bluuebunny/crossref_metadata_2025 huggingface dataset:
Alpha-crystallin/small heat shock protein has autokinase activity.
Authors: Kantorow M , Piatigorsky J
Published: May 2006

The alpha-crystallins (alpha A and alpha B) are major water-soluble proteins of the transparent eye lens that are expressed in a variety of tissues and can function as molecular chaperones. alpha B-crystallin is also a small heat shock protein associated with numerous degenerative diseases and abnormal growth patterns. Previous experiments have shown that alpha A-and alpha B-crystallin are phosphorylated on specific serine residues by a cAMP-dependent pathway. Here we provide evidence that either total bovine alpha-crystallin or its isolated polypeptides can autophosphorylate serine by a cAMP-independent mechanism in the presence of Mg2+ and [gamma-32P]ATP; the autophosphorylated products isoelectrically focus with the authentic phosphorylated forms of the alpha-crystallin polypeptides. Thus, the alpha A- and alpha B-crystallin/small heat shock protein polypeptides are enzyme-crystallins which may be involved in metabolic pathways important for the development, maintenance, or pathology of the lens and other tissues.
Anticarcinogenic activities of sulforaphane and structurally related synthetic norbornyl isothiocyanates.
Authors: Zhang Y , Kensler T W , Cho C G , Posner G H , Talalay P
Published: May 2006

Sulforaphane [1-isothiocyanato-4-(methyl-sulfinyl)butane] was recently isolated from one variety of broccoli as the major and very potent inducer of phase 2 detoxication enzymes in murine hepatoma cells in culture. Since phase 2 enzyme induction is often associated with reduced susceptibility of animals and their cells to the toxic and neoplastic effects of carcinogens and other electrophiles, it was important to establish whether sulforaphane could block chemical carcinogenesis. In this paper we report that sulforaphane and three synthetic analogues, designed as potent phase 2 enzyme inducers, block the formation of mammary tumors in Sprague-Dawley rats treated with single doses of 9,10-dimethyl-1,2-benzanthracene. The analogues are exo-2-acetyl-exo-6-isothiocyanatonorbornane, endo-2-acetyl-exo-6-isothiocyanatonorbornane, and exo-2-acetyl-exo-5-isothiocyanatonorbornane. When sulforaphane and exo-2-acetyl-exo-6-isothiocyanatonorbornane were administered by gavage (75 or 150 mumol per day for 5 days) around the time of exposure to the carcinogen, the incidence, multiplicity, and weight of mammary tumors were significantly reduced, and their development was delayed. The analogues endo-2-acetyl-exo-6-isothiocyanatonorbornane and exo-2-acetyl-exo-5-isothiocyanatonorbornane were less potent protectors. Thus, a class of functionalized isothiocyanates with anticarcinogenic properties has been identified. These results validate the thesis that inducers of phase 2 enzymes in cultured cells are likely to protect against carcinogenesis.
Electron micrographic studies of transport of oligodeoxynucleotides across eukaryotic cell membranes.
Authors: Zamecnik P , Aghajanian J , Zamecnik M , Goodchild J , Witman G
Published: May 2006

Unmodified oligodeoxynucleotides (ODNs) were synthesized and tested for their ability to cross external eukaryotic cell membranes and to enter the cytosol and nucleus in tissue cultures. The ODNs were labeled with high-specific-activity [3H]thymidine (> or = 100 Ci/mmol), or [ alpha-32P]ATP or [ gamma-32P]ATP (300-1000 Ci/mmol; 1 Ci = 37 GBq), and the label was either in the central portion of the molecule or at the 3' or 5' end. The cells employed were for the most part 3T6 murine fibroblasts, grown in monolayers, either semiconfluent or confluent, but some experiments were carried out with chicken embryo fibroblasts or human HeLa cells. Parallel wells in the same experiment were prepared for electron microscopy or for cell fractionation and radioactivity assays. Electron microscopic autoradiography indicated that ODNs cross the external cell membrane, traverse the cytosol, and begin to enter the cell nucleus within a few seconds to 5 min at 37 degrees C in Dulbecco's medium without added serum. After 30-60 min of incubation with ODNs, abundant silver grains were observed at or just inside the nuclear membrane or well distributed across the nucleus, particularly in association with euchromatin. There was a paucity of silver grains associated with nucleoli. Cell entry of oligomer was related to cell cycling events and was energy dependent. Degradation of oligomer to monomers, with reincorporation into DNA, does not appear to explain these results. No sequestration of labeled oligomer in cytoplasmic vesicles en route from the exterior of the cell to the nucleus was observed. The observations are more suggestive of internalization of oligonucleotide by a mechanism as yet unclear or, alternatively, by a caveolar, potocytotic mechanism rather than by endocytosis.
Mutational processes of simple-sequence repeat loci in human populations.
Authors: Di Rienzo A , Peterson A C , Garza J C , Valdes A M , Slatkin M , Freimer N B
Published: May 2006

