Identification of a specific region of low molecular weight phospholipases A2 (residues 21-40) as a potential target for structure-based design of inhibitors of these enzymes.

We have identified a specific region of porcine pancreatic phospholipase A2 (residues 21-40) which interacts with a neutralizing antibody causing a dramatic inhibition of its enzymatic activity (Ki in the order of 10(-8) M). The binding equilibrium of the antibody-phospholipase A2 complex is reached in < 3 min at 37 degrees C. Fab fragments are equally effective phospholipase A2 inhibitors, as are intact IgG molecules.

Human Clara cell 10-kDa protein is the counterpart of rabbit uteroglobin.

Human Clara cell 10-kDa protein has been suggested to be a counterpart of rabbit uteroglobin, an immunomodulatory and antiinflammatory secretory protein. Since this human protein is not readily available in substantial quantity for detailed characterization of its biochemical, biological, and pharmacological properties, we sought to express it in Escherichia coli in order to study its structure-function relationship. However, bacterial overproduction of homodimeric proteins with interchain disulfide bonds, such as Clara cell 10-kDa protein, was thought to be impossible until we achieved expression of native uteroglobin (Miele, L.

Transglutaminase-catalyzed incorporation of polyamines into phospholipase A2.

We have previously demonstrated that when phospholipase A2 is treated with either tissue transglutaminase or human plasma Factor XIIIa, a striking increase of its catalytic activity is observed, due to the formation of an intramolecular epsilon-(gamma-glutamyl)-lysine crosslink [Cordella-Miele et al. (1990) J. Biol.

Amino acid composition and N-terminal sequence of purified cystine binding protein of Escherichia coli.

Cystine Binding Protein (CBP), a commercially available crude protein extract obtained by osmotic shock of Escherichia coli (E. coli), was studied to characterize further its cystine binding properties and to elucidate its cystine transport activity. We report here the amino acid composition, the N-terminal amino acid sequence analysis and some binding characteristics of the purified cystine binding component of CBP.

Initial improvement as a criterion for drug choice in acute schizophrenia.

Several investigators have found that the initial improvement of acute schizophrenia after some days of neuroleptic treatment is correlated in a statistically significant way to the outcome after four weeks. In all these studies the question arises as to whether the correlation between early response and subsequent outcome is due to a specific response to a certain neuroleptic, or whether patients who respond early simply have a better prognosis. In order to isolate the specific drug effect from prognostic influences we performed a controlled double-blind study in 50 newly admitted schizophrenic inpatients.

Regulation of extracellular phospholipase A2 activity: implications for inflammatory diseases.

Phospholipases A2 (PLA2s; E.C.3.

Effects of antiflammins on endotoxin-induced uveitis in rats.

Antiflammins are phospholipase A2-inhibitory, anti-inflammatory, synthetic oligopeptides derived from the region of the highest amino-acid sequence similarity between uteroglobin and lipocortin I. Endotoxin-induced uveitis is a model for anterior uveitis of the eye, which has been suggested to be induced through phospholipase A2 activation. In a preliminary report we demonstrated that topical administration of antiflammins could inhibit endotoxin-induced uveitis in rats.

Inhibition of pancreatic phospholipase A2 activity by uteroglobin and antiflammin peptides: possible mechanism of action.

We investigated the possible mechanism of inhibition of porcine pancreatic phospholipase A2 in vitro by rabbit uteroglobin and by the antiflammin peptides. We optimized the conditions of phospholipase A2 assay using a deoxycholate-phosphatidylcholine mixed micellar substrate and established the activity of these inhibitors under optimized conditions. The results of fluorescence studies and crosslinking experiments indicate that the inhibitors interact with the enzyme in solution and affect the increase in intrinsic fluorescence of phospholipase A2 observed upon interaction with a mixed micellar substrate.

Gene organization and nucleotide sequence of the primase region of IncP plasmids RP4 and R751.

The primase genes of RP4 are part of the primase operon located within the Tra1 region of this conjugative plasmid. The operon contains a total of seven transfer genes four of which (traA, B, C, D) are described here. Determination of the nucleotide sequence of the primase region confirmed the existence of an overlapping gene arrangement at the DNA primase locus (traC) with in-phase translational initiation signals.

A novel transglutaminase-mediated post-translational modification of phospholipase A2 dramatically increases its catalytic activity.

