Conversion, by limited proteolysis, of an archaebacterial citrate synthase into essentially a citryl-CoA hydrolase.

1. Limited proteolysis of citrate synthase from Sulfolobus solfataricus by trypsin reduced the rate of the overall reaction (acetyl-CoA + oxaloacetate + H2O----citrate + CoASH) to 4% but did not affect the hydrolysis of citryl-CoA. Experimental results indicate that a connecting link between the enzyme's ligase and hydrolase activity becomes impaired specifically on treatment with trypsin. Other proteolytic enzymes like chymotrypsin and subtilisin inactivated catalytic functions of citrate synthase, ligase and hydrolase, equally well. 2. Tryptic hydrolysis occurs at the N-terminal region of citrate synthase, but a study by SDS/PAGE revealed no difference in molecular mass between native and proteolytically nicked citrate synthase. The peptide removed from the enzyme by trypsin, therefore, contains less than about 15 amino acid residues. 3. The Km values of the substrates for both native and nicked enzyme were identical, as was the state of aggregation (dimeric) of the two enzyme species. These could be separated by affinity chromatography on Blue-Sepharose and differentiated by their isoelectric points (pI = 6.68 +/- 0.08 and pI = 6.37 +/- 0.03 for native citrate synthase and the large tryptic peptide, respectively) as well as by the N-terminus which is blocked in the native enzyme only. 4. Edman degradation of the large tryptic fragment yielded the N-terminal sequence GLEDVYIKSTSLTYIDGVNGVLRY, which is 71% identical to the N-terminal region (positions 9-32) of citrate synthase from Thermoplasma acidophilum. 5. The conversion of citrate synthase into essentially a citryl-CoA hydrolase is considered the consequence of a conformational change thought to occur on tryptic removal of the N-terminal small peptide.     

Protein LG: a hybrid molecule with unique immunoglobulin binding properties.

Immunoglobulin (Ig)-binding bacterial proteins have attracted theoretical interest for their role in molecular host-parasite interactions, and they are widely used as tools in immunology, biochemistry, medicine, and biotechnology. Protein L of the anaerobic bacterial species Peptostreptococcus magnus binds Ig light chains, whereas streptococcal protein G has affinity for the constant (Fc) region of IgG. In this report, Ig binding parts of protein L and protein G were combined to form a hybrid molecule, protein LG, which was found to bind a large majority of intact human Igs as well as Fc and Fab fragments, and Ig light chains. Binding to Ig was specific, and the affinity constants of the reactions between protein LG and human IgG, IgGFc fragments, and kappa light chains, determined by Scatchard plots, were 5.9 x 10(9), 2.2 x 10(9), and 2.0 x 10(9) M-1, respectively. The binding properties of protein LG were more complete as compared with previously described Ig-binding proteins when also tested against mouse and rat Igs. This hybrid protein thus represents a powerful tool for the binding, detection, and purification of antibodies and antibody fragments.     

Liver-specific drug targeting by coupling to bile acids.

