Bleomycin stimulates lung fibroblasts to release neutrophil and monocyte chemotactic activity

We determined whether human lung fibroblasts might release chemotactic activity for neutrophils (NCA) and monocytes (MCA) in response to bleomycin. The human lung fibroblasts supernatant fluids were evaluated for chemotactic activity by a blind well chamber technique. Human lung fibroblasts released NCA and MCA in a dose- and time-dependent manner in response to bleomycin. Checkerboard analysis of supernatant fluids revealed that both NCA and MCA were chemotactic. Partial characterization revealed that NCA was partly heat labile, trypsin sensitive, and predominantly ethyl acetate extractable. In contrast, MCA was partly trypsin sensitive and ethyl acetate extractable. The release of chemotactic activity was inhibited by lipoxygenase inhibitors and cycloheximide. Molecular sieve column chromatography revealed that both NCA and MCA had multiple chemotactic peaks. NCA was inhibited by leukotriene B4 receptor antagonist and anti-IL-8 and G-CSF Abs. MCA was attenuated by leukotriene B4 receptor antagonist, and monocyte chemoattractant protein-1, GM-CSF, and TGF-beta Abs. Leukotriene B4 receptor antagonist and these Abs inhibited the corresponding m.w. chemotactic activity separated by column chromatography. The concentrations of IL-8, G-CSF, monocyte chemoattractant protein-1, GM-CSF, and TGF-beta in the supernatant fluids significantly increased in response to bleomycin. These data suggest that lung fibroblasts may modulate inflammatory cell recruitment into the lung by releasing NCA and MCA in response to bleomycin.     

Increased numbers of B-1 cells and enhanced responses against TI-2 antigen in mice lacking APS, an adaptor molecule containing PH and SH2 domains

APS (adaptor molecule containing PH and SH2 domains) is an intracellular adaptor protein that forms an adaptor family along with Lnk and SH2-B. While experiments using cultured cell lines have demonstrated that APS is phosphorylated in response to various stimuli, its in vivo functions remain unclear. We attempted to determine the physiological roles of APS by generating APS-deficient (APS(-/-)) mice. APS(-/-) mice were viable and fertile and showed no abnormalities or growth retardation. Immunologically, APS(-/-) mice showed normal development and distribution of lymphocytes and myeloid cells, except for increased numbers of B-1 cells in the peritoneal cavity. APS(-/-) mice exhibited an enhanced humoral immune response against trinitrophenol-Ficoll, a thymus-independent type 2 antigen, while APS(-/-) B-2 cells exhibited normal proliferative responses and tyrosine phosphorylation of intracellular proteins upon B-cell receptor (BCR) cross-linking. APS colocalized with filamentous actin (F-actin) accumulated during the capping of BCRs in APS-transgenic B cells. After BCR stimulation, F-actin contents were lower in APS(-/-) B-1 cells than in wild-type B-1 cells. Our results indicate that APS might have a novel regulatory role in actin reorganization and control of B-1 cell compartment size.     

Development of definitive endoderm from embryonic stem cells in culture

The cellular and molecular events regulating the induction and tissue-specific differentiation of endoderm are central to our understanding of the development and function of many organ systems. To define and characterize key components in this process, we have investigated the potential of embryonic stem (ES) cells to generate endoderm following their differentiation to embryoid bodies (EBs) in culture. We found that endoderm can be induced in EBs, either by limited exposure to serum or by culturing in the presence of activin A (activin) under serum-free conditions. By using an ES cell line with the green fluorescent protein (GFP) cDNA targeted to the brachyury locus, we demonstrate that endoderm develops from a brachyury(+) population that also displays mesoderm potential. Transplantation of cells generated from activin-induced brachyury(+) cells to the kidney capsule of recipient mice resulted in the development of endoderm-derived structures. These findings demonstrate that ES cells can generate endoderm in culture and, as such, establish this differentiation system as a unique murine model for studying the development and specification of this germ layer.     

Molecular characterization of dextranase from Streptococcus rattus

The complete nucleotide sequence of the dextranase gene of Streptococcus rattus ATCC19645 was determined. An open reading frame of the dextranase gene was 2,760 bp long and encoded a dextranase protein consisting of 920 amino acids with a molecular weight of 100,163 Da and an isoelectric point of 4.67. The S. rattus dextranase purified from recombinant Escherichia coli cells showed dextran-hydrolyzing activity with optimal pH (5.0) and temperature (40 C) similar to those of dextranases from Streptococcus mutans and Streptococcus sobrinus. The deduced amino acid sequence of the S. rattus dextranase revealed that the dextranase molecule consists of two variable regions and a conserved region. The variable regions contained an N-terminal signal peptide and a C-terminal cell wall sorting signal; the conserved region contained two functional domains, catalytic and dextran-binding sites. This structural feature of the S. rattus dextranase is quite similar to that of other cariogenic species such as S. mutans, S. sobrinus, and Streptococcus downei.     

