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1.
Neuroscience ; 317: 1-11, 2016 Mar 11.
Article in English | MEDLINE | ID: mdl-26762802

ABSTRACT

This study examined whether peripheral inflammatory injury increases the levels or changes the disposition of substance P (SubP) in the rostral ventromedial medulla (RVM), which serves as a central relay in bulbospinal pathways of pain modulation. Enzyme immunoassay and reverse transcriptase quantitative polymerase chain reaction were used to measure SubP protein and transcript, respectively, in tissue homogenates prepared from the RVM and the periaqueductal gray (PAG) and cuneiform nuclei of rats that had received an intraplantar injection of saline or complete Freund's adjuvant (CFA). Matrix-Assisted Laser Desorption/Ionization Time of Flight analysis confirmed that the RVM does not contain hemokinin-1 (HK-1), which can confound measurements of SubP because it is recognized equally well by commercial antibodies for SubP. Levels of SubP protein in the RVM were unchanged four hours, four days and two weeks after injection of CFA. Tac1 transcripts were similarly unchanged in the RVM four days or two weeks after CFA. In contrast, the density of SubP immunoreactive processes in the RVM increased 2-fold within four hours and 2.7-fold four days after CFA injection; it was unchanged at two weeks. SubP-immunoreactive processes in the RVM include axon terminals of neurons located in the PAG and cuneiform nucleus. SubP content in homogenates of the PAG and cuneiform nucleus was significantly increased four days after CFA, but not at four hours or two weeks. Tac1 transcripts in homogenates of these nuclei were unchanged four days and two weeks after CFA. These findings suggest that there is an increased mobilization of SubP within processes in the RVM shortly after injury accompanied by an increased synthesis of SubP in neurons that project to the RVM. These findings are consonant with the hypothesis that an increase in SubP release in the RVM contributes to the hyperalgesia that develops after peripheral inflammatory injury.


Subject(s)
Inflammation/complications , Medulla Oblongata/metabolism , Pain/etiology , Pain/pathology , Substance P/metabolism , Actin Cytoskeleton/genetics , Actin Cytoskeleton/metabolism , Animals , Disease Models, Animal , Freund's Adjuvant/toxicity , Functional Laterality , Gene Expression Regulation , Hyperalgesia/physiopathology , Inflammation/chemically induced , Male , Mitogen-Activated Protein Kinase 6/metabolism , Pain Measurement , Pain Threshold/physiology , Periaqueductal Gray/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Tachykinins/genetics , Tachykinins/metabolism
2.
Ann Rheum Dis ; 69(5): 903-9, 2010 May.
Article in English | MEDLINE | ID: mdl-19933746

ABSTRACT

BACKGROUND: Rheumatoid arthritis is an inflammatory disease marked by intra-articular decreases in pH, aberrant hyaluronan regulation and destruction of bone and cartilage. Acid-sensing ion channels (ASICs) are the primary acid sensors in the nervous system, particularly in sensory neurons and are important in nociception. ASIC3 was recently discovered in synoviocytes, non-neuronal joint cells critical to the inflammatory process. OBJECTIVES: To investigate the role of ASIC3 in joint tissue, specifically the relationship between ASIC3 and hyaluronan and the response to decreased pH. METHODS: Histochemical methods were used to compare morphology, hyaluronan expression and ASIC3 expression in ASIC3+/+ and ASIC3-/- mouse knee joints. Isolated fibroblast-like synoviocytes (FLS) were used to examine hyaluronan release and intracellular calcium in response to decreases in pH. RESULTS: In tissue sections from ASIC3+/+ mice, ASIC3 localised to articular cartilage, growth plate, meniscus and type B synoviocytes. In cultured FLS, ASIC3 mRNA and protein was also expressed. In FLS cultures, pH 5.5 increased hyaluronan release in ASIC3+/+ FLS, but not ASIC3-/- FLS. In FLS from ASIC3+/+ mice, approximately 50% of cells (25/53) increased intracellular calcium while only 24% (14/59) showed an increase in ASIC3-/- FLS. Of the cells that responded to pH 5.5, there was significantly less intracellular calcium increases in ASIC3-/- FLS compared to ASIC3+/+ FLS. CONCLUSION: ASIC3 may serve as a pH sensor in synoviocytes and be important for modulation of expression of hyaluronan within joint tissue.


