Demonstration of extensive GABA synthesis in the small population of GAD positive neurons in cerebellar cultures by the use of pharmacological tools

Cultures of dissociated cerebella from 7-day-old mice were maintained in vitro for 1-13 days. GABA biosynthesis and degradation were studied during development in culture and pharmacological agents were used to identify the enzymes involved. The amount of GABA increased, whereas that of glutamate was unchanged during the first 5 days and both decreased thereafter. The presence of aminooxyacetic acid (AOAA, 10 microM) which inhibits transaminases and other pyridoxal phosphate dependent enzymes including GABA-transaminase (GABA-T), in the culture medium caused an increase in the intracellular amount of GABA and a decrease in glutamate. The GABA content was also increased following exposure to the specific GABA-T inhibitor gamma-vinyl GABA. From day 6 in culture (day 4 when cultured in the presence of AOAA) GABA levels in the medium were increased compared to that in medium from 1-day-old cultures. Synthesis of GABA during the first 3 days was demonstrated by the finding that incubation with either [1-(13)C]glucose or [U-(13)C]glutamine led to formation of labeled GABA. Synthesis of GABA after 1 week in culture, when the enzymatic machinery is considered to be at a more differentiated level, was shown by labeling from [U-(13)C]glutamine added on day 7. Altogether the findings show continuous GABA synthesis and degradation throughout the culture period in the cerebellar neurons. At 10 microM AOAA, GABA synthesis from [U-(13)C]glutamine was not affected, indicating that transaminases are not involved in GABA synthesis and thus excluding the putrescine pathway. At a concentration of 5 mM AOAA GABA labeling was, however, abolished, showing that glutamate decarboxylase, which is inhibited at this level of AOAA, is responsible for GABA synthesis in the cerebellar cultures. In conclusion, the present study shows that GABA synthesis is taking place via GAD in a subpopulation of the cerebellar neurons, throughout the culture period.     

Ultrastructure of interstitial cells of Cajal in myenteric plexus of human colon

The role of the interstitial cells of Cajal (ICC) associated with the myenteric plexus (ICC-MP) as regulators of the motility of the colonic external muscle remains unclear. Ultrastructural studies of myenteric interstitial cells are lacking in human colon. We therefore characterized the distinctive ultrastructure of these cells in the myenteric region of the colon by transmission electron microscopy of the region between the main muscle layers in all parts of the colon in unaffected areas of resected specimens from nine adult human patients. ICC-MP were similar in various colonic regions and had myoid features such as scattered caveolae, prominent intermediate filaments, and cytoplasmic dense bodies. We found characteristic dense membrane-associated bands with a patchy basal lamina, invaginating cellular protrusions (peg and socket junctions) between ICC and between ICC and muscle cells, and close contacts (<100 nm) between ICC and nerves. No gap junctions were observed. Fibroblast-like cells (FLC) were abundant showing well-developed secretory organelles, including coated vesicles, but lacked prominent intermediate filaments and caveolae. FLC had a patchy basal lamina, and peg and socket junctions were observed between them. Macrophage-like cells frequently occurred in close apposition with FLC and, more seldomly, with ICC-MP. The ultrastructure of ICC and FLC in the myenteric region of the human colon thus differs characteristically, but significant overlaps in the ultrastructure between ICC and FLC might complicate any interpretation in pathological ultrastructural studies of the human colonic muscle layer. An erratum to this article can be found at     

Molecular dynamics simulations and functional characterization of the interactions of the PAR2 ectodomain with factor VIIa

Signaling of the tissue factor‐FVIIa complex regulates angiogenesis, tumor growth, and inflammation. TF‐FVIIa triggers cell signaling events by cleavage of protease activated receptor (PAR2) at the Arg36‐Ser37 scissile bond. The recognition of PAR2 by the FVIIa protease domain is poorly understood. We perform molecular modeling and dynamics simulations to derive the PAR2‐FVIIa interactions. Docking of the PAR2 Arg36‐Ser37 scissile bond to the S1 site and subsequent molecular dynamics leads to interactions of the PAR2 ectodomain with P and P′ sites of the FVIIa catalytic cleft as well as to electrostatic interactions between a stably folded region of PAR2 and a cluster of basic residues remote from the catalytic cleft of FVIIa. To address the functional significance of this interaction for PAR2 cleavage, we employed two antibodies with epitopes previously mapped to this cluster of basic residues. Although these antibodies do not block the catalytic cleft, both antibodies completely abrogated PAR2 activation by TF‐FVIIa. Our simulations indicate a conformation of the PAR2 ectodomain that limits the cleavage site to no more than 33 Å from its membrane proximal residue. Since the active site of FVIIa in the TF‐FVIIa complex is ～75 Å above the membrane, cleavage of the folded conformation of PAR2 would require tilting of the TF‐FVIIa complex toward the membrane, indicating that additional cellular factors may be required to properly align the scissile bond of PAR2 with TF‐FVIIa. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Eutrophication and macrophyte species richness in the large shallow North-European Lake Peipsi

