The betagamma subunit of heterotrimeric G proteins interacts with RACK1 and two other WD repeat proteins

A yeast two-hybrid approach was used to discern possible new effectors for the betagamma subunit of heterotrimeric G proteins. Three of the clones isolated are structurally similar to Gbeta, each exhibiting the WD40 repeat motif. Two of these proteins, the receptor for activated C kinase 1 (RACK1) and the dynein intermediate chain, co-immunoprecipitate with Gbetagamma using an anti-Gbeta antibody. The third protein, AAH20044, has no known function; however, sequence analysis indicates that it is a WD40 repeat protein. Further investigation with RACK1 shows that it not only interacts with Gbeta(1)gamma(1) but also unexpectedly with the transducin heterotrimer Galpha(t)beta(1)gamma(1). Galpha(t) alone does not interact, but it must contribute to the interaction because the apparent EC(50) value of RACK1 for Galpha(t)beta(1)gamma(1) is 3-fold greater than that for Gbeta(1)gamma(1) (0.1 versus 0.3 microm). RACK1 is a scaffold that interacts with several proteins, among which are activated betaIIPKC and dynamin-1 (1). betaIIPKC and dynamin-1 compete with Gbeta(1)gamma(1) and Galpha(t)beta(1)gamma(1) for interaction with RACK1. These findings have several implications: 1) that WD40 repeat proteins may interact with each other; 2) that Gbetagamma interacts differently with RACK1 than with its other known effectors; and/or 3) that the G protein-RACK1 complex may constitute a signaling scaffold important for intracellular responses.     

Mapping QTLs for field resistance to the rice blast pathogen and evaluating their individual and combined utility in improved varieties

Lines from a Lemont x Teqing recombinant inbred population were evaluated for dilatory resistance to rice blast disease using: (1) the Standard Evaluation System (SES) for rating leaf blast, (2) the percentage diseased leaf area (%DLA), and (3) the area under a disease progress curve (AUDPC). RFLP mapping using 175 well-distributed loci revealed nine QTLs, one each on chromosomes 1, 2, 3, 4, 6, 7 and 9, with two loci on chromosome 12. All nine putative QTLs were associated with AUDPC, six with both a %DLA and a SES rating. Teqing contributed the resistance allele for all these loci except for the one located on chromosome 4. Individual QTLs accounted for 5-32% of the observed phenotypic variation, and combined QTL models accounted for 43-53%. Three QTLs were located near three of the four major resistance genes previously identified in this population. The resistances of both Lemont and Teqing were attributable to a combination of both major genes capable of inducing hypersensitive reactions and minor genes causing less-distinctive phenotypic differences. Interactions were noted between QTLs and major genes. Our findings are in support of the strategy of pyramiding major genes and QTLs in carefully selected combinations to develop improved varieties with resistance to the blast fungus that is both broad in spectrum and durable.     

Complete structure of an increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom. ICPP is angiogenic via vascular endothelial growth factor receptor signalling

The partial sequence of the increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom revealed a strong homology to vascular endothelial growth factor (VEGF)-A. We now report its complete amino acid sequence determined by Edman degradation and its biological effects on mouse and human vascular endothelial cells. ICPP is a homodimeric protein linked by cysteine disulfide bonds of 25115 Da revealed by mass spectrometry. Each monomer is composed of 110 amino acids including eight cysteine residues and a pyroglutamic acid at the N-terminal extremity. ICPP shares 52% sequence identity with human VEGF but lacks the heparin binding domain and Asn glycosylation site. Besides its strong capillary permeability activity, ICPP was found to be a potent in vitro angiogenic factor when added to mouse embryonic stem cells or human umbilical vein endothelial cells. ICPP was found to be as potent as human VEGF165 in activating p42/p44 MAPK, in reinitiation of DNA synthesis in human umbilical vein endothelial cells, and in promoting in vitro angiogenesis of mouse embryonic stem cells. All these biological actions, including capillary permeability in mice, were fully inhibited by 1 microm of a new specific VEGF receptor tyrosine kinase inhibitor (ZM317450) from AstraZeneca that belongs to the anilinocinnoline family of compounds. Indeed, up to a 30 times higher concentration of inhibitor did not affect platelet-derived growth factor, epidermal growth factor, FGF-2, insulin, alpha-thrombin, or fetal calf serum-induced p42/p44 MAPK and reinitiation of DNA synthesis. Therefore, we conclude that this venom-derived ICPP exerts its biological action (permeability and angiogenesis) through activation of VEGF receptor signaling (VEGF-R2 and possibly VEGF-R1).     

