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PubMedDate not supplied

Functional analysis of conserved motifs in type III restriction-modification enzymes.

EcoP1I and EcoP15I are members of type III restriction-modification enzymes. EcoPI and EcoP15I DNA methyltransferases transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the N6 position of the second adenine residues in their recognition sequences, 5'-AGACC-3' and 5'-CAGCAG-3' respectively. We have altered various residues in two highly conserved sequences, FxGxG (motif I) and DPPY (motif IV) in these proteins by site-directed mutagenesis. Using a mixture of in vivo and in vitro assays, our results on the mutational analysis of these methyltransferases demonstrate the universal role of motif I in AdoMet binding and a role for motif IV in catalysis. All six cysteine residues in EcoP15I DNA methyltransferase have been substituted with serine and the role of cysteine residues in EcoP15I DNA methyltransferase catalysed reaction assessed. The Res subunits of type III restriction enzymes share a distant sequence similarity with and contain the motifs characteristic of the DEAD box proteins. We have carried out site-directed mutagenesis of the conserved residues in two of the helicase motifs of the EcoP1I restriction enzyme in order to investigate the role of motifs in DNA cleavage by this enzyme. Our findings indicate that certain conserved residues in these motifs are involved in ATP hydrolysis while the other residues are involved in coupling restriction of DNA to ATP hydrolysis. Taken collectively, these results form the basis for a detailed structure-function analysis of EcoP1I and EcoP15I restriction enzymes.

Adenosine Triphosphate↗
PubMed2005

Form-function analysis of the effect of canopy morphology on leaf self-shading in the seagrass Thalassia testudinum.

The variation in seagrass morphology and the magnitude of leaf self-shading within the canopy of Thalassia testudinum, were compared among nine sites in a fringing reef lagoon. We found a significant variation in the growth-form of T. testudinum reflected in a 5.4-fold variation in the attenuation coefficient (K (d)) within the canopy. The largest morphological variation was observed in shoot density. Leaf biomass, leaf area index (LAI), and shoot density were positively associated with canopy-K (d) and with the percentage of surface irradiance received by the top of the seagrass canopy (% Es). These results provide an explanation for the consistent pattern of depth reduction in seagrass leaf biomass and shoot density reported in the literature. Shoot density and shoot size are two descriptors of the growth-form of T. testudinum related to its clonal life-form. Shoot size was not significantly correlated with canopy-K (d), nevertheless, it showed a significant effect on the slope of the relationship between shoot density and canopy-K (d). According to this model, shoot size also contributes to light attenuation within the seagrass canopy by increasing the effect of shoot density. This form-function analysis suggests that light may have a relevant role in the regulation of the optimal plant balance between horizontal (variation in shoot density) and vertical (variation in shoot size) growth of seagrasses. Other environmental factors and interactions also need to be examined to fully understand the mechanistic bases of the morphological responses of seagrasses to the environment.

Biomass↗
PubMed2000

Functional analysis of androgen receptor N-terminal and ligand binding domain interacting coregulators in prostate cancer.

Several new androgen receptor (AR) coregulators, including ARA70, ARA55, ARA54, ARA160 and ARA24, associated with the N-terminal or the ligand-binding domain (LBD) of AR, have been identified by our group. We first identified the AR-LBD coregulators ARA70, ARA55, and ARA54. Our previous reports suggest that ARA70 can enhance the androgenic activity of 17 beta-estradiol (E2) and antiandrogens toward AR. It is of interest to compare and determine if the specificity of sex hormones and antiandrogens can be modulated by different coregulators. Our results indicate that, ARA70 is the best coregulator for increasing the androgenic activity of E2. Only ARA70 and ARA55 were able to significantly increase the androgenic activity of hydroxyflutamide, the active metabolite of a widely-used antiandrogen for the treatment of prostate cancer. Furthermore, our results suggest that among the LBD coregulators, ARA70 has a relatively high specificity for AR in the human prostate cancer cell line DU145. Together, our data suggest that the androgenic activity of some sex hormones and antiandrogens can be modulated by selective AR coactivators. In addition to the AR-LBD associated proteins, ARA24 and ARA160 have been identified as AR coregulators, interacting with the AR N-terminal instead of the LBD. Functional analysis revealed that the AR N-terminal coregulator ARA160 could cooperate with the AR LBD-associated coregulator ARA70. Our data indicate that ARA24 could also interact with AR, and that this binding is decreased by an expanding poly-glutamine (Q) length within AR. The length of the poly-Q stretch in the AR N-terminal domain is inversely correlated with the transcriptional activity of AR. Our data suggest that optimal AR transactivation may require interaction of AR with AR coregulators. The identification of factors or peptides that can interrupt androgen-mediated AR-ARA interactions may be useful in the development of better antiandrogens for treating androgen-related diseases, such as prostate cancer.

