Number Of Specific Binding Sites Research Articles

Drying and rewetting induce changes in biofilm characteristics and the subsequent release of metal ions

Drying and rewetting can markedly influence the microbial structure and function of river biofilm communities and potentially result in the release of metal ions from biofilms containing metals. However, little information is available on the response of metal-enriched biofilms to drying and rewetting over time. In this study, natural biofilms were allowed to develop in four rotating annular bioreactors for 2–11 weeks, followed by drying for 5 days and rewetting for another 5 days. Subsequently, we assessed Zn, Cd, and As desorption from the biofilms and other related parameters (microbial community structure, biofilm morphology, enzyme activity, and surface components as well as characteristics). High-throughput sequencing of the 16 S rRNA gene and confocal laser scanning microscopy revealed that the biofilm architecture and bacterial communities were distinct in different growth phases and under drying and rewetting conditions (permutational multivariate analysis of variance; p = 0.001). Proteobacteria was the dominant bacterial phylum, accounting for 69.7–90.1% of the total content. Kinetic experiments revealed that the drying and rewetting process increased metal desorption from the biofilm matrix. The desorption of heavy metals was affected by the age of the biofilm, with the maximum amount of metal ions released from 2-week-old biofilms (one-way ANOVA, Zn: p < 0.001; Cd: p = 0.008; As: p < 0.001). The modifications in biofilm properties and decreased diversity of the bacterial community (paired t-test, p < 0.05) after drying and rewetting decreased the number of specific binding sites for metal ions. In addition, negatively charged arsenate and other anions in the liquid phase could compete with As ions for adsorption sites to promote the release of As(V) and/or reductive desorption of As(III). The results of this study and their interpretation are expected to help refine the behaviors of heavy metals in the aquatic environment.

Sensitive lateral flow immunoassay for the detection of hepatitis B virus surface antigen

Bisphenol A molecular imprinted adsorbent (BMIA) was successfully synthesized by a sol-gel process and showed a good specific binding performance in the water. The further studies showed that the mass transfer process was controlled by in-diffusion, and the synthesis conditions would effect on the amount of imprinting sites. Scatchard model analysis evidenced that the high binding affinity sites and the low binding affinity sites were both on BMIA, and the high binding affinity sites played a key role in the specific binding process. Scatchard model analysis of temperature effect experiments and dosage effect experiments proved that the specific binding sites with high binding affinity and the unexpressed specific binding sites with low binding affinity were the two different states of the imprinting binding sites. The conversion between the two different states depended on the reaction driving force, and the increasing reaction driving force would increase the number of specific binding sites. Especially, the temperature showed a linear positive correlation with the amount of specific binding sites. Finally, a possible model was put forward to explain the two different states conversion mechanism of the imprinting sites.

Two different states conversion mechanism of the imprinting sites

Bisphenol A molecular imprinted adsorbent (BMIA) was successfully synthesized by a sol-gel process and showed a good specific binding performance in the water. The further studies showed that the mass transfer process was controlled by in-diffusion, and the synthesis conditions would effect on the amount of imprinting sites. Scatchard model analysis evidenced that the high binding affinity sites and the low binding affinity sites were both on BMIA, and the high binding affinity sites played a key role in the specific binding process. Scatchard model analysis of temperature effect experiments and dosage effect experiments proved that the specific binding sites with high binding affinity and the unexpressed specific binding sites with low binding affinity were the two different states of the imprinting binding sites. The conversion between the two different states depended on the reaction driving force, and the increasing reaction driving force would increase the number of specific binding sites. Especially, the temperature showed a linear positive correlation with the amount of specific binding sites. Finally, a possible model was put forward to explain the two different states conversion mechanism of the imprinting sites.

