bluuebunny/crossref_metadata_2025 huggingface dataset:
Emergence of human immunodeficiency virus type 1 variants with resistance to multiple dideoxynucleosides in patients receiving therapy with dideoxynucleosides.
Authors: Shirasaka T , Kavlick M F , Ueno T , Gao W Y , Kojima E , Alcaide M L , Chokekijchai S , Roy B M , Arnold E , Yarchoan R
Published: May 2006

A set of mutations [Ala-62-->Val(A62V), V75I, F77L, F116Y, and Q151M] in the polymerase domain of reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) confers on the virus a reduced sensitivity to multiple antiretroviral dideoxynucleosides and has been seen in HIV-1 variants isolated from patients receiving combination chemotherapy with 3'-azido-3'-deoxythymidine (AZT) plus 2',3'-dideoxycytidine (ddC) or 2',3'-dideoxyinosine (ddI). The IC50 values of AZT, ddC, ddI, 2',3'-dideoxyguanosine, and 2',3'-didehydro-3'-deoxythymidine against an infectious clone constructed to include the five mutations were significantly higher than those of a wild-type infectious clone. The K1 value for AZT 5'-triphosphate determined for the virus-associated RT from a posttherapy strain was 35-fold higher than that of RT from a pretherapy strain. Detailed analysis of HIV-1 strains isolated at various times during therapy showed that the Q151M mutation developed first in vivo, at the time when the viremia level suddenly increased, followed by the F116Y and F77L mutations. All five mutations ultimately developed, and the viremia level rose even further. Analyses based on the three-dimensional structure of HIV-1 RT suggest that the positions where at least several of the five mutations occur are located in close proximity to the proposed dNTP-binding site of RT and the first nucleotide position of the single-stranded template.
Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding yeast.
Authors: Dalton S , Whitbread L
Published: May 2006

The CDC47 gene was isolated by complementation of a cdc47 temperature-sensitive mutant in Saccharomyces cerevisiae and was shown to encode a predicted polypeptide, Cdc47, of 845 aa. Cdc47 belongs to the Cdc46/Mcm family of proteins, previously shown to be essential for initiation of DNA replication. Using indirect immunofluorescence microscopy and subcellular fractionation techniques, we show that Cdc47 undergoes cell cycle-regulated changes in its subcellular localization. At mitosis, Cdc47 enters the nucleus, where it remains until soon after the initiation of DNA replication, when it is rapidly exported back into the cytoplasm. Cdc47 protein levels do not vary with the cell cycle, but expression of CDC47 and nascent synthesis of Cdc47 occur late in the cell cycle, coinciding with mitosis. Together, these results show that Cdc47 is not only imported into the nucleus at the end of mitosis but is also exported back into the cytoplasm at the beginning of S phase. The observation that Cdc47 is exported from the nucleus at the beginning of S phase has important implications for how initiation of DNA replication is controlled.
Aceruloplasminemia: molecular characterization of this disorder of iron metabolism.
Authors: Harris Z L , Takahashi Y , Miyajima H , Serizawa M , MacGillivray R T , Gitlin J D
Published: May 2006

Ceruloplasmin is an abundant alpha 2-serum glycoprotein that contains 95% of the copper found in the plasma of vertebrate species. We report here on the identification of a genetic defect in the ceruloplasmin gene in a patient previously noted to have a total absence of circulating serum ceruloplasmin in association with late-onset retinal and basal ganglia degeneration. In this patient T2 (transverse relaxation time)-weighted magnetic resonance imaging of the brain revealed basal ganglia densities consistent with iron deposition, and liver biopsy confirmed the presence of excess iron. Although Southern blot analysis of the patient's DNA was normal, PCR amplification of 18 of the 19 exons composing the human ceruloplasmin gene revealed a distinct size difference in exon 7. DNA sequence analysis of this exon revealed a 5-bp insertion at amino acid 410, resulting in a frame-shift mutation and a truncated open reading frame. The validity of this mutation was confirmed by analysis of DNA from the patient's daughter, which revealed heterozygosity for this same 5-bp insertion. The presence of this mutation in conjunction with the clinical and pathologic findings demonstrates an essential role for ceruloplasmin in human biology and identifies aceruloplasminemia as an autosomal recessive disorder of iron metabolism. These findings support previous studies that identified ceruloplasmin as a ferroxidase and are remarkably consistent with recent studies on the essential role of a homologous copper oxidase in iron metabolism in yeast. The clinical and laboratory findings suggest that additional patients with movement disorders and nonclassical Wilson disease should be examined for ceruloplasmin gene mutations.
The Menkes/Wilson disease gene homologue in yeast provides copper to a ceruloplasmin-like oxidase required for iron uptake.
Authors: Yuan D S , Stearman R , Dancis A , Dunn T , Beeler T , Klausner R D
Published: May 2006

