bluuebunny/crossref_metadata_2025 huggingface dataset:
Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism).
Authors: Daughaday W H , Trivedi B
Published: May 2006

It has recently been recognized that human serum contains a protein that specifically binds human growth hormone (hGH). This protein has the same restricted specificity for hGH as the membrane-bound GH receptor. To determine whether the GH-binding protein is a derivative of, or otherwise related to, the GH receptor, we have examined the serum of three patients with Laron-type dwarfism, a condition in which GH refractoriness has been attributed to a defect in the GH receptor. The binding of 125I-labeled hGH incubated with serum has been measured after gel filtration of the serum through an Ultrogel AcA 44 minicolumn. Nonspecific binding was determined when 125I-hGH was incubated with serum in the presence of an excess of GH. Results are expressed as percent of specifically bound 125I-hGH and as specific binding relative to that of a reference serum after correction is made for endogenous GH. The mean +/- SEM of specific binding of sera from eight normal adults (26-46 years of age) was 21.6 +/- 0.45%, and the relative specific binding was 101.1 +/- 8.6%. Sera from 11 normal children had lower specific binding of 12.5 +/- 1.95% and relative specific binding of 56.6 +/- 9.1%. Sera from three children with Laron-type dwarfism lacked any demonstrable GH binding, whereas sera from 10 other children with other types of nonpituitary short stature had normal relative specific binding. We suggest that the serum GH-binding protein is a soluble derivative of the GH receptor. Measurement of the serum GH-binding protein may permit recognition of other abnormalities of the GH receptor.
Analysis of the tissue-specific enhancer at the 3' end of the chicken adult beta-globin gene.
Authors: Emerson B M , Nickol J M , Jackson P D , Felsenfeld G
Published: May 2006

In an earlier paper we identified a tissue-specific enhancer in the 3' flanking region of the chicken adult beta-globin gene. In this paper we analyze the properties of this enhancer. Deletion analysis and transient expression assays show that the domain responsible for activation of transcription is at most 136 base pairs long. Specific factors that bind to discrete sequences within the enhancer DNA are found in extracts of embryonic and adult erythrocytes and in brain. These factors are specific for the tissue or the erythrocyte developmental stage and protect at least five discrete regions in or near the enhancer against DNase I digestion in "footprinting" experiments. Four of these regions reside wholly within the 136-base-pair functional enhancer domain, which also comprises a site in chromatin that is hypersensitive to nucleases. The nature of the binding sites and the program of appearance of the factors during development suggest that a subset of these interactions may be responsible for the developmental specificity of the enhancer.
Effect of premature termination of translation on mRNA stability depends on the site of ribosome release.
Authors: Nilsson G , Belasco J G , Cohen S N , von Gabain A
Published: May 2006

Translational stop codons were introduced at various locations in the protein-coding regions of the monocistronic bla and ompA gene transcripts of Escherichia coli, and the decay characteristics of the upstream and downstream mRNA segments were analyzed. Premature termination of translation at codon position 26 reduced the stability of both the translated and ribosome-free segments of bla mRNA, whereas release of ribosomes just 30 codons further downstream resulted in normal stability for both segments. Normal stability of an untranslated bla gene mRNA segment required its linkage to a ribosome-bound segment of bla gene mRNA. These findings indicate that depriving an mRNA segment of ribosomes does not necessarily render it more susceptible to degradation. However, premature termination of translation at a location that allows ribosomes to traverse only a short segment of bla mRNA can lead to destabilization of the entire transcript.
Two human homeobox genes, c1 and c8: structure analysis and expression in embryonic development.
Authors: Simeone A , Mavilio F , Acampora D , Giampaolo A , Faiella A , Zappavigna V , D'Esposito M , Pannese M , Russo G , Boncinelli E
Published: May 2006

Two human cDNA clones (HHO.c1.95 and HHO.c8.5111) containing a homeobox region have been characterized, and the respective genomic regions have been partially analyzed. Expression of the corresponding genes, termed c1 and c8, was evaluated in different organs and body parts during human embryonic/fetal development. HHO.c1.95 apparently encodes a 217-amino acid protein containing a class I homeodomain that shares 60 out of 61 amino acid residues with the Antennapedia homeodomain of Drosophila melanogaster. HHO.c8.5111 encodes a 153-amino acid protein containing a homeodomain identical to that of the frog AC1 gene. Clones HHO.c1 and HHO.c8 detect by blot-hydridization one and two specific polyadenylylated transcripts, respectively. These are differentially expressed in spinal cord, backbone rudiments, limb buds (or limbs), heart, and skin of human embryos and early fetuses in the 5- to 9-week postfertilization period, thus suggesting that the c1 and c8 genes play a key role in a variety of developmental processes. Together, the results of the embryonic/fetal expression of c1 and c8 and those of two previously analyzed genes (c10 and c13) indicate a coherent pattern of expression of these genes in early human ontogeny.
Multiple point mutations in a shuttle vector propagated in human cells: evidence for an error-prone DNA polymerase activity.
Authors: Seidman M M , Bredberg A , Seetharam S , Kraemer K H
Published: May 2006

