bluuebunny/crossref_metadata_2025 huggingface dataset:
Allosteric changes in the cAMP receptor protein of Escherichia coli: hinge reorientation.
Authors: Kim J , Adhya S , Garges S
Published: May 2006

The cAMP receptor protein (CRP) of Escherichia coli is a dimer of a two-domain subunit. It requires binding of cAMP for a conformational change in order to function as a site-specific DNA-binding protein that regulates gene activity. The hinge region connecting the cAMP-binding domain to the DNA-binding domain is involved in the cAMP-induced allosteric change. We studied the structural changes in CRP that are required for gene regulation by making a large number of single and double amino acid substitutions at four different positions in or near the hinge. To achieve cAMP-independent transcription by CRP, amino acid residues 138 (located within the hinge region) and 141 (located in the D alpha-helix adjacent to the hinge) must be polar. This need for polar residues at positions 138 and 141 suggests an interaction that causes the C and D alpha-helices to come together. As a consequence, the F alpha-helix is released from the D alpha-helix and can interact with DNA. At position 144 in the D alpha-helix and within interacting distances of the F alpha-helix, replacement of alanine by an amino acid with a larger side chain, regardless of its nature, allows cAMP independence. This result indicates that pushing against the F alpha-helix may be a way of making the helix available for DNA binding. We believe that the cAMP-induced allosteric change involves similar hinge reorientation to adjust the C and D alpha-helices, allowing outward movement of the F alpha-helix.
CD22-mediated stimulation of T cells regulates T-cell receptor/CD3-induced signaling.
Authors: Aruffo A , Kanner S B , Sgroi D , Ledbetter J A , Stamenkovic I
Published: May 2006

Interaction between B lymphocytes and other cell types is mediated in part by the B-cell adhesion molecule CD22. Recent work has suggested one of the T-cell ligands of B cells to be CD45RO, an isoform of the receptor-linked phosphotyrosine phosphatase CD45. Here we demonstrate direct interaction between CD22 and several isoforms of CD45, including CD45RO, and propose that the interaction may participate in regulation of lymphocyte signaling. Cross-linking of CD3 and CD22 T-cell ligands with anti-CD3 antibody and soluble CD22 is shown to block anti-CD3-induced intracellular calcium increase and to inhibit tyrosine phosphorylation of phospholipase C gamma 1. These effects are consistent with those observed upon coligation of CD3 and CD45 with antibody, providing support to the possibility that ligand-mediated stimulation of CD45 may result in modulation of substrate phosphorylation and lymphocyte activation.
Determination of the orientation of a DNA binding motif in a protein-DNA complex by photocrosslinking.
Authors: Pendergrast P S , Chen Y , Ebright Y W , Ebright R H
Published: May 2006

We have developed a straightforward biochemical method to determine the orientation of the DNA binding motif of a sequence-specific DNA binding protein relative to the DNA site in the protein-DNA complex. The method involves incorporation of a photoactivatable crosslinking agent at a single site within the DNA binding motif of the sequence-specific DNA binding protein, formation of the derivatized protein-DNA complex, UV-irradiation of the derivatized protein-DNA complex, and determination of the nucleotide(s) at which crosslinking occurs. We have applied the method to catabolite gene activator protein (CAP). We have constructed and analyzed two derivatives of CAP: one having a phenyl azide photoactivatable crosslinking agent at amino acid 2 of the helix-turn-helix motif of CAP, and one having a phenyl azide photoactivatable crosslinking agent at amino acid 10 of the helix-turn-helix motif of CAP. The results indicate that amino acid 2 of the helix-turn-helix motif is close to the top-strand nucleotides of base pairs 3 and 4 of the DNA half site in the CAP-DNA complex, and that amino acid 10 of the helix-turn-helix motif is close to the bottom-strand nucleotide of base pair 10 of the DNA half site in the CAP-DNA complex. The results define unambiguously the orientation of the helix-turn-helix motif relative to the DNA half site in the CAP-DNA complex. Comparison of the results to the crystallographic structure of the CAP-DNA complex [Schultz, S., Shields, S. & Steitz, T. (1991) Science 253, 1001-1007] indicates that the method provides accurate, high-resolution proximity and orientation information.
Chromosomal organization of the heavy chain variable region gene segments comprising the human fetal antibody repertoire.
Authors: Willems van Dijk K , Milner L A , Sasso E H , Milner E C
Published: May 2006

The adult repertoire of antibody specificities is acquired in a developmentally programmed fashion that, in mouse and man, parallels the ordered rearrangement of a limited number of germ-line heavy chain variable region (VH) gene segments during development. It has been hypothesized that this developmental bias is a consequence of gene organization. In the mouse, rearrangement of VH gene segments proximal to the heavy chain joining region (JH) locus precedes rearrangement of genes located more distal to the JH locus. Similarly, in man, two VH elements located proximal to JH are expressed during fetal development. To test further this hypothesis in man, we have determined in a single individual the positions of an additional eight distinct VH elements known to comprise a significant fraction of the human developmental repertoire. These developmentally expressed VH elements were found to be dispersed over a region of 890 kilobases of the VH locus and were interspersed with other VH elements that are not known to be developmentally expressed. Thus, the ordered developmental expression of VH gene segments in man must involve mechanisms beyond physical proximity to the JH locus. Further, these results support the notion that fetal expression of VH gene segments is a regulated process and suggest that this regulation is important in the acquisition of immunocompetence.
A synthetic peptide inhibitor of human immunodeficiency virus replication: correlation between solution structure and viral inhibition.
Authors: Wild C , Oas T , McDanal C , Bolognesi D , Matthews T
Published: May 2006

