Mitochondria in activated microglia <Emphasis Type="Italic">in vitro</Emphasis>

In the CNS, microglia become activated, i.e. change their functional state and phenotype, in response to a wide variety of pathological stimuli. Since this activation is triggered at a very low threshold and at the same time remains territorially restricted, the spatial distribution of activated microglia can be used as a sensitive, generic measure of the anatomical localisation of ongoing disease processes. One protein complex, undetectable in resting microglia but highly up-regulated upon activation in vivo and in vitro, is the ‘peripheral benzodiazepine binding site’, as measured by binding of the isoquinoline derivate PK11195. Particularly numerous in the outer membrane of mitochondria, this binding site has also been referred to as the ‘mitochondrial benzodiazepine receptor’. The de novo expression of this receptor by activated microglia suggests that the process of activation may be associated with important qualitative changes in the state of mitochondria. Here, we provide confocal light- and electron microscopic evidence that the activation of microglia indeed entails conspicuous mitochondrial alterations. In cultured rat microglia stained with the fluorescent probe, JC-1, a sensitive indicator of mitochondrial membrane potential, we demonstrate that stimulation by bacterial lipopolysaccharide and interferon-γ increases the number of microglial mitochondrial profiles and leads to marked changes in their morphology. Prominent elongated, “needle-like” mitochondria are a characteristic feature of activated microglia in vitro. Electron microscopically, an abundance of abnormal profiles, including circular cristae or ring- and U-shaped membranes, are found. Our observations support the notion that the previously reported increase in microglial binding of PK11195, that labelled with carbon-11 ([¹¹C] (R)-PK11195) has clinical use for the visualisation of activated microglia in vivo by positron emission tomography, may at least in part relate to an increased number and altered functional state of microglial mitochondria.     

Microscopy-based multicolor tissue cytometry at the single-cell level.

Cytomics is a novel perspective from which to look at life. As with genomics and proteomics before, this discipline requires novel and innovative techniques and technologies to focus on its substrate of research--the cytome. With cytomics being the discipline that analyzes cellular systems and their interdependencies, advanced microscopy represents a key technology in cytomics research. Yet, conventional microscopy-based investigations, i.e., "look and conclude" analyses, do not meet the major cytomics criteria of 1) relating multiple parameters to each other, 2) within large populations of cells, 3) on a single-cell basis, and 4) in a quantitative and observer-independent manner. However, emerging improvements in the fields of fluorophore technology, sensitive fluorescence detection devices, and sophisticated image analysis procedures, are important and necessary steps into the cytomics era. Tissue represents an important class of cytomes, hence tissue cytometry--on the single cell level--can be expected to become an important cytomics technology. In this report, the techniques and technologies of microscopy-based multicolor tissue cytometry (MMTC) are outlined and applications are discussed, including the phenotypic characterization of tissue infiltrating leukocytes, in situ quantification of proliferation markers and tumor suppressors, and in situ quantification of apoptosis.     

Protein kinase-independent activation of CFTR by phosphatidylinositol phosphates

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed in many epithelia and in the heart. Phosphorylation of CFTR by protein kinases is thought to be an absolute prerequisite for the opening of CFTR channels. In addition, nucleoside triphosphates were shown to regulate the opening of phosphorylated CFTR. Here, we report that phosphatidylinositol 4,5-bisphosphate (PIP₂) activates human CFTR, resulting in ATP responsiveness of PIP₂-treated CFTR. PIP₂ alone is not sufficient to open CFTR, but ATP opens nonphosphorylated CFTR after application of PIP₂. The effect of PIP₂ is independent of protein kinases, as PIP₂ activates CFTR in the complete absence of Mg. Phosphatidylinositol and phosphatidylinositol monophosphate activate CFTR less efficiently than PIP₂. PIP₂ application to phosphorylated CFTR may inhibit the CFTR chloride current. We suggest that regulation of CFTR by PIP₂ is a previously unrecognized, alternative mechanism to control chloride conductance.     

Mycoplasma haemocanis infection--a kennel disease?

