bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the human alpha-globin gene cluster in transgenic mice.
Authors: Sharpe J A , Wells D J , Whitelaw E , Vyas P , Higgs D R , Wood W G
Published: May 2006

A 350-bp segment of DNA associated with an erythroid-specific DNase I-hypersensitive site (HS-40), upstream of the alpha-globin gene cluster, has been identified as the major tissue-specific regulator of the alpha-globin genes. However, this element does not direct copy number-dependent or developmentally stable expression of the human genes in transgenic mice. To determine whether additional upstream hypersensitive sites could provide more complete regulation of alpha gene expression we have studied 17 lines of transgenic mice bearing various DNA fragments containing HSs -33, -10, -8, and -4, in addition to HS -40. Position-independent, high-level expression of the human zeta- and alpha-globin genes was consistently observed in embryonic erythroid cells. However, the additional HSs did not confer copy-number dependence, alter the level of expression, or prevent the variable down-regulation of expression in adults. These results suggest that the region upstream of the human alpha-globin genes is not equivalent to that upstream of the beta locus and that although the two clusters are coordinately expressed, there may be differences in their regulation.
Importance of independence in ribozyme reactions: kinetic behavior of trimmed and of simply connected multiple ribozymes with potential activity against human immunodeficiency virus.
Authors: Ohkawa J , Yuyama N , Takebe Y , Nishikawa S , Taira K
Published: May 2006

The kinetic behavior of ribozymes derived from two types of multiple-ribozyme expression vector were examined. In some cases, multiple ribozymes were expressed as a single RNA molecule and all the ribozymes were simply connected in tandem (connected type). In other cases, multiple ribozymes were flanked by cis-acting ribozymes at both their 5' and 3' ends so that, upon transcription, multiple ribozymes were trimmed at both their 5' and 3' ends, with resultant liberation of multiple independent ribozymes (shotgun type). When levels of ribozyme expression were examined for the shotgun-type vector, the level of the ribozyme transcript was found to be proportional to the number of units (n) connected in tandem. Accordingly, the activities of the shotgun-type ribozymes, in terms of the cleavage of HIV-1 RNA in vitro, were also found to be proportional to the number of units connected in tandem (n). By contrast, the activities of the connected-type ribozymes reached plateau values at around n = 3. These results indicate that, when the shotgun-type expression system is used, it is theoretically possible to generate various independent ribozymes, each specific for a different target site, without sacrificing the activity of any individual ribozyme.
Association and phosphorylation-dependent dissociation of proteins in the insulin receptor complex.
Authors: Zhang W , Johnson J D , Rutter W J
Published: May 2006

Receptor tyrosine kinases have been found to interact with a variety of specific signaling molecules. To detect molecules that interact with the insulin receptor, we have produced a modified insulin receptor with an additional epitope allowing rapid purification under mild conditions of the insulin receptor complex. By this method we have found multiple proteins (including the p85 subunit of phosphatidylinositol 3'-kinase and the ras GTPase-activating protein) that specifically associate with the activated (phosphorylated) insulin receptor (insulin receptor complex proteins) but are released from the complex after they are phosphorylated on tyrosine residues. We have also shown that tyrosine phosphorylation of p85 by the activated insulin receptor blocks binding to the activated receptor. These results suggest that association of proteins with the insulin receptor complex is controlled by phosphorylation of the receptor, while dissociation of insulin receptor complex proteins is controlled in turn by phosphorylation of the proteins in the insulin receptor complex. This process results in the dispersion of phosphorylated insulin receptor complex proteins into the cell.
Importance of receptor occupancy, concentration differences, and ligand exchange in the insulin-like growth factor I receptor system.
Authors: Zhong P , Cara J F , Tager H S
Published: May 2006

We have investigated by use of placental membranes the mechanisms through which insulin-like growth factor I (IGF-I) comes to be associated with its alpha 2 beta 2 receptor heterotetramer. Our results suggest that (i) at low ligand concentrations, the formation and disruption of IGF-I--receptor complexes are consistent with ligand binding de novo to empty receptors but not with equilibria involving ligand dissociation; (ii) at higher ligand concentrations, rapid exchange arising from the formation and collapse of bis-liganded receptors leads to a transiently perturbed receptor state; (iii) these nonclassical IGF-I receptor interactions depend on close communication between the alpha beta halves of the alpha 2 beta 2 holo-IGF-I receptor; and (iv) related processes based on ligand exchange have the potential for serving as biological sensors of changes in ligand concentration, while ordinary binding processes serve as sensors of ligand concentrations themselves. A model is presented in which one or two molecules of ligand can be bound to an alpha 2 beta 2 IGF-I receptor heterotetramer, new ligand becomes associated with receptor by exchanging for a previously bound molecule of IGF-I, and fluctuating changes in free-ligand concentration might lead to enhanced IGF-I function.
Age-dependent appearance of non-major histocompatibility complex-restricted helper T cells.
Authors: Russo C , Cherniack E P , Wali A , Weksler M E
Published: May 2006