A peptide designated DP-107 was synthesized containing amino acid residues 558-595 of the envelope glycoprotein gp160 of human immunodeficiency virus type 1 strain LAI (HIV-1LAI). Algorithms for secondary structure have predicted that this region of the envelope transmembrane protein should form an extended alpha-helix. Consistent with this prediction, analysis by circular dichroism (CD) indicated that, under physiological conditions, DP-107 is approximately 85% helical. The high degree of stable secondary structure in a synthetic peptide of this size suggests self-association typical of a coiled coil or leucine zipper. In biological assays, the peptide efficiently blocked virus-mediated cell-cell fusion processes as well as infection of peripheral blood mononuclear cells by both prototypic and primary isolates of HIV-1. A single amino acid substitution in the peptide greatly destabilized its solution structure as measured by CD and abrogated its antiviral activity. An analogue containing a terminal cysteine was oxidized to form a dimer, and this modification lowered the dose required for antiviral effect from 5 to about 1 microgram/ml. These results suggest that both oligomerization and ordered structure are necessary for biological activity. They provide insights also into the role of this region in HIV infection and the potential for development of a new class of antiviral agents.
Distribution of mRNA of a Na(+)-independent neutral amino acid transporter cloned from rat kidney and its expression in mammalian tissues and Xenopus laevis oocytes.
Authors: Yan N , Mosckovitz R , Udenfriend S , Tate S S
Published: May 2006

The Na(+)-independent neutral amino acid transporter (NAA-Tr) that we had previously cloned from rat kidney has been investigated with respect to its distribution in mammalian tissues and cells. By Northern blot analysis and RNase protection assay, a 2.4-kilobase (kb) mRNA in rat intestine was found to be identical to that in rat kidney. Of the other rat tissues examined, only brain and heart were found to contain mRNAs related to kidney NAA-Tr by Northern assay. However, these were larger (approximately 5 and approximately 7 kb). Mouse and rabbit kidney also contain mRNAs of 2.4 kb that exhibited a high degree of homology with rat kidney NAA-Tr. Of the several cultured cells investigated that demonstrated considerable Na(+)-independent neutral amino acid transport activity, only human colon carcinoma (Caco) cells were positive by Northern assay. The failure to detect NAA-Tr mRNA in many cells and tissues that carry out Na(+)-independent transport indicates that unrelated transporters must also exist. Cells and tissues that were negative with respect to rat kidney NAA-Tr as well as those that were positive transported leucine and tryptophan equally well. However, when mRNA from the same cells and tissues was expressed in oocytes, in all cases tryptophan was transported far less efficiently than leucine. This defect in tryptophan transport is apparently due to aberrant expression of neutral amino acid transporters in general in Xenopus oocytes.
Productive nonlytic human immunodeficiency virus type 1 replication in a newly established human leukemia cell line.
Authors: Banerjee R , Bekesi J G , Tarcsafalvi A , Sperber K , Deak G , Choi H S , Paronetto F , Holland J F , Acs G
Published: May 2006

We have isolated a lymphoid cell line, MDS, from the pleural exudate of a patient with chronic myelomonocytic leukemia. The cells are biphenotypic, containing various T-cell and myeloid markers, and are surface negative for CD4 and CD8 but have low CD4 mRNA. The cells grow in suspension with a doubling time of 15 hr, have been karyotyped as trisomy 21, are negative for human immunodeficiency virus type 1 (HIV-1), and are tumorigenic in the nude mouse. We have isolated two stable HIV-1-producing cell lines, MDS-T, by transfecting MDS cells with pHXBc2, and MDS-I, by infecting MDS cells with HIV-1IIIB. In 24 hr, 1 x 10(5) MDS-T or MDS-I cells produce 46 ng of p24 per ml and reverse transcriptase that is capable of incorporating 0.2 pmol of [32P]TTP into oligo(dT).poly(A). Ultrastructural studies showed numerous mature viral particles in MDS-T and MDS-I cells that are capable of infecting T cells. HIV-1 infection could be inhibited by 25% in the MDS cells with the anti-CD4 antibody Leu 3a. For over a year MDS-T and MDS-I cells have been producing high concentrations of HIV-1 in culture. A subclone derived from the MDS cells behaves like the parent cells when transfected or infected with HIV-1. In contrast to other T-cell lines, neither phorbol 12-myristate 13-acetate nor tumor necrosis factor alpha stimulated the replication of HIV-1, whereas bromoadenosine 3',5'-cyclic monophosphate or interferon alpha caused 50% and 80% inhibition of reverse transcriptase production, respectively. These chronically infected T-cell lines are a useful model system to study the effect of anti-HIV agents and cellular factors required for HIV-1 replication.
Kindred S thyroid hormone receptor is an active and constitutive silencer and a repressor for thyroid hormone and retinoic acid responses.
Authors: Baniahmad A , Tsai S Y , O'Malley B W , Tsai M J
Published: May 2006

