Establishment from mouse growth cartilage of clonal cell lines with responsiveness to parathyroid hormone, alkaline phosphatase activity, and ability to produce an endothelial cell growth inhibitor.

Three clonal cell lines with differences in responsiveness to parathyroid hormone (PTH), alkaline phosphatase activity, and ability to produce an endothelial cell growth inhibitor(s) during more than 3 years, more than 58 passages, in culture were established from growth cartilage (GC) of mouse ribs. In sparse cultures the three clonal cell lines, MGC/T1.4, MGC/T1.17, and MGC/T1.18, all showed fibroblast-like morphology. However, as they became confluent, MGC/T1.4 cells became polygonal and then multilayered. MGC/T1.18 cells also became polygonal, but showed contact inhibition. MGC/T1.17 cells remained fibroblastic in confluent cultures and formed nodules when cultured for more than 7 days after they became confluent. These nodules calcified in the presence of beta-glycerophosphate. Glycosaminoglycan (GAG) synthesis in the parent uncloned line, MGC/T1 cells, at early passages was about 50-75% of that of primary cultures of mouse GC cells. The GAG syntheses in the three clonal lines were much lower than that of primary cultures of GC cells. Moreover, the sizes of proteoglycan monomers synthesized by these cells were not the same as that of cartilage-specific proteoglycan. The three clonal lines mainly synthesized type I collagen. PTH increased the intracellular cyclic AMP level in MGC/T1, MGC/T1.4, T1.17, and T1.18 cells: their maximal levels, observed after 2 minutes, were, respectively, about 160, 150, 70, and 200 times that of controls. The activity of alkaline phosphatase in MGC/T1.17 cells was higher than that in primary cultures of mouse GC cells, whereas those in MGC/T1 and T1.4 cells were comparable with that of GC cells, and that in MGC/T1.18 was lower. The three clonal lines, and especially MGC/T1.4, secreted a heat-stable, nondializable growth inhibitor(s) of endothelial cells into the culture medium. Because of their different properties, these cell lines should be useful for studies on endochondral ossification, the actions of PTH on skeletal cells, and anti-angiogenesis factors.

Cephalic phase response of pancreatic exocrine secretion in conscious dogs.

The effects of three taste solutions on the cephalic phase of pancreatic secretion were studied in conscious dogs. Male beagle dogs weighing 9-11 kg were prepared with gastric and duodenal fistulas. Gustatory receptors were stimulated for 5 min with 100 ml of 0.5% agar solutions containing 0.05, 0.12, and 0.3 M of either sodium chloride, sucrose, or monosodium glutamate (MSG). Pancreatic juice was collected every 5 min before and after stimulation, and volume flow and protein output were measured. Pancreatic secretory responses were found to vary with the type of taste stimulus. Sucrose was a better stimulus than MSG for both protein output and volume flow. Taste stimulation with sodium chloride produced a lower pancreatic response than those with sucrose or MSG.

[Suppressive effect of lentinan on pulmonary metastases in murine tumors].

Lentinan has been shown to have a suppressive effect on the natural pulmonary metastasis of Lewis lung carcinoma, though its effect seems to depend on the timing of the administration. Administration of lentinan after the amputation of tumor was found to be more effective than before administration. Additionally, lentinan also has shown a marked suppressive effect on the pulmonary metastasis of MC-CS-1 fibrosarcoma, a high sensitive tumor for lentinan in any administration examined. These date suggest the usefulness of lentinan in suppressing tumor metastasis.

[Age-dependent anti-tumor activity of lentinan in mice].

During the course of aging, the weight percentage of organs such as kidney to body weight remain almost unchanged, whereas the weight of the thymus decreases significantly. The antitumor activity of Lentinan is constant in mice younger than 1 year (52 weeks). From 52 weeks, aging has a suppressive effect on antitumor activity. Even in aged mice, administration of Lentinan was shown to be effective, and better results were obtained through chronic administration, even with a smaller dose. Lentinan enhanced delayed cutaneous hypersensitivity in aged mice similarly to that in young mice. Thymus homogenate showed synergism with Lentinan in 2-year-old mice for suppression of tumor growth, although thymus homogenate alone showed no effect. These results show that Lentinan is effective as an anticancer immunopotentiator in aged animals in a similar way to young animals.

Effects of ornithine or arginine administration on serum amino acid levels.

In healthy humans after overnight fasting, an oral administration of ornithine induced a new steady state: an accumulation of serum alanine and proline, a decrease in serum valine concentration, transient reductions in serum urea and urinary urea contents, and then an increased urea excretion. On the other hand, an oral administration of arginine resulted in an anabolic state: decreases in serum leucine and isoleucine concentrations, reductions in serum glucose and free fatty acid contents and a rapid increase in serum insulin level. It was assumed that the effect of ornithine administration may be exerted through an activation of hepatic System A transport and that of arginine is an insulin-mediated action.

