The binding of C5-alkynyl and alkylfurano[2,3-d]pyrimidine glucopyranonucleosides to glycogen phosphorylase b: synthesis, biochemical and biological assessment.

C5-alkynyl and alkylfurano[2,3-d]pyrimidine glucopyranonucleosides have been synthesized and studied as inhibitors of glycogen phosphorylase b (GPb). Kinetic experiments have shown that most of these compounds were low micromolar inhibitors of the enzyme. The best inhibitor was 1-(ß-D-glucopyranosyl)-5-ethynyluracil (K(i)=4.7 µM). Crystallographic analysis of these compounds in complex with GPb revealed that inhibitors with a long C5-alkynyl group exploited interactions with ß-pocket of the active site and induced significant conformational changes of the 280s loop compared to GPb in complex with compounds with a short C5-alkynyl group. The results highlight the importance in the length of the aliphatic groups used to enhance inhibitory potency for the exploitation of the hydrophobic ß-pocket. The best of the inhibitors had also a moderate effect on glycogenolysis in the cellular lever with an IC(50) value of 291.4 µM.

Differential distribution of glucocorticoid and estrogen receptor isoforms: localization of GRbeta and ERalpha in nucleoli and GRalpha and ERbeta in the mitochondria of human osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cell lines.

The localization of glucocorticoid and estrogen receptors alpha (GRalpha, ERalpha) and beta (GRbeta, ERbeta) in osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cells was studied by immunofluorescence labelling and confocal laser scanning microscopy, as well as by subcellular fractionation and immunoblotting of the proteins of the fractions with respective antibodies. In HepG2 and SaOS-2 cells GRbeta and ERalpha were localized mainly in the nucleus, particularly concentrated in nuclear structures, which on the basis of their staining with antibody against C23-nucleolin, were characterized as nucleoli. A faint, diffuse GRbeta and ERalpha staining was also observed in the cytoplasm. GRalpha and ERbeta were specifically enriched at the site of cell mitochondria, which were visualized by labelling with the vital dye CMX. Immunoblotting experiments corroborated the immunofluorescence labelling distribution of glucocorticoid and estrogen receptor isoforms in the cell lines studied. These findings support the concept of a direct action of steroid/thyroid hormones on mitochondrial functions by way of their cognate receptors and also suggest a direct involvement of GRbeta and ERalpha in nucleolar-related processes in HepG2 and SaOS-2 cells.

The mitochondrion as a primary site of action of regulatory agents involved in neuroimmunomodulation.

A major system of neuroimmunomodulation is the hypothalamic-pituitary-adrenocortical (HPA) axis, acting through glucocorticoids and their intracellular signaling components, exerting both stimulatory and inhibitory effects on the immune reaction. Glucocorticoids inhibit the production of proinflammatory cytokines by interacting with nuclear transcription factors (nuclear factor [NF]-kappaB, activated protein [AP]-1) and induce the production of several anti-inflammatory cytokines by gene activation. In some cells and/or in extreme stress conditions, apoptosis is evoked. In most processes related to neuroimmunomodulation a prominent role is emerging for mitochondria. These organelles generate more than 90% of the cell's energy requirements through oxidative phosphorylation (OXPHOS), which is regulated by several agents, including steroid and thyroid hormones. These hormones are inducers of nuclear and mitochondrial OXPHOS gene transcription and they exert a primary action not only on nuclear but also on mitochondrial genes by way of cognate receptors. Recently, additional nuclear transcription factors involved in neuroimmunomodulation have been detected in mitochondria (NF-kappaB, AP-1, p53, calcium/cAMP response element binding protein [CREB]), and binding sites of these and putative binding sites of other nuclear transcription factors have been identified in the mitochondrial genome. The interaction of these factors with mitochondrial regulatory proteins, with receptors and with the genome has been shown and, in some cases, modulation of mitochondrial transcription was observed with possible effects on energy yield. The mitochondria store a host of critical apoptotic activators and inhibitors in their intermembrane space and the release of these factors could be another possible mode of action of the mitochondrially translocated regulatory agents and receptors.

