Fe(III) Reduction and U(VI) Immobilization by Paenibacillus sp. Strain 300A,Isolated from Hanford 300A Subsurface Sediments

A facultative iron-reducing [Fe(III)-reducing] Paenibacillus sp. strain was isolated from Hanford 300A subsurface sediment biofilms that was capable of reducing soluble Fe(III) complexes [Fe(III)-nitrilotriacetic acid and Fe(III)-citrate] but unable to reduce poorly crystalline ferrihydrite (Fh). However, Paenibacillus sp. 300A was capable of reducing Fh in the presence of low concentrations (2 μM) of either of the electron transfer mediators (ETMs) flavin mononucleotide (FMN) or anthraquinone-2,6-disulfonate (AQDS). Maximum initial Fh reduction rates were observed at catalytic concentrations (<10 μM) of either FMN or AQDS. Higher FMN concentrations inhibited Fh reduction, while increased AQDS concentrations did not. We also found that Paenibacillus sp. 300A could reduce Fh in the presence of natural ETMs from Hanford 300A subsurface sediments. In the absence of ETMs, Paenibacillus sp. 300A was capable of immobilizing U(VI) through both reduction and adsorption. The relative contributions of adsorption and microbial reduction to U(VI) removal from the aqueous phase were ∼7:3 in PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)] and ∼1:4 in bicarbonate buffer. Our study demonstrated that Paenibacillus sp. 300A catalyzes Fe(III) reduction and U(VI) immobilization and that these reactions benefit from externally added or naturally existing ETMs in 300A subsurface sediments.     

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). Download video file.^{(49M, mov)}     

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.Download video file.^{(121M, mp4)}     

Design, synthesis, and studies of small molecule STAT3 inhibitors

A series of small molecule STAT3 inhibitors originally derived from our lead compound STA 21 were synthesized and evaluated. The most potent compound in this series, compound 1, exhibited the same anti-proliferative activities as STA 21 against prostate cancer cell lines that express constitutively active STAT3. Molecular docking showed compound 1 bound to the STAT3beta SH2 domain in a similar manner as STA 21.     

Structure–activity relationship studies of curcumin analogues

Two series of curcumin analogues, a total of twenty-four compounds, were synthesized and evaluated. The most potent compound, compound 23, showed potent growth inhibitory activities on both prostate and breast cancer lines with IC₅₀ values in sub-micromolar range, fifty times more potent than curcumin. Curcumin analogues might be potential anti-tumor agents for breast and prostate cancers.     

Proteome profiling of aging in mouse models: Differential expression of proteins involved in metabolism,transport, and stress response in kidney

Aging is a time‐dependent complex biological phenomenon observed in various organs and organelles of all living organisms. To understand the molecular mechanism of age‐associated functional loss in aging kidneys, we have analyzed the expression of proteins in the kidneys of young (19–22 wk) and old (24 months) C57/BL6 male mice using 2‐DE followed by LC‐MS/MS. We found that expression levels of 49 proteins were upregulated (p ≤ 0.05), while that of only ten proteins were downregulated (p ≤ 0.05) due to aging. The proteins identified belong to three broad functional categories: (i) metabolism (e.g., aldehyde dehydrogenase family, ATP synthase β‐subunit, malate dehydrogenase, NADH dehydrogenase (ubiquinone), hydroxy acid oxidase 2), (ii) transport (e.g., transferrin), and (iii) chaperone/stress response (e.g., Ig‐binding protein, low density lipoprotein receptor‐related protein associated protein 1, selenium‐binding proteins (SBPs)). Some proteins with unknown functions were also identified as being differentially expressed. ATP synthase β subunit, transferrin, fumarate hydratase, SBPs, and albumin are present in multiple forms, possibly arising due to proteolysis or PTMs. The above functional categories suggest specific mechanisms and pathways for age‐related kidney degeneration.     

Co-localisation of membranes and actin in growing infected cells of Glycine max root nodules

The root nodule of Glycine max (L.) Merr. is almost spherical at maturity, and its central tissue consists of infected cells filled with numerous symbiosomes containing bacteroids, interspersed with uninfected cells. During the growth of the nodule, the volume of each infected cell and the number of bacteroids per cell increases, and thus abundant membranes are required for the proliferation of symbiosomes. In expanding infected cells, there are areas adjacent to the nucleus that are devoid of bacteroids, but these areas are filled with numerous membranes and actin filaments, surrounded by endoplasmic reticulum membranes, indicating a perinuclear reservoir of newly formed membranes and a role for actin in delivering membranes to proliferating symbiosomes.     

Structure–activity relationship studies of thalidomide analogs with a taxol-like mode of action

Anti-microtubule agents such as paclitaxel and docetaxel have played an important role in the treatment of cancer for many years. Recently, a small molecule that has a taxol-like mode of action (5HPP-33) was reported. Herein, the detailed structure–activity relationship (SAR) studies of 5HPP-33 analogs that are substituted at the isoindole and phenyl rings are described. Bulky substitutions (such as di-isopropyl groups) on the phenyl ring result in the isoindole and phenyl rings being perpendicular to each other. It was found that this conformation is critical for anti-microtubule activity. These studies have provided valuable information, which will be helpful in the design of more potent analogs.     

Rg3 inhibits gemcitabine-induced lung cancer cell invasiveness through ROS-dependent,NF-κB- and HIF-1α-mediated downregulation of PTX3

PTX3, a member of the long pentraxin subfamily, associated with innate immunity is indispensable for resistance to some cancer. Gemcitabine, an analog of cytosine arabinoside, has shown restrained benefits because of profound chemoresistance. The PTX3 expression on GEM in human lung cancer cells have not yet been clarified; the present study aimed to show reactive oxygen species (ROS) mediatory PTX3 expression through distinct mechanisms. Whereas ginsenoside Rg3 is a herbal medicine with strong antitumor activity. Furthermore, we tested the hypothesis; Rg3 abrogates GEM-induced production of ROS-mediated activation of Akt and extracellular signal-regulated kinase (ERK) pathways and inhibits nuclear piling-up of nuclear factor kappa B (NF-κB) and HIF-1α. On the basis of time and dose-dependent manner, our data demonstrated that GEM-induced PTX3 expression was dependent on ROS generation as it was abrogated by pretreatment of lung cancer cells with the free radical scavenger N-acetyl-l -cysteine. Our data demonstrated that PTX3 upregulation by GEM correlated with the time-dependent escalation of NF-κB and HIF-1α in the nucleus resulted from phosphorylation-induced degradation of IκBα, whereas HIF-1α upregulation was NF-κB-dependent. Increase in ROS expression in