bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeted mutations that ablate either the adenylate cyclase or hemolysin function of the bifunctional cyaA toxin of Bordetella pertussis abolish virulence.
Authors: Gross M K , Au D C , Smith A L , Storm D R
Published: May 2006

Bordetella pertussis, the causative agent of whooping cough, secretes several toxins implicated in this disease. One of these putative virulence factors is the adenylate cyclase (AC) toxin that elevates intracellular cAMP in eukaryotic cells to cytotoxic levels. This toxin is a bifunctional protein comprising both AC and hemolysin (HLY) enzymatic domains. The gene encoding the AC toxin (cyaA) is expressed as part of an operon that includes genes required for secretion or activation of the toxin. Because of this genetic organization, it is difficult to create B. pertussis mutants of cyaA that are ablations of a single enzyme function by conventional means, such as transposon mutagenesis. Therefore, to clarify the role of individual toxin functions in the virulence of B. pertussis, we have used site-directed or deletion mutagenesis and genetic recombination to specifically target the cyaA gene of B. pertussis to produce mutants that lack only the AC or HLY activity of this toxin. A point mutant of B. pertussis with abolished AC catalytic activity was greater than 1000 times less pathogenic to newborn mice than wild-type bacteria, directly demonstrating the importance of the AC toxin in pertussis virulence. Similarly, an in-frame deletion mutant of B. pertussis that lacks HLY is equally avirulent, supporting observations that the HLY domain plays a critical role in AC toxin entry into cells. Furthermore, the genetically inactivated AC toxin produced by the point mutant is antigenically similar to the native toxin, suggesting that this strain may be useful in the development of pertussis component vaccines.
Sliding and intermolecular transfer of the lac repressor: kinetic perturbation of a reaction intermediate by a distant DNA sequence.
Authors: Ruusala T , Crothers D M
Published: May 2006

The lac repressor associates with its operator at a rate faster than conventional diffusion allows, either because of one-dimensional diffusion of a captured repressor along the DNA (sliding) or because the tetrameric repressor can be rapidly transferred between DNA sites distant from each other in the primary sequence (direct transfer). We report measurements of relative repressor-operator association rates at physiological operator concentrations. We assay for the ability of DNA targets of equal length (approximately 200 base pairs) containing one or six operator segments to compete for repressor; as the sliding range decreases, the hexameric operator fragment should react up to six times faster than the monomeric operator fragment. We find that the advantage enjoyed by the hexameric fragment varies from little to none at low operator concentration (depending on ionic strength) to more than 3-fold at high concentration. We ascribe this behavior to sliding at low operator concentration and to an increasing contribution of bimolecular direct transfer events as concentration increases. The observations require a "semibound" intermediate state of the protein at operator sites. This species can either undergo a relatively slow (tau greater than 1 sec) unimolecular isomerization to the final complex, or the isomerization can occur in a bridged complex with another operator site, accompanied by transfer to the second operator with probability of 0.5. Bridging alters one or more rate constants in the complex.
Translocation of spectrin and protein kinase C to a cytoplasmic aggregate upon lymphocyte activation.
Authors: Gregorio C C , Kubo R T , Bankert R B , Repasky E A
Published: May 2006

We have previously reported that mammalian tissue lymphocytes exhibit significant heterogeneity with respect to the subcellular distribution of spectrin and that this phenomenon may result from a dynamic behavior of spectrin in response to activation signals. Here, we further characterize the involvement of spectrin in lymphocyte activation by examining its relationship with protein kinase C (PKC). PKC isoenzymes are a family of cytosolic kinases that translocate from the soluble to particulate fraction upon cell stimulation. It is reported here that activation of lymph node T cells through the antigen-specific receptor, or direct activation of PKC by phorbol esters, results in a striking increase in cells expressing a cytoplasmic aggregate of spectrin. Additionally, a concurrent increase in cells expressing aggregates of the beta II isozyme of PKC is observed. Immunofluorescence staining revealed that spectrin and PKC beta II are colocalized in untreated lymphocytes and that these two proteins are coincidently translocated to the same focal aggregate within the cytoplasm following stimulation. This redistribution of spectrin and PKC beta is blocked by pretreatment with calphostin C, a specific inhibitor of PKC. Solubility studies showed that there is an increase of both proteins in the detergent-insoluble fraction of lymphocytes upon activation, and immunoprecipitation studies indicated that the soluble form of these molecules may be associated directly or indirectly as part of a complex of proteins. These data indicate that the positioning of the spectrin-based cytoskeleton is sensitive to activation signals and may play a role in the function or positioning of PKC beta II.
The rad3+ gene of Schizosaccharomyces pombe is involved in multiple checkpoint functions and in DNA repair.
Authors: Jimenez G , Yucel J , Rowley R , Subramani S
Published: May 2006

A number of important molecular checkpoints are believed to control the orderly progression of cell cycle events. We have found that the radiation-sensitive Schizosaccharomyces pombe mutant rad3-136 is deficient in two molecular checkpoint functions. Unlike wild-type cells, the mutant cells are unable to arrest in the G2 phase of the cell cycle after DNA damage by gamma-irradiation and are also incapable of maintaining the dependence of mitosis upon the completion of DNA synthesis. An S. pombe genomic clone that complements the UV sensitivity of the rad3-136 mutant completely restores the missing checkpoint functions. The rad3+ gene is also likely to play a role in DNA repair.
A single polypeptide catalyzing the conversion of phytoene to zeta-carotene is transcriptionally regulated during tomato fruit ripening.
Authors: Pecker I , Chamovitz D , Linden H , Sandmann G , Hirschberg J
Published: May 2006

