Haemoglobin messenger RNA in spleen of anaemic rabbits

Haemoglobin meseenger RNA was isolated from spleen erythroïd cells of anaemic rabbits. The mRNA fraction looks quite homogeneous when analyzed by sucrose gradient centrifugation; it sediments in the 9S region. When added to an ascites cell free system, spleen 9S RNA stimulates the incorporation of radioactive leucine into protein. The synthesized product has been characterized and identified as and chains of rabbit globin.     

Characterization of a messenger RNA transport protein

A cytoplasmic protein which facilitates the energy-dependent transport of mRNA from isolated nuclei to a specified medium has been further characterized, since it could have relevance to the mechanism of mRNA nucleo-cytoplasmic transport in vivo. This protein is now shown, by cDNA hybridization analysis using appropriate recombinant probes, to be obligatory for the transport of alpha 2u-globulin and albumin mRNA from male rat liver nuclei. It is concentrated in the cytoplasm. When isolated under conditions where they retain nuclear proteins, the nuclei contain less than 2% of the total mRNA transport activity. Approx. 20% is recovered in the cytosol, while the rest (80%) copurifies with the messenger ribonucleoproteins in the polyribosome fraction. The protein is eluted from the poly A-messenger ribonucleoproteins between 0.25 and 0.50 M NaCl. The activities of the cytosolic- and messenger ribonucleoprotein-derived transport proteins were mutually additive below saturation of the transport system. Further, the activities of both fractions were increased when they were fortified with the catalytic subunit of the cAMP-dependent protein kinase in the presence of ATP. On the other hand, protein kinase-induced thiophosphorylation of the protein with ATP[S] decreased transport activity. The molecular weight of the transport protein from either cell compartment as judged by molecular sieving is approx. 35,000. It has now been purified 2000-fold and requires manganese ions and serum albumin for stabilization of activity. The highly purified transport factor from the cytosol is tentatively assigned a molecular weight of 32,000 by SDS-polyacrylamide gel electrophoresis.     

Upregulation of hepatic LDL transport by n-3 fatty acids in LDL receptor knockout mice

We determined the effects of dietary n-6 and n-3 polyunsaturated fatty acids (PUFA) on parameters of plasma lipoprotein and hepatic lipid metabolism in LDL receptor (LDLr) knockout mice. Dietary n-3 PUFA decreased the rate of appearance and increased the hepatic clearance of IDL/LDL resulting in a marked decrease in the plasma concentration of these particles. Dietary n-3 PUFA increased the hepatic clearance of IDL/LDL through a mechanism that appears to involve apolipoprotein (apo)E but is independent of the LDLr, the LDLr related protein (LRP), the scavenger receptor B1, and the VLDLr. The decreased rate of appearance of IDL/VLDL in the plasma of animals fed n-3 PUFA could be attributed to a marked decrease in the plasma concentration of precursor VLDL. Decreased plasma VLDL concentrations were due in part to decreased hepatic secretion of VLDL triglyceride and cholesteryl esters, which in turn was associated with decreased concentrations of these lipids in liver. Decreased hepatic triglyceride concentrations in animals fed n-3 PUFA were due in part to suppression of fatty acid synthesis as a result of a decrease in sterol regulatory element binding protein-1 (SREBP-1) expression and processing. In conclusion, these studies indicate that n-3 PUFA can markedly decrease the plasma concentration of apoB-containing lipoproteins and enhance hepatic LDL clearance through a mechanism that does not involve the LDLr pathway or LRP.     

Fuel metabolism in fasted newborn rabbits

Newborn rabbits delivered by Caesarean section at term were fasted for 72 h at 36 degrees C. Despite the abrupt interruption of maternal supply of energy substrates, glycaemia remains stable for 4 h after birth. This can be related to glucose production via rapid liver glycogenolysis; however, indirect evidence suggests that gluconeogenesis could also contribute to glucose production during this period. There is a selective decrease in the concentrations of gluconeogenic substrates and a suitable hormonal environment for gluconeogenesis as decreased insulin and increased glucagon concentration just after birth. The relative hypoglycaemia which develops after 6 h of life (2.6 mM at 72 h), despite high blood concentrations of non-esterified fatty acids and ketone bodies is not due to a deficient gluconeogenesis per se, as injection of gluconeogenic substrates to 72 h fasted newborns produces a three-fold increase in plasma glucose concentration. It is suggested that this relative hypoglycaemia is secondary to limited gluconeogenic substrate availability in the form of low circulting concentrations of gluconeogenic amino acids.     

Immunological detection of the messenger RNA cap-binding protein

The 24-kilodalton messenger RNA cap-binding protein (CBP) was purified from the rabbit reticulocyte postribosomal supernatant fraction using an affinity resin consisting of the p-aminophenyl gamma-ester of m7GTP coupled to Sepharose. The affinity-purified CBP was used to raise a goat antiserum. Anti-CBP antibodies were purified by adsorption to CBP coupled to either Controlled-Pore Glass or diazobenzyloxymethyl paper. The affinity-purified antibodies reacted specifically with only the 24-kilodalton polypeptide in whole reticulocyte lysate and in initiation factors prepared from the same source. During a conventional (nonaffinity) purification of CBP from a high salt extract of the ribosomal pellet, immunological reactivity paralleled the ability to reverse cap analogue inhibition of translation, indicating that the 24-kilodalton polypeptide present in the postribosomal supernatant fraction is immunologically cross-reactive with the CBP purified from ribosomes. Fractionation of whole reticulocyte lysate by sucrose gradient sedimentation followed by immunoblotting revealed that CBP was present in the supernatant fraction and the region of the gradient corresponding to ribosomal subunits but not in mono- or polysomes. The CBP to ribosome ratio was found to be approximately 0.02, assuming that the m7GTP-Sepharose retains all of the protein. This is considerably lower than that of other initiation factors and suggests that CBP may be the limiting polypeptide factor involved in the initiation of protein synthesis. The antibodies also inhibited the translation of a capped messenger RNA (globin). Inhibition of the translation of an uncapped RNA (satellite tobacco necrosis virus) was also observed, but to a lesser degree than with globin mRNA.     

