Genomic instability and tumor-specific alterations in oral squamous cell carcinomas assessed by inter- (simple sequence repeat) PCR

Purpose: Genomic instability plays a major role in the genesis and progression of tumors, and in the evolution of tumor heterogeneity. To determine the role of genomic instability in the genesis and progression of oral cancer, we assessed the extent of genomic alterations in oral squamous cell carcinomas (OSCCs). Experimental Design: We used the recently developed inter-(simple sequence repeat) PCR technique to quantitate genomic instability using matched tumor and normal OSCC samples (n = 25). The inter-repeat region bands of similar molecular size observed to be altered in more than one case were sequenced and analyzed to identify probable OSSC-associated specific genetic lesions. Results: Of the four base-anchored, dinucleotide repeat-based primers used for the study, the most informative profile in OSCCs was generated by the (CA)8RG primer. Measurement of genomic instability index using the (CA)8RG primer revealed a high incidence of genomic instability in OSCCs. No significant correlation between the extent of alterations and stage or location of the tumor was observed. Sequencing analysis of the altered bands revealed gains/losses in several chromosomal regions. Of the matched tumor and corresponding normal tissue DNA studied, hitherto unreported losses were seen in 11p15 and 17q25 chromosomal regions. Sequencing of some of the tumor-specific altered regions indicated that they code for regions of UDP-GalNAc and hRAD 17 genes, which were lost (deleted) in oral cancer. Conclusions: Our results indicate that the extent of genomic instability in OSCC is not correlated to the tumor stage or location. For the first time, we have shown that chromosomal alterations detected by inter-(simple sequence repeat) PCR could be correlated to genes associated with cancer development

Overexpression of branched-chain amino acid aminotransferases rescues the growth defects of cells lacking the Barth syndrome-related gene TAZ1.

The yeast protein Taz1 is the orthologue of human Tafazzin, a phospholipid acyltransferase involved in cardiolipin (CL) remodeling via a monolyso CL (MLCL) intermediate. Mutations in Tafazzin lead to Barth syndrome (BTHS), a metabolic and neuromuscular disorder that primarily affects the heart, muscles, and immune system. Similar to observations in fibroblasts and platelets from patients with BTHS or from animal models, abolishing yeast Taz1 results in decreased total CL amounts, increased levels of MLCL, and mitochondrial dysfunction. However, the biochemical mechanisms underlying the mitochondrial dysfunction in BTHS remain unclear. To better understand the pathomechanism of BTHS, we searched for multi-copy suppressors of the taz1Δ growth defect in yeast cells. We identified the branched-chain amino acid transaminases (BCATs) Bat1 and Bat2 as such suppressors. Similarly, overexpression of the mitochondrial isoform BCAT2 in mammalian cells lacking TAZ improves their growth. Elevated levels of Bat1 or Bat2 did not restore the reduced membrane potential, altered stability of respiratory complexes, or the defective accumulation of MLCL species in yeast taz1Δ cells. Importantly, supplying yeast or mammalian cells lacking TAZ1 with certain amino acids restored their growth behavior. Hence, our findings suggest that the metabolism of amino acids has an important and disease-relevant role in cells lacking Taz1 function. KEY MESSAGES: Bat1 and Bat2 are multi-copy suppressors of retarded growth of taz1Δ yeast cells. Overexpression of Bat1/2 in taz1Δ cells does not rescue known mitochondrial defects. Supplementation of amino acids enhances growth of cells lacking Taz1 or Tafazzin. Altered metabolism of amino acids might be involved in the pathomechanism of BTSH

Menthol stereoisomers exhibit different effects on α4β2 nAChR upregulation and dopamine neuron spontaneous firing