The cDNA of the gene pds from tomato, encoding the carotenoid biosynthesis enzyme phytoene desaturase, was cloned, and its nucleotide sequence was determined. Cells of Escherichia coli that expressed the tomato pds gene could convert phytoene to zeta-carotene. This result suggests that one polypeptide, the product of the pds gene, can carry out phytoene desaturation in the carotenoid biosynthetic pathway. Transcripts of the pds gene accumulate in orange tomato fruit, indicating transcriptional control of pds expression during fruit ripening. The deduced amino acid sequence of phytoene desaturase indicates that this enzyme in tomato contains 583 amino acids that are highly conserved with respect to the homologous enzymes in cyanobacteria and algae. The deduced amino acid sequences of the phytoene desaturases from other microorganisms (purple bacteria and fungi) appear to be evolutionarily unrelated to those from green photosynthetic organisms.
Negative regulation of Rb expression by the p53 gene product.
Authors: Shiio Y , Yamamoto T , Yamaguchi N
Published: May 2006

Mutation of the p53 gene is one of the most frequent genetic changes found in human cancers. Recent experiments indicated that p53 might contain a transcription-activating domain, which functions when directed to a promoter. This study shows that wild-type p53 suppresses transcription of the retinoblastoma (Rb) gene. From deletion and mutagenesis experiments, a cis-acting element (GGAAGTGA) susceptible to regulation by p53 was mapped within the Rb promoter. This element overlaps the basal transcription unit of the Rb promoter, suggesting that p53 suppresses Rb transcription through inhibition of the basal promoter activity. The N-terminal acidic and C-terminal basic domains of p53 were both required for this suppression. These findings indicate that p53 can act as a transcriptional regulator in vivo.
BEN, a surface glycoprotein of the immunoglobulin superfamily, is expressed in a variety of developing systems.
Authors: Pourquié O , Corbel C , Le Caer J P , Rossier J , Le Douarin N M
Published: May 2006

We have previously identified a 95- to 100-kDa cell surface glycoprotein, which we named BEN (for bursal epithelium and neurons), that is widely expressed during chicken embryonic development. In the central nervous system, it is restricted to subsets of neurons including the motoneurons and the inferior olivary nucleus neurons (which provide the cerebellum with the climbing fibers) where its expression occurs during the phase of axonogenesis and synaptogenesis. In the present work, we show that BEN expression extends to a variety of tissues originating from the three embryonic germ layers. We have found that BEN immunopurified from neural, epithelial, and hemopoietic tissues is differently glycosylated and may or may not carry the HNK-1 epitope. We then cloned a full-length cDNA encoding this protein. Analysis of its sequence reveals that BEN is a member of the immunoglobulin superfamily. Two molecules with an identical cDNA sequence were recently reported: DM-GRASP [Burns, F. R., Von Kannen, S., Guy, L., Raper, J. A., Kamholz, J. & Chang, S. (1991) Neuron 7, 209-220] and SC1 [Tanaka, H., Matsui, T., Agata, A., Tomura, M., Kubota, I., Mcfarland, K. C., Kohr, B., Lee, A., Phillips, H. S. & Shelton, D. L. (1991) Neuron 7, 535-545]. Their pattern of expression and structural properties are consistent with those reported for BEN. Therefore BEN, DM-GRASP, and SC1 are likely to be the same molecule of the immunoglobulin superfamily.
Meiotic recombination and segregation of human-derived artificial chromosomes in Saccharomyces cerevisiae.
Authors: Sears D D , Hegemann J H , Hieter P
Published: May 2006

We have developed a system that utilizes human DNA-derived yeast artificial chromosomes (YACs) as marker chromosomes to study factors that contribute to the fidelity of meiotic chromosome transmission. Since aneuploidy for the YACs does not affect spore viability, different classes of meiotic missegregation can be scored accurately in four-viable-spore tetrads including precocious sister separation, meiosis I nondisjunction, meiotic chromatid loss, and meiosis II nondisjunction. Segregation of the homologous pair of 360-kilobase marker YACs was shown to occur with high fidelity in the first meiotic division and was associated with a high frequency of recombination within the human DNA segment. By using this experimental system, a series of YAC deletion derivatives ranging in size from 50 to 225 kilobases was analyzed to directly assess the relationship between meiotic recombination and meiosis I disjunction in a genotypically wild-type background. The relationship between physical distance and recombination frequency within the human DNA segment was measured to be comparable to that of endogenous yeast chromosomal DNA--ranging from less than 2.0 to 7.7 kilobases/centimorgan. Physical analysis of recombinant chromosomes detected no unequal crossing-over at dispersed repetitive elements distributed along the YACs. Recombination between YACs containing unrelated DNA segments was not observed. Furthermore, the segregational data indicated that meioses in which YAC pairs failed to recombine exhibited dramatically increased levels of meiosis I missegregation, including both precocious sister chromatid separation and nondisjunction.
On the relationship between bird and woody plant species diversity in the Uttara Kannada district of south India.
Authors: Daniels R J , Joshi N V , Gadgil M
Published: May 2006

