runx-2 and OPG, but downregulating RANKL. This study suggest that RA should be applied as an effective functional and therapeutic substance to enhance osseointegration of osteoblast cells by increasing differentiation, mineralization, and bone formation through the RANKL/RANK/OPG pathway during the differentiation in MC3T3-E1 osteoblastic cells on the Ti surface.

Effect of Korean red ginseng extract in a steroid-induced polycystic ovary murine model.

Experimental induction of polycystic ovary (PCO) in rodent resembling some aspects of human PCO syndrome was produced using the long-acting compound estradiol valerate (EV). Our previous study on the role of Korean red ginseng total saponins in a steroid-induced PCO rat model demonstrated that electro-acupuncture modulates nerve growth factor (NGF) concentration in the ovaries. In fact, the involvement of a neurogenic component in the pathology of PCO-related ovarian dysfunction is preceded by an increase in sympathetic outflow to the ovaries. In the present study, we tested the hypothesis that Korean red ginseng extract (KRGE) administration modulates sympathetic nerve activity in PCO-induced rats. This was done by analyzing NGF protein and NGF mRNA expression involved in the pathophysiological process underlying steroid-induced PCO. EV injection resulted in significantly higher ovarian NGF protein and NGF mRNA expression in PCO-induced rats compared to control rats, and PCO ovaries were counteracted by KRGE administration with significantly lower expression of NGF protein and NGF mRNA compared to EV treated ovaries. These results indicate that EV modulates the neurotrophic state of the ovaries, which may be a component of the pathological process by which EV induces cyst formation and anovulation in rodents.

Thymosin ß4 is associated with bone sialoprotein expression via ERK and Smad3 signaling pathways in MDPC-23 odontoblastic cells.

Thymosin ß4 (Tß4) regulates the expression of molecules associated with dentinogenesis, including bone sialoprotein (BSP). BSP regulates the initiation of mineralization and the direction of dentin growth. However, the association between Tß4 signaling and BSP expression in odontoblasts remains unclear. Therefore, the aim of the present study was to investigate Tß4 mRNA expression in odontoblasts during dentinogenesis and the association between the Tß4 signaling pathway and BSP expression in MDPC­23 odontoblastic cells. Expression and localization of Tß4 mRNA was determined by in situ hybridization during mouse tooth development. The effect of Tß4 signaling on BSP expression was investigated by reverse transcription polymerase chain reaction, western blot analysis, immunofluorescence and a luciferase reporter assay in the presence or absence of specific inhibitors of mitogen activated protein kinase kinase (PD98059) and mothers against decapentaplegic homolog 3 (Smad3; SIS3) in MDPC­23 cells. The expression of Tß4 mRNA in the odontoblast layer was highest at postnatal day 5, known as the advanced bell stage, when odontoblasts actively secrete dentin matrix proteins. Tß4 increased BSP mRNA and protein levels in MDPC­23 cells, but this was inhibited by PD98059 or SIS3 treatment. Tß4 increased levels of phosphorylated (p) extracellular signal­regulated kinase (ERK)1/2, pSmad3, pß­catenin, and runt­related transcription factor 2 (Runx2) protein, but these effects were inhibited by PD98059 or SIS3. Tß4 induced the nuclear translocation of Runx2 and pSmad3, while nuclear translocation of ß­catenin was decreased. Tß4 significantly increased BSP promoter activity, which was decreased by PD98059 or SIS3 treatment. Tß4 induced BSP expression in MDPC­23 cells via ERK and Smad3 signaling pathways, suggesting its role as a signaling molecule in odontoblasts for regulating BSP secretion during dentinogenesis.

Estrogen modulates cutaneous wound healing by downregulating macrophage migration inhibitory factor.

Characteristic of both chronic wounds and acute wounds that fail to heal are excessive leukocytosis and reduced matrix deposition. Estrogen is a major regulator of wound repair that can reverse age-related impaired wound healing in human and animal models, characterized by a dampened inflammatory response and increased matrix deposited at the wound site. Macrophage migration inhibitory factor (MIF) is a candidate proinflammatory cytokine involved in the hormonal regulation of inflammation. We demonstrate that MIF is upregulated in a distinct spatial and temporal pattern during wound healing and its expression is markedly elevated in wounds of estrogen-deficient mice as compared with intact animals. Wound-healing studies in mice rendered null for the MIF gene have demonstrated that in the absence of MIF, the excessive inflammation and delayed-healing phenotype associated with reduced estrogen is reversed. Moreover, in vitro assays have shown a striking estrogen-mediated decrease in MIF production by activated murine macrophages, a process involving the estrogen receptor. We suggest that estrogen inhibits the local inflammatory response by downregulating MIF, suggesting a specific target for future therapeutic intervention in impaired wound-healing states.

Effects of aucubin on the healing of oral wounds.

Aucubin is an iridoid glycoside with a variety of pharmacological effects, such as antimicrobial and antiinflammatory, whilst also promoting dermal wound healing. This study examined the effects of 0.1% aucubin on oral wound healing. ICR male mice were divided into two groups: an untreated control group (n=18) and an aucubin-treated group (n=18). Saline or 0.1% aucubin solution was injected and artificial full thickness wounds were made on either side of the buccal mucosa. Specimens were taken on days 1, 3 and 5, and light microscopic examination and quantitative histological analysis were performed to determine the extent of re-epithelization, inflammatory cell infiltration and matrix formation. Re-epithelization and matrix formation of the aucubin-treated group occurred earlier than that of the control group. In addition, the number of inflammatory cells of the aucubin-treated group was fewer than that of the control group. In conclusion, aucubin may be useful for oral wound healing and can be applied as a topical agent to oral wounds.

