Synergistic growth stimulation of corneal fibroblasts by components of mesodermal growth factor from murine submaxillary glands

Recycled mesodermal growth factor (R-MGF) is a pool of proteins of 26,000 molecular weight obtained by recycling gel chromatography of male murine submaxillary gland extracts. R-MGF strikingly accelerates corneal stromal wound healing in vivo, fibroblast growth and migration in cultured corneal buttons and is shown here to stimulate stromal fibroblast growth and division in tissue culture. Chromatographic fractionation of R-MGF has yielded several components, none of which has a greater biological potency than the parent R-MGF. In contrast, two components, MGF-I and -II, when recombined synergistically stimulate fibroblast response in tissue culture and organ culture in excess of those obtained with the parent R-MGF. Three MGF components (I, III, and IV) have been purified and are inactive at 10–15 m?g/ml in organ culture but potently stimulate fibroblast responses when combined in pairs containing 7.5 m?g of each component. The striking synergism in organ culture suggests that the stimulation of wound healing by R-MGF in vivo may also reflect synergistic action of more than one R-MGF component. Procedures for isolating gram quantities of R-MGF and for the purification of different R-MGF components by ion exchange chromatography are detailed.     

Localization of a mitochondrial type of NADP-dependent isocitrate dehydrogenase in kidney and heart of rat: an immunocytochemical and biochemical study.

We studied the subcellular localization of the mitochondrial type of NADP-dependent isocitrate dehydrogenase (ICD1) in rat was immunofluorescence and immunoelectron microscopy and by biochemical methods, including immunoblotting and Nycodenz gradient centrifugation. Antibodies against a 14-amino-acid peptide at the C-terminus of mouse ICD1 was prepared. Immunoblotting analysis of the Triton X-100 extract of heart and kidney showed that the antibodies developed a single band with molecular mass of 45 kD. ICD1 was highly expressed in heart, kidney, and brown fat but only a low level of ICD1 was expressed in other tissues, including liver. Immunofluorescence staining showed that ICD1 was present mainly in mitochondria and, to a much lesser extent, in nuclei. Low but significant levels of activity and antigen of ICD1 were found in nuclei isolated by equilibrium sedimentation. Immunoblotting analysis of subcellular fractions isolated by Nycodenz gradient centrifugation from rat liver revealed that ICD1 signals were exclusively distributed in mitochondrial fractions in which acyl-CoA dehydrogenase was present. Immunofluorescence staining and postembedding electron microscopy demonstrated that ICD1 was confined almost exclusively to mitochondria and nuclei of rat kidney and heart muscle. The results show that ICD1 is expressed in the nuclei in addition to the mitochondria of rat heart and kidney. In the nuclei, the enzyme is associated with heterochromatin. In kidney, ICD1 distributes differentially in the tubule segments.     

Effects of X-ray irradiation on male sperm transfer ability and fertility in the sweetpotato weevils <Emphasis Type="Italic">Euscepes postfasciatus</Emphasis> (Coleoptera: Curculionidae) and <Emphasis Type="Italic">Cylas formicarius</Emphasis> (Coleoptera: Brentidae)

Gamma radiation from isotopic sources has been used in sterile insect technique (SIT) programs worldwide, but it might be difficult to continue using these sources in future SIT programs because of social issues. Therefore, an alternative sterilization source to gamma rays, such as X-rays, needs to be developed. The physical properties of radiation are different between gamma rays and X-rays: for example, X-rays have a shorter penetration depth than gamma rays. Therefore, X-rays may not fully confer male sterility, depending on the target pest insects. The present study investigated whether the West-Indian sweetpotato weevil Euscepes postfasciatus (Fairmaire) and the sweetpotato weevil Cylas formicarius (Fabricius) are sterilized by X-rays generated in a low-energy X-ray irradiator, without deterioration of male mating ability, at the doses currently used in the eradication programs for E. postfasciatus (150 Gy) and C. formicarius (200 Gy) using gamma rays at Okinawa, Japan. The results demonstrated that it is possible to use X-rays in future SIT programs for E. postfasciatus and C. formicarius, because X-ray irradiated males were almost completely sterilized without deterioration of their mating ability.     