Bird species richness is inversely related to woody plant species diversity and vertical stratification in the natural vegetation of Uttara Kannada, the district with the largest contiguous tract of humid tropical forest in peninsular India. This inverse relationship may be explained by the fact that although the peninsular Indian evergreen forests are rich in woody plant species when compared with the drier vegetation, they harbor an impoverished bird fauna due to their smaller overall extent and greater isolation. Much of this impoverishment is accounted for by the absence of many species of understory timaliids characteristic of the humid evergreen forests of the Eastern Himalayas and Southeast Asia. The plantations of Uttara Kannada largely derive their bird fauna from the drier vegetation and exhibit the commoner trend of a positive correlation between bird species richness and vertical stratification of the vegetation.
Purification of a distinctive form of endotoxin-induced nitric oxide synthase from rat liver.
Authors: Evans T , Carpenter A , Cohen J
Published: May 2006

An endotoxin-induced form of nitric oxide synthase (EC 1.14.23) was purified to homogeneity from rat liver by sequential anion-exchange chromatography and affinity chromatography using 2',5'-ADP-Sepharose. The enzyme has a subunit molecular mass of 135 kDa as determined by SDS/PAGE, a maximum specific activity of 462 nmol of citrulline formed from arginine per min per mg, and a Km for arginine of 11 microM. The enzyme was strongly stimulated by the addition of calmodulin with an EC50 of 2 nM, but removal of free calcium from the assay medium only reduced activity by 15%. Calmodulin inhibitors significantly reduced the enzyme activity. Tetrahydrobiopterin, FAD, and FMN were all required for full enzyme activity. This form of endotoxin-induced nitric oxide synthase from liver differs from the inducible enzyme found in macrophages and is unusual in that it is stimulated by calmodulin with little dependence on the calcium ion concentration.
Gene conversion in Neisseria gonorrhoeae: evidence for its role in pilus antigenic variation.
Authors: Zhang Q Y , DeRyckere D , Lauer P , Koomey M
Published: May 2006

Antigenic variation of gonococcal pili results from the unidirectional transfer of genetic information from variant-encoding partial pilin genes to an active expression locus. Two potential mechanisms that may result in the observed alterations of gene linkage and organization are conversion and transformation. To determine the relative contributions of these two distinct pathways of recombination to pilus variation, gonococcal strains carrying defined frameshift, missense, and nonsense mutations within the pilin expression locus were constructed. Reversion to a piliated state required correction of the lesions and provided a simple means of scoring productive recombination and antigenic variation. Examination of the mutants revealed a lack of correspondence between the frequencies with which they could be transformed (10(-6) per recipient) and the incidence with which they gave rise to revertants (greater than 10(-4) per colony-forming unit per generation). Further, the rates of reversion demonstrated by these mutants were not altered by growth in the presence of DNase I, conditions that abolished intercellular transfer of chromosomal markers during cultivation. Through the use of a pilin mutant in which a frameshift mutation encompassed the introduction of a restriction endonuclease site, the symmetry of recombination that resulted in reversion could be scored by Southern hybridization. In all cases examined, the DNA alterations responsible for pilin variation were nonreciprocal events. The results favor the model that productive pilin gene rearrangements in gonococci arise by gene conversion.
Phosphorylation-dependent epitopes of neurofilament antibodies on tau protein and relationship with Alzheimer tau.
Authors: Lichtenberg-Kraag B , Mandelkow E M , Biernat J , Steiner B , Schröter C , Gustke N , Meyer H E , Mandelkow E
Published: May 2006

We have studied the phosphorylation of tau protein from Alzheimer paired helical filaments, of tau from normal human brain, and of recombinant tau isoforms. As a tool we used monoclonal antibodies against neurofilament protein [Sternberger, N., Sternberger, L. & Ulrich, J. (1985) Proc. Natl. Acad. Sci. USA 82, 4274-4276] that crossreact with tau in a phosphorylation-dependent manner. This allowed us to deduce the state of phosphorylation in normal and pathological tau, as well as antibody epitopes. The epitope of antibody SMI33 is at the first Lys-Ser-Pro sequence motif (residues 234-236) and requires an unphosphorylated Ser-235. Antibody SMI31 binds between Ser-396 (in the second Lys-Ser-Pro motif) and Ser-404, both of which must be phosphorylated. SMI34 has a conformational epitope that depends on the interaction between regions on either side of the microtubule-binding region; it also requires phosphorylation. The phosphorylatable serines detected by the SMI antibodies are part of Ser-Pro motifs and can be phosphorylated by a protein kinase activity that can be used to induce a paired helical filament-like state in human brain tau in vitro. The phosphates are incorporated in several stages that can be identified by antibody reactivity and gel shift. This suggests a role for the phosphorylation sites in Alzheimer disease, as well as the involvement of a Ser-Pro-directed protein kinase.
Preferential repair and strand-specific repair of benzo[a]pyrene diol epoxide adducts in the HPRT gene of diploid human fibroblasts.
Authors: Chen R H , Maher V M , Brouwer J , van de Putte P , McCormick J J
Published: May 2006