Mycoplasma haemocanis (formerly Haemobartonella canis) is a red blood cell parasite that causes disease mainly in immunosuppressed and splenectomized dogs. Clinical outbreak of the disease resulted in failure of a large experimental project. We aimed to identify whether M. haemocanis has increased prevalence in kennel-raised dogs. In a prospective study, we compared the prevalence of M. haemocanis in whole blood (anti-coagulated by use of EDTA) collected from pet dogs (University of Illinois, Urbana Champaign, Ill.; n = 60) with that in blood from dogs raised in three distinct kennels in western Europe (WE; n = 23), eastern Europe (EE; n = 20), and North America (NA; n = 20). Screening included antibody testing and microscopy of blood smears. The presence of M. haemocanis was identified using a polymerase chain reaction (PCR) assay for specific DNA of the organism. None of the pet dogs (0%) was test positive for M. haemocanis DNA. Mycoplasma haemocanis was found in dogs tested at all of the kennels. Infection rate in the three kennels was 30, 35, and 87%, respectively (all P < 0.001 versus control, chi2-test). Latent infection with M. haemocanis was not a single observation in kennel-raised dogs. Prevalence may be higher than that in a pet dog population. The potential exists for these latent infections to adversely affect or confound research results.     

Fluorescent epsilon-ATP analogues for probing physicochemical properties of proteins. Synthesis, biochemical evaluation, and sensitivity to properties of the medium

Despite the significance of the elucidation of proteins' physicochemical parameters to understand various molecular phenomena, direct methods for measuring these parameters are not readily available. Here, we propose the use of 8-[p-amino-Ph]-epsilon-ATP, 3b, as a fluorescent probe for the elucidation of physicochemical parameters of binding sites in certain proteins. We synthesized novel fluorescent nucleotide analogues based on an extension of the epsilon-ATP scaffold. These analogues bear a primary or tertiary p-amino-phenyl moiety on the etheno-bridge. We explored the recognition of the fluorescent analogues by the target proteins: P2Y(1)-receptor (P2Y(1)-R) and NTPDase1. Based on the high affinity to the P2Y(1)-R (EC(50) 100nM), 3b proved a suitable probe for the investigation of this receptor. Next, we elucidated the dependencies of the absorption and emission spectra of 3b on environmental parameters, for establishing correlation equations. These equations will help determine the properties of the ATP-binding site from the spectral data of the protein-bound 3b. For this purpose, the sensitivity of the probe to acidity, dielectricity, H-bonding, viscosity, and to correlation between these parameters was determined. Thus, the pH-dependence of 3b emission intensity is bell shaped. At pH2.8 the quantum yield (phi) is enhanced 150-fold, as compared to neutral pH. The basic nitrogen atoms of 3b were assigned and pK(a) values were determined. A linear relationship was found between log phi and log viscosity, however, emission maxima (lambda(max)) remained constant. A linear relationship was found between both phi and lambda(max) and dielectricity, as measured in protic or aprotic solvents of comparable viscosity. pK(a)-like values were measured in acid-titrated alcohols with varying dielectricity but comparable viscosity, or with varying viscosity but comparable dielectricity. An inverse relationship and a linear relationship were found between the pK(a) values of 3b and the medium dielectricity and viscosity, respectively. These correlations help the calibration of properties of a protein ATP-binding site.     

A new purple sulfur bacterium isolated from a littoral microbial mat,<Emphasis Type="Italic"> Thiorhodococcus drewsii</Emphasis> sp. nov.

