Anti-inflammatory actions of endogenous and exogenous interleukin-10 versus glucocorticoids on macrophage functions of the newly born.

OBJECTIVE: To determine whether specific macrophage immune functions of the newly born are insensitive to the actions of therapeutic levels of dexamethasone (DEX), previously measured in infants with bronchopulmonary dysplasia (BPD), compared with betamethasone (BETA) and exogenous or endogenous interleukin-10 (IL-10). STUDY DESIGN: Macrophages were differentiated from cord blood monocytes (N=18). A serial dose-response (around 10(-8 )M), in vitro study was used to examine the effect of DEX, BETA and IL-10, on proinflammatory (PI) cytokine release, phagocytosis and respiratory burst. RESULT: Exogenous IL-10 (10(-8 )M) significantly (P<0.05) inhibited the endotoxin-stimulated release of IL-6, IL-8 and tumor necrosis factor by 63 to 82% with no significant effect by DEX and BETA. There was no inhibition by these three agents at 10(-8 )M on phagocytosis and respiratory burst. Inhibition of endogenous IL-10 with a monoclonal antibody significantly increased endotoxin-stimulated cytokine release by at least fourfold. CONCLUSION: Macrophages were relatively insensitive to therapeutic levels of DEX and BETA with regard to PI cytokine release. This study provides rationale for translational and preclinical research using airway instillation of IL-10 for the treatment of BPD.

NF-kappa B activation in tumor necrosis factor alpha-stimulated neutrophils is mediated by protein kinase Cdelta. Correlation to nuclear Ikappa Balpha.

The transcription factor NF-kappaB is critical for the expression of multiple genes involved in inflammatory responses and apoptosis. However, the signal transduction pathways regulating NF-kappaB activation in human neutrophils in response to stimulation with tumor necrosis factor-alpha (TNFalpha) are undefined. Since recent studies implicated activation of NF-kappaB as well as protein kinase C-delta (PKCdelta) in neutrophil apoptosis, we investigated involvement of PKCdelta in the activation of NF-kappaB in TNFalpha-stimulated neutrophils. Specific inhibition of PKCdelta by rottlerin prevented IkappaBalpha degradation and NF-kappaB activation in TNFalpha-stimulated neutrophils. This regulation of NF-kappaB activation by PKCdelta was specific only for TNFalpha signaling, since lipopolysaccharide- or interleukin-1beta-induced NF-kappaB activation and IkappaBalpha degradation were not inhibited by rottlerin. In addition, we show that in human neutrophils, but not monocytes, IkappaBalpha localizes in significant amounts in the nucleus of unstimulated cells, and the amount of IkappaBalpha in the nucleus, as well as in the cytoplasm, correlates with the NF-kappaB DNA binding. These results suggest that in human neutrophils, the presence of IkappaBalpha in the nucleus may function as a safeguard against initiation of NF-kappaB dependent transcription of pro-inflammatory and anti-apoptotic genes, and represents a distinct and novel mechanism of NF-kappaB regulation.

Activation of nuclear factor-kappaB and its suppression by dexamethasone in polymorphonuclear leukocytes: newborn versus adult.

Polymorphonuclear leukocytes (PMN) of the newborn, to a greater extent than those of the adult, have the ability to amplify PMN recruitment to an inflammatory site by their own release of IL-8, and this process is inhibited by dexamethasone. The aim of the present study was to determine whether the regulation of nuclear factor-kappaB (NF-kappaB) could explain the previous observations. NF-kappaB is a transcription factor pivotal for expression of genes encoding inflammatory cytokines such as IL-8, but NF-kappaB has not been previously studied in the PMN of the newborn. NF-kappaB activation was measured by an electrophoretic mobility shift assay in nuclear extracts prepared from PMN isolated from adults and cord blood from newborns. Two distinct molecular forms of NF-kappaB were identified after tumor necrosis factor-alpha stimulation; this included the previously characterized p50/65 heterodimer and a newly identified p50/50 homodimer. Both NF-kappaB dimers were activated by tumor necrosis factor-alpha to significantly higher levels in the neutrophil of the newborn versus adult. An additional new finding was that pretreatment of PMN with dexamethasone (10(-7) M, therapeutic range) inhibited activation of both NF-kappaB complexes in both the newborn and the adult PMN. We conclude that the increased activation of NF-kappaB by the PMN of the newborn may play an important role in neonatal inflammatory reactions. Eventually, specific targeting of NF-kappaB activation in the neutrophil may be an effective molecular approach for the treatment of neutrophil-mediated disorders in the newborn.