T cells which recognize antigen in association with self major histocompatibility complex (MHC) molecules are positively selected within the thymus. This results in skewing of the T-cell repertoire toward the recognition of antigenic peptides presented by self MHC molecules. While the thymus gland involutes at a relatively young age, bone marrow function and the size of the peripheral T-cell pool are maintained with age. We have investigated the MHC restriction of helper T-cell function for B-cell production of specific antibody in mice of different ages. Splenic helper T cells from 2- to 3-month old mice immunized with phosphocholine-keyhole limpet hemocyanin conjugate were MHC-restricted as defined by their capacity to induce phosphocholine-specific antibody synthesis by syngeneic but not by allogeneic B cells. In contrast, splenic T cells from immunized 18- to 20-month-old mice induced specific anti-phosphocholine antibodies by both syngeneic and allogeneic B cells. No evidence of polyclonal immunoglobulin synthesis was detected. The ability of T cells from old mice immunized with phosphocholine-keyhole limpet hemocyanin to induce phosphocholine-specific antibody synthesis by B cells from allogeneic mice was inhibited by T cells from immunized young mice. These findings suggest that non-MHC-restricted T-cell helper activity in old mice results from the loss of T cells, present in young mice, which suppress non-MHC-restricted helper cells.
The accessory bacteriochlorophyll: a real electron carrier in primary photosynthesis.
Authors: Arlt T , Schmidt S , Kaiser W , Lauterwasser C , Meyer M , Scheer H , Zinth W
Published: May 2006

The primary electron transfer in reaction centers of Rhodobacter sphaeroides is studied by subpicosecond absorption spectroscopy with polarized light in the spectral range of 920-1040 nm. Here the bacteriochlorophyll anion radical has an absorption band while the other pigments of the reaction center have vanishing ground-state absorption. The transient absorption data exhibit a pronounced 0.9-ps kinetic component which shows a strong dichroism. Evaluation of the data yields an angle between the transition moments of the special pair and the species related with the 0.9-ps kinetic component of 26 +/- 8 degrees. This angle compares favorably with the value of 29 degrees expected for the reduced accessory bacteriochlorophyll. Extensive transient absorbance data are fully consistent with a stepwise electron transfer via the accessory bacteriochlorophyll.
A large cell-adhesive scatter factor secreted by human gastric carcinoma cells.
Authors: Miyazaki K , Kikkawa Y , Nakamura A , Yasumitsu H , Umeda M
Published: May 2006

Human gastric carcinoma cell line STKM-1 secretes a large protein that induces scattering of a rat liver epithelial cell line (BRL) into disconnected individual cells in monolayer culture. This cell-scattering factor was purified from serum-free conditioned medium of STKM-1 cells and found to be composed of three disulfide-linked subunits of 140, 150, and 160 kDa. The 140-kDa peptide contains an amino acid sequence homologous to that of the laminin B2t chain. The native protein has an apparent molecular mass of > 1000 kDa and a pI of 5.0. In addition to the cell-scattering activity, the purified protein stimulates attachment of BRL cells to substrate and their migration. Similar effects have been observed toward various cell lines, including nontumorigenic epithelial, endothelial, and fibroblastic cell lines and human cancer cell lines. Similar cell-scattering activity was secreted by human squamous carcinoma and gastric carcinoma cell lines and nontumorigenic epithelial and endothelial cell lines. These results indicate that the protein, named "ladsin," is probably an extracellular matrix protein that regulates cell-cell and cell-substrate interactions and cell migration.
Primer-mediated in situ detection of the B-hordein gene cluster on barley chromosome 1H.
Authors: Abbo S , Dunford R P , Miller T E , Reader S M , King I P
Published: May 2006

In situ hybridization methods allow the detection of specific DNA sequences on whole chromosomes. The technique has been widely used as a diagnostic and research tool by animal cytogeneticists, for whom detection of unique sequences on mammalian chromosomes is routinely achieved. However, detection of unique sequences on plant chromosomes is less reliable. The recently developed primer-induced in situ hybridization (PRINS) technique allows rapid and reliable in situ detection by the hybridization of primers to denatured target DNA, followed by extension with DNA polymerase in the presence of a labeled nucleotide. The use of short oligonucleotide primers could allow improved penetration of debris and highly condensed chromatin common in preparations of plant chromosomes, thus increasing the sensitivity of in situ detection. The feasibility of this approach is demonstrated by the oligonucleotide primer-mediated detection of the B-hordein gene cluster on a barley chromosome. Applications of the PRINS technique for plant cytogeneticists are discussed.
Metabolic correction and cross-correction of mucopolysaccharidosis type II (Hunter syndrome) by retroviral-mediated gene transfer and expression of human iduronate-2-sulfatase.
Authors: Braun S E , Aronovich E L , Anderson R A , Crotty P L , McIvor R S , Whitley C B
Published: May 2006

To explore the possibility of using gene transfer to provide iduronate-2-sulfatase (IDS; EC 3.1.6.13) enzyme activity for treatment of Hunter syndrome, an amphotropic retroviral vector, L2SN, containing the human IDS coding sequence was constructed and studied for gene expression in vitro. Lymphoblastoid cell lines (LCLs) from patients with Hunter syndrome were transduced with L2SN and expressed high levels of IDS enzyme activity, 10- to 70-fold higher than normal human peripheral blood leukocytes or LCLs. Such L2SN-transduced LCLs failed to show accumulation of 35SO4 into glycosaminoglycan (35SO4-GAG), indicating that recombinant IDS enzyme participated in GAG metabolism. Coculture of L2SN-transduced LCLs with fibroblasts from patients with Hunter syndrome reduced the accumulation of 35SO4-GAG. These results demonstrated retroviral-mediated IDS gene transfer into lymphoid cells and the ability of such cells to provide recombinant enzyme for intercellular metabolic cross-correction.
Tyrosine kinase activity of CD4-associated p56lck may not be required for CD4-dependent T-cell activation.
Authors: Collins T L , Burakoff S J
Published: May 2006

