bluuebunny/crossref_metadata_2025 huggingface dataset:
Calpain (Ca(2+)-dependent thiol protease) in erythrocytes of young and old individuals.
Authors: Glaser T , Schwarz-Benmeir N , Barnoy S , Barak S , Eshhar Z , Kosower N S
Published: May 2006

Limited proteolysis by calpain (Ca(2+)-activated protease; EC 3.4.22.17) is believed to regulate the function of membrane enzymes and modify the behavior of membrane structural proteins. Calpain is activated by autolysis. The degradation of band 3 protein by mu-calpain is known to be enhanced in erythrocyte membranes from human individuals > 70 years old (old) as compared with that from individuals 20-30 years old (young). In the present study, monoclonal antibody to mu-calpain was used to study the behavior of calpain in erythrocytes of young and old individuals. Less calpain was found in erythrocyte cytosol and membranes from old than in those from young. Increasing the erythrocyte Ca2+ induced translocation of calpain to the cell membrane and autolysis of the enzyme. Alkylation of erythrocyte thiols also promoted translocation of calpain to the membrane, especially in the presence of Ca2+. When calpain was added to erythrocyte membranes, initial binding was greater and subsequent autolysis faster in old than in young individuals, possibly arising from alterations in cell membranes of old individuals. The enhanced calpain autolysis was accompanied by enhanced degradation of band 3 protein in the old. The results suggest that calpain in old individuals is translocated to the cell membrane and is activated by autolysis, resulting in degradation of certain membrane proteins and loss of calpain. Enhanced calpain-induced membrane proteolysis may play a role in abnormal cell destruction (e.g., shortening the life span of erythrocytes in the aged, neuronal degeneration, etc). The erythrocyte membrane provides a convenient model for the study of age-associated alterations in cell membranes and in calpain behavior.
Phosphorylation enhances the target gene sequence-dependent dimerization of thyroid hormone receptor with retinoid X receptor.
Authors: Bhat M K , Ashizawa K , Cheng S Y
Published: May 2006

To understand the molecular basis of the phosphorylation-enhanced transcriptional activity of human thyroid hormone nuclear receptor subtype beta 1 (hTR beta 1), we studied the effect of phosphorylation on the interaction of hTR beta 1 with the retinoid X receptor beta (RXR beta), we studied the effect of phosphorylation on the interaction of hTR beta 1 with the retinoid X receptor beta (RXR beta). In vitro, the extent of hTR beta 1.RXR beta heterodimer bound to various thyroid hormone response elements (TREs) was compared before and after phosphorylation of hTR beta 1. Without phosphorylation, hTR beta 1.RXR beta heterodimer was barely detectable under the experimental conditions. After phosphorylation of hTR beta 1, heterodimer bound to (i) the chicken lysozyme gene TRE, (ii) a TRE consisting of direct repeats of half-site binding motifs separated by four gaps, and (iii) a palindromic TRE was enhanced by approximately 10-, 7-, and 6-fold, respectively. The effect of phosphorylation on hTR beta 1.RXR beta heterodimerization was reversible. Dephosphorylation of the phosphorylated hTR beta 1 by alkaline phosphatase led to loss of the ability of hTR beta 1 to form a heterodimer with RXR beta in either the absence or the presence of DNA. These results indicate that the heterodimerization is enhanced by phosphorylation. To evaluate the effect of phosphorylation on the interaction of hTR beta 1 with RXR beta in vivo, we cotransfected hTR beta 1, RXR beta and TRE-chloramphenicol acetyltransferase (CAT) expression plasmids into CV-1 cells. CAT activity was assessed in the presence or absence of okadaic acid. Okadaic acid is a potent inhibitor of phosphatases 1 and 2A and increases the in vivo phosphorylation of hTR beta 1 by approximately 10-fold. Using the CAT reporter gene under control of the TRE from the malic enzyme gene, we found that RXR beta increased the okadaic acid-enhanced hTR beta 1-mediated CAT activity by 2- to 3-fold in the presence of 3,3',5-triiodo-L-thyronine. However, 9-cis-retinoic acid did not enhance the effect of okadaic acid. Our results indicate that phosphorylation is essential for the interaction of hTR beta 1 with RXR beta. Thus, phosphorylation plays a pivotal role in the gene-regulating activity of hTR beta 1.
Matrix decompositions of two-dimensional nuclear magnetic resonance spectra.
Authors: Havel T F , Najfeld I , Yang J X
Published: May 2006

Two-dimensional NMR spectra are rectangular arrays of real numbers, which are commonly regarded as digitized images to be analyzed visually. If one treats them instead as mathematical matrices, linear algebra techniques can also be used to extract valuable information from them. This matrix approach is greatly facilitated by means of a physically significant decomposition of these spectra into a product of matrices--namely, S = PAPT. Here, P denotes a matrix whose columns contain the digitized contours of each individual peak or multiple in the one-dimensional spectrum, PT is its transpose, and A is an interaction matrix specific to the experiment in question. The practical applications of this decomposition are considered in detail for two important types of two-dimensional NMR spectra, double quantum-filtered correlated spectroscopy and nuclear Overhauser effect spectroscopy, both in the weak-coupling approximation. The elements of A are the signed intensities of the cross-peaks in a double quantum-filtered correlated spectrum, or the integrated cross-peak intensities in the case of a nuclear Overhauser effect spectrum. This decomposition not only permits these spectra to be efficiently simulated but also permits the corresponding inverse problems to be given an elegant mathematical formulation to which standard numerical methods are applicable. Finally, the extension of this decomposition to the case of strong coupling is given.
Agonists and antagonists bind to different domains of the cloned kappa opioid receptor.
Authors: Kong H , Raynor K , Yano H , Takeda J , Bell G I , Reisine T
Published: May 2006