A new strain of purple sulfur bacterium was isolated from a marine microbial mat sampled in Great Sippewissett Salt Marsh at the Atlantic coast (Woods Hole, Mass., USA). Single cells of strain AZ1 were coccus-shaped, highly motile by means of a single flagellum, and did not contain gas vesicles. Intracellular membranes were of the vesicular type. However, additional concentric membrane structures were present. The photosynthetic pigments were bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series, with rhodopin as the dominant carotenoid. Hydrogen sulfide (up to 11 mM), sulfur, thiosulfate, and molecular hydrogen were used as electron donors during anaerobic phototrophic growth. During growth on sulfide, elemental sulfur globules were transiently stored inside the cells. Strain AZ1 is much more versatile than most other Chromatiaceae with respect to electron donor and organic substrates. In the presence of CO(2), it is capable of assimilating C(1)-C(5) fatty acids, alcohols, and intermediates of the tricarboxylic acid cycle. Strain AZ1 could also grow photoorganotrophically with acetate as the sole photosynthetic electron donor. Chemotrophic growth in the dark under microoxic conditions was not detected. Optimum growth occurred at pH 6.5-6.7, 30-35 degrees C, > or =50 micro mol quanta m(-2) s(-1), and 2.4-2.6% NaCl. The DNA base composition was 64.5 mol% G+C. Comparative sequence analysis of the 16S rRNA gene confirmed that the isolate is a member of the family Chromatiaceae. Sequence similarity to the most closely related species, Thiorhodococcus minor DSMZ 11518(T), was 97.8%; however, the value for DNA-DNA hybridization between both strains was only 20%. Because of the low genetic similarity and since strain AZ1 physiologically differs considerably from all other members of the Chromatiaceae, including Trc. minor, the new isolate is described as a new species of the genus Thiorhodococcus, Thiorhodococcus drewsii sp. nov.     

Crystal structure of the avilamycin resistance-conferring methyltransferase AviRa from Streptomyces viridochromogenes

The emergence of antibiotic-resistant bacterial strains is a widespread problem in contemporary medical practice and drug design. It is therefore important to elucidate the underlying mechanism in each case. The methyltransferase AviRa from Streptomyces viridochromogenes mediates resistance to the antibiotic avilamycin, which is closely related to evernimicin, an oligosaccharide antibiotic that has been used in medical studies. The structure of AviRa was determined by X-ray diffraction at 1.5A resolution. Phases were obtained from one selenomethionine residue introduced by site-directed mutagenesis. The chain-fold is similar to that of most methyltransferases, although AviRa contains two additional helices as a specific feature. A putative-binding site for the cofactor S-adenosyl-L-methionine was derived from homologous structures. It agrees with the conserved pattern of interacting amino acid residues. AviRa methylates a specific guanine base within the peptidyltransferase loop of the 23S ribosomal RNA. Guided by the target, the enzyme was docked to the cognate ribosomal surface, where it fit well into a deep cleft without contacting any ribosomal protein. The two additional alpha-helices of AviRa filled a depression in the surface. Since the transferred methyl group of the cofactor is in a pocket beneath the enzyme surface, the targeted guanine base has to flip out for methylation.     

Expansion of the genetic code enables design of a novel "gold" class of green fluorescent proteins

Much effort has been dedicated to the design of significantly red shifted variants of the green fluorescent protein (GFP) from Aequoria victora (av). These approaches have been based on classical engineering with the 20 canonical amino acids. We report here an expansion of these efforts by incorporation of an amino substituted variant of tryptophan into the "cyan" GFP mutant, which turned it into a "gold" variant. This variant possesses a red shift in emission unprecedented for any avFP, similar to "red" FPs, but with enhanced stability and a very low aggregation tendency. An increasing number of non-natural amino acids are available for chromophore redesign (by engineering of the genetic code) and enable new general strategies to generate novel classes of tailor-made GFP proteins.     

The role of thrombin and thrombin receptors in ischemic,hemorrhagic and traumatic brain injury: deleterious or protective?

In the last two decades it has become apparent that thrombin has many extravascular effects that are mediated by a family of protease-activated receptors (PARs). PAR-1, -3 and -4 are activated via cleavage by thrombin. The importance of extravascular thrombin in modulating ischemic, hemorrhagic and traumatic injury in brain has recently become clear. Thus, in vitro, thrombin at low concentration protects neurons and astrocytes from cell death caused by a number of different insults. In vivo, pretreating the brain with a low dose of thrombin (thrombin preconditioning), attenuates the brain injury induced by a large dose of thrombin, an intracerebral hemorrhage or by focal cerebral ischemia. Thrombin may also be an important mediator of ischemic preconditioning. In contrast, high doses of thrombin kill neurons and astrocytes in vitro and cause disruption of the blood-brain barrier, brain edema and seizures in vivo. This review examines the role of thrombin in brain injury and the molecular mechanisms and signaling cascades involved.