Functional disturbances in brain following injury

It was shown previously that local cerebral glucose utilization is less than 50% of normal in all cortical areas of rat brain 3 days following a focal freeze-lesion and that this effect of trauma is significantly diminished by dexamethasone (0.25 mg/Kg/day), and by indomethacin (7.5 mg/Kg single dose). To elucidate the mechanism of action of steroids and non-steroidal antiinflammatory drugs in traumatized brain, the effects of dexamethasone and indomethacin on arachidonic acid release, malondialdehyde production and prostaglandin synthesis in the lesion area were investigated. Five seconds after a freezing lesion arachidonic acid was significantly increased in the lesion area of untreated animals. Neither dexamethasone nor indomethacin had any effect on this release. The thiobarbituric acid reaction, as an estimate of malondialdehyde and non-enzymatic free radical lipoperoxide formation from unsaturated free fatty acids showed no change in the control and lesion areas of untreated and both dexamethasone and indomethacin treated groups. There was a marked increase in PGF2 alpha, PGE2, PGD2 in the lesion area of untreated animals. Indomethacin prevented the formation of prostaglandins by more than 90% while dexamethasone had no effect. These results suggest that some components of the arachidonic acid metabolism must be involved in functional disturbances resulting from trauma while steroid action is mediated in injured brain independently from the prostaglandin cascade.     

Presence of coenzyme M derivatives in the prosthetic group (coenzyme MF430) of methylcoenzyme M reductase from Methanobacterium thermoautotrophicum

2-Mercaptoethanesulfonic acid (coenzyme M), or a derivative of it, and a yellow chromophore, known as the nickel-containing tetrapyrrole factor F₄₃₀, occur in the prosthetic group of methylcoenzyme M reductase in an equimolar amount, and bound to each other; this enzyme catalyzes the final step of methane production. The prosthetic group, which is called coenzyme MF₄₃₀, was isolated from the purified enzyme and was extracted from cells. The presence of coenzyme M was confirmed by a bioassay using Methanobrevibacter ruminantium and by the use of chemical and physicochemical analyses.     

Murine H-2Dd-reactive monoclonal antibodies recognize shared antigenic determinant(s) on human HLA-B7 or HLA-B27 molecules or both

We have evaluated the serological relationships between the murine H-2Dd and human HLA molecules using four H-2Dd-reactive monoclonal antibodies (mAbs) produced in the A.BY (KbIbDb) anti-A.TL (KsIkDd) combination. In the mouse, these reagents exhibited three distinct reactivity patterns: Dd, Ks, and H-2u (mAb 81.L); Dd, H-2p, and H-2u (mAb 81.R); and Dd, Kd, H-2p, H-2u, and H-2v (mAbs 97.G and 97.H). Sequential immunoprecipitation and cross-competitive mAb binding experiments revealed that these mAbs recognized determinants in two spatially distinct polymorphic domains on the H-2Dd molecule of B10.A(5R) cells (defined by mAbs 81.L and 81.R, 97.H, and 97.G, respectively). MAbs 81.R, 97.G, and 97.H, but not 81.L, also defined an HLA-linked polymorphism in the human, the main characteristics of which can be summarized as follows: (i) on B lymphoblastoid cell lines, mAbs 81.R and 97.H bound to cells expressing the HLA-B7, HL-B27 or Bw40 cross-reacting specificities, (ii) on peripheral blood lymphocyte (PBL) panel mAb 81.R exerted C dependent cytotoxicity to 118 of 400 cells tested, including almost all HLA-B7 or HLA-B27 cells or both (r: 0.952), (iii) the expression of the 81.R cross-reacting determinant segregated in an informative family with the parental haplotype carrying the HLA-B7 allele, and (iv) mAbs 81.R, 97.G, and 97.H recognized topologically related determinants on the same class I molecule(s) of the human B lymphoblastoid cells JY (HLA-A2,2, -B7,7). These data support the view that some, but not all H-2Dd allotopes have been conserved throughout evolution and are associated in the human with the HLA-B7, -B27 cross-reacting specificities.     

Early stages of vocal ontogeny in the magpie (Pica pica)

