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Although immobilization of antigen-specific immunoglobulins onto matrix-assisted laser desorption/ionization (MALDI) targets allows the specific detection and enrichment of an antigen from complex biological fluids, the process of antibody immobilization is not optimal. The principal reason is that the antibody can bind to the template in various orientations, many of which block antigen recognition. An affinity capture MALDI mass spectrometry methodology was developed by covalently immobilizing an [F.sub.c] receptor (recombinant protein G) onto MALDI gold targets for the purpose of orientating an immunoglobulin G, with the [F.sub.ab] domains pointing away from the target surface. The pregnancy and cancer marker, human chorionic gonadotropin [beta] core fragment (hCG[beta]cf), was our chosen test substance. To optimize the methodology, different surface densities of protein G and immunoglobulin were achieved by employing varying concentrations for immobilization. Captured amounts of hCG[beta]cf were compared using an external standard (cytochrome c). Orientation of immunoglobulin resulted in an ~3-fold increase in MALDI signal compared to using randomly immobilized antibody. Higher antibody concentrations resulted in diminished MALDI signals, which were explained by steric hindrance. Purification and enrichment of hCG[beta]cf was achieved from a test solution containing contaminant peptides and proteins using oriented immunoglobulins on-target.

Byline: R. Matyal (1), Y. Vin (1), R. Delude (1), C. Lee (1), A. Creasey (2), M. Fink (1) Keywords: Peritonitis Coagulation Escherichia coli Disseminated intravascular coagulation Tissue factor pathway inhibitor Abstract: Objective: We sought to determine the lowest dose of recombinant human tissue factor pathway inhibitor (TFPI) that can provide protection from lethality in a rabbit model of septic shock. Methods: Sepsis was induced in New Zealand white rabbits by intraperitoneal implantation of 7.0 ml of a solution containing hemoglobin (4.8 g/dl), porcine mucin (6 g/dl), and 0.8--1.4x10.sup.4 viable Escherichia coli (strain O:18 K+). Gentamicin (5 mg/kg) was administered 4 h following surgery, and this dose was repeated every 12 h for 3 days. Beginning 4 h following the induction of sepsis, animals were treated with a bolus (1 ml) plus a continuous infusion (100 ml over 24) of either TFPI (various doses) or its vehicle. Four different doses of TFPI were studied, and each experiment included a contemporaneous control group. The primary outcome parameter was survival time. Results were analyzed using the Wilcoxen log rank test. Results: The average survival time for rabbits treated with the highest dose of TFPI tested (50 Aug/kg bolus and 0.5 Aug/kg per minute infusion) was 118 h, as compared to 81 h in vehicle-treated controls). The average survival time for septic rabbits treated with a much lower dose of TFPI (100 ng/kg bolus and 1.0 ng/kg per minute infusion) was 119 h as compared to 57 h in surviving vehicle-treated controls. Treatment with an even lower dose of TFPI (10 ng/kg bolus and 0.1 ng/kg per minute infusion) still produced a marginally significant prolongation of average survival time (80 h) relative to contemporaneously studied controls (47 h). When the dose of TFPI was decreased still further (1.0 ng/kg bolus and 0.01 ng/kg per minute infusion), average survival times were not significantly different between TFPI-treated and vehicle-treated rabbits (77 and 51 h, respectively). Conclusions: Delayed infusion with remarkably low doses of recombinant human TFPI prolongs survival in a rabbit model of antibiotic-treated Gram-negative bacterial sepsis. In planning human trials of TFPI as an adjuvant treatment for sepsis it may be reasonable to use much lower doses of the agent than were heretofore contemplated. Author Affiliation: (1) Department of Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA (2) Chiron Corporation, Emeryville, CA 94608, USA (3) Mailing address: Division of Crit Care Med, 616 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261, USA Article History: Received Date: 28/06/2000 Accepted Date: 30/04/2001 Article note: Final revision received: 4 April 2001 Electronic Publication

A combination of immunological, histological, tissue culture and molecular biological techniques were employed to study the molecular basis of the neural tropism of Mycobacterium leprae. The data revealed that M. leprae exhibits specific binding to the G domain of the laminin-alpha-2 (LN-alpha-2) chain on Schwann cell-axon units, thus forming an LN-alpha-2G bridge. Such findings provide evidence for the novel role of the G domain of LN-2 in infection and that of a host-derived bridging molecule for the determination of nerve tropism of a pathogen.

Recombinant forms of the Origin Recognition Complex (ORC) protein prepared with the use of the Baculovirus-infected SF9 cells were used to study the role of ORC in ATP binding and hydrolysis. The results revealed that the Orc1 and Orc5 subunits of the ORC bind ATP. Orc1 was also found to take part in the hydrolysis of ATP. The data showed that the occupancy of the Orc1p ATP-binding site has a profound effect on ORC function and that ATP hydrolysis by Orc1p can drive transitions between different functional states of ORC.

