Global DNA and p53 region-specific hypomethylation in human colonic cells is induced by folate depletion and reversed by folate supplementation.

There is increasing evidence to suggest that reduced folate status may be a causative factor in carcinogenesis, particularly colorectal carcinogenesis. Folate is essential for the synthesis of S-adenosylmethionine, the methyl donor required for all methylation reactions in the cell, including the methylation of DNA. Global DNA hypomethylation appears to be an early, and consistent, molecular event in carcinogenesis. We have examined the effects of folate depletion on human-derived cultured colon carcinoma cells using 2 novel modifications to the Comet (single cell gel electrophoresis) assay to detect global DNA hypomethylation and gene region–specific DNA hypomethylation. Colon cells cultured in folate-free medium for 14 d showed a significant increase in global DNA hypomethylation compared with cells grown in medium containing 3µmol/L folic acid. This was also true at a gene level, with folate-deprived cells showing significantly more DNA hypomethylation in the region of the p53 gene. In both cases, the effects of folate depletion were completely reversed by the reintroduction of folic acid to the cells. These results confirm that decreased folate levels are capable of inducing DNA hypomethylation in colon cells and particularly in the region of the p53 gene, suggesting that a more optimal folate status in vivo may normalize any DNA hypomethylation, offering potential protective effects against carcinogenesis. This study also introduces 2 novel functional biomarkers of DNA hypomethylation and demonstrates their suitability to detect folate depletion–induced molecular changes.

Copper/zinc superoxide dismutase is phosphorylated and modulated specifically by granulocyte-colony stimulating factor in myeloid cells.

Using two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) of 32P-labeled cytosolic and membrane extracts, we identified a 21.5 kDa phosphoprotein with an isoelectric point of 6.0 in NFS-60 cells that was phosphorylated maximally at 15 min by treatment with granulocyte-colony stimulating factor (G-CSF) but not with interlevkin-3 (IL-3) or colony-stimulating factor-1 (macrophage-colony stimulating factor (CSF-1 (M-CSF)). The phosphorylation of this protein, designated 21.5/6.0, was unaffected by a series of antiproliferative agents [32]. These findings suggested that the 21.5/6.0 phosphoprotein may be involved in specific G-CSF-mediated biological responses such as activation and/or differentiation. We sought to characterize this 21.5/6.0 by a novel combination of 2-D SDS-PAGE and hydroxyapatite (HTP)-chromatography. Amino acid sequence determination of 21.5/6.0 revealed it to share a high level of homology with copper/zinc superoxide dismutase (Cu/Zn-SOD), indicating that a Cu/Zn-SOD is phosphorylated following treatment with G-CSF. This is the first report of the phosphorylation and possible involvement of Cu/Zn-SOD protein in granulocyte activation/differentiation events. In addition, Cu/Zn-SOD levels and activity were diminished by G-CSF but not IL-3 treatment. This new protocol combining 2-D SDS-PAGE and HTP-chromatography allows the characterization of low abundance phosphoproteins involved in the cellular responses to G-CSF and presumably to other cytokines/growth factors.

An improved purification procedure for cyclosporine synthetase

We have developed expedient and reliable methods to isolate cyclosporin synthetase for in vitro biosynthesis of cyclosporins. We have examined enzyme purification strategies suited to large-scale processing and present a chromatographic sequence that serves as a pilot model for industrial scale preparation of cyclosporin synthetase from cyclosporin producing fungi. A chromatographic sequence consisting of ammonium sulfate precipitation → gel filtration → hydrophobic interaction chromatography → anion exchange chromatography, yielded an electrophoretically homogeneous cyclosporin synthetase preparation (Coomassie G-250 brilliant blue staining). Furthermore, a native polyacrylamide gel electrophoresis system was developed for the isolation of active cyclosporin synthetase enzyme from crude extracts of cyclosporin producing fungi. The environmental factors affecting enzyme stability and the continuity of the in vitro cyclosporin biosynthetic reaction-temperature, pH, and substrate depletion were assessed and manageable conditions have been defined for sustainable cyclosporin biosynthesis with enzyme isolates. Cyclosporin synthetase exhibited an optimal temperature range of 24–29 °C and a pH optimum of 7.6. The native enzyme displayed a pI of 5.7, as determined by isoelectric focusing. The industrial implementation of an in vitro biosynthetic approach could potentially prove useful for the production of important therapeutic cyclosporins which occur as only minor fermentation by-products.