Effect of the hepatocarcinogen ethionine on hepatic messenger RNA synthesis.

An experiment was conducted to determine if any changes resulted in the proportion of hepatic messenger RNA following treatment with ethionine, a hepatocarcinogen. The relative specific activity of the total RNA isolated from nontumor-like tissues was increased in Sprague-Dawley rats fed ethionine. However, the percentage of total RNA that was message was found to be decreased in the ethionine-treated rats.     

Cellular retinol-binding protein messenger RNA levels in normal and retinoid-deficient rats

A study was conducted to determine the levels of cellular retinol-binding protein (CRBP) mRNA and protein in various tissues of the rat, to explore relationship between CRBP mRNA and protein levels in different tissues, and to examine the effects of changes in retinol nutritional status on the tissue distribution and levels of CRBP mRNA. Previous studies have shown that tissue CRBP protein levels are reduced in totally retinoid-deficient rats, but are otherwise minimally affected by changes in retinoid status. Three groups of male rats were compared: normal controls, retinoid-deficient, and retinol-repleted deficient rats. CRBP mRNA levels were measured by RNase protection assay and CRBP protein levels by radioimmunoassay in seven tissues. High levels of both CRBP mRNA and CRBP protein were found in the proximal epididymis, kidney, and liver; lower levels were seen in lung, testis, spleen, and small intestine. Tissue CRBP mRNA and protein levels were highly correlated (P less than 0.01) with each other. Retinoid deficiency did not alter the levels of CRBP mRNA found in the proximal epididymis, kidney, and liver. In contrast, CRBP mRNA levels in the lung, testis, spleen, and small intestine were reduced substantially in retinoid-deficient rats, to values that were only 23% to 50% of the corresponding values in the tissues of control rats. After oral repletion with retinol (4-18 h earlier), CRBP mRNA levels for these latter four tissues were found to have risen to control or near-control levels. The suggestion is raised that retinol repletion may have directly induced the expression of the CRBP gene in these particular tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormonal regulation of epidermis-specific protein and messenger RNA synthesis in amphibian metamorphosis

Experiments using a monospecific antibody directed against one type of epidermis-specific keratin from adult skin of the amphibian Xenopus laevis have demonstrated that polysomes synthesizing this protein first appear within larval skin during natural metamorphosis. Further experiments demonstrated that the synthesis of keratin within larval skin could be induced precociously by the thyroid hormone, 3,3′,5-triiodo-l-thyronine, both in vivo and when the isolated larval skin is cultured in vitro. The earliest developmental age responsive to such hormone induction appeared to be Stage $\text{50}\text{52}$ of larval development. This is about 20–24 days before keratin would normally make its appearance within the skin during natural metamorphosis. Hormone treatment of tadpoles at this age will also cause a precocious increase in the amount of keratin messenger RNA present within larval skin. This has been demonstrated directly by the isolation of poly(A)-containing messenger RNA from hormone-treated larvae and its translation in a wheat germ cell-free system to give immunoprecipitable keratin. Peptide analysis of the in vitro translation product indicates that the hormone-induced mRNA probably codes for an initial protein product that is slightly larger than keratin itself.     

Down-regulation of ribosomal protein S15A mRNA with a short hairpin RNA inhibits human hepatic cancer cell growth in vitro

Ribosomal protein s15a (RPS15A) is a highly conserved protein that promotes mRNA/ribosome interactions early in translation. Recent evidence showed that RPS15A could stimulate growth in yeast, plant and human lung carcinoma. Here we report that RPS15A knockdown could inhibit hepatic cancer cell growth in vitro. When transduced with shRPS15A-containing lentivirus, we observed inhibited cell proliferation and impaired colony formation in both HepG2 and Bel7404 cells. Furthermore, cell cycle analysis showed that HepG2 cells were arrested at the G0/G1 phase when transduced with Lv-shRPS15A. In conclusion, our findings provide for the first time the biological effects of RPS15A in hepatic cancer cell growth. RPS15A may play a prominent role in heptocarcinogenesis and serve as a potential therapeutic target in hepatocellular carcinoma.     

Bistability in gene transcription: interplay of messenger RNA, protein, and nonprotein coding RNA

The author proposes a kinetic model describing the interplay of messenger ribonucleic acid (mRNA), protein, produced via translation of this RNA, and nonprotein coding RNA (ncRNA). The model includes association of mRNA and ncRNA and regulation of the ncRNA production by protein. In the case of positive feedback between the production of protein and ncRNA, the steady state of the system is found to be unique. For negative feedback, the model predicts in the mean-field case either unique steady state or bistable kinetics. With incorporation of fluctuations, the bistability is manifested in the form of kinetic bursts provided that the number of reactants is low. Basically, the model describes the simplest biological switch operating with participation of ncRNA. Although the results obtained are applicable to ncRNSs in general, the presentation is focused primarily on microRNAs (miRNAs) which form a large important subclass of ncRNAs and are thought to regulate up to one third of all human genes.