Distribution and Properties of Chromatin-Associated Nucleoside Triphosphate Diphosphatase Purified by a New Method

Chromatin-associated nucleoside triphosphate diphosphatase (NTDPase)activity was detected in Alaska pea plant only at the germinationstage in a study of the plant's development over 5 month fromthe dormant to fruiting stages. This enzyme activity was alsodetected in seedlings of several dicotyledonous plants includingsoybean, Usui pea, cowpea and cucumber, but almost none wasfound in monocotyledonous corn and rice plants. When the chromatin of pea epicotyl was fractionated into template-activeand -inactive forms by DNase II digestion, most of the NTDPaseactivity was found in the active chromatin. A new method to rapidly isolate and purify NTDPase from peaepicotyl chromatin was developed in which NTDPase was elutedwith a linear gradient of NaCl on a column packed with cellulosepowder and chromatin. DNA remained on the column and the elutedNTDPase was purified further by chromatography using trimethylamino-2-hydroxypropylcellulose (TMAP-cellulose) and a Sephadex G-100 column, whichgive chromatin 18-fold purer than the starting sample. This purified NTDPase was adsorbed by DNA-cellulose, which isfurther evidence that NTDPase is a kind of non-histone proteinassociated with DNA. Several cations including Ca^2$, Mg^2$, Mn^2$and Zn^2$ stimulated the enzyme activity, with the maximal eightfoldactivation by Ca^2$. NTDPase activity was clearly inhibited byKCN and pyrophosphate, but not by SH-blocking inhibitors andvarious metal chelators at 1 mM.     

Rambutan genome revealed gene networks for spine formation and aril development

Rambutan is a popular tropical fruit known for its exotic appearance, has long flexible spines on shells, extraordinary aril growth, desirable nutrition, and a favorable taste. The genome of an elite rambutan cultivar Baoyan 7 was assembled into 328 Mb in 16 pseudo-chromosomes. Comparative genomics analysis between rambutan and lychee revealed that rambutan chromosomes 8 and 12 are collinear with lychee chromosome 1, which resulted in a chromosome fission event in rambutan (n = 16) or a fusion event in lychee (n = 15) after their divergence from a common ancestor 15.7 million years ago. Root development genes played a crucial role in spine development, such as endoplasmic reticulum pathway genes, jasmonic acid response genes, vascular bundle development genes, and K⁺ transport genes. Aril development was regulated by D-class genes (STK and SHP1), plant hormone and phenylpropanoid biosynthesis genes, and sugar metabolism genes. The lower rate of male sterility of hermaphroditic flowers appears to be regulated by MYB24. Population genomic analyses revealed genes in selective sweeps during domestication that are related to fruit morphology and environment stress response. These findings enhance our understanding of spine and aril development and provide genomic resources for rambutan improvement.     

Intermediate filaments with novel protein composition from certain goldfish cells

Using the conditions for vimentin filament recycling, intermediate filaments (approximately 10 nm) were prepared from the cytoskeleton of a goldfish tumor cell line (erythrophoroma or xanthophoroma). 2-D analysis showed unusual protein composition, with four proteins of molecular weights of 60, 45, 56 and 51 kilodaltons in ratios of approximately 4:4:1:1. These correspond to four of the major cytoskeletal proteins of both the tumor cells and normal xanthophores.     

Regulation of protein degradation in muscle by calcium. Evidence for enhanced nonlysosomal proteolysis associated with elevated cytosolic calcium

Calcium-dependent regulation of intracellular protein degradation was studied in isolated rat skeletal muscles incubated in vitro in the presence of a large variety of agents known to affect calcium movement and distribution. A23187, KC1, sucrose, and 8-(diethylamino)octyl-3,4, 5-trimethoxybenzoate hydrochloride increase proteolysis while tetracaine, verapamil, and low extracellular calcium caused significant decreases. Additionally, dantrolene decreases proteolysis in the presence of depolarizing levels of potassium, while it has no effect on degradation in normal media. The dose dependence of calcium ionophore A23187 on proteolysis and contracture tension are parallel. Furthermore, excess KC1 and hypertonic solutions increased protein degradation at doses reported to cause tension. Thus, the parallel increase in proteolysis and tension in response to various agents supports the hypothesis that protein degradation in muscle is regulated by calcium. To determine the responsible proteolytic systems involved in calcium-dependent degradation, the effect of different classes of protease inhibitors was tested. Addition of the inhibitors leupeptin and E-64-c blocked the A23187-induced increase in degradation. Since proteases sensitive to these agents are present in both the sarcoplasm and lysosomes, known lysosomotropic agents, methylamine and chloroquine, as well as 3-methyladenine, a specific autophagy inhibitor, were used in combination with A23187. These agents did not inhibit calcium ionophore-induced proteolysis, although these three agents selectively inhibited enhanced degradation seen in the absence of insulin, demonstrating an autophagic/lysosomal pathway in these muscles. Thus, our results suggest that nonlysosomal leupeptin- and E-64-c-sensitive proteases are responsible for calcium-dependent proteolysis in muscle.     