Menthol contributes to poor cessation rates among smokers, in part because menthol enhances nicotine reward and reinforcement. Mentholated tobacco products contain (−)-menthol and (+)-menthol, in varying proportions. We examined these two menthol stereoisomers for their ability to upregulate α4β2 nAChRs and to alter dopamine neuron firing frequency using long-term, low-dose (≤ 500 nM) exposure that is pharmacologically relevant to smoking. We found that (−)-menthol upregulates α4β2 nAChRs while (+)-menthol does not. We also found that (−)-menthol decreases dopamine neuron baseline firing and dopamine neuron excitability, while (+)-menthol exhibits no effect. We then examined both stereoisomers for their ability to inhibit α4β2 nAChR function at higher concentrations (>10 µM) using the Xenopus oocyte expression system. To probe for the potential binding site of menthol, we conducted flooding simulations and site-directed mutagenesis. We found that menthol likely binds to the 9’ position on the TM2 helix. We found that menthol inhibition is dependent on the end-to-end distance of the side chain at the 9’ residue. Additionally, we have found that (−)-menthol is only modestly (∼25%) more potent than (+)-menthol at inhibiting wildtype α4β2 nAChRs and a series of L9’ mutant nAChRs. These data reveal that menthol exhibits a stereoselective effect on nAChRs and that the stereochemical effect is much greater for long-term, sub µM exposure in mice than for acute, higher level exposure. We hypothesize that of the two menthol stereoisomers, only (−)-menthol plays a role in enhancing nicotine reward through nAChRs on dopamine neurons

A Link between Integral Membrane Protein Expression and Simulated Integration Efficiency

Integral membrane proteins (IMPs) control the flow of information and nutrients across cell membranes, yet IMP mechanistic studies are hindered by difficulties in expression. We investigate this issue by addressing the connection between IMP sequence and observed expression levels. For homologs of the IMP TatC, observed expression levels vary widely and are affected by small changes in protein sequence. The effect of sequence changes on experimentally observed expression levels strongly correlates with the simulated integration efficiency obtained from coarse-grained modeling, which is directly confirmed using an in vivo assay. Furthermore, mutations that improve the simulated integration efficiency likewise increase the experimentally observed expression levels. Demonstration of these trends in both Escherichia coli and Mycobacterium smegmatis suggests that the results are general to other expression systems. This work suggests that IMP integration is a determinant for successful expression, raising the possibility of controlling IMP expression via rational design

Moltemplate: A Tool for Coarse-Grained Modeling of Complex Biological Matter and Soft Condensed Matter Physics

Coarse-grained models have long been considered indispensable tools in the investigation of biomolecular dynamics and assembly. However, the process of simulating such models is arduous because unconventional force fields and particle attributes are often needed, and some systems are not in thermal equilibrium. Although modern molecular dynamics programs are highly adaptable, software designed for preparing all-atom simulations typically makes restrictive assumptions about the nature of the particles and the forces acting on them. Consequently, the use of coarse-grained models has remained challenging. Moltemplate is a file format for storing coarse-grained molecular models and the forces that act on them, as well as a program that converts moltemplate files into input files for LAMMPS, a popular molecular dynamics engine. Moltemplate has broad scope and an emphasis on generality. It accommodates new kinds of forces as they are developed for LAMMPS, making moltemplate a popular tool with thousands of users in computational chemistry, materials science, and structural biology. To demonstrate its wide functionality, we provide examples of using moltemplate to prepare simulations of fluids using many-body forces, coarse-grained organic semiconductors, and the motor-driven supercoiling and condensation of an entire bacterial chromosome

Suv4-20h Histone Methyltransferases Promote Neuroectodermal Differentiation by Silencing the Pluripotency-Associated Oct-25 Gene

