Effect of drug efflux blockers on vital staining of cellular DNA with Hoechst 33342

The present study shows that staining of certain live cells, e.g., adriamycin-resistant P388 cells, by Hoechst 33342 is difficult because of the presence of a rapid efflux pump, which reduces intracellular dye concentration. Coincubation of these refractory cells in the presence of efflux blockers such as phenothiazines (trifluoperazine) or Ca++ channel blockers (verapamil) enhances dye retention and thus leads to generation of normal DNA distribution histograms. Laser flow cytometric data is confirmed by fluorometric assays, which show that P388/R cells retain one-third the amount of Hoechst 33342, and coincubation with efflux blockers increases Hoechst retention to values similar to those of drug-sensitive P388 cells. DNA histograms of mouse splenocytes incubated with Hoechst 33342 alone have a bimodal distribution possibly because of the presence of subpopulations that do not retain the fluorochrome owing to rapid efflux. Coincubation with an efflux blocker results in the generation of unimodal DNA histograms from these cells. These preliminary studies suggest that reduced retention of Hoechst 33342 in certain cell types (because of rapid efflux) can be blocked by efflux blockers, thus leading to generation of typical DNA distribution histograms.     

Irreversible platelet aggregation does not depend on lipoxygenase metabolites

Previous investigations in our laboratory demonstrated the existence of an intrinsic mechanism, termed membrane modulation, capable of restoring sensitivity to aspirin treated platelets, resulting in irreversible aggregation in response to arachidonic acid (AA). The mechanism underlying correction of aspirin induced inhibition of platelet function, however, was not clear. In the present study we have evaluated the role of lipoxygenase (LO) metabolites of AA in securing irreversible aggregation of drug induced cyclooxygenase (CO) deficient platelets. Platelets treated with aspirin or Ibuprofen did not convert radiolabeled AA to thromboxane, but generated significant quantities of hydroxy acids via the LO pathway. However, drug exposed platelets, when stirred with epinephrine first and then challenged with AA, aggregated irreversibly. Eicosatetraynoic acid (ETYA 1, U53119) inhibited AA conversion by the LO pathway, whereas 5,8,11,14-eicosatetraynoic acid (ETYA 2) inhibited AA conversion by both CO and LO enzymes. Yet, at the inhibitory concentration these fatty acids failed to prevent AA induced irreversible aggregation of CO deficient, alpha adrenergic receptor stimulated platelets. Results of four studies show that the generation of LO metabolites of AA are not essential for securing irreversible aggregation of platelets.     

Cell surface tubulin in leukemic cells: molecular structure, surface binding, turnover, cell cycle expression, and origin

We report here new characteristics of cell surface tubulin from a human leukemia cell line. These cells (CEM cells) possess tubulin that is readily iodinated on the surface of living cells, turns over at a rate identical to that of other surface proteins, and is present throughout the cell cycle. When removed with trypsin, it rapidly returns to the surface. Peptide mapping of iodinated surface tubulin indicates that it possesses a similar, but not identical, primary structure to total CEM and rat brain tubulin. Living CEM cells are able to bind specifically a subfraction of CEM tubulin from metabolically labeled high speed supernatants of lysed CEM cells. Surface tubulin is more basic than the total tubulin pool. The binding, which is saturable, is inhibited by unlabeled CEM high speed supernatants but not by excess thrice-cycled rat or bovine brain tubulin. Surface tubulin is also shown to bind to living nontransformed normal rat kidney cells but not to normal, circulating, mononuclear white cells. Activated lymphocytes produce a tubulin that binds to CEM cells. Since CEM tubulin was detected in the media of 6-h cultures of CEM cells, we must conclude that at least some of the surface tubulin comes from the media. We further conclude that these leukemic cells produce an unusual tubulin that may bind specifically to any membrane. The presence of iodinatable surface tubulin, however, appears to require both the production of a unique tubulin and the presence of a "receptor-like" surface binding component.     

