Murine acute leukemia cell line with megakaryocytic differentiation (MK-8057) induced by whole-body irradiation in C3H/He mice: cytological properties and kinetics of its leukemic stem cells

Five cases of murine leukemia with megakaryocytic differentiation were observed among the 417 cases of radiation-induced leukemias which developed in 30% of C3H/HeMs mice exposed at 8 to 10 weeks to 0.5 to 5 gy total body irradiation. Cells from individual leukemic colonies in the spleen of the irradiated mice, and cells from colonies in methylcellulose (MC) culture in vitro, derived from one of these leukemias, MK-8057, were injected into mice; both types of cells caused the deaths of the recipient mice by inducing the same type of leukemia. MK-8057 can be maintained in Dexter-type liquid culture with a feeder layer of irradiated bone marrow cells. There was a linear reciprocal relationship between the increasing number of MK-8057 cells injected versus the survival of the recipient mice. A reciprocal relationship also was seen between an increasing number of leukemic stem cells, corresponding to the number of MK-8057 cells, and the survival of mice injected with MK-8057. Giant nuclear megakaryocytes developed during the course of colony growth in the spleen as they did in the MC culture. Such megakaryocytes were acetylcholinesterase positive, whereas leukemic cells in the peripheral blood showed no sign of platelet production nor of a positive reaction to acetylcholinesterase. Cells maintained in culture were entirely positive in platelet glycoprotein IIb/IIIa when anti-human antibody was used. The larger cells in a splenic cell suspension derived from a moribund mouse were separated and enriched by velocity sedimentation using centrifugal elutriation (CE), and then subjected to flow cytometry using propidium iodide staining. Cells with up to 32N-DNA content were detected. After separating MK-8057 by counter-flow CE, the larger cell fraction (mode at 540 microns3) produced more leukemic colonies when injected into irradiated mice than did the small cell fraction (mode at 240 microns3). A higher percent of the larger cell fraction (61.9%) was killed by the addition of tritiated thymidine cytocide than in the smaller cell fraction (14.9%). Thus, the smaller cell fraction is considered to have more leukemic spleen colony-forming units (L-CFU-s) in the resting state.     

Mechanism of insulin resistance in the post receptor events in sheep: 3-O-methylglucose transport in ovine adipocytes

A severe resistance to the stimulatory action of insulin on glucose metabolism has been shown in ruminant adipose tissue or isolated adipocytes as compared to that of rats. To elucidate the mechanism of insulin resistance in ruminants, we measured the stimulatory effect of insulin on 3-O-methylgulose transport and on intracellular glucose metabolism in isolated adipocytes from sheep and rats. At a glucose concentration (0.1 mM) where transport is thought to be rate-limiting for metabolism, lipogenesis from [U-14C]glucose by ovine adipocytes was markedly less than by rat adipocytes in both the basal state and at all insulin concentrations. The responsiveness to insulin assessed by percent increase above basal was reduced to about 15% of that in rat adipocytes, but the insulin sensitivity was similar, because the insulin concentration giving half-maximal stimulation, ED50, did not differ significantly between ovine and rat adipocytes. The maximal insulin-stimulated 3-O-methylglucose transport in ovine adipocytes per cell was less than 20% of that in rat adipocytes, with a significant lowering in basal rates of transport. However, when data was expressed per 3-O-methylglucose equilibrium space no significant differences were found between ovine and rat in the basal transport rates, but a lowered ability of insulin to stimulate glucose transport was still seen in ovine adipocytes. The dose-response curve for glucose transport was slightly shifted to the right in ovine adipocytes compared to rat adipocytes, indicating a small decrease in insulin sensitivity. The decrease in glucose transport was due to 60% reduction in the maximum velocity in the insulin--stimulated state, with no change in the Km.     

Synthesis and structural stability of helichrome as an artificial hemeproteins

A detailed procedure is described for the synthesis of helichrome, which is the first successful example of polypeptide-based artificial hemeprotein. The segment synthesis-condensation approach used for the assembly of small proteins has proven to be extremely useful for protein mimetics as well. The final deprotection was performed using the TMSOTf-thioanisole method instead of the less-convenient hydrogen fluoride method. The unfolding transition of the alpha-helical conformation of helichrome induced by guanidine hydrochloride was studied to understand the stability and dynamics of the folded structure. The resulting parameters (C0.5 = 5.2 M and delta GH2O = -4.4 kcal mol-1) characterizing helichrome denaturation were comparable to that of native globular proteins.     

Observation of the proximal portions of axons of anterior-horn cells in the human spinal cord

The proximal portions of axons of large anterior-horn cells were investigated in the lumbar cords of 10 normal human autopsy cases. Light-microscopically, 81 myelinated axons were observed to be connected with the cell body. Of the 81 axons, 78 emanated from the cell body and 3 others originated in the proximal part of primary dendrites. As for normal-looking neurons (n = 77), the length of the axon hillock plus initial segment was 64.0 +/- 12.3 microns (average +/- SEM), ranging from 47.5 to 110.0 microns, while the diameter of the thinnest portion of the initial segment was 2.40 +/- 0.30 microns (average +/- SEM), ranging from 1.32 to 3.92 microns. Electron-microscopically, the predominant organelles of the axon hillock were mitochondria, neurofilaments which merged into the axon and occasional granular endoplasmic reticulum. A few synaptic boutons were found on the surface of the axon hillock. The cell membrane of the initial segment consisted of a layer of electron-dense material (undercoating). The cytoplasm contained many neurofilaments, running parallel to the longitudinal axis of the initial segment. Among the neurofilaments, lysosomes, smooth endoplasmic reticulum, dense bodies and vesicular profiles as well as mitochondria were seen. At the beginning of the myelin sheath, the axoplasm contained mitochondria, many neurofilaments and occasional lysosomes.     

Purification and properties of an endo-1,4-beta-glucanase translated from a Clostridium josui gene in Escherichia coli.

An endoglucanase encoded by a gene of Clostridium josui was expressed in Escherichia coli and purified. The homogeneous enzyme, with a molecular weight of 39,000, revealed maximum endoglucanase activity at pH 7.2 to 7.5 and a temperature of 65 to 70 degrees C. The enzyme was stable at a temperature lower than 45 degrees C (the growth temperature of the bacterium) in the range of pH 4.5 to 9.0. The amino acid sequence of the enzyme at the N terminus was Val-Glu-Glu-Asp-Ser-Ser-His-Leu-Ile-Thr-Asn-Gln-Ala-Lys-Lys----. The enzyme hydrolyzed cellotetraose to cellobiose and then transferred cellobiose to the residual cellotetraose. The resulting cellohexaose was cleaved to cellotriose.     

Interrelationship between serum muscle enzymes and a low T3 in anorexia nervosa

To determine the interrelationship between muscle dysfunction and a low T3 state, both seen in anorexia nervosa, we studied the relationship between the degree of muscle involvement, as assessed by the circulating concentration of the three muscle indicators (CPK, GOT and LDH), and serum T3 in thirty-three patients when they were admitted to the hospital. We also studied the malnutritional state, as assessed by their body weight or serum GH, serum potassium and the degree of hyperactivity exhibited. Additionally, another twelve patients were studied in order to explore the mounding phenomenon which is typically elicited in hypothyroidism. The logarithms of serum CPK and GOT correlated only with the serum T3 concentration (r = -0.35, p less than 0.05; r = -0.41, p less than 0.05; respectively). The logarithm of serum LDH highly correlated with serum T3, the percentage of ideal body weight, and the logarithm of serum GH (r = -0.55, p less than 0.01; r = -0.66, p less than 0.001; r = 0.43, p less than 0.05; respectively). The mounding phenomenon was elicited in ten out of twelve patients. In conclusion, it was implied that a low T3 state was associated with an increase in serum muscle indicators and thus with muscle dysfunction encountered in anorexia nervosa.     

Localization of aldosterone-producing adenoma: venous sampling in primary aldosteronism

Adrenal venous sampling of blood was performed for nine patients with aldosterone-producing adenoma (APA). Measurement of adrenal venous aldosterone is useful for localization of APA but difficult, because catheterization of the right adrenal vein is not easy, and the blood is diluted by nonadrenal flow. To solve these problems, levels of aldosterone (A; ng/dl) and cortisol (C; micrograms/dl) were measured in samples from the left adrenal vein (LAV) and the inferior vena cava (IVC), and the LAV A/C and (LAV A/C)/(IVC A/C) ratios were calculated. These ratios were also obtained for 16 patients with essential hypertension. The adenoma could be localized in three of the nine cases by the measurement of aldosterone alone, but the use of a LAV A/C ratio greater than 5 x 10(-3) and a (LAV A/C)/(IVA A/C) ratio less than 1.0 as criteria separated the patients into those with a left APA, right APA, or essential hypertension. Consequently, adrenal venous sampling and the calculation of these ratios enables preoperative localization of APA with more accuracy, especially when the tumor is small or the result of CT and adrenal scintigraphy is not consistent.     

The late-replicating X chromosome in digynous mouse triploid embryos

Using BrdU-labeling and acridine orange staining, the behavior of X-chromosome replication was studied in 28 XXX and 19 XXY digynous mouse triploids. In some of these the paternal and maternal X chromosome could by cytologically distinguished. Such embryos were obtained by mating chromosomally normal females with males carrying Cattanach's X chromosome which contains an autosomal insertion that substantially increases the length of this chromosome. In the XXX triploids there were two distinct cell lines, one with two late-replicating X chromosomes, and the other with only one late-replicating X. The XXY triploids were also composed of two cell populations, one with a single late-replicating X and the other with no late replicating X chromosome. Assuming that the late-replicating X is genetically inactive, in both XXX and XXY triploids, cells from the embryonic region tended to have only one active X chromosome, whereas those from the extra-embryonic membranes tended to have two active X chromosomes. The single active X chromosome was either paternal or maternal in origin, but two active X chromosomes were overwhelmingly maternal in origin, suggesting paternal X-inactivation in extra-embryonic tissues.     

Simultaneous determination of triazolo-benzophenone [2′,5-dichloro-2-(3-glycylaminomethyl-5-methyl-4H-1,2,4-triazol-4-yl)-benzophenone] and its major blood metabolite, triazolam, in monkey plasma by electron-capture gas—liquid chromatography

A gas—liquid chromatographic method for the simultaneous determination of triazolobenzophenone [2′,5-dichloro-2-(3-glycylaminomethyl-5-methyl-4H-1,2,4-triazol-4-yl)-benzophenone, TB] and its major blood metabolite, triazolam, 8-chloro-6-(o-chlorophenyl)-1-methyl-4H-s-triazolo[4,3-a][1,4]benzodiazepine (TZ), in monkey plasma was developed. Decomposition of TB was observed during gas—liquid chromatography. In alkaline medium, TB in plasma was submitted to ring closure reaction to yield triazolo-aminoquinoline, [4-amino-7-chloro-5-(2-chlorophenyl)-1-methyl-4H-s-triazolo[4,3-a]quinoline (TAQ), while TZ remained unaffected, and TAQ and TZ in the benzene extract were assayed by gas—liquid chromatography using an electron-capture detector. The concentration ranges studied were from 5 to 40 ng of TB per 0.5 ml of plasma and from 2 to 20 ng of TZ per 0.5 ml of plasma. This method could be applied to the determination of the plasma levels of TB and TZ in monkeys following intravenous administration of a single 0.2 mg/kg dose of TB.     

Induction of sister-chromatid exchanges by indirect mutagens/carcinogens in cultured rat hepatoma and esophageal tumor cells and in Chinese hamster Don cells co-cultivated with rat cells

2 rat cell lines originated from ascites hepatoma AH66-B and esophageal tumor R1 were examined for their inducibility of sister-chromatid exchanges (SCEs) after treatment with 14 kinds of indirect mutagens/carcinogens, including 6 amine derivatives, 4 azo compounds, 3 aromatic hydrocarbons and 1 steroid. Of the 14 chemicals tested, 2-acetylaminofluorene (AAF), butylbutanolnitrosamine (BBN), dimethylnitrosamine (DMN), cyclophosphamide (CP), urethane, 2-methyl-4-dimethylaminoazobenzene (2-MeDAB), 3′-methyl-4-dimethylaminoazobenzene (3′-MeDAB), 4-o-tolylazo-o-toluidine (4-TT), benzo[a]pyrene (BP), 7,12-dimethyl-benz[a]anthracene (DMBA) and diethylstilbestrol (DES) were estimated to be effective inducers of SCEs in AH66-B and/or R1 cells, without the use of exogenous activating systems. Cell-mediated SCE tests with 6 selected chemicals, CP, 2-MeDAB, 4-TT, BP, DMBA and DES, showed a significant increase of SCEs in Chinese hamster Don-6 cells co-cultivated with AH66-B or R1 cells, depending on the number and sensitivity of AH66-B or R1 cells, as well as on the dose of chemicals tested, whereas singly cultured Don-6 cells were much less sensitive or almost insensitive to these chemicals. The above findings suggest that AH66-B and R1 cells may retain metabolic activities to convert a wide range of indirect mutagens/carcinogens into their active forms to induce SCEs, and that these cell lines provide simple and reliable screening systems in vitro, including the cell-mediated SCE assay, for detection of genotoxic agents, without the use of exogenous activation systems.