Studies on the structure of NADH: ubiquinone oxidoreductase complex: topography of the subunits of the iron-sulfur protein component

Resolution of the mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) by chaotropic agents result in the separation of three building blocks of the enzyme, designated FP (flavoprotein), IP (iron-sulfur protein), and HP (hydrophobic protein). FP contains three subunits of Mr 51, 24, and 9 kDa; one FMN; and two iron-sulfur clusters. Immunochemical studies with monospecific antibodies to the FP subunits have indicated that all three subunits of FP protrude from the inner mitochondrial membrane on the matrix side, whereas no reactive epitopes from these subunits were found exposed on the cytosolic side [A.-L. Han, T. Yagi, and Y. Hatefi (1988) Arch. Biochem. Biophys. 267, 490-496]. IP contains six subunits of Mr 75, 49, 30, 18, 15, and 13 kDa and four iron-sulfur clusters. In the present study, immunochemical experiments (enzyme-linked immunosorbent assays and 125I-protein A labeling) were carried out with monospecific antibodies to the above IP subunits and with bovine Complex I, submitochondrial particles, mitoplasts, and intact mitochondria as sources of antigens. Results have indicated that all six IP subunits protrude from the inner mitochondrial membrane into the matrix, and that the 75-kDa subunit, and possibly the 15-kDa subunit, protrude in mitoplasts from the cytosolic side as well. No epitopes reactive toward the monospecific antibodies to the 49-, 30-, 18-, and 13-kDa subunits were detected in mitoplasts.     

Resonance Raman studies of Escherichia coli sulfite reductase hemoprotein. 2. Fe4S4 cluster vibrational modes

Resonance Raman (RR) spectra from the hemoprotein subunit of Escherichia coli sulfite reductase (SiR-HP) are examined in the low-frequency (200-500 cm-1) region where Fe-S stretching modes are expected. In spectra obtained with excitation in the siroheme Soret or Q bands, this region is dominated by siroheme modes. Modes assignable to the Fe4S4 cluster are selectively enhanced, however, with excitation at 488.0 or 457.9 nm. The assignments are confirmed by observation of the expected frequency shifts in SiR-HP extracted from E. coli grown on 34S-labeled sulfate. The mode frequencies and isotopic shifts resemble those seen in RR spectra of other Fe4S4 proteins and analogues, but the breathing mode of the cluster at 342 cm-1 is higher than that observed in the other species. Spectra of various ligand complexes of SiR-HP reveal only slight sensitivity of the cluster terminal ligand modes to the presence of exogenous heme ligands, at variance with a model of ligand binding in a bridged mode between heme and cluster. Close examination of RR spectra obtained with siroheme Soret-band excitation reveals additional 34S-sensitive features at 352 and 393 cm-1. These may be attributed to a bridging thiolate ligand.     

Relation of phosphatidylinositol metabolism to glycolytic pathway in skeletal muscle membranes

Skeletal muscle triads are possessing the whole set of enzymes of the phosphatidylinositol (PI)-linked signal generating pathway, PI-kinase, PI(4)P-kinase, and PI(4,5)P₂-phospholipase C (PLC). The activities of these enzymes are comparable to those found in other cell types for which a functional role of the PI-pathway in intracellular signal transduction has been established. For skeletal muscle an unequivocal function and an initiating signal for Ins(1,4,5)P₃-liberation is still unknown. However, the observed Ca-dependency of PLC activity suggests that here Ins(1,4,5)P₃ production is a consequence rather than a cause of increasing cytosolic Ca²⁺. Recently, the glycolytic enzyme aldolase, whose activity can be modulated by inositol polyphosphates, has been localized in the triadic structure. The enzyme which has a high affinity to Ins(1,4)P₂, Ins(1,4,5)P₃ and Ins(1,3,4,5)P₄, seems to be compartmentalized to the junctional foot structure from which it is released upon binding of these molecules. This phenomenon could reflect a capability for regulation of the glycolytic flux even for aldolase, especially if a non steady-state situation in the junctional gap is considered. Meanwhile we have accumulated evidence for the operation of a partial glycolytic sequence in the junctional region established by the enzymes aldolase, glyceraldehyde-3-P (GAP) dehydrogenase and phosphoglycerate kinase. This system is able to produce ATP upon oxidation of GAP and could be, because of the inositol polyphosphate-sensing abilities of aldolase, a target for the membrane associated PI-pathway. The ATP production is however transient which indicates the coupling to an ATP hydrolyzing reaction. Thus, it appears that the ATP produced by the membrane associated system is effectively utilized by an ATP consuming membrane localized system like PI-metabolism or protein kinases. There are indications that exogeneously added ATP does not equilibrate with the ATP synthesized in the junctional region which suggests an effective structural or kinetical compartmentalization of this system. Therefore it is hypothesized that the ATP synthesized by the membrane associated glycolytic sequence is utilized in membrane localized reactions.     

