bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular mapping within the mouse albino-deletion complex.
Authors: Johnson D K , Hand R E , Rinchik E M
Published: May 2006

Induced germ-line deletion mutations in the mouse provide a malleable experimental system for in-depth molecular and functional analysis of large segments of the mammalian genome. To obtain an initial bank of molecular probes for the region of mouse chromosome 7 associated with the albino-deletion complex, random anonymous DNA clones, derived from a library constructed from flow-sorted chromosomes, were screened on DNAs from Mus musculus-Mus spretus F1 hybrids carrying large, multilocus, lethal albino deletions. Clones falling within a given deletion interval can easily be recognized because hybridization bands that represent restriction fragment length polymorphisms specific for the mutant (deleted) chromosome inherited from the M. musculus parent will be absent. Among 72 informative clones used as probes, one, which defines the locus D7OR1, mapped within two deletions that are 6-11 centimorgans in length. Submapping of this anonymous clone across a panel of 27 smaller deletions localized D7OR1 distal to a chromosomal subregion important for survival of the preimplantation embryo, proximal to globin [beta-chain (Hbb)], and near the shaker-1 (sh-1) locus. The results of these deletion-mapping experiments were also confirmed by standard three-point linkage analysis. This strategy for selection and rapid mapping of anonymous DNA probes to chromosomal segments corresponding to germ-line deletion mutations should contribute to the generation of more detailed physical and functional maps of genomic regions associated with mutant developmental phenotypes.
Human major histocompatibility complex contains a minimum of 19 genes between the complement cluster and HLA-B.
Authors: Spies T , Bresnahan M , Strominger J L
Published: May 2006

A 600-kilobase (kb) DNA segment from the human major histocompatibility complex (MHC) class III region was isolated by extension of a previous 435-kb chromosome walk. The contiguous series of cloned overlapping cosmids contains the entire 555-kb interval between C2 in the complement gene cluster and HLA-B. This region is known to encode the tumor necrosis factors (TNFs) alpha and beta, B144, and the major heat shock protein HSP70. Moreover, a cluster of genes, BAT1-BAT5 (HLA-B-associated transcripts) has been localized in the vicinity of the genes for TNF alpha and TNF beta. An additional four genes were identified by isolation of corresponding cDNA clones with cosmid DNA probes. These genes for BAT6-BAT9 were mapped near the gene for C2 within a 120-kb region that includes a HSP70 gene pair. These results, together with complementary data from a similar recent study, indicated the presence of a minimum of 19 genes within the C2-HLA-B interval of the MHC class III region. Although the functional properties of most of these genes are yet unknown, they may be involved in some aspects of immunity. This idea is supported by the genetic mapping of the hemopoietic histocompatibility locus-1 (Hh-1) in recombinant mice between TNF alpha and H-2S, which is homologous to the complement gene cluster in humans.
Experimental pancreatitis is mediated by low-affinity cholecystokinin receptors that inhibit digestive enzyme secretion.
Authors: Saluja A K , Saluja M , Printz H , Zavertnik A , Sengupta A , Steer M L
Published: May 2006

Rats infused with a supramaximally stimulating dose of the cholecystokinin (CCK) analog caerulein develop acute edematous pancreatitis. Using CCK-JMV-180, a recently developed CCK analog that acts as an agonist at high-affinity CCK receptors but antagonizes the effect of CCK at low-affinity receptors, we have determined that caerulein induces pancreatitis by interacting with low-affinity CCK receptors. Those low-affinity receptors mediate CCK-induced inhibition of digestive enzyme secretion from the pancreas. Our observations, therefore, suggest that this form of experimental pancreatitis results from the inhibition of pancreatic digestive enzyme secretion.
Cloning and characterization of band 3, the human erythrocyte anion-exchange protein (AE1).
Authors: Lux S E , John K M , Kopito R R , Lodish H F
Published: May 2006

The human erythrocyte anion-exchange protein (band 3 or AE1) was cloned from a fetal liver cDNA library. Three overlapping clones, encompassing 3637 nucleotides, were analyzed in detail. These encode a 911-amino acid protein (Mr 101,791) and detect a single 4.7-kilobase species in human reticulocyte RNA. The corresponding gene is located on chromosome 17. The protein is similar in structure to other anion exchangers and is divided into three regions: a hydrophilic, cytoplasmic domain that interacts with a variety of membrane and cytoplasmic proteins (residues 1-403); a hydrophobic, transmembrane domain that forms the anion antiporter (residues 404-882); and an acidic, C-terminal domain of unknown function (residues 883-911). The N-terminal domain contains several conserved sections (e.g., residues 57-86, 102-164, 219-347, and 375-403), some of which may contribute to binding sites for ankyrin, protein 4.1, or protein 4.2. The membrane domain is highly conserved with the exception of a single segment (residues 543-567) that contains several sites for cleavage of the protein by extracellular proteases. Based on hydropathy analyses and the wealth of available topographical and functional data, a model is proposed in which the protein crosses the membrane 14 times.
Genetic evidence that RNA polymerase associated with sigma A factor uses a sporulation-specific promoter in Bacillus subtilis.
Authors: Kenney T J , York K , Youngman P , Moran C P
Published: May 2006

