bluuebunny/crossref_metadata_2025 huggingface dataset:
Global control in Pseudomonas fluorescens mediating antibiotic synthesis and suppression of black root rot of tobacco.
Authors: Laville J , Voisard C , Keel C , Maurhofer M , Défago G , Haas D
Published: May 2006

Pseudomonas fluorescens CHA0 colonizes plant roots, produces several secondary metabolites in stationary growth phase, and suppresses a number of plant diseases, including Thielaviopsis basicola-induced black root rot of tobacco. We discovered that mutations in a P. fluorescens gene named gacA (for global antibiotic and cyanide control) pleiotropically block the production of the secondary metabolites 2,4-diacetylphloroglucinol (Phl), HCN, and pyoluteorin. The gacA mutants of strain CHA0 have a drastically reduced ability to suppress black root rot under gnotobiotic conditions, supporting the previous observations that the antibiotic Phl and HCN individually contribute to the suppression of black root rot. The gacA gene is directly followed by a uvrC gene. Double gacA-uvrC mutations render P. fluorescens sensitive to UV irradiation. The gacA-uvrC cluster is homologous to the orf-2 (= uvrY)-uvrC operon of Escherichia coli. The gacA gene specifies a trans-active 24-kDa protein. Sequence data indicate that the GacA protein is a response regulator in the FixJ/DegU family of two-component regulatory systems. Expression of the gacA gene itself was increased in stationary phase. We propose that GacA, perhaps activated by conditions of restricted growth, functions as a global regulator of secondary metabolism in P. fluorescens.
Neuronal-type alpha-bungarotoxin receptors and the alpha 5-nicotinic receptor subunit gene are expressed in neuronal and nonneuronal human cell lines.
Authors: Chini B , Clementi F , Hukovic N , Sher E
Published: May 2006

alpha-Bungarotoxin (alpha Bgtx) is a toxin known to interact with muscle nicotinic receptors and with some neuronal nicotinic receptors. We show that alpha Bgtx binding sites are also expressed in nonmuscle and nonneuronal human cells, including small cell lung carcinoma and several epithelial cell lines. These receptors are immunologically related to the alpha Bgtx receptors of unknown function described in the nervous system and in the IMR32 neuroblastoma cell line and are distinct from muscle nicotinic receptors. We have also cloned from IMR32 cells the human alpha 5-nicotinic receptor subunit, which is supposed to participate in the formation of alpha Bgtx receptors. Transcripts corresponding to the alpha 5-subunit gene were found not only in neuroblastoma cells but also in all the cell lines expressing alpha Bgtx receptors, with the exception of the TE671 cell line, whose nicotinic receptor subunits are of the muscle type. We conclude that both alpha Bgtx receptors and the alpha 5-nicotinic subunit gene are not neuron-specific, as previously thought, but are expressed in a number of human cell lines of various origin.
Activation of the hippocampus in normal humans: a functional anatomical study of memory.
Authors: Squire L R , Ojemann J G , Miezin F M , Petersen S E , Videen T O , Raichle M E
Published: May 2006

We studied regional cerebral blood flow using the H2(15)O method while normal subjects performed four similar tasks involving three-letter word beginnings (stems). Prior to each task, subjects studied a list of words. Local blood flow was then monitored during a 40-sec period while subjects (i) silently viewed word stems, (ii) completed stems to form the first words to come to mind, but the stems were not the beginnings of any study words (baseline), (iii) completed stems and half of them could form study words (priming), or (iv) tried to recall study words, and half of the stems could form these words (memory). There were three major findings. (i) The memory task engaged the right hippocampal region when the memory task was compared to either the baseline or the priming condition. The right hemispheric locus suggests that performance is driven by the visual characteristics of the words rather than by semantic or phonetic analysis. (ii) In the priming-minus-baseline comparison, there was reduction in blood flow in the right posterior cortex. (iii) Right prefrontal cortex was activated in the memory-minus-baseline condition. The results provide evidence for selective activation of the human hippocampal region in association with memory function. The results also lead to a suggestion about the neural basis of repetition priming: following presentation of a stimulus, less neural activity is required to process the same stimulus.
Altering the insertional specificity of a Drosophila transposable element.
Authors: Kassis J A , Noll E , VanSickle E P , Odenwald W F , Perrimon N
Published: May 2006

Vectors derived from the Drosophila P element transposon are widely used to make transgenic Drosophila. Insertion of most P-element-derived vectors is nonrandom, but they exhibit a broad specificity of target sites. During experiments to identify cis-acting regulatory elements of the Drosophila segmentation gene engrailed, we identified a fragment of engrailed DNA that, when included within a P-element vector, strikingly alters the specificity of target sites. P-element vectors that contain this fragment of engrailed regulatory DNA insert at a high frequency near genes expressed in stripes.
Cloning and nucleotide sequence of the cDNA encoding human 2-oxoglutarate dehydrogenase (lipoamide).
Authors: Koike K , Urata Y , Goto S
Published: May 2006