Opium and its derivatives are potent analgesics that can also induce severe side effects, including respiratory depression and addiction. Opioids exert their diverse physiological effects through specific membrane-bound receptors. Three major types of opioid receptors have been described, termed delta, kappa, and mu. The recent molecular cloning of these receptor types opens up the possibility to identify the ligand-binding domains of these receptors. To identify the ligand-binding domains of the kappa and delta receptors, we have expressed in COS-7 cells the cloned mouse delta and kappa receptors and chimeric delta/kappa and kappa/delta receptors in which the NH2 termini have been exchanged. The opioid antagonist naloxone binds potently to wild-type kappa receptor but not to wild-type delta receptor. The kappa/delta chimera bound [3H]naloxone with high affinity. In contrast, the kappa-specific agonist [3H]U-69,593 did not bind to the kappa/delta chimera. These findings indicate that selective agonists and antagonists interact with different recognition sites in the kappa receptor and localize the antagonist-binding domain to the NH2 terminus. Consistent with the results of radioligand-binding studies, the kappa/delta chimera did not mediate kappa-agonist inhibition of cAMP formation. In contrast, the delta/kappa chimera did mediate kappa-agonist inhibition of cAMP formation, but this effect was not blocked by naloxone. Furthermore, a truncated kappa receptor lacking its NH2 terminus was able to mediate agonist inhibition of cAMP accumulation in a naloxone-insensitive manner. This result further indicates that the NH2 terminus of the kappa receptor contains the selective antagonist-binding domain. The ability to dissociate agonist- and antagonist-binding sites will facilitate the development of more specific kappa agonists, which could have analgesic properties devoid of side effects.
Inhibition of proopiomelanocortin expression by an oligodeoxynucleotide complementary to beta-endorphin mRNA.
Authors: Spampinato S , Canossa M , Carboni L , Campana G , Leanza G , Ferri S
Published: May 2006

Gene expression in mammalian cells can be suppressed by oligonucleotides complementary to the target mRNA. This strategy was explored as a means of arresting translation of the prohormone precursor proopiomelanocortin (POMC), used as a model system of peptide messengers that are synthesized and released from endocrine and neuronal cells. The synthesis of the POMC-derived peptides adrenocorticotropin (ACTH) and beta-endorphin (beta-END) was markedly reduced by an oligodeoxynucleotide (ODN) complementary to a region of beta-END mRNA in AtT-20 cells, which retain many of the differentiated phenotypes of corticotrophs; this treatment did not affect the steady-state levels of POMC mRNA. Antisense ODN was stable in cell culture medium for 24 h, and cellular uptake was low (approximately 2.5% of the added ODN); however, the intracellular levels of the ODN were sufficient to form a ribonuclease-resistant duplex with complementary cellular mRNA. Addition of ODN to the cell culture did not affect the cellular levels of chromogranin A-(264-314)/pancreastatin or cell viability and proliferation, as evidenced by bromodeoxyuridine incorporation and ornithine decarboxylase activity. Microinfusion of the antisense ODN in the rat hypothalamic arcuate nucleus, where the majority of POMC-positive brain perikarya are located, significantly reduced ACTH- and beta-END-immunopositive neurons, and antisense ODN-treated rats showed substantially less of the grooming behavior usually observed in a novel environment.
Universal signals control slime mold stalk formation.
Authors: van Es S , Nieuwenhuijsen B W , Lenouvel F , van Deursen E M , Schaap P
Published: May 2006

The primitive slime mold Dictyostelium minutum does not display oscillations during aggregation, cannot form migrating slugs, and does not form a prestalk/prespore pattern, all of which are characteristic for development of its advanced relative Dictyostelium discoideum. We used D. minutum to investigate whether slime molds share common mechanisms controlling development. In D. discoideum, the morphogen differentiation inducing factor (DIF) can induce stalk-cell differentiation in vitro. However, stalk formation in vivo is supposedly triggered by local depletion of DIF antagonists such as ammonia or cAMP. A homologue of the D. discoideum stalk gene ecmB was cloned in D. minutum that encodes a 3.4-kb mRNA, and its deduced amino acid sequence shows repeats of 24 amino acids that are characteristic for the D. discoideum ecmB gene. Remarkably, DIF effectively induces expression of the D. minutum ecmB gene and ammonia inhibits its expression. D. discoideum cells were transformed with a construct of the D. minutum ecmB promoter fused to the lacZ reporter gene and showed expression in the stalk, but not in the upper and lower cup of the fruiting body, which also express the D. discoideum ecmB gene. In D. discoideum, the D. minutum ecmB and the ecmB promoter are similarly activated by DIF and repressed by both cAMP and ammonia, suggesting that additional signaling is required for ecmB expression in upper and lower cup cells. Our data indicate that the extracellular signals controlling stalk formation and their intracellular signaling cascades including gene regulatory proteins remained highly conserved during slime mold evolution.
Transcriptional photoregulation of cell-type-preferred expression of maize rbcS-m3: 3' and 5' sequences are involved.
Authors: Viret J F , Mabrouk Y , Bogorad L
Published: May 2006