The construction of allele-specific suppressor mutations has enabled us to demonstrate that a sporulation-specific transcription unit in Bacillus subtilis, the spoIIG operon, is transcribed by a form of RNA polymerase associated with sigma A, the principal sigma factor in vegetative cells. The spoIIG operon encodes sporulation-specific factor sigma E, and its transcription is directed from a promoter that is activated about 1 hr after the onset of endospore formation. This promoter contains sequences that are similar to those found at the -10 and -35 regions of promoters that are used by sigma A-associated RNA polymerase, but these sigma A-like recognition sequences are separated by 22 base pairs rather than the typical 17 or 18 base pairs. We have found that substitution of an arginyl residue for the glutamyl residue at position 196 of sigma A (Glu-196----Arg) suppresses the deleterious effect of a thymidine-to-cytidine base substitution at position -11 in the spoIIG promoter. This suppression was allele-specific, since it did not suppress the effects of base substitutions in other positions in the spoIIG promoter or the effects of a thymidine-to-guanosine change at -11. These results support a model in which a form of RNA polymerase containing sigma A is utilized in an unusual manner to activate the transcription of the spoIIG operon well after the onset of endospore formation.
Transfection of Leishmania enriettii and expression of chloramphenicol acetyltransferase gene.
Authors: Laban A , Wirth D F
Published: May 2006

We report a transient expression transfection system in Leishmania enriettii. A hybrid gene containing an intergenic region of the alpha-tubulin cluster and the bacterial chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) gene is expressed after transfection of L. enriettii with the hybrid plasmid. The expression of the CAT gene is dependent on the presence of sequences from the alpha-tubulin gene. The hybrid gene is also active in Leishmania braziliensis and Leishmania major.
Specific gene suppression by engineered ribozymes in monkey cells.
Authors: Cameron F H , Jennings P A
Published: May 2006

Short catalytic RNAs possessing specific endoribonuclease activity (ribozymes) have recently been designed that can potentially shear any chosen target RNA in trans at a specific site. Here, engineered ribozymes targeted against chloramphenicol acetyltransferase (CAT), derived from Tn9, have been cloned into a mammalian expression vector and tested in transient transfection experiments for their effects on CAT expression in monkey (COS1) cells. The ribozymes contained the catalytic domain of the satellite RNA from tobacco ringspot virus and were targeted to three sites in the CAT mRNA by flanking antisense sequences. These ribozymes, which were previously shown to accurately cleave CAT message in vitro, were cloned into a replicating plasmid vector under the control of the highly active simian virus 40 early promoter. The ribozyme gene sequence was incorporated into the 3' untranslated region of the gene for firefly luciferase as it was ineffective when expressed as a short RNA. Each ribozyme construction gave a similar level of suppression of CAT activity when the target was transcribed from the herpes virus thymidine kinase promoter. One of the three (ribozyme 2) was chosen for further study and tested after it had been modified by the addition of extra flanking bases. The reporter gene for luciferase was used to monitor ribozyme level and to function as a specificity control, and the human growth hormone gene was cotransfected as an independent reporter for specificity of the ribozyme against the intended target CAT. At high (approximately 1000-fold) molar excess this ribozyme was demonstrated to consistently and specifically suppress CAT expression (up to approximately 60%) in COS1 cells relative both to a plasmid clone with the ribozyme inserted in the reversed (inactive) orientation and to a control corresponding to the relevant 26-nucleotide antisense segment of CAT.
ExoMeth sequencing of DNA: eliminating the need for subcloning and oligonucleotide primers.
Authors: Sorge J A , Blinderman L A
Published: May 2006

A method is reported for sequencing DNA based on exonuclease III digestion and strand protection by using modified nucleoside triphosphates. Up to 10 kilobases of sequence information may be obtained from each strand of a given template without subcloning. Prior knowledge of the restriction map is not important; prior knowledge of any of the sequence is not required. Nor are oligonucleotide primers needed. Double-stranded cosmids, plasmids, lambda phage, or linear molecules (including amplified molecules) may be used as starting material. The method creates a single-stranded template from these starting molecules, thus generating high-quality sequence ladders. Most commonly used DNA polymerases may be utilized, including reverse transcriptase and T7 DNA polymerase. The approach is "ordered", so little time is wasted on redundant sequencing.
Magnitude of the solvation pressure depends on dipole potential.
Authors: Simon S A , McIntosh T J
Published: May 2006

As polar surfaces in solvent are brought together, they experience a large repulsive interaction, termed the solvation pressure. The solvation pressure between rough surfaces, such as lipid bilayers, has been shown previously to decay exponentially with distance between surfaces. In this paper, we compare measured values of the solvation pressure between bilayers and the dipole potential for monolayers in equilibrium with bilayers. For a variety of polar solvents and lipid phases, we find a correlation between the measured solvation pressures and dipole potentials. Analysis of the data indicates that the magnitude of the solvation pressure is proportional to the square of the dipole potential. Our experiments also show that the oriented dipoles in the lipid head-group region, including those of both the lipid and solvent molecules, contribute to the dipole potential. We argue that (i) the field produced by these interfacial dipoles polarizes the interbilayer solvent molecules giving rise to the solvation pressure and (ii) both the solvation pressure and the dipole potential decay exponentially with distance from the bilayer surface, with a decay constant that depends on the packing density of the interbilayer solvent molecules (1-2 A in water). These results may have importance in cell adhesion, adsorption of proteins to membranes, characteristics of channel permeability, and the interpretation of electrokinetic experiments.
Identification and characterization of receptors for granulocyte colony-stimulating factor on human placenta and trophoblastic cells.
Authors: Uzumaki H , Okabe T , Sasaki N , Hagiwara K , Takaku F , Tobita M , Yasukawa K , Ito S , Umezawa Y
Published: May 2006

