TetL tetracycline efflux protein from Bacillus subtilis is a dimer in the membrane and in detergent solution

The TetL antiporter from the Bacillus subtilis inner membrane is a tetracycline-divalent cation efflux protein that is energized by the electrochemical proton gradient across the membrane. In this study, we expressed tetL in Escherichia coli and investigated the oligomeric state of TetL in the membrane and in detergent solution. Evidence for an oligomeric state of TetL emerged from SDS-PAGE and Western blot analysis of membrane samples as well as purified protein samples from cells that expressed two differently tagged TetL species. Furthermore, no formation or restoration of TetL oligomers occurred upon detergent solubilization of the membrane. Rather, oligomeric forms established in vivo persisted after solubilization. Mass spectrometry of the purified protein showed the absence of proteolysis and posttranslational modifications. Analytical size-exclusion chromatography of the purified protein revealed a dimeric TetL in dodecyl-maltoside solution. In addition, TetL dimers were found in a number of other detergents and over a wide pH range. It is therefore likely that the oligomeric form of the protein in the membrane is also a dimer.     

Escherichia coli LPS-induced LV dysfunction: role of toll-like receptor-4 in the adult heart

The precise molecular mechanisms responsible for sepsis-induced myocardial dysfunction remain undefined. Toll-like receptor-4 (TLR-4) engages lipopolysaccharide (LPS) and activates signaling pathways leading to the expression of proinflammatory cytokines implicated in myocardial dysfunction. We determined whether TLR-4 was necessary for LPS-induced myocardial dysfunction in vivo. The effects of LPS on left ventricular (LV) function were studied in mice with defective TLR-4 signaling (C3H/HeJ, TLR-4 deficient) and wild-type mice (C3HeB/FeJ). Mice (n = 5/group) were injected with LPS or diluent, and LV function was examined by using two-dimensional echocardiography and conductance catheters. LPS significantly decreased all indexes of LV function in wild-type mice when compared with controls; LV function was not depressed in the LPS-treated TLR-4-deficient mice relative to controls. LPS increased myocardial nitric oxide synthase-2 expression and cGMP only in wild-type mice. This study suggests that TLR-4 mediates the LV dysfunction that occurs in LPS-induced shock. Therefore, TLR-4 might be a therapeutic target for attenuating the effects of LPS on the heart.     

Temperature influence on embryonic development of Anopheles albitarsis and Anopheles aquasalis

Temperature influence on the embryonic development of Anopheles aquasalis and An. albitarsis was investigated. At 26 degrees C, 75% and 60% of respectively An. aquasalis and An. albitarsis eggs hatched, with one peak of eclosion, between the 2nd and 3rd day after oviposition. At 20 +/- 2 degrees C, around 66-70% of An. aquasalis eggs hatched, with one eclosion peak, on the 5th day. On the other hand, An. albitarsis eclosion at 21+/- 2 degrees C decreased to 10-22%, with two eclosion peaks, on the 4th-5th day and on the 9th-12th day. These data indicate a stronger temperature influence over An.albitarsis than over An. aquasalis embryos.     

Age-related hematologic changes in captive-reared houbara,white-bellied,and rufous-crested bustards

Blood samples were obtained at monthly intervals between April 1994 and March 1996 from captive-bred houbara (Chlamydotis undulata macqueenii), rufous-crested (Eupodotis ruficrista gindiana), and white-bellied (Eupodotis senegalensis) bustards from 4-24 wk of age. Hematology investigations were conducted to determine age-related changes and to establish reference values for growing chicks of these species. There were significant age-related changes in hematocrit, hemoglobin, and red cell count in young birds compared with those of adults. White cell counts (lymphocytes and monocytes) were higher in juvenile birds, compared with adult values.     

Susceptibility to T cell-mediated injury in immune complex disease is linked to local activation of renin-angiotensin system: the role of NF-AT pathway

FcR provides a critical link between ligands and effector cells in immune complex diseases. Emerging evidence reveals that angiotensin (Ang)II exerts a wide variety of cellular effects and contributes to the pathogenesis of inflammatory diseases. In anti-glomerular basement membrane Ab-induced glomerulonephritis (GN), we have previously noted that FcR-deficient mice (gamma(-/-)) surviving from lethal initial damage still developed mesangial proliferative GN, which was drastically prevented by an AngII type 1 receptor (AT1) blocker. We further examined the mechanisms by which renin-Ang system (RAS) participates in this immune disease. Using bone marrow chimeras between gamma(-/-) and AT1(-/-) mice, we found that glomerular injury in gamma(-/-) mice was associated with CD4(+) T cell infiltration depending on renal AT1-stimulation. Based on findings in cutaneous delayed-type hypersensitivity, we showed that AngII-activated renal resident cells are responsible for the recruitment of effector T cells. We next examined the chemotactic activity of AngII-stimulated mesangial cells, as potential mechanisms coupling RAS and cellular immunity. Chemotactic activity for T cells and Th1-associated chemokine (IFN-gamma-inducible protein-10 and macrophage-inflammatory protein 1alpha) expression was markedly reduced in mesangial cells from AT1(-/-) mice. Moreover, this activity was mainly through calcineurin-dependent NF-AT. Although IFN-gamma-inducible protein-10 was NF-kappaB-dependent, macrophage-inflammatory protein 1alpha was dominantly regulated by NF-AT. Furthermore, AT1-dependent NF-AT activation was observed in injured glomeruli by Southwestern histochemistry. In conclusion, our data indicate that local RAS activation, partly via the local NF-AT pathway, enhances the susceptibility to T cell-mediated injury in anti-glomerular basement membrane Ab-induced GN. This novel mechanism affords a rationale for the use of drugs interfering with RAS in immune renal diseases.     

