Effect of pressure on refolding of recombinant pentameric cholera toxin B.

The production of recombinant proteins is an essential tool for the expansion of modern biological research and biotechnology. The expression of heterologous proteins in Escherichia coli often results in an incomplete folding process that leads to the accumulation of inclusion bodies (IB), aggregates that hold a certain degree of native-like secondary structure. High hydrostatic pressure (HHP) impairs intermolecular hydrophobic and electrostatic interactions, leading to dissociation of aggregates under non-denaturing conditions and is therefore a useful tool to solubilize proteins for posterior refolding. Cholera toxin (CT) is composed of a non-toxic pentamer of B subunits (CTB), a useful adjuvant in vaccines, and a toxic subunit A (CTA). We studied the process of refolding of CTB using HHP. HHP was shown to be effective for dissociation of CTB monomers from IB. Posterior incubation at atmospheric pressure of concentrated CTB (1mg/ml) is necessary for the association of the monomers. Pentameric CTB was obtained when suspensions of CTB IB were compressed at 2.4kbar for 16h in the presence of Tween 20 and incubated at 1bar for 120h. Soluble and biologically active pentameric CTB was obtained, with a yield of 213mg CTB/liter of culture. The experience gained in this study can be important to improve the refolding of proteins with quaternary structure.

Regulation of IGFBP3 gene expression in short children born small for gestational age.

OBJECTIVE: Approximately 6% of newborns at term are small for gestational age (SGA) and present a birth weight and/or length less than -2SD from the mean. SGA infants are at increased risk for perinatal morbidity, associated psychological and/or mental problems, persistent short stature (about 15% of subjects) and metabolic alterations. Insulin-like growth factors (IGFs), their common receptor (IGF1R) and their binding proteins (IGFBPs) play a critical role in fetal and postnatal growth. In these genes common polymorphisms, such as single nucleotide polymorphisms and variable number of tandem repeats, have been investigated with conflicting results with respect to SGA-related outcomes, and the functional role of these gene variants remains to be elucidated. DESIGN: The study group consisted of 100 pre-pubertal short children born SGA and 94 healthy controls, matched for sex and age, recruited at the Department of Biomedicine of Development Age of the Bari University and at the Paediatric Department of the Messina Hospital. In the present study we analyzed the allelic frequency of the polymorphisms -795 G/A, -667 G/A, -396 C/T in the IGFBP3 in SGA children and their influence on the basal and insulin-stimulated transcriptional activity of the gene. RESULTS: We found that the polymorphisms -667 G/A and -396 C/T in the IGFBP3 promoter region are capable of having an effect on the transcriptional activity of the gene, although with opposing effects. Interestingly, the -667 G/A polymorphism has a negative impact on the IGFBP3 transcription, while the -396 C/T polymorphism determines an increase of the transcriptional activity of the IGFBP3 gene promoter. Interestingly, we found that the -396 C/T polymorphism correlates with lower birth length in SGA children. Most importantly, while the diminished IGFBP3 transcriptional activity induced by the -667A polymorphism was significantly recovered after insulin administration (p-value<0.05), the increased transcriptional activity caused by the -396T polymorphism was not restored to baseline levels by insulin. CONCLUSIONS: Altogether our results demonstrated that the -667 G/A and the -396 C/T polymorphisms in IGFBP3 promoter region influence the basal transcriptional activity of the gene.

Effect of radiation protraction in intensity-modulated radiation therapy with direct aperture optimization: a phantom study.

The effect of radiation protraction in step-and-shoot IMRT is investigated for treatment plans created with the help of direct aperture optimization. The latter approach can be used during inverse planning for all clinical linear accelerators with conventional MLC. Direct aperture optimization significantly shortens fraction time for IMRT plans as compared to that for plans obtained by using the conventional inverse planning approach. By analyzing several IMRT plans obtained with direct aperture optimization we found that for alpha/beta ratio of 10 Gy (characteristic of fast growing tumors) the protraction effect is probably clinically insignificant for both conventional and large fraction sizes of 1.9 Gy and 5.7 Gy, respectively. For small alpha/beta of 1-1.5 Gy and conventional fraction size the effect of protraction is still small; however, this effect can be significant for hypofractionated treatments. Based on the obtained results it is recommended that, when possible, IMRT for slow growing prostate cancers be performed with small number of beams (e.g., 5) and small number of segments (e.g., 5-7 segments per beam) to reduce delivery time and, as a result, the associated effect of radiation protraction.

