Mutated polyadenylation signals for controlling expression levels of multiple genes in mammalian cells

A set of mutated SV40 early polyadenylation signals (SV40pA) with varying strengths is generated by mutating the AATAAA sequence in the wild-type SV40pA. They are shown to control the expression level of a gene over a 10-fold range using luciferase reporter genes in transient transfection assays. The relative strength of these SV40pA variants remains similar under three commonly used mammalian promoters and in five mammalian cell lines. Application of SV40pA variants for controlling expression level of multiple genes is demonstrated in a study of monoclonal antibody (mAb) synthesis in mammalian cells. By using SV40pA variants of different strengths, the expression of light chain (LC) and heavy chain (HC) genes encoded in a single vector is independently altered which results in different ratios of LC to HC expression spanning a range from 0.24 to 16.42. The changes in gene expression are determined by measuring mRNA levels and intracellular LC and HC polypeptides. It is found that a substantial decrease of HC expression, which increases the LC/HC mRNA ratio, only slightly reduces mAb production. However, reducing the LC expression by a similar magnitude, which decreases the LC/HC mRNA ratio results in a sharp decline of mAb production to trace amounts. This set of SV40pA variants offers a new tool for accurate control of the relative expression levels of multiple genes. It will have wide-ranging applications in fields related to the study of biosynthesis of multi-subunit proteins, proteomic research on protein interactions, and multi-gene metabolic engineering.     

A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease

In drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure–activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC₅₀ values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC₅₀ values with steep dose–response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay’s utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other protein–ligand interactions.     

Sequence and analysis of a plasmid-encoded mercury resistance operon from Mycobacterium marinum identifies MerH, a new mercuric ion transporter

In this study, we report the DNA sequence and biological analysis of a mycobacterial mercury resistance operon encoding a novel Hg²⁺ transporter. MerH was found to transport mercuric ions in Escherichia coli via a pair of essential cysteine residues but only when coexpressed with the mercuric reductase.     

Genetic characterization of <Emphasis Type="Italic">Perna viridis</Emphasis> L. in peninsular Malaysia using microsatellite markers

A total of 19 polymorphic microsatellite loci were used to analyse levels of genetic variation for 10 populations of Perna viridis L. collected from all over peninsular Malaysia. The populations involved in this study included Pulau Aman in Penang, Tanjung Rhu in Kedah, Bagan Tiang in Perak, Pulau Ketam in Selangor, Muar, Parit Jawa, Pantai Lido and Kampung Pasir Puteh in Johore, and Kuala Pontian and Nenasi in Pahang state. The number of alleles per locus ranged from two to seven, with an average of 3.1. Heterozygote deficiencies were observed across all the 10 populations. Characterization of the populations revealed that local populations of P. viridis in peninsular Malaysia were genetically similar enough to be used as a biomonitoring agent for heavy metal contamination in the Straits of Malacca. Cluster analysis grouped the P. viridis populations according to their geographical distributions with the exception of Parit Jawa. The analysis also revealed that P. viridis from the northern parts of peninsular Malaysia were found to be the most distant populations among the populations of mussels investigated and P. viridis from the eastern part of peninsular Malaysia were closer to the central and southern populations than to the northern populations.     

Bacterial Communities Associated with the Digestive Tract of the Predatory Ground Beetle, <Emphasis Type="Italic">Poecilus chalcites,</Emphasis> and Their Modification by Laboratory Rearing and Antibiotic Treatment

Ground beetles such as Poecilus chalcites (Coleoptera: Carabidae) are beneficial insects in agricultural systems where they contribute to the control of insect and weed pests. We assessed the complexity of bacterial communities occurring in the digestive tracts of field-collected P. chalcites using terminal restriction fragment length polymorphism analyses of polymerase chain reaction-amplified 16S rRNA genes. Bacterial identification was performed by the construction of 16S rRNA gene clone libraries and sequence analysis. Intestinal bacteria in field-collected beetles were then compared to those from groups of beetles that were reared in the lab on an artificial diet with and without antibiotics. Direct cell counts estimated 1.5 × 10⁸ bacteria per milliliter of gut. The digestive tract of field-collected P. chalcites produced an average of 4.8 terminal restriction fragments (tRF) for each beetle. The most abundant clones were affiliated with the genus Lactobacillus, followed by the taxa Enterobacteriaceae, Clostridia, and Bacteriodetes. The majority of the sequences recovered were closely related to those reported from other insect gastrointestinal tracts. Lab-reared beetles produced fewer tRF, an average of 3.1 per beetle, and a reduced number of taxa with a higher number of clones from the family Enterobacteriaceae compared to the field-collected beetles. Antibiotic treatment significantly (p < 0.05) reduced the number of tRF per beetle and selected for a less diverse set of bacterial taxa. We conclude that the digestive tract of P. chalcites is colonized by a simple community of bacteria that possess autochthonous characteristics. Laboratory-reared beetles harbored the most common bacteria found in field-collected beetles, and these bacterial communities may be manipulated in the laboratory with the addition of antibiotics to the diet to allow study of functional roles.     

