Apoptotic effect of cisplatin and cordycepin on OC3 human oral cancer cells.

OBJECTIVE: To evaluate apoptotic effects of cisplatin and cordycepin as single agent or in combination with cytotoxicity in oral cancer cells. METHODS: The influences of cisplatin (2.5 µg/mL) and/or cordycepin treatment (10 or 100 µmol/L) to human OC3 oral cancer cell line were investigated by morphological observation for cell death appearance, methylthiazoletetrazolium (MTT) assay for cell viability, flow cytometry assay for cell apoptosis, and Western blotting for apoptotic protein expressions. RESULTS: Data demonstrated that co-administration of cisplatin (2.5 µg/mL) and cordycepin (10 or 100 µmol/L) resulted in the enhancement of OC3 cell apoptosis compared to cisplatin or cordycepin alone treatment (24 h), respectively (P <0.05). In flow cytometry assay, percentage of cells arrested at subG1 phase with co-treatment of cordycepin and cisplatin (30%) was significantly higher than cisplatin (5%) or cordycepin (12%) alone group (P <0.05), confirming a synergistically apoptotic effect of cordycepin and cisplatin. In cellular mechanism study, co-treatment of cordycepin and cisplatin induced more stress-activated protein kinase/Jun terminal kinase (JNK), the expressions of caspase-7, and the cleavage of poly ADP-ribose polymerase (PARP) as compared to cisplatin or cordycepin alone treatment (P <0.05). CONCLUSION: Cisplatin and cordycepin possess synergistically apoptotic effect through the activation of JNK/caspase-7/PARP pathway in human OC3 oral cancer cell line.

Unfolding analysis of the mature and unprocessed forms of Bacillus licheniformis γ-glutamyltranspeptidase.

Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) undergoes an autocatalytic process to generate 44.9 and 21.7 kDa subunits; however, a mutant protein (T399A) loses completely the processing ability and mainly exists as a precursor. For a comprehensive understanding of their structural features, the biophysical properties of these two proteins were investigated by circular dichroism and fluorescence spectroscopy. Tryptophan fluorescence and circular dichroism spectra were nearly identical for BlGGT and T399A, but unfolding analyses revealed that these two proteins had a different sensitivity towards temperature- and guanidine hydrochloride (GdnHCl)-induced denaturation. BlGGT and the unprocessed T399A displayed T(m) values of 61.4°C and 68.1°C, respectively, and thermal unfolding of both proteins was found to be highly irreversible. Fluorescence quenching analysis showed that T399A had a dynamic quenching constant similar to that of the wild-type enzyme. BlGGT started to unfold beyond ∼2.14 M GdnHCl and reached an unfolded intermediate, [GdnHCl](0.5, N - U), at 2.85 M, corresponding to free energy change [Formula: see text] of 12.34 kcal mol( - 1), whereas the midpoint of the denaturation curve for T399A was approximately 3.94 M, corresponding to a [Formula: see text] of 4.45 kcal mol( - 1). Taken together, it can be concluded that the structural stability of BlGGT is superior to that of T399A.

Immobilization of Escherichia coli novablue gamma-glutamyltranspeptidase in Ca-alginate-kappa-carrageenan beads.

The recombinant Escherichia coli gamma-glutamyltranspeptidase (EcGGT) was immobilized in Ca-alginate-kappa-carrageenan beads. Effects of alginate concentration, amount of loading enzyme, and bead size on the entrapped activity were investigated. Optimum alginate concentration for EcGGT immobilization was found to be 2% (w/v). Using a loading enzyme concentration of 1.5 mg/g alginate, maximum enzyme activity was observed. With increase in bead size from 1.9 to 3.1 mm, the immobilization efficiency was decreased significantly because of mass transfer resistance. Thermal stability of the free EcGGT was increased as a result of the immobilization. Ca-alginate-kappa-carrageenan-EcGGT beads were suitable for up to six repeated uses, losing only 45% of their initial activity. Upon 30 days of storage the preserved activity of free and immobilized enzyme were found as 4% and 68%, respectively. The synthesis of L: -theanine was performed in 50 mM Tris-HCl buffer (pH 10) containing 25 mM L: -glutamine, 40 mM ethylamine, and 1.5 mg EcGGT/g alginate at 40 degrees C for 12 h, and a conversion rate of 27% was achieved.

Intrathecal midazolam combined with low-dose bupivacaine improves postoperative recovery in diabetic mellitus patients undergoing foot debridement.

BACKGROUND: Intrathecal midazolam acts synergically with other anesthetics to relieve surgical pain, and the drug combination may decrease complications attributable to each component drug. This prospective study was to determine the spinal effects of low-dose of bupivacaine (5 mg) combined with intrathecal midazolam (2 mg) in diabetes mellitus (DM) patients undergoing foot debridement. METHODS: Sixty diabetic patients were admitted for foot debridement under spinal anesthesia were equally divided into two groups. Group 1 (M) received 7.5 mg of hyperbaric bupivacaine; group 2 (M+M) received 5 mg of hyperbaric bupivacaine combined with 2 mg of midazolam intrathecally. The intensity of motor block was assessed with modified Bromage scale 20 minutes after injection, and at 0, 30, 60, 90 and 120 min after arriving at the post anesthesia care unit (PACU). Pain score was assessed with a 10 cm visual analog scale (VAS, 0 = no pain and 10 = intolerable pain) at 0, 1, 2, 6 h and 24 h postoperatively. RESULTS: Anesthesia was smooth in all patients except one in group M, whose analgesia was inadequate and general anesthesia was given to complete the surgery. The number of patients who sustained moderate to severe pain (VAS > 5) was significantly less in the M+M group than in M group as accessed 6 and 24 h postoperatively. The requirement of additional analgesic as reinforcement was significantly less in the M+M group than in the M group within the space of 24 h postoperatively. Recovery of motor function was significantly faster in the M+M group. CONCLUSIONS: The combination of intrathecal midazolam and bupivacaine was a safe and effective anesthetic technique, and it also provided early recovery of motor function and reduced the requirement of analgesics postoperatively.