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PubMed1993

Identification of connective tissue gene transcripts in freshly isolated parenchymal, endothelial, Kupffer and fat-storing cells by northern hybridization analysis.

The aim of the present study was to identify the cell types that express collagen alpha 1(I), alpha 1(III) and alpha 1(IV), fibronectin and laminin B1 genes in normal rat liver. Parenchymal, sinusoidal endothelial, Kupffer and fat-storing (Ito) cells were isolated and purified. Total RNA of the freshly isolated cells was subjected to Northern hybridization analysis. We also compared the steady state levels of specific mRNAs in freshly isolated fat-storing cells to the levels in myofibroblast-like cells obtained from purified fat-storing cells cultured for two passages. The average purity of each cell preparation, and the percentage of contaminating cells, were determined by transmission electron microscopy and by examining the presence of vitamin A-autofluorescent cells. Fibronectin and collagen alpha 1(III) mRNAs were detected in total RNA of purified parenchymal cells. In poly(A)+ enriched RNA, small amounts of collagen alpha 1(I) mRNA were also present. In total RNA of freshly isolated fat-storing cells, collagen alpha 1(III), alpha 1(IV), and laminin B1 transcripts were found, whereas collagen alpha 1(I) and fibronectin mRNAs were not detected. Cultured fat-storing cells, however, did contain high levels of collagen alpha 1(I) and fibronectin mRNAs. The molecular size of the latter transcript was larger than the fibronectin transcript found in parenchymal cells and the whole liver. Endothelial cells contained small amounts of alpha 1(IV) mRNA. Kupffer cells did not contain the investigated transcripts. We conclude that normal parenchymal, fat-storing and endothelial cells each express a typical pattern of connective tissue molecules. When fat-storing cells are allowed to differentiate into myofibroblast-like cells, they express high levels of collagen alpha 1(I) and fibronectin mRNAs, in addition to collagen alpha 1(III) and alpha 1(IV), and laminin B1 chain mRNAs.

Adipocytes↗
PubMed2006

Photoperiod affects gene expression of leptin and leptin receptors in adipose tissue from lactating dairy cows.

Leptin is mainly secreted by adipocytes and is implicated in the regulation of metabolic status, feed intake, and body condition. Day length (DL) can affect leptin gene expression and secretion. The aim of the study was to evaluate the effect of DL on gene expression of leptin and leptin receptors in adipose tissue (AT). Four lactating and pregnant Holstein cows were housed in a climate-controlled chamber for 51 d. The first 30 d were used to adapt animals to the new housing conditions. During that period the DL adopted was 12 h light:12 h dark (12:12). The experimental period included 3 different and consecutive phases: 7 d of neutral DL (12:12); 7 d of long DL (18 h light:6 h dark); and 7 d of short DL (6 h light:18 h dark). Subcutaneous AT biopsies were performed at the end of each phase. Prolactin, growth hormone, cortisol, leptin, glucose, nonesterified fatty acids, beta-OH-butyrate, and cholesterol were determined in plasma samples. Abundance of leptin mRNA, and Ob-Ra and Ob-Rb leptin receptor mRNA were determined in AT samples by ribonuclease protection assay. Day length did not affect feed intake or body condition score. Exposure to short DL significantly reduced milk yield (13.1 +/- 2.2 vs. 15.8 +/- 1.7 and 16.0 +/- 2.0 kg/d for short vs. neutral and long DL, respectively). Plasma leptin, growth hormone, cortisol, nonesterified fatty acids, beta-OH-butyrate, and glucose were not affected by DL; cholesterol was lowest under short DL (3.93 +/- 0.38 vs. 4.36 +/- 0.39 and 4.07 +/- 0.38 mmol/L for short vs. neutral and long DL, respectively). Prolactin increased under long DL (134.82 +/- 16.94 vs. 81.98 +/- 20.25 and 96.16 +/- 0.38 ng/mL for long vs. neutral and short DL, respectively). Gene expression of leptin and its receptors was affected by DL. Leptin mRNA increased under long DL (11.91 +/- 0.84 vs. 7.82 +/- 0.84 and 7.56 +/- 0.84 pg of mRNA/microg of total RNA for long vs. neutral and short DL, respectively). Leptin receptors Ob-Ra and Ob-Rb mRNA were higher under long DL, whereas Ob-Ra and Ob-Rb mRNA were lower under short DL (Ob-Ra: 1.91 +/- 0.41, 2.49 +/- 0.41, and 0.65 +/- 0.41 pg of mRNA/microg of total RNA for neutral, long, and short DL, respectively; Ob-Rb: 5.29 +/- 0.79, 5.98 +/- 0.68, and 2.02 +/- 0.70 pg of mRNA/microg of total RNA for neutral, long, and short DL, respectively). Results of the present study appear to exclude an effect of feed intake and metabolic status on leptin gene expression. A prolactin-mediated effect of photoperiod on AT leptin modulation may be proposed in lactating dairy cows.

