bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell surface oligosaccharides participate in cohesion during aggregation of Dictyostelium discoideum.
Authors: Ziska S E , Henderson E J
Published: May 2006

Plasma membrane glycoproteins from Dictyostelium discoideum amoebae at three stages of early development were digested with Pronase and endoglycosidase H and fractionated by gel filtration. This gave three classes of glycans (polysaccharides, endoglycosidase H-resistant glycopeptides, and endoglycosidase H-released oligosaccharides), which were tested for their ability to block agglutination of amoebae from vegetative, aggregation (8-hr), and late-aggregation (13-hr) stages of development. The endoglycosidase H-resistant glycopeptides from 8-hr cells inhibited agglutination of disaggregated 8-hr cells but not vegetative or 13-hr cells. The 8-hr polysaccharide and endo H-sensitive oligosaccharides did not inhibit. The glycopeptides from 8-hr cells were resolved into five species by electrophoresis in borate-containing buffer. Two of these had agglutination-inhibiting activity, and three did not. None of the glycan fractions from vegetative or 13-hr cells inhibited agglutination of vegetative, 8-, or 13-hr cells. These data implicate specific cell surface glycans in aggregation-stage intercellular cohesion and suggest that both these glycans and receptors for them are developmentally regulated.
Number and continuous proliferative pattern of transplanted primitive immunohematopoietic stem cells.
Authors: Harrison D E , Astle C M , Lerner C
Published: May 2006

We estimated numbers of transplantable primitive stem cells (PSCs) and found evidence that the same PSC continuously produced circulating erythrocytes and lymphocytes. These estimations used the binomial formula on data from recipients of identical portions of marrow mixtures containing two distinguishable cell types. Analysis of variance was used to compare repeated tests within each recipient. Values of pi s or pi c, probabilities that two independently sampled cells were descended from the same PSC, were also estimated, as this does not require the unverified condition that all PSCs contribute equally to the differentiated cell population. Several months after transplantation, erythrocytes were descended from only a single PSC per 1-2 X 10(5) marrow cells injected, several times rarer than previously reported. Percentages of erythrocyte and lymphocyte types in each recipient were closely correlated, with r values ranging from 0.86 to 0.94, in groups receiving 2-8 X 10(5) marrow cells; apparently the same precursors repopulated both myeloid and lymphoid lines in each recipient, as expected of true PSCs. Our data did not fit the clonal succession model, which predicts sequential activation of new PSCs and deactivation of old. Between 76 and 154 days, differentiated erythrocyte precursors were probably exhausted, with no evidence for new precursor activation or for further change between 154 and 250 days. The percentage of newly produced erythrocytes (reticulocytes) of each donor type varied little when individual recipients were followed between 165 and 295 days after transplantation, and variances within recipients were similar at marrow doses from 8 to 200 X 10(5) cells, further contradicting models of sequential activation and deactivation of PSC clones. Thus, transplanted PSCs were continually active during much of the recipient's lifespan.
Introns increase transcriptional efficiency in transgenic mice.
Authors: Brinster R L , Allen J M , Behringer R R , Gelinas R E , Palmiter R D
Published: May 2006

Experiments were designed to test the effect of introns on gene expression in transgenic mice. Four different pairs of gene constructs, which were identical except that one member of each pair lacked all introns, were compared for expression of mRNA after introduction into the murine germ line by microinjection of fertilized eggs. The expression of two chimeric genes, made by fusing either the mouse metallothionein I or the rat elastase 1 promoter/enhancer to the rat growth hormone gene, was assayed in fetal liver or pancreas, respectively, while two natural genes, an oligonucleotide-marked mouse metallothionein I gene and the human beta-globin gene, were assayed in fetal liver. In each case there was, on average, 10- to 100-fold more mRNA produced from the intron-containing construct. Moreover, mRNA levels were proportional to the relative rates of transcription that were measured in isolated nuclei. However, when the expression of the two mouse metallothionein I gene-based constructs was tested after transfection into cultured cells, little difference was observed. These observations suggest that introns play a role in facilitating transcription of microinjected genes and that this effect may be manifest only on genes exposed to developmental influences.
Gene transfer system for the phytopathogenic fungus Ustilago maydis.
Authors: Wang J , Holden D W , Leong S A
Published: May 2006

A selectable marker for transformation was constructed by transcriptional fusion of a Ustilago maydis heat shock gene promoter with the hygromycin B phosphotransferase gene of Escherichia coli. U. maydis was transformed to hygromycin B resistance by polyethylene glycol-induced fusion of spheroplasts following exposure to plasmid DNA that carried the marker gene. Transformation frequencies of 50 and 1000 transformants per microgram of DNA per 2 x 10(7) spheroplasts were obtained for circular and linear vector DNA, respectively. In the majority of transformants, the vector was integrated at a single chromosomal site, in either single copy or tandem duplication, as determined by Southern hybridization analysis of electrophoretically separated chromosomes and of restriction-endonuclease-cleaved DNA. The predominant form (82%) of vector integration was by nonhomologous recombination; the remainder carried the plasmid at the homologous heat shock gene locus. No evidence for gene conversion or gene replacement was obtained in 28 transformants. Hygromycin B phosphotransferase activity and resistance to hygromycin B were roughly correlated with the copy number of the integrated vector at the homologous location. Transforming DNA was stably maintained during mitosis and meiosis. This transformation procedure and associated vector should permit the cloning of genes by direct complementation in U. maydis.
Detection of Anaplasma-marginale-infected tick vectors by using a cloned DNA probe.
Authors: Goff W , Barbet A , Stiller D , Palmer G , Knowles D , Kocan K , Gorham J , McGuire T
Published: May 2006

