Oestrogen metabolism in lymphangioleiomyomatosis: catechol-O-methyltransferase pathway is not involved.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is an uncommon lung disease for which no effective method of treatment has been found. The predilection of LAM for premenopausal women has led to the assumption that hormonal factors play an important role in the pathogenesis of this disease. The aim of this study was to determine if women with LAM manifest alterations in the catechol-O-methyltransferase (COMT) pathway which is essential for preventing the generation of oestrogen derived reactive oxygen species (ROS). METHODS: Blood samples were collected from 15 women with LAM and compared with appropriate controls. The distribution of high and low activity alleles of COMT was determined with a PCR based RFLP assay. The enzymatic activity of COMT was measured in each sample and the potential presence of a circulating inhibitor of COMT was determined. Since an alteration in the COMT pathway could increase the oxidative stress, the plasma concentration of malondialdehyde (MDA), a secondary product generated from lipid peroxidation, has been used as an internal marker. RESULTS: The distribution of high and low activity alleles of COMT (named COMT(HH), COMT(LL), and COMT(HL)) was similar in the two groups with proportions of 40%, 7%, and 53%, respectively, in the women with LAM and 38%, 6%, and 56% in the control subjects. The mean (SD) COMT activity was 24.2 (12.3) pmol/min/mg protein in women with LAM and 24.1 (6.3) pmol/min/mg protein in the control group. Incubation of plasma from women in the two groups with a preparation of commercial COMT showed that no detectable COMT inhibitor was present. The plasma concentration of MDA in the women with LAM was also not significantly different from control subjects. CONCLUSIONS: This study shows that there are no significant alterations in the COMT pathway of women with LAM. It is therefore unlikely that alterations in oestrogen mediated cell signalling pathways are mediated by oxidants derived from an excess of catecholoestrogens in LAM.

Modulation of amyloid precursor protein processing by compounds with various mechanisms of action: detection by liquid phase electrochemiluminescent system.

beta-Amyloid peptide (A beta) is a major component of senile plaques formed in the brain of Alzheimer disease patients. We describe here a new method of quantitating A beta in biological material using liquid phase electrochemiluminescent system (LPECL). We used both enzyme-linked immunosorbent assay (ELISA) and LPECL methods to measure A beta and APPs alpha levels in conditioned medium of beta-amyloid precursor protein (APP)-transfected CHO cells treated with known modulators of APP processing, and in CSF and plasma of guinea pigs. Our results indicate that while maintaining the accuracy and sensitivity of ELISA, LPECL is a significantly taster and less labor-intensive method for measuring of A beta and APPs alpha levels in biological fluids.

Alpha2-macroglobulin associates with beta-amyloid peptide and prevents fibril formation.

We have used the yeast two-hybrid system to isolate cDNAs encoding proteins that specifically interact with the 42-aa beta-amyloid peptide (Abeta), a major constituent of senile plaques in Alzheimer's disease. The carboxy terminus of alpha2-macroglobulin (alpha2M), a proteinase inhibitor released in response to inflammatory stimuli, was identified as a strong and specific interactor of Abeta, utilizing this system. Direct evidence for this interaction was obtained by co-immunoprecipitation of alpha2M with Abeta from the yeast cell, and by formation of SDS-resistant Abeta complexes in polyacrylamide gels by using synthetic Abeta and purified alpha2M. The association of Abeta with alpha2M and various purified amyloid binding proteins was assessed by employing a method measuring protein-protein interactions in liquid phase. The dissociation constant by this technique for the alpha2M-Abeta association using labeled purified proteins was measured (Kd = 350 nM). Electron microscopy showed that a 1:8 ratio of alpha2M to Abeta prevented fibril formation in solution; the same ratio to Abeta of another acute phase protein, alpha1-antichymotrypsin, was not active in preventing fibril formation in vitro. These results were corroborated by data obtained from an in vitro aggregation assay employing Thioflavine T. The interaction of alpha2M with Abeta suggests new pathway(s) for the clearance of the soluble amyloid peptide.

