bluuebunny/crossref_metadata_2025 huggingface dataset:
Initiation of simian virus 40 DNA replication in vitro: pulse-chase experiments identify the first labeled species as topologically unwound.
Authors: Bullock P A , Seo Y S , Hurwitz J
Published: May 2006

A distinct unwound form of DNA containing the simian virus 40 (SV40) origin is produced in replication reactions carried out in mixtures containing crude fractions prepared from HeLa cells. This species, termed form UR, comigrates on chloroquine-containing agarose gels with the upper part of the previously described heterogeneous highly unwound circular DNA, form U. As with form U, formation of form UR is dependent upon the SV40 tumor (T) antigen. Pulse-chase experiments demonstrate that the first species to incorporate labeled deoxyribonucleotides comigrates with form UR. Restriction analyses of the products of the pulse-chase experiments show that initiation occurs at the SV40 origin and then proceeds outward in a bidirectional manner. These experiments establish form UR as the earliest detectable substrate for SV40 DNA replication and suggest that SV40 DNA replication initiates on an unwound species.
Translational coupling of the maize chloroplast
atpB
and
atpE
genes
Authors: Gatenby Anthony A. , Rothstein Steven J. , Nomura Masayasu
Published: May 2006

The genes for the β and ε subunits of maize chloroplast ATP synthase are encoded by the organelle genome, are cotranscribed, and have overlapping translation initiation and termination codons. To determine whether the
atpB
and
atpE
genes are translationally coupled, they were transformed into
Escherichia coli
on a multicopy plasmid. Synthesis of full-length β and ε polypeptides demonstrated correct initiation of translation by the bacterial ribosomes. To assay for translational coupling, the promoter-distal
atpE
gene was fused to
lacZ
, resulting in the synthesis of an active hybrid β-galactosidase. A frameshift mutation was introduced into the promoter-proximal
atpB
gene, and its effect on the transcription and translation of the
atpE
::
lacZ
fusion was measured. The mutation resulted in a 1000- to 2000-fold reduction in β-galactosidase activity, but only a 2-fold decrease in LacZ mRNA synthesis rates or galactoside transacetylase levels. Similar results were obtained when the
atpB
/
atpE
::
lacZ
fusion and the
atpB
frameshift mutation were introduced into the photosynthetic cyanobacterium
Synechocystis
sp. PCC6803. We show that >99% of
atpE
translation depends on successful translation of
atpB
and, thus, conclude that the two genes are translationally coupled.
Genetic analysis of Schizosaccharomyces pombe 7SL RNA: a structural motif that includes a conserved tetranucleotide loop is important for function.
Authors: Liao X B , Brennwald P , Wise J A
Published: May 2006

We have studied the effects of mutations in a 6-base segment of Schizosaccharomyces pombe 7SL RNA, which lies within a 35-nucleotide domain whose sequence and secondary structure are conserved in RNAs from many divergent organisms, including the 7SL component of human signal recognition particle (SRP). Surprisingly, many changes in this region can be tolerated under normal growth conditions. An exception is the lethality of several mutations at positions 159 and 160, 2 nucleotides previously shown to be protected from RNase digestion by the 19-kDa canine SRP protein. Nucleotide 160 is, in addition, the most highly conserved base in a consensus sequence for the most common tetranucleotide loop in ribosomal RNAs. Mutations that are likely to affect the stability and/or conformation of the RNA give rise to a conditional phenotype: when osmolarity of the medium is raised, the RNAs become partially or completely defective in function at high temperature.
Direct observation of glycogen synthesis in human muscle with 13C NMR.
Authors: Jue T , Rothman D L , Shulman G I , Tavitian B A , DeFronzo R A , Shulman R G
Published: May 2006

On the basis of previous indirect measurements, skeletal muscle has been implicated as the major site of glucose uptake and it has been suggested that muscle glycogen formation is the dominant pathway. However, direct measurements of the rates of glycogen synthesis have not been possible by previous techniques. We have developed 13C NMR methods to measure directly the rate of human muscle glycogen formation from infused, isotopically labeled [1-13C]glucose. We show that under conditions of imposed hyperglycemia and hyperinsulinemia, a majority of the infused glucose was converted to muscle glycogen in a normal man. This directly shows that muscle is the major site of glucose disposal under these conditions, and provides quantitation of the glucose flux to muscle glycogen.
Survival of hemopoietic progenitors in the G0 period of the cell cycle does not require early hemopoietic regulators.
Authors: Leary A G , Hirai Y , Kishimoto T , Clark S C , Ogawa M
Published: May 2006