Anaplasmosis is the most widely distributed of several important tick-borne diseases that constrain cattle production throughout much of the world. Evaluation of the effectiveness of disease control strategies that integrate vaccination with tick control requires the ability to monitor tick and cattle infection rates. To detect Anaplasma marginale in ticks and bovine erythrocytes, a 2-kilobase DNA fragment from a cloned A. marginale gene coding for a surface protein having a Mr of 105,000 was prepared and evaluated as a probe. The probe was species specific and detected A. marginale DNA derived from infected bovine erythrocytes and adult Dermacentor ticks infected either as nymphs or adults. Tick infection was confirmed by microscopy and test feeding on a susceptible calf. The sensitivity of the probe is suitable for detecting infected ticks in experimental and field epizootiology studies.
Human promyelocytic leukemia HL-60 cell proliferation and c-myc protein expression are inhibited by an antisense pentadecadeoxynucleotide targeted against c-myc mRNA.
Authors: Wickstrom E L , Bacon T A , Gonzalez A , Freeman D L , Lyman G H , Wickstrom E
Published: May 2006

The human promyelocytic leukemia cell line HL-60 overexpresses the c-myc protooncogene. A calculated secondary structure for c-myc mRNA placed the initiation codon in a bulge of a weakly base-paired region. Treatment of HL-60 cells with 5' d(AACGTTGAGGGGCAT) 3', complementary to the initiation codon and the next four codons of c-myc mRNA, inhibited c-myc protein expression in a dose-dependent manner. However, treatment of HL-60 cells with 5' d(TTGGGATAACACTTA) 3', complementary to nucleotides 17-31 of vesicular stomatitis virus matrix protein mRNA, displayed no such effects. These results agree with analogous studies of normal human T lymphocytes [Heikkila, R., Schwab, G., Wickstrom, E., Loke, S. L., Pluznik, D. H., Watt, R. & Neckers, L. M. (1987) Nature (London) 328, 445-449], except that only one-third as much oligomer was needed for a comparable effect. Proliferation of HL-60 cells in culture was inhibited in a sequence-specific, dose-dependent manner by the c-myc-complementary oligomer, but neither the oligomer complementary to vesicular stomatitis virus matrix protein mRNA nor 5' d(CATTTCTTGCTCTCC) 3', complementary to nucleotides 5399-5413 of human immunodeficiency virus tat gene mRNA, inhibited proliferation. It thus appears that antisense oligodeoxynucleotides added to myc-transformed cells via culture medium are capable of eliciting sequence-specific, dose-dependent inhibition of c-myc protein expression and cell proliferation.
Possible evolution of splice-junction signals in eukaryotic genes from stop codons.
Authors: Senapathy P
Published: May 2006

Splice-junction sequence signals are strongly conserved structural components of eukaryotic genes. These sequences border exon/intron junctions and aid in the process of removing introns by the RNA splicing machinery. Although substantial research has been undertaken to understand the mechanism of splicing, little is known about the origin and evolution of these splice signal sequences. Based on the previously published theory that the primitive genes evolved in pieces from primordial genetic sequences to avoid the interfering stop codons, a "stop-codon walk" mechanism is proposed in this paper to have assisted in the evolution of coding genes. This mechanism predicts the presence of stop codons in splice-junction signals inside the introns. Evidence of the consistent presence of stop codons in the splice-junction signals, in a position where they are expected, is shown by the analysis of codon statistics in these signal sequences in the GenBank databank. The results suggest that the splice-junction signals may have evolved from stop codons as a consequence of a selective pressure to avoid stop codons during the original evolution of coding genes. They also suggest that other splice signals within the introns, such as the branch-point sequence, may have evolved from stop codons for similar reasons.
Alpha/beta T-cell antigen receptor gene and protein expression occurs at early stages of thymocyte differentiation.
Authors: Richie E R , McEntire B , Crispe N , Kimura J , Lanier L L , Allison J P
Published: May 2006

Alterations in gene expression that orchestrate eukaryotic cellular differentiation often require appropriate interactions between differentiating cells and a specialized microenvironment. During T-lymphocyte differentiation, immature thymocytes undergo a stringent intrathymic selection process that requires intimate contact with thymic stromal elements. Since this selection process generates T cells that are self-tolerant and recognize nominal antigen only within the context of self-major histocompatibility antigen complex molecules, it is possible that thymocyte/stromal cell interactions are mediated, in part, by antigen-specific receptors expressed on differentiating thymocytes. However, the developmental stage at which alpha/beta antigen-specific receptors are expressed during T-cell maturation has been a matter of debate. To address this issue, we have studied alpha/beta T-cell antigen receptor gene and protein expression on normal thymocyte subsets of AKR/J mice, as well as on a panel of AKR/J primary thymic lymphomas characterized for CD4 (L3T4) and CD8 (Lyt-2) differentiation antigen expression. The data unequivocally demonstrate that alpha/beta heterodimers are expressed not only on phenotypically mature thymocytes but also on the majority of CD4+8+ double-positive cells that comprise the predominant nonmature thymocyte subset. Furthermore, a fraction of thymocytes in the CD4-8- double-negative compartment, known to contain progenitor cells, also expresses readily detectable cell-surface alpha/beta receptors. Therefore, during the process of intrathymic selection, interactions between nonmature thymocytes and stromal cells via the antigen-receptor complex may play a pivotal role in T-cell differentiation and should be considered in formulating schemes for functional T-cell selection.
Gating kinetics of the cyclic-GMP-activated channel of retinal rods: flash photolysis and voltage-jump studies.
Authors: Karpen J W , Zimmerman A L , Stryer L , Baylor D A
Published: May 2006