Subject(s)
Chondrocytes/metabolism , Hyaluronic Acid/metabolism , Sodium Channels/physiology , Synovial Membrane/metabolism , Acid Sensing Ion Channels , Animals , Calcium/metabolism , Cartilage, Articular/metabolism , Cells, Cultured , Fibroblasts/metabolism , Hydrogen-Ion Concentration , Male , Mice , Mice, Inbred C57BL , Reverse Transcriptase Polymerase Chain Reaction/methods , Sodium Channels/metabolism , Synovial Membrane/cytology
3.
Pain ; 137(3): 662-669, 2008 Jul 31.
Article in English | MEDLINE | ID: mdl-18343037

ABSTRACT

The acid sensing ion channel 3 (ASIC3) is critical for the development of secondary hyperalgesia as measured by mechanical stimulation of the paw following muscle insult. We designed experiments to test whether ASIC3 was necessary for the development of both primary and secondary mechanical hyperalgesia that develops after joint inflammation. We used ASIC3 -/- mice and examined the primary (response to tweezers) and secondary hyperalgesia (von-Frey filaments) that develops after joint inflammation comparing to ASIC3 +/+ mice. We also examined the localization of ASIC3 to the knee joint afferents innervating the synovium using immunohistochemical techniques before and after joint inflammation. We show that secondary mechanical hyperalgesia does not develop in ASIC3 -/- mice. However, the primary mechanical hyperalgesia of the inflamed knee joint still develops in ASIC3 -/- mice and is similar to ASIC3 +/+ mice. In knee joint synovium from ASIC3 +/+ mice without joint inflammation, ASIC3 was not localized to joint afferents that were stained with an antibody to protein gene product (PGP) 9.5 or calcitonin gene-related peptide (CGRP). ASIC3 was found, however, in synoviocytes of the knee joint of uninflamed mice. In ASIC3 +/+ mice with joint inflammation, ASIC3 co-localized with PGP 9.5 or CGRP in joint afferents innervating the synovium. We conclude that the decreased pH that occurs after inflammation would activate ASIC3 on primary afferent fibers innervating the knee joint, increasing the input to the spinal cord resulting in central sensitization manifested behaviorally as secondary hyperalgesia of the paw.


Subject(s)
Afferent Pathways/metabolism , Arthritis/complications , Arthritis/metabolism , Hyperalgesia/etiology , Hyperalgesia/metabolism , Knee Joint/innervation , Knee Joint/metabolism , Sodium Channels/metabolism , Acid Sensing Ion Channels , Animals , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic
4.
Am J Hum Genet ; 62(2): 355-61, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9463315

ABSTRACT

Bartter syndrome (BS) is a family of disorders manifested by hypokalemic hypochloremic metabolic alkalosis with normotensive hyperreninemic hyperaldosteronism. We evaluated a unique, inbred Bedouin kindred in which sensorineural deafness (SND) cosegregates with an infantile variant of the BS phenotype. Using a DNA-pooling strategy, we screened the human genome and successfully demonstrated linkage of this unique syndrome to chromosome 1p31. The genes for two kidney-specific chloride channels and a sodium/hydrogen antiporter, located near this region, were excluded as candidate genes. Although the search for the disease-causing gene in this family continues, this linkage further demonstrates the genetic heterogeneity of BS. In addition, the cosegregation of these phenotypes allows us to postulate that a single genetic alteration may be responsible for the SND and the BS phenotype. The identification and characterization of this gene would lead to a better understanding of the normal physiology of the kidney and the inner ear.


Subject(s)
Bartter Syndrome/genetics , Chromosomes, Human, Pair 1 , Deafness/genetics , Genetic Linkage , Chromosome Mapping , DNA Primers , Family , Female , Genetic Markers , Genotype , Humans , Male , Pedigree , Repetitive Sequences, Nucleic Acid
5.
Biochemistry ; 37(13): 4325-35, 1998 Mar 31.
Article in English | MEDLINE | ID: mdl-9521753

