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1.  Staging of Alzheimer's Pathology in Triple Transgenic Mice: A Light and Electron Microscopic Analysis 
The age-related pathological cascade underlying intraneuronal tau formation in 3xTg-AD mice, which harbor the human APPSwe, PS1M126V , and TauP301L gene mutations, remains unclear. At 3 weeks of age, AT180, Alz50, MC1, AT8, and PHF-1 intraneuronal immunoreactivity appeared in the amygdala and hippocampus and at later ages in the cortex of 3xTg-AD mice. AT8 and PHF-1 staining was fixation dependent in young mutant mice. 6E10 staining was seen at all ages. Fluorescent immunomicroscopy revealed CA1 neurons dual stained for 6E10 and Alz50 and single Alz50 immunoreactive neurons in the subiculum at 3 weeks and continuing to 20 months. Although electron microscopy confirmed intraneuronal cytoplasmic Alz50, AT8, and 6E10 reaction product in younger 3xTg-AD mice, straight filaments appeared at 23 months of age in female mice. The present data suggest that other age-related biochemical mechanisms in addition to early intraneuronal accumulation of 6E10 and tau underlie the formation of tau filaments in 3xTg-AD mice.
PMCID: PMC2925282  PMID: 20798886
2.  Innervated Cross-Finger Pulp Flap for Reconstruction of the Fingertip 
Archives of Plastic Surgery  2012;39(6):637-642.
Fingertip injuries involving subtotal or total loss of the digital pulp are common types of hand injuries and require reconstruction that is able to provide stable padding and sensory recovery. There are various techniques used for reconstruction of fingertip injuries, but the most effective method is functionally and aesthetically controversial. Despite some disadvantages, cross-finger pulp flap is a relatively simple procedure without significant complications or requiring special techniques.
This study included 90 patients with fingertip defects who underwent cross-finger pulp flap between September 1998 and March 2010. In 69 cases, neurorrhaphy was performed between the pulp branch from the proper digital nerve and the recipient's sensory nerve for good sensibility of the injured fingertip. In order to evaluate the outcome of our surgical method, we observed two-point discrimination in the early (3 months) and late (12 to 40 months) postoperative periods.
Most of the cases had cosmetically and functionally acceptable outcomes. The average defect size was 1.7×1.5 cm. Sensory return began 3 months after flap application. The two-point discrimination was measured at 4.6 mm (range, 3 to 6 mm) in our method and 7.2 mm (range, 4 to 9 mm) in non-innervated cross-finger pulp flaps.
The innervated cross-finger pulp flap is a safe and reliable procedure for lateral oblique, volar oblique, and transverse fingertip amputations. Our procedure is simple to perform under local anesthesia, and is able to provide both mechanical stability and sensory recovery. We recommend this method for reconstruction of fingertip injuries.
PMCID: PMC3518008  PMID: 23233890
Finger injuries; Subcutaneous tissue; Microsurgery; Nerve transfer
3.  Cholinotrophic basal forebrain system alterations in 3xTg-AD transgenic mice 
Neurobiology of disease  2010;41(2):338-352.
The cholinotrophic system, which is dependent upon nerve growth factor and its receptors for survival, is selectively vulnerable in Alzheimer's disease (AD). But, virtually nothing is known about how this deficit develops in relation to the hallmark lesions of this disease, amyloid plaques and tau containing neurofibrillary tangles. The vast majority of transgenic models of AD used to evaluate the effect of beta amyloid (Aβ) deposition upon the cholinotrophic system over-express the amyloid precursor protein (APP). However, nothing is known about how this system is affected in triple transgenic (3xTg)-AD mice, an AD animal model displaying Aβ plaque- and tangle-like pathology in the cortex and hippocampus, which receive extensive cholinergic innervation. We performed a detailed morphological and biochemical characterization of the cholinotrophic system in young (2-4 months), middle-aged (13-15 months), and old (18-20 months) 3xTg-AD mice. Cholinergic neuritic swellings increased in number and size with age, and were more conspicuous in the hippocampal-subicular complex in aged female than in 3xTg-AD male mice. Stereological analysis revealed a reduction in choline acetyltransferase (ChAT) positive cells in the medial septum/vertical limb of the diagonal band of Broca in aged 3xTg-AD mice. ChAT enzyme activity levels decreased significantly in the hippocampus of middle-aged 3xTg-AD mice compared to age-matched ntg mice. ProNGF protein levels increased in the cortex of aged 3xTg-AD mice, whereas TrkA protein levels were reduced in a gender-dependent manner in aged mutant mice. In contrast, p75NTR protein cortical levels were stable but increased in the hippocampus of aged 3xTg-AD mice. These data demonstrate that cholinotrophic alterations in 3xTg-AD mice are age and gender dependent and more pronounced in the hippocampus, a structure more severely affected with Aβ plaque pathology.
PMCID: PMC3014453  PMID: 20937383
Alzheimer's disease; amyloid; cholinergic; nerve growth factor; ChAT; proNGF; p75NTR; transgenic mice; TrkA; aging
4.  Destabilization of Rb by human papillomavirus E7 is cell cycle dependent: E2-25K is involved in the proteolysis 
Virology  2009;396(1):118-124.
The HPV-oncoprotein, E7 promotes proteasomal degradation of the tumor suppressor protein, Rb. In this study, we analyzed the regulation of E7-induced Rb proteolysis in HPV-containing Caski cervical cancer cells. We show that the Rb proteolysis is cell cycle dependent; in S phase Rb is stable while in post-mitotic early G1 phase cells and in differentiated cells, Rb is unstable. Similarly, the in vivo Rb/E7 interaction is not detected in S phase cells, but is readily detected in differentiating Caski cells. The ubiquitinating enzymes involved in Rb proteolysis have not been identified. We find that the E3 ligase MDM2 is not involved in the Rb proteolysis in Caski cells. An in vivo analysis using multiple catalytic-site mutant dominant negative E2-enzymes show that the C92A E2-25K most effectively blocks E7-induced Rb proteolysis. Taken together, these results show that E7 induces Rb proteolysis in growth-arrested cells and E2-25K is involved in the proteolysis.
