Proteolytic Remodeling of the Synaptic Cell Adhesion Molecules (CAMs) by Metzincins in Synaptic Plasticity

Cell adhesion molecules participate in the formation, maturation, function and plasticity of synaptic connections. The growing body of evidence indicates that in the regulation of the synaptic plasticity, in which these molecules play pivotal role, also the proteolytic processes are involved. This review focuses on extracellular proteolysis of the cell adhesion molecules by specific subgroup of the matrix metalloproteinases, a disintegrin and metalloproteases and a disintegrin and metalloproteinase with thrombospondin motifs, jointly referred to as metzincins, in driving coordinated synaptic structural and functional modifications underlying synaptic plasticity in the adult brain.     

Mycobacterium tuberculosis ClpX Interacts with FtsZ and Interferes with FtsZ Assembly

FtsZ assembly at the midcell division site in the form of a Z-ring is crucial for initiation of the cell division process in eubacteria. It is largely unknown how this process is regulated in the human pathogen Mycobacterium tuberculosis. Here we show that the expression of clpX was upregulated upon macrophage infection and exposure to cephalexin antibiotic, the conditions where FtsZ-ring assembly is delayed. Independently, we show using pull-down, solid-phase binding, bacterial two-hybrid and mycobacterial protein fragment complementation assays, that M. tuberculosis FtsZ interacts with ClpX, the substrate recognition domain of the ClpXP protease. Incubation of FtsZ with ClpX increased the critical concentration of GTP-dependent polymerization of FtsZ. Immunoblotting revealed that the intracellular ratio of ClpX to FtsZ in wild type M. tuberculosis is approximately 1∶2. Overproduction of ClpX increased cell length and modulated the localization of FtsZ at midcell sites; however, intracellular FtsZ levels were unaffected. A ClpX-CFP fusion protein localized to the cell poles and midcell sites and colocalized with the FtsZ-YFP protein. ClpX also interacted with FtsZ mutant proteins defective for binding to and hydrolyzing GTP and possibly for interactions with other proteins. Taken together, our results suggest that M. tuberculosis ClpX interacts stoichiometrically with FtsZ protomers, independent of its nucleotide-bound state and negatively regulates FtsZ activities, hence cell division.     

Synthesis and separation of diastereoisomers of 5'-O-(4,4'-dimethoxytrimethylphenyl)dithymidyl-(3,5')-4,4' -dimethoxytriphenylmethanephosphonate

"In solution" synthesis and separation of diastereoisomers of 5'-O-(4,4'-dimethoxytriphenylmethyl)dithymidyl (3',5') 4,4'-dimethoxytriphenylmethanephosphonate (DMTTDMTT) is described. One of diastereoisomers was successfully crystallized and characterized by means of HPLC.     

Diastereomers of thymidine 3'-O-(methanephosphonothioate): synthesis, absolute configuration and reaction with 3'-methoxyacetylthymidine under conditions of triester approach to oligonucleotide synthesis

Diastereomers of the title compound were obtained and absolute configuration was assigned by means of stereochemical correlation. Their reaction with 3'-O-methoxyacetylthymidine in the presence of triisopropylbenzenesulphonyl (4-nitro) triazole is neither chemo- nor stereo-selective and leads to diastereomeric pairs of dithymidyl (3',5')methanephosphonate and -methanephosphonothioate. Obtained results are discussed in terms of mechanism of activation of phosphodiesters under conditions known as "phosphotriester approach to oligonucleotide synthesis".     

Some aspects of activity regulation of Galleria mellonella PTTH cells

Using the Galleria prothoracicotropic bioassay, five small neurosecretory cells occurring in each dorsolateral part of protocerebrum of Galleria mellonella brain were identified as prothoracicotropic hormone (PTTH) cells. It was found that the critical period for the release of PTTH from a brain implanted in neck-ligated larva lasts up to the third day after implantation. The content of paraldehyde-fuchsin positive neurosecretory material (NSM) in PTTH cells was determined during the penultimate and last larval instar, during pupal instar, and in starved or poststarvation fed or space-deprived last instar larvae. Two peaks of NSM in PTTH cells were found in the penultimate instar (in freshly molted, and 76-h-old larvae), four peaks in the last instar larvae (in freshly molted, and in 67-, 132-, and 174-h-old larvae), and one peak in the pupal instar (in 56-76-h-old pupae). It was also observed that upon starvation NSM accumulated in PTTH cells, while after 3 h of poststarvation feeding it was released. In permanent space-deprived last instar larvae no NSM occurred in PTTH cells. In all investigated larval instars a rapid release of NSM from PTTH cells was found a few hours after molt associated with the beginning of the feeding period. The significance of the NSM content in PTTH cells is discussed in relation to ecdysteroid titer.     

