bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective inhibition of interleukin 2 gene function following thymocyte antigen/major histocompatibility complex receptor crosslinking: possible thymic selection mechanism.
Authors: Ramarli D , Fox D A , Reinherz E L
Published: May 2006

Considerable evidence now exists to support the notion that the 50-kDa sheep erythrocyte binding protein, T11, represents an essential cell surface component of a human T-cell-lineage activation pathway. Furthermore, it is known that the human T-cell antigen-major histocompatibility complex (MHC) receptor complex T3-Ti is capable of regulating cell growth mediated by the T11 structure. Here we show that, within the T3+ thymocyte compartment, T3-Ti crosslinking rapidly inhibits T11-initiated interleukin 2 (IL-2) gene transcription and translation. This inhibition is restricted to the IL-2 gene (IL2) as transcription of both the IL-2 receptor gene (IL2R) and the Ti beta-chain gene (TCRB) are not affected (human gene designations are in parentheses). Perhaps more importantly, T3-Ti-mediated IL-2 inhibition of this type is not operational in peripheral T lymphocytes. The results imply that the majority of T3+ thymocytes are functionally distinct from peripheral T lymphocytes despite their T3+ phenotype and may possess a unique endogenous regulatory component for suppressing IL-2 gene activity. Moreover, since IL-2 is likely rate-limiting for growth within the thymus, the findings provide one plausible mechanism for thymic selection--namely, T3-Ti crosslinking of thymocytes upon interaction with self-major histocompatibility complex inhibits clonal expansion of high-affinity autoreactive cells.
Human aldolase A deficiency associated with a hemolytic anemia: thermolabile aldolase due to a single base mutation.
Authors: Kishi H , Mukai T , Hirono A , Fujii H , Miwa S , Hori K
Published: May 2006

Fructose-1,6-bisphosphate aldolase A (fructose-bisphosphate aldolase; EC 4.1.2.13) deficiency is an autosomal recessive disorder associated with hereditary hemolytic anemia. To clarify the molecular mechanism of the deficiency at the nucleotide level, we have cloned aldolase A cDNA from a patient's poly(A)+ RNA that was expressed in cultured lymphoblastoid cells. Nucleotide analysis of the patient's aldolase A cDNA showed a substitution of a single nucleotide (adenine to guanine) at position 386 in a coding region. As a result, the 128th amino acid, aspartic acid, was replaced with glycine (GAT to GGT). Furthermore, change of the second letter of the aspartic acid codon extinguished a F ok I restriction site (GGATG to GGGTG). Southern blot analysis of the genomic DNA showed the patient carried a homozygous mutation inherited from his parents. When compared with normal human aldolase A, the patient's enzyme from erythrocytes and from cultured lymphoblastoid cells was found to be highly thermolabile, suggesting that this mutation causes a functional defect of the enzyme. To further examine this possibility, the thermal stability of aldolase A of the patient and of a normal control, expressed in Escherichia coli using expression plasmids, was determined. The results of E. coli expression of the mutated aldolase A enzyme confirmed the thermolabile nature of the abnormal enzyme. The Asp-128 is conserved in aldolase A, B, and C of eukaryotes, including an insect, Drosophila, suggesting that the Asp-128 of the aldolase A protein is likely to be an amino acid residue with a crucial role in maintaining the correct spatial structure or in performing the catalytic function of the enzyme.
Sperm-associated retroviruses in the mouse epididymis.
Authors: Kiessling A A , Crowell R C , Connell R S
Published: May 2006

Sperm adsorbed with retrovirus particles were recovered from the epididymis of apparently normal male mice. Epididymal semen from all four mouse strains examined was positive for retrovirus (10(5) to 10(8) particles per microgram of protein) indicating that epididymal fluids and sperm may be important vehicles for murine retrovirus spread. Immunoblot analyses revealed that the banding patterns of electrophoretically separated epididymal viral proteins from the four strains of males were more similar to each other than to either xenotropic New Zealand Black virus or ecotropic Rauscher leukemia virus proteins. The results indicate that retrovirus particles, possibly a unique strain, are commonly expressed at relatively high titers in the reproductive tract of male mice and are sperm-associated.
Mutants of Escherichia coli formylmethionine tRNA: a single base change enables initiator tRNA to act as an elongator in vitro.
Authors: Seong B L , RajBhandary U L
Published: May 2006

We show that the absence of a Watson-Crick base pair at the end of the amino acid acceptor stem, which is a hallmark of all prokaryotic initiator tRNAs, is one of the key features that prevents them from acting as an elongator in protein synthesis. We generated mutants of Escherichia coli formylmethionine tRNA that have a base pair at the end of the acceptor stem. The mutants generated were C1----T1, which had a U.A base pair, A72----G72, which had a C.G base pair, and the C1A72----T1G72 double mutant, which lacked the base pair. After aminoacylation, the activity of these and other mutant initiator methionyl-tRNAs (Met-tRNAs) in elongation were assayed in a MS2 RNA-directed E. coli protein-synthesizing system and in binding to the elongation factor Tu (EF-Tu). Unlike wild-type initiator tRNA or the T1G72 double mutant, the T1 and G72 mutant Met-tRNAs were active in elongation, the G72 mutant being more active than the T1 mutant. The T1 and G72 mutant Met-tRNAs also formed a ternary complex with elongation factor EF-Tu.GTP, and their relative affinities for EF-Tu.GTP paralleled their activities in elongation. Combination of the T1 or G72 mutation with mutations in the GGG.CCC sequence conserved in the anticodon stem of initiator tRNAs led to a further increase in the activities of these mutant tRNAs in elongation such that one of these mutants was now almost as good an elongator as E. coli elongator methionine tRNA.
ATP-dependent formation of a specialized nucleoprotein structure by simian virus 40 (SV40) large tumor antigen at the SV40 replication origin.
Authors: Dean F B , Dodson M , Echols H , Hurwitz J
Published: May 2006

