Hodgkin's lymphoma cell lines express a fusion protein encoded by intergenically spliced mRNA for the multilectin receptor DEC-205 (CD205) and a novel C-type lectin receptor DCL-1

Classic Hodgkin's lymphoma (HL) tissue contains a small population of morphologically distinct malignant cells called Hodgkin and Reed-Sternberg (HRS) cells, associated with the development of HL. Using 3'-rapid amplification of cDNA ends ( RACE) we identified an alternative mRNA for the DEC-205 multilectin receptor in the HRS cell line L428. Sequence analysis revealed that the mRNA encodes a fusion protein between DEC-205 and a novel C-type lectin DCL-1. Although the 7.5-kb DEC-205 and 4.2-kb DCL-1 mRNA were expressed independently in myeloid and B lymphoid cell lines, the DEC-205/DCL-1 fusion mRNA (9.5 kb) predominated in the HRS cell lines ( L428, KM-H2, and HDLM-2). The DEC-205 and DCL-1 genes comprising 35 and 6 exons, respectively, are juxtaposed on chromosome band 2q24 and separated by only 5.4 kb. We determined the DCL-1 transcription initiation site within the intervening sequence by 5'-RACE, confirming that DCL-1 is an independent gene. Two DEC-205/DCL-1 fusion mRNA variants may result from cotranscription of DEC-205 and DCL-1, followed by splicing DEC-205 exon 35 or 34-35 along with DCL-1 exon 1. The resulting reading frames encode the DEC-205 ectodomain plus the DCL-1 ectodomain, the transmembrane, and the cytoplasmic domain. Using DCL-1 cytoplasmic domain-specific polyclonal and DEC-205 monoclonal antibodies for immunoprecipitation/Western blot analysis, we showed that the fusion mRNA is translated into a DEC-205/DCL-1 fusion protein, expressed in the HRS cell lines. These results imply an unusual transcriptional control mechanism in HRS cells, which cotranscribe an mRNA containing DEC-205 and DCL-1 prior to generating the intergenically spliced mRNA to produce a DEC-205/DCL-1 fusion protein.

5 '-Untranslated regions with multiple upstream AUG codons can support low-level translation via leaky scanning and reinitiation

Upstream AUGs (uAUGs) and upstream open reading frames (uORFs) are common features of mRNAs that encode regulatory proteins and have been shown to profoundly influence translation of the main ORF. In this study, we employed a series of artificial 5'-untranslated regions (5'-UTRs) containing one or more uAUGs/uORFs to systematically assess translation initiation at the main AUG by leaky scanning and reinitiation mechanisms. Constructs containing either one or two uAUGs in varying contexts but without an in-frame stop codon upstream of the main AUG were used to analyse the leaky scanning mechanism. This analysis largely confirmed the ranking of different AUG contextual sequences that was determined previously by Kozak. In addition, this ranking was the same for both the first and second uAUGs, although the magnitude of initiation efficiency differed. Moreover, similar to10% of ribosomes exhibited leaky scanning at uAUGs in the most favourable context and initiated at a downstream AUG. A second group of constructs containing different numbers of uORFs, each with optimal uAUGs, were used to measure the capacity for reinitiation. We found significant levels of initiation at the main ORF even in constructs containing four uORFs, with nearly 10% of ribosomes capable of reinitiating five times. This study shows that for mRNAs containing multiple uORFs/uAUGs, ribosome reinitiation and leaky scanning are efficient mechanisms for initiation at their main AUGs.

