bluuebunny/crossref_metadata_2025 huggingface dataset:
Pst I restriction fragment length polymorphism of human placental alkaline phosphatase gene: Mendelian segregation and localization of mutation site in the gene.
Authors: Tsavaler L , Penhallow R C , Sussman H H
Published: May 2006

The pattern of inheritance of a Pst I restriction fragment length polymorphism (RFLP) of the human placental alkaline phosphatase gene was studied in nine nuclear families by Southern blot hybridization analysis of genomic DNA. The dimorphic RFLP is defined by the presence of allelic fragments 1.0 kilobase and 0.8 kilobase long. The results of this study show that the two alleles of the PstI RFLP of the placental alkaline phosphatase gene segregate as codominant traits according to Mendelian expectations. For a polymorphism to be useful as a genetic marker the probability that an offspring is informative (PIC) must be at least 0.15. The allelic frequency of the 1.0-kilobase allele is 0.21, which correlates to a probability that an offspring is informative of 0.275 and is indicative of a useful polymorphism. By using probes derived from different regions of the placental alkaline phosphatase cDNA, the mutated Pst I site causing the RFLP was located in the penultimate intron 2497 base pairs downstream from the transcriptional initiation site.
T-cell receptor alpha-chain variable-region haplotypes of normal and autoimmune laboratory mouse strains.
Authors: Singer P A , McEvilly R J , Balderas R S , Dixon F J , Theofilopoulos A N
Published: May 2006

We used Southern blotting and mRNA analysis to characterize allelic polymorphisms among genes of the T-cell antigen receptor (TCR) alpha-chain variable-region (V alpha) locus in a large panel of normal and autoimmune-susceptible or autoimmune-contributing strains of laboratory mice. Four major V alpha haplotypes were defined on the basis of multiple restriction fragment length polymorphisms for each of nine V alpha subfamily probes used. Southern blotting also revealed haplotype-specific loss of bands within some V alpha subfamilies, consistent with the deletion of particular V alpha genes or sets of genes from haplotype to haplotype. In contrast to the situation in the V beta locus, however, deletion of entire V alpha subfamilies was not observed. The nature of V alpha allelic variability was further explored by using an RNase protection assay to analyze expressed V alpha mRNA sequences in thymocyte RNA. Such analysis revealed both shared and unique patterns of V alpha mRNA expression among the different haplotypes and supported the conclusion that haplotype differences sometimes involve V alpha gene deletions. Interestingly, a disproportionate number of, but not all, autoimmune-susceptible strains, including NZB, SJL, SWR, PL/J, and NOD, share a common V alpha haplotype. The identification of murine TCR V alpha haplotypes should provide a basis for understanding the role of TCR diversity in normal immunoregulatory and immune-response phenomena, as well as autoimmune-disease predisposition.
Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications.
Authors: Bazan J F , Fletterick R J
Published: May 2006

Proteases that are encoded by animal picornaviruses and plant como- and potyviruses form a related group of cysteine-active-center enzymes that are essential for virus maturation. We show that these proteins are homologous to the family of trypsin-like serine proteases. In our model, the active-site nucleophile of the trypsin catalytic triad, Ser-195, is changed to a Cys residue in these viral proteases. The other two residues of the triad, His-57 and Asp-102, are otherwise absolutely conserved in all the viral protease sequences. Secondary structure analysis of aligned sequences suggests the location of the component strands of the twin beta-barrel trypsin fold in the viral proteases. Unexpectedly, the 2a and 3c subclasses of viral cysteine proteases are, respectively, homologous to the small and large structural subclasses of trypsin-like serine proteases. This classification allows the molecular mapping of residues from viral sequences onto related tertiary structures; we precisely identify amino acids that are strong determinants of specificity for both small and large viral cysteine proteases.
Recognition helices of lac and lambda repressor are oriented in opposite directions and recognize similar DNA sequences.
Authors: Lehming N , Sartorius J , Oehler S , von Wilcken-Bergmann B , Müller-Hill B
Published: May 2006

Exchanges in positions 1 and 2 of the putative recognition helix allow lac repressor to bind to ideal lac operator variants in which base pair 4 has been replaced. We show here that an Arg-22----Asn exchange in position 6 of the putative recognition helix of lac repressor abolishes lac repressor binding to ideal lac operator. This lac repressor variant, however, binds to a variant of the ideal lac operator 5' TTTGAGCGCTCAAA 3' in which the original G.C of position 6 has been replaced by T.A. This result and our previous data confirm our suggestion that the N terminus of the recognition helix of lac repressor enters the major groove close to the center of symmetry of lac operator and that its C terminus leaves the major groove further away from the center of symmetry. The consequences of this model are discussed in regard to various phage and bacterial repressor operator systems.
Gene transfer from targeted liposomes to specific lymphoid cells by electroporation.
Authors: Machy P , Lewis F , McMillan L , Jonak Z L
Published: May 2006

