Causal relationship between the loss of RUNX3 expression and gastric cancer

Runx3/Pebp2alphaC null mouse gastric mucosa exhibits hyperplasias due to stimulated proliferation and suppressed apoptosis in epithelial cells, and the cells are resistant to growth-inhibitory and apoptosis-inducing action of TGF-beta, indicating that Runx3 is a major growth regulator of gastric epithelial cells. Between 45% and 60% of human gastric cancer cells do not significantly express RUNX3 due to hemizygous deletion and hypermethylation of the RUNX3 promoter region. Tumorigenicity of human gastric cancer cell lines in nude mice was inversely related to their level of RUNX3 expression, and a mutation (R122C) occurring within the conserved Runt domain abolished the tumor-suppressive effect of RUNX3, suggesting that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer.     

Norepinephrine triggers Ca2+-dependent exocytosis of 5-hydroxytryptamine from rat pinealocytes in culture

5-hydroxytryptamine (5-HT) is a precursor and a putative modulator for melatonin synthesis in mammalian pinealocytes. 5-HT is present in organelles distinct from l-glutamate-containing synaptic-like microvesicles as well as in the cytoplasm of pinealocytes, and is secreted upon stimulation by norepinephrine (NE) to enhance serotonin N-acetyltransferase activity via the 5-HT2 receptor. However, the mechanism underlying the secretion of 5-HT from pinealocytes is unknown. In this study, we show that NE-evoked release of 5-HT is largely dependent on Ca2+ in rat pinealocytes in culture. Omission of Ca2+ from the medium and incubation of pineal cells with EGTA-tetraacetoxymethyl-ester inhibited by 59 and 97% the NE-evoked 5-HT release, respectively. Phenylephrine also triggered the Ca2+-dependent release of 5-HT, which was blocked by phentolamine, an alpha antagonist, but not by propranolol, a beta antagonist. Botulinum neurotoxin type E cleaved 25 kDa synaptosomal-associated protein and inhibited by 50% of the NE-evoked 5-HT release. Bafilomycin A1, an inhibitor of vacuolar H+-ATPase, and reserpine and tetrabenazine, inhibitors of vesicular monoamine transporter, all decreased the storage of vesicular 5-HT followed by inhibition of the NE-evoked 5-HT release. Agents that trigger L-glutamte exocytosis such as acetylcholine did not trigger any Ca2+-dependent 5-HT release. Vice versa neither NE nor phenylephrine caused synaptic-like microvesicle-mediated l-glutamate exocytosis. These results indicated that upon stimulation of a adrenoceptors pinealocytes secrete 5-HT through a Ca2+-dependent exocytotic mechanism, which is distinct from the exocytosis of synaptic-like microvesicles.     

Fungal melanin inhibitor and related compounds from Penicillium decumbens

In Silene vulgaris (M.) G. cell culture three growth phases were distinguished, namely, a lag phase, an exponential phase and a stationary phase. Pectin termed silenan and an acidic arabinogalactan were isolated as cell wall polysaccharides of S. vulgaris callus at the different growth phases during culture. Production of silenan as the galacturonan (or rhamnogalacturonan) core was observed at the beginning of the exponential phase and at the stationary phase of the callus growth. Arabinogalactan, containing the galacturonic acid residues, is formed at the exponential phase followed by attachment to the core of silenan in the middle of the exponential phase. The arabinogalactan constituent of silenan appeared to be destroyed gradually at the stationary growth phase. The monosaccharide compositions of silenan and arabinogalactan were determined at various phases of the callus growth. Silenan was found to be formed in maximum amounts at the exponential phase of the cell growth. Insignificant alterations of the yields of acidic arabinogalactan were found during culture while total productivity per litre of medium and rate of production per day of arabinogalactan were found to be maximal at the exponential phase of growth.     

Effects of chemically modified heparin on Chlamydia trachomatis serovar L2 infection of eukaryotic cells in culture

The mechanism and inhibitors of Chlamydia trachomatis serovar L2 infection of eukaryotic host cells were studied using a tissue culture model infection system. Potent inhibition of infectivity was observed when elementary bodies (EBs) were exposed to heparin or when HeLa 229 cells were treated with heparinase. No significant inhibition was seen the other way around. The same potent inhibition was observed when EBs were exposed to chemically 2-O-desulfated heparin (2-ODS heparin), which is composed of repeating disaccharide units of IdoA-GlcNS(6S), but not when exposed to chemically 6-ODS heparin or completely desulfated and N-resulfated heparin, which is composed of repeating disaccharide units of IdoA(2S)-GlcNS or IdoA-GlcNS, respectively. The inhibitory effects of 2-ODS heparin could be seen only with oligosaccharides longer than dodecasaccharides. The mutant Chinese hamster ovary (CHO) cell line 677, which is deficient in the biosynthesis of heparan sulfate, was less sensitive to C. trachomatis infection than were wild-type CHO cells. F-17 cells, deficient in 2-O-sulfation of heparan sulfate, had the same sensitivity to infection as wild-type CHO cells did. These data suggest that infection of host cells by EBS results from the specific binding of ligand molecules with affinity for heparin on the EB surface to heparan sulfate proteoglycans on the host cell surface. This binding may depend on host cell heparan sulfate chains that are 6-O-sulfated and longer than dodecasaccharides. The 2-ODS heparin oligosaccharides may be a potential agent for the prevention of C. trachomatis infection.     