The lymphoid-specific tyrosine kinase p56lck (Lck) is critical for the development and activation of T lymphocytes, and Lck kinase activity has been implicated in both T-cell antigen receptor/CD3- and CD4-mediated signaling. CD4-dependent T-cell activation has been demonstrated to be dependent upon the association of CD4 with Lck. To examine the role of the kinase activity of Lck in CD4-dependent T-cell activation, we have generated several kinase-deficient mutants of Lck. When transfected into CD4+ murine T-cell hybridoma cells, these mutants cause approximately 90% diminution in CD4-associated Lck kinase activity. Specifically, upon CD4 crosslinking there is decreased Lck autophosphorylation and decreased phosphorylation of an exogenous substrate. When CD4 is crosslinked to the T-cell antigen receptor-CD3 complex, decreased phosphorylation of associated substrates is also observed. In spite of this striking inhibition of Lck kinase function, cells expressing the kinase-deficient mutants demonstrate normal or enhanced CD4-dependent antigen responsiveness. These data demonstrate that the level of Lck kinase activity does not correlate with its CD4-associated function and suggest that the kinase activity of Lck may not be required for CD4-mediated signaling.
Cloning and characterization of subunit genes of ribonucleotide reductase, a cell-cycle-regulated enzyme, from Plasmodium falciparum.
Authors: Chakrabarti D , Schuster S M , Chakrabarti R
Published: May 2006

Ribonucleotide reductase (EC 1.17.4.1; RNR), a cell-cycle-regulated enzyme, catalyzes the rate-limiting step in the de novo synthesis of deoxyribonucleotides by the reduction of the corresponding ribonucleotides. The important role of the RNR in DNA synthesis and cell division makes this enzyme an excellent target for chemotherapy. However, nothing is known about this enzyme from the malaria parasite Plasmodium falciparum. We have isolated cDNA clones encoding both the large and small RNR subunits. The sequences of full-length clones of the large and small RNR subunits revealed an open reading frame encoding 806 and 349 amino acids, respectively, and showed significant identity with other RNR sequences in the data base. RNA blot analysis showed that the size of the large and small RNR subunit transcripts are 5.4 kb and 2.2 kb, respectively. Both the RNR subunit transcripts fluctuate in level during the cell cycle, reaching a peak preceding maximal DNA synthesis activity. An oligodeoxynucleotide phosphorothioate that is complementary to sequences around the translational initiation codon of the small RNR subunit showed significant inhibition of growth, as measured by the inhibition in DNA synthesis.
Escherichia coli cell division protein FtsZ is a guanine nucleotide binding protein.
Authors: Mukherjee A , Dai K , Lutkenhaus J
Published: May 2006

FtsZ is an essential cell division protein in Escherichia coli that forms a ring structure at the division site under cell cycle control. The dynamic nature of the FtsZ ring suggests possible similarities to eukaryotic filament forming proteins such as tubulin. In this study we have determined that FtsZ is a GTP/GDP binding protein with GTPase activity. A short segment of FtsZ is homologous to a segment in tubulin believed to be involved in the interaction between tubulin and guanine nucleotides. A lethal ftsZ mutation, ftsZ3 (Rsa), that leads to an amino acid alteration in this homologous segment decreased GTP binding and hydrolysis, suggesting that interaction with GTP is essential for ftsZ function.
Cyclic amphipathic peptide-DNA complexes mediate high-efficiency transfection of adherent mammalian cells.
Authors: Legendre J Y , Szoka F C
Published: May 2006

A DNA transfection protocol has been developed that makes use of the cyclic cationic amphipathic peptide gramicidin S and dioleoyl phosphatidylethanolamine. The DNA complex is formed by mixing gramicidin S with DNA at a 1:1 charge ratio and then adding phosphatidylethanolamine at a lipid/peptide molar ratio of 5:1. The complex mediates rapid association of DNA with cells and leads to transient expression levels of beta-galactosidase ranging from 1 to 30% of the transfected cells, with long-term expression being about an order of magnitude lower. The respective roles of peptide and phospholipid are not yet resolved but optimal transfection requires both the cyclic peptide and the hexagonal phase-competent phospholipid PtdEtn. Transfection in CV-1 cells is not affected by lysomotrophic agents, which suggests that DNA entry into the cell is via the plasma membrane. This technique that is simple, economical, and reproducible mediates transfection levels up to 20-fold higher than cationic liposomes in adherent mammalian cells.
sid1, a gene initiating siderophore biosynthesis in Ustilago maydis: molecular characterization, regulation by iron, and role in phytopathogenicity.
Authors: Mei B , Budde A D , Leong S A
Published: May 2006

