Biomolecular study and conjugation of two para-aminobenzoic acid derivatives with serum proteins: drug binding efficacy and protein structural analysis.

Two aminobenzoic acid derivatives DAB-0 and DAB-1 showed distinct biological properties on murine bladder cancer (BCa) cell line MB49-I. In contrast to DAB-1, DAB-0 does not possess any anti-inflammatory activity and is less toxic. Furthermore, DAB-0 does not interfere with INFγ-induced STAT1 activation and TNFα-induced IκB phosphorylation, while DAB-1 does. In order to rationalize these results, the binding efficacy of DAB-0 and DAB-1 with serum proteins such a human serum albumin (HSA), bovine serum albumin (BSA) and beta-lactoglobulin (ß-LG) was investigated in aqueous solution at physiological pH. Multiple spectroscopic methods and thermodynamic analysis were used to determine the binding efficacy of DAB-0 and DAB-1 with serum proteins. Drug-protein conjugation was observed via through ionic contacts. DAB-1 forms stronger adducts than DAB-0, while ß-LG shows more affinity with the order of stability ß-LG > BSA > HSA. The stronger complexation of DAB-1 with serum proteins might account for its biological potential and transport in the blood. The binding efficacy ranged from 40 to 60%. Major alterations of protein secondary structures were detected upon drug complexation. Serum proteins are capable of delivering DAB-1 in vitro.Communicated by Ramaswamy H. Sarma.

Structural robustness of the gut mucosal microbiota is associated with Crohn's disease remission after surgery.

OBJECTIVES: Preventing postoperative recurrence after ileocolonic resection (ICR) for Crohn's disease (CD) is challenging. Defining the disturbances of the microbial composition and community structure after ICR and their link with early disease recurrence is crucial. DESIGN: Microbiota composition (fingerprinting and 16S rDNA sequencing) and community structure (correlation networks of bacterial species) were assessed from ileal mucosa sampled in 20 patients undergoing ICR and 6 months later during endoscopy from above (neoterminal ileum) and below (subanastomotic colon) the surgical anastomosis. RESULTS: ICR had a dramatic effect on gut microbial ecosystem. At surgery, CD mucosa harboured a dysbiotic microbiota with high proportions of α/ß Proteobacteria and Bacilli. Six months later, half of the patients had recurrent lesions at ileocolonoscopy and presented higher numbers of Lachnospiraceae. Recurrence of endoscopic lesions was associated with enrichment in Enterococcus durans while patients in remission had increased proportions of Dorea longicatena and Bacteroides plebeius. Structural differences were striking between recurrence and remission microbiota; while the microbiota of patients with CD recurrence exhibited a loose community structure, the microbiota of patients in remission displayed communities that were robustly correlated to each other. Microbiota colonising the neoterminal ileum and subanastomotic colon 6 months after ICR only differed in patients with recurrence. CONCLUSIONS: ICR modifies the gut microbiome. Remission after 6 months was associated with homogenous bacterial distribution around the anastomosis. Community structure and bacterial networks highlight target species, including Faecalibacterium prausnitzii and Ruminococcus gnavus, which may allow precise modulations of the overall microbial ecosystem towards remission pattern.

Combination of C-reactive protein, infliximab trough levels, and stable but not transient antibodies to infliximab are associated with loss of response to infliximab in inflammatory bowel disease.

BACKGROUND: Antibodies to infliximab [ATI] and trough levels to infliximab [TRI] are associated with loss of response in inflammatory bowel diseases [IBD]. The best way to predict loss of response [LOR] to infliximab [IFX] is unknown. METHODS: We conducted a prospective observational cohort study enrolling all IBD patients who were in clinical remission at Week 14 after IFX treatment initiation. TRI, ATI and C-reactive protein [CRP] level were measured at Week 22 [T1] and thereafter at every other IFX infusion. Loss of clinical response was defined by a flare requiring therapeutic change [IFX dose intensification, initiation of another drug class, and/or surgery]. RESULTS: A total of 93 patients [59 Crohn's disease, mean duration of follow-up 17.2 months] were included; 32 patients [34.4%] lost clinical response during follow-up. Cumulative probability of LOR was 50% at 20 months. Mean TRI at T1 was significantly lower in IBD patients with stable ATI as compared with those with transient ATI or without ATI [0.052, 3.34 ,and 4.29 µg/ml, respectively; p = 0.001 between no ATI vs stable ATI, and p = 0.005 between stable and transient ATI] [p = 0.0001]. Three independent factors were predictive of LOR after Cox proportional hazards modelling: TRI > 5.5 µg/ml (hazard ratio [HR]: 0.21; 95% confidence interval [CI]: 0.05-0.89;p = 0.034) at T1, CRP > 5mg/l [HR: 2.5; 95% CI: 1.16-5.26; p = 0.019] at T1, and stable ATI defined by two consecutive ATI > 20ng/ml [HR: 3.77; 95% CI: 1.45-10.0; p = 0.007]. Transient ATI did not influence LOR. CONCLUSIONS: LOR can be predicted based on a combination of CRP, TRI and stable ATI with a high degree of accuracy.

