bluuebunny/crossref_metadata_2025 huggingface dataset:
Major histocompatibility complex class II DR alleles DRB1*1501 and those encoding HLA-DR13 are preferentially associated with a diminution in maternally transmitted human immunodeficiency virus 1 infection in different ethnic groups: determination by an automated sequence-based typing method.
Authors: Winchester R , Chen Y , Rose S , Selby J , Borkowsky W
Published: May 2006

Transmission of human immunodeficiency virus 1 (HIV-1) from an infected women to her offspring during gestation and delivery was found to be influenced by the infant's major histocompatibility complex class II DRB1 alleles. Forty-six HIV-infected infants and 63 seroreverting infants, born with passively acquired anti-HIV antibodies but not becoming detectably infected, were typed by an automated nucleotide-sequence-based technique that uses low-resolution PCR to select either the simpler Taq or the more demanding T7 sequencing chemistry. One or more DR13 alleles, including DRB1*1301, 1302, and 1303, were found in 31.7% of seroreverting infants and 15.2% of those becoming HIV-infected [OR (odds ratio) = 2.6 (95% confidence interval 1.0-6.8); P = 0.048]. This association was influenced by ethnicity, being seen more strongly among the 80 Black and Hispanic children [OR = 4.3 (1.2-16.4); P = 0.023], with the most pronounced effect among Black infants where 7 of 24 seroreverters inherited these alleles with none among 12 HIV-infected infants (Haldane OR = 12.3; P = 0.037). The previously recognized association of DR13 alleles with some situations of long-term nonprogression of HIV suggests that similar mechanisms may regulate both the occurrence of infection and disease progression after infection. Upon examining for residual associations, only only the DR2 allele DRB1*1501 was associated with seroreversion in Caucasoid infants (OR = 24; P = 0.004). Among Caucasoids the DRB1*03011 allele was positively associated with the occurrence of HIV infection (P = 0.03).
The BALB/c mouse B-cell response to pigeon cytochrome c initiates as a heteroclitic response specific for the self antigen mouse cytochrome c.
Authors: Minnerath J M , Wakem L P , Comfort L L , Sherman F , Jemmerson R
Published: May 2006

Direct evidence is presented in support of the longstanding but unproven hypothesis that B lymphocytes specific for self antigens (Ags) can be used in the immune response to foreign Ags. We show that the B cells in BALB/c mic responding early to pigeon cytochrome c (CYT) produce antibodies that recognize and bind the major antigenic site on mouse CYT with greater affinity than they bind pigeon CYT i.e., they are heteroclitic for the self Ag. Furthermore, these B cells express the same combination of immunoglobulin variable region (V) genes that are known to be used in B-cell recognition of mouse CYT. Over time, the response to pigeon CYT becomes more specific for the foreign Ag through the recruitment of B cells expressing different combinations of V genes and, possibly, somatic mutation of the mouse CYT specific B cells from early in the response. Cross-recognition of pigeon CYT by mouse CYT-specific B cells results from the sharing of critical amino acid residues by the two Ags. Although B-cell recognition of the self Ag, mouse CYT, is very specific, which limits the extent to which foreign Ags can cross-activate the autoreactive B cells, it is possible that polyreactive B cells to other self Ags may be used more frequently in response to foreign Ags.
Peptide synthesis using unprotected peptides through orthogonal coupling methods.
Authors: Tam J P , Lu Y A , Liu C F , Shao J
Published: May 2006

We describe an approach to the synthesis of peptides from segments bearing no protecting groups through an orthogonal coupling method to capture the acyl segment as a thioester that then undergoes an intramolecular acyl transfer to the amine component with formation of a peptide bond. Two orthogonal coupling methods to give the covalent ester intermediate were achieved by either a thiol-thioester exchange mediated by a trialkylphosphine and an alkylthiol or a thioesterification by C alpha-thiocarboxylic acid reacting with a beta-bromo amino acid. With this approach, unprotected segments ranging from 4 to 37 residues were coupled to aqueous solution to give free peptides up to 54 residues long with high efficiency.
T cells induce terminal differentiation of transformed B cells to mature plasma cell tumors.
Authors: Hilbert D M , Shen M Y , Rapp U R , Rudikoff S
Published: May 2006

Major interest in the analysis of mature plasma cell neoplasias of mice and humans has focused on identification of precursor cells that give rise to mature malignant plasma cells. Although several laboratories have recently suggested that such cells are present in the granulomas of pristane-treated mice and the bone marrow of some multiple myeloma patients, the in vivo cellular interactions required for their differentiation into mature plasma cell tumors remains unclear. Given the extensive interactions of peripheral T cells and normal B cells, we assessed the potential role of T cells in plasma-cell tumor development, by using a myc, raf-containing retrovirus, J3V1, to induce plasmacytomas in normal BALB/c mice, T-cell-deficient nude mice, and T-cell-reconstituted nude mice. The B-lineage tumors arising in normal BALB/c mice were uniformly mature plasmacytomas, most of which secreted immunoglobulin. In contrast, nude mice yielded predominantly non-immunoglobulin-secreting B-cell lymphomas with a phenotype characteristic of peripheral B cells. T-cell reconstitution of nude mice prior to tumor induction resulted in a shift from B-cell lymphomas to plasmacytomas. These results imply that transformation can occur prior to terminal differentiation of B cells and that such transformed cells can be driven to terminal differentiation by peripheral T cells. These findings further suggest that, in human multiple myeloma, the ability of T cells to influence the differentiation state of transformed B cells may provide a mechanism by which malignant plasma cells found in the bone marrow could arise from clonotypically related less-mature B cells found in both the bone marrow and periphery.
Protein-tyrosine-phosphatase SHPTP2 is a required positive effector for insulin downstream signaling.
Authors: Yamauchi K , Milarski K L , Saltiel A R , Pessin J E
Published: May 2006