A factor in conditioned medium of rabbit costal chondrocytes inhibits the proliferation of cultured endothelial cells and angiogenesis induced by B16 melanoma: its relation with cartilage-derived anti-tumor factor (CATF).

Serum-free medium conditioned by exposure to rabbit costal chondrocytes in culture inhibited the proliferation and DNA synthesis of bovine pulmonary endothelial cells in culture. The factor in conditioned medium did not inhibit DNA synthesis in B16 melanoma cells, L1210 cells or 3T3 fibroblasts in culture, but it inhibited angiogenesis in the chorioallantoic membrane of chick embryos induced by B16 melanoma and growth of the tumor transplanted onto the membrane. These findings strongly suggest that rabbit costal chondrocytes produce an anti-angiogenesis factor that is similar to cartilage-derived anti-tumor factor (CATF).

An anabolic state in the heart induced by arginine intubation.

Using rat heart perfusion, we found that an anabolic state can be induced with a medium which includes glucose, carnitine, branched-chain amino acids and arginine after arginine intubation at a dose of 250 mg/kg body weight. It showed diminished levels of glutamine, glutamate, branched-chain oxoacids and phenylalanine (a marker of heart protein metabolism) release, reflecting anabolic changes occurring in the myocardium. While ornithine intubation caused a catabolic state in which the release of alanine and glutamate was increased but phenylalanine release was unchanged. This anabolic state may be a useful model providing for myocardial protection.

An improved procedure of chronic intravenous infusion in unrestrained rats.

A continuous infusion technique for rats is described which is a significant improvement upon existing procedures with regard to the degree of physiological and psychological stress it provokes. The system was assembled with a head attachment, a practical swivel and a special metabolic cage, permitting long-term tube feeding of unrestrained rats.

Cartilage-derived anti-tumor factor (CATF) inhibits the proliferation of endothelial cells in culture.

Previous studies showed that a cartilage-derived anti-tumor factor (CATF), which was extracted from bovine cartilage with 1 M guanidine hydrochloride, inhibited the growth of solid sarcoma 180, but not the growth of ascites tumors of sarcoma 180 [Suzuki, F. et al. Jpn. J. Bone Metab., 2, 231-235 (1984)]. This study showed that CATF inhibited the proliferation and DNA synthesis of endothelial cells isolated from bovine pulmonary artery, but not the proliferations of sarcoma 180, L1210 and L cells. These results suggest that CATF has anti-angiogenic properties, thereby inhibiting the growth of solid tumors.

A new orally active hypotensive peptide, N-(dibenzyloxyphosphinoyl)-L-alanyl-L-prolyl-L-proline (PAPP).

The antihypertensive effects and mechanisms of a newly developed orally active tripeptide, N-(dibenzyloxyphosphinoyl)-L-alanyl-L-prolyl-L-proline (PAPP), were investigated. When PAPP (1-30 mg/kg orally) was administered to spontaneously hypertensive rats (SHR), systolic blood pressure (SBP) reached a maximum reduction after 8 h and this effect lasted over 24 h. In two-kidney, one clip hypertensive rats (2KIC rats) and DOCA-salt hypertensive rats, PAPP also reduced SBP. In normotensive Wistar rats, however, PAPP did not have a hypotensive effect. PAPP showed low toxicity in Sprague-Dawley rats. The depressor response to bradykinin (BK) was potentiated, but the pressor response to angiotensin I (ANG I) was not inhibited by PAPP. PAPP significantly relaxed isolated SHR aortic strips treated with KC or norepinephrine. Angiotensin converting enzyme (ACE) inhibition by PAPP was relatively low in vivo and in vitro.

Marinactan, antitumor polysaccharide produced by marine bacteria.

Extracellular polysaccharides of marine bacteria were screened for their antitumor activity against sarcoma-180 solid tumor in mice. An active polysaccharide was purified and named marinactan. The producing microorganism has a typical marine bacterial nature requiring sea water for growth and was identified as Flavobacterium uliginosum. Marinactan is a novel heteroglycan consisting of glucose, mannose and fucose in a ratio of approximately 7:2:1. Marinactan, 10-50 mg mg/kg daily for 10 days i.p., produced 70-90% inhibition of the growth of solid sarcoma 180. Complete regression of the tumor was observed in some treated mice. Its administrations before and after tumor transplantation showed almost the same inhibitory effect. Marinactan prolonged markedly the survival period of mice bearing ascites sarcoma 180.

Antitumor activity of a new compound, ethyl O-[N-(p-carboxyphenyl)-carbamoyl]-mycophenolate, against various experimental tumors upon oral administration.