椭圆斜羽叶的解剖研究及斜羽叶的系统分类位置

本文根据对椭圆斜羽叶(Plagiozamites oblongifolius)钙质石化化石的研究,认为其主要特征如下:羽状复叶,叶肉无海绵组织和栅栏组织分化。羽片叶脉维管束外韧式,木质部外始式,与现代苏铁属叶脉特征相近。羽轴维管束为“U”形,外始式木质部、梯状纹孔管胞,与现代苏铁属的羽轴维管束相似。根据上述特征,目前可将斜羽叶属归入原始苏铁类植物。但是,如果斜羽叶的生殖器官同 Noeggerathia 一样,为异孢型孢子囊穗,则斜羽叶可能为一种与苏铁类起源有关的原裸子植物。     

Alzheimer's disease: study of the distribution of tau proteins constituting helical filament pairs in human central nervous tissue

Tau proteins are the major components of Paired Helical Filaments (PHF) of Alzheimer's disease. Using the immunoblot technique and an antiserum against PHF, we have studied the distribution of Tau proteins in the different areas of normal human brains and Alzheimer brains. Tau proteins were clearly present in cortical grey matter but were difficult to detect in the white matter. In Alzheimer brains, we observed two differences: first, there is an important background due to the partial dissociation of the lesions containing Tau aggregates. Second, the profile of Tau proteins is modified, due to abnormal phosphorylation. Thus, Tau proteins are found in large amounts in the grey matter of the cortical areas and are not exclusively distributed in the axonal domain. The normal cortical distribution of Tau in the human brain correlates well with the distribution of histological lesions that contain PHF (neurofibrillary tangles and neuritic plaques) in the Alzheimer cortex.     

In situ hybridization using 32P labelled oligodeoxyribonucleotides for the cellular localisation of mRNA in neuronal and endocrine tissue

Summary Methodological variables for in situ hybridization using ³²P labelled oligodeoxyribonucleotides (oligomers) have been examined. Four different oligomers directed against proglucagon messenger RNA (mRNA) and two different oligomers against prosomatostatin mRNA have been used. Specific hybridization was obtained in adult rat brain, stomach and pancreas and in neonatal rat ileum. Tissue was perfusion fixed with 4% paraformaldehyde 0.2% glutaraldehyde and hybridization was carried out in 50% formamide for 72 h at 42° C. Using hybridization conditions of lower stringency (33% formamide) labelling was also obtained in guinea pig tissue. Other variables which affected hybridization signal intensity were the inclusion of a prehybridization dehydration stage, the probe concentration, the inclusion of ammonium acetate in the posthybridization dehydrating ethanols and in the autoradiographic emulsion, and the exposure time. The localisation of proglucagon mRNA in rat pancreas using a 20mer was used as a model tissue for testing these methodological variables and the results were found generally also to apply to the other probes and tissues tested. The methods described provide single cell resolution and show that ³²P labelled oligomers may be used to localise neuropeptide and endocrine mRNAs in different types of tissue and in different mammalian species.     

Mobilization of calcium from intracellular stores as one of the mechanisms underlying the antiopioid effect of cholecystokinin octapeptide.

In enzymatically dissociated brain cells prepared from neonatal rats, KCl produced a significant increase in [Ca2+]i and this increase could be prevented by verapamil or nifedipine, known to block voltage-sensitive calcium channels. The opioid receptor agonists ohmefentanyl (OMF, mu agonist), [D-Pen2,D-Pen5]enkephalin (DPDPE, delta agonist), and 66A-078 (kappa agonist) produced a marked suppression of the Ca2+ influx induced by high K+ depolarization. The suppressive effect of OMF, DPDPE, and 66A-078 on the high K(+)-induced increase in [Ca2+]i was markedly reversed by their respective antagonists beta-funaltrexamine (beta-FNA), ICI174864, and nor-binaltorphimine (nor-BNI). Cholecystokinin octapeptide (CCK-8), at concentrations of 0.3, 3.0, and 30 nM, dose-dependently mobilized Ca2+ from intracellular stores. While CCK-8 30 nM did not affect significantly the increase of [Ca2+]i following high K+, it did reverse the suppression of the high K(+)-induced increase in [Ca2+]i by the mu agonist OMF and the kappa agonist 66A-078, but not that by the delta agonist DPDPE. The results suggested that while opioid ligands suppress [Ca2+]i by blocking voltage-operated Ca2+ influx, the antiopioid effect of CCK-8 seems to be operated via mobilization of Ca2+ from intracellular stores.     

