The mechanism of placental alkaline phosphatase induction in vitro

The mechanism of placental alkaline phosphatase (PLAP) induction by prednisolone in a uterine cervical epidermoid cancer cell line SKG-IIIa was investigated in vitro by enzyme-cytochemistry, enzyme immunoassay, Northern and Southern blot analysis, and in situ hybridization. Enzyme-cytochemical alkaline phosphatase (ALP) staining and immunoassay revealed increased levels of PLAP (heat-stable ALP) in prednisolone-treated cells. Northern blot analysis and in situ hybridization showed increased amounts of PLAP mRNA. Southern blot analysis indicated that PLAP was not a product of an amplified or rearranged gene. These findings suggest that the induction of PLAP mRNA in SKG-IIIa cells by prednisolone in turn increased the levels of PLAP.     

Specific refractoriness of adenylate cyclase in skin to epinephrine,prostaglandin E,histamine and AMP

The cyclic AMP level in pig skin (epidermis) increases markedly after incubation with epinephrine, prostaglandin E, histamine or adenosine 5′-monophosphate. This increase is transient and “spiking” is the consistent response to these four stimulators. The “spiking” is due to a non-responsiveness or refractoriness which develops within minutes and is specific to any one stimulating hormone but not to the others. The addition of inhibitors of protein syntheses did not prevent the development of the refractoriness. Adenylate cyclase and phosphodiesterase activities measured in skin homogenates prepared from skin samples taken before, during and after the “spiking” did not change significantly. The hormone-induced refractoriness in this skin system appears to be due to a specific, localized loss of function of the adenylate cyclase system.     

Translation-Competent Extracts fromSaccharomyces cerevisiae:Effects of L-A RNA, 5′ Cap, and 3′ Poly(A) Tail on Translational Efficiency of mRNAs

Yeast genetics has proven fruitful in the identification of key players that are involved in translational initiation. However, the exact roles of many translation initiation factors in translation initiation remain unknown. This has been due to lack of a suitablein vitrotranslation system in which the mode of action of certain translation factors can be studied. This report describes the preparation of cell-freeSaccharomyces cerevisiaelysates that can mediate the translation of exogenously added mRNAs. Optimal translation required the absence of viral L-A RNA in the lysate and the presence of both a 5′ cap and a 3′ poly(A) tail on the mRNAs. A cooperative effect of cap and poly(A) tail on translation initiation was observed, a property that has been found to operate in intact yeast cells as well. In addition, the yeast lysates mediated translational initiation through several viral internal ribosome entry sites, demonstrating that the yeast translation apparatus can perform internal initiation. Thus, these lysates may be useful in the biochemical analysis of cap-dependent and cap-independent translation events.     

Induced pluripotent stem cell‐derived melanocyte precursor cells undergoing differentiation into melanocytes

Induced pluripotent stem cell (iPSC) technology offers a novel approach for conversion of human primary fibroblasts into melanocytes. During attempts to explore various protocols for differentiation of iPSCs into melanocytes, we found a distinct and self‐renewing cell lineage that could differentiate into melanocytes, named as melanocyte precursor cells (MPCs). The MPCs exhibited a morphology distinctive from that of melanocytes, in lacking either the melanosomal structure or the melanocyte‐specific marker genes MITF, TYR, and SOX10. In addition, gene expression studies in the MPCs showed high‐level expression of WNT5A, ROR2, which are non‐canonical WNT pathway markers, and its related receptor TGFβR2. In contrast, MPC differentiation into melanocytes was achieved by activating the canonical WNT pathway using the GSK3β inhibitor. Our data demonstrated the distinct characteristic of MPCs' ability to differentiate into melanocytes, and the underlying mechanism of interfacing between canonical WNT signaling pathway and non‐canonical WNT signaling pathway.     

Changes in Inner and Outer Retinal Layer Thicknesses after Vitrectomy for Idiopathic Macular Hole: Implications for Visual Prognosis

Purpose

To investigate sequential post-operative thickness changes in inner and outer retinal layers in eyes with an idiopathic macular hole (MH).

