bluuebunny/crossref_metadata_2025 huggingface dataset:
Phospholipase A2 activity of low density lipoprotein: evidence for an intrinsic phospholipase A2 activity of apoprotein B-100.
Authors: Parthasarathy S , Barnett J
Published: May 2006

During oxidative modification of low density lipoprotein (LDL) there is extensive degradation of phosphatidylcholine (PtdCho) to lysophosphatidylcholine (lyso-PtdCho), with the removal of fatty acids from the 2 position. The phospholipase A2 (PLA2) activity responsible for hydrolysis is closely associated with LDL. By use of lipoxygenase-oxidized 2-[1-14C]linoleoyl PtdCho as the substrate and delipidated apoprotein B (apo-B), evidence is presented to show that (i) the activity is destroyed progressively during the oxidative modification of LDL; (ii) p-bromophenacyl bromide (pBPB), a histidine modifier that inhibits the oxidative modification of LDL, also substantially inhibits the PLA2 activity; and (iii) photooxidation of LDL in the presence of Rose Bengal completely inactivates the enzyme with concomitant loss of apo-B histidine residues. High molecular weight proteins from delipidated LDL, separated by polyacrylamide gel electrophoresis, showed PLA2 activity. It is suggested that apo-B itself may possess PLA2 activity.
Method for identifying microbial antigens that stimulate specific lymphocyte responses: application to Salmonella.
Authors: Warren R L , Lu D , Sizemore D R , Baron L S , Kopecko D J
Published: May 2006

Vaccine development and understanding of cellular immune stimulatory mechanisms have been impeded by the paucity of data on microbial antigens that stimulate protective immunity. We describe here a general method for identifying and isolating peptide antigens that specifically stimulate sensitized lymphocytes. First, Salmonella typhimurium C5 genomic DNA fragments were subcloned into Escherichia coli by use of the lambda gt11 expression vector. Next, antigens expressed by recombinant phage from this genomic library were tested for their capacity to stimulate proliferative responses in pooled lymphocytes obtained from BALB/c mice infected 14 days earlier with S. typhimurium. Of 2000 recombinant phages tested, 5 stimulated a polypeptide-antigen-specific proliferative response. Physical analyses of these 5 recombinant phages revealed cloned inserts of 0.5-2.4 kilobase pairs representing nonoverlapping regions of the C5 chromosome. Four of the five insert DNAs hybridized at high stringency to both S. typhimurium and Salmonella typhi total chromosomal DNA, suggesting that these pathogens of different host specificity share several antigenic determinants. Use of sensitized primary polyclonal lymphocytes provides a rapid and simple method for screening recombinant DNA libraries for clones that stimulate specific immune responses and avoids the use of cloned lymphocyte cell lines. This approach should be generally applicable to similar studies in different hosts of many other microbial pathogens.
Site-specific cleavage of duplex DNA by a semisynthetic nuclease via triple-helix formation.
Authors: Pei D , Corey D R , Schultz P G
Published: May 2006

A Lys-84----Cys mutant staphylococcal nuclease was selectively linked to the 5' and/or 3' terminus of a thiol-containing polypyrimidine oligonucleotide via a disulfide bond. The oligonucleotide-staphylococcal nuclease adduct is capable of binding to a homopurine-homopyrimidine region of Watson-Crick duplex DNA by the formation of a triple-helical structure. Upon the addition of Ca2+, the nuclease cleaves DNA at sites adjacent to the homopurine tract. Specific double-strand cleavage occurred predominantly at A + T-rich sites to the 5' side of the homopurine tract for both the 5'-derivatized and the 5',3'-diderivatized nucleases; the 3'-derivatized nuclease gave no cleavage. The cleavage pattern is asymmetric and consists of multiple cleavage sites shifted to the 5' side on each strand, centered at the terminal base pair of the binding site. Microgram amounts of plasmid pDP20 DNA (4433 base pairs) containing a homopurine-homopyrimidine tract were selectively cleaved by a semisynthetic nuclease with greater than 75% efficiency at room temperature within 1 hr. Cleavage reaction conditions were optimized with respect to pH, temperature, reaction times, and reaction components. Semisynthetic nucleases of this type should provide a powerful tool in chromosomal DNA manipulations.
Another mechanism for creating diversity in gamma-aminobutyrate type A receptors: RNA splicing directs expression of two forms of gamma 2 phosphorylation site.
Authors: Whiting P , McKernan R M , Iversen L L
Published: May 2006

Diversity of gamma-aminobutyrate type A (GABAA) receptors has recently been proposed to be achieved by assembly of receptor subtypes from a multitude of subunits (alpha 1-6, beta 1-3, gamma 1-2, and delta) encoded by different genes. Here we report a further mechanism for creating GABAA receptor diversity: alternative RNA splicing. Two forms of bovine gamma 2 subunit cDNA were isolated (gamma 2S and gamma 2L) that differed by the presence or absence of a 24-base-pair (8-amino acid) insertion in the cytoplasmic domain between the third and fourth putative membrane-spanning regions. Polymerase chain reaction from RNA demonstrated that the two forms of gamma 2 subunit are expressed in bovine, human, and rat brain. Sequencing of genomic DNA clones encoding the gamma 2 subunit demonstrated that the 24-base-pair insert is organized as a separate exon. Analysis of the sequence of the 8-amino acid insert revealed that it contains a protein kinase C consensus phosphorylation site. Expression of the large cytoplasmic loop domains of gamma 2S and gamma 2L in Escherichia coli, followed by phosphorylation of the recombinant proteins by protein kinase C, demonstrated that gamma 2L, but not gamma 2S, could be phosphorylated. Thus the two forms of gamma 2 subunit differ by the presence or absence of a protein kinase C phosphorylation site. This mechanism for creating GABAA receptor diversity may allow differential regulation of the function of receptor subtypes.
Immunohistochemical localization of renal mineralocorticoid receptor by using an anti-idiotypic antibody that is an internal image of aldosterone.
Authors: Lombès M , Farman N , Oblin M E , Baulieu E E , Bonvalet J P , Erlanger B F , Gasc J M
Published: May 2006