Summary The vocal repertoire of magpie (Pica pica) chicks consists of six calls: Begging Trill (BT), Soft Whistle (SW), Begging Scream (BS), Alarm Call (AC), Distress Call (DC) and Brief Contact Note (BCN). Both BT and SW have a tonal structure and their occurrence is restricted to the nestling period. At fledging, there is a gradual change from BT into BS and a sudden appearance of harsh calls similar to those of adult birds (AC, DC, BCN), without evident transitional forms with preceding tonal calls. Both the existence and the structural design of calls seem to be adapted for providing nestlings with immediate benefits linked to the two major chapters of allocation of parental care. Emission rates of BT increase with hunger motivation under laboratory conditions. Their structure suggests that they are easily located but liable to suffer from environmental degradation. BS of fledglings may be more resistant to degradation, a trait which may facilitate the identification by parents of their own offspring. Both AC and DC attract parents to defend the nest against potential predators, and their structure make them to be easily located and detectable at long distances. BCN are given by fledglings during bouts of locomotory activity (exploration and play) and they probably help in maintaining the cohesion of the group under conditions of poor visibility. In accordance, this call may be fairly located at short distances. The function of SW was unclear. It is given during periods of nestling inactivity between begging bouts, and could be easily elicited by tactile and auditory stimuli. After laboratory experiments, it is concluded that SW serve to indicate parents that nestlings are in good condition, hence to benefit from the parental willingness to invest in a brood with high prospects of survival. Since (i) there is a widespread lack of continuity in the development of adult vocalizations starting from nestling calls, and (ii) nestling calls seem to have evolved to provide birds with benefits in the short-term, these facts argue against the prevailing idea that the main function of calls early in ontogeny is to act as precursors of adult vocalizations.

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Zusammenfassung Das Lautrepertoire von Elsternestlingen besteht aus 6 Lautäußerungen: Betteltrillern (BT), Sanftes Pfeifen (SP), Bettelkreischen (BK), Alarmruf (AR), Angstschreien (AS) und kurzer Kontaktruf (KR). BT und SP sind tonal und treten nur während der Nestlingsperiode auf. Zum Zeitpunkt des Ausfliegens geht BT graduell in BK über und plötzlich treten auch ohne vorausgehende tonale Übergangsformen rauhe Rufe ähnlich denen der Altvögel auf (AR, AS, KR). Sowohl die Existenz als auch die Struktur der Lautäußerungen scheinen als Anpassungen im Zusammenhang mit einer Optimierung der Brutpflege zu interpretieren zu sein. Die Emissionsrate von BT steigt unter Laborbedingungen mit der Hungermotivation. Die Struktur legt nahe, daß BT leicht geortet werden kann, aber auch leicht von der Umgebung verschluckt wird. BS der flüggen Jungen scheint davon weniger betroffen zu sein, so daß die Eltern leichter ihre Jungen identifizieren können. AR und AS veranlassen die Altvögel, ihr Nest gegenüber potentiellen Prädatoren zu verteidigen; die Struktur der Laute begünstigt ihre Ortung und Wahrnehmung über größere Entfernungen. KR werden von den flüggen Jungen bei lebhafter lokomotorischer Aktivität (Exploration, Spiel) geäußert; sie tragen vielleicht dazu bei, die Gruppe auch unter erschwerten optischen Kontaktmöglichkeiten zusammenzuhalten. Die Funktion von SP blieb unklar. SP war während inaktiver Perioden zwischen Bettelverhalten zu hören und konnte leicht durch taktile und akustische Reize ausgelöst werden. Nach Laborexperimenten ist zu schließen, daß SP den Eltern Wohlbefinden der Jungen anzeigt und so Bereitschaft zur Investition in eine Brut mit hoher Überlebenswahrscheinlichkeit fördert. Daß (1) zwischen den Rufen der Altvögel und dem Repertoire der Nestlingen weitgehend keine kontinuierlichen Übergänge festzustellen und (2) Nestlingsrufe offensichtlich im Hinblick auf kuzfristige Gewinne zu interpretieren sind, spricht gegen die allgemein vorherrschende Ansicht, das Lautrepertoire in frühen Stadien der Ontogenie sei hauptsächlich ein Vorläufer der Adultlaute.

     

Lipolysis is a prerequisite for lipid accumulation in HepG2 cells induced by large hypertriglyceridemic very low density lipoproteins.

Lipoprotein kinetic studies have demonstrated that a large proportion of Sf 60-400 very low density lipoprotein (VLDL) is cleared directly from the circulation in Type IV hypertriglyceridemic subjects, at an unknown tissue site. The present studies were designed to investigate the role of hepatocytes in this process and to define the conditions, whereby Type IV Sf 60-400 VLDL would induce lipid accumulation in HepG2 cells. Type IV VLDL (Sf 60-400) failed to augment the total cholesterol, esterified cholesterol, or triglyceride content of HepG2 cells following 24-h incubations. Coincubation of bovine milk lipoprotein lipase (LPL) and Type IV VLDL with HepG2 cells induced a 3-fold increment in cellular esterified cholesterol mass (p less than 0.005) and a 7-fold increase in cellular triglyceride mass (p less than 0.005), compared to VLDL alone. The increased cellular lipid mass was associated with increased oleate incorporation into cellular cholesterol esters and triglycerides. Exogenous LPL hydrolyzed 76% of the VLDL triglyceride over 24 h. LPL action on Type IV VLDL was sufficient to promote cellular uptake of these lipoproteins, while elevated media-free fatty acid levels were not. Although HepG2 cells secrete apolipoprotein (apo) E, we assessed the role of VLDL-associated apoE in the lipid accumulation induced by VLDL plus LPL. ApoE-rich and apoE-poor Type IV VLDL subfractions induced similar increments in cellular esterified cholesterol in the presence of LPL, despite a 4-fold difference in apoE content. Sf 60-400 VLDL, from subjects homozygous for the defective apoE2, plus LPL, behaved identically to Type IV VLDL plus LPL. Type IV VLDL plus LPL, preincubated with anti-apoE (1D7) and apoB (5E11) monoclonal antibodies, known to block the binding of apoE and -B, respectively, to the LDL receptor failed to block lipid accumulation. In contrast, apoE-poor Type IV VLDL, apoE2 VLDL, and VLDL plus 1D7 were taken up poorly by J774 cells, cells that secrete LPL, but not apoE. These studies suggest that lipolytic remodeling of large Type IV VLDL by LPL is a prerequisite for their uptake by HepG2 cells and that HepG2 cell-secreted apoE rather than VLDL-associated apoE is the ligand involved in uptake.     