SHPTP2 is a ubiquitously expressed tyrosine-specific protein phosphatase that contains two amino-terminal Src homology 2 (SH2) domains responsible for its association with tyrosine-phosphorylated proteins. In this study, expression of dominant interfering mutants of SHPTP2 was found to inhibit insulin stimulation of c-fos reporter gene expression and activation of the 42-kDa (Erk2) and 44-kDa (Erk1) mitogen-activated protein kinases. Cotransfection of dominant interfering SHPTP2 mutants with v-Ras or Grb2 indicated that SHPTP2 regulated insulin signaling either upstream of or in parallel to Ras function. Furthermore, phosphotyrosine blotting and immunoprecipitation identified the 125-kDa focal adhesion kinase (pp125FAK) as a substrate for insulin-dependent tyrosine dephosphorylation. These data demonstrate that SHPTP2 functions as a positive regulator of insulin action and that insulin signaling results in the dephosphorylation of tyrosine-phosphorylated pp125FAK.
The salivary gland-specific apyrase of the mosquito Aedes aegypti is a member of the 5'-nucleotidase family.
Authors: Champagne D E , Smartt C T , Ribeiro J M , James A A
Published: May 2006

The saliva of hematophagous insects contains a variety of pharmacologically active substances that counteract the normal hemostatic response to injury in vertebrate hosts. The yellow-fever mosquito, Aedes aegypti, secretes an apyrase that inhibits ADP-dependent platelet aggregation. Apyrase was purified as an active enzyme from adult female salivary glands and subjected to tryptic digestion, and the resulting peptides were sequenced. The amino acid sequences obtained match the conceptual translation product of a cDNA clone isolated from an adult female salivary gland library. Sequence comparisons indicate similarities with a ubiquitous family of 5'-nucleotidases. The mosquito protein differs from other members of the family by lacking a carboxyl-terminal hydrophobic domain. The apparent conversion of a gene encoding an enzyme involved in a common metabolic event at the cellular level to a gene involved in the antihemostatic response of mosquitoes illustrates one way this particular insect has adapted to the challenges of bloodfeeding.
Induction of vascular endothelial growth factor expression by hypoxia and by glucose deficiency in multicell spheroids: implications for tumor angiogenesis.
Authors: Shweiki D , Neeman M , Itin A , Keshet E
Published: May 2006

Perfusion insufficiency, and the resultant hypoxia, often induces a compensatory neovascularization to satisfy the needs of the tissue. We have used multicellular tumor spheroids, simulating avascular microenvironments within a clonal population of glioma tumor cells, in conjunction with in situ analysis of gene expression, to study stress inducibility of candidate angiogenic factors. We show that expression of vascular endothelial growth factor (VEGF) is upregulated in chronically hypoxic niches (inner layers) of the spheroid and that expression is reversed when hypoxia is relieved by hyperoxygenation. Acute glucose deprivation--another consequence of vascular insufficiency--also activates VEGF expression. Notably, glioma cells in two distinct regions of the spheroid upregulated VEGF expression in response to hypoxia and to glucose starvation. Experiments carried out in cell monolayers established that VEGF is independently induced by these two deficiencies. Upon implantation in nude mice, spheroids were efficiently neovascularized. Concomitant with invasion of blood vessels and restoration of normoxia to the spheroid core, VEGF expression was gradually downregulated to a constitutive low level of expression, representing the output of nonstressed glioma cells. These findings show that stress-induced VEGF activity may compound angiogenic activities generated through the tumor "angiogenic switch" and suggest that stress-induced VEGF should be taken into account in any attempt to target tumor angiogenesis.
Hydrogen bonding in water using synthetic receptors.
Authors: Kato Y , Conn M M , Rebek J
Published: May 2006

Four water-soluble adenine receptors were synthesized to study the influence of hydrophobic interactions and hydrogen bonding on molecular recognition in aqueous solution. Association constants were measured in aqueous solution at five temperatures from 3-27 degrees C (pH 6, 51 mM ionic strength). For the mono(imide) receptors, delta H was -5.8 kcal/mol (carbazole) and -9.2 kcal/mol (naphthalene). The entropy of association for these was -13 cal.mol-1.K-1 (carbazole) and -26 cal.mol-1.K-1 (naphthalene). The carbazole bis(imide) receptor showed a binding enthalpy of -7.4 kcal/mol and entropy of -18 cal.mol-1.K-1. From this the free energy at 298 K of a single hydrogen bond is estimated to be only 0.2 kcal/mol. The enthalpy of a single hydrogen bond in this solvent-exposed system is estimated to be, at most, 0.8 kcal/mol, indicating that enthalpy just compensates for the unfavorable entropy in this system. These values reflect stronger hydrophobic interactions with the more polarizable naphthalene, as well as enthalpy-entropy compensation effects.
Amino acid sequence of rat kidney glutathione synthetase.
Authors: Huang C S , He W , Meister A , Anderson M E
Published: May 2006

