Kaposi's Sarcoma-Associated Herpesvirus LANA-Adjacent Regions with Distinct Functions in Episome Segregation or Maintenance.

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that mediates episome persistence of viral genomes. LANA binds the KSHV terminal-repeat (TR) sequence through its carboxy-terminal domain to mediate DNA replication. LANA simultaneously binds mitotic chromosomes and TR DNA to segregate virus genomes to daughter cell nuclei. Amino-terminal LANA attaches to chromosomes by binding histones H2A/H2B, and carboxy-terminal LANA contributes to mitotic-chromosome binding. Although amino- and carboxy-terminal LANA are essential for episome persistence, they are not sufficient, since deletion of all internal LANA sequence renders LANA highly deficient for episome maintenance. Internal LANA sequence upstream of the internal repeat elements contributes to episome segregation and persistence. Here, we investigate this region with a panel of LANA deletion mutants. Mutants retained the ability to associate with mitotic chromosomes and bind TR DNA. In contrast to prior results, deletion of most of this sequence did not reduce LANA's ability to mediate DNA replication. Deletions of upstream sequence within the region compromised segregation of TR DNA to daughter cells, as assessed by retention of green fluorescent protein (GFP) expression from a replication-deficient TR plasmid. However, deletion of this upstream sequence did not reduce episome maintenance. In contrast, deletions that included an 80-amino-acid sequence immediately downstream resulted in highly deficient episome persistence. LANA with this downstream sequence deleted maintained the ability to replicate and segregate TR DNA, suggesting a unique role for the residues. Therefore, this work identifies adjacent LANA regions with distinct roles in episome segregation and persistence.IMPORTANCE KSHV LANA mediates episomal persistence of viral genomes. LANA binds the KSHV terminal-repeat (TR) sequence to mediate DNA replication and tethers KSHV DNA to mitotic chromosomes to segregate genomes to daughter cell nuclei. Here, we investigate LANA sequence upstream of the internal repeat elements that contributes to episome segregation and persistence. Mutants with deletions within this sequence maintained the ability to bind mitotic chromosomes or bind and replicate TR DNA. Deletion of upstream sequence within the region reduced segregation of TR DNA to daughter cells, but not episome maintenance. In contrast, mutants with deletions of 80 amino acids immediately downstream were highly deficient for episome persistence yet maintained the ability to replicate and segregate TR DNA, the two principal components of episome persistence, suggesting another role for the residues. In summary, this work identifies adjacent LANA sequence with distinct roles in episome segregation and persistence.

Kaposi's sarcoma-associated herpesvirus LANA recruits the DNA polymerase clamp loader to mediate efficient replication and virus persistence.

Kaposi's sarcoma-associated herpesvirus (KSHV) latently infects tumor cells and persists as a multiple-copy, extrachromosomal, circular episome. To persist, the viral genome must replicate with each cell cycle. The KSHV latency-associated nuclear antigen (LANA) mediates viral DNA replication and persistence, but little is known regarding the underlying mechanisms. We find that LANA recruits replication factor C (RFC), the DNA polymerase clamp [proliferating cell nuclear antigen (PCNA)] loader, to drive DNA replication efficiently. Mutated LANA lacking RFC interaction was deficient for LANA-mediated DNA replication and episome persistence. RFC depletion had a negative impact on LANA's ability to replicate and maintain viral DNA in cells containing artificial KSHV episomes or in infected cells, leading to loss of virus. LANA substantially increased PCNA loading onto DNA in vitro and recruited RFC and PCNA to KSHV DNA in cells. These findings suggest that PCNA loading is a rate-limiting step in DNA replication that is incompatible with viral survival. LANA enhancement of PCNA loading permits efficient virus replication and persistence, revealing a previously unidentified mechanism for KSHV latency.

Extraction of functional ingredients from spinach (Spinacia oleracea L.) using liquid solvent and supercritical CO2 extraction.

