High affinity beta-2-adrenergic receptors in mononuclear leucocytes: similar density in young and old normal subjects

In normal subjects beta-adrenergic responsiveness in the cardiovascular system has been shown to be impaired with increasing age. In order to correlate reduced hormonal responsiveness to an age-related defect at the receptor level, high affinity beta-adrenergic receptors in homogenates of human mononuclear leucocytes have been studied with a (?)-³H-dihydroalprenolol (³H-DHA) binding assay. The binding sites have been characterized by rapid kinetics, saturability, structural and sterospecificity. Binding equilibrium was obtained within 16 minutes at 37° and was reversed by 50% within 10.6 minutes. In 22 healthy subjects a binding capacity of 60 ± 8 fmol/mg protein and an equilibrium dissociation constant (K_D) of 0.6 ± 0.05 nM was found. Beta-adrenergic agonists displaced ³H-DHA binding with a potency order of isoproterenol > adrenaline > noradrenaline. The (?) isomers of beta-adrenergic agonists and antagonists were one to two orders of magnitude more potent as inhibitors of ³H-DHA binding than their corresponding (+) isomers. The binding capacity and affinity of the beta-adrenergic receptors did not differ in old, as compared to young normal subjects. Leucocytes from 14 individuals 18–40 years old had an average density of 53 ± 4 fmol/mg protein, while the average density in leucocytes from 8 individuals aged 53–65 years was 67 ± 8 fmol/mg protein. The K_D was 0.6 ± 0.05 nM in both groups. In conclusion, an age-related decrease of beta-adrenergic receptor-mediated cardiovascular functions does not seem to be reflected in the properties of beta-adrenergic receptors of mononuclear leucocytes.     

Studies on protein kinase activity and the binding of adenosine 3'5-monophosphate by membranes of hereditary spherocytosis erythrocytes.

Evidence has been presented recently of a deficiency of an endogenous membrane-associated protein kinase in erythrocytes of patients with hereditary spherocytosis (HS). We have measured endogenous protein kinase activity in erythrocyte membranes of 4 HS subjects using different membrane isolation and reaction conditions and find that the phosphorylation of the spectrin component (mean ± S.E. 17.1 ± 1.2 pmoles/10 mins per mg protein) is not significantly different to that of 4 normal controls (mean ± S.E. 20.7 ± 1.1 pmoles/10 mins per mg protein). Phosphorylation of exogenous proteins such as casein and protamine is also not deficient in HS erythrocyte membranes. Adenosine 3′5-monophosphate (cyclic AMP) binding to normal and HS erythrocyte membranes was also studied using a Millipore filtration assay. The affinity of cyclic AMP for erythrocyte membranes as determined by Hill plots of binding data from 4 HS subjects (K_D mean ± S.E. = 2.2 ± 0.2 nM) was not significantly different to 4 normal controls (K_D mean ± S.E. = 2.8 ± 0.6 nM). The rate of dissociation of bound cyclic AMP from HS membranes was also similar to control membranes. We thus cannot confirm the prediction by others that an abnormality of cyclic AMP interaction with the erythrocyte membrane underlies HS.     

人血清载脂蛋白CII酶联免疫测定法的研究

 本文介绍一种特异、灵敏、简便人血清载脂蛋白CⅡ(apoCⅡ)竞争性酶兔疫测定法(Competitive Enzyme Immunoassay,CEIA)。单价特异抗体由免疫家兔获得。采用部分纯化的apoCs包被聚苯乙烯板。羊抗兔γ-球蛋白酶交联物用辣根过氧化物酶按简化过碘酸钠法制备。apoCⅠ、AⅠ、AⅡ以及LDL无交叉反应。本法最小检测量为25ng,标准曲线工作范围是1.5～30.0mg％,板内及板间变异系数分别为6.5～7.8％及6.6～11.0％,回收率为107.5％;185例正常人血清apoCⅡ含量,男5.1±1.9mg％(n=95),女4.8±1.7mg％(n=90)。     