Androgen Antagonists↗
PubMed1993

Functional display of human plasminogen-activator inhibitor 1 (PAI-1) on phages: novel perspectives for structure-function analysis by error-prone DNA synthesis.

The synthesis of the human plasminogen-activator inhibitor 1 (PAI-1) protein in the cytoplasm of transformed Escherichia coli cells results in inactive protein preparations that can be activated by denaturation and renaturation. We have used the phagemid pComb3, designed for combinatorial immunoglobulin repertoire cloning, for routing of PAI-1 to the periplasm and subsequent exposure on the surface of filamentous phages. Phage-displayed PAI-1 specifically binds to immobilized polyclonal and monoclonal anti-human PAI-1 antibodies. In addition, PAI-1 retains its capacity to form equimolar complexes with its target serine protease tissue-type plasminogen activator (t-PA), as well as its ability to inhibit t-PA activity. Finally, we have explored and manipulated the error-prone property of TaqI DNA polymerase during PCR amplification of the full-length PAI-1 cDNA to generate a large library of predominantly single, random PAI-1 mutants. In addition, a computer simulation program has been devised that converts the number of mutations per codogenic region (in this case PAI-1) into actual mutant proteins. The PAI-1-phage mutant library is composed of 46% single and 34% double mutants and 20% wild-type PAI-1 and can be employed to isolate mutants defective in interactions of PAI-1 with other components. The method described here is applicable to other studies on the structure-function analysis of eukaryotic proteins.

Antibodies, Monoclonal↗
PubMed1998

Functional analysis of Gscl in the pathogenesis of the DiGeorge and velocardiofacial syndromes.

Gscl encodes a Goosecoid-related homeodomain protein that is expressed during mouse embryogenesis. In situ hybridization and immunohistochemistry studies show that Gscl is expressed in the pons region of the developing central nervous system and primordial germ cells. Gscl expression is also detected in a subset of adult tissues, including brain, eye, thymus, thyroid region, stomach, bladder and testis. Gscl is located within a region of the mouse genome that is syntenic with the region commonly deleted in DiGeorge and velocardiofacial syndrome (DGS/VCFS) patients. DGS/VCFS patients have craniofacial abnormalities, cardiac outflow defects and hypoplasia of the parathyroid gland and thymus due to haploinsufficiency of a gene or genes located within the deleted region. Thus, the genomic location of Gscl and its expression in a subset of the tissues affected in DGS/VCFS patients suggest that Gscl may contribute to the pathogenesis of DGS/VCFS. To determine the role of Gscl during mouse embryogenesis and in DGS/VCFS, we have deleted Gscl by gene targeting in mouse embryonic stem cells. Both Gscl heterozygous and Gscl null mice were normal and fertile, suggesting that Gscl is not a major factor in DGS/VCFS. Interestingly, expression of the adjacent Es2 gene in the pons region of Gscl null fetuses was absent, suggesting that mutations within the DGS/VCFS region can influence expression of adjacent genes. In addition, embryos that lacked both Gscl and the related Gsc gene appeared normal. These studies represent the first functional analysis of a DGS/VCFS candidate gene in vivo. These Gscl null mice will be an important genetic resource for crosses with other mouse models of the DGS/VCFS.

