bluuebunny/crossref_metadata_2025 huggingface dataset:
Physical association and functional antagonism between the p65 subunit of transcription factor NF-kappa B and the glucocorticoid receptor.
Authors: Ray A , Prefontaine K E
Published: May 2006

Glucocorticoids, which are widely used as antiinflammatory agents, downregulate the expression of the interleukin 6 gene and of additional cytokine genes involved in inflammatory responses. Conversely, the transcription factor NF-kappa B, a member of the Rel family of transcription factors, has been implicated in the induction of multiple genes involved in the early processes of immune and inflammatory responses. This prompted us to investigate whether one of the mechanisms by which glucocorticoids exert their antiinflammatory activities is through inhibition of gene activation mediated by NF-kappa B. We report that, in intact cells, activation of the interleukin 6 promoter by a combination of the factor NF-IL6 and the p65 subunit of NF-kappa B is inhibited by dexamethasone (ligand)-activated glucocorticoid receptor. Conversely, activation of the mouse mammary tumor virus promoter by a combination of dexamethasone and glucocorticoid receptor is inhibited by overexpression of p65. Furthermore, we provide evidence for physical association between glucocorticoid receptor and p65 in protein crosslinking and coimmunoprecipitation experiments, using either in vitro translated proteins or those present in cell extracts. These studies suggest that direct interactions between NF-kappa B and glucocorticoid receptor may partly account for the antiinflammatory properties of glucocorticoids in vivo.
Facilitation of glutamate receptors enhances memory.
Authors: Staubli U , Rogers G , Lynch G
Published: May 2006

A benzamide drug that crosses the blood-brain barrier and facilitates DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated synaptic responses was tested for its effects on memory in three behavioral tasks. The compound reversibly increased the amplitude and prolonged the duration of field excitatory postsynaptic potentials in hippocampal slices and produced comparable effects in the dentgate gyrus in situ after intraperitoneal injections. Rats injected with the drug 30 min prior to being given a suboptimal number of training trials in a two-odor discrimination task were more likely than controls to select the correct odor in a retention test carried out 96 hr later. Evidence for improved memory was also obtained in a water maze task in which rats were given only four trials to find a submerged platform in the presence of spatial cues; animals injected with the drug 30 min before the training session were significantly faster than vehicle-injected controls in returning to the platform location when tested 24 hr after training. Finally, the drug produced positive effects in a radial maze test of short-term memory. Well trained rats were allowed to retrieve rewards from four arms of an eight-arm maze and then tested for reentry errors 8 hr later. The number of such errors was substantially reduced on days in which the animals were injected with the drug before initial learning. These results indicate that a drug that facilitates glutamatergic transmission enhances the encoding of memory across tasks involving different sensory cues and performance requirements. This may reflect an action on the cellular mechanisms responsible for producing synaptic changes since facilitation of AMPA receptors promotes the induction of the long-term potentiation effect.
Predicting Ca(2+)-binding sites in proteins.
Authors: Nayal M , Di Cera E
Published: May 2006

The coordination shell of Ca2+ ions in proteins contains almost exclusively oxygen atoms supported by an outer shell of carbon atoms. The bond-strength contribution of each ligating oxygen in the inner shell can be evaluated by using an empirical expression successfully applied in the analysis of crystals of metal oxides. The sum of such contributions closely approximates the valence of the bound cation. When a protein is embedded in a very fine grid of points and an algorithm is used to calculate the valence of each point representing a potential Ca(2+)-binding site, a typical distribution of valence values peaked around 0.4 is obtained. In 32 documented Ca(2+)-binding proteins, containing a total of 62 Ca(2+)-binding sites, a very small fraction of points in the distribution has a valence close to that of Ca2+. Only 0.06% of the points have a valence > or = 1.4. These points share the remarkable tendency to cluster around documented Ca2+ ions. A high enough value of the valence is both necessary (58 out of 62 Ca(2+)-binding sites have a valence > or = 1.4) and sufficient (87% of the grid points with a valence > or = 1.4 are within 1.0 A from a documented Ca2+ ion) to predict the location of bound Ca2+ ions. The algorithm can also be used for the analysis of other cations and predicts the location of Mg(2+)- and Na(+)-binding sites in a number of proteins. The valence is, therefore, a tool of pinpoint accuracy for locating cation-binding sites, which can also be exploited in engineering high-affinity binding sites and characterizing the linkage between structural components and functional energetics for molecular recognition of metal ions by proteins.
Weak male-driven molecular evolution in rodents.
Authors: Chang B H , Shimmin L C , Shyue S K , Hewett-Emmett D , Li W H
Published: May 2006

In humans and rodents the male-to-female ratio of mutation rate (alpha m) has been suggested to be extremely large, so that the process of nucleotide substitution is almost completely male-driven. However, our sequence data from the last intron of the X chromosome-linked (Zfx) and Y chromosome-linked (Zfy) zinc finger protein genes suggest that alpha m is only approximately 2 in rodents with a 95% confidence interval from 1 to 3. Moreover, from published data on oogenesis and spermatogenesis we estimate the male-to-female ratio of the number of germ cell divisions per generation to be approximately 2 in rodents, confirming our estimate of alpha m and suggesting that errors in DNA replication are the primary source of mutation. As the estimated alpha m for rodents is only one-third of our previous estimate of approximately 6 for higher primates, there appear to be generation-time effects--i.e., alpha m decreases with decreasing generation time.
A tRNA-like structure is present in 10Sa RNA, a small stable RNA from Escherichia coli.
Authors: Komine Y , Kitabatake M , Yokogawa T , Nishikawa K , Inokuchi H
Published: May 2006