Although it is generally held that hemopoietic stem cells in steady-state marrow are dormant in the cell cycle, the direct proof for this concept has been lacking. In the present study, we have documented the development of human multipotential blast cell colonies from single cells by daily observation of the growth of candidate progenitors. The results clearly demonstrated that early hemopoietic progenitors may remain as single cells for more than 2 weeks of incubation. Once the progenitors began proliferation, the subsequent growth was characterized by steady cell doubling. Next, we tested the survival of blast cell colony progenitors in the presence of neutralizing antibodies prepared against early acting hemopoietic factors including interleukin (IL) 1 alpha, IL-1 beta, IL-3, IL-6, and granulocyte colony-stimulating factor. Cultures were initiated with individual antibodies, and, on day 14, IL-3 and the corresponding growth factor in concentrations that neutralize the antibodies were added. On days 18-27 of culture, blast cell colonies containing 25 or more cells were identified and replated for analysis of their ability to form secondary colonies. The cumulative frequency of the blast cell colonies in cultures containing antibody did not differ significantly from that of the control group containing rabbit IgG. A combination of anti-IL-1 alpha, anti-IL-1 beta, anti-IL-6, and anti-granulocyte colony-stimulating factor did not affect the survival of dormant blast cell colony-forming cells. These results indicate that survival of hemopoietic stem cells in the G0 period of the cell cycle is independent of early hemopoietic regulators.
Silent genes in the mouse major urinary protein gene family.
Authors: Shi Y , Son H J , Shahan K , Rodriguez M , Costantini F , Derman E
Published: May 2006

To date, two classes of mouse major urinary protein (MUP)-encoding genes have been described, the expressed genes and the intervening-sequence-containing pseudogenes. The data presented in this paper define a third class, the silent Mup genes, which are potentially functional but appear not to be expressed under normal circumstances. We describe a MUP subfamily (Mup-1.5) containing two genes, Mup-1.5a and Mup-1.5b, that are nearly identical, differing at only three positions (greater than 99.9% identity) over the entire 4-kilobase (kb) transcription unit and approximately 1 kb of flanking DNA. The similarity between these two genes extends over greater than 35 kb. Using specific oligonucleotides, we have shown that the 5a gene is expressed in BALB/cByJ mice, primarily in the submaxillary gland, whereas the 5b gene is not expressed. However, we found that when a 9.4-kb DNA fragment containing the Mup-1.5b gene was introduced into the mouse germ line, mice in two of the four transgenic lines expressed this gene at a high level and with the tissue-specificity characteristic of the Mup-1.5a gene. These results suggest that the inactivity of the endogenous Mup-1.5b gene is due not to a lack of functional positive regulatory elements, but to long-range, inhibitory position effects.
Nucleotide changes in sequential variants of influenza virus hemagglutinin genes and molecular structures of corresponding monoclonal antibodies specific for each variant.
Authors: Meek K , Johansson B , Schulman J , Bona C , Capra J D
Published: May 2006

We have generated four monoclonal antibodies specific for one or more members of a series of two sequentially derived PR8 influenza virus variants. Three of these antibodies share cross-reactive idiotypes. The amino acid sequences of these antibodies were determined, and it was found that two of these antibodies use genes from the VH7183 family, whereas the third uses a gene from the VHJ558 family. All four monoclonal antibodies derive from different families of genes encoding the variable region of the kappa chain. The RNA sequence of the parent PR8 virus as well as the RNA sequences of the sequential variants were also determined, and it was demonstrated that the variant hemagglutinin molecules differed from the parent molecule by only a single amino acid interchange. Despite these subtle differences in antigenic structures of hemagglutinin, and the cross-reactive idiotype of the antibodies, their primary structures were very different. These data reinforce the idea that a wide variety of antibody structures exist which are directed against subtly different structures in biologically important antigens.
Serine proteases from nematode and protozoan parasites: isolation of sequence homologs using generic molecular probes.
Authors: Sakanari J A , Staunton C E , Eakin A E , Craik C S , McKerrow J H
Published: May 2006

Serine proteases are one of the biologically most important and widely distributed families of enzymes. Isolation of serine protease genes from organisms of widely diverged phylogenetic groups would provide a basis for studying their biological function, the relationship between structure and function, and the molecular evolution of these enzymes. Serine proteases for which little structural information is known are those that are important in the pathogenesis of parasitic nematode and protozoan diseases. Identification and isolation of protease genes from these organisms is a critical first step in understanding their function for the parasite and possibly suggesting innovative approaches to arresting parasitic diseases. Serine protease gene fragments were isolated from genomic DNA of the parasitic nematode Anisakis simplex by using degenerate oligonucleotide primers and the polymerase chain reaction. Primers were designed based upon the consensus sequence of amino acids flanking the active site serine and histidine residues of eukaryotic serine proteases. Four serine protease gene fragments from this parasite were sequenced and one is 67% identical to the rat trypsin II gene. Alignment of these two genes revealed that the intron-exon junctions are conserved between nematode and rat suggesting that this Anisakis serine protease is structurally and functionally similar to rat trypsin. The generality of this approach to identify serine protease genes from genomic DNA of two very divergent species, a parasitic protozoan and a mammal, was also confirmed. Genes for other enzymes or any protein with conserved structural motifs can be identified and isolated using this technology. Using a similar strategy, a cathepsin B-like cysteine (thiol) protease gene fragment was isolated from Caenorhabditis elegans DNA.
Comparison of the chromosomal localization of murine and human glucocerebrosidase genes and of the deduced amino acid sequences.
Authors: O'Neill R R , Tokoro T , Kozak C A , Brady R O
Published: May 2006

