bluuebunny/crossref_metadata_2025 huggingface dataset:
Grammatical model of the regulation of gene expression.
Authors: Collado-Vides J
Published: May 2006

Based on a formal proof that justifies the search for generative grammars in the study of gene regulation, a linguistic formalization of an exhaustive data base of Escherichia coli sigma 70 promoters and their regulatory binding sites has been initiated. The grammar presented here generates all the arrays of the collection plus those that are predicted as consistent with the principles of regulation of sigma 70 promoters. "Systems of regulation," sets of regulatory sites that collaborate in a mechanism of regulation, are represented by means of syntactic categories. A small set of phrase structure rules restricted by an X-bar principle and by a hierarchical, c-command relation generates a representation of arrays of sites of regulation where the selection of the protein(s) identifying the system(s) of regulation occurs. Based on the features of the proteins, optional duplicated proximal and remote sites are generated by means of transformational rules. Consistency with the data, the predictions that the grammar generates, and important similarities and differences with some aspects of the generative theory of natural language are discussed.
Initiation of phi 29 DNA replication occurs at the second 3' nucleotide of the linear template: a sliding-back mechanism for protein-primed DNA replication.
Authors: Méndez J , Blanco L , Esteban J A , Bernad A , Salas M
Published: May 2006

Bacteriophage phi 29 DNA replication is initiated when a molecule of dAMP is covalently linked to a free molecule of the terminal protein, in a reaction catalyzed by the viral DNA polymerase. We demonstrate that single-stranded DNA molecules are active templates for the protein-primed initiation reaction and can be replicated by phi 29 DNA polymerase. Using synthetic oligonucleotides, we carried out a mutational analysis of the phi 29 DNA right end to evaluate the effect of nucleotide changes at the replication origin and to determine the precise initiation site. The results indicate that (i) there are no strict sequence requirements for protein-primed initiation on single-stranded DNA; (ii) initiation of replication occurs opposite the second nucleotide at the 3' end of the template; (iii) a terminal repetition of at least two nucleotides is required to efficiently elongate the initiation complex; and (iv) all the nucleotides of the template, including the 3' terminal one, are replicated. A sliding-back model is proposed in which a special transition step from initiation to elongation can account for these results. The possible implication of this mechanism for the fidelity of the initiation reaction is discussed. Since all the terminal protein-containing genomes have some sequence reiteration at the DNA ends, this proposed sliding-back model could be extrapolable to other systems that use proteins as primers.
A fraction of the mRNA 5' cap-binding protein, eukaryotic initiation factor 4E, localizes to the nucleus.
Authors: Lejbkowicz F , Goyer C , Darveau A , Neron S , Lemieux R , Sonenberg N
Published: May 2006

The 5' cap structure m7GpppN (where N is any nucleotide) is a ubiquitous feature of cellular eukaryotic mRNAs. The cap is multifunctional as it is involved in translation, nucleocytoplasmic transport, splicing, and stabilization of mRNA against 5' exonucleolytic degradation. The cap binding protein, eukaryotic initiation factor 4E (eIF-4E), is a translation initiation factor that binds to the cap structure and is part of a complex (eIF-4F) that promotes mRNA binding to ribosomes. Overexpression of eIF-4E in fibroblasts results in cell transformation. To test the hypothesis that some of the biological effects of eIF-4E might be effected by a nuclear function, we determined the cellular distribution of eIF-4E. By means of indirect immunofluorescence experiments using polyclonal and monoclonal antibodies against eIF-4E as well as transfected epitope-tagged eIF-4E, we demonstrate that a fraction of eIF-4E localizes to the nucleus. These results suggest that eIF-4E is also involved in a nuclear function.
Conformational substates in azurin.
Authors: Ehrenstein D , Nienhaus G U
Published: May 2006

Azurin is a small blue copper protein in the electron transfer chain of denitrifying bacteria. It forms a photolabile complex with nitric oxide (NO) at low temperatures. We studied the temperature dependence of the ligand binding equilibrium and the kinetics of the association reaction after photodissociation over a wide range of temperature (80-280 K) and time (10(-6)-10(2) s). The nonexponential rebinding below 200 K is independent of the NO concentration and is interpreted as internal recombination. The rebinding can be modeled with the Arrhenius law by using a single preexponential factor of 6.3 x 10(8) s-1 and a Gaussian distribution of enthalpy barriers centered at 23 kJ/mol with a width of 11 kJ/mol. Above 200 K, a slower, exponential rebinding process appears. The dependence of the kinetics on the NO concentration characterizes this reaction as bimolecular rebinding. The binding kinetics of NO to azurin show impressive analogies to the binding of carbon monoxide to myoglobin. We conclude that conformational substates occur not only in heme proteins but also in proteins with different active sites and secondary structures.
Asexual metazoans undergo senescence.
Authors: Martínez D E , Levinton J S
Published: May 2006

August Weismann popularized the notion that metazoans have a potentially immortal germ line separated from a mortal soma, and evolutionary biologists regard senescence as an evolved characteristic of the soma. Many have claimed that metazoans that do not sequester their germ line have no clear distinction between germ line and soma, and consequently they should lack senescence. Here we present experimental evidence that senescence occurs in the asexually reproducing marine oligochaete Paranais litoralis. We also analyze data reported in Sonneborn's classical study and show that the rhabdocoel Stenostomum incaudatum undergoes senescence. We argue that the stability of commitment to somatic function and the fact that asexual metazoans form their germ cells from undifferentiated stem cells are sufficient to allow for senescence of the asexual metazoan's soma. Thus the evolution of somatic differentiation, and not germ-line sequestration, would be the necessary condition for the evolution of senescence.
Leishmania infections damage the feeding mechanism of the sandfly vector and implement parasite transmission by bite.
Authors: Schlein Y , Jacobson R L , Messer G
Published: May 2006

