The promoter of filamentation (POF1) protein from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> is an ATPase involved in the protein quality control process

Background

The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene.

Results

Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo.

Conclusions

Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Monocarbonyl Analogs of Curcumin with Potential to Treat Colorectal Cancer

Curcumin has a plethora of biological properties, making this compound potentially effective in the treatment of several diseases, including cancer. However, curcumin clinical use is compromised by its poor pharmacokinetics, being crucial to find novel analogs with better pharmacokinetic and pharmacological properties. Here, we aimed to evaluate the stability, bioavailability and pharmacokinetic profiles of monocarbonyl analogs of curcumin. A small library of monocarbonyl analogs of curcumin 1a–q was synthesized. Lipophilicity and stability in physiological conditions were both assessed by HPLC-UV, while two different methods assessed the electrophilic character of each compound monitored by NMR and by UV-spectroscopy. The potential therapeutic effect of the analogs 1a–q was evaluated in human colon carcinoma cells and toxicity in immortalized hepatocytes. Our results showed that the curcumin analog 1e is a promising agent against colorectal cancer, with improved stability and efficacy/safety profile.     

Modulation of G protein-coupled adenosine receptors by strategically functionalized agonists and antagonists immobilized on gold nanoparticles

Gold nanoparticles (AuNPs) allow the tuning of pharmacokinetic and pharmacodynamic properties by active or passive targeting of drugs for cancer and other diseases. We have functionalized gold nanoparticles by tethering specific ligands, agonists and antagonists, of adenosine receptors (ARs) to the gold surface as models for cell surface interactions with G protein-coupled receptors (GPCRs). The AuNP conjugates with chain-extended AR ligands alone (PEGylated nucleosides and nonnucleosides, anchored to the Au via thioctic acid) were found to be insoluble in water due to hydrophobic entities in the ligand. Therefore, we added a second, biologically inactive pendant moiety to increase the water solubility, consisting of a PEGylated chain terminating in a carboxylic or phosphate group. The purity and stability of the immobilized biologically active ligand were examined by ultrafiltration and HPLC. Pharmacological receptor binding studies on these GPCR ligand-derivatized AuNPs (2–5 nm in diameter), performed using membranes of mammalian cells stably expressing human A₁, A_2A, and A₃ARs, showed that the desired selectivity was retained with K _i values (nanomolar) of A₃AR agonist 21b and A_2AAR antagonists 24 and 26a of 14 (A₃), 34 (A_2A), and 69 (A_2A), respectively. The corresponding monomers displayed K _i values of 37, 61, and 1,420 nM, respectively. In conclusion, we have synthesized stable, water-soluble AuNP derivatives of tethered A₃ and A_2AAR ligands that retain the biological properties of their monomeric ligands and are intended for therapeutic and imaging applications. This is the first prototypical application to gold carriers of small molecule (nonpeptide) GPCR ligands, which are under investigation for treatment of cancer and inflammatory diseases.     

Synthesis and evaluation of 1,2,4-triazolo[1,5-c]pyrimidine derivatives as A2A receptor-selective antagonists

Movement disorders such as Parkinson’s disease and Huntington’s disease are serious life-limiting and debilitating movement disorders. Their onset typically occurs from mid-life to late in life, and effective diagnostic techniques for detecting and following the disease course are lacking. Our goal is to develop receptor imaging agents for positron emission tomography (PET) that selectively target the most relevant subtype of adenosine receptors (AR) that are highly expressed in the striatum, that is, the A_2A AR. To further this goal, we have synthesized and characterized pharmacologically a family of high affinity A_2A AR ligands, based on the known antagonist, SCH 442416 (R = –Me), which have structural variability on the terminus (R = –Et, –i-Pr, –allyl, and others). A O-fluoroethyl analogue suitable for use as a PET tracer had a K_i value of 12.4 nM and was highly selective for the A_2A AR in comparison to the A₁ and A₃ ARs.     

Prevalence of genetic variants associated with cardiovascular disease risk and drug response in the Southern Indian population of Kerala

BACKGROUND AND AIM:

This study reports the prevalence of five clinically significant variants associated with increased risk of cardiovascular disorders, and variable responses of individuals to commonly prescribed cardiovascular drugs in a South Indian population from the state of Kerala.

MATERIALS AND METHODS:

Genomic DNA isolated from 100 out-patient samples from Kerala were sequenced to examine the frequency of clinically relevant polymorphisms in the genes MYBPC3 (cardiomyopathy), SLCO1B1 (statin-induced myopathy), CYP2C9, VKORC1 (response to warfarin) and CYP2C19 (response to clopidogrel).

RESULTS:

Our analyses revealed the frequency of a 25 bp deletion variant of MYBPC3 associated with risk of cardiomyopathy was 7%, and the SLCO1B1 “C” allele associated with risk for statin-induced myopathy was 15% in this sample group. Among the other variants associated with dose-induced toxicity of warfarin, VKORC1 (c.1639G>A), was detected at 22%, while CYP2C9*3 and CYP2C9*2 alleles were present at a frequency of 15% and 3% respectively. Significantly, the tested sample population showed high prevalence (66%) of CYP2C19*2 variant, which determines response to clopidogrel therapy.

CONCLUSIONS:

We have identified that certain variants associated with cardiovascular disease and related drug response in the five genes, especially those in VKORC1, CYP2C19 and MYBPC3, are highly prevalent in the Kerala population, with almost 2 times higher prevalence of CYP2C19*2 variant compared with other regions in the country. Since the variants chosen in this study have relevance in disease phenotype and/or drug response, and are detected at a higher frequency, this study is likely to encourage clinicians to perform genetic testing before prescribing therapy.     

Principal component analysis and artificial neural network analysis of oral tissue fluorescence spectra: classification of normal premalignant and malignant pathological conditions

Pulsed laser-induced autofluorescence spectroscopic studies of pathologically certified normal, premalignant, and malignant oral tissues were carried out at 325 nm excitation. The spectral analysis and classification for discrimination among normal, premalignant, and malignant conditions were performed using principal component analysis (PCA) and artificial neural network (ANN) separately on the same set of spectral data. In case of PCA, spectral residuals, Mahalanobis distance, and scores of factors were used for discrimination among normal, premalignant, and malignant cases. In ANN, parameters like mean, spectral residual, standard deviation, and total energy were used to train the network. The ANN used in this study is a classical multiplayer feed-forward type with a back-propagation algorithm for the training of the network. The specificity and sensitivity were determined in both classification schemes. In the case of PCA, they are 100 and 92.9%, respectively, whereas for ANN they are 100 and 96.5% for the data set considered.