bluuebunny/crossref_metadata_2025 huggingface dataset:
Sperm capacitation in humans is transient and correlates with chemotactic responsiveness to follicular factors.
Authors: Cohen-Dayag A , Tur-Kaspa I , Dor J , Mashiach S , Eisenbach M
Published: May 2006

In humans, only a small fraction (2-12%) of a sperm population can respond by chemoattraction to follicular factors. This recent finding led to the hypothesis that chemotaxis provides a mechanism for selective recruitment of functionally mature spermatozoa (i.e., of capacitated spermatozoa, which possess the potential to undergo the acrosome reaction and fertilize the egg). This study aimed to examine this possibility. Capacitated spermatozoa were identified by their ability to undergo the acrosome reaction upon stimulation with phorbol 12-myristate 13-acetate. Under capacitating conditions, only a small portion (2-14%) of the spermatozoa were found to be capacitated. The spermatozoa were then separated according to their chemotactic activity, which resulted in a subpopulation enriched with chemotactically responsive spermatozoa and a subpopulation depleted of such spermatozoa. The level of capacitated spermatozoa in the former was approximately 13-fold higher than that in the latter. The capacitated state was temporary (50 min < life span < 240 min), and it was synchronous with the chemotactic activity. A continuous process of replacement of capacitated/chemotactic spermatozoa within a sperm population was observed. Spermatozoa that had stopped being capacitated did not become capacitated again, which indicates that the capacitated state is acquired only once in a sperm's lifetime. A total sperm population depleted of capacitated spermatozoa stopped being chemotactic. When capacitated spermatozoa reappeared, chemotactic activity was restored. These observations suggest that spermatozoa acquire their chemotactic responsiveness as part of the capacitation process and lose this responsiveness when the capacitated state is terminated. We suggest that the role of sperm chemotaxis in sperm-egg interaction in vivo may indeed be selective recruitment of capacitated spermatozoa for fertilizing the egg.
Restriction-modification systems as genomic parasites in competition for specific sequences.
Authors: Kusano K , Naito T , Handa N , Kobayashi I
Published: May 2006

Restriction-modification (RM) systems are believed to have evolved to protect cells from foreign DNA. However, this hypothesis may not be sufficient to explain the diversity and specificity in sequence recognition, as well as other properties, of these systems. We report that the EcoRI restriction endonuclease-modification methylase (rm) gene pair stabilizes plasmids that carry it and that this stabilization is blocked by an RM of the same sequence specificity (EcoRI or its isoschizomer, Rsr I) but not by an RM of a different specificity (PaeR7I) on another plasmid. The PaeR7I rm likewise stabilizes plasmids, unless an rm gene pair with identical sequence specificity is present. Our analysis supports the following model for stabilization and incompatibility: the descendants of cells that have lost an rm gene pair expose the recognition sites in their chromosomes to lethal attack by any remaining restriction enzymes unless modification by another RM system of the same specificity protects these sites. Competition for specific sequences among these selfish genes may have generated the great diversity and specificity in sequence recognition among RM systems. Such altruistic suicide strategies, similar to those found in virus-infected cells, may have allowed selfish RM systems to spread by effectively competing with other selfish genes.
Antibodies against the T61 antigen inhibit neuronal migration in the chick optic tectum.
Authors: Meyer S U , Henke-Fahle S
Published: May 2006

Cell migration in the central nervous system depends, in part, on receptors and extracellular matrix molecules that likewise support axonal outgrowth. We have investigated the influence of T61, a monoclonal antibody that has been shown to inhibit growth cone motility in vitro, on neuronal migration in the developing optic tectum. Intraventricular injections of antibody-producing hybridoma cells or ascites fluid were used to determine the action of this antibody in an in vivo environment. To document alterations in tectal layer formation, a combination of cell-nuclei staining and axonal immunolabeling methods was employed. In the presence of T61 antibody, cells normally destined for superficial layers accumulated in the ventricular zone instead, leading to a reduction of the cell-dense layer in the tectal plate. Experiments with 5-bromo-2'-deoxyuridine labeling followed by antibody staining confirmed that the nonmigrating cells remaining in the ventricular zone were postmitotic and had differentiated. The structure of radial glial cells, as judged by staining with a glia-specific antibody and the fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), remained intact in these embryos. Our findings suggest that the T61 epitope is involved in a mechanism underlying axonal extension and neuronal migration, possibly by influencing the motility of the leading process.
Spatial heterogeneity of inhibitory surrounds in the middle temporal visual area.
Authors: Xiao D K , Raiguel S , Marcar V , Koenderink J , Orban G A
Published: May 2006

A recurrent theme in the organization of vertebrate visual cortex is that of receptive fields with an associated "silent" opponency component. In the middle temporal area (area MT), a cortical visual area involved in the analysis of retinal motion in primates, this opponency appears in the form of a region outside the classical receptive field (CRF) that in itself gives no response but suppresses responses to motion evoked within the CRF. This antagonistic motion surround has been described as very large and symmetrically arrayed around the CRF. On the basis of this view, the primary function of the surround has long been thought to consist of simple figure-ground segregation based on movement. We have made use of small stimulus patches to map the form and extent of the surround and find evidence that the surround inhibition of many MT cells is in fact confined to restricted regions on one side or on opposite sides of the CRF. Such regions endow MT cells with the ability to make local-to-local motion comparisons, capable of extracting more complex features from the visual environment, and as such, may be better viewed as intrinsic parts of the receptive field, rather than as separate entities responsible for local-to-global comparisons.
Inhibition of ascorbate peroxidase by salicylic acid and 2,6-dichloroisonicotinic acid, two inducers of plant defense responses.
Authors: Durner J , Klessig D F
Published: May 2006

