An in situ assay for induced diphtheria-toxin-resistant mutants of diploid human fibroblasts

The sensitivity of diploid human fibroblasts to the cytotoxic effects of diphtheria toxin (DT) depended on the cell growth status. Exponentially growing cells treated with 10^?3-1 lethal flocculating units (LF) of DT/ml for 4 days survived with a frequency of 4 × 10^?4. However, the DT-resistant phenotype of colonies isolated under these conditions was not stable. When the growth of the cells had been arrested by confluence or deprivation of serum growth factors prior to treatment with DT (4 days, 10^?3-0.6 LF/ml), the survival decreased to 2 × 10^?6 and the resistance of isolated colonies was stable. An in situ assay for induced DT-resistant mutants was developed in order to avoid problems associated with the possible reduced viability of the mutants relative to that of wild-type cells. A reproducible and linear dose response was obtained for the induction of DT-resistant mutants by ethylnitrosourea. The mutants were induced with high frequency by this compound (e.g., 10^?3 mutants/viable cell at a 37% survival dose); complete expression of the mutant phenotype occurred after 6 generations of growth under nonselective conditions. Isolated mutant colonies showed stable resistance to DT and were cross-resistant to Pseudomonas aeruginosa exotoxin A.     

2-β-d-Glucopyranosyloxy-2-methylpropanol in Acacia sieberana var. woodii

A new diol glucoside, 2-β-d-glucopyranosyloxy-2-methylpropanol, the first reported naturally occurring monoglucoside of an aliphatic dihydric alcohol, was isolated from pods of Acacia sieberana var. woodii. Structure elucidation was based on ¹ H and ¹³C NMR spectroscopy, and enzymatic analyses. The compound was hydrolysed very slowly by almond β-glucosidase, but cleaved by a β-glucuronidase enzyme complex from Helix pomatia.     

Types of sesquiterpenes from Artemisia douglasiana

Artemisia douglasiana afforded, in addition to known compounds, two new C₁₄-acetylenes, five longipinene derivatives, three nerolidol derivatives, a lactone and a ketone with a new carbon skeleton and lavendulol-2-methylbutyrate. The structures were elucidated by spectroscopic methods and some chemical transformations. The configurations of several oxo longipinene-7, 9-di- and 7, 8, 9-triesters isolated previously were corrected. The biogenesis of the new lactones is discussed briefly.     

A sensitive fluorimetric microassay for the determination of glutathione peroxidase activity. Application to human blood platelets

The method proposed for measuring glutathione peroxydase (GSH-Px) activity is based on the determination of oxidized glutathione (GSSG) using o-phtalaldehyde (OPT) as a fluorescent reagent. This method makes it possible to study the kinetics of both substrates (peroxide and reduced glutathione, GSH), and allosteric kinetics were found for GSH, with human platelets as the source of GSH-Px. Different methods for platelet disruption were compared. The reference values obtained for GSH-Px activity in human blood platelets by this fluorimetric procedure and the conventional enzymatic method were very similar and significantly higher than those previously reported; the reasons for this difference are discussed.     

The mechanism of deuterium oxide-induced protein degradation in Lemna minor

Transfer of Lemna minor fronds to culture medium containing 50% (v/v) deuterium oxide induces a large increase in the rate of protein breakdown, which is not due to an increase in the activity of acidic or neutral proteolytic enzymes or peptidases. Biochemical and ultrastructural evidence indicates that deuterium oxide affects the properties of certain membranes, particularly the tonoplast, and allows vacuolar proteolytic enzymes to pass into the cytoplasm and cause the increased protein breakdown.Abbreviations BAPA benzylarginine-p-nitroanilide - LPA leucine-p-nitroanilide - TCA trichloroacetic acid     

Anti-microtubular herbicides and fungicides affect Ca2+ transport in plant mitochondria

The herbicides amiprophosmethyl (APM) trifluralin, and oryzalin as well as the fungicides methylbenzimidazolyl carbamate (MBC), O-isopropyl N-phenyl carbamate (IPC), and chlorisopropyl N-phenyl carbamate (CIPC), which are known to cause the destruction of microtubules in vivo but do not interfere with tubulin polymerization in vitro, have been examined with respect to their ability to affect Ca²⁺ transport in isolated cell organelles. In contrast to colchicine which has no effect on Ca²⁺ transport in isolated mitochondrial and microsomal fractions, all of the substances investigated caused considerable reduction of ca²⁺ net uptake into mitochondrial but not into microsomal fractions. This reduction has been shown to be due to an increase in passive Ca²⁺ efflux. These results have been extrapolated to in vivo situations where they are postulated to act by raising cytoplasmic Ca²⁺ levels.Abbreviations APM amiprophosmethyl - CIPC chlorisopropyl N-phenyl carbamate - IPC O-isopropyl N-phenyl carbamate - MBC methylbenzimidazolyl carbamate - Mops 3-(N-Morpholino) propanesulfonic acid - DMSO dimethylsulfoxide     

Oxygen inhibition of photosynthesis in the C4 species Amaranthus graecizans L.

