Effects of anesthetic alcohols on membrane transport processes in human erythrocytes

1. 1. Anesthetic alcohols (pentanol, hexanol and heptanol) were found to increase the fluidity of red cell membrane lipids as monitored by the fluorescence depolarization of diphenylhexatriene. The relative potency of the alcohols was found to be parallel to their relative membrane/water partition coefficients.

2. 2. Hexanol had biphasic effect on erythritol uptake by simple diffusion by red cells. At concentrations less than 9 mM, hexanol had no significant effect. At concentrations greater than 9 mM, there was an approximately linear increase in erythritol permeability with increasing alcohol concentration.

3. 3. The facilitated transport of uridine was markedly inhibited by hexanol. Hexanol at 6 mM produced a 65% inhibition of uridine (4 mM) uptake. Hexanol decreased both the apparent K_m and V values for the equilibrium exchange of uridine.

4. 4. The facilitated transport of galactose was only slightly inhibited by hexanol.

5. 5. Hexanol was without effect on the passive and active fluxes of Na⁺ and K⁺ in red cells with altered cation contents. Cells that were slightly depleted of K⁺ and cells that were highly K⁺-depleted were both insensitive to hexanol.

(p-Amidinophenyl)methanesulfonyl fluoride, an irreversible inhibitor of serine proteases

p-(Amidinophenyl)methanesulfonyl fluoride (p-APMSF) has been synthesized and shown to be a specific, irreversible inhibitor of the class of plasma serine proteases which demonstrate substrate specificity for the positively charged side chains of the amino acid lysine or arginine. In equimolar concentration, this compound causes immediate and complete irreversible inhibition of bovine trypsin and human thrombin. A 5-10-fold molar excess of reagent over enzyme is required to achieve complete irreversible inhibition of bovine Factor Xa, human plasmin, human C1-r, and human C1-s. the Ki of p-APMSF for all of the above-mentioned proteases is between 1 and 2 microM. In contrast, p-APMSF in large molar excess does not inactivate chymotrypsin or acetylcholinesterase. The unique reactivity of p-APMSF has been further shown in comparison with the related compound p-nitrophenyl (p-amidinophenyl)methanesulfonate which is an active-site titrant for thrombin but reacts poorly with Factor Xa, C1-r, and C1-s and is not hydrolyzed by bovine trypsin or human plasmin. Similarly, (p-amidinophenyl)methanesulfonate has a Ki of 30 microM for thrombin but is a poor inhibitor of trypsin, Factor Xa, C1-r, C1-s, and plasmin. Studies with bovine trypsin have demonstrated that the inhibitory activity of p-APMSF is the result of its interaction with the diisopropyl fluorophosphate reactive site. The unique reactivity of this inhibitor classifies it as one of the most effective active site directed reagents for this class of serine proteases. Collectively, these results suggest that the primary substrate binding site of these enzymes, which share a high degree of structural homology, do in fact significantly differ from each other in their ability to interact with low molecular weight inhibitors and synthetic substrates.     

Two isoaccepting seryl tRNAs coded by separate mitochondrial genes in yeast

Summary In S. cerevisiae four isoacceptor mitochondrial tRNAs for serine have been separated by reversed phase chromatography. At least two of these species are products of different genes. In this work the deletion mapping technique has been used to locate two genes for tRNA^ser. The gene for tRNA^ser previously localized in the oli I region of the mitochondrial genome has been found to code for tRNA _ser ² , and another gene coding for tRNA _ser ¹ has been detected in the region where most of other tRNA genes are found. Results of fine mapping experiments allowed to localize this gene in the proximity of the gene for tRNA^arg.     

In vitro testing and the carcinogenic potential of several nitrosated indole compounds

4-chloro-methoxyindole is a naturally occurring compound in Vicia faba which can easily react with nitrite to form a N-nitroso compound. In this in vitro study, the potential genotoxic effects of nitrosated 4-chloro-6-methoxyindole and its structural analogue 4-chloroindole were evaluated for the first time by using both Salmonella and Chinese hamster V79 cells. Additionally, the inhibition of gap junctional intercellular communication in V79 cells by these compounds was determined; this is a validated parameter for tumor-promoting activity. Most assays were also performed with nitrosated indole-3-acetonitrile, a naturally occurring compound in brassicas. Both nitrosated chloroindoles were highly mutagenic to Salmonella typhimurium TA100 without the need of exogenous metabolic activation and were potent inducers of Sister Chromatid Exchanges. Nitrosated indole-3-acetonitrile generated the same effects, although at much higher concentrations. Equivocal results were obtained for the nitrosated chloroindoles in a forward mutation assay using the hypoxanthine guaninephosphoribosyltransferase locus. All nitrosated indole compounds significantly inhibited gap junctional intercellular communication. These results indicate that nitrosated chloroindoles and nitrosated indole-3-acetonitrile should be considered as mutagens and agents with potential tumor-promoting capacity.Abbreviations BrdU 5-bromo-2-deoxyuridine - 4Cl 4-chloroindole - 4C6MI 4-chloro-6-methoxy-indole - DMSO dimethyl sulfoxide - EBSS Earle's balanced salt solution - EMS ethyl methanesulfonate - GJIC gap junctional intercellular communication - HBSS Hanks balanced salt solution - HGPRT hypoxanthine guaninephosphoribosyl transferase - I₃A indole-3-acetonitrile - MNNG 1-methyl-1-nitroso-3-nitroguanidine - NOC N-nitroso compounds - NQO 4-nitroquinolone-N-oxide - SCE sister chromatid exchange - 6TG 6-thioguanine - TPA 12-O-tetradecanoylphorbol-13-acetate     

