Leukocyte flow properties, polymorphonuclear membrane fluidity and cytosolic Ca2+ content in subjects with vascular atherosclerotic disease

Hereditary predisposition to thrombosis due to activated protein C resistance (APCR) has been attributed to a missense mutation in the factor V gene at nucleotide 1691 (G to A), causing replacement of arginine at codon 506 with glutamine. Using an RFLP-PCR assay to detect this mutation, we measured a prevalence of 3.3% in healthy Caucasians and 1.25% in healthy African-Americans. In addition, we evaluated a total of 90 consecutive specimens submitted to the coagulation laboratory at the Medical College of Virginia for the presence of this mutation. We compared our results for 78 of these specimens with the values measured by a modified partial thromboplastin assay, the COATEST. Twelve of the 90 samples could not be tested using the COATEST because the patients were undergoing anticoagulant therapy. One of the latter 12 specimens was positive by the RFLP-PCR test. Using the genetic test as the definitive assay and the cutoff value established for distinguishing between normal and abnormal results by the COATEST, the COATEST had a sensitivity of 50% and specificity of 93% for the detection of factor V mutation. Analysis of the 90 samples stratified by ethnic groups revealed a frequency of mutation of 13.3% for Caucasians and 6.88% for African-Americans, although with the present sample size, the difference was not statistically significant. Although the COATEST is technically simpler to perform than the genetic test for diagnosing the presence of the factor V mutation, its use for this purpose is limited due to low sensitivity. Thus where this disorder is clinically suspected, submission of the specimen directly for genetic testing by RFLP-PCR or equivalent assay should be considered.     

Polymorphonuclear leukocyte membrane fluidity and cytosolic Ca2+ concentration in diabetes mellitus

OBJECTIVE: To establish a rapid and efficient technique of constructing human chromosomal band specific probe pools and their libraries. METHODS: A modified method of combining chromosome microdissection with degenerate oligonucleotide primed PCR(DOP-PCR) was used. 3p23-p26, 3q21-q22 and 4p12- p16 band from human chromosomes were microdissected and amplified as probe pools. The origins of the PCR products were determined by chromosome fluorescence in situ hybridization. The PCR products and pUC19 were digested by Xho I and Sal I respectively, and linke up. The DH5alpha were transformed by the recombinated vectors as the specific band libraries. The inserts were digested by EcoR I and Hind III, then measured by electrophoretic analysis. And the copies of inserts were identified by in situ bacterial colony hybridization with genomic DNA. RESULTS: All the three probe pools showed the special yellow-green signals in their microdissection responsible bands. The sizes of DOP-PCR products ranged from 300bp to 1800bp. 3q21-q22 probe pool generated about 1.2 x 10(4) clones. The average size of inserts was about 420bp by analysis of 30 positive clones. The rate of single-copy and low-repeated sequences was about 81%(178/220), while the rate of middle-repeated and high- repeated sequences was about 19%(42/220). CONCLUSION: The results proved that the modified microdissection combining DOP-PCR technique provided a simple and efficient method to construct the human chromosome band-specific probe pools and might contribute to gene cloning and complete sequencing of human genome.     

Behavior of the polymorphonuclear leukocyte membrane fluidity and cystolic Ca2+ content in vascular atherosclerotic disease with and without non-insulin-dependent diabetes mellitus

In 71 subjects with vascular atherosclerotic disease (VAD), in 32 VAD subjects with non-insulin-dependent diabetes mellitus (NIDDM) and in 31 normal controls, we evaluated polymorphonuclear leukocyte (PMN) membrane fluidity and PMN cytosolic Ca2+ content. The PMN membrane fluidity was obtained by marking intact and unstimulated PMN cells with fluorescent probe 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and the PMN cytosolic Ca2+ content was obtained by marking intact and unstimulated PMN cells with the fluorescent probe Fura 2-AM. From the obtained results, it is evident that PMN membrane fluidity does not differentiate normals from VAD subjects and VAD subjects with NIDDM, and normals from subjects with monovascular disease (MVAD) and polyvascular disease (PVAD) with and without NIDDM. The PMN cytosolic Ca2+ content is significantly increased in VAD subjects and VAD subjects with NIDDM, and also in MVAD and PVAD subjects with and without NIDDM. A positive correlation is present between PMN membrane fluidity and PMN cytosolic Ca2+ content in normals and VAD subjects, but not in VAD subjects with NIDDM. In conclusion, in VAD subjects with and without NIDDM, an increase of the PMN cytosolic Ca2+ content is present; this increase might be related to the PMN spontaneous activation.     