The gating kinetics of the cGMP-activated cation channel of salamander retinal rods have been studied in excised membrane patches. Relaxations in patch current were observed after two kinds of perturbation: (i) fast jumps of cGMP concentration, generated by laser flash photolysis of a cGMP ester ("caged" cGMP), and (ii) membrane voltage jumps, which perturb activation of the channel by cGMP. In both methods the speed of activation increased with the final cGMP concentration. The results are explained by a simple kinetic model in which activation involves three sequential cGMP binding steps with bimolecular rate constants close to the diffusion-controlled limit; fully liganded channels undergo rapid open-closed transitions. Voltage perturbs activation by changing the rate constant for channel closing, which increases with hyperpolarization. Intramolecular transitions of the fully liganded channel limit the kinetics of activation at high cGMP concentrations (greater than 50 microM), whereas at physiological cGMP concentrations (less than 5 microM), the kinetics of activation are limited by the third cGMP binding step. The channel appears to be optimized for rapid responses to changes in cytoplasmic cGMP concentration.
Block of N-methyl-D-aspartate-activated current by the anticonvulsant MK-801: selective binding to open channels.
Authors: Huettner J E , Bean B P
Published: May 2006

Whole-cell and single-channel recording techniques were used to study the action of the anticonvulsant drug MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]- cyclohepten-5,10-imine maleate) on responses to excitatory amino acids in rat neocortical neurons in cell culture. MK-801 caused a progressive, long-lasting blockade of current induced by N-methyl-D-aspartate (N-Me-D-Asp). However, during the time that N-Me-D-Asp responses were inhibited, there was no effect on responses to quisqualate or kainate, suggesting that N-Me-D-Asp receptors and kainate/quisqualate receptors open separate populations of ion channels. Binding and unbinding of MK-801 seems to be possible only if the N-Me-D-Asp-operated channel is in the transmitter-activated state: MK-801 was effective only when applied simultaneously with N-Me-D-Asp, and recovery from MK-801 blockade was speeded by continuous exposure to N-Me-D-Asp [time constant (tau) approximately equal to 90 min at -70 to -80 mV]. Recovery from block during continuous application of N-Me-D-Asp was strongly voltage dependent, being faster at positive potentials (tau approximately equal to 2 min at +30 mV). Mg2+, which is thought to block the N-Me-D-Asp-activated ion channel, inhibited blockade by MK-801 at negative membrane potentials. In single-channel recordings from outside-out patches. MK-801 greatly reduced the channel activity elicited by application of N-Me-D-Asp but did not significantly alter the predominant unitary conductance. Consistent with an open-channel blocking mechanism, the mean channel open time was reduced by MK-801 in a dose-dependent manner.
Aspartic acid-484 of nascent placental alkaline phosphatase condenses with a phosphatidylinositol glycan to become the carboxyl terminus of the mature enzyme.
Authors: Micanovic R , Bailey C A , Brink L , Gerber L , Pan Y C , Hulmes J D , Udenfriend S
Published: May 2006

A carboxyl-terminal chymotryptic peptide from mature human placental alkaline phosphatase was purified by HPLC and monitored by a specific RIA. Sequencing and amino acid assay showed that the carboxyl terminus of the peptide was aspartic acid, representing residue 484 of the proenzyme as deduced from the corresponding cDNA. Further analysis of the peptide showed it to be a peptidoglycan containing one residue of ethanolamine, one residue of glucosamine, and two residues of neutral hexose. The inositol glycan is apparently linked to the alpha carboxyl group of the aspartic acid through the ethanolamine. Location of the inositol glycan on Asp-484 of the proenzyme indicates that a 29-residue peptide is cleaved from the nascent protein during the post-translational condensation with the phosphatidylinositol-glycan.
Cloning and cDNA sequence of the dihydrolipoamide dehydrogenase component human alpha-ketoacid dehydrogenase complexes.
Authors: Pons G , Raefsky-Estrin C , Carothers D J , Pepin R A , Javed A A , Jesse B W , Ganapathi M K , Samols D , Patel M S
Published: May 2006

cDNA clones comprising the entire coding region for human dihydrolipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4) have been isolated from a human liver cDNA library. The cDNA sequence of the largest clone consisted of 2082 base pairs and contained a 1527-base open reading frame that encodes a precursor dihydrolipoamide dehydrogenase of 509 amino acid residues. The first 35-amino acid residues of the open reading frame probably correspond to a typical mitochondrial import leader sequence. The predicted amino acid sequence of the mature protein, starting at the residue number 36 of the open reading frame, is almost identical (greater than 98% homology) with the known partial amino acid sequence of the pig heart dihydrolipoamide dehydrogenase. The cDNA clone also contains a 3' untranslated region of 505 bases with an unusual polyadenylylation signal (TATAAA) and a short poly(A) track. By blot-hybridization analysis with the cDNA as probe, two mRNAs, 2.2 and 2.4 kilobases in size, have been detected in human tissues and fibroblasts, whereas only one mRNA (2.4 kilobases) was detected in rat tissues.
Accessory pathway for sound transfer in a neotropical frog.
Authors: Narins P M , Ehret G , Tautz J
Published: May 2006