A monoclonal antibody (H10E), generated by an auto-anti-idiotypic procedure and directed at the aldosterone-binding site of mineralocorticoid receptor (MR), was used in immunohistochemical studies to localize MR in rabbit kidney preparations. In agreement with earlier physiological and biochemical observations, MR was detected in connecting and cortical collecting tubules. Additionally, MR was detected in the distal tubules, the medullary and papillary collecting ducts, and in the epithelial cells lining the papilla. The internal image properties of the antibody were exploited to assess the specificity of MR detection by means of competition studies with hormones and antihormones. Immunostaining was completely abolished by preincubation with aldosterone but not with RU 486, a steroid antagonist that does not bind MR. On the cellular level, immunostaining occurred in the cytoplasm and, in the majority of cells, in the nucleus as well. The nucleocytoplasmic distribution of MR was unaffected by adrenalectomy or by administration of aldosterone. The availability of this specific monoclonal antibody makes it feasible to study MR expression in other target tissues and in pathological disorders.
Characterization of chicken octamer-binding proteins demonstrates that POU domain-containing homeobox transcription factors have been highly conserved during vertebrate evolution.
Authors: Petryniak B , Staudt L M , Postema C E , McCormack W T , Thompson C B
Published: May 2006

The DNA sequence motif ATTTGCAT (octamer) or its inverse complement has been identified as an evolutionarily conserved element in the promoter region of immunoglobulin genes. Two major DNA-binding proteins that bind in a sequence-specific manner to the octamer DNA sequence have been identified in mammalian species--a ubiquitously expressed protein (Oct-1) and a lymphoid-specific protein (Oct-2). During characterization of the promoter region of the chicken immunoglobulin light chain gene, we identified two homologous octamer-binding proteins in chicken B cells. When the cloning of the human gene for Oct-2 revealed it to be a member of a distinct family of homeobox genes, we sought to determine if the human Oct-2 cDNA could be used to identify homologous chicken homeobox genes. Using a human Oct-2 homeobox-specific DNA probe, we were able to identify 6-10 homeobox-containing genes in the chicken genome, demonstrating that the Oct-2-related subfamily of homeobox genes exists in avian species. Low-stringency screening of a chicken embryonic cDNA library allowed us to clone one of these genes. DNA sequence analysis revealed it to be the chicken homologue of the human Oct-1 gene. The predicted protein sequence of the chicken Oct-1 gene demonstrated that the gene for Oct-1 has been highly conserved during vertebrate evolution with an overall 96% amino acid sequence identity between the chicken and human proteins. The previously described POU domain (termed POU for its presence in the Pit-1, Oct-1/Oct-2, and Unc-86 genes) and homeobox domain are 100% conserved between the two protein products. Together, our data show that the POU-containing subfamily of homeobox genes have been highly conserved during vertebrate evolution, apparently as a result of selection for their DNA-binding and transcriptional regulatory properties.
Transient analysis for antiproliferative gene activity.
Authors: Fordis C M , Helmly B , Novotny E , Holter W , Barker J L , Howard B H
Published: May 2006

A subset of tumor suppressor genes presumably functions by the inhibition of cellular proliferation; however, antiproliferative activity after transfection with putative suppressor genes has been difficult to demonstrate and often requires lengthy selection either in nude mice or in vitro. A rapid alternative is presented here that utilizes a gene encoding a surface marker protein to identify transfectants in a transient expression assay. In this assay the labeling index, rate of DNA synthesis, cell-cycle distribution, and surface receptor display are measured by flow cytometry. Human beta-interferon, a gene with proven antiproliferative activity, was studied using the transient analysis system. The beta-interferon gene was introduced into human tumor cells along with the marker gene encoding the 55-kDa subunit of the human interleukin 2 receptor. Within a few days of transfection, analysis of transfectants by flow cytometry revealed a decrease in the fraction of cells in G2/M and an increase in the fraction of cells in G1/G0 and S phases. The distortion of the cell cycle was accompanied by as much as a 69% reduction in the rate of DNA synthesis and, in some experiments, an unanticipated increase in the labeling index. Therefore, cells accumulating in S phase were not blocked but continued to synthesize DNA although at a reduced rate. These studies on DNA synthetic rates revealed the caveat that screening for antiproliferative candidate genes with a labeling index alone could, in certain circumstances, exclude potentially interesting sequences from further consideration. Although this transient analysis system was developed for studies on cellular proliferation, it may prove suitable for phenotypic assays on other genes as well.
Differential expression and enzymatic properties of the Na+,K(+)-ATPase alpha 3 isoenzyme in rat pineal glands.
Authors: Shyjan A W , Ceña V , Klein D C , Levenson R
Published: May 2006

