Biomedical subjects

C Almonte

Publications and source records attributed to C Almonte.

17 recordsLinked to original sources

PubMed1999

Subcellular trafficking of the cytoplasmic expression system.

Cationic liposomes have provided many advantages over viral vector formulations; however, the problem of inefficient gene expression remains. This is due in part to the nuclear membrane, which limits DNA entry into the nucleus. Cytoplasmic expression systems using T7 RNA polymerase have been developed to express genes in the cytoplasm and avoid the need for nuclear import of DNA. Although these systems show improved transgene expression, little is known about how they function in transfected cells. Direct comparisons between a cytoplasmic and nuclear expression system were carried out with a 293 cell line stably expressing T7 RNA polymerase. A formulation for optimal reporter gene expression was developed and used in conjunction with a variety of subcellular trafficking inhibitors to study the process of DNA endocytosis. Transfected cells were also studied at different stages of the cell cycle to determine the dependence of each system on mitosis. These results showed that cytoplasmic and nuclear expression systems utilize similar endocytosis pathways to the point of endosomal release. Once DNA is released into the cytoplasm, the cytoplasmic expression system shows immediate expression that is proportional to the amount of DNA released. In contrast, DNA targeted for nuclear expression requires additional time for nuclear entry. The level of nuclear expression is also restricted by the limited amount of DNA that is imported into the nucleus. Finally, mitosis is required for effective nuclear expression but not for cytoplasmic expression. Therefore, the cytoplasmic expression system has considerable advantages over traditional nuclear expression systems and may be an effective method for transfecting nondividing cells. Efficient expression of genes delivered by nonviral vectors is hindered owing to poor nuclear transport of plasmid DNA. A potential solution to this problem would be to use a cytoplasmic expression system. Previous studies have shown that this method produces enhanced gene expression when compared with traditional nuclear expression systems; however, the actual mechanisms by which the cytoplasmic expression system works remains unknown. This article focuses on a direct comparison between cytoplasmic and nuclear expression in terms of optimal DNA delivery formulations, intracellular trafficking of DNA, and cell cycle dependence. These results indicate that the cytoplasmic expression system has two primary advantages over nuclear expression in that it does not rely on nuclear DNA transport or mitosis for efficient expression.

Anti-Bacterial Agents↗

PubMed1999

Human bleomycin hydrolase binds ribosomal proteins.

Bleomycin hydrolase (BH) is a cysteine proteinase that inactivates the anticancer drug bleomycin. Yeast BH forms a homohexameric structure that resembles a 20S proteasome and binds to single-stranded RNA and DNA. We now demonstrate that human BH (hBH) interacts and colocalizes with ribosomal proteins. Using a yeast two-hybrid system, we found hBH bound to human homologues of rat ribosomal proteins L11 and L29. The N-terminus of hBH (amino acids 14-175), which contains a catalytic Cys93, was critical for the binding to L11 in the two-hybrid environment. hBH precipitated 35S-labeled L11 and L29 in vitro, and hBH colocalized with L11 and L29 as determined by immunofluorescence. In addition to cytosolic bleomycin hydrolase, we found abundant bleomycin hydrolase activity associated with the ribosomal subcellular fraction by differential centrifugation. hBH was also detected by Western immunoblotting in a high-speed particulate fraction, where the majority of L11 and L29 were found. In vitro experiments showed recombinant hBH binds to Chinese hamster ovary cell microsomes. Thus, our data strongly suggest that hBH exists as both a free cytosolic and ribosome-associated protein.

Animals↗

PubMed1999

Std1 and Mth1 proteins interact with the glucose sensors to control glucose-regulated gene expression in Saccharomyces cerevisiae.

The Std1 protein modulates the expression of glucose-regulated genes, but its exact molecular role in this process is unclear. A two-hybrid screen for Std1-interacting proteins identified the hydrophilic C-terminal domains of the glucose sensors, Snf3 and Rgt2. The homologue of Std1, Mth1, behaves differently from Std1 in this assay by interacting with Snf3 but not Rgt2. Genetic interactions between STD1, MTH1, SNF3, and RGT2 suggest that the glucose signaling is mediated, at least in part, through interactions of the products of these four genes. Mutations in MTH1 can suppress the raffinose growth defect of a snf3 mutant as well as the glucose fermentation defect present in cells lacking both glucose sensors (snf3 rgt2). Genetic suppression by mutations in MTH1 is likely to be due to the increased and unregulated expression of hexose transporter genes. In media lacking glucose or with low levels of glucose, the hexose transporter genes are subject to repression by a mechanism that requires the Std1 and Mth1 proteins. An additional mechanism for glucose sensing must exist since a strain lacking all four genes (snf3 rgt2 std1 mth1) is still able to regulate SUC2 gene expression in response to changes in glucose concentration. Finally, studies with green fluorescent protein fusions indicate that Std1 is localized to the cell periphery and the cell nucleus, supporting the idea that it may transduce signals from the plasma membrane to the nucleus.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Potentiation of E7 antisense RNA-induced antitumor immunity by co-delivery of IL-12 gene in HPV16 DNA-positive mouse tumor.

