Reproducibility and complications in gene searches: linkage on chromosome 6, heterogeneity, association, and maternal inheritance in juvenile myoclonic epilepsy

Evidence for genetic influences in epilepsy is strong, but reports identifying specific chromosomal origins of those influences conflict. One early study reported that human leukocyte antigen (HLA) markers were genetically linked to juvenile myoclonic epilepsy (JME); this was confirmed in a later study. Other reports did not find linkage to HLA markers. One found evidence of linkage to markers on chromosome 15, another to markers on chromosome 6, centromeric to HLA. We identified families through a patient with JME and genotyped markers throughout chromosome 6. Linkage analysis assuming equal male-female recombination probabilities showed evidence for linkage (LOD score 2.5), but at a high recombination fraction (theta), suggesting heterogeneity. When linkage analysis was redone to allow independent male-female thetas, the LOD score was significantly higher (4.2) at a male-female theta of.5,.01. Although the overall pattern of LOD scores with respect to male-female theta could not be explained solely by heterogeneity, the presence of heterogeneity and predominantly maternal inheritance of JME might explain it. By analyzing loci between HLA-DP and HLA-DR and stratifying the families on the basis of evidence for or against linkage, we were able to show evidence of heterogeneity within JME and to propose a marker associated with the linked form. These data also suggest that JME may be predominantly maternally inherited and that the HLA-linked form is more likely to occur in families of European origin.     

Probing Structural Elements of Thermal Stability in Bacterial Oligomeric Alcohol Dehydrogenases. I. Construction and Characterization of Chimeras Consisting of Secondary ADHs from Thermoanaerobacter brockii and Clostridium beijerinckii

Two tetrameric secondary alcohol dehydrogenases (ADHs), one from the mesophile Clostridium beijerinckii (CBADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TBADH), share 75% sequence identity but differ by 26 °C in thermal stability. To explore the role of linear segments of these similar enzymes in maintaining the thermal stability of the thermostable TBADH, a series of 12 CBadh and TBadh chimeric genes and the two parental wild-type genes were expressed in Escherichia coli, and the enzymes were isolated, purified and characterized. The thermal stability of each chimeric enzyme was approximately exponentially proportional to the content of the amino acid sequence of the thermophilic enzyme, indicating that the amino acid residues contributing to the thermal stability of TBADH are distributed along the whole protein molecule. It is suggested that major structural elements of thermal stability may reside among the nine discrepant amino acid residues between the N-terminal 50-amino acid residues of TBADH and CBADH.     

The influence of alcohol-containing and alcohol-free beverages on lipid levels and lipid peroxides in serum of rats

It is an established fact that moderate consumption of alcoholic beverages leads to some positive biochemical changes in blood that are widely regarded as indicators of improved prevention of atherosclerosis. However, at present, there are different opinions regarding the biologically active compounds of alcoholic beverages that bring about these changes. This experiment was conducted on 60 male Wistar rats, which were divided into five groups, each of which contained 12 rats: four experimental groups (EG1, EG2, EG3, EG4) and one control group (CG). During 4 weeks, all groups of rats were fed basal diet (BD) supplemented with dry red wine (EG1), beer (EG2), lyophilized dry red wine (EG3), or lyophilized beer (EG4). The rats of the CG were fed BD only. The rats of EG1 and EG2 were fed BD supplemented daily with 2.0 mL of wine and 6.0 mL of beer, respectively. The rats of EG3 and EG4 were fed BD supplemented daily with lyophilized wine and lyophilized beer at a concentration corresponding to an intake of 2.0 mL of original wine and 6.0 mL of original beer, respectively. Before and after completion of the trial, a wide range of laboratory tests including lipids and lipid peroxides were performed. The results of this investigation reveal that both original and lyophilized wine and beer exercise statistically significant beneficial lipidemic and antioxidant effects by reducing total cholesterol (TC), low density lipoprotein cholesterol, triglycerides, and lipid peroxides (P < 0.05 for all) and by elevating the high density lipoprotein cholesterol:TC ratio. There were no statistically significant differences in the results between groups fed BD supplemented with original wine and beer versus groups fed BD supplemented with lyophilized wine and beer. Therefore, it can be concluded that the biologically active compound of these beverages is their dry matter containing inter alia polyphenols in relatively high concentrations.     