In Vivo Characterization of Two 18F-Labeled PDE10A PET Radioligands in Nonhuman Primate Brains

Positron emission tomography (PET) with phosphodiesterase 10A (PDE10A) specific radioligands provides a noninvasive and quantitative imaging tool to access the expression of this enzyme in vivo under normal and diseased conditions. We recently reported two potent 18F-labeled PDE10A radioligands (18F-TZ19106B and 18F-TZ8110); initial evaluation in rats and nonhuman primates indicated stable metabolic profiles and excellent target-to-nontarget ratio (striatum/cerebellum) for both tracers. Herein, we focused on in vivo characterization of 18F-TZ19106B and 18F-TZ8110 to identify a suitable radioligand for imaging PDE10A in vivo. We directly compared microPET studies of these two radiotracers in adult male Macaca fascicularis nonhuman primates (NHPs). 18F-TZ19106B had higher striatal uptake and tracer retention in NHP brains than 18F-TZ8110, quantified by either standardized uptake values (SUVs) or nondisplaceable binding potential (BPND) estimated using reference-based modeling analysis. Blocking and displacement studies using the PDE10A inhibitor MP-10 indicated the binding of 18F-TZ19106B to PDE10A was specific and reversible. We also demonstrated sensitivity of 18F-TZ19106B binding to varying number of specific binding sites using escalating doses of MP-10 blockade (0.3, 0.5, 1.0, 1.5, and 2.0 mg/kg). Pretreatment with a dopamine D2-like receptor antagonist enhanced the striatal uptake of 18F-TZ19106B. Our results indicate that 18F-TZ19106B is a promising radioligand candidate for imaging PDE10A in vivo and it may be used to determine target engagement of PDE10A inhibitors and serve as a tool to evaluate the effect of novel antipsychotic therapies.

Preparation and adsorption properties of molecularly imprinted polymer via RAFT precipitation polymerization for selective removal of aristolochic acid I

The molecularly imprinted polymers (MIP) were prepared via aqueous RAFT precipitation polymerization, with aristolochic acid I (AAI) as the template molecule, AA as the functional monomer, EGDMA as the cross-linker, AIBN as the chain initiator, CTP as the chain transfer agent and 80% (g/g) DMF-aqueous solution as the porogen. The differential UV–vis spectra revealed that a cooperative hydrogen-bonding complex between AAI and AA might be formed at the molar ratio of 1:3 in prepolymerized system. The synthesized MIPs were characterized by FTIR spectra, solid UV–visible absorption spectra, nitrogen adsorption-desorption isotherms and scanning electron microscope, which proved that the MIPs and NIPs have the similar chemical structures and the adding of AAI could affect the size and morphology of the microspheres. UV–visible absorption spectra and High-performance liquid chromatography (HPLC) was used to investigate the adsorption and recognition properties of the MIPs. The Scatchard isotherm model described that the binding sites independently acted. The Langmuir isotherm model suggested an excellent imprinting effect owing to the presence of a large number of specific binding sites on the MIP. The Freundlich model indicated that the AAI could be readily absorbed by MIP. Selective absorption of the template molecule was demonstrated in presence of its analogous compounds, benzoic acid and nitrobenzene. The recycling experiments implied that the MIP could be reused to further selective recognition and separation to AAI for six times at least. MIP was developed for removal of AAI from the Aristolochia manshuriensis extraction. The results indicated that 25mg of MIP could remove the AAI below the HPLC detection limits (6.47ngmL-1) from 5.0mL of the extraction (CAAI=0.0018mgmL-1) with the recovery of AAI to 91.50% (n=3, SD=4.24%). Therefore, it is clearly revealed that the MIP can be a useful tool to remove toxic compounds from natural products.