Leishmania parasites are transmitted by the bites of infected female sandflies by a mechanism that has not been clarified. Leishmania infections in the vector develop only in the gut, and the parasites' exit is through the food channel in the proboscis. The problem is how during the bite, when blood flows in, parasites are emitted through the same channel in the opposite direction. It is well documented that infected sandflies maintained on sugar diets are potent vectors, whereas transmission fails after constant feeding on blood. Hence to study the mechanism of transmission, we fed these diets to Phlebotomus papatasi infected with L. major. Histological examination demonstrated that only in the sugar-fed flies did the cuticle lining of the cardiac valve detach and other valve tissues degenerate gradually. The injury of the main valve of the food pumps hindered gorging of most flies when force-fed from capillaries, and they regurgitated the gut contents with fluids from the capillaries. We suggest that infections are caused by parasites regurgitated from the stomach that are deposited in the host tissue. We found that secretion of chitinolytic enzymes by cultured L. major parasites is inhibited by blood or hemoglobin, and hence these enzymes are apparently absent from the blood-fed infected flies, where the cardiac valve appears undamaged. We therefore presume that lysis of the chitin in the cuticle lining of the valve leads to exposure and degeneration of the underlying tissues.
Large, identical, tandem repeating units in the C protein alpha antigen gene, bca, of group B streptococci.
Authors: Michel J L , Madoff L C , Olson K , Kling D E , Kasper D L , Ausubel F M
Published: May 2006

Group B Streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis in the United States. The surface-associated C protein alpha antigen of GBS is thought to have a role in both virulence and immunity. We previously cloned the C protein alpha antigen structural gene (named bca for group B, C protein, alpha) into Escherichia coli. Western blots of both the native alpha antigen and the cloned gene product demonstrate a regularly laddered pattern of heterogeneous polypeptides. The nucleotide sequence of the bca locus reveals an open reading frame of 3060 nucleotides encoding a precursor protein of 108,705 Da. Cleavage of a putative signal sequence of 41 amino acids yields a mature protein of 104,106 Da. The 20,417-Da N-terminal region of the alpha antigen shows no homology to previously described protein sequences and is followed by a series of nine tandem repeating units that make up 74% of the mature protein. Each repeating unit is identical and consists of 82 amino acids with a molecular mass of 8665 Da, which is encoded by 246 nucleotides. The size of the repeating units corresponds to the observed size differences in the heterogeneous ladder of alpha C proteins expressed by GBS. The C-terminal region of the alpha antigen contains a membrane anchor domain motif that is shared by a number of Gram-positive surface proteins. The large region of identical repeating units in bca defines protective epitopes and may play a role in generating phenotypic and genotypic diversity of the alpha antigen.
The dorsal premammillary nucleus: an unusual component of the mammillary body.
Authors: Canteras N S , Swanson L W
Published: May 2006

The results of anterograde and retrograde axonal transport experiments in the rat indicate that the dorsal premammillary nucleus (PMd) gives rise to a branched pathway ending in the anterior thalamic group and brainstem, like the medial and lateral mammillary nuclei. However, unlike these nuclei, the ascending PMd projection courses through and to the anterior hypothalamic nucleus, and the descending PMd projection ends in the periaqueductal gray, superior colliculus, and adjacent parts of the reticular formation. Also unlike the traditional mammillary nuclei, the PMd does not receive a direct input from the columns of the fornix; instead, it receives a bilateral input from the anterior hypothalamic nucleus, which in turn receives inputs from areas related to the prefrontal cortex, amygdala, and hippocampus. The results provide interesting perspectives on the organization of medial hypothalamic circuits underlying the goal-oriented behaviors associated with hunger, thirst, and reproduction.
Cloning and expression of an NADP(+)-dependent alcohol dehydrogenase gene of Entamoeba histolytica.
Authors: Kumar A , Shen P S , Descoteaux S , Pohl J , Bailey G , Samuelson J
Published: May 2006

Ethanol is the major metabolic product of glucose fermentation by the protozoan parasite Entamoeba histolytica under the anaerobic conditions found in the lumen of the colon. Here an internal peptide sequence determined from a major 39-kDa amoeba protein isolated by isoelectric focusing followed by SDS/PAGE was used to clone the gene for the E. histolytica NADP(+)-dependent alcohol dehydrogenase (EhADH1; EC 1.1.1.2). The EhADH1 clone had an open reading frame that was 360 amino acids long and encoded a protein of approximately 39 kDa (calculated size). EhADH1 showed a 62% amino acid identity with the tetrameric NADP(+)-dependent alcohol dehydrogenase of Thermoanaerobium brockii. In contrast, EhADH1 showed a 15% amino acid identity with the closest human alcohol dehydrogenase. EhADH1 contained 18 of the 22 amino acids conserved in other alcohol dehydrogenases, including glycines involved in binding NAD(P)+ as well as histidine and cysteine residues involved in binding the catalytic zinc ion. Like the T. brockii alcohol dehydrogenase, EhADH1 lacked a 23-amino acid stretch present in other alcohol dehydrogenases that includes four cysteines that bind a second noncatalytic zinc ion. An EhADH1-glutathione-S-transferase fusion protein showed the expected NADP(+)-dependent alcohol dehydrogenase and NADPH-dependent acetaldehyde reductase activities. The enzymatic activities of the EhADH1 fusion protein were inhibited by pyrazole and 4-methylpyrazole.
A role for Quox-8 in the establishment of the dorsoventral pattern during vertebrate development.
Authors: Takahashi Y , Monsoro-Burq A H , Bontoux M , Le Douarin N M
Published: May 2006