Large unilamellar liposomes, coated with protein A and encapsulating the gene that confers resistance to mycophenolic acid, were used as a model system to demonstrate gene transfer into specific lymphoid cells. Protein A, which selectively recognizes mouse IgG2a antibodies, was coupled to liposomes to target them specifically to defined cell types coated with IgG2a antibody. Protein A-coated liposomes bound human B lymphoblastoid cells preincubated with a mouse IgG2a anti-HLA monoclonal antibody but failed to adhere to cells challenged with an irrelevant (anti-H-2) antibody of the same isotype or to cells incubated in the absence of antibody. Transfection of target cells bound to protein A-coated liposomes was achieved by electroporation. This step was essential since only electroporated cells survived in a selective medium containing mycophenolic acid. Transfection efficiency with electroporation and targeted liposomes was as efficient as conventional procedures that used unencapsulated plasmids free in solution but, in the latter case, cell selectivity is not possible. This technique provides a methodology for introducing defined biological macromolecules into specific cell types.
Experimental control of the differentiation of Leydig cells in the rat fetal testis.
Authors: Jost A , Perlman S , Valentino O , Castanier M , Scholler R , Magre S
Published: May 2006

In the developing fetal testis, in vitro as well as in vivo, two kinds of endocrine cells differentiate successively: Sertoli cells, which produce the Müllerian inhibitor (or anti-Müllerian hormone) and aggregate with germ cells into seminiferous cords; and Leydig cells, which release androgens. Serum added to the synthetic culture medium prevents the morphogenesis of the seminiferous cords but not the cytodifferentiation of the endocrine cells. L-Azetidine 2-carboxylic acid (LACA), a proline competitor, introduced into the medium also prevents differentiation of seminiferous cords. In the present experiments, the effects of LACA on the endocrine cells were studied. It did not suppress production of the Müllerian inhibitor, but it opposed differentiation of Leydig cells. Histochemically detectable 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was virtually absent and the release of testosterone, delta 4-androstenedione, 17-hydroxyprogesterone, or progesterone into the medium became undetectable. Moreover, dibutyryl cAMP added to the medium during the final day in vitro had very little effect on the parameters of steroidogenesis. An excess of proline added to the LACA-containing medium permitted normal morphogenesis of seminiferous cords, normal steroidogenesis, and normal response to cAMP. LACA did not prevent the appearance of 3 beta-HSD activity in the adrenals, nor did it reduce the expression of laminin and fibronectin (data not shown) in the mesonephric structures as much as in the testes. The differentiation of the testis and especially of the Leydig cells appears to have special requirements for proline.
Unequal partitioning of bovine mitochondrial genotypes among siblings.
Authors: Laipis P J , Van de Walle M J , Hauswirth W W
Published: May 2006

Two polymorphic mitochondrial DNA genomes, differing by a single Hpa II restriction site, are present at significantly different levels in tissue of three sibling dairy cows. The relative ratio of the two heteroplasmic molecules varies 3-fold among these three animals and documents a rapid segregation of mitochondrial genotypes in mammals. DNA sequencing shows the difference is due to a single guanine at position 364 in bovine mitochondrial DNA. A model involving unequal partitioning of the two amplified mitochondrial DNA species during the early cell divisions of the embryo can explain the appearance of such variation in heteroplasmic sibling animals. The model provides a basis for understanding the rapid DNA sequence variation observed in vertebrate mitochondrial DNA despite its high copy number and strict maternal inheritance.
Lysolecithins as endothelium-dependent vascular smooth muscle relaxants that differ from endothelium-derived relaxing factor (nitric oxide)
Authors: Saito T , Wolf A , Menon N K , Saeed M , Alves C , Bing R J
Published: May 2006

The effects of lysolecithin (lysophosphatidylcholine) derived from egg yolk as well as of synthetic lysolecithins with different aliphatic chain lengths on tension development of rabbit aortic strips were investigated. Lysolecithins caused slowly progressing, dose-dependent relaxation that was inhibited by hemoglobin, methylene blue, and nordihydroguiaretic acid. Indomethacin caused no inhibition of relaxation. The degree of relaxation was endothelium-dependent and appeared to be related to the activation of guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2]. Superoxide dismutase failed to influence relaxation. Lysolecithins with the longest aliphatic chain were the most potent relaxants of aortic strips. The experiments suggest a role of lysolecithins through their weak detergent action on membrane dynamics of endothelial cells, resulting in the production of cyclic GMP and the relaxation of arterial smooth muscle. Lysolecithins differ in several respects from endothelium-derived relaxing factor. Endothelium-derived relaxing factor is an unstable humoral substance released from endothelium and is identical to nitric oxide, itself a labile substance causing vascular relaxation and cyclic GMP accumulation. Lysolecithins may represent a different type of endothelium-dependent muscle relaxant.
Transcriptional activation of homologous viral long terminal repeats by the human immunodeficiency virus type 1 or the human T-cell leukemia virus type I tat proteins occurs in the absence of de novo protein synthesis.
Authors: Jeang K T , Shank P R , Kumar A
Published: May 2006

The genomes of human retroviruses [human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus (HTLV-I)] encode positive trans-activator proteins, named tat. In the presence of tat, the transcriptional activity of the homologous HIV-1 or HTLV-I long terminal repeat (LTR) promoter is markedly increased. We have constructed mammalian cell lines that contain stably integrated copies of a HIV-1 or a HTLV-I LTR-chloramphenicol acetyltransferase (CAT) gene. When presynthesized HIV-1 or HTLV-I tat proteins were separately introduced into these cells in the presence of cycloheximide, we found a strong increase in the steady-state expression of the homologous viral LTR. Nuclear "run-on" assays verified that this tat-mediated enhancement, occurring in the absence of de novo cellular protein synthesis, was due to increased transcriptional initiation at the LTR promoter. We conclude that one aspect of transcriptional trans-activation of viral LTR by the HIV-1 and HTLV-I tat proteins does not require the production of new cellular proteins.
A serine peptidase responsible for the inactivation of endogenous cholecystokinin in brain.
Authors: Rose C , Camus A , Schwartz J C
Published: May 2006