Peptide regulation of specific ligand-receptor interactions of GABA with the plasma membranes of nerve cells

Here we studied the role of peptide anxiolytics (Selank and its short fragment, Arg-Pro-Gly-Pro) in the occurrence of direct and indirect changes in the basic parameters of the specific ligand-receptor interactions of γ-aminobutyric acid (GABA) with the plasma membranes of nerve cells in the brain. We found that in the presence of the studied peptides the amount of the specifically bound ligand, [3H] GABA, varied. Preliminary intranasal administration of Selank also induced changes in the number of specific binding sites of [3H] GABA but did not affect the affinity of the receptors. More complex and delayed effects were caused by the preliminary intranasal administration of Arg-Pro-Gly-Pro (RPGP). The data that were obtained in the research also indicate the impact of the peptides on cellular regulatory mechanisms that are not associated with the ligand-receptor interaction of studied peptides in the places of their action. Thus, the biological effects of Selank were apparently due to a combination of the direct modulating action of the peptide on the target receptor and the initiation of the biochemical cellular mechanisms that determine the molecular basis of the physiological effect of the peptide.

Structure-Function Relationships of the Collagen-Specific Chaperone Hsp47

Hsp47 is an essential collagen specific chaperone that is crucial for proper formation of the collagen triple helix. KO-mice die at an early emybronic state and missense mutations are linked to severe forms of osteogenesis imperfecta (Ishida &amp; Nagata, 2011). On the other hand, Hsp47 is strongly upregulated in fibrotic pathogenies and has in proof-of-principle studies been successfully targeted . It is a very unusual chaperone as it specifically recognises the folded conformation and releases ist client upon changes in pH and not in an ATP-cycle dependent manner. We have deterined the crystal structure of Hsp47 alone and in complex with homotrimeric collagen model peptides (Widmer et al., 2012), revealing a 2:1 stoichiometry Hsp47:collagen-trimer. The specific recognition of an arginine at the Y-position of the triple helix is explained by a saltbridge to an aspartic acid of Hsp47. Based on these structures we have undergone further investigation in order to shed light on the pH-triggered client release and to gain further insight into client recognition. We have undergone a systematic site-directed mutagenesis study of histidine residues in the binding interface. Interestingly, only few side-chains are predicted to change significantly their protonation state by a pH change from 7 to 6. We have furthermore investigated the influence of the reactive center loop, which is disordered in most crystal structures. An open question is how many binding sites for Hsp47 exist in procollagen. EM studies reveal a large number of specific binding sites, explaining how Hsp47 clamps and stabilises the triple helix and prevents lateral aggregation.

Effects of pH on the Molecular Binding between β-Lactoglobulin and Bixin

Binding between β-lactoglobulin (β-Lg), the major whey protein, and bixin, the major carotenoid in annatto, was studied at pH 3.0-10.0. The Fourier transform infrared spectroscopy and UV-vis absorption spectroscopy results showed that the binding involved a complex formation. The tryptophan quenching fluorescence and analytical ultracentrifugation data showed that there were two specific binding sites and that the binding affinity increased significantly with an increase in pH. A higher efficiency of energy transfer from tryptophan fluorescence to bixin was observed at higher pH. Thermodynamic parameters and the number of specific binding sites obtained from isothermal titration calorimetry and analytical ultracentrifugation suggested that binding involved mostly hydrophobic interactions for the two specific binding sites. The impacts of pH on binding were correlated to the conformation of β-Lg, the hydrophobic pocket of which becomes more available at higher pH and ionic strength.

Dopamine receptor binding sites in the rat superior colliculus.

Following intravenous administration of [3H]spiperone or [3H]N,n-propylnorapomorphine (NPA) to rats, radioactivity derived from the ligands accumulated in the striatum and superior colliculus when compared with cerebellar levels. The accumulation of [3H]spiperone in both areas was prevented by intraperitoneal administration of (+)-butaclamol, haloperidol and sulpiride but not by (-)-butaclamol, cinanserin, propranolol or prazosin. The accumulation of [3H]NPA was prevented by administration of (+)-butaclamol, haloperidol and apomorphine but not by (-)-butaclamol. In in-vitro experiments, membrane preparations from the superior colliculus showed a small number of specific binding sites for both [3H]spiperone and [3H]NPA. The dissociation constant (KD) for [3H]NPA was not different from that for striatal preparations but that for [3H]spiperone was 10-fold higher. We conclude that dopamine receptors may be present within the superior colliculus.