A portion of the lateral body wall overlying the lung cavity of the arboreal frog, Eleutherodactylus coqui, vibrates in response to free-field sound. Peak displacement amplitude of the body wall in response to a natural call note presented at 73 decibels sound pressure level is 1.70 X 10(-9) m, roughly 8 decibels less than that of the ipsilateral eardrum, as measured by laser Doppler vibrometry. We show that the vibration magnitude varies predictably across the body profile and is posture and frequency dependent. Two routes to the inner ear are described for sounds impinging on the body wall; either of these accessory pathways could modify direct input from the peripheral auditory system and enhance sound localization in these small vertebrates.
Mutation in the CYP21B gene (Ile-172----Asn) causes steroid 21-hydroxylase deficiency.
Authors: Amor M , Parker K L , Globerman H , New M I , White P C
Published: May 2006

Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia. It results from a deficiency in a specific cytochrome P450, P450c21 (P450XXIA). The gene encoding this protein (CYP21B) and a closely linked pseudogene (CYP21A) are located in the HLA complex on chromosome 6p. Many mutant alleles are associated with deletions of CYP21B; we report the cloning and characterization of a nondeleted mutant CYP21B gene. This mutant gene is expressed on transfection into mouse Y1 adrenal cells, producing mRNA levels similar to those seen after transfection of the normal CYP21B gene. In codon 172 of the mutant gene, the normal codon ATC, encoding isoleucine, has been changed to AAC, encoding asparagine. This mutation is normally present in the CYP21A pseudogene, so that it may have been transferred to the mutant CYP21B gene by gene conversion. Hybridization of oligonucleotide probes corresponding to this and two other mutations normally present in CYP21A demonstrated that 4 out of 20 patients carried the codon 172 mutation; in one of these patients, the mutation was present as part of a larger gene conversion involving at least exons 3-6. Gene conversion may be a frequent cause of 21-hydroxylase deficiency alleles due to the presence of six chi-like sequences (GCTGGGG) in the CYP21 genes and the close proximity of the CYP21A pseudogene, which has several potentially deleterious mutations.
Identification of a 24-kDa cytokine that is required for development of cytolytic T lymphocytes.
Authors: Cernetti C , Steinman R M , Granelli-Piperno A
Published: May 2006

It is known that the production of cytolytic T lymphocytes requires growth factors such as interleukins 2 and 4 (IL-2 and IL-4). Elsewhere we have described bioassays that detect a cytokine that operates in concert with growth factor to generate cytolytic T lymphocytes. The factor that is termed cytolytic T-lymphocyte differentiation factor (CDF), together with IL-2 and lectin, mediates the formation of CD8+ killer cells in 2 days from thymocyte or peripheral lymphoid precursors. CDF is not mimicked by natural or recombinant sources of interferons, colony-stimulating factors, and IL-1 to IL-4. Here we use these bioassays to isolate and further characterize a single 24-kDa CDF protein from the conditioned medium of stimulated human blood mononuclear cells. CDF is first enriched by three successive chromatographic procedures that utilize anion exchange, hydroxyapatite, and phenyl-Superose. A single 24-kDa band with CDF activity is then isolated on 12% NaDodSO4/PAGE and clearly distinguished from the 17-kDa band of IL-2. The apparent molecular mass is similar under reducing and nonreducing conditions. After elution from NaDodSO4/PAGE the cytokine is maximally active at 0.25 nM in the CDF assay and has no growth factor activity for T lymphoblasts. To generate cytolytic CD8+, CD4- cells from spleen and lymph node T lymphocytes, IL-2 and small numbers of accessory dendritic cells must be applied together with CDF.
An Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat.
Authors: Kenney S , Kamine J , Markovitz D , Fenrick R , Pagano J
Published: May 2006

Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, we demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBV gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses.
Macrophage-mediated myelin-related mitogenic factor for cultured Schwann cells.
Authors: Baichwal R R , Bigbee J W , DeVries G H
Published: May 2006

Conditioned medium from cultured peritoneal macrophages that have phagocytosed a myelin membrane fraction is mitogenic for cultured Schwann cells. Production of the mitogenic supernatant was time- and dose-dependent with a maximal Schwann cell-proliferative response from supernatants after 48-hr incubation of cultured macrophages with myelin-enriched fraction (200 micrograms of protein per ml). The response was specific for myelin membrane: supernatants derived from macrophages incubated with axolemma, liver microsomes, polystyrene beads, or lipopolysaccharide were not mitogenic. Lysosomal processing of the myelin membrane was necessary for the production of the mitogenic factor, which was shown to be heat labile and trypsin sensitive. There was no species specificity because myelin membranes isolated from the central and peripheral nervous systems of rat, bovine, and human were equally potent in eliciting mitogenic supernatant. However, supernatants derived from central nervous system myelin membranes were two to three times more mitogenic than those obtained from peripheral nervous system fractions of the same species. Previous observations that myelin is mitogenic for cultured Schwann cells may, in part, involve the intermediate processing of myelin by macrophages that are present in Schwann cell cultures. These results suggest that macrophages play a crucial role in Schwann cell proliferation during Wallerian degeneration.
Isolation and characterization of the gene encoding the heavy chain of Drosophila kinesin.
Authors: Yang J T , Saxton W M , Goldstein L S
Published: May 2006

