Genes differentially expressed under photoinhibition stress in flag leaves of super-hybrid rice Liangyoupeijiu (<Emphasis Type="Italic">Oryza sativa</Emphasis>) and their genetic origins

Genes differentially expressed under high irradiance (HI) stress in mature flag leaves of super-hybrid rice Liangyoupeijiu (Oryza sativa ssp. indica) were studied by the silver-staining mRNA differential display technique. We obtained 167 differentially displayed bands on silver-stained polyacrylamide gels and searched for their genetic origins. Five cDNA fragments, which were upregulated by HI stress, were cloned and sequenced. The clones of G25×320, A31×380, and G29×280 putatively encode a cytosolic monodehydroascorbate reductase (MDAR), a phosphatidylinositol 4-kinase (PI-4-K), and a DEAH-box RNA helicase, respectively. Most differentially expressed genes in hybrid rice were inherited from parents and many of them were related with both parents, although some were derived from one parent only. The differentially expressed cDNA fragments having no distinct genetic origins indicated the involvement of some unclear mechanisms in the inheritance processes from parents to hybrid.This research was supported by the State Key Basic Research and Development Plan of China (G1998010100).     

Cdk pathway: Cyclin-dependent kinases and cyclin-dependent kinase inhibitors

Many mechanisms either activate or inhibit the cdks and thereby either promote or arrest progression through the mitotic cell cycle. Since the signal transduction pathways emanating from extracellular mitogens and the agents controlling these pathways are complicated there may yet be novel mechanisms of cell cycle regulation remaining to be elucidated. In this article we outline the different techniques used to study the cell cycle and its regulation. These include: establishing that the cell cycle is arrested by propidium iodide staining followed by FACS analysis or by measuring ³H-thymidine incorporation into DNA; measuring the amount of cyclin/cdk associated kinase activity; assessing the steady-state expression profiles of cyclins, cdks and ckis by immunoblotting; and investigating the formation of complexes between these proteins by coimmunoprecipitations. Caveats and advantages of each technique are discussed. Following this paradigm yielded the discovery of the cell cycle inhibitors p27^Kip1 and p21^Cip1 and could very well lead to the discovery or novel cell cycle regulatory mechanisms.     

Bacterial infection and tissue-specific Hsp72, -73 and -90 expression in western painted turtles

Heat shock proteins (Hsps) are molecular chaperones that assist intracellular folding, assembly and translocation of proteins in prokaryotic and eukaryotic cells. A variety of stresses including hyperthermia, radiation, heavy metals, ischemia, anoxia and reoxygenation have been shown to increase the expression of Hsps. Likewise, bacterial infection represents a stress for the host cell. In this study, expression of the constitutive (Hsp73) and inducible (Hsp72) isoforms of Hsp70 and Hsp90 was monitored in brain, heart, liver and skeletal muscle from the western painted turtle Chrysemys picta bellii diagnosed with Septicemic Cutaneous Ulcerative Dermatitis (SCUD). This disease is caused by a gram-negative bacterium probably belonging to the Citrobacter spp. The expression of Hsp73 increased 1.8-fold in brain and liver, 2.2-fold in heart but did not change in skeletal muscle; Hsp72 expression increased 5.5-fold in brain and 3-fold in liver but did not change in heart or skeletal muscle; Hsp90 expression increased 9-fold in brain, 2.7-fold in heart and 2.4-fold in skeletal muscle but did not change in liver. These results suggest a tissue-specific Hsp response during bacterial infection and a role for Hsps in immunopathological events in reptiles.     

Endogenous expression of nNOS protein in several neuronal cell lines

Several neuronal cell lines were screened for endogenous expression of neuronal nitric oxide synthase (nNOS) protein using Western blot analysis. Detectable levels of the nNOS protein were evident in the SK-N-SH, SH-SY5Y, and N1E-115 neuroblastoma cell lines, as well as the NG108-15 neuroblastoma x glioma hybrid. Only trace amounts were visible in Neuro2A human neuroblastoma cells. The presence of endogenously expressed nNOS in these cells may allow for the study of the interaction between nNOS and the endogenous receptor systems expressed in the same cells.     

