Abstract: Homeodomain only protein, Hop, is an unusual small protein that modulates target gene transcription without direct binding to DNA. Here we show that Hop interacts with Enhancer of Polycomb1 (Epc1), a homolog of a Drosophila polycomb group gene product that regulates transcription, to induce the skeletal muscle differentiation. Yeast two-hybrid assay with the human adult heart cDNA library revealed that Hop can associate with Epc1. The amino-terminal domain of Epc1 as well as full Epc1 physically interacted with Hop in mammalian cells and in yeast. Epc1 is highly expressed in the embryonic heart and adult skeletal muscles. Serum deprivation induced differentiation of H9c2, a myoblast cell line, into skeletal myocytes, and Epc1 was up-regulated. Differentiation of H9c2 was induced by Epc1 overexpression, although it was severely impaired in Epc1-knockdown cells. Co-transfection of Hop potentiated Epc1-induced transactivation of myogenin and myotube formation. Hop knock-out mice elicited a decrease in myosin heavy chain and myogenin expressions in skeletal muscle and showed delay in hamstring muscle healing after injury. Differentiation was impaired in skeletal myoblasts from Hop knock-out mice. These results suggest that Epc1 plays a role in the initiation of skeletal muscle differentiation, and its interaction with Hop is required for the full activity.

Abstract: AIMS: To investigate whether the regulation of aquaporin (AQP) channels is altered by inhibition of type 2 11beta-hydroxysteroid dehydrogenase (11betaHSD2). METHODS: Male Sprague-Dawley rats were treated with glycyrrhizic acid (GA, 2 g/l drinking water) for 7 days. The expression of AQP2 and AQP3 was determined in the kidney by immunoblotting and immunohistochemistry. The expression of Gsalpha and type VI adenylyl cyclase, and the activity of adenylyl cyclase were also determined. Results: Following the GA treatment, the expression of 11betaHSD2 was significantly decreased in the kidney. The expression of AQP3 was increased, while that of AQP2 remained unchanged. Plasma renin activity and serum aldosterone levels were decreased. Plasma arginine vasopressin (AVP) levels were comparable between the groups. Neither the forskolin-stimulated cAMP generation nor the expression of Gsalpha and type VI adenylyl cyclase was altered significantly. CONCLUSION: A decreased expression of 11betaHSD2 may result in an upregulation of AQP3, in which AVP/cAMP-dependent mechanisms are unlikely to be involved.

Abstract: BACKGROUND: A number of distinct stress signaling pathways in myocardium cause cardiac hypertrophy and heart failure. Class II histone deacetylases (HDACs) antagonize several stress-induced pathways and hypertrophy. However, cardiac hypertrophy induced by transgenic overexpression of the homeodomain only protein, HOP, can be prevented by the nonspecific HDAC inhibitors trichostatin A and valproic acid, suggesting that alternate targets that oppose class II HDAC function might exist in myocardium. We tested the effects of several HDAC inhibitors, including a class I HDAC-selective inhibitor, SK-7041, on cardiac hypertrophy induced by angiotensin II (Ang II) treatment or aortic banding (AB). METHODS AND RESULTS: Cardiac hypertrophy was induced by chronic infusion of Ang II or by AB in mice or rats and evaluated by determining the ratio of heart weight to body weight or to tibia length, cross-sectional area, or echocardiogram. Cardiac hypertrophy induced by Ang II or AB for 2 weeks was significantly reduced by simultaneous administration of trichostatin A, valproic acid, or SK-7041. Echocardiogram revealed that exaggerated left ventricular systolic dimensions were relieved by HDAC inhibitors. HDAC inhibitors partially reversed preestablished cardiac hypertrophy and improved survival of AB mice. The expressions of atrial natriuretic factor, alpha-tubulin, beta-myosin heavy chain, and interstitial fibrosis were reduced by HDAC inhibition. CONCLUSIONS: These results suggest that the predominant effect of HDAC inhibition, mainly mediated by class I HDACs, is to prevent cardiac hypertrophy in response to a broad range of agonist and stretch stimuli.

Abstract: Resection of the infraorbital fat is performed in blepharoplasty of the lower eyelid, however, the previous anatomical reports on its compartmentalization have been in disagreement. The aim of this study was to classify the infraorbital fat based on the extent of compartmentalization, and to clarify its topographic relationship with the surrounding structures. Sixty orbits from 30 cadavers were dissected. The infraorbital fat was classified into four types based on its compartmentalization. In type I, which was the most common type (60.0%), the infraorbital fat was compartmentalised into three encapsulated medial, central, and lateral parts, which were side by side. In type II (11.7%), the medial or lateral compartment, or both compartments were under the central fat compartment. In type III (26.7%), there were two compartments, the medial and remaining part or the lateral and remaining part. In type IV (1.7%), the fat was not compartmentalised, but presented as a single pad. The average heights from the inferior orbital rim, the average widths, and the average distances from the fornix were 7.3, 17.2, and 7.1 mm in the medial compartment, 8.9, 24.2, and 8.0mm in the central compartment, and 8.1, 17.2, and 6.9 mm in the lateral compartment, respectively. The average distance from the end of the margin of the stretched lower eyelid to the most cephalic point in the compartments was 8.6 mm. These results are relevant to blepharoplasty with removal of the infraorbital fat.