BACKGROUND: In this work three different techniques were applied to extract dry leaves of spinach (Spinacia oleracea): solid-liquid extraction (SLE), pressurised liquid extraction (PLE) and supercritical fluid extraction (SFE) to investigate the influence of extraction solvent and technique on extracts composition and antioxidant activity. Moreover, the influence of carotenoids and phenolic compounds on the antioxidant and anti-inflammatory activities of spinach extracts was also studied. RESULTS: The higher concentrations of carotenoids and the lower content of phenolic compounds were observed in the supercritical CO2 extracts; whereas water and/or ethanol PLE extracts presented low amounts of carotenoids and the higher concentrations of phenolic compounds. PLE extract with the highest content of phenolic compounds showed the highest antioxidant activity, although SFE carotenoid rich extract also showed a high antioxidant activity. Moreover, both extracts presented an important anti-inflammatory activity. CONCLUSION: PLE seems to be a good technique for the extraction of antioxidant and anti-inflammatory compounds from spinach leaves. Moreover, spinach phenolic compounds and carotenoids present a high antioxidant activity, whereas spinach carotenoids seem to show a higher anti-inflammatory activity than phenolic compounds. It is worth noting that of our knowledge this is the first time the anti-inflammatory activity of lipophilic extracts from spinach leaves is reported.

Identification of Kaposi's sarcoma-associated herpesvirus LANA regions important for episome segregation, replication, and persistence.

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that mediates the maintenance of episomal viral genomes in latently infected cells. The two central components of episome persistence are DNA replication with each cell division and the segregation of DNA to progeny nuclei. LANA self-associates to bind KSHV terminal-repeat (TR) DNA and to mediate its replication. LANA also simultaneously binds to TR DNA and mitotic chromosomes to mediate the segregation of episomes to daughter nuclei. The N-terminal region of LANA binds histones H2A and H2B to attach to mitotic chromosomes, while the C-terminal region binds TR DNA and also associates with chromosomes. Both the N- and C-terminal regions of LANA are essential for episome persistence. We recently showed that deletion of all internal LANA sequences results in highly deficient episome maintenance. Here we assess independent internal LANA regions for effects on episome persistence. We generated a panel of LANA mutants that included deletions in the large internal repeat region and in the unique internal sequence. All mutants contained the essential N- and C-terminal regions, and as expected, all maintained the ability to associate with mitotic chromosomes in a wild-type fashion and to bind TR DNA, as assessed by electrophoretic mobility shift assays (EMSA). Deletion of the internal regions did not reduce the half-life of LANA. Notably, deletions within either the repeat elements or the unique sequence resulted in deficiencies in DNA replication. However, only the unique internal sequence exerted effects on the ability of LANA to retain green fluorescent protein (GFP) expression from TR-containing episomes deficient in DNA replication, consistent with a role in episome segregation; this region did not independently associate with mitotic chromosomes. All mutants were deficient in episome persistence, and the deficiencies ranged from minor to severe. Mutants deficient in DNA replication that contained deletions within the unique internal sequence had the most-severe deficits. These data suggest that internal LANA regions exert critical roles in LANA-mediated DNA replication, segregation, and episome persistence, likely through interactions with key host cell factors.

The internal Kaposi's sarcoma-associated herpesvirus LANA regions exert a critical role on episome persistence.

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that acts on viral terminal repeat (TR) DNA to mediate KSHV episome persistence. The two essential components of episome persistence are DNA replication prior to cell division and episome segregation to daughter nuclei. These functions are located within N- and C-terminal regions of LANA. N- and C-terminal regions of LANA are sufficient for TR DNA replication. In addition, N- and C-terminal regions of LANA tether episomes to mitotic chromosomes to segregate episomes to progeny cell nuclei. To generate a tethering mechanism, N-terminal LANA binds histones H2A/H2B to attach to mitotic chromosomes, and C-terminal LANA binds TR DNA and also associates with mitotic chromosomes. Here, we test the importance of the internal LANA sequence for episome persistence. We generated LANA mutants that contain N- and C-terminal regions of LANA but have most of the internal sequence deleted. As expected, the LANA mutants bound mitotic chromosomes in a wild-type pattern and also bound TR DNA as assayed by electrophoretic mobility shift assays (EMSA). The mutants mediated TR DNA replication, although with reduced efficiency compared with LANA. Despite the ability to replicate DNA and exert the chromosome and DNA binding functions necessary for segregating episomes to daughter nuclei, the mutants were highly deficient for the ability to mediate both short- and long-term episome persistence. These data indicate that internal LANA sequence exerts a critical effect on its ability to maintain episomes, possibly through effects on TR DNA replication.