In the C4 plant maize, members of the rbcS gene family, encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, are not expressed in mesophyll cells (MC) but are expressed strongly in the adjacent bundle sheath cells (BSC). Expression of genes in an in situ transient expression assay indicates that the photostimulated expression seen in BSC during the first 24 h that leaves of dark-grown seedlings are illuminated requires rbcS-m3 sequences lying between -211 bp and +434 bp of the transcription start site. Photoregulated partial suppression of rbcS-m3 expression in MC, on the other hand, requires gene sequences that lie between -907 bp and -445 bp together with sequences that lie between +720 and +957 bp within the 3' transcribed region of the gene. Suppression in MC occurs during the second 24-h period that dark-grown seedlings have been illuminated, but not during the first 24 h. The 3' +720- to +957-bp region is also effective in lowering MC expression when it is relocated to a position > 2 kbp upstream of the transcription start site. Thus, suppression of rbcS-m3 expression in MC has, at the least, a substantial transcriptional component. As reported earlier, a converse pattern of suppression in BSC and stimulation of expression in MC is seen in the control of cab-m1 in maize leaves.
NADPH-binding protein of the neutrophil superoxide-generating oxidase of guinea pigs.
Authors: Ge F , Guillory R J
Published: May 2006

Polymorphonuclear neutrophil membranes, activated in a cell-free system, contain all of the essential components required for superoxide formation including the NADPH-binding component. Arylazido-beta-alanyl- [32P]NADPH--3'-O-(3-[N-(4-azido-2-nitrophenyl)amino] propionyl)-[32P]NADPH--an NADPH analogue and photoaffinity probe, has been used to identify the specific NADPH binding component of the oxidase in activated membranes. A protein of about 52 kDa was photodependently labeled in the activated membranes by arylazido-beta-alanyl-[32P]NADPH. Specificity of labeling was indicated by the absence of such labeling in nonactivated membranes. The 52-kDa-labeled protein was the only isotopically labeled protein extracted from the labeled membranes with the chaotrope sodium perchlorate. Sodium perchlorate extraction of the 52-kDa protein from activated membranes correlates with the loss of the membranes' superoxide-generating capability. Reconstitution of the lost activity for sodium perchlorate-extracted membranes was accomplished by reincubating the extracted membranes with cytosol. It is proposed that the arylazido- beta-alanyl-[32P]NADPH-labeled protein of 52- to 57-kDa present on the activated membranes is the NADPH-binding protein of the neutrophil superoxide-generating oxidase.
Regulation of receptor internalization by the major histocompatibility complex class I molecule.
Authors: Olsson L , Goldstein A , Stagsted J
Published: May 2006

We showed previously that peptides derived from the alpha 1 domain of the major histocompatibility complex class I protein (MHC-I) inhibit internalization of some receptors, thereby increasing the steady-state number of active receptors on the cell surface. In consequence, sensitivity to hormone (e.g., insulin) is enhanced, transport (e.g., of glucose by GLUT-4) is increased, and carrier proteins (e.g., transferrin) operate less efficiently. Now we report that a bioactive peptide (but not closely related inactive ones) binds to MHC-I on the cell surface, not in the groove but apparently to the alpha 1 helix. The binding is saturable, and the number of peptide binding sites on the cell surface approximately equals the number of MHC-I molecules. Antibodies to MHC-I inhibit peptide binding. Most significant, antibodies to MHC-I mimic the effect of a bioactive peptide, inhibiting receptor internalization. These results indicate that MHC-I participates in the regulation of cell surface receptor activity.
Enhancer 1 binding factor (E1BF), a Ku-related protein, is a growth-regulated RNA polymerase I transcription factor: association of a repressor activity with purified E1BF from serum-deprived cells.
Authors: Niu H , Jacob S T
Published: May 2006

Previous studies from this laboratory have demonstrated that the enhancer 1 binding factor (E1BF), a Ku-related protein, purified from the serum-enriched cells functions as a positive factor in an RNA polymerase (pol I) transcription system. We have now shown that E1BF purified from the serum-deprived cells (E1BFs) can inhibit rDNA transcription completely in a fractionated extract from the cells grown in serum-enriched medium. The suppression of transcription was overcome by the addition of control E1BF (E1BFc). Immunoprecipitation of purified E1BFs by the anti-Ku monoclonal antibody and addition of the supernatant to the transcription reaction mixture prevented the inhibition significantly, whereas immunoprecipitation with the control mouse IgG did not restore the transcription. The transcriptional repressor activity associated with the final DNA affinity column fractions copurified with E1BF. Neither the amount of E1BF nor its promoter binding activity was altered following serum depletion. E1BFs selectively inhibited the initiation of rDNA transcription. The inhibitory activity of E1BFs was not due to a nonspecific RNase activity. These data suggest that E1BF is post-translationally modified following serum starvation of cells, and that the repressor activity of E1BFs is largely responsible for the down-regulation of pol I transcription in serum-deprived cells.
A yeast TCP-1-like protein is required for actin function in vivo.
Authors: Vinh D B , Drubin D G
Published: May 2006