A serine endopeptidase was characterized as a major inactivating enzyme for endogenous cholecystokinin (CCK) in brain. CCK-8 released by depolarization of slices of rat cerebral cortex, as measured by its immunoreactivity (CCK-ir), undergoes extensive degradation (approximately 85% of the amount released) before reaching the incubation medium. However, recovery of CCK-ir is enhanced up to 3-fold in the presence of serine-alkylating reagents (i.e., phenylmethylsulfonyl fluoride) as well as selected active site-directed inactivators (i.e., peptide chloromethyl ketones) or transition-state inhibitors (i.e., peptide boronic acids) of serine peptidases. Among these compounds, elastase inhibitors were the most potent protecting agents, whereas trypsin or chymotrypsin inhibitors were ineffective. HPLC analysis of endogenous CCK-ir recovered in media of depolarized slices indicated that endogenous CCK-5 [CCK-(29-33)-pentapeptide] was the most abundant fragment and that its formation was strongly decreased in the presence of an elastase inhibitor. HPLC analysis of fragments formed upon incubation of exogenous CCK-8 [CCK-(26-33)-octapeptide] with brain slices showed CCK-5, Gly-Trp-Met, and Trp-Met to be major metabolites of CCK-8 whose formation was prevented or at least diminished in the presence of the elastase inhibitor. It is concluded that there is an elastase-like serine endopeptidase in brain that cleaves the two peptide bonds of CCK-8 where the carboxyl group is donated by a methionine residue and constitutes a major inactivation ectoenzyme for the neuropeptide.
Manipulation of stretch-induced atriopeptin prohormone release and processing in the perfused rat heart.
Authors: Ito T , Toki Y , Siegel N , Gierse J K , Needleman P
Published: May 2006

Atriopeptin (AP) is stored as the prohormone AP-126 [atrial natriuretic factor-(1-26)] in atrial granules. Cultured atrial myocytes synthesize and release only prohormone into the medium. HPLC analysis of the coronary venous effluent of media from perfused rat hearts subjected to right atrial stretch indicated the presence of the C-terminal mature hormone AP-28 [atrial natriuretic factor-(99-126)] and little or no prohormone. Absence of calcium from the perfusion medium increased total AP release and surprisingly blocked the proteolytic cleavage of the prohormone. Similarly, addition of the proteolytic inhibitor aprotinin to the perfusion medium suppressed the processing of the endogenous AP-126 released by atrial stretch. Aprotinin would be restricted to the extracellular space, which is therefore implicated as the site of prohormone processing. This suggestion was validated by the demonstration that the perfused rat heart could readily cleave exogenous prohormone to mature hormone, a process blocked by aprotinin. Hypothetically, the stimulus-release-processing event initiated by atrial stretch may require the concerted action of the synthetic cell (i.e., atrial myocyte) and a processing cell or site (e.g., the adjacent atrial mesenchymal cell) for the production of the mature AP-28, which is the circulating molecular form of this endocrine system.
Involvement of distinct murine T-cell receptors in the autoimmune encephalitogenic response to nested epitopes of myelin basic protein.
Authors: Sakai K , Sinha A A , Mitchell D J , Zamvil S S , Rothbard J B , McDevitt H O , Steinman L
Published: May 2006

The peptide p89-101 (Val-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro-Arg-Thr-Pro) of myelin basic protein is encephalitogenic in mice expressing H-2q and H-2s antigens. Six of 13 encephalitogen-specific T-cell clones were shown to express the variable beta-chain (V beta) 17a gene product (KJ23a+), whereas seven clones were KJ23a-. Both KJ23a+ and KJ23a- subpopulations were encephalitogenic in SJL/J mice when adoptively transferred. Depletion of KJ23a+ cells in vivo with the administration of the antibody KJ23a suppresses experimental allergic encephalomyelitis induced with KJ23a+ T-cell lines. However, experimental allergic encephalomyelitis induced with either (i) encephalitogenic peptide p89-101, (ii) intact myelin basic protein, or (iii) KJ23a- T cells reactive to p89-101 cannot be prevented with monoclonal antibody KJ23a. These data indicate that in spite of the V beta 17a gene expression in a relatively large proportion of p89-101-specific T cells, such V beta gene use is not essential for the induction of experimental allergic encephalomyelitis in SJL/J mice. These results contrast with the predominance of V beta gene use (V beta 8.2) in T cells reactive to the encephalitogenic fragment (pR1-11) in PL/J mice. One reason for this lack of dominant use of a particular T-cell receptor V beta gene family in the autoimmune response to myelin basic protein in SJL/J mice stems from the observation that two encephalitogenic epitopes exist in p89-101. KJ23a- T cells are stimulated by the deleted peptide p89-100, whereas KJ23a+ T cells are not. Thus, in the response to an encephalitogenic fragment of myelin basic protein containing two nested epitopes, at least two distinct T-cell receptor V beta genes are expressed. These distinct T-cell subpopulations can each trigger experimental allergic encephalomyelitis. These findings have implications for therapy of autoimmune disease with antibodies to the T-cell receptor gene products.
Stochastic development of invasive potential in rat mammary tumors induced by N-methyl-N-nitrosourea.
Authors: Dulbecco R , Armstrong B
Published: May 2006

