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S Polo

Publications and source records attributed to S Polo.

At least 19 recordsLinked to original sources
PubMed2004

Signaling through monoubiquitination.

Ubiquitination is a post-translational modification in which a small conserved peptide, ubiquitin, is appended to target proteins in the cell, through a series of complex enzymatic reactions. Recently, a particular form of ubiquitination, monoubiquitination, has emerged as a nonproteolytic reversible modification that controls protein function. In this review, we highlight recent findings on monoubiquitination as a signaling-induced modification, controlled, among others, by pathways originating from active receptor tyrosine kinases. Furthermore, we review the major cellular processes controlled by ubiquitin modification, including membrane trafficking, histone function, transcription regulation, DNA repair, and DNA replication.

Animals↗
PubMed2002

[Influence of Gilbert's syndrome on serum bilirubin levels and gallstone formation in children with chronic hemolytic disease].

To determine whether Gilbert's syndrome increases the risk of gallstone formation in children with chronic hemolytic disease, we studied 44 children with this diagnosis. Gallstones were detected by abdominal ultrasonography. This took place annually in scheduled examinations or in the context of acute abdominal pain. In all patients, the mean values of hemoglobin, reticulocyte and serum bilirubin in the chronic phase were recorded. In addition, TA insertion in the A(TA)nTATAA motif within the promoter region of the enzyme uridine-diphosphate-glucuronyl transferase (UGT1A1) was screened, since this is typically associated with GS.We found 10 (22.7 %) homozygotes for the mutated allele TA*7/TA*7, 12 (27.3 %) TA*6/TA*6 heterozygotes and 22 (50 %) homozygotes for the wild-type allele TA*6/TA*6. No statistically significant differences were found in the values of hemoglobin (Kruskal-Wallis test 2.496; p > 0.05) or in reticulocyte count (Kruskal-Wallis test 1.696; p > 0,05) between the three groups of patients, suggesting a similar degree of hemolysis. Patients with the UGT1A1 TA*7/TA*7 genotype showed higher mean serum bilirubin levels than did patients who were homozygous for the wild-type allele (Mann-Whitney test 35.5; p < 0.05). None of the patients with the TA*6/TA*6 genotype developed gallstones, whereas this complication was found in 2 of 12 (16.6 %) heterozygotes and 6 of 10 (60 %) homozygotes for the allele with TA insertion. In this latter group, 4 patients presented acute pancreatitis as a consequence of gallstone formation.The association between increased bilirubin load due to chronic hemolytic disease and diminished hepatic conjugation leads to raised serum bilirubin levels and consequently to an increased risk of gallstone formation. Therefore, we recommend screening for Gilbert's syndrome in children in the initial phases of chronic hemolytic diseases.

Bilirubin↗
PubMed2001

Nucleocytoplasmic shuttling of endocytic proteins.

Many cellular processes rely on the ordered assembly of macromolecular structures. Here, we uncover an unexpected link between two such processes, endocytosis and transcription. Many endocytic proteins, including eps15, epsin1, the clathrin assembly lymphoid myeloid leukemia (CALM), and alpha-adaptin, accumulate in the nucleus when nuclear export is inhibited. Endocytosis and nucleocytoplasmic shuttling of endocytic proteins are apparently independent processes, since inhibition of endocytosis did not appreciably alter nuclear translocation of endocytic proteins, and blockade of nuclear export did not change the initial rate of endocytosis. In the nucleus, eps15 and CALM acted as positive modulators of transcription in a GAL4-based transactivation assay, thus raising the intriguing possibility that some endocytic proteins play a direct or indirect role in transcriptional regulation.

Active Transport, Cell Nucleus↗
PubMed2001

Structural determinants of CCR5 recognition and HIV-1 blockade in RANTES.

Certain chemokines act as natural antagonists of human immunodeficiency virus (HIV) by blocking key viral coreceptors, such as CCR5 and CXCR4, on the surface of susceptible cells. Elucidating the structural determinants of the receptor-binding and HIV-inhibitory functions of these chemokines is essential for the rational design of derivative molecules of therapeutic value. Here, we identify the structural determinants of CCR5 recognition and antiviral activity of the CC chemokine RANTES, showing that critical residues form a solvent-exposed hydrophobic patch on the surface of the molecule. Moreover, we demonstrate that the biological function is critically dependent on dimerization, resulting in the exposure of a large ( approximately 180 A2), continuous hydrophobic surface. Relevant to the development of novel therapeutic approaches, we designed a retroinverted RANTES peptide mimetic that maintained both HIV- and chemotaxis-antagonistic functions.

