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PubMed1986

Effects of continuous LHRH infusion on plasma levels of LH and FSH in males, before and after oestrogen or anti-oestrogen treatment.

In order to study the role of oestrogens on gonadotrophin release in the human male, LHRH was administered as an infusion at a constant rate of 0.5 micrograms/minute for 4 hours to 7 normogonadotrophic oligozoospermic men, 6 eugonadal male-to-female transsexuals and 9 eugonadal male volunteers. In agreement with in vitro data a biphasic release pattern of both LH and FSH was observed in eugonadal transsexuals as well as in normogonadotrophic oligozoospermic men. In the latter the release of LH was greater than in eugonadal transsexual males and volunteers, which points to a different functioning of the hypothalamic-pituitary unit in normogonadotrophic oligozoospermic men. On the other hand the FSH response to LHRH stimulation was normal in these men. Three months' treatment with the oestrogen-receptor antagonist tamoxifen (TAM) (10 mg twice daily) in the normogonadotrophic oligozoospermic men stimulated basal LH, FSH and testosterone (T) levels. The fact that gonadotrophin levels rose in spite of increased T levels, suggests a role of endogenous oestrogens in the negative feedback regulation of gonadotrophin release in these men. Upon TAM treatment the first phase, the plateau and the second phase of LH release were augmented, whereas only the plateau and the second phase of FSH release were increased. Six weeks' administration of the oestrogen ethinyloestradiol (EE) (10 micrograms three times a day) in the eugonadal transsexual males suppressed basal T and oestradiol (E2) levels without affecting basal gonadotrophin levels significantly. In EE-treated males the first phase of LH release tended to be lower, whereas the plateau of LH had decreased significantly. The second phase of LH was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed2001

4-Hydroxytamoxifen binds to and deactivates the estrogen-related receptor gamma.

The estrogen-related receptors (ERR alpha, ERR beta, and ERR gamma) form a family of orphan nuclear receptors that share significant amino acid identity with the estrogen receptors, but for which physiologic roles remain largely unknown. By using a peptide sensor assay, we have identified the stilbenes diethylstilbestrol (DES), tamoxifen (TAM), and 4-hydroxytamoxifen (4-OHT) as high-affinity ligands for ERR gamma. In direct binding assays, 4-OHT had a K(d) value of 35 nM, and both DES and TAM displaced radiolabeled 4-OHT with K(i) values of 870 nM. In cell-based assays, 4-OHT binding caused a dissociation of the complex between ERR gamma and the steroid receptor coactivator-1, and led to an inhibition of the constitutive transcriptional activity of ERR gamma. ERR alpha did not bind 4-OHT, but replacing a single amino acid predicted to be in the ERR alpha ligand-binding pocket with the corresponding ERR gamma residue allowed high-affinity 4-OHT binding. These results demonstrate the existence of high-affinity ligands for the ERR family of orphan receptors, and identify 4-OHT as a molecule that can regulate the transcriptional activity of ERR gamma.

Amino Acid Sequence↗
PubMed1986

Extinction of Harrington's mountain goat.

Keratinous horn sheaths of the extinct Harrington's mountain goat, Oreamnos harringtoni, were recovered at or near the surface of dry caves of the Grand Canyon, Arizona. Twenty-three separate specimens from two caves were dated nondestructively by the tandem accelerator mass spectrometer (TAMS). Both the TAMS and the conventional dates indicate that Harrington's mountain goat occupied the Grand Canyon for at least 19,000 years prior to becoming extinct by 11,160 +/- 125 radiocarbon years before present. The youngest average radiocarbon dates on Shasta ground sloths, Nothrotheriops shastensis, from the region are not significantly younger than those on extinct mountain goats. Rather than sequential extinction with Harrington's mountain goat disappearing from the Grand Canyon before the ground sloths, as one might predict in view of evidence of climatic warming at the time, the losses were concurrent. Both extinctions coincide with the regional arrival of Clovis hunters.

