Endocannabinoids Are Expressed in Bone Marrow Stromal Niches and Play a Role in Interactions of Hematopoietic Stem and Progenitor Cells with the Bone Marrow Microenvironment

Endocannabinoids are lipid signaling molecules that act via G-coupled receptors, CB₁ and CB₂. The endocannabinoid system is capable of activation of distinct signaling pathways on demand in response to pathogenic events or stimuli, hereby enhancing cell survival and promoting tissue repair. However, the role of endocannabinoids in hematopoietic stem and progenitor cells (HSPCs) and their interaction with hematopoietic stem cells (HSC) niches is not known. HSPCs are maintained in the quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signaling. We report that HSPCs express the CB₁ receptors and that BM stromal cells secrete endocannabinoids, anandamide (AEA) (35 pg/10⁷ cells), and 2-AG (75.2 ng/10⁷ cells). In response to the endotoxin lipopolysaccharide (LPS), elevated levels of AEA (75.6 pg/10⁷ cells) and 2-AG (98.8 ng/10⁷ cells) were secreted from BM stromal cells, resulting in migration and trafficking of HSPCs from the BM niches to the peripheral blood. Furthermore, administration of exogenous cannabinoid CB₁ agonists in vivo induced chemotaxis, migration, and mobilization of human and murine HSPCs. Cannabinoid receptor knock-out mice Cnr1^−/− showed a decrease in side population (SP) cells, whereas fatty acid amide hydrolase (FAAH)^−/− mice, which have elevated levels of AEA, yielded increased colony formation as compared with WT mice. In addition, G-CSF-induced mobilization in vivo was modulated by endocannabinoids and was inhibited by specific cannabinoid antagonists as well as impaired in cannabinoid receptor knock-out mice Cnr1^−/−, as compared with WT mice. Thus, we propose a novel function of the endocannabinoid system, as a regulator of HSPC interactions with their BM niches, where endocannabinoids are expressed in HSC niches and under stress conditions, endocannabinoid expression levels are enhanced to induce HSPC migration for proper hematopoiesis.     

Mycobacterium tuberculosis cells growing in macrophages are filamentous and deficient in FtsZ rings

FtsZ, a bacterial homolog of tubulin, forms a structural element called the FtsZ ring (Z ring) at the predivisional midcell site and sets up a scaffold for the assembly of other cell division proteins. The genetic aspects of FtsZ-catalyzed cell division and its assembly dynamics in Mycobacterium tuberculosis are unknown. Here, with an M. tuberculosis strain containing FtsZ(TB) tagged with green fluorescent protein as the sole source of FtsZ, we examined FtsZ structures under various growth conditions. We found that midcell Z rings are present in approximately 11% of actively growing cells, suggesting that the low frequency of Z rings is reflective of their slow growth rate. Next, we showed that SRI-3072, a reported FtsZ(TB) inhibitor, disrupted Z-ring assembly and inhibited cell division and growth of M. tuberculosis. We also showed that M. tuberculosis cells grown in macrophages are filamentous and that only a small fraction had midcell Z rings. The majority of filamentous cells contained nonring, spiral-like FtsZ structures along their entire length. The levels of FtsZ in bacteria grown in macrophages or in broth were comparable, suggesting that Z-ring formation at midcell sites was compromised during intracellular growth. Our results suggest that the intraphagosomal milieu alters the expression of M. tuberculosis genes affecting Z-ring formation and thereby cell division.     

Analysis of the Influences of Short-Term Levosimendan Exposure on Oxidant/Antioxidant Status and Trace-Element Levels in the Physiological Status of the Thoracic Aorta of Rats

The objective of this study was to evaluate the effect of levosimendan (chemical formula C₁₄H₁₂N₆O) exposure on oxidant/antioxidant status and trace-element levels in the thoracic aorta of rats. Eighteen male Wistar albino rats were randomly divided into two groups of eight animals each. Group 1 was not exposed to levosimendan and served as a control. Levosimendan (12???g/kg) diluted in 10 ml 0.5?% dextrose was administered intraperitoneally to group 2. Animals of both groups were killed after 3?days, and their thoracic aortae were harvested for determination of changes in tissue oxidant/antioxidant status and trace-element levels. The animals in both groups were killed 72?h after levosimendan exposure, and thoracic aortae were harvested for determination of the lipid peroxidation product MDA and antioxidant GSH levels and the activities of antioxidant enzymes such as SOD, GSH-Px and CAT. It was found that MDA, GSH and CAT enzyme levels increased in thoracic aortae of rats after levosimendan administration. SOD and CA enzyme activities and the level of antioxidant GSH decreased in thoracic aortae of rats after levosimendan treatment. Pb, Cd and Fe levels of thoracic aortae were significantly higher (P?<?0.001) and Mg, Mn, Zn and Cu were significantly lower (P?<?0.001) in the levosimendan group compared to the control group. These results suggest that short-term levosimendan treatment caused an increase in free radical production and a decrease in antioxidant enzyme activity in thoracic aortae of levosimendan-treated rats. It also causes a decrease or increase in many mineral levels of the thoracic aorta, which is an undesirable condition for normal pharmacological function.     

