Cytochalasin B: effect on phospholipid metabolism and lysosomal enzyme release by leukocytes;.

The effect of cytochalasin B on phospholipid metabolism and beta-glucuronidase extrusion by polymorphonuclear leukocytes from guinea pid peritoneal exudates has been studied. Cytochalasin B inhibited the engulfing of starch granules by leukocytes, but it enhanced the incorporation of 32-Pi into phosphatidic acid and the phosphoinositidesmit also stimulated the release of beta-glucuronidase into the incubation medium in the presence or absence of starch granulesmkinetic studies showed that the effects of cytochalasin B on 32-Pi incorporation into phosphatidic acid and the phosphoinositides, and the release of beta-glucuronidase into the extracellular medium were comparablempulse-chase experiment revealed that cytochalasin B did not stimulate the isotopic decay of prelabeled lipids, indicating that cytochalasin B increased the radiophosphorus activity of phosphatidic acid and the phosphoinositides by increasing the synthesis of these lipidsmthe incorporation of myo-[2-3H]inositol into the phosphoinositides was also enhanced in the presence of cytochalasin B, but the incorporation of [methyl-14-C] choline into phosphatidylcholine and sphingogonyelin was unchanged;

Effects of insulin on prenylation as a mechanism of potentially detrimental influence of hyperinsulinemia.

To investigate the cause and effect relationship between hyperinsulinemia and the increased amounts of farnesylated p21Ras, we performed hyperinsulinemic euglycemic clamps in normal weight volunteers as well as in normal mice and dogs. Insulin infusions significantly raised the amounts of farnesylated p21Ras in the white blood cells of humans, in liver samples of mice and dogs, and in aorta samples of mice. Obese hyperinsulinemic individuals and dogs (made hyperinsulinemic by surgical diversion of the pancreatic outflow from the portal vein into the vena cava) displayed increased amounts of farnesylated p21Ras before the hyperinsulinemic clamps. Infusions of insulin did not alter the already increased levels of farnesylated p21Ras in these experimental models. To further investigate the role of acquired insulin resistance in modulating insulin's effect on p21Ras prenylation, we induced insulin resistance in rats by glucosamine infusion. Insulin-resistant glucosamine-treated animals displayed significantly increased farnesylated p21Ras in response to insulin infusion compared to that in control saline-treated animals. Transgenic models of insulin resistance (heterozygous insulin receptor substrate-1 knockout mice, A-ZIP/F-1 fatless mice, and animals overexpressing glutamine:fructose-6-phosphate amidotransferase) contained increased amounts of farnesylated p21Ras. We conclude that hyperinsulinemia, either endogenous (a prominent feature of insulin resistance) or produced by infusions of insulin, increases the amounts of farnesylated p21Ras in humans, mice, and dogs. This aspect of insulin action may represent one facet of the molecular mechanism of the potentially detrimental influence of hyperinsulinemia.

Electron paramagnetic resonance investigation of high-spin iron (III) in cancer.

The concentration of iron (III)-transferrin (IT) in whole blood and serum, along with another high-spin (five unpaired electrons) iron complex (probably IT) accumulated by tumor tissue, was investigated by electron paramagnetic resonance (EPR) spectroscopy during the development of Murphy-Sturm rat lymphosarcoma. The observed changes in concentration (microgram/ml) of IT in sera/blood were generally complementary to those from tissue and the character of the modifications suggested the existence of three distinct phases of systemic response to the implantation: (1) an initial response, evidenced by a sharp reduction in serum IT and somewhat high tissue-IT concentration (microgram/g); (2) a period in which the tumor is (2) a period in which the tumor is becoming established, indicated by relatively constant tissue IT levels and near normal serum IT; and (3) the onset of rapid cell multiplication, characterized by increased total tissue-IT accumulation that rises to above 200% of normal available serum iron, increasing tissue-IT concentration, and rapidly declining serum-IT concentration. The results suggest that, in the face of an implanted tumor there are two detectable abnormal serum-IT responses: (1) an initial change, probably due to systemic blockage of iron reutilization; and (2) extraction of IT from serum by multiplying tumor cells, which is probably a major contributor to reduced serum-IT levels and ultimately anemia.