The mitochondrion as a primary site of action of steroid and thyroid hormones: presence and action of steroid and thyroid hormone receptors in mitochondria of animal cells.

Mitochondria are key cellular organelles that regulate events related to energy production and apoptosis. These processes are modulated, in turn, by steroid and thyroid hormones in the course of their actions on metabolism, growth and development. In this context, a direct effect of these hormones on the mitochondrial-linked processes, possibly by way of cognate mitochondrial receptors, has been proposed. In this paper we review data from the literature and present new findings supporting this concept. Receptors for steroid hormones, glucocorticoids and estrogens, and for T(3), have been detected in mitochondria by immunofluorescence labeling and confocal laser microscopy, by Western blotting of mitochondrial proteins and by immunogold electron microscopy. Furthermore, the mitochondrial genome contains nucleotide sequences with high similarity to known hormone-responsive elements, which interact with the appropriate receptors to confer hormone-dependent activation of reporter genes in transfection experiments. Thus, thyroid hormone stimulates mitochondrial transcription mediated by the cognate receptor when added to an in organello mitochondrial system, capable of faithful transcription.

Estrogen receptors alpha and beta (ERalpha and ERbeta) and androgen receptor (AR) in human sperm: localization of ERbeta and AR in mitochondria of the midpiece.

BACKGROUND: The central role of estrogens and androgens in the male reproductive system has focused attention on the presence and distribution of their cognate receptors [estrogen receptor (ER) alpha, ERbeta and androgen receptor (AR)] in male reproductive tissues and cells. Since the presence of steroid hormone receptors in mitochondria of mammalian cells has been well documented, we investigated the possibility of mitochondrial localization of sex steroid hormone receptors in sperm. METHODS AND RESULTS: Applying immunofluorescence labelling and confocal laser scanning microscopy we show that the estrogen receptor beta and the AR of human sperm are specifically enriched in the midpiece, at the site of the mitochondria, which were visualized by labelling with the vital dye CMX. Nuclear and mitochondrial localization of AR was also detected in LnCap human prostate cancer cells. Differentially, most of the ERalpha immunostaining is in the form of a compact zone at a region corresponding to the equatorial segment of the upper post-acrosomal region of the sperm head. Immunoblotting experiments using sperm extracts revealed the presence of a 66 and a 45 kDa protein reacting with the ERalpha antibody, one 64 kDa protein reacting with the ERbeta antibody and a 110 and a 90 kDa protein reacting with the antibody against AR. CONCLUSIONS: Our findings suggest that the differential localization of AR and ER isoforms in human sperm reveals distinct roles of these receptors in the physiology of sperm cells and, perhaps, also in the process of fertilization.

Differential subcellular distribution of estrogen receptor isoforms: localization of ERalpha in the nucleoli and ERbeta in the mitochondria of human osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cell lines.

The localization of estrogen receptors alpha (ERalpha) and beta (ERbeta) in osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cells was studied by immunofluorescence labelling and confocal laser scanning microscopy, as well as by subcellular fractionation and immunoblotting of the proteins of the fractions with respective antibodies. In both cell types, ERalpha was localized mainly in the nucleus, particularly concentrated on nuclear structures, which on the basis of their staining with pyronin and with antibodies against the nucleoli-specific Ki67 antigen and C23-nucleolin, were characterized as nucleoli. A faint, diffuse ERalpha staining was also observed in the cytoplasm. ERbeta was specifically enriched at the site of the mitochondria, visualized by labelling with the vital dye CMX and antibody against the mitochondrial-specific cytochrome oxidase subunit I. Immunoblotting experiments corroborated the immunofluorescence labelling distribution of ERalpha and ERbeta. These findings support the concept of a direct action of steroid/thyroid hormones on mitochondrial functions by way of their cognate receptors and also suggest a direct involvement of ERalpha in nucleolar-related processes.

Early antiplatelet and antithrombotic therapy in patients with a history of recurrent miscarriages of known and unknown aetiology.