Formaldehyde exposure induces airway inflammation by increasing eosinophil infiltrations through the regulation of reactive oxygen species production.

Formaldehyde (FA) is a well-known cytotoxic irritant to the airways, but the mechanism of airway inflammation due to FA has not been clarified. In the present study, C57BL/6 mice were exposed to two concentrations (5 and 10ppm) of FA for 6h/day, 5days/week, for 2 weeks. The FA-exposed mice had much higher number of CCR3(+) eosinophils than control mice, and showed upregulated gene expression of CC-chemokine receptor-3 (CCR3), eotaxin and intercellular adhesion molecules-1 (ICAM-1) as well as an increased expression of proinflammatory and Th2 cytokines, such as interleukin (IL)-1ß, IL-4 and IL-5. In addition, FA exposure revealed a considerable increase in the serum levels of IgG1, IgG3, IgA and IgE compared to controls. Histopathological analysis of the lung tissues demonstrated eosinophils and mononuclear cell infiltration of the alveolar cell walls and alveolar spaces. Gene expression of thioredoxin (TRX), redox-regulating antioxidant proteins, was markedly suppressed in FA-exposed mice, and thereby intracellular ROS levels were increased along with increased FA concentration. These results were consistent with an increase in the number of CCR3-expressing eosinophils, and indicate that FA-induced ROS was generated from eosinophils recruited to the inflammatory sites of the airways.

Antioxidant and cytoprotective effects of morin against hydrogen peroxide-induced oxidative stress are associated with the induction of Nrf-2­mediated HO-1 expression in V79-4 Chinese hamster lung fibroblasts.

Natural phytochemicals of plant origin, including flavonoids, have been found to be potent antioxidants providing beneficial effects against oxidative stress-related diseases. The present study was carried out to investigate the antioxidant properties of morin, a flavonoid originally isolated from the flowering plants of the Moraceae family. Superoxide dismutase (SOD)­like activity and 2,2'­azino­bis­(3­ethylbenzothiazoline­6­sulfonic acid) (ABTS•+) radical scavenging activity were determined. We also investigated the cytoprotective effects of morin against hydrogen peroxide (H2O2)­induced DNA damage and apoptosis in V79­4 Chinese hamster lung fibroblasts. Our results demonstrated that morin had strong scavenging effects against ABTS•+ radicals with enhanced SOD activity, which varied in a dose-dependent manner. Morin was found to reduce H2O2­induced intracellular reactive oxygen species generation and nuclear DNA damage, and it recovered cell viability damaged by H2O2 via inhibition of mitochondrial dysfunction­mediated apoptosis. Notably, the treatment of V79­4 cells with morin markedly enhanced the expression of heme oxygenase­1 (HO­1) but not quinone oxidoreductase-1, which was associated with the increased expression and phosphorylation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and the downregulation of Kelch­like ECH­associated protein 1 expression. Based on our findings, we conclude that morin effectively ameliorated oxidative stress­induced DNA damage through intrinsic free radical scavenging activity and activation of the Nrf2/HO-1 pathway.

Ultrastructural changes of the gemifloxacin on Achilles tendon in immature rats: comparison with those of ciproxacin and ofloxacin.

Gemifloxacin is a synthetic fluoroquinolone antimicrobial agent that exhibits potent activity against most Gram-negative and Gram-positive organisms, and has a comparatively low chondrotoxic potential in immature animals. This study examined the effects of gemifloxacin on the Achilles tendons in immature Sprague-Dawley rats treated by oral intubation once daily for 5 consecutive days from postnatal week 4 onward at doses of 0 (vehicle), and 600 mg/kg body weight. Ofloxacin or ciprofloxacin were used for comparison. The Achilles tendon specimens were examined by electron microscopy. In comparison with the vehicle-treated controls, there were ultrastructural changes in all samples from the gemifloxacin-, ofloxacin-, and ciprofloxacin-treated rats. Degenerative changes were observed in the tenocytes, and the cells that detached from the extracellular matrix were recognizable. The degree of degenerative changes and the number of degenerated cells in the Achilles tendon were significantly higher in the treated group than in the control group. Moreover, among the quinolone-treated groups, these findings were most significant in the ofloxacin-treated group, and least significant in the gemifloxacin-treated group. It is unclear what these findings mean with respect to the possible risk in juvenile patients treated with gemifloxacin or other quinolones. However, these results show that gemifloxacin causes less changes in the connective tissue structures.

Comparative fine structure of the epididymal spermatozoa from three Korean shrews with considerations on their phylogenetic relationships.

This study examined the fine structures of epididymal spermatozoa on the lesser white-toothed shrew (Crocidura suaveolens), the Japanese white-toothed shrew (C. dsinezumi) and the big white-toothed shrew (C. lasiura) belonging to the subfamily Crocidurinae living in Korea. In the spermatozoa of C. suaveolens, the head has a large acrosome, a smooth inner acrosomal membrane and a wavy, finger-like, electron-dense apical body. The neck has a solid proximal centriole that is filled with electron-dense material. These results showed the spermatozoa of C. suaveolens possess the characteristics of both Crocidurinae and Soricinae. In C. dsinezumi and C. lasiura, the head has a large acrosome, a serrated inner acrosomal membrane and a common apical body. The neck has a fistulous proximal centriole with slightly dense electron granules. These results showed the typical characteristics of Crocidurinae. Although C. suaveolens belongs to the subfamily Crocidurinae, the spermatozoan morphology is different from C. dsinezumi and C. lasiurai because it has conserved characteristics of the subfamily Soricinae.