Mutagenesis was studied at the DNA-sequence level in human fibroblast and lymphoid cells by use of a shuttle vector plasmid, pZ189, containing a suppressor tRNA marker gene. In a series of experiments, 62 plasmids were recovered that had two to six base substitutions in the 160-base-pair marker gene. Approximately 20-30% of the mutant plasmids that were recovered after passing ultraviolet-treated pZ189 through a repair-proficient human fibroblast line contained these multiple mutations. In contrast, passage of ultraviolet-treated pZ189 through an excision-repair-deficient (xeroderma pigmentosum) line yielded only 2% multiple base substitution mutants. Introducing a single-strand nick in otherwise unmodified pZ189 adjacent to the marker, followed by passage through the xeroderma pigmentosum cells, resulted in about 66% multiple base substitution mutants. The multiple mutations were found in a 160-base-pair region containing the marker gene but were rarely found in an adjacent 170-base-pair region. Passing ultraviolet-treated or nicked pZ189 through a repair-proficient human B-cell line also yielded multiple base substitution mutations in 20-33% of the mutant plasmids. An explanation for these multiple mutations is that they were generated by an error-prone polymerase while filling gaps. These mutations share many of the properties displayed by mutations in the immunoglobulin hypervariable regions.
Cytogenetic and molecular studies on a recombinant human X chromosome: implications for the spreading of X chromosome inactivation.
Authors: Mohandas T , Geller R L , Yen P H , Rosendorff J , Bernstein R , Yoshida A , Shapiro L J
Published: May 2006

A pericentric inversion of a human X chromosome and a recombinant X chromosome [rec(X)] derived from crossing-over within the inversion was identified in a family. The rec(X) had a duplication of the segment Xq26.3----Xqter and a deletion of Xp22.3----Xpter and was interpreted to be Xqter----Xq26.3::Xp22.3----Xqter. To characterize the rec(X) chromosome, dosage blots were done on genomic DNA from carriers of this rearranged X chromosome using a number of X chromosome probes. Results showed that anonymous sequences from the distal end of the long arm to which probes 4D8, Hx120A, DX13, and St14 bind as well as the locus for glucose-6-phosphate dehydrogenase (G6PD) were duplicated on the rec(X). Mouse-human cell hybrids were constructed that retained the rec(X) in the active or inactive state. Analyses of these hybrid clones for markers from the distal short arm of the X chromosome showed that the rec(X) retained the loci for steroid sulfatase (STS) and the cell surface antigen 12E7 (MIC2); but not the pseudoautosomal sequence 113D. These molecular studies confirm that the rec(X) is a duplication-deficiency chromosome as expected. In the inactive state in cell hybrids, STS and MIC2 (which usually escape X chromosome inactivation) were expressed from the rec(X), whereas G6PD was not. Therefore, in the rec(X) X chromosome inactivation has spread through STS and MIC2 leaving these loci unaffected and has inactivated G6PD in the absence of an inactivation center in the q26.3----qter region of the human X chromosome. The mechanism of spreading of inactivation appears to operate in a sequence-specific fashion. Alternatively, STS and MIC2 may have undergone inactivation initially but could not be maintained in an inactive state.
Characterization of c-Ki-ras oncogene alleles by direct sequencing of enzymatically amplified DNA from carcinogen-induced tumors.
Authors: McMahon G , Davis E , Wogan G N
Published: May 2006

Activated c-Ki-ras genes in liver tumors from rats exposed to the potent hepatocarcinogen aflatoxin B1 were analyzed to determine the nature of their activation by characterization of two c-Ki-ras alleles present in tumor-derived NIH 3T3 mouse transformants. Using selective hybridization of synthetic oligonucleotides to transformant DNA, we have determined that a single G X C to A X T base transition in either the first or second position of the 12th codon is associated with activation of the gene. Such mutations would lead to amino acid substitutions of aspartate or serine for glycine in the mutant proteins. To confirm these findings, we applied a technique for direct sequence analysis of a 90-base-pair region of the rat c-Ki-ras gene produced by primer-directed enzymatic amplification. Findings produced by this approach, which provides a convenient method to characterize mutations in multiple alleles without the necessity to clone individual genes, confirmed the presence and identity of the 12th codon mutations in the activated oncogene, as initially determined by the oligonucleotide hybridization technique.
Construction of an extended three-dimensional idiotope map by electron microscopic analysis of idiotope-anti-idiotope complexes.
Authors: Roux K H , Monafo W J , Davie J M , Greenspan N S
Published: May 2006

A three-dimensional map of the positions of four idiotypic determinants (idiotopes or Ids) and an isotypic determinant was derived by transmission electron microscopy of negatively stained immune complexes. Each complex was composed of a monoclonal Id-expressing IgG and one or two varieties of monoclonal anti-Id (or anti-isotype) Fab fragment or IgG. Data from the various combinations of Id and anti-Id (and anti-isotype) were used to construct a low-resolution three-dimensional model that revealed not only the approximate locations of Ids on the surface of the antibody variable domains but also details of the geometry of Id-anti-Id interactions not otherwise available. The Ids were shown to be dispersed over the variable domains, extending from the complementarity-determining region to near the variable-constant switch region. Thus, immunoelectron microscopy is a useful complement to serologic, biochemical, and genetic strategies for the topographical analysis of immunoglobulin Ids or other epitopes. This same approach should be of broader applicability in the study of epitopes and receptor sites on other macromolecules.
Primary structure of bovine pituitary secretory protein I (chromogranin A) deduced from the cDNA sequence.
Authors: Ahn T G , Cohn D V , Gorr S U , Ornstein D L , Kashdan M A , Levine M A
Published: May 2006

