The influence of oxygen concentrations on denitrification in soil

Summary The influence of different O₂ concentrations on denitrification was studied in an agricultural soil. In both nitrate and nitrite amended soil, denitrification was not observed until the O₂ concentration decreased to 0.20 and 0.21 mol/ml, respectively. Denitrification was not observed in soil samples with O₂ concentrations above 0.28 mol/ml in the gas phase. These findings suggest that a completely anoxic environment is not required for denitrification to occur in soil.     

Analysis of the sheep MHC using HLA class I,II, and C4 cDNA probes

Four cDNA probes for the human major histocompatibility complex (MHC) were used to investigate the sheep MHC, in conjunction with serological typing for ovine lymphocyte antigen (OLA). Lymphocytes from a family (two parents and five offspring) of Romanov sheep were subjected to genomic DNA digestion by the restriction endonuclease Eco RI, followed by gel electrophoresis. A single Southern blot representing all seven individuals was then consecutively hybridized with the class I, alpha-DC, beta-DR, and C4 probes, which were originally designed to identify HLA class I, class II (DC and DR), and C4 products, respectively. Using each of the three class I/class II probes, several bands showing DNA polymorphism were detected. The segregation of these bands in the five offspring exactly paralleled the OLA haplotype segregation established by serological typing. A further eight individuals carrying haplotypes which were phenotypically identical to those in the above-mentioned family showed bands in the corresponding positions when tested with the same three probes. Using the C4 probe, no polymorphism was detected in these fifteen individuals.Abbreviations used in this paper MHC major histocompatibility complex - OLA ovine lymphocyte antigen - kbp kilobase pair(s) - MLR mixed lymphocyte reaction - RFLP restriction fragment length polymorphism     

Operation of the glycolate pathway in isolated bundle sheath strands of maize and Panicum maximum

Operation of the glycolate pathway in isolated bundle sheath (BS) strands of two C₄ species was demonstrated from ¹⁴C incorporation into two intermediates, glycine and serine, under conditions favourable for photorespiratory activity. Isolated BS strands fixing ¹⁴CO₂ under light at physiological rates incorporate respectively 3% (Zea mays L., cv. INRA 258) and 7% (Panicum maximum Jacq.) of total ¹⁴C fixed into glycine + serine, at low bicarbonate levels (less than the Km for CO₂ fixation, 0.8 mM). Higher bicarbonate concentrations depressed the percentage of incorporation into the two amino acids. No labelling was observed in the absence of added glutamate. Oxygen was required for glycine + serine labelling, since ¹⁴C incorporation into glycine was largely depressed by argon flushing, and labelling of the two amino acids was nearly suppressed by the addition of the strong reductant, dithionite, especially in maize. Two inhibitors of the glycolate pathway were tested. With α-hydroxypyridine-methanesulfonic acid, an inhibitor of glycolate oxidase, labelling of glycine and serine remained minimal whereas glycolate was accumulated. Isoniazid, an inhibitor of the transformation of glycine to serine induced a 50% increased labelling of glycine in maize BS, and a large decrease in serine labelling. In Panicum, the increase in [¹⁴C]-glycine was 90%. These results suggest that the pathway glycolate → glycine → serine operates in these plants. However, leakage of metabolites occurs in BS cells, especially in maize and a large part of newly formed glycolate, glycine and serine is exported out of the cells. Operation of ribulose-1,5-bisphosphate oxygenase activity in competition with ribulose-1,5-bisphosphate carboxylase is demonstrated by the lowering of total ¹⁴CO₂ fixation when O₂ is increased at low bicarbonate concentration. An interesting feature observed in maize BS, at low bicarbonate concentration, was an increase in ribulose-1,5-bisphosphate labelling when the O₂ level was decreased. This was accompanied by an increase in CO₂ fixation. This could indicate an increased rate in synthesis of ribulose-1,5-bisphosphate (which accumulated) due to a stimulation of ATP synthesis by cyclic photophosphorylation under anaerobic conditions.     

A diffusion model of forest succession

Based on a tree by tree replacement mechanism, a diffusion model of forest stand canopy composition is formulated and analyzed. The model is used to explore composition dichotomies by estimating coefficients from forest stand data and interpreting the results in terms of mechanisms for succession. The model yields a concrete characterization of the succession phenomenon known as the climax state.     