Glutathione (GSH) synthetase [gamma-L-glutamyl-L-cysteine:glycine ligase (ADP-forming), EC 6.3.2.3], an enzyme present in almost all cells, catalyzes the ATP-dependent synthesis of GSH from gamma-L-glutamyl-L-cysteine and glycine. Highly purified preparations of the enzyme have been obtained from rat kidney and several lower forms. The rat kidney enzyme (M(r), 118,000), which contains approximately 2% carbohydrate, is composed of two apparently identical subunits. The cDNA encoding rat kidney GSH synthetase was isolated from a rat kidney lambda gt11 cDNA library by immunoscreening with an antibody prepared against the isolated enzyme. The cDNA contains 1905 nucleotides and an open reading frame of 1422 nucleotides coding for 474 amino acids. The cDNA has a 3' untranslated region of 439 nucleotides, which includes a poly(A) tail. The deduced amino acid sequence (M(r), 52,344) contains all five of the peptide sequences that were independently determined by Edman degradation. The cDNA was expressed in Escherichia coli. The amino acid sequence of the rat kidney enzyme has no significant similarity to that of the enzyme from E. coli and shows some similarity to those deduced for the yeast and frog enzymes. Knowledge of this amino acid sequence is expected to facilitate elucidation of the sequence of the corresponding human enzyme and to lead to studies on the biochemical mechanisms involved in human GSH synthetase deficiency as well as to development of improved methods for prenatal diagnosis of these inborn diseases.
Transfection of Plasmodium falciparum within human red blood cells.
Authors: Wu Y , Sifri C D , Lei H H , Su X Z , Wellems T E
Published: May 2006

Plasmodium falciparum malaria parasites within human red blood cells (RBCs) have been successfully transfected to produce chloramphenicol acetyltransferase (CAT). Electroporation of parasitized RBCs was used to introduce plasmids that have CAT-encoding DNA flanked by 5' and 3' untranslated sequences of the P. falciparum hsp86, hrp3, and hrp2 genes. These flanking sequences were required for expression as their excision abolished CAT activity in transfected parasites. Transfection signals from native CAT-encoding DNA compared well with those from a synthetic DNA sequence adapted to the P. falciparum major codon bias, demonstrating effective expression of the bacterial sequence despite its use of rare P. falciparum codons. Transfected ring-stage parasites produced CAT signals at least as strong as transfected schizont-stage parasites even though ring stages are surrounded by more RBC cytoplasm than schizonts. The transfection of erythrocyte-stage P. falciparum parasites advances our ability to pursue genetic analysis of this major pathogen.
Biochemical evidence for the autophosphorylation and transphosphorylation of transforming growth factor beta receptor kinases.
Authors: Chen F , Weinberg R A
Published: May 2006

Transforming growth factor beta (TGF-beta) signals through a receptor complex containing the type I (TGF-beta RI) and type II (TGF-beta RII) receptors. We describe here biochemical studies on early events in the TGF-beta signaling pathways. TGF-beta RII is highly phosphorylated when expressed alone in COS-1 cells; its autophosphorylation occurs via an intramolecular (cis) mechanism that is independent of ligand binding. TGF-beta RI is also highly phosphorylated when expressed alone in COS-1 cells. Both wild-type TGF-beta RI and a kinase-deficient mutant thereof are transphosphorylated by the coexpressed TGF-beta RII kinase in a ligand-independent fashion in these cells. We propose that the association of TGF-beta RI and TGF-beta RII, induced by ligand binding or over-expression, leads to transphosphorylation of the TGF-beta RI by the TGF-beta RII kinase. This represents a mechanism of activation of receptors distinct from that of tyrosine kinase receptors and may apply to other serine/threonine kinase receptors.
Knockout of the mouse apolipoprotein B gene results in embryonic lethality in homozygotes and protection against diet-induced hypercholesterolemia in heterozygotes.
Authors: Farese R V , Ruland S L , Flynn L M , Stokowski R P , Young S G
Published: May 2006

Apolipoprotein B is synthesized by the intestine and the liver in mammals, where it serves as the main structural component in the formation of chylomicrons and very low density lipoproteins, respectively. Apolipoprotein B is also expressed in mammalian fetal membranes. To examine the consequences of apolipoprotein B deficiency in mice, we used gene targeting in mouse embryonic stem cells to generate mice containing an insertional disruption of the 5' region of the apolipoprotein B gene. Mice that were heterozygous for the disrupted apolipoprotein B allele had an approximately 20% reduction in plasma cholesterol levels, markedly reduced plasma concentrations of the pre-beta and beta-migrating lipoproteins, and an approximately 70% reduction in plasma apolipoprotein B levels. When fed a diet rich in fat and cholesterol, heterozygous mice were protected from diet-induced hypercholesterolemia; these mice, which constitute an animal model for hypobetalipoproteinemia, should be useful for studying the effects of decreased apolipoprotein B expression on atherogenesis. The breeding of heterozygous mice yielded no viable homozygous apolipoprotein B knockout mice. Most homozygous embryos were resorbed by midgestation (before gestational day 11.5); several embryos that survived until later in gestation exhibited exencephalus. The embryonic lethal phenotype was rescued by complementation with a human apolipoprotein B transgene--i.e., human apolipoprotein B transgenic mice that were homozygous for the murine apolipoprotein B knockout mutation were viable. Our findings indicate that apolipoprotein B plays an essential role in mouse embryonic development.
Immunophilin FK506 binding protein associated with inositol 1,4,5-trisphosphate receptor modulates calcium flux.
Authors: Cameron A M , Steiner J P , Sabatini D M , Kaplin A I , Walensky L D , Snyder S H
Published: May 2006

