Trypanosoma brucei: trypanocidal and cell swelling activities of lasalocid acid.

Chemotherapeutic treatment of human and animal trypanosomiasis is unsatisfactory because only a few drugs are available. As these drugs have poor efficacy and cause adverse reactions, more effective and tolerable medications are needed. As the polyether ionophore antibiotic lasalocid acid is used as medicated feed additive in cattle, the compound was tested for its trypanocidal and cytotoxic activity against bloodstream forms of Trypanosoma brucei and human myeloid HL-60 cells. The concentrations required of lasalocid acid to reduce the growth rate of trypanosomes by 50% and to kill the parasites were 1.75 and 10 µM, respectively. The ionophore displayed also cytotoxic activity against HL-60 cells but the human cells were about 10 to 14 times less sensitive indicating moderate selectivity. As the trypanocidal mechanism of action of polyether ionophore antibiotics is due to a sodium influx-induced cell swelling, the effect of lasalocid acid on cell volume change in bloodstream-form trypanosomes was investigated. Interestingly, lasalocid acid induced a much faster cell swelling in trypanosomes than the more trypanocidal related ionophore salinomycin. These results support further investigations of lasalocid acid and derivatives thereof as potential agents against African trypanosomiasis.

Lasalocid induces cytotoxic apoptosis and cytoprotective autophagy through reactive oxygen species in human prostate cancer PC-3 cells.

Lasalocid is an antibiotic from the group of carboxylic ionophores, produced by Streptomyces lasaliensis. But there was limited information of lasalocid on human prostate cancer cells. In the present studies, to better understand its effect in human prostate cancer cells, apoptosis and autophagy associated with possible signal pathways in vitro was examined. Our study showed that lasalocid mediated cell cycle arrest in G0/G1 phase by reducing G1 phase dependent proteins, indicating entering into apoptotic cell death pathway. Lasalocid-induced apoptosis was involved with reactive oxygen species (ROS) production, and mitochondrial hyperpolarization. In addition, lasalocid induced autophagy through microtubule-associated protein 1 light chain 3 (LC-3)-II conversion, acidic vesicular organelles formation and GFP-LC-3 punctuate, which was inhibited by 3-methyladenine (3-MA), a widely used pharmacological inhibitor of autophagy. Furthermore, the autophagic phenomena were mediated by production of ROS, confirming that inhibition of ROS with N-acetyl-l-cysteine, a ROS inhibitor, attenuated lasalocid-triggered autophagy. Inhibition of autophagy with 3-MA enhanced the lasalocid-induced apoptosis through enhanced ROS generation. Taken together, lasalocid should be useful in the search for new potential chemotherapeutic agents for understanding the molecular mechanisms of anticancer in prostate cancer cells.

Modification of plant cell wall structure accompanied by enhancement of saccharification efficiency using a chemical, lasalocid sodium.

The cell wall is one major determinant of plant cell morphology, and is an attractive bioresource. Here, we report a novel strategy to modify plant cell wall property by small molecules. Lasalocid sodium (LS) was isolated by chemical screening to identify molecules that affect the cell morphology of tobacco BY-2 cells. LS treatment led to an increase in cell wall thickness, whilst the quantity and sugar composition of the cell wall remained unchanged in BY-2 cells. The chemical also disordered the cellular arrangement of hypocotyls of Arabidopsis plants, resulting in a decrease in hypocotyl length. LS treatment enhanced enzymatic saccharification efficiency in both BY-2 cells and Arabidopsis plants. Microarray analysis on Arabidopsis showed that exposure to LS upregulated type III peroxidase genes, of which some are involved in lignin biogenesis, and jasmonic acid response genes, and phloroglucinol staining supported the activation of lignification by the LS treatment. As jasmonic acid-mediated lignification is a typical reaction to cell wall damage, it is possible that LS induces cell wall loosening, which can trigger cell wall damage response. Thus, LS is a unique chemical for modification of cell wall and morphology through changes in cell wall architecture.

One-pot synthesis and cytotoxicity studies of new Mannich base derivatives of polyether antibiotic--lasalocid acid.