The CCC2 gene of the yeast Saccharomyces cerevisiae is homologous to the human genes defective in Wilson disease and Menkes disease. A biochemical hallmark of these diseases is a deficiency of copper in ceruloplasmin and other copper proteins found in extracytosolic compartments. Here we demonstrate that disruption of the yeast CCC2 gene results in defects in respiration and iron uptake. These defects could be reversed by supplementing cells with copper, suggesting that CCC2 mutant cells were copper deficient. However, cytosolic copper levels and copper uptake were normal. Instead, CCC2 mutant cells lacked a copper-dependent oxidase activity associated with the extracytosolic domain of the FET3-encoded protein, a ceruloplasmin homologue previously shown to be necessary for high-affinity iron uptake in yeast. Copper restored oxidase activity both in vitro and in vivo, paralleling the ability of copper to restore respiration and iron uptake. These results suggest that the CCC2-encoded protein is required for the export of copper from the cytosol into an extracytosolic compartment, supporting the proposal that intracellular copper transport is impaired in Wilson disease and Menkes disease.
Functional estrogen receptors in a human preosteoclastic cell line.
Authors: Fiorelli G , Gori F , Petilli M , Tanini A , Benvenuti S , Serio M , Bernabei P , Brandi M L
Published: May 2006

The primary biological effect of the estrogen estradiol-17 beta (17 beta E2) on bone is to decrease bone resorption. However, whether 17 beta E2 affects osteoclast differentiation or function directly or through its action on osteoblasts is unclear. To investigate this question we examined the human preosteoclastic cell line FLG 29.1 for evidence of functional estrogen receptors (ERs). Southern blotting of reverse transcription-PCR amplification products with a 32P-labeled cDNA probe for the human ER mRNA demonstrated that FLG 29.1 cells express ER mRNA. Binding of [3H]17 beta E2 to nuclear ERs was steroid specific with approximately 400 saturable, high affinity (Kd approximately 1 nM) binding sites per cell nucleus. Nuclear ERs covalently labeled with [3H]tamoxifen aziridine showed an apparent molecular weight of 65,000 by SDS/PAGE and Western blotting with the D75 monoclonal antibody to human ER. Pretreatment of cells with 0.1, 1.0, or 10 nM 17 beta E2 induced a dose- and time-dependent specific binding of progesterone to FGL 29.1 cells, and stimulation of the cells with 10 nM and 100 nM 17 beta E2 significantly (P < 0.05) reduced cell proliferation. Transcriptional activity of the ER gene was detected by transient transfection of cells with the pERE-BLCAT plasmid containing the estrogen response element for the vitellogenin A2 gene and the bacterial chloramphenicol acetyltransferase reporter gene. Treatment of FLG 29.1 cells with 10 nM 17 beta E2 increased chloroamphenicol acetyltransferase expression from 5- to 29-fold compared to controls. These observations suggest a potential role for estrogen in osteoclastogenesis.
In vitro sulfotransferase activity of Rhizobium meliloti NodH protein: lipochitooligosaccharide nodulation signals are sulfated after synthesis of the core structure.
Authors: Schultze M , Staehelin C , Röhrig H , John M , Schmidt J , Kondorosi E , Schell J , Kondorosi A
Published: May 2006

The Rhizobium common nod gene products NodABC are involved in the synthesis of the core lipochitooligosaccharide (Nod factor) structure, whereas the products of the host-specific nod genes are necessary for diverse structural modifications, which vary in different Rhizobium species. The sulfate group attached to the Rhizobium meliloti Nod signal is necessary for activity on the host plant alfalfa, while its absence renders the Nod factor active on the non-host plant vetch. This substituent is therefore a major determinant of host specificity. The exact biosynthetic pathway of Nod factors has not been fully elucidated. In particular, it is not known why some chemical modifications are introduced with high fidelity whereas others are inaccurate, giving rise to a family of different Nod factor structures produced by a single Rhizobium strain. Using protein extracts and partially purified recombinant NodH protein obtained from Escherichia coli expressing the R. meliloti nodH gene, we demonstrate here NodH-dependent in vitro sulfotransferase activity. Kinetic analyses with Nod factors, chitooligosaccharides, and their deacetylated derivatives revealed that Nod factors are the preferred substrate for the sulfate transfer. Moreover, the tetrameric Nod factor, NodRm-IV, was a better substrate than the trimer, NodRm-III, or the pentamer, NodRm-V. These data suggest that the core lipochitooligosaccharide structure must be synthesized prior to its host-specific modification with a sulfate group. Since in R. meliloti tetrameric Nod factors are the most abundant and the most active ones, high affinity of NodH for the appropriate tetrameric substrate guarantees its modification and thus contributes to the fidelity of host-specific behavior.
Serological response patterns of melanoma patients immunized with a GM2 ganglioside conjugate vaccine.
Authors: Kitamura K , Livingston P O , Fortunato S R , Stockert E , Helling F , Ritter G , Oettgen H F , Old L J
Published: May 2006

