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PubMed1973

Control of 5S RNA synthesis during early development of anucleolate and partial nucleolate mutants of Xenopus laevis.

Ribosomes of all eukaryotes contain a single molecule of 5S, 18S, and 28S RNA. In the frog Xenopus laevis the genes which code for 18S and 28S RNA are located in the nucleolar organizer, but these genes are not linked to the 5S RNA genes. Therefore the synthesis of the three ribosomal RNAs provides a model system for studying interchromosomal aspects of gene regulation. In order to determine if the synthesis of the three ribosomal RNAs are interdependent, the relative rate of 5S RNA synthesis was measured in anucleolate mutants (o/o), which do not synthesize any 18S or 28S RNA, and in partial nucleolate mutants (p(l-1)/o), which synthesize 18S and 28S RNA at 25% of the normal rate. Since the o/o and p(l-1)/o mutants have a complete and partial deletion of 18S and 28S RNA genes respectively, but the normal number of 5S RNA genes, they provide a unique system in which to study the dependence of 5S RNA synthesis on the synthesis of 18S and 28S RNA. Total RNA was extracted from embryos labeled during different stages of development and analyzed by polyacrylamide gel electrophoresis. Quite unexpectedly it was found that 5S RNA synthesis in o/o and p(l-1)/o mutants proceeds at the same rate as it does in normal embryos. Furthermore, 5S RNA synthesis is initiated normally at gastrulation in o/o mutants in the complete absence of 18S and 28S RNA synthesis.

Animals↗

PubMed2004

Pretranslational markers of contractile protein expression in human skeletal muscle: effect of limb unloading plus resistance exercise.

Previously, it has been shown that the human ground-based model consisting of unilateral limb suspension (ULLS) induces atrophy and reduced strength of the affected quadriceps muscle group. Resistance exercise (RE) involving concentric-eccentric actions, in the face of ULLS, is effective in ameliorating these deficits. The goal of the present study was to determine whether alterations in contractile protein gene expression, e.g., myosin heavy chain and actin, as studied at the pretranslational level, provide molecular markers concerning the deficits that occur in muscle mass/volume during ULLS, as well as its maintenance in response to ULLS plus RE. Muscle biopsies were obtained from the vastus lateralis muscle of 31 middle-aged men and women before and after 5 wk of ULLS, ULLS plus RE, or RE only. The RE paradigm consisted of 12 sessions of 4 sets of 7 concentric-eccentric knee extensions. Our findings show that there were net deficits in total RNA, total mRNA, and actin and myosin heavy chain mRNA levels of expression after ULLS (P < 0.05), whereas these alterations were blunted in the two groups receiving RE. Additional observations involving IGF-I and its associated receptor and binding proteins suggest that RE postures the skeletal muscle for signaling processes that favor a greater anabolic state relative to that observed in the ULLS group. Collectively, these findings suggest that molecular markers of contractile protein gene expression serve as useful subcellular indicators for ascertaining the underlying mechanisms regulating alterations in muscle mass in human subjects in response to altered loading states.

Adult↗

PubMed1997

Intercellular adhesion molecule-1 is expressed on human granulosa cells and mediates their binding to lymphoid cells.

Some immune cellular components have been recently demonstrated to play a critical role in ovarian physiology. Resident ovarian white blood cells are known to produce cytokines that modulate granulosa cell (GC) functions and differentiation. Moreover, it has been postulated that, during the formation of the corpus luteum and luteolysis, human luteal cells are able to interact with lymphocytes and macrophages through some adhesion molecules. This study was designed to examine, at messenger RNA and protein levels, whether intercellular adhesion molecule (ICAM)-1, known to be involved in leukocyte-cell binding, is expressed by human GCs. Furthermore, we also investigated whether this molecule could be involved in the complex events that allow the interaction between the ovary and the immune system. GCs, obtained from women undergoing in vitro fertilization procedures, were enzymatically dispersed with collagenase and cultured for different time periods. To assess the presence of ICAM-1 messenger RNA, total RNA obtained from freshly aspirated GCs and GCs luteinized in culture was reverse transcribed and then amplified using two oligonucleotide primers specific for the human ICAM-1 gene. A single major DNA band of the expected size (943 bp) was obtained. The identity of this material with the human ICAM-1 sequence was further confirmed by restriction enzyme analysis. Surface ICAM-1 protein was detected by flow cytometric analysis on luteinized GCs cultured for 7 and 15 days. Finally, to evaluate a possible functional activity of ICAM-1, a 51Cr-release-binding assay between peripheral blood lymphocytes and luteinized GCs was performed in the presence and absence of a monoclonal antibody against ICAM-1. As a result, lymphocyte adhesion to GC monolayers was significantly, but not completely, inhibited by the anti-ICAM-1 monoclonal antibody. These findings demonstrate that intercellular interactions between GCs and the immune system are, at least in part, mediated by the adhesion molecule ICAM-1. Based on this data, we might speculate that this molecule could participate in the remodeling processes of the ovarian endocrine compartment.