Mutational processes of simple sequence repeats (SSRs) in complex genomes are poorly understood. We examined these processes by introducing a two-phase mutation model. In this model, most mutations are single-step changes, but infrequent large jumps in repeat number also occur. We used computer simulations to determine expected values of statistics that reflect frequency distributions of allele size for the two-phase model and two alternatives, the one-step and geometric models. The theoretical expectations for each model were tested by comparison with observed values for 10 SSR loci genotyped in the Sardinian population, whose genetic and demographic histories have been previously reconstructed. The two-phase model provided the best fit to the data for most of these loci in this population. In the analysis we assumed that the loci were neutral and that this population had undergone rapid population growth. Recent observations made for unstable trinucleotide repeats support our suggestion that frequent small changes and rare large changes in repeat number represent two distinct classes of mutation at SSR loci. We genotyped the same 10 loci in Egyptian and sub-Saharan African samples to assess the utility of SSRs for studying the divergence of populations and found that estimates of interpopulation distances from SSRs were similar to those derived from analysis of mitochondrial DNA.
Immunosuppressant FK506 promotes neurite outgrowth in cultures of PC12 cells and sensory ganglia.
Authors: Lyons W E , George E B , Dawson T M , Steiner J P , Snyder S H
Published: May 2006

The immunosuppressant drug FK506 acts by binding to receptor proteins, FK506-binding proteins (FKBPs), which in turn can bind to and regulate a Ca(2+)-dependent phosphatase, calcineurin, and a Ca2+ release channel, the ryanodine receptor. Based on our findings in regeneration models that levels of FKBPs during neural regeneration parallel those of growth-associated protein GAP43, a calcineurin substrate that regulates neurite extension, we examined effects of FK506 in PC12 rat pheochromocytoma cells and in rat sensory ganglia. FK506 enhances neurite outgrowth in both systems by increasing sensitivity to nerve growth factor. Blockade of FK506 actions in sensory ganglia by rapamycin, an FK506 antagonist, establishes that these effects involve FKBPs. Rapamycin itself stimulates neurite outgrowth in PC12 cells. These drug effects are detected at subnanomolar concentrations, suggesting therapeutic application in diseases involving neural degeneration.
The Arabidopsis thaliana apurinic endonuclease Arp reduces human transcription factors Fos and Jun.
Authors: Babiychuk E , Kushnir S , Van Montagu M , Inzé D
Published: May 2006

An Arabidopsis thaliana cDNA encoding an analogue, referred to as Arp for apurinic endonuclease-redox protein, of the human redox factor REF has been cloned. Arp stimulates in vitro DNA-binding activity of the human transcription factor Jun and Fos by the reduction of a cysteine residue located in the DNA-binding domain. Based on amino acid sequence homology, this redox activity is probably confined to the small internal domain of the Arp protein. In analogy to REF, we show that the Arabidopsis Arp protein also functions as an apurinic/apyrimidinic class II endonuclease. This base-free endonuclease activity resides in the carboxyl-terminal domain, and this part of the protein has significant sequence similarity to bacterial (Escherichia coli exonuclease III and Streptococcus pneumoniae exonuclease A) and animal (Drosophila Rrp1 and human REF/HAP) apurinic/apyrimidinic endonucleases. The amino-terminal domain of the Arp protein is highly charged and apparently increases the affinity of the protein for DNA. Therefore, the Arabidopsis Arp protein is multifunctional and may be involved both in DNA repair and in the regulation of transcription.
Three-dimensional spectral-spatial EPR imaging of free radicals in the heart: a technique for imaging tissue metabolism and oxygenation.
Authors: Kuppusamy P , Chzhan M , Vij K , Shteynbuk M , Lefer D J , Giannella E , Zweier J L
Published: May 2006

It has been hypothesized that free radical metabolism and oxygenation in living organs and tissues such as the heart may vary over the spatially defined tissue structure. In an effort to study these spatially defined differences, we have developed electron paramagnetic resonance imaging instrumentation enabling the performance of three-dimensional spectral-spatial images of free radicals infused into the heart and large vessels. Using this instrumentation, high-quality three-dimensional spectral-spatial images of isolated perfused rat hearts and rabbit aortas are obtained. In the isolated aorta, it is shown that spatially and spectrally accurate images of the vessel lumen and wall could be obtained in this living vascular tissue. In the isolated rat heart, imaging experiments were performed to determine the kinetics of radical clearance at different spatial locations within the heart during myocardial ischemia. The kinetic data show the existence of regional and transmural differences in myocardial free radical clearance. It is further demonstrated that EPR imaging can be used to noninvasively measure spatially localized oxygen concentrations in the heart. Thus, the technique of spectral-spatial EPR imaging is shown to be a powerful tool in providing spatial information regarding the free radical distribution, metabolism, and tissue oxygenation in living biological organs and tissues.
Redox regulation of signal transduction: tyrosine phosphorylation and calcium influx.
Authors: Staal F J , Anderson M T , Staal G E , Herzenberg L A , Gitler C , Herzenberg L A
Published: May 2006

Studies presented here show that altering the intracellular redox balance by decreasing glutathione levels profoundly affects early signal transduction events in human T cells. In a T-cell receptor (TCR) signaling model, short-term pretreatment with buthionine sulfoximine, which specifically decreases intracellular glutathione, essentially abrogates the stimulation of calcium influx by anti-CD3 antibodies without significantly impairing other aspects of TCR-initiated signal transduction, such as overall levels of TCR-stimulated tyrosine phosphorylation. In an inflammatory-cytokine signaling model, the failure of tumor necrosis factor alpha to stimulate more than minimal tyrosine phosphorylation in lymphocytes is overcome by buthionine sulfoximine pretreatment--i.e., tumor necrosis factor alpha stimulates extensive tyrosine phosphorylation in glutathione-depleted lymphocytes. These redox-dependent changes in T-cell responsiveness suggest that the glutathione deficiency that we and others have demonstrated in human immunodeficiency virus-infected individuals may contribute significantly to the immunodeficiency and the increased inflammatory reactions in these individuals.
Rapid cell variation can determine the establishment of a persistent viral infection.
Authors: Martín Hernández A M , Carrillo E C , Sevilla N , Domingo E
Published: May 2006

