Basic Consideration for Facial Aging: Age-Related Changes of the Bony Orbit and Orbicularis Oculi Muscle in East Asians.

BACKGROUND: Age-related changes in the periocular areas are mainly caused by anatomic changes of the bony orbit and orbicularis oculi muscle (OOM). To achieve effective rejuvenation, it is necessary to understand the age-related aspects of these anatomic changes. OBJECTIVES: The aim of this study was to analyze the configuration of the bony orbit and OOM with computed tomography (CT) and to evaluate the effects of aging on these structures. METHODS: A total of 220 orbits and OOMs of 110 Japanese participants (55 males, 55 females) aged 20 to 87 years were enrolled. The long diameter of the orbits, orbital ellipticity, OOM thickness, and OOM attachment to the inferior orbital rim were analyzed. These variables were statistically evaluated for their relationship with age. RESULTS: The long diameter of the orbit was significantly longer in those over than in those under 60 years, with a moderate and significant positive correlation between orbital ellipticity and age. OOM thickness and age showed a strong negative correlation. The degree of OOM attachment to the inferior orbital rim decreased significantly with age. CONCLUSIONS: This study showed that age-related changes of the bony orbit in Japanese individuals tended to be the same as those in Caucasians, but there were differences in the degree of changes observed. As a new finding in the Japanese population, the OOM not only thins with aging, but also gradually loosens from the facial bone. In the elderly, only the nasal side of the OOM was attached to the bone. In clinical applications, this knowledge could contribute to the development of cosmetic surgeries.

A vertebrate-specific qPCR assay as an endogenous internal control for robust species identification.

The identification of vertebrate species is important in numerous fields including archaeology, ecology, as well as food and forensic sciences. Real-time quantitative PCR (qPCR) assays specific for one vertebrate species are promising approaches for species identification, although there are several drawbacks such as difficulty determining whether the detected DNA is authentic or a contaminant. Here, we describe a qPCR assay specific for vertebrate mitochondrial DNA (mtDNA) which can overcome these drawbacks. Since we found that mitochondrial 16S rRNA contains regions that are perfectly (not highly) conserved across virtually all vertebrates, but are variable in invertebrates, we were able to design a vertebrate-specific qPCR assay by placing primers/probe within these regions. The specificity and accuracy of this assay were validated with representative vertebrate and invertebrate samples. This assay detected DNA from all vertebrate samples, but not from any invertebrate samples. In addition, this assay was able to quantify vertebrate mtDNAs as accurately as previously reported species-specific qPCR assays. The results demonstrated it is feasible to quantify vertebrate mtDNA specifically and accurately in a sample. This means that it is possible to determine the ratio of specific vertebrate species mtDNA to total vertebrate mtDNA in a sample. In conjunction with this assay as an endogenous internal control, species-specific qPCR assays will allow for the robust identification of vertebrate species.

Automatic landmark annotation in 3D surface scans of skulls: Methodological proposal and reliability study.

BACKGROUND AND OBJECTIVES: Craniometric landmarks are essential in many biomedical applications, such as morphometric analysis or forensic identification. The process of locating landmarks is usually a manual and slow task, highly influenced by fatigue, skills and the experience of the practitioner. Localization errors are propagated and magnified in subsequent steps, which can result in incorrect measurements or assumptions. Thereby, standardization, reliability and reproducibility lay the foundations for the necessary accuracy in subsequent measurements or anatomical analysis. In this paper, we present an automatic method to annotate 3D surface skull models taking into account anatomical and geometrical features. METHODS: The proposed method follows a hybrid structure where a deformable template is used to initialize the landmark positions. Then, a refinement stage is applied using prior anatomical knowledge to ensure a correct placement. Our proposal is validated over thirty 3D skull scans of male Caucasians, acquired by hand-held surface scanning, and a set of 58 craniometric landmarks. A statistical analysis was carried out to analyze the inter- and intra-observer variability of manual annotations and the automatic results, along with a visual assessment of the final results. RESULTS: Inter-observer errors show significant differences, which are reflected in the expert consensus used as reference. The average localization error was 2.19±1.5 mm when comparing the automatic landmarks to the reference location. The subsequent visual analysis confirmed the reliability of the refinement method for most landmarks. CONCLUSIONS: Repeated manual annotations show a high variability depending on both skills and expertise of the observer, and landmarks' location and characteristics. In contrast, the automatic method provides an accurate, robust and reproducible alternative to the tedious and error-prone task of manual landmarking.

