Inhibition of neutrophil superoxide production by human plasma alpha 1-antitrypsin.

We report here that human plasma alpha 1-antitrypsin (alpha 1-AT) inhibited human neutrophil O2.- release elicited by a variety of stimulants. In comparison, the inhibitory capacities of two serine protease inhibitors, L-1-tosylamide 2-phenylethyl chloromethyl ketone (TPCK) and soybean trypsin inhibitor (SBTI), and the human recombinant alpha 1-AT mutant, alpha 1-AT-Arg358 were in the order: alpha 1-AT = TPCK much greater than alpha 1-AT-Arg358 greater than SBTI when cells were stimulated with concanavalin A plus cytochalasin E. These data suggest that, in human inflammatory fluids containing relatively high concentrations of alpha 1-AT (such as rheumatoid arthritis synovial fluid), (i) alpha 1-AT may down-regulate the inflammatory process by inhibiting the neutrophil respiratory burst and (ii) serpin oxidation by neutrophil-released reactive oxygen species is unlikely to occur.

In vivo effects of thymic hormones (Imunotim) on immunologic parameters of patients with secondary immunodeficiency diseases.

In previous papers we revealed the immuno-restorative capacity of thymic hormones both in vitro and in vivo. This paper is concerned with the investigation in a phase I trial of the effects of a thymic extract (Imunotim) prepared in the Cantacuzino Institute, Bucharest upon patients with immunodeficiencies manifest as recurrent or persistent infections resistant to classical treatments. Twenty five testings were performed on 23 patients with secondary immunodepressions: carcinomas with immunodepressions following the acute phase of the disease or cytoreductive treatments (10 cases), allergic and autoimmune conditions (3 cases) and frequent, recurrent infections with prolonged evolution. In 73.9% of cases (17/23) after administration of Imunotim per os the immunologic parameters showed a tendency to return to normal values. In patients with low pre-therapy values the following were noticed: a remarkable increase of the number of lymphocytes from 1317.2 +/- 506.40 to 1961.1 +/- 899.11 (p < 0.01), of PMN from 1401.7 +/- 444.21 to 2651.0 +/- 755.31 (p < 0.01), of the absolute number of B lymphocytes from 192.0 +/- 79.00 to 444.8 +/- 299.75 (p < 0.05); the rise in the rate and absolute number of total T lymphocytes (628.2 +/- 192.81, 1041.0 +/- 441.84, respectively; p < 0.05), T helper (from 29.84 +/- 12.75% to 44.14 +/- 18.76%, respectively from 421.6 +/- 127.29 to 1058.7 +/- 411.05; p < 0.01 respectively p < 0.02) and the rise of the absolute number of T suppressor/cytotoxic lymphocytes from 92.8 +/- 97.11 to 368.3 +/- 368.53 (p < 0.05). In some patients with lymphocytosis and adenopathies the absolute lymphocyte number and the maturation of the lymphocyte populations and subpopulations in blood showed a decline. Individual variations could be noticed even within one and the some primary affection.

Zinc aspartate in vivo and in vitro modulation of reactive oxygen species production by human neutrophils and monocytes.

The involvement of zinc ions in cell metabolic processes and the immunopathologic consequences of zinc deficiency are well known. We investigated the effect of zinc aspartate upon production of reactive oxygen species (ROS) by monocytes and polymorphonuclear cells isolated from healthy subjects and patients with leukemia and rheumatoid arthritis. The cells were stimulated in vitro with serum-treated zymosan, aggregated IgG, aggregated and opsonized IgG and digitonin. Zinc concentrations of 10(-4)M do not influence the in vitro release of ROS by polymorphonuclears but moderately activate the monocytes. Following treatment with orally administered zinc aspartate, monocytes from leukemia patients reveal an increased capacity to release ROS after in vitro stimulation. In patients with rheumatoid arthritis monocytes usually produce abnormally high ROS amounts after in vitro stimulation; the treatment with zinc aspartate does not induce considerable alterations of this capacity; however a tendency to restore the normal values is manifest.

Substitution of an alanine residue for glycine 146 in TMP kinase from Escherichia coli is responsible for bacterial hypersensitivity to bromodeoxyuridine.

The wild-type TMP kinases from Escherichia coli and from a strain hypersensitive to 5-bromo-2'-deoxyuridine were characterized comparatively. The mutation at codon 146 causes the substitution of an alanine residue for glycine in the enzyme, which is accompanied by changes in the relative affinities for 5-Br-UMP and TMP compared to those of the wild-type TMP kinase. Plasmids carrying the wild-type tmk gene from Escherichia coli or Bacillus subtilis, but not the defective tmk gene, restored the resistance to bromodeoxyuridine of an E. coli mutant strain.

Mutational analysis of UMP kinase from Escherichia coli.