The immunophilin FK506 binding protein 12 (FKBP12) is associated with and modulates the ryanodine receptor calcium release channel of skeletal muscle. Ryanodine receptor has amino acid homology and functional similarity with another intracellular Ca2+ release channel, the inositol 1,4,5-trisphosphate receptor (IP3R). In the present study we show that highly purified preparations of IP3R contain FKBP12. The complex of these two proteins is disrupted by the immunosuppressants FK506 and rapamycin, both of which are known to bind FKBP12 with high affinity. Disrupting the IP3R-FKBP12 interaction increases Ca2+ flux through IP3R, an effect that is reversed by added FKBP12. FKBP12 appears to be physiologically linked to IP3R, regulating its Ca2+ conductance.
Use, disuse, and growth of the brain.
Authors: Wong R O
Published: May 2006

It is well known that across species, the relative size of the cortical area representing a particular sensory surface is proportional to how important that sense is for the animal. Furthermore, we are commonly aware of the observation that the loss of one sense, such as sight, appears to lead to an increase in sensitivity of the remaining senses, although the physiological basis for this is not entirely clear. Now, several studies, including that of Zheng and Purves (11), have suggested that the cortical area devoted to a particular sensory system can be modulated by neuronal activity during development. The fact that use, or disuse, of a sensory organ can lead to significant changes in its area of representation in the developing cortex is intriguing and calls for further investigations aimed at understanding the functional significance and the mechanisms underlying these changes. What remains to be determined is whether enhanced "growth" also means enhanced performance by that sensory system and, if so, whether this is the result of selective changes in neuronal connectivity and/or synaptic efficacy. It is too early to tell, but, whatever the outcome, it is refreshing to consider neuronal growth in the light of enhanced neural activity, in parallel to the results of activity deprivation, to which we are more accustomed.
Transposon-induced promoter scrambling: a mechanism for the evolution of new alleles.
Authors: Kloeckener-Gruissem B , Freeling M
Published: May 2006

We have studied a germinal revertant of the Mutator (Mu3)-induced mutation (Adh1-3F1124) of the maize alcohol dehydrogenase 1 gene (adh1). Transposon Mu3 was inserted at the TATA box of the promoter. The excision of Mu3 caused a complex, multibreakpoint DNA rearrangement with deletion, inverted duplication, and inversions affecting 430 nucleotides in the promoter region. These changes led to an unusual pattern of adh1 gene expression: increased levels of enzyme activity in one organ, decreased levels in another, and almost unchanged levels in a third organ. The evolutionary impact of transposon-induced promoter scrambling on generation of allelic diversity is discussed. We present a fragmentation model to help explain how transposon excision could induce multiple breakpoint aberrations without involving a homologous chromosome.
Structural and functional similarities between the promoters for mouse tenascin and chicken cytotactin.
Authors: Copertino D W , Jenkinson S , Jones F S , Edelman G M
Published: May 2006

Cytotactin/tenascin is an extracellular matrix glycoprotein expressed in a restricted anteroposterior pattern during vertebrate development and is reexpressed in the adult during wound healing, tumorigenesis, and nerve regeneration. Previously, we have characterized the chicken cytotactin promoter and have shown its regulation by homeobox gene products in vitro. We have now isolated the promoter for the mouse tenascin gene in order to determine whether common or different DNA regulatory elements control the expression of this gene in these two species. Like the chicken cytotactin gene, the mouse tenascin gene has a single RNA start site that lies 27 bp downstream of a TATA box. A 4028-bp region of DNA upstream of the mouse tenascin gene was sequenced and examined for regulatory motifs in common with the upstream sequence from the chicken cytotactin promoter. Two hundred thirty base pairs of the proximal promoter regions from both genes had an extended sequence similarity and contained common regulatory motifs such as two tracts of homopolymeric dA.dT sequence, an octamer motif, an ATTA (TAAT) motif which is a common core sequence for binding of homeodomain transcription factors, and a TATA-box/cap-site region. Reporter gene constructs with various 5' deletions of the mouse tenascin upstream sequence were tested in transient transfections of mouse NIH 3T3 and chicken embryo fibroblasts. The conserved proximal promoter region of tenascin was responsible for most of the positive regulatory activity. In addition, an upstream region (-2478 to -247) repressed proximal promoter activity in mouse fibroblasts and also in chicken embryo fibroblasts. These data indicate that both the structure and function of the cytotactin/tenascin proximal promoters have remained conserved over 250 million years.
Partial rescue of human carbonic anhydrase II frameshift mutation by ribosomal frameshift.
Authors: Hu P Y , Waheed A , Sly W S
Published: May 2006

A single-base-pair deletion in exon 7 of the human carbonic anhydrase II gene was found to be the molecular defect in a group of independently ascertained, clinically heterogeneous, Hispanic carbonic anhydrase II-deficient patients, all of whom had ancestors from the Caribbean islands. This mutation predicts a +1 frameshift at codon 227 and incorporation of 12 missense amino acids before an early stop codon at position 239 produces a 27-kDa truncated carbonic anhydrase II. Expression of the Hispanic mutant cDNA in bacteria produced predominantly the 27-kDa protein, which was inactive. However, a minor 29-kDa polypeptide species was also produced that had 10% the specific activity of the wild-type enzyme after affinity purification. Amino acid sequencing showed that the 29-kDa mutant protein was produced by two frameshift events: a +1 frameshift at codon 227 due to the single-base deletion and a -1 ribosomal frameshift at codon 237 that restored the original reading frame after 11 missense amino acids were incorporated. Antibody against the 11-amino acid frameshift peptide detected the 29-kDa mutant protein in lysates of transfected COS cells. These results indicate that ribosomal frameshift can partially rescue the human carbonic anhydrase II frameshift mutation and suggest a mechanism whereby a compensatory ribosomal frameshift can ameliorate the consequences of certain frameshift mutations. Whether individual differences in efficiency of ribosomal frameshift contribute to clinical heterogeneity in patients with such mutations deserves further study.
Molecular mimicry: histone H3 and mycobacterial protein epitopes.
Authors: Eriksen N , Kumar S B , Fukuchi K , Martin G M , Benditt E P
Published: May 2006

