CAM-cytarabine, aclarubicin plus macrophage colony-stimulating factor in the treatment of acute myelogenous leukemia with trilineage dysplasia: usefulness of in vitro apoptosis in leukemic cells.

A 67-year-old woman was treated for acute myelogenous leukemia with trilineage dysplasia (AML-TLD) by combination chemotherapy with cytarabine, aclarubicin plus macrophage colony-stimulating factor (M-CSF) (referred to as CAM therapy). Complete remission was achieved after two courses of CAM therapy. After coculture of her bone marrow mononuclear cells with M-CSF in vitro, differentiation of leukemic cells into macrophages with apoptotis was observed. This case confirms an earlier report that an effect of M-CSF inducible by differentiation with apoptotic phenomena, against human leukemic cells was shown both in vitro and in vivo when achieving complete remission.

Identification of functional domains of the human thrombopoietin receptor required for growth and differentiation of megakaryocytic cells.

To identify the functional domains of the human thrombopoietin (TPO) receptor essential for proliferation and megakaryocytic differentiation, we introduced human wild type c-mpl cDNA and deletion mutants of c-mpl cDNA into the human erythropoietin (EPO)-dependent cell line UT-7/EPO that does not express endogenous c-Mpl. TPO induced the proliferation and megakaryocytic differentiation of UT-7/EPO expressing wild type c-Mpl, as evidenced by increased levels of the CD41 antigen specific for cells of the megakaryocytic lineage and by changes in morphology. Mutational analysis of the cytoplasmic domain of c-Mpl identified four functional regions: (a) two C-terminal regions (amino acids 575-586 and 615-630) containing a domain essential for cell proliferation and megakaryocytic differentiation but not for DNA synthesis; (b) a region (amino acids 587-614) containing a negative domain for TPO-induced cell proliferation and megakaryocytic differentiation; and (c) a region (amino acids 565-574) including a box2 motif that is required for DNA synthesis. These deletion mutants will provide useful materials for analyzing the signals specific for TPO-induced proliferation and megakaryocytic differentiation.

Effects of the contents of dietary crude protein on growth rate and NK activity.

The relationship between body weight gain and food intake was examined using 3 strains of mice, C3H/He, C57BL/6 and A/J mice, that were fed 4 kinds of diets differing in crude protein contents. Body weight gain of the mice fed the diet containing 3% protein was extremely small, while that of the mice fed the diet containing 40% protein was smaller than that of the mice fed the diet containing 10% or 20% protein. Mice were shown to possess a characteristic ability to intake an almost constant amount of calories irrespective of crude protein contents in diets, with some exceptions. These experimental results indicate that crude protein intake plays an important role for body weight gain. It was also assumed that energy consumption in C57BL/6 mice fed the 40% protein diet was accelerated because of excessive protein intake. Normal NK activity and antibody responses to SRBC were maintained at the age of 12 weeks in all the strains of mice fed the different diets.

Effects of feeding a protein-free diet on the mitochondrial respiration and on phospholipid fatty acids of mitochondrial and microsomal membranes in the rat liver.

In our previous report, we have described morphological changes in hepatocytes, e.g., enlargement of mitochondria and a change in the lamellar formation of rough endoplasmic reticulum, produced by short-term feeding of a protein-free diet to rats. In order to examine whether or not these morphological changes in the subcellular organella are accompanied by any functional and compositional changes, we measured the P/O ratio and respiratory rate of mitochondria, and the phospholipid fatty acids of the mitochondrial and microsomal membranes in the liver of rats fed a protein-free diet for a short period. Feeding rats the protein-free diet for 4 days or 27-28 days had no effect on the rate of hepatic mitochondrial oxygen consumption (State 3 respiration). The diet significantly decreased the P/O ratio on the 4th day, but did not affect it on the 27-28th days. The decreased P/O ratio observed on the 4th day returned to the control level after overnight refeeding of a 20% casein diet. Main compositional changes induced by feeding rats the protein-free diet for 2 days were significant decreases in the phospholipid/protein ratios of the total liver and mitochondrial inner membrane, a tendency of an increase in the ratio of phosphatidylcholine (PC) to total phospholipids in the mitochondrial outer membrane, a significant decrease and a tendency of decrease in the arachidonate/linoleate ratio in the phosphatidylethanolamine (PE) and PC, respectively, in the mitochondrial outer membrane. Some of these results were discussed in relation to the morphological changes in mitochondria and rough endoplasmic reticulum produced by short-term feeding of the protein-free diet which we previously reported.

[Ammonia in the intestinal contents from germfree rats].

The values of pH and the concentrations of nitrogen (N) and ammonia in the middle part of the small intestinal and cecal contents of germfree (GF) and conventionalized (CVZ) seven-week-old rats were compared. The pH of the small intestinal and cecal contents of GF rats was higher than that of CVZ rats. There was no difference in total N per fresh weight in contents from the middle part of the small intestine between GF and CVZ, whereas total N per fresh weight of the cecal contents was higher in CVZ than in GF rats. The ammonia concentrations per fresh weight or per total N in the intestinal and cecal contents of CVZ rats were higher than those of GF rats.

Influence of diets low in protein or lysine on the cecal flora of rats with reference to cecal contents.