UMP kinase from Escherichia coli is one of the four regulatory enzymes involved in the de novo biosynthetic pathway of pyrimidine nucleotides. This homohexamer, with no counterpart in eukarya, might serve as a target for new antibacterial drugs. Although the bacterial enzyme does not show sequence similarity with any other known nucleoside monophosphate kinase, two segments between amino acids 35 to 78 and 145 to 194 exhibit 28% identity with phosphoglycerate kinase and 30% identity with aspartokinase, respectively. Based on these similarities, a number of residues of E. coli UMP kinase were selected for site-directed mutagenesis experiments. Biochemical, kinetic, and spectroscopic analysis of the modified proteins identified residues essential for catalysis (Asp146), binding of UMP (Asp174), and interaction with the allosteric effectors, GTP and UTP (Arg62 and Asp77).

Application of methionine as a detector molecule for the assessment of oxygen radical generation by human neutrophils and endothelial cells.

Diverse cell types can generate reactive oxygen species (ROS) which are implicated in many disease processes and are ascribed both beneficial and deleterious roles. In vitro studies of this phenomenon indicate that properties of the microenvironment in culture influence the cells' behaviour with regard to ROS generation in vivo. To date, however, the assessment of cellular ROS generation has been limited to techniques which are invasive of the culture environment, or require cells to be in suspension. This study describes the application of NMR spectroscopy to the detection of ROS generation, a technique which is non-invasive of the cell culturing environment.

Structural properties of UMP-kinase from Escherichia coli: modulation of protein solubility by pH and UTP.

UMP-kinase from Escherichia coli, unlike the analogous enzyme from eukaryotic organisms, is an oligomeric protein subjected to complex regulatory mechanisms in which UTP and GTP act as allosteric effectors. While the enzyme has an unusually low solubility at neutral pH (< or = 0.1 mg of protein/ mL), its solubility increases markedly above pH 8 and below pH 4. Furthermore, the solubility of the bacterial UMP-kinase at neutral pH is greatly enhanced in the presence of Mg-free UTP. Thermal denaturation experiments have demonstrated that UTP also increases the stability of the protein. Fourier-transform infrared spectroscopy and circular dichroism show that the secondary structure of the protein is the same at neutral and at alkaline pH. These data indicate that variations in enzyme solubility must be related to subtle changes in the tertiary and/or quaternary structure which modulate the exposure of hydrophobic surfaces in the protein molecule. A variant of UMP-kinase, obtained by site-directed mutagenesis (Asp159Asn), which is similar to the wild-type enzyme in its stability and kinetic properties, has a much increased water solubility (> 5 mg protein/mL) even at neutral pH. This suggests that salt bridges may be involved in the equilibrium between the soluble and aggregated forms of the wild-type enzyme, and that conformational changes induced upon binding of UTP increase the protein solubility by disrupting these salt bridges.

CMP kinase from Escherichia coli is structurally related to other nucleoside monophosphate kinases.

CMP kinase from Escherichia coli is a monomeric protein of 225 amino acid residues. The protein exhibits little overall sequence similarities with other known NMP kinases. However, residues involved in binding of substrates and/or in catalysis were found conserved, and sequence comparison suggested conservation of the global fold found in adenylate kinases or in several CMP/UMP kinases. The enzyme was purified to homogeneity, crystallized, and analyzed for its structural and catalytic properties. The crystals belong to the hexagonal space group P6(3), have unit cell parameters a = b = 82.3 A and c = 60.7 A, and diffract x-rays to a 1.9 A resolution. The bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 328 nm upon excitation at 295 nm, which suggests that the single tryptophan residue (Trp30) is located in a hydrophobic environment. Substrate specificity studies showed that CMP kinase from E. coli is active with ATP, dATP, or GTP as donors and with CMP, dCMP, and arabinofuranosyl-CMP as acceptors. This is in contrast with CMP/UMP kinase from Dictyostelium discoideum, an enzyme active on CMP or UMP but much less active on the corresponding deoxynucleotides. Binding of CMP enhanced the affinity of E. coli CMP kinase for ATP or ADP, a particularity never described in this family of proteins that might explain inhibition of enzyme activity by excess of nucleoside monophosphate.

Genetic and biochemical characterization of Salmonella enterica serovar typhi deoxyribokinase.

We identified in the genome of Salmonella enterica serovar Typhi the gene encoding deoxyribokinase, deoK. Two other genes, vicinal to deoK, were determined to encode the putative deoxyribose transporter (deoP) and a repressor protein (deoQ). This locus, located between the uhpA and ilvN genes, is absent in Escherichia coli. The deoK gene inserted on a plasmid provides a selectable marker in E. coli for growth on deoxyribose-containing medium. Deoxyribokinase is a 306-amino-acid protein which exhibits about 35% identity with ribokinase from serovar Typhi, S. enterica serovar Typhimurium, or E. coli. The catalytic properties of the recombinant deoxyribokinase overproduced in E. coli correspond to those previously described for the enzyme isolated from serovar Typhimurium. From a sequence comparison between serovar Typhi deoxyribokinase and E. coli ribokinase, whose crystal structure was recently solved, we deduced that a key residue differentiating ribose and deoxyribose is Met10, which in ribokinase is replaced by Asn14. Replacement by site-directed mutagenesis of Met10 with Asn decreased the V(max) of deoxyribokinase by a factor of 2.5 and increased the K(m) for deoxyribose by a factor of 70, compared to the parent enzyme.