Characterization of new polyclonal antibodies specific for 40 and 42 amino acid-long amyloid beta peptides: their use to examine the cell biology of presenilins and the immunohistochemistry of sporadic Alzheimer's disease and cerebral amyloid angiopathy cases.

BACKGROUND: In Alzheimer's disease (AD), the main histological lesion is a proteinaceous deposit, the senile plaque, which is mainly composed of a peptide called A beta. The aggregation process is thought to occur through enhanced concentration of A beta 40 or increased production of the more readily aggregating 42 amino acid-long A beta 42 species. MATERIALS AND METHODS: Specificity of the antibodies was assessed by dot blot, Western blot, ELISA, and immunoprecipitation procedures on synthetic and endogenous A beta produced by secreted HK293 cells. A beta and p3 production by wild-type and mutated presenilin 1-expressing cells transiently transfected with beta APP751 was monitored after metabolic labeling and immunoprecipitation procedures. Immunohistochemical analysis was performed on brains of sporadic and typical cerebrovascular amyloid angiopathy (CAA) cases. RESULTS: Dot and Western blot analyses indicate that IgG-purified fractions of antisera recognize native and denaturated A beta s. FCA3340 and FCA 3542 display full specificity for A beta 40 and A beta 42, respectively. Antibodies immunoprecipitate their respective synthetic A beta species but also A beta s and their related p3 counterparts endogenously secreted by transfected human kidney 293 cells. This allowed us to show that mutations on presenilin 1 triggered similar increased ratios of A beta 42 and its p 342 counterpart over total A beta and p3. ELISA assays allow detection of about 25-50 pg/ml of A beta s and remain linear up to 750 to 1500 pg/ml without any cross-reactivity. FCA18 and FCA3542 label diffuse and mature plaques of a sporadic AD case whereas FCA3340 only reveals the mature lesions and particularly labels their central dense core. In a CAA case, FCA18 and FCA3340 reveal leptomeningeal and cortical arterioles whereas FCA3542 only faintly labels such structures. CONCLUSIONS: Polyclonal antibodies exclusively recognizing A beta 40 (FCA 3340) or A beta 42 (FCA3542) were obtained. These demonstrated that FAD-linked presenilins similarly affect both p342 and A beta 42, suggesting that these mutations misroute the beta APP to a compartment where gamma-secretase, but not alpha-secretase, cleavages are modified. Overall, these antibodies should prove useful for fundamental and diagnostic approaches, as suggested by their usefulness for biochemical, cell biological, and immunohistochemical techniques.

Cyclic AMP potentiation of cytokine-induced nitric oxide synthase activity in a murine astrocyte cell line.

Astrocytes in culture have been previously shown to express inducible nitric oxide synthase (iNOS) following treatment with cytokines such as interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma). We report here on the effects of the cyclic nucleotide analogues 8-bromo-cyclic AMP and 8-bromo-cyclic GMP on cytokine-stimulated iNOS gene expression in a cultured murine astrocyte cell line. In these cells, neither 8-bromo-cyclic AMP nor 8-bromo-cyclic GMP alone was able to stimulate iNOS activity. Similarly, neither IL-1 beta nor IFN-gamma was capable of independently stimulating iNOS expression. Co-stimulation with both cytokines, however, resulted in measurable increases in iNOS activity, and correlated to increases in iNOS mRNA levels. The addition of 8-bromo-cyclic AMP, but not 8-bromo-cyclic GMP, was found to further enhance the expression of iNOS activity induced by IL-1 beta and IFN-gamma co-stimulation. This potentiation effect of 8-bromo-cyclic AMP correlated to a further elevation in iNOS mRNA levels over that produced by cytokine co-stimulation alone. However, 8-bromo-cyclic AMP co-treatment with either cytokine alone did not stimulate iNOS activity, indicating that the signal transduction pathway(s) involved in the potentiation effect of 8-bromo-cyclic AMP is functional only in the presence of both cytokines. These results indicate that cyclic AMP-mediated processes can participate in modulating the expression of astrocyte iNOS when the appropriate combinations of stimulatory cytokines are present.