Synthesis and structure-activity relationships of M(2)-selective muscarinic receptor ligands in the 1-[4-(4-arylsulfonyl)-phenylmethyl]-4-(4-piperidinyl)-piperazine family

The synthesis and muscarinic binding properties of compounds based on the 1-[4-(4-arylsulfonyl)phenylmethyl]-4-(1-aroyl-4-piperidinyl)-piperazine skeleton are described. For compounds, substituted with appropriately configured methyl groups at the benzylic center and at the piperazine 2-position, high levels of selective, M(2) subtype affinity could be obtained, particularly when the terminal N-aroyl residue was ortho-substituted.     

The relationship between innervation density and dynamic balance function

Functional reach (FR) significantly increases with additional sensory information from the fifth metacarpal surface of the dominant hand. The purpose of this study was to determine the relationship between the innervation density of the skin above the fifth metacarpal surface of the palm and the increase of FR with an additional light touch. The results show a moderate negative correlation between innervation density and the increase of FR with light touch.     

Effects of combined selenium and vitamin E administration on DNA in Walker tumor bearing Wistar rat exposed to cytostatic acute treatment

Previous studies have showed that organic Se and Vitamin E have a significant protective effect when administered in combination with cytostatics. This paper reports the investigation on effects of mixed administration Orgasel 50 and Vitamin E in Wistar rat with experimentally induced Walker tumor under acute cytostatic treatment, with emphasis on two aspects: a) the influence of antioxidants upon liver unscheduled DNA biosynthesis under cytostatic (Lomustin) acute aggression; and b) the potential improvement of cytostatic effects by antioxidants treatment in tumor. Two lots of animals were used: lot 1 - Orgasel 50 and Vitamin E administered 7 days before the initiation of tumor induction and lot 2 - the antioxidants were concomitantly administered with tumor cell inoculation. The Walker tumor (an epithelial carcinoma) cells were subcutaneously injected (5 x 10(6) cells/0.5 ml in isotonic saline solution); the first tumor nodules appeared in 4 days; the tumor has reached the appropriate dimensions in 12 days. The unscheduled DNA biosynthesis caused by Lomustin in rat liver as well as the replicative DNA biosynthesis taking place in Walker tumor cells were assessed radioisotopically by measuring the uptake of 3H-Thymidine (200 microCi / 100 g.b.w.). Our observations regarding the role of antioxidant treatment suggest: 1) a benefic effect on DNA alkylant-induced lesions, expressed by a decrease in the level of 3H-Thymidine uptake in liver and, 2) an increase of the inhibitory activity of cytostatic on DNA replication biosynthesis in tumor cells, suggested by lower 3H-Thymidine incorporation in tumor cells. The most significant results were showed in both analyzed tissues, when the Orgasel 50 + Vitamin E administration begins at the same time with the tumor cell inoculation. These findings clearly show the organic Se salts and Vitamin E constitute a valuable adjuvant in anticancer medication, increasing the interest for the application of these antioxidants in cancer therapy and prevention.     