Since radioiodination of human granulocyte colony-stimulating factor (G-CSF) is difficult, we synthesized a mutein of human G-CSF that retains full biological activity and receptor-binding capacity for at least 2 weeks after radioiodination. Receptors for human G-CSF were characterized in the plasma membrane fraction from the human term placenta (human placental membranes) and trophoblastic cells by using the 125I-labeled mutein of human G-CSF (KW-2228). The specific binding of 125I-labeled KW-2228 to placental membranes was pH-dependent, with maximal specific binding at pH 7.8; it increased linearly with protein to 3.7 mg of protein per ml and was both time- and temperature-dependent, with maximal binding at 4 degrees C after a 24-hr incubation. When we examined the ability of hematopoietic growth factors to inhibit 125I-labeled KW-2228 binding, we found that KW-2228 and intact human G-CSF inhibited 125I-labeled KW-2228 binding, whereas erythropoietin or granulocyte-macrophage colony-stimulating factor did not. Scatchard analysis revealed a single receptor type with a Bmax of 210 fmol/mg of protein and a Kd of 480 pM. The human G-CSF receptors on human placental membranes were shown to consist of two molecular species of 150 kDa and 120 kDa that could be specifically cross-linked to 125I-labeled KW-2228. Human trophoblastic cells, T3M-3, also possessed a single receptor for G-CSF with a Bmax of 533 receptors per cell and a Kd of 390 pM. Thus we have identified the receptor for human G-CSF on human placental membranes and trophoblastic cells, and the presence of this receptor in these membranes suggests that human G-CSF plays some role in the feto-placental unit during human development.
Bleaching in reef corals: Physiological and stable isotopic responses
Authors: Porter James W. , Fitt William K. , Spero Howard J. , Rogers Caroline S. , White Michael W.
Published: May 2006

During the late summer to fall of 1987, Caribbean reef corals experienced an intense and widespread discoloration event described as bleaching. Contrary to initial predictions, most bleached corals did not die. However, energy input from zooxanthellae decreased, as estimated from: (
i
) δ
13
C values, a measure of the discrimination against
13
C in
12
C/
13
C assimilation, of skeletal aragonite; (
ii
)
in situ
photosynthesis-irradiance measurements; (
iii
) and tissue biomass parameters of
Montastraea annularis
and
Agaricia lamarcki
. The δ
18
O signal, a measure of the discrimination against
18
O in
16
O/
18
O assimilation, from
M. annularis
skeletons demonstrated that this event coincided with abnormally elevated water temperatures.
Mitochondrial DNA sequences in single hairs from a southern African population.
Authors: Vigilant L , Pennington R , Harpending H , Kocher T D , Wilson A C
Published: May 2006

Hypervariable parts of mitochondrial DNA (mtDNA) were amplified enzymatically and sequenced directly by using genomic DNA from single plucked human hairs. This method has been applied to study mtDNA sequence variation among 15 members of the !Kung population. A genealogical tree relating these aboriginal, Khoisan-speaking southern Africans to 68 other humans and to one chimpanzee has the deepest branches occurring amongst the !Kung, a result consistent with an African origin of human mtDNA. Fifteen cases of unrelated individuals having identical sequences in the most variable parts of the mtDNA control region were found within populations of !Kung, Western Pygmies, and Eastern Pygmies, but no cases of identity were evident among these populations. This and other evidence of geographic structuring of the mitochondrial diversity in Africa, together with knowledge of the rate of accumulation of base changes in human mtDNA, implies that the average rate at which female lineages have moved their home bases during hunter-gatherer times could be as low as 13 meters per year. The technique of enzymatic amplification and direct sequencing applied to readily collected, highly stable biological materials such as hairs makes it possible to examine with high resolution many representatives of virtually any population.
Circular RNAs: relics of precellular evolution?
Authors: Diener T O
Published: May 2006

The demonstration of enzymatic capabilities of certain RNAs, in addition to their well-known template properties, has led to the recognition that RNAs are the only biological macromolecules that can function both as genotype and phenotype, hence raising the possibility of Darwinian selection and precellular evolution at the RNA level in the absence of DNA or protein. Recent models of such precellular RNA systems are patterned after the properties of intron-derived ribozymes. On the basis of a phylogenetic analysis and known properties of certain small plant pathogenic RNAs (viroids and viroid-like satellite RNAs), I suggest that these plant RNAs are more plausible candidates than introns as "living fossils" of a precellular RNA world. Their small size and circularity would have enhanced probability of their survival in error-prone, primitive self-replicating RNA systems and assured complete replication without the need for initiation or termination signals. All of these RNAs possess efficient mechanisms for the precise cleavage of monomers from oligomeric replication intermediates. Some (most viroids) require a host factor, but others (viroid-like satellite RNAs and one viroid) function as self-cleaving RNA enzymes far smaller and simpler than those derived from introns. The question is raised whether introns could have evolved from viroids or viroid-like satellite RNAs rather than vice versa, as has been widely speculated.
Mouse genes coding for "zinc-finger"-containing proteins: characterization and expression in differentiated cells.
Authors: Passananti C , Felsani A , Caruso M , Amati P
Published: May 2006