An improved method for the purification of rat liver-type fatty acid binding protein from Escherichia coli

We have developed expedient and reliable methods to isolate cyclosporin synthetase for in vitro biosynthesis of cyclosporins. We have examined enzyme purification strategies suited to large-scale processing and present a chromatographic sequence that serves as a pilot model for industrial scale preparation of cyclosporin synthetase from cyclosporin producing fungi. A chromatographic sequence consisting of ammonium sulfate precipitation → gel filtration → hydrophobic interaction chromatography → anion exchange chromatography, yielded an electrophoretically homogeneous cyclosporin synthetase preparation (Coomassie G-250 brilliant blue staining). Furthermore, a native polyacrylamide gel electrophoresis system was developed for the isolation of active cyclosporin synthetase enzyme from crude extracts of cyclosporin producing fungi. The environmental factors affecting enzyme stability and the continuity of the in vitro cyclosporin biosynthetic reaction-temperature, pH, and substrate depletion were assessed and manageable conditions have been defined for sustainable cyclosporin biosynthesis with enzyme isolates. Cyclosporin synthetase exhibited an optimal temperature range of 24–29 °C and a pH optimum of 7.6. The native enzyme displayed a pI of 5.7, as determined by isoelectric focusing. The industrial implementation of an in vitro biosynthetic approach could potentially prove useful for the production of important therapeutic cyclosporins which occur as only minor fermentation by-products.

Purification and characterization of naringinase from a newly isolated strain of Aspergillus niger 1344 for the transformation of flavonoids

An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg2+, SDS, p-chloromercuribenzoate, Cu2+ and Mn2+ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca2+, Co2+ and Mg2+ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg2+ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.

Fresh garlic : a possible vehicle for Salmonella Virchow

A sustained increase in Salmonella enterica serovar Virchow notifications in South Eastern Australia between September 1997 and May 1998 instigated a case-control study and environmental investigations. Cases were defined as having locally acquired culture-confirmed S. Virchow phage-type 8 infection and diarrhoeal disease. Matched controls were selected by progressive digit dialling based on cases telephone numbers. An exposure and food history questionnaire was administered by telephone. Phage typing and pulse field gel electrophoresis were performed on case and environmental isolates. Thirty-two notifications of S. Virchow infection met the case definition, 37% reported bloody diarrhoea and S. Virchow was isolated from blood in 13% of cases. Twelve patients were admitted to hospital and one died. Fresh garlic (OR 4·1, 95% CI 1·3-12·8) and semi-dried tomatoes (OR 12·6, 95% CI 1·5-103·1) were associated with these cases. The associations remained significant after adjusting for sex and age. S. Virchow (PT 8) was cultured from two brands of semi-dried tomatoes associated with cases in two different states. We provide sufficient evidence for semi-dried tomatoes and fresh garlic to be considered as potential risk foods in future Salmonella outbreak investigations.

Zinc and DHA have opposing effects on the expression levels of histones H3 and H4 in human neuronal cells

Zn and DHA have putative neuroprotective effects and these two essential nutrients are known to interact biochemically. We aimed to identify novel protein candidates that are differentially expressed in human neuronal cell line M17 in response to Zn and DHA that would explain the molecular basis of this interaction. Two-dimensional gel electrophoresis and MS were applied to identify major protein expression changes in the protein lysates of human Ml7 neuronal cells that had been grown in the presence and absence of Zn and DHA. Proteomic findings were further investigated using Western immunoblot and real-time PCR analyses. Four protein spots, which had significant differential expression, were identified and selected for in-gel trypsin digestion followed by matrix-assisted laser desorption ionisation MS analysis. The resultant peptide mass fingerprint for each spot allowed their respective identities to be deduced. Two human histone variants H3 and H4 were identified. Both H3 and H4 were downregulated by Zn in the absence of DHA (Zn effect) and upregulated by DHA (DHA effect) in the presence of Zn (physiological condition). These proteomic findings were further supported by Western immunoblot and real-time PCR analyses using H3- and H4-specific monoclonal antibodies and oligonucleotide primers, respectively. We propose that dietary Zn and DHA cause a global effect on gene expression, which is mediated by histones. Such novel information provides possible clues to the molecular basis of neuroprotection by Zn and DHA that may contribute to the future treatment, prevention and management of neurodegenerative diseases such as Alzheimer's disease.