We have studied the progression of breast tumors induced by N-methyl-N-nitrosourea in Wistar/Furst rats during serial passages through isogeneic animals, using characteristic markers previously defined. The tumors contained various types of structure, which evolve from intraductal nodes to duct-like structures and then to invasive strands or cells. Most primary tumors were not invasive but became so in subsequent transfers. The distribution of invasive and noninvasive tumors in the various lineages was not random, implying that the tumors differed from their inception in the propensity toward invasion. In different lineages invasive structures appeared repeatedly and independently but with different frequencies, suggesting that they were produced by a stochastic mechanism with different probability of occurrence. The possible nature of this mechanism is discussed. This experimental system is useful for further studies of tumor invasion; the results obtained are relevant for understanding and evaluating the evolution of human breast cancer.
A human 200-kDa protein binds selectively to DNA fragments containing G.T mismatches.
Authors: Jiricny J , Hughes M , Corman N , Rudkin B B
Published: May 2006

G.T mispairs, the sole mismatch type that can arise in "resting" mammalian DNA (through spontaneous hydrolytic deamination of 5-methylcytosine), are corrected in vivo with high efficiency and mostly to a G.C. We identified a protein factor, present in HeLa cell extracts, that binds selectively to DNA substrates containing this mismatch. The partially purified protein was shown by gel-filtration chromatography and UV cross-linking experiments to have an apparent molecular mass of 200 kDa. Its binding to G.T mispairs was not influenced by sequences flanking the mismatch, but methylation of guanines either within the mismatch itself or in its immediate vicinity abolished the formation of the protein-DNA complex. The protein appears to lack both endo- and exonuclease activities and requires neither magnesium nor zinc nor ATP for binding. We discuss the possible role of this protein in a repair pathway, which helps mammalian cells counter the mutagenic effect of the hydrolytic deamination of 5-methylcytosine.
Reexamination of the kinetics of the transfer of NADH between its complexes with glycerol-3-phosphate dehydrogenase and with lactate dehydrogenase.
Authors: Chock P B , Gutfreund H
Published: May 2006

Srivastava and Bernhard [Srivastava, D. K. & Bernhard, S. A. (1986) Science 234, 1081-1086] have proposed that glycolytic enzymes form multienzyme complexes for the direct transfer of metabolites from the producing enzyme to the utilizing one. We have reinvestigated the evidence for direct transfer of NADH between its complexes with alpha-glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) and L-lactate dehydrogenase (LDH; EC 1.1.1.27). The results reveal the following. (i) Proper treatment of the kinetics of and equilibrium data for the transfer of NADH between GPDH and LDH indicates that NADH transfer proceeds by a free-diffusion mechanism and not by direct transfer through a ternary complex. (ii) The koff for NADH from its GPDH complex is 60 sec-1 rather than 9.4 sec-1 in Tris.HCl buffer (pH 7.4) at 25 degrees C. With this value one can explain kcat = 50 sec-1 for LDH-catalyzed hydrogenation of pyruvate with GPDH-bound NADH as coenzyme. (iii) Steady-state kinetics show that LDH inhibits the GPDH-catalyzed reaction simply by reducing the concentration of free NADH. Similarly, aldolase inhibits the GPDH-catalyzed reduction of dihydroxyacetone phosphate to glycerol-3-phosphate by binding to the substrate. The proposed direct transfer of NADH between GPDH and LDH is therefore mainly based on a misinterpretation of the experimental data.
Phosphorylation of nitrogen regulator I (NRI) of Escherichia coli.
Authors: Weiss V , Magasanik B
Published: May 2006

It has previously been shown that phosphorylated nitrogen regulator I (NRI-phosphate) is the activator responsible for increasing the transcription of glnA, the structural gene for glutamine synthetase, and that NRII catalyzes the transfer of the gamma-phosphate of ATP to NRI. We have now shown that the reaction of ATP with NRII results in the reversible transfer of the gamma-phosphate of ATP to a histidine residue of NRII. In turn, NRII-phosphate transfers its phosphate reversibly to an aspartic residue of NRI. NRI-phosphate is hydrolyzed to NRI and inorganic phosphate in a divalent cation-requiring autocatalytic reaction.
Isolation of a full-length cDNA insert encoding human aromatase system cytochrome P-450 and its expression in nonsteroidogenic cells.
Authors: Corbin C J , Graham-Lorence S , McPhaul M , Mason J I , Mendelson C R , Simpson E R
Published: May 2006

The isolation and cloning of a full-length cDNA insert complementary to mRNA encoding human aromatase system cytochrome P-450 is reported. The insert contains an open reading frame encoding a protein of 503 amino acids. This gene is clearly a member of the cytochrome P-450 gene superfamily, because the sequence contains regions of marked homology to those of other members, notably a putative membrane-spanning region, I helix, Ozols, and heme-binding regions. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to human placental aromatase system cytochrome P-450, as detected by immunoblot analysis, and catalyzed the aromatization of androstenedione, testosterone, and 16 alpha-hydroxyandrostenedione. This activity was inhibited by the known aromatase inhibitors, 4-hydroxyandrostenedione and econazole. Thus the several steps involved in the aromatization reaction appear to be catalyzed by a single polypeptide chain, which can metabolize the three major physiological substrates.
Purified secB protein of Escherichia coli retards folding and promotes membrane translocation of the maltose-binding protein in vitro.
Authors: Weiss J B , Ray P H , Bassford P J
Published: May 2006