Evaluation of Parameters for Estimating the Postmortem Interval of Skeletal Remains Using Bovine Femurs: A Pilot Study.

The postmortem interval (PMI) of victims is a key parameter in criminal investigations. However, effective methods for estimating the PMI of skeletal remains have not been established because it is determined by various factors, including environmental conditions. To identify effective parameters for estimating the PMI of skeletal remains, we investigated the change in bone focusing on the amount of DNA, element concentrations, and bone density that occurred in the bone samples of bovine femurs, each maintained under one of five simulated environmental conditions (seawater, freshwater, underground, outdoors, and indoors) for 1 year. The amount of extracted mitochondrial DNA (mtDNA; 404 bp fragment) decreased over time, and significant DNA degradation (p < 0.01), as estimated by a comparison with amplification results for a shorter fragment (128 bp), was detected between 1 month and 3 months. Eleven of 30 elements were detected in samples by inductively coupled plasma optical emission spectrometry, and Na and Ba showed significant quantitative differences in terms of environmental conditions and time (p < 0.01). This preliminary study suggests that the level of DNA degradation determined by real-time polymerase chain reaction and element concentrations determined by inductively coupled plasma optical emission may be useful indices for estimating the PMI of victims under a wide range of environmental conditions. However, this study is a limited experimental research and not applicable to forensic cases as it is. Further studies of human bone with longer observation periods are required to verify these findings and to establish effective methods for PMI estimation.

Three-dimensional shape variation and sexual dimorphism of the face, nose, and mouth of Japanese individuals.

Three-dimensional (3D) shape variations of the face and facial parts in Japanese adults were examined to collect basic data to be used for facial comparison in forensics. In total, 1000 3D facial scans (500 males, 500 females) of Japanese individuals were re-meshed into anatomically homologous shape models and analyzed by principal component analysis (PCA) after Procrustes superimposition. Facial parts (the nose and the mouth) were segmented from homologous face models and analyzed by PCA, too. Among all kinds of objects (the face, the nose, and the mouth), the most predominant shape variation represented by the first principal component (PC1) was the height-width proportion. The second largest variation (PC2) in the face and the nose was depth; for the mouth, it was the relative protrusion of the upper and lower lips. We interpreted predominant shape variations represented by the first five principal components (PCs) in each object. Asymmetric shape variations were observed within these PCs for the nose and the mouth. Sexual dimorphism of the face and the facial parts was also examined by testing the significance of sex-linked differences in PC scores. A significant difference was found between males and females for many PCs. Sexual dimorphism was examined also by emphasizing the shape difference between average male and female faces. Our results revealed predominant 3D shape variations and sexual dimorphism of the face and facial parts. The results may be informative for performing facial comparison in police investigations, an increasingly used technique.

Anti-Inflammatory Effect of ETAS®50 by Inhibiting Nuclear Factor-κB p65 Nuclear Import in Ultraviolet-B-Irradiated Normal Human Dermal Fibroblasts.