Iron uptake in Ustilago maydis is mediated by production of extracellular hydroxamate siderophores. L-Or-nithine N5-oxygenase catalyzes hydroxylation of L-ornithine, which is the first committed step of ferrichrome and ferrichrome A biosynthesis in U. maydis. We have characterized sid1, a gene coding for this enzyme, by complementation in trans, gene disruption, and DNA sequence analysis. A comparison of genomic DNA and cDNA sequences has shown that the gene is interrupted by three introns. The putative amino acid sequence revealed similarity with Escherichia coli lysine N6-hydroxylase, which catalyzes the hydroxylation of lysine, the first step in biosynthesis of aerobactin. Two transcription initiation points have been determined, both by PCR amplification of the 5' end of the mRNA and by primer extension. A 2.3-kb transcript which accumulates in cells grown under low iron conditions was detected by Northern hybridization. A less abundant 2.7-kb transcript was observed in cells grown in iron-containing medium. By contrast, constitutive accumulation of the 2.3-kb transcript was observed in a mutant carrying a disruption of urbs1, a gene involved in regulation of siderophore biosynthesis. Analysis of the pathogenicity of mutants carrying a null allele of sid1 suggests that the biosynthetic pathway of siderophores does not play an essential role in the infection of maize by U. maydis.
Inhibitors of two-component signal transduction systems: inhibition of alginate gene activation in Pseudomonas aeruginosa.
Authors: Roychoudhury S , Zielinski N A , Ninfa A J , Allen N E , Jungheim L N , Nicas T I , Chakrabarty A M
Published: May 2006

Pseudomonas aeruginosa strains infecting cystic fibrosis patients often produce copious amounts of the exopolysaccharide alginate. Expression of alginate genes in P. aeruginosa is regulated by several proteins including members of the two-component bacterial signal transduction systems. Two of these regulatory proteins are AlgR1, the DNA-binding response regulator that transcriptionally activates alginate gene expression, and AlgR2, the kinase that modifies AlgR1 via phosphorylation to enhance its activity. In this paper, we report the identification of compounds that inhibit alginate gene expression by inhibiting (i) the phosphorylation/dephosphorylation of AlgR2 and (ii) the DNA-binding activity of AlgR1. Compounds with these activities may have potential as components of therapy for eliminating P. aeruginosa infection from the cystic fibrosis lung. In addition, we describe the effect of these compounds on the autophosphorylation activity of other known two-component kinases and show the ability of one compound to significantly inhibit the kinase activities of CheA, NRII, and KinA.
A genetic approach to the generation of antibodies with enhanced catalytic activities.
Authors: Lesley S A , Patten P A , Schultz P G
Published: May 2006

A hydrolytic catalytic antibody, generated against a nitrophenyl phosphonate transition state analogue, has been cloned and expressed in Escherichia coli for use as a model system to demonstrate the feasibility of using genetic selections to enhance catalytic activity. Conditions were found that permit the secretion of active recombinant antibody into the periplasm of a strain of E. coli deficient in the biotin biosynthetic genes (delta bio-gal). A number of substrates were synthesized that, upon hydrolysis by the antibody, yield free biotin, which is required for cell growth. The substrates and selections can be used to identify mutants of the antibody with altered activities. This approach should be generalizable to a wide number of hydrolytic reactions including the selective cleavage of peptide, polysaccharide, phosphodiester, and ester bonds.
Histidine-226 is part of the pH sensor of NhaA, a Na+/H+ antiporter in Escherichia coli.
Authors: Gerchman Y , Olami Y , Rimon A , Taglicht D , Schuldiner S , Padan E
Published: May 2006

The nhaA gene of Escherichia coli, which encodes a pH-activated Na+/H+ antiporter, has been modified; six of its eight histidine codons were mutated to arginine codons by site-directed mutagenesis, yielding the mutations H254R-H257R (a double mutant), H226R, H39R, H244R, and H319R. In addition a deletion (delta nhaA1-14) lacking the remaining two histidines, His-3 and His-5, has been constructed. By comparing the phenotypes conferred by plasmids bearing the various mutations to the phenotype of the wild type upon transformation of strains NM81 (delta nhaA) or EP432 (delta nhaA, delta nhaB) we found that none of the NhaA histidines are essential for the Na+/H+ antiporter activity of the NhaA protein. However, the replacement of His-226 by Arg markedly changes the pH dependence of the antiporter. All mutants except H226R confer to NM81 and EP432 Na+ resistance up to pH 8.5 as well as Li+ resistance. Cells bearing H226R are resistant to Li+ and to Na+ at neutral pH, but they become sensitive to Na+ above pH 7.5. Analysis of the Na+/H+ antiporter activity of membrane vesicles derived from H226R cells shows that the mutated protein is activated by pH to the same extent as the wild type. However, whereas the activation of the wild-type NhaA occurs between pH 7 and pH 8, that of H226R antiporter occurs between pH 6.5 and pH 7.5. Furthermore, while the wild-type antiporter remains almost fully active at least up to pH 8.5, H226R is reversibly inactivated above pH 7.5, reaching 10-20% of the maximal activity at pH 8.5. We suggest that His-226 is part of a pH-sensitive site that regulates the activity of NhaA.
Histidine residue in the zinc-binding motif of aminopeptidase A is critical for enzymatic activity.
Authors: Wang J , Cooper M D
Published: May 2006