We have determined that 10Sa RNA (one of the small stable RNAs found in Escherichia coli) has an interesting structural feature: the 5' end and the 3' end of 10Sa RNA can be arranged in a structure that is equivalent to a half-molecule (acceptor stem and TFC stem-loop) of alanine tRNA of E. coli. Primer-extension analysis of 10Sa RNA extracted from a bacterial mutant with temperature-sensitive RNase P function revealed that the precursor to 10Sa RNA (pre-10Sa RNA) is folded into a pre-tRNA-like structure in vivo such that it can be cleaved by RNase P to generate the 5' end of the mature 10Sa RNA. The purified 10Sa RNA can be charged with alanine in vitro. Disruption of the gene encoding 10Sa RNA (ssrA) caused a reduction in the rate of cell growth, which was especially apparent at 45 degrees C, and a reduction in motility on semisolid agar. These phenotypic characteristics of the deletion strain (delta ssrA) allowed us to investigate the effects of some mutations in 10Sa RNA in vivo, although the exact function of 10Sa RNA still remains unclear. When the G.U pair (G3.U357) in 10Sa RNA, which may be equivalent to the determinant G.U pair of alanine tRNA, was changed to a G.A or G.C pair, the ability to complement the phenotypic mutations of the delta ssrA strain was lost. Furthermore, this inability to complement the mutant phenotypes that was caused by the substitution of the determinant bases by a G.A pair could be overcome by the introduction of a gene encoding alanyl-tRNA synthetase (alaS) on a multicopy plasmid. The evidence suggests that the proposed structural features of 10Sa RNA are indeed manifested in vivo.
Glutamine synthetase of Mycobacterium tuberculosis: extracellular release and characterization of its enzymatic activity.
Authors: Harth G , Clemens D L , Horwitz M A
Published: May 2006

We have investigated the activity and extracellular release of glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] of Mycobacterium tuberculosis. The purified, homogeneous M. tuberculosis glutamine synthetase appears to consist of 12 most likely identical subunits of M(r) 58,000, arranged in two superimpose hexagons. In the catalysis of L-glutamine, the enzyme has an apparent Km for L-glutamate of approximately 3 mM at the pH optimum of 7.5. M. tuberculosis releases a large proportion (approximately 30%) of its total measurable enzyme activity into the culture medium, a feature that is highly specific for pathogenic mycobacteria. Immunogold electron microscopy revealed that M. tuberculosis also releases the enzyme into its phagosome in infected human monocytes. Two potentially important roles for glutamine synthase in the pathogenesis of M. tuberculosis infection are (i) the synthesis of L-glutamine, a major component of the cell wall of pathogenic but not nonpathogenic mycobacteria, and (ii) the modulation of the ammonia level in the M. tuberculosis phagosome, which may in turn influence phagosomal pH and phagosomelysosome fusion.
Brain neurons and glial cells express Neu differentiation factor/heregulin: a survival factor for astrocytes.
Authors: Pinkas-Kramarski R , Eilam R , Spiegler O , Lavi S , Liu N , Chang D , Wen D , Schwartz M , Yarden Y
Published: May 2006

Neu differentiation factor (NDF, also called heregulin) was isolated from mesenchymal cells on the basis of its ability to elevate phosphorylation of ErbB proteins. Earlier in situ hybridization analysis showed that NDF was transcribed predominantly in the central nervous system during embryonic development. To gain insights into the role of NDF in brain we analyzed its distribution by immunohistochemistry and in situ hybridization. Late-gestation (day 17) rat embryos displayed high NDF immunoreactivity in both motor (e.g., putamen) and limbic (e.g., septum) regions. Lower levels of the factor were exhibited by adult brain, except for the cerebellum, where NDF expression was increased postnatally. Both neurons and glial cells were identified by immunohistochemistry as NDF-producing cells (e.g., pyramidal neurons in the cerebral cortex and glial cells in the corpus callosum). By establishment of primary cultures of rat brain cells we confirmed that NDF was expressed in neurons as well as in astrocytes. In addition, by using such primary cultures we observed that NDF treatment exerted only a limited mitogenic effect, which was accompanied by significant acceleration of astrocyte maturation. Furthermore, long-term incubation with the factor specifically protected astrocytes from apoptosis, implying that NDF functions in brain as a survival and maturation factor for astrocytes.
Cerebrodiene: a brain lipid isolated from sleep-deprived cats.
Authors: Lerner R A , Siuzdak G , Prospero-Garcia O , Henriksen S J , Boger D L , Cravatt B F
Published: May 2006

We report the isolation of a heretofore unrecognized brain lipid that is present in cerebrospinal fluid of sleep-deprived cats. The molecule appears to be a long-chain base structurally related to sphingosine and sphinganine in which a second unsaturated bond has been introduced. An increase in the degree of unsaturation of a key membrane component is expected to have important physiological consequences.
6-Hydroxydopamine lesions of rat substantia nigra up-regulate dopamine-induced phosphorylation of the cAMP-response element-binding protein in striatal neurons.
Authors: Cole D G , Kobierski L A , Konradi C , Hyman S E
Published: May 2006