Ultraviolet (UV) irradiation induces proinflammatory responses in skin cells, including dermal fibroblasts, accelerating premature skin aging (photoaging). ETAS 50, a standardized extract from the Asparagus officinalis stem, is a novel and unique functional food that suppresses proinflammatory responses of hydrogen peroxide-stimulated skin fibroblasts and interleukin- (IL-) 1ß-stimulated hepatocytes. To elucidate its antiphotoaging potencies, we examined whether ETAS 50 treatment after UV-B irradiation attenuates proinflammatory responses of normal human dermal fibroblasts (NHDFs). UV-B-irradiated NHDFs showed reduced levels of the cytosolic inhibitor of nuclear factor-κB α (IκBα) protein and increased levels of nuclear p65 protein. The nuclear factor-κB nuclear translocation inhibitor JSH-23 abolished UV-B irradiation-induced IL-1ß mRNA expression, indicating that p65 regulates transcriptional induction. ETAS 50 also markedly suppressed UV-B irradiation-induced increases in IL-1ß mRNA levels. Immunofluorescence analysis revealed that ETAS 50 retained p65 in the cytosol after UV-B irradiation. Western blotting also showed that ETAS 50 suppressed the UV-B irradiation-induced increases in nuclear p65 protein. Moreover, ETAS 50 clearly suppressed UV-B irradiation-induced distribution of importin-α protein levels in the nucleus without recovering cytosolic IκBα protein levels. These results suggest that ETAS 50 exerts anti-inflammatory effects on UV-B-irradiated NHDFs by suppressing the nuclear import machinery of p65. Therefore, ETAS 50 may prevent photoaging by suppressing UV irradiation-induced proinflammatory responses of dermal fibroblasts.

Regular Voluntary Exercise Potentiates Interleukin-1ß and Interleukin-18 Secretion by Increasing Caspase-1 Expression in Murine Macrophages.

Moderate-intensity regular exercise improves proinflammatory responses of lipopolysaccharide- (LPS-) stimulated macrophages. However, intracellular events that mediate the beneficial effects of exercise were unclear. This study aimed to clarify the mechanism by which regular voluntary exercise (VE) improves proinflammatory cytokine production by macrophages challenged with LPS. Peritoneal macrophages from VE mice secreted considerably higher amounts of interleukin- (IL-) 1ß and IL-18 than did cells from sedentary control (SC) mice in the presence and absence of LPS, although tumor necrosis factor-α and IL-10 secretion were comparable between both groups. The mRNA levels of these cytokines increased significantly in response to LPS; similar levels were noted in macrophages from both SC and VE mice. Moreover, LPS evoked similar levels of degradation of inhibitor of κB (IκB) α and phosphorylation of IκB kinase ß, c-Jun N-terminal kinase, and p38 in macrophages from SC and VE mice. These results indicate that the increased IL-1ß and IL-18 secretion in VE mice are regulated posttranscriptionally. On the other hand, macrophages from VE mice showed higher amounts of caspase-1 protein than did cells from SC mice. These results suggest that regular VE potentiates IL-1ß and IL-18 secretion in LPS-challenged macrophages by increasing caspase-1 levels.

Study on the criteria for assessing skull-face correspondence in craniofacial superimposition.

Craniofacial superimposition has the potential to be used as an identification method when other traditional biological techniques are not applicable due to insufficient quality or absence of ante-mortem and post-mortem data. Despite having been used in many countries as a method of inclusion and exclusion for over a century it lacks standards. Thus, the purpose of this research is to provide forensic practitioners with standard criteria for analysing skull-face relationships. Thirty-seven experts from 16 different institutions participated in this study, which consisted of evaluating 65 criteria for assessing skull-face anatomical consistency on a sample of 24 different skull-face superimpositions. An unbiased statistical analysis established the most objective and discriminative criteria. Results did not show strong associations, however, important insights to address lack of standards were provided. In addition, a novel methodology for understanding and standardizing identification methods based on the observation of morphological patterns has been proposed.

Enzyme-Treated Asparagus Extract Attenuates Hydrogen Peroxide-Induced Matrix Metalloproteinase-9 Expression in Murine Skin Fibroblast L929 Cells.

