Development and implementation of an electrochemiluminescence immunoassay for the determination of an angiogenic polypeptide in dog and rat plasma.

A quantitative method based on electrochemiluminescence immunoassay for the determination of the angiogenic agent aFGF-S117 has been developed and validated. Two polyclonal antibodies specific to aFGF-S117 and a wild-type aFGF antibody were selected for the analysis. The assay was based on the non-competitive sandwich immunoassay principle in which the drug is trapped with a biotinylated antibody that is immobilized on a streptavidin magnetic particle. The drug is then sandwiched with a ruthenium chelated second antibody. The assay demonstrates good accuracy and reproducibility at plasma concentration of 0.5 ng/ml.

The pro domain of beta-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain.

beta-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4' Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k(cat)/K(m) of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4' Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.

Photoactivated gamma-secretase inhibitors directed to the active site covalently label presenilin 1.

Cleavage of amyloid precursor protein (APP) by the beta- and gamma-secretases generates the amino and carboxy termini, respectively, of the A beta amyloidogenic peptides A beta40 and A beta42--the major constituents of the amyloid plaques in the brain parenchyma of Alzheimer's disease patients. There is evidence that the polytopic membrane-spanning proteins, presenilin 1 and 2 (PS1 and PS2), are important determinants of gamma-secretase activity: mutations in PS1 and PS2 that are associated with early-onset familial Alzheimer's disease increase the production of A beta42 (refs 4-6), the more amyloidogenic peptide; gamma-secretase activity is reduced in neuronal cultures derived from PS1-deficient mouse embryos; and directed mutagenesis of two conserved aspartates in transmembrane segments of PS1 inactivates the ability of gamma-secretase to catalyse processing of APP within its transmembrane domain. It is unknown, however, whether PS1 (which has little or no homology to any known aspartyl protease) is itself a transmembrane aspartyl protease or a gamma-secretase cofactor, or helps to colocalize gamma-secretase and APP. Here we report photoaffinity labelling of PS1 (and PS2) by potent gamma-secretase inhibitors that were designed to function as transition state analogue inhibitors directed to the active site of an aspartyl protease. This observation indicates that PS1 (and PS2) may contain the active site of gamma-secretase. Interestingly, the intact, single-chain form of wild-type PS1 is not labelled by an active-site-directed photoaffinity probe, suggesting that intact wild-type PS1 may be an aspartyl protease zymogen.

Presenilin 1 is linked with gamma-secretase activity in the detergent solubilized state.

gamma-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C termini of the two Abeta peptide isoforms, Abeta40 and Abeta42. Here we report the detergent solubilization and partial characterization of gamma-secretase. The activity of solubilized gamma-secretase was measured with a recombinant substrate, C100Flag, consisting largely of the C-terminal fragment of amyloid precursor protein downstream of the beta-secretase cleavage site. Cleavage of C100Flag by gamma-secretase was detected by electrochemiluminescence using antibodies that specifically recognize the Abeta40 or Abeta42 termini. Incubation of C100Flag with HeLa cell membranes or detergent-solubilized HeLa cell membranes generates both the Abeta40 and Abeta42 termini. Recovery of catalytically competent, soluble gamma-secretase critically depends on the choice of detergent; CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) but not Triton X-100 is suitable. Solubilized gamma-secretase activity is inhibited by pepstatin and more potently by a novel aspartyl protease transition-state analog inhibitor that blocks formation of Abeta40 and Abeta42 in mammalian cells. Upon gel exclusion chromatography, solubilized gamma-secretase activity coelutes with presenilin 1 (PS1) at an apparent relative molecular weight of approximately 2.0 x 10(6). Anti-PS1 antibody immunoprecipitates gamma-secretase activity from the solubilized gamma-secretase preparation. These data suggest that gamma-secretase activity is catalyzed by a PS1-containing macromolecular complex.

Use of pentastarch solution in the treatment of patients with hemorrhagic hypovolemia: randomized phase II study in the emergency room.

This study evaluates the hemodynamic effects of the administration of 10% pentastarch solution (PS) during the initial treatment of hypovolemia in trauma patients. This prospective randomized phase II study included trauma patients admitted to the emergency room with hemorrhagic hypovolemia: systolic blood pressure (SBP) < 90 mmHg. Upon admission, the patients were randomized to receive 10% PS (n = 12) or isotonic 0.9% NaCl solution (IS) (n = 11), infused intravenously in 250-ml boluses, repeated until SBP > 100 mmHg. Blood pressure, infused volumes necessary to maintain SBP, and overall survival rates were determined and compared between groups. SBP increased significantly following either IS (from 64.4 +/- 9.2 mmHg to 111.1 +/- 6.3 mmHg), or PS (from 63.7 +/- 10.6 mmHg to 108.1 +/- 9.8 mmHg) when compared to admission values (p < 0.05). Endovenous volumes infused were greater (p = 0.001) in IS patients (1420 +/- 298 ml) than in PS patients (356 +/- 64 ml). No blood was transfused into PS patients, compared to 370 +/- 140 ml of red blood cells transfused into IS patients (p = 0.015). Mortality rates were similar in the two groups (p = 0.725). We concluded that PS is a safe, efficient method for inducing hemodynamic recovery of hypovolemic trauma patients, with a clear reduction in the intravenous volumes required for acute resuscitation.

