Regulation of pollen tube growth by Rac-like GTPases

Plant Rac-like GTPases have been classified phylogenetically into two major groups-class I and class II. Several pollen-expressed class I Rac-like GTPases have been shown to be important regulators of polar pollen tube growth. The functional participation by some of the class I and all of the class II Arabidopsis Rac-like GTPases in pollen tube growth remains to be explored. It is shown that at least four members of the Arabidopsis Rac GTPase family are expressed in pollen, including a class II Rac, AtRac7. However, when over-expressed as fusion proteins with GFP, both pollen- and non-pollen-expressed AtRacs interfered with the normal pollen tube tip growth process. These observations suggest that these AtRacs share similar biochemical activities and may integrate into the pollen cellular machinery that regulates the polar tube growth process. Therefore, the functional contribution by individual Rac GTPase to the pollen tube growth process probably depends to a considerable extent on their expression characteristics in pollen. Among the Arabidopsis Racs, GFP-AtRac7 showed association with the cell membrane and Golgi bodies, a pattern distinct from all previously reported localization for other plant Racs. Over-expressing GFP-AtRac7 also induced the broadest spectrum of pollen tube growth defects, including pollen tubes that are bifurcated, with diverted growth trajectory or a ballooned tip. Transgenic plants with multiple copies of the chimeric Lat52-GFP-AtRac7 showed severely reduced seed set, probably many of these defective pollen tubes were arrested, or reduced in their growth rates that they did not arrive at the ovules while they were still receptive for fertilization. These observations substantiate the importance of Rac-like GTPases to sexual reproduction.     

The N-terminal domain of hepatocyte growth factor inhibits the angiogenic behavior of endothelial cells independently from binding to the c-met receptor

Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an important role in complex biological processes such as embryogenesis, tissue regeneration, cancerogenesis, and angiogenesis. HGF promotes cell proliferation, survival, motility, and morphogenesis through binding to its receptor, a transmembrane tyrosine kinase encoded by the MET proto-oncogene (c-met). Structurally speaking, HGF is a polypeptide related to the enzymes of the blood coagulation cascade. Thus, it comprises kringle domains that in some other proteins have been shown to be responsible for the anti-angiogenic activity. To check whether the isolated kringles of HGF were able to inhibit angiogenesis, we produced them as recombinant proteins and compared their biological activity with that of the recombinant HGF N-terminal domain (N). We showed that (i) none of the isolated HGF kringle exhibits an anti-angiogenic activity; (ii) N is a new anti-angiogenic polypeptide; (iii) the inhibitory action of N is not specific toward HGF, because it antagonized the angiogenic activity of other growth factors, such as fibroblast growth factor-2 and vascular endothelial growth factor; and (iv) in contrast with full-length HGF, N does not bind to the c-met receptor in vitro, but fully retains its heparin-binding capacity. Our results suggest that N inhibits angiogenesis not by disrupting the HGF/c-met interaction but rather by interfering with the endothelial glycosaminoglycans, which are the secondary binding sites of HGF.     

RhoA interaction with inositol 1,4,5-trisphosphate receptor and transient receptor potential channel-1 regulates Ca2+ entry. Role in signaling increased endothelial permeability

We tested the hypothesis that RhoA, a monomeric GTP-binding protein, induces association of inositol trisphosphate receptor (IP3R) with transient receptor potential channel (TRPC1), and thereby activates store depletion-induced Ca2+ entry in endothelial cells. We showed that RhoA upon activation with thrombin associated with both IP3R and TRPC1. Thrombin also induced translocation of a complex consisting of Rho, IP3R, and TRPC1 to the plasma membrane. IP3R and TRPC1 translocation and association required Rho activation because the response was not seen in C3 transferase (C3)-treated cells. Rho function inhibition using Rho dominant-negative mutant or C3 dampened Ca2+ entry regardless of whether Ca2+ stores were emptied by thrombin, thapsigargin, or inositol trisphosphate. Rho-induced association of IP3R with TRPC1 was dependent on actin filament polymerization because latrunculin (which inhibits actin polymerization) prevented both the association and Ca2+ entry. We also showed that thrombin produced a sustained Rho-dependent increase in cytosolic Ca2+ concentration [Ca2+]i in endothelial cells overexpressing TRPC1. We further showed that Rho-activated Ca2+ entry via TRPC1 is important in the mechanism of the thrombin-induced increase in endothelial permeability. In summary, Rho activation signals interaction of IP3R with TRPC1 at the plasma membrane of endothelial cells, and triggers Ca2+ entry following store depletion and the resultant increase in endothelial permeability.     

Adaptive response to alkylating agents in the Drosophila sex-linked recessive lethal assay

The adaptive response to alkylating agents was studied in Drosophila assays under various treatment procedures. Pre-treatment of males as well as treatment of females with low doses of EMS (0.05-0.1 mM) did not affect sex-linked recessive lethal (SLRL) rates induced by high doses of this mutagen (10 mM, various feeding duration) in mature sperm cells. Pre-treatment of males with a low dose of MMS (0.1 mM) enhanced mutagenesis induced by the high dose of EMS (10 mM) at different stages of spermatogenesis, the observed effects exceeding the additive action of both mutagens. On the contrary, larval pre-treatment with the adaptive dose of EMS (0.05 mM) resulted in resistance of their germ cells to higher doses of EMS (1 mM). Specifically, offspring production increased while dominant lethality in F(1) as well SLRL frequency in F(2) was significantly reduced as compared with the effects of larval exposure to the challenge dose. Under the conditions tested, the adaptive response of germ cells to alkylating agents was demonstrated in larvae, but not in adult flies.     