Evidence for a mechanism of initiation of viral persistence in which the cell, and not the virus, plays a critical role has been obtained using the important animal pathogen foot-and-mouth disease virus (FMDV). We have developed a virulence assay consisting of quantification of the ability of virus to kill cells and of cells to divide in the presence of virus and to initiate a carrier state. Cells were cured of FMDV at early times following a cytolytic infection of BHK-21 monolayers with FMDV. When cured cells were subjected to the virulence assay they showed an increased ability to survive a second infection by FMDV but not by other RNA viruses. This altered phenotype was maintained as a stable genetic trait. When the virus present in such early surviving cells was used to infect BHK-21 cells, it proved to be as virulent as the initial cytolytic FMDV and, furthermore, its ability to kill BHK-21 cells increased upon replication in the surviving cells. Both the level of genetic heterogeneity and the rate of evolution of FMDV were similar to those previously documented during acute and persistent FMDV infections. The results suggest that, in contrast to most other viral systems, the critical element in the establishment of a persistent infection of BHK-21 cells with FMDV is the ability of the host cells to vary genetically and phenotypically, which promotes selection of cells with increased resistance to virus. The possible relevance of this mechanism to viral persistence in vivo is discussed.
N-nitroso-N-methylurea-induced rat mammary tumors arise from cells with preexisting oncogenic Hras1 gene mutations.
Authors: Cha R S , Thilly W G , Zarbl H
Published: May 2006

GGA to GAA mutations in the 12th codon of the Hras gene are frequently observed in rat mammary tumors induced by N-nitroso-N-methylurea (NMU). We developed an assay to measure point mutations present in tissues at a frequency of 10(-5) and have now applied this assay to measure the specific G to A transition of the Hras gene in rat mammary epithelium. We find that (i) 70% of untreated rats contain detectable levels of Hras mutants; (ii) these mutants are clustered within the gland as sectors in a manner consistent with their origin as a mutation arising during early organ development; and (iii) treatment with a carcinogenic dose of NMU did not result in a significant increase in the number of such mutants, the fraction of organ sectors with mutant cells, or the fraction of animals containing detectable levels of ras mutants. We conclude that the NMU-induced mammary tumors carrying the G to A transition at the 12th codon of the Hras gene arose from preexisting ras mutants and that an independent effect of NMU was directly or indirectly responsible for tumor formation.
A requirement for extracellular signal-regulated kinase (ERK) function in the activation of AP-1 by Ha-Ras, phorbol 12-myristate 13-acetate, and serum.
Authors: Frost J A , Geppert T D , Cobb M H , Feramisco J R
Published: May 2006

The role of ERK-1 and ERK-2 in wild-type (wt) Ha-Ras, phorbol 12-myristate 13-acetate (PMA), and serum-induced AP-1 activity was studied. Microinjection of ERK-specific substrate peptides inhibited the induction of AP-1 activity by all three stimuli, whereas a control peptide had no effect. By using eukaryotic expression constructs encoding wt ERK-1 and kinase-deficient mutants of ERKs 1 and 2, it was found that ERK-1 and ERK-2 activities are required for AP-1 activation stimulated by either wt Ha-Ras, PMA, or serum. Overexpression of ERK-1 augmented wt Ha-Ras stimulation of AP-1, while having no effect upon PMA or serum stimulation. Overexpression of either kinase-deficient ERK-1 or kinase-deficient ERK-2 partially inhibited AP-1 activation by wt Ha-Ras but had no effect on PMA or serum-induced activation. Coexpression of both interfering mutants abolished AP-1 induction by wt Ha-Ras, PMA, or serum. We conclude that ERKs are necessary components in the pathway leading to the activation of AP-1 stimulated by these agents.
Cellular latency in human immunodeficiency virus-infected individuals with high CD4 levels can be detected by the presence of promoter-proximal transcripts.
Authors: Adams M , Sharmeen L , Kimpton J , Romeo J M , Garcia J V , Peterlin B M , Groudine M , Emerman M
Published: May 2006

We have investigated the molecular basis of human immunodeficiency virus type 1 (HIV-1) latency in a tissue culture model and in HIV-infected people. We show that increased levels of Tat, but not Rev, can release the proviruses from latency in U1 cells. The absence of Tat in these cells is manifested by the accumulation of promoter-proximal viral transcripts, whereas the presence of Tat correlates with increased expression of viral proteins and an increase in promoter-distal transcripts. The presence of promoter-proximal transcripts also serves as a marker for latency in humans. We observed the exclusive presence of promoter-proximal viral transcripts in peripheral mononuclear cells from the majority (10/11) of asymptomatic HIV-infected individuals examined. Activation of these cells in vitro, and viremia in vivo, correlated with a switch from promoter-proximal transcription to promoter-distal transcription. These results suggest that the control between latency and replication of HIV in vivo is at the level of transcription elongation.
Metaphase and anaphase in the artificially induced monopolar spindle.
Authors: Ito K , Masuda M , Fujiwara K , Hayashi H , Sato H
Published: May 2006

