bluuebunny/crossref_metadata_2025 huggingface dataset:
Coordinate tropic hormone regulation of mRNAs for insulin-like growth factor II and the cholesterol side-chain-cleavage enzyme, P450scc [corrected], in human steroidogenic tissues.
Authors: Voutilainen R , Miller W L
Published: May 2006

Insulin-like growth factors (IGFs) are single-chain polypeptides important for cell proliferation and growth. IGFs are produced in several tissues, suggesting that they function in a paracrine or autocrine fashion as well as functioning as endocrine hormones. We studied the hormonal regulation of IGF-I and IGF-II mRNA in human steroidogenic tissues. In cultured human ovarian granulosa cells, follicle-stimulating hormone, human chorionic gonadotropin, and dibutyryl cAMP increased IGF-II mRNA, but corticotropin [adrenocorticotropic hormone (ACTH)], chorionic somatomammotropin, growth hormone, prolactin, dexamethasone, estradiol, and progesterone had no effect. In cultured human fetal adrenal cells, ACTH and dibutyryl cAMP increased IGF-II mRNA accumulation, but human chorionic gonadotropin and angiotensin II did not. The same five size species of IGF-II mRNA were detected in transfer blots of RNA from granulosa cells and fetal adrenal cells, and all of these increased after hormonal stimuli. Dibutyryl cAMP also increased IGF-II mRNA accumulation in cultured human placental cells. Accumulation of mRNA for the cholesterol side-chain-cleavage monooxygenase [P450scc [corrected]; cholesterol, reduced-adrenal-ferredoxin:oxygen oxidoreductase (side-chain-cleaving), EC 1.14.15.6] was regulated in parallel with IGF-II mRNA in all these steroidogenic tissues. IGF-I mRNA was not detected in transfer blots of these RNAs, and the minimal amounts detected in dot blots showed no detectable change after any of the hormonal stimuli studied. The data indicate that the IGF-II gene is expressed in human steroidogenic tissues and is regulated by cAMP. These data suggest that IGF-II may act in an autocrine or paracrine fashion to stimulate the adrenal and gonadal growth stimulated by ACTH and gonadotropins, respectively.
Sea urchin prosome: characterization and changes during development.
Authors: Akhayat O , Grossi de Sa F , Infante A A
Published: May 2006

A cytoplasmic particle displaying properties in common with a structure present in duck erythroblasts, termed the prosome, has been isolated from eggs and embryos of two species of sea urchin. This particle was partially purified by sedimentation in sucrose gradients containing 0.5 M KCl, and some of its physical properties and its behavior during early development were determined. The prosome sediments between 16 and 19 S and has a buoyant density of 1.30 g/cm3 in Cs2SO4 gradients. Biochemically, the particle is characterized as 20-25 polypeptides of molecular size 24-35 kDa with about 10 small RNAs. A monoclonal antibody directed against the 27-kDa protein of duck erythroblast prosome recognizes a 27-kDa protein of the sea urchin prosome. We have used this protein, as representative of the prosome, to immunologically determine the level and the subcellular localization of the particle during development. Immunoblotting and cellular fractionation studies show that the 27-kDa prosome polypeptide is present almost entirely in the postribosomal supernatant of unfertilized egg lysates. After fertilization and during early development, the total amount of 27-kDa protein per embryo remains constant, but the amount in the postribosomal supernatant decreases; there is a concomitant increase in the level of the 27-kDa protein in a rapidly sedimenting, particulate fraction containing nuclei. Immunofluorescence studies further show that the 27-kDa protein is located mainly in the cytoplasm of eggs and two-cell embryos. The subcellular location of the prosome, therefore, appears to change during development. In vivo labeling experiments have failed to detect the synthesis of either the prosome proteins or RNAs in eggs and embryos up to 48 hr of development, suggesting that this cytoplasmic particle is not synthesized de novo in early embryogenesis and thus is metabolically stable. The prosome is thus a normal cellular constituent of the sea urchin and is most likely synthesized during oogenesis and stored in the unfertilized egg.
Restriction fragment length polymorphism of the HLA-DP subregion and correlations to HLA-DP phenotypes.
Authors: Hyldig-Nielsen J J , Morling N , Odum N , Ryder L P , Platz P , Jakobsen B , Svejgaard A
Published: May 2006

The restriction fragment length polymorphism (RFLP) of the class II HLA-DP subregion of the major histocompatibility complex (MHC) of humans has been unraveled by Southern blotting using DP alpha and DP beta probes in a study of 46 unrelated individuals with known HLA-DP types. Contrary to earlier preliminary findings with a limited number of enzymes, the RFLP appears to be quite extensive both with the DP beta (14 different DNA markers defined by individual fragments or clusters thereof) and the DP alpha (8 markers) probes, especially when enzymes recognizing only four base pairs were used. A few markers were absolutely or strongly associated with individual DP antigens, whereas most were associated with two or more DP antigens as defined by primed lymphocyte typing. Thus, Southern blotting seems feasible for typing for most DP determinants by specific fragments or subtraction between the various more broadly reactive DNA markers, and the RFLP provides further information on the DP subregion in addition to that provided by primed lymphocyte typing. In two recombinant families, the DP beta and DP alpha DNA markers segregated with DP antigens, whereas the DR beta, DQ beta, DQ alpha, and DX alpha markers followed the DR and DQ antigens.
Application of a protein-blotting procedure to the study of human glucocorticoid receptor interactions with DNA.
Authors: Silva C M , Tully D B , Petch L A , Jewell C M , Cidlowski J A
Published: May 2006