The efficient export of a subset of Escherichia coli envelope proteins is dependent upon the product of the secB gene. Previous studies indicated that SecB promotes the export of the periplasmic maltose-binding protein (MBP) by preventing premature folding of the precursor MBP in the cytoplasm into an export-incompetent form. In this study, SecB has been purified to homogeneity and shown to be a soluble, cytoplasmic, multimeric protein composed of identical 17-kDa subunits. SecB was required for efficient in vitro translocation of MBP into inverted membrane vesicles. The addition of purified SecB to an in vitro system prepared from SecB- cells significantly enhanced MBP translocation. The purified protein also quantitatively retarded folding of precursor MBP into a stable, protease-resistant conformation in the absence of membranes. Finally, the inclusion of excess purified SecB in a SecB+ in vitro system significantly prolonged the time in which precursor MBP remained competent for posttranslational import into membrane vesicles.
Decreased de novo synthesis of proteoglycans in drug-induced renal cystic disease.
Authors: Lelongt B , Carone F A , Kanwar Y S
Published: May 2006

Cellular and extracellular (tubular basement membrane, TBM) alterations in the proteoglycans (PGs) of the rat renal tubules in diphenylthiazole-induced cystic disease were investigated. The PGs of normal and cystic kidneys were labeled with [35S]sulfate in an organ-perfusion system. Extracted cellular and TBM PGs were characterized by Sepharose CL-6B chromatography before or after treatment with heparitinase (degrades heparan sulfate) or chondroitinase ABC (degrades chondroitin sulfate). Total radioactivities in cellular, TBM, and medium fractions of cystic kidneys were reduced by factors of 9, 7, and 3, respectively. The PGs obtained from cystic and normal kidneys had similar profiles, namely, two peaks of radioactivity with Kav values of 0.26 (Mr = 130,000-150,000) and 0.40 (Mr = 50,000-55,000). The peaks had variable proportions of radioactivity for cellular and TBM fractions. Besides heparan sulfate, an additional 15-20% of chondroitin sulfate was synthesized in all three fractions obtained from cystic kidneys. The PGs synthesized by cystic kidneys had lower charge-density characteristics as compared to controls by DEAE-Sephacel chromatography. The medium fractions contained mostly glycosaminoglycan chains (Kav = 0.47, Mr = 24,000-26,000) of heparan sulfate. Autoradiograms of tissue samples revealed approximately 50% and approximately 60% decreases of grain densities over the cellular and TBM compartments, respectively. This decrease in de novo PG synthesis may have some relationship in the pathogenesis of polycystic kidney disease.
Isolation and sequence determination of a cDNA clone for rat peroxisomal urate oxidase: liver-specific expression in the rat.
Authors: Reddy P G , Nemali M R , Reddy M K , Reddy M N , Yuan P M , Yuen S , Laffler T G , Shiroza T , Kuramitsu H K , Usuda N
Published: May 2006

Urate oxidase (UOxase; urate:oxygen oxidoreductase, EC 1.7.3.3), which catalyzes the oxidation of uric acid to allantoin, is present in most mammals but is absent in humans and certain primates. A cDNA clone for UOxase containing an insert of 1.3 kilobases (kb) was isolated from a lambda gt11 cDNA library prepared from rat liver mRNA. This recombinant clone with a 1283-nucleotide insert has sequence for 97% of the coding region together with 401 nucleotides of the 3'-untranslated region of the mRNA. The identity of UOxase cDNA clone was verified by analyzing the fusion protein, immunocytochemical localization with epitope-selected antibody, and hybrid-select translation analysis and by comparing sequences of four CNBr-cleaved peptides of the protein. Blot analysis revealed that the probe hybridizes to a single 1.5-kb mRNA species in the rat liver and a transplantable hepatocellular carcinoma. No UOxase mRNA was detected in 11 nonhepatic tissues of rat, suggesting tissue specificity of expression of this UOxase gene. Blot analysis of RNA from livers of rats treated with a peroxisome proliferator showed 2- to 3-fold increase in UOxase mRNA content, whereas the fatty acyl-CoA oxidase mRNA increased over 30-fold. Southern blot analysis of restriction enzyme digests of rat DNA suggests that there is a single copy of UOxase gene. Analysis of human genomic DNA revealed restriction fragments that are homologous to rat UOxase cDNA, although no UOxase mRNA was detected in human liver.
Amino acid changes linked to pyrimethamine resistance in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum.
Authors: Cowman A F , Morry M J , Biggs B A , Cross G A , Foote S J
Published: May 2006

We describe the isolation and the sequence of the gene for the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS; EC 1.5.1.3 and EC 2.1.1.45, respectively) from two pyrimethamine-resistant clones of Plasmodium falciparum, HB3 and 7G8. We have also derived the sequence of the DHFR portion of the gene, by amplification using polymerase chain reaction, for the pyrimethamine-sensitive clone 3D7 and the pyrimethamine-resistant strains V-1, K-1, Csl-2, and Palo-alto. The deduced protein sequence of the resistant DHFR portion of the enzyme from HB3 contained a single amino acid difference from the pyrimethamine-sensitive clone 3D7. It is highly likely that this difference is involved in the mechanism of drug resistance in HB3. The sequence of the DHFR gene from other pyrimethamine-resistant strains contains the same amino acid difference from the sensitive clone 3D7. However, they all differ at one other site that may influence pyrimethamine resistance. The DHFR-TS gene is present as a single copy on chromosome 4 in all pyrimethamine-sensitive and pyrimethamine-resistant isolates tested. Therefore, the molecular basis of pyrimethamine resistance in the parasites tested is not amplification of the DHFR-TS gene.
High frequency of mosaic mutants produced by N-ethyl-N-nitrosourea exposure of mouse zygotes.
Authors: Russell L B , Bangham J W , Stelzner K F , Hunsicker P R
Published: May 2006

