Suppressed in vitro blastogenic responsiveness of rat spleen cells after continuous infusion of endotoxin by an implanted osmotic pump

Continuous infusion of a gram-negative bacterial endotoxin in relatively small doses into rats by means of an implanted osmotic pump was studied. The model system was designed to examine the effects of endotoxin on the blastogenic response of spleen cells to the endotoxin itself and to a nonspecific T-cell mitogen, concanavalin A (Con A). Rats were implanted with an osmotic pump which delivered saline for the first 42 hr to provide postsurgical recovery before the onset of endotoxin infusion. Previous studies had shown that during the first 1-4 days after administration of endotoxin marked alterations of metabolism and some changes in physiologic parameters such as blood pressure and in vitro myocardial performance occurred. In the present study the blastogenic responsiveness of spleen cells to endotoxin itself as well as to the nonspecific T-cell mitogen Con A was markedly decreased after several days of continuous administration of endotoxin. Control animals receiving only saline for the same period of time showed a similar depression of blastogenic responsiveness to the lipopolysaccharide (LPS), as well as to Con A, however, with a delay of 2-4 days before comparable levels of suppression became evident. These results indicate that marked alterations of immune competence as measured by blastogenesis of spleen cells to Escherichia coli LPS and to a mitogen such as Con A may occur after implantation of an osmotic pump, with or without continuous infusion of endotoxin. Further studies seem warranted to determine the role of the foreign body reaction to the osmotic pump as well as to the endotoxin administered by the pump.     

High colony-forming efficiency of primary human tumor cells cultured in the adhesive-tumor-cell culture system: improvements with medium and serum alterations

The colony-forming efficiency (CFE) of primary human tumor cells cultured in the adhesive-tumor-cell culture system (ATCCS) using Ham's F12 (F12) or Eagle's minimum essential medium, alpha modification (alphaMEM) and culture medium supplemented with either swine, equine or bovine sera were compared. AlphaMEM supplemented with equine serum provided the highest CFE of the combinations. The CFE increase due to the change from F12 to alphaMEM was approximately 5-fold, and the increase due to the change in serum from swine to equine was approximately 2-fold. Cytokeratin staining showed that this increase was not due to fibroblast growth. The high-average CFE with alphaMEM, approximately 3%, means that an inoculum of only 2 X 10(3) cells is needed to achieve formation of approximately 65 colonies in control cultures, thereby increasing the performance of this system when used in a chemosensitivity assay.     

Lipoxygenase and cyclooxygenase blockade by BW755C prevents endotoxin-induced hypotension but not changes in glucose metabolism

Arachidonic acid metabolites are mediators of various pathophysiologic events following endotoxin administration. However, their role in the endotoxin-induced increase in glucose metabolism has not been examined. Rats were administered either saline or BW755C (an inhibitor of both the cyclooxygenase and lipoxygenase pathways) 30 min prior to injection of E. coli endotoxin and whole body glucose kinetics assessed using a constant iv infusion of [6-3H] glucose. Treatment with BW755C prevented the endotoxin-induced hypotension and tachycardia. Endotoxin produced characteristic increases in the plasma glucose (23-70%) and lactate (2- to 9-fold) concentrations, as well as elevations in the rate of glucose appearance (34-63%) and metabolic clearance (40-92%). In contrast to the amelioration in hemodynamics, pretreatment with BW755C did not prevent these alterations in glucose metabolism normally seen after endotoxin. BW755C markedly reduced the endotoxin-induced increase in plasma catecholamine concentrations, but levels were still elevated 2- to 4-fold compared to control values. The results suggest that arachidonic acid metabolites mediate the early hypotensive response following endotoxin, but are not by themselves responsible for the elevated rates of glucose production and utilization.     

Molecular defect in factor IXBm Lake Elsinore. Substitution of Ala390 by Val in the catalytic domain

Earlier studies with factor IXBm Lake Elsinore (IXBmLE), a nonfunctional variant of factor IX, suggested that the defect in this protein may reside in the catalytic domain of the molecule (Usharani, P., Warn-Cramer, B. J., Kasper, C. K., and Bajaj, S. P. (1985) J. Clin. Invest. 75, 76-83). In this report, genomic DNA fragments from normal IX and IXBmLE alleles were cloned into phage lambda EMBL3 and the recombinant phage identified using normal IX cDNA and synthetic oligonucleotides. Exons VI, VII, and VIII of normal IX and IXBmLE gene were also amplified using a newly developed primer-directed polymerase chain reaction method. All eight exons and flanking regions of the normal IX and IXBmLE gene were sequenced by the dideoxy chain termination method. Comparison of the normal IX and IXBmLE sequences revealed a single base substitution (C----T) in the exon VIII of the BmLE variant, which results in the replacement of Ala390 by Val in the variant molecule. Although this mutation is in the catalytic domain of the molecule, purified factor IXaBmLE is indistinguishable from normal IXa in its activity toward a small synthetic substrate, L-tosylarginine methyl ester. These data, coupled with the previous data, identify a region (around residue 390) in the normal factor IXa which appears to play a major role in the extended macromolecular substrate binding site.     

