Platelet endothelial cell adhesion molecule (PECAM-1), a member of the Ig superfamily, is found on endothelial cells and neutrophils and has been shown to be involved in the migration of leukocytes across the endothelium. Adhesion is mediated, at least in part, through binding interactions involving its first N-terminal Ig-like domain, but it is still unclear which sequences in this domain are required for in vivo function. Therefore, to identify functionally important regions of the first Ig-like domain of PECAM-1 that are required for the participation of PECAM-1 in in vivo neutrophil recruitment, a panel of mAbs against this region of PECAM-1 was generated and characterized in in vitro adhesion assays and in an in vivo model of cutaneous inflammation. It was observed that mAbs that disrupted PECAM-1-dependent homophilic adhesion in an L cell aggregation assay also blocked TNF-α-induced intradermal accumulation of neutrophils in a transmigration model using human skin transplanted onto SCID mice. Localization of the epitopes of these Abs indicated that these function-blocking Abs mapped to specific regions on either face of domain 1. This suggests that these regions of the first Ig-like domain may contain or be close to binding sites involved in PECAM-1-dependent homophilic adhesion, and thus may represent potential targets for the development of antiinflammatory reagents.

Endotoxic shock follows a cascade of events initiated by release of lipopolysaccharide during infection with Gram-negative organisms. Two overlapping 15-mer peptides were identified, corresponding to residues 91-108 of human lipopolysaccharide binding protein that specifically bound the lipid A moiety of lipopolysaccharide with high affinity. The peptides inhibited binding of lipopolysaccharide to lipopolysaccharide binding protein, inhibited the chromogenic Limulus amebocyte lysate reaction, and blocked release of tumor necrosis factor alpha following lipopolysaccharide challenge both in vitro and in vivo. These results suggest lipopolysaccharide binding protein residues 91-108 form at least part of the lipopolysaccharide binding site. Moreover, derivatives of lipopolysaccharide binding protein residues 91-108 might modulate lipopolysaccharide toxicity in the clinical setting.

Modern on-road diesel engine systems incorporate flexible fuel injection, variable geometry turbocharging, high pressure exhaust gas recirculation, oxidation catalysts, particulate filters, and selective catalytic reduction systems in order to comply with strict tailpipe-out NOx and soot limits. Fuel consuming strategies, including late injections and turbine-based engine exhaust throttling, are typically used to increase turbine-outlet temperature and flow rate in order to reach the aftertreatment component temperatures required for efficient reduction of NOx and soot. The same strategies are used at low load operating conditions to maintain aftertreatment temperatures. This paper demonstrates that cylinder deactivation (CDA) can be used to maintain aftertreatment temperatures in a more fuel-efficient manner through reductions in airflow and pumping work. The incorporation of CDA to maintain desired aftertreatment temperatures during idle conditions is experimentally demonstrated to result in fuel savings of 3.0% over the HD-FTP drive cycle. Implementation of CDA at non-idle portions of the HD-FTP where BMEP is below 3 bar is demonstrated to reduce fuel consumption further by an additional 0.4%, thereby resulting in 3.4% fuel savings over the drive cycle.

SummaryWe have analyzed the structural and genetic basis for T cell recognition of the complex formed between antigen and class II products of the major histocompatibility complex by performing sequence analysis of T cell receptors (TCRs) induced in response to the helper T cell site 1 of the influenza virus hemagglutinin . The results demonstrate, first, that structurally highly diverse TCRs can be utilized in recognition of the same antigen/1-Ed complex: 12 of 13 TCRs utilize unique Vct/VS gene segment combinations, suggesting that -70 different Vtx/V# combinations are available to BALB/c mice in response to this determinant . Second, comparison of these sequences with the ability of each hybridoma to recognize a panel of peptide analogues suggests that a and (3 chains of these TCRs frequently determine specificity for the NH2-terminal and the COOH terminal portions, respectively, of the site 1 determinant .