Stimulating the production of homoisoflavonoids in cell suspension cultures of Caesalpinia pulcherrima using cork tissue

It has previously been demonstrated that cork tissue increases the efficiency of the production of lipophilic secondary metabolites in diverse plant cell suspension cultures. In the present study, three new homoisoflavonoids--named dihydrobonducellin, 2'-methoxydihydrobonducellin, and 2'-methoxybonducellin--and bonducellin and isobonducellin were isolated from Caesalpinia pulcherrima cultured cells coincubated with cork tissue. Cork tissue increased the production of 2'-methoxybonducellin by about 7-fold relative to control cells, and more than 80% of the product was recoverable from the cork tissue. When cork tissue and methyl jasmonate or yeast extract were added simultaneously to the medium, the amount of 2'-methoxybonducellin produced increased further. The production of the other four homoisoflavonoids was enhanced by variable amounts. Our results indicate that the addition of cork tissue would be an effective technique for investigating formation of secondary metabolites that usually accumulate only in trace amounts.     

Orally active anti-proliferation agents: novel diphenylamine derivatives as FGF-R2 autophosphorylation inhibitors

(6,7-Disubstituted-quinolin-4-yloxy-phenyl)(4-substituted-phenyl)amine derivatives were synthesized and evaluated by a cellular autophosphorylation assay for FGF-R2 in the human scirrhous gastric carcinoma cell line, OCUM-2MD3. We also performed metabolic stability studies showing that substitutions at the 7-position of quinoline affect its biological stability. In this study, we achieved a remarkable improvement in the solubility and metabolic stability of the diphenylamine derivative. The most promising compound 15e showed a significant decrease in tumor volume when orally administered.     

The cytoplasmic male-sterile type and normal type mitochondrial genomes of sugar beet share the same complement of genes of known function but differ in the content of expressed ORFs

The complete nucleotide sequence (501,020 bp) of the mitochondrial genome from cytoplasmic male-sterile (CMS) sugar beet was determined. This enabled us to compare the sequence with that previously published for the mitochondrial genome of normal, male-fertile sugar beet. The comparison revealed that the two genomes have the same complement of genes of known function. The rRNA and tRNA genes encoded in the CMS mitochondrial genome share 100% sequence identity with their respective counterparts in the normal genome. We found a total of 24 single nucleotide substitutions in 11 protein genes encoded by the CMS mitochondrial genome. However, none of these seems to be responsible for male sterility. In addition, several other ORFs were found to be actively transcribed in sugar beet mitochondria. Among these, Norf246 was observed to be present in the normal mitochondrial genome but absent from the CMS genome. However, it seems unlikely that the loss of Norf246 is causally related to the expression of CMS, because previous studies on mitochondrial translation products failed to detect the product of this ORF. Conversely, the CMS genome contains four transcribed ORFs (Satp6presequence, Scox2-2 , Sorf324 and Sorf119) which are missing from the normal genome. These ORFs, which are potential candidates for CMS genes, were shown to be generated by mitochondrial genome rearrangements.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by R. Hagemann     

SOCS proteins in T helper cell differentiation: implications for allergic disorders?

Asthma, allergic rhinitis and atopic dermatitis are allergic immune disorders characterised by a predominance of T helper 2 (Th2) cells, the resulting elevation of allergen-specific IgE, and mast-cell- and basophil-associated inflammation. The cytokine environment at the site of the initial antigen stimulation determines the direction of Th-cell differentiation into Th1 or Th2 cells. The SOCS (suppressor of cytokine signalling) proteins are implicated in the control of the balance between Th1 and Th2 cells in this process. SOCS3 is predominantly expressed in Th2 cells and inhibits Th1 differentiation; conversely, SOCS5 is expressed predominantly in Th1 cells and inhibits Th2 differentiation. Here, we discuss the role of SOCS proteins in Th-cell differentiation and explore the potential of SOCS proteins as targets for therapeutic strategies in allergic disorders.     

Potent and orally active ET(A) selective antagonists with 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acid structures

The synthesis and structure-activity relationships of a series of 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids are described. Our efforts have been focused on modification of the aryl ring at the 5-position and the alkyl substituent at the 2-position of the bottom 4-methoxyphenyl ring in an effort to develop orally available ET(A) selective antagonists with safer profiles in terms of the P-450 enzyme inhibitory activity. Incorporation of a hydroxymethyl group as an alkyl substituent in methylenedioxyphenyl and 6-dihydrobenzofuran derivatives led to the identification of orally bioavailable ET(A) selective antagonists 1f and 7f. These compounds also showed not only excellent binding affinity (IC(50) < 0.10nM, more than 800-fold selectivity for the ET(A) receptor over the ET(B) receptor) but also sufficient oral bioavailability, 48% and 56%, respectively, in rats. Furthermore, these compounds did not exhibit either competitive or mechanism-based inhibition of human cytochrome P450 enzymes.