Proteomic analysis of the response of Arabidopsis chloroplast proteins to high light stress

Light is an essential environmental factor in the progression of plant growth and development but prolonged exposure to high levels of light stress can cause cellular damage and ultimately result in the death of the plant. Plants can respond defensively to this stress for a limited period and this involves changes to their gene expression profiles. Proteomic approaches were therefore applied to the study of the response to high light stress in the Arabidopsis thaliana plant species. Wild-type Arabidopsis was grown under normal light (100 micromol photons.m(-2).s(-1)) conditions and then subjected to high light (1000 micromol photons.m(-2).s(-1)) stress. Chloroplasts were then isolated from these plants and both soluble and insoluble proteins were extracted and subjected to two-dimensional (2-D) gel electrophoresis. The resolved proteins were subsequently identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and comparative database analysis. 64 protein spots, which were identified as candidate factors that responded to high light stress, were then selected for analysis and 52 of these were successfully identified using MALDI-TOF-MS analysis. 35 of the 52 identified proteins were found to decrease their expression levels during high light stress and a further 14 of the candidate proteins had upregulated expression levels under these conditions. Most of the proteins that were downregulated during high light stress are involved in photosynthesis pathways. However, many of the 14 upregulated proteins were identified as previously well-known high light stress-related proteins, such as heat shock proteins (HSPs), dehydroascorbate reductase (DHAR), and superoxide dismutase (SOD). Three novel proteins that were more highly expressed during periods of high light stress but had no clear functional relationship to these conditions, were also identified in this study.     

Nitric oxide synthase and calcium-binding protein-containing neurons in the hamster visual cortex

The distribution and morphology of neurons containing neuronal nitric oxide synthase (NOS), and calcium-binding proteins calbindin D28K and calretinin in the hamster visual cortex were compared by immunocytochemistry. Staining for NOS, calbindin D28K and calretinin was seen both in the specific layers and in the selective cell types. The densest concentration of anti-NOS-immunoreactive (IR) neurons was found in layer VI. Most of the calbindin D28K-IR neurons were located in layers II/III and V while the calretinin-IR neurons were predominantly located in layers II/III. The labeled neurons varied in morphology. The large majority of NOS-IR neurons were round or oval cells with many dendrites coursing in all directions. The majority of the calbindin D28K-IR neurons were stellate and round or oval cells with multipolar dendrites. The majority of the calretinin-IR neurons were vertical fusiform cells with long processes traveling perpendicular to the pial surface. Our study showed that 14.7% and 27.5% of the NOS-IR cells in the hamster visual cortex contained calbindin D28K or calretinin, respectively. These results indicate that NOS, calbindin and calretinin are located in specific layers and specific cell types and the vast majority of NOS-containing neurons are limited to neurons that do not express calbindin D28K or calretinin.     

Inhibitory effects of glycoprotein-120 (G-120) from Ulmus davidiana Nakai on cell growth and activation of matrix metalloproteinases

Excessive breakdown of extracellular matrix by metalloproteinases (MMPs) occurs in many pathological conditions. Consequently, methods for inhibiting MMP activity have therapeutic potential. In this study, we investigated the effect of G-120, a 120 kDa glycoprotein purified from the Oriental herbal plant, Ulmus davidiana Nakai (UDN), on the activity and production of several MMPs by evaluating its growth inhibitory effect on NIH 3T3 cells. Tritium uptake assays showed that proliferation of NIH 3T3 cells was strongly suppressed, and G-120-mediated inhibition of DNA synthesis proved to involve a cytostatic, rather than a cytotoxic, effect, as shown by cytotoxicity and apoptosis assays. More importantly, G-120 strongly reduced the gelatinolytic and collagenase activities of MMP proteins, as well as expression of MMP-2 and MMP-9. Electrophoretic mobility shift assays revealed that it suppressed the DNA binding activity of NF-kappaB. Collectively, our observations show that G-120 strongly inhibits the activation of MMPs and NF-kappaB.     

Retrovirus-mediated gene transfer and expression of EGFP in chicken

Here, we successfully demonstrate expression of the EGFP (enhanced green fluorescence protein) gene in chickens using replication-defective MLV (murine leukemia virus)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G glycoprotein). The recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). After 12 days incubation, all of the eight living embryos assayed were found to express this vector-encoded EGFP gene, which was under the control of the RSV (Rous Sarcoma Virus) promoter, in diverse organ tissues, including head, beak, neck, wing, hock, tail, toes, heart, amnion, and yolk sac. Surprisingly, despite the presumed cytotoxicity of EGFP, some embryos hatched and survived and these had prominent green fluorescent spots, both in internal organs and externally.     