An antiserum that recognizes the heavy chain of Drosophila kinesin was used to isolate Drosophila cDNA clones. Immunoblot analysis of the proteolytic fragments of the protein produced by one of the cDNA clones has demonstrated that the cDNA clones encode the heavy chain of Drosophila kinesin. The in vitro-synthesized product of the largest cDNA comigrates with Drosophila kinesin heavy chain on NaDodSO4/polyacrylamide gels and binds to taxol-stabilized microtubules in the presence of the nonhydrolyzable analogue of ATP, 5'-adenylyl imidodiphosphate, but not in the presence of ATP or 0.1 M KCl. Analysis of the cDNA clones suggests that there is a single gene encoding kinesin heavy chain in Drosophila located at polytene chromosome position 53A. However, Southern hybridization analyses suggest the presence of related sequences in the Drosophila genome.
Growth regulation of skin cells by epidermal cell-derived factors: implications for wound healing.
Authors: Eisinger M , Sadan S , Silver I A , Flick R B
Published: May 2006

Epidermal cell-derived factors (EDF), present in extracts and supernatant fluids of cultured epidermal cells, were found to stimulate the proliferation of keratinocytes but to inhibit fibroblasts. In vitro, the effect of EDF on epidermal cells resulted in an increased number of rapidly proliferating colonies composed mainly of basal keratinocytes. Control cultures grown in the absence of EDF had a high proportion of terminally differentiated cells. In fibroblast cultures EDF inhibited the ability of fibroblasts to cause contraction of collagen sponges by 90%. Epidermal growth factor, basic fibroblast growth factor, platelet-derived growth factor, transforming growth factor beta, nerve growth factor, and extracts of WI-38 cells (human embryonic lung fibroblasts) did not have this inhibitory activity. Application of EDF to surgical wounds stimulated extensive migration and proliferation of keratinocytes from remnants of glands, hair follicles, and wound edges. The restoration of complete epidermal coverage of wounds treated with EDF occurred twice as rapidly as that of control wounds. In addition, regenerating dermis in the EDF-treated wounds contained 1/5th to 1/15th as many cells as wounds treated with epidermal growth factor, urogastrone, transforming growth factor, or phosphate-buffered saline. The use of EDF to enhance re-epithelization and to prevent scar formation is proposed.
N-ethyl-N-nitrosourea-induced null mutation at the mouse Car-2 locus: an animal model for human carbonic anhydrase II deficiency syndrome.
Authors: Lewis S E , Erickson R P , Barnett L B , Venta P J , Tashian R E
Published: May 2006

Electrophoretic screening of (C57BL/6J x DBA/2J)F1 progeny of male mice treated with N-ethyl-N-nitrosourea revealed a mouse that lacked the paternal carbonic anhydrase II (CA II). Breeding tests showed that this trait was heritable and due to a null mutation at the Car-2 locus on chromosome 3. Like humans with the same inherited enzyme defect, animals homozygous for the new null allele are runted and have renal tubular acidosis. However, the prominent osteopetrosis found in humans with CA II deficiency could not be detected even in very old homozygous null mice. A molecular analysis of the deficient mice shows that the mutant gene is not deleted and is transcribed. The CA II protein, which is normally expressed in most tissues, could not be detected by immunodiffusion analysis in any tissues of the CA II-deficient mice, suggesting a nonsense or a missense mutation at the Car-2 locus.
Classical conditioning induces long-term translocation of protein kinase C in rabbit hippocampal CA1 cells.
Authors: Bank B , DeWeer A , Kuzirian A M , Rasmussen H , Alkon D L
Published: May 2006

The role of the Ca2+/phospholipid-dependent, diacylglycerol-activated enzyme protein kinase C (PKC) in rabbit eyelid conditioning was examined. PKC was partially purified from the CA1 region of hippocampal slices from naive, pseudoconditioned, and conditioned rabbits 24 hr after the rabbits were well conditioned. Crude membrane and cytosol fractions were prepared. In conditioned rabbits, significantly more PKC activity (63.3%) was associated with the membrane fraction (and significantly less with the cytosol fraction) compared to naive (42.0%) and pseudoconditioned (44.7%) animals. These differences in distribution of enzyme activity were paralleled by differences in stimulation of enzyme activity by Ca2+, phospholipid, and diacylglycerol. There were no between-group differences in basal protein kinase activity. These results suggest that there is a long-term translocation of PKC from cytosol to membrane as a result of conditioning. Autoradiographic binding of radioactive phorbol 12,13-dibutyrate to PKC demonstrated that almost all specific binding was in the stratum radiatum, a region containing the proximal apical dendrites of CA1 pyramidal neurons. Therefore, this may be the site of the conditioning-specific PKC translocation, a locus well-suited to underlie the biophysical effects of conditioning.
Isolation and characterization of the human Gs alpha gene.
Authors: Kozasa T , Itoh H , Tsukamoto T , Kaziro Y
Published: May 2006

