Effect of medium-chain triglycerides on calbindin-D9k expression in the intestine.

These studies determined the effect of the saturated fat source in infant formula on the expression of calbindin-D9k (CaBP-9k). Piglets were fed from birth to 8 d with milk or formula containing saturated fatty acids as medium-chain triglycerides (MCT), coconut oil, palm oil (Palm 1), or synthesized triglycerides with 16:0 directed to the sn-2 position (Palm 2). Levels of intestinal CaBP-9k mRNA were significantly (P < 0.01) higher in piglets fed formula with MCT than in piglets fed the other formula or milk; and higher in piglets fed the Palm-1 than in piglets fed Palm-2 formula. This is the first evidence that MCT alter piglet intestinal CaBP-9k mRNA.

The baboon expresses the calbindin-D9k gene in intestine but not in uterus and placenta: implication for conservation of the gene in primates.

Expression of the Calbindin-D9k (CaBP-9k) gene was studied in the baboon. Northern blot analysis using a human CaBP-9k cDNA probe detected expression in duodenum but not in uterus and placenta. Reverse transcription/polymerase chain reaction (RT/PCR) confirmed this expression pattern and indicated a high degree of identity between the baboon and human CaBP-9k mRNAs. PCR was employed to amplify the intron A region of the baboon CaBP-9k gene using human-derived primers and baboon genomic DNA. The baboon intron was closely related to the human CaBP-9k intron A, including the presence a complete Alu-repetitive element. Most significantly, a 13 nucleotide long element at the 5' end of the baboon intron matched exactly the human sequence. This element represents a nonfunctional variation of an estrogen response element found at the same location in the rat CaBP-9k gene. The rat element functions as an enhancer and mediates uterine and possibly placental CaBP-9k expression in the rat and probably most other mammals. The finding of a modified ERE in baboon as in human suggests that during primate evolution the expression of this mammalian-specific gene has been eliminated in uterus and placenta. This scenario raises the question of the role of CaBP-9k in these reproductive tissues.

The human gonadotropin-releasing hormone receptor gene: complete structure including multiple promoters, transcription initiation sites, and polyadenylation signals.

The interaction of gonadotropin-releasing hormone and its receptor is a critical event in the endocrine regulation of reproduction. We have recently cloned the gene encoding for the human gonadotropin-releasing hormone receptor (hGnRHR). Partial sequence analysis revealed a structural organization consisting of three exons and two introns. Exon II contains only 219 bp and the remainder of the approximately 5 kb transcript is distributed between exons I and III. The complete coding region for the hGnRHR represented only 987 bp leaving an extensive 5' and 3' non-translated region and potentially additional exons unaccounted for. This report provides the complete sequence of exon I and III and demonstrates that further exons are unlikely to be contained within this gene. Sequencing of the 5' end of the gene revealed the presence of five consensus TATA sequences distributed within a 700 nucleotide region. Primer extension analysis detected multiple transcription initiation sites associated with this cluster of TATA sequences. Transcription of this region up to the most 5' initiation site was demonstrated by the reverse transcription-polymerase chain reaction (RT-PCR) method. The 5' non-translated region stretches between 703 and 1393 bp, depending on which initiation site is used. Several consensus cis-acting regulatory sequences were identified within the 5' end. These include, among others, sites for PEA-3, AP-1, and Pit-1. In addition, cAMP response element (CRE)-like and glucocorticoid/progesterone response element (GRE/PRE)-like sequences were found.(ABSTRACT TRUNCATED AT 250 WORDS)

Porcine calbindin-D9k gene: expression in endometrium, myometrium, and placenta in the absence of a functional estrogen response element in intron A.