Rapid and quantitative assessment of KSHV LANA-mediated DNA replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) mediates DNA replication of terminal repeat (TR) DNA to enable viral episome persistence in latently infected cells. Southern blotting is routinely used to detect LANA-replicated DNA. We developed and validated a real-time PCR assay for TR-associated DNA and compared it with Southern blot analysis. Both PCR and Southern blot detected LANA-replicated DNA, but the PCR assay was more rapid and did not require radioisotope. PCR detection at 24 and 72 hours post-transfection demonstrated rapid loss of transfected TR DNA. LANA, and to a lesser extent a moderately deficient LANA mutant, reduced the rate of DNA loss through addition of replicated TR DNA and reduction in the loss of non-replicated DNA, the latter of which is consistent with LANA's nuclear segregation function. Therefore, this work develops a rapid, sensitive, and quantitative PCR (qPCR) assay to detect KSHV LANA-replicated DNA and demonstrates that LANA reduces TR DNA loss after transfection through replication and nuclear partitioning of TR DNA.

Role of Kaposi's sarcoma-associated herpesvirus C-terminal LANA chromosome binding in episome persistence.

Kaposi's sarcoma-associated herpesvirus (KSHV) LANA is an 1,162-amino-acid protein that tethers terminal repeat (TR) DNA to mitotic chromosomes to mediate episome persistence in dividing cells. C-terminal LANA self-associates to bind TR DNA. LANA contains independent N- and C-terminal chromosome binding regions. N-terminal LANA binds histones H2A/H2B to attach to chromosomes, and this binding is essential for episome persistence. We now investigate the role of C-terminal chromosome binding in LANA function. Alanine substitutions for LANA residues (1068)LKK(1070) and (1125)SHP(1127) severely impaired chromosome binding but did not reduce the other C-terminal LANA functions of self-association or DNA binding. The (1068)LKK(1070) and (1125)SHP(1127) substitutions did not reduce LANA's inhibition of RB1-induced growth arrest, transactivation of the CDK2 promoter, or C-terminal LANA's inhibition of p53 activation of the BAX promoter. When N-terminal LANA was wild type, the (1068)LKK(1070) and (1125)SHP(1127) substitutions also did not reduce LANA chromosome association or episome persistence. However, when N-terminal LANA binding to chromosomes was modestly diminished, the substitutions in (1068)LKK(1070) and (1125)SHP(1127) dramatically reduced both LANA chromosome association and episome persistence. These data suggest a model in which N- and C-terminal LANA cooperatively associates with chromosomes to mediate full-length LANA chromosome binding and viral persistence.

Nivel de conocimiento sobre VPH en estudiantes de la Universidad del Pacífico

Introducción. El virus del papiloma humano (VPH) es una infección viral más habitual del aparato reproductor y causa diversos trastornos, tanto en hombres como en mujeres, y se considera como el principal agente para el desarrollo del cáncer cervicouterino. Objetivo. Determinar el nivel de conocimiento sobre el VPH de las alumnas del segundo año de las diferentes carreras de la Universidad del Pacífico Sede Asunción en el año 2023. Materiales y Métodos. Se realizó un estudio observacional, descriptivo de corte transversal en alumnas del segundo año de las diferentes carreras de la Universidad del Pacifico en el 2023, mediante una encuesta de conocimiento, influencia y métodos para evitar el contagio del VPH. Se aplicó la estadística descriptiva utilizando el programa EpiDat 4.2. Resultados. Participaron 95 alumnas, el 49,92% conoce que el VPH es una infección viral, el 87,37% que su síntoma más común son las verrugas genitales, el 91,58% sabe que la infección por VPH afecta a ambos sexos, el 90,53% que se puede transmitir por vía sexual, 71,58% sabe que puede producir cáncer de cuello uterino y el 86,32% conoce que la vacunación es la principal medida de prevención. El 48% de las estudiantes tuvieron un buen nivel de conocimiento global sobre el VPH. Conclusión. El nivel insuficiente de conocimiento sobre VHP refleja la necesidad de realizar fortalecer el programa educativo universitario y campañas educativas con respecto al VPH y el cáncer de cuello uterino.

