Seletividade, eficiência de controle de plantas daninhas e persistência no solo de imazamox aplicado na cultura do feijoeiro

O presente trabalho teve como objetivo avaliar a seletividade, o controle de plantas daninhas e a persistência no solo de imazamox aplicado na cultura do feijoeiro, durante os anos de 1995 a 1997. Nos experimentos de seletividade foram testados: imazamox (20, 30 e 40 g i.a./ha), imazamox + bentazon (30+480 g i.a./ha), imazamox + fomesafen (30+125 g i.a./ha) e testemunha nas cultivares Pérola, Jalo Precoce, Novo Jalo e Xamego. Os experimentos foram mantidos livres de plantas daninhas e foram avaliadas a fitotoxicidade visual e a produção de grãos. Nos experimentos de eficiência de controle de plantas daninhas foram testados: imazamox (20, 30 e 40 g i.a./ha), bentazon (480 g i.a./ha), fomesafen (125 e 250 g i.a./ha), imazamox + bentazon (30+480 g i.a./ha), imazamox + fomesafen (30 + 125 g i.a./ha), bentazon / imazamox (480/30 g i.a/ha, aplicação seqüencial), fomesafen/imazmox (125/30 g i.a./ha, aplicação seqüencial), fomesafen/fomesafen (100/100 g i.a./ha, aplicação seqüencial) e testemunha. Foram avaliadas as porcentagens de controle de Euphorbia heterophylla e Bidens pilosa. Para o estudo da persistência de imazamox no solo, foram conduzidos experimentos em dois locais: Goiânia, GO (argiloso) e Jussara, GO (arenoso). Imazamox (40 g i.a./ha) causou injúrias à cultura do feijoeiro que acarretaram redução de 15% na produtividade (média dos experimentos). A mistura de imazamox e bentazon causou menor grau de injúrias no feijoeiro, obtendo-se ganhos de produtividade de 8% (médias dos experimentos). Imazamox mostrou-se ineficiente para o controle de Bidens pilosa, enquanto para Euphorbia heterophylla observou-se controle eficiente a partir da dose de 40 g i.a./ha. Aplicações seqüenciais de fomesafen/imazamox (125/30 g i.a/ha) apresentaram controle eficiente de Euphorbia heterophylla e Bidens pilosa. A persistência de imazamox no solo, para ambos os locais, foi maior em 1995 que 1996. Isto foi devido à maior umidade do solo em 1996. A sensibilidade das culturas sucedâneas aos resíduos de imazamox no solo foi, em ordem decrescente: sorgo, milho e arroz. O período entre a aplicação do herbicida e o plantio da cultura sucedânea (INP) variou de acordo com a sensibilidade das culturas aos resíduos de imazamox no solo e à sua persistência. Considerando ambos os locais e anos, o INP variou de 68 a 111 dias para milho, 78 a 139 dias para sorgo e 25 a 75 dias para o arroz.

Selectivity of clomazone and S-metolachlor applied after cassava pruning

The objective of this work was to evaluate the selectivity of clomazone in two formulations and S-metolachlor applied on shoots of different sizes after pruning of 'Baianinha' cassava. The experiment was arranged in a randomized block design in a factorial 5 x 2 (5 treatments x 2 sizes of shoots after pruning - 10 and 33 cm) with four replications. The herbicides evaluated were: clomazone (encapsulated suspension - 900 g h-1), clomazone (encapsulated suspension - 1,080 g ha-1), clomazone (emulsifiable concentrate 900 g ha-1), S-metolachlor (1,920 g ha-1) and an untreated control. During the experiment, all plots were kept free of weed interference by hand weeding. It was concluded that both the formulations of clomazone and S-metolachlor were selective when applied on both the shoot sizes evaluated. However, the application of S-metolachlor on 33 cm shoots gave higher selectivity to 'Baianinha' cassava plants.

Poly-DL-lactide-co-glycolide microspheres as a controlled release antigen delivery system

Successful vaccine application means maximum protection with minimal number of administrations. A rational development of vaccines involves studies of the nature of the antigen as well as of the adjuvant to be used to improve the immune responses. This has provided the impetus for studies to design the degradable devices and for different approaches to antigen delivery by different routes of administration. The development of controlled release systems based on polymeric devices that permit a sustained or pulsed release of encapsulated antigens has attracted much interest. Polymeric delivery systems consist of polymers that release their content continuously in a controlled manner over a period of time. The development of a biocompatible delivery system for parenteral administration offers several advantages in terms of immunoadjuvanticity over other compounds. It was found that, in contrast to other carriers, microspheres are more stable, thus permitting administration by the oral or parenteral route. In the present study, we describe the main characteristics and potentialities of this new immunoadjuvant for oral and parenteral administration.