The expression of Calbindin-D9k (CaBP-9k) in the pig uterus and placenta was measured by Northern blot analysis and reverse transcription polymerase chain reaction (PCR), respectively. Progesterone (P4) administration to ovariectomized pigs decreased CaBP-9k mRNA levels. Expression of endometrial CaBP-9k mRNA was high on pregnancy Days 10-12 and below the detection limit on Days 15 and 18. On Day 60, expression could be detected at low levels. In myometrium and placenta, CaBP-9k mRNA expression was not detectable by Northern analysis using total RNA. Reverse-transcribed RNA from both tissues demonstrated the presence of CaBP-9k transcripts by means of PCR. The partial CaBP-9k gene was amplified by PCR and cloned to determine the sequence of intron A. In contrast to the rat CaBP-9k gene, the pig gene does not contain a functional estrogen response element (ERE) within this region. A similar ERE-like sequence located at the identical location was examined by gel retardation analysis and failed to bind the estradiol receptor. A similar disruption of this ERE-like sequence has been described in the human CaBP-9k gene, which is not expressed at any level in placenta, myometrium, or endometrium. It is concluded that the pig CaBP-9k gene is regulated in these reproductive tissues in a manner distinct from that in rat and human tissues. The regulation is probably due to a regulatory region outside of intron A, which in the rat gene contains the key cis element for uterine expression of the CaBP-9k gene.

The human gonadotropin-releasing hormone (GnRH) receptor gene: cloning, genomic organization and chromosomal assignment.

The cDNA encoding the gonadotropin-releasing hormone (GnRH) receptor has recently been cloned and characterized in several species, including human. To determine the structure of the gene encoding the human GnRH receptor, we have screened a human genomic library and isolated seven positive clones, using cDNA probes derived from a human pituitary cDNA library. The isolated genomic clone contains the entire protein coding region of the GnRH receptor which is distributed between three exons and spans over 18.9 kb. Sequence analysis and restriction endonuclease mapping revealed the presence of two introns of 4.2 and 5.0 kb, respectively, both located within the open reading frame, designating the human GnRH receptor gene to the intron-containing class of the G-protein coupled receptor superfamily. Genomic Southern blot analysis indicated the presence of a single copy of the gene encoding for the GnRH receptor within the human genome. Using DNA from human-hamster somatic hybrid cell lines, the GnRH receptor gene was assigned to human chromosome 4, by means of PCR. The present study represents the first report on the GnRH receptor gene and its partial characterization should facilitate further investigation of the mechanisms by which expression of this gene is regulated.

Expression of calbindin-D9k in the early pregnant rat uterus: effects of RU 486 and correlation to estrogen receptor mRNA.

Calbindin-D9k (CaBP-9k) is a calcium binding protein expressed at high levels in the rat uterus. The CaBP-9k gene carries an estrogen response element which is involved in the steroid hormone regulation of the gene during the estrous cycle and gestation. The present study was aimed at determining expression of the gene during the first half of pregnancy and to assess the role of progesterone (P4) and the estrogen receptor (ER). Expression of CaBP-9k mRNA was determined by Northern blot analysis during the first 10 days of pregnancy. On pregnancy day 1 (P1), CaBP-9k mRNA levels were relatively high. On P2, 3 and 5 CaBP-9k mRNA decreased to the detection limit using 10 micrograms total RNA probed with a random primed cDNA. On P10, CaBP-9k transcripts began to reappear at levels of about 30% of P1. Expression of beta-actin mRNA displayed a continuous increase during this period with a rapid rise of 240% between P2 and P3. The typical increase of P4 accompanied by moderate changes of estradiol (E2) was determined in serum of experimental groups. When RU 486 at 10 mg/kg was administered as a single s.c. injection on P3, the CaBP-9k down-regulation was rapidly interrupted and mRNA expression became extremely high. The effect was seen maximally at 24 h post injection and was maintained at 48 and 72 h. Expression of beta-actin mRNA was increased only moderately at 24 h and was unchanged at 48 and 72 h. Serum P4 remained unaffected by the treatment and E2 displayed a slight increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadotropin-releasing hormone (GnRH) and cyclic AMP positively regulate inhibin subunit messenger RNA levels in human placental cells.