Journal Article↗
PubMed1987

Radiocarbon dates on bones of extinct birds from Hawaii.

Bones from a stratified sedimentary deposit in the Puu Naio Cave site on Maui, Hawaiian Islands, reveal the late Holocene extinction of 19 species of birds. The age of the sediment and associated fauna was determined by direct radiocarbon dating (tandem particle accelerator-mass spectrometer; TAMS) of amino acids extracted from bones weighing as little as 450 mg. The 14C dates indicate that sediment has been accumulating in the lava tube for at least the last 7750 years, a suitable time frame for testing the hypothesis that Holocene extinction on islands began after human colonization. Despite growing evidence that a worldwide wave of extinctions coincided with human colonization of oceanic islands, little radiometric data have been available to date the extinction of most small fossil vertebrates on islands. The TAMS technique of dating purified collagen from the bones of small vertebrates could lead to vastly improved chronologies of extinction for oceanic islands where catastrophic mid- to late-Holocene extinction is expected or known to have occurred. Chronologies derived from nonarcheological sites that show continuous sedimentation, such as the Puu Naio Cave deposit, may also yield key evidence on the timing of earliest human settlement of Oceania.

Animals↗
PubMed1999

The activation of the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase signaling pathways protects HeLa cells from apoptosis following photodynamic therapy with hypericin.

In this study, we elucidate signaling pathways induced by photodynamic therapy (PDT) with hypericin. We show that PDT rapidly activates JNK1 while irreversibly inhibiting ERK2 in several cancer cell lines. In HeLa cells, sustained PDT-induced JNK1 and p38 mitogen-activated protein kinase (MAPK) activations overlap the activation of a DEVD-directed caspase activity, poly(ADP-ribose) polymerase (PARP) cleavage, and the onset of apoptosis. The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zDEVD-fmk) protect cells against apoptosis and inhibit DEVD-specific caspase activity and PARP cleavage without affecting JNK1 and p38 MAPK activations. Conversely, stable overexpression of CrmA, the serpin-like inhibitor of caspase-1 and caspase-8, has no effect on PDT-induced PARP cleavage, apoptosis, or JNK1/p38 activations. Cell transfection with the dominant negative inhibitors of the c-Jun N-terminal kinase (JNK) pathway, SEK-AL and TAM-67, or pretreatment with the p38 MAPK inhibitor PD169316 enhances PDT-induced apoptosis. A similar increase in PDT-induced apoptosis was observed by expression of the dual specificity phosphatase MKP-1. The simultaneous inhibition of both stress kinases by pretreating cells with PD169316 after transfection with either TAM-67 or SEK-AL produces a more pronounced sensitizing effect. Cell pretreatment with the p38 inhibitor PD169316 causes faster kinetics of DEVD-caspase activation and PARP cleavage and strongly oversensitizes the cells to apoptosis following PDT. These observations indicate that the JNK1 and p38 MAPK pathways play an important role in cellular resistance against PDT-induced apoptosis with hypericin.

Anthracenes↗
PubMed1999

A novel methyltransferase catalyzes the methyl esterification of trans-aconitate in Escherichia coli.

