Identification of cDNA clones for ligninase from Phanerochaete chrysosporium using synthetic oligonucleotide probes

Four cDNA clones for ligninase were isolated from the cDNA library (constructed into the PstI site of E. coli vector pUC9) representing 6 day-old lignin degrading culture of Phanerochaete chrysosporium by the use of three synthetic oligonucleotide probes corresponding to partial amino acid sequences of tryptic peptides of the ligninase. Each of the three probes, 14.1, 14.2 and 25, represents a mixture of 32 12- or 14-base long oligonucleotides. Three cDNA clones hybridized with probe 14.1 but not with probe 25 or 14.2, but one cDNA clone hybridized with all of the three probes. Differential hybridization studies showed that these clones are unique to 6-day poly(A) RNA, but not to 2-day poly(A) RNA.     

Characterization and immunohistochemical localization of rat salivary cobalamin-binding protein and comparison with human salivary haptocorrin

Rat saliva contains a cobalamin-binding protein that binds cobalamin as well as cobinamide. The protein binds cobalamin with an affinity constant of 8 X 10(10) l X mol-1, and it binds cobalamin over a more narrow pH range (pH 7.5-10) than does human haptocorrin. It has a Stokes radius of 2.45 nm as compared to the Stokes radius of 4.50 nm for human haptocorrin. Upon isoelectricfocusing it dissociates into four strong bands with pI between 7 and 8, while human haptocorrin dissociates into acid isoproteins. Since human haptocorrin binds to concanavalin A while rat haptocorrin does not, we suggest that rat haptocorrin lacks carbohydrate. The substance concentration of rat saliva haptocorrin is 0.04-12.9 nmol X l-1 (median 7.5 nmol X l-1, n = 9) for control animals. After stimulation with isoproterenol, a beta-adrenergic agent, the substance concentration is 46.4-96.6 nmol X l-1 (median 69.7 nmol X l-1, n = 8). Immunohistochemical studies show haptocorrin in the secretory acini of the submandibular and parotid glands of the rat. In the human submandibular gland, the protein is detected both in the mucous secretory acini and in the intercalated ducts.     

Bone mineral content of limb bones of male weanling rats subjected to 30 and 60 days of simulated increases in body weight

The purpose of this study was to subject groups of male rats each to a specific 10% simulated increase in body weight, ranging from 1.1 to 2.0 g. Constant centrifugation was employed. After 30 and 60 days, rats were sacrificed and perfused with 10% BNF. The humerus, radius, ulna, femur, and tibia were removed, cleared of all soft tissues, weighed, decalcified with EDTA, and reweighed. Bone mineral content (BMC) was determined using the formula: [(Wu - Wd) divided by Wu] X 100. Tukey's multiple range test was used. The data suggest that male weanling rats subjected to simulated increases in body weight, within the range used in this study, undergo enhanced BMC, a bimodal curve describing the relationship between BMC and simulated increases in body weight.     

Carbon isotopic fractionation in heterotrophic microbial metabolism.

Differences in the natural-abundance carbon stable isotopic compositions between products from aerobic cultures of Escherichia coli K-12 were measured. Respired CO2 was 3.4% depleted in 13C relative to the glucose used as the carbon source, whereas the acetate was 12.3% enriched in 13C. The acetate 13C enrichment was solely in the carboxyl group. Even though the total cellular carbon was only 0.6% depleted in 13C, intracellular components exhibited a significant isotopic heterogeneity. The protein and lipid fractions were -1.1 and -2.7%, respectively. Aspartic and glutamic acids were -1.6 and +2.7%, respectively, yet citrate was isotopically identical to the glucose. Probable sites of carbon isotopic fractionation include the enzyme, phosphotransacetylase, and the Krebs cycle.     

Methanogenesis in an Upflow Anaerobic Sludge Blanket Reactor at pH 6 on an Acetate-Propionate Mixture

High-rate anaerobic digestion can be applied in upflow anaerobic sludge blanket reactors for the treatment of various wastewaters. In upflow anaerobic sludge blanket reactors, sludge retention time is increased by a natural immobilization mechanism (viz. the formation of a granular type of sludge). When this sludge is cultivated on acid-containing wastewater, the granules mainly consist of an acetoclastic methanogen resembling Methanothrix soehngenii. This organism grows either in rods or in long filaments. Attempts to cultivate a stable sludge consisting predominantly of Methanosarcina sp. on an acetate-propionate mixture as substrate by lowering the pH from 7.5 during the start-up to approximately 6 failed. After 140 days of continuous operation of the reactor a filamentous organism resembling Methanothrix soehngenii prevailed in the sludge. The specific methanogenic activity of this sludge on acetate-propionate was optimal at pH 6.6 to 6.8 and 7.0 to 7.2, respectively.     