Binding and internalization of lipopolysaccharide by Cla-1, a human orthologue of rodent scavenger receptor B1

Scavenger receptor, class B, type I (SR-BI) mediates selective uptake of high density lipoprotein (HDL) cholesteryl ester. SR-BI recognizes HDL, low density lipoprotein (LDL), exchangeable apolipoproteins, and protein-free lipid vesicles containing negatively charged phospholipids. Lipopolysaccharides (LPS) are highly glycosylated anionic phospholipids contributing to septic shock. Despite significant structural similarities between anionic phospholipids and LPS, the role of SR-BI in LPS uptake is unknown. Cla-1, the human SR-BI orthologue, was determined to be a LPS-binding protein and endocytic receptor mediating the binding and internalization of lipoprotein-free, monomerized LPS. LPS strongly competed with HDL, lipidfree apoA-I and apoA-II for HDL binding to the mouse RAW cells. Stably transfected HeLa cells expressing Cla-1-bound LPS with a Kd of about 16 microg/ml, and had a 3-4-fold increase in binding capacity and LPS uptake. Bodipy-labeled LPS uptake was found to initially accumulate in the plasma membrane and subsequently in a perinuclear region identified predominantly as the Golgi complex. Bodipy-LPS and Alexa-apoA-I had staining that colocalized on the cell surface and intracellularly indicating similar transport mechanisms. When associated with HDL, LPS uptake was increased in Cla-1 overexpressing HeLa cells by 5-10-fold. Cla-1-associated 3H-LPS uptake exceeded 125I-apolipoprotein uptake by 5-fold indicating a selective LPS uptake. Upon interacting with Cla-1 overexpressing HeLa cells, the complex (Bodipy-LPS/Alexa 488 apolipoprotein-labeled HDL) bound and was internalized as a holoparticle. Intracellularly, LPS and apolipoproteins were sorted to different intracellular compartments. With LPS-associated HDL, intracellular LPS co-localized predominantly with transferrin, indicating delivery to an endocytic recycling compartment. Our study reveals a close similarity between Cla-1-mediated selective LPS uptake and the recently described selective lipid sorting by rodent SR-BI. In summary, Cla-1 was found to bind and internalize monomerized and HDL-associated LPS, indicating that Cla-1 may play important role in septic shock by affecting LPS cellular uptake and clearance.     

Modulation of neuronal evoked responses to the medial septal area of hibernating ground squirrels by some neuropeptides after chronic disconnection between septum and preoptico-hypothalamic structures

Effects of some neuropeptides identified in the brain of hibernators (TSKYR, TSKY, DY) and of monoaminergic neurotransmitters (noradrenaline and serotonin) on responses of the medial septal neurons evoked by intraseptal electrical stimulation were analyzed in slices taken from the ground squirrels with chronic basal undercutting of the septum. Despite the elimination of direct contacts with the preoptic area and afferents ascending in the medial forebrain bundle, the neurons retained almost normal level of reactivity and distribution of the reaction types. The neuropeptides effectively modulated neuronal responses of various types, including oligosynaptic short-latency single-spike responses. The latter were strongly facilitated by the neuropeptides. As a rule, changes in the responses to electrical stimulation were independent of the spontaneous activity shifts (in 78% of the tests). It was suggested that the neuropeptides exert a double influence on the septal neurons: direct nonsynaptic effects on the pacemaker potential responsible for the background activity and modulation of synaptic processes. Our experiments showed that descending influences of the septo-hippocampal system are not crucial for the entrance into the hibernation state and its tonic maintenance. The influences of the thermoregulatory--circadian structures of the preoptico-hypothalamic area determine the paradoxically increased latent excitability of septal neurons that allows the septo-hippocampal system to gate external stimuli and organize arousal of the forebrain during hibernation in case of emergency.     

Crystals of tryptophan indole-lyase and tyrosine phenol-lyase form stable quinonoid complexes

The binding of substrates and inhibitors to wild-type Proteus vulgaris tryptophan indole-lyase and to wild type and Y71F Citrobacter freundii tyrosine phenol-lyase was investigated in the crystalline state by polarized absorption microspectrophotometry. Oxindolyl-lalanine binds to tryptophan indole-lyase crystals to accumulate predominantly a stable quinonoid intermediate absorbing at 502 nm with a dissociation constant of 35 microm, approximately 10-fold higher than that in solution. l-Trp or l-Ser react with tryptophan indole-lyase crystals to give, as in solution, a mixture of external aldimine and quinonoid intermediates and gem-diamine and external aldimine intermediates, respectively. Different from previous solution studies (Phillips, R. S., Sundararju, B., & Faleev, N. G. (2000) J. Am. Chem. Soc. 122, 1008-1114), the reaction of benzimidazole and l-Trp or l-Ser with tryptophan indole-lyase crystals does not result in the formation of an alpha-aminoacrylate intermediate, suggesting that the crystal lattice might prevent a ligand-induced conformational change associated with this catalytic step. Wild-type tyrosine phenol-lyase crystals bind l-Met and l-Phe to form mixtures of external aldimine and quinonoid intermediates as in solution. A stable quinonoid intermediate with lambda(max) at 502 nm is accumulated in the reaction of crystals of Y71F tyrosine phenol-lyase, an inactive mutant, with 3-F-l-Tyr with a dissociation constant of 1 mm, approximately 10-fold higher than that in solution. The stability exhibited by the quinonoid intermediates formed both by wild-type tryptophan indole-lyase and by wild type and Y71F tyrosine phenol-lyase crystals demonstrates that they are suitable for structural determination by x-ray crystallography, thus allowing the elucidation of a key species of pyridoxal 5'-phosphate-dependent enzyme catalysis.