scholarly journals Aldehyde dehydrogenase from adult human brain that dehydrogenates γ-aminobutyraldehyde: purification, characterization, cloning and distribution

1996 ◽  
Vol 316 (1) ◽  
pp. 317-324 ◽  
Author(s):  
Alexandra KIKONYOGO ◽  
Regina PIETRUSZKO
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Human Brain ◽  
Aldehyde Dehydrogenase ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Mrna Levels ◽  
Adult Human Brain ◽  
Adult Human

Enzyme purification and characterization, cDNA cloning and Northern blot analysis were the techniques utilized during this investigation to determine the identity and occurrence of the aldehyde dehydrogenase that metabolizes γ-aminobutyraldehyde in adult human brain. The purification yielded one major protein which was active with γ-aminobutyraldehyde. It had the physicochemical and kinetic properties of the human liver E3 isoenzyme of aldehyde dehydrogenase (EC 1.2.1.3), and also interacted with an anti-(liver E3 isoenzyme) antibody. Tryptic peptides derived from the purified brain protein matched the amino acid sequence of the liver E3 isoenzyme. Employing liver E3 cDNA, a human cerebellar cDNA library was screened and a 2.0 kb cDNA fragment was isolated. The cerebellar cDNA yielded a derived primary structure which differed from the liver E3 amino acid sequence by a single serine-to-cysteine substitution at position 88 (position 84 in the liver sequence). Thus the γ-aminobutyraldehyde-metabolizing enzyme from human brain can be identified as E3′, a variant of the E3 isoenzyme. The catalytic properties of the brain variant were indistinguishable from those of E3, and so the functional importance of this variant is at present unknown. The distribution of this enzyme in brain was investigated by Northern blot analysis, which demonstrated the presence of E3′ mRNA in all regions of the human brain. mRNA levels were variable in the different brain areas, with the highest levels in the spinal cord and the lowest in the occipital pole.

Gene expression analysis of the pro-oestrous-stage rat uterus reveals neuroligin 2 as a novel steroid-regulated gene

2004 ◽  
Vol 16 (8) ◽  
pp. 763 ◽  
Author(s):  
Han-Seung Kang ◽  
Chae-Kwan Lee ◽  
Ju-Ran Kim ◽  
Seong-Jin Yu ◽  
Sung-Goo Kang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Oestrous Cycle ◽  
Mrna Levels ◽  
Rat Uterus ◽  
Ovx Rats

In the present study, differential gene expression in the uteri of ovariectomised (OVX) and pro-oestrous rats (OVX v. pro-oestrus pair) was investigated using cDNA expression array analysis. Differential uterine gene expression in OVX rats and progesterone (P4)-injected OVX rats (OVX v. OVX + P4 pair) was also examined. The uterine gene expression profiles of these two sets of animals were also compared for the effects of P4 treatment. RNA samples were extracted from uterine tissues and reverse transcribed in the presence of [α32P]-dATP. Membrane sets of rat arrays were hybridised with cDNA probe sets. Northern blot analysis was used to validate the relative gene expression patterns obtained from the cDNA array. Of the 1176 cDNAs examined, 23 genes showed significant (>two-fold) changes in expression in the OVX v. pro-oestrus pair. Twenty of these genes were upregulated during pro-oestrus compared with their expression in the OVX rat uterus. In the OVX v. OVX + P4 pair, 22 genes showed significant (>two-fold) changes in gene expression. Twenty of these genes were upregulated in the OVX + P4 animals. The genes for nuclear factor I–XI, afadin, neuroligin 2, semaphorin Z, calpain 4, cyclase-associated protein homologue, thymosin β-4X and p8 were significantly upregulated in the uteri of the pro-oestrus and OVX + P4 rats of both experimental pairs compared with the OVX rat uteri. These genes appear to be under the control of P4. One of the most interesting findings of the present study is the unexpected and marked expression of the neuroligin 2 gene in the rat uterus. This gene is expressed at high levels in the central nervous system and acts as a nerve cell adhesion factor. According to Northern blot analysis, neuroligin 2 gene expression was higher during the pro-oestrus and metoestrus stages than during the oestrus and dioestrus stages of the oestrous cycle. In addition, neuroligin 2 mRNA levels were increased by both 17β-oestradiol (E2) and P4, although P4 administration upregulated gene expression to a greater extent than injection of E2. These results indicate that neuroligin 2 gene expression in the rat uterus is under the control of both E2 and P4, which are secreted periodically during the oestrous cycle.