Bioactive and immunodetectable levels of both inhibin and activin are present in the placenta, raising questions as to the regulatory control of their synthesis. This study was designed to determine the effect of cyclic AMP (cAMP) and gonadotropin-releasing hormone (GnRH) on inhibin subunit gene expression in short-term incubations of placental cells. A semi-quantitative polymerase chain reaction (PCR) technique was used after isolation of total RNA and first strand cDNA synthesis from mechanically dispersed trophoblast-enriched cells obtained from human placentae at term. The level of gene expression of inhibin subunits was higher for beta A and alpha-subunits mRNA compared to the beta B-subunit mRNA as determined by PCR in combination with Southern blotting or Northern hybridization. Steady-state levels of beta-actin mRNA did not change throughout the 6-h incubation period and was used as a control of PCR amplification of the respective inhibin subunit gene transcripts following treatments with 8-bromo cAMP or GnRH. 8-bromo cAMP dose-dependently increased all three inhibin subunit gene transcripts with maximal responses seen at 150 microM (alpha-subunit mRNA 2.3-fold, beta A-subunit mRNA 1.8-fold and beta B-subunit mRNA 2.8-fold over control). GnRH (100 nM) significantly increased inhibin alpha and beta B-subunit mRNA levels 2.9-fold and 2.0-fold, respectively (P < 0.01), but not beta A-subunit mRNA. Collectively, the present findings demonstrate that in human term placental cells, gene expression of all inhibin subunits is under the direct influence of cAMP and further support a modulatory role of local GnRH in placental trophoblasts during late pregnancy.

The human calbindin-D9k gene. Complete structure and implications on steroid hormone regulation.

The gene encoding the human calbindin-D9k has been cloned and the complete sequence established. The gene spans about 5.5 kilobases and is localized on the X-chromosome, consists of three exons and carries four Alu repeats. The promoter and 1300 base-pairs of 5' flanking region have been characterized. Besides a TATA box and two CAAT-like motifs a sequence related to a vitamin D response element was detected about 1.1 kilobases upstream from the promoter. A sequence positioned 50 nucleotides downstream from the promoter showed extensive homology to the estrogen response element at the same location within the rat calbindin-D9k gene. Two essential nucleotides within this region are changed when the rat and human sequences are compared. The human element failed to bind the estrogen receptor as determined by gel retardation assay. It is proposed that a two-nucleotide change within this region causes the gene to lack expression in human uterus and possibly placenta.

Calbindin-D9k gene expression during the perinatal period in the rat: correlation to estrogen receptor expression in uterus.

Calbindin-D9k (CaBP-9k) is a cytosolic calcium binding protein mainly expressed in duodenum, placenta and uterus. The gene encoding the rat CaBP-9k is subject to tissue specific induction by 1,25 dihydroxyvitamin D3 (intestine) and estradiol (E2) (uterus). Control of placental expression remains unknown. The expression of CaBP-9k mRNA during the perinatal period was studied (pregnancy day 21 (P21)-lactation day 4 (L4)). In uterus, maximal expression levels were found at P21 and maintained until L1. With the transition to L2, the CaBP-9k mRNA concentration dropped drastically below the detection limit as quantitated by Northern blot analysis. Measurements of E2 and progesterone (P) levels showed a gradual decrease at late pregnancy (P21; birth). Post partum E2 levels continued to decline and P concentrations increased slightly. Uterine estrogen receptor (ER) mRNA levels determined by cDNA/PCR analysis revealed close correlation between expression of ER and CaBP-9k mRNAs. ER mRNA levels were maximal at P22 and declined at parturition and with onset of lactation. At L2 and L3 ER mRNA levels were minimal and had decreased 5-fold compared to late pregnancy. CaBP-9k protein concentrations fluctuated only slightly dependent on the stage of the estrous cycle: estrus > proestrus > diestrus. During the perinatal period CaBP-9k concentration was overall lower than in non-pregnant uterus and revealed only a moderate increase at birth and decrease in early lactation. Similar to the uterine levels, placental CaBP-9k mRNA was highest at P21 and remained high until birth. Fetal duodenal CaBP-9k rose sharply just prior to birth and plateaued in the early postpartal period.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning of the porcine Calbindin-D9k complementary deoxyribonucleic acid by anchored polymerase chain reaction technique.