Alanine↗
PubMed2001

Photodamage induced by Zinc(II)-phthalocyanine to microtubules, actin, alpha-actinin and keratin of HeLa cells.

We have studied the photosensitizing effects of zinc(II)-phthalocyanine (ZnPc) on the cytoskeleton of HeLa cells using sublethal (10(-7) M, followed by 1 or 3 min of red light to induce 20%, LD20, or 60%, LD60, cell death, respectively) or lethal (5 x 10(-6) M and 15 min of irradiation, LD100) experimental conditions. The immunofluorescent analysis of the cytoskeleton showed a variable photodamage to microtubules (MT), actin microfilaments (AF) and intermediate filaments of keratin (KF), as well as on alpha-actinin, which was dependent on treatment conditions. Both sublethal treatments induced deep alterations on interphase and mitotic MT. The mitotic index increased with time with the maximum at 18 h (12%) or 24 h (14%) after LD20 or LD60, respectively. The alterations on AF and alpha-actinin were much more severe than those observed on KF at any evaluated time. With the exception of the KF, which remained partially organized, the MT and AF network was severely damaged by the lethal treatment. Western blot analysis for alpha-tubulin, G-actin and alpha-actinin from soluble and insoluble fractions confirmed the results observed by immunofluorescence, thus indicating that these cytoskeletal components are involved in cell damage and death by ZnPc photosensitization.

Actinin↗
PubMed2000

Multi-step purification strategy for RANTES wild-type and mutated analogues expressed in a baculovirus system.

RANTES (regulated on activation, normal T cell expressed and secreted), a C-C chemokine, is one of the major HIV-suppressive factors produced by CD8+ T cells. Wild-type RANTES and genetically modified analogues were expressed in a baculovirus system and purified from cell culture supernatants employing a multi-step strategy based on affinity and RP-HPLC. Quantification and purity control of the final proteins were carried out by capillary electrophoresis using the synthetic or the recombinant wild-type RANTES as a reference. The procedure here reported requires only three days to obtain 0.016-0.270 mg of the pure and characterised proteins, starting from 370-900 ml of culture media, and is suitable for the analysis of a large number of RANTES analogues.

Baculoviridae↗
PubMed2000

Enhancement of the HIV-1 inhibitory activity of RANTES by modification of the N-terminal region: dissociation from CCR5 activation.

Although selected chemokines act as natural inhibitors of human immunodeficiency virus (HIV) infection, their inherent proinflammatory activity may limit a therapeutic use. To elucidate whether the antiviral and signaling functions of RANTES can be dissociated, several recombinant analogues mutated at the N terminus were generated and functionally compared with the wild-type (WT) molecule, as well as with three previously described mutants. Substitution of selected residues within the N-terminal region caused a marked loss of antiviral potency. By contrast, two unique analogues (C1.C5-RANTES and L-RANTES) exhibited an increased antiviral activity against different CXCR4-negative HIV-1 isolates grown in primary mononuclear cells or in macrophages. This enhanced HIV-blocking activity was associated with an increased binding affinity for CCR5. Both C1.C5-RANTES and L-RANTES showed a dramatically reduced ability to trigger intracellular calcium mobilization via CCR3 or CCR5, while potently antagonizing the action of the WT chemokine. By contrast, two previously described analogues (RANTES(3-68) and AOP-RANTES) maintained a WT ability to trigger CCR5-mediated signaling, while a third one (RANTES(9-68)) showed a dramatic loss of antiviral activity. These data demonstrate that the antiviral and signaling functions of RANTES can be uncoupled, opening new perspectives for the development of chemokine-based therapeutic approaches for HIV infection.

Anti-HIV Agents↗
PubMed2000

Construction of a full length infectious clone for dengue-1 virus Western Pacific,74 strain.

The flavivirus dengue 1 Western Pacific,74 (DEN1 WP) virus has a positive-stranded RNA genome of 10,735 nucleotides. DEN1 WP genomic RNA was amplified into three overlapping fragments by RT-PCR. These fragments were assembled into a full-length cDNA clone in the yeast-E. coli shuttle vector pRS424, using homologous recombination in yeast. RNA produced by in vitro transcription of this clone was infectious upon electroporation into LLCMK2 cells, as shown by cytopathic effects and detection of viral antigens by indirect immunofluorescence, and by propagation of the virus released into the culture media. Biological properties of the transcript-derived virus, such as the pattern of dengue-specific protein synthesis and growth rate in LLCMK2 or C6/36 cells, resembled those of the parent DEN1 WP virus.