Intracellular localization of human respiratory syncytial virus L protein.

Replication and transcription of the human respiratory syncytial virus (hRSV) genome is carried out by the ribonucleocapsid complex (RNA together with N, P, M2-1 and L proteins), with the L protein being responsible for all enzymatic activities. In the present study, we obtained anti-L polyclonal sera in mice. These antibodies were functional in immunofluorescence and Western blotting assays in hRSV-infected HEp-2 cells. In the immunofluorescence assays, we detected inclusion bodies in the anti-L staining, similar to the ones seen by anti-N or anti-P staining. The results presented here provide the first evidence of the intracellular localization of the hRSV L protein.

Construction of a recombinant plasmid pSH-G containing the rabies-virus glycoprotein G gene

A plasmid named pSH-G was constructed with the rabies-virus G-gene insert. This plasmid was transfected into eukaryotic BHK-21 cells and its stability tested. The presence of the pSH-G plasmid was confirmed by means of polymerase chain reaction (PCR) after each of ten cell passages, and the results were positive. The stable BHK-21/pSH-G+ clone obtained can be used in the study of rabies as well as in the production of vaccines.

Insertion of an exogenous domain in the adenovirus type 2 fiber globular region.

Adenoviruses have been used for gene therapy or immunization due to their ability to efficiently infect a broad range of cells and tissues. These applications as well as specificity could be improved further by redirecting binding of the virus to specific cell types. In this regard, modification of viral genes encoding coat proteins is an option to achieve improvement in retargeting. In this report, we describe a substitution in the adenovirus type 2 fiber globular region by the 44 amino acid C4 domain of human immunodeficiency virus type 1 gp120. In vitro translation analysis and immunoprecipitation assays show that the incorporation of the C4 domain into the fiber protein does not ablate its trimerization property and demonstrates the availability of the C4 epitope for interaction with monoclonal anti-C4 antibody. The recombinant adenovirus containing this modified fiber was also characterized by immunoprecipitation with the same antibody, showing the viability of such kind of modification.

An adenovirus vector containing the suicide gene thymidine kinase for a broad application in cancer gene therapy.

Treatment of cancer using gene therapy is based on adding a property to the cell leading to its elimination. One possibility is the use of suicide genes that code for enzymes that transform a pro-drug into a cytotoxic product. The most extensively used is the herpes simplex virus thymidine kinase (TK) gene, followed by administration of the antiviral drug ganciclovir (GCV). The choice of the promoter to drive the transcription of a transgene is one of the determinants of a given transfer vector usefulness, as different promoters show different efficiencies depending on the target cell type. In the experiments presented here, we report the construction of a recombinant adenovirus carrying TK gene (Ad-TK) driven by three strong promoters (P CMV IE, SV40 and EN1) and its effectiveness in two cell types. Human HeLa and mouse CCR2 tumor cells were transduced with Ad-TK and efficiently killed after addition of GCV. We could detect two sizes of transcripts of TK gene, one derived from the close together P CMV IE/SV40 promoters and the other from the 1.5 Kb downstream EN1 promoter. The relative amounts of these transcripts were different in each cell type thus indicating a higher flexibility of this system.

An adenovirus vector containing the suicide gene thymidine Kinase for a broad application in cancer gene therapy

Treatment of cancer using gene therapy is based on adding a property to the cell leading to its elimination. One possibility is the use of suicide genes that code for enzymes that transform a pro-drug into a cytotoxic product. The most extensively used is the herpes simplex virus thymidine kinase (TK) gene, followed by administration of the antiviral drug ganciclovir (GCV). The choice of the promoter to drive the transcription of a transgene is one of the determinants of a given transfer vector usefulness, as different promoters show different efficiencies depending on the target cell type. In the experiments presented here, we report the construction of a recombinant adenovirus carrying TK gene (Ad-TK) driven by three strong promoters (P CMV IE, SV40 and EN1) and its effectiveness in two cell types. Human HeLa and mouse CCR2 tumor cells were transduced with Ad-TK and efficiently killed after addition of GCV. We could detect two sizes of transcripts of TK gene, one derived from the close together P CMV IE/SV40 promoters and the other from the 1.5 Kb downstream EN1 promoter. The relative amounts of these transcripts were different in each cell type thus indicating a higher flexibility of this system

[IgG antibody response against Plasmodium vivax in children exposed to malaria, before and after specific treatment] / Resposta de anticorpos IgG anti-Plasmodium vivax em crianças expostas à malária, antes e após tratamento específico.