Ca2+/calmodulin-dependent microtubule-associated protein 2 kinase: broad substrate specificity and multifunctional potential in diverse tissues

In previous studies, we described a soluble Ca2+/calmodulin-dependent protein kinase which is the major Ca2+/calmodulin-dependent microtubule-associated protein 2 (MAP-2) kinase in rat brain [Schulman, H. (1984) J. Cell Biol. 99, 11-19; Kuret, J. A., & Schulman, H. (1984) Biochemistry 23, 5495-5504]. We now demonstrate that this protein kinase has broad substrate specificity. Consistent with a multifunctional role in cellular physiology, we show that in vitro the enzyme can phosphorylate numerous substrates of both neuronal and nonneuronal origin including vimentin, ribosomal protein S6, synapsin I, glycogen synthase, and myosin light chains. We have used MAP-2 to purify the enzyme from rat lung and show that the brain and lung kinases have nearly indistinguishable physical and biochemical properties. A Ca2+/calmodulin-dependent protein kinase was also detected in rat heart, rat spleen, and in the ring ganglia of the marine mollusk Aplysia californica. Partially purified MAP-2 kinase from each of these three sources displayed endogenous phosphorylation of a 54 000-dalton protein. Phosphopeptide analysis reveals a striking homology between this phosphoprotein and the 53 000-dalton autophosphorylated subunit of the major rat brain Ca2+/calmodulin-dependent protein kinase. The enzymes phosphorylated MAP-2, synapsin I, and vimentin at peptides that are identical with those phosphorylated by the rat brain kinase. This enzyme may be a multifunctional Ca2+/calmodulin-dependent protein kinase with a widespread distribution in nature which mediates some of the effects of Ca2+ on microtubules, intermediate filaments, and other cellular constituents in brain and other tissues.     

A comparative description of mitochondrial DNA differentiation in selected avian and other vertebrate genera

Levels of mitochondrial DNA (mtDNA) sequence divergence between species within each of several avian (Anas, Aythya, Dendroica, Melospiza, and Zonotrichia) and nonavian (Lepomis and Hyla) vertebrate genera were compared. An analysis of digestion profiles generated by 13-18 restriction endonucleases indicates little overlap in magnitude of mtDNA divergence for the avian versus nonavian taxa examined. In 55 interspecific comparisons among the avian congeners, the fraction of identical fragment lengths (F) ranged from 0.26 to 0.96 (F = 0.46), and, given certain assumptions, these translate into estimates of nucleotide sequence divergence (p) ranging from 0.007 to 0.088; in 46 comparisons among the fish and amphibian congeners, F values ranged from 0.00 to 0.36 (F = 0.09), yielding estimates of P greater than 0.070. The small mtDNA distances among avian congeners are associated with protein-electrophoretic distances (D values) less than approximately 0.2, while the mtDNA distances among assayed fish and amphibian congeners are associated with D values usually greater than 0.4. Since the conservative pattern of protein differentiation previously reported for many avian versus nonavian taxa now appears to be paralleled by a conservative pattern of mtDNA divergence, it seems increasingly likely that many avian species have shared more recent common ancestors than have their nonavian taxonomic counterparts. However, estimates of avian divergence times derived from mtDNA- and protein-calibrated clocks cannot readily be reconciled with some published dates based on limited fossil remains. If the earlier paleontological interpretations are valid, then protein and mtDNA evolution must be somewhat decelerated in birds. The empirical and conceptual issues raised by these findings are highly analogous to those in the long-standing debate about rates of molecular evolution and times of separation of ancestral hominids from African apes.      

Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes

Statistical methods for computing the standard errors of the branching points of an evolutionary tree are developed. These methods are for the unweighted pair-group method-determined (UPGMA) trees reconstructed from molecular data such as amino acid sequences, nucleotide sequences, restriction-sites data, and electrophoretic distances. They were applied to data for the human, chimpanzee, gorilla, orangutan, and gibbon species. Among the four different sets of data used, DNA sequences for an 895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979) gave the least reliable one. The DNA sequence data suggested that the chimpanzee is the closest and that the gorilla is the next closest to the human species. The orangutan and gibbon are more distantly related to man than is the gorilla. This topology of the tree is in agreement with that for the tree obtained from chromosomal studies and DNA-hybridization experiments. However, the difference between the branching point for the human and the chimpanzee species and that for the gorilla species and the human-chimpanzee group is not statistically significant. In addition to this analysis, various factors that affect the accuracy of an estimated tree are discussed.      

Regulation of substrate oxidation in isolated myocardial cells by beta-hydroxybutyrate

The role of ketone bodies in myocardial substrate oxidation was examined using freshly isolated Ca2+-tolerant heart myocytes, beta-hydroxybutyrate (beta OHB) inhibited lactate oxidation by the myocytes by 30-60%, and the inhibition was concentration dependent. Palmitate oxidation was also markedly decreased, whereas octanoate oxidation was only minimally affected by the presence of beta OHB. Lactate, octanoate, or palmitate had little, if any, effect on beta OHB oxidation. beta OHB oxidation was reduced by 22-28% in myocytes isolated from chronically diabetic rats, whereas the oxidation of palmitate remained similar to the controls. However, beta OHB still inhibited palmitate oxidation to the same extent as in the control cells. Our data support the role of beta OHB as a physiologic regulator of myocardial substrate metabolism.