rDrak1, a novel kinase related to apoptosis, is strongly expressed in active osteoclasts and induces apoptosis

This is the first report of a novel serine/threonine kinase, rabbit death-associated protein (DAP) kinase-related apoptosis-inducing protein kinase 1 (rDRAK1), involved in osteoclast apoptosis. We searched for osteoclast-specific genes from a cDNA library of highly enriched rabbit osteoclasts cultured on ivory. One of the cloned genes has a high homology with human DRAK1 (hDRAK1), which belongs to the DAP kinase subfamily of serine/threonine kinases. By screening a rabbit osteoclast cDNA library and 5'-RACE (rapid amplification of cDNA ends), we obtained a full length of this cDNA, termed rDRAK1. The sequencing data indicated that rDRAK1 has 88.0, 44.6, 38.7, and 42.3% identity with hDRAK1, DAP kinase, DRP-1, and ZIP (zipper-interacting protein) kinase, respectively. To clarify the role of DRAK1 in osteoclasts, we examined the effect of three osteoclast survival factors (interleukin-1, macrophage colony-stimulating factor, and osteoclast differentiation-inducing factor) on rDRAK1 mRNA expression and the effect of rDRAK1 overexpression on osteoclast apoptosis. The results suggested that these three survival factors were proved to inhibit rDRAK1 expression in rabbit osteoclasts. After transfection of a rDRAK1 expression vector into cultured osteoclasts, overexpressed rDRAK1 was localized exclusively to the nuclei and induced apoptosis. Hence, rDRAK1 may play an important role in the core apoptosis program in osteoclast.     

The adapter protein Crkl links Cbl to C3G after integrin ligation and enhances cell migration

Crkl, an SH2-SH3-SH3 adapter protein, is one of the major tyrosine phosphoproteins detected in cells from patients with chronic myelogenous leukemia. Crkl binds to BCR/ABL through its N-terminal SH3 domain and is known to interact with several signaling proteins that have been implicated in integrin signaling, including Cbl, Cas, Hef-1, and paxillin. We have previously shown that overexpression of Crkl enhances adhesion to extracellular matrix proteins through beta(1) integrins. In this study, the effects of Crkl on spontaneous and chemokine-directed migration of the hematopoietic cell line Ba/F3 were examined. Full-length, SH2-, and SH3(N)-domain deletion mutants of Crkl were expressed transiently as fusion proteins with green fluorescent protein. Successfully transfected cells were isolated by fluorescence-activated cell sorting. The ability of these cells to migrate across a fibronectin-coated membrane, either spontaneously or in response to the chemokine stromal-derived factor-1alpha, was determined. Cells expressing green fluorescent protein alone were not distinguishable from untransfected or mock transfected Ba/F3 cells. However, Ba/F3 cells overexpressing full-length Crkl were found to have an increase in spontaneous migration of 2.8 +/- 0.6-fold in seven independent assays. The enhancement of migration required both the SH2 domain and the N-terminal SH3 domain. Migration in response to stromal-derived factor-1alpha was not significantly enhanced by overexpression of Crkl. Overexpression of Crkii also augmented spontaneous migration but to a lesser degree than did Crkl. Because the SH2 domain was required for enhanced migration, we looked for changes in phosphotyrosine containing proteins coprecipitating with Crkl, but not Crkl DeltaSH2, after integrin cross-linking. Full-length Crkl, but not CrklDeltaSH2, coprecipitated with a single major tyrosine phosphoprotein with an M(r) of approximately 120 kDa, identified as Cbl. The major Crkl SH3-binding protein in these cells was found to be the guanine nucleotide exchange factor, C3G. Interestingly, overexpression of C3G also enhanced migration, suggesting that a Cbl-Crkl-C3G complex may be involved in migration signaling in Ba/F3 cells. These data suggest that Crkl is involved in signaling pathways that regulate migration, possibly through a complex with Cbl and C3G.     

Regeneration and maturation of daughter cell walls in the autospore-forming green alga<Emphasis Type="Italic"> Chlorella vulgaris</Emphasis> (Chlorophyta,Trebouxiophyceae)

Cell-wall synthesis in Chlorella vulgaris, an autospore-forming alga, was observed using the cell wall-specific fluorescent dye Fluostain I. The observation suggested two clearly distinguishable stages in cell-wall synthesis: moderate synthesis during the cell-growth process and rapid synthesis at the cell-division stage. We used electron microscopy to examine the structural changes that occurred with growth in the premature daughter cell wall during the cell-growth and cell-division phases. The cell began to synthesize a new daughter cell wall shortly after its release from the autosporangium. A very thin daughter cell wall, with a thickness of about 2 nm, was formed inside the mother cell wall and completely enveloped the outer surface of the plasma membrane of the cell. The daughter cell wall gradually increased in thickness from 2 to 3.8 nm. During the protoplast-division phase in the cell-division stage, the daughter cell wall expanded on the surface of the invaginating plasma membrane of the cleavage furrow, accompanied by active synthesis of the cell wall, which increased in thickness from 3.8 to 6.1 nm. The daughter cell matured into an autospore while completely enclosed by its own thickening (from 6.1 to 17 nm) wall. Finally, the released daughter cell was enclosed by its own cell wall after the mother cell wall burst. The daughter cell with mature wall thickness (17–21 nm) emerged as a small, but complete, autospore.     