Gangliosides, such as GM2, GD2, GD3, and 9-O-acetyl GD3, are receiving attention as targets for antibody-based and vaccine-based therapies of melanoma. GM2 appears to be a particularly immunogenic ganglioside in humans, as indicated by the presence of naturally occurring IgM anti-GM2 antibodies in approximately 5% of humans and the fact that immunization with irradiated GM2-expressing melanoma cells or purified GM2 adherent to bacillus Calmette-Guérin elicits GM2 antibodies of low to moderate titers in a high proportion of vaccinated patients. To develop vaccines that consistently induce high titers of IgM as well as IgG anti-GM2 antibodies, vaccines containing GM2 conjugated to keyhole limpet hemocyanin as the carrier protein and QS-21 as the adjuvant have been constructed. The serological response of vaccinated patients was monitored by ELISA using purified GM2 ganglioside for IgM and IgG anti-GM2 antibodies and for GM2 cell surface-reactive antibodies by immune adherence assays and cytotoxic tests (IgM antibodies) and mixed hemadsorption assays (IgG antibodies). The majority of vaccinated patients developed IgM and IgG antibodies detectable by ELISA. In most cases, the results of IgM ELISA correlated with assays for cell surface-reactive IgM antibodies. This was not true for IgG anti-GM2 antibodies, where strong discrepancies were seen between high titers in ELISA and little or no reactivity in mixed hemadsorption tests for cell surface-reactive antibodies. These IgG antibodies (and the less frequent IgM antibodies that show similar discrepancies) may be directed against GM2 determinants that are buried, hidden, or not present on GM2-expressing target cells. With regard to a major objective of ganglioside vaccines--i.e., generation of cytotoxic antibodies--the GM2-keyhole limpet hemocyanin/QS-21 vaccine is clearly superior to the previously tested GM2/bacillus Calmette-Guérin vaccine. However, variability in patient response and lack of persistence of high-titered IgM cytotoxic antibodies in many patients are problems that remain to be solved.
Detection of one slowly exchanging substrate water molecule in the S3 state of photosystem II.
Authors: Messinger J , Badger M , Wydrzynski T
Published: May 2006

The exchangeability of the substrate water molecules at the catalytic site of water oxidation in photosystem II has been probed by isotope-exchange measurements using mass spectrometric detection of flash-induced oxygen evolution. A stirred sample chamber was constructed to reduce the lag time between injection of H2(18)O and the detecting flash by a factor of more than 1000 compared to the original experiments by R. Radmer and O. Ollinger [(1986) FEBS Lett. 195, 285-289]. Our data show that there is a slow (t1/2 approximately 500 ms, 10 degrees C) and a fast (t1/2 <25 ms, 10 degrees C) exchanging substrate water molecule in the S3 state of photosystem II. The slow exchange is coupled with an activation energy of about 75 kJ/mol and is discussed in terms of a terminal manganese oxo ligand, while the faster exchanging substrate molecule may represent a water molecule not directly bound to the manganese center.
Overexpression of the c-Myc oncoprotein blocks the growth-inhibitory response but is required for the mitogenic effects of transforming growth factor beta 1.
Authors: Alexandrow M G , Kawabata M , Aakre M , Moses H L
Published: May 2006

One of the more intriguing aspects of transforming growth factor beta 1 (TGF beta 1) is its ability to function as both a mitogenic factor for certain mesenchymal cells and a potent growth inhibitor of lymphoid, endothelial, and epithelial cells. Data are presented indicating that c-myc may play a pivotal role in both the mitogenic and antiproliferative actions of TGF beta 1. In agreement with previous studies using C3H/10T1/2 fibroblasts constitutively expressing an exogenous c-myc cDNA, we show that AKR-2B fibroblasts expressing a chimeric estrogen-inducible form of c-myc (mycER) are able to form colonies in soft agar in the presence of TGF beta 1 only when c-myc is activated by hormone. Whereas these findings support a synergistic role for c-myc in mitogenic responses to TGF beta 1, we also find that c-myc can antagonize the growth-inhibitory response to TGF beta 1. Mouse keratinocytes (BALB/MK), which are normally growth-arrested by TGF beta 1, are rendered insensitive to the growth-inhibitory effects of TGF beta 1 upon mycER activation. This ability of mycER activation to block TGF beta 1-induced growth arrest was found to occur only when the fusion protein was induced with hormone in the early part of G1. Addition of estradiol late in G1 had no suppressive effect on TGF beta 1-induced growth inhibition.
Addiction protein Phd of plasmid prophage P1 is a substrate of the ClpXP serine protease of Escherichia coli.
Authors: Lehnherr H , Yarmolinsky M B
Published: May 2006

Plasmid-encoded addiction genes augment the apparent stability of various low copy number bacterial plasmids by selectively killing plasmid-free (cured) segregants or their progeny. The addiction module of plasmid prophage P1 consists of a pair of genes called phd and doc. Phd serves to prevent host death when the prophage is retained and, should retention mechanisms fail, Doc causes death on curing. Doc acts as a cell toxin to which Phd is an antidote. In this study we show that host mutants with defects in either subunit of the ClpXP protease survive the loss of a plasmid that contains a P1 addiction module. The small antidote protein Phd is fully stable in these two mutant hosts, whereas it is labile in a wild-type host. We conclude that the role of ClpXP in the addiction mechanism of P1 is to degrade the Phd protein. This conclusion situates P1 among plasmids that elicit severe withdrawal symptoms and are able to do so because they encode both a cell toxin and an actively degraded macromolecule that blocks the synthesis or function of the toxin.
Gene therapy for diabetes mellitus in rats by hepatic expression of insulin.
Authors: Kolodka T M , Finegold M , Moss L , Woo S L
Published: May 2006

Type 1 diabetes mellitus is caused by severe insulin deficiency secondary to the autoimmune destruction of pancreatic beta cells. Patients need to be controlled by periodic insulin injections to prevent the development of ketoacidosis, which can be fatal. Sustained, low-level expression of the rat insulin 1 gene from the liver of severely diabetic rats was achieved by in vivo administration of a recombinant retroviral vector. Ketoacidosis was prevented and the treated animals exhibited normoglycemia during a 24-hr fast, with no evidence of hypoglycemia. Histopathological examination of the liver in the treated animals showed no apparent abnormalities. Thus, the liver is an excellent target organ for ectopic expression of the insulin gene as a potential treatment modality for type 1 diabetes mellitus by gene therapy.
Protein structure-based design of potent orally bioavailable, nonpeptide inhibitors of human immunodeficiency virus protease.
Authors: Reich S H , Melnick M , Davies J F , Appelt K , Lewis K K , Fuhry M A , Pino M , Trippe A J , Nguyen D , Dawson H
Published: May 2006