To exert their effects, glucocorticoid receptor complexes interact selectively with DNA sequences known as glucocorticoid regulatory elements. We have studied the interaction between human glucocorticoid receptors and mouse mammary tumor virus (MMTV) DNA by means of a procedure that permits analysis after immobilization of the receptor on nitrocellulose. Proteins from crude cytosolic or nuclear extracts were electrophoresed on NaDodSO4/PAGE gels, soaked in a urea buffer to remove NaDodSO4, transferred to nitrocellulose, and probed with nick-translated MMTV [32P]DNA in a 5% nonfat dry milk buffer, which minimizes nonselective DNA-protein interactions. We present evidence that MMTV [32P]DNA interacts selectively with the glucocorticoid receptor. These data include comigration of [3H]dexamethasone mesylate-labeled band and bound MMTV [32P]DNA on gel electrophoresis systems; localization of DNA-binding activity in the cytosol of cells incubated with steroid at 0 degrees C and in the nucleus and cytosol of cells incubated at 37 degrees C; binding of the MMTV DNA to highly purified receptor; and absence of MMTV DNA binding activity in extracts from cells whose receptor has been down-regulated. Furthermore, glucocorticoid receptors analyzed under these conditions exhibit selective binding to DNA fragments that contain glucocorticoid regulatory elements.
Inhibition of replication forks exiting the terminus region of the Escherichia coli chromosome occurs at two loci separated by 5 min.
Authors: de Massy B , Béjar S , Louarn J , Louarn J M , Bouché J P
Published: May 2006

The replication cycle of Escherichia coli strains duplicating their chromosome from the same plasmid origin placed at various locations or of strains having undergone a major inversion event along the origin-to-terminus axis was studied by marker-frequency analysis. It was observed that replication forks are unidirectionally inhibited at two loci of the termination region: counterclockwise-moving forks are inhibited at terminator T1 (28.5 min), and forks moving in the opposite direction are inhibited at terminator T2 (33.5 min). By determining the strand preference of Okazaki fragments that are specific for markers from the T1-T2 interval, it was shown that this interval is replicated in either direction, depending upon the strain analyzed. In addition, we also observed that forks moving in the "unnatural" direction along each oriC-T1 or -T2 arm are very slow, especially in the one-third portion of the chromosome around the terminators. We propose that this phenomenon is a consequence of nucleoid organization, which is proposed to be symmetrical on the two oriC-T1 or -T2 arms and polarized with respect to the direction of replication. We also propose that T1 and T2 are the terminal limits of these two polarized half-nucleoid bodies.
Labeling quinone-binding sites in photosynthetic reaction centers: A 38-kilodalton protein associated with the acceptor side of photosystem II
Authors: Worland Stephen T. , Yamagishi Akihiko , Isaacs Stephen , Sauer Kenneth , Hearst John E.
Published: May 2006

2-Acetoxymethyl-1,4-naphthoquinone (2-AcOMeNQ) binds with rapid kinetics and high affinity to the primary quinone Q
A
site of reaction centers from
Rhodopseudomonas capsulata
. Binding of 2-AcOMeNQ fully restores electron-transfer activity with kinetics that is similar, but not identical, to that seen with ubiquinone-50. When bound at the Q
A
site, 2-AcOMeNQ preferentially labels the L subunit. This preference suggests that 2-AcOMeNQ labels primarily the region of a quinone-binding site that is close to the first isoprenoid unit of the side chain, which is expected from the location and structure of the reaction region of the molecule. In photosystem II particles from
Synechococcus
sp., 2-AcOMeNQ primarily labels two polypeptides with apparent molecular masses of 38 and 19 kDa. Labeling of only the 38-kDa polypeptide is sufficiently sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to conclude that it is involved in binding quinones on the acceptor side of photosystem II. Although we have not yet identified the 38-kDa protein, its properties suggest that it is the D2 protein. From the DCMU-sensitive labeling and from homologies to functionally important regions of the bacterial reaction-center subunits, we propose that the 38-kDa protein is intimately involved in binding the cofactors that mediate primary photochemistry.
Sporulation-specific sigma factor sigma 29 of Bacillus subtilis is synthesized from a precursor protein, P31.
Authors: LaBell T L , Trempy J E , Haldenwang W G
Published: May 2006

Evidence is presented that a sporulation-essential sigma factor of Bacillus subtilis, sigma 29, is synthesized as an inactive precursor (P31) and that its activation occurs by a developmentally regulated cleavage of 29 amino acids from the P31 amino terminus. A pulse-chase experiment demonstrated that sigma 29 was derived from a preexisting protein, with appearance of radioactively labeled sigma 29 paralleling the disappearance of labeled P31. The disappearance of pulse-labeled P31 did not occur when the experiment was done with a B. subtilis strain carrying a mutation in a locus (spoIIE) required for sigma 29, but not P31, synthesis. Microsequencing of sigma 29 protein revealed that its amino terminus originates at amino acid 30 of the P31 amino acid sequence. In order to test whether a proteolytic event alone could activate P31 to a protein with sigma 29-like properties, a fusion protein (P31*) containing most of P31 was overproduced in Escherichia coli and converted in vitro into a protein with the electrophoretic mobility of sigma 29 by limited treatment with Staphylococcus aureus V8 protease. Protease-treated P31*, but not untreated P31*, was capable of directing B. subtilis core RNA polymerase to specifically initiate RNA synthesis at a sigma 29-recognized promoter in vitro.
Cloning of genes developmentally regulated during plant embryogenesis
Authors: Choi Jung H. , Liu Liang-shi , Borkird Chumpol , Sung Z. R.
Published: May 2006