The murine BP-1 antigen (also called 6C3) is a homodimeric, phosphorylated cell surface glycoprotein that is expressed on immature B-lineage cells, bone marrow stromal cell lines, thymic cortical epithelial cells, endothelial cells, enterocytes, and renal proximal tubular cells. The amino acid sequence deduced from a BP-1 cDNA predicted a type II integral membrane protein with a zinc-binding motif (His-Glu-Xaa-Xaa-His) found in zinc-dependent metallopeptidases, and functional analysis suggested that BP-1 is aminopeptidase A [APA; L-alpha-aspartyl(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7]. Here we constructed an expression vector in which the BP-1 cDNA was placed downstream from the SR alpha promoter and used this construct to transfect COS-7 and Ltk- cells. Both transfectants expressed the BP-1 antigen on the cell surface and APA activity. The enzymatic activity of recombinant APA was increased by Ca2+ and inhibited by Zn2+, consistent with previous reports with purified APA. Point mutation of one of the histidine residues in the zinc-binding motif to phenylalanine completely abolished APA enzymatic activity, suggesting the structure of the zinc-binding motif of APA is critical for catalytic activity. Both wild-type and mutant BP-1 were glycosylated, transported to the cell surface, and possessed molecular weights similar to native BP-1 molecules on the murine 18.81 pre-B-cell line. The successful expression of both wild-type and mutant APA should allow more precise analysis of the diverse physiological roles of this ectoenzyme.
Daidzin: a potent, selective inhibitor of human mitochondrial aldehyde dehydrogenase.
Authors: Keung W M , Vallee B L
Published: May 2006

Human mitochondrial aldehyde dehydrogenase (ALDH-I) is potently, reversibly, and selectively inhibited by an isoflavone isolated from Radix puerariae and identified as daidzin, the 7-glucoside of 4',7-dihydroxyisoflavone. Kinetic analysis with formaldehyde as substrate reveals that daidzin inhibits ALDH-I competitively with respect to formaldehyde with a Ki of 40 nM, and uncompetitively with respect to the coenzyme NAD+. The human cytosolic aldehyde dehydrogenase isozyme (ALDH-II) is nearly 3 orders of magnitude less sensitive to daidzin inhibition. Daidzin does not inhibit human class I, II, or III alcohol dehydrogenases, nor does it have any significant effect on biological systems that are known to be affected by other isoflavones. Among more than 40 structurally related compounds surveyed, 12 inhibit ALDH-I, but only prunetin and 5-hydroxydaidzin (genistin) combine high selectivity and potency, although they are 7- to 15-fold less potent than daidzin. Structure-function relationships have established a basis for the design and synthesis of additional ALDH inhibitors that could both be yet more potent and specific.
DNA homology requirements for mitotic gap repair in Drosophila.
Authors: Nassif N , Engels W
Published: May 2006

We used P transposable-element mobilization to study the repair of double-strand DNA breaks in Drosophila melanogaster premeiotic germ cells. Distribution of conversion tracts was found to be largely unaffected by changes in the length of sequence homology between the broken ends and the template, suggesting that only a short match is required. However, the frequency of repair was highly sensitive to single-base mismatches within the homologous region, ranging from 19% reversion when there were no mismatches to 5% when 15 mismatches were present over a 3455-bp span.
Single-cell transplantation determines the time when Xenopus muscle precursor cells acquire a capacity for autonomous differentiation.
Authors: Kato K , Gurdon J B
Published: May 2006

We have used a single-cell transplantation technique to find out whether there is a stage in development when a single cell can reach and maintain its differentiated state in the absence of its neighbors. Muscle precursor cells from early, mid-, and late gastrula stages of Xenopus laevis embryos were isolated and transplanted singly into the ventral region of late gastrula hosts. Single cells from late gastrulae differentiated into muscle when surrounded by nonmuscle cells. Similar cells from early or mid-gastrulae did not, unless they were transplanted as a group of adjacent cells taken from the same region of an embryo. These results show that single embryonic cells in a tissue can complete their differentiation without interacting with their normal neighbors and that, in the case of Xenopus muscle precursor cells, they acquire this capacity at the late gastrula stage. Our results also suggest that, in addition to mesoderm induction, further cell interactions during gastrulation are required for Xenopus muscle cell differentiation.
Calcium binding proteins distinguish large and small cells of the ventral posterior and lateral geniculate nuclei of the prosimian galago and the tree shrew (Tupaia belangeri).
Authors: Diamond I T , Fitzpatrick D , Schmechel D
Published: May 2006

Two different cell types were identified in the thalamus of galago and Tupaia by using antibodies to two calcium binding proteins, calbindin and parvalbumin. In each species studied, the lateral geniculate nucleus consists of six layers, two of which have smaller relay cells. Previous studies have shown that the small cell layers receive fibers from the superior colliculus and project to the superficial layers of the striate cortex. These are the only geniculate layers that react to a calbindin antibody but not parvalbumin. The ventral posterior nucleus was included in the study and the results for both nuclei show that calbindin is a marker for thalamic cells that receive small fibers and project to superficial layers of koniocortex.
Reduction of exogenous vasopressin RNA poly(A) tail length increases its effectiveness in transiently correcting diabetes insipidus in the Brattleboro rat.
Authors: Maciejewski-Lenoir D , Jirikowski G F , Sanna P P , Bloom F E
Published: May 2006

Magnocellular hypothalamic neurons in Brattleboro rats can accumulate, transport, and translate exogenous [Arg8]vasopressin (AVP) mRNA after injection in the hypothalamo-hypophysial tract in amounts sufficient to reverse transiently the animals' characteristic diabetes insipidus. In the present study, different preparations of hypothalamic RNA extracted from normal rats or synthetic AVP RNA were injected into the lateral hypothalamus of Brattleboro rats. Poly(A)- RNA and poly(A)+ RNA from which tails were removed by RNase H digestion were much more effective than poly(A)+ RNA in expressing AVP in the magnocellular hypothalamic neurons and in raising urine osmolarity. Synthetic AVP RNA lacking a poly(A) tail also produced a very potent dose-dependent diabetes insipidus reversal. Our results suggest that a short or absent poly(A) tail may facilitate the accumulation, transport, or expression of exogenous AVP mRNA by magnocellular neurons.
Molecular basis of transabdominal--a sexually dimorphic mutant of the bithorax complex of Drosophila.
Authors: Celniker S E , Lewis E B
Published: May 2006