Antibodies, Monoclonal↗

PubMed1994

Stage-specific expression of c-myc messenger ribonucleic acid in porcine granulosa cells early in follicular growth.

In the present study, the expression of c-myc mRNA in porcine granulosa cells harvested from varying follicle stages was analyzed using northern blot hybridization with a c-myc probe, and compared during follicular growth. Northern blot analysis revealed that porcine granulosa cell RNA obtained from small (1-2 mm) follicles contained an abundance of c-myc transcript of 2.4 kb, the size of which was identical to the c-myc transcript obtained with human early placental RNA used as the control. In contrast to small-follicle granulosa cell RNA, total RNA preparations from either medium (3-5 mm)-follicle granulosa cells or large (6-11 mm)-follicle granulosa cells exhibited no detectable transcripts with the c-myc probe. These results suggest that expression of c-myc mRNA in porcine granulosa cells is limited only at the early stage of follicular growth. The stage-specific expression of c-myc mRNA in granulosa cells early in follicular growth may play a physiological role in the autonomous growth of immature granulosa cells in early folliculogenesis, seemingly independent of pituitary gonadotropic stimulation.

Animals↗

PubMed2005

RT-nested PCR detection of Mourilyan virus in Australian Penaeus monodon and its tissue distribution in healthy and moribund prawns.

Mourilyan virus (MoV) is a newly identified virus of Penaeus monodon prawns that is genetically related to the Uukuniemi virus and other phleboviruses of the Bunyaviridae. This paper describes an RT-nested PCR test that can reliably detect between 2 and 6 copies of a synthetic MoV RNA. Total RNA isolated from the lymphoid organ, gills and haemocytes of P. monodon with moderate infections gave comparable amplicon yields in the RT-PCR step of the test. However, in prawns with extremely low-level infections, haemocytes and gill tissue proved slightly more reliable in detecting MoV RNA following nested PCR. The distribution of MoV in tissues of healthy and moribund P. monodon was examined by in situ hybridisation (ISH) using a digoxigenin-labelled DNA probe to a approximately 0.8 kb M RNA segment cDNA insert in clone pMoV4.1. The DNA probe targeted a region in the MoV M RNA segment containing a coding sequence with homology to the C-terminus of the G2 glycoprotein of phleboviruses. In healthy prawns harbouring an unapparent MoV infection, ISH signal primarily occurred in the lymphoid organ, where it was more prominent in hypertrophied cells of 'spheroids' than within cells of normal tubules. ISH signal was also sometimes detected in cells of cuticular epithelium, segmental nerve ganglion and the antennal and tegmental glands. MoV was distributed widely throughout these and other cephalothoracic tissues of mesodermal and ectodermal origin in moribund P. monodon following experimental infection or collected from farm pond edges during disease episodes. Transmission electron microscopy of gill of moribund, captive-reared P. monodon identified spherical (approximately 85 nm diameter) to ovoid MoV particles (approximately 85 x 100 nm) in and around highly necrotic cells in which the nucleus and other organelles had disintegrated. MoV virions co-existed with rod-shaped virions of gill-associated virus and were often seen clustered within cytoplasmic vacuoles or associated with the outer rim of concentric ring-shaped structures comprised of endoplasmic membranes likely to represent degenerated Golgi.

Animals↗

PubMed1987

A temperature-compensated ultradian clock explains temperature-dependent quantal cell cycle times.