Seven Mannich base derivatives of polyether antibiotic Lasalocid acid (2a-2g) were synthesized and screened for their antiproliferative activity against various human cancer cell lines. A novel chemoselective one-pot synthesis of these Mannich bases was developed. Compounds 2a-2c and 2g with sterically smaller dialkylamine substituent, displayed potent antiproliferative activity (IC50: 3.2-7.3 µM), and demonstrated higher than twofold selectivity for specific type of cancer. The nature of Mannich base substituent on C-2 atom at the aromatic ring may be critical in the search for selectivity towards a particular cancer cell.

Suplemento múltiplo com ionóforos para novilhos em pasto: desempenho / Multiple supplement with ionophores for grazing steers: performance

Avaliou-se o desempenho de 25 novilhos Holandês x Zebu, castrados, com média de peso vivo inicial de 265±50 kg, sob pastejo emBrachiaria decumbens, distribuídos em cinco grupos e em cinco piquetes, segundo os tratamentos: controle - suplementação múltipla sem ionóforos (CONT); suplementação múltipla com 100mg/cab/dia de monensina (M100); suplementação múltipla com 200mg/cab/dia de monensina (M200); suplementação múltipla com 100mg/cab/dia de lasalocida (L100); suplementação múltipla com 200mg/cab/dia de lasalocida (L200). O período experimental foi de 105 dias, com rotação dos grupos nos piquetes a cada 21 dias. A suplementação foi fornecida ad libitum. A avaliação de desempenho ocorreu mediante a pesagem dos animais, em jejum de alimento e água de 14 horas, no início de cada período e término do experimento. Os animais alimentados com suplementos com ionóforos apresentaram maior ganho de peso em relação aos do controle (0,357 vs. 0,268; P = 0,0068). Entre os ionóforos, os animais alimentados com lasalocida ganharam mais peso (0,398 vs. 0,333; P=0,0175). O melhor desempenho pode ser explicado pelo maior consumo dos suplementos pelos animais alimentados com lasalocida (0,53 vs. 0,42; P<0,0001).

The performance of 25 castrated Holstein x Zebu crossbred steers averaging 265±50kgBW, grazing on Brachiaria decumbens, during dry season was evaluated. The experiment was carried out in a completed randomized design and the animals were grouped in five different paddocks with the following treatments: control - multiple supplement without ionophores (CONT); multiple supplement with 100mg of monensin/animal/day (M100); multiple supplement with 200mg of monensin/animal/day (M200); multiple supplement with 100mg of lasalocid/animal/day (L100); and multiple supplement with 200mg of lasalocid/animal/day (L200). The experimental period was 105 days, with changing groups on paddocks every 21 days. The multiple supplement was offered ad libitum. Body weight was evaluated after 14 hours of fasting. Animals fed multiple supplement with ionophores showed higher average daily weight gain than control (0.357 vs 0.268; P= 0.0068), as well as steers fed lasalocid in comparision to monensin (0.398 vs. 0.333; P= 0.0175). Animals suplemented with lasalocid had higher intakes and higher average daily gain (0.53 vs. 0.42; P<0.0001).

Suplemento múltiplo com ionóforos para novilhos em pasto: consumo, fermentação ruminal e degradabilidade in situ / Multiple supplement with ionophores for grazing steers: intake, ruminal fermentation, and in situ degradability

Avaliou-se o efeito do suplemento múltiplo com ionóforos sobre o consumo, a fermentação ruminal e a degradabilidade in situ da matéria seca da forragem. Utilizaram-se cinco novilhos Holandês x Zebu fistulados no rúmen, com peso vivo médio de 350kg, em delineamento em quadrado latino. Os tratamentos foram: suplementação múltipla sem ionóforos (CONT); suplementação múltipla com 100mg/cab/dia de monensina (M100); suplementação múltipla com 200mg/cab/dia de monensina (M200); suplementação múltipla com 100mg/cab/dia de lasalocida (L100); e suplementação múltipla com 200mg/cab/dia de lasalocida (L200). O uso de ionóforos no suplemento não influenciou o consumo de forragem, que foi, em média, 7,24kg MS/dia. A presença de ionóforos resultou em ligeiro aumento do pH ruminal em relação à ausência desses aditivos (P<0,05). Houve diferença na concentração do N-NH3 apenas para os teores de ionóforos em que 200mg/cab/dia reduziu a quantidade de N-NH3. As concentrações de acetato, propionato e butirato não foram influenciadas pela inclusão, pelo tipo ou pelos teores de ionóforos. A fração solúvel média (A) da Brachiaria decumbens foi igual a 22 por cento, e a fração insolúvel potencialmente degradável média (B) igual a 65 por cento, resultando em degradação potencial média de 87 por cento. A taxa de degradação média (c) foi de 0,03/hora. Os ionóforos não alteraram a degradação in situ da matéria seca.