Adipose Tissue↗
PubMed1993

Expression of the genes encoding bombesin-related peptides and their receptors in anterior pituitary tissue.

The bombesin-related peptides gastrin-releasing peptide (GRP) and neuromedin B (NMB) have been demonstrated in the anterior pituitary (AP) on an immunological basis. We studied the presence of mRNAs for these peptides and for their receptors by RNAse protection assay using fresh adult male rat AP, AP cell reaggregates cultured in the presence of estradiol and the rat AP derived GH3 cell line. In total RNA from fresh AP we detected high amounts of NMB mRNA and much smaller amounts of GRP mRNA, while finding a weak signal for GRP-receptor (GRP-R) and NMB-receptor (NMB-R) mRNAs. In total RNA from the reaggregate cell cultures we detected high levels of NMB mRNA as well as a strong signal for GRP-R mRNA. Finally, in GH3 cells, high levels of NMB mRNA and GRP-R mRNA were found, while GRP mRNA and NMB-R mRNA remained undetectable even in high amounts (200 micrograms) of total RNA. We conclude that mRNAs encoding both bombesin-related peptides and each of the mRNAs encoding their receptors are expressed in rat AP tissue. NMB mRNA is more prominent than GRP mRNA in all AP-like tissues examined (fresh AP, estradiol-treated reaggregate AP cell cultures and GH3 cells). NMB-R mRNA and GRP-R mRNA are both present in low levels in fresh AP whereas the GRP-R mRNA is predominant in GH3 cells and estradiol treated AP reaggregate cell cultures. Compared to fresh AP tissue, NMB mRNA and GRP-R mRNA expression is enhanced in estradiol-treated reaggregate cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Expression of growth factor ligand and receptor genes in rat cancellous bone trabeculae and marrow.

The technique of reverse transcription-PCR for mRNA phenotyping was applied to total RNA isolated from the two compartments of cancellous bone, namely trabecular bone and hematopoitic tissue or marrow. The pattern of gene expression for ten different growth factor ligands and five growth factor receptors was examined in total RNA isolated from the two compartments of cancellous bone of the female rat distal femur. Our results show that transcripts encoding IGF-I, IGF-II, transforming growth factor-beta 1 (TGF-beta 1), TGF-alpha, basic fibroblast growth factor, platelet-derived growth factor A and osteocalcin are detectable in samples from both trabeculae and marrow. Expression of epidermal growth factor (EGF) was confined to samples from trabeculae while nerve growth factor expression was only detected in marrow. Transcipts encoding insulin were not detected in any of the bone-derived samples in this study. Samples from cancellous bone trabeculae and marrow both showed evidence of expression of the genes encoding receptors for IGF-I, parathyroid hormone (PTH)/PTH-related protein and insulin. Neither compartment of cancellous bone contained transcripts encoding the receptor for IGF-II. Transcripts encoding the EGF receptor were detected in samples from cancellous bone marrow and not trabeculae as has been previously reported. These patterns of growth factor ligand and receptor gene expression suggest that it is likely that both autocrine and paracrine regulatory circuits are established in cancellous bone. This study also demonstrated the feasibility of assessing the expression of multiple genes from the small samples of total RNA obtained from separated tissues of cancellous bone. This is the first time that growth factor gene expression has been examined in separated trabeculae and marrow from cancellous bone and this approach will allow a more detailed analysis of molecular events in cancellous bone as opposed to whole bone or extracts of isolated and cultured bone cells.