Region-specific activity of the plasma membrane Ca2+ pump and delayed activation of Ca2+ entry characterize the polarized, agonist-evoked Ca2+ signals in exocrine cells

The initial release of Ca2+ from the intracellular Ca2+ stores is followed by a second phase during which the agonist-dependent Ca2+ response becomes sensitive to the extracellular Ca2+, indicating the involvement of the plasma membrane (PM) Ca2+ transport systems. The time course of activation of these transport systems, which consist of both Ca2+ extrusion and Ca2+ entry pathways, is not well established. To investigate the participation of these processes during the agonist-evoked Ca2+ response, isolated pancreatic acinar cells were exposed to maximal concentrations of an inositol 1,4,5-trisphosphate-mobilizing agonist (acetylcholine, 10 microM) in different experimental conditions. Following the increase of [Ca2+]i, there was an almost immediate activation of the PM Ca2+ extrusion system, and maximal activity was reached within less than 2s. The rate of Ca2+ extrusion was dependent on the level of [Ca2+]i, with a steep activation at values just above the resting [Ca2+]i and reached a plateau value at 700 nM Ca2+. In contrast, the PM Ca2+ entry pathway was activated with a much slower time course. There was also a delay of 3-4 s between the maximal effective depletion of the intracellular Ca2+ stores and the activation of this entry pathway. By use of digital imaging data, the PM Ca2+ transport systems were also analyzed independently in two regions of the cells, the lumenal and the basal poles. With respect to the activation of the Ca2+ entry pathways, no significant difference existed between these two regions. In contrast, the PM Ca2+ pump displayed a different pattern of activity in these regions. In the basal pole, the pump activity was more sensitive to changes of [Ca2+]i and had a higher maximal activity. Also, in the lumenal pole, the pump became saturated at values of [Ca2+]i around 700 nM, whereas at the basal pole [Ca2+]i had a biphasic effect on the pump activity, and higher [Ca2+]i inhibited the pump. It is argued that these differences in sensitivity to the levels of [Ca2+]i and the different relationship between [Ca2+]i and the rate of extrusion at the two functional poles of the pancreatic acinar cells indicate that the plasma membrane Ca2+ ATPase might play an important role in the polarization of the Ca2+ response.     

Excitation-contraction-relaxation cycle: role of Ca2+-regulatory membrane proteins in normal, stimulated and pathological skeletal muscle (review)

Extremely large protein complexes involved in the Ca2+-regulatory system of the excitation-contraction-relaxation cycle have been identified in skeletal muscle, i.e. clusters of the Ca2+-binding protein calsequestrin, apparent tetramers of Ca2+-ATPase pump units and complexes between the transverse-tubular alpha1-dihydropyridine receptor and ryanodine receptor Ca2+-release channel tetramers of the sarcoplasmic reticulum. While receptor interactions appear to be crucial for signal transduction during excitation-contraction coupling, avoidance of passive disintegration of junctional complexes and stabilization of receptor interactions may be mediated by disulfide-bonded clusters of triadin. Oligomerization of Ca2+-release, Ca2+-sequestration and Ca2+-uptake complexes appear to be an intrinsic property of these muscle membrane proteins. During chronic low-frequency stimulation, the expression of triad receptors is decreased while conditioning has only a marginal effect on Ca2+-binding proteins. In contrast, muscle stimulation induces a switch from the fast-twitch Ca2+-ATPase to its slow-twitch/cardiac isoform. These alterations in Ca2+-handling might reflect early functional adaptations to electrical stimulation. Studying Ca2+-homeostasis in transformed muscles is important regarding the evaluation of new clinical applications such as dynamic cardiomyoplasty. Studies of Ca2+-handling in skeletal muscle fibers have not only increased our understanding of muscle regulation, but have given important insights into the molecular pathogenesis of malignant hyperthermia, hypokalemic periodic paralysis and Brody disease.     