To study structure-function relationships and molecular evolution, we determined the nucleotide sequence and chromosomal location of the gene encoding murine glucocerebrosidase (glucosylceramidase; D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45). In the protein coding region of the murine cDNA, the nucleotide sequence and the corresponding deduced amino acid sequences were 82% and 86% identical to the respective human sequences. All five amino acids presently known to be essential for normal enzymatic activity were conserved between mouse and man. The murine enzyme had a single deletion relative to the human enzyme at amino acid number 273. One ATG translation initiation signal was present in the mouse sequence in contrast to the human sequence, where two start codons have been reported. Nucleotide sequencing of a clone derived from murine genomic DNA revealed that the murine signal for translation initiation was located in exon 2. The locations of all 10 introns were conserved among mouse and man. We mapped the genetic locus for glucocerebrosidase to mouse chromosome 3, at a position 7.6 +/- 3.2 centimorgans from the locus for the beta subunit of nerve growth factor. Comparison of linkage relationships in the human and murine genome indicates that these closely linked mouse genes are also syntenic on human chromosome 1 but in positions that span the centromere.
Penetration of human intestinal epithelial cells by Salmonella: molecular cloning and expression of Salmonella typhi invasion determinants in Escherichia coli.
Authors: Elsinghorst E A , Baron L S , Kopecko D J
Published: May 2006

Salmonella typhi, the causative agent of typhoid fever, must invade the human gastrointestinal tract and multiply within the host to cause disease. We have cloned from S. typhi Ty2 a chromosomal region that confers upon Escherichia coli HB101 the ability to invade cultured human intestinal epithelial cells. Three invasion-positive recombinant cosmids were isolated and restriction endonuclease analyses of the inserts showed a 33-kilobase region of identity. Transmission electron microscopy of epithelial cells invaded by S. typhi Ty2 or E. coli HB101 carrying an invasion cosmid showed intracellular bacteria contained within endocytic vacuoles. One of the invasion cosmids was mutagenized with transposon Tn5 to identify the cloned sequences that are required for the invasive phenotype. Seven of 92 independent Tn5 insertions within the common 33-kilobase region eliminated invasive ability and revealed at least four separate loci that are required for invasion. Penetration of epithelial cells by Ty2 and HB101 carrying the cloned invasion determinants was inhibited by cytochalasin B and D, indicating that epithelial cell endocytosis of S. typhi is a microfilament-dependent event. The invasion cosmids were found to carry the recA and srlC genes indicating that the cloned invasion determinants are located at about 58 minutes on the S. typhi chromosome. With a segment of the cloned S. typhi invasion region used as a probe, homologous sequences were isolated from Salmonella typhimurium. Two independent S. typhimurium recombinant cosmids containing the entire 33-kilobase common region identified in S. typhi were isolated, but these cosmids did not confer upon HB101 the ability to invade epithelial cells.
Human placenta protein-tyrosine-phosphatase: amino acid sequence and relationship to a family of receptor-like proteins.
Authors: Charbonneau H , Tonks N K , Kumar S , Diltz C D , Harrylock M , Cool D E , Krebs E G , Fischer E H , Walsh K A
Published: May 2006

The amino acid sequence of the cytosolic human placenta protein-tyrosine-phosphatase 1B (PTPase 1B; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) has been determined. It consists of a single chain of 321 residues with an N-acetylated N-terminal methionine and an unusually proline-rich C-terminal region. The enzyme is structurally related to the two cytoplasmic domains of both the leukocyte common antigen CD45 and LAR, a CD45-like molecule with an external segment that resembles a neural cell adhesion molecule. A low molecular weight protein encoded by a cDNA clone from T cells also shows extensive sequence similarities. The present study defines homologous domains common to this diverse family of PTPases that includes both soluble and receptor-like transmembrane forms. The cysteinyl residues 121 and 215 of PTPase 1B are conserved among all members of the family and are candidates for involvement in catalysis since PTPase 1B is inactivated by thiol modifying reagents. Two segments rich in positively charged residues (residues 33-47 and 227-238) may provide sites of interaction with inhibitory anionic polymers such as heparin or poly(Glu/Tyr).
A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions.
Authors: Cupples C G , Miller J H
Published: May 2006

We describe the construction of six strains of Escherichia coli with different mutations at the same coding position in the lacZ gene, which specifies the active site glutamic acid residue at position 461 in beta'-galactosidase. Each strain is Lac- and reverts to Lac+ only by restoring the glutamic acid codon. The strains have been designed so that each reverts via one of the six base substitutions. The set of strains allows detection of each transition and transversion simply by monitoring the Lac- to Lac+ frequency, as demonstrated here with characterized mutagens and mutator alleles. These strains are useful for rapidly determining the mutagenic specificity of mutagens at a single site, for detecting low levels of stimulation of certain base substitutions, for monitoring specific base changes in response to various experimental conditions or strain backgrounds, and for isolating new mutator strains.
Development of biologically active peptides based on antibody structure.
Authors: Williams W V , Moss D A , Kieber-Emmons T , Cohen J A , Myers J N , Weiner D B , Greene M I
Published: May 2006