Methods

Retrospective case series. Twenty-four eyes of 23 patients who had received pars plana vitrectomy (PPV) for the closure of MH were included in the study. Spectral domain optical coherence tomography C-scan was used to automatically measure the mean thickness of the inner and outer retinal layers pre-operatively and up to 6 months following surgery. The photoreceptor outer segment (PROS) length was measured manually and was used to assess its relationship with best-corrected visual acuity (BCVA).

Results

Compared with the pre-operative thickness, the inner layers significantly thinned during follow-up (P = 0.02), particularly in the parafoveal (P = 0.01), but not perifoveal, area. The post-operative inner layer thinning ranged from the ganglion cell layer to the inner plexiform layer (P = 0.002), whereas the nerve fiber layer was unaltered. Outer layer thickness was significantly greater post-operatively (P = 0.002), and especially the PROS lengthened not only in the fovea but also in the parafovea (P < 0.001). Six months after surgery, BCVA was significantly correlated exclusively with the elongated foveal PROS (R = 0.42, P = 0.03), but not with any of the other thickness parameters examined.

Conclusions

Following PPV for MH, retinal inner layers other than the nerve fiber layer thinned, suggestive of subclinical thickening in the inner layers where no cyst was evident pre-operatively. In contrast, retinal outer layer thickness significantly increased, potentially as a result of PROS elongation linking tightly with favorable visual prognosis in MH eyes.     

Yeast Cell Wall Extract Induces Disease Resistance against Bacterial and Fungal Pathogens in Arabidopsis thaliana and Brassica Crop

Housaku Monogatari (HM) is a plant activator prepared from a yeast cell wall extract. We examined the efficacy of HM application and observed that HM treatment increased the resistance of Arabidopsis thaliana and Brassica rapa leaves to bacterial and fungal infections. HM reduced the severity of bacterial leaf spot and anthracnose on A. thaliana and Brassica crop leaves with protective effects. In addition, gene expression analysis of A. thaliana plants after treatment with HM indicated increased expression of several plant defense-related genes. HM treatment appears to induce early activation of jasmonate/ethylene and late activation of salicylic acid (SA) pathways. Analysis using signaling mutants revealed that HM required SA accumulation and SA signaling to facilitate resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola and the fungal pathogen Colletotrichum higginsianum. In addition, HM-induced resistance conferred chitin-independent disease resistance to bacterial pathogens in A. thaliana. These results suggest that HM contains multiple microbe-associated molecular patterns that activate defense responses in plants. These findings suggest that the application of HM is a useful tool that may facilitate new disease control methods.     

RAD18 Activates the G2/M Checkpoint through DNA Damage Signaling to Maintain Genome Integrity after Ionizing Radiation Exposure

The ubiquitin ligase RAD18 is involved in post replication repair pathways via its recruitment to stalled replication forks, and its role in the ubiquitylation of proliferating cell nuclear antigen (PCNA). Recently, it has been reported that RAD18 is also recruited to DNA double strand break (DSB) sites, where it plays novel functions in the DNA damage response induced by ionizing radiation (IR). This new role is independent of PCNA ubiquitylation, but little is known about how RAD18 functions after IR exposure. Here, we describe a role for RAD18 in the IR-induced DNA damage signaling pathway at G2/M phase in the cell cycle. Depleting cells of RAD18 reduced the recruitment of the DNA damage signaling factors ATM, γH2AX, and 53BP1 to foci in cells at the G2/M phase after IR exposure, and attenuated activation of the G2/M checkpoint. Furthermore, depletion of RAD18 increased micronuclei formation and cell death following IR exposure, both in vitro and in vivo. Our data suggest that RAD18 can function as a mediator for DNA damage response signals to activate the G2/M checkpoint in order to maintain genome integrity and cell survival after IR exposure.