In recent years, it has become apparent that salicylic acid (SA) plays an important role in plant defense responses to pathogen attack. Previous studies have suggested that one of SA's mechanisms of action is the inhibition of catalase, resulting in elevated levels of H2O2, which activate defense-related genes. Here we demonstrate that SA also inhibits ascorbate peroxoidase (APX), the other key enzyme for scavenging H2O2. The synthetic inducer of defense responses, 2,6-dichloroisonicotinic acid (INA), was also found to be an effective inhibitor of APX. In the presence of 750 microM ascorbic acid (AsA), substrate-dependent IC50 values of 78 microM and 95 microM were obtained for SA and INA, respectively. Furthermore, the ability of SA analogues to block APX activity correlated with their ability to induce defense-related genes in tobacco and enhance resistance to tobacco mosaic virus. Inhibition of APX by SA appears to be reversible, thus differing from the time-dependent, irreversible inactivation by suicide substrates such as p-aminophenol. In contrast to APX, the guaiacol-utilizing peroxidases, which participate in the synthesis and crosslinking of cell wall components as part of the defense response, are not inhibited by SA or INA. The inhibition of both catalase and APX, but not guaiacol peroxidases, supports the hypothesis that SA-induced defense responses are mediated, in part, through elevated H2O2 levels or coupled perturbations of the cellular redox state.
Structure and evolution of the compact radio source in NGC 1275.
Authors: Romney J D , Benson J M , Dhawan V , Kellermann K I , Vermeulen R C , Walker R C
Published: May 2006

Investigations of the fine-scale structure in the compact nucleus of the radio source 3C 84 in NGC 1275 (New General Catalogue number) are reported. Structural monitoring observations beginning as early as 1976, and continuing to the present, revealed subluminal motions in a jet-like relatively diffuse region extending away from a flat-spectrum core. A counterjet feature was discovered in 1993, and very recent nearly simultaneous studies have detected the same feature at five frequencies ranging from 5 to 43 GHz. The counterjet exhibits a strong low-frequency cutoff, giving this region of the source an inverted spectrum. The observations are consistent with a physical model in which the cutoff arises from free-free absorption in a volume that surrounds the core but obscures only the counterjet feature. If such a model is confirmed, very-long-baseline radio interferometry observations can then be used to probe the accretion region, outside the radio jet, on parsec scales.
Evolution of powerful extragalactic radio sources.
Authors: Readhead A C
Published: May 2006

Observations of complete flux density limited samples of powerful extragalactic radio sources by very-long-baseline interferometry enable us to study the evolution of these objects over the range of linear scales from 1 parsec to 15 kiloparsees (1 parsec = 3.09 x 10(18) cm). The observations are consistent with the unifying hypothesis that compact symmetric objects evolve into compact steep-spectrum doubles, which in turn evolve into large-scale Fanaroff-Riley class II objects. It is suggested that this is the primary evolutionary track of powerful extragalactic radio sources. In this case there must be significant luminosity evolution in these objects, but little velocity evolution, as they expand from 1 parsec to several hundred kiloparsecs in overall size.
Microsatellite variability and genetic distances.
Authors: Zhivotovsky L A , Feldman M W
Published: May 2006

We analyze the within- and between-population dynamics of the distribution of the number of repeats at multiple microsatellite DNA loci subject to stepwise mutation. Analytical expressions for moments up to the fourth order within a locus and the variance of between-locus variance at mutation-drift equilibrium have been obtained. These statistics may be used to test the appropriateness of the one-step mutation model and to detect between-locus variation in the mutation rate. Published data are compatible with the one-step mutation model, although they do not reject the two-step model. Using both multinomial sampling and diffusion approximations for the analysis of the genetic distance introduced by Goldstein et al. [Goldstein, D. B., Linares, A. R., Cavalli-Sforza, L. L. & Feldman, M. W. (1995) Proc. Natl. Acad. Sci. USA 92, 6723-6727], we show that this distance follows a chi 2 distribution with degrees of freedom equal to the number of loci when there is no variation in mutation rates among the loci. In the presence of such variation, the variance of the distance is obtained. We conclude that the number of microsatellite loci required for the construction of phylogenetic trees with reliable branch lengths may be several hundred. Also, mutations that change repeat scores by several units, even though extremely rare, may dramatically influence estimates of population parameters.
Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas.
Authors: Suzich J A , Ghim S J , Palmer-Hill F J , White W I , Tamura J K , Bell J A , Newsome J A , Jenson A B , Schlegel R
Published: May 2006