Analysis of plume emissions after papovavirus irradiation with the carbon dioxide laser.

This study was undertaken to evaluate potential inhalation hazards to operating room personnel after irradiation of tumors with the carbon dioxide laser. Cellular debris was analyzed for viability using labeled nucleotides and labeled glucose. In this way the plume was investigated for the presence of material with oncogenic potential. Most surgeons who have ablated venereal warts or certain tumors with the carbon dioxide laser have worried about possible hazards of inhaling the vapor that is produced as a result of their work. We utilized three methods to determine whether viable particles exist in the laser plume. Fortunately, it is most comforting that the metabolic studies, DNA and RNA studies and cytologic studies seem to indicate that the plume is biologically inactive.

Carbohydrate metabolism in leukocytes. VII. Metabolism of glucose, acetate, and propionate by human plasma cells.

Plasma cells obtained from the peripheral blood of a patient with multiple myeloma was incubated in serum and Krebs-Ringer bicarbonate buffer with (14)C-labeled glucose, acetate, and propionate. Glucose utilization by these cells amounted to 0.5 mumole per hr per 10(8) cells and was mainly via the Embden-Meyerhof pathway, and only 6% or less traversed the hexose monophosphate shunt. The presence of Krebs cycle activity was demonstrated by direct isolation of several labeled intermediates after incubation with either (14)C-acetate or (14)C-propionate. The distribution of (14)C in lactate, succinate, fumarate, malate, aspartate, and glutamate indicate a complete Krebs cycle. Acetate was metabolized via the Krebs cycle to the extent of 0.15 mumoles per hr per 10(8) cells, and the rate of propionate utilization was 0.17 mumoles per hr per 10(8) cells.

Preliminary evaluation of platinum transferrin (MPTC-63) as a potential nontoxic treatment for breast cancer.

Transferrin receptors on proliferating and malignant cells are well documented. Faulk et al. demonstrated transferrin receptors in breast carcinoma by immunofluorescence. Malignant cells requiring more iron modulate a transferrin receptor and the iron transporting protein transferrin delivers iron to the cell. We have developed a physiologically active platinum transferrin complex that has been tested on several cell lines in culture, a tumor model in the Fischer rat, and five human patients with advanced breast carcinoma. The complex slowed the rate of growth of feline lymphoma cells to one-half that of controls and killed human HeLa cell cultures in 7 days. Growth of the rat tumor was slightly impaired, but treated rats never got systemic disease and controls died. Two patients had dramatic responses to treatment. One had systemic disease and the other advanced locoregional disease. Both patients were on Tamoxifen, as receptors were positive for estrogen. Disease was progressing in the former with little improvement in the latter. After treatment both had a marked response. We postulate that MPTC-63 may work synergistically with Tamoxifen and be an effective nontoxic antitumor agent. More studies are indicated.

Factors affecting filamentation in Candida albicans: relationship of the uptake and distribution of proline to morphogenesis.

When glucose was present in high concentration, Candida albicans formed filaments in a phosphate-buffered medium, regardless of the nitrogen source. In lower concentrations of glucose, filamentation occurred only when various members of the glutamate, succinyl, or acetoacetyl-coenzyme A families of amino acids were used as sole nitrogen sources. Yeast morphology could be maintained either by replacing the amino acids in the medium with ammonium chloride or by making the medium high in phosphate or biotin. Studies using [U-14C]proline indicated that proline was catabolized in a manner consistent with the generation of increased cellular reducing potential and that the proline label entered into the Kreb's cycle. A reduction in Kreb's cycle activity was evidenced by an initial increase and then a rapid drop of the total organic acid content of the cells as well as in specific Kreb's cycle intermediates. Filamentation under conditions of low phosphate, high glucose, and increased cellular reduction potential, accompanied by a decrease in Kreb's cycle activity, suggests that morphogenesis in C. albicans is correlated with a Crabtree-like effect, i.e., repression of mitochondrial activity.