Phospholipase activity in human mesenteric ischaemia and infarction

Currently, diagnostic tests for mesenteric ischaemia and infarction are inadequate due to poor sensitivity and specificity. In addition, many potential markers appear too late to be clinically useful. At present, definitive diagnosis can only be made at the time of surgery, which is not ideal as surgery is often to be avoided in critically ill and elderly patients. A clinically useful, minimally invasive test is likely to decrease the currently very high mortality rate and allow monitoring of 'at risk' patients during their hospital stay. A two-dimensional electrophoresis based proteomic approach was undertaken to assess plasma protein differences between patients with surgically confirmed bowel infarction and control Intensive Care patients. The major protein differences were found to be members or variants of acute phase proteins. Serum amyloid A showed the largest difference between the two patient groups, and this protein was investigated in greater depth. An analysis was performed to compare the diagnostic ability of several commonly used indicators of critical illness and bowel infarction with serum amyloid A and phospholipase A2. Although none of the variables were ideal for clinical use, plasma phospholipase A2 activity showed the best discriminatory power, as determined by Receiver Operating Characteristic curves. From a review of the literature, phospholipase AI (PLA2) appeared to be increased in the bowel as a result of ischaemia and infarction. In one patient, matched tissues were obtained, and PLA2 activity was found to be significantly higher in infarcted bowel tissue compared to ischaemic bowel tissue. PLA2 activity was significantly greater in bowel lumen than tissue, suggesting that the protein was being released, and may enter the circulation. PLA2 activity was increased in the plasma of bowel infarction patients compared with control patients, though the difference was not significant. The phospholipase activity exhibited a number of similarities to typical phospholipase A2 proteins, but also showed a number of inconsistent characteristics. For this reason, we wished to identify the protein responsible for the increased phospholipase activity in infarcted human bowel. The PLA2 activity in human bowel could not be abolished by immunoprecipitation of the PLA2 isoforms IIA (well described in bowel) and V (a closely related isoform). To investigate these proteins, a native urea protein gel devised for snake venom phospholipase A2 was modified for use with mammalian phospholipase AI. The modified gel was used to show that the protein with phospholipase activity from infarcted gut was different from normal gut PLA2 and type IIA PLA2. A number of extensions were devised for these native gels and were found to be useful both in this investigation and for venom investigations. Protein purification was undertaken to identify the protein responsible for the increased phospholipase activity in infarcted bowel. Protein was purified from infarcted human bowel using a number of techniques that exploited unusual characteristics of the protein. The purification techniques each retained the native activity of the protein and the purification could therefore be monitored with a phospholipid hydrolysis assay at each stage. The protein identified by mass spectrometry was an excellent match for cyclophilin B, an inflammatory protein that had previously been identified in rat bowel at the mRNA level (Hasel et al, 1991, Kainer & Doris, 2000). As the purification progress had been monitored throughout with a phospholipid hydrolysis assay, cyclophilin B was an unexpected identification, as it is not known to have phospholipase activity. Cyclophilin B was removed from the highly purified samples via immunoprecipitation and this process abolished all phospholipase activity. The addition of cyclosporin A, (the pharmaceutical ligand of cyclophilin B), did not effect the phospholipase activity. Cyclophilin B protein was found in normal and infarcted human bowel using Western blotting. Cyclophilin B protein also appeared to be present in the bowel lumen and plasma of several patients with bowel infarction, but not in control patients. Immunohistochemistry confirmed the ubiquitous nature of cyclophilin B that had been reported by other groups. This project has investigated the use of two dimensional gel electrophoresis based proteomics to identify proteins present in the plasma of patients with confirmed bowel infarction and control intensive care patients. The major protein classes observed were members of the acute phase proteins, which highlights the need for pre-fractionation of plasma to identify lower abundance, disease associated proteins. A series of potential plasma markers were compared using Receiver Operating Characteristic Curves. Although no ideal marker was clear from this analysis, phospholipase activity appeared to warrant further investigation. Phospholipase activity was investigated in human infarcted bowel. Protein purification identified cyclophilin B as a bowel protein that showed unusual phospholipid hydrolysing activity. Cyclophilin B is a ubiquitous protein in intestinal cell types in both normal and infarcted tissue. There appears to be release of cyclophilin B into bowel lumen and plasma under conditions of mesenteric ischaemia and infarction.

Methionine regulates copper/hydrogen peroxide oxidation products of Aβ

Metal-catalysed oxidation (MCO) may play a causative role in the pathogenesis of Alzheimer's disease (AD). Amyloid peptide (A), the major biomarker of AD, in the presence of copper ions reduces Cu2+ to Cu+ and catalyses the formation of H2O2 that subsequently induces radicals through Fenton chemistry. A is also subject to attack by free radicals, where the presence of Cu2+ in conjunction with H2O2 catalyses oxygenation, primarily at the methionine sulfur atom. This work investigates MCO of A, to gain further insight into the role of oxidative stress in AD. By combining a fluorescence assay with gel electrophoresis to monitor MCO reactions of A (1-28) in the presence and absence of methionine it was determined that methionine can both protect some residues against MCO and promote the oxidation of Tyr(10) specifically. Electrospray ionization mass spectrometric analysis of methionine MCO products indicated the formation of methionine sulfoxide, methionine sulfone and related hydroxylated products. Similar products could be formed from the oxidation of Met(35) of A and may relate to changes in properties of the peptide following MCO.