Effects of inhibitors of RNA and protein synthesis on the subcellular distribution of the eukaryotic translation initiation factor, eIF-5A, and the HIV-1 Rev protein.

The subcellular distributions of the endogenous eukaryotic translation initiation factor, eIF-5A, and Rev, a protein of the human immunodeficiency virus proposed to interact with eIF-5A, were studied in COS-7 cells treated with inhibitors of RNA or protein synthesis. We have previously shown that transiently expressed Rev is localized in the nucleolus, whereas eIF-5A is primarily in the cytoplasm. The subcellular localization of Rev was not affected by treatment with protein synthesis inhibitors (cycloheximide, CHX, 10 micrograms/ml; puromycin, 10 micrograms/ml), although its location changed from predominantly the nucleolus to the cytoplasm after treatment with RNA synthesis inhibitors (actinomycin D, 4 micrograms/ml, and 5,6-dichloro-1 beta-D-ribofuranosylbenzimidazole, DRB; 0.1 mM), as previously reported. In contrast, none of the RNA synthesis inhibitors (alpha-amanitin, 10 micrograms/ml; actinomycin D, 4 micrograms/ml, and DRB, 0.1 mM) caused any significant changes in the subcellular distribution pattern of eIF-5A. However, treatment with puromycin, a protein synthesis inhibitor known to dissociate ribosomes, dramatically altered the subcellular distribution pattern of eIF-5A in 30% of the cell population. In these cells, the staining of eIF-5A was changed from an endoplasmic reticulum (ER) net work-like perinuclear structure to a patched dotted pattern dispersed throughout the cytoplasm. This change was not observed in the same cells stained for calnexin, an ER resident protein, nor in cells treated with CHX, which freezes the ribosomes to block protein synthesis. Our data suggest that eIF-5A does not shuttle between the nucleus and cytoplasm in the same way as Rev. Our findings are consistent with our previous conclusion that eIF-5A is associated with the ER through ribosomes and support a role for eIF-5A in protein synthesis.

The subcellular distribution of eukaryotic translation initiation factor, eIF-5A, in cultured cells.

To gain insight into the role of the eukaryotic translation initiation factor, eIF-5A, we investigated the subcellular distribution of this protein in several cultured cell types and at different stages of the cell cycle using a highly potent monospecific polyclonal antibody to eIF-5A. Studies using indirect immunofluorescence and confocal microscopy in conjunction with subcellular fractionation demonstrate that eIF-5A is primarily localized in the cytoplasm of cells. This cytoplasmic location of eIF-5A is not significantly altered in different stages of the cell cycle and the subcellular distribution pattern of eIF-5A is not changed by viral oncogene transformation. Cell fractionation experiments identified two populations of eIF-5A in the cytoplasm, a soluble fraction and a fraction bound to internal membranes. By double immunofluorescence staining with an antibody against calnexin, a resident protein of the endoplasmic reticulum (ER), we demonstrate that the membrane-bound fraction of eIF-5A colocalizes with the ER and not with the cytoskeleton. Expression of Rev, a regulatory protein of human immunodeficiency virus type 1 (HIV-1), does not alter the subcellular distribution of endogenous eIF-5A in these cells. eIF-5A is detected in all tissues and cells examined including extracts prepared from Xenopus oocytes. Our results indicate that eIF-5A is a ubiquitous cytoplasmic protein and suggest that a site of eIF-5A function is likely to be in association with the ER.

Effects of N1-guanyl-1,7-diaminoheptane, an inhibitor of deoxyhypusine synthase, on the growth of tumorigenic cell lines in culture.

N1-guanyl-1,7-diaminoheptane (GC7) is a potent inhibitor of deoxyhypusine synthase (DHS), the enzyme that catalyzes the first step in the hypusination of eukaryotic translation initiation factor 5A (eIF-5A). Since eIF-5A is the only known cellular substrate for DHS and GC7 has been reported to block the proliferation of CHO cells, it has been suggested that DHS may be a novel target for anti-cancer therapy. In the present study we investigated the antiproliferative effect of GC7 on several tumorigenic cell lines under various growth conditions. We found that this compound inhibits the proliferation of H9 cells in suspension culture and the growth of HeLa cells and v-src-transformed NIH3T3 cells under both anchorage-dependent and anchorage-independent conditions. Moreover, studies with NIH3T3 cells and v-src-transformed NIH3T3 cells show that GC7 inhibits the growth of both cell lines in monolayer culture with similar potency and could not reverse the transformed phenotype. In addition, the v-src-transformed cells grown under both anchorage-dependent and anchorage-independent conditions showed similar sensitivity toward GC7. These data indicate that GC7 acts as a general antiproliferative agent and does not appear to preferentially target tumorigenic cell types. Cell cycle analysis show that GC7 reduces the CHO-K1 cell population in the G1-phase of the cell cycle by 42% and increases the number of cells in the S-phase by 44%. This cell cycle distribution profile strikingly resembles the distribution of cells treated with puromycin. This result supports the hypothesis that the synthesis of a subset of proteins important for the S-phase progression of CHO-K1 cells might be dependent upon hypusinated eIF-5A. Thus the antiproliferative effect of GC7 appears to be related to its interference with the progression of cell cycle, which also provides a possible explanation for the lack of selectivity of GC7 between nontransformed and transformed cell types tested in this study.