Study on Ca8Mg(SiO4)4Cl2:Eu2+ Doped with Sr2+

Ca8Mg(SiO4)4Cl2:Eu2 phosphor doped with Sr2 cation for Ca2 partially, was synthesized by solid-state reaction at high temperature under reducing atmosphere, and its luminescent properties were investigated. The experimental results indicate that the emission intensity of the phosphor increases after being doped with a few amount of Sr2 ion. The emission peak of the phosphor blue shift to about 464 nm when the phosphor is doped with large quantity of Sr2 ions. The excitation spectrum indicates that the phosphor can be well excited by UV and blue light from 300 to 460 nm, and the phosphor was fitted well for the excitation by UV or blue-LED.     

Induction of thymocyte apoptosis by Ca2+-independent protein kinase C (nPKC) activation and its regulation by calcineurin activation

Glucocorticoids appear to participate in apoptosis of unselected CD4(+)CD8(+) thymocytes. Activation of Ca2+-independent novel protein kinase C (nPKC) precedes glucocorticoid-induced thymocyte apoptosis, while proper levels of Ca2+-dependent protein kinase C (cPKC) and calcineurin activities contribute to rescue thymocytes. To clarify the role of nPKC in thymocyte apoptosis, murine thymocytes were stimulated with the diterpene diester, ingenol 3, 20-dibenzoate (IDB). IDB induced selective translocation of nPKC-delta, -epsilon, and -theta and PKC-mu from the cytosolic fraction to the particulate fraction and induced morphologically typical apoptosis through de novo synthesis of macromolecules. The apoptosis was also induced by thymeleatoxin, a diterpene ester, at relatively high concentrations that induced translocation of cPKC, nPKC-theta, and PKC-mu. The IDB- or thymeleatoxin-induced death was inhibited by non-isoform-selective PKC inhibitors, but not by their structural analogs with weak PKC-inhibitory activity or the selective inhibitor of cPKC and PKC-mu, G? 6976. The death was also inhibited by calcium ionophore ionomycin at concentrations within a narrow range. The range corresponded to the concentration range that contributes to the inhibition of glucocorticoid-induced apoptosis. The antiapoptotic effect was canceled by the immunosuppressant FK506 but not by rapamycin. These results indicate that activation of nPKC, especially nPKC-theta, induces apoptosis in thymocytes and that calcineurin activation regulates the apoptosis.     

Luminescence studies of MBrCl： Eu2＋ （M=Ca, Sr, Ba）

Europium doped MBrCl (M=Ca, Sr, and Ba) phosphors were prepared by solid state reaction in reductive atmosphere. Photolu-minescence (PL), photostimulated luminescence (PSL) after X-ray irradiation and optical absorption studies of MBrCl:Eu2+ (M=Ca, Sr, and Ba) revealed that: (1) blue light emission, under the excitation of 300 nm, was observed in all these phosphors; (2) the shape of the emission spectra in CaBrCl:Eu2+ could be changed by varying the bromine/chlorine ratio during synthesis, while that in SrBrCl:Eu2+ and BaBrCl:Eu2+ showed no change; and (3) PSL was observed in SrBrCl:Eu2+ and BaBrCI:Eu2+ after X-ray irradiation. Difference absorption spectrum (DAS) in SrBrCl:Eu2+ showed two broad bands centered at about 470 and 570 nm, and DAS in BaBrCI:Eu2+ showed two bands at about 550 and 675 nm, respectively. This enabled the use of He-Ne laser (633 nm) or even semiconductor light-emitting diodes (LED) instead of gas lasers for photostimulation.     

