Genetic Analysis of Resistance to Benzimidazoles in Physarum: Differential Expression of β-Tubulin Genes

Physarum displays two vegetative cell types, uninucleate myxamoebae and multinucleate plasmodia. Mutant myxamoebae of Physarum resistant to the antitubulin drug methylbenzimidazole-2-yl-carbamate (MBC) were isolated. All mutants tested were cross-resistant to other benzimidazoles but not to cycloheximide or emetine. Genetic analysis showed that mutation to MBC resistance can occur at any one of four unlinked loci, benA, benB, benC or benD. MBC resistance of benB and benD mutants was expressed in plasmodia, but benA and benC mutant plasmodia were MBC sensitive, suggesting that benA and benC encode myxamoeba-specific products. Myxamoebae carrying the recessive benD210 mutation express a β-tubulin with noval electrophoretic mobility, in addition to a β-tubulin with wild-type mobility. This and other evidence indicates that benD is a structural gene for β-tubulin, and that at least two β-tubulin genes are expressed in myxamoebae. Comparisons of the β-tubulins of wildtype and benD210 strains by gel electrophoresis revealed that, of the three (or more) β-tubulin genes expressed in Physarum, one, benD, is expressed in both myxamoebae and plasmodia, one is expressed specifically in myxamoebae and one is expressed specifically in plasmodia. However, mutation in only one gene, benD, is sufficient to confer MBC resistance on both myxamoebae and plasmodia.     

VARICOSE VEINS—A Practical Approach to Treatment

Adequate treatment of varicose veins requires thorough mapping of perforating veins, communicating veins and “blow out” areas. Combined ligations, stripping and injection of sclerotic substances after operation is the most effective regimen of therapy.The technique of stripping is facilitated by isolating the saphenous vein at the ankle, inserting the stripper from below upward, then making a transverse groin incision over the palpable stripper. The tip of the stripper should be twice the diameter of the vein to be removed. Stripping should be done with the patient in the Trendelenburg position.All patients must be examined at regular intervals after operation and injection of sclerosing material carried out as necessary.     

Genetic Counseling—A Health Department Service to Physicians

Questions about inheritance in all kinds of diseases and defects are commonly asked of nearly all physicians. In attempting to answer these questions, however, the physician is often hampered by lack of formal instruction in clinical genetics.Since the health department, if it is to carry out its epidemiologic function, must be as concerned over the increasing identification of genetic agents in disease as it is and has been over environmental disease agents, it should come to represent a source of assistance not now generally available to the physician. In short, as it carries out those activities by which its store of general genetic information is increased, and until other sources of genetic consultation become reasonably available, the health department can be of real service to physicians as a resource to which they may turn for help when dealing with families wanting genetic information.Such a service has been provided experimentally for the last two years by the Contra Costa County Health Department.This program calls for the taking of family pedigrees by public health nurses on families with questions of a genetic nature who are health department clients and on families who are referred by their private physicians for this service. An interpretation of each pedigree is made by the department''s physician in charge of the program and submitted to the family''s physician for his use in counseling the family. Evidence to date suggests the process can be a highly useful service to the practitioner and his patient.     

B-Raf Regulation of Integrin α4β1-mediated Resistance to Shear Stress through Changes in Cell Spreading and Cytoskeletal Association in T Cells