The large tumor antigen (T antigen) specified by simian virus 40 (SV40) is required for viral DNA replication. To carry out its function, T antigen binds to duplex DNA at the origin of replication (oriSV40) and exerts a helicase activity that unwinds the two DNA strands. Previous work has defined two binding sites for T antigen near oriSV40, designated sites I and II; site II is within the 64-base-pair core sequence absolutely required for viral DNA replication. We have used electron microscopy and gel electrophoresis to characterize the interaction of T antigen with the origin region. We have found that effective binding to site II under conditions that support DNA replication requires ATP or a nonhydrolyzable analog. In the absence of ATP, T antigen binds mainly to site I; in the presence of ATP, both sites I and II are occupied, and binding is markedly increased. The ATP-dependent reaction generates a complex multimeric structure for T antigen. We conclude that T antigen forms an ATP-dependent nucleoprotein structure at oriSV40. We suggest that this nucleoprotein complex provides for the precise initiation of SV40 DNA replication.
Artificial combination of two cis-regulatory elements generates a unique pattern of expression in transgenic plants.
Authors: Strittmatter G , Chua N H
Published: May 2006

We show that a 36-base-pair-long upstream fragment from the soybean hsp17.3-B gene comprising two partly overlapping heat-shock element (HSE)-like sequences can confer heat inducibility to a reporter gene in transgenic tobacco. The heat-shock response does not display organ specificity and is not affected by light. Insertion of these HSE-like elements into the pea rbcS-3A 5' flanking fragment (position -410 to +15) either at position -410 (5' to the enhancer) or at position -49 (between the enhancer and the "TATA" box) renders the transcript level of the reporter gene light-inducible and organ-specific under heat-shock conditions. These results demonstrate the possibility of generating a unique pattern of expression (e.g., light-dependent and organ-specific heat-shock response) by artificial combination of appropriate cis-acting regulatory elements. Moreover, by using the HSE-like sequences as a weak heat-inducible enhancer in the chimeric regulatory regions we uncover the function of negative elements within the pea rbcS-3A upstream region. These negative elements are responsible for a repressed transcript level in roots as well as in dark-adapted leaves. Therefore, the upstream fragment containing two HSE-like elements can be considered a useful tool to test the function of other cis-acting elements.
A conserved domain regulates interactions of the v-fps protein-tyrosine kinase with the host cell.
Authors: DeClue J E , Sadowski I , Martin G S , Pawson T
Published: May 2006

All cytoplasmic protein-tyrosine kinases (PTKs) share a noncatalytic domain, termed SH2, which comprises approximately 100 residues located immediately N-terminal to the kinase domain. A linker in the AX9m mutant of Fujinami avian sarcoma virus (FSV) introduces a dipeptide insertion into the SH2 domain of the P130gag-fps PTK, which abolishes its ability to transform Rat-2 cells. However, at 36 degrees C AX9m FSV elicits focus formation and agar colony formation in infected chicken embryo fibroblasts (CEF) with single hit kinetics. At 41.5 degrees C AX9m FSV is nontransforming for CEF, and the mutant is therefore both host and temperature dependent for transforming activity. Both in vitro and in vivo, the specific kinase activity of AX9m FSV P130gag-fps, measured by autophosphorylation and phosphorylation of exogenous substrates, correlated with transforming activity. The consequences of the AX9m mutation for enzymatic function and transforming activity therefore depend on the cellular environment in which the altered v-fps protein is expressed. We conclude that the SH2 domain directs the interaction of the P130gag-fps catalytic domain with cellular proteins such as substrates for phosphorylation or regulators of kinase activity important for its transforming ability.
Nucleotide sequence of a bean (Phaseolus vulgaris) U1 small nuclear RNA gene: implications for plant pre-mRNA splicing.
Authors: van Santen V L , Spritz R A
Published: May 2006

We have isolated a U1 small nuclear RNA (snRNA) gene from common bean (Phaseolus vulgaris). The haploid bean genome contains only one or a few copies of the U1 snRNA gene. The bean and human U1 snRNA genes are 65% homologous but share no significant similarity in the 5' or 3' flanking regions. The predicted secondary structure of bean U1 snRNA is identical to that of other U1 snRNAs, and the sequences of the single-stranded regions have been highly conserved. Thus, both the sequence of the single-stranded regions and the secondary structure of U1 snRNA appear to be important for its function. The role of U1 snRNA in pre-mRNA splicing is probably similar in plants and animals.
Genetic analysis of tumorigenesis: a conserved region in the human and Chinese hamster genomes contains genetically identified tumor-suppressor genes.
Authors: Stenman G , Sager R
Published: May 2006

Regional chromosome homologies were found in a comparison of human 11p with Chinese hamster 3p. By use of probes that recognize six genes of human 11p (INS, CAT, HBBC, CALC, PTH, and HRAS), the corresponding genes were localized by in situ hybridization on Chinese hamster chromosome 3. INS and CAT were located close to the centromere on 3p, whereas HBBC, CALC, and PTH were at 3q3-4 and HRAS at 3q4. Extensive prior data from chromosome studies of tumorigenic and tumor-derived Chinese hamster cells have suggested the presence of a tumor-suppressor gene on 3p. Two tumor-suppressor genes have been described on human 11p, one linked to CAT and one to INS. The present study raises the possibility that the Chinese hamster suppressor may be closely linked to INS or CAT.
Site-specific insertion of DNA into a pseudorabies virus vector.
Authors: Sauer B , Whealy M , Robbins A , Enquist L
Published: May 2006