We have used immunoblotting and biochemical techniques to analyze expression of Na+,K(+)-ATPase alpha and beta subunits in rat pineal glands. Western blot analysis of pineal microsomal membrane fractions with antisera specific for each of the three rat alpha and two rat beta subunits revealed similar levels of expression of alpha 1 and alpha 3 subunits in pineal glands of 5-day-old rats. High levels of alpha 3 and beta 2 subunits and low levels of alpha 1 subunits were detected in adult glands. No alpha 2 or beta 1 subunits were detectable at either developmental stage. Examination of the enzymatic properties of the pineal gland alpha 3 isoform suggests that this enzyme is a ouabain-sensitive ATPase whose activity is dependent upon Na+ and K+. This ATPase exhibited a lower apparent Km for Na+ than the kidney alpha 1 isoenzyme and did not show positive cooperative Na+ activation. Our results suggest that the activity of the Na+,K(+)-ATPase alpha 3 isoenzyme may be adapted to function under conditions of hyperpolarizing transmembrane potentials.
A cluster of hematopoietic serine protease genes is found on the same chromosomal band as the human alpha/delta T-cell receptor locus.
Authors: Hanson R D , Hohn P A , Popescu N C , Ley T J
Published: May 2006

The chymotrypsin-like family of serine protease genes includes several members that are expressed exclusively in subsets of hematopoietic cells. For example, human neutrophil elastase and cathepsin G are expressed only in myelomonocytic precursors, and cytotoxic-T-cell serine proteases are found only in cytotoxic lymphocytes. We have used a cathepsin G cDNA probe to clone two cathepsin G-like genes (designated CGL-1 and CGL-2) from a human genomic library. We have determined that CGL-1 is identical to a previously identified gene (known as CCPI, CTLA I, or cytotoxic serine protease B) that is expressed only in activated cytotoxic T lymphocytes. We show here that cathepsin G, CGL-1, and CGL-2 are linked on an approximately 50-kilobase locus found on human chromosome 14 at band q11.2. This gene cluster maps to the same chromosomal band as the alpha and delta T-cell receptor genes; this region is involved in most chromosomal translocations and inversions that are specifically associated with T-cell malignancies.
An unusual insertion/deletion in the gene encoding the beta-subunit of propionyl-CoA carboxylase is a frequent mutation in Caucasian propionic acidemia.
Authors: Tahara T , Kraus J P , Rosenberg L E
Published: May 2006

Propionic acidemia is an inherited disorder of organic acid metabolism that is caused by deficiency of propionyl-CoA carboxylase (PCC; EC 6.4.1.3). Affected patients fall into two complementation groups, pccA and pccBC (subgroups B, C, and BC), resulting from deficiency of the nonidentical alpha and beta subunits of PCC, respectively. We have detected an unusual insertion/deletion in the DNA of patients from the pccBC and pccC subgroups that replaces 14 nucleotides in the coding sequence of the beta subunit with 12 nucleotides unrelated to this region of the gene. This results in elimination of an Msp I restriction site, a 2-base-pair (bp) deletion, a frameshift, and a stop codon in the new frame approximately 100 amino acid residues proximal to the normal carboxyl terminus. Among 14 unrelated Caucasian patients in the pccBC complementation group, this unique mutation was found in 8 of 28 mutant alleles examined. Mutant allele-specific oligonucleotide hybridization to amplified genomic DNAs revealed that the inserted 12 nucleotides do not originate in an approximately 1000-bp region around the mutation. In the course of our investigation, we identified another mutation in the same exon: a 3-bp in-frame deletion that eliminates one of two isoleucine codons immediately preceding the Msp I site. Two unrelated patients were compound heterozygotes for this single-codon deletion and for the insertion/deletion described above. We conclude that either there is a propensity for the PCC beta-subunit gene to undergo mutations of this sort at this position or, more likely, the mutations in all of the involved Caucasian patients have a common origin in preceding generations.
Nuclear proteins bind conserved elements in the abscisic acid-responsive promoter of a rice rab gene.
Authors: Mundy J , Yamaguchi-Shinozaki K , Chua N H
Published: May 2006

We have previously shown that the expression of a rice gene, rab-16A, is responsive to abscisic acid (ABA) and osmotic stress in plant tissues and cultured suspension cells. We demonstrate here that transcriptional elements between -294 and -52 of this gene are sufficient to confer ABA-dependent expression on the chloramphenicol acetyltransferase reporter gene in rice protoplasts. Sequence motifs within this 242-base-pair region of the rab-16A gene are conserved among the 5' upstream regions of other ABA-responsive genes. Gel retardation and DNAse I experiments show nuclear factor(s) binding to these sequences. This correlative data indicate that these motifs are involved in the transcription of the rab genes and suggest that they may be ABA-responsive-elements (ABREs).
Application of fluorescence energy transfer and polarization to monitor Escherichia coli cAMP receptor protein and lac promoter interaction.
Authors: Heyduk T , Lee J C
Published: May 2006