Introduction. Human papillomavirus (HPV) is the most common viral infection of the reproductive tract, causes various disorders in both men and women, and it is considered to be the main agent for the development of cervical cancer. Objective. To determine the level of knowledge about HPV among students in the second year of the different careers of the Universidad del Pacífico, Asunción, 2023. Materials and Methods. A cross-sectional, observational, descriptive study was carried out with 2nd year students of the different careers of the Universidad del Pacifico in 2023, by means of a survey of knowledge about HPV, influence, and methods to prevent infection. Descriptive statistics using EpiDat 4.2 software were applied. Results. Of the 95 female students surveyed, 49.92% knew that HPV was a viral infection, 87.37% knew that the most common symptom was genital warts, 91.58% knew that HPV disease affects both sexes, 90.53% knew that it can be transmitted sexually, 71.58% knew that it could cause cervical cancer, and 86.32% knew that vaccination was the main prevention measure, 48% of the students have a good global knowledge about HPV. Conclusion. The insufficient level of knowledge about HPV reflects the need to strengthen the university educational program and educational campaigns regarding HPV and cervical cancer.

Supercritical fluid extraction of heather (Calluna vulgaris) and evaluation of anti-hepatitis C virus activity of the extracts.

Previous studies using lipid extracts of heather (Calluna vulgaris) leaves showed the presence of high concentrations of ursolic and oleanolic acid. These two compounds have been reported to present antiviral activity against hepatitis C virus (HCV). In this work, the supercritical fluid extraction of heather was studied with the aim of assessing a potential anti-HCV activity of the extracts owing to their triterpenic acid content. Supercritical extraction assays were carried out exploring the pressure range of 20-50 MPa, temperatures of 40-70°C and 0-15% of ethanol cosolvent. The content of oleanolic and ursolic acid in the extracts were determined, and different samples were screened for cellular cytotoxicity and virus inhibition using a HCV cell culture infection system. Antiviral activity was observed in most extracts. In general, superior anti-HCV activity was observed for higher contents of oleanolic and ursolic acids in the extracts.

Pharmacological profile of the 5-HT-induced inhibition of cardioaccelerator sympathetic outflow in pithed rats: correlation with 5-HT1 and putative 5-ht5A/5B receptors.

