bluuebunny/crossref_metadata_2025 huggingface dataset:
A cloned DNA segment from the telomeric region of human chromosome 4p is not detectably rearranged in Huntington disease patients.
Authors: Pritchard C , Casher D , Bull L , Cox D R , Myers R M
Published: May 2006

Genetic linkage studies have mapped the Huntington disease (HD) mutation to the distal region of the short arm of human chromosome 4. Analysis of recombination events in this region has produced contradictory locations for HD. One possible location is in the region distal to the D4S90 marker, which is located within 300 kilobases of the telomere. Other crossover events predict a more centromeric position for HD. Here we analyze the telomeric region of 4p in detail. Cloned DNA segments were derived from this region by utilizing a radiation-induced somatic cell hybrid as a source of DNA combined with preparative pulsed-field gel electrophoresis to enrich for the telomeric fraction. Additional DNA was obtained by using the cloned segments as multiple start points for cosmid walks. This strategy proved to be an effective method for cloning 250 kilobases of DNA in the region telomeric to D4S90. Hybridization analysis with the cloned DNA did not provide any evidence for the presence of rearrangements of 100 base pairs or greater in the DNA of individuals affected with HD. We also found no change in the size or structure of the 4p telomere in these samples.
Molecular cloning of a human macrophage lectin specific for galactose.
Authors: Cherayil B J , Chaitovitz S , Wong C , Pillai S
Published: May 2006

The murine Mac-2 protein is a galactose- and IgE-binding lectin secreted by inflammatory macrophages. We describe here the cloning and characterization of a cDNA representing the human homolog of Mac-2 (hMac-2). The amino acid sequence derived from the hMac-2 cDNA indicates that the protein is evolutionarily highly conserved, with 85% of its amino acid residues being similar to those in the murine homolog. This conservation is especially marked in the carboxyl-terminal lectin domain. The amino-terminal half of the protein is less conserved but still contains the repetitive proline-glycine-rich motif seen in the mouse protein. hMac-2 synthesized in vitro is recognized by the M3/38 monoclonal antibody to Mac-2 and binds to the desialylated glycoprotein asialofetuin and to laminin, a major component of basement membranes. These findings are discussed in the context of the potential functions of hMac-2.
Human kidney amiloride-binding protein: cDNA structure and functional expression.
Authors: Barbry P , Champe M , Chassande O , Munemitsu S , Champigny G , Lingueglia E , Maes P , Frelin C , Tartar A , Ullrich A
Published: May 2006

Phenamil, an analog of amiloride, is a potent blocker of the epithelial Na+ channel. It has been used to purify the porcine kidney amiloride-binding protein. Synthetic oligonucleotides derived from partial sequences have been used to screen a human kidney cDNA library and to isolate the cDNA encoding the human amiloride-binding protein. The primary structure was deduced from the DNA sequence analysis. The protein is 713 residues long, with a 19-amino acid signal peptide. The mRNA was expressed in 293-S and NIH 3T3 cells, yielding a glycoprotein (i) that binds amiloride and amiloride analogs with affinities similar to the amiloride receptor associated with the apical Na+ channel in pig kidney membranes and (ii) that is immunoprecipitated with monoclonal antibodies raised against pig kidney amiloride-binding protein.
Hydrolysis of inositol phospholipids precedes cellular proliferation in asbestos-stimulated tracheobronchial epithelial cells.
Authors: Sesko A , Cabot M , Mossman B
Published: May 2006

Metabolism of inositol phospholipids and phosphatidylcholine was investigated in tracheobronchial epithelial cells exposed to mitogenic concentrations of crocidolite asbestos. Alterations in levels of diacylglycerol, the endogenous activator of protein kinase C, and inositol polyphosphates, presumed mobilizers of intracellular calcium, were examined. Cultures labeled with [3H]glycerol and exposed to proliferative concentrations of crocidolite asbestos demonstrated significant elevations in [3H]diacylglycerol. In contrast, crocidolite-exposed cells labeled with [3H]myristic acid or [3H]choline did not display elevated production of [3H]diacylglycerol or release of [3H]choline metabolites--i.e., evidence of phosphatidylcholine hydrolysis. The soluble tumor promoter phorbol 12-myristate 13-acetate catalyzed both of these changes. myo-[3H]Inositol-labeled cells exposed as briefly as 10 min to mitogenic concentrations of crocidolite demonstrated elevations in [3H]inositol mono-, tris-, and terakisphosphates, phenomena indicating turnover of inositol phospholipids. The detection of diacylglycerol and inositol phosphates in crocidolite asbestos-exposed cells suggests that this fibrous tumor promoter activates phospholipase C as it stimulates cellular proliferation.
Purification and characterization of recombinant Rev protein of human immunodeficiency virus type 1.
Authors: Nalin C M , Purcell R D , Antelman D , Mueller D , Tomchak L , Wegrzynski B , McCarney E , Toome V , Kramer R , Hsu M C
Published: May 2006

Recombinant Rev protein of human immunodeficiency virus type 1 has been expressed in Escherichia coli and purified by ion-exchange and gel-filtration chromatography. Specific binding of the purified protein to the Rev-responsive element of the viral RNA is demonstrated. Physical characterization of the purified protein by circular dichroism and intrinsic fluorescence spectroscopy indicate that the protein preparation is suitable for structural analysis. Circular dichroism measurements show that the protein is approximately 40-45% alpha-helix. Tryptophan fluorescence measurements suggest that the single tryptophan residue is located near the surface of the protein. Gel-filtration chromatography of the protein indicates that it has an apparent molecular mass of 33,000 daltons. This suggests that the protein in solution forms a stable tetramer consisting of monomers having molecular mass of 13,000 daltons.
Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis.
Authors: de Smit M H , van Duin J
Published: May 2006