On the O2 flash yields of two cyanophytes

We explored O₂ flash yield in two cyanophytes, Anacystis nidulans and Agmenellum quadruplicatum. On a rate-measuring electrode, a single flash gave a contour of O₂ evolution with a peak at about 10 ms which was maximum (100) for 680 nm background light. On 625 nm illumination the peak was smaller (62) but was followed by an increased tail of O₂ attributed to enhancement of the background. After a period of darkness, repetitive flashes (5 Hz) gave a highly damped initial oscillation in individual flash yields which finally reached steady state at 94% of the yield for 680 nm illumination. When O₂ of repetitive flashes was measured as an integrated flash yield the results was distinctive and similar to that for a continuous light 1 (680 nm). An apparent inhibition of respiration which persisted into the following dark period was taken as evidence for the Kok effect. With a concentration-measuring electrode, integrated flash yield vs. flash rate showed the same nonlinear behavior as O₂ rate vs. intensity of light 1. We draw three conclusions about the two cyanophytes. (a) The plastoquinone pool is substantially reduced in darkness. (b) Because of a high ratio of reaction centers, reaction center 1 / reaction center 2, for the two photoreactions, saturating flashes behave as light 1. (c) Because repetitive flashes are light 1, they also give a Kok effect which must be guarded against in measurements designed to count reaction centers.     

Catecholamine Secretion by Chemically Skinned Cultured Chromaffm Cells

The secretory system of intact chromaffin cells is not accessible to direct chemical manipulation because of the selective permeability of the plasmalemma. We have devised a simple procedure for chemically "skinning" (permeabilizing) cultured adrenal medullary chromaffin cells by brief exposure to the detergent saponin. This procedure disrupts the continuity of the plasmalemma, thus allowing us to bypass those aspects of the secretory process controlled by the cell membrane and giving direct access to exogenous substances to the cellular secretory machinery. We report here that the skinned cells retain a fully competent secretory mechanism dependent only on exogenous calcium and MgATP. Saponin treatment had no significant effect on the total catecholamine content of the cells. Secretion could be initiated by either MgATP or calcium as long as the other was present in the medium. Catecholamine and dopamine-beta-hydroxylase release by the skinned cells was dependent on the calcium concentration of the medium. The ratio of secreted catecholamine and enzyme was similar to that of the cells, indicating that secretion occurred by an exocytotic mechanism. About half the total cellular content of the cytoplasmic enzyme lactic dehydrogenase was released during the permeabilization process and subsequent incubations, indicating plasmalemma permeability to molecules as large as protein. Calcium-induced secretion was unaffected by several drugs known to affect catecholamines and granule function. Saponin treatment of chromaffin cells in culture appears to be a simple means for allowing access to exogenous substances to the cells' secretory machinery. Therefore, it offers the opportunity to use chemical treatments, and perhaps specific antibodies to cellular components, to determine the role of these elements in the secretory process. These techniques should also be applicable to other cells known to secrete by an exocytotic mechanism.     

A calcium antagonist drug binding site in skeletal muscle sarcoplasmic reticulum: Evidence for a calcium channel

The sarcoplasmic reticulum (S.R.) of rabbit skeletal muscle has been found to contain a single, high affinity binding site for the Ca antagonist drug [³H] -nitrendipine. Two subfractions of the reticulum were studied, the heavy (HSR) and light (LSR) preparations, which exhibited similar nitrendipine equilibrium dissociation constants (K_D) of 1nM. Crude cardiac and brain membranes assayed under the same conditions exhibited K_D values of 0.2–0.3nM. The concentration of binding sites per mg. protein (B_max) in HSR was found to be very high, namely 6.7 picomoles/mg, some four times greater than that of LSR. [³H] -nitrendipine binding to HSR was reversible and inhibited by the Ca antagonists flunarizine and verapamil, and by the intracellular Ca release antagonist TMB-8 (8-diethylamino-octyl 3,4,5- trimethylbenzoate hydrochloride). However, unlabelled nitrendipine at 2 × 10^?5M had no effect on contraction of isolated electrically stimulated rabbit lumbrical or rat diaphragm muscles, nor did it affect the neuromuscular junction as studied in rat phrenic nerve-diaphragm preparations. Also, little effect of 2 × 10^?5M nitrendipine was seen on net ⁴⁵Ca uptake by HSR. These results suggest that [³H] -nitrendipine binding to skeletal muscle S.R. resembles that of brain membranes, which also contain a high affinity binding site for [³H] -nitrendipine and which similarly are pharmacologically insensitive to this dihydropyridine type of Ca channel blocking agent. Since HSR is also enriched in calsequestrin and terminal cysternae from which Ca is released in vivo, it seems likely that the [³H]- nitrendipine binding sites in S.R. are associated with Ca channels in the S.R.     