2-Oxoglutarate dehydrogenase (lipoamide) (( OGDH: 2-oxoglutarate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-succinylating), EC 1.2.4.2 )) is a component enzyme of the 2-oxoglutarate dehydrogenase complex. We have cloned a human cDNA encoding OGDH from a fetal liver cDNA library by plaque hybridization with a mixture of oligonucleotide probes designed from the amino acid sequences of porcine OGDH. This cDNA spans 4156 bases and contains an open reading frame of 3009 nucleotides encoding a presequence of 40 amino acid residues and a mature protein of 963 amino acid residues (Mr = 108,642). The size of the mRNA is approximately 4.2 kilobases. Comparison of the deduced amino acid sequence of the human OGDH with experimentally determined segments of porcine OGDH comprising 308 amino acid residues shows 93% sequence identity. The human OGDH has 37% sequence identity with 933 amino acid residues of the Escherichia coli OGDH and 40% sequence identity with 1014 residues of the yeast OGDH.
c-fos gene induction by interleukin 2: identification of the critical cytoplasmic regions within the interleukin 2 receptor beta chain.
Authors: Hatakeyama M , Kawahara A , Mori H , Shibuya H , Taniguchi T
Published: May 2006

Interleukin 2 (IL-2) plays a critical role in the growth and differentiation of lymphoid cells. The IL-2 signal is delivered intracellularly by the IL-2 receptor beta chain (IL-2R beta); however, the mechanism by which the signal reaches the nucleus remains unclear. In this study, we demonstrate the rapid activation of c-fos protooncogene transcription by IL-2 and provide evidence that the serum-responsive element (SRE) within the c-fos promoter is responsible for the activation in a murine pro-B-cell line, BAF-B03, expressing the human IL-2R beta cDNA. Interestingly, the same SRE is also responsible for c-fos gene activation by interleukin 3 or erythropoietin. Further, we show that the activation of c-fos by IL-2 requires defined cytoplasmic regions of IL-2R beta--i.e., the "serine-rich" region, which is known to be essential for growth-signal transduction in BAF-B03 cells, and the "acidic region," which is located more distal to the cell membrane. These results indicate the functional importance of the two distinct regions within the IL-2R beta cytoplasmic domain in IL-2-induced c-fos gene activation and point to a potential role of the acidic region in IL-2 signal transduction that could not be adequately assessed in a previous study.
A retroviral-like metal binding motif in an aminoacyl-tRNA synthetase is important for tRNA recognition.
Authors: Miller W T , Schimmel P
Published: May 2006

The gag genes of retroviruses encode nucleocapsid proteins that package genomic RNA and are essential for viral infectivity. These RNA binding proteins have a Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys zinc binding motif that is distinct from the typical zinc-finger motif Cys-Xaa2-Cys-Xaa12-14-His-Xaa2-His that is found in some transcriptional activators. Escherichia coli alanyl-tRNA synthetase contains a zinc-binding Cys-Xaa2-Cys-Xaa6-His-Xaa2-His motif that resembles that of retroviral nucleic acid binding proteins. We show here that, for alanyl-tRNA synthetase, the metal bound at the retroviral-like metal binding motif is important specifically for tRNA recognition and not for amino acid activation. Moreover, the enzyme-tRNA interaction is strongly dependent on the geometry of metal coordination to the protein. These and additional experiments collectively suggest a role for the retroviral-like metal binding motif in RNA recognition and, further, raise the possibility that the protein-bound metal itself participates in an RNA interaction.
Cable properties of arborized Retzius cells of the leech in culture as probed by a voltage-sensitive dye.
Authors: Fromherz P , Vetter T
Published: May 2006

Retzius cells of Hirudo medicinalis were cultivated on extracellular matrix protein so that extended arborizations were formed. The propagation of voltage transients along 1-microns-thick neurites was observed at a resolution of 8 microns at 10 kHz by use of a voltage-sensitive dye. Delay and width of the fluorescence transients caused by hyperpolarization of the soma are described by passive spread of voltage in a homogeneous cable (time constant, 10 ms; space constant, 320 microns). The local sensitivity of the dye was determined from a comparison of the amplitudes of fluorescence and of fitted voltage. The fluorescence transients caused by depolarization were scaled using the sensitivity profile. Action potentials were found to pervade the neurites without significant change of amplitude but with enhanced pulse width.
Induction of nitric oxide synthase activity by toxic shock syndrome toxin 1 in a macrophage-monocyte cell line.
Authors: Zembowicz A , Vane J R
Published: May 2006

Toxic shock syndrome toxin 1 (TSST-1) is a Mr 22,000 protein produced by Staphylococcus aureus. It is thought to be the cause of toxic shock syndrome. We investigated the hypothesis that TSST-1 induces nitric oxide (NO) synthase and that the NO formed may be involved in the pathogenesis of toxic shock syndrome. We used the murine monocyte-macrophage cell line J744.2 that responds to TSST-1 and also expresses NO synthase activity upon immunological stimulation. J774.2 macrophages stimulated with TSST-1 (10-100 nM) generated nitrite, a breakdown product of NO, and induced concentration-dependent elevations of cGMP in the pig kidney epithelial cell line (LLC-PK1). This latter effect was due to the generation of L-arginine-derived NO for it was (i) abolished by oxyhemoglobin (10 microM), a scavenger of NO, or by methylene blue (10 microM), an inhibitor of NO-activated guanylate cyclase; (ii) potentiated by superoxide dismutase (100 units/ml), which prolongs the life of NO; (iii) inhibited by NG-monomethyl-L-arginine (0.3 mM), an inhibitor of NO synthase; (iv) significantly decreased when L-arginine (0.4 mM) in the medium was replaced by D-arginine (0.4 mM). Moreover, TSST-1 (100 nM) enhanced the activity of cytosolic NO synthase in J774.2 cells. Hydrocortisone (1 microM) but not indomethacin (5 micrograms/ml) or salicylic acid (5 micrograms/ml) prevented the generation of NO2- and the increases in cGMP levels in LLC-PK1 cells induced by J774.2 cells stimulated with TSST-1. The effects of hydrocortisone were partially reversed by coincubation with RU 486 (1 microM), an antagonist of glucocorticoid receptors. Thus, TSST-1 and perhaps other exotoxins produced by Gram-positive bacteria induce NO synthase and the increased NO formation may contribute to toxic shock syndrome and possibly to changes in the immune responses that accompany infection.
cDNA sequence of a second fibrinogen alpha chain in lamprey: an archetypal version alignable with full-length beta and gamma chains.
Authors: Pan Y , Doolittle R F
Published: May 2006

