[Lys61]N-Ras is able to induce full activation and nuclear accumulation of Cdk4 in NIH3T3 cells.

The elements of the cell cycle regulatory machinery activated by the oncogenic form of Ras, [Lys61]N-Ras, have been analysed in NIH3T3 cells. We demonstrate that [Lys61]N-Ras expression is able to induce full cdk4 activation. As already reported, oncogenic Ras expression was sufficient to induce cyclin D1 and p21cip1 expression and their association with cdk4. Furthermore, serum-starved [Lys61]N-Ras NIH3T3 cells showed nuclear accumulation of cyclin D1 and cdk4 not observed in serum-starved NIH3T3 cells. This accumulation of cdk4 into the cell nucleus observed in serum-starved [Lys61]N-Ras NIH3T3 cells was inhibited by a microinjection of neutralizing anti-Ras antibodies. Thus, active [Lys61]N-Ras was a sufficient signal to induce nuclear accumulation of cyclin D1/cdk4, leading to its full activation. Transfection of [Lys61]N-Ras NIH3T3 cells with an inactive form of MEK or their treatment with PD 98059, showed that nuclear translocation of cdk4 was MEK dependent. Interestingly, cells constitutively expressing [Lys61]N-Ras did not inactivate pRb and did not proliferate in the absence of serum. This may be due to the fact that although association of cdk2 with cyclin E and the translocation of those complexes to the nucleus were achieved, [Lys61]N-Ras expression was not sufficient to induce cdk2 activation. The high levels of p27(kip1) that were found in cyclin E/cdk2 complexes may be responsible for the inability of oncogenic Ras to activate this kinase. In consequence, oncogenic alterations that lead to a decrease in p27kip1 bound to cyclin E may cooperate with Ras to induce full cdk2 activation, pRb inactivation and thus cell proliferation.

New nuclear functions for calmodulin.

The data reported here summarize a series of results which reveal new functions for nuclear calmodulin (CaM). The addition of CaM inhibitors to cultures of proliferating NRK cells blocked the activity of the cyclin-dependent protein kinases 4 (cdk4) and 2 (cdk2), which are enzymes implicated in the progression of G1 and in the onset of DNA replication, respectively. CaM modulates the activity of cdk4 by regulating the nuclear location of both cdk4 and cyclin D, its associated regulatory subunit. By using CaM-affinity chromatography, we have recently identified two new nuclear CaM-binding proteins: (i) the protein La/SSB, which is an autoantigen implicated in several autoimmune diseases such as lupus erythematosus and Sjögren's syndrome (since La/SSB participates in the process of transcription mediated by RNA polymerase III, CaM could be involved in the regulation of this process); and (ii) the protein SAP145, a member of the spliceosome-associated proteins (SAPs) which is a subunit of the splicing factor SF3(b). This finding suggests the involvement of CaM in pre-mRNA splicing. Finally, a screening for new CaM-binding proteins in the fission yeast performed by using the phage display analysis, revealed that several nucleolar-ribosomal proteins associate to CaM, suggesting that CaM modulates ribosomal assembly and/or function.

Catalonia WHO Demonstration Project on Palliative Care Implementation 1990-1995: results in 1995.

A global, comprehensive, publicly planned and financed program to implement palliative care was designed by the Department of Health of Catalonia (6 million inhabitants. Planned in collaboration with the cancer unit of the WHO in 1989, the program was implemented in 1990-1995. It included specific services, measures general resources, education and training, organizational and educational standards, opioid availability, legislation and evaluation. The aims included coverage for cancer, AIDS, geriatric and other conditions, equity, quality, reference, and satisfaction for patients, families, and professionals. The results in 1995 include the implementation of 18 hospital support teams and 19 Units, with a total of 350 beds, 42 home-care teams. The coverage for cancer and AIDS is around 40%, and 44/55 (80%) districts have a specific team. Palliative care implementation has been completely publicly financed, with a total yearly investment of 2,200 million ptas. Eighty percent of this has been saved through radical changes in costs and the pattern of the use of resources. Palliative care implementation has demonstrated efficacy in the care of the patients and families, efficiency in the provision of care, and cost-benefit in the regional global approach. It adds qualitative and organizational values to the health-care system. Its implementation must be prioritized and planned by the health administration, not only to improve the quality of care for advanced and terminal patients, but also to improve the global efficiency and appropriate use of resources in the public health system.

