The Effect ofTinospora crispaon Serum Glucose and Insulin Levels in Patients with Type 2 Diabetes Mellitus

Objective. To determine the effects ofTinospora crispaon serum glucose and insulin levels in healthy subjects and patients with type 2 diabetes mellitus.Method. Serum from 10 healthy subjects and 10 diabetic participants, who had fasted overnight, were obtained every 30–60 minutes during the 3 hours of continued fasting and during the 3 hours after ingestion of 75 g of glucose with or without ingestion of 125 or 250 g ofTinospora crispadry powder capsule. Glucose and Insulin levels were analyzed and the areas under the curve for mean serum glucose and insulin levels were calculated.Result. The areas under the curve of mean serum glucose and insulin levels in both healthy and diabetic participants were not significantly different between with or withoutTinospora crispadry powder capsule. In diabetic participants the area under the curve of glucose was slightly lesser when 250 mg ofTinospora crispawas ingested, but not reaching statistical significance (478 and 444 mg min/ml, resp.,P=0.57).Conclusion. The results suggest thatTinospora crispaingestion cannot affect serum glucose and insulin levels in healthy subjects or patients with type 2 diabetes mellitus.

Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher’s patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr−) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa) and secreted (63–69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources.

Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained insideE. coliallows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method inE. colifor molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects forin vitrohomologous recombination for genetic cloning.

Antioxidant, Antinociceptive, and Anti-Inflammatory Effects of Carotenoids Extracted from Dried Pepper (Capsicum annuumL.)

Carotenoids extracted from dried peppers were evaluated for their antioxidant, analgesic, and anti-inflammatory activities. Peppers had a substantial carotenoid content: guajillo3406±4 μg/g, pasilla2933±1 μg/g, and ancho1437±6 μg/g of sample in dry weight basis. A complex mixture of carotenoids was discovered in each pepper extract. The TLC analysis revealed the presence of chlorophylls in the pigment extract from pasilla and ancho peppers. Guajillo pepper carotenoid extracts exhibited good antioxidant activity and had the best scavenging capacity for the DPPH+cation (24.2%). They also exhibited significant peripheral analgesic activity at 5, 20, and 80 mg/kg and induced central analgesia at 80 mg/kg. The results suggest that the carotenoids in dried guajillo peppers have significant analgesic and anti-inflammatory benefits and could be useful for pain and inflammation relief.

Protegrin-1 Inhibits Dengue NS2B-NS3 Serine Protease and Viral Replication in MK2 Cells

Dengue diseases have an economic as well as social burden worldwide. In this study, the antiviral activity of protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR) peptide towards dengue NS2B-NS3pro and viral replication in Rhesus monkey kidney (MK2) cells was investigated. The peptide PG-1 was synthesized by solid-phase peptide synthesis, and disulphide bonds formation followed by peptide purification was confirmed by LC-MS and RPHPLC. Dengue NS2B-NS3pro was produced as a single-chain recombinant protein inE. coli. The NS2B-NS3pro assay was carried out by measuring the florescence emission of catalyzed substrate. Real-time PCR was used to evaluate the inhibition potential of PG-1 towards dengue serotype-2 (DENV-2) replication in MK2 cells. The results showed that PG-1 inhibited dengue NS2B-NS3pro at IC50of 11.7 μM. The graded concentrations of PG-1 at nontoxic range were able to reduce viral replication significantly (P<0.001) at 24, 48, and 72 hrs after viral infection. However, the percentage of inhibition was significantly (P<0.01) higher at 24 hrs compared to 48 and 72 hrs. These data show promising therapeutic potential of PG-1 against dengue infection, hence it warrants further analysis and improvement of the peptide features as a prospective starting point for consideration in designing attractive dengue virus inhibitors.

Toward Personalized Cell Therapies by Using Stem Cells: Seven Relevant Topics for Safety and Success in Stem Cell Therapy

Stem cells, both embryonic and adult, due to the potential for application in tissue regeneration have been the target of interest to the world scientific community. In fact, stem cells can be considered revolutionary in the field of medicine, especially in the treatment of a wide range of human diseases. However, caution is needed in the clinical application of such cells and this is an issue that demands more studies. This paper will discuss some controversial issues of importance for achieving cell therapy safety and success. Particularly, the following aspects of stem cell biology will be presented: methods for stem cells culture, teratogenic or tumorigenic potential, cellular dose, proliferation, senescence, karyotyping, and immunosuppressive activity.

Eucalyptus Biodiesel as an Alternative to Diesel Fuel: Preparation and Tests on DI Diesel Engine

Nowadays, the increasing oil consumption throughout the world induces crucial economical, security, and environmental problems. As a result, intensive researches are undertaken to find appropriate substitution to fossil fuels. In view of the large amount of eucalyptus trees present in arid areas, we focus in this study on the investigation of using eucalyptus biodiesel as fuel in diesel engine. Eucalyptus oil is converted by transesterification into biodiesel. Eucalyptus biodiesel characterization shows that the physicochemical properties are comparable to those of diesel fuel. In the second phase, a single cylinder air-cooled, DI diesel engine was used to test neat eucalyptus biodiesel and its blends with diesel fuel in various ratios (75, 50, and 25 by v%) at several engine loads. The engine combustion parameters such as peak pressure, rate of pressure rise, and heat release rate are determined. Performances and exhaust emissions are also evaluated at all operating conditions. Results show that neat eucalyptus biodiesel and its blends present significant improvements of carbon monoxide, unburned hydrocarbon, and particulates emissions especially at high loads with equivalent performances to those of diesel fuel. However, the NOx emissions are slightly increased when the biodiesel content is increased in the blend.

Cloning of Acyl-ACP Thioesterase FatA fromArachis hypogaeaL. and Its Expression inEscherichia coli

In this study, a full-length cDNA of the acyl-ACP thioesterase,AhFatA, was cloned from developing seeds ofArachis hypogaeaL. by 3′-RACE. Sequence analysis showed that the open reading frame encodes a peptide of 372 amino acids and has 50–70% identity with FatA from other plants. Real-time quantitative PCR analysis revealed thatAhFatA was expressed in all tissues ofA. hypogaeaL., but most strongly in the immature seeds harvested at 60 days after pegging. Heterologous expression ofAhFatA inEscherichia coliaffected bacterial growth and changed the fatty acid profiles of the membrane lipid, resulting in directed accumulation towards palmitoleic acid and oleic acid. These results indicate that AhFatA is at least partially responsible for determining the high palmitoleic acid and oleic acid composition ofE. coli.