Adenosine A2A Receptor Signaling and Golf Assembly Show a Specific Requirement for the γ7 Subtype in the Striatum

The adenosine A(2A) receptor (A(2A)R) is increasingly recognized as a novel therapeutic target in Parkinson disease. In striatopallidal neurons, the G-protein α(olf) subtype is required to couple this receptor to adenylyl cyclase activation. It is now well established that the βγ dimer also performs an active role in this signal transduction process. In principal, sixty distinct βγ dimers could arise from combinatorial association of the five known β and 12 γ subunit genes. However, key questions regarding which βγ subunit combinations exist and whether they perform specific signaling roles in the context of the organism remain to be answered. To explore these questions, we used a gene targeting approach to specifically ablate the G-protein γ(7) subtype. Revealing a potentially new signaling paradigm, we show that the level of the γ(7) protein controls the hierarchial assembly of a specific G-protein α(olf)β(2)γ(7) heterotrimer in the striatum. Providing a probable basis for the selectivity of receptor signaling, we further demonstrate that loss of this specific G-protein heterotrimer leads to reduced A(2A)R activation of adenylyl cyclase. Finally, substantiating an important role for this signaling pathway in pyschostimulant responsiveness, we show that mice lacking the G-protein γ(7) subtype exhibit an attenuated behavioral response to caffeine. Collectively, these results further support the A(2A)R G-protein α(olf)β(2)γ(7) interface as a possible therapeutic target for Parkinson disease.

Molecularly imprinted hydrogel displaying reduced non‐specific binding and improved protein recognition

A novel approach for enhancing protein recognition in molecularly imprinted hydrogel (MIH) is presented. This approach was developed based on the hypothesis that the number of specific binding sites created in the previously described MIH is very small, thus attempts to enhance the capacity result in most cases in additional non-specific binding and loss of selectivity. Thus, blocking the non-specific binding sites could lead to higher capacities and better selectivity. To test this hypothesis, MIH interpenetrating networks designed to block non-specific binding sites were synthesized using two separate stages of polymerization. Re-binding of the template protein (lysozyme) and a competitor protein (cytochrome C) was measured, and the results were compared with the similar experiment performed using a control non-imprinted hydrogel and a "conventional" MIH. The imprinting efficacy of the MIH interpenetrating network was found to be much higher than that of the controls. Furthermore, competitive adsorption assays have demonstrated the superiority of the new formulation.

Elevated expression of urotensin II and its receptor in skeletal muscle of diabetic mouse

Urotensin II (UII) is a somatostatin-like peptide recently identified to be involved in metabolic regulation and to play a significant role in diabetes and its complications. In the present study, we investigated the expression of UII and its receptor UT in the soleus muscle of male diabetic KK/upj-AY/J mice (2DM group) and the effects of UII on glucose uptake by the skeletal muscle to explore the role of skeletal muscle-derived UII in the pathogenesis of insulin resistance and diabetes. Radioimmunoassay, RT-PCR, immunohistochemistry and radio-ligand binding assay were used in this study. Compared with C57BL/6J mice (control group), 2DM mice showed increased UII content, by 34.0% in plasma, 15.4% in skeletal muscle tissue and 30.6% in medium containing UII from muscle (all P < 0.05 or P < 0.01). UII protein and UT mRNA expression were significantly enhanced in the skeletal muscle of 2DM mice. On [ 125I]UII binding to muscle sarcolemma, UT binding exhibited a saturable single-component characteristic in a specific and time-dependent manner. Scatchard plot analysis showed higher maximal number of specific binding sites ( Bmax) in skeletal muscle, by 42.9% ( P < 0.01), and a lower dissociation constant ( Kd), by 26.4% ( P < 0.01), in the 2DM group than in controls. On in vitro tissue pre-incubation with UII (10 − 9 , 10 − 8 and 10 − 7 mol/L), the insulin-stimulated [ 3H]-2-DG uptake by split soleus muscle was lower, by 9.5%, 33.4% and 39.7% (all P < 0.01), respectively, than without UII incubation. UII/UT upregulated in skeletal muscle of 2DM mice suggests that UII derived from skeletal muscle might induce the pathogenesis of skeletal muscle insulin resistance as an autocrine factor.

