Metabolism of benz[alpha]anthracene by human bone marrow in vitro.

The metabolism of polycyclic aromatic hydrocarbons by bone marrow, mononuclear cells from normal donors and leukaemia patients in remission has been investigated. When benz[alpha]anthracene (BA) was included with marrow under cell culture conditions, it was converted to materials which were resolved into three peaks by normal phase HPLC, and which had the chromatographic characteristics of BA-dihydrodiols. Formation of hydroxymethyl-or dihydrodiol-derivatives of 7, 12-dimethylbenz[alpha]anthracene were not detected under the same conditions. The BA-metabolites were identified as BA-5,6-dihydrodiol, BA-10,11-dihydrodiol and BA-8,9-dihydrodiol. The identification was based upon chromatographic properties of the metabolites during normal and reverse phase chromatography and on UV spectral and fluorometric characterization. It was not possible to detect the formation of BA-3,4-dihydrodiol since this dihydrodiol co-elutes with BA-8,9-dihydrodiol and BA-10,11-dihydrodiol during normal phase and reverse phase chromatography, respectively. the UV spectra of BA-3,4-dihydrodiol does not have features which enable it to be readily identified in the presence of these other compounds. Formation of the dihydrodiol-metabolites was dependent on cell number and temperature. Two general cytochrome P450 inhibitors, carbon monoxide and piperonyl butoxide, blocked the formation of metabolites but the cyclooxygenase inhibitor, indomethacin had no effect. Large variations were observed in the capacity of marrow from different individuals to form benz[alpha]anthracene-dihydrodiols but, in each sample where dihydrodiols were formed, the relative amount of each metabolite was BA-8,9-dihydrodiol >> BA-5,6-dihydrodiol > BA-10,11-dihydrodiol. Factors which may contribute to this variation, including disease status, genetic and environmental agents, are considered.

Alterations in the activity of adenylate cyclase and high affinity GTPase in Alzheimer's disease.

The aim of this study was to assess the effect of Alzheimer's disease has on the functional integrity of several signal transduction proteins. The relative levels of the G-protein alpha subunits Gs alpha-L, Gs alpha-S, Gi alpha-2 and G(o) alpha were measured by western blotting and found to be unchanged in membranes prepared from Alzheimer-diseased frontal cortex or hippocampus compared to control brains. However the activity of the G-protein associated enzyme, high affinity GTPase, was found to be reduced in the frontal cortex (reduced by 25%) and by a similar magnitude in the hippocampus (reduced by 27%) of Alzheimer subjects. The same membrane preparations were also assayed for the activity of adenylate cyclase. Basal enzyme activity was not significantly altered in Alzheimer diseased hippocampus, but was markedly reduced (by 45%) in the frontal cortex. The ability of fluoride and aluminium ions to stimulate adenylate cyclase was not significantly changed in either brain region. This suggests that G-proteins, especially Gs, are still able to interact with this enzyme. These results indicate that although the presence of Alzheimer's disease does not significantly alter G-protein levels, changes have taken place in the overall activity of these proteins. However this alteration does not affect their ability to stimulate adenylate cyclase activity.

On the stability of messenger RNA and ribosomal RNA in the brains of control human subjects and patients with Alzheimer's disease.

The levels of the mRNAs encoding the G protein subunits GS alpha, G beta 1, and G beta 2 were measured by northern blotting in the frontal cortex and hippocampus of control subjects and of patients with a clinical and histopathological diagnosis of dementia of the Alzheimer type (DAT). There was no significant difference, in either brain region, between the control and DAT groups for any of the G protein mRNAs measured. The degree of intersubject variability was very high, e.g., GS alpha mRNA in the frontal cortex (mean optical density +/- SD) was 405 +/- 342 in the control group versus 305 +/- 207 in the DAT group. The extent of generalised RNA degradation was assessed by detecting the breakdown products of 28S rRNA. RNA degradation was present in tissue samples from every human subject studied. The extent of 28S rRNA degradation in each subject was found to be related to the levels of G protein mRNA detected. The degree of RNA degradation in human subjects was found to be very variable and unaffected by the presence of DAT. RNA degradation correlated poorly with postmortem interval and this was confirmed by a controlled study of postmortem degradation in rat tissue. The possibility that the relative hypoxia and ischaemia in patients immediately before death could influence RNA degradation is discussed. The variable extent of RNA degradation means that great care must be taken to ensure the validity of RNA analyses undertaken in human postmortem brain, particularly when techniques are employed (such as in situ hybridisation) that themselves give no indication of RNA integrity.

Tissue distribution and change in potato starch phosphorylase mRNA levels in wounded tissue and sprouting tubers.