Abnormalities, Multiple↗
PubMed2005

Functional analysis of highly defined, FACS-isolated populations of human regulatory CD4+ CD25+ T cells.

The importance of CD4+ CD25+ regulatory T cells (Treg) in maintaining immune homeostasis has been directly demonstrated in vivo by their manipulation in a number of autoimmune disease models in the mouse. In the study of human regulatory cells, we have found that the cells that consistently demonstrate the in vitro regulatory activity most similar to that described for murine cells in vitro are best identified by restricting the isolation of CD25+ CD4 T cells to those cells expressing only the highest levels of CD25, representing approximately 2-3% of total CD4 T cells. Thus, it is the CD4+ CD25high subset that exhibits the in vitro characteristics that are identical to the CD4+ CD25+ regulatory cells initially characterized in mice. Furthermore, the cells expressing medium to low levels of CD25 not only do not exhibit suppressive activity directly ex vivo, but also actually contain a significant proportion of CD62L- CD4 T cells which are believed to be in vivo activated T cells. Due to the inherent difficulties in using CD25 as a marker for the purification of Treg cells, the finding that selection of the CD25high subset of CD4+ CD25+ T cells minimizes the co-isolation of contaminating activated CD4 T cells is important for future studies of these Treg cells in human disease. In order to perform these studies, we first had to establish a highly reproducible 'micro in vitro co-culture' assay system to enable the functional analysis of high-purity, but low-yield regulatory populations derived from FACS sorting. With this system in place, we are poised to dissect the potential heterogeneity of mechanisms employed by highly specific subpopulations of CD4+ CD25+ cells.

Antigens, CD↗
PubMed2000

Identification and functional analysis of mutations in the hepatocyte nuclear factor-1alpha gene in anti-islet autoantibody-negative Japanese patients with type 1 diabetes.

Mutations in the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene are the cause of maturity-onset diabetes of the young type 3 (MODY 3), which is characterized by a severe impairment of insulin secretion and early onset of the disease. Although the majority of patients with type 1 diabetes have type 1A, immune-mediated diabetes, there is a significant percentage of the patients who have no evidence of an autoimmune disorder at the onset of disease. The aim of this study was to estimate the prevalence of MODY 3 in antiislet autoantibody negative patients with type 1 diabetes. From a large population-based sample of unrelated Japanese patients with type 1 diabetes, 28 patients who lacked autoantibodies to glutamic acid decarboxylase, islet cell antigen 512/insulinoma-associated antigen-2, phogrin (phosphate homolog of granules of insulinoma)/insulinoma-associated antigen-2beta, and insulin at the onset of type 1 diabetes were examined by PCR-based direct sequencing of the 10 exons, flanking introns, and the promoter region of the HNF-1alpha gene. Two (7.1%) of 28 autoantibody-negative patients with type 1 diabetes were identified as carrying mutations in the HNF-1alpha gene. One patient carried a frameshift mutation (Pro379fsdelCT) in exon 6, and another patient carried a novel 2-bp substitution at nucleotides +45 (G to A) and +46 (C to A) from the transcriptional site of the promoter region. These mutations were identified in heterozygous form and were not identified in 64 unrelated healthy control subjects or 54 unrelated islet autoantibody-positive patients with type 1 diabetes. Functional analysis of the mutant HNF-1alpha gene indicated that the Pro379fsdelCT mutation had no transcriptional trans-activation activity and acted in a dominant negative manner. The +45/46 GC to AA mutation in the promoter region showed reduced promoter activity by 10-20% compared to the wild-type sequence. In conclusion, about 7% of Japanese diabetic patients lacking antiislet autoantibodies initially classified as having type 1 diabetes could have diabetes caused by mutations in the HNF-1alpha gene.

Adolescent↗
PubMed1996

Induction of the glucokinase gene by insulin in cultured neonatal rat hepatocytes. Relationship with DNase-I hypersensitive sites and functional analysis of a putative insulin-response element.

