Schizophyllan (SPG)-treated macrophages and anti-tumor activities against syngeneic and allogeneic tumor cells

We tested anti-tumor activities of macrophages treated with a neutral polysaccharide, schizophyllan (SPG), against syngeneic and allogeneic tumor cell lines. SPG was a macrophage stimulant which was not mitogenic to lymphocytes. That made a sharp contrast with the data that Corynebacterium parvum, BCG, and muramyl dipeptide (MDF) were macrophage stimulants which had lymphocyte-activating properties. Treatment of SPG-treated PEC with Thy12 monoclonal antibody and guinea pig complement did not affect the capabilities of tumor-cell-growth suppression by the treated PEC. Thus, the effector cells were peritoneal adherent cells (macrophages morphologically) and effector-to-target contact seemed to be necessary for effective tumor-cell-growth inhibition, although contradictory data exist for this. Murine peritoneal adherent cells harvested 4 days after a single IP injection of SPG at a dose of 100 mg/kg body weight of mouse showed the most prominent cytostatic and cytotoxic activities against syngeneic and allogeneic tumor cells. The distribution of anti-tumor activity in macrophages of various sizes followed the same pattern as macrophages treated with C. Parvum, i.e., larger macrophages showed more remarkable anti-tumor activity. Crude nonadherent peritoneal cells incubated with SPG at a concentration of 10 micrograms/ml, 100 micrograms/ml, or 1 mg/ml did not secrete lymphokine that rendered macrophages cytotoxic, while ConA-treated nonadherent cells did so. Furthermore, spleen cells treated with SPG in vivo did not secrete macrophage-activating lymphokine in the presence of SPG. On the other hand, addition of 1 mg/ml of SPG-treated peritoneal adherent cells and bone-marrow-derived macrophages in vitro rendered them cytotoxic to a moderate degree. This implies that SPG may activate macrophages directly, allowing them to become cytotoxic in the peritoneal cavity. Lastly, SPG could induce production of II-1-like factor to a moderate degree. SPG, whose molecular structure is well elucidated, will provide us with a strong tool to analyze the mechanism of macrophage activation both in vitro and in vivo.     

Choline kinase and ethanolamine kinase are separate, soluble enzymes in rat liver.

Choline kinase and ethanolamine kinase are located in the cytosol from rat liver and have been copurified more than 500-fold by affinity chromatography [P. J. Brophy and D. E. Vance (1976) FEBS Lett. 62, 123-125]. Kinetic properties of the two activities were determined. Choline kinase had a Km for choline of 0.033 mM and ethanolamine was a competitive inhibitor (Ki = 6.2 mM). Ethanolamine kinase had a Km for ethanolamine of 7.7 mM and choline was a 'mixed' type of inhibitor with a Ki of 0.037 mM. Both enzymes activities responded in a similar fashion to the adenylate energy charge. Betaine and choline phosphate partially inhibited both kinases with a 93% inhibition of the ethanolamine kinase by 5 mM choline phosphate. CTP and ethanolaminephosphate partially inhibited the ethanolamine kinase, but not the choline kinase. Other metabolites tested had negliglible effects on both kinases. The affinity-column-purified enzyme was analyzed by disc gel electrophoresis which resolved the two activities. Hence, although many of the properties of the two activities are similar, choline kinase and ethanolamine kinase must be separate enzymes. Analysis of rat liver cytosol by disc gel electrophoresis indicated four isoenzymes for choline kinase and ethanolamine kinase.     

