Kinetic and molecular orbital studies on the rate of oxidation of monosubstituted phenols and anilines by horseradish peroxidase compound II

The second-order rate constant (k4) for the oxidation of a series of aromatic donor molecules (monosubstituted phenols and anilines) by horseradish peroxidase (HRP) compound II was examined with a stopped-flow apparatus. The electronic states of these substrates were calculated by an ab initio molecular orbital method. It was found that in both phenols and anilines log k4 values correlate well with the highest occupied molecular orbital (HOMO) energy level and the lowest unoccupied molecular orbital (LUMO) energy level, but not with the net charge or frontier electron density on atoms of these molecules. The HOMO and LUMO energy levels of phenols and anilines further showed linear relationships with Hammett's sigma values with negative slopes. Similar results were obtained in the oxidation of substrates by HRP compound I, except that the rate of reaction was much higher than in the case of HRP compound II. In addition, the rates of oxidation of phenols by compound I or II were found to be about 1000 times higher than those of anilines with similar HOMO energy levels. On the basis of these results, the mechanism of electron transfer from the substrate to the heme iron of HRP compound II is discussed.     

A nucleotide-binding domain of porcine liver annexin VI. Proteolysis of annexin VI labelled with 8-azido-ATP,purification by affinity chromatography on ATP-agarose,and fluorescence studies

Porcine liver annexin VI (AnxVI) of M_r 68.000 is an ATP-binding protein as evidenced by specific and saturable UV-dependent labelling with 8-azido-[-³²P]ATP or the fluorescent analog of ATP, 2-(or 3)-O-(2,4,6-trinitrophenyl)adenosine triphosphate and by binding of AnxVI to ATP-agarose. These characteristics of purified AnxVI were used to identify and characterize preliminary nucleotide-binding domain of the protein. AnxVI labelled with 8-azido-ATP was subjected to limited proteolysis and the proteolytic fragments of AnxVI that retained the covalently-bound nucleotide were separated by means of gel electrophoresis and visualized by exposure of the gel to a phosphor storage screen. It was found that the AnxVI proteolytic fragments of M$$_r 34-36.000 and smaller retained the nucleotide. In a reciprocal experiment, AnxVI was digested with proteolytic enzymes and in an ATP eluate from an ATP-agarose column protein fragments of similar M_r to these labelled with 8-azido-ATP were identified. The extent of AnxVI labelling with 8-azido-ATP and the distribution of proteolytic fragments varied upon calcium concentration. These results lead to the conclusion that there is a nucleotide-binding domain within the AnxVI molecule that is functionally similar to the nucleotide-binding domains of other nucleotide-binding proteins. The nucleotide-binding domain is located close to the tryptophan residue 343 of AnxVI and in close vicinity to the Ca²⁺- and phospholipid-binding sites of the protein. This is confirmed by the observation that the tryptophan fluorescence intensity of AnxVI decreases in the presence of a fluorescence analog of ATP in a calcium-dependent manner, due to the quenching properties of the nucleotide and/or fluorescence energy transfer from AnxVI tryptophan to fluorophore. Both processes were modulated by the presence of phospholipid molecules.     

Chromosome and isoenzyme studies on cells derived from protoplast fusion of Nicotiana glauca with glycine max-Nicotiana glauca cell hybrids

Summary The somatic hybrids of Glycine max (L)Merr.-Nicotiana glauca Grah. exhibited a preferential loss of N. glauca chromosomes. When protoplasts from such hybrid cells were back fused twice to N. glauca protoplasts, a considerable increase in stability of the N. glauca chromosomes was observed. Gel electrophoresis studies of aspartate aminotransferase showed that the chromosome(s) responsible for this enzyme was stabilized in the back fused hybrid cell lines. The data suggest that the back fusion technique described in this study might aid in stabilizing somatic hybrids.NRCC No. 18040     

A critical evaluation of the predicted and X-ray structures of alpha-lactalbumin

The rapidly increasing availability of protein amino-acid sequences, many of which have been determined from the corresponding gene sequences, has intensified interest in the prediction of related protein structures when the three-dimensional structure of another member of the family is known. The study of bovine -Lactalbumin provides a classic example in which the three-dimensional structure was predicted, first by Browneet al. (1969) and later by Warmeet al. (1974), from the three-dimensional structure of hen-egg-white lysozyme (Blakeet al., 1965), taking into account the striking relationship between the amino acid sequences of the two proteins. A comprehensive comparison of these models with the structure of baboon -Lactalbumin derived from X-ray crystallography (Acharyaet al., 1989) is presented. The models mostly compare well with the experimentally determined structure except in the flexible C-terminal region of the molecule (rms deviation on C of residues 1–95, 1.1 Å).     