We previously identified the ANC2 gene in a screen for mutations that enhance the defects caused by yeast actin mutations. Here we report that ANC2 is an essential gene that encodes a member of the TCP-1 family. TCP-1-related proteins are subunits of cytosolic heteromeric protein complexes referred to as chaperonins. These complexes can bind to newly synthesized actin and tubulin in vitro and can convert these proteins into an assembly-competent state. We show that anc2-1 mutants contain abnormal and disorganized actin structures, are defective in cellular morphogenesis, and are hypersensitive to the microtubule inhibitor benomyl. Furthermore, overexpression of wild-type Anc2p ameliorates defects in actin organization and cell growth caused by actin overproduction. Mutations in BIN2 and BIN3, two other genes that encode TCP-1-like proteins, also enhance the phenotypes of actin mutants. Taken together, these findings demonstrate that TCP-1-like proteins are required for actin and tubulin function in vivo.
Protein farnesyltransferase inhibitors block the growth of ras-dependent tumors in nude mice.
Authors: Kohl N E , Wilson F R , Mosser S D , Giuliani E , deSolms S J , Conner M W , Anthony N J , Holtz W J , Gomez R P , Lee T J
Published: May 2006

The posttranslational addition of a farnesyl moiety to the Ras oncoprotein is essential for its transforming activity. Cell-active inhibitors of the enzyme that catalyzes this reaction, protein farnesyltransferase, have been shown to selectively block ras-dependent transformation of cells in culture. Here we describe the protein farnesyltransferase inhibitor 2(S)-[2(S)-[2(R)-amino-3-mercapto]propylamino-3(S)-methyl] pentyloxy-3-phenylpropionylmethioninesulfone methyl ester (L-739,749), which suppressed the anchorage-independent growth of Rat1 cells transformed with viral H-ras and the human pancreatic adenocarcinoma cell line PSN-1, which harbors altered K-ras, myc, and p53 genes. This compound also suppressed the growth of tumors arising from ras-transformed Rat1 cells in nude mice by 66%. Under the same conditions, doxorubicin inhibited tumor growth by 33%. Control tumors formed by v-raf- or v-mos-transformed Rat1 cells were unaffected by L-739,749. Furthermore, mice treated with L-739,749 exhibited no evidence of systemic toxicity. This is a demonstration of antitumor activity in vivo using a synthetic small molecule inhibitor of protein farnesyltransferase.
Mapping of the C5a receptor signal transduction network in human neutrophils.
Authors: Buhl A M , Avdi N , Worthen G S , Johnson G L
Published: May 2006

Human neutrophils respond to chemoattractants, resulting in their accumulation at an inflammatory site. Chemoattractants such as the C5a peptide, derived from the C5 complement factor, bind to inhibitory guanine nucleotide binding protein (Gi)-coupled seven membrane-spanning receptors expressed in neutrophils. C5a receptor activation results in the Gi-dependent activation of the mitogen-activated protein (MAP) kinase pathway in human neutrophils. C5a receptor ligation activates both B-Raf and Raf-1, with B-Raf activation overlapping but temporally distinct from that of Raf-1. B-Raf and Raf-1 both efficiently phosphorylate MAP kinase kinase (MEK-1). C5a also stimulates guanine nucleotide exchange and activation of Ras. Ras and Raf activation in response to C5a involves protein kinase C-dependent and -independent pathways. Activation of both Raf-1 and B-Raf was inhibited by protein kinase A stimulation, consistent with the inhibitory effects of increased cAMP levels on neutrophil function. The findings define a functional signal transduction pathway linking the neutrophil C5a chemoattractant receptor to the regulation of Ras, B-Raf, Raf-1, and MAP kinase.
Identification of dynamin 2, an isoform ubiquitously expressed in rat tissues.
Authors: Cook T A , Urrutia R , McNiven M A
Published: May 2006

Dynamin is a 100-kDa microtubule-activated GTPase originally isolated from mammalian brain that has been proposed to be crucial in the early steps of endocytosis. Previous studies on the primary structure, biochemical properties, and functional role of dynamin indicated that it was neuron-specific. However, using an antibody against a synthetic peptide representing an enzymatic region of rat brain dynamin (D100), we identified a 100-kDa protein doublet in rat liver, suggesting that dynamin exists as different isoforms that are distinct from the brain counterpart. We then initiated a search for distinctive dynamin isoforms with antibodies and cDNA probes. A 500-bp PCR-generated cDNA probe corresponding to the enzymatic region of the rat brain dynamin-encoding gene was used to isolate six overlapping clones from a rat liver cDNA library that together span the complete coding sequence of another dynamin gene, "Dyn2." Sequence analyses reveal that dynamin 2 (Dyn2) is 75% identical to brain dynamin at the DNA level and is 79% identical at the protein level. By Northern blot analysis and isoform-specific PCR, Dyn2 was found ubiquitously in adult rat tissues as two transcripts of 3.5 kb and 4 kb; the highest levels were found in testis. These results indicate that dynamin proteins are encoded by at least two genes expressed differentially in mammalian tissues and that the expression of Dyn2, and not of brain dynamin, accounts for the ubiquitous distribution of dynamin in rat tissues.
CD8+ T cells in psoriatic lesions preferentially use T-cell receptor V beta 3 and/or V beta 13.1 genes.
Authors: Chang J C , Smith L R , Froning K J , Schwabe B J , Laxer J A , Caralli L L , Kurland H H , Karasek M A , Wilkinson D I , Carlo D J
Published: May 2006