Transcriptional inhibition of the beta-amyloid precursor protein by interferon-gamma.

Attenuating beta-amyloid precursor protein (beta-APP) gene expression may have relevance in diseases such as Alzheimer's disease, where beta-APP has been implicated in neuropathological processes. We report here on the transcriptional down-regulation of beta-APP by interferon-gamma (IFN-gamma) in SKNMC human neuroblastoma cells. Treatment of the cells with IFN-gamma resulted in a 85% dose-dependent inhibition of beta-APP promoter activity after 24 h of exposure, with no changes observed at 5 h. For comparison, additional cytokines and signaling agents were also investigated for effects on beta-APP promoter activity. Elevated levels of activity were observed after treatment with phorbol 12-myristate 13-acetate and basic fibroblast growth factor whereas no significant effects were seen after treatment with lipopolysaccharide or interleukin-1 beta. Thus, IFN-gamma was shown here to be a suppressor of beta-APP promoter activity and is the first cytokine reported to possess such down-regulating effects.

Interleukin-6 mRNA expression by cortical neurons in culture: evidence for neuronal sources of interleukin-6 production in the brain.

In this study, we investigated the capacity of murine cortical neurons to express interleukin-6 (IL-6) mRNA and protein in culture. Using in situ hybridization techniques, IL-6 mRNA was localized to neuronal cells in these cultures. Moreover, IL-6 mRNA expression as measured by in situ and PCR was shown to be upregulated by the proinflammatory cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha). This was consistent with the dose and time-dependent increases in IL-6 secreted protein observed from cultures stimulated with IL-1 beta and TNF-alpha. Taken together, the data suggest that neurons are capable of participating more directly in the CNS cytokine network than previously thought and may play an important role in the inflammatory response activities in the brain.

Quantitative analysis of specific mRNA transcripts using a competitive PCR assay with electrochemiluminescent detection.

Reverse transcriptase-polymerase chain reaction (RT--PCR) has been widely utilized for both the qualitative and quantitative assessment of the levels of specific mRNA transcripts in living systems. Quantitation of specific transcripts has often proved to be problematic because of the difficulty associated with relating the PCR-amplified product to the starting cDNA representing the mRNA of interest. We have overcome these difficulties and have developed a competitive PCR assay employing the property of electrochemiluminescence for the detection of PCR products. This assay possesses the dual advantage of being both nonradioactive and highly sensitive.

Characterization and regulation of corticotropin-releasing factor receptors in the central nervous, endocrine and immune systems.

Corticotropin-releasing factor (CRF) plays a major role in coordinating the endocrine, autonomic, behavioural and immune responses to stress through actions in the brain and in the periphery. CRF receptors identified in brain, pituitary and spleen have comparable kinetic and pharmacological characteristics, guanine nucleotide sensitivity and adenylate cyclase-stimulating activity. Differences were observed in the molecular mass of the CRF receptor complex between brain (58,000 Da) and pituitary and spleen (75,000 Da), which appeared to be due to differential glycosylation of the receptor proteins. In autoradiographic studies, CRF receptors were localized in highest densities in anterior and intermediate lobes of the pituitary, olfactory bulb, cerebral cortex, amygdala, cerebellum and the macrophage-rich marginal zones and red pulp regions of the spleen. CRF can modulate the number of CRF receptors in both the brain and pituitary in a reciprocal manner. The demonstration of functional CRF receptors in brain, pituitary and spleen suggests the importance of this neuropeptide in integrating the responses of the CNS, endocrine and immune systems to physiological, psychological and immunological stimuli.

Autoradiographic identification and characterization of sigma receptors in guinea pig brain using [3H]1(cyclopropylmethyl)-4-(2'-(4''-fluorophenyl)-2'-oxoethyl) piperidine ([3H]DuP 734), a novel sigma receptor ligand.