By using the zinc-finger region of human cHF.10 cDNA as a probe at low-stringency hybridization conditions, several individual phages from a mouse skeletal muscle cDNA library have been isolated. The amino acid sequences of the "zinc-finger" domains derived from the DNA sequences of three cDNA clones are shown. The expression of the corresponding mRNAs in three cell lines (NIH 3T3, F9 teratocarcinoma, and C2 myoblast cells) at different stages of differentiation and in eight adult mouse tissues has been analyzed. The transcription of these genes is induced during the in vitro differentiation of the cell lines tested. These three genes are widely and evenly expressed in adult mouse tissues, with the remarkable exception of one that is expressed predominantly in the testis.
Long-term culture and fine specificity of human cytotoxic T-lymphocyte clones reactive with human immunodeficiency virus type 1.
Authors: Walker B D , Flexner C , Birch-Limberger K , Fisher L , Paradis T J , Aldovini A , Young R , Moss B , Schooley R T
Published: May 2006

The definition of human immunodeficiency virus type 1 (HIV-1) immunogenic epitopes is central to the rational design of AIDS vaccine strategies. In this study, we have generated seven HIV-1 reverse transcriptase-specific cytotoxic T-lymphocyte (CTL) clones from the peripheral blood of two seropositive subjects. Epitopes recognized by these CTL clones were identified by using target cells infected with recombinant HIV-1-vaccinia virus vectors expressing truncated reverse transcriptase proteins and further defined by using target cells incubated with overlapping 25-amino acid synthetic reverse transcriptase peptides. Five different CTL epitopes were identified, and in each case recognition was restricted by class I human leukocyte antigens (HLA). Clones maintained specific cytolytic function in continuous culture for up to 11 months, requiring only periodic restimulation with a CD3-specific monoclonal antibody. These results indicate that HIV-1-specific, major histocompatibility class I-restricted CTL recognize multiple epitopes of a single viral gene product in conjunction with different host HLA antigens. In addition, they demonstrate that human virus-specific CTL can be grown in long-term culture without the need for reexposure to viral antigen.
An explanation for the purported excitation of piriform cortical neurons by N-acetyl-L-aspartyl-L-glutamic acid (NAAG).
Authors: Whittemore E R , Koerner J F
Published: May 2006

The excitation of piriform cortical neurons by iontophoresis of N-acetyl-L-aspartyl-L-glutamic acid (NAAG) isolated from rat brain is frequently cited as major support for the possible neurotransmitter role of NAAG in the CNS [ffrench-Mullen, J. M. H., Koller, K., Zaczek, R., Coyle, J. T., Hori, N. & Carpenter, D. O. (1985) Proc. Natl. Acad. Sci. USA 82, 3897-3900]. However, we have been unable to reproduce this observation using synthetic NAAG, and instead we offer an alternative explanation. In our experiments, iontophoresis of the sodium salt of synthetic NAAG did not induce single-unit spiking at sites in slices of rat piriform cortex that responded vigorously to L-glutamate. In contrast, iontophoresis of the potassium salt of synthetic NAAG or of potassium ions alone induced single unit activity. The responses to both NAAG/KCl and KCl alone were inhibited by L-2-amino-4-phosphonobutanoic acid and desensitized rapidly, as previously reported for NAAG. These results suggest that residual potassium ions, remaining after the original purification of NAAG, were responsible for the excitations attributed to NAAG.
HLA-DR4 subtype frequencies in rheumatoid arthritis indicate that DRB1 is the major susceptibility locus within the HLA class II region.
Authors: Wordsworth B P , Lanchbury J S , Sakkas L I , Welsh K I , Panayi G S , Bell J I
Published: May 2006

Susceptibility to rheumatoid arthritis (RA) may be due to the presence of shared functional epitopes common to the HLA-DR beta chains of several RA-associated haplotypes. We have obtained direct evidence for this hypothesis by using the polymerase chain reaction and sequencing the DRB1 and DQB1 genes from RA patients. A highly conserved epitope present on DR beta chains of DR4 and DR1 haplotypes was found in 83% of 149 patients with classical or definite RA but was found in only 46% of 100 control individuals (P less than 0.0001). Two Dw subtypes of DR4 (Dw4 and Dw14) were associated with disease susceptibility but two other subtypes (Dw10 and Dw13) were not. Sequence differences between these subtypes implicate those residues around the putative antigen binding site of the DR beta molecule in the pathogenesis of RA. These data provide a basis for understanding host susceptibility to RA at a molecular level.
Regulation of immunoglobulin production in hyperimmunoglobulin E recurrent-infection syndrome by interferon gamma.
Authors: King C L , Gallin J I , Malech H L , Abramson S L , Nutman T B
Published: May 2006