The effect of multiple supplement with ionophores was evaluated on intake, ruminal fermentation, and in situ degradability of dry matter (DM) of the pasture forage. Five rumen fistulated Holstein x Zebu steers averaging 350kg of BW were used. The animals were grouped in five different paddocks under Latin Square experimental design. The treatments were multiple supplement without ionophores (CONT); multiple supplement with 100mg of monensin/animal/day (M100); multiple supplement with 200mg of monensin/animal/day (M200); multiple supplement with 100mg of lasalocid/animal/day (L100); multiple supplement with 200mg of lasalocid/animal/day (L200). The pasture intake was 7.24kg DM/day and it was not affected by ionophores. The average pH was influenced (P<0.05) by the presence of the ionophores in the supplements. There rumen N-NH3 concentration was negatively influenced by the ionophores levels in the multiple supplement. The molar concentrations of acetate, propionate, and butirate in the rumen were not affect by the presence, type, or level of ionophores. The mean soluble fraction A of Brachiaria decumbens was 22 percent, the mean potential degradable insoluble fraction (B) was 65 percent, and the degradability was 87 percent. The mean degradation rate (c) was 0.03/h. The ionophores did not affect DM in situ degradability.

Effect of dietary supplementation with oregano essential oil on performance of broilers after experimental infection with Eimeria tenella.

A study was carried out to examine the effect of dietary supplementation of oregano essential oil on performance of broiler chickens experimentally infected with Eimeria tenella at 14 days of age. A total of 120 day-old Cobb-500 chicks separated into 4 equal groups with three replicates each, were used in this study. Two groups, one infected with 5 x 10(4) sporulated oocysts of E. tenella and the other not, were given a basal diet and served as controls. The other two groups also infected with E. tenella were administered diets supplemented with oregano essential oil at a level of 300 mg/kg, or with the anticoccidial lasalocid at 75 mg/kg. Following this infection, survival rate, bloody diarrhoea and oocysts excretion as well as lesion score were determined. Throughout the experimental period of 42 days, body weight gain and feed intake were recorded weekly, and feed conversion ratios were calculated. Two weeks after the infection with E. tenella supplementation with dietary oregano oil resulted in body weight gains and feed conversion ratios not differing from the non-infected group, but higher than those of the infected control group and lower than those of the lasalocid group. These parameters correspond with the extent of bloody diarrhoea, survival rate, lesion score and oocyst numbers and indicated that oregano essential oil exerted an anticoccidial effect against E. tenella, which was, however, lower than that exhibited by lasalocid.

Effects of lasalocid on circulating concentrations of leptin and insulin-like growth factor-I and reproductive performance of postpartum Brahman cows.

Objectives were to determine effects of lasalocid on reproductive performance and serum concentrations of leptin and IGF-I, and to correlate concentrations of leptin and IGF-I with reproductive performance of beef cows. Forty-one purebred, multiparous Brahman cows were blocked to control (C; n = 20) or lasalocid (L; n = 21) treatments by BW, BCS, and predicted calving date. Treatment began 21 d before expected calving. Cows were each fed 1.4 kg daily of an 11:1 corn:soybean meal supplement, with the L group receiving 200 mg of lasalocid/cow daily. Cows and calves were weighed, and cow BCS was assessed at calving and at 28-d intervals thereafter. Blood samples were collected weekly precalving, at parturition, and twice weekly thereafter. Sterile marker bulls were maintained with cows for estrous detection. Six days after estrus, ovaries were evaluated for corpus luteum formation, and blood samples from d 6, 7, and 8 after estrus were collected. Serum samples were assayed for progesterone (P4), IGF-I, and leptin concentration. Progesterone concentrations > 1 ng/mL were considered indicative of a functional corpus luteum. Treatment ended after completion of a normal estrous cycle, and cows removed from treatment were placed with a fertile bull equipped with a chinball marker. There were no treatment differences in calving date, calf sex, cow BW, BCS, calf BW, calf ADG, or in serum concentrations of P4, IGF-I, or leptin. Prepartum cow ADG was increased (P < 0.01) in L cows and tended (P < 0.011) to be increased from calving to d 56 after calving in L cows. Postpartum interval (PPI) was not affected by treatment; however, a greater percentage (P < 0.05) of L cows conceived by 90 d after calving (43% L vs. 15% C). First-service conception rate tended (P < 0.08) to be greater in L vs. C cows (68 vs. 40%), but pregnancy rate was not different (P < 0.12; 86% for L vs. 65% for C). There were no treatment differences (P > 0.18) for serum IGF-I concentrations. At calving, leptin was positively correlated with IGF-I (P < 0.04; r = 0.32), BCS (P < 0.06; r = 0.29), and cow BW (P < 0.02; r = 0.36), and was negatively correlated with PPI (P < 0.06; r = -0.29). These results provide evidence that feeding an ionophore before calving and during the postpartum period may increase the number of cows that rebreed to maintain a yearly calving interval. Cows with higher concentrations of leptin postpartum may exhibit shorter PPI.