Animals↗
PubMed1997

Endothelin receptor mRNAs in human fetal membranes, chorionic vessels, and decidua parietalis.

Endothelin-1 (ET-1) is synthesized in avascular human amnion; and, immunoreactive ET-1 is present in amniotic fluid in concentrations 10 to 100 times those in blood. ET-1 acts, most commonly, in a local or paracrine manner; therefore, it is possible that amnion/amniotic fluid ET-1 acts on contiguous tissues, namely chorion laeve or placental surface (chorionic) vessels, or in an autocrine fashion on amnion cells. To address these possibilities, the levels of ET(A) and ET(B) receptor mRNAs were evaluated in amnion, chorion laeve, decidua parietalis, placenta, and chorionic vessel tissues. By Northern analysis of total RNA (20 microg), ET(A) and ET(B) receptor mRNAs were detected in decidua (n = 18), placenta (n = 14), and chorionic vessels (n = 13). In chorion laeve (n = 24), ET(B) receptor mRNA but not ET(A) receptor mRNA was detected by Northern analysis of total RNA. Northern analysis of chorion laeve poly(A)+ mRNA (1.5-2.5 microg) revealed ET(A) receptor mRNA at low levels. Neither ET(A) nor ET(B) receptor mRNAs were detected in amnion tissue by Northern analysis of total RNA (n = 30; placental and reflected amnion from 15 pregnancies) or by Northern analysis of poly(A)+ mRNA (1.5-2.5 microg). Moreover, there was no demonstrable dose-dependent effect of ET-1 on prostaglandin E2 production or DNA synthesis in amnion epithelial cells in primary culture. The findings of this investigation are indicative that ET-1 in amniotic fluid or secreted from amnion may act in a paracrine fashion on chorion laeve by way of the ET(B) receptor and on chorionic vessels by way of ET(A) and ET(B) receptors.

Amnion↗
PubMed1999

Quantitative reverse transcription-PCR measurement of thyroglobulin mRNA in peripheral blood of healthy subjects.

BACKGROUND: Thyroglobulin mRNA can be detected qualitatively in the peripheral blood of patients with metastatic thyroid cancer, thyroid cancer patients with residual thyroid bed uptake, and individuals with no known thyroid disease with intact thyroid glands by use of a lengthy, highly sensitive extraction technique. To improve and broaden the clinical usefulness of this assay, we developed a quantitative reverse transcription (RT)-PCR assay for thyroglobulin mRNA, using RNA recovered from whole blood with a simplified extraction technique. METHODS: Whole blood was drawn from 32 healthy subjects in standard EDTA blood collection tubes. Total RNA was extracted from whole blood, using the PUREscript RNA Isolation Kit. RT-PCR using intron-spanning primers was used to quantitatively amplify thyroglobulin mRNA, using the ABI PRISM 7700 Sequence Detection System with a fluorescent-labeled, thyroglobulin-specific oligonucleotide probe. Thyroid RNA calibration curves were created using total RNA recovered from a single nondiseased thyroid gland. RESULTS: Qualitative RT-PCR demonstrated the presence of thyroglobulin mRNA in the whole blood sample of each healthy subject. The mean concentration of thyroglobulin mRNA detected in these subjects was 433 +/- 69 ng of total thyroid RNA per liter of whole blood (range, 26-1502 ng/L). Overall assay imprecision (CV) was 24% for five samples analyzed 10 times each in separate analytical runs on different days. CONCLUSIONS: Thyroglobulin mRNA can be accurately detected and quantified in peripheral blood from healthy subjects. This new quantitative technique may improve the clinical utility of circulating thyroglobulin mRNA detection in patients with thyroid disease.

Humans↗
PubMed1991

Quantitative determination of mRNA phenotypes by the polymerase chain reaction.