Sustained changes in lung expansion alter tropoelastin mRNA levels and elastin content in fetal sheep lungs

2003 ◽  
Vol 284 (4) ◽  
pp. L643-L649 ◽  
Author(s):  
Belinda J. Joyce ◽  
Megan J. Wallace ◽  
Richard A. Pierce ◽  
Richard Harding ◽  
Stuart B. Hooper
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Fetal Lung ◽  
Elastic Fibers ◽  
Mrna Levels ◽  
Fetal Sheep ◽  
Time Points ◽  
Tracheal Obstruction ◽  
Lung Expansion

Our objective was to determine the effects of sustained alterations in fetal lung expansion on pulmonary elastin synthesis. In fetal sheep, lung expansion was either decreased between 111 and 131 days' gestation (term ∼147 days) by tracheal drainage or increased for 2, 4, 7, or 10 days by tracheal obstruction, ending at 128 days' gestation. Lung tropoelastin mRNA levels were assessed by Northern blot analysis, total elastin content was measured biochemically, and staining of lung sections was used to assess the localization and form of elastic fibers. Tracheal obstruction significantly elevated pulmonary tropoelastin mRNA levels 2.5-fold at 2 days, but values were not different from controls at 4, 7, and 10 days; elastin content tended to be increased at all time points. A sustained decrease in lung expansion by tracheal drainage reduced pulmonary tropoelastin mRNA levels 2.5-fold; elastin content was also decreased compared with controls, and tissue localization was altered. Our results indicate that the degree of lung expansion in the fetus influences elastin synthesis, content, and tissue deposition.

Prognostic Significance of the Combined Expression of Matrix Metalloproteinase-9, Urokinase Type Plasminogen Activator and its Receptor in Breast Cancer as Measured by Northern Blot Analysis

2001 ◽  
Vol 16 (1) ◽  
pp. 62-68 ◽  
Author(s):  
M.M. Pacheco ◽  
I.N. Nishimoto ◽  
M. Mourão Neto ◽  
E.B. Mantovani ◽  
M.M. Brentani
Keyword(s):  
Breast Cancer ◽  
Overall Survival ◽  
Plasminogen Activator ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Prognostic Significance ◽  
Mrna Levels ◽  
Upa Mrna ◽  
The Impact

Using Northern blot analysis we have measured the co-expression of the matrix metalloprotease MMP-9, plasminogen activator urokinase type (uPA) and its receptor (uPAR) mRNAs in 81 biopsies of breast carcinomas with the objective of analyzing the impact of these factors on the overall survival probability of the patients (median follow-up time: 4 years). Individual mRNA levels of either uPA or uPAR showed parallel variations with MMP-9 mRNA, suggesting a coordinate transcription of these markers. When median values were used as cutoff points to discriminate between high and low factor expression, no association was found with patient outcome and MMP-9 or uPA mRNA distribution. However, increased uPAR mRNA levels were associated with poor prognosis (p = 0.01). The combination of MMP-9 and uPAR mRNA measurements has not enhanced prognostic information compared to information supplied by the receptor alone (p = 0.01). The combination of MMP-9 and high levels of uPA mRNA led to a significant association with poor outcome (p = 0.03). Multivariate analysis supported the notion that increased uPAR mRNA production in primary breast cancer may be a predictor of overall survival.

scholarly journals Three mRNA transcripts of the proopiomelanocortin gene in human placenta at term

