Upregulation of TET activity with ascorbic acid induces epigenetic modulation of lymphoma cells.

The Ten Eleven Translocation (TET) enzymes have been found to be mutated in both diffuse large B-cell (DLBCL) and peripheral T-cell (PTCL) lymphomas resulting in DNA hypermethylation. Recent studies in embryonal stem cells showed that ascorbic acid (AA) is a cofactor for TET with a binding site at the catalytic domain, and enhances TET activity. We hypothesized that AA could potentially enhance TET activity in lymphoma cells to cause DNA demethylation, reactivate expression of tumor suppressor genes and enhance chemosensitivity. We demonstrate in vitro that AA treatment of DLBCL and PTCL cells using AA concentrations achievable intravenously increased TET activity leading to DNA demethylation. This epigenetic effect is independent of hydrogen peroxide. AA treatment increased the expression of SMAD1, a tumor suppressor gene known to be suppressed by methylation, and increased chemosensitivity of lymphoma cells. Twenty-nine percent (10/34) of unselected lymphoma patients had plasma AA levels that were deficient suggesting an additional clinical mechanism of TET hypofunction. These data indicate that AA has the potential to modify TET function in lymphoma and enhance chemosensitivity. In addition, the AA deficiency seen in some patients may further impair TET function and contribute to resistance. Clinical trials testing intravenous AA with chemotherapy are warranted.

Sequential pathogenesis of metastatic VHL mutant clear cell renal cell carcinoma: putting it together with a translational perspective.

Clear cell renal cell carcinoma (ccRCC) accounts for ∼80% of all RCC, and biallelic Von Hippel-Lindau (VHL) gene defects occur in ∼75% of sporadic ccRCC. The etiopathogenesis of VHL mutant metastatic RCC, based on our understanding to date of molecular mechanisms involved, is a sequence of events which can be grouped under the following: (i) loss of VHL activity (germline/somatic mutation + inactivation of the wild-type copy); (ii) constitutive activation of the hypoxia-inducible factor (HIF) pathway due to loss of VHL activity and transcription of genes involved in angiogenesis, epithelial-mesenchymal transition, invasion, metastasis, survival, anaerobic glycolysis and pentose phosphate pathway; (iii) interactions of the HIF pathway with other oncogenic pathways; (iv) genome-wide epigenetic changes (potentially driven by an overactive HIF pathway) and the influence of epigenetics on various oncogenic, apoptotic, cell cycle regulatory and mismatch repair pathways (inhibition of multiple tumor suppressor genes); (v) immune evasion, at least partially caused by changes in the epigenome. These mechanisms interact throughout the pathogenesis and progression of disease, and also confer chemoresistance and radioresistance, making it one of the most difficult metastatic cancers to treat. This article puts together the sequential pathogenesis of VHL mutant ccRCC by elaborating these mechanisms and the interplay of oncogenic pathways, epigenetics, metabolism and immune evasion, with a perspective on potential therapeutic strategies. We reflect on the huge gap between our understanding of the molecular biology and currently accepted standard of care in metastatic ccRCC, and present ideas for better translational research involving therapeutic strategies with combinatorial drug approach, targeting different aspects of the pathogenesis.

Halitosis: much beyond oral malodor.

Oral malodor one of the most common complaints with which patients approaches us thinking it can be detrimental to his self-image and confidence. Even though majority of oral malodor is of oral origin, there are multiple other systemic causes that have to be addressed while we diagnose and treat this condition. Most of these patients look up to oral care physicians for expert advice, it is critical for us to have the knowledge base and communication techniques to provide quality clinical assessment and implement effective intervention programs. This article reviews the various causes and the diagnostic modalities which will help us treat this multifaceted condition.

Knowledge about heart attack and hypertension among individuals attending a cardiac camp in Pokhara city.

