Expression of GC-C, a receptor-guanylate cyclase, and its endogenous ligands uroguanylin and guanylin along the rostrocaudal axis of the intestine

Members of the receptor-guanylate cyclase (rGC) family possess an intracellular catalytic domain that is regulated by an extracellular receptor domain. GC-C, an intestinally expressed rGC, was initially cloned by homology as an orphan receptor. The search for its Ligands has yielded three candidates: STa (a bacterial toxin that causes traveler's diarrhea) and the endogenous peptides uroguanylin and guanylin. Here, by performing Northern and Western blots, and by measuring [I-125]STa binding and STa-dependent elevation of cGMP levels, we investigate whether the distribution of GC-C matches that of its endogenous ligands in the rat intestine. We establish that 1) uroguanylin is essentially restricted to small bowel; 2) guanylin is very low in proximal small bowel, increasing to prominent levels in distal small bowel and throughout colon; 3) GC-C messenger RNA and STa-binding sites are uniformly expressed throughout the intestine; and 4) GC-C-mediated cGMP synthesis peaks at the proximal and distal extremes of the intestine (duodenum and colon), but is nearly absent in the middle (ileum). These observations suggest that GC-C's activity may be posttranslationally regulated, demonstrate that the distribution of GC-C is appropriate to mediate the actions of both uroguanylin and guanylin, and help to refine current hypotheses about the physiological role(s) of these peptides.

Regulation of cytochrome P-450b/e gene expression by a heme- and phenobarbitone-modulated transcription factor

The cloned DNA fragment of the cytochrome P-450b/e gene containing the upstream region from position -179 through part of the first exon is faithfully transcribed in freeze-thawed rat liver nuclei. Phenobarbitone treatment of the animal strikingly increases this transcription, and the increase is blocked by cycloheximide (protein synthesis inhibitor) or CoCl2 (heme biosynthetic inhibitor) treatment of animals. This picture correlates very well with the reported cytochrome P-450b/e mRNA levels in vivo and run-on transcription rates in vitro under these conditions. The upstream region (from position -179) was assessed for protein binding with nuclear extracts by nitrocellulose filter binding, gel retardation, DNase I treatment ("footprinting"), and Western blot analysis. Phenobarbitone treatment dramatically increases protein binding to the upstream region, an increase once again blocked by cycloheximide or CoCl2 treatments. Addition of heme in vitro to heme-deficient nuclei and nuclear extracts restores the induced levels of transcription and protein binding to the upstream fragment, respectively. Thus, drug-mediated synthesis and heme-modulated binding of a transcription factor(s) appear involved in the transcriptional activation of the cytochrome P-450b/e genes, and an 85-kDa protein may be a major factor in this regard.

Gene expression profile of epithelial cells and mesenchymal cells derived from limbal explant culture

Purpose: Limbal stem cell deficiency is a challenging clinical problem and the current treatment involves replenishing the depleted limbal stem cell (LSC) pool by either limbal tissue transplantation or use of cultivated limbal epithelial cells (LEC). Our experience of cultivating the LEC on denuded human amniotic membrane using a feeder cell free method, led to identification of mesenchymal cells of limbus (MC-L), which showed phenotypic resemblance to bone marrow derived mesenchymal stem cells (MSC-BM). To understand the transcriptional profile of these cells, microarray experiments were carried out.Methods: RNA was isolated from cultured LEC, MC-L and MSC-BM and microarray experiments were carried out by using Agilent chip (4x44 k). The microarray data was validated by using Realtime and semiquntitative reverse transcription polymerase chain reaction. Results: The microarray analysis revealed specific gene signature of LEC and MC-L, and also their complementary role related to cytokine and growth factor profile, thus supporting the nurturing roles of the MC-L. We have also observed similar and differential gene expression between MC-L and MSC-BM.Conclusions: This study represents the first extensive gene expression analysis of limbal explant culture derived epithelial and mesenchymal cells and as such reveals new insight into the biology, ontogeny, and in vivo function of these cells.

Expression of drug resistance markers by spheroplasts ofMycobacterium smegmatis after fusion

Mycobacterial spheroplasts were prepared by treatment of the glycinesensitized cells with a combination of lipase and lysozyme. They were stable for several hours at room temperature but were lysed on treatment with 0.1% sodium dodecyl sulfate. The spheroplasts could be regenerated on a suitable medium. Fusion and regeneration of the spheroplasts were attempted using drug resistant mutant strains ofM. smegmalis. Recombinants were obtained from spheroplast fusion mediated by polyethylene glycol and dimethyl sulfoxide. Simultaneous expression of rccombinant properties was observed only after an initial lag in the isolated clones. This has been explained as due to “chromosome inactivation” in the fused product.