Previous, in vivo experiments have shown that an appropriate hormonal environment (high plasma insulin, low plasma glucagon) was unable to induce the accumulation of glucokinase mRNA in term fetal rat liver, whereas it was very efficient in the newly born rat. We have confirmed in the present study that insulin induced the accumulation of glucokinase mRNA in cultured hepatocytes from 1-day-old newborn rats, but not in cultured hepatocytes from 21-day-old fetuses. To identify regulatory regions of the glucokinase gene involved in the insulin response, we have scanned the glucokinase locus for DNase I hypersensitive sites in its in vivo conformation. We confirmed the presence of four liver-specific DNase I hypersensitive sites located in the 5' flanking region of the gene. Moreover, two additional hypersensitive sites, located at 2.5 kb and 3.5 kb upstream of the cap site were found but none of these new sites displayed inducibility by insulin. Finally, an increase of the sensitivity of hypersensitive site-1 and hypersensitive site-2 to DNase I correlates with the ability of insulin to induce glucokinase gene expression in cultured hepatocytes from 1-day-old rats, as observed in previous in vivo studies. This suggests that neither a prior exposure to insulin nor a simple aging of the fetal cells in the presence of the hormone in culture are instrumental for the full DNase-I hypersensitivity of the two proximal sites necessary for the neonatal response of the glucokinase gene to insulin. The proximal hypersensitive site-1, which is close to the transcription start site in the liver, does coincide with a sequence (designated IRSL) that is 80% identical to the phosphoenolpyruvate carboxykinase IRS and with a DNase-I footprint that has been identified overlapping this sequence. Nevertheless, functional analysis of this sequence suggested that it is unlikely that the insulin-response sequence like alone is sufficient to mediate the transcriptional effect of insulin on the hepatic glucokinase gene.

Animals↗
PubMed2004

Expression profiling with arrays of randomly disrupted genes in mouse embryonic stem cells leads to in vivo functional analysis.

DNA arrays are capable of profiling the expression patterns of many genes in a single experiment. After finding a gene of interest in a DNA array, however, labor-intensive gene-targeting experiments sometimes must be performed for the in vivo analysis of the gene function. With random gene trapping, on the other hand, it is relatively easy to disrupt and retrieve hundreds of genes/gene candidates in mouse embryonic stem (ES) cells, but one could overlook potentially important gene-disruption events if only the nucleotide sequences and not the expression patterns of the trapped DNA segments are analyzed. To combine the benefits of the above two experimental systems, we first created approximately 900 genetrapped mouse ES cell clones and then constructed arrays of cDNAs derived from the disrupted genes. By using these arrays, we identified a novel gene predominantly expressed in the mouse brain, and the corresponding ES cell clone was used to produced mice homozygous for the disrupted allele of the gene. Detailed analysis of the knockout mice revealed that the gene trap vector completely abolished gene expression downstream of its integration site. Therefore, identification of a gene or novel gene candidate with an interesting expression pattern by using this type of DNA array immediately allows the production of knockout mice from an ES cell clone with a disrupted allele of the sequence of interest.

Adenosine↗
PubMed1988

Functional analysis of mononuclear cells infiltrating into tumors. IV. Purification and functional characterization of cytotoxic cell-generating factor.