The air-blood barrier in the human lung

Summary The air-blood barrier was studied in replicas of freeze-fractured lung biopsies collected from healthy human subjects. Adjacent pneumocytes display a belt-like network composed of 3–7 superimposed ridges (fibrils) on the P face and complementary grooves on the E face, i.e., a structure corresponding to a tight junction. On the other hand, adjacent capillary endothelial cells show a continuous system of 2–4 membrane foldings. These appear mainly as smooth surfaced crests on the P face; on the E face furrows are seen, at the bottom of which a row of particles is situated. This arrangement suggests a leaky type of junction. Discontinuous occluding junctions are located in the pericytic venular segment of the alveolar vessels. The present findings are in agreement with previous physiological and ultrastructural tracer studies locating the main part of the diffusion barrier for small polar solutes and proteins in the alveolar epithelium. Communicating junctions are demonstrated between type I and type II pneumocytes, indicating intercellular cooperation between these cells of common embryonic origin, but which fulfill different functions in the adult. In the endothelium of the non-muscular alveolar vessels communicating junctions are lacking. Desmosomes occur in the epithelium between type I and type II pneumocytes; square arrays of particles characterize the plasma membrane of type I pneumocytes.A portion of this work was presented in partial fulfillment for the degree of Dr. med., Hannover Medical School     

Attenuation of changes in sarcoplasmic reticular gene expression in cardiac hypertrophy by propranolol and verapamil

The prothymosin a kinase (ProTK) is an apparently novel enzyme that is responsible for the phosphorylation of prothymosin (ProT), involved in the proliferation of mammalian cells. The present study investigated the properties of this enzyme. ProTK is more effectively activated by Mn²⁺ than by other divalent cations, and its activity is unaffected by RNA. Its principal substrate in proliferating cells appears to be ProTa. Both in vivo and in vitro, it is unable to phosphorylate the peptides thymosin ₁ and thymosin ₁₁, derived from the amino terminus of ProT, despite the fact that the sites of phosphorylation of ProT are contained within this part of its sequence. In trials in vivo, inhibition of gene expression abolished both phosphorylation of ProT and ProTK activity. ProTK is located in the cytosolic fractions throughout the cell cycle. Its activity, which is dependent on cell proliferation, increases markedly during S phase and begins to decline as the cell enters G2. Studies of the effects of activators and inhibitors of protein kinases involved in signal transduction pathways suggest that ProTK is activated by phosphorylation in a mitogen-initiated pathway that is dependent on PKC; however, PKC does not itself phosphorylate ProTK, which is therefore presumably phosphorylated by another kinase.     

Ultrastruktur des Neurohaemalorgans im Nervus corporis allati II vonAcheta domesticus

Summary InAcheta domesticus the proximal part of the nervus corporis allati II (Nca II) is differentiated as a neurohemal organ und consists of several hundred fiber profiles. The neurosecretory region is confined to the peripheral layer and contains axons with different vesicular inclusions. The liberation of neurohormones is accomplished by exocytosis and the formation of synaptoids. Structures resembling synaptic ribbons were observed in contact with axons, glial profiles and interstitial stroma. The central area of the nerve contains only non-neurosecretory axons of various sizes. Connection with the subesophageal ganglion is attained by only 9 large axons of the central region.Zusammenfassung BeiAcheta domesticus ist der proximale Abschnitt des Nervus corporis allati II (Nca II) als Neurohaemalorgan mit mehreren hundert Faserprofilen ausgebildet. Die neurosekretorische Region ist auf die Peripherie des Nerven begrenzt und enthält Fasern mit unterschiedlichen vesikulären Einschlüssen. Die Freisetzung der Neurohormone erfolgt exocytotisch und durch Bildung von Synaptoiden. Es werden stiftchenförmige synapsenähnliche Kontaktstellen mit Axonen, Gliaprofilen und dem interzellulären Stroma beschrieben. Der zentrale Teil des Nerven führt nicht-neurosekretorische Axone unterschiedlichen Durchmessers, von denen lediglich 9 große Fasern die Verbindung mit dem Unterschlundganglion herstellen.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Synthesis of peptides employing Fmoc-/Boc-/Z-amino acid fluorides and activated commercial zinc dust

The coupling of urethane protected amino acidfluorides is accomplished in the presence ofactivated, commercial zinc dust to synthesize severaldi- and tripeptides.The coupling was fast and racemization free. The yieldas well as purity of the peptides was satisfactory.The method was extended for the incorporation ofsterically hindered ,-dialkylaminoacids and N-methylamino acids as well.     

Rna-mediated regulation of Receptor-Ck gene in human platelets

The study addressed to understand the regulation of Receptor-C_k gene atthe translational level revealed that exogenous cholesterol has the inherentcapacity to regulate the endogenous synthesis of Receptor-C_k by initiatingintracellular targeting of the Receptor-C_k to the mRNP pool within humanplatelets and this effect could be reversed when the platelets wereincubated with cholesterol coupled with either dB cAMP or dB cGMP. Basedupon these observations, we propose that Receptor-C_k initiated signalling,which involves second messengers like PA, cAMP and cGMP, may be responsiblefor the autoregulation of Receptor-C_k gene expression at the translationallevel.     