Association of inflammation and cytotoxin-associated gene a positive strains of helicobacter pylori in cardiac syndrome x

Background: Cardiac syndrome X (CSX) is a condition in which patients have the pain of angina despite normal coronary angiogram. Helicobacter pylori (H. pylori) infection causes chronic inflammation which may play a pathogenic role in CSX. We surveyed the association of inflammation with H. pylori and its virulent strain (cytotoxin‐associated gene A positive; CagA+) infections with CSX. Material and Methods: Sixty patients with CSX (38 women/22 men; mean age: 51.8 ± 12.3) and 60 age‐ and gender‐matched healthy controls (39 women/21 men; mean age: 48.9 ± 6.3) were enrolled. Plasma samples were tested for the presence of IgG antibody to H. pylori using enzyme linked immunosorbent assay (ELISA) method. IgG‐ positive patients were determined by the presence of IgG antibody to CagA, also by ELISA method. Also, plasma levels of interleukin‐6 (IL‐6) and tumor necrosis factor‐alpha (TNF‐α) were measured by ELISA method. Results: Patients with CSX were detected to have significantly higher plasma IL‐6 and TNF‐α level in comparison with normal controls (33.6 ± 3.5 vs 3.2 ± 0.4 and 24.2 ± 2.3 vs 3.1 ± 0.4, respectively; p < 0.01). The plasma levels of these inflammatory factors in CgA+ were significantly higher than those in CagA? (CSX: IL‐6: 43.05 ± 5.04 vs 23.97 ± 4.58 and TNF‐α: 31.43 ± 3.13 vs 16.47 ± 2.93, Controls: IL‐6: 3.52 ± 1.39 vs 2.90 ± 0.67 and TNF‐α: 5.39 ± 1.17 vs 2.22 ± 0.43, respectively; p < 0.05). Conclusion: The CagA+ strain of H. pylori, can not only be a trigger, and may also have a role via chronic inflammation in the pathogenesis of CSX.     

Benzodiazepine receptor and neurotransmitter studies in the brain of suicides

The characteristics of benzodiazepine binding sites (affinity, number heterogeneity) were studied on frozen sections of hippocampus of 7 suicides and 5 controls subjects, using biochemical and autoradiographic techniques. 3H flunitrazepam was used as ligand, clonazepam and CL 218,872 as displacing agents. Some neurotransmitters or their derivatives (GABA, catecholamines, hydroxy-indols) were evaluated quantitatively in parallel in the hippocampal tissue by liquid chromatography. We observed mainly an increase in the Ki of CL 218,872 subtype I binding sites in suicides, (7.48±1.7 to 17.24±1.7 nM, P < 0.01), (m±SEM) and an increase in % of type I binding sites (30±4.2 to 42±2.5, P=0.01). Among neurotransmitters, only norepinephrine differed significantly between controls and suicides (11.34±1.9 to 24.34 ng/gtissue, P=0.02).     

Apparent “negative cooperativity” kinetics in the absence of a nonlinear Scatchard plot of thyrotropin-receptor interaction in a human thyroid adenoma

A human thyroid adenoma (benign nodule) was identified which exhibited a linear Scatchard plot of ¹²⁵I-TSH binding, characteristic of a single class of binding site with high affinity (K_d = 0.5±0.1 nM) and low binding capacity (0.8±0.2 pmol/mg protein). In contrast, Scatchard analysis of binding to adjacent normal thyroid was nonlinear, suggesting the presence of high and low-affinity binding sites with K_d's of 0.4±0.2 and of 27.9±11.0 nM and capacities of 0.7±0.3 and 1.8±1.0 pmol/mg protein, respectively. Dissociation of bound ¹²⁵I-TSH from membranes of both adenoma and normal tissue revealed identical enhancement of dissociation in the presence of excess native hormone, thought to be evidence for the “negative cooperativity” model of hormone-receptor interaction. Furthermore, adenylate cyclase from both tissues was equally responsive to TSH. Thus, a thyroid adenoma which contains TSH-responsive adenylate cyclase still exhibited enhanced dissociation by native hormone, even though Scatchard analysis yielded a single, non-cooperative class of binding sites. This suggests that enhanced dissociation of bound hormone does not provide a demonstration of negatively-cooperative site-site interaction. Furthermore, nonlinear Scatchard plots, typical of TSH binding in normal thyroid, represent two classes of binding sites, of which the high affinity type is responsible for stimulation of adenylate cyclase.     