An ectonucleotide ATP-diphosphohydrolase activity in Trichomonas vaginalis stimulated by galactose and its possible role in virulence

This work describes the ability of living Trichomonas vaginalis to hydrolyze extracellular ATP (164.0 +/- 13.9 nmol Pi/h x 10(7) cells). This ecto-enzyme was stimulated by ZnCl2, CaCl2 and MgCl2, was insensitive to several ATPase and phosphatase inhibitors and was able to hydrolyze several nucleotides besides ATP. The activity was linear with cell density and with time for at least 60 min. The optimum pH for the T. vaginalis ecto-ATPase lies in the alkaline range. D-galactose, known to be involved in adhesion of T. vaginalis to host cells, stimulated this enzyme by more than 90%. A comparison between two strains of T. vaginalis showed that the ecto-ATPase activity of a fresh isolate was twice as much as that of a strain axenically maintained in culture, through daily passages, for several years. The results suggest a possible role for this ecto-ATPase in adhesion of T. vaginalis to host cells and in its pathogenicity.     

β1 Integrin triggering affects leukemic cell line sensitivity to natural killer cells

The role of beta1 (CD29) integrins in natural killer (NK) cell-target cell conjugation and cytotoxicity has not been clearly established. Ligation of beta1 integrins in NK cells can modulate the lytic capacity in both a positive and a negative manner; however, the contribution of the beta1 integrins present on target cells remains to be evaluated. Here, we analyzed the effect of beta1 integrins expressed by potential tumor target cells on conjugation and cytotoxicity. Using normalized flow cytometry binding assays, we demonstrated that the pretreatment of MOLT-4, K562, U-937 and HL-60 human leukemia target cell lines with selected anti-beta1 monoclonal antibodies (mAb) increased conjugation to human NK cell line NKL as well as to purified NK cells. Only mAb recognizing residues 207-218 of the beta1 subunit and functionally involved in the induction of homotypic adhesion (functional epitope A1) increased conjugation of all the target cells. Moreover, mAb to adhesion molecules different from beta1 but also inducers of homotypic adhesion of the target cells, i.e. CD43 and CD50 (ICAM-3), failed to increase conjugation to NKL cells. Cytotoxicity assays demonstrated that lysis of NK-sensitive target cells (MOLT-4) also increased after pretreatment with anti-beta1 epitope A1 mAb. Importantly, pretreatment of NK-resistant target cells (U-937 and HL-60) with anti-beta1 mAb was not able to outweigh the cytotoxic inhibitory mechanisms controlled by HLA class I molecules. However, simultaneous masking of HLA class I molecules with mAb and pretreatment with anti-beta1 mAb rendered NK-resistant cells susceptible to lysis, as predicted by the missing self hypothesis. Triggering of tumor target cells through beta1 integrins may thus play a role in conjugation to NK cells as well as in co-stimulation of cell-mediated cytotoxicity.     

Thyrotropin secretagogues reduce rat pituitary neuromedin B, a local thyrotropin release inhibitor

Neuromedin B (NB), a bombesin-like peptide, highly concentrated in rat pituitary gland, has been shown to act as an autocrine/paracrine inhibitor of thyrotropin (TSH) release. Here it is shown that a single injection of thyrotropin-releasing hormone (TRH, 1.5 microg/animal, ip), the most important stimulator of thyrotropin secretion, induced approximately 35%-45% decrease in pituitary NB content in rats, as well as an important decrease in NB mRNA at 15 and 30 min (P < 0.05). Acute cold exposure, which induced higher serum TSH with a peak at 30 min, was associated with progressive decrease in pituitary NB, starting at 15 min although only reaching statistical significance after 2 hr (P < 0.05). Although not involved in the early peak, the decrease in NB may be contributing to maintenance of higher serum TSH in cold-exposed animals compared with those at room temperature. Fed rats, 2 hr after being subcutaneously injected with mouse recombinant leptin (8 microg /100 g body wt), showed a x2 increase in serum TSH and 38% reduction in pituitary NB (P < 0.05). In conclusion, TRH and leptin rapidly decreased pituitary NB and it is first proposed that the reduction of the inhibitory tonus of NB on TSH release will ultimately contribute to the amplification of TSH secretion elicited by TSH secretagogues.