A fluorescence method was developed to study DNA-protein interactions in solution. A 32-base-pair (bp) DNA fragment of the lac promoter containing the primary binding site for Escherichia coli cAMP receptor protein (CRP) was chemically synthesized and labeled specifically at the 5' end with fluorescent probe. Binding of cAMP receptor protein to this fragment can be conveniently followed by measuring changes in polarization of fluorescence of the labeled DNA or by measuring fluorescence energy transfer from protein tryptophan residues to the DNA label. Formation of protein-DNA complex was monitored as a function of cAMP concentration. Various equilibrium constants can be resolved to characterize the binding of cAMP to CRP and the subsequent binding of CRP-cAMP and CRP-(cAMP)2 to DNA. These binding studies showed that the two ligated forms of CRP have significantly different affinities for specific-site DNA. These results show that, in principle, the fluorescence technique can yield thermodynamically valid equilibrium constants under essentially any solution conditions. This technique also has the potential of providing information regarding the structure of protein-DNA complexes.
The SpoOA protein of Bacillus subtilis is a repressor of the abrB gene.
Authors: Strauch M , Webb V , Spiegelman G , Hoch J A
Published: May 2006

The spoOA gene of Bacillus subtilis is critical for the initial stages in the developmental cycle leading to the formation of an endospore. We show that one function of the SpoOA protein is to negatively regulate another regulatory locus, abrB, which controls the expression of many genes associated with the onset of sporulation. Purified SpoOA protein binds to a specific region of the abrB promoter and functions as a repressor of transcription in an in vitro assay. The binding of the SpoOA protein is independent of the binding of the AbrB protein, which is known to autoregulate its expression. This independence mirrors the temporal sequence of events in abrB control.
Prediction of the three-dimensional structure of Escherichia coli 30S ribosomal subunit: a molecular mechanics approach.
Authors: Malhotra A , Tan R K , Harvey S C
Published: May 2006

We introduce a computer-assisted procedure for folding large RNA chains into three-dimensional conformations consistent with their secondary structure and other known experimental constraints. The RNA chain is modeled using pseudoatoms at different levels of detail--from a single pseudoatom per helix to a single pseudoatom for each nucleotide. A stepwise procedure is used, starting with a simple representation of the macromolecule that is refined and then extrapolated into higher resolution for further refinement. The procedure is capable of folding different random-walk chains by using energy minimization, allowing generation of a range of conformations consistent with given experimental data. We use this procedure to generate several possible conformations of the 16S RNA in the 30S ribosomal subunit of Escherichia coli by using secondary structure and the neutron-scattering map of the 21 proteins in the small subunit. The RNA chain is modeled using a single pseudoatom per helix. RNA-RNA and RNA-protein crosslinks, reported in current literature, are included in our model. Footprinting data for different ribosomal proteins in the 16S RNA are also used. Several conformations of the 16S RNA are generated and compared to predict gross structural features of the 30S subunit as well as to identify regions of the 16S RNA that cannot be well-defined with current experimental data.
Charge isoforms of the adrenocortical pregnenolone-binding protein: influence of phosphorylation on isoformation and binding activity.
Authors: Lee Y C , Driscoll W J , Strott C A
Published: May 2006

Isoelectric focusing of the Mr 34,000 pregnenolone-binding protein (PBP) isolated from the guinea pig adrenal cortex has revealed multiple charge isoforms. Alkaline phosphatase treatment resulted in the disappearance of the pI 5.4 isoform associated with the appearance of pI 5.9 and pI 6.1 isoforms; this alteration in the charge-isoform pattern of the PBP correlated with a loss in pregnenolone-binding activity. This finding appears to be novel for intracellular steroid-binding proteins and has not been demonstrated for steroid receptors, a well-studied group of phosphoproteins. Resolution of the PBP by nondenaturing polyacrylamide gel electrophoresis produced two radioactive peaks of [3H]pregnenolone in an equilibrium system, while only one peak was present in a nonequilibrium system, suggesting high- and low-binding affinity forms of PBP. Isoelectric focusing of highly purified PBP resolved multiple forms of Mr 34,000 proteins with pI values ranging from 6.4 to 5.2. Two of the Mr 34,000 charge isoforms were isolated, and each was used to generate polyclonal antibodies; both antisera were crossreactive against all forms of Mr 34,000 PBP. Western blot analysis revealed that the PBP was present in both the fasciculata and reticularis of the adrenal cortex, though the isoform patterns were not identical for the two zones. Additionally, the pregnenolone-binding activity was approximately 10-fold greater in the zona reticularis. In vitro alkaline phosphatase treatment of the PBP abolished pregnenolone-binding activity and caused an alteration in the charge-isoform pattern for PBP in the zona reticularis, where pregnenolone binding is high, to resemble the pattern found for the zona fasciculata, where pregnenolone binding is low. The results indicate that phosphorylation/dephosphorylation regulates pregnenolone-binding activity and influences the pattern of the PBP isoformation. The data further suggest that the pI 5.4 isoform may be the active steroid-binding molecule.
Genesis by meiotic unequal crossover of a de novo deletion that contributes to steroid 21-hydroxylase deficiency.
Authors: Sinnott P , Collier S , Costigan C , Dyer P A , Harris R , Strachan T
Published: May 2006