The message for a second fibrinogen alpha chain has been cloned from a lamprey liver cDNA library. The sequence is unique in that the amino-terminal half is homologous to all other known alpha chains, including another from lamprey, but its carboxyl-terminal half is homologous to the carboxyl-terminal portions of beta and gamma chains, segments that compose the distal globular regions of fibrinogen. The structural pattern of this newly discovered alpha chain suggests that it could be a direct descendant of the archetypal chain that existed prior to the gene duplications that led to unique beta and gamma chains and before the dislocating events that gave rise to contemporary alpha chains.
Cell adhesion molecules as targets for Hox genes: neural cell adhesion molecule promoter activity is modulated by cotransfection with Hox-2.5 and -2.4.
Authors: Jones F S , Prediger E A , Bittner D A , De Robertis E M , Edelman G M
Published: May 2006

In an effort to determine whether homeobox genes modulate the activity of the promoter of the mouse neural cell adhesion molecule (N-CAM) gene, we have carried out a series of cotransfection experiments using NIH 3T3 cells. Plasmids were constructed containing Xenopus laevis Hox-2.5 and -2.4 coding sequences linked to a human cytomegalovirus promoter (CMV-Hox-2.5 and CMV-Hox-2.4). A 4.9-kilobase DNA fragment containing 5' flanking and first exon sequences of the mouse N-CAM gene was linked to a chloramphenicol acetyltransferase (CAT) reporter gene (N-CAM-Pro-CAT). Cotransfection with CMV-Hox-2.5 and N-CAM-Pro-CAT resulted in a strong induction of CAT activity. The N-CAM promoter contained two potential homeodomain binding sites (sites I and II) within a 47-base-pair segment (512-559 base pairs upstream of the ATG codon in the first exon of the N-CAM gene). This segment was linked to a minimal promoter (simian virus 40 early) and a downstream CAT gene. Although this construct was transcriptionally active at a low level in NIH 3T3 cells, cotransfection of CMV-Hox-2.5 resulted in CAT activity that was greatly elevated. Mutational studies revealed that it was the homeodomain binding site II sequence that was required for this regulation. In contrast, cotransfection with CMV-Hox-2.4 eliminated the CAT activity that was driven by the CMV-Hox-2.5 construct. Thus, the products of two related Hox genes, which are located adjacent to each other in the Hox-2 complex, can differentially modulate transcription from the promoter of a cell adhesion molecule gene. The results suggest that the N-CAM gene is likely to be a target for regulation by Hox gene products.
T-cell activation by autologous human T-cell leukemia virus type I-infected T-cell clones.
Authors: Wucherpfennig K W , Höllsberg P , Richardson J H , Benjamin D , Hafler D A
Published: May 2006

A unique feature of both human T-cell leukemia virus type I (HTLV-I) carriers and subjects with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic inflammatory disease of the nervous system, is the presence of large numbers of activated T cells that spontaneously proliferate in vitro. We have investigated the mechanisms of T-cell activation by HTLV-I in freshly isolated blood T cells and in naturally infected T-cell clones obtained by direct single-cell cloning from patients with HAM/TSP. Both CD4+ and CD8+ HTLV-I-infected T-cell clones showed the unusual ability to proliferate in the absence of exogenous interleukin 2 (IL-2). Nevertheless, HTLV-I-infected clones were not transformed, as they required periodic restimulation with phytohemagglutinin and feeder cells for long-term growth. Irradiated or fixed HTLV-I-infected clones were found to induce the proliferation of blood T cells when cocultured, which we refer to as THTLV-1-T cell activation. This THTLV-1-T cell-mediated activation was blocked by monoclonal antibodies (mAbs) against CD2/lymphocyte function-associated molecule 3 (LFA-3), LFA-1/intercellular cell-adhesion molecule (ICAM), and the IL-2 receptor but not by mAbs against class I or class II major histocompatibility complex molecules, HTLV-I gp46, or a high-titer HAM/TSP serum. Spontaneous proliferation of blood T cells from HAM/TSP patients could also be inhibited by mAbs to CD2/LFA-3, LFA-1/ICAM and to the IL-2 receptor (CD25). These results show at the clonal level that HTLV-I infection induces T-cell activation and that such activated T cells can in turn stimulate noninfected T cells by cognate THTLV-1-T cell interactions involving the CD2 pathway.
Isolation of a candidate gene for choroideremia.
Authors: Merry D E , Jänne P A , Landers J E , Lewis R A , Nussbaum R L
Published: May 2006

Choroideremia is an X chromosome-linked retinal dystrophy of unknown pathogenesis. We have isolated cDNAs from a human retinal library with a genomic probe located at the X chromosomal breakpoint in a female with choroideremia and an X;13 translocation. This cDNA spans the breakpoint in the X;13 translocation female and is deleted in males who have choroideremia as part of a complex phenotype including mental retardation and deafness. However, this cDNA detects no alterations in the DNA of 34 males with isolated choroideremia. Nonetheless, the cDNA does detect reduced or absent levels of mRNA in three-quarters of male patients with an apparently intact gene. These data support the hypothesis that this cDNA represents the gene in which mutations cause choroideremia.
Thrombomodulin gene regulation by cAMP and retinoic acid in F9 embryonal carcinoma cells.
Authors: Weiler-Guettler H , Yu K , Soff G , Gudas L J , Rosenberg R D
Published: May 2006