A method for quantitative determination of specific cellular mRNA is described. The mRNA in a dilution series of total RNA was reverse transcribed by an oligo-dT primer and the cDNA was amplified by the polymerase chain reaction (PCR) using sets of specific primers. A 32P- or biotin-labeled specific probe was hybridized to the PCR products immobilized on nitrocellulose membrane. The intensity of the hybridization signals was evaluated for quantification of the PCR products. A standard curve was produced by the known amount of the in vitro transcribed cRNA, which contained the same sequence as the mRNA. The series of standard cRNA dilutions were reverse transcribed, amplified and hybridized in the same manner. The amount of the specific RNA was deduced by fitting to the standard curve. Two tissue specimens of intestinal tumors, evaluated on the basis of hybridization signals by three different methods, were shown to contain similar amounts of beta-actin mRNA. Furthermore, a Chinese hamster ovary (CHO) cell line transfected with platelet-derived growth factor (PDGF) beta-receptor cDNA was found to contain similar amounts of beta-actin mRNA as the untransfected CHO cell line. However, the transfected CHO cell line contained over 10(11) copies of the PDGF beta-receptor mRNA per microgram of total RNA, while the untransfected one showed no detectable RNA, indicating that the latter contained less than 10(6) copies per microgram of total RNA in this assay.

Animals↗
PubMed1981

Total content of RNA in individual nerve cells of spinal ganglion during ischemia in dogs.

The total RNA content in the individual nerve cells of the lumbal spinal ganglia in the dog after the 80 minutes lasting partial ischemia was followed. The spinal ganglion neurons did not react in ischemia in the same way. The total RNA content in the individual neurons of the control animals occurred in the range 132-990 arbitrary units, after the ischemia in the range 116-670 arbitrary units. In both groups the cells with the low or high RNA content were seen. The decreases in total RNA content after ischemia was at the higher level of the statistical significance in the group with the largest and middle large neurons giving the evidence about the greater sensitivity to the oxygen shortage in comparison with the group of the smallest spinal ganglion nerve cells.

Animals↗
PubMedDate not supplied

Molecular cloning, sequence, and expression of cynomolgus monkey cholesteryl ester transfer protein. Inverse correlation between hepatic cholesteryl ester transfer protein mRNA levels and plasma high density lipoprotein levels.

A cDNA clone containing the coding region for cynomolgus monkey cholesteryl ester transfer protein (CETP) was isolated by the polymerase chain reaction with primers based on the human CETP cDNA sequence and cDNA synthesized from liver poly (A+) RNA. Analysis of that cDNA indicated that the nucleotide and amino acid sequences of cynomolgus monkey CETP were greater than 95% homologous with the human sequences. A fragment of the cDNA was used to develop an internal-standard/RNAse protection assay that allowed precise quantification of CETP mRNA levels. Analysis of total RNA from various tissues with this assay revealed that the liver and thoracic aorta expressed high levels of CETP mRNA; the mesenteric fat, adrenal gland, spleen, and abdominal aorta had low but detectable levels of the mRNA; and the brain, kidney, intestine, and skeletal muscle had undetectable levels of that mRNA. When the monkeys were made hypercholesterolemic by a high-fat, high-cholesterol (HFHC) diet, hepatic levels of CETP mRNA increased from 1.6 +/- 0.4 pg/micrograms total RNA (mean +/- SEM) to 4.1 +/- 0.8 pg/micrograms (p less than 0.005); mesenteric fat CETP mRNA increased from 0.4 +/- 0.1 pg/micrograms total RNA to 5.3 +/- 2.2 pg/micrograms (p less than 0.05); and plasma CET activity increased approximately fourfold. The CETP mRNA levels in the thoracic and abdominal aortas were not significantly increased in monkeys fed the HFHC diet, even though those animals had gross atherosclerosis. The apoprotein E mRNA levels, however, were markedly increased in the aortas of monkeys with atherosclerosis, with the largest increase occurring in the abdominal aorta. Taken together, these data suggest that lipid deposition in the artery was not accompanied by increased expression of the CETP gene in that tissue. Statistical analysis showed that a strong, negative correlation existed between hepatic CETP mRNA levels and both high density lipoprotein cholesterol (r = -0.85, p less than 0.001) and apoprotein A-I (r = -0.84, p less than 0.001). These data suggest that HFHC diet-induced changes in high density lipoprotein metabolism may be linked to altered expression of a function CETP gene.