We have been interested in the possible involvement of the gene Quox-8, a member of the Hox-7/msh family of homeobox-containing genes in the patterning of the neural tube and the somitic-derived mesoderm. We demonstrate that in the embryonic day 2-6 avian embryo, the expression of Quox-8 in the roof plate depends upon (i) the normal dorsoventral orientation of the neural primordium and (ii) interactions between the dorsal neural tube and the superficial ectoderm. We also show that Quox-8 is expressed in the dorsal mesenchyme located between the neural tube and the superficial ectoderm that yields the spinous processes of the vertebrae. Experimental inhibition of Quox-8 expression in these areas of the dorsal mesenchyme results in the failure of its differentiation into vertebral cartilage. We conclude, therefore, that this gene is involved in the patterning of vertebral structures through a cascade of tissue interactions primarily occurring between the dorsal neural tube and the overlying ectoderm.
A periplasmic protein disulfide oxidoreductase is required for transformation of Haemophilus influenzae Rd.
Authors: Tomb J F
Published: May 2006

The mutated gene in JG16, a Haemophilus influenzae strain deficient in competence-induced DNA binding and uptake, was cloned and the wild-type allele was sequenced. The gene was shown by Northern analysis to be constitutively expressed on a 1.7-kilobase transcript. The gene product was identified as a 20.6-kDa protein targeted to the periplasm. The protein contains the sequence Cys-Pro-His-Cys (CPHC) and is highly similar to two other periplasmic CPHC motif-containing proteins: DsbA, an Escherichia coli protein (45% identity, 87% homology) and TcpG, a Vibrio cholerae protein (32% identity, 74% homology). Both DsbA and TcpG promote disulfide bond formation in periplasmic proteins, are required for pilus biogenesis, and, like thioredoxin, are capable of reducing insulin in vitro. The Haemophilus protein was shown to complement an E. coli mutation in DsbA and was named Por (periplasmic oxidoreductase). In JG16 the competence-dependent redistribution of inner membrane proteins did not occur. These findings suggest that Por is required for the correct assembly and/or folding of one or more disulfide-containing cell envelope protein involved either in competence development or in the DNA-binding and -uptake machinery.
Localization of Epstein-Barr virus-encoded RNAs EBER-1 and EBER-2 in interphase and mitotic Burkitt lymphoma cells.
Authors: Schwemmle M , Clemens M J , Hilse K , Pfeifer K , Tröster H , Müller W E , Bachmann M
Published: May 2006

The subcellular distribution of the small Epstein-Barr virus-encoded RNAs EBER-1 and EBER-2 has been investigated by using a high-resolution in situ hybridization technique. The distribution patterns in Raji cells of fluorescent oligodeoxynucleotides complementary to each RNA were detected by confocal laser scanning microscopy. Both RNAs were found in the cytoplasm as well as in the nuclei of interphase cells. In contrast, use of the same technique indicated an exclusively nuclear location for cellular U2 RNA. In the cytoplasm distribution of the EBERs was similar to that of the double-stranded RNA-dependent protein kinase, to which these RNAs can bind, and was coincident with the rough endoplasmic reticulum. In cells undergoing mitosis the EBERs became localized around the chromosomes, whereas the protein kinase remained uniformly distributed in the cytoplasm. A cytoplasmic location for EBER-1 and EBER-2 in interphase cells is consistent with the evidence for a role for these small RNAs in translational control.
Activation of human peripheral blood T lymphocytes by pharmacological induction of protein-tyrosine phosphorylation.
Authors: O'Shea J J , McVicar D W , Bailey T L , Burns C , Smyth M J
Published: May 2006

Protein-tyrosine kinase and protein-tyrosine phosphatase (PTPase) activities are essential for T-cell antigen receptor-mediated signaling. To assess the functional consequences of alteration of the levels of tyrosine phosphorylation in normal human T cells, the effects of vanadate and hydrogen peroxide were studied. In combination, these agents induced tyrosine phosphorylation of cellular substrates, elevated cytosolic free calcium, and induced interleukin 2 receptor (IL-2R) alpha chain expression but not IL-2 secretion. However, anti-CD28 antibody in combination with vanadate and hydrogen peroxide induced IL-2 secretion, consistent with the requirement for a costimulatory signal in the induction of this gene. The effects of vanadate and hydrogen peroxide were enhanced in the absence of the T-cell PTPase, CD45. Thus, acute pharmacologic manipulation of the level of tyrosine phosphorylation in normal T cells correlates with partial, but not full, activation of these cells; in concert with a costimulatory signal provided by perturbation of the CD28 molecule, the complete program of activation is initiated. These agents should prove useful in dissecting signaling pathways involved in the regulation of genes critical to the immune response.
Basonuclin: a keratinocyte protein with multiple paired zinc fingers.
Authors: Tseng H , Green H
Published: May 2006