Psoriasis is an inflammatory skin disorder characterized by epidermal keratinocyte hyperproliferation in association with a cellular infiltrate. There is evidence that activated T cells play a role in psoriatic plaque formation. We examined the T-cell receptor beta-chain variable gene segment (V beta) use of epidermal T cells in shave biopsies of psoriatic lesions. Our results show increased expression of V beta 3 and/or V beta 13.1 messages in the CD8+, but not CD4+, T cells in the lesions of a majority of patients studied. Sequence analysis of complementarity-determining region 3 (CDR3) of these two V beta genes from the skin demonstrated monoclonality or marked oligoclonality. A second biopsy from the same or different lesions, performed 3.5-8 months later in four patients, again revealed increased V beta 3 and/or V beta 13.1 expression and clonality. Moreover, in three of the four patients, the same V beta CDR3 rearrangement was found in both biopsies, although there was no V beta CDR3 homology between patients. In two patients in which V beta 3 and/or V beta 13.1 was not increased, an increase in V beta 17 gene use and clonality was found. The clonality of V beta sequence data indicates these cells are recruited and expanded in situ. The persistence of V beta 3-and/or V beta 13.1-bearing CD8+ T cells in lesions that did not undergo resolution suggests their role as effector cells rather than as regulatory cells.
Ligand-induced formation of nucleic acid triple helices.
Authors: Pilch D S , Breslauer K J
Published: May 2006

We demonstrate that ligand binding can be used to induce the formation of triplex structures that would not otherwise form. Specifically, we show that binding of berenil or 4',6-diamidino-2-phenylindole DAPI) induces formation of the poly(rA).poly(rA).poly(dT) triplex, providing an example of an RNA(purine).RNA(purine).DNA(pyrimidine) triplex. We also show that binding of berenil, DAPI, ethidium, or netropsin can induce formation of the poly(dT).poly(rA).poly(dT) triplex, thereby overcoming a practical limitation to the formation of DNA.RNA.DNA triplexes with a purine RNA strand. Based on the enhanced thermal stabilities of the drug-bound poly(dT).poly(rA).poly(dT) complexes at 18 mM Na+, we define the relative triplex-inducing efficiencies of these four ligands to be: berenil > DAPI > ethidium > netropsin. Our results demonstrate that ligand binding can be used to induce the formation of triplex structures that do not form in the absence of the ligand. This triplex-inducing capacity has potentially important implications in the design of novel antisense, antigene, and diagnostic strategies.
Definition of a human suppressor T-cell epitope.
Authors: Mutis T , Cornelisse Y E , Datema G , van den Elsen P J , Ottenhoff T H , de Vries R R
Published: May 2006

The quality of the response produced by regulatory or helper T (Th) cells presently receives much attention because of its possible implications for vaccine development and immunomodulation. Apart from cytokines and so-called costimulatory signals, antigens and the presenting major histocompatibility complex (MHC) molecules may play a role in determining the type of T-cell response generated toward antigens. To examine the role of antigen and/or HLA in control of T-cell subset activation, we have studied a special case, namely CD4+ suppressor T (Ts) cells in leprosy. Mycobacterium leprae-induced Ts cell clones have been previously isolated from peripheral blood and skin lesions of lepromatous leprosy patients and were shown to specifically down-regulate mycobacterium-specific Th cell responses. Despite considerable effort, the antigens recognized by these Ts cells have thus far not been identified. Here we report that all HLA-DR2-restricted CD4+ Ts cell clones derived from a lepromatous leprosy patient recognize an epitope that maps between the amino acid residues 439 and 448 of the mycobacterial hsp65. The peptide was presented to these Ts cells by HLA-DRB1*1503, a recently discovered HLA-DR2 variant. Non-suppressor T-cell clones derived from the same patient recognized antigens other than the hsp65 and were also stimulated by other HLA-DR2 variants. In independent cloning experiments peptide 435-449 and recombinant hsp65 induced exclusively Ts cells in this lepromatous leprosy patient. The Ts clones recognizing this particular epitope were derived from at least seven different progenitors, as they expressed different T-cell receptor alpha and beta chains. Thus, our data indicate that a specific peptide-HLA class II combination may exclusively activate Ts cells.
Cloning and characterization of a Neurospora crassa gene required for (1,3) beta-glucan synthase activity and cell wall formation.
Authors: Enderlin C S , Selitrennikoff C P
Published: May 2006

The glucan synthase 1 gene (gs-1) is required for (1,3) beta-glucan synthase activity [E.C. 2.4.1.34; UDP glucose:1,3-beta-D-glucan 3-beta-D-glucosyltransferase] and for cell wall formation. The gs-1 gene was cloned by functional complementation of the cell-wall-less defect of the (1,3) beta-glucan synthase-deficient mutant, TM1, by using a genomic Neurospora crassa cosmid library. A 2568-nucleotide gs-1 cDNA sequence revealed a 532-amino acid open reading frame encoding a polypeptide of 59 kDa. The predicted gs-1 gene product has no obvious signal peptide cleavage sites or transmembrane domains. A gs-1 null mutant is defective for cell wall formation and (1,3) beta-glucan synthase activity. The predicted GS-1 protein is weakly homologous to a putative Saccharomyces cerevisiae transcriptional regulatory protein.
Cloning of a cDNA encoding an ectoenzyme that degrades thyrotropin-releasing hormone.
Authors: Schauder B , Schomburg L , Köhrle J , Bauer K
Published: May 2006