We have identified a new type of S-adenosyl-L-methionine-dependent methyltransferase in the cytosol of Escherichia coli that is expressed in early stationary phase under the control of the RpoS sigma factor. This enzyme catalyzes the monomethyl esterification of trans-aconitate at high affinity (Km = 0.32 mM) and cis-aconitate, isocitrate, and citrate at lower velocities and affinities. We have purified the enzyme to homogeneity by gel-filtration, anion-exchange, and hydrophobic chromatography. The N-terminal amino acid sequence was found to match that expected for the o252 open reading frame at 34.57 min on the E. coli genomic sequence whose deduced amino acid sequence contains the signature sequence motifs of the major class of S-adenosyl-L-methionine-dependent methyltransferases. Overexpression of the o252 gene resulted in an overexpression of the methyltransferase activity, and we have now designated it tam for trans-aconitate methyltransferase. We have generated a knock-out strain of E. coli lacking this activity, and we find that its growth and stationary phase survival are similar to that of the parent strain. We demonstrate the endogenous formation of trans-aconitate methyl ester in extracts of wild type but not tam- mutant cells indicating that trans-aconitate is present in E. coli. Since trans-aconitate does not appear to be a metabolic intermediate in these cells but forms spontaneously from the key citric acid cycle intermediate cis-aconitate, we suggest that its methylation may limit its potential interference in normal metabolic pathways. We have detected trans-aconitate methyltransferase activity in extracts of the yeast Saccharomyces cerevisiae, whereas no activity has been found in extracts of Caenorhabditis elegans or mouse brain.

Amino Acid Sequence↗
PubMed2000

Potentiation of Smad transactivation by Jun proteins during a combined treatment with epidermal growth factor and transforming growth factor-beta in rat hepatocytes. role of phosphatidylinositol 3-kinase-induced AP-1 activation.

Cross-talk between Smad and mitogen-activated protein kinase pathways has been described recently, and evidence for Smad cooperation with AP-1 is emerging. Here we report that epidermal growth factor (EGF) potentializes transforming growth factor beta (TGF-beta)-induced Smad3 transactivation in rat hepatocytes, an effect abrogated by TAM-67, a dominant negative mutant of AP-1. Antisense transfection experiments indicated that c-Jun and JunB were involved in the synergistic effect, and endogenous c-Jun physically associated with Smad3 during a combined EGF/TGF-beta treatment. We next investigated which signaling pathway transduced by EGF was responsible for the Jun-induced synergism. Whereas inhibition of JNK had no effect, inhibition of the phosphatidylinositol-3' kinase (PI3-kinase) pathway by LY294002 or by expression of a dominant negative mutant of PI3-kinase reduced EGF/TGF-beta-induced Smad3 transcriptional activity. Transfection of an activated Ras with a mutation enabling the activation of the PI3-kinase pathway alone mimicked the EGF/TGF-beta potentiation of Smad3 transactivation, and TAM-67 abolished this effect, suggesting that the PI3-kinase pathway stimulates Smad3 via AP-1 stimulation. The EGF/TGF-beta-induced activation of Smad3 correlated with PI3-kinase and p38-dependent but not JNK-dependent phosphorylation of c-Jun. Since potentiation of a Smad-binding element-driven gene was also induced by EGF/TGF-beta treatment, this novel mechanism of Jun/Smad cooperation might be crucial for diversifying TGF-beta responses.

Animals↗
PubMed2003

Amelogenin interacts with cytokeratin-5 in ameloblasts during enamel growth.

The enamel protein amelogenin binds to GlcNAc (Ravindranath, R. M. H., Moradian-Oldak, R., and Fincham, A.G. (1999) J. Biol. Chem. 274, 2464-2471) and to the GlcNAc-mimicking peptide (GMp) (Ravindranath, R. M. H., Tam, W., Nguyen, P., and Fincham, A. G. (2000) J. Biol. Chem. 275, 39654-39661). The GMp motif in the N-terminal region of the cytokeratin 14 of ameloblasts binds to trityrosyl motif peptide (ATMP) of amelogenin (Ravindranath, R. M. H., Tam, W., Bringas, P., Santos, V., and Fincham, A. G. (2001) J. Biol. Chem. 276, 36586 - 36597). K14 (Type I) pairs with K5 (Type II) in basal epithelial cells; GlcNAc-acylated K5 is identified in ameloblasts. Dosimetric analysis showed the binding affinity of amelogenin to K5 and to GlcNAc-acylated-positive control, ovalbumin. The specific binding of [3H]ATMP with K5 or ovalbumin was confirmed by Scatchard analysis. [3H]ATMP failed to bind to K5 after removal of GlcNAc. Blocking K5 with ATMP abrogates the K5-amelogenin interaction. K5 failed to bind to ATMP when the third proline was substituted with threonine, as in some cases of human X-linked amelogenesis imperfecta or when tyrosyl residues were substituted with phenylalanine. Confocal laser scan microscopic observations on ameloblasts during postnatal (PN) growth of the teeth showed that the K5-amelogenin complex migrated from the cytoplasm to the periphery (on PN day 1) and accumulated at the apical region on day 3. Secretion of amelogenin commences from day 1. K5, similar to K14, may play a role of chaperone during secretion of amelogenin. Upon secretion of amelogenin, K5 pairs with K14. Pairing of K5 and K14 commences on day 3 and ends on day 9. The pairing of K5 and K14 marks the end of secretion of amelogenin.