A simple, efficient method for introducing recombinant DNA into a herpesvirus vector and retrieving it at a later time has been developed. By using the Cre-lox site-specific recombination system of coliphage P1, DNA can be readily inserted in vitro into a pseudorabies virus (PRV) vector containing the lox recombination site. The vector PRV42 contains a lox site within the nonessential gIII gene, which encodes a virion envelope glycoprotein. Incubation in vitro of PRV42 DNA with Cre protein and a circular plasmid containing a lox site generates approximately 5% recombinant molecules having the plasmid integrated into the PRV genome at the lox site. Transfection of the reaction mixture into cultured cells allows recovery of the infectious recombinant virus, which is readily identified by a nondestructive "black-plaque assay" using a gIII-specific monoclonal antibody. PRV42 plaques stain black when treated with the gIII monoclonal antibody and a peroxidase-linked second anti-antibody because the lox site placed within the gIII gene of PRV42 does not destroy the gIII epitope. However, Cre-mediated integration of heterologous DNA at the lox site disrupts the gIII epitope so that the resulting recombinant virus produces white plaques. The recombinant virus is infectious, stable, and grows as well as the parental PRV42 vector. The inserted plasmid can be efficiently excised (greater than 50%) from viral DNA by Cre and recovered by transformation of Escherichia coli.
Activation of resting human B cells by helper T-cell clone supernatant: characterization of a human B-cell-activating factor.
Authors: Diu A , Gougeon M L , Moreau J L , Reinherz E L , Thèze J
Published: May 2006

The effects of helper T-cell clone supernatants on resting human B cells were investigated. Four different helper T-cell clones (two T4+ and two T8+) were stimulated by anti-T3 monoclonal antibodies on Sepharose beads or anti-T11(2) plus anti-T11(3) monoclonal antibodies. The supernatants from these activated clones induced the proliferation of highly purified resting B lymphocytes from the peripheral blood. The B cells exhibited a cell size and a surface-antigen pattern (4F2 antigen and transferrin receptor) of phase G0 B cells, and they were functionally resting. In response to T-cell supernatants a large fraction of the B cells enlarged and expressed 4F2 antigens and transferrin receptors. In gel filtration, the corresponding activity migrated with an apparent Mr of 12,000-15,000. Our findings strongly support the existence of a human B-cell-activating factor acting on resting B cells and causing them to enter phase G1 of the cell cycle.
Structure and expression of the human and mouse T4 genes.
Authors: Maddon P J , Molineaux S M , Maddon D E , Zimmerman K A , Godfrey M , Alt F W , Chess L , Axel R
Published: May 2006

The T4 molecule may serve as a T-cell receptor recognizing molecules on the surface of specific target cells and also serves as the receptor for the human immunodeficiency virus. To define the mechanisms of interaction of T4 with the surface of antigen-presenting cells as well as with human immunodeficiency virus, we have further analyzed the sequence, structure, and expression of the human and mouse T4 genes. T4 consists of an extracellular segment comprised of a leader sequence followed by four tandem variable-joining (VJ)-like domains, a transmembrane domain, and a cytoplasmic segment. The structural domains of the T4 protein deduced from amino acid sequence are precisely reflected in the intron-exon organization of the gene. Analysis of the expression of the T4 gene indicates that T4 RNA is expressed not only in T lymphocytes, but in B cells, macrophages, and granulocytes. T4 is also expressed in a developmentally regulated manner in specific regions of the brain. It is, therefore, possible that T4 plays a more general role in mediating cell recognition events that are not restricted to the cellular immune response.
Structure of a third murine immunoglobulin lambda light chain variable region that is expressed in laboratory mice.
Authors: Sanchez P , Marche P N , Le Guern C , Cazenave P A
Published: May 2006

Recently, we reported evidence for the existence of an immunoglobulin lambda light chain (lambda x) whose variable region differs from those encoded by the known V lambda gene segments V lambda 1 and V lambda 2. Expression of lambda x was detected in some hybridomas elicited by treatment of a BALB/c mouse with rabbit anti-lambda 2 antibodies coupled to bacterial lipopolysaccharide [Sanchez, P. & Cazenave, P.-A. (1987) J. Exp. Med. 166, 265-270]. We constructed a cDNA clone from one hybridoma (B6) that expresses the lambda x chain and determined the complete nucleotide sequence. The deduced amino acid sequence of V lambda x is 30-33% identical with those encoded by V lambda 1 and V lambda 2 and by V kappa gene segments. The third hypervariable region of V lambda x is four codons longer than those of the other murine variable gene segments. The expression of lambda x requires a genomic rearrangement that juxtaposes the V lambda x gene with the J lambda 2-C lambda 2 joining-constant gene pair. Rabbit anti-V lambda x antibodies detected the lambda x light chain in the normal sera of all laboratory mice tested. Lambda x expression seems to be independent of lambda 1 expression, since both SJL and SJA strains, which are defective in lambda 1 production, express normal levels of lambda x chain.
Molecular basis of the cell-surface expression of immunoglobulin mu chain without light chain in human B lymphocytes.
Authors: Pollok B A , Anker R , Eldridge P , Hendershot L , Levitt D
Published: May 2006