Morphology-oriented epigenetic research

Cytosine methylation plays a major role in the regulation of sequential and tissue-specific expression of genes. De novo aberrant DNA methylation and demethylation are also crucial processes in tumorigenesis and tumor progression. The mechanisms of how and when such aberrant methylation and demethylation occur in tumor cells are still obscure, however. To evaluate subtle epigenetic alteration among minor subclonal populations, morphology-oriented epigenetic analysis is requisite, especially where heterogeneity and flexibility are as notable as in the process of cancer progression and cellular differentiation at critical stages. Therefore, establishment of reliable morphology-oriented epigenetic studies has become increasingly important in not only the experimental but also the diagnostic field. By selecting a subset of cells based on characteristic morphological features disclosed by microdissection or in situ hybridization, we discovered how methylation at certain CpG sites outside of CpG islands would play a crucial epigenetic role in the versatility and flexibility of gene expression during cancer progression. In this review, we first introduce technical aspects of two morphology-oriented epigenetic studies: (1) histoendonuclease-linked detection of methylated sites of DNA (HELMET), and (2) padlock probe and rolling circle amplification (RCA) for in situ identification of methylated cytosine in a sequence-dependent manner. We then present our observation of a novel MeCP2-mediated gene-silencing mechanism through the addition of methylation to a single-CpG-locus upstream of the TATA-box of the receptor activator of NF-κB ligand (RANKL) and of secreted frizzled-related protein 4 (SFRP4) gene promoters.     

Suppression of human interferon-gamma production by a 17 amino acid peptide homologous to the transmembrane envelope protein of retroviruses: evidence for a primary role played by monocytes.

CKS-17, a synthetic amino acid peptide homologous to a highly conserved region of retroviral transmembrane protein exerts a suppressive action on staphylococcal enterotoxin A (SEA)-induced the production of IFN-gamma by human peripheral blood mononuclear cells (PBMC) (Ogasawara et al., J. Immunol. 141, 615, 1988). This action has been shown in the present study to be preceded by dramatic clustering of PBMC. Clusters appear within 3 hr of exposure of PBMC to CKS-17; they are dose dependent, inhibited by cycloheximide, and require a temperature of 37 degrees C. The cells in the clusters are predominantly monocytes. Although it has been previously shown that CKS-17 inhibits monocyte-mediated killing by inactivating IL-1 (Kleinerman et al., J. Immunol. 139, 2329, 1987) and production of IL-2 by murine thymoma cells treated with IL-1 (Gottlieb et al., J. Immunol. 142, 4321, 1989), in the present study we show that IL-1 does not prevent clustering of PBMC by CKS-17. Using CKS-17 and highly purified monocytes or lymphocytes, profound alterations occur only with monocytes, as revealed by light or electron microscopy. SEA- or staphylococcal enterotoxin B-induced production of IFN-gamma is inhibited when highly purified monocytes pretreated with CKS-17 are cocultured with highly purified T lymphocytes. Thus, CKS-17 induces dramatic clustering of cells apparently by inducing alterations of monocytes but not lymphocytes, suggesting that CKS-17 may interfere with the capacity of monocytes to facilitate production of IFN-gamma by T lymphocytes.     

Three novel mutations in the liver-type arginase gene in three unrelated Japanese patients with argininemia.

Argininemia is caused by a hereditary deficiency of liver-type arginase (E.C.3.5.3.1) and is characterized by psychomotor retardation and spastic tetraplegia. We examined findings in three Japanese patients with argininemia, by using the PCR, cloning, and sequencing procedures. We found three different mutations--G-to-A-365 in exon 4, G-to-C-703 in exon 7, and C-del-842 in exon 8--thereby leading to mutant arginase proteins of W122X, G235R, and L282FS, respectively. Patient 1 was a compound heterozygote, inheriting the allele with G-to-A-365 from his mother and the allele with G-to-C-703 from his father. Patients 2 and 3 were homozygotes of the allele with G-to-C-703 and of the allele with C-del-842, respectively. Expression tests of these mutant arginases in Escherichia coli indicated that the mutant arginase of W122X did not remain a stable product. The other two mutant arginases--G235R and L282FS--were detected by immunoblot analyses. There was no evidence of activity of the three mutant arginases expressed in E. coli. We tentatively conclude that argininemia is heterogeneous, at the molecular level.     

