Importance of region-specific epithelial rearrangements in mouse rugae development

Epithelial appendages on palatal rugae develop during mouse palatogenesis through epithelial thickening and pattern formation. Recently, the patterned formation of nine rugae was observed together with the specific expression patterns of Shh in rodents. However, no crucial evidence was found for a direct association between Shh expression and the distinct structural formation of rugae. In order to reveal possible relationships, we investigated the morphological changes of rugae and expression patterns of Shh directly by in vitro organ culture at embryonic day 13 (E13) for 2 days. To compare and examine the diverse growing aspects of the palate and rugae, we carefully observed the detailed morphogenesis, with cell proliferation of the rugae occurring between E13 and E14.5. After 2 days of cultivation at E13, DiI micro-injections revealed that the middle part of the palate, adjacent to the upper molar-forming region, contributed to the formation of the subsequent structure of rugae by extensive cell rearrangement and proliferation within the epithelium in the preferred anteroposterior direction. The results also defined the intimate relationship between Shh expression and rugae formation.     

The parthenogenetic activation of canine oocytes with Ca-EDTA by various culture periods and concentrations

In the present study, canine oocytes were exposed to various concentrations of and durations of exposure to EDTA saturated with Ca(2+) (Ca-EDTA), a cell membrane-impermeable metal ion chelator, to determine if parthenogenetic activation could be induced. When oocytes were cultured for 48 or 72 h in parthenogenetic activation medium (PAM) without Ca-EDTA (control) or PAM supplemented with 1 or 5mM Ca-EDTA, the highest rate of pronuclear formation (PN) was obtained in oocytes cultured in 1mM Ca-EDTA for 48 h (8.0%; P<0.05). There was no pronuclear formation in the control group (PAM without Ca-EDTA). Oocytes treated with 5mM Ca-EDTA for 48 h or 1mM Ca-EDTA for 72 h formed a parthenogenetic pronucleus (3.1 and 4.5, respectively). However, there was no pronuclear formation in oocytes treated with 5mM Ca-EDTA for 72 h. In summary, exposure to Ca-EDTA can induce pronuclear formation in canine oocytes.     

Prognosis Prediction of Measurable Enhancing Lesion after Completion of Standard Concomitant Chemoradiotherapy and Adjuvant Temozolomide in Glioblastoma Patients: Application of Dynamic Susceptibility Contrast Perfusion and Diffusion-Weighted Imaging

Purpose

To assess the prognosis predictability of a measurable enhancing lesion using histogram parameters produced by the normalized cerebral blood volume (nCBV) and normalized apparent diffusion coefficient (nADC) after completion of standard concomitant chemoradiotherapy (CCRT) and adjuvant temozolomide (TMZ) medication in glioblastoma multiforme (GBM) patients.

Materials and Methods

This study was approved by the institutional review board (IRB), and the requirement for informed consent was waived. A total of 59 patients with newly diagnosed GBM who received standard CCRT with TMZ and adjuvant TMZ for six cycles underwent perfusion-weighted and diffusion-weighted imaging. Twenty-seven patients had a measurable enhancing lesion and 32 patients lacked a measurable enhancing lesion based on the Response Assessment in Neuro-Oncology (RANO) criteria in the follow-up MRI, which was performed within 3 months after adjuvant TMZ therapy was completed. We measured the nCBV and nADC histogram parameters based on the measurable enhancing lesion. The progression free survival (PFS) was analyzed by the Kaplan-Meier method with the use of the log-rank test.

Results

The median PFS of patients lacking measurable enhancing lesion was longer than for those with measurable enhancing lesions (17.6 vs 3.3 months, P<.0001). There was a significant, positive correlation between the 99^th percentile nCBV value of a measurable enhancing lesion and the PFS (P = .044, R² = .152). In addition, the median PFS was longer in patients with a 99^th percentile nCBV value ≧4.5 than it was in those with a value <4.5 (4.4 vs 3.1 months, P = .036).

Conclusion

We found that the nCBV value can be used for the prognosis prediction of a measurable enhancing lesion after the completion of standard treatment for GBM, wherein a high 99^th percentile nCBV value (≧4.5) suggests a better PFS for GBM patients.     