Four distinct human B-lymphoid cell lines possess the ability to circumvent the mechanism regulating intracellular transport of immunoglobulin protein. These cells do not produce light chains, yet they express mu heavy chains on the cell surface at comparable levels to B-cell lines that produce native forms of both proteins. The mu-chain mRNA produced in all four cell lines was found to contain an identical deletion of most of the heavy-chain variable (VH) region (75% of the 3' portion), with no apparent alteration in constant (C) region structure. The truncated mu (mu*)-chain mRNA in these cells was created through the use of a cryptic splice donor site found within the human VH gene(s) utilized by these B-cell lines. The truncated mu chains exhibited a decreased ability to associate with the intracellular transport regulatory protein, heavy-chain binding protein (BiP). This result indicates that VH region structure, in addition to C mu 1 region structure, influences the formation of the BiP recognition site on the heavy chain. Furthermore, it suggests that the mechanism allowing for cell-surface expression of the mu* chains in the absence of light-chain pairing is the inability of BiP to bind to the mu* chains and hence prevent their intracellular transport. The high frequency with which the mu-only surface immunoglobulin positive phenotype is present in our collection of human B-cell lines and the isolation of one of the cell lines from a healthy individual also suggest that B cells of this type may represent a significant subpopulation among the normal human B-cell repertoire.
Cloning, sequence analysis, and expression of the large subunit of the human lymphocyte activation antigen 4F2.
Authors: Lumadue J A , Glick A B , Ruddle F H
Published: May 2006

Among the earliest expressed antigens on the surface of activated human lymphocytes is the surface antigen 4F2. We have used DNA-mediated gene transfer and fluorescence-activated cell sorting to obtain cell lines that contain the gene encoding the large subunit of the human 4F2 antigen in a mouse L-cell background. Human DNAs cloned from these cell lines were subsequently used as hybridization probes to isolate a full-length cDNA clone expressing 4F2. Sequence analysis of the coding region has revealed an amino acid sequence of 529 residues. Hydrophobicity plotting has predicted a probable structure for the protein that includes an external carboxyl terminus, an internal leader sequence, a single hydrophobic transmembrane domain, and two possible membrane-associated domains. The 4F2 cDNA detects a single 1.8-kilobase mRNA in T-cell and B-cell lines. RNA gel blot analysis of RNA derived from quiescent and serum-stimulated Swiss 3T3 fibroblasts reveals a cell-cycle modulation of 4F2 gene expression: the mRNA is present in quiescent fibroblasts but increases 8-fold 24-36 hr after stimulation, at the time of maximal DNA synthesis.
Tandem quadrupole Fourier-transform mass spectrometry of oligopeptides and small proteins.
Authors: Hunt D F , Shabanowitz J , Yates J R , Zhu N Z , Russell D H , Castro M E
Published: May 2006

Modifications to the newly developed tandem quadrupole Fourier-transform mass spectrometer have made it possible to record mass spectra on oligopeptides and small proteins in the mass range between 2 and 13 kDa.
Amino acid sequence of S-adenosyl-L-homocysteine hydrolase from rat liver as derived from the cDNA sequence.
Authors: Ogawa H , Gomi T , Mueckler M M , Fujioka M , Backlund P S , Aksamit R R , Unson C G , Cantoni G L
Published: May 2006

Rat liver cDNA libraries constructed in lambda gt11 were screened for reactivity with polyclonal antibodies to native S-adenosyl-L-homocysteine (AdoHcy) hydrolase (adenosylhomocysteinase; EC 3.3.1.1). Five clones were isolated and sequenced. The amino acid sequence, deduced from the cDNA sequence, contained the sequence of eight peptides obtained by tryptic and cyanogen bromide fragmentation of rat liver AdoHcy hydrolase. Identification of the amino- and carboxyl-terminal peptides in the amino acid sequence showed that the complete sequence was obtained. A "fingerprint" sequence was found that is characteristic of dinucleotide-binding domains of many proteins. For AdoHcy hydrolase, this region from the lysine at position 213 to the aspartate at position 244, containing the sequence Gly-Xaa-Gly-Xaa-Xaa-Gly at positions 219-224, is presumably the site of binding for NAD+, which is required for the activity of the enzyme.
Nucleotide sequence and characterization of the 5' flanking region of the rat Ha-ras protooncogene.
Authors: Damante G , Filetti S , Rapoport B
Published: May 2006

Thyrotropin and cAMP stimulate growth of FRTL5 rat thyroid cells and increase c-Ha-ras mRNA levels. To study the mechanism by which thyrotropin enhances c-Ha-ras expression in the thyroid, we constructed a genomic library of FRTL5 DNA in the bacteriophage vector EMBL3. Using a v-Ha-ras probe (0.7-kilobase Sst I-Pst I fragment), we isolated eight clones containing portions of the FRTL5 c-Ha-ras gene. Restriction mapping of one of these clones revealed a structure very similar to that previously reported for the rat c-Ha-ras gene. We determined the nucleotide sequence of exon 1 as well as 1.15 kilobases upstream from exon 1. Blot-hybridization analysis of FRTL5 thyroid cell mRNA was performed with three DNA fragments upstream of exon 1. Two of these probes were Pst I-Pst I fragments 0.4 and 0.55 kilobases long, 1.15 and 0.6 kilobases upstream of exon 1, respectively. The third probe, a 0.6-kilobase Pst I-HindIII fragment, was immediately upstream of exon 1 and included 54 base pairs of the 5' end of exon 1. The data revealed an upstream ("-1") exon, consistent with the homology between the nucleotide sequences of our clone and the human c-Ha-ras gene in this region. Primer extension of a synthetic 30-mer oligonucleotide probe complementary to exon +1 on a FRTL5 mRNA template revealed three transcription start (cap) sites, 182, 169, and 153 bases upstream of the ATG codon. "CCAAT boxes" are present 65 and 100 bases upstream from the initial cap site. Three "GC boxes" and two C + G-rich inverted repeats characteristic of the binding site for the transcription regulation factor Sp1 are present in the 177 base pairs upstream of the initial cap site. Comparison of the rat and human c-Ha-ras -1 exons showed an area of poor homology immediately upstream of the human cap sites, followed further upstream by a region of close homology (approximately equal to 60 base pairs). The nucleotide sequence of the rat -1 exon is more similar to the v-ras sequence than is the human. The v-ras gene is truncated relative to both human and rat -1 exons.
Potentiating and suppressive IgE-binding factors are expressed by a single cloned gene.
Authors: Martens C L , Jardieu P , Trounstine M L , Stuart S G , Ishizaka K , Moore K W
Published: May 2006

