Uptake of ferrous iron histidinate, a promoter of lipid peroxidation, by Ehrlich ascites tumor cells

The kinetics of the uptake of Fe(II)-histidinate, a known promoter of lipid peroxidation, into Ehrlich ascites tumor (EAT) cells and the intracellular binding of iron were studied in vitro. EAT cells (27.10(6)/ml) were incubated in Hanks' balanced salts solution at 37 degrees C for various time intervals in the presence of FeSO4 (1 mM) and L-histidine (10 mM). Total iron was determined by the 1,10-phenanthroline/ascorbate method and ferric iron by reaction with 5-sulfosalicylic acid; the difference was ascribed to ferrous iron. Total iron decreased rapidly in the medium (242 nmol within the first 10 min), and a corresponding increase of total iron (saturation value 376 nmol after 60 min) was determined within the cells, after the cellular proteins had been solubilized with 6 M urea. In the absence of EAT cells, Fe(II)-histidinate was readily oxidized to Fe(III)-histidinate by oxygen, but this reaction was strongly retarded by the tumor cells. The uptake of iron histidinate occurred in the oxidized state, while an uptake of ferrous iron could not be proven unambiguously. When EAT cells were saturated with iron, it was found that 93% of intracellular iron was bound to water-insoluble proteins and 7% was associated with soluble proteins, while no unbound iron was detectable by the method used. It was concluded that, despite the high uptake of total iron, only a very small portion of the intracellular iron was available as a redox catalyst for lipid peroxidation.     

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

Relative apparent synapomorphy analysis (RASA). I: The statistical measurement of phylogenetic signal

We have developed a new approach to the measurement of phylogenetic signal in character state matrices called relative apparent synapomorphy analysis (RASA). RASA provides a deterministic, statistical measure of natural cladistic hierarchy (phylogenetic signal) in character state matrices. The method works by determining whether a measure of the rate of increase of cladistic similarity among pairs of taxa as a function of phenetic similarity is greater than a null equiprobable rate of increase. Our investigation of the utility and limitations of RASA using simulated and bacteriophage T7 data sets indicates that the method has numerous advantages over existing measures of signal. A first advantage is computational efficiency. A second advantage is that RASA employs known methods of statistical inference, providing measurable sensitivity and power. The performance of RASA is examined under various conditions of branching evolution as the number of characters, character states per character, and mutations per branch length are varied. RASA appears to provide an unbiased and reliable measure of phylogenetic signal, and the general approach promises to be useful in the development of new techniques that should increase the rigor and reliability of phylogenetic estimates.      

Variation in heat-shock proteins among species of desert fishes (Poeciliidae, Poeciliopsis)

Analysis of the heat-shock proteins (hsps) of six closely related species of Poeciliopsis demonstrated the existence of biochemical diversity in the hsp100, hsp70, hsp60, and hsp30 protein families among species. Each species expressed five to seven hsp70-related isoforms. Constitutive 70-kD isoforms were identical among species, but four different patterns of heat-inducible isoforms were seen in these six species. Members of the hsp70 family of molecular chaperones are included among the most highly conserved proteins known, and the possibility of variation in hsp70 among closely related species has rarely been addressed. The hsp30 family is known to be less conserved than the hsp70 family, and, as expected, the Poeciliopsis hsp30 patterns showed more variation. Most of the hsp30 isoforms characteristic of a particular species were unique to that species. Hsp100 and hsp60 were identical in five of the species, but alternate isoforms were found in P. monacha. The small size and limited geographical distribution of the P. monacha population have probably contributed to the uniqueness of the monacha pattern. Two of the species were shown to acquire thermotolerance, the ability to withstand normally lethal temperatures when subjected to a gradual temperature increase. Rapid-heating protocols commonly used to establish critical thermal maxima of organisms do not include this inducible component of thermoresistance and therefore do not adequately assess an organism's capacity to withstand thermal stress.      

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.      

Interaction of the pyridoindole stobadine with alkoxyl and stable free radicals

Stobadine, a pyridoindole derivative which has been synthesized in search of new antiarrhytmic drugs, was able to interact with alpha-diphenyl-beta-picrylhydrazyl (DPPH^.) a stable free radical. Its scavenging potential was in the same order of magnitude as that of well known strong antioxidants. The scavenging effect of antioxidants tested increased in the order: stobadine, ascorbic acid, trolox, cysteine. Stobadine exerted a scavenging effect also towards alkoxyl radicals in a nonpolar solvent in the canthaxantin bleaching test. In this test stobadine was more effective than trolox.     

Mechanisms of iron-induced oxidative modifications of creatine kinase in rat brain in vitro. possible involvement of HNE

The model of oxidative stress induced by Fe/ascorbate in rat brain in vitro was used to compare the antioxidant capacity of known antioxidants. Creatine kinase (CK) was selected as a marker of protein injury in such studies. Of the antioxidant enzymes (catalase, superoxide dismutase), oxygen radical scavengers (mannitol, glutathione), and the chelator (EDTA) tested in this work and this system, only catalase and glutathione prevented the injury induced by oxidative stress, indicating that H2O2 and the glutathione peroxidase reaction were involved in the preventive effect. Additionally, the preventive effect of glutathione may be caused also by the fact that glutathione easily reacts with 4-hydroxynonenal (HNE), generated in rat brain homogenate, thus protecting CK from inactivation by this aldehyde. To find out whether and if at which concentrations CK may be oxidatively modified by HNE, pure CK was incubated in the presence of 10 and 64 micromol/l HNE for 30 min at 37 degrees C. The activity of CK incubated with HNE decreased significantly. Simultaneously, the protein carbonyls, determined by electrophoresis and immunoblotting increased at 10 micromol/l HNE or disappeared probably due to crosslinking of CK at 64 micromol/l HNE. The concentration of HNE in rat brain homogenates after oxidative stress was determined by HPLC and was in the range of 10-16 nmol/mg prot., corresponding to a concentration of 10-16 micromol/l HNE. This indicates that CK of rat brain homogenates oxidized by Fe/ascorbate may be impaired not only directly by oxygen radicals but also secondarily by HNE.