Transabdominal (Tab) is a dominant gain-of-function mutation that results in islands of sexually dimorphic abdominal cuticle in the dorsal thorax of the adult fly. This phenotype has complete penetrance and constant expressivity, and we show that it results from ectopic expression of ABD-BII, one of two proteins derived from the Abdominal B (Abd-B) domain of the bithorax complex (BX-C) and one that is normally expressed only in terminal portions of the abdomen. In Tab/+ animals ABD-BII is ectopically expressed in the relevant imaginal "wing" disc as three islands of cells whose location on the fate map corresponds to the three islands of transformed cuticle in each half of the adult thorax. Tab is associated with an inseparable inversion bringing sequences in 90E next to sequences in the transcription unit encoding ABD-BII in 89E. That 90E sequences drive ectopic expression of ABD-BII is indicated by our finding that such sequences in a P-element transformant express the reporter gene's product (beta-galactosidase) in the same three islands of wing disc cells. On morphological grounds, the transformed islands in the adult thorax correspond to subsets of muscle attachment cells. Ectopic expression of a homeodomain protein thus creates a unique and invariant pattern of sexual dimorphism.
Normal development and growth of mice carrying a targeted disruption of the alpha 1 retinoic acid receptor gene.
Authors: Li E , Sucov H M , Lee K F , Evans R M , Jaenisch R
Published: May 2006

Three unlinked genes encode receptors for retinoic acid (RAR alpha, -beta, and -gamma). Each gene expresses two major protein isoforms differing in the amino terminal A domain by alternative promoter use, fused to common exons encoding most of the receptor protein. The two RAR alpha transcripts (RAR alpha 1 and -alpha 2) are differentially expressed and evolutionarily conserved, as are the RAR beta and -gamma transcripts, suggesting that each isoform may have specific functions in the development of animals. To address the biological function of the alpha 1 receptor, we have disrupted the portion of the RAR alpha gene encoding this isoform by homologous recombination in mouse embryonic stem cells. Surprisingly, offspring homozygous for this mutation were viable and showed no apparently altered phenotype. RNA analysis confirmed that the RAR alpha 1 transcript was absent in homozygous tissues, and no evidence for a compensatory increase of RAR alpha 2 or of another RAR gene was obtained to account for the vitality of the mutant animals. These results clearly demonstrate that loss of RAR alpha 1 function does not disrupt embryonic development and argue for combinatorial or overlapping functions among the RAR isoforms.
Stimulation of T cells by antigenic peptide complexed with isolated chains of major histocompatibility complex class II molecules.
Authors: Nag B , Wada H G , Deshpande S V , Passmore D , Kendrick T , Sharma S D , Clark B R , McConnell H M
Published: May 2006

Major histocompatibility complex (MHC) class II molecules are heterodimeric glycoproteins with one alpha and one beta polypeptide chain of similar molecular size. In this report, we describe the binding of an acetylated N-terminal peptide of myelin basic protein, [Ala4]MBP-(1-14), to purified individual alpha and beta chains of murine I-Ak molecules. Purified complexes of isolated single chains and antigenic peptide bind to cloned T cells restricted by I-Ak and [Ala4]MBP-(1-14) tetradecapeptide. The binding is blocked by alpha/beta anti-T-cell receptor (TCR) monoclonal antibody. Cell triggering as measured by an increase in extracellular acidification rate is observed when cloned T cells are exposed to purified complexes of isolated chains and antigenic peptide. This increase in the extracellular acidification rate is antigen specific and MHC-restricted, as chains alone or irrelevant chain-peptide complexes do not trigger an increase in the metabolic acidification rate. These results together demonstrate that in vitro cloned T cells are triggered by complexes of specific antigenic peptides and isolated individual chains of their cognate MHC proteins.
Tissue-specific regulation of bovine heart cytochrome-c oxidase activity by ADP via interaction with subunit VIa.
Authors: Anthony G , Reimann A , Kadenbach B
Published: May 2006

The activity of reconstituted cytochrome-c oxidase (EC 1.9.3.1) from bovine heart is stimulated by intraliposomal ADP but not by NaCl of the same ionic strength. A monoclonal antibody which reacts with subunits VIa-H (heart-type) and VIc, due to the evolutionary relationship between these subunits, also stimulates the activity of the enzyme from bovine heart but not from bovine liver. The antibody induces a conformational change in the heart enzyme but not in the liver enzyme, as shown by the visible difference spectrum. Preincubation of heart cytochrome-c oxidase with the antibody prevents stimulation of activity by intraliposomal ADP after reconstitution in liposomes. Reconstituted liver cytochrome c oxidase is not stimulated by intraliposomal ADP. The data suggest tissue-specific regulation of the activity of cytochrome-c oxidase by ADP via interaction with the matrix domain of subunit VIa-H.
Dominant lethal phenotype of a mutation in the -35 recognition region of Escherichia coli sigma 70.
Authors: Keener J , Nomura M
Published: May 2006

