STUDIES ON THE MECHANISM OF ASSEMBLY OF MURINE LEUKEMIA VIRUS

The purpose of this research was to elucidate the mechanism of assembly of retroviruses, specifically of murine leukemia virus, as studied through the treatment of virus-infected cells with interferon and through the use of temperature sensitive (ts) mutants. Our studies have shown a rapid and specific association of Rauscher murine leukemia virus (R-MuLV) precursor polyprotein Pr65('gag) with cytoskeletal elements in infected mouse fibroblasts. The Pr65('gag) associated with Nonidet P-40 (NP40)-insoluble cytoskeletal structures appeared to be subphosphorylated in comparison to NP40-soluble Pr65('gag). The association of Pr65('gag) with skeletal elements could be disrupted by extraction of the cytoskeleton with sodium deoxycholate, an ionic detergent. Both the skeleton-associated Pr65('gag) and its NP40-soluble counterpart were labeled with {('3)H}-palmitate, indicating their probable association with lipids presumably in the plasma membrane. Pr65('gag) molecules bound to skeletal elements in the infected cell appeared to be more stable to proteolytic processing than NP40-soluble Pr65('gag). Our studies with certain ts mutants of murine leukemia virus, defective in virus assembly, including Mo-MuLV ts3 and R-MuLV ts17, ts24, ts25 and ts26, have shown that virions released at 39(DEGREES)C (nonpermissive temperature) had high levels of uncleaved Pr65('gag) relative to that seen in virions released at 33(DEGREES)C (permissive temperature). Examination of cell extracts revealed that Pr54('gag) was more stable to processing at 39(DEGREES)C than at 33(DEGREES)C, whereas the 'env' and glycosylated 'gag' proteins were processed to the same extent at both temperatures. Detergent extraction of pulse-labeled cells to generate an NP40-insoluble cytoskeleton-enriched fraction showed that in ts3-, ts17- and ts24-infected cells, Pr65('gag) accumulated in the cytoskeleton-enriched fraction. In contrast, cells infected with ts25 or ts26 showed no preferential localization of Pr65('gag) in the cytoskeleton in a short pulse, but instead, Pr65('gag) accumulated in both the NP40-soluble and -insoluble fractions during a chase-incubation. The association of Pr65('gag) with cytoskeletal elements in the cell was neither increased nor decreased by blocking virus assembly and release with interferon. Based on these and other results, we have proposed a model for the active role of cytoskeleton-associated Pr65('gag) in retrovirus assembly.^

Functional differences of beta-tubulin isotypes: A study of microtubule assembly and antimitotic drug resistance

Mammalian cells express 7 β-tubulin isotypes in a tissue specific manner. This has long fueled the speculation that different isotypes carry out different functions. To provide direct evidence for their functional significance, class III, IVa, and VI β-tubulin cDNAs were cloned into a tetracycline regulated expression vector and stably transfected Chinese hamster ovary cell lines expressing different levels of ectopic β-tubulin were compared for effects on microtubule organization, microtubule assembly and sensitivity to antimitotic drugs. It was found that all three isotypes coassembled with endogenous β-tubulin. βVI expression caused distinct microtubule rearrangements including microtubule dissociation from the centrosome and accumulation at the cell periphery; whereas expression of βIII and βVIa caused no observable changes in the interphase microtubule network. Overexpression of all 3 isotypes caused spindle malformation and mitotic defects. Both βIII and βIVa disrupted microtubule assembly in proportion to their abundance and thereby conferred supersensitivity to microtubule depolymerizing drugs. In contrast, βVI stabilized microtubules at low stoichiometry and thus conferred resistance to many microtubule destabilizing drugs but not vinblastine. The 3 isotypes caused differing responses to microtubule stabilizing drugs. Expression of βIII conferred paclitaxel resistance while βVI did not. Low expression of βIVa caused supersensitivity to paclitaxel, whereas higher expression resulted in the loss of supersensitivity. The results suggest that βIVa may possess an enhanced ability to bind paclitaxel that increases sensitivity to the drug and acts substoichiometrically. At high levels of βVIa expression, however, microtubule disruptive effects counteract the assembly promoting pressure exerted by increased paclitaxel binding, and drug supersensitivity is lost. From this study, I concluded that β-tubulin isotypes behave differently from each other in terms of microtubule organization, microtubule assembly and dynamics, and antimitotic drug sensitivity. The isotype composition of cell can impart subtle to dramatic effects on the properties of microtubules leading to potential functional consequences and opening the opportunity to exploit differences in microtubule isotype composition for therapeutic gain. ^