The psychotomimetic effects of certain cycloalkyls and benzomorphans that interact with sigma receptors has led to the hypothesis that these sites may be important in the etiology of schizophrenia. DuP 734 [1-(cyclopropylmethyl)-4-(2'-(4''-fluoro-phenyl)-2'-oxoethyl) piperidine HBr] is a novel sigma receptor ligand. The receptor binding specificity and neuroanatomical distribution of [3H]DuP 734-labeled sigma receptors in guinea pig brain were examined using quantitative autoradiography. [3H]DuP 734 binding (10 microM haloperidol displaceable) to slide-mounted sections of guinea pig brain was saturable and of high affinity (Ki = 3.9 nM). Competition studies, under conditions identical to those used to visualize the receptor, yielded the following rank order of potency: DuP 734 > haloperidol > (+)-pentazocine > (-)-butaclamol > DTG > (+)-SKF 10,047 > (+)-3-PPP > (-)-pentazocine > (+)-butaclamol > U50,488H > (-)-SKF 10,047 > cinanserin > PCP >> MK801, sulpiride. High densities of [3H]DuP 734 binding sites displaceable by haloperidol were present in the limbic system, in particular the dorsal and ventral bands of Broca as well as the ventral pallidum. Within the hippocampus, the pyramidal layers were sparsely labeled, while higher densities of binding sites were evident in the dentate gyrus. The frontal cortex, the mammillary complex of the hypothalamus, the central gray and red nucleus of the midbrain, the pontine reticular nucleus, the Purkinje cell layer of the cerebellum and dorsal and ventral horns, as well as the central gray matter of the spinal cord, all showed enrichments of [3H]DuP 734 binding sites. Lower levels of binding were present in the other regions of the cerebral cortex including parietal, pyriform, occipital, cingulate cortex, as well as the basal ganglia, and negligible specific binding was present in the white matter tracts. The kinetic and pharmacological characteristics and distribution of [3H]DuP 734 binding sites in brain are similar to those previously reported for sigma receptors.

Peritoneal dialysis patient completes successful pregnancy.

This patient was able to meet seven of the expected outcomes. Her weight and nutritional status improved after initiating CAPD, which she performed safely and effectively. She closely monitored her vital signs and appropriately notified the obstetrician when her blood pressure became elevated. She maintained proper fluid balance. The patient was successfully treated for exit site infection with antibiotics. She self-administered intraperitoneal antibiotics for peritonitis successfully. In spite of the infections, hypertension, and poor nutrition, the patient was able to complete her pregnancy to the 35th week and delivered a small, but healthy infant. Frequent monitoring and a team approach to S.B.'s well-being contributed greatly to her delivery of a viable infant. The education and training provided by the nephrology nurses was a key element in the successful management of the patient.

Spectrofluorimetric and image recordings of spontaneous and lectin-induced cytosolic calcium oscillations in Jurkat T cells.

Intracellular variations in Ca2+ concentrations have been measured in single Jurkat T lymphocyte variants (77 6.8 and E6.1) using Fura-2 as a probe. Under basal conditions, the cytosolic Ca2+ level is stable but some cells show spontaneous Ca2+ oscillations (frequency, 0.30 +/- 0.06 Hz). These oscillations are sensitive to the external concentration of Ca2+ since they can no longer be observed when the bathing solution is replaced (superfusion) with a Ca(2+)-free medium or when a Ca2+ chelator (EGTA) is added. Various changes in the cytosolic concentration of Ca2+ ([Ca2+]i) can be observed when the cells are exposed to the mitogenic lectin phytohemagglutinin (PHA, 80 nM). For instance, in the case of non-oscillating cells, the lectin induces either a rapid increase in [Ca2+]i that is followed by a sustained response (plateau) or it triggers Ca2+ spikes. In the case of experiments done in Ca(2+)-free medium, only the initial spike was observed. In the case of spontaneously oscillating cells, PHA induces a rapid increase in [Ca2+]i that is followed by a plateau where oscillations are absent. In every case, the PHA-dependent Ca2+ response is abrogated in a Ca(2+)-free medium. Computer simulations based on the model of Goldbeter et al. [27] show that the various Ca2+ responses of Jurkat cells are related to the cytosolic level of free Ca2+. Video imaging analyses show that the cellular Ca2+ responses are not homogeneous whether the observations are made in spontaneously oscillating Jurkat cells or when they are exposed to PHA.

