Hypotonic swelling-induced Ca2+ release by an IP3-insensitive Ca2+ store

Hypotonicswelling increases the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). The source of this Ca²⁺ is not clear. To study thesource of increase in [Ca²⁺]_i in response tohypotonic swelling, we measured [Ca²⁺]_i infura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a40.7-nM increase in [Ca²⁺]_i that was notinhibited by EGTA but was inhibited by 1 µM thapsigargin. Priordepletion of inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores with vasopressin did not inhibit the increasein [Ca²⁺]_i in response to hypotonic swelling.Exposure of ⁴⁵Ca²⁺-loaded intracellular storesto hypotonic swelling in permeabilized VSMC produced an increase in⁴⁵Ca²⁺ efflux, which was inhibited by 1 µMthapsigargin but not by 50 µg/ml heparin, 50 µM ruthenium red, or25 µM thio-NADP. Thus hypotonic swelling of VSMC causes a release ofCa²⁺ from the intracellular stores from a novel sitedistinct from the IP₃-, ryanodine-, and nicotinic acidadenine dinucleotide phosphate-sensitive stores.

     

Avidity analysis of the human immune response to a chitin binding protein of Toxoplasma gondii.

A 30-33 kDa electroeluted fraction of T. gondii tachyzoites improved discrimination between acute and chronic phase sera when used instead of the whole tachyzoite extract in an avidity-ELISA. In order to identify the components of these fractions, crude tachyzoite antigen was fractionated by anionic exchange chromatography. The 30-33 kDa antigen cluster eluted in the not-bound fraction could account for a large proportion of the antibody response against the 30-33 kDa electroeluted fraction. According to the N-terminal sequence data, this antigen fraction is composed mainly of SAG1 and another protein with high homology to chitin binding proteins from plants.     

High extracellular concentrations of potassium and rubidium modulate the synaptic transmission in the frog labyrinth

High extracellular K or Rb levels (20 mM) produce an increase in the resting EPSP and spike frequencies recorded intra cellularly from single fibres of the posterior nerve in the isolated frog labyrinth. The afferent discharge facilitation proved to be inversely related to the fibre's initial resting activity. The K effect is systematically larger than the Rb effect. High sensitive and scarcely sensitive units may be identified with respect to K and Rb action. The present findings suggest that, according to previous models of hair cell functioning, the K and Rb effects are mediated by a raise in intracellular Ca concentration which sustains an increased transmitter release at the cyto-neural junction.     

Conformation and ion binding properties of peptides related to calcium binding domain III of bovine brain calmodulin

The conformational and ion binding properties of the sequences 93-104, 96-104, and 93-98 of domain III of bovine brain calmodulin (CaM) have been studied by CD and Tb3+-mediated fluorescence. In aqueous solution the interaction of all fragments with Ca2+ and Mg2+ ions is very weak and without any effect on the peptide conformation, which remains always random. In trifluoroethanol the interaction is very strong and the different fragments exhibit very distinct binding properties. In particular, the dodecapeptide fragment 93-104, and its N-terminal hexapeptide 98-104, bind calcium and magnesium with a very high binding constant (Kb greater than 10(5) M-1), undergoing a substantial conformational change. The structural rearrangement is particularly evident in the hexapeptide fragment, which tend to form a beta-bend. The C-terminal nonapeptide fragment 96-104 interacts with calcium and magnesium more weakly, and the binding process causes a decrease of ordered structure. These results suggest that, even in the entire dodecapeptide sequence corresponding to the loop of domain III of CaM, the calcium binding site is shifted toward the N-terminal hexapeptide segment. This interpretation is consistent with the results of crystallographic studies of CaM, which show that the calcium ions are located toward the amino terminal portion of the loop.     