Regulation of innate immune responses by transmembrane interactions: Lessons from the TLR family

The mammalian innate immune response is responsible for the early stages of defense against invading pathogens. One of the major receptor families facilitating innate immune activation is the Toll-like receptor (TLR) family. These receptors are type 1 membrane proteins spanning the membrane with a single transmembrane domain (TMD). All TLRs form homo- and hetero-dimers within membranes and new data suggest that the single transmembrane domain of some of these receptors is involved in their dimerization and function. Newly identified TLR dimers are continuously reported but only little is known about the importance of the TMDs for their dimer assembly and signaling regulation. Uncontrolled or untimely activation of TLRs is related to a large number of pathologies ranging from cystic fibrosis to sepsis and cancer. In this review we will focus on the contribution of the TMDs of innate immune receptors – specifically TLR2–to their regulation and function. In addition, we will address the current issues remaining to be solved regarding the mechanistic insights of this regulation. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

The effects of erythropoietin signaling on telomerase regulation in non-erythroid malignant and non-malignant cells

Treatment with erythropoietin (EPO) in several cancers is associated with decreased survival due to cancer progression. Due to the major importance of telomerase in cancer biology we hypothesized that some of these effects may be mediated through EPO effect on telomerase. For this aim we explored the possible effects of EPO on telomerase regulation, cell migration and chemosensitivity in non-erythroid malignant and non-malignant cells.     

Oral cancer,cigarette smoke and mitochondrial 18 kDa translocator protein (TSPO) — In vitro,in vivo,salivary analysis

Oral cancer features high rates of mortality and morbidity, and is in dire need for new approaches. In the present study we analyzed 18 kDa translocator protein (TSPO) expression in oral (tongue) cancer tumors by immunohistochemistry. We also assayed TSPO binding in human tongue cancer cell lines and in the cellular fraction of saliva from tongue cancer patients, heavy cigarette smokers, and non-smoking healthy people as controls. Concurrently, TSPO protein levels, cell viability, mitochondrial membrane potential (Δψ_m), and general protein levels were analyzed. TSPO expression could be significantly enhanced in oral cancer tumors, compared to unaffected adjacent tissue. We also found that five-year survival probability dropped from 65% in patients with TSPO negative tumors to 7% in patients with highly expressed TSPO (p < 0.001). TSPO binding capacity was also pronounced in the human oral cancer cell lines SCC-25 and SCC-15 (3133 ± 643 fmol/mg protein and 6956 ± 549 fmol/mg protein, respectively). Binding decreased by 56% and 72%, in the SCC-25 and SCC-15 cell lines, respectively (p < 0.05) following CS exposure in cell culture. In the cellular fraction of saliva of heavy smokers TSPO binding was lower than in non-smokers (by 53%, p < 0.05). Also the cellular fraction of saliva exposed to CS in vitro showed decreased TSPO binding compared to unexposed saliva (by 30%, p < 0.001). Interestingly, oral cancer patients also displayed significantly lower TSPO binding in the cellular fraction of saliva compared to healthy controls (by 40%, p < 0.01). Our results suggest that low TSPO binding found in the cellular fraction of saliva may depend on genetic background as well as result from exposure to CS. We suggest that this may be related to a predisposition for occurrence of oral cancer.     