We have quantitatively analyzed the relationship between translational efficiency and the mRNA secondary structure in the initiation region. The stability of a defined hairpin structure containing a ribosome binding site was varied over 12 kcal/mol (1 cal = 4.184 J) by site-directed mutagenesis and the effects on protein yields were analyzed in vivo. The results reveal a strict correlation between translational efficiency and the stability of the helix. An increase in its delta G0 of -1.4 kcal/mol (i.e., less than the difference between an A.U and a G.C pair) corresponds to the reduction by a factor of 10 in initiation rate. Accordingly, a single nucleotide substitution led to the decrease by a factor of 500 in expression because it turned a mismatch in the helix into a match. We find no evidence that exposure of only the Shine-Dalgarno region or the start codon preferentially favors recognition. Translational efficiency is strictly correlated with the fraction of mRNA molecules in which the ribosome binding site is unfolded, indicating that initiation is completely dependent on spontaneous unfolding of the entire initiation region. Ribosomes appear not to recognize nucleotides outside the Shine-Dalgarno region and the initiation codon.
Dietary pesticides (99.99% all natural).
Authors: Ames B N , Profet M , Gold L S
Published: May 2006

The toxicological significance of exposures to synthetic chemicals is examined in the context of exposures to naturally occurring chemicals. We calculate that 99.99% (by weight) of the pesticides in the American diet are chemicals that plants produce to defend themselves. Only 52 natural pesticides have been tested in high-dose animal cancer tests, and about half (27) are rodent carcinogens; these 27 are shown to be present in many common foods. We conclude that natural and synthetic chemicals are equally likely to be positive in animal cancer tests. We also conclude that at the low doses of most human exposures the comparative hazards of synthetic pesticide residues are insignificant.
Molecular relapse in chronic myelogenous leukemia patients after bone marrow transplantation detected by polymerase chain reaction.
Authors: Sawyers C L , Timson L , Kawasaki E S , Clark S S , Witte O N , Champlin R
Published: May 2006

Relapse of chronic myelogenous leukemia after bone marrow transplantation can be detected by using clinical, cytogenetic, or molecular tools. A modification of the polymerase chain reaction can be used in patients to detect low levels of the BCR-ABL-encoded mRNA transcript, a specific marker for chronic myelogenous leukemia. Early detection of relapse after bone marrow transplantation could potentially alter treatment decisions. We prospectively evaluated 19 patients for evidence of molecular relapse, cytogenetic relapse, and clinical relapse after bone marrow transplantation. We used the polymerase chain reaction to detect residual BCR-ABL mRNA in patients followed up to 45 months after treatment (median, 15 months; range, 6-45 months) and found 4 patients with BCR-ABL mRNA expression following bone marrow transplantation. In 2 patients BCR-ABL mRNA was detected in all samples, and both have developed cytogenetic relapse. In 1 patient BCR-ABL mRNA was detected transiently during the first month after transplant but was undetectable thereafter. The fourth patient had BCR-ABL mRNA 6 months after bone marrow transplantation but not in prior samples. Fifteen patients did not express detectable BCR-ABL mRNA. All 19 patients remain in clinical remission. In this prospective study of chronic myelogenous leukemia patients treated with bone marrow transplantation, molecular relapse preceded cytogenetic relapse in those patients who persistently express BCR-ABL mRNA. We recommend using standard clinical and cytogenetic testing to make patient care decisions until further follow-up determines the clinical outcome of those patients with residual BCR-ABL mRNA transcripts detected by polymerase chain reaction.
Acid-induced folding of proteins.
Authors: Goto Y , Calciano L J , Fink A L
Published: May 2006

The addition of HCl, at low ionic strength, to the native state of apomyoglobin, beta-lactamase, and cytochrome c caused these proteins to adopt an essentially fully unfolded conformation in the vicinity of pH 2. However, contrary to expectation, the addition of further acid resulted in refolding to a compact conformation with the properties of a molten globule. The major factor responsible for the refolding is believed to be the binding of the anion, which minimizes the intramolecular charge repulsion that initially brought about the unfolding.
Supersymmetric Hilbert space.
Authors: Rota G C , Stein J A
Published: May 2006

A generalization is given of the notion of a symmetric bilinear form over a vector space, which includes variables of positive and negative signature ("supersymmetric variables"). It is shown that this structure is substantially isomorphic to the exterior algebra of a vector space. A supersymmetric extension of the second fundamental theorem of invariant theory is obtained as a corollary. The main technique is a supersymmetric extension of the standard basis theorem. As a byproduct, it is shown that supersymmetric Hilbert space and supersymplectic space are in natural duality.
A nonsense mutation causing decreased levels of insulin receptor mRNA: detection by a simplified technique for direct sequencing of genomic DNA amplified by the polymerase chain reaction.
Authors: Kadowaki T , Kadowaki H , Taylor S I
Published: May 2006