ABSTRACT

Four variants of human beta globin in which the Trp at position 37 has been replaced with a Tyr, Ala, Gly, or Glu have been expressed in Escherichia coli. These globins have been combined with normal human alpha chains and heme to form tetrameric hemoglobin molecules. A technique for the preparation of alpha chain dimers, which are cross-linked between their alpha99 lysine residues, has been developed, and these alpha dimers were combined with two of the beta globins, betaW37G and betaW37E, to form the corresponding cross-linked variants. The kinetics of CO binding to the deoxygenated derivatives following rapid mixing and of CO rebinding following flash photolysis have been examined as functions of pH in the presence and absence of the organic phosphate inositol hexaphosphate, IHP. The kinetic measurements indicate that replacement of the tryptophan with other residues destabilizes the hemoglobin tetramer, resulting in considerable dissociation of even the deoxygenated hemoglobins into alphabeta dimers at micromolar protein concentrations. Substitutions at beta37 also alter the properties of the deoxygenated hemoglobin tetramer. The alteration of the functional properties of the T states of these variants as well as the tendency of the deoxygenated derivatives to dissociate into alphabeta dimers increases in the order HbA < betaW37Y < betaW37A < betaW37G < betaW37E. Stabilizing the betaW37G or betaW37E tetramers by addition of IHP or by cross-linking does not restore the normal functional properties of the T state. Measurements of the geminate rebinding of CO establish a kinetic difference between the normal R state tetramer and the alphabeta dimer consistent with quaternary enhancement, the greater affinity of oxygen for the R state tetramer than for the alphabeta dimer. Kinetics of geminate rebinding also suggest that quaternary enhancement may be altered by substitutions at the beta37 position.


Subject(s)
Hemoglobin A/metabolism , Protein Conformation , Amino Acid Substitution , Carbon Monoxide/metabolism , Carbon Monoxide/radiation effects , Cross-Linking Reagents/chemistry , Escherichia coli/metabolism , Globins/biosynthesis , Globins/chemistry , Globins/genetics , Globins/metabolism , Hemoglobin A/biosynthesis , Hemoglobin A/chemistry , Hemoglobin A/genetics , Humans , Hydrogen-Ion Concentration , Kinetics , Mass Spectrometry , Mutation , Photolysis , Phytic Acid/metabolism , Protein Binding , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Tryptophan/genetics
6.
Mamm Genome ; 9(12): 1013-21, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9880670

ABSTRACT

In an effort to generate a genome-wide set of high-quality polymorphic markers for the rat, we used the marker-selection method, which has already been proven useful for the development of markers, especially for the human genome. Small-insert (300-900 bp) rat genomic libraries were constructed with an estimated complexity of three genome equivalents and enriched for short tandem repeat sequences (STRs). The enriched libraries were found to contain 45% (CA)n and 27% (GATA)n, representing at least a 50-fold enrichment over unselected small insert genomic libraries. A subset of 2160 STR-containing clones, primarily of the (GATA)n class of repeats, were sequenced. PCR primers flanking the repeats were synthesized from some of the sequences from the (CA)n and (GATA)n classes of STRs and tested for polymorphism in a panel of eight inbred rat strains. This strategy yielded 147 polymorphic markers, which mapped with high odds to all chromosomes by linkage in three F2 populations. The integration of these STR markers with other rat genetic markers and mapping reagents will facilitate the mapping of disease genes in the rat and the identification of loci associated with complex mammalian phenotypes.


Subject(s)
Genetic Markers/genetics , Genome , Genomic Library , Tandem Repeat Sequences/genetics , Animals , Base Sequence , Chromosome Mapping , Chromosomes/genetics , Cloning, Molecular , DNA/chemistry , DNA/genetics , Dinucleotide Repeats/genetics , Microsatellite Repeats/genetics , Molecular Sequence Data , Polymorphism, Genetic , Rats , Rats, Inbred BN , Rats, Inbred Lew , Rats, Inbred SHR , Rats, Inbred WKY , Rats, Sprague-Dawley
7.
Hum Mol Genet ; 6(9): 1491-7, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9285786

ABSTRACT

Familial hypomagnesemia with secondary hypocalcemia (HSH) (MIM 307600) was studied in three inbred Bedouin kindreds from Israel. The three kindreds, one extended and two nuclear families, contained 13 affected individuals, 11 males and two females. Assuming that the individuals affected with hypomagnesemia shared a chromosomal region inherited from a common ancestor, we used a DNA pooling strategy in a genome-wide search for loci which show homozygosity for shared alleles in affected individuals. DNA samples from affected individuals within a single kindred were pooled and used as the template for PCR amplification of short tandem repeat polymorphic markers (STRPs). Pooled DNA from unaffected siblings and parents were used as controls. A shift towards homozygosity was observed in the affected DNA pool compared with the control pools with D9S301 (GATA7D12). Genotyping of individual DNA samples with D9S301 and several flanking markers confirmed linkage to chromosome 9 with maximum LOD scores of 3.4 (theta = 0.05), 3.7 (theta = 0) and 2.3 (theta = 0) for the three families. We have identified a 14 cM interval on chromosome 9 (9q12-9q22.2), flanked by proximal marker D9S1874 and distal marker D9S1807, within which all affected individuals from the three kindreds are homozygous for a shared haplotype. The disease segregates with a common affected haplotype in the three families, suggesting that hypomagnesemia is caused by a common ancestral mutation in these families. Although HSH has been previously reported to be X linked, these linkage data demonstrate that the disorder is an autosomal recessive disease in these kindreds. Mapping of a chromosomal breakpoint in a somatic cell line established from a patient with HSH and a balanced X;9 translocation placed the chromosomal breakpoint in a 500 kb region flanked by D9S1844 and D9S273. Identification of the gene responsible for hypomagnesemia will provide insight into the regulation of this essential cation.