Rat cytotoxic cell-generating factor (CGF) was purified from cell-free supernatants of a T cell hybridoma (6B2-B8) that constitutively produces CGF. CGF activity was assessed by its ability to generate cytotoxic cells against 51Cr-labeled T-9 cells from spleen cells of T-9-immunized rats. The purification scheme consisted of ammonium sulfate precipitation, AcA 54 gel permeation, Mono Q anion exchange chromatography, Superose 12HR 10/30 gel permeation, SDS-PAGE with subsequent electroelution, and ProRPC HR5/10 reverse phase column chromatography. Overall, CGF was purified approximately 13,000-fold, with a maximum 2.5% recovery of activity, and the sp. act. of the purified CGF was approximately 19,000 U/mg. The purified CGF is distinct from the other lymphokines such as IL-1, IL-2, IL-3, IL-4, T cell-replacing factor/IL-5, IL-6, and IFN-gamma. It is capable of promoting the generation of cytotoxic T cells from R1-10B5 (+) spleen cells of T-9-immunized rats and also stimulates a W3/25 (+) T cell hybridoma to express the IL-2R. The CGF has an apparent m.w. of 28,000 under non-reducing and 14,000 and 16,000 under reducing conditions. 125I-labeled CGF binds to normal thymocytes as well as splenic T cells. The highest level of binding of CGF was detected on splenic T cells derived from T-9-immunized rats that were previously shown to contain CTL precursors. The binding analysis with 125I-labeled CGF demonstrated that CGF binds to a specific cell surface molecule with an approximate m.w. of 60,000 to 70,000.

Animals↗
PubMed1998

Functional analysis of mature hematopoietic cells from mice lacking the betac chain of the granulocyte-macrophage colony-stimulating factor receptor.

Mice with a null mutation of the betac chain of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors (betac-null mice) develop an alveolar proteinosis-like lung disease. The pathogenesis of this disease is uncertain and, although a defect in alveolar macrophage function has been postulated, no previous analysis of mature hematopoietic cells in mice with alveolar proteinosis has been reported. Therefore, we undertook a functional analysis of the mature hematopoietic cell compartment in betac-null mice. In addition, we reexamined the roles of the GM-CSF receptor chain and the betac chain in signaling by GM-CSF. Neutrophils and macrophages from betac-null mice were capable of normal survival and phagocytosis in the absence of stimulus and of similar levels of nitric oxide production in response to interferon-gamma and lipopolysaccharide. GM-CSF-mediated augmentation of survival, phagocytosis, and hydrogen-ion production were absent in neutrophils from betac-null mice. Interestingly, we were unable to show any ability of the GM-CSF receptor -chain alone to mediate glucose transport in these cells. In keeping with the betac-null mice lung pathology, examination of lavage fluid from the lungs of betac-null mice showed increased cellularity. This was caused by an increase in the number of lymphocytes, neutrophils, and macrophages. Large foamy cells in the lavage fluid from betac-null mice were identified as macrophages using immunohistochemistry. Functional analysis showed that these betac-null alveolar macrophages were capable of phagocytosis but uptake of colloidal carbon and cellular adhesion were reduced. In summary, mature hematopoietic cells with a null mutation of the betac receptor were unable to perform GM-CSF-mediated hematopoietic cell functions including glucose transport, but responded normally to a range of other ligands.

Animals↗
PubMed1988

Functional analysis of alternatively spliced tyrosinase gene transcripts.

Three different cDNA clones (pmcTyr1, pmcTyr2 and pmcTyr3) representing mRNAs originating by alternative splicing of the primary transcript of mouse tyrosinase gene, were identified and characterized by sequence analysis and by a functional assay. These cDNAs were subcloned into the newly constructed expression vector pHD. After electroporation of these hybrid clones into tyrosinase negative cells, protein extracts were prepared and tested for tyrosinase enzyme activity. Only the cDNA insert of pmcTyr1 was able to confer tyrosinase enzyme activity. This cDNA encodes a protein 533 amino acid residues in length containing a putative leader peptide of 18 amino acids and six putative glycosylation sites. Comparisons of the deduced amino acid sequence of the cDNA clone pmcTyr1 with the protein sequence of tyrosinases from man, Streptomyces, Neurospora and with haemocyanin subunits from a spider showed two regions of sequence conservation. One of these regions is known to be involved in copper binding. Since this gene with the coding capacity for tyrosinase is absent in all studied c-locus lethal deletion mutant mice, we have evidence that albinism in mice is caused by mutations of the tyrosinase gene.

Amino Acid Sequence↗
PubMed2003

Functional analysis of the human Sprouty2 gene promoter.