The role of folate in colorectal cancer : human studies into folate in those at risk of colorectal cancer

Colorectal cancer is one of the most common cancers worldwide and specific nutients have been associated with the risk of developing it, one of which is folate. As cancer starts at a cellular level, it is important to look at known markers or precursors of cancerous changes to see what effect a nutrient or toxin may have. It is also important to define the nutrient of concern within the exact tissue of interest as well as more easily available samples like blood. This text seeks to define folate concentrations within human colonic tissue and blood and then using a specialised technique known as single cell gel electrophoresis examine the level of damage seen in precursors of cancerous change associated with folate status. An intervention trial will also be discussed whereby folic acid supplementation was conducted in a double blind placebo controlled environment in those at risk of developing colorectal cancer.

Detection of brassinosteroids in pollen of Lolium perenne L. by immunocytochemistry

Bioactive brassinosteroids have been localized in developing and mature pollen of anhydrously fixed rye-grass (Lolium perenne) by immunocytochemistry using polyclonal antibodies to castasterone generated in rabbits. Tricellular pollen fixed by freeze-substitution was also labelled in the starch granules. Study of the developmental sequence of the pollen through the microsporocyte, microspore, bicellular and tricellular stages showed that the brassinosteroids were increasingly sequestered in starch granules as the amyloplasts matured, supporting the view that these are storage organelles for these potent plant growth promoters. In bicellular pollen, heavy labelling was seen in the zone within 0.5 μm of the starch granule, where stromal tissue remains. Thus, the stroma may be the site of synthesis of these compounds. During aqueous fixation, the brassinosteroids leached from the starch granules of tricellular pollen, indicating that they would be quickly available after imbibition to influence the physiology of germinating pollen. The results from high-performance liquid chromatography of dansylaminophenylboronates from partially purified extracts of freshly dehisced tricellular pollen of rye-grass showed 25-methylcastasterone may be a minor component, together with two unknown peaks. No specific binding of brassinolide to any soluble proteins extracted from tricellular rye-grass pollen was observed using the antibodies in gel electrophoresis or enzyme-linked immunosorbent assays.

Identification of tRNAs incorporated into wild-type and mutant human immunodeficiency virus type 1

We have identified the tRNAs which are incorporated into both wild-type human immunodeficiency virus type 1 strain IIIB (HIV-1IIIB) produced in COS-7 cells transfected with HIV-1 proviral DNA and mutant, noninfectious HIV-1Lai particles produced in a genetically engineered Vero cell line. The mutant proviral DNA contains nucleotides 678 to 8944; i.e., both long terminal repeats and the primer binding site are absent. As analyzed by two-dimensional polyacrylamide gel electrophoresis, both mutant and wild-type HIV-1 contain four major-abundance tRNA species, which include tRNA(1,2Lys), tRNA(3Lys) (the putative primer for HIV-1 reverse transcriptase) and tRNA(Ile). Identification was accomplished by comparing the electrophoretic mobilities and RNase T1 digests with those of tRNA(3Lys) and tRNA(1,2Lys) purified from human placenta and comparing the partial nucleotide sequence at the 3' end of each viral tRNA species with published tRNA sequences. Thus, the absence of the primer binding site in the mutant virus does not affect tRNA(Lys) incorporation into HIV-1. However, only the wild-type virus contains tRNA(3Lys) tightly associated with the viral RNA genome. The identification of the tightly associated tRNA as tRNA(3Lys) is based upon an electrophoretic mobility identical to that of tRNA(3Lys) and the ability of this RNA to hybridize with a tRNA(3Lys)-specific DNA probe. In addition to the four wild-type tRNA species, the mutant HIV-1-like particle contains two tRNA(His) species and three tRNA-sized species that we have been unable to identify. Their absence in wild-type virus makes it unlikely that they are required for viral infectivity.

