bluuebunny/crossref_metadata_2025 huggingface dataset:
Megalin-mediated endocytosis of transcobalamin-vitamin-B12 complexes suggests a role of the receptor in vitamin-B12 homeostasis.
Authors: Moestrup S K , Birn H , Fischer P B , Petersen C M , Verroust P J , Sim R B , Christensen E I , Nexø E
Published: July 2002

Kidney cortex is a main target for circulating vitamin B12 (cobalamin) in complex with transcobalamin (TC). Ligand blotting of rabbit kidney cortex with rabbit 125I-TC-B12 and human TC-57Co-B12 revealed an exclusive binding to megalin, a 600-kDa endocytic receptor present in renal proximal tubule epithelium and other absorptive epithelia. The binding was Ca2+ dependent and inhibited by receptor-associated protein (RAP). Surface plasmon resonance analysis demonstrated a high-affinity interaction between purified rabbit megalin and rabbit TC-B12 but no measurable affinity of the vitamin complex for the homologous alpha 2-macroglobulin receptor (alpha 2MR)/low density lipoprotein receptor related protein (LRP). 125I-TC-B12 was efficiently endocytosed in a RAP-inhibitable manner in megalin-expressing rat yolk sac carcinoma cells and in vivo microperfused rat proximal tubules. The radioactivity in the tubules localized to the endocytic compartments and a similar apical distribution in the proximal tubules was demonstrated after intravenous injection of 125I-TC-B12. The TC-B12 binding sites in the proximal tubule epithelium colocalized with megalin as shown by ligand binding to cryosections of rat kidney cortex, and the binding was inhibited by anti-megalin polyclonal antibody, EDTA, and RAP. These data show a novel nutritional dimension of megalin as a receptor involved in the cellular uptake of vitamin B12. The expression of megalin in absorptive epithelia in the kidney and other tissues including yolk sac and placenta suggests a role of the receptor in vitamin B12 homeostasis and fetal vitamin B12 supply.
Overexpression of the transcription factor UBF1 is sufficient to increase ribosomal DNA transcription in neonatal cardiomyocytes: implications for cardiac hypertrophy.
Authors: Hannan R D , Stefanovsky V , Taylor L , Moss T , Rothblum L I
Published: July 2002

The accelerated protein accumulation characteristic of cardiomyocyte hypertrophy results from increased cellular protein synthetic capacity (elevated ribosome content). The rate limiting step in ribosome accumulation is transcription of the rRNA genes. During neonatal cardiomyocyte hypertrophy induced by norepinephrine or spontaneous contraction, changes in the expression of a ribosomal DNA transcription factor, UBF, correlated with increased rates of ribosome biogenesis. We hypothesized that elevated expression of UBF was part of the mechanism by which these hypertrophic stimuli effected increases in the rate of transcription from the rDNA promoter. In this study, we have examined directly the effect of overexpressing UBF on rDNA transcription in neonatal cardiomyocytes in culture. In control experiments, a novel reporter construct for rDNA transcription (pSMECAT) showed similar increases in activity in response to hypertrophic stimuli (10(-4) M phenylephrine, 10(-7) M endothelin, and spontaneous contraction) as did the endogenous rRNA genes. When contraction-arrested cardiomyocytes were cotransfected with pSMECAT and increasing amounts of a UBF1 expression vector; a dose-dependent (3-5 fold) increase in rDNA transcription was observed. Western blot analysis confirmed that the overexpressed, FLAG-tagged UBF accumulated in the cardiomyocyte nuclei. The observation that overexpression of UBF1 is sufficient to increase rDNA transcription in neonatal cardiomyocytes provides evidence in support of the hypothesis that the regulation of UBF is a key component of the increased ribosome biogenesis and protein accumulation associated with cardiomyocyte hypertrophy.
Heightened expression of tumor necrosis factor alpha, interleukin 1 alpha, and glial fibrillary acidic protein in experimental Creutzfeldt-Jakob disease in mice.
Authors: Kordek R , Nerurkar V R , Liberski P P , Isaacson S , Yanagihara R , Gajdusek D C
Published: July 2002

The ultrastructural pathology of myelinated axons in mice infected experimentally with the Fujisaki strain of Creutzfeldt-Jakob disease (CJD) virus is characterized by myelin sheath vacuolation that closely resembles that induced in murine spinal cord organotypic cultures by tumor necrosis factor alpha (TNF-alpha), a cytokine produced by astrocytes and macrophages. To clarify the role of TNF-alpha in experimental CJD, we investigated the expression of TNF-alpha in brain tissues from CJD virus-infected mice at weekly intervals after inoculation by reverse transcription-coupled PCR, Northern and Western blot analyses, and immunocytochemical staining. Neuropathological findings by electron microscopy, as well as expression of interleukin 1 alpha and glial fibrillary acidic protein, were concurrently monitored. As determined by reverse transcription-coupled PCR, the expression of TNF-alpha, interleukin 1 alpha, and glial fibrillary acidic protein was increased by approximately 200-fold in the brains of CJD virus-inoculated mice during the course of disease. By contrast, beta-actin expression remained unchanged. Progressively increased expression of TNF-alpha in CJD virus-infected brain tissues was verified by Northern and Western blot analyses, and astrocytes in areas with striking myelin sheath vacuolation were intensely stained with an antibody against murine TNF-alpha. The collective findings of TNF-alpha overexpression during the course of clinical disease suggest that TNF-alpha may mediate the myelin sheath vacuolation observed in experimental CJD.
Analysis of estrogen receptor transcriptional enhancement by a nuclear hormone receptor coactivator.
Authors: McInerney E M , Tsai M J , O'Malley B W , Katzenellenbogen B S
Published: July 2002

