bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo mutations of calmodulin: a mutant Paramecium with altered ion current regulation has an isoleucine-to-threonine change at residue 136 and an altered methylation state at lysine residue 115.
Authors: Lukas T J , Wallen-Friedman M , Kung C , Watterson D M
Published: May 2006

The Paramecium tetraurelia mutants termed pantophobiacs have altered behavior due to perturbed calcium activation of ion channel activity. The calmodulin from pantophobiac A1 (pntA1) was shown in previous studies to have a single amino acid change at residue 101 that is selective in its effects on activity. This change has no effect on posttranslational modifications. However, the calmodulin from the phenotypically related mutant pantophobiac A2 (pntA2) has a threonine residue at position 136, in the fourth calcium-binding domain, instead of an isoleucine or valine like all other calmodulins. This region of the calmodulin structure is within 4 A of a complementary hydrophobic structure in the third calcium-binding domain, raising the possibility of a perturbation of interdomain interactions in the pntA2 mutant. This possibility is supported by the heterogenous methylation state of lysine-115 in the pntA2 calmodulin. This lysine residue, located in the peptide connecting calcium-binding domains three and four, is fully trimethylated in the wild-type and pntA1 calmodulins. The functional selectivity of these structural changes is demonstrated by the conservation of calmodulin activator activity with a calmodulin-regulated protein kinase that has been used as a standard of comparison. Overall, these results indicate the degree to which the calmodulin can be mutated in vivo without being lethal to the organism, and they provide genetic evidence suggesting that the post-translational methylation state of residue 115 requires the appropriate conformation in addition to the local amino acid sequence.
Obesity-linked regulation of the adipsin gene promoter in transgenic mice.
Authors: Platt K A , Min H Y , Ross S R , Spiegelman B M
Published: May 2006

The mouse adipsin gene encodes a member of the serine protease family that is expressed predominantly in adipose tissue and is secreted into the bloodstream. Adipsin expression is sharply down-regulated in several models of genetic and acquired obesity, representing the first example of an adipocyte gene whose expression is greatly altered in this disorder. In this study, we have asked whether a DNA fragment from the adipsin gene can direct tissue-specific expression of a heterologous gene and mediate the suppression of this expression in genetic and chemically induced obesity. Transgenic mice have been constructed with 950 bases of DNA from the 5' flanking region of the adipsin gene linked to the bacterial chloramphenicol acetyltransferase (CAT) gene in a mouse strain bearing a recessive obesity gene (diabetes, db). By crossing db/+ transgenic mice with nontransgenic db/+ mice, we obtained progeny that allowed a direct comparison of CAT expression in the tissues of lean and obese littermates. The lean mice express CAT activity predominantly in adipose tissue, while the obese mice show a marked reduction in CAT expression relative to the lean controls. When similar experiments are performed with an adipsin-CAT fusion gene containing a heterologous AKV (AKR mouse leukemia virus) enhancer, the tissue specificity of CAT expression in lean mice is broadened to include the thymus, spleen, brain, and other tissues; down-regulation occurs in all of these tissues in mice homozygous for the obesity gene or in mice that have been injected with monosodium glutamate (MSG), which induces obesity. These results indicate that 950 bases of the 5' flanking region of the adipsin gene carry information that specifies both expression in adipose tissue and a response to a gene or chemical that induces obesity. These results also suggest that the trans-acting factors that are regulated aberrantly in these forms of obesity are not restricted to adipose tissue and could play a role in obesity-linked dysfunctions observed in other tissues as well.
Dramatic thermostabilization of yeast iso-1-cytochrome c by an asparagine----isoleucine replacement at position 57.
Authors: Das G , Hickey D R , McLendon D , McLendon G , Sherman F
Published: May 2006

Two Saccharomyces cerevisiae yeast mutants, cyc1-73 and cyc1-190, contain nonfunctional and presumably unstable forms of iso-1-cytochrome c due to Gly-34----Ser and His-38----Pro replacements, respectively. Second-site reversions that produced Asn-57----Ile replacements at least partially restored function, presumably by alleviating the instability of these two altered iso-1-cytochromes c. Introduction of the Ile-57 replacement by site-directed mutagenesis in an otherwise normal protein resulted in a 17 degrees C increase in the transition temperature (Tm), corresponding to over a 2-fold increase in the free energy change (delta G degrees) for thermal unfolding.
Isolation, sequence, and bacterial expression of a cDNA for (S)-tetrahydroberberine oxidase from cultured berberine-producing Coptis japonica cells.
Authors: Okada N , Koizumi N , Tanaka T , Ohkubo H , Nakanishi S , Yamada Y
Published: May 2006

cDNA clones for the (S)-tetrahydroberberine (H4Ber) oxidase of cultured berberine-producing Coptis japonica cells were isolated by screening a C. japonica cDNA library with synthetic nucleotides that can encode the NH2-terminal sequence of this enzyme. Analyses of the nucleotide sequences of the cloned cDNA inserts revealed a 759-base-pair open reading frame that encoded a 253-amino acid polypeptide with a Mr of 27,089 and NH2-terminal and internal sequences identical with those of the (S)-H4Ber oxidase, as determined by microsequencing methods. Escherichia coli were transformed with an expression vector carrying (S)-H4Ber oxidase cDNA. The transformed bacteria were induced to overproduce a 28-kDa protein that reacted with Coptis (S)-H4Ber oxidase-specific antibody. A comparison of the derived amino acid sequence of (S)-H4Ber oxidase with sequences in the protein data base of the Protein Research Foundation showed a marked similarity between (S)-H4Ber oxidase and the NH2-terminal portion of mouse P1-450, which is encoded by a single exon of the mouse P1-450 gene. The availability of cloned cDNA for (S)-H4Ber oxidase allows use of the methods of molecular biology to study the regulation of (S)-H4Ber oxidase gene expression in cultured C. japonica cells in relation to berberine biosynthesis.
Endogenous ADP-ribosylation of elongation factor 2 in polyoma virus-transformed baby hamster kidney cells.
Authors: Fendrick J L , Iglewski W J
Published: May 2006

