The use of mutagens to increase the efficiency of the androgenic progeny production in <Emphasis Type="Italic">Solanum nigrum</Emphasis>

Pollen embryogenesis was successfully induced in Solanum nigrum L. (2n=6×=72). Stimulation of androgenesis expressed as the frequency of androgenic responsive anthers was observed after 10 and 20 mM ethyl methanesulphonate (EMS), 10 and 20 mM sodium azide (NaN₃) and 0.2 mM N-nitroso-N-methylurea (MNU) treatment applied on seeds for 24 h. The frequency of androgenesis on the medium with sucrose was higher than on the medium with maltose. Androgenic regenerants originated also in the anthers collected from donor plants where survival after mutagenic treatment was lower than 50 %. Green haploid (3x), aneuploid (to 8x) and dihaploid (6x) plants were obtained. The high frequency of aneuploids among androgenic plants is explained by cell division irregularities in microsporial calli.     

Evidence for proprotein convertase activity in the endoplasmic reticulum/early Golgi

Processing of precursor proteins by the proprotein convertases is thought to occur mainly in the trans-Golgi network or post-Golgi compartments. Such cleavage is inhibited by the prosegment of the convertases. During our studies of the use of the inhibitory prosegment of PC1, we noticed that a construct containing the prosegment fused to the C-terminal secretory granule sorting domain was cleaved in the endoplasmic reticulum (ER) at a pair of basic residues, best recognized by furin and PC7. This was further confirmed when this construct was fused at the C-terminus with a KDEL ER-retention signal. This suggests that the convertases could cleave some substrates within the ER, possibly by displacing the inhibitory prosegment associated with them.     

N-terminal glycine-specific protein conjugation catalyzed by microbial transglutaminase

Here, we report the N-terminal glycine (Gly) residue of a target protein can be a candidate primary amine for site-specific protein conjugation catalyzed by microbial transglutaminase (MTG) from Streptomyces mobaraensis. Gly5-enhanced green fluorescent protein (EGFP) (EGFP with five additional Gly residues at its N-terminus) was cross-linked with Myc-dihydrofolate reductase (DHFR) (DHFR with the myc epitope sequence at its N-terminus) to yield DHFR-EGFP heterodimers. The reactivities of additional peptidyl linkers were investigated and the results obtained suggested that at least three additional Gly residues at the N-terminus were required to yield the EGFP-DHFR heterodimeric form. Site-directed mutagenesis analysis revealed marked preference of MTG for amino acids adjacent to the N-terminal Gly residue involved in the protein conjugation. In addition, peptide-protein conjugation was demonstrated by MTG-catalyzed N-terminal Gly-specific modification of a target protein with the myc epitope peptide.     

Polyomavirus EGFP-pseudocapsids: analysis of model particles for introduction of proteins and peptides into mammalian cells

A vector for preparation of mouse polyomavirus capsid-like particles for transfer of foreign peptides or proteins into cells was constructed. Model pseudocapsids carrying EGFP fused with the C-terminal part of the VP3 minor protein (EGFP-VLPs) have been prepared and analysed for their ability to be internalised and processed by mouse cells and to activate mouse and human dendritic cells (DC) in vitro. EGFP-VLPs entered mouse epithelial cells, fibroblasts and human and mouse DC efficiently and were processed by both, lysosomes and proteasomes. Surprisingly, they did not induce upregulation of DC co-stimulation molecules or maturation markers in vitro; however, they did induce interleukin 12 secretion.     

Structural analysis of biological aliphatic compounds using surface-enhanced Fourier transform Raman spectroscopy

The surface-enhanced Raman scattering (SERS) technique for Fourier transform Raman spectrometry is employed to reveal the chemical structure of biological aliphatic compounds consisting of folded, long aliphatic chains. The structural analysis is performed via the measurements of the accordion-vibration modes generated in the ordered, long aliphatic chain. The SERS spectra after subtraction of a background spectrum give segment lengths that are almost perfectly consistent with the chemical structures studied by mass spectrometry. The agreement of the SERS results with those of mass spectrometry suggests the positions of kinks in the long hydrocarbon chain. The combination technique of SERS and mass spectrometry is useful to discuss the structure of folded, long biological lipids.     

超声彩色血流成像的计算机快速仿真方法

研究超声彩色血流成像的快速仿真方法,克服原先仿真方法非常耗时的缺点。方法超声彩色血流成像计算机仿真中,血流信号是对成像区间内所有点散射体的回波信号累加而得到的。通过引入新的等效散射体模型,可以大大降低散射体的密度,从而减少计算回波信号所需时间。在计算机上用Matlab编程来进行仿真实验,对以往仿真方法和基于等效散射体模型方法的性能进行比较。结果实验表明:基于等效散射体模型的仿真,在保证相同流速精度的前提下,仿真速度比传统方法提高了10倍以上。结论基于等效散射体模型的仿真方法能极大地提高超声彩色血流成像的仿真速度,可以为超声彩色血流成像的方法研究提供便利。     

Engineering of an Orthogonal Aminoacyl-tRNA Synthetase for Efficient Incorporation of the Non-natural Amino Acid O-Methyl-L-tyrosine using Fluorescence-based Bacterial Cell Sorting