A dominant lethal mutation in the Escherichia coli rpoD gene, which encodes sigma 70, the promoter recognition subunit of RNA polymerase, was isolated after random mutagenesis. The lethal gene was maintained under control of the lac repressor on a low copy plasmid. An amount of lethal sigma 70 that was nearly equimolar with the chromosomally encoded sigma 70 was sufficient to cause cessation of growth. RNA synthesis per unit cell mass was unaffected, but protein synthesis was inhibited by the mutant sigma 70. The amino acid change (Glu-585 to Gln) was in a region of sigma 70 thought to bind the -35 hexamer of the promoter, and the mutant sigma 70 caused increased expression from promoters with nonconsensus bases in the third position of the -35 hexamer. A null mutation of the fis gene could partially suppress the mutant phenotype. These properties are consistent with those expected of a sigma 70 insensitive to growth rate control of rRNA and tRNA promoters.
Glucose sensing in pancreatic islet beta cells: the key role of glucokinase and the glycolytic intermediates.
Authors: German M S
Published: May 2006

The beta cells of the pancreatic islets of Langerhans respond to changes in glucose concentration by varying the rate of insulin synthesis and secretion. Beta cells sense glucose concentration by the levels of the products of glucose catabolism. Distinctive beta-cell proteins glucose transporter 2 and glucokinase catalyze the first two steps in beta-cell glucose catabolism. To test whether either protein controls the sensitivity of the beta cell to glucose by controlling the rate of glucose catabolism, we used gene-transfer techniques to express the isoenzymes glucose transporter 1 and hexokinase I in beta cells and measured the response to glucose of the insulin gene promoter. Cells expressing glucose transporter 1 do not differ significantly from control cells, but in cells expressing hexokinase I, insulin promoter activity increases, reaches a maximum by 1 mM glucose, and does not respond to changes in glucose concentration within the physiologic range. We conclude that glucokinase catalyzes the rate-limiting step of glucose catabolism in beta cells and, therefore, acts as the glucose sensor. Pyruvate, the end product of anaerobic glycolysis, is readily oxidized by mitochondria in normal beta cells but cannot substitute for glucose as a stimulator of insulin synthesis and secretion. We found that pyruvate can stimulate the insulin promoter in cells expressing the bacterial gluconeogenic enzyme phosphoenolpyruvate carboxykinase, which allows the conversion of pyruvate to phosphoenolpyruvate and the earlier intermediates of glycolysis. We conclude that the intermediates of anaerobic glycolysis between fructose 1,6-diphosphate and phosphoenolpyruvate are essential for beta-cell glucose sensing.
Lack of correlation between interspecific divergence and intraspecific polymorphism at the suppressor of forked region in Drosophila melanogaster and Drosophila simulans.
Authors: Langley C H , MacDonald J , Miyashita N , Aguadé M
Published: May 2006

Levels of DNA sequence polymorphism at the suppressor of forked [su(f)] region in natural populations of Drosophila melanogaster and Drosophila simulans are estimated by restriction map analysis. su(f) is located at the base of the euchromatic portion of the X chromosome where the level of crossing-over per physical length is extremely low. In a survey of 55 alleles from three natural populations of D. melanogaster, only 2 restriction sites of 27 hexanucleotide and 108 tetranucleotide restriction sites scored are polymorphic. Among 103 alleles from three natural populations of D. simulans, just one polymorphic restriction site is found in 109 tetranucleotide-recognizing restriction sites scored. The few polymorphisms in these surveys yield estimates of per site heterozygosities (0.00, 0.0002, and 0.0005, respectively) at least a factor of 10 less than the average observed at loci located in regions of the genome with normal levels of crossing-over. Because under a broad category of models of molecular evolution (including the neutral theory) a correlation between levels of polymorphism and interspecific divergence is expected, the DNA sequence divergence is examined for the su(f) region. Contrary to the predicted correlation, the estimated divergence (0.12 substitution per silent site) is, in fact, greater than that observed at loci in regions of normal crossing-over. According to an alternative hypothesis (hitchhiking effect model) intraspecific polymorphism is swept out of the population in regions of the genome closely linked to rare but selectively favored variants as they quickly go to fixation; the rate of divergence is, however, unaffected by these rare hitchhiking events. Thus, the observed paucity of polymorphism and lack of correlation with divergence are in accord with the theory of the hitchhiking effect and several recent reports of polymorphism and divergence in other genomic regions with reduced crossing-over per physical length.
Roles of Ser101, Asp236, and His237 in catalysis of thioesterase II and of the C-terminal region of the enzyme in its interaction with fatty acid synthase.
Authors: Tai M H , Chirala S S , Wakil S J
Published: May 2006

Thioesterase II (TE II), present in specialized tissues, catalyzes the chain termination and release of medium-chain fatty acids from fatty acid synthase [FAS; acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl- and enoyl-reducing and thioester-hydrolyzing), EC 2.3.1.85]. We have expressed rat mammary gland TE II in Escherichia coli and created several site-directed mutants. Replacing both Ser101 and His237 with Ala yielded inactive proteins, suggesting that these residues are part of the catalytic triad as in FAS thioesterase (TE I). Mutating the conserved Asp236 or modifying it with Woodward's reagent K caused partial loss (40%) of TE II activity and reduced reactivity of Ser101 and His237 toward their specific inhibitors, phenylmethylsulfonyl fluoride and diethylpyrocarbonate, respectively. These results suggested that Asp236 enhances, but is not essential for, the reactivity of Ser101 and His237. Mutation analyses revealed that, at the C terminus, Leu262 is critical for TE II to interact with FAS. Hydrophobic interactions seem to play a role, since the interaction of TE II with FAS is enhanced by polyethylene glycol but reduced by salt. The Ser101 and His237 mutants and a synthetic C-terminal decapeptide did not compete in the interaction. These results suggest that a TE II-acyl FAS complex forms first, which then is stabilized by the interaction of the hydrophobic C terminus of TE II with FAS, leading ultimately to hydrolysis and release of fatty acid.
Immunoglobulin D-deficient mice can mount normal immune responses to thymus-independent and -dependent antigens.
Authors: Nitschke L , Kosco M H , Köhler G , Lamers M C
Published: May 2006

