Zeenathul Nazariah Allaudin

Regulation of low-density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression by thymoquinone-rich fraction and thymoquinone in HepG2 cells.

Background and Aim: Nigella sativa and its active constituent thymoquinone (TQ) have been exploited for their various health benefits. This work was aimed to investigate the regulatory effects of TQ-rich fraction (TQRF) and commercial TQ on the low-density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) genes in HepG2 cells. Methods and Results: TQRF was extracted from N. sativa seeds using supercritical fluid extraction. The regulatory effects of TQRF at 80 μg/ml and TQ at 2 μg/ml on LDLR and HMGCR gene expression were investigated in HepG2 cells using quantitative real-time PCR. The TQ content in TQRF was 2.77% (w/w) and was obtained at a temperature of 40°C and a pressure of 600 bar. Treatment of cells with TQRF and TQ resulted in a 7- and 2-fold upregulation of LDLR mRNA level, respectively, compared with untreated cells. The mRNA level of HMGCR was downregulated by 71 and 12%, respectively, compared with untreated cells. Conclusion: TQRF and TQ regulated genes involved in cholesterol metabolism by two mechanisms, the uptake of low-density lipoprotein cholesterol via the upregulation of the LDLR gene and inhibition of cholesterol synthesis via the suppression of the HMGCR gene.

Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology

BackgroundCell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg) from Pichia pastoris expression cells were optimized using response surface methodology (RSM) based on the central composite design (CCD). The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells.ResultsThe proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW) and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR) and HBsAg concentration of 29.20 mg/L respectively.ConclusionsThe model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing.

Scaling-up recombinant plasmid DNA for clinical trial: current concern, solution and status.

Gene therapy and vaccines are rapidly developing field in which recombinant nucleic acids are introduced in mammalian cells for enhancement, restoration, initiation or silencing biochemical function. Beside simplicity in manipulation and rapid manufacture process, plasmid DNA-based vaccines have inherent features that make them promising vaccine candidates in a variety of diseases. This present review focuses on the safety concern of the genetic elements of plasmid such as propagation and expression units as well as their host genome for the production of recombinant plasmid DNA. The highlighted issues will be beneficial in characterizing and manufacturing plasmid DNA for save clinical use. Manipulation of regulatory units of plasmid will have impact towards addressing the safety concerns raised in human vaccine applications. The gene revolution with plasmid DNA by alteration of their plasmid and production host genetics will be promising for safe delivery and obtaining efficient outcomes.

Isolation, density purification, and in vitro culture maintenance of functional caprine islets of Langerhans as an alternative islet source for diabetes study.

Hani H, TengkuAzmi TI, Abas MO, Mohd‐Azmi ML, Zeenathul NA. Isolation, density purification, and in vitro culture maintenance of functional caprine islets of Langerhans as an alternative islet source for diabetes study. Xenotransplantation 2010; 17: 469–480.

Engineering an L-cell line that expresses insulin under the control of the glucagon-like peptide-1 promoter for diabetes treatment.

BackgroundDiabetes mellitus is a complicated disease with a pathophysiology that includes hyperinsulinemia, hyperglycemia and other metabolic impairments leading to many clinical complications. It is necessary to develop appropriate treatments to manage the disease and reduce possible acute and chronic side effects. The advent of gene therapy has generated excitement in the medical world for the possible application of gene therapy in the treatment of diabetes. The glucagon-like peptide-1 (GLP-1) promoter, which is recognised by gut L-cells, is an appealing candidate for gene therapy purposes. The specific properties of L-cells suggest that L-cells and the GLP-1 promoter would be useful for diabetes therapy approaches.ResultsIn this study, L-cells were isolated from a primary intestinal cell line to create suitable target cells for insulin expression studies. The isolated cells displayed L-cell properties and were therefore used as an L-cell surrogate. Next, the isolated L-cells were transfected with the recombinant plasmid consisting of an insulin gene located downstream of the GLP-1 promoter. The secretion tests revealed that an increase in glucose concentration from 5 mM to 25 mM induced insulin gene expression in the L-cells by 2.7-fold. Furthermore, L-cells quickly responded to the glucose stimulation; the amount of insulin protein increased 2-fold in the first 30 minutes and then reached a plateau after 90 minutes.ConclusionOur data showed that L-cells efficiently produced the mature insulin protein. In addition, the insulin protein secretion was positively regulated with glucose induction. In conclusion, GLP-1 promoter and L-cell could be potential candidates for diabetes gene therapy agents.