The HLA-linked human steroid 21-hydroxylase gene CYP21B and its closely homologous pseudogene CYP21A are each normally located centromeric to a fourth component of complement (C4) gene, C4B and C4A, respectively, in an organization suggesting tandem duplication of a ca. 30-kilobase DNA unit containing a CYP21 gene and a C4 gene. Such an organization has been considered to facilitate gene deletion and addition events by unequal crossover between the tandem repeats. We have identified a steroid 21-hydroxylase [steroid, hydrogen-donor:oxygen oxidoreductase (21-hydroxylating), EC 1.14.99.10] deficiency patient who has a maternally inherited disease haplotype that carries a de novo deletion of a ca. 30-kilobase repeat unit including the CYP21B gene and associated C4B gene. This disease haplotype appears to have been generated as a result of meiotic unequal crossover between maternal homologous chromosomes. One of the maternal haplotypes is the frequently occurring HLA-DR3, B8, A1 haplotype that normally carries a deletion of a ca. 30-kilobase unit including the CYP21A gene and C4A gene. Haplotypes of this type may possibly act as premutations, increasing the susceptibility of developing a 21-hydroxylase deficiency mutation by facilitating unequal chromosome pairing.
Expression of striatal D1 dopamine receptors coupled to inositol phosphate production and Ca2+ mobilization in Xenopus oocytes.
Authors: Mahan L C , Burch R M , Monsma F J , Sibley D R
Published: May 2006

Expression of central nervous system receptors for dopamine was examined by injection of poly(A)+ RNA (mRNA) from rat striatum into oocytes from Xenopus laevis. Electrophysiological measurements in mRNA-injected oocytes indicated that addition of 100 microM dopamine induced an inward current (40-100 nA) that was consistent with the activation of endogenous Ca2(+)-dependent Cl- channels. This current was also elicited by addition of the selective D1 agonist SKF 38393 but not by the selective D2 agonist quinpirole. Prior addition of the dopaminergic antagonist cis-piflutixol completely abolished dopamine-induced currents but had no effect on currents produced by serotonin. Using 45Ca2+ efflux assays, addition of 100 microM dopamine to injected oocytes stimulated efflux 2- to 3-fold. This increase was mimicked by SKF 38393 and was blocked by the D1-selective antagonist (+)SCH 23390 but not by the D2-selective antagonist domperidone. No increase in 45Ca2+ efflux was seen with 100 microM quinpirole. Size fractionation of striatal mRNA yielded a single peak (2.5-3.0 kilobases) of D1 receptor-mediated 45Ca2+ efflux activity in injected oocytes. In addition, dopamine stimulation of oocytes injected with peak fractions and prelabeled with myo-[3H]inositol caused a 3-fold increase in [3H]inositol 1,4,5-triphosphate [( 3H]InsP3) formation. No effect on [3H]InsP3 production or 45Ca2+ efflux was observed, however, in injected oocytes incubated with 1 mM N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate. Thus, in addition to D1 receptors that stimulate adenylyl cyclase, rat striatum contains D1 receptors that can couple to InsP3 formation and mobilization of intracellular Ca2+.
Guanosine 3',5'-cyclic nucleotide binding proteins of bovine retina identified by photoaffinity labeling.
Authors: Thompson D A , Khorana H G
Published: May 2006

Cyclic GMP-binding proteins present in membrane fractions of bovine retina and, in particular, rod outer segments (ROS) were identified by photoaffinity labeling with 8-azido-[32P]cGMP. Two soluble proteins and two membrane-associated proteins were specifically labeled. The soluble proteins, 93 and 72 kDa, corresponded respectively to the alpha subunit of ROS cGMP phosphodiesterase and cGMP-dependent protein kinase. One of the two membrane-associated proteins, 53 kDa, was present in all particulate retinal fractions. Its function is unknown. It is distinct from cAMP-dependent protein kinase or the 63-kDa cGMP-activated channel from ROS. The second membrane-associated protein, 37 kDa, was present only in fractions that did not contain ROS. The molecular mass of this protein was similar to that of a cGMP-binding protein previously attributed to rod cells.
Regulation of icosahedral virion capsid size by the in vivo activity of a cloned gene product.
Authors: Agarwal M , Arthur M , Arbeit R D , Goldstein R
Published: May 2006

Determination of icosahedral virion capsid size can be directly studied during helper-dependent lytic development of satellite P4 because the assembly pathway specified by the P2 helper virus is altered to yield smaller-sized capsids. Size determination (sid) mutations identify a P4-encoded function regulating this process. To determine whether the sid gene product is necessary and sufficient to redirect the assembly pathway, we (i) cloned the sid structural gene in a plasmid vector (pMA30) under the control of an inducible promoter and (ii) constructed a packaging substrate (pMA1), a P4 genome-sized plasmid containing only that region of P4, the cos site, necessary for encapsidation. Superinfection by P2 of a host carrying pMA30 under induced conditions resulted in a shift from large to small capsid production. P2 superinfection of a host carrying the cos plasmid pMA1 plus pMA30 under induced conditions yielded pMA1-transducing particles of P4 capsid size. These cloning-based analyses directly demonstrate that sid protein is the only P4 gene product required for small-capsid size determination. In the absence of the P2 O gene product no capsids of any size are assembled during solo infection by P2. Nevertheless, P2 Oam mutant superinfection of a host carrying pMA1 and pMA30 under induced conditions yielded small P4-sized transducing particles. We therefore propose that (i) the sid gene product competes with the O gene product to determine the assembly of small vs. large capsid sizes and (ii) both gene products probably function as temporary scaffolding proteins.
Transcriptional activities in dry seed nuclei indicate the timing of the transition from embryogeny to germination.
Authors: Comai L , Harada J J
Published: May 2006