Genes specifically induced during somatic embryogenesis may play key roles in plant embryo development. An antiserum against an extract of carrot somatic embryos revealed a few rare antigens induced at the onset of embryogenesis. Through differential immunoadsorption techniques, we purified antibodies against the embryo-specific antigens and probed a phage λ gt11 library of cDNA from carrot somatic embryos. This paper describes three distinct cDNA clones that hybridize to embryo-specific RNAs. Monospecific antibodies, purified by affinity to the recombinant phage fusion proteins, confirm that the cloned cDNAs encode unique embryo-specific peptide antigens. One 50-kDa protein correlates with embryogenic ability in cultures of other plant species, including cereals.
The congenital goiter mutation is linked to the thyroglobulin gene in the mouse.
Authors: Taylor B A , Rowe L
Published: May 2006

Rat thyroglobulin (TG) cDNA clones were used to identify DNA restriction fragment variants among inbred mouse strains. One of these variants was shown to be closely linked to the recessive mutation congenital goiter (cog), which had previously been mapped to mouse chromosome 15. These results indicate that the structural gene for thyroglobulin is on chromosome 15 and suggest that a mutation at the site of the TG gene is the basis of the cog defect. No differences were observed between cog/cog and +/+ DNA in Southern blots using TG cDNA probes corresponding to 88% of the coding sequences, suggesting that the cog mutation is not due to a large deletion of this portion of the gene. Neither was there any obvious qualitative or quantitative difference between mutant and normal TG mRNA as judged by blot hybridization of electrophoretically fractionated thyroid RNAs. The thyroglobulin gene locus (Tgn) was mapped near the glutamic-pyruvic transaminase isoenzyme locus Gpt-1. The Tgn locus is syntenic with the c-myc protooncogene locus (Myc) in the mouse as in the rat and man.
Long-term inhibition of human T-lymphotropic virus type III/lymphadenopathy-associated virus (human immunodeficiency virus) DNA synthesis and RNA expression in T cells protected by 2',3'-dideoxynucleosides in vitro.
Authors: Mitsuya H , Jarrett R F , Matsukura M , Di Marzo Veronese F , DeVico A L , Sarngadharan M G , Johns D G , Reitz M S , Broder S
Published: May 2006

We report that 2',3'-dideoxyadenosine and 2',3'-dideoxycytidine inhibit retroviral DNA synthesis and mRNA expression in T cells exposed to the virus that causes acquired immunodeficiency syndrome, and afford such cells long-term protection in vitro under conditions of substantial viral excess. Both 2',3'-dideoxyadenosine and 2',3'-dideoxycytidine appear to completely block reverse transcription from viral RNA to viral DNA. Viral mRNA expression is also not detected in cells protected by the drugs throughout 30 days of culture following exposure to the virus. Purine and pyrimidine analogues as 2',3'-dideoxynucleoside-5'-triphosphate serve as substrates for the human T-lymphotropic virus type III/lymphadenopathy-associated virus reverse transcriptase to elongate a DNA chain by one residue, after which the chain is terminated. Cloned normal helper/inducer T cells exposed to a cytopathic dose of the virus, but protected by the drugs, respond normally to specific antigen in vitro. These results suggest that the drugs could be promising agents for further studies in the experimental treatment of patients infected with retroviruses.
Transport of conjugated bilirubin and other organic anions in bile: relation to biliary lipid structures.
Authors: Tazuma S , Holzbach R T
Published: May 2006

Using gel-permeation chromatography, we studied associative relationships between conjugated bilirubin and various biliary lipid particle species, including lecithin/cholesterol vesicles, mixed-lipid micelles, and simple bile salt micelles. Five other organic anions were comparably studied: phenol red, Evans blue, sulfobromophthalein, rose bengal, and indocyanine green. For compounds of intermediate hydrophobicity, including conjugated bilirubin, the dominant association was with a bile salt/organic anion hybrid particle of dimensions larger than that of a simple pure bile salt micelle. Vesicular association was found to be dominant only for the most hydrophobic organic anions, indocyanine green and rose bengal; conversely, the most hydrophilic anion, phenol red, showed no vesicular association. Accordingly, a strong positive correlation (P less than 0.001) was found between percent vesicular association and degree of hydrophobicity of the organic anion. Alkaline conditions (eluant pH 9) decreased or prevented vesicular hydrophobic interaction with all anions. We conclude that two important particulate mechanisms for transport in bile of conjugated bilirubin and other water-soluble anions are bile salt/organic anion hybrid particles and vesicles. For most organic anions of intermediate hydrophobicity, including conjugated bilirubin, the bile salt/organic anion hybrid particle is the dominant transport vehicle.
Nonmetric test of the minimax theory of two-person zerosum games.
Authors: O'Neill B
Published: May 2006

As an experimental test of the minimax theory for two-person zerosum games, subjects played a game that was especially easy for them to understand and whose minimax-prescribed solution did not depend on quantitative assumptions about their utilities for money. Players' average relative frequencies for the moves and their proportions of wins were almost exactly as predicted by minimax, but subject-to-subject variability was too high. These results suggest that people can deviate somewhat from minimax play since their opponents have limited information-processing ability and are imperfect record keepers, but they do not stray so far that the difference will be noticed and their own payoffs will be diminished.
Pathway for inositol 1,3,4-trisphosphate and 1,4-bisphosphate metabolism.
Authors: Inhorn R C , Bansal V S , Majerus P W
Published: May 2006