Noncovalent imprinted microspheres: Preparation, evaluation and selectivity of DBU template

Abstract1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU) imprinted polymer was prepared as microspheres by precipitation polymerization method to obtain molecular recognition systems based on the noncovalent interactions between DBU template, methacrylic acid (MAA), and ethylene .glycol dimethacrylate (EDMA) in acetonitrile. 1H NMR analysis of DBU/MAA mixture has been performed and hydrogen bonding interactions have been established. Microspheres have been characterized by FTIR studies with evidence of DBU linkage in polymer particles and by Scanning Electron Microscopy (SEM) to study their morphological properties. How pH values affect the binding capacity of imprinted polymer during the binding stage has been also discussed and results suggest that imprinted poly‐(MAA‐EDMA) behavior is related to the influence of DBU basicity during rebinding processes and the optimum pH value for binding has been found around neutral range. Binding ability of the imprinted polymer towards different concentration of DBU buffered solutions has been evaluated and compared with binding ability of the non‐imprinted polymer. A more sensitive response to the template in the imprinted system suggests that a reasonable number of specific binding sites is formed. Finally, differential selectivity towards other less strong than DBU nitrogen bases, such as pyridine, imidazole, and 1,5‐diazabicyclo[4.3.0]non‐5‐ene (DBN) has been also discussed. Our results indicate that both specific sites and basic properties are involved in the rebinding process. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007

Albumin up-regulates the type II transforming growth factor-beta receptor in cultured proximal tubular cells

Clinical and experimental observations suggest that proteinuria is not merely a marker of chronic nephropathies, but may also be involved in the progression to end-stage renal failure. Filtered proteins are taken up by tubular cells, and overwhelming this system may lead to tubular synthesis of various proinflammatory and profibrogenic cytokines, including transforming growth factor-beta (TGF-beta). TGF-beta acts by first binding to specific receptors. We studied in an in vitro system using a well-defined mouse proximal tubular cell line (MCT cells) whether fatty acid-free bovine albumin modulates expression of specific receptors for TGF-beta. MCT (and LLC-PK1) cells were challenged in serum-free medium with different concentrations of albumin. Activation of a local renin-angiotensin system was tested by real-time polymerase chain reaction (PCR) for renin and angiotensinogen transcripts and determination of secreted angiotensin II (Ang II) by enzyme-linked immunosorbent assay (ELISA). Some cells were also treated with the AT1 receptor antagonist losartan. TGF-beta receptor types I and II mRNA levels were determined by Northern analysis whereas protein abundance was measured by Western blots. To test for a functional consequence of up-regulated TGF-beta receptors, MCT cells were preincubated with albumin and subsequently treated with low-dose TGF-beta that normally does not induce collagen type IV expression by itself. Downstream signaling events were detected by Western blots for phosphorylated Smad2. Scatchard assays with [125I]TGF-beta1 were performed to estimate affinity and number of specific binding sites. Different length TGF-beta type II promoter constructs linked to CAT reporter were transiently transected into MCT cells to determine transcriptional activity. Incubation of MCT cells with 0.5 to 10 mg/mL albumin leads to an increase in type II TGF-beta receptor mRNA and protein expression without influencing type I receptors. An increase in type II TGF-beta receptor protein expression was detected after 12 hours of albumin incubation and was still detectable after 48 hours. The albumin-mediated increase in type II TGF-beta receptor mRNA was attenuated in the presence of 1 micromol/L losartan, suggesting involvement of a local renin-angiotensin system. MCT cells treated with albumin significantly increased expression of angiotensinogen and renin transcripts and also secreted more Ang II into the culture supernatant. Analysis of transcriptional activity showed that promoter segments containing activating protein (AP-1)-binding sites are necessary for albumin-induced transcription of the TGF-beta type II receptor. Binding assays revealed that albumin treatment significantly increased the overall binding sites as well as the affinity for TGF-beta. This effect had functional consequences because MCT cells pretreated with albumin reacted with a stronger TGF-beta-mediated phosphorylation of down-stream Smad2 and also increased collagen IV expression compared with control cells. Our findings indicate that albumin up-regulates ligand-binding TGF-beta receptors on cultured proximal tubular cells. Albumin-induced activation of local Ang II production appears to be responsible for this effect. This may amplify the matrix-stimulatory actions of TGF-beta on tubular cells and could be a novel mechanism for how proteinuria exhibits pathophysiologic effects on tubular cells ultimately leading to tubulointerstitial fibrosis.

