Colchicine Neurotoxicity Demonstrates the Cholinergic Projection from the Supracommissural Septum to the Habenula and the Nucleus Interpeduncularis in the Rat

Colchicine injections in the supracommissural septum of the rat caused degeneration of several neurons in the nucleus triangularis septi and the nucleus septofimbrialis. The lesions resulted in significant decreases of choline acetyltransferase in the habenula (-34%) and in the nucleus interpeduncularis (-36%), thus demonstrating the existence of a major cholinergic projection to these nuclei from the supracommissural septum. A large fall in choline acetyltransferase was also noticed in the dorsal hippocampus as a consequence of colchicine damage to the fimbria-fornix fibers crossing the injected area.     

Biochemical Properties and Substrate Specificities of a Recombinantly Produced Azotobacter vinelandii Alginate Lyase

Alginate is a polysaccharide composed of β-d-mannuronic acid (M) and α-l-guluronic acid (G). An Azotobacter vinelandii alginate lyase gene, algL, was cloned, sequenced, and expressed in Escherichia coli. The deduced molecular mass of the corresponding protein is 41.4 kDa, but a signal peptide is cleaved off, leaving a mature protein of 39 kDa. Sixty-three percent of the amino acids in this mature protein are identical to those in AlgL from Pseudomonas aeruginosa. AlgL was partially purified, and the activity was found to be optimal at a pH of 8.1 to 8.4 and at 0.35 M NaCl. Divalent cations are not necessary for activity. The pI of the enzyme is 5.1. When an alginate rich in mannuronic acid was used as the substrate, the K_m was found to be 4.6 × 10⁻⁴ M (sugar residues). AlgL was found to cleave M-M and M-G bonds but not G-M or G-G bonds. Bonds involving acetylated residues were also cleaved, but this activity may be sensitive to the extent of acetylation.

Alginate is a family of 1-4-linked copolymers of β-d-mannuronic acid (M) and α-l-guluronic acid (G). It is produced by brown algae and by some bacteria belonging to the genera Azotobacter and Pseudomonas (8, 17, 18, 31). The polymer is widely used in industry and biotechnology (36, 44), and the genetics of its biosynthesis in Pseudomonas aeruginosa has been extensively studied due to its role in the disease cystic fibrosis (33). In bacterial alginates, some of the M residues may be O-2- and/or O-3-acetylated (42). The polymer is initially synthesized as mannuronan, and the G residues are introduced at the polymer level by mannuronan C-5-epimerases (13, 22, 23). The epimerized alginates contain a mixture of blocks of consecutive G residues (G blocks), consecutive M residues (M blocks), and alternating M and G residues (MG blocks). Alginates from Pseudomonas sp. do not contain G blocks (42).Alginate lyases catalyze the depolymerization of alginates by β-elimination, generating a molecule containing 4-deoxy-l-erythro-hex-4-enepyranosyluronate at the nonreducing end. Such lyases have been found in organisms using alginate as a carbon source, in bacteriophages specific for alginate-producing organisms, and in alginate-producing bacteria (45). An alginate molecule may contain four different glycosidic bonds, M-M, G-M, M-G, or G-G, and the relative rates at which each of these bonds are cleaved vary among different lyases (36a). The lyases also differ in the extent to which they are affected by acetylation (35, 43, 46).Davidson et al. (10) described an Azotobacter vinelandii lyase which preferred M blocks as a substrate. Kennedy et al. (28) later reported the purification of periplasmic alginate lyases from A. vinelandii and from Azotobacter chroococcum which also seemed to prefer deacetylated, M-rich alginate. The activities of these enzymes were found to be optimal at pH 6.8 and 7.2, respectively, while the enzyme reported by Davidson et al. (10) was found to display optimal activity at pH 7.8.A gene, algL, encoding an alginate lyase has been cloned from P. aeruginosa (2, 41). The gene was found to be located in a cluster containing most of the genes necessary for the biosynthesis of alginate. A homologous gene cluster has recently been identified in A. vinelandii (38) and shown to encode an alginate lyase (32). In our previous report, we showed that plasmid pHE102, which contains a part of this gene cluster, contains a DNA sequence sharing homology with algL from P. aeruginosa (38). We have now subcloned, sequenced, and expressed this gene in Escherichia coli. The lyase was shown to preferentially cleave deacetylated M-M and M-G bonds, but acetylated substrates were also cleaved.     

Comparison of the Properties of γ-Aminobutyric Acid and L-Glutamate Uptake into Synaptic Vesicles Isolated from Rat Brain

Rat brain synaptic vesicles exhibit ATP-dependent uptake of gamma-[3H]amino-n-butyric acid ([3H]GABA) and L-[3H]glutamate. After hypotonic shock, the highest specific activities of uptake of both L-glutamate and GABA were recovered in the 0.4 M fraction of a sucrose gradient. The uptakes of L-glutamate and GABA were inhibited by similar, but not identical, concentrations of the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone and the ionophores nigericin and gramicidin, but they were not inhibited by the K+ carrier valinomycin. N,N'-Dicyclohexyl-carbodiimide and N-ethylmaleimide, Mg2+-ATPase inhibitors, inhibited the GABA and L-glutamate uptakes similarly. Low concentrations of Cl- stimulated the vesicular uptake of L-glutamate but not that of GABA. The uptakes of both L-glutamate and GABA were inhibited by high concentrations of Cl-. These results indicate that the vesicular GABA and L-glutamate uptakes are driven by an electrochemical proton gradient generated by a similar Mg2+-ATPase. The vesicular uptake mechanisms are discussed in relation to other vesicle uptake systems.     

