Neuronal porosome in the rat and cat brain

It is well established that during cell secretion, membrane-bound secretory vesicles dock and fuse at the base of supramolecular cup-shaped structures at the cell plasma membrane called "porosomes", to expel intra-vesicular contents to the outside. In neurons, it has been demonstrated that 12-17 nm cup-shaped lipoprotein structure possessing a central plug are present at the presynaptic membrane, where 50 nm in diameter synaptic vesicles transiently dock and fuse to release neurotransmitter. In the past decade, the neuronal porosome has been isolated and its major chemical composition determined. Additionally, the porosome has been both structurally and functionally reconstituted into artificial lipid membrane, establishing its role as the secretory portal in neurons. Studies utilizing atomic force and electron microscopy, combined with electron density and 3D contour mapping, provide at the nanoscale, the structure and assembly of proteins within the neuronal porosome. In the current study, ultrahigh resolution imaging of the presynaptic membrane of isolated brains from both rats and cats, demonstrate for the first time, the presence of neuronal porosomes in cat brain, and further confirms the presence of porosomes at the presynaptic membrane in rat brain synaptosomes. Results from the present study further confirm the cup-shaped morphology of porosomes in the rat brain, and demonstrates their similar shape and size in the cat nerve terminal. The study also demonstrates for the first time, the universal presence of similar porosomes in different species of mammals.     

Method for isolation of intact titin (connectin) molecules from mammalian cardiac muscle

Cardiac titin was isolated from rabbit and ground squirrel ventricular muscles by a method that was used earlier to obtain myofibrils with intact minor proteins located in A-bands of sarcomeres (Podlubnaya, Z. A., et al. (1989) J. Mol. Biol., 210, 655–658). Small pieces of cardiac muscle were incubated for 2–3 weeks at 4°C in Ca²⁺-depleting solution before their homogenization to decrease activity of Ca²⁺-dependent proteases. Then the muscle was homogenized, and titin was isolated by the method of Soteriou, A., et al. (1993) J. Cell Sci., 14, 119–123. In control experiments, titin was isolated from cardiac muscle without its preincubation in Ca²⁺-depleting solution. Sometimes control titin preparations contained only T2-fragment, but generally they contained ～5–20% N2B-isoform of titin along with its T2-fragment. Preparations of titin obtained from rabbit cardiac muscle by our method contained ～30–50% of N2BA- and N2B-titin isoforms along with its T2-fragment. The content of α-structures in titin isolated by our method was increased. Actomyosin ATPase activity in vitro increased in the presence of titin preparations containing more intact molecules. This result confirms the significant role of titin in the regulation of actin-myosin interaction in muscles. The method used by us to preserve titin might be used for isolation of other proteins that are substrates of Ca²⁺-dependent proteases.     

The Genus Syringa: Molecular Markers of Species and Cultivars

RAPD analysis was carried out with 22 accessions of the genus Syringa, including six species, one interspecific hybrid, and 15 cultivars. In total, 512 polymorphic fragments were detected; species-specific and cultivar-specific markers were identified. For the first time, genetic polymorphism and genome similarity coefficients were estimated and phylogenetic relationships were established for the genus Syringa.     

Changes in titin and myosin heavy chain isoform composition in skeletal muscles of Mongolian Gerbil (Meriones unguiculatus) after 12-day spaceflight

Changes of titin and myosin heavy chain isoform composition in skeletal muscles (m. soleus, m. gastrocnemius, m. tibialis anterior, m. psoas major) in Mongolian Gerbil (Meriones unguiculatus) were investigated after 12-day spaceflight on board of Russian space vehicle “Foton-M3.” In m. psoas and m. soleus in the gerbils from “Flight” group the expected increase in the content of fast myosin heavy chain isoforms (IIxd and IIa, respectively) were observed. No significant differences were found in the content of IIxd and IIa isoforms of myosin heavy chain in m. tibialis anterior in the gerbils from control group as compared to that in “Flight” group. An unexpected increase in the content of slow myosin heavy chain I isoform and a decrease in the content of fast IIx/d isoform in m. gastrocnemius of the gerbils from “Flight” group were observed. In skeletal muscles of the gerbils from “Flight” group the relative content of titin N2A-isoform was reduced (by 1.2–1.7 times), although the content of its NT-isoform, which was revealed in striated muscles of mammals in our experiments earlier, remained the same. When the content of titin N2A-isoform was decreased, no predictable abnormalities in sarcomeric structure and contractile ability of skeletal muscles in the gerbils from “Flight” group were found. An assumption on the leading role of titin NT-isoform in maintenance of structural and functional properties of striated muscles of mammals was made.     

Antiamyloid properties of fullerene C60 derivatives

A comparative estimation of the ability of complexes of fullerene C60 with polyvinylpyrrolidone and fullerene C60 derivatives (the sodium salt of the polycarboxylic derivative of fullerene C60, sodium fullerenolate), has been carried out. The fullerenes destroyed amyloid fibrils of the Abeta(1-42) peptide of the brain and the muscle X-protein. A study of the effect of fullerenes on muscle actin showed that complexes of fullerene C60 with polyvinylpyrrolidone and sodium fullerenolate did not prevent the filament formation of actin, nor did they destroy its filaments in vitro. Conversely, sodium salt of the polycarboxylic derivative of fullerene C60 destroyed actin filaments and prevented their formation. It was concluded that sodium fullerenolate and complexes of fullerene C60 with polyvinylpyrrolidone are the most effective antiamyloid compounds among the fullerenes examined.     

