A comparative study on immunosuppressive effects of cyclosporin A and FK 506 on peripheral blood lymphocytes in dogs

Immunosuppressive effects of cyclosporin A (CsA) and FK 506 (FK) on peripheral blood lymphocytes were studied in dogs in respect to mixed lymphocyte reaction, proliferative responses to recombinant interleukin-2 (rIL-2), phytohemagglutinin (PHA) and concanavalin-A (Con-A); phenotypes of OKIa1, CD3, CD8 and surface IgM; cytotoxic activity against xenogeneic tumor cells. CsA (2.0 or 5.0 mg/kg, intravenously) or FK (0.16 mg/kg, intramuscularly) was given to mongrel dogs every morning for serial 21 days. The blood concentrations of CsA, measured as trough levels by fluorescence polarization method, ranged from 37 to 350 ng/ml in dogs administered at 2.0 mg/kg and from 170 to 894 ng/ml in dogs administered at 5.0 mg/kg during treatment, respectively. In dogs treated with FK at a dose of 0.16 mg/kg, the drug concentrations in the plasma during treatment ranged from 0.16 to 1.8 ng/ml. Mixed lymphocyte reaction and proliferative responses to rIL-2, PHA and Con-A, which were declined by CsA, were not affected by FK. In contrast, the proportion of OKIa1+ cells was not affected by CsA, whereas FK decreased the proportion of OKIa1+ cells progressively during the course of treatment. Cytotoxic activity was suppressed by both CsA and FK. These results possibly indicate that CsA and FK exert their immunosuppressive effects via different mechanisms.     

Binding of [3H]befunolol to beta-adrenoceptors in cardiac muscles of fetal and neonatal rat

Characterization of beta-adrenoceptors was studied in heart muscles of rat fetus and neonate. The results of binding assay with [3H]befunolol, a beta-adrenergic partial agonist, to membrane fractions from rat heart muscles indicate that beta-adrenoceptors contain two different affinity sites. In the presence of 5'-guanylylimidodiphosphate, the low affinity site was reduced, while the high affinity site was not affected. The dissociation constants for both sites did not change during pre- and post-natal development. But the maximum binding sites for both sites decreased slightly but significantly (p less than 0.05) during development. A 10-fold decrease in norepinephrine sensitivity and isoprenaline sensitivity during pre- and post-natal development was not explained by the slight decrease in the maximum binding sites.     

Complete 1H-NMR spectral assignments for globotriaosyl-Z- and isoglobotriaosyl-E-ceramide

Two-dimensional scalar-correlated (COSY) 1H-NMR spectra of the title compounds, and phase-sensitive COSY spectrum of lactosylceramide, have been fully assigned and some spectral reassignments for related structures suggested. Glycosylation-induced shifts, and shielding by Z- and E-ceramide residues are discussed.     

Enhancement of ATPase Activity by a Lipid Peroxide of Arachidonic Acid in Rat Brain Microvessels

The effects of 15-hydroperoxyarachidonic acid (15-HPAA) on Na+, K+- and Mg+-ATPase activities in the blood-brain barrier (BBB) were examined using rat brain microvessels (MV). 15-HPAA markedly stimulated these ATPase activities in MV at low concentrations whereas the synaptosomal Na+, K+-ATPase activity was inhibited in a dose-dependent manner. Further neurochemical analysis revealed that this stimulatory effect of 15-HPAA in MV was not due to a simple detergent-like action of the compound on the membranes but rather to stimulation of the phospholipase A2 and lipoxygenase activity within MV. In addition, it was shown that free radical reactions were involved in the mechanism. Since such anti-edema drugs as 1,2-bis(nicotinamido)propane were proved to be potent suppressors of the enhanced ATPase activity, further speculations on the role of this effect for ischemic brain edema are offered.     