Transglutaminases (TG), which include coagulation Factor XIIIa, are calcium-dependent ubiquitous enzymes. TGs catalyze the formation of an isopeptide bond by cross-linking a specific glutamine and a lysine residue between two proteins or within the same protein molecule. Phospholipase A2 (PLA2) is a key enzyme in the regulation of prostaglandin and leukotriene biosynthetic pathways, which catalyzes the release of free fatty acids from the sn-2 position of membrane glycerophospholipids.

High level bacterial expression of uteroglobin, a dimeric eukaryotic protein with two interchain disulfide bridges, in its natural quaternary structure.

Bacterial expression of eukaryotic proteins is a tool of ever-increasing importance in biochemistry and molecular biology. However, the majority of the recombinant eukaryotic proteins that have been expressed in bacteria are produced as fusion proteins and not in their native conformation. In particular, correct formation of quaternary structures by recombinant proteins in bacterial hosts has been reported very rarely.

Novel peptides derived from a region of local homology between uteroglobin and lipocortin-1 inhibit platelet aggregation and secretion.

The active site for uteroglobin inhibition of phospholipase A2 has been localized to a nonapeptide (P1) which is partially homologous to a nonapeptide (P2) in lipocortin, which also inhibits phospholipase A2. P1 and P2 share an identical tetrapeptide (P4) which is required for inhibition, although P4 alone does not inhibit this enzyme. We found the mechanism of inhibition of platelet aggregation and secretion by the nonapeptides and P4 varied depending on whether platelets were thrombin- or ADP-activated.

The molecular localization of non-tryptophan chromophores in calf lens crystallins.

A single-step separation of calf lens gamma-crystallin into six protein components is described. UV absorption spectra, characterized by the presence of high absorbance in the 240-250 nm and 310-360 nm spectral regions as well as by fluorescence emission above 400 nm, are shown by six components. alpha-, beta and beta S crystallins have been compared with the gamma-fraction for the presence of non-tryptophan fluorescence.

Novel anti-inflammatory peptides from the region of highest similarity between uteroglobin and lipocortin I.

Significant future developments in the effective treatment of inflammatory diseases may arise from non-toxic dual inhibitors of both cyclooxygenase and lipoxygenase pathways in the arachidonate cascade. Inhibition of phospholipase A2(PLA2)(EC3.1.

Presence of nontryptophan fluorophores specifically bound to gamma 2-crystallin.

alpha-, beta-, and gamma-crystallins have been purified from nonpathological lenses of calves. The pure proteins have been examined for nontryptophan fluorescence and fluorescent compounds have been found specifically bound to gamma 2-crystallin. The protein has been unfolded by 6 M guanidine hydrochloride (Gdn-HCl) and a separation of the fluorescent compounds has been obtained by gel chromatography in the presence of 6 M Gdn-HCl.

Uteroglobin: structure, molecular biology, and new perspectives on its function as a phospholipase A2 inhibitor.

UG or blastokinin is a low molecular weight protein which is secreted by the endometrium of the rabbit during early pregnancy. Its synthesis and secretion by the endometrium are regulated by ovarian steroids, especially P. However, the protein is also produced by tracheo-bronchial, gastrointestinal, prostatic, and seminal vesicular epithelium.

Nucleotide sequence of the kanamycin resistance determinant of plasmid RP4: homology to other aminoglycoside 3'-phosphotransferases.

The kanamycin resistance determinant of the broad-host-range plasmid RP4 encodes an aminoglycoside 3'-phosphotransferase of type I. The nucleotide sequence of the kanamycin resistance gene (Kmr) and the right end of the insertion element IS8 of plasmid RP4 has been determined. The gene (816 bp) is located between IS8 and the region (Tra 1) encoding plasmid factors mediating bacterial conjugation.

Alteration of serum tryptophan metabolism in patients suffering from senile cataract.

In vitro experimental evidence suggests tryptophan (TRP) is involved in protein modifications which could cause cataract formation in vivo. Previous studies of tryptophan plasma and serum metabolism are conflicting. In this study free and bound TRP plasma levels were measured in patients with senile cataract and in controls after an oral load of L-TRP (20 mg/kg b.

Serum tryptophan in humans with senile cataracts.

Serum free and total tryptophan levels have been measured in patients with cataracts and compared with the same levels in controls with clear lenses. No statistically significant differences have been demonstrated between the two groups of examined fasting subjects. Preliminary results seem to indicate that differences could be evident after L-tryptophan oral load.