The estrogen receptor (ER), a member of a large superfamily of nuclear hormone receptors, is a ligand-inducible transcription factor that regulates the expression of estrogen-responsive genes. The ER, in common with other members of this superfamily, contains two transcription activation functions (AFs)--one located in the amino-terminal region (AF-1) and the second located in the carboxyl-terminal region (AF-2). In most cell contexts, the synergistic activity of AF-1 and AF-2 is required for full estradiol (E2)-stimulated activity. We have previously shown that a ligand-dependent interaction between the two AF-containing regions of ER was promoted by E2 and the antiestrogen trans-hydroxytamoxifen (TOT). This interaction, however, was transcriptionally productive only in the presence of E2. To explore a possible role of steroid receptor coactivators in transcriptional synergism between AF-1 and AF-2, we expressed the amino terminal (AF-1-containing) and carboxyl-terminal (AF-2-containing) regions of ER as separate polypeptides in mammalian cells, along with the steroid receptor coactivator-1 protein (SRC-1). We demonstrate that SRC-1, which has been shown to significantly increase ER transcriptional activity, enhanced the interaction, mediated by either E2 or TOT, between the AF-1-containing and AF-2-containing regions of the ER. However, this enhanced interaction resulted in increased transcriptional effectiveness only with E2 and not with TOT, consistent with the effects of SRC-1 on the full-length receptor. Our results suggest that after ligand binding, SRC-1 may act, in part, as an adapter protein that promotes the integration of amino- and carboxyl-terminal receptor functions, allowing for full receptor activation. Potentially, SRC-1 may be capable of enhancing the transcriptional activity of related nuclear receptor superfamily members by facilitating the productive association of the two AF-containing regions in these receptors.
The Chlorella hexose/H+ symporter is a useful selectable marker and biochemical reagent when expressed in Volvox.
Authors: Hallmann A , Sumper M
Published: July 2002

The multicellular obligately photoautotrophic alga Volvox is composed of only two types of cells, somatic and reproductive. Therefore, Volvox provides the simplest model system for the study of multicellularity. Metabolic labeling experiments using radioactive precursors are crucial for the detection of stage- and cell-type-specific proteins, glycoproteins, lipids, and carbohydrates. However, wild-type Volvox lacks import systems for sugars or amino acids. To circumvent this problem, the hexose/H+ symporter (HUP1) gene from the unicellular alga Chlorella was placed under the control of the constitutive Volvox beta-tubulin promoter. The corresponding transgenic Volvox strain synthesized the sugar transporter in a functional state and was able to efficiently incorporate 14C from labeled glucose or glucosamine. Sensitivity toward the toxic glucose/mannose analogue 2-deoxy-glucose increased by orders of magnitude in transformants. Thus we report the successful transformation of Volvox with a gene of heterologous origin. The chimeric gene may be selected for in either a positive or a negative manner, because transformants exhibit both prolonged survival in the dark in the presence of glucose and greatly increased sensitivity to the toxic sugar 2-deoxyglucose. The former trait may make the gene useful as a dominant selectable marker for use in transformation studies, whereas the latter trait may make it useful in development of a gene-targeting system.
Surface hydrophobic residues of multiubiquitin chains essential for proteolytic targeting.
Authors: Beal R , Deveraux Q , Xia G , Rechsteiner M , Pickart C
Published: July 2002

Ubiquitin conjugation is a signal for degradation of eukaryotic proteins by the 26S protease. Conjugation of a homopolymeric multiubiquitin chain to a substrate lysine residue results in 10-fold faster degradation than does conjugation of monoubiquitin, but the molecular basis of enhanced targeting by chains is unknown. We show that ubiquitin residues L8, I44, and V70 are critical for targeting. Mutation of pairs of these residues to alanine had little effect on attachment of ubiquitin to substrates but severely inhibited degradation of the resulting conjugates. The same mutations blocked the binding of chains to a specific subunit (S5a) of the regulatory complex of the 26S protease. The side chains implicated in this binding--L8, I44, and V70--form repeating patches on the chain surface. Thus, hydrophobic interactions between these patches and S5a apparently contribute to enhanced proteolytic targeting by multiubiquitin chains.
Ecological factors rather than temporal factors dominate the evolution of vesicular stomatitis virus
Authors: Rodríguez Luis L. , Fitch Walter M. , Nichol Stuart T.
Published: July 2002

Vesicular stomatitis New Jersey virus (VSV-NJ) is a rhabdovirus that causes economically important disease in cattle and other domestic animals in endemic areas from southeastern United States to northern South America. Its negatively stranded RNA genome is capable of undergoing rapid evolution, which allows phylogenetic analysis and molecular epidemiology studies to be performed. Previous epidemiological studies in Costa Rica showed the existence of at least two distinct ecological zones of high VSV-NJ activity, one located in the highlands (premontane tropical moist forest) and the other in the lowlands (tropical dry forest). We wanted to test the hypothesis that the viruses circulating in these ecological zones were genetically distinct. For this purpose, we sequenced the hypervariable region of the phosphoprotein gene for 50 VSV-NJ isolates from these areas. Phylogenetic analysis showed that viruses from each ecological zone had distinct genotypes. These genotypes were maintained in each area for periods of up to 8 years. This evolutionary pattern of VSV-NJ suggests an adaptation to ecological factors that could exert selective pressure on the virus. As previous data indicated an absence of virus adaptation to factors related to the bovine host (including immunological pressure), it appears that VSV genetic divergence represents positive selection to adapt to specific vectors and/or reservoirs at each ecological zone.
Involvement of stress-activated protein kinase and p38 mitogen-activated protein kinase in mIgM-induced apoptosis of human B lymphocytes
Authors: Graves J. D. , Draves K. E. , Craxton A. , Saklatvala J. , Krebs E. G. , Clark E. A.
Published: July 2002