A class of potent nonpeptidic inhibitors of human immunodeficiency virus protease has been designed by using the three-dimensional structure of the enzyme as a guide. By employing iterative protein cocrystal structure analysis, design, and synthesis the binding affinity of the lead compound was incrementally improved by over four orders of magnitude. An inversion in inhibitor binding mode was observed crystallographically, providing information critical for subsequent design and highlighting the utility of structural feedback in inhibitor optimization. These inhibitors are selective for the viral protease enzyme, possess good antiviral activity, and are orally available in three species.
Transmembrane signaling characterized in bacterial chemoreceptors by using sulfhydryl cross-linking in vivo.
Authors: Lee G F , Lebert M R , Lilly A A , Hazelbauer G L
Published: May 2006

Transmembrane signaling by bacterial chemoreceptors is thought to involve conformational changes within a stable homodimer. We investigated the functional consequences of constraining movement between pairs of helices in the four-helix structure of the transmembrane domain of chemoreceptor Trg. Using a family of cysteine-containing receptors, we identified oxidation treatments for intact cells that catalyzed essentially complete sulfhydryl cross-linking at selected positions and yet left flagellar and sensory functions largely unperturbed. Constraining movement by cross-links between subunits had little effect on tactic response, but constraining movement between transmembrane segments of the monomer drastically reduced function. We deduce that transmembrane signaling requires substantial movement between transmembrane helices of a monomer but not between interacting helices across the interface between subunits.
Ethanol inhibits luteinizing hormone-releasing hormone (LHRH) secretion by blocking the response of LHRH neuronal terminals to nitric oxide.
Authors: Canteros G , Rettori V , Franchi A , Genaro A , Cebral E , Faletti A , Gimeno M , McCann S M
Published: May 2006

It has previously been shown that alcohol can suppress reproduction in humans, monkeys, and small rodents by inhibiting release of luteinizing hormone (LH). The principal action is via suppression of the release of LH-releasing hormone (LHRH) both in vivo and in vitro. The present experiments were designed to determine the mechanism by which alcohol inhibits LHRH release. Previous research has indicated that the release of LHRH is controlled by nitric oxide (NO). The proposed pathway is via norepinephrine-induced release of NO from NOergic neurons, which then activates LHRH release. In the present experiments, we further evaluated the details of this mechanism in male rats by incubating medial basal hypothalamic (MBH) explants in vitro and examining the release of NO, prostaglandin E2 (PGE2), conversion of arachidonic acid to prostanoids, and production of cGMP. The results have provided further support for our theory of LHRH control. Norepinephrine increased the release of NO as measured by conversion of [14C]arginine to [14C]citrulline, and this increase was blocked by the alpha 1 receptor blocker prazosin. Furthermore, the release of LHRH induced by nitroprusside (NP), a donor of NO, is related to the activation of soluble guanylate cyclase by NO since NP increased cGMP release from MBHs and cGMP also released LHRH. Ethanol had no effect on the production of NO by MBH explants or the increased release of NO induced by norepinephrine. Therefore, it does not act at that step in the pathway. Ethanol also failed to affect the increase in cGMP induced by NP. On the other hand, as might be expected from previous experiments indicating that LHRH release was brought about by PGE2, NP increased the conversion of [14C]arachidonic acid to its metabolites, particularly PGE2. Ethanol completely blocked the release of LHRH induced by NP and the increase in PGE2 induced by NP. Therefore, the results support the theory that norepinephrine acts to stimulate NO release from NOergic neurons. This NO diffuses to the LHRH terminals where it activates guanylate cyclase, leading to an increase in cGMP. At the same time, it also activates cyclooxygenase. The increase in cGMP increases intracellular free calcium, activating phospholipase A2 to provide arachidonic acid, the substrate for conversion by the activated cyclooxygenase to PGE2, which then activates the release of LHRH. Since alcohol inhibits the conversion of labeled arachidonic acid to PGE2, it must act either directly to inhibit cyclooxygenase or perhaps it may act by blocking the increase in intracellular free calcium induced by cGMP, which is crucial for activation of of both phospholipase A2 and cyclooxygenase.
Mos overexpression in Swiss 3T3 cells induces meiotic-like alterations of the mitotic spindle.
Authors: Fukasawa K , Vande Woude G F
Published: May 2006

High levels of mos protooncogene product are expressed during oocyte meiotic maturation and Mos has been implicated in formation of the spindle and spindle pole. Here, we show that in Swiss 3T3 cells with 4N DNA content, high levels of Mos lead to the production of binucleated cells. The Swiss 3T3 cells in mitosis, before binucleation occurs, are anastral and the spindle poles are juxtaposed to the cell membrane. These phenotypes may be related to the meiotic process of attachment of the spindle pole to the oocyte membrane during polar body formation. The production of binucleated somatic cells could result from attachment of the altered mitotic spindle pole to the cell membrane that interferes with cytokinesis but not karyokinesis. This can explain at least one form of genetic instability that leads to altered DNA content in tumor cells.
Identification of a DNA transformation gene required for com101A+ expression and supertransformer phenotype in Haemophilus influenzae.
Authors: Zulty J J , Barcak G J
Published: May 2006