Mutations in the gene encoding the human thyroid hormone receptor beta (hTR beta) have been associated with generalized thyroid hormone resistance (GTHR). However, the molecular basis by which the receptor mutants cause the clinical symptoms is largely unknown. We show here that the beta form of the human receptor possesses, in addition to hormone-dependent activation, the ability to repress basal-level activity of a target promoter. This silencing function is localized in the carboxyl-terminal part of the receptor and can be transferred to a heterologous DNA binding domain. This mode of silencing is therefore distinct from inhibition by competition with activator proteins on DNA. We show that two receptor mutants isolated from patients with GTHR are impaired in transcriptional activation but fully retain the silencing function, which enforces dominant negative regulation by the receptor. Interestingly, the kindred S receptor (hTR delta 332) acts as a constitutive repressor with a strong silencing ability similar to that of the v-erbA oncogene product. We also provide evidence for distinct transcriptional regulatory properties of both proteins. Finally, we show that both thyroid hormone- and retinoic acid-responsive genes are potentially repressed to generate the clinical manifestations of the GTHR syndrome. Our findings suggest that silencing plays an important role in the phenotypic expression of the symptoms in patients with GTHR.
Preferential repair of cyclobutane pyrimidine dimers in the transcribed strand of a gene in yeast chromosomes and plasmids is dependent on transcription.
Authors: Sweder K S , Hanawalt P C
Published: May 2006

While preferential repair of the transcribed strands within active genes has been demonstrated in organisms as diverse as humans and Escherichia coli, it has not previously been shown to occur in chromosomal genes in the yeast Saccharomyces cerevisiae. We found that repair of cyclobutane pyrimidine dimers in the transcribed strand of the expressed RPB2 gene in the chromosome of a repair-proficient strain is much more rapid than that in the nontranscribed strand. Furthermore, a copy of the RPB2 gene borne on a centromeric ARS1 plasmid showed the same strand bias in repair. To investigate the relation of this strand bias to transcription, we studied repair in a yeast strain with the temperature-sensitive mutation, rpb1-1, in the largest subunit of RNA polymerase II. When exponentially growing rpb1-1 cells are shifted to the nonpermissive temperature, they rapidly cease mRNA synthesis. At the permissive temperature, both rpb1-1 and the wild-type, parental cells exhibited rapid, proficient repair in the transcribed strand of chromosomal and plasmid-borne copies of the RPB2 gene. At the nonpermissive temperature, the rate of repair in the transcribed strand in rpb1-1 cells was reduced to that in the nontranscribed strand. These findings establish the dependence of strand bias in repair on transcription by RNA polymerase II in the chromosomes and in plasmids, and they validate the use of plasmids for analysis of the relation of repair to transcription in yeast.
Mechanism of interferon action: autoregulation of RNA-dependent P1/eIF-2 alpha protein kinase (PKR) expression in transfected mammalian cells.
Authors: Thomis D C , Samuel C E
Published: May 2006

The expression of a molecular cDNA clone (P1 KIN) of the human RNA-dependent P1/eIF-2 alpha protein kinase (PKR) was examined in transfected monkey cells and in cell-free protein-synthesizing systems. Expression of the wild-type (wt) P1 KIN cDNA, which encodes an active protein kinase, was compared with that of the phosphotransfer catalytic domain II Lys-296-->Arg (K296R) mutant cDNA, which does not encode an active kinase. wt and K296R mutant P1 mRNAs prepared by transcription in vitro with T7 RNA polymerase programmed the cell-free synthesis of P1 ribosome-associated protein with comparable efficiency in the rabbit reticulocyte system. The K296R mutant P1 protein was also efficiently synthesized in vivo in transfected COS monkey cells. However, synthesis of the wt P1 protein was reduced about 30-fold in transfected COS cells as compared with the K296R mutant P1 protein. Cotransfection of wt P1 KIN cDNA with either K296R mutant P1 KIN cDNA or reovirus S4 cDNA greatly reduced the synthesis of K296R mutant P1 protein and reovirus sigma 3 protein, respectively. Although the wt and K296R mutant P1 KIN plasmid expression vectors replicated with comparable efficiencies in COS cells, the steady-state amount of P1 mRNA was about 3-fold less in COS cells transfected with the wt as compared with the K296R mutant P1 KIN cDNA. These results suggest that RNA-dependent P1 protein kinase expression is autoregulated in vivo in transfected mammalian cells primarily at the level of translation by a mechanism that is likely dependent upon catalytically active P1 kinase.
Gene therapy via primary myoblasts: long-term expression of factor IX protein following transplantation in vivo.
Authors: Dai Y , Roman M , Naviaux R K , Verma I M
Published: May 2006

We have explored the use of primary myoblasts as a somatic tissue for gene therapy of acquired and inherited diseases where systemic delivery of a gene product may have therapeutic effects. Mouse primary myoblasts were infected with replication-defective retroviruses expressing canine factor IX cDNA under the control of a mouse muscle creatine kinase enhancer and human cytomegalovirus promoter. The infected myoblasts were injected into the hindlegs of recipient mice and levels of secreted factor IX protein were monitored in the plasma. We report sustained expression of factor IX protein for over 6 months without any apparent adverse effect on the recipient mice.
Human platelet-derived growth factor A chain is transcriptionally repressed by the Wilms tumor suppressor WT1.
Authors: Gashler A L , Bonthron D T , Madden S L , Rauscher F J , Collins T , Sukhatme V P
Published: May 2006