Changes in isoform composition,structure, and functional properties of titin from mongolian gerbil (<Emphasis Type="Italic">Meriones unguiculatus</Emphasis>) cardiac muscle after space flight

Changes in isoform composition, secondary structure, and titin phosphorylation in Mongolian gerbil (Meriones unguiculatus) cardiac muscle were studied after 12-day-long space flight onboard the Russian spacecraft Foton-M3. The effect of titin on the actin-activated myosin ATPase activity at pCa 7.5 and 4.6 was also studied. Almost twofold increase in titin long N2BA isoform content relative to that of short N2B isoform was found on electrophoregrams of cardiac muscle left ventricle of the flight group gerbils. Differences in secondary structure of titin isolated from cardiac muscle of control and flight groups of gerbils were found. An increase in phosphorylation (1.30–1.35-fold) of titin of cardiac muscle of the flight group gerbils was found. A decrease in activating effect of titin of cardiac muscle of the flight group gerbils on actomyosin ATPase activity in vitro was also found. The observed changes are discussed in the context of M. unguiculatus cardiac muscle adaptation to conditions of weightlessness.     

Neuronal porosome in the rat and cat brain

It is well established that during cell secretion, membrane-bound secretory vesicles dock and fuse at the base of supramolecular cup-shaped structures at the cell plasma membrane called “porosomes”, to expel intra-vesicular contents to the outside. In neurons, it has been demonstrated that 12–17 nm cupshaped lipoprotein structure possessing a central plug are present at the presynaptic membrane, where 50 nm in diameter synaptic vesicles transiently dock and fuse to release neurotransmitters. In the past decade, the neuronal porosome has been isolated and its major chemical composition determined. Additionally, the porosome has been both structurally and functionally reconstituted into artificial lipid membrane, establishing its role as the secretory portal in neurons. Studies utilizing atomic force and electron microscopy, combined with electron density and 3D contour mapping, provide at the nanoscale, the structure and assembly of proteins within the neuronal porosome. In the current study, ultrahigh resolution imaging of the presynaptic membrane of isolated brains from both rats and cats, demonstrate for the first time, the presence of neuronal porosomes in cat brain, and further confirms the presence of porosomes at the presynaptic membrane in rat brain synaptosomes. Results from the present study further confirm the cup-shaped morphology of porosomes in the rat brain, and demonstrates their similar shape and size in the cat nerve terminal. The study also demonstrates for the first time, the universal presence of similar porosomes in different species of mammals.     

Interaction of a cationic polymer with negatively charged proteoliposomes

In the present study, we have analyzed a previously identified constitutively active pituitary adenylate cyclase activating polypeptide (PACAP) type I (PAC1) receptor with a deletion of the single amino acid residue Glu(261) (Y.-J. Cao, G. Gimpl, F. Fahrenholz, A mutation of second intracellular loop of pituitary adenylate cyclase activating polypeptide type I receptor confers constitutive receptor activation, FEBS Lett. 469 (2000)). This glutamic acid residue is highly conserved within the second intracellular loop of class II G protein-coupled receptors and may thus be of importance for many members of this receptor class. To explore the molecular characteristics of this mutant receptor, we performed photoaffinity labeling using previously defined photoreactive PACAP analogues. In COS cells, the PAC1 receptor was expressed in two differently glycosylated forms: a M(r) 75,000 and a M(r) 55,000 form. According to partial deglycosylation, at least three carbohydrate chains may exist in the rat PAC1 receptor expressed in COS cells. The constitutively active PAC1 receptor was expressed at the surface of COS-7 cells at the same density as the wild-type receptor. With respect to the different photoreactive PACAP analogues, the labeling specificity was the same for the wild-type versus mutant receptor: (125)I-[Lys(15)(pBz(2))]-PACAP-27 and (125)I-[Bpa(22)]-PACAP-27 were efficiently incorporated into each of the receptors, whereas (125)I-[Bpa(6)]-PACAP-27 labeled each of the receptors only to a negligible extent. This suggests that both receptors have the same or at least a very similar hormone binding site which is in close contact to Tyr(22) and Lys(15) located in the carboxy-terminal alpha-helical region of the PACAP-27 molecule. However, in comparison with the wild-type PAC1 receptor, the constitutively active receptor showed a markedly (approx. 6--8-fold) enhanced photoaffinity labeling efficiency in particular of the high glycosylated form. The enzymatically deglycosylated rat PAC1 receptor was efficiently labeled by photoreactive PACAP analogues. In contrast, nonglycosylated PAC1 receptors produced by tunicamycin treatment of the transfected COS-7 cells showed a 30-fold lower affinity for PACAP-27 and were capable of signal transduction with 30--50-fold lower potency as compared with the glycosylated PAC1 receptors.