Highlighting new phylogenetic specificities of Crohn's disease microbiota.

BACKGROUND: Recent studies suggest that gastrointestinal (GI) microbes play a part in the pathogenesis of Crohn's disease (CD). METHODS: Fecal samples were collected from 16 healthy individuals and 16 CD patients (age- and sex-matched). The DNA extracted from these samples were subjected to two different methods of microbiome analysis. Specific bacterial groups were quantified by real-time polymerase chain reaction (PCR) methods using primers designed using a high-throughput in-house bioinformatics pipeline. The same DNA extracts were also used to produce fluorescently labeled cRNA amplicons to interrogate a custom-designed phylogenetic microarray for intestinal bacteria. RESULTS: Even though the intersubject variability was high, differences in the fecal microbiomes of healthy and CD patients were detected. Faecalibacterium prausnitzii and Escherichia coli were more represented in healthy and ileal CD patients, respectively. Additionally, probes specific for Ruminococcus bromii, Oscillibacter valericigenes, Bifidobacterium bifidum, and Eubacterium rectale produced stronger hybridization signals with the DNA samples from healthy subjects. Conversely, species overrepresented in CD patients were E. coli, Enterococcus faecium, and species from the Proteobacteria not normally found in the healthy human GI tract. Furthermore, we detected "healthy specific" molecular species or operational taxonomic units (OTUs) that are not closely related to any known species (Faecalibacterium, Subdoligranulum, and Oscillospora species), indicating that the phylogenetic dysbiosis is broader than at strain or species level. CONCLUSIONS: These two techniques of microbiome analysis provided a statistically robust new picture of the dysbiosis in fecal microbiota from ileal CD patients. Specifically, we identified a set of six species discriminant for CD, which provides a preliminary diagnostic tool.

Evolutionary relationships between 15 Plasmodium species from new and old world primates (including humans): an 18S rDNA cladistic analysis.

We present a new phylogenetic analysis of 15 primate Plasmodium species based on 18S rDNA sequences including new sequences of Plasmodium coatneyi, P. fieldi, P. gonderi, P. hylobati and P. simium. The results are discussed in the context of the parasite host species and their geographical distribution. Contrary to other phylogenies constructed with this 18S rDNA molecule, we observed that the topology of phylogenetic trees was not affected either by the quality of the nucleotide matrices, or by the species present in the outgroup. This analysis showed the following. (1) The polyphyly of human Plasmodium is confirmed. (2) The monophyly of Plasmodium from Old World monkeys is confirmed by the new added sequences and P. gonderi, an African species, possibly could be at the root of this group. (3) The most parsimonious biogeographical hypothesis is that P. vivax originated in Asia; thus, its related species P. simium appears to be derived through a transfer from the human P. vivax to New World monkey species in South America. (4) Sampling efforts of non-human primate Plasmodium could permit improvement of the knowledge of primate Plasmodium phylogeny and also consideration of the risks of malaria emergence from monkey reservoirs.

Alternating stereospecific copolymerization of ethylene and propylene with metallocene catalysts.