Osteoclastogenesis is decreased by cysteine proteinase inhibitors

The effects of cystatin C and other cysteine proteinase inhibitors on osteoclast formation and differentiation have been investigated. Cystatin C decreased osteoclast formation stimulated by parathyroid hormone (PTH), 1,25(OH) 2-vitamin D3 or interleukin-6 (IL-6) (in the presence of its soluble receptor) as assessed by the number of tartrate-resistant acid phosphatase (TRAP +) multinucleated cells in mouse bone marrow cultures. The inhibitory effect was associated with decreased mRNA expression for the calcitonin receptor as well as decreased number of specific binding sites for 125I-calcitonin, and without any effect on the mRNA expression of receptor activator of nuclear factor κB (NF-κB) ligand (RANKL). Similarly, the cysteine proteinase inhibitors leupeptin, E-64 and benzyloxycarbonyl-Phe-Ala-diazomethane (Z-FA-CHN 2) decreased PTH-stimulated formation of TRAP + multinucleated cells and binding of 125I-calcitonin. A peptidyl derivative synthesized to mimic part of the proteinase-binding site of cystatin C (benzyloxycarbonyl-Arg-Leu-Val-Gly-diazomethane, or Z-RLVG-CHN 2) also decreased PTH-stimulated osteoclast formation. In a 9-day culture, addition of cystatin C during the last 5 days was sufficient to cause substantial inhibition of osteoclast formation. Cystatin C-induced decrease of osteoclast formation was associated with enhanced number of F4/80-positive macrophages and increased mRNA expression of the macrophage receptor c-fms in the bone marrow culture. Osteoclast formation in mouse bone marrow cultures as well as in mouse spleen cell cultures, stimulated by macrophage colony-stimulating factor (M-CSF) and RANKL was also decreased by different cysteine proteinase inhibitors. In addition, cystatin C inhibited M-CSF/RANKL induction of calcitonin receptor mRNA in spleen cell cultures. The inhibitory effect by cystatin C in spleen cells was associated with decreased mRNA expression of RANK and the transcription factor NFAT2. It is concluded that cysteine proteinase inhibitors decrease formation of osteoclasts by interfering at a late stage of pre-osteoclast differentiation.

Loss of mRNA levels, binding and activation of GTP-binding proteins for cannabinoid CB 1 receptors in the basal ganglia of a transgenic model of Huntington’s disease

