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.     

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.     

Influence of experimental diets on cholesterol and triglyceride levels of rabbit blood serum lipoproteins

Groups of rabbits were fed for six weeks various diets: standard died + ethanol, high-cholesterol diet and a high-cholesterol + ethanol one. During the next six weeks every diet was supplemented with a fresh vegetable (carrot). Cholesterol and triglycerides were determined in the whole serum and in lipoprotein fractions. In rabbits fed standard diet ethanol caused a moderate elevation of VLDL cholesterol and triglyceride and LDL cholesterol levels. In animals on high-cholesterol diet cholesterol and triglyceride concentrations in these fractions were very high. Simultaneous consumption of large amounts of cholesterol and of ethanol resulted in a greater rise of cholesterol concentration in the whole serum and in VLDL and LDL fraction than did high-cholesterol diet alone. Addition of carrot caused a pronounced reduction of serum cholesterol concentration in animals fed all kinds of diets. The reduction concerned mainly VLDL.     

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.     

Iron-induced lipid peroxidation and inhibition of dopamine synthesis in striatum synaptosomes

Crude striatum synaptosomes (P₂ fraction) from Fisher 344 female rats were incubated in the presence of ADP-chelated Fe³⁺ (0.5–50 M) and ascorbate (250 M). Intrasynaptosomal conversion of tyrosine to dopamine (DA) was measured by¹⁴CO₂ evolution froml-[1-¹⁴C]tyrosine in the absence of added cofactors and DOPA decarboxylase. Malondialdehyde (MDA) was measured as an index of lipid peroxidation. A concentration-dependent inhibition of DA synthesis by ADP-Fe³⁺/ascorbate was found with 50% inhibition occurring at 2.5 M Fe³⁺ concentration. This was accompanied by marked accumulation of MDA. Ascorbate or ADP alone did not affect DA synthesis and ADP-Fe³⁺ in the absence of exogenous ascorbate was effective only above 25 M. Exogenously added MDA did not inhibit DA synthesis. Purified synaptosomes were isolated from peroxidized and control P₂ fractions using sucrose gradients. Membrane microviscosity of the purifled synaptosomes was assessed by nitroxyl spin labels of stearic acid using electron paramagetic resonance techniques. There was a significant increase in membrane microviscosity as a result of ADP-Fe³⁺/ascorbate induced peroxidation. Maleimide nitroxide spin-label binding to protein sulhydryls was significantly modified by peroxidation of striatum synaptosomes. The weakly immobilized component of the sulhydryl spin-label (w) was drastically decreased whereas the strongly immobilized component (s) was modified less, thus leading to a marked reduction of w/s ratio. The exposure of striatum synaptosomes to the peroxidizing system resulted in a significant increase in total iron and in a 25% decrease in protein sulhydryl content. It is concluded that ironinduced damage to the DA synthetic system is mediated by alterations of the structural properties of nerve ending membranes.     

Some events of mitosis and cytokinesis in the generative cell of Ornithogalum virens L.

The organization of the microtubule (Mt) cytoskeleton during mitosis and cytokinesis of the generative cell (GC) in Ornithogalum virens L. (bicellular pollen type, chromosome number, n = 3) from prophase to telophase/sperm formation was investigated by localization of -tubulin immunofluorescence using a conventional fluorescence microscope and a confocal laser scanning microscope. Chromosomes were visualized with DNA-binding fluorochrome dyes (ethidium bromide and 46-diamino-2-phenyl-indole). The GC of O. virens is characterized by G2/M transition within the pollen grain and not in the pollen tube as occurs in the majority of species with bicellular pollen. It was found that prophase in the GC starts before anthesis and prometaphase takes place after 10 min of pollen germination. The prophase Mts are organized into three prominent bundles, located near the generative nucleus. The number of these Mt bundles is the same as the number of GC chromosomes, a relation which has not previously been considered in other species. The most evident feature in the prophase/ prometaphase transition of O. virens GC is a direct rapid rearrangement of Mt bundles into a network which appears to interact with kinetochores and form a typical prometaphase Mt organization. The metaphase chromosomes are arranged into a conventional equatorial plate, and not in tandem as is thought to be characteristic of GC metaphase. The metaphase spindle consists of kinetochore fibres and a few interzonal fibres which form dispersed poles. Anaphase is characterized by a significant elongation of the mitotic spindle concomitant with the extension of the distance between the opposite poles. At anaphase the diffuse poles converge. Cytokinesis is realized by cell plate formation in the equatorial plane of the GC. The phragmoplast Mts between two future sperm nuclei appear after Mts of the mitotic spindle have disappeared.Abbreviations DAPI 46-diamino-2-phenyl-indole - GC generative cell - GN generative nucleus - Mt microtubule This research was made possible in part due to TEMPUS Programme and Global Network for Cell and Molecular Biology UNESCO grants to Magorzata Bana. The experimental part of the work was done in Siena University. M. Banas is very grateful to Prof. Mauro Cresti and his group for scientific interest, offering the excellent laboratory facilities, and kind reception.     

Oscillations in the electrical resistance of Nitellopsis obtusa nodes

Oscillatory changes of the electrical resistance across the nodal complex of Nitellopsis obtusa (Desv. in Lois) J. Gr. were observed in experiments performed for 40–150 min with the use of external electrodes and microelectrodes. Three main patterns of node resistance oscillations were similar to those found for membrane potential and resistance. The presented findings indicate an oscillatory behaviour of the plasmodesmata system at the node, which may be connected with e.g. pulsatile variations in the number of open plasmodesmata.     

Different UmuC requirements for generation of different kinds of UV-induced mutations in Escherichia coli

An Escherichia coli strain bearing the dnaQ49 mutation, which results in a defective s subunit of DNA polymerase III, and carrying the lexA71 mutation, which causes derepression of the SOS regulon, is totally unable to maintain high-copy-number plasmids containing the umuDC operon. The strain is also unable to maintain the pAN4 plasmid containing a partial deletion of the umuD gene but retaining the wild-type umuC gene. These results suggest that a high cellular level of UmuC is exceptionally harmful to the defective DNA polymerase III of the dnaQ49 mutant. We have used this finding as a basis for selection of new plasmid umuC mutants. The properties of two such mutants, bearing the umuC61 or umuC95 mutation, are described in detail. In the umuC122:: Tn 5 strain harbouring the mutant plasmids, UV-induced mutagenesis is severely decreased compared to that observed with the parental umuDC ⁺ plasmid. Interestingly, while the frequency of UV-induced GC → AT transitions is greatly reduced, the frequency of AT → TA transversions is not affected. Both mutant plasmids bear frameshift mutations within the same run of seven A residues present in umuC ⁺; in umuC61 the run is shortened to six A whereas in umuC95 is lengthened to eight A. We have found in both umuC61 and umuC95 that translation is partially restored to the proper reading frame. We propose that under conditions of limiting amounts of UmuC, the protein preferentially facilitates processing of only some kinds of UV-induced lesions.