Search

In the Tekorsh Sub-Watershed, East Gojjam Zone, Ethiopia, researchers investigated the impact of land use patterns and slope position on selected soil physico-chemical parameters. The study area was arbitrarily divided into three slope positions (higher, middle, and lower), two land uses types (grazing and cultivated land), and two soil depths (0–20 and 20–40 cm) with three replications, based on the in situ field survey. For laboratory analysis, a total of 36 composite samples were obtained. Sand, clay, and silt fraction were highly significantly (P ≤ 0.05) affected by the interaction effect of the three factors. Clay and clay loam were the textural classes of the soil in the study area. The interaction effects of the three factors were highly significant (P ≤ 0.001) affected bulk density (D b), total porosity (TP), organic carbon (OC), available phosphorus (AP), exchangeable (Mg2+, Ca2+, and acidity), cation exchange capacity (CEC), and micronutrients (Fe, Mn, Cu, and Zn). The soils were medium to high (1.22–1.44 g/cm3) in D b, very high (45.66–63.13%) in TP, medium to high (15.72–19.56% v/v) in available water holding capacity, low to medium (1.37–2.91%) in OC, very low (1.65–7.68 mg/kg) in AP, high (4.62–5.36 cmol(+)/kg) in exchangeable Mg2+, very high in CEC (43.60–51.06 cmol(+)/kg), Fe (25.20–52.91 mg/kg), Mn (37.29–105.55 mg/kg), Cu (4.04–7.87 kg/kg), and Zn (0.83 2.53 kg/kg). In general, it was discovered that the majority of the assessed soil properties were better in grazing land than in soils utilized for cultivated land uses, and that the lower slope position was preferable to the upper and middle ones.

The importance of Mg for the immune function is well recognized; however, there is no information available about the effect of Mg intake on the modulation of local immune response in the intestine. Thus, in the present study the hypothesis that short periods of Mg deprivation can affect intestinal mucosa and local immune response was tested. For this purpose, OF1 female mice were fed a semipurified diet (1000 mg Mg/kg diet). For 3 d before immunization and 1 d after, half of the animals were fed a Mg-deficient diet (30 mg Mg/kg diet), three immunizations per os were performed every 3 weeks with Escherichia coli producing the CS31A capsule-like protein (1010or 2??109 bacteria per animal). Mice were killed 10 d after the last immunization. The level of specific anti CS31A immunoglobulin (Ig) G and IgA in the serum and secretory IgA in the intestinal secretions and faeces were measured by ELISA. The results indicated that administration of a high dose of immunogen with a low-Mg diet led to lower specific IgA levels in the intestinal mucus and serum. Administration of a low dose of immunogen with a low-Mg diet led to lower IgA and IgG levels in the serum and secretory IgA coproantibodies. To assess alterations of intestinal mucosa caused by a low-Mg diet for a short period, histological and scanning electron microscopy analyses were performed on samples from mice (not submitted to the vaccination protocol) after 3 d on the Mg-deficient diet. These analyses showed several alterations, suggesting perturbations in the growth of the intestinal mucosa. These changes were accompanied by modifications in the expression of several genes involved in cell growth and stress response. From this present work, it may be concluded that short periods of Mg deprivation can affect the intestinal mucosa and local immune response of the intestine. [ABSTRACT FROM PUBLISHER]

There is a lack of agreement on index of Cu status and reliable and sensitive biomarkers are still required. The purpose of this present work was to assess in rats the sensitivity of diamine oxidase (DAO) activity, a recently proposed biomarker, to modifications in dietary Cu intake in comparison with other plasma biomarkers of Cu status. We also evaluated the effect of Cu dietary level on Cu and Zn intestinal absorption. Results showed that plasma Cu and plasma caeruloplasmin were significantly decreased at day 8 compared with the control group (7·4 mg Cu/kg diet) while DAO activity was significantly decreased at day 12 of the deficient diet (0·61 mg Cu/kg diet). Cu supplementation (35 mg Cu/kg diet) had no effect on any of the studied biomarkers of Cu status. In Cu-deficient rats plasma Cu and DAO activities were normalized 4 d after return to the control diet while caeruloplasmin was normalized later, at day 11. Apparent absorption values (%) of total Cu or 65Cu isotope were significantly increased in the Cu-deficient rats compared with the other groups and similar in the control and the Cu-supplemented groups. The urinary excretion of total Cu or 65Cu isotope were increased in the Cu-supplemented group compared with the other two groups. Both apparent absorption and urinary excretion of total Zn or 67Zn isotope remained unchanged in the three experimental groups. In conclusion, DAO activity seemed to be less sensitive to Cu deficiency than plasma Cu or caeruloplasmin concentrations. The present study also showed a significant increase in Cu intestinal absorption with dietary Cu restriction but no decrease with Cu supplementation in the rat. [ABSTRACT FROM PUBLISHER]

Since experimental Se deficiency results in a significant increase in plasma cholesterol concentration the present investigation was undertaken to assess further the influence of this deficiency on the expression of proteins involved in hepatic lipid metabolism. Se deficiency was induced by feeding weanling male Wistar rats on a deficient diet for 6 weeks. Hypercholesterolaemia associated with Se deficiency was related to increased 3-hydroxy-3-methylglutaryl-coA (HMG-CoA) reductase (EC 1.1.1.34) activity in liver microsomes as compared with control animals. Hepatic lipoprotein receptor levels (LDL-receptor and HDL-binding proteins, HB1 and HB2) were not significantly affected by Se deficiency, as assessed by immunoblotting. Plasma triacylglycerol concentrations tended to decrease in Se-deficient rats in concert with their reduced post-Triton secretion. There was no significant effect of Se deficiency on the hepatic synthesis of apolipoproteins. These results point to the need for further investigations into the mechanism related to the increased activity of HMG-CoA reductase and the enhanced cholesterogenesis in the liver of Se-deficient rats likely to result from this.Selenium: Cholesterol: Triacylglycerol: HMG-CoA reductase [ABSTRACT FROM PUBLISHER]

Perceptual cognitive skills in real game settings, under conditions of fatigue, such as the ability to gather relevant visual information, are key factors in achieving motor goals in sports. The objectives were to evaluate the effects of acute physical fatigue on gaze behavior during a badminton game (Study 1) and in an unfavorable force ratio situation (Study 2). Six international-level badminton players played two sets and unfavorable force ratio situations while wearing eye-tracking glasses before and after a fatiguing task. During the set, fatiguing physical exercise led to fewer fixations per exchange and more fixations on one area of interest. During unfavorable force ratio situations, fatiguing physical exercise led to shorter fixation durations per exchange, shorter fixation durations on two areas of interest, and longer fixation durations on one area of interest. The results showed that gaze behaviors were adapted in acute physical fatigue conditions to maintain performance.