Secretory protein I (SP-I), also referred to as chromogranin A, is an acidic glycoprotein that has been found in every tissue of endocrine and neuroendocrine origin examined but never in exocrine or epithelial cells. Its co-storage and co-secretion with peptide hormones and neurotransmitters suggest that it has an important endocrine or secretory function. We have isolated cDNA clones from a bovine pituitary lambda gt11 expression library using an antiserum to parathyroid SP-I. The largest clone (SP4B) (approximately equal to 1.6 kilobases) hybridized to a transcript of 2.1 kilobases in RNA from parathyroid, pituitary, and adrenal medulla. Immunoblots of bacterial lysates derived from SP4B lysogens demonstrated specific antibody binding to an SP4B/beta-galactosidase fusion protein (160 kDa) with a cDNA-derived component of 46 kDa. Radioimmunoassay of the bacterial lysates with SP-I antiserum yielded parallel displacement curves of 125I-labeled SP-I by the SP4B lysate and authentic SP-I. SP4B contains a cDNA of 1614 nucleotides that encodes a 449-amino acid protein (calculated mass, 50 kDa). The nucleotide sequences of the pituitary SP-I cDNA and adrenal medullary SP-I cDNAs are nearly identical. Analysis of genomic DNA suggests that pituitary, adrenal, and parathyroid SP-I are products of the same gene.
Hexahydro-1,4-thiazepine-3,5-dicarboxylic acid and thiomorpholine-3,5-dicarboxylic acid are present in normal human urine.
Authors: Matarese R M , Pecci L , Ricci G , Nardini M , Antonucci A , Cavallini D
Published: May 2006

Hexahydro-1,4-thiazepine-3,5-dicarboxylic acid and thiomorpholine-3,5-dicarboxylic acid, simply referred to as cyclothionine and TMDA, respectively, are two cyclic sulfur-containing imino acids detected in bovine brain. Human urine has been investigated to establish the occurrence of these imino acids as common constituents under normal conditions. The morning urine of healthy subjects has been analyzed for enrichment of these compounds by using an ion-exchange procedure. Gas/liquid chromatography of the final extracts revealed the presence of peaks coeluting with authentic cyclothionine and TMDA. The latter compound eluted very close to an unknown sulfur-containing compound. A resolved peak of TMDA has been obtained by high-performance liquid chromatography of the final extracts derivatized with phenylisothiocyanate. Selected ion monitoring with multiple-ion detection applied to the compounds separated by gas chromatography revealed the presence of the respective molecular ions and of the decarboxylated fragments, thus confirming the identification of cyclothionine and TMDA in human urine.
Isolation of cDNA clones coding for human tissue factor: primary structure of the protein and cDNA.
Authors: Spicer E K , Horton R , Bloem L , Bach R , Williams K R , Guha A , Kraus J , Lin T C , Nemerson Y , Konigsberg W H
Published: May 2006

Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. lambda Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin K-dependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC).
Regulation of cell shape in the Cloudman melanoma cell line.
Authors: Preston S F , Volpi M , Pearson C M , Berlin R D
Published: May 2006

We show that Cloudman melanoma cells undergo rapid arborization in response to [Nle4,D-Phe7]alpha-melanocyte-stimulating hormone, a potent analogue of alpha-melanocyte stimulating hormone (alpha-MSH). The arbors were established by extension of processes and resembled dendrites. We used this system to study the regulation of cell shape. alpha-MSH is known to induce increases in cAMP levels, and agents such as forskolin and isobutylmethylxanthine that led to increased cAMP also caused arborization. However, equally dramatic arbors were formed after incubation with the protein kinase C inhibitor H-7 [1-(5-isoquinolinesulfonyl)-alpha-methyl-piperazine]. Phorbol diesters that activate protein kinase C led to cell rounding and antagonized alpha-MSH. The actions of protein kinase C cannot be rationalized in terms of indirect effects on cAMP: neither H-7 nor phorbol diesters alone altered cAMP levels, nor did they affect the increase in cAMP induced by MSH. We show also that MSH produced longer-term effects that cannot be mimicked by cAMP. Specifically, even in the continued presence of alpha-MSH, arborization was followed by morphological reversal to the unstimulated flattened configuration within 2 hr. (This did not occur with other agents that increase cAMP or with H-7.) Most importantly, whereas MSH-induced arborization occurred in the presence of cycloheximide, actinomycin D, or in enucleated cells, the reversal of arborization did not. Thus, MSH induced a program of rapid shape change that was dependent on new protein synthesis and gene transcription.
Primary structure and nuclear localization of a murine homeodomain protein.
Authors: Kessel M , Schulze F , Fibi M , Gruss P
Published: May 2006