Thyrotropin-releasing hormone (TRH) is an important extracellular signal substance that acts as a hypothalamic-releasing factor, which stimulates the release of adenohypophyseal hormones and functions as a neurotransmitter/neuromodulator in the central and peripheral nervous system. The inactivation of TRH after its release is catalyzed by an ectoenzyme localized preferentially on neuronal cells in the brain and on lactotrophic pituitary cells. This enzyme exhibits a very high degree of substrate specificity as well as other unusual properties. The activity of the adenohypophyseal enzyme is stringently controlled by estradiol and thyroid hormones, indicating that this enzyme itself may serve regulatory functions. Fragments of the enzyme isolated from rat or pig brain were generated by enzymatic digestion or cyanogen bromide cleavage, purified by reverse-phase HPLC, and sequenced. PCR amplification and screening of cDNA libraries from rat brain and pituitary led to the identification and isolation of a cDNA that encodes a protein of 1025 amino acids. The analysis of the deduced amino acid sequence was consistent with the identification of the enzyme as a glycosylated, membrane-anchored Zn metallopeptidase. Furthermore, Northern blot analysis demonstrated that the mRNA levels paralleled the tissue distribution of the enzyme and that in pituitary tissue the transcript levels rapidly increased when the animals were treated with triiodothyronine. Finally, transient transfection of COS-7 cells with this cDNA led to the expression of an active ectopeptidase that displayed the characteristics of the TRH-degrading ectoenzyme.
A physical map of the Myxococcus xanthus chromosome.
Authors: He Q , Chen H , Kuspa A , Cheng Y , Kaiser D , Shimkets L J
Published: May 2006

A physical map of the 9.2-Mbp Myxococcus xanthus DK1622 chromosome at a resolution of 25 kbp was constructed by using a strategy that is applicable to virtually all microorganisms. Segments of the chromosome were used as hybridization probes to subdivide a yeast artificial chromosome (YAC) library into groups of linked clones. The clones were aligned by comparing their EcoRI restriction patterns. The groups of YAC clones ("contigs") were oriented and aligned with the genomic restriction map by means of common genetic and physical markers such as rare restriction sites and transposon insertions. Over 95% of the genome is represented by cloned DNA. Sixty genetic loci including > 100 genes, many of which play a role in fruiting body development, have been mapped in this way. Additional genes can now be located on the chromosome map by hybridization of their sequences to the ordered set of YAC chromosomes. The mapped genetic loci account for approximately 2% of the genome.
Community structure of a microbial mat: the phylogenetic dimension.
Authors: Risatti J B , Capman W C , Stahl D A
Published: May 2006

Traditional studies of microbial communities are incomplete because of the inability to identify and quantify all contributing populations. In the present study, we directly determine the abundance and distribution of sulfate-reducing bacterial populations in a microbial mat community by using hybridization probes complementary to the 16S-like rRNAs of major phylogenetic groups. Most of the major groups were found in this single community, distributed for the most part in nonoverlapping depth intervals of the mat. The reflection of the phylogenetic structure in the community structure suggests that those species making up the major phylogenetic groups perform specific interrelated metabolic functions in the community. Comparison of population profiles to previously observed rates of sulfate reduction suggests there are additional populations of sulfate-reducing bacteria both within the photooxic zone and deeper in the mat.
Cell interactions in the control of size in Drosophila wings.
Authors: García-Bellido A , Cortés F , Milán M
Published: May 2006

The vein locus (vn) includes lethal alleles (designated also defective dorsal discs) that prevent growth of dorsal discs and in viable genetic combinations reduce the number of cells of the adult wing. Those effects are prominent in genetic mosaics. Cell proliferation is reduced in all regions of the wing blade in a local autonomous way. These effects are more extreme when mutant clones occupy full intervein regions bordering veins. Clones have, in addition, nonautonomous effects (accommodation) in the proliferation of wild-type cells of the same wing. These effects are more extreme in double mutant vn (ddd) and ve (rhomboid) allelic combinations. Developmental analysis shows that cell proliferation stops earlier in larval development the stronger the vn allele considered. A model is discussed of how cell proliferation is controlled by cellular interactions.
The primer tRNA sequence is not inherited during Ty1 retrotransposition.
Authors: Lauermann V , Boeke J D
Published: May 2006

Yeast retrotransposon Ty1 transposes through an RNA intermediate by a mechanism resembling retroviral replication. Long terminal repeat retroelements require primers for initiation of reverse transcription. The primer for minus-strand DNA synthesis is the 3' end of a cellular tRNA that base pairs to the complementary region of genomic RNA (the primer binding site). The genomic RNA of retroviruses and retrotransposons is shorter than its proviral DNA counterpart, lacking complete long terminal repeats. A variety of models have been proposed to describe how complete long terminal repeats are regenerated during reverse transcription. A common feature of these models is the requirement that the 3' portion of the primer tRNA be reverse-transcribed and then utilized in a strand-transfer reaction. We introduced a silent mutation into the Ty1 primer binding site and followed its fate during a single cycle of reverse transcription to directly test this aspect of the reverse transcription model. We demonstrate that the tRNA sequence is not inherited by progeny Ty1 elements during reverse transcription.
A strategy for the analysis of gene expression during neural development.
Authors: Arnold D , Feng L , Kim J , Heintz N
Published: May 2006