2000 ◽  
pp. 533-536 ◽  
Author(s):  
SI Grigorakis ◽  
E Anastasiou ◽  
K Dai ◽  
A Souvatzoglou ◽  
M Alevizaki
Keyword(s):  
Pregnant Women ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Mode Of Delivery ◽  
Mrna Levels ◽  
Functional Protein ◽  
Mrna Transcripts ◽  
Gene Transcripts ◽  
Pomc Gene

The proopiomelanocortin (POMC) gene whose normal pituitary specific mRNA product is 1200 bases (b) is also expressed in placenta and its peptide derivatives such as ACTH and beta-endorphin may play an important role in the initiation of labor. So far, two mRNA transcripts, one small (800b) and one large (1380b) have been reported in placenta by Northern blot analysis, similar to other endocrine tissues and various extrapituitary tumors; however, it is questionable whether both of these transcripts are effectively translated to a functional protein. We examined by Northern blot analysis the size and the differential expression of placental POMC gene transcripts in pregnant women with different modes of delivery. Placental tissues were collected from two groups of pregnant women, six with vaginal delivery (VD) and five with cesarean section (CS). In both groups of placentae three POMC gene transcripts were detected of 800, 1200 and 1380 bases; the 1200b pituitary specific species often predominated and was always present. The 800b transcript was also always present, while the large transcript (1380b) was expressed in 3/6 VD and 2/5 CS placental tissues. No differences in the relative levels of any of these mRNA species showing effect of the mode of delivery were observed. We conclude that POMC gene transcription in placental tissue at term gives rise to three mRNA transcripts, thus resembling extrapituitary tumors. The reported changes in the levels of the derivative peptides according to the mode of delivery do not reflect changes in POMC mRNA levels and could be attributed to a post-translational effect.

scholarly journals Acetylcholine receptor alpha-subunit mRNA is increased by ascorbic acid in cloned L5 muscle cells: Northern blot analysis and in situ hybridization.

1989 ◽  
Vol 108 (5) ◽  
pp. 1823-1832 ◽  
Author(s):  
O Horovitz ◽  
D Knaack ◽  
T R Podleski ◽  
M M Salpeter
Keyword(s):  
Ascorbic Acid ◽  
In Situ Hybridization ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Alpha Subunit ◽  
Mrna Levels ◽  
Surface Receptors ◽  
Subunit Mrna

Ascorbic acid is the major factor in brain extract responsible for increasing the average acetylcholine receptor (AChR) site density on the cloned muscle cell line L5. In the present study, we show that this effect of ascorbic acid requires mRNA synthesis, and that the mRNA level for the AChR alpha-subunit is increased to about the same level as are the surface receptors. We have found no increase in the mRNA levels of the beta-, gamma-, and delta-subunits, or in the mRNAs of other muscle-specific proteins, such as that of light chain myosin 2, alpha-actin, and creatine kinase. By in situ hybridization, we further show that the increase in alpha-mRNA in response to ascorbic acid is exclusively in myotubes and is located near clusters of nuclei. mRNA levels for the alpha-subunit in mononucleated cells are very low and do not significantly increase in response to ascorbic acid. The mononucleated cells are thus excluded as a possible source for the increase in alpha-subunit mRNA detected by Northern blot analysis. Our results indicate that there is a very specific action of ascorbic acid on the regulation of AChR alpha-mRNA in the L5 muscle cells, and that the expression of surface receptors in these cells is limited by the amount of AChR alpha-subunit mRNA.