A cDNA clone has been prepared from mRNA of cultured human keratinocytes. The sequence of the cDNA reveals that in the C-terminal two-thirds of the corresponding protein (basonuclin), there are three separated pairs of adjacent zinc fingers. The amino acid sequence of each pair is homologous to that of the single pair of zinc fingers of the Drosophila transcription factor encoded by disco. Near the C-terminal end of basonuclin and on the surface of a putative alpha-helix, there is a stripe of serine residues similar to that of the transcription factor PRDII-BF1. Basonuclin possesses a sequence of six amino acids quite similar to one present in the myogenic family of proteins, including Myf5; this sequence is located in the omega loop of the myogenic proteins but within a zinc finger of the keratinocyte protein. As basonuclin is present mainly in the nuclei of the basal cell layer, its regulatory function is likely to be exerted prior to the process of terminal differentiation.
Macrophages in Drosophila embryos and L2 cells exhibit scavenger receptor-mediated endocytosis.
Authors: Abrams J M , Lux A , Steller H , Krieger M
Published: May 2006

Mammalian macrophage scavenger receptors exhibit unusually broad binding specificity and are implicated in atherosclerosis and host defense. Scavenger receptor-like endocytosis was observed in Drosophila melanogaster embryos and in primary embryonic cell cultures. This receptor activity was expressed primarily by macrophages. The Drosophila Schneider L2, but not the Kc, cell line also exhibited a scavenger receptor-mediated endocytic pathway similar to its mammalian counterpart. L2 receptors mediated high-affinity internalization and subsequent temperature- and chloroquine-sensitive degradation of 125I-labeled acetylated low density lipoprotein and displayed characteristic ligand specificity. These findings suggest that scavenger receptors mediate important, well-conserved functions and raise the possibility that they may be pattern recognition receptors that arose early in the evolution of host defense mechanisms. They also establish additional systems for the investigation of endocytosis in Drosophila and scavenger receptor function in disease, host defense, and development.
A region of group I introns that contains universally conserved residues but is not essential for self-splicing.
Authors: Williams K P , Fujimoto D N , Inoue T
Published: May 2006

The catalytic core of the self-splicing group I intron RNAs is composed of six paired regions together with their connecting sequences; these are thought to form two elongated domains, with paired regions P5, P4, and P6 aligned along one axis and P8, P3, and P7 along the other. Most of the very highly conserved residues of the group I introns lie in or near P7, but two occur in L4, the internal loop connecting P4 and P5. It is generally believed that such bases are conserved because they are essential for splicing. Mutants were created in a member of each of the two major subclasses of group I introns, in which P5, L4, and the distal portion of P4 were deleted. Splicing activity was still detected in these mutants, albeit substantially weakened; splicing was accurate and occurred by the normal group I mechanism, with addition of a guanosine molecule to the intron. Thus the deleted region, containing two universally conserved bases, is not essential but facilitates splicing. Another reaction characteristic of group I introns, hydrolysis of the 3' splice site, was less severely affected by the deletions. The results are discussed in terms of the prevailing three-dimensional model for the core structure of the group I introns.
The (4;11)(q21;q23) chromosome translocations in acute leukemias involve the VDJ recombinase.
Authors: Gu Y , Cimino G , Alder H , Nakamura T , Prasad R , Canaani O , Moir D T , Jones C , Nowell P C , Croce C M
Published: May 2006

Chromosomal region 11q23 is frequently rearranged in acute lymphocytic leukemias (ALLs) and in acute myeloid leukemias (AMLs), mostly in reciprocal exchanges with various translocation partners. The most common of these translocations is t(4;11)(q21;q23). It is present in approximately 10% of ALL patients, most frequently in very young children. We have recently cloned a region of chromosome 11, the ALL-1 locus, found to be rearranged in malignant cells from patients with the t(4;11), t(9;11), t(11;19), t(1;11), t(6;11), t(10;11), and del(11q23) chromosomal abnormalities. Here we report the cloning and characterization of chromosomal breakpoints from leukemic cells with t(4;11) aberrations. The breakpoints cluster in regions of 7-8 kilobases on both chromosomes 4 and 11. The presence of heptamer- and nonamer-like sequences at the sites of breakage suggests that the VDJ recombinase utilized for immunoglobulin gene rearrangement is also directly involved in these translocations. We also show that leukemic cells with t(4;11) express altered RNAs transcribed from the derivative chromosomes 11 and 4.
Blocked early-stage latency in the peripheral blood cells of certain individuals infected with human immunodeficiency virus type 1.
Authors: Seshamma T , Bagasra O , Trono D , Baltimore D , Pomerantz R J
Published: May 2006