The delineation of cis-acting elements regulating stage-specific gene expression has played a critical role in the definition of the transcriptional circuitry regulating differentiation. In complex tissues such as the central nervous system, differentiation often requires temporally and spatially dynamic epigenetic cues that cannot be reproduced in cell culture. In these cases, identification of critical regulatory sequences and subsequent characterization of cognate transcription factors have been limited by lack of a rapid and efficient assay system for gene expression. We report a methodology that combines particle-bombardment transfection procedures and organotypic slice culture techniques to provide an acute assay system for transcriptional control in the developing central nervous system. Using this system, we demonstrate that cell-specific regulation of the brain lipid-binding protein (BLBP) gene in Bergmann glia, astrocytes, and migrating granule cells is conferred by 1.7 kb of 5' flanking sequences and that Purkinje cell-specific expression of the calbindin D28k gene in cerebellar cortex can be achieved with 1.1 kb of flanking DNA. Nearly 100% cotransfection of multiple DNAs can be achieved, allowing the design of precisely internally controlled experiments and providing the potential for rapid and efficient genetic analysis of gene function in single cells in a wild-type environment.
Inducing differentiation of transformed cells with hybrid polar compounds: a cell cycle-dependent process.
Authors: Marks P A , Richon V M , Kiyokawa H , Rifkind R A
Published: May 2006

Transformed cells do not necessarily lose their capacity to differentiate. Various agents can induce many types of neoplastic cells to terminal differentiation. Among such inducers, a particularly potent group consists of hybrid polar compounds; hexamethylene bisacetamide (HMBA) is the prototype of this group. With virus-transformed murine erythroleukemia cells as a model, HMBA was shown to cause these cells to arrest in G1 phase and express globin genes. This review focuses on HMBA-induced modulation of factors regulating G1-to-S phase progression, including a decrease in the G1 cyclin-dependent kinase cdk4, associated with inhibition of phosphorylation of the retinoblastoma protein pRB and possibly other related proteins that, in turn, sequester factors required for initiation of DNA synthesis; this provides a possible mechanism for HMBA-induced terminal cell division. Evidence that hybrid polar compounds have therapeutic potential for cancer treatment will also be reviewed.
Specific alteration of the oxidation potential of the electron donor in reaction centers from Rhodobacter sphaeroides.
Authors: Lin X , Murchison H A , Nagarajan V , Parson W W , Allen J P , Williams J C
Published: May 2006

The effects of multiple changes in hydrogen bond interactions between the electron donor, a bacteriochlorophyll dimer, and histidine residues in the reaction center from Rhodobacter sphaeroides have been investigated. Site-directed mutations were designed to add or remove hydrogen bonds between the 2-acetyl groups of the dimer and histidine residues at the symmetry-related sites His-L168 and Phe-M197, and between the 9-keto groups and Leu-L131 and Leu-M160. The addition of a hydrogen bond was correlated with an increase in the dimer midpoint potential. Measurements on double and triple mutants showed that changes in the midpoint potential due to alterations at the individual sites were additive. Midpoint potentials ranging from 410 to 765 mV, compared with 505 mV for wild type, were achieved by various combinations of mutations. The optical absorption spectra of the reaction centers showed relatively minor changes in the position of the donor absorption band, indicating that the addition of hydrogen bonds to histidines primarily destabilized the oxidized state of the donor and had little effect on the excited state relative to the ground state. Despite the change in energy of the charge-separated states by up to 260 meV, the mutant reaction centers were still capable of electron transfer to the primary quinone. The increase in midpoint potential was correlated with an increase in the rate of charge recombination from the primary quinone, and a fit of these data using the Marcus equation indicated that the reorganization energy for this reaction is approximately 400 meV higher than the change in free energy in wild type. The mutants were still capable of photosynthetic growth, although at reduced rates relative to the wild type. These results suggest a role for protein-cofactor interactions--in particular, histidine-donor interactions--in establishing the redox potentials needed for electron transfer in biological systems.
Role of c-myc in simian virus 40 large tumor antigen-induced DNA synthesis in quiescent 3T3-L1 mouse fibroblasts.
Authors: Hermeking H , Wolf D A , Kohlhuber F , Dickmanns A , Billaud M , Fanning E , Eick D
Published: May 2006

Stably transfected NIH 3T3-L1 mouse fibroblasts (L1 cells) expressing the simian virus 40 large tumor antigen (LTAg) maintain c-myc expression and proliferation in low serum, whereas cells expressing the mutant form LTAg-K1, defective in binding of the retinoblastoma suppressor gene product pRb, showed reduced levels of c-myc RNA and only background levels of DNA synthesis in low serum. The role of the c-Myc protein in LTAg-induced DNA synthesis was studied in microinjection experiments. Expression of LTAg induced cellular DNA synthesis in > 95% of microinjected serum-starved L1 cells, whereas the mutant LTAg-K1 could not induce DNA synthesis. Coexpression of dominant negative c-Myc or Max mutants with LTAg inhibited DNA synthesis, indicating that functional c-Myc is necessary for induction of DNA synthesis by LTAg. Expression of c-Myc induced programmed cell death (apoptosis) in serum-starved L1 cells. Coexpression of c-Myc with LTAg-K1 restored induction of DNA synthesis without apoptosis. Expression of a truncated LTAg, LTAg-(1-259), defective in binding of the tumor suppressor gene product p53, failed to prevent c-Myc-induced apoptosis. The data indicate that c-Myc can restore the ability of LTAg-K1 to induce DNA synthesis and that LTAg-K1 prevents c-Myc-induced apoptosis in serum-starved L1 cells by its interaction with p53.
A transcribing RNA polymerase molecule survives DNA replication without aborting its growing RNA chain.
Authors: Liu B , Wong M L , Alberts B
Published: May 2006