The hyperimmunoglobulin E recurrent-infection (Job) syndrome (HIE) is a congenital disorder characterized by high serum IgE, chronic eczematoid dermatitis, and recurrent infections. We examined the effect of interferon gamma (IFN-gamma) on excessive IgE production in HIE patients. Spontaneous in vitro production of IgE by peripheral blood mononuclear cells from HIE patients was elevated compared to normal individuals and correlated with serum IgE. In 9 of 13 patients, IgE production by peripheral blood mononuclear cells was inhibited by 50% by IFN-gamma at 100-1000 units/ml, whereas inhibition by IFN-gamma at 10(4) units/ml ranged from 67 to 93% for these 9 patients. IFN-gamma also inhibited IgG1, IgG3, and IgG4 production by B lymphocytes without inhibiting IgG2 production. IFN-gamma was administered subcutaneously to 5 HIE patients. After 2 weeks of treatment with IFN-gamma (0.05 mg/m2) at three doses per week given on alternate days, peripheral blood mononuclear cells from all 5 HIE patients decreased spontaneous in vitro IgE production (27-62% decrease) with no change in IgG and IgM. One patient had a 58% decrease in serum IgE and another patient had a 50% decrease in serum IgE after the IFN-gamma was increased to 0.1 mg/m2 for three doses per week for a month. In both patients, serum IgE returned to pre-IFN-gamma-challenge levels 1-3 months after completion of treatment, and in vivo IFN-gamma did not affect serum IgG and IgM, although serum IgG4 decreased with changes in serum IgE. Our studies demonstrate that IFN-gamma can regulate production of IgE and some IgG subclasses in humans.
A gene required for transfer of T-DNA to plants encodes an ATPase with autophosphorylating activity.
Authors: Christie P J , Ward J E , Gordon M P , Nester E W
Published: May 2006

The virB operon of the Agrobacterium tume-faciens pTiA6NC plasmid likely plays a role in directing T-DNA transfer events at the bacterial membrane, as determined previously by mutagenesis and cellular fractionation studies and by DNA sequence analysis of the approximately 12-kilobase-pair operon. The DNA sequence analysis also revealed consensus mononucleotide binding domains in the deduced virB5 and virB11 gene products, suggesting that one or both of these proteins couple energy, by means of nucleotide triphosphate (NTP) hydrolysis, to T-DNA transport. In this report, the product of virB11, an essential virulence gene, was overproduced in Escherichia coli and purified by using immunoaffinity chromatography. The immunoaffinity purified protein, as well as NaDodSO4/polyacrylamide gel-eluted protein, bound and hydrolyzed ATP in the absence of DNA effectors. VirB11 protein also demonstrated in vitro autophosphorylation activity. VirB11 protein was localized primarily to the cytoplasmic membrane by immunoblot analysis of membrane fractions. The deduced VirB11 protein exhibits sequence similarity to comG ORF1, a protein required for uptake of DNA by competent Bacillus subtilis cells. These findings suggest that phosphorylation may serve to activate a component(s) of the A. tumefaciens T-DNA transport apparatus and may also represent a general activation mechanism of other bacterial DNA transport systems.
Multiple functions of human single-stranded-DNA binding protein in simian virus 40 DNA replication: single-strand stabilization and stimulation of DNA polymerases alpha and delta.
Authors: Kenny M K , Lee S H , Hurwitz J
Published: May 2006

The human single-stranded-DNA binding protein (human SSB) is required for simian virus 40 (SV40) DNA replication in vitro. SV40 large tumor antigen and human SSB can support extensive unwinding of SV40 origin-containing DNA in the presence of ATP and a topoisomerase that relieves positive superhelicity. Although SSBs from viral and prokaryotic sources substituted for human SSB in the DNA-unwinding reaction, they did not substitute in the replication of SV40 DNA. The specificity for human SSB in SV40 DNA replication can be explained, at least in part, by the finding that DNA polymerase alpha was stimulated 10-fold by human SSB but not by other SSBs. Human SSB also stimulated proliferating-cell nuclear antigen-dependent DNA polymerase delta; however, other SSBs stimulated this polymerase as well.
Primary structures of the heterogeneous nuclear ribonucleoprotein A2, B1, and C2 proteins: a diversity of RNA binding proteins is generated by small peptide inserts.
Authors: Burd C G , Swanson M S , Görlach M , Dreyfuss G
Published: May 2006

We have isolated cDNAs for the major heterogeneous nuclear ribonucleoprotein (hnRNP) A2, B1, and C2 proteins and determined their nucleotide and deduced amino acid sequences. The A2 and B1 cDNAs are identical except for a 36-nucleotide in-frame insert in B1. Similarly, the sequence of the C2 protein cDNA is related to that of C1 in that C2 contains an extra 39 in-frame nucleotides. Therefore, the B1 amino acid sequence is identical to A2 except for the insertion of 12 amino acids near its amino terminus, and C1 and C2 are also identical to each other except for an extra 13 amino acids near the middle of C2. All three proteins are members of a large family of RNA binding proteins that contain the consensus sequence-type RNA binding domain (CS-RBD). The A2 and B1 proteins have a modular structure similar to that of the hnRNP protein A1: they contain two CS-RBDs and a glycine-rich auxiliary domain at the carboxyl terminus. The CS-RBDs of A2 and B1 have approximately 80% amino acid identity with those of A1, whereas the glycine-rich auxiliary domain is considerably more divergent with less than 30% of the amino acids being identical. These findings indicate that the addition of small peptides, probably by alternative pre-mRNA splicing, generates some of the diversity apparent among hnRNP proteins.
Mapping the active-site tyrosine of vaccinia virus DNA topoisomerase I.
Authors: Shuman S , Kane E M , Morham S G
Published: May 2006