Novel methods for the encapsulation of meglumine antimoniate into liposomes

The antimonial drug, meglumine antimoniate, was successfully encapsulated in dehydration-rehydration vesicles and in freeze-dried empty liposomes (FDELs). High encapsulation efficiencies (from 28 to 58%) and low weight ratios of lipids to encapsulated antimony (from 1:0.15 to 1:0.3) were achieved. These formulations, contrary to those obtained by conventional methods, can be stored as intermediate lyophilized forms and reconstituted just before use. The efficacy of FDEL-encapsulated meglumine antimoniate was evaluated in hamsters experimentally infected with Leishmania chagasi. A significant reduction of liver parasite burdens was observed in animals treated with this preparation, when compared to control animals treated with empty liposomes. In contrast, free meglumine antimoniate was found to be inefficient when administered at a comparable dose of antimony. This novel liposome-based meglumine antimoniate formulation appears to be promising as a pharmaceutical product for the treatment of visceral leishmaniasis.

Immunohistochemical demonstration of TGF-ß and decorin in paracoccidioidal granulomas

Different patterns of granulomas have been observed in 6- to 8-week-old mice after ip inoculation with 5 x 10(6) yeast cells of Paracoccidioides brasiliensis. Transforming growth factor-ß (TGF-ß) is a cytokine that has been shown to participate in fibrosis and granuloma formation; its activities seem to be modulated by the small proteoglycan decorin. In the present study, TGF-ß and decorin expression in epiploon granulomas was assessed by immunohistochemistry in susceptible (B10.A) and resistant (A/J) mice after 15, 30, 120 and 150 days of P. brasiliensis ip infection. The epiploon was collected, fixed in Methacarn solution and embedded in paraffin, and 5-µm thick sections were used for immunohistochemical analysis employing the streptavidin-biotin-peroxidase technique. The former mouse strain developed fatal disease with many disseminated lesions increasing in size and number during the infection and the latter developed mild disease with the presence of encapsulated granulomas. In the epiploon, TGF-ß was present on macrophages, giant cells, lymphocytes and fibroblasts, and absent on neutrophils. It was also detected in areas of fibrosis and necrosis, as well as disperse in amorphous extracellular matrix, mostly in resistant mice. Decorin was present circumscribing macrophages and giant cells containing fungi, but absent on these cells. In both mouse strains, decorin was found at the periphery of the lesions, and markedly in milky spot granulomas. In resistant mice, positivity was found around fibrotic and necrotic areas of encapsulated and residual lesions containing lysed fungi. Decorin was found associated with thick fibers around encapsulated lesions. In susceptible mice, the size and number of lesions increased with the progression of the disease and were correlated with the weaker expression of decorin. We suggest an association of decorin with the fibrogenic process observed in paracoccidioidal granulomas.

Antagonist G-mediated targeting and cytotoxicity of liposomal doxorubicin in NCI-H82 variant small cell lung cancer

The aim of the present study was to characterize the interactions of antagonist G (H-Arg-D-Trp-NmePhe-D-Trp-Leu-Met-NH 2)-targeted sterically stabilized liposomes with the human variant small cell lung cancer (SCLC) H82 cell line and to evaluate the antiproliferative activity of encapsulated doxorubicin against this cell line. Variant SCLC tumors are known to be more resistant to chemotherapy than classic SCLC tumors. The cellular association of antagonist G-targeted (radiolabeled) liposomes was 20-30-fold higher than that of non-targeted liposomes. Our data suggest that a maximum of 12,000 antagonist G-targeted liposomes were internalized/cell during 1-h incubation at 37ºC. Confocal microscopy experiments using pyranine-containing liposomes further confirmed that receptor-mediated endocytosis occurred, specifically in the case of targeted liposomes. In any of the previously mentioned experiments, the binding and endocytosis of non-targeted liposomes have revealed to be negligible. The improved cellular association of antagonist G-targeted liposomes, relative to non-targeted liposomes, resulted in an enhanced nuclear delivery (evaluated by fluorimetry) and cytotoxicity of encapsulated doxorubicin for incubation periods as short as 2 h. For an incubation of 2 h, we report IC50 values for targeted and non-targeted liposomes containing doxorubicin of 5.7 ± 3.7 and higher than 200 µM doxorubicin, respectively. Based on the present data, we may infer that receptors for antagonist G were present in H82 tumor cells and could mediate the internalization of antagonist G-targeted liposomes and the intracellular delivery of their content. Antagonist G covalently coupled to liposomal drugs may be promising for the treatment of this aggressive and highly heterogeneous disease.