A 15-kDa protein detected initially in amyloidotic ileum from a transgenic mouse and subsequently in control (nontransgenic) ileum by various polyclonal rabbit antiserums applied to electroblots of extracts derived from these tissues was identified by partial sequence analysis as histone H3. Antiserums were made against immunogens unrelated to the histone, but they recognized calf thymus histone H3 (14.7 kDa) on Western blots. The bacterial component of the Freund's medium used as an adjuvant for the immunogens was either Mycobacterium butyricum or Mycobacterium smegmatis. Absorption tests with histone H3 and sonicated M. butyricum substantiated the presence of anti-histone H3 activity in the antiserums. These findings indicate that the two mycobacterium species make a protein with epitopes perceived as nonself by recipient rabbits but sufficiently similar to epitopes of mammalian histone H3 that the rabbits produced antibodies cross-reactive with the histone.
Selection of peptide inhibitors of interactions involved in complex protein assemblies: association of the core and surface antigens of hepatitis B virus.
Authors: Dyson M R , Murray K
Published: May 2006

As an example for studies of contacts involved in complex biological systems, peptide ligands that bind to the core antigen of hepatitis B virus (HBcAg) have been selected from a random hexapeptide library displayed on filamentous phage. Affinity-purified phage bearing aa sequence LLGRMK, or some related sequences, bound full-length or truncated HBcAg but did not bind denatured HBcAg. The long (L), but not the short (S), hepatitis B virus envelope polypeptide, when synthesized in an in vitro system, bound firmly to HBcAg, indicating that interaction between HBcAg and the pre-S region of the L polypeptide is critical for virus morphogenesis. This interaction was inhibited by peptide ALLGRMKG, suggesting that this and related small molecules may inhibit viral assembly.
Expression of the zinc-finger gene PLZF at rhombomere boundaries in the vertebrate hindbrain.
Authors: Cook M , Gould A , Brand N , Davies J , Strutt P , Shaknovich R , Licht J , Waxman S , Chen Z , Gluecksohn-Waelsch S
Published: May 2006

To investigate the potential biological role(s) of the PLZF gene, discovered as a fusion with the RARA locus in a patient with acute promyelocytic leukemia harboring a t(11;17) chromosomal translocation, we have isolated its murine homologue (mPLZF) and studied its patterns of developmental expression. The levels of mPLZF mRNAs increased perinatally in the liver, heart, and kidney, but with the exception of the heart, they were either absent or very low in the adult tissues. In situ analysis of mPLZF expression in mouse embryos between 7.0 and 10.5 days of development revealed that mPLZF mRNAs and proteins were coexpressed in spatially restricted and temporally dynamic patterns in the central nervous system. In the hindbrain region, a segmental pattern of expression correlated with the development of the rhombomeres. From 9.0 days of development, starting first in rhombomeres 3 and 5, there was an ordered down-regulation of expression in the center of each rhombomere, so that 1 day later elevated levels of mPLZF mRNAs and proteins were restricted to cells surrounding the rhombomeric boundaries. The chicken homologue of the PLZF gene, which we have also cloned, demonstrated a similar segmental pattern of expression in the hindbrain. To date, PLZF represents the only example of a transcription factor with elevated expression at rhombomeric boundaries. The high degree of evolutionary conservation between the patterns of PLZF expression during mammalian and avian central nervous system development suggests that it has an important functional role in the regionalization of the vertebrate hindbrain, potentially regulating boundary cell interactions.
Membrane-associated and soluble granulocyte/macrophage-colony-stimulating factor receptor alpha subunits are independently regulated in HL-60 cells.
Authors: Heaney M L , Vera J C , Raines M A , Golde D W
Published: May 2006

The effects of granulocyte/macrophage-colony-stimulating factor (GM-CSF) are mediated by interaction with its composite receptor (GMR), which consists of a unique alpha subunit (GMR alpha) and a beta subunit (GMR beta) that is common to the receptors for GM-CSF, interleukin 3, and interleukin 5. GMR beta is required for high-affinity binding, cell proliferation, and protein phosphorylation but has no intrinsic GM-CSF-binding activity. GMR alpha in isolation binds to GM-CSF with low affinity and can signal for increased glucose uptake. In addition to the membrane-bound receptor (mGMR alpha), there is a naturally occurring soluble isoform (sGMR alpha) that is released free into the pericellular milieu. Analysis of genomic sequences reveals that the soluble GMR alpha isoform comes about by alternative mRNA splicing. To examine GMR alpha expression, we developed a quantitative reverse transcription-polymerase chain reaction assay based on serial dilutions of in vitro transcribed GMR alpha RNA. This assay provides a strict log-log measure of GMR alpha RNA expression, distinguishes transcripts related to the soluble and membrane-associated isoforms, and quantitatively detects 0.1 fg of GMR alpha-related mRNA. There was little or no GMR alpha expression in two human lymphoid cell lines and in the erythroblastic leukemia cell line K562, but all myeloid cell lines tested expressed both the membrane-associated and soluble isoforms of GMR alpha. Baseline level of expression of both isoforms varied > 20-fold among the myeloid cell lines studied. Differentiation of HL-60 cells to neutrophils with dimethyl sulfoxide led to a 2-fold downregulation of sGMR alpha and a 20-fold upregulation of mGMR alpha. These differentiation-induced transcriptional changes were unrelated to changes in mRNA stability. These findings indicate that sGMR alpha is differentially expressed from mGMR alpha in human hematopoietic cells and that programmed downregulation of sGMR alpha may be important in myeloid maturation.
Association of a polynuclear iron-sulfur center with a mutant FNR protein enhances DNA binding.
Authors: Khoroshilova N , Beinert H , Kiley P J
Published: May 2006

