Hepatocyte odd protein shuttling (HOPS) is a bridging protein in the nucleophosmin-p19 Arf network.

Nucleophosmin (NPM), a ubiquitously and abundantly expressed protein, occurs in the nucleolus, shuttling between the nucleoplasm and cytoplasm. The NPM gene is mutated in almost 30% of human acute myeloid leukemia cells. NPM interacts with p53 and p19(Arf), directs localization of p19(Arf) in the nucleolus and protects the latter from degradation. Hepatocyte odd protein shuttling (HOPS) is also a ubiquitously expressed protein that moves between the nucleus and cytoplasm. Within the nucleus of resting cells, HOPS overexpression causes cell cycle arrest in G0/G1. HOPS knockdown causes centrosome hyperamplification leading to multinucleated cells and the formation of micronuclei. We demonstrate a direct interaction of HOPS with NPM and p19(Arf), resulting in a functionally active trimeric complex. NPM appeared to regulate HOPS half-life, which, in turn, stabilized p19(Arf) and controlled its localization in the nucleolus. These findings suggest that HOPS acts as a functional bridge in the interaction between NPM and p19(Arf), providing new mechanistic insight into how NPM and p19(Arf) will oppose tumor cell proliferation.

Promoter characterization and expression of the gene coding for the human GM2 activator protein.

Genomic clones of the human GM2 activator protein have been isolated and analyzed. The 5' region of the gene demonstrated promoter activity as ascertained by its ability to drive luciferase gene expression in transfected COS cells. This sequence contains GC rich region and several putative promoter elements were present, including Sp1, AP2, cAMP-responsive element, and B-cell-specific activating protein. Analysis of tissue distribution of the GM2 activator protein gene revealed tissue-specific variations in transcript levels. Placenta, bone marrow, mammary gland, bladder, lymph node, and spleen had the highest mRNA levels.

Differential expression of CD44 isoforms during liver regeneration in rats.

BACKGROUND: CD44 is a transmembrane glycoprotein known to bind hyaluronic acid (HA). This molecule is a multifunctional cell surface glycoprotein involved in lymphocyte homing and activation, tumor growth and metastasis. We have investigated the qualitative modification of CD44 in the regenerating liver as a model for studying cellular proliferation in vivo. Molecules involved in cell adhesion and the extracellular matrix (ECM), which influence differentiation, growth, cell-cell interactions and cellular polarity, play an important role in the liver regeneration. We studied the modulation of CD44 gene expression and its post-transcriptional modifications, analyzing the expression of different isoforms containing exon v6 in the regenerating liver, in sham operated liver and in the hepatoma cells H-35. METHODS: The expression of CD44 and CD44v6 were analyzed in RNA extracted from regenerating liver at different times after partial hepatectomy (PH), and H-35 hepatoma cells by Northern blot, RT-PCR and Southern blot, and in protein extracts from regenerating liver by Western blot. H-35 hepatoma cells were assayed with the antibody cross-linked technique with CD44 antibodies. RESULTS: The standard CD44 form is expressed in regenerating liver and its levels were not modified following PH. However, our analysis revealed CD44 isoforms containing v6 in the first hours after PH as well as in the H-35 hepatoma cell line. H-35 cells treated with cross-linked anti-CD44 antibodies or HA show an increased rate of incorporation of [3H]thymidine (30 and 25%, respectively) with respect to the control. CONCLUSION: These findings suggest that CD44 may play a role in the proliferation of residual hepatocytes following PH.

Transcription factor CREM coordinates the timing of hepatocyte proliferation in the regenerating liver.

The liver regenerates upon partial hepatectomy (PH) as terminally differentiated hepatocytes undergo a tremendous proliferative process. CREM gene expression is powerfully induced during liver regeneration. We show that cell proliferation is significantly reduced upon PH in CREM-/- mice. There is a reduction in DNA synthesis, in the number of mitosis and of phosphorylated histone H3-positive cells. The post-PH proliferation peak is delayed by 10 hr, indicating an altered hepatocyte cell cycle. Expression of cyclins A, B, D1, E, and cdc2, of c-fos and tyrosine aminotransferase is deregulated. CREM mutation results in delayed S-phase entry, impairing the synchronization of proliferation.

Stress-induced expression of transcriptional repressor ICER in the adrenal gland.