We have investigated expression of an IgE-binding factor (IgE-BF) cDNA in both COS-7 monkey kidney cells and Chinese hamster ovary cells. Transient expression of the IgE-BF clone in either cell type yielded IgE-BF, which potentiated an in vitro IgE response and had an affinity for lentil lectin. In contrast, when the transient expression experiments were carried out in the presence of tunicamycin, the factors no longer bound to lentil lectin. Moreover, IgE-BF expressed under these conditions suppressed an in vitro IgE response. IgE-BF lacking affinity for lentil lectin and suppressing the IgE response also resulted from transient expression of the IgE-BF gene in the presence of glycosylation inhibiting factor, a phospholipase inhibitory protein. Thus, IgE-BF that either potentiate or suppress the IgE response can be expressed from a single cloned gene; the difference in biological activities appears to be determined principally by the type of glycosylation of the common polypeptide chain. Previous work showed that IgE-BF bears an antigenic determinant recognized by the anti-Ia monoclonal antibody OX3. IgE-BF produced in the presence of tunicamycin, and IgE-BF expressed from a mutant cDNA lacking one of two carbohydrate-attachment sites, lacked the OX3 determinant. Thus, the OX3 determinant on IgE-BF appears to be associated with a site of N-linked glycosylation.
Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA.
Authors: Sells M A , Chen M L , Acs G
Published: May 2006

The hepatoblastoma cell line Hep G2 was transfected with a plasmid carrying the gene that confers resistance to G418 and four 5'-3' tandem copies of the hepatitis B virus (HBV) genome positioned such that two dimers of the genomic DNA are 3'-3' with respect to one another. Cells of one clone that grew in the presence of G418 produce high levels of hepatitis B e antigen and of hepatitis B surface antigen. HBV DNA is carried by these cells as chromosomally integrated sequences and episomally as relaxed circular, covalently closed, and incomplete copies of the HBV genome. Viral DNA was detected also in conditioned growth medium at the buoyant densities characteristic for infectious Dane and immature core particles. Finally, HBV-specific components morphologically identical to the 22-nm spherical and filamentous hepatitis B surface antigen particles as well as 42-nm Dane particles were visualized by immunoelectron microscopic analysis. Therefore, we have demonstrated that the Hep G2 cell line can support the assembly and secretion not only of several of the replicative intermediates of HBV DNA but also of Dane-like particles. This in vitro system can now be used to study the life cycle of HBV and the reaction of immunocompetent cells with cells carrying HBV.
Conformational change in a viral glycoprotein during maturation due to disulfide bond disruption.
Authors: Morrison T G , Peeples M E , McGinnes L W
Published: May 2006

The fusion glycoprotein of Newcastle disease virus is synthesized as an inactive precursor, F0. During intracellular transport and maturation, F0 undergoes a conformational change resulting from the loss of intramolecular disulfide bonds. F0 is also cleaved to yield F1, F2, the active, membrane-fusing form of the protein. Two monoclonal antibodies were used to explore this conformational change and its relationship to cleavage. These antibodies failed to precipitate the pulse-labeled fusion protein but did precipitate the F0 and the F1, F2 forms of the "chase" fusion protein. Use of the inhibitors carbonylcyanide m-chlorophenylhydrazone and monensin showed that the fusion protein acquired the ability to react with the monoclonal antibodies after it left the rough endoplasmic reticulum but before it left the medial Golgi membranes and before it was cleaved. The acquisition of antigenicity correlates with the disruption of intramolecular disulfide bonds during transit through the cell. This correlation was directly confirmed. The pulse-labeled fusion protein could be recognized by both monoclonal antibodies if the protein was first reduced. The formation and disruption of intramolecular disulfide bonds as a posttranslational modification of glycoproteins is discussed.
The role of class I histocompatibility antigens in the regulation of T-cell activation.
Authors: Dasgupta J D , Cemach K , Dubey D P , Yunis E J , Amos D B
Published: May 2006

Class I major histocompatibility antigens in humans (HLA antigens) were found to participate in the regulation of T-cell activation and proliferation induced by phytohemagglutinin. W6/32, a monomorphic antibody directed against class I HLA-A,B,C antigens, significantly inhibited the phytohemagglutinin-induced cell proliferation of peripheral blood lymphocytes. Almost complete suppression of cell activation was achieved on a subfraction of peripheral blood lymphocytes enriched in Mo1+ monocyte/macrophage cells. This inhibition of cell proliferation takes place at an early stage of activation and was found to be adherent cell dependent. Removal of monocyte/macrophage type cells from peripheral blood lymphocytes completely abrogated the inhibitory influence of anti-HLA-class I antibody, and, upon adding them back, suppression reappeared. Indirect immunofluorescence demonstrated that the expression of receptors for interleukin 2 and transferrin was impaired in the presence of antibody. Although the amount of interleukin 2 synthesized by these cells was also reduced, the addition of exogenous purified interleukin 2 did not restore cell proliferation. Mitogenesis induced by the Ca2+ ionophore A23187 was similarly suppressed, but mitogenesis induced by the phorbol diester phorbol myristate acetate, which activates cells by directly stimulating protein kinase C, was not suppressed. These results are consistent with a hypothesis that HLA class I antigens regulate an early event(s) of the Ca2+-dependent pathway of activation of T lymphocytes and that this event(s) apparently occurs before protein kinase C stimulation.
Hoogsteen base pairs proximal and distal to echinomycin binding sites on DNA.
Authors: Mendel D , Dervan P B
Published: May 2006