Destruction of the substantia nigra produces striatal D1 dopamine receptor supersensitivity without increasing receptor number or affinity, thus implicating postreceptor mechanisms. The nature of these mechanisms is unknown. Increased striatal c-fos expression ipsilateral to a unilateral lesion of the substantia nigra in rats treated with appropriate dopamine agonists provides a cellular marker of D1 receptor supersensitivity. D1 receptors are positively linked to adenylate cyclase and therefore to cAMP-dependent protein kinase. Because expression of the c-fos gene in response to cAMP- and Ca2+/calmodulin-regulated protein kinases depends on phosphorylation of cAMP-response element-binding protein (CREB) at Ser-133, we examined CREB phosphorylation after dopaminergic stimulation in cultured striatal neurons and in the striatum of rats after unilateral 6-hydroxydopamine ablation of the substantia nigra. Using an antiserum specific for CREB phosphorylated at Ser-133, we found that dopamine increases CREB phosphorylation in cultured striatal neurons. This effect was blocked by a D1 antagonist. L-Dopa produced marked CREB phosphorylation in striatal neurons in rats ipsilateral, but not contralateral, to a 6-hydroxydopamine lesion. This response was blocked by a D1 antagonist, but not a D2 antagonist, and was reproduced by a D1 agonist, but not a D2 agonist. These findings are consistent with the hypothesis that D1 receptor supersensitivity is associated with upregulated activity of cAMP-dependent or Ca2+/calmodulin-dependent protein kinases, or both, following dopamine denervation of striatal neurons.
P-type ATPase from the cyanobacterium Synechococcus 7942 related to the human Menkes and Wilson disease gene products.
Authors: Phung L T , Ajlani G , Haselkorn R
Published: May 2006

DNA encoding a P-type ATPase was cloned from the cyanobacterium Synechococcus 7942. The cloned ctaA gene encodes a 790-amino acid polypeptide related to the CopA Cu(2+)-uptake ATPase of Enterococcus hirae, to other known P-type ATPases, and to the candidate gene products for the human diseases of copper metabolism, Menkes disease and Wilson disease. Disruption of the single chromosomal gene in Synechococcus 7942 by insertion of an antibiotic-resistance cassette results in a mutant cell line with increased tolerance to Cu2+ compared with the wild type.
Dual regulation of Ca2+/calmodulin-dependent kinase II activity by membrane voltage and by calcium influx.
Authors: Xiao R P , Cheng H , Lederer W J , Suzuki T , Lakatta E G
Published: May 2006

Calcium entry through voltage-gated Ca2+ channels is critical in cardiac excitation-contraction coupling and calcium metabolism. In this report, we demonstrate both spatially resolved and temporally distinct effects of Ca2+/calmodulin-dependent protein kinase II (CaMKII) on L-type Ca2+ channel current (ICa) in rat cardiac myocytes. Either depolarization alone or calcium influx can increase the amplitude and slow the inactivation of ICa. The distinct voltage- and Ca(2+)-dependent effects persist with time constants of approximately 1.7 sec and 9 sec, respectively. Both effects are completely abolished by a specific peptide inhibitor of CaMKII. This CaMKII inhibitor also suppresses the prolongation of ICa induced by depolarizing holding potentials. Furthermore, using an antibody specific for the autophosphorylated (activated) CaMKII, we find that this kinase is localized close to sarcolemmal membranes and that the profile of CaMKII activation correlates qualitatively with the changes in ICa under various conditions. Therefore, we conclude that the action of CaMKII on ICa is dually regulated by membrane depolarization and by Ca2+ influx; the latter directly activates CaMKII, whereas the former likely promotes the interaction between constitutive CaMKII and the membrane-channel proteins. These regulatory mechanisms provide positive-feedback control of Ca2+ channels and are probably important in the regulation of cardiac contractility and other intracellular Ca(2+)-regulated processes.
Phylogeny of hard- and soft-tick taxa (Acari: Ixodida) based on mitochondrial 16S rDNA sequences.
Authors: Black W C , Piesman J
Published: May 2006

Ticks are parasitiform mites that are obligate hematophagous ectoparasites of amphibians, reptiles, birds, and mammals. A phylogeny for tick families, subfamilies, and genera has been described based on morphological characters, life histories, and host associations. To test the existing phylogeny, we sequenced approximately 460 bp from the 3' end of the mitochondrial 16S rRNA gene (rDNA) in 36 hard- and soft-tick species; a mesostigmatid mite, Dermanyssus gallinae, was used as an outgroup. Phylogenies derived using distance, maximum-parsimony, or maximum-likelihood methods were congruent. The existing phylogeny was largely supported with four exceptions. In hard ticks (Ixodidae), members of Haemaphysalinae were monophyletic with the primitive Amblyomminae and members of Hyalomminae grouped within the Rhipicephalinae. In soft ticks (Argasidae), the derived phylogeny failed to support a monophyletic relationship among members of Ornithodorinae and supported placement of Argasinae as basal to the Ixodidae, suggesting that hard ticks may have originated from an Argas-like ancestor. Because most Argas species are obligate bird octoparasites, this result supports earlier suggestions that hard ticks did not evolve until the late Cretaceous.
Constitutive and inducible nitric oxide synthase gene expression, regulation, and activity in human lung epithelial cells.
Authors: Asano K , Chee C B , Gaston B , Lilly C M , Gerard C , Drazen J M , Stamler J S
Published: May 2006