Abstract: OBJECTIVE: This study was designed to examine whether mesenchymal stem cells (MSCs) transduced with Akt enhance cardiac repair after transplantation into the ischemic porcine heart. METHODS: MSCs isolated from porcine bone marrow and transduced with myr-Akt were transplanted into porcine hearts after experimental myocardial infarction (MI) using intracoronary injection [Group I, vehicle; Group II, MSCs; Group III, Akt-MSCs]. Myocardial single photon emission tomography (M-SPECT) was performed to assess myocardial function and the infarcted area. Pigs were also sacrificed for immunohistochemical characterization and histologic analysis. In addition, in vitro assays were performed to examine the resistance of Akt-MSCs to H(2)O(2) stimulation. RESULTS: Transplantation of MSCs into the ischemic porcine myocardium (Group II) increased the left ventricular ejection fraction (DeltaLV EF; -6.3 +/- 15.1% versus 0.5 +/- 6.4%, P < 0.001) and decreased the Deltaarea of MI (6.8+/-5.6% versus -5.0+/-5.3%, P < 0.001) compared with the vehicle control (Group I). Transplantation of MSCs transduced with myr-Akt (Group III) resulted in further improvement in DeltaLV EF (-6.3 +/- 15.1% versus 5.8 +/- 11.3%, P < 0.001) and in Deltaarea of MI (6.8 +/- 5.6% versus -17.0 +/- 7.6%, P < 0.001). Akt-MSCs were more resistant to apoptosis, and the levels of extracellular signal-regulated protein kinase (ERK) activation and vascular endothelial growth factor (VEGF) were higher in H(2)O(2)-stimulated Akt-MSCs. CONCLUSION: Cellular transplantation of Akt-MSCs further enhances the repair of injured myocardium compared to MSC transplantation alone by increasing the number of viable MSCs after cellular transplantation.

Abstract: The tyramide signal amplification (TSA) technique has been shown to detect scarce tissue antigens in light and electron microscopy. In this study we applied the TSA technique at the electron microscopic level to pre-embedding immunocytochemistry. This protocol was compared to the non-amplified protocol. With the TSA protocol, the labeling of GM130, a cis-Golgi matrix protein, was tested in a cell line and found to be highly sensitive and more enhanced than that with the simple protocol. Moreover, the gold particles were well localized to the cis-side of the Golgi apparatus in both the TSA and the simple protocol.

Abstract: To obtain greater insight into atrial remodeling at the molecular level we analyzed the changes in gene expression in human atrial tissue between patients with chronic atrial fibrillation (AF) and those with normal sinus rhythm (NSR). cDNA microarray analysis was used to identify genes differentially expressed during sustained AF of more than 6 months (n = 9, mean age, 45 +/- 12, 6 males and 3 females) as compared to those with NSR (n = 9, mean age, 47 +/- 13, 6 males and 3 females). Western blot analysis was performed to confirm the altered gene expression and to establish the changes in protein expression. DNA gel electrophoresis to establish DNA ladder formation, which was associated with apoptosis in response to chronic AF, was performed. Microscopic findings were observed via electron microscopy. In the microarray analysis, out of 8,167 candidate genes, 66 genes showed a significant change in the expression level in the patients with chronic AF, which was in contrast to those with NSR. Among those, 31 genes were consistently down-regulated and 35 up-regulated more than 2-fold. The relative amounts of the Bcl-2 and p27 in the atrial tissue were decreased and angiotensin II type 2 (AT2) receptor and p21 were increased in the patients with chronic AF as compared to those with NSR. The atrial cardiomyocytes in chronic AF showed a prominent DNA ladder, which is a biochemical hallmark of apoptosis. The expression of Bcl-2, AT2 receptor, p21, and p27 were consistent with a significant role in the apoptosis of cardiac myocytes in the patients with chronic AF.