The effects of sublethal heat pulses on cell division have provided insights into possible molecular mechanisms. Thus Zeuthen's findings of 'set-backs' up to a transition point provides the basis for the idea that the continuous accumulation of a compound needed for cell division spans a major portion of the cell cycle. The accumulating substance is a 'division protein' which forms part of a structure which is unstable until completely assembled at the transition point. Experiments showing phase resetting of mammalian cells by temperature perturbation indicate limit-cycle oscillator control of the cell cycle with a phase-response curve with a repeat interval equal to the period of the clock. As well as providing a method for establishing synchronized cultures these observations have found application in the selective effects of hyperthermia as an antitumour agent. Circadian rhythms display several unique features distinguishing them from other periodic processes. Only recently has it been recognized that some of these characteristics may be properties of ultradian rhythms as well. The probably most striking feature of circadian timekeeping, i.e. independence of ambient temperature, was found for ultradian rhythmicity even at the level of the unicellular organization. Synchronous cultures of some lower eukaryotes were prepared by centrifugal size selection methods. Experiments with asynchronous control cultures substantiated the view that the conditions employed were such as to minimize any perturbative effects: most importantly the organisms were never removed from their culture medium. Whereas the control cultures showed smoothly increasing respiration rates, total RNA, total protein, enzyme activities and enzyme protein (e.g. for cytochrome aa3, ATPase, catalase), in synchronous cultures all these parameters showed oscillatory behaviour. Different periods were observed in different organisms: thus in Acanthamoeba castellanii the period was about 70 min, in Tetrahymena pyriformis strain ST it was about 50 min, in T. pyriformis AII it was 30 min, and in Candida utilis it was about 30 min (all measurements at 30 degrees C). In A. castellanii the periods of both the oscillations in rate of respiration and the total cell protein were hardly affected by the temperature of growth over the range 20 to 30 degrees C. The oscillations show no damping during experiments lasting 12 h: these properties suggest that we are observing temperature-compensated endogenous rhythms which presumably serve a timekeeping function in cells undergoing growth and division.(ABSTRACT TRUNCATED AT 400 WORDS)

Activity Cycles↗

PubMed1993

Effect of excess phenylacetate diet during pregnancy on fetal brain growth in rats.

The effects of phenylacetate (PA) on fetal brain growth were examined in pregnant rats receiving a 20% casein diet with 1.0, 1.5, 2.0 or 2.5% PA. Control rats were fed the 20% casein diet ad libitum or restricted to daily consumption of 9 and 6 g. In experimental groups of rats total food intake during pregnancy decreased, however the decrease was not so large as we had expected. By plotting the fetal brain weight (Y, mg) against maternal food intake (X, g/21 days) in control groups the following hyperbolic regression equation was obtained: Y = -4243/X + 124.6 (n: 18, r = 0.80, p < 0.001). Similar plots for excess PA rats fell below this line, indicating that prenatal fetal brain growth was impaired by an excess in PA per se specifically, as well as by decreased food intake nonspecifically. Administering the excess PA diets resulted in decreases in total RNA, total protein and RNA/DNA ratio in the fetal brain, whereas total DNA was unchanged, showing the impairment of protein synthesis, not proliferation. No remarkable changes in concentration and pattern of free amino acids in maternal plasma in excess PA groups were observed. Using a relation between total food intake during pregnancy and fetal brain weight, the extent of specific and nonspecific effects of excess PA and phenylalanine on brain growth was compared.

Amino Acids↗

PubMed2004

Utility of linearly amplified RNA for RT-PCR detection of chromosomal translocations: validation using the t(2;5)(p23;q35) NPM-ALK chromosomal translocation.

The requirement for sufficient quantities of starting RNA has limited the ability to evaluate multiple transcripts using reverse transcriptase-polymerase chain reaction (RT-PCR). In this study, we demonstrate the utility of linear RNA amplification for RT-PCR analysis of multiple gene transcripts including a chromosomal translocation, using the t(2;5)(p23;q35) as a model. RNA from the t(2;5)-positive cell line, SU-DHL-1, and the t(2;5)-negative cell line, HUT-78, was extracted and exposed to two rounds of linear amplification. RT-PCR using cDNA from the resultant amplified (a) RNA and total RNA resulted in the 177 bp NPM-ALK fusion gene product from the SU-DHL-1 cell line, but not from aRNA or total RNA from the HUT-78 cell line. DNA sequencing of the RT-PCR products from total and aRNA of SU-DHL-1 cells demonstrated identical sequences corresponding to the NPM-ALK fusion gene. Evaluation of 25 snap-frozen tissue samples, including eight NPM-ALK-positive ALCLs demonstrated 100% concordance of t(2;5) detection between cDNA from total RNA and that from aRNA. Our results show that linear amplification of RNA can enhance starting RNA greater than 200-fold and can be used for rapid and specific detection of multiplex gene expression from a variety of sources. This method can generate a renewable archive of representative cDNA, which can be used for retrospective screening of stored samples as well as positive controls for the clinical molecular diagnostic laboratory.

