Klebsiella pneumoniae OmpA, the 40-kDa major protein of the outer membrane, was cloned and expressed in Escherichia coli. The recombinant protein was produced intracellularly in E. coli as inclusion bodies. Fusion of a short peptide to the N-terminus of native P40 facilitated high-level expression of the recombinant protein. Purified recombinant P40 was analyzed to verify purity and structural integrity. The molecular mass of purified recombinant P40 determined by electrospray mass spectrometry was 37 061 Da, in agreement with the theoretical mass deduced from the DNA sequence. Specific proliferation of recombinant-P40-primed murine lymph node cells in response to recombinant P40 stimulation in vitro indicated the presence of a T-cell epitope on recombinant P40. The induction of high serum antibody titers to a synthetic peptide derived from the attachment protein G of the respiratory syncytial virus when chemically coupled to recombinant P40 indicated that the protein had potent carrier properties. OmpA [20, 21] have been demonstrated to mediate expression of gram-negative bacteria, present at about 10 5 copies/cell. It is believed to occur in a monomeric form in its native state. A of peptides on the surface of live enterobacterial vectors. OmpC, the outer-membrane protein complex of Neisseria meningitidis, typical feature of OmpA is that it can be modified by heat: the mobility of Escherichia coli OmpA on SDS/PAGE decreases has been shown to be effective in humans as a conjugate vaccine with Haemophilus influenzae, pneumococcal and meningococcal when it is heated in the presence of SDS [1]. OmpA is highly conserved among gram-negative bacteria and is thought to con-capsular polysaccharides [22Ϫ25]. Other clinically useful carrier proteins have been derived from bacteria: diphteria and tetanus sist of two domains. The N terminus, including amino acid residues 1Ϫ170, forms a membrane-spanning domain and crosses toxoids are both successfully used in conjugated H. influenzae vaccines to transform the capsular polysaccharide, which is a Tthe outer membrane eight times in antiparallel β-strands, leading to a typical amphiphilic β-barrel [2Ϫ4]. The C-terminal moiety independent antigen, into a T-dependent antigen [26]. KeywordsIn this paper we describe the expression and production in of the protein is thought to be periplasmic [5]. The protein seems to be multifunctional. In addition to non-physiological functions, E. coli and purification of Klebsiella pneumoniae OmpA [27].Using various analytical criteria, the purity and structural integsuch as serving as a receptor for phages and colicins [6], it serves as a mediator in F-factor-dependent conjugation [7]. It is rity of the protein were evaluated. We demonstrate the presence of at least one T-cell epitope on the molecule and its potential also required for the structural integrity of the outer membrane and the generation of normal cell shape [8]. The capacity to use as a carrier protein for conjugated peptides. The immunological carrier properties of the recombin...

The somatostatin receptor subtype sst2 mediates both activation of a tyrosine phosphatase activity and inhibition of cell proliferation induced by somatostatin analogues.

We have previously shown that somatostatin promotes the stimulation of a membrane tyrosine phosphatase activity in pancreatic cells. To gain insight into the mechanism of somatostatin action, we purified somatostatin-receptor complexes from somatostatin 28-prelabelled rat pancreatic plasma membranes by immunoaffinity chromatography using immobilized antibodies raised against the N-terminal part of somatostatin 28, somatostatin 28 (1-14), which is not involved in receptor-binding-site recognition. After SDS gel electrophoresis a band with a molecular mass of 87 kDa was identified in the affinity-purified material as the somatostatin receptor. The 87 kDa protein was not observed when the membrane receptors were solubilized in a free unoccupied or somatostatin 14-occupied form, or when nonimmune serum replaced the anti-[somatostatin 28 (1-14)] anti-serum. Somatostatin 14 inhibited the appearance of the 87 kDa protein in the same range of concentrations that inhibit radioligand binding on pancreatic membranes. After somatostatin 28 treatment of membranes, purified somatostatin receptor preparations exhibited an elevated tyrosine phosphatase activity that dephosphorylated phosphorylated epidermal growth factor receptor and poly(Glu,Tyr). This activity was related to the presence of somatostatin receptors in purified material. It was increased by dithiothreitol and inhibited by orthovanadate. In purified material containing somatostatin receptors, anti-[Src homology 2 domains (SH2)]-containing tyrosine phosphatase SHPTP1 polyclonal antibodies identified a protein of 66 kDa which was not detected in the absence of somatostatin receptor. Furthermore, the anti-SHPTP1 antibodies immunoprecipitated specific somatostatin receptors from somatostatin-prelabelled pancreatic membranes and from untreated membranes. These results indicate that a 66 kDa tyrosine phosphatase related to SHPTP1 co-purifies with the pancreatic somatostatin receptors, and suggest that this protein is associated with somatostatin receptors at the membrane level.