Post-transcriptional regulation of the cystic fibrosis gene in cardiac development and hypertrophy

Eukaryotic gene expression, reflected in the amount of steady-state mRNA, is regulated at the post-transcriptional level. The 5'-untranslated regions (5'-UTRs) of some transcripts contain cis-acting elements, including upstream open reading frames (uORFs), that have been identified as being fundamental in modulating translation efficiency and mRNA stability. Previously, we demonstrated that uORFs present in the 5'-UTR of cystic fibrosis transmembrane conductance regular (CFTR) transcripts expressed in the heart were able to modulate translation efficiency of the main CFTR ORF. Here, we show that the same 5'-UTR elements are associated with the differential stability of the 5'-UTR compared to the main coding region of CFTR transcripts. Furthermore, these post-transcriptional mechanisms are important factors governing regulated CFTR expression in the heart, in response to developmental and pathophysiological stimuli. (C) 2004 Elsevier Inc. All rights reserved.

Communicating continuous quantum variables between different Lorentz frames

We show how to communicate Heisenberg-limited continuous (quantum) variables between Alice and Bob in the case where they occupy two inertial reference frames that differ by an unknown Lorentz boost. There are two effects that need to be overcome: the Doppler shift and the absence of synchronized clocks. Furthermore, we show how Alice and Bob can share Doppler-invariant entanglement, and we demonstrate that the protocol is robust under photon loss.

A survey of the intestinal transcriptomes of the hookworms, Necator americanus and Ancylostoma caninum, using tissues isolated by laser microdissection microscopy

The gastrointestinal tracts of multi-cellular blood-feeding parasites are targets for vaccines and drugs. Recently, recombinant vaccines that interrupt the digestion of blood in the hookworm gut have shown efficacy, so we explored the intestinal transcriptomes of the human and canine hookworms, Necator americanus and Ancylostoma caninum, respectively. We used Laser Microdissection Microscopy to dissect gut tissue from the parasites, extracted the RNA and generated cDNA libraries. A total of 480 expressed sequence tags were sequenced from each library and assembled into contigs, accounting for 268 N. americanus genes and 276 A. caninum genes. Only 17% of N. americanus and 36% of A. caninum contigs were assigned Gene Ontology classifications. Twenty-six (9.8%) N. americanus and 18 (6.5%) A. caninum contigs did not have homologues in any databases including dbEST-of these novel clones, seven N. americanus and three A. caninum contigs had Open Reading Frames with predicted secretory signal peptides. The most abundant transcripts corresponded to mRNAs encoding cholesterol-and fatty acid-binding proteins, C-type lectins, Activation-Associated Secretory Proteins, and proteases of different mechanistic classes, particularly astacin-like metallopeptidases. Expressed sequence tags corresponding to known and potential recombinant vaccines were identified and these included homologues of proteases, anti-clotting factors, defensins and integral membrane proteins involved in cell adhesion. (c) 2006 Australian Society for Parasitology Inc Published by Elsevier Ltd. All fights reserved.

Diversity of polyketide synthase genes from bacteria associated with the marine sponge Pseudoceratina clavata: culture-dependent and culture-independent approaches

Diverse ketosynthase (KS) genes were retrieved from the microbial community associated with the Great Barrier Reef sponge Pseudoceratina clavata. Bacterial isolation and metagenomic approaches were employed. Phylogenetic analysis of 16S rRNA of culturable sponge-associated bacterial communities comprised eight groups over four phyla. Ten KS domains were amplified from four genera of isolates and phylogenetics demonstrated that these KS domains were located in three clusters (actinobacterial, cyanobacterial and trans-AT type). Metagenomic DNA of the sponge microbial community was extracted to explore community KS genes by two approaches: direct amplification of KS domains and construction of fosmid libraries for KS domain screening. Five KS domains were retrieved from polymerase chain reaction (PCR) amplification using sponge metagenome DNA as template and five fosmid clones containing KS domains found using multiplex PCR screening. Analysis of selected polyketide synthase (PKS) from one fosmid showed that the PKS consists of two modules. Open reading frames located up- and downstream of the PKS displayed similarity with membrane synthesis-related proteins such as cardiolipin synthase. Metagenome approaches did not detect KS domains found in sponge isolates. All KS domains from both metagenome approaches formed a single cluster with KS domains originating from metagenomes derived from other sponge species from other geographical regions.