Mouse zygotes containing one multiple-recessive parental genome (a, b; p cch; d se; s) and the corresponding wild-type alleles in the other were exposed to N-ethyl-N-nitrosourea (ENU) at various stages in vivo. At weaning age, the resulting mice were examined for mutations at the marked loci as well as at others producing externally visible phenotypes. Because of viability problems in one of two reciprocal crosses, the bulk of the mutagenesis data are derived from the cross that detects recessive mutations in the maternal genome. The mutation rate was approximately 8 times higher in groups treated 2.5-3 hr postmating (sperm entry, completion of second meiotic division) than in those injected 5-6 hr postmating (pronuclear formation). In the former more sensitive zygote population, the mutation rate is about an order of magnitude greater than that induced by the same ENU exposure (50 mg/kg) to spermatogonial stem cells. Of 11 mutants recovered, 8 were mosaics. Progeny tests have demonstrated germ-line involvement for most of the mosaics, and the average fraction of the germ line carrying the mutation is close to 50%. The nature of the mutations indicates (i) that the mosaicism results not from misassortment at the first cleavage but from mutation affecting one DNA strand of the maternal chromosome, and (ii) that the mutations are intragenic lesions rather than multilocus deletions, thus resembling ENU-induced mutations in spermatogonia. The finding that mosaicism for presumed point mutations is readily inducible by ENU treatment of zygotes may provide a means of generating genetic materials that can be of use for developmental studies.
M13 repeat probe detects DNA minisatellite-like sequences in gymnosperms and angiosperms.
Authors: Rogstad S H , Patton J C , Schaal B A
Published: May 2006

Several kinds of minisatellite DNA, all of which are composed of low to moderately repetitive DNA, have been identified in tetrapod genomes. While the repeating oligonucleotide elements (subrepeats) of a given minisatellite are virtually identical, subrepeat nucleotide composition differs between different minisatellites. Several minisatellites have exhibited moderate to high levels of restriction length polymorphism in a number of tetrapods. Such hypervariable markers provide powerful tools for genetic analyses in several fields of biology. Minisatellite applications have been restricted to tetrapods, but here we demonstrate that one probe, the M13 repeat probe previously used to detect minisatellites in humans and bovines, also reveals minisatellite-bearing endonuclease fragments in gymnosperms and angiosperms. While the plant minisatellites appear to be somatically stable within an individual, they often vary within species in potentially useful ways. These results demonstrate that minisatellite-like families may be distributed over a wide taxonomic range in eukaryotes, opening the possibility of a commensurately wide utility of minisatellite probes in genetic analyses.
Analysis of temperature-sensitive mutations in the simian virus 40 gene encoding virion protein 1.
Authors: Behm M , Lowman H , Ng S C , Bina M
Published: May 2006

Temperature-sensitive (ts) assembly mutants of the tumorigenic virus simian virus 40 (SV40) fail to follow the normal pathway of virion morphogenesis at 40 degrees C. The mutations were previously mapped to the gene coding for the major virion protein VP1 and fall into three groups: tsB, tsBC, and tsC. We have determined the tsB/C mutations by DNA sequence analysis and deduced the corresponding amino acid substitutions. We find that the mutations are global and span 68% of the VP1 gene. They result predominantly in single amino acid substitutions. The B mutations are localized between nucleotides 1667 and 2091, spanning the VP1 amino acid residues 54-195. With the exception of one mutation in tsC260, the C group mutations occur between the nucleotides 2141 and 2262, spanning VP1 residues 212-252. The tsBC substitutions are not localized within a distinct region. We present a model for the VP1 structure. The model correlates the distribution of ts assembly mutations in the SV40 VP1 gene with the VP1 functional domains, deduced form the phenotypes exhibited by the assembly mutants, and the VP1 structural domains, deduced recently from the cryoelectron microscopic studies of the SV40 virions. We summarize the behavior of the SV40 ts mutants and discuss the possible relationship between the ts phenotype and amino acid substitutions.
Purification and characterization of other distinct bone-inducing factors.
Authors: Wang E A , Rosen V , Cordes P , Hewick R M , Kriz M J , Luxenberg D P , Sibley B S , Wozney J M
Published: May 2006

We purified a factor that induces bone formation greater than 300,000-fold from guanidinium chloride extracts of demineralized bone. Fifty nanograms of highly purified protein was active in an in vivo cartilage and bone-formation assay. The activity resided in a single gel band, corresponding to a molecular mass of approximately 30 kDa, which yielded proteins of 30, 18, and 16 kDa on reduction. The partial amino acid sequence obtained from these proteins confirmed our identification of specific factors that induce new bone formation in vivo.
Transgenic mice containing intestinal fatty acid-binding protein-human growth hormone fusion genes exhibit correct regional and cell-specific expression of the reporter gene in their small intestine.
Authors: Sweetser D A , Hauft S M , Hoppe P C , Birkenmeier E H , Gordon J I
Published: May 2006