[Effects of veterinary drugs on beta-hexosaminidase release from rat basophilic leukemia cells (RBL-2H3)].

Little is known about the effects of residual veterinary drugs on the allergic reaction, except for the antigenicity of antibiotics and synthetic antimicrobials. Therefore, 59 kinds of veterinary drugs were investigated for their effects on the IgE receptor-mediated beta-hexosaminidase release from RBL-2H3 cells as an index of immediate allergic reaction. We found that the antibiotics chlorotetracycline, doxycycline, monensin, the synthetic antimicrobial pyrimethamine and the steroid hormone testosterone inhibited beta-hexosaminidase release. Most of the veterinary drugs showed no action, though the ionophores lasalocid, salinomycin and the steroid hormone hexestrol promoted beta-hexosaminidase release from injured cells. Based on the residual levels of these drugs and the frequencies of detection in actual food samples, it seems unlikely that these drugs have any immediate allergic effect in practice.

Secretion of the trefoil factor TFF3 from the isolated vascularly perfused rat colon.

The trefoil factor TFF3 is a peptide predominantly produced by mucus-secreting cells in the small and large intestines. It has been implicated in intestinal protection and repair. The mechanisms that govern TFF3 secretion are poorly understood. The aim of this study was, therefore, to evaluate the influence of neurotransmitters, hormonal peptides and mediators of inflammation on the release of TFF3. For this purpose, an isolated vascularly perfused rat colon preparation was used. After a bolus administration of 1 ml isotonic saline into the lumen, TFF3 secretion was induced by a 30-min intra-arterial infusion of the compounds to be tested. TFF3 was evaluated in the luminal effluent using a newly developed radioimmunoassay. TFF3 was barely detected in crude luminal samples. In contrast, dithiothreitol (DTT) treatment of the effluent revealed TFF3 immunoreactivity, which amounted to about 0.3 pmol min(-1) cm(-1) in the basal state. Gel chromatography of DTT-treated luminal samples revealed a single peak that co-eluted with the monomeric form of TFF3. TFF3 was not detected in the portal effluent. Bethanechol (10(-6)-10(-4) M), vasoactive intestinal peptide (VIP, 10(-8)-10(-7) M) or bombesin (10(-8)-10(-7) M) induced a dose-dependent release of TFF3. In contrast, substance P evoked a modest release of TFF3, whereas calcitonin gene-related peptide (CGRP), somatostatin, neurotensin or peptide YY (PYY) did not modify TFF3 secretion. The degranulator compound bromolasalocid, 16,16-dimethyl PGE2 (dmPGE2) or interleukin-1-beta (IL-1-beta) also evoked a marked release of TFF3. In conclusion, TFF3 in the colonic effluent is present in a complex. This association presumably involves a disulfide bond. Additionally, the present results suggest a role for enteric nervous system and resident immune cells in mediation of colonic TFF3 secretion.

Systemic lipopolysaccharide influences rectal sensitivity in rats: role of mast cells, cytokines, and vagus nerve.