Polyoma virus-transformed baby hamster kidney (pyBHK) cells were cultured in medium containing [32P]orthophosphate and 10% (vol/vol) fetal bovine serum. A 32P-labeled protein with an apparent molecular mass of 97 kDa was immunoprecipitated from cell lysates with antiserum to ADP-ribosylated elongation factor 2 (EF-2). The 32P labeling of the protein was enhanced by culturing cells in medium containing 2% serum instead of 10% serum. The 32P label was completely removed from the protein by treatment with snake venom phosphodiesterase and the digestion product was identified as [32P]AMP, indicating the protein was mono-ADP-ribosylated. HPLC analysis of tryptic peptides of the 32P-labeled 97-kDa protein and purified EF-2, which was ADP-ribosylated in vitro with diphtheria toxin fragment A and [32P]NAD, demonstrated an identical labeled peptide in the two proteins. The data strongly suggest that EF-2 was endogenously ADP-ribosylated in pyBHK cells. Maximum incorporation of radioactivity in EF-2 occurred by 12 hr and remained constant over the subsequent 12 hr. It was estimated that 30-35% of the EF-2 was ADP-ribosylated in cells cultured in medium containing 2% serum. When 32P-labeled cultures were incubated in medium containing unlabeled phosphate, the 32P label was lost from the EF-2 within 30 min.
The computation of classical constants
Authors: Chudnovsky D. V. , Chudnovsky G. V.
Published: May 2006

Hypergeometric representations of classical constants and efficient algorithms for their calculation are discussed. Particular attention is devoted to algorithms for computing π.
Energy coupling to periplasmic binding protein-dependent transport systems: stoichiometry of ATP hydrolysis during transport in vivo.
Authors: Mimmack M L , Gallagher M P , Pearce S R , Hyde S C , Booth I R , Higgins C F
Published: May 2006

Periplasmic binding protein-dependent transport systems mediate the accumulation of many diverse substrates in prokaryotic cells. Similar transport systems, including the P-glycoprotein responsible for multidrug resistance in human tumors, are also found in eukaryotes. The mechanism by which energy is coupled to the accumulation of substrate by these transport systems has been controversial. In this paper we demonstrate that ATP hydrolysis occurs in vivo concomitantly with transport. These data strongly suggest that ATP hydrolysis directly energizes substrate accumulation by these transport systems. The apparent stoichiometry is one to two molecules of ATP hydrolyzed per molecule of substrate transported.
Search for deep slabs in the Northwest Pacific mantle
Authors: Zhou Hua-wei , Anderson Don L.
Published: May 2006

A residual sphere is formed by projecting seismic ray travel-time anomalies, relative to a reference Earth model, onto an imaginary sphere around an earthquake. Any dominant slab-like fast band can be determined with spherical harmonic expansion. The technique is useful in detecting trends associated with high-velocity slabs beneath deep earthquakes after deep-mantle and near-receiver effects are removed. Two types of corrections are used. The first uses a tomographic global mantle model; the second uses teleseismic station averages of residuals from many events over a large area centered on the events of interest. Under the Mariana, Izu-Bonin, and Japan trenches, the dominant fast bands are generally consistent with seismicity trends. The results are unstable and differ from the seismicity trend for Kurile events. The predominant fast band for most deep earthquakes under Japan is subhorizontal rather than near vertical. We find little support for the deep slab penetration hypothesis.
Stimulation of the expression of osteogenic and chondrogenic phenotypes in vitro by osteogenin.
Authors: Vukicevic S , Luyten F P , Reddi A H
Published: May 2006

Osteogenin was recently purified and the amino acid sequences of tryptic peptides were determined. Osteogenin in conjunction with insoluble collagenous bone matrix induces cartilage and bone formation in vivo. To understand the mechanism of action of osteogenin, we examined its influence on periosteal cells, osteoblasts, fibroblasts, chondrocytes, and bone marrow stromal cells in vitro. Osteogenin stimulated alkaline phosphatase activity and collagen synthesis in periosteal cells. The cAMP response to parathyroid hormone in periosteal cells was increased by osteogenin. In primary cultures of calvarial osteoblasts, osteogenin stimulated alkaline phosphatase activity, the cAMP response to parathyroid hormone, and the synthesis of collagenous and noncollagenous proteins; however, cell proliferation was not affected. Osteogenin increased the production of sulfated proteoglycans in fetal rat chondroblasts and in rabbit articular chondrocytes. The present experiments demonstrate the significant influence of osteogenin in the stimulation of osteogenic and chondrogenic phenotypes in vitro.
Animal model for ultraviolet radiation-induced melanoma: platyfish-swordtail hybrid.
Authors: Setlow R B , Woodhead A D , Grist E
Published: May 2006