The Calbindin-D9k (CaBP-9k) is a cytosolic calcium binding protein expressed in the mammalian intestine, placenta, and uterus. The protein is probably involved in calcium transport across the intestinal and placental epithelia. In uterus, a function in controlling myometrial activity involving intracellular calcium has been postulated. The amino acid sequence of the porcine CaBP-9k has been determined from intestine. The cDNAs for the bovine, murine, and rat CaBP-9k have been cloned. The objective of this study was the cloning of the porcine cDNA encoding the CaBP-9k. We performed the anchored polymerase chain reaction (PCR) technique using rat and bovine cDNA sequence-derived primers for amplification of intestinal cDNA. Both 5' and 3' amplification products were cloned and sequenced. The sequences revealed the full-length cDNA encoding the porcine CaBP-9k, coding region for 79 amino acids, 57 nucleotides 5' and 149 nucleotides 3' noncoding region. The inferred amino acid sequence is identical to the published amino acid sequence, except for one residue. The porcine CaBP-9k cDNA is 82.8% and 69.1% homologous with the bovine and rat sequences, respectively. Both bovine and porcine cDNAs contain a stretch of approximately 50 nucleotides not found in the rat sequence. Northern analysis showed a 600 nucleotide transcript in intestine, kidney, and uterus.

Calbindin-D9k gene expression during pregnancy and lactation in the rat.

Calbindin-D9k (CaBP-9k) is a calcium binding protein expressed in mammalian intestine, uterus and placenta. It is believed to be involved in transepithelial calcium transport in intestine and placenta and regulation of cytosolic calcium concentration in uterus. CaBP-9k mRNA levels were measured by Northern blot analysis in maternal duodenum, uterus, placenta and fetal/neonatal duodenum during pregnancy and lactation. In maternal duodenum a maximal increase occurred at day 15 of lactation (2.3-fold) and 20 days post-lactation levels decrease to 30.3% of non-pregnant controls. In non-pregnant uterus a 10-fold variation of CaBP-9k mRNA levels was observed between individual animals despite a uniform expression of beta-actin. During pregnancy high CaBP-9k expression is found, averaging about 20% of duodenal levels, which abruptly drops below detection during early lactation. At late lactation CaBP-9k mRNA levels are again subject to great variation ranging from no expression to maximal levels found in the non-pregnant uterus. Placental CaBP-9k is maximally expressed at the end of pregnancy (day 20) reaching about 2.5% of duodenal levels. Fetal intestinal CaBP-9k mRNA was detectable in 20 micrograms total RNA at day 18 of pregnancy and rose sharply in early lactation reaching about 50% of adult duodenal levels at day 20 lactation. The profound changes of uterine CaBP-9k mRNA in non-pregnant (cycling), pregnant, and lactating rats indicate a rapid hormonal regulation of gene expression, most likely involving 17 beta-estradiol.

Calbindin-D9k mRNA is tightly regulated during the estrous cycle in the rat uterus.

Calbindin-D9k (CaBP-9k) is a cytosolic calcium binding protein with a molecular weight of 9000. CaBP-9k is mainly expressed in intestine, uterus and placenta, with intestinal levels controlled by vitamin D and uterine levels controlled by estrogens. CaBP-9k mRNA levels were measured in rat uterus throughout the estrous cycle. On the morning of proestrus, estrus and diestrus animals were sacrificed. Serum 17 beta-estradiol concentrations were determined using a radioimmunoassay. Whole uterus was used for preparation of total RNA. Northern blot analysis was performed to quantify CaBP-9k and beta-actin mRNA. CaBP-9k levels were highest at proestrus, dropped 10-fold at estrus and were not detectable at diestrus. beta-Actin levels did not change significantly throughout the estrous cycle. Peak 17 beta-estradiol concentrations coincided with maximum CaBP-9k mRNA expression at proestrus. Despite minimal concentrations of 17 beta-estradiol at estrus, CaBP-9k mRNA was still present at 10% of the proestrus level. At diestrus, CaBP-9k mRNA was not detectable despite increasing 17 beta-estradiol. It is concluded that CaBP-9k is subject to 17 beta-estradiol regulation during the estrous cycle. Correlation between CaBP-9k mRNA and 17 beta-estradiol levels indicates a lag period for CaBP-9k induction in diestrus following a rise in steroid hormone levels.

Molecular cloning of the full-length cDNA encoding the human calbindin-D9k.