Antibody molecules are composed of several functional domains, including a variable domain that contacts antigen and a constant domain. The hypervariable regions of antibody molecules play an integral role in determining their specificity. However, the delineation of specific residues most critical in binding is difficult. We have been studying a monoclonal antibody (87.92.6) that binds to the reovirus type 3 receptor on a number of cell types, down-modulates the receptor, and inhibits DNA synthesis in the cells. We have shown that a peptide derived from the second complementarity-determining region of the monoclonal antibody 87.92.6 light-chain variable region can reproduce both of these effects. We were also able to demonstrate specific amino acid residues and structural features involved in producing these effects. The study of antibody structure, coupled with molecular synthetic techniques, can lead to the development of biologically active substances with potential clinical use.
Interleukin 3 activates human blood basophils via high-affinity binding sites.
Authors: Valent P , Besemer J , Muhm M , Majdic O , Lechner K , Bettelheim P
Published: May 2006

Pure populations of human basophilic granulocytes were obtained from chronic myeloid leukemia (CML) blood by negative selection using a mixture of monoclonal antibodies and complement. 125I-radiolabeled recombinant human interleukin 3 (rhIL-3) bound to purified basophils in a specific manner. Quantitative binding studies and Scatchard plot analyses performed on samples from two donors revealed the presence of a single class of high-affinity IL-3 binding sites (500 and 2100 sites per cell; dissociation constant at equilibrium, 230 and 160 pmol/liter, respectively). Purified CML basophils maintained in suspension in the presence of rhIL-3 (100 units/ml) incorporated up to 12 times more [3H]thymidine than basophils in control cultures. Furthermore, after preincubation in vitro with rhIL-3 (100 units/ml) for 30 min, normal blood basophils released 2- to 3-fold more histamine than basophils pretreated with control medium when exposed to various concentrations of an anti-IgE antibody. Together, these results show that rhIL-3 binds to a specific receptor on blood basophils and is a regulator of basophil function.
Actin polymerization induces a shape change in actin-containing vesicles.
Authors: Cortese J D , Schwab B , Frieden C , Elson E L
Published: May 2006

We have encapsulated actin filaments in the presence and absence of various actin-binding proteins into lipid vesicles. These vesicles are approximately the same size as animal cells and can be characterized by the same optical microscopic and mechanical techniques used to study cells. We demonstrate that the initially spherical vesicles can be forced into asymmetric, irregular shapes by polymerization of the actin that they contain. Deformation of the vesicles requires that the actin filaments be on average at least approximately 0.5 micron long as shown by the effects of gelsolin, an actin filament-nucleating protein. Filamin, a filament-crosslinking protein, caused the surfaces of the vesicles to have a smoother appearance. Heterogeneous distribution of actin filaments within the vesicles is caused by interfilament interactions and modulated by gelsolin and filamin. The vesicles provide a model system to study control of cell shape and cytoskeletal organization, membrane-cytoskeleton interactions, and cytomechanics.
Alteration in membrane protein band 3 associated with accelerated erythrocyte aging.
Authors: Kay M M , Flowers N , Goodman J , Bosman G
Published: May 2006

We report a human band 3 alteration that is associated with anemia as determined by a reticulocyte count of 20%. Erythrocyte defects included increased IgG binding, increased breakdown products of band 3, and altered anion- and glucose-transport activity in middle-aged cells. These changes were observed during normal erythrocyte aging in situ. Binding of ankyrin to band 3 was normal. Serum/cell crossover studies indicated that a neoantigen appears on the propositus' erythrocytes to which IgG from both propositus and control serum binds as measured with a protein A binding assay. IgG eluted from the propositus' erythrocytes appeared to have a specificity for senescent cell antigen as determined by a phagocytosis inhibition assay. Immunoelectron microscopy showed that antibodies to band 3, which do not normally bind to intact erythrocytes, bound to the propositus' erythrocytes. Antibody 980 binds to normal old cells but not young or middle-aged cells. It also binds to a distinct region of band 3 in immunoblots of membranes from the propositus' middle-aged cells. Cells from both of the propositus' parents exhibited increased IgG binding and altered anion and glucose transport. The results of these studies suggest that (i) band 3 is aging prematurely in erythrocytes from the propositus, (ii) senescent cell antigen appears on the propositus' middle-aged red cells, and (iii) band 3 alterations observed in the propositus may have a genetic component.
Developmentally restricted immunoglobulin heavy chain variable region gene expressed at high frequency in chronic lymphocytic leukemia.
Authors: Kipps T J , Tomhave E , Pratt L F , Duffy S , Chen P P , Carson D A
Published: May 2006