Bile acids are selectively taken up from portal blood into the liver by specific transport systems in the hepatocyte plasma membrane. Therefore, studies were performed to evaluate the potential of bile acids as shuttles to deliver drugs specifically to the liver. The alkylating cytostatic drug chlorambucil and the fluorescent prolyl-4-hydroxylase inhibitor 4-nitrobenzo-2-oxa-1,3-diazol-beta-Ala-Phe-5-oxaproline-Gly were covalently linked via an amide bond to 7 alpha, 12 alpha,-dihydroxy-3 beta- (omega-aminoalkoxy)-5-beta-cholan-24-oic acid. The chlorambucil-bile acid conjugates S 2521, S 2539, S 2567, and S 2576 inhibited Na(+)-dependent [3H]taurocholate uptake in a concentration-dependent manner both into isolated rat hepatocytes and rabbit ileal brush border membrane vesicles, whereas the parent drug chlorambucil showed no significant inhibitory effect. The chlorambucil-bile acid conjugates were able to prevent photoaffinity labeling of bile acid binding proteins in rat hepatocytes by the photolabile [3H]7,7-azo derivative of taurocholic acid indicating their bile acid character. The chlorambucil-bile acid conjugate S 2577 was able to alkylate proteins demonstrating the drug character conserved in the hybrid-molecules. Liver perfusion experiments revealed a secretion profile of the chlorambucil-bile acid conjugate S 2576 into bile very similar to taurocholate compared to chlorambucil which is predominantly excreted by the kidney. 4-Nitrobenzo-2-oxa-1,3-diazol-beta-Ala-Phe-5-oxaproline-Gly- t-butylester (S 4404), a fluorescent peptide inhibitor of prolyl-4-hydroxylase, was not transported in intact form from portal blood into bile in contrast to its bile acid conjugate S 3744; about 25% of the peptide-bile acid conjugate S 3744 was secreted in intact form into bile within 40 min compared with less than 4% of the parent oxaprolylpeptide S 4404. In conclusion, these studies reveal that modified bile acid molecules can be used as "Trojan horses" to deliver a drug molecule specifically into the liver and the biliary system. This offers important pharmacological options for the development of liver-specific drugs.     

Site-specific frameshift mutagenesis by a propanodeoxyguanosine adduct positioned in the (CpG)4 hot-spot of Salmonella typhimurium hisD3052 carried on an M13 vector.

Malondialdehyde induces frameshift mutations in Salmonella typhimurium strain hisD3052. The ability of propanodeoxyguanosine (PdG), a structural analog of the major malondialdehyde-deoxyguanosine adduct, to induce site-specific frameshift mutations was tested in the (CpG)4 hot-spot of hisD3052 carried on an M13 vector (M13MB102). PdG was introduced at position 6248 of duplex M13MB102 by ligation of the oligonucleotide 5'-CGC(PdG)CGGCATG-3' into a heteroduplex containing an 11-nucleotide gap in the (-)-strand between the SphI and BssHII restriction sites and deoxyuridine in place of thymidine in the (+)-strand. Ligation proceeded with 70% efficiency, and closed circular duplex DNA molecules were isolated in 40% yield. The adducted genome was sensitive to cleavage by SphI but resistant to cleavage by BssHII. Transformation of Escherichia coli strain JM105 with adducted M13MB102 led to 25% reduced survival relative to unadducted M13MB102 and produced frameshift mutations in 2.5% of the progeny phage. All of the mutations were deletions, and 70% occurred by deletion of CpG. Unadducted genomes exhibited a 40-fold lower mutation frequency, and all the mutations were single-base deletions at the sites of ligation of the 11-mer. These results illustrate that PdG, a structural analog of the major malondialdehyde-deoxyguanosine adduct, induces frameshift mutations in M13MB102 and that single-stranded nicks are efficient premutagenic lesions in this recombinant bacteriophage.     

Identification and differential expression of a novel alternative splice isoform of the beta A4 amyloid precursor protein (APP) mRNA in leukocytes and brain microglial cells.

The gene for the beta A4-amyloid precursor protein (APP) consists of 19 exons which code for a typical N- and O-glycosylated transmembrane protein with four extracellular domains followed by the transmembrane domain and a short cytoplasmic domain. The beta A4-amyloid sequence is part of exons 16 and 17. Several APP isoforms can be generated by alternative splicing of exons 7 and 8, encoding domains with homologies to Kunitz-type protease inhibitors and the MRC OX-2 antigen, respectively. The mechanism by which the pathological beta A4 is generated is unknown, it is however a critical event in Alzheimer's disease and is distinct from the normally occurring cleavage and secretion of APPs within the beta A4 sequence. We report here for the first time considerable APP mRNA expression by rat brain microglial cells. In addition we showed by S1 nuclease protection and polymerase chain reaction analysis of reverse transcribed RNA (RT-PCR) that T-lymphocytes, macrophages, and microglial cells expressed a new APP isoform by selection of a novel alternative splice site and exclusion of exon 15 of the APP gene. This leads to a transmembrane, beta A4 sequence containing APP variant, lacking 18 amino acid residues close to the amyloidogenic region. The use of this novel alternative splice site alters the structure of APP in close proximity to the beta A4 region and thus may determine a variant, potentially pathogenic processing of leukocyte-derived APP in brain.     