Mutations in the insulin receptor gene can render the cell resistant to the biological action of insulin. We have studied a patient with leprechaunism (leprechaun/Minn-1), a genetic syndrome associated with intrauterine growth retardation and extreme insulin resistance. Genomic DNA from the patient was amplified by the polymerase chain reaction catalyzed by Thermus aquaticus (Taq) DNA polymerase, and the amplified DNA was directly sequenced. A nonsense mutation was identified at codon 897 in exon 14 in the paternal allele of the patient's insulin receptor gene. Levels of insulin receptor mRNA are decreased to less than 10% of normal in Epstein-Barr virus-transformed lymphoblasts and cultured skin fibroblasts from this patient. Thus, this nonsense mutation appears to cause a decrease in the levels of insulin receptor mRNA. In addition, we have obtained indirect evidence that the patient's maternal allele of the insulin receptor gene contains a cis-acting dominant mutation that also decreases the level of mRNA, but by a different mechanism. The nucleotide sequence of the entire protein-coding domain and the sequences of the intron-exon boundaries for all 22 exons of the maternal allele were normal. Presumably, the mutation in the maternal allele maps elsewhere in the insulin receptor gene. Thus, we conclude that the patient is a compound heterozygote for two cis-acting dominant mutations in the insulin receptor gene: (i) a nonsense mutation in the paternal allele that reduces the level of insulin receptor mRNA and (ii) an as yet unidentified mutation in the maternal allele that either decreases the rate of transcription or decreases the stability of the mRNA.
Higher order structural elements in ribosomal RNAs: pseudo-knots and the use of noncanonical pairs.
Authors: Gutell R R , Woese C R
Published: May 2006

The data base of prokaryotic small subunit ribosomal RNAs alone now numbers more than 400 sequences, while that for the large subunit rRNAs numbers more than 70 when eukaryotic, mitochondrial, and plastid sequences are also included. Comparisons among these rRNA sequences reveal a number of positions that covary in composition, suggestive of higher order structural elements; 5 such structures are reported for the small subunit rRNA and 15 for the large subunit rRNA. While some of these are properly (small) secondary structural elements, the majority would have to be classified as more complex "tertiary" interactions, which in some cases bring together diverse areas in the secondary structural diagram. A number of the covariances are not of the canonical type, indicating non-Watson-Crick interactions.
Dopamine modulates the kinetics of ion channels gated by excitatory amino acids in retinal horizontal cells.
Authors: Knapp A G , Schmidt K F , Dowling J E
Published: May 2006

Upon exposure to dopamine, cultured teleost retinal horizontal cells become more responsive to the putative photoreceptor neurotransmitter L-glutamate and to its analog kainate. We have recorded unitary and whole-cell currents to determine the mechanism by which dopamine enhances ion channels activated by these agents. In single-channel recordings from cell-attached patches with agonist in the patch pipette, the frequency of 5- to 10-pS unitary events, but not their amplitude, increased by as much as 150% after application of dopamine to the rest of the cell. The duration of channel openings also increased somewhat, by 20-30%. In whole-cell experiments, agonists with and without dopamine were applied to voltage-clamped horizontal cells by slow superfusion. Analysis of whole-cell current variance as a function of mean current indicated that dopamine increased the probability of channel opening for a give agonist concentration without changing the amount of current passed by an individual channel. For kainate, noise analysis additionally demonstrated that dopamine did not alter the number of functional channels. Dopamine also increased a slow spectral component of whole-cell currents elicited by kainate or glutamate, suggesting a change in the open-time kinetics of the channels. This effect was more pronounced for currents induced by glutamate than for those induced by kainate. We conclude that dopamine potentiates the activity of horizontal cell glutamate receptors by altering the kinetics of the ion channel to favor the open state.
Independent and disparate evolution in nature of influenza A virus hemagglutinin and neuraminidase glycoproteins.
Authors: Kilbourne E D , Johansson B E , Grajower B
Published: May 2006

The hemagglutinin (HA) and neuraminidase (NA) external glycoprotein antigens of H1N1 and H3N2 subtypes of epidemiologically important influenza A viruses prevalent during recent decades were subjected to intensive antigenic analysis by four different methods. Prior to serological analysis with polyclonal rabbit antisera, HA and NA antigens of four viruses of each subtype were segregated by genetic reassortment to forestall nonspecific steric hindrance during antigen-antibody combination. This analysis has demonstrated that with respect to antigenic phenotype, HA and NA proteins have evolved at different rates. With H1N1 viruses, an arrest of significant evolution of the NA discordant with the continuing antigenic drift of HA was found in the 1980-1983 period. It is probable that the different and independent rates of evolution of HA and NA reflect the greater selective pressure of HA antibodies, which forces the more rapid emergence of HA escape mutants. The slower antigenic change found for NA further supports the potential for NA-specific infection-permissive immunization as a useful stratagem against influenza.
Isolated dystrophin molecules as seen by electron microscopy.
Authors: Pons F , Augier N , Heilig R , Léger J , Mornet D , Léger J J
Published: May 2006