Animals↗
PubMed1999

Nonproductive human immunodeficiency virus type 1 infection of human fetal astrocytes: independence from CD4 and major chemokine receptors.

Human immunodeficiency virus type 1 (HIV-1) infection of the brain is associated with neurological manifestations both in adults and in children. The primary target for HIV-1 infection in the brain is the microglia, but astrocytes can also be infected. We tested 26 primary HIV-1 isolates for their capacity to infect human fetal astrocytes in culture. Eight of these isolates, independent of their biological phenotype and chemokine receptor usage, were able to infect astrocytes. Although no sustained viral replication could be demonstrated, the virus was recovered by coculture with receptive cells such as macrophages or on stimulation with interleukin-1beta. To gain knowledge into the molecular events that regulate attachment and penetration of HIV-1 in astrocytes, we investigated the expression of several chemokine receptors. Fluorocytometry and calcium-mobilization assay did not provide evidence of expression of any of the major HIV-1 coreceptors, including CXCR4, CCR5, CCR3, and CCR2b, as well as the CD4 molecule on the cell surface of human fetal astrocytes. However, mRNA transcripts for CXCR4, CCR5, Bonzo/STRL33/TYMSTR, and APJ were detected by RT-PCR. Furthermore, infection of astrocytes by HIV-1 isolates with different chemokine receptor usage was not inhibited by the chemokines SDF-1beta, RANTES, MIP-1beta, or MCP-1 or by antibodies directed against the third variable region or the CD4 binding site of gp120. These data show that astrocytes can be infected by primary HIV-1 isolates via a mechanism independent of CD4 or major chemokine receptors. Furthermore, astrocytes are potential carriers of latent HIV-1 and on activation may be implicated in spreading the infection to other neighbouring cells, such as microglia or macrophages.

Adult↗
PubMed1999

Longitudinal analysis of serum chemokine levels in the course of HIV-1 infection.

OBJECTIVES: To investigate the correlation between the serum levels of the CC-chemokines RANTES, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, and the progression of HIV-1 disease. DESIGN: Retrospective analysis of serial serum samples from HIV-1 seroconverters selected according to clinical outcome. METHODS: Twenty-one patients, derived from a cohort recruited between 1985 and 1996 for a prospective study of the natural history of HIV infection, were analysed. All patients had at least one HIV-1-seronegative sample within 1 year prior to the first seropositive test and were followed longitudinally throughout the course of HIV-1 infection (mean follow-up, 73.5 months). Nine were rapid progressors (RP; patients who developed AIDS within 60 months of antibody seroconversion), seven were slow progressors (SP; patients who developed AIDS after 60 months), and five were long-term asymptomatic (LTA; patients with circulating CD4+ cells higher than 400 x 10(6)/l, no signs of HIV disease, no antiretroviral therapy for more than 96 months). A total of 339 serum samples was studied (mean, 16.1 per patient). The levels of RANTES, MIP-1alpha and MIP-1beta were measured by enzyme-linked immunosorbent assay and correlated with different immunological and clinical parameters. RESULTS: Over the entire follow-up period, the geometric mean of serum RANTES was significantly higher in RP [68.6 ng/ml; 95% confidence interval (CI), 56.9-82.7] than in SP (23.7 ng/ml; 95% CI, 20.0-28.2; P < 0.001) and LTA (19.5 ng/ml; 95% CI, 15.5-24.5; P < 0.001). This difference was already significant during the early clinical stages, when patients had peripheral blood CD4+ cell counts still greater than 400 x 10(6)/l (P < 0.001). By contrast, the mean serum levels of MIP-1alpha and MIP-1beta did not differ significantly between the three study groups. Multivariate analysis using the Cox proportional hazard model demonstrated that the mean serum concentration of RANTES before the development of AIDS was independently associated with the time to AIDS (relative risk, 4.5; 95% CI, 1.1-18.2; P = 0.035). In patients with low versus high mean serum RANTES before the fall of CD4+ cells below 400 x 10(6)/l, the median AIDS-free time was 117.5 and 42.7 months, respectively (P = 0.037). CONCLUSION: These data suggest that an elevation of serum RANTES predicts a rapid progression of the disease since the early stages of HIV-1 infection.