The rat intestinal fatty acid binding protein (I-FABP) gene exhibits cell-specific as well as regional differences in its expression within the continuously regenerating small intestinal epithelium. To investigate the underlying mechanisms, we linked portions of its 5' nontranscribed domain to the human growth hormone (hGH) gene and analyzed expression of the hGH reporter in transgenic mice by RNA blot, solution hybridization, and immunocytochemical techniques. Sequences located within 277 nucleotides of the start site of I-FABP transcription are sufficient to limit hGH expression to the intestine. Although the absolute levels of hGH mRNA in the duodenum and proximal jejunum of these transgenic mice were similar to those of I-FABP mRNA, steady-state hGH mRNA concentrations were approximately 100 times lower in their distal small intestine. Addition of nucleotides -278 to -1178 of the I-FABP gene "restored" hGH mRNA concentrations in the distal jejunum and ileum to levels comparable to murine I-FABP mRNA. Serum hGH levels were 1000 times lower in the "short promoter" transgenic mice compared to animals with the "long promoter" transgene, indicating that efficient distal small intestinal hGH expression is required to produce elevated hGH concentrations in serum. The distribution of hGH in villus-associated enterocytes and goblet cells and its lack of expression in the crypts of Lieberkuhn mimicked that of the endogenous I-FABP gene product in all transgenic pedigrees. However, bands of hGH-negative cells extending from the base to the tips of villi were frequently observed in mice that were heterozygous for the short promoter transgene. This mosaic staining was not observed for I-FABP. These data suggest that (i) different cis-acting sequences may be required for complete expression of proximal-distal I-FABP gradients than for recapitulation of its normal crypt-villus tip distribution; (ii) differences may exist in the export pathways of secreted proteins within enterocytes located in various regions of the small intestine; and (iii) there may be subtle genetic differences among various crypt stem cells that can be detected in vivo by observing mosaic patterns of transgene expression along the villus epithelium.
Role of interleukin 1 in the activation of T lymphocytes.
Authors: Lichtman A H , Chin J , Schmidt J A , Abbas A K
Published: May 2006

The activation of T lymphocytes requires their stimulation via clonotypic antigen receptors as well as nonantigen-specific costimulators, the best defined of which is the cytokine interleukin 1 (IL-1). Recent studies have shown that murine CD4+ helper T lymphocytes consist of two nonoverlapping subsets that selectively utilize interleukin 2 (IL-2) or interleukin 4 as their autocrine growth factors and are called Th1 and Th2 cells, respectively. We now show that IL-1 functions as a costimulator for the proliferation of Th2 but not of Th1 clones and only Th2 cells express high-affinity receptors for IL-1. Secretion of autocrine growth-promoting lymphokines by Th1 and Th2 cells occurs after stimulation via the antigen receptor-CD3 complex and is neither dependent on nor affected by IL-1. These findings suggest that the activation of T lymphocytes can be divided into two stages, lymphokine secretion and proliferation, and only proliferation requires costimulators such as IL-1. Moreover, the prevailing view that IL-1 functions as a costimulator by inducing secretion of IL-2 or expression of IL-2 receptors may not be generally applicable, because IL-2-producing Th1 clones do not express receptors for IL-1 and are insensitive to this cytokine.
Identification, cloning, and characterization of an immune activation gene.
Authors: Lipes M A , Napolitano M , Jeang K T , Chang N T , Leonard W J
Published: May 2006

We have identified an immune activation gene, denoted Act-2, by differential hybridization screening of an activated T-cell library. The gene is induced rapidly after T-cell activation with phytohemagglutinin, B-cell activation with Staphylococcus aureus Cowan I, and monocyte activation with lipopolysaccharide. We have isolated a cDNA containing the full-length coding region. The deduced amino acid sequence predicts an open reading frame of 92 amino acids, including a very hydrophobic N terminus, which by weight matrix score is predicted to be a signal peptide. Using a baculovirus expression system, we have shown that this gene encodes a secreted product. It is therefore possible that Act-2 represents a newly discovered cytokine.
Restricted production of interleukin 4 by activated human T cells.
Authors: Lewis D B , Prickett K S , Larsen A , Grabstein K , Weaver M , Wilson C B
Published: May 2006

Interleukin 4 (IL-4) is secreted by activated T cells and pleiotropically modulates both B- and T-lymphocyte function. In murine helper (CD4+) T-cell clones IL-4 production appears to be regulated independently of interferon gamma and interleukin 2. To determine whether production of these lymphokines is also differentially regulated in uncloned human T cells, we studied lymphokine production by normal human peripheral T cells and T-cell subsets after in vitro polyclonal activation. After maximal induction of lymphokine expression, IL-4 mRNA was detectable in less than 5% of CD4+ and 1-2% of unfractionated T cells, whereas approximately 33% and 60% of CD4+ cells expressed detectable mRNA for interferon gamma and interleukin 2, respectively. This finding correlated with dramatically lower production of IL-4 mRNA and protein than of interferon gamma and interleukin 2 by peripheral blood and tonsillar T cells. The helper-inducer (CD4+ CD45R-) T-cell subset, which significantly enhances in vitro immunoglobulin production, accounted for the preponderance of IL-4 mRNA accumulation and protein production by CD4+ T cells; nevertheless, cells with detectable IL-4 mRNA constituted less than 10% of the CD4+ CD45R- subset. Limitation of IL-4 production to a comparatively small population of normal human T cells could selectively regulate the effects of this lymphokine in T-cell-mediated immune responses; such selective regulation may be a fundamental mechanism for restricting the potentially pleiotropic effects of certain lymphokines to appropriate responder cells.
Characterization, purification, and affinity labeling of the brain [3H]glibenclamide-binding protein, a putative neuronal ATP-regulated K+ channel.
Authors: Bernardi H , Fosset M , Lazdunski M
Published: May 2006