Activation of a low specific activity form of DNA polymerase alpha by inositol-1,4-bisphosphate

A low activity form of DNA polymerase alpha immunoaffinity-purified from adult-derived human fibroblasts was activated by interaction with phosphatidylinositol-4-monophosphate, while a high activity form of the enzyme did not interact with phosphatidylinositol-4-monophosphate or its derivatives. Phosphatidylinositol-4-monophosphate was apparently hydrolyzed in the presence of a highly purified low activity form of DNA polymerase alpha, effecting the release of diacylglycerol and the retention of inositol-1,4-bisphosphate by the enzyme complex. The resulting inositol-1,4-bisphosphate/protein complex exhibited increased affinity of binding to DNA template/primer and increased deoxynucleotidyltransferase activity. These data indicate that inositol-1,4-bisphosphate may function as an effector molecule in the activation of a low activity form of human DNA polymerase alpha and suggest that it may function as a second messenger during the initiation of mitosis in stimulated cells.     

Duplications of the X chromosome in males: evidence that most parts of the X chromosome can be active in two copies

Summary We have analysed two duplications of the X chromosome in male patients using chromosome replication and DNA methylation patterns as determinants of the functional status of the duplicated segments. In both cases, the large duplicated regions, Xq12-q22 and Xq26.3-qter, were not inactivated. A review of previously reported male cases revealed that these duplications were also not subject to inactivation. Taken together, the examined duplications cover almost the entire X chromosome except the pericentromeric region and Xq25–26. Thus, most regions of the X chromosome can be present in two functional copies without lethal consequences.     

Activity of acid deoxyribonuclease towards diastereoisomers of thymidyl 3'-(4-nitrophenyl phosphorothioate). Stereochemistry of transnucleotidylation reaction

The (Rp)- and (Sp)-diastereoisomers of thymidyl 3'-(4-nitrophenyl phosphorothioate) (1) were found to act as unusual substrates for acid deoxyribonuclease (DNase II). Instead of the expected thymidine 3'-phosphorothioate, the product resulting from the reaction of (Rp)-1 catalyzed by DNase II was identified as (Sp, Rp)-thymidyl (3'-5')thymidyl phosphorothioate 3'-(4-nitrophenyl phosphorothioate), while that from (Sp)-1 has been recognized as a 10:1 mixture of (Sp, Rp)-thymidyl (3'-5')thymidyl phosphorothioate 5'-(4-nitrophenyl phosphorothioate) and (Rp, Sp)-thymidyl (3'-5')-thymidyl phosphorothioate 3'-(4-nitrophenyl phosphorothioate), respectively. Both types of transnucleotidylations were found to occur with retention of configuration at phosphorus. Stereochemical results may be interpreted in terms of two step mechanisms involving the formation of the intermediate, covalent substrate enzyme complexes.     

Inhibition of glycogenolysis in isolated rat hepatocytes by the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate

The diastereomeric forms of adenosine cyclic 3',5'-phosphorothioate, Rp cAMPS and Sp cAMPS, were studied in isolated hepatocytes from fed rats for their ability to interact with the intracellular cAMP-dependent protein kinase and to affect the phosphorylase kinase-phosphorylase glycogenolytic cascade. Incubation of the cells with increasing concentrations of Sp cAMPS produced a concentration-dependent activation of cAMP-dependent protein kinase with a concomitant increase in the glycogenolytic rate. Half-maximal and maximal velocities of glycogenolysis were reached at 8 X 10(-7) and 1 X 10(-5) M Sp cAMPS, respectively. Incubation of the cells with 10(-9) to 10(-4) M Rp cAMPS had no effect on basal glucose production or on cAMP-dependent protein kinase activity. Incubation of the cells simultaneously with 3 X 10(-6) M Sp cAMPS and increasing concentrations of Rp cAMPS produced half-maximal inhibition of glycogenolysis at 1 X 10(-5) M Rp cAMPS and maximal inhibition at 1 X 10(-4) M. The concentrations of Sp cAMPS required for half-maximal and maximal activation of glycogenolysis were increased 10-fold when 1 X 10(-5) M Rp cAMPS was present. These data imply that Sp cAMPS is a cAMP-agonist while Rp cAMPS is a cAMP-antagonist.