The different effects of recombinant human tumor necrosis factor on rat fibrosarcoma sublines

Summary The antitumor effect of recombinant human tumor necrosis factor (rH-TNF) on two clones of rat fibrosarcoma with different metastatic potential to lymph nodes was examined. The colony formation of clone A, which has high metastatic potential, was completely inhibited by continuous exposure to rH-TNF at 50 U/ml. In contrast, colony formation of clone G, which has low metastatic potential, was not inhibited by high concentrations of rH-TNF (10,000 U/ml). The inhibitory effect of rH-TNF on colony formation by clone A was also observed with a 1-h exposure to rH-TNF. This effect was time and concentration dependent, as determined by the colony assay, ³H-thymidine uptake assay, and ⁵¹Cr-release assay. ³H-thymidine and ³H-uridine uptake per cell of clone A exposed to rH-TNF was not decreased. This suggests that the mechanisms of the antitumor effect of rH-TNF were not due to inhibition of DNA and RNA synthesis of tumor cells. In vivo growth and lymph node metastases of clone A inoculated i.p. to Donryu strain rats were completely suppressed by 14 consecutive i.p. injections of 10⁵ or 10⁶ U/kg per day of rH-TNF. On the other hand the growth of clone G was not influenced by rH-TNF administration.     

Proteomic study for the cellular responses to Cd2+ in Schizosaccharomyces pombe through amino acid-coded mass tagging and liquid chromatography tandem mass spectrometry

Cadmium (Cd(2+)) is one of well-known toxic heavy metal ions. To gain a global understanding how Cd(2+) affects cells at the molecular level, we systematically studied the cellular response of the fission yeast Schizosaccharomyces pombe to Cd(2+) using our integrated proteomic strategy of amino acid-coded mass tagging (AACT) and liquid chromatography-tandem mass spectrometry. Our proteome-wide investigation unequivocally identified 1133 S. pombe proteins. Of which, the AACT-based quantitative analysis revealed 106 up-regulated and 55 down-regulated proteins on the Cd(2+) exposure. The most prevalent functional class in the up-regulated proteins, approximately 28% of our profile, was the proteins involved in protein biosynthesis, showing a time-dependent biphasic expression pattern characteristic with rapid initial induction and later repression. Most significantly, 27 proteins functionally classified as cell rescue and defense were up-regulated for oxygen and radical detoxification, heat shock response, and other stress response. Furthermore, the large precursor sequence coverage of our AACT approach allowed us to unequivocally identify and quantitate different isozymes for glutathione S-transferase, which have close similarity in their amino acid sequence. Our quantitative dataset also showed that 80% of the up-regulated proteins found in the S. pombe response were different from those in the Saccharomyces cerevisiae response. The function of some of the key identifications was validated through biochemical assays. It is very interesting that the induction of cysteine synthase expression was not observed in our study, although it has been proven as a critical enzyme to supply free cysteines for the enhancing synthesis of Cd(2+)-sequestering molecules such as glutathione and phytochelatins in plants and some yeasts. Our quantitative proteomic result instead suggested that, as an alternative mechanism for the detoxification of Cd(2+), S. pombe produced significantly higher level of inorganic sulfide to immobilize cellular Cd(2+) as a form of CdS nanocrystallites capped with glutathione and/or phytochelatins.     

Flavobacterium cheonhonense sp. nov., isolated from a freshwater reservoir

A novel bacterium, designated strain ARSA-15(T), was isolated from a freshwater sample collected from the Cheonho reservoir, Cheonan, Republic of Korea. The isolate was deep-yellow pigment, Gram-negative, rod-shaped, non-motile, and catalase- and oxidase-positive. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate belongs to the genus Flavobacterium, and shared less than 97% sequence similarity with recognized Flavobacterium species. The novel species was able to grow at 10-37°C, pH 6.5-10.0, and in 0-0.5% (w/v) NaCl concentrations. Chemotaxonomically, iso-C(15:1), iso-C(15:0), and iso-C(16:0) were observed to be the predominant cellular fatty acid, and menaquinone-6 (MK-6) was the predominant respiratory quinone. The major polar lipid patterns of strain ARSA-19(T) was phosphatidylethanolamine, unknown aminolipid (AL1 and AL2), and unidentified polar lipids (L1, L2, and L3). The genomic DNA G+C content of the isolate was 39.2 mol%. On the basis of polyphasic approach, strain ARSA-15(T) represents a novel species of the genus Flavobacterium, for which the name Flavobacterium cheonhonense sp. nov. is proposed. The type strain is ARSA-15(T) (=KACC 14967(T) =KCTC 23180(T) =JCM 17064(T)).     