Comparison of response of two C3 species to leaf water relation,proline synthesis,gas exchange and water use under periodic water stress

Water relations, proline content and gas exchange of leaf were investigated under periodic water stress for two C3 plants (eggplant and tomato) in a greenhouse to study comparative adaptive responses. Although both species showed reduced water content of leaf and increased osmolality and proline content under low soil water potential, the recovery capacity after the stress was better in eggplant than tomato. Both species over-accumulated proline under low soil water potential and returned to its initial concentration during the recovery, indicating that proline may act as an osmoprotectant during drought. Proline was directly corresponding with osmolality during stress, and dehydration stress reduced the gas exchange parameters such as transpiration rate (ET), stomatal conductance (GS), and photosynthesis rate (Pn). At the final stage of the experiment both species showed 2.6 and 3.3 times lower Pn and 27 and 19 times lower GS for eggplant and tomato, respectively, as compared to control. But after stress was relieved by rewatering, both plants increased GS for 2 to 3 times and Pn for 4.5 times. Eggplant showed better water use efficiency (WUE) in relation to fruit production under the stress than tomato. Higher biomass allocation at root and fruit parts in eggplant indicated more efficient recovery than that of tomato. These findings inferred that both C3 plants developed internal complementary drought survival mechanism by lowering relative water content, increasing proline, and decreasing stomatal conductance but eggplants withstood the periodic draughting better than tomato, mainly due to its ability to recover from a water stress condition.     

Proteomics of the rice cell: systematic identification of the protein populations in subcellular compartments

Despite recent progress in sequencing the complete genome of rice (Oryza sativa), the proteome of this species remains poorly understood. To extend our knowledge of the rice proteome, the subcellular compartments, which include plasma membranes (PM), vacuolar membranes (VM), Golgi membranes (GM), mitochondria (MT), and chloroplasts (CP), were purified from rice seedlings and cultured suspension cells. The proteins of each of these compartments were then systematically analyzed using two-dimensional (2D) electrophoresis, mass spectrometry, and Edman sequencing, followed by database searching. In all, 58 of the 464 spots detected by 2D electrophoresis in PM, 43 of the 141 spots in VM, 46 of the 361 spots in GM, 146 in the 672 spots in MT, and 89 of the 252 spots in CP could be identified by this procedure. The characterized proteins were found to be involved in various processes, such as respiration and the citric acid cycle in MT; photosynthesis and ATP synthesis in CP; and antifungal defense and signal systems in the membranes. Edman degradation revealed that 60–98% of N-terminal sequences were blocked, and the ratios of blocked to unblocked proteins in the proteomes of the various subcellular compartments differed. The data on the proteomes of subcellular compartments in rice will be valuable for resolving questions in functional genomics as well as for genome-wide exploration of plant function.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. Jürgens     

Dual effects of acupuncture on gastric motility in conscious rats

The effects of manual acupuncture on gastric motility were investigated in 35 conscious rats implanted with a strain gauge transducer. Twenty (57.1%) rats showed no cyclic groupings of strong contractions (type A), whereas 15 (42.9%) rats showed the phase III-like contractions of the migrating motor complex (type B) in the fasting gastric motility. Acupuncture at the stomach (ST)-36 (Zusanli), but not on the back [Weishu, bladder (BL)-21], increased the peak amplitude of contractions to 172.4 +/- 25.6% of basal in the type A rats (n = 20, P < 0.05). On the other hand, the motility index for 60 min after the acupuncture was not affected by the acupuncture in this group. On the contrary, acupuncture decreased the peak amplitude and motility index to 72.9 +/- 14.0% and 73.6 +/- 16.2% in the type B rats (n = 15, P < 0.05), respectively. The stimulatory and inhibitory effects of acupuncture observed in each type were reproducible on the separate days. In 70% of type A rats, acupuncture induced strong phase III-like contractions lasting for over 3 h that were abolished by atropine, hexamethonium, atropine methyl bromide, and vagotomy. Naloxone significantly shortened the duration of the stimulatory effects from 3.52 +/- 0.21 to 1.02 +/- 0.15 h (n = 3, P < 0.05). These results suggest that acupuncture at ST-36 induces dual effects, either stimulatory or inhibitory, on gastric motility. The stimulatory effects are mediated in part via vagal efferent and opioid pathways.     

