A centrifugation-free high-throughput protein purification system using in-line microfluidization

The majority of current high-throughput protein purification protocols include rate-limiting centrifugation steps. A column and nozzle assembly was developed that can be used in-line with microfluidization for the purification of bacterially-overexpressed, His-tagged proteins directly from bacterial cultures. Yields and purity are comparable with standard protocols. This large-scale protein purification protocol is easy to use and widely-applicable.     

Liquid overlaying improves somatic embryogenesis in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Somatic embryos were induced from in vitro germinating seed-hypocotyls of Catharanthus roseus. The process of embryogenesis has been categorized into a few distinct stages (induction, proliferation, maturation and germination) in which liquid overlaying at varying levels 0 ml (T₀), 0.25 ml (T₁), 0.5 ml (T₂)_, 0.75 ml (T₃) and 1.0 ml (T₄) was applied on solid medium. It was found that liquid overlaying improved proliferation; maturation, germination of embryos in C. roseus. In proliferation stage, particularly in T₂, torpedo embryo number increased significantly (i.e. 129.6%) as compared to control. Liquid overlaying (T₂, T₃ and T₄) also improved embryo maturation and showed early germination even in maturation medium. It also accelerated normal embryo germination frequency particularly in treatment with T₂ and shortened ‘embryo—plantlet’ recovery time. Biochemical analyses revealed more proline, protein and amino acid with increasing level of liquid overlaying as it improved embryo induction, development and faster germination.     

A dominant-negative mutant of androgen receptor coregulator ARA54 inhibits androgen receptor-mediated prostate cancer growth.

The ligand-bound androgen receptor (AR) regulates target genes via a mechanism involving coregulators such as androgen receptor-associated 54 (ARA54). We investigated whether the interruption of the AR coregulator function could lead to down-regulation of AR activity. Using in vitro mutagenesis and a yeast two-hybrid screening assay, we have isolated a mutant ARA54 (mt-ARA54) carrying a point mutation at amino acid 472 changing a glutamic acid to lysine, which acts as a dominant-negative inhibitor of AR transactivation. In transient transfection assays of prostate cancer cell lines, the mt-ARA54 suppressed endogenous mutated AR-mediated and exogenous wild-type AR-mediated transactivation in LNCaP and PC-3 cells, respectively. In DU145 cells, the mt-ARA54 suppressed exogenous ARA54 but not other coregulators, such as ARA55-enhanced or SRC-1-enhanced AR transactivation. In the LNCaP cells stably transfected with the plasmids encoding the mt-ARA54 under the doxycycline inducible system, the overexpression of the mt-ARA54 inhibited cell growth and endogenous expression of prostate-specific antigen. Mammalian two-hybrid assays further demonstrated that the mt-ARA54 can disrupt the interaction between wild-type ARA54 molecules, suggesting that ARA54 dimerization or oligomerization may play an essential role in the enhancement of AR transactivation. Together, our results demonstrate that a dominant-negative AR coregulator can suppress AR transactivation and cell proliferation in prostate cancer cells. Further studies may provide a new therapeutic approach for blocking AR-mediated prostate cancer growth.     

Identification of somatic embryogenesis (SE) related proteins through label-free shotgun proteomic method and cellular role in Catharanthus roseus (L.) G. Don

Plant Cell, Tissue and Organ Culture (PCTOC) - Somatic embryogenesis (SE) is an intricate in vitro multi-step biotechnological tool used to develop embryos/plants from a single or a group of...     

Tim-3 negatively regulates cytotoxicity in exhausted CD8+ T cells in HIV infection