If excision repair-proficient human cells are allowed time for repair before onset of S phase, the premutagenic lesions formed by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene diol epoxide, BPDE) are lost from the transcribed strand of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene faster than from the nontranscribed strand. No change in strand distribution is seen with repair-deficient cells. These results suggest strand-specific repair of BPDE-induced DNA damage in human cells. To test this, we measured the initial number of BPDE adducts formed in each strand of the actively transcribed HPRT gene and the rate of repair, using UvrABC excinuclease in conjunction with Southern hybridization and strand-specific probes. We also measured the rate of loss of BPDE adducts from the inactive 754 locus. The frequencies of adducts formed by exposure to BPDE (1.0 or 1.2 microM) in either strand of a 20-kilobase fragment that lies entirely within the transcription unit of the HPRT gene were similar; the frequency in the 14-kilobase 754 fragment was approximately 20% lower. The rates of repair in the two strands of the HPRT fragment differed significantly. Within 7 hr after treatment with 1.2 microM BPDE, 53% of the adducts had been removed from the transcribed strand, but only 26% from the nontranscribed strand; after 20 hr, these values were 87% and 58%, respectively. In contrast, only approximately 14% of the BPDE adducts were lost from the 754 locus in 20 hr, a value even lower than the rate of loss from the overall genome (i.e., 38%). These results demonstrate strand-specific and preferential repair of BPDE adducts in human cells. They suggest that the heterogeneous repair of BPDE adducts in the human genome cannot be accounted for merely by the greatly increased rate of the repair specific to the transcribed strand of the active genes, and they point to a role for the chromatin structure.
Evidence for in vitro replication of hepatitis C virus genome in a human T-cell line.
Authors: Shimizu Y K , Iwamoto A , Hijikata M , Purcell R H , Yoshikura H
Published: May 2006

A human T-cell line, MOLT-4, either uninfected or infected with murine retroviruses, was tested for its susceptibility to hepatitis C virus (HCV) infection. The cell cultures were inoculated with a serum containing HCV and then examined for the presence of viral sequences by cDNA/PCR. In murine retrovirus-infected MOLT-4 (MOLT-4 Ma) cells, intracellular minus-strand viral RNA, a putative replication intermediate, was first detected 3 days after inoculation, and the maximum signal was seen on day 7. When the cells were continuously subcultured in fresh medium, HCV sequences were intermittently detected in cells over a period of 3 weeks. In MOLT-4 cells free of retroviruses, replication of minus-strand HCV RNA appeared less efficient than in MOLT-4 Ma cells. The presence of minus-strand viral RNA in MOLT-4 Ma cells inoculated with HCV was confirmed by in situ hybridization with a strand-specific RNA probe. Immunofluorescence tests with antibodies specific for HCV core and NS4 antigens showed that MOLT-4 Ma cells were positive for viral antigen 7 days after inoculation. Thus, it appears likely that the HCV genome can replicate in the human T-cell line MOLT-4.
Nonrandom integration of retroviral DNA in vitro: effect of CpG methylation.
Authors: Kitamura Y , Lee Y M , Coffin J M
Published: May 2006

We have developed a PCR-based system that allows us to assess the relative frequency of use of specific bases as targets for the avian leukosis virus in vitro integration system. Using this system, we tested the effect of 5-methylation of cytosine in runs of CpG on the distribution of integration target sites. We found that the distribution of preferred integration sites was not uniform along the target DNA; rather, there was a distinct and reproducible pattern of frequently used sites. This pattern was independent of orientation of the integrated DNA, and of overall structure and sequence of the target and fragment amplified. Methylation did not inhibit integration into CpG dinucleotides; on the contrary, this modification created highly preferred targets within runs of alternating CpG. Finally, similar but not identical specificity was observed by using preintegration complexes in infected extracts or purified integrase and DNA as enzyme and substrate. Thus, most of the specificity observed is conferred by interaction of integrase and targets, although it may be modified by other viral and/or cellular components.
CD4 and CD8 regulate interleukin 2 responses of T cells.
Authors: Takahashi K , Nakata M , Tanaka T , Adachi H , Nakauchi H , Yagita H , Okumura K
Published: May 2006

To characterize the T-cell surface molecules involved in regulation of T-cell interleukin 2 (IL-2) responses, we established several monoclonal antibodies (mAbs) that inhibit IL-2 responses of freshly isolated CD8+ T cells and the IL-2-dependent cell line CTLL-2. Here we show that two inhibitory mAbs are directed against Lyt-2 (CD8 alpha). In fact, all anti-Lyt-2 mAbs tested were able to inhibit the IL-2 response of the Lyt-2- and L3T4-deficient cell line HT-2 after transfection with a Lyt-2 cDNA clone. Similarly, anti-L3T4 mAbs inhibited the IL-2 response of CD4-transfected HT-2 cells. These inhibitory effects of anti-CD4 and anti-CD8 mAbs occur on normal T lymphocytes, since they also were observed with CD4+ and CD8+ T-cell blasts, and are specific for IL-2 responses, since IL-4 responses of CD4- and CD8-transfected HT-2 cells were not affected by the anti-CD4 and anti-CD8 mAbs. The inhibitory effects of anti-CD4 or anti-CD8 mAbs could not be explained by interference with IL-2 binding and depended on CD4 and CD8 crosslinking, because F(ab')2 or Fab plus crosslinking second antibody, but not Fab alone, were effective. A mutant Lyt-2 molecule lacking the cytoplasmic region that mediates p56lck binding could not mediate the inhibitory effect upon crosslinking. These results suggest that CD4 and CD8 mediate negative regulation of T-cell IL-2 responses via cytoplasmically associated p56lck.
Intervening sequences in an Archaea DNA polymerase gene.
Authors: Perler F B , Comb D G , Jack W E , Moran L S , Qiang B , Kucera R B , Benner J , Slatko B E , Nwankwo D O , Hempstead S K
Published: May 2006