The regulation of integrin-mediated adhesion is of vital importance to adaptive and innate immunity. Integrins are versatile proteins and mediate T cell migration and trafficking by binding to extracellular matrix or other cells as well as initiating intracellular signaling cascades promoting survival or activation. The MAPK pathway is known to be downstream from integrins and to regulate survival, differentiation, and motility. However, secondary roles for canonical MAPK pathway members are being discovered. We show that chemical inhibition of RAF by sorafenib or shRNA-mediated knockdown of B-Raf reduces T cell resistance to shear stress to α4β1 integrin ligands vascular cell adhesion molecule 1 (VCAM-1) and fibronectin, whereas inhibition of MEK/ERK by U0126 had no effect. Microscopy showed that RAF inhibition leads to significant inhibition of T cell spreading on VCAM-1. The association of α4β1 integrin with the actin cytoskeleton was shown to be dependent on B-Raf activity or expression, whereas α4β1 integrin affinity for soluble VCAM-1 was not. These effects were shown to be specific for α4β1 integrin and not other integrins, such as α5β1 or LFA-1, or a variety of membrane proteins. We demonstrate a novel role for B-Raf in the selective regulation of α4β1 integrin-mediated adhesion.     

A Syntenic Cross Species Aneuploidy Genetic Screen Links RCAN1 Expression to β-Cell Mitochondrial Dysfunction in Type 2 Diabetes

Type 2 diabetes (T2D) is a complex metabolic disease associated with obesity, insulin resistance and hypoinsulinemia due to pancreatic β-cell dysfunction. Reduced mitochondrial function is thought to be central to β-cell dysfunction. Mitochondrial dysfunction and reduced insulin secretion are also observed in β-cells of humans with the most common human genetic disorder, Down syndrome (DS, Trisomy 21). To identify regions of chromosome 21 that may be associated with perturbed glucose homeostasis we profiled the glycaemic status of different DS mouse models. The Ts65Dn and Dp16 DS mouse lines were hyperglycemic, while Tc1 and Ts1Rhr mice were not, providing us with a region of chromosome 21 containing genes that cause hyperglycemia. We then examined whether any of these genes were upregulated in a set of ~5,000 gene expression changes we had identified in a large gene expression analysis of human T2D β-cells. This approach produced a single gene, RCAN1, as a candidate gene linking hyperglycemia and functional changes in T2D β-cells. Further investigations demonstrated that RCAN1 methylation is reduced in human T2D islets at multiple sites, correlating with increased expression. RCAN1 protein expression was also increased in db/db mouse islets and in human and mouse islets exposed to high glucose. Mice overexpressing RCAN1 had reduced in vivo glucose-stimulated insulin secretion and their β-cells displayed mitochondrial dysfunction including hyperpolarised membrane potential, reduced oxidative phosphorylation and low ATP production. This lack of β-cell ATP had functional consequences by negatively affecting both glucose-stimulated membrane depolarisation and ATP-dependent insulin granule exocytosis. Thus, from amongst the myriad of gene expression changes occurring in T2D β-cells where we had little knowledge of which changes cause β-cell dysfunction, we applied a trisomy 21 screening approach which linked RCAN1 to β-cell mitochondrial dysfunction in T2D.     

ADVANCED PERIPHERAL ARTERIOSCLEROSIS—A Physiological Approach to Management

When peripheral arteriosclerosis progresses to the stage where blood flow through both primary and collateral circulations is impaired seriously, arteriolar relaxants, physical therapeutic, pharmacological and surgical, may aggravate the nutritional deficit. Although tissue viability is maintained precariously, limited activity may be tolerated unless the delicate balance between blood supply and metabolic activity is upset by the vicious circle of rest pain and dependent edema. This vicious circle constitutes an immediate threat to the structural integrity of the limb. Rest pain must be controlled if the delicate balance between blood supply and metabolic activity is to be restored and the threat of amputation mitigated. Intravenous injections of procaine may be of value.     