We have demonstrated elsewhere that a precisely placed, stalled Escherichia coli RNA polymerase ternary transcription complex (polymerase-RNA-DNA) stays on the DNA template after passage of a DNA replication fork. Moreover, the bypassed complex remains competent to resume elongation of its bound RNA chain. But the simplicity of our experimental system left several important questions unresolved: in particular, might the observation be relevant only to the particular ternary complex that we studied, and can the finding be generalized to a transcribing instead of a stalled RNA polymerase? To address these issues, we have created three additional ternary transcription complexes and examined their fates after passage of a replication fork. In addition, we have examined the fate of moving RNA polymerase molecules during DNA replication. The results suggest that our previous finding applies to all transcription intermediates of the E. coli RNA polymerase.
A composite intragenic silencer domain exhibits negative and positive transcriptional control of the bone-specific osteocalcin gene: promoter and cell type requirements.
Authors: Frenkel B , Montecino M , Stein J L , Lian J B , Stein G S
Published: May 2006

The osteocalcin (OC) silencer is a unique example of exonic sequences contributing to negative transcriptional control of mammalian gene expression. In this paper we demonstrate, using a reporter transfection assay, that multiple elements reside within the OC +24/+151 domain. Thirty-fold repression is mediated by the +49/+104 fragment, experimentally relocated 3' of the poly(A) signal. Deletion of either the +49/+54 protein-coding sequence or the +98/+104 intronic part of this fragment results in loss of repression activity, suggesting a bipartite organization of the +49/+104 silencer. Of particular interest, we have mapped an antisilencer activity to the ACCCTCTCT motif (+40/+48), found in silencers associated with several other genes. Extension of the +49/+104 silencer to include the +24/+48 and/or the +105/+151 sequences results in increased silencer activity up to 170-fold, suggesting the presence of additional silencer elements within these sequences. The activity of the silencer contained within the +24/+151 OC sequence is directed to the basal promoter and is not dependent on 5' distal enhancer elements, including those that mediate responsiveness of OC transcription to vitamin D. The OC silencer represses the heterologous thymidine kinase promoter and is operative in osseous (normal diploid osteoblasts, ROS 17/2.8 osteosarcoma) as well as HeLa cells. Our results, which suggest the presence of at least five regulatory elements downstream of the OC transcription start site, indicate the complexity of sequences that mediate repression of OC promoter activity.
Major histocompatibility complex class I genes of the coelacanth Latimeria chalumnae.
Authors: Betz U A , Mayer W E , Klein J
Published: May 2006

The coelacanth fish Latimeria chalumnae is the sole surviving species of a phylogenetic lineage that was founded more than 400 million years ago and that has changed morphologically very little since that time. Little is known about the molecular evolution of this "living fossil," considered by some taxonomists to be the closest living relative of tetrapods. Here we describe the isolation and characterization of L. chalumnae major histocompatibility complex (MHC) class I genes. The exon-intron organization of these genes is the same as that of their mammalian counterparts. The genes fall into four families, which we designate Lach-UA through Lach-UD. There are multiple loci in all of the families. Genes of the first two families are transcribed. The Lach-UA family bears the characteristics of functional, polymorphic class I genes; the other three families may be represented by nonclassical genes. All the Lach loci arose by duplication from an ancestral gene after the foundation of the coelacanth lineage. Intergenic variation is highest at positions corresponding to the mammalian peptide-binding region. The closest relatives of the Lach genes among the MHC genes sequenced thus far are those of the amphibian Xenopus.
The replication terminator protein of the gram-positive bacterium Bacillus subtilis functions as a polar contrahelicase in gram-negative Escherichia coli.
Authors: Kaul S , Mohanty B K , Sahoo T , Patel I , Khan S A , Bastia D
Published: May 2006

The replication terminator protein (RTP) of Bacillus subtilis is a dimer with a monomeric molecular mass of 14.5 kDa. The protein terminates DNA replication at a specific binding site. Although the protein has been crystallized and its crystal structure has been solved, the lack of an in vitro replication system in B. subtilis has been a serious impediment to the analysis of the mechanism of action of this protein. We have discovered that the protein is functional in the Gram-negative bacterium Escherichia coli in vivo and in vitro. RTP blocked replication forks initiated from a ColE1 replication origin at the cognate DNA-binding site (BS3) in a polar mode. The protein did not block rolling circle replication initiated from the pT181 origin in cell extracts of Staphylococcus aureus. RTP antagonized the helicase activity of DnaB but not that of helicase II of E. coli. Thus, RTP functioned as a polar contrahelicase blocking a helicase that participates in symmetric DNA replication but it did not impede rolling circle replication nor the action of a helicase involved in DNA repair.