OBJECTIVES: To assess the efficacy of early thromboprophylaxis throughout pregnancy in women with previous history of first trimester recurrent miscarriages of unknown aetiology. METHODS: Low dose aspirin and low molecular weight heparin (LMWH) were administered from the day of detection of the fetal heart up to the 37th week, in two groups of patients of known (Group A, n = 24) and unknown aetiology recurrent miscarriages (Group B, n = 27). RESULTS: The success rate (viable pregnancy >24 weeks) was high and equally effective in both Groups A and B (83.3% and 85.1%, respectively). The complications recorded (pre-eclampsia, IUGR, placenta abruptio, injection site heamatomas and skin reactions) were more prevalent in Group A but of no significant difference. No abnormal bleeding was observed during vaginal delivery or caesarean section. CONCLUSIONS: Our results reaffirm previous reports that the use of LMWH in combination with low dose aspirin throughout pregnancy is safe and effective. It was also shown that the treatment is equally effective against recurrent miscarriages in both groups of patients, of known and unknown aetiology.

Asynchronous delivery of twins and triplets with an interval period ranging from 48 hours to 19 weeks.

OBJECTIVE: To improve perinatal survival rates by prolonging the rest of the pregnancy after an abortion or extremely premature birth of one fetus in multiple pregnancies, especially in women with low fertility potential. METHODS: Following the expulsion of one fetus a cervical cerclage was applied to all patients. The placenta of the expelled fetus including a small portion of its cord after it was ligated close to the external os, was left in situ. The patients were invariably kept on bed rest until the pregnancy was completed under close observation, tocolysis and preventive antibiosis. After the 24th week of gestation corticosteroids were administered. RESULTS: The delivery interval achieved ranged between two and 135 days, the longest reported. Although the survival rate was relatively low (40%) all but one of the women (83%) managed eventually to have a live child, one with twins. CONCLUSIONS: In selected multiple pregnancies the attempt to prolong the rest or the pregnancy, following the abortion or the extremely premature birth of one fetus, seems efficacious and justified especially in women with a history of long-term infertility.

The relevance of peripheral blood T-helper 1 and 2 cytokine pattern in the evaluation of patients with mycosis fungoides and Sézary syndrome.

BACKGROUND: There is evidence that a T-helper (Th) 2 cytokine pattern dominates in the peripheral blood as well as in tissue of patients with Sézary syndrome (SS), and that the malignant clone is of Th2 phenotype. However, there are conflicting studies on the cytokine pattern in the peripheral blood in different stages of cutaneous T-cell lymphoma (CTCL). OBJECTIVES: To examine, by means of flow cytometry (FC), the Th1/Th2 cytokine profile [cytoplasmic interferon (IFN)-gamma/interleukin (IL)-4] in peripheral blood T cells from patients with mycosis fungoides (MF) and SS, the most common forms of CTCL, and to correlate their expression with clinical stage, clonality and T-cell immunophenotype changes in order to evaluate their relevance in CTCL progression. METHODS: We investigated by FC the percentage of CD3+ T cells expressing cytoplasmic IFN-gamma and IL-4 after stimulation in blood specimens of 43 CTCL patients (32 stage I-II and 11 stage III-IV), eight of whom were erythrodermic. Next, we compared cytoplasmic IFN-gamma and IL-4 expression between patients of different stages and controls, and correlated our findings to T-cell receptor (TCR)-gamma gene rearrangement, used as a marker of clonality, and changes in T-cell immunophenotype (CD4+, CD8+, CD4+/CD7-, CD4+/CD25+) and natural killer cells. Polymerase chain reaction amplification of the TCR-gamma gene was performed in 41 blood and 26 skin specimens. We also examined the cytokine expression pattern in patients with erythrodermic MF and SS. RESULTS: A significantly higher frequency of CD3+/IL-4+ T cells was found in late (III-IV) compared with early (I-II) CTCL patients (P = 0.002) or controls (P < 0.001). There were significant positive correlations between the percentages of CD3+/IL-4+ and the percentages of CD3+/CD4+ T cells (r = 0.385, P = 0.05), CD4+/CD7- T cells (r = 0.335, P < 0.05) and CD4+/CD25+ T cells (r = 0.433, P = 0.01); there was a negative correlation between the percentages of CD3+/IL-4+ and CD3+/CD8+ T cells (r = -0.463, P = 0.005) and a positive correlation between the percentages of CD3+/IFN-gamma+ and CD3+/CD8+ T cells (r = 0.368, P = 0.02). Increased percentages of CD3+/IL-4+, CD3+/CD4+ and CD4+/CD7- T lymphocytes were associated with the presence of clonality (P = 0.025, P < 0.001 and P = 0.0031, respectively). All independent variables showed a statistically significant difference between SS and erythrodermic MF patients, or controls, apart from cytoplasmic IL-4, which was high both in erythrodermic MF and SS patients compared with controls (P = 0.003 and P = 0.008, respectively). In multiple regression logistic analysis, the probability of belonging to advanced CTCL stages was associated only with increased cytoplasmic IL-4 (P = 0.007, odds ratio 1.13, 95% confidence interval 1.033-1.229). CONCLUSIONS: Increased T-cell cytoplasmic IL-4 is more frequent in late CTCL stages, correlates with T-cell immunophenotype changes found in advanced disease and is associated with clonality. Increased cytoplasmic IL-4 is frequent both in erythrodermic MF and SS patients, in contrast to other variables found increased only in SS, suggesting that IL-4 may be an early indicator of disease progression. Moreover, our results show that increased cytoplasmic IL-4 is the sole predictor of advanced CTCL disease and confirm the relevance of FC determination of IL-4 in the routine evaluation of CTCL cases.