Population Genetic Diversity in the Australian ‘Seascape’: A Bioregion Approach

Genetic diversity within species may promote resilience to environmental change, yet little is known about how such variation is distributed at broad geographic scales. Here we develop a novel Bayesian methodology to analyse multi-species genetic diversity data in order to identify regions of high or low genetic diversity. We apply this method to co-distributed taxa from Australian marine waters. We extracted published summary statistics of population genetic diversity from 118 studies of 101 species and > 1000 populations from the Australian marine economic zone. We analysed these data using two approaches: a linear mixed model for standardised data, and a mixed beta-regression for unstandardised data, within a Bayesian framework. Our beta-regression approach performed better than models using standardised data, based on posterior predictive tests. The best model included region (Integrated Marine and Coastal Regionalisation of Australia (IMCRA) bioregions), latitude and latitude squared. Removing region as an explanatory variable greatly reduced model performance (delta DIC 23.4). Several bioregions were identified as possessing notably high genetic diversity. Genetic diversity increased towards the equator with a ‘hump’ in diversity across the range studied (−9.4 to −43.7°S). Our results suggest that factors correlated with both region and latitude play a role in shaping intra-specific genetic diversity, and that bioregion can be a useful management unit for intra-specific as well as species biodiversity. Our novel statistical model should prove useful for future analyses of within species genetic diversity at broad taxonomic and geographic scales.     

Molecular Genetic Testing in Endocrinology—A Practical Guide

ObjectiveTo discuss the factors to consider when evaluating patients with a suspected genetic endocrine disorder, so as to guide practicing endocrinologists through the process of genetic testing and result interpretation.MethodsThe author’s experience and review of appropriate literature have been used to give a personal perspective on the role of genetic testing in hereditary endocrine disorders.ResultsRecent advances in our understanding of genetics and genomics have uncovered that they have a far more important role in the pathogenesis of endocrine disease than previously appreciated. Not only are we expanding our understanding of rare mendelian disorders such as multiple endocrine neoplasia type 1 and 2, but we are also beginning to understand the clinical significance of genetic factors in the pathogenesis of common disorders such as obesity and dyslipidemia.ConclusionsIt can be difficult to appreciate the clinical significance and utility of genetic testing that is currently available, and the interpretation of genetic test results can be challenging. Decisions on whether genetic testing is needed should be made on a case-by-case basis, with the endocrinologist and geneticist working together from the outset. (Endocr Pract. 2012;18:85-89)     

Genetic Influences on Hand Osteoarthritis in Finnish Women – A Replication Study of Candidate Genes

Objectives

Our aims were to replicate some previously reported associations of single nucleotide polymorphisms (SNPs) in five genes (A2BP1, COG5, GDF5, HFE, ESR1) with hand osteoarthritis (OA), and to examine whether genes (BCAP29, DIO2, DUS4L, DVWA, HLA, PTGS2, PARD3B, TGFB1 and TRIB1) associated with OA at other joint sites were associated with hand OA among Finnish women.

Design

We examined the bilateral hand radiographs of 542 occupationally active Finnish female dentists and teachers aged 45 to 63 and classified them according to the presence of OA by using reference images. Data regarding finger joint pain and other risk factors were collected using a questionnaire. We defined two hand OA phenotypes: radiographic OA in at least three joints (ROA) and symptomatic DIP OA. The genotypes were determined by PCR-based methods. In statistical analysis, we used SNPStats software, the chi-square test and logistic regression.

Results

Of the SNPs, rs716508 in A2BP1 was associated with ROA (OR = 0.7, 95% CI 0.5–0.9) and rs1800470 in TGFB1 with symptomatic DIP OA (1.8, 1.2–2.9). We found an interaction between ESR1 (rs9340799) and occupation: teachers with the minor allele were at an increased risk of symptomatic DIP OA (2.8, 1.3–6.5). We saw no association among the dentists. We also found that the carriage of the COG5 rs3757713 C allele increased the risk of ROA only among women with the BCAP29 rs10953541 CC genotype (2.6; 1.1–6.1). There was also a suggestive interaction between the HFE rs179945 and the ESR1 rs9340799, and the carriage of the minor allele of either of these SNPs was associated with an increased risk of symptomatic DIP OA (2.1, 1.3–2.5).

Conclusions

Our results support the earlier findings of A2BP1 and TBGF1 being OA susceptibility genes and provide evidence of a possible gene-gene interaction in the genetic influence on hand OA predisposition.     