The DNA polymerase gene from the Archaea Thermococcus litoralis has been cloned and expressed in Escherichia coli. It is split by two intervening sequences (IVSs) that form one continuous open reading frame with the three polymerase exons. To our knowledge, neither IVS is similar to previously described introns. However, the deduced amino acid sequences of both IVSs are similar to open reading frames present in mobile group I introns. The second IVS (IVS2) encodes an endonuclease, I-Tli I, that cleaves at the exon 2-exon 3 junction after IVS2 has been deleted. IVS2 self-splices in E. coli to yield active polymerase, but processing is abolished if the IVS2 reading frame is disrupted. Silent changes in the DNA sequence at the exon 2-IVS2 junction that maintain the original protein sequence do not inhibit splicing. These data suggest that protein rather than mRNA splicing may be responsible for production of the mature polymerase.
Stimulation of system y(+)-like amino acid transport by the heavy chain of human 4F2 surface antigen in Xenopus laevis oocytes.
Authors: Bertran J , Magagnin S , Werner A , Markovich D , Biber J , Testar X , Zorzano A , Kühn L C , Palacin M , Murer H
Published: May 2006

A kidney cortex cDNA clone (rBAT) has recently been isolated, which upon in vitro transcription and capping complementary RNA (cRNA) and injection into Xenopus laevis oocytes induces a system b0,(+)-like amino acid transport activity. This cDNA encodes a type II membrane glycoprotein that shows significant homology to another type II membrane glycoprotein, the heavy chain of the human and mouse 4F2 surface antigen (4F2hc). Here we demonstrate that injection of human 4F2hc cRNA into oocytes results in the activation of a cation-preferring amino acid transport system that appears to be identical to the y(+)-like transport already present in the oocyte. This is based on the following results: (i) Injection of in vitro transcripts from 4F2hc cDNA (4F2hc cRNA) into oocytes stimulates up to 10-fold the sodium-independent uptake of L-arginine and up to 4.1-fold the sodium-dependent uptake of L-leucine. In contrast, 4F2hc cRNA does not increase the basal sodium-independent uptake of L-leucine. (ii) Basal and 4F2hc cRNA-stimulated sodium-independent uptake of L-arginine is completely inhibited by L-leucine in the presence of sodium. Similarly, the basal and 4F2hc cRNA-stimulated sodium-dependent uptake of L-leucine is entirely inhibited by L-arginine. (iii) The stimulation of sodium-independent uptake of L-arginine and the stimulation of sodium-dependent uptake of L-leucine induced by injection of 4F2hc cRNA are both completely inhibited by dibasic L amino acids and to a lesser extent by D-ornithine. (iv) Both basal and 4F2hc cRNA-stimulated sodium-independent uptake of L-arginine show two additional characteristics of the system y+ transport activity: inhibition of L-arginine uptake by L-homoserine only in the presence of sodium and an increase in the inhibition exerted by L-histidine as the extracellular pH decreased. Our results allow us to propose that an additional family of type II membrane glycoproteins (composed by rBAT and 4F2hc) is involved in amino acid transport, either as specific activators or as components of amino acid transport systems.
Predominant expression and activation-induced tyrosine phosphorylation of phospholipase C-gamma 2 in B lymphocytes.
Authors: Coggeshall K M , McHugh J C , Altman A
Published: May 2006

The triggering of T- or B-cell antigen-specific receptors is accompanied by rapid tyrosine phosphorylation of distinct cellular substrates, one of which is the gamma 1 isoform of inositol phospholipid-specific phospholipase C (PLC-gamma 1). This phosphorylation event, mediated by a putative protein tyrosine kinase coupled to the antigen receptor, probably stimulates the enzymatic activity of PLC-gamma 1, thereby promoting inositol phospholipid hydrolysis and other downstream signal transduction events. Recently, another ubiquitously expressed PLC isoform, PLC-gamma 2 (which shares 50.2% amino acid homology with PLC-gamma 1), has been identified. PLC-gamma 2-specific antibodies were used to evaluate the distribution and potential signaling role of this isoform in lymphocytes. Here, we report that, in contrast to T lymphocytes that express predominantly PLC-gamma 1, the major isoform expressed in murine and human resting B cells is PLC-gamma 2. Among B-cell tumor lines, all five murine B-lymphoma lines tested and one of six human B-lymphoblastoid cell lines also expressed predominantly PLC-gamma 2. However, three other human lines preferentially expressed PLC-gamma 1, and two others displayed similar levels of the two PLC-gamma isoforms. Furthermore, the triggering of B-cell surface immunoglobulin by anti-receptor antibodies was accompanied by a rapid tyrosine phosphorylation of PLC-gamma 2, which peaked after 5 min of stimulation. Conversely, and in agreement with recent reports, triggering of the T-cell antigen receptor complex led to the predominant phosphorylation of PLC-gamma 1 on tyrosine. These findings identify PLC-gamma 2 as a substrate for a B-cell putative protein tyrosine kinase coupled to the antigen receptor and suggest that its tyrosine phosphorylation constitutes a critical and early event in B-cell activation and, furthermore, that PLC-gamma 1 and PLC-gamma 2 may participate in similar but distinct signal transduction pathways in lymphocytes.
Dynamic magnetic resonance imaging of human brain activity during primary sensory stimulation.
Authors: Kwong K K , Belliveau J W , Chesler D A , Goldberg I E , Weisskoff R M , Poncelet B P , Kennedy D N , Hoppel B E , Cohen M S , Turner R
Published: May 2006