Enzyme-treated asparagus extract (ETAS) exerts a wide variety of beneficial biological actions including facilitating anti-cortisol stress and neurological anti-aging responses. However, the anti-skin aging effects of ETAS remain to be elucidated. Reactive oxygen species (ROS) play pivotal roles in skin aging. Increased ROS levels in fibroblasts in response to ultraviolet irradiation activate c-Jun N-terminal kinase (JNK) and its downstream transcription factor activator protein-1 (AP-1), and the resultant gene expression of matrix metalloproteinase (MMP) isoforms accelerates collagen breakdown in the dermis. Therefore, we explored whether ETAS has anti-skin aging effects by attenuating the oxidative stress responses in fibroblasts. Simultaneous treatment of murine skin L929 fibroblasts with hydrogen peroxide (H2O2) and either ETAS or dextrin showed that ETAS significantly suppressed H2O2-induced expression of MMP-9 mRNA as measured by real-time polymerase chain reaction. ETAS also clearly suppressed H2O2-stimulated phosphorylation of c-Jun (AP-1 subunit) and JNK as determined by Western blot. However, ETAS did not affect the increased amounts of carbonyl proteins in response to H2O2, also as determined by Western blotting. These results suggest that ETAS diminishes cellular responsiveness to ROS but does not scavenge ROS. Thus, ETAS has the potential to prevent skin aging through attenuating the oxidative stress responses in dermal fibroblasts.

Enzyme-Treated Asparagus Extract Prevents'Hydrogen Peroxide-Induced Pro-Inflammatory Responses by Suppressing p65 Nuclear Translocation in Skin L929 Fibroblasts.

We recently reported that enzyme-treated asparagus extract (ETAS) attenuates hydrogen peroxide (H(2)0(2))-stimulated matrix metalloproteinase-9 expression in skin fibroblast L929 cells. To further elucidate the anti-aging effects of ETAS on skin, we examined whether ETAS has preventive effects on H202-induced pro-inflammatory responses of skin fibroblasts. H(2)0(2) induced Ser536 phosphorylation and nuclear accumulation of nuclear factor-κB (NF-κB) p65, and increased the mRNA levels .of interleukin-12α (IL-12α)-and inducible nitric oxide synthase (iNOS) in L929 cells. Pretreatment of the cells with JSH-23, an inhibitor of NF-κB nuclear translocation, abolished the H(2)(0(2)-induced expression of IL-12α and iNOS, indicating that the increased transcription is regulated by p65. The H(2)0(2)-stimulated nuclear accumulation of p65 and-induction of IL12a and iNOS mRNA were significantly attenuated after pretreatment with ETAS for 3 h, and these responses were completely abolished when the duration was extended to 24 h. However, ETAS did not affect the H(2)0(2)-stimulated degradation of IκBα and phosphorylation of p65. On the other hand, ETAS treatment for 24 h resulted in decreased protein levels of importin-α. These results suggest that ETAS prevents pro-inflammatory responses by suppressing the p65 nuclear translocation in skin fibroblasts induced by H202.

Three-dimensional analyses of aging-induced alterations in facial shape: a longitudinal study of 171 Japanese males.

We sought to generate data to facilitate forensic facial comparisons. Specifically, we conducted a longitudinal study of alterations in face shape induced by aging. We obtained two three-dimensional facial shape measurements in 171 Japanese males at intervals of approximately 10 years. With this data, we created a homologous model consisting of 10,741 data points for each face based on 33 anatomical landmarks. We averaged the movements of corresponding data points between the two homologous models for each individual and used this data to predict up to 30 years of face aging in an average Japanese male. We clearly identified aging-induced shape changes, such as drooping and denting of the facial folds, drooping of the upper lip, and projection of the lower eyelid, in the virtually aged model. A quantitative comparison of aging-induced shape alterations among three age groups (individuals in their 20's, 30's, and 40-50's) showed that these alterations accelerated more quickly as age increased. Using our predictive model, we conducted a preliminary study focused on facial shape alterations induced by reductions in body weight. Our findings indicated that our proposed method would also be valid for this purpose.

Direct and indirect suppression of interleukin-6 gene expression in murine macrophages by nuclear orphan receptor REV-ERBα.