Dual effect of the anti-allergic astemizole on Ca2+ fluxes in rat basophilic leukemia (RBL-2H3) cells: release of Ca2+ from intracellular stores and inhibition of Ca2+ release-activated Ca2+ influx

The antiallergic drugs astemizole and norastemizole inhibit exocytosis in mast cells, which might be relevant for their therapeutic action. From previous studies, it appeared that the drugs inhibited 45Ca2+ influx. Here, we present a more detailed study on the effects of astemizole and norastemizole on Ca2+ fluxes. Fura-2-loaded rat basophilic leukemia (RBL-2H3) cells were activated through the high-affinity receptor for IgE (FcepsilonRI) with antigen or by the endoplasmatic reticulum ATPase inhibitor thapsigargin, bypassing direct FcepsilonRI-related events. It appeared that astemizole (>15 microM), in contrast to norastemizole, showed a dual effect on intracellular calcium concentration ([Ca2+]i): a rise in intracellular calcium concentration was induced, which originated in the release of intracellular Ca2+ stores, whereas Ca2+ influx via store-operated Ca2+ (SOC) channels was inhibited. Ca2+ influx was further characterized using Ba2+ influx, whereas processes in the absence of Ca2+ influx were studied using Ni2+ or EGTA. It was concluded that the drugs most likely affect the store-operated Ca2+ channels in RBL cells directly. The two effects of astemizole on Ca2+ fluxes had opposing influences on exocytosis, thereby accounting for the biphasic effect of increasing astemizole concentration on mediator release in RBL cells.     

Ca2+ channels, ryanodine receptors and Ca(2+)-activated K+ channels: a functional unit for regulating arterial tone

Local calcium transients ('Ca2+ sparks') are thought to be elementary Ca2+ signals in heart, skeletal and smooth muscle cells. Ca2+ sparks result from the opening of a single, or the coordinated opening of many, tightly clustered ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In arterial smooth muscle, Ca2+ sparks appear to be involved in opposing the tonic contraction of the blood vessel. Intravascular pressure causes a graded membrane potential depolarization to approximately -40 mV, an elevation of arterial wall [Ca2+]i and contraction ('myogenic tone') of arteries. Ca2+ sparks activate calcium-sensitive K+ (KCa) channels in the sarcolemmal membrane to cause membrane hyperpolarization, which opposes the pressure induced depolarization. Thus, inhibition of Ca2+ sparks by ryanodine, or of KCa channels by iberiotoxin, leads to membrane depolarization, activation of L-type voltage-gated Ca2+ channels, and vasoconstriction. Conversely, activation of Ca2+ sparks can lead to vasodilation through activation of KCa channels. Our recent work is aimed at studying the properties and roles of Ca2+ sparks in the regulation of arterial smooth muscle function. The modulation of Ca2+ spark frequency and amplitude by membrane potential, cyclic nucleotides and protein kinase C will be explored. The role of local Ca2+ entry through voltage-dependent Ca2+ channels in the regulation of Ca2+ spark properties will also be examined. Finally, using functional evidence from cardiac myocytes, and histological evidence from smooth muscle, we shall explore whether Ca2+ channels, RyR channels, and KCa channels function as a coupled unit, through Ca2+ and voltage, to regulate arterial smooth muscle membrane potential and vascular tone.     

Limbic system-associated membrane protein (LAMP) induces neurite outgrowth and intracellular Ca2+ increase in primary fetal neurons

The ability of cell adhesion molecules (CAMs) to transduce cell surface signals into intracellular responses is critical for developing neurons, particularly during axonal pathfinding and targeting. It has been suggested that different CAMs can promote neuronal outgrowth via activation of common neuronal CAM-specific second-messenger pathways, although the elements involved in this cascade could differ. Limbic system-associated membrane protein (LAMP), a member of the Ig superfamily, is a molecule that promotes cell adhesion and neurite outgrowth from specific populations of fetal neurons. In the present study, we show that LAMP can induce several types of calcium (Ca2+) signals. Neurite outgrowth is promoted if fetal hippocampal neurons are grown on lamp-transfected CHO cells. This LAMP-induced outgrowth of neurons is mediated in part through activation of L-type Ca channels. Application of soluble LAMP to cultures of fetal hippocampal neurons caused a sustained (up to 60 min) elevation of intracellular Ca2+ as measured by fluo-3 fluorescence on a confocal microscope. The number of responding hippocampal neurons was initially low, but increased with age in culture and the [Ca2+]i elevation was only partially decreased by an L-type Ca(2+)-channel blocker. In contrast, at all times in culture, only a small fraction of neurons from visual cortex responded to LAMP application and only with transient elevation of cytosolic Ca2+ (< 15 min). Based on these observations, LAMP appears to function primarily through homophilic interactions and acts in part by modulating intracellular Ca2+ levels during neurite outgrowth by increasing the Ca2+ influx through L-type calcium channels, but has additional effects on intracellular Ca2+ signaling at later developmental stages.     