OBJECTIVE: To assess the IgG antibody response against P. vivax (IgG anti-PV), and cytophilic (IgG1 and IgG3) and noncytophilic (IgG2) IgG subclasses in 34 children aged 0 to 15 years old infected with P. vivax malaria. METHODS: IgG levels were determined by indirect fluorescent antibody technique during the acute and therapeutic control phase. Patients were distributed into 2 groups according to the presence or absence of a previous malaria episode. IgG anti-PV levels were measured on the first and last day of treatment, and compared by Studentacute;s t test. Antibody levels were correlated with asexual parasitemia (Spearmans correlation test). RESULTS AND CONCLUSIONS: Increased IgG levels were observed on the first and on the last day of treatment. The geometric means for IgG subclass titers found in patients with previous history of malaria were: IgG1 (806.35) > IgG3 (28.28) > IgG2 (20). In patients infected for the first time, the responses obtained were: IgG1 (709.21) > IgG3 (39.3) > IgG2 (10.7). There was no association between asexual parasitemia and antibody levels on the first day of treatment. Cytophilic antibodies (IgG1, IgG3) predominated over noncytophilic antibodies (IgG2).

HIV-1 long terminal repeat modulation by glucocorticoids in monocytic and lymphocytic cell lines.

Glucocorticoid gene regulation can be carried out through direct binding of glucocorticoid receptor to glucocorticoid responsive elements (GRE), regulating directly gene transcription and modulating some signaling pathways. The human immunodeficiency virus type 1 (HIV-1) expression can be activated by different immunomodulators through binding of particular nuclear factors to its long terminal repeat (LTR). In order to investigate the effect of glucocorticoids in pathways that activate HIV-1 expression, we transfected promonocyte (U937) and T lymphocyte (CEM-T4) cell lineages with a plasmid containing the chloramphenicol acetyl transferase (CAT) reporter gene under the control of the HIV-1 LTR. In U937 cells, dexamethasone (DEX) downregulates CAT expression induced by either phorbol myristate acetate (PMA), tumor necrosis factor alpha (TNFalpha) or granulocyte/macrophage-colony stimulating factor (GM-CSF). In CEM-T4 cells the CAT activity was slightly upregulated by DEX following the induction by either PMA or TNFalpha. Interestingly, in both cell lines transactivation of this reporter gene by transactivator protein (TAT) was downregulated by DEX. When the CAT gene was under control of HIV-1 enhancer isolated from its LTR background, the CAT activity induced by PMA was not affected by the presence of glucocorticoids. In all experiments, comparable data were obtained when DEX was replaced by hydrocortisone (HC). Our results show that, depending on the cell line, glucocorticoids can differently affect HIV-1 expression, probably by interfering in cellular pathways involved in virus expression. Moreover, the target of this regulation in LTR is probably not the enhancer region itself.

[Plasmodium vivax malaria in children and adolescents - epidemiological, clinical and laboratory features] / Malária por Plasmodium vivax em crianças e adolescentes - aspectos epidemiológicos, clínicos e laboratoriais.

OBJECTIVE: Evaluation of epidemiological, clinical and laboratory features of Plasmodium vivax malaria in children and adolescents. METHODS: This study was carried out in the Malaria Program of the Evandro Chagas Institute (Belém, Pará), from January 1995 to November 1996. 100 children and adolescents with the diagnosis of P. vivax malaria (thick blood film) were randomly enrolled. A protocol was created to assess epidemiological, clinical and laboratory parameters of this pathology. RESULTS: Malaria occurred in both sexes, and had a prevailing incidence among adolescents (37%). Most of the children and adolescents (92%) had been infected in the State of Pará. Autochthonous cases in the metropolitan area of Belém accounted for 34 % of the sample. Primary infection was seen in 80% of the patients. Fever was the major onset clinical symptom (88%). A history of typical febrile paroxysm was recorded in only 25% of the casuistic. In the first day of treatment (D0) fever (97%), chills (91%), pallor (85%), splenomegaly (46%) and hepatomegaly (29%) were some of the clinical features observed. Pallor (clinical signal) was found to be significantly (p=0.0004) associated with anemia (hemoglobin rate). There was a high significant negative correlation (p=0.0001) between delay of diagnosis (mean 12,5 days) and hemoglobin values. Regarding parasitological examination, just children and adolescents with positive results to hookworms were significantly (p=0.0133, p=0.0075) more anemic than those who had a positive parasitological examination to other helminths and/or protozoa species. CONCLUSIONS: Malaria affected children and adolescents from both sexes. An emphasis on epidemiological and clinical data is an important tool to the precocious diagnosis of the disease. Delay on diagnosis made anemia worse.