[Smoking prevalence trends in Catalonia, Spain, 1982-1994]. / La evolución del hábito tabáquico en Cataluña, 1982-1994.

BACKGROUND: Smoking prevalence trends from 1982 to 1994 of adult population in Catalonia (Spain) are described. SUBJECTS AND METHODS: Four population surveys have been carried out periodically using the same questionnaires and definitions for smoking status. Surveys in 1982, 1986 and 1990 have been implemented taking samples of Catalonia population through a multistage sampling with random stratified selection by province and habitat. Individuals were chosen through a random route process. In 1994, a survey with a complex probabilistic sample design with 8 geographical areas (health regions) and 2 basic units (towns and individuals) was implemented. RESULTS: Among the 15-64 years old adults, a decrease of 6.9% in smoking prevalence has been observed. The initial prevalence in 1982 was 37.9% (CI 95%: 35.4; 40.3); in 1994 this prevalence was 35.3% (CI 95%: 34.4; 36.2). In 1994, the prevalence of current smokers in population over 14 years old was 30.6% (CI 95%: 29.8; 31.4). We have observed a significant decrease in smoking prevalence in all age groups among male population (-20.6% for the 12-year period) whereas prevalence has increased among female (+28.0%) mainly among those between 25 and 54 years old. The main percentual decrease in smoking prevalence has been observed among young people aged 15-24 years old for both genders. The proportion of former smokers has remained stable (11.4% in 1982, 12.9% in 1994) during the period studied. The proportion of former smokers increases with age among man over 25 years. CONCLUSIONS: Smoking habit is still very prevalent in Catalonia, even higher than in other Western European countries. In spite of the increase among women, the significant dectines of smoking prevalence among men and youngsters (of both genders) could represent encouraging findings in order to pursue the efforts aimed at reducing the morbi-mortality burden of smoking in our society.

Effect of alpha 1-adrenergic blockade on nucleolar growth, chromatin relaxation, and histone H1(0) content in regenerating liver.

alpha 1-Adrenergic agonists are known to be involved in the regulation of hepatocyte proliferation after a partial hepatectomy. The blockade of alpha 1-adrenergic receptors with the specific antagonist prazosin inhibits DNA synthesis which peaks at 24 h after surgery. In this report we have studied the effects of prazosin administration on several events occurring during liver regeneration. The results show that the nuclear volume and nucleolar volume density of hepatocytes were increased and that the relative amount of heterochromatin decreased at 24 h. The increase in hepatocyte nucleolar volume density and the decrease in the relative amount of heterochromatin were partially abolished by prazosin administration while the increase in the nuclear volume was not affected. The relative amount of the histone H1 variant H1(0) was reduced in 24-h regenerating liver and prazosin treatment prevented this reduction.

[Inhibitory effect of human alpha 2-macroglobulin and a peptide released by trypsin, on the G1-S transition of hepatocytes in vivo]. / Effet inhibiteur de l'alpha 2 macroglobuline humaine et d'un peptide libéré par la trypsine, sur la transition G1-S des hépatocytes, in vivo.

Highly purified human alpha 2 M inhibits hepatocyte proliferation. 1 mg of alpha 2 M corresponds to 1 baby rat unit (BRU). alpha 2 M is bound to a low molecular weight glycopeptide, which is released during trypsinization of alpha 2 M. 3 micrograms of trypsin-treated alpha 2 M release 1 BRU. alpha 2 M and the glycopeptide have been shown to be identical, respectively, to high and low molecular weight components present in normal human plasma. Both components inhibit the G1-S transition of the hepatocyte cycle. alpha 2 M acts as an antagonist to the inhibitory effect of the glycopeptide when the molar ratio of trypsin to alpha 2 M is greater than 2.