Continuous infusions of 5-hydroxytryptamine (5-HT) inhibit the tachycardiac responses to preganglionic (C7-T1) sympathetic stimulation in pithed rats pretreated with desipramine. The present study identified the pharmacological profile of this inhibitory action of 5-HT. The inhibition induced by intravenous (i.v.) continuous infusions of 5-HT (5.6 microg x kg-1x min-1) on sympathetically induced tachycardiac responses remained unaltered after i.v. treatment with saline or the antagonists GR 127935 (5-HT1B/1D), the combination of WAY 100635 (5-HT1A) plus GR 127935, ritanserin (5-HT2), tropisetron (5-HT3/4), LY215840 (5-HT7) or a cocktail of antagonists/inhibitors consisting of yohimbine (alpha2), prazosin (alpha1), ritanserin, GR 127935, WAY 100635 and indomethacin (cyclooxygenase), but was abolished by methiothepin (5-HT1/2/6/7 and recombinant 5-ht5A/5B). These drugs, used in doses high enough to block their respective receptors/mechanisms, did not modify the sympathetically induced tachycardiac responses per se. I.v. continuous infusions of the agonists 5-carboxamidotryptamine (5-CT; 5-HT1/7 and recombinant 5-ht5A/5B), CP 93129 (r5-HT1B), sumatriptan (5-HT1B/1D), PNU-142633 (5-HT1D) and ergotamine (5-HT1B/1D and recombinant 5-ht5A/5B) mimicked the above sympatho-inhibition to 5-HT. In contrast, the agonists indorenate (5-HT1A) and LY344864 (5-ht1F) were inactive. Interestingly, 5-CT-induced cardiac sympatho-inhibition was abolished by methiothepin, the cocktail of antagonists/inhibitors, GR 127935 or the combination of SB224289 (5-HT1B) plus BRL15572 (5-HT1D), but remained unchanged when SB224289 or BRL15572 were given separately. Therefore, 5-HT-induced cardiac sympatho-inhibition, being unrelated to 5-HT2, 5-HT3, 5-HT4, 5-ht6, 5-HT7 receptors, alpha1/2-adrenoceptor or prostaglandin synthesis, seems to be primarily mediated by (i). 5-HT1 (probably 5-HT1B/1D) receptors and (ii). a novel mechanism antagonized by methiothepin that, most likely, involves putative 5-ht5A/5B receptors.

Further characterization of the 5-HT1 receptors mediating cardiac sympatho-inhibition in pithed rats: pharmacological correlation with the 5-HT1B and 5-HT1D subtypes.

Serotonin (5-hydroxytryptamine; 5-HT) is capable of inhibiting the tachycardic responses elicited by sympathetic stimulation, but not by exogenous noradrenaline, in pithed rats pre-treated with desipramine. More recently, it has been shown that this cardiac sympatho-inhibitory response to 5-HT, mediated by prejunctional 5-HT1 receptors as well as putative 5-ht5A/5B receptors, is mimicked dose-dependently by the agonists CP 93,129 (r5-HT1B), sumatriptan (5-HT1B/1D) and PNU-142633 (5-HT1D). This study analysed further the pharmacological profile of the above 5-HT1 receptors. Continuous i.v. infusions of CP 93,129, sumatriptan or PNU-142633 (30 micro g kg(-1)min(-1) each) failed to modify the tachycardic responses to exogenous noradrenaline but inhibited those elicited by preganglionic (C7-T1) stimulation of the cardiac sympathetic outflow. These sympatho-inhibitory responses were unaltered after i.v. administration of physiological saline (1 ml kg(-1)) or the 5-HT1A receptor antagonist WAY 100635 (10 micro g kg(-1)). In contrast, the antagonist GR 127935 (5-HT1B/1D; 100 micro g kg(-1), i.v.) abolished the responses to CP 93,129, sumatriptan and PNU-142633, whilst SB224289 (5-HT1B; 300 micro g kg(-1), i.v.) abolished the responses to CP 93,129 without affecting those to sumatriptan and PNU-142633. Interestingly, BRL15572 (5-HT1D; 300 micro g kg(-1), i.v.) abolished the responses to PNU-142633 and attenuated those to sumatriptan, but not those to CP 93,129. WAY 100635, GR 127935, SB224289 and BRL15572, given alone at the above doses, failed to modify the sympathetically induced tachycardic responses. The 5-HT1 receptors producing cardiac sympatho-inhibition in pithed rats thus display the pharmacological profile of the 5-HT1B and 5-HT1D receptor subtypes.

A short sequence immediately upstream of the internal repeat elements is critical for KSHV LANA mediated DNA replication and impacts episome persistence.