Development of the intestinal SGLT1 transporter in rats.

Glucose absorption from the small intestine is largely mediated via the sodium-coupled glucose transporter (SGLT1). The goal of this study was to investigate the ontogenesis of the SGLT1, using the rat as an animal model at three stages of development: during lactation, at weaning, and at physiologic maturity. The techniques involved upper small intestinal perfusions with solutions containing 200 mM glucose and 50 mM NaCl, with or without 1 mM phloridzin (Phl), as an inhibitor of SGLT1. Molecular expression of the SGLT1 was also investigated via Western blot analysis from intestinal specimens of the three growth periods. Glucose absorption in weanling rats, in the absence of Phl, was several times higher than in sucklings and approximately double that of mature animals, and the effects of Phl were the greatest in weanlings. Furthermore, the physiologic data correlate to the molecular analysis of the SGLT1 which showed an increase in expression of the SGLT1 in both the weanlings and the adults compared to the sucklings. At all three stages of development Phl abolished Na absorption, and in sucklings there was a net outflow of Na. Due to the coupling between Na and water transport, net water absorption and the influx/efflux ratio, a more sensitive indicator of changes in unidirectional fluid movement, were similarly affected by Phl at the three stages of development. Net water absorption was highest in weanling animals. These findings are consistent with an early development of SGLT1 in rat small intestine and an apparent burst of activity at weaning. Less than complete maturity of other absorptive mechansims is occurring at this time.

Regulation of phosphatidylinositol 4-phosphate 5-kinase from Schizosaccharomyces pombe by casein kinase I.

Phosphatidylinositol ()P 5-kinase (PtdIns(4)P 5-kinase) catalyzes the last step in the synthesis of phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2). PtdIns(4,5)P2 is a precursor of diacylglycerol and inositol 1,4,5-trisphosphate and is also involved in regulation of actin cytoskeleton remodeling and membrane traffic. To satisfy such varied demands in several aspects of cell physiology, synthesis of PtdIns(4,5)P2 must be stringently regulated. In this paper we describe extraction, purification, and characterization of PtdIns(4)P 5-kinase from the plasma membranes of Schizosaccharomyces pombe. We also provide evidence that PtdIns(4)P 5-kinase is phosphorylated and inactivated by Cki1, the S. pombe homolog of casein kinase I. Phosphorylation by Cki1 in vitro decreases the activity of PtdIns(4)P 5-kinase. In addition, and most importantly, overexpression of Cki1 in S. pombe results in a reduced synthesis of PtdIns(4,5)P2 and in a lower activity of PtdIns(4)P 5-kinase associated with the plasma membrane. These results suggest that PtdIns(4)P 5-kinase is a target of Cki1 in S. pombe and that Cki1 is involved in regulation of PtdIns(4, 5)P2 synthesis by phosphorylating and inactivating PtdIns(4)P 5-kinase.

A domain distinct from nucleoplasmin's nuclear localization sequence influences its transport.

We constructed mutants of the prototypical, nuclear-accumulating protein nucleoplasmin and used them in both in vivo and in vitro nuclear transport assays to search for transport-influencing domains distinct from this protein's recognized nuclear localization sequence. We identified the polyglutamic acid tract on the amino flank of the nuclear localization sequence as being involved in two stages of nuclear transport. This poly-glu tract is required for the facilitated translocation of nucleoplasmin through the nuclear pore complex, and it also enhances the subsequent binding of nucleoplasmin within the nucleus.

Distinct phosphorylation sites differentially influence facilitated transport of an NLS-protein and its subsequent intranuclear binding.

We measured the nuclear transport of radiolabeled fusion proteins consisting of variants of the Simian Virus 40 large T antigen's nuclear localization sequence region linked to beta-galactosidase, itself a cytoplasmic protein. We microinjected the fusion protein variants into the cytoplasm of living Xenopus oocytes or supplied them to the surface of oil-isolated oocyte nuclei via paired beads or cytoplasm. Presence of the cdc2 kinase site (124T) on the amino flank of the nuclear localization sequence (126PKKKRKV132) greatly enhances facilitated transport through the nuclear pore complex; additional presence of the casein kinase II site (112S) enhances subsequent intranuclear binding.