The prognostic role of CD5 negativity in B-cell chronic lymphocytic leukaemia: a case-control study.

B cells in chronic lymphocytic leukaemia (CLL) usually express the CD5 antigen, which appears to participate in the pathogenesis of autoimmune phenomena. However, 7-20% of B-CLL patients are CD5-. The aim of this study was to assess whether CD5 expression could be used as a discriminating factor for two subgroups of B-CLL. Twenty-nine CD5- B-CLL patients were compared in terms of clinico-biological characteristics and survival with a control group of 29 sex- and age-matched, consecutive CD5+ B-CLL subjects. B-CLL was considered to be CD5- when less than 5% of mononuclear cells expressed CD5 after subtraction of the number of T cells. Splenomegaly, lymph node involvement, and haemolytic anemia were found in CD5+ patients in a significantly higher proportion than in their CD5- counterparts, who presented with an earlier stage of disease. CD5- patients had a median survival of 97.2 (22-130) months, exceeding CD5+ subjects significantly [84.0 (19-120) months, p = 0.0025]. CD5- patients seemingly present with milder disease and have a favourable prognosis compared with the vast majority of B-CLL patients who express CD5.

Thromboprophylaxis throughout pregnancy in women with previous history of recurrent miscarriages of unknown aetiology.

The purpose of this prospective preliminary clinical study was to assess the efficacy of thromboprophylaxis throughout pregnancy in women with a history of unexplained first trimester recurrent miscarriages. From the 53 patients originally assigned to the study 15 were excluded. The remaining 38 were treated with low molecular weight heparin (LMWH-natroparine calcium 0.3 ml twice daily) and low dose aspirin from the day the fetal heart motion was detected until the 37th week or earlier at the onset of premature labor. Among the patients treated (n = 38) thrombophilia screening was positive in 16 patients and in the remaining 22 no causative factor was detected. The overall success rate (viable pregnancy > or = 24 weeks) was 92.2% with no significant difference between patients with positive or negative thrombophilia screening. The most significant complications were: preeclampsia (21%), IUGR (26%), placenta abruptio (5.2%), injection site haematoma (44%) and skin reaction (15.7%). No abnormal bleeding was observed during vaginal or caesarean section. The results of this study suggest that thromboprophylaxis during pregnancy, which has already been successfully tried in patients with recurrent miscarriages with a causative factor, may be similarly effective in patients with such a pregnancy complication but of unknown aetiology.

Localization of the glucocorticoid receptor in rat brain mitochondria.

