Inhibitory effects of transforming growth factor-beta1 pretreatment on experimental pulmonary metastasis of MCS-1 Chinese hamster mesenchymal chondrosarcoma cells.

Recent studies have suggested that transforming growth factor(TGF)-beta1 acts as a multifunctional regulator of cell growth, and also modifies tumor progression and metastasis. In the present study, we investigated the effects of TGF-beta1 on the proliferation and experimental pulmonary metastasis of MCS-1. MCS-1 are undifferentiated type cloned tumor cells established from a mesenchymal chondrosarcoma which spontaneously occurred in the soft tissue of a female Chinese hamster. MCS-1 cells were pretreated with TGF-beta1 (0, 0.05, 0.5, 2, 10 ng/ml) for 72 hours in a medium containing 1% fetal bovine serum, then tested for in vitro growth by the MTT method, in vivo growth by subcutaneous inoculation into athymic nude mice (1 x 10(6) cells/mouse) and experimental pulmonary metastasis by injection into the lateral tail vein of athymic nude mice (5 x 10(4) cells/mouse). TGF-beta1 significantly inhibited in vitro growth of MCS-1, depending on its concentrations, and also experimental metastasis with maximal inhibition at 0.5 or 2 ng/ml treatment compared to untreated controls. TGF-beta1, however, was ineffective for in vivo subcutaneous growth of MCS-1. These results indicated that TGF-beta1 might be an inhibitor of metastasis of mesenchymal chondrosarcomas including other types of non-epitherial cartilage or bone formation tumors.

[Experimental pulmonary metastasis of Chinese hamster mesenchymal chondrosarcoma cell lines].

Pulmonary metastases of chinese hamster mesenchymal chondrosarcoma cell lines, MCS-1 (undifferentiated type) and MCS-8 (differentiated type), were examined by intravenous transplantation into athymic nude mice. The incidence of pulmonary metastasis of MCS-1 was 100% and that of MCS-8 was 33% at the 23rd day after transfer. Mean number of metastatic nodules in the lung was 41 in the former and only 3 in the latter. Mean survival time of mice with MCS-1 injection (5 x 10(4) cells) was 27 days and that with MCS-8 (5 x 10(4) cells) was 48 days after transfer. At the 42nd day after transfer of MCS-8, however, the incidence of pulmonary metastasis was 100%. These data suggest that the tumor growth rate in the metastatic lesion, as well as the affinity to the target organ, is very important for evaluation of experimental metastasis.

Mouse Ly-gene haplotypes and subcutaneous regression of Ehrlich ascites tumor.

Ehrlich ascites tumor cells (EAT cells) lack most cell surface H-2 antigens and grow in any mouse strain following intraperitoneal injection of 10(5) cells. With subcutaneous injection of 2 x 10(7) cells, however, EAT regressed in C57BL/10 (H-2b), BALB/c (H-2d), NZB (H-2d), A (H-2a) and SJL (H-2s) but progressed and formed a solid tumor in DBA/2 (H-2d), C3H (H-2k), AKR (H-2k), CBA (H-2k) and DBA/1 (H-2q). By genotype analysis, Ly haplotypes of EAT-regressive mouse strains were found to have Ly-1.2 and Ly-2.2 as common features, while EAT-progressive mouse strains had Ly-1.1 (except AKR) and Ly-2.1.

Nephrotoxic serum nephritis in nude rats: the roles of host immune reactions in the accelerated type.

By immunization with rabbit immunoglobulins and the injection of a subnephritogenic dose of rabbit nephrotoxic serum (NTS), accelerated-type nephrotoxic serum nephritis (NTN) was induced in heterozygous (rnu/+) rats but not in athymic nude (rnu/rnu) rats. By transferring rat antibody against rabbit immunoglobulins, marked proteinuria was induced also in nude rats (202.0 +/- 98.4 mg/day on day 3) as in rnu/+ rats (122.6 +/- 35.3 mg/day on day 3). No marked differences in histological findings could be found between both groups. The most marked increase in the number of intraglomerular infiltrating cells was observed in heterozygous rats indicating that the presence of thymus-derived cells leads to the accumulation of more cells in glomeruli. We conclude that humoral immunity alone is enough to accelerate the pathogenic mechanism which induces glomerular injury with heavy proteinuria in this model.

Tumor dormancy and the effect of selected drugs on the tumor-dormant state.

An animal model for studying tumor dormancy was established by two-way selection of tumor-progressive and nonprogressive (tumor-dormant state) ddY mice in the same basal stock. In the tumor-progressive (prg) substrain of mice, Ehrlich ascites tumor cells (2 x 10(7)) subcutaneously inoculated into the dorsal skin formed a progressive solid tumor. In the tumor-dormant substrain (drm) of mice, the same tumor cells did not grow at all but formed a small caked nodule (1 to 3 mm in diameter) within 1 week. Some of the tumor cells persisted in the nodule at least 3 months without apparent mitotic figures. Such dormant tumor cells emerged and revealed outgrowth to overt solid tumors after they were transplanted into the dorsal skin of prg mice or into athymic mice (C57BL/6J nu/nu). To estimate the predisposition of drm mice to form tumors, the effect of certain drugs on the tumor-dormant state was examined. Estradiol, progesterone, dexamethasone, prostaglandin E2, Cyclosporin A, and collagenase all failed to promote the emergence of dormant tumor cells in drm mice.