Intranuclear binding of nucleoplasmin.

Many proteins--including not only structural proteins, but also enzymes, hormone receptors, and other transcription factors--accumulate to much higher nuclear than cytoplasmic concentrations. Nuclear localization sequences or signals (NLSs) within their primary structures entrain specific transport of these proteins through the nuclear pore complexes. This transport process is energy-dependent, but evidence for a true active transport mechanism is not conclusive. An alternative mechanism--facilitated transport of NLS proteins followed by their intranuclear binding--has been implicated by experiments with oil-isolated nuclei. However, there has been no agreement as to a role for binding in the in vivo nuclear accumulation of NLS-containing proteins. We demonstrate herein that a prototypical NLS protein, nucleoplasmin (Np), binds within the nucleus of the living Xenopus oocyte and that this binding accounts for its nuclear accumulation.

Nucleoplasmin associates with and is phosphorylated by casein kinase II.

Nucleoplasmin is a phosphorylated nuclear-accumulating protein. We report herein that the kinetics of its cytoplasm-->nucleus transport are affected by its degree of phosphorylation. Therefore, we sought to identify any protein kinase which specifically associates with nucleoplasmin. We discovered that nucleoplasmin co-isolates by two independent methods (immunoabsorption and chromatography) in a complex including a kinase which phosphorylates nucleoplasmin. The co-purifying kinase is casein kinase II-like because: (i) it phosphorylates casein; (ii) its phospho-transferase activity can be competed out by GTP; (iii) it is stimulated by polylysine; and (iv) it is inhibited by heparin. Moreover, a polyclonal antibody to the alpha (38 kDa) and alpha' (36 kDa) catalytic subunits of casein kinase II specifically recognizes 38 and 36 kDa polypeptides in the nucleoplasmin-complex, and a specific inhibitor of casein kinase II inhibits nucleoplasmin's nuclear transport. Additionally, we found that phosphorylation of nucleoplasmin by its associated casein kinase II is strongly inhibited by histones and that, in addition to nucleoplasmin, another protein (p100) in the nucleoplasmin-complex is phosphorylated by casein kinase II.

An NLS is sufficient to engage facilitated translocation by the nuclear pore complex and subsequent intranuclear binding.

We investigated the nuclear transport of a fusion protein consisting of a nuclear localization signal linked to beta-galactosidase, normally a cytoplasmic protein. We microinjected the radiolabeled fusion protein into the cytoplasm of living Xenopus oocytes or supplied it directly to the surface of the oil-isolated oocyte nucleus and measured its transport into the nucleus. Our data confirm that a nuclear localization signal is sufficient to entrain a protein's facilitated transport through the nuclear pore complex and its subsequent nuclear accumulation. Moreover, nuclear envelope micropuncture experiments determine that the fusion protein's accumulation results from its intranuclear binding, demonstrating that no specific region of a transported protein--other than the nuclear localization signal itself--is required for facilitated transport and intranuclear binding. Finally, we present evidence that the intranuclear binding of a transported protein requires not only its nuclear localization signal, but also its prior facilitated transport through the nuclear pore complex.

Nucleoplasmin uptake by facilitated transport and intranuclear binding.

Specific proteins are selectively translocated into the cell nucleus and accumulated therein, but the molecular mechanisms underlying this fundamental eukaryotic transport process remain obscure. We have employed a new experimental system with notable advantages for resolving protein translocation and accumulation mechanisms. Individual nuclei are isolated from oocytes under mineral oil and conjoined under the oil with either an aqueous bead or a similar volume of oocyte cytoplasm to form closed transport pairs. Using these pairs one can (i) present transportant proteins via the bead or cytoplasm to a minimally disturbed nucleus, (ii) monitor the intactness of the nuclear envelope, and (iii) separate pairs at various times after formation and measure the amount of transportant in each compartment. In addition, it is uniquely possible with these pairs to determine whether or not a transportant's concentration gradient constitutes a chemical activity gradient. This is done by puncturing the envelope, thus eliminating its normal sieving restrictions on diffusion, and measuring the effect on the transportant distributions. We demonstrate that a prototypical nuclear-accumulating protein, nucleoplasmin (Np), is translocated through the nuclear pore complex by a mechanism of facilitated transport, rather than active transport. We further show that Np's high accumulation results from subsequent intranuclear binding. Np's facilitated transport and intranuclear binding are both ATP-dependent, and the latter requires cytoplasmic protein(s).

