Stain Technology: A Simplified Aluminum Stain in Paraffin Sections of Bone from Hemodialysis Patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

Prostacyclin contributes to inhibition of hypoxic pulmonary vasoconstriction by alkalosis

The mechanism by which extracellular alkalosis inhibits hypoxic pulmonary vasoconstriction is unknown. We investigated whether the inhibition was due to intrapulmonary production of a vasodilator prostaglandin such as prostacyclin (PGI₂). Hypoxic vasoconstriction in isolated salt-solution-perfused rat lungs was blunted by both hypocapnic and NaHCO₃_induced alkalosis (perfusate pH increased from 7.3 to 7.7). The NaHCO₃-induced alkalosis was accompanied by a significant increase in the perfusate level of 6-keto-prostaglandin F_1α (6-keto-PGF_1α), an hydrolysis product of PGI₁. Meclofenamate, an inhibitor of cyclooxygenase, counteracted both the blunting of hypoxic vasoconstriction and the increased level of 6-keto-PGF_1α. In intact anesthetized dogs, hypocapnic alkalosis (blood pH increased from 7.4 to 7.5) blunted hypoxic pulmonary vasoconstriction before but not after administration of meclofenamate. In separate cultures of bovine pulmonary artery endothelial and smooth muscle cells stimulated by bradykinin, the incubation medium levels of 6-keto-PGF_1α were increased by both hypocapnia and NaHCO₃-induced alkalosis (medium pH increased from 7.4 to 7.7). These results suggest that inhibition of hypoxic pulmonary vasoconstriction by alkalosis is mediated at least partly by PGI_2.     

Stimulating effects of insulin and low-density lipoprotein on cell growth and macromolecular syntheses of HeLa cells cultured in K(+)-depleted medium

The influence of the intracellular K+ concentration on the effects of growth factors (insulin, EGF, hydrocortisone, and transferrin) and LDL on growth of HeLa cells was investigated. Upon replacement of K+ in a chemically defined medium (K(+)-CDM) by Rb+ (Rb(+)-CDM), about 80% of the intracellular K+ was replaced by Rb+ within 24 h, but showed no further change in the next 24 h, irrespective of addition of dialyzed calf serum (5%) or growth factors to the medium. In Rb(+)-CDM, cell growth and DNA synthesis were greatly suppressed, although cell viability was not significantly altered for 72 h. The suppression of cell growth was partially restored by addition of serum, insulin (5 micrograms/ml), or LDL (2.5 mg/ml) to Rb(+)-CDM. A combination of serum and insulin or insulin and LDL stimulated cell growth to approximately the level in K(+)-CDM without any addition, but a combination of serum and LDL did not have more effect than that of serum alone. Unexpectedly, other factors were ineffective in stimulating growth in Rb(+)-CDM. In Rb(+)-CDM, the effect of insulin was lost in 24-48 h, whereas that of LDL persisted for at least 96 h. Insulin and LDL also enhanced growth in K(+)-CDM. After cessation of cell growth in Rb(+)-CDM for 24 h, addition of insulin and/or LDL markedly restored cell growth and DNA synthesis. Therefore, insulin and LDL may stimulate certain mechanisms required for cell growth that can operate in K(+)-deficient conditions.     

Role of carbohydrate moiety of IL-5. Effect of tunicamycin on the glycosylation of IL-5 and the biologic activity of deglycosylated IL-5

IL-5 is a T cell-derived lymphokine that induces B cell growth and differentiation in murine systems. In this study, we examined the role of carbohydrate moiety of IL-5 in the expression of biological function. IL-5 polypeptides translated in Xenopus oocytes were heterogeneous in terms of isoelectric point (pI 4.7 to 8.0) and m.w. (45,000 to 60,000 under nonreducing conditions) and yielded m.w. of 25,000 to 30,000 under reducing conditions. Treatment of rIL-5 with N-glycanase under reducing conditions yielded an IL-5 monomer of m.w. 12,000 to 14,000. Furthermore, deglycosylated rIL-5 that had been translated in the presence of tunicamycin showed very limited heterogeneity by two-dimensional gel electrophoresis (first dimension, nonequilibrium pH gradient electrophoresis; second dimension, SDS-PAGE). The m.w. was 27,000 to 28,000 under non-reducing conditions and migrated to m.w. 13,000 to 14,000 under reducing conditions. These results indicate that IL-5 is a glycoprotein carrying the N-glycosidically-linked carbohydrates. Treatment of IL-5 with sialidase caused the decrease in the heterogeneity in isoelectric point of IL-5. Deglycosylated rIL-5 that had been obtained from tunicamycin-treated oocytes could bind to IL-5-responding cells (T88-M), which express both high- and low-affinity IL-5 receptors, as efficient as intact rIL-5 under high-affinity conditions. Scatchard plot analysis of equilibrium binding of 35S-labeled rIL-5 to T88-M cells revealed that the dissociation constants (Kd) of glycosylated rIL-5 and deglycosylated rIL-5 were 127 pM and 110 pM, respectively. IL-5 activities determined by both B cell growth and differentiation assays were not affected by deglycosylation. These results indicate that N-linked glycoside moiety of IL-5 molecules may not play an essential role in the expression of its activity.     