Subject(s)
Chromosomes, Human, Pair 9/genetics , Genetic Markers , Magnesium/blood , Metabolism, Inborn Errors/genetics , Translocation, Genetic , Arabs , Cell Culture Techniques , Chromosome Mapping , Female , Genetic Linkage , Humans , Infant , Infant, Newborn , Male , Pedigree , X Chromosome/genetics
8.
Hypertension ; 27(3 Pt 2): 546-51, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8613201

ABSTRACT

In 1994, it was reported that Dahl salt-sensitive SS/Jr rats supplied by Harlan Sprague Dawley were genetically contaminated and resistant to the pressor effects of a high salt diet. Harlan Sprague Dawley subsequently developed new pedigree expansion and production colonies from their foundation colony to supply new, purportedly inbred, Harlan Sprague Dawley SS/Jr (S(HSD)). To evaluate the genetic integrity and salt sensitivity of thse new S(HSD), we performed genotyping (microsatellite DNA markers) and phenotyping (radiotelemetric arterial pressure) of 12 S(HSD), 16 "authentic" SS/Jr from the inbred colony of John Rapp (S(Rapp)), 9 Harlan Sprague Dawley salt-resistant SR/Jr (R(HSD)), and (genotyping only) 6 known "contaminated" Harlan Sprague Dawley Dahl SS/Jr (S*). In the genotyping studies, 20 of 22 markers revealed polymorphisms between S(Rapp) and S* and 18 were polymorphic between S(Rapp) and R(Rapp), but none of the 22 markers revealed polymorphisms between S(Rapp) and the new S(HSD). The phenotyping studies showed that during an ultra-low salt diet, mean arterial pressure was higher (P < .05) in both authentic S(Rapp) (129 +/- 2 mm Hg; mean +/- SE) and new S(HSD) (120 +/- 2 mm Hg) than in R(HSD) (93 +/- 1 mm Hg). A high salt diet increased mean arterial pressure in every S(HSD) and S(Rapp). Increases in mean arterial pressure after 4 weeks of a high salt diet were significantly (P < 0.05) greater in authentic S(Rapp) (+51 +/- 3 mm Hg) than in new S(HSD) (+39 +/- 3 mm Hg). In addition, salt-induced mortality was significantly greater in S(Rapp) (62.5%) than S(HSD) (8.3%) after 8 weeks (P < 0.01). S(HSD) were genotypically indistinguishable from S(Rapp), had an elevated arterial pressure on a low salt diet, and had a pressor response to salt. Thus, the new S(HSD) supplied to us had several characteristics of inbred Dahl SS/Jr and did not have evidence of the previously detected genetic contamination. However, phenotypic characteristics such as body weight, salt-induced hypertension, and mortality were significantly different in S(HSD) compared with S(Rapp). This may reflect genetic differences between these two strains or differences in environmental factors and suggests that the S(HSD) and S(Rapp) may now constitute distinct substrains of Dahl SS/Jr.


Subject(s)
Blood Pressure/genetics , Rats, Sprague-Dawley/genetics , Sodium, Dietary/administration & dosage , Animals , Blood Pressure/drug effects , Female , Genotype , Male , Phenotype , Rats
10.
Nucleic Acids Res ; 21(18): 4339-43, 1993 Sep 11.
Article in English | MEDLINE | ID: mdl-8414989

ABSTRACT

Cross-contamination with previously amplified products poses a serious limitation in the use of PCR for clinical testing and in certain research applications as well. In the present study we report the use of novel primers containing a 3'-terminal ribose residue to circumvent this problem. Extension of the primer by Taq DNA polymerase generates a cleavable ribonucleotide linkage within the amplified product. Cleavage of the primer by base or with a ribonuclease interferes with further replication of the product should carry over to another sample occur. Primers terminating in any of the 4 ribose residues function equally well as all DNA primers. Taq DNA polymerase is thus able to both efficiently extend and copy the single ribose residue. In translating from all DNA primers to ones containing a 3'-ribose residue no modification of the PCR protocol is required. The products formed can be used in all applications of the PCR. Since neither the original sample DNA, the primers or the extension products are modified by base or ribonuclease treatment both pre- and post-amplification sterilization can be carried out. Pre-amplification treatment with RNase A can yield as high as 10(4)-fold sterilization. Under these conditions the addition of beta-mercaptoethanol or other sulfhydryl reducing agent is necessary to inactivate the enzyme during thermocycling. Post-amplification treatment with NaOH readily yields at least 10(6)-fold sterilization. This alone is sufficient for most, if not all, applications of PCR. It is especially useful for quantitative RT-PCR, since the original target RNA sequence, which may be present in high copy numbers, is also destroyed.