N-glycosylation is required for human CD2 immunoadhesion functions.

The T-lymphocyte glycoprotein receptor, CD2, mediates cell-cell adhesion by binding to the surface molecule CD58 (LFA-3) on many cell types including antigen presenting cells. Two domains comprise the CD2 extracellular segment, with all adhesion functions localized to the amino-terminal domain that contains a single N-glycosylation site at Asn65. We have defined an important role for the N-linked glycans attached to Asn65 of this domain in mediating CD2-CD58 interactions and also characterize its N-glycotype structure. Analysis of deglycosylated soluble recombinant CD2 as well as a mutant transmembrane CD2 molecule containing a single Asn65-Gln65 substitution demonstrates that neither deglycosylated CD2 nor the mutant CD2 transmembrane receptor binds CD58 or monoclonal antibodies directed at native CD2 adhesion domain epitopes. Electrospray ionization-mass spectrometry demonstrates that high mannose oligosaccharides ((Man)nGlcNAc2, n = 5-9) are the only N-glycotypes occupying Asn65 when soluble CD2 is expressed in Chinese hamster ovary cells. Based on a model of human CD2 secondary structure, we propose that N-glycosylation is required for stabilizing domain 1 in the human receptor. Thus, N-glycosylation is essential for human CD2 adhesion functions.     

Influence of hypothalamic-pituitary-adrenocortical hormones on reproductive hormones in gray wolves (Canis lupus).

The release of hypothalamic-pituitary-adrenocortical hormones was studied in intact and neutered gray wolves (Canis lupus) to determine how these hormones interact and affect reproductive hormones. Experiments were performed on adult wolves anesthetized with 400 mg ketamine and 50 mg promazine. Intravenous (i.v.) injections with 50 micrograms ovine corticotropin releasing factor (oCRF) significantly increased adrenocorticotropin (ACTH; P < or = 0.01), cortisol (CORT; P < or = 0.004), and progesterone (P < or = 0.036), but not beta-endorphin (P > or = 0.52). Since neutered wolves demonstrated dose-dependent elevations in response to ACTH, it was concluded that the progesterone was secreted from the adrenal gland. Basal luteinizing hormone (LH) concentrations in neutered wolves were similar before and 60 min after i.v. injection of 1, 5, or 25 IU ACTH (P > or = 0.36) or 2.2 mg/kg cortisol (P = 0.42). Neither 25 IU ACTH (P = 0.55) nor 0.22 mg/kg dexamethasone (P = 0.49) altered the LH response to injection of LH releasing hormone in neutered wolves. Chronic administration of 0.22 mg/kg/day dexamethasone for 3 d did not alter baseline LH concentrations (P = 0.75). Injection of 1.0 mg/kg naloxone (NAL), however, increased LH concentrations relative to baseline values in both intact (P = 0.032) and neutered (P = 0.0005) female wolves, but not in intact (P = 0.19) or neutered males (P = 0.07). These results indicated that in gray wolves (1) oCRF stimulated the release of pituitary and adrenal hormones in a fashion similar to that of other mammals; (2) the adrenal cortex was capable of secreting progesterone into the systemic circulation; (3) exogenous glucocorticoids did not alter LH concentrations; and (4) endogenous opioids may modulate LH secretion in female wolves.     

The diagnostic value of haematuria and proteinuria in Schistosoma haematobium infection in southern Nigeria.