Cytochemical evaluation of gamma-glutamyl-transpeptidase activity in cervical smears

The cervicovaginal smears of 43 patients attending an outpatient service for early cancer detection were cytochemically studied for the presence of gamma-glutamyl-transpeptidase (GGT) in epithelial cells. This was done in order to evaluate such an enzyme phenotype as a marker for cancer development. The results showed that 70% of the 38 patients with a cytologic diagnosis of "inflammatory" or preneoplastic/neoplastic conditions had GGT-positive cells in their smears. None of the five cytologically normal cases showed any epithelial cells with GGT activity. Although most of the GGT-positive cells were metaplastic, some morphologically normal, dysplastic or neoplastic cells also expressed the enzyme. The data suggest that cytochemically detectable transpeptidase activity appears whenever alterations of the normal epithelial microenvironment occurs, but is not necessarily linked to the carcinogenic process. Therefore, cytochemically GGT-positive cells should not be used as an indicator of neoplastic transformation of the cervical epithelium.     

Cyclic nucleotide phosphodiesterases in somatic and germ cells of mouse seminiferous tubules

The distribution of phosphodiesterase forms in somatic and germ cells, and their variations during testicular development and germ cell differentiation have been investigated. Seminiferous tubules from immature mice and Sertoli cells in culture possessed two enzyme activities which were comparable to forms described for different tissues and species: (a) a calcium-calmodulin-dependent enzyme with high affinity for guanosine 3',5'-(cyclic)-monophosphate (cGMP), and (b) a calcium-calmodulin-independent enzyme with high affinity for adenosine 3',5'-(cyclic)-monophosphate (cAMP) the activity of which increased in cultured Sertoli cells after treatment with FSH or dibutyryl cAMP. Seminiferous tubules from adult animals and germ cells at the meiotic and post-meiotic stage of differentiation possessed two enzyme forms that could be distinguished from those present in somatic cells of the seminiferous tubules: (a) a calcium-calmodulin-dependent form with high affinity for both cAMP and cGMP, similar to forms described in other tissues from different species, and (b) a calcium-calmodulin-independent phosphodiesterase with high affinity for cAMP and present only in post-meiotic cells, previously identified also in germ cells of the rat.     

"In vitro" effect of CCl4, PG and EDTA on GSH levels at different time of incubation of rat liver homogenates

During CCl4-induced lipid peroxidation GSH content in total homogenate from rat liver falls very rapidly in the first 30 min. of incubation "in vitro". CCl4 does not enhance the decrease in total glutathione (TG) during the incubation time, so GSH loss is mainly due to its oxidation to GSSG. On the contrary PG and EDTA, two substances decreasing lipid peroxidation rate, are able to decrease GSH oxidation, without affecting TG content. At 25 degrees C EDTA and PG completely prevent GSH decrease at pH 7.4, while at pH 6 PG affords only a partial prevention. At 37 degrees C both compounds are able to limit GSH decrease at a large extent. Lipid peroxidation seems to have a great importance in the kinetics of GSH decrease and GSSG formation, at least "in vitro". It is noteworthy that PG which inhibits lipid peroxidation stimulated by CCl4 is also able to limit the high GSH loss observed in the homogenates incubated in the presence of halogeno-alkane.     

Specific photoaffinity labeling of the digitalis binding site of the sodium and potassium ion activated adenosinetriphosphatase induced by energy transfer

A ouabain p-aminobenzenediazonium derivative with a high specific radioactivity has been synthesized from ouabain and used as a photolabel for the (sodium plus potassium)-activated adenosinetriphosphatase from Electrophorus electricus electric organ and from dog kidney. In the dark it binds reversibly to the digitalis receptor site, with binding characteristics comparable to those of ouabain. The photoactivation of the ouabain derivative to produced covalent labeling of the receptor was obtained by energy transfer from a tryptophan residue in the (Na+,K+)ATPase to the ouabain p-aminobenzenediazonium molecule bound at the active site. The great advantage of this procedure compared to previous methods is that free molecules of the photoactivatable derivative are not photodecomposed. Analysis of the photolabeled polypeptides on sodium dodecyl sulfate gel electrophoresis showed that over 90% of the total radioactivity incorporated was found in the large molecular weight alpha-chain of the kidney enzyme (Mr 93 000). The same specific labeling of the alpha-subunit was obtained with a crude microsomal fraction from Electrophorus electricus. A mild tryptic fragmentation of the subunit into two peptide fragments of Mr 58 000 and 41 000, respectively, shows that the digitalis receptor is located in the N-terminal 41 000 fragment.