The copolymerization of ethylene and propylene with bridged metallocenes Me(2)E(3-RCp)(Flu)X(2)/MAO (E = C, X = Me; E = Si, X = Cl; R = H or alkyl) was investigated. Ethylene/propylene copolymerization with metallocenes having heterotopic active sites (R =Me, i-Pr) yield alternating, isotactic ethylene/propylene copolymers with percentages of alternating EPE+PEP triads in the range of 61-76% at 50% ethylene incorporation. Both the nature of the substituent R and the bridge E influence the copolymerization behavior including the copolymerization activity, copolymer sequence distribution, molecular weight, and stereochemistry. Silicon-bridged metallocenes produce copolymers with higher activity and molecular weight but lower propylene incorporation at similar feeds than the carbon-bridged analogues. Isotactic PEPEP sequences were observed for all metallocenes, while the tacticities of the EPPE sequences varied with the bridge and the substituent on the metallocene ligand. Isotactic PEPEP sequences and atactic EPPE sequence errors in the alternating copolymers are consistent with a mechanism where the comonomers are enchained alternately at the heterotopic coordination sites of the metallocenes. Isotactic EPPE sequences are indicative of occasional multiple insertions at the stereospecific site, caused by an isomerization of the chain prior to monomer insertion (backskip).

Effect of estrogen on calcium and sodium transport by the nephron luminal membranes.

Estrogens are widely used for contraception and osteoporosis prevention. The aim of the present study was to investigate the effect of 17 beta-estradiol on calcium (Ca(2+)) transport by the nephron luminal membranes, independently of any other Ca(2+)-regulating hormones. Proximal and distal tubules of rabbit kidneys were incubated with 17 beta-estradiol or the carrier for various periods of time, and the luminal membranes of these tubules were purified and vesiculated. Ca(2+) uptake by membrane vesicles was measured using the Millipore filtration technique. Incubation of proximal tubules with the hormone did not influence Ca(2+) uptake by the luminal membranes. In contrast, incubation of distal tubules with 10(-8) M 17 beta-estradiol for 30 min decreased the initial uptake of 0.5 mM Ca(2+) from 0.34+/-0.04 (s.e.m. ) to 0.17+/-0.04 pmol/microg per 5 s (P<0.05). In the presence of 100 mM Na(+), 0.5 mM Ca(2+) uptake was strongly diminished and the effect of 17 beta-estradiol disappeared (0.17+/-0.01 and 0.21+/-0.07 pmol/microg per 5 s in vesicles from the control and treated tubules). Direct incubation of the membranes with 17 beta-estradiol, however, failed to show any influence of the hormone on Ca(2+) transport. The action of 17 beta-estradiol was dose-dependent, with a half-maximal effect at approximately 10(-9) M. Ca(2+) uptake by the distal tubule membranes presents dual kinetics. 17 beta-Estradiol decreased the V(max) value of the high-affinity component from 0.42+/-0.02 to 0.31+/-0.03 pmol/microg per 10 s (P<0.02). In contrast with the effect of the hormone on Ca(2+) transport, estradiol increased Na(+) uptake by both the proximal and distal tubule luminal membranes. In conclusion, incubation of proximal and distal tubules with estrogen decreases Ca(2+) reabsorption by the high-affinity Ca(2+) channels of the distal luminal membranes, and enhances Na(+) transport by the membranes from proximal and distal nephrons.

Effect of angiotensin II on calcium reabsorption by the luminal membranes of the nephron.

In the rat and the rabbit, a number of studies have reported the effects of angiotensin II (ANG II) on Na(+) reabsorption by the proximal (PT) and distal (DT) convoluted tubules of the kidney. The aim of the present study was to examine the effect of ANG II on Ca(2+) uptake by the luminal membranes of the PT and DT of the rabbit. Incubation of PT and DT with 10(-12) M ANG II enhanced the initial Ca(2+) uptake in the two segments. Dose-response experiments revealed, for Ca(2+) as well as for Na(+) transport, a biphasic action with a maximal effect at 10(-12) M. Ca(2+) transport by the DT luminal membrane presents a dual kinetic. ANG II action influenced the high-affinity Ca(2+) channel, increasing maximal velocity from 0.72 +/- 0.03 to 0.90 +/- 0.05 pmol x microg(-1) x 10 s(-1) (P < 0.05, n = 3) and leaving the Michaelis-Menten constant unchanged. The effect of ANG II was abolished by losartan, suggesting that the hormone is acting through AT1 receptors. In the PT, calphostin C inhibited the effect of the hormone. It is therefore probable that protein kinase C is involved as a messenger. In the DT, however, neither Rp cAMP, calphostin C, nor econazole (a phospholipase A inhibitor) influenced the hormone action. Therefore, the mechanisms involved in the hormone action remain undetermined. Finally, we questioned whether ANG II acts in the same DT segment as does parathyroid hormone on Ca(2+) transport. The two hormones increased Ca(2+) transport, but their actions were not additive, suggesting that they both influence the same channels in the same segment of the distal nephron, i.e., the segment responsible for the high-affinity calcium channel.