We prepared [3H]inositol-,3-[32P]phosphate-and 4-[32P]phosphate-labeled inositol phosphate substrates to investigate the metabolism of inositol 1,3,4-trisphosphate and inositol 1,4-bisphosphate. In crude extracts of calf brain, inositol 1,3,4-trisphosphate is first converted to inositol 3,4-bisphosphate, then the inositol 3,4-bisphosphate intermediate is further converted to inositol 3-phosphate. Similarly, inositol 1,4-bisphosphate is converted to inositol 4-phosphate, and no inositol 1-phosphate is formed. We partially purified an enzyme that we tentatively name inositol polyphosphate 1-phosphatase. This cytosolic enzyme converts inositol 1,3,4-trisphosphate to inositol 3,4-bisphosphate and also converts inositol 1,4-bisphosphate to inositol 4-phosphate. The enzyme does not utilize inositol 1,3,4,5-tetrakisphosphate, inositol 1,4,5-trisphosphate, or inositol 1-phosphate as substrates. Thus we propose a new scheme for inositol phosphate metabolism. According to this pathway inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate are degraded to inositol 4-phosphate. Inositol 1-phosphate, which is the major inositol monophosphate formed in stimulated brain, is derived either from phospholipase C cleavage of phosphatidylinositol or from the degradation of inositol cyclic phosphates.
Identification of atrial natriuretic factor gene transcripts in the central nervous system of the rat.
Authors: Gardner D G , Vlasuk G P , Baxter J D , Fiddes J C , Lewicki J A
Published: May 2006

Atrial natriuretic factor (ANF) gene transcripts have been identified in the hypothalamus, brainstem, and cerebral cortex of the rat. The hypothalamic transcripts are similar in overall size (approximately 1100 nucleotides), 5' terminus, 3' terminus, and nucleotide sequence to their counterparts synthesized in the cardiac atria. The 5' terminus of the hypothalamic transcripts, determined by both S1 nuclease and primer-extension analysis, was located approximately 20 nucleotides downstream from the TATAAAA sequence that is thought to dictate the start of transcription. Similar 5' termini were identified in RNA from the brainstem and cerebral cortex. The 3' termini of the hypothalamic transcripts mapped approximately 10 nucleotides downstream from each of two successive AATAAA sequences that are believed to provide the cleavage signal required for subsequent polyadenylylation. The same 3' termini were present in atrial, ventricular, lung, and pituitary RNA. To confirm the identity of the hypothalamic gene transcript, a partial-length cDNA clone encoding ANF was isolated from a hypothalamic cDNA library and sequenced. This hypothalamic clone was identical to an analogous atrial cDNA clone at each of its 612 nucleotide positions. These findings suggest that ANF peptides, previously identified in specific loci of the hypothalamus and pontine tegmentum, are synthesized locally in these tissues and support a role for this peptide in the central regulation of cardiovascular physiology.
Diphtheria toxin prevents catecholamine desensitization of A431 human epidermoid carcinoma cells.
Authors: DeBernardi M , Brooker G
Published: May 2006

We proposed that a rapidly turning over protein, induced in response to catecholamine stimulation of C6-2B rat astrocytoma cells, inhibits subsequent hormonal activation of adenylate cyclase. Studies upon which our hypothesis is based and confirmatory work in a variety of other cell lines and in vivo have utilized actinomycin D and cycloheximide to inhibit RNA and protein synthesis, respectively. These inhibitors, however, are not specific and have been reported also to interfere with other cellular processes. Diphtheria toxin is a specific protein synthesis inhibitor that acts only by ADP-ribosylating elongation factor 2, thus preventing peptide chain elongation. We thus tested whether diphtheria toxin could prevent catecholamine-induced desensitization in A431 human epidermoid carcinoma cells. The toxin inhibited protein synthesis and altered the time course of isoproterenol-stimulated cAMP accumulation as did the less-specific protein synthesis inhibitor cycloheximide. Cellular cAMP content after a 30-min exposure to isoproterenol was similar in control and in toxin-treated cells. However, after 4 hr of treatment with isoproterenol, toxin-treated cells accumulated up to six times more cAMP than controls. When cells or cell-free adenylate cyclase preparations were rechallenged with agonists, toxin-mediated inhibition of protein synthesis prevented desensitization. These results show that diphtheria toxin, a specific inhibitor of protein synthesis, can interfere with the normal physiological regulation of cAMP metabolism in eukaryotic cells and provide compelling evidence that catecholamine stimulation of adenylate cyclase promotes the synthesis of a protein(s) that, in some way, inhibits hormone-stimulated adenylate cyclase.
Translocation of an unusual cAMP receptor to the nucleus during development of Dictyostelium discoideum.
Authors: Kay C A , Noce T , Tsang A S
Published: May 2006

cAMP has been implicated in the control of the expression of developmental genes in Dictyostelium discoideum. To determine the potential role of cAMP receptors as regulators of gene expression, we have used immunocytochemical and immunoblotting techniques to reveal the subcellular localization of a cAMP binding protein CABP1. Most of the CABP1 antigen in early developing cells is localized near the cell periphery, with a small amount found in the nucleus. The level of CABP1 in the nucleus increases approximately 30-fold during development. Moreover, immunofluorescence studies showed that CABP1 can also be detected on the cell surface. Binding of anti-CABP1 to intact cells followed by reaction with 125I-labeled secondary antibody revealed that the cell-surface CABP1 activity peaks during aggregation and culmination. In addition, several proteins related to CABP1 are found mainly in the nuclear fraction of developing cells. The possible role of these proteins in the regulation of developmental gene activity is discussed.
Incomplete Y chromosomes promote magnification in male and female Drosophila.
Authors: Komma D J , Endow S A
Published: May 2006