Kinetic analysis of the binding of immunoglobulins IgG1 and IgG2 to bovine mammary cells

Previous reports were confirmed that specific binding sites exist on bovine mammary cells near parturition presumably involved in the transfer of immunoglobulins IgG1 and IgG2 across the mammary gland at the time of colostrum formation. Determination of the kinetic parameters of these binding sites using ¹²⁵I-labeled IgG1 and IgG2 immunoglobulins indicated the presence of sites with association constants (K_a) of about 5 · 10⁸?10 · 10⁸ M^?1 for both subclasses during normal lactation with about 9000 and 3000 sites per cell for each, respectively. The number of IgG1 sites tended to increase as the time of parturition approached. In addition, a new group of sites numbering about 5000 per cell with very strong binding of IgG1 (K_a about 45 · 10⁸ M^?1) appeared on the cells about a week before parturition. The numbers and affinity of the IgG1 and IgG2 binding sites bear a relationship to the approximate 7:1 ratio of these immunoglobulin subclasses found in colostrum and normal milk and to the time of maximum colostrum formation. The results support the premise that a highly selective transport mechanism exists in the bovine mammary epithelial cell for the transfer of IgG1 and IgG2 immunoglobulins from blood to the lacteal secretions.     

Radioimmunoassay of urinary testosterone glucuronoside

A simple method is described for the determination of testosterone glucuronoside in urine without prior hydrolysis or extraction. Appropriate amounts (5 μl male, 50 μ1 female urine) are dried at 100 °C to eliminate nonspecific binding by urinary proteins. The residues are cooled, redissolved in buffer and equilibrated with antiserum to testosterone-17β-glucosiduronate-bovine serum albumin and tritiated testosterone glucuronoside. The unbound steroid is removed with dextran-coated charcoal. The total random theoretical percentage error was calculated as 7 %. The inter and intra assay precision were 18 and 9 % respectively. Daily urine collections from 8 complete menstrual cycles have been analysed and the results related to the peak of urinary LH. In seven subjects, there was a visible peak of testosterone glucuronoside at mid cycle (LH peak ± 1 day), in six a distinct peak in the luteal phase, and in five a smaller peak during the follicular phase. The levels of testosterone glucuronoside in groups of healthy men and women were 273 ± 169 and 19 ± 10 μg/24 hrs. The corresponding range of values by a gas-liquid chromatographic method were 204 ± 102 and 14 ± 8. The values are discussed.     

Microbiota of Initial Periodontitis in Adults

This paper reviews our recent studies of the microbiota and host response of initial periodontitis. Understanding the initial stages of periodontitis will allow appropriate early treatment and prevention strategies. Out studies aimed to determine the major bacterial species that differentiated initial periodontitis from health, and evaluate whether subjects with initial periodontitis differed in serum IgG reactivity to putative initial periodontitis pathogens compared with healthy subjects. Initial periodontitis was characterized clinically using longitudinal periodontial attachment level measurements. Progressing periodontal loss was detected at interproximal (initial periodontitis), and buccal (progressing recession) locations from the study population of minimally periodontally diseased subjects. Initial periodontitis was characterized microbiologically by elevated proportions of Bacteroides forsythus, Selenomonas noxia and Campylobacter rectus when compared with non-periodontitis sites. The immunological checkerboard assay did not detect differences in serum IgG reactivity among healthy, gingivitis or initial periodontitis subjects, or changes in reactivity co-incident with detection of initial peridontitis. Clinical, microbiological and immunological characterization of initial periodontitis was consistent with infection-associated Gram-negative anaerobic periodontal species. Progressing recession sites were colonized byActinomyces and Streptococcus species, as were healthy sites. Progressing recession sites demonstrated periodontal loss that appeared unrelated to infection and appeared to be consistent with a traumatic tooth brushing etiology. Different types of lesions will require different approaches to therapy and prevention.     