Sunlight exposure is strongly indicated as one of the important etiologic agents in human cutaneous malignant melanoma. However, because of the absence of good animal models, it has not been possible to estimate the wavelengths or wavelength regions involved. We have developed a useful animal model from crosses and backcrosses of platyfish (Xiphophorus maculatus) and swordtails (Xiphophorus helleri). Two strains of these fish are susceptible to invasive melanoma induction by exposure to filtered radiation from sunlamps in the wavelength ranges lambda greater than 290 nm and lambda greater than 304 nm. Multiple exposures on 5-20 consecutive days beginning on day 5 after birth or a single exposure of approximately 200 J/(m2.day) of lambda greater than 304 nm result in a tumor prevalence of 20% to 40% at 4 months of age compared with a background rate of 12% in one strain and 2% in another. Exposure of the fish to visible light after UV exposure reduces the prevalence to background. The melanomas are similar in many respects to mammalian melanomas, as judged by light and electron microscopy. The genetics of the crosses determined by others and the high sensitivity of the hybrids to melanoma induction indicate that the UV radiation probably inactivates the one tumor repressor gene (or a small number of tumor repressor genes) in the hybrid fish. The small size of the animals and their high susceptibility to melanoma induction make them ideal for action spectroscopy.
Tumor suppression involves down-regulation of interleukin 3 expression in hybrids between autocrine mastocytoma and interleukin 3-dependent parental mast cells.
Authors: Diamantis I D , Nair A P , Hirsch H H , Moroni C
Published: May 2006

Interleukin 3 (IL-3)-dependent PB-3c mouse mastocytes can be transformed by the v-Ha-ras oncogene to generate autocrine IL-3-producing mastocytomas. Hybrid cell lines were constructed by fusing an IL-3-producing mastocytoma cell line with its IL-3-dependent normal parental cell. Unlike the mastocytoma parent cell line, hybrid cell lines required growth factor for in vitro proliferation, indicating that the IL-3-dependent phenotype is dominant. IL-3 mRNA, expressed at high levels in the tumor cells, appeared down-regulated in the cell hybrids. In contrast, p21v-Ha-ras levels were not reduced in the hybrids. The hybrid lines generated tumors in vivo with drastically prolonged latency times when compared to the tumor parent (10 versus 2 weeks). We propose that down-regulation of IL-3 mRNA production after cell fusion is responsible for the loss of growth autonomy in the hybrids and is likely to play a role in the partial suppression of tumor formation in vivo. Our data are consistent with the hypothesis that a tumor suppressor, present in PB-3c cells, acts as a negative regulator of IL-3 expression.
Chromosome-breaking structure in maize involving a fractured Ac element.
Authors: Ralston E , English J , Dooner H K
Published: May 2006

Chromosome breakage in maize can result from an interaction between certain transposable elements. When an Ac (Activator) element and a state I Ds (Dissociation) element are present together in the genome, either linked or unlinked, breaks occur regularly at the locus of the Ds element. We show here that breaks occur with high frequency at or near the locus of a structure consisting of a 2.5-kilobase (kb) terminally deleted or fractured Ac element very tightly linked to a second, intact 4.6-kb Ac element. This structure has the features of a macrotransposon and may behave like one. Loss of the tight linkage abolishes chromosome breakage. A model based on transposition of the macrotransposon is proposed to explain the chromosome-breaking properties of Ac and Ds.
v-src induces clonal sarcomas and rapid metastasis following transduction with a replication-defective retrovirus.
Authors: Stoker A W , Sieweke M H
Published: May 2006

v-src is an effective carcinogen when expressed from Rous sarcoma virus (RSV) in vivo. Whereas RSV tumors require sustained oncogene expression, their growth is largely a balance between viral recruitment of tissues and host immune destruction of infected cells. We have therefore examined the tumorigenic potential of v-src in the absence of viral recruitment and viral antigen expression. v-src was introduced with high efficiency into chicken wing web tissues using replication-defective (rd) retroviral vectors. Clonal sarcomas were induced rapidly, and, furthermore, v-src potentiated metastatic progression in approximately 0.1%-1% of tumor clones with unexpectedly short latency. rd vectors proved effective not only in transducing v-src into tissues but also as insertional markers of tumor clonality. The rd vector present in most primary and metastatic tumors was a highly truncated form of RSV derived by viral transmission of spliced v-src mRNA; this vector should thus avoid viral recruitment and host anti-viral immune reaction through its complete lack of viral structural genes. Under such conditions v-src maintains strong carcinogenicity in vivo when restricted to clonal tumor growth and can confer rapid metastatic potential on a discrete subset of tumor clones.
Isolation, partial amino acid sequence, and immunohistochemical localization of a brain-specific calcium-binding protein.
Authors: Winsky L , Nakata H , Martin B M , Jacobowitz D M
Published: May 2006

A calcium-binding protein (protein 10) having a molecular mass of 29 kDa and an isoelectric point of 5.3 was purified from guinea pig brain. The amino acid sequence of fragments from proteolytic digestion of protein 10 revealed an 86% sequence identity with a calcium-binding protein (calretinin) found in chicken retina. Polyclonal antibodies against protein 10 revealed a specific distribution of this protein within sensory neurons of auditory, visual, olfactory, nociceptive, and gustatory systems as well as other discrete neuronal circuits in rat and guinea pig brain, whereas no specific label was observed in any of several peripheral tissues examined.
Characterization of the mouse insulin receptor gene promoter.
Authors: Sibley E , Kastelic T , Kelly T J , Lane M D
Published: May 2006