Whereas agonist-directed differential signaling at a single receptor subtype has become an accepted pharmacological concept, distinct behaviors by ligands that are assumed to be antagonists is less documented. The intrinsic activity and capacity of antagonism for a new series of imidazoline-derived adrenergic ligands analogous to dexefaroxan were investigated by measuring two distinct signaling pathways at the recombinant human ␣ 2 -Adrenoceptors (␣ 2 ARs) are cell surface G protein-coupled receptors that bind native catecholamines and couple preferentially to the G i/o family of G proteins (Limbird, 1988). They are widely expressed in both the central and peripheral nervous systems (Eason and Liggett, 1993;Handy et al., 1993;Tavares et al., 1996) and have been shown to participate in a broad spectrum of physiological functions, which include inhibition of neurotransmitter release, regulation of blood pressure (both centrally and within the vasculature), sedation, analgesia, regulation of insulin release and lipolysis, renal function, and multiple behavioral and cognitive functions (Small and Liggett, 2001). The cellular effects of ␣ 2 AR activation include inhibition of adenylate cyclase, activaArticle, publication date, and citation information can be found at

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We have recently cloned a new protein, recombinant P40 (rP40). When tested in vivo after conjugation to a B-cell epitope, rP40 induces an important antibody response without the need for adjuvant. To characterize its potency, this carrier protein was coupled to a peptide derived from respiratory syncytial virus attachment G protein (G1′). After immunization of mice with the rP40-G1′ conjugate, strong antipeptide antibodies were detected, whereas peptide alone was not immunogenic. To emphasize the carrier properties of rP40, a polysaccharide derived from Haemophilus influenzae type b (Hib) was coupled to it. Immunoglobulin G responses against the Hib polysaccharide were observed after coupling to rP40. Interestingly, an antipeptide antibody response was observed despite preexisting anti-rP40 antibodies generated by preimmunization with rP40. In addition, rP40 compares well with the reference carrier protein, tetanus toxoid (TT), since antibody responses of equal intensity were observed when a peptide or a polysaccharide was coupled to TT and rP40. Moreover, rP40 had advantages compared to TT; e.g., it induced a mixed Th1/Th2 response, whereas TT induced only a Th2 profile. Together, the results indicate that rP40 is a novel carrier protein with potential for use as an alternative carrier for human vaccination.

The effect of glucocorticoids, known to induce inhibition of growth and differentiation of pancreatic cells, has been examined on the tyrosine phosphatase containing two src homology 2 domains, PTP1C, in rat pancreatic cancer AR42J cells. Immunoblotting analysis revealed that PTP1C protein was present in AR42J cells as two PTP1C species of 66 and 31 kilodaltons (kDa), the 31-kDa species representing a proteolytic product of the larger form. Dexamethasone increased the level of the two PTP1C species by 2 to 3 times. Nearly 80% of the PTP1C molecules were found in the particulate fraction in control cells and dexamethasone did not change the distribution of PTP1C. The increase of PTP1C protein was also detected by immunohistochemical analysis. Dexamethasone increased the tyrosine phosphatase activity of immunoprecipitated PTP1C. In addition, dexamethasone raised the level of expression of PTP1C messenger RNA in a time- and dose-dependent manner in relation with its effect on cell growth and differentiation. This effect was selective, the messenger RNA levels of the other tyrosine phosphatase containing two src homology 2 domains (SH2), PTP1D, and that of the cytosolic PTP1 being not affected. This is the first report of glucocorticoid increase of PTP1C expression, suggesting that PTP1C may be involved in the glucocorticoid-mediated pancreatic cell differentiation.

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