A method to eliminate odor from recirculating air in the animal house.

A new method to eliminate odors from air recirculating in an animal house is described. The new system consists of an ozonizer and titania silica ozone decomposition catalyst in the terminal of a recirculating ventilation system. The principle underlying the elimination of odors is the oxidative reaction of malodorous components on the surface of the catalyst caused by coupling with ozonolysis. This method appeared superior in terms of its durability, efficiency and lack of resistance to air flow.

Establishment of clonal cell lines, with or without cartilage phenotypes, from a hamster mesenchymal chondrosarcoma.

A hamster mesenchymal chondrosarcoma was found in the soft tissues of the cheek pouch and has been successfully transplanted. The tumor was composed principally of two cell types: poorly differentiated mesenchymal cells and focal areas of cells in islands showing cartilaginous differentiation. The clonal cell lines, MCS-1 and MCS-8 which closely correspond to the respective cell types in the original tumor were also established. MCS-1 cells formed an undifferentiated sarcoma in the subcutaneous layer of nude mice and MCS-8 cells formed a chondrosarcoma of a common type.

Effects of antibody to 5 S-RNA-binding protein on protein synthesis in Artemia salina ribosomes.

The effects of an affinity-purified polyclonal antibody to Artemia salina ribosomal protein L5 on protein synthesis in vitro were examined. The antibody interacted with 60 S subunits more strongly than with 80 S ribosomes, and inhibited reassociation of ribosomal subunits to some extent at 5 mM-Mg2+ but not at 10 mM. Polyphenylalanine synthesis in vitro at 10 mM-Mg2+ was significantly inhibited, especially when the antibody was first preincubated with 60 S subunits prior to the assay. The incorporation of amino acid directed by globin mRNA was inhibited only when the preincubation with 60 S subunits was carried out. On the other hand, no effect was detected on elongation factor 2- and 60 S subunit-dependent uncoupled GTPase activity. These results suggest that L5 is probably located at or near the subunit interface and may play an important role in protein synthesis.

Assay of nucleoside-diphosphate kinase activity by the coupled nucleotidyltransferase.

A method coupled with Escherichia coli RNA polymerase is described for assaying nucleoside-diphosphate kinase activity. The principle of the procedure is indirect but allows processing of a large number of samples by employing filter-paper disk techniques.

Possible regulation by nucleosidediphosphate kinase involvement of the synthesis of tRNA 3'-terminal -pCpCpA in mammalian cells.

When the cytosol of Ehrlich ascites tumor cells was fractionated by chromatofocusing in the pH range of 9 to 6, two active peaks (I and II) of tRNA nucleotidyltransferase were obtained. Fraction I was a multiple complex with a high molecular weight (M.W. greater than 300K) and fraction II comprised components derived from fraction I. Fraction II was separated into tRNA nucleotidyltransferase (M.W., ca. 46,000) and nucleosidediphosphate kinase (M.W., ca. 74,000) by subsequent Sephacryl S-200 chromatography. The two enzymes appeared to be associated loosely with each other. Using the above fraction II or a mixture of the purified tRNA nucleotidyltransferase and nucleosidediphosphate kinase, it was possible to effectively synthesize the 3'-terminal -pCpCpA of tRNA in a reaction mixture containing [3H]-CDP plus XTP or [3H]ADP plus XTP as substrate. Among the XTPs investigated, dTTP was most effective. In addition, it was found that [3H]AMP + XTP also serves as a substrate. [14C]CMP plus XTP, however, was not utilized. From the antagonism of cold CDP against [3H]CTP, and that of cold ADP and AMP against [3H]ATP with the purified tRNA nucleotidyltransferase, the affinity of CDP to the enzyme was estimated to be 1/100 of that of CTP, while the affinities of ADP and AMP to the enzyme were 3 and 30 times higher, respectively, than that of ATP, suggesting that the subsite which binds ATP also binds ADP or AMP. The tRNA nucleotidyltransferase, which had bound ADP or AMP, could not completely synthesize the 3'-terminus of tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of vascular permeability-increasing component isolated from solid tumors and the effect of highly polymerized dextran sulfate on its activity.

Increase in vascular permeability is usually seen at the growth site of a tumor implant in murine dermal tissue. Increased vascular permeability was inducible by the subcutaneous injection of a solid tumor extract rich in protein precipitable at 20-50% saturation of ammonium sulfate. The vascular permeability-increasing activity of the tumor extract was reducible in the presence of highly polymerized dextran sulfate (DS-500) which showed a strong anticomplementary activity, but not by other substances such as dextran sulfate with a low molecular weight, non-sulfated dextran, chondroitin sulfate or heparin. As the tumor extract includes gamma-globulins in aggregated or bound form and adsorbs complements, it is probable that the aggregated gamma-globulins increase vascular permeability by triggering the complement activation system in the skin. DS-500 might antagonize the process.