Is There a Twelfth Protein-Coding Gene in the Genome of Influenza A? A Selection-Based Approach to the Detection of Overlapping Genes in Closely Related Sequences

Protein-coding genes often contain long overlapping open-reading frames (ORFs), which may or may not be functional. Current methods that utilize the signature of purifying selection to detect functional overlapping genes are limited to the analysis of sequences from divergent species, thus rendering them inapplicable to genes found only in closely related sequences. Here, we present a method for the detection of selection signatures on overlapping reading frames by using closely related sequences, and apply the method to several known overlapping genes, and to an overlapping ORF on the negative strand of segment 8 of influenza A virus (NEG8), for which the suggestion has been made that it is functional. We find no evidence that NEG8 is under selection, suggesting that the intact reading frame might be non-functional, although we cannot fully exclude the possibility that the method is not sensitive enough to detect the signature of selection acting on this gene. We present the limitations of the method using known overlapping genes and suggest several approaches to improve it in future studies. Finally, we examine alternative explanations for the sequence conservation of NEG8 in the absence of selection. We show that overlap type and genomic context affect the conservation of intact overlapping ORFs and should therefore be considered in any attempt of estimating the signature of selection in overlapping genes.     

Lateral Femoral Hernias in a Line of FVB/NHsd Mice: A New Confounding Lesion Linked to Genetic Background?

Several strains of transgenic mice derived from an inbred FVB/NHsd colony developed large masses on 1 or both flanks. Although originally suspected to be a phenotypic anomaly related to genetic modifications, nontransgenic littermates subsequently were affected with equal frequency, inculpating the FVB/NHsd founder colony. The masses were subcutaneous, soft, and exophytic and appeared over the course of a few weeks. Female mice were affected more frequently than males. Gross examination revealed the masses to consist of uni- or bilateral hernias of variable size, occasionally containing small or large intestine (or both), cecum, mesenteric adipose tissue, male reproductive organs, and ureters. All hernial sacs pouched through the femoral triangle laterally to the femoral vessels and therefore were classified as lateral femoral hernias. Lateral femoral hernias have not previously been described in the veterinary literature and have never been described as background lesions in a strain of mice. Our findings suggest likely genetic drift in this strain of FVB/NHsd mice, causing a background lesion that confounded phenotypic analyses of transgenic mice derived from this strain.     

Decoding Input Signals in Time Domain—A Model Approach

From an observation of efferent interspike intervals of a neuron, we consider how to decode the input temporal information. It is found that the integrate-and-fire model is blind in the temporal domain due to the fact that its efferent firing rate is independent of the input temporal frequency. The conclusion is then confirmed for the integrate-and-fire model with correlated inputs, with reversal potentials, with a nonlinear leakage and with a subthreshold oscillation. For the Hodgkin-Huxley model, however, in terms of efferent firing rates alone, it is possible to read out the input temporal information.     

Increasing ω-3 Desaturase Expression in Tomato Results in Altered Aroma Profile and Enhanced Resistance to Cold Stress