Intraperitoneal lipopolysaccharide (LPS) produces somatic hyperalgesia, releases interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha), and activates vagal afferents. The aim of this study was to evaluate the effect of peripheral LPS on rectal sensitivity and to specify the mechanisms involved. Abdominal muscle contractions were recorded in conscious rats equipped with intramuscular electrodes. Rectal distension (RD) was performed at various times after LPS or experimental treatments. In controls, RD significantly increased the number of abdominal contractions from a threshold volume of distension of 0.8 ml. At the lowest volume (0.4 ml), this number was increased after administration of LPS (3, 9, and 12 h later), recombinant human IL-1beta (from 3 to 9 h), recombinant bovine TNF-alpha (from 6 to 9 h), and BrX-537A (from 6 to 12 h), a mast cell degranulator. The effect of LPS was reduced by doxantrazole, Lys-D-Pro-Thr, and soluble recombinant TNF receptor. Vagotomy selectively amplified the response to LPS. We conclude that, in vivo, intraperitoneal LPS lowers visceral pain threshold (allodynia) through a mechanism involving mast cell degranulation and IL-1beta and TNF-alpha release and that the vagus nerve may exert a tonic protective role against LPS-induced rectal allodynia.

Effects of neurotransmitters, gut hormones, and inflammatory mediators on mucus discharge in rat colon.

The effect of potential mediators of mucus secretion was investigated in the isolated vascularly perfused rat colon by using a sandwich enzyme-linked immunosorbent assay for rat colonic mucin and by histochemical analysis. Bethanechol (100-200 microM), bombesin (100 nM), and vasoactive intestinal peptide (VIP, 100 nM) provoked a dramatic mucin discharge (maximal response at 900, 900, and 600% of control loops, respectively). VIP-stimulated mucin secretion was abolished by tetrodotoxin, whereas atropine was without effect. In contrast, both tetrodotoxin and atropine significantly decreased mucin release induced by bombesin. Isoproterenol or calcitonin gene-related peptide was without effect. Serotonin (1-5 microM) and peptide YY (10 nM) evoked mucin discharge, whereas glucagon-like peptide-1 did not release mucin. Finally, bromolasalocid (20 microM), interleukin-1beta (0.25 nM), sodium nitroprusside (1 mM), and dimethyl-PGE2 (2.5 microM) induced mucus discharge. The results demonstrated a good correlation between the immunological method and histological analysis. In conclusion, these findings suggest a role for the enteric nervous system, the enteroendocrine cells, and resident immune cells in mediation of colonic mucus release.

Lasalocid effects on ruminal degradation of protein and postruminal supply of amino acids in Holstein steers.

Five ruminally and duodenally cannulated Holstein steers (305 kg) were used in a switchback experiment with three periods to evaluate two experimental treatments: a basal diet with or without 45 ppm of lasalocid. The basal diet contained approximately 43% rolled corn, 45% alfalfa hay, and 10% soybean meal (DM basis). Lasalocid did not affect feed intake or ruminal digestion of OM and NDF. Ruminal digestion of ADF tended to increase with supplemental lasalocid. Total tract digestion of OM, NDF, ADF, and N and intestinal flow of amino acids were not affected by lasalocid. Also, the ratio of microbial to nonmicrobial N fractions at the duodenum remained unchanged. Ruminal pH and concentrations of NH3, VFA, peptides, and amino acids were not affected by lasalocid. Ruminal protease activity decreased with supplemental lasalocid, but this decrease was not reflected in other variables, such as ruminal concentrations of peptides and amino acids. Ruminal deaminase activity remained unchanged. Thus, we concluded that dietary lasalocid did not alter ruminal protein degradation or postruminal flow of amino acids.

The tryptophan fluorescence change upon conformational transition of the phosphoenzyme intermediate in sarcoplasmic reticulum Ca(2+)-ATPase is revealed in the absence of K+ and the presence of lasalocid.

ATP-induced changes in the tryptophan fluorescence of the Ca(2+)-ATPase were determined with sarcoplasmic reticulum vesicles at pH 7.0 and 0 degrees C by steady-state measurements in the presence of Ca2+ and the absence of K+ with and without added lasalocid (a carboxylic ionophore, 50 microM), which was previously shown to cause a predominant accumulation of the ADP-insensitive form of the phosphoenzyme intermediate (EP) (Kawashima, T., Hara, H., and Kanazawa, T. (1990) J. Biol. Chem. 265, 10993-10999). When ATP was added in the absence of lasalocid, the fluorescence decreased by 1.7%. The addition of lasalocid quenched 71% of the fluorescence but did not reduce the ATP-induced fluorescence drop. The fluorescence drop and the EP formation were also determined in the presence of lasalocid by stopped-flow spectrometry and continuous-flow rapid quenching. The observed fluorescence drop was biphasic. The first phase coincided with the formation of EP, which was largely ADP-sensitive in this early stage of the reaction. The second phase was much slower than the first phase and coincided with the accumulation of ADP-insensitive EP. When the transition of EP from the ADP-sensitive form to the ADP-insensitive form was blocked by N-ethylmaleimide treatment, the second phase disappeared, and the fluorescence drop entirely coincided with the formation of ADP-sensitive EP. These findings demonstrate that the first phase of the fluorescence drop is attributed to the formation of ADP-sensitive EP, the second phase being attributed to the transition of EP from the ADP-sensitive form to the ADP-insensitive form. The present results reveal the conditions that definitely discriminate these two phases.