Human melanoma TrkC: its association with a purine-analog-sensitive kinase activity

The various members of the Trk tyrosine kinase family and p75 neurotrophin receptor (p75(NTR)) have been identified as signaling receptors for the structurally related members of the neurotrophins (NT) family. We have previously reported that NT treatment of murine and human brain-metastatic melanoma cells affects their invasive capacities and increases the production of extracellular-matrix degradative enzymes. These cells express aberrant levels of functional p75(NTR) and TrkC, the putative high-affinity receptor for the neurotrophin NT-3. Here we demonstrate that, by using sensitive immune-complex kinase assays in human brain-metastatic (70W) melanoma cells, TrkC receptors associate with a kinase activity exhibiting a dose-dependent susceptibility to inhibition by the purine-analogs 6-thioguanine and 2-aminopurine. The activity of this purine-analog-sensitive kinase (PASK) was induced by NT-3 in a time-dependent fashion, phosphorylating exogenous myelin basic protein (MBP) but not denatured enolase. It is similar to the one reported to relate with p75(NTR) and TrkA receptors and stimulated by the prototypic NT, nerve growth factor. Thus, PASKs may represent unique signaling components common to NT receptors that could engage joint downstream signaling effectors in brain-metastatic melanoma.     

Regulation of human Cdc25A stability by Serine 75 phosphorylation is not sufficient to activate a S phase checkpoint

Degradation of Cdc25A phosphatase is an ubiquitous feature of stress. There are some discrepancies in the reported roles for different phosphorylation sites in the regulation of Cdc25A stability. Using a panel of doxycycline-inducible phosphorylation mutants we show that the stability of human Cdc25A protein is dependent upon phosphorylation at S75. In non-stressed conditions and in non-mitotic cells, Cdc25A is unstable and its stability is regulated in a Chk1-dependent manner. During mitosis, Cdc25A becomes stable and does not undergo degradation after DNA damage. We further show that Chk1 kinase regulates Cdc25A stability after UV irradiation. Similar to Chk1 kinase, p38 MAPK controls Cdc25A protein level after osmotic stress. Using phospho-specific antibodies, we find that both kinases can phosphorylate S75 and S123 in vitro. Inactivation of either Chk1 after UV-irradiation or p38 MAPK after osmotic stress prevents activation of a S phase checkpoint and S75 and S123 phosphorylation. However, introduction of stable Cdc25A (S75A or S75/123A) proteins is not sufficient to overcome this checkpoint. We propose that regulation of human Cdc25A stability by its phosphorylation at S75 may contribute to S phase checkpoint activation only in cooperation with other regulatory mechanisms.     

Cellular compartmentation of lysozyme and alpha-amylase in the mouse salivary glands. Immunogold approaches at light and electron microscopy level

The research was planned to study the subcellular distribution of enzymatic secretory products within the secretory structures of the mouse major salivary glands at light and electron microscopy level by immunogold silver stain (IGSS) technique and double-sided post-embedding immunogold binding and silver amplification in order to speculate about their compartmentation. In particular, we experimented the above immunogold labeling approaches to localize the lysozyme and to verify its distribution patterns in relation to another secretion enzyme, alpha-amylase. Co-presence of lysozyme and alpha-amylase was observed in the convoluted granular tubule cells of the submandibular gland and in the demilunar cells of the sublingual gland as well as in the electron-dense regions of the mottled secretory granules in the parotid gland. Exclusive binding patterns of lysozyme were observed in the acinar cells of the submandibular and sublingual glands where alpha-amylase did not occur.     

Egg rejection in a passerine bird: size does matter

Avian brood parasites reduce the reproductive success of their hosts, selecting for the evolution of egg discrimination by the host, and potentially creating a coevolutionary arms race between host and parasite. Host egg discrimination ability is crucial in determining whether the arms race results in extinction (of the parasite on a particular host) or stable coevolutionary equilibrium of the host-parasite pair. I examined egg discrimination behaviour in the yellow-browed leaf warbler, Phylloscopus humei, a presumed former host of parasitic cuckoos, to show how discrimination ability has become very strong. Field experiments using model eggs demonstrate that rejection decisions are based on the relative size of eggs in the clutch. Individuals do not learn the particular size of their own eggs, but will accept both large and small eggs as long as all eggs in the clutch are of similar size. Host rejection decisions are continuously modified based on assessment of variation in egg sizes currently in the clutch, making it a difficult strategy for a cuckoo to defeat. Copyright 2000 The Association for the Study of Animal Behaviour.