Neural oscillations contribute to speech parsing via cortical tracking of hierarchical linguistic structures, including syllable rate. While the properties of neural entrainment have been largely probed with speech stimuli at either normal or artificially accelerated rates, the important case of natural fast speech has been largely overlooked. Using magnetoencephalography, we found that listening to naturally-produced speech was associated with cortico-acoustic coupling, both at normal (∼6 syllables/s) and fast (∼9 syllables/s) rates, with a corresponding shift in peak entrainment frequency. Interestingly, time-compressed sentences did not yield such coupling, despite being generated at the same rate as the natural fast sentences. Additionally, neural activity in right motor cortex exhibited stronger tuning to natural fast rather than to artificially accelerated speech, and showed evidence for stronger phase-coupling with left temporo-parietal and motor areas. These findings are highly relevant for our understanding of the role played by auditory and motor cortex oscillations in the perception of naturally produced speech., (Copyright © 2021. Published by Elsevier Inc.)

In badminton, the ability to quickly gather relevant visual information is one of the most important determinants of performance. However, gaze behavior has never been investigated in a real-game setting (with fatigue), nor related to performance. The aim of this study was to evaluate the effect of fatigue on gaze behavior during a badminton game setting, and to determine the relationship between fatigue, performance and gaze behavior. Nineteen novice badminton players equipped with eye-tracking glasses played two badminton sets: one before and one after a fatiguing task. The duration and number of fixations for each exchange were evaluated for nine areas of interest. Performance in terms of points won or lost and successful strokes was not impacted by fatigue, however fatigue induced more fixations per exchange on two areas of interest (shuttlecock and empty area after the opponent's stroke). Furthermore, two distinct gaze behaviors were found for successful and unsuccessful performance: points won were associated with fixations on the boundary lines and few fixation durations on empty area before the participant's stroke; successful strokes were related to long fixation durations, few fixation durations on empty area and a large number of fixations on the shuttlecock, racket, opponent's upper body and anticipation area. This is the first study to use a mobile eye-tracking system to capture gaze behavior during a real badminton game setting: fatigue induced changes in gaze behavior, and successful and unsuccessful performance were associated with two distinct gaze behaviors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Loiseau-Taupin, Ruffault, Slawinski, Delabarre and Bayle.)

Chronic Mg 2+ deficiency is the underlying cause of a broad range of health dysfunctions. As 25% of body Mg 2+ is located in the skeletal muscle, Mg 2+ transport and homeostasis systems (MgTHs) in the muscle are critical for whole-body Mg 2+ homeostasis. In the present study, we assessed whether Mg 2+ deficiency alters muscle fiber characteristics and major pathways regulating muscle physiology. C57BL/6J mice received either a control, mildly, or severely Mg 2+ -deficient diet (0.1%; 0.01%; and 0.003% Mg 2+ wt/wt, respectively) for 14 days. Mg 2+ deficiency slightly decreased body weight gain and muscle Mg 2+ concentrations but was not associated with detectable variations in gastrocnemius muscle weight, fiber morphometry, and capillarization. Nonetheless, muscles exhibited decreased expression of several MgTHs ( MagT1 , CNNM2 , CNNM4 , and TRPM6 ). Moreover, TaqMan low-density array (TLDA) analyses further revealed that, before the emergence of major muscle dysfunctions, even a mild Mg 2+ deficiency was sufficient to alter the expression of genes critical for muscle physiology, including energy metabolism, muscle regeneration, proteostasis, mitochondrial dynamics, and excitation-contraction coupling.

Food matrix interactions with polyphenols can affect their bioavailability and as a consequence may modulate their biological effects. The aim of this study was to determine if the matrix and its processing would modulate the bioavailability and the postprandial nutrigenomic response to a dietary inflammatory stress of apple flavan-3-ol monomers. We carried out an acute randomized controlled study in minipigs challenged with a high fat meal (HFM) supplemented with raw fruit, puree, or apple phenolic extract with matched content of flavan-3-ol monomers. Fasting and postprandial blood samples were collected over 3 h to quantify flavan-3-ol monomers in sera by UPLC-Q-TOF/MS and to isolate peripheral blood mononuclear cells (PBMCs) for assessing the changes in the gene expression profile using a microarray analysis. When compared to the extract-supplemented meal, the peak of the total flavan-3-ol concentration was reduced by half with both raw apple and puree supplements. The apple matrices also affected the gene expression profile as revealed by the Principal Component Analysis of the microarray data from PBMCs which discriminated the supplementation of HFM with the polyphenol extract from those with raw apples or puree. A total of 309 genes were identified as differentially expressed by the apple-derived products compared to HFM, with 63% modulated only in the presence of the food matrix (apple and puree). The number of differentially modulated genes was higher with the puree (246) than with the unprocessed apple (182). Pathway enrichment analyses revealed that genes affected by the apple-derived products control inflammation and leukocyte transendothelial migration both involved in the onset of atherosclerotic processes. Overall, this study showed that the two apple matrices reduce the postprandial serum concentration of flavon-3-ols whereas they increase the nutrigenomic response of PBMCs. The biological processes identified as modulated by the apple products suggest an attenuation of the transient pro-inflammatory response induced by a HFM. The differences observed between the nutrigenomic responses support that the apple matrix and its processing affect the nutrigenomic response, probably by increasing the bioavailability of other apple phytochemicals. To conclude, this study raises awareness for considering the impact of the food matrix and its processing on the biological response of polyphenols in nutritional studies.