DNA sequencing, RNA mapping, and protein expression experiments revealed the presence of a gene, tfoX+, encoding a 24.9-kDa polypeptide, that is transcribed divergently from a common promoter region with the Haemophilus influenzae rec-1+ gene. H. influenzae strains mutant for tfoX failed to bind transforming DNA and were transformation deficient. Primer extension experiments utilizing in vivo total RNA from precompetent and competent H. influenzae cells demonstrated that transcription of tfoX+ increased immediately upon competence induction, suggesting that tfoX+ is an early competence gene. Similar experiments showed that the expression of the late competence-specific gene, com101A+, was tfoX+ dependent. Moreover, expression of plasmid-borne tfoX+ in H. influenzae resulted in constitutive competence. The addition of cyclic adenosine monophosphate (cAMP) to strains carrying a tfoX::lacZ operon fusion resulted in an immediate increase in beta-galactosidase activity that correlated with an increase in genetic transformability. Collectively, our results suggest that TfoX may play a key role in the development of genetic competence by regulating the expression of late competence-specific genes.
Molecular and cytotoxic effects of camptothecin, a topoisomerase I inhibitor, on trypanosomes and Leishmania.
Authors: Bodley A L , Shapiro T A
Published: May 2006

Parasites pose a threat to the health and lives of many millions of human beings. Among the pathogenic protozoa, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani are hemoflagellates that cause particularly serious diseases (sleeping sickness, Chagas disease, and leishmaniasis, respectively). The drugs currently available to treat these infections are limited by marginal efficacy, severe toxicity, and spreading drug resistance. Camptothecin is an established antitumor drug and a well-characterized inhibitor of eukaryotic DNA topoisomerase I. When trypanosomes or leishmania are treated with camptothecin and then lysed with SDS, both nuclear and mitochondrial DNA are cleaved and covalently linked to protein. This is consistent with the existence of drug-sensitive topoisomerase I activity in both compartments. Camptothecin also inhibits the incorporation of [3H]thymidine in these parasites. These molecular effects are cytotoxic to cells in vitro, with EC50 values for T. brucei, T. cruzi, and L. donovani, of 1.5, 1.6, and 3.2 microM, respectively. For these parasites, camptothecin is an important lead for much-needed new chemotherapy, as well as a valuable tool for studying topoisomerase I activity.
Structural interpretation of the mutations in the beta-cardiac myosin that have been implicated in familial hypertrophic cardiomyopathy.
Authors: Rayment I , Holden H M , Sellers J R , Fananapazir L , Epstein N D
Published: May 2006

In 10-30% of hypertrophic cardiomyopathy kindreds, the disease is caused by > 29 missense mutations in the cardiac beta-myosin heavy chain (MYH7) gene. The amino acid sequence similarity between chicken skeletal muscle and human beta-cardiac myosin and the three-dimensional structure of the chicken skeletal muscle myosin head have provided the opportunity to examine the structural consequences of these naturally occurring mutations in human beta-cardiac myosin. This study demonstrates that the mutations are related to distinct structural and functional domains. Twenty-four are clustered around four specific locations in the myosin head that are (i) associated with the actin binding interface, (ii) around the nucleotide binding site, (iii) adjacent to the region that connects the two reactive cysteine residues, and (iv) in close proximity to the interface of the heavy chain with the essential light chain. The remaining five mutations are in the myosin rod. The locations of these mutations provide insight into the way they impair the functioning of this molecular motor and also into the mechanism of energy transduction.
Receptor usage and differential downregulation of CD46 by measles virus wild-type and vaccine strains.
Authors: Schneider-Schaulies J , Schnorr J J , Brinckmann U , Dunster L M , Baczko K , Liebert U G , Schneider-Schaulies S , ter Meulen V
Published: May 2006

Recently, two cell surface molecules, CD46 and moesin, have been found to be functionally associated with measles virus (MV) infectivity of cells. We investigated the receptor usage of MV wild-type, subacute sclerosing panencephalitis, and vaccine strains and their effect on the down-regulation of CD46 after infection. We found that the infection of human cell lines with all 19 MV strains tested was inhibitable with antibodies against CD46. In contrast, not all strains of MV led to the downregulation of CD46 following infection. The group of CD46 non-downregulating strains comprised four lymphotropic wild-type isolates designated AB, DF, DL, and WTF. Since the downregulation of CD46 is caused by interaction with newly synthesized MV hemagglutinin (MV-H), we tested the capability of recombinant MV-H proteins to downregulate CD46. Recombinant MV-H proteins of MV strains Edmonston, Halle, and CM led to the down-regulation of CD46, whereas those of DL and WTF did not. This observed differential downregulation by different MV strains has profound consequences, since lack of CD46 on the cell surface leads to susceptibility of cells to complement lysis. These results suggest that lymphotropic wild-type strains of MV which do not downregulate CD46 may have an advantage for replication in vivo. The relatively weak immune response against attenuated vaccine strains of MV compared with wild-type strains might be related to this phenomenon.
D-serine, an endogenous synaptic modulator: localization to astrocytes and glutamate-stimulated release.
Authors: Schell M J , Molliver M E , Snyder S H
Published: May 2006