We describe a strategy for the rapid selection of mutant aminoacyl-tRNA synthetases (aaRS) with specificity for a novel amino acid based on fluorescence-activated cell sorting of transformed Escherichia coli using as reporter the enhanced green fluorescent protein (eGFP) whose gene carries an amber stop codon (TAG) at a permissive site upstream of the fluorophore. To this end, a one-plasmid expression system was developed encoding an inducible modified Methanocaldococcus jannaschii (Mj) tyrosyl-tRNA synthetase, the orthogonal cognate suppressor tRNA, and eGFP_UAG in an individually regulatable fashion. Using this system a previously described aaRS with specificity for O-methyl-L-tyrosine (MeTyr) was engineered for 10-fold improved incorporation of the foreign amino acid by selection from a mutant library, prepared by error-prone as well as focused random mutagenesis, for MeTyr-dependent eGFP fluorescence. Applying alternating cycles of positive and negative fluorescence-activated bacterial cell sorting in the presence or in the absence, respectively, of the foreign amino acid was crucial to select for high specificity of MeTyr incorporation. The optimized synthetase was used for the preparative expression of a modified uvGFP carrying MeTyr at position 66 as part of its fluorophore. This biosynthetic protein showed quantitative incorporation of the non-natural amino acid, as determined by mass spectrometry, and it revealed a unique emission spectrum due to the altered chemical structure of its fluorophore. Our combined genetic/selection system offers advantages over earlier approaches that relied wholly or in part on antibiotic selection schemes, and it should be generally useful for the engineering and optimization of orthogonal aaRS/tRNA pairs to incorporate non-natural amino acids into recombinant proteins.     

Optimization of ultrasound contrast agents with computational models to improve selection of ligands and binding strength

Diagnosis of cardiovascular disease is currently limited by the testing modality. Serum tests for biomarkers can provide quantification of severity but lack the ability to localize the source of the cardiovascular disease, while imaging technology such as angiography and ultrasound can only determine areas of reduced flow but not the severity of tissue ischemia. Targeted imaging with ultrasound contrast agents offers the ability to locally image as well as determine the degree of ischemia by utilizing agents that will cause the contrast agent to home to the affected tissue. Ultrasound molecular imaging via targeted microbubbles (MB) is currently limited by its sensitivity to molecular markers of disease relative to other techniques (e.g., radiolabeling). We hypothesize that computational modeling may provide a useful first approach to maximize microbubble binding by defining key parameters governing adhesion. Adhesive dynamics (AD) was used to simulate the fluid dynamic and stochastic molecular binding of microbubbles to inflamed endothelial cells. Sialyl Lewis^X (sLe^x), P‐selectin aptamer (PSA), and ICAM‐1 antibody (abICAM) were modeled as the targeting receptors on the microbubble surface in both single‐ and dual‐targeted arrangements. Microbubble properties (radius [R_c], kinetics [k_f, k_r], and densities of targeting receptors) and the physical environment (shear rate and target ligand densities) were modeled. The kinetics for sLe^x and PSA were measured with surface plasmon resonance. R_c, shear rate, and densities of sLe^x, PSA, or abICAM were varied independently to assess model sensitivity. Firm adhesion was defined as MB velocity <2% of the free stream velocity. AD simulations revealed an optimal microbubble radius of 1–2 µm and thresholds for (>10² s^?1) and (<10^?3 s^?1) for firm adhesion in a multi‐targeted system. State diagrams for multi‐targeted microbubbles suggest sLe^x and abICAM microbubbles may require 10‐fold more ligand to achieve firm adhesion at higher shear rates than sLe^x and PSA microbubbles. The AD model gives useful insight into the key parameters for stable microbubble binding, and may allow flexible, prospective design, and optimization of microbubbles to enhance clinical translation of ultrasound molecular imaging. Biotechnol. Bioeng. 2010;107: 854–864. © 2010 Wiley Periodicals, Inc.     

Effects of infection of EGFP-expressing Escherichia coli on haemocytes in Ciona intestinalis

The effects of infection of EGFP-expressing Escherichia coli on the haemocytes of the ascidian Ciona intestinalis were investigated. The results showed that THC of the infected individuals changed significantly. Hyaline amoebocytes phagocytosed E. coli in 5 min and excreted lysosome particles that attached to the surface of the bacteria. Granular amoebocytes released lots of particles for humoral immunity while stem-cell-like haemocytes remained intact. With the increase in THC, the stem-cell-like haemocytes showed division and proliferation. A small portion of hyaline amoebocytes was at early apoptosis stage 1 h after infection and typical apoptosis bodies emerged in granular amoebocytes. A few of the infected haemocytes showed DNA damage using SCGE assay. Flow cytometry analysis revealed an obvious apoptosis peak in infected haemocytes. In conclusion, apoptosis was found to be an important immune response of ascidian haemocytes response to bacterial infection. To our best knowledge, this is the first report of the occurrence of apoptosis of haemocytes in ascidians.     

Monoclonal antibody A7 coupled to magnetic particles as a contrast enhancing agent for magnetic resonance imaging of human colorectal carcinoma

Background: Local recurrence, the most frequent pattern of recurrence of rectal carcinoma, is almost always fatal. The difficulty of diagnosing local recurrence contributes importantly to the poor prognosis. Methods: We coupled monoclonal antibody (Mab) A7, which reacts specifically with human colorectal carcinoma, to ferromagnetic lignosite (FML) particles to distinguish rectal carcinoma from other tissues by magnetic resonance (MR) imaging. We examined retention of immunoreactivity by the A7-FML complexes in vitro, and also their distribution in vivo according to radiolabeling and MR imaging when injected into nude mice bearing human colorectal carcinoma xenografts. Results: A7-FML retained binding activity nearly identical to that of Mab A7. Significantly more ¹²⁵I-labeled A7-FML accumulated in engrafted tumors than did ¹²⁵I-labeled normal mouse IgG-FML complexes (P<0.05). A7-FML disappeared rapidly from the blood. Normal tissues accumulated less ¹²⁵I-labeled A7-FML than tumors; this accumulation decreased linearly with time. In MR imaging, signal intensity was reduced in the tumor by the injection of A7-FML. Conclusions: A7-FML is potentially useful as a MR contrast enhancing agent for human colorectal carcinoma xenografts implanted subcutaneously.