Amelogenin↗
PubMedDate not supplied

Evaluation and functional assessment of flexor tendon repair in the hand.

Seventeen patients with 28 flexor tendon injuries were examined after tendon repair. The current most frequently used evaluation systems, including grip and pinch strength, were compared with functional outcome as assessed by a questionnaire, evaluating Disabilities of Arm, Shoulder and Hand (DASH). Good correlation was found between Total Active Motion (TAM) and the Original Strickland test (kappa = 0.85), however with reduced categories. Only limited correlation was found between the DASH-score and TAM (r = -0.33) as well as between the DASH-score and pinch strength (r = -0.35). We suggest reporting the average Range of Motion (ROM) of the complete finger as a percentage of the contralateral finger, instead of reporting the classified result, and to include assessment of pinch strength. It would be very useful to have an accurate functional outcome assessment, but DASH proves to be insufficiently sensitive.

Adolescent↗
PubMed1989

Effect of embryonic and neonatal administration of tamoxifen on adiposity in the broiler chicken.

1. The effect of early exposure of heavy breed (HB) chicks to an anti-oestrogen (tamoxifen--TAM) on later adiposity was studied. 2. TAM administration at the embryonic stage, but not at the day of hatching, reduced adiposity in females but not in males, at 8 to 9 weeks of age. This reduction in adiposity in females minimised or even alleviated the excess of fat in females compared to males.

Adipose Tissue↗
PubMed1990

The response of broilers' adiposity to testosterone after embryonic exposure to androgen and tamoxifen.

1. The effects of early exposure of heavy breed (HB) chicks to an anti-oestrogen (tamoxifen--TAM) and to an androgen which cannot be aromatised (5 alpha-dihydrotestosterone--DHT) on subsequent adiposity and its response to testosterone were studied. 2. Embryonic TAM administration reduced adiposity in females but not in males at 8 weeks of age. Embryonic DHT produced similar responses but to a lesser extent. 3. Testosterone propionate (TP) administration during growth had no effect on adiposity in any of the treated groups but TP reduced adiposity in males which had been exposed to DHT at the embryonic stage.

Adipose Tissue↗
PubMed1991

Sexual differentiation of copulatory behaviour in the male chick requires gonadal steroids.

1. Embryonic injections of 0.3 mg/egg of tamoxifen (TAM), 0.2 mg/egg CI-628 (both antioestrogens), 0.5 mg/egg (ATD (aromatisation inhibitor), or antibodies to oestradiol (E), all suppressed male copulatory activity (MCA) in young male chicks. 2. Embryonic injections with either flutamide (F, androgen antagonist) or high dose of antibodies to testosterone (T) only slightly suppressed MCA. 3. TAM had no effect on embryonic plasma LH levels, 24 and 48 h after injection. 4. It seems that at the embryonic stage oestradiol is required for the normal differentiation of MCA.

Animals↗
PubMedDate not supplied

Recent developments in the molecular, biochemical and functional characterization of GPI8 and the GPI-anchoring mechanism [review].

