Nonbenzamidine tetrazole derivatives as factor Xa inhibitors

Factor Xa (fXa) is an important serine protease that holds the central position linking the intrinsic and extrinsic activation mechanisms in the blood coagulation cascade. Therefore, inhibition of fXa has potential therapeutic applications in the treatments of both arterial and venous thrombosis. Herein we describe a series of tetrazole fXa inhibitors containing benzamidine mimics as the P(1) substrate, of which the aminobenzisoxazole moiety was found to be the most potent benzamidine mimic. SR374 (12) inhibits fXa with a K(i) value of 0.35 nM and is very selective for fXa over thrombin and trypsin.     

Ras/MAPK pathway confers basement membrane dependence upon endoderm differentiation of embryonic carcinoma cells

The formation of extraembryonic endoderm is one of the earliest steps in the differentiation of pluripotent cells of the inner cell mass during the early stages of embryonic development. The primitive endoderm cells and the derived parietal and visceral endoderm cells gain the capacity to produce collagen IV and laminin. The deposition of these components results in the formation of basement membrane and epithelium of the endoderm, with polarized cells covering the inner surface of the blastocoels. We used retinoic acid-induced endoderm differentiation of stem cell-like F9 embryonic carcinoma cells to study the role of the Ras pathway and its regulation in the formation of the visceral endoderm. Upon endoderm differentiation of F9 cells induced by retinoic acid, c-Fos expression, the downstream target of the Ras pathway, is suppressed by uncoupling Elk-1 phosphorylation/activation to MAPK activity. However, attachment to matrix gel greatly enhances the activation of MAPK in endoderm cells but not in undifferentiated F9 cells. Enhanced MAPK activation as a result of contact with basement membrane is able to compensate for reduced Elk-1 phosphorylation and c-Fos expression. We conclude that endoderm differentiation renders the activation of the Ras pathway basement membrane dependent, contributing to the epithelial organization of the visceral endoderm.     

Immune Control of Chlamydial Growth in the Human Epithelial Cell Line RT4 Involves Multiple Mechanisms That Include Nitric Oxide Induction,Tryptophan Catabolism and Iron Deprivation

The antimicrobial activity of T cell-derived cytokines, especially interferon (IFN)-γ, against intracellular pathogens, such as Chlamydia trachomatis, involves the induction of 3 major biochemical processes: tryptophan catabolism, nitric oxide (NO) induction and intracellular iron (Fe) deprivation. Since the epithelial cell is the natural target of chlamydial infection, the presence of these antimicrobial systems in the cell would suggest that they may be involved in T cell control of intracellular multiplication of Chlamydia. However, the controversy over whether these 3 antimicrobial processes are present in both mice and humans has precluded the assessment of the relative contribution of each of the 3 mechanisms to chlamydial inhibition in the same epithelial cell from either mice or humans. In the present study, we identified a Chlamydia-susceptible human epithelial cell line, RT4, that possesses the 3 antimicrobial systems, and we examined the role of nitric oxide (NO) induction, and deprivation of tryptophan or Fe in cytokine-induced inhibition of chlamydiae. It was found that the 3 antimicrobial systems contributed to cytokine-mediated inhibition of the intracellular growth of Chlamydia. NO induction accounted for ~20% of the growth inhibition; tryptophan catabolism contributed approximately 30%; iron deprivation was least effective; but the combination of the 3 systems accounted for greater than 60% of the inhibition observed. These results indicate that immune control of chlamydial growth in human epithelial cells may involve multiple mechanisms that include NO induction, tryptophan catabolism and Fe deprivation.     

The circular dichroism of ribosomal ribonucleic acids.

1. The c.d. (circular dichroism) of Drosophila melanogaster rRNA (42% G+C) and of G+C-rich fragments (78% G+C) obtained by partial hydrolysis of rabbit L-rRNA (the largest RNA species isolated from the large subribosomal particle) were measured and found to differ substantially. 2. To interpret these spectra a relation between c.d. of bihelical RNA and % G+C was derived, namely delta epsilonfG = AFG2+bfG+c, where deltaepsilonfG is the c.d. of RNA characterized by a mole fraction, fG, of guanine nucleotides and a, b and c are constants. 3. A frame of reference was established by studying the c.d. of a range of rRNA species, including S-rRNA (the RNA species isolated from the smaller subribosomal particle) and L-rRNA of Escherichia coli. 4. It was found for the rRNA species studied that 0.60+/-0.05 of residues appear to form bihelical secondary structure. 5. A higher helical content, 0.66+/-0.05, was found for the G+C-rich fragment of L-rRNA. The difference in the c.d. of rabbit L-rRNA and of D. melanogaster rRNA is attributable to the dependence of c.d. of the bihelical parts on %G+C. 6. The minimum in c.d. at 295 nm increases with increasing %G+C. The c.d. of rRNA was compared with that of the parent subparticle in this region of the spectrum, where high precision may be attained.     