Dystrophin, the protein product of the Duchenne muscular dystrophy locus [Hoffman, E. P., Brown, R. H., Jr., & Kunkel, L. M. (1987) Cell 51, 919-928], is expressed in striated and smooth muscles as well as in non-muscle tissues. Examination of its primary structure has revealed that the molecule is composed of four domains, three of which share many features with the membrane cytoskeletal proteins spectrin and actinin. Dystrophin has thus been predicted to adopt a rod shape [Koenig, M., Monaco, A. P. & Kunkel, L. M. (1988) Cell 53, 219-228]. In the present study, we describe its isolation from the chicken gizzard smooth muscle and present electron microscopic images of the molecule. Polyclonal antibodies were first prepared from a dystrophin fragment derived from the chicken skeletal muscle gene (residues 1173-1728). A dystrophin-enriched membrane preparation from chicken gizzard muscle was then purified by passing it through an affinity chromatography column made with the anti-dystrophin antibodies. Electron microscopy of isolated and rotatory-shadowed dystrophin molecules revealed that the lengths measured for the dystrophin monomers (175 +/- 15 nm) are compatible with a structural arrangement of the repeat sequence segments in triple-barrel alpha-helices connected by short-turn regions, as was earlier postulated for the repeat domains of spectrin and actinin. Electron microscopic images indicate that in addition the dystrophin molecules could present the same capacity of self-association in oligomeric structures as these cytoskeletal proteins and may thus be a part of a complex molecular meshwork essential to muscle cell function.
A monoclonal antibody that neutralizes Epstein-Barr virus, human cytomegalovirus, human herpesvirus 6, and bacteriophage T4 DNA polymerases.
Authors: Tsai C H , Williams M V , Glaser R
Published: May 2006

A monoclonal antibody (mAb) designated 55H3 was produced by using chemically induced Epstein-Barr virus genome-positive B95-8 cells. mAb 55H3, which reacted with an 85- to 80-kDa polypeptide, neutralized Epstein-Barr virus-encoded DNA polymerase activity in crude extracts of chemically induced M-ABA, HR-1, and B95-8 cells, as well as the partially purified Epstein-Barr virus DNA polymerase in a dose-dependent manner. The mAb also neutralized the virus-encoded DNA polymerase activity from cells infected with human cytomegalovirus, human herpesvirus 6, and the purified bacteriophage T4 DNA polymerases. However, mAb 55H3 did not neutralize the DNA polymerase activities encoded for by herpes simplex virus types 1 and 2, the reverse transcriptase of avian myeloblastosis virus, or Escherichia coli DNA polymerase 1 (Klenow fragment). These results suggest that mAb 55H3 recognizes an epitope common to some herpesviruses and T4 DNA polymerases and further supports the hypothesis that these organisms are evolutionarily related.
Central prolactin infusions stimulate maternal behavior in steroid-treated, nulliparous female rats.
Authors: Bridges R S , Numan M , Ronsheim P M , Mann P E , Lupini C E
Published: May 2006

A series of experiments were conducted to determine whether and under what conditions central prolactin (PRL) administration would stimulate the onset of maternal behavior in female rats and to identify possible neural sites of PRL action. In each experiment ovariectomized, nulliparous rats whose endogenous PRL levels were suppressed with bromocriptine were tested for maternal behavior toward foster young. In experiments 1, 2, and 4, females were also exposed to pregnancy-like levels of progesterone (days 1-11) followed by estradiol (days 11-17). In experiment 1 infusions (days 11-13) of four doses of ovine PRL (400 ng, 2 micrograms, 10 micrograms, or 50 micrograms, but not 80 ng) into the lateral ventricle resulted in a rapid onset of maternal behavior (behavioral testing, days 12-17). The stimulatory action of these doses of PRL appears to be central, since subcutaneous injections of 50 micrograms of ovine PRL failed to affect maternal responsiveness (experiment 2). Experiment 3 indicated that the stimulatory effect of intracerebroventricularly administered PRL is steroid dependent. Infusions of either 10 micrograms of ovine PRL or 10 micrograms of rat PRL failed to induce maternal behavior in nonsteroid-treated animals. In the final experiment (no. 4) bilateral infusions of 40 ng of ovine PRL into the medial preoptic area of steroid-treated rats resulted in a pronounced stimulation of maternal behavior. These findings demonstrate a central site of PRL action in the stimulation of maternal responsiveness and point to the medial preoptic area as a key neural site for PRL regulation of maternal behavior.
E1a-dependent expression of adenovirus genes in OTF963 embryonal carcinoma cells: role of E1a-induced differentiation.
Authors: Nelson C C , Braithwaite A W , Silvestro M , Bellett A J
Published: May 2006

Some undifferentiated F9 embryonal carcinoma cells allow adenovirus genes to be expressed independently of the E1a oncogene normally required for their activation; this has been attributed to a cellular equivalent of E1a in F9 cells. However, transcription of all early genes was low in undifferentiated OTF963 embryonic carcinoma cells during the first 48 hr after infection with adenovirus type 5 (Ad5). Transcription then increased to about the level seen 16 hr after infection of cells induced to differentiate by retinoic acid (RA) (referred to as RA-dF9 cells), but this increase did not occur in cells infected by the E1a deletion mutant dl312. Addition of E1a in trans, or of RA, had no immediate effect on viral transcription in OTF963 cells, but viral transcription increased about 48 hr after these additions. Ad5 induced transcription of several differentiation-specific genes in OTF963 cells with about the same kinetics as their induction by RA. These genes were superinduced in RA-dF9 cells by cAMP or infection by adenovirus. We suggest the small amount of E1a produced early in infection of OTF963 cells activates cellular genes, some of which are differentiation specific and required for efficient transcription of viral genes, so that E1a both induces and is induced by differentiation. The simple hypothesis of a cellular equivalent to E1a does not adequately explain the complex interactions between viral and cellular genes in OTF963 embryonic carcinoma cells.
A single amino acid substitution in the variable region of the light chain specifically blocks immunoglobulin secretion.
Authors: Dul J L , Argon Y
Published: May 2006

