Rapid microwave-assisted CNBr cleavage of bead-bound peptides

Large libraries of peptides, cyclic peptides, and other molecules are standard tools for the discovery of drugs, molecular probes, and affinity reagents. In particular, one-bead-one-compound (OBOC) libraries,(1) prepared by the split-and-mix method,(2) provide access to a broad chemical space with a minimum of reagents. Once such a library has been screened against the target of interest, the chemical identity of the library elements on the hit beads is identified. For peptide libraries and their variants, mass spectrometry (MS) based peptide sequencing provides the most rapid method for such analysis. OBOC libraries are constructed in a number of ways to facilitate MS analysis,(3-5) but one common feature is that the peptide must be cleaved from the bead prior to being introduced into the mass spectrometer. While a number of chemical(6) and photochemical(7) cleavage strategies have been developed, the most common strategy is to incorporate a CNBr-cleavable methionine-linker group at the C-terminus of the peptide.(8) CNBr cleavage has also been widely used in proteomics to cleave proteins.(9) With such chemistry, up to 100 beads from an OBOC peptide library can be sequenced in a 24 h period.(10) A large fraction of that time, however, is devoted to the CNBr cleavage step. Standard literature protocols describe CNBr cleavage as requiring between 12 and 24 h, using 20−30 μL of 0.25 M CNBr in 70% aqueous formic acid at room temperature.(11) Although the CNBr cleavage time may be reduced to 2−4 h at elevated temperatures (47 °C), significant side-products may be generated.(12) All reports that we have found that describe CNBr cleavage chemistry from single beads have used the same conditions as for proteomics, although the two chemical processes are not necessarily equivalent

Rubisco activity and gene expression of tropical tree species under light stress

Tropical rain forests contain an ecologically and physiologically diverse range of vegetation and habitats. Sun-acclimated plants can be divided into two groups, shade-tolerant and shade-intolerant, according to the plant’s physiological and genetic responses. Some tropical species have potential capacity for light damage in a shaded environment as well as shade-tolerance to compensate for the impaired light harvesting complex. In particular, ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) is regulated by the Calvin cycle, which participated in protein synthesis. Rubisco plays a role in CO2 fixation, which helps supply the energy to regulate Rubisco for ribulose 1,5-bisphosphate (RuBP) reduction. Light intensity is associated with the photosynthetic rate and genetic response to moderate growth environments.Keywords: Gene expression, growth, light intensity, Rubisco activityAfrican Journal of Biotechnology Vol. 12(20), pp. 2764-276

Synthesis and bioactivity of a conjugate composed of green tea catechins and hyaluronic acid

(-)-Epigallocatechin-3-gallate (EGCG) is a green tea polyphenol that has several biological activities, including anti-cancer activity and anti-inflammation. Hyaluronic acid (HA) is a naturally-occurring polysaccharide that is widely used as a biomaterial for drug delivery and tissue engineering due to its viscoelastic, biocompatible and biodegradable properties. By conjugating HA with EGCG, the resulting HA-EGCG conjugate is expected to exhibit not only the inherent properties of HA but also the bioactivities of EGCG. Toward this end, we report the synthesis of an amine-functionalized EGCG as an intermediate compound for conjugation to HA. EGCG was reacted with 2,2-diethoxyethylamine (DA) under acidic conditions, forming ethylamine-bridged EGCG dimers. The EGCG dimers were composed of four isomers, which were characterized by HPLC, high-resolution mass spectrometry and NMR spectroscopy. The amine-functionalized EGCG dimers were conjugated to hyaluronic acid (HA) through the formation of amide bonds. HA-EGCG conjugates demonstrated several bioactivities which were not present in unmodified HA, including resistance to hyaluronidase-mediated degradation, inhibition of cell growth and scavenging of radicals. The potential applications of HA-EGCG conjugates are discussed

The effect of various design codes and dynamic magnification on buildings with torsional irregularity

Seismic provisions have utilized design eccentricities to reduce planar irregularities in lateral stiffness of buildings. In calculating a design eccentricity, the torsional amplification factor may be applied either to accidental eccentricity or to both inherent and accidental eccentricities according to design codes. In this paper, different code provisions and their impact on torsional responses of buildings are investigated using example buildings with various aspect ratios and inherent eccentricities. It was found that the design eccentricity in KBC-2009 using torsional amplification factor for only accidental eccentricity reflects the dynamic magnification more accurately than that in KBC-2006 using this factor for both inherent and accidental eccentricity. And dynamic magnification of a torsionally imbalanced building is affected by the size of seismic design force of response spectrum analysis than design eccentricity of equivalent static analysis in KBC-2009. In other words, design eccentricity including torsional amplification factor in KBC-2009 do not reflect the dynamic magnification accurately