The murine homeobox Hox 1.1 (m6) is the first of a cluster of six boxes on chromosome 6. Using probes and synthetic peptides derived from the Hox 1.1 sequence, we were able to isolate cDNAs and antibodies that allowed us to characterize the product of this homeobox-containing gene. From the open reading frame on the cDNA clone B21, a protein could be predicted, made up of 229 amino acids and having a calculated molecular weight of 25,740. A unique feature of this protein is that it has 15 glutamic acid residues as its carboxyl terminus, which gives it a very hydrophilic and acidic carboxyl terminal structure, most probably folding onto an alpha-helix. A second domain of six amino acids is present on the Hox 1.1 protein, which is conserved in other homeodomain proteins. Antibodies generated against synthetic peptides from the homeobox region were used in the immunoblotting procedure and revealed a major protein band of Mr 31,000 in extracts from 3T3 cells and F9 teratocarcinoma cells induced by retinoic acid and cAMP. The nuclear location of the protein was established by immunofluorescence. The presence of this protein in F9 cell nuclei is in faithful accordance with the kinetics established for the 2.4-kilobase Hox 1.1 transcript during differentiation into parietal endoderm cells.
Differential effects of base-pair mismatch on intrachromosomal versus extrachromosomal recombination in mouse cells.
Authors: Waldman A S , Liskay R M
Published: May 2006

To initially determine the effect that base-pair mismatch has on homologous recombination in mammalian cells, we have studied genetic recombination between thymidine kinase (tk) gene sequences from herpes simplex virus 1 and 2. These tk genes are approximately 81% homologous at the nucleotide level. We observed that, in mouse LTK- cells, intrachromosomal recombination between type 1 and type 2 tk sequences is reduced by a factor of at least 1000 relative to the rate of intrachromosomal recombination between homologous type 1 tk sequences. In sharp contrast, the rate of intermolecular or intramolecular extrachromosomal recombination between the heterologous tk sequences introduced by calcium phosphate or microinjection was reduced only by a factor of 3 to 15 compared with extrachromosomal homologous tk crosses. Our results suggest differences between the mechanisms of extrachromosomal and intrachromosomal recombination in mammalian cells.
Alternative use of 5' exons in the specification of Ly-5 isoforms distinguishing hematopoietic cell lineages.
Authors: Saga Y , Tung J S , Shen F W , Boyse E A
Published: May 2006

Previous inferences that Ly-5 glycoprotein isoforms of murine hematopoietic cells are generated by alternative splicing of primary transcripts of a single Ly-5 gene are supported by the present study. A cDNA library was prepared from B cells by extension from primer representing a known T-cell cDNA sequence. Three different Ly-5 clones from this library included sequences missing in T-cell cDNA clones. From the constitution of cDNA clones and of the Ly-5 gene, and from S1 nuclease mapping, it is concluded that at least two exons, provisionally numbered Ex-6(B) and Ex-7(B), in the 5'-proximal region are mainly represented in mRNA of the B-cell lines examined but not of the T-cell lines examined. Also, exons 1 and 2 appear to be used alternatively in different species of B-cell mRNA and probably also in different species of T-cell mRNA.
Mu and delta receptors belong to a family of receptors that are coupled to potassium channels.
Authors: North R A , Williams J T , Surprenant A , Christie M J
Published: May 2006

The effects of agonists at mu and delta opioid receptors were compared by measuring membrane currents under voltage clamp from neurons of the rat nucleus locus coeruleus and guinea pig submucous plexus. In each tissue, the appropriate selective agonist (Tyr-D-Ala-Gly-MePhe-Gly-ol for mu receptors in locus coeruleus or Tyr-D-Pen-Gly-Phe-D-Pen for delta receptors in submucous plexus) increased the conductance of an inwardly rectifying potassium conductance and strongly hyperpolarized the membrane. The properties of the potassium conductance affected by the two opioids could not be distinguished. Experiments with intracellular application of guanosine 5'-[gamma-thio]triphosphate indicated that a guanine nucleotide-binding regulatory protein was involved in the coupling between opioid receptor and potassium channel, but there was no evidence for activation of either cAMP-dependent protein kinase or protein kinase C. It is noted that a number of vertebrate neurotransmitter receptors are coupled to potassium channels. The potassium conductance associated with these channels has properties similar to the conductance activated by mu and delta opioids; this family includes the following receptors: acetylcholine M2, norepinephrine alpha 2, dopamine D2, 5-hydroxytryptamine 5-HT1, adenosine A1, gamma-aminobutyric acid GABAB, and somatostatin. It is suggested that this conductance is a conserved neuronal effector coupled to one of the receptor types that mediates the effects of each of several major transmitters. The mu and delta opioid receptors appear to be unusual in that both utilize this same effector mechanism.
Topography and stoichiometry of acidic proteins in large ribosomal subunits from Artemia salina as determined by crosslinking.
Authors: Uchiumi T , Wahba A J , Traut R R
Published: May 2006

The 60S subunits isolated from Artemia salina ribosomes were treated with the crosslinking reagent 2-iminothiolane under mild conditions. Proteins were extracted and fractions containing crosslinked acidic proteins were obtained by stepwise elution from CM-cellulose. Each fraction was analyzed by "diagonal" (two-dimensional nonreducing-reducing) NaDodSO4/polyacrylamide gel electrophoresis. Crosslinked proteins below the diagonal were radioiodinated and identified by two-dimensional acidic urea-NaDodSO4 gel electrophoresis. Each of the acidic proteins P1 and P2 was crosslinked individually to the same third protein, P0. The fractions containing acidic proteins were also analyzed by two-dimensional nonequilibrium isoelectric focusing-NaDodSO4/polyacrylamide gel electrophoresis. Two crosslinked complexes were observed that coincide in isoelectric positions with monomeric P1 and P2, respectively. Both P1 and P2 appear to form crosslinked homodimers. These results suggest the presence in the 60S subunit of (P1)2 and (P2)2 dimers, each of which is anchored to P0. Protein P0 appears to play the same role as L10 in Escherichia coli ribosomes and may form a pentameric complex with the two dimers in the 60S subunits.
Agents that elevate the concentration of cAMP in platelets inhibit the formation of a NaDodSO4-resistant complex between thrombin and a 40-kDa protein.
Authors: Lerea K M , Glomset J A
Published: May 2006