PMCID: PMC2796474  PMID: 19906396
5.  Deregulation of eIF4E: 4E-BP1 in Differentiated Human Papillomavirus-Containing Cells Leads to High Levels of Expression of the E7 Oncoprotein 
Journal of Virology  2006;80(14):7079-7088.
Infections with high-risk human papillomaviruses (HPVs) are linked to more than 95% of cervical cancers. HPVs replicate exclusively in differentiated cells and the function of the HPV E7 oncoprotein is essential for viral replication. In this study, we investigated the mechanism that regulates E7 expression in differentiated cells. The level of E7 protein was strongly induced in HPV-containing Caski, HOK-16B, and BaP-T cells during growth in methylcellulose-containing medium, a condition that induces differentiation. Enhanced expression of E7 was observed between 4 and 8 h of culturing in methylcellulose and was maintained for up to 24 h. The increase was not due to altered stability of the E7 protein or an increase in the steady-state level of the E7 mRNA. Instead, the translation of the E7 mRNA was enhanced during differentiation. More than 70 to 80% of the E7 mRNA was found in the polysome fractions in the differentiated cells. Consistent with this observation, higher levels of the phosphorylated translator inhibitor 4E-BP1 were observed in differentiated HPV-containing cells but not in differentiated non-HPV tumor cells or primary keratinocytes. The mTOR kinase inhibitor rapamycin blocked phosphorylation of 4E-BP1 and significantly decreased the level of E7 protein in Caski cells, suggesting that phosphorylation of 4E-BP1 is linked to E7 expression. Prevailing models for the molecular mechanisms underlying E7 expression have focused largely on transcriptional regulation. The results presented in this study demonstrate a significant role of the cellular translation machinery to maintain a high level of E7 protein in differentiated cells.
PMCID: PMC1489051  PMID: 16809313
6.  Evidence for Cyclooxygenase-2 Association with Caveolin-3 in Primary Cultured Rat Chondrocytes 
Journal of Korean Medical Science  2006;21(1):100-106.
The purpose of this study was to demonstrate the cellular localization of cyclooxygenase-2 (COX-2) and caveolin-3 (Cav-3) in primarily cultured rat chondrocytes. In normal rat chondrocytes, we observed relatively high levels of Cav-3 and a very low level of COX-2 mRNA and protein. Upon treating the chondrocytes with 5 µM of CdCl2 (Cd) for 6 hr, the expressions of COX-2 mRNA and protein were increased with the decreased Cav-3 mRNA and protein expressions. The detergent insoluble caveolae-rich membranous fractions that were isolated from the rat chondrocytes and treated with Cd contained the both proteins of both COX-2 and Cav-3 in a same fraction. The immuno-precipitation experiments showed complex formation between the COX-2 and Cav-3 in the rat chondrocytes. Purified COX-2 with glutathione S-transferase-fused COX-2 also showed complex formation with Cav-3. Confocal and electron microscopy also demonstrated the co-localization of COX-2 and Cav-3 in the plasma membrane. The results from our current study show that COX-2 and Cav-3 are co-localized in the caveolae of the plasma membrane, and they form a protein-protein complex. The co-localization of COX-2 with Cav-3 in the caveolae suggests that the caveolins might play an important role for regulating the function of COX-2.
PMCID: PMC2733955  PMID: 16479074
Chondrocytes; Cyclooxygenase 2; Caveolins; Microscopy, Confocal; Microscopy, Electron
7.  The Papillomavirus E7 Oncoprotein Is Ubiquitinated by UbcH7 and Cullin 1- and Skp2-Containing E3 Ligase 
Journal of Virology  2004;78(10):5338-5346.
Recurrent infections with high-risk human papillomaviruses (HPVs) are associated with human cervical cancers. All HPV-associated cancer tissues express the viral oncoproteins E6 and E7, which stimulate cell growth. The expression of E7 is crucial for both the initiation and the maintenance of HPV-associated cancer. Recent studies showed that the level of E7 in cancer cells is regulated by ubiquitin-dependent proteolysis through the 26S proteasome. In this study, we characterized the enzymes involved in the ubiquitin-dependent proteolysis of E7. We show that UbcH7, an E2 ubiquitin-conjugating enzyme, is specifically involved in the ubiquitination of E7. Furthermore, we show that E7 interacts with the SCF (Skp-Cullin-F box) ubiquitin ligase complex containing Cullin 1 (Cul1) and Skp2 and can be ubiquitinated by the Cul1-containing ubiquitin ligase in vitro. Coimmunoprecipitation analyses revealed that E7 interacts with Skp2 and Cul1 in vivo. Finally, the half-life of E7 was found to be significantly longer in Skp2−/− mouse embryo fibroblasts (MEFs) than in wild-type MEFs. Taken together, these results suggest that the Cul1- and Skp2-containing ubiquitin ligase plays a role in the ubiquitination and proteolysis of E7. In HPV type 16-containing cervical carcinoma cell line Caski, E7 localizes to both the cytoplasm and the nucleus. Brief treatment of Caski cells with MG132 (a proteasome inhibitor) causes the accumulation of E7 in discrete nuclear bodies. These nuclear bodies are detergent insoluble and contain polyubiquitinated E7. We suggest that E7 relocates to specific nuclear bodies for proteolysis in HPV-containing epithelial cells.
PMCID: PMC400333  PMID: 15113913

Results 1-7 (7)