Second messenger cyclic AMP plays a central role in signalling within the hypothalamo-pituitary-adrenal (HPA) axis. Changes in gene expression are central to long-term adaptations made in response to stress in the adrenal gland. Here we demonstrate that expression of the cAMP inducible transcriptional repressor, ICER (Inducible cAMP Early Repressor), is rapidly and powerfully induced in response to surgical stress in the rat adrenal gland. Hypophysectomisation blocks stress-induced ICER expression. Finally we demonstrate that injection of the pituitary hormone ACTH (Adrenocorticotropin Hormone) induces robust ICER expression in the adrenal cortex. Thus, induction of the transcriptional repressor ICER is coupled to the HPA axis response to stress.

Hexosaminidase in Trichinella spiralis is a single protein with alpha- and beta-subunits catalytic activities.

Beta-N-acetylhexosaminidase is expressed as a single protein in Trichinella spiralis and has catalytic properties similar to the alpha- and beta-subunits of human and mouse isoenzymes A and B. It can hydrolyze the artificial substrates, 4-methylumbelliferyl-beta-D-glucosamine and 4-methylumbelliferyl-beta-D-glucosamine-6-sulphate which are respectively hydrolyzed by the beta- and alpha-subunits. The enzyme is thermostable, has a basic isoelectric point, and thus is similar to the B isoenzyme. Northern blotting experiments indicate that the enzyme is encoded by a single gene. Hexosaminidase from Trichinella spiralis shows that the substrate specificities of alpha- and beta-subunits precede the duplication of their genes.

Lysosomal alpha-mannosidases of mouse tissues: characteristics of the isoenzymes, and cloning and expression of a full-length cDNA.

Lysosomal alpha-d-mannosidase from mouse tissues was separated into its constituent isoenzymes by DEAE-cellulose chromatography. Forms corresponding to the human isoenzymes B and A were present in testis, brain, spleen and kidney, whereas in epididymis and liver only the B form was present. Murine alpha-mannosidases A and B are glycoproteins and have pH optima, thermal stabilities and molecular masses similar to those of the human isoenzymes. A full-length cDNA (3.1 kb) containing the complete coding sequence for alpha-mannosidase was isolated from a mouse macrophage cDNA library. Comparison of the deduced amino acid sequences of human and mouse alpha-mannosidases showed that they had 75% identity and 83% similarity. Expression of this cDNA in COS cells showed that both the A and the B isoenzymes can arise from a single transcript. Northern blotting analysis showed a 10-fold range in the abundance of alpha-mannosidase mRNA in mouse tissues, with the highest levels found in epididymis, and the lowest in liver.

Cyclic AMP signalling and cellular proliferation: regulation of CREB and CREM.

In eukaryotes, transcriptional regulation upon stimulation of the adenylyl cyclase signalling pathway is mediated by a family of cAMP-responsive nuclear factors. This family consists of a large number of members which may act as activators or repressors. These factors contain the basic domain/leucine zipper motifs and bind as dimers to cAMP-response elements (CRE). The function of CRE-binding proteins (CREB) is modulated by phosphorylation by the cAMP-dependent protein kinase. The ICER (inducible cAMP early repressor) protein is the only inducible member of this family and is a product of the CREM gene. The induction of this powerful repressor is likely to be important for the transient nature of cAMP-induced gene expression. CREB proteins have been found to play an important role in the physiology of neuroendocrine functions. In addition, recent results indicate that CREB and CREM could be involved in the proliferation of hepatocytes which follows partial hepatectomy.

Cyclic AMP signalling pathway and cellular proliferation: induction of CREM during liver regeneration.

The CREM gene encodes both activators and repressors of cAMP-induced gene expression. An isoform of CREM encodes the powerful transcriptional repressor ICER (Inducible cAMP Early Repressor), which has been shown to be inducible by virtue of an alternative, intronic promoter. The CREM gene belongs to the early response class and displays a characteristic neuroendocrine cell- and tissue-specific expression. To date ICER inducibility has been described in non-replicating, terminally differentiated tissues. In this paper we document a robust induction of CREM expression in the regenerating rat liver after partial hepatectomy. This represents the first link of inducible CREM expression to the phenomenon of cellular proliferation. Furthermore, it represents the first example of transcriptional activation of a cAMP-responsive factor in the regenerating liver. This has significant physiological relevance since the adenylate cyclase signalling pathway is strongly implicated in liver regeneration. Finally, we show that the repressor ICER is inducible in the hepatoma cell line H35 upon activation of the adenylate cyclase and phosphorylation of the activator CREB.

GM2 activator protein expression in mouse tissues.