One of the drawbacks in improving the aroma properties of tomato (Solanum lycopersicum) fruit is the complexity of this organoleptic trait, with a great variety of volatiles contributing to determine specific quality features. It is well established that the oxylipins hexanal and (Z)-hex-3-enal, synthesized through the lipoxygenase pathway, are among the most important aroma compounds and impart in a correct proportion some of the unique fresh notes in tomato. Here, we confirm that all enzymes responsible for the synthesis of these C6 compounds are present and active in tomato fruit. Moreover, due to the low odor threshold of (Z)-hex-3-enal, small changes in the concentration of this compound could modify the properties of the tomato fruit aroma. To address this possibility, we have overexpressed the ω-3 fatty acid desaturases FAD3 and FAD7 that catalyze the conversion of linoleic acid (18:2) to linolenic acid (18:3), the precursor of hexenals and its derived alcohols. Transgenic OE-FAD tomato plants exhibit altered fatty acid composition, with an increase in the 18:3/18:2 ratio in leaves and fruits. These changes provoke a clear variation in the C6 content that results in a significant alteration of the (Z)-hex-3-enal/hexanal ratio that is particularly important in ripe OE-FAD3FAD7 fruits. In addition to this effect on tomato volatile profile, OE-FAD tomato plants are more tolerant to chilling. However, the different behaviors of OE-FAD plants underscore the existence of separate fatty acid fluxes to ensure plant survival under adverse conditions.Tomato (Solanum lycopersicum) breeding has often focused on improving yield, fruit size, and disease resistance, while organoleptic properties have largely been neglected. However, consumer demand for higher nutritional and flavor characteristics in tomato fruits is growing. Despite the complexity of this trait, with multiple biosynthetic pathways contributing, quantitative trait loci that affect volatile composition have been recently identified (Tieman et al., 2006; Mathieu et al., 2009). While proper tomato flavor requires low sugar and acid concentrations, tomato aroma is determined by the contribution of over 400 volatile compounds. The importance of each volatile is determined by both its concentration and its odor threshold (Baldwin et al., 2000). A group of approximately 30 compounds participate, either in a positive or a negative manner, in the properties of tomato aroma. Among them, straight-chain C6 aldehydes and alcohols, such as hexanal, (Z)-hex-3-enal, its isomer (E)-hex-2-enal, and (Z)-hex-3-enol, are the most important to tomato flavor, imparting in a correct proportion some of the unique fresh notes to tomato fruit aroma. Indeed, most appreciated tomato varieties have a higher (Z)-hex-3-enal/hexanal ratio than others less demanded by consumers (Carbonell-Barrachina et al., 2006). Therefore, modifying the (Z)-hex-3-enal/hexanal ratio may be important in the aroma perception of tomato fruits, and since the odor threshold for (Z)-hex-3-enal is low, small changes in the concentration of this compound may exert an important variation in the tomato fruit aroma.These C6 aldehydes and alcohols belong to the complex group of oxylipins, biologically active compounds derived from the oxygenation of unsaturated fatty acids. From the different fatty acids present in plants, hexanal is produced from linoleic acid (18:2), while linolenic acid (18:3) is the precursor of hexenals and derived alcohols. 18:2 and 18:3 are the most abundant fatty acids in plant membrane lipids. In contrast to the biosynthetic pathways of other components of the tomato aroma, the enzymes that participate in the biosynthesis of hexenals and hexanal have been identified and characterized to a large extent (Feussner and Wasternack, 2002). The high specificity of many of the enzymes involved is a feature of this pathway that determines the final products obtained. The first step of this pathway is the production by a specific lipoxygenase (LOX) of the fatty acid hydroperoxide (HPO), derived either from 18:2 or 18:3. According to the position of oxygen insertion, either at the carbon atom 9 or at the carbon atom 13 of the fatty acid backbone, LOXs are classified as 9-LOX or 13-LOX, respectively. In tomato, there are five genes that encode LOXs (TomLoxAto -E) that are differentially expressed during fruit ripening (Chen et al., 2004). TomLoxA, TomLoxB, and TomLoxE are mainly found in fruits and, although their substrate and product specificity is not clear, likely belong to the 9-LOX group based on their sequence similarities and