Infection of mucosal epithelium by papillomaviruses is responsible for the induction of genital and oral warts and plays a critical role in the development of human cervical and oropharyngeal cancer. We have employed a canine model to develop a systemic vaccine that completely protects against experimentally induced oral mucosal papillomas. The major capsid protein, L1, of canine oral papillomavirus (COPV) was expressed in Sf9 insect cells in native conformation. L1 protein, which self-assembled into virus-like particles, was purified on CsCl gradients and injected intradermally into the foot pad of beagles. Vaccinated animals developed circulating antibodies against COPV and became completely resistant to experimental challenge with COPV. Successful immunization was strictly dependent upon native L1 protein conformation and L1 type. Partial protection was achieved with as little as 0.125 ng of L1 protein, and adjuvants appeared useful for prolonging the host immune response. Serum immunoglobulins passively transferred from COPV L1-immunized beagles to naive beagles conferred protection from experimental infection with COPV. Our results indicate the feasibility of developing a human vaccine to prevent mucosal papillomas, which can progress to malignancy.
The LAR/PTP delta/PTP sigma subfamily of transmembrane protein-tyrosine-phosphatases: multiple human LAR, PTP delta, and PTP sigma isoforms are expressed in a tissue-specific manner and associate with the LAR-interacting protein LIP.1.
Authors: Pulido R , Serra-Pagès C , Tang M , Streuli M
Published: May 2006

The transmembrane protein-tyrosine-phosphatases (PTPases) LAR, PTP delta, and PTP sigma each contain two intracellular PTPase domains and an extracellular region consisting of Ig-like and fibronectin type III-like domains. We describe the cloning and characterization of human PTP sigma (HPTP sigma) and compare the structure, alternative splicing, tissue distribution, and PTPase activity of LAR, HPTP delta, and HPTP sigma, as well their ability to associate with the intracellular coiled-coil LAR-interacting protein LIP.1. Overall, these three PTPases are structurally very similar, sharing 64% amino acid identity. Multiple isoforms of LAR, HPTP delta, and HPTP sigma appear to be generated by tissue-specific alternative splicing of up to four mini-exon segments that encode peptides of 4-16 aa located in both the extracellular and intracellular regions. Alternative usage of these peptides varies depending on the tissue mRNA analyzed. Short isoforms of both HPTP sigma and HPTP delta were also detected that contain only four of the eight fibronectin type III-like domains. Northern blot analysis indicates that LAR and HPTP sigma are broadly distributed whereas HPTP delta expression is largely restricted to brain, as is the short HPTP sigma isoform containing only four fibronectin type III-like domains. LAR, HPTP delta, and HPTP sigma exhibit similar in vitro PTPase activities and all three interact with LIP.1, which has been postulated to recruit LAR to focal adhesions. Thus, these closely related PTPases may perform similar functions in various tissues.
Cytochrome P450 1A1 promoter as a genetic switch for the regulatable and physiological expression of a plasma protein in transgenic mice.
Authors: Smith J D , Wong E , Ginsberg M
Published: May 2006

Transgenic and gene knockout techniques allow for in vivo study of the consequences of adding or subtracting specific genes. However, in some instances, such as the study of lethal mutations or of the physiological consequences of changing gene expression, turning on and off an introduced gene at will would be advantageous. We have used cytochrome p450 1A1 promoter to drive expression of the human apolipoprotein E (apoE) gene in transgenic mice. In six independent lines, robust expression of the transgene depended upon injection of the inducer beta-naphthoflavone, whereas the seventh line had high basal expression that was augmented further by the inducer. The low level of basal expression in an inducer-dependent line was confirmed upon breeding the transgene onto the hypercholesterolemic apoE-deficient background. In the basal state transgene expression was physiologically insignificant, as these mice were as hypercholesterolemic as their nontransgenic apoE-deficient littermates. When injected with the inducer, plasma cholesterol levels of the transgenic mice decreased dramatically as apoE expression was induced to yield greater than physiological levels in plasma. The inducer could pass transplacentally from an injected mother to her fetuses with concomitant induction of fetal transgene mRNA. Inducer could also pass via breast milk from an injected mother to her suckling neonatal pups, giving rise to the induction of human apoE in neonate plasma. These finding suggest a strategy to temporarily ameliorate genetic deficiencies that would otherwise lead to fetal or neonatal lethality.
Targeting p53 as a general tumor antigen.
Authors: Theobald M , Biggs J , Dittmer D , Levine A J , Sherman L A
Published: May 2006

A major barrier to the design of immunotherapeutics and vaccines for cancer is the idiosyncratic nature of many tumor antigens and the possibility that T cells may be tolerant of broadly distributed antigens. We have devised an experimental strategy that exploits species differences in protein sequences to circumvent tolerance of high-affinity T cells. HLA transgenic mice were used to obtain cytotoxic T lymphocytes specific for peptides from the human p53 tumor-suppressor molecule presented in association with HLA-A2.1. Although such p53-specific cytotoxic T cells did not recognize nontransformed human cells, they were able to lyse a wide variety of human tumor cells lines, thus confirming the existence of broadly distributed determinants that may serve as targets for immunotherapy.
Cocaine: mechanism of inhibition of a muscle acetylcholine receptor studied by a laser-pulse photolysis technique.
Authors: Niu L , Abood L G , Hess G P
Published: May 2006