Age-related decreases in corticotropin-releasing factor (CRF) receptors in rat brain and anterior pituitary gland.

Corticotropin-releasing factor (CRF) receptors were measured in discrete areas of brain and in anterior pituitary of 4-, 12-, 18-, and 24-month-old male Fischer rats. No significant age-related alterations in [125I]ovine CRF binding were observed in the olfactory bulb, cerebral cortex, hippocampus, brainstem, and cerebellum; there was a trend for CRF binding to decrease in the striatum as a consequence of aging. Significant age-related decreases were observed in 125I-ovine CRF binding in the anterior pituitary and hypothalamus with maximal reductions of 60 and 27%, respectively. Saturation analysis in the anterior pituitary indicated an age-related reduction in the density of CRF receptors (i.e. Bmax) without an alteration in the affinity (i.e. Kd) of CRF for its binding site. Northern analysis of proopiomelanocortin (POMC) mRNA in the anterior pituitary indicated no significant differences in the levels of POMC mRNA between 4- and 24-month-old rats. These and other data suggest that the age-related decrease in anterior pituitary CRF receptors may be due to hypersecretion of hypothalamic CRF rather than a loss of corticotropes in the anterior pituitary.

Purification and characterization of human liver phenol-sulfating phenol sulfotransferase.

The phenol-sulfating form of phenol sulfotransferase (P-PST) was purified and characterized from human liver cytosol using DEAE-cellulose, Sephacryl S-200, and 3',5'-diphosphoadenosine-agarose affinity chromatography. During the purification procedure, P-PST was resolved from the monoamine-sulfating form of phenol sulfotransferase (M-PST) and dehydroepiandrosterone sulfotransferase, which are also present in human liver cytosol. P-PST activity was purified 560-fold as compared to liver cytosol and the purified enzyme possessed a specific activity of 340 nmol phenol sulfated per minute per milligram protein. Enzymatically active P-PST has an apparent molecular size of 68,000 Da as determined by Sephacryl S-200 chromatography and a subunit molecular weight of 32,000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that P-PST exists in vivo as a homodimer. Antibodies raised to human platelet M-PST cross-reacted strongly with pure P-PST suggesting the two PSTs are structurally closely related. Two types of P-PST activity have been identified in different human livers by their thermostability and elution during anion-exchange chromatography. Each of the livers examined possessed only one type of P-PST activity. Both types of P-PST were shown to possess the same subunit molecular weight and immunoreactivity, whereas the differences in thermostability of the two P-PST activities appeared to be related to the method of preparation of liver cytosol. Both types of P-PST activity were inhibited to similar extents by incubation with 50 microM N-ethylmaleimide or 5 mM phenylglyoxal. These results suggest that the two types of P-PST in different human livers are very similar and probably represent different allelic forms of the enzyme.

Immunological characterization of human phenol sulfotransferase.

The immunological characterization of the different forms of phenol sulfotransferase (PST) in a variety of human and nonhuman tissues is described. Immunoblotting techniques revealed that polyclonal antibodies raised to human platelet MII-PST reacted with polypeptides of 32 and 34 kDa from human platelet 100,000 x g supernatant solution. Immunoblot analysis of platelet 100,000 x g supernatant solution that was fractionated over a DEAE-cellulose column indicated a close correspondence of P-PST activity, as measured by phenol sulfation, and M-PST activity, as assessed by dopamine sulfation, with the 32 and 34 kDa polypeptides, respectively. Examination of various human tissues revealed the presence of immunologically detectable levels of P-PST in liver and adrenal gland whereas both M- and P-PST were detected in placenta at a 1/10,000 dilution of the antisera. Under these conditions, PST was undetectable in human frontal cortex, pituitary gland, kidney, lung, and jejunum. Further evaluation of human liver samples from four individuals indicated a strong correlation (r = 0.94) between the amount of 32-kDa immunoreactive protein and P-PST activity. Analysis of liver samples from several animal species (monkey, rat, mouse, guinea pig, and frog) revealed the presence of immunoreactive proteins of various molecular masses, suggesting that considerable homology may exist between human and nonhuman forms of PST.