It is now evident that many nuclear hormone receptors can modulate target gene expression. REV-ERBα, one of the nuclear hormone receptors with the capacity to alter clock function, is critically involved in lipid metabolism, adipogenesis, and the inflammatory response. Recent studies suggest that REV-ERBα plays a key role in the mediation between clockwork and inflammation. The purpose of the current study was to investigate the role of REV-ERBα in the regulation of interleukin-6 (il6) gene expression in murine macrophages. REV-ERBα agonists, or overexpression of rev-erb α in the murine macrophage cell line RAW264 cells, suppressed the induction of il6 mRNA following a lipopolysaccharide (LPS) endotoxin challenge. Also, rev-erb α overexpression decreased LPS-stimulated nuclear factor κB (NFκB) activation in RAW264 cells. We showed that REV-ERBα represses il6 expression not only indirectly through an NFκB binding motif but also directly through a REV-ERBα binding motif in the murine il6 promoter region. Furthermore, peritoneal macrophages from mice lacking rev-erb α increased il6 mRNA expression. These data suggest that REV-ERBα regulates the inflammatory response of macrophages through the suppression of il6 expression. REV-ERBα may therefore be identified as a potent anti-inflammatory receptor and be a therapeutic target receptor of inflammatory diseases.

Posttranscriptional Suppression of Lipopolysaccharide-Stimulated Inflammatory Responses by Macrophages in Middle-Aged Mice: A Possible Role for Eukaryotic Initiation Factor 2 α.

The intensities of macrophage inflammatory responses to bacterial components gradually decrease with age. Given that a reduced rate of protein synthesis is a common age-related biochemical change, which is partially mediated by increased phosphorylation of eukaryotic initiation factor-2 α (eIF-2 α ), we investigated the mechanism responsible for the deterioration of macrophage inflammatory responses, focusing specifically on the age-related biochemical changes in middle-aged mice. Peritoneal macrophages isolated from 2-month-old (young) and 12-month-old (middle-aged) male BALB/c mice were stimulated with lipopolysaccharide (LPS). Although LPS-stimulated secretion of tumor necrosis factor- α (TNF- α ) by the macrophages from middle-aged mice was significantly lower than that from young mice, LPS caused marked increases in levels of TNF- α mRNA in macrophages from middle-aged as well as young mice. Moreover, LPS evoked similar levels of phosphorylation of c-Jun N-terminal kinase (JNK) and nuclear factor- κ B (NF- κ B) in young and middle-aged mice. In contrast, the basal level of phosphorylated eIF-2 α in macrophages from middle-aged mice was higher than that in macrophages from young mice. Salubrinal, an inhibitor of the phosphatase activity that dephosphorylates eIF-2 α , suppressed the LPS-stimulated inflammatory responses in a murine macrophage cell line RAW264.7. These results suggest that posttranscriptional suppression of macrophage inflammatory responses during middle age requires phosphorylation of eIF-2 α .

A circadian clock gene, Rev-erbα, modulates the inflammatory function of macrophages through the negative regulation of Ccl2 expression.

Disruption of the circadian rhythm is a contributory factor to clinical and pathophysiological conditions, including cancer, the metabolic syndrome, and inflammation. Chronic and systemic inflammation are a potential trigger of type 2 diabetes and cardiovascular disease and are caused by the infiltration of large numbers of inflammatory macrophages into tissue. Although recent studies identified the circadian clock gene Rev-erbα, a member of the orphan nuclear receptors, as a key mediator between clockwork and inflammation, the molecular mechanism remains unknown. In this study, we demonstrate that Rev-erbα modulates the inflammatory function of macrophages through the direct regulation of Ccl2 expression. Clinical conditions associated with chronic and systemic inflammation, such as aging or obesity, dampened Rev-erbα gene expression in peritoneal macrophages from C57BL/6J mice. Rev-erbα agonists or overexpression of Rev-erbα in the murine macrophage cell line RAW264 suppressed the induction of Ccl2 following an LPS endotoxin challenge. We discovered that Rev-erbα represses Ccl2 expression directly through a Rev-erbα-binding motif in the Ccl2 promoter region. Rev-erbα also suppressed CCL2-activated signals, ERK and p38, which was recovered by the addition of exogenous CCL2. Further, Rev-erbα impaired cell adhesion and migration, which are inflammatory responses activated through the ERK- and p38-signaling pathways, respectively. Peritoneal macrophages from mice lacking Rev-erbα display increases in Ccl2 expression. These data suggest that Rev-erbα regulates the inflammatory infiltration of macrophages through the suppression of Ccl2 expression. Therefore, Rev-erbα may be a key link between aging- or obesity-associated impairment of clockwork and inflammation.