Using an antibody highly specific for D-serine conjugated to glutaraldehyde, we have localized endogenous D-serine in rat brain. Highest levels of D-serine immunoreactivity occur in the gray matter of the cerebral cortex, hippocampus, anterior olfactory nucleus, olfactory tubercle, and amygdala. Localizations of D-serine immunoreactivity correlate closely with those of D-serine binding to the glycine modulatory site of the N-methyl-D-aspartate (NMDA) receptor as visualized by autoradiography and are inversely correlated to the presence of D-amino acid oxidase. D-Serine is enriched in process-bearing glial cells in neuropil with the morphology of protoplasmic astrocytes. In glial cultures of rat cerebral cortex, D-serine is enriched in type 2 astrocytes. The release of D-serine from these cultures is stimulated by agonists of non-NMDA glutamate receptors, suggesting a mechanism by which astrocyte-derived D-serine could modulate neurotransmission. D-Serine appears to be the endogenous ligand for the glycine site of NMDA receptors.
Founder-effect speciation theory: failure of experimental corroboration.
Authors: Moya A , Galiana A , Ayala F J
Published: May 2006

The theory of founder-effect speciation proposes that colonization by very few individuals of an empty habitat favors rapid genetic changes and the evolution of a new species. We report here the results obtained in a 10-year-long and large-scale experiment with Drosophila pseudoobscura designed to test the theory. In our experimental protocol, populations are established with variable numbers of very few individuals and allowed to expand greatly for several generations until conditions of severe competition for resources are reached and the population crashes. A few random survivors are then taken to start a new population expansion and thus initiate a new cycle of founding events, population flushes, and crashes. Our results provide no support for the theories proposing that new species are very likely to appear as by-products of founder events.
Expression cloning of dSR-CI, a class C macrophage-specific scavenger receptor from Drosophila melanogaster.
Authors: Pearson A , Lux A , Krieger M
Published: May 2006

Mammalian class A macrophage-specific scavenger receptors (SR-A) exhibit unusually broad binding specificity for a wide variety of polyanionic ligands. The properties of these receptors suggest that they may be involved in atherosclerosis and host defense. We have previously observed a similar receptor activity in Drosophila melanogaster embryonic macrophages and in the Drosophila macrophage-like Schneider L2 cell line. Expression cloning was used to isolate from L2 cells a cDNA that encodes a third class (class C) of scavenger receptor, Drosophila SR-CI (dSR-CI). dSR-CI expression was restricted to macrophages/hemocytes during embryonic development. When expressed in mammalian cells, dSR-CI exhibited high affinity and saturable binding of 125I-labeled acetylated low density lipoprotein and mediated its chloroquine-dependent, presumably lysosomal, degradation. Although the broad polyanionic ligand-binding specificity of dSR-CI was similar to that of SR-A, their predicted protein sequences are not similar. dSR-CI is a 609-residue type I integral membrane protein containing several well-known sequence motifs, including two complement control protein (CCP) domains and somatomedin B, MAM, and mucin-like domains. Macrophage scavenger receptors apparently mediate important, well-conserved functions and may be pattern-recognition receptors that arose early in the evolution of host-defense mechanisms. Genetic and physiologic analysis of dSR-CI function in Drosophila should provide further insights into the roles played by scavenger receptors in host defense and development.
IκBε provides negative feedback to control NF-κB oscillations, signaling dynamics, and inflammatory gene expression
Authors: Kearns Jeffrey D. , Basak Soumen , Werner Shannon L. , Huang Christine S. , Hoffmann Alexander
Published: May 2006

NF-κB signaling is known to be critically regulated by the NF-κB–inducible inhibitor protein IκBα. The resulting negative feedback has been shown to produce a propensity for oscillations in NF-κB activity. We report integrated experimental and computational studies that demonstrate that another IκB isoform, IκBε, also provides negative feedback on NF-κB activity, but with distinct functional consequences. Upon stimulation, NF-κB–induced transcription of IκBε is delayed, relative to that of IκBα, rendering the two negative feedback loops to be in antiphase. As a result, IκBε has a role in dampening IκBα-mediated oscillations during long-lasting NF-κB activity. Furthermore, we demonstrate the requirement of both of these distinct negative feedback regulators for the termination of NF-κB activity and NF-κB–mediated gene expression in response to transient stimulation. Our findings extend the capabilities of a computational model of IκB–NF-κB signaling and reveal a novel regulatory module of two antiphase negative feedback loops that allows for the fine-tuning of the dynamics of a mammalian signaling pathway.
Block of Muscle Nicotinic Receptors by Choline Suggests that the Activation and Desensitization Gates Act as Distinct Molecular Entities
Authors: Purohit Yamini , Grosman Claudio
Published: May 2006