OBJECTIVES: Previous studies in other countries had shown lacunae in patients' and general publics' understanding of cardiovascular diseases. Such studies are lacking in Nepal. Hence the present study was carried out to: a) note the respondent's knowledge regarding myocardial infarction and hypertension and b) note the association, if any, of the knowledge with demographic and personal characteristics. MATERIALS AND METHODS: Respondents attending a cardiac camp organized in the Manipal Teaching hospital during September 2002 were interviewed by previously briefed seventh semester students using a structured questionnaire. Basic demographic information and knowledge about myocardial infarction and hypertension was collected. The median score was calculated. Differences in scores among different subgroups of respondents were noted using appropriate statistical tests (p<0.05). RESULTS: Sixty-six respondents were interviewed; 44 were male. The knowledge scores for heart attack and hypertension were 6 (maximum score 8) and 11 (maximum score 14) respectively. The scores were significantly lower among respondents with a monthly family income below 2000 rupees and was higher among respondents/family members suffering from cardiovascular disease. CONCLUSION: The respondents were aware of the basic facts regarding myocardial infarction and hypertension. However, lacunae in knowledge were noted. Further studies are required.

A survey of drug use patterns in western Nepal.

BACKGROUND: In Nepal, self-treatment is common and complementary medicine practitioners play an important role in providing health services. Previous studies on drug use patterns have been mainly carried out in the Kathmandu valley. Studies in the Pokhara valley, western Nepal are lacking. The objectives of our study were to obtain: 1) baseline information on drug use patterns in the preceding six-month period, 2) reasons for using complementary and self-medication and 3) any association of drug use patterns with demographic variables. METHODS: Health workers of the community medicine department carried out the study in Pokhara city and Bedabari village using a semi-structured questionnaire. Differences in the proportion of patients using self-medication and complementary medicines according to sex, age, place of residence and socioeconomic status of the family were analysed by the z test of proportions (p < 0.05). FINDINGS AND CONCLUSIONS: One hundred and twenty individuals from 112 households had used prescribed allopathic or complementary remedies. Seventy-one point six percent of the respondents had used allopathic medicines. The commonest allopathic medicines prescribed were antibiotics and paracetamol. Complementary medicine use was more common among older respondents (> 30 years). Thirty-nine families practiced self-medication with home remedies accounting for 18.9% of the drugs used. Self-medication was more common among rural households. Complementary practitioners should be integrated into the health care system to provide health care in the rural areas. Studies on drug use patterns and on factors influencing drug use in the remote areas of Nepal are urgently required.

Self-medication and non-doctor prescription practices in Pokhara valley, Western Nepal: a questionnaire-based study.

BACKGROUND: Self-medication and non-doctor prescribing of drugs is common in developing countries. Complementary and alternative medications, especially herbs, are also commonly used. There are few studies on the use of these medications in Pokhara Valley, Western Nepal. METHODS: Previously briefed seventh semester medical students, using a semi-structured questionnaire, carried out the study on 142 respondents. Demographic information and information on drugs used for self-medication or prescribed by a non-allopathic doctor were collected. RESULTS: Seventy-six respondents (54%) were aged between 20 to 39 years. The majority of the respondents (72 %) stayed within 30 minutes walking distance of a health post/medical store. 59% of these respondents had taken some form of self-medication in the 6-month period preceding the study. The common reasons given for self-medication were mild illness, previous experience of treating a similar illness, and non-availability of health personnel. 70% of respondents were prescribed allopathic drugs by a non-allopathic doctor. The compounder and health assistant were common sources of medicines. Paracetamol and antimicrobials were the drugs most commonly prescribed. A significantly higher proportion of young (<40 years) male respondents had used self-medication than other groups. CONCLUSIONS: Self-medication and non-doctor prescribing are common in the Pokhara valley. In addition to allopathic drugs, herbal remedies were also commonly used for self-medication. Drugs, especially antimicrobials, were not taken for the proper duration. Education to help patients decide on the appropriateness of self-medication is required.

SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors.