Biogeography revisited with network theory: retracing the history of hydrothermal vent communities

Defining biogeographic provinces to understand the history and evolution of communities associated with a given kind of ecosystem is challenging and usually requires a priori assumptions to be made. We applied network theory, a holistic and exploratory method, to the most complete database of faunal distribution available on oceanic hydrothermal vents, environments which support fragmented and unstable ecosystems, to infer the processes driving their worldwide biogeography. Besides the identification of robust provinces, the network topology allowed us to identify preferential pathways that had hitherto been overlooked. These pathways are consistent with the previously proposed hypothesis of a role of plate tectonics in the biogeographical history of hydrothermal vent communities. A possible ancestral position of the Western Pacific is also suggested for the first time. Finally, this work provides an innovative example of the potential of network tools to unravel the biogeographic history of faunal assemblages and to supply comprehensive information for the conservation and management of biodiversity.     

Vascular endothelial growth factor modulates neutrophil transendothelial migration via up-regulation of interleukin-8 in human brain microvascular endothelial cells.

Hypoxia, a strong inducer for vascular endothelial growth factor (VEGF)/vascular permeable factor (VPF) expression, regulates leukocyte infiltration through the up-regulation of adhesion molecules and chemokine release. To determine whether VEGF/VPF is directly involved in chemokine secretion, we analyzed its effects on chemokine expression in human brain microvascular endothelial cells (HBMECs) by using a human cytokine cDNA array kit. Cytokine array analysis revealed a significant increase in expression of monocyte chemoattractant protein-1 and the chemokine receptor CXCR4 in HBMECs, a result similar to that described previously in other endothelial cells. Interestingly, we also observed that VEGF/VPF induced interleukin-8 (IL-8) expression in HBMECs and that IL-8 mRNA was maximal after 1 h of VEGF/VPF treatment of the cells. Enzyme-linked immunosorbent assay data and immunoprecipitation analysis revealed that although VEGF/VPF induced IL-8 expression at the translational level in HBMECs, basic fibroblast growth factor failed to induce this protein expression within 12 h. VEGF/VPF increased IL-8 production in HBMECs through activation of nuclear factor-KB via calcium and phosphatidylinositol 3-kinase pathways, whereas the ERK pathway was not involved in this process. Supernatants of the VEGF/VPF-treated HBMECs significantly increased neutrophil migration across the HBMEC monolayer compared with those of the untreated control. Furthermore, addition of anti-IL-8 antibody blocked this increased migration, indicating that VEGF/VPF induced the functional expression of IL-8 protein in HBMECs. Taken together, these data demonstrate for the first time that VEGF/VPF induces IL-8 expression in HBMECs and contributes to leukocyte infiltration through the expression of chemokines, such as IL-8, in endothelial cells.     

Study of asparagine 353 in aminopeptidase A: characterization of a novel motif (GXMEN) implicated in exopeptidase specificity of monozinc aminopeptidases.