Neuronal activity causes local changes in cerebral blood flow, blood volume, and blood oxygenation. Magnetic resonance imaging (MRI) techniques sensitive to changes in cerebral blood flow and blood oxygenation were developed by high-speed echo planar imaging. These techniques were used to obtain completely noninvasive tomographic maps of human brain activity, by using visual and motor stimulus paradigms. Changes in blood oxygenation were detected by using a gradient echo (GE) imaging sequence sensitive to the paramagnetic state of deoxygenated hemoglobin. Blood flow changes were evaluated by a spin-echo inversion recovery (IR), tissue relaxation parameter T1-sensitive pulse sequence. A series of images were acquired continuously with the same imaging pulse sequence (either GE or IR) during task activation. Cine display of subtraction images (activated minus baseline) directly demonstrates activity-induced changes in brain MR signal observed at a temporal resolution of seconds. During 8-Hz patterned-flash photic stimulation, a significant increase in signal intensity (paired t test; P less than 0.001) of 1.8% +/- 0.8% (GE) and 1.8% +/- 0.9% (IR) was observed in the primary visual cortex (V1) of seven normal volunteers. The mean rise-time constant of the signal change was 4.4 +/- 2.2 s for the GE images and 8.9 +/- 2.8 s for the IR images. The stimulation frequency dependence of visual activation agrees with previous positron emission tomography observations, with the largest MR signal response occurring at 8 Hz. Similar signal changes were observed within the human primary motor cortex (M1) during a hand squeezing task and in animal models of increased blood flow by hypercapnia. By using intrinsic blood-tissue contrast, functional MRI opens a spatial-temporal window onto individual brain physiology.
A simple explanation of the electrostatics of the B-to-Z transition of DNA.
Authors: Guéron M , Demaret J P
Published: May 2006

Whereas the phosphates of B-DNA jut out into the solution, those of Z-DNA, being closer to DNA matter, are less subject to electrostatic screening by counterions. We present simple planar models of B- and Z-DNA that reflect these geometric features. The ionic strength dependence of the difference in the Poisson-Boltzmann electrostatic free energy of the models agrees with that measured by Pohl [Pohl, F. M. (1983) Cold Spring Harbor Symp. Quant. Biol. 47, 113-118]. This indicates that the electrostatics of the B-to-Z transition are primarily controlled by a qualitative geometrical difference and not by details of the DNA geometry or by complex electrostatic properties of the ionic solution.
Efficient inhibition of P-glycoprotein-mediated multidrug resistance with a monoclonal antibody.
Authors: Mechetner E B , Roninson I B
Published: May 2006

P-glycoprotein (Pgp), encoded by the MDR1 gene, is an active efflux pump for many structurally diverse lipophilic compounds. Cellular expression of Pgp results in multidrug resistance (MDR) in vitro and is believed to be a clinically relevant mechanism for tumor resistance to chemotherapy. We have developed a mouse monoclonal antibody, UIC2, that recognizes an extracellular epitope of human Pgp. UIC2 inhibited the efflux of Pgp substrates from MDR cells and significantly increased the cytotoxicity of Pgp-transported drugs, under the conditions where no effect was detectable with other anti-Pgp antibodies. Potentiation of cytotoxicity by UIC2 was observed with all the tested drugs associated with MDR (vinblastine, vincristine, colchicine, taxol, doxorubicin, etoposide, actinomycin D, puromycin, and gramicidin D) but not with any of the drugs to which MDR cells are not cross-resistant (methotrexate, 5-fluorouracil, cis-platinum, G418, and gentamicin). The inhibitory effect of UIC2 in vitro was as strong as that of verapamil (a widely used Pgp inhibitor) at its highest clinically achievable concentrations. Our results suggest that UIC2 or its derivatives provide an alternative or supplement to chemical agents for the reversal of MDR in clinical cancer.
Fumarase C, the stable fumarase of Escherichia coli, is controlled by the soxRS regulon.
Authors: Liochev S I , Fridovich I
Published: May 2006

Fumarase C was strongly induced by paraquat in a parental strain of Escherichia coli but was not induced in a strain lacking the soxRS response. Moreover, a strain that constitutively expresses the soxRS regulon contained more fumarase C than did the parental strain. The Mn-containing superoxide dismutase and glucose-6-phosphate dehydrogenase, members of the soxRS regulon, were similarly induced by paraquat. Mutational defects in glucose-6-phosphate dehydrogenase increased the induction of fumarase C by paraquat. For Mn-containing superoxide dismutase, responsiveness to paraquat was also enhanced in the glucose-6-phosphate dehydrogenase-defective strains. Overproduction of the Mn-containing superoxide dismutase, elicited by isopropyl beta-D-thiogalactoside in a tac-sodA fusion strain, did not diminish induction of fumarase C or of glucose-6-phosphate dehydrogenase by paraquat, and induction of these enzymes was more sensitive to paraquat when the cells were growing on succinate rather than on LB medium. These results indicate that fumarase C is a member of the soxRS regulon and that this regulon does not respond to changes in O2- concentration but perhaps does respond to some consequence of a decrease in the ratio of NADPH to NADP+.
Intrinsic signal changes accompanying sensory stimulation: functional brain mapping with magnetic resonance imaging.
Authors: Ogawa S , Tank D W , Menon R , Ellermann J M , Kim S G , Merkle H , Ugurbil K
Published: May 2006

We report that visual stimulation produces an easily detectable (5-20%) transient increase in the intensity of water proton magnetic resonance signals in human primary visual cortex in gradient echo images at 4-T magnetic-field strength. The observed changes predominantly occur in areas containing gray matter and can be used to produce high-spatial-resolution functional brain maps in humans. Reducing the image-acquisition echo time from 40 msec to 8 msec reduces the amplitude of the fractional signal change, suggesting that it is produced by a change in apparent transverse relaxation time T*2. The amplitude, sign, and echo-time dependence of these intrinsic signal changes are consistent with the idea that neural activation increases regional cerebral blood flow and concomitantly increases venous-blood oxygenation.
Surface-bound optical probes monitor protein translocation and surface potential changes during the bacteriorhodopsin photocycle.
Authors: Heberle J , Dencher N A
Published: May 2006