The GM2 activator protein is an essential cofactor of hexosaminidase A in the degradation of GM2 ganglioside and it is responsible for the variant AB of GM2 gangliosidosis in man. In this study GM2 activator protein and its mRNA were determined in different mouse tissues. It was found that this protein is expressed mostly in the spleen and testis followed by brain and kidney which represent the main source in man. It is also interesting that in mouse testis there is a higher expression of the alpha subunit of hexosaminidase, thus suggesting a relationship between alpha subunit and GM2 activator protein. Furthermore the results indicate that the expression of GM2 activator protein is regulated, at least in part, at the transcriptional level.

Changes in oncogene expression in ascite tumour cells during ageing.

The expression of two oncogenes, c-myc and c-fos, was studied in an ascitic tumour (ATPC+) at different times after implantation. The specific mRNA synthesis was analysed by Northern blot analysis. The presence of the oncogene proteins was shown by immunofluorescence using flow cytometry and referred to the distribution of the cells in the different cell phases. The results show that both oncogenes are expressed by ATPC+ tumour cells. c-myc is expressed 5, 8 and 12 days after implantation, although with a different intensity, and the protein is mainly present in S or S+G2 phase cells. The c-fos oncogene is expressed only 12 days after tumour implantation and the cells labelled with the specific antibody are mainly in G1 phase. We conclude that c-myc is principally correlated with proliferative activity, whereas c-fos is expressed by non-cycling cells.

Different expression of beta-N-acetylhexosaminidase in mouse tissues.

The expression of genes encoding for the alpha and beta-subunits of the lysosomal enzyme beta-N-acetylhexosaminidase was investigated in different mouse tissues. It was found, using fluorogenic substrates, that the amounts of alpha and beta subunits were not the same in different tissues: alpha-subunit was more abundant in the brain, beta-subunit in epididymis and brain. The different isoenzyme patterns and specific activities in mouse tissues are due to the differences in the amount of hexosaminidase subunits. The mRNA, evaluated by Northern blotting analyses, revealed a greater expression of alpha-subunit in the testis and of beta-subunit in the brain and epididymis. The results indicate, therefore, that gene expression and the amount of subunits are in good relationship for beta-subunit, whereas there is no correlation for alpha-subunit.

Tyrosine aminotransferase gene regulation during aging.

Tyrosine aminotransferase activity (TAT) and its expression were measured in rats of different ages (3, 12 and 24 months). The enzyme activity showed a circadian rhythm with a peak at midnight due to different levels of transcription during the day. The circadian rhythm was present at all ages studied but showed some differences: an age-related shift of the peak was more evident in the actual levels of transcript. Both specific mRNA and enzyme activity analyses provide better insight into the complex modifications in gene-expression of aging animals.

Different expression of tyrosine aminotransferase and serine deydratase in rat livers after partial hepatectomy.

Tyrosine aminotransferase activity in rat liver increases during the first 24 hrs after partial hepatectomy with two peaks, one at 10 hrs and another at 18 hrs. This behaviour is due to an increase in TATmRNA synthesis. Expression of serine deydratase is also enhanced during the first 5 hrs after hepatectomy. It is suggested that the enhanced expression of the two genes is due to an increase in hormone incretion particularly glucagon and glucocorticoids.

[Abnormal origin of the left coronary artery from the pulmonary artery. Description of a rare case in adulthood]. / Origine anomala della coronaria sinistra dall'arteria polmonare. Descrizione di un raro caso in età adulta.

The anomalous origin of the left coronary artery from the pulmonary artery is a rare and usually fatal congenital malformation. The Authors present a case of anomalous left coronary artery arising from the pulmonary artery diagnosed in an adult patient.

Variation of tyrosine aminotransferase expression during the day in rats of different ages.

The activity of the enzyme tyrosine aminotransferase and the synthesis of its specific mRNA were evaluated at different hours of the day in the liver of 3-, 12- and 24-month old BN rats. The enzyme activity has a circadian rhythm with a peak at midnight in 3- and 12-month old, which shifts to 03.00 hrs in 24-month old animals, in agreement with previous results. The expression of TATmRNA also changes during the day indicating circadian fluctuations which change with age. In 3-month old rats the TATmRNA peak is at 19.00 hrs, preceding that of the enzyme activity. In 12-month old rats the TATmRNA synthesis reaches a maximum at midnight and in 24-month old rats at 03.00 hrs. The results show that the circadian rhythm of tyrosine aminotransferase activity is due to a different gene expression throughout the day, which is influenced by age.