The 5' flanking region of the mouse insulin proreceptor gene was isolated, and the 5' boundary of the minimal promoter was mapped. Genomic clones encompassing greater than 30 kilobases of the gene contain the promoter and exons 1 and 2 interrupted by an approximately 20-kilobase intron at the codon for amino acid 7 of the alpha subunit. The nucleotide sequence of a 1.3-kilobase fragment containing 766 base pairs of the 5' flanking region and the entire first exon was determined. Two major transcription start sites were mapped by S1 nuclease analysis to sites located 469 and 424 nucleotides upstream from the initiation codon for translation. The 5' terminus of an insulin proreceptor cDNA, isolated from a mouse 3T3-L1 adipocyte cDNA library, corresponds to the 3'-most major start site of transcription. The 5' deletion mutants of the 5' flanking region of the proreceptor gene, linked upstream of the bacterial chloramphenicol acetyltransferase reporter gene, were transfected into 3T3-L1 preadipocytes and assayed for promoter activity. The 5' boundary of the minimal promoter, which directs unexpectedly high levels of reporter gene expression, maps to a region 22 base pairs upstream from the 3'-most major transcription start site.
Growth restriction of influenza A virus by M2 protein antibody is genetically linked to the M1 protein.
Authors: Zebedee S L , Lamb R A
Published: May 2006

The M2 protein of influenza A virus is a 97-amino acid integral membrane protein expressed at the surface of infected cells. Recent studies have shown that a monoclonal antibody (14C2) recognizes the N terminus of M2 and restricts the replication of certain influenza A viruses. To investigate the mechanism of M2 antibody growth restriction, 14C2 antibody-resistant variants of strain A/Udorn/72 have been isolated. Most of the variant viruses are not conventional antigenic variants as their M2 protein is still recognized by the 14C2 antibody. A genetic analysis of reassortant influenza viruses prepared from the 14C2 antibody-resistant variants and an antibody-sensitive parent virus indicates that M2 antibody growth restriction is linked to RNA segment 7, which encodes both the membrane protein (M1) and the M2 integral membrane protein. Nucleotide sequence analysis of RNA segment 7 from the variant viruses predicts single amino acid substitutions in the cytoplasmic domain of M2 at positions 71 and 78 or at the N terminus of the M1 protein at residues 31 and 41. To further examine the genetic basis for sensitivity and resistance to the 14C2 antibody, the nucleotide sequences of RNA segment 7 of several natural isolates of influenza virus have been obtained. Differences in the M1 and M2 amino acid sequences for some of the naturally resistant strains correlate with those found for the M2 antibody variant viruses. The possible interaction of M1 and M2 in virion assembly is discussed.
DNA binding specificity of the Arc and Mnt repressors is determined by a short region of N-terminal residues.
Authors: Knight K L , Sauer R T
Published: May 2006

The Arc and Mnt repressors of phage P22 are related proteins that bind to different operator DNA sites. By creating a hybrid Arc-Mnt protein, we show that the binding specificity of Mnt can be switched to that of Arc by replacing six residues at the N terminus of Mnt with the corresponding nine residues from Arc.
Localization to the inner surface of the cytoplasmic membrane by immunoelectron microscopy of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.
Authors: Ghosh B K , Owens K , Pietri R , Peterkofsky A
Published: May 2006

The phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli constitutes a major pathway for sugar translocation. It is composed of integral membrane proteins (enzyme II components) that recognize specific extracellular sugars as well as phosphocarrier proteins, one of which is called enzyme I. While enzyme I plays a role in energizing the enzyme II for sugar transfer, its precise cellular distribution had not previously been defined. This study was designed to elucidate the cellular location of this protein by immunoelectron microscopy. Enzyme I antibody bound to E. coli cryosections was visualized with protein A-gold. The gold particles in sections of wild-type E. coli were found primarily associated with the surface of the inner membrane. A strain of E. coli harboring a plasmid encoding the gene for enzyme I was also tested for its distribution of enzyme I. Consistent with the biochemically established overproduction of enzyme I, this strain showed an approximately 80-fold higher density of gold particles per unit cell volume than the wild-type cells. The substantial overproduction of immunoreactive enzyme I was associated with a significant (approximately 20-fold) increase in the amount of that protein bound to the inner membrane. In addition, a substantial fraction of the total enzyme I accumulated within a 60-nm-wide zone in the vicinity of the inner membrane. A model to explain the zonal distribution of enzyme I under conditions of overexpression of the protein is presented.
Mutants of human colon adenocarcinoma, selected for thymidylate synthase deficiency.
Authors: Houghton P J , Germain G S , Hazelton B J , Pennington J W , Houghton J A
Published: May 2006

GC3/c1 human colon adenocarcinoma cells were treated with the mutagen ethyl methanesulfonate, and three clones deficient in thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1. 45) activity were selected and characterized. Growth in medium deficient in thymidine caused cell death in two clones (TS- c1 and TS- c3), whereas one clone (TS- c2) showed limited growth. Growth correlated with thymidine synthase activity and 5-fluoro-2'-deoxyuridine 5'-monophosphate-binding capacity and with incorporation of 2'-deoxy[6-3H]uridine into DNA. In the presence of optimal thymidine, growth rates were only 5-18% that of the parental clone (GC3/c1), which grew equally well in thymidine-deficient or -replete medium. Analysis of poly(A)+ RNA showed normal levels of a 1.6-kilobase transcript in TS- c1 and TS- c2 but decreased levels (approximately 6% control) in TS- c3. Clone TS- c3 was 32-, 750-, and greater than 100,000-fold more resistant than the parental clone to 5-fluorouracil, 5-fluoro-2'-deoxyuridine, and methotrexate, respectively. When inoculated into athymic nude mice, each TS- clone produced tumors, demonstrating continued ability to proliferate in vivo.
jun-D: a third member of the jun gene family.
Authors: Ryder K , Lanahan A , Perez-Albuerne E , Nathans D
Published: May 2006