Although immunoglobulin light chains are usually secreted in association with heavy chains, free light chains can be secreted by lymphocytes. To identify the structural features of light chains that are essential for their secretion, we mutated a conserved sequence in the variable domain of a lambda I light chain. The effects of the mutations on secretion were assayed by transient expression in COS-1 cells. One mutant (AV60), which replaced Ala-60 with Val, was secreted as efficiently as wild-type lambda I by transfected COS-1 cells. This result was not surprising because secreted lambda II chains contain valine in this position. However, a second lambda I mutant (AV60FS62), which replaced Phe-62 with Ser as well as Ala-60 with Val, was not secreted. This mutant was arrested in the endoplasmic reticulum, as judged by immunofluorescence and by its association with a lumenal endoplasmic reticulum protein, immunoglobulin heavy chain binding protein (BiP). The defect in secretion was not due to gross misfolding of the lambda I chain, since cells cotransfected with AV60FS62 and an immunoglobulin heavy chain gene produced functional antigen-binding antibodies. These assembled IgM molecules were still not secreted. Hence, the replacement of Phe-62 with Ser specifically affects a determinant on the lambda I light chain that is necessary for the intracellular transport of this molecule.
Interchangeable RNA polymerase I and II enhancers.
Authors: Lorch Y , Lue N F , Kornberg R D
Published: May 2006

The RNA polymerase I (pol I) enhancer of Saccharomyces cerevisiae contains at least three elements commonly associated with RNA polymerase II (pol II) enhancers, binding sites for the transcriptional activators general regulatory factor 2 and autonomously replicating sequence-binding factor I, and a thymidine-rich element. When the particular form of the thymidine-rich element found in the pol I enhancer was placed in front of a pol II promoter, transcription was stimulated 43-fold, comparable to the effect of a powerful pol II activator such as Gal4. Conversely, when two copies of a thymidine-rich element from a pol II enhancer were placed upstream of a pol I promoter, transcription was stimulated 38-fold. This functional reciprocity of pol I and II enhancers may reflect similarities in the mechanisms of transcriptional activation. The pol I enhancer also contains an element that appears to be pol I-specific and prevent the activation of pol II.
Electrophoretic karyotype for Dictyostelium discoideum.
Authors: Cox E C , Vocke C D , Walter S , Gregg K Y , Bain E S
Published: May 2006

This paper reports on the separation of the Dictyostelium discoideum chromosomes by pulse-field electrophoresis and the correlation of the electrophoretic pattern with linkage groups established by classical genetic methods. In two commonly used laboratory strains, five chromosome-sized DNA molecules have been identified. Although the majority of the molecular probes used in this study can be unambiguously assigned to established linkage groups, the electrophoretic karyotype differs between the closely related strains AX3k and NC4, suggesting that chromosomal fragmentation may have occurred during their maintenance and growth. The largest chromosome identified in this study is approximately 9 million base pairs. To achieve resolution with molecules of this size, programmed voltage gradients were used in addition to programmed pulse times.
A putative protein kinase gene (kin1+) is important for growth polarity in Schizosaccharomyces pombe.
Authors: Levin D E , Bishop J M
Published: May 2006

Mixed synthetic oligonucleotides encoding a sequence conserved among tyrosine-specific protein kinases were used to probe the genome of the fission yeast Schizosaccharomyces pombe. A single gene (kin1+) was isolated that encodes a putative protein kinase closely related to the KIN1- and KIN2-encoded serine/threonine-specific protein kinases of Saccharomyces cerevisiae. kin1+ is transcribed into a 3.5-kilobase mRNA that contains an uninterrupted open reading frame encoding a polypeptide of 98 kDa. In contrast to results obtained with kin mutants of S. cerevisiae, disruption of the Sc. pombe kin1+ gene resulted in recessive morphological and growth defects. kin1-disrupted cells grew slowly on enriched medium and grew as spheres, in contrast to wild-type Sc. pombe cells, which grow as rods. Relative to kin1+ cells, kin1-disrupted cells were differentially sensitive to lysis by treatment with alpha- and beta-glucanases, suggesting an alteration in either the composition or the organization of their cell walls.
Regulation of the fibroblast growth factor receptor in early Xenopus embryos.
Authors: Musci T J , Amaya E , Kirschner M W
Published: May 2006

Recent evidence suggests that fibroblast growth factor (FGF) is a primary mesoderm inducer in Xenopus development. We have isolated a full-length cDNA clone for the Xenopus FGF receptor. Like other FGF receptors, the Xenopus homolog is a membrane-spanning protein with a split intracellular tyrosine kinase domain. The Xenopus FGF receptor mRNA is present as a maternal message whose levels are constant through early development. There is no specific regional localization of the transcript by analysis of FGF receptor mRNA levels in microdissected embryonic tissue. In isolated animal-pole blastomeres, FGF receptor mRNA declines over 16 hr in culture and this loss can be prevented by incubation with FGF or activin. Despite the presence of the FGF receptor mRNA in the oocyte, oocytes in culture do not respond to added FGF. However, injection of exogenous Xenopus FGF receptor transcripts into oocytes does generate a functional response to FGF. Our data suggest that posttranscriptional response to FGF. Our data suggest that posttranscriptional mechanisms regulate the FGF receptor in the oocyte and early embryo and further suggest that mesoderm-inducing factors influence receptor mRNA levels during the time of early tissue formation.
Parasitoid virus-like particles destroy Drosophila cellular immunity.
Authors: Rizki R M , Rizki T M
Published: May 2006