Myogenic vector expression of insulin-like growth factor I stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice.

The avian skeletal alpha-actin gene was used as a template for construction of a myogenic expression vector that was utilized to direct expression of a human IGF-I cDNA in cultured muscle cells and in striated muscle of transgenic mice. The proximal promoter region, together with the first intron and 1.8 kilobases of 3'-noncoding flanking sequence of the avian skeletal alpha-actin gene directed high level expression of human insulin-like growth factor I (IGF-I) in stably transfected C2C12 myoblasts and transgenic mice. Expression of the actin/IGF-I hybrid gene in C2C12 muscle cells increased levels of myogenic basic helix-loop-helix factor and contractile protein mRNAs and enhanced myotube formation. Expression of the actin/IGF-I hybrid gene in mice elevated IGF-I concentrations in skeletal muscle 47-fold resulting in myofiber hypertrophy. IGF-I concentrations in serum and body weight were not increased by transgene expression, suggesting that the effects of transgene expression were localized. These results indicate that sustained overexpression of IGF-I in skeletal muscle elicits myofiber hypertrophy and provides the basis for manipulation of muscle physiology utilizing skeletal alpha-actin-based vectors.

Human erythroleukemic (HEL) cells express a platelet P2T-like ADP receptor.

Treatment of Fura-2 loaded HEL cells, a human megakaryocyte-like cell line, with P2-purinoceptor nucleotide ligands (ADP, ATP, UTP, 2-methylthio-ATP) evoked a rise in cytosolic calcium. Homologous- and cross-desensitization studies using sequential addition of nucleotides showed that the ADP-induced calcium response in HEL cells was mediated mainly by purinoceptor(s) for which ADP but not ATP was an agonist. There were also minor contributions from purinoceptors for which ATP and ADP are both agonists (probably P2U and P2Y). ATP inhibited the ADP-induced calcium response in HEL cells. This inhibition was overcome by raising the ADP concentration, thus indicating that ATP was an antagonist for the HEL cell ADP receptor. AMP was also an antagonist, albeit weak, for the HEL cell ADP receptor. Antagonism of the ADP-induced calcium response by ATP was similarly observed in MEG-01 cells, another human megakaryocyte-like cell line, but not in 293 cells, a nonhematopoietic cell line. These studies suggest that HEL cells express an ADP receptor for which ATP and AMP are antagonists. This characteristic of the HEL cell ADP receptor is also displayed by the platelet P2T receptor. Thus, HEL cells appear to be a surrogate source of the platelet ADP receptor.

Elements of the smooth muscle alpha-actin promoter required in cis for transcriptional activation in smooth muscle. Evidence for cell type-specific regulation.

To assess the role of cis-acting elements within the smooth muscle alpha-actin gene in smooth muscle cells (SMC), we transfected chicken smooth muscle alpha-actin promoter-chloramphenicol acetyltransferase gene fusion plasmids into SMC derived from rat and chicken aortas. In marked contrast to effects in chicken skeletal myoblasts and fibroblasts, p122CAT (positions -122 to +19), containing two conserved CArG elements, elicited a modest increase in chloramphenicol acetyltransferase reporter activity in chicken SMC. Addition of upstream sequences between -122 and -151 (p151CAT) increased activity in adult chicken SMC. Addition of sequence between positions -151 and -257 (p257CAT) resulted in a 7-fold increase in chloramphenicol acetyltransferase activity over that of p151CAT in rat SMC, but not in chicken SMC. A genomic clone encoding the rat smooth muscle alpha-actin gene was isolated, and the 5'-flanking region was partially characterized. Comparison of primary sequence between rat and chicken promoters showed a conserved E box motif at position -214 in the chicken gene and at position -213 in the rat gene. Results of these studies demonstrate that regions upstream of the conserved CArG elements exert potent regulatory effects on transcription and that SMC require different cis-acting elements than other cell types to transcriptionally regulate this gene.

Nucleotide sequence of the Escherichia coli replication gene dnaZX.

The Escherichia coli 2.2 kilobase dnaZX region contains one 1929 nucleotide reading frame which directs the synthesis of two protein products involved in DNA polymerization. The larger consists of 643 amino acids in a deduced 71,114 dalton chain which could be the tau subunit of DNA polymerase III. The smaller, the DNA polymerase III gamma subunit, is encoded by the same reading frame as the larger. The dnaZX sequence contains a region homologous to ATP binding sites, suggesting that these replication factors are adenine nucleotide binding proteins.