Effects of cocaine on the muscle nicotinic acetylcholine receptor were investigated by using a chemical kinetic technique with a microsecond time resolution. This membrane-bound receptor regulates signal transmission between nerve and muscle cells, initiates muscle contraction, and is inhibited by cocaine, an abused drug. The inhibition mechanism is not well understood because of the lack of chemical kinetic techniques with the appropriate (microsecond) time resolution. Such a technique, utilizing laser-pulse photolysis, was recently developed; by using it the following results were obtained. (i) The apparent cocaine dissociation constant of the closed-channel receptor form is approximately 50 microM. High carbamoylcholine concentration and, therefore, increased concentrations of the open-channel receptor form, decrease receptor affinity for cocaine approximately 6-fold. (ii) The rate of the receptor reaction with cocaine is at least approximately 30-fold slower than the channel-opening rate, resulting in a cocaine-induced decrease in the concentration of open receptor channels without a concomitant decrease in the channel-opening or -closing rates. (iii) The channel-closing rate increases approximately 1.5-fold as the cocaine concentration is increased from 20 to 60 microM but then remains constant as the concentration is increased further. The results are consistent with a mechanism in which cocaine first binds rapidly to a regulatory site of the receptor, which can still form transmembrane channels. Subsequently, a slow step (t1/2 approximately 70 ms) leads to a receptor form that cannot form transmembrane channels, and acetylcholine receptor-mediated signal transmission is, therefore, blocked. Implications for the search for therapeutic agents that alleviate cocaine poisoning are mentioned.
Three distinct structural environments of a transmembrane domain in the inwardly rectifying potassium channel ROMK1 defined by perturbation.
Authors: Choe S , Stevens C F , Sullivan J M
Published: May 2006

To probe the protein environment of an ion channel, we have perturbed the structure of a transmembrane domain by substituting side chains with those of two different sizes by using site-specific mutagenesis. We have used Trp and Ala as a high- and a low-impact perturbation probe, respectively, to replace each of 18 consecutive residues within the putative second transmembrane segment, M2, of an inwardly rectifying potassium channel, ROMK1. Our rationale is that a change in the channel function as a consequence of these mutations at a particular position will reflect the structural environment of the altered side chain. Each position can then be assigned to one of three classes of environments, as grated by different levels of perturbation: very tolerant (channel functions with both Trp and Ala substitutions), tolerant (function preserved with Ala but not with Trp substitution), and intolerant (either Ala or Trp substitution destroys function). We identify the very tolerant environment as being lipid-facing, tolerant as protein-interior-facing, and intolerant as pore-facing. We observe a strikingly ordered pattern of perturbation of all three environmental classes. This result indicates that M2 is a straight alpha-helix.
Active site topology and reaction mechanism of GTP cyclohydrolase I.
Authors: Nar H , Huber R , Auerbach G , Fischer M , Hösl C , Ritz H , Bracher A , Meining W , Eberhardt S , Bacher A
Published: May 2006

GTP cyclohydrolase I of Escherichia coli is a torus-shaped homodecamer with D5 symmetry and catalyzes a complex ring expansion reaction conducive to the formation of dihydroneopterin triphosphate from GTP. The x-ray structure of a complex of the enzyme with the substrate analog, dGTP, bound at the active site was determined at a resolution of 3 A. In the decamer, 10 equivalent active sites are present, each of which contains a 10-A deep pocket formed by surface areas of 3 adjacent subunits. The substrate forms a complex hydrogen bond network with the protein. Active site residues were modified by site-directed mutagenesis, and enzyme activities of the mutant proteins were measured. On this basis, a mechanism of the enzyme-catalyzed reaction is proposed. Cleavage of the imidazole ring is initiated by protonation of N7 by His-179 followed by the attack of water at C8 of the purine system. Cystine Cys-110 Cys-181 may be involved in this reaction step. Opening of the imidazole ring may be in concert with cleavage of the furanose ring to generate a Schiff's base from the glycoside. The gamma-phosphate of GTP may be involved in the subsequent Amadori rearrangement of the carbohydrate side chain by activating the hydroxyl group of Ser-135.
The stem cell antigen CD34 functions as a regulator of hemopoietic cell adhesion.
Authors: Healy L , May G , Gale K , Grosveld F , Greaves M , Enver T
Published: May 2006

Although the CD34 antigen is widely used in the identification and purification of hemopoietic stem and progenitor cells, its function within hemopoiesis is unknown. We have investigated this issue by ectopically expressing human (hu) CD34 on the surface of murine hemopoietic cells. Forced expression of hu-CD34 in the thymocytes of transgenic mice did not appear to affect the development, maturation, or distribution of murine T cells but did significantly increase their ability to adhere to bone marrow stromal layers of human but not mouse origin. Ectopic expression of hu-CD34 on murine 416B cells, a multipotential progenitor that expresses murine CD34, yielded similar results. In both cases hu-CD34-dependent adhesion was enhanced by molecular engagement of the hu-CD34 protein using anti-CD34 antibodies. These results provide evidence that CD34 promotes the adhesive interactions of hemopoietic cells with the stromal microenvironment of the bone marrow thereby implicating CD34 in regulation and compartmentalization of stem cells. We propose that CD34 regulates these processes in part via an indirect mechanism, signaling changes in cellular adhesion in response to molecular recognition of an as yet unidentified stromal CD34 counterreceptor or ligand.
The sigma factor sigma s affects antibiotic production and biological control activity of Pseudomonas fluorescens Pf-5.
Authors: Sarniguet A , Kraus J , Henkels M D , Muehlchen A M , Loper J E
Published: May 2006