Roles of ATP and cytoskeleton in the regulation of Na+/H+ exchanger along the nephron luminal membrane.

Although in LLC-PK cells ATP depletion has been shown to result in alterations of cytoskeleton actin and an inhibition of Na+/H+ exchanger activity, there is little information concerning the regulation of this exchanger in the distal luminal membrane by ATP and actin filaments. The present study examined the direct effect of ATP and cytochalasin B on the Na+/H+ exchanger activity in the proximal and distal tubule luminal membranes. The presence of 100 microM ATP in the luminal membrane vesicles from rabbit proximal tubules did not influence the Ethyl Isopropyl Amiloride sensitive Na+ uptake by these membranes. In contrast, the same treatment of luminal membranes from distal tubules significantly enhanced the exchanger activity from 0.22 +/- 0.04 to 0.39 +/- 0.08 pM/microg/10 sec (P < 0.02). When ATP was replaced by its nonhydrolysable form, ATPgammas, the effect on the distal luminal membrane was strongly diminished suggesting that the action of the nucleotide implicates a phosphorylation step. Confirming this hypothesis, addition of 300-microM-Rp cAMP, a protein kinase A inhibitor, completely abolished the effect of ATP. In view of the fact that a tight relationship has been described between ATP, the cytoskeleton complex and the exchanger activity, we studied the effect of cytochalasin B on this activity. The presence of 20 microM cytochalasin B in the distal luminal membrane vesicles induced, as observed with ATP, a significant increase in the Na+ uptake. However, the actions of ATP and cytochalasin B were not additive. These results suggest that firstly, ATP and short actin filaments of the cytoskeleton regulate the distal luminal isoform through an intramembranous mechanism and secondly, a phosphorylation mechanism is, at least partially, implicated in the action of ATP. In contrast, the proximal tubule exchanger is regulated through different mechanisms.

The paradoxical effect of adrenomedullin on Na+ transport by the renal distal tubule luminal membrane.

Adrenomedullin (ADM) is a potent hypotensive and natriuretic peptide which is synthetized in several mammalian tissues including the kidney. The purpose of this study was to investigate whether the natriuresis was due to a change in Na+ transport by either the proximal (PT) or the distal tubule (DT) luminal membrane, and to characterize this effect, if present. PT and DT suspensions were incubated with human ADM for 20 min at 37 degrees C and luminal membranes of these tubules were purified using the Mg2+ precipitation technique. Na+ uptake was measured by the Millipore filtration technique. A volume of 10(-8) M ADM had no effect on Na+ uptake by the PT luminal membranes. In contrast and unexpectedly, the hormone increased Na+ transport by the DT membranes from 0.28 +/- 0.03 to 0.68 +/- 0.06 pmol/microg per 5 s (P < 0.01). The dose-response curve of this effect showed a maximal response with 10(-7) M ADM. The hormone influenced exclusively the Na+/H+ exchanger, leaving the N-ethyl-N-isopropyl-amiloride (EIPA) insensitive transport intact. The addition of Rp cAMPs to the preparations completely abolished the effect of the hormone on Na+ transport suggesting that cAMP was the messenger involved in this action. Finally, incubation of the DT suspensions with aldosterone also stimulated 1 mM Na+ uptake by the luminal membrane and the two hormone actions were not additive. We conclude that, although ADM is a natriuretic hormone probably through its vasodilating action, it increases distal Na+ reabsorption by the stimulation of the Na+/H+ exchanger activity, as does aldosterone at the same site.

Taxonomic and phylogenetic analysis of Saprolegniaceae (Oomycetes) inferred from LSU rDNA and ITS sequence comparisons.