During fetal development, murine and human B-lineage cells rearrange and express a highly restricted set of immunoglobulin heavy chain variable region genes (VH genes). We noted that a VH gene of the restricted human fetal repertoire, designated 51p1, potentially could encode the VH region of two human IgM rheumatoid factor proteins. These rheumatoid factors share a cross-reactive idiotype (CRI) defined by reactivity with G6, a murine monoclonal antibody that recognizes an antibody heavy chain determinant present on many human IgM autoantibodies, particularly rheumatoid factors. Recently, we found that the G6 CRI also is expressed frequently by neoplastic CD5 (Leu1) B cells from patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma. However, neoplastic CD5-negative B cells from patients with lymphomas of follicular center cell origin rarely express this CRI. Here, we report that G6-reactive leukemic cells from two unrelated CLL patients express a VH gene that shares greater than 99% homology with a rearranged VH gene previously isolated from the leukemic cell DNA of another CLL patient and that is identical to VH 51p1. Using the polymerase chain reaction, we find that this VH gene is rearranged, and presumably expressed, in the genomic DNA of all examined cases of G6-reactive CLL or small lymphocytic lymphoma. Thus these data indicate that the autoantibody-associated G6 CRI is a serologic marker for a conserved and developmentally restricted VH1 gene that is expressed at high frequency in CD5 B-cell malignancies and early B-cell ontogeny.
Vasoactive intestinal polypeptide stimulates cell proliferation and adenylate cyclase activity of cultured human keratinocytes.
Authors: Haegerstrand A , Jonzon B , Dalsgaard C J , Nilsson J
Published: May 2006

An increasing body of evidence has suggested trophic effects of peripheral nerves. In this study, the growth stimulatory properties of the sensory neuropeptides vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin generelated peptide (CGRP), and somatostatin (SOM) on cultured human keratinocytes were investigated. It was shown that VIP, in the presence of lethally treated 3T3 fibroblast feeder cells and epidermal growth factor (EGF), stimulated proliferation of keratinocytes in a dose-dependent manner, whereas SP, CGRP, and SOM were ineffective. VIP stimulated adenylate cyclase activity in membranes obtained from cultured keratinocytes in a dose-dependent manner, indicating an involvement of cAMP as second messenger in this reaction. Furthermore, 125I-labeled VIP was shown to bind to cultured keratinocytes and this binding could be displaced by addition of unlabeled VIP, suggesting the presence of specific receptors. It is therefore possible that VIP, released from sensory nerve endings in the skin, may act as a local mitogenic factor for human keratinocytes by stimulating adenylate cyclase activity via specific VIP receptors.
Phosphorylation of OmpR by the osmosensor EnvZ modulates expression of the ompF and ompC genes in Escherichia coli.
Authors: Forst S , Delgado J , Inouye M
Published: May 2006

EnvZ and OmpR, the regulatory proteins for ompF and ompC expression in Escherichia coli, belong to a modulator-effector family of regulatory proteins which are essential for the response to environmental signals. We show that the soluble cytoplasmic domain of the transmembrane modulator protein EnvZ is phosphorylated in vitro by [gamma-32P]-ATP. We also demonstrate that the phosphate group can, in turn, be transferred to the transcription activator protein OmpR. The pH stability properties of the phosphate groups linked to EnvZ indicate that this molecule contains histidyl phosphate. The invariant His-243 of EnvZ corresponds to the phosphorylated His-48 of the chemotactic modulator protein CheA. Substitution of His-243 with valine produces an EnvZ that is refractory to phosphorylation and can no longer catalyze the transfer of phosphate to OmpR. Furthermore, in a delta envZ strain of E. coli, containing the envZ Val-243 plasmid, ompC expression is elevated 7-fold relative to that found in cells carrying the wild-type envZ plasmid. Based on these results we propose a model in which the phosphorylated state of OmpR modulates the expression of the ompF and ompC genes.
Mechanism by which cellulose triggers cellobiohydrolase I gene expression in Trichoderma reesei.
Authors: el-Gogary S , Leite A , Crivellaro O , Eveleigh D E , el-Dorry H
Published: May 2006

The expression of cellobiohydrolase I mRNA from Trichoderma reesei, measured by Northern blot hybridization, is controlled by the nature of carbon sources used in the culture medium. Cellulose and the soluble disaccharide sophorose, but not glycerol or glucose, act as inducers. Cellobiohydrolase I mRNA was undetectable when antibodies to the major members of the cellulolytic system were present in the culture medium prior to the addition of cellulose. These antibodies had no repressive effect if sophorose was used as an inducer. The results strongly suggest that a low constitutive cellulolytic system catalyzes the formation of a soluble inducer from cellulose and that this inducer triggers the expression of the cellobiohydrolase I gene transcript, most probably at the transcription level.
Generalized immunological recognition of the major merozoite surface antigen (gp195) of Plasmodium falciparum.
Authors: Chang S P , Hui G S , Kato A , Siddiqui W A
Published: May 2006

