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PubMed1996

The genes YNI1 and YNR1, encoding nitrite reductase and nitrate reductase respectively in the yeast Hansenula polymorpha, are clustered and co-ordinately regulated.

The nitrite reductase-encoding gene (YNI1) from the yeast Hansenula polymorpha was isolated from a lambda EMBL3 H. polymorpha genomic DNA library, using as a probe a 481 bp DNA fragment from the gene of Aspergillus nidulans encoding nitrite reductase (niiA). An open reading frame of 3132 bp, encoding a putative protein of 1044 amino acids with high similarity with nitrite reductases from fungi, was located by DNA sequencing in the phages lambdaNB5 and lambdaJA13. Genes YNI1 and YNR1 (encoding nitrate reductase) are clustered, separated by 1700 bp. Northern blot analysis showed that expression of YNI1 and YNR1 is co-ordinately regulated; induced by nitrate and nitrite and repressed by sources of reduced nitrogen, even in the presence of nitrate. A mutant lacking nitrite reductase activity was obtained by deletion of the chromosomal copy of YNI1. The mutant does not grow in nitrate or in nitrite; it exhibits a similar level of transcription of YNR1 to the wild type, but the nitrate reductase enzymic activity is only about 50% of the wild type. In the presence of nitrate the delta ynil::URA3 mutant extrudes approx. 24 nmol of nitrite/h per mg of yeast (wet weight), about five times more than the wild type.

Amino Acid Sequence↗
PubMed1998

Structure and activity of mouse S-adenosylmethionine decarboxylase gene promoters and properties of the encoded proteins.

The promoter regions of two S-adenosylmethionine decarboxylase genes (AMD genes) were isolated from a mouse genomic library. One promoter was that of the bona fide mouse AMD gene (AMD1) whereas the other was that of the intronless AMD gene (AMD2). There was no sequence identity between the two promoters. The sequence of the AMD1 promoter was highly homologous to the human AMD1 and rat Amd1B promoters. After transient transfection in various cell lines, the AMD1 promoter was one to two orders of magnitude stronger than the AMD2 promoter. Similar results were obtained by using stably transfected mouse FM3A cells. In S-adenosylmethionine decarboxylase (AdoMetDC)-overproducing SAM-1 cells, the AMD1 gene was amplified over 5-fold. AdoMetDC encoded by the intronless AMD2 gene had two amino acid replacements (Met to Ile at codon 70 and Ala to Val at codon 139), compared with the protein encoded by the AMD1 gene, and exhibited decreased catalytic activity (<50%) and decreased processing activity when expressed in AdoMetDC-deficient Escherichia coli cells. When Ile-70 of the protein encoded by AMD2 was converted into Met, both the catalytic and processing activities recovered markedly, indicating that Met-70 adjacent to the proenzyme-processing site is important for both activities. The third AMD locus (AMD3) in FM3A cells contains a pseudogene, in which deletion of two bases generates a premature termination codon at position 57. Since the AMD2 promoter had only 1-10% of the strength of the bona fide AMD1 gene and AMD2 protein possessed lower specific activity, the relative contribution of the AMD2-encoded enzyme to total AdoMetDC activity is small. Thus AdoMetDC activity in murine cells is thought to be due mainly to the product of the AMD1 gene.

Adenosylmethionine Decarboxylase↗
PubMed2002

Codominant expression of the polymorphic MICA alloantigens encoded by genes in the HLA region.

