Bacterial Activity Associated with the Decomposition of Woody Substrates in a Stream Sediment

Ground bark and heartwood from Alnus rubra and Pseudotsuga menziesii were added to a muddy sediment from a small Oregon stream and incubated in situ. Carbon dioxide and methane production rates were increased by all amendments, the biggest increase being shown with A. rubra wood. Except for sediment amended with A. rubra wood, nitrogen fixation rates from all treatments (including the control) were approximately 0.1 nmol/g per h throughout the 6-month study period. Contrary to expectations, neither bark had a noticeable adverse effect on microbial activity, but the A. rubra wood promoted nitrogen fixation. These results help to explain the faster rate of decomposition of A. rubra wood in water compared with that of P. menziesii described in the literature. The uptake kinetics of glucose (V_max) did not follow the same pattern as gas evolution.     

Stimulation of eicosapentaenoic acid metabolism in washed human platelets by 12-hydroperoxyeicosatetraenoic acid

Washed human platelets were not able to convert eicosapentaenoic acid (EPA) to thromboxane B3 (TXB3) and 12-hydroxyeicosapentaenoic acid (AA) to washed human platelets induced conversion of EPA to TXB3 and 12-HEPE. Esculetin, a specific inhibitor of 12-lipoxygenase, prevented the effect of AA, but cyclooxygenase inhibitor did not. The conversion of AA to TXB2 was not affected by the same dose of esculetin. These data suggest that products of AA formed by 12-lipoxygenase in human platelets have stimulatory effects on EPA metabolism. When AA was preincubated with washed human platelets, its effect on EPA conversion was reduced, suggesting that a labile product of AA formed by 12-lipoxygenase is involved in the facilitation of EPA metabolism. Addition of 12-hydroperoxyeicosatetraenoic acid directly to washed human platelets caused dose-dependent synthesis of TXB3 and 12-HEPE, while addition of 12-hydroxyeicosatetraenoic acid had no effect. Thus, 12-hydroperoxyeicosatetraenoic acid formed from AA promotes the metabolism of EPA in washed human platelets.     

Growth Regulation in Dwarf Barley Coleoptiles by the Minor Cell Wall Components, Galactose and Mannose

Sugar compositions of cell walls of dark-grown coleoptiles from12 barley strains, 11 of which were coleoptilar semi-dwarf strains,were analyzed on days 2 and 3 after germination. Major wallcomponents of the 12 strains were arabinose, xylose, and glucosein hemicellulose and cellulose; minor components were galactoseand mannose. The sugar content of each wall component per unit length wasnot correlated to any growth parameters calculated from a logisticequation simulating coleoptile growth, but the relative contentsof galactose and mannose in relation to the total wall sugarcontent was correlated to the growth rate on day 3 and the growthcontinuing period. These facts suggest that growth of these12 barley strains in the late growth stage is regulated by theminor wall components, galactose and mannose. ¹ Dedicated to the late Professor Joji Ashida. (Received October 12, 1982; Accepted January 12, 1983)     

Dichroism of bacteriochlorophyll in cells of the photosynthetic bacterium Rhodopseudomonas palustris

1. The dichroism was measured at varied wavelengths of polarized light in “intact” cells of Rhodopseudomonas palustris and in films of air-dried lamellar fragments of the bacterium.     

Localization of Myoglobin in Striated Muscle of the Domestic Pig; Benzidine and NADH2-TR Reactions

Tissue blocks 1 cm³ from longissimus (white) and trapezius (red) muscles of adult pigs were fixed in phosphate-buffered 2.5% glutaraldehyde, pH 7.4, for 4 hr at about 25 C; washed 4 hr in running tap water, and immersed in 30% w/v sucrose solution for 16 hr or more. After freezing in liquid N₂, cryostat sections were cut and floated into saturated aqueous benzidine containing 0.15% H₂O₂ at 25 C for 30 min. Stained sections were washed in distilled water and mounted on slides with glycerol jelly. Three distinguishable gradiations of color intensity were found: strong, intermediate, and negative. The trapezius had a greater number of myoglobin-positive fibers than the longissimus muscle. Myoglobin-positive and myoglobin-negative staining occurred in red and white fibers, respectively; intermediates were apparently more closely related to the red than to the white fibers. The NADH₂TR reaction showed the same sites as did the benzidine reaction.     

Elektronenmikroskopische Untersuchungen am Nucleus praeopticus der Kröte (Bufo vulgaris formosus)