Selective actions of growth hormone on rat liver estrogen binding proteins

Levels of hepatic estrogen receptor were 9.0 ± 2.4 fmoles/mg cytosol protein in intact females compared to 3.4 ± 2.2 in hypophysectomized females. Likewise, levels of receptor were 9.8 ± 1.5 fmoles/mg cytosol protein in intact males and 2.7 ± 1.8 in hypophysectomized males. Hypophysectomy abolished the sex differences in a second class of binding sites termed higher capacity lower affinity binding sites by increasing female levels and decreasing male levels. Treatment of hypophysectomized male or female rats with growth hormone (2 units/kg body wt, two times daily) restored normal levels of hepatic estrogen receptor. Administration of growth hormone to hypophysectomized rats did not reverse the effects of hypophysectomy on higher capacity lower affinity binding sites. These studies demonstrate that growth hormone exerts selective actions on different forms of hepatic estrogen binding proteins.     

Establishment and clinical application of a highly sensitive enzyme immunoassay for determination of N‐acetyl‐seryl‐aspartyl‐lysyl‐proline

N‐acetyl‐seryl‐aspartyl‐lysyl‐proline (AcSDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. Because AcSDKP is hydrolyzed by the N‐terminal active site of angiotensin converting enzyme and partially eliminated in urine, its plasma level is a result of a complex balance between its production, hydrolysis by ACE, and renal elimination. In this study, we attempted to establish an enzyme immunoassay (EIA) for quantifying AcSDKP‐like immunoreactive substance (IS), which is applicable for monitoring plasma AcSDKP levels in healthy subjects and patients with chronic renal failure. Using β‐ d ‐galactosidase‐labeled Gly‐γAbu‐SDKP as a marker antigen, an anti‐rabbit IgG‐coated immunoplate as a bound/free separator and 4‐methylumbelliferyl‐β‐ d ‐galactopyranoside as a fluorogenic substrate, a highly sensitive and specific EIA was developed for the quantification of AcSDKP‐IS in human plasma. The lower limit of quantification was 0.32 fmol/well, and the sharp inhibition competitive EIA calibration curve obtained was linear between 8.0 and 513 fmol/ml. This EIA was so sensitive that only 10 µl plasma sample was required for a single assay. The coefficients of variation (reproducibility) for human plasma concentrations of 0.2 and 2.1 pmol/ml were 7.2 and 7.7%, respectively, for inter‐assay and 13.3 and 7.8% for intra‐assay comparisons. Plasma AcSDKP‐IS level was significantly higher in patients with chronic renal failure (0.92 ± 0.39 pmol/ml) compared with healthy subjects (0.29 ± 0.07 pmol/ml). These results suggest that our EIA may be useful to evaluate plasma AcSDKP level as a biomarker in various patients. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Specific and saturable binding of albumin to rat adipocytes: Modulation by epinephrine and possible role in free fatty acid transfer

Specific and saturable binding of ¹²⁵I-bovine albumin to rat adipocytes in suspension was observed (apparent K_d 2.09 ± 0.52 × 10^?6 M; 8.58 ± 2.49 × 10⁶ sites per cell; mean ± SEM). The binding was rapid and reversible for at least 10 min, suggesting that endocytosis of albumin was minor under assay conditions. Pre-incubation of cells with epinephrine bitartrate caused an apparent increase in number and decrease in affinity of the adipocyte binding sites for albumin. These findings suggest that a specific and saturable interaction of albumin with the adipocyte surface may play a role in the cellular uptake and release of free fatty acids.     