Histochemical activity and immunoreactivity of nitric oxide synthase (NOS, EC 1.14.13.39) have been recently demonstrated in human lung epithelium. However, the molecular nature of NOS and the regulation and function of the enzyme(s) in the airway is not known. A549 cells (human alveolar type II epithelium-like), BEAS 2B cells (transformed human bronchial epithelial cells), and primary cultures of human bronchial epithelial cells all exhibited constitutive NOS activity that was calcium dependent and inhibitable by the NOS inhibitor NG-monomethyl-L-arginine. Nitric oxide production by epithelial cells was enhanced by culture in the presence of interferon gamma, interleukin 1 beta, tumor necrosis factor alpha, and lipopolysaccharide; the NOS activity expressed under these conditions showed less dependence on calcium, reminiscent of other inducible forms of NOS. Two distinct NOS mRNA species, homologous to previously identified constitutive brain (type I) and inducible hepatic (type II) NOS, were demonstrated by reverse transcription-polymerase chain reaction in all cell lines. Northern analysis confirmed the expression of inducible NOS mRNA. Cell culture with epidermal growth factor, a principal regulator of epithelial cell function, decreased inducible NOS activity by posttranscriptional action but did not affect constitutive NOS activity. The coexistence of constitutive and inducible NOS in human alveolar and bronchial epithelial cells is consistent with a complex mechanism evolved by epithelial cells to protect the host from microbial assault at the air/surface interface while shielding the host from the induction of airway hyperreactivity.
Hypoxia induces voltage-dependent Ca2+ entry and quantal dopamine secretion in carotid body glomus cells.
Authors: Ureña J , Fernández-Chacón R , Benot A R , Alvarez de Toledo G A , López-Barneo J
Published: May 2006

We have investigated the changes of cytosolic [Ca2+] and the secretory activity in single glomus cells dispersed from rabbit carotid bodies during exposure to solutions with variable O2 tension (Po2). In normoxic conditions (Po2 = 145 mmHg; 1 mmHg = 133 Pa), intracellular [Ca2+] was 58 +/- 29 nM, and switching to low Po2 (between 10 and 60 mmHg) led to a reversible increase of [Ca2+] up to 800 nM. The response to hypoxia completely disappeared after removal of external Ca2+ or with the addition of 0.2 mM Cd2+ to the external solution. These same solutions also abolished both the Ca2+ current of the cells and the increase of internal [Ca2+] elicited by high external K+. Elevations of cytosolic [Ca2+] in response to hypoxia or to direct membrane depolarization elicited the release of dopamine, which was detected by amperometric techniques. Dopamine secretion occurred in episodes of spike-like activity that appear to represent the release from single secretory vesicles. From the mean charge of well-resolved secretory events, we estimated the average number of dopamine molecules per vesicle to be approximately 140,000, a value about 15 times smaller than a previous estimate in chromaffin granules of adrenomedullary cells. These results directly demonstrate in a single-cell preparation the secretory response of glomus cells to hypoxia. The data indicate that the enhancement of cellular excitability upon exposure to low Po2 results in Ca2+ entry through voltage-gated channels, which leads to an increase in intracellular [Ca2+] and exocytotic transmitter release.
Killing of primary CD4+ T cells by non-syncytium-inducing macrophage-tropic human immunodeficiency virus type 1.
Authors: Yu X , McLane M F , Ratner L , O'Brien W , Collman R , Essex M , Lee T H
Published: May 2006

Understanding the mechanism by which human immunodeficiency virus type 1 (HIV-1) kills CD4+ T lymphocytes is important to the development of therapeutic and prophylactic strategies. Recent studies have indicated that, in some cases, progression to AIDS is associated with the appearance of syncytium-inducing, T cell line-tropic HIV-1 variants. Nevertheless, approximately 50% of subjects with AIDS harbor only non-syncytium-inducing, macrophage-tropic (NSI-M) variants of HIV-1. In most asymptomatic patients, NSI-M HIV-1 isolates are the predominant virus type found. We report here that cytopathicity of NSI-M HIV-1 for primary CD4+ T lymphocytes can be directly detected in vitro. The extent of CD4+ T-cell killing was not completely correlated with the rate of viral replication, suggesting that other characteristics of HIV-1 contribute to its cytopathicity. Our findings suggest that: (i) direct killing by NSI-M HIV-1 may contribute to CD4+ T-lymphocyte depletion in vivo, and (ii) the determinants of HIV-1 cytopathicity for CD4+ T lymphocytes and cell tropism or syncytia-forming ability are not necessarily tightly linked.
Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age.
Authors: Vaziri H , Dragowska W , Allsopp R C , Thomas T E , Harley C B , Lansdorp P M
Published: May 2006

The proliferative life-span of the stem cells that sustain hematopoiesis throughout life is not known. It has been proposed that the sequential loss of telomeric DNA from the ends of human chromosomes with each somatic cell division eventually reaches a critical point that triggers cellular senescence. We now show that candidate human stem cells with a CD34+CD38lo phenotype that were purified from adult bone marrow have shorter telomeres than cells from fetal liver or umbilical cord blood. We also found that cells produced in cytokine-supplemented cultures of purified precursor cells show a proliferation-associated loss of telomeric DNA. These findings strongly suggest that the proliferative potential of most, if not all, hematopoietic stem cells is limited and decreases with age, a concept that has widespread implications for models of normal and abnormal hematopoiesis as well as gene therapy.
Microsatellite alterations as clonal markers for the detection of human cancer.
Authors: Mao L , Lee D J , Tockman M S , Erozan Y S , Askin F , Sidransky D
Published: May 2006

