A serotonergic system in the brain of the Antarctic fish, Trematomus bernacchii

The immunohistochemical distribution of serotonin-containing nerve fibres and cells has been described in the brain of the Antarctic fish, Trematomus bernacchii. The largest serotonergic system was associated with the diencephalic and rhombencephalic ventricles. In particular, serotonin-positive cells have been found in the lateral recess and neuropile zone of the diencephalic ventricle, where we have identified the serotonergic portion of the paraventricular organ. Numerous serotonin cells were localized in the dorsal nucleus of the raphe, the dorsal tegmental nucleus and the central gray. Two large cell groups, arranged in a pair of well-defined columns and connecting the central gray with the dorsal reticular formation, were immunostained in the region of the trigeminal nuclei. In addition, few positive cells have been found in the preoptic area and the cerebellar valvula, and few serotonergic nerve fibres, probably belonging to the lateral lemniscus, have been identified. The distribution of serotonin elements in the brain of T. bernacchii has been compared with that described in other fish, where it showed some modifications in the immunoreactive pattern. Finally, the lack of a serotonergic system at the level of the reticular superior formation has been reported; however, it was not possible to rule out a phylogenetic or environmental explanation.     

Entry of [(1,2-13C2)acetyl]-L-carnitine in liver tricarboxylic acid cycle and lipogenesis: a study by 13C NMR spectroscopy in conscious, freely moving rats.

The biochemical pathways involved in acetyl-L-carnitine utilization were investigated in conscious, freely moving rats by 13C NMR spectroscopy. Following 4-h [(1,2-13C2)acetyl]-L-carnitine infusion in fasted animals, the free carnitine levels in serum were increased, and an efflux of unlabelled acetyl-L-carnitine from tissues was observed. [(1,2-13C2)Acetyl]-L-carnitine was found to enter biosynthetic pathways in liver, and the acetyl moiety was incorporated into both cholesterol and 3-hydroxybutyrate carbon skeleton. In accord with the entry of [(1,2-13C2)acetyl]-L-carnitine in the mitochondrial acetylCoA pool associated with tricarboxylic acid cycle, the 13C label was also found in liver glutamate, glutamine, and glutathione. The analysis of the 13C-labelling pattern in 3-hydroxybutyrate and cholesterol carbon skeleton provided evidence that the acetyl-L-carnitine-derived acetylCoA pool used for ketone bodies synthesis in mitochondria was homogeneous, whereas cholesterol was synthesized from two different acetylCoA pools located in the extra- and intramitochondrial compartment, respectively. Furthermore, cholesterol molecules were shown to be preferentially synthesized by the metabolic route involving the direct channelling of CoA-activated mitochondria-derived ketone bodies into 3-hydroxy-3-methylglutarylCoA pathway, prior to equilibration of their acyl groups with extramitochondrial acetylCoA pool via acetoacetylCoA thiolase.     

C-terminal peptide identification by fast atom bombardment mass spectrometry.

A previously described technique [Rose, Simona, Offord, Prior, Otto & Thatcher (1983) Biochem. J. 215, 273-277] permits the identification of the C-terminal peptide of a protein as the only peptide that does not incorporate any 18O upon partial enzymic hydrolysis in 18O-labelled water. Formation of chemical derivatives followed by combined g.l.c.-m.s. was used in this earlier work. We now describe the isolation from protein digests, by reversed-phase h.p.l.c., of labelled and unlabelled polypeptides and their direct analysis by fast atom bombardment mass spectrometry. Under the conditions used, the 18O label is retained throughout the separation and analysis, thus permitting assignments of C-terminal peptides to be made. Enzyme-catalysed exchange of label into the terminal carboxy group was found to occur in some cases without hydrolysis of a peptide bond. This effect, which may be exploited to prepare labelled peptides, does not prevent application of the method (two separate digests must then be used). We have applied our method to the analysis of enzymic partial hydrolysates of glucagon, insulin and of several proteins produced by expression of recombinant DNA.     

On the association of DNA polymerase alpha activity with the nuclear matrix in HeLa cells

The association of DNA polymerase alpha activity with the nuclear matrix has been reinvestigated in HeLa cells. Isolated nuclei were extracted with 2M NaCl and then digested with Dnase I and the final structures were recovered by centrifugation through a sucrose cushion. Typically over 98% of the total DNA synthesized in the matrix fraction on either endogenous matrix-associated DNA or activated calf thymus DNA was due to DNA polymerase alpha as defined by inhibition to n-ethylmaleimide or aphidicolin. DNA polymerase beta activity was absent or recovered in only trace amounts. Matrix-bound DNA polymerase alpha activity demonstrated a remarkable degree of stability: DNA synthesis was essentially linear up to 3 hours at 37 degrees C. Overall, these results substantiate previous findings from regenerating rat liver, unlike other data obtained from tissue culture cells.     