We studied the influence of prostaglandin E1 and theophylline on the ability of rabbit or human platelets to form NaDodSO4-resistant complexes between 125I-labeled thrombin and a platelet protein of approximately equal to 40 kDa. Control platelets formed two types of these complexes, one that sedimented with the platelets and one that was found in the suspension medium. There were 30-40 sedimentable complexes per platelet and about three times this number of soluble complexes. Pretreatment of rabbit or human platelets with prostaglandin E1 and theophylline decreased the formation of both types of complex by as much as 60-80%. The pretreatment was particularly effective when low doses of thrombin were used. When added to control platelets, such doses of thrombin caused the formation of sedimentable complexes and the concomitant formation of inositol trisphosphate. However, when added to platelets that had been pretreated with prostaglandin E1 and theophylline, low doses of thrombin had little or no effect on either reaction.
Differential expression of opioid peptide genes by testicular germ cells and somatic cells.
Authors: Kilpatrick D L , Borland K , Jin D F
Published: May 2006

Spermatogenic cells have been previously shown to be a major site of testicular proenkephalin gene expression. Using RNA gel-blot analysis of purified mouse and hamster germ cells and of testes from prepuberal and germ cell-deficient mutant mice, we now have demonstrated that, in addition to its previously described expression by somatic (Leydig) cells, the gene for a second opioid peptide precursor, pro-opiomelanocortin (POMC), is also expressed by spermatogenic cells. Of particular significance is the finding that the RNAs for proenkephalin and POMC are differentially regulated during spermatogenesis. Two forms of POMC RNA were detected in mouse testis, a larger component 675- to 750-nucleotides (nt) in size common to somatic and spermatogenic cells and a smaller 625-nt RNA found only in pachytene spermatocytes. Two distinct, cell-specific proenkephalin RNAs were also shown to be present in mouse testis: a 1700-nt transcript previously shown to be expressed by spermatogenic cells and a 1450-nt form associated with somatic cells. These data suggest that proenkephalin- and POMC-derived peptides are produced by both somatic cells and germ cells in the testis and in germ cells these two families of opioid peptides may function at different stages of spermatogenesis.
Coclustering of CD4 (L3T4) molecule with the T-cell receptor is induced by specific direct interaction of helper T cells and antigen-presenting cells.
Authors: Kupfer A , Singer S J , Janeway C A , Swain S L
Published: May 2006

Blocking studies with monoclonal antibodies have suggested that helper T cell recognition and triggering involve the CD4 (L3T4) accessory molecule as well as the T-cell receptor (TCR) that is linked to the T3 complex. We have investigated the surface distribution of L3T4 and TCR during the direct interaction of a cloned murine helper T-cell line with an antigen-presenting B-cell line. Using immunofluorescence microscopy, we show that in 1:1 cell couples formed between the two cells, in which a specific interaction can be demonstrated, the L3T4 and the TCR become redistributed on the T-cell surface so that they are concentrated in the cell-cell contact region. This coclustering of L3T4 with TCR occurs only when the relevant antigen and appropriate major histocompatibility class II molecules are presented to the T cell, and it therefore requires the specific interaction of the TCR with its complex ligand on the antigen-presenting cell.
Cell transformation and tumor induction by Abelson murine leukemia virus in the absence of helper virus.
Authors: Green P L , Kaehler D A , Risser R
Published: May 2006

We investigated the role of the Moloney helper virus, Moloney murine leukemia virus (Mo-MuLV), in cell transformation and tumor induction by the defective Abelson murine leukemia virus (Ab-MuLV). A molecular clone of Ab-MuLV (P160 strain) was transfected into the psi 2 packaging cell line, and helper virus-free Ab-MuLV (psi 2) was harvested from the supernatant medium. Ab-MuLV (psi 2) was as efficient as helper virus-containing Ab-MuLV (Mo-MuLV) in the transformation of primary bone marrow cells in vitro. Inoculation of weanling BALB/c mice with Ab-MuLV (psi 2) induced nonthymic pre-B-cell lymphomas with high efficiency and short latency (28 days). Adult BALB/c mice were less sensitive to tumor induction by a factor of 100. Ab-MuLV (psi 2) did not induce tumors in weanling C57BL/6 mice, unlike Ab-MuLV (Mo-MuLV). Examination of the proviral integration pattern in Ab-MuLV (psi 2)-induced tumor cell DNA revealed that each of the tumors contained a single integrated provirus. Immunoprecipitation of viral-encoded proteins in helper virus-free tumor cell lines detected the P160 Ab-MuLV-transforming protein; however, no trace of the gag, pol, and env helper virus-encoded proteins was found. Our results indicate that integration and expression of a single Ab-MuLV genome is sufficient for efficient transformation of primary bone marrow cells by Ab-MuLV in vitro and tumor induction in susceptible mice. However, the helper virus may contribute to tumor induction in weanling resistant mice.
Molecular cloning of a brain-specific calcium/calmodulin-dependent protein kinase.
Authors: Lin C R , Kapiloff M S , Durgerian S , Tatemoto K , Russo A F , Hanson P , Schulman H , Rosenfeld M G
Published: May 2006