Distribution and developmental pattern of neuromedin U expression in the rat gastrointestinal tract

1994 ◽  
Vol 12 (3) ◽  
pp. 257-263 ◽  
Author(s):  
C Austin ◽  
M Oka ◽  
K A Nandha ◽  
S Legon ◽  
N Khandan-Nia ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Postnatal Development ◽  
Food Deprivation ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Developmental Pattern ◽  
Mrna Levels ◽  
First Time

ABSTRACT This study has quantified, for the first time, the relative levels of neuromedin U (NmU) mRNA in the rat gastrointestinal tract using Northern blot analysis. NmU message was detected in all regions of the gastrointestinal tract from the oesophagus to the rectum. The greatest levels were found in the duodenum and jejunum, the principal sites for absorption, which were 2·5- and 3-fold respectively above ileal levels. Quantification of NmU mRNA and peptide contents in the duodenum, jejunum and ileum during postnatal development of the rat showed message and peptide levels to be greater in the maturing rat than in neonates. Message levels in the duodenum, jejunum and ileum showed 14-, 7- and 4-fold increases respectively between 1 and 56 days after birth, whilst the corresponding peptide levels in the duodenum, jejunum and ileum showed 33-, 14- and 25-fold increases respectively. Food deprivation caused a small, but significant, decrease in message levels in the jejunum and colon, but there was no change in the duodenum or ileum. This shows that the presence of food has little effect on NmU mRNA levels in the gut.

scholarly journals Towards Quantitative In Situ Hybridization

1997 ◽  
Vol 45 (3) ◽  
pp. 413-423 ◽  
Author(s):  
Ard Jonker ◽  
Piet A. J. de Boer ◽  
Maurice J. B. van den Hoff ◽  
Wouter H. Lamers ◽  
Antoon F. M. Moorman
Keyword(s):  
In Situ Hybridization ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Photographic Emulsion ◽  
Mrna Levels ◽  
Intestinal Tissue ◽  
Tissue Sections ◽  
Silver Grains

In situ hybridization analysis of tissue mRNA concentrations remains to be accepted as a quantitative technique, even though exposure of tissue sections to photographic emulsion is equivalent to Northern blot analysis. Because of the biological importance of in situ quantification of RNA sequences within a morphological context, we evaluated the quantitative aspects of this technique. In calibrated microscopic samples, autoradiographic signal (density of silver grains) was proportionate to the radioactivity present, to the exposure time, and to time of development of the photographic emulsion. Similar results were obtained with tissue sections, showing that all steps of the in situ hybridization protocol, before and including the detection of the signal, can be reproducibly performed. Furthermore, the integrated density of silver grains produced in liver and intestinal sections by the in situ hybridization procedure using 35S-labeled riboprobes is directly proportionate to the signal obtained by quantitative Northern blot analysis. The significance of this finding is that in situ quantification of RNA can be realized with high sensitivity and with the additional advantage of the possibility of localizing mRNA within the cells of interest. Application of this procedure on fetal and adult intestinal tissue showed that the carbamoylphosphate synthetase (CPS)-expressing epithelial cells of both tissues accumulated CPS mRNA to the same level but that whole-organ CPS mRNA levels decreased four- to fivefold in the same period, owing to a comparable decrease in the number of CPS-expressing cells in total intestinal tissue. (J Histochem Cytochem 45:413–423, 1997)

Sequence analysis of the turkey LH β subunit and its regulation by gonadotrophin-releasing hormone and prolactin in cultured pituitary cells

1995 ◽  
Vol 14 (1) ◽  
pp. 117-129 ◽  
Author(s):  
S You ◽  
L K Foster ◽  
J L Silsby ◽  
M E El Halawani ◽  
D N Foster
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Pituitary Cells ◽  
Β Subunit ◽  
Spontaneous Release ◽  
Gonadotrophin Releasing Hormone ◽  
Prl Gene

