Discovery of a potent, selective and orally active canine COX-2 inhibitor, 2-(3-difluoromethyl-5-phenyl-pyrazol-1-yl)-5-methanesulfonyl-pyridine

Structure-activity relationship (SAR) studies of 2-[3-di(and tri)fluoromethyl-5-arylpyrazol-1-yl]-5-methanesulfonylpyridine derivatives for canine COX enzymes are described. This led to the identification of 12a as a lead candidate for further progression. The in vitro and in vivo activity of 12a for the canine COX-2 enzyme as well as its in vivo efficacy and pharmacokinetic properties in dog are highlighted.     

Maintenance of long term gamma-herpesvirus B cell latency is dependent on CD40-mediated development of memory B cells

It has been proposed that the gamma-herpesviruses maintain lifelong latency in B cells by gaining entry into the memory B cell pool and taking advantage of host mechanisms for maintaining these cells. We directly tested this hypothesis by kinetically monitoring viral latency in CD40(+) and CD40(-) B cells from CD40(+)CD40(-) mixed bone marrow chimera mice after infection with a murine gamma-herpesvirus, MHV-68. CD40(+) B cells selectively entered germinal centers and differentiated into memory B cells. Importantly, latency was progressively lost in the CD40(-) B cells and preferentially maintained in the long-lived, isotype-switched CD40(+) B cells. These data directly demonstrate viral exploitation of the normal B cell differentiation pathway to maintain latency.     

CD40, but not CD154, expression on B cells is necessary for optimal primary B cell responses

CD40 is an important costimulatory molecule for B cells as well as dendritic cells, monocytes, and other APCs. The ligand for CD40, CD154, is expressed on activated T cells, NK cells, mast cells, basophils, and even activated B cells. Although both CD40(-/-) and CD154(-/-) mice have impaired ability to isotype switch, form germinal centers, make memory B cells, and produce Ab, it is not entirely clear whether these defects are intrinsic to B cells, to other APCs, or to T cells. Using bone marrow chimeric mice, we investigated whether CD40 or CD154 must be expressed on B cells for optimal B cell responses in vivo. We demonstrate that CD40 expression on B cells is required for the generation of germinal centers, isotype switching, and sustained Ab production, even when other APCs express CD40. In contrast, the expression of CD154 on B cells is not required for the generation of germinal centers, isotype switching, or sustained Ab production. In fact, B cell responses are completely normal when CD154 expression is limited exclusively to Ag-specific T cells. These results suggest that the interaction of CD154 expressed by activated CD4 T cells with CD40 expressed by B cells is the primary pathway necessary to achieve B cell activation and differentiation and that CD154 expression on B cells does not noticeably facilitate B cell activation and differentiation.     

Perkinsus marinus, a protozoan parasite of the Eastern oyster (Crassostrea virginica): effects of temperature on the uptake and metabolism of fluorescent lipid analogs and lipase activities

The effects of temperature on the uptake and metabolism of fluorescent labeled palmitic acid (FLC16) and phosphatidylcholine (FLPC) and lipase activities in the oyster protozoan parasite, Perkinsus marinus, meront stage were tested at 10, 18, and 28 degrees C. Temperature significantly affected not only the uptake, assimilation, and metabolism of both FLC16 and FLPC in P. marinus, but also its triacylglycerol (TAG) lipase activities. The incorporation of both FLC16 and FLPC increased with temperature and paralleled the increase in the amount of total fatty acids in P. marinus meront cultures. The incorporation of FLC16 was higher than FLPC at all temperatures. The percentage of FLC16 metabolized to TAG was significantly higher at higher temperatures. Trace amounts of incorporated FLC16 were detected in monoacylglycerol (MAG) and PC at 18 and 28 degrees C. P. marinus meronts metabolized FLPC to TAG, diacylglycerol (DAG), monoacylglycerol (MAG), free fatty acids (FFA), phosphatidylethanolamine (PE), and cardiolipin (CL). The conversion of FLPC to TAG and PE was highest at 28 degrees C. The relative proportions of individual fatty acids and total saturated, monounsaturated and polyunsaturated fatty acids changed with temperatures. While total saturated fatty acids (SAFAs) increased with temperature, total monounsaturated fatty acids (MUFAs) decreased with temperature. Total polyunsaturated fatty acids (PUFAs) increased from 28 to 18 degrees C. The findings of increase of total SAFAs and decrease of total MUFAs with the increase of temperatures and upward shift of total PUFAs from 28 to 18 degrees C suggest that, as in other organisms, P. marinus is capable of adapting to changes in environmental temperatures by modifying its lipid metabolism. Generally, higher lipase activities were noted at higher cultivation temperatures. Both TAG lipase and phospholipase activities were detected in P. marinus cells and their extra cellular products (ECP), but phospholipase activities in both the cell pellets and ECP were very low. Also, lipase activities were much lower in ECP than in the cells. The observations of low metabolism, bioconversion of incorporated fluorescent lipid analogs and lipase activities at low temperatures are consistent with the low in vitro growth rate and low infectivity of P. marinus at low temperatures.     