A comparison of the dominant cataract and recessive specific-locus mutation rates induced by treatment of male mice with ethylnitrosourea

Mice were derived from parental males treated with 250 mg ethylnitrosourea per kg body weight. The mice were screened simultaneously for induced dominant cataract and recessive specific-locus mutations. In the spermatogonial treatment group, 16 dominant cataract, 1 dominant corneal opacity and 60 recessive specific-locus mutations were recovered and genetically confirmed in 9352 offspring observed. This lower yield of dominant cataract mutations, when compared with the yield of recessive specific-locus mutations, is similar to results observed by Kratochvilova in a series of experiments on dominant cataract mutations induced by radiation treatment. These results taken with reported results from other dominant mutation test systems, suggest a lower per-locus mutation rate to dominant than to recessive alleles. A corollary to the hypothesis that most dominantly expressed alleles code for an alteration in the function of the normal gene product is that a limited subset of mutations could normally lead to a dominantly expressed mutation. This may explain the lower per-locus mutation rate to dominant than to recessive alleles.

Genetic confirmation tests of recovered presumed dominant cataract mutations indicate that a certain category of phenotypic variants (bilateral, severe or unique lens opacity) is likely to be a true mutation but only represents 7 of the 19 mutations recovered. A second category of phenotypic variants (unilateral, neither severe nor unique lens opacity) has an extremely low probability of being a true mutation. Only 1 confirmed mutation in 181 phenotypic variants was obtained. The remaining category of phenotypic variants (either unilateral severe or unique, or bilateral neither severe nor unique lens opacity) represented the majority, 11, of the confirmed mutations obtained. However, 266 presumed mutations in this category were recovered. If a sub-class of phenotypic variants within this category could be identified that could be ignored owing to a very low probability of being a true mutation, the efficiency of recovery of confirmed dominant cataract mutations would be greatly increased with no sacrifice in the accuracy of the observed mutation rate.

Finally, the 17 confirmed dominant cataract mutations obtained included a class of 7 that produced significantly fewer than the Mendelian expectation of offspring exhibiting the mutant phenotype. This class probably represents both mutations with penetrance effects and mutations with viability effects.

The present experiments represent the first systematic comparison of induced genetically confirmed dominant and recessive mutations for a chemical mutagen in mice. Such results contribute to our limited understanding of the mutation process to dominant alleles.     

Neocarzinostatin chromophore: Presence of a highly strained ether ring and its reaction with mercaptan and sodium borohydride

Spectroscopic evidence suggests the presence of a highly strained ether ring (Fig. 1) (possibly an epoxide) in the C₁₂-subunit of the previously determined partial structure $\text{2a}$ (Fig. 2) of the major neocarzinostatin chromophore (NCS-Chrom $\text{A}$) which completes assignment of all the oxygens in the molecule. The main product from mercaptan treatment suggests opening of the ether ring involving the addition of one molecule of mercaptan as well as reduction of the C₁₂-substructure, whereas a parallel two-step reduction occurs on NaBH₄ treatment. Both reactions occur with rearrangement of the C₁₂-substructure and the implication for the mechanism of action of NCS-Chrom $\text{A}$ in DNA strand scission activity is discussed. The evidence suggests a downward revision of the molecular formula for NCS-Chrom $\text{A}$ as well as minor components $\text{B}$ and $\text{C}$ by two protons.     

Growth of Chikungunya Virus in Baby Hamster Kidney Cell (BHK-21-Clone 13) Suspension Cultures

This report describes the methods used to obtain high titers of chikungunya virus with suspension cultures of BHK-21-clone 13 cells. The cells were grown at 37 C to a cell concentration of 10(6) to 2 x 10(6) per ml. After maximum cell growth, the cells were inoculated with chikungunya virus at a multiplicity of 1 to 2 50% suckling mouse intracerebral lethal doses (SMICLD(50)) per cell in the spent Eagle's minimum essential medium for suspension cultures (MEMS), or the cell cultures were centrifuged at 200 x g and resuspended in either fresh MEMS or medium 199 prior to inoculation. The medium used had no effect on virus titer. The inoculated cultures were incubated at 34 C until the cell viability dropped to 30%, which usually occurred 28 to 30 hr postinoculation. After these procedures, chikungunya virus titers of log(10) 10.3 to 11.8 SMICLD(50) per ml were obtained.