Curcumin is a phenolic compound that exhibits beneficial properties for cardiometabolic health. We previously showed that curcumin reduced the infiltration of immune cells into the vascular wall and prevented atherosclerosis development in mice. This study aimed to investigate the effect of curcumin on monocyte adhesion and transendothelial migration (TEM) and to decipher the underlying mechanisms of these actions. Human umbilical vein endothelial cells (HUVECs) were exposed to curcumin (0.5-1μM) for 3h prior to their activation by Tumor Necrosis Factor alpha (TNF-α). Endothelial permeability, monocyte adhesion and transendothelial migration assays were conducted under static condition and shear stress that mimics blood flow. We further investigated the impact of curcumin on signaling pathways and on the expression of genes using macroarrays. Pre-exposure of endothelial cells to curcumin reduced monocyte adhesion and their transendothelial migration in both static and shear stress conditions. Curcumin also prevented changes in both endothelial permeability and the area of HUVECs when induced by TNF-α. We showed that curcumin modulated the expression of 15 genes involved in the control of cytoskeleton and endothelial junction dynamic. Finally, we showed that curcumin inhibited NF-κB signaling likely through an antagonist interplay with several kinases as suggested by molecular docking analysis. Our findings demonstrate the ability of curcumin to reduce monocyte TEM through a multimodal regulation of the endothelial cell dynamics with a potential benefit on the vascular endothelial function barrier., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Whereas the anti-inflammatory properties and mechanisms of action of long chain ω3 PUFAs have been abundantly investigated, research gaps remain regarding the respective contribution and mechanisms of action of their oxygenated metabolites collectively known as oxylipins. We conducted a dose-dependent and comparative study in human primary macrophages aiming to compare the anti-inflammatory activity of two types of DHA-derived oxylipins including the well-described protectins (NPD1 and PDX), formed through lipoxygenase pathway and the neuroprostanes (14-A 4t - and 4-F 4t -NeuroP) formed through free-radical mediated oxygenation and expected to be new anti-inflammatory mediators. Considering the potential ability of these DHA-derived oxylipins to bind PPARs and knowing the central role of these transcription factors in the regulation of macrophage inflammatory response, we performed transactivation assays to compare the ability of protectins and neuroprostanes to activate PPARs. All molecules significantly reduced mRNA levels of cytokines such as IL-6 and TNF-α, however not at the same doses. NPD1 showed the most effect at 0.1µM (-14.9%, p<0.05 for IL-6 and -26.7%, p<0.05 for TNF-α) while the three other molecules had greater effects at 10µM, with the strongest result due to the cyclopentenone neuroprostane, 14-A 4t -NeuroP (-49.8%, p<0.001 and -40.8%, p<0.001, respectively). Part of the anti-inflammatory properties of the DHA-derived oxylipins investigated could be linked to their activation of PPARs. Indeed, all tested oxylipins significantly activated PPARγ, with 14-A 4t -NeuroP leading to the strongest activation, and NPD1 and PDX also activated PPARα. In conclusion, our results show that neuroprostanes and more especially cyclopentenone neuroprostanes have potent anti-inflammatory activities similar or even more pronounced than protectins supporting that neuroprostanes should be considered as important contributors to the anti-inflammatory effects of DHA., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The orbitofrontal cortex (OFC) plays a key role not only in processing emotions but also in monitoring performance outcome. Although the neuroanatomical substrates underlying each of the two processes have been extensively investigated, they have predominantly been probed separately and therefore a precise knowledge of the functional overlap within the multiple OFC sub-portions involved is still lacking. Here, we explore the neural dynamics mediating performance monitoring and emotional processing using direct intracranial EEG (iEEG) recordings from multiple OFC sites of an epileptic patient. Neural activity was recorded during two experiments. The first task required processing of emotional faces and the second investigated action outcome evaluation based on a visual feedback on the subject's performance. Task-related neural dynamics were assessed using modulations of high frequency responses in the gamma-band (50-150Hz). Our results reveal that processing negative facial emotions as well as receiving negative feedback both elicited gamma-band responses in the lateral OFC. By contrast, the mid-OFC was selectively activated for positive feedback. Furthermore, we also found significant gamma-band deactivation in the gyrus rectus during processing of negative feedback. Our findings provide novel evidence for an intricate valence-selective interaction between the networks mediating emotion processing and performance monitoring in human OFC and support the hypothesis of a tight relationship between gamma-band activity and behavior., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Objective: The aim of this study was to disclose the dynamics of the frontal processes involved in a task shifting between two attentional states., Methods: Magnetoencephalographic activities were recorded during a Wisconsin Card Sorting Test where subjects had to match card stimuli according to one of three possible dimensions ("maintained condition"). The matching dimension was intermittently changed and subjects, after feedback presentation, had to identify the new correct dimension ("shifted condition")., Results: Source activations following the feedback to the subject's response in these two attentional conditions did not differ before 350 ms post feedback. After 350 ms, in the shifted condition, a lateral/posterior frontal activation was maintained later, while a medial/anterior frontal activation appeared up to 450 ms., Conclusions: The dynamics of activities corresponding to the two conditions disclose a spread of activation from posterior lateral frontal in the "maintained condition" to anterior medial frontal in the "shifted condition"., Significance: These results are consistent with fMRI results concerning the major involvement of medial frontal cortex in tasks involving reasoning and choice making., (2009 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.)

The magnesium concentrations in plasma and cells could be affected by diet, disease and genetic factors. To characterise the genetic factors involved in the regulation of magnesium homeostasis, Low (MgL) and High (MgH) magnesium status mice were developed by bidirectional selective breeding. The effects of a low-magnesium diet on the magnesium status parameters were analysed in these mice. Using a cDNA array, a screen for differential gene expression was performed in kidneys from these animals, fed either a magnesium adequate or deficient diet. The magnesium-deficient diet significantly affected the plasma, erythrocyte and urine magnesium concentrations in'both strains, in similar proportions in the two strains. Furthermore, in response to the magnesium-deficient diet, both strains showed changes of the expression of genes belonging, for the majority of them, to the family of transcription and growth factors (down-regulated). Of the identified genes, five were of particular interest because they were differently expressed in response to the deficient diet in these two strains: osteopontin, the cholecystokinin A receptor, connexin 45, a growth hormone receptor and BAG1. These results suggest that the two strains exhibit different physiopathological responses to magnesium deficiency.