Animals↗
PubMed2003

Donor corneoscleral buttons: a new source of trabecular meshwork for research.

The human trabecular meshwork (TM) is the major site of resistance for aqueous humor outflow. The purpose of this investigation was to determine the suitability of TMs harvested from donor corneoscleral buttons (buttons) for laboratory studies. Histologic examination using light and electron microscopy was performed. Additionally, the effect of incubation in serum free media on histologic appearance was studied. Total RNA extraction was performed on 45 buttons and four whole eyes. Some TMs were used as a source for establishing primary cell cultures of TM endothelial cells. Compared with donor whole eyes, light microscopy showed comparable TM cellularity in the juxtacanalicular and corneoscleral TM; there was some loss of cells in the inner portion of uveal TM that was adjacent to the anterior chamber. The TM cells appeared healthy and structurally normal on transmissive electron microscopy. Usable quantities of total RNA could be extracted from buttons that had been stored up to 5 weeks. The amount of total RNA extracted correlated well with the histologic appearance. Incubation in serum free media did not have an effect on the histologic appearance. All attempts at establishing primary cell cultures were successful. Unused, donor corneoscleral buttons are an excellent source of TM.

Adult↗
PubMed2000

Fatty acid transport protein-1 mRNA expression in skeletal muscle and in adipose tissue in humans.

Fatty acid transporter protein (FATP)-1 mRNA expression was investigated in skeletal muscle and in subcutaneous abdominal adipose tissue of 17 healthy lean, 13 nondiabetic obese, and 16 obese type 2 diabetic subjects. In muscle, FATP-1 mRNA levels were higher in lean women than in lean men (2.2 +/- 0.1 vs. 0.6 +/- 0.2 amol/microg total RNA, P < 0.01). FATP-1 mRNA expression was decreased in skeletal muscle in obese women both in nondiabetic and in type 2 diabetic patients (P < 0.02 vs. lean women in both groups), and in all women there was a negative correlation with basal FATP-1 mRNA level and body mass index (r = -0.74, P < 0.02). In men, FATP-1 mRNA was expressed at similar levels in the three groups both in skeletal muscle (0.6 +/- 0.2, 0.6 +/- 0.2, and 0.8 +/- 0.2 amol/microg total RNA in lean, obese, and type 2 diabetic male subjects) and in adipose tissue (0.9 +/- 0.2 amol/microg total RNA in the 3 groups). Insulin infusion (3 h) reduced FATP-1 mRNA levels in muscle in lean women but not in lean men. Insulin did not affect FATP-1 mRNA expression in skeletal muscle in obese nondiabetic or in type 2 diabetic subjects nor in subcutaneous adipose tissue in any of the three groups. These data show a gender-related difference in the expression of the fatty acid transporter FATP-1 in skeletal muscle of lean individuals and suggest that changes in FATP-1 expression may not contribute to a large extent to the alterations in fatty acid uptake in obesity and/or type 2 diabetes.

Abdomen↗
PubMedDate not supplied

Pregnancy induces an increase in the expression of glyceraldehyde-3-phosphate dehydrogenase in uterine artery endothelial cells.