The gene for Gs alpha (the alpha subunit of the guanine nucleotide-binding protein Gs) was isolated from human genomic libraries using rat Gs alpha cDNA as a probe. Comparison of the nucleotide sequence of the human gene with that of the rat cDNA revealed that the human Gs alpha gene spans approximately equal to 20 kilobases and is composed of 13 exons and 12 introns. Genomic Southern blot analysis suggests that the human haploid genome contains a single Gs alpha gene. Previous reports indicated the presence of multiple species of Gs alpha cDNA. The structure of the human Gs alpha gene suggests that four types of Gs alpha mRNAs may be generated from a single Gs alpha gene by alternate use of exon 3 and/or of two 3' splice sites of intron 3, where an unusual splice junction sequence (TG) instead of the consensus (AG) is used. S1 nuclease mapping analysis of human Gs alpha mRNA identified multiple transcriptional initiation sites. The promoter region of the human Gs alpha gene has extremely high G + C content (85%). It contains 4 "GC" boxes, but no typical "TATA" or "CAAT" box sequence. In the 5' flanking region, there are several blocks of sequences that are similar to the sequences of the 5' flanking region of the human c-Ki-ras2 gene.
Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5' untranslated region of ferritin heavy- and light-subunit mRNAs.
Authors: Leibold E A , Munro H N
Published: May 2006

The mRNAs for the heavy and light subunits of the iron-storage protein ferritin occur in cells largely as inactive ribonucleoprotein particles, which are recruited for translation when iron enters the cell. Cytoplasmic extracts from rat tissues and hepatoma cells were shown by an electrophoretic separation procedure to form RNA-protein complexes involving a highly conserved sequence in the 5' untranslated region of both ferritin heavy- and light-subunit mRNAs. The pattern of complex formation was affected by pretreatment of rats or cells with iron. Crosslinking by UV irradiation showed that the complexes contained an 87-kDa protein interacting with the conserved sequence of the ferritin mRNA. We propose that intracellular iron levels regulate ferritin synthesis by causing changes in specific protein binding to the conserved sequence in the ferritin heavy- and light-subunit mRNAs.
Alloreactive immune responses of transgenic mice expressing a foreign transplantation antigen in a soluble form.
Authors: Arnold B , Dill O , Küblbeck G , Jatsch L , Simon M M , Tucker J , Hämmerling G J
Published: May 2006

Transfection of cells with the H-2Kk gene lacking the transmembrane and cytoplasmic segments resulted in secretion of the H-2Kk protein, as determined by immunoprecipitation with monoclonal anti-H-2Kk antibodies. Transgenic (H-2b X H-2d)F1 mice were established carrying integrated copies of the modified H-2Kk gene. Expression of the soluble H-2Kk antigen in the transgenic mice was demonstrated in cell supernatants of biosynthetically labeled splenic and thymic Con A blasts as well as bone marrow-derived macrophages. Soluble H-2Kk molecules were also present in the sera of the transgenic animals. No cell-surface expression of the H-2Kk antigen could be observed. In spite of the presence of the soluble H-2Kk molecules in the transgenic mice, the animals were able to generate H-2Kk-specific cytolytic T cells as well as antibody responses when stimulated with cell-surface-bound H-2Kk antigens. These responses were indistinguishable from those of the nontransgenic littermates. Possible explanations for the observed lack of tolerance are discussed.
Selective differentiation and proliferation of hematopoietic cells induced by recombinant human interleukins.
Authors: Saito H , Hatake K , Dvorak A M , Leiferman K M , Donnenberg A D , Arai N , Ishizaka K , Ishizaka T
Published: May 2006

Effects of recombinant human interleukins on hematopoiesis were explored by using suspension cultures of mononuclear cells of human umbilical-cord blood and bone marrow. The results showed that interleukin 5 induced the selective differentiation and proliferation of eosinophils. After 3 weeks in culture with interleukin 5, essentially all nonadherent cells in both bone marrow and cord blood cell cultures became eosinophilic myelocytes. Culture of the same cells with interleukin 4 resulted in the selective growth of OKT3+ lymphocytes. However, OKT3+ cells did not develop if the bone marrow cells were depleted of OKT3+/OKT11+ cells prior to the culture, indicating that interleukin 4 induced the proliferation of a subpopulation of resting T cells present in cord blood and bone marrow cell preparations. In suspension cultures of bone marrow cells and cord blood cells grown in the presence of interleukin 3, basophilic, eosinophilic, and neutrophilic myelocytes and macrophages developed within 2 weeks. By 3 weeks, however, the majority of nonadherent cells became eosinophilic myelocytes. In contrast to mouse bone marrow cell cultures, neither interleukin 3 nor a combination of interleukins 3 and 4 induced the differentiation of mast cells in human bone marrow or cord blood cell cultures.
Receptor- and heparin-binding domains of basic fibroblast growth factor.
Authors: Baird A , Schubert D , Ling N , Guillemin R
Published: May 2006

Two functional domains in the primary structure of basic fibroblast growth factor (FGF) have been identified on the basis of their ability to interact with the FGF receptor, bind radiolabeled heparin, and modulate the cellular response to FGF. Peptides derived from these two functional domains can act as partial agonists and antagonists in biological assays of FGF activity. Peptides related to the sequences of FGF-(24-68)-NH2 and FGF-(106-115)-NH2 inhibit thymidine incorporation into 3T3 fibroblasts when they are stimulated by FGF but have no effect when the cells are treated with either platelet-derived growth factor or epidermal growth factor. They also possess partial agonist activity and can stimulate DNA synthesis when tested in the absence of exogenous FGF. The active peptides have no effect on the binding of epidermal growth factor to its receptor on A431 cells and they can modulate the effects of FGF, but not fibronectin, on endothelial cell adhesion. The results suggest the possibility of designing specific analogs of FGF that are capable of inhibiting the biological effects of FGF.
Protective effects of analogs of luteinizing hormone-releasing hormone against chemotherapy-induced testicular damage in rats.
Authors: Karashima T , Zalatnai A , Schally A V
Published: May 2006