The HLA-related, polymorphic MHC class I-related chain A (MICA) gene encodes a 383-amino acid polypeptide, with three extracellular domains (alpha1, alpha2 and alpha3), a transmembrane region and a cytoplasmic tail. We have previously shown that freshly isolated endothelial cells, fibroblasts, keratinocytes and monocytes express MICA, while peripheral blood CD4+, CD8+ or CD19+ lymphocytes do not. This polymorphic MICA molecule is a target for specific alloantibodies in sera from kidney, heart and lung transplant recipients, although its possible role during graft rejection remains to be demonstrated. In this study we investigated whether there is codominance in the expression of MICA. We isolated RNA from a heterozygous cell line (HCT116), previously shown by sequencing-based typing to be MICA*001/MICA*00902, as well as 12 clones derived from it. Thereafter, we retrotranscribed the RNA into cDNA, and performed a molecular typing using MICA-sequence specific oligonucleotides (SSOP). Using this approach, we detected the RNA encoding MICA*001 and MICA*00902 in all the clones and in the parental cell line, indicating that MICA is codominantly expressed. This codominant expression was further confirmed by cloning and sequencing plasmids encoding these two alleles produced from the same HCT116 RNA preparation. We also produced the two recombinant MICA proteins (MICA*001 and MICA*00902). They reacted with rabbit anti-MICA polyclonal antibodies by ELISA and Western blot, indicating that the plasmids carrying the cDNA inserts probably encode functional MICA proteins. This strongly suggests that, like the HLA class I and class II proteins, MICA is codominantly expressed. The codominant expression of the polymorphic, HLA-like MICA alloantigens may have implications for the immune response elicited by the allograft in organ transplantation.

DNA Probes↗
PubMed2003

Molecular characterization of Oenococcus oeni genes encoding proteins involved in arginine transport.

AIMS: This work was carried out to complete the sequence of the arc cluster involved in arginine catabolism in Oenococcus oeni, and particularly to characterize the genes encoding proteins involved in arginine transport. METHODS AND RESULTS: Using molecular cloning, two loci encoding proteins involved in the arginine-ornithine antiport were isolated. Their expression patterns were monitored by RT-PCR to study the influence of arginine on their transcription. Polycistronic mRNAs were detected. PCR performed directly on colonies with primer pairs specific of arc genes was used to discriminate strains able/unable to degrade arginine. CONCLUSIONS: Oenococcus oeni contains two arcD loci encoding similar proteins. Their expression is not influenced by arginine and polycistronic messengers were detected. The inability to use arginine is due to a lack of genetic information encoding proteins of the arginine deiminase pathway. SIGNIFICANCE AND IMPACT OF THE STUDY: The constitutive expression of arcD genes points to the positive role of arginine on O. oeni cell growth. The occasional presence of all the arc ABCD genes together in O. oeni strains might provide insights into the growth rate variability within this species.

Amino Acid Sequence↗
PubMed1998

Circularization of Tn916 is required for expression of the transposon-encoded transfer functions: characterization of long tetracycline-inducible transcripts reading through the attachment site.

A detailed transcriptional analysis of the conjugative transposon Tn916 was carried out, which revealed that transcription of the transfer functions requires excision of the element and dramatically increases in the presence of tetracycline. The key components of this regulatory system are two contiguous transposon-borne genes, orf7 and orf8, located downstream from and having the same polarity of transcription as the tetracycline resistance determinant tetM. The gene orf7 encodes a 140-amino-acid (aa) protein exhibiting limited homology with sigmaF of Bacillus subtilis, whereas orf8 encodes a 76-aa peptide that does not share any sequence homology with any cognate proteins. In the presence of tetracycline, an attenuation mechanism enables the transcription of orf7 and orf8 from the tetM promoter. The resulting increased synthesis of ORF7 and ORF8 activates the promoter Porf7 located upstream from orf7, which then directs the expression of the transfer functions in the transposon circular intermediate through long transcripts encompassing the attachment site. The apparently non-regulated promoter Pxis located upstream of the excisionase encoding gene xis could also participate in the expression of the tra genes. We also demonstrate that Tn916 carries another regulated promoter, Porf9, which directs transcription of a single gene, orf9, located downstream from and transcribed counterclockwise to tetM. This gene encodes a 117-aa putative transcriptional repressor, but the exact role of this protein in the mobility of Tn916, as well as the regulation of its expression, remains to be elucidated. Our results constitute the molecular basis for the observation that tetracycline increased the transfer frequency of this type of element.

Bacillus subtilis↗
PubMed2000

Early lateral transfer of genes encoding malic enzyme, acetyl-CoA synthetase and alcohol dehydrogenases from anaerobic prokaryotes to Entamoeba histolytica.