By using monopolar spindles artificially induced in sea urchin embryos, we examined whether or not the presence of two opposing poles was an indispensable condition for keeping chromosomes at a fixed distance from the pole at metaphase and for the anaphase chromosome movement. Chromosomes were stained with Hoechst dye 33342 and their behavior was followed in the monopolar and the control bipolar spindles. In the monopolar spindle, chromosomes were first arranged on a curved metaphase plate and then spread on a part of the imaginary surface of a sphere whose center was the monopole. The estimated chromosome-to-pole distance was similar to that of bipolar spindles at metaphase and remained fixed until chromosomes started to move toward the pole. The average duration of metaphase in the monopolar spindle was 6 times longer than that in the bipolar spindle. The poleward movement of chromosomes in the monopolar spindle was similar to the anaphase A (chromosome-to-pole movement) in the bipolar spindle with respect to the velocity, duration, distance, and synchronization of migration. These results show that even half of the normal spindle has capacities for the arrangement of chromosomes at metaphase and for the anaphase A chromosome movement. Based on these results, we were able to exclude some existing theories of metaphase, such as the one based on the balance of forces between the two poles.
Heterogeneous activation thresholds to cytokines in genetically distinct endothelial cells: evidence for diverse transcriptional responses.
Authors: Bender J R , Sadeghi M M , Watson C , Pfau S , Pardi R
Published: May 2006

It is well accepted that the induction of endothelial cell (EC) adhesion molecules is a critical component in acute inflammatory responses as well as allogeneic interactions in vascularized allografts and, possibly, atherogenesis. The "inflammatory triad" of interleukin 1 (IL-1), tumor necrosis factor, and lipopolysaccharide are potent stimulators of the EC activation/adhesion molecules intercellular adhesion molecule 1 (ICAM-1), endothelial-leukocyte adhesion molecule 1 (ELAM-1), and vascular cell adhesion molecule 1 (VCAM-1). To address whether there exist differing thresholds to cytokine-mediated EC activation, we utilized a panel of genetically distinct human umbilical vein EC lines, assessing their modulated EC surface expression and transcriptional responses to cytokines, with regard to the cell adhesion molecules (CAMs) ELAM-1, ICAM-1, and VCAM-1. With submaximal concentrations of cytokine, EC ELAM-1 surface expression varied from negligible to marked. This phenotypic response was maintained over numerous passages in culture and was observed in ex vivo organ culture analyses with cytokine-treated umbilical vein sections. Relative patterns of ELAM-1, ICAM-1, and VCAM-1 induction were similar in response to multiple stimuli (IL-1, tumor necrosis factor, and lipopolysaccharide, but not phorbol 12-myristate 13-acetate). Nuclear run-off experiments demonstrated that the "high responder" phenotype is a consequence of enhanced transcriptional activation of the CAM genes in response to IL-1 (1 unit/ml), whereas transcriptional responses in "low responders" are minimal. Despite the known involvement of NF-kappa B in endothelial CAM transcription, gel shift assays failed to demonstrate a correlation between the levels of IL-1-mediated nuclear NF-kappa B expression and CAM induction in high and low responding lines. We postulate that varying EC activation thresholds to cytokines observed here, in vitro, may be a critical determinant in the susceptibility to vasculopathic states.
Consistent intergenic splicing and production of multiple transcripts between AML1 at 21q22 and unrelated genes at 3q26 in (3;21)(q26;q22) translocations.
Authors: Nucifora G , Begy C R , Kobayashi H , Roulston D , Claxton D , Pedersen-Bjergaard J , Parganas E , Ihle J N , Rowley J D
Published: May 2006

Two genes have been implicated in leukemias of patients with abnormalities of chromosome 3, band q26: EVI1, which can be activated over long distances by chromosomal rearrangements involving 3q26, and EAP, a ribosomal gene that fuses with AML1 in a therapy-related myelodysplasia patient with a t(3;21)(q26.2;q22). AML1 was identified by its involvement in the t(8;21)(q22;q22) of acute myeloid leukemia. Here we report the consistent identification of fusion transcripts between AML1 and EAP or between AML1 and previously unidentified sequences that we named MDS1 (MDS-associated sequences) in the leukemic cells of four patients with therapy-related myelodysplasia/acute myeloid leukemia and in one patient with chronic myelogenous leukemia in blast crisis, all of whom had a t(3;21). In addition, we have identified a third chimeric transcript, AML1/EVI1, in one of the therapy-related acute myeloid leukemia patients. Pulsed-field gel electrophoresis established the order of the genes as EAP, the most telomeric, and EVI1, the most centromeric, gene. The results indicate that translocations could involve multiple genes and affect gene expression over long distances.
Underlying order in protein sequence organization.
Authors: Berman A L , Kolker E , Trifonov E N
Published: May 2006

The idea of a possible standard modular structure of proteins has been known since 1929 when it was introduced by Svedberg. It still remains an idea with no quantitative confirmation of universality of such hypothetical organization. From a large collection of nonredundant protein sequences representing > 100 eukaryotic and prokaryotic species, we have obtained the protein sequence length distributions. Mere inspection of these distributions, as well as spectral analysis, shows that 15-30% of proteins, depending on species and sequence types, indeed appear to be made of sequence units with characteristic lengths of approximately 125 aa for eukaryotes and approximately 150 aa for prokaryotes. This underlying order in protein sequence organization is shown to be universal--that is, the weak regularity observed is not caused by a particular dominant species or protein group. Possible mechanisms are discussed that may be responsible for the observed regularity, including a hypothesis about the recombinational nature of such protein sequence organization.
Functional coupling of Ca2+ channels and ryanodine receptors in cardiac myocytes.
Authors: Sham J S , Cleemann L , Morad M
Published: May 2006

