James S.C. Gilchrist

Distribution of omega-3 fatty acids in tissues of rabbits fed a flaxseed-supplemented diet

Diets rich in omega-3 polyunsaturated fatty acids are associated with decreased incidences of cardiovascular disease. The extent of incorporation and distribution of these beneficial fats into body tissues is uncertain. Rabbits were fed regular rabbit chow or a diet containing 10% ground flaxseed that is highly enriched with the omega-3 polyunsaturated fatty acid alpha-linolenic acid (ALA). The high-flaxseed diet resulted in an incorporation of ALA in all tissues, but mostly in the heart and liver with little in the brain. Docosahexaenoic and eicosapentaenoic acid levels were also selectively increased in some tissues, and the effects were not as large as ALA. Arachidonic acid and the ratio of omega-6/omega-3 fatty acids were decreased in all tissues obtained from the flax-supplemented group. Consumption of dietary flaxseed appears to be an effective means to increase ALA content in body tissues, but the degree will depend upon the tissues examined.

Nucleolin is a calcium‐binding protein

We have purified a prominent 110‐kDa protein (p110) from 1.6 M NaCl extracts of rat liver nuclei that appears to bind Ca2+. p110 was originally identified by prominent blue staining with ‘Stains‐All’ in sodium dodecyl sulfate–polyacrylamide gels and was observed to specifically bind ruthenium red and 45Ca2+ in nitrocellulose blot overlays. In spin‐dialysis studies, purified p110 saturably bound approximately 75 nmol Ca2+/mg protein at a concentration of 1 mM total Ca2+ with half‐maximal binding observed at 105 μM Ca2+. With purification, p110 became increasingly susceptible to proteolytic (likely autolytic) fragmentation, although most intermediary peptides between 40 and 90 kDa retained “Stains‐All”, ruthenium red, and 45Ca2+ binding. N‐terminal sequencing of intact p110 and a 70‐kDa autolytic peptide fragment revealed a strong homology to nucleolin. Two‐dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)/IEF revealed autolysis produced increasingly acidic peptide fragments ranging in apparent pIs from 5.5 for intact p110 to 3.5 for a 40 kDa peptide fragment. Intact p110 and several peptide fragments were immunostained with a highly specific anti‐nucleolin antibody, R2D2, thus confirming the identity of this protein with nucleolin. These annexin‐like Ca2+‐binding characteristics of nucleolin are likely contributed by its highly acidic argyrophilic N‐terminus with autolysis apparently resulting in largely selective removal of its basic C‐terminal domain. Although the Ca2+‐dependent functions of nucleolin are unknown, we discuss the possibility that like the structurally analogous HMG‐1, its Ca2+‐dependent actions may regulate chromatin structure, possibly during apoptosis. J. Cell. Biochem. 85: 268–278, 2002.

Oxidation of Nuclear Membrane Cholesterol Inhibits Nucleoside Triphosphatase Activity

Oxygen derived free radicals can oxidize membrane cholesterol. We have previously shown that cholesterol in the nuclear membrane can modulate nuclear nucleoside triphosphatase (NTPase) activity. Nucleocytoplasmic transport of peptides and mRNA via the nuclear pore complex may be regulated by the NTPase. The purpose of the present study was to determine if oxidation of nuclear cholesterol could alter NTPase activity. Nuclear membrane cholesterol was oxidized in situ with cholesterol oxidase (to selectively oxidize cholesterol) and NTPase activity measured. HPLC analysis confirmed the formation of cholesterol oxides. The activity of the NTPase was strikingly inhibited by cholesterol oxidase treatment. The Vmax of the NTPase was significantly decreased after cholesterol oxidase treatment but the Km value was unchanged. The sensitivity of NTPase activity to varying cholesterol oxidase concentrations also suggested that cholesterol located in the inner leaflet of the nuclear membrane appeared to be more important in the modulation of NTPase activity than that in the cytoplasmic leaflet. Our results indicate that oxidation of nuclear membrane cholesterol inhibits NTPase activity. These results have implications for peptide and mRNA flux across the nuclear membrane during conditions where lipid oxidation may be expected.