Vascular endothelial growth factor, fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) and their cognate receptor tyrosine kinases are strongly implicated in angiogenesis associated with solid tumors. Using rational drug design coupled with traditional screening technologies, we have discovered SU6668, a novel inhibitor of these receptors. Biochemical kinetic studies using isolated Flk-1, FGF receptor 1, and PDGF receptor beta kinases revealed that SU6668 has competitive inhibitory properties with respect to ATP. Cocrystallographic studies of SU6668 in the catalytic domain of FGF receptor 1 substantiated the adenine mimetic properties of its oxindole core. Molecular modeling of SU6668 in the ATP binding pockets of the FIk-1/KDR and PDGF receptor kinases provided insight to explain the relative potency and selectivity of SU6668 for these receptors. In cellular systems, SU6668 inhibited receptor tyrosine phosphorylation and mitogenesis after stimulation of cells by appropriate ligands. Oral or i.p. administration of SU6668 in athymic mice resulted in significant growth inhibition of a diverse panel of human tumor xenografts of glioma, melanoma, lung, colon, ovarian, and epidermoid origin. Furthermore, intravital multifluorescence videomicroscopy of C6 glioma xenografts in the dorsal skinfold chamber model revealed that SU6668 treatment suppressed tumor angiogenesis. Finally, SU6668 treatment induced striking regression of large established human tumor xenografts. Investigations of SU6668 activity in cancer patients are ongoing in Phase I clinical trials.

Destabilization of osteogenesis imperfecta collagen-like model peptides correlates with the identity of the residue replacing glycine.

Mutations resulting in replacement of one obligate Gly residue within the repeating (Gly-Xaa-Yaa)(n) triplet pattern of the collagen type I triple helix are the major cause of osteogenesis imperfecta (OI). Phenotypes of OI involve fragile bones and range from mild to perinatal lethal. In this study, host-guest triple-helical peptides of the form acetyl-(Gly-Pro-Hyp)(3)-Zaa-Pro-Hyp-(Gly-Pro-Hyp)(4)-Gly-Gly-amide are used to isolate the influence of the residue replacing Gly on triple-helix stability, with Zaa = Gly, Ala, Arg, Asp, Glu, Cys, Ser, or Val. Any substitution for Zaa = Gly (melting temperature, T(m) = 45 degrees C) results in a dramatic destabilization of the triple helix. For Ala and Ser, T(m) decreases to approximately 10 degrees C, and for the Arg-, Val-, Glu-, and Asp-containing peptides, T(m) < 0 degrees C. A Gly --> Cys replacement results in T(m) < 0 degrees C under reducing conditions but shows a broad transition (T(m) approximately 19 degrees C) in an oxidizing environment. Addition of trimethylamine N-oxide increases T(m) by approximately 5 degrees C per 1 M trimethylamine N-oxide, resulting in stable triple-helix formation for all peptides and allowing comparison of relative stabilities. The order of disruption of different Gly replacements in these peptides can be represented as Ala

Growth inhibition of psoriatic keratinocytes by quinazoline tyrosine kinase inhibitors.

Psoriasis is characterized by hyperproliferation of keratinocytes associated with an inflammatory infiltrate in the epidermis. Among factors which may be related to hyperplasia of psoriatic keratinocytes is the persistent autocrine stimulation of the epidermal growth factor receptor (EGFR) by transforming growth factor-alpha. Owing to the pivotal role of the EGFR in driving the growth of human psoriatic keratinocytes, we examined two selective inhibitors of EGFR kinase activity: 4-(3-bromophenylamino)-6, 7-dimethoxyquinazoline (AG1517/SU5271) and 4-(3-chlorophenylamino)-6, 7-dimethoxyquinazoline (AG1478) on psoriatic keratinocytes. SU5271 potently inhibits ligand-induced autophosphorylation of EGFR, and downstream signal transduction events, including DNA replication and cell cycle progression. SU5271, at micromolar concentrations, inhibited the proliferation of keratinocytes isolated from psoriatic lesions in excellent correlation with its EGFR kinase inhibitory activity in these cells. Biologically active concentrations of SU5271 penetrated human cadaver skin, suggesting that this compound is a strong candidate as an antipsoriatic agent.