Some interactions ofSerratia marcescens nucleopolyhedrosis virus andBlepharipa pratensis [Dip.: Tachinidae] inLymantria dispar [Lep.: Lymantriidae]

The entomopathogensSerratia marcescens Bizio and nucleopolyhedrosis virus were each fed alone and in combination with the parasiteBlepharipa pratensis (Meigen) to 4th-instar gypsy moth,Lymantria dispar, (L.) larvae. At LD₃₀ for NPV, the presence of the parasite enhanced polyhedrosis about 30%, but the total number of gypsy moth larvae and pupae killed (85%) was not significantly different from the number killed by the parasite alone (93%). When the parasite was combined withS. marcencens, a strain nonpathogenic inL. dispar, total mortality was not significantly different from that in insects exposed only to the parasite (89 and 86%, respectively), but parasite survival was reduced about 12%. However, deaths not attributable to the parasite could not be ascribed to the bacterium either.     

Prion-like domains drive CIZ1 assembly formation at the inactive X chromosome

CIZ1 forms large assemblies at the inactive X chromosome (Xi) in female fibroblasts in an Xist lncRNA-dependent manner and is required for accurate maintenance of polycomb targets genome-wide. Here we address requirements for assembly formation and show that CIZ1 undergoes two direct interactions with Xist, via independent N- and C-terminal domains. Interaction with Xist, assembly at Xi, and complexity of self-assemblies formed in vitro are modulated by two alternatively spliced glutamine-rich prion-like domains (PLD1 and 2). PLD2 is dispensable for accumulation at existing CIZ1–Xi assemblies in wild-type cells but is required in CIZ1-null cells where targeting, assembly, and enrichment for H3K27me3 and H2AK119ub occur de novo. In contrast, PLD1 is required for both de novo assembly and accumulation at preexisting assemblies and, in vitro, drives formation of a stable fibrillar network. Together they impart affinity for RNA and a complex relationship with repeat E of Xist. These data show that alternative splicing of two PLDs modulates CIZ1’s ability to build large RNA–protein assemblies.     

Jumping Ship: A Stepping Stone Event Mediating Transfer of a Non-indigenous Species Via a Potentially Unsuitable Environment

The smooth shelled blue mussel, Mytilus galloprovincialis Lmk (Bivalvia: Mollusca) arrived in Pearl Harbor, Oahu, Hawai'i on 22 June 1998 as a member of the fouling community of the USS Missouri, and mussel spawning activity was observed within 2h of the vessel's arrival. Small mussels (<10mm shell length, approximately 6 weeks post-metamorphosis) were collected on 30 September 1998 from a submarine ballast tank in Pearl Harbor, indicating that a successful recruitment event had taken place very soon after the first arrival of the species at this location. We suggest that even if M. galloprovincialis is not able to establish permanently within Pearl Harbor, the fact that it has been able to successfully spawn and recruit to another shipping vector within the Harbor indicates that a stepping stone model of range expansion from temperate to temperate region via an intermediary subtropical environment is quite feasible for this species. Data from worldwide distributions of mussels of the family Mytilidae indicate that preferred habitats are eutrophic continental shelf regions, which suggests that successful establishment within Pearl Harbor is possible. However, oceanic coral-reef environments are not preferred habitat types, suggesting that M. galloprovincialis is not likely to become widely distributed in the Hawaiian Islands.     

<Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> infected macrophages exhibit enhanced plasma membrane fluidity and show increased adherence to endothelial cells

Chlamydia pneumoniae, an intracellular prokaryote, is known to have requirement for some lipids which it is incapable of synthesizing, and these lipids have important fluidizing roles in plasma membrane. We decided to examine if the trafficking of these lipids to C. pneumoniae alters the physicochemical properties of macrophage plasma membrane, affects the expression of genes and proteins of enzymes associated with metabolism of some of these lipids and assess if Ca²⁺ signaling usually induced in macrophages infected with C. pneumoniae modulates the genes of these selected enzymes. Chlamydia pneumoniae induced the depletion of macrophage membrane cholesterol, phosphatidylinositol and cardiolipin but caused an increase in phosphotidylcholine resulting in a relative increase in total phospholipids. There was increased membrane fluidity, enhanced macrophage fragility and heightened adherence of macrophages to endothelial cells despite the application of inhibitor of adhesion molecules. Also, there was impairment of macrophage 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase gene and protein expression independent of Ca²⁺ signaling, while phospholipase C gene and protein were up-regulated in a manner minimally dependent on Ca²⁺ signaling. The implications of these findings are that macrophages infected with C. pneumoniae have altered membrane physicochemical characteristics which may render them atherogenic. (Mol Cell Biochem 269: 69–84, 2005)