Regulation of Histamine Turnover via Muscarinic and Nicotinic Receptors in the Brain

To clarify the regulation of central histaminergic (HAergic) activity by cholinergic receptors, the effects of drugs that stimulate the cholinergic system on brain histamine (HA) turnover were examined, in vivo, in mice and rats. The HA turnover was estimated from the accumulation of tele-methylhistamine (t-MH) during the 90-min period after administration of pargyline (65 mg/kg, i.p.). In the whole brain of mice, oxotremorine, at doses higher than 0.05 mg/kg, s.c., significantly inhibited the HA turnover, this effect being completely antagonized by atropine but not by methylatropine. A large dose of nicotine (10 mg/kg, s.c.) also significantly inhibited the HA turnover. This inhibitory effect was antagonized by mecamylamine but not by atropine or hexamethonium. A cholinesterase inhibitor, physostigmine, at doses higher than 0.1 mg/kg, s.c., significantly inhibited the HA turnover. This effect was antagonized by atropine but not at all by mecamylamine. None of these cholinergic antagonists used affected the steady-state t-MH level or HA turnover by themselves. In the rat brain, physostigmine (0.1 and 0.3 mg/kg, s.c.) also decreased the HA turnover. This inhibitory effect of physostigmine was especially marked in the striatum and cerebral cortex where muscarinic receptors are present in high density. Oxotremorine (0.2 mg/kg, s.c.) and nicotine (1 mg/kg, s.c.) also decreased the HA turnover in the rat brain. However, these effects showed no marked regional differences. These results suggest that the stimulation of central muscarinic receptors potently inhibits the HAergic activity in the brain and that strong stimulation of central nicotinic receptors can also induce a similar effect.     

Crystallization and preliminary X-ray studies of glutathione synthetase from Escherichia coli B

The glutathione synthetase from Escherichia coli B has been crystallized from 27% saturated ammonium sulfate solution (pH 5.5). The crystals are hexagonal, space group P6(2)22 or P6(4)22. The cell dimensions are a = b = 88.0 A, c = 164.2 A, and gamma = 120 degrees. The enzyme is a tetramer (Mr = 143,000) with 222 symmetry, and the asymmetric unit contains one subunit molecule (Mr = 35,600). The crystals diffract to at least 2.5 A resolution.     

Effects of temperature and pH on hemoglobin release from hydrostatic pressure-treated erythrocytes

The release of hemoglobin from human erythrocytes hemolyzed beforehand by hydrostatic pressure, osmotic pressure, and freeze-thaw methods was examined as a function of temperature (0-45 degrees C) and pH (5.5-8.8) at atmospheric pressure. Only in the case of high pressure (2,000 bar) did the release of hemoglobin increase significantly with decreasing temperature and pH. Maleimide spin label studies showed that the temperature and pH dependences of hemoglobin release were qualitatively explicable in terms of those of the conformational changes of membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins showed the diminution of band intensities corresponding to spectrin, ankyrin, and actin in the erythrocytes hemolyzed by high pressure. Cross-linking of cytoskeletal proteins by diamide stabilized the membrane structure against high pressure and suppressed hemoglobin release. These results indicate that the disruption of cytoskeletal apparatus by high pressure makes the membrane more leaky.     

Synthetic inositol trisphosphate analogs and their effects on phosphatase, kinase, and the release of Ca2+

A series of inositol 1,4,5-trisphosphate (IP3) analogs and positional isomers was examined to explore the structure-activity relationships among IP3 5-phosphatase, IP3 3-kinase, and the release of Ca2+. All analogs with additional groups on the 2nd position of IP3 inhibited the hydrolysis of [5-32P]IP3 catalyzed by erythrocyte ghosts, with a lower Ki value than seen with IP3. IP3 dehydroxylated at the 2nd position also had a lower Ki, while 2,4,5-IP3 or cyclic(1:2), 4,5-IP3 had higher Ki values. Among these compounds 2-deoxy-IP3 was as potent as IP3 in inhibiting the phosphorylation by [3H] IP3-3-kinase in rat brain cytosol. The other compounds, except for 2,4,5-IP3 inhibited the phosphorylation, however, 2-30 times higher concentrations were required. By lowering free Ca2+, the concentrations required for half-maximal inhibition were low, while those of IP3, 2-deoxy-IP3, and positional isomers remained unchanged. These compounds acted as full agonists in releasing Ca2+ from permeabilized macrophages, although 1.6-50-fold higher concentrations than IP3 were required. These compounds also inhibited the binding of [3H]IP3 to rat cerebellum and bovine adrenal cortex microsomes, but the potencies were 2.9-33 times less than that of IP3. Thus, the 2nd position of IP3 can be modified with only a slight loss of biological activity.