Data obtained from the basal ganglia of postmortem Huntington’s disease (HD) brains have revealed that the level of cannabinoid CB 1 receptors in striatal efferent neurons decreases in parallel to the dysfunction and subsequent degeneration of these neurons. These findings, and others from rat models of HD generated by lesions with mitochondrial toxins, suggest that the loss of CB 1 receptors may be involved in the pathogenesis of the disease. To explore further the changes in the endocannabinoid system, as well as the potential of endocannabinoid-related compounds, we examined the status of CB 1 receptors in the HD94 transgenic mouse model of HD. These mice express huntingtin exon 1 with a polyglutamine tract of 94 repeats in a tissue-specific and conditional manner using the tet regulatable system. They develop many features of HD, such as striatal atrophy, intraneuronal aggregates and progressive dystonia. In these animals, we analyzed mRNA levels for the CB 1 receptor, in addition to the number of specific binding sites and the activation of GTP-binding proteins by CB 1 receptor agonists. mRNA transcripts of the CB 1 receptor were significantly decreased in the caudate-putamen of HD transgenic mice compared to age-matched littermate controls. The decrease concurred with a marked reduction in receptor density in both the caudate-putamen and its projection areas such as the globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata. Furthermore, the efficacy of CB 1 receptor activation was reduced in the globus pallidus, as determined by agonist-induced [ 35S]GTPγS binding, and tended towards a decrease in the substantia nigra. None of these changes was seen in the cerebral cortex and hippocampus, despite high levels of expression of the mutant protein in these regions. The decrease in CB 1 receptor levels was accompanied by a decrease in the proenkephalin-mRNA levels but not in substance P-mRNA levels. Taken together, these results suggest that the loss of CB 1 receptor might be preferential to the enkephalinergic CB 1 receptor-containing striatopallidal neurons, and further implicate the CB 1 receptor to the subsequent HD symptomatology and neuropathology.

Effect of chronic hypoxia on contents of urotensin II and its functional receptors in rat myocardium.

The cyclic peptide urotensin II (UII) has recently been cloned in mammals and reported to constrict rat pulmonary arteries potently. An enhanced maximal response was shown in rats exposed to chronic hypoxia. The aim of this study was to investigate changes in plasma and myocardial UII levels and its receptor sites in crude sarcolemma of ventricles from chronic hypoxic rats. We observed that rats exposed to chronic hypoxia for 4 weeks developed pulmonary hypertension and right ventricular hypertrophy. Compared with controls, the UII content in hypoxic rats was increased by 97.5% (45.24 +/- 7.1 vs. 22.9 +/- 3.24pg/mg protein, P < 0.01) in the right ventricle and 33.2% (24.89 +/- 0.99 vs. 18.68 +/- 2.04pg/mg protein, P < 0.01) in the left ventricle, respectively. However, there was no significant difference in plasma (27.44 +/- 3.11 vs. 27.82 +/- 5.57pg/ml, P > 0.05) and lung tissue levels (34.03 +/- 4.63 vs. 33.74 +/- 4.06 pg/ mg protein, P > 0.05) between the control and hypoxic groups. The time course of the binding of [125I]UII to crude ventricular sarcolemma was specific and time dependent. Scatchard plot analysis of the data demonstrated that the maximal number of specific binding sites (Bmax) in both the right and left ventricles was upregulated in the hypoxic group. Moreover, Bmax in the right ventricular specimens was upregulated to a greater extent than in the left ventricle (increased by 114% and 25% in the right and left ventricles, respectively, compared with control group, P < 0.01). In contrast, the UII binding affinity in right and left ventricular membranes from hypoxic rats was decreased (the dissociation constant Kd) increased by 20% and 33%, respectively compared with controls, P < 0.01). These results indicate that UII may act as an autocrine and/or paracrine hormone rather than as a circulating hormone, playing important roles in the development of ventricular hypertrophy induced by chronic hypoxia, and that the pathophysiological significance of UII in pulmonary and cardiovascular alteration induced by chronic hypoxia deserves further investigation.