Microsatellite instability has been reported to be an important feature of tumors from hereditary nonpolyposis colorectal carcinoma (HNPCC) patients. The recent discovery of genetic instability in small cell lung carcinoma, a neoplasm not associated with HNPCC, led us to investigate the possible presence of microsatellite alterations in other tumor types. We examined 52 microsatellite repeat sequences in the DNA of normal and tumor pairs from 100 head and neck, bladder, and lung cancer patients by the polymerase chain reaction. Although alterations were rare in dinucleotide repeats, larger (tri- or tetranucleotide) repeats were found to be more prone to expansion or deletion. We screened 100 tumors with a panel of nine tri- and tetranucleotide repeat markers and identified 26 (26%) that displayed alterations in at least one locus. This observation prompted us to examine the possibility of using microsatellite alterations as markers to detect clonal tumor-derived cell populations in pathologic samples. The identical microsatellite alterations detected in the primary tumors were successfully identified in corresponding urine, sputum, and surgical margins from affected patients. This study demonstrates that appropriately selected microsatellite loci are commonly altered in many cancers and can serve as clonal markers for their detection.
Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region.
Authors: Moi P , Chan K , Asunis I , Cao A , Kan Y W
Published: May 2006

Hypersensitive site 2 located in the beta-globin locus control region confers high levels of expression to the genes of the beta-globin cluster. A tandem repeat of the consensus sequence for the transcription factors AP1 and NF-E2 (activating protein 1 and nuclear factor erythroid 2, respectively) is present within hypersensitive site 2 and is absolutely required for strong enhancer activity. This sequence binds, in vitro and in vivo, to ubiquitous proteins of the AP1 family and to the recently cloned erythroid-specific transcription factor NF-E2. Using the tandem repeat as a recognition site probe to screen a lambda gt11 cDNA expression library from K562 cells, we isolated several DNA binding proteins. Here, we report the characterization of one of the clones isolated. The gene, which we named Nrf2 (NF-E2-related factor 2), is encoded within a 2.2-kb transcript and predicts a 66-kDa protein with a basic leucine zipper DNA binding domain highly homologous to that of NF-E2. Although Nrf2 is expressed ubiquitously, a role of this protein in mediating enhancer activity of hypersensitive site 2 in erythroid cells cannot be excluded. In this respect, Nrf2 contains a powerful acidic activation domain that may participate in the transcriptional stimulation of beta-globin genes.
Evolution of social organization: a reappraisal for primates by using phylogenetic methods.
Authors: Di Fiore A , Rendall D
Published: May 2006

For many animal taxa, the extent to which phylogeny can account for the form of species' social systems has seldom been investigated formally. A quantitative phylogenetic analysis of social systems in the order Primates reveals that social organization may be strongly conserved in some lineages, even in the face of considerable ecological variability. This result has important implications for efforts to understand the evolution of animal societies and for attempts to reconstruct the social organization of early humans.
Inhibition of fatty acid synthesis by expression of an acetyl-CoA carboxylase-specific ribozyme gene.
Authors: Ha J , Kim K H
Published: May 2006

We describe the construction of ribozyme genes that are specific to acetyl-CoA carboxylase [ACC; acetyl-CoA: carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] mRNAs and the effects of their expression on long-chain fatty acid synthesis. In a cell-free system, these ribozymes precisely cleave ACC mRNA at the expected sites. 30A5 preadipocyte cells stably transfected with the ribozyme gene show a substantial reduction in the amount of ACC mRNA as compared to non-ribozyme-expressing cells. The decrease in ACC mRNA was associated with a significant decrease in ACC enzyme activity, and the rate of fatty acid synthesis fell to about 30-70% of the control. When these cells are induced to differentiate into adipocytes, lipid accumulation is very slow in comparison with control cells. The activity of fatty acid synthase and the mRNA level of beta-actin were not affected. These data indicate that ribozymes designed to specifically target ACC mRNA under in vivo conditions act by decreasing the ACC mRNA level, which, in turn, decreases fatty acid synthesis.
Left-handed Z-DNA and in vivo supercoil density in the Escherichia coli chromosome.
Authors: Lukomski S , Wells R D
Published: May 2006

A system for studying Z-DNA formation in the Escherichia coli chromosome was developed. Prior investigations in recombinant plasmids showed that alternating (Pur-Pyr) sequences can adopt a left-handed Z-DNA conformation both in vitro and in vivo. We constructed mobile, transposon-based cassettes carrying cloned (Pur-Pyr) sequences containing an EcoRI site in the center. These cassettes were subsequently inserted into different locations in the E. coli chromosome in a random fashion. A number of stable insertions were characterized by Southern analysis and pulsed-field gel electrophoresis mapping. A cloned temperature-sensitive MEcoRI methylase was expressed in trans as the probe to study Z-DNA formation in vivo. In this system, the control EcoRI sites were quickly methylated when cells were placed at the permissive temperature. Strong inhibition of the methylation was observed, however, only for the EcoRI sites embedded in a 56-bp run of (C-G). In contrast, the shorter sequence of 32 bp did not show this behavior. Prior in vitro determinations revealed that the longer tract required less energy to stabilize the Z-helix than the shorter block. We conclude that the observed inhibition of methylation is due to Z-DNA formation in the E. coli chromosome. In vitro, these sequences undergo the B- to Z-DNA transition at a supercoil density of -0.026 for the 56-bp insert and -0.032 for the 32-bp block. Since only the longer (C-G) tract but not the shorter run adopted the left-handed conformation in the chromosome, we propose that these densities establish the boundaries in the different chromosomal loci investigated; these boundaries are in good agreement with the extremes found in plasmids.
Food restriction eliminates preneoplastic cells through apoptosis and antagonizes carcinogenesis in rat liver.
Authors: Grasl-Kraupp B , Bursch W , Ruttkay-Nedecky B , Wagner A , Lauer B , Schulte-Hermann R
Published: May 2006