Subject(s)
DNA Primers/chemistry , Polymerase Chain Reaction/methods , Ribose/chemistry , Base Sequence , Molecular Sequence Data , Ribonuclease, Pancreatic/metabolism
11.
Biochimie ; 75(1-2): 123-6, 1993.
Article in English | MEDLINE | ID: mdl-8389211

ABSTRACT

Recently we have shown that the major isoform of RNase H in human cells, RNase H1, is able to cleave DNA substrates containing a single RNA-DNA base pair, an activity which appears to be involved in an excision repair system for the removal of ribose residues misincorporated into DNA. In the present work we have further characterized the substrate specificity of the enzyme. DNA substrates containing all four ribonucleotides are cleaved by the enzyme. A RNA-DNA base pair is not required for substrate recognition. RNA residues present within a mismatch or in a RNA-RNA base pair are also cleaved. The principal structural feature for recognition by the enzyme may simply be the presence of the 2'-OH group of the ribose residue adjacent to the cleavage site.


Subject(s)
DNA Repair/physiology , Ribonuclease H/physiology , Ribose/chemistry , Base Sequence , Humans , Kinetics , Molecular Sequence Data , Substrate Specificity
12.
Biochemistry ; 31(36): 8619-28, 1992 Sep 15.
Article in English | MEDLINE | ID: mdl-1390646

ABSTRACT

The overexpression of a nonfusion product of human beta-globin in Escherichia coli from its cDNA sequence has been accomplished for the first time. Expression of beta-globin from its native cDNA required the use of the strong bacteriophage T7 promoter. In this system, beta-globin accumulated to approximately 10% of total E. coli proteins. alpha-Globin was not expressed in the T7 system using the native cDNA. For the expression of alpha-globin, synthetic genes containing optimal E. coli codons were constructed. Neither synthetic alpha- nor beta-globin gene alone was expressed from the lac or tac promoter. Globin expression was achieved when the two synthetic alpha- and beta-globin genes were combined as an operon downstream of the lac promoter. The two proteins combined intracellularly with endogenous heme, which was concomitantly overproduced to yield tetrameric hemoglobin as roughly 5-10% of total E. coli protein. Cloning the alpha- and beta-globin cDNAs in a construct identical with the lac promoter did not yield globin production, establishing the requirement for optimal codon usage. The recombinant beta-globin from the T7 expression system was purified and reconstituted in vitro with heme and native alpha chains. N-terminal analyses showed that the beta-globin produced in the T7 system and the tetrameric hemoglobin produced from the synthetic genes contained an additional beta 1 methionine residue. Two additional mutants, beta 1 Val----Met and beta 1 Val----Ala were produced using the T7 system. Functional and structural properties of the purified hemoglobins will be discussed in the following papers.


Subject(s)
Hemoglobins/genetics , Amino Acid Sequence , Base Sequence , Codon/genetics , Escherichia coli/genetics , Genes, Synthetic , Globins/biosynthesis , Globins/genetics , Hemoglobins/biosynthesis , Humans , Molecular Sequence Data , Mutagenesis , Operon , Promoter Regions, Genetic/genetics , Protein Engineering/methods , Protein Processing, Post-Translational , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , T-Phages/genetics
13.
J Biol Chem ; 265(10): 5561-6, 1990 Apr 05.
Article in English | MEDLINE | ID: mdl-1690712