Wilms tumor, an embryonic kidney malignancy, accounts for approximately 6% of all pediatric neoplasms. A gene implicated in the genesis of this tumor, the Wilms tumor suppressor gene (WT1), encodes a zinc-finger DNA-binding protein (WT1) that functions as a transcriptional repressor. In certain Wilms tumors, the platelet-derived growth factor A chain (PDGF-A) is overexpressed; it has therefore been suggested that it may play an autocrine role in development of these neoplasms. Since the PDGF-A promoter contains putative binding sites for WT1, we explored the role of WT1 in regulating A-chain expression. The major PDGF-A promoter activity was localized in transient transfection assays to a region spanning from -643 to + 8 relative to the transcription start site. WT1 bound to several sites in this region of the promoter, as demonstrated by gel-shift analysis and DNase I footprinting, and functioned as a powerful repressor of PDGF-A transcription in vivo. Maximal repression (> 50-fold) of the PDGF-A promoter was dependent on the presence of multiple WT1 binding sites in transient transfection assays. Our observations suggest a mechanism for normal downregulation of a growth factor gene and of an autocrine growth process of import in kidney development and other biological systems.
Allelic deletions in the long arm of chromosome 12 identify sites of candidate tumor suppressor genes in male germ cell tumors.
Authors: Murty V V , Houldsworth J , Baldwin S , Reuter V , Hunziker W , Besmer P , Bosl G , Chaganti R S
Published: May 2006

Human male germ cell tumors (GCTs) result from malignant transformation of premeiotic or early meiotic germ cells and exhibit embryonal-like differentiation of the three germinal layers. The genetic basis of origin and expression of differentiated phenotypes by GCTs are poorly understood. Our recent cytogenetic analysis of a large series of GCTs has shown that two chromosome 12 abnormalities, an isochromosome for the short arm [i(12p)] and deletions in the long arm [del(12q)], characterize these tumors, which led us to suggest that the deletions represent loss of one or more candidate tumor suppressor genes whose products regulate the normal proliferation of the spermatogonial stem cells. We undertook a molecular mapping of the deletions by comparing germ-line and tumor genotypes of eight polymorphic loci in paired normal/tumor DNA samples from 45 GCT patients. Analysis of loss of constitutional heterozygosity at these loci revealed two regions of frequent loss (> 40%), one at 12q13 and the other at 12q22, identifying the sites of the postulated tumor suppressor genes. One tumor (no. 143A) exhibited a homozygous deletion of a region of 12q22, which included the MGF gene. The KIT and MGF genes have been shown to play key roles in embryonal and postnatal development of germ cells; therefore, we evaluated their expression by Northern blot analysis in a panel of three GCT cell lines and 24 fresh GCT biopsies. Deregulated expression of MGF and KIT, which was discordant between seminomatous and nonseminomatous lesions, was observed.
Cornifin, a cross-linked envelope precursor in keratinocytes that is down-regulated by retinoids.
Authors: Marvin K W , George M D , Fujimoto W , Saunders N A , Bernacki S H , Jetten A M
Published: May 2006

In this study, we have characterized the cDNA clone SQ37 that was isolated previously from a rabbit squamous cell library. The gene encodes a 14-kDa protein that appears to function as a component of the cross-linked envelope in squamous differentiating cells. The protein, which has been named cornifin, has a high content of proline (31%), glutamine (20%), and cysteine (11%) and contains 13 repeats of an octapeptide (consensus sequence, EPCQPKVP) at its C terminus. SQ37 mRNA and protein are induced during squamous differentiation of rabbit tracheal (RbTE) cells and human epidermal keratinocytes. This induction is repressed by retinoids. Immunohistochemical studies reveal SQ37 immunoreactivity in fragmented cross-linked envelopes from squamous-differentiated RbTE cells and in the suprabasal layers of the epidermis. In situ hybridization analysis showed that the presence of SQ37 mRNA is restricted to the suprabasal layers. Treatment of RbTE cells with a Ca2+ ionophore induces cross-linking of the SQ37 protein into higher molecular weight complexes. This cross-linking reaction appears to be mediated by transglutaminase type I. Our observations suggest that the protein encoded by SQ37 participates in the assembly of the cross-linked envelope.
Active complete in vitro replication of nodavirus RNA requires glycerophospholipid.
Authors: Wu S X , Ahlquist P , Kaesberg P
Published: May 2006

