Cross-regulation of carbon monoxide and the adenosine A2a receptor in macrophages

Adenosine and heme oxygenase-1 (HO-1) exert a wide range of anti-inflammatory and immunomodulatory actions, making them crucial regulatory molecules. Despite the diversity in their modes of action, the similarity of biological effects of adenosine and HO-1 led us to hypothesize a possible interrelationship between them. We assessed a potential role for HO-1 in the ability of adenosine or 5'-N-ethylcarboxamidoadenosine (NECA), a stable adenosine analog, to modify the response of LPS-stimulated macrophages. Adenosine and NECA markedly induced HO-1 and blocked LPS-induced TNF-alpha production via adenosine A2aR-mediated signaling; blocking of HO-1 by RNA interference abrogated the effects of adenosine and NECA on TNF-alpha. HO-1 overexpression or exposure to carbon monoxide (CO), a product of HO-1 enzymatic activity, resulted in augmented A2aR mRNA and protein levels in RAW264.7 cells and primary macrophages. The induction of A2aR expression by HO-1 or CO resulted in an increase in the sensitivity to the anti-inflammatory effects of adenosine and NECA, which was lost in macrophages isolated from A2aR-deficient mice. Moreover, a decrease in cAMP levels upon NECA stimulation of naive macrophages was counterbalanced by CO exposure to up-regulate A2aR levels. This implies adenosine receptor isoform switch as a selective modification in macrophage phenotype. Taken together, these data suggest the existence of a positive feedback loop among adenosine, HO-1, CO, and the A2aR in the chronological resolution of the inflammatory response.     

Emergence of distinct genetic variants in the population of primary Bartonella henselae isolates

Bartonella henselae isolates from different hosts display a marked genetic heterogeneity, as determined by pulsed-field gel electrophoresis (PFGE). The aim of the present study was to determine whether different genetic variants may coexist within the population of distinct B. henselae isolates and could be detected by PFGE. Three primary B. henselae isolates and the B. henselae reference strains ATCC 49793 and 49882 were subjected as single colony derived cultures in quadruplicate to PFGE analysis upon restriction with SmaI or NotI. Up to 4 fragment differences were found among the cultures obtained from each primary isolate, indicating the coexistence of genetic variants in the population of primary B. henselae isolates. The clonal relatedness of the genetic variants was confirmed by arbitrarily primed PCR and multi-locus sequence typing. In contrast to the primary isolates, no variants were detected among the single colony derived cultures of the high-passage ATCC strains. We hypothesized that the coexistence of different genetic variants may represent a feature that is restricted to primary or low-passage B. henselae isolates. The primary isolates were serially passed in vitro and then subjected as single colony derived cultures to PFGE analysis, which now revealed identical patterns among the quadruplicate cultures of each high-passage isolate. These results suggest that the population of a primary B. henselae isolate is composed of distinct genetic variants, which may disappear upon repeated passages on artificial culture media. Generation of genetic variants by B. henselae may represent an escape mechanism to circumvent the host specific immune responses.     

Multi-Locus Sequence Typing of a Geographically and Temporally Diverse Sample of the Highly Clonal Human Pathogen Bartonella quintana

Bartonella quintana is a re-emerging pathogen and the causative agent of a variety of disease manifestations in humans including trench fever. Various typing methods have been developed for B. quintana, but these tend to be limited by poor resolution and, in the case of gel-based methods, a lack of portability. Multilocus sequence typing (MLST) has been used to study the molecular epidemiology of a large number of pathogens, including B. henselae, a close relative of B. quintana. We developed a MLST scheme for B. quintana based on the 7 MLST loci employed for B. henselae with two additional loci to cover underrepresented regions of the B. quintana chromosome. A total of 16 B. quintana isolates spanning over 60 years and three continents were characterized. Allelic variation was detected in five of the nine loci. Although only 8/4270 (0.002%) of the nucleotide sites examined were variable over all loci, these polymorphisms resolved the 16 isolates into seven sequence types (STs). We also demonstrate that MLST can be applied on uncultured isolates by direct PCR from cardiac valve tissue, and suggest this method presents a promising approach for epidemiological studies in this highly clonal organism. Phylogenetic and clustering analyses suggest that two of the seven STs form a distinct lineage within the population.     

Identification of [1-14C]pantothenic-acid-mediated modified mitochondrial proteins

The in vivo administration of [1-14C]pantothenic acid, which is the precursor of coenzyme A, resulted in the radioactive labelling of several mitochondrial proteins in rat liver. The incorporated radioactivity could be released by glutathione or 2-mercaptoethanol. Two mitochondrial matrix proteins acetyl-CoA acetyltransferase (liver and heart), an enzyme involved in the biosynthesis or degradation of ketone bodies, and 3-oxoacyl-CoA thiolase (liver), a protein participating in fatty acid oxidation were identified as modified proteins. The radioactivity was localized exclusively in forms A1 and A2 indicating that these forms represent the modified states of the acetyl-CoA acetyltransferase protein. Kinetics of incorporation of radioactivity revealed an accumulation of the modified forms. The ratio of specific radioactivities of A2 compared to A1 was 2.41 +/- 0.15 (n = 10). After in vivo labelling with [14C]leucine, the specific radioactivity of acetyl-CoA acetyltransferase depended on the state of the enzyme protein. The unmodified enzyme exhibited a lower specific radioactivity than its modified forms suggesting different turnover rates of these proteins.     