Ion channel block in muscle acetylcholine nicotinic receptors (AChRs) is an extensively reported phenomenon. Yet, the mechanisms underlying the interruption of ion flow or the interaction of the blocker with the channel's gates remain incompletely characterized. In this paper, we studied fast channel block by choline, a quaternary-ammonium cation that is also an endogenous weak agonist of this receptor, and a valuable tool in structure–function studies. Analysis of the single-channel current amplitude as a function of both choline concentration and voltage revealed that extracellular choline binds to the open-channel pore with millimolar apparent affinity (KB ≅ 12 mM in the presence of ∼155 mM monovalent and 3.5 mM divalent, inorganic cations), and that it permeates the channel faster than acetylcholine. This, together with its relatively small size (∼5.5 Å along its longest axis), suggests that the pore-blocking choline binding site is the selectivity filter itself, and that current blockages simply reflect the longer-lived sojourns of choline at this site. Kinetic analysis of single-channel traces indicated that increasing occupancy of the pore-blocking site by choline (as judged from the reduction of the single-channel current amplitude) is accompanied by the lengthening of (apparent) open interval durations. Consideration of a number of possible mechanisms firmly suggests that this prolongation results from the local effect of choline interfering with the operation of the activation gate (closure of blocked receptors is slower than that of unblocked receptors by a factor of ∼13), whereas closure of the desensitization gate remains unaffected. Thus, we suggest that these two gates act as distinct molecular entities. Also, the detailed understanding gained here on how choline distorts the observed open-time durations can be used to compensate for this artifact during activation assays. This correction is necessary if we are to understand how choline binds to and gates the AChR.
Calcium Release Domains in Mammalian Skeletal Muscle Studied with Two-photon Imaging and Spot Detection Techniques
Authors: Gómez José , Ñeco Patricia , DiFranco Marino , Vergara Julio L.
Published: May 2006

The spatiotemporal characteristics of the Ca2+ release process in mouse skeletal muscle were investigated in enzymatically dissociated fibers from flexor digitorum brevis (FDB) muscles, using a custom-made two-photon microscope with laser scanning imaging (TPLSM) and spot detection capabilities. A two-microelectrode configuration was used to electrically stimulate the muscle fibers, to record action potentials (APs), and to control their myoplasmic composition. We used 125 μM of the low-affinity Ca2+ indicator Oregon green 488 BAPTA-5N (OGB-5N), and 5 or 10 mM of the Ca2+ chelator EGTA (pCa 7) in order to arrest fiber contraction and to constrain changes in the [Ca2+] close to the release sites. Image and spot data showed that the resting distribution of OGB-5N fluorescence was homogeneous along the fiber, except for narrow peaks (∼23% above the bulk fluorescence) centered at the Z-lines, as evidenced by their nonoverlapping localization with respect to di-8-ANEPPS staining of the transverse tubules (T-tubules). Using spot detection, localized Ca2+ transients evoked by AP stimulation were recorded from adjacent longitudinal positions 100 nm apart. The largest and fastest ΔF/F transients were detected at sites flanking the Z-lines and colocalized with T-tubules; the smallest and slowest were detected at the M-line, whereas transients at the Z-line showed intermediate features. Three-dimensional reconstructions demonstrate the creation of two AP-evoked Ca2+ release domains per sarcomere, which flank the Z-line and colocalize with T-tubules. In the presence of 10 mM intracellular EGTA, these domains are formed in ∼1.4 ms and dissipate within ∼4 ms, after the peak of the AP. Their full-width at half-maximum (FWHM), measured at the time that Ca2+ transients peaked at T-tubule locations, was 0.62 μm, similar to the 0.61 μm measured for di-8-ANEPPS profiles. Both these values exceed the limit of resolution of the optical system, but their similarity suggests that at high [EGTA] the Ca2+ domains in adult mammalian muscle fibers are confined to Ca2+ release sites located at the junctional sarcoplasmic reticulum (SR).
Costertonia aggregata gen. nov., sp. nov., a mesophilic marine bacterium of the family Flavobacteriaceae, isolated from a mature biofilm
Authors: Kwon Kae Kyoung , Lee Yoo Kyung , Lee Hong Kum
Published: May 2006

A marine bacterium, strain KOPRI 13342T, was isolated from a mature marine biofilm, including various marine algae, covering a rock-bed of the East Sea, Korea (also known as the Sea of Japan). Colonies of the isolate were orange-coloured on marine agar 2216. The isolate showed relatively high 16S rRNA gene sequence similarities to members of the genera Maribacter (91.2–92.4 % similarity), Zobellia (90.7–91.5 %) and Muricauda (90.7–91.4 %). Phylogenetic analysis based on the nearly complete 16S rRNA gene sequence revealed that the isolate formed a phyletic lineage with members of the genus Muricauda. Cells were aerobic, motile, Gram-negative rods and they produced non-diffusible carotenoid pigments. Optimal growth was observed at pH 7.5–8.0 and 26–32 °C and required the presence of 3 % (w/v) sea salt. The strain required Ca2+ and K+ ions in addition to NaCl for growth. The dominant fatty acids were i-15 : 0, i-15 : 1ω10, 15 : 0 and 16 : 1ω9. The major respiratory quinone was MK-6. The DNA G+C content was 35.8 mol%. On the basis of this polyphasic taxonomic evidence, strain KOPRI 13342T should be classified as a representative of a novel species in a new genus in the family Flavobacteriaceae; the name Costertonia aggregata gen. nov., sp. nov. is proposed. The type strain of Costertonia aggregata is KOPRI 13342T (=KCCM 42265T=JCM 13411T).
‘Building a new normality’: mothers’ experiences of caring for a child with acute lymphoblastic leukaemia
Authors: Earle E. A. , Clarke S. A. , Eiser C. , Sheppard L.
Published: May 2006

Background  Treatment of childhood cancer occurs over a 2‐ to 3‐year period, with initial intense phases of chemotherapy followed by less severe treatment periods. From first diagnosis, families are encouraged by healthcare professionals (following government guidelines) to try to maintain a normal life. The aim of this paper is to contribute to our understanding of how ‘normal’ family life is compromised from the perspective of the families themselves during this period of extreme stress and concern for the health and well‐being of their child.