Cytotoxic CD8(+) T cells (CTLs) contain virus infections through the release of granules containing both perforin and granzymes. T cell 'exhaustion' is a hallmark of chronic persistent viral infections including HIV. The inhibitory regulatory molecule, T cell Immunoglobulin and Mucin domain containing 3 (Tim-3) is induced on HIV-specific T cells in chronic progressive infection. These Tim-3 expressing T cells are dysfunctional in terms of their capacities to proliferate or to produce cytokines. In this study, we evaluated the effect of Tim-3 expression on the cytotoxic capabilities of CD8(+) T cells in the context of HIV infection. We investigated the cytotoxic capacity of Tim-3 expressing T cells by examining 1) the ability of Tim-3(+) CD8(+) T cells to make perforin and 2) the direct ability of Tim-3(+) CD8(+) T cells to kill autologous HIV infected CD4(+) target cells. Surprisingly, Tim-3(+) CD8(+) T cells maintain higher levels of perforin, which was mainly in a granule-associated (stored) conformation, as well as express high levels of T-bet. However, these cells were also defective in their ability to degranulate. Blocking the Tim-3 signalling pathway enhanced the cytotoxic capabilities of HIV specific CD8(+) T cells from chronic progressors by increasing; a) their degranulation capacity, b) their ability to release perforin, c) their ability to target activated granzyme B to HIV antigen expressing CD4(+) T cells and d) their ability to suppress HIV infection of CD4(+) T cells. In this latter effect, blocking the Tim-3 pathway enhances the cytotoxcity of CD8(+) T cells from chronic progressors to the level very close to that of T cells from viral controllers. Thus, the Tim-3 receptor, in addition to acting as a terminator for cytokine producing and proliferative functions of CTLs, can also down-regulate the CD8(+) T cell cytotoxic function through inhibition of degranulation and perforin and granzyme secretion.     

Influence of freezing and non-freezing temperature on somatic embryogenesis and vinblastine production in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don.

Catharanthus roseus (L.) G. Don. (Apocynaceae) is an important dicotyledonous medicinal plant. It produces vinblastine and vincristine, two alkaloids that are being used against a variety of cancers. In the present study, the freezing (−196, 4, 15°C) and non-freezing (25°C) temperature was imposed on embryogenic cultures, and later in vitro embryogeny and vinblastine production in C. roseus was studied. Somatic embryo (SE) production was maximum at 15°C, but the SE maturation was high at 4°C. The SEs, grown at 25°C, showed highest germination and plantlet conversion. Quantitative estimation of vinblastine was carried out using high-performance liquid chromatography in various in vitro raised tissues (embryogenic callus), embryo stages (proliferated, matured and germinated embryos)], and SE-derived plantlets (leaf, shoot, root and whole plant) after various freezing- and non-freezing temperature treatments. Vinblastine synthesis was temperature dependent in C. roseus that has been discussed in this present article.     

Somatic embryogenesis and two embryo specific proteins (38 and 33 kD) in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

In the present study, the regeneration pathway, especially the different events of somatic embryogenesis (SE) have been studied morphologically and biochemically in Catharanthus roseus. Firstly, the calluses were induced from different explant sources (hypocotyl, epicotyl and root) by using various auxins. Embryogenic and non-embryogenic calluses were identified based on their morphology, colour and dry weight. Embryogenic callus was later cultivated on MS added with 0.45 μM 2,4-D, 6.62 μM BAP and 1.44 μM GA3 for obtaining various developmental stages of embryos. Different stages of embryos have been assayed for the establishment of marker based embryogenesis, particularly on embryo specific proteins whose presence or absence will ensure a rapid and efficient production of embryos that has a special application to clonal biotechnology. Two embryo specific proteins (38 and 33 kD) have been identified for the first time in C. roseus during torpedo stage of embryogenesis. Besides, multiple shoot formation from in vitro raised emblings was also attempted to examine the role of BAP and kinetin for shoot proliferation. The shoots were rooted with 5.37 μM NAA and 5.71 μM IAA before transplantation.     

Alliin production in various tissues and organs of <Emphasis Type="Italic">Allium sativum</Emphasis> grown under normal and sulphur-supplemented in vitro conditions

Garlic extract contains alliin (S-allyl-l-cysteine sulfoxide), an organosulphur compound that contributes to its therapeutic value and pharmacological importance. Alliin production in plant organs (leaf and root), plantlet, callus (non-embryogenic and embryogenic), embryo (proliferated, matured and germinated embryos), grown under in-vitro conditions was evaluated after 8 weeks. Highest alliin content was recorded in leaves. Clove callus and germinated embryos also showed alliin production. Evaluation of alliin content of in-vitro grown tissues both in normal (control) and sulphur supplemented conditions (4, 8, 16, 32 mg l⁻¹) showed that sulphur treatment at supply of 16 mg l⁻¹ gypsum (CaSO₄) significantly enhanced the production of alliin content in all in-vitro grown tissues and organs. These findings suggest that production of alliin is dependent upon type and age of tissue and can be altered by growth conditions.