Protonated state of methotrexate, trimethoprim, and pyrimethamine bound to dihydrofolate reductase

13C nuclear magnetic resonance (NMR) of methotrexate, trimethoprim, and pyrimethamine enriched 90% with 13C at C2 has provided a sensitive means of detecting the state of protonation of the heterocyclic rings of these inhibitors. In each case, protonation of N1 causes an upfield movement of the chemical shift of C2 by more than 6 ppm. By this method it has been shown that, at pH values up to 9.2, methotrexate is bound to bovine liver dihydrofolate reductase with N1 of the inhibitor protonated, just as in the case of the complex with reductase from Streptococcus faecium and Lactobacillus casei. Furthermore, trimethoprim bound to reductase from any of the three sources, and pyrimethamine bound to either of the bacterial reductases also have N1 protonated even at pH values up to 10. This implies that in all cases there is a strong interaction between protonated N1 of the inhibitor and the carboxylate group of the active site aspartate or glutamate. In every case pKa of the bound inhibitor is increased by several units, a finding in accord with crystallographic evidence that inhibitor bound to L. casei reductase is in a hydrophobic environment and that N1 is not hydrogen-bonded to water. It was confirmed by titration of protein fluorescence that trimethoprim has greater affinity for bacterial reductase than for vertebrate (bovine) reductase, and that this selectivity is more marked in ternary complexes in which NADPH is also bound to the active site. However, the data cited above indicate that this difference in affinities is not due to a weaker ionic interaction between protonated N1 of trimethoprim and the bovine enzyme. Instead, binding of the trimethoprim side chain to hydrophobic sites on the enzyme must provide less binding energy in the case of the mammalian enzyme.     

The native state of apomyoglobin described by proton NMR spectroscopy: interaction with the paramagnetic probe HyTEMPO and the fluorescent dye ANS.

Proton NMR experiments were carried out on apomyoglobin from sperm whale and horse skeletal muscle. Two small molecules, the paramagnetic relaxation agent 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO) and the fluorescent dye 8-anilino-1-naphthalenesulfonic acid (ANS), were used to alter and simplify the spectrum. Both were shown to bind in the heme pocket by docking onto the hydrophobic residues lining the distal side. Only 1 extensive region of the apoprotein structure, composed of hydrophobic residues, is not affected by HyTEMPO. It includes the 2 tryptophans (located in the A helix), other nonpolar residues of the A helix and side chains from the E, G, and GH helices. The spectral perturbations induced by ANS allowed assignment of the distal histidine (His-64) in horse apomyoglobin. This residue was previously reported to titrate with a pKa below 5 and tentatively labeled as His-82 on the basis of this value (Cocco MJ, Kao YH, Phillips AT, Lecomte JTJ, 1992, Biochemistry 31:6481-6491). The packing of the side chains and the low pKa of His-64 reinforce the idea that the distal side of the binding site is folded in a manner closely related to that in the holoprotein. ANS was found to sharpen the protein signals and the improvement of the spectral resolution facilitated the assignment of backbone amide resonances. Secondary structure, as manifested in characteristic inter-amide proton NOEs, was detected in the A, B, C, E, G, and H helices. The combined information on the hydrophobic cores and the secondary structure composes an improved representation of the native state of apomyoglobin.     

Multiple attractors,catastrophes and chaos in seasonally perturbed predator-prey communities

A classical predator-prey model is considered in this paper with reference to the case of periodically varying parameters. Six elementary seasonality mechanisms are identified and analysed in detail by means of a continuation technique producing complete bifurcation diagrams. The results show that each elementary mechanism can give rise to multiple attractors and that catastrophic transitions can occur when suitable parameters are slightly changed. Moreover, the two classical routes to chaos, namely, torus destruction and cascade of period doublings, are numerically detected. Since in the case of constant parameters the model cannot have multiple attractors, catastrophes and chaos, the results support the conjecture that seasons can very easily give rise to complex populations dynamics.     

The indris of Anjanaharibe-Sud,northeastern Madagascar

During a short field trip to the Special Reserve of Anjanaharibe-Sud in northeastern Madagascar, data concerning pelage coloration, behavior (especially vocalization), and ecology of indris were collected. Anjanaharibe-Sud is the northernmost locality of indri distribution. In comparison to the better-known indris from the southern part of their distribution, the indris in this region show different pelage coloration. Several types of loud vocalizations are analyzed, based on a small sample of tape recordings. Their song structure is more complicated than previously reported, containing distinct sequences of duetting. Data on behavior and ecology were collected by interviewing guides and local inhabitants. Some information contrasts with reports on the more southern indri populations. The conservation status of indris in Anjanaharibe-Sud and the future of the reserve are outlined.     

Successful synthesis of a glial-specific blood–brain barrier shuttle peptide following a fragment condensation approach on a solid-phase resin

Successful manual synthesis of the TD2.2 peptide acting as a blood–brain barrier shuttle was achieved. TD2.2 was successfully synthesised by sequential condensation of four protected peptide fragments on solid-phase settings, after several unsuccessful attempts using the stepwise approach. These fragments were chosen to minimise the number of demanding amino acids (in terms of coupling, Fmoc removal) in each fragment that are expected to hamper the overall synthetic process. Thus, the hydrophobic amino acids as well as Arg(Pbf) were strategically spread over multiple fragments rather than having them congested in one fragment. This study shows how a peptide that shows big challenges in the synthesis using the common stepwise elongation methodology can be synthesised with an acceptable purity. It also emphasises that choosing the right fragment with certain amino acid constituents is key for a successful synthesis. It is worth highlighting that lower amounts of reagents were required to synthesise the final peptide with an identical purity to that obtained by the automatic synthesiser.