We have recently shown that magnification, an increase in the number of ribosomal RNA genes (rDNA) in gametes produced by rDNA-deficient flies, can occur in female Drosophila if they have a Y chromosome. We now have tested several X-Y translocation and recombinant chromosomes to determine which parts of the Y chromosome are necessary for magnification to occur in females. Our data indicate that the required region is the distal part of the long arm of the Y chromosome, YL. We have also used X-Y translocation chromosomes to study magnification of rDNA-deficient X chromosomes in males. Our data show that the region of the Y chromosome from the distal end of the nucleolus organizer through the centromere is not required for magnification in males. The frequency of magnification in males with rDNA-deficient Y fragments is comparable to that produced by Ybb-, a chromosome that has often been used to produce magnification in males. These results demonstrate that the Ybb-chromosome is not uniquely effective in causing magnification to occur in males. The results of these studies imply that sequences present on YL are required for magnification to occur in females; these sequences are probably also required for magnification in males. Since unequal sister chromatid exchange has been implicated as the major mechanism of ribosomal gene increase during magnification, the YL sequences required for magnification may be involved in encoding or regulating products needed for sister chromatid recombination in germ-line cells.
Transmission of mitochondrial and chloroplast genomes in crosses of Chlamydomonas.
Authors: Boynton J E , Harris E H , Burkhart B D , Lamerson P M , Gillham N W
Published: May 2006

Physical differences between organelle genomes of the interfertile species Chlamydomonas reinhardtii and Chlamydomonas smithii have been used to demonstrate that sexual zygotes transmit chloroplast and mitochondrial DNA from opposite mating types. Processes responsible can be separated functionally and genetically, although both are controlled by mating type. In vegetative diploids, chloroplast and mitochondrial genomes are transmitted biparentally, but a 1-kilobase insert present in the C. smithii mitochondrial genome spreads unidirectionally to all C. reinhardtii genomes in a manner reminiscent of the intron found in the mitochondrial 21S rRNA gene of omega + strains of yeast.
Mutations that inactivate a yeast transcriptional regulatory protein cluster in an evolutionarily conserved DNA binding domain.
Authors: Johnston M , Dover J
Published: May 2006

The protein encoded by the GAL4 gene of the yeast Saccharomyces cerevisiae binds to DNA upstream of several genes and activates transcription. To try to understand these processes, we have undertaken a genetic analysis of GAL4. Here we report that nearly all missense mutations in GAL4, selected in vivo to lack function of the protein, cluster in the small region of the gene that encodes the DNA binding domain. About half of these mutations alters a cysteine-rich region of the protein highly homologous to several eukaryotic DNA binding proteins; the other half alters some of the 20 amino acids adjacent to the cysteine-rich region. Nearly all of the missense mutations that alter the DNA binding domain abolish the DNA binding activity of GAL4 protein measured in vitro. In contrast, nearly all of the mutations that alter the 3' 95% of the gene that encodes the transcription activation function are nonsense or frameshift mutations. These results support the idea that the conserved cysteine-rich sequence motif is directly involved in binding of several eukaryotic transcriptional regulatory proteins to DNA.
Chemical and serological investigations on the genus-specific lipopolysaccharide epitope of Chlamydia.
Authors: Brade H , Brade L , Nano F E
Published: May 2006

Members of the bacterial genus Chlamydia are responsible for widespread disease among humans and animals, including endemic trachoma in developing countries, venereal disease in developed countries, and a variety of other diseases such as infantile pneumonia and lymphogranuloma venereum. Although there is little genetic relatedness between and large antigenic diversity between and among the two chlamydial species, one antigenic determinant has been preserved among all serovars: the genus-specific lipopolysaccharide epitope. In this report, the tools of molecular genetics, monoclonal antibodies, and analytical and synthetic chemistry have been combined to determine the structure of this epitope. This epitope is attributed to the presence of a trisaccharide of 3-deoxy-D-manno-octulosonic acid (KDO) of the sequence KDOp-(2----8)-KDOp-(2----4)-KDO. The structure includes a unique linkage of two KDO residues through a 2.8-linkage.
Mammalian alcohol dehydrogenases of separate classes: intermediates between different enzymes and intraclass isozymes.
Authors: Jörnvall H , Höög J O , von Bahr-Lindström H , Vallee B L
Published: May 2006

A comparison of the structure of class II human liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) (containing pi subunits) with those of the human class I isozymes (containing alpha, beta, and gamma subunits) reveals differences at about 40% of all positions. Variations are large for active-site regions, the segment around the second zinc atom, and for segments involved in subunit interactions. The two classes of alcohol dehydrogenase have diverged to exhibit structural differences to about half the extent of those between alcohol and polyol dehydrogenases. Hence, the two classes of alcohol dehydrogenase represent steps in enzyme rather than isozyme divergence. An evolutionary scheme that relates different types of zinc-containing mammalian dehydrogenases to one another encompasses at least three levels of gene duplication subsequent to the early step(s) of assembly of building unit(s). The first level of duplication results in the formation of now clearly different enzymes. The second level concerns the various classes of alcohol dehydrogenase, forming steps between typical enzymes and isozymes. The third level encompasses recent and multiple duplications in isozyme evolution of alcohol dehydrogenases. This scheme, linking zinc-containing dehydrogenases at different levels, resembles that in other protein families and reflects general patterns in protein relationships.
Conversion of isoaspartyl peptides to normal peptides: implications for the cellular repair of damaged proteins.
Authors: McFadden P N , Clarke S
Published: May 2006

