Molecular cloning and stress-induced expression of paralichthys olivaceus heme-regulated initiation factor 2 alpha kinase

The heme-regulated initiation factor 2 alpha kinase (HRI) is acknowledged to play an important role in translational shutoff in reticulocytes in response to various cellular stresses. In this study, we report its homologous cDNA cloning and characterization from cultured flounder embryonic cells (FEC) after treatment with UV-inactivated grass carp haemorrhagic virus (GCHV). The full-length cDNA of Paralichthys olivaceus HRI homologue (PoHRI) has 2391 bp and encodes a protein of 651 amino acids. The putative PoHRI protein exhibits high identity with all members of eIF2 alpha kinase family. It contains 12 catalytic subdomains located within the C-terminus of all Ser/Thr protein kinases, a unique kinase insertion of 136 amino acids between subdomains IV and V, and a relatively conserved N-terminal domain (NTD). Upon heat shock, virus infection or Poly PC treatment, PoHRI mRNA and protein are significantly upregulated in FEC cells but show different expression patterns in response to different stresses. In healthy flounders, PoHRI displays a wide tissue distribution at both the mRNA and protein levels. These results indicate that PoHRI is a ubiquitous eIF2a kinase and might play an important role in translational control over nonheme producing FEC cells under different stresses. (c) 2006 Elsevier Ltd. All rights reserved.

alr0117, a two-component histidine kinase gene, is involved in heterocyst development in Anabaena sp PCC 7120

Anabaena sp. PCC; 7120 was mutagenized by transposon Tn5-1087b, generating a mutant whose heterocysts lack the envelope polysaccharide layer. The transposon was located between nucleotides 342 and 343 of alr0117, a 918 bp gene encoding a histidine kinase for a two-component regulatory system. Complementation of the mutant with a DNA fragment containing alr0117 and targeted inactivation of the gene confirmed that alr0117 is involved in heterocyst development. RT-PCR showed that alr0117 was constitutively expressed in the presence or absence of a combined-nitrogen source. hepA and patB, the two genes turned on during wild-type heterocyst development, were no longer activated in an alr0117-null mutant. The two-component signal transduction system involving alr0117 may control the formation of the envelope polysaccharide layer and certain late events essential to the function of heterocysts.

pkn22 (alr2502) encoding a putative Ser/Thr kinase in the cyanobacterium Anabaena sp PCC 7120 is induced by both iron starvation and oxidative stress and regulates the expression of isiA

In cyanobacteria, the isiA gene is required for cell adaptation to oxidative damage caused by the absence of iron. We show here that a putative Ser/Thr kinase gene, pkn22 (alr2052), is activated by iron deficiency and oxidative damage in Anabaena sp. PCC 7120. A pkn22 insertion mutant is unable to grow when iron is limiting. pkn22 regulates the expression of isiA (encoding CP43') but not of isiB (encoding flavodoxin) and psbC (CP43). Fluorescence measurement at 77 K reveals the absence of the typical signature of CP43' associated with photosystem I in the mutant under iron-limiting conditions. We propose that Pkn22 is required for the function of isiA/CP43' and constitutes a regulatory element necessary for stress response. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Oscillatory change of SR-protein kinase activities during oocyte maturation meiosis in fish

The SR-protein kinase activity was analyzed and the cytological changes were observed during oocyte maturation in bisexual transparent color crucian carp ( Carassius auratus color variety). The results revealed that the SR-protein kinase activity was sensitive to the artificially induced spawning hormones, and the change of oscillatory activity was similar to that of the maturation-promoting factor (MPF) kinase that regulates meiotic cell cycle in fish.

PURIFICATION AND CHARACTERIZATION OF A KINASE SPECIFIC FOR THE SERINE-RICH AND ARGININE-RICH PRE-MESSENGER-RNA SPLICING FACTORS

Members of the SR family of pre-mRNA splicing factors are phosphoproteins that share a phosphoepitope specifically recognized by monoclonal antibody (mAb) 104. Recent studies have indicated that phosphorylation may regulate the activity and the intracellular localization of these splicing factors. Here, we report the purification and kinetic properties of SR protein kinase 1 (SRPK1), a kinase specific for SR family members. We demonstrate that the kinase specifically recognizes the SR domain, which contains serine/arginine repeats. Previous studies have shown that dephosphorylated SR proteins did not react with mAb 104 and migrated faster in SDS gels than SR proteins from mammalian cells. We show that SRPK1 restores both mobility and mAB 104 reactivity to a SR protein SF2/ASF (splicing factor 2/alternative splicing factor) produced in bacteria, suggesting that SRPK1 is responsible for the generation of the mAb 104-specific phosphoepitope in vivo. Finally, we have correlated the effects of mutagenesis in the SR domain of SF2/ASF on splicing with those on phosphorylation of the protein by SRPK1, suggesting that phosphorylation of SR proteins is required for splicing.

