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.     

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.     

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.     

Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors

In the presence of complementing adeno-associated virus type 2 (AAV-2) Rep proteins, AAV-2 genomes can be pseudotyped with the AAV-5 capsid to assemble infectious virions. Using this pseudotyping strategy, the involvement of the ubiquitin-proteasome system in AAV-5 and AAV-2 capsid-mediated infections was compared. A recombinant AAV-2 (rAAV-2) proviral luciferase construct was packaged into both AAV-2 and AAV-5 capsid particles, and transduction efficiencies in a number of cell lines were compared. Using luciferase expression as the end point, we demonstrated that coadministration of the viruses with proteasome inhibitors not only increased the transduction efficiency of rAAV-2, as previously reported, but also augmented rAAV-5-mediated gene transfer. Increased transgene expression was independent of viral genome stability, since there was no significant difference in the amounts of internalized viral DNA in the presence or absence of proteasome inhibitors. Western blot assays of immunoprecipitated viral capsid proteins from infected HeLa cell lysates and in vitro reconstitution experiments revealed evidence for ubiquitin conjugation of both AAV-2 and AAV-5 capsids. Interestingly, heat-denatured virus particles were preferential substrates for in vitro ubiquitination, suggesting that endosomal processing of the viral capsid proteins is a prelude to ubiquitination. Furthermore, ubiquitination may be a signal for processing of the capsid at the time of virion disassembly. These studies suggest that the previously reported influences of the ubiquitin-proteasome system on rAAV-2 transduction are also active for rAAV-5 and provide a clearer mechanistic framework for understanding the functional significance of ubiquitination.     

Long-term circulation of vaccine-derived poliovirus that causes paralytic disease

Successful implementation of the global poliomyelitis eradication program raises the problem of vaccination against poliomyelitis in the posteradication era. One of the options under consideration envisions completely stopping worldwide the use of the Sabin vaccine. This strategy is based on the assumption that the natural circulation of attenuated strains and their derivatives is strictly limited. Here, we report the characterization of a highly evolved derivative of the Sabin vaccine strain isolated in a case of paralytic poliomyelitis from a 7-month-old immunocompetent baby in an apparently adequately immunized population. Analysis of the genome of this isolate showed that it is a double (type 1-type 2-type 1) vaccine-derived recombinant. The number of mutations accumulated in both the type 1-derived and type 2-derived portions of the recombinant genome suggests that both had diverged from their vaccine predecessors approximately 2 years before the onset of the illness. This fact, along with other recent observations, points to the possibility of long-term circulation of Sabin vaccine strain derivatives associated with an increase in their neurovirulence. Comparison of genomic sequences of this and other evolved vaccine-derived isolates reveals some general features of natural poliovirus evolution. They include a very high preponderance and nonrandom distribution of synonymous substitutions, conservation of secondary structures of important cis-acting elements of the genome, and an apparently adaptive character of most of the amino acid mutations, with only a few of them occurring in the antigenic determinants. Another interesting feature is a frequent occurrence of tripartite intertypic recombinants with either type 1 or type 3 homotypic genomic ends.     

Electron microscopy and ultracytochemistry of blood lymphocytes containing Gall bodies in healthy individuals

The authors have conducted electron microscopic and ultracytochemical studies of blood lymphocytes of healthy individuals containing Gall bodies (GB) which are typical for CD4+ cell subpopulation. It has been established that GB have quite a complex submicroscopic structure and together with its derivates, granules-satellites, as well as mitochondria, microfilaments, rough endoplasmic reticulum canaliculi, and Golgi complex it forms a unique active functional complex. GB show a high activity of acid phosphatase and positive reaction to glycogen. The data obtained suggest the leading role of GB in the production of cytokins and other biologically active substances.