Light-induced H+ release and reuptake as well as surface potential changes inherent in the bacterio-rhodopsin reaction cycle were measured between 10 degrees C and 50 degrees C. Signals of optical pH indicators covalently bound to Lys-129 at the extracellular surface of bacteriorhodopsin were compared with absorbance changes of probes residing in the aqueous bulk phase. Only surface-bound indicators monitor the kinetics of H+ ejection from bacteriorhodopsin and allow the correlation of the photocycle with the pumping cycle. During the L550----M412 transition the H+ appears at the extracellular surface of bacteriorhodopsin. Surface potential changes detected by bound fluorescein or by the potentiometric probe 4-[2-(di-n-butylamino)-6-naphthyl]vinyl-1-(3-sulfopropyl)pyridinium betaine (di-4-ANEPPS) occur in milliseconds concomitantly with the formation and decay of the N intermediate. pH indicators residing in the aqueous bulk phase reflect the transfer of H+ from the membrane surface into the bulk but do not probe the early events of H+ pumping. The observed retardation of H+ at the membrane surface for several hundred microseconds is of relevance for energy conversion of biological membranes powered by electrochemical H+ gradients.
Rapid fitness losses in mammalian RNA virus clones due to Muller's ratchet.
Authors: Duarte E , Clarke D , Moya A , Domingo E , Holland J
Published: May 2006

Muller's ratchet is an important concept in population genetics. It predicts that when mutation rates are high and a significant proportion of mutations are deleterious, a kind of irreversible ratchet mechanism will gradually decrease the mean fitness of small populations of asexual organisms. In contrast, sexual recombination may stop or reverse this mutational ratchet by recombinational repair of genetic damage. Experimental support for Muller's ratchet has previously been obtained in protozoa and in a tripartite RNA bacteriophage. We now show clear evidence that Muller's ratchet can operate on a nonsegmented nonrecombining pathogenic RNA virus of animals and humans. We did genetic bottleneck passages (plaque-to-plaque transfers) of vesicular somatitis virus (VSV) and then quantitated relative fitness of the bottleneck clones by allowing direct replication competition in mixed infections in cell culture. We document variable fitness drops (some severe) following only 20 plaque-to-plaque transfers of VSV. In some clones no fitness changes (or only insignificant changes) were observed. Surprisingly, the most regular and severe fitness losses occurred during virus passages on a new host cell type. These results again demonstrate the extreme genetic and biological variability of RNA virus populations. Muller's ratchet could have significant implications for variability of disease severity during virus outbreaks, since genetic bottlenecks must often occur during respiratory droplet transmissions and during spread of low-yield RNA viruses from one body site to another (as with human immunodeficiency virus). Likewise, the lower-probability generation of increased-fitness clones during repeated genetic bottleneck transfers of RNA viruses in nature might also affect disease pathogenesis in infected individuals and in host populations. Whenever genetic bottlenecks of RNA viruses occur, enhanced biological differences among viral subpopulations may result.
Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes.
Authors: Wagner E , Zatloukal K , Cotten M , Kirlappos H , Mechtler K , Curiel D T , Birnstiel M L
Published: May 2006

We are developing efficient methods for gene transfer into tissue culture cells. We have previously shown that coupling of a chimeric adenovirus with polylysine allowed the construction of an adenovirus-polylysine-reporter-gene complex that transferred the transporter gene with great efficiency into HeLa cells. We have now explored simpler, biochemical means for coupling adenovirus to DNA/polylysine complexes and show that such complexes yield virtually 100% transfection in tissue culture cell lines. In these methods adenovirus is coupled to polylysine, either enzymatically through the action of transglutaminase or biochemically by biotinylating adenovirus and streptavidinylating the polylysine moiety. Combination complexes containing DNA, adenovirus-polylysine, and transferrin-polylysine have the capacity to transfer the reporter gene into adenovirus-receptor- and/or transferrin-receptor-rich cells.
Structures of apo and complexed Escherichia coli glycinamide ribonucleotide transformylase.
Authors: Almassy R J , Janson C A , Kan C C , Hostomska Z
Published: May 2006

The three-dimensional structure of phosphoribosylglycinamide formyltransferase (10-formyltetrahydrofolate:5'-phosphoribosylglycinamide formyltransferase, EC 2.1.2.2) has been solved both as an apoenzyme at 2.8-A resolution and as a ternary complex with the substrate glycinamide ribonucleotide and a folate inhibitor at 2.5-A resolution. The structure is a modified doubly wound alpha/beta sheet with flexibility in the active site, including a disordered loop in the apo structure, which is ordered in the ternary complex structure. This enzyme is a target for anti-cancer therapy and now for structure-based drug design.
Vitamin D-responsive protein-DNA interactions at multiple promoter regulatory elements that contribute to the level of rat osteocalcin gene expression.
Authors: Bortell R , Owen T A , Bidwell J P , Gavazzo P , Breen E , van Wijnen A J , DeLuca H F , Stein J L , Lian J B , Stein G S
Published: May 2006