A SERINE KINASE REGULATES INTRACELLULAR-LOCALIZATION OF SPLICING FACTORS IN THE CELL CYCLE

Small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing factors containing a serine/arginine-rich domain (SR proteins) concentrate in 'speckles' in the nucleus of interphase cells(1). It is believed that nuclear speckles act as storage sites for splicing factors while splicing occurs on nascent transcripts(2). Splicing factors redistribute in response to transcription inhibition(3,4) or viral infection(5), and nuclear speckles break down and reform as cells progress through mitosis(6). We have now identified and cloned a kinase, SRPK1, which is regulated by the cell cycle and is specific for SR proteins; this kinase is related to a Caenorhabditis elegans kinase and to the fission yeast kinase Dsk1 (ref. 7). SRPK1 specifically induces the disassembly of nuclear speckles, and a high level of SRPK1 inhibits splicing in vitro. Our results indicate that SRPK1 mag have a central role in the regulatory network for splicing, controlling the intranuclear distribution of splicing factors in interphase cells, and the reorganization of nuclear speckles during mitosis.

Development of solar-blind AlGaN 128x128 ultraviolet focal plane arrays

This paper reports the development of solar-blind aluminum gallium nitride (AlGaN) 128x128 UV Focal Plane Arrays (FPAs). The back-illuminated hybrid FPA architecture consists of an 128x128 back-illuminated AlGaN PIN detector array that is bump-mounted to a matching 128x128 silicon CMOS readout integrated circuit (ROIC) chip. The 128x128 p-i-n photodiode arrays with cuton and cutoff wavelengths of 233 and 258 nm, with a sharp reduction in response to UVB (280-320 nm) light. Several examples of solar-blind images are provided. This solar-blind band FPA has much better application prospect.

Diffractive microlens with a cascade focal plane fabricated by single mask UV-photolithography and common KOH:H2O etching

A diffractive microlens with a cascade focal plane along the main optical axis of the device is fabricated using a low-cost technique mainly including single mask ultraviolet (UV) photolithography and dual-step KOH:H2O etching. Based on the evolutionary behavior of converse pyramid-shaped microholes (CPSMs) preshaped over a {100}-oriented silicon wafer in KOH etchant, the first-step KOH etching is performed to transfer initial square micro-openings in a SiO2 film grown by plasma enhanced chemical vapor deposition (PECVD) and patterned by single mask UV-photolithography, into CPSMs with needed dimension. After completely removing a thinned SiO2 mask, basic annular phase steps with a relatively steep sidewall and scheduled height can be shaped in the overlapped etching region between the neighboring silicon concave-arc microstructures evolved from CPSMs through the second-step KOH etching. Morphological measurements demonstrate a desirable surface of the silicon microlens with a roughness in nanometer scale and the feature height of the phase steps formed in the submicrometer range. Conventional optics measurements of the plastic diffractive microlens obtained by further hot embossing the fine microrelief phase map over the nickel mask made through a common electrochemical method indicate a highly efficient cascaded focusing performance.

The Effects of Roughness on Adhesion Hysteresis

Adhesion hysteresis is defined as the difference between the work needed to separate two surfaces and that originally gained on bringing them together. Adhesion hysteresis is a common phenomenon in most surface/interface interactions. This paper studies the effects of surface roughness on adhesion hysteresis. We assumed that the surface asperity height distribution is Gaussian. Numerical simulations based on Fuller's model showed that adhesion hysteresis depended upon a single dimensionless parameter, the adhesion parameter, which represents the statistical average of a competition between the compressive forces exerted by the higher asperities, which are trying to separate the surfaces, and the adhesion forces of the lower asperities which are trying to hold the surfaces together. (C) Koninklijke Brill NV, Leiden, 2010

Size-Dependent Adhesion Strength of a Single Viscoelastic Fiber

Nano-fibrillar adhesives can adhere strongly to surfaces as a gecko does. The size of each fiber has significant effects on the adhesion enhancement, especially on rough surfaces. In the present study, we report the size effects on the normal and shear strength of adhesion for a single viscoelastic fiber. It is found that there exists a limited region of the critical sizes under which the interfacial normal or tangential tractions uniformly attain the theoretical adhesion strength. The region for a viscoelastic fiber under tension with similar material constants to a gecko's spatula is 135-255 nm and that under torque is 26.5-52 nm. This finding is significant for the development of artificial biomimetic attachment systems.