Preparation and cytotoxicity of cisplatin-containing liposomes

We encapsulated cisplatin into stealth pH-sensitive liposomes and studied their stability, cytotoxicity and accumulation in a human small-cell lung carcinoma cell line (GLC4) and its resistant subline (GLC4/CDDP). Since reduced cellular drug accumulation has been shown to be the main mechanism responsible for resistance in the GLC4/CDDP subline, we evaluated the ability of this new delivery system to improve cellular uptake. The liposomes were composed of dioleoylphosphatidylethanolamine (DOPE), cholesteryl hemisuccinate (CHEMS), and distearoylphosphatidylethanolamine-polyethyleneglycol 2000 (DSPE-PEG2000) and were characterized by determining the encapsulation percentage as a function of lipid concentration. Among the different formulations, DOPE/CHEMS/DSPE-PEG liposomes (lipid concentration equal to 40 mM) encapsulated cisplatin more efficiently than other concentrations of liposomes (about 20.0%, mean diameter of 174 nm). These liposomes presented good stability in mouse plasma which was obtained using a 0.24-M EDTA solution (70% cisplatin was retained inside the liposomes after 30 min of incubation). Concerning cytotoxic effects, they are more effective (1.34-fold) than free cisplatin for growth inhibition of the human lung cancer cell line A549. The study of cytotoxicity to GLC4 and GLC4/CDDP cell lines showed similar IC50 values (approximately 1.4 µM), i.e., cisplatin-resistant cells were sensitive to this cisplatin formulation. Platinum accumulation in both sensitive and resistant cell lines followed the same pattern, i.e., approximately the same intracellular platinum concentration (4.0 x 10-17 mol/cell) yielded the same cytotoxic effect. These results indicate that long-circulating pH-sensitive liposomes, also termed as stealth pH-sensitive liposomes, may present a promising delivery system for cisplatin-based cancer treatment. This liposome system proved to be able to circumvent the cisplatin resistance, whereas it was not observed when using non-long-circulating liposomes composed of phosphatidylcholine, phosphatidylserine, and cholesterol.

The effect of down-regulation of Smad3 by RNAi on hepatic stellate cells and a carbon tetrachloride-induced rat model of hepatic fibrosis

Searching for effective Smad3 gene-based gene therapies for hepatic fibrosis, we constructed siRNA expression plasmids targeting the rat Smad3 gene and then delivered these plasmids into hepatic stellate cells (HSCs). The effect of siRNAs on the mRNA levels of Smad2, Smad3, Smad4, and collagens I-α1, III-α1 and IV-α1 (Colα1, Col3α1, Col4α1, respectively) was determined by RT-PCR. Eighty adult male Sprague-Dawley rats were randomly divided into three groups. Twice a week for 8 weeks, the untreated hepatic fibrosis model (N = 30) and the treated group (N = 20) were injected subcutaneously with 40% (v/v) carbon tetrachloride (CCl4)-olive oil (3 mL/kg), and the normal control group (N = 30) was injected with olive oil (3 mL/kg). In the 4th week, the treated rats were injected subcutaneously with liposome-encapsulated plasmids (150 µg/kg) into the right liver lobe under general anesthesia once every 2 weeks, and the untreated rats were injected with the same volume of buffer. At the end of the 6th and 8th weeks, liver tissue and sera were collected. Pathological changes were assessed by a semi-quantitative scoring system (SSS), and a radioimmunoassay was used to establish a serum liver fibrosis index (type III procollagen, type IV collagen, laminin, and hyaluronic acid). The mRNA expression levels of the above cited genes were reduced in the HSCs transfected with the siRNA expression plasmids. Moreover, in the treated group, fibrosis evaluated by the SSS was significantly reduced (P < 0.05) and the serum indices were greatly improved (P < 0.01). These results suggest that Smad3 siRNA expression plasmids have an anti-fibrotic effect.