The relationship of mutations in alpha- and beta-tubulin to microtubule assembly and anti-mitotic drug resistance

The mechanisms responsible for anti-cancer drug (including Taxol) treatment failure have not been identified. In cell culture model systems, many β-tubulin, but very few α-tubulin, mutations have been associated with resistance to Taxol. To test what, if any, mutations in α-tubulin can cause resistance, we transfected a randomly mutagenized α-tubulin cDNA into Chinese hamster ovary (CHO) cells and isolated drug resistant cell lines. A total of 12 mutations were identified in this way and all of them were confirmed to confer Taxol resistance. Furthermore, all cells expressing mutant α-tubulin had less microtubule polymer. Some cells also had abnormal nuclei and enlarged cell bodies. The data indicate that α-tubulin mutations confer Taxol resistance by disrupting microtubule assembly, a mechanism consistent with a large number of previously described β-tubulin mutations. ^ Because α- and β-tubulin are almost identical in their three dimensional structure, we hypothesized that mutations discovered in one subunit, when introduced into the other, would produce similar effects on microtubule assembly and drug resistance. 9 α- and 2 β-tubulin mutations were tested. The results were complex. Some mutations produced similar changes in microtubule assembly and drug resistance irrespective of the subunit in which they were introduced, but others produced opposite effects. Still one mutation produced resistance when present in one subunit, yet had no effect when present on the other; and one mutation that produced Taxol resistance when present in α-tubulin, resulted in assembly-defective tubulin when it was present in β-tubulin. The results suggest that in most cases, the same amino acid modification in α- and β-tubulin affects the microtubule structure and assembly in a similar way. ^ Finally, we tested whether three β-tubulin mutations found in patient tumors could confer resistance to Taxol by recreating the mutations in a β-tubulin cDNA and transfecting it into CHO cells. We found that all three mutations conferred Taxol resistance, but to different extents. Again, microtubule assembly in the transfectants was disrupted, suggesting that mutations in β-tubulin are a potential problem in cancer therapeutics. ^

Contribution of transmembrane and cytoplasmic domains to membrane protein association

Membrane proteins are critical to every aspect of cell physiology, with their association mediating important biological functions. The transmembrane and cytoplasmic domains are known to be important for their association. In order to characterize their role in detail, we have applied different biophysical techniques in detergent micelles to two model systems. The first study involves FcRγ, a single transmembrane domain protein existing as a disulfide linked homodimer. We investigated the role of a conserved transmembrane polar residue and the cytoplasmic tail in FcRγ homo-interactions. Our results by various biophysical techniques including SDS-PAGE, circular dichroism and sedimentation equilibrium in detergent micelles indicate importance of both the transmembrane polar residue and cytoplasmic tail in maintaining proper conformation for FcRγ homo-interactions. A contrasting second study was on L-selectin, another single transmembrane domain protein with a large extracellular domain and a short cytoplasmic tail. Previous cross-linking experiments indicate its possible dimerization. However, the purified fragment of L-selectin and corresponding mutants did not dimerize when analyzed by TOXCAT assay, sedimentation equilibrium and fluorescence resonance energy transfer. It was likely that the presence of juxtamembrane positively charged residues led to decreased migrational rates in SDS PAGE. In conclusion, complementary biophysical techniques should be used with care when studying membrane protein association in detergent micelles. As an extension to our study on L-selectin, we also investigated its interaction with Calmodulin (CaM) in detergent micelles. CaM was found to interact with different detergents. We applied fluorescence and NMR spectroscopy to characterize the interaction of both the apo and Ca 2+ bound form of CaM, with commonly used detergents, below and above their respective critical micelle concentrations. The interaction of apo-CaM with detergents was found to vary with the nature of the detergent head group, whereas Ca2+-CaM interacted with individual detergent molecules irrespective of the nature of their head group. NMR titration experiments of CaM with detergents indicated involvement of specific residues from the N-lobe, linker and C-lobe of CaM. ^