Thrombomodulin (TM) expression was investigated during differentiation of F9 embryonal carcinoma cells into primitive or parietal endoderm. Exposure of F9 cells to retinoic acid (RA) triggers differentiation into primitive endoderm and induces the appearance of barely detectable amounts of TM mRNA, whereas treatment with dibutyryl cAMP plus theophylline (CT) augments the levels of TM mRNA to a 4-fold greater extent than RA. Exposure of F9 cells to RA plus CT initiates differentiation into parietal endoderm and synergistically increases the levels of TM mRNA by 10- to 12-fold compared with CT. The time-dependent establishment of cooperativity between RA and CT appears to be secondary to RA-induced differentiation to primitive endoderm. The above alterations in TM mRNA levels occur by a transcriptional mechanism as judged by nuclear run-on experiments. Transient gene expression experiments show that the human TM promoter is transactivated by coexpression of the human RA receptor beta. Thus, the mechanism of induction of TM expression in F9 cells undergoing differentiation to parietal endoderm appears to be similar, but not identical, to that noted for other late response genes.
Chromosomal position of rearranging gene segments influences allelic exclusion in transgenic mice.
Authors: Costa T E , Suh H , Nussenzweig M C
Published: May 2006

Formation of a complete immunoglobulin heavy-chain transcription unit involves the ordered rearrangement of variable (V), diversity (D), and joining (J) region gene segments. In antibody-producing cells, this process is regulated such that only one of two antibody genes is expressed. Experiments with transgenic mice suggest that this mechanism, known as allelic exclusion, is mediated through the membrane-bound form of the immunoglobulin heavy chain. However, in all transgenic lines produced to date exclusion of the endogenous genes by the transgene is incomplete. To characterize the molecular basis for this escape from regulation, we have examined the rearrangements of endogenous immunoglobulin heavy-chain genes. We find that a transgene that encodes the membrane-bound form of human IgM efficiently inhibits rearrangements of endogenous gene segments located at the 5' end of the heavy-chain locus. However, recombining elements found at the 3' end of the locus escape and continue to undergo recombination. A transgene that encodes the secreted form of the same immunoglobulin protein has no effect on recombination, regardless of position of the recombining segment in the chromosome. These results have important implications for our understanding of the control of allelic exclusion.
Translational frameshifting mediated by a viral sequence in plant cells.
Authors: Brault V , Miller W A
Published: May 2006

It has been proposed that the polymerase gene of barley yellow dwarf virus and related viruses is expressed by a ribosomal frameshift event during translation. The 5' end of this gene overlaps with the 3' end of an upstream gene that is in a different reading frame. The region of overlap is similar to sequences in retro- and coronaviruses that are known to express their polymerase genes by frameshifting. This overlap region includes a "shifty" heptanucleotide, followed by a highly structured region that may contain a pseudoknot. Sequences of 115 or 144 base pairs that span this region from barley yellow dwarf virus (PAV serotype) genomic RNA were introduced into a plasmid, so that a reporter gene could be expressed in plant cells only if a minus one (-1) frameshift event occurred. Frameshifting was detected at a rate of approximately 1%. This frameshifting was abolished when the stop codon at the 3' end of the upstream open reading frame was deleted. A sequence expected to form a strong stem-loop immediately upstream of the frameshift site was unnecessary for frameshifting, and initiation at AUG codons within the stem-loop appeared to be inhibited. Like viruses that infect hosts in other kingdoms, plant viruses also can induce frameshifting in translation of their genes.
Functional expression of human mdr1 in the yeast Saccharomyces cerevisiae.
Authors: Kuchler K , Thorner J
Published: May 2006

Development of multiple drug resistance in tumor cells involves amplification of the mdr1 gene product, a 170-kDa plasma membrane glycoprotein that is an ATP-driven pump that extrudes the drugs. Human mdr1 (also designated as PGY1) cDNA was expressed in yeast cells by using the promoter and translational initiation signal of a related yeast gene, STE6. Immunoblotting of subcellular fractions showed that all of the Mdr1 (also known as P glycoprotein) was associated with the particulate material. Immunofluorescence microscopy revealed that the majority of the Mdr1 was localized to the plasma membrane (although a significant amount was also found in the endoplasmic reticulum). In contrast to mammalian cells, Mdr1 was not glycosylated in yeast. Nevertheless, some, if not all, of the Mdr1 made in yeast was properly folded and functional because it could be photoaffinity labeled specifically with 8-azido-ATP and because cells overexpressing Mdr1 displayed increased resistance towards valinomycin, an ionophore known to interact with Mdr1 in animal cells. Hence, a human polytopic membrane protein was correctly inserted into the yeast plasma membrane, and glycosylation was not required for its function.
A conditional yeast mutant deficient in mRNA transport from nucleus to cytoplasm.
Authors: Kadowaki T , Zhao Y , Tartakoff A M
Published: May 2006