Effect of phorbol ester, 12-deoxyphorbol 13-isobutylate (DPB), on muscle tension and cytosolic Ca2+ in rat anococcygeus muscle

Effects of phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB), on muscle tension and cytosolic Ca2+ ([Ca2+]i) level was investigated in rat anococcygeus muscle in comparison with other smooth muscles. 1) DPB (10(-6) M) induced a large contraction and an elevation of [Ca2+]i level in rat aorta and small and rhythmic changes in tension and [Ca2+]i level in guinea pig ileum. However, DPB did not change either of the parameters in rat anococcygeus muscle. 2) DPB caused tension development without changing the [Ca2+]i level elevated by high K+, ionomycin or beta-escin in the anococcygeus muscle. 3) In the beta-escin permeabilized muscles of guinea pig ileum and urinary bladder, rabbit mesenteric artery and rat anococcygeus muscle, DPB enhanced the Ca(2+)-developed tension. Moreover, the enhancement was inhibited by H-7 (3 x 10(-5) M). 4) DPB did not cause muscle tension to develop in the muscle of rat aorta, guinea pig ileum and rat anococcygeus muscle, pretreated with phorbol 12-myristate 13-acetate for 24 hr. In conclusion, DPB showed different contractile effects on the aorta, ileum and anococcygeus muscle, respectively. The initiation of muscle tension by DPB probably requires [Ca2+]i and the DPB-induced enhancement may be due to a Ca2+ sensitization of contractile elements in the anococcygeus muscle. Therefore, the difference between the DPB-induced response of the anococcygeus muscle and those of the other muscles seems to be due to a different Ca2+ movement caused by DPB. Moreover, it is suggested that DPB develops muscle tension by increasing [Ca2+]i and enhances it through the mediation of protein kinase C in the anococcygeus muscle as well as the other smooth muscles.     

Effects of Cd2+, Pb2+ and CH3Hg+ on high voltage-activated calcium currents in pheochromocytoma (PC12) cells: potency, reversibility, interactions with extracellular Ca2+ and mechanisms of block

The present study attempted to determine the relationship between affective disposition and the favorability of letters of reference. We hypothesized that individuals with positive dispositions would write more favorable letters than would individuals with less positive or neutral dispositions. In addition, we also hypothesized that length of letter would partly mediate the relationship between affective disposition and letter favorability. To test these hypotheses, two studies were conducted. In order to present letter writers with a controlled stimulus, Study 1 entailed having 110 undergraduate students write letters of reference for two hypothetical job candidates in a laboratory setting. In order to test the generalizability of the laboratory study, Study 2 involved 95 faculty members who submitted three recent letters of reference they had written. Results showed that affective disposition was related to the favorability of letters of reference in both studies. Results also revealed that length of letter partly mediated the relationship between affective disposition and letter favorability. Copyright 1998 Academic Press.     

Effects of semotiadil fumarate (SD-3211) and its enantiomer, SD-3212, on the changes in cytosolic Ca2+ and tension caused by KCl and norepinephrine in isolated rat aortas