We present an overview of long-term changes in the floristic composition and growth areas in L. Peipsi (3555 km², unregulated water level) that have occurred since the 1960s and a list of plant taxa containing 140 species of higher plants and 4 charophytes. A significant correlation was found between the relative abundance and frequency per stations (Fs) (Rs = 0.93). Data on five inhabitants of the eulittoral revealed significant (p < 0.05) inter-annual differences in Fs. Comparison of the data of Fs for 67 taxa for 1970-1980 (87 stations) and 1997-2007 (139 stations) showed a significant change in the Fs distribution (p < 0.03) and a decline (p < 0.05) for 20 taxa; for 15 species Fs had decreased two times or more. However, 14 of the markedly declined taxa, e.g. the long-term dominating submergent Potamogeton perfoliatus, belong still among the top 33 in the list. A significant (χ² = 11.8; p < 0.028) change was observed in the species number of different frequency classes. The number of taxa in the Fs class 46-100 (92)% was 17 in 1970-1980 but only 3 in 1997-2007. The top of the list of macrophytes is dominated by circumpolar species and vicariants. Impoverishment of the flora in the course of eutrophication is expressed by the decrease in Fs; at the same time, the total number of species had not changed. Among the 20 declined taxa 14 are characteristic of the temporarily flooded and/or shallow-water zone of eutrophic water bodies (amphibious and emergent plants); the remaining taxa are shallow-water submergents. The simpliest explanation for their decrease is the expansion of thick reeds occupying suitable eulittoral habitats.     

Readers of PCNA modifications

The eukaryotic sliding clamp, proliferating cell nuclear antigen (PCNA), acts as a central coordinator of DNA transactions by providing a multivalent interaction surface for factors involved in DNA replication, repair, chromatin dynamics and cell cycle regulation. Posttranslational modifications (PTMs), such as mono- and polyubiquitylation, sumoylation, phosphorylation and acetylation, further expand the repertoire of PCNA’s binding partners. These modifications affect PCNA’s activity in the bypass of lesions during DNA replication, the regulation of alternative damage processing pathways such as homologous recombination and DNA interstrand cross-link repair, or impact on the stability of PCNA itself. In this review, we summarise our current knowledge about how the PTMs are “read” by downstream effector proteins that mediate the appropriate action. Given the variety of interaction partners responding to PCNA’s modified forms, the ensemble of PCNA modifications serves as an instructive model for the study of biological signalling through PTMs in general.     

Glucose,Lactate and Glutamine but not Glutamate Support Depolarization-Induced Increased Respiration in Isolated Nerve Terminals

Synaptosomes prepared from various aged and gene modified experimental animals constitute a valuable model system to study pre-synaptic mechanisms. Synaptosomes were isolated from whole brain and the XFe96 extracellular flux analyzer (Seahorse Bioscience) was used to study mitochondrial respiration and glycolytic rate in presence of different substrates. Mitochondrial function was tested by sequentially exposure of the synaptosomes to the ATP synthase inhibitor, oligomycin, the uncoupler FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone) and the electron transport chain inhibitors rotenone and antimycin A. The synaptosomes exhibited intense respiratory activity using glucose as substrate. The FCCP-dependent respiration was significantly higher with 10 mM glucose compared to 1 mM glucose. Synaptosomes also readily used pyruvate as substrate, which elevated basal respiration, activity-dependent respiration induced by veratridine and the respiratory response to uncoupling compared to that obtained with glucose as substrate. Also lactate was used as substrate by synaptosomes but in contrast to pyruvate, mitochondrial lactate mediated respiration was comparable to respiration using glucose as substrate. Synaptosomal respiration using glutamate and glutamine as substrates was significantly higher compared to basal respiration, whereas oligomycin-dependent and FCCP-induced respiration was lower compared to the responses obtained in the presence of glucose as substrate. We provide evidence that synaptosomes are able to use besides glucose and pyruvate also the substrates lactate, glutamate and glutamine to support their basal respiration. Veratridine was found to increase respiration supported by glucose, pyruvate, lactate and glutamine and FCCP was found to increase respiration supported by glucose, pyruvate and lactate. This was not the case when glutamate was the only energy substrate.     