Staphylococcus aureus alpha-toxin attack on human platelets promotes assembly of the prothrombinase complex

alpha-Toxin, the major cytolysin of Staphylococcus aureus, promotes blood coagulation by its attack on human platelets (Bhakdi S., Muhly, M., Mannhardt, U., Hugo, F., Klapettek, K., Mueller-Eckhardt, C., and Roka, L. (1988) J. Exp. Med. 168, 527-542). In the present study we demonstrate that toxin attack on gel-filtered human platelets initiates the assembly of prothrombinase complexes at rates up to 10-fold of controls. Treatment of platelets with 0.1 microgram/ml alpha-toxin resulted in generation of 1.4 units of thrombin/10(8) platelets. A similar rate of thrombin generation was noted when platelets were subjected to three cycles of freezing and thawing. However, the alpha-toxin-induced prothrombinase activity was not due to platelet lysis, since less than 1% of total cellular lactate dehydrogenase was released by this alpha-toxin concentration. Two distinct and dissociable processes contributed to enhanced prothrombinase assembly. First, alpha-toxin promoted the exocytotic release of factor V from alpha-granules, which was accompanied by co-secretion of platelet factor 4. This process was calcium-dependent. Second, toxin-treated platelets exhibited an enhanced capacity to bind external factor V(a), a phenomenon that was not linked to Ca2(+)-dependent factor V secretion. Assembly of prothrombinase complexes via these two mechanisms together accounts for the procoagulant action of S. aureus alpha-toxin.     

Electrochemical spectroscopic investigations on the interaction of an ytterbium complex with DNA and their analytical applications such as biosensor

Metal ion-DNA interactions are important in nature, often changing the genetic material's structure and function. A new Yb complex of YbCl₃ (tris(8-hydroxyquinoline-5-sulfonic acid) ytterbium) was synthesized and utilized as an electrochemical indicator for the detection of DNA oligonucleotide based on its interaction with Yb(QS)₃. Cyclic voltammetry (CV) and fluorescence spectroscopy were used to investigate the interaction of Yb(QS)₃ with ds-DNA. It was revealed that Yb(QS)₃ presented an excellent electrochemical activity on glassy carbon electrode (GCE) and could intercalate into the double helix of double-stranded DNA (ds-DNA). The binding mechanism of interaction was elucidated on glassy carbon electrode dipped in DNA solution and DNA modified carbon paste electrode by using differential pulse voltammetry and cyclic voltammetry. The binding ratio between this complex and ds-DNA was calculated to be 1:1. The extent of hybridization was evaluated on the basis of the difference between signals of Yb(QS)₃ with probe DNA before and after hybridization with complementary DNA. With this approach, this DNA could be quantified over the range from 1 × 10⁻⁸ to 1.1 × 10⁻⁷ M. The interaction mode between Yb(QS)₃ and DNA was found to be mainly intercalative interaction. These results were confirmed with fluorescence experiments.     

Apoptosis induced by cytoskeletal disruption requires distinct domains of MEKK1

MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the MAPK JNK and is required for microtubule inhibitor-induced apoptosis in B cells. Here, we find that apoptosis induced by actin disruption via cytochalasin D and by the protein phosphatase 1/2A inhibitor okadaic acid also requires MEKK1 activation. To elucidate the functional requirements for activation of the MEKK1-dependent apoptotic pathway, we created mutations within MEKK1. MEKK1-deficient cells were complemented with MEKK1 containing mutations in either the ubiquitin interacting motif (UIM), plant homeodomain (PHD), caspase cleavage site or the kinase domain at near endogenous levels of expression and tested for their sensitivity to each drug. We found that both the kinase activity and the PHD domain of MEKK1 are required for JNK activation and efficient induction of apoptosis by drugs causing cytoskeletal disruption. Furthermore, we discovered that modification of MEKK1 and its localization depends on the integrity of the PHD.     

Multi-locus sequence typing of Bartonella henselae isolates from three continents reveals hypervirulent and feline-associated clones

Bartonella henselae is a zoonotic pathogen and the causative agent of cat scratch disease and a variety of other disease manifestations in humans. Previous investigations have suggested that a limited subset of B. henselae isolates may be associated with human disease. In the present study, 182 human and feline B. henselae isolates from Europe, North America and Australia were analysed by multi-locus sequence typing (MLST) to detect any associations between sequence type (ST), host species and geographical distribution of the isolates. A total of 14 sequence types were detected, but over 66% (16/24) of the isolates recovered from human disease corresponded to a single genotype, ST1, and this type was detected in all three continents. In contrast, 27.2% (43/158) of the feline isolates corresponded to ST7, but this ST was not recovered from humans and was restricted to Europe. The difference in host association of STs 1 (human) and 7 (feline) was statistically significant (P< or =0.001). eBURST analysis assigned the 14 STs to three clonal lineages, which contained two or more STs, and a singleton comprising ST7. These groups were broadly consistent with a neighbour-joining tree, although splits decomposition analysis was indicative of a history of recombination. These data indicate that B. henselae lineages differ in their virulence properties for humans and contribute to a better understanding of the population structure of B. henselae.