Factors affecting filamentation in Candida albicans: changes in respiratory activity of Candida albicans during filamentation.

Glucose metabolism and respiration of Candida albicans were compared under conditions which permitted either maximal filamentous or maximal yeast growth. Changes in metabolism were monitored by comparing the quantities of ethanol produced, CO2 evolved, and oxygen consumed. Filamenting cultures produced more ethanol and less CO2 than yeasts, with oxygen consumption in the former concomitantly slower than that of the latter. Studies involving cofactors and inhibitors associated with electron transport imply that a transfer of electrons away from flavoprotein is required for maintenance of yeast morphology. Conditions consistent with a buildup of reduced flavoprotein, however, favored filament formation. These changes were expressed metabolically as a shift from an aerobic to a fermentative metabolism. The results presented are consistent with hypotheses correlating filament production with changes in carbohydrate metabolism and an interruption of electron transfer within the cell.

Binding of platinum to human transferrin.

A complex of platinum and human transferrin has been formed by appropriately combining apotransferrin (metal free protein) and potassiumchloroplatinate (K2PtCl4). Atomic absorption spectroscopy indicated that both primary bind sites on the protein participated in the complex. Electron paramagnetic resonance (EPR) examination showed that the bound platinum was not paramagnetic, and thus it is highly probable that the Pt ion is in the +2 oxidation state. The results suggest a possible mechanism for physiological distribution of third-transition-series metals.

Impaired chemiluminescence during phagocytosis of opsonized bacteria.

A simple technique is presented for rapid screening of leukocytes of patients suspected of having a defective intracellular mechanism for the bactericidal and digestive disposal of bacteria.

Potentiation of Rho-A-mediated lysophosphatidic acid activity by hyperinsulinemia.

We have shown previously that insulin promotes phosphorylation and activation of farnesyltransferase and geranylgeranyltransferase (GGTase) II. We have now examined the effect of insulin on geranylgeranyltransferase I in MCF-7 breast cancer cells. Insulin increased GGTase I activity 3-fold and augmented the amounts of geranylgeranylated Rho-A by 18%. Both effects of the insulin were blocked by an inhibitor of GGTase I, GGTI-286. The insulin-induced increases in the amounts of geranylgeranylated Rho-A resulted in potentiation of the Rho-A-mediated effects of lysophosphatidic acid (LPA) on a serum response element-luciferase construct. Preincubation of cells with insulin augmented the LPA-stimulated serum response element-luciferase activation to 12-fold, compared with just 6-fold for LPA alone (p < 0.05). The potentiating effect of insulin was dose-dependent, inhibited by GGTI-286 and not mimicked by insulin-like growth factor-1. We conclude that insulin activates GGTase I, increases the amounts of geranylgeranylated Rho-A protein, and potentiates the Rho-A-dependent nuclear effects of LPA in MCF-7 breast cancer cells.

Correlation of metabolic and chemiluminescent responses of granulocytes from three female siblings with chronic granulomatous disease.

Polymorphonuclear leukocytes (PMNL) from children with atypical chronic granulomatous disease (CGD), their mother and siblings, and normal controls were studied in regard to glycolytic and hexose monophosphate shunt activities in the resting, methylene blue-stimulated, and phagocytizing states. PMNL from the patients with CGD had normal glycolytic and hexose monophophate shunt activities in the resting state and after stimulation with methylene blue. However, stimulation of the hexose monophosphate shunt after phagocytosis was greatly decreased. These data were correlated with studies of both initial rate and integral counts of chemiluminescence. The chemiluminescent response from patients with atypical CGD was also greatly decreased. This decreased response probably reflects a defect in the oxidative destruction of the phagocytized microbe and correlates well with the decreased activity of the phagocytically activated hexose monophosphate shunt. The defect in generation of radical species of oxygen, singlet oxygen, and chemiluminescence by leukocytes from patients with CGD is discussed.