Nuclear membrane cholesterol can modulate nuclear nucleoside triphosphatase activity

Previous work has suggested that changes in nuclear membrane cholesterol may induce a stimulation in nuclear nucleoside triphosphatase (NTPase) activity. The purpose of the present study was to directly investigate if nuclear membrane cholesterol can stimulate nuclear NTPase activity. The cholesterol content of nuclei was altered with a liposomal methodology. The cholesterol content of nuclei isolated from hepatic tissue was relatively low in comparison to that typically exhibited by other membrane fractions. Because of this, it was difficult to further deplete the nuclear membrane of cholesterol, but we could successfully increase the cholesterol content after exposure to cholesterol‐enriched liposomes. Nuclear NTPase activity was potently stimulated (∼︁ 150–200% of control) by an increase in the nuclear membrane cholesterol content. The Vmax of the NTPase activity in the presence of ATP or GTP was significantly increased after cholesterol enrichment without altering the affinity of the enzyme for these moieties. Mg2+ dependency of NTPase activity was also altered by cholesterol incorporation into the nuclear membrane. Cholesterol enrichment of the nuclear membrane also left the nuclei more susceptible to damage by salt‐induced lysis than control nuclei. Our results clearly demonstrate that the cholesterol content of the nuclear membrane will have significant, direct effects on nuclear integrity and NTPase activity.

Spectroscopic determination of sarcoplasmic reticulum Ca2+ uptake and Ca2+ release

In this report we describe the application of spectroscopic methods to the study of Ca2+ release by isolated native sarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle. To date, dual-wavelength spectroscopy of arsenazo III and antipyrylazo III difference absorbance have been the most common spectroscopic methods for the assay of SR Ca2+ transport. The utility of these methods is the ability to manipulate intraluminal Ca2+ loading of SR vesicles. These methods have also been useful for studying the effect of both agonists and antagonists upon SR Ca2+ release and Ca2+ uptake. In this study, we have developed the application of Calcium Green-2, a long-wavelength excitable fluorescent indicator, for the study of SR Ca2+ uptake and release. With this method we demonstrate how ryanodine receptor Ca2+ channel opening and closing is regulated in a complex manner by the relative distribution of Ca2+ between extraluminal and intraluminal Ca2+ compartments. Intraluminal Ca2+ is shown to be a key regulator of Ca2+ channel opening. However, these methods also reveal that the intraluminal Ca2+ threshold for Ca2+-induced Ca2+ release varies as a function of extraluminal Ca2+ concentration. The ability to study how the relative distribution of a finite pool of Ca2+ across the SR membrane influences Ca2+ uptake and Ca2+ release may be useful for understanding how the ryanodine receptor is regulated, in vivo.

Caspase 3 activity in isolated fetal rat lung fibroblasts and rat periodontal ligament fibroblasts: cigarette smoke induced alterations

BackgroundCigarette smoking is the leading cause of preventable death and has been implicated in pathogenesis of pulmonary, oral and systemic diseases. Smoking during pregnancy is a risk factor for the developing fetus and may be a major cause of infant mortality. Moreover, the oral cavity, and all cells within are the first to be exposed to cigarette smoke and may be a possible source for the spread of toxins to other organs of the body. Fibroblasts in general are morphologically heterogeneous connective tissue cells with diverse functions. Apoptosis or programmed cell death is a crucial process during embryogenesis and for the maintenance of homeostasis throughout life. Deregulation of apoptosis has been implicated in abnormal lung development in the fetus and disease progression in adults. Caspases are proteases which belong to the family of cysteine aspartic acid proteases and are key components for downstream amplification of intracellular apoptotic signals. Of 14 known caspases, caspase-3 is the key executioner of apoptosis. In the present study we explored the hypothesis that cigarette smoke (CS) extract activates caspase-3 in two types of fibroblasts, both of which would be exposed directly to cigarette smoke, isolated fetal rat lung fibroblasts and adult rat periodontal ligament (PDL) fibroblasts.MethodsIsolated fetal rat lung fibroblasts and adult PDLs were used. Cells were exposed to different concentrations of CS for 60 min. Caspase-3 activity and its inhibition by Z-VAD-fmk were measured by caspase-3 fluorometric assay. The effect of CSE on cellular viability was measured using the MTT formazan assay. Caspase-3 expression was detected by western blot analysis and cellular localization of caspase-3 was determined by immunofluorescence using fluorescence microscopy.ResultsIt was observed in fetal rat lung fibroblast cells that CSE extract significantly (p<0.05) increased caspase-3 activity and decrease cell proliferation. However, no significant changes in activity or viability were observed in PDLs.ConclusionsThis indicates CS activates caspase-3 the key regulatory point in apoptosis in fetal rat lung fibroblast cells suggesting that smoking during pregnancy may alter the developmental program of fetal lung, jeopardizing the establishment of critical cellular mechanisms necessary to expedite pulmonary maturation at birth.of critical cellular mechanisms necessary to expedite pulmonary maturation at birth.