OBJECTIVE: We determined the effects of pregnancy on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in ovine uterine and omental (systemic control) artery endothelial cells (UAEC, OAEC). We also determined in primary cultures of UAEC the effects of exposure to either exogenous nitric oxide (NO) or angiogenic growth factors (basic fibroblast growth factor [bFGF], vascular endothelial growth factor [VEGF], and epidermal growth factor [EGF]) on UAEC GAPDH expression. METHODS: We isolated UAEC to high purity from both nonpregnant (NP; n = 4) and pregnant ewes (P; 110-120 days' gestation, n = 4) by limited collagenase dispersion and immediately extracted total RNA. In additional experiments performed in vitro, ovine UAEC isolated from NP ewes and maintained in culture (n = 3) were exposed to 1) 100 mumol/L sodium nitro-prusside for 0, 6, 12, or 24 hours or 2) 10 ng/mL bFGF, VEGF, EGF, or vehicle for 24 hours. Total RNA was then immediately extracted. A partial ovine GAPDH (oGAPDH) cDNA was isolated by reverse transcriptase polymerase chain reaction (RT/PCR) and sequenced. A one-tube semiquantitative RT/PCR amplification assay was established, and GAPDH mRNA was subsequently quantified in all samples of total RNA. The PCR products were separated by size, quantified by Southern hybridization analysis, and normalized to 28S rRNA content. Expression of GAPDH protein was also measured by Western analysis of endothelium-derived protein from omental (n = 14) and uterine (n = 16) arteries from NP and P ewes. RESULTS: Pregnancy was associated with a 4.5-fold increase in GAPDH mRNA levels in UAEC, although in vitro exposure of primary cultures of UAEC from NP ewes to NO or angiogenic growth factors did not significantly change GAPDH mRNA expression. A 1.6-fold increase in GAPDH protein was detected in the uterine artery endothelium of P versus NP ewes, but no corresponding increase was found in omental artery endothelium. CONCLUSION: Pregnancy increases the expression of both GAPDH mRNA and protein in UAEC. Furthermore, the pregnancy-induced increase in GAPDH protein in the endothelium of the uterine artery appears specific, as it is not observed in the omental (systemic) artery. This induction is not, however, reproduced in vitro with exogenous NO or angiogenic growth factor treatment (up to 24 hours).

Animals↗
PubMed1978

alpha-Lactalbumin mRNA in 4-day lactating rat mammary gland.

Rat alpha-Lactalbumin (alpha-LA) mRNA has been purified from the total RNA of 4-day lactating mammary gland by poly(U)-Sepharose chromatography and by fractionation of the poly(A)+ RNA on sucrose gradients. alpha-LA mRNA was homogeneous as judged by electrophoresis in urea/agarose gel and in 1.5% agarose gel under glyoxal-denaturation conditions, which gave a molecular weight of 210,000. The chymotryptic fingerprints of the protein synthesized with this mRNA, in a translational system derived from wheat germ, were similar to those of purified rat alpha-LA. In rat milk or lactating gland, the content of Mr 42,000 casein is 6 times greater than that of Mr 29,000 casein and 10 times greater than that of alpha-LA. However, alpha-LA mRNA (Mr 210,000) and two casein mRNas (Mr 460,000 and 390,000), coding for Mr 42,000 and 29,000 caseins, were present in equal proportions when measured with cDNA probes in the total RNA of 4- to 5-day lactating gland. Moreover, alpha-LA and total casein were synthesized in a ratio fo 1:2.5 in a wheat-germ translational system using the total RNA of the lactating gland. The results suggest post-transcription controls in the synthesis of these proteins.

Animals↗
PubMed2001

Developmental regulation of vascular endothelial growth/permeability factor messenger ribonucleic acid levels in and vascularization of the villous placenta during baboon pregnancy.

