F-actin in guinea pig spermatozoa: its role in calmodulin translocation during acrosome reaction.

The presence of actin has been determined in mammalian spermatozoa. However, its function in these cells is still almost unknown. Only in boar spermatozoa has evidence for F-actin and a possible function for it been presented. In this work, actin distribution and F-actin were determined in uncapacitated, capacitated, and acrosomal-reacted guinea pig spermatozoa, by means of monoclonal and polyclonal antibodies, using an indirect immunoperoxidase technique, and by the use of rhodamine-phalloidin. With the last probe we found filamentous actin in these cells. By both techniques, actin was detected in the acrosome and in the entire tail. In some cells with acrosomal reaction, actin was also detected in the equatorial and in the postacrosomal regions. SDS-PAGE and Western blots immunostained with monoclonal and polyclonal anti-actin antibodies confirmed the presence of actin in extracts of guinea pig spermatozoa. Actin was also detected in preparations of Percoll-purified spermatozoa. We have communicated that guinea pig spermatozoa show a change on calmodulin location during the acrosome reaction. They present it first in the equatorial region and later in the postacrosomal region. To determine if F-actin participates in this calmodulin translocation, we studied the effect of cytochalasin D. It was found that the number of cells with calmodulin in the equatorial region increased in the presence of cytochalasin D while the number of cells with calmodulin in the postacrosomal region decreased. We also found that after cytochalasin D treatment acrosome loss was increased and sperm motility was slightly inhibited. Our results suggest that actin participate in calmodulin translocation to the postacrosomal region during acrosome reaction, in maintaining the acrosome structure, and perhaps also in sperm motility.     

A limnological reconnaissance of Lake Tebenquiche,Salar de Atacama,Chile

A number of expeditions to the area of Salar de Atacama, Chile, 68° 15'W, 20° 30'S, have involved studies of the biological and chemical features of Lake Tebenquiche, situated in the interior of the salar. Chemically, Tebenquiche is hypersaline, with practically anoxic waters dominated by sodium and chloride ions but with high concentrations of sulphate also. The lake is surrounded and invaded by macrophytes, dominated by Scirpus olmeyi and Juncus, which provide organic material for the formation of bacterial mats. The fauna of limnetic crustaceans is almost exclusively of Artemia salina. The most important genera of bacteria are: Marinomonas, Halobacterium, Acinetobacter and the sulphur reductors Vibrio and Bacillus. The Cyanobacteria are represented exclusively by Oscillatoria.     

Calcium binding to the cytosol and calcium extrusion mechanisms in intact synaptosomes and their alterations with aging.

A simple method to measure cytosolic calcium binding in intact presynaptic nerve terminals (synaptosomes) from rat brain, which is based on the simultaneous determination of [Ca2+]i and total [45Ca2+] in quin2-loaded synaptosomes undergoing a switch from high- to low-calcium containing medium, is presented. Binding to the cytosolic compartment alone was obtained following depletion of calcium storing organelles in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone/oligomycin plus caffeine. Synaptosomes, as compared to various cells types, have a high calcium binding capacity to the cytosolic compartment; maximum binding, Ca.Bmax, was 4.76 mM and calculated s0.5 was 218 nM. Calcium binding to the cytosolic compartment as a function of aging was also determined; Ca.Bmax was reduced to 1.84 mM and s0.5 increased to 492 nM in 30-month-old rats, indicating that the buffering of high calcium loads is impaired in old animals. The results obtained for binding of calcium to mitochondria and caffeine-sensitive calcium stores are consistent with an age-dependent reduction in calcium bound to mitochondria, whereas caffeine-sensitive calcium stores were unaffected. Finally, we have estimated the net rates of calcium extrusion in intact synaptosomes, and found that calcium efflux through the Na/Ca exchanger and Ca(2+)-ATPase was markedly reduced in old rats.     

Epidermal growth factor receptor in synaptic fractions of the rat central nervous system.