Taxonomic studies of Streptomyces virginiae mutants overproducing virginiamycin M1.

By using both the traditional International Streptomycetes Project methods and chemical approaches followed by a hierarchical cluster analysis, Streptomyces virginiae mutants A-1 and B-43 (yielding higher amounts of the M1 component of virginiamycin complex), their wild ancestor ATCC 13161, and another virginiamycin producer, S. pristinaespiralis NRRL 2958, were subjected to taxonomic studies to find kinship or differences among the strains. Of the methods used, only the test of carbon utilization, investigation of spore surface and analysis of sugar constituents of cell walls proved to be reliable enough to demonstrate the species identity of S. virginiae strains and to distinguish them from S. pristinaespiralis. L,L-2,6-Diaminopimelic acid was present in all strains. Analysis of fatty acids and total proteins as well as investigations of morphology and pigmentation of agar cultures led to confusing results.

A DNA-activated protein kinase from HeLa cell nuclei.

A DNA-activated protein kinase (DNA-PK) was purified from nuclei of HeLa cells. Activity was associated with a single high-molecular-mass (approximately-300,000 Da) polypeptide when analyzed by gel filtration, denaturing polyacrylamide gel electrophoresis, and Western immunoblotting using a monoclonal antibody that also inhibits enzyme activity. Nuclear localization was indicated by subcellular fractionation and confirmed by immunofluorescence on whole cells. Double-stranded DNA stimulated phosphorylation of the 300-kDa polypeptide in purified preparations as well as phosphorylation of the exogenous substrates alpha-casein, simian virus 40 large T antigen, and the human heat shock protein hsp90. Autophosphorylation led to inactivation of the enzyme. The phosphorylation of casein was stimulated over 30-fold by DNA and was specific for serine and threonine residues. Bovine serum albumin and histone H1 were poor substrates for DNA-PK, and no phosphorylation of immunoglobulin G or histones other than H1 was observed. Supercoiled or heat-denatured DNA and synthetic double-stranded RNA or RNA-DNA copolymers did not stimulate casein phosphorylation by DNA-PK. Interaction of the enzyme with DNA in the absence of exogenous substrates was demonstrated by thermal inactivation and gel mobility shifts. These characteristics identify DNA-PK as distinct from other protein kinases described in the literature and suggest that activation by DNA is an important feature of the enzyme's in vivo function.

Purification and properties of NADP-dependent glutamate dehydrogenase from Streptomyces fradiae.

Streptomyces fradiae has two chromatographically distinct forms of glutamate dehydrogenase (GDH): one GDH utilizes NAD as coenzyme, the other uses NADP. The intracellular level of both GDHs is strongly regulated by the nitrogen source in the growth medium. NADP-dependent GDH was purified to homogeneity from crude extracts of S. fradiae. The Mr of the native enzyme was determined to be 200,000 by size-exclusion high-performance liquid chromatography whereas after sodium dodecyl sulphate-polyacrylamide gel electrophoresis one major band of Mr 49,000 was found, suggesting that the enzyme is a tetramer. The enzyme was highly specific for the substrates 2-oxoglutarate and L-glutamate, and required NADP, which could not be replaced by NAD, as a cofactor. The pH optimum was 9.2 for oxidative deamination of glutamate and 8.4 for reductive amination of 2-oxoglutarate. The Michaelis constants (Km) were 28.6 mM for L-glutamate and 0.12 mM for NADP. Km values for reductive amination were 1.54 mM for 2-oxoglutarate, 0.07 mM for NADPH and 30.8 mM for NH+4. The enzyme activity was significantly reduced by adenine nucleotides, particularly ATP.

A direct-injection reversed-phase liquid chromatographic micromethod for studying the kinetics of terminal reactions of tetracycline biosynthesis.

A new micromethod for measuring enzyme-catalyzed reactions was developed. The method involves a number of consecutive direct injections of aliquots of the reaction mixture onto a microbore column and permits the determination of the time dependence of the decrease of substrate or increase of product concentrations. The reactions proceed in a microvial placed in the autosampler, and as the starting volume can be as low as 10 microliters, the requirement for the amount of enzyme is very low. The autosampler backed by the liquid chromatographic software allows automation of the analyses including data processing and easy quantitation of the enzymatic reaction(s). The method was applied to a system of two consecutive terminal reactions of tetracycline biosynthesis in Streptomyces aureofaciens, catalyzed by anhydrotetracycline oxygenase and tetracycline dehydrogenase. The usage of a diode-array detector facilitated the quantification of the reactions as the product and substrate could be monitored at their optimal wavelengths.