The aim of this study was to improve our knowledge about the taxonomy and phylogeny of the family Saprolegniaceae, a group of water molds including several pathogens of plants, fish and crustacea. ITS and LSU rDNA were sequenced for representatives of forty species corresponding to ten genera (Achlya, Aphanomyces, Brevilegnia, Dictyuchus, Leptolegenia, Plectospira, Pythiopsis, Saprolegnia, Thraustotheca). Phenetic and cladistic analyses were then carried out. The species Brevilegnia bispora does not appear to belong to the family Saprolegniaceae. Plectospira myrianda clusters with Aphanomyces spp. and they constitute an ancestral group. (Thraustotheca clavata is closely related to the eccentric species of the genus Achlya. The genus Achlya appears polyphyletic, corroborating more or less the three known subgroups, defined by their sexual spore type (eccentric, centric and subcentric). The achlyoid type of spore dehiscence, shared by Aphanomyces and Achlya genera, is shown to be an ancestral character. The saprolegnioid, dictyoid and thraustothecoid types of spore dehiscence are derived characters but their relative evolutionary positions are not resolved.

Identification, characterization, and immunolocalization of a nucleoside triphosphate diphosphohydrolase in pig liver.

Different isoforms of nucleoside triphosphate diphosphohydrolases (NTPDases; EC 3.6.1.5), also identified as ATP diphosphohydrolases, have been previously described in mammalian tissues. We report here the biochemical characterization of NTPDases in the pig liver. Optimum pH of catalysis is more acidic for this enzyme than for NTPDases (neutral or alkaline pH) found in other mammalian tissues. It is less sensitive to bile salts than the bovine spleen NTPDase. Calculated Km values for ATP and ADP (31 and 21 microM, respectively) are slightly higher than those reported for the latter enzyme. Electrophoretograms of these enzymes also show different migration patterns. Western blots with Ringo, an antibody that recognizes the different isoforms of mammalian NTPDases, show a small but reproducible difference in estimated molecular masses (75 kDa for liver vs 78 kDa for spleen NTPDase). A second antibody, generated against a different sequence of NTPDase I, does not recognize the liver enzyme, thereby indicating some differences in primary structure. Immunolocalization produced a strong signal on hepatocytes, epithelial cells of the bile duct system, and vascular cells. Immunoreactivity was variable among hepatocytes of different lobules and among hepatocytes within a given lobule. In general, those located in the perilobular zone were more reactive than those located in the central zone and in the periphery of the centrolobular vein.

Characterization of the Na+/H+ exchanger in the luminal membrane of the distal nephron.

In the rabbit as well as the rat, a Na+/H+ exchanger is expressed in the apical membrane of both the proximal and distal tubules of the renal cortex. Whereas the isoform derived from the proximal tubule has been extensively studied, little information is available concerning the distal luminal membrane isoform. To better characterize the latter isoform, we purified rabbit proximal and distal tubules, and examined the ethylpropylamiloride (EIPA)-sensitive 22Na uptake by the luminal membrane vesicles from the two segments. The presence of 100 micron EIPA in the membrane suspension decreased the 15 sec Na+ uptake to 75.70 +/- 4.70% and 50.30 +/- 2.23% of the control values in vesicles from proximal and distal tubules, respectively. The effect of EIPA on 35 mM Na+ uptake was concentration dependent, with a IC50 of 700 micron and 75 micron for the proximal and distal luminal membranes. Whereas the proximal tubule membrane isoform was insensitive to cimetidine and clonidine up to a concentration of 2 mM, the 35 mM Na+ uptake by the distal membrane was strongly inhibited by cimetidine (IC50 700 micron) and modestly inhibited by clonidine (IC50 1.6 mM). The incubation of proximal tubule suspensions with 1 mM (Bu2) cAMP decreased the 15-sec EIPA-sensitive Na+ uptake by the brush border membranes to 24.1 +/- 2.38% of the control values. Unexpectedly, the same treatment of distal tubules enhanced this uptake by 46.5 +/- 10.3%. Finally, incubation of tubule suspensions with 100 nm phorbol 12-myristate 13-acetate (PMA) decreased the exchanger activity to 58.6 +/- 3.04% and 79.7 +/- 3.21% of the control values in the proximal and distal luminal membranes, respectively. In conclusion, the high sensitivity of the distal luminal membrane exchanger to various inhibitors, and its stimulation by cAMP-dependent protein kinase A, indicate that this isoform differs from that of the proximal tubule and probably corresponds to isoform 1.