To investigate processes regulating the reinitiation of growth and differentiation during seed germination, we have studied the transcriptional activities of sets of genes that are expressed at specific stages of embryogeny and post-germination in the higher plant Brassica napus L. We show that transcripts from a subset of the genes are elongated in nuclei isolated from dry seeds, indicating that these genes are competent to be transcribed in desiccated and quiescent mature embryos. Analysis of the specific transcripts produced in dry seed nuclei indicates that the changes in gene expression patterns associated with germination are not initiated during late embryogeny. The results suggest that the transition from an embryonic to a postgerminative program of development occurs after seeds are rehydrated.
Evidence for the double-strand break repair model of bacteriophage lambda recombination.
Authors: Takahashi N , Kobayashi I
Published: May 2006

We have obtained evidence for the repair of double-strand gaps promoted by the Red function of bacteriophage lambda. A double-strand gap was made in one of the two regions of homology in an inverted orientation on a plasmid DNA molecule. The gapped plasmid was introduced into Escherichia coli cells expressing the red alpha (exo) and red beta (bet) genes of lambda. The gap was repaired by DNA synthesis copying an intact duplex. This gap repair was sometimes accompanied by reciprocal recombination (crossing over). The gap stimulated recombination about 100-fold. Our results are compatible with previous proposals that lambda homologous recombination involves the following early steps: (i) generation of double-stranded ends by the packaging machinery or by the replication machinery; (ii) production of a single-stranded tail with a 3'-hydroxyl end by 5'----3' degradation by lambda exonuclease (red alpha gene product); (iii) pairing of the single-stranded tail with a complementary strand from a homologous duplex with the help of beta protein (red beta gene product); (iv) priming of DNA synthesis at this 3'-hydroxyl end to copy the second DNA molecule.
AlgR3, a protein resembling eukaryotic histone H1, regulates alginate synthesis in Pseudomonas aeruginosa.
Authors: Kato J , Misra T K , Chakrabarty A M
Published: May 2006

A regulatory mutation (alg52) in a Pseudomonas aeruginosa alginate-negative mutant (strain 8882) is complemented efficiently by the gene algR2 and somewhat inefficiently by a second gene termed algR3. algR3 and algR2 are located on a 4.4-kilobase-pair HindIII-BamHI fragment, which has been completely sequenced. algR2 has previously been characterized. Introduction of kanamycin-resistance cassettes and deletion-subcloning experiments involving various open reading frames in the HindIII-BamHI fragment have localized the algR3 gene, which encodes a 340-amino acid polypeptide. This highly basic regulatory protein contains 17% lysine and 36% alanine. The predicted amino acid sequence shows no significant similarity with any bacterial proteins and yet is highly similar to the sea urchin Lytechinus pictus histone H1 subtype of protein. Promoter localization by reverse transcriptase mapping of the algR3 gene shows the presence of Escherichia coli sigma 70 recognition sequences, and coupled transcription/translation experiments in E. coli demonstrate the presence of a 39-kDa polypeptide encoded by the cloned algR3 gene.
Hormonal regulation of potassium currents in single myometrial cells.
Authors: Toro L , Stefani E , Erulkar S
Published: May 2006

Three potassium currents (IK) were recorded from myometrial cells isolated from the uterus of rats at estrus and diestrus and kept in culture for 1-6 days. IK were differentiated by their modulation with norepinephrine and/or by their onset kinetics. At +50 mV the activation time constants were about 0.7 ms, 6 ms, and 15 ms for the fast, the intermediate, and the slow IK, respectively. Norepinephrine (1 microM) potentiated the fast IK and reduced the intermediate IK. In addition, differences were found with respect to cells from animals at estrus and diestrus. The fast IK was preferentially expressed in cultures from animals at estrus, whereas the intermediate IK was more frequent in cells from rats at diestrus. These results indicate that K+ channels from myometrial cells are multiregulated. Regulation may occur by short-term signals (neurotransmitters) and/or by preferentially expressing distinct types of channels depending on the hormonal status of the animal.
Pattern of interleukin 6 gene expression in vivo suggests a role for this cytokine in angiogenesis.
Authors: Motro B , Itin A , Sachs L , Keshet E
Published: May 2006

Interleukin 6 (IL-6) is a cytokine that acts on various cell types. Here we show that IL-6 mRNA is produced in vivo in two self-limiting physiologic angiogenic processes: (i) the formation of the vascular system accompanying development of ovarian follicles and (ii) the formation of a capillary network in the maternal decidua following embryonic implantation. In situ and RNA blot hybridization analyses detected transient expression of IL-6 mRNA in gonadotropin-primed hyperstimulated ovaries, with maximal mRNA levels coinciding with the period of formation of a capillary network around follicles. Expression of IL-6 mRNA was detected in the vasculature extending from the ovarian medulla to the forming capillary sheath in the thecal layer of individual growing follicles. No expression was detected in more-developed preovulatory follicles once angiogenesis had been completed. IL-6 mRNA was also detected in the uterus of pregnant mice 9.5 days postcoitum, and there was no appreciable IL-6 mRNA at later stages of embryonic development. Expression in the uterus was confined to cords of endothelial cells in the process of formation of an anastomosing network that traversed the maternal decidua towards the developing embryo. The expression of IL-6 mRNA in two independent physiological angiogenic processes and the transient nature of its expression in endothelial cells suggest a role for IL-6 in angiogenesis.
Human monoclonal antibodies to the human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein gp41 enhance HIV-1 infection in vitro.
Authors: Robinson W E , Kawamura T , Gorny M K , Lake D , Xu J Y , Matsumoto Y , Sugano T , Masuho Y , Mitchell W M , Hersh E
Published: May 2006