Molecular adjuvant interleukin-33 enhances the antifertility effect of Lagurus lagurus zona pellucida 3 DNA vaccine administered by the mucosal route

It has been shown that cytokines can act as molecular adjuvant to enhance the immune response induced by DNA vaccines, but it is unknown whether interleukin 33 (IL-33) can enhance the immunocontraceptive effect induced by DNA vaccines. In the present study, we explored the effects of murine IL-33 on infertility induced by Lagurus lagurus zona pellucida 3 (Lzp3) contraceptive DNA vaccine administered by the mucosal route. Plasmid pcD-Lzp3 and plasmid pcD-mIL-33 were encapsulated with chitosan to generate the nanoparticle chi-(pcD-Lzp3+pcD-mIL-33) as the DNA vaccine. Sixty female ICR mice, divided into 5 groups (n=12/group), were intranasally immunized on days 0, 14, 28, and 42. After intranasal immunization, the anti-LZP3-specific IgG in serum and IgA in vaginal secretions and feces were determined by ELISA. The results showed that chi-(pcD-Lzp3+pcD-mIL-33) co-immunization induced the highest levels of serum IgG, secreted mucosal IgA, and T cell proliferation. Importantly, mice co-immunized with chi-(pcD-Lzp3+pcD-mIL-33) had the lowest birth rate and mean litter size, which correlated with high levels of antibodies. Ovaries from infertile female mice co-immunized with chi-(pcD-Lzp3+pcD-mIL-33) showed abnormal development of ovarian follicles, indicated by atretic follicles and loss of oocytes. Our results demonstrated that intranasal delivery of the molecular adjuvant mIL-33 with chi-pcD-Lzp3significantly increased infertility by enhancing both systemic and mucosal immune responses. Therefore, chi-(pcD-Lzp3+pcD-mIL-33) co-immunization could be a strategy for controlling the population of wild animal pests.

Effect of JJYMD-C, a novel synthetic derivative of gallic acid, on proliferation and phenotype maintenance in rabbit articular chondrocytes in vitro

Tissue engineering encapsulated cells such as chondrocytes in the carrier matrix have been widely used to repair cartilage defects. However, chondrocyte phenotype is easily lost when chondrocytes are expanded in vitro by a process defined as “dedifferentiation”. To ensure successful therapy, an effective pro-chondrogenic agent is necessary to overcome the obstacle of limited cell numbers in the restoration process, and dedifferentiation is a prerequisite. Gallic acid (GA) has been used in the treatment of arthritis, but its biocompatibility is inferior to that of other compounds. In this study, we modified GA by incorporating sulfamonomethoxine sodium and synthesized a sulfonamido-based gallate, JJYMD-C, and evaluated its effect on chondrocyte metabolism. Our results showed that JJYMD-C could effectively increase the levels of the collagen II, Sox9, and aggrecan genes, promote chondrocyte growth, and enhance secretion and synthesis of cartilage extracellular matrix. On the other hand, expression of the collagen I gene was effectively down-regulated, demonstrating inhibition of chondrocyte dedifferentiation by JJYMD-C. Hypertrophy, as a characteristic of chondrocyte ossification, was undetectable in the JJYMD-C groups. We used JJYMD-C at doses of 0.125, 0.25, and 0.5 µg/mL, and the strongest response was observed with 0.25 µg/mL. This study provides a basis for further studies on a novel agent in the treatment of articular cartilage defects.

Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells

Recent advances have raised hope that transplantation of adherent somatic cells could provide dramatic new therapies for various diseases. However, current methods for transplanting adherent somatic cells are not efficient enough for therapeutic applications. Here, we report the development of a novel method to generate quasi-natural cell blocks for high-efficiency transplantation of adherent somatic cells. The blocks were created by providing a unique environment in which cultured cells generated their own extracellular matrix. Initially, stromal cells isolated from mice were expanded in vitro in liquid cell culture medium followed by transferring the cells into a hydrogel shell. After incubation for 1 day with mechanical agitation, the encapsulated cell mass was perforated with a thin needle and then incubated for an additional 6 days to form a quasi-natural cell block. Allograft transplantation of the cell block into C57BL/6 mice resulted in perfect adaptation of the allograft and complete integration into the tissue of the recipient. This method could be widely applied for repairing damaged cells or tissues, stem cell transplantation, ex vivo gene therapy, or plastic surgery.