Despite intensive efforts, the intracellular signaling pathways
that mediate apoptosis remain unclear. The human B lymphoma
cell line, B104, possesses characteristics that make it an attractive
model for analysis of receptor-mediated apoptosis. Although
these cells express both membrane IgM (mIgM) and membrane IgD (mIgD)
crosslinking mIgM results in significant apoptosis while
crosslinking mIgD does not. Our results show that crosslinking mIgM but
not mIgD induced a delayed and sustained activation of the
mitogen-activated protein kinase (MAPK) family members stress-activated
protein kinase (SAPK) and p38 MAPK. The calcium ionophore ionomycin,
which also induces apoptosis in B104 cells, stimulated a
similar SAPK and p38 MAPK response. Cyclosporin A, a potent inhibitor
of apoptosis induced by either mIgM or ionomycin, inhibited
activation of both SAPK and p38 MAPK, suggesting that stimulation of
these kinases may be required for induction of apoptosis.
Collectively, our results indicate that SAPK and p38 MAPK may be
downstream targets during mIgM-induced, calcium-mediated,
apoptosis in human B lymphocytes.
Identification of a second transmembrane protein tyrosine phosphatase, IA-2beta, as an autoantigen in insulin-dependent diabetes mellitus: precursor of the 37-kDa tryptic fragment.
Authors: Lu J , Li Q , Xie H , Chen Z J , Borovitskaya A E , Maclaren N K , Notkins A L , Lan M S
Published: July 2002

A novel cDNA, IA-2beta, was isolated from a mouse neonatal brain library. The predicted protein sequence revealed an extracellular domain, a transmembrane region, and an intracellular domain. The intracellular domain is 376 amino acids long and 74% identical to the intracellular domain of IA-2, a major autoantigen in insulin-dependent diabetes mellitus (IDDM). A partial sequence of the extracellular domain of IA-2beta indicates that it differs substantially (only 26% identical) from that of IA-2. Both molecules are expressed in islets and brain tissue. Forty-six percent (23 of 50) of the IDDM sera but none of the sera from normal controls (0 of 50) immunoprecipitated the intracellular domain of IA-2beta. Competitive inhibition experiments showed that IDDM sera have autoantibodies that recognize both common and distinct determinants on IA-2 and IA-2beta. Many IDDM sera are known to immunoprecipitate 37-kDa and 40-kDa tryptic fragments from islet cells, but the identity of the precursor protein(s) has remained elusive. The current study shows that treatment of recombinant IA-2beta and IA-2 with trypsin yields a 37-kDa fragment and a 40-kDa fragment, respectively, and that these fragments can be immunoprecipitated with diabetic sera. Absorption of diabetic sera with unlabeled recombinant IA-2 or IA-2beta, prior to incubation with radiolabeled 37-kDa and 40-kDa tryptic fragments derived from insulinoma or glucagonoma cells, blocks the immunoprecipitation of both of these radiolabeled tryptic fragments. We conclude that IA-2beta and IA-2 are the precursors of the 37-kDa and 40-kDa islet cell autoantigens, respectively, and that both IA-2 and IA-2beta are major autoantigens in IDDM.
Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial merA gene.
Authors: Rugh C L , Wilde H D , Stack N M , Thompson D M , Summers A O , Meagher R B
Published: July 2002

With global heavy metal contamination increasing, plants that can process heavy metals might provide efficient and ecologically sound approaches to sequestration and removal. Mercuric ion reductase, MerA, converts toxic Hg2+ to the less toxic, relatively inert metallic mercury (Hg0) The bacterial merA sequence is rich in CpG dinucleotides and has a highly skewed codon usage, both of which are particularly unfavorable to efficient expression in plants. We constructed a mutagenized merA sequence, merApe9, modifying the flanking region and 9% of the coding region and placing this sequence under control of plant regulatory elements. Transgenic Arabidopsis thaliana seeds expressing merApe9 germinated, and these seedlings grew, flowered, and set seed on medium containing HgCl2 concentrations of 25-100 microM (5-20 ppm), levels toxic to several controls. Transgenic merApe9 seedlings evolved considerable amounts of Hg0 relative to control plants. The rate of mercury evolution and the level of resistance were proportional to the steady-state mRNA level, confirming that resistance was due to expression of the MerApe9 enzyme. Plants and bacteria expressing merApe9 were also resistant to toxic levels of Au3+. These and other data suggest that there are potentially viable molecular genetic approaches to the phytoremediation of metal ion pollution.
Intracellular misrouting and abnormal secretion of adrenocorticotropin and growth hormone in
Cpe

fat

mice associated with a carboxypeptidase E mutation
Authors: Shen Fu-Sheng , Loh Y. Peng
Published: July 2002