[Clinical efficacy of four schemes for vivax malaria treatment in children] / Eficácia de quatro esquemas terapêuticos para malaria vivax em crianças.

OBJECTIVES: In the treatment of vivax malaria, an important factor affecting the occurrence of relapse is the duration of treatment. In Belém, a number of patients with vivax malaria were found to be cured despite failure to complete the standard course of treatment. In Belém, a number of patients with vivax malaria were found to be cured despite failure to complete the standard course of treatment. This observation suggested the present study, investigating more practicable courses of treatment for children with vivax malaria.METHODS: A randomized prospective clinical trial was conducted in 200 outpatient children with vivax malaria. Parasite clearance time and response to four therapeutic schedules were investigated: a) chloroquine*, 10 mg/kg in a single dose (chloroquine SD) + primaquine, 0.50 mg/kg/dose for 7 days; b) chloroquine SD + primaquine, 0.25 mg/kg/dose for 7 days; c) chloroquine SD + primaquine, 0.50 mg/kg/dose for 5 days; d) chloroquine SD + primaquine, 0.25 mg/kg/dose for 5 days. Fisherss Exact test was used to compare the responses to the schedules.RESULTS: All 144 children who completed the study had clearance of asexual parasitemia by the fourth day of treatment. Significant differences were observed between schedules A/D (p= 0.022) and C/D (p= 0.005). A doubled dose of primaquine (schedules A and C) proved to be significantly more effective (p=0.0042) than the standard dose (B and D). However, duration of treatment had no significant effect (p = 0.6104).CONCLUSIONS: In this study, complete cure of vivax malaria was better achieved with a doubled dose of primaquine than with standard doses. Effectiveness of the doubled dose was independent of the duration of treatment. Treatment schedule D is not recommended.

Laminin-binding proteins in EJ-ras-transformed fibroblasts.

Malignant transformation is accompanied by changes in cell-matrix interactions. Upon transfection with EJ-ras oncogene, transformed fibroblasts acquired a migratory phenotype towards laminin-1. The increase in integrin expression was responsible for the migratory activity of transformed fibroblasts. In addtion alpha 6 beta 1 integrins, both galectin-3 and an unidentified laminin-binding polypeptide had their expression pattern altered upon transformation. Here, we review these two classes of laminin-binding proteins and their possible roles in cell-laminin interactions.

Laminin-binding proteins in EJ-ras-transformed fibroblasts

Malignant transformation is accompanied by changes in cell-matrix interations. Upon transfection with EJ-ras oncogene, transformed fibroblasts, acquired a migratory phenotype towards laminin-1. The increase in integrin expression was responsible for the migratory activity of transformed fibroblasts. In addition alpha(6)beta(1) integrins, both galectin-3 and an unidentified laminin-binding polypeptide had their expression pattern altered upon transformation. Here, we review these two classes of laminin-binding proteins and their possible roles in cell-laminin interactions.

alpha6beta1-Integrin, a major cell surface carrier of beta1-6-branched oligosaccharides, mediates migration of EJ-ras-transformed fibroblasts on laminin-1 independently of its glycosylation state.