Starch phosphorylase has been cloned from a lambda gt10 cDNA library of potato tuber mRNA. Selected recombinants have been used to demonstrate that phosphorylase mRNA is most abundant in tubers but is also detectable in stolon, root, stem and leaf tissue. The level of phosphorylase mRNA was greatly reduced in wounded stem and tuber tissue. The wounding-induced decrease in phosphorylase mRNA levels is not reversed in the presence of sucrose or mannitol. Regional differences are described in the levels of phosphorylase and patatin mRNA in different parts of the tuber and in the shoot of sprouting potatoes.

Robustness of G proteins in Alzheimer's disease: an immunoblot study.

Many of the neurotransmitter systems that are altered in senile dementia of the Alzheimer type are known to mediate their effects via G proteins, yet the integrity of guanine nucleotide-binding proteins (G proteins) in Alzheimer's diseased brains has received minimal investigation. The aim of this study was to establish whether the level of G alpha subunits of five G proteins was altered in Alzheimer's disease. We used immunoblotting (Western blotting) to compare the amounts of Gi1, Gi2, GsH (heavy molecular weight), GsL (light molecular weight), and Go in the frontal cortex and hippocampus, two regions severely affected by the disease, and the cerebellum, which is less severely affected. The number of senile plaques was also quantified. We report that there was no significant difference in the level of these G alpha subunits between Alzheimer's diseased and age-matched postmortem brains. These results suggest that alterations in the amount of G protein alpha subunits are not a feature of Alzheimer's disease.

Diabetes-induced changes in guanine-nucleotide-regulatory-protein mRNA detected using synthetic oligonucleotide probes.

Synthetic oligonucleotide probes were designed to detect the alpha-subunits of the guanine-nucleotide-regulatory proteins (G-proteins) Gi-1, Gi-2, Gi-3 and Gs (Gi is inhibitory and Gs is stimulatory). Each probe detected a single major mRNA species in Northern blots of RNA extracted from a variety of tissues. A probe was designed to identify the two forms of G-protein beta-subunits, beta 1 and beta 2. This probe hybridised with a single 1.8-kb transcript (beta 2) in RNA from all tissues studied except for brain, where a less-abundant 3.4-kb transcript (beta 1) was also detected. These probes were used to assess whether the induction of diabetes, using streptozotocin, altered the levels of mRNA coding for specific G-protein components. In hepatocytes, diabetes caused a significant reduction in the number of transcripts coding for alpha-Gs, alpha-Gi-2 and alpha-Gi-3; mRNA for alpha-Gi-1 was undectable. In adipocytes, diabetes increased dramatically the mRNA coding for alpha-Gi-1 and alpha-Gi-3, whilst no significant changes occurred in the fractions coding for alpha-Gi-2 and alpha-Gs. No significant changes in the mRNA coding for G-protein alpha-subunits were observed in either brain, heart, skeletal muscle or kidney. Diabetes did not cause any significant changes in the mRNA coding for beta 2 in any tissue or cell population studied. Such results on the relative levels of mRNA encoding G-protein components was obtained by comparing equal amounts of total RNA from tissues of control and diabetic animals. G-protein mRNA levels were expressed relative to ribosomal 28S RNA levels and, in some instances, relative to transcripts for a structural protein called CHO-B. The total cellular levels of both RNA and DNA were assessed in the various tissues and cells studied. Major falls in RNA levels/cell appeared to occur in hepatocytes and to a lesser extent in adipocytes and skeletal muscle. Thus major reductions in G-protein transcripts occurred in hepatocytes. The detected changes in G-protein mRNA are discussed in relation to the available evidence on G-protein expression. We suggest that diabetes causes tissue-specific changes in the levels of mRNA for particular G-protein species; this may have consequences for the functioning of cellular signal-transduction mechanisms in the affected tissues.

Diabetes-induced alterations in the expression, functioning and phosphorylation state of the inhibitory guanine nucleotide regulatory protein Gi-2 in hepatocytes.