Regulation of Lck and Fyn tyrosine kinase activities by transmembrane protein tyrosine phosphatase leukocyte common antigen-related molecule

Leukocyte common antigen-related molecule (LAR) is a receptor-like protein tyrosine phosphatase (PTPase) with two PTPase domains. In the present study, we detected the expression of LAR in the brain, kidney, and thymus of mice using anti-LAR PTPase domain subunit monoclonal antibody (mAb) YU1. In the thymus, LAR was expressed on CD4(-)CD8(-) and CD4(-)CD8(low) thymocytes. The development of thymocytes in CD45 knockout mice is blocked partially in the maturation of CD4(-)CD8(-) to CD4(+)CD8(+). We postulated that LAR regulates Lck and Fyn in the immature thymocytes. Transfection of wild-type LAR activated extracellular signal-regulated kinase signal transduction pathway in CD45-deficient Jurkat cells stimulated with anti-CD3 mAb. LAR mutants, with Cys to Ser mutation in the catalytic center of PTPase D1, bound to tyrosine-phosphorylated Lck and Fyn, and LAR PTPase domain 2 was tyrosine phosphorylated by Fyn tyrosine kinase. The phosphorylated LAR was associated with Fyn Src homology 2 domain. Moreover, LAR dephosphorylated phosphorylated tyrosine residues in both the COOH terminus and kinase domain of Fyn in vitro. Our results indicate that Lck and Fyn would be substrates of LAR in immature thymocytes and that each LAR PTPase domain plays distinct functional roles in phosphorylation and dephosphorylation.     

Pallidin is a component of a multi-protein complex involved in the biogenesis of lysosome-related organelles

The Hermansky–Pudlak syndrome defines a group of genetic disorders characterized by defective lysosome-related organelles such as melanosomes and platelet dense bodies. Hermansky–Pudlak syndrome can be caused by mutations of at least four genes in humans and 15 genes in mice. One of these genes is mutated in the pallid mouse strain and encodes a novel protein named pallidin (L. Huang, Y. M. Kuo and J. Gitschier, Nat Genet 1999; 23: 329–332). Pallidin has no homology to any other known protein and no recognizable functional motifs. We have conducted a biochemical characterization of human pallidin using a newly developed polyclonal antibody. We show that pallidin is a ubiquitously expressed ∼ 25 kDa protein found both in the cytosol and peripherally associated to membranes. Sedimentation velocity analyses show that native pallidin has a sedimentation coefficient of ∼ 5.1 S, much larger than expected from the molecular mass of the pallidin polypeptide. In line with this observation, cosedimentation and coprecipitation analyses reveal that pallidin is part of a hetero-oligomeric complex. One of the subunits of this complex is the product of another Hermansky–Pudlak syndrome gene, muted. Fibroblasts derived from the muted mouse strain exhibit reduced levels of pallidin, suggesting that the absence of the muted protein destabilizes pallidin. These observations indicate that pallidin is a subunit of a novel multi-protein complex involved in the biogenesis of lysosome-related organelles.     

A missense mutant myostatin causes hyperplasia without hypertrophy in the mouse muscle

Myostatin, which is a member of the TGF-beta superfamily, is a negative regulator of skeletal muscle formation. Double-muscled Piedmontese cattle have a C313Y mutation in myostatin and show increased skeletal muscle mass which resulted from an increase of myofiber number (hyperplasia) without that of myofiber size (hypertrophy). To examine whether this mutation in myostatin gene affects muscle development in a dominant negative manner, we generated transgenic mice overexpressing the mutated gene. The transgenic mice exhibited dramatic increases in the skeletal muscle mass resulting from hyperplasia without hypertrophy. In contrast, it has been reported that a myostatin mutated at its cleavage site produces hypertrophy without hyperplasia in the muscle. Thus, these results suggest that (1) the myostatin containing the missense mutation exhibits a dominant negative activity and that (2) there are two types in the dominant negative form of myostatin, causing either hypertrophy or hyperplasia.     

A plant growth retardant related to chlamydocin and its proposed mechanism of action

A comparison of the plant growth retardant activity of the chlamydocin analogues, compound 1, six derivatives from 1 and 2, and two synthetic analogues revealed that there are two types of retardant in chlamydocin analogues. One, for example in compound 1, requires an oxygen atom at C-8 of the 2-aminodecanoic acid moiety to show retardant activity. The other, for example in compound 8, requires no oxygen atom at C-8 but requires a specific alkyl group chain length for activity. To determine the differences in mode of action of both types of retardant, rice seedlings were separately treated with compounds 1 and 8, and after appearance of dwarfism, their endogenous ABA and GA(1) levels were determined and compared to those of the control. Treatment with 1 (10 nmol/plant) increased ABA levels 4 times higher than that of the control and decreased GA(1) levels to 20% of that of the control. Treatment with 8 (30 nmol/plant) did not affect the ABA level but decreased GA(1) content to 5% of that of the control.