Phenylboronic acid-salicylhydroxamic acid bioconjugates. 2. Polyvalent immobilization of protein ligands for affinity chromatography

Phenylboronic acid bioconjugates prepared from alkaline phosphatase by reaction with either 2,5-dioxopyrrolidinyl 3-[N-[3-(1,3,2-dioxaboran-2-yl)phenyl]carbamoyl]propanoate (PBA-XX-NHS) or 2,5-dioxopyrrolidinyl 6-[[3,5-di-(1,3,2-dioxaboran-2-yl)phenyl]carbonylamino]hexanoate (PDBA-X-NHS) were compared with respect to the efficiency with which they were immobilized on salicylhydroxamic acid-modified Sepharose (SHA-X-Sepharose) by boronic acid complex formation. When immobilized on moderate capacity SHA-X-Sepharose (5.4 micromol of SHA/mL of gel), PDBA-alkaline phosphatase conjugates were shown to be stable with respect to both the alkaline (pH 11.0) and acidic (pH 2.5) buffers utilized to recover anti-alkaline phosphatase during affinity chromatography. Boronic acid complex formation was compared to covalent immobilization of alkaline phosphatase on Affi-Gel 10 and Affi-Gel 15. PDBA-AP.SHA-X-Sepharose was shown to afford superior performance to both Affi-Gel 10 and Affi-Gel 15 with respect to immobilization of alkaline phosphatase, retention of anti-alkaline phosphatase and recovery of anti-alkaline phosphatase under alkaline conditions. High capacity SHA-X-Sepharose (> or = 7 micromol of SHA/mL of gel) was shown to afford superior performance to moderate capacity SHA-X-Sepharose (4.5 micromol of SHA/mL of gel) with respect to stability at pH 11.0 and pH 2.5 when a PDBA-alphaHuman IgG conjugate with a low incorporation ratio of only 1.5:1 was immobilized on SHA-X-Sepharose and subsequently utilized for affinity chromatography of Human IgG. The results are interpreted in terms of either a bivalent or trivalent interaction involving boronic acid complex formation.     

Increased mRNA expression and protein secretion of interleukin-6 in primary human osteoblasts differentiated in vitro from rheumatoid and osteoarthritic bone

We have investigated the expression and synthesis of potential bone-resorbing cytokines, interleukin-6 (IL-6), interleukin-1 (IL-1), and tumor necrosis factor (TNF) in rheumatoid arthritic (RA) and osteoarthritic (OA) bone, two common diseases which are associated with bone loss. Primary human osteoblast (hOB) cultures were established to determine the temporal mRNA expression of IL-6, IL-1 (alpha and beta), and TNF (alpha and beta) in relation to osteoblast growth and phenotypic genes. IL-6 mRNA levels were found to be significantly higher (P < 0.04) in both OA hOB (17 patients) and RA hOB (10 patients) compared to normal (NO) hOB (9 patients) and reached five-fold increases in OA hOB and 13-fold increases in RA hOB. Maximal levels of IL-6 are expressed at Day 21 which corresponds to the mineralization stage reflected by decreasing collagen I (alpha(1)), osteopontin, bone sialoprotein, alkaline phosphatase mRNA levels, while osteocalcin (OC) mRNA levels increased. IL-6 protein levels also were significantly higher (P < 0.05) in OA hOB and RA hOB compared to NO hOB. These increases were not attributable to sex or age of the donor bone. Neither the mRNA encoding IL-1(alpha and beta) and TNF(alpha and beta) nor the related proteins were detectable. These results indicate that differentiated OA hOB and RA hOB within a bone tissue-like matrix constitutively express and secrete high levels of IL-6. This inherent property suggests that these osteoblasts, independent of local inflammatory parameters, can contribute to enhanced recruitment of osteoclast progenitors and thereby bone resorption.     

Characterization of erythrocyte carbonic anhydrase in an ancient fish,the longnose gar ( Lepisosteus osseus)

This study investigates the evolutionary history of vertebrate red blood cell carbonic anhydrase (CA) by characterizing the isozyme properties and nucleotide sequence of an ancient fish, the longnose gar ( Lepisosteus osseus). The inhibitor sensitivities of gar rbc CA closely resembled those for mammalian CA II, as well as those for CAs from more recently evolved fishes. The kinetic properties of gar rbc CA were not closely aligned with either mammalian CA I and CA II, but fit well into an emerging phylogenetic pattern for early vertebrates. Gar rbc CA cDNA was also amplified from mRNA using 5' and 3'-RACE and the open reading frame consisted of 786 bp. This sequence shares approximately 65% identity with the nucleotide and amino acid sequences of both mammalian CA I and CA II. When the amino acid sequences within the active site are compared, gar rbc CA differs from mammalian CA I, CA II and CA VII by 9, 4 and 3 of the 36 amino acids, respectively. Phylogenetic analyses suggest that gar rbc CA diverged before the amniotic CAs (CA I, CA II and CA III), but after CA V and CA VII.     

Use of fluorescent sequence-specific polyamides to discriminate human chromosomes by microscopy and flow cytometry

In this paper, we demonstrate the use of synthetic polyamide probes to fluorescently label heterochromatic regions on human chromosomes for discrimination in cytogenetic preparations and by flow cytometry. Polyamides bind to the minor groove of DNA in a sequence-specific manner. Unlike conventional sequence-specific DNA or RNA probes, polyamides can recognize their target sequence without the need to subject chromosomes to harsh denaturing conditions. For this study, we designed and synthesized a polyamide to target the TTCCA-motif repeated in the heterochromatic regions of chromosome 9, Y and 1. We demonstrate that the fluorescently labeled polyamide binds to its target sequence in both conventional cytogenetic preparations of metaphase chromosomes and suspended chromosomes without denaturation. Chromosomes 9 and Y can be discriminated and purified by flow sorting on the basis of polyamide binding and Hoechst 33258 staining. We generate chromosome 9- and Y-specific ‘paints’ from the sorted fractions. We demonstrate the utility of this technology by characterizing the sequence of an olfactory receptor gene that is duplicated on multiple chromosomes. By separating chromosome 9 from chromosomes 10–12 on the basis of polyamide fluorescence, we determine and differentiate the haplotypes of the highly similar copies of this gene on chromosomes 9 and 11.