It has been well documented that experimental hypomagnesemia in rodents evokes, as an early consequence, an inflammatory response. This also leads to the activation of cells producing reactive species of oxygen and, as a result, to the oxidative damage of tissues. Several studies have shown that lungs might be a specific target of Mg deficiency. Here, we report that 3 weeks of Mg deficiency in mice resulted in inflammatory processes in the lungs, including interstitial and perivascular pneumonia, manifested by the infiltration of leukocytes, plasmocytes and histiocytes, as well as the phenomenon of disseminated intravascular coagulation (DIC). These phenomena were accompanied by changes in gene expression assessed by cDNA array. In this study we identified 26 genes significantly changed by Mg deficiency, mostly involved in the anti-oxidative response, regulation of cell cycle and growth, apoptosis as well as cell-cell and cell-matrix interactions. We conclude that these changes are related to the phenomena of inflammatory and oxidative processes and consecutive remodeling occurring in the tissues as a result of Mg deficiency. This may have implications for at least several lung pathologies, including allergies, asthma, SIDS (Sudden Infant Death Syndrome) or facilitate formation of lung metastases.

Recently discovered peptide-hepcidin (Hepc) may be a central player in the communication of iron body stores to the intestinal absorptive cells and thus involved in the maintenance of iron homeostasis. The aim of this study was to determine the effects of the level of dietary iron on Hepc gene expression in the liver. OF1 male mice were fed for 3 weeks either control diet (35 mg iron/kg diet), low-iron diet (1 mg iron/kg diet), or high-iron diet (500 mg iron/kg diet), and Hepc 1 and 2 mRNA abundance in the liver was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Results clearly showed that Hepc gene expression is upregulated by high dietary iron and downregulated when the dietary iron level is low. Both Hepc 1 and Hepc 2 expression responds coordinately to dietary iron. This work provides additional evidence of the key role of Hepc in the regulation of iron homeostasis.

Magnesium deficiency in experimental animals leads to inflammation, exacerbated immune stress response and a decrease of specific immune response. It also results in a significant increase in free radical species and subsequent tissue injury. An accelerated thymus involution was observed in Mg-deficient rats in relation to enhanced apoptosis and enhanced susceptibility to oxidative stress. To examine the stress-inducing effects of low Mg status on thymocytes, cDNA arrays were used to evaluate changes in gene expression in weaning rats submitted to Mg deficiency of short duration (2 days). Several genes exhibited changes in their expression caused by Mg deficiency before any perceptible modification in cell integrity and functions. The up-regulated genes included cytochrome c oxidase, glutathione transferase, CuZn superoxide dismutase, genes associated with the stress response (HSP70 and HSP84) and a gene involved in DNA synthesis and repair (GADD45). The down-regulated genes included Na/P cotransporter 1. These findings are consistent with altered cell growth, modifications of ion fluxes and oxidative stress described during Mg deficiency. The observation of induction of genes involved in protection and repair in cells from Mg-deficient animals provides additional evidence of the role of oxidative stress in the pathobiology of this deficiency.

The transmembrane topology of Na(+)/H(+) exchanger NHE3 has been studied using in vitro transcription/translation of two types of fusion vectors designed to test membrane insertion properties of cDNA sequences encoding putative NHE3 membrane spanning domains (msds). These vectors encode N-terminal 101 (HKM0) or 139 (HKM1) amino acids of the H,K-ATPase alpha-subunit, a linker region and a reporter sequence containing five N-linked glycosylation consensus sites in the C-terminal 177 amino acids of the H,K-ATPase beta-subunit. The glycosylation status of the reporter sequence was used as a marker for the analysis of signal anchor and stop transfer properties of each putative msd in both the HKM0 and the HKM1 vectors. The linker region of the vectors was replaced by sequences that contain putative msds of NHE3 individually or in pairs. In vitro transcription/translation was performed using [(35)S]methionine in a reticulocyte lysate system +/- microsomes, and the translation products were identified by autoradiography following separation using SDS-PAGE. We propose a revised NHE3 topology model, which contains a cleaved signal peptide followed by 11 msds, including extracellular orientation of the N-terminus and intracellular orientation of the C-terminus. The presence of a cleavable signal peptide in NHE3 was demonstrated by its cleavage from NHE3 during translational processing of full-length and truncated NHE3 in the presence of microsomes. Of 11 putative msds, six (msds 1, 2, 4, 7, 10, and 11) acted as both signal anchor and stop transfer sequences, while five (msds 3, 5, 6, 8, and 9) had signal anchor activities when tested alone. Of the latter, 3, 5, 6, and 9 were shown to act as stop transfer sequences after C-terminal extension. The actual membrane orientation of each sequential transmembrane segment of NHE3 was deduced from the membrane location of the N- and C-termini of NHE3. The regions between putative msds 8 and 9 and between msds 10 and 11, which correspond to the fourth and fifth extracellular loops, did not act as msds when tested alone. However, the extension of the fifth extracellular loop with adjacent putative msds showed some membrane-associated properties suggesting that the fifth extracellular loop might be acting as a "P-loop"-like structure.

On adhesion to host cells the flagellate Trichomonas vaginalis switches to an amoeboid form rich in actin microfilaments. We have undertaken the identification of actin-associated proteins that regulate actin dynamics. A monoclonal antibody 4C12 raised against a cytoskeletal fraction of T. vaginalis labeled a protein doublet at circa 50 kDa. These two bands were recognized by the antibody against Dictyostelium discoideum coronin. During cell extraction and actin polymerization, T. vaginalis coronin cosedimented with F-actin. By two-dimensional gel electrophoresis, the protein doublet was separated into two sets of isoforms covering two Ip zones around 6 and 7. By screening a T. vaginalis library with 4C12, two clones Cor 1 and Cor 2 were isolated. This gene duplicity is a particularity among unicellular organisms examined. The complete sequence of the gene Cor 1 encodes a 435-residue protein with a calculated molecular mass of 48 kDa and Ip of 5.58. The incomplete sequence Cor 2 was very similar but with a more basic calculated Ip than Cor 1 on the same region. T. vaginalis coronin had 50% similarity with the coronin family, possessing the five WD-repeats and a leucine zipper in its C-terminal part. Double immunofluorescence labeling showed that coronin mainly colocalized with actin at the periphery of the adherent amoeboid cells. However, coronin labeling displayed patches within a reticular array. Immunogold electron microscopy confirmed the coronin labeling in the actin-rich microfilamentous fringe beneath the plasma membrane, with accumulation in phagocytic zones and pseudopodial extensions. In T. vaginalis, one of the first emerging lineage of eukaryotes, coronin seems to play an important role in actin dynamics and may be a downstream target of a signaling mechanism for the cytoskeleton reorganization.