Sprouty2 plays a key role in negatively modulating the fibroblast growth factor signaling pathway, which is required for early branching events in embryonic development. The expression of the murine Sprouty2 gene shows a temporally and spatially restricted pattern in developing lung. In order to clarify the molecular mechanisms governing the transcription of the Sprouty2 gene, we first characterized the genomic organization of the human Sprouty2 (hSpry2) gene and mapped its transcription start sites by 5'-rapid amplification of cDNA ends. Subsequently, a 4-kb sequence from the 5'-flanking region of the gene was cloned and determined to contain promoter activity. Detailed truncation analysis of the hSpry2 promoter revealed the presence of context-specific suppressor activity in the distal upstream region. More importantly, we demonstrated that all the elements necessary to achieve strong basal transcription activity were located within the proximal 0.4-kb region. Sequence analysis revealed that this functionally important proximal region contains neither TATA nor CAAT box but an initiator element around the transcription start site. Several cis-acting elements (including AP2, CREB, SP1 and Ets-1) were found to be present in the proximal region, and their interactions with specific nuclear proteins were confirmed by electrophoretic mobility shift assays. To our knowledge, this is the first promoter study of any mammalian Sprouty gene. We propose that the high-level basal expression of hSpry2 is controlled by multiple transcription factors binding to its proximal promoter.

5' Flanking Region↗
PubMed2006

Wave function analysis of MHC-peptide interactions.

We have carried out an analysis of the wave function data for three MHC-peptide complexes: HLA-DRbeta1*0101-HA, HLA-DRbeta1*0401-HA and HLA-DRbeta1*0401-Col. We used quantum chemistry computer programs to generate wave function coefficients for these complexes, from which we obtained both molecular and atomic orbital data for both pocket and peptide amino acids within each pocket region. From these discriminated data, interaction molecular orbitals (IMOs) were identified as those with large and similar atomic orbital coefficient contributions from both pocket and peptide amino acids. The present results correlate well with our previous research where only electrostatic moments were used to explore molecular component interactions. Furthermore, we show a quantum chemical methodology to produce more fine-grained results concerning amino acid behavior in the MHC-peptide interaction.

Amino Acids↗
PubMed2001

Functional analysis of 14 genes that constitute the purine catabolic pathway in Bacillus subtilis and evidence for a novel regulon controlled by the PucR transcription activator.

The soil bacterium Bacillus subtilis has developed a highly controlled system for the utilization of a diverse array of low-molecular-weight compounds as a nitrogen source when the preferred nitrogen sources, e.g., glutamate plus ammonia, are exhausted. We have identified such a system for the utilization of purines as nitrogen source in B. subtilis. Based on growth studies of strains with knockout mutations in genes, complemented with enzyme analysis, we could ascribe functions to 14 genes encoding enzymes or proteins of the purine degradation pathway. A functional xanthine dehydrogenase requires expression of five genes (pucA, pucB, pucC, pucD, and pucE). Uricase activity is encoded by the pucL and pucM genes, and a uric acid transport system is encoded by pucJ and pucK. Allantoinase is encoded by the pucH gene, and allantoin permease is encoded by the pucI gene. Allantoate amidohydrolase is encoded by pucF. In a pucR mutant, the level of expression was low for all genes tested, indicating that PucR is a positive regulator of puc gene expression. All 14 genes except pucI are located in a gene cluster at 284 to 285 degrees on the chromosome and are contained in six transcription units, which are expressed when cells are grown with glutamate as the nitrogen source (limiting conditions), but not when grown on glutamate plus ammonia (excess conditions). Our data suggest that the 14 genes and the gde gene, encoding guanine deaminase, constitute a regulon controlled by the pucR gene product. Allantoic acid, allantoin, and uric acid were all found to function as effector molecules for PucR-dependent regulation of puc gene expression. When cells were grown in the presence of glutamate plus allantoin, a 3- to 10-fold increase in expression was seen for most of the genes. However, expression of the pucABCDE unit was decreased 16-fold, while expression of pucR was decreased 4-fold in the presence of allantoin. We have identified genes of the purine degradation pathway in B. subtilis and showed that their expression is subject to both general nitrogen catabolite control and pathway-specific control.