Flockhouse virus (FHV) is a member of the nodavirus group of positive-strand RNA viruses. In the absence of additional compounds, a template-dependent RNA-dependent RNA polymerase extracted from FHV-infected cells synthesizes complementary (-)-strand copies of added FHV RNA to yield a double-stranded RNA product. Upon addition of glycerophospholipid (GPL), this system reproducibly carries out complete highly active replication of added FHV RNA, producing newly synthesized (+)-strand RNA in predominantly single-stranded RNA form. This accounts for previously observed effects of Lipofectin (a mixture of GPL and cationic lipid) in the system. All tested neutral and negatively charged GPLs except phosphatidic acid support complete FHV RNA replication in this in vitro system, as do phospholipid extracts from uninfected and FHV-infected cells. Neither sphingomyelin, a membrane phospholipid that is not derived from glycerol, nor cholesterol supported FHV RNA replication. Testing of compounds derived from GPL shows that the ability of active GPL to support FHV (+)-strand RNA synthesis is dependent on the structures of both the head group and the acyl chains. Neither the phosphorylated head group nor the diacylglycerol lipid moiety alone supports RNA replication. The length and saturation of acyl chains strongly influence the ability of GPL to support RNA replication. Other characteristics of this in vitro RNA replication system and the possible role played by membranes and their components in FHV RNA replication are discussed.
Increased levels of trkB mRNA and trkB protein-like immunoreactivity in the injured rat and cat spinal cord.
Authors: Frisén J , Verge V M , Cullheim S , Persson H , Fried K , Middlemas D S , Hunter T , Hökfelt T , Risling M
Published: May 2006

Expression of neurotrophins and neurotrophin receptors was examined with in situ hybridization and immunohistochemical techniques 10 days to 6 weeks after ventral or dorsal funiculus spinal cord lesions in adult rats and cats, lesions that have previously been shown to allow axon regrowth. Strongly elevated levels of trkB mRNA were seen in the scar tissue formed in the white matter after both types of lesions. Only small increases were detected for nerve growth factor, brain-derived neurotrophic factor, neurotrophin 3, neurotrophin 4, trk, and trkC mRNA in response to the injuries. trkB protein-like immunoreactivity was increased in the regions that showed elevated levels of trkB mRNA. EM localized this immunoreactivity to neurons, astrocytes, and leptomeningeal cells. Neurofilament immunolabeling and axonal tracing demonstrated that nerve fibers in the scar tissue were concentrated to areas that showed strong trkB protein-like immunoreactivity. The findings implicate a role for neurotrophin receptors in axonal sprouting and glial reactions in the injured spinal cord.
Yeast RNA polymerase II initiation factor e: isolation and identification as the functional counterpart of human transcription factor IIB.
Authors: Tschochner H , Sayre M H , Flanagan P M , Feaver W J , Kornberg R D
Published: May 2006

Yeast RNA polymerase II initiation factor e was purified to homogeneity and identified by biochemical criteria as the counterpart of human transcription factor IIB. Factor e was essential for initiation of transcription from yeast and mammalian promoters in a reconstituted yeast transcription system. Activity resided in a single polypeptide of approximately 41 kDa, identified by peptide sequence analysis as the product of the SUA7 gene. Factor e interacted specifically with RNA polymerase II, consistent with a proposed role in determining the start site of transcription.
Effect of Ca2+ on weak cross-bridge interaction with actin in the presence of adenosine 5'-[gamma-thio]triphosphate).
Authors: Kraft T , Yu L C , Kuhn H J , Brenner B
Published: May 2006

In the presence of the nucleotide analog adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]), effects of Ca2+ on stiffness and equatorial x-ray diffraction patterns of single skinned fibers of the rabbit psoas muscle were studied. It is shown that cross-bridges in the presence of ATP[gamma S] have properties of the weak-binding states of the ATP hydrolysis cycle. Raising the Ca2+ concentration up to pCa 4.5 has little effect on actin affinity of cross-bridges in the presence of ATP[gamma S]. However, the rate constants for cross-bridge dissociation and reassociation from and to actin are reduced by about 2 orders of magnitude. In addition, nucleotide affinity of the cross-bridge is much smaller at high Ca2+ concentrations. Implications for interpretation of fiber stiffness recorded during isotonic shortening and the rising phase of a tetanus are discussed.
A proposed function for spermine and spermidine: protection of replicating DNA against damage by singlet oxygen.
Authors: Khan A U , Mei Y H , Wilson T
Published: May 2006

Like all aliphatic amines, the polyamines spermine and spermidine are physical quenchers of singlet molecular oxygen (1O2*). The rate constants of these processes were determined in vitro with photochemically generated 1O2* and the hydrocarbon rubrene as substrate, in pyridine. At millimolar concentration, spermine and spermidine should quench 1O2* in vivo and prevent it from damaging DNA. It is proposed that a biological function of polyamines is the protection of replicating DNA against oxidative damage.
Selective endothelial binding of interleukin-2-dependent human T-cell lines derived from different tissues.
Authors: Salmi M , Granfors K , Leirisalo-Repo M , Hämäläinen M , MacDermott R , Leino R , Havia T , Jalkanen S
Published: May 2006