ABSTRACT

The kinetic properties of Escherichia coli ribonuclease H (RNase H) were investigated using oligonucleotide substrates that consist of a short stretch of RNA, flanked on either side by DNA (DNA-RNA-DNA). In the presence of a complementary DNA strand, RNase H cleavage is restricted to the short ribonucleotide stretch of the DNA/RNA heteroduplex. The DNA-RNA-DNA substrate utilized for kinetic studies: (formula; see text) is cleaved at a single site (decreases) in the presence of a complementary DNA strand, to generate (dT)7-(rA)2-OH and p-(rA)2-(dT)9. Anion exchange high performance liquid chromatography was used to separate and quantitate the cleavage products. Under these conditions, RNase H-specific and nonspecific degradation products could be resolved. Kinetic parameters were measured under conditions of 100% hybrid formation (1.2-1.5 molar excess of complementary DNA, T much less than Tm). A linear double reciprocal plot was obtained, yielding a Km of 4.2 microM and a turnover number of 7.1 cleavages per s per RNase H monomer. The kinetic properties of substrate analogs containing varying lengths of RNA (n = 3-5) and 2'-O-methyl modifications were also investigated. Maximal turnover was observed with DNA-RNA-DNA substrates containing a minimum of four RNA residues. Kcat for the rA3 derivative was decreased by more than 100-fold. The Km appeared to decrease with the size of the internal RNA stretch (n = 3-5). No significant difference in turnover number of Km was observed when the flanking DNA was replaced with 2'-O-methyl RNA, suggesting that RNase H does not interact with this region of the heteroduplex.


Subject(s)
DNA/metabolism , Endoribonucleases/metabolism , Escherichia coli/enzymology , Nucleic Acid Heteroduplexes/metabolism , RNA/metabolism , Base Sequence , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Kinetics , Molecular Sequence Data , Ribonuclease H , Substrate Specificity , Uridine/analogs & derivatives , Uridine/metabolism
14.
Proc Natl Acad Sci U S A ; 85(14): 5011-5, 1988 Jul.
Article in English | MEDLINE | ID: mdl-2839827

ABSTRACT

The mechanism of hybrid-arrested translation by antisense oligodeoxynucleotides has been investigated with the rabbit reticulocyte lysate system. The oligonucleotides studied were directed against different regions of mouse alpha- or beta-globin mRNAs. Freshly prepared reticulocyte lysates were found to contain 1-2% of the level of RNase H in nucleated cells. This level of activity was sufficient to cleave nearly 100% of the targeted mRNA at the site of hybridization with a complementary oligodeoxynucleotide in 1 hr under conditions of active translation. Using poly(rA).oligo(dT) as a competitive inhibitor of the enzyme, hybrid arrest by oligodeoxynucleotides complementary to the sequence spanning the initiation codon or to a sequence in the coding region was found to be due entirely to cleavage of mRNA by RNase H. Hybridization of oligodeoxynucleotides adjacent to the cap site of beta-globin mRNA, but not the alpha-globin mRNA, also inhibited protein synthesis directly. Even in this case, however, cleavage of the mRNA by RNase H was the predominant pathway of inhibition.


Subject(s)
Endoribonucleases/metabolism , Nucleic Acid Hybridization , Oligonucleotides/pharmacology , Protein Biosynthesis/drug effects , RNA, Messenger/metabolism , Animals , Codon , DNA/genetics , Endoribonucleases/antagonists & inhibitors , Female , Globins/genetics , Mice , Mice, Inbred BALB C , Oligodeoxyribonucleotides/pharmacology , Oligonucleotides/genetics , Oligonucleotides, Antisense , Poly A/pharmacology , RNA, Messenger/genetics , Rabbits , Reticulocytes/metabolism , Ribonuclease H
15.
Proc Natl Acad Sci U S A ; 84(20): 7280-4, 1987 Oct.
Article in English | MEDLINE | ID: mdl-3478694

ABSTRACT

Under deoxygenated conditions, bis(3,5-dibromosalicyl) fumarate reacts with hemoglobin selectively to cross-link the alpha subunits between Lys-alpha 1 99 and Lys-alpha 2 99. We have characterized further the properties of this recently described hemoglobin and have demonstrated its utility as a blood substitute. The oxygen transport characteristics of the cross-linked derivative are very similar to those of whole blood. Under physiological conditions, the partial pressure of oxygen at half-saturation of hemoglobin is increased to 29 mm Hg (1 mm Hg = 133.3 kPa), compared to 12 mm Hg for hemoglobin A, fully compensating for the absence of 2,3-bisphosphoglycerate outside of the erythrocyte. The Hill coefficient is 2.9. The dependence of the oxygen affinity of HbXL99 alpha on CO2 is also identical to that of hemoglobin A. The cross-link between the alpha subunits blocks dissociation of oxyhemoglobin into alpha beta dimers and thereby prevents renal excretion of the modified hemoglobin. In the rat, the half-life of HbXL99 alpha in plasma, at a 15% volume exchange, is increased to 3.3 hr, compared to 90 min for hemoglobin A. Cross-linking HbXL99 alpha intermolecularly with bis(sulfosuccinimidyl) suberate to form predominantly a mixture of dimers and trimers further increased the half-life of the hemoglobin within the circulation by about 2-fold. The rate of autooxidation of the transfused hemoglobin was found to be markedly reduced because of the presence of an endogenous reducing system in plasma.