Effects of modifying the tRNA(3Lys) anticodon on the initiation of human immunodeficiency virus type 1 reverse transcription

tRNA(3Lys) is a primer for reverse transcription in human immunodeficiency virus type 1 (HIV-1), and the anticodon of tRNA(3Lys) has been implicated in playing a role in both its placement onto the HIV-1 genome and its interaction with HIV-1 reverse transcriptase (RT). In this work, the anticodon in a tRNA(3Lys) gene was changed from UUU to CUA (tRNA(3Lys)Su+) or, in addition, G-73 was altered to A (tRNA(3Lys)Su+G73A). COS-7 cells were transfected with either wild-type or mutant tRNA(3Lys) genes, and both the wild-type and mutant tRNA(3Lys) produced were purified by using immobilized tRNA-specific hybridization probes. Each mutant tRNA(3Lys) was tested for its ability to prime reverse transcription in vitro, either alone or in competition with wild-type tRNA(3Lys). Short RT extensions of wild-type and mutant tRNALys could be distinguished from each other by their different mobilities in one-dimensional single-stranded conformation polymorphism polyacrylamide gel electrophoresis. These reverse transcription products show that heat-annealed tRNA(3Lys)Su+ has the same ability as heat-annealed wild-type tRNA(3Lys) to prime RT and competes equally well with wild-type tRNA(3Lys) for priming RT. tRNA(3Lys)Su+G73A has 60% of the wild-type ability to prime RT but competes poorly with wild-type tRNA(3Lys) for priming RT. However, the priming abilities of wild-type and mutant tRNA(3) are quite different when in vivo-placed tRNA is examined. HIV-1 produced in COS cells transfected with a plasmid containing both the HIV-1 proviral DNA and DNA coding for tRNA(3Lys)Su+ contains both endogenous, cellular wild-type tRNA(3Lys) and mutant tRNA(3Lys). When total viral RNA is used as the source of primer tRNA placed onto the genomic RNA in vivo, only wild-type tRNA(3Lys) is used as a primer. If the total viral RNA is first heated and exposed to hybridizing conditions, then both the wild-type and mutant tRNA(3Lys) act as primers for RT. These results indicate that the tRNA(3Lys)Su+ packaged into the virions is unable to act as a primer for RT, and a model is proposed to explain the disparate results between heat-annealed and in vivo-placed primer tRNA.

Purification and characterization of a low molecular mass alkaliphilic lipase of Bacillus cereus MTCC 8372

A low molecular mass alkaliphilic extra-cellular lipase of Bacillus cereus MTCC 8372 was purified 35-fold by hydrophobic interaction (Octyl-Sepharose) chromatography. The purified enzyme was found to be electrophoretically pure by denaturing gel electrophoresis and possessed a molecular mass of approximately 8 kDa. It is a homopentamer of 40 kDa as revealed by native-PAGE. The lipase was optimally active at 55 °C and retained approximately half of its original activity after 40 min incubation at 55 °C. The enzyme was maximally active at pH 8.5. Mg ²⁺ , Cu ²⁺ , Ca ²⁺ , Hg ²⁺ , Al ³⁺ and Fe ³⁺ at 1 mM enhanced hydrolytic activity of the lipase. Interestingly, Hg ²⁺ ions synergized and Zn ²⁺ and Co ²⁺ ions antagonized the lipase activity. Among surfactants, Tween 80 promoted the lipase activity. Phenyl methyl sulfonyl fluoride (PMSF, 15 mM) decreased 98% of original activity of lipase. The lipase was highly specific towards p -nitrophenyl palmitate and showed a V _max and K _m of 0.70 mmol.mg ⁻¹ .min ⁻¹ and 32 mM for hydrolysis of p NPP.

Anti-metastatic and differential effects on protein expression of epigallocatechin-3-gallate in HCCLM6 hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third highest cause of cancer-related mortality in humans. Epigallocatechin-3-gallate (EGCG) has been shown to inhibit the metastatic activity of certain cancer cells. The aim of this study was to determine the effects and molecular mechanism(s) of action of EGCG in human HCC cells. A migration and invasion assay for the metastatic behavior of HCCLM6 cells was performed. The anti-metastatic effects of EGCG were investigated by RT-PCR and gelatin zymography. A total cellular protein profile was obtained using 2-dimensional gel electrophoresis (2-DE), followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) analyses of proteins with significant differences in expression following treatment with EGCG. The results revealed that EGCG induced apoptosis and inhibited the metastasis of HCCLM6 cells. The anti-metastatic effects of EGCG were associated with the inhibition of matrix metalloproteinase (MMP)-2 and MMP-9 activity. The expression levels of far upstream element (FUSE) binding protein 1 (FUBP1), heat shock protein beta 1 (HSPB1), heat shock 60 kDa protein 1 (chaperonin) (CH60) and nucleophosmin (NPM) proteins, which are associated with metastasis, were significantly altered in the EGCG-treated HCCLM6 cells. The data from the present study suggest that EGCG has potential as a therapeutic agent for the treatment of HCC.