Purification and characterization of a cytosolic insulin-stimulated serine kinase from rat liver.

A cytosolic insulin-sensitive serine kinase has been purified to apparent homogeneity in parallel from livers of control or acutely insulin-treated rats. The kinase is labile and requires rapid purification for stability. The kinase migrates as a band of apparent Mr = 90,000 on denaturing gels and elutes as a monomer on Superose 12 gel filtration. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renaturation, the 90-kDa band presumed to be the kinase shows kinase activity toward myelin basic protein in situ. Substrates of the kinase include Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide), ribosomal protein S6, S6 peptide, a proline-rich peptide substrate, microtubule-associated protein 2, and myelin basic protein. The kinase also phosphorylates histones H1 and H2B, but does not autophosphorylate to a significant stoichiometry. The activity of the kinase is inhibited by fluoride, glycerophosphate, p-nitrophenyl phosphate, p-nitrophenol, heparin, quercetin, poly-L-lysine, and potassium phosphate, but is unaffected by calcium, cAMP, spermine, protein kinase inhibitor peptide, phorbol myristate acetate, calcium plus phosphatidylserine, or vanadate. The kinase will utilize magnesium (10 mM) as well as manganese (1 mM) as a cofactor for maximal phosphotransferase activity. The kinase is not detected by immunoblotting with antibodies directed against protein kinase C or type II S6 kinase. Taken together, these properties distinguish this kinase from other insulin-sensitive kinases that have been described previously. The purified kinase from livers of insulin-treated rats shows a 5-20-fold higher specific activity compared to enzyme prepared from control rats, suggesting a covalent modification as the mechanism of activation. Incubation of purified, insulin-stimulated kinase with purified phosphatase 2A leads to deactivation of the kinase activity, and the phosphatase inhibitor nitrophenyl phosphate blocks this deactivation. The insulin-activated kinase fails to immunoblot with anti-tyrosine phosphate antibodies. Taken together, these results indicate that insulin activates this novel cytosolic protein kinase by a mechanism that causes its phosphorylation on serine or threonine residues.     

Plant size,geitonogamy and seed set in Ipomopsis aggregata

Summary We used powdered fluorescent dyes to estimate receipt of self vs. outcross pollen in the self-incompatible species Ipomopsis aggregata (Polemoniaceae). Flowers on small and large plants received equal amounts of outcross pollen, whereas flowers on large plants received more self pollen, so the proportion of self pollen delivered through geitonogamy increased with plant size. In natural populations emasculation of all flowers on a plant raised average seed set per flower from 5.19 to 6.99 and also raised fruit set, though not significantly. From these results one expects a negative correlation between plant size and seeds per flower. The opposite trend was observed in a sample of plants in the field, suggesting that deleterious effects of geitonogamy on female fecundity in large plants can be overruled by other factors such as size-related fruit or seed abortion. Results are discussed in relation to the evolution of gynodioecy.     

Cytokinins and leaf development in sweet pepper (Capsicum annuum L.)

Stimulation of leaf expansion by an exogenous cytokinin was studied in isolated leaf discs of sweet pepper with emphasis on the assimilate utilization of the tissue. Leaf discs were floated on solutions containing sucrose and plant growth regulators. Benzyladenine (BA) promoted the area expansion rate of the leaf discs. Sucrose at 100 mM resulted in increased area expansion rate compared with 10 mM sucrose. However, the increased sucrose concentration had no influence on the effect of BA. Over a period of 24 h, treatment with BA did not result in any change of sucrose uptake nor of the partitioning of assimilated carbon in the leaf discs. Neither did BA treatment affect the activity of acid invertase (EC 3.2.1.26) or pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) in the leaf discs. We conclude that the observed promotion of leaf area expansion by exogenous BA is not mediated through the uptake of sucrose or the carbohydrate metabolism of the leaf tissue.Abbreviations BA N⁶-benzyladenine - GA₃ gibberellic acid - PPi-PFK pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) This study was supported by grants from the Danish Research Counsil (SJVF 13-4148 and 13-4547 to P.U. SJVF 13-4146 and 13-4494 to T.H.N.) and from The Research Center for Plant Biotechnology to P.U.