Site-directed mutagenesis of the vaccinia virus gene encoding a type I DNA topoisomerase implicates Tyr-274 as the active-site residue that forms a covalent adduct with DNA during cycles of DNA-strand breakage and reunion. Replacement of Tyr-274 by phenylalanine results in loss of the ability of the enzyme to relax negatively supercoiled DNA as well as to form the covalent DNA-protein intermediate. Substitution of phenylalanine for tyrosine at nine other sites in the protein has no apparent effect on enzyme activity. Amino acid sequence alignment reveals Tyr-274 to be homologous to Tyr-727 and Tyr-771, respectively, of the type I topoisomerases from Saccharomyces cerevisiae and Saccharomyces pombe; Tyr-727 and Tyr-771 have been shown to represent the active-site tyrosines of those enzymes. Sequence comparison of the active-site regions defines a motif Ser-Lys-Xaa-Xaa-Tyr common to the viral and cellular type I topoisomerases, including the human enzyme.
Internal dynamics of lactose permease.
Authors: Dornmair K , Jähnig F
Published: May 2006

The transport protein lactose permease was reconstituted in vesicles of dimyristoylphosphatidylcholine, and the internal dynamics were studied by measuring the fluorescence anisotropy decay of the tryptophan residues and of a covalently bound pyrene label. For the tryptophans three relaxation processes and for the pyrene two relaxation processes with relaxation times in the nanosecond range were observed. The slowest process, of approximately 50 ns, is assigned to orientational fluctuations of membrane-spanning helices. When the temperature is decreased below the lipid-phase transition, this relaxation process is slowed down and restricted in amplitude. Because the transport rate is known to also decrease below the phase transition, this observation suggests a coupling between internal dynamics and transport. This coupling is analyzed on the basis of the Kramers relation for chemical reactions.
In vivo 23Na and 31P NMR measurement of a tonoplast Na+/H+ exchange process and its characteristics in two barley cultivars.
Authors: Fan T W , Higashi R M , Norlyn J , Epstein E
Published: May 2006

A Na+ uptake-associated vacuolar alkalinization was observed in roots of two barley cultivars (Arivat and the more salt-tolerant California Mariout) by using 23Na and 31P in vivo NMR spectroscopy. A NaCl uptake-associated broadening was also noted for both vacuolar Pi and intracellular Na NMR peaks, consistent with Na+ uptake into the same compartment as the vacuolar Pi. A close coupling of Na+ with H+ transport (presumably the Na+/H+ antiport) in vivo was evidenced by qualitative and quantitative correlations between Na+ accumulation and vacuolar alkalinization for both cultivars. Prolongation of the low NaCl pretreatment (30 mM) increased the activity of the putative antiport in Arivat but reduced it in California Mariout. This putative antiport also showed a dependence on NaCl concentration for California Mariout but not for Arivat. No cytoplasmic acidification accompanied the antiporter activity for either cultivar. The response of adenosine phosphates indicated that ATP utilization exceeded the capacity for ATP synthesis in Arivat, but the two processes seemed balanced in California Mariout. These comparisons provide clues to the role of the tonoplast Na+/H+ antiport and compensatory cytoplasmic adjustments including pH, osmolytes, and energy phosphates in governing the different salt tolerance of the two cultivars.
Cellular responses to Ca2+ from extracellular and intracellular sources are different as shown by simultaneous measurements of cytosolic Ca2+ and secretion from bovine chromaffin cells.
Authors: Kim K T , Westhead E W
Published: May 2006

Bovine adrenal medullary cells, cultured on quartz plates, were superfused with buffer to which pulses of stimulant were added. Cytosolic Ca2+ was measured by the fura-2 fluorescence method and the simultaneously released catecholamine was measured electrochemically. When stimulant concentrations were adjusted to given equivalent elevations of cytosolic Ca2+, secretion depended entirely on whether Ca2+ came from internal stores or from the extracellular medium. Calcium from internal stores did not support secretion under these conditions. This nonequivalence of the two sources of cytosolic Ca2+ points to important differences in the physiological roles of the two sources of calcium. Dimethylphenylpiperazinium (a cholinergic agonist) and elevated K+ increased cytosolic Ca2+ and caused secretion only in the presence of external Ca2+. Bradykinin, muscarine, and ATP elevated cytosolic Ca2+ in the presence and absence of extracellular Ca2+ but caused secretion only in the presence of extracellular Ca2+. UTP, which in the absence of extracellular Ca2+ elevated cytosolic Ca2+ as effectively as ATP, did not cause detectable secretion under any circumstance. Because of the high Ca2+-buffering capacity of the cytosol, we expected that Ca2+ gradients, perhaps quite steep, would be produced by a pulse of Ca2+ entering the cytosol. Fura-2 fluorescence measures only the average free cytosolic Ca2+. Our data show that Ca2+ entering across the plasma membrane was much more effective at triggering exocytosis than was Ca2+ released from internal stores, suggesting that the two sources of Ca2+ are effectively compartmentalized, probably by concentration gradients in the cytosol.
Cloning of murine interferon gamma receptor cDNA: expression in human cells mediates high-affinity binding but is not sufficient to confer sensitivity to murine interferon gamma.
Authors: Hemmi S , Peghini P , Metzler M , Merlin G , Dembic Z , Aguet M
Published: May 2006

