Radially Interrupted Viscous Fingers in a Lifting Hele-Shaw Cell

Viscous fingers have been produced in the lifting Hele-Shaw cell, with concentric circular grooves etched onto the lower plate. The invading fluid (air) enters the defending newtonian fluid - olive oil as fingers proceeding radially inwards towards the centre. The fingers are interrupted at the circular groove, and reform as secondary fingers. The effect of the grooves is to speed up the fingering process considerably and the fingers now reach the centre much faster. We explain this by comparing the variation in velocity of the fingers in the normal HS cell and the grooved cells with time. In the normal HS cell the fingers move fastest on initial formation and slow down later. Since in case of the grooved plate, the fingers reform and receive a boost in their speed each time they encounter a groove, the fingers proceed to the centre faster. PACS nos. 47.20.Gv, 47.54.+r, 68.03.-gComment: 4 pg. 2 fi

Targeting the active site of the placental isozyme of alkaline phosphatase by phage-displayed scFv antibodies selected by a specific uncompetitive inhibitor

BACKGROUND: The isozymes of alkaline phosphatase, the tissue non-specific, intestinal and placental, have similar properties and a high degree of identity. The placental isozyme (PLAP) is an oncofetal antigen expressed in several malignancies including choriocarcinoma, seminoma and ovarian carcinoma. We had earlier attempted to isolate PLAP-specific scFv from a synthetic human immunoglobulin library but were unable to do so, presumably because of the similarity between the isozymes. In this work, we have employed a PLAP-specific uncompetitive inhibitor, L-Phe-Gly-Gly, to select isozyme specific scFvs. An uncompetitive inhibitor binds to the enzyme in the presence of substrate and stabilizes the enzyme-substrate complex. Several uncompetitive inhibitors have varying degrees of isozyme specificity for human alkaline phosphatase isozymes. A specific uncompetitive inhibitor would be able to unmask conformational differences between the otherwise very similar molecules. Also, such inhibitors would be directed to regions at/close to the active site of the enzyme. In this work, the library was first incubated with PLAP and the bound clones then eluted by incubation with L-Phe-Gly-Gly along with the substrate, para-nitro phenyl phosphate (pNPP). The scFvs were then studied with regard to the biochemical modulation of their binding, isozyme specificity and effect on enzyme activity. RESULTS: Of 13 clones studied initially, the binding of 9 was inhibited by L-Phe-Gly-Gly (with pNPP) and 2 clones were inhibited by pNPP alone. Two clones had absolute and 2 clones had partial specificity to PLAP. Two clones were cross-reactive with only one other isozyme. Three scFv clones, having an accessible His6-tag, were purified and studied for their modulation of enzyme activity. All the three scFvs inhibited PLAP activity with the kinetics of competitive inhibition. Cell ELISA could demonstrate binding of the specific scFvs to the cell surface expressed PLAP. CONCLUSION: The results demonstrate the biochemical modulation of scFv binding. Also, the scFvs bound to the active site and denied the access to the substrate. The selection strategy could generate specific anti-enzyme antibodies to PLAP that can potentially be used for targeting, for modulating enzyme activity in in vitro and in vivo and as probes for the active site. This strategy also has a general application in selecting antibodies from combinatorial libraries to closely related molecules and conformations

High affinity mouse-human chimeric Fab against hepatitis B surface antigen

Aim: Passive immunotherapy using antibody against hepatitis B surface antigen (HBsAg) has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal (5S) against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody. Methods: We cloned the VH and VL genes of this mouse antibody, and fused them with CH1 domain of human IgG1 and CL domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. coli. Results: The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. This chimeric antibody fragment was further expressed in different strains of E. coli to increase the yield. Conclusion: We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a full-length chimeric antibody for therapeutic uses

Regulation of sonic hedgehog-GLI1 downstream target genes PTCH1, Cyclin D2, Plakoglobin, PAX6 and NKX2.2 and their epigenetic status in medulloblastoma and astrocytoma

Abstract Background The Sonic hedgehog (Shh) signaling pathway is critical for cell growth and differentiation. Impairment of this pathway can result in both birth defects and cancer. Despite its importance in cancer development, the Shh pathway has not been thoroughly investigated in tumorigenesis of brain tumors. In this study, we sought to understand the regulatory roles of GLI1, the immediate downstream activator of the Shh signaling pathway on its downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6 in medulloblastoma and astrocytic tumors. Methods We silenced GLI1 expression in medulloblastoma and astrocytic cell lines by transfection of siRNA against GLI1. Subsequently, we performed RT-PCR and quantitative real time RT-PCR (qRT-PCR) to assay the expression of downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6. We also attempted to correlate the pattern of expression of GLI1 and its regulated genes in 14 cell lines and 41 primary medulloblastoma and astrocytoma tumor samples. We also assessed the methylation status of the Cyclin D2 and PTCH1 promoters in these 14 cell lines and 58 primary tumor samples. Results Silencing expression of GLI1 resulted up-regulation of all target genes in the medulloblastoma cell line, while only PTCH1 was up-regulated in astrocytoma. We also observed methylation of the cyclin D2 promoter in a significant number of astrocytoma cell lines (63%) and primary astrocytoma tumor samples (32%), but not at all in any medulloblastoma samples. PTCH1 promoter methylation was less frequently observed than Cyclin D2 promoter methylation in astrocytomas, and not at all in medulloblastomas. Conclusions Our results demonstrate different regulatory mechanisms of Shh-GLI1 signaling. These differences vary according to the downstream target gene affected, the origin of the tissue, as well as epigenetic regulation of some of these genes.http://deepblue.lib.umich.edu/bitstream/2027.42/78313/1/1471-2407-10-614.xmlhttp://deepblue.lib.umich.edu/bitstream/2027.42/78313/2/1471-2407-10-614.pdfPeer Reviewe

Inhibition of preS1-hepatocyte interaction by an array of recombinant human antibodies from naturally recovered individuals

Neutralizing monoclonal antibodies are being found to be increasingly useful in viral infections. In hepatitis B infection, antibodies are proven to be useful for passive prophylaxis. The preS1 region (21–47a.a.) of HBV contains the viral hepatocyte-binding domain crucial for its attachment and infection of hepatocytes. Antibodies against this region are neutralizing and are best suited for immune-based neutralization of HBV, especially in view of their not recognizing decoy particles. Anti-preS1 (21–47a.a.) antibodies are present in serum of spontaneously recovered individuals. We generated a phage-displayed scFv library using circulating lymphocytes from these individuals and selected four preS1-peptide specific scFvs with markedly distinct sequences from this library. All the antibodies recognized the blood-derived and recombinant preS1 containing antigens. Each scFv showed a discrete binding signature, interacting with different amino acids within the preS1-peptide region. Ability to prevent binding of the preS1 protein (N-terminus 60a.a.) to HepG2 cells stably expressing hNTCP (HepG2-hNTCP-C4 cells), the HBV receptor on human hepatocytes was taken as a surrogate marker for neutralizing capacity. These antibodies inhibited preS1-hepatocyte interaction individually and even better in combination. Such a combination of potentially neutralizing recombinant antibodies with defined specificities could be used for preventing/managing HBV infections, including those by possible escape mutants

Glial tumorigenesis: molecular alterations and identification of targets

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