Regulation of DNA polymerase alpha activity by the alpha 1-adrenergic receptors in proliferatively activated rat liver cells.

The administration of the alpha 1-adrenergic antagonist prazosin to hepatectomized rats inhibited DNA synthesis induced in the remaining hepatocytes. This inhibitory effect could be reversed by the simultaneous injection of the agonist phenylephrine. In order to establish how the alpha 1-adrenergic receptors can regulate DNA replication, the effect of prazosin administration on DNA polymerase alpha was examined. At 24 h after partial hepatectomy, the activity of DNA polymerase alpha increased 5, 7 and 9 fold in the homogenates, nuclei and nuclear matrix, respectively. This increase was inhibited by 70%-80% when prazosin was injected at 1, 8 or 11 h after surgery. Kinetic studies revealed that the Km for DNA was 2 fold lower in hepatectomized than in control animals. The administration of prazosin to hepatectomized rats increased the Km to the control values. These results indicate that the alpha 1-adrenergic receptors are involved in the regulation of DNA synthesis through the activation of DNA polymerase alpha and that this activation could be produced by increasing its affinity for DNA.

[Demonstration in human plasma of 2 factors inhibiting hepatocyte multiplication by blocking the cell cycle at the level of G1-S transition]. / Mise en évidence dans le plasma humain de deux facteurs inhibiteurs de la multiplication des hépatocytes par blocage du cycle cellulaire au niveau de la transition G1-S.

A factor which inhibits the G1-S transition of synchronized proliferating hepatocytes is detected in human serum or plasma. This activity is associated with an electronegatively charged component of high molecular weight in the native serum. Serum and plasma submitted to trypsin hydrolysis or to liver microsomes, release an ultrafiltrable component of low molecular weight which displays the same activity as the high molecular weight one from the native serum. This inhibitory system of hepatocyte proliferation is similar to that already described in Rat serum. It is species independent.

Calmodulin is essential for cyclin-dependent kinase 4 (Cdk4) activity and nuclear accumulation of cyclin D1-Cdk4 during G1.

Although it is known that calmodulin is involved in G1 progression, the calmodulin-dependent G1 events are not well understood. We have analyzed here the role of calmodulin in the activity, the expression, and the intracellular location of proteins involved in G1 progression. The addition of anti-calmodulin drugs to normal rat kidney cells in early G1 inhibited cyclin-dependent kinase 4 (Cdk4) and Cdk2 activities, as well as retinoblastoma protein phosphorylation. Protein levels of cdk4, cyclin D1, cyclin D2, cyclin E, p21, and p27 were not affected after CaM inhibition, whereas decreases in the amount of cyclin A and Cdc2 were observed. The decrease of Cdk4 activity was due neither to changes in its association to cyclin D1 nor to changes in the amount of p21 or p27 bound to cyclin D1-Cdk4 complexes. Calmodulin inhibition also produced a translocation of nuclear cyclin D1 and Cdk4 to the cytoplasm. This translocation could be responsible for the decreased Cdk4 activity upon calmodulin inhibition. Immunoprecipitation, calmodulin affinity chromatography, and direct binding experiments indicated that calmodulin associates with Cdk4 and cyclin D1 through a calmodulin-binding protein. The facts that Hsp90 interacts with Cdk4 and that its inhibition induced Cdk4 and cyclin D1 translocation to the cytoplasm point to Hsp90 as a good candidate for being the calmodulin-binding protein involved in the nuclear accumulation of Cdk4 and cyclin D1.

Evidence for the Involvement of annexin 6 in the trafficking between the endocytic compartment and lysosomes.