Leucobacter denitrificans sp. nov., isolated from cow dung

The bacterial strain M1T8B10^T was isolated from cow dung in Suwon, Republic of Korea. The strain was a Gram stain-positive rod, nonmotile, and non-spore-forming. According to 16S rRNA gene sequence analysis, the strain fell within the clade of the genus Leucobacter, showing the highest sequence similarities with Leucobacter aridicollis L-9^T (98.7%), Leucobacter iarius 40^T (98.4%), and Leucobacter komagatae JCM 9414^T (98.2%). Cell-wall peptidoglycan contained the diagnostic diamino acid 2,4-diaminobutyric acid of the genus Leucobacter, showing B-type cross-linked peptidoglycans. The major fatty acids were anteiso-C_15:0, iso-C_16:0, and anteiso-C_17:0. The quinone system consisted of the menaquinones MK-11 (78%) and MK-10 (22%). The polar lipid profiles contained diphosphatidylglycerol, phosphatidylglycerol, and an unidentified glycolipid. Differences in several physiological features including nitrate reduction enabled the isolate to be differentiated from all recognized Leucobacter species. Based on these phylogenetic, chemotaxonomic, and phenotypic results, the isolate represents a novel species, for which the name Leucobacter denitrificans sp. nov. is proposed. The type strain is M1T8B10^T (=KACC 14055^T =NBRC 106309^T).     

Deciphering the proteome of the in vivo diagnostic reagent "purified protein derivative" from Mycobacterium tuberculosis

Purified protein derivative (PPD) has served as a safe and effective diagnostic reagent for 60 years and is the only broadly available material to diagnose latent tuberculosis infections. This reagent is also used as a standard control for a number of in vitro immunological assays. Nevertheless, the molecular composition and specific products that contribute to the extraordinary immunological reactivity of PPD are poorly defined. Here, a proteomic approach was applied to elucidate the gene products in the U.S. Food and Drug Administration (FDA) standard PPD-S2. Many known Mycobacterium tuberculosis T-cell antigens were detected. Of significance, four heat shock proteins (HSPs) (GroES, GroEL2, HspX, and DnaK) dominated the composition of PPD. The chaperone activities and capacity of these proteins to influence immunological responses may explain the exquisite solubility and immunological potency of PPD. Spectral counting analysis of three separate PPD reagents revealed significant quantitative variances. Gross delayed-type hypersensitivity (DTH) responses in M. tuberculosis infected guinea pigs were comparable among these PPD preparations; however, detailed histopathology of the DTH lesions exposed unique differences, which may be explained by the variability observed in the presence and abundance of early secretory system (Esx) proteins. Variability in PPD reagents may explain differences in DTH responses reported among populations.     

Lycopene inhibits Helicobacter pylori-induced ATM/ATR-dependent DNA damage response in gastric epithelial AGS cells

Oxidative stress linked to DNA damage is involved in the pathogenesis of Helicobacter pylori-associated gastric diseases. The DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair, and apoptosis through the activation of ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) and their target proteins. However, neither H. pylori-induced DDR nor the effects of antioxidants on the DNA damage have been established. This study aimed to investigate the detailed process of H. pylori-induced DNA damage and to examine whether lycopene, a natural antioxidant, inhibits DNA damage and cellular response of gastric epithelial AGS cells infected with H. pylori. AGS cells were cultured with H. pylori in Korean isolates and treated with or without lycopene. Cell viability, DNA damage indices, levels of 8-OH-dG, and reactive oxygen species (ROS) as well as cell-cycle distributions were determined. The activation of ATM, ATR, Chk1, and Chk2; histone H2AX focus formation; activation and induction of p53; and levels of Bax and Bcl-2 and poly(ADP-ribose) polymerase-1 (PARP-1) were assessed. The results showed that H. pylori induced apoptosis in AGS cells with increased Bax and decreased Bcl-2 expression as well as PARP-1 cleavage. Culture with H. pylori led to increases in intracellular ROS, 8-OH-dG, double-strand DNA breaks (DSBs), and DNA fragmentation. H. pylori induced activation of the ATM/Chk2 and ATR/Chk1 pathways, phosphorylation of H2AX and p53, and a delay in the progression of the cells entering the S phase. Lycopene inhibited H. pylori-induced increases in ROS, apoptosis, alterations in cell-cycle distribution, DSBs, and ATM- and ATR-mediated DDR in AGS cells. In conclusion, lycopene may be beneficial for treatment of H. pylori-induced gastric diseases associated with oxidative DNA damage.     