Intrathoracic sources of caudal mediastinal lymph node efferent lymph in sheep

We investigated the intrathoracic contributions to the caudal mediastinal lymph node (CMN) efferent lymph in 12 anesthetized sheep after removing possible systemic contributions from below the diaphragm. We interrupted various pathways that may send lymph to the CMN (chest wall, esophagus, lung). Because the experiment is destructive, we did the resections in various combinations and waited 1 h between steps. Base-line CMN efferent lymph flow averaged 0.90 +/- 0.52 g/15 min (mean +/- SD). Cutting the pulmonary ligaments bilaterally caused lymph flow to decrease by an average of 58%. In five sheep, cauterizing around the lung hila reduced lymph flow by 16% of base line, cauterizing along the esophagus reduced lymph flow by 18% of base line, and cauterizing along the chest wall increased lymph flow by 6% of base line. After complete isolation of the node, except for the bronchoesophageal artery, dorsal mediastinal vein, and CMN efferent duct, 14% of base-line flow remained. The lymph-to-plasma total protein concentration ratios did not change significantly with any procedure. Under the conditions of our experiments, approximately 74% of base-line intrathoracic CMN efferent flow comes from the lung.     

Relationship between dopamine content and its secretion in PC12 cells as a function of cell growth

The PC12 cell line derived from a rat adrenal medullary tumor is known to synthesize dopamine and to release it in response to cholinergic agonists or depolarizing agents. In this report, we have studied the relationship between dopamine biosynthesis and its stimulus-induced secretion in PC12 cells as a function of cell growth. The endogenous dopamine content was found to depend on cell growth, and reached a maximum in the stationary phase. This increase was associated both with an increase in the specific activity of tyrosine 3-monooxygenase, and with an increase of DOPA-decarboxylase in the cells. On the other hand, the maximal release of dopamine occurred in the late exponential phase before the endogenous dopamine was maximally synthesized in the cells. Moreover, the uptake of 45Ca2+ stimulated with either carbamylcholine or high K+ was also regulated by cell division: the maximal uptake took place in the same period of culture in which the maximal release of dopamine was observed. Thus, this report offers new evidence that the biosynthesis and secretion of dopamine are separately regulated in PC12 cells.     

Nucleosidediphosphate kinase from Ehrlich ascites tumor cells

A phosphate-incorporating protein has been highly purified from the cytosol of Ehrlich ascites tumor cells (EAT cells). The nitrocellulose membrane method was used to follow the progress of the purification by quantitation of the [32P]phosphorylated form of the protein. The purified protein was identified as an NDP-kinase since it exhibited NDP-kinase activity and had enzyme characteristics in common with other NDP-kinases from various mammalian cells. The purified NDP-kinase was found to have a molecular weight of approximately 76,000 daltons. Moreover, the enzyme appears to consist of two distinct polypeptides (18,000 and 20,000 daltons). This enzyme contained 19 amino acids, with high levels of glycine (9.8%) and lysine (9.0%). The enzyme rapidly formed a [32P]phosphoenzyme when incubated with [gamma-32P]ATP in the presence of Mg2+ (1 mM) at the optimum pH of 7.5 even at low temperature (below 4 degrees C). This phosphoenzyme is an enzyme-bound, high-energy-phosphate intermediate, because ATP was formed from it on incubation with ADP in the presence of Mg2+ (1 mM). This finding suggests that the phosphoenzyme functions as an intermediate in NDP-kinase action.     

Isolation of an autonomously replicating sequence (ARS) from satellite DNA of Drosophila melanogaster

Summary Autonomously replicating sequences (ARSs) were cloned from the 1.688 satellite DNA of D. melanogaster using YIp5, consisting of pBR322 and the yeast ura3 gene, as the cloning vector and YNN27, a Ura^– yeast strain as the recipient. Three out of six clones contained an ARS and the average frequncy of the occurrence of ARS was thus calculated to be approximately one per 14 kbp of the satellite DNA. A 500 bp ARS fragment (BgHS500) was obtained from one of the resultant clones (pYDS57). BgHS500 does not hybridize with the major repeating unit (370 bp) but it does with the minor unique sequence of the satellite. The sequence of BgHS500 was determined and found to be rich in AT and to contain the sequence, 5AAAACATAAAA3, a sequence common to yeast ARSs. However, a smaller fragment (150 bp) isolated from BgHS500 and containing the 11 bp sequence did not exhibit the characteristics of an ARS. The average copy number in the transformants of pBgHS500, a recombinant molecule of BgHS500 and YIp5, ranged from 0.05–0.5, while that of the parent plasmid, pYDS57, was about 2–10. On the basis of these results, it is postulated that the sequence 5AAAACATAAAA3 may possibly consiitute the core of ARSs and certain other sequences may also be necessary to insure that the ARS consistently undergoes at least one complete replication in each cell cycle. The role of ARSs in the genome of D. melanogaster is discussed.