Three distinct P type pumps were cloned from H. pylori 69A. Two of these pumps, ATPase 439 and ATPase 948 (CopA), were isolated by gene library screening using DNA oligonucleotide primers. Amino acid similarities found for the predicted proteins were about 50% to Cd2+/Cu2+ pumps. Gene disruption mutagenesis rendered the H. pylori knockout mutants more sensitive to Zn2+ and Cd2+ (ATPase 439) or Cu2+ (CopA). Some of the ATPase 439-deficient mutants were negative for urease activity while the majority of the mutants remained positive. Functional diversity of the pumps was also reflected by the ion affinities found for N-terminal peptides of CopA to Cu2+ and of ATPase 439 to Ni2+, Cu2+ and CO2+. The membrane domain of the two pumps were experimentally shown to consist of eight membrane spans. When ATPase 439 was expressed under control of a tac promoter in Escherichia coli, vanadate-sensitive phosphate accumulation was observed cytochemically along the membrane of the host cells. The third P type pump (ATPase 115) which also exhibited homology to transition metal ATPase was identified by sequencing a library of H. pylori membrane genes. The hydropathy plot of this pump was very similar to the former H. pylori ATPases whereas the N-terminal ion binding region was distinct. It was concluded that, in H. pylori, the presence of three transition metal ATPases with distinct ion specificity contributes to the adaptive mechanisms for gastric survival.

The alpha subunit of eukaryotic P-type ATPases has ten experimentally defined transmembrane or membrane inserted segments. The fifth and sixth of these are short, not predicted by hydropathy analysis, do not insert independently into microsomal membranes, and are readily removed after tryptic digestion and therefore may be membrane inserted sequences. Acid transport by the gastric H, K-ATPase is covalently inhibited by several substituted pyridyl methylsulfinyl benzimidazoles, such as omeprazole. These act as probes of accessible extracytoplasmic thiols because they are accumulated in the acid transporting gastric vesicles and then convert to thiol reactive, cationic tetracyclic sulfenamides. Inhibition is due mainly to disulfide formation with Cys813 or Cys822 in M5/6 and perhaps with a contribution from Cys892 in the loop between transmembrane segment (TM) 7 and TM8. Identification of the specific cysteine responsible for inhibition should be able to define the turn between M5 and M6. The gastric H,K-ATPase alpha-beta heterodimer was expressed as a fusion protein in HEK 293 cells. Transient transfection resulted in most of the protein being retained in the endoplasmic reticulum with only core glycosylation and minor activity of the ATPase evident. Stable transfection resulted in plasma membrane localization of the protein and complex glycosylation. The transfected but not the control cells displayed cation-stimulated, SCH 28080-inhibited ATPase activity and SCH 28080- and omeprazole-inhibited 86Rb uptake. The two cysteines in M5/6 and Cys892 in the TM7/8 loop were mutated to the amino acids found in the Na,K-ATPase in order to determine which of the three cysteine residues were important for benzimidazole inhibition. Mutation of one, two, or all three cysteines did not alter enzyme activity, 86Rb transport, or SCH 28080 inhibition. Only removal of Cys822 blocked omeprazole inhibition of 86Rb transport. These data suggest that Cys822 is present in a region of the enzyme most easily accessed by the cationic sulfenamide formed by omeprazole, presumably the turn between M5 and M6.

The cop operons of Helicobacter pylori and Helicobacter felis were cloned by gene library screening. Both operons contain open reading frames for a P-type ion pump (CopA) with homology to Cd2+ and Cu2+ ATPases and a putative ion binding protein (CopP), the latter representing a CopZ homolog of the copYZAB operon of Enterococcus hirae. The predicted CopA ATPases contained an N-terminal GMXCXXC ion binding motif and a membrane-associated CPC sequence. A synthetic N-terminal peptide of the H. pylori CopA ATPase bound to Cu2+ specifically, and gene disruption mutagenesis of CopA resulted in an enhanced growth sensitivity of H. pylori to Cu2+ but not to other divalent cations. As determined experimentally, H. pylori CopA contains four pairs of transmembrane segments (H1 to H8), with the ATP binding and phosphorylation domains lying between H6 and H7, as found for another putative transition metal pump of H. pylori (K. Melchers, T. Weitzenegger, A. Buhmann, W. Steinhilber, G. Sachs, and K. P. Schäfer, J. Biol. Chem. 271:446-457, 1996). The corresponding transmembrane segments of the H. felis CopA pump were identified by hydrophobicity analysis and via sequence similarity. To define functional domains, similarly oriented regions of the two enzymes were examined for sequence identity. Regions with high degrees of identity included the N-terminal Cu2+ binding domain, the regions of ATP binding and phosphorylation in the energy transduction domain, and a transport domain consisting of the last six transmembrane segments with conserved cysteines in H4, H6, and H7. The data suggest that H. pylori and H. felis employ conserved mechanisms of ATPase-dependent copper resistance.

