Effects of Salvia mirzayanii extract administration on hyperglycemia improvement in diabetic rats: The role of GLUT4, PEPCK and G6Pase genes.

Diabetes is a dangerous metabolic disorder by increasing incidence in human societies worldwide. Recently, much attention has been focused on the development of hypoglycemic agents, particularly the derivatives of herbal drugs, in the treatment of diabetes. This research aimed to study the anti-diabetic effect of Salvia mirzayanii in the diabetic rat models. First, the plant material was extracted from the leaves, and orally administered to the rats. After treating the animals with the aqueous extract of S. mirzayanii at a dose of 600 mg/kg, animal body weight for 12 weeks, fasting blood glucose, oral glucose tolerance test (OGTT), and body weight changes were examined. To analyze the anti-diabetic function of S. mirzayanii, we measured the expression of glucose transporter-4 (GLUT4), phosphoenolpyruvate carboxykinase (PEPCK), and glucose 6-phosphatase (G6Pase) genes in healthy and streptozotocin (STZ)-diabetic rats. The expression levels of the genes of interest in muscle and liver tissues were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). There were no significant differences in fasting blood glucose and OGTT between normal control (NC) group and the diabetic control (DC) group treated with S. mirzayanii. In contrast, there was a significant difference with the untreated DC (P < 0.05). The treatment of diabetic rats with S. mirzayanii significantly increased the expression of GLUT4 in the muscle and decreased the expression levels of PEPCK and G6Pase in the liver compared to the DC group (P < 0.05). These findings clearly show that S. mirzayanii can improve hyperglycemia by increasing the GLUT4 expression, and inhibiting the gluconeogenesis pathway in the liver. In general, the obtained results provided a new insight into the efficacy of S. mirzayanii aqueous extract as an anti-diabetic herbal medicine.

Expression and response surface optimization of the recovery and purification of recombinant D-galactose dehydrogenase from Pseudomonas fluorescens.

The enzyme D-galactose dehydrogenase (GalDH) has been used in diagnostic kits to screen blood serum of neonates for galactosemia. It is also a significant tool for the measurement of ß-D-galactose, α-D-galactose and lactose as well. In this study, response surface methodology (RSM) was used to identify the suitable conditions for recovery of recombinant GalDH from Pseudomonas fluorescens in aqueous two-phase systems (ATPS). The identified GalDH gene was amplified by PCR and confirmed by further cloning and sequencing. E. coli BL-21 (DE3) containing the GalDH gene on a plasmid (pET28aGDH) was used to express and purify the recombinant enzyme. The polyethylene glycol (PEG) and ammonium sulfate concentrations and pH value were selected as variables to analyze purification of GalDH. To build mathematical models, RSM with a central composite design was applied based on the conditions for the highest separation. The recombinant GalDH enzyme was expressed after induction with IPTG. It showed NAD'-dependent dehydrogenase activity towards D-Galactose. According to the RSM modeling, an optimal ATPS was composed of PEG-2000 14.0% (w/w) and ammonium sulfate 12.0% (w/w) at pH 7.5. Under these conditions, GalDH preferentially concentrated in the top PEG-rich phase. The enzyme activity, purification factor (PF) and recovery (R) were 1400 U/ml, 60.0% and 270.0%, respectively. The PEG and salt concentrations were found to have significant effect on the recovery of enzyme. Briefly, our data showed that RSM could be an appropriate tool to define the best ATPS for recombinant P. fluorescens GalDH recovery.

Expression, Purification and Characterization of the Proline Dehydrogenase Domain of PutA from Pseudomonas putida POS-F84.

The objective of the present work was to express a truncated form of Pseudomonas putida PutA that shows proline dehydrogenase (ProDH) activity. The putA gene encoding ProDH enzyme was cloned into pET23a vector and expressed in Escherichia coli strain BL-21 (DE3) plysS. The recombinant P. putida enzyme was biochemically characterized and its three dimensional structure was also predicted. ProDH encoding sequence showed an open reading frame of 1,035-bp encoding a 345 amino acid residues polypeptide chain. Purified His-tagged enzyme gave a single band with a molecular mass of 40 kDa on SDS-PAGE. The molecular mass of the isolated enzyme was found to be about 40 kDa by gel filtration. This suggested that the enzyme of interest consists of one subunit. The K m and V max values of recombinant P. putida ProDH were estimated to be 31 mM and 132 µmol/min, respectively. The optimum pH and temperature for the catalytic activity of the enzyme was about pH 8.5 and 30 °C. The modeling analysis of the three dimensional structure elucidated that Ser-165, Lys-195 and Ala-252 were key residues for the ProDH activity. This study provides data on the cloning, sequencing and recombinant expression of PutA ProDH domain from P. putida POS-F84.

Isolation and characterization of Leucine dehydrogenase from a thermophilic Citrobacter freundii JK-91strain Isolated from Jask Port.

BACKGROUND AND OBJECTIVES: Leucine dehydrogenase (LeuDH; EC 1.4.1.9) belongs to the amino acid dehydrogenase family and isused as a biocatalyst in medical and pharmaceutical industries (1). This study reported deals with the isolation and characterization of LeuDH from a thermophilic bacterium isolated from Jask Port in the Province of Hormozgan. MATERIALS AND METHODS: Aliquots of soil and water samples were cultured in LEU specific medium and thermophilc bacteria that exhibited LeuDH activity were isolated and characterized biochemically. The LeuDH was purified and characterized in regard to the effects of pH and temperature on the activity, as well as its molecular weight determination. RESULTS: A thermophilic bacterium, Citrobacter freundii strain JK-9 was identified and found to exhibit LeuDH activity. The enzyme characterization revealed that LeuDH exhibits higher activity at temperature range of 60 to 75°C (optimum of 60°C) and an optimum pH of activity at pH 10.5. The K m value of LeuDH is 1.2 mM, while its molecular weight is about 320 kDa, and consisted of eight subunits identical in molecular mass (40 kDa). CONCLUSION: Briefly, a thermostableLeuDH enzyme from a strain of C. freundii was isolated and characterized. Our data indicate that the C. freundii enzyme has potential for use in biotechnological applications.