A calcium/calmodulin-dependent protein kinase type II (CaM-K) alpha-subunit cDNA has been cloned from rat brain. This enzyme is encoded by a 5.1-kilobase mRNA expressed exclusively in the brain. Hybridization histochemistry reveals that the CaM-K mRNA expression corresponds to the distribution of the immunoreactive alpha-subunit protein, suggesting that the high enzyme levels in specific brain areas reflect regional differences in gene expression. The sequence of CaM-K alpha-subunit cDNA indicates a 478-amino acid (54-kDa) protein with three functional domains. The domain organization suggests a structural model for calcium/calmodulin-dependent and independent states that might subserve short- and long-term responses to transient stimuli.
Two yeast genes that encode unusual protein kinases.
Authors: Levin D E , Hammond C I , Ralston R O , Bishop J M
Published: May 2006

Mixed synthetic oligonucleotides encoding sequences conserved among tyrosine-specific protein kinases were used to probe the genome of the budding yeast Saccharomyces cerevisiae. Two genes with homology to protein kinases were isolated and characterized by DNA sequence analysis. These genes, designated KIN1 and KIN2, are closely related to each other. Among previously characterized protein kinases, the products of KIN1 and KIN2 are most closely related to the bovine cAMP-dependent protein kinase (30% amino acid identities) and the protein encoded by the v-src oncogene (27% and 25% identities with KIN1 and KIN2, respectively) within their putative kinase domains. KIN1 and KIN2 are transcribed into 3.5-kilobase mRNAs that contain uninterrupted open reading frames encoding polypeptides of 117 kDa and 126 kDa, respectively. The predicted proteins are unusual in two respects: (i) their catalytic domains are carried near the N termini of relatively large proteins, in contrast to the majority of characterized protein kinases, and (ii) these catalytic domains are structural mosaics, with some features characteristic of tyrosine-specific protein kinases and other elements that are distinctive of serine/threonine-specific enzymes.
Behavioral and sensory basis of courtship success in Drosophila melanogaster.
Authors: Markow T A
Published: May 2006

In Drosophila some individuals are more successful at mating than others. Reproductive fitness is strongly dependent upon the ability to recognize and compete for members of the opposite sex. Experiments were designed to answer two questions. (i) What behavioral components are characteristic or predictive of successful courtship? and (ii) How important is the information transmitted in the different sensory channels for courtship success in each sex? These questions were approached by two experimental procedures. Flies having a sensory deficiency (olfactory, auditory, or visual) competed with wild-type flies of the same sex for mates. Males were found to rely upon sensory channels different from those used by females in order to court successfully. In addition, the courtships of pairs of various genotypes were recorded and subjected to multivariate analysis. The multivariate courtship profiles deviated most widely from those of successful wild-type pairs when the male or female was unable to receive information in the sensory channel most important for successful mating by that sex. Both sequential and quantitative courtship properties were altered when one participant was deficient in ability to receive certain sensory information.
DNA sequence analysis suggests that expression of flagellar and chemotaxis genes in Escherichia coli and Salmonella typhimurium is controlled by an alternative sigma factor.
Authors: Helmann J D , Chamberlin M J
Published: May 2006

Biosynthesis of bacterial flagella involves the coordinated expression of 30 or more genes in several separate operons. We have recently shown that in Bacillus subtilis, the sigma 28 factor is essential for flagellar synthesis, suggesting that transcription of these genes is directly under the control of this alternative sigma factor. In enteric bacteria structural genes for flagellar, chemotaxis, and motility operons appear to be under coordinate control, however, the nature of the regulatory factors has not been determined. Sequence analysis of many such genes has failed to reveal plausible promoter sequences for the predominant bacterial RNA polymerase, and several such genes are not transcribed effectively in vitro by the Escherichia coli sigma 70 RNA polymerase. However, all of the sequenced flagellar, chemotaxis, and motility operons from the enteric bacteria are preceded by DNA sequences highly homologous to B. subtilis sigma 28 promoters. We propose that an alternative sigma factor controls expression of the flagellar regulon in both B. subtilis and in the enteric bacteria.
Proton/hydroxide conductance and permeability through phospholipid bilayer membranes.
Authors: Gutknecht J
Published: May 2006

Proton/hydroxide (H+/OH-) permeability of phospholipid bilayers is several orders of magnitude higher than alkali or halide ion permeabilities at pH 7. The objective of this study was to determine the mechanism(s) of H+/OH- conductance and permeability through planar phospholipid bilayer membranes. Membranes were formed from decane solutions of bacterial phosphatidylethanolamine, diphytanoyl phosphatidylcholine, or egg phosphatidylcholine plus cholesterol. At pH 7, H+/OH- conductance (GH/OH) ranged from 2 to 6 nS.cm-2, corresponding to H+/OH- "net" permeabilities of (0.4-1.6) X 10(-5) cm.sec-1. GH/OH was inhibited by serum albumin (fatty acid-free), phloretin, and low pH. GH/OH was increased by chlorodecane, long-chain fatty acids, and voltages greater than 80 mV. Water permeability and GH/OH were not correlated. The results suggest that the H+/OH- charge carrier (i) is primarily anionic, (ii) crosses the membrane via nonpolar pathway(s), and (iii) can be removed from the membrane by "washing" with serum albumin. The simplest explanation is that the phospholipids contain weakly acidic contaminants that act as proton carriers at neutral pH. However, at low pH or in the presence of inhibitors, a "background" GH/OH remains that may be due to other mechanisms.
Cranin: a laminin-binding protein of cell membranes.
Authors: Smalheiser N R , Schwartz N B
Published: May 2006