Restriction of dietary calories reduces cancer formation in experimental animals and probably also in humans. This effect is generally attributed to the inhibitory effect of fasting on cell proliferation. Here we studied the effect of fasting on physiological cell death through apoptosis by using rat liver as a model. (i) In normal liver, involution of hyperplasia by apoptosis was reinforced by food withdrawal and suppressed by feeding. Complete food withdrawal for 8 days or food reduction by 40% for 3 months eliminated 20-30% of normal liver cells through apoptosis. (ii) Putative preneoplastic liver foci exhibited severalfold higher rates of DNA replication and apoptosis than unaltered liver. Food restriction lowered DNA replication but increased apoptosis, which reduced the number and volume of putative preneoplastic liver foci by 85% within 3 months. Subsequent return to ad libitum feeding normalized cell replication and apoptosis but clear differences in the volume and number of putative preneoplastic liver foci persisted throughout the following 17 months. Treatment of animals after food restriction with nafenopin, a peroxisome proliferator and potent tumor promoter, produced only half as many hepatocellular adenomas and carcinomas as in animals fed unrestrictedly throughout their lifetime. This indicates that food restriction had actually eliminated a part of the initiated cells. This study demonstrates that food restriction preferentially enhances apoptosis of preneoplastic cells. This effect in combination with lowered cell replication provides protection from carcinogenesis.
A truncated bone morphogenetic protein receptor affects dorsal-ventral patterning in the early Xenopus embryo.
Authors: Suzuki A , Thies R S , Yamaji N , Song J J , Wozney J M , Murakami K , Ueno N
Published: May 2006

Bone morphogenetic proteins (BMPs), which are members of the transforming growth factor beta (TGF-beta) superfamily, have been implicated in bone formation and the regulation of early development. To better understand the roles of BMPs in Xenopus laevis embryogenesis, we have cloned a cDNA coding for a serine/threonine kinase receptor that binds BMP-2 and BMP-4. To analyze its function, we attempted to block the BMP signaling pathway in Xenopus embryos by using a dominant-negative mutant of the BMP receptor. When the mutant receptor lacking the putative serine/threonine kinase domain was expressed in ventral blastomeres of Xenopus embryos, these blastomeres were respecified to dorsal mesoderm, eventually resulting in the formation of a secondary body axis. These findings suggest that endogenous BMP-2 and BMP-4 are involved in the dorsal-ventral specification in the embryo and that ventral fate requires induction rather than resulting from an absence of dorsal specification.
The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system DnaK, DnaJ, and GrpE.
Authors: Szabo A , Langer T , Schröder H , Flanagan J , Bukau B , Hartl F U
Published: May 2006

Molecular chaperones of the Hsp70 class bind unfolded polypeptide chains and are thought to be involved in the cellular folding pathway of many proteins. DnaK, the Hsp70 protein of Escherichia coli, is regulated by the chaperone protein DnaJ and the cofactor GrpE. To gain a biologically relevant understanding of the mechanism of Hsp70 action, we have analyzed a model reaction in which DnaK, DnaJ, and GrpE mediate the folding of denatured firefly luciferase. The binding and release of substrate protein for folding involves the following ATP hydrolysis-dependent cycle: (i) unfolded luciferase binds initially to DnaJ; (ii) upon interaction with luciferase-DnaJ, DnaK hydrolyzes its bound ATP, resulting in the formation of a stable luciferase-DnaK-DnaJ complex; (iii) GrpE releases ADP from DnaK; and (iv) ATP binding to DnaK triggers the release of substrate protein, thus completing the reaction cycle. A single cycle of binding and release leads to folding of only a fraction of luciferase molecules. Several rounds of ATP-dependent interaction with DnaK and DnaJ are required for fully efficient folding.
Disproportionate growth in mice with Igf-2 transgenes.
Authors: Ward A , Bates P , Fisher R , Richardson L , Graham C F
Published: May 2006

Injection transgenesis was used to study the long-term effects of excess insulin-like growth factor II on mouse growth and differentiation. By using a construct in which the coding region of the mouse insulin like growth factor II gene (Igf-2) was placed under the control of a keratin gene promoter, four transgenic lines were established, all of which displayed overgrowth of the skin as judged by wrinkling. In addition to high levels of expression in the skin, transgene transcripts were also present in the alimentary canal and uterus. At most of the sites of transgene expression the cell number (DNA content) was greatly increased, indicating a local action of the excess insulin-like growth factor II on cell multiplication. Adult total live weight was slightly increased and there was no macroscopic evidence of tumor formation. The characteristics of these transgenic mice indicate distinct local and systemic actions for insulin-like growth factor II.
The prolactin gene is expressed in the hypothalamic-neurohypophyseal system and the protein is processed into a 14-kDa fragment with activity like 16-kDa prolactin.
Authors: Clapp C , Torner L , Gutiérrez-Ospina G , Alcántara E , López-Gómez F J , Nagano M , Kelly P A , Mejía S , Morales M A , Martínez de la Escalera G
Published: May 2006