Cpe

fat

mice carry a mutation in the carboxypeptidase E/H gene which encodes an exopeptidase that removes C-terminal basic residues from endoproteolytically cleaved hormone intermediates. These mice have endocrine disorders including obesity, infertility, and hyperproinsulinemia–diabetes syndrome, but the etiology remains an enigma. Because studies have identified membrane carboxypeptidase E as a sorting receptor for targeting prohormones to the regulated secretory pathway for processing and secretion, the intracellular routing and secretion of pro-opiomelanocortin/adrenocorticotropin and growth hormone from anterior pituitary cells were investigated in
Cpe

fat

mice. In
Cpe

fat

mice, pro-opiomelanocortin was accumulated 24-fold above normal animals in the pituitary and it was poorly processed to adrenocorticotropin. Furthermore, pro-opiomelanocortin was secreted constitutively at high levels, showing no response to stimulation by corticotropin-releasing hormone. Similarly, growth hormone release was constitutive and did not respond to high K
+
stimulation. Both pro-opiomelanocortin and growth hormone levels were elevated in the circulation of
Cpe

fat

mice versus normal mice. These data provide evidence that the lack of carboxypeptidase E, the sorting receptor, results in the intracellular misrouting and secretion of pro-opiomelanocortin and growth hormone via the constitutive pathway in the pituitary of
Cpe

fat

mice.
Pattern changes of pituitary peptides in rat after salt-loading as detected by means of direct, semiquantitative mass spectrometric profiling
Authors: Jiménez C. R. , Li K. W. , Dreisewerd K. , Mansvelder H. D. , Brussaard A. B. , Reinhold B. B. , Van der Schors R. C. , Karas M. , Hillenkamp F. , Burbach J. P. H. , Costello C. E. , Geraerts W. P. M.
Published: July 2002

We have established a differential peptide display method, based on a mass spectrometric technique, to detect peptides that show semiquantitative changes in the neurointermediate lobe (NIL) of individual rats subjected to salt-loading. We employed matrix-assisted laser desorption/ionization mass spectrometry, using a single-reference peptide in combination with careful scanning of the whole crystal rim of the matrix-analyte preparation, to detect in a semiquantitative manner the molecular ions present in the unfractionated NIL homogenate. Comparison of the mass spectra generated from NIL homogenates of salt-loaded and control rats revealed a selective and significant decrease in the intensities of several molecular ion species of the NIL homogenates from salt-loaded rats. These ion species, which have masses that correspond to the masses of oxytocin, vasopressin, neurophysins, and an unidentified putative peptide, were subsequently chemically characterized. We confirmed that the decreased molecular ion species are peptides derived exclusively from propressophysin and prooxyphysin (i.e., oxytocin, vasopressin, and various neurophysins). The putative peptide is carboxyl-terminal glycopeptide. The carbohydrate moiety of the latter peptide was determined by electrospray tandem MS as bisected biantennary Hex
3
HexNAc
5
Fuc. This posttranslational modification accounts for the mass difference between the predicted mass of the peptide based on cDNA studies and the measured mass of the mature peptide.
Dendritic calcium conductances generate high-frequency oscillation in thalamocortical neurons
Authors: Pedroarena Christine , Llinás Rodolfo
Published: July 2002

Cortical-projecting thalamic neurons, in guinea pig brain slices, display high-frequency membrane potential oscillations (20–80 Hz), when their somata are depolarized beyond −45 mV. These oscillations, preferentially located at dendritic sites, are supported by the activation of P/Q type calcium channels, as opposed to the expected persistent sodium conductance responsible for such rhythmic behavior in other central neurons. Short hyperpolarizing pulses reset the phase and transiently increase the amplitude of these oscillations. This intrinsic thalamic electroresponsiveness may serve as a cellular-based temporal binding mechanism that sharpens the temporal coincidence of cortical-feedback synaptic inputs, known to distribute at remote dendritic sites on thalamic neurons.
Inhibition of nuclear factor κB by prostaglandin A1: An effect associated with heat shock transcription factor activation
Authors: Rossi Antonio , Elia Giuliano , Santoro M. Gabriella
Published: July 2002

Prostaglandins (PGs) function as intracellular signal mediators in the regulation of a variety of physiological and pathological processes, including inflammation and immune responses. Cyclopentenone PGs are characterized by antiviral activity against several viruses, including human immunodeficiency virus type 1 (HIV-1), and by the ability to induce heat shock protein expression through activation of the heat shock transcription factor. Here we report that PGA1is a potent inhibitor of nuclear factor-κB (NF-κB) activation in human cells and of NF-κB-dependent HIV-1 transcription in long terminal repeat-chloramphenicol acetyltransferase transient transfection experiments. PGA1acts by inhibiting phosphorylation and preventing degradation of the NF-κB inhibitor IκB-α. Inhibition does not require protein synthesis, is dependent on the presence of a reactive cyclopentenonic moiety, and is associated with heat shock transcription factor activation. Because NF-κB is critically involved in the activation of immunoregulatory and viral genes, inhibition of its activity could be a major component of the immunosuppressive and antiviral activity of PGs.
Glu-tRNA
Gln
amidotransferase: A novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation
Authors: Curnow Alan W. , Hong Kwang-won , Yuan Robert , Kim Sung-il , Martins Orlando , Winkler Wade , Henkin Tina M. , Söll Dieter
Published: July 2002

The three genes,
gatC
,
gatA
, and
gatB
, which constitute the transcriptional unit of the
Bacillus subtilis
glutamyl-tRNA
Gln
amidotransferase have been cloned. Expression of this transcriptional unit results in the production of a heterotrimeric protein that has been purified to homogeneity. The enzyme furnishes a means for formation of correctly charged Gln-tRNA
Gln
through the transamidation of misacylated Glu-tRNA
Gln
, functionally replacing the lack of glutaminyl-tRNA synthetase activity in Gram-positive eubacteria, cyanobacteria, Archaea, and organelles. Disruption of this operon is lethal. This demonstrates that transamidation is the only pathway to Gln-tRNA
Gln
in
B. subtilis
and that glutamyl-tRNA
Gln
amidotransferase is a novel and essential component of the translational apparatus.
Is sexual selection and species recognition a continuum? Mating behavior of the stalk-eyed fly
Drosophila heteroneura
Authors: Boake Christine R. B. , DeAngelis Matthew P. , Andreadis Debra K.
Published: July 2002