Annexins are a family of calcium-dependent phospholipid-binding proteins, which have been implicated in a variety of biological processes including membrane trafficking. The annexin 6/lgp120 prelysosomal compartment of NRK cells was loaded with low-density lipoprotein (LDL) and then its transport from this endocytic compartment and its degradation in lysosomes were studied. NRK cells were microinjected with the mutated annexin 6 (anx6(1-175)), to assess the possible involvement of annexin 6 in the transport of LDL from the prelysosomal compartment. The results indicated that microinjection of mutated annexin 6, in NRK cells, showed the accumulation of LDL in larger endocytic structures, denoting retention of LDL in the prelysosomal compartment. To confirm the involvement of annexin 6 in the trafficking and the degradation of LDL we used CHO cells transfected with mutated annexin 6(1-175). Thus, in agreement with NRK cells the results obtained in CHO cells demonstrated a significant inhibition of LDL degradation in CHO cells expressing the mutated form of annexin 6 compared to controls overexpressing wild-type annexin 6. Therefore, we conclude that annexin 6 is involved in the trafficking events leading to LDL degradation.

Antineutrophil antibodies associated with ulcerative colitis interact with the antigen(s) during the process of apoptosis.

BACKGROUND: Cell death by apoptosis seems to be an important mechanism for translocation to the cell surface of a variety of intracellular components capable of inducing autoantibody production. AIMS: To identify the cellular location of antigen (Ag)-antineutrophil cytoplasmic antibodies (ANCA) in non-apoptotic human neutrophils, and to assess if ANCA associated with ulcerative colitis reacts with neutrophil antigen(s) during neutrophil apoptosis. The cellular distribution of Ag-ANCA in apoptotic neutrophils was also investigated. METHODS: Sera from 18 ulcerative colitis patients known to be positive for perinuclear IgG-ANCA (titre > or =1/320), as assessed by indirect immunofluorescence (IIF), were analysed by immunofluorescent confocal laser scanning microscopy. ANCA were identified with fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) in non-apoptotic and apoptotic neutrophils, respectively. Apoptotic and non-apoptotic DNA was labelled with FITC and propidium iodide, respectively. Cycloheximide was added to polymorphonuclear leucocyte culture to induce apoptosis. RESULTS: Three patterns of scanning laser immunofluorescence microscopy in non-apoptotic neutrophils were observed with respect to cellular ulcerative colitis associated ANCA distribution: (1) diffuse nuclear localisation (16.7%); (2) nuclear localisation in the nuclear periphery (50%); and (3) mixed nuclear and cytoplasmic localisation (33.4%). In all sera ANCA fluorescence colocalised almost completely with apoptotic DNA, with persistence of a diffuse and intense fluorescence. No significant changes in ANCA titres were found in non-apoptotic neutrophils. CONCLUSIONS: The antigen(s) of ANCA associated with ulcerative colitis seems to be localised in most cases in the neutrophil nucleus. The almost identical colocalisation of ANCA and apoptotic cleaved DNA suggests that intracellular DNA redistribution during neutrophil apoptosis may play a role in antigen exposure to the immune system and ANCA production in ulcerative colitis.

Quantitative and qualitative changes of plasma membrane glycoproteins in the early period of liver regeneration.

Alterations of cell surface glycoconjugates have been observed in many developing systems and may be important in the physiological control of growth and differentiation. Liver regeneration after partial hepatectomy is a suitable model in which to study the regulatory mechanisms of cell proliferation in vivo. We have isolated the sinusoidal plasma membrane of hepatocytes at different times after partial hepatectomy. The sialic acid content and the SDS-polyacrylamide gel electrophoresis pattern of glycoproteins were determined. A decrease of periodic acid-Schiff-profiles, a change in the binding capacities of 125I-concanavalin A, a reduction of the sialic acid content and the appearance and disappearance of specific components have been observed during the pre-replicative phase of liver regeneration. These findings during this early period are consistent with the active involvement of the plasma membrane glycoproteins in the transition of cells to the proliferative state.

Dialysis pruritus: effect of cimetidine.

The effect of oral Cimetidine compared to a classic antihistaminic and to a placebo was evaluated, under the double hypothesis of its anti-PTH and anti-pruritus action, in a population of patients recently included in Hemodialysis that were asked about the incidence and intensity of pruritus according to a scored questionnaire made for this purpose. No different efficacy of Cimetidine versus the antihistaminic classic and the placebo was observed in our study.