Transport of mRNA from nucleus to cytoplasm is critical for eukaryotic gene expression; however, the mechanism of export is unknown. Selection and screening procedures have therefore been used to obtain a family of temperature-sensitive conditional mutants of Saccharomyces cerevisiae that accumulate poly(A)+ RNA in the nucleus when incubated at 37 degrees C, as judged by in situ hybridization. In one such mRNA transport mutant, mtr1-1, RNA synthesis continues, the export of poly(A)+ RNA is inhibited, intranuclear poly(A)+ is remarkably stable, and protein synthesis gradually stops. Thus, there is no tight coupling between RNA synthesis and export. The export lesion is reversible. Although mRNA export is clearly not a default option, neither inhibition of protein synthesis, inhibition of mRNA splicing, nor inhibition of poly(A)-binding protein function blocks export of the average poly(A)+, as judged by in situ hybridization. Further analysis of the family of mtr mutants should help map the path of RNA transport.
The rat trk protooncogene product exhibits properties characteristic of the slow nerve growth factor receptor.
Authors: Meakin S O , Suter U , Drinkwater C C , Welcher A A , Shooter E M
Published: May 2006

Two distinct nerve growth factor receptor (NGFR) complexes are present on NGF-responsive cell types; these correspond to 100 kDa and 158 kDa for the fast (fNGFR) and the slow (sNGFR) NGFRs, respectively. Previous studies indicate that each complex is derived from a separate gene product and that the sNGFR contains tyrosine kinase activity. The cDNA encoding the fNGFR has previously been cloned. In this report, a rat trk protooncogene cDNA has been isolated from PC12 cells and Trk has been shown to bind NGF, generating a complex of 158 kDa. Characterization of NGF-Trk interactions indicates that Trk and NGF dissociate more slowly than do NGF and the fNGFR. Moreover, NGF-bound Trk is not destroyed by trypsin digestion whereas the NGF-fNGFR complex is sensitive to trypsin digestion. These observations suggest that the trk protooncogene product, expressed in the absence of the fNGFR, binds NGF with properties characteristic of the sNGFR, which was identified as the high-affinity NGFR on primary neurons and PC12 cells.
Differential low density lipoprotein receptor-dependent formation of eicosanoids in human blood-derived monocytes.
Authors: Salbach P B , Specht E , von Hodenberg E , Kossmann J , Janssen-Timmen U , Schneider W J , Hugger P , King W C , Glomset J A , Habenicht A J
Published: May 2006

We studied the ability of low density lipoproteins (LDLs) to provide arachidonic acid (AA) for eicosanoid biosynthesis in human blood-derived monocytes. When incubated in the presence of reconstituted LDL that contained cholesteryl [1-14C]arachidonate (recLDL-[14C]AA-CE), resting monocytes formed three labeled products of the prostaglandin (PG) H synthase pathway: 6-keto-PGF1 alpha, thromboxane B2, and PGE2. The amounts of these eicosanoids in response to recLDL-[14C]AA-CE were comparable to or exceeded those that were produced in response to the addition of 10 microM unesterified [1-14C]AA. By contrast, resting monocytes formed only small amounts of products of the 5-lipoxygenase pathway, leukotriene (LT) B4 and LTC4 from either recLDL-[14C]AA-CE or [14C]AA, indicating preferential utilization of AA in the PGH synthase reaction. However, they converted LDL-derived [14C]AA efficiently into LTB4 and LTC4, when they were first incubated with recLDL-[14C]AA-CE and subsequently stimulated with the chemotactic peptide N-formylmethionylleucylphenylalanine or the Ca2+ ionophore A23187. The classical LDL receptor pathway mediated the synthesis of all of the above eicosanoids from LDL but not from unesterified AA. These results demonstrate that the LDL receptor pathway preferentially promotes the synthesis of PGH synthase products in resting human blood-derived monocytes and that an additional mechanism is required to promote effective synthesis of 5-lipoxygenase pathway products from AA that originates in LDL cholesteryl esters.
Cloning of a D-type cyclin from murine erythroleukemia cells.
Authors: Kiyokawa H , Busquets X , Powell C T , Ngo L , Rifkind R A , Marks P A
Published: May 2006

We report the complete coding sequence of a cDNA, designated CYL2, derived from a murine erythroleukemia cell library. CYL2 is considered to encode a D-type cyclin because (i) there is cross hybridization with CYL1 (a murine homolog of human cyclin D1) and the encoded protein has 64% amino acid sequence identity with CYL1 and (ii) murine erythroleukemia cell-derived CYL2 contains an amino acid sequence identical to that previously reported for the C-terminal portion of a partially sequenced CYL2. Transcripts of murine erythroleukemia cell CYL2 undergo alternative polyadenylylation like that of human cyclin D1. A major 6.5-kilobase CYL2 transcript changes its expression during the cell cycle with a broad peak through G1 and S phases and a decrease in G2/M phases. The present findings suggest that CYL2 plays a role in the G1 to S phase progression.
High frequency of single-base transitions and extreme frequency of precise multiple-base reversion mutations in poliovirus.
Authors: de la Torre J C , Giachetti C , Semler B L , Holland J J
Published: May 2006