Purification and characterization of metallothionein from liver of cadmium exposed Rhesus monkeys (Macaca mulatta)

Metallothionein (MT) a low molecular weight, Cd-binding, cysteine rich, cytosolic protein has been isolated, purified and characterized from cadmium exposed Rhesus monkeys maintained on protein calorie malnourished (PCM) diet. Metallothionein was resolved into three isoforms i.e. MT_a, MT_b and MTV_c. The ratio of Cd, Zn and Cu varied in these isometallothioneins. MT_c was the major isometallothionein. UV Spectra of MT_c revealed the presence of mercaptide bonds and absence of aromatic amino acids. These observations were further confirmed by amino acid analysis of MT_c which demonstrated high cysteine content (22.6) followed by serine, glycine and lysine. The molecular weight of MT_c as determined by gel filtration and amino acid analysis was 13000 and 6398 daltons respectively. This demonstrates that MT_c is a non-globular ellipsoid polypeptide. MT_c showed a unique property of binding selenium. Monkey liver metallothionein was immunologically identical with human metallothionein. All the characteristics of MT_c obtained in the present study reveal a similarity between monkey and human metallothionein probably due to closer phylogenetic relationship between the two species.     

Evaluation of Salmonella Porins as a Broad Spectrum Vaccine Candidate

Porins were prepared from smooth strain of Salmonella typhi 0–901 and chemotype of rough mutant of S. typhimurium Ra-30. Mice were immunized with both the porin preparations in different groups and challenged with S. typhimurium LT2–71 and S. enteritidis SH-1269. Porin immunized mice showed significant protection (P <0.01) against challenge with homologous as well as heterologous strains. Hence, the use of porins may be attempted in future to protect against salmonellosis.     

In vitro effect of prostaglandins,prostaglandin endoperoxides and thromboxane on rat intestinal alkaline phosphatase and (Ca2+-Mg2+) adenosine triphosphatase

The activity of alkaline phosphate and²⁺-Mg²⁺ adenosine triphosphatase, two of the enzymes involved in limpid and calcium uptake across the intestinal membrane, were increased in experimental atherosclerosis. Administration ofAnnapavala sindhooram, an antiatherosclerotic drug, lowers these enzyme levels to near normal values. Prostaglandin E2 stimulated the enzyme activitiesin vitro, while prostaglandin endoperoxide inhibited the activity. Thromboxane and other prostaglandins had no effect on the enzyme activities. Addition of the antiatherosclerotic drug to thein vitro assay system reversed the effect of both prostaglandin E₂ and prostaglandin endoperoxide.     

Hexokinase receptors: Preferential enzyme binding in normal cells to nonmitochondrial sites and in transformed cells to mitochondrial sites

Hexokinase plays an important role in normal glucose-utilizing tissues like brain and kidney, and an even more important role in highly malignant cancer cells where it is markedly overexpressed. In both cell types, normal and transformed, a significant portion of the total hexokinase activity is bound to particulate material that sediments upon differential centrifugation with the crude mitochondrial fraction. In the case of brain, particulate binding may constitute most of the total hexokinase activity of the cell, and in highly malignant tumor cells as much as 80 percent of the total. When a variety of techniques are rigorously applied to better define the particulate location of hexokinase within the crude mitochondrial fraction, a striking difference is observed between the distribution of hexokinase in normal and transformed cells. Significantly, particulate hexokinase found in rat brain, kidney, or liver consistently distributes with nonmitochondrial membrane markers whereas the particulate hexokinase of highly glycolytic hepatoma cells distributes with outer mitochondrial membrane markers. These studies indicate that within normal tissues hexokinase binds preferentially to non-mitochondrial receptor sites but upon transformation of such cells to yield poorly differentiated, highly malignant tumors, the overexpressed enzyme binds preferentially to outer mitochondrial membrane receptors. These studies, taken together with the well-known observation that, once solubilized, the particulate hexokinase from a normal tissue can bind to isolated mitochondria, are consistent with the presence in normal tissues of at least two different types of particulate receptors for hexokinase with different subcellular locations. A model which explains this unique transformation-dependent shift in the intracellular location of hexokinase is proposed.