Human immunodeficiency virus type 1 (HIV-1) infections of humans have a natural history characterized by a variable but usually slow progression to an immunodeficient state. We have described a molecular model of HIV-1 proviral latency in certain cell lines, characterized by extremely low or undetectable levels of unspliced genomic HIV-1-specific RNA but significant levels of multiply spliced HIV-1-specific RNA. We have utilized a quantitative reverse transcriptase-initiated polymerase chain reaction to measure the levels of various HIV-1 RNA species in peripheral blood mononuclear cells. The median level of multiply spliced HIV-1 RNA was dramatically higher than the median level of unspliced viral RNA in asymptomatic individuals. In addition, HIV-1 RNA patterns characterized by at least a 10-fold excess of multiply spliced to unspliced viral RNA were significantly more common in asymptomatic individuals than in patients with the acquired immunodeficiency syndrome. We suggest that asymptomatic clinical HIV-1 infection is characterized by a preponderance of HIV-1-infected peripheral blood cells blocked at an early stage of HIV-1 infection. This viral expression pattern, which we have called blocked early-stage latency, may constitute a reservoir of latently infected cells in certain HIV-1-infected persons.
Slp is an essential component of an EDTA-resistant activation pathway of mouse complement.
Authors: van den Berg C W , Démant P , Aerts P C , Van Dijk H
Published: May 2006

Slp (sex-limited protein) is a mouse serum protein encoded by a major histocompatibility complex class III gene. It is considered to be a product of a duplicated complement component C4 gene, but without functional activity. Originally it has been found expressed only in adult males with the S region of the H-2d or H-2s haplotype. In this report we present evidence that Slp is involved in a form of mouse complement activation that occurs after fractionation of serum by polyethylene glycol precipitation. This activation pathway is EDTA-resistant (i.e., independent of classical and alternative pathway activation), is regulated by C1 inhibitor, and leads to the generation of hemolytically active membrane attack complexes. A positive correlation between this EDTA-resistant mouse complement activity and reported Slp levels was found. Direct evidence for a functional role of Slp came from substitution experiments in which purified Slp induced hemolytic activity in polyethylene glycol-fractionated, Slp-deficient mouse serum. Selective depletion of other complement components suggested a role for C1s-, C2, and C5, but not C3, in the Slp-dependent complement activation. A model for this type of mouse complement activation is presented.
Fatty acids regulate hepatic low density lipoprotein receptor activity through redistribution of intracellular cholesterol pools.
Authors: Daumerie C M , Woollett L A , Dietschy J M
Published: May 2006

When the intake of dietary cholesterol in the hamster is constant, feeding the saturated 14:0 fatty acid (n-tetradecanoic acid) elevates the plasma low density lipoprotein (LDL) cholesterol concentration from 72 to 204 mg/dl, while the monounsaturated 18:1 fatty acid (cis-9-octadecenoic acid) lowers this level to 28 mg/dl. The 14:0 fatty acid lowers the hepatic cholesteryl ester concentration from 12 to 5 mg/g, while the abundance of this fatty acid in the ester fraction is increased 13-fold. Hepatic LDL receptor activity is depressed to 41% of control, while the LDL cholesterol production rate is increased to 132%. These changes account for the 3-fold increase in the plasma LDL cholesterol concentration. In contrast, feeding the 18:1 fatty acid increases hepatic cholesteryl ester concentration to 21 mg/g, and the abundance of this acid in the esters is increased 1.4-fold. Hepatic receptor activity is increased to 145%, while the production rate is suppressed to 68% of control. These changes account for the decrease in plasma LDL cholesterol level to 28 mg/dl. Despite these marked changes in LDL metabolism, however, the 14:0 and 18:1 fatty acids cause no change in net cholesterol balance across the liver. These results suggest that there are two fundamentally different mechanisms regulating hepatic LDL metabolism. One involves changes in net sterol balance across the liver brought about by alterations in the rate of cholesterol or bile acid absorption across the intestine, while the second is articulated through a redistribution of the putative sterol regulatory pool within the hepatocyte that is dictated by the type of long-chain fatty acid that reaches the liver.
Inhibition of human immunodeficiency virus type 1 expression by a hairpin ribozyme.
Authors: Ojwang J O , Hampel A , Looney D J , Wong-Staal F , Rappaport J
Published: May 2006

Ribozymes are RNAs that possess the dual properties of RNA sequence-specific recognition, analogous to conventional antisense molecules, and RNA substrate destruction via site-specific cleavage. The cleavage reaction is catalytic in that more than one substrate molecule is processed per ribozyme molecule. We have designed a hairpin ribozyme that cleaves human immunodeficiency virus type 1 (HIV-1) RNA in the leader sequence (at nucleotides +111/112 relative to the transcription initiation site). The ribozyme was tested in vitro and gave efficient and specific cleavage of RNA containing the leader sequence. To test the antiviral efficacy of this ribozyme, we have cotransfected into HeLa cells HIV-1 proviral DNA and a plasmid expressing the ribozyme from the human beta-actin promoter. HIV-1 expression was inhibited as measured by p24 antigen levels and reduced Tat activity. The antiviral effect of the ribozyme appears to be specific and results from directed RNA cleavage; activity requires both a target sequence and a functional RNA catalytic center. These results suggest that this HIV-1-directed hairpin ribozyme may be useful as a therapeutic agent.
The optimal number of major histocompatibility complex molecules in an individual.
Authors: Nowak M A , Tarczy-Hornoch K , Austyn J M
Published: May 2006

A straightforward argument is presented to calculate the number of different major histocompatibility complex (MHC) molecules in an individual that maximizes the probability of mounting immune responses against a large number of foreign peptides. It is assumed that increasing the number of MHC molecules per individual, n, has three different effects: (i) it increases the number of foreign peptides that can be presented; (ii) it increases the number of different T-cell receptors (TCRs) positively selected in the thymus; but (iii) it reduces the number of TCRs by negative selection. The mathematical analysis shows that n = 1/f maximizes the number of different TCRs that pass through positive and negative selection and that n = 2/f maximizes the probability to mount immune responses against a large fraction of foreign peptides. Here f is the fraction of TCRs deleted by one MHC molecule. Both results depend on approximations that are discussed in the paper. The model presented has implications for our understanding of the evolutionary forces acting on the MHC.
A cluster of hydrophobic amino acid residues required for fast Na(+)-channel inactivation.
Authors: West J W , Patton D E , Scheuer T , Wang Y , Goldin A L , Catterall W A
Published: May 2006