In vitro transcription assay and Western blotting were performed to determine the role of MeCP2, an abundant chromosomal protein with high binding affinity for methyl-CpG pairs, in the transcription process. The results revealed that MeCP2 bears an efficient transcriptional repression domain. It was shown that the potency of such repressor function is dependent on the density and proximity of methyl-CpGs to a promoter.

Hepatits C virus (HCV)-encoded polypeptides, such as C100-3 may be used to capture and measure reactive antibodies circulating in infected patients, making them valuable clinical and diagnostic tools. The immunoreactivities of recombinant HCV polypeptides derived from different regions of the HCV polyprotein were assessed. The results showed that one of the recombinant polypeptides, designated C25, could be used for an antibody capture assay more sensitive than the conventional C100-3 assay. Using this more sensitive assay, it was shown that HCV has had a greater role in liver disease than previously thought.

The secretion of Erwinia chrysanthemi protease B depends on a C-terminal signal sequence located downstream from a glycine-rich tandem repeat sequence. The role of the repeat sequence was investigated by gene fusion experiments involving either direct fusions to the signal sequence or fusions separated from the glycine-rich domain. The results showed that the glycine-rich domain is required for the secretion of high-molecular-weight fusion proteins.

The c-Jun protein is a regulator of transactivation whose suppressor function may be elicited when its truncated forms saturate the AP-1 binding sites of ras products. A LexA-Jun zipper fusion protein was constructed to investigate the minimal m-Jun segment involved in the suppression of ras transformation. The LexA-Jun zipper fusion was able to dimerize efficiently to the wild-type Jun and Fos leucine-zipper peptides, resulting in protein complexes unable to bind to DNA, leading to suppressed transformation. Therefore, the LexA-Jun fusion suppresses with cell transformation by interfering with the dimerization of transcription factors.

The conditions for the biotinylation of recombinant aequorin with minimal activity loss was determined. Aequorin is a calcium-binding protein responsible for bioluminescence of the hydromedusan Aequorea victoria. Recombinant aequorin from Escherichia coli was purified by gel filtration on Sephadex G-100 and subsequent anion-exchange chromatography on DEAE-Sepahrose. The use of the biotinylated derivative with streptavidin to detect biotinylated targets immobilized on microtiter wells or membrane supports was demonstrated.

Byline: M.M.M. Ahmed, Elsayed E. Hafez, M.A. El-Saadani Alasan, the bioemulsifier of Acinetobacter radioresistens KA53, is a high molecular-mass complex of an alanine-containing polysaccharide and three proteins. This gene was isolated from the A. radioresistens using PCR and the amplified product (850 bp) was cloned using PGEMT cloning kit. DNA sequence was carried out for the amplification and sequence analysis showed that the PCR product was highly similar to the OMPA precursor protein which called Alan. Subcloning was carried out into pTRAC expression vector and the purified protein was used in mineralisation of the polyaromatic hydrocarbon. As well as the recombinant E. coli cells were cultured directly on MSM containing four polyaromatic hydrocarbon using real time method (phenol, naphthalene, phenantherene and antherathene). The obtained results showed that no difference between the purified protein and the using of the recombinant cells directly with the presence of the inducer like IPTG. Both of the purified protein and the recombinant cells showed high degradation rates for (anthrathene and phenantherene).

The crustacean CHH/MIH/GIH neuropeptide family constitutes a group of important hormone that regulates important processes such as molting, glucose metabolism and reproduction. The characterization of these neuropeptides has attracted much attention due to their potential importance in aquaculture. By RT-PCR and library screening approach, we have isolated and cloned four cDNAs from the eyestalk of the shrimp Metapenaeus ensis. Two of the cDNAs encoded for proteins are homologous to the CHH-subtypes and two of the cDNA encoded for proteins belong to the MIH/GIH subtypes. The organizations of these genes are very similar. They consist of three exons and two introns with conserved location of boundaries between the intronexon. There are more than 20 CHH-A genes, 5-6 CHH-B genes and 1-2 copies of MIH and GIH gene in the shrimp genome. CHH-A, CHH-B, MIH- and GIH RNA transcripts can be detected in the eyestalk of animal at most stages. Only CHH-A and GIH transcripts are detected in ventral nerve cord. Recombinant proteins for these neuropeptides have been produced and used for bioassay. Additionally, the recombinant proteins were purified for antibody generation. Specific antibodies against these recombinant proteins were generated and had been used to detect these neuropeptides by immunochemistry. Supported in part by a HKU institutional grant (CRCG) and the RGC of the HKSAR, China (HKU 7229/00M)

Research was conducted to examine the mechanism of Cdk2/Cyclin E inhibition by p27 and p27 phosphorylation using purified, recombinant p27 and CAK-phosphorylated Cdk2/Cyclin E. It was observed that Cdk2/Cyclin E has 60-fold higher specificity for pRb than for histone H1. Results also demonstrate that the Cdk2/Cyclin E/p27 ternary complex is kinetically inactive as an enzyme and acts as a substrate for Cdk2/Cyclin E.