Quantitation of RNA polymerase subunits in Escherichia coli during exponential growth and after bacteriophage T4 infection

Summary A method was developed to measure the amounts of RNA polymerase subunits, , , and in crude extracts of Escherichia coli. The proteins were labelled by growing the cells in ³⁵S-sulphate containing media. For measuring and , the cell lysate was electrophoresed on 6% polyacrylamide gels containing SDS and the and bands cut out and counted. For measuring and , the cell lysate was co-electrophoresed with dansylated RNA polymerase on 8% polyacrylamide gels containing SDS. The fluorescent bands were cut out, the proteins eluted, and the and subunits further purified on polyacrylamide gels containing 8 molar urea.The results are: (1) is the subunit of the core RNA polymerase which is present in limiting amount. (2) The core enzyme, as measured by , constitutes a constant fraction of total cellular protein (0.9%), independent of the bacterial growth rate. (3) The subunit is made in excess and is probably regulated independently. (4) The subunit is present in 0.3–0.4 times the amount of the core enzyme. (5) All four subunits are fully conserved after bacteriophage T4 infection.     

Nitric oxide donors selectively potentiate thrombin-stimulated p70(S6k) activity and morphological changes in Swiss 3T3 cells

Thrombin stimulates both DNA synthesis and cell morphological changes in Swiss 3T3 cells, although the mechanism of signal coordination leading to these responses is unknown. We report here that nitric oxide (NO) donors selectively enhance thrombin-stimulated p70(S6k) activity by 40-60%, an effect that was sustained for 24 h. Potentiation of p70(S6k) also was observed with cGMP analogues indicating that this effect is mediated by cGMP-activated protein kinase. NO donors also induced morphological changes characterized by spindle-shaped cells in confluent, nondividing cells or by extended protrusions from the trailing edge in subconfluent, polarized cells. NO donors had no significant effects on intracellular Ca(2+) mobilization, DNA synthesis, proliferation, or ERKs 1 and 2 and p90RSK activities, indicating that mitogenic responses and cell division are not altered by NO donors. We conclude that NO donors modulate the morphological changes associated with cellular motility in response to thrombin stimulation through selective enhancement of p70(S6k) activity.     

Influence of experimental set-up and methodology for measurements of metabolic rates and critical swimming speed in Atlantic salmon Salmo salar

In this study, swim-tunnel respirometry was performed on Atlantic salmon Salmo salar post-smolts in a 90 l respirometer on individuals and compared with groups or individuals of similar sizes tested in a 1905 l respirometer, to determine if differences between set-ups and protocols exist. Standard metabolic rate (SMR) derived from the lowest oxygen uptake rate cycles over a 20 h period was statistically similar to SMR derived from back extrapolating to zero swim speed. However, maximum metabolic rate (MMR) estimates varied significantly between swimming at maximum speed, following an exhaustive chase protocol and during confinement stress. Most notably, the mean (±SE) MMR was 511 ± 15 mg O₂ kg⁻¹ h⁻¹ in the swim test which was 52% higher compared with 337 ± 9 mg O₂ kg⁻¹ in the chase protocol, showing that the latter approach causes a substantial underestimation. Performing group respirometry in the larger swim tunnel provided statistically similar estimates of SMR and MMR as for individual fish tested in the smaller tunnel. While we hypothesised a larger swim section and swimming in groups would improve swimming performance, U_crit was statistically similar between both set-ups and statistically similar between swimming alone v. swimming in groups in the larger set-up, suggesting that this species does not benefit hydrodynamically from swimming in a school in these conditions. Different methods and set-ups have their own respective limitations and advantages depending on the questions being addressed, the time available, the number of replicates required and if supplementary samplings such as blood or gill tissues are needed. Hence, method choice should be carefully considered when planning experiments and when comparing previous studies.     

Membrane and membrane-associated proteins in Triton X-114 extracts of Mycobacterium bovis BCG identified using a combination of gel-based and gel-free fractionation strategies

Tuberculosis is an ancient disease that remains a significant global health problem. Because many membrane and membrane-associated proteins of this pathogen represent potential targets for drugs, diagnostic probes or vaccine components, we have analysed Mycobacterium bovis, bacillus Calmette-Guérin (BCG) substrain Moreau, using Triton X-114 for extraction of lipophilic proteins, followed by identification with LC coupled MS/MS. We identified 351 different proteins in total, and 103 (29%) were predicted as integral membrane proteins with at least one predicted transmembrane region and another 84 (23.9%) proteins had a positive grand average of hydropathicity (GRAVY) value, indicating increased probability for membrane association. Altogether 43 predicted lipoproteins (Lpps) were identified which is close to 50% of the total number of Lpps in the genome. Fifty-four proteins, including twenty-four predicted integral membrane proteins and seven predicted Lpps are described for the first time. The proportion of hydrophobic membrane and membrane-associated proteins shows that Triton X-114 is a highly efficient method for extraction of membrane proteins from bacteria, without the need for preisolation of membranes. ATP synthase, NAD(P) transhydrogenase, ubiquinone oxidoreductase and ubiquinol-cytochrome C reductase appear to represent major enzyme complexes in the membrane of Mycobacterium tuberculosis complex organisms.     

Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation,platelet aggregation,and P-selectin expression

In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.