Aminopeptidase A (EC 3.4.11.7, APA) is a 160 kDa membrane-bound zinc enzyme that contains the HEXXH consensus sequence found in members of the zinc metalloprotease family, the zincins. In addition, the monozinc aminopeptidases are characterized by another conserved motif, GXMEN, the glutamate residue of which has been shown to be implicated in the exopeptidase specificity of aminopeptidase A [Vazeux G. (1998) Biochem. J. 334, 407-413]. In carboxypeptidase A (EC 3.4.17.1, CPA), the exopeptidase specificity is conferred by an arginine residue (Arg-145) and an asparagine residue (Asn-144). Thus, we hypothesized that Asn-353 of the GXMEN motif in APA plays a similar role to Asn-144 in CPA and contributes to the exopeptidase specificity of APA. We investigated the functional role of Asn-353 in APA by substituting this residue with a glutamine (Gln-353), an alanine (Ala-353) or an aspartate (Asp-353) residue by site-directed mutagenesis. Expression of wild-type and mutated APAs revealed that Gln-353 and Ala-353 are similarly routed and glycosylated to the wild-type APA, whereas Asp-353 is trapped intracellularly and partially glycosylated. Kinetic studies, using alpha-L-glutamyl-beta-naphthylamide (GluNA) as a substrate showed that the K(m) values of the mutants Gln-353 and Ala-353 were increased 11- and 8-fold, respectively, whereas the k(cat) values were decreased (2-fold) resulting in a 24- and 14-fold reduction in cleavage efficiency. When alpha-L-aspartyl-beta-naphthylamide or angiotensin II were used as substrates, the mutations had a greater effect on k(cat), leading to a similar decrease in cleavage efficiencies as that observed with GluNA. We then measured the inhibitory potencies of several classes of inhibitors, glutamate thiol, glutamine thiol and two isomers (L- or D-) of glutamate phosphonate to explore the functional role of Asn-353. The data indicate that Asn-353 is critical for the integrity and catalytic activity of APA. This residue is involved in substrate binding via interactions with the free N-terminal part and with the P1 carboxylate side chain of the substrate. In conclusion, Asn-353 of the GXMEN motif, together with Glu-352, contributes to the exopeptidase specificity of APA and plays an equivalent role to Asn-144 in CPA.     

Type I nitric oxide synthase (NOS) is the predominant NOS in rat small intestine. Regulation by platelet-activating factor.

Constitutive nitric oxide synthase (cNOS) may play an important protective role in the intestine, since our previous study has shown that the degree of bowel injury induced by platelet-activating factor (PAF), a potent inflammatory mediator, is inversely related to the cNOS content of the intestine. This study aims to examine the composition of the cNOS system in rat small intestine, and its regulation by PAF. We found that an approximately 120 kDa NOS I (neuronal NOS) is the predominant NOS in rat intestine, as evidenced by the following: (a) immunoblotting with specific antibodies detected a NOS I of approximately 120 kDa, but little NOS III; (b) the Ca(2+)-dependent, constitutive NOS (cNOS) activity of the rat intestine was removed by immunoprecipitation with the anti-NOS I, but not anti-NOS II or anti-NOS III antibodies; (c) RT-PCR revealed constitutive expression of NOS I in the intestinal tissue, but only a minute amount of NOS III. Immunofluorescent staining with anti-NOS I located NOS in the Auerbach plexus and nerve fibers in the muscle layer. We also found that this 120 kDa NOS I is rapidly (within 1 h) down-regulated in response to PAF administration. The protein level, enzyme activity as well as mRNA of nNOS were all decreased in the intestine.     

Trace element levels in mollusks from clean and polluted coastal marine sites in the Mediterranean, Red and North Seas

The trace element contamination levels in mollusks were evaluated for different marine coastal sites in the Mediterranean (Israeli coast), Red (Israeli coast) and North (German coast) Seas. Three bivalve species (Mactra corallina, Donax sp, and Mytilus edulis) and two gastropod species (Patella sp.and Cellana rota) were sampled at polluted and relatively clean sites, and their soft tissue analyzed for Hg, Cd, Zn, Cu, Mn and Fe concentrations. Representative samples were screened for organic contaminants [(DDE), polychlorinated biphenyls PCBs and polycyclic aromatic hydrocarbons (PAHs)] which exhibited very low concentrations at all sites. In the Red Sea, the gastropod C. rota showed low levels of Hg (below detection limit) and similar Cd concentrations at all the examined sites, while other trace elements (Cu, Zn, Mn, Fe) were slightly enriched at the northern beach stations. Along the Mediterranean coast of Israel, Hg and Zn were enriched in two bivalves (M. corallina and Donax sp.) from Haifa Bay, both species undergoing a long-term decrease in Hg based on previous studies. Significant Cd and Zn enrichment was detected in Patella sp. from the Kishon River estuary at the southern part of Haifa Bay. In general, Patella sp. and Donax sp. specimens from Haifa Bay exhibited higher levels of Cd compared to other sites along the Israeli Mediterranean coast, attributed to the enrichment of Cd in suspended particulate matter. Along the German coast (North Sea) M. edulis exhibited higher concentrations of Hg and Cd at the Elbe and Eider estuaries, but with levels below those found in polluted sites elsewhere. Received: 25 February 1999 / Received in revised form: 22 April 1999 / Accepted: 30 April 1999