Low divergence in rDNA ITS sequences among five species of Fucus (Phaeophyceae) suggests a very recent radiation.

Sequences from the two ribosomal DNA internal transcribed spacers (ITS1 and ITS2) were compared among five species of Fucus. Based on the present taxon sampling, parsimony analysis showed that Fucus serratus is the sister-group of the remaining Fucus species when Ascophyllum nodosum was used as an outgroup. The topology of the tree was (Fucus serratus (F. lutarius (F. vesiculosus (F. spiralis + F. ceranoides)))). The extremely low variation observed suggests a very recent radiation of the genus which supports the view widely accepted that the Fucales are among the most evolutionarily advanced of the brown algae. We further note that sequence differences between Fucus and Ascophyllum were 28%: this does not rule out the utility of ITS sequences within the Fucaceae. The very low number of informative positions allows to demonstrate empirically that distance matrix methods group on the basis of symplesiomorphies.

Effect of calcitonin on calcium transport by the luminal and basolateral membranes of the rabbit nephron.

In the rabbit, calcitonin has been shown to enhance calcium (Ca2+) reabsorption in the early distal tubule. The aim of the present study was to investigate the mechanism of this action, using isolated luminal and basolateral membranes of distal tubules. The tubule suspensions were preincubated in the presence or absence of 10(-7) M calcitonin. The luminal or basolateral membranes were subsequently purified and 45Ca transport through the vesicles was measured using the rapid filtration technique. Results were compared with those obtained from proximal tubule membranes. In the proximal tubules, calcitonin had no effect on Ca2+ uptake by luminal membranes. In the distal tubules, the presence of Na+ in the incubation medium strongly decreased the uptake of Ca2+ by luminal membranes. Preincubation of distal tubules with calcitonin partially restored this uptake. We previously reported a dual kinetics of Ca2+ uptake by the distal luminal membranes. Calcitonin enhanced Ca2+ transport by the low affinity component, increasing the Vmax and leaving the K(m) unchanged. Renal calcitonin receptors usually couple to both adenylate cyclase and phospholipase C. To determine through which messenger(s) calcitonin enhances Ca2+ transport by the distal tubules, we first confirmed that the hormone stimulates cAMP and IP3 release. Incubation of the distal tubules with 10(-7) M calcitonin significantly increased both messengers. In contrast, calcitonin did not influence the IP3 nor the cAMP content of proximal tubules. Therefore, we studied the actions of cAMP and phorbol 12-myristate 13 acetate (PMA) on Ca2+ transport by the distal luminal membranes. Incubation of distal tubule suspensions with dibutyryl cAMP significantly increased Ca2+ uptake by the luminal membranes. However, incubation of these tubules with various concentrations of PMA (10 nM, 100 nM and 1 microM) had no effect on this uptake. Calcitonin also influenced Ca2+ transport by the distal basolateral membrane. Incubation of distal tubule suspensions with 10(-7) M calcitonin activated the Na+/Ca2+ exchanger activity, almost doubling the Na+ dependent Ca2+ uptake. Here again this action was mimicked by cAMP. We conclude that calcitonin increases Ca2+ transport by the distal tubule through two mechanisms: the opening of low affinity Ca2+ channels in the luminal membrane and the stimulation of the Na+/Ca2+ exchanger in the basolateral membrane, both actions depending on the activation of adenylate cyclase.

Ca2+ transport by the luminal membrane of the distal nephron: action and interaction of protein kinases A and C.

We previously reported that parathyroid hormone and calcitonin increase Ca2+ uptake by purified distal luminal membranes. This effect is mimicked by high concentrations of cAMP. However, both hormones stimulate adenylate cyclase and phospholipase C. The purpose of the present study was to investigate the role of the phospholipase C pathway in the hormone action, and the interrelationship between the two messengers. Distal tubules from rabbit kidneys were incubated with dibutyryl cAMP (dbcAMP) or PMA, or both, and Ca2+ uptake by purified luminal membranes was measured by the rapid filtration technique. Incubation of the distal tubules with 1 mM dbcAMP significantly increased Ca2+ transport by the luminal membranes. A dose-response curve showed a half-maximal stimulation with 0.82 mM dbcAMP. In contrast, treatment of the tubules with 10 nM, 100 nM or 1 microM PMA did not influence Ca2+ uptake by these membranes. However, the addition of 100 nM PMA to low concentrations of dbcAMP strongly increased this uptake. The presence of cAMP or protein kinase C inhibitors prevented the effects of either a high concentration of dbcAMP alone or a low concentration of dbcAMP combined with 100 nM PMA. Our laboratory has already reported that Ca2+ uptake by the distal luminal membranes displays two-component kinetics. dbcAMP increased the Vmax of the low-affinity component, whereas a combination of the two messengers stimulated the Vmax of both the low- and high-affinity components. From these results, we conclude that: (1) in the distal tubule cells, activation of both protein kinases A and C is necessary for the stimulation of Ca2+ transport by the luminal membrane; (2) the combined effect of protein kinases A and C involves both components of the Ca2+-transport kinetics.