The full-length cDNA encoding the human calbindin-D9k (CaBP-9k) has been cloned using reverse transcription/PCR methodology with rat- and bovine-derived primers and intestinal RNA. A core product, and both a 5' and 3' product encompassing the full-length cDNA were obtained. The clones include coding region for 79 amino acids, 57 nucleotides 5'-and 159 nucleotides 3'-non-coding region, and a poly(A) tail. The deduced protein sequence is homologous to other mammalian CaBPs. Northern analysis revealed the mRNA in human duodenum to be about 600 nucleotides in length. Expression levels in adult human tissue were substantially lower than in child, rat or porcine intestine.

1,25-Dihydroxyvitamin D and not calcium is the major regulator of calbindin-D 9-kDa mRNA levels in vivo.

A possible role of calcium in vivo on intestinal calbindin-D 9-kDa mRNA levels has been studied in rats. In vitamin D-deficient rats, a marked increase in dietary calcium has a small but significant effect on calbindin-D 9-kDa mRNA levels, despite a dramatic increase in serum calcium concentration that clearly resulted from increased intestinal absorption of calcium. On the other hand, vitamin D under all circumstances increased calbindin-D 9-kDa mRNA levels, with the greatest levels found in animals on a low calcium diet where little or no calcium is available for absorption. These results strongly support the idea that 1,25-dihydroxyvitamin D is directly responsible for the induction of calbindin-D 9-kDa.

Isolation of a mRNA that encodes a putative intestinal alkaline phosphatase regulated by 1,25-dihydroxyvitamin D-3.

Using differential hybridization techniques to screen a rat intestinal cDNA library we isolated a cDNA whose predicted amino acid sequence exhibits a high degree of homology to the alkaline phosphatases. The predicted cDNA sequence has 79% identity at the amino acid level to the rat intestinal alkaline phosphatase, and shows approx. 70% homology to other human and rat alkaline phosphatases. The corresponding mRNA is markedly increased by 6 h after a single dose of 1,25-dihydroxyvitamin D-3. The mRNA is also increased by 24-homologated analogs of 1,25-dihydroxyvitamin D-3 that do not increase calcium transport.

Induction of calbindin-D 9k mRNA but not calcium transport in rat intestine by 1,25-dihydroxyvitamin D3 24-homologs.

The function and precise mechanism of regulation of calbindin-D 9k in intestine is largely unknown. It is suggested that this calcium binding protein is involved in active intestinal calcium transport and that its expression is mainly mediated by 1,25-dihydroxyvitamin D3. We examined the effect of two side chain modified analogs of 1,25-dihydroxyvitamin D3 as compared to 1,25-dihydroxyvitamin D3 itself on the regulation of the calbindin-D 9k at the mRNA level and on intestinal calcium transport in the rat. delta 22-24,24-dihomo-1,25-dihydroxyvitamin D3 at a single dose of 500, 1,000, and 2,000 pmol caused greater than 7.0-fold increase in calbindin-D 9k mRNA without stimulating intestinal calcium transport. A 10,000-pmol dose of delta 22-24,24,24-trihomo-1,25-dihydroxyvitamin D3 caused a 7.6-fold increase in calbindin-D 9k mRNA without significantly increasing intestinal absorption of calcium. In contrast, 1,25-dihydroxyvitamin D3 caused a parallel increase in calbindin-D 9k mRNA and intestinal absorption of calcium. Thus, calbindin 9k is not by itself responsible for 1,25-dihydroxyvitamin D3-mediated increase in intestinal absorption of calcium.

An estrogen-responsive element mediates the transcriptional regulation of calbindin D-9K gene in rat uterus.