Functional evidence for an extracellular calcium receptor mechanism triggering tyrosine kinase activation associated with mouse keratinocyte differentiation.

Calcium-induced keratinocyte differentiation is associated with tyrosine phosphorylation of a p62 protein which associates with the ras-GTPase activating protein (GAP). We have examined the nature of the calcium signal triggering p62 phosphorylation. EGTA, a specific chelator of calcium, was able to completely block calcium-induced p62 phosphorylation, even after using conditioned medium from calcium-treated keratinocytes. Preventing calcium-induced cell-cell contacts by anti-cadherin antibodies did not inhibit tyrosine phosphorylation. Slight increases in extracellular calcium concentrations (0.15 or 0.30 mM) were already sufficient to induce p62 phosphorylation. Other divalent cations, such as magnesium, zinc, nickel, and cobalt, but not the trivalent cation lanthanum, induced p62 phosphorylation to a similar extent as calcium. There was no close correlation between the ability of the various ions to induce p62 phosphorylation and increase free intracellular calcium. Similarly, treatment of primary keratinocytes with the calcium ionophores A23187 or X537A did not induce p62 phosphorylation, although it increased free intracellular calcium levels. Finally, blockers of potassium uptake, which is induced by calcium, did not inhibit p62 phosphorylation. Thus, in keratinocyte differentiation, calcium is likely to provide the primary signal for p62 tyrosine phosphorylation and may act directly at the cell membrane through a "cationic receptor mechanism" analogous to that described in other cell types.

Interleukin-1 beta maturation and release in response to ATP and nigericin. Evidence that potassium depletion mediated by these agents is a necessary and common feature of their activity.

Lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages produce large quantities of interleukin (IL)-1 beta but in the absence of a secondary stimulus little of this cytokine is proteolytically processed to its mature biologically active state and externalized. The potassium-proton ionophore nigericin and ATP are known to promote the maturation and release of IL-1 beta from LPS-stimulated cells. We investigated the mechanisms by which these agents act in an attempt to understand requirements of the post-translational processing. Like nigericin, the ionophores A204 and lasalocid induced the release and maturation of IL-1 beta. The electrogenic potassium ionophore valinomycin, however, did not stimulate these post-translational events. Addition of nigericin or lasalocid to LPS-stimulated cells produced a rapid intracellular acidification; A204, however, did not alter pH, indicating that an acidification was not necessary for activation of IL-1 beta maturation. Macrophages treated with ATP became rounded and swollen, and after 30 min of treatment their appearance was comparable with cells treated with nigericin. Post-translational maturation and release of IL-1 beta began immediately after ATP addition. The majority of the 17-kDa mature IL-1 beta produced within the first 30 min of treatment was recovered extracellularly; in contrast, during this same time period the 35-kDa IL-1 beta precursor and the cytoplasmic marker enzyme lactate dehydrogenase and the lysosomal enzyme beta-N-acetylglucosaminidase remained cell-associated. ATP, therefore, promoted both the proteolytic maturation of IL-1 beta and the release of the biologically active species in the absence of cell lysis. Longer incubations with ATP caused cytolysis as judged by the release of the cytoplasmic enzymes. ADP was less active than ATP at initiating the post-translational maturation and release of IL-1 beta and AMP, GTP, and UTP were totally inactive, ATP, nigericin, A204, and lasalocid promoted a rapid and complete loss of the potassium analog 86Rb+ from cells that were preloaded with this cation; valinomycin-treated cells released only a portion of the radiolabeled cation. Agents that promoted the maturation and release of IL-1 beta from LPS-stimulated macrophages, therefore, shared an ability to mobilize intracellular potassium. Macrophages treated with ATP or nigericin in medium that contained KCl rather than NaCl failed to proteolytically activate and to release IL-1 beta. These data suggest that ATP and nigericin induce a net decrease in intracellular levels of K+ which is necessary for activation of the post-translational maturation of IL-1 beta.

