Identification of active site residues in mevalonate diphosphate decarboxylase: implications for a family of phosphotransferases

A combination of sequence homology analyses of mevalonate diphosphate decarboxylase (MDD) proteins and structural information for MDD leads to the hypothesis that Asp 302 and Lys 18 are active site residues in MDD. These residues were mutated to replace acidic/basic side chains and the mutant proteins were isolated and characterized. Binding and competitive displacement studies using trinitrophenyl-ATP, a fluorescent analog of substrate ATP, indicate that these mutant enzymes (D302A, D302N, K18M) retain the ability to stoichiometrically bind nucleotide triphosphates at the active site. These observations suggest the structural integrity of the mutant MDD proteins. The functional importance of mutated residues was evaluated by kinetic analysis. The 10(3) and 10(5)-fold decreases in k(cat) observed for the Asp 302 mutants (D302N and D302A, respectively) support assignment of a crucial catalytic role to Asp 302. A 30-fold decrease in activity and a 16-fold inflation of the K(m) for ATP is documented for the K18M mutant, indicating that Lys 18 influences the active site but is not crucial for reaction chemistry. Demonstration of the influence of conserved aspartate 302 appears to represent the first documentation of the functional importance of a residue in the MDD catalytic site and affords insight into phosphotransferase reactions catalyzed by a variety of enzymes in the galactokinase, homoserine kinase, mevalonate kinase, phosphom-evalonate kinase (GHMP kinase) family.     

Kindling fluorescent proteins for precise in vivo photolabeling

Photobleaching of green fluorescent protein (GFP) is a widely used approach for tracking the movement of subcellular structures and intracellular proteins. Although photobleaching is a powerful technique, it does not allow direct tracking of an object's movement and velocity within a living cell. Direct tracking becomes possible only with the introduction of a photoactivated fluorescent marker. A number of previous studies have reported optically induced changes in the emission spectra of fluorescent proteins. However, the ideal photoactivated fluorescent marker should be a nonfluorescent tag capable of "switching on" (i.e., becoming fluorescent) in response to irradiation by light of a particular wavelength, intensity, and duration. In this report, we generated a mutant of Anemonia sulcata chromoprotein asCP. The mutant protein is capable of unique irreversible photoconversion from the nonfluorescent to a stable bright-red fluorescent form ("kindling"). This "kindling fluorescent protein" (KFP1) can be used for precise in vivo photolabeling to track the movements of cells, organelles, and proteins. We used KFP1 for in vivo cell labeling in mRNA microinjection assays to monitor Xenopus laevis embryo development and to track mitochondrial movement in mammalian cells.     

Calcium microdomains in aspiny dendrites

Dendritic spines receive excitatory synapses and serve as calcium compartments, which appear to be necessary for input-specific synaptic plasticity. Dendrites of GABAergic interneurons have few or no spines and thus do not possess a clear morphological basis for synapse-specific compartmentalization. We demonstrate using two-photon calcium imaging that activation of single synapses on aspiny dendrites of neocortical fast spiking (FS) interneurons creates highly localized calcium microdomains, often restricted to less than 1 microm of dendritic space. We confirm using ultrastructural reconstruction of imaged dendrites the absence of any morphological basis for this compartmentalization and show that it is dependent on the fast kinetics of calcium-permeable (CP) AMPA receptors and fast local extrusion via the Na+/Ca2+ exchanger. Because aspiny dendrites throughout the CNS express CP-AMPA receptors, we propose that CP-AMPA receptors mediate a spine-free mechanism of input-specific calcium compartmentalization.     

