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.     

Velocity of Lordosis Angle during Spinal Flexion and Extension

The importance of functional parameters for evaluating the severity of low back pain is gaining clinical recognition, with evidence suggesting that the angular velocity of lordosis is critical for identification of musculoskeletal deficits. However, there is a lack of data regarding the range of functional kinematics (RoKs), particularly which include the changing shape and curvature of the spine. We address this deficit by characterising the angular velocity of lordosis throughout the thoracolumbar spine according to age and gender. The velocity of lumbar back shape changes was measured using Epionics SPINE during maximum flexion and extension activities in 429 asymptomatic volunteers. The difference between maximum positive and negative velocities represented the RoKs. The mean RoKs for flexion decreased with age; 114°/s (20–35 years), 100°/s (36–50 years) and 83°/s (51–75 years). For extension, the corresponding mean RoKs were 73°/s, 57°/s and 47°/s. ANCOVA analyses revealed that age and gender had the largest influence on the RoKs (p<0.05). The Epionics SPINE system allows the rapid assessment of functional kinematics in the lumbar spine. The results of this study now serve as normative data for comparison to patients with spinal pathology or after surgical treatment.     

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.     

Entrapment of high-molecular-mass DNA molecules in liposomes for the genetic transformation of animal cells.

We modified the Ca/EDTA procedure for the production of liposomes [Papahadjopoulos, Vail, Jacobson & Poste (1975) Biochim. Biophys. Acta 394, 483-491] to entrap intact DNA molecules of very high molecular mass into large unilamellar phospholipid vesicles. The use of DNA-protein complexes and phage particles instead of naked linear DNA increases the efficiency of entrapment and protects the integrity of DNA molecules. We investigated the interaction of mammalian cells with liposome-encapsulated recombinant lambda bacteriophages carrying marker genes. The liposomes bind surprisingly fast to the cellular surface and are taken up by the cells. A significant proportion of the encapsulated DNA is transported to and soon located in or around the nuclei. Experiments prove that these liposomes can be used for the genetic transformation of mammalian cells.     

Thermoanaerobium lactoethylicum spec. nov. a new anaerobic bacterium from a hot spring of Kamchatka

A new anaerobic thermophilic Gram-positive, nonsporeforming bacterium strain ZE-1 was isolated from a hot spring of Kamchatka (USSR). The cells are rod-shaped, (0.5–0.8 · 2.0–20 m), non-motile. The bacterium can grow between 42 and 75°C; the optimal temperature is 65°C. The growth is possible between pH values 5.0 and 8.5; optimal pH is 7.0. The cultures grow on the media containing peptone, yeast extract, or casein hydrolysate as nitrogen sources in the presence of glucose or some other sugars, mannitol or starch. The main fermentation products of glucose are ethanol, acetate, lactate, H₂, CO₂; byproducts are propionic, butyric and isovaleric acids. Glucose is metabolized via Embden-Meyerhoff-Parnas pathway. Molecular hydrogen does not inhibit growth. The bacterium does not reduce aceton to isopropanol, but is able to form H₂S from elemental sulfur. The bacterium contains a soluble hydrogenase. This enzyme catalyzes both evolution and uptake of H₂ and is active in the presence of methyl viologen. The DNA-base composition is 34.6 mol%; the genome size 2.08x10⁹ D. The name proposed for the isolated bacterium strain ZE-1 is Thermoanaerobium lactoethylicum spec. nov.     

Genetic evidence for α2-adrenergic receptor subtypes in rat brain,heart and adrenal gland

Summary A complementary DNA (cDNA) clone - cA₂-47 - corresponding to a new ₂-adrenergic receptor subtype has been isolated from a rat brain cDNA library and used as a hybridization probe to scrutinize the ₂-receptor poly(A⁺) RNAs in rat brain, heart and adrenal gland. Hybridization of the 5 half of the coding region of this cDNA at 37°C to rat brain poly(A+) RNA revealed a single band at 5.8 kb as the size of its corresponding mRNA. Under identical hybridization conditions, a human platelet ₂-receptor genomic probe failed to hybridize to any rat brain mRNAs.Under lower stringency conditions, hybridization of the full-length cDNA, cA₂-47, to selected rat tissue poly(A⁺) RNA showed the presence of four different sized mRNAs in brain and three in both heart and adrenal gland. Messages of 1.3 kb and 2.1 kb were common in all three tissues (although the band at 2.1 kb was slightly higher in the heart and adrenal gland). A 5.8 kb mRNA was unique to the brain and a slightly higher band at 6.0 kb was consistently present in heart and adrenal gland but was absent in the brain. A fourth message at 3.4 kb was found predominantly in the brain and was either absent or present at very low levels in the other tissues examined. Under the same conditions, a human platelet ₂-receptor probe hybridized to similar sized messages of 2.1 and 5.8 kb in rat brain and 2.2 and 6.0 kb in rat heart and adrenal gland. This probe, however, failed to detect the abundant 1.3 kb mRNA common to all tissues or the 3.4 kb message in rat brain. The extent of homology of these messages with cA₂-47 is not confined to limited regions of the cDNA since similar hybridization patterns were observed using either 5-noncoding or 5-coding regions of the probe.These results provide the first direct evidence of a surprisingly large range of mRNA sizes for members of the ₂-receptor family in brain, heart, and adrenal gland. The unique nature of certain members of the family in each of the tissues examined raises the curious possibility that these members might contribute to some of the individualized functions of the brain, cardiovasculature and adrenal gland.     

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.     

Refolding of serine proteinases

Bovine trypsinogen and chymotrypsinogen were successfully refolded as the mixed disulfide of glutathione using cysteine as the disulfide interchange catalyst. The native structures were regenerated with yields of 40%-50% at pH 8.6 and 4 degrees C, and the half-time for the refolding was approximately 60-75 min. We then refolded threonine-neochymotrypsinogen, which is a two-chain structure held together by disulfide bonds and produced on cleavage of Tyr 146-Thr 147 in native chymotrypsinogen [Duda CT, Light A, J Biol Chem 257 9866-9871, 1982]. Neochymotrypsinogen was denatured and fully reduced, and the thiols were converted to the mixed disulfide of glutathione. The two polypeptide fragments, representing the amino- and carboxyl-terminal domains, were separated on Sephadex G-75. Mixtures of the polypeptide fragments varying in the ratio of their concentration from 1:5 to 5:1 were refolded with yields of 21-28%. The lack of dependence on the concentration of either fragment and the relatively high yields suggest independent folding of the amino- and carboxyl-terminal domains. When the globular structures of the domains formed, they then interacted with one another and produced the native intermolecular disulfide bridge and the proper geometry of the active site.