NSAIDs do not require the presence of a carboxylic acid to exert their anti-inflammatory effect – why do we keep using it?

The carboxylic acid group (–COOH) present in classical NSAIDs is partly responsible for the gastric toxicity associated with the administration of these drugs. This concept has been extensively proven using NSAID prodrugs. However, the screening of NSAIDs with no carboxylic acid at all has been neglected. The goal of this work was to determine if new NSAID derivatives devoid of acidic moieties would retain the anti-inflammatory activity of the parent compound, without causing gastric toxicity. To test this concept, we replaced the carboxylic acid group in ibuprofen, flurbiprofen, and naproxen with three ammonium moieties. We tested the resulting water-soluble NSAID derivatives for anti-inflammatory and ulcerogenic activity in vitro and in vivo. In this regard, we observed that all non-acidic NSAIDs exerted a potent anti-inflammatory activity, suggesting that the acid group in commercial 2-phenylpropionic acid NSAIDs not be an essential requirement for anti-inflammatory activity. These data provide complementary evidence supporting the discontinuation of ulcerogenic acidic NSAIDs.     

Detection of high levels of heparin binding growth factor-1 (acidic fibroblast growth factor) in inflammatory arthritic joints

The synovium from patients with rheumatoid arthritis (RA) and LEW/N rats with streptococcal cell wall (SCW) arthritis, an experimental model resembling RA, is characterized by massive proliferation of synovial connective tissues and invasive destruction of periarticular bone and cartilage. Since heparin binding growth factor (HBGF)-1, the precursor of acidic fibroblast growth factor (FGF), is a potent angiogenic polypeptide and mitogen for mesenchymal cells, we sought evidence that it was involved in the synovial pathology of RA and SCW arthritis. HBGF-1 mRNA was detected in RA synovium using the polymerase chain reaction technique, and its product was immunolocalized intracellularly in both RA and osteoarthritis (OA) synovium. HBGF-1 staining was more extensive and intense in synovium of RA patients than OA and correlated with the extent and intensity of synovial mononuclear cell infiltration. HBGF-1 staining also correlated with c-Fos protein staining. In SCW arthritis, HBGF-1 immunostaining was noted in bone marrow, bone, cartilage, synovium, ligamentous and tendinous structures, as well as various dermal structures and developed early in both T-cell competent and incompetent rats. Persistent high level immunostaining of HBGF-1 was only noted in T-cell competent rats like the disease process in general. These observations implicate HBGF-1 in a multitude of biological functions in inflammatory joint diseases.     

Identification of the product of dnaB gene in Bacillus subtilis

In order to detect the product of dnaB gene in B. subtilis, a gene which is involved in the initiation of DNA replication and the formation of the DNA-membrane complex, we synthesized an origopeptide of 15 amino acids which corresponds to a region near the carboxyl-terminal of the gene product, and raised antibody against the synthetic peptide. We have also employed a filter binding assay to measure the predicted DNA binding activity of the product of the dnaB gene, using the plasmid pUB110. The binding activity was detected after fractionation of cell lysates of B. subtilis on sucrose-density gradients. When the active fraction was prepared from a mutant which was temperature-sensitive for the dnaB gene, the DNA binding activity in the fraction showed significant thermolability. Furthermore, the binding activity was inhibited by the purified antibody raised against the synthetic peptide. These results suggest that the product of the dnaB gene does indeed have DNA binding activity, and that the filter binding assay and the antibody can be used for the detection and characterization of the gene product.     

Genetics of Familial Combined Hyperlipidemia (FCHL) Disorder: An Update

Biochemical Genetics - Familial combined hyperlipidemia (FCHL) is one of the most common familial lipoprotein disorders of the lipoproteins, with a prevalence of 0.5% to 2% in different...     

Enhanced persistence in the colonic microbiota of Escherichia coli strains belonging to phylogenetic group B2: role of virulence factors and adherence to colonic cells