Slostridium homopropionicum sp. nov., a new strict anaerobe growing with 2-, 3-, or 4-hydroxybutyrate

From anoxic sewage sludge a new strictly anaerobic, spore-forming bacterium was isolated with 2-hydroxybutyrate as sole substrate. 2-, 3-, and 4-hydroxybutyrate, 4-chlorobutyrate, crotonate, vinylacetate, and pyruvate were fermented to acetate and butyrate. Fructose was converted to acetate, butyrate, butanol, and H₂. Lactate and acrylate were fermented to acetate and propionate. Cells pregrown with lactate fermented 2-hydroxybutyrate to butyrate, propionate and acetate. No inorganic electron acceptors were reduced. The DNA base ratio was 32.0±1.0 mol % and was similar to that of Clostridium propionicum, which was determined to be 35.3±0.5 mol %. Strain LuHBu1 is described as type strain of a new species, Clostridium homopropionicum sp. nov. Another isolate obtained from marine sediment degraded 2-and 3-hydroxybutyrate to acetate and butyrate and was in some respects similar to the known species Ilyobacter polytropus.     

Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus

The ability of four exoglycosidases (-galactosidase, -glucosidase, -glucosidase and invertase) from the termite Macrotermes subhyalinus to catalyse tranglycosylation reactions was tested using lactose, cellobiose, maltose and sucrose as glycosyl donors and 2-phenylethanol as glycosyl acceptor. The experimental conditions were optimized in relation to the time course of the reaction, pH and concentrations of glycosyl donor and acceptor. Whereas the hydrolytic activity was largely predominant over the transferase activity with -galactosidase and -glucosidase, the transglycosylation activity represented 68% with -glucosidase. In addition, as demonstrated by the transglycosylation product formed, the hydrolysis of sucrose was catalysed by -glucosidase and not by invertase. On the basis of this work, -glucosidase from M. subhyalinus appears to be a valuable tool for the preparation of neoglycoconjugates.     

Afferent bursting activity of ruff lateral line induced by background noise stimulation

Summary Single unit resting activities were recorded from fibres innervating a neuromast of the supra-orbital canal of the lateral line system of the ruff (Acerina cernua). The interval histogram of 1 of the 4 types of resting activity had a bimodal distribution (bursting activity). The resting activity of these fibres was compared with the measured vibration of the experimental table. The conclusion that the bursting activity is not spontaneous but is caused by small background vibrations of the table was supported by recording of extracellular hair cell responses.Abbreviation ISI Interspike interval     

Induction of cellulose- and xylan-degrading enzyme complex in the yeast Trichosporon cutaneum

The specificity of induction of cellulose- and xylan-degrading enzymes was investigated on the yeast strain Trichosporon cutaneum CCY 30-5-4 using series of compounds structurally related to cellulose and xylan, including monosaccharides, glycosides, glucooligosaccharides and xylooligosaccharides. Determination of activities of secreted cellulase and -xylanase, intracellular, cell wall bound and extracellular -glucosidase and -xylosidase revealed that: (1) The synthesis of xylan-degrading enzymes is induced in the cell only by xylosaccharides, 1,3--xylobiose, 1,2--xylobiose, 1,4--xylosyl-L-arabinose, 1,4--xylobiose and thioxylobiose being the best inducers. The xylan-degrading enzymes show different pattern of development in time and discrete cellular localization, i.e. intracellular -xylosidase precedes extracellular -xylanase. (2) A true cellulase is not inducible by glucosaccharides and cellulose. Negligible constitutive cellulase activity was detected which was about two orders lower than an induced cellulase in the typical cellulolytic fungus Trichoderma reesei QM 9414. (3) The best inducer of intracellular -glucosidase splitting cellobiose was thiocellobiose in a wide range of concentration (0.1–10 mM), whereas xylosaccharides at high concentrations induced -xylosidase of xylobiose type and a non-specific aryl -D-glucosidase.The results were confirmed by growing cells on cellulose and xylan. T. cutaneum was found to be a xylan-voracious yeast, unable to grow on cellulose.     