Potential role of DNA-dependent protein kinase in cellular resistance to ionizing radiation

In this paper, we study the ability of DNA-PK-deficient (M059J) and -proficient (M059K) cells to undergo the rate of cellular proliferation, cell cycle distribution and apoptosis after 10 Gy X-ray irradiation, and the role of DNA-PK in radiosensitivity. The results showed that M059J cells exhibited hyper-radiosensitivity compared with M059K cells. A strong G2 phase arrest was observed in M059J cells post irradiation. Significant accumulation in the G2 phase in M059J cells was accompanied by apoptosis at 12 h. Altogether, the data suggested that DNA-PK may have two roles in mammalian cells after DNA damage, a role in DNA DSB repair and a second role in DNA-damaged cells to traverse a G2 checkpoint, by which DNA-PK may affect cellular sensitivity to ionizing radiation. 地址: [Li Ning; Zhang Hong; Wang Yanling; Hao Jifang] Chinese Acad Sci, Inst Modern Phys, Lanzhou 730000, Peoples R China; [Li Ning; Zhang Hong; Wang Yanling; Hao Jifang] Key Lab Heavy Ion Radiat Med Gansu Prov, Lanzhou 730000, Peoples R China; [Li Ning; Wang Yanling] Chinese Acad Sci, Grad Sch, Beijing 100039, Peoples R China; [Wang Xiaohu] Gansu Tumor Hosp, Dept Radiotherapy, Lanzhou 730050, Peoples R China

Influence of Inter-Fiber Spacing and Interfacial Adhesion on Failure of Multi-Fiber Model Composites: Experiment and Numerical Analysis

The uniaxial tension experiments on glass-fiber-reinforced epoxy matrix composites reveal that the fragmentations of fibers display vertically aligned fracture, clustered fracture, coordinated fracture, and random fracture with the increase of inter-fiber spacing. The finite element analysis indicates that the fragmentations of fibers displaying different phenomena are due to the stress concentration as well as the inherent randomness of fiber defects, which is the dominant factor. The experimental results show that matrices adjacent to the fiber breakpoints all exhibit birefringent-whitening patterns for the composites with different interfacial adhesion strengths. The larger the extent of the interfacial debonding, the less the domain of the birefringent-whitening patterns. The numerical analysis indicates that the orientation of the matrix adjacent to a fiber breakpoint is caused by the interfacial shear stress, resulting in the birefringent-whitening patterns. The area of shear stress concentrations decides on the domain of the birefringent-whitening patterns.

Layer-by-layer assembly of viral capsid for cell adhesion

Cowpea mosaic virus (CPMV)-based thin films are biologically active for cell culture. Using layer-by-layer assembly of CPMV and poly(diallyldimethylammonium chloride), quantitatively scalable biomolecular surfaces were constructed, which were well characterized using quartz crystal microbalance, UV-vis and atomic force microscopy. The surface coverage of CPMV nanoparticles depended on the adsorption time and pH of the virus solution, with a greater amount of CPMV adsorption occurring near its isoelectric point. It was found that the adhesion and proliferation of NIH-3T3 fibroblasts can be controlled by the coverage of viral particles using this multilayer technique.

Microarray-based Raman spectroscopic assay for kinase inhibition by gold nanoparticle probes

In this paper, a microarray-based surface-enhanced Raman spectroscopic (SERS) assay for detection of kinase functionality and inhibition has been reported. Biotinylated anti-phosphoserinen antibodies mark the phosphorylation and inhibition events and gold nanoparticles are attached to the antibodies by standard avidin-biotin chemistry, followed by silver deposition for SERS signal enhancement. The avidin conjugated fluorescein is used as SERS probe. The alpha-catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP) dependent protein kinase (PKA), its well known substrate, kemptide, and three inhibitors, H89, HA1077, and KN62 have been chosen here to establish the SERS assay. As expected, highly selective inhibition of PKA is demonstrated with the inhibitor H89 and the inhibition assay enable to detect kinase inhibition as well as derive IC50 (half maximal inhibitory concentration) plots.