Escherichia coli segregates into four phylogenetic groups, A, B1, B2 and D. B2 and D strains usually possess virulence factors, cause most extra-intestinal infections and have superior capacity to persist in the infantile colonic microbiota. Here, we investigated 24 resident and 37 transient E. coli strains from the colonic microbiota of 13 Swedish schoolgirls sampled in the 1970s with respect to phylogenetic group identity, carriage of virulence factor genes, O and K antigens and mannose-sensitive and -resistant adherence to the colonic cell line HT-29. Resident strains more often belonged to phylogenetic group B2 than transient strains (38% vs 5% p=0.004). In contrast, transient strains more often than resident strains belonged to group A (57% vs 29%, p=0.04) or B1 (24% vs 13%, p=0.33). Most B2 strains belonged to uropathogenic O serogroups, carried genes for P fimbriae, K5 capsule and hemolysin and adhered in higher numbers to HT-29 cells via mannose-resistant mechanisms than strains from the other groups. Further, among strains carrying genes for P or S fimbriae, those belonging to group B2 adhered in highest numbers. In logistic regression, genes for P fimbriae and aerobactin predicted persistence in the colonic microbiota (p=0.050 and 0.056, respectively), while B2 origin did not reach significance as an independent variable (p=0.16). Our results indicate that virulence factors carried by group B2 strains contribute to their strong colonizing capacity. These factors may actually be regarded as fitness factors in the human gut.     

Eph B4 receptor signaling mediates endothelial cell migration and proliferation via the phosphatidylinositol 3-kinase pathway

The goals of this study were 2-fold: 1) to determine whether stimulation of Eph B4 receptors promotes microvascular endothelial cell migration and/or proliferation, and 2) to elucidate signaling pathways involved in these responses. The human endothelial cells used possessed abundant Eph B4 receptors with no endogenous ephrin B2 expression. Stimulation of these receptors with ephrin B2/Fc chimera resulted in dose- and time-dependent phosphorylation of Akt. These responses were inhibited by LY294002 and ML-9, blockers of phosphatidylinositol 3-kinase (PI3K) and Akt, respectively. Eph B4 receptor activation increased proliferation by 38%, which was prevented by prior blockade with LY294002, ML-9, and inhibitors of protein kinase G (KT5823) and MEK (PD98059). Nitrite levels increased over 170% after Eph B4 stimulation, indicating increased nitric oxide production. Signaling of endothelial cell proliferation appears to be mediated by a PI3K/Akt/endothelial nitric-oxide synthase/protein kinase G/mitogen-activated protein kinase cascade. Stimulation with ephrin B2 also increased migration by 63% versus controls. This effect was inhibited by blockade with PP2 (Src inhibitor), LY294002 or ML-9 but was unaffected by the PKG and MEK blockers. Eph B4 receptor stimulation increased activation of both matrix metalloproteinase-2 and -9. The results from these studies indicate that Eph B4 stimulates migration and proliferation and may play a role in angiogenesis.     

Overexpression of a secretory form of FGF-1 promotes MMP-1-mediated endothelial cell migration

A coordinated interaction between fibroblast growth factors (FGFs) and matrix metalloproteinases (MMPs) is implicated in migration of microvascular endothelial cells (ECs), an early stage of angiogenesis. Specifically, we investigated microvascular ECs migration in vitro, which can be initiated by the overexpression of a secretory form of the angiogenic fibroblast growth factor-1 (FGF-1) and mediated through the enzymatic activity of matrix metalloproteinase-1 (MMP-1). MMP-1 is a member of the MMP family with a propensity for degradation of interstitial type I collagen. We stably overexpressed a chimeric FGF-1 construct composed of the FGF-4 signal-peptide gene, linked in-frame to the FGF-1 coding frame gene (sp-FGF-1), in cultured postcapillary venular ECs. The presence of the biologically active form of FGF-1 was readily detected in the conditioned medium of ECs transfected with sp-FGF-1 construct as demonstrated by DNA synthesis assay. The sp-FGF-1-, but not the plasmid vector alone-transfected ECs, exhibited an altered morphology as demonstrated by their conversion from a classic cobblestone form to a fibroblastlike shape that featured prominent neuritelike extensions. Addition of the anti-FGF receptor 1 antibody (FGFR1 Ab) reverted the transformed phenotype of sp-FGF-1 transfectants. This suggests that the resulting phenotypic transformation in sp-FGF-1 transfectants requires an uninterrupted interaction between the FGF-1 ligand and its receptor. We studied migration of cells through matrices of either highly pure collagen I or reconstituted basement membrane (matrigel) and found that sp-FGF-1-transfected cells migrated two times and six times faster than the vector control transfectants in the respective matrices. We further demonstrated that the enhanced migration rate of sp-FGF-1-transfected EC coincided with the induction of their MMP-1 mRNA level and increased enzymatic activity. The enhanced migratory activity of sp-FGF-1 could be blocked with a selective inhibitor of MMP-1. These results suggest that the multipotent FGF-1 plays a key role in the early stages of angiogenesis, by mediating MMP-1 proteolytic activity.