Genetic analysis of housekeeping genes of members of the genus Acholeplasma: phylogeny and complementary molecular markers to the 16S rRNA gene

The partial nucleotide sequences of the rpoB and gyrB genes as well as the complete sequence of the 16S-23S rRNA intergenic transcribed spacer (ITS) were determined for all known Acholeplasma species. The same genes of Mesoplasma and Entomoplasma species were also sequenced and used to infer phylogenetic relationships among the species within the orders Entomoplasmatales and Acholeplasmatales. The comparison of the ITS, rpoB, and gyrB phylogenetic trees with the 16S rRNA phylogenetic tree revealed a similar branch topology suggesting that the ITS, rpoB, and gyrB could be useful complementary phylogenetic markers for investigation of evolutionary relationships among Acholeplasma species. Thus, the multilocus phylogenetic analysis of Acholeplasma multilocale sequence data (ATCC 49900 (T) = PN525 (NCTC 11723)) strongly indicated that this organism is most closely related to the genera Mesoplasma and Entomoplasma (family Entomoplasmataceae) and form the branch with Mesoplasma seiffertii, Mesoplasma syrphidae, and Mesoplasma photuris. The closest genetic relatedness of this species to the order Entomoplasmatales was additionally supported by the finding that A. multilocale uses UGA as the tryptophan codon in its gyrB and gyrA sequences. Use of the UGA codon for encoding tryptophan was previously reported as a unique genetic feature of Entomoplasmatales and Mycoplasmatales but not of Acholeplasmatales. These data, as well as previously published data on metabolic features of A. multilocale, leads to the proposal to reclassify A. multilocale as a member of the family Entomoplasmataceae.     

Development of the downstream process in the production of the recombinant histone H1.3 variant

It was shown that H1 type histones possess anticancer activity and could be utilized in therapy of acute myeloid leukemia. We developed an experimental pilot-scale technology of the recombinant histone H1.3 variant production. The downstream process includes acidic extraction of the target protein from the culture broth, ion-exchange, reversed-phase and size-exclusion chromatography, and freeze-drying. The accent was made on the reduction of bacterial endotoxin contamination of the active pharmaceutical ingredient. Highly efficient downstream strategy of the target protein depyrogenation is discussed in the paper. The developed technology allows the production of the protein with high yield (approx. 110 g per 1000 L of the culture broth) and of high purity (estimated productivity is 75–100 g/month). The activity and purity of the active pharmaceutical ingredients produced for clinical trials were confirmed by different tests including ultra performance liquid chromatography, size exclusion chromatography, ELISA, SDS–PAGE, gel-clot LAL-test. Clinical trials were started in the Russian Federation.     

Regulation of immune cells by local-tissue oxygen tension: HIF1 alpha and adenosine receptors

Immune cells are often exposed to low oxygen tensions, which markedly affect cellular metabolism. We describe how activated T cells adapt to the changing energy supplies in hypoxic areas of inflamed tissues by using hypoxia-inducible factor 1 (HIF1) to switch to glycolysis as the main source of energy and by signalling through extracellular-adenosine receptors. This hypoxic regulation might alter the balance between T helper 1 cells and T helper 2 cells and might alter the activities of cells of the innate immune system, thereby qualitatively and quantitatively affecting immune responses. This regulatory mechanism should be taken into account in the design and interpretation of in vitro and in vivo studies of immune-cell effector functions.     

Balance between survivin,a key member of the apoptosis inhibitor family,and its specific antibodies determines erosivity in rheumatoid arthritis