Syngeneic monoclonal antibody against melanoma antigen with interspecies cross-reactivity recognizes GM3, a prominent ganglioside of B16 melanoma

It has previously been reported that a mouse (C57BL/6) monoclonal antibody, M2590, was established against syngeneic melanoma B16 cells, which was shown to react only with melanoma cells from various species but not with other tumor cells or normal tissues (Taniguchi, M., and Wakabayashi, S. (1984) Gann 75, 418-426). In the present study, the specificity of M2590 antibody was shown to be directed to a saccharide arrangement (NeuAc alpha 2-3Gal beta 1-4Glc (or -GlcNAc)) of gangliosides by three different assay systems including enzyme immunostaining on thin layer plates, sandwich radioimmunoassay, and enzyme-linked immunoadsorbent assays using a variety of glycolipids with known structures. Neither gangliosides having NeuGc terminus, including NeuGc alpha 2-3Gal beta 1-4Glc-ceramide and NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-ceramide, nor ganglio series gangliosides carrying NeuAc reacted with the antibody. An M2590 antibody-reactive antigen was isolated from B16 melanoma cells, and its structure was determined to be NeuAc alpha 2-3Gal beta 1-4Glc-ceramide by fast atom bombardment mass spectrometry, methylation analysis, and exoglycosidase treatment. The ceramide was composed of d18:1 as its long-chain base and C16:0, C24:1, and C24:0 as major fatty acids. The same ganglioside was also detected in the culture supernatant of the melanoma cells as shedding antigen.     

Modulation of growth and differentiation in normal human keratinocytes by transforming growth factor-beta

The effect of transforming growth factor-type beta 1(TGF-beta) on the growth and differentiation of normal human skin keratinocytes cultured in serum-free medium was investigated. TGF-beta markedly inhibited the growth of keratinocytes at the concentrations greater than 2 ng/ml under low Ca2+ conditions (0.1 mM). Growth inhibition was accompanied by changes in cell functions related to proliferation. Remarkable inhibition of DNA synthesis was demonstrated by the decrease of [3H]thymidine incorporation. The decrease of [3H]thymidine incorporation was observed as early as 3 hr after addition of TGF-beta. TGF-beta also decreased c-myc messenger RNA (mRNA) expression 30 min after addition of TGF-beta. This rapid reduction of c-myc mRNA expression by TGF-beta treatment is possibly one of the main factors in the process of TGF-beta-induced growth inhibition of human keratinocytes. Since growth inhibition and induction of differentiation are closely related in human keratinocytes, the growth-inhibitory effect of TGF-beta under high Ca2+ conditions (1.8 mM Ca2+, differentiation-promoting culture environment) was examined. TGF-beta inhibited the growth of keratinocytes under high Ca2+ conditions in the same manner as under low Ca2+ conditions, suggesting that it is a strong growth inhibitor in both low and high Ca2+ environments. The induction of keratinocyte differentiation was evaluated by measuring involucrin expression and cornified envelope formation: TGF-beta at 20 ng/ml increased involucrin expression from 9.3% to 18.8% under high Ca2+ conditions, while it decreased involucrin expression from 7.0% to 3.3% under low Ca2+ conditions. Cornified envelope formation was modulated in a similar way by addition of TGF-beta: TGF-beta at 20 ng/ml decreased cornified envelope formation by 53% under low Ca2+ conditions, while it enhanced cornified envelope formation by 30.7% under high Ca2+ conditions. Thus, the effect of TGF-beta on keratinocyte differentiation is Ca2+ dependent. It enhances differentiation of human keratinocytes under high Ca2+ conditions, but inhibits differentiation under low Ca2+ conditions. Taken together, there is a clear discrepancy between TGF-beta effects on growth inhibition and induction of differentiation in human keratinocytes. These data indicate that growth inhibition of human keratinocytes by TGF-beta is direct and not induced by differentiation.     