Vascular endothelial growth/permeability factor (VEG/PF) has an important role in angiogenesis; however, very little is known about the developmental regulation of VEG/PF and the vascular system within the placenta during human pregnancy. In the present study, therefore, a developmental approach was used in the baboon to determine the placental source of VEG/PF and its fms-like tyrosine kinase (flt-1) and kinase-insert domain containing (KDR/flk-1) receptors, and whether the rise in estrogen with advancing pregnancy was associated with a corresponding increase in placental VEG/PF expression and vascularization. VEG/PF messenger RNA (mRNA) levels were determined by competitive RT-PCR in villous cell fractions isolated by Percoll gradient centrifugation from placentas obtained on days 45 and 54 (very early), 60 (early), 100 (mid), and 165-170 (late) of baboon pregnancy (term = 184 days). Maternal peripheral serum estradiol increased from very low concentrations early in gestation (0.15-0.20 ng/ml) to an early surge of over 2.5 ng/ml on days 60-85, and peak levels of 4-6 ng/ml late in baboon pregnancy. VEG/PF mRNA was expressed in low level in the syncytiotrophoblast (<2,000 attomol/microgram total RNA), and values in this fraction did not change significantly with advancing gestation. VEG/PF mRNA expression was slightly greater in the inner villous core cell fraction; however, levels decreased (P < 0.05) between early and late gestation. Cytotrophoblasts were a major source of VEG/PF mRNA and levels increased (P < 0.01) from 3,631 +/- 844 attomol/microgram total RNA on day 45 to 25,807 +/- 5,873 attomol/microgram total RNA on day 170. VEG/PF protein expression determined by immunocytochemistry was abundant in cytotrophoblasts and lower in the syncytiotrophoblast and inner villous core cells. The flt-1 and KDR/flk-1 receptors were expressed in the vascular endothelial cells of the baboon villous placenta. The percentage of villous placenta occupied by blood vessels and the number of vessels/mm(2) villous tissue, determined by image analysis, progressively increased (P < 0.001; r = 0.97) from 3.4 +/- 0.2% and 447 +/- 29, respectively, on day 54 to 15.9 +/- 0.9% and 1,375 +/- 71, respectively, on day 170. In summary, the present study shows that villous cytotrophoblasts were a major source of VEG/PF mRNA and protein in the baboon villous placenta, and that cytotrophoblast VEG/PF mRNA levels and vascularization of the villous placenta closely paralleled the increase in estradiol concentrations of advancing pregnancy. These results are consistent with the concept that estrogen has an important role in establishing the new vascular system within the developing placenta during primate pregnancy and that VEG/PF mediates this process.

Animals↗
PubMed1995

In situ localization of ribosomal RNAs is a reliable reference for hybridizable RNA in tissue sections.

Assessments of RNA integrity and its hybridizability are essential for successful implementation of in situ hybridization (ISH) for mRNA or viral RNA, particularly when paraffin-embedded specimens from surgical, biopsy, and autopsy cases are used. In this study, we examined the suitability of ISH of 28S ribosomal RNA (rRNA) for this purpose. Oligo-DNA with nucleotide sequences complementary to a well-conserved segment of 28S rRNA with auxiliary adenine-thymine-thymine (ATT) repeats at the 3' and 5' ends was synthesized. The oligo-DNA was made antigenic by converting the adjacent thymines to T-T dimers by UV irradiation and was used as a probe for ISH of 28S rRNA. The T-T dimers were detected by enzyme immunohistochemistry. When the results of ISH rRNA staining and that of total RNA staining by methyl green/pyronin Y were compared for various types of sections prepared from rat and human tissues, the staining intensities of total RNA did not always match those of ISH rRNA staining. In paraffin sections of formalin-fixed tissues, the degree of proteinase digestion influenced the ISH rRNA staining intensity, whereas it had no effect on the total RNA staining intensity. The intensities of ISH rRNA staining agreed well with those of various types of mRNA staining by ISH in 10 cases of paraffin-embedded pathological specimens. We therefore believe that ISH rRNA staining is a convenient parameter for evaluation of levels of hybridizable RNAs in tissue sections.

Animals↗
PubMed1983

Polysomal organization in growing and resting cultures and its relation to protein synthesis in Tetrahymena.

Cytosol from Tetrahymena cells growing at different rates was isolated and separated by centrifugation into polysomal and non-polysomal fractions. The RNAs of either fraction were separated chromatographically into poly(A)+ RNA and poly(A)-RNA. It was found that in resting cultures the total RNA per cell is only about half of that of rapidly growing cultures. All fractions of RNA were reduced proportionally. Thus, the percentage of polysomally bound total RNA (70% of cytosol RNA) and polysomally bound poly(A)+ RNA (72% of cytosol poly(A)+ RNA) is the same in growing and resting cultures. Differences, however, were found in the polysomal structure. Polysomes from resting cultures contained significantly fewer ribosomes. The amounts of RNA bound to polysomes were related to the rate of protein synthesis under different growth conditions. The decrease in cellular RNA corresponded well with the reduction in amino acid incorporation in resting cells. The rate of protein accumulation in resting cells, on the other hand, was considerably less, suggesting that polypeptides in resting cultures are less stable.