Responsiveness of antennal taste hairs of the apterygotan insect, Thermobia domestica (Zygentoma); an electrophysiological investigation

Antennal taste hairs of Thermobia domestica were investigated by a modified tip recording technique. They are sensitive to aqueous salt solutions but do not show the characteristic features of a water and a salt receptor cell. Sucrose and fructose stimulated approximately half of the tested hairs. As revealed by spike amplitude histograms, in most cases two sense cells responded. Proline stimulated the hairs with a threshold concentration of about 2 mM. Again, in most cases, two sense cells responded. In relation to sucrose the major sensitivities to both fructose and proline were not found in clearly defined receptor cells as in flies, but were realized in different cells of either a higher or smaller spike amplitude. This suggests, that the number of specific binding sites varies among the receptor cells from one taste hair to the other. As in cockroaches, but unlike flies, spike series are mainly generated in the apical dendritic segments. It is suggested that these features of the sense cells in thermobian taste hairs represent a primitive condition in relation to the specification of taste hairs cells in higher insect taxa

Inhibitory properties of ruthenium amine complexes on mitochondrial calcium uptake.

The recent finding that the inhibition of Ca2+-stimulated respiration by ruthenium red is mainly due to a binuclear ruthenium complex (Ru360) present in the commercial samples of the classical inhibitor ruthenium red (Ying et. al., 1991), showed that this complex is the more potent and specific inhibitor of the mitochondrial calcium uniporter. This work was aimed to provide insights into the mechanism by which Ru360 and other ruthenium-related compounds inhibits calcium uptake. Ruthenium red and a synthesized analog (Rrphen) were compared with Ru360. The inhibition by this binuclear complex was noncompetitive, with a Ki of 9.89 nM. The number of specific binding sites for Ru360 was 6.2 pmol/mg protein. Ruthenium red and Ru360 were mutually exclusive inhibitors. Bound La3+ was not displaced by Ru360. Rrphen was the least effective for inhibiting calcium uptake. The results support the notion of a specific binding site in the uniporter for the polycationic complexes and a negative charged region from the phospholipids in the membrane, closely associated with the uniporter inhibitor-binding site.

Helicobacter pylori upregulates expression of epidermal growth factor-related peptides, but inhibits their proliferative effect in MKN 28 gastric mucosal cells.

Acute exposure to Helicobacter pylori causes cell damage and impairs the processes of cell migration and proliferation in cultured gastric mucosal cells in vitro. EGF-related growth factors play a major role in protecting gastric mucosa against injury, and are involved in the process of gastric mucosal healing. We therefore studied the acute effect of H. pylori on expression of EGF-related growth factors and the proliferative response to these factors in gastric mucosal cells (MKN 28) derived from gastric adenocarcinoma. Exposure of MKN 28 cells to H. pylori suspensions or broth culture filtrates upregulated mRNA expression of amphiregulin (AR) and heparin-binding EGF-like growth factor (HB-EGF), but not TGFalpha. This effect was specifically related to H. pylori since it was not observed with E. coli, and was independent of VacA, CagA, PicA, PicB, or ammonia. Moreover, H. pylori broth culture filtrates stimulated extracellular release of AR and HB-EGF protein by MKN 28 cells. AR and HB-EGF dose-dependently and significantly stimulated proliferation of MKN 28 cells in the absence of H. pylori filtrate, but had no effect in the presence of H. pylori broth culture filtrates. Inhibition of AR- or HB-EGF- induced stimulation of cell growth was not mediated by downregulation of the EGF receptor since EGF receptor protein levels, EGF binding affinity, number of specific binding sites for EGF, or HB-EGF- or AR-dependent tyrosine phosphorylation of the EGF receptor were not significantly altered by incubation with H. pylori broth culture filtrates. Increased expression of AR and HB-EGF were mediated by an H. pylori factor > 12 kD in size, whereas antiproliferative effects were mediated by both VacA and a factor < 12 kD in size. We conclude that H. pylori increases mucosal generation of EGF-related peptides, but in this acute experimental model, this event is not able to counteract the inhibitory effect of H. pylori on cell growth. The inhibitory effect of H. pylori on the reparative events mediated by EGF-related growth factors might play a role in the pathogenesis of H. pylori-induced gastroduodenal injury.