In the facultative anaerobe Escherichia coli, the transcription factor FNR (fumarate nitrate reduction) regulates gene expression in response to oxygen deprivation. To investigate how the activity of FNR is regulated by oxygen availability, two mutant proteins, DA154 and LH28-DA154, which have enhanced in vivo activity in the presence of oxygen, were purified and compared. Unlike other previously examined FNR preparations, the absorption spectrum of LH28-DA154 had two maxima at 324 nm and 419 nm, typical of iron-sulfur (Fe-S)-containing proteins. Consistent with these data, metal analysis showed that only the LH28-DA154 protein contained a significant amount of iron and acid-labile sulfide, and, by low temperature EPR spectroscopy, a signal typical of a [3Fe-4S]+ cluster was detected. The LH28-DA154 protein that contained the Fe-S cluster also contained a higher proportion of dimers and had a 3- to 4-fold higher apparent affinity for the target DNA than the DA154 protein. In agreement with this, we found that when the LH28-DA154 protein was treated with an iron chelator (alpha,alpha'-dipyridyl), it lost its characteristic absorption and the apparent affinity for DNA was reduced 6-fold. However, increased DNA binding and the characteristic absorption spectrum could be restored by in vitro reconstitution of the Fe-S center. DNA binding of the LH28-DA154 protein was also affected by the redox state of the Fe-S center, since protein exposed to oxygen bound 1/10th as much DNA as the protein reduced anaerobically with dithionite. The observation that DNA binding is enhanced when the Fe-S center is reduced indicates that the redox state of the Fe-S center affects the DNA-binding activity of this protein and suggests a possible mechanism for regulation of the wild-type protein.
Core lipid structure is a major determinant of the oxidative resistance of low density lipoprotein.
Authors: Schuster B , Prassl R , Nigon F , Chapman M J , Laggner P
Published: May 2006

The influence of thermally induced changes in the lipid core structure on the oxidative resistance of discrete, homogeneous low density lipoprotein (LDL) subspecies (d, 1.0297-1.0327 and 1.0327-1.0358 g/ml) has been evaluated. The thermotropic transition of the LDL lipid core at temperatures between 15 degrees C and 37 degrees C, determined by differential scanning calorimetry, exerted significant effects on the kinetics of copper-mediated LDL oxidation expressed in terms of intrinsic antioxidant efficiency (lag time) and diene production rate. Thus, the temperature coefficients of oxidative resistance and maximum oxidation rate showed break points at the core transition temperature. Temperature-induced changes in copper binding were excluded as the molecular basis of such effects, as the saturation of LDL with copper was identical below and above the core transition. At temperatures below the transition, the elevation in lag time indicated a greater resistance to oxidation, reflecting a higher degree of antioxidant protection. This effect can be explained by higher motional constraints and local antioxidant concentrations, the latter resulting from the freezing out of antioxidants from crystalline domains of cholesteryl esters and triglycerides. Below the transition temperature, the conjugated diene production rate was decreased, a finding that correlated positively with the average size of the cooperative units of neutral lipids estimated from the calorimetric transition width. The reduced accessibility and structural hindrance in the cluster organization of the core lipids therefore inhibits peroxidation. Our findings provide evidence for a distinct effect of the dynamic state of the core lipids on the oxidative susceptibility of LDL and are therefore relevant to the atherogenicity of these cholesterol-rich particles.
Strong natural selection causes microscale allozyme variation in a marine snail.
Authors: Johannesson K , Johannesson B , Lundgren U
Published: May 2006

Natural selection is one of the most fundamental processes in biology. However, there is still a controversy over the importance of selection in microevolution of molecular traits. Despite the general lack of data most authors hold the view that selection on molecular characters may be important, but at lower rates than selection on most phenotypic traits. Here we present evidence that natural selection may contribute substantially to molecular variation on a scale of meters only. In populations of the marine snail Littorina saxatilis living on exposed rocky shores, steep microclines in allele frequencies between splash and surf zone groups are present in the enzyme aspartate aminotransferase (allozyme locus Aat; EC. 2.6.1.1). We followed one population over 7 years, including a period of strong natural perturbation. The surf zone part of the population dominated by the allele Aat100 was suddenly eliminated by a bloom of a toxin-producing microflagellate. Downshore migration of splash zone snails with predominantly Aat120 alleles resulted in a drastic increase in surf zone frequency of Aat120, from 0.4 to 0.8 over 2 years. Over the next four to six generations, however, the frequency of Aat120 returned to the original value. We estimated the coefficient of selection of Aat120 in the surf zone to be about 0.4. Earlier studies show similar or even sharper Aat clines in other countries. Thus, we conclude that microclinal selection is an important evolutionary force in this system.
Benzodiazepine receptors mediate regional blood flow changes in the living human brain.
Authors: Matthew E , Andreason P , Pettigrew K , Carson R E , Herscovitch P , Cohen R , King C , Johanson C E , Greenblatt D J , Paul S M
Published: May 2006