Latent Transforming Growth Factor ��-binding Proteins and Fibulins Compete for Fibrillin-1 and Exhibit Exquisite Specificities in Binding Sites

Latent transforming growth factor (TGF) β-binding proteins (LTBPs) interact with fibrillin-1. This interaction is important for proper sequestration and extracellular control of TGFβ. Surface plasmon resonance interaction studies show that residues within the first hybrid domain (Hyb1) of fibrillin-1 contribute to interactions with LTBP-1 and LTBP-4. Modulation of binding affinities by fibrillin-1 polypeptides in which residues in the third epidermal growth factor-like domain (EGF3) are mutated demonstrates that the binding sites for LTBP-1 and LTBP-4 are different and suggests that EGF3 may also contribute residues to the binding site for LTBP-4. In addition, fibulin-2, fibulin-4, and fibulin-5 bind to residues contained within EGF3/Hyb1, but mutated polypeptides again indicate differences in their binding sites in fibrillin-1. Results demonstrate that these protein-protein interactions exhibit “exquisite specificities,” a phrase commonly used to describe monoclonal antibody interactions. Despite these differences, interactions between LTBP-1 and fibrillin-1 compete for interactions between fibrillin-1 and these fibulins. All of these proteins have been immunolocalized to microfibrils. However, in fibrillin-1 (Fbn1) null fibroblast cultures, LTBP-1 and LTBP-4 are not incorporated into microfibrils. In contrast, in fibulin-2 (Fbln2) null or fibulin-4 (Fbln4) null cultures, fibrillin-1, LTBP-1, and LTBP-4 are incorporated into microfibrils. These data show for the first time that fibrillin-1, but not fibulin-2 or fibulin-4, is required for appropriate matrix assembly of LTBPs. These studies also suggest that the fibulins may affect matrix sequestration of LTBPs, because in vitro interactions between these proteins are competitive.Fibrillin microfibrils are ubiquitous structural elements in the connective tissue. Fibrillin microfibrils provide organs with tissue-specific architectural frameworks designed to support the mature functional integrity of the particular organ. In addition, fibrillin microfibrils contribute to proper developmental patterning of organs by targeting growth factors to the right location in the extracellular matrix (1, 2).Molecules of fibrillin-1 (3), fibrillin-2 (4, 5), and fibrillin-3 (6) polymerize to form the backbone structure of microfibrils. Latent TGFβ-binding protein (LTBP)³-1 associates with fibrillin microfibrils in the perichondrium and in osteoblast cultures (7, 8), and LTBP-1 and LTBP-4 interact with fibrillin (9). Other proteins associated with fibrillin microfibrils include the fibulins (10, 11), microfibril-associated glycoprotein-1 and -2 (12, 13), decorin (14), biglycan (15), versican (16), and perlecan (17). It is likely that one function of these associated extracellular matrix molecules is to connect the fibrillin microfibril scaffold to other architectural elements in tissue- and organ-specific patterns.In addition to performing architectural functions, fibrillins bind directly to prodomains of bone morphogenetic proteins and growth and differentiation factors (18, 19) and LTBPs bring with them the small latent TGFβ complex (20), suggesting that the microfibril scaffold may position, concentrate, and control growth factor signaling. Studies of fibrillin-1 (Fbn1) and fibrillin-2 (Fbn2) mutant mice demonstrate that loss of fibrillins results in phenotypes associated with dysregulated TGFβ (21–23) or bone morphogenetic protein (24) signaling. Microfibril-associated glycoprotein-1 (Magp-1) null mice reveal phenotypes that may also be related to abnormal TGFβ signaling (25).In a previous study (9), we determined that the binding site for LTBP-1 and -4 is contained within a specific four-domain region of fibrillin-1. In this study, we performed additional experiments to more precisely define the LTBP binding site. At the same time, we compared binding of fibulins to fibrillin, because the region in fibrillin-1 that was suggested to contain the fibulin binding site (11) was very close to our region of interest for LTBP binding. Our results demonstrate that LTBPs and fibulins compete for binding to fibrillin-1. However, the proteins tested (LTBP-1, LTBP-4, fibulin-2, fibulin-4, and fibulin-5) displayed “exquisite specificities” in their interactions with fibrillin-1.To test the potential significance of these interactions with fibrillin-1, we investigated matrix incorporation of LTBPs in cell cultures obtained from wild type, Fbn1 null, Fbn2 null, fibulin-2 (Fbln-2) null, and fibulin-4 (Fbln-4) null mice. In addition, we examined the distribution of LTBPs in Fbn1 null and Fbn2 null mice.     