Dose-dependent effects of diallyl disulfide on plasma glucose and free fatty acid levels in rats.

The acute effects of oral administration of diallyl disulfide (DADS), the major organosulfur compound of garlic, on plasma glucose and free fatty acid (FFA) concentrations were examined in rats. Male, 10-week-old Sprague-Dawley rats were divided into DADS-free and DADS-administered (dose = 10, 20, and 40 mg/kg body weight [BW]) groups. Plasma samples were prepared from whole blood drawn from the tail vein 0, 1, 2, 4, and 6 hr after administration. The stomachs were isolated, and the contents were measured 8 hr after administration. In DADS-administered groups, plasma glucose concentrations were increased in a dose-dependent manner 1 hr after the administration. The increase was transient, except in groups administered 40 mg/kg BW of DADS, in which plasma glucose levels remained significantly higher than the DADS-free levels throughout the experimental period. Similar patterns were observed in the plasma FFA concentrations, although the significant differences were lower than those observed in the plasma glucose concentrations. The gastric contents were dose-dependently elevated after DADS administration. The increase was significant when 20 or 40 mg/kg BW of DADS was administered. These results suggest that oral administration of DADS can mobilize energy substrates into the blood, although a higher dose of DADS slows gastric emptying.

An evaluation of the effect of microwave irradiation on bone decalcification aimed to DNA extraction.

An effect of intermittent microwave irradiation on decalcification of compact bone followed by DNA extraction was verified. In order to perform quantitative analysis regarding the degree of decalcification, Cubic bone specimens were prepared from bovine metacarpal bone and micro-focus X-ray CT imaging was applied to measure precise volume of decalcified area in the cubes. Microwave irradiation was performed under strict control of temperature using commercially available experimental device which is designed for advancing tissue fixation, decalcification, and antigen-antibody reaction by intermittent microwave. The integrity of the DNA obtained from irradiated specimen was also examined by PCR analysis. The results of morphological analysis with CT imaging showed that microwave irradiation has a positive effect on decalcification though that effect is not so drastic. The results obtained from PCR analysis showed that microwave irradiation decrease amplifiable DNA, suggesting that we should be careful to use microwave for the purpose of bone DNA extraction.

Discriminant functions for sex estimation of modern Japanese skulls.

The purpose of this study is to generate a set of discriminant functions in order to estimate the sex of modern Japanese skulls. To conduct the analysis, the anthropological measurement data of 113 individuals (73 males and 40 females) were collected from recent forensic anthropological test records at the National Research Institute of Police Science, Japan. Birth years of the individuals ranged from 1926 to 1979, and age at death was over 19 years for all individuals. A total of 10 anthropological measurements were used in the discriminant function analysis: maximum cranial length, cranial base length, maximum cranial breadth, maximum frontal breadth, basion-bregmatic height, upper facial breadth, bizygomatic breadth, bicondylar breadth, bigonial breadth, and ramal height. As a result, nine discriminant functions were established. The classification accuracy ranged from 79.0 to 89.9% when the measurements of the 113 individuals were substituted into the established functions, from 77.8 to 88.1% when a leave-one-out cross-validation procedure was applied to the data, and from 86.7 to 93.0% when the measurements of 50 new individuals (25 males and 25 females), unrelated to the establishment of the discriminant functions, were used.

Intracellular ß2-adrenergic receptor signaling specificity in mouse skeletal muscle in response to single-dose ß2-agonist clenbuterol treatment and acute exercise.