The hypothesis that cellular protein carboxyl-methylation reactions recognize altered aspartyl residues as part of a protein repair pathway has been tested in an in vitro system using tetragastrin (Trp-Met-Asp-Phe-NH2) as a model sequence. The L-isoaspartyl form of tetragastrin, where the phenylalanine residue is linked to the side-chain carboxyl group of the aspartate residue ([iso-Asp3]tetragastrin), is a substrate for the erythrocyte protein carboxyl methyltransferases, while the normal form is not. The enzymatically produced alpha-methyl ester of [iso-Asp3]tetragastrin, [iso-Asp(OMe)3]tetragastrin, is unstable at pH 7.4 and 37 degrees C and spontaneously demethylates with a half-time of 41 min to an intermediate L-succinimide form ([Asu3]tetragastrin) that, in turn, spontaneously hydrolyzes with a half time of 116 min to give a mixture of normal tetragastrin (20%) and [iso-Asp3]tetragastrin (80%). This sequence of enzymatic and nonenzymatic reactions can be coupled in a single reaction mixture; the [iso-Asp3]tetragastrin that is produced upon succinimide hydrolysis can reenter the reaction sequence by enzymatic methylation, and the net result of the process is the conversion of the isomerized peptide to the normal peptide. The efficiency of this "repair" reaction is limited by a side reaction of racemization at the alpha-carbon of the succinimide (half-time = 580 min). In a 24-hr time period, normal L-aspartyl-containing tetragastrin is obtained in about 50% yield from the coupled reaction mixture; other products include [D-iso-Asp3]tetragastrin and [D-Asp3]tetragastrin. The versatile chemistry of succinimide peptides suggests that methylated L-isoaspartyl sites (and possibly methylated D-aspartyl sites) in cellular polypeptides can eventually yield "repaired" normal L-aspartyl sites through succinimide intermediates.
Opposing effects of a ras oncogene on growth factor-stimulated phosphoinositide hydrolysis: desensitization to platelet-derived growth factor and enhanced sensitivity to bradykinin.
Authors: Parries G , Hoebel R , Racker E
Published: May 2006

Expression of a transforming Harvey or Kirsten ras gene caused opposing effects in the ability of platelet-derived growth factor (PDGF) and bradykinin to activate phospholipase C-mediated phosphoinositide hydrolysis. In [3H]inositol-labeled rat-1 fibroblasts, PDGF (5 ng/ml) resulted in a 2-fold increase in the level of [3H]inositol trisphosphate (InsP3) after 2 min and, in the presence of LiCl, a 3- to 8-fold increase in the level of [3H]inositol monophosphate (InsP1) after 30 min. However, in EJ-ras-transfected rat-1 cells, which exhibit near normal levels of PDGF receptors, PDGF resulted in little or no accumulation of either [3H]InsP3 or [3H]InsP1. Similarly, marked stimulations by PDGF were observed in NIH 3T3 cells, as well as in v-src-transformed 3T3 cells, but not in 3T3 cells transformed by Kirsten sarcoma virus or by transfection with v-Ha-ras DNA. This diminished phosphoinositide response in ras-transformed cells was associated with a markedly attenuated mitogenic response to PDGF. On the other hand, both phosphoinositide metabolism and DNA synthesis in ras-transformed fibroblasts were stimulated several-fold by serum. In NIH 3T3 cells carrying a glucocorticoid-inducible v-Ha-ras gene, a close correlation was found between the expression of p21ras and the loss of PDGF-stimulated [3H]InsP1 accumulation. In contrast to this ras-induced desensitization to PDGF, ras-transformed NIH 3T3 cells exhibited an enhanced sensitivity to bradykinin; this effect was associated with an elevated level of high-affinity [3H]bradykinin binding. We propose that a ras gene product (p21) can, directly or indirectly, influence growth factor-stimulated phosphoinositide hydrolysis, as well as DNA synthesis, via alterations in the properties of specific growth factor receptors.
Induction of superoxide dismutase in Escherichia coli by heat shock.
Authors: Privalle C T , Fridovich I
Published: May 2006

Exposure of midlogarithmic-phase cultures of Escherichia coli B to 48 degrees C for 1 hr elicited an induction of the manganese-containing superoxide dismutase (MnSOD), which became more pronounced during 1 hr of recovery at 37 degrees C. This induction required protein biosynthesis, since it was suppressed by chloramphenicol. Induction of MnSOD appeared to be a response to a heat-mediated increase in O2- production because it was dioxygen-dependent and because heating to 48 degrees C doubled the cyanide-resistant fraction of the total respiration.
Molecular cloning and expression of T11 cDNAs reveal a receptor-like structure on human T lymphocytes.
Authors: Sayre P H , Chang H C , Hussey R E , Brown N R , Richardson N E , Spagnoli G , Clayton L K , Reinherz E L
Published: May 2006

