Exposure of cryptic domains in the alpha 1-chain of laminin-1 by elastase stimulates macrophages urokinase and matrix metalloproteinase-9 expression

Degradation of the extracellular matrix leads to the release of fragments, which elicit biological responses distinct from intact molecules. We have reported that alpha1:Ser(2091)-Arg(2108), a peptide derived from the alpha1-chain of laminin-1, triggers protein kinase C-dependent activation of MAPK(erk1/2), leading to the up-regulation of macrophage urokinase type plasminogen activator and matrix metalloproteinase (MMP)-9 expression. Since intact laminin-1 failed to trigger these events, we hypothesized that alpha1:Ser(2091)-Arg(2108) is cryptic or assumes a conformation not recognized by macrophages. Here we demonstrate that elastase cleavage of laminin-1 generates fragments, which stimulate proteinase expression by RAW264.7 macrophages and peritoneal macrophages. In contrast, fragments generated by MMP-2, MMP-7, or plasmin had no effect on macrophage proteinase expression. Elastase-generated laminin-1 fragments were fractionated by heparin-Sepharose chromatography. Heparin-binding fragments stimulated macrophages' proteinase expression severalfold greater than nonbinding fragments. The heparin binding fragments reacted with antibodies directed against regions of the alpha1-chain including alpha1:Ser(2091)-Arg(2108) and the globular domain. A peptide from the first loop of the globular domain (alpha1:Ser(2179)-Ser(2198)) triggered the phosphorylation of MAPK(erk1/2) and stimulated the expression of macrophage urokinase type plasminogen activator and MMP-9. Moreover, a heparin-binding fraction isolated from an aortic aneurysm contained fragments of alpha1-chain and stimulated macrophages' proteinase expression. Based on these data, we conclude that cryptic domains in the COOH-terminal portion of the alpha1-chain of laminin are exposed by proteolysis and stimulate macrophages' proteinase expression.     

Effect of phosphocreatine on H+ extrusion,pHi and dimorphism in Candida albicans

Candida albicans is an opportunistic pathogen. Its proliferation in human hosts is believed to be controlled by immunologic mechanisms. The plasma membrane of the fungus possesses an H(+)-ATPase (PM-ATPase) which actively extrudes protons to generate an electrochemical gradient which is used in co-transport of nutrients. This ATPase is associated with the growth, dimorphism and pathogenicity of the fungus. The physiological concentration of phosphocreatine (PCr) is 20-35 mM in skeletal muscles. H(+)-extrusion in Candida cells was strongly inhibited by PCr; 44% at 20 mM and 69% at 40 mM. H(+)-extrusion was stimulated 6.2-fold in the presence of 10 mM glucose. This glucose stimulated extrusion was inhibited significantly by PCr; 36% at 20 mM and 53% at 40 mM. The intracellular pH pattern of cells destined to differentiate was greatly altered in the presence of PCr. Evagination time for control cells was between 90-120 min. PCr, delayed dimorphism, reduced the population of cells differentiating to hyphae and also reduced the length of hyphae after each time interval. Only 60% differentiation was observed with 10 mM PCr and 40% for higher PCr concentration even after 210 min. Direct interaction of PM-ATPase and PCr has been demonstrated by difference spectrum measurement employing stopped flow spectrophotometer. It can be concluded that PCr may be playing a significant role in checking growth and pathogenesis of C. albicans.     

Requirement of Skp1-Bub1 interaction for kinetochore-mediated activation of the spindle checkpoint

The spindle checkpoint transiently prevents cell cycle progression of cells that have incurred errors or failed to complete steps during mitosis, including those involving kinetochore function. The molecular nature of the primary signal transmitted from defective kinetochores and how it is detected by the spindle checkpoint are unknown. We report biochemical evidence that Bub1, a component of the spindle checkpoint, associates with centromere (CEN) DNA via Skp1, a core kinetochore component in budding yeast. The Skp1's interaction with Bub1 is required for the mitotic delay induced by kinetochore tension defects, but not for the arrest induced by spindle depolymerization, kinetochore assembly defects, or Mps1 overexpression. We propose that the Skp1-Bub1 interaction is important for transmitting a signal to the spindle checkpoint pathway when insufficient tension is present at kinetochores.     

Terpenes from Inula verbascifolia

The aerial parts of Inula verbascifolia afforded two new xanthanes and a new germacranolide derivative, together with the known compounds inusoniolide, 4-O-dihydroinusoniolide and 9beta-hydroxyparthenolide. The structures were determined by spectral methods (IR, HRMS,1H NMR, 13C NMR, DEPT, 1H-1H COSY, HMQC and HMBC).     