The association of allergies and attention deficit hyperactivity disorder in children

To investigate the association between allergies and attention deficit hyperactivity disorder (ADHD), a case-control study was conducted using the National Longitudinal Survey of Youth population. Cases were between the ages of 4 and 11 years and were classified either by a maternal-reported diagnosis or by the Behavior Problems Index Hyperactivity Scale. Controls were chosen from the same age group but had a score of less than 14 on the overall Behavior Problems Index. A history of allergies was considered positive if any of the following conditions were reported as requiring treatment by a doctor or other health professional: asthma, allergic conditions, or food allergies. A strong association was observed between allergies and a maternal-reported diagnosis while controlling for demographic, socioeconomic, perinatal, and environmental factors (adjusted odds ratio = 2.85 (95% CI = 1.49-5.42)). Other risk factors found to be important risk factors for a diagnosis of ADHD were gender (male), gestational age ($<$36 weeks), and maternal education ($\leq$high school). No association between allergies and cases classified as ADHD based on the hyperactivity symptom scale was observed. This study confirms other studies that reported an allergy/ADHD association in diagnoses populations. Further investigation confirming the association and explanation of the reasons and underlying mechanisms of the observed association is warranted. These studies should use validated diagnostic criteria for the diagnosis of ADHD symptoms and allergies, adequate sample sizes, and control for confounding. ^

Systematic review of current trends of cardiovascular disease (CVD) and association between health insurance coverage and CVD mortality among women aged 60--65 in Texas in 2000--2011

Cardiovascular disease (CVD) is highly preventable, yet it is a leading cause of death among women in Texas. The primary goals of this research were to examine past and current trends of CVD, as well as identify whether there is an association between the insurance coverage and mortality from CVD among women aged 60–65 in Texas between 2000 and 2011. ^ The systematic review of the research is based on the guidelines and recommendations set by the Centre for Reviews and Dissemination for conducting reviews in health care. Over 47 citations of peer-reviewed articles from Ovid MEDLINE and PubMed databases and five websites were identified, of which 7 studies met inclusion criteria for the first systematic review to examine the trends of CVD in Texas. Ten citations of peer-reviewed articles from Ovid MEDLINE and PubMed databases and five web sites were reviewed for the second systematic review (to study the association between insurance coverage and cardiovascular health among Texas women 60–64 years of age), of which 3 studies met inclusion criteria and were included in the research. The results of the study highlighted key gaps in the existing literature and important areas for the further research, as well as determined directions for future public health CVD prevention programs in Texas. ^ Based on the conducted research, the major determinants of premature mortality among women attributed to cardiovascular disease are based on individual level characteristics, more specifically sex, age, race/ethnicity, and education. The results indicate that African American and non-Hispanic white women are more likely to have higher CVD mortality rates than Hispanic women due to higher prevalence of cardiac risk factors. The data also shows higher levels of mortality from CVD in the southeastern United States, with Texas ranking as the third state with the highest prevalence of CVD among women. According to the Texas Department of State Health Services, there are approximately 56,000 deaths caused by CVD annually in Texas, which represents about one death every ten minutes. Coronary artery disease and stroke were the causes of 31.2 percent of all female deaths in Texas in 2009, meaning that approximately 68 women die from any form of cardiac disease in Texas each day. ^ The data of the reviewed studies indicate that women' lack of health insurance was significantly associated with a higher prevalence of cardiovascular disease. The uninsured women were more likely to be unaware of their risk factors and more likely to have undiagnosed diabetes—a co-morbidity factor of CVD. One of the studies also reports strong correlation between state rates of uninsured and lower rates of preventive care. Given these strong correlations, those who were chronically uninsured were at a higher risk of mortality than the insured, due to prolonged periods of time without basic access to preventive and medical care. ^ Suggested recommendations to decrease CVD mortality rates in Texas are consistent with the existing literature and include state policy development that addresses elimination of health disparities, consideration of potential benefits of universal health coverage by the legislative policymakers, and maintenance of solid partnerships between public health agencies and hospitals to educate on, diagnose, and treat CVD among the female population in Texas. ^