Cell Line, Tumor↗

PubMed2007

[Primary investigation of the relationship between glucocorticoid induced leucine zipper and inflammatory reaction].

OBJECTIVE: To investigate the mechanism of the action of glucocorticoid induced leucine zipper (GILZ) in inflammatory reaction. METHODS: Human monocyte cell line THP-1 cells were divided into two groups and cultured in non-serum RPMI1640 medium.In one group the cells were treated with dexamethasone (DEX). Twelve hours later total RNA and total protein were abstracted in both two groups. The mRNA encoding for expression of GILZ was semiquantitatively detected by reserve transcriptase-polymerase chain reaction (RT-PCR). Protein expression of nuclear factor-KappaB (NF-KappaB) p65 and activator protein-1 (AP-1) were assessed by Western blotting. Peripheral blood of 10 trauma patients [injury severity score (ISS) >or=16 scores] were collected and the leukocytes were isolated within 24 hours after trauma. The leukocytes were divided into two groups and cultured in non-serum medium. In one group the cells were treated with DEX. Twelve hours later total RNA and total protein were abstracted in both two groups. The mRNA encoding for expression of GILZ was semiquantitatively detected by RT-PCR. Protein expression of NF-KappaB p65 and AP-1 were assessed by Western blotting. RESULTS: Stimulated by DEX, the expression of GILZ mRNA was increased both in THP-1 cells and the leukocytes of trauma patients compared with those of control groups (both P<0.01). Whereas, protein expressions of NF-KappaB p65 and AP-1 of THP-1 cells and leukocytes in peripheral blood of trauma patients were decreased in the stimulation groups compared with those of control groups (all P<0.01). CONCLUSION: The expression of GILZ gene is up-regulated by glucocorticoid. Overexpression of GILZ inhibits NF-KappaB and AP-1 activities, suggesting that GILZ possesses anti-inflammatory function.

Adult↗

PubMed1986

Effect of safe application rate (SAR) concentrations of some biocides on the gonads of the fresh water murrel, Channa punctatus (Bl.)--a biochemical study.

The effect of safe application rate (SAR) concentrations of fenitrothion 50% EC--an organo-phosphate and carbofuran 3% G--a carbamate on the gonads (ovaries/testes) of Channa punctatus was studied for 120 days (mid-April through mid-August) extending over the preparatory, prespawning, and spawning phases of annual reproductive cycle. The studies have revealed that the levels of total proteins, RNA, total lipids, and ascorbic acid decreased, while those of the cholesterol and the phospholipids increased following exposure to the two biocides. Furthermore, it was observed that in both treatments the decline in the levels of total total proteins, RNA, total lipids, and ascorbic acid was relatively more marked in the testes than in the ovaries. On the contrary, increase in the levels of cholesterol was relatively more in the ovaries, while increase in the levels of phospholipids was more in the testes in both treatments. The results suggest that the decrease/increase in the levels of the above-mentioned biochemical parameters was more in the fenitrothion treatment than in the carbofuran treatment and, therefore, may be considered relatively more toxic to this species.

Animals↗

PubMed1999

Affinity separation of messenger RNA by thermo-responsive polymer carrying oligo(dT).

The conjugate between oligo(dT)16 and thermo-responsive polymer, poly(N-isopropylacrylamide), was prepared for isolation of poly(A)+ RNA from total RNA. The hybridization reaction between the conjugate and poly(A) (average length: 320 base) was equilibrated in 10 min, and all the poly(A) (16 nmol base for 24 nmol base of conjugate) was precipitated when raising the solution temperature to 35 degrees C. The precipitate was dissolved in water, and poly(A) was dissociated from the conjugate by heating to 65 degrees C. This separation system was successfully applied to the isolation of poly(A)+ RNA from total RNA.

Acrylic Resins↗

PubMed1992

Divergent regulation of the Glut 1 and Glut 4 glucose transporters in isolated adipocytes from Zucker rats.