The T11 (CD2) sheep-erythrocyte-binding protein is a T-cell surface molecule involved in activation of T lymphocytes and thymocytes, including those lacking the T3-Ti antigen-receptor complex. The primary structure of T11 was deduced from protein microsequencing and cDNA cloning. The mature human protein appears to be divided into three domains: a hydrophilic 185 amino acid external domain bearing only limited homology to the T-cell surface protein T4 and the immunoglobulin kappa light chain variable region, a 25 amino acid hydrophobic transmembrane segment, and a 126 amino acid cytoplasmic domain rich in prolines and basic residues. Transfection of cDNAs encoding either the 1.7- or the 1.3-kilobase T11 mRNA into COS-1 cells resulted in expression of surface T11 epitopes as well as sheep-erythrocyte-binding capacity. The predicted structure is consistent with the possibility that T11 functions in signal transduction.
Superoxide dismutase protects cultured neurons against death by starvation.
Authors: Sáez J C , Kessler J A , Bennett M V , Spray D C
Published: May 2006

Brief substrate deprivation resulted in high mortality of superior cervical ganglion neurons in culture, assayed 2 hr later by trypan blue exclusion. Involvement of superoxide anions was indicated by several observations. Survival was increased significantly by prior treatment that induced cells to take up superoxide dismutase. During starvation, neurons reduced nitroblue tetrazolium to form the blue precipitate formazan, and the color change was blocked in neurons preloaded with superoxide dismutase. The incidence of staining was comparable to the mortality. In many cells, brief starvation caused the appearance of fluorescence due to oxidation of 2',7'-dichlorofluorescin to dichlorofluorescein, which indicates that oxidants were generated intracellularly. In some cells fluorescence was transient, as would be caused by membrane breakdown, and these cells were then shown to be dead. Superoxide generation caused by substrate deprivation may contribute importantly to cell damage in a variety of pathological conditions.
Mechanical relaxation of the hair bundle mediates adaptation in mechanoelectrical transduction by the bullfrog's saccular hair cell.
Authors: Howard J , Hudspeth A J
Published: May 2006

Mechanoelectrical transduction by hair cells of the frog's internal ear displays adaptation: the electrical response to a maintained deflection of the hair bundle declines over a period of tens of milliseconds. We investigated the role of mechanics in adaptation by measuring changes in hair-bundle stiffness following the application of force stimuli. Following step stimulation with a glass fiber, the hair bundle of a saccular hair cell initially had a stiffness of approximately equal to 1 mN X m-1. The stiffness then declined to a steady-state level near 0.6 mN X m-1 with a time course comparable to that of adaptation in the receptor current. The hair bundle may be modeled as the parallel combination of a spring, which represents the rotational stiffness of the stereocilia, and a series spring and dashpot, which respectively, represent the elastic element responsible for channel gating and the apparatus for adaptation.
Tree species richness of upper Amazonian forests
Authors: Gentry Alwyn H.
Published: May 2006

Upper Amazonian data for tree species richness in 1-hectare plots are reported. All plants ≥10 cm diameter were censused and identified in six plots in Amazonian Peru and one on the Venezuela-Brazil border. The two plots from the everwet forests near Iquitos, Peru, are the most species-rich in the world, with ≈300 species ≥10 cm diameter in single hectares; all of the Peruvian plots are among the most species-rich ever reported. Contrary to accepted opinion, upper Amazonian forest, and perhaps Central African ones, have as many or more tree species as comparable Asian forests. Very high tree species richness seems to be a general property of mature lowland evergreen forests on fertile to moderately infertile soils on all three continents.
Distribution of glutamine synthetase and carbamoyl-phosphate synthetase I in vertebrate liver.
Authors: Smith D D , Campbell J W
Published: May 2006

Mitochondrial glutamine synthetase (EC 6.3.1.2) is the primary ammonia-detoxifying enzyme in avian liver and is therefore analogous in function to carbamoyl-phosphate synthetase I (ammonia) (EC 6.3.4.16) in mammalian liver. In mammalian liver, glutamine synthetase is cytosolic and its distribution is restricted to a few hepatocytes around the terminal venules. These cells do not express carbamoyl-phosphate synthetase I. Using immunocytochemistry, we show here that there is little or no zonation of glutamine synthetase in avian liver. Rather, it is broadly distributed to most hepatocytes, much like carbamoyl-phosphate synthetase I in mammalian liver. In situ hybridization with a cloned glutamine synthetase cDNA probe showed the distribution of glutamine synthetase mRNA in both mammalian and avian liver to correspond to the distribution of immunoreactive protein. Neither glutamine synthetase nor carbamoyl-phosphate synthetase I and ornithine transcarbamoylase (EC 2.1.3.3) are strictly zoned in liver of the Texas tortoise or of an Argentine tree frog, both of which possess a complete urea cycle but which may also rely on glutamine synthetase for ammonia detoxication. These latter results suggest that the mutually exclusive expression of either carbamoyl-phosphate synthetase I or glutamine synthetase may be unique to mammalian liver.
Characterization of the TPR-MET oncogene p65 and the MET protooncogene p140 protein-tyrosine kinases.
Authors: Gonzatti-Haces M , Seth A , Park M , Copeland T , Oroszlan S , Vande Woude G F
Published: May 2006

The proteins encoded by the human TPR-MET oncogene (p 65tpr-met) and the human MET protooncogene (p140met) have been identified. The p65tpr-met and p140met, as well as a truncated TPR-MET product expressed in Escherichia coli, p50met, are autophosphorylated in vitro on tyrosine residues. Using the immunocomplex kinase assay, p140met activity was detected in various human tumor epithelial cell lines. In vivo, p65tpr-met is phosphorylated on both serine and tyrosine residues, while p140met is phosphorylated on serine and threonine. p140met is labeled by cell-surface iodination procedures, suggesting that it is a receptor-like transmembrane protein-tyrosine kinase.
Autoimmune antibody (IgG Kansas) against the fibrin stabilizing factor (factor XIII) system.
Authors: Lorand L , Velasco P T , Rinne J R , Amare M , Miller L K , Zucker M L
Published: May 2006