μ-Calpain-mediated deregulation of cardiac, brain, and kidney NCX1 splice variants

μ-Calpain is a Ca(2+)-activated protease abundant in mammalian tissues. Here, we examined the effects of μ-calpain on three alternatively spliced variants of NCX1 using the giant, excised patch technique. Membrane patches from Xenopus oocytes expressing either heart (NCX1.1), kidney (NCX1.3), or brain (NCX1.4) variants of NCX1 were exposed to μ-calpain and their Na(+)-dependent (I(1)) and Ca(2+)-dependent (I(2)) regulatory phenotypes were assessed. For these exchangers, I(1) inactivation is evident as a Na(+)(i)-dependent decay of peak outward currents whereas I(2) regulation manifests as outward current activation by micromolar Ca(2+)(i) concentrations. Notably, with NCX1.1 and NCX1.4 but not in NCX1.3, higher Ca(2+)(i) levels alleviate I(1) inactivation. Our results show that (i) μ-calpain selectively ablates Ca(2+)-dependent (I(2)) regulation leading to a constitutive activation of exchange current, (ii) μ-calpain has much smaller effects on Na(+)-dependent (I(1)) regulation, produced by a slight destabilization of the I(1) state, and (iii) Ca(2+)-dependent regulation (I(2)) and Ca(2+)-mediated alleviation of I(1) appear to be functionally distinct mechanisms, the latter of which is left largely intact after μ-calpain treatment. The ability of μ-calpain to selectively and constitutively activate Na(+)-Ca(2+) exchange currents may have important pathophysiological implications in tissue where these splice variants are expressed.

Functional Significance of Ryanodine Receptor-Mediated Calcium Leaks in Sarcoplasmic Reticulum Membranes

Since their original purification and identification as the main Ca2+ efflux pathway from sarcoplasmic reticulum (SR) membranes, ryanodine receptors (RyRs) have attracted an enormous level of research interest. This is not surprising for several reasons. First, they sit at the epicentre of regulatory mechanisms involved in excitation-contraction coupling. Second, RyRs represent the main building blocks of a huge multimeric protein assembly with a molecular mass estimated to be around 2.3 mega Daltons. Third, RyRs are able to bind an unprecedented number of regulatory proteins, toxins and other molecules producing, in many cases, quite complex effects upon its Ca2+ ion channel properties. Much remains to be determined about how in vitro kinetic and features of RyR activation and inactivation account for observed Ca2+ release properties in vivo. Some studies favor the idea that in intact SR membranes ATP-dependent Ca2+ pumps (SERCA) can also influence both activation and inactivation of Ca2+ release. More recently, however, we have come to appreciate the significant leakiness of SR membranes to Ca2+ at rest and the possibility that this occurs primarily through activation of RyRs. A number of more recent lines of evidence suggest this leakiness has some physiological significance in activating RyRs to initiate Ca2 + sparks and trigger Ca2+ release. Because of the potential importance of this to RyR function a great deal of interest has been generated in an attempt to reveal underlying regulatory mechanism of RyR leakiness. A class of immunophillin binding proteins, known as FKBP12/12.6, is now thought to play a very important role in determining RyR leaks in both normal and pathological settings. However, species and tissue differences in FKBP12/12.6 distribution make it hard to make any firm generalizations, at this point, regarding in vivo RyR leak regulation.

Age- and sex-related differences in nuclear lipid content and nucleoside triphosphatase activity in the JCR:LA-cp corpulent rat

The putative role of the nuclear nucleoside triphosphatase (NTPase) is to provide energy to the nuclear pore complex for poly A(+) mRNA export. Previous work has demonstrated that liver nuclear NTPase activity is greater in 6 month old corpulent (cp/cp) female JCR:LA rats, a hyperlipidemic rat model, compared to lean (+/?) animals. This increase appeared to be related to increases in nuclear membrane cholesterol content. The current study extended these initial data to compare NTPase activity as a function of age and sex in isolated JCR:LA-cp rat liver nuclei, to further test the hypothesis that nuclear membrane cholesterol may modulate NTPase activity. NTPase activity was increased in cp/cp female animals compared to +/? females at all ages studied, with Vmax values increased by 60-176%. Membrane integrity of cp/cp female nuclei was reduced compared to +/? female nuclei. Nuclear membrane cholesterol levels increased linearly with age by 50, 150 and 250% in 3, 6 and 9 month old cp/cp females over leans. In contrast, nuclei from cp/cp males exhibited only minor, isolated changes in NTPase activity. Furthermore, there were no significant changes in nuclear cholesterol content or membrane integrity in the less hyperlipidemic male animals at any age. These data suggest that altered lipid metabolism may lead to changes in nuclear membrane structure, which in turn may alter NTPase activity and functioning of the nuclear pore complex.