In skeletal muscle, dihydropyridine receptors are functionally coupled to ryanodine receptors of the sarcoplasmic reticulum in triadic or diadic junctional complexes. In cardiac muscle direct physical or functional couplings have not been demonstrated. We have tested the hypothesis of functional coupling of L-type Ca2+ channels and ryanodine receptors in rat cardiac myocytes by comparing the efficacies of Ca2+ in triggering Ca2+ release when the ion enters the cell via the Ca2+ channels or the Na+/Ca2+ exchanger. Ca2+ transported through the Ca2+ channels was 20-160 times more effective than Ca2+ influx via the Na+/Ca2+ exchanger in gating Ca2+ release from the sarcoplasmic reticulum, suggesting privileged communication between Ca2+ channels and ryanodine receptors. In support of this hypothesis we found that Ca2+ channels were inactivated by Ca2+ release from the sarcoplasmic reticulum, even though the myoplasmic Ca2+ concentrations were buffered with 10 mM EGTA. The data thus suggest privileged cross signaling between the dihydropyridine and ryanodine receptors such that Ca2+ flux through either the Ca2+ channel or the ryanodine receptor alters the gating kinetics of the other channel.
FALL-39, a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis.
Authors: Agerberth B , Gunne H , Odeberg J , Kogner P , Boman H G , Gudmundsson G H
Published: May 2006

PR-39, a proline/arginine-rich peptide antibiotic, has been purified from pig intestine and later shown to originate in the bone marrow. Intending to isolate a clone for a human counterpart to PR-39, we synthesized a PCR probe derived from the PR-39 gene. However, when this probe was used to screen a human bone marrow cDNA library, eight clones were obtained with information for another putative human peptide antibiotic, designated FALL-39 after the first four residues. FALL-39 is a 39-residue peptide lacking cysteine and tryptophan. All human peptide antibiotics previously isolated (or predicted) belong to the defensin family and contain three disulfide bridges. The clone for prepro-FALL-39 encodes a cathelin-like precursor protein with 170 amino acid residues. We have postulated a dibasic processing site for the mature FALL-39 and chemically synthesized the putative peptide. In basal medium E, synthetic FALL-39 was highly active against Escherichia coli and Bacillus megaterium. Residues 13-34 in FALL-39 can be predicted to form a perfect amphiphatic helix, and CD spectra showed that medium E induced 30% helix formation in FALL-39. RNA blot analyses disclosed that the gene for FALL-39 is expressed mainly in human bone marrow and testis.
Human immunodeficiency virus (HIV) antigens: structure and serology of multivalent human mucin MUC1-HIV V3 chimeric proteins.
Authors: Fontenot J D , Gatewood J M , Mariappan S V , Pau C P , Parekh B S , George J R , Gupta G
Published: May 2006

Molecular modeling and two-dimensional NMR techniques enable us to identify structural features in the third variable region (V3) loop of the human immunodeficiency virus (HIV) surface glycoprotein gp120, in particular the principal neutralizing determinant (PND), that remain conserved despite the sequence variation. The conserved structure of the PND is a solvent-accessible protruding motif or a knob, structurally isomorphous with the immunodominant knobs in the tandem repeat protein of human mucin 1 (MUC1) (a tumor antigen for breast, pancreatic, and ovarian cancer). We have replaced the mucin antigenic knobs by the PND knobs of the HIV MN isolate in a set of chimeric human MUC1/HIV V3 antigens. This produced multivalent HIV antigens in which PNDs are located at regular intervals and separated by extended mucin spacers. In this article we show by two-dimensional NMR spectroscopy that the multivalent antigens preserve the PNDs in their native structure. We also demonstrate by ELISA that the antigens correctly present the PNDs for binding to monoclonal antibodies or polyclonal antisera from HIV-infected patients.
DNA alterations detected in the progeny of paternally irradiated Japanese medaka fish (Oryzias latipes).
Authors: Kubota Y , Shimada A , Shima A
Published: May 2006

A nonmammalian test system for germ-cell mutagenesis has been developed by using the Japanese medaka fish. We describe a system for detecting DNA alterations in F1 progeny descended from the gamma-irradiated male medaka that uses an arbitrarily primed polymerase chain reaction and fingerprinting. A combination of these two methods has some advantages for screening changes in genomic DNA of individual progeny because this detection system can (i) screen for mutational events before embryos with dominant lethal mutations are eliminated during development and (ii) detect DNA changes in the progeny of irradiated males without functional selection and bias, such as resistance to chemicals. DNA alterations are detected as changes in patterns (i.e., band loss and/or band gain) of DNA fingerprints of progeny descended from males whose spermatozoa or spermatids were gamma-irradiated (4.75 or 9.50 Gy). We determined the frequency of gamma-irradiation-induced band loss in arbitrarily primed polymerase chain reaction fingerprints of DNA from severely malformed embryos with dominant lethal mutations and hatched viable embryos. The frequency of band loss in both dominant lethal embryos and hatched viable embryos increased with increasing gamma-ray dose, although more so in the former. We detected a new band in the fingerprints as a heritable DNA alteration but not a viability- or phenotype-affecting DNA alteration in two viable mutants recovered after gamma-irradiation experiments. A cloned amplified fragment of the new band contained a repeated sequence of p(ATGT)n.
The chemistry of signal transduction.
Authors: Clardy J
Published: May 2006