To determine which amino acid sequences account for transmembrane folding of G7 receptors, the membrane domain of the rabbit cholecystokinin-A (CCK-A) G-protein-coupled receptor has been investigated by in vitro transcription/translation of two types of fusion vectors containing sequences that include putative transmembrane segments. First, the seven putative transmembrane domains of the CCK-A receptor were inserted individually into pGEM vectors beginning with the cDNA encoding the first 101 (HK-M0) or 139 (HK-M1) amino acids of the alpha subunit of the gastric H, K-ATPase. These were separated by the cDNA for the inserted transmembrane domains from the cDNA encoding the last 177 amino acids of the beta subunit of the H,K-ATPase containing five N-linked glycosylation consensus sequences (Bamberg, K., and Sachs, G. (1994) J. Biol. Chem. 269, 16909-16919). Transcription/translation of these fusion vectors in rabbit reticulocyte lysate +/- dog pancreatic microsomes followed by SDS-polyacrylamide gel electrophoresis defined the presence of signal anchor sequences in HK-M0 by glycosylation and stop transfer sequences in HK-M1 by inhibition of glycosylation. Six out of the seven putative transmembrane domains had membrane insertion signals, but no membrane insertion activity was found for the H3 segment in these vectors. To test the effect of specific upstream and downstream sequences on membrane insertion, vectors were also made starting with the cDNA encoding the N terminus of the CCK-A receptor separated from the last 177 amino acids of the H,K-ATPase beta subunit by cDNA encoding CCK-A receptor sequences of different lengths. In addition to transcription/translation, endoglycosidase H treatment was used to verify glycosylation when multiple bands were found in the presence of microsomes. The four positive charges in the loop between H1 and H2 were required for the correct orientation of the first transmembrane domain. The H3 segment acted as a stop transfer sequence only when the whole N terminus and H3 were followed by the positive charges in the cytoplasmic loop between H3 and H4. The activity of H6 as a signal anchor sequence depended on preceding positive charges. These translation data using two types of fusion vectors establish a seven-transmembrane folding model using only in vitro translation for the CCK-A receptor beginning with two signal anchor sequences and then alternating stop transfer and signal anchor insertions. Positive charges between H1 and H2, H3 and H4, and H5 and H6 function as cytoplasmic anchors in the membrane folding of this receptor.

A method of in vitro translation scanning was applied to a variety of polytopic integral membrane proteins, a transition metal P type ATPase from Helicobacter pylori, the SERCA 2 ATPase, the gastric H+,K+ ATPase, the CCK-A receptor and the human ileal bile acid transporter. This method used vectors containing the N terminal region of the gastric H+,K+ ATPase or the N terminal region of the CCK-A receptor, coupled via a linker region to the last 177 amino acids of the beta-subunit of the gastric H+,K+ ATPase. The latter contains 5 potential N-linked glycosylation sites. Translation of vectors containing the cDNA encoding one, two or more putative transmembrane domains in the absence or presence of microsomes allowed determination of signal anchor or stop transfer properties of the putative transmembrane domains by the molecular weight shift on SDS PAGE. The P type ATPase from Helicobacter pylori showed the presence of 8 transmembrane segments with this method. The SERCA 2 Ca2+ ATPase with this method had 9 transmembrane co-translational insertion domains and coupled with other evidence these data resulted in a 11 transmembrane segment model. Translation of segments of the gastric H+,K+ ATPase provided evidence for only 7 transmembrane segments but coupled with other data established a 10 membrane segment model. The G7 protein, the CCK-A receptor showed the presence of 6 of the 7 transmembrane segments postulated for this protein. Translation of segments of the human ileal bile acid transporter showed the presence of 8 membrane insertion domains. However, translation of the intact protein provided evidence for an odd number of transmembrane segments, resulting in a tentative model containing 7 or 9 transmembrane segments. Neither G7 type protein appeared to have an arrangement of sequential topogenic signals consistent with the final assembled protein. This method provides a useful addition to methods of determining membrane domains of integral membrane proteins but must in general be utilized with other methods to establish the number of transmembrane alpha-helices.

The membrane topology of the rat endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR) Ca2+ ATPases were investigated using in vitro transcription/translation of fusion vectors containing DNA sequences encoding putative membrane-spanning domains. The sequences of these Ca2+ ATPases are identical except for the COOH-terminal end, which contains an additional predicted transmembrane segment in the ER ATPase. The M0 and M1 fusion vectors (Bamberg, K., and Sachs, G. (1994) J. Biol. Chem. 269, 16909-16919) encode the NH2-terminal 101 (M0 vector) or 139 (M1 vector) amino acids of the H,K-ATPase alpha subunit followed by a linker region for insertion of putative transmembrane sequences and, finally, the COOH-terminal 177 amino acids of the H,K-ATPase beta subunit containing five N-linked glycosylation consensus sequences. The linker region was replaced by the putative transmembrane domains of the Ca2+ ATPases, either individually or in pairs. Transcription and translation were performed using [35S]methionine in a reticulocyte lysate system in the absence or presence of canine pancreatic microsomes. The translated fusion protein was identified by autoradiography following separation using SDS-polyacrylamide gel electrophoresis. When testing single transmembrane segments, this method detects signal anchor activity with M0 or stop transfer activity with M1. The first four predicted SERCA transmembrane domains acted as both signal anchor and stop transfer sequences. A construct containing the fifth predicted transmembrane segment was able to act only as a stop transfer sequence. The sixth transmembrane segment did not insert cotranslationally into the membrane. The seventh was able to act as both a signal anchor and stop transfer sequence, and the eighth showed stop transfer ability in the M1 vector. The ninth transmembrane segment had both signal anchor and stop transfer capacity, whereas the tenth transmembrane segment showed only stop transfer sequence properties. The eleventh transmembrane sequence, unique to the ER Ca2+ ATPase, had both signal anchor and stop transfer properties. These translation data provide direct experimental evidence for 8 or 9 of the 10 or 11 predicted transmembrane sequences in the current topological models for the SR or ER Ca2+ ATPases, respectively.

Early chick embryos contain two beta-galactoside-binding lectins of 16 kDa and 14 kDa. using several antisera to these proteins, we have studied lectin expression at embryonic stages when the segregation and early differentiation of organ primordia are taking place. With antisera to the 16 kDa lectin that display similar immunoreactivity in immunoblot analysis, we show that these antisera exhibit varying immunoreactivity in embryo sections. One antiserum reacts preferentially with a matrix form of lectin while another detects mainly a cellular form of this protein. During early development, galactoside-binding lectins of the matrix type are expressed in the vitelline membrane, the outer and inner limiting membranes of the neural tube, the surface of the notochord and the coelomic surface of the cardiac rudiments. The cellular form of the lectin occurs in the intracellular yolk of early embryos, in the primordial germ cells, the myocardium, in the early myotome, and in a cohort of cells which are presumed to belong to the neural crest. Our results indicate that, although all of the antisera recognize the intracellular lectin of the extraembryonic endoderm, some antisera to the 16 kDa lectin exhibit preferential reactivity with different lectin isoforms. The extracellular matrix form of lectin is transiently expressed during early development at the stages when the segregation of organ primordia is occurring. It's expression could be related to the acquisition of polarity in developing epithelia. Results also suggest that various versions of the same protein may perform distinct developmental roles in the embryo.