Adult↗
PubMed1999

Microscopical and spectroscopic studies on the fluorescence of a daunomycin-aluminum complex.

In this study, the spectroscopic features and microscopical applications of the fluorescent daunomycin-Al3+ complex have been analyzed. In the presence of Al3+, the absorption spectrum of daunomycin showed a deep bathochromic shift and new peaks at 529 and 566 nm, whereas the fluorescence emission was considerably modified. The emission of daunomycin alone (peak at 560 nm under optimal excitation at 470 nm) decreased continuously from 0.5 to 24h after addition of Al3+ ions, and a new emission peak appeared at 580 nm (optimal excitation at 530 nm). Under the fluorescence microscope using green exciting light, nuclei from chicken blood smears and paraffin sections of rat embryos stained with daunomycin showed a weak emission, which greatly increased after treatment with Al3+ ions. The bright and stable fluorescence of chromatin DNA induced by daunomycin-Al3+ could be a valuable labelling method in fluorescence microscopy and DNA cytochemistry.

Aluminum Compounds↗
PubMed1999

Translation of two nested genes in bacteriophage P4 controls immunity-specific transcription termination.

In phage P4, transcription of the left operon may occur from both the constitutive PLE promoter and the regulated PLL promoter, about 400 nucleotides upstream of PLE. A strong Rho-dependent termination site, timm, is located downstream of both promoters. When P4 immunity is expressed, transcription starting at PLE is efficiently terminated at timm, whereas transcription from PLL is immunity insensitive and reads through timm. We report the identification of two nested genes, kil and eta, located in the P4 left operon. The P4 kil gene, which encodes a 65-amino-acid polypeptide, is the first translated gene downstream of the PLE promoter, and its expression is controlled by P4 immunity. Overexpression of kil causes cell killing. This gene is the terminal part of a longer open reading frame, eta, which begins upstream of PLE. The eta gene is expressed when transcription starts from the PLL promoter. Three likely start codons predict a size between 197 and 199 amino acids for the Eta gene product. Both kil and eta overlap the timm site. By cloning kil upstream of a tRNA reporter gene, we demonstrated that translation of the kil region prevents premature transcription termination at timm. This suggests that P4 immunity might negatively control kil translation, thus enabling transcription termination at timm. Transcription starting from PL proceeds through timm. Mutations that create nonsense codons in eta caused premature termination of transcription starting from PLL. Suppression of the nonsense mutation restored transcription readthrough at timm. Thus, termination of transcription from PLL is prevented by translation of eta.

Amino Acid Sequence↗
PubMed1999

Photokilling mechanisms induced by zinc(II)-phthalocyanine on cultured tumor cells.

The photosensitizing effects of liposomal zinc(II)-phthalocyanine (ZnPc) on HeLa cells, with emphasis on morphological changes and mechanisms for cell death, have been studied. No dark toxicity for ZnPc alone was found. Incubation for 1 h with ZnPc followed by red light irradiation induced a variable decrease in the surviving of cells, which was related to both drug concentration and irradiation time. A lethal photodynamic effect (100% of the cells are killed: LD100) was induced by 5 x 10-6 M ZnPc and 5-min irradiation, whereas a sublethal effect (60% of the cells are killed: LD60) was detected with 10 7 M ZnPc and 3 min of red light. Toluidine blue and Hoechst 33258 staining showed characteristic alterations of cell morphology. Numerous bubbles on the plasma membrane were found immediately after an LD100 treatment, and a necrotic morphology appeared 24 h later. On the contrary, severe cell shrinkage with nuclear fragmentation. characteristic of apoptosis. was observed 8 and 24 h after LD60 treatments. In this case, propidium iodide-acridine orange labeling and the TUNEL assay confirmed the occurrence of apoptosis. The highest amount of apoptotic cells appeared 24 h after LD60 treatments, particularly in detached cells, as revealed by cell counting and DNA electrophoresis. Both apoptotic and necrotic mechanisms for cell death occur in HeLa cells in dependence on the experimental protocol of ZnPc photodynamic treatments.

Apoptosis↗
PubMed1998

CXCR4 is a functional coreceptor for infection of human macrophages by CXCR4-dependent primary HIV-1 isolates.