Improved Strains and Plasmid Vectors for Conditional Overexpression of His-Tagged Proteins in Haloferax volcanii

Research into archaea will not achieve its full potential until systems are in place to carry out genetics and biochemistry in the same species. Haloferax volcanii is widely regarded as the best-equipped organism for archaeal genetics, but the development of tools for the expression and purification of H. volcanii proteins has been neglected. We have developed a series of plasmid vectors and host strains for conditional overexpression of halophilic proteins in H. volcanii. The plasmids feature the tryptophan-inducible p.tnaA promoter and a 6×His tag for protein purification by metal affinity chromatography. Purification is facilitated by host strains, where pitA is replaced by the ortholog from Natronomonas pharaonis. The latter lacks the histidine-rich linker region found in H. volcanii PitA and does not copurify with His-tagged recombinant proteins. We also deleted the mrr restriction endonuclease gene, thereby allowing direct transformation without the need to passage DNA through an Escherichia coli dam mutant.Over the past century, our understanding of fundamental biological processes has grown exponentially, and this would have been impossible without the use of organisms that are amenable to experimental manipulation. Model species, such as Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Arabidopsis thaliana, have become a byword for scientific progress (15). The rational choice of a model organism is critically important, and certain features are taken for granted, such as ease of cultivation, a short generation time, and systems for genetic manipulation. This list has now grown to include a genome sequence and methods for biochemical analysis of purified proteins in vitro.Research into archaea has lagged behind work on bacteria and eukaryotes but has nonetheless yielded profound insights (2). One hurdle has been the paucity of archaeal organisms suitable for both biochemistry and genetics. For example, Methanothermobacter thermautotrophicus is a stalwart of archaeal biochemistry but has proved resistant to even the most rudimentary genetic manipulation (2). Progress has recently been made with another biochemical workhorse, Sulfolobus spp., and a few genetic tools are now available (6, 13, 37). Methanosarcina spp. and Thermococcus kodakaraensis offer alternative systems with an increasing array of techniques (16, 35, 36), but sophisticated genetics has traditionally been the preserve of haloarchaea, of which Haloferax volcanii is the organism of choice (39). It is easy to culture, the genome has been sequenced (19), and there are several selectable markers and plasmids for transformation and gene knockout (3, 7, 31), including a Gateway system (14), as well as reporter genes (20, 33) and a tightly controlled inducible promoter (26).The genetic prowess of H. volcanii is not yet fully matched by corresponding systems for protein overexpression and purification. Like other haloarchaea, H. volcanii grows in high salt concentrations (2 to 5 M NaCl), and to cope with the osmotic potential of such environments, it accumulates high intracellular concentrations of potassium ions (12). Consequently, halophilic proteins are adapted to function at high salt concentrations and commonly feature a large excess of acidic amino acids; the negative surface charge is thought to be critical to solubility (28). This can pose problems for expression in heterologous hosts, such as E. coli, since halophilic proteins can misfold and aggregate under conditions of low ionic strength. The purification of misfolded halophilic enzymes from E. coli has relied on the recovery of insoluble protein from inclusion bodies, followed by denaturation and refolding in hypersaline solutions (8, 11). This approach is feasible only where the protein is well characterized and reconstitution of the active form can be monitored (for example, by an enzymatic assay). Furthermore, archaeal proteins expressed in heterologous bacterial hosts lack posttranslational modifications, such as acetylation or ubiquitination (4, 22), which are critical to understanding their biological function.Systems for expression of halophilic proteins in a native haloarchaeal host are therefore required. A number of studies have successfully purified recombinant proteins with a variety of affinity tags after overexpression in H. volcanii. For example, Humbard et al. employed tandem affinity tagging to purify 20S proteasomal core particles from the native host (23). However, the protein expression constructs used in these studies were custom made and somewhat tailored to the application in question. We report here the development of “generic” plasmid vectors and host strains for conditional overexpression of halophilic proteins in H. volcanii. The plasmids feature a tryptophan-inducible promoter derived from the tnaA gene of H. volcanii (26). We demonstrate the utility of these vectors by overexpressing a hexahistidine-tagged recombinant version of the H. volcanii RadA protein. Purification was greatly facilitated by a host strain in which the endogenous pitA gene was replaced by an ortholog from Natronomonas pharaonis. The latter protein lacks the histidine-rich linker region found in H. volcanii PitA (5) and therefore does not copurify with His-tagged recombinant proteins. Finally, we deleted the mrr gene of H. volcanii, which encodes a restriction enzyme that cleaves foreign DNA methylated at GATC residues. The mrr deletion strain allows direct transformation of H. volcanii without the need to passage plasmid DNA through an E. coli dam mutant (21).