Repeatability of subject/bed interface pressure measurements

The repeatability of a technique for measuring interface pressures has been assessed. Pressure was measured using a Talley SA500 Pressure Evaluator under six anatomical sites (occiput, scapula, elbow, sacrum, buttock, and heel) of six healthy subjects lying supine on a Clinifloat mattress (trademark, SSI). For each site of each subject, four repeat readings were taken per day on four separate days. Mean pressures varied significantly between subjects (p < 0.02), though differences in mean pressures between sites were greater. Pressure was not significantly related to subject mass. The overall repeatability of the technique was ± 0.77 kPa (± 5.8 mm Hg) which was much smaller than the range of pressures found under different sites (2.72 kPa or 20.4 mm Hg at the sacrum to 9.00 kPa or 67.5 mm Hg at the heel). Repeatability varied from site to site, from ± 0.47 kPa (± 3.5 mm Hg) at the buttocks to ± 1.20 kPa (± 9.0 mm Hg) at the heel. Measurements were found to vary significantly more between days than between repeats on the same day (p < 0.02).     

Chemiluminescent immunoassay (CLIA) for urinary albumin

A simple chemiluminescent immunoassay (CLIA) for urinary albumin has been developed based on the use of a chemiluminescent acridinium ester-labelled human albumin and a commercially available antiserum. It includes two incubation steps and a second polyethylene glycol-assisted antibody separation. The sensitivity of detection is 0.016 mg/l, the assay working range is 0.1-5 mg/l, and the inter-assay CVs are ≤ 15%. Using 10? and 50-fold sample dilutions in assay buffer, a wide working range (1-250 mg/l) is obtained covering normal and pathological conditions. Timed overnight urine samples (bed rest conditions) were collected on three consecutive days for each patient. Albumin excretion rate (AER) was 4.7 ± 2.7 μg/min (x ± SD), range 1-15.9 μg/min in 36 healthy subjects (17♂, 19♀, ages 4-56 years), with day-to-day variations of 28.5 ± 20% (x ± SD), range 3.3-76.1%. The use of an acridinium ester as a chemiluminescent (CL) label overcomes the disadvantages of short shelf-life and health and safety hazards associated with radioisotopes. Results compare favourably with those obtained using a commercially available RIA kit.     

Differential Regulation of Transferrin Receptor Gene Expression in Human Hemopoietic Cells: Molecular and Cellular Aspects

Abstract

As we have shown earlier (-)¹²⁵lodocyanopindolol (¹²⁵ICYP) binding to β-adrenoceptors (β-AR) in human mononuclear leucocytes (MNL) yields evidence for the existence of high affinity (B_hiaff) and low affinity (B_loaff) binding sites. We studied the regulation of these 2 classes of binding sites during 240 min of (-)-epinephrine (EPI) infusion (0.1 μg/kg/min) (n=8) in male healthy volunteers. Saturation experiments were performed on MNL membranes with ¹²⁵ICYP over a large concentration range (1–550 pmol/l). Binding parameters were calculated by computer analysis assuming 2 classes of binding sites. We found a preinfusion value of 830±50 [sites/cell] (K_D=1.5±0.2 pmol/l) of B_hiaff binding sites and 5210±510 [sites/cell] (K_D=420±80 pmol/l) of B_loaff. During EPI infusion we observed biphasic modulation of the B_hiaff and an inverse modulation of the B_loaff. After 40 min of EPI B_hiaff increased to 1970±280 [sites/cell] (K_D=4.2±0.8 pmol/l), whereas B_loaff decreased to 2720±280 [sites/cell] (K_D=140±70 pmol/l); despite constant plasma epinephrine concentration (PEC) after 240 min of EPI B_hiaff changed to 1310±240 [sites/cell] (K_D=2.8±1.0 pmol/l) vs. 4370±760 [sites/cell] (K_D=190±100 pmol/l) B_loaff. These results suggest an interdependent inverse modulation of the 2 classes of binding sites for ¹²⁵ICYP on MNL during EPI infusion.     