The ability of lymphocytes to recognize and bind to high endothelial venules (HEVs) is essential for lymphocyte migration from the blood into lymphoid tissues and into sites of inflammation. Endothelial cell binding capacity also critically determines the clinical usefulness of T-cell lines and clones in immunotherapy. In the present study, interleukin-2-dependent T-cell lines were derived from the blood, lamina propria of the gut, inflamed synovium, synovial fluid, and peripheral lymph nodes. After 3-8 weeks of culture, the expression of homing-associated molecules and binding to mucosal, synovial, and peripheral lymph node HEVs were analyzed. Cell lines derived from the blood and mucosal sites bound significantly better to mucosal and synovial HEVs than to peripheral lymph node HEVs. Three out of seven synovial T-cell lines showed preferential binding to synovial HEVs, whereas the rest bound almost equally well to synovial and mucosal HEVs. T-cell lines from peripheral lymph nodes bound preferentially to lymph node HEVs despite the lack of L-selectin (the peripheral lymph node homing receptor). Expression of the known homing-associated molecules did not predict the HEV-binding specificity of these lines. Importantly, two cell lines bound well to synovial venules, but poorly, if at all, to mucosal or peripheral lymph node HEVs, supporting the concept that synovial-specific HEV recognition mechanisms exist. In conclusion, the tissue origin of T-cell lines critically determines their selectivity for endothelial cell recognition, and besides the known "homing receptors," other molecules may also mediate tissue-specific HEV-binding of interleukin-2-activated T cells.
DNA binding and heteromerization of the Drosophila transcription factor chorion factor 1/ultraspiracle.
Authors: Christianson A M , King D L , Hatzivassiliou E , Casas J E , Hallenbeck P L , Nikodem V M , Mitsialis S A , Kafatos F C
Published: May 2006

The Drosophila chorion factor 1/ultraspiracle (CF1/USP) transcription factor, a homologue of the retinoid X receptor, is a developmentally important member of the family of nuclear (steroid) hormone receptors. Using newly developed monoclonal antibodies and a full-length bacterially produced protein, we have studied in detail the in vitro DNA-binding properties of this factor and aspects of its distribution in vivo. During oogenesis, CF1/USP is present both in germline cells and in the somatic follicular epithelium. We have determined the optimal binding site of partially purified bacterially produced CF1/USP by an in vitro selection procedure and also have characterized its binding to the follicular-specific chorion s15 promoter. In vitro this bacterially produced factor is unusual in binding to a single element ("half-site"); simultaneous but noncoordinate binding to a second half-site is possible if these repeated elements are organized in direct orientation and spaced adequately. However, the factor interacts synergistically with several other nuclear hormone receptors: notably, it can form in vitro heteromers with mammalian thyroid and retinoic acid receptors, binding to two half-sites that are organized in either direct or inverted orientation. In vivo the factor most probably functions as a heterodimer, but its partner(s) remains to be determined.
Apparent nonlinearity of the redshift-distance relation in infrared astronomical satellite galaxy samples.
Authors: Segal I E , Nicoll J F
Published: May 2006

The Hubble (linear) redshift-distance law predicts values for directly observed quantities that are quite deviant from their actual values in infrared astronomical satellite (IRAS) galaxy samples. These samples are objectively defined, have modern measurements, are presently the largest such samples to which the Hubble law is theoretically applicable, and are otherwise generally considered to be statistically appropriate. The Hubble law predicts in particular that the dispersion in log flux will be much greater than it is observed to be. This type of deviation is fundamentally incapable of explanation via the assumption of any physically known type of perturbation. The Lundmark (quadratic) redshift-distance law predicts values for these directly observed quantities that are consistent with, and in fact quite close to, their actual values in the same samples. The predictions of a cubic law are typically deviant from observation but somewhat less so than those of the Hubble law. The Lundmark law accurately predicts the deviations from observation of statistical estimates predicated on either the Hubble or the cubic law. Parallel predictions for the latter laws for the results of statistical estimation predicated on the alternative laws are typically quite inaccurate. The Hubble and Lundmark laws are predicted at the low redshifts of the IRAS galaxy samples by generic big bang cosmology (BBC) and chronometric cosmology (CC), respectively. The present results confirm earlier studies of a variety of objectively defined samples of discrete sources in other wave bands that were contraindicative of BBC and indicative of CC.
Cellular regulation of the iron-responsive element binding protein: disassembly of the cubane iron-sulfur cluster results in high-affinity RNA binding.
Authors: Haile D J , Rouault T A , Harford J B , Kennedy M C , Blondin G A , Beinert H , Klausner R D
Published: May 2006

The translation of ferritin mRNA and degradation of transferrin receptor mRNA are regulated by the interaction of an RNA-binding protein, the iron-responsive element binding protein (IRE-BP), with RNA stem-loop structures known as iron-responsive elements (IREs) contained within these transcripts. IRE-BP produced in iron-replete cells has aconitase (EC 4.2.1.3) activity. The protein shows extensive sequence homology with mitochondrial aconitase, and sequences of peptides prepared from cytosolic aconitase are identical with peptides of IRE-BP. As an active aconitase, IRE-BP is expected to have an Fe-S cluster, in analogy to other aconitases. This Fe-S cluster has been implicated as the region of the protein that senses intracellular iron levels and accordingly modifies the ability of the IRE-BP to interact with IREs. Expression of the IRE-BP in cultured cells has revealed that the IRE-BP functions either as an active aconitase, when the cells are iron-replete, or as an active RNA-binding protein, when the cells are iron-depleted. We compare properties of purified authentic cytosolic aconitase from beef liver with those of IRE-BP from tissue culture cells and establish that characteristics of the physiologically relevant form of the protein from iron-depleted cells resemble those of cytosolic aconitase apoprotein. We demonstrate that loss of the labile fourth iron atom of the Fe-S cluster results in loss of aconitase activity, but that more extensive cluster alteration is required before the IRE-BP acquires the capacity to bind RNA with the affinity seen in vivo. These results are consistent with a model in which the cubane Fe-S cluster is disassembled when intracellular iron is depleted.
Resistance to host antimicrobial peptides is necessary for Salmonella virulence.
Authors: Groisman E A , Parra-Lopez C , Salcedo M , Lipps C J , Heffron F
Published: May 2006