To examine the in vivo function of IgD we generated mice deficient for IgD by gene targeting. The IgD-mice show a reduced B-cell compartment with 30-50% less B cells in the spleen and lymph nodes but show a normal pre-B-cell compartment. The surface-IgD- B cells express two to three times more surface IgM than B cells of control animals. Serum concentrations of the immunoglobulin isotypes of IgD- mice are almost normal, indicating that surface-IgD expression is not necessary for class switching of B cells. Immunization experiments showed that IgD- mice could respond well to thymus-dependent and -independent antigens. After immunization normal germinal centers developed in the IgD- mice. These data suggest that IgD is not necessary for the induction of immune responses but may be important in homeostasis of cells in the B-cell compartment.
Gene replacement in Halobacterium halobium and expression of bacteriorhodopsin mutants.
Authors: Krebs M P , Mollaaghababa R , Khorana H G
Published: May 2006

A gene replacement method has been developed to express bacteriorhodopsin mutants in the archaeon Halobacterium halobium. Selectable plasmids carrying the bacterioopsin gene (bop) were integrated at the chromosomal bop locus of H. halobium. Under nonselective conditions, recombinants were isolated that had lost the integrated plasmid and retained a single chromosomal copy of the bop gene. This approach was used to construct a bop deletion strain. By using this strain, recombinants were obtained that express wild-type bacteriorhodopsin and mutants known to be defective in proton translocation. The expressed proteins were purified in a membrane fraction similar to purple membrane and were characterized in this form. UV/visible spectra of dark- and light-adapted bacteriorhodopsin from wild-type and Asp-96 mutants were identical to those of purple membrane. Arg-82, Asp-85, and Asp-212 mutants had 10- to 50-nm red shifts in their absorption maxima and showed altered light adaptation. The proton translocation activity of the wild-type samples and purple membrane was comparable, whereas the mutants had 0-60% of wild-type activity. These results support earlier studies of proton translocation mutants expressed in Escherichia coli.
Amplification of nitric oxide signaling by interstitial cells isolated from canine colon.
Authors: Publicover N G , Hammond E M , Sanders K M
Published: May 2006

The effects of nitric oxide (NO) on intracellular Ca2+ concentration ([Ca2+]i) were studied in enzymatically dispersed interstitial cells (ICs) and smooth muscle cells (SMCs) isolated from canine colon. [Ca2+]i was monitored by using fluo-3 and video fluorescence imaging techniques. Exogenous NO caused an increase in [Ca2+]i in ICs and a decrease in [Ca2+]i in SMCs. Effects of NO on ICs were not blocked by removal of extracellular Ca2+ but were blocked by ryanodine, suggesting that NO caused release of Ca2+ from intracellular stores. When [Ca2+]i was elevated in an IC by micropressure ejection of Bay K 8644, [Ca2+]i decreased in nearby SMCs, suggesting release of a diffusible substance. The diffusible substance may be NO or an NO-related substance based on blockade of transmission by NG-nitro-L-arginine methyl ester, NG-monomethyl-L-arginine, or oxyhemoglobin. The elevation of [Ca2+]i in ICs by NO, which, in turn, might cause further release of NO and elevation of [Ca2+]i, suggests a positive feedback and amplification mechanism in these cells. Elevation of [Ca2+]i in SMCs had no effect on adjacent SMCs. Our data suggest that ICs may play a central role in amplification of NO signaling and propagation of inhibitory wave fronts.
Metal-binding site in a class I tRNA synthetase localized to a cysteine cluster inserted into nucleotide-binding fold.
Authors: Landro J A , Schimmel P
Published: May 2006

The 10 class I aminoacyl-tRNA synthetases share a common N-terminal nucleotide-binding fold. Idiosyncratic polypeptide insertions into this fold introduce residues important for activity, including those that interact with the tRNA acceptor helix. The class I Escherichia coli methionyl-tRNA synthetase (L-methionine:tRNA(Met) ligase, EC 6.1.1.10), a 676-amino acid homodimer, was shown previously by others to contain zinc and to have an activity dependent on its presence. We show here by atomic absorption spectroscopy and zinc titrations the presence of 1 mol of zinc per polypeptide. Replacement of zinc with cobalt yields an active enzyme with a visible absorption spectrum characteristic of tetrahedral coordination to sulfur ligands and an intense metal-to-sulfur charge-transfer band at 340 nm. Mapping of the metal-binding site by zinc blotting of recombinant and proteolytic fragments localized the site to a polypeptide insertion between two strands and a beta-sheet in the N-terminal nucleotide-binding fold that contains the catalytic site. Beginning at Cys-145, this insertion contains a Cys-Xaa2-Cys-Xaa9-Cys-Xaa2-Cys motif. Site-directed substitution of these cysteines with serines yielded proteins that were stable but generally devoid of activity. With this result there is now at least one example of a class I and of a class II E. coli tRNA synthetase with a metal-binding domain important for activity inserted into the catalytic domain.