A further characterization of alanine dehydrogenase from Streptomyces aureofaciens.

Homogeneous alanine dehydrogenase isolated from Streptomyces aureofaciens, a producer of tetracycline, was characterized from the point of its molecular and catalytic properties. Using analytical ultracentrifugation the molecular weight of alanine dehydrogenase was found to be 198,000. The enzyme could use as cofactors apart from NAD+ also 1,N6-etheno-NAD+, 3-acetylpyridine-NAD+, deamino-NAD+ and nicotinamide guanine dinucleotide. The enzyme activity in the direction of oxidative deamination was not affected by the addition of nonsubstrate amino acids, however, it was sensitive to inhibitors of SH-groups. Reductive amination of pyruvate was inhibited by L-alanine, L-serine and D-alanine. The inhibition by L-alanine and L-serine was uncompetitive with respect to NADH and noncompetitive with regard to pyruvate and ammonium ions.

Alanine dehydrogenase from Streptomyces fradiae. Purification and properties.

Alanine dehydrogenase was purified to homogeneity from a cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1180-fold to give a 21% yield, using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The relative molecular mass of the native enzyme was determined to be 210,000 or 205,000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of Mr 51,000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The Km were 10.0 mM for L-alanine and 0.18 mM for NAD+. Km values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH4+ and 0.05 mM for NADH. Oxidative deamination of L-alanine proceeds through a sequential-ordered binary-ternary mechanism in which NAD+ binds first to the enzyme, followed by alanine, and products are released in the order ammonia, pyruvate and NADH.

Valine dehydrogenase from Streptomyces fradiae: purification and properties.

Valine dehydrogenase (VDH) was purified to homogeneity from cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1508-fold in a 17.7% yield using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The Mr of the native enzyme was determined to be 218,000 and 215,000, by equilibrium ultracentrifugation and size-exclusion high-performance liquid chromatography, respectively. The enzyme is composed of 12 subunits of Mr 18,000. Using analytical isoelectric focusing the isoelectric point of VDH was found to be 4.7. Oxidative deamination of L-valine was optimal at pH 10.6. Reductive amination of 2-oxoisovalerate was optimal at pH 8.8. The Michaelis constants (Km) were 1 mM for L-valine and 0.029 mM for NAD+. Km values for reductive amination were 0.80 mM for 2-oxoisovalerate, 0.050 mM for NADH and 22 mM for NH4+.

Isolation and characterization of valine dehydrogenase from Streptomyces aureofaciens.

Valine dehydrogenase was purified to homogeneity from the crude extracts of Streptomyces aureofaciens. The molecular weight of the native enzyme was 116,000 by equilibrium ultracentrifugation and 118,000 by size exclusion high-performance liquid chromatography. The enzyme was composed of four subunits with molecular weights of 29,000. The isoelectric point was 5.1. The enzyme required NAD+ as a cofactor, which could not be replaced by NADP+. Sulfhydryl reagents inhibited the enzyme activity. The pH optimum was 10.7 for oxidative deamination of L-valine and 9.0 for reductive amination of alpha-ketoisovalerate. The Michaelis constants were 2.5 mM for L-valine and 0.10 mM for NAD+. For reductive amination the Km values were 1.25 mM for alpha-ketoisovalerate, 0.023 mM for NADH, and 18.2 mM for NH4Cl.

Isolation of pure anhydrotetracycline oxygenase from Streptomyces aureofaciens.

Anhydrotetracycline oxygenase was purified to homogeneity from Streptomyces aureofaciens, a producer of tetracycline. The enzyme was purified 60-fold in a 40% yield by a two-step procedure using a combination of hydrophobic chromatography and ion-exchange h.p.l.c. Purified anhydrotetracycline oxygenase was homogeneous according to SDS/polyacrylamide-gel electrophoresis, isoelectric focusing, ion-exchange h.p.l.c. on a Mono Q HR 5/5 column and size-exclusion h.p.l.c. on a TSK G 3000 SW column. The enzyme consists of two subunits of Mr 57,500, as determined by SDS/polyacrylamide-gel electrophoresis.