Pseudomonas fluorescens Pf-5, a rhizosphere-inhabiting bacterium that suppresses several soilborne pathogens of plants, produces the antibiotics pyrrolnitrin, pyoluteorin, and 2,4-diacetylphloroglucinol. A gene necessary for pyrrolnitrin production by Pf-5 was identified as rpoS, which encodes the stationary-phase sigma factor sigma s. Several pleiotropic effects of an rpoS mutation in Escherichia coli also were observed in an RpoS- mutant of Pf-5. These included sensitivities of stationary-phase cells to stresses imposed by hydrogen peroxide or high salt concentration. A plasmid containing the cloned wild-type rpoS gene restored pyrrolnitrin production and stress tolerance to the RpoS- mutant of Pf-5. The RpoS- mutant overproduced pyoluteorin and 2,4-diacetyl-phloroglucinol, two antibiotics that inhibit growth of the phytopathogenic fungus Pythium ultimum, and was superior to the wild type in suppression of seedling damping-off of cucumber caused by Pythium ultimum. When inoculated onto cucumber seed at high cell densities, the RpoS- mutant did not survive as well as the wild-type strain on surfaces of developing seedlings. Other stationary-phase-specific phenotypes of Pf-5, such as the production of cyanide and extracellular protease(s) were expressed by the RpoS- mutant, suggesting that sigma s is only one of the sigma factors required for the transcription of genes in stationary-phase cells of P. fluorescens. These results indicate that a sigma factor encoded by rpoS influences antibiotic production, biological control activity, and survival of P. fluorescens on plant surfaces.
Ceramide formation during heat shock: a potential mediator of alpha B-crystallin transcription.
Authors: Chang Y , Abe A , Shayman J A
Published: May 2006

Ceramide has been identified as a potential second messenger that may mediate cell differentiation and apoptosis after exposure to hormonal agonists such as 1 alpha, 25-dihydroxyvitamin D3, tumor necrosis factor alpha, or gamma-interferon. The secondary cellular events that follow ceramide generation remain undefined. We report that in NIH WT-3T3 cells, ceramide induces an enhancement of gene transcription of alpha B-crystallin, a small heat shock protein. The levels of alpha B-crystallin, as measured by Northern blot and immunoblot analyses, were increased by the addition of an exogenous short-chain ceramide, N-acetylsphingosine, or by increasing endogenous intracellular ceramide by inhibition of glucosylceramide synthase. Similar effects were not seen in the expression of the closely related gene, Hsp25. To ascertain whether ceramide-mediated gene transcription was a feature of the heat shock response, cell ceramide was measured in heat shocked cells and observed to be elevated 2-fold immediately upon the return of cells to 37 degrees C. Thus ceramide formed after heat shock treatment of 3T3 cells may mediate the transcription events associated with the cell stress response.
Parvovirus B19 promoter at map unit 6 confers autonomous replication competence and erythroid specificity to adeno-associated virus 2 in primary human hematopoietic progenitor cells.
Authors: Wang X S , Yoder M C , Zhou S Z , Srivastava A
Published: May 2006

The pathogenic human parvovirus B19 is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells. Although the target cell specificity for B19 infection has been suggested to be mediated by the erythrocyte P-antigen receptor (globoside), a number of nonerythroid cells that express this receptor are nonpermissive for B19 replication. To directly test the role of expression from the B19 promoter at map unit 6 (B19p6) in the erythroid cell specificity of B19, we constructed a recombinant adeno-associated virus 2 (AAV), in which the authentic AAV promoter at map unit 5 (AAVp5) was replaced by the B19p6 promoter. Although the wild-type (wt) AAV requires a helper virus for its optimal replication, we hypothesized that inserting the B19p6 promoter in a recombinant AAV would permit autonomous viral replication, but only in erythroid progenitor cells. In this report, we provide evidence that the B19p6 promoter is necessary and sufficient to impart autonomous replication competence and erythroid specificity to AAV in primary human hematopoietic progenitor cells. Thus, expression from the B19p6 promoter plays an important role in post-P-antigen receptor erythroid-cell specificity of parvovirus B19. The AAV-B19 hybrid vector system may also prove to be useful in potential gene therapy of human hemoglobinopathies.
Steroid receptor heterodimerization demonstrated in vitro and in vivo.
Authors: Liu W , Wang J , Sauter N K , Pearce D
Published: May 2006

The mineralocorticoid and glucocorticoid receptors (MR and GR, respectively) are members of the intracellular receptor superfamily that bind as homodimers to the same hormone response elements (HREs). Physiological evidence suggests that MR and GR interact with each other in cells that express both receptors, implying that they might directly interact in the regulation of transcription initiation. Indeed, we have found that coexpressed MR and GR interact functionally at the transcriptional level and furthermore that they interact physically through heterodimer formation at a shared HRE in vitro and in vivo. We suggest from these findings that heterodimerization may play an important role in steroid receptor transcriptional regulation.
Congenital myasthenic syndrome caused by prolonged acetylcholine receptor channel openings due to a mutation in the M2 domain of the epsilon subunit.
Authors: Ohno K , Hutchinson D O , Milone M , Brengman J M , Bouzat C , Sine S M , Engel A G
Published: May 2006

