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An investigation of the specific localization of estrogen receptors in frozen sections of mammary tumors using an estrogen-BSA-FITC complex is presented. The results are discussed in relation to the existing literature. Although the fluorescence is certainly not due to interaction with type I receptors, the reaction is not as non-specific as is suggested sometimes in the literature. It is likely that type II receptors are involved, which are probably localized at the same site as type I receptors. [ABSTRACT FROM AUTHOR]

Glutamine synthetase (GS) is expressed at various levels in a wide range of tissues, suggesting that a complex network of modules regulates its expression. We explored the interactions between the upstream enhancer, regulatory regions in the first intron, and the 3'-untranslated region and immediate downstream genomic sequences of the GS gene (the GS "tail"), and compared the results with those obtained previously in conjunction with the bovine growth hormone (bGH) tail. The statistical analysis of these interactions revealed that the GS tail was required for full enhancer activity of the combination of the upstream enhancer and either the middle or the 3'-intron element. The GS tail also prevented a productive interaction between the upstream enhancer and the 5'-intron element, whereas the bGH tail did not, suggesting that the 5'-intron element is a regulatory element that needs to be silenced for full GS expression. Using the CMV promoter/enhancer and transfection experiments, we established that the 2.8 kb GS mRNA polyadenylation signal is approximately 10-fold more efficient than the 1.4 kb mRNA signal. Because the steady-state levels of both mRNAs are similar, the intervening conserved elements destabilize the long mRNA. Indeed, one but not all constructs containing these elements had a shorter half life in FTO-2B cells. A construct containing only 300 bases before and 100 bases after the 2.8 kb mRNA polyadenylation site sufficed for maximal expression. A stretch of 21 adenines inside this fragment conferred, in conjunction with the upstream enhancer and the 3'-part of the first intron, sensitivity of GS expression to ambient glutamine.

The pronounced increase in the protein/mRNA ratio of ammonia-metabolising enzymes in rat liver in the last prenatal week represents a clear example of a post-transcriptional level of control of gene expression. Both the underlying mechanism, namely an increase in translational efficiency of the mRNA and/or enhanced stability of the protein, and its importance for perinatal adaptation are unknown. We investigated this process in spiny mouse liver, because the comparison of rat and spiny mouse can discriminate adaptively from developmentally regulated processes in the perinatal period. We focused on glutamine synthetase (GS) because of the conveniently small size of its mRNA. Prenatally, GS enzyme activity slowly accumulated to approximately 1.3 U x g-1 liver at birth and postnatally more rapidly to 5.5 U x g-1 at 2 weeks. Both phases of enzyme accumulation obeyed exponential functions. Western-blot analysis showed that changes in GS activity reflected changes in GS protein content. GS mRNA content of the liver was 45 fmol x g-1 at 2 weeks before birth and slowly declined to approximately 25 fmol x g-1 at 2 weeks after birth. The GS protein/mRNA ratio increased 2.5-fold prenatally and sixfold postnatally. Analysis of prenatal and postnatal polysome profiles revealed no evidence of GS mRNA-containing ribonucleoprotein particles. Instead, GS mRNAs were (fully) occupied by 12 ribosomes, indicating regulation at the level of elongation. The kinetics of GS protein accumulation, in conjunction with GS mRNA content, are consistent with an approximately sixfold increase in its rate of synthesis at birth as the result of a corresponding stimulation of the rate of elongation.

In previous studies of the glutamine synthetase gene, the promoter and two enhancer elements, one in the upstream region and one within the first intron, were identified. To analyze the role of the far-upstream enhancer element in the regulation of the expression of the glutamine synthetase gene, two classes of transgenic mice were generated. In GSK mice, the basal promoter directs the expression of the chloramphenicol acetyltransferase reporter gene. In GSL mice reporter gene expression is driven, in addition, by the upstream regulatory region, including the far-upstream enhancer. Whereas chloramphenicol acetyltransferase expression was barely detectable in GSK mice, high levels were detected in GSL mice. By comparing chloramphenicol acetyltransferase expression with that of endogenous glutamine synthetase in GSL mice, three groups of organs were distinguished in which the effects of the upstream regulatory region on the expression of glutamine synthetase were quantitatively different. The chloramphenicol acetyltransferase mRNA in the GSL mice was shown to be localized in the pericentral hepatocytes of the liver. The developmental changes in chloramphenicol acetyltransferase enzyme activity in the liver were similar to those in endogenous glutamine synthetase. These results show that the upstream region is a major determinant for three characteristics of glutamine synthetase expression: its organ specificity, its pericentral expression pattern in the liver, and its developmental appearance in the liver.