expression (Griffiths et al., 1999; Chen et al., 2004). On the other hand, TomLoxC and TomLoxD are 13-LOX and show differential expression. While TomLoxC is found in fruits, TomLoxD is mainly expressed in leaves and in response to wounding (Heitz et al., 1997; Chen et al., 2004). Interestingly, the major LOX activity in tomato fruit, close to 95%, has 9-LOX specificity (Hatanaka et al., 1992), and no further enzymatic processing of 9-HPOs has been reported. Since the enzymes responsible for HPO modification in fruits have a preference for 13-HPOs, 9-HPOs accumulate in tomato fruits (Matthew et al., 1977). However, minor 13-LOX activity produces a small quantity of 13-HPOs in the fruits that are further cleaved to C6 aldehydes by the action of 13-hydroperoxide lyases (HPLs). From the aldehydes produced by 13-HPL, (Z)-hex-3-enal, derived from 18:3, contributes the most valuable notes to tomato fruit aroma (Boukobza et al., 2001).Addition of exogenous 18:3 increases the level of (Z)-hex-3-enal produced by tomato fruit homogenates (Boukobza et al., 2001), suggesting that the enzymes required for the synthesis of this aroma compound are fully functional in fruit tissues and that the abundance of 18:3 may be a limiting step in (Z)-hex-3-enal production. Contrary to the situation in leaves, tomato fruit is more abundant in 18:2, precursor of hexanal, which may represent up to 80% of its fatty acid content (Galliard et al., 1977). Conversion of 18:2 to 18:3 is carried out by membrane-bound ω-3 desaturases. In Arabidopsis (Arabidopsis thaliana), three genes, FAD3, FAD7, and FAD8, encode the enzymes that participate in the synthesis of hexadecatrienoic acid (16:3) and 18:3 from dienoic fatty acids. FAD3 catalyzes the desaturation reaction of 18:2 that takes place in the endoplasmic reticulum. It uses phospholipids as acyl substrates and NADH, NADH-cytochrome b₅ reductase, and cytochrome b₅ as electron donors. In contrast, FAD7 and FAD8 are located at the chloroplast, providing the majority of the trienoic fatty acids present in the chloroplastic membranes (Wallis and Browse, 2002). They use primarily glycolipids as acyl carriers and NAD(P)H, ferredoxin-NAD(P) reductase, and ferredoxin as electron donors.Metabolic engineering offers an ideal solution to improve the aroma in tomato fruit by increasing the levels of (Z)-hex-3-enal that provides the highly valued fresh notes. To this end, one possible strategy would be to increase the 13-LOX activity specifically involved in the generation of short-chain aldehyde precursors. However, several independent efforts to overexpress the responsible 13-LOX gene led to cosuppression and the consequent depletion of this specific activity (Leon et al., 2002; Chen et al., 2004). A different approach to address this question is to alter the balance between hexenals and hexanal by overexpressing the ω-3 desaturase to increase the content of 18:3, the hexenal precursor. In addition, tomato being a cold-sensitive crop, modifying the unsaturation level of fatty acids present in membrane lipids could contribute to improve the cold tolerance of tomato plants. It is known that modification of the unsaturation degree of the fatty acids is a significant adaptive feature in response to temperature stress (Somerville and Browse, 1991; Iba, 2002). This increase in the trienoic fatty acids present in membrane lipids upon exposure to chilling temperatures is supposed to maintain the required membrane fluidity and to reduce membrane damage, thus ensuring the numerous processes that take place at cell membranes. This capacity of the plants to withstand chilling temperature is not constant but increases noticeably upon exposure to progressively lower temperatures (Guy, 1990). Interestingly, this cold acclimation increases the desaturase activity and the percentage of unsaturated fatty acids (Steponkus et al., 1993). Since most trienoic acids are present in the thylakoid membranes, where the photosynthetic machinery is found, variation of their unsaturation degree at low temperatures could play an important role in maintaining the photosynthetic capacity of the plants.We report here that overexpression of ω-3 desaturases FAD3 and FAD7 in transgenic tomato plants results in a modification of the fatty acid composition, with a major increase of the unsaturation ratio 18:3/18:2 in leaves and fruits. This altered fatty acid profile leads to changes in the ratio of the aroma compounds (Z)-hex-3-enal/hexanal in both tissues. Moreover, transgenic tomato plants with higher levels of FAD3 and FAD7 desaturases are more tolerant to chilling temperatures.