Gene-gene interaction and gene-pathway analysis of genome-wide association for schizophrenia

Schizophrenia (SZ) is a complex disorder with high heritability and variable phenotypes that has limited success in finding causal genes associated with the disease development. Pathway-based analysis is an effective approach in investigating the molecular mechanism of susceptible genes associated with complex diseases. The etiology of complex diseases could be a network of genetic factors and within the genes, interaction may occur. In this work we argue that some genes might be of small effect that by itself are neither sufficient nor necessary to cause the disease however, their effect may induce slight changes to the gene expression or affect the protein function, therefore, analyzing the gene-gene interaction mechanism within the disease pathway would play crucial role in dissecting the genetic architecture of complex diseases, making the pathway-based analysis a complementary approach to GWAS technique. ^ In this study, we implemented three novel linkage disequilibrium based statistics, the linear combination, the quadratic, and the decorrelation test statistics, to investigate the interaction between linked and unlinked genes in two independent case-control GWAS datasets for SZ including participants of European (EA) and African (AA) ancestries. The EA population included 1,173 cases and 1,378 controls with 729,454 genotyped SNPs, while the AA population included 219 cases and 288 controls with 845,814 genotyped SNPs. We identified 17,186 interacting gene-sets at significant level in EA dataset, and 12,691 gene-sets in AA dataset using the gene-gene interaction method. We also identified 18,846 genes in EA dataset and 19,431 genes in AA dataset that were in the disease pathways. However, few genes were reported of significant association to SZ. ^ Our research determined the pathways characteristics for schizophrenia through the gene-gene interaction and gene-pathway based approaches. Our findings suggest insightful inferences of our methods in studying the molecular mechanisms of common complex diseases.^

Characterization of a Novel Role for the Tousled- like Kinase in Kinetochore Assembly and Function in Caenorhabditis elegans

Chromosome segregation is a critical step during cell division to avoid aneuploidy and promote proper organismal development. Correct sister chromatid positioning and separation during mitosis helps to achieve faithful transmission of genetic material to daughter cells. This prevents improper chromosome partitioning that can potentially result in extrachromosomal fragments, increasing the tumorigenic potential of the cells. The kinetochore is a protenaicious structure responsible for the initiation and orchestration of chromosome movement during mitosis. This highly conserved structure among eukaryotes is required for chromosome attachment to the mitotic spindle and failure to assemble the kinetochore results in aberrant chromosome segregation. Thus elucidating the mechanism of kinetochore assembly is important to have a better understanding of the regulation that controls chromosome segregation. Our previous work identified the C. elegans Tousled-like kinase (TLK-1) as a mitotic kinase and depletion of TLK-1 results in embryonic lethality, characterized by nuclei displaying poor mitotic chromosome alignment, lagging chromosome, and chromosome bridges during anaphase. Additionally, previous studies from our group revealed that TLK-1 is phosphorylated independently by Aurora B at serine 634, and by CHK-1 at threonine T610. The research presented herein reveals that both phosphorylated forms of TLK-1 associate with the kinetochore during mitosis. Moreover, by systematic depletion of kinetochore proteins, I uncovered that pTLK-1 is bona fide kinetochore component that is located at the outer kinetochore layer, influencing the microtubule-binding interface. I also demonstrated that TLK-1 is necessary for the kinetochore localization of the microtubule interacting proteins CLS-2 and LIS-1 and I show that embryos depleted of TLK-1 presented an aberrant twisted kinetochore pattern. Furthermore, I established that the inner kinetochore protein KNL-2 is an in vitro substrate of TLK-1 indicating a possible role of TLK-1 in regulating centromeric assembly. Collectively, these results suggest a novel role for the Tousled-like kinase in regulation of kinetochore assembly and microtubule dynamics and demonstrate the necessity of TLK-1 for proper chromosome segregation in C. elegans.

