Characterization of mouse cysteinyl leukotriene receptors mCysLT1 and mCysLT2: differential pharmacological properties and tissue distribution

Cysteinyl leukotrienes (LTs) are important proinflammatory mediators. Their precise roles in mice need to be elucidated to interpret mouse models of inflammatory diseases. For this purpose, we cloned and characterized mouse receptors for cysteinyl LTs, mCysLT(1) and mCysLT(2). mCysLT(1) and mCysLT(2) were composed of 339 amino acids with 87.3% identity and 309 amino acids with 73.4% identity to human orthologues, respectively. A pharmacological difference was noted between mouse and human CysLT(2). Pranlukast, a specific inhibitor for human CysLT(1), antagonized mCysLT(2) responses as determined by Ca(2+) elevation and receptor-induced promoter activation. The mRNA expressions of both mCysLTs were higher in C57BL/6 mice than in 129 mice. mCysLT(1) mRNA was expressed mainly in skin, lung, and small intestine. mCysLT(2) was seen more ubiquitously with high expressions in spleen, lung, and small intestine. By in situ hybridization we demonstrated for the first time that mCysLT(1) and mCysLT(2) were expressed in subcutaneous fibroblasts. The different pharmacological characteristics of CysLT(2) between human and mouse and the different distributions of CysLTs between mouse strains suggest that careful choice and interpretation are necessary for a study of CysLTs using animal models.     

Dual functions of fractalkine/CX3C ligand 1 in trafficking of perforin+/granzyme B+ cytotoxic effector lymphocytes that are defined by CX3CR1 expression

Fractalkine/CX3C ligand 1 and its receptor CX3CR1 are known to mediate both cell adhesion and cell migration. Here we show that CX3CR1 defines peripheral blood cytotoxic effector lymphocytes commonly armed with intracellular perforin and granzyme B, which include NK cells, gammadelta T cells, and terminally differentiated CD8(+) T cells. In addition, soluble fractalkine preferentially induced migration of cytotoxic effector lymphocytes. Furthermore, interaction of cytotoxic effector lymphocytes with membrane-bound fractalkine promoted subsequent migration to the secondary chemokines, such as macrophage inflammatory protein-1beta/CC ligand 4 or IL-8/CXC ligand 8. Thus, fractalkine expressed on inflamed endothelium may function as a vascular regulator for cytotoxic effector lymphocytes, regardless of their lineage and mode of target cell recognition, through its ability to capture them from blood flow and to promote their emigration in response to other chemokines.     

Inhibitory action of a macrolide antibiotic,roxithromycin, on co-stimulatory molecule expressions in vitro and in vivo

OBJECTIVE: The influence of a macrolide antibiotic, roxithromycin (RXM), on co-stimulatory molecule expression was examined in vitro and in vivo. MATERIALS AND METHODS: Spleen cells obtained from BALB/c mice 10 days after immunization with 8.0 microg of hemocyanin absorbed to 4.0 mg of aluminum hydroxide were cultured in the presence of 100.0 microg/ml of hemocyanin and various concentrations of RXM. We first examined the influence of RXM on cell activation by examining the proliferative response of cells and cytokine production. We also examined the influence of RXM on co-stimulatory molecule (CD40, CD80 and CD86) expressions on cultured splenic B-lymphocytes induced by in vitro antigenic stimulation using flow cytometry. In the second part of experiments, non-immunized and immunized mice were treated orally with 2.5 mg/kg of RXM once a day for 4 or 8 weeks. Splenic B lymphocytes were obtained from these mice 24 h after antigenic challenge, and co-stimulatory molecule expressions were examined by flow cytometer. RESULTS: Cell activation induced by in vitro antigenic stimulation was suppressed by RXM when cells were cultured in the presence of more than 5.0 microg/ml of the agent. Addition of RXM at a concentration of 5.0 microg/ml into cell cultures also suppressed co-stimulatory molecule (CD40, CD80 and CD86) expressions on splenic B lymphocytes, which was enhanced by antigenic stimulation in vitro. Oral RXM administration for 4 weeks clearly suppressed the enhancement of CD40 and CD86 (but not CD80) expressions on splenic B lymphocytes induced by antigenic stimulation in vivo. This suppressive activity of RXM on co-stimulatory molecule (CD40 and CD86) expressions was further strengthened by the treatment of mice for 8 weeks. Long-term treatment with oral RXM also suppressed CD80 expressions, which was not suppressed by 4-week treatment. CONCLUSION: The present results suggest that RXM exerts its immunomodulating effects through suppression of both cell activation and co-stimulatory molecule expressions induced by antigenic stimulation. These suppressive activities of RXM might contribute, in part, to the therapeutic mode of action of RXM on inflammatory diseases.     

Aggregation and neurotoxicity of mutant amyloid beta (A beta) peptides with proline replacement: importance of turn formation at positions 22 and 23

Aggregation of the amyloid beta peptides (A beta 1-42 and A beta 1-40) plays a pivotal role in pathogenesis of Alzheimer's disease. Although it is widely accepted that the aggregates of A betas mainly consist of beta-sheet structure, the precise aggregation mechanism remains unclear. To identify amino acid residues that are important for the beta-sheet formation, a series of proline-substituted mutants of A beta 1-42 peptides at positions 19-26 was synthesized in a highly pure form and their aggregation ability and neurotoxicity on PC12 cells were investigated. All proline-substituted A beta 1-42 mutants except for 22P- and 23P-A beta 1-42 were hard to aggregate and showed weaker cytotoxicity than wild-type A beta 1-42, suggesting that the residues at positions 19-21 and 24-26 are important for the beta-sheet formation. In contrast, 22P-A beta 1-42 extensively aggregated with stronger cytotoxicity than wild-type A beta 1-42. Since proline has a propensity for beta-turn structure as a Pro-X corner, these data implicate that beta-turn formation at positions 22 and 23 plays a crucial role in the aggregation and neurotoxicity of A beta peptides.     