Parasitoid wasps must avoid the destructive effects of the host's cellular defense system in order to exploit the host hemocoel as a suitable environment for their survival. To protect their eggs from encapsulation by Drosophila melanogaster blood cells, Leptopilina heterotoma females inject a factor that selectively destroys lamellocytes, the type of Drosophila blood cell involved in recognition and encapsulation of large foreign objects. Other types of host blood cells, including the phagocytic plasmatocytes, remain functional. This report demonstrates that the destructive factor for lamellocytes is a virus-like particle (VLP) stored in the reservoir of an accessory gland associated with the female wasp reproductive system. We show that VLPs enter Drosophila blood cells in vitro. VLPs are found free in the cytoplasm of lamellocytes but are confined to phagocytic vesicles of plasmatocytes. As lamellocytes are susceptible to the VLP infection and plasmatocytes are not, we conclude that the mode of VLP entry and its disposition in the cytoplasm determine the fate of the infected host blood cell.
Molecular cloning and characterization of the immunologically protective surface glycoprotein GP46/M-2 of Leishmania amazonensis.
Authors: Lohman K L , Langer P J , McMahon-Pratt D
Published: May 2006

Immunization of mice with the GP46/M-2 membrane glycoprotein has been demonstrated to elicit protection against infection with the parasitic protozoan Leishmania amazonensis. As this molecule is important for future vaccine studies of leishmaniasis, the gene encoding the GP46/M-2 surface membrane glycoprotein of Leishmania amazonensis has been cloned and sequenced. The protein sequence derived from the DNA sequence data is consistent with the known biochemical and immunochemical properties of the protein and indicates a number of structural areas of interest. A repetitive sequence (24 amino acids repeated four times) occurs within the amino-terminal portion of the molecule and constitutes approximately 22% of the total mature protein. The protease-resistant immunodominant carboxyl-terminal domain of the protein comprises approximately half of the molecule and consists of proline-rich and cysteine-rich areas of sequence; the distribution of cysteine residues is suggestive of metal binding motifs. The sequence predicts a hydrophobic leader peptide, and a putative attachment site for a glycosyl-phosphatidylinositol anchor is indicated at the carboxyl terminus, consistent with the membrane location of the protein. Southern blot analyses also indicate the presence of a GP46/M-2 gene family.
Cytochrome c oxidase: decay of the primary oxygen intermediate involves direct electron transfer from cytochrome a.
Authors: Han S H , Ching Y C , Rousseau D L
Published: May 2006

The decay of the primary intermediate generated in the reaction of oxygen with cytochrome c oxidase is nearly one order of magnitude faster in the fully reduced form of the enzyme than it is in the mixed valence form. To account for this observation, we propose a model describing the early molecular events in the reaction. In this model the decay of the primary Fe-O2 intermediate in the fully reduced enzyme is a consequence of direct electron transfer from cytochrome a. To test the model we measured the time dependence of the oxidation of cytochrome a by monitoring the resonance Raman scattering intensity of its vibrational modes. A rapid oxidation of cytochrome a was detected that quantitatively agrees with the model. These results indicate that the mechanism of oxygen reduction and proposed frameworks for proton translocation must be reexamined.
Cloning and sequence of the human adrenodoxin reductase gene.
Authors: Lin D , Shi Y F , Miller W L
Published: May 2006

Adrenodoxin reductase (ferrodoxin:NADP+ oxidoreductase, EC 1.18.1.2) is a flavoprotein mediating electron transport to all mitochondrial forms of cytochrome P450. We cloned the human adrenodoxin reductase gene and characterized it by restriction endonuclease mapping and DNA sequencing. The entire gene is approximately 12 kilobases long and consists of 12 exons. The first exon encodes the first 26 of the 32 amino acids of the signal peptide, and the second exon encodes the remainder of signal peptide and the apparent FAD binding site. The remaining 10 exons are clustered in a region of only 4.3 kilobases, separated from the first two exons by a large intron of about 5.6 kilobases. Two forms of human adrenodoxin reductase mRNA, differing by the presence or absence of 18 bases in the middle of the sequence, arise from alternate splicing at the 5' end of exon 7. This alternately spliced region is directly adjacent to the NADPH binding site, which is entirely contained in exon 6. The immediate 5' flanking region lacks TATA and CAAT boxes; however, this region is rich in G + C and contains six copies of the sequence GGGCGGG, resembling promoter sequences of "housekeeping" genes. RNase protection experiments show that transcription is initiated from multiple sites in the 5' flanking region, located about 21-91 base pairs upstream from the AUG translational initiation codon.
Dioxin-inducible, Ah receptor-dependent transcription in vitro.
Authors: Wen L P , Koeiman N , Whitlock J P
Published: May 2006