Candida Arthritis after Arthroscopic Arthroplasty in a Patient without Predisposing Factors

Because candidiasis is usually associated with immunosuppression, candida arthritis in an immunocompetent patient is rare. The symptoms of candidiasis are similar to bacterial infections, tuberculosis, and autoimmune diseases. In our patient with no predisposing factors, candida arthritis was initially excluded because the probability of occurrence was low. The patient had no leukocytosis, the acid-fast bacteria (AFB) stain was negative, and the autoimmune antibody screen was negative. After Candida parapsilosis was cultured in the synovial fluid, the patient was treated with amphotericin B (0.7 mg/kg/day) and oral fluconazole (400 mg/day). The treatment was successful and there were no side effects of the medications

Thin cell fringe-field-switching liquid crystal display with a chiral dopant

A fringe-field-switching (FFS) liquid crystal display (LCD) using a thin cell doped with a reverse-handed chiral compound is proposed. Such a FFS LCD exhibits a fast response time (similar to 8 ms), high transmittance ( \u3e 90%), low operating voltage (5 V(rms)), and intrinsically wide viewing angle. Its application for LCD televisions in order to reduce image blurring is emphasized. (C) 2008 American Institute of Physics

Bucillamine prevents cisplatin-induced ototoxicity through induction of glutathione and antioxidant genes.

Bucillamine is used for the treatment of rheumatoid arthritis. This study investigated the protective effects of bucillamine against cisplatin-induced damage in auditory cells, the organ of Corti from postnatal rats (P2) and adult Balb/C mice. Cisplatin increases the catalytic activity of caspase-3 and caspase-8 proteases and the production of free radicals, which were significantly suppressed by pretreatment with bucillamine. Bucillamine induces the intranuclear translocation of Nrf2 and thereby increases the expression of γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GSS), which further induces intracellular antioxidant glutathione (GSH), heme oxygenase 1 (HO-1) and superoxide dismutase 2 (SOD2). However, knockdown studies of HO-1 and SOD2 suggest that the protective effect of bucillamine against cisplatin is independent of the enzymatic activity of HO-1 and SOD. Furthermore, pretreatment with bucillamine protects sensory hair cells on organ of Corti explants from cisplatin-induced cytotoxicity concomitantly with inhibition of caspase-3 activation. The auditory-brainstem-evoked response of cisplatin-injected mice shows marked increases in hearing threshold shifts, which was markedly suppressed by pretreatment with bucillamine in vivo. Taken together, bucillamine protects sensory hair cells from cisplatin through a scavenging effect on itself, as well as the induction of intracellular GSH

Transcriptional regulatory networks underlying the reprogramming of spermatogonial stem cells to multipotent stem cells

Spermatogonial stem cells (SSCs) are germline stem cells located along the basement membrane of seminiferous tubules in testes. Recently, SSCs were shown to be reprogrammed into multipotent SSCs (mSSCs). However, both the key factors and biological networks underlying this reprogramming remain elusive. Here, we present transcriptional regulatory networks (TRNs) that control cellular processes related to the SSC-to-mSSC reprogramming. Previously, we established intermediate SSCs (iSSCs) undergoing the transition to mSSCs and generated gene expression profiles of SSCs, iSSCs and mSSCs. By comparing these profiles, we identified 2643 genes that were up-regulated during the reprogramming process and 15 key transcription factors (TFs) that regulate these genes. Using the TF-target relationships, we developed TRNs describing how these TFs regulate three pluripotency-related processes (cell proliferation, stem cell maintenance and epigenetic regulation) during the reprogramming. The TRNs showed that 4 of the 15 TFs (Oct4/Pou5f1, Cux1, Zfp143 and E2f4) regulated cell proliferation during the early stages of reprogramming, whereas 11 TFs (Oct4/Pou5f1, Foxm1, Cux1, Zfp143, Trp53, E2f4, Esrrb, Nfyb, Nanog, Sox2 and Klf4) regulated the three pluripotency-related processes during the late stages of reprogramming. Our TRNs provide a model for the temporally coordinated transcriptional regulation of pluripotency-related processes during the SSC-to-mSSC reprogramming, which can be further tested in detailed functional studies.111Ysciescopuskc