Reverse-phase High-pressure Chromatography Research Articles

Brevinin-2R and Derivatives as Potential Anticancer Peptides: Synthesis, Purification, Characterization and Biological Activities

Brevinin-2R (BR-2R), a novel antimicrobial and anti-cancer peptide and two derivatives, BR-C and BR-D, were well designed, synthesized and purified by RP-HPLC (reverse phase high pressure chromatography). Accuracy was confirmed through mass spectrometry. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay was carried out to determine cytotoxicity of various concentrations of peptides and their anti-proliferative potential. Results indicate that these compounds have significant anticancer activities in vitro on both MCF-7 (human breast adenocarcinoma) and A549 (human lung carcinoma) cancer cells in a concentration-dependent manner, although showed less cytotoxicity on human red blood cells. Further investigation verified that apoptosis induction, confirmed by Annexin V/PI staining and strong sub G1 cell cycle arrest, was observed in treated MCF-7 and A549 cells. Induction of apoptosis and anti-proliferating activity were closely associated with augmentation of caspase-3/7 activity, surging the apoptotic cells with no change in caspase-8 activity. The growth inhibition of BR-2R was exceedingly stronger than two derivatives; on the other hand, MCF-7 was more susceptible to treatment than A549. Findings prominently suggested development of Brevinin-2R and derivatives as novel promising compounds for cancer therapy, which may conquer drug resistance.

English

Proteolysis of UHT milk during storage is one of the problems affecting the dairy industry worldwide. Native enzymes have been implicated as being the main cause of spoilage of such milk. In the current study, reverse phase high pressure chromatography (RP-HPLC) method was used to detect proteolysis by native enzymes in high temperature heated milk. The aim of this research was to assess susceptibility of milk to proteolysis by native enzymes, after being subjected to various temperature-time processing conditions. Samples of raw and heated milk were clarified prior to analysis. Clarification was carried out to obtain pH 4.6 and 6% TCA soluble extracts, which were injected into RP-HPLC after filtration. This method confirmed that raw milk and milk processed at 85°C /15 s were the most proteolysed, indicating that the high temperatures employed during this study (110, 120, 130 and 142°C for 2s) inactivated the native enzymes. The RP-HPLC method is a useful method for the detection of proteolysis in milk. Key words: Reverse phase-high performance chromatography (RP-HPLC), proteolysis, milk, plasmin, heat-treatments.

Purification and analysis of a 5 kDa component of enamel matrix derivative

High performance liquid chromatography (HPLC) methods were used to analyse a 5 kDa component purified from enamel matrix derivative (EMD), the active ingredient in Emdogain ®, a commercial product for periodontal tissue regeneration. After initial purification by size-exclusion chromatography (SEC) on a 100 cm × 5 cm column (Bio-Gel P-30 Fine, 280 nm), collected fractions were analysed by size-exclusion HPLC (SE HPLC; TSK-Gel Super SW2000, 220 nm). The fractions containing only the 5 kDa component were analysed by reversed-phase high-pressure chromatography (RP HPLC; YMC-Pack ODS-A, 200 nm), revealing four peaks of the 5 kDa component. From 1200 mg of EMD (of which 9% is the 5 kDa component), approximately 65 mg of lyophilised 5 kDa component were obtained, corresponding to a recovery of 60%. The SE HPLC method was mainly suitable for qualitative analysis, whereas the RP HPLC method was appropriate for both qualitative and quantitative analysis.

Digestion of Streptococcus pneumoniae Cell Walls with Its Major Peptidoglycan Hydrolase Releases Branched Stem Peptides Carrying Proinflammatory Activity