Expression of swelling- and/or pH-regulated chloride channels (ClC-2, 3, 4 and 5) in human leukemic and normal immune cells

Chloride channels on immune cells reportedly play important roles in cell volume regulation, cell proliferation and immune functions, but they are not well characterized at the molecular level. We examined the expression of swelling-and/or pH-regulated chloride channels (ClC-2, 3, 4 and 5) in human leukemic cell lines [Jurkat and Hut-78 (T cells), Raji and Daudi (B cells), K-562 and HL-60 (myeloid cells)] and T cells, B cells and neutrophils from 8 normal subjects to clarify the difference of their expression among different cell types and maturity. Semi-quantitative RT-PCR and Northern blot analysis showed that ClC-3 was most abundantly expressed in all cells regardless of the cell types and maturity, while expression of ClC-2 was weak in these cells. Expression of ClC-4 was observed mainly in leukemic B cell lines, and in B cells and neutrophils from normal subjects. ClC-5 was expressed in all cell lines, while it was observed in only T and B cells but not in neutrophils from normal subjects. Thus, these chloride channels (ClC-2, 3, 4 and 5) showed distinct distribution among human immune cells, suggesting that they have specific roles in these cells. Molecular identification of chloride channels in leukocytes of different types and maturity may provide a new approach for the treatment of leukemia.     

Regulation of Xenopus embryonic cell adhesion by the small GTPase,rac

TGF-beta family signalling pathways are important for germ layer formation and gastrulation in vertebrate embryos and have been studied extensively using embryos of Xenopus laevis. Activin causes changes in cell movements and cell adhesion in Xenopus animal caps and dispersed animal cap cells. Rho family GTPases, including rac, mediate growth factor-induced changes in the actin cytoskeleton, and consequently, in cell adhesion and motility, in a number of different cell types. Ectopic expression of mutant rac isoforms in Xenopus embryos was combined with animal cap adhesion assays and a biochemical assay for rac activity to investigate the role of rac in activin-induced changes in cell adhesion. The results indicate that (1) the perturbation of rac signalling disrupts embryonic cell-cell adhesion, (2) that rac activity is required for activin-induced changes in cell adhesive behavior on fibronectin, and (3) that activin increases endogenous rac activity in animal cap explants.     

A gene located at 71F in Drosophila melanogaster encodes a novel zinc-finger protein that interacts with protein kinase CK2

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are two hormones produced and secreted by the heart to control blood pressure, body fluid homeostasis and electrolyte balance. Each peptide binds to a common family of 3 receptors (GC-A, GC-B and C-receptor) with varying degrees of affinity. The proANP gene disrupted mouse model provides an excellent opportunity to examine the regulation and expression of BNP in the absence of ANP. A new radioimmunoassay (RIA) was developed in order to measure mouse BNP peptide levels in the plasma, atrium and ventricle of the mouse. A detection limit of 3–6 pg/tube was achieved by this assay. Results show that plasma and ventricular level of BNP were unchanged among the three genotypes of mice. However, a significant decrease in the BNP level was noted in the atrium. The homozygous mutant (ANP–/–) had undetectable levels of BNP in the atrium, while the heterozygous (ANP+/–) and wild-type (ANP+/+) mice had 430 and 910 pg/mg in the atrium, respectively. Northern Blot analysis shows the ANP–/– mice has a 40% reduction of BNP mRNA level in the atrium and a 5-fold increase in the ventricle as compared with that of the ANP+/+ mouse. Our data suggest that there is a compensatory response of BNP expression to proANP gene disruption. Despite the changes in the atrial and ventricular tissue mRNA and peptide levels, the plasma BNP level remains unaltered in the ANP–/– mice. We conclude that the inability of BNP to completely compensate for the lack of ANP eventually leads to chronic hypertension in the proANP gene disrupted mice.     

Expression of the gene for large subunit of m-calpain is elevated in skeletal muscle from Duchenne muscular dystrophy patients

Calpain is an intracellular nonlysosomal protease involved in essential regulatory or processing functions of the cell, mediated by physiological concentrations of Ca²⁺. However, in an environment of abnormal intracellular calcium, such as that seen in Duchenne muscular dystrophy (DMD), calpain is suggested to cause degeneration of muscle owing to enhanced activity. To test whether the reported increase in calpain activity in DMD results fromde novo synthesis of the protease, we have assessed the quantitative changes in mRNA specific for m-calpain. mRNA isolated from DMD and control muscle was analysed by dot blot hybridization using a cDNA probe for the large subunit of m-calpain. Compared to control a four-fold increase in specific mRNA was observed in dystrophic muscle. This enhanced expression of the m-calpain gene in dystrophic condition suggests that the reported increase in m-calpain activity results fromde novo synthesis of protease and underlines the important role of m-calpain in DMD.