A full-length cDNA encoding the murine interferon gamma (IFN-gamma) receptor was isolated from a lambda gt11 library using a human IFN-gamma receptor cDNA probe. The deduced amino acid sequence of the murine IFN-gamma receptor shows approximately 53% homology to its human counterpart but no homology to other known proteins. Murine IFN-gamma receptor cDNA was expressed in human HEp-2 cells, which do not bind murine IFN-gamma and are insensitive to its action. Transfectants displayed the same binding properties as mouse cells. The biological responsiveness of such transfectants to various biological effects of both human and murine IFN-gamma was investigated, including modulation of major histocompatibility complex class I and class II antigen expression, inhibition of cell growth, and antiviral activity. Like parental HEp-2 cells, these transfectants responded only to human, but not to murine, IFN-gamma. Inversely, mouse L929 cells transfected with human IFN-gamma receptor cDNA were insensitive to human IFN-gamma. These results confirm and extend previous findings, suggesting that species-specific cofactors are needed for IFN-gamma-mediated signal transduction.
Flat reversion by okadaic acid of raf and ret-II transformants.
Authors: Sakai R , Ikeda I , Kitani H , Fujiki H , Takaku F , Rapp U , Sugimura T , Nagao M
Published: May 2006

Okadaic acid is a non-phorbol 12-myristate 13-acetate (PMA)-type tumor promoter on mouse skin and known to be a potent inhibitor of serine/threonine protein phosphatases. Contrary to expectation from its tumor-promoting activity, okadaic acid was shown to have a potential to revert the phenotypes of cells transformed by raf and ret-II to that of normal cells. Two to 3 days after addition of 8 ng of okadaic acid per ml to the culture medium, raf and ret-II transformants changed to flat cells and gained contact inhibition. The amount of fibronectin, which was decreased in malignant transformed cells, was increased in the flat revertants. Moreover, okadaic acid caused a dose-dependent loss of ability to grow in soft agar. The morphology of the cells reverted to malignant phenotype within 1 week after removal of okadaic acid. The levels of mRNA and protein of activated c-raf in flat revertants were similar to those in parental transformed cells. The level of mRNA of ret-II was also not changed by flat reversion. No induction of flat reversion was observed with okadaic acid tetramethyl ether, an inactive compound, or a phorbol ester, PMA. As okadaic acid is a potent inhibitor of protein phosphatases 1 and 2A, the possibility that these phosphatases are involved in signal transduction from the raf and ret-II oncogenes is suggested.
2-Acetylpyridine 5-[(dimethylamino)thiocarbonyl]-thiocarbonohydrazone (A1110U), a potent inactivator of ribonucleotide reductases of herpes simplex and varicella-zoster viruses and a potentiator of acyclovir.
Authors: Spector T , Harrington J A , Morrison R W , Lambe C U , Nelson D J , Averett D R , Biron K , Furman P A
Published: May 2006

2-Acetylpyridine 5-[(dimethylamino)thiocarbonyl]thiocarbonohydrazone (A1110U) was found to be a potent inactivator of the ribonucleotide reductases (EC 1.17.4.1) encoded by herpes simplex virus types 1 and 2 and by varicella-zoster virus and to be a weaker inactivator of human ribonucleotide reductase. It also markedly potentiated the antiherpetic activity of acyclovir against these viruses in tissue culture. A1110U both decreased the dGTP pool that builds up when infected cells are treated with acyclovir and induced a large increase in the pool of acyclovir triphosphate. The resultant 100-fold increase in the ratio of the concentrations of acyclovir triphosphate to dGTP should facilitate the binding of the fraudulent nucleotide to its target enzyme, herpes virus-encoded DNA polymerase, and could account for the synergy between A1110U and acyclovir. A similar change in the acyclovir triphosphate-to-dGTP ratio was previously reported to be induced by another ribonucleotide reductase inhibitor, 2-acetylpyridine 4-(2-morpholinoethyl)thiosemicarbazone (A723U). However, A1110U is considerably more potent and may have better clinical potential. Synergistic toxic interactions between A1110U and acyclovir were not detected in uninfected cells.
Topology of cell adhesion molecules.
Authors: Becker J W , Erickson H P , Hoffman S , Cunningham B A , Edelman G M
Published: May 2006

The neural cell adhesion molecule (N-CAM) exists in two major forms [ld (large cytoplasmic domain) peptide and sd (small cytoplasmic domain) peptide] that contain transmembrane segments and different cytoplasmic domains and in a third form [ssd (small surface domain) peptide] that lacks transmembrane and cytoplasmic regions. All forms have the same extracellular region of more than 600 amino acid residues, a region also found in a fragment (Fr2) that can be released from cells by proteolysis. The liver cell adhesion molecule (L-CAM) is expressed as a single species that is distinct from N-CAM, but its extracellular region can also be obtained as a proteolytic fragment (Ft1). Examination of the various forms of N-CAM and the Ft1 fragment of L-CAM by electron microscopy of rotary shadowed molecules indicated that they all have rod-shaped structures that contain a hinge region which is apparently flexible. Both the ssd chain and the Fr2 fragment of N-CAM are single rods bent into arms approximately 18 and 10 nm long. The ld and sd chains are longer bent rods that form rosettes comprising two to six branches; detergent treatment disrupts these rosettes into single rods. Specific antibodies that block homophilic N-CAM binding labeled the distal ends of the branches of the ld/sd rosettes and the ends of the longer arm of both the ssd chain and the Fr2 fragment. Antibodies that bind to the sialic acid-rich region of N-CAM bound near the hinge. These data indicate that the N-CAM rosettes are formed by interaction between their transmembrane or cytoplasmic domains and not by interactions involving their homophilic binding sites. The L-CAM Ft1 fragment is also a bent rod with an apparently flexible hinge; like the ssd chain and the Fr2 fragment of N-CAM, it does not form aggregates. The similarities between L-CAM and N-CAM, despite their differences in amino acid sequence, suggest that their general configuration and the presence of a flexible hinge are important elements in assuring effective and specific cell-cell adhesion.
Secondary sigma factor controls transcription of flagellar and chemotaxis genes in Escherichia coli.
Authors: Arnosti D N , Chamberlin M J
Published: May 2006