We studied the effects of a high-affinity gamma-aminobutyric acid (GABA)-benzodiazepine-receptor agonist (lorazepam) and an antagonist (flumazenil) in humans, using H2(15)O positron-emission tomography. Administration of lorazepam to healthy volunteers caused time- and dose-dependent reductions in regional cerebral blood flow and self-reported alterations in behavioral/mood parameters. Flumazenil administration reversed these changes. These observations indicated that benzodiazepine-induced effects on regional cerebral blood flow and mood/behavior are mediated at some level through GABA-benzodiazepine receptors, although the specific mechanism remains unclear. The approach described here provides a method for quantifying GABA-benzodiazepine-receptor-mediated neurotransmission in the living human brain and may be useful for studying the role of these receptors in a variety of neuropsychiatric disorders.
Identification of the porcine intestinal accessory factor that enables DNA sequence recognition by vitamin D receptor.
Authors: Munder M , Herzberg I M , Zierold C , Moss V E , Hanson K , Clagett-Dame M , DeLuca H F
Published: May 2006

The nuclear accessory protein in porcine intestinal nuclear extracts that activates the binding of the vitamin D receptor to its vitamin D response elements has been highly purified. It contains a protein that binds 9-cis-[3H]retinoic acid, was detected on immunoblots with an anti-retinoid X receptor (RXR) peptide antibody, and supports the binding of retinoic acid receptor gamma to the retinoic acid receptor beta gene response element. Most important, the two specific complexes formed by porcine nuclear extract with the vitamin D response elements from either the osteocalcin gene or the rat 24-hydroxylase gene are shifted to a larger complex by both an anti-vitamin D receptor antibody and an anti-RXR antibody, leaving no doubt that in vivo the nuclear accessory factor for the vitamin D receptor in the intestine is an RXR protein.
Scrapie prions selectively modify the stress response in neuroblastoma cells.
Authors: Tatzelt J , Zuo J , Voellmy R , Scott M , Hartl U , Prusiner S B , Welch W J
Published: May 2006

The fundamental event underlying scrapie infection seems to be a conformational change in the prion protein. To investigate proteins that might feature in the conversion of the cellular prion protein (PrPC) into the scrapie isoform (PrPSc), we examined mouse neuroblastoma N2a cells for the expression and cellular distribution of heat shock proteins (Hsps), some of which function as molecular chaperones. In scrapie-infected N2a (ScN2a) cells, Hsp72 and Hsp28 were not induced by heat shock, sodium arsenite, or an amino acid analog, in contrast to uninfected control N2a cells, while other inducible Hsps were increased by these treatments. Following heat shock of the N2a cells, constitutively expressed Hsp73 was translocated from the cytoplasm into the nucleus and nucleolus. In contrast, the distribution of Hsp73 in ScN2a cells was not altered by heat shock; the discrete cytoplasmic structures containing Hsp73 were largely resistant to detergent extraction. These alterations in the expression and subcellular translocation of specific Hsps in ScN2a cells may reflect the cellular response to the accumulation of PrPSc. Whether any of these Hsps feature in the conversion of PrPC into PrPSc or the pathogenesis of prion diseases remains to be established.
Bidirectional regulation of osteoclast function by nitric oxide synthase isoforms.
Authors: Brandi M L , Hukkanen M , Umeda T , Moradi-Bidhendi N , Bianchi S , Gross S S , Polak J M , MacIntyre I
Published: May 2006

Nitric oxide (NO) produces rapid osteoclast detachment and contraction in vitro, and this effect is accompanied by a profound inhibition of bone resorption. Work by others has confirmed these findings in vivo: inhibition of NO synthase [NOS; L-arginine, NADPH: oxygen oxidoreductase (NO-forming), EC 1.14.13.39] in normal rats is followed by increased bone resorption reflected by a marked loss in bone mineral density. In our present study, immunocytochemistry and Northern blotting show the presence of the constitutive calcium-sensitive NOS isoform (cNOS) in normal rat osteoclasts and in the human preosteoclast cell line (FLG 29.1). The inducible NOS isoform (iNOS) was also clearly demonstrable in the rat cells especially after treatment with gamma interferon (IFN-gamma) and bacterial wall products [lipopolysaccharide (LPS)], while a basal level of transcript was detected in the untreated human preosteoclast line. However NADPH-diaphorase activity was intense only in neonatal rat osteoclasts attached to bone, perhaps reflecting either enhancement of cNOS activity by calcium or increased amounts of the inducible isoform in activated osteoclasts in situ compared with isolated neonatal rat osteoclasts. These actively resorb devitalized bone but the untreated cells contain relatively low levels of NOS; they are extremely sensitive to inhibition by NO. The iNOS inhibitor aminoguanidine markedly enhances in vitro resorption by activated NOS-rich chick osteoclasts and by normal rat osteoclasts treated with LPS or IFN-gamma. In contrast, the nonselective NOS inhibitor NG-monomethyl-L-arginine inhibits resorption by untreated neonatal rat osteoclasts. Thus, osteoclast function may require intermittent calcium-stimulated increases in NO production by cNOS against a basal inhibitory background activity of the iNOS isoform. However, bone resorption depends on precursor replication and on the activity of the mature cells, and we found that the NO donor 3-morpholinosydnonimine (SIN-1) (50 microM) profoundly depressed replication in the human preosteoclast line. Taken together, these results strongly suggest that NO maintains a central control of bone resorption in both avian and mammalian species by exerting a powerful tonic restraint of osteoclast numbers and activity. The presence of NOS in human cells implies a similar function in man and that conventional views of calcium homoeostasis and skeletal metabolism will need substantial revision. Since NO also influences behavior of the osteoblast, the bone-forming cell, in vitro, a similar effect in vivo might imply a general influence on bone remodeling.
A single residue can modify target-binding affinity and activity of the functional domain of the Rho-subfamily GDP dissociation inhibitors.
Authors: Platko J V , Leonard D A , Adra C N , Shaw R J , Cerione R A , Lim B
Published: May 2006