Haematuria and proteinuria as detected by chemical reagent strips correlated moderately (r = 0.7) with prevalence and intensity of infection with Schistosoma haematobium in an area of Anambra State, Nigeria. Differences attributable to age and sex were also reflected in a similar pattern, all peaks occurring in the 5-14 year age group. The differences observed with varying levels of intensity and haematuria at both 10 and 50 erythrocytes/microliter (p < 0.001) and proteinuria at 0.3 g/dl (p < 0.01) were statistically significant. At a proteinuria level of 1 g/dl, the observed differences were however not statistically significant (p > 0.5). The percentage of specimens from children (0-14 years) positive for S. haematobium eggs and with at least traces of haematuria and proteinuria (63.4% and 95%, respectively) was higher than in adults (33.3% and 80.2%, respectively). All individuals with more than 50 eggs/10 ml of urine were correctly identified using both indices either separately or in combination. For egg counts of less than 50 eggs/10 ml of urine, false diagnosis occurred in only 5% of all specimens examined. The sensitivity and specificity of haematuria and proteinuria at trace quantities was very high, but haematuria had a higher predictive value for a positive test (PvPt) and was considered the overall better indicator. A combination of both indices did not significantly increase the PvPt. When trace haematuria and moderate proteinuria were combined, both the sensitivity, specificity and PvPt were all above 90%, giving the best overall values in all the combinations made.     

Synthetic peptides anchor T cell-specific TNP epitopes to MHC antigens.

Several TNP-specific, H-2Kb-restricted mouse CTL clones were identified which specifically lysed target cells in the presence of tryptic digests of TNP-modified BSA. Glutaraldehyde fixation of cells revealed that the tryptic fragments did not require further cellular processing. Chromatographic fractionation of digested TNP-BSA identified the peptide TNP-BSA222-231, containing a TNP-modified lysine at BSA position 227, as the antigenic entity. The corresponding synthetic peptide was immunologically cross-reactive with the digest. All clones reactive with TNP-BSA222-231 cross-reacted with a similar peptide from mouse serum albumin (TNP-MSA126-135), favoring the assumption that TNP-BSA222-231 represents an artificial determinant, cross-reacting with some as yet unidentified, TNP-modified, Kb-associated self-peptides. Some of our clones also cross-reacted with tryptic digests of TNP-OVA or TNP-keyhole limpet hemocyanin. We interpret these findings to indicate that 1) a significant proportion of hapten (TNP) determinants for T cells are anchored to MHC via peptides; and 2) the amino acid sequence of these peptides may only partly define the specificity of the T cell-relevant hapten epitope, implying a particularly repetitive nature of these determinants. The production of T cell-antigenic hapten-peptide conjugates will hopefully open new roads to study immune responses to environmental allergens.     

Human endothelial cells are target for platelet-activating factor. II. Platelet-activating factor induces platelet-activating factor synthesis in human umbilical vein endothelial cells.

Platelet-activating factor (PAF), a phospholipid mediator with broad and potent biologic activities, is synthesized by several inflammatory cells including endothelial cells (EC). PAF is also an effective stimulating agent for EC leading to increased cell permeability and adhesivity. We examined the synthesis of PAF in human umbilical cord vein EC after stimulation of EC with PAF or with its nonmetabolizable analog 1-O-alkyl-2-N-methyl-carbamyl-sn-glycero-3-phosphocholine (C-PAF). PAF (1 to 100 nM) induced a dose- and time-dependent increase of PAF synthesis as detected by [3H]acetate incorporation into PAF fraction. Stimulation of PAF synthesis occurred via activation of the "remodeling pathway" as the 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase was dose-dependently increased after PAF treatment. The de novo pathway of PAF synthesis was not activated under these conditions. C-PAF was able to mimic the effect of authentic PAF on [3H] acetate incorporation. The inactive metabolite lyso-PAF (100 nM) had no influence on PAF synthesis in EC. CV-3988, BN 52021, and WEB 2086, potent and specific antagonists of PAF suppressed PAF effects on the remodeling pathway completely. The PAF- and C-PAF-induced [3H]PAF remained 93% cell-associated and was not degraded up to 10 min after stimulation. Characterization of the [3H]acetate-labeled material co-migrating with authentic PAF revealed that a significant proportion (approximately 57%) was actually 1-acyl-2-acetyl-sn-glycero-3-phosphocholine. PAF-induced PAF synthesis might be an important mechanism for amplifying original PAF signals and potentiating adhesive interactions of circulating cells with the endothelium.