The fermentation enzymes, which enable the microaerophilic protist Entamoeba histolytica to parasitize the colonic lumen and tissue abscesses, closely resemble homologues in anaerobic prokaryotes. Here, genes encoding malic enzyme and acetyl-CoA synthetase (nucleoside diphosphate forming) were cloned from E. histolytica, and their evolutionary origins, as well as those encoding two alcohol dehydrogenases (ADHE and ADH1), were inferred by means of phylogenetic reconstruction. The E. histolytica malic enzyme, which decarboxylates malate to pyruvate, closely resembles that of the archaeon Archaeoglobus fulgidus, strongly suggesting a common origin. The E. histolytica acetyl-CoA synthetase, which converts acetyl-CoA to acetate with the production of ATP, appeared to be closely related to the Plasmodium falciparum enzyme, but it was no more closely related to the Giardia lamblia acetyl-CoA synthetase than to those of archaea. Phylogenetic analyses suggested that the adh1 and adhe genes of E. histolytica and Gram-positive eubacteria share a common ancestor. Lateral transfer of genes encoding these fermentation enzymes from archaea or eubacteria to E. histolytica probably occurred early, because the sequences of the amoebic enzymes show considerable divergence from those of prokaryotes, and the amoebic genes encoding these enzymes are in the AT-rich codon usage of the parasite.

Acetate-CoA Ligase↗
PubMed2004

Differential transcriptome profiling identifies Plasmodium genes encoding pre-erythrocytic stage-specific proteins.

Invasive sporozoite and merozoite stages of malaria parasites that infect mammals enter and subsequently reside in hepatocytes and red blood cells respectively. Each invasive stage may exhibit unique adaptations that allow it to interact with and survive in its distinct host cell environment, and these adaptations are likely to be controlled by differential gene expression. We used suppression subtractive hybridization (SSH) of Plasmodium yoelii salivary gland sporozoites versus merozoites to identify stage-specific pre-erythrocytic transcripts. Sequencing of the SSH library and matching the cDNA sequences to the P. yoelii genome yielded 25 redundantly tagged genes including the only two previously characterized sporozoite-specific genes encoding the circumsporozoite protein (CSP) and thrombospondin-related anonymous protein (TRAP). Twelve novel genes encode predicted proteins with signal peptides, indicating that they enter the secretory pathway of the sporozoite. We show that one novel protein bearing a thrombospondin type 1 repeat (TSR) exhibits an expression pattern that suggests localization in the sporozoite secretory rhoptry organelles. In addition, we identified a group of four genes encoding putative low-molecular-mass proteins. Two proteins in this group exhibit an expression pattern similar to TRAP, and thus possibly localize in the sporozoite secretory micronemes. Proteins encoded by the differentially expressed genes identified here probably mediate specific interactions of the sporozoite with the mosquito vector salivary glands or the mammalian host hepatocyte and are not used during merozoite-red blood cell interactions.

Amino Acid Sequence↗
PubMed1992

Molecular characterization of cDNA encoding for adenylate kinase of rice (Oryza sativa L.).

Two types of genes (Adk-a, and Adk-b) encoding for adenylate kinase (AK, EC 2.7.4.3.) were isolated from the cDNA library constructed from poly(A)+ RNA of rice (Oryza sativa L.). Two cDNAs were heterogeneous at 5' and 3' ends of non-coding sequences and had possible polyadenylation signals. One of the genes, Adk-a, had 1154 bp sequences encoding 241 amino acid residues, while the other type, Adk-b, contained 1085 bp sequences encoding for 243 amino acid residues. Homology between Adk-a and Adk-b was 73.7% in nucleotide sequences, and 90.8% in amino acid level. Two genes showed about 53% homology to bovine mitochondrial adenylate kinase (AK2) at nucleotide and amino acid levels. Concerning the codon usage of rice AK genes, T was abundant at the third position of a codon in the reading frames. In order to examine the enzyme activity of the protein encoded by the rice cDNA, Adk-a was cloned into an expression vector, pUC119, which was introduced into Escherichia coli strain CV2, a temperature-sensitive mutant of adenylate kinase. We found that the transformant carrying the rice Adk-a gene in the sense orientation recovered cell growth at non-permissive high temperature (42 degrees C) and expressed enzyme activities higher than the untransformed CV2 and the transformant possessing Adk-a cDNA in the antisense orientation. These observations suggest that rice Adk-a codes a biologically active enzyme. Furthermore, sucrose was found to regulate the transcription of AK genes in rice cell cultures. Organ related accumulation of mRNA in whole plants was also found.