We report that a 120-kDa glycoprotein is the predominant laminin-binding protein detected within plasma membranes of rodent NG108-15 neural hybrid cells, embryonic chicken brain, and mouse 3T3 fibroblasts. This protein was detected when membrane extracts were separated by PAGE, transferred to nitrocellulose, and incubated with laminin at concentrations as low as 2.8 X 10(-11) M, under conditions of physiological ionic strength and pH and in the presence of calcium ions. It behaves as an integral membrane component, and its laminin-binding moiety is accessible to the external face of the cell surface. Moreover, it appears to bind to a site on laminin that is very sensitive to proteolysis. The properties of this protein, which we have termed "cranin," distinguish it from other known laminin receptors and make it a candidate to mediate some of the effects of laminin upon cells.
Detection of disease-specific restriction fragment length polymorphisms in pemphigus vulgaris linked to the DQw1 and DQw3 alleles of the HLA-D region.
Authors: Szafer F , Brautbar C , Tzfoni E , Frankel G , Sherman L , Cohen I , Hacham-Zadeh S , Aberer W , Tappeiner G , Holubar K
Published: May 2006

Pemphigus vulgaris in Israeli Ashkenazi and non-Ashkenazi Jews and in Austrian non-Jewish patients is strongly associated with the DR4 and DRw6 alleles of the HLA-D region class II genes. Restriction fragment length polymorphism analysis was undertaken with DQ beta, DQ alpha, and DR beta cDNA probes. Hybridization with the DQ beta probe identifies Pvu II, BamHI, and EcoRV fragments that absolutely discriminate pemphigus vulgaris patients from healthy DR-, DQ-, and ethnic-matched controls. In contrast the DQ alpha and DR beta probes failed to identify disease-specific restriction fragment length polymorphism fragments. These studies indicate that DQw1 and DQw3 polymorphisms carried by pemphigus vulgaris patients may be directly involved in predisposition to the disease or may be tightly linked to the susceptibility gene itself. To our knowledge, this is the first example of an HLA restriction fragment length polymorphism that is highly associated with susceptibility to autoimmune disease.
Poly(gamma-glutamylcysteinyl)glycine: its role in cadmium resistance in plant cells.
Authors: Jackson P J , Unkefer C J , Doolen J A , Watt K , Robinson N J
Published: May 2006

Angiosperms can be selected for the ability to grow in the presence of normally toxic concentrations of certain trace metal ions. Addition of Cd and Cu to Cd-resistant Datura innoxia cell cultures results in the rapid synthesis and accumulation of sulfur-rich, metal-binding polypeptides. The structure of these compounds was determined using amino acid analysis, 13C NMR, and site-specific enzymic digestion. These compounds are poly(gamma-glutamylcysteinyl)glycines. Greater than 80% of the cellular Cd is bound to the bis and tris forms in Cd-resistant cells. There is a direct correlation between the maximum accumulation of the metal-binding polypeptides and the concentration of toxic ions to which the cells are resistant. In the presence of metal ions, the polypeptides form multimeric aggregates that can be resolved by gel chromatography. Cd binds to both the high and low molecular weight aggregates, whereas Cu preferentially binds to the higher molecular weight forms. The presence of gamma-carboxamide linkages between glutamyl and adjacent cysteinyl residues indicates that these polypeptides are products of biosynthetic pathways. Poly(gamma-glutamylcysteinyl)glycines bind metals and, in this respect, appear to be functional analogs of the protein metallothionein. However, in the absence of supraoptimal concentrations of trace metal ions, the functions of metallothionein in animals and microorganisms and poly(gamma-glutamylcysteinyl)glycines in plants may differ.
The major clotting protein from guinea pig seminal vesicle contains eight repeats of a 24-amino acid domain.
Authors: Moore J T , Hagstrom J , McCormick D J , Harvey S , Madden B , Holicky E , Stanford D R , Wieben E D
Published: May 2006

The complete amino acid sequence of the major clotting protein from the guinea pig seminal vesicle (SVP-1) has been determined by nucleotide sequencing of cDNA clones corresponding to the 3' terminus of an mRNA that codes for a protein precursor to SVP-1. The first 40 amino acids of the derived protein sequence are identical to those determined by N-terminal sequencing of SVP-1 isolated from the lumen of the seminal vesicle. This finding confirms that SVP-1 is cleaved from the C terminus of a larger precursor protein. The portion of the nucleotide sequence that codes for SVP-1 contains eight highly homologous but imperfect repeats of a 72-nucleotide domain. This repeated structure is also evident at the amino acid level. The consensus 24-amino acid repeat unit contains two lysine and three glutamine residues. Since the clotting of SVP-1 is known to involve the formation of gamma-glutamyl-epsilon-lysine crosslinks, it is likely that the 24-amino acid repeating unit is the unit of function of SVP-1.
Antigen-presenting B cells and helper T cells cooperatively mediate intravirionic antigenic competition between influenza A virus surface glycoproteins.
Authors: Johansson B E , Moran T M , Kilbourne E D
Published: May 2006