We have studied the relationship between glucose uptake rate and Glut 1 and Glut 4 protein and mRNA levels per fat cell in lean (FA/FA) and obese (fa/fa) Zucker rats at 5, 10, and 20 wk of age, and after induction of acute diabetes with streptozotocin. 5 wk obese rats exhibit insulin hyperresponsive glucose uptake, whereas 20 wk obese rats show insulin resistant glucose uptake. The relative abundance of Glut 1 and Glut 4 mRNA and protein per equal amount of total RNA and total membrane protein, respectively, is lower in adipocytes from obese rats. However, at all ages the enlargement of fat cells from obese rats is accompanied by a severalfold increase in total RNA and total membrane protein per cell. Thus, on a cellular basis, mRNA and protein levels of Glut 4 increases in young obese rats and gradually declines as a function of age. Basal glucose uptake is increased severalfold in fat cells from obese rats, and in parallel Glut 1 expression per cell in obese rats is two- to threefold increased over lean rats at all ages. Acute diabetes in 20 wk obese rats causes a profound downregulation of glucose uptake and a concomitant reduction of both Glut 1 and Glut 4 protein levels. Thus, changes in Glut 4 expression are a major cause of alteration in insulin-stimulated glucose uptake of adipocytes during evolution of obesity and diabetes in Zucker rats.

Adipose Tissue↗

PubMed1992

Cholecystokinin upregulation during intestinal repair.

To determine whether cholecystokinin (CCK), a small intestinal hormone, may have autocrine or paracrine functions, gene regulation in the rat stomach and duodenum has been evaluated following cytotoxic injury. We quantified total RNA, CCK messenger RNA (mRNA), total protein, small and large forms of CCK peptides and gastrin. The stomach and the intestine respond differently. Following cytotoxic injury duodenal total RNA falls (1.5 +/- 0.1 vs 0.18 +/- 0.04 mg/g P less than or equal to 0.0001), and CCK mRNA content is depleted (260 +/- 23 vs 41 +/- 8 pg CCK mRNA/duodenum P less than or equal to 0.0001), yet there is a paradoxical increase in CCK mRNA concentration (176 +/- 20 vs 303 +/- 38 pg CCK mRNA/mg total RNA P less than or equal to 0.01). Increases occurred in both molecular species of CCK peptides evaluated: CCK8 (8 +/- 7 vs 26 +/- 2 pmole/g P less than or equal to 0.0001), large forms of CCK (42 +/- 4 vs 250 +/- 27 pmole/g P less than or equal to 0.0001). By contrast, in the stomach, only decreases were observed. These data identify sites of anatomical and biosynthetic upregulation during gastrointestinal repair. Changes are dependent upon the length of the period of recovery, differ between stomach and duodenum, and may be age related. Intestinal CCK may have para- and or autocrine roles in addition to its hormone function.

Animals↗

PubMed1979

Isolation and partial purification of the major abundant class rat seminal vesicle poly(A+)-messenger RNA.

Total poly(A(+))-RNA (poly(A(+))-RNA(tot)) was isolated from rat seminal vesicle and its size distribution determined by 70% formamide 5-25% sucrose density analysis. One major peak was resolved in the 10-13 S region and accounted for approximately 35% of the total poly(A(+))-RNA applied. Preparative 1% SDS, 5-20% linear sucrose density gradients also resolved a single major peak in the 11S region (poly(A(+))(11S). Analysis of poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S) under denaturing conditions on 2% agarose gel electrophoresis demonstrated two major components in both poly(A(+))-RNA populations. Size estimations for these components are 620 and 540 NT respectively. (3)H-cDNA was made to both poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S). Back-hybridization of poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S) to their respective (3)H-cDNA revealed a highly abundant class representing 41% and 85% of the sequences in their respective (3)H-cDNA's. The highly abundant class corresponded to 3-5 sequences present in 30,000-50,000 copies/cell. Invitro translation of poly(A(+))-RNA(11S) resulted in two major polypeptides coded for by the 620 NT long and 540 NT long poly(A(+))-RNA respectively.Images

Animals↗

PubMed1982

Mechanism of host defense suppression induced by viral infection: mode of action of inosiplex as an antiviral agent.

The mechanism of influenza virus (INFV)-induced immunosuppression and the mode of inosiplex action against INFV infection were studied. INFV suppressed both anti-lipopolysaccharide and anti-sheep erythrocyte antibody production in mice. INFV infection caused viral mRNA synthesis and increased total RNA synthesis in lymphocytes, but total mRNA synthesis was decreased. The translational ability of INFV-infected lymphocytes was also suppressed. Thus, INFV seemed to cause suppression of both mRNA synthesis and the translational ability of lymphocytes, resulting in suppression of lymphocyte functions. Inosiplex potentiated antibody production against sheep erythrocytes but not against lipopolysaccharide in normal and INFV-infected mice. Adamantanamine did not produce such a potentiating effect. The lymphocytes obtained from INFV-immunized and inosiplex-treated mice conferred resistance against INFV infection. This resistance was partially inhibited by anti-Thy 1.2 antibody treatment of the lymphocytes. In an adoptive cell transfer system, inosiplex treatment of T-cell donors potentiated antibody production when a non-immunosuppressive carrier (human serum albumin) was used. When an immunosuppressive carrier (INFV) was used, inosiplex treatment of either B-cell donors or T-cell donors increased antibody production. Direct introduction of inosiplex into lymphocytes by a cell fusion technique stimulated anti-sheep erythrocyte antibody production more effectively than the addition of inosiplex to cultures. Inosiplex increased total RNA and total mRNA syntheses in phytohemagglutinin-treated lymphocytes. In INFV-infected lymphocytes, inosiplex decreased syntheses of total RNA, total mRNA, and viral mRNA and restored translational ability. From these results, we concluded that inosiplex penetrates into lymphocytes and suppresses viral RNA synthesis and that it supports lymphocyte functions by promoting RNA synthesis and translational ability, both of which are necessary for hosts.