Abstract: We isolated a human gene encoding keratinocyte proline-rich protein (hKPRP). hKPRP gene is located in the region of epidermal differentiation complex on chromosome 1q21, and its approximately 2.5 kb mRNA encodes 579 amino acid protein with high proline content (18%). The mRNA level of hKPRP was markedly increased at both 7 and 14 d after treatment with 1.2 mM calcium in cultured normal human epidermal keratinocytes. In situ hybridization demonstrated that hKPRP was expressed in upper granular layer of normal epidermis with characteristic intermittent pattern. In psoriatic lesion, hKPRP expression was increased as compared with normal skin and showed continuous pattern. Immunohistochemical analysis also confirmed the expression of hKPRP at the protein level. Western blot analysis showed that hKPRP protein of approximately 70 kDa size was significantly increased by calcium in a time-dependent manner. In mouse tissue blot assays, the expression of KPRP was detected in stomach and skin tissues, and began at 17.5 embryonic days. Additionally, hKPRP expression was detected in the periderm of human fetal skin from 16 wk estimated gestational age. Together, these results suggest that hKPRP is an epidermal marker expressed in stratified squamous epithelia and has a potential role in keratinocytes differentiation.

Abstract: Previously, phytanoyl-CoA alpha-hydroxylase-associated protein 1 (PAHX-AP1) was isolated as a novel neuron-specific protein to interact with Refsum disease (RfD) gene PAHX. Its expression in the brain increased after eyelid opening, and the elevated level was maintained through adulthood. In this report, to verify the hypothesis that light could trigger this increase, we have examined the developmental distribution pattern of PAHX-AP1 in rat retina and visual cortex, and changes of its expression by binocular deprivation. Northern blot analyses demonstrated PAHX-AP1 expression reached its highest level in the visual cortex and eyeball at 4 weeks after birth, and these levels were maintained through adult life. Two weeks after visual deprivation, its expression in the eyeball and visual cortex decreased compared with the control. In situ hybridization analyses of the retina showed that PAHX-AP1 expression was limited to the ganglionic cell layer at 10 days after birth, but expressed in the inner nuclear cell layer and extended to the outer nuclear cell layer at 2 and 3 weeks after birth, respectively. Two weeks after visual deprivation, however, it decreased in the ganglionic and inner nuclear cell layer, and disappeared in the rod and cone cell layers. In the visual cortex, strong signals of PAHX-AP1 were detected in layers IV and VI, and II-VI at 10 days and 2 weeks after birth, respectively. Its expression decreased after 2 weeks of visual deprivation. These results indicate that visual stimulation is essential for the maintenance of PAHX-AP1 expressions in the retina, especially in the rod and cone cell layers, and visual cortex, and suggest that PAHX-AP1 may be involved in the developmental regulation of the photoreceptor's function.

Abstract: The purpose of this study was to evaluate the anastomotic relationships between the accessory nerve and the posterior root of the first cervical nerve and to determine the course of the posterior root nerve fibers after anastomosis. The relationships between these two nerves were studied in 100 sides of the spinal cord and then classified into four types. In the most common type of anastomosis (38% of cases), either a branch from the posterior C1 root was seen to course cranially and join the accessory nerve, or the posterior root and accessory nerve fused as they coursed orthogonal to one other. In the second most common type (36% of cases), the accessory nerve anastomosed with a posterior C1 root that had no direct connection with the spinal cord; the nerve fibers of the posterior root were observed in stained samples to course caudally along the accessory nerve and join the posterior C2 root. In the least common type (6% of cases), no connection was observed between the accessory nerve and the posterior C1 root. In the next least common type (20% of cases), the posterior C1 root was absent and a connecting branch was sometimes observed between the accessory nerve and the anterior C1 root.

Abstract: The cornified cell envelope is formed during the terminal differentiation of epidermis through cross-linking of specific proteins by transglutaminases. The specific arrangement of individual protein in the cornified cell envelope and participation of individual protein in the cornified cell envelope at different regions of skin, i.e., palm, foreskin, lips, etc. are not clearly understood. In order to understand the pattern and expression schedule of each individual precursor protein during the differentiation and formation of cornified cell envelope, the expression of precursor proteins in developing human fetal skins from the first to the third trimester were examined by immunohistochemical studies. Involucrin was found in the periderm and intermediate layer from 14 wk estimated gestational age, while loricrin and small proline-rich protein 1 were found in the periderm from 16 wk estimated gestational age. Filaggrin and trichohyalin that are absent in the adult cornified cell envelope were found in the granular and horny layers from 24 wk estimated gestational age. The precursor proteins except trichohyalin did not change their patterns after the onset of initial expression during development. Trichohyalin was transiently expressed in the granular and horny layers of the epidermis from 24 wk estimated gestational age with peak expression at 27 wk estimated gestational age, but was not detected in adult skin. In hair follicles, trichohyalin expression was stable without change from 20 wk estimated gestational age. These findings suggest that fetal skin may have different sets of barriers from the second trimester; the immature cornified cell envelope is formed in the early second trimester and the mature cornified cell envelope is formed in the late second or early third trimester when filaggrin and trichohyalin appear.