The GDP dissociation inhibitors (GDIs) represent an important class of regulatory proteins for the Rho- and Rab-subtype GTP-binding proteins. As a first step toward identifying the key functional domain(s) on the Rho-subtype GDI, truncations of the amino and carboxyl termini were performed. Deletion of the final four amino acids from the carboxyl terminus of Rho GDI or the removal of 25 amino acids from the amino terminus had no significant effect on the ability of the GDI to inhibit GDP dissociation from the Rho-like protein Cdc42Hs or on its ability to release Cdc42Hs from membrane bilayers. However, the deletion of 8 amino acids from the carboxyl terminus of Rho GDI eliminated both activities. To further test the importance of the carboxyl-terminal domain of the Rho GDI molecule, chimeras were constructed between this GDI and a related protein designated LD4, which is 67% identical to Rho GDI but is less potent by a factor of 10-20 than Rho GDI in functional assays with the Cdc42Hs protein. Two sets of chimeras were constructed that together indicated that as few as 6 amino acids near the carboxyl terminus of Rho GDI could impart full GDP dissociation inhibition and membrane dissociation activities on the LD4 molecule. Further analysis of this region by site-directed mutagenesis showed that a single change at residue 174 of LD4 to the corresponding residue of Rho GDI (i.e., Asp-174-->Ile) could impart nearly full (70%) Rho GDI activity on the LD4 molecule.
Immunoregulatory activity of a T-cell receptor alpha chain demonstrated by in vitro transcription and translation.
Authors: Onda T , Brunner T , Messier H , Bissonnette R , Echeverri F , Baier G , Fotedar A , Green D R
Published: May 2006

Previous studies from our laboratory and those of others suggested the possibility that the T-cell antigen receptor alpha (TCR alpha) chain from some T cells can be released in a soluble form and can have antigen-specific immunoregulatory activity. We have analyzed this phenomenon by in vitro transcription and translation (IVTT) of a cDNA encoding a TCR alpha chain (A1.1 TCR alpha) suspected of having such activity. We found that TCR alpha, but not TCR beta, protein produced in this way showed antigen-specific regulatory activity in an in vitro immune-response assay. Protein derived from truncated forms of the A1.1 TCR alpha cDNA had activity providing that, in addition to the variable (V) and joining (J) regions of the alpha chain (VJ alpha), at least the first 25 amino acids of the alpha chain of the constant (C) region (C alpha) were present. Addition of an irrelevant protein sequence to the VJ alpha failed to impart activity to the molecule, suggesting that the C alpha requirement is not simply for stabilization of the resulting protein. These results are discussed in the context of other recent studies on the immunoregulatory activity of soluble TCR alpha molecules, and the possible physiological relevance of these observations is considered.
Retinoic acid inhibition of ex vivo human immunodeficiency virus-associated apoptosis of peripheral blood cells.
Authors: Yang Y , Bailey J , Vacchio M S , Yarchoan R , Ashwell J D
Published: May 2006

T cells from human immunodeficiency virus (HIV)-infected individuals undergo spontaneous and activation-induced ex vivo apoptosis. Here we report that peripheral blood mononuclear cells (PBMCs) obtained from six HIV-infected individuals exhibited reduced ex vivo DNA fragmentation and cell death after ingestion of all-trans-retinoic acid (tRA). These effects were attenuated with continued daily RA administration, which correlated with a > 5-fold decrease in serum peak RA concentrations. Incubation of PBMCs from HIV+ individuals with tRA in vitro resulted in decreased DNA fragmentation in a subset of patients, especially those having < 500 CD4+ T cells per mm3. tRA also inhibited apoptosis of preactivated normal PBMCs induced to die by restimulation, which raises the possibility of a common mechanism between activation-induced apoptosis of activated normal PBMCs and apoptosis associated with HIV infection. Whether HIV-associated apoptosis of PBMCs, and its prevention by RA, has an impact on T-cell survival or the course of disease in patients infected with HIV will require further evaluation.
Pex1, a pollen-specific gene with an extensin-like domain.
Authors: Rubinstein A L , Broadwater A H , Lowrey K B , Bedinger P A
Published: May 2006

We report here the identification of a pollen-specific gene from Zea mays that contains multiple Ser-(Pro)n repeats, the motif found in the cell wall-associated extensins. Sequence analysis reveals that the encoded protein has a putative globular domain at the N terminus and an extensin-like domain at the C terminus. The Pex1 (pollen extensin-like) gene is expressed exclusively in pollen, not in vegetative or female tissues, and is not induced in leaves upon wounding. We propose that the encoded protein may have a role in reproduction, either as a structural element deposited in the pollen tube wall during its rapid growth or as a sexual recognition molecule that interacts with partner molecules in the pistil.