The peptidoglycan of Gram-positive bacteria is known to trigger cytokine release from peripheral blood mononuclear cells (PBMCs). However, it requires 100-1000 times more Gram-positive peptidoglycan than Gram-negative lipopolysaccharide to release the same amounts of cytokines from target cells. Thus, either peptidoglycan is poorly active or only part of it is required for PBMC activation. To test this hypothesis, purified Streptococcus pneumoniae walls were digested with their major autolysin N-acetylmuramoyl-L-alanine amidase, and/or muramidase. Solubilized walls were separated by reverse phase high pressure chromatography. Individual fractions were tested for their PBMC-stimulating activity, and their composition was determined. Soluble components had a Mr between 600 and 1500. These primarily comprised stem peptides cross-linked to various extents. Simple stem peptides (Mr <750) were 10-fold less active than undigested peptidoglycan. In contrast, tripeptides (Mr >1000) were >/=100-fold more potent than the native material. One dipeptide (inactive) and two tripeptides (active) were confirmed by post-source decay analysis. Complex branched peptides represented </=2% of the total material, but their activity (w/w) was almost equal to that of LPS. This is the first observation suggesting that peptidoglycan stem peptides carry high tumor necrosis factor-stimulating activity. These types of structures are conserved among Gram-positive bacteria and will provide new material to help elucidate the mechanism of peptidoglycan-induced inflammation.

Iron-induced lipid peroxidation and inhibition of proliferation in Ehrlich ascites tumor cells

The purpose of this study was to find further experimental evidence for the postulated negative association between the extent of lipid peroxidation in tumor cells and their proliferative behavior. After incubation of Ehrlich ascites tumor cells at 37 degrees C for 30 min with increasing concentrations of Fe(II) histidinate (Fe/His) the following parameters were determined: the formation of lipid hydroperoxides was measured fluorimetrically after reaction with dichlorofluorescein; 4-hydroxynonenal was determined by reversed-phase high-pressure chromatography after derivatization with dinitrophenylhydrazine; as a third parameter of lipid peroxidation the formation of 2-thiobarbituric-acid-reactive substances was determined. The proliferative activity was determined by measuring the growth rate in vivo after reimplantation i.p. of the tumor cells into mice. Trypan-blue exclusion tests for viability were performed before reimplantation. The reliability of the trypan-blue exclusion tests was checked by comparing the results with another parameter of viability, the release of the cytosolic enzyme lactate dehydrogenase. The concentration both of lipid hydroperoxides and of 2-thiobarbituric-acid-reactive substances showed a biphasic dependence on the concentration of Fe/His with maximal increase at iron concentrations of 0.25 mM and 0.1 mM respectively. 4-Hydroxynonenal, in contrast, showed a continuous increase up to 41.1 nM (corresponding to 0.58 pmol/10(9) cells) with increasing iron concentration in the range from 0.1 mM to 0.6 mM. The total number of tumor cells, when determined 5 days after reimplantation, continuously decreased with increasing iron concentration, showing half-maximal inhibition at about 0.22 mM Fe. The exclusion of the trypan-blue dye was unaffected by the presence of iron at any concentration used. Similarly, iron had no influence on the release of lactate dehydrogenase. The results support the hypothesis that 4-hydroxynonenal may act as an inhibiting messenger between endogenic lipid peroxidation and proliferation.

Presence of TRF immunoreactivity in marginal islet cells in rat pancreas.

Isolated rat pancreatic islet extracts were submitted to reverse phase high pressure chromatography (HPLC) and assayed for thyrotrophin releasing factor (TRF), glucagon and insulin in radioimmunoassays (RIA). Our anti-TRF serum was prepared by immunizing rabbits with TRF conjugated to bovine thyroglobulin (bTG) by bisdiazobenzidine. Anti-glucagon and antiinsulin were from a commercial source (Novo, Denmark). The concentrations of TRF, glucagon and insulin were 0.05 +/- 0.02, 0.06 +/- 0.02 and 1.0 +/- 0.2 pmol/islet, respectively. Formalin-fixed pancreatic sections were stained for TRF, glucagon and insulin by peroxidase, antiperoxidase (PAP) complex method. When the adjacent sections were stained for glucagon or insulin, it was observed that TRF and glucagon-specific peroxidase reactions were confined to the marginal islet cells, but insulin reactions to the central cells. The TRF-specific peroxidase reaction was clearly reduced when the anti-TRF serum was unchanged when the antiserum was pre-incubated with synthetic TRF. The present HPLC results suggest that the islets contained TRF. Immunocytochemical studies show that the TRF-immunoreactive material, either synthesized or bound, is localized in the marginal islet cells.