The antibody response to the Plasmodium falciparum major merozoite surface antigen (gp195) of congenic mouse strains differing in H-2 haplotype has been examined. All seven strains of mice were capable of producing gp195-specific antibodies. Generalized immune recognition of gp195 by mice of diverse H-2 haplotypes distinguished gp195 from the P. falciparum circumsporozoite protein and the 230-kDa and 48/45-kDa gamete surface antigens. However, the H-2 genetic locus appeared to influence the specificity of gp195-specific antibodies. Immunoblot patterns of mouse sera with parasite antigens revealed a complex pattern of reactivity with terminal and intermediate processing fragments of gp195. The majority of immunoblot bands observed were similar for all of the mouse strains; however, there were several strains that additionally recognized a few unique fragments or displayed more intense reactivities with specific processing fragments. These results suggest that while individuals of diverse major histocompatibility complex makeup are capable of recognizing the gp195 antigen, the recognition of specific gp195 B-cell and T-cell epitopes may be under control of the major histocompatibility complex.
Cloning and molecular characterization of genes whose products allow Salmonella typhimurium to penetrate tissue culture cells.
Authors: Galán J E , Curtiss R
Published: May 2006

Invasion of the intestinal epithelium is thought to be an important step in the pathogenesis of Salmonella infections. Using an in vitro system, we have isolated a genetic locus, inv, that confers to a noninvasive strain of Salmonella typhimurium the ability to penetrate tissue culture cells. Highly virulent S. typhimurium strains carrying inv mutations were defective for entry into Henle-407 cells while remaining unaffected in their ability to attach to cultured cells. When administered perorally to BALB/c mice, inv mutants of S. typhimurium had higher 50% lethal doses (LD50) than their wild-type parent strains. To the contrary, there were no differences in the observed LD50 when strains were administered intraperitoneally. In addition, inv mutants presented decreased ability to colonize the Peyer's patches, the small intestinal wall, and the spleen when administered perorally, although when administered intraperitoneally, they showed no difference in their ability to colonize the spleen compared to the wild-type parent strain.
Early rearrangements of genes encoding murine immunoglobulin kappa chains, unlike genes encoding heavy chains, use variable gene segments dispersed throughout the locus.
Authors: Lawler A M , Kearney J F , Kuehl M , Gearhart P J
Published: May 2006

Immunoglobulin heavy-chain variable region (TH) gene segments located closest to the joining (JH) gene segments are preferentially rearranged during ontogeny, indicating that chromosomal position influences the frequency of rearrangement. In addition, certain VH gene segments are repeatedly rearranged, suggesting that the DNA sequence or structure surrounding these segments may increase the probability of rearrangement. To determine whether there is similar based rearrangement of kappa variable (V kappa) gene segments, 25 rearrangements were sequenced from murine fetal and neonatal B-cell hybridomas and from subclones of a pre-B cell line that rearranged V kappa genes during in vitro culture. Four gene segments were isolated twice and one gene segment was isolated three times, suggesting that the process that targets individual variable gene segments for repeated rearrangement operates on both the VH and V kappa loci. Based on a current map of the V kappa locus, the rearranged gene segments belong to nine families that are dispersed throughout the locus. Thus, in these cell types, V kappa rearrangements use germ-line gene segments located across the entire locus, whereas the corresponding VH rearrangements use gene segments proximal to the JH gene segments. Heterogeneity of V kappa rearrangements would add diversity to the biased pool of VH rearrangements, producing a broad repertoire of antibodies early in development.
Criteria for the validity of clinical trials of treatments of cohorts of cancer patients based on the Hardin Jones principle.
Authors: Pauling L , Herman Z S
Published: May 2006

With the assumption of the validity of the Hardin Jones principle that the death rate of members of a homogeneous cohort of cancer patients is constant, three criteria for the validity of clinical trials of cancer treatments are formulated. These criteria are satisfied by most published clinical trials, but one trial was found to violate all three, rendering the validity of its reported results uncertain.
The amino-terminal domain of LexA repressor is alpha-helical but differs from canonical helix-turn-helix proteins: a two-dimensional 1H NMR study.
Authors: Lamerichs R M , Padilla A , Boelens R , Kaptein R , Ottleben G , Rüterjans H , Granger-Schnarr M , Oertel P , Schnarr M
Published: May 2006

The amino-terminal DNA binding domain of LexA repressor consisting of 84 amino acid residues has been studied by two-dimensional 1H NMR. Sequence-specific 1H resonance assignments were made for the first 60 amino acid residues. The secondary structure of this part of the protein contains three alpha-helices in the peptide segments 8-20, 28-35, and 41-54. The last helix has a distortion around residues 47-48. The peptide segment 28-47 shows weak homology with other helix-turn-helix proteins. To investigate the spatial structure of this region of the molecule distance-geometry calculations were performed based on proton-proton distance constraints from nuclear Overhauser effects. The resulting structure shows that the segment 28-47 contains two helices with a loop region between them. The relative orientation of the two helices is similar to that found in helix-turn-helix proteins, but the helices are further apart, with the phenyl ring of Phe-37 located between them. The Brookhaven Protein Data Bank was searched for structurally homologous peptide segments in other proteins. The result of this search was that the two-helical structure of LexA is not more closely related to the canonical helix-turn-helix motif than it is to similar substructures found in other classes of proteins.
A strategy for high-volume sequencing of cosmid DNAs: random and directed priming with a library of oligonucleotides.
Authors: Studier F W
Published: May 2006

