Expression and synthesis of cross-reactive idiotypic antibodies by peripheral blood lymphocytes and their suppression by anti-idiotypes in patients with IgA nephropathy.

We have recently described that patients with IgA nephropathy present high serum levels of anti-BSA idiotypic antibodies that were well correlated with the existence of hematuria. Furthermore, these Id were found in circulating and renal deposited immune complexes. In the present work, we examined the expression of surface idiotypic determinants on PBL by flow cytometry and their in vitro production, using as reagent anti-idiotypic antibodies previously well characterized. The presence of cross-reactive Id-bearing cells was observed in 5 out of 6 patients studied, with frequencies ranging from 3 to 12% of lymphocytes. After 7 days of culture, the spontaneous synthesis of idiotypic antibodies by PBL was found elevated in 6 out of 13 (46%) patients. A major Id cell expression and production was noted in patients with active disease as defined by hematuria. The preincubation of PBL with 20 and 50 micrograms of anti-idiotypic antibodies/2 x 10(6) cells for 3 days induced a significant inhibition of cross-reactive Id production in a dose-dependent fashion, with a degree of suppression between 12 and 50% in five out of six patients studied. In the above assays, as negative controls, we used the anti-Id antibodies previously adsorbed on an Id-Sepharose column. On the whole, these results suggest that patients with IgA nephropathy present dysfunctions in the Id-Anti-Id network that could play an important role in the pathogenesis of this disease.     

Stress induced changes in testis function

The mechanism through which chronic stress inhibits the hypothalamic-pituitary-testicular axis has been investigated. Chronic restraint stress decreases testosterone secretion, an effect that is associated with a decrease in plasma gonadotropin levels. In chronically stressed rats there was a decrease in hypothalamic luteinizing hormone-releasing hormone (LHRH) content and the response on plasma gonadotropins to LHRH administration was enhanced. Thus the inhibitory effect of chronic stress on plasma LH and FSH levels seems not to be due to a reduction in pituitary responsiveness to LHRH, but rather to a modification in LHRH secretion. It has been suggested that beta-endorphin might interfere with hypothalamic LHRH secretion during stress. Chronic immobilization did not modify hypothalamic beta-endorphin, while an increase in pituitary beta-endorphin secretion was observed. Since we cannot exclude that changes in beta-endorphin secreted by the pituitary or other opioids may play some role in the stress-induced decrease in LHRH secretion, the effect of naltrexone administration on plasma gonadotropin was studied in chronically stressed rats. Naltrexone treatment did not modify the decrease in plasma concentrations of LH or FSH. These findings suggest that the inhibitory effect of restraint on the testicular axis is exerted at hypothalamic level by some mechanism other than opioids.     

In vitro studies on the growth of Shigella sonnei by Lactobacillus casei and Lact. acidophilus.

The inhibitory effect of lactobacilli on growth of Shigella sonnei was studied. The effect was not due to pH alone, as addition of hydrochloric, lactic or acetic acids to culture media did not inhibit the normal growth of the shigellas. The degree of inhibition was measured by disc assay and showed that the inhibitory substance(s) can be extracellular and diffusible, varying the degrees of inhibition depending on the media tested. When broth was inoculated with mixed cultures of Lactobacillus and Shigella strains, the inhibition began at 6 h and the death phase at 9 h. The higher inhibition was produced by the mixture of lactobacilli (35.5 +/- 2.5% at 6 h culture, 57.4 +/- 1.9% at 9 h and 91.2 +/- 1.2% at 14 h). The degree of inhibition was higher when the relationship pathogen : lactobacilli was 1:10(3). The specific growth rate of lactobacilli and shigella was different in pure or mixed cultures. When the lactobacillus alone was grown for 12 h and the shigellas then added, the numbers of shigellas began to decrease immediately at 37 degrees C. This work shows that the Lactobacillus strains employed in fermented milk can be used to inhibit the growth of Sh. sonnei.     

Prothymosin alpha is phosphorylated by casein kinase-2.

Prothymosin alpha (ProT alpha) is a 12.5 kDa acidic polypeptide that is considered to have a nuclear function related to cell proliferation. Inspection of its amino acid sequence revealed the presence of sequences that may serve as targets for phosphorylation by casein kinase-2 (CK-2). ProT alpha isolated from calf thymocytes was phosphorylated in vitro by CK-2. The phosphorylation sites are Ser and Thr residues located among the first 14 amino acid residues in the ProT alpha sequence. Another site that is theoretically suitable for phosphorylation by CK-2, at the C-terminus of the polypeptide, is not, in fact, phosphorylated. Thymosin alpha 1 (T alpha 1), a peptide whose sequence corresponds to the first 28 amino acids of ProT alpha, is also phosphorylated by CK-2 at the same phosphorylation sites as ProT alpha. In cultured splenic lymphocytes ProT alpha was phosphorylated at Thr residues located at positions 7, 12 and/or 13. Based on these observations we conclude that CK-2, or another cellular kinase with similar sequence specificity, is responsible for phosphorylation of ProT alpha in vivo.     