The 23-kDa form of prolactin (PRL) has been proposed to function as both a mature hormone and a prohormone precursor for different uniquely bioactive forms of the molecule. We have shown that the 16-kDa N-terminal fragment of PRL (16K PRL) inhibits angiogenesis via a specific receptor. In addition, 16K PRL stimulates natriuresis and diuresis in the rat, and kidney membranes contain high-affinity specific binding sites for this PRL fragment. 16K PRL can be derived from an enzymatically cleaved form of PRL (cleaved PRL). With the use of a specific 16K PRL antiserum, we have localized a 14-kDa immunoreactive protein in the paraventricular and supraoptic nuclei of the hypothalamus and in the neurohypophysis. Reverse transcription-polymerase chain reaction of RNA from isolated paraventricular nuclei showed the expression of the full-length PRL mRNA. The neurohypophysis was found to contain the enzymes that produce cleaved PRL, small amounts of PRL, and cleaved PRL. Medium conditioned by neurohypophyseal cultures, enriched with the 14-kDa immunoreactive protein, has antiangiogenic effects that are blocked by the 16K PRL antiserum. These results are consistent with the expression of PRL in the hypothalamic-neurohypophyseal system, and the preferential processing of the protein into a 14-kDa fragment with biological and immunological properties of 16K PRL.
Expression of cAMP-dependent protein kinase in prespore cells is sufficient to induce spore cell differentiation in Dictyostelium.
Authors: Mann S K , Richardson D L , Lee S , Kimmel A R , Firtel R A
Published: May 2006

The activity of cAMP-dependent protein kinase (PKA) is required for proper development at several stages during the Dictyostelium life cycle. We present evidence that activation of PKA is rate-limiting for the differentiation of prespore cells to spores and that PKA activation may be the developmental trigger for sporulation. Strains that overexpress the gene encoding the catalytic subunit of PKA (PKAcat) or lack a functional regulatory subunit (rdeC strains) undergo rapid, heterochronic development. We show that overexpression of PKAcat in prespore cell is sufficient to directly induce expression of the spore maturation marker spiA and differentiation to spores, in a cell-autonomous manner. Moreover, overexpression of PKAcat in prespore cells can bypass a mutation that blocks an earlier developmental step to induce spiA expression. Our results suggest that the regulatory pathway in prespore cells between the activation of PKA and spiA induction/spore maturation is quite short and that PKAcat expression in prespore cells may mediate spore differentiation at the level of transcription. This induction of sporulation requires the prior activation of the prespore cell pathway. In addition, we show that beta-galactosidase activity expressed from a PKAcat promoter/lacZ reporter construct is highly enriched in the anterior prestalk A region during the tipped aggregate, slug, and early culminant stages and that this pattern switches abruptly to a prespore pattern at the time of spore maturation, supporting the proposed role of PKA in this process.
Localization and functional properties of a rat brain alpha 1A calcium channel reflect similarities to neuronal Q- and P-type channels.
Authors: Stea A , Tomlinson W J , Soong T W , Bourinet E , Dubel S J , Vincent S R , Snutch T P
Published: May 2006

Functional expression of the rat brain alpha 1A Ca channel was obtained by nuclear injection of an expression plasmid into Xenopus oocytes. The alpha 1A Ca current activated quickly, inactivated slowly, and showed a voltage dependence typical of high voltage-activated Ca channels. The alpha 1A current was partially blocked (approximately 23%) by omega-agatoxin IVA (200 nM) and substantially blocked by omega-conotoxin MVIIC (5 microM blocked approximately 70%). Bay K 8644 (10 microM) or omega-conotoxin GVIA (1 microM) had no significant effect on the alpha 1A current. Coexpression with rat brain Ca channel beta subunits increased the alpha 1A whole-cell current and shifted the current-voltage relation to more negative values. While the beta 1b and beta 3 subunits caused a significant acceleration of the alpha 1A inactivation kinetics, the beta 2a subunit dramatically slowed the inactivation of the alpha 1A current to that seen typically for P-type Ca currents. In situ localization with antisense deoxyoligonucleotide and RNA probes showed that alpha 1A was widely distributed throughout the rat central nervous system, with moderate to high levels in the olfactory bulb, in the cerebral cortex, and in the CA fields and dentate gyrus of the hippocampus. In the cerebellum, prominent alpha 1A expression was detected in Purkinje cells with some labeling also in granule cells. Overall, the results show that alpha 1A channels are widely expressed and share some properties with both Q- and P-type channels.
Purification and properties of double-stranded RNA-specific adenosine deaminase from calf thymus.
Authors: O'Connell M A , Keller W
Published: May 2006

A double-stranded RNA-specific adenosine deaminase, which converts adenosine to inosine, has been purified to homogeneity from calf thymus. The enzyme was purified approximately 340,000-fold by a series of column chromatography steps. The enzyme consists of a single polypeptide with a molecular mass of 116 kDa as determined by electrophoresis on a SDS/polyacrylamide gel. The native protein sediments at 4.2 s in glycerol gradients and has a Stokes radius of 42 A upon gel-filtration chromatography. This leads to an estimate of approximately 74,100 for the native molecular weight, suggesting that the enzyme exists as a monomer in solution. Enzyme activity is optimal at 0.1 M KCl and 37 degrees C. Divalent metal ions or ATP is not required for activity. The Km for double-stranded RNA substrate is approximately 7 x 10(-11) M. The Vmax is approximately 10(-9) mol of inosine produced per min per mg and the Kcat is 0.13 min-1.
Phorbol ester-induced stimulation and phosphorylation of adenylyl cyclase 2.
Authors: Jacobowitz O , Iyengar R
Published: May 2006