Several disciplines, including chemical ecology, seek to understand the molecular basis of information transfer in biological systems, and general molecular strategies are beginning to emerge. Often these strategies are discovered by a careful analysis of natural products and their biological effects. Cyclosporin A, FK506, and rapamycin are produced by soil microorganisms and are being used or considered as clinical immunosuppressive agents. They interrupt the cytoplasmic portion of T-cell signaling by forming a complex with a binding protein--FKBP12 in the case of FK506 and rapamycin and cyclophilin A (CyPA) in the case of cyclosporin A (CsA). This complex in turn inhibits a protein target, and the best understood target is calcineurin, which is inhibited by FK506-FKBP12 and CyPA-CsA. Mutational and structural studies help define how FK506-FKBP12 interacts with calcineurin, and the results of these studies are summarized. The existence of strong FK506-FKBP12 binding suggests that FK506 is mimicking some natural ligand for FKBP12. Synthetic and structural studies to probe this mimicry are also described.
Conformational implications of asparagine-linked glycosylation.
Authors: Imperiali B , Rickert K W
Published: May 2006

The effects of cotranslational protein modification on the process of protein folding are poorly understood. Time-resolved fluorescence energy transfer has been used to assess the impact of glycosylation on the conformational dynamics of flexible oligopeptides. The peptide sequences examined are selected from glycoproteins of known three-dimensional structure. The energy transfer modulation associated with N-linked glycosylation is consistent with the glycopeptides sampling different conformational profiles in water. Results show that glycosylation causes the modified peptides to adopt a different ensemble of conformations, and for some peptides this change may lead to conformations that are more compact and better approximate the conformation of these peptides in the final folded protein. This result further implies that cotranslational glycosylation can trigger the timely formation of structural nucleation elements and thus assist in the complex process of protein folding.
Primary structure of hepatocyte nuclear factor/forkhead homologue 4 and characterization of gene expression in the developing respiratory and reproductive epithelium.
Authors: Hackett B P , Brody S L , Liang M , Zeitz I D , Bruns L A , Gitlin J D
Published: May 2006

Members of the winged helix/forkhead family of transcription factors are believed to play a role in cell-specific gene expression. A cDNA encoding a member of this family of proteins, termed hepatocyte nuclear factor/forkhead homologue 4 (HFH-4), has been isolated from rat lung and rat testis cDNA libraries. This cDNA contains an open reading frame of 421 amino acids with a conserved DNA binding domain and several potential transactivating regions. During murine lung development, a single species of HFH-4-specific transcript (2.4 kb long) is first detected precisely at the start of the late pseudoglandular stage (embryonic day 14.5) and, by in situ hybridization, is specifically localized to the proximal pulmonary epithelium. The unique temporal and spatial pattern of HFH-4 gene expression in the developing lung defines this protein as a marker for the initiation of bronchial epithelial cell differentiation and suggests that it may play an important role in cell fate determination during lung development. In addition to expression in the pulmonary epithelium, RNA blot analysis reveals 2.4-kb HFH-4 transcripts in the testis and oviduct. By using mice with genetic defects in spermatogenesis, HFH-4 expression in the testis is found to be associated with the appearance of haploid germ cells and in situ hybridization studies indicate that HFH-4 expression is confined to stages I-VII of spermatogenesis. This pattern of HFH-4 gene expression during the early stages of differentiation of haploid germ cells suggests that HFH-4 may play a role in regulating stage-specific gene expression and cell-fate determination during lung development and in spermatogenesis.
Nonneuronal expression of Rab3A: induction during adipogenesis and association with different intracellular membranes than Rab3D.
Authors: Baldini G , Scherer P E , Lodish H F
Published: May 2006

Rab3A is a small GTP-binding protein expressed predominantly in brain and neuroendocrine cells, in which it is associated with synaptic and synaptic-like vesicles, respectively. Here we report that adult mouse fat cells and 3T3-L1 adipocytes also express Rab3A mRNA and protein. They do not express synaptophysin, an abundant protein in synaptic vesicles or synaptic-like vesicles. The amount of Rab3A mRNA and protein, like that of the highly homologous isoform Rab3D, increases severalfold during differentiation of 3T3-L1 fibroblasts into mature adipocytes. In fat cells, most Rab3D and Rab3A protein is bound to membrane, irrespective of insulin addition. Rab3A and Rab3D are localized in different subcellular compartments, since about half of the Rab3A, but none of the Rab3D, is associated with a low-density organelle(s). Rab3D and Rab3A may be involved in different pathways of regulated exocytosis in adipocytes. Moreover, in adipocytes Rab3A may define an exocytic organelle that is different from synaptic vesicles or synaptic-like microvesicles found in neuronal and endocrine cells.
Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle.
Authors: Sawin K E , Mitchison T J
Published: May 2006

Eg5, a member of the bimC subfamily of kinesin-like microtubule motor proteins, localizes to spindle microtubules in mitosis but not to interphase microtubules. We investigated the molecular basis for spindle localization by transient transfection of Xenopus A6 cells with myc-tagged derivatives of Eg5. Expressed at constitutively high levels from a cytomegalovirus promoter, mycEg5 protein is cytoplasmic throughout interphase, begins to bind microtubules in early prophase, and remains localized to spindle and/or midbody microtubules through mitosis to the end of telophase. Both N- and C-terminal regions of Eg5 are required for this cell-cycle-regulated targeting. Eg5 also contains within its C-terminal domain a sequence conserved among bimC subfamily proteins that includes a potential p34cdc2 phosphorylation site. We show that mutation of a single threonine (T937) within this site to nonphosphorylatable alanine abolishes localization of the mutant protein to the spindle, whereas mutation of T937 to serine preserves spindle localization. We hypothesize that phosphorylation of Eg5 may regulate its localization to the spindle in the cell cycle.
Neural cell adhesion molecule (N-CAM) inhibits astrocyte proliferation after injury to different regions of the adult rat brain.
Authors: Krushel L A , Sporns O , Cunningham B A , Crossin K L , Edelman G M
Published: May 2006