Remarkable sequence signatures in archaeal genomes

Complete archaeal genomes were probed for the presence of long (> or = 25 bp) oligonucleotide repeats (words). We detected the presence of many words distributed in tandem with narrow ranges of periodicity (i.e., spacer length between repeats). Similar words were not identified in genomes of non-archaeal species, namely Escherichia coli, Bacillus subtilis, Haemophilus influenzae, Mycoplasma genitalium and Mycoplasma pneumoniae. BLAST similarity searches against the GenBank nucleotide sequence database revealed that these words were archaeal species-specific, indicating that they are of a signature character. Sequence analysis and genome viewing tools showed these repeats to be restricted to non-coding regions. Thus, archaea appear to possess a non-coding genomic signature that is absent in bacterial species. The identification of a species-specific genomic signature would be of great value to archaeal genome mapping, evolutionary studies and analyses of genome complexity.     

T cell substance P receptor governs antigen-elicited IFN-gamma production

Substance P (SP) enhances antigen-dependent T cell IFN-gamma production. It was determined if a T cell neurokinin-1 receptor (NK-1R) was critical for IFN-gamma regulation. T cells from schistosome-infected mice were mixed with splenocytes from uninfected NK-1R knockout (KO) animals. Thus only the schistosome egg antigen-specific T cells expressed NK-1R. The cells were cultured 18 h with or without SP. SP enhanced antigen-induced IFN-gamma production fourfold without affecting IL-4 or IL-5 secretion. NK-1R inhibitor blocked this stimulation. Neither purified T cells nor naive KO splenocytes cultured alone responded to antigen. To further define the importance of T cell NK-1R, we developed a T cell-selective NK-1R KO mouse by reconstituting T cell-deficient Rag mice with NK-1R KO T cells. These mice challanged with schistosomiasis developed abnormal liver granulomas. Granuloma size was smaller in T cell-selective NK-1R KO mice compared with granulomas in Rag reconstituted with normal T cells. Splenocytes and granuloma cells from NK-1R KO mice made less IFN-gamma. The mice also made less IgG2a. Thus T cell NK-1R is important for IFN-gamma regulation.     

Bioluminometric assay of ATP in mouse brain: Determinant factors for enhanced test sensitivity

Firefly luciferase bioluminescence (FLB) is a highly sensitive and specific method for the analysis of adenosine-5-triphosphate (ATP) in biological samples. Earlier attempts to modify the FLB test for enhanced sensitivity have been typically based onin vitro cell systems. This study reports an optimized FLB procedure for the analysis of ATP in small tissue samples. The results showed that the sensitivity of the FLB test can be enhanced several fold by using ultraturax homogenizer, perchloric acid extraction, neutralization of acid extract and its optimal dilution, before performing the assay reaction.     

The role of antioxidant enzymes in TCDD-induced oxidative stress in various brain regions of rats after subchronic exposure

The induction of oxidative stress by TCDD in various brain regions of rats has been investigated after subchronic exposure. TCDD was administered by gavage to female Sprague-Dawley rats at daily doses of 0, 10, 22, and 46 ng/kg for 13 weeks. The brains were dissected to cerebral cortex (Cc), hippocampus (H), cerebellum (C), and brain stem (Bs); the production of superoxide anion (SA) and lipid peroxides and the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px) were determined in those regions. TCDD caused dose-dependent increases in the production of SA and lipid peroxidation in Cc and H and those were associated with dose-dependent suppressions of SOD. While a TCDD dose of 10 ng/kg/d resulted in significant increases in catalase and GSH-Px activities in Cc and H, doses of 22 and 46 ng/kg/d resulted in dose-dependent suppressions of these two enzymes in the same regions. In the C and Bs, TCDD treatment did not result in significant production of SA and lipid peroxidation but it resulted in dose-dependent increases in the activities of various antioxidant enzymes. These results suggest that Cc and H are vulnerable to TCDD-induced oxidative stress after subchronic exposure, and that C and Bs are protected against that effect.     

Synthesis and DNA-binding affinity of A-C8/C-C2 alkoxyamido-linked pyrrolo[2,1-c][1,4]benzodiazepine dimers

The synthesis of new A-C8/C-C2 alkoxyamido-linked pyrrolo[2,1-c][1,4]-benzodiazepine dimers have been described in this report. These dimers exhibit significant DNA-binding ability with moderate anticancer activity.