Rheumatoid arthritis (RA) is a highly heterogeneous disease with respect to its joint destructivity. The reasons underlying this heterogeneity are unknown. Deficient apoptosis in rheumatoid synovial tissue has been recently demonstrated. We have therefore decided to study the synovial expression of survivin, a key member of the apoptosis inhibitor family. The levels of survivin and antibodies against survivin were assessed by an ELISA in matched blood and synovial fluid samples collected from 131 RA patients. Results were related to joint erosivity at the time of sampling. Monocytes were transfected with survivin anti-sense oligonucleotides and were assessed for their ability to produce inflammatory cytokines. Survivin levels were significantly higher in patients with destructive disease as compared with in RA patients displaying a non-erosive disease. High survivin levels were an independent prognostic parameter for erosive RA. In contrast, high levels of antibodies against survivin were found in patients with non-erosive RA, and were negatively related to erosivity. Survivin levels in RA patients were influenced by treatment, being significantly lower among patients treated with disease-modifying anti-rheumatic drugs. Specific suppression of survivin mRNA resulted in downregulation of IL-6 production. We conclude that survivin determines the erosive course of RA, whereas survivin antibodies lead to a less aggressive course of the disease. These findings together with decreased survivin levels upon disease-modifying anti-rheumatic drug treatment, and the downregulation of inflammatory response using survivin anti-sense oligonucleotides, suggest that extracellular survivin expression mediates the erosive course of joint disease whereas autoimmune responses to the same molecule, manifested as survivin targeting antibodies, mediate protection.     

Differential modulation of human melanoma cell metalloproteinase expression by alpha2beta1 integrin and CD44 triple-helical ligands derived from type IV collagen

Tumor cell binding to components of the basement membrane is well known to trigger intracellular signaling pathways. Signaling ultimately results in the modulation of gene expression, facilitating metastasis. Type IV collagen is the major structural component of the basement membrane and is known to be a polyvalent ligand, possessing sequences bound by the alpha1beta1, alpha2beta1, and alpha3beta1 integrins, as well as cell surface proteoglycan receptors, such as CD44/chondroitin sulfate proteoglycan (CSPG). The role of alpha2beta1 integrin and CD44/CSPG receptor binding on human melanoma cell activation has been evaluated herein using triple-helical peptide ligands incorporating the alpha1(IV)382-393 and alpha1(IV)1263-1277 sequences, respectively. Gene expression and protein production of matrix metalloproteinases-1 (MMP-1), -2, -3, -13, and -14 were modulated with the alpha2beta1-specific sequence, whereas the CD44-specific sequence yielded significant stimulation of MMP-8 and lower levels of modulation of MMP-1, -2, -13, and -14. Analysis of enzyme activity confirmed different melanoma cell proteolytic potentials based on engagement of either the alpha2beta1 integrin or CD44/CSPG. These results are indicative of specific activation events that tumor cells undergo upon binding to select regions of basement membrane collagen. Based on the present study, triple-helical peptide ligands provide a general approach for monitoring the regulation of proteolysis in cellular systems.     

Fatty liver in familial hypobetalipoproteinemia: roles of the APOB defects, intra-abdominal adipose tissue, and insulin sensitivity

Fatty liver is frequent in the apolipoprotein B (apoB)-defective genetic form of familial hypobetalipoproteinemia (FHBL), but interindividual variability in liver fat is large. To explain this, we assessed the roles of metabolic factors in 32 affected family members with apoB-defective FHBL and 33 related and unrelated normolipidemic controls matched for age, sex, and indices of adiposity. Two hour, 75 g oral glucose tests, with measurements of plasma glucose and insulin levels, body mass index, and waist-hip ratios were obtained. Abdominal subcutaneous, intraperitoneal (IPAT), and retroperitoneal adipose tissue masses were quantified by MR imaging, and hepatic fat was quantified by MR spectroscopy. Mean +/- SD liver fat percentage values of FHBL and controls were 14.8 +/- 12.0 and 5.2 +/- 5.9, respectively (P = 0.001). Means for these measures of obesity and insulin action were similar in the two groups. Important determinants of liver fat percentage were FHBL-affected status, IPAT, and area under the curve (AUC) insulin in both groups, but the strongest predictors were IPAT in FHBL (partial R(2) = 0.55, P < 0.0002) and AUC insulin in controls (partial R(2) = 0.59, P = 0.0001). Regression of liver fat percentage on IPAT fat was significantly greater for FHBL than for controls (P < 0.001). In summary, because apoB-defective FHBL imparts heightened susceptibility to liver triglyceride accumulation, increasing IPAT and insulin resistance exert greater liver fat-increasing effects in FHBL.