Three of 16 human monoclonal antibodies (hu-mAbs) enhanced human immunodeficiency virus type 1 (HIV-1) infection of MT-2 target cells by means of a mechanism that is dependent on complement. Enhanced infections are characterized by an increase in cytopathic effects and antigen synthesis as well as an increase in the production of progeny virus as detected by release of reverse transcriptase activity and infectious virus into the culture medium. Analyses by radioimmunoprecipitation, Western blot, and ELISA using the pENV9 envelope fragment localize the antigenic specificities of these three hu-mAbs to the N-terminal two-thirds of the transmembrane protein gp41. Competitive binding experiments indicate that the hu-mAbs are reactive with immunodominant epitopes of gp41 recognized by sera from essentially all HIV-1-infected subjects. Combination dose-effect experiments demonstrate that these hu-mAbs can act synergistically in vitro to enhance HIV-1 infection. These data demonstrate that hu-mAbs directed against the HIV-1 transmembrane glycoprotein gp41 can enhance HIV-1 infection in vitro. The availability of these reagents allows for the mapping of enhancing epitopes on HIV-1 and provides a means for studying whether deletion of such enhancing epitopes from candidate HIV-1 vaccines might improve the protective immune response to HIV-1 in immunized humans and chimpanzees.
Glucose transporters serve as water channels.
Authors: Fischbarg J , Kuang K Y , Vera J C , Arant S , Silverstein S C , Loike J , Rosen O M
Published: May 2006

Water traverses the plasma membranes of some eukaryotic cells faster than can be explained by the water permeability of their lipid bilayers. This has led to a search for a water channel. Our previous work identified glucose transporters as candidates for such a channel. We report here that Xenopus laevis oocytes injected with mRNA encoding the brain/Hep G2, adult skeletal muscle/adipocyte, or liver forms of the glucose transporter exhibit an osmotic water permeability of their plasma membranes larger than that of untreated oocytes. The osmotic water permeability component attributable to glucose transporters increased an average of 4.8-fold in the injected oocytes. These studies provide direct evidence that the facilitative, sodium-independent mammalian glucose transporters serve as membrane water channels.
Persistent induction of cyclooxygenase in p60v-src-transformed 3T3 fibroblasts.
Authors: Han J W , Sadowski H , Young D A , Macara I G
Published: May 2006

A BALB/c 3T3 cell line infected with the temperature-sensitive Rous sarcoma virus strain LA90 has been used to investigate early, p60v-src-dependent changes in gene expression (protein synthesis). Giant two-dimensional electrophoresis, which can resolve greater than 3000 polypeptides from [35S]methionine-labeled cell lysates, was used to detect the induction of a p72-74 (72-74 kDa) doublet (pI 7.5) after activation of p60v-src at 35 degrees C. Antiserum against cyclooxygenase (prostaglandin synthase or prostaglandin endoperoxide synthase) specifically immunoprecipitated the p72-74 doublet. The p72-74 doublet was also induced by platelet-derived growth factor and by phorbol 12-myristate 13-acetate and was elevated in an NIH 3T3 cell line transformed by wild-type src. Activation of p60v-src caused a persistent increase in p72-74, whereas the effect of the growth factor was transient. These dissimilar kinetics of induction were paralleled by changes in cyclooxygenase activity. Down-regulation of protein kinase C inhibited subsequent induction of cyclooxygenase by phorbol myristate acetate but did not block induction by p60v-src. The glucocorticoid agonist dexamethasone inhibited induction of cyclooxygenase by p60v-src. Although induction of this enzyme may not be directly involved in transformation, the data support the view that oncogenic transformation may result, not from expression of transformation-specific genes, but from persistent changes in the expression of genes normally induced only transiently during passage from the G0 stage of the cell cycle.
Rat brain expresses a heterogeneous family of calcium channels.
Authors: Snutch T P , Leonard J P , Gilbert M M , Lester H A , Davidson N
Published: May 2006

We describe the isolation and characterization of several rat brain cDNAs that are homologous to the alpha 1 subunit of heart and skeletal muscle dihydropyridine-sensitive Ca channels. Northern blot analysis of 32 cDNAs shows that they can be grouped into four distinct classes (A, B, C, and D), each corresponding to a distinct hybridization pattern of brain mRNAs. Southern blot and DNA sequencing suggest that each class of cDNA represents a distinct gene or gene family. In the regions sequenced, the rat brain class C and D gene products share approximately 75% amino acid identity with the rabbit skeletal muscle Ca channel. In addition, the class C polypeptide is almost identical to the rabbit cardiac Ca channel (97% identity). In contrast, the rat brain class A and B cDNAs are more distantly related to dihydropyridine-sensitive Ca channels (47-64% amino acid identity) and to the brain class C and D genes (51-55% amino acid identity). To examine the functional significance of the isolated brain cDNAs, hybrid depletion experiments were performed in Xenopus oocytes. Antisense oligonucleotides against class A and B cDNAs each partially inhibited, and a class C oligonucleotide almost fully inhibited, the expression of Ba current in rat brain mRNA injected oocytes; but none of the oligonucleotides affected the expression of voltage-gated Na or K conductances. The clone characterization and sequencing results demonstrate that a number of distinct, yet related, voltage-gated Ca-channel genes are expressed in the brain. The antisense oligonucleotide experiments specifically show that one or several of the Ca-channel classes are related to the Ca channels observed in rat brain mRNA injected oocytes.
Human interleukin 2 receptor beta-chain gene: chromosomal localization and identification of 5' regulatory sequences.
Authors: Gnarra J R , Otani H , Wang M G , McBride O W , Sharon M , Leonard W J
Published: May 2006