The identification of HIV-1 coreceptors has provided a molecular basis for the tropism of different HIV-1 strains. CXC chemokine receptor-4 (CXCR4) mediates the entry of both primary and T cell line-adapted (TCLA) syncytia-inducing strains. Although macrophages (M phi) express CXCR4, this coreceptor is assumed to be nonfunctional for HIV-1 infection. We addressed this apparent paradox by infecting human monocyte-derived M phi with primary and TCLA isolates that were rigorously characterized for coreceptor usage and by adding the natural CXCR4 ligand, stem cell differentiation factor-1, to specifically block CXCR4-mediated entry. Our results show that primary HIV-1 isolates that selectively use CXCR4 productively infected both normal and C-C chemokine receptor-5-null M phi. By contrast, M phi supported the entry of CXCR4-dependent TCLA strains with variable efficiency but were not productively infected. Thus, the tropism of HIV isolates results from complex virus/host cell interactions both at the entry and postentry levels.

Anti-HIV Agents↗
PubMed1998

Meso-tetraphenylporphyrin: photosensitizing properties and cytotoxic effects on cultured tumor cells.

In this study we analyzed photosensitizing and photodamaging properties of the hydrophobic meso-tetraphenylporphyrin (TPP, incorporated into liposomes) on HeLa cells. Under the fluorescence microscope, red fluorescence by TPP was detected on the cell surface. TPP followed by violet-blue or red irradiation led to cell death, blebs and plasma membrane deformations appearing immediately after photodynamic treatment. Production of singlet oxygen by TPP was studied by analyzing tryptophan photodegradation, which increased in the presence of D2O and was abolished by NaN3. Present results suggest that the plasma membrane is the main cellular target for TPP, which could be a valuable photosensitizing drug in studies on photodynamic therapy of cancer.

HeLa Cells↗
PubMed1997

C-C chemokines released by lipopolysaccharide (LPS)-stimulated human macrophages suppress HIV-1 infection in both macrophages and T cells.

Human immunodeficiency virus-1 (HIV-1) expression in monocyte-derived macrophages (MDM) infected in vitro is known to be inhibited by lipopolysaccharide (LPS). However, the mechanisms are incompletely understood. We show here that HIV-1 suppression is mediated by soluble factors released by MDM stimulated with physiologically significant concentrations of LPS. LPS-conditioned supernatants from MDM inhibited HIV-1 replication in both MDM and T cells. Depletion of C-C chemokines (RANTES, MIP-1 alpha, and MIP-1 beta) neutralized the ability of LPS-conditioned supernatants to inhibit HIV-1 replication in MDM. A combination of recombinant C-C chemokines blocked HIV-1 infection as effectively as LPS. Here, we report an inhibitory effect of C-C chemokines on HIV replication in primary macrophages. Our results raise the possibility that monocytes may play a dual role in HIV infection: while representing a reservoir for the virus, they may contribute to the containment of the infection by releasing factors that suppress HIV replication not only in monocytes but also in T lymphocytes.

Cells, Cultured↗
PubMed1997

Infectious RNA transcripts from full-length dengue virus type 2 cDNA clones made in yeast.

The dengue virus type 2 genomic RNA was amplified by reverse transcription-PCR and cloned as four cDNA fragments. We could not assemble these four fragments into full-length cDNA in Escherichia coli. The full-length dengue virus cDNA was constructed by homologous recombination in yeast, either as part of a yeast artificial chromosome or in a yeast-E. coli shuttle vector. Full-length cDNA clones were propagated once in E. coli to prepare useful quantities of DNA. In vitro transcription of these clones produced full-length RNA transcripts. Introduction of these transcripts into LLC-MK2 cells produced typical dengue infection, as judged by cytopathic effects and indirect immunofluorescence. Infectivity was sensitive to RNase digestion and was dependent on the presence of cap analog in the transcription reaction mixture. Virus in the medium was passaged on C6-36 cells to produce stocks, and these stocks had titers and plaque morphologies similar to those of the parental dengue virus type 2. Intracellular dengue virus RNA from cells infected with transcript-derived virus contained an introduced BstEII site, proving that infectivity was derived from RNA transcripts and not from contamination with parental dengue virus. Transcript-derived virus was comparable to dengue virus type 2 for growth and protein expression in tissue culture cells. Sequence analysis of the dengue virus cDNA in one full-length clone revealed only one unexpected silent mutation. By using yeast technology, it will be easy to introduce specific mutations into the dengue virus cDNA, allowing analysis of the virus phenotype in cells transfected with mutant transcripts.

Aedes↗