The protooncogene c-jun encodes a component of the transcription factor AP-1. Both murine c-jun and a related gene (jun-B) are rapidly activated in BALB/c3T3 cells by serum growth factors. We report here the cloning and analysis of a cDNA encoding a third member of the murine jun family, jun-D. The amino acid sequence encoded by jun-D has two extensive regions of homology with the other Jun proteins. One homology region includes the DNA-binding domain and sequences required for dimer formation and interaction with the Fos oncoprotein; the other includes the acidic sequence thought to be involved in gene activation. All three jun mRNAs are present in a variety of murine tissues and cell lines. In resting 3T3 cells, jun-D is expressed at a higher level compared to c-jun and jun-B, and its transcription is stimulated only slightly by serum growth factors. Thus, jun-D appears to be regulated differently than c-jun and jun-B.
Conjugative transfer of promiscuous IncP plasmids: interaction of plasmid-encoded products with the transfer origin.
Authors: Fürste J P , Pansegrau W , Ziegelin G , Kröger M , Lanka E
Published: May 2006

To characterize protein-DNA interactions involved in the initiation of conjugative transfer replication, we isolated and sequenced the transfer origins (oriT) of the promiscuous IncP plasmids RP4 and R751. The central initiating event at the transfer origin of a conjugative plasmid is the cleavage at a unique site (nic) of the strand to be transferred to a recipient cell. This process can be triggered after the assembly of "relaxosomes" (plasmid DNA-protein relaxation complexes), requiring plasmid-encoded gene products. We analyzed the nicking reaction for plasmid RP4 and demonstrated that one of the plasmid strands is specifically cleaved within oriT. The fully functional oriT of RP4 represents an intergenic DNA region of approximately 350 base pairs. Dissection of oriT revealed that a portion carrying nic and symmetric sequence repeats determines oriT specificity. This part of oriT is contiguous to a region that is essential for efficient mobilization of oriT plasmids. In addition, oriT contains potential promoter sites allowing divergent transcription of two operons flanking oriT. We over-produced gene products and, from analyzing the products of defined deletion mutants, deduced the gene arrangements. Formation of RP4 relaxosomes is likely to depend on the presence of at least two plasmid-encoded components, which act in trans. Corresponding genes map on one side of oriT. Purification of the traJ product revealed it to be an 11-kDa polypeptide that binds to oriT DNA in vitro. The protein recognizes the part of oriT that is responsible for oriT specificity.
Transactivation of both early and late simian virus 40 promoters by large tumor antigen does not require nuclear localization of the protein.
Authors: Wildeman A G
Published: May 2006

The early gene product of simian virus 40, large tumor antigen (T antigen), is required for the onset of viral replication. This protein has also been reported to transactivate viral late gene expression, independently of replication. In this study I have used a vector that permits simultaneously a precise quantitation of simian virus 40 early and late promoter activity with a single nuclease S1 mapping probe. The results show that T antigen can activate the early promoter as well as the late promoter and that only on replicating templates does a shift occur in the ratio of late-to-early transcription. This simultaneous transactivation of early and late promoters occurs in human (HeLa) and monkey (CV-1) cells but does not occur in mouse embryonal carcinoma cells. It is seen with either wild-type T antigen or with a T antigen protein that carries a mutation in the nuclear localization signal. The mutant protein cannot bring about an early-to-late shift, consistent with its inability to support viral replication.
In vitro duplication and in vivo cure of mast-cell deficiency of Sl/Sld mutant mice by cloned 3T3 fibroblasts.
Authors: Fujita J , Onoue H , Ebi Y , Nakayama H , Kanakura Y
Published: May 2006

Sl/Sld mutant mice are profoundly deficient in tissue mast cells as a result of a defect in the microenvironment promoting the development of these cells. To facilitate the analysis of the Sl mutation, we attempted to establish an in vitro system in which the in vivo defect of Sl/Sld mice could be reproduced. 3T3 cell lines were established from 17-day-old embryos of Sl/Sld and congenic +/+ genotypes and were cocultured with mast cells obtained in vitro from the bone marrow of +/+ mice. All eight 3T3 cell lines derived from +/+ of T-cell-derived growth factors. By contrast, none of eight 3T3 cell lines from Sl/Sld embryos supported mast cells under similar conditions. The defect in Sl/Sld 3T3 cells was further characterized as a failure to induce the G1-to-S transition in synchronized mast cells upon contact, suggesting that the Sl gene product is indispensable for this activity. When 3T3 cells of +/+ genotype, grown on pieces of cellulose acetate membrane, were transplanted into the peritoneal cavity of Sl/Sld mice, mast cells appeared locally in the transplanted 3T3 cell layers. These results suggested an essential role of fibroblasts in vivo as the tissue microenvironment promoting the development of mast cells and that they are defective in Sl/Sld mice. The present coculture system duplicated mast-cell deficiency of Sl/Sld mice in vitro and should prove useful for analysis of the Sl gene product.
Estimating the superiority of a drug to a placebo when all and only those patients at risk are treated with the drug.
Authors: Robbins H , Zhang C H
Published: May 2006