EJ-ras oncogene-induced malignant transformation is characterized by a series of changes in cell surface carbohydrates and cell-cell and cell-matrix interactions. Here, we show that EJ-ras-transformed NIH-3T3 fibroblasts acquired a migratory phenotype on laminin-1 surfaces. Such a phenotype was accompanied by overexpression of: (a) functional alpha6beta1, but not other laminin binding beta1-integrins; and (b) glycoconjugates on the cell surface bearing large oligosaccharides recognized by leukoagglutinin from Phaseolus vulgaris (L-PHA). The internal pool of pre-beta1-integrins was differently regulated in EJ-ras-transformed cells compared with nontransfected fibroblasts. Conversion of pre-beta1- into mature beta1-integrins was faster in EJ-ras-transformed cells, a process associated with the overexpression of the alpha6-chain. Overexpression of L-PHA-reactive oligosaccharides is dependent on the activity of N-acetylglucosaminyltransferase V, which is increased in transformed cells [J. W. Dennis et al., Science (Washington DC), 236: 582-585, 1987]. We show that beta1-integrins were the major carriers of L-PHA-reactive oligosaccharides on the cell surface. This glycosylation pattern, however, was not necessary for either the cell surface expression of beta1-integrins or their functional activity in the migratory response to laminin-1. Moreover, EJ-ras-transformed fibroblasts aggregated spontaneously. These effects were not observed in c-jun-transfected fibroblasts, which were unable to migrate on laminin, did not overexpress either beta1-integrins or L-PHA-reactive oligosaccharides, and did not self-aggregate.

Plasmid pSH: a strong mammalian expression vector.

Plasmid pSH is an expression vector enabling high level expression of inserted genes in a variety of cell types compared to SV40 early promoter. Here, constructs made with pSH have been tested in human primary or immortalized keratinocytes, primary human foreskin fibroblasts and mouse fibroblasts.

Silencing of human immunodeficiency virus long terminal repeat expression by an adenovirus E1a mutant.

Gene expression from the human immunodeficiency virus (HIV) long terminal repeat (LTR) is strongly stimulated by the viral tat gene. The HIV LTR is also activated by several physical and chemical agents and heterologous viral genes, including adenovirus E1a. As E1a has separable transcriptional activation and repression functions, we examined the negative regulatory effects of E1a on the expression of the HIV LTR by using a trans-dominant E1a mutant. Mutant hr5 strongly suppressed the basal activity of the LTR as well as trans-activation of the LTR by heterologous agents such as the cytomegalovirus immediate early gene or DNA-damaging agents such as mitomycin C and UV irradiation. In addition, hr5 also caused significant suppression of tat gene-mediated trans-activation. The suppression of HIV LTR expression by hr5 appears to be mediated, at least in part, by the repression of the HIV enhancer, as the activity of an enhancer test system composed of the human T-cell leukemia virus I LTR containing an HIV-1 enhancer substitution was severely repressed by hr5. Cotransfection of HIV-1 proviral DNA with hr5 DNA resulted in a significant reduction of HIV production.

Recovery of DNA synthesis from inhibition by ultraviolet light in mammalian cells.

In general mammalian cells recover from DNA synthesis inhibition by ultraviolet light (u.v.) before most of the pyrimidine dimers have been removed from the genome. This is a complex phenomenon whose biological significance has not been fully assessed. In Chinese hamster V79 cells this recovery seems to be directly coupled to an enhanced rate of double-stranded DNA elongation. The presence of the DNA polymerase alpha inhibitor, aphidicolin, after u.v. irradiation produces two different responses. At low concentration, sufficient to inhibit 95% of DNA replication but having no effect on excision repair, the drug has no effect on the recovery. This shows that ongoing replicative DNA synthesis is not required for recovery. At higher concentrations of aphidicolin, sufficient to block excision repair, the recovery phenomenon was prevented. The recovery was also prevented by actinomycin D at a concentration that inhibits 60% of RNA synthesis. In quantitative autoradiography experiments in which previously irradiated cells were fused with unirradiated cells the nuclei of the latter exhibited a higher resistance to inhibition by u.v. than nuclei from non-fused cells. These results indicate that: (1) even the low repair rate exhibited by V79 cells (relative to human cells) is important for recovery; although most of the dimers remain in the V79 genome after recovery of DNA synthesis, either the removal of lesions from some important region of chromatin or the activity of the repair process itself is important for the recovery; (2) the recovery mechanism is induced and depends on RNA synthesis and the production of specific factors. Finally, we have observed that cells previously treated with fluorodeoxyuridine become more resistant to inhibition by u.v. After irradiation these cells replicate DNA faster than untreated cells. Since it has been shown that this drug activates unused origins of replication in Chinese hamster cells, reducing the average replicon size, we assume that the acquired resistance has to do with the operation of a larger number of smaller replicons. This may also be the mechanism whereby recovery from inhibition occurs after u.v. irradiation.