We have developed a homologous in vitro transcription system that requires (i) 2,3,7,8-tetrachlorodibenzo-p-dioxin (called TCDD or dioxin), (ii) the Ah receptor, and (iii) a dioxin-responsive enhancer for activity. Unfractionated nuclear extracts from mouse hepatoma cells contain an inhibitor and fail to direct transcription in vitro. However, following phosphocellulose chromatography and reconstitution, the fractionated nuclear extract directs accurate transcription in vitro, using as a template the promoter/enhancer region from the mouse cytochrome P1-450 gene (Cyp1a1) linked to a "G-free cassette" (which generates a transcript with no guanosine residues). Extracts from TCDD-treated cells exhibit higher activity than extracts from untreated cells when transcribing a template containing both the promoter and enhancer but not when transcribing a template containing the promoter alone. Extracts from Ah receptor-defective cells fail to direct in vitro transcription in a TCDD-inducible fashion. A regulatory element that contains two binding sites for the liganded Ah receptor plus a truncated Cyp1a1 promoter suffices to direct TCDD-inducible, Ah receptor-dependent transcription in vitro. The inducible, receptor-dependent, enhancer-dependent properties of this system make it appropriate for analyzing in vitro the mechanism of dioxin action and the function of the Ah receptor.
Actin cores of hair-cell stereocilia support myosin motility.
Authors: Shepherd G M , Corey D P , Block S M
Published: May 2006

The actin cores of hair-cell stereocilia were tested as a substrate for the movement of myosin-coated beads in an in vitro assay. Large numbers of stereocilia from bullfrog sacculi and semicircular canals were isolated by blotting onto coverglasses and were demembranated to expose the polar actin tracks of their cytoskeletal cores. Silica or polystyrene beads, coated with thick filaments of chicken skeletal muscle myosin, were added to this core preparation in the presence of ATP. Myosin-coated beads could reach some of the cores by diffusion alone, but the efficiency and precision of the assay were improved considerably by the use of "optical tweezers" (a gradient-force optical trap) to deposit the beads directly on the cores. Beads applied in this fashion bound and moved unidirectionally at 1-2 microns/s, escaping the retarding force of the trap. Actin filaments within the stereocilia are cross-linked by fimbrin, but this did not appear to interfere with the motility of myosin. Beads coated with optic-lobe kinesin were also tested for movement; these bound and moved unidirectionally at 0.1-0.2 microns/s when applied to microtubule-based kinociliary cores, but not when applied to actin-based stereociliary cores. Our results are consistent with, and lend support to, a model for hair cell adaptation in which a molecular motor such as myosin maintains tension on the mechanically gated transduction channels. Optical tweezers and video-enhanced differential interference contrast optics provide high efficiency and improved optical resolution for the in vitro analysis of myosin motility.
Incorporation of Paramecium axonemal tubulin into higher plant cells reveals functional sites of microtubule assembly.
Authors: Vantard M , Levilliers N , Hill A M , Adoutte A , Lambert A M
Published: May 2006

Incorporation of Paramecium axonemal tubulin into lysed endosperm cells of the higher plant Haemanthus enabled us to identify sites of microtubule assembly. This exogenous Paramecium tubulin could be traced by specific antibodies that do not stain endogenous plant microtubules. Intracellular copolymerization of protozoan and higher plant tubulins gave rise to hybrid polymers that were visualized by immunofluorescence and by immunoelectron microscopy. The addition of exogenous tubulin revealed many free ends of endogenous microtubules that were competent to assemble ciliate tubulin. The functional roles of the nuclear surface and the equatorial region of the phragmoplast as plant microtubule-organizing centers, which were revealed by the intense incorporation of exogenous tubulin, are discussed. These data shed light on the present debate on higher plant microtubule organizing centers.
Lovastatin decreases coenzyme Q levels in humans.
Authors: Folkers K , Langsjoen P , Willis R , Richardson P , Xia L J , Ye C Q , Tamagawa H
Published: May 2006

Lovastatin is clinically used to treat patients with hypercholesterolemia and successfully lowers cholesterol levels. The mechanism of action of lovastatin is inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, an enzyme involved in the biosynthesis of cholesterol from acetyl-CoA. Inhibition of this enzyme could also inhibit the intrinsic biosynthesis of coenzyme Q10 (CoQ10), but there have not been definitive data on whether lovastatin reduces levels of CoQ10 as it does cholesterol. The clinical use of lovastatin is to reduce a risk of cardiac disease, and if lovastatin were to reduce levels of CoQ10, this reduction would constitute a new risk of cardiac disease, since it is established that CoQ10 is indispensable for cardiac function. We have conducted three related protocols to determine whether lovastatin does indeed inhibit the biosynthesis of CoQ10. One protocol was done on rats, and is reported in the preceding paper [Willis, R. A., Folkers, K., Tucker, J. L., Ye, C.-Q., Xia, L.-J. & Tamagawa, H. (1990) Proc. Natl. Acad. Sci. USA 87, 8928-8930]. The other two protocols are reported here. One involved patients in a hospital, and the other involved a volunteer who permitted extraordinary monitoring of CoQ10 and cholesterol levels and cardiac function. All data from the three protocols revealed that lovastatin does indeed lower levels of CoQ10. The five hospitalized patients, 43-72 years old, revealed increased cardiac disease from lovastatin, which was life-threatening for patients having class IV cardiomyopathy before lovastatin or after taking lovastatin. Oral administration of CoQ10 increased blood levels of CoQ10 and was generally accompanied by an improvement in cardiac function. Although a successful drug, lovastatin does have side effects, particularly including liver dysfunction, which presumably can be caused by the lovastatin-induced deficiency of CoQ10.
Oscillatory activity of single units in a somatosensory cortex of an awake monkey and their possible role in texture analysis.
Authors: Ahissar E , Vaadia E
Published: May 2006