Animals↗

PubMed1976

The metabolism of high-molecular-weight ribonucleic acid including polyadenylated species, in the developing rat brain.

High-molecular-weight RNA was isolated from rat brain at various times after the intracranial administration of [32P]Pi. The synthesis of 28S and 18S rRNA could be detected within 1h of the injection of the radioactive precursor and appeared to be more pronounced, relative to other high-molecular-weight RNA, in the brains of older rats compared with those of newborn rats. Polyadenylated RNA, representing most mRNA and their precursors, was isolated by chromatography on oligo(dT)-cellulose. The contribution of this polyadenylated RNA to total RNA synthesis was investigated in the cerebral cortex and the phylogenetically older brain stem at different stages in the development of the rats by using a 5h period of labelling as an arbitrary index of transcription. In the brain stem the proportion of labelled polyadenylated RNA comprised 27-30% of the total RNA. The corresponding values for the cortex decreased from 34% in newborn rats to 23% in 40-150-day-old rats. These data indicated that proportionately more polyadenylated RNA is synthesized in the cortex of the newborn than in the adult rat and that there is a progressive decrease in the synthesis of polyadenylated RNA relative to rRNA during development.

Animals↗

PubMed1979

Comparison of poly(A)-containing RNAs in different cell types of the lower eukaryote Schizophyllum commune.

Poly(A)-containing RNAs were isolated from morphologically different cells of the fungus Schizophyllum commune. Using mRNA markers the number-average length of poly(A)-containing RNA in total RNA and in purified poly(A)-containing RNA was estimated as 1100 nucleotides. Number-average length of poly(A)-tracts was 33 nucleotides. 2.5% of total RNA is poly(A)-containing RNA and probably up to 7.5% are non-polyadenylated polydisperse RNA sequences. Saturation hybridization of poly(A)-containing RNA to gap-translated [3H]DNA resulted in 16% of the reactive single-copy DNA to become S1 nuclease resistant. It was found that purified poly(A)-containing RNA represented the entire RNA complexity, i.e. 10 000 different RNA sequences in S. commune. RNA sequences isolated from morphologically different mycelia and from fruiting and non-fruiting mycelia were identical for at least 90%.

Agaricales↗

PubMed1993

Decrease in Gs protein expression may impair adenylate cyclase activation in old kidneys.

The possibility that alteration in stimulatory guanine nucleotide-binding protein (Gs) expression may contribute to the blunting of renal parathyroid hormone (PTH)-stimulated adenylate cyclase in aged rats was examined. Using rat cDNA probe, we identified a Gs alpha-subunit (Gs alpha) of 1.9 kb. Age did not alter the size of Gs alpha mRNA. The level of Gs alpha mRNA [normalized to poly(A)+ RNA] was decreased 23%, which was consistent with our previous report that Gs alpha protein decreased in senescence. In contrast, mRNA level of Gi alpha 2 increased with age. Level of beta-actin mRNA did not change with age. Nuclear runoff assay was performed to determine the transcription rate of Gs mRNA. Synthesis of poly(A)+ RNA and total RNA was reduced 39% and 37%, respectively, in nuclei prepared from old kidney, which suggested a general decline in RNA synthesis capacity in old rats. Our results also showed the transcription rate of Gs alpha mRNA in aged rats was reduced 89%, a decrease far exceeding that observed for total RNA or poly(A)+ RNA. We concluded that the decrease in steady-state level of Gs alpha mRNA was specific and probably was due to a reduction in the transcription activity. Thus alteration in Gs transcription may contribute, at least in part, to the impaired renal adenylate cyclase activation in aged rats.

Adenylyl Cyclases↗