Levels of the G-protein alpha-subunits alpha-Gi-2, alpha-Gi-3 and the 42 kDa, form of alpha-Gs were markedly decreased in hepatocyte membranes from streptozotocin-diabetic animals as compared with normals. In contrast, no detectable changes in alpha-Gi subunits were seen in liver plasma membranes of streptozotocin-diabetic animals, although levels of the 45 kDa form of Gs were increased. G-protein beta subunits in plasma membranes were unaffected by diabetes induction. Analysis of whole-liver RNA indicated that the induction of diabetes had little effect on transcript levels of Gi-3, caused an increase in Gs transcripts and decreased transcript number for Gi-2, albeit to a much lesser extent than was observed upon analysis of hepatocyte RNA. In both hepatocyte and liver plasma membranes, immunoblot analysis showed that levels of the catalytic unit of adenylate cyclase were increased upon induction of diabetes. Under basal conditions, alpha-Gi-2 from hepatocytes of diabetic animals was found to be both phosphorylated to a greater extent than alpha-Gi-2 isolated from hepatocytes of normal animals, and furthermore was resistant to any further phosphorylation upon challenge of hepatocytes with angiotensin, vasopressin or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. Treatment of isolated plasma membranes from normal, but not diabetic, animals with purified protein kinase C caused the phosphorylation of alpha-Gi-2. Treatment of membranes from diabetic animals with alkaline phosphatase caused the dephosphorylation of alpha-Gi-2 and rendered it susceptible to subsequent phosphorylation with protein kinase C. Low concentrations of the non-hydrolysable GTP analogue guanylyl 5'-imidodiphosphate inhibited adenylate cyclase activity in both hepatocyte and liver plasma membranes from normal, but not diabetic, animals.

Role of ribonuclease and ribonuclease inhibitor activities in Alzheimer's disease.

It has been suggested that defects in the relationship between ribonuclease and its proteinaceous inhibitor could be a contributory factor in Alzheimer's disease. We have investigated this possibility further by analysing free and bound enzyme activities and the activity of the inhibitor in nine regions of diseased and normal brain. These were chosen to include areas known to be affected by the disease, regions not histologically affected but thought to be involved in the disease process, and areas not thought to be involved in the disease. Neither the enzyme nor its inhibitor is defective in its activities in the chosen areas of Alzheimer's disease brain when compared with those of carefully age-matched controls.

Guanine-nucleotide-binding proteins expressed in rat white adipose tissue. Identification of both mRNAs and proteins corresponding to Gi1, Gi2 and Gi3.

Considerable debate has focused on the molecular identity of the guanine-nucleotide-binding proteins (G-proteins) in adipose tissue which can be detected following pertussis-toxin-catalysed ADP-ribosylation [Rapiejko, Northup, Evans, Brown & Malbon (1986) Biochem. J. 240, 35-40; Hinsch, Rosenthal, Spicher, Binder, Gausepohl, Frank, Schultz & Joost (1988) FEBS Lett. 238, 191-196]. We have used a panel of selective anti-peptide antisera which are able to discriminate between the different pertussis-toxin-sensitive G-proteins to assess which of these are expressed in rat adipose tissue. We demonstrate that plasma membranes of rat white adipocytes contain alpha subunits corresponding to each of Gi1, Gi2 and Gi3. Furthermore, using synthetic oligonucleotides complimentary to unique regions of each of the three polypeptides, we demonstrate that the mRNAs for the three G-protein alpha subunits can also be detected in adipose tissue.

The isolation of recombinant RNA species responsive to oestrogen and tamoxifen in rat uterus and MCF-7 cells.

We have previously shown that oestrogen elicits profound effects on the mRNA population of the immature rat uterus. In order to follow these responses in a more precise manner, we have prepared a cDNA library to uterine mRNA and screened this for sequences most responsive to the hormone. The stimulatory effect of oestrogen and tamoxifen on the levels of selected uterine mRNA species has been compared with the effect of the hormone and its agonist on total uterine poly(A)+ RNA levels. Two of the selected recombinant species were also expressed in the MCF-7 breast carcinoma cell line where their relative levels were again stimulated by oestrogen. Tamoxifen greatly reduced the poly(A)+ RNA levels of MCF-7 cells.

Purification and characterisation of ribonuclease activities that interact with the cytoplasmic inhibitor protein of rat tissues.

Evidence is presented that a number of different ribonuclease activities interact with the cytoplasmic ribonuclease inhibitor of rat and that these enzymes vary in their distribution in different tissues. Two ribonuclease activities were purified from rat liver and three from rat uterus. They were characterised with respect to their activity, substrate preference and pH optima.

Two endogenous protein kinase activities in heterogeneous nuclear ribonucleoprotein particles (hnRNP).

Two protein kinase activities which differ in their catalytic activity towards endogenous and exogenous substrates have been detected and partially purified from heterogeneous nuclear ribonucleoprotein particles (hnRNP).

Oestrogen-induced expression of oncogenes in the immature rat uterus.

4 h after a single precocious administration of oestrogen there was a considerable but short-lived surge in the uterine levels of myc-encoded polyadenylated mRNA. This was followed by a further peak 28 h after hormone administration. The expression of rasHa showed a totally different time course with a build up of hybridizable message that peaked 8 h after oestrogen administration.

Oestrogen-induced changes in the relative concentrations of ribonuclease and ribonuclease inhibitor in rat uterus.

