Epithelial Membrane Protein-2 (EMP2) Activates Src Protein and Is a Novel Therapeutic Target for Glioblastoma

Despite recent advances in molecular classification, surgery, radiotherapy, and targeted therapies, the clinical outcome of patients with malignant brain tumors remains extremely poor. In this study, we have identified the tetraspan protein epithelial membrane protein-2 (EMP2) as a potential target for glioblastoma (GBM) killing. EMP2 had low or undetectable expression in normal brain but was highly expressed in GBM as 95% of patients showed some expression of the protein. In GBM cells, EMP2 enhanced tumor growth in vivo in part by up-regulating αvβ3 integrin surface expression, activating focal adhesion kinase and Src kinases, and promoting cell migration and invasion. Consistent with these findings, EMP2 expression significantly correlated with activated Src kinase in patient samples and promoted tumor cell invasion using intracranial mouse models. As a proof of principle to determine whether EMP2 could serve as a target for therapy, cells were treated using specific anti-EMP2 antibody reagents. These reagents were effective in killing GBM cells in vitro and in reducing tumor load in subcutaneous mouse models. These results support the role of EMP2 in the pathogenesis of GBM and suggest that anti-EMP2 treatment may be a novel therapeutic treatment.     

A high-throughput genome-walking method and its use for cloning unknown flanking sequences

We developed a PCR-based high-throughput genome-walking protocol. The novelty of this protocol is in the random introduction of unique walker primer binding sites into different regions of the genome efficiently by taking advantage of the rolling circle mode of DNA synthesis by Phi29 DNA polymerase after annealing the partially degenerate primers to the denatured genomic DNA. The inherent strand-displacement activity of the Phi29 DNA polymerase displaces the 5′ ends of downstream strands and DNA synthesis continues, resulting in a large number of overlapping fragments that cover the whole genome with the unique walker adapter attached to the 5′ end of all the genomic DNA fragments. The directional genome walking can be performed using a locus-specific primer and the walker primer and Phi29 DNA polymerase-amplified genomic DNA fragments as template. The locus-specific primer will determine the position and direction of the genome walk. Two rounds of successive PCR amplifications by locus-specific and walker primers and their corresponding nested primers effectively amplify the flanking DNA fragments. The desired PCR fragment can be either cloned or sequenced directly using another nested, locus-specific primer. We successfully used this protocol to isolate and sequence 5′ flanking regions/promoters of selected plant genes.     

Stability of sheath blight resistance in transgenic ASD16 rice lines expressing a rice <Emphasis Type="Italic">chi11</Emphasis> gene encoding chitinase

Development of transgenic plants by introducing defense genes is one of the strategies to engineer disease resistance. Transgenic ASD16 rice plants harbouring rice chitinase chi11 gene, belonging to a PR-3 group of defense gene conferring sheath blight (Rhizoctonia solani Kuhn) resistance, were used in this study. Three T₂ homozygous lines (ASD16-4-1-1, 5-1-1, and 6-1-1) were identified from seven putative (T₀) transgenic lines expressing chi11 using Western blotting analysis. The inheritance of sheath blight resistance in those lines was studied over generations. The stability of chi11 expression up to T₄ generation in all the three homozygous lines was proved by Western blot and the stability of sheath blight resistance in the homozygous lines was proved up to T₄ generation using detached leaf and intact leaf sheath assays. Among the three homozygous lines tested, ASD16-4-1-1 showed consistent results in all the generations and gave a better protection against the sheath blight pathogen than the other two lines.     

Glycan mapping of recombinant human follicle stimulating hormone by mass spectrometry

In humans, regulation of reproductive functions are carried out mainly by glycoprotein hormones namely follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH) and chorionic gonadotropin (CG). Since glycans play an important role in binding of gonadotropins with their respective receptors, it is important to identify associated glycans and their pharmacological properties not only for the disease manipulation but also for making more efficacious and safer recombinant versions. With the advancement of mass spectrometry, it is possible to identify minute quantity of associated glycans. Here, we studied the N-glycans of the FSH based on mass spectrometry and report one more complex glycan species in addition to twenty four previously reported glycans. The new glycan was a tetra antennary species that may have important role in binding of FSH with receptor with higher biological activity as well as lower clearance rate and higher half-life.     