The aim of this study was to clarify the intracellular ß2-adrenergic receptor signaling specificity in mouse slow-twitch soleus and fast-twitch tibialis anterior (TA) muscles, resulting from single-dose ß2-agonist clenbuterol treatment and acute exercise. At 1, 4, and 24 h after single-dose treatment with clenbuterol or after acute running exercise, the soleus and TA muscles were isolated and subjected to analysis. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) increased after single-dose clenbuterol treatment and acute exercise in the soleus muscle but not in the TA muscle. Although there was no change in the phosphorylation of Akt after acute exercise in either muscle, phosphorylation of Akt in the soleus muscle increased after single-dose clenbuterol treatment, whereas that in the TA muscle remained unchanged. These results suggest that p38 MAPK and Akt pathways play a functional role in the adaptation to clenbuterol treatment and exercise, particularly in slow-twitch muscles.

Predominant expression of H3K9 methyltransferases in prehypertrophic and hypertrophic chondrocytes during mouse growth plate cartilage development.

Histone lysine methylation (HKM) is an epigenetic change that establishes cell-specific gene expression and determines cell fates. In this study, we investigated the expression patterns of histone H3 lysine 9 methyltransferases (H3K9MTases) G9a (euchromatic histone lysine N-methyltransferase 2, Ehmt2), GLP (euchromatic histone lysine N-methyltransferase 1, Ehmt1), SETDB1 (SET domain, bifurcated 1), PRDM2 (PR domain containing 2), SUV39H1 (suppressor of variegation 3-9 homolog 1), and SUV39H2, as well as the distribution of 3 types of HKM at histone H3 lysine 9: mono- (H3K9me1), di- (H3K9me2), or tri-methylation (H3K9me3), during mouse growth plate development. In the forelimb cartilage primordial at embryonic day 12.5 (E12.5), none of the H3K9MTases were detected and H3K9me1, H3K9me2, and H3K9me3 were scarcely detected. At E14.5, the H3K9MTases were expressed at low levels in proliferating chondrocytes and at high levels in prehypertrophic and hypertrophic chondrocytes. Among the H3K9 methylations, H3K9me1 and H3K9me3 were markedly noted in these chondrocytes. At E16.5, G9, GLP, SETDB1, PRDM2, SUV39H1, and SUV39H2, as well as H3K9me1, H3K9me2, and H3K9me3, were detected in prehypertrophic and hypertrophic chondrocytes in the growth plate. Western blotting and real-time quantitative polymerase chain reaction analysis revealed the distributions of G9 and GLP proteins and the expression of all the H3K9MTase mRNAs in prehypertrophic and hypertrophic chondrocytes. These data suggest that H3K9 methyltransferases are predominantly expressed in prehypertrophic and hypertrophic chondrocytes, and that they could be involved in the regulation of gene expression and progression of chondrocyte differentiation by affecting the methylation state of histone H3 lysine 9 in the mouse growth plate.

ß2-agonist clenbuterol suppresses bacterial phagocytosis of splenic macrophages expressing high levels of macrophage receptor with collagenous structure.

Splenic marginal zone macrophages expressing macrophage receptor with collagenous structure (MARCO) contribute to the clearance of blood-borne pathogens. We determined a splenic adherent cell fraction abundantly containing cells expressing a higher level of MARCO by flow cytometry, and examined the effects of daily administration of an anabolic dose of ß2-agonist clenbuterol on the phagocytic capacity of the cells in mice. After 6 weeks of clenbuterol (1.0 mg/kg body weight/d) or vehicle administration to the mice, splenic adherent cells were isolated. These cells were separated into three cell-size subpopulations. Among them, the small-cell subpopulation contained abundantly the cells with markedly higher levels of MARCO and exhibited more intense phagocytic capacity against Escherichia coli, as compared with the other subpopulations. The phagocytic capacity of the small cells was significantly reduced after clenbuterol administration. These results suggest that the utilization of clenbuterol as doping drug impairs bacterial clearance in the spleen.