We employed independent clones of a temperature-sensitive mutant of type 1 poliovirus, 3AB-310/4, to quantitate the frequency of specific U----C transitions at nucleotide 5310, within the genomic region encoding polypeptide 3AB, which is involved in the initiation of RNA replication. Only this U----C base substitution restores the wild-type phenotypic ability to form plaques at 39 degrees C; the other two base substitutions at this site are lethal. The observed frequency of this specific transition averaged 2 x 10(-5), and all revertant viruses forming plaques at 39 degrees C contained the expected cytidine at nucleotide 5310. Incredibly, only 3 of 10 revertants exhibited this one specific U----C transition whereas 7 of 10 exhibited this same transition plus four additional base substitutions that precisely reverted temperature-sensitive 3AB-310/4 to wild-type poliovirus sequence (these latter four mutations had been introduced into 3AB-310/4 as silent third base mutations to provide new restriction sites in infectious cDNAs). No other mutations were detected in this polypeptide 3AB domain in either the single-base or the precise 5-base revertants. No intermediates were seen; all revertants exhibited either the single U----C transition at nucleotide 5310 or the same transition plus four precise reversions to the wild-type sequence at sites 8, 11, 43, and 46 bases distant from nucleotide 5310. Similar results were obtained after transfection of cDNA-derived transcripts. We discuss possible mechanisms for our data. These include (but may not be limited to) error-prone polymerase activity, sequential RNA recombination events joining independent mutations, or some unusual RNA editing process.
Decreased levels of soluble amyloid beta-protein precursor in cerebrospinal fluid of live Alzheimer disease patients.
Authors: Van Nostrand W E , Wagner S L , Shankle W R , Farrow J S , Dick M , Rozemuller J M , Kuiper M A , Wolters E C , Zimmerman J , Cotman C W
Published: May 2006

The amyloid beta-protein is deposited in senile plaques and the cerebrovasculature in Alzheimer disease (AD). Since it is derived from proteolytic processing of its parent protein, the amyloid beta-protein precursor (APP), we investigated whether levels of the secreted forms of APP are altered in cerebrospinal fluid (CSF) of AD patients. Quantitative immunoblotting studies with the anti-APP monoclonal antibody P2-1 revealed that probable AD patients had markedly lower CSF APP levels than did demented non-Alzheimer-type patients and healthy control subjects. Using antibody P2-1 in an enzyme-linked immunosorbent assay, we measured CSF levels of APP in a larger population consisting of 13 patients diagnosed with probable AD, 18 patients diagnosed with dementia (non-Alzheimer type), and 16 nondemented, healthy controls. Mean CSF levels of APP were approximately 3.5-fold lower in the live patients diagnosed with probable AD compared to the demented non-Alzheimer-type controls or the nondemented, healthy individuals. These findings suggest that abnormal metabolism of APP is reflected in the extracellular fluids of the central nervous system and that CSF levels of soluble APP provide a useful biochemical marker to assist in the clinical diagnosis of AD.
A uridine-rich sequence required for translation of prokaryotic mRNA.
Authors: Zhang J , Deutscher M P
Published: May 2006

Binding of 30S ribosomal subunits to mRNA during the initiation of prokaryotic translation is known to be influenced by the initiation codon and the Shine-Dalgarno sequence. Site-directed mutagenesis of rnd, the Escherichia coli gene encoding RNase D, has now shown that a U8 sequence upstream of the Shine-Dalgarno region is also essential for expression of this mRNA. Alteration of two to five uridine residues within this sequence has no effect on mRNA levels but decreases RNase D protein and activity by as much as 95%, indicating that the U-rich sequence acts as an enhancer of translation. Moreover, mutant transcripts bind to 30S ribosomes in vitro with lower affinity than their wild-type counterparts, suggesting that the role of the U8 sequence is in the initial binding of ribosomes to the translation initiation region of the message. These data demonstrate that sequences other than those previously recognized can be essential for translation initiation.
Ligand and DNA-dependent phosphorylation of human progesterone receptor in vitro.
Authors: Bagchi M K , Tsai S Y , Tsai M J , O'Malley B W
Published: May 2006

The progesterone receptor (PR), like other members of the steroid receptor family, is a ligand-induced transcription factor. We have demonstrated previously that progesterone-induced binding of PR to a progesterone response element (PRE)-linked promoter stimulates RNA synthesis from that promoter in a cell-free transcription extract. It has been established that a hormone-mediated activation of PR beyond the removal of associated heat shock proteins is essential for efficient transactivation of the target gene. We now report that treatment with hormone leads rapidly to multiple phosphorylations of both the A and B forms of human PR in a HeLa nuclear extract. The putative kinase is present in the transcriptional extract but fails to phosphorylate the receptor significantly in the absence of specific hormone or DNA. Efficient phosphorylation of the PR occurs only in the presence of PREs, indicating that ligand-induced binding of PR to its cognate DNA response element makes it a preferred substrate for the kinase. The kinetics of the phosphorylation reaction overlap the kinetics of hormone-dependent RNA synthesis from a PRE-containing target promoter in vitro. We postulate that ligand and DNA-dependent phosphorylation of PR is an important functional event in the process leading to receptor-mediated transactivation of target genes.
Cloning and physical mapping of the HLA class I region spanning the HLA-E-to-HLA-F interval by using yeast artificial chromosomes.
Authors: Geraghty D E , Pei J , Lipsky B , Hansen J A , Taillon-Miller P , Bronson S K , Chaplin D D
Published: May 2006

The HLA class I genes are located within a 2-million-base pair (2-Mbp) region constituting the telomeric half of the human major histocompatibility complex. The large majority of the class I sequences, including the HLA-A, -E, -F, and -G genes, is found within the telomeric 1 Mbp. We report here the isolation and characterization of yeast artificial chromosome (YAC) clones that span a contiguous region of greater than 1.2 Mbp and include 14 of the 18 characterized class I sequences. Restriction enzyme mapping and the use of locus-specific probes have allowed all of the class I genes and sequences to be ordered and positioned within the region. In addition, the transcriptional orientation of the four class I genes has been determined. Using probes derived from the ends of YAC inserts and from class I pseudogenes, we describe a highly polymorphic region between the HLA-A and HLA-G genes. This region appears to be deleted in certain HLA haplotypes, shortening the distance between HLA-A and HLA-G by greater than 50 kilobase pairs (kbp). As part of the characterization of the YAC clones, unique sequence probes derived from the ends of each YAC insert were identified. When combined with probes derived from HLA genes and pseudogenes, 25 locus-specific probes spanning the 1.2-Mbp region have been identified for an average of 1 probe every 48 kbp.
Transport and anchoring of beta-lactamase to the external surface of Escherichia coli.
Authors: Francisco J A , Earhart C F , Georgiou G
Published: May 2006