Sequence dependence of the folding of collagen-like peptides. Single amino acids affect the rate of triple-helix nucleation.

The refolding of thermally denatured model collagen-like peptides was studied for a set of 21 guest triplets embedded in a common host framework: acetyl-(Gly-Pro-Hyp)3-Gly-Xaa-Yaa-(Gly-Pro-Hyp)4-Gly-Gly-amide. The results show a strong dependence of the folding rate on the identity of the guest Gly-Xaa-Yaa triplet, with the half-times for refolding varying from 6 to 110 min (concentration = 1 mg/ml). All triplets of the form Gly-Xaa-Hyp promoted rapid folding, with the rate only marginally dependent on the residue in the Xaa position. In contrast, triplets of the form Gly-Pro-Yaa and Gly-Xaa-Yaa were slower and showed a wide range of half-times, varying with the identity of the residues in the triplet. At low concentrations, the folding can be described by third-order kinetics, suggesting nucleation is rate-limiting. Data on the relative nucleation ability of different Gly-Xaa-Yaa triplets support the favorable nature of imino acids, the importance of hydroxyproline, the varying effects of the same residue in the Xaa position versus the Yaa position, and the difficulties encountered when leucine or aspartic acid are in the Yaa position. Information on the relative propensities of different tripeptide sequences to promote nucleation of the triple-helix in peptides will aid in identification of nucleation sites in collagen sequences.

Influence of handedness on the visual and auditory reaction time.

Visual Reaction Time (VRT) and Auditory Reaction Time (ART) were measured in right-handed and left-handed human volunteers, using an Electronic Response Timer Unit. The study demonstrated that a group of left-handed women reacted faster (P < 0.05) with their left hand to an auditory stimulus than their counterpart using their right hand. VRT did not show any significant change within, and between, the groups and was not influenced by sex or age. The observed differences in ART may be due to central phenomenon.

Studies in C-terminal sequencing: new reagents for the synthesis of peptidylthiohydantoins.

In previous studies aimed at the sequencing of peptides and proteins from the carboxy terminus, we have derivatized the C-terminus to a thiohydantoin using acetic anhydride and trimethylsilylisothiocyanate (TMS-ITC) and subsequently hydrolyzed it to form a shortened peptide capable of further degradation and an amino acid thiohydantoin which can be identified by reverse-phase HPLC. Current limitations to this chemistry include an inability to derivatize proline and low yields with asparagine and aspartic acid residues (Bailey et al., 1992). In an attempt to solve some of these problems, we have investigated the use of reagents other than acetic anhydride for the activation of the C-terminal carboxylic acid. These include 2-fluoro-1-methylpyridinium tosylate, 2-chloro-1-methylpyridinium iodide, and acetyl chloride. Addition of TMS-ITC to peptides activated by the 2-halo-pyridinium salts formed the expected peptidylthiohydantoin, but in addition formed a peptide chemically modified at the C-terminus which was blocked to C-terminal sequence analysis. This derivative was not obtained when either acetic anhydride or acetyl chloride was used for activation. Formation of this derivative was found to require the presence of an isothiocyanate reagent in addition to the halo-pyridinium salt. Sodium thiocyanate, TMS-ITC, and a new reagent for thiohydantoin synthesis, tributyltinisothiocyanate (TBSn-ITC), were all found to be capable of forming this analogue. Structural elucidation of the C-terminally modified amino acid revealed it to be a 2-imino-pyridinium analogue. Formation of this C-terminally blocked peptide could be minimized by the use of the 2-chloro-pyridinium reagent, rather than the 2-fluoro reagent, and by performing the reaction at a temperature of 50 degrees C or lower. The 2-halo-pyridinium reagents offer potential advantages over the use of acetic anhydride for activation of the C-terminal carboxylic acid. These include: milder reaction conditions, faster reaction times, and the ability to sequence through C-terminal aspartic acid. The TBSn-ITC reagent was found to be comparable to TMS-ITC for formation of peptidylthiohydantoins.