The production of antibacterial peptides is a host defense strategy used by various species, including mammals, amphibians, and insects. Successful pathogens, such as the facultative intracellular bacterium Salmonella typhimurium, have evolved resistance mechanisms to this ubiquitous type of host defense. To identify the genes required for resistance to host peptides, we isolated a library of 20,000 MudJ transposon insertion mutants of a virulent peptide-resistant S. typhimurium strain and screened it for hypersensitivity to the antimicrobial peptide protamine. Eighteen mutants had heightened susceptibility to protamine and 12 of them were characterized in detail. Eleven mutants were attenuated for virulence in vivo when inoculated into BALB/c mice by the intragastric route, and 8 of them were also avirulent following intraperitoneal inoculation. The mutants fell into different phenotypic classes with respect to their susceptibility to rabbit defensin NP-1, frog magainin 2, pig cecropin P1, and the insect venom-derived peptides mastoparan and melittin. The resistance loci mapped to eight distinct locations in the genome. Characterization of the mutants showed that one had a defective lipopolysaccharide and another mutant harbored a mutation in phoP, a locus previously shown to control expression of Salmonella virulence genes. Our data indicate that the ability to resist the killing effect of host antimicrobial peptides is a virulence property and that several resistance mechanisms operate in S. typhimurium.
Detection of glutamine synthetase in the cerebrospinal fluid of Alzheimer diseased patients: a potential diagnostic biochemical marker.
Authors: Gunnersen D , Haley B
Published: May 2006

In this report, 8- and 2-azidoadenosine 5'-[gamma-32P]triphosphate were used to examine cerebrospinal fluid (CSF) samples for the presence of an ATP binding protein unique to individuals with Alzheimer disease (AD). A 42-kDa ATP binding protein was found in the CSF of AD patients that is not observed in CSF from normal patients or other neurological controls. The photolabeling is saturated with 30 microM 2-azidoadenosine 5'-[gamma-32P]triphosphate. Photoinsertion can be totally prevented by the addition of 25 microM ATP. Photoinsertion of 2-azidoadenosine 5'-triphosphate into the protein is only weakly protected by other nucleotides such as ADP and GTP, indicating that this is a specific ATP binding protein. A total of 83 CSF samples were examined in a blind manner. The 42-kDa protein was detected in 38 of 39 AD CSF samples and in only 1 of 44 control samples. This protein was identified as glutamine synthetase [GS; glutamate-ammonia ligase; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] based on similar nucleotide binding properties, comigration on two-dimensional gels, reaction with a polyclonal anti-GS antibody, and the presence of significant GS enzyme activity in AD CSF. In brain, GS plays a key role in elimination of free ammonia and also converts the neurotransmitter and excitotoxic amino acid glutamate to glutamine, which is not neurotoxic. The involvement of GS, if any, in the onset of AD is unknown. However, the presence of GS in the CSF of terminal AD patients suggests that this enzyme may be a useful diagnostic marker and that further study is warranted to determine any possible role for glutamate metabolism in the pathology of AD.
The alternative sigma factor katF (rpoS) regulates Salmonella virulence.
Authors: Fang F C , Libby S J , Buchmeier N A , Loewen P C , Switala J , Harwood J , Guiney D G
Published: May 2006

Nutrient limitation is a critical signal in Salmonella virulence gene regulation. The katF (rpoS) gene mediates the expression of the Salmonella spv plasmid virulence genes during bacterial starvation. A katF Salmonella mutant has increased susceptibility to nutrient deprivation, oxidative stress, acid stress, and DNA damage, conditions which are relevant to the intraphagosomal environment of host macrophages. Moreover, the katF mutant has significantly reduced virulence in mice. katF encodes an alternative sigma factor of RNA polymerase which coordinately regulates Salmonella virulence.
Nitric oxide synthase in macula densa regulates glomerular capillary pressure.
Authors: Wilcox C S , Welch W J , Murad F , Gross S S , Taylor G , Levi R , Schmidt H H
Published: May 2006