Actin organization and fibrin-clot retractile activity of cultured mouse fibroblasts

Summary It is known that human and animal fibroblasts are able to induce the retraction of a fibrin clot. In the present study the correlation between (i) fibrinclot retractile (FCR) activity, (ii) the number of actin stress-lines in mouse fibroblasts during growth in culture, and (iii) the sensitivity of actin stress-lines to a powerful actin-depolymerizing factor (ADF), present in plasma and serum of humans and laboratory animals was investigated. Fibroblasts at early passages (2–4) were tested for these parameters at various intervals after seeding (24, 96, and 168 h). The number of actin stress-lines was progressively higher, while the sensitivity to ADF action was progressively lower in cells cultured from 24 to 168 h; the FCR capacity was significantly decreased at 168 h. These data suggest that cells containing weakly polymerized and/or stabilized actin are more active than those containing highly polymerized and/or stabilized actin in triggering fibroblast contraction.     

Differential stimulation of pancreatic-polypeptide and gastrin secretion by bombesin in man

Bombesin, besides many other actions on the mammalian gastroentero-pancreatic tract, strongly stimulates the release of pancreatic-polypeptide (PP) in dogs. In 8 healthy human volunteers (5 males, 3 females), the PP response during bombesin infusion was low (25.7 ± 6.3 peak vs. 5.0 ± 2.0 basal pmol/1) compared to the effect of a protein meal (144.1 ± 13.4 pmol/1) or to the gastrin response to the same dose of the amphibian polypeptide (140.0 ± 23.6 pmol/1 eq SHG 17 I). The response pattern of PP and gastrin was different as PP concentrations peaked 10 min after cessation of bombesin infusion (32.0 ± 4.9 pmol/1) when gastrin concentrations already were down to one third of the maximal response. Atropine inhibited the PP response to bombesin but did not abolish it completely. It is concluded that in man, the total effect of bombesin on PP secretion is minor compared both to the effect of the peptide on gastrin secretion in man and to the effect of bombesin in dogs. It is suggested that bombesin might have a dual, inhibitory-stimulatory, effect on PP secretion in man.     

Complex and simple sequences in human repeated DNAs

Highly repeated human DNA sequences were isolated by isopycnic centrifugation, or were eluted from gels after restriction enzyme cleavage. High molecular weight DNA peaks separable from the bulk of the DNA in a variety of gradients were shown to consist of very simple sequences characteristic of simple satellite DNAs; DNA fingerprint studies indicated each of these peaks could consist of tandem repeats of a specific oligonucleotide sequence as low as 10 base pairs (bp) long. All the gradient peaks could be assigned to one of two sequence groups and several different buoyant density peaks revealed the same sequence. — Restriction fragment multimers did not share common sequences with the satellite DNAs as judged by hybridization data. They could not be separated by isopycnic centrifugation. Furthermore these highly repeated DNAs were more complex in sequence and more variable than the satellites. Even the smallest (50 bp) fragments by depurination and other direct sequencing methods were shown to be more complex than the high molecular weight satellite peaks. — The idea that subsets of repeated DNAs may be defined by sequence complexity, possibly with discrete or separable functions, is proposed.     

Effects of alkali salts on carbon and nitrogen mineralization of organic matter in soil

Summary In an incubation experiment the mineralization of gulmohur leaves (Delonix regia L.) added to soil was studied at increased levels of exchangeable sodium percentage (ESP). CO₂ evolution, total mineralization of carbon and humic/fulvic acid carbon ratio increased with increased ESP and the process of nitrification was inhibited completely between 70–92 ESP. The mineralization and losses of nitrogen were equal to control up to 70 ESP but were very high at 92 ESP. The extractable carbon varied from 26% in case of control to 31% at 48.6 ESP.     

Annulate lamellae in hindgut epithelial cells ofIsopoda

Summary Hindgut epithelial cells of the aquatic isopod,Asellus communis, and the terrestrial isopod,Armadillidium vulgare, possess annulate lamellae. The organelle is present in non-dividing cells of intermolt adult organisms. The lamellae exhibit dense pore areas traversed by diaphragms, and the lamellae are associated with elements of endoplasmic reticulum.This research was supported in part by a grant to E. R. Witkus from the Irene Heinz and John La Porte Given Foundation.