Functional relationships between epidermal growth factor (EGF) and neural tissues have of late attracted increasing interest. However, in spite of reported EGF effects on neurons, the expression of the EGF receptor (EGF-R) has not yet been unambiguously demonstrated in these cells. This 170-kDa protein bears an intracellular tyrosine kinase domain in which activity is ligand-dependent. We give definitive evidence here for its presence in neonatal and adult rat neurons showing also, for the first time, its binding and functional tyrosine kinase activities in the synaptic region. Immunohistochemistry using a polyclonal antibody prepared against the receptor purified from rat liver showed positive staining localized exclusively to neurons without regionalization to any particular brain zone. Binding studies made in Percoll-obtained synaptosomes revealed specific high affinity 125I-EGF binding sites (Kd, 1.42 x 10(-10) +/- 0.58 M) accounting for 17% of total binding and a great majority of low affinity (Kd, 2.55 x 10(-9) +/- 0.35 M) binding sites. Higher binding capacity was found in synaptosomal fractions obtained from newborn rats. The identity of the synaptosomal EGF binding activity with the 170-kDA EGF-R protein was demonstrated by cross-linking experiments. Furthermore, EGF-Affi-Prep affinity chromatography adsorbs a 170-kDa protein with EGF-R immunoreactivity from whole homogenates of adult rat brain. Phosphorylation assays made in freeze-thawed or intact synaptosomes showed EGF-induced tyrosine phosphorylation in the range of 170-, 126-150-, 124-, 113-, 98-, and 70-kDa proteins including the EGF-R. Thus, the EGF-R/EGF regulatory system could have a role in synaptic function that remains to be explored.     

Nigericin forms highly stable complexes with lithium and cesium

Nigericin is a monocarboxylic polyether molecule described as a mobile K⁺ ionophore unable to transport Li⁺ and Cs⁺ across natural or artificial membranes. This paper shows that the ion carrier molecule forms complexes of equivalent energy demands with Li⁺, Cs⁺, Na⁺, Rb⁺, and K⁺. This is in accordance with the similar values of the complex stability constants obtained from nigericin with the five alkali metal cations assayed. On the other hand, nigericinalkali metal cation binding isotherms show faster rates for Li⁺ and Cs⁺ than for Na⁺, K⁺, and Rb⁺, in conditions where the carboxylic proton does not dissociate. Furthermore, proton NMR spectra of nigericin-Li⁺ and nigericin-Cs⁺ complexes show wide broadenings, suggesting strong cation interaction with the ionophore; in contrast, the complexes with Na⁺, K⁺, and Rb⁺ show only clear-cut chemical shifts. These latter results support the view that nigericin forms highly stable complexes with Li⁺ and Cs⁺ and contribute to the explanation for the inability of this ionophore to transport the former cations in conditions where it catalyzes a fast transport of K⁺>Rb⁺>Na⁺.Part of the results of this paper were presented at the 14th International Congress of Biochemistry in Prague, Czechoslovakia.     

Effect of stobadine on indomethacin- and ethanol-induced stomach lesions and gastric secretion.

Stobadine was found to inhibit the ulcerogenic activity of indomethacin in relation to the dose but was ineffective against the direct necrotizing action of ethanol. It also inhibited gastric acid secretion when administered intraduodenally. Although stobadine is considered to be a scavenger of free radicals, our results indicate that, under the given experimental conditions, it is rather the inhibition of gastric acid secretion that is responsible for its antiulcerogenic effect. The preliminary results do not allow the exclusion of other mechanisms for explaining its antiulcerogenic effect.     

The Roberts syndrome

Summary Four siblings with the autosomal recessive Roberts syndrome are reported, and we discuss the phenotypic overlap of this syndrome with other similar radial, aplasia syndromes.     

Permeabilization of yeast for in situ determination of α-glucosidase

A quantitative in situ assay of yeast α-glucosidase involving permeabilization of the cells by freezing and thawing is described. The assay was applied to different strains in different physiological states and was shown to give results comparable to those obtained with total cell homogenates. The primary advantage of the in situ assay was the possibility of analyzing a large number of samples from the same culture during a growth curve using a very reduced cell mass.