Carbohydrate metabolism during fruit development in sweet pepper (Capsicum annuum) plants

Growth, accumulation of sugars and starch, and the activity of enzymes involved in sucrose mobilization were determined throughout the development of sweet pepper fruits. Fruit development was roughly divided into three phases: (1) an initial phase with high relative growth rate and hexose accumulation, (2) a phase with declining growth rate and accumulation of sucrose and starch, and (3) a ripening phase with no further fresh weight increase and with accumulation of hexoses, while sucrose and starch were degraded. Acid and neutral invertase (EC 3.2.1.26) were closely correlated to relative growth rate until ripening and inversly correlated to the accumulation of sucrose. Acid invertase specifically increased during ripening, concurrently with the accumulation of hexoses. Sucrose synthase (EC 2.4.1.13) showed little correlation to fruit development, and in periods of rapid growth the activity of sucrose synthase was low compared to the invertases. However, during late fruit growth sucose synthase was more active than the invertases. We conclude that invertase activities determine the accumulation of assimilates in the very young fruits, and a reactivation of acid invertase is responsible for the accumulation of hexoses during ripening. During late fruit growth, before ripening, sucrose synthase is transiently responsible for the sucrose breakdown in the fruit tissue. Results also indicate that pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) and its activator fructose-2,6-bisphosphate (Fru2,6bisP) are involved in the regulation of the sink metabolism of the fruit tissue.     

A defective cell surface collagen-binding protein in dermatosparactic sheep fibroblasts

Fibroblasts from dermatosparactic sheep fail to contract collagen gels and show a reduced attachment to collagenous substrates. By comparing collagen-binding membrane proteins of normal (+/+), homozygote (-/-), and heterozygote (+/-) fibroblasts, we present evidence that the interaction of normal fibroblasts with native type I collagen involves a protein of apparent Mr = 34,000 which is absent from dermatosparactic fibroblasts and seems to be related to anchorin CII. This conclusion was reached from the following experiments: (a) On a blot of membrane proteins from normal fibroblasts radioactively labeled type I collagen bound predominantly to a protein band of 34 kD; dermatosparactic membranes revealed only a small amount of binding to a component with a molecular mass of 47 kD. (b) After separation of normal fibroblast membrane proteins on type I collagen-Sepharose, a collagen-binding component of 34 kD was found which was absent from the corresponding fraction of dermatosparactic membranes. (c) Antibodies to anchorin CII stained the surface of normal (+/+), but not of dermatosparactic (-/-) fibroblasts and labeled a 34-kD component after immunoblotting of normal fibroblast membrane proteins. (d) After metabolic labeling of fibroblasts with [35S]methionine and immunoprecipitation with anti-anchorin CII, 40- and 34-kD components were precipitated from extracts of normal fibroblasts, while the latter component was absent from affected cells. Similar differences were found after immunoblotting of membranes from whole normal or affected skin. These data indicate that dermatosparaxis of sheep involves a molecular defect of a collagen-binding protein. Therefore this disease represents a model to study the complex interaction of cells with the extracellular matrix on a molecular level.     

Maximum growing depth of submerged macrophytes in European lakes

Submerged macrophytes are important elements for the structure and functioning of lake ecosystems. In this study, we used chemical and maximum colonisation depth (C_max) data from 12 European countries in order to investigate how suitable C_max may describe the impact by eutrophication. The analyses include data from 757 lakes and 919 lake years covering oligotrophic to eutrophic lakes. Overall, C_max was closely related to Secchi depth (R ² = 0.58) and less closely to chlorophyll a (R ² = 0.31), TP (R ² = 0.31) and total nitrogen, TN (R ² = 0.24). The low coefficients of determination between C_max and nutrient concentrations suggest that other response factors than nutrient-phytoplankton-light conditions are important for C_max and that it will be difficult to establish strong relationships between external nutrient loading and C_max. Yearly monitoring for 13–16 years in eight Danish lakes showed considerable year-to-year variability in C_max, which for the individual lakes only related weakly to changes in Secchi depth. The use of C_max as an eutrophication indicator is especially relevant in not very shallow lakes (maximum depth >4–5 m), not too turbid lakes (C_max >1 m) and not very humic lakes (colour <60 mg Pt/l).