Possible protective effects of analogs of luteinizing hormone-releasing hormone (LH-RH) against testicular damage caused by various cytotoxic agents were investigated in rats. The agonist [D-Trp6]LH-RH (in which Gly-6 is replaced by D-tryptophan) and the antagonist N-Ac-[D-Phe(pCl)1,2,D-Trp3,D-Arg6,D-Ala10]LH-RH were administered for 12 weeks: [D-Trp6]LH-RH was given once a month in the form of long-acting microcapsules liberating 25 micrograms of agonist per day, and the antagonist was injected s.c. at a dose of 1000 micrograms per kg of body weight per day for the first 3 weeks and, thereafter, at a dose of 500 micrograms per kg of body weight per day. After a recovery period of 3 months, most seminiferous tubules in the antagonist-treated group showed a normal morphology, while patches of tubules in the agonist-treated group continued to show some suppression of spermatogenesis. Administration of busulfan, cisplatin, or cyclophosphamide produced only a reversible testicular injury, and pretreatment with LH-RH analogs seemed to temporarily enhance the tubular damage. Administration of procarbazine (200 mg per kg of body weight per week for 6 weeks) resulted in decreased testicular weights and increased serum LH levels 1 and 3 months after the discontinuation of treatment. The histology showed severe diffuse damage to seminiferous tubules. The germinal cells completely disappeared and the Sertoli cells were markedly degenerated. This damage was not restored even after a recovery period of 5 months. Some animals were pretreated for 6 weeks with the agonist or antagonist and then received procarbazine for 6 weeks while administration of analogs was continued. In these animals, the decrease in testicular weights and increase in serum LH levels after procarbazine were less marked than in the group not pretreated with the analogs. Three and 5 months after cessation of treatment, a large number of tubules showed a complete restoration of structural morphology in 30-45% of the animals that received procarbazine and the LH-RH agonist or antagonist. These results indicate that pretreatment with LH-RH analogs may protect testes against damage caused by some cytotoxic agents.
Acidic fibroblast growth factor enhances regeneration of processes by postnatal mammalian retinal ganglion cells in culture.
Authors: Lipton S A , Wagner J A , Madison R D , D'Amore P A
Published: May 2006

Postnatal rat retinal ganglion cells (RGCs) were identified with specific fluorescent labels and placed in culture. Under these conditions, the outgrowth of processes by RGCs was found to be promoted to a far greater degree by acidic fibroblast growth factor (aFGF) than by basic fibroblast growth factor (bFGF). The effect of aFGF and bFGF on process extension by solitary RGCs was quantified after 24 hr in culture, a time when neither a FGF nor bFGF enhanced RGC survival. The action of aFGF on process outgrowth was markedly potentiated by the addition of heparin (10 micrograms/ml) to the medium, but heparin alone had no effect. In the presence of heparin, half-maximal process outgrowth occurred at an aFGF concentration of less than 20 pg/ml (1 pM). Since all of the centrally projecting processes have already been formed in the living animal prior to use (at 7-12 days of age), at least a portion of the process outgrowth in culture appears to represent a regenerative phenomenon. Statistical analysis of the increase in process growth revealed that aFGF with heparin contributed to both neurite initiation and elongation. The mean number of glial cells, identified with polyclonal antiserum against glial fibrillary acidic protein, was slightly increased in cultures receiving aFGF plus heparin, but this effect was variable, and these glial cells were not in contact with the solitary RGCs that were scored for regeneration of processes. Thus, glial cells probably did not exert a direct physical influence on the degree of process outgrowth observed in the solitary RGCs, although a humoral effect cannot be totally excluded. These results suggest that aFGF has a potent influence on the outgrowth of processes by a neuron in the mammalian central nervous system. The potentiation of this effect by heparin leads us to speculate that the interaction of aFGF with a heparin-like molecule located in the extracellular matrix (such as heparan sulfate proteoglycan) may produce physiological effects in vivo. Furthermore, the lack of a substantial effect of bFGF in this system under these conditions shows that a specific population of mammalian central neurons may be differentially influenced by these two closely related peptide growth factors.
Spatial distribution of calcium channels and cytosolic calcium transients in growth cones and cell bodies of sympathetic neurons.
Authors: Lipscombe D , Madison D V , Poenie M , Reuter H , Tsien R Y , Tsien R W
Published: May 2006

Ca2+ imaging and single-channel recording were used to study the regulation of cytosolic free Ca2+ ([Ca2+]i) in local regions of frog sympathetic neurons. Digital imaging with the fluorescent Ca2+ indicator fura-2 demonstrated: (i) resting [Ca2+]i of 70-100 nM; (ii) significant increases in [Ca2+]i in growth cones and cell bodies following depolarization induced by extracellular electrical stimulation or increased external K+; (iii) in cell bodies, large transient increases in [Ca2+]i following exposure to caffeine and sustained oscillations in [Ca2+]i in the presence of elevated K+ and caffeine; and (iv) in growth cones, smaller and briefer changes in [Ca2+]i in response to caffeine. The nature of the depolarization-induced Ca2+ entry was studied with cell-attached patch recordings (110 mM Ba2+ in recording pipette). Ca2+ channel activity was observed in 18 of 20 patches on cell bodies, 3 of 5 patches along neurites, and 36 of 41 patch recordings from growth cones. We observed two types of Ca2+ channels: L-type channels, characterized by a 28-pS slope conductance, sensitivity to dihydropyridine Ca2+ channel agonist, and availability even with depolarizing holding potentials; and N-type channels, characterized by a 15-pS slope conductance, resistance to dihydropyridines, and inactivation with depolarized holding potentials. Both types of channels were found on growth cones and along neurites as well as on cell bodies; channels often appeared concentrated in local hot spots, sometimes dominated by one channel type.
Sequence-specific recognition and cleavage of DNA by metallobleomycin: minor groove binding and possible interaction mode.
Authors: Kuwahara J , Sugiura Y
Published: May 2006