The cytoplasm of the immature rat uterus contains an excess of ribonuclease inhibitor over endogenous cytoplasmic ribonuclease. The ratio of free inhibitor to bound inhibitor is approximately 3:1. After treatment with oestrogen, there is a marked increase in the level of cytoplasmic ribonuclease such that the enzyme is present in excess of the inhibitor. The increase in activity appears to result from at least 2 distinct ribonucleases with molecular masses of approximately 14000 and 18000. The changes observed after oestrogen treatment are also evident during normal development of the rat uterus. The implications of these results on the proposed role of the inhibitor/ribonuclease system are discussed.

Post-transcriptional regulation of rat liver gene expression by glucocorticoids.

We have investigated the mechanisms whereby glucocorticoids control the expression of specific genes in the livers of adult male rats. Construction and differential screening of a cDNA library representing normal rat liver polysomal poly(A)+ RNAs allowed selection of probes for hormonally regulated genes. The mechanism of this regulation was analysed by studying the changes in relative abundance of the RNA sequences homologous to four selected recombinants in RNA from subcellular fractions of liver, and comparing them with that of albumin mRNA. The relative abundance of these four RNA sequences increased to varying degrees in the nucleus, whilst that of three of them was concomitantly depleted in polysomal RNA when circulating levels of glucocorticoids were negligible, i.e. 14 days after adrenalectomy. One of the sequences was identified as alpha 2U-globulin mRNA. Within 2 hours of injecting Dexamethasone (a synthetic glucocorticoid) into rats that had been adrenalectomised 14 days previously, the relative abundances of alpha 2U-globulin RNA in nuclear and polysomal RNA returned to those found in normal rat liver. The data indicate that reduced glucocorticoid levels lead to sequence specific retention of RNA in the nucleus and that the RNA retained is released to the cytoplasm following glucocorticoid injection. Our results provide an example, for the first time, of glucocorticoid regulation of gene expression at the post-transcriptional level of nucleo-cytoplasmic transport.

The synthesis of ribosomal RNA and ribosomal protein and their incorporation into ribosomes in the uterus of the oestrogen-stimulated immature rat.

The effect of oestrogen on the synthesis of ribosomal proteins in the uterus of the immature rat has been investigated. Stimulated synthesis peaks, at 6-7-times control levels, 12 h after a single administration of the hormone. The stimulated synthesis and incorporation of newly made proteins into ribosomal particles exhibit very similar kinetics. The incorporation of newly made rRNA into ribosomes mirrors that of ribosomal protein but lags several hours behind the peak of oestrogen-stimulated rRNA synthesis.

The effects of oestradiol-17 beta on the synthesis and modification of ribosomal proteins in the uterus of the immature rat.

The effects of oestrogen on the incorporation of newly-made ribosomal proteins into the ribosomes of the immature rat uterus has been investigated. Different newly-made proteins were shown to enter ribosomes at different rates and there was some evidence that the hormone exerted differential effects. Oestradiol also stimulated the phosphorylation of ribosomal protein S6 but the effect could be explained by hormone-induced changes in the precursor pools.

Tamoxifen-induced changes in the polyribosomes and associated mRNA of rat uterus.

We have analysed the effect of tamoxifen on the accumulation of polysomes and their associated mRNA populations in the uterus of the immature rat. Tamoxifen caused the aggregation of ribosomes into polysomes but, when compared with the effects of oestrogen, the accumulation was delayed, of smaller magnitude and resulted in smaller polysomes. Tamoxifen did not inhibit oestrogen-induced polysome aggregation. Tamoxifen and monohydroxytamoxifen caused oestrogen-like changes in the polyadenylated mRNA population of uterine polysomes, but some quantitative and qualitative as well as kinetic differences were found in the response to the inhibitors when compared with the hormone. The reasons for these differences are discussed.

Protein synthesis in the uterus of the immature rat: the effects of short exposures to oestradiol-17 beta.

Three methods were used to study the effect of oestrogen on the incorporation of radioactive precursor into uterine protein. Intact tissue was incubated in vitro. Isolated uterine epithelial, stromal and myometrial cells were labelled in vitro. Isolated polysomes were translated in cell free protein synthesising systems. In all of these systems, minor qualitative changes in protein synthesis were detected where the uteri were derived from oestrogen-treated rats. These changes were most dramatic in isolated stromal cells and were accompanied by a marked overall increase in protein synthesis. The translation of mRNA on isolated uterine polysomes revealed a sequence of minor, but reproducible, oestradiol-induced changes. It was difficult, however, to relate these changes to those detected in incubated tissue or cells, possibly because the cell free translation products were not subject to normal post-translational modification and processing.