Noninertial observers in special relativity and clock synchronization debates

Selleri's arguments that a consideration of noninertial reference frames in the framework of special relativity identify absolute simultaneity as being Nature's choice of synchronization are considered. In the case of rectilinearly accelerating rockets, it is argued by considering two rockets which maintain a fixed proper separation rather than a fixed separation relative to the inertial frame in which they start from rest, that what seems the most natural choice for a simultaneity convention is problem-dependent and that Einstein's definition is the most natural (though still conventional) choice in this case. In addition, the supposed problems special relativity has with treating a rotating disk, namely how a pulse of light traveling around the circumference of the disk can have a local speed of light equal to c everywhere but a global speed not equal to c, and how coordinate transformations to the disk can give the Lorentz transformations in the limit of large disk radius but small angular velocity, are addressed. It is shown that the theory of Fermi frames solves both of these problems. It is also argued that the question of defining simultaneity relative to a uniformly rotating disk does riot need to be resolved in order to resolve Ehrenfest's paradox.

Crisis and institutional change in urban governance

Recent studies of new institutional spaces typically underplay the uneven and contested process of institutional change by undervaluing the role of inherited institutions and discourses. This is a critical issue as neoliberal networked forms of governance interact with inherited institutional arrangements, characterised by important path dependencies that guide actors. Contradiction and tensions can emerge, culminating in crisis tendencies, and producing both discursive and material contestation between actors. It is with an understanding of path dependencies, ideas (structured into discourses), and (perceived and actual) crisis tendencies that this paper examines contested institutional change through a case-study analysis of one city, and a critical engagement with neoinstitutionalism. The purpose is to examine, firstly, the significance of inherited path-dependent arrangements in fostering conflict and crisis tendencies during interaction with emergent state action; secondly, the extent to which crisis is evident in processes of institutional change and the form that this takes; and, thirdly, the importance of ideas in producing institutional transformation. It is found that institutional conflict is evident between inherited institutions and emergent state action, and stems both from the way agents are organised by the state and from certain path dependencies, but that this does not lead to an actual material crisis. Rather, the nation-state, in partnership with senior city government actors, use ideational/discursive ‘crisis talk’ as a means by which to induce institutional change. The role of ideas has been in critical in this process as the nation-state frames problems and solutions in line with its existing policy paradigm and institutional arrangements, and with discourses further reinforcing existing material power relations.

Sequence analysis and distribution in Salmonella enterica serovars of IS3-like elements

The genome of Salmonella enterica serovar Enteritidis was shown to possess three IS3-like insertion elements, designated IS1230A, B and C, and each was cloned and their respective deoxynucleotide sequences determined. Mutations in elements IS1230A and B resulted in frameshifts in the open reading frames that encoded a putative transposase to be inactive. IS1230C was truncated at nucleotide 774 relative to IS1230B and therefore did not possess the 3' terminal inverted repeat. The three IS1230 derivatives were closely related to each other based on nucleotide sequence similarity. IS1230A was located adjacent to the sef operon encoding SEF14 fimbriae located at minute 97 of the genome of S. Enteritidis. IS1230B was located adjacent to the umuDC operon at minute 42.5 on the genome, itself located near to one terminus of an 815-kb genome inversion of S. Enteritidis relative to S. Typhimurium. IS1230C was located next to attB, the bacteriophage P22 attachment site, and proB, encoding gamma-glutamyl phosphate reductase. A truncated 3' remnant of IS1230, designated IS1230T, was identified in a clinical isolate of S. Typhimurium DT193 strain 2391. This element was located next to attB adjacent to which were bacteriophage P22-like sequences. Southern hybridisation of total genomic DNA from eighteen phage types of S. Enteritidis and eighteen definitive types of S. Typhimurium showed similar, if not identical, restriction fragment profiles in the respective serovars when probed with IS1230A.