In a congenital myasthenic syndrome with a severe endplate myopathy, patch-clamp studies revealed markedly prolonged acetylcholine receptor (AChR) channel openings. Molecular genetic analysis of AChR subunit genes demonstrated a heterozygous adenosine-to-cytosine transversion at nucleotide 790 in exon 8 of the epsilon-subunit gene, predicting substitution of proline for threonine at codon 264 and no other mutations in the entire coding sequences of genes encoding the alpha, beta, delta, and epsilon subunits. Genetically engineered mutant AChR expressed in a human embryonic kidney fibroblast cell line also exhibited markedly prolonged openings in the presence of agonist and even opened in its absence. The Thr-264-->Pro mutation in the epsilon subunit involves a highly conserved residue in the M2 domain lining the channel pore and is likely to disrupt the putative M2 alpha-helix. Our findings indicate that a single mutation at a critical site can greatly alter AChR channel kinetics, leading to a congenital myasthenic syndrome. This observation raises the possibility that mutations involving subunits of other ligand-gated channels may also exist and be the basis of various other neurologic or psychiatric disorders.
Coordinate regulation of tetrahydrobiopterin turnover and phenylalanine hydroxylase activity in rat liver cells.
Authors: Mitnaul L J , Shiman R
Published: May 2006

This work had two purposes: (i) to determine in vivo whether liver phenylalanine hydroxylase (PAH) is regulated by its substrates phenylalanine and tetrahydrobiopterin (BH4) as studies with purified enzyme suggest and (ii) to investigate in vivo the relationship between PAH activity and BH4 turnover. We found there are two BH4 pools in hepatocytes, one that is metabolically available (free BH4) and one that is not (bound BH4). Bound BH4 appears bound to PAH; the PAH-BH4 complex has much less catalytic activity and is less readily phenylalanine activated than uncomplexed enzyme. Interconversion of activated and unactivated PAH and bound and free BH4 is driven by phenylalanine; and free BH4 concentration is determined by the state of activation and activity of PAH. In hepatocytes, BH4 and PAH (subunit) concentrations are equal, all intracellular BH4 appears to be available to PAH, and free BH4 turns over rapidly (t1/2 approximately 1 hr). There is no evidence for feedback inhibition of BH4 synthesis; the BH4 synthetic rate appears high when free BH4 concentration is high and low when free BH4 is low. The data provide support in vivo that phenylalanine and BH4 are positive and negative regulators of the activity and activation state of PAH in the proposed manner; they also imply that regulation of BH4 turnover and PAH activity are linked processes, which are both controlled by phenylalanine concentration.
Complementation of the ionizing radiation sensitivity, DNA end binding, and V(D)J recombination defects of double-strand break repair mutants by the p86 Ku autoantigen.
Authors: Boubnov N V , Hall K T , Wills Z , Lee S E , He D M , Benjamin D M , Pulaski C R , Band H , Reeves W , Hendrickson E A
Published: May 2006

Two ionizing radiation-sensitive (IRs) and DNA double-strand break (DSB) mutants, sxi-3 and sxi-2, were shown to be severely deficient in a DNA end binding activity, similar to a previously described activity of the Ku autoantigen, correlating with the xrs (XRCC5) mutations. Cell fusions with xrs-6, another IRs, DSB repair-deficient cell line, defined these sxi mutants in the XRCC5 group. sxi-3 cells have low expression levels of the p86Ku mRNA. Introduction of the Ku p86 gene, but not the p70 Ku gene, complemented the IRs, DNA end binding, and variable (diversity) joining [V(D)J] recombination signal and coding junction deficiencies of sxi-3. Thus, the p86 Ku gene product is essential for DSB repair and V(D)J recombination.
Intracellular promiscuity in Schistosoma mansoni: nuclear transcribed DNA sequences are part of a mitochondrial minisatellite region.
Authors: Pena H B , de Souza C P , Simpson A J , Pena S D
Published: May 2006

It has been shown that the mtDNA of the parasitic trematode Schistosoma mansoni is hypervariable in size. We report here that this length variation is due to a large polymorphic minisatellite composed of two types of repeated sequences of 558 bp and 62 bp. Each minisatellite repeat is made up of a large 558-bp component and a variable tandem array of the small 62-bp unit. Of more fundamental interest was the finding that both the 558-bp and 62-bp components have significant homology with a gene, SM750, previously identified in the nuclear genome of S. mansoni. The small 62-bp unit is identical to the nuclear polymorphic repeat element, which is apparently spread throughout the nuclear genome and is abundant among transcripts, in addition to being present in five tandem copies in SM750. The presence, in the S. mansoni mtDNA, of fragments of genes that are present in and transcribed from the nuclear genome raises the question of the origin of these sequences. The arrangement and the variability that the mtDNA minisatellite embodies were explored as an identity test for S. mansoni based on the use of PCR for tallying the relative abundance of the several repeat numbers of the tandem arrays of the 62-bp unit within the minisatellite structure.
Application of chiral gas chromatography with electroantennographic detection to the determination of the stereochemistry of a cockroach sex pheromone.
Authors: Leal W S , Shi X , Liang D , Schal C , Meinwald J
Published: May 2006

The coupling of an electroantennographic detector to a chiral capillary gas chromatographic system provides a highly sensitive technique for the determination of pheromone stereochemistry. Electronic modification of the usual electroantennographic detector enables the detector to respond to the relatively broad peaks produced by chiral gas chromatographic systems. By using this methodology, supellapyrone, a female sex pheromone of the brownbanded cockroach Supella longipalpa (Orthoptera: Blattellidae), is shown to be 5-(2'R,4'R-dimethylheptanyl)-3-methyl-2H-pyran-2-one.
Determination of absolute amounts of GDP and GTP bound to Ras in mammalian cells: comparison of parental and Ras-overproducing NIH 3T3 fibroblasts.
Authors: Scheele J S , Rhee J M , Boss G R
Published: May 2006