Serum from a patient who died from massive hemorrhage within 4 months after onset of an acquired bleeding disorder at age 85 contained a potent inhibitor of fibrin stabilization. Other parameters of coagulation and fibrinolysis and his bleeding time were within normal limits. The inhibitor was shown to be an IgG with kappa light chains (IgG Kansas); its specific target was the factor XIII system itself. Although IgG Kansas combined with the virgin [ab] form of the zymogen, it did not block the thrombin-catalyzed conversion to [a'b]. However, IgG Kansas prevented the subsequent Ca2+-mediated activation of [a'b] to a + b, where a denotes the catalytically competent factor XIIIa species. IgG Kansas, in contrast to a previously studied autoimmune antibody from a similar bleeding disorder (IgG Warsaw), could also inhibit the transamidating activity of the preactivated a enzyme.
Differential expression of insulin-like growth factor genes in rat central nervous system.
Authors: Rotwein P , Burgess S K , Milbrandt J D , Krause J E
Published: May 2006

A sensitive solution-hybridization assay was used to investigate the expression of genes encoding insulin-like growth factors I and II (IGF-I and -II) in the rat central nervous system (CNS). mRNAs for both IGFs are synthesized throughout the CNS of adult rats but exhibit distinct regional differences for each growth factor. IGF-I mRNA is 8-10 times more abundant in the cervical-thoracic spinal cord and in the olfactory bulb than in whole brain and is enriched 3-fold in the midbrain and cerebellum. IGF-II mRNA is minimally enriched in the medulla-pons and cerebellum but is 3-5 times less abundant in the midbrain and corpus striatum than in total brain. During CNS development the content of IGF-I and IGF-II mRNAs is highest at embryonic day 14 and declines by a factor of 3-4 at birth, to values found in adult brain. Embryonic neurons and glia synthesize IGF-I mRNA during short-term cell culture; only glia produce IGF-II mRNA. These observations show that IGF-I and IGF-II are differentially expressed in the developing and adult CNS and suggest that each growth factor may play a unique role in the mammalian nervous system.
Amino acid sequence of the BSC-1 cell growth inhibitor (polyergin) deduced from the nucleotide sequence of the cDNA.
Authors: Hanks S K , Armour R , Baldwin J H , Maldonado F , Spiess J , Holley R W
Published: May 2006

The complete amino acid sequence of the BSC-1 cell growth inhibitor, including its precursor polypeptide, is reported. The sequence was deduced from the nucleotide sequence of the cDNA. The N-terminal amino acid sequence of the mature bioactive BSC-1 cell growth inhibitor is identical with the N-terminal sequences of the factors that have been called type beta 2 transforming growth factor and cartilage-inducing factor B, suggesting that these are identical. The complete amino acid sequence of the mature BSC-1 cell growth inhibitor differs from that of human type beta transforming growth factor in 32 of the 112 amino acids. Polyergin is proposed as the name for the BSC-1 cell growth inhibitor.
Proposed structure for the zinc-binding domains from transcription factor IIIA and related proteins.
Authors: Berg J M
Published: May 2006

Several proteins, including the gene regulatory protein transcription factor IIIA, have been shown to contain tandem repeats of sequences of approximately equal to 30 amino acids that are believed to form structural domains around bound zinc ions ("zinc fingers"). The consensus sequence for these repeats is (Phe, Tyr)-Xaa-Cys-(Xaa)2 or 4-Cys-(Xaa)3-Phe-(Xaa)5-Leu-(Xaa)2-His-(Xaa)3-His- (Xaa)5, where Xaa is any amino acid. Comparisons with metalloproteins with known structures have allowed the development of a detailed three-dimensional model for these domains consisting of an antiparallel beta-sheet followed by an alpha-helix. The proposed structure provides a basis for understanding the detailed roles of the conserved residues and allows construction of a model for the interaction of these proteins with nucleic acids in which the proteins wrap around the nucleic acids in the major groove.
Calmodulin-dependent multifunctional protein kinase in Aspergillus nidulans.
Authors: Bartelt D C , Fidel S , Farber L H , Wolff D J , Hammell R L
Published: May 2006

A Ca2+/calmodulin (CaM)-dependent multifunctional protein kinase has been isolated from Aspergillus nidulans and purified to homogeneity. Unlike any CaM-dependent multifunctional protein kinase described previously, the native enzyme from Aspergillus behaves as a monomer. The calculated molecular weight is 41,200. NaDodSO4/PAGE reveals a single protein band with an apparent Mr of 51,000. Two-dimensional isoelectric focusing/NaDodSO4/PAGE of the purified enzyme showed one major and one minor more acidic Coomassie blue-stained spot, both of which bind 125I-labeled calmodulin in a calcium-dependent manner. The kinase is autophosphorylated in a calcium- and CaM-dependent manner, yielding an increase in the amount and number of more acidic forms of the enzyme. The Aspergillus kinase catalyzes the Ca2+/CaM-dependent phosphorylation of known substrates of type II Ca2+/CaM-dependent protein kinases, including glycogen synthase, microtubule-associated protein 2, synapsin, tubulin, gizzard myosin light chain, and casein. Cross-reactivity between antiserum raised against native rat brain protein kinase II and 125I-labeled Aspergillus kinase has been detected. Two forms of CaM have been isolated from Aspergillus nidulans, both of which activate the Aspergillus kinase at lower concentrations than that required for activation by bovine brain CaM.