The membrane insertion of colicins.

Pore-forming toxins, such as colicin A, are water-soluble proteins that insert into lipid bilayers. The water-soluble structure of Colicin A is known at a high resolution and this review describes the kinetic and structural steps involved in its soluble-to-membrane bound transformation.     

Phylogenetic analysis of the thiolase family. Implications for the evolutionary origin of peroxisomes

Summary The thiolase family is a widespread group of proteins present in prokaryotes and three cellular compartments of eukaryotes. This fact makes this family interesting in order to study the evolutionary process of eukaryotes. Using the sequence of peroxisomal thiolase from Saccharomyces cerevisiae recently obtained by us and the other known thiolase sequences, a phylogenetic analysis has been carried out. It shows that all these proteins derived from a primitive enzyme, present in the common ancestor of eubacteria and eukaryotes, which evolved into different specialized thiolases confined to various cell compartments. The evolutionary tree obtained is compatible with the endosymbiotic theory for the origin of peroxisomes. Offprint requests to: J.E. Pérez-Ortín     

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.     

Reconstitution of sodium channels in large liposomes formed by the addition of acidic phospholipids and freeze-thaw sonication

Phosphatidylcholine (PC) alone or with phosphatidylethanolamine (PE) are sufficient for the reconstitution of Na+ channels in planar lipid bilayers. However, when Na+ channels were first reconstituted into liposomes using the freeze-thaw-sonication method, addition of acidic phospholipids, such as phosphatidylserine (PS), to the neutral phospholipids was necessary to obtain a significant toxin-modulated 22Na uptake. To further investigate the acidic phospholipid effect on reconstitution into liposomes, Na+ channels purified from Electrophorus electricus electrocytes were reconstituted into liposomes of different composition by freeze-thaw sonication and the effect of batrachotoxin and tetrodotoxin on the 22Na flux was measured. The results revealed that, under our experimental conditions, the presence of an acidic phospholipid was also necessary to obtain a significant neurotoxin-modulated 22Na influx. Though neurotoxin-modulated 22Na fluxes have been reported in proteoliposomes made with purified Na+ channels and PC alone, the 22Na fluxes were smaller than those found using lipid mixtures containing acidic phospholipids. Electron microscopy of negatively stained proteoliposomes prepared with PC, PC/PS (1:1 molar ratio), and PS revealed that the acidic phospholipid increases the size of the reconstituted proteoliposomes. The increment in size caused by the acidic phospholipid, due to the associated increase in internal volume for 22Na uptake and in area for Na+ channel incorporation, appears to be responsible for the large neurotoxin-modulated 22Na fluxes observed.     

Proliferation index in bone marrow cells from severely malnourished rats during lactation.

The aim of this study was to determine if severe malnutrition affects the proportion of proliferating bone marrow cells in malnourished rats during lactation. Sister chromatid staining of metaphases was used as a parameter, as well as the morphology, size and color of bromodeoxyuridine labeled interphase nuclei. The BrdU proliferation labeling index was statistically lower in malnourished rats (20.4%), than in well-nourished controls (35.1%). A difference was also found between the two groups in the proportion of metaphases in first, second and third or successive cell cycle. The average generation time was longer in the malnourished group: 15.3 h, against 11.8 h for the controls. These results indicate that severe malnutrition affects both the proportion of bone marrow proliferating cells and their cell kinetics.     

Modulation of the affinity of the vasopressin receptor by magnesium ions.

The binding of [3H]vasopressin (AVP) and the 125I-labelled vasopressin antagonist (VP-AT) d(CH2)5[Tyr2(Me),Tyr9(NH2)]AVP to rat liver membranes was examined with or without the addition of milimolar concentrations of divalent cations. The binding of vasopressin was enhanced by Mg2+ and Co2+ and markedly decreased by EGTA. The addition of EGTA and Mg2+ together restored the binding to a value similar to that of Mg2+ alone. On the contrary, the addition of Mg2+, Co2+, EGTA, and the combination of EGTA and Mg2+ decreased the binding of VP-AT to rat liver membranes. Kinetic analyses showed that Mg2+ increased the Kd twofold for VP-AT; that is from 0.13 nM to 0.28 nM. Moreover, it showed that the receptor with or without the addition of Mg2+ consists of a single population of binding sites, indicating that the receptor is switched from a high affinity to a low affinity state for VP-AT in the presence of 10 mM Mg2+. GTP gamma S was unable to block the effect of Mg2+ on the binding of VP-AT. These results suggest that this divalent cation interacts with receptor itself producing a conformational changes which thus modulates the affinity of the receptor.