正常人卵巢绒毛膜促性腺激素(hCG)受体的研究

23例正常卵巢制备之细胞膜,可与绒毛膜促性腺激素(hCG)发生特异性结合,最大结合率为13.2±2.24％,Scatchard作图分析,得一直线。23例正常卵巢之受体量为O.66±0.142×10~(-10)M/μg膜蛋白,Kd值为10.16±5.5×10~(-9)M。     

The influence of normal human IgG and of thyroid-stimulating antibody (TSAb) on the binding of thyrotropin to thyroid plasma membranes.

The influence of serum IgG from normal and Graves' disease subjects on the binding of 125I-thyrotropin to isolated thyroid membranes was studied. IgG from either source inhibited binding in a concentration -dependent fashion. Human membranes were more sensitive to the human IgG than were bovine thyroid membranes and, when membranes were purified by discontinuous sucrose gradient centrifugation, they were more sensitive than were those less-purified, obtained by differential centrifugation. Using a Dixon plot, inhibition by normal IgG showed non-competitive kinetics. Fab fragments were more effective, on an equimolar basis, than was intact normal IgG, but were less potent when Graves' disease IgG was the parent molecule; this difference implies distinct modes of inhibition of thyrotropin-binding. The degree of inhibition by normal IgG was variable so that multiple control preparations are required to assess the additional effect characteristic of IgG from the subject with Graves' disease.     

Serum zinc levels in healthy subjects from southeastern Spain

The serum zinc (Zn) concentrations of 80 healthy subjects (48 male, 32 female) from southeastern Spain were determined by atomic absorption spectrometry. The samples were digested by heating in a 4:1 mixture of nitric and perchloric acids. The concentration of Zn was determined against a Contox Trace Metal Serum Control Panel A standard reference. Zn concentrations in the standard were found to be 2.332 ±0.489 mg/L, with a mean recovery of 102.7%. In the serum samples, the relative standard deviation was <6% for the range of concentrations determined: 0.420-1.540 mg/L for women (mean value 0.947 ±0.265 mg/L) and 0.490-1.480 mg/L for men (mean value 0.951 ±0.243 mg/L). In healthy subjects, no statistically significant differences were observed in the Zn levels with respect to their sex (p > 0.05) or the location where they lived (mountainous vs coastal zones). It is concluded that the dietary Zn intake and Zn status for healthy adults in this region of Spain are within normal values.     

Changes in fucosylation of human seminal IgG and secretory component of IgA in leukocytospermic patients

Our study compares the status of human seminal plasma immunoglobulin G (IgG) and IgA secretory component (SC) fucosylation between infertile leukocytospermic and normal, fertile normozoospermic patients. The seminal IgG and SC are decorated with AAL-reactive core fucose, and antennary UEA- and LTA-reactive fucose of Lewis^y and Lewis^x structures, respectively. However, a correlation between IgG core fucosylation and IgG concentration (r?=??0.52; p?<?0.0003) was observed. The IgG present in leukocytospermic samples is characterized by lower expression of core fucose than in the normal group (0.82?±?0.3 AU and 1.2?±?0.3 AU, respectively; p?<?0.002). In seminal plasma the SC is present in two forms: 78-kDa and 63-kDa. The present study has also shown a higher AAL and LTA specific reactivity of glycans expressed in 63-kDa SC, in comparison to 78-kDa SC, in the normal group. In leukocytospermia, the values of specific lectin reactivity for core fucose, fucose α(1-2)- and α(1-3)- linked, were similar for both SC bands. Moreover, the present study has shown that in leukocytospermic samples the mean concentrations of IgG and S-IgA are twice as high (131.68?±?102.6 mg/l and 36?±?27 mg/l, respectively) as in the normal group (67.68?±?29.2 mg/l; p?<?0.02, and 19?±?18 mg/l, p?<?0.019, respectively). The analysis of IgG and SC fucosylation status and the determination of IgG and S-IgA concentrations in seminal plasma might constitute a valuable diagnosis tools for the evaluation of male infertility associated with leukocytospermia with accompanying inflammation.