The rat calbindin (molecular mass 9 kDa) gene sequence revealed the presence of an imperfect palindromic estrogen-responsive-like element (ERE) located at position +51 from the transcriptional initiation site. This element has the sequence AGGTCAGGGTGATCT which differs by one nucleotide from the vitellogenin ERE palindromic sequence AGGTCACTGTGACCT. The activity of this element to induce transcription in response to estrogen and the ability to bind estrogen receptor was investigated. This sequence confers estrogen-dependent transcriptional activity when cloned upstream from the tkp-CAT construct in pBLCAT2 plasmid and transfected into T47D cells, similar to the vitellogenin gene ERE activity but to a lesser extent. This element binds to the estrogen receptor in vitro as assessed by gel retardation assay similar to the vitellogenin gene ERE. No such binding was detected when a mutant sequence AGATCACTGTGATCT was used. The specificity of the complex was confirmed using polyclonal antibodies, ER712 raised against the estrogen receptor. Furthermore, competition assays showed that both sequences were able to compete for binding to the estrogen receptor. The in vivo transcriptional regulation of the calbindin D-9K gene by estrogen was also investigated in the rat. Female animals maintained on a vitamin D-sufficient or a vitamin D-deficient diet were ovariectomized, housed for 3 weeks, and then injected with 17 beta-estradiol (0.5 micrograms/kg body weight/day). Slot blot analysis of total RNA showed a marked increased in calbindin D-9K mRNA levels in the uterus but not in the intestine by 26 and 50 h post-injection. These results demonstrate that the calbindin D-9K gene is transcriptionally regulated by estrogen in the uterus mediated by an estrogen-responsive element identified in the gene.

The vitamin D system: 1990.

Many new aspects of vitamin D have made their appearance. New roles for vitamin D in reproduction, parathyroid function, skin proliferation, and differentiation, in myeloid cell differentiation, and as anti-cancer agents will be new areas of investigation. At the basic science level, much effort will be expended to understand how the vitamin D hormone and its receptor bring about gene expression and how these expressed genes bring about cellular functions of the vitamin D system. Similarly, future work will center on the molecular mechanism of regulation of the hydroxylases of the vitamin D endocrine system. All of this is based on the original discovery that vitamin D must be first metabolically altered before function, producing the vitamin D hormone 1 alpha,25-(OH)2D3 in the proximal convoluted tubule cells of the kidney. There is no doubt that the vitamin D area will continue to expand in the next decade as new functions of the vitamin D system are uncovered.

Methylation of the vitamin D-dependent CaBP gene (calbindin 9 Kd) does not mediate tissue or vitamin D regulation.

The vitamin D-dependent intestinal calcium binding protein gene is predominantly expressed in the intestine. In this report we have examined the possibility that methylation of the gene might play a role in its tissue-specific expression employing genomic Southern analysis. None of the Hpa II and Hha I sites examined by the indicated probes in and around the gene were found to be methylated in the intestine, kidney and liver. No change in the methylation of these sites was detected in response to 1,25-dihydroxy-vitamin D3 administration to vitamin D-deficient rats under conditions which stimulate the expression of the gene. These results indicate that the rat intestinal calcium binding protein gene is not methylated in these tissues, at the indicated sites and, therefore, methylation seems not to be involved in the regulation of this gene's expression.

[The effect of benzenecarboxylic acids--in particular mellitic acid--in in vitro biological system]. / Uber den Einfluss von Benzolcarbonsäuren--insbesondere Mellitsäure--auf biologische In-vitro-Systeme.

Influence of Benzenecarboxylic Acids, Particularly Mellitic Acid, on Biological in vitro Systems. In a first part of the present paper influences of mellitic acid and some other benzenecarboxylic acids on reduplication of Ehrlich ascites tumor cells cultured in vitro are reported. Mellitic acid in the concentration range between 1 and 5 mmol/l caused an increase, in concentrations of 10 mmol/l and above an inhibition of cell multiplication. Benzoic acid, phthalic acid, hemimellitic acid and pyromellitic acid showed in concentrations above 6 to 10 mmol/l inhibitory effects on cell multiplication. The second part reports studies on potential influences of mellitic acid on growth and mesenchymal metabolism of explanted murine fetal tibiae cultured in vitro for six days. Mellitic acid effected in concentrations between 0.5 and 15 mmol/l as compared with control cultures a concentration dependent inhibition of calcification and in concentrations above 2 mmol/l significant increases of the glycosaminoglycan content and growth of the explants, whereas DNA-, total protein- and hydroxyprolin content were not significantly influenced by concentrations up to 10 mmol/l.