Direct sequencing of cosmid DNAs using a library of oligonucleotide primers of length 8, 9, or 10 is proposed. The statistics of priming indicate that a primer library sufficient for determining the sequence of the entire human genome (100,000 cosmids) would be small enough to be assembled and managed. Such a library would greatly reduce the cost and effort of high-volume sequencing: primers would be instantly available; the sequence of each cosmid DNA could be determined from a single DNA preparation without the necessity for mapping or subcloning; and, because each primer would be used repeatedly, the cost of primers would become a negligible fraction of other costs. A combination of random and directed priming could determine the sequence of a cosmid DNA in 1.2-1.5 times the minimum number of sequencing reactions required, and completely directed priming would be even more efficient. The success of this strategy requires that a considerable fraction of octamers, nonamers, or decamers be able to prime selectively in double-stranded DNAs 45,000 base pairs (bp) long; initial results indicate that this is likely to be the case. The strategy is not limited to cloned DNAs and would be useful for rapid identification and direct sequencing of viral nucleic acids.
Mandelonitrile lyase from Ximenia americana L.: stereospecificity and lack of flavin prosthetic group.
Authors: Kuroki G W , Conn E E
Published: May 2006

A mandelonitrile lyase (EC 4.1.2.10) that catalyzes the dissociation of (S)-(-)-mandelonitrile to benzaldehyde and hydrogen cyanide has been purified to apparent homogeneity from leaves of Ximenia americana L. (Olacaceae). The lyase was purified 122-fold with 38% yield by chromatography on carboxymethyl-cellulose and chromatofocusing. The enzyme had a pH optimum of 5.5, with a Km value of 280 microM. Activity toward 4-hydroxy-(R,S)-mandelonitrile was 77% of that observed with the endogenous substrate; no activity was observed toward the aliphatic substrate acetone cyanohydrin. The enzyme was stable at 4 degrees C and at room temperature for at least 1 month. Native and subunit molecular weights of 38,000 and 36,500, respectively, suggest the enzyme is a monomer. The isoelectric point was pH 3.9 as determined by isoelectric focusing. Staining with periodic acid-Schiff and fluorescein-labeled concanavalin A reagents indicate this enzyme is a glycoprotein. In contrast to (R)-mandelonitrile lyases isolated from Prunus species, the Ximenia lyase does not appear to be a flavoprotein. A second enzyme that eluted from the chromatofocusing column at pH 4.0 was also active toward mandelonitrile. However, this form accounted for less than 10% of the total activity, and its specific activity was only 6% of that of the major component. Additional physical and kinetic studies suggested this activity may be due to a nonspecific enzyme that is active toward mandelonitrile.
Different 3'-end processing produces two independently regulated mRNAs from a single H1 histone gene.
Authors: Cheng G H , Nandi A , Clerk S , Skoultchi A I
Published: May 2006

We describe the isolation of a mouse H1 histone gene that encodes two mRNA transcripts. One mRNA ends just beyond the coding region, near a highly conserved palindrome sequence typical of cell cycle-regulated histone genes. The level of this transcript is coupled to DNA replication. The second mRNA ends nearly 1 kilobase downstream near a polyadenylation signal. This mRNA is polyadenylylated, and its accumulation is not coupled to DNA replication. The two mRNAs are regulated independently and in some circumstances in opposite directions under several physiological conditions. The production of a polyadenylylated mRNA from an otherwise cell cycle-regulated histone gene may allow for continued synthesis of the histone protein when DNA synthesis ceases in nondividing cells.
Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human neutrophil respiratory burst oxidase.
Authors: Volpp B D , Nauseef W M , Donelson J E , Moser D R , Clark R A
Published: May 2006

Neutrophil NADPH oxidase is a multicomponent enzyme that is activated to generate superoxide anion and is defective in the cells of patients with chronic granulomatous disease. It requires both membrane and cytosolic components, the latter including 47- and 67-kDa proteins recognized by the polyclonal antiserum B-1. Immunoscreening of an induced HL-60 lambda ZAP cDNA library yielded seven cross-hybridizing cDNAs encoding the 47-kDa component. Fusion proteins of 22-50 kDa were recognized by B-1. Antiserum against a fusion protein recognized a 47-kDa protein in normal neutrophils but not in those from patients with autosomal chronic granulomatous disease who lack the 47-kDa cytosolic oxidase component. In a cell-free NADPH oxidase system full-length and C-terminal fusion proteins augmented superoxide generation and reconstituted the cytosolic defect of a patient missing the 47-kDa protein. The cDNA hybridized with a 1.4-kilobase mRNA from induced HL-60 cells. The longest cDNA contained an open reading frame encoding a protein of 41,440 Da with a calculated pI of 10.4, an N-terminal glycine, sites favorable for phosphorylation, a nucleotide binding domain, and a region of homology to the src protein kinases, phospholipase C, and alpha-fodrin. These structural features are pertinent to proposed functional roles of the protein in the respiratory burst oxidase.
Retroviral transfer of a human tyrosine hydroxylase cDNA in various cell lines: regulated release of dopamine in mouse anterior pituitary AtT-20 cells.
Authors: Horellou P , Guibert B , Leviel V , Mallet J
Published: May 2006