If behavioral isolation between species can evolve as a consequence of sexual selection within a species, then traits that are both sexually selected and used as a criterion of species recognition by females should be identifiable. The broad male head of the Hawaiian picture-winged fly
Drosophila heteroneura
is a novel sexual dimorphism that may be sexually selected and involved in behavioral isolation from
D. silvestris
. We found that males with broad heads are more successful in sexual selection, both through female mate choice and through aggressive interactions. However, female
D. heteroneura
do not discriminate against hybrids on the basis of their head width. Thus, this novel trait is sexually selected but is not a major contributor to species recognition. Our methods should be applicable to other species in which behavioral isolation is a factor.
Parasite-mediated nuclear factor κB regulation in lymphoproliferation caused byTheileria parva infection
Authors: Palmer Guy H. , Machado Joel , Fernandez Paula , Heussler Volker , Perinat Therese , Dobbelaere Dirk A. E.
Published: July 2002

Infection of cattle with the protozoanTheileria parvaresults in uncontrolled T lymphocyte proliferation resulting in lesions resembling multicentric lymphoma. Parasitized cells exhibit autocrine growth characterized by persistent translocation of the transcriptional regulatory factor nuclear factor κB (NFκB) to the nucleus and consequent enhanced expression of interleukin 2 and the interleukin 2 receptor. HowT. parvainduces persistent NFκB activation, required for T cell activation and proliferation, is unknown. We hypothesized that the parasite induces degradation of the IκB molecules which normally sequester NFκB in the cytoplasm and that continuous degradation requires viable parasites. UsingT. parva-infected T cells, we showed that the parasite mediates continuous phosphorylation and proteolysis of IκBα. However, IκBα reaccumulated to high levels in parasitized cells, which indicated thatT. parvadid not alter the normal NFκB-mediated positive feedback loop which restores cytoplasmic IκBα. In contrast,T. parvamediated continuous degradation of IκBβ resulting in persistently low cytoplasmic IκBβ levels. Normal IκBβ levels were only restored followingT. parvakilling, indicating that viable parasites are required for IκBβ degradation. Treatment ofT. parva-infected cells with pyrrolidine dithiocarbamate, a metal chelator, blocked both IκB degradation and consequent enhanced expression of NFκB dependent genes. However treatment using the antioxidantN-acetylcysteine had no effect on either IκB levels or NFκB activation, indicating that the parasite subverts the normal IκB regulatory pathway downstream of the requirement for reactive oxygen intermediates. Identification of the critical points regulated byT. parvamay provide new approaches for disease control as well as increase our understanding of normal T cell function.
Telomerase activity: A biomarker of cell proliferation, not malignant transformation
Authors: Belair Cassandra D. , Yeager Thomas R. , Lopez Patricia M. , Reznikoff Catherine A.
Published: July 2002

Telomerase activity is readily detected in most cancer biopsies, but not in premalignant lesions or in normal tissue samples with a few exceptions that include germ cells and hemopoietic stem cells. Telomerase activity may, therefore, be a useful biomarker for diagnosis of malignancies and a target for inactivation in chemotherapy or gene therapy. These observations have led to the hypothesis that activation of telomerase may be an important step in tumorigenesis. To test this hypothesis, we studied telomerase activity in isogeneic samples of uncultured and cultured specimens of normal human uroepithelial cells (HUCs) and in uncultured and cultured biopsies of superficial and myoinvasive transitional cell carcinoma (TCC) of the bladder. Our results demonstrated that four of four TCC biopsies, representing both superficial and myoinvasive TCCs, were positive for telomerase activity, but all samples of uncultured HUC were telomerase negative. However, when the same normal HUC samples were established as proliferating culturesin vitro, telomerase activity was readily detected but usually at lower levels than in TCCs. Consistent with the above observation of the telomerase activity in HUCs, telomeres did not shorten during the HUCin vitrolifespan. Demonstration of telomerase in proliferating human epithelial cellsin vitrowas not restricted to HUCs, because it was also present in prostate and mammary cell cultures. Notably, telomerase activity was relatively low or undetectable in nonproliferating HUC cultures. These data do not support a model in which telomerase is inactive in normal cells and activated during tumorigenic transformation. Rather, these data support a model in which the detection of telomerase in TCC biopsies, but not uncultured HUC samples, reflects differences in proliferation between tumor and normal cellsin vivo.
Structure and function in rhodopsin: Rhodopsin mutants with a neutral amino acid at E134 have a partially activated conformation in the dark state
Authors: Kim Jong-Myoung , Altenbach Christian , Thurmond Robin L. , Khorana H. Gobind , Hubbell Wayne L.
Published: July 2002