Carbamoylphosphate synthetase I (CbmPS) is first expressed in rat hepatocytes shortly before birth. After birth, expression of CbmPS gradually becomes confined to the hepatocytes surrounding the portal veins. To obtain insight into the spatiotemporal regulation of its expression, the rat CbmPS gene was isolated and characterized. The gene is 110 kb in length and contains 38 exons. The basal promoter comprises the first 161 nucleotides upstream of the transcription-initiation site. Determination of the state of methylation of the 5' portion of the gene identified a CCGG sequence at -6.3 kb that is selectively demethylated in adult tissues which express CbmPS. This site remains methylated before birth, however, despite recruitment of all hepatocytes for CbmPS synthesis, indicating that its demethylation is a consequence of rather than a condition for expression of CbmPS. Transient expression assays revealed that the region surrounding the CCGG site at 6.3 kb functions as an enhancer. In FTO-2B hepatoma cells and Rat-1 fibroblasts, this enhancer is constitutively active when tested in front of the basal viral thymidine kinase promoter. When tested in front of the basal CbmPS promoter in hepatoma cells, however, the activity of this enhancer is dependent on the presence of glucocorticoids. In Rat-1 fibroblasts, the presence of both glucocorticoids and cyclic AMP is required for full activity, suggesting that the hepatocyte-specific expression of CbmPS is related to tissue-specific differences in the sensitivity to cyclic AMP. Matrix-attachment regions (MAR) are present upstream and downstream of the CbmPS gene. The downstream MAR defines the 3' boundary of the gene. The upstream MAR is located midway between the basal promoter and the enhancer, and may function as a hinge point to facilitate the positioning of the enhancer in the vicinity of the basal promoter.

From a rat genomic library in phage lambda Charon4A, a complete glutamine synthetase-encoding gene was isolated. The gene is 9.5-10 kb long, consists of seven exons, and codes for two mRNA species of 1375 nucleotides (nt) and 2787 nt, respectively. For both mRNAs, full-length cDNAs containing a short poly(A) tract were identified. The sequences of the entire mRNA and of the exon-intron transitions were determined. The smaller mRNA is identical to the 5' 1375 nt of the long mRNA and contains the entire protein-coding region. The position of the transcription start point was mapped. Within the first 118 bp of promoter sequence, a (T)ATAA-box, a CCAAT-box and an SP1-binding site were identified.

The distribution pattern of creatine kinase (E.C 2.7.3.2) isozymes in prenatal rat heart and skeletal muscle was studied by immunohistochemistry. Between embryonic day (ED) 12-18, creatine kinase M (CK-M) is heterogeneously expressed in the heart: a pronounced staining of CK-M is first observed in the outflow tract and the trabeculae of the right ventricle (ED12-14), and subsequently in the venous valves, the interatrial septum and the sinoatrial node. From ED18 onwards, a homogeneous expression of CK-M is observed due to an increase in isozyme concentration in the remaining part of the myocardium. By contrast, the developmental appearance of creatine kinase B (CK-B) occurs almost homogeneously throughout the heart between ED11-14. Thereafter, a decrease of the CK-B is first observed in the inflow tract (in particular in the sinoatrial node), in the inner part of those atrial walls that are adjacent to the atrioventricular junction, and temporarily in a band in the upper part of the interventricular septum. From ED18, a selective disappearance of CK-B is found in the papillary muscle of the left ventricle. At birth, a considerable amount of CK-B remains present in the ventricular walls. Although some of the stage-dependent regional differences in expression of the creatine kinase isozymes, in particular those of the M-subunit, are shared by other mammalian and avian species, their significance for the developmental changes in the physiology of the heart is speculative at present.