Effects of diet type,developmental stage,and gut compartment in the gut bacterial communities of two <Emphasis Type="Italic">Cerambycidae species</Emphasis> (Coleoptera)

The gut bacterial community of wood-feeding beetles has been examined for its role on plant digestion and biocontrol method development. Monochamus alternatus and Psacothea hilaris, both belonging to the subfamily Lamiinae, are woodfeeding beetles found in eastern Asia and Europe and generally considered as destructive pests for pine and mulberry trees, respectively. However, limited reports exist on the gut bacterial communities in these species. Here, we characterized gut bacterial community compositions in larva and imago of each insect species reared with host tree logs and artificial diets as food sources. High-throughput 454 pyrosequencing of bacterial 16S rRNA gene revealed 225 operational taxonomic units (OTUs) based on a 97% sequences similarity cutoff from 138,279 sequence reads, the majority of which were derived from Proteobacteria (48.2%), Firmicutes (45.5%), and Actinobacteria (5.2%). The OTU network analysis revealed 7 modules with densely connected OTUs in specific gut samples, in which the distributions of Lactococcus-, Kluyvera-, Serratia-, and Enterococcus-related OTUs were distinct between diet types or developmental stages of the host insects. The gut bacterial communities were separated on a detrended correspondence analysis (DCA) plot and by c-means fuzzy clustering analysis, according to diet type. The results from this study suggest that diet was the main determinant for gut bacterial community composition in the two beetles.     

The species range‐size patterns for vascular plants of Seorak Mountain (Korea): Relationship between group of life forms and phytogeography affinity along the elevational gradient

Research on species richness patterns and the advanced elevational Rapoport rule (ERR) has been widespread in recent years; however, there is a lack of such research for the temperate mountainous regions in northeast Asia. Here, we collected plant species from the Seorak Mountain in northeast Asia through field surveys. The species were divided into 11 groups according to the life‐form types and phytogeography affinities of each species. The ERR was evaluated using Steven''s method and by examining the species richness patterns of each group. The species richness patterns revealed a positive multimodal pattern along the elevation gradient, but phytogeography affinities (increasing trend) and life‐form analysis (unimodal) exhibited different patterns. The elevation gradients (1,350 m for the mean elevation–range relationships), which are affected by the boundary effect and different life forms, did not consistently support the ERR. However, herbs as well as rare, endemic, and red list species showed consistent support for the ERR, which could be attributed to the influence by phytogeography affinities. Therefore, the results from Seorak Mountain showed that the ERR was not consistent for different plant life forms in the same area; however, phytogeography affinities could support and explain ERR.     

Glucosamine Treatment-mediated O-GlcNAc Modification of Paxillin Depends on Adhesion State of Rat Insulinoma INS-1 Cells

Protein-protein interactions and/or signaling activities at focal adhesions, where integrin-mediated adhesion to extracellular matrix occurs, are critical for the regulation of adhesion-dependent cellular functions. Although the phosphorylation and activities of focal adhesion molecules have been intensively studied, the effects of the O-GlcNAc modification of their Ser/Thr residues on cellular functions have been largely unexplored. We investigated the effects of O-GlcNAc modification on actin reorganization and morphology of rat insulinoma INS-1 cells after glucosamine (GlcN) treatment. We found that paxillin, a key adaptor molecule in focal adhesions, could be modified by O-GlcNAc in INS-1 cells treated with GlcN and in pancreatic islets from mice treated with streptozotocin. Ser-84/85 in human paxillin appeared to be modified by O-GlcNAc, which was inversely correlated to Ser-85 phosphorylation (Ser-83 in rat paxillin). Integrin-mediated adhesion signaling inhibited the GlcN treatment-enhanced O-GlcNAc modification of paxillin. Adherent INS-1 cells treated with GlcN showed restricted protrusions, whereas untreated cells showed active protrusions for multiple-elongated morphologies. Upon GlcN treatment, expression of a triple mutation (S83A/S84A/S85A) resulted in no further restriction of protrusions. Together these observations suggest that murine pancreatic β cells may have restricted actin organization upon GlcN treatment by virtue of the O-GlcNAc modification of paxillin, which can be antagonized by a persistent cell adhesion process.