Subject(s)
Blood Substitutes/metabolism , Hemoglobins/metabolism , Animals , Aspirin/analogs & derivatives , Blood Substitutes/pharmacokinetics , Blood Transfusion , Cross-Linking Reagents , Half-Life , Hemoglobin A2/metabolism , Hemoglobins/pharmacokinetics , Kidney/metabolism , Male , Methemoglobin/metabolism , Oxidation-Reduction , Oxygen/metabolism , Oxyhemoglobins/metabolism , Rats , Rats, Inbred Strains
17.
Science ; 233(4763): 569-71, 1986 Aug 01.
Article in English | MEDLINE | ID: mdl-3523758

ABSTRACT

In Trypanosomatidae the messenger RNA's (mRNA's) that code for the variant surface glycoproteins (VSG's), tubulins, calmodulin, and at least a subset of other proteins contain a common 35-nucleotide leader sequence at their 5' ends. Hybrid-arrested in vitro translation has been used to show that all mRNA's in both African and South American trypanosomes contain this 35-nucleotide sequence. Oligonucleotides complementary to this sequence blocked translation of all trypanosome mRNA's in a rabbit reticulocyte lysate system, but did not inhibit translation of mRNA's from other organisms lacking this sequence. An oligonucleotide complementary to the VSG mRNA downstream from the spliced leader sequence arrested only VSG synthesis. Thus, the 35-nucleotide leader sequence is a general feature of all trypanosome mRNA's. The high specificity of oligonucleotides complementary to the spliced leader for their target sequence suggests that analogues permeable to the cell membrane may be useful in the treatment of trypanosomal infections.


Subject(s)
Protein Sorting Signals/genetics , RNA, Messenger/genetics , Trypanosoma/genetics , Base Sequence , Protein Biosynthesis , Trypanosoma brucei brucei/genetics , Trypanosoma cruzi/genetics
18.
J Biol Chem ; 261(21): 9929-37, 1986 Jul 25.
Article in English | MEDLINE | ID: mdl-3090027

ABSTRACT

Bis(3,5-dibromosalicyl) fumarate and a number of related bifunctional reagents react preferentially with oxyhemoglobin to cross-link the beta chains within the 2,3-diphosphoglycerate-binding site. In this report we describe a new derivative cross-linked between the alpha chains which is formed specifically in the reaction with deoxyhemoglobin. X-ray crystallographic studies show that the cross-link lies between Lys-99 alpha 1 and Lys-99 alpha 2, spanning the central cavity of the tetramer. Lys-99 alpha 1 and Lys-99 alpha 2 are located within a cluster of charged residues very near the middle of the hemoglobin molecule. In oxyhemoglobin, this site is completely inaccessible to the cross-linking agent. Competition experiments with inositol hexaphosphate indicate that the compound enters the central cavity in deoxyhemoglobin through the cleft between the alpha chains. Despite the presence of the cross-link between the alpha chains, the modified hemoglobin remains highly cooperative. The Hill coefficient for HbXL99 alpha is 2.6. The oxygen affinity of the cross-linked derivative is decreased by approximately 2-fold; at pH 7.0 in the presence of 0.1 M NaCl the P50 is 13.9 mm Hg compared to 6.6 mm Hg for HbA. This difference appears to be due to relatively small changes in both KR, the association constant for binding of oxygen to the R state, and the allosteric constant L. Surprisingly, the isoelectric point of oxyHbXL99 alpha is almost identical to that of oxyHbA, whereas in the deoxy form the isoelectric point of the cross-linked derivative is decreased relative to native hemoglobin as expected due to the loss of the two positive charges of the modified amino groups. In agreement with these findings, the alkaline Bohr effect of HbXL99 alpha is decreased by more than 50%. Earlier studies argue strongly against the possibility that Lys-99 alpha is directly responsible for this large fraction of the Bohr effect in HbA. Analysis of the structure suggests that in the cross-linked derivative Glu-101 beta, which is in close proximity to Lys-99 alpha in oxyhemoglobin, becomes an acid Bohr group.