The inward Na+ current underlying the action potential in nerve is terminated by inactivation. The preceding report shows that deletions within the intracellular linker between domains III and IV remove inactivation, but mutation of conserved basic and paired acidic amino acids has little effect. Here we show that substitution of glutamine for three clustered hydrophobic amino acids, Ile-1488, Phe-1489, and Met-1490, completely removes fast inactivation. Substitution of Met-1490 alone slows inactivation significantly, substitution of Ile-1488 alone both slows inactivation and makes it incomplete, and substitution of Phe-1489 alone removes inactivation nearly completely. These results demonstrate an essential role of Phe-1489 in Na(+)-channel inactivation. It is proposed that the hydrophobic cluster of Ile-1488, Phe-1489, and Met-1490 serves as a hydrophobic latch that stabilizes the inactivated state in a hinged-lid mechanism of Na(+)-channel inactivation.
Cloned, expressed rat cerebellar nitric oxide synthase contains stoichiometric amounts of heme, which binds carbon monoxide.
Authors: McMillan K , Bredt D S , Hirsch D J , Snyder S H , Clark J E , Masters B S
Published: May 2006

The endogenous formation of nitric oxide (NO) has become an area of intense interest as evidence for its biological functions has been obtained in three distinct tissues: circulating macrophages, in which it exerts cytotoxic effects; blood vessels, in which it has been identified as endothelium-derived relaxing factor; and neuronal cells, in which it functions as a neurotransmitter. The formation of NO in brain extracts has been shown to be catalyzed by an enzyme, termed NO synthase, which generates the NO responsible for stimulation of cGMP formation, the highest levels of which occur in the cerebellum. NO synthase catalyzes the formation of citrulline from arginine with the coincident production of NO and has been shown to be a flavoprotein, containing 1 mol each of FAD and FMN, tetrahydrobiopterin, and iron. It is also reported to contain an alpha-helical, calmodulin-binding consensus sequence consistent with its stimulation by calmodulin in the presence of Ca2+. The formation of NO requires incorporation of one of the atoms of molecular oxygen into one of the guanidinium nitrogen atoms of arginine with the coincident formation of citrulline. This communication reports that rat cerebellar NO synthase, cloned and stably expressed in human kidney 293 cells, contains heme in amounts stoichiometric with the flavins FAD and FMN as evidenced by the appearance of a pyridine hemochrome and a reduced CO difference spectrum with an absorbance maximum at approximately 445 nm. The finding of a CO-binding heme moiety explains the presence of iron in the enzyme and suggests a role for prosthetic heme as an oxygenase reaction center. This report also presents evidence for incorporation of delta-[14C]aminolevulinate specifically into immunoprecipitable NO synthase in stably transfected human kidney 293 cells but not in nontransfected cells. Simultaneously, K. A. White and M. A. Marletta [(1992) Biochemistry 31, 6627-6631] have demonstrated a CO-binding heme prosthetic group in purified murine macrophage NO synthase and have suggested the identity of these reaction centers in both the constitutive (cerebellar) and inducible (macrophage) forms of NO synthase.
Marked replicative advantage of human mtDNA carrying a point mutation that causes the MELAS encephalomyopathy.
Authors: Yoneda M , Chomyn A , Martinuzzi A , Hurko O , Attardi G
Published: May 2006

The segregation of mutant and wild-type mtDNA was investigated in transformants constructed by transferring human mitochondria from individuals belonging to four pedigrees with the MELAS encephalomyopathy-associated mtDNA mutation (MELAS is mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) into human mtDNA-less (rho 0) cells. Five of 13 clonal cell lines containing mixtures of wild-type and mutant mtDNAs were found to undergo a rapid shift of their genotype toward the pure mutant type. The other 8 cell lines, which included 6 exhibiting nearly homoplasmic mutant mtDNA, on the contrary, maintained a stable genotype. Subcloning experiments and growth rate measurements clearly indicated that an intracellular replicative advantage of mutant mtDNA was mainly responsible for the dramatic shift toward the mutant genotype observed in the unstable cell lines.
Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco.
Authors: Cahoon E B , Shanklin J , Ohlrogge J B
Published: May 2006