Identification and expression of the 20 kd structural protein gene of colitis bacteriophage

The 2.3 kb BamHI fragment from the colitis bacteriophage DNA was transcribed and translated into a 20 kd structural protein P6, in a coupled transcription-translation system derived from Escherichia coli. This protein was expressed in vivo by the 2.3 kb DNA cloned in pBR322. The gene with the regulatory elements for this protein was located on the 680 bp AvaII fragment of the insert DNA. It hybridized with two RNAs of sizes 520 and 1630 nucleotides indicating that both are messengers for the 20 kd protein. Dot-blot hybridization showed that the transcripts for P6 reached a maximum level at 12 min after phage infection.

A matrix identity and perturbation expressions

An identity expressing formally the diagonal and off-diagonal elements of an inverse of a matrix is deduced employing operator techniques. Several well-known perturbation expressions for the self-energy are deduced as special cases. A new approximation and other applications following from the above formalism are briefly indicated through illustrations from a perturbed harmonic oscillator, chemisorption approximations and Kelly's result in the problem of electron correlation.

High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2

The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-1R). These actions are modulated by a family of six IGF-binding proteins (ICFBP-1-6; 22-31 kDa) that via high affinity binding to the IGFs (K-D similar to 300-700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access In recent years, IGFBPs have been implicated in a variety of cancers However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in Eschericha coli Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E coli and first structural characterization of a full-length IGFBP (C) 2010 Elsevier Inc. All rights reserved

Microscopic expression for frequency and wave vector dependent dielectric constant of a dipolar liquid

A microscopic expression for the frequency and wave vector dependent dielectric constant of a dense dipolar liquid is derived starting from the linear response theory. The new expression properly takes into account the effects of the translational modes in the polarization relaxation. The longitudinal and the transverse components of the dielectric constant show vastly different behavior at the intermediate values of the wave vector k. We find that the microscopic structure of the dense liquid plays an important role at intermediate wave vectors. The continuum model description of the dielectric constant, although appropriate at very small values of wave vector, breaks down completely at the intermediate values of k. Numerical results for the longitudinal and the transverse dielectric constants are obtained by using the direct correlation function from the mean‐spherical approximation for dipolar hard spheres. We show that our results are consistent with all the limiting expressions known for the dielectric function of matter.

Genome-wide expression profiling identifies deregulated miRNAs in malignant astrocytoma

Malignant astrocytoma includes anaplastic astrocytoma (grade III) and glioblastoma (grade IV). Among them, glioblastoma is the most common primary brain tumor with dismal responses to all therapeutic modalities. We performed a large-scale, genome-wide microRNA (miRNA) (n=756) expression profiling of 26 glioblastoma, 13 anaplastic astrocytoma and 7 normal brain samples with an aim to find deregulated miRNA in malignant astrocytoma. We identified several differentially regulated miRNAs between these groups, which could differentiate glioma grades and normal brain as recognized by PCA. More importantly, we identified a most discriminatory 23-miRNA expression signature, by using PAM, which precisely distinguished glioblastoma from anaplastic astrocytoma with an accuracy of 95%. The differential expression pattern of nine miRNAs was further validated by real-time RT-PCR on an independent set of malignant astrocytomas (n-72) and normal samples (n=7). Inhibition of two glioblastoma-upregulated miRNAs (miR-21 and miR-23a) and exogenous overexpression of two glioblastoma-downregulated miRNAs (miR-218 and miR-219-5p) resulted in reduced soft agar colony formation but showed varying effects on cell proliferation and chemosensitivity. Thus we have identified the miRNA expression signature for malignant astrocytoma, in particular glioblastoma, and showed the miRNA involvement and their importance in astrocytoma development. Modern Pathology (2010) 23, 1404-1417; doi:10.1038/modpathol.2010.135; published online 13 August 2010

Chaperone expression profiles correlate with distinct physiological states of Plasmodium falciparum in malaria patients

Background: Molecular chaperones have been shown to be important in the growth of the malaria parasite Plasmodium falciparum and inhibition of chaperone function by pharmacological agents has been shown to abrogate parasite growth. A recent study has demonstrated that clinical isolates of the parasite have distinct physiological states, one of which resembles environmental stress response showing up-regulation of specific molecular chaperones. Methods: Chaperone networks operational in the distinct physiological clusters in clinical malaria parasites were constructed using cytoscape by utilizing their clinical expression profiles. Results: Molecular chaperones show distinct profiles in the previously defined physiologically distinct states. Further, expression profiles of the chaperones from different cellular compartments correlate with specific patient clusters. While cluster 1 parasites, representing a starvation response, show up-regulation of organellar chaperones, cluster 2 parasites, which resemble active growth based on glycolysis, show up-regulation of cytoplasmic chaperones. Interestingly, cytoplasmic Hsp90 and its co-chaperones, previously implicated as drug targets in malaria, cluster in the same group. Detailed analysis of chaperone expression in the patient cluster 2 reveals up-regulation of the entire Hsp90-dependent pro-survival circuitries. In addition, cluster 2 also shows up-regulation of Plasmodium export element (PEXEL)-containing Hsp40s thought to have regulatory and host remodeling roles in the infected erythrocyte. Conclusion: In all, this study demonstrates an intimate involvement of parasite-encoded chaperones, PfHsp90 in particular, in defining pathogenesis of malaria.