Characterization of Ca2+ and K+ currents in the human Jurkat T cell line: effects of phytohaemagglutinin.

1. Inward and outward currents were recorded in the human Jurkat T cell line using the whole-cell configuration of the patch-clamp technique. 2. The transient outward current was activated at membrane potentials positive to -60 mV. The activation time constant-voltage relationship decreased from 17 ms to 2 ms for membrane potentials ranging from -40 to +40 mV. The inactivation phase could be fitted by a single-exponential function and the inactivation time constant decreased from 250 ms to 150 ms for membrane potentials ranging from -20 to +100 mV. 3. The steady-state inactivation-voltage relationship showed a mid-point potential of -32 +/- 2.6 mV, and the slope factor was 10.8 +/- 1.8 mv (n = 3). 4. The calcium ionophore A23187 provoked a decrease in the amplitude of the outward current, suggesting a dependence of this current on the cytosolic concentration of Ca2+. 5. The K+ outward current was blocked by tetraethylammonium (TEA, Michaelis-Menten constant (Km), 6 mM) and by the calcium channel blockers Ni2+, Co2+, Mn2+ and Cd2+. 6. Forty per cent (n = 120) of the patched Jurkat cells displayed an inward current. In a physiological medium containing Ca2+ (2.2 mM), the inward current threshold voltage was -60 mV, the maximum current was observed at -40 mV and the zero current voltage was positive to +20 mV. At negative membrane potentials, the time required to reach 50% of the maximum amplitude was 60 ms and grew shorter with increasing depolarization, reaching a value of 5 ms at -5 mV. The inactivation of the inward current was very slow and the time constant varied from 1200 ms at -35 mV to approximately 250 ms for potentials positive to -10 mV. 7. The current availability had a value of one for potentials negative to -50 mV and zero for potentials positive to -15 mV. The mid-point potential was -31 +/- 3.4 mV and the slope factor was 3.3 +/- 0.2 mV (n = 3). 8. The inward channels were permeable to Sr2+, but were blocked by classical Ca2+ channel inhibitors such as Co2+, Mn2+ and Ni2+. 9. Phaseolus vulgaris phytohaemagglutinin (PHA), an inducer of interleukin-2 production in Jurkat cells, increased the inward current amplitude by 32 +/- 20% (n = 4). This increase was concomitant with a decrease (45 +/- 12%) in the amplitude of the outward current, but only when the current was carried by Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Physical characterization of a monoamine-sulfating form of phenol sulfotransferase from human platelets.

The purification to homogeneity and physical characterization of a monoamine-sulfating form of phenol sulfotransferase (PST) from human platelets is described. DEAE-cellulose chromatography of a 100,000 x g supernatant solution of homogenized human platelets revealed the presence of two peaks of both dopamine- and phenol-sulfating activity, termed M- and P-PST, respectively. The latter dopamine-sulfating form eluting from the ion exchange column, MII-PST, was purified approximately 10,000-fold to electrophoretic homogeneity by Sephacryl S-200 HR and 3'-phosphoadenosine-5'-phosphate-agarose chromatography. The final specific activity of the enzyme was 930 nmol/min/mg of protein. As determined by the hydrodynamic properties of MII-PST, the native Mr was approximately 69,000. The frictional ratio (f/fo) was estimated to be 1.28, indicating that the enzyme possesses a relatively low degree of asymmetry. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the affinity-purified enzyme revealed the presence of single Mr species of approximately 34,000, suggesting that MII-PST exists as a homodimer in vivo. Isoelectric focusing of purified MII-PST yielded a single protein species with a pl of 4.7. The sulfhydryl-modifying reagent N-ethylmaleimide (50 microM) was found to inactivate MII-PST in a time-dependent manner. This inactivation was totally prevented by saturating concentrations of 3'-phosphoadenosine-5'-phosphosulfate, whereas dopamine bestowed only partial protection to the enzyme. These results suggest that at least one sulfhydryl moiety is present at the active site of MII-PST.