Adenylate Kinase↗
PubMed1995

The aspartate aminotransferase gene family of Arabidopsis encodes isoenzymes localized to three distinct subcellular compartments.

Here, a complete study is described of all the genes and isoenzymes for aspartate aminotransferase (AspAT) present in Arabidopsis thaliana. Four classes of cDNAs representing four distinct AspAT genes (ASP1-ASP4) have been cloned from Arabidopsis. Sequence analysis of the cDNAs suggests that the encoded proteins are targeted to different subcellular compartments. ASP1 encodes a mitochondrial form of AspAT, ASP3 encodes a chloroplastic/plastidic form of AspAT, whereas ASP2 and ASP4 each encode cytosolic forms of AspAT. Three distinct AspAT holoenzymes (AAT1-AAT3) were resolved by activity gel analysis. Organelle isolation reveals that AAT1 is mitochondrial-localized, AAT3 is plastid-localized, and AAT2 is cytosolic. Gene-specific Northern analysis reveals that each Asp mRNA accumulates differentially with respect to organ-type. However, the individual Asp mRNAs show no dramatic fluctuations in response to environmental stimuli such as light. Southern analysis reveals that four distinct nuclear genes probably represent the entire AspAT gene family in Arabidopsis. These molecular studies shed light on the subcellular synthesis of aspartate in Arabidopsis and suggest that some of the AspAT isoenzymes may play overlapping roles in plant nitrogen metabolism.

Amino Acid Sequence↗
PubMed1997

Regulation of expression of a cDNA from barley roots encoding a high affinity sulphate transporter.

A cDNA encoding a high-affinity sulphate transporter has been isolated from barley by complementation of a yeast mutant. The cDNA, designated HVST1, encodes a polypeptide of 660 amino acids (M(r) = 72,550), which is predicted to have 12 membrane-spanning domains and has extensive sequence homology with other identified eukaryotic sulphate transporters. The K(m) for sulphate was 6.9 microM when the HVST1 cDNA was expressed in a yeast mutant deficient in the gene encoding for the yeast SUL1 sulphate transporter. The strong pH-dependency of sulphate uptake when HVST1 was expressed heterologously in yeast suggests that the HVST1 polypeptide is a proton/sulphate co-transporter. The gene encoding HVST1 is expressed specifically in root tissues and the abundance of the mRNA is strongly influenced by sulphur nutrition. During sulphur-starvation of barley, the abundance of mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, both increase. Upon re-supply of sulphate, the abundance of the mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, decrease rapidly, concomitant with rises in tissue sulphate, cysteine and glutathione contents. Addition of the cysteine precursor, O-acetylserine, to plants grown with adequate sulphur supply, leads to increases in sulphate transporter mRNA, sulphate uptake rates and tissue contents of glutathione and cysteine. It is suggested, that whilst sulphate, cysteine and glutathione may be candidates for negative metabolic regulators of sulphate transporter gene expression, this regulation may be overridden by O-acetylserine acting as a positive regulator.

Amino Acid Sequence↗
PubMed1998

Cloning and functional expression of AtCOQ3, the Arabidopsis homologue of the yeast COQ3 gene, encoding a methyltransferase from plant mitochondria involved in ubiquinone biosynthesis.