Adenylyl cyclase 2 was expressed in Sf9 cells by recombinant baculovirus infection. Phorbol 12-myristate 13-acetate (PMA) treatment of cells expressing adenylyl cyclase 2 (AC2) increased basal activity. This increase was blocked by staurosporine, a protein kinase C inhibitor. PMA treatment increased Vmax without affecting Km. Greatest increase in basal activity was seen at physiologically relevant Mg2+ concentrations. PMA treatment did not alter sensitivity to guanine nucleotide stimulatory factor (Gs) but enhanced stimulation at all concentrations of activated Gs alpha subunit tested. AC2 was tagged at the N terminus with an 8-amino acid epitope. Epitope-tagged AC2 was purified to apparent homogeneity in a single step by using an antiepitope antibody-affinity column. The eluate was resolved by SDS/PAGE. Silver staining of the gel showed a 106-kDa band. The purified protein was recognized by antipeptide antibody against a region common to all mammalian adenylyl cyclases. The epitope-tagged enzyme expressed in Sf9 cells was also stimulated by PMA. When cells were labeled with 32P and treated with PMA, a 3-fold increase in 32P incorporation of purified epitope-tagged AC2 was observed. We conclude that activation of protein kinase C results in phosphorylation and stimulation of AC2, a cell-surface G protein effector enzyme. Thus, covalent modification of cell-surface effectors may provide an independent mode for signal transmission through G protein pathways.
Progressive de novo DNA methylation at the bcr-abl locus in the course of chronic myelogenous leukemia.
Authors: Zion M , Ben-Yehuda D , Avraham A , Cohen O , Wetzler M , Melloul D , Ben-Neriah Y
Published: May 2006

De novo methylation of CpG islands is a rare event in mammalian cells. It has been observed in the course of developmental processes, such as X chromosome inactivation and genomic imprinting. The methylation of DNA, an important factor in the epigenetic control of gene expression, may also be involved in tumorigenesis. After the t(9;22) chromosomal translocation and generation of the Philadelphia chromosome, the initiating event in chronic myelogenous leukemia (CML), most of the abl coding sequence is fused to the 5' region of the bcr gene. Expression of the hybrid bcr-abl gene is, therefore, regulated by the bcr promoter. In most cases of CML, one of the two abl promoters (Pa) is nested within the bcr-abl transcriptional unit and should be able to transcribe the type Ia 6-kb normal abl mRNA from the Philadelphia chromosome. However, we have found that the 6-kb transcript is present only in CML cell lines containing a normal abl allele and that the apparent inactivation of the nested Pa promoter is associated with allele-specific methylation. Furthermore, we have noticed that the Pa promoter is contained within a CpG island and undergoes progressive de novo methylation in the course of the disease. This is attested to by the fact that DNA samples from CML patients that are methylation-free at the time of diagnosis invariably become methylated in advanced CML. Since tumor progression in CML cannot always be inferred from the clinical presentation, assessment of de novo CpG methylation may prove to be of critical value in management of the disease. It could herald blastic transformation at a stage when bone marrow transplantation, the only potentially curative therapeutic procedure in CML, is still effective.
In vivo suppression of injury-induced vascular smooth muscle cell accumulation using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene.
Authors: Guzman R J , Hirschowitz E A , Brody S L , Crystal R G , Epstein S E , Finkel T
Published: May 2006

Restenosis, a process characterized in part by excessive smooth muscle cell (SMC) proliferation in areas of vascular injury, occurs in up to 50% of patients undergoing balloon angioplasty. In an effort to develop a treatment strategy for restenosis, we constructed a replication-deficient recombinant adenovirus (AdMLP.HSTK) containing the herpes simplex virus thymidine kinase gene (HSV tk). This viral gene product phosphorylates the prodrug ganciclovir to form a nucleoside analog that inhibits DNA synthesis. Cultured primary rat SMCs infected with AdMLP.HSTK were completely growth-inhibited by incubation in ganciclovir-containing medium. In addition, when only a portion of the SMC population received the HSV tk transgene, an inhibitory effect on neighboring SMCs was evident. Evaluation of this strategy in vivo using a rat carotid balloon injury model demonstrated that local infection of injured arteries with AdMLP.-HSTK followed by 2 weeks of systemic ganciclovir treatment significantly (P < 0.01) reduced injury-induced SMC accumulation. In contrast, there was no suppression of injury-induced SMC accumulation in animals infected with AdMLP.HSTK but not receiving ganciclovir or in those animals infected with a control adenovirus and either treated or not treated with ganciclovir. These results demonstrate the potential utility of adenovirus-mediated gene transfer for treatment of restenosis after balloon injury.
On the nature and differential distribution of mRNAs in hippocampal neurites: implications for neuronal functioning.
Authors: Miyashiro K , Dichter M , Eberwine J
Published: May 2006

Neurons are highly polarized cells with a mosaic of cytoplasmic and membrane proteins differentially distributed in axons, dendrites, and somata. In Drosophila and Xenopus, mRNA localization coupled with local translation is a powerful mechanism by which regionalized domains of surface or cytoplasmic proteins are generated. In neurons, there is substantial ultrastructural evidence positing the presence of protein synthetic machinery in neuronal processes, especially at or near postsynaptic sites. There are, however, remarkably few reports of mRNAs localized to these regions. We now present direct evidence that an unexpectedly large number of mRNAs, including members of the glutamate receptor family, second messenger system, and components of the translational control apparatus, are present in individual processes of hippocampal cells in culture.