Amino Acids↗
PubMed1997

No evidence for involvement of the growth hormone/insulin-like growth factor-1 axis in psoriasis.

We have examined whether alterations in the growth hormone/insulin-like growth factor-1 axis play a role in the pathogenesis of psoriasis. Serum, urine, full skin biopsies, and suction blister roofs were obtained from patients with psoriasis and from healthy controls. Serum concentrations of insulin-like growth factor-1 and insulin-like growth factor binding protein-3 were measured by radioimmunoassay. Growth hormone-binding protein was measured by ligand-mediated immunofunctional assay. Growth hormone concentration in urine was measured by an immunometric assay, and growth hormone receptor-gene expression was measured by RNase protection assay or by quantitative reverse transcriptase polymerase chain reaction in total RNA isolated from epidermal suction blister roofs. Serum concentrations of insulin-like growth factor-1 (249 +/- 12 micrograms per liter, mean +/- SEM, n = 42, and 277 +/- 21 micrograms per liter, n = 9, for psoriatic patients and controls, respectively), insulin-like growth factor binding protein-3 (3.1 +/- 0.08 mg per liter, n = 42, and 3.3 +/- 0.22 mg per liter, n = 9), growth hormone-binding protein (344 +/- 65 pmol per liter, n = 10, and 311 +/- 83 pmol per liter, n = 9), urinary growth hormone excretion during 24 h (12.8 +/- 2.7 microIU per 24 h, n = 12, and 12.3 +/- 1.6 microIU per 24 h, n = 9), and epidermal growth hormone receptor gene expression [32 +/- 12 x 10(3) mRNA transcripts per microgram total RNA (involved skin), n = 11, and 47 +/- 14 x 10(3) mRNA transcripts per microgram total RNA, n = 9] were similar in patients and controls. For insulin-like growth factor-1 and insulin-like growth factor binding protein-3 the values in psoriatic patients were also similar to those in larger control groups, n = 195 and n = 400, respectively. In addition, we found no evidence of local expression of growth hormone or prolactin in full skin punch biopsies from psoriatic involved skin by reverse transcriptase polymerase chain reaction. In conclusion, our results suggest that alterations in the growth hormone/ insulin-like growth factor-1 axis do not play a major role in the pathogenesis of psoriasis.

Adult↗
PubMed2000

Reverse transcription - 3' rapid amplification of cDNA ends-nested PCR of ACT1 and SAP2 mRNA as a means of detecting viable Candida albicans in an in vitro cutaneous candidiasis model.

The presence of viable cells of Candida albicans, in broth or in a reconstructed living skin equivalent, was determined by the detection of amplicons of partial mRNA sequences of the genes encoding fungal actin (ACT1) and secreted aspartyl proteinase 2 (SAP2). The mRNA of both genes were amplified by reverse transcription-3' rapid amplification of cDNA ends-nested polymerase chain reaction. Single bands of ACT1 (315 bp) and SAP2 (162 bp) mRNA were amplified from total RNA extracts of C. albicans grown in yeast carbon base-albumin broth or in living skin equivalent tissue; only the former was amplified from Sabouraud broth-grown organisms. Primer pairs targeted for ACT1 and SAP2 were Candida genus-specific and C. albicans-specific, respectively. The sensitivity limits of the assay were 100 fg of total RNA or 10 cells of C. albicans, by ethidium bromide staining. When C. albicans-infected living skin equivalent was exposed to amorolfine, amplicons of ACT1 and SAP2 mRNA were not detected in total RNA extracts. Non-amplification of the mRNA correlated with the absence of C. albicans growth in Sabouraud agar cultures of living skin equivalent samples. Reverse transcription-3' rapid amplification of cDNA ends-nested polymerase chain reaction of the mRNA encoding specific proteins of an organism has potential application in determining the viability of the organism in tissue, thus monitoring the efficacy of an antimicrobial therapy, and in detecting mRNA expressed in very little amounts in tissue.

Actins↗