Regulation by a novel protein of the bimodal distribution of lipopolysaccharide in the outer membrane of Escherichia coli.

We report on the cloning and characterization of the rfb gene cluster of the O75 lipopolysaccharide from a urinary tract isolate of Escherichia coli. Deletion cloning defined the minimum region of DNA that expressed the O75 antigen in E. coli host strains to be on a 12.4-kb insert. However, the E. coli strain expressing this region did not produce a polymerized O chain as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. A slightly larger DNA clone of 13.4 kb produced a polymerized O chain in E. coli S phi 874 but was found to be abnormal in its distribution over the surface membrane. Normal wild-type E. coli, as with Salmonella spp., has a bimodal distribution of the lipopolysaccharide on the surface which is seen as an abundance of long and short O chains attached to the lipid A-core structure. We found in a region adjacent to the cloned rfb region, and on the opposite side from where the putative polymerase (rfc) is encoded, a novel protein of 35.5 kDa expressed from a 1.75-kb DNA fragment. This protein was shown to complement in trans the E. coli strains carrying plasmids that expressed abnormal, unregulated lipopolysaccharides. The expression of these complemented strains was bimodal in distribution. Mutation of the gene encoding this protein destroyed its ability to regulate O-chain distribution. We propose to call this regulator gene rol, for regulator of O length.     

Partial characterization of a glucocorticoid suppressible mitogenic activity secreted from a rat hepatoma cell line hypersensitive to the antiproliferative effects of glucocorticoids

We have previously shown that glucocorticoids suppress the proliferation of Fu5 hepatoma cells and have selected subclones which are either hypersensitive (BDS1) or resistant (EDR3) to the antiproliferative effects of dexamethasone, a synthetic glucocorticoid. BDS1 cells externalize a glucocorticoid suppressible mitogenic activity (denoted GSM) which stimulated [3H]thymidine incorporation in quiescent, serum-starved Balb/c 3T3 cells. Glucocorticoid treatment of BDS1 cells reduced the secreted levels of GSM activity by approximately 20-fold in comparison to untreated cells. The GSM activity was constitutively secreted from a glucocorticoid receptor minus variant (EDR3) demonstrating that the suppression of this mitogenic activity is a new glucocorticoid hormone response which required a functional receptor. GSM activity was sensitive to sulfhydryl reducing agents or trypsin, stable to heat and acid treatments and fractionated in gel filtration columns with a native molecular weight of approximately Mr 30,000. The persistence of this size for mitogenic activity after electrophoretic fractionation in nonreducing sodium dodecyl sulfate-poly-acrylamide gels suggested that the GSM activity is comprised of a single protein. Total secreted protein isolated from untreated BDS1, but not dexamethasone-treated BDS1, stimulated 3T3 cells to grow in transformed-appearing large colonies in soft agar and to display multiple layering and elongated spindle-like morphology on solid substratum. The addition of both insulin and EGF to conditioned medium protein isolated from glucocorticoid-treated BDS1 cells restored full induction of 3T3 cell anchorage-independent growth while insulin restored full and EGF partial mitogenic stimulation of these fibroblasts. These results suggest that the GSM activity acts in a pathway common to that of insulin or EGF in fibroblasts.     

Detection and localization of a new enzyme catalyzing the beta-aryl ether cleavage in the soil bacterium (Pseudomonas paucimobilis SYK-6)

Cleavage of the arylglycerol-beta-aryl ether linkage is the most important process in the biological degradation of lignin. We determined the activity of the enzyme cleaving the beta-aryl ether linkage in membranes of Pseudomonas paucimobilis SYK-6. This enzyme was tightly associated with the cellular membrane and catalyzed the unique and reductive cleavage of compound II but not cleavage of compound I. This enzymatic activity was stimulated by addition of NADH. On the basis of this evidence, we present a model of the specific cellular assimilation of beta-aryl ether by P. paucimobilis SYK-6.     

Introduction of an Azido Group to the C-6 Position of Uridine by the Use of a 6-Iodouridine Derivative

Abstract

Nucleophilic addition-elimination reaction of azide ion to 6-iodo-2′,3′-O-isopropylidene-5′-O-methoxy-methyluridine proceeded under mild conditions to give a 6-azidouridine derivative.