A mutant of Saccharomyces cerevisiae deleted for the COQ3 gene was constructed. COQ3 encodes a 3,4-dihydroxy-5-hexaprenylbenzoate (DHHB) methyltransferase that catalyses the fourth step in the biosynthesis of ubiquinone from p-hydroxybenzoic acid. A full length cDNA encoding a homologue of DHHB-methyltransferase was cloned from an Arabidopsis thaliana cDNA library by functional complementation of a yeast coq3 deletion mutant. The Arabidopsis thaliana cDNA (AtCOQ3) was able to restore the respiration ability and ubiquinone synthesis of the mutant. The product of the 1372 bp cDNA contained 322 amino acids and had a molecular mass of 35,360 Da. The predicted amino acid sequence contained all consensus regions for S-adenosyl methionine methyltransferases and presented 26% identity with Saccharomyces cerevisiae DHHB-methyltransferase and 38% identity with the rat protein, as well as with a bacterial (Escherichia coli and Salmonella typhimurium) methyltransferase encoded by the UBIG gene. Southern analysis showed that the Arabidopsis thaliana enzyme was encoded by a single nuclear gene. The NH2-terminal part of the cDNA product contained features consistent with a putative mitochondrial transit sequence. The cDNA in Escherichia coli was overexpressed and antibodies were raised against the recombinant protein. Western blot analysis of Arabidopsis thaliana and pea protein extracts indicated that the AtCOQ3 gene product is localized within mitochondrial membranes. This result suggests that at least this step of ubiquinone synthesis takes place in mitochondria.

Amino Acid Sequence↗
PubMed1998

Reassignment of the gene encoding the Escherichia coli hydrogenase 2 small subunit--identification of a soluble precursor of the small subunit in a hypB mutant.

An active tryptic fragment of hydrogenase 2 from Escherichia coli has been isolated from the periplasmic face of the cytoplasmic membrane, and the large and small subunits N-terminally sequenced. The large subunit is encoded by the hybC gene and shows no N-terminal processing, other than removal of the initiator methionine during its biosynthesis. Both N-terminal and the subsequent internal tryptic-fragment amino acid sequence indicate that the small subunit is neither encoded by hybA, a gene previously identified as encoding the small subunit [Menon et al. (1994) J. Bacteriol. 176, 4416-4423], nor any of the remaining genes in the hyb operon. Genome sequence analysis revealed the presence of an open reading frame which could potentially encode the peptide sequences of the proteolysed small subunit. The gene, designated hyb0, lies directly upstream of, and is separated by two nucleotides from, the start of the hybA gene. Hyb0, which shares an approximate 40% identity with other hydrogenase small subunit amino acid sequences, is synthesised with an N-terminal signal sequence containing a twin-arginine motif which is probably required for export of the enzyme. In the mature enzyme the small subunit is proteolytically cleaved after Ala37. Immunological analysis of strains overproducing either recombinant Hyb0 or HybA using antibodies specific for hydrogenase 2, readily identified Hyb0 as the small subunit. In a pleiotropic hypB mutant, which is unable to insert nickel into the active site, both the large and small subunits accumulate as unprocessed, soluble forms, consistent with the two subunits being assembled and processed in a coordinated manner during biosynthesis.

Amino Acid Sequence↗
PubMed1998

A gene cluster encoding malonyl-CoA decarboxylase (MatA), malonyl-CoA synthetase (MatB) and a putative dicarboxylate carrier protein (MatC) in Rhizobium trifolii--cloning, sequencing, and expression of the enzymes in Escherichia coli.

A gene cluster consisting of three consecutive genes, matABC, was isolated using a probe prepared from amino acid sequence information of Rhizobium trifolii malonyl-CoA synthetase, and was subsequently sequenced. The sequences of matA and matB were overlapped by four base pairs, whereas the intergenic region between matB and matC had 95 base pairs. The upstream region contained DNA sequences which are typical for an Escherichia coli sigma70 promoter, and no other open reading frame was found within 400 bp downstream of matC. The ribosome-binding sites were found 7 to 12 base pairs upstream of each gene. MatA gene encoded a polypeptide of 462 amino acid residues, with deduced molecular mass of 51414 Da. A glutathione-S-transferase-MatA fusion protein has been purified and MatA was shown to have an intrinsic malonyl-CoA decarboxylase activity (Km = 0.47 mM; Vmax = 52 micromol x min(-1) x mg(-1)). MatB encoded a polypeptide of 504 amino acid residues with deduced molecular mass of 54612 Da. MatB was also purified from E. coli transformant carrying the gene cluster. The enzyme was essentially indistinguishable from the wild-type malonyl-CoA synthetase of R. trifolii by the criteria of polyacrylamide gel electrophoresis and biochemical properties. MatC encoded a 46453-Da protein with a high content of hydrophobic residues. The deduced amino acid sequences of matC showed identity to some extent with anaerobic C4-dicarboxylate carrier proteins from E. coli (25%) and Haemophilus influenzae (17%). MatC protein appears to be an integral membrane protein that could function as a malonate carrier. The formation of acetyl-CoA and malonyl-CoA from malonate was confirmed by thin-layer chromatographic analysis. These results strongly suggest that the gene cluster encodes proteins involved in the malonate-metabolizing system, malonate-->malonyl-CoA-->acetyl-CoA, in R. trifolii and that the metabolic pathway in the malonate-rich clover nodule might play an important role in symbiosis.