An immunosuppressed rat model for evaluation of anti-Cryptosporidium activity of sinefungin.

Cryptosporidium parvum causes life-threatening diarrhoea in immunocompromised, especially AIDS patients and the efficiency of proposed anti-cryptosporidial therapies is limited or doubtful. An immunosuppressed adult rat model of C. parvum infection was developed for screening molecules candidate for curative and preventive activity in human cryptosporidiosis. Among 31 drugs tested, lasalocid (2-10 mg/kg/24 h), and sinefungin (2-10 mg/kg/24 h), exhibited some activity against C. parvum infection. Oral sinefungin therapy resulted in a dose related suppression of oocysts shedding, which correlated with oocyst disappearance from ileum sections and was also efficient in preventing infection. Relapses were observed after discontinuation of curative sinefungin therapy, which suggests that the biliary tract, a major location and parasite reservoir which sustains persisting infection, was not cleared of parasites by the drug. Improved therapeutic procedures with sinefungin (or analogues) will result from current pharmacological studies.

Proton ejection as a major feature of the Ca(2+)-pump.

H+ ejection and Ca2+ uptake promoted by the sarcoplasmic reticulum (SR) Ca(2+)-pump are similarly stimulated by millimolar Mg2+. This cannot be assigned to enhanced Ca2+ uptake and H+ displacement from internal metal binding sites since: (1) loading SR vesicles with high Mg2+ concentrations does not impair H+ ejection; (2) loading SR vesicles with Mn2+ does not depress H+ ejection occurring during Mn2+ uptake; (3) H+ ejection occurs even when Ca2+ accumulation inside the vesicles is prevented with Ca2+ ionophores. It is concluded that the Ca(2+)-pump promotes an active Ca2+/H+ countertransport stimulated by Mg2+. Finally, a mechanism for Ca2+ translocation is proposed in basic physico-chemical terms.

Involvement of the 3',5'-cyclic AMP pathway in the induction of calmodulin synthesis in myoblasts by 1,25(OH)2-vitamin D3.

The participation of second messenger pathways in 1,25(OH)2D3-induced stimulation of protein synthesis in chick embryo myoblasts undergoing proliferation was studied. Double-labelling experiments with 14C- and 3H-leucine showed the induction by the hormone of proteins with apparent molecular masses (treatment interval) of 60 kDa (1 to 2 h). 70 kDa (2 to 4 h), 80 kDa (4 h) and a 19 kDa protein (6 to 12 h) previously identified as calmodulin. The PKC activator TPA and the Ca2+ ionophore X-537 A did not mimic the effects of the sterol on protein synthesis whereas similar double-labelling patterns were obtained with forskolin, an adenylate cyclase activator. Dot-blot and Northern hybridization analysis revealed increased calmodulin mRNA levels in response to both the hormone and forskolin. These results involve the cAMP messenger system in 1,25(OH)2D3 stimulation of calmodulin synthesis and may be relevant to understand hormone regulation of muscle cell proliferation.

Cornifin, a cross-linked envelope precursor in keratinocytes that is down-regulated by retinoids.

In this study, we have characterized the cDNA clone SQ37 that was isolated previously from a rabbit squamous cell library. The gene encodes a 14-kDa protein that appears to function as a component of the cross-linked envelope in squamous differentiating cells. The protein, which has been named cornifin, has a high content of proline (31%), glutamine (20%), and cysteine (11%) and contains 13 repeats of an octapeptide (consensus sequence, EPCQPKVP) at its C terminus. SQ37 mRNA and protein are induced during squamous differentiation of rabbit tracheal (RbTE) cells and human epidermal keratinocytes. This induction is repressed by retinoids. Immunohistochemical studies reveal SQ37 immunoreactivity in fragmented cross-linked envelopes from squamous-differentiated RbTE cells and in the suprabasal layers of the epidermis. In situ hybridization analysis showed that the presence of SQ37 mRNA is restricted to the suprabasal layers. Treatment of RbTE cells with a Ca2+ ionophore induces cross-linking of the SQ37 protein into higher molecular weight complexes. This cross-linking reaction appears to be mediated by transglutaminase type I. Our observations suggest that the protein encoded by SQ37 participates in the assembly of the cross-linked envelope.