Zusammenfassung Die Feinstruktur der neurosekretorischen Nervenzellen des Nucleus praeopticus magnocellularis der Kröte (Bufo vulgaris formosus) und ihre Umgebung wurde untersucht.Die neurosekretorischen Zellen enthalten drei Arten von osmiophilen Gebilden: die neurosekretorischen Elementargranula, die neurosekretorischen Kügelchen und die Einschlußkörper.Die neurosekretorischen Elementargranula besitzen einen Durchmesser von 1000–3000 Å, durchschnittlich von 1300–1500 Å. Sie entstehen im Golgi-Apparat (Perikaryon) der betreffenden Zellen wie bei den schon beschriebenen anderen Tierarten.Die neurosekretorischen Kügelchen haben einen Durchmesser von 4000 Å bis zu mehreren . Sie kommen zuerst in den Ergastoplasmacisternen des Perikaryons vor und wandern dann innerhalb des Axons caudalwärts ab, ebenso wie die Elementargraunla, verlieren sich aber vor dem Erreichen der Neurohypophyse. Nach Lage und Gestalt entsprechen sie den Kolloidtropfen, die von vielen Lichtmikroskopikern für die neurosekretorischen Zellen niederer Vertebraten beschrieben wurden.Die Einschlußkörper treten vornehmlich im zentralen Bezirk des Perikaryons in Erscheinung. Sie sind so groß wie die Mitochondrien und besitzen verschiedene Innenstrukturen. Auf Grund der Struktur und der histochemischen Reaktion möchten wir diese Einschlußkörper den Lipofuscingranula mit saurer Phosphatase zuordnen.Die neurosekretorischen Nervenzellen schmiegen sich an den die Kapillare umgebenden Perivaskularraum unmittelbar an, innerhalb dessen die Basalmembran unvollkommen ausgebildet ist oder ganz fehlt.Stellenweise dehnt sich ein Abschnitt des Endothels durch den Perivaskularraum hindurch entlang der Außenfläche des Perivaskularraums aus, wobei sich die Endothelzellen der Kapillare und die neurosekretorischen Nervenzellen direkt berühren können. Eine poröse Bauweise des Endothels wurde nicht nachgewiesen. Zwischen den Ependymzellen des III. Ventrikels und den darunterliegenden neurosekretorischen Nervenzellen sind oftmals auffallend große Extrazellularräume zu beobachten, die durch den Spaltraum der benachbarten Ependymzellen mit dem Ventrikellumen kommunizieren. Sie enthalten mikrovilliartige Ausläufer der Ependymzellen und die geschilderten, neurosekretorische Bildungen führenden Axone. Eine Ausstoßung dieser Axone in den Ventrikel wurde nicht festgestellt.Diese Untersuchung wurde zum Teil mit finanzieller Unterstützung durch das Japanische Unterrichtsministerium im Jahre 1963 durchgeführt.Der kurze Inhalt dieser Arbeit wurde unter dem Thema 'Electron microscopic studies on the praeoptic nucleus in the toad am 5. und 6. September 1963 auf dem Kongreß für Endokrinologie in Gunma, Japan, vorgetragen.     

Improved maintenance of adult rat hepatocytes in a new serum-free medium in the presence or absence of barbiturates

Summary For serum-free primary culture of adult rat hepatocytes, a synthetic medium DM-160 and rat-tail collagen were selected for the basal medium and for the culture substratum, respectively. Barbiturates, such as phenobarbital and 1-ethyl-5-isobutylbarbiturate, efficiently supported survival of hepatocytes and maintained their morphologic features at lower concentrations under the serum-free conditions than under the serum-supplemented conditions. However, the hepatocyte survival rates under the serum-free conditions were lower than those under the serum-supplemented conditions in the presence or absence of barbiturates. Supplementation of the basal medium with a combination of five groups of factors (5Fs), such as eight amino acids (Ala, Arg, Gly, Ile, Met, Phe, Pro, and Trp), two unsaturated fatty acids (linoleate and oleate), a protease inhibitor (aprotinin), three vitamins (A, C, and E), and five trace elements (Mn, Fe, Cu, Zn, and Se), improved the hepatocyte survival under the serum-free conditions in the presence or absence of barbiturates. In other words, the serum could be completely substituted by the 5Fs. Hepatocyte cultures maintained in the 5Fs-suppelemented basal medium showed excellent induction of tyrosine aminotransferase activity in response to dexamethasone in the presence or absence of barbiturates. The efficiency of the 5Fs-supplemented basal medium for maintaining hepatocytes was not inferior to those of other media in common use with hepatocytes, such as Williams' medium E and Waymouth's medium MB-752/1. In conclusion, maintenance of functional hepatocytes in serum-free primary culture could be improved by use of the new medium preparation (the 5Fs-supplemented DM-160) in the presence of barbiturates. This work was supported by a grant no. 61771923 from the Ministry of Education, Science and Culture of Japan.     

The beta subunit of tryptophan synthase. Clarification of the roles of histidine 86, lysine 87, arginine 148, cysteine 170, and cysteine 230

Our studies, which are aimed at understanding the catalytic mechanism of the beta subunit of tryptophan synthase from Salmonella typhimurium, use site-directed mutagenesis to clarify the functional roles of several putative active site residues. Although previous chemical modification studies have suggested that histidine 86, arginine 148, and cysteine 230 are essential residues in the beta subunit, our present findings that beta subunits with single amino acid replacements at these positions have partial activity show that these 3 residues are not essential for catalysis or substrate binding. These conclusions are consistent with the recently determined three-dimensional structure of the tryptophan synthase alpha 2 beta 2 complex. Amino acid substitution of lysine 87, which forms a Schiff base with pyridoxal phosphate in the wild type beta subunit, yields an inactive form of the beta subunit which binds alpha subunit, pyridoxal phosphate, and L-serine. We also report a rapid and efficient method for purifying wild type and mutant forms of the alpha 2 beta 2 complex from S. typhimurium from an improved enzyme source. The enzyme, which is produced by a multicopy plasmid encoding the trpA and trpB genes of S. typhimurium expressed in Escherichia coli, is crystallized from crude extracts by the addition of 6% poly(ethylene glycol) 8000 and 5 mM spermine. This new method is also used in the accompanying paper to purify nine alpha 2 beta 2 complexes containing mutant forms of the alpha subunit.