A newly synthesized mycophenolic acid (MPA) derivative, ethyl O-[N-(p-carboxyphenyl)-carbamoyl]-mycophenolate (CAM, NSC-297879D) was tested for antitumor activity, when given orally, against transplantable murine tumors. The compound was markedly effective against transplantable murine tumors. The compound was markedly effective against leukemia P388 and L1210, lymphoma L5178Y, mastocytoma P815 and sarcoma Meth-A, moderately effective against sarcoma-180, C3MC2 and BAMC1, Ehrlich carcinoma, Lewis lung carcinoma and melanoma B16 and marginally effective against hepatoma MH134. The antitumor effects were manifested not only in growth inhibitory effects on subcutaneously transplanted tumors but also in the prolongation of life span of mice int which the tumors had been inoculated intraperitoneally or subcutaneously. The growth of primary transplants of a mammary tumor which developed spontaneously in a C3H/He mouse was inhibited by consecutive administration of CAM frm the 34th day after the transplantation. Oral CAM was more potent than its mother compound, MPA, in the tumor models examined. These results indicate that orally administered CAM has a wide antitumor spectrum.

Comparison of the nutritive values of L-, DL- and D-tryptophan in the rat and chick.

Experiments were designed to compare the nutritive values of L-, DL- and D-tryptophan in rats and chicks. Growing rats and chicks were fed for 19 and 21 days, respectively, diets containing amino acid mixtures with graded levels of either L-, DL- or D-tryptophan so that the regression of weight gain or protein retention on tryptophan intake could be established. After the end of the experiments, plasma free L-tryptophan was estimated by a microbiological method. The nutritive values of DL- and D-tryptophan relative to the L-isomer were 47 and 21%, respectively in chicks and close to 100% in rats. In chicks, plasma free L-tryptophan concentration increased with the increase of L- and DL-tryptophan levels in the diet, but remained at a low level regardless of the D-tryptophan level in the diet. In rats, however, a good correlation was observed between plasma free L-tryptophan and tryptophan level in the diet.

Inversion of D-tryptophan to L-tryptophan and excretory patterns in the rat and chick.

The behavior of D-tryptophan in the blood plasma and the pattern of tryptophan excretion in the urine were studied in the rat and the chick. When D-tryptophan was administered orally to rats and chicks, both showed D-tryptophan in the plasma. Conversion of D-tryptophan to the L-isomer in the rat was found by an examination of plasma from the posterior vena cava and the portal vein, following stomach intubation of 100 micromoles D-tryptophan/100 g of body weight. The peak in the increase of L-tryptophan was approximately 150 nmoles/ml plasma when measured at 30 minutes after administration and the peak was 200 nmoles when measured 2 hours after administration. No conversion of D-tryptophan to the L-isomer was found in chicks. Under similar conditions, D-tryptophan was measured in the urine of rats and chicks. In rats the D-tryptophan excreted was at most 1% of the amount administered; while in chicks most of the D-tryptophan was excreted.

Protective effect of arginine, leucine and preinjection of insulin on glutamate neurotoxicity in mice.

The simultaneous intubation of 10-day-old mice with either 2.28 g/kg body weight arginine hydrochloride or 0.2 g/kg b.wt. leucine significantly reduced the number of necrotic neurons in the arcuate nucleus (AN) of the hypothalamus resulting from 2 g/kg b. wt. monosodium L-gultamate (MSG). The prior injection of 0.02 units per capita of insulin suppressed the MSG-induced lesions to an even great extent. These findings are believed to form a basis for the ineffectiveness of dietary MSG in producing hypothalamic lesions.

The influence of carrier of gustatory stimulation on the cephalic phase of canine pancreatic secretion.

The pancreatic secretory responses of dogs to various taste stimuli were examined in this study. Additionally, taste preferences were examined in 24-hour exposure tests to taste stimulus solutions as well as in short exposure tests to taste solutions mixed with commercial stock diet. The liquid and solid food preference tests produced quite different results. In dogs with cannulated gastric and duodenal fistulas, gustatory receptors were stimulated orally with 100 ml of taste stimulus solution (water 0.05 M monosodium glutamate (MSG), 0.05 M citric acid or 0.3 M sucrose) mixed with 25 g of a carrier (commercial stock diet, purified diet or cellulose). Pancreatic secretory responses to the taste stimuli varied with the type of carrier. Stock diet carrier was a better stimulant than the purified diet for both protein output and volume flow. Taste stimuli with a cellulose carrier did not produce any pancreatic response at all. The differences in responses to the different carriers were greater than the differences between taste stimuli when the same carrier was used. This experiment indicates that gustatory stimulation does influence the function of pancreatic secretion depending on the carriers used.