Subject(s)
Aspirin/analogs & derivatives , Cross-Linking Reagents/pharmacology , Hemoglobins/isolation & purification , Lysine , Amino Acid Sequence , Aspirin/pharmacology , Crystallography , Humans , Isoelectric Focusing , Mathematics , Molecular Conformation , Oxyhemoglobins/analysis , Stereoisomerism , X-Ray Diffraction
19.
Gene ; 42(2): 133-9, 1986.
Article in English | MEDLINE | ID: mdl-3015727

ABSTRACT

In this report we describe a highly efficient method for site-specific mutagenesis using the yeast transformation system. The method is based on the observation that Saccharomyces cerevisiae can be transformed at high frequency with single-stranded circular DNA vectors [Singh et al., Gene 20 (1982) 441-449]. The model system studied was the TRP1 gene of S. cerevisiae cloned into a derivative of the phage M13mp9 vector containing the yeast URA3 gene. ARS1, located adjacent to the TRP1 gene, allows the plasmid to replicate autonomously in yeast. Synthetic 5'P-oligodeoxynucleotides, 19 and 35 nucleotides (nt) in length, designed to produce an A----T transversion mutation within the TRP1 gene, were annealed to ss DNA of the M13 vector at a molar ratio of 30:1 and directly transformed into yeast. The intended single nt mutation was obtained at frequencies of 24 and 43%, respectively. The latter approaches the theoretical limit of 50%. In the absence of the 5'-terminal phosphate, both the transformation frequency and the efficiency of mutagenesis by the synthetic oligodeoxynucleotide (oligo) were decreased by 2-4 fold. This procedure completely obviates the need for any enzymatic manipulations in vitro after forming the heteroduplex with the oligo primer containing the desired mutation. For yeast genes, direct phenotypic selection is possible in the recipient strain.


Subject(s)
Mutation , Oligodeoxyribonucleotides/pharmacology , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Base Sequence , DNA Restriction Enzymes , Genes , Genes, Fungal , Genetic Vectors , Saccharomyces cerevisiae/drug effects , Transformation, Genetic
20.
J Biol Chem ; 259(23): 14863-73, 1984 Dec 10.
Article in English | MEDLINE | ID: mdl-6501320

ABSTRACT

Bis(3,5-dibromosalicyl)fumarate (I) reacts preferentially with oxyhemoglobin to cross-link the two beta 82 lysine residues within the 2,3-diphosphoglycerate (DPG) binding site and as a result markedly increases the solubility of deoxyhemoglobin S. The cross-link acts by perturbing the acceptor site for Val 6 within the sickle cell fiber (Chatterjee, R., Walder, R. Y., Arnone, A., and Walder, J. A. (1982) Biochemistry 21, 5901-5909). In the present studies we have compared a large number of analogs of I to determine the structural features of the reagent required for specificity and for transport into the red cell. Both electrostatic and hydrophobic interactions contribute to the binding of these compounds at the DPG site. The optimal position for the negatively charged groups on the cross-linking agent for productive binding is adjacent to the ester as in the original salicylic acid derivatives. There is a direct correlation between the reactivity toward hemoglobin and the hydrophobicity of the substituent attached at the para position. Phenyl and substituted phenyl derivatives as in the analgesic, antiinflammatory drug diflunisal are particularly effective. These groups probably interact with hydrophobic residues of the amino-terminal tripeptide and the EF corner of the beta chains adjacent to the DPG binding site. Although bis(3,5-dibromosalicyl)fumarate is very reactive toward hemoglobin in solution, it is much less effective in modifying hemoglobin within the red cell. The reaction with intracellular hemoglobin was shown to be limited by competing hydrolysis of the reagent catalyzed at the outer surface of the erythrocyte membrane. Inactivation of the red cell membrane acetylcholinesterase with phenylmethylsulfonyl fluoride did not inhibit this reaction. Introduction of a single methyl group onto the carbon-carbon double bond of the fumaryl moiety decreases the lability of the ester 10-fold, due to steric effects, and allows the reagent to be taken up by the red cell and modify intracellular hemoglobin. The kinetics of transport of the methylfumarate derivative, bis(3,5-dibromosalicyl)mesaconate, are first-order, consistent with passive diffusion. The attachment of larger alkyl groups onto the cross-link bridge further enhances the transport of the reagent into the red cell. The solubility of deoxyhemoglobin S cross-linked with the butylfumarate derivative was found to be increased by almost 10% compared to the original fumarate diester.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Antisickling Agents/blood , Aspirin/analogs & derivatives , Cross-Linking Reagents/metabolism , Diphosphoglyceric Acids/blood , Hemoglobin, Sickle/metabolism , 2,3-Diphosphoglycerate , Antisickling Agents/chemical synthesis , Aspirin/blood , Aspirin/chemical synthesis , Binding Sites , Biological Transport , Humans , Indicators and Reagents , Kinetics , Protein Binding , Structure-Activity Relationship
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