Forms of the DNA double helix containing non-Watson-Crick base-pairing have been discovered recently based on x-ray diffraction analysis of quinoxaline antibiotic-oligonucleotide complexes. In an effort to find evidence for Hoogsteen base-pairing at quinoxaline-binding sites in solution, chemical "footprinting" (differential cleavage reactivity) of echinomycin bound to DNA restriction fragments was examined. We report that purines (A greater than G) in the first and/or fourth base-pair positions of occupied echinomycin-binding sites are hyperreactive to diethyl pyrocarbonate. The correspondence of the solid-state data and the sites of diethyl pyrocarbonate hyperreactivity suggests that diethyl pyrocarbonate may be a sensitive reagent for the detection of Hoogsteen base-pairing in solution. Moreover, a 12-base-pair segment of alternating A-T DNA, which is 6 base pairs away from the nearest strong echinomycin-binding site, is also hyperreactive to diethyl pyrocarbonate in the presence of echinomycin. This hyperreactive segment may be an altered form of right-handed DNA that is entirely Hoogsteen base-paired.
Partial characterization of a low molecular weight human collagen that undergoes alternative splicing.
Authors: Pihlajaniemi T , Myllylä R , Seyer J , Kurkinen M , Prockop D J
Published: May 2006

A cDNA library prepared from RNA isolated from a cultured human tumor cell line, HT-1080, was screened with a mouse cDNA clone coding for part of the -Gly-Xaa-Yaa- domain of the alpha 2(IV) collagen chain. Four overlapping cDNA clones were characterized that coded for a low molecular weight human collagen. The cDNA clones did not, however, code for the short-chain collagens, types IX and X. The amino acid sequences derived from the clones resembled type IV collagen in that there were short interruptions in the repeating -Gly-Xaa-Yaa- sequence. The noncollagenous, carboxyl-terminal domain was, however, much shorter and contained only 18 amino acid residues. Interestingly, one of the cDNA clones contained an additional 36 nucleotides not found in an overlapping clone. The 36 nucleotides encoded four -Gly-Xaa-Yaa- repeats without changing the reading frame. Nuclease S1 mapping demonstrated that the difference between the clones was due to existence of two different mRNAs. A synthetic 24-residue peptide corresponding to the last two -Gly-Xaa-Yaa- triplets and the entire carboxyl-terminal domain was used to generate polyclonal antibodies. Electrophoretic transfer blot analysis of HT-1080 cells and normal human skin fibroblasts identified two polypeptides, Mr 67,000 and Mr 62,000, that were sensitive to bacterial collagenase.
3-Phosphoglycerate-dependent protein phosphorylation.
Authors: Ueda T , Plagens D G
Published: May 2006

The importance of protein phosphorylation in biological regulation is widely recognized; thus, a number of extracellular signal- and second messenger-linked protein kinases have been well characterized, and various substrates for these kinases have been identified. We present evidence that the glycolytic intermediate 3-phosphoglycerate can cause a marked stimulation of phosphorylation of specific proteins in mammalian tissues. In the brain, there are at least two types of 3-phosphoglycerate-dependent protein phosphorylation systems, which differ in activator specificity, substrate specificity, and sensitivity to the activator. Both of these phosphorylation systems are, however, insensitive to cAMP, cGMP, Ca2+, calmodulin, and protein kinase C activators and have characteristics different from those of casein kinase II-catalyzed protein phosphorylation. The most prominent endogenous substrates in the bovine brain are rather minor proteins with minimal molecular masses of 72 and 155 kDa, which were not detected in "nonnervous" tissues such as heart, liver, lung, and kidney. In nonnervous tissues, 3-phosphoglycerate affected the state of phosphorylation of other proteins. Evidence provided suggests the occurrence of an as yet undescribed type of protein kinase, which may be designated 3-phosphoglycerate-dependent protein kinase, in mammalian tissues.
Altered protein kinase C in a mast cell variant defective in exocytosis.
Authors: Mazurek N , Regazzi R , Borner C , Wyss R , Conscience J F , Erne P , Eppenberger U , Fabbro D
Published: May 2006

The murine mast cell line PB-3c is dependent on interleukin 3 (IL-3) with respect to survival and proliferation. These cells also require IL-3 to display antigen-mediated serotonin release, which is coupled to a transient increase of cytosolic free calcium ([Ca2+]i). The antigen-mediated exocytosis is inhibited by phorbol 12-tetradecanoate 13-acetate (PTA), an activator of phospholipid/Ca2+-sensitive protein kinase. In contrast, the malignant mast cell variant PB-1 is IL-3 independent with respect to proliferation but is unable to undergo antigen-mediated exocytosis. Yet this cell line exhibits basal levels of [Ca2+]i, serotonin content, and numbers of IgE receptors comparable to those of PB-3c cells. Subcellular distribution studies revealed that the specific activity of cytosolic protein kinase C of PB-1 cells was only 40% of that found in PB-3c cells. Furthermore, the PB-1 cells showed a significantly higher specific activity of membrane-bound protein kinase C than PB-3c cells. Scatchard plot analysis of [3H]-phorbol 12,13-dibutyrate binding to intact PB-1 cells demonstrated the presence of 20% high-affinity (Kd = 6 nM) and 80% low-affinity (Kd = 60 nM) phorbol ester receptors, whereas PB-3c cells displayed only the low-affinity phorbol ester binding. Immunological characterization of protein kinase C from both cell lines revealed the presence of a normal 77-kDa protein kinase C holoenzyme in both cell lines. In addition, a 72-kDa protein kinase C-related protein band was found mainly in the membrane fraction of the PB-1 variant. It is suggested that this altered and membrane-bound form of protein kinase C may be involved in the blockage of the antigen-mediated exocytosis of PB-1 cells.
Ha-ras oncogene expression directed by a milk protein gene promoter: tissue specificity, hormonal regulation, and tumor induction in transgenic mice.
Authors: Andres A C , Schönenberger C A , Groner B , Hennighausen L , LeMeur M , Gerlinger P
Published: May 2006