The Glu-134–Arg-135 residues in rhodopsin, located near the cytoplasmic end of the C helix, are involved in G protein binding, or activation, or both. Furthermore, the charge-neutralizing mutation Glu-134 to Gln-134 produces hyperactivity in the activated state and produces constitutive activity in opsin. The Glu/Asp-Arg charge pair is highly conserved in equivalent positions in other G protein-coupled receptors. To investigate the structural consequences of charge-neutralizing mutations at Glu-134 and Arg-135 in rhodopsin, single spin-labeled side chains were introduced at sites in the cytoplasmic domains of helices C (140), E (227), F (250), or G (316) to serve as “molecular sensors” of the local helix bundle conformation. In each of the spin-labeled rhodopsins, a Gln substitution was introduced at either Glu-134 or Arg-135, and the electron paramagnetic resonance spectrum of the spin label was used to monitor the structural response of the helix bundle. The results indicate that a Gln substitution at Glu-134 induces a photoactivated conformation around helices C and G even in the dark state, an observation of potential relevance to the hyperactivity and constitutive activity of the mutant. In contrast, little change is induced in helix F, which has been shown to undergo a dominant motion upon photoactivation. This result implies that the multiple helix motions accompanying photoactivation are not strongly coupled and can be induced to take place independently. Gln substitution at Arg-135 produces only minor structural changes in the dark- or light-activated conformation, suggesting that this residue is not a determinant of structure in the regions investigated, although it may be functionally important.
An essential component of a C-terminal domain phosphatase that interacts with transcription factor IIF in
Saccharomyces cerevisiae
Authors: Archambault Jacques , Chambers Ross S. , Kobor Michael S. , Ho Yuen , Cartier Mireille , Bolotin Diana , Andrews Brenda , Kane Caroline M. , Greenblatt Jack
Published: July 2002

One of the essential components of a phosphatase that specifically dephosphorylates the
Saccharomyces cerevisiae
RNA polymerase II (RPII) large subunit C-terminal domain (CTD) is a novel polypeptide encoded by an essential gene termed
FCP1
. The Fcp1 protein is localized to the nucleus, and it binds the largest subunit of the yeast general transcription factor IIF (Tfg1).
In vitro
, transcription factor IIF stimulates phosphatase activity in the presence of Fcp1 and a second complementing fraction. Two distinct regions of Fcp1 are capable of binding to Tfg1, but the C-terminal Tfg1 binding domain is dispensable for activity
in vivo
and
in vitro
. Sequence comparison reveals that residues 173–357 of Fcp1 correspond to an amino acid motif present in proteins of unknown function predicted in many organisms.
Glutamate transporter currents in Bergmann glial cells follow the time course of extrasynaptic glutamate
Authors: Bergles Dwight E. , Dzubay Jeffrey A. , Jahr Craig E.
Published: July 2002

Glutamate transporters in the central nervous system are expressed in both neurons and glia, they mediate high affinity, electrogenic uptake of glutamate, and they are associated with an anion conductance that is stoichiometrically uncoupled from glutamate flux. Although a complete cycle of transport may require 50–100 ms, previous studies suggest that transporters can alter synaptic currents on a much faster time scale. We find that application of
l
-glutamate to outside-out patches from cerebellar Bergmann glia activates anion-potentiated glutamate transporter currents that activate in <1 ms, suggesting an efficient mechanism for the capture of extrasynaptic glutamate. Stimulation in the granule cell layer in cerebellar slices elicits all or none α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor and glutamate transporter currents in Bergmann glia that have a rapid onset, suggesting that glutamate released from climbing fiber terminals escapes synaptic clefts and reaches glial membranes shortly after release. Comparison of the concentration dependence of both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor and glutamate transporter kinetics in patches with the time course of climbing fiber-evoked responses indicates that the glutamate transient at Bergmann glial membranes reaches a lower concentration than attained in the synaptic cleft and remains elevated in the extrasynaptic space for many milliseconds.
Interconversion of red opsin isoforms by the cyclophilin-related chaperone protein Ran-binding protein 2
Authors: Ferreira Paulo A. , Nakayama Tomoko A. , Travis Gabriel H.
Published: July 2002

Ran-binding protein 2 (RanBP2) (type II) is a retinal cyclophilin-related protein that binds Ran-GTPase. Type I cyclophilin is a shorter, alternatively spliced isoform of RanBP2. Recently, we showed that the Ran-binding domain 4 (RBD4)/cyclophilin (CY) supradomain of RanBP2 acts both
in vitro
and
in vivo
as a specific chaperone for bovine red/green opsin (R/G opsin). R/G opsin undergoes a stable modification of its electrophoretic mobility upon binding to RanBP2. This modification is likely due to
cis–trans
isomerization of one or more proline residues in the opsin protein. Here, we show that expression of human red opsin in
Escherichia coli
and COS cells results in the production of still a third electrophoretic variant of this protein. This variant was converted to the RBD4 binding-competent form of opsin through direct interaction with RBD4/CY, both
in vivo
and
in vitro
. We suggest that these distinct opsin species may represent kinetically or thermodynamically trapped prolyl conformers that can be interconverted by concerted action of the RBD4 and CY domains of RanBP2. We also show that the C-terminal half of RBD4 is the binding domain for bovine R/G opsin and that coexpression of human red opsin with type I cyclophilin
in vivo
enhances the production of functional visual pigment. These observations imply that prolyl isomerization may have importance beyond its role in protein folding, possibly as a molecular switch modulated by cyclophilin for the loading of opsin onto RanBP2 during visual pigment processing in cones.
Analysis of HIV-1envgene sequences reveals evidence for a low effective number in the viral population
Authors: Brown Andrew J. Leigh
Published: July 2002

Selection is usually considered to be the dominant force controlling viral variation; the large population sizes suggest that deterministic population genetic models are appropriate. To investigate their validity for HIV, samples ofenvgene sequences were tested for departure from neutrality because of mutation–selection balance. None of the samples departed significantly when tested as nucleotide sequences. At the amino acid level, significantly elevated diversity was detected in two samples within, but not outside, the V3 loop. The effective population number has been estimated (using a phylogenetic method) to be close to 103. Estimates from nucleotide diversity are about 2-fold lower. The low value of the effective population number might arise from high variability in progeny number between infected cells, from the expansion in population number from a small inoculum as the virus is transmitted between hosts, or from variable selection at linked sites. These results suggest that the population genetics of HIV are best described by stochastic models.
Blockage of 5HT2Cserotonin receptors by fluoxetine (Prozac)
Authors: Ni Y. G. , Miledi R.
Published: July 2002

