Coronavirus mRNA synthesis: identification of novel transcription initiation signals which are differentially regulated by different leader sequences.

The mRNA synthesis of mouse hepatitis virus (MHV) has been proposed to be the result of interaction between the leader RNA and the intergenic sites. Previously, we have identified a transcription initiation site (for mRNA 2-1), which is more efficiently transcribed by viruses containing two copies of UCUAA sequence in the leader RNA than by those with three copies. In this study, we have identified several sites which are regulated in the opposite way, namely, they are efficiently transcribed by the leader RNA with three UCUAA copies but not by those with two copies. These sites were characterized by primer extension and amplification by polymerase chain reaction. One of these sites is in the gene 3 region of a recombinant virus between A59 and JHM strains of MHV. Another is in the gene 2 region of MHV-1 strain. Both of these sites have a sequence similar to but different from the consensus transcription initiation signal (UCUAAUCUAUC and UUUAAUCUU, as opposed to UCUAAAC). These two novel intergenic sequences are not present in the genome of the JHM strain, consistent with the absence of these mRNAs in the JHM-infected cells. The discovery of this type of transcription initiation site provides additional evidence for the importance of the leader RNA in the transcription initiation of MHV mRNAs

Biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus.

We have previously shown that gp65 (E3) is a virion structural protein which varies widely in quantity among different strains of mouse hepatitis virus (MHV). In this study, the biosynthetic pathway and possible biological activities of this protein were examined. The glycosylation of gp65 in virus-infected cells was inhibited by tunicamycin but not by monensin, suggesting that it contains an N-glycosidic linkage. Glycosylation is cotranslational and appears to be complete before the glycoprotein reaches the Golgi complex. Pulse-chase experiments showed that this protein decreased in size after 30 min of chase, suggesting that the carbohydrate chains of gp65 undergo trimming during its transport across the Golgi. This interpretation is supported by the endoglycosidase treatment of gp65, which showed that the peptide backbone of gp65 did not decrease in size after pulse-chase periods. This maturation pathway is distinct from that of the E1 or E2 glycoproteins. Partial endoglycosidase treatment indicated that gp65 contains 9 to 10 carbohydrate side chains; thus, almost all of the potential glycosylation sites of gp65 were glycosylated. In vitro translation studies coupled with protease digestion suggest that gp65 is an integral membrane protein. The presence of gp65 in the virion is correlated with the presence of an acetylesterase activity. No hemagglutinin activity was detected

Membrane Computing Schema: A New Approach to Computation Using String Insertions

In this paper, we introduce the notion of a membrane computing schema for string objects. We propose a computing schema for a membrane network (i.e., tissue-like membrane system) where each membrane performs unique type of operations at a time and sends the result to others connected through the channel. The distinguished features of the computing models obtained from the schema are: 1. only context-free insertion operations are used for string generation, 2. some membranes assume filtering functions for structured objects (molecules), 3. generating model and accepting model are obtained in the same schema, and both are computationally universal, 4. several known rewriting systems with universal computability can be reformulated by the membrane computing schema in a uniform manner. The first feature provides the model with a simple uniform structure which facilitates a biological implementation of the model, while the second feature suggests further feasibility of the model in terms of DNA complementarity. Through the third and fourth features, one may have a unified view of a variety of existing rewriting systems with Turing computability in the framework of membrane computing paradigm.Ministerio de Educación y Ciencia TIN2006-13425Junta de Andalucía TIC-58

Role of Ucp1 enhancer methylation and chromatin remodelling in the control of Ucp1 expression in murine adipose tissue

Aims/hypothesis Increasing the expression of the brown adipose tissue-specific gene uncoupling protein-1 (Ucp1) is a potential target for treating obesity. We investigated the role of DNA methylation and histone modification in Ucp1 expression in adipose cell lines and ex vivo murine adipose tissues. Methods Methylation state of the Ucp1 enhancer was studied using bisulphite mapping in murine adipose cell lines, and tissue taken from cold-stressed mice, coupled with functional assays of the effects of methylation and demethylation of the Ucp1 promoter on gene expression and nuclear protein binding. Results We show that demethylation of the Ucp1 promoter by 5-aza-deoxycytidine increases Ucp1 expression while methylation of Ucp1 promoter–reporter constructs decreases expression. Brown adipose tissue-specific Ucp1 expression is associated with decreased CpG dinucleotide methylation of the Ucp1 enhancer. The lowest CpG dinucleotide methylation state was found in two cyclic AMP response elements (CRE3, CRE2) in the Ucp1 promoter and methylation of the CpG in CRE2, but not CRE3 decreased nuclear protein binding. Chromatin immunoprecipitation assays revealed the presence of the silencing DiMethH3K9 modification on the Ucp1 enhancer in white adipose tissue and the appearance of the active TriMethH3K4 mark at the Ucp1 promoter in brown adipose tissue in response to a cold environment. Conclusions/interpretation The results demonstrate that CpG dinucleotide methylation of the Ucp1 enhancer exhibits tissue-specific patterns in murine tissue and cell lines and suggest that adipose tissue-specific Ucp1 expression involves demethylation of CpG dinucleotides found in regulatory CREs in the Ucp1 enhancer, as well as modification of histone tails

Lymphatic marker podoplanin/D2-40 in human advanced cirrhotic liver- Re-evaluations of microlymphatic abnormalities