The distribution of glucocorticoid receptor in subcellular fractions of brain cortex and hippocampus, two regions rich in glucocorticoid receptor, has revealed its presence in nuclei, cytosol, mitochondria, synaptosomes, and synaptosomal mitochondria. The identification of glucocorticoid receptor has been accomplished both by Western blotting using antibodies recognizing the carboxy and the amino terminus of the glucocorticoid receptor and by immunogold electron microscopy using the same anti-glucocorticoid receptor antibodies. Antibody-glucocorticoid receptor interaction is abolished by preincubation of each antibody with its competing peptide. In addition to the intact 95-kDa glucocorticoid receptor in all fractions, lower molecular weight glucocorticoid receptor fragments have been also detected by Western blotting. The presence of glucocorticoid receptor in brain mitochondria supports the concept of a direct action of glucocorticoids on mitochondrial gene transcription, parallel to the established primary actions of the hormones on nuclear gene transcription, as a mechanism of coordinate regulation of respiratory enzyme biosynthesis by steroid hormones.

Expression of the glucocorticoid receptor in early and late passage C-6 glioma cells and in normal astrocytes derived from aged mouse cerebral hemispheres.

The presence of the glucocorticoid receptor in early and late passage C-6 glioma cells 2B clone and in astrocytes derived from aged mouse cerebral hemispheres has been documented by immunoblotting and/or immunofluorescence labelling. All cell types studied express the glucocorticoid receptor of molecular weight 97 KDa. In addition, in astrocytes derived from aged mouse cerebral hemispheres a smaller molecular weight polypeptide reacting with anti-glucocorticoid receptor antibody was also demonstrated. No difference in the amount of the 97 KDa glucocorticoid receptor between early and late C-6 2B cells was observed, whereas the astrocytes from aged cerebral hemispheres contained considerably reduced amounts of the glucocorticoid receptor compared to C-6 2B cells. Late passage C-6 2B cells were immunofluorescence labelled with the anti-glucocorticoid antibody, the receptor being almost exclusively present in the cytoplasm, with particular concentration in the perinuclear region. The presence of glucocorticoid receptor of molecular weight 97 KDa in glial cells corroborates and expands the existing data based on radioligand binding and immunocytochemical studies. These cell populations can be exploited as a model system for the study of the effects of glucocorticoids on senescence and brain aging.

Immunocytochemical localization of glycogen phosphorylase kinase in rat brain sections and in glial and neuronal primary cultures.

The physiological function of brain glycogen and the role of phosphorylase kinase as a regulatory enzyme in the cascade of reactions associated with glycogenolysis in the brain have not been fully elucidated. As a first step toward elucidating such a function, we studied the localization of phosphorylase kinase in glial and neuronal primary cell cultures, and in adult rat brain slices, using a rabbit polyclonal antibody against skeletal muscle glycogen phosphorylase kinase. Immunocytochemical examination of rat astroglia-rich primary cultures revealed that a large number of cells were positive for glycogen phosphorylase kinase immunoreactivity. These cells were also positive for vimentin, a marker for immature glia, while they were negative for glial fibrillary acidic protein, a marker for mature astroglia, and for galactocerebroside, an oligodendroglial marker. Neurons in rat neuron-rich primary cultures did not show any kinase-positive staining. In paraformaldehyde-fixed adult rat brain sections, phosphorylase kinase immunoreactivity was detected in glial-like cells throughout the brain, with relatively high staining found in the cerebral cortex, the cerebellum, and the medulla oblongata. Phosphorylase kinase immunoreactivity could not be detected in neurons, with the exception of a group of large neurons in the brain stem, most likely belonging to the mesencephalic trigeminal nucleus. Phosphorylase kinase was also localized in the choroid plexus and to a lesser degree in the ependymal cells lining the ventricles. Phosphorylase kinase thus appears to have the same cellular distribution in nervous tissue as its substrates, i.e. glycogen phosphorylase and glycogen, which suggests that the physiological role of brain phosphorylase kinase is the mobilization of glycogen stores to fuel the increased metabolic demands of neurons and astrocytes.

Prenatal determination of chorionicity in triplet pregnancy by ultrasonographic examination of the ipsilon zone.