The outer membrane of Gram-negative bacteria presents an effective barrier that restricts the release of proteins from the cell. Virtually all extracellular proteins of Gram-negative bacteria are exported by specialized systems requiring the action of several gene products. We have constructed a tripartite fusion consisting of (i) the signal sequence and first nine N-terminal amino acids of the mature major Escherichia coli lipoprotein, (ii) amino acids 46-159 of the outer membrane protein OmpA, and (iii) the complete mature beta-lactamase (EC 3.5.2.6) sequence. This protein had an enzymatically active beta-lactamase and was found predominantly in the outer membrane. Immunofluorescence microscopy, the accessibility of the fusion protein to externally added proteases, and the rates of hydrolysis of nitrocefin and penicillin G by whole cells demonstrated that a substantial fraction (20-30%) of the beta-lactamase domain of the fusion protein was exposed on the external surface of E. coli. In cells grown at 24 degrees C the localization of beta-lactamase on the cell surface was almost quantitative (greater than 80% of the enzymatically active protein was exposed to the extracellular fluid) as determined by nitrocefin and penicillin G hydrolysis and trypsin accessibility. These results demonstrated that a soluble protein, beta-lactamase, can be transported through--and become anchored on--the outer membrane by fusion to the proper targeting and localization signals.
Photoaffinity labeling of the primary fibrin polymerization site: isolation and characterization of a labeled cyanogen bromide fragment corresponding to gamma-chain residues 337-379.
Authors: Shimizu A , Nagel G M , Doolittle R F
Published: May 2006

Human fibrinogen and the plasmin-generated fibrinogen fragment D were photoaffinity labeled specifically with the peptide [14C]Gly-Pro-Arg-N(4-azido-2-nitrophenyl)Lys amide. In the case of fibrinogen, greater than 85% of the incorporated radioactivity was found in the gamma chain. Similarly, when fragment D (Mr, 90,000) was labeled with the same derivatized peptide, virtually all the radioactivity was found in the gamma-chain portion. The labeled fragment D was treated with CNBr and an initial purification was achieved by two gel-filtration steps. The labeled material was purified further by HPLC and was also compared with CNBr digests of unlabeled material. Amino acid analysis and gas-phase sequencing showed the labeled fragment to be gamma-chain residues 337-379.
Photoaffinity labeling of the primary fibrin polymerization site: localization of the label to gamma-chain Tyr-363.
Authors: Yamazumi K , Doolittle R F
Published: May 2006

Fragment D prepared from human fibrinogen was labeled specifically by photoactivation of the peptide [14C]Gly-Pro-Arg-N-(4-azido-2-nitrophenyl)Lys amide. The preparation was freed of excess labeling reagents and then reduced and alkylated. The component alpha, beta, and gamma chains were purified by chromatography on carboxymethylcellulose and the radioactivity was found to be restricted to the gamma chain. Isolated gamma chains were digested with various endopeptidases, both alone and in tandem, and the products were fractionated by gradient HPLC. The amino acid compositions of all labeled peptides led to the conclusion that the modification occurs exclusively on gamma-chain Tyr-363.
CDK2 encodes a 33-kDa cyclin A-associated protein kinase and is expressed before CDC2 in the cell cycle.
Authors: Elledge S J , Richman R , Hall F L , Williams R T , Lodgson N , Harper J W
Published: May 2006

Critical cell cycle transitions are controlled by the coordinate actions of the p34cdc2 protein kinase and its regulatory subunits, cyclins. Recently we identified another human p34 homolog, cyclin-dependent kinase 2 (CDK2) by complementation of a cdc28-4 mutation in Saccharomyces cerevisiae using a lambda YES human cDNA expression library. CDK2 is 66% identical to CDC2Hs and 89% identical to the Xenopus Eg1 gene, forming a distinct subfamily of CDC2-related protein kinases. We have found that CDK2 encodes a 33-kDa cyclin A-associated protein kinase that contains phosphotyrosine, two characteristics it shares with CDC2Hs. However, we show that the subunit composition of these two protein kinase complexes can vary in different cell types, that they have different in vitro substrate preferences, and that CDK2 mRNA is observed much earlier than CDC2Hs mRNA when lymphocytes are stimulated to enter the cell cycle. We suggest that cells in different developmental or transformed states may have different mechanisms of cell cycle regulation.
A transcriptional silencer downstream of the promoter in the osmotically controlled proU operon of Salmonella typhimurium.
Authors: Overdier D G , Csonka L N
Published: May 2006