Low concentrations of dexamethasone (Dex) stimulate the initial rate of radioactive choline (Ch) accumulation in the endplate-rich area (EPA) of indirectly stimulated hemidiaphragms, while higher concentrations (greater than 0.6 microM) inhibit. This biphasic concentration-effect curve is found even in the presence of 26 microM hemicholinium-3 (HC-3), an inhibitor of Ch accumulation. In incubations (3 min) where the total hemidiaphragm acetylcholine (ACh) content is not altered by 26 microM HC-3, the inhibition by HC-3 of both the Ch accumulation rate and the incorporation of radioactive Ch into ACh in the EPA of stimulated tissues is less in the presence of 0.2 microM Dex. In 120 min incubations with 15 microM HC-3 and without added Ch, the tissue ACh content is depleted in both stimulated and unstimulated hemidiaphragms. In both cases the depletion of ACh is significantly less in the presence of 0.2 microM Dex. In stimulated tissues a comparable depletion of ACh due to 15 microM HC-3 is also found with 1 and 10 microM Ch added to the medium. It is significantly less when 0.2 microM Dex and 1 microM Ch are added to the medium. In 120 min incubations with stimulated tissue, the amount of "bound' ACh is increased by addition of 30 microM Ch to the medium, decreased in the presence of 0.2 microM Dex, and greatly decreased in the presence of 15 microM HC-3. In the presence of Dex plus HC-3, the decrease in the amount of "bound' ACh due to either Dex or HC-3 alone, is abolished provided that 30 microM Ch is also present.(ABSTRACT TRUNCATED AT 250 WORDS)

Low concentrations of dexamethasone (up to 200 nM) increase the accumulation of choline (Ch) and its incorporation into acetylcholine (ACh) in the endplate rich area (EPA) of stimulated and unstimulated diaphragms in the presence of 10 microM Ch. Tissue ACh is not significantly altered, even after 140 min incubation. The specific radioactivity of the ACh in the EPA is thus increased by dexamethasone (Dex). The corticosteroid has no effects on acetylcholinesterase or choline acetyltransferase in diaphragm extracts. In the same medium, the amplitudes of the MEPPs, MEPCs and EPCs are also increased by Dex. Neither the quantal content of the EPCs nor the MEPP frequency, nor the half decay time of the MEPCs are altered. Therefore Dex (200 nM) increases both the resting and evoked output, and turnover of ACh in rat diaphragm. Beta-bungarotoxin (beta-BuTx) antagonizes the Dex-induced increase in Ch accumulation and its incorporation into ACh, and abolishes the increases in MEPC- and EPC-amplitudes, providing further argument for a presynaptic effect of Dex. In continuously-stimulated diaphragms, beta-BuTx causes an accumulation of ACh which is much greater than in unstimulated tissue. This accumulation of ACh is less in the presence of Dex, provided that Dex is added before beta-BuTx. The interaction of Dex and beta-BuTx is discussed in terms of their possible presynaptic sites of action.

A recently developed method for inducing fatal, IgE-mediated, bronchial and cardiovascular anaphylaxis in the rat was used to compare the effects of exogenously administered, purified alpha M-foetoprotein (alpha M FP) and BaSO4 pretreatment (as mean to induce an acute phase reaction with increased alpha M FP serum levels) with regard to mortality, bronchoconstriction and cardiovascular events. The BaSO4 pretreatment protected the rats almost completely against mortality, whereas exogenously administered alpha M FP offered no protection at all. With respect to the antigen-induced bronchoconstriction alpha M FP greatly inhibited the increase of the pulmonary resistance (RI), whereas the BaSO4 pretreatment suppressed either the dynamic lung compliance (Cdyn) or RI considerably. The cardiovascular events were only influenced by the BaSO4 pretreatment demonstrating a small but highly significant reduction of the initial fall in blood pressure together with a remarkable recovery within almost I h in the majority (91%) of the animals. Both exogenously administered alpha M FP and BaSO4 pretreatment increased the alpha M FP serum levels from a normal value of 59 +/- 4 micrograms/ml (n = 22), to 2732 +/- 252 micrograms/ml (n = 9) and 855 +/- 200 micrograms/ml (n = 22), respectively. From these data we conclude that the antianaphylactic activity of alpha M FP is limited to bronchoprotection of the more central parts of the lungs, whereas BaSO4 pretreatment covers a much broader antianaphylactic profile. This implies that BaSO4 pretreatment does not only induce alpha M FP but also other endogenous antianaphylactic factors.

Two paralysing toxins (A-MTX and B-MTX) from extracts of Microbracon hebetor (Say) wasps were isolated and purified by gel chromatography, ion exchange chromatography and gel electrophoresis. Both toxins are labile proteins with molecular weights of 43,700 and 56,700, respectively, as estimated by gel chromatography, and with isoelectric pH of 6.85 and 6.62, respectively. Both toxins are inactivated by proteolytic enzymes and by dithiothreitol, but A-MTX appears to be more resistant than B-MTX. The relative amino acid compositions of both toxins show great similarity. The biological effects of the two toxins were identical to those previously found for crude toxin preparations.