Fluoxetine (Prozac) inhibited the membrane currents elicited by serotonin (5-hydroxytryptamine; 5HT) inXenopusoocytes expressing either cloned 5HT2Creceptors or 5HT receptors encoded by rat cortex mRNA. Responses of 5HT2Creceptors, elicited by nM concentrations of 5HT, were rapidly and reversibly blocked by micromolar concentrations of fluoxetine. For responses elicited by 1 μM 5HT, the IC50of fluoxetine inhibition was ≈20 μM. In accord with the electrophysiological results, fluoxetine inhibited the binding of [3H]5HT to 5HT2Creceptors expressed in HeLa cells (Ki≈ 65–97 nM), and the binding to 5HT receptors in rat cortex membranes was also inhibited but less efficiently (Ki≈ 56 μM). Our results show that fluoxetine is a competitive and reversible antagonist of 5HT2Creceptors and suggest that some therapeutic effects of fluoxetine may involve blockage of 5HT receptors, in addition to its known blockage of 5HT transporters. Similar work may help to design more selective compounds for use in the treatment of brain disorders.
The ultrastructural localization of 60-kDa Ro protein and human cytoplasmic RNAs: Association with novel electron-dense bodies
Authors: Farris A. Darise , Puvion-Dutilleul Francine , Puvion Edmond , Harley John B. , Lee Lela A.
Published: July 2002

The 60-kDa Ro ribonucleoprotein is an important target of humoral autoimmune responses. However, the ultrastructural locations of the 60-kDa Ro protein and its associated small cytoplasmic RNAs (Y RNAs) have not been previously determined, and the functions of the Ro protein and RNAs are not known. In this study, the cellular locations of the 60-kDa Ro protein and the Ro Y1 and Y4 RNAs are determined by immunoelectron microscopy and
in situ
hybridization electron microscopy, respectively. Both Ro protein and Y RNAs are concentrated in discrete areas of the nucleoplasm, nucleolus, and cytoplasm of cultured cells and human skin sections. The 60-kDa Ro protein and Y RNAs are also present diffusely in the cytoplasm, where they occur in ribosome-rich regions, and in the nucleus. The presence of Ro ribonucleoprotein components in nucleoli and in ribosome-rich cytoplasmic areas suggests a potential for the involvement of Y RNAs and/or 60-kDa Ro protein in ribosome synthesis, assembly, or transport. Double labeling experiments show that Ro protein and Y RNAs colocalize in the nucleoplasm, nucleolus, and cytoplasm. In addition, aggregates of Y RNA occur unassociated with 60-kDa Ro protein, and aggregates of 60-kDa Ro protein occur unassociated with Y RNA. Aggregates of both Ro protein and Y RNAs label previously unreported nuclear and cytoplasmic electron-dense bodies. We propose that these distinctive Ro-associated electron-dense bodies may represent structure(s) important for cellular transport and/or Ro function.
Induction of dopamine D
3
receptor expression as a mechanism of behavioral sensitization to levodopa
Authors: Bordet Régis , Ridray Sophie , Carboni Suzanna , Diaz Jorge , Sokoloff Pierre , Schwartz Jean-Charles
Published: July 2002

In rats with unilateral lesions of the nigrostriatal dopamine pathway with 6-hydroxydopamine, the motor stimulating effects of levodopa, an indirect dopamine receptor agonist, evidenced by contraversive rotations, become enhanced upon repeated intermittent administration. However, the mechanisms of this behavioral sensitization are essentially unknown. We show that development of sensitization is accompanied by a progressive appearance of D
3
receptor mRNA and binding sites, visualized by
in situ
hybridization and 7-[
3
H]hydroxy-
N
,
N
-di-
n
-propyl-2-aminotetralin autoradiography, respectively, occurring in the denervated caudate putamen, a brain area from which this receptor subtype is normally absent. Development and decay of these two processes occur with closely parallel time courses, whereas there were no marked changes in D
1
or D
2
receptor mRNAs. D
3
receptor induction by levodopa is mediated by repeated D
1
receptor stimulation, since it is prevented by the antagonist SCH 33390 and mimicked by the agonist SKF 38393, but not by two D
2
receptor agonists. The enhanced behavioral response to levodopa is mediated by the newly synthesized D
3
receptor, since it is antagonized by nafadotride, a preferential D
3
receptor antagonist, in low dosage, which has no such effect before D
3
receptor induction. D
3
receptor induction and behavioral sensitization are also accompanied by a sustained enhancement of prodynorphin mRNA level and a progressively decreasing expression of the preprotachykinin gene. We propose that imbalance between dynorphin and substance P release from the same striatonigral motor efferent pathway, related to D
3
receptor induction, is responsible for behavioral sensitization.
A new approach to determining the rates of recruitment of circulating leukocytes into tissues: Application to the measurement of leukocyte recruitment into atherosclerotic lesions
Authors: Steinberg Daniel , Khoo John C. , Glass Christopher K. , Palinski Wulf , Almazan Felicidad
Published: July 2002