Influence of field bed position,ground surface color,mycorrhizal fungi,and high root-zone temperature in woody plant container production

High root-zone temperatures can stress plants and reduce nursery productivity of container produced crops. Field studies were conducted to study position of containers in field beds, ground surface color, mycorrhizal fungi and high root-zone temperatures in the production of selected woody plants. Root-zone temperature profiles in containers were established to determine nursery production conditions for white and black ground bed surfaces. White surfaces increased container medium temperatures in beds of plants with open canopies by 2–4°C compared to full canopied plants. Under field conditions with container medium temperatures as high as 40–50°C, the open canopiedBerberis thunbergii DC. Atropurpurea,Pinus eldarica Medw. andBuxus microphylla Seibold and Zucc. were more susceptible to temeprature stress compared to the more close canopiedPittosporum tobira (Thunb.) Ait. Wheeler's dwarf. When compared to controls,P. tobira colonized with mycorrhizal fungi [Glomus etunicatus Baker and Gerd. andGlomus fasciculatum (Thax.sensu Gerd.) Gerd. and Trappe] had increased shoot growth in all bed areas except the western exposure, and increased root growth in western and eastern bed regions. Greatest root damage generally occurred in containers of colonized and noncolonizedB. thunbergii in southern and western bed exposures. Mycorrhizal colonization did not improve plant growth of the more high temperature susceptibleB. thunbergii.     

Repeated DNA sequences isolated by microdissection. II. Comparative analysis in Hordeum vulgare and Triticum aestivum

The genomic organization of two satellite DNA sequences, pHvMWG2314 and pHvMWG2315, of barley (Hordeum vulgare, 2n=14, HH) was studied by comparative in situ hybridization (ISH) and PCR analysis. Both sequences are members of different RsaI families. The sequence pHvMWG2314 is a new satellite element with a monomer unit of 73 bp which is moderately amplified in different grasses and occurs in interstitial clusters on D-genome chromosomes of hexaploid wheat (Triticum aestivum, 2n=42, AABBDD). The 331-bp monomer pHvMWG2315 belongs to a tandemly amplified repetitive sequence family that is present in the Poaceae and preferentially amplified in Aegilops squarrosa (2n=14, DD), H. vulgare and Agropyron elongatum. (2n=14, EE). The first described representative of this family was pAs 1 from Ae. squarrosa. Different sequences of one satellite DNA family were amplified from Ae. squarrosa, A. elongatum and H. vulgare using PCR. Characteristic differences between members of the D and H genome occurred in a variable region which is flanked by two conserved segments. The heterogeneity within this element was exploited for the cytogenetic analysis of Triticeae genomes and chromosomes. Comparative ISH with pHvMWG2315 identified individual wheat and barley chromosomes under low (75%) and high (85%) hybridization stringency in homologous and heterologous systems. We propose the designation Tas330 for the Triticeae amplified sequence (Tas) satellite family with a 330 bp average monomer length.     

The role of macrophages in the tissues regeneration stimulated by the biomaterials

Allogenic grafted tissues are subjected to biodegradation and replaced by the regenerate. To minimize the immune response and improve the rebuilding of tissues there was developed a technology to treat tissues with a cells elimination and dosed out extraction of proteoglycanes (Alloplant^®). With aim to clarify the role of macrophages in the tissues regeneration resulting implantation the biomaterials 112 rats were injected the allogenic and xenogenic (rabbits) pulverized biomaterials in the form of suspension. Injections were performed subcutaneously into the animals back by the base of the tail. The control group (14 rats) were injected a physiologic saline. Animals were killed by ether inhalation on day 2, 4, 7, 14, 30, 90 and 180 and tissue sections were studied by light and electron microscopy. The study showed the key role of the macrophages in resorption of the allogenic biomaterial and formation of the newly-formed tissue. Implantation of the biomaterial induced activity a great number of the mature macrophages, which completely lysed and resorbed the biomaterial particles. Expression TNF was significantly higher whereas expression TGF-1 was significantly lower. With xenogenic biomaterial implantation there were less macrophages, their activity was restricted. Macrophages containing large vacuoles with an active endo- and exocytosis were revealed in the allogenic biomaterial implantation and were named matrix-forming macrophages. We may suppose that these macrophages synthesize (or re-synthesize) proteoglycan component of the newly-formed collagen fibers. There was put forward a hypothesis about the two component mechanism of the collagen fibers formation.     

Tremorgenic mycotoxins produced by strains of Penicillium spp. isolated from toxic Poa huecu parodi

Seventeen strains of Penicillium spp. have been isolated from Poa huecu Parodi from the Zapala zone, exhibiting toxicity to sheet. The following strains have been identified: P. crustosum, cyclopium, notatum, palitans, puberulum, verrucosum, viridicatum and Penicillium spp. The toxigenic capacity of the strains was studied after growing them under suitable conditions. Toxins produced were analysed by thin layer chromatography (TLC). Penitrem A (PA) and Penitrem B (PB) neurotoxins were identified and quantitated in twelve strains; verruculogen (VERR) and fumitremorgen B (FTB) being present in one of them. The effect of these mycotoxins was studied in mice. Neurological symptoms characteristic of the intoxication by tremorgenic toxins and similar to those observed in sheep suffering from huecu's disease were observed. The possible role of these toxins as causative agents of huecu's disease is discussed.