Drosophila BRUCE inhibits apoptosis through non-lysine ubiquitination of the IAP-antagonist REAPER

Active caspases execute apoptosis to eliminate superfluous or harmful cells in animals. In Drosophila, living cells prevent uncontrolled caspase activation through an inhibitor of apoptosis protein (IAP) family member, dIAP1, and apoptosis is preceded by the expression of IAP-antagonists, such as Reaper, Hid and Grim. Strong genetic modifiers of this pathway include another IAP family gene encoding an E2 ubiquitin conjugating enzyme domain, dBruce. Although the genetic effects of dBruce mutants are well documented, molecular targets of its encoded protein have remained elusive. Here, we report that dBruce targets Reaper for ubiquitination through an unconventional mechanism. Specifically, we show that dBruce physically interacts with Reaper, dependent upon Reaper's IAP-binding (IBM) and GH3 motifs. Consistently, Reaper levels were elevated in a dBruce -/- background. Unexpectedly, we found that dBruce also affects the levels of a mutant form of Reaper without any internal lysine residues, which normally serve as conventional ubiquitin acceptor sites. Furthermore, we were able to biochemically detect ubiquitin conjugation on lysine-deficient Reaper proteins, and knockdown of dBruce significantly reduced the extent of this ubiquitination. Our results indicate that dBruce inhibits apoptosis by promoting IAP-antagonist ubiquitination on unconventional acceptor sites.     

Peripheral benzodiazepine receptor regulates vascular endothelial activations via suppression of the voltage-dependent anion channel-1

Peripheral benzodiazepine receptor (PBR) is a multifunctional protein mainly found on the outer mitochondrial membrane. PBR expression is increased by tumor necrosis factor-α (TNF-α) in endothelial cells. Adenoviral overexpression of PBR inhibits monocyte adhesion, VCAM-1, and ICAM-1 expression in TNF-α-activated endothelial cells. Rotenone, cyclosporine A, and bongkrekic acid suppress TNF-α-induced VCAM-1 expression. Overexpression of PBR inhibits voltage-dependent anion channel-1 (VDAC-1) expression and the silencing of PBR increases VDAC-1 expression in endothelial cells. Moreover, TNF-α-induced VCAM-1 expression is suppressed by VDAC-1 gene silencing. PBR overexpression significantly decreases TNF-α-induced mitochondrial reactive oxygen species and MnSOD expression. These results suggest that PBR can inhibit endothelial activation and this action is related to the inhibition of mitochondrial ROS and/or VDAC-1 expression in endothelial cells.     

CDK5 and MEKK1 mediate pro-apoptotic signalling following endoplasmic reticulum stress in an autosomal dominant retinitis pigmentosa model

Chronic stress in the endoplasmic reticulum (ER) underlies many degenerative and metabolic diseases involving apoptosis of vital cells. A well-established example is autosomal dominant retinitis pigmentosa (ADRP), an age-related retinal degenerative disease caused by mutant rhodopsins. Similar mutant alleles of Drosophila Rhodopsin-1 also impose stress on the ER and cause age-related retinal degeneration in that organism. Well-characterized signalling responses to ER stress, referred to as the unfolded protein response (UPR), induce various ER quality control genes that can suppress such retinal degeneration. However, how cells activate cell death programs after chronic ER stress remains poorly understood. Here, we report the identification of a signalling pathway mediated by cdk5 and mekk1 required for ER-stress-induced apoptosis. Inactivation of these genes specifically suppressed apoptosis, without affecting other protective branches of the UPR. CDK5 phosphorylates MEKK1, and together, they activate the JNK pathway for apoptosis. Moreover, disruption of this pathway can delay the course of age-related retinal degeneration in a Drosophila model of ADRP. These findings establish a previously unrecognized branch of ER-stress response signalling involved in degenerative diseases.     

Stem cell recovering effect of copper‐free GHK in skin

The peptide Gly‐His‐Lys (GHK) is a naturally occurring copper(II)‐chelating motifs in human serum and cerebrospinal fluid. In industry, GHK (with or without copper) is used to make hair and skin care products. Copper‐GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. We also reported that copper‐GHK promotes the survival of basal stem cells in the skin. However, the effects of copper‐free GHK (GHK) have not been investigated well. In this study, the effects of GHK were studied using cultured normal human keratinocytes and skin equivalent (SE) models. In monolayer cultured keratinocytes, GHK increased the proliferation of keratinocytes. When GHK was added during the culture of SE models, the basal cells became more cuboidal than control model. In addition, there was linear and intense staining of α6 and β1 integrin along the basement membrane. The number of p63 and proliferating cell nuclear antigen positive cells was also significantly increased in GHK‐treated SEs than in control SEs. Western blot and slide culture experiment showed that GHK increased the expression of integrin by keratinocytes. All these results showed that GHK increased the stemness and proliferative potential of epidermal basal cells, which is associated with increased expression of integrin. In conclusion, copper‐free GHK showed similar effects with copper‐GHK. Thus, it can be said that copper‐free GHK can be used in industry to obtain the effects of copper‐GHK in vivo. Further study is necessary to explore the relationship between copper‐free GHK and copper‐GHK. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.