Neuronal activity was extracellularly recorded in the cortex of an awake monkey (Macaca fascicularis). Single units displaying oscillatory firing patterns were found in the upper bank of the lateral sulcus in a region where most of the neurons responded to somatosensory stimuli. The spectral energies of the oscillating activity were distributed in a trimodal fashion--0-15, 15-50, and 80-250 Hz--with the most common frequencies around 30 Hz. The oscillatory activity was not affected by anesthesia, but it was often reduced by tactile stimulation or self-initiated movements. Analysis of the spike trains suggests that the majority of oscillatory activity was intrinsically generated by the neurons. A neural model of texture analysis is offered based on a corticothalamic phase-locked loop. The newly identified oscillators play a key role in this model. The relevance of the model to physiological, anatomical, and psychophysical data, as well as testable predictions, are discussed.
Site-directed mutagenesis of conserved cysteine residues in Escherichia coli fumarate reductase: modification of the spectroscopic and electrochemical properties of the [2Fe-2S] cluster.
Authors: Werth M T , Cecchini G , Manodori A , Ackrell B A , Schröder I , Gunsalus R P , Johnson M K
Published: May 2006

Site-directed mutants of Escherichia coli fumarate reductase in which each of the four N-terminal cysteine residues in the FrdB subunit, residues 57, 62, 65, and 77, was mutated individually to serine have been constructed, overexpressed, and investigated in terms of enzymatic activity as well as the EPR and redox properties of the iron-sulfur centers. In each case, the mutant contains a functional fumarate reductase in which all three of the constituent iron-sulfur clusters (i.e., center 1, [2Fe-2S]; center 2, [4Fe-4S]; center 3, [3Fe-4S]) have been assembled. The mutations affect the properties of center 1 only and demonstrate that the anomalously high redox potential of this [2Fe-2S] center is essential for optimal enzymatic activity. The results are consistent with cysteines 57, 62, 65, and 77 providing the ligands to center 1 but leave open the possibility of noncysteinyl coordination for the localized valence Fe(III) site of the reduced cluster. The implications of the results for the role of center 1 in the electron-transfer pathway and the valence localization of reduced center 1 are discussed.
Advanced protein glycosylation induces transendothelial human monocyte chemotaxis and secretion of platelet-derived growth factor: role in vascular disease of diabetes and aging.
Authors: Kirstein M , Brett J , Radoff S , Ogawa S , Stern D , Vlassara H
Published: May 2006

Diabetes and aging are commonly accompanied by arterio- and atherosclerosis. Infiltration of the arterial subendothelial intima by macrophages/monocytes is an important early event preceding the development of atheromatous lesions; these macrophages are known to produce mitogenic factors in early atherosclerotic lesions. It has been previously shown that, over time, vascular matrix accumulates proteins nonenzymatically modified by advanced glycosylation end products (AGEs). In view of the fact that macrophages/monocytes have AGE-specific receptors associated with the expression of several growth factors, we investigated the possibility that AGEs mediate initial monocyte-vessel wall interactions that occur before overt formation of vascular lesions. This study demonstrates that (i) in vitro- and in vivo-formed AGEs are chemotactic for human blood monocytes, (ii) sub-endothelial AGEs can selectively induce monocyte migration across an intact endothelial cell monolayer, and (iii) subsequent monocyte interaction with AGE-containing matrix results in the expression of platelet-derived growth factor. These results support the existing hypothesis that in vivo-forming glucose-derived protein adducts can act as signals for the normal turnover of senescent tissue protein by means of the AGE-specific receptor system. Time-dependent glucose-induced deposition of AGEs on matrix proteins may promote monocyte infiltration into the subendothelium. Subsequent AGE-triggered macrophage activation and consequent elaboration of proliferative factors may normally coordinate remodeling but may also lead to the diverse pathogenic changes typical of arterio- and atherosclerosis in diabetic or aging populations.
N epsilon-acetyl-beta-lysine: an osmolyte synthesized by methanogenic archaebacteria.
Authors: Sowers K R , Robertson D E , Noll D , Gunsalus R P , Roberts M F
Published: May 2006

Methanosarcina thermophila, a nonmarine methanogenic archaebacterium, can grow in a range of saline concentrations. At less than 0.4 M NaCl, Ms. thermophila accumulated glutamate in response to increasing osmotic stress. At greater than 0.4 M NaCl, this organism synthesized a modified beta-amino acid that was identified as N epsilon-acetyl-beta-lysine by NMR spectroscopy and ion-exchange HPLC. This beta-amino acid derivative accumulated to high intracellular concentrations (up to 0.6 M) in Ms. thermophila and in another methanogen examined--Methanogenium cariaci, a marine species. The compound has features that are characteristic of a compatible solute: it is neutrally charged at physiological pH and it is highly soluble. When the cells were grown in the presence of exogenous glycine betaine, a physiological compatible solute, N epsilon-acetyl-beta-lysine synthesis was repressed and glycine betaine was accumulated. N epsilon-acetyl-beta-lysine was synthesized by species from three phylogenetic families when grown in high solute concentrations, suggesting that it may be ubiquitous among the methanogens. The ability to control the biosynthesis of N epsilon-acetyl-beta-lysine in response to extracellular solute concentration indicates that the methanogenic archaebacteria have a unique beta-amino acid biosynthetic pathway that is osmotically regulated.