Parenteral vaccination of BALB/c mice primed by infection with H3N2 variants of influenza A virus results in a reduced production of N2 antibody in response to homologous (H3N2) vaccine compared with the response to an H7N2 vaccine equal in N2 immunologenicity. We now have studied the interaction in vitro of purified splenic B and T lymphocytes from variably immunized mice to ascertain the cellular basis of the hemagglutinin (HA)-influenced antibody response to neuraminidase (NA). Assay of the proliferative response of T cells in B/T-cell mixtures stimulated by H3N1 (HA-specific) and H6N2 (NA-specific) reassortant (recombinant) viruses in vitro has enabled us to differentiate cellular responses to HA and NA antigens. Using a factorial design in analysis of B/T-cell mixtures, we have shown that: (i) intravirionic HA is dominant over NA in both B- and T-cell priming; (ii) an increase in H3-specific B cells occurs in mice administered boosters of H3N2 vaccine, and an increase in N2-specific B cells occurs in those given a booster of H7N2 vaccine; and (iii) memory B cells function as antigen-presenting cells and interact with memory helper T cells in the mediation of intravirionic HA-NA antigenic competition in favor of HA. The damping of response to the NA antigen in favor of HA with reinfection prohibits balanced immunologic response to the two antigens. The present studies define further the complex immunology of influenza virus infection.
Diversity and structure of human T-cell receptor alpha-chain variable region genes.
Authors: Klein M H , Concannon P , Everett M , Kim L D , Hunkapiller T , Hood L
Published: May 2006

The nucleotide sequences of 27 T-cell receptor alpha-chain variable region (V alpha)-containing cDNA clones isolated from a cDNA library derived from human peripheral blood lymphocytes were determined. Eighteen different V alpha and 26 different joining (J alpha) gene segments are utilized in these clones. The V alpha gene segments belong to 12 different subfamilies, each containing from one to seven members. Comparisons with the 16 different V alpha and 21 different J alpha sequences previously reported suggest that the germ-line repertoires for these gene segments are greater than previously estimated. Flexibility in the sites of gene segment joining and possibly N-region diversification also contribute to human alpha-chain diversity. Comparisons of human V alpha regions indicate a high degree of variability spread uniformly across the entire V alpha region without obvious hypervariable regions. However, amino acids important for the maintenance of V gene structure are conserved.
Long-term physiological effects of enhanced O2 release by inositol hexaphosphate-loaded erythrocytes.
Authors: Teisseire B , Ropars C , Villeréal M C , Nicolau C
Published: May 2006

A continuous lysing and resealing procedure with erythrocytes permitted incorporation in these cells of inositol hexaphosphate (InsP6), a strong allosteric effector of Hb. This leads to significant rightward shifts of the HbO2 dissociation curves with in vitro P50 (partial pressure of O2 at 50% Hb saturation), values increasing from 32.2 +/- 1.8 torr for control erythrocytes to 86 +/- 60 torr (pH 7.40; PCO2 40 torr at 37 degrees C; 1 torr = 1.333 X 10(2) Pa). The shape of the dissociation curve was still sigmoidal, although the Hill coefficient was decreased. The life span of InsP6-loaded erythrocytes equaled that of control erythrocytes. The long-term physiological effects of the InsP6-loaded erythrocytes on piglets were increased O2 release and reduced cardiac output. The reduced O2 affinity of the InsP6-loaded erythrocytes was still effective 20 days after transfusion in awake piglets. The electrolyte concentration appeared stable over the 5-day observation period except for a transient, but significant, hyperkalemia immediately after transfusion. The reductions in the O2 affinity of Hb reported here are large compared with previously reported values. Introduction of InsP6 into viable erythrocytes improves tissue oxygenation when, for any reason, normal blood flow is impaired.
Retinal transplants can drive a pupillary reflex in host rat brains.
Authors: Klassen H , Lund R D
Published: May 2006

Retinae taken from embryonic rats were transplanted over the midbrain of neonatal rats, from whom one eye had been removed. After 5 months, the optic nerve of the remaining eye was cut, and the transplant was exposed. Illumination of the transplant caused pupilloconstriction of the host eye, a response abolished by damaging the transplant. Thus neural transplants are capable of driving specific reflexes in response to natural stimuli.
beta-Hydroxyasparagine in domains homologous to the epidermal growth factor precursor in vitamin K-dependent protein S.
Authors: Stenflo J , Lundwall A , Dahlbäck B
Published: May 2006

Vitamin K-dependent protein S is involved in the regulation of blood coagulation. It is a 75-kDa single chain protein with an NH2-terminal gamma-carboxyglutamic acid-containing domain followed by a thrombin-sensitive region and four domains arranged in tandem, each of which is homologous to the epidermal growth factor (EGF) precursor. The NH2-terminal EGF-like domain contains beta-hydroxyaspartic acid, which has been identified in vitamin K-dependent proteins. The following EGF-like repeat has a very pronounced sequence homology (10 consecutive residues identical) to one of the EGF-like units in the EGF precursor. We now show that, in protein S, this EGF-like repeat has one beta-hydroxyasparagine residue formed by hydroxylation of asparagine. The two COOH-terminal EGF-like repeats also contain beta-hydroxyasparagine, an amino acid not previously found in proteins. Sequence comparisons have enabled us to identify a consensus sequence that seems to be required by the hydroxylase(s).