Little is known about the molecular events mediating neurotransmitter release, a crucial step in synaptic transmission. In this paper, the biosynthesis and release of L-beta-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine were analyzed in three heterologous cell lines after retroviral-mediated gene transfer of tyrosine hydroxylase (EC 1.14.16.2), the rate-limiting enzyme in catecholamine synthesis. A recombinant retrovirus encoding human tyrosine hydroxylase type I as well as neomycin-resistance gene was used to infect a fibroblast (NIH 3T3), a neuroblastoma (NS20 Y), and a neuroendocrine (AtT-20) cell line. After selection in the presence of neomycin and in tyrosine-free medium, high levels of exogenous tyrosine hydroxylase activity were detected in extracts of the three cell lines. High-performance liquid chromatography of cell extracts and culture supernatants confirmed that the three cell lines hydroxylated tyrosine to form L-DOPA and released this metabolite into the culture medium. Interestingly, the neuroendocrine cell line AtT-20 synthesized not only L-DOPA but also dopamine. Evoked secretion studies established that AtT-20 cells released the transmitter upon depolarization in a regulated, calcium-dependent way. We discuss the implication of this approach for the analyses of neurotransmitter release as well as in the context of degenerative disorders such as Parkinson disease.
T-cell nuclei contain a protein that binds upstream of the murine granulocyte-macrophage colony-stimulating factor gene.
Authors: James R , Kazenwadel J
Published: May 2006

Stimulation of a murine T-cell line (O18A) by Con A has been shown to cause a large increase in the cytoplasmic granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA level. Using run-on transcription experiments in isolated nuclei, we have shown that some of this response is from enhanced transcription of the GM-CSF gene. Changes in the transcription rate of this gene can be seen as early as 30 min after adding the Con A. With a DNA fragment mobility-shift assay on nuclear extracts from these cells we detected a protein that binds upstream of the murine GM-CSF gene. Partial purification and concentration of this protein by DEAE-Sephacel and phosphocellulose chromatography enabled us to examine its interaction with DNA in more detail. Competition and methylation interference experiments have shown that the protein binds to the sequence 3'-TCCATCAAGGGG-5' (positions -90 to -82). This sequence is contained within a region found to be involved in regulating inducible GM-CSF transcription in a human T-cell line [Miyatake, S., Seiki, M., Yoshida, M. & Arai, K. (1988) Mol. Cell. Biol. 8, 5581-5587].
Developmental regulation of the rat insulin-like growth factor I receptor gene.
Authors: Werner H , Woloschak M , Adamo M , Shen-Orr Z , Roberts C T , LeRoith D
Published: May 2006

We have investigated the developmental regulation of the rat insulin-like growth factor I (IGF-I) receptor gene in various tissues using a sensitive and specific solution hybridization/RNase protection assay. For this purpose we characterized rat IGF-I receptor cDNAs that were cloned from a simian virus 40-transformed rat granulosa cell cDNA library. The specific cDNA clone used in these studies encoded the putative signal peptide and the first 53 amino acids of the alpha subunit and was approximately 94% homologous to its human counterpart. IGF-I receptor gene expression was studied during the perinatal period and at various intervals until early adulthood. Overall, steady-state IGF-I receptor mRNA levels decreased dramatically during postnatal development; however, the extent of the decrease differed among the various tissues studied. In contrast to receptor mRNA levels, IGF-I mRNA levels increased in some of the same tissues. The molecular mechanisms underlying this apparent divergent transcriptional control of the IGF-I and IGF-I receptor genes warrant further study.
Conservation of an immunoglobulin variable-region gene family indicates a specific, noncoding function.
Authors: Tutter A , Riblet R
Published: May 2006

Blot-hybridization and DNA sequence analyses reveal the particular evolutionary conservation of a group of immunoglobulin heavy-chain variable-region (VH) genes in all mammalian species examined. These particular genes are group III genes--the VH7183 family in the mouse and the homologous VH III family in human. This conservation is localized to sequences encoding framework regions 1 and 3 of the antibody variable region and is exerted at the nucleotide level. Because selection acting at the amino acid level alone cannot explain the conservation of these sequences, these sequences must have a noncoding function. The preferential rearrangement of VH7183 and VH III genes, together with the similarity of the conserved sequences to elements implicated in recombination in other systems, suggest that these sequences function to target the series of rearrangements that assemble complete immunoglobulin genes.
Z-DNA-forming sequences are spontaneous deletion hot spots.
Authors: Freund A M , Bichara M , Fuchs R P
Published: May 2006

Z-DNA-forming sequences are shown to elicit a biological response in Escherichia coli. Plasmids containing sequences capable of adopting the Z conformation (GC and CA/GT) are shown to be hot spots for spontaneous deletions. All the deletions involve an even number of base pairs. The distribution of the deletion events shows that the process ends when the Z-DNA-forming sequence has been reduced to a size no longer capable of adopting the Z conformation at natural superhelical density.