It is shown that, under certain assumptions, one can estimate the difference between the effect of a treatment and that of a placebo even when the treatment has been given to all and only those patients who are at risk (as evidenced by a screening examination).
Decreased susceptibility of a 70-kDa protein to cathepsin L after phosphorylation by protein kinase C.
Authors: Laumas S , Abdel-Ghany M , Leister K , Resnick R , Kandrach A , Racker E
Published: May 2006

A 70-kDa protein is phosphorylated in cell-free preparations from rat or mouse fibroblasts by an endogenous protein kinase. This protein is immunologically related to a group of 68-kDa to 87-kDa proteins described in the literature as substrates for protein kinase C (PK-C). Although the phosphorylation of the 70-kDa protein by isolated plasma membranes takes place in the presence of EGTA, we conclude that the reaction is catalyzed by PK-C based on its inhibition by staurosporin. As shown previously, pure PK-C phosphorylates a synthetic random polymer of arginine and serine in the absence of Ca2+ and lipids, a reaction markedly stimulated by an endogenous unidentified activator of PK-C. When the 70-kDa protein from normal fibroblasts was exposed to the cytosol of chemically or ras-transformed fibroblasts, it disappeared as measured by phosphorylation by added PK-C. Cytosol of normal fibroblasts was much less effective (ca. 20%). Cathepsin L purified from rat kidney or from the medium of transformed cells had an effect similar to that of the cytosol of transformed cells. When the 70-kDa protein was phosphorylated by PK-C prior to exposure to cathepsin L or to the cytosol of transformed cells, there was a marked protection of the 70-kDa protein. We conclude that the 70-kDa protein is degraded by cathepsin L as ascertained by both immunological and biochemical assays and that it is protected by prior phosphorylation with PK-C. The possible role of this effect in signal transduction is discussed.
The virD operon of Agrobacterium tumefaciens Ti plasmid encodes a DNA-relaxing enzyme.
Authors: Ghai J , Das A
Published: May 2006

The virD locus of Agrobacterium tumefaciens Ti plasmid encodes functions necessary for endonucleolytic cleavage of transferred DNA (T-DNA) prior to its transfer to plant cells. For the overproduction of the VIRD proteins in Escherichia coli a tac-virD operon fusion was constructed. A significant increase in the accumulation of VIRD proteins was observed in a lon protease-deficient E. coli host. The presence of an overlapping open reading frame (ORF) upstream of the VIRD1 coding sequence had a negative effect on VIRD1 production. A preparation containing VIRD proteins catalyzes the conversion of supercoiled (form I) DNA to relaxed (form IV) DNA. This activity is similar to that of a DNA topoisomerase. The relaxation activity lacks DNA sequence specificity, requires magnesium ion, and has no requirement for an energy source. Studies with plasmids that had lost defined DNA segments encompassing various virD coding regions showed that VIRD1 is the DNA-relaxing enzyme. In a density gradient centrifugation experiment, the DNA-relaxing activity sedimented as a 21-kDa polypeptide. Earlier studies of Jayaswal et al. [Jayaswal, R., Veluthambi, K., Gelvin, S. & Slightom, J. (1987) (J. Bacteriol. 169, 5035-5045] have shown that in E. coli VIRD2 alone is not sufficient for endonucleolytic cleavage of T-DNA and requires VIRD1 for its activity.
Inhibition of insulin receptor phosphorylation by peptides derived from major histocompatibility complex class I antigens.
Authors: Hansen T , Stagsted J , Pedersen L , Roth R A , Goldstein A , Olsson L
Published: May 2006

Peptides from the alpha 1 region (residues 61-85) of the D and K molecules of the major histocompatibility complex class I antigens inhibit insulin-induced tyrosine kinase activity of the purified human insulin receptors (IRs) as measured both by autophosphorylation and IR-mediated substrate [poly(Glu,Tyr)] phosphorylation. Half-maximal effect of the Dk-(61-85) peptide on IR autophosphorylation is obtained at 1.2 microM, and almost complete inhibition of IR kinase activity is obtained at 10 microM peptide. The corresponding K kappa-(61-85) peptide has a significantly weaker effect on autophosphorylation. No such effects are observed with nine peptides of similar length, but unrelated to major histocompatibility complex class I antigens. Neither of the major histocompatibility complex class I-derived peptides has any effect on the constitutively active kinase of a genetically engineered cytoplasmic IR domain. Further, insulin binding to IR is unaltered in the presence of the major histocompatibility complex class I-derived peptides. The inhibitory activity of the peptides on insulin-induced IR phosphorylation facilitated the observation that IRs require insulin to become substrate for an independent tyrosine kinase. In the presence of an inhibitory peptide, the constitutively active cytoplasmic IR kinase domain only phosphorylates the intact IR in the presence of insulin. We conclude that the tyrosine kinase activity of IRs may be altered by peptide interaction at an allosteric site and, moreover, IRs require insulin to assume a conformation permitting phosphorylation by an independent kinase.
denV gene of bacteriophage T4 restores DNA excision repair to mei-9 and mus201 mutants of Drosophila melanogaster.
Authors: Banga S S , Boyd J B , Valerie K , Harris P V , Kurz E M , de Riel J K
Published: May 2006