Gas-phase disproportionation of nitric oxide at elevated pressures

T.P. Melia's chemical kinetics study of the disproportionation of nitric oxide (NO), 3NO →NO₂ + N₂O , (Melia, T.P. (1965) J. Inorg. Nucl. Chem., 27, 95-98), which is the most quoted quantitative investigation presently available, revealed a rather strong dependence of the effective rate constant, Kk ', of Melia's third-order rate law,- d[NO]/d t = Kk'[NO]₃, on the initial pressure of NO. In order to estimate extent of accumulation of NO₂ and N₂O as a function of time by integration of the rate law, we have evaluated the dependence of the effective rate constant as a function of pressure and thus as a function of time on the basis of the non-ideality of NO gas. Although our approach is crude in that the non-idealities of NO₂ and N₂O and other NO_x products and a probable deviation of the gas mixture from the Dalton's law have not been considered, it provides a means for approximately estimating the rate of accumulation of NO₂ and N₂O based on Melia's data. According to these calculations, the extent of the disproportionation is generally negligible at low initial pressures, e.g. 5 atm or less, while at 200 atm, the mole fractions of NO₂ and N₂O can become as high as 12-13% only after 10 days. These values are alarmingly high for handling pressured NO- in N₂-mixture in either research or clinical settings. This information must be borne in mind when compressed NO in commercial cylinders is employed in high precision experiments. Disproportionation of NO under pressure also deserves attention in inhalation of low doses of NO in the treatment of diseases characterized by pulmonary hypertension and hypoxemia.     

Site-specific PEGylation of a lysine-deficient TNF-alpha with full bioactivity

Addition of polyethylene glycol to protein (PEGylation) to improve stability and other characteristics is mostly nonspecific and may occur at all lysine residues, some of which may be within or near an active site. Resultant PEGylated proteins are heterogeneous and can show markedly lower bioactivity. We attempted to develop a strategy for site-specific mono-PEGylation using tumor necrosis factor-alpha (TNF-alpha). We prepared phage libraries expressing TNF-alpha mutants in which all the lysine residues were replaced with other amino acids. A fully bioactive lysine-deficient mutant TNF-alpha (mTNF-alpha-Lys(-)) was isolated by panning against TNF-alpha-neutralizing antibody despite reports that some lysine residues were essential for its bioactivity. mTNF-alpha-Lys(-) was site-specifically mono-PEGylated at its N terminus. This mono-PEGylated mTNF-alpha-Lys(-), with superior molecular uniformity, showed higher bioactivity in vitro and greater antitumor therapeutic potency than randomly mono-PEGylated wild-type TNF-alpha. These results suggest the usefulness of the phage display system for creating functional mutant proteins and of our site-specific PEGylation approach.     

Synthesis of a poly(vinylpyrrolidone-co-dimethyl maleic anhydride) co-polymer and its application for renal drug targeting

We have synthesized a polymeric drug carrier, polyvinylpyrrolidone-co-dimethyl maleic anhydride [poly(VP-co-DMMAn)], for use in renal drug delivery. About 80% of the 10-kDa poly(VP-co-DMMAn) selectively accumulated in the kidneys 24 h after intravenous administration to mice. Although this accumulated poly(VP-co-DMMAn) was gradually excreted in the urine, about 40% remained in the kidneys 96 h after treatment. Poly(VP-co-DMMAn) was taken up by the renal proximal tubular epithelial cells and no cytotoxicity was noted. Higher doses did not produce toxicity in the kidneys or other tissues. In contrast, polyvinylpyrrolidone of the same molecular weight did not show any tissue-specific distribution. Poly(VP-co-DMMAn)-modified superoxide dismutase accumulated in the kidneys after intravenous administration and accelerated recovery from acute renal failure in a mouse model. In contrast, polyvinylpyrrolidone-modified superoxide dismutase and native superoxide dismutase were not as effective. Thus, poly(VP-co-DMMAn) is a useful candidate as a targeting carrier for renal drug delivery systems.     

Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis

Species of the genus Streptomyces are of major pharmaceutical interest because they synthesize a variety of bioactive secondary metabolites. We have determined the complete nucleotide sequence of the linear chromosome of Streptomyces avermitilis. S. avermitilis produces avermectins, a group of antiparasitic agents used in human and veterinary medicine. The genome contains 9,025,608 bases (average GC content, 70.7%) and encodes at least 7,574 potential open reading frames (ORFs). Thirty-five percent of the ORFs (2,664) constitute 721 paralogous families. Thirty gene clusters related to secondary metabolite biosynthesis were identified, corresponding to 6.6% of the genome. Comparison with Streptomyces coelicolor A3(2) revealed that an internal 6.5-Mb region in the S. avermitilis genome was highly conserved with respect to gene order and content, and contained all known essential genes but showed perfectly asymmetric structure at the oriC center. In contrast, the terminal regions were not conserved and preferentially contained nonessential genes.