Little is known about the metabolic origin of petroselinic acid (18:1 delta 6cis), the principal fatty acid of the seed oil of most Umbelliferae, Araliaceae, and Garryaceae species. To examine the possibility that petroselinic acid is the product of an acyl-acyl carrier protein (ACP) desaturase, Western blots of coriander and other Umbelliferae seed extracts were probed with antibodies against the delta 9-stearoyl-ACP desaturase of avocado. In these extracts, proteins of 39 and 36 kDa were detected. Of these, only the 36-kDa peptide was specific to tissues which synthesize petroselinic acid. A cDNA encoding the 36-kDa peptide was isolated from a coriander endosperm cDNA library, placed under control of the cauliflower mosaic virus 35S promoter, and introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Expression of this cDNA in transgenic tobacco callus was accompanied by the accumulation of petroselinic acid and delta 4-hexadecenoic acid, both of which were absent from control callus. These results demonstrate the involvement of a 36-kDa putative acyl-ACP desaturase in the biosynthetic pathway of petroselinic acid and the ability to produce fatty acids of unusual structure in transgenic plants by the expression of the gene for this desaturase.
Identification of a human src homology 2-containing protein-tyrosine-phosphatase: a putative homolog of Drosophila corkscrew.
Authors: Freeman R M , Plutzky J , Neel B G
Published: May 2006

src homology 2 (SH2) domains direct binding to specific phosphotyrosyl proteins. Recently, SH2-containing protein-tyrosine-phosphatases (PTPs) were identified. Using degenerate oligonucleotides and the PCR, we have cloned a cDNA for an additional PTP, SH-PTP2, which contains two SH2 domains and is expressed ubiquitously. When expressed in Escherichia coli, SH-PTP2 displays tyrosine-specific phosphatase activity. Strong sequence similarity between SH-PTP2 and the Drosophila gene corkscrew (csw) and their similar patterns of expression suggest that SH-PTP2 is the human corkscrew homolog. Sequence comparisons between SH-PTP2, SH-PTP1, corkscrew, and other SH2-containing proteins suggest the existence of a subfamily of SH2 domains found specifically in PTPs, whereas comparison of the PTP domains of the SH2-containing PTPs with other tyrosine phosphatases suggests the existence of a subfamily of PTPs containing SH2 domains. Since corkscrew, a member of the terminal class signal transduction pathway, acts in concert with D-raf to positively transduce the signal generated by the receptor tyrosine kinase torso, these findings suggest several mechanisms by which SH-PTP2 may participate in mammalian signal transduction.
Thymic depletion and peripheral activation of class I major histocompatibility complex-restricted T cells by soluble peptide in T-cell receptor transgenic mice.
Authors: Mamalaki C , Norton T , Tanaka Y , Townsend A R , Chandler P , Simpson E , Kioussis D
Published: May 2006

Injection of mice transgenic for a class I major histocompatibility complex-restricted T-cell receptor with a soluble peptide antigen from influenza virus nucleoprotein results in clonal depletion of double-positive immature thymocytes in the thymus and activation of mature T cells in the periphery, accompanied by a transient up-regulation of the T-cell receptor and CD3 and CD8 coreceptor molecules.
Introduction of a retinoid reporter gene into the urodele limb blastema.
Authors: Brockes J P
Published: May 2006

After amputation of the limb of an adult urodele amphibian at any point along the proximodistal axis, blastemal cells (the progenitor cells of the regenerate) give rise only to the missing structures. Retinoic acid (RA) is able to respecify the positional identity of the blastema to a more proximal value, thus raising the possibility that the RA response system is activated during limb regeneration. Cultured newt (Notophthalmus viridescens) limb cells were transfected by nuclear microinjection of plasmids which provided RA-sensitive reporter activity that could be normalized for differences in cell recovery and transfection efficiency. Such cells showed a dose-dependent response to RA in culture, and this required a functional RA response element. The cells were implanted under the wound epidermis of newt hindlimb blastemas. After injection of a proximalizing dose of RA there was a significant difference in the level of reporter activity dependent on a functional response element. When cells were implanted into contralateral proximal and distal hindlimb blastemas the proximal-to-distal ratio for activation of the reporter through the response element was approximately 3.5-fold, suggesting that a gene whose expression is regulated by RA could be differentially activated along the proximodistal axis during limb regeneration.
Transmembrane signaling by the high-affinity IgE receptor on membrane preparations.
Authors: Pribluda V S , Metzger H
Published: May 2006

Aggregating the receptor with high affinity for IgE (Fc epsilon RI) stimulates a variety of phenomena in mast cells. Previous efforts to reproduce some of these events in broken-cell preparations such as isolated membranes have had limited success, possibly because the phenomena being monitored were too distal from the initial events. One of the earliest responses is now known to be the phosphorylation of tyrosine residues on several proteins, including the beta and gamma subunits of Fc epsilon RI. We show that in cell sonicates or on partially purified membranes derived from tumor mast cells, aggregating Fc epsilon RI stimulates phosphorylation of receptor tyrosine residues. As in the intact cells, receptor-mediated phosphorylation occurs only on receptors that are themselves aggregated. Because even in the unfractionated sonicates the phosphorylation of other cellular components was not detectably enhanced, and because the evidence is against the receptor itself being a kinase, our results suggest that phosphorylation of Fc epsilon RI is one of the earliest events stimulated by the receptor--an event that can now be investigated on simpler biological preparations than previously available.
Activation of estrogen receptor transfected into a receptor-negative breast cancer cell line decreases the metastatic and invasive potential of the cells.
Authors: Garcia M , Derocq D , Freiss G , Rochefort H
Published: May 2006