The DNase I cleavage-inhibition analysis shows binding sites of approximately 2 or 3 base pairs--in particular, 5' N-G-C sequences--for the green-colored CoIII and fully oxidized FeIII complexes of bleomycin. The apparent binding constant of the bleomycin-CoIII complex is quite similar for glucosylated and nonglucosylated phage T4 DNAs, whereas poly[d(I-C)] clearly gives a smaller binding constant than does poly[d(G-C)]. In contrast to the covalent attachment of the guanine N-7 with aflatoxin B1, the modification of the guanine 2-amino group with anthramycin remarkable inhibits the DNA cleavages at 5' G-C and 5' G-T sites by the FeIII and CoIII complex systems of bleomycin. These results strongly indicate that metallobleomycin binds in the minor groove of B-DNA and that the 2-amino group of guanine adjacent to the 5' side of the cleaved pyrimidine base is one key element of the specific 5' G-C or G-T recognition by the bleomycin-metal complex. A possible binding mode of metallobleomycin in the DNA helix has been proposed by computer-constructed model building.
Purification of an angiotensin II binding protein by using antibodies to a peptide encoded by angiotensin II complementary RNA.
Authors: Elton T S , Dion L D , Bost K L , Oparil S , Blalock J E
Published: May 2006

We have generated a monospecific antibody to a synthetic peptide encoded by an RNA complementary to the mRNA for angiotensin II (AII) and determined whether this antibody recognizes the AII receptor. We demonstrate that the antibody competes specifically with 125I-labeled AII for the same binding site on rat adrenal membranes. Furthermore, we show that this antibody inhibits the secretion of aldosterone from cultured rat adrenal cells, suggesting that the antibody recognizes the biologically relevant AII receptor. Finally, we demonstrate that antibody to the complementary peptide can be used to immunoaffinity-purify a protein of Mr 66,000 that specifically binds radiolabeled AII.
Structure of rho factor: an RNA-binding domain and a separate region with strong similarity to proven ATP-binding domains.
Authors: Dombroski A J , Platt T
Published: May 2006

The domain structure of rho protein, a transcription termination factor of Escherichia coli, was analyzed by oligonucleotide site-directed mutagenesis and chemical modification methods. The single cysteine at position 202, previously thought to be essential for rho function, was changed to serine or to glycine with no detectable effects on the protein's hexameric structure, RNA-binding ability, or ATPase, helicase, and transcription termination activities. A 151-residue amino-terminal fragment (N1), generated by hydroxylamine cleavage, and its complementary carboxyl-terminal fragment of 268 amino acids (N2) were extracted from NaDod-SO4/polyacrylamide gels and renatured. The N1 fragment binds poly(C) and mRNA corresponding to the rho-dependent terminator sequence trp t', but not RNA unrecognized by rho; hence, this small renaturable domain retains not only the binding ability but also the specificity of the native protein. Uncleaved rho renatures to regain its RNA-dependent ATPase activity, but neither N1 nor N2 exhibits any detectable ATP hydrolysis. Similarly, the two fragments, isolated separately but renatured together, are unable to hydrolyze ATP. Sequence homology to the alpha subunit of the E. coli F1 membrane ATPase, and to consensus elements of other nucleotide-binding proteins, strongly suggests a structural domain for ATP binding that begins after amino acid 164. The implications of discrete domains for RNA and nucleotide binding are discussed in the context of requirements for specific interactions between RNA-binding and ATP-hydrolysis sites during transcription termination.
cDNA cloning of human DNA topoisomerase I: catalytic activity of a 67.7-kDa carboxyl-terminal fragment.
Authors: D'Arpa P , Machlin P S , Ratrie H , Rothfield N F , Cleveland D W , Earnshaw W C
Published: May 2006

cDNA clones encoding human topoisomerase I were isolated from an expression vector library (lambda gt11) screened with autoimmune anti-topoisomerase I serum. One of these clones has been expressed as a fusion protein comprised of a 32-kDa fragment of the bacterial TrpE protein linked to 67.7 kDa of protein encoded by the cDNA. Three lines of evidence indicate that the cloned cDNA encodes topoisomerase I. (i) Proteolysis maps of the fusion protein and human nuclear topoisomerase I are essentially identical. (ii) The fusion protein relaxes supercoiled DNA, an activity that can be immunoprecipitated by anti-topoisomerase I serum. (iii) Sequence analysis has revealed that the longest cDNA clone (3645 base pairs) encodes a protein of 765 amino acids that shares 42% identity with Saccharomyces cerevisiae topoisomerase I. The sequence data also show that the catalytically active 67.7-kDa fragment is comprised of the carboxyl terminus.