Tubular-fluid reabsorption by specialized cells of the nephron at the junction of the ascending limb of the loop of Henle and the distal convoluted tubule, termed the macula densa, releases compounds causing vasoconstriction of the adjacent afferent arteriole. Activation of this tubuloglomerular feedback response reduces glomerular capillary pressure of the nephron and, hence, the glomerular filtration rate. The tubuloglomerular feedback response functions in a negative-feedback mode to relate glomerular capillary pressure to tubular-fluid delivery and reabsorption. This system has been implicated in renal autoregulation, renin release, and longterm body fluid and blood-pressure homeostasis. Here we report that arginine-derived nitric oxide, generated in the macula densa, is an additional intercellular signaling molecule that is released during tubular-fluid reabsorption and counters the vasoconstriction of the afferent arteriole. Antibody to rat cerebellar constitutive nitric oxide synthase stained rat macula densa cells specifically. Microperfusion of the macula densa segment of single nephrons with N omega-methyl-L-arginine (an inhibitor of nitric oxide synthase) or with pyocyanin (a lipid-soluble inhibitor of endothelium-derived relaxation factor) showed that generation of nitric oxide can vasodilate the afferent arteriole and increase glomerular capillary pressure; this effect was blocked by drugs that prevent tubular-fluid reabsorption. We conclude that nitric oxide synthase in macula densa cells is activated by tubular-fluid reabsorption and mediates a vasodilating component to the tubuloglomerular feedback response. These findings imply a role for arginine-derived nitric oxide in body fluid-volume and blood-pressure homeostasis, in addition to its established roles in modulation of vascular tone by the endothelium and in neurotransmission.
Involvement of cytochrome P450 in host-plant utilization by Sonoran Desert Drosophila.
Authors: Frank M R , Fogleman J C
Published: May 2006

The four Drosophila species endemic to the Sonoran Desert (Drosophila mettleri, Drosophila mojavensis, Drosophila nigrospiracula, and Drosophila pachea) utilize necrotic cactus tissue or soil soaked by rot exudate as breeding substrates. Each Drosophila species uses a different cactus species as its primary host. D. pachea is limited to senita cactus by a biochemical dependency on unusual sterols available only in that cactus. For the other Drosophila species, no such chemical dependencies exist to explain the relationships with their primary host plants. Each cactus species has a different array of allelochemicals that have detrimental effects on non-resident fly species. We have hypothesized that the desert fly-cactus associations are due, in part, to differences between the fly species in their allelochemical detoxication enzymes, the cytochrome P450 system. To test whether P450s are involved in the detoxication of cactus allelochemicals, several experiments were done. (i) The effect of a specific P450 inhibitor, piperonyl butoxide, on larval survival through eclosion on each cactus substrate was investigated. (ii) In vitro metabolism of cactus alkaloids was determined for each Drosophila species. The effects of specific inducers and inhibitors were included in these experiments. (iii) The basal and induced content of cytochrome P450 in each species was determined. The results support the hypothesis that P450 enzymes are involved in host-plant utilization by these Sonoran Desert Drosophila species.
Wild-type p53 binds to the TATA-binding protein and represses transcription.
Authors: Seto E , Usheva A , Zambetti G P , Momand J , Horikoshi N , Weinmann R , Levine A J , Shenk T
Published: May 2006

p53 activates transcription of genes with a p53 response element, and it can repress genes lacking the element. Here we demonstrate that wild-type but not mutant p53 inhibits transcription in a HeLa nuclear extract from minimal promoters. Wild-type but not mutant p53 binds to human TATA-binding protein (TBP). p53 does not bind to yeast TBP, and it cannot inhibit transcription in a HeLa extract where yeast TBP substitutes for human TBP. These results suggest a model in which p53 binds to TBP and interferes with transcriptional initiation.
The Saccharomyces cerevisiae ARG4 initiator of meiotic gene conversion and its associated double-strand DNA breaks can be inhibited by transcriptional interference.
Authors: Rocco V , de Massy B , Nicolas A
Published: May 2006

In the yeast Saccharomyces cerevisiae, as in other eukaryotes, some regions of the genome have a much higher level of meiotic gene conversion than others. Previous deletion analysis indicated that the sequence necessary for the high level of gene conversion within the ARG4 region defined an initiation site located between positions -316 and -37 [relative to the first base pair (+1) of the ARG4 coding sequence] of the ARG4 promoter. To test whether this sequence is sufficient to promote gene conversion in a novel chromosomal context, we inverted on the chromosome various DNA fragments including the implicated region and the ARG4 coding sequence. Surprisingly, these inversions resulted in the loss of the normal recombination properties and double-strand-break formation associated with this process. By Northern analysis, we found that a transcript traverses the ARG4 initiation site in these inversion mutants but not in the wild type. When transcription through this region was prevented by a transcription terminator, the activity of the initiation site and the formation of double-strand breaks were restored. From these results and from complementary deletion analysis in the normal ARG4 orientation, we conclude that the activity of the ARG4 initiation site requires protection from transcriptional interference.
Organization of the human skeletal myosin heavy chain gene cluster.
Authors: Yoon S J , Seiler S H , Kucherlapati R , Leinwand L
Published: May 2006

Myosin is an important structural and enzymatic component of skeletal muscle. Multiple myosin isoforms are encoded by a multigene family and are expressed in different developmental stages and fiber types. In humans and mice, skeletal myosin heavy chain (MYH) genes are clustered on a single chromosome (17p and 11, respectively). Since the structural organization of the gene cluster may affect its expression as well as shed light on MYH genetic alterations, a physical map of the human MYH gene cluster was constructed. Nine yeast artificial chromosomes containing MYH genes were isolated and used to construct a contiguous set (contig) of overlapping yeast artificial chromosomes. This contig encompasses a genetic marker mapped to 17p13.1. Six MYH genes were located within a 500-kilobase segment of human DNA. The order of the genes within this cluster does not correspond to the developmental pattern of expression of individual members.