Calmodulin-binding proteins in the nuclei of quiescent and proliferatively activated rat liver cells.

alpha-Spectrin, myosin light chain kinase (MLCK), and caldesmon have been detected in the nuclei of rat liver cells by 125I-calmodulin overlay, immunoblotting, and immunocytochemical methods. alpha-Spectrin is localized in the nuclear matrix, nuclear envelope, and nuclear pores. It has also been detected inside the nuclei in the form of small aggregates. MLCK is present in the nuclear matrix, envelope, nucleoli, and in a nuclease extract (S1 subfraction) but not in the nuclear pores. Caldesmon shows a diffuse distribution pattern inside the nuclei but it is not present in the nucleoli. Since all these proteins are components of the actin-myosin motility systems the presence of actin in the different nuclear subfractions has also been investigated: actin is present in the nuclear matrix, nuclear envelope, nucleoli, and nuclear pores. Proliferative activation of rat liver cells in vivo by partial hepatectomy induces the increase of alpha-spectrin, MLCK, and actin in different nuclear subfractions. This, together with the increase of nuclear calmodulin at the same time after hepatectomy (Pujol, M. J., Soriano, M., Aligúe, R., Carafoli, E., and Bachs, O. (1989) J. Biol. Chem. 264, 18863-18865), indicates that nuclear calmodulin could activate a nuclear contractile system during proliferative activation. A 62-kDa protein (p62) which binds to calmodulin columns and shows immunological similarities to caldesmon is specifically located in the region surrounding the nuclear envelope and is associated with the heterochromatin.

Nuclear calmodulin/62 kDa calmodulin-binding protein complexes in interphasic and mitotic cells.

We report here that a 62 kDa calmodulin-binding protein (p62), recently identified in the nucleus of rat hepatocytes, neurons and glial cells, consists of four polypeptides showing pI values between 5.9 and 6.1. By using a DNA-binding overlay assay we found that the two most basic of the p62 polypeptides bind both single- and double-stranded DNA. The intranuclear distribution of calmodulin and p62 was analysed in hepatocytes and astrocyte precursor cells, and in proliferating and differentiated astrocytes in primary cultures by immunogold-labeling methods. In non-dividing cells nuclear calmodulin was mostly localized in heterochromatin although it was also present in euchromatin and nucleoli. A similar pattern was observed for p62, with the difference that it was not located in nucleoli. p62/calmodulin complexes, mainly located over heterochromatin domains were also observed in interphasic cells. These complexes remained associated with the nuclear matrix after in situ sequential extraction with nucleases and high-salt containing buffers. In dividing cells, both calmodulin and p62 were found distributed over all the mitotic chromosomes but the p62/calmodulin aggregates were disrupted. These results suggest a role for calmodulin and p62 in the condensation of the chromatin.

Calmodulin binds to p21(Cip1) and is involved in the regulation of its nuclear localization.

p21(Cip1), first described as an inhibitor of cyclin-dependent kinases, has recently been shown to have a function in the formation of cyclin D-Cdk4 complexes and in their nuclear translocation. The dual behavior of p21(Cip1) may be due to its association with other proteins. Different evidence presented here indicate an in vitro and in vivo interaction of p21(Cip1) with calmodulin: 1) purified p21(Cip1) is able to bind to calmodulin-Sepharose in a Ca(2+)-dependent manner, and this binding is inhibited by the calmodulin-binding domain of calmodulin-dependent kinase II; 2) both molecules coimmunoprecipitate when extracted from cellular lysates; and 3) colocalization of calmodulin and p21(Cip1) can be detected in vivo by electron microscopy immunogold analysis. The carboxyl-terminal domain of p21(Cip1) is responsible for the calmodulin interaction, since p21(145-164) peptide is also able to bind calmodulin and to compete with full-length p21(Cip1) for the calmodulin binding. Because treatment of cells with anti-calmodulin drugs decreases the nuclear accumulation of p21(Cip1), we hypothesize that calmodulin interaction with p21(Cip1) is important for p21(Cip1), and in consequence for cyclin D-Cdk4, translocation into the cell nucleus.