ABSTRACT cDNAs encoding the precursor molecule of the turkey LH β subunit (tLHβ) were cloned from a turkey pituitary cDNA library. The nucleotide sequence of the longest of two different tLHβ cDNA clones contained 592 bp, and included 23 bp of the 5′ untranslated region (UTR) and 92 bp of the 3′ UTR in addition to a 477 bp open reading frame that encoded a 39 amino acid leader polypeptide and a 120 amino acid mature apoprotein. Turkey and chicken LHβ sequences shared approximately 92 and 93% nucleotide and amino acid sequence similarities respectively. Northern blot analysis of total cellular anterior pituitary RNA showed that an approximate 800 base transcript hybridized to a 32P-labelled tLHβ cDNA probe. The gonadotrophin-releasing hormone (GnRH)-and prolactin (PRL)-regulated expression of LH and PRL in dispersed pituitary cells was determined by Northern blot analysis of tLHβ and PRL steady-state mRNA levels and by RIA analysis of secreted LH and PRL. GnRH-treated cells showed increased levels of both tLHβ mRNA and secreted LH, whereas mRNA and secreted levels of PRL did not change significantly. Cells treated with PRL showed lower levels of tLHβ and PRL mRNA as well as decreased release of LH and PRL. When cells were treated with both PRL and GnRH, increases in tLHβ mRNA and secreted levels of LH observed with GnRH alone were negated, whereas the decreases in mRNA and secreted levels of PRL observed with PRL alone were abrogated. These findings suggest that PRL can down-regulate tLHβ gene expression and spontaneous release of LH as well as autoregulate PRL gene expression and spontaneous release of PRL, while GnRH appears capable of modulating the effects of PRL-regulated LH and PRL gene expression and spontaneous release.

scholarly journals Evidence for the existence of a pristanoyl-CoA oxidase gene in man

1997 ◽  
Vol 325 (3) ◽  
pp. 593-599 ◽  
Author(s):  
Johannes C. T. VANHOOREN ◽  
Peter MARYNEN ◽  
Guy P. MANNAERTS ◽  
Paul P. VAN VELDHOVEN
Keyword(s):  
Amino Acid ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Developmental Stages ◽  
Genomic Library ◽  
Immunoblot Analysis ◽  
Human Tissues ◽  
Oxidase Gene ◽  
Activity Measurements

In the rat, 2-methyl branched fatty acids and the bile acid intermediates di- and tri-hydroxycoprostanic acids are desaturated by pristanoyl-CoA oxidase and trihydroxycoprostanoyl-CoA oxidase respectively. In the human, these compounds are oxidized by a single enzyme, branched-chain acyl-CoA oxidase, which according to its amino acid sequence is the human homologue of rat trihydroxycoprostanoyl-CoA oxidase. Pristanoyl-CoA oxidase is apparently absent from human tissues as indicated by immunoblot analysis [Van Veldhoven, Van Rompuy, Fransen, de Béthune and Mannaerts (1994) Eur. J. Biochem. 222, 795–801] and Northern-blot analysis [Vanhooren, Fransen, de Béthune, Baumgart, Baes, Torrekens, Van Leuven, Mannaerts and Van Veldhoven (1996) Eur. J. Biochem. 239, 302–309] of human tissues. In this paper we present evidence, however, that at least the gene for pristanoyl-CoA oxidase is present in the human. A human liver cDNA encoding a protein of 700 amino acids, showing 75% amino acid identity with rat pristanoyl-CoA oxidase and harbouring a peroxisomal C-terminal-targeting signal (SKL), was isolated. Bacterial expression of the cDNA resulted in a fusion protein that was cross-reactive with antibodies directed against rat pristanoyl-CoA oxidase and the C-terminal SKL sequence. Screening of a genomic library with the isolated cDNA as a probe resulted in a genomic clone in which four introns were localized. By means of fluorescence in situhybridization the gene for human pristanoyl-CoA oxidase was mapped at chromosome position 4p15.3. We conclude that a gene for pristanoyl-CoA oxidase is present in the human genome. The gene appears to be expressed to such a low extent in liver that its mRNA cannot be detected by routine Northern-blot analysis and that its product remains undetected by standard immunoblotting or by enzyme activity measurements. We speculate that the gene may be expressed under special (e.g. certain developmental stages) conditions or in certain specialized tissues not examined thus far.

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