Down-regulation of oncogene expression by antisense-based gene therapy has been extensively studied, and in some cases, therapeutic effects have been demonstrated. We have previously shown that down-regulation of HPV16 E6 and E7 gene expression inhibited HPV DNA-positive C3 mouse tumor growth. Although not all of the tumor cells were transfected by pU6E7AS plasmid, complete tumor regression was achieved if the tumor size was small at the start of therapy in a syngeneic host. This suggests that some other antitumor mechanisms may be involved in addition to the direct down-regulation of HPV16 E7 oncogene expression by the antisense effect of E7AS. In the current study, we demonstrated that E7AS induces tumor cell apoptosis. More importantly, a strong antitumor immune response was elicited in the pU6E7AS-treated and tumor-regressed mice. There was no tumor growth after rechallenging the tumor-regressed mice with 1 million C3 cells. This E7AS-induced antitumor immune response was augmented by co-delivery of mIL-12 cytokine gene. The combination therapy strategy resulted in complete regression of 26 of 28 (93%) tumors. Only 12 of 31 (38%) tumors from the group treated with pU6E7AS alone and 14 of 28 (50%) tumors from the group treated with pCMVmIL-12 alone had completely regressed. Complete regression was also demonstrated in tumors located 1 cm from the treated tumors, which indicates that a systemic antitumor effect was induced by E7AS and mIL-12. Immunohistochemistry demonstrated that a significant amount of CD4+ and CD8+ cells infiltrated into tumors treated with pU6E7AS, pCMVmIL-12 and pU6E7AS+pCMVmIL-12. These data indicate that host immunity is an important factor for antisense-based gene therapy approach which can be further enhanced by combination with cytokine gene therapy.

Animals↗

PubMed1995

Modulation of a volume-regulated chloride current by F-actin.

We have examined whether F-actin integrity is involved in activation of a volume-regulated Cl- current (VRChlC) in B-lymphocytes. VRChlC activation was initiated in response to establishing a whole cell recording in the presence of a hyposmotic gradient. Parallel confocal microscopy experiments using Rhodamine-Phalloidin (R-P) as a specific marker of F-actin showed that the submembrane actin ring is reversibly disrupted in response to an hyposmotic gradient. Disruptions of cortical F-actin integrity by 50 microM cytochalasin B (CB) does not trigger activation of VRChlC under isosmotic conditions or potentiate the rate of activation when the osmolarity of the extracellular solution was decreased by 75%. However, incubation with CB increased the rate of VRChlC activation in response to a 90% hyposmotic gradient. Phalloidin, a stabilizer of F-actin, decreases the rate of VRChlC activation in response to a 90% gradient, but has no effect in response to a 75% gradient. These observations suggest that disassembly of cortical F-actin is not critical for VRChlC activation in B-lymphocytes. The integrity of cortical F-actin, however, can exert a modulatory effect on the rate of VRChlC activation in the presence of a hyposmotic gradient.

Actins↗

PubMed1986

[Infanto-juvenile stuttering: a multidisciplinary approach].

The authors have conducted a clinical, psychopathological, exploratory research, with a multidisciplinary approach, applied to a sample of 52 children and adolescents stutterers, whose ages ranged between 3.6 and 19.8 years. They made a comparison of two groups, according to the moment in which they began stuttering and the level of development of their previous language. They emphasized the importance of studying the disorders of psychomotor and perceptive-motor functions; of learning disabilities, and the difficulties of interpersonal relationships in the family group and at school. They concluded that stuttering would be a symptom referred to a disorder in the rhythm of speaking, frequently associated to developmental deviations of different areas of language, psychomotor and/or perceptive-motor functions. Stuttering presents itself in normal as well as abnormal personality structures. Its beginning, persistence, and aggravation is frequently associated with unsatisfactory verbal and non verbal communication patterns.

Adolescent↗

PubMedDate not supplied

[Parental socialization styles and psychosocial development in 16 to 19 year old adolescents].

To study relationships between perceptions that 16 to 19 year old adolescents have about socialization styles and disciplinary methods used by their parents, with their view of the world, interpersonal relations, moral development, use of psychotropic substances and maladjusted behaviours, 241 adolescents--123 males--which belong to a follow up study on growth and development from northern metropolitan Santiago, Chile, were asked to answer a questionary of psychosocial development, previously elaborated by two of the authors (GS and CA). The most used socialization styles by these fathers and mothers were the negative power one (39.2% and 38.5%) and the inductive one (23.8% and 30.0%), while permissive styles occurred at much lower frequency (1.5% and 1.1%). Coincidence among parents in socialization styles was found in 47.7% and disagreement in 19.2% of cases. The inductive style and coincidence in it's use by both parents, were frequently associated to idealistic world views, autonomous and conventional moral development, satisfactory interpersonal relationships and low frequency of psychotropic consumption, and maladjusted behaviours while negative power based styles by both parents and disagreement of styles among them were rather related to realistic or negative world view, preconventional moral development, higher frequency of relational problems, psychotropic consumption and maladjusted behaviours.

Adolescent↗

PubMedDate not supplied