In a previous work, resting and acid-secreting rabbit gastric mucosa were freeze-fractured and shadowed at 45 degrees with Pt-C. The shadow widths of proteic particles of tubulovesicle and canaliculus membranes were measured and compared. It was concluded that the frequency distributions of widths are significantly different in resting and secreting membranes and that each distribution accounts for several subpopulations of homogeneous particles. In the present study, an attempt is made to describe the experimental distributions as a mixture of those of two major proteins, say A and B and their aggregates (AA, AB and BB). The modelling, although simple, gave a very satisfactory statistical fit between observed and computed distributions. The comparison of parameters calculated from histamine and ranitidine experimental data further improves the fits and finally, component A accounts for 69% of the particles. Most replica of A particles are heart-shaped and the median shadow widths are 6.1 and 6.8 nm in canaliculus and tubulovesicles respectively. The component B accounts for 31% of the particles. They mainly appear as small barrels and the median shadow widths are 8.8 and 10.3 nm in canaliculus and tubulovesicles respectively. According to calculated parameters and observed particle replica, the onset of secretion does not change the relative ratio of proteins but changes their shapes. Component A should be the (H+,K+)-ATPase whereas debate on the identity of B is wide open.

The location of soluble lactose-binding proteins (S-lac lectins) has been studied by immunohistochemical methods during morphogenesis of the chick embryo, when segregation and early differentiation of organ primordia was occurring. Using a panel of polyclonal antisera raised to various purified lectin preparations, we observed striking differences in the antigenic properties of these antisera, indicating that diverse versions of the lectins may be expressed during development. The antisera referred to as anti-L-16, anti-M-16, anti-S-14 and anti-I-14 were respectively raised to native or denatured 16 kDa lectins from adult liver and embryonic muscle and to 14 kDa lectins from embryonic skin and adult intestine. Having determined the optimal immunohistochemical conditions in the preparation of embryo sections (fixation, embedding, sectioning) we show that anti-L-16, anti-S-14 and anti-I-14 mostly bind the lectins expressed at the cell surface, in the extracellular matrix and in some released secretion. As previously shown, anti-L-16 and anti-S-14 are also able to recognize the cytoplasmic form of some migrative lectin-rich cells (primitive streak, neural crest cells, germ cells). Anti-M-16 was bound exclusively to the cytoplasmic form of the 16 kDa lectin in the same cell lines as above and also in some others, such as in the notochord, the myotomal part of the somites, the pharyngeal endoderm and the cardiac muscle. These different antigenic properties may be applied to the accurate mapping of various lectin isoforms and evaluation of the respective contribution of their intra- and extracellular variants during development and differentiation.

Several antibodies against the gastric H+/K(+)-ATPase were analysed for the topological and sequence location of their epitopes. Topological mapping was done by comparing indirect immunofluorescent staining in intact and permeabilised rat parietal cells. Epitope definition was by Western analysis of intact and of trypsin or V8-proteinase-fragmented hog gastric ATPase combined with N-terminal sequencing of the fragments; by Western analysis of fragments of rabbit alpha subunit expressed in Escherichia coli; by analysis of rabbit alpha and beta subunits expressed in baculovirus-transfected SF 9 cells and by ELISA assay of synthetic octamers of one region of the hog alpha subunit. It was confirmed that the monoclonal antibody, mAb 95-111, recognised a cytoplasmic region between M4 and M5, close to the ATP-binding domain. The major epitope for monoclonal antibody mAb 12-18 was also in this region, but a second epitope was confirmed to be present in the M7/M8 region. The monoclonal antibody, mAb 146-14, was shown to recognise an extracytoplasmic epitope dependent on intact disulfide bonds, present in the rat and the rabbit, but absent in the hog beta subunit, due to non-conservative amino-acid substitutions. This antibody also recognised an epitope present in the alpha subunit of the H+/K(+)-ATPase at the M7 extracytoplasmic interface, perhaps indicating structural association of these two regions. The polyclonal antibody, pAb39, raised against the C-terminal portion of the enzyme, reacted only with the cytoplasmic surface of the H+/K(+)-ATPase, showing that the alpha subunit of the enzyme has an even number of membrane spanning segments.

In gastrinomas, as well as in other endocrine tumors whose hormone overproduction is responsible for clinical syndromes, antibodies against the bioactive form(s) of hormones can fail to detect immunoreactivity. Moreover, tumor secretory granule morphology may fail to allow tumor type identification. The use of anti-pre-pro-gastrin antibodies has been proposed as an alternative to identify gastrinomas. The aim of the present study was to demonstrate that in situ detection of gastrin mRNA may represent another possibility. A 35S-labeled cDNA probe encoding the human gastrin pre-pro-hormone was used to localize gastrin gene transcripts in antral mucosa and digestive endocrine tumors from patients with a Zollinger-Ellison syndrome characterized by high serum gastrin levels. In situ hybridization was combined with light and electron microscopic immunostaining of the bioactive gastrin 17/34 form and morphological study of secretory granules. Gastrin mRNAs were detected in antral gastrin cells and in a variable proportion of tumor cells in all endocrine tumor studied. Transcript expression correlated well with immunohistochemical staining and granule ultrastructure for most of the tumors, and provided crucial evidence for identifying as gastrinomas two tumors with weak immunoreactivity and poorly granulated cells. Our data show that in situ hybridization is a sensitive method for gastrin mRNA detection and represents a valuable tool for the identification of gastrinomas.

1. The tubulovesicles of hog and rabbit gastric parietal cells were immunopurified from microsomes using monoclonal antibodies against the (H+, K+)-ATPase. 2. The best yields of immunoprecipitation were obtained with an ATPase/mAb molar ratio of 0.3: the immunoprecipitate contained 79 and 90% of the hog and rabbit microsomal PNPPase activity respectively and K(+)-stimulated ATPase specific activity was 221 +/- 29 mumoles Pi per hr and per mg of membrane protein. 3. The immunoprecipitate contained vesicles that were 85% cytoplasmic-side out, like tubulovesicles in vivo, demonstrating that the epitopes were cytoplasmic. 4. The alpha-beta protomer of (H+, K+)-ATPase accounted for 80 +/- 12% of the immunopurified proteins. 5. The major other proteins ran at 80, 75, 69, 57, 47, 44, 39, 34 and 32 kDa on the SDS-PAGE. 6. Comparative analysis between sucrose-gradient purified fractions and immunopurified tubulovesicles demonstrated that carbonic anhydrase and actin were contaminants and that the 53 kDa and presumably the 50 kDa bands of the gradient fraction were alpha and beta subunits of F1 ATPase.

