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Fulltext Effective refolding of a cysteine rich glycoside hydrolase family 19 recombinant chitinase from Streptomyces griseus by reverse dilution and affinity chromatography

Oyeleye AO, Mohd Yusoff SF, Abd Rahim IN, Leow ATC, Saidi NB, Normi YM

PLoS One, 2020;15(10):e0241074.
PMID: 33091044 DOI: 10.1371/journal.pone.0241074

Conventional refolding methods are associated with low yields due to misfolding and high aggregation rates or very dilute proteins. In this study, we describe the optimization of the conventional methods of reverse dilution and affinity chromatography for obtaining high yields of a cysteine rich recombinant glycoside hydrolase family 19 chitinase from Streptomyces griseus HUT6037 (SgChiC). SgChiC is a potential biocontrol agent and a reference enzyme in the study and development of chitinases for various applications. The overexpression of SgChiC was previously achieved by periplasmic localization from where it was extracted by osmotic shock and then purified by hydroxyapatite column chromatography. In the present study, the successful refolding and recovery of recombinant SgChiC (r-SgChiC) from inclusion bodies (IB) by reverse dilution and column chromatography methods is respectively described. Approximately 8 mg of r-SgChiC was obtained from each method with specific activities of 28 and 52 U/mg respectively. These yields are comparable to that obtained from a 1 L culture volume of the same protein isolated from the periplasmic space of E. coli BL21 (DE3) as described in previous studies. The higher yields obtained are attributed to the successful suppression of aggregation by a stepwise reduction of denaturant from high, to intermediate, and finally to low concentrations. These methods are straight forward, requiring the use of fewer refolding agents compared with previously described refolding methods. They can be applied to the refolding of other cysteine rich proteins expressed as inclusion bodies to obtain high yields of actively folded proteins. This is the first report on the recovery of actively folded SgChiC from inclusion bodies.
Evaluation on the Effectiveness of High Cholesterol Diet Feeding in Inducing Early and Established Atherosclerotic Lesions in New Zealand White Rabbits

Abd Rahim IN, Mohd Kasim NA, Omar E, Abd Muid S, Nawawi H

Front Biosci (Landmark Ed), 2023 Apr 06;28(4):70.
PMID: 37114545 DOI: 10.31083/j.fbl2804070

BACKGROUND: Various methods were used to induce atherosclerosis in rabbits. One of the most common methods used is high-cholesterol diet (HCD) feeding. However, the exact amount and duration of HCD feeding to induce early and established atherosclerosis in New Zealand white rabbits (NZWR) continue to be debated among researchers. Therefore, this study aims to evaluate the effectiveness of 1% HCD feeding in inducing early and established atherosclerosis lesions in NZWR.
METHODS: A total of 50 g/kg/day of 1% HCD was fed to three to four months old male rabbits weighing 1.8 to 2.0 kg for four and eight weeks to induce early and established atherosclerosis respectively. The body weight and lipid profile were measured at baseline and post-HCD intervention. Following euthanasia, the aorta was excised and prepared for histology and immunohistochemical analysis to confirm the stages of atherosclerosis.
RESULTS: The mean body weight of the rabbits in early and established atherosclerosis groups increased significantly up to 17.5% (p = 0.026) and 19.75% (p = 0.019) respectively compared to baseline. The total cholesterol level dramatically elevated up to 13-fold (p = 0.005) and 38-fold (p = 0.013) compared to baseline, after four and eight weeks of 1% HCD feeding respectively. The low-density lipoprotein level significantly increased up to 42-fold (p = 0.006) and 128-fold (p = 0.011) compared to baseline, after four and eight weeks of 1% HCD feeding respectively. Rabbits fed with four and eight weeks 1% HCD significantly developed 5.79% (p = 0.008) and 21.52% (p = 0.008) aortic lesion areas compared to the control group. Histological evaluation in the aorta showed accumulation of foam cells in early atherosclerosis group and formation of fibrous plaque and lipid core in the established atherosclerosis group. Rabbits fed with eight weeks HCD showed higher tissue expressions of ICAM-1, VCAM-1, e-selectin, IL-6, IL-8, NF-κBp65, and MMP-12 compared to four weeks of HCD intervention.
CONCLUSIONS: A total of 50 g/kg/day of 1% HCD for four and eight weeks is sufficient to induce early and established atherosclerosis in NZWR respectively. The consistent results through this method could facilitate researchers in inducing early and established atherosclerosis in NZWR.
Fulltext Safety evaluation of saffron extracts in early and established atherosclerotic New Zealand white rabbits

Abd Rahim IN, Mohd Kasim NA, Omar E, Abdul Muid S, Nawawi H

PLoS One, 2024;19(1):e0295212.
PMID: 38207245 DOI: 10.1371/journal.pone.0295212

Previous research has shown that natural medications pose health risks, especially in subjects with comorbidities. This study aimed to evaluate the safety of saffron ethanolic extract (SEE) administration in early and established atherosclerotic rabbits. Rabbits were given a high-cholesterol diet (HCD) for 4 and 8 weeks to induce early and established atherosclerosis respectively, and then they were treated with 50 and 100 mg/kg/day SEE. The body weight of the animals was recorded. Blood samples were collected at baseline, pre-treatment, and post-treatment for hematological studies, lipid profiles, and biochemical profiles. Tissue specimens of the vital organs were subjected to histological examination. The above parameters were significantly altered post-intervention with 4 and 8 weeks of HCD. No significant differences in body weight were observed in all the groups post-treatment with 50 and 100mg/kg of SEE compared to pre-treatment. However, low-density lipoprotein cholesterol, total cholesterol, serum urea, and glucose significantly decreased post-treatment with 50 and 100mg/kg/day SEE compared to pre-treatment in early and established atherosclerosis groups. Hematological parameters that were affected post-intervention with HCD returned to their baseline values post-treatment with 50 and 100mg/kg/day SEE. There was a significant improvement in the vital organs post-treatment with 50 and 100mg/kg SEE. SEE can safely be administered without causing harmful effects on the hematological, biochemical profiles, and vital organs. Notably, SEE exerts hypolipidemic and hypoglycemic effects on atherosclerotic conditions. Further clinical trials are warranted to ensure the safety of saffron administration in patients with atherosclerosis-related diseases.

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