Purification of retinol dehydrogenase from bovine retinal rod outer segments

We purified retinol dehydrogenase from bovine rod outer segments using polyethylene glycol precipitation and hydroxylapatite, concanavalin A-Sepharose CL-4B, and Sepharose CL-6B column chromatography in the presence of NADP. We obtained 13-fold purification of retinol dehydrogenase with specific activity of 61.8 nmol/min/mg and 3.8% recovery. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that retinol dehydrogenase had a molecular mass of 37,000 daltons. The Km values of purified retinol dehydrogenase for all-trans retinol and all-trans retinal were 6.6 mM and 0.085 mM, respectively. The purified enzyme reacted with the all-trans retinal but not with 13-, 11-, and 9-cis compounds. In addition, we prepared antibody to retinol dehydrogenase using rat. The anti-retinol dehydrogenase antibody precipitated retinol dehydrogenase activity and was confirmed to bind to 37-kDa protein by Western blotting. We also found that anti-retinol dehydrogenase antibody bound to bovine rod outer segments specifically by immunohistochemical technique. The molar ratio of retinol dehydrogenase to opsin in rod outer segments estimated by enzyme-linked immunosorbent assay was 1:140.     

Azidopine noncompetitively interacts with vinblastine and cyclosporin A binding to P-glycoprotein in multidrug resistant cells.

It is believed that P-glycoprotein (P-gp) is an energy-dependent drug efflux pump responsible for decreased drug accumulation in multidrug resistant (MDR) cells. In this study, we investigated whether azidopine, a photoactive dihydropyridine calcium channel blocker, is transported by P-gp in MDR Chinese hamster lung cells, DC-3F/VCRd-5L, and whether its binding site(s) on P-gp are distinct from those of Vinca alkaloids and cyclosporins. The efflux of azidopine from MDR cells was energy-dependent and inhibited by the cytotoxic agent vinblastine (VBL). Cyclosporin A (CsA), a modulator of MDR, also increased azidopine accumulation in MDR cells by decreasing the energy-dependent efflux of azidopine. P-gp in these cells was the only protein specifically bound to [3H]azidopine in photoaffinity experiments. The specific photoaffinity labeling of P-gp by [3H]azidopine was inhibited by CsA, SDZ 33-243, nonradioactive azidopine, and VBL with median concentrations (IC50) of 0.5, 0.62, 1.7, and 25 microM, respectively. The equilibrium binding of azidopine to plasma membranes of MDR variant DC-3F/VCRd-5L cells showed a single class of specific binding sites having a dissociation constant of 1.20 microM and a maximum binding capacity of 4.47 nmol/mg of protein. Kinetic analysis indicated that the inhibitory effect of VBL and CsA on azidopine binding to plasma membranes of MDR cells was noncompetitive, indicating that azidopine binds to P-gp at a binding site(s) different from the binding site(s) of these drugs.     

Micronucleus induction in mouse peripheral reticulocytes by 7,12-dimethylbenz[a]anthracene.

Micronucleus assays using mouse peripheral blood stained vitally on acridine orange (AO)-coated slides were evaluated at two laboratories with 7,12-dimethylbenz[a]anthracene (DMBA) and compared with the standard bone marrow assay. DMBA was administered by single intraperitoneal injection to CD-1 mice at doses ranging from 5 to 80 mg/kg, then 5 microliters of peripheral blood was sampled from a tail vein at 24, 48, 72, 96, and 120 h after treatment. Similar incidences of micronucleated young erythrocytes were observed in peripheral blood reticulocytes and bone marrow polychromatic erythrocytes. The dose response of micronucleated reticulocytes was delayed compared to that of micronucleated polychromatic erythrocytes. The dose-response curves after treatment with DMBA differed depending on the sampling times, which revealed the difficulty of obtaining accurate dose-response relations in the micronucleus assay. The present result demonstrated that the simple and rapid AO supravital staining method is a valuable and easier method for obtaining dose- and time-response data for quantification of micronucleus induction by chemicals.     

Proof of plasmatic imbibition in rat musculocutaneous grafts: enzymatic proof using peroxidase.

At present, the concept of plasmatic imbibition is generally accepted. That is, observation of weight change or pigmentation methods of the grafts were established to support it. In the present study, we investigated plasmatic imbibition in rat musculocutaneous grafts histologically using peroxidase, which is one of the reductases. Tissue pigmentation by peroxidase became an insoluble sediment that indicated the sites of peroxidase activity. The entire contact surface of the graft with the recipient bed was stained brown within a few minutes after the operation. At 30 minutes, the panniculus carnosus and dermis were stained dark brown diffusely, extending toward the epidermis. This condition continued until the sixth day at least. As a result, peroxidase that was dissolved with the exudate between the graft and recipient bed was imbibed into the musculocutaneous graft.     