Semotiadil fumarate (SD-3211), a Ca2+ channel blocker of benzothiazine derivative and its (S)-(-)-enantiomer (SD-3212), inhibited K(+)- and norepinephrine (NE)-induced contractions in isolated rat aortas. Inhibition of NE contraction induced by both drugs was greater than that induced by diltiazem or bepridil, whereas inhibition of K(+)-contraction was similar to that induced by diltiazem or bepridil. Semotiadil and SD-3212 (10 microM) inhibited the increase in cytosolic Ca2+ ([Ca2+]i) induced by 65.4 mM K+ in fura-2-loaded preparations as well as diltiazem and bepridil (10 microM). On the other hand, semotiadil and SD-3212 (10 microM) inhibited only the early phase of increase in [Ca2+]i induced by 1 microM NE. After 5 min, no significant effect on [Ca2+]i was observed with these compounds despite the significant decrease in the contraction. In contrast to these compounds, diltiazem and bepridil 10 microM affected neither the increase in [Ca2+]i nor the contraction induced by NE. Semotiadil and SD-3212 inhibited the transient contraction induced by 1 microM NE in the absence of external Ca2+. Both compounds partially but significantly inhibited the NE-induced contraction in nifedipine-treated muscles. These results suggest that semotiadil and SD-3212 inhibit contractions of vascular smooth muscle (VSM) not only through blockade of voltage-dependent Ca2+ channels but also through other mechanisms, such as inhibition of Ca2+ release from Ca2+ stores or decrease in sensitivity of the contractile elements to Ca2+.     

Luminescence and Preparation of LED Phosphor Ca8Mg(SiO4)4Cl2:Eu2+

Calcium magnesium chlorosilicate doped by europium, Ca8Mg(SiO4)4Cl2:Eu2 , was prepared by the solid state reaction at high temperature. The compound obtained is pure Ca8Mg(SiO4)4Cl2 phase with cubic structure. Its average particle size is 5 μm, and it has good dispersity and morphological form. The excitation spectrum of Ca8Mg(SiO4)4Cl2:Eu2 is a wide band, which covers from 270 to 480 nm. The emission spectrum is also a wide band peaked at 510 nm. The luminescent intensity reaches to the maximum when the concentration of Eu2 is 2%. The wavelength of emission and excitation of the phosphor with various Eu2 contents keeps constant. This spectrum range matches violet and blue LED chips very well, and its strong luminescence intensity is suitable for a green phosphor of tricolor phosphor of white light LED.     

Stimulation of primed neutrophils by soluble immune complexes: priming leads to enhanced intracellular Ca2+ elevations, activation of phospholipase D, and activation of the NADPH oxidase

Soluble immune complexes activate a rapid burst of reactive oxidant secretion from neutrophils that have previously been primed with GM-CSF. Binding of these complexes to the cell surface of unprimed neutrophils results in the generation of intracellular Ca2+ transients, but the NADPH oxidase fails to become activated. No phospholipase D activity was observed following the addition of soluble immune complexes to unprimed cells. Upon priming with GM-CSF, the intracellular Ca2+ signal generated following soluble complex binding was greatly extended and phospholipase D was activated: there was also increased phosphorylation of proteins on tyrosine residues and the NADPH oxidase was activated. When Ca2+ influx was prevented, this phospholipase D activity was not observed. This primed oxidase activity was completely inhibited by erbstatin. Treatment of unprimed neutrophils with pervanadate (to inhibit protein tyrosine phosphatases) mimicked the effects of priming in that pervanadate-treated neutrophils secreted reactive oxidants in response to soluble immune complexes. The data indicate that during priming a new signaling pathway is activated that involves Ca2+ influx, phosphorylation on tyrosine residues, phospholipase D activity, and NADPH oxidase activation.     

Simultaneous activation of Ca(2+)-dependent K+ and Cl- currents by various forms of stimulation in the membrane of smooth muscle cells from the rabbit basilar artery

In 1997, the Harvard School of Public Health College Alcohol Study resurveyed colleges that participated in a 1993 study. The findings revealed little change in binge drinking: a slight decrease in percentage of binge drinkers and slight increases in percentages of abstainers and frequent binge drinkers. Two of 5 students were binge drinkers (42.7%); 1 in 5 (19.0%) was an abstainer, and 1 in 5 was a frequent binge drinker (20.7%). As was true in 1993, 4 of 5 residents of fraternities or sororities were binge drinkers (81.1%). Asian students showed a greater increase and White students a greater decrease in binge drinking from 1993 to 1997, compared with all other students. Among students who drank alcohol, increases in frequency of drinking; drunkenness; drinking to get drunk; and alcohol-related problems, including drinking and driving, were reported. Binge drinkers in both 1993 and 1997 were at increased risk of alcohol-related problems, and nonbingers at colleges with high binge drinking rates had increased risks of encountering secondhand effects of binge drinking.