Glycoconjugates are utilized by eukaryotic organisms ranging from yeast to humans for the cell surface expression of a wide variety of proteins and lipids. These glycoconjugates are expressed as enzymes or receptors and serve a diversity of functions, including cell signaling and cell survival. In parasitic protozoans, glycoconjugates play roles in infectivity, survival, virulence and immune evasion. Among the alternate glycoconjugate structures that have been identified, glycosylphosphatidylinositols (GPIs) represent a universal structure for the anchorage of proteins, lipids, and phosphosaccharides to cellular membranes. Biosynthesis of the GPI is a multi-step process that culminates in the attachment of the assembled GPI to a precursor protein. This final step in the transfer of the GPI to a protein is catalyzed by GPI8 of the putative transamidase complex (TAM). GPI8 functions dually to perform the proteolytic cleavage of the C-terminal signal sequence of the precursor protein, followed by the formation of an amide bond between the protein and the ethanolamine phosphate of the GPI. This review summarizes the current aggregate of biochemical, gene-disruption and active site mutagenesis studies, which provide evidence that GPI8 is responsible for the protein-GPI anchoring reaction. We describe recently published studies that have identified other potential components of the TAM complex and that have elucidated their likely role in protein-GPI attachment. Further, we discuss the biochemical, molecular and functional differences between protozoan and mammalian GPI8 and the protein-GPI anchoring machinery. Finally, we will present the implications of these studies for the development of anti-parasite drug therapies.

Acyltransferases↗
PubMed2000

c-Jun activation-dependent tumorigenic transformation induced paradoxically by overexpression or block of S-adenosylmethionine decarboxylase.

All mammalian cells absolutely require polyamines (putrescine, spermidine, and spermine) for growth. Here we show that the overexpression of cDNA for S-adenosylmethionine decarboxylase (AdoMetDC), the main regulatory enzyme in the biosynthesis of higher polyamines, induces transformation of rodent fibroblasts when expressed in the sense or the antisense orientation. Both transformants were able to induce invasive tumors in nude mice. Neither transformation was associated with activation of the mitogen-activated protein kinases Erk1 and Erk2. Instead, the AdoMet DC sense, but not antisense, transformants displayed constitutive activation of the c-Jun NH(2)-terminal kinase (JNK) pathway. However, both transformations converged on persistent phosphorylation of endogenous c-Jun at Ser73. The phenotype of the AdoMetDC sense transformants was reversed by expression of dominant-negative mutants of SEK1 (MKK4), JNK1, and c-Jun (TAM-67), which were also found to impair cytokinesis. Similarly, TAM-67 reverted the morphology of the AdoMetDC-antisense expressors. This report is the first demonstration of a protein whose overexpression or block of synthesis can induce cell transformation. In addition, we show that the polyamine biosynthetic enzymes require c-Jun activation for eliciting their biological effects.

3T3 Cells↗
PubMed2001

Optimization of field-cycled PEDRI for in vivo imaging of free radicals.

A numerical model of the behaviour of the magnetization in a field-cycled dynamic nuclear polarization (DNP) experiment is presented, with the aim of optimizing pulse sequence parameters in field-cycled proton-electron double-resonance free radical imaging. The model is used to predict the observed enhancement of the NMR signal as a function of the magnetic field strength, EPR irradiation frequency and pulse sequence timing, as well as the properties of the sample including the NMR and EPR relaxation times. The model allowed optimization of parameters in the field-cycled DNP experiment, in particular the EPR irradiation frequency, to find the value which would give the largest difference between NMR signals recorded with and without EPR irradiation. Experiments to verify the model were carried out using aqueous solutions of TEMPOL, which exhibits three hyperfine lines in its EPR spectrum and triarylmethyl (TAM), which has a single, narrow line. It was found that the model predicted very well the variation in DNP enhancement with EPR irradiation power for both samples. The behaviour of the NMR signal with EPR irradiation frequency in studies using TEMPOL was also accurately modelled, with the optimum frequency lying between 60 and 80 MHz, depending on the EPR irradiation power. The optimum frequency obtained from the model also agreed with the experimental data obtained using the TAM free radical, but with this sample the theoretical curves tended to deviate from the experimental data at irradiation frequencies below 70 MHz.