Protective mechanism of sodium molybdate against the acute toxicity of cadmium in rats. II. Prevention of cytoplasmic acidification

In order to clarify the protective mechanism of sodium molybdate against the acute toxicity of cadmium chloride in rat, the effect of in vivo sodium molybdate pretreatment on the cytotoxic action of cadmium in isolated hepatocytes was studied. The cytosolic pH of hepatocytes isolated from untreated rats immediately decreased with incubation in either neutral Hank's balanced salt solution (HBS), pH 7.4, containing 5 µM cadmium chloride minimum or acidic HBS (pH 7.1, 6.8, 6.5, and 6.2). The presence of 5 µM cadmium in HBS adjusted to pH 7.1 aggravated cytosalic acidification induced by the acidic medium alone. Cell viability of hepatocytes incubated in HBS at pH 6.2 was significantly reduced as compared to that of control cells in HBS at pH 7.4, but the presence of cadmium in the acidic HBS had no aggravating action against such a toxic action of the acidic medium although cellular uptake of the metal in the medium increased, as compared to that in HBS at pH 7.4. Molybdenum pretreatment alleviated cytoplasmic acidification induced by the treatment with HBS at pH 7.4 or 7.1 containing cadmium or by extracellular acid load wothout cadmium. This pretreatment also prevented the loss of cell viability induced by the treatment with HBS at pH 6.2 but could not attenuate that when cadmium was present in the medium.These facts suggest that molybdenum pretreatment alleviated the acute toxicity of cadmium in rat by preventing cytoplasmic acidification caused by the harmful metal.     

The alpha 1-adrenergic photoaffinity probe [125I]arylazidoprazosin binds to a specific peptide of P-glycoprotein in multidrug-resistant cells

Much evidence suggests that P-glycoprotein (P-gp) confers multidrug-resistance (MDR) in tumor cells by energy-dependent efflux of hydrophobic cytotoxic agents. In this study, we have used the alpha 1-adrenergic photoaffinity probe, [125I]arylazidoprazosin ([125I]AAP), and identified P-gp as a specific acceptor for prazosin. Drugs to which MDR cells are resistant, including vincristine, vinblastine, doxorubicin, actinomycin D and colchicine as well as agents reversing MDR, including verapamil, nicardipine, prenylamine, diltiazem, trifluoperazine, dibucaine, reserpine, monensin, and progesterone, differentially reduced [125I]AAP photolabeling of P-gp. We also analyzed the influence of alpha 2-adrenergic drugs and dopaminergic drugs on [125I]AAP photolabeling of P-gp. Limited proteolysis of [125I]AAP photolabeled P-gp with Staphylococcus aureus V8 protease revealed that prazosin binds to a single 8 kDa fragment of P-gp.     

Scanning Electron Microscopy of Rhizobium trifolii Infection Sites on Root Hairs of White Clover

White clover root hairs which were inoculated with Rhizobium trifolii 4S (infectious strain) contained infection threads which were observed by light microscopy and scanning electron microscopy. Three morphological types of root hairs retaining infection threads were recognized. The bacteria were strongly attached between the surfaces of two plant cell walls as follows: between surfaces of a root hair tip curled back on itself, between a protuberance from a root hair and its cell surface, or between two root hair tips clinging together. An anatomical analysis documented the attachment site of the infection thread sheath from the inside of the root hair cell.     

Biochemical characterization of the submicrosomal membrane of the rat brain. Selective solubilization of the components of the light smooth-surfaced membrane by lysophosphatidylcholine

The light smooth-surfaced membrane, one of the three membrane fractions derived from the rat brain microsomal fraction, was fractionated into its soluble and insoluble parts by the use of lysophosphatidylcholine and the chemical composition of these was investigated. Under the condition whereby the maximal amount of the membrane protein was solubilized by lysophosphatidylcholine (0.5% lysophosphatidylcholine at 37 degrees C for 10 min), the insoluble residue, which accounted for approximately 30% of the membrane protein, was ultracentrifugally homogeneous and showed a granular structure under the electron microscope. The lipid composition of the soluble and insoluble fractions, as well as their protein composition, revealed a preferential and limited solubilization of the constituents of the membrane by lysophosphatidylcholine.