白腹锦鸡机体营养成分的分析与探讨

本文报道笼养和野生白腹锦鸡机体营养成分及其差异。分析表明,笼养的比野生种营养成分含量高的有:腿肌蛋白质高11％,胸肌、腿肌、全血的氨基酸分别高2.64％,1.39％和4.68％,胸肌、腿肌和肝脏的碳水化合物分别高0.076％、0.092％和3.962％,胸肌和腿肌的维生素A分别高188.63和84.09 I.U.,胸肌和腿肌的维生素D分别高47.2和12.8 I.U.。但是胸肌蛋白质含量笼养的比野生的低26％。     

Gastric effects of thyrotropin-releasing hormone microinjected into the dorsal vagal nucleus in cats

We investigated the gastric acid secretory and motility responses to microinjection of thyrotropin-releasing hormone (TRH) into the dorsal motor nucleus of the vagus (DMV) in anesthetized cats. Gastric acid output was collected every 15 min through a gastric cannula after saline flush and titrated to pH 7.0. Antral and corpus contractions were continuously recorded by extraluminal force transducers. TRH dissolved in 200 nl of saline and microinjected unilaterally into the DMV induced a dose-dependent (50-200 ng) increase in gastric acid secretion. The acid secretory response began in the first 15 min collection and lasted 45 min. TRH frequently increased the force of contractions of the antrum and corpus within one minute of microinjection. The minimal effective dose for eliciting increased motility was lower than for inducing acid secretion. These results demonstrate that TRH acts in the DMV of cats to stimulate gastric acid secretion and contractions.     

Paracrinology of growth regulation

Embryonic and fetal growth is dependent on genetic factors and epigenetic factors such as peptide growth factors. We describe here the interactions of several peptide growth factors during the growth and function of two cell types, growth plate chondrocytes from the ovine fetus and astroglial cells from the newborn rat cerebral cortex. Isolated chondrocytes released two endogenous growth factors, basic fibroblast growth factor (bFGF) and insulin-like growth factor II (IGF II). Although the latter was released in greater abundance, as detected by radioimmunoassay, exogenous bFGF was more than a thousand fold more active as a mitogen. Insulin was also able to increase chondrocyte replication at physiological concentrations, and bFGF, insulin and IGFs were additive in their effects on DNA and protein synthesis. Transforming growth factor beta (TGF beta), which is abundant in bone, had little effect on chondrocyte DNA or total protein synthesis alone, but blocked the stimulatory actions of insulin and IGFs on these parameters. However, TGF beta when alone or in combination caused an increase in the collagen: non collagenous protein ratio of new proteins synthesized by chondrocytes. Adult rat brain is a rich source of IGF II, and both IGF I and II are present during neurogenesis and gliagenesis in the fetal and neonatal rat respectively. We have cultured astroglial cells isolated from neonatal rat cerebral cortex to examine the production and interaction of peptide growth factors during their growth. Isolated astroglial cells contained mRNAs encoding both IGF I and II but abundance was not regulated by other hormones or growth factors. Using affinity cross-linking we found that cultured cells also released two species of IGF binding protein (IGF-BP) of 33 kDa and 38 kDa. Northern blot analysis using homologous cDNA probes showed that astroglial cells expressed IGF-BP2 and BP3, but little BP1. Both IGF I and II were mitogenic for astroglial cells, as was insulin at physiologic concentrations. Exogenous IGF-BP2 was able to modulate the mitogenic actions of exogenous IGF I. These two very different cell models show many similarities of endogenous growth control. Both release IGFs and IGF-BPs which regulate mitogenic rate. In addition, in both insulin functions as a growth factor at physiologic concentrations. These findings suggest common principles governing embryonic and fetal growth and development. Studies have shown that fetal and neonatal growth is independent of regulation by classic hormones (e.g. growth hormones) synthesized by the mother or the fetus. It is believed that embryonic and fetal growth is controlled by two major mechanisms, namely, (i) the genetic factors as determined by the embryonic and fetal genome, and (ii) the epigenetic and environmental factors that alter the expression of the embryonic or fetal genome.(ABSTRACT TRUNCATED AT 400 WORDS)