Cell cycle traverse and protein metabolism in human NHIK 3025 cells: the role of anchorage

We have studied the effect of cell anchorage on the human cell line NHIK 3025 in vitro, to see whether the growth regulating effect of cell anchorage primarily affected DNA division cycle or mass growth cycle. It was found that cell to cell anchorage had the same effect on cell cycle progression as anchorage to a solid surface, which indicates that it is anchorage per se and not cell shape that is important for growth control in NHIK 3025 cells. When NHIK 3025 cells were grown without attachment to a solid surface, both G1 and cell cycle duration was prolonged by 6 h, which means that the prolonged cell cycle was due to a prolonged G1. During the first part of the cell cycle the rate of protein synthesis and degradation was constant, and at the same level in cells grown with and without attachment. This means that the prolonged G1 was not due to a reduced protein accumulation or mass growth. Towards the end of the cell cycle protein accumulation was reduced. This effect was either due to a size control before cell division or a secondary effect of the prolonged G1. We therefore conclude that cell anchorage as a growth regulator primarily affects the DNA/cell division cycle.     

Organic free radical levels in seeds and pollen: The effects of hydration and aging

In view of their possible role in oxidative deterioration of seeds and pollen, organic free radicals were measured by electron spin resonance in embryonic axes and cotyledons of soybean [ Glycine max (L.) Merr], embryo and endosperm fractions of corn [ Zea mays L.] and pollen of cattail [ Typha latifolia L.]. A pronounced decline in the radical signal ensued when hydration increased above about 7% (wet weight basis) in both the seed materials and in pollen. Moderate hydration of the soybean axis followed by drying led to a small decrease in organic free radicals compared to untreated material, especially if the desiccation step was performed under nitrogen. In a comparison of soybeans of various ages under normal storage, organic free radical levels in the axis showed little or no increase with age. In marked contrast, over 5 days of accelerated aging at 40°C and near-saturating humidity, organic radical levels approximately doubled in the axis. This pronounced increase in free radical content was not associated with a decrease in the proportion of polyunsaturated fatty acids. The data suggest that hydration of seed and pollen causes a release of free radicals from the trapped state.     

Cloning of genes specifying carbohydrate catabolism in Pseudomonas aeruginosa and Pseudomonas putida.

A 6.0-kilobase EcoRI fragment of the Pseudomonas aeruginosa PAO chromosome containing a cluster of genes specifying carbohydrate catabolism was cloned into the multicopy plasmid pRO1769. The vector contains a unique EcoRI site for cloning within a streptomycin resistance determinant and a selectable gene encoding gentamicin resistance. Mutants of P. aeruginosa PAO transformed with the chimeric plasmid pRO1816 regained the ability to grow on glucose, and the following deficiencies in enzyme or transport activities corresponding to the specific mutations were complemented: glcT1, glucose transport and periplasmic glucose-binding protein; glcK1, glucokinase; and edd-1, 6-phosphogluconate dehydratase. Two other carbohydrate catabolic markers that are cotransducible with glcT1 and edd-1 were not complemented by plasmid pRO1816: zwf-1, glucose-6-phosphate dehydrogenase; and eda-9001, 2-keto-3-deoxy-6-phosphogluconate aldolase. However, all five of these normally inducible activities were expressed at markedly elevated basal levels when transformed cells of prototrophic strain PAO1 were grown without carbohydrate inducer. Vector plasmid pRO1769 had no effect on the expression of these activities in transformed mutant or wild-type cells. Thus, the chromosomal insert in pRO1816 contains the edd and glcK structural genes, at least one gene (glcT) that is essential for expression of the glucose active transport system, and other loci that regulate the expression of the five clustered carbohydrate catabolic genes. The insert in pRO1816 also complemented the edd-1 mutation in a glucose-negative Pseudomonas putida mutant but not the eda-1 defect in another mutant. Moreover, pRO1816 caused the expression of high specific activities of glucokinase, an enzyme that is naturally lacking in these strains of Pseudomonas putida.     

Modulation of [3H]Diazepam Binding in Rat Cortical Membranes by GABAA Agonists

GABAA receptor agonists modulate [3H]diazepam binding in rat cortical membranes with different efficacies. At 23 degrees C, the relative potencies for enhancement of [3H]diazepam binding by agonists parallel their potencies in inhibiting [3H]gamma-aminobutyric acid [( 3H]GABA) binding. The agonist concentrations needed for enhancement of [3H]diazepam binding are up to 35 times higher than for [3H]GABA binding and correspond closely to the concentrations required for displacement of [3H]bicuculline methochloride (BMC) binding. The maximum enhancement of [3H]diazepam varied among agonists: muscimol = GABA greater than isoguvacine greater than 3-aminopropane sulphonic acid (3APS) = imidazoleacetic acid (IAA) greater than 4,5,6,7-tetrahydroisoxazolo (4,5,6)-pyridin-3-ol (THIP) = taurine greater than piperidine 4-sulphonic acid (P4S). At 37 degrees C, the potencies of agonists remained unchanged, but isoguvacine, 3 APS, and THIP acquired efficacies similar to GABA, whereas IAA, taurine, and P4S maintained their partial agonist profiles. At both temperatures the agonist-induced enhancement of [3H]diazepam binding was reversible by bicuculline methobromide and by the steroid GABA antagonist RU 5135. These results stress the importance of studying receptor-receptor interaction under near-physiological conditions and offer an in vitro assay that may predict the agonist status of putative GABA receptor ligands.