In an attempt to elucidate the role of the dietary fermentable fiber in reduction of hyperlipidemia, we substituted 30% wheat starch with 30% sugar-beet fiber in rats fed a fructose-based (41% fructose), low-fat (2% corn oil) diet. Male Wistar rats ate the test diets for 3 weeks. Feeding the sugar-beet fiber (SBF) diet resulted in a significant enlargement of the cecum; it also increased the concentration of volatile fatty acids compared with rats fed a fiber-free (FF) diet. Feeding SBF decreased plasma triglyceride and cholesterol concentrations in the postprandial as well as the postabsorptive period. In the liver, triglyceride levels were depressed in concert with the decreased liver lipogenesis and the post-Triton triglyceride secretion. Liver cholesterol levels were unaffected by SBF diet feeding. SBF-fed animals were markedly less fat compared with fiber-free-diet-fed rats. Adipose tissue lipogenesis was depressed in the postprandial period in SBF-fed animals. In short, this study suggests that substitution of easily digested carbohydrates by certain fermentable fibers may play an interesting role in the reduction of hyperlipidemia and obesity.

The effect of the dietary fiber on apo B mRNA level was studied in the intestine of rats that were fed either fiber-free or high-fiber (30% sugar-beet fiber) low-fat diets for 3 weeks. The fiber diet studied does not affect jejunal apo B mRNA levels but decreases the level of ileal apo B mRNA. In the rat cecum, in both fiber-free and fiber groups, we failed to detect the apo B mRNA. The test fiber diet feeding markedly increased fecal bile salt and cholesterol excretions. We suggest that dietary fiber can modify apo B expression in the intestine. The increased fecal bile salt excretion might be involved in such a modification.

The fundic mucosa of resting and acid-secreting rabbit stomachs were freeze-fractured and replicated to compare the intramembranous particles on the parietal cell tubulovesicles (rest) and canaliculus (secretion). The particles were counted and their shadow diameters were measured using an image analysis program. The tubulovesicles bore 9,726 +/- 400 particles per microns2 (mean +/- SD), having a mean diameter of 8.4 nm (n = 2,571). The canaliculus bore 8,369 +/- 430 particles per microns2, having a mean diameter of 7.7 nm (n = 3,259). The data were reproducible: three fractures of tubulovesicles and canaliculus gave essentially the same distributions of particle diameters. By contrast, the frequency distributions of tubulovesicle and of canaliculus particle diameters were significantly different (P less than 0.0005). Neither the opposite curvatures of tubulovesicle and canaliculus microvillus fractures nor subpopulations of tubulovesicles with different particle diameters, were the cause of the difference, since there was only one population of tubulovesicles. We therefore postulate that the diameters of intramembranous particles of tubulovesicles and canaliculus are different and suggest, as a working hypothesis, that the difference could be due to a conformational change of the major intramembranous protein, the (H+,K+)-ATPase.

A monoclonal antibody (mAb 95-111) was used to titrate the amounts of H+/K(+)-ATPase in subcellular fractions of the fundus of rats, pigs, rabbits and humans. All four had similar amounts of H+/K(+)-ATPase: 2.1 +/- 0.5 (human), 1.9 +/- 0.4 (rabbit), 4.4 +/- 0.5 (rat) and 4.2 +/- 0.8 (hog) mg ATPase/g wet tissue. The antigen concentrations and H+/K(+)-ATPase enzymatic activities of subcellular fractions were linearly correlated in all species but rat suggesting that human, rabbit and hog H+/K(+)-ATPases have similar rates of catalysis (223 mumol Pi/h per mg ATPase). The non-correlation of rat data probably reflects the known lability of the rat enzyme. Hence, immuno-titration promises to be a more reliable method of estimating rat ATPase than the currently used enzymatic assay. The H+/K(+)-ATPase content of human biopsies was 20-fold higher than previously-published (Smolka, A. and Weinstein, W.M. (1986) Gastroenterology 90, 532-539) suggesting that the previous immuno-titration underestimated the H+/K(+)-ATPase content of the human fundus.

Testosterone (T) and dihydrotestosterone (DHT) have been quantitated by means of radioimmunoassay in Müllerian ducts (CM) from control quail embryos (6 to 8-day male and 6 to 15-day female) and from female embryos injected with 50 nanograms of testosterone propionate (PT) on the 8th day. These hormones are demonstrable in undifferentiated CM from 6-day control embryos. In control males although a highly significant decrease of the CM weight occurs during the CM involution, the detected amounts of androgens remain at a constant level. In control females, the right CM shows a slight increase of the androgen content during the rudimentation, i.e. from day 8 on; in the left CM: the highest steroid levels are found on the 6th day; while the CM differentiate, concentrations decrease and become similar to those found in neutral tissues. Given testosterone propionate on day 8 female embryos: right CM: both testosterone and DHT levels highly increase until day 14; the CM of treated embryos contain 8 times as much steroid as control; left CM: testosterone and DHT increase after injection until day 9,5; they slightly decrease between days 9,5 and 12, and then remain constant on day 14. Differences in concentrations are highly significant between CM of control and of PT - injected embryos. It seems that the binding sites of these androgens are more numerous during the involution and that bound testosterone or DHT could take a part in CM regression in male embryos and right CM rudimentation in female embryos.

A lectin activity inhibitable by thiodigalactose, N-acetyllactosamine, lactulose, lactose and by an antibody raised against CLL I (chicken-lactose lectin I) has been investigated in the chick embryo developing kidney. At post-induction stages this activity was found in both mesonephros and metanephros. In immunofluorescence and immunoelectron microscopy, the extracellular distribution of CLL I was similar in the mesonephros and the metanephros. The lectin was never found intracellularly; cultured kidney cells did not express any endogenous lectin but were rich in lectin-receptor sites, which led to the hyphothesis that CLL I is not produced in situ but could be adsorbed on renal cells. Potential physiological roles for embryonic lectins are discussed.