Automated carboxy-terminal sequence analysis of peptides and proteins using diphenyl phosphoroisothiocyanatidate.

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on the automation of the thiocyanate chemistry using acetic anhydride and trimethylsilylisothiocyanate (TMS-ITC) to derivatize the C-terminal amino acid to a thiohydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C-terminal amino acid (Bailey, J.M., Shenoy, N.R., Ronk, M., & Shively, J.E., 1992, Protein Sci. 1, 68-80). A major limitation of this approach was the need to activate the C-terminus with acetic anhydride. We now describe the use of a new reagent, diphenyl phosphoroisothiocyanatidate (DPP-ITC) and pyridine, which combines the activation and derivatization steps to produce peptidylthiohydantoins. Previous work by Kenner et al. (Kenner, G.W., Khorana, H.G., & Stedman, R.J., 1953, Chem. Soc. J., 673-678) with this reagent demonstrated slow kinetics. Several days were required for complete reaction. We show here that the inclusion of pyridine was found to promote the formation of C-terminal thiohydantoins by DPP-ITC resulting in complete conversion of the C-terminal amino acid to a thiohydantoin in less than 1 h. Reagents such as imidazole, triazine, and tetrazole were also found to promote the reaction with DPP-ITC as effectively as pyridine. General base catalysts, such as triethylamine, do not promote the reaction, but are required to convert the C-terminal carboxylic acid to a salt prior to the reaction with DPP-ITC and pyridine. By introducing the DPP-ITC reagent and pyridine in separate steps in an automated sequencer, we observed improved sequencing yields for amino acids normally found difficult to derivatize with acetic anhydride/TMS-ITC. This was particularly true for aspartic acid, which now can be sequenced in yields comparable to most of the other amino acids. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene and proteins (200 pmol to 5 nmol) noncovalently applied to Zitex (porous Teflon). The generality of our automated C-terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids tested were found to sequence in good yield except for proline, which was found not to be capable of derivatization. In spite of this limitation, the methodology should be a valuable tool for the C-terminal sequence analysis of peptides and proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibitory effect of diet related sulphydryl compounds on the formation of carcinogenic nitrosamines.

N-Nitroso compounds (NOCs) are known to be strong carcinogens in various animals including primates (Preussman and Stewart, (1984) N-Nitroso Compounds). Human exposure to these compounds can be by ingestion or inhalation of preformed NOCs or by endogenous nitrosation from naturally occurring precursors (Bartsch and Montesano, Carcinogenesis, 5 (1984) 1381-1393; Tannebaum (1979) Naturally Occuring Carcinogens, Mutagens and Modulators of Carcinogenesis; Shephard et al., Food Chem. Toxicol., 25 (1987) 91-108). Several factors present in the diet can modify levels of endogenously formed nitrosamines by acting as catalysts or inhibitors. Compounds in the human diet that alter nitrosamine formation would thus play an important role in carcinogenesis study. Earlier researchers have reported the nitrite scavenging nature of sulphydryl compounds (Williams, Chem. Soc. Rev., 15 (1983) 171-196). We therefore studied the modifying effect of sulphydryl compounds viz., cysteine (CE), cystine (CI), glutathione (GU), cysteamine (CEA), cystamine (CEI), cysteic acid (CIA) and thioglycolic acid (TGA) on the nitrosation of model amines viz., pyrrolidine (PYR), piperidine (NPIP) and morpholine (NMOR). Many of these compounds are present in the food we consume. The present work also describes the inhibitory effect of onion and garlic juices on the nitrosation reactions. Both onion and garlic are known to contain sulphur compounds (Block, Sci. Am., 252 (1985) 114-119). Most of these compounds behave as antinitrosating agents and their inhibitory activity towards formation of carcinogenic nitrosamines, under different conditions is described.

Characterization of potentially mutagenic products from the nitrosation of piperine.