A novel approach to design of cis-acting DNA structural element for regulation of gene expression in vivo

Taking advantage of the degeneracy of the genetic code we have developed a novel approach to introduce, within a gene, DNA sequences capable of adopting unusual structures and to investigate the role of such sequences in regulation of gene expression in vivo. We used a computer program that generates alternative codon sequences for the same amino-acid sequence to convert a stretch of nucleotides into an inverted-repeat sequence with the potential to adopt cruciform structure. This approach was used to replace a 51-base-pair EcoRI-HindIII segment in the N-terminal region of the beta-galactosidase gene in plasmid pUC19 with a 51-bp synthetic oligonucleotide sequence with the potential to adopt a cruciform structure with 18 bp in the stem region. In selecting the 51-bp sequence, care was taken to include those codons that are preferred in E. coli. E. coli DH5-alpha cells harbouring the plasmid containing the redesigned sequence showed drastic reduction in expression of the beta-galactosidase gene compared to cells harbouring the plasmid with the native sequence. This approach demonstrates the possibility of introducing DNA secondary-structure elements to alter regulation of gene expression in vivo.

Over-expression and purification strategies for recombinant multi-protein oligomers: A case study of Mycobacterium tuberculosis sigma/anti-sigma factor protein complexes

The function of a protein in a cell often involves coordinated interactions with one or several regulatory partners. It is thus imperative to characterize a protein both in isolation as well as in the context of its complex with an interacting partner. High resolution structural information determined by X-ray crystallography and Nuclear Magnetic Resonance offer the best route to characterize protein complexes. These techniques, however, require highly purified and homogenous protein samples at high concentration. This requirement often presents a major hurdle for structural studies. Here we present a strategy based on co-expression and co-purification to obtain recombinant multi-protein complexes in the quantity and concentration range that can enable hitherto intractable structural projects. The feasibility of this strategy was examined using the sigma factor/anti-sigma factor protein complexes from Mycobacterium tuberculosis. The approach was successful across a wide range of sigma factors and their cognate interacting partners. It thus appears likely that the analysis of these complexes based on variations in expression constructs and procedures for the purification and characterization of these recombinant protein samples would be widely applicable for other multi-protein systems. (C) 2010 Elsevier Inc. All rights reserved.

Development stage-specific expression of fibroin in the silk worm Bombyx mori is regulated translationally

The contents of fibroin H RNA as a function of development have been quantitated in the posterior silk glands of Bombyx mori larvae on different days of 4th and 5th instars. The fibroin RNA levels increased during the feeding stages of larvae and the RNA got completely degraded during the interim moult. The patterns of accumulation of fibroin RNA were similar in both the instars. Although there was considerable increase in the fibroin RNA content during the 5th larval instar, the relative abundance of fibroin RNA in the total RNA was fairly constant during the 4th and 5th instars. The increased content of fibroin RNA in 5th instar was the consequence of an overall increase in transcription accompanying the development progress, rather than specific increase only in fibroin transcription. The contents of fibroin protein in the 4th and 5th instars of development have also been quantitated making use of a sensitive radioimmune assay with a purified, antifibroin antibody. There were substantial differences between 4th and 5th instars in the absolute fibroin contents as well as the relative proportion of fibroin in the total proteins. These results implied that although the fibroin gene was transcribed at the same efficiency during the 4th and 5th instars, the translational efficiency was much lower during the 4th instar. The extent of polyadenylation of fibroin RNA was similar in both instars. However, there was a two-fold increase in the polysome association of fibroin RNA in the 5th instar. Over and above this, there was substantial increase during the 5th instar in the contents of those tRNAs. (e.g. Gly, Ala and Ser) which are abundantly represented in fibroin and therefore directly related to the expression of fibroin. The increased polysome association of fibroin mRNA and the adequate supply of cognate tRNAs in the 5th instar, together contributes to the translational regulation of fibroin in a developmental stage-specific manner. Based on these observations, we propose that translational regulation plays a major role in the development stage-specific synthesis of fibroin in Bombyx mori.