The denV gene of bacteriophage T4 was fused to a Drosophila hsp70 (70-kDa heat shock protein) promoter and introduced into the germ line of Drosophila by P-element-mediated transformation. The protein product of that gene (endonuclease V) was detected in extracts of heat-shocked transformants with both enzymological and immunoblotting procedures. That protein restores both excision repair and UV resistance to mei-9 and mus201 mutants of this organism. These results reveal that the denV gene can compensate for excision-repair defects in two very different eukaryotic mutants, in that the mus201 mutants are typical of excision-deficient mutants in other organisms, whereas the mei-9 mutants exhibit a broad pleiotropism that includes a strong meiotic deficiency. This study permits an extension of the molecular analysis of DNA repair to the germ line of higher eukaryotes. It also provides a model system for future investigations of other well-characterized microbial repair genes on DNA damage in the germ line of this metazoan organism.
Differences in fatty acid composition between vegetative cells and N
2
-fixing vesicles of
Frankia
sp. strain CpI1
Authors: Tunlid Anders , Schultz Nancy A. , Benson David R. , Steele D. Bernie , White David C.
Published: May 2006

When growing on N
2
, actinomycetes from the genus
Frankia
form multicellular structures that contain nitrogenase. The structures are referred to as vesicles and are indistinguishable from vesicles formed when
Frankia
sp. are in root-nodule symbioses. Vesicles isolated from N
2
-grown cells of
Frankia
sp. strain CpI1 had a significantly higher amount and different composition of fatty acids than did vegetative cells recovered from NH
4
+
-containing medium. Lipids from vesicles, whole cells grown on N
2
, and whole cells grown on NH
4
+
were fractionated by silicic acid chromatography into neutral lipids, glycolipids, and polar lipids. The fatty acids were transesterified by methanolysis and analyzed by gas chromatography and mass spectrometry. Vesicles had considerably higher amounts of fatty acids in the neutral and glycolipid fractions but lower amounts of polar lipid fatty acids than did vegetative cells. Polar lipids from vesicles had a higher proportion of mono-unsaturated and cyclopropane fatty acids and a lower proportion of isobranched fatty acids than did polar lipids from NH
4
+
-grown or N
2
-grown cells. The neutral lipid and glycolipid fractions contained several long-chain compounds with molecular ions at
m/z
408 and 410. The proportions of these compounds were significantly higher in the lipids from vesicles than from vegetative cells. These results suggest that lipids in vesicles might be involved in the protection of nitrogenase from O
2
and suggest a parallel with the glycolipids involved in protecting nitrogenase from O
2
in the cyanobacterial heterocysts.
Major cold shock protein of Escherichia coli.
Authors: Goldstein J , Pollitt N S , Inouye M
Published: May 2006

When exponentially growing Escherichia coli cell cultures were transferred from 37 degrees C to 10 degrees C or 15 degrees C, the production of a 7.4-kDa cytoplasmic protein (CS7.4) was prominently induced. The rate of CS7.4 production reached 13% of total protein synthesis within 1-1.5 hr after a shift to 10 degrees C and subsequently dropped to a lower basal level. Regulation of CS7.4 expression was very strict, such that synthesis of the protein was undetectable at 37 degrees C. We have cloned the gene encoding this protein and have completed the nucleotide sequence analysis, which revealed that the gene encodes a hydrophilic protein of 70 amino acid residues.
Selective alterations of RNA in rat hippocampus after entorhinal cortex lesioning.
Authors: Poirier J , May P C , Osterburg H H , Geddes J , Cotman C , Finch C E
Published: May 2006

In vitro translation products from RNA of rat hippocampus after deafferentation by entorhinal cortex lesions were analyzed by two-dimensional gel electrophoresis. Although hippocampal total RNA yield was not affected 14 days after the lesion, analysis of the gels showed reproducible changes in the steady-state level of several transcripts. Glial fibrillary acidic protein RNA increased 2-fold over control hippocampi RNA. Moreover, seven other transcripts of unknown identity had increased prevalence in the denervated hippocampus. The changes, which ranged from 2- to 20-fold, involved mRNA encoding small slightly acidic polypeptides: 12 kDa (pI 5.6), 13 kDa (pI 6.1), 20 kDa (pI 5.8), 31 kDa (pI 5.7), 33 kDa (pI 5.7), 35 kDa (pI 5.6), and 53 kDa (pI 5.4). These results suggest new molecular markers for analyzing the complex mechanisms of synaptic reorganization in the dentate gyrus after deafferentation.
Innervation patterns of single physiologically identified geniculocortical axons in the striate cortex of the tree shrew.
Authors: Fitzpatrick D , Raczkowski D
Published: May 2006

We examined the termination patterns of single geniculocortical axons in the striate cortex of the tree shrew by using intracellular recording and horseradish peroxidase staining methods. Axons were classified by whether they responded to light onset (ON center) or light offset (OFF center) and whether they were driven by the ipsi- or contralateral eye. Afferents with ON-center responses end in the upper part of layer IV (IVa) whereas afferents with OFF-center responses end in the lower part of layer IV (IVb). Within each tier, axons driven by the ipsilateral and contralateral eye overlap. These results suggest that binocular convergence occurs within layer IV without mixing the information from the ON- and OFF-center pathways and we consider the significance of this arrangement for visual cortical function.
The N-terminal region of the human immunodeficiency virus envelope glycoprotein gp120 contains potential binding sites for CD4.
Authors: Syu W J , Huang J H , Essex M , Lee T H
Published: May 2006