Acetyl Coenzyme A↗
PubMed2000

Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase.

During the course of a search for cDNAs encoding plant sterol acyltransferases, an expressed sequence tag clone presenting substantial identity with yeast and animal acyl CoA:cholesterol acyltransferases was used to screen cDNA libraries from Arabidopsis and tobacco. This resulted in the isolation of two full-length cDNAs encoding proteins of 520 and 532 amino acids, respectively. Attempts to complement the yeast double-mutant are1 are2 defective in acyl CoA:cholesterol acyltransferase were unsuccessful, showing that neither gene encodes acyl CoA:cholesterol acyltransferase. Their deduced amino acid sequences were then shown to have 40 and 38% identity, respectively, with a murine acyl CoA:diacylglycerol acyltransferase and their expression in are1 are2 or wild-type yeast resulted in a strong increase in the incorporation of oleyl CoA into triacylglycerols. Incorporation was 2-3 times higher in microsomes from yeast transformed with these plant cDNAs than in yeast transformed with the void vector, clearly showing that these cDNAs encode acyl CoA:diacylglycerol acyltransferases. Moreover, during the preparation of microsomes from the Arabidopsis DGAT-transformed yeast, a floating layer was observed on top of the 100 000 g supernatant. This fraction was enriched in triacylglycerols and exhibited strong acyl CoA:diacylglycerol acyltransferase activity, whereas almost no activity was detected in the corresponding clear fraction from the control yeast. Thanks to the use of this active fraction and dihexanoylglycerol as a substrate, the de novo synthesis of 1,2-dihexanoyl 3-oleyl glycerol by AtDGAT could be demonstrated. Transformation of tobacco with AtDGAT was also performed. Analysis of 19 primary transformants allowed detection, in several individuals, of a marked increase (up to seven times) of triacylglycerol content which correlated with the AtDGAT mRNA expression. Furthermore, light-microscopy observations of leaf epidermis cells, stained with a lipid-specific dye, showed the presence of lipid droplets in the cells of triacylglycerol-overproducer plants, thus illustrating the potential application of acyl CoA:diacylglycerol acyltransferase-transformed plants.

Acyl Coenzyme A↗
PubMed2000

Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris.

Laccases are multicopper-containing enzymes which catalyse the oxidation of phenolic and nonphenolic compounds with the concomitant reduction of molecular oxygen. In this study, a full-length cDNA coding for laccase (lac1) from Pycnoporus cinnabarinus I-937 was isolated and characterized. The corresponding open reading frame is 1557 nucleotides long and encodes a protein of 518 amino acids. The cDNA encodes a precursor protein containing a 21 amino-acid signal sequence corresponding to a putative signal peptide. The deduced amino-acid sequence of the encoded protein was similar to that of other laccase proteins, with the residues involved in copper coordination sharing the greatest extent of similarity. The cDNA encoding for laccase was placed under the control of the alcohol oxidase (Aox 1) promoter and expressed in the methylotropic yeast Pichia pastoris. The laccase leader peptide, as well as the Saccharomyces cerevisiae alpha-factor signal peptide, efficiently directed the secretion into the culture medium of laccase in an active form. Moreover, the laccase activity was directly detected in plates. The identity of the recombinant product was further confirmed by protein immunoblotting. The expected molecular mass of the mature protein is 81 kDa. However, the apparent molecular mass of the recombinant protein is 110 k Da, thus suggesting that the protein expressed in P. pastoris may be hyperglycosylated.