Kinetics of biosurfactant production by Pseudomonas aeruginosa strain BS2 from industrial wastes

Summary Batch kinetic studies were carried out on rhamnolipid biosurfactant production from synthetic medium, industrial wastes viz. distillery and whey waste as substrates. The results indicated that the specific growth rates ( _max) and specific product formation rates (V _max) from both the wastes are comparatively better than the synthetic medium, revealing that both the industrial wastes (distillery and whey) can be successfully utilized as substrates for biosurfactant production.     

Biotransformation of ferulic acid and vanillylamine to capsaicin and vanillin in immobilized cell cultures of Capsicum frutescens

Plant tissue culture medium which contained FeEDTA as sole iron source was incubated aseptically in light (16-h photoperiod, 100 mol m^-2 s^-1 PAR) at 20°C without plant tissue. Soluble iron dropped from an initial concentration of 4 mg 1^-1 to less than 0.1 mg 1^-1 in 4 weeks. This occurred in both glass and plastic culture vessels. No loss occurred when medium was incubated at 20°C in darkness. A further experiment showed that soluble iron concentration fell to <0.2 mg 1^-1 in only 4 days but the loss was slower at lower irradiances.Effects of the loss of soluble iron on plantlet growth were assessed by culturing single node stem segments of in vitro potato (Solanum tuberosum L. cv. Arran Banner) plantlets on medium previously exposed to light. Pre-exposure sufficient to reduce soluble iron concentration to <0.1 mg 1^-1 had no inhibitory effect on plantlet development in solidified medium or in liquid medium, except when the liquid medium had been centrifuged before inoculation to remove iron precipitated during pre-exposure to light. The plantlets then became chlorotic.     

Furanoid sugar amino acids as dipeptide mimics in design of analogs of vasoactive intestinal peptide receptor binding inhibitor.

In this study we describe the development of peptidomimetic analogs of the potent vasoactive intestinal peptide receptor binding inhibitor, Leu(1) -Met(2) -Tyr(3) -Pro(4) -Thr(5) -Tyr(6) -Leu(7) -Lys(8) -OH 1, by incorporating furanoid sugar amino acids (SAAs) 2-4 into the molecule. The furanoid SAAs 2-4 were used as dipeptide isosteres to replace Tyr(3) -Pro(4) or Pro(4) -Thr(5) in sequence 1. The resulting analogs 5-9 were tested for their anti-cancer activities in vitro, following the standard MTT assay on a panel of human cancer cell lines. One of the potent analogs, 6a was tested in vivo for tumor regression on primary colon tumor xenografted nude mice. Our experimental results suggest that many of these analogs show either retention or enhancement of biological activity.     

A promising RVO4:Eu3+,Li+@SiO2 (R = Gd,Y and Gd/Y) red‐emitting phosphor with improved luminescence (cd/m2) and colour purity for optical display applications

Red emission intensity was optimized in three stages, by investigating the effects of: (i) host composition (Gd, Y and Gd/Y), (ii) codoping Li⁺ as a sensitizer and, finally, (iii) with a SiO₂ shell coating as a protecting layer. Lanthanide vanadate powder phosphors were synthesized using a modified colloidal precipitation technique. The effects of SiO₂ coating on phosphor particles were characterized using scanning electron microscopy (SEM)‐EDAX, transmission electron microscopy (TEM), Fourier transform infrared (FTIR) and photoluminescence (PL) measurements. An improvement in the PL intensity on Li codoping was due to improved crystallinity, which led to higher oscillating strengths for the optical transitions, and also a lowering of the inversion symmetry of Eu³⁺ ions. Red emission intensity due to ⁵D₀ → ⁵D₂ transition of the phosphor Y_0.94VO₄:Eu³⁺_0.05,Li⁺_0.01 was enhanced by 22.28% compared with Y_0.95VO₄:Eu³⁺_0.05, and was further improved by 58.73% with SiO₂ coating. The luminescence intensity (I) and colour coordinates (x, y) of the optimized phosphor Y_0.94VO₄:Eu³⁺_0.05,Li⁺_0.01@SiO₂, where I = 13.07 cd/m² and (x = 0.6721, y = 0.3240), were compared with values for a commercial red phosphor (Y₂O₂S:Eu³⁺), where I = 27 cd/m² and (x = 0.6522, y = 0.3437). The measured colour coordinates are superior to those of the commercial red phosphor, and moreover, match well with standard NTSC values (x = 0.67, y = 0.33). Copyright © 2015 John Wiley & Sons, Ltd.