Post-translational modifications (PTMs) of histones exert fundamental roles in regulating gene expression. During development, groups of PTMs are constrained by unknown mechanisms into combinatorial patterns, which facilitate transitions from uncommitted embryonic cells into differentiated somatic cell lineages. Repressive histone modifications such as H3K9me3 or H3K27me3 have been investigated in detail, but the role of H4K20me3 in development is currently unknown. Here we show that Xenopus laevis Suv4-20h1 and h2 histone methyltransferases (HMTases) are essential for induction and differentiation of the neuroectoderm. Morpholino-mediated knockdown of the two HMTases leads to a selective and specific downregulation of genes controlling neural induction, thereby effectively blocking differentiation of the neuroectoderm. Global transcriptome analysis supports the notion that these effects arise from the transcriptional deregulation of specific genes rather than widespread, pleiotropic effects. Interestingly, morphant embryos fail to repress the Oct4-related Xenopus gene Oct-25. We validate Oct-25 as a direct target of xSu4-20h enzyme mediated gene repression, showing by chromatin immunoprecipitaton that it is decorated with the H4K20me3 mark downstream of the promoter in normal, but not in double-morphant, embryos. Since knockdown of Oct-25 protein significantly rescues the neural differentiation defect in xSuv4-20h double-morphant embryos, we conclude that the epistatic relationship between Suv4-20h enzymes and Oct-25 controls the transit from pluripotent to differentiation-competent neural cells. Consistent with these results in Xenopus, murine Suv4-20h1/h2 double-knockout embryonic stem (DKO ES) cells exhibit increased Oct4 protein levels before and during EB formation, and reveal a compromised and biased capacity for in vitro differentiation, when compared to normal ES cells. Together, these results suggest a regulatory mechanism, conserved between amphibians and mammals, in which H4K20me3-dependent restriction of specific POU-V genes directs cell fate decisions, when embryonic cells exit the pluripotent state

Menthol stereoisomers exhibit different effects on α4β2 nAChR upregulation and dopamine neuron spontaneous firing

Menthol contributes to poor cessation rates among smokers, in part because menthol enhances nicotine reward and reinforcement. Mentholated tobacco products contain (−)-menthol and (+)-menthol, in varying proportions. We examined these two menthol stereoisomers for their ability to upregulate α4β2 nAChRs and to alter dopamine neuron firing frequency using long-term, low-dose (≤ 500 nM) exposure that is pharmacologically relevant to smoking. We found that (−)-menthol upregulates α4β2 nAChRs while (+)-menthol does not. We also found that (−)-menthol decreases dopamine neuron baseline firing and dopamine neuron excitability, while (+)-menthol exhibits no effect. We then examined both stereoisomers for their ability to inhibit α4β2 nAChR function at higher concentrations (>10 µM) using the Xenopus oocyte expression system. To probe for the potential binding site of menthol, we conducted flooding simulations and site-directed mutagenesis. We found that menthol likely binds to the 9’ position on the TM2 helix. We found that menthol inhibition is dependent on the end-to-end distance of the side chain at the 9’ residue. Additionally, we have found that (−)-menthol is only modestly (∼25%) more potent than (+)-menthol at inhibiting wildtype α4β2 nAChRs and a series of L9’ mutant nAChRs. These data reveal that menthol exhibits a stereoselective effect on nAChRs and that the stereochemical effect is much greater for long-term, sub µM exposure in mice than for acute, higher level exposure. We hypothesize that of the two menthol stereoisomers, only (−)-menthol plays a role in enhancing nicotine reward through nAChRs on dopamine neurons

A Link between Integral Membrane Protein Expression and Simulated Integration Efficiency

Integral membrane proteins (IMPs) control the flow of information and nutrients across cell membranes, yet IMP mechanistic studies are hindered by difficulties in expression. We investigate this issue by addressing the connection between IMP sequence and observed expression levels. For homologs of the IMP TatC, observed expression levels vary widely and are affected by small changes in protein sequence. The effect of sequence changes on experimentally observed expression levels strongly correlates with the simulated integration efficiency obtained from coarse-grained modeling, which is directly confirmed using an in vivo assay. Furthermore, mutations that improve the simulated integration efficiency likewise increase the experimentally observed expression levels. Demonstration of these trends in both Escherichia coli and Mycobacterium smegmatis suggests that the results are general to other expression systems. This work suggests that IMP integration is a determinant for successful expression, raising the possibility of controlling IMP expression via rational design