Size‐related assessment on viability and insulin secretion of caprine islets in vitro

The successful isolation, purification, and culture of caprine islets has recently been reported. The present study shows arange of size distribution in caprine islet diameter from 50 to 250 μm, in which 80% of the total islet yield was comprised of small islets.

Observation of risk factors, clinical manifestations and genetic characterization of recent Newcastle Disease Virus outbreak in West Malaysia

BackgroundNewcastle disease virus remains a constant threat in commercial poultry farms despite intensive vaccination programs. Outbreaks attributed to ND can escalate and spread across farms and states contributing to major economic loss in poultry farms.ResultsPhylogenetic analysis in our study showed that eleven of the samples belonged to genotype VIId. All farms were concurrently positive with two immunosuppressive viruses; Infectious Bursal Disease Virus (IBDV) and Marek’s Disease Virus (MDV). Amino acid sequence analysis confirmed that eleven of the samples had sequence motifs for velogenic/mesogenic strains; three were lentogenic.ConclusionIn conclusion, no new NDV genotype was isolated from the 2011 NDV outbreak. This study suggests that the presence of other immunosuppressive agents such as IBD and MDV could have contributed to the dysfunction of the immune system of the chickens, causing severe NDV outbreaks in 2011. Risk factors related to biosecurity and farm practices appear to have a significant role in the severity of the disease observed in affected farms.

Caprine pancreatic islet xenotransplantation into diabetic immunosuppressed BALB/c mice.

Type 1 diabetes mellitus is a devastating disease for which there is currently no cure, but only lifetime management. Islet xenotransplantation is a promising technique for the restoration of blood glucose control in patients with diabetes mellitus. The purpose of this study was to explore the potential use of caprine (goat) islet cells as xenogeneic grafts in the treatment for diabetes in a mouse model.

An approach towards optimal usage of immobilized sensor chips in Surface Plasmon Resonance based biosensor

In recent decades, there was a surge of interest in Surface Plasmon Resonance (SPR) biosensor based bimolecular interaction analysis. The unique characteristics of this technique made it possible to measure real time unlabelled bimolecular interactions with great sensitivity. However, the major challenge in SPR is providing the ability to re-use the surface of the chip. The main goal of this study was to address the problem faced in establishing an ideal regeneration condition and removing non-covalently bound analyt without disturbing ligand. Considering four different types of proteins including virus, hormone, cells and antibody, a comprehensive regeneration protocol for protein-protein interaction was developed and compared with common regeneration methods. The presented protocol screened five multi-ingredient stock solutions that represented the five most common chemical properties such as acidic, basic, ionic, chelating and non-polar water soluble solvent solutions employed as regeneration agents. Upon three cycles of screening, it was found out that enveloped virus–antibody complexes could be effectively regenerated via a combination of acidic and chelating solution whilst non-enveloped viruses needed a basic solution for successful regeneration. Both insulin-antibody and cell-enveloped virus complexes could be detached efficiently using acidic solutions. Regenerations using non-polar water soluble solvents presented a harsh reaction, whilst ionic solutions were too mild. Thus, incomplete regeneration occurred. In summary, this study will serve as a platform of reference for multiple regenerations for a cluster of protein-protein complexes. Key words: Surface Plasmon Resonance (SPR), regeneration, virus, insulin, monoclonal antibody, cell.

Insulin secreted from genetically engineered intestinal cells reduces blood glucose levels in diabetic mice

Poorly controlled diabetes mellitus can result in serious complications. Gene therapy is increasingly being considered as an alternative approach to treat diabetes, because of its ability to induce physiological insulin secretion and it allows patients to escape insulin injections. The properties of gut K and L-cells, including glucose sensitivity, the ability to process insulin and a regulated secretion pathway support their use as surrogate β-cells. Previous in vitro studies have provided sufficient evidence supporting the use of these cells for gene therapy studies. Therefore, we examined the ability of K and L-cells to produce insulin in diabetic mice. Chitosan nanoparticles were used to transfer the insulin gene into intestinal cells via oral administration. The efficiency of chitosan as a gene vehicle was investigated through the use of reporter gene. Insulin mRNA and protein expression levels were measured by RT-PCR and ELISA, respectively. Blood glucose testing revealed that this treatment reduced glucose levels in diabetic mice. The decrease in blood glucose level in the first week of treatment was greater in mice with K-cell specific insulin expression compared with mice with L-cell-specific insulin expression. These results indicate that inducing insulin secretion in K-cells conferred a quicker response to gene therapy.