<p>Abstract</p> <p>Background</p> <p>From the morphological appearance, it was impossible to distinguish terminal portal venules from small lymphatic vessels in the portal tract even using histochemical microscopic techniques. Recently, D2-40 was found to be expressed at a high level in lymphatic endothelial cells (LECs). This study was undertaken to elucidate hepatic lymphatic vessels during progression of cirrhosis by examining the expression of D2-40 in LECs.</p> <p>Methods</p> <p>Surgical wedge biopsy specimens were obtained from non-cirrhotic portions of human livers (normal control) and from cirrhotic livers (LC) (Child A-LC and Child C-LC). Immunohistochemical (IHC), Western blot, and immunoelectron microscopic studies were conducted using D2-40 as markers for lymphatic vessels, as well as CD34 for capillary blood vessels.</p> <p>Results</p> <p>Imunostaining of D2-40 produced a strong reaction in lymphatic vessels only, especially in Child C-LC. It was possible to distinguish the portal venules from the small lymphatic vessels using D-40. Immunoelectron microscopy revealed strong D2-40 expression along the luminal and abluminal portions of the cell membrane of LECs in Child C-LC tissue.</p> <p>Conclusion</p> <p>It is possible to distinguish portal venules from small lymphatic vessels using D2-40 as marker. D2-40- labeling in lymphatic capillary endothelial cells is related to the degree of fibrosis in cirrhotic liver.</p

Computational genes: a tool for molecular diagnosis and therapy of aberrant mutational phenotype

<p>Abstract</p> <p>Background</p> <p>A finite state machine manipulating information-carrying DNA strands can be used to perform autonomous molecular-scale computations at the cellular level.</p> <p>Results</p> <p>We propose a new finite state machine able to detect and correct aberrant molecular phenotype given by mutated genetic transcripts. The aberrant mutations trigger a cascade reaction: specific molecular markers as input are released and induce a spontaneous self-assembly of a wild type protein or peptide, while the mutational disease phenotype is silenced. We experimentally demostrated in <it>in vitro </it>translation system that a viable protein can be autonomously assembled.</p> <p>Conclusion</p> <p>Our work demostrates the basic principles of computational genes and particularly, their potential to detect mutations, and as a response thereafter administer an output that suppresses the aberrant disease phenotype and/or restores the lost physiological function.</p

DNA Specificity Determinants Associate with Distinct Transcription Factor Functions

To elucidate how genomic sequences build transcriptional control networks, we need to understand the connection between DNA sequence and transcription factor binding and function. Binding predictions based solely on consensus predictions are limited, because a single factor can use degenerate sequence motifs and because related transcription factors often prefer identical sequences. The ETS family transcription factor, ETS1, exemplifies these challenges. Unexpected, redundant occupancy of ETS1 and other ETS proteins is observed at promoters of housekeeping genes in T cells due to common sequence preferences and the presence of strong consensus motifs. However, ETS1 exhibits a specific function in T cell activation; thus, unique transcriptional targets are predicted. To uncover the sequence motifs that mediate specific functions of ETS1, a genome-wide approach, chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), identified both promoter and enhancer binding events in Jurkat T cells. A comparison with DNase I sensitivity both validated the dataset and also improved accuracy. Redundant occupancy of ETS1 with the ETS protein GABPA occurred primarily in promoters of housekeeping genes, whereas ETS1 specific occupancy occurred in the enhancers of T cell–specific genes. Two routes to ETS1 specificity were identified: an intrinsic preference of ETS1 for a variant of the ETS family consensus sequence and the presence of a composite sequence that can support cooperative binding with a RUNX transcription factor. Genome-wide occupancy of RUNX factors corroborated the importance of this partnership. Furthermore, genome-wide occupancy of co-activator CBP indicated tight co-localization with ETS1 at specific enhancers, but not redundant promoters. The distinct sequences associated with redundant versus specific ETS1 occupancy were predictive of promoter or enhancer location and the ontology of nearby genes. These findings demonstrate that diversity of DNA binding motifs may enable variable transcription factor function at different genomic sites

Effects of Time of Day and Sleep Deprivation on Motorcycle-Driving Performance

The aim of this study was to investigate whether motorcycle handling capabilities – measured by means of the efficiency of emergency manoeuvres – were dependent on prior sleep deprivation and time of day. Twelve male participants voluntarily took part in four test sessions, starting at 6 a.m., 10 a.m., 2 p.m., and 6 p.m., following a night either with or without sleep. Each test session comprised temperature and sleepiness measurements, before three different types of motorcycling tests were initiated: (1) stability in straight ahead riding at low speed (in “slow motion” mode and in “brakes and clutch” mode), (2) emergency braking and (3) crash avoidance tasks performed at 20 kph and 40 kph. The results indicate that motorcycle control at low speed depends on time of day, with an improvement in performance throughout the day. Emergency braking performance is affected at both speeds by time of day, with poorer performance (longer total stopping distance, reaction time and braking distance) in the morning, and also by sleep deprivation, from measurements obtained at 40 kph (incorrect initial speed). Except for a tendency observed after the sleepless night to deviate from the initial speed, it seems that crash avoidance capabilities are quite unaffected by the two disturbance factors. Consequently, some motorcycle handling capabilities (stability at low speed and emergency braking) change in the same way as the diurnal fluctuation observed in body temperature and sleepiness, whereas for others (crash avoidance) the participants were able to maintain their initial performance level despite the high levels of sleepiness recorded after a sleepless night. Motorcycle riders have to be aware that their handling capabilities are limited in the early morning and/or after sleep deprivation. Both these situations can increase the risk of falls and of being involved in a road accident