The activated human Ha-ras oncogene was subjected to the control of the promoter region of the murine whey acidic protein (Wap) gene, which is expressed in mammary epithelial cells in response to lactogenic hormones. The Wap-ras gene was stably introduced into the mouse germ line of five transgenic mice (one male and four females). Wap-ras expression was observed in the mammary glands of lactating females in two lines derived from female founders. The tissue-directed and hormone-dependent Wap expression was conferred on the Ha-ras oncogene. The signals governing Wap expression are located within 2.5 kilobases of 5' flanking sequence. The other two lines derived from female founders did not express the chimeric gene. In the line derived from the male founder, the Wap-ras gene is integrated into the Y chromosome. Expression was found in the salivary gland of male animals only. After a long latency, Wap-ras-expressing mice developed tumors. The tumors arose in tissues expressing Wap-ras--i.e., mammary or salivary glands. Compared to the corresponding nonmalignant tissues, Wap-ras expression was enhanced in the tumors.
Rhizobium
symbiotic genes required for nodulation of legume and nonlegume hosts
Authors: Marvel Deborah J. , Torrey John G. , Ausubel Frederick M.
Published: May 2006

Parasponia
, a woody member of the elm family, is the only nonlegume genus whose members are known to form an effective nitrogen-fixing symbiosis with
Bradyrhizobium
or
Rhizobium
species. The
Bradyrhizobium
strain Rp501, isolated from
Parasponia
nodules, also nodulates the legumes siratro (
Macroptilium atropurpureum
) and cowpea (
Vigna unguiculata
). To test whether some of the same genes are involved in the early stages of legume and nonlegume nodulation, we generated transposon Tn
5
insertions in the region of three evolutionarily conserved genes (
nodA, nodB
, and
nodC
) required for legume nodulation in several
Rhizobium
and
Bradyrhizobium
species. Assays of these mutant Rp501 strains on legume hosts and
Parasponia
seedlings established that
nodABC
are required for nodulation of legume and nonlegume hosts, indicating that nonlegumes and legumes can respond to the same bacterial signal(s). In addition, a strain carrying a Tn
5
insertion adjacent to the
nodABC
genes vigorously nodulated Rp501 legume hosts but was incapable of nodulating
Parasponia
, possibly identifying a nonlegume-specific nodulation function.
Altered cell-surface protein(s), crossreactive with DNA, on spleen cells of autoimmune lupic mice.
Authors: Jacob L , Lety M A , Monteiro R C , Jacob F , Bach J F , Louvard D
Published: May 2006

A murine monoclonal anti-DNA antibody, PME77, with specificity for double-stranded DNA, has previously been shown to react with a protein(s) present at the surface of such cells involved in lupus pathogenesis as human glomeruli, T and B lymphocytes, erythrocytes, and platelets. Mild elastase treatment of lymphoid cells from non-autoimmune (CBA/ca or BALB/c) mice releases a series of crossreactive polypeptides (34, 33, 17, 16, and 14 kDa) recognized by PME77. These polypeptides are not formed after treatment of the same cells with papain or trypsin. When lymphoid cells from autoimmune [MRL-lpr/lpr or (NZB X NZW)F1 B/W] mice are treated with elastase, trypsin, or papain, PME77 detects, in all supernatants, a single polypeptide of about 55 kDa. Antibodies present in the sera of autoimmune MRL-lpr/lpr and B/W mice and IgG eluted from kidneys of MRL-lpr/lpr mice react with the same polypeptides. Neither sera nor eluted IgG of normal BALB/c mice react with these polypeptides. These results suggest that an altered cell-surface protein(s), which we call LAMP [for lupus-associated membrane protein(s)], may be involved in lupus pathogenesis.
Pharmacokinetics of aspirin and salicylate in relation to inhibition of arachidonate cyclooxygenase and antiinflammatory activity.
Authors: Higgs G A , Salmon J A , Henderson B , Vane J R
Published: May 2006