Production and characterization of lipid microparticles produced by spray cooling encapsulating a low molar mass hydrophilic compound

The objective of this research was to produce and characterize lipid particles (MpLs) that may be used as carriers of high amounts of hydrophilic core and evaluate the influence of the core amount on the performance of lipid microparticles. The MpLs were produced by spray cooling from solid and liquid lipid mixtures (stearic and oleic fatty acids and partly hydrogenated vegetable fat) containing glucose solution as core and soy lecithin as surfactant. The performance of MpLs was evaluated by means of the effective amount of encapsulated core, the core amount present on the surface of MpLs (superficial glucose) and the core release profile in aqueous solution. Morphological observations showed that MpLs presented spherical shape and a rugged and continuous surface, and an average diameter between 25 and 32 µm. The effective amount of encapsulated core was greater than 78% for all formulations evaluated. Larger amounts of superficial glucose were found in formulations in which more concentrated glucose solutions were used, regardless of the glucose lipid-solution ratio. The release results showed that core retention was significantly influenced by the glucose solution concentration, whereas release modulation was influenced by the glucose lipid-solution ratio.

Microbial profile of a kefir sample preparations: grains in natura and lyophilized and fermented suspension

Probiotics are supplementary foods developed by microbial strains that improve animal health beyond basic nutrition. Probiotics are consumed orally, regardless of being considered as normal inhabitants of the intestines, able to survive in enzimatic and biliary secretions. Kefir is a probiotic originated from the old continent, fermented by several bacteria and yeasts, encapsulated in a polyssacharide matrix, and resembles jelly grains. Kefir is also presented as its sourish product both in sugary or milky suspensions containing vitamins, aminoacids, peptides, carbohydrates, ethanol, and volatile compounds. Kefir is known to have a diverse microbial content depending on the country and fermentative substrates, which cause distinct probiotic effects. In this sense, the purpose of this work was to isolate, identify, and quantify the microbial content of a native sugary kefir sample (fermented suspension and lyophilized natural grains). Serial dilutions were plated on Rogosa agar (AR) and De Man, Rogosa and Sharpe (MRS), for Lactobacillus; Brain Heart Infusion (BHI), for total bacteria; Sabouraud-Dextrose-Agar (SDA), for yeasts and filamentous fungi; Thioglycolate Agar (TA), for Streptococcus, Acetobacteria and Leuconostoc; and Coconut Water Agar (CWA), and CWA supplemented with yeast extract (CWAY), for various genera. Genera and species for all strains were identified through biochemical reactions and specific API systems. The microbial profile of kefir was different from other sources of grains despite the presence of similar microorganisms and others which have not been reported yet. The data obtained with the CWA and CWAE media suggest that both substrates are alternative and salutary media for culture of kefir strains.

Docosahexaenoic acid ethyl esther (DHAEE) microcapsule production by spray-drying: optimization by experimental design

Docosahexaenoic acid is an essential polyunsaturated fatty acid with important metabolic activities. Its conjugated double bonds make it susceptible to decomposition. Its stability may be improved through fatty acid entrapment with a spray-drying technique; however, the many parameters involved in this technique must be considered to avoid affecting the final product quality. Therefore, this study aimed to evaluate the entrapment conditions and yields of fish oil enriched with docosahexaenoic acid ethyl ester. Microcapsules were obtained from Acacia gum using a spray-drying technique. The experimental samples were analyzed by chromatography and delineated by Statistica software, which found the following optimum entrapment conditions: an inlet temperature of 188 °C; 30% core material; an N2 flow rate of 55 mm; and a pump flow rate of 12.5 mL/minute. These conditions provided a 66% yield of docosahexaenoic acid ethyl ester in the oil, corresponding to 19.8% of entrapped docosahexaenoic acid ethyl ester (w/w). This result was considered significant since 30% corresponded to wall material.

Retention of short chain fatty acids under drying and storage conditions

Cheese whey permeate was used as a substrate for the fermentation of Propionibacterium freudenreichi PS1 for the production of short chain fatty acids, components of the bio-aroma of Swiss cheese. The liquid bio-aroma was encapsulated by spray drying under different conditions of air inlet temperature and feed rate. A study was carried out on the stability of the bio-aroma during storage in laminated packages at 30 °C for 96 days using the product showing the greatest retention of acetic and propionic acids. The results showed that the best drying conditions were an air entrance temperature of 180 °C and a feed rate of 24 g/min resulting in particles with a smooth surface and few invaginations and micro-fissures. However, 72% of the acetic acid and 80% of the propionic acid were lost during storage showing that the wall material used was inadequate to guarantee product stability.