OBJECTIVE: To determine chorionicity in triplet pregnancies by ultrasonographic assessment of the ipsilon zone, the junction of the three interfetal membranes. METHODS: The thickness of the component membranes in the ipsilon zone was studied to determine chorionicity in 28 triplet pregnancies, by retrospective examination of the ultrasonographic images taken at 9-24 weeks' gestation, and in 20 consecutive triplet pregnancies followed prospectively by targeted ultrasonography at 8-21 weeks' gestation. Prenatal ultrasonographic findings were compared with those obtained from the records of the infertility centers or referring hospitals (the number of gestational sacs and live embryos in each sac seen by transvaginal scanning at 6-7 weeks' gestation). RESULTS: Of the 28 triplet pregnancies with appropriate images demonstrating the ipsilon zone, 22 were classified as trichorionic, five dichorionic, and one monochorionic. This classification was correct in all but one trichorionic pregnancy, which was misclassified as dichorionic. In the prospective subset (n = 20) there were 16 trichorionic and four dichorionic triplet pregnancies. The ipsilon zone was not present in one case in which the interfetal membrane did not intersect. In the remaining 19 pregnancies, there was a complete correlation between the findings at the ipsilon zone and transvaginal ultrasonography at 6-7 weeks. CONCLUSION: Ultrasonographic assessment of the ipsilon zone is useful for predicting chorionicity in triplet pregnancies.

Fetal karyotyping in twin pregnancies: selection of technique by measurement of fetal nuchal translucency.

OBJECTIVE: To examine the usefulness of selecting the appropriate technique for fetal karyotyping in twin pregnancies by using maternal age and fetal nuchal translucency thickness to determine risk for chromosomal defects in each fetus. SETTING: Fetal Medicine Centre, London, United Kingdom. SUBJECTS: Sixty-seven twin pregnancies identified at the time of an ultrasound scan for determination of fetal nuchal translucency thickness, where the parents requested karyotyping. INTERVENTION: The risk for chromosomal defects in each fetus was calculated from the maternal age and fetal nuchal translucency thickness at 10 to 14 weeks of gestation. If the estimated risk for either fetus was 1 in 50 or greater, chorion villus sampling was the method of choice, whereas if the risk was less than 1 in 50 second trimester amniocentesis was performed. RESULTS: The estimated risk for trisomies was more than 1 in 50 in 34 pregnancies and 23.5% of these fetuses were found to be chromosomally abnormal. In contrast, in the 33 low risk pregnancies chromosomal abnormalities were found in only 1.5% of the fetuses. CONCLUSIONS: In twin pregnancies the technique for fetal karyotyping may by selected by calculating the risk for chromosomal abnormality based on maternal age and fetal nuchal translucency thickness.

Subcellular distribution of phosphorylase kinase in rat brain. Association of the enzyme with mitochondria and membranes.

The evaluation of glycogen phosphorylase kinase in rat brain subcellular fractions was undertaken in order to get further insight into the association of this kinase with specific neuronal cell compartments. The enzyme was found to be primarily soluble, but considerable latent specific activities were observed in particulate fractions, especially in microsomes, mitochondria and synaptosomes, which could be unmasked by treatment with Triton-X-100. The submitochondrial and subsynaptic distribution patterns of phosphorylase kinase revealed high overt activity in the mitochondrial intermembrane space and high latent activities in mitochondrial membranes, and synaptic vesicles, membranes and mitochondria. The Ca(2+)-dependency of soluble phosphorylase kinase was similar to that of microsomal enzyme but higher than that of other particulate enzyme forms. Mitochondrial phosphorylase kinase showed a higher pH 6.8:8.2 activity ratio than the soluble and the microsomal enzyme. The rate of inactivation of cytosolic phosphorylase kinase by proteinase K was higher than that of microsomal and mitochondrial enzymes. Antibodies against rabbit skeletal muscle phosphorylase kinase effectively inhibited both cytosolic and microsomal enzyme but failed to significantly affect the kinase activity present in intact mitochondria and intermembrane space. Western blotting with anti-phosphorylase kinase showed that rat brain mitochondria exhibited a significantly lower immunoreactivity compared to soluble cytosol. In conclusion, the presence of phosphorylase kinase activity in a variety of particulate fractions of rat brain suggests a multiplicity of actions of this kinase in neuronal tissues.