Detection of different adenosine triphosphatases in human placental brush border membranes.

The microvillous membrane of human placental syncytiotrophoblast cells contains a high ATPase activity. The purpose of this study was to characterize this activity and to investigate the presence of vacuolar type H+ ATPase in this membrane. Intact brush border membrane vesicles strongly hydrolyzed ATP, reflecting the presence of ATPase on the external side of the membrane. The ATPase activity was entirely Mg2+ dependent and increased with pH. At pH 7.5, Vmax was 31.0 +/- 1.7 mumol/mg/20 min and Km 0.18 +/- 0.03 mM ATP. Hydrolysis of ATP was not influenced by the presence of bicarbonate or alkaline phosphatase inhibitors, but at pH 8 it decreased by half following addition of 100 microM dicyclohexylcarbodiimide (DCCD). At pH 7.5, 1 mM N-ethylmaleimide (NEM) depressed this activity by less than 5%. Opening the membrane vesicles with 0.1% desoxycholate (DOC) or Triton-X neither revealed any additional ATPase activity nor altered the low sensitivity to NEM. Treatment of these membranes with 1% cholate decreased the ATPase activity by more than 70% and did not enhance the sensitivity of ATP hydrolysis to NEM. 10(-7) M Bafilomycin, which reduced by 56 +/- 9% the ATPase activity in dog kidney brush border membranes treated with 0.1% DOC, had no effect on placental brush border membranes subjected to the same procedure. Finally, neither immunocytochemical staining using monoclonal antibody to the M(r) 31000 subunit of V-type H+ ATPase, nor electron microscopic examination detected the presence of H(+) ATPase in placental membranes. In conclusion, the placental brush border membrane is the site of a strong "ecto" ATPase activity which is partially DCCD sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)

Depression of cytochrome P-450 in mouse liver induced by fractions from Nocardia opaca.

We measured the liver cytochrome P-450 content of mice 24 h after they had been injected with the following immunoadjuvants: Nocardia opaca derivatives and peptidoglycans from several bacterial strains. The cell wall fraction was not active, the others diminished liver cytochrome P-450 levels. The dose-response activity varied with the bacterial origin of the peptidoglycans. These findings indicate that the toxicity and efficiency of immunochemotherapeutic protocols can be modified by altering drug metabolism.

Adrenalectomy-induced increase of brain protein synthesis is antagonized by corticosterone replacements in free-moving rats.

The autoradiographic method with L-[35S]-methionine was used to determine whether changes in glucocorticoid circulating levels were associated with changes in local rates of protein synthesis in rat brain. Chronic bilateral adrenalectomy induced an increase of methionine incorporation rates into proteins in 60 of the 62 brain regions examined (mean effect, +50%). This effect was confirmed biochemically and quantified by correcting for the relative contribution of methionine derived from protein degradation to the precursor pool for protein synthesis in the whole brain. Acute or chronic administration of corticosterone, at doses that normalize basal levels of adrenocorticotrophic hormone, reversed or prevented the adrenalectomy-induced increase of protein synthesis in most regions. However, in nearly all the regions studied (59 of 62), acute corticosterone administration to sham-operated rats did not change the apparent rate of protein synthesis. These results demonstrate that glucocorticoids exert a generalized inhibitory action on brain protein synthesis, because the stimulatory and persistent effect of adrenalectomy on protein synthesis was antagonized by corticosterone replacements at physiological doses. Thus, the regulation of overall brain protein synthesis by glucocorticoids emphasizes the role of neuroendocrine events on long-term neurochemical processes.