The genes specifying chemotaxis, motility, and flagellar function in Escherichia coli are coordinately regulated and form a large and complex regulon. Despite the importance of these genes in controlling bacterial behavior, little is known of the molecular mechanisms that regulate their expression. We have identified a minor form of E. coli RNA polymerase that specifically transcribes several E. coli chemotaxis/flagellar genes in vitro and is likely to carry out transcription of these genes in vivo. The enzyme was purified to near homogeneity based on its ability to initiate transcription of the E. coli tar chemotaxis gene at start sites that are used in vivo. Specific tar transcription activity is associated with a polypeptide of apparent 28-kDa molecular mass that remains bound to the E. coli RNA polymerase throughout purification. This peptide behaves as a secondary sigma factor--designated sigma F--because it restores specific tar transcription activity when added to core RNA polymerase. The sigma F holoenzyme also transcribes the E. coli tsr and flaAI genes in vitro as well as several Bacillus subtilis genes that are transcribed specifically by the sigma 28 form of B. subtilis RNA polymerase. The latter holoenzyme is implicated in transcription of flagellar and chemotaxis genes in B. subtilis. Hence E. coli sigma F holoenzyme appears to be analogous to the B. subtilis sigma 28 RNA polymerase, both in its promoter specificity and in the nature of the regulon it controls.
Identification of androgen receptors in normal human osteoblast-like cells.
Authors: Colvard D S , Eriksen E F , Keeting P E , Wilson E M , Lubahn D B , French F S , Riggs B L , Spelsberg T C
Published: May 2006

The sex steroids, androgens and estrogens, are major regulators of bone metabolism. However, whether these hormones act on bone cells through direct or indirect mechanisms has remained unclear. A nuclear binding assay recently used to demonstrate estrogen receptors in bone [Eriksen, E.F., Colvard, D.S., Berg, N.J., Graham, M.L., Mann, K.G., Spelsberg, T.C. & Riggs, B.L. (1988) Science 241, 84-86] was used to identify specific nuclear binding of a tritiated synthetic androgen, [3H]R1881 (methyltrienolone), in 21 of 25 (84%) human osteoblast-like cell strains and a concentration of bound steroid receptors of 821 +/- 140 (mean +/- SEM) molecules per cell nucleus. Binding was saturable and steroid-specific. Androgen receptor gene expression in osteoblasts was confirmed by RNA blot analysis. Relative concentrations of androgen and estrogen receptors were compared by measuring specific nuclear estrogen binding. Nuclear binding of [3H]estradiol was observed in 27 of 30 (90%) cell strains; the concentration of bound estradiol receptor was 1537 +/- 221 molecules per cell nucleus. The concentrations of nuclear binding sites were similar in males and females for both [3H]R1881 and [3H]estradiol. We conclude that both androgens and estrogens act directly on human bone cells through their respective receptor-mediated mechanisms.
Estrogen receptor selectively binds the "coding strand" of an estrogen responsive element.
Authors: Lannigan D A , Notides A C
Published: May 2006

An initial step in the transcriptional activation of the prolactin gene by estrogen is the binding of the estrogen-receptor complex to a specific nucleotide sequence [estrogen responsive element (ERE)]. Using the gel mobility assay, we examined the binding mechanism of purified estrogen receptor to the ERE contained on a 255-base-pair fragment from the upstream region between nucleotides -1784 to -1531 of the rat prolactin gene. Remarkably, specific high-affinity binding was detected to the dissociated "coding strand" but not to the "noncoding strand" of the ERE-containing fragment. The dissociated strands of this fragment possess unusual secondary structure, as indicated by their anomalous migration in the gel mobility assay. The estrogen receptor binds to the coding strand of the ERE with a 60-fold higher affinity than to the double-stranded ERE. Furthermore, the receptor binds with a 1000-fold greater affinity to the coding strand of the ERE than to a double-stranded nonspecific DNA fragment. We propose that, in vivo, the estrogen receptor initially binds to the double-stranded ERE. Subsequently, the DNA strands separate due to transitory strand separation and supercoiling, allowing folding of the coding strand of the ERE into a structure that is then bound more tightly by the receptor. The formation of this receptor-ERE coding strand complex may be a crucial step in the mechanism of estrogen-stimulated transcription.
Growing and developing Dictyostelium cells express different ras genes.
Authors: Robbins S M , Williams J G , Jermyn K A , Spiegelman G B , Weeks G
Published: May 2006

The expression of ras-related protein in the cellular slime mold Dictyostelium discoideum is developmentally regulated. It was previously reported that Dictyostelium possesses a single ras gene (Ddras) that is maximally expressed during the pseudoplasmodial stage of development. We have isolated a series of cDNA clones derived from a second ras gene, DdrasG. It encodes a protein that is very similar to the protein encoded by Ddras, but in contrast to Ddras, DdrasG is only expressed during growth and early development. Although other eukaryotic organisms possess more than one ras gene, Dictyostelium is thus far unique in expressing different ras genes at different stages of development. In Dictyostelium the two ras proteins may fulfill different functions, with the DdrasG protein playing a role during cell growth and the Ddras protein playing a role in signal transduction during multicellular development.