Electron Spin Resonance Spectroscopy↗
PubMed2006

A BPF-type algorithm for CT with a curved PI detector.

Helical cone-beam CT is used widely nowadays because of its rapid scan speed and efficient utilization of x-ray dose. Recently, an exact reconstruction algorithm for helical cone-beam CT was proposed (Zou and Pan 2004a Phys. Med. Biol. 49 941-59). The algorithm is referred to as a backprojection-filtering (BPF) algorithm. This BPF algorithm for a helical cone-beam CT with a flat-panel detector (FPD-HCBCT) requires minimum data within the Tam-Danielsson window and can naturally address the problem of ROI reconstruction from data truncated in both longitudinal and transversal directions. In practical CT systems, detectors are expensive and always take a very important position in the total cost. Hence, we work on an exact reconstruction algorithm for a CT system with a detector of the smallest size, i.e., a curved PI detector fitting the Tam-Danielsson window. The reconstruction algorithm is derived following the framework of the BPF algorithm. Numerical simulations are done to validate our algorithm in this study.

Algorithms↗
PubMed2004

Simultaneous measurement of oxygenation in intracellular and extracellular compartments of lung microvascular endothelial cells.

A new technique is described for simultaneous determination of intra- and extracellular oxygen concentrations [partial pressure of O(2) (pO(2))] in bovine lung microvascular endothelial cells (BLMVECs) using electron paramagnetic resonance (EPR) oximetry. The measurements were performed in BLMVEC suspensions of a 20- micro l volume containing 4,000 cells. The extracellular pO(2) was measured using a trityl EPR probe [triarylmethyl (TAM), 10 micro M], a tricarboxylate anion radical, that stays exclusively in the extracellular space. The intracellular oxygen was measured using a pre-internalized particulate spin probe, lithium 5,9,14,18,23,27,32,36-octa-n-butoxy-naphthalocyanine (LiNc-BuO). Because there is a wide discrepancy in the reported values of cellular oxygenation by and large due to differences in the methods employed, we utilized the dual EPR probe technique to measure the oxygen gradient that apparently exists across the cell membrane. The intra- and extracellular pO(2) values were 139 +/- 2.5 and 157 +/- 3.6 mm Hg, respectively, for cells exposed to room air. A fairly smaller gradient of oxygen was observed in cells exposed to 7.5% oxygen (pO(2) = 57 mm Hg). In summary, this study confirms the feasibility of simultaneous and accurate measurements of intra- and extracellular pO(2) using LiNc-BuO and TAM EPR oximetry probes.

Animals↗
PubMed1988

Ultrastructural studies of peripheral blood of neonates with Down's syndrome and transient abnormal myelopoiesis.

Ultrastructural studies were performed on blood samples from five neonates with Down's syndrome and transient abnormal myelopoiesis (TAM). Three methods of fixation were employed to detect diaminobenzidine (DAB) reactivity. The first method used glutaraldehyde solution, while the second and third methods used tannic acid-glutaraldehyde mixtures. Before fixation by the second method, specimens were washed in order to eliminate plasma, and before fixation by the third were diluted to lessen the effects of plasma protein. The latter two methods were more sensitive than the first for detection of DAB reactivity, while the third also resulted in better preservation of morphology than did the second. Even the first method was able to detect DAB reactivity in cells of megakaryocyte-platelet series in appropriate sections. Although the majority of blasts appeared with light microscopy to be undifferentiated, their ultrastructural morphology and ultrastructural cytochemistry were in fact found to be quite heterogeneous, consisting of cells of the megakaryocyte-platelet and granulocytic series, including basophils, and erythroid precursors. This finding supported the view that TAM was the result of unstable hematopoiesis rather than true leukemia.

3,3'-Diaminobenzidine↗