Human monocyte colony-stimulating factor enhances the clearance of lipoproteins containing apolipoprotein B-100 via both low density lipoprotein receptor-dependent and -independent pathways in rabbits

To investigate the effects of recombinant human monocyte colony-stimulating factor (M-CSF) on plasma cholesterol metabolism, we injected M-CSF intravenously into New Zealand White rabbits (n = 13) at a dose of 100 micrograms/day for 7 days. After the treatment, the plasma cholesterol levels fell by 33.2% from 61.4 +/- 25.9 to 41.0 +/- 10.2 mg/dl (mean +/- S.D.). We also injected a large dose of M-CSF (500 micrograms/day) for 6 days into Watanabe Heritable Hyperlipidemic rabbits, which are deficient in low density lipoprotein (LDL) receptors. Again, there was a significant reduction in plasma cholesterol levels by 36.2% from 730.5 +/- 176.4 to 466.0 +/- 104.9 mg/dl (n = 4). In the kinetic studies in New Zealand White rabbits with very low density lipoprotein, LDL, and methylated LDL, the removal rates of those lipoproteins were increased 1.9-, 1.7-, and 2.0-fold, respectively, after the treatment. Immunoblot analysis of LDL receptors in the treated rabbits showed no significant changes in LDL receptor proteins in livers but a great increase in spleens and bone marrows compared with the controls. Messenger RNA was also estimated by Northern blotting in both groups, and the results were compatible with those from the immunoblot. The data suggest that M-CSF stimulates the clearance of lipoproteins containing apolipoprotein B-100 via both LDL receptor-dependent and -independent pathways in target cells of M-CSF and reduces plasma cholesterol.     

Destrin, a mammalian actin-depolymerizing protein, is closely related to cofilin. Cloning and expression of porcine brain destrin cDNA

Destrin is a mammalian 19-kDa protein that rapidly depolymerizes F-actin in a stoichiometric manner. In this study, we isolated cDNA clones coding for destrin from a porcine brain cDNA library. The deduced amino acid sequence of destrin is 165 residues long and is very similar (71% identical) to that of cofilin, a widely distributed, pH-sensitive actin-modulating protein. Destrin contains a sequence nearly identical with the putative nuclear transport signal sequence of cofilin and a hexapeptide sequence identical with the amino-terminal sequence (residues 2-7) of tropomyosin, which is shown to be involved in cofilin binding to actin. Destrin, like cofilin, also has in its carboxyl-terminal portion a region homologous to the sequence shared by gelsolin, fragmin, and Acanthamoeba profilin. We have expressed destrin as well as cofilin in Escherichia coli, purified them, and examined their function in vitro. The two proteins were found to differ in their interaction with actin, like destrin and cofilin isolated from porcine brain. This suggests that the difference in the function of the two proteins results from the subtle difference in their amino acid sequence rather than possible differences in post-translational modifications. Northern blot analyses indicated that both destrin mRNA and cofilin mRNA are widely distributed in various tissues, but both mRNAs differ in their relative abundance among tissues.     

Changes in diacylglycerol and cyclic GMP during the differentiation of human myeloid leukemia K562 cells

When the human myeloid leukemia cell line, K562, was induced to differentiate along the erythroid lineage by a 4 day treatment with 10 microM tiazofurin, the cellular content of diacylglycerol decreased to 35% of the value in untreated control cells. Under the same conditions the content of cGMP decreased to 61% of the control value. Tiazofurin inhibits guanine nucleotide biosynthesis and lowers cellular GTP. When guanosine and adenine were added together with tiazofurin, the differentiation of K562 was prevented, the concentration of diacylglycerol was maintained at control values, and the reduction in the concentration of cGMP was partially prevented. Other inducers of differentiation which acted by different mechanisms, caused similar changes in the concentrations of diacylglycerol and cGMP.     

Effect of a platelet activating factor antagonist (CV6209) on shock caused by temporary hepatic inflow occlusion

Platelet activating factor (PAF) is a newly discovered inflammatory chemical mediator, which was reported to play a pivotal role in various types of shock. There is also a great possibility that PAF plays an important role in the shock caused by hepatic inflow occlusion. In the present study, the effect of CV6209, a PAF antagonist, on the shock caused by the occlusion was investigated. Intravenous 3 micrograms/kg of PAF caused hypotension in Wistar rats (n=6), and pretreatment with intravenous 3 mg/kg of CV6209 significantly (p less than 0.01) prevented the hypotension (n=6). Forty-five minutes of hepatic inflow occlusion caused hypotension in rats during the occlusion period, and the hypotension continued even after restoration of blood flow in control group (pretreated with saline i.v. only, n=5). In contrast, this hypotension was significantly (p less than 0.01) reversed in PAF antagonist group (pretreated with 3 mg/kg of CV6209 i.v., n=5). In sham-operated rats (n=6), arterial pressure remained unchanged and not hypotensive during the monitoring period. The survival rate of rats 90 minutes after declamp was 30% in control group (n=20), and that was significantly (p less than 0.05) improved to be 65% in PAF antagonist group (n=20). In conclusion, PAF plays an important role in the shock and death caused by temporary hepatic inflow occlusion, and a PAF antagonist could be a therapeutic drug against temporary hepatic inflow occlusion.