Systematic analysis of the combinatorial nature of epitopes recognized by TCR leads to identification of mimicry epitopes for glutamic acid decarboxylase 65-specific TCRs

Accumulating evidence indicates that recognition by TCRs is far more degenerate than formerly presumed. Cross-recognition of microbial Ags by autoreactive T cells is implicated in the development of autoimmunity, and elucidating the recognition nature of TCRs has great significance for revelation of the disease process. A major drawback of currently used means, including positional scanning synthetic combinatorial peptide libraries, to analyze diversity of epitopes recognized by certain TCRs is that the systematic detection of cross-recognized epitopes considering the combinatorial effect of amino acids within the epitope is difficult. We devised a novel method to resolve this issue and used it to analyze cross-recognition profiles of two glutamic acid decarboxylase 65-autoreactive CD4(+) T cell clones, established from type I diabetes patients. We generated a DNA-based randomized epitope library based on the original glutamic acid decarboxylase epitope using class II-associated invariant chain peptide-substituted invariant chains. The epitope library was composed of seven sublibraries, in which three successive residues within the epitope were randomized simultaneously. Analysis of agonistic epitopes indicates that recognition by both TCRs was significantly affected by combinations of amino acids in the antigenic peptide, although the degree of combinatorial effect differed between the two TCRs. Protein database searching based on the TCR recognition profile proved successful in identifying several microbial and self-protein-derived mimicry epitopes. Some of the identified mimicry epitopes were actually produced from recombinant microbial proteins by APCs to stimulate T cell clones. Our data demonstrate the importance of the combinatorial nature of amino acid residues of epitopes in molecular mimicry.     

Non-steroidal anti-inflammatory drugs affect the methotrexate transport in IEC-6 cells

Methotrexate (MTX) is used not only for the cancer chemotherapy but also for the treatment of rheumatic disease, often together with non-steroidal anti-inflammatory drugs (NSAIDs). MTX is actively cotransported with H(+) in the small intestine, mediated by a reduced folate carrier (RFC). The coadministration of some NSAIDs with MTX to rats caused a decrease of MTX absorption through the small intestine. This may be due to the uncoupling effect of oxidative phosphorylation of the NSAIDs. The present study investigated whether flufenamic acid, diclofenac and indomethacin, NSAIDs, decreased ATP content of rat-derived intestinal epithelial cell line IEC-6 cells and affected the MTX transport in IEC-6 cells. The MTX uptake in IEC-6 cells was dependent on medium pH and maximum around pH 4.5-5.5. The MTX uptake was composed of a transport inhibited by 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS) and a non-saturable one. The DIDS-sensitive component in the MTX uptake showed a saturation kinetics (Michaelis-Menten constant (Km): 3.91 +/- 0.52 microM, Maximum velocity (Vmax): 94.66 +/- 6.56 pmol/mg protein/5 min). The cellular ATP content in IEC-6 cells decreased significantly at 30 min after the cells were started to incubate with the NSAIDs (250 microM flufenamic acid, 500 microM diclofenac and 500 microM indomethacin). The MTX uptake in IEC-6 cells in the presence of the NSAIDs decreased with the reduction of cellular ATP content and showed a good correlation with the ATP content (correlation coefficient: 0.982). Thus it seems likely that the ATP content in IEC-6 cells with the NSAIDs decreased due to the uncoupling effect of oxidative phosphorylation of the NSAIDs, resulting in the inhibition of the secondary active transport of MTX in IEC-6 cells. The present results also suggest that IEC-6 cells are useful to evaluate the drug interaction relating to this carrier system.     

High-cut characteristics of the baroreflex neural arc preserve baroreflex gain against pulsatile pressure

A transfer function from baroreceptor pressure input to sympathetic nerve activity (SNA) shows derivative characteristics in the frequency range below 0.8 Hz in rabbits. These derivative characteristics contribute to a quick and stable arterial pressure (AP) regulation. However, if the derivative characteristics hold up to heart rate frequency, the pulsatile pressure input will yield a markedly augmented SNA signal. Such a signal would saturate the baroreflex signal transduction, thereby disabling the baroreflex regulation of AP. We hypothesized that the transfer gain at heart rate frequency would be much smaller than that predicted from extrapolating the derivative characteristics. In anesthetized rabbits (n = 6), we estimated the neural arc transfer function in the frequency range up to 10 Hz. The transfer gain was lost at a rate of -20 dB/decade when the input frequency exceeded 0.8 Hz. A numerical simulation indicated that the high-cut characteristics above 0.8 Hz were effective to attenuate the pulsatile signal and preserve the open-loop gain when the baroreflex dynamic range was finite.