Stimulated indole alkaloid release from Catharanthus roseus immobilized cultures. Initial studies.

Vacuolar sequestration of valuable secondary metabolites remains the major limitation to the use of immobilization technology for large scale plant-cell-based bioprocesses, which otherwise may be a more efficient culture system than suspension for this biomass. In this initial study, the release of indole alkaloids produced by immobilized Catharanthus roseus cells cultured in Zenk's Alkaloid Production Medium was evaluated. Unstimulated alkaloid release in immobilized cultures reached levels of 10 to 50% of total production or 3 to 100% of known alkaloid content (30 to 4700 micrograms l-1), which was higher than that found for suspension cultures of the cell line used (10 to 25% of total production) without apparent cell lysis. Modifications of the medium pH value of immobilized cultures were explored in order to improve this release. Periodical additions of acid (HCl 0.1 N) or base (KOH 0.1 N) solutions (2% v/v) to different cultures resulted in rapid (less than 3 h) and transient variations in extracellular pH value from 5.5 to 4.3, and 5.8 to 8.5, respectively. In both cases, these variations provoked significant increase in total alkaloid (from approximately 5-10 mg l-1 to 15 mg l-1), ajmalicine (from 0 to approximately 0.29 mg l-1) and serpentine (from 0 to approximately 0.20 mg l-1) release, without apparent cell lysis or decrease in the culture viability. This product release was estimated to represent 100% of alkaloids produced.     

Anatomical variation of orthotropic elastic moduli of the proximal human tibia

The anatomical variation of orthotropic elastic moduli of the cancellous bone from three human proximal tibiae was investigated using an ultrasonic technique. With this technique, it was possible to measure three orthogonal elastic moduli and three shear moduli from cubic specimens of cancellous bone as small as 8 mm per side. Correlation with mechanical tensile testing has shown this technique to offer a precise measure of cancellous modulus (Eten = 0.94Eult + 144.6 MPa, r2 = 0.96, n = 34). The cancellous bone of the proximal tibia was found to be very inhomogeneous, with the axial modulus ranging between 340 and 3350 MPa. A course map is presented, showing measured Young's moduli as a function of anatomical position. The anisotropy of the cancellous bone, determined by the relative differences between the three orthogonal moduli, was shown to be relatively constant over the entire range of cancellous densities tested. The relationship between the axial elastic modulus and the apparent density was found to be approximately linear, as reported by others for proximal tibial cancellous bone.     

Association of adenovirus type 2 early proteins with a soluble complex that synthesizes adenovirus DNA in vitro

A soluble Ad2 DNA synthesizing complex was prepared from Ad2-infected KB cell nuclei and purified by exclusion chromatography on a BioGel A-50m column. The purified complex was able to synthesize DNA from all regions of the virus genome, as indicated by $\text{Eco}$RI restriction endonuclease analysis of $\text{in vitro}$ labeled DNA. Experiments were performed to identify Ad2-induced early polypeptides present in the complex. Ad2-infected and mock-infected cells were labeled with [³⁵S]methionine 7–10 h postinfection, then incubated for 8 h to allow the ³⁵S-labeled early polypeptides to become associated with the complex. The polypeptides in the purified complex and each of the cell fractions were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The major components of the purified complex were the 73K DNA binding phosphoprotein and 11K, two adenovirus 2-induced early polypeptides. The 11K has a preferred nuclear location. Small quantities of other Ad2-induced early proteins, 21K, 15K, and possibly 8.3K were also associated with the complex.     

Direct fluorometric analysis of benzo(a)pyrene metabolite formation by mouse liver microsomes

We have examined the metabolites produced by in vitro incubation of benzo(a)pyrene with 3-methylcholanthrene-induced mice liver microsomes. Our objective was to observe directly a possible difference in microsomal enzyme systems of animal models having different susceptibility to chemical carcinogens. The metabolites produced by the two animal models,$\text{C57BL}\text{6J}$ and $\text{DBA}\text{2}$ mice, were analyzed by a highly sensitive, “three-dimensional” fluorescence plotting technique. The fluorescence spectra of the total ethyl acetate-soluble metabolites clearly indicate that the metabolites produced by $\text{DBA}\text{2}$ enzymes were predominantly monohydroxylated benzo(a)pyrene while those produced by the liver microsomes of $\text{C57BL}\text{6J}$ were highly enriched with the 7,8-dihydrodihydroxybenzo(a)pyrene type.     

From proteomics toward systems biology: integration of different types of proteomics data into network models

Living organisms are comprised of various systems at different levels, i.e., organs, tissues, and cells. Each system carries out its diverse functions in response to environmental and genetic perturbations, by utilizing biological networks, in which nodal components, such as, DNA, mRNAs, proteins, and metabolites, closely interact with each other. Systems biology investigates such systems by producing comprehensive global data that represent different levels of biological information, i.e., at the DNA, mRNA, protein, or metabolite levels, and by integrating this data into network models that generate coherent hypotheses for given biological situations. This review presents a systems biology framework, called the 'Integrative Proteomics Data Analysis Pipeline' (IPDAP), which generates mechanistic hypotheses from network models reconstructed by integrating diverse types of proteomic data generated by mass spectrometry-based proteomic analyses. The devised framework includes a serial set of computational and network analysis tools. Here, we demonstrate its functionalities by applying these tools to several conceptual examples.     