Kaposi's sarcoma-associated herpesvirus LANA (1162 residues) mediates episomal persistence of viral genomes during latency. LANA mediates viral DNA replication and segregates episomes to daughter nuclei. A 59 residue deletion immediately upstream of the internal repeat elements rendered LANA highly deficient for DNA replication and modestly deficient for the ability to segregate episomes, while smaller deletions did not. The 59 amino acid deletion reduced LANA episome persistence by ~14-fold, while sequentially smaller deletions resulted in ~3-fold, or no deficiency. Three distinct LANA regions reorganized heterochromatin, one of which contains the deleted sequence, but the deletion did not abolish LANA's ability to alter chromatin. Therefore, this work identifies a short internal LANA sequence that is critical for DNA replication, has modest effects on episome segregation, and substantially impacts episome persistence; this region may exert its effects through an interacting host cell protein(s).

Isolation of essential oil from different plants and herbs by supercritical fluid extraction.

Supercritical fluid extraction (SFE) is an innovative, clean and environmental friendly technology with particular interest for the extraction of essential oil from plants and herbs. Supercritical CO(2) is selective, there is no associated waste treatment of a toxic solvent, and extraction times are moderate. Further, supercritical extracts were often recognized of superior quality when compared with those produced by hydro-distillation or liquid-solid extraction. This review provides a comprehensive and updated discussion of the developments and applications of SFE in the isolation of essential oils from plant matrices. SFE is normally performed with pure CO(2) or using a cosolvent; fractionation of the extract is commonly accomplished in order to isolate the volatile oil compounds from other co-extracted substances. In this review the effect of pressure, temperature and cosolvent on the extraction and fractionation procedure is discussed. Additionally, a comparison of the extraction yield and composition of the essential oil of several plants and herbs from Lamiaceae family, namely oregano, sage, thyme, rosemary, basil, marjoram and marigold, which were produced in our supercritical pilot-plant device, is presented and discussed.

Angiotensin II-dependent induction of AT(2) receptor expression after renal ablation.

Angiotensin (ANG) II can be associated with gene expression regulation. Thus we studied the possible role of ANG II in the regulation of AT(2) mRNA and protein expression. We utilized sham-operated renal ablation rats as well as renal ablation rats pretreated during the first 7 days of the development of renal damage with either the angiotensin-converting inhibitor ramipril, the AT(1) receptor antagonist losartan, or the AT(2) receptor antagonist PD-123319. Renal tissue was analyzed for histological changes and expression of AT(2) receptor mRNA (by RT-PCR) and protein (by immunohistochemistry). To explore the physiological role of AT(2) receptor overexpression in the development of renal damage, blood pressure, urinary protein excretion, and renal damage were evaluated. A time-dependent increase in the expression of AT(2) receptor mRNA and protein was observed at 7, 15, and 30 days after renal ablation. Because these effects were already evident at day 7, the effects of ramipril, losartan, or PD-123319 were tested at this time. The ramipril group and the PD-123319-pretreated group showed inhibition of AT(2) receptor expression, whereas the losartan-pretreated group showed a further increase in AT(2) receptor expression. Inhibition of the AT(2) receptor during renal ablation was associated with increased renal damage and a further increase in the blood pressure. This suggests that overexpression of AT(2) receptors after renal ablation is modulated by ANG II through its own AT(2) receptor and that functional expression of this effect may represent a counterregulatory mechanism to modulate the renal damage induced by renal ablation.

Morbilidad atendida y derivada por médicos generales en atención primaria: consultorio Bellavista, servicio sur-oriente, 1985 / Morbidity assisted and refered by general practitioners in primary health care level

En 1985 en un consultorio de atención primaria de la Región Metropolitana, se efectuaron 48.536 consultas, de las cuales el 50.1% correspondió a morbilidad infantil y el restante a adultos. De 46.214 consultas solicitadas, los médicos generales atendieron el 93.9% de estas, derivando las restantes mediante interconsultas al nivel secundario. De todas las atenciones ambulatorias, las enfermedades del aparato respiratorio tuvieron una importancia relativa de un 26.3%. Las enfermedades del oído ocuparon en su grupo el mayor porcentaje de derivación con un 47.9%, mientras que desde el punto de vista de todas las interconsultas, las enfermedades del sistema nervioso y órganos de los sentidos tuvieron una importancia relativa de un 20.6% de derivación