Dynamic Studies of Shell and Spatial Structures

For the past 20 years, dynamic analysis of shells has been one of the most fascinating fields for research. Using the new light materials the building engineer soon discovered that the subsequent reduction of gravity forces produced not only the desired shape freedom but the appearance of ecologic loads as the first factor of design; loads which present strong random properties and marked dynamic influence. On the other hand, the technological advance in the aeronautical and astronautical field placed the engineers in front of shell structures of nonconventional shape and able to sustain substantialy dynamic loads. The response to the increasingly challenger problems of the last two decades has been very bright; new forms, new materials and new methods of analysis have arosen in the design of off-shore platforms, nuclear vessels, space crafts, etc. Thanks to the intensity of the lived years we have at our disposition a coherent and homogeneous amount of knowledge which enable us to face problems of inconceivable complexity when IASS was founded. The open minded approach to classical problems and the impact of the computer are, probably, important factors in the Renaissance we have enjoyed these years, and a good proof of this are the papers presented to the previous IASS meetings as well as that we are going to consider in this one. Particularly striking is the great number of papers based on a mathematical modeling in front of the meagerness of those treating laboratory experiments on physical models. The universal entering of the computer into almost every phase of our lifes, and the cost of physical models, are –may be- reasons for this lack of experimental methods. Nevertheless they continue offering useful results as are those obtained with the shaking-table in which the computer plays an essential role in the application of loads as well as in the instantaneous treatment of control data. Plates 1 and 2 record the papers presented under dynamic heading, 40% of them are from Japan in good correlation with the relevance that Japanese research has traditionally showed in this area. Also interesting is to find old friends as profesors Tanaka, Nishimura and Kostem who presented valuable papers in previous IASS conferences. As we see there are papers representative of all tendencies, even purely analytical! Better than discuss them in detail, which can be done after the authors presentation, I think we can comment in the general pattern of the dynamical approach are summarized in plate 3.

UPM Activities on Sensitivity and Uncertainty Analysis of Assembly Depletion

UPM Activities on Sensitivity and Uncertainty Analysis of Assembly Depletion

De novo assembly and annotation of a transcriptome during xylogenesis in Pinus canariensis Chr. Sm. ex DC

Nowadays, there is a great amount of genomic and transcriptomic data available about forest species, including ambitious projects looking for complete sequencing and annotation of different gymnosperm genomes [1, 2]. Pinus canariensis is an endemic conifer of the Canary Islands with re-sprouting capability and resilience against fire and mechanical damage, as result of an adaptation to volcanic environments. Additionally, this species has a high proportion of axial parenchyma compared with other conifers, and this tissue connects with radial parenchyma allowing transport of reserves. The most internal tracheids stop accumulating water [3], and get filled of resins and polyphenols synthesized by the axial parenchyma; this is the so-called ?torch-heartwood? [4], which avoids decay. This wood achieves very high prices due to its particular resistance to rot. These features make P. canariensis an interesting model species for the analysis of these developmental processes in conifers. In this study we aim to perform a complete transcriptome annotation during xylogenesis in Pinus canariensis, using next-generation sequencing (NGS) -Roche 454 pyrosequencing-, in order to provide a genomic resource for further analysis, including expression profiling and the identification of candidate genes for important adaptive features.

Supramolecular organization of immature and mature murine leukemia virus revealed by electron cryo-microscopy: Implications for retroviral assembly mechanisms

We have used electron cryo-microscopy and image analysis to examine the native structure of immature, protease-deficient (PR−) and mature, wild-type (WT) Moloney murine leukemia virus (MuLV). Maturational cleavage of the Gag polyprotein by the viral protease is associated with striking morphological changes. The PR− MuLV particles exhibit a rounded central core, which has a characteristic track-like shell on its surface, whereas the WT MuLV cores display a polygonal surface with loss of the track-like feature. The pleomorphic shape and inability to refine unique orientation angles suggest that neither the PR− nor the WT MuLV adheres to strict icosahedral symmetry. Nevertheless, the PR− MuLV particles do exhibit paracrystalline order with a spacing between Gag molecules of ≈45 Å and a length of ≈200 Å. Because of the pleomorphic shape and paracrystalline packing of the Gag–RNA complexes, we raise the possibility that assembly of MuLV is driven by protein–RNA, as well as protein–protein, interactions. The maturation process involves a dramatic reorganization of the packing arrangements within the ribonucleoprotein core with disordering and loosening of the individual protein components.

The association between older age and receipt of care and outcomes in patients with acute coronary syndromes : a cohort study of the Myocardial Ischaemia National Audit Project (MINAP)

Peer reviewed