Interleukin 2 (IL-2) binds to and stimulates activated T cells through high-affinity IL-2 receptors (IL-2Rs). Such receptors represent a complex consisting of at least two proteins, the 55-kDa IL-2R alpha chain and the 70-kDa IL-2R beta chain. The low-affinity, IL-2R alpha chain cannot by itself transduce a mitogenic signal, whereas IL-2 stimulates resting lymphocytes through the intermediate-affinity, IL-2R beta receptor. We report here identification of the genomic locus for IL-2R beta. The exons are contained on four EcoRI fragments of 1.1, 9.2, 7.2, and 13.7 kilobases. The 1.1-kilobase EcoRI fragment lies at the 5'-most end of the genomic locus and contains promoter sequences. The promoter contains no TATA box-like elements but does contain the d(GT)n class of middle repetitive elements, which may play an interesting regulatory role. The IL-2R beta gene is localized to chromosome 22q11.2-q12, a region that is the locus for several lymphoid neoplasias.
Disruption of genes encoding subunits of yeast vacuolar H(+)-ATPase causes conditional lethality.
Authors: Nelson H , Nelson N
Published: May 2006

The main function of vacuolar H(+)-ATPases in eukaryotic cells is to generate proton and electrochemical gradients across the membranes of the vacuolar system. The enzyme is composed of a catalytic sector with five subunits (A-E) and a membrane sector containing at least two subunits (a and c). We disrupted two genes of this enzyme, in yeast cells, one encoding a subunit of the membrane sector (subunit c) and another encoding a subunit of the catalytic sector (subunit B). The resulting mutants did not grow in medium with a pH value higher than 6.5 and grew well only within a narrow pH range around 5.5. Transformation of the mutants with plasmids containing the corresponding genes repaired the mutations. Thus failure to lower the pH in the vacuolar system of yeast, and probably other eukaryotic cells, is lethal and the mutants may survive only if a low external pH allows for this acidification by fluid-phase endocytosis.
Nerve growth factor binding domain of the nerve growth factor receptor.
Authors: Welcher A A , Bitler C M , Radeke M J , Shooter E M
Published: May 2006

A structural analysis of the rat low-affinity nerve growth factor (NGF) receptor was undertaken to define the NGF binding domain. Mutant NGF receptor DNA constructs were expressed in mouse fibroblasts or COS cells, and the ability of the mutant receptor to bind NGF was assayed. In the first mutant, all but 16 amino acid residues of the intracellular domain of the receptor were removed. This receptor bound NGF with a Kd comparable to that of the wild-type receptor. A second mutant contained only the four cysteine-rich sequences from the extracellular portion of the protein. This mutant was expressed in COS cells and the resultant protein was a secreted soluble form of the receptor that was able to bind NGF. Two N-terminal deletions, in which either the first cysteine-rich sequence of the first and part of the second cysteine-rich sequences were removed, bound NGF. However, a mutant lacking all four cysteine-rich sequences was unable to bind NGF. These results show that the four cysteine-rich sequences of the NGF receptor contain the NGF binding domain.
Selection of suppressor methionyl-tRNA synthetases: mapping the tRNA anticodon binding site.
Authors: Meinnel T , Mechulam Y , Le Corre D , Panvert M , Blanquet S , Fayat G
Published: May 2006

Accurate aminoacylation of a tRNA by Escherichia coli methionyl-tRNA synthetase (MTS) is specified by the CAU anticodon. A genetic screening procedure was designed to isolate MTS mutants able to aminoacylate a methionine amber tRNA (CUA anticodon). Selected suppressor MTS enzymes all possess one or several mutations in the vicinity of Trp-461, a residue that is the major contributor to the stability of complexes formed with tRNAs having the cognate CAU anticodon. Analysis of catalytic properties of purified suppressor enzymes shows that they have acquired an additional specificity toward the amber anticodon without complete disruption of the methionine anticodon site. It is concluded that both positive and negative discrimination toward the binding of tRNA anticodon sequences is restricted to a limited region of the synthetase, residues 451-467.
Binding of small acid-soluble spore proteins from Bacillus subtilis changes the conformation of DNA from B to A.
Authors: Mohr S C , Sokolov N V , He C M , Setlow P
Published: May 2006

Small acid-soluble spore proteins (SASPs) appear 3-4 hr after the onset of sporulation in Gram-positive bacteria and constitute up to 20% of the protein of mature spores. Previous studies using Bacillus subtilis deletion mutants lacking SASP-alpha and -beta have shown that such mutations abolish the elevated resistance of spores to UV radiation. Analyses using circular dichroism and Fourier-transform infrared spectroscopy now demonstrate that binding alpha/beta-type SASPs to DNA in vitro causes a structural change in DNA, from the B to the A conformation. This may provide the basis whereby alpha/beta-type SASPs confer increased spore UV resistance in vivo--by changing spore DNA conformation, they alter DNA photochemistry such that UV irradiation produces spore photoproduct instead of the more lethal cyclobutane-type thymine dimers.