Amino acid uptake in the peripheral nerve of the rat

When rat sciatic nerves were incubated with C¹⁴l-lysine, l- or d-glutamate, or d-l γ-aminoisobutyrate, the labeled compounds penetrated the nerve, and the level of lysine and leucine after 1 hr was higher in the nerve than in the medium. The level increased with time, and at 24 hr glutamate levels also were higher in the nerve than in the medium. Lowering the temperature strongly inhibited uptake, while other conditions such as absence of glucose, absence of sodium, or the presence of cyanide inhibited uptake by nerve less than uptake by brain slices. The uptake against a concentration gradient, and inhibitions of this uptake by metabolic inhibitors and by structural analogs, were interpreted as showing the presence of transport processes for amino acids in peripheral nerves with characteristics similar to such transport processes in the central nervous system.     

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.     

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.