After a penetrating lesion in the central nervous system, astrocytes enlarge, divide, and participate in creating an environment that adversely affects neuronal regeneration. We have recently shown that the neural cell adhesion molecule (N-CAM) partially inhibits the division of early postnatal rat astrocytes in vitro. In the present study, we demonstrate that addition of N-CAM, the third immunoglobulin-like domain of N-CAM, or a synthetic decapeptide corresponding to a putative homophilic binding site in N-CAM partially inhibits astrocyte proliferation after a stab lesion in the adult rat brain. Animals were lesioned in the cerebral cortex, hippocampus, or striatum with a Hamilton syringe and needle at defined stereotaxic positions. On one side, the lesions were concomitantly infused with N-CAM or with one of the N-CAM-related molecules. As a control, a peptide of the same composition as the N-CAM decapeptide but of random sequence was infused on the contralateral side of the brain. We consistently found that the population of dividing astrocytes was significantly smaller on the side in which N-CAM or one of the N-CAM-related molecules was infused than on the opposite side. The inhibition was greatest in the cortical lesion sites (approximately 50%) and was less pronounced in the hippocampus (approximately 25%) and striatum (approximately 20%). Two weeks after the lesion, the cerebral cortical sites infused with N-CAM continued to exhibit a significantly smaller population of dividing astrocytes than the sites on the opposite side. When N-CAM and basic fibroblast growth factor, which is known to stimulate astrocyte division in vitro, were coinfused into cortical lesion sites, astrocyte proliferation was still inhibited. These results suggest the hypothesis that, by reducing glial proliferation, N-CAM or its peptides may help create an environment that is more suitable for neuronal regeneration.
Immunolocalization of the mercurial-insensitive water channel and glycerol intrinsic protein in epithelial cell plasma membranes.
Authors: Frigeri A , Gropper M A , Turck C W , Verkman A S
Published: May 2006

Two water channel homologs were cloned recently from rat kidney, mercurial-insensitive water channel (MIWC) and glycerol intrinsic protein (GLIP). Polyclonal antibodies were raised against synthetic C-terminal peptides and purified by affinity chromatography. MIWC and GLIP antibodies recognized proteins in rat kidney with an apparent molecular mass of 30 and 27 kDa, respectively, and did not cross-react. By immunofluorescence, MIWC and GLIP were expressed together on the basolateral plasma membrane of collecting duct principal cells in kidney. By immunohistochemistry, MIWC and GLIP were expressed on tracheal epithelial cells with greater expression of GLIP on the basal plasma membrane and MIWC on the lateral membrane; only MIWC was expressed in bronchial epithelia. In eye, GLIP was expressed in conjunctival epithelium, whereas MIWC was found in iris, ciliary body, and neural cell layers in retina. MIWC and GLIP colocalized on the basolateral membrane of villus epithelial cells in colon and brain ependymal cells. Expression of MIWC and GLIP was not detected in small intestine, liver, spleen, endothelia, and cells that express water channels CHIP28 or WCH-CD. These studies suggest water/solute transporting roles for MIWC and GLIP in the urinary concentrating mechanism, cerebrospinal fluid absorption, ocular fluid balance, fecal dehydration, and airway humidification. The unexpected membrane colocalization of MIWC and GLIP in several tissues suggests an interaction at the molecular and/or functional levels.
Physical mapping, cloning, and identification of genes within a 500-kb region containing BRCA1.
Authors: Brown M A , Jones K A , Nicolai H , Bonjardim M , Black D , McFarlane R , de Jong P , Quirk J P , Lehrach H , Solomon E
Published: May 2006

BRCA1 is a breast/ovarian cancer susceptibility gene on human chromosome 17q21. We describe a complete and detailed physical map of a 500-kb region of genomic DNA containing the BRCA1 gene and the partial cloning in phage P1 artificial chromosomes. Approximately 70 exons were isolated from this region, 11 of which were components of the BRCA1 gene. Analysis of the other exons revealed a rho-related G protein and the interferon-induced leucine-zipper protein IFP-35.
Roles of nitric oxide in tumor growth.
Authors: Jenkins D C , Charles I G , Thomsen L L , Moss D W , Holmes L S , Baylis S A , Rhodes P , Westmore K , Emson P C , Moncada S
Published: May 2006