We devised enzyme-based methods to measure fmol amounts of GDP and GTP and applied these methods to measure absolute amounts of Ras-bound GDP and GTP in NIH 3T3 fibroblasts. We found that parental NIH 3T3 cells contained 509 and 1.3 fmol of Ras-bound GDP and GTP, respectively, per mg of cellular protein and that stable transfectants of NIH 3T3 cells overexpressing wild-type Ha-Ras contained 7008 and 21.3 fmol of Ras-bound GDP and GTP, respectively, per mg of cellular protein; thus, in both cell types < 0.3% of Ras was in the active GTP-bound state. In contrast, NIH 3T3 cells overexpressing an activated form of Ha-Ras contained 5013 and 2049 fmol of Ras-bound GDP and GTP, respectively, per mg of protein, yielding 29% of Ras in the GTP-bound state. Since intracellular Ras is probably all in a guanine-nucleotide bound state, this method allows one to calculate the number of Ras molecules in each cell: parental NIH 3T3 cells and the Ha-Ras overproducing cells contain approximately 20,000 and approximately 275,000 Ras molecules per cell, respectively. We also incubated parental NIH 3T3 cells with 32PO4 and determined the radioactivity in Ras-bound GDP and GTP and the specific activity of cytosolic GDP and GTP; these experiments indicated that Ras-bound GTP may not be in equilibrium with the total cytosolic GTP pool.
Modulation of protein tyrosine phosphorylation in rat insular cortex after conditioned taste aversion training.
Authors: Rosenblum K , Schul R , Meiri N , Hadari Y R , Zick Y , Dudai Y
Published: May 2006

Protein tyrosine phosphorylation is a major signal transduction pathway involved in cellular metabolism, growth, and differentiation. Recent data indicate that tyrosine phosphorylation also plays a role in neuronal plasticity. We are using conditioned taste aversion, a fast and robust associative learning paradigm subserved among other brain areas by the insular cortex, to investigate molecular correlates of learning and memory in the rat cortex. In conditioned taste aversion, rats learn to associate a novel taste (e.g., saccharin) with delayed poisoning (e.g., by LiCl injection). Here we report that after conditioned taste aversion training, there is a rapid and marked increase in tyrosine phosphorylation of a set of proteins in the insular cortex but not in other brain areas. A major protein so modulated, of 180 kDa, is abundant in a membrane fraction and remains modulated for more than an hour after training. Exposure of the rats to the novel taste alone results in only a small modulation of the aforementioned proteins whereas administration of the malaise-inducing agent per se has no effect. To the best of our knowledge, this is the first demonstration of modulation of protein tyrosine phosphorylation in the brain after a behavioral experience.
Paternal investment affects prevalence of malaria.
Authors: Richner H , Christe P , Oppliger A
Published: May 2006

Both reproduction and parasite defense can be costly, and an animal may face a trade-off between investing in offspring or in parasite defense. In contrast to the findings from nonexperimental studies that the poorly reproducing individuals are often the ones with high parasite loads, this life-history view predicts that individuals with high reproductive investment will show high parasite prevalence. Here we provide an experimental confirmation of a positive association between parental investment levels of male great tits Parus major and the prevalence of Plasmodium spp, a hematozoa causing malaria in various bird species. We manipulated brood size, measured feeding effort of both males and females, and assessed the prevalence of the hemoparasite from blood smears. In enlarged broods the males, but not the females, showed significantly higher rates of food provisioning to the chicks, and the rate of malarial infection was found to be more than double in male, but not female, parents of enlarged broods. The findings show that there may be a trade-off between reproductive effort and parasite defense of the host and also suggest a mechanism for the well documented trade-off between current reproductive effort and parental survival.
Adherence of human immunodeficiency virus-infected lymphocytes to fetal placental cells: a model of maternal --> fetal transmission.
Authors: Schwartz D H , Sharma U K , Perlman E J , Blakemore K
Published: May 2006

The precise timing and mechanism of in utero human immunodeficiency virus (HIV) infection are unknown, but transplacental transmission is likely. Term placentas from HIV+ pregnancies contain only rare HIV-infected cells whose origins and phenotypes remain controversial, and no correlation has been found between the presence of HIV in term placentas and transmission to offspring. Reports of trophoblast infectibility have not been reproducible and do not address the question of infection in the placental stroma, the cells in direct contact with fetal circulation. We report that primary cultures of fetal placental chorionic villus stromal cells, while not infectable in vitro, do support lethally irradiated HIV-infected peripheral blood mononuclear cells (PBMCs) in a form that permits rescue of HIV by activated PBMCs weeks later. Infected PBMCs adhere and become intimately associated with placental cells by a mechanism that is LFA-1 and CD4 independent but can be blocked by antibodies or soluble CD4 binding to cell surface-expressed HIV envelope. The ability to sustain infected irradiated cells was not shared by several trophoblast, fibroblast, or epithelial cell lines. This model has several features that are compatible with in utero transmission and allow testing of various agents proposed as interventions to block maternal-->fetal transmission. Placental stromal cells appear to inhibit apoptosis of HIV-infected, irradiated lymphocytes.
Nitric oxide signaling to iron-regulatory protein: direct control of ferritin mRNA translation and transferrin receptor mRNA stability in transfected fibroblasts.
Authors: Pantopoulos K , Hentze M W
Published: May 2006