In the C₄ plant, Amaranthus graecizans, increasing [O₂] from 2% up to 100% inhibited photosynthesis, quantum yield, and the carboxylation efficiency, and increased the CO₂ compensation point () from 2 to about 12 l/l. The O₂ inhibition of photosynthesis was fully reversible. When changing from 2.5 to 40% O₂ and vice versa, about 1 h was required for full equilibration with an O₂ inhibition of 18%; whereas in wheat, a C₃ species, inhibition of photosynthesis and its reversal occurs within minutes after changing [O₂], resulting in 63% inhibition of photosynthesis by 45% O₂. These differences in O₂ inhibition between a C₄ and C₃ species can be explained by high diffusive resistance across bundle-sheath cells of C₄ plants and the increased CO₂/O₂ ratio in bundle-sheath cells which is the consequence of the C₄ cycle. In A. graecizans, increased with increasing [O₂] but tended to reach a maximum at relatively high O₂ levels. The lack of a linear increase in as previously observed for C₃ species indicates that a considerable amount of photorespired CO₂ may be re-fixed with increasing levels of O₂. In comparison to previous reports with other C₄ species, photosynthesis of A. graecizans shows greater sensitivity to O₂, with a noticeable inhibition occurring with shifts from 2 to 21% O₂. A. graecizans has characteristics of other C₄ species with respect to Kranz anatomy, localization of PEP carboxylase in mesophyll cells and RuBP carboxylase in bundle-sheath cells, and little fractionation among carbon isotopes during CO₂ fixation. The basis for the higher sensitivity of photosynthesis of A. graecizans to O₂ may be based upon a lower diffusive resistance of gases across bundle-sheath cells than in some other C₄ species.Abbreviations CE carboxylation efficiency - RuBP ribulose-1,5-bisphosphate - CO₂ compensation point     

Effects of taurine and γ-aminobutyric acid on akinesia and analgesia induced by D-Ala2-Met-enkephalinamide in rats

Effects of taurine or γ-aminobutyric acid (GABA) on akinesia and analgesia induced by D-Ala²-Met-enkephalinamide were investigated in rats. Administration of taurine (dose range: 2.375×10^?2 M–9.5×10^?2 M/10 μl) into the left lateral ventricle 10 min prior to the injection of D-Ala²-Met-enkephalinamide (50 μg/10 μl) produced a dose-dependent reduction in the duration of akinesia and to some extent of analgesia, as estimated at 30 min and 60 min following the enkephalinamide injection; at the first estimation-time (10 min), taurine did not alter the duration of akinesia or that of analgesia. The median effective dose (ED₅₀) for akinesia determined at 60 min after D-Ala²-Met-enkephalinamide was 5 times greater and that for analgesia assessed at the same time was 1.7 times greater in taurine-treated rats than the respective doses in control animals. Administration of GABA under similar experimental conditions produced a dose-dependent reduction in the duration of analgesia from the initial estimation time (10 min) following the injection of D-Ala²-Met-enkephalinamide. The ED₅₀ for analgesia determined at 30 min after D-Ala²-Met-enkephalinamide was 3 times greater in GABA-treated rats than in control animals. Unlike the effects of taurine, GABA did not alter the duration of akinesia. Neither the duration of akinesia nor that of analgesia was modified by taurine or GABA alone in rats tested 9 min after the injection of each amino acid. These findings suggest that taurine may promote a recovery from both akinesia and analgesia, while GABA decreases only the analgesia induced by D-Ala²-Met-enkephalinamide.     

Abnormalities in the fine structure of the spermatids of rats injected with cadmium

The degenerative changes in the spermatids as measured by changes in fine structure abnormalities increased with time following injection of Cd²⁺ into rat testis. The spermatids in the twelve hours group appear as peculiarly club shaped and elongated structures with one or two small but perceptible vacuoles. The subacrosomal area and the space between the nucleus and the middle piece are seen abnormally dilated. In the 30 day group, the central filaments are the most susceptible unit of 9+2 axoneme complex. The plasma membrane, the cytoplasmic matrix, the mitochondria of the middle piece and the fibrous sheath appear shrunken, discontinuous and degenerative.     

Effect of sciatectomy and induced ammonia stress on Mg2+_adenosine triphosphatase activity in frog tissues

Mg² + dependent —adenosine triphosphatase activity has been studied in the muscle, brain, kidney and liver tissues of frog,Rana hexadactyla (Lesson) after sciatectomy and induced chronic ammonia stress. The enzyme activity decreased in the tissues of the denervated frog. The activity of the enzyme increased in all the tissues of the normal and denervated frogs except in the denervated muscle when ammonium lactate was infused intraperitoneally.