The proU operon of Salmonella typhimurium is induced by conditions of high osmolality. The cis-acting sequences that mediate osmotic control of transcription were characterized by deletion analysis. The nucleotide sequence between -60 and +274 (relative to the transcription start point) is sufficient for normal osmotic control. Deletions that removed sequences upstream of position +274 but left the promoter intact resulted in greatly increased expression from the proU promoter in the absence of osmotic stress. Thus, the transcription control region of the proU operon consists of two discrete components: (i) the promoter and (ii) a negatively acting site that overlaps the coding sequence of the first structural gene of the operon, proV. That this negative regulatory element is a transcriptional terminator or mRNA processing site was ruled out. Our results suggest that the negative regulatory element behaves as a transcriptional silencer that inhibits transcription initiation at the proU promoter in medium of low osmolality by some action at a distance. We propose several possible mechanisms for the function of this regulatory site.
Induction of heat shock protein closely correlates with protection against Toxoplasma gondii infection.
Authors: Nagasawa H , Oka M , Maeda K , Jian-Guo C , Hisaeda H , Ito Y , Good R A , Himeno K
Published: May 2006

Heat shock proteins (HSPs) are evolutionarily highly conserved polypeptides that appear to be produced by many cells to preserve cellular functions under a variety of conditions of stress, including infections. We report that a 65-kDa HSP is present in mouse peritoneal cells that have been infected with a low-virulence (Beverley) strain of Toxoplasma gondii, as determined by electroblot assay using a monoclonal antibody specific for microbial HSP65. This HSP is, however, not expressed when infection occurs with the high-virulence RH strain of T. gondii. Furthermore, HSP was demonstrable in mice that acquired resistance against infection with a lethal dose of bradyzoites of the Beverley strain or even of an inoculum of a highly virulent strain of T. gondii (RH). From these results, it can be suggested that HSPs play an important role in developing effective defenses that include effective immune responses against infection with Toxoplasma parasites in vivo.
The N-terminal 22 amino acid residues in the lactose permease of Escherichia coli are not obligatory for membrane insertion or transport activity.
Authors: Bibi E , Stearns S M , Kaback H R
Published: May 2006

When the lactose (lac) permease of Escherichia coli is expressed from the lac promoter at relatively low rates, deletion of amino acid residues 2-8 (delta 7) or 2-9 (delta 8) from the hydrophilic N terminus has a relatively minor effect on the ability of the permease to catalyze active lactose transport. Activity is essentially abolished, however, and the permease is hardly detected in the membrane when two additional amino acid residues are deleted (delta 10), and mutants deleted of residues 2-23 (delta 22) or 2-39 (delta 38) also exhibit no activity and are not inserted into the membrane. Dramatically, when the defective deletion mutants are overexpressed at high rates via the T7 promoter, delta 10 and delta 22 are inserted into the membrane in a stable form and catalyze active lactose transport in a highly significant manner, whereas delta 38 is hardly detected in the membrane and exhibits no activity. Interestingly, a fusion protein consisting of delta 38 and the ompA leader peptide is inserted into the membrane but exhibits no transport activity. The results indicate that the N-terminal hydrophilic domain of lac permease and the N-terminal half of the first putative transmembrane alpha-helix are not mandatory for either membrane insertion or transport activity.
Reaction kinetic model of a proposed plasma membrane two-cycle H(+)-transport system of Chara corallina.
Authors: Fisahn J , Hansen U P , Lucas W J
Published: May 2006

Biophysical and numerical analysis methods were used to characterize and model the transport protein that gives rise to the acid and alkaline regions of Chara. A measuring system that permits the detection of area-specific current-voltage curves was used. These current-voltage curves, obtained from the inward current regions of Chara, underwent a parallel shift when the alkaline region was inverted by means of an acid pH treatment. In this situation the reversal potential of this area shifted from -120 mV to -340 mV. Together with data obtained from experiments using a divided chamber system, these results suggest that a common transport protein generates inward and outward current regions of Chara. On the basis of these experimental findings, a reaction kinetic model is proposed that assigns two operational modes to the proposed transport protein. Switching between these modes generates either acid or alkaline behavior. Since the observed pH dependence of the postulated transporter is rather complex, a reaction kinetic saturation mechanism had to be incorporated into the model. This final 10-state reaction kinetic model provides an appropriate set of mathematical relations to fit the measured current-voltage curves by computer.
FDC-P1 myeloid cells engineered to express fibroblast growth factor receptor 1 proliferate and differentiate in the presence of fibroblast growth factor and heparin.
Authors: Li M , Bernard O
Published: May 2006

Full-length murine fibroblast growth factor (FGF) receptor 1 (FGFR-1L) cDNA was introduced into the FDC-P1 mouse myeloid progenitor cell line, which lacks FGF receptors and depends on interleukin 3 (IL-3) or granulocyte/macrophage colony-stimulating factor (GM-CSF) for its proliferation and survival. The expression of the FGFR-1L gene in FDC-P1 cells allowed these cells to grow in the presence of FGF and heparin. The resulting cell line, designated FD FGFR-1L.A, exhibited a more mature myeloid phenotype than did the parental FD FGFR-1L cells or uninfected FDC-P1 cells. They formed mainly dispersed colonies in soft-agar cultures when grown in the presence of FGF and heparin, suggestive of myeloid differentiation. The cells can be switched between growth on FGF/heparin and IL-3. Northern blot analysis and cytochemical staining demonstrated that FD FGFR-1L.A cells expressed myeloperoxidase mRNA and protein, biochemical markers specifically expressed during differentiation from the promyelocytic to the granulocytic stages, whereas the parental FD FGFR-1L cells and FDC-P1 cells failed to express this marker. These results indicate that the expression of FGFR-1L by FDC-P1 cells transmitted signals for growth in the presence of FGF and heparin and generated an additional signal for early myeloid differentiation but failed to commit FD FGFR-1L.A cells to terminal differentiation. This in vitro culture system can be used for molecular analysis of the regulation of cellular growth and differentiation mediated by the FGFs and their receptors.