96 shRNAs designed for maximal coverage of HIV-1 variants

Background

Extensive studies of primary infection are crucial to our understanding of the course of HIV disease. In SIV-infected macaques, a model closely mimicking HIV pathogenesis, we used a combination of three markers -- viral RNA, 2LTR circles and viral DNA -- to evaluate viral replication and dissemination simultaneously in blood, secondary lymphoid tissues, and the gut during primary and chronic infections. Subsequent viral compartmentalization in the main target cells of the virus in peripheral blood during the chronic phase of infection was evaluated by cell sorting and viral quantification with the three markers studied.

Results

The evolutions of viral RNA, 2LTR circles and DNA levels were correlated in a given tissue during primary and early chronic infection. The decrease in plasma viral load principally reflects a large decrease in viral replication in gut-associated lymphoid tissue (GALT), with viral RNA and DNA levels remaining stable in the spleen and peripheral lymph nodes. Later, during chronic infection, a progressive depletion of central memory CD4+ T cells from the peripheral blood was observed, accompanied by high levels of viral replication in the cells of this subtype. The virus was also found to replicate at this point in the infection in naive CD4+ T cells. Viral RNA was frequently detected in monocytes, but no SIV replication appeared to occur in these cells, as no viral DNA or 2LTR circles were detected.

Conclusion

We demonstrated the persistence of viral replication and dissemination, mostly in secondary lymphoid tissues, during primary and early chronic infection. During chronic infection, the central memory CD4+ T cells were the major site of viral replication in peripheral blood, but viral replication also occurred in naive CD4+ T cells. The role of monocytes seemed to be limited to carrying the virus as a cargo because there was an observed lack of replication in these cells. These data may have important implications for the targeting of HIV treatment to these diverse compartments.     

An exploration of how clinician attitudes and beliefs influence the implementation of lifestyle risk factor management in primary healthcare: a grounded theory study

Background

Given the current emphasis on networks as vehicles for innovation and change in health service delivery, the ability to conceptualise and measure organisational enablers for the social construction of knowledge merits attention. This study aimed to develop a composite tool to measure the organisational context for evidence-based practice (EBP) in healthcare.

Methods

A structured search of the major healthcare and management databases for measurement tools from four domains: research utilisation (RU), research activity (RA), knowledge management (KM), and organisational learning (OL). Included studies were reports of the development or use of measurement tools that included organisational factors. Tools were appraised for face and content validity, plus development and testing methods. Measurement tool items were extracted, merged across the four domains, and categorised within a constructed framework describing the absorptive and receptive capacities of organisations.

Results

Thirty measurement tools were identified and appraised. Eighteen tools from the four domains were selected for item extraction and analysis. The constructed framework consists of seven categories relating to three core organisational attributes of vision, leadership, and a learning culture, and four stages of knowledge need, acquisition of new knowledge, knowledge sharing, and knowledge use. Measurement tools from RA or RU domains had more items relating to the categories of leadership, and acquisition of new knowledge; while tools from KM or learning organisation domains had more items relating to vision, learning culture, knowledge need, and knowledge sharing. There was equal emphasis on knowledge use in the different domains.

Conclusion

If the translation of evidence into knowledge is viewed as socially mediated, tools to measure the organisational context of EBP in healthcare could be enhanced by consideration of related concepts from the organisational and management sciences. Comparison of measurement tools across domains suggests that there is scope within EBP for supplementing the current emphasis on human and technical resources to support information uptake and use by individuals. Consideration of measurement tools from the fields of KM and OL shows more content related to social mechanisms to facilitate knowledge recognition, translation, and transfer between individuals and groups.