Sequence analysis and distribution of an IS3-like insertion element isolated from Salmonella enteritidis

The nucleotide sequence of a 3 kb region immediately upstream of the sef operon of Salmonella enteritidis was determined. A 1230 base pair insertion sequence which shared sequence identity (> 75%) with members of the IS3 family was revealed. This element, designated IS1230, had almost identical (90% identity) terminal inverted repeats to Escherichia coli IS3 but unlike other IS3-like sequences lacked the two characteristic open reading frames which encode the putative transposase. S. enteritidis possessed only one copy of this insertion sequence although Southern hybridisation analysis of restriction digests of genomic DNA revealed another fragment located in a region different from the sef operon which hybridised weakly which suggested the presence of an IS1230 homologue. The distribution of IS1230 and IS1230-like elements was shown to be widespread amongst salmonellas and the patterns of restriction fragments which hybridised differed significantly between Salmonella serotypes and it is suggested that IS1230 has potential for development as a differential diagnostic tool.

Design of sway frames

The aim of the work presented in this thesis is to produce a direct method to design structures subject to deflection constraints at the working loads. The work carried out can be divided into four main parts. In the first part, a direct design procedure for plane steel frames subjected to sway limitations is proposed. The stiffness equations are modified so that the sway in each storey is equal to some specified values. The modified equations are then solved by iteration to calculate the cross-sectional properties of the columns as well as the other joint displacements. The beam sections are selected initially and then altered in an effort to reduce the total material cost of the frame. A linear extrapolation technique is used to reduce this cost. In this design, stability functions are used so that the effect of axial loads in the members are taken into consideration. The final reduced cost design is checked for strength requirements and the members are altered accordingly. In the second part, the design method is applied to the design of reinforced concrete frames in which the sway in the columns play an active part in the design criteria. The second moment of area of each column is obtained by solving the modified stiffness equations and then used to calculate the mlnlmum column depth required. Again the frame has to be checked for all the ultimate limit state load cases. In the third part, the method is generalised to design pin-jointed space frames for deflection limitatlions. In these the member areas are calculated so that the deflection at a specified joint is equal to its specified value. In the final part, the Lagrange multiplier technique is employed to obtain an optimum design for plane rigidly jointed steel frames. The iteration technique is used here to solve the modified stiffness equations as well as derivative equations obtained in accordance to the requirements of the optimisation method.

Die load and stresses in press forging

Several axi-symmetric EN3B steel components differing in shape and size were forged on a 100 ton joint knuckle press. A load cell fitted under the lower die inserts recorded the total deformation forces. Job parameters were measured off the billets and the forged parts. Slug temperatures were varied and two lubricants - aqueous colloidal graphite and oil - were used. An industrial study was also conducted to check the results of the laboratory experiments. Loads were measured (with calibrated extensometers attached to the press frames) when adequately heated mild steel slugs were being forged in finishing dies. Geometric parameters relating to the jobs and the dies were obtained from works drawings. All the variables considered in the laboratory study could not, however, be investigated without disrupting production. In spite of this obvious limitation, the study confirmed that parting area is the most significant geometric factor influencing the forging load. Multiple regression analyses of the laboratory and industrial results showed that die loads increase significantly with the weights and parting areas of press forged components, and with the width to thickness ratios of the flashes formed, but diminish with increasing slug temperatures and higher billet diameter to height ratios. The analyses also showed that more complicated parts require greater loads to forge them. Die stresses, due to applied axial loads, were investigated by the photoelastic method. The three dimensional frozen stress technique was employed. Model dies were machined from cast araldite cylinders, and the slug material was simulated with plasticene. Test samples were cut from the centres of the dies after the stress freezing. Examination of the samples, and subsequent calculations, showed that the highest stresses were developed in die outer corners. This observation partly explains why corner cracking occurs frequently in industrial forging dies. Investigation of die contact during the forging operation revealed the development of very high stresses.