The observation that vitamin D-mediated enhancement of osteocalcin (OC) gene expression is dependent on and reciprocally related to the level of basal gene expression suggests that an interaction of the vitamin D responsive element (VDRE) with basal regulatory elements of the OC gene promoter contributes to both basal and vitamin D-enhanced transcription. Protein-DNA interactions at the VDRE of the rat OC gene (nucleotides -466 to -437) are reflected by direct sequence-specific and antibody-sensitive binding of the endogenous vitamin D receptor present in ROS 17/2.8 osteosarcoma nuclear protein extracts. In addition, a vitamin D-responsive increase in OC gene transcription is accompanied by enhanced non-vitamin D receptor-mediated protein-DNA interactions in the "TATA" box region (nucleotides -44 to +23), which also contains a potential glucocorticoid responsive element. Evidence for proximity of the VDRE with the basal regulatory elements is provided by two features of nuclear architecture. (i) Nuclear matrix attachment elements in the rat OC gene promoter that bind nuclear matrix proteins with sequence specificity may impose structural constraints on promoter conformation. (ii) Limited micrococcal nuclease digestion and Southern blot analysis indicate that three nucleosomes can be accommodated in the sequence spanning the OC gene VDRE, the OC/CCAAT box (nucleotides -99 to -76), and the TATA/glucocorticoid responsive element, and thereby the potential distance between the VDRE and the basal regulatory elements can be reduced. A model is presented for the contribution of both the VDRE and proximal promoter elements to the enhancement of OC gene transcription in response to vitamin D. The vitamin D receptor plus accessory proteins may function cooperatively with basal regulatory factors to modulate the extent to which the OC gene is transcribed.
Androgen receptor gene mutations in human prostate cancer.
Authors: Newmark J R , Hardy D O , Tonb D C , Carter B S , Epstein J I , Isaacs W B , Brown T R , Barrack E R
Published: May 2006

We screened human prostate cancer tissues for the presence of somatic mutations in the hormone binding domain of the androgen receptor (AR) gene. Exons E-H were amplified from genomic DNA using the polymerase chain reaction and analyzed by denaturing gradient gel electrophoresis (DGGE), which separates DNA fragments that differ by only a single base. We detected a mutation in exon E of the hormone binding domain in 1 of 26 specimens of untreated organ-confined stage B prostate cancer. The mutation was not detectable in peripheral blood lymphocyte DNA. Lymphocyte DNA (wild-type AR) migrated in DGGE as a single band. The tumor DNA migrated in DGGE as four bands, consistent with the presence of cells with mutant AR plus cells with wild-type AR and indicating that the tumor contained a somatic mutation. To our knowledge, a somatic AR gene mutation has not been reported previously. Sequencing revealed a G----A substitution in codon 730, changing valine to methionine. Codon 730 is in a region highly conserved among all steroid receptors. The abundance of the mutated fragment (about 50% of the DNA in the specimen) indicates its presence in cells with a growth advantage. A somatic mutation could be detected by DGGE if it represented at least 10% of the sample. Failure to detect mutations in other specimens analyzed may be due to this limit of sensitivity, the presence of mutations in other parts of the AR, or a low frequency of mutations in early stage disease.
Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity.
Authors: Kato K , Cox A D , Hisaka M M , Graham S M , Buss J E , Der C J
Published: May 2006

We have introduced a variety of amino acid substitutions into carboxyl-terminal CA1A2X sequence (C = cysteine; A = aliphatic; X = any amino acid) of the oncogenic [Val12]Ki-Ras4B protein to identify the amino acids that permit Ras processing (isoprenylation, proteolysis, and carboxyl methylation), membrane association, and transformation in cultured mammalian cells. While all substitutions were tolerated at the A1 position, substitutions at A2 and X reduced transforming activity. The A2 residue was important for both isoprenylation and AAX proteolysis, whereas the X residue dictated the extent and specificity of isoprenoid modification only. Differences were observed between Ras processing in living cells and farnesylation efficiency in a cell-free system. Finally, one farnesylated mutant did not undergo either proteolysis or carboxyl methylation but still displayed efficient membrane association (approximately 50%) and transforming activity, indicating that farnesylation alone can support Ras transforming activity. Since both farnesylation and carboxyl methylation are critical for yeast a-factor biological activity, the three CAAX-signaled modifications may have different contributions to the function of different CAAX-containing proteins.
ADP-ribosylation factor, a small GTP-binding protein, is required for binding of the coatomer protein beta-COP to Golgi membranes.
Authors: Donaldson J G , Cassel D , Kahn R A , Klausner R D
Published: May 2006

The coatomer is a cytosolic protein complex that reversibly associates with Golgi membranes and is implicated in modulating Golgi membrane transport. The association of beta-COP, a component of coatomer, with Golgi membranes is enhanced by guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), a nonhydrolyzable analogue of GTP, and by a mixture of aluminum and fluoride ions (Al/F). Here we show that the ADP-ribosylation factor (ARF) is required for the binding of beta-COP. Thus, beta-COP contained in a coatomer fraction that has been resolved from ARF does not bind to Golgi membranes, whereas binding can be reconstituted by the addition of recombinant ARF. Furthermore, an N-terminal peptide of ARF, which blocks ARF binding to Golgi membranes, inhibits GTP[gamma S]- as well as the Al/F-enhanced binding of beta-COP. We show that Golgi coat protein binding involves a sequential reaction where an initial interaction of ARF and GTP[gamma S] with the membrane allows subsequent binding of beta-COP to take place in the absence of free ARF and GTP[gamma S]. The fungal metabolite brefeldin A, which is known to prevent the association of coat proteins with Golgi membrane, is shown to exert this effect by interfering with the initial ARF-membrane interaction step.