Growth experiment of Hantaan virus in A549 cells: an attempt to improve the immunofluorescent antibody technique for hemorrhagic fever with renal syndrome.

An assay method for the infectivity of Hantaan virus, a causative agent of HFRS (hemorrhagic fever with renal syndrome), was developed by the use of IFA (immunofluorescent antibody technique). With the aid of this method, the growth characteristics of Hantaan virus, 76-118 strain, were followed in A549 cells. At a maximal MOI (multiplicity of infection) of 1.6 VAIU (viral antigen-inducing units) per cell, the conventionally available value, plateau level potencies of the viral antigen and virus infectivity were attained at eight and ten days postinfection, respectively, and most of the infective virus produced accumulated in the culture fluids of infected cells. When infections were defined with MOI values in terms of VAIU per cell, development of the viral antigen was highly consistent and followed a given pattern of kinetics. Based on these findings, a protocol for preparation of the viral antigen in IFA was presented, wherein spot culture and FBS treatment were emphasized as effective procedures to minimize non-specific staining.

[Histochemical demonstration of proteins, lipids and polysaccharides in Japanese monkey oocytes].

A histochemical study of proteins, lipids and polysaccharides was carried out in the oocytes of Japanese monkey (Macaca fuscata) during development of follicles. There were a small number of protein granules reactive to acrolein-Schiff in the cytoplasm of oocytes from primordial, secondary and vesicular follicles, while there were no lipid droplets, granules of neutral polysaccharides or acid polysaccharides in the cytoplasm. Proteins reactive to acrolein-Schiff, neutral polysaccharides reactive to periodic acid-Schiff and acid polysaccharides stainable with alcian blue were observed in the zona pellucida of the oocytes of secondary and vesicular follicles. The zona pellucida contained sudanophilic lipids composed of neutral fats and lipoids, besides the proteins and polysaccharides.

Characterization of tumor-induced inflammation and the effect of some anti-inflammatory drugs on the increased vascular permeability.

The vascular bed in a murine dermal tissue responded to inoculated tumor cells by two-phased changes in the vascular permeability. The initial increase in the vascular permeability was seen in an early stage (1 to 3 day post tumor cells inoculation), and the inflammation was sensitive to glutathione (GSH). Glucocorticoids reduced the increased vascular permeability, but neither acetylsalicylic acid nor indomethacin did. The later vascular response was produced by a growing solid tumor in a continuous mode beginning at 5th to 10th day post inoculation. The degree of the increased vascular permeability in this chronic phase was in direct proportion to the wet weight of the solid tumor, and the inflammation was insensitive to glutathione. Glucocorticoids reduced the increased vascular permeability, but neither acetylsalicylic acid nor indomethacin did. The action of glucocorticoids on the tumor-induced vascular hyper-permeability was discussed in connection with a tumor factor possibly responsible for the vasoexudation.

[Evaluation of amputation techniques for the study of limb regeneration in the newt and toad].

A comparison between forelimb amputations through the radius-ulna and the humerus was made in the adult newt, Cynops pyrrhogaster pyrrhogaster and the young toad, Xenopus laevis. Newts regenerated their forelimbs after the amputations and the new parts resembled the old in structure and function. Toads regenerated their forelimbs incompletely after the amputations. The new parts lacked joints and fingers showing a simply elongate pattern. The effect of re-amputation of exposed bone on the regeneration was evaluated in the adult newt. A left forelimb was amputated through the humerus and the exposed bone which protruded from the cut surface due to the relationships of the soft tissues, was then re-amputated a few minutes later. A right forelimb of the same individual was simply amputated without re-amputation of the exposed bone. Newts regenerated both forelimbs simultaneously. The new parts resembled the old in structure and function.

Effect of nucleoside 5'-triphosphates on tRNA nucleotidyltransferase activity in cytoplasmic fractions of various types of mammalian cells.

The tRNA nucleotidyltransferase activity (3H-CMP incorporation into 3'-terminus of tRNApC) in cytoplasmic fractions of various types of cells such as Ehrlich ascites tumor cells, mouse liver and spleen cells, rat spleen, lymph node, and macrophages cells was found to be dependent on the concentrations of nucleoside 5'-triphosphates (ATP, GTP, UTP, dATP, dGTP, dCTP, and/or dTTP). The purified tRNA nucleotidyltransferase did not show such dependency. The dependency of the enzyme activity on nucleoside 5'triphosphates in the crude cytoplasmic fractions was possibly due to the presence of inhibitors which interfere with the repair system of defective 3'-termini of tRNA. Two kinds of inhibitors were distinguishable in the cytoplasmic fractions. One was unstable on heat treatment at 55 decrees C and showed ribonuclease activity for the tRNA 3'-terminus. The other which lacked ribonuclease activity was rather stable to the heat treatment and inhibited purified tRNA nucleotidyltransferase. The actions of both inhibitors were suppressed by nucleoside 5'-triphosphates.