Immunohistochemical localization of Bis protein in the rat central nervous system

We studied the distribution of Bis (Bcl-2 interacting death suppressor) protein in the adult rat brain and spinal cord using immunohistochemistry. Immunoreactivity was observed in specific neuronal populations in distinct nuclei. The most intensely labeled cells were associated with the motor system, including most cranial nerve motor nuclei, Purkinje cells of the cerebellum, the red nucleus, and the ventral motor neurons of the spinal cord. Bis protein was also expressed in several structures associated with the ventricular system, including the subventricular zone of the lateral ventricle and its rostral extension, in the subcommissural organ, and in tanycytes, radial glial cells in the hypothalamus. Using double-labeling techniques, Bis-immunoreactive cells in the rostral migratory stream, coexpressing Bcl-2, were confirmed as glial fibrillary acidic protein-positive astrocytes comprising the glial tubes. The widespread distribution of Bis suggests that this protein has broader functions in the adult rat central nervous system than previously thought, and that it could be associated with a particular role in the rostral migratory system.J.-H. Lee and M.-Y. Lee contributed equally to this study. This work was supported by the KOSEF through the Cell Death Disease Research Center of MRC at the Catholic University of Korea (R13-2002-005-01001-0) and the Catholic Medical Center Research Foundation grant made in the program year of 2002     

Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR

Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (> 45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.     

An intramolecular spin of the LDL receptor beta propeller

Study of the LDL receptor as a model system has led to insights into general principles underlying receptor-mediated endocytosis of bound ligands. The recently published structure of the entire LDL receptor ectodomain, determined at pH 5.3, now suggests an elegant model to explain how lipoprotein ligands are released from the receptor by exposure to the low-pH environment of the endosome.     

Immunocytochemical localization of neurons containing the AMPA GluR2/3 subunit in the hamster visual cortex

AMPA glutamate receptors play a crucial role in brain functions such as synaptic plasticity and development. We have studied the chemo-architecture of the AMPA glutamate receptor subtype GluR2/3 in the hamster visual cortex by immunocytochemistry and compared it with the distribution of the calcium-binding proteins, calbindin D28K and calretinin. Anti-GluR2/3-immunoreactive (IR) neurons were predominantly located in layers II/III, V, and VI, and the majority of the labeled neurons were round or oval. However, many pyramidal cells in layer V were also labeled. Two-color immunofluorescence revealed that none of the GluR2/3-IR neurons contained calbindin D28 K or calretinin. Thus specific layers of neurons express the GluR2/3 subunit and these do not correlate with expression of calbindin D28K and calretinin.     

A functional interaction between chromogranin B and the inositol 1,4,5-trisphosphate receptor/Ca2+ channel

Chromogranins A and B (CGA and CGB) are high capacity, low affinity calcium (Ca2+) storage proteins found in many cell types most often associated with secretory granules of secretory cells but also with the endoplasmic reticulum (ER) lumen of these cells. Both CGA and CGB associate with inositol 1,4,5-trisphosphate receptor (InsP3R) in a pH-dependent manner. At an intraluminal pH of 5.5, as found in secretory vesicles, both CGA and CGB bind to the InsP3R. When the intraluminal pH is 7.5, as found in the ER, CGA totally dissociates from InsP3R, whereas CGB only partially dissociates. To investigate the functional consequences of the interaction between the InsP3R and CGB monomers or CGA/CGB heteromers, purified mouse InsP3R type I were fused to planar lipid bilayers and activated by 2 microM InsP3. In the presence of luminal CGB monomers or CGA/CGB heteromers the InsP3R/Ca2+ channel open probability and mean open time increased significantly. The channel activity remained elevated when the pH was changed to 7.5, a reflection of CGB binding to the InsP3R even at pH 7.5. These results suggest that CGB may play an important modulatory role in the control of Ca2+ release from the ER. Furthermore, the difference in the ability of CGA and CGB to regulate the InsP3R/Ca2+ channel and the variability of CGA/CGB ratios could influence the pattern of InsP3-mediated Ca2+ release.     

Leukotactin-1-induced ERK activation is mediated via Gi/Go protein/PLC/PKC delta/Ras cascades in HOS cells

Recently cloned leukotactin-1 (Lkn-1) that belongs to CC chemokine family has not been characterized. To understand the intracellular events following Lkn-1 binding to CCR1, we investigated the activities of signaling molecules in response to Lkn-1 in human osteogenic sarcoma cells expressing CCR1. Lkn-1-stimulated cells showed elevated phosphorylation of extracellular signal-related kinases (ERK1/2) with a distinct time course. ERK activation was peaked in 30 min and 12 h showing biphasic activation of ERK. Pertussis toxin, an inhibitor of G(i)/G(o) protein, and phospholipase C (PLC) inhibitor blocked Lkn-1-induced activation of ERK. Protein kinase C delta (PKC delta) specific inhibitor rottlerin inhibited ERK activation in Lkn-1-stimulated cells. The activities of PLC and PKC delta were also enhanced by Lkn-1 stimulation. Dominant negative Ras inhibited activation of ERK. Immediate early response genes such as c-fos and c-myc were induced by Lkn-1 stimulation. Lkn-1 affected the cell cycle progression by cyclin D(3) induction. These results suggest that Lkn-1 activates the ERK pathway by transducing the signal through G(i)/G(o) protein, PLC, PKC delta and Ras, and it may play a role for cell proliferation, differentiation, and regulation of gene expression for other cellular processes.