Recruitment of circulating monocytes into the artery wall is an important feature of early atherogenesis.
In vitro
studies have identified a number of adhesion molecules and chemokines that may control this process but very little work has been done to evaluate their relative importance
in vivo
, in part because there have been no methods available of sufficient sensitivity and reliability. This paper proposes a new approach in which advantage is taken of naturally occurring or transgenically induced mutations to “mark” donor cells and to follow their fate in recipient animals using highly sensitive PCR methods. The feasibility of the approach is demonstrated by preliminary studies of monocyte recruitment into atherosclerotic lesions. However, the method should in principle be applicable to the study of any of the circulating leukocytes and their rate of entry into any tissue or tissues of interest.
Measurement and modeling of the transient difference between blood and subcutaneous glucose concentrations in the rat after injection of insulin
Authors: Schmidtke David W. , Freeland Angela C. , Heller Adam , Bonnecaze Roger T.
Published: July 2002

The kinetics of the fall in subcutaneous fluid glucose concentration in anesthetized rats (
n
= 7) after intravenous injection of insulin (0.5 units/kg) was studied by using 5 × 10
−4
cm
2
active area, <150-sec 10–90% response time, amperometric glucose sensors. The onset of the decline in the subcutaneous glucose concentration was delayed and statistically different (
P
< 0.001) from that in blood (8.9 ± 2.1 min vs. 3.3 ± 0.5 min). Similarly, the rate of drop in glucose concentration between 6 and 20 min after the insulin injection was different for subcutaneous tissue (3.9 ± 1.3 mg⋅dl
−1
⋅ min
−1
) and blood (6.8 ± 2.0 mg⋅dl
−1
⋅min
−1
) (
P
= 0.003). The hypoglycemic nadir in subcutaneous fluid occurred 24.5 ± 6.8 min after that in the blood (
P
< 0.001). A “forward” mass-transfer model, predicting the subcutaneous glucose concentration from the blood glucose concentrations and an “inverse” model, predicting the blood glucose concentration from the subcutaneous glucose concentration were derived. By using an algorithm based on the latter, the average discrepancy between the measured blood glucose concentration and that estimated from the subcutaneous measurement through the entire 4-hr experiment was reduced from 22.9% to 11.1% (
P
= 0.025). The maximum discrepancy during the 40-min period after the injection of insulin was reduced from 84.1% to 29.3% (
P
= 0.006).
From estrogen to androgen receptor: A new pathway for sex hormones in prostate
Authors: Yeh Shuyuan , Miyamoto Hiroshi , Shima Hiroki , Chang Chawnshang
Published: July 2002

While all three coactivators ARA
70
, steroid receptor coactivator 1, and RAC3/ACTR can enhance androgen receptor (AR) transcriptional activity at 1 nM dihydrotestosterone, we here demonstrate that only ARA
70
can induce AR transcriptional activity >30-fold in the presence of 10 nM 17β-estradiol (E2), but not diethylstilbestrol. The significance of this newly described E2-induced AR transcriptional activity in DU145 human prostate cancer cells was further strengthened by finding patients with Reifenstein partial-androgen-insensitive syndrome that fail in the E2-AR-ARA
70
pathway. Together, our data suggest, for the first time, testosterone/dihydrotestosterone may not be the only ligands for the AR. E2 represents another important natural ligand for AR that may play an essential role for the AR function and the development of the male reproductive system.
Huntingtin aggregation monitored by dynamic light scattering
Authors: Georgalis Yannis , Starikov E. B. , Hollenbach Birgit , Lurz Rudi , Scherzinger Eberhard , Saenger Wolfram , Lehrach Hans , Wanker Erich E.
Published: July 2002

An initial stage of fibrillogenesis in solutions of glutathioneS-transferase-huntingtin (GST-HD) fusion proteins has been studied by using dynamic light scattering. Two GST-HD systems with poly-l-glutamine (polyGln) extensions of different lengths (20 and 51 residues) have been examined. For both systems, kinetics of z-average translation diffusion coefficients (Dapp) and their angular dependence have been obtained. Our data reveal that aggregation does occur in both GST-HD51 and GST-HD20 solutions, but that it is much more pronounced in the former. Thus, our approach provides a powerful tool for the quantitative assay of GST-HD fibrillogenesisin vitro.
Inactivation of the mouse sperm receptor, mZP3, by site-directed mutagenesis of individual serine residues located at the combining site for sperm
Authors: Chen Jie , Litscher Eveline S. , Wassarman Paul M.
Published: July 2002

To initiate fertilization, mouse sperm bind to Ser- (
O
-) linked oligosaccharides located at the sperm combining site of zona pellucida glycoprotein mZP3. Apparently, the oligosaccharides are present on one or more of five Ser residues clustered in the carboxyl-terminal region of the mZP3 polypeptide. Here, each of the Ser residues, as well as an intervening Asn residue, was converted to a small, nonhydroxy amino acid by site-directed mutagenesis. Mouse embryonal carcinoma (EC) cells were then stably transfected with the wild-type and mutated
mZP3
genes. In each case, transfected cells synthesized and secreted recombinant EC-mZP3 into the culture medium. The glycoproteins were partially purified and assayed for their ability to inhibit binding of sperm to ovulated eggs
in vitro.
As compared with wild-type EC-mZP3, mutations of Ser-329, Ser-331, or Ser-333 had no effect on sperm receptor activity. Mutation of Asn-330, a potential
N
-linked glycosylation site, also had no effect on sperm receptor activity. On the other hand, mutation of either Ser-332 or Ser-334, or mutation of Ser-332, Ser-333, and Ser-334, resulted in complete inactivation of EC-mZP3 as a sperm receptor. These results suggest that Ser-332 and Ser-334, residues conserved in mouse, hamster, and human ZP3, are essential for sperm receptor activity.