Among the nonsteroid antiinflammatory drugs there is generally a close correlation between the potency of their inhibition of arachidonate cyclooxygenase, and thus prostaglandin production, and their antiinflammatory activity. One anomaly in this generalization is that whereas aspirin and salicylate are equipotent as antiinflammatory agents, salicylate is less active than aspirin in inhibiting prostaglandin production in vitro. Using rats, we have now measured the concentrations of aspirin and salicylate in plasma and in inflammatory exudates after their oral administration and determined their effects on thromboxane B2 production in clotting blood and prostaglandin (PG) E2 concentrations in the exudates. We have also investigated the effects of both drugs, at concentrations achieved in the exudates, on PGE2 production by nonproliferative explants of acutely inflamed tissues. Aspirin is rapidly metabolized, resulting in peak concentrations of salicylate in the plasma and exudate that exceeded peak concentrations of aspirin by 30- to 50-fold. Furthermore, concentrations of aspirin rapidly declined, whereas high concentrations of salicylate persisted in the plasma and in the exudate for up to 6 hr after a single administration of aspirin. Both drugs reduced PGE2 concentrations in inflammatory exudates by 50-70%, but aspirin was considerably more potent than salicylate in inhibiting thromboxane B2 production in clotting blood. The concentration of salicylate found in inflammatory exudates 6 hr after the administration of aspirin was sufficient to reduce PGE2 production in explants by more than 50%. We conclude that the antiinflammatory action of both drugs depends on the inhibition of PGE2 synthesis by salicylate.
Terbium luminescence-lifetime heterogeneity and protein equilibrium conformational dynamics.
Authors: Austin R H , Stein D L , Wang J
Published: May 2006

The fluorescence decay of the rare earth terbium when bound to the protein calmodulin changes from a simple exponential decay to a complex nonexponential decay as the temperature is lowered below 200 K. We have fit the observed decay curves by assuming that the terbium emission is a forced electric dipole transition and proteins have a distribution of continuous conformational states. Quantitative fits to the data indicate that the root-mean-square configurational deviation of the atoms surrounding the terbium ion is 0.2 A, in good agreement with other measurements. We further point out that because the protein seems to undergo a glass transition yet retains configurational order at room temperature, the proper name for the physical state of a protein at room temperature is the rubber-like state.
Carboxyl-terminal sequence of entactin deduced from a cDNA clone.
Authors: Durkin M E , Carlin B E , Vergnes J , Bartos B , Merlie J , Chung A E
Published: May 2006

Entactin is a widely distributed basement membrane sulfated glycoprotein of approximately equal to 150 kDa. The entactin gene is expressed early in mouse embryogenesis. Two cDNA clones complementary to rat entactin mRNA were isolated by antibody screening of an oligo(dT)-primed cDNA library constructed in the lambda gt11 expression vector. One of the clones, lambda 1E, was subcloned into plasmid pBR322 and further characterized. The clone contained sequences complementary to an mRNA species 6 kilobases in length. This mRNA was translated in rabbit reticulocyte lysates to yield a polypeptide of 143 kDa that was precipitated with anti-entactin antiserum. The cDNA insert, 1328 base pairs long, was sequenced and found to contain an open reading frame of 729 base pairs that coded for 243 amino acids at the carboxyl terminus of entactin. Analysis of the peptide revealed no extended alpha-helical or beta-sheet secondary structures. Radiolabeled probes prepared by nicktranslation of p lambda 1E were used to monitor the steady-state levels of entactin mRNA in F9 embryonal carcinoma cells that were induced to differentiate by exposure to retinoic acid and dibutyryl cyclic AMP. The increase in steady-state levels of entactin mRNA lagged behind the increase in mRNA for the B2 chain of laminin, suggesting that laminin and entactin are independently rather than coordinately regulated.
Cloning and sequence of the human nuclear protein cyclin: homology with DNA-binding proteins.
Authors: Almendral J M , Huebsch D , Blundell P A , Macdonald-Bravo H , Bravo R
Published: May 2006

A full-length cDNA clone for the human nuclear protein cyclin has been isolated by using polyclonal antibodies and sequenced. The sequence predicts a protein of 261 amino acids (Mr 29,261) with a high content of acidic (41, aspartic and glutamic acids) versus basic (24, lysine and arginine) amino acids. The identity of the cDNA clone was confirmed by in vitro hybrid-arrested translation of cyclin mRNA. Blot-hybridization analysis of mouse 3T3 and human MOLT-4 cell RNA revealed a mRNA species of approximately the same size as the cDNA insert. Expression of cyclin mRNA was undetectable or very low in quiescent cells, increasing after 8-10 hr of serum stimulation. Inhibition of DNA synthesis by hydroxyurea in serum-stimulated cells did not affect the increase in cyclin mRNA but inhibited 90% the expression of H3 mRNA. These results suggest that expression of cyclin and histone mRNAs are controlled by different mechanisms. A region of the cyclin sequence shows a significant homology with the putative DNA binding site of several proteins, specially with the transcriptional-regulator cAMP-binding protein of Escherichia coli, suggesting that cyclin could play a similar role in eukaryotic cells.
Expression of the proopiomelanocortin gene is developmentally regulated and affected by germ cells in the male mouse reproductive system.
Authors: Gizang-Ginsberg E , Wolgemuth D J
Published: May 2006

Proopiomelanocortin (POMC), a major pituitary product, is also present in the adult mouse testis. We have shown previously that POMC mRNAs are most abundant in a subpopulation of Leydig cells associated with tubules in specific stages of the cycle of the seminiferous epithelium. In the present study, we examined the expression of the gene encoding POMC during testicular development and in other tissues of the male reproductive system. We also analyzed the effects of cellular interactions on POMC gene expression in the testis. Blot-hybridization analysis revealed that POMC transcripts of approximately equal to 800 nucleotides were present in enriched populations of meiotic prophase spermatocytes and in caput epididymis but were absent in cauda epididymis and vas deferens. POMC transcripts were present in fetal testis (day 17 of gestation to newborn), could not be detected in prepuberal testis (days 7-8 postpartum), but reappeared in the adult testis. No difference in the size or abundance of POMC transcripts was seen in testes from mouse mutant strains in which spermatogenesis is arrested in early spermiogenesis. In contrast, POMC transcripts were virtually undetectable in testes that are devoid of germ cells. These results emphasize the importance of interactions between germ cells and interstitial cells and the regulation of the POMC gene in the mammalian testis.