Minimization of chemical modifications during the production of proteins for pharmaceutical and medical applications is of fundamental and practical importance. The gluconoylation of heterologously expressed protein which is observed in Escherichia coli BL21(DE3) constitutes one such undesired posttranslational modification. We postulated that formation of gluconoylated/phosphogluconoylated products of heterologous proteins is caused by the accumulation of 6-phosphogluconolactone due to the absence of phosphogluconolactonase (PGL) in the pentose phosphate pathway. The results obtained demonstrate that overexpression of a heterologous PGL in BL21(DE3) suppresses the formation of the gluconoylated adducts in the therapeutic proteins studied. When this E. coli strain was grown in high-cell-density fed-batch cultures with an extra copy of the pgl gene, we found that the biomass yield and specific productivity of a heterologous 18-kDa protein increased simultaneously by 50 and 60%, respectively. The higher level of PGL expression allowed E. coli strain BL21(DE3) to satisfy the extra demand for precursors, as well as the energy requirements, in order to replicate plasmid DNA and express heterologous genes, as metabolic flux analysis showed by the higher precursor and NADPH fluxes through the oxidative branch of the pentose phosphate shunt. This work shows that E. coli strain BL21(DE3) can be used as a host to produce three different proteins, a heterodimer of liver X receptors, elongin C, and an 18-kDa protein. This is the first report describing a novel and general strategy for suppressing this nonenzymatic modification by metabolic pathway engineering.

In this study, we used proteomics to better understand the growth on glucose of Escherichia coli in high cell density, fed-batch cultures and the response to overexpression of plasmid-encoded 6-phosphogluconolactonase (PGL). Using liquid chromatography coupled to electrospray mass spectrometry, at least 300 proteins were identified in the cytosolic fraction of the six time points used to monitor the fermentation. The relative abundance changes of selected proteins were obtained by comparing the peak area of the corresponding peptides at a particular m/z (mass over charge ratio) value. During the time course of samples collected during the rapid growth achieved under batch and fed-batch conditions, both the control and recombinant E. coli strains showed up-regulation of proteins participating in the tricarboxylic acid (TCA) cycle, particularly acetyl-CoA synthetase (AcCoAS), malate dehydrogenase (MDH), and succinyl-CoA synthetase (SuccCoAS). In the recombinant strain culture, fumarase was up-regulated until 35 h after inoculation but was not in the control strain culture. In addition, the proteomic measurement detected up-regulation of three well-characterized binding transport proteins in both control and recombinant strains. The up-regulation of TCA cycle enzymes is consistent with the increase in growth rate observed in the cell culture. In addition, up-regulation of these proteins demonstrated the importance of both the pentose-phosphate shunt and TCA cycle to the increased biosynthetic activity required by a high level protein synthesis. This study shows the potential of proteomics using shotgun sequencing (LC/MS of tryptic digests) to measure global changes in protein abundance during a fermentation process and will facilitate the development of robust manufacturing systems.

We previously described a monoclonal antibody (Y8-10C2) that binds influenza virus hemagglutinin (HA) monomers but not native trimers. In this study, we demonstrated that Y8-10C2 binds to the globular domain of HA and found evidence that its epitope is located at the interface of adjacent subunits. We further showed

Eight nonoverlappimg regions of the hemagglutinin (HA) molecule of influenza virus A/PR/8/34 (PR8), which serve as recognition sites for class II-restricted T cells (TH) from BALB/c mice, have been identified in the form of 10to 15-amino-acid-long synthetic peptides. These TH determinants are located between residues 110 to 313 of the HA1 polypeptide. From a total of 36 HA-specific TH clones and limiting-dilution cultures of independent clonal origins, 33 (90%) responded to stimulation with one of these peptides. The residual three TH clones appeared to recognize a single additional determinant on the HA1 polypeptide which could not be isolated, however, in the form of a stimulatory peptide. None of the motifs that have been proposed to typify TH determinants were displayed by more than half of these recognition sites. Most unexpected was the finding that none of the TH determinants was located in the ectodomain of the HA2 polypeptide that makes up roughly one-third of the HA molecule. Possible reasons for the preferential recognition of HA1 as opposed to HA2 by TH are discussed.