To determine the effect of a certain diet on the intestinal flora of rats, the cecal flora of rats fed a low protein or low lysine diet was examined. Total counts of bacteria in the cecal contents of rats given the four kinds of diet (a normal protein diet, a low protein diet, a normal lysine diet and a low lysine diet) were not significantly different. The counts of Streptococcus, Enterobacteriaceae, and Clostridium perfringens in the cecal contents from rats fed the low protein diet were significantly lower than those from rats fed the normal protein diet. The count of Lactobacillus in the cecal contents of the low protein group were significantly lower than those of the control. But no significant difference was found between the levels of most of the free amino acids in the cecal contents of the low lysine group and those of the control group.

Influence of diets low in protein or lysine on the intestinal flora of chicks with reference to cecal contents.

To determine the effect of a certain diet on the intestinal flora of chicks, the cecal flora of chicks fed on a low protein or low lysine diet was examined. The cecal flora of chicks fed on the low protein diet was similar to that of chicks fed on a normal protein diet, but the total count of bacteria, Eubacterium and Enterobacteriaceae in the cecal content of chicks fed on the low lysine diet containing a formulated amino acid mixture minus lysine was significantly lower than that of chicks fed on the control diet. The total count of Lactobacillus in the cecum was remarkably reduced by feeding the amino acid diet, especially the low lysine diet. Levels of most free amino acids in the cecal contents of the low protein group were significantly lower than those of the control. Lysine, leucine, phenylalanine, methionine, histidine, glycine and tyrosine of the cecal contents in the low lysine group were significantly lower than those of the control group.

A comparison of the gastrointestinal tract in germ-free and conventional mice fed an amino acid mixture or purified whole-egg protein.

An amino acid mixture diet(AA) simulated with purified whole-egg protein and a purified whole-egg protein diet(WE) were given to ICR strain male germ-free(GF) and conventional(CV) mice for three weeks from five to eight weeks of age. All mice were killed at eight weeks of age and the gastrointestinal tracts were removed. The lengths of the small intestine were measured and six parts, i.e., the stomach; upper, middle and lower parts of the small intestine; cecum; and colon and rectum were separated, and each part (with contents) was weighed immediately. The contents were removed from each part by washing with distilled water. For the gut without contents, only total nitrogen(TN) was estimated and for the gut contents, TN, protein nitrogen(PN) and water-insoluble nitrogen(WIN) were estimated. The fresh weight of cecum with contents per 100 g of body weight of GF mice fed on AA diet, 2.07 +/- 0.11 g (mean +/- SE), was lighter than that of GF mice fed on WE diet, which was 4.51 +/- 0.28 g. The weight and length of the small intestine of AA diet groups were smaller than those of WE diet group. TN and PN(mg) in whole gut contents per 100 g of body weight in the WE diet group were higher than those in the AA diet group and high TN and PN were observed in GF mice. The PN per TN and WIN per PN of gut contents were affected more by the presence of the flora than by the difference in diets, and CV mice showed high values. The TN of the intestinal tract excluding contents showed no differences in GF and CV mice fed on either diet.

Influence of intestinal microbes on heat production in germ-free, gnotobiotic and conventional mice.

To observe the influence of intestinal microbes on energy metabolism, male ICR strain germ-free (GF) mice, gnotobiotic (GB) mice, produced from GF mice monocontaminated with Staphylococcus epidermidis (Staph.) at three weeks of age, and conventional (CV) mice were used. Metabolizable energy, heat production and net energy (gain) per mouse were measured for three weeks when the mice were given 5 Mrad 60Co irradiated purified whole-egg protein diet from 5 to 8 weeks of age. With respect to metabolizable energy and heat production per mouse or per kg of metabolic body size for three weeks, CV mice gave the highest values, followed by GB and GF mice. GV mice showed the highest net energy (gain) followed by GF mice, with GB mice having the lowest value.

Isolation of diacylglyceryl-2-aminoethylphosphonate from bovine liver.

Diacylglyceryl-2-aminoethylphosphonate was isolated from bovine liver by a combination of silicic acid column and silica gel thin-layer chromatographic techniques, and was identified from the results of elementary analysis, the infrared spectrum, chemical properties, and chromatographic behavior. This is the first isolation of a lipid-bound form of ciliatine in mammals.

A new bile acid conjugate, ciliatocholic acid, from bovine gall bladder bile.

This study was carried out to investigate the occurrence of ciliatocholic acid in bovine gall bladder bile. Ciliatocholic acid was synthesized according to the method described by Bergstrôm and Norman for the synthesis of taurocholic acid. Elemental analysis, melting point, and the infrared spectrum of this substance were determined. An isolation procedure for ciliatocholic acid was established by stepwise elution with an HCl-ethanol solvent system using a Dowex-1 anion exchange resin column chromatographic technique. Ciliatocholic acid amounting to 158 mug (as ciliatine) per 100 ml of gall bladder bile was found in the fraction eluted with 0.01 N HCl in 50% ethanol. This coumpound was purified by preparative thin-layer chromatography and confirmed to be ciliatocholic acid from the hydrolytic stability, phosphorus determination, and chromatographic behavior. Thus, bovine gall bladder bile contains a small amount of ciliatocholic acid.