A subclone of the human colon adenocarcinoma cell line DLD-1, which grew reproducibly as subcutaneous tumors in nude mice, was isolated. Such cells, when engineered to generate nitric oxide (NO) continuously, grew more slowly in vitro than the wild-type parental cells. This growth retardation was reversed by the addition of N-iminoethyl-L-ornithine. In nude mice, however, the tumors from these cells grew faster than those derived from wild-type cells and were markedly more vascularized, suggesting that NO may act as part of a signaling cascade for neovascularization. Recent observations that the generation of NO in human breast and gynecological cancers correlates positively with tumor grade are consistent with this hypothesis. We suggest that NO may have a dual pro- and antitumor action, depending on the local concentration of the molecule.
Early Alzheimer disease-like histopathology increases in frequency with age in mice transgenic for beta-APP751.
Authors: Higgins L S , Rodems J M , Catalano R , Quon D , Cordell B
Published: May 2006

beta-Amyloid deposition and neurofibrillary tangle formation are two histopathological features of Alzheimer disease. We have previously reported that beta-amyloid immunoreactive deposits form in the brains of transgenic mice programmed for neuronal expression of the 751-amino acid isoform of human beta-amyloid precursor protein (beta-APP751) and now describe that these animals also display Alz50 intraneuronal immunoreactivity similar to that seen in early Alzheimer disease. This suggests that abnormal beta-APP expression and/or beta-amyloid deposition promotes pathogenic alterations in tau protein. The frequency of both beta-amyloid deposition and Alz50-positive neurons was twice as prevalent in brains from old (22 months) as compared to young (2-3 months) beta-APP751 transgenic mice. This increase in histopathology with age in beta-APP751 transgenic mice parallels the time-dependent progression seen in the human disease.
Metallothionein protects against the cytotoxic and DNA-damaging effects of nitric oxide.
Authors: Schwarz M A , Lazo J S , Yalowich J C , Allen W P , Whitmore M , Bergonia H A , Tzeng E , Billiar T R , Robbins P D , Lancaster J R
Published: May 2006

In inflammatory states, nitric oxide (.NO) may be synthesized from precursor L-arginine via inducible .NO synthase (iNOS) in large amounts for prolonged periods of time. When .NO acts as an effector molecule under these conditions, it may be toxic to cells by inhibition of iron-containing enzymes or initiation of DNA single-strand breaks. In contrast to molecular targets of .NO, considerably less is known regarding mechanisms by which cells become resistant to .NO. Metallothionein (MT), the major protein thiol induced in cells exposed to cytokines and bacterial products, is capable of forming iron-dinitrosyl thiolates in vitro. Therefore, we tested the hypothesis that overexpression of MT reduces the sensitivity of NIH 3T3 cells to the .NO donor, S-nitrosoacetylpenicillamine (SNAP), and to .NO released from cells (NIH 3T3-DFG-iNOS) after infection with a retroviral vector expressing human iNOS gene. There was a 4-fold increase in MT in cells transfected with the mouse MT-1 gene (NIH 3T3/MT) compared to cells transfected with the promoter-free inverted gene (NIH 3T3/TM). NIH 3T3/MT cells were more resistant than NIH 3T3/TM cells to the cytotoxic effects of SNAP (0.1-1.0 mM) or .NO released from NIH 3T3-DFG-iNOS cells. A brief (1 h) exposure to 10 mM SNAP caused DNA single-strand breaks that were 9-fold greater in NIH 3T3/TM compared to NIH 3T3/MT cells. Electron paramagnetic resonance spectroscopy of NIH 3T3 cells revealed a greater peak at g = 2.04 (e.g., iron-dinitrosyl complex) in NIH 3T3/MT than NIH 3T3/TM cells. These data are consistent with a role for cytoplasmic MT in interacting with .NO and reducing .NO-induced cyto- and nuclear toxicity.
G1 phase arrest induced by Wilms tumor protein WT1 is abrogated by cyclin/CDK complexes.
Authors: Kudoh T , Ishidate T , Moriyama M , Toyoshima K , Akiyama T
Published: May 2006

WT1, the Wilms tumor-suppressor gene, maps to the human chromosomal region 11p13 and encodes a transcriptional repressor, WT1, implicated in controlling normal urogenital development. Microinjection of the WT1 cDNA into quiescent cells or cells in early to mid G1 phase blocked serum-induced cell cycle progression into S phase. The activity of WT1 varied significantly depending on the presence or absence of an alternatively spliced region located upstream of the zinc finger domain. The inhibitory activity of WT1 was abrogated by the overexpression of cyclin E/CDK2 as well as cyclin D1/CDK4. Furthermore, both CDK4- and CDK2-associated kinase activities were downregulated in cells overexpressing WT1, whereas the levels of CDK4, CDK2, and cyclin D1 expression were unchanged. These findings suggest that inhibition of the activity of cyclin/CDK complexes may be involved in mediating the WT1-induced cell cycle block.
L-arginine may mediate the therapeutic effects of low protein diets.
Authors: Narita I , Border W A , Ketteler M , Ruoslahti E , Noble N A
Published: May 2006

We have previously shown beneficial effects of dietary protein restriction on transforming growth factor beta (TGF-beta) expression and glomerular matrix accumulation in experimental glomerulonephritis. We hypothesized that these effects result from restriction of dietary L-arginine intake. Arginine is a precursor for three pathways, the products of which are involved in tissue injury and repair: nitric oxide, an effector molecule in inflammatory and immunological tissue injury; polyamines, which are required for DNA synthesis and cell growth; and proline, which is required for collagen production. Rats were fed six isocaloric diets differing in L-arginine and/or total protein content, starting immediately after induction of glomerulonephritis by injection of an antibody reactive to glomerular mesangial cells. Mesangial cell lysis and monocyte/macrophage infiltration did not differ with diet. However, restriction of dietary L-arginine intake, even when total protein intake was normal, resulted in decreased proteinuria, decreased expression of TGF-beta 1 mRNA and TGF-beta 1 protein, and decreased production and deposition of matrix components. L-Arginine, but not D-arginine, supplementation to low protein diets reversed these effects. These results implicate arginine as a key component in the beneficial effects of low protein diet.