Methods  This study was longitudinal and involved a cross section of 32 mothers of children recently diagnosed with acute lymphoblastic leukaemia (ALL) currently participating in the Medical Research Council ALL‐97 randomized control trial. Mothers were interviewed at three time points (3–4 months post diagnosis, 15 and 27 months) using a semi‐structured format with open‐ended questions. A qualitative methodology was employed to analyse interview data using Thematic analysis.

Results  Mothers reported understanding the importance of achieving normal life, but described how this was difficult to realize. At first interview, mothers were optimistic that they could achieve the ‘normal life’ as advised by healthcare workers. At 12 and 24 months, although all mothers reported that life was not back to normal, there were differences in how they perceived this lack of normality. Whereas some families experienced frustration and disappointment, others had adjusted and managed to accept the new order.

Conclusions  Families felt encouraged on diagnosis to be told that despite the severity of the disease and treatment regime, a normal life was possible and should be pursued. Our findings indicate that over time, more concrete information is needed to guide parents through the treatment process in order to help them achieve this.
FOXP3 Identifies Regulatory CD25brightCD4+ T Cells in Rheumatic Joints
Authors: Cao D. , Börjesson O. , Larsson P. , Rudin A. , Gunnarsson I. , Klareskog L. , Malmström V. , Trollmo C.
Published: May 2006

Regulatory T cells have recently been implicated in a number of human diseases, including rheumatoid arthritis. To investigate whether the presence of CD25+CD4+ regulatory T cells is a general finding in arthritic joints, synovial fluid of patients with different rheumatic diseases such as undifferentiated arthritides, systemic rheumatic diseases and reactive arthritis were investigated for the presence of such cells. In 95% of the patients, a higher frequency of CD25brightCD4+ T cells was found in synovial fluid as compared with peripheral blood. Both in vitro suppression experiments and FOXP3 mRNA analysis confirmed these cells to be natural regulatory T cells. Together with our previous data, we conclude that arthritic joints, irrespective of precise diagnosis and disease duration, are enriched with natural regulatory T cells. These results suggest that suppressor cells migrate to and/or multiply at the sites of inflammation as part of the immune responses' effort to combat injurious inflammation.
A novel strategy for preventing PERV transmission to human cells by remodeling the viral envelope glycoprotein
Authors: Miyagawa Shuji , Nakatsu Shino , Hazama Kenji , Nakagawa Takatoshi , Kondo Akihiro , Matsunami Katsuyoshi , Yamamoto Aki , Yamada Junko , Miyazawa Takayuki , Shirakura Ryota
Published: May 2006

Miyagawa S, Nakatsu S, Hazama K, Nakagawa T, Kondo A, Matsunami K, Yamamoto A, Yamada J, Miyazawa T, Shirakura R. A novel strategy for preventing PERV transmission to human cells by remodeling the viral envelope glycoprotein. Xenotransplantation 2006; 13: 258–263. © Blackwell Munksgaard, 2006

Abstract:  Background:  Porcine endogenous retrovirus (PERV) released from pig cells is a main problem associated with clinical xenotransplantation. In a previous study, we demonstrated that the high mannose type of N‐glycan of the envelope glycoprotein is closely related to PERV infectivity with respect to human cells. In this study, we addressed the effects of reducing the high mannose type of N‐glycan on PERV infectivity.

Methods:  Pig endothelial cells (PEC) were transduced with the LacZ gene by a pseudotype infection to produce PEC(Z). The PEC(Z)s were then further infected with PERV subtype B (PERV‐B) to produce PEC(Z)/PB. The PEC(Z)/PBs were next transfected with the α 1,2 mannosidase Ib (Man Ib), N‐acetylglucosaminyltransferase I (GnT‐I) or α‐mannosidase II (Man II) gene in order to reduce the levels of high mannose type of N‐glycan. HEK293 cells were inoculated with the PERV in each of the culture supernatants. The inoculated cells were histochemically stained and the LacZ‐positive cells were counted.

Results:  In experiment I, PERV transmission from the PEC(Z)/PB with GnT‐I or Man II to HEK 293 cells was significantly reduced in comparison with control PEC(Z)/PB, while the PEC(Z)/PB with Man Ib was not. However, in experiment II, PERV transmission from the PEC(Z)/PB with ManIb to HEK 293 cells was also significantly reduced in comparison with control PEC(Z)/PB.

Conclusion:  The transfection of these genes to pig cells is effective in reducing the susceptibility of human cells to PERV infection. The results suggest that this represents a potentially useful strategy for further decreasing the likelihood of PERV infections.
Evidence‐based decision making in transfusion medicine
Authors: Heddle N. M.
Published: May 2006

Some of the basic principles behind evidence‐based medicine have been known for many years; however, the concept and approach to integrating evidenced‐based decision making into clinical practice on a day‐to‐day basis has only evolved over the past 15 years. This paper focuses on five important steps in evidence‐based decision making: (1) the importance of a well‐defined question; (2) ways to effectively search the scientific literature; (3) the process of critical appraisal for an article about therapy; (4) the role of clinical expertise, patient values, clinical circumstances and society's expectations in the decision‐making process; and (5) continuous quality improvement of the process.