Human immunodeficiency virus (HIV) vaccines targeted at blocking HIV-CD4 interactions are expected to be less affected by the sequence heterogeneity of HIV than those targeted at variable regions of the envelope outercoat glycoprotein, gp120. All potential CD4 binding sites identified thus far in HIV are localized in the C-terminal region of gp120. In this study we demonstrate that the N-terminal region of gp120 also contains conserved residues critical for binding to CD4 and that gp120-CD4 interactions can be blocked by an antiserum with binding specificity to an N-terminal region of gp120. These results suggest that not all potential CD4 binding sites are present in the C-terminal region of gp120 and that an alternative HIV vaccine development strategy may have to include the N-terminal gp120 region as a component to raise effective CD4-blocking antibodies.
Isolation of cellular promoters by using a retrovirus promoter trap.
Authors: von Melchner H , Reddy S , Ruley H E
Published: May 2006

A retrovirus vector has been used to isolate transcriptional promoters from mammalian cells. The virus contains a selectable gene encoding histidinol dehydrogenase (his) in the U3 region of the 3' long terminal repeat (LTR). When the virus is passaged, duplication of LTRs places his sequences just 30 nucleotides from the adjacent cellular DNA. As a result, selection for histidinol resistance generates cell clones in which his is expressed on transcripts initiating in the flanking cellular DNA. Upstream cellular sequences, cloned after amplification by polymerase chain reaction, hybridized to RNA from uninfected cells, indicating that the adjacent promoters were transcriptionally active prior to virus integration. Two cloned transcribed flanking sequences also contained highly active transcriptional promoters, as estimated by their ability to activate expression of a linked reporter gene. Thus, U3His vectors provide a rapid and efficient means to isolate promoters active in different cell types. Moreover, by selecting for cell clones containing proviruses integrated in expressed genes, the virus may make an effective insertional mutagen.
Autogenous regulation of the Bordetella pertussis bvgABC operon.
Authors: Roy C R , Miller J F , Falkow S
Published: May 2006

The bvgABC operon of the bacterial pathogen Bordetella pertussis encodes a sensory transduction system that regulates the expression of several virulence genes in response to environmental stimuli. In this study we have examined the transcriptional regulation of the bvgABC operon. Transcriptional bvg::lacZYA fusions in Escherichia coli show that the bvgABC operon is autogenously activated. Autoactivation is inhibited by the same environmental stimuli that result in the lack of expression of bvg-activated genes in B. pertussis. These observations were confirmed in B. pertussis using a chromosomal chloramphenicol acetyltransferase transcriptional fusion in bvgC. Transcriptional initiation sites upstream of bvgA were mapped by primer extension analysis in E. coli and B. pertussis. Two differentially regulated bvg promoters were identified. The bvgP1 promoter is a positively autoregulated promoter located 90 base pairs upstream of bvgA. The bvgP2 promoter is located 141 base pairs upstream of bvgA and does not appear to require any positive regulatory factors for activity. These results suggest a model describing the regulatory events that take place upstream of the bvgABC operon.
Ultrasensitivity and heavy-metal selectivity of the allosterically modulated MerR transcription complex.
Authors: Ralston D M , O'Halloran T V
Published: May 2006

The MerR metalloregulatory protein is a heavy-metal receptor that functions as the repressor and Hg(II)-responsive transcription activator of the prokaryotic mercury-resistance (mer) genes. We demonstrate that this allosterically modulated regulatory protein is sensitive to HgCl2 concentrations of 1.0 +/- 0.3 x 10(-8) M in the presence of 1.0 x 10(-3) M dithiothreitol for half-maximal induction of transcription of the mer promoter by Escherichia coli RNA polymerase in vitro. Transcription mediated by MerR increases from 10% to 90% of maximum in response to a 7-fold change in concentration of HgCl2, consistent with a threshold phenomenon known as ultrasensitivity. In addition, MerR exhibits a high degree of selectivity. Cd(II), Zn(II), Ag(I), Au(I), and Au(III) have been found to partially stimulate transcription in the presence of MerR, but concentrations at least two to three orders of magnitude greater than for Hg(II) are required. The molecular basis of the ultrasensitivity and selectivity phenomena are postulated to arise from the unusual topology of the transcription complex and a rare trigonal mercuric ion coordination environment, respectively. This mercuric ion-induced switch is to our knowledge the only known example of ultrasensitivity in a signal-responsive transcription mechanism.
Discrimination between activators and nonactivators of the alternative pathway of complement: regulation via a sialic acid/polyanion binding site on factor H.
Authors: Meri S , Pangburn M K
Published: May 2006

The alternative complement pathway is capable of discriminating human cells and tissues from a wide variety of potential pathogens. It has been recently demonstrated that attachment of complement component C3b to activator-derived molecules (e.g., small polysaccharides) restricts inactivation of C3b by factors H and I in a manner similar to activator surfaces. It is now shown that restriction is reversed by certain soluble polyanions (e.g., sialoglycopeptides, heparin, or dextran sulfate) that mimic the effects of sialic acid and glycosaminoglycans on human cells and tissues. Fluid-phase polyanions enhanced binding of factor H to C3b attached to activating particles, indicating that the effect resulted from increased affinity between C3b and factor H. The enhancement was specific for activator-bound C3b since no enhancement was observed on nonactivating particles. While several polyanions could cause this effect, some polyanions could not, indicating specificity. The active polyanions also inhibited lysis of cells via the alternative pathway. The binding site for sialic acid appears to reside on factor H, since factor H bound to heparin-agarose and to sialic acid-bearing fetuinagarose, whereas C3b bound to neither under the same conditions. These observations suggest that occupation of a specific site on factor H by polyanions induces an increase in the C3b-H affinity, resulting in discrimination of host cells and tissues from alternative pathway-activating foreign cells.