Breast cancers containing estrogen receptors are responsive to antiestrogen treatment and have a better prognosis than estrogen receptor-negative tumors. The loss of estrogen and progesterone receptors appears to be associated with a progression to less-differentiated tumors. We transfected the human estrogen receptor into the estrogen receptor-negative metastatic breast cancer cell line MDA-MB-231 in an attempt to restore their sensitivity to antiestrogens. Two stable sublines of MDA-MB-231 cells (HC1 and HE5) expressing functional estrogen receptors were studied for their ability to grow and invade in vitro and to metastasize in athymic nude mice. The number and size of lung metastases developed by these two sublines in ovariectomized nude mice was not markedly altered by tamoxifen but was inhibited 3-fold by estradiol. Estradiol also significantly inhibited in vitro cell proliferation of these sublines and their invasiveness in Matrigel, a reconstituted basement membrane, whereas the antiestrogens 4-hydroxytamoxifen and ICI 164,384 reversed these effects. These results show that estradiol inhibits the metastatic ability of estrogen receptor-negative breast cancer cells following transfection with the estrogen receptor, whereas estrogen receptor-positive breast cancers are stimulated by estrogen, indicating that factors other than the estrogen receptor are involved in progression toward hormone independence. Reactivation or transfer of the estrogen receptor gene can therefore be considered as therapeutic approaches to hormone-independent cancers.
Subpicosecond equilibration of excitation energy in isolated photosystem II reaction centers.
Authors: Durrant J R , Hastings G , Joseph D M , Barber J , Porter G , Klug D R
Published: May 2006

Photosystem II reaction centers have been studied by femtosecond transient absorption spectroscopy. We demonstrate that it is possible to achieve good photoselectivity between the primary electron donor P680 and the majority of the accessory chlorins. Energy transfer can be observed in both directions between P680 and these accessory chlorins depending on which is initially excited. After excitation of either P680 or the other chlorins, the excitation energy is observed to equilibrate between the majority of these pigments at a rate of 100 +/- 50 fs-1. This energy-transfer equilibration takes place before any electron-transfer reactions and must therefore be taken into account in studies of primary electron-transfer reactions in photosystem II. We also show further evidence that the initially excited P680 excited singlet state is delocalized over at least two chlorins and that this delocalization lasts for at least 200 fs.
Honeybee colony integration: worker-worker interactions mediate hormonally regulated plasticity in division of labor.
Authors: Huang Z Y , Robinson G E
Published: May 2006

Adult workers in honeybee (Apis mellifera) colonies exhibit plasticity in hormonally regulated, age-based division of labor by altering their pattern of behavioral development in response to changes in colony conditions. One form of this plasticity is precocious development: levels of juvenile hormone increase prematurely and bees begin foraging as much as 2 weeks earlier than average. We used two experimental paradigms inspired by developmental biology to study how bees obtain information on changing colony needs that results in precocious foraging. An analog of "cell culture," with bees reared outside of colonies in different sized groups, revealed that worker-worker interactions exert quantitative effects on endocrine and behavioral development. "Transplants" of older bees to colonies otherwise lacking foragers demonstrated that worker-worker interactions also affect behavioral development in whole colonies. These results provide insights to a long-standing problem in the biology of social insects and further highlight similarities in the integration of activity that exist between individuals in insect colonies and cells in metazoans.
Insulin-like growth factor I accelerates recovery from ischemic acute tubular necrosis in the rat.
Authors: Miller S B , Martin D R , Kissane J , Hammerman M R
Published: May 2006

The effects of administering insulin-like growth factor I (IGF-I) were examined in a model of ischemic acute tubular necrosis in rats. Injury was induced by 75 min of bilateral renal artery occlusion. Compared to rats administered vehicle, rats administered IGF-I (100 micrograms/day via continuous subcutaneous infusion) had significantly lower serum creatinine and blood urea nitrogen levels over the course of 7 days postocclusion. Glomerular filtration rate as determined by inulin clearance was examined on day 2 postocclusion and was significantly increased in IGF-I-treated animals (0.16 +/- 0.02 ml per min per 100 g of body weight) compared to vehicle-treated controls (0.08 +/- 0.02 ml per min per 100 g of body weight). The weight loss that occurred during the course of acute tubular necrosis was ameliorated by IGF-I. Mortality was reduced from 36.7% in vehicle-treated rats to 7.1% in rats administered IGF-I. Histologically, there was much less renal injury evident at day 7 postocclusion in the IGF-I-treated rats compared to vehicle-treated controls. In contrast, growth hormone (200 micrograms administered subcutaneously for 4 days) did not affect recovery of renal function or reduce mortality postreperfusion. This report demonstrates a beneficial effect of IGF-I administration in the setting of acute tubular necrosis. Several properties of IGF-I render it a pharmacological agent with excellent potential for treatment of this condition in humans.
Major histocompatibility complex class II genes of zebrafish.
Authors: Ono H , Klein D , Vincek V , Figueroa F , O'hUigin C , Tichy H , Klein J
Published: May 2006

Twenty cDNA clones derived from beta-chain-encoding class II genes of the zebrafish (Brachydanio rerio) major histocompatibility complex (MHC) have been sequenced. They fall into three groups identifying three loci of expressed genes. The length and organization of these genes are similar to those of their mammalian homologs. Amplification by polymerase chain reaction and sequencing of genomic DNA from zebrafish collected at different locations in India indicate the existence of a fourth group of sequences (fourth locus). A high degree of polymorphism at the B. rerio MHC loci and concentration of variability to the putative peptide-binding region of the beta 1-domain-encoding part of the gene are also indicated. Large genetic distances between alleles suggest trans-specific evolution of fish MHC polymorphism. Zebrafish genes appear to be derived from a different ancestor than the various class II gene families of other vertebrates. In spite of great sequence divergence between fish and mammalian MHC genes, there seems to be a striking conservation in their overall organization.