Sulfonylurea and particularly glibenclamide are potent blockers of ATP-regulated K+ channels in insulin-secreting cells. A very good correlation exists between binding of sulfonylurea to brain and insulinoma cell membranes. The [3H]glibenclamide-binding component from pig brain microsomes was solubilized with digitonin with a complete retention of its properties of interaction with glibenclamide and other sulfonylureas. A four-step purification was achieved that used (i) hydroxylapatite chromatography, (ii and iii) affinity chromatographies on ADP-agarose and wheat germ agglutinin-agarose columns, and (iv) a final chromatographic step on a mixture of AMP-agarose/GMP-agarose/hydroxylapatite. This procedure led to a 2500-fold purification. NaDodSO4/polyacrylamide gel electrophoresis of the purified material in reducing and nonreducing conditions showed that the sulfonylurea-binding component is made of a single major polypeptide chain of Mr 150,000 +/- 10,000. Direct photoaffinity labeling of the receptor with [3H]glibenclamide at different steps of the purification also showed that radioactivity was specifically incorporated into a polypeptide of Mr 150,000 +/- 5000, thus confirming the subunit structure indicated by the purification.
Observations and comments on the mechanism of epoxidation of alkenes by manganese(III) porphyrins with hypochlorite.
Authors: Lee R W , Nakagaki P C , Balasubramanian P N , Bruice T C
Published: May 2006

The kinetics of olefin epoxidation in a methylene chloride/water biphase containing a phase-transfer agent with hypochlorite as oxygen transfer reagent and manganese(III) tetraphenylporphyrin catalysts has been reinvestigated. The results do not support the accumulation of an intermediate composed of an alkene and a hypervalent metaloxo porphyrin. Second-order rate constants for oxygen transfer from hypochlorite to the manganese porphyrin, comproprotionation of the hypervalent manganese-oxo porphyrin with manganese(III) porphyrin, and olefin epoxidation by the hypervalent manganese-oxo porphyrin were obtained from experiments in a wet organic monophase.
Complex between carboxypeptidase A and a hydrated ketomethylene substrate analogue.
Authors: Shoham G , Christianson D W , Oren D A
Published: May 2006

The complex of carboxypeptidase A (CPA) with 5-amino-(N-t-butoxycarbonyl)-2-benzyl-4-oxo-6-phenylhexanoic acid (BBP), the ketomethylene substrate analogue of the peptide substrate N-(t-butoxycarbonyl)-L-phenylalanyl-L-phenylalanine, was studied by x-ray crystallographic methods. Interestingly, the enzyme specifically binds only one of four stereoisomers of BBP that were present in the buffer solution in which the CPA crystals were soaked. Furthermore, the species observed to bind to the enzyme is the hydrated form of the ketone. This is rather surprising since the hydrated form of BBP is expected to be present in aqueous solution at a concentration of less than 0.2%. Hence, the enzyme-inhibitor complex is most stable with a species resembling a structure along the reaction coordinate of a chemical reaction rather than a species resembling a reactant or a product. Important structural information regarding the catalytic conformations of active-site residues spanning the S'1-S2 subsites of the enzyme is provided from the results of these x-ray diffraction experiments. The structure of the CPA-hydrated BBP complex provides support for a promoted-water hydrolytic mechanism, although it is not certain whether the enzyme has actually participated in the hydration reaction at the ketone carbonyl of BBP.
Toward an analogue of alcoholism in mice: analysis of nongenetic variance in consumption of alcohol.
Authors: Dole V P , Ho A , Gentry R T , Chin A
Published: May 2006

Drinking behavior of the isogenic mouse strain C57BL/6J was analyzed into nongenetic components: stochastic fluctuations, responses to fluctuations in the current environment, and persistent differences between individual animals. The latter accounted for the major part of the variance. The variance was neither increased by differences in diet during the postweaning rapid growth period (prior to assay for drinking choice) nor diminished by uniformity of treatment during this period, suggesting that significant differentiation had occurred prior to weaning. The large variance between animals could be explained by assuming that the genetic role in consumption of alcohol by C57BL mice is permissive--a relative insensitivity to the aversive orosensory and pharmacological effects of 10% alcohol--rather than a specific drug-seeking predisposition.
Low density lipoprotein causes general cellular activation with increased phosphatidylinositol turnover and lipoprotein catabolism.
Authors: Block L H , Knorr M , Vogt E , Locher R , Vetter W , Groscurth P , Qiao B Y , Pometta D , James R , Regenass M
Published: May 2006

Low density lipoprotein (LDL), at concentrations high enough for receptor binding but not high enough to saturate the receptor, induces activation of phosphatidylinositol (PtdIns) turnover in a variety of cell types with various biological functions. Using both biochemical and electron microscopic studies, we have shown that blood platelets take up and degrade LDL in a manner reminiscent of phagocytic cell types. The activation of both PtdIns turnover and LDL metabolism is inhibited by high density lipoprotein. Thus, LDL at hormonal concentrations causes general cellular activation. Since all cell types studied responded to LDL with increased PtdIns turnover and uptake of LDL cholesterol, the PtdIns cycle may also be involved in the cellular regulation of LDL cholesterol metabolism.