Evaluation of GABA Production and Probiotic Activities of Enterococcus faecium BS5

Probiotics and Antimicrobial Proteins - Gamma-aminobutyric acid (GABA) is a principal inhibitory neurotransmitter in the central nervous system and is produced by irreversible decarboxylation of...     

In vitro antiviral activity of phlorotannins isolated from Ecklonia cava against porcine epidemic diarrhea coronavirus infection and hemagglutination

Despite the prepdominat agent causing severe entero-pathogenic diarrhea in swine, there are no effective therapeutical treatment of porcine epidemic diarrhea virus (PEDV). In this study, we evaluated the antiviral activity of five phlorotannins isolated from Ecklonia cava (E. cava) against PEDV. In vitro antiviral activity was tested using two different assay strategies: (1) blockage of the binding of virus to cells (simultaneous-treatment assay) and (2) inhibition of viral replication (post-treatment assay). In simultaneous-treatment assay, compounds 2–5 except compound 1 exhibited antiviral activities of a 50% inhibitory concentration (IC₅₀) with the ranging from 10.8 ± 1.4 to 22.5 ± 2.2 μM against PEDV. Compounds 1–5 were completely blocked binding of viral spike protein to sialic acids at less than 36.6 μM concentrations by hemagglutination inhibition. Moreover, compounds 4 and 5 of five phlorotannins inhibited viral replication with IC₅₀ values of 12.2 ± 2.8 and 14.6 ± 1.3 μM in the post-treatment assay, respectively. During virus replication steps, compounds 4 and 5 exhibited stronger inhibition of viral RNA and viral protein synthesis in late stages (18 and 24 h) than in early stages (6 and 12 h). Interestingly, compounds 4 and 5 inhibited both viral entry by hemagglutination inhibition and viral replication by inhibition of viral RNA and viral protein synthesis, but not viral protease. These results suggest that compounds isolated from E. cava have strong antiviral activity against PEDV, inhibiting viral entry and/or viral replication, and may be developed into natural therapeutic drugs against coronavirus infection.     

Use of somatic cell nuclear transfer to study meiosis in female cattle carrying a sex-dependent fertility-impairing X-chromosome abnormality

Animal models have played an important part in establishing our knowledge base on reproduction, development, and the occurrence and impact of chromosome abnormalities. Translocations involving the X chromosome and an autosome are unique in that they elicit sex-dependent infertility, with male carriers rendered sterile by synaptic anomalies during meiosis, whereas female carriers conceive but repeatedly abort. Until now the limited access to relevant fetal oocytes has precluded direct study of meiotic events in female carriers. Because somatic cell nuclear transfer (SCNT) circumvents meiotic problems associated with fertility disturbances in translocation carriers, we used SCNT to generate embryos, fetuses, and calves from a cell line derived from a deceased subfertile X-autosome translocation carrier cow to study the meiotic configurations in carrier oocytes. Data from 33 replicates involving 2470 oocyte-donor-cell complexes were assessed for blastocyst development and of these, 42 blastocysts were transferred to 21 recipients. Fourteen pregnancies were detected on day 35 of gestation. One of these was sacrificed for ovary retrieval on day 94 and three went to term. Features of oocytes from the fetal ovary and from the newborn ovaries were examined. Of the pachytene spreads analyzed, 16%, 82%, and 1.5% exhibited quadrivalent, trivalent/univalent, and bivalent/univalent/univalent structures, respectively, whereas among the diakinesis/metaphase I spreads, 16% ring, 75% chain, and 8.3% bivalent/bivalent configurations were noted, suggesting that the low fertility among female carriers may be related to synaptic errors in a predominant proportion of oocytes. Our results indicate that fibroblasts carrying the X-autosome translocation can be used for SCNT to produce embryos, fetuses, and newborn clones to study such basic aspects of development as meiosis and to generate carriers that cannot easily be reproduced by conventional breeding.     

Assessment of substrate specificity of hepatitis G virus NS3 protease by a genetic method

The RNA genome of hepatitis G virus (HGV) encodes a large polyprotein that is processed to mature proteins by viral-encoded proteases. The HGV NS3 protease is responsible for the cleavage of the HGV polyprotein at four different locations. No conserved sequence motif has been identified for the cleavage sites of the NS3 protease. To determine the substrate specificity of the NS3 protease, amino acid sequences cleaved by the NS3 protease were obtained from randomized sequence libraries by using a screening method referred to as GASP (Genetic Assay for Site-specific Proteolysis). Based on statistical analyses of the obtained cleavable sequences, a consensus substrate sequence was deduced: Gln-Glu-Thr-Leu-Val downward arrow Ser, with the scissile bond located between Val and Ser. The relevance of this peptide as a cleavable substrate was further supported by molecular modeling of the NS3 protease. Our result would provide an insight on the molecular activity of the NS3 protease and may be useful for the design of substrate-based inhibitors.