Alcohol Oxidoreductases↗
PubMed1999

Network analysis in episodic encoding and retrieval of word-pair associates: a PET study.

The involvement of distributed brain regions in declarative memory has been hypothesized based on studies with verbal memory tasks. To characterize episodic declarative memory function further, 14 right-handed volunteers performed a visual verbal learning task using paired word associates. The volunteers underwent positron emission tomography. 15O-butanol was used as a tracer of regional cerebral blood flow (rCBF). Inter-regional functional interactions were assessed based on within-task, across-subject inter-regional rCBF correlations. Anatomical connections between brain areas were based on known anatomy. Structural equation modelling was used to calculate the path coefficients representing the magnitudes of the functional influences of each area on the ones to which it is connected by anatomical pathways. The encoding and the retrieval network elicit similarities in a general manner but also differences. Strong functional linkages involving visual integration areas, parahippocampal regions, left precuneus and cingulate gyrus were found in both encoding and retrieval; the functional linkages between posterior regions and prefrontal regions were more closely linked during encoding, whereas functional linkages between the left parahippocampal region and posterior cingulate as well as extrastriate areas and posterior cingulate gyrus were stronger during retrieval. In conclusion, these findings support the idea of a global bihemispheric, asymmetric encoding/retrieval network subserving episodic declarative memory. Our results further underline the role of the precuneus in episodic memory, not only during retrieval but also during encoding.

Adult↗
PubMed1998

Increased expression of substance P receptor-encoding mRNA in bladder biopsies from patients with interstitial cystitis.

OBJECTIVES: To determine whether substance P (SP, also known as neurokinin 1, NK1) receptors are differentially expressed in bladder biopsies from patients with interstitial cystitis (IC) compared with matched controls. MATERIALS AND METHODS: Cold-cup biopsies were taken during routine diagnostic cystoscopy. NK1-receptor expression was assessed using a quantitative analysis of NK1-receptor-encoding mRNA in bladder biopsies from patients and controls using in situ hybridization histochemistry (ISHH) combined with autoradiographic image analysis. RESULTS: Autoradiographic signal indicating the presence of NK1-encoding mRNA was localized to detrusor muscle, urothelium and vascular structures. In the bladder vasculature, the signal was predominantly associated with endothelial cells. NK1 receptor-encoding mRNA within the vascular endothelium was increased in the biopsies obtained from patients with IC. CONCLUSION: Increased levels of NK1 receptor-encoding mRNA within the bladder vascular endothelium suggests the up-regulation of NK1 receptor as a putative factor in the pathogenesis of pain related to IC. Increased responsiveness to SP released from the perivascular sensory terminals may result in a local cascade of neurogenic inflammatory responses which trigger the pathophysiological changes, including pain, characteristic of IC.

Adult↗
PubMed1994

B1 and B2 bradykinin receptors encoded by distinct mRNAs.

Bradykinin receptors have been subdivided into at least two major pharmacological subtypes, B1 and B2. The cDNAs encoding functional B2 receptors have recently been cloned, but no molecular information exists at present on the B1 receptor. In this article, we describe experiments examining the possible relationship between the mRNAs encoding the B1 and B2 types of receptor. We showed previously that the human fibroblast cell line WI38 expresses both B1 and B2 receptors. In this report, we describe oocyte expression experiments showing that the B1 receptor in WI38 human fibroblast cells is encoded by a distinct mRNA approximately 2 kb shorter than that encoding the B2 receptor. We have used an antisense approach in conjunction with the oocyte expression system to demonstrate that the two messages differ in sequence at several locations throughout the length of the B2 sequence. Taken together with the mixed pharmacology exhibited in some expression systems by the cloned mouse receptor, the data indicate that B1-type pharmacology may arise from two independent molecular mechanisms.

Amino Acid Sequence↗