Bacillus subtilis↗
PubMed2006

Structural and functional analysis of Rv3214 from Mycobacterium tuberculosis, a protein with conflicting functional annotations, leads to its characterization as a phosphatase.

The availability of complete genome sequences has highlighted the problems of functional annotation of the many gene products that have only limited sequence similarity with proteins of known function. The predicted protein encoded by open reading frame Rv3214 from the Mycobacterium tuberculosis H37Rv genome was originally annotated as EntD through sequence similarity with the Escherichia coli EntD, a 4'-phosphopantetheinyl transferase implicated in siderophore biosynthesis. An alternative annotation, based on slightly higher sequence identity, grouped Rv3214 with proteins of the cofactor-dependent phosphoglycerate mutase (dPGM) family. The crystal structure of this protein has been solved by single-wavelength anomalous dispersion methods and refined at 2.07-Angstroms resolution (R = 0.229; R(free) = 0.245). The protein is dimeric, with a monomer fold corresponding to the classical dPGM alpha/beta structure, albeit with some variations. Closer comparisons of structure and sequence indicate that it most closely corresponds with a broad-spectrum phosphatase subfamily within the dPGM superfamily. This functional annotation has been confirmed by biochemical assays which show negligible mutase activity but acid phosphatase activity with a pH optimum of 5.4 and suggests that Rv3214 may be important for mycobacterial phosphate metabolism in vivo. Despite its weak sequence similarity with the 4'-phosphopantetheinyl transferases (EntD homologues), there is little evidence to support this function.

Amino Acid Sequence↗
PubMed1986

Striato-nigral dynorphin and substance P pathways in the rat. II. Functional analysis.

In the present study the functional role of the striato-nigral dynorphin and substance P pathways in rat brain has been studied using the rotational behavioural model and an intracerebral dialysis technique complemented with brain lesions and immunohistochemical analysis. Attempts were made to evaluate whether these striato-nigral neurons have a feed-back modulatory action on the dopaminergic nigro-striatal system, or whether they represent an outflow pathway conveying motor information from the striatum. Unilateral injection of dynorphin A into the substantia nigra reticulata of naive rats induced contralateral rotational behaviour. This effect was dose-dependent and mimicked by the kappa-opioid receptor agonist, U50,488H. Intranigral injection of substance P, as well as substance K, also produced dose-dependent contralateral rotational behaviour. Unilateral injections of ibotenic acid into various sites of the striatum were used to destroy the striato-nigral pathways. The lesions produced a depletion of dynorphin- and substance P-like immunoreactivity in the pars reticulata of the substantia nigra ipsilateral to the lesion and markedly affected the behavioural responses to intranigral peptide injections. Dynorphin A more potently induced contralateral rotation in the lesioned compared to naive non-lesioned rats, suggesting development of supersensitivity for this peptide. Substance P on the other hand, was markedly less potent in inducing rotation in lesioned animals. The rotational responses to both dynorphin A and substance P were potentiated by injection of amphetamine 1 h later, suggesting that both peptides act via nigro-striatal dopamine neurons. However, in rats with unilateral nigro-striatal dopamine denervation, produced with 6-hydroxy-dopamine, dynorphin A retained its potency to induce rotational behaviour; substance P was again much less potent. Thus, both the ibotenic acid and 6-hydroxy-dopamine lesions differently affect the action of dynorphin A and substance P in the zona reticulata of the substantia nigra. The data suggests that substance P requires an intact dopamine pathway to produce the rotational response, while dynorphin A does not. Direct evidence that behavioural activation produced by dynorphin A is not dependent upon dopamine stimulation was obtained by intrastriatal dialysis experiments in which changes in striatal dopamine release were measured following intranigral injection of dynorphin A or substance P. Intranigral dynorphin A in fact reduced, while substance P increased the release of dopamine.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