Iron-regulatory protein (IRP) is a master regulator of cellular iron homeostasis. Expression of several genes involved in iron uptake, storage, and utilization is regulated by binding of IRP to iron-responsive elements (IREs), structural motifs within the untranslated regions of their mRNAs. IRP-binding to IREs is controlled by cellular iron availability. Recent work revealed that nitric oxide (NO) can mimic the effect of iron chelation on IRP and on ferritin mRNA translation, whereas the stabilization of transferrin receptor mRNA following NO-mediated IRP activation could not be observed in gamma-interferon/lipopolysaccharide-stimulated murine macrophages. In this study, we establish the function of NO as a signaling molecule to IRP and as a regulator of mRNA translation and stabilization. Fibroblasts with undetectable levels of endogenous NO synthase activity were stably transfected with a cDNA encoding murine macrophage inducible NO synthase. Synthesis of NO activates IRE binding, which in turn represses ferritin mRNA translation and stabilizes transferrin receptor mRNA against targeted degradation. Furthermore, iron starvation and NO release are shown to be independent signals to IRP. The posttranscriptional control of iron metabolism is thus intimately connected with the NO pathways.
Retinoblastoma gene product as a downstream target for a ceramide-dependent pathway of growth arrest.
Authors: Dbaibo G S , Pushkareva M Y , Jayadev S , Schwarz J K , Horowitz J M , Obeid L M , Hannun Y A
Published: May 2006

Ceramide, a lipid mediator, has been most closely associated with antiproliferative activities. In this study, we examine the mechanism by which ceramide induces growth suppression and the role of the retinoblastoma gene product (Rb) in this process. Withdrawal of serum from the serum-dependent MOLT-4 cells resulted in significant dephosphorylation of Rb, correlating with the induction of G0/G1 cell cycle arrest. Serum withdrawal resulted in marked elevation in the levels of endogenous ceramide (3-fold at 24 h and 8-fold at 96 h) with little changes in the endogenous levels of sphingosine. The addition of exogenous C6-ceramide resulted in a concentration- and time-dependent dephosphorylation of Rb. Exogenous ceramide was active at levels comparable to endogenous levels achieved with serum withdrawal. Peak activity of exogenous ceramide (at 6 h) correlated with the uptake of C6-ceramide by MOLT-4 cells. Next, a number of studies were conducted to determine whether Rb plays a role in ceramide-induced growth suppression. (i) C6-Ceramide was poorly active in growth suppression of retinoblastoma cells that lack Rb. (ii) Mink lung epithelial cells in which Rb had been sequestered by overexpression of large tumor antigen were resistant to the action of ceramide compared to cells transfected with large tumor antigen mutated in the Rb-binding pocket. (iii) Overexpression of the EIA adenoviral protein, which binds and sequesters Rb, resulted in protection from growth suppression and cell cycle arrest induced by ceramide. Thus, these studies demonstrate that Rb is a downstream target for ceramide and may function in a growth suppressor pathway resulting in cell cycle arrest.
The Drosophila yolkless gene encodes a vitellogenin receptor belonging to the low density lipoprotein receptor superfamily.
Authors: Schonbaum C P , Lee S , Mahowald A P
Published: May 2006

Sequence comparisons of vitellogenins from a wide range of organisms have identified regions of similarity not only to each other but also to vertebrate apolipoproteins (e.g. apoB-100 and apoE). Furthermore, the chicken vitellogenin receptor, which also binds apolipoproteins receptor (LDLR) superfamily [Bujo, H., Hermann, M., Kaderli, M. O., Jacobsen, L., Sugawara, S., Nimpf, J., Yamamoto, T. & Schneider, W. J. (1994) EMBO J. 13, 5165-5175]. The yolk proteins of higher dipterans are exceptional, however, and instead show similarity to lipoprotein lipases. The molecular characterization of the putative Drosophila melanogaster vitellogenin receptor gene, yolkless (yl), described in this report reveals that the protein it encodes (Yl), is also a member of the LDLR superfamily. The ovary-specific 6.5-kb yl RNA codes for a protein of approximately 210 kDa which contains all three motifs common to the LDLR class of proteins. Within this superfamily, Yl may be related more to the LDLR-related proteins (LRPs), which bind both apolipoproteins and lipoprotein lipases. The similarity of Yl to the other LDLR proteins is restricted to the putative extracellular domain. Most noticeably, the cytoplasmic domain of Yl lacks the typical NPXY sequence which is involved in receptor internalization.
Identification of a neural-specific cDNA, NPDC-1, able to down-regulate cell proliferation and to suppress transformation.
Authors: Galiana E , Vernier P , Dupont E , Evrard C , Rouget P
Published: May 2006

Immortalized neural precursor cell lines carrying the polyoma large tumor (T) gene have been shown previously to retain a clear-cut contact inhibition of growth and to differentiate in vitro. In the present study, we have identified and isolated cDNA clones corresponding to RNA expressed preferentially when these cells reach confluence. One of them, NPDC-1, is expressed specifically in the nervous system. The transfection of dividing cells with a NPDC-1 expression vector results in the inhibition of cell proliferation. In addition, the stable introduction of NPDC-1 into transformed cells, even of nonneural origin, leads to the suppression of transformed characteristics.