Piperine is the main pungent principle of pepper, a spice consumed by people all over the world. It is the trans-trans isomer of 1-piperoylpiperidine and contains the methylene dioxy moiety. It is known to give unidentified mutagenic products on reaction with nitrite. The nitrosation reaction of piperine is of concern as endogenous nitrosation could take place in the human stomach from ingested precursors, piperine and nitrite. Nitrites can be ingested directly by consuming cured foods or indirectly as nitrates, which could be converted to nitrites under appropriate conditions. We have nitrosated piperine using aqueous nitrous acid and have isolated and identified some N-nitroso and C-nitro compounds. Their isolation, characterization and potential mutagenicity has been discussed.

Carboxylic acid-modified polyethylene: a novel support for the covalent immobilization of polypeptides for C-terminal sequencing.

We have developed a method for the covalent immobilization of peptides, for the purpose of C-terminal sequencing, to a novel solid support, carboxylic acid-modified polyethylene (PE-COOH) film. The peptides are attached by coupling the N-terminal amino group to the activated carboxyl groups of the film. Reagents for carboxyl group activation, including 1,3-dicyclohexylcarbodiimide (DCC), 1,1'-carbonyldiimidazole (CDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1,3-diisopropylcarbodiimide (DICD) were compared. The best yields were obtained with DCC for a variety of tested peptides and averaged approximately 50%. The covalent attachment at pH 6.7 of peptides was shown to occur predominantly thorough the alpha-amino group for the peptide, SIGSLAK, which after attachment to the PE-COOH support permitted the C-terminal lysine residue to be sequenced in good yield, indicating that the epsilon-amino group of lysine is not covalently attached. This support offers a number of advantages over other solid supports, such as silica and polyvinylidene difluoride, for C-terminal sequencing including (1) stability to base and the high temperatures (65 degrees C) employed for C-terminal sequencing, (2) wettability with both aqueous and organic solvents, (3) a high capacity (1.6 nmol/mm2) for covalent coupling of polypeptides, and (4) easy divisibility into 1 x 5-mm pieces for use in our continuous flow reactor (CFR), which is also used for automated N-terminal sequencing (Shively, J.E., Miller, P., & Ronk, M., 1987, Anal. Biochem. 163, 517-529). Automated C-terminal sequencing on these supports is described in the companion paper (Bailey, J.M., Shenoy, N.R., Ronk, M., & Shively, J.E., 1992, Protein Sci. 1, 68-80).

Automated carboxy-terminal sequence analysis of peptides.

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on solution phase conditions for formation of the peptidylthiohydantoins with trimethylsilylisothiocyanate (TMS-ITC) and for hydrolysis of these peptidylthiohydantoins into an amino acid thiohydantoin derivative and a new shortened peptide capable of continued degradation (Bailey, J. M. & Shively, J. E., 1990, Biochemistry 29, 3145-3156). The current study is a continuation of this work and describes the construction of an instrument for automated C-terminal sequencing, the application of the thiocyanate chemistry to peptides covalently coupled to a novel polyethylene solid support (Shenoy, N. R., Bailey, J. M., & Shively, J. E., 1992, Protein Sci. I, 58-67), the use of sodium trimethylsilanolate as a novel reagent for the specific cleavage of the derivatized C-terminal amino acid, and the development of methodology to sequence through the difficult amino acid, aspartate. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene. The chemistry involves activation with acetic anhydride, derivatization with TMS-ITC, and cleavage of the derivatized C-terminal amino acid with sodium trimethylsilanolate. The thiohydantoin amino acid is identified by on-line high performance liquid chromatography using a Phenomenex Ultracarb 5 ODS(30) column and a triethylamine/phosphoric acid buffer system containing pentanesulfonic acid. The generality of our automated C-terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids were found to sequence in high yield (90% or greater) except for asparagine and aspartate, which could be only partially removed, and proline, which was found not be capable of derivatization. In spite of these current limitations, the methodology should be a valuable new tool for the C-terminal sequence analysis of peptides.