Biochemical and genetic characterization of three hamster cell mutants resistant to diphtheria toxin

We describe here three different hamster cell mutants which are resistant to diphtheria toxin and which provide models for investigating some of the functions required by the toxin inactivates elongation factor 2 (EF-2). Cell-free extracts from mutants Dtx(r)-3 was codominant. The evidence suggests that the codominant phenotype is the result of a mutation in a gene coding for EF-2. The recessive phenotype might arise by alteration of an enzyme which modifies the structure of EF-2 so that it becomes a substrate for reaction with the toxin. Another mutant, Dtx(r)-2, contained EF-2 that was sensitive to the toxin and this phenotype was recessive. Pseudomonas aeruginosa exotoxin is known to inactivate EF-2 as does diphtheria toxin and we tested the mutants for cross-resistance to pseudomonas exotoxin. Dtx(r)-1 and Dtx(r)-3 were cross-resistant while Dtx(r)-2 was not. It is known that diphtheria toxin does not penetrate to the cytoplasm of mouse cells and that these cell have a naturally occurring phenotype of diphtheria toxin resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance of the hybrid cells to diphtheria toxin. Intraspecies hybrids containing the genome of mutants Dtx(r)-1 and Dtx(r)-3 had some resistance while those formed with Dtx(r)-2 were as sensitive as hybrids derived from fusions between wild-type hamster cells and mouse 3T3 cells.     

Two-locus inbreeding measures for recurrent selection

Summary For a population undergoing recurrent selection, a method is presented for determining the average inbreeding coefficients at the end of each breeding cycle. The coefficients are derived in terms of probability measures that genes are identical by descent. For the one-locus case, two digametic measures are defined and employed in the derivation of a recurrence formula for the inbreeding coefficient. Two further classes of measures, trigametic and quadrigametic, are required for transition from one cycle to the previous one to allow the calculation of the inbreeding function for the two-locus case. Numerical values of the average probability of double identity by descent for populations with various imposed assumptions are listed to illustrate the effects of linkage and population size on the accrual of inbreeding and hence of homozygosity.Paper number 5018 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina. This investigation was supported in part by NIH research grant number GM 11546 from the National Institute of General Medical Sciences.     

Effects of excess selenomethionine on selenium status indicators in pregnant long-tailed macaques (Macaca fascicularis)

Forty pregnant long-tailed macaques were treated daily for 30 d with 0, 25, 150 or 300 μg selenium as L-selenomethionine/kg body weight. Erythrocyte and plasma selenium and glutathione peroxidase specific activities, hair and fecal selenium, and urinary selenium excretion were increased by and were linearly related to L-selenomethionine dose. Hair selenium was most sensitive to L-selenomethionine dose, with an 84-fold increase in the 300 μg selenium/(kg-d) group relative to controls (r=0.917). Daily urinary selenium excretion (80-fold,r=0.958), plasma selenium (22-fold,r=0.885), erythrocyte selenium (24-fold,r=0.920), and fecal selenium (18-fold,r=0.911) also responded strongly to L-selenomethionine. Erythrocyte and plasma glutathione peroxidase specific activities increased 154% and 69% over controls, respectively. Toxicity was associated with erythrocyte selenium >2.3 μg/mL, plasma selenium >2.8 μg/mL, and hair selenium >27 μg/g. Plasma, erythrocyte, and hair selenium concentrations may be useful for monitoring and preventing the toxicity of L-selenomethionine administered to humans in cancer chemoprevention trials.     

Solubilization of rat heart sarcolemma 5′-nucleotidase by phosphatidylinositol-specific phospholipase C

The influence of a phosphatidylinositol-specific phospholipase C treatment on rat heart sarcolemmal 5′-nucleotidase was investigated. Upon complete hydrolysis of all phosphatidylinositol in the sarcolemma, 75% of 5′-nucleotidase activity was found in the solubilized form. The insolubilized enzyme after this treatment has the same K_m for AMP as the untreated, sarcolemmal-bound enzyme (0.04 mM), whereas the solubilized enzyme has a 40-fold increase in K_m for AMP (0.16 mM). Other sarcolemmal-bound enzymes were not affected by the same treatment. Hence, the specific involvement of phosphatidylinositol in the binding of 5′-nucleotidase to the sarcolemma of the rat heart is clearly demonstrated.     

Genome-wide haploinsufficiency screen reveals a novel role for γ-TuSC in spindle organization and genome stability

How subunit dosage contributes to the assembly and function of multimeric complexes is an important question with implications in understanding biochemical, evolutionary, and disease mechanisms. Toward identifying pathways that are susceptible to decreased gene dosage, we performed a genome-wide screen for haploinsufficient (HI) genes that guard against genome instability in Saccharomyces cerevisiae. This led to the identification of all three genes (SPC97, SPC98, and TUB4) encoding the evolutionarily conserved γ-tubulin small complex (γ-TuSC), which nucleates microtubule assembly. We found that hemizygous γ-TuSC mutants exhibit higher rates of chromosome loss and increases in anaphase spindle length and elongation velocities. Fluorescence microscopy, fluorescence recovery after photobleaching, electron tomography, and model convolution simulation of spc98/+ mutants revealed improper regulation of interpolar (iMT) and kinetochore (kMT) microtubules in anaphase. The underlying cause is likely due to reduced levels of Tub4, as overexpression of TUB4 suppressed the spindle and chromosome segregation defects in spc98/+ mutants. We propose that γ-TuSC is crucial for balanced assembly between iMTs and kMTs for spindle organization and accurate chromosome segregation. Taken together, the results show how gene dosage studies provide critical insights into the assembly and function of multisubunit complexes that may not be revealed by using traditional studies with haploid gene deletion or conditional alleles.     

Alterations in osteoclast morphology following osteoprotegerin administration in the magnesium-deficient mouse

In the present study, we used osteoprotegerin (OPG), which blocks osteoclastogenesis, to correct and thus explain the hypercalcemia that is seen during dietary Mg deficiency in the mouse. Control and Mg-deficient mice received injections for 12 days of either OPG or vehicle only. Serum Ca was similar in Mg-deficient mice treated with OPG and in control mice receiving OPG (9.2±0.3 mg/dl vs. 9.2±0.5). Both groups had significantly higher serum Ca than controls or Mg-deficient animals receiving vehicle alone. Surprisingly, Mg-depleted mice that received OPG in doses that inhibit osteoclastic bone resorption remained hypercalcemic. Because mature osteoclasts still present in the marrow might be hyperactive, we examined osteoclast morphology at the light microscopic and ultrastructural level. Light microscopic examination of trabecular bone showed few osteoclasts in OPG-treated mice. Ultrastructural examination revealed that osteoclasts in OPG-treated mice have decreased contact with the endosteal bone surface and absence of a ruffled border. Because the morphology of the existing pool of mature osteoclasts did not enhance resorption, another mechanism, such as increased intestinal absorption of Ca in Mg-deficient mice, likely contributes to the hypercalcemia observed during Mg deficiency.     

Slow rise of Ca2+ and slow release of reactive oxygen species are two cross-talked events important in tumour necrosis factor-α-mediated apoptosis

Tumour necrosis factor-α(TNF-α) was found to be a cell cycle-independent apoptogenic cytokine in cultured fibroblast L929 cells. This assertion is based on the observations (1) TNF-α increased the number of cells with hypo-diploid DNA in a time dependent manner as revealed by flow cytometry, and (2) TNF-α induced DNA fragmentation as resolved by agarose gel electrophoresis. When cells were exposed to TNF-α (50ng/ml), a slow rise in intracellular free Ca²⁺ level and a delayed increase in the production of reactive oxygen species (ROS) (both observed 3h after the addition of TNF-α) were observed in fluo-3 and furared or dichlorofluorescein loaded cells, respectively. Interestingly, challenge of cells with TNF-α in the presence of BAPTA/AM, an intracellular Ca²⁺ chelator, decreased the release of ROS. Removal of ROS by 4-hydroxy 2,2,6,6-tetra-methyl-piperidinooxy (4OH-TEMPO) blocked the TNF-α-mediated Ca²⁺ rise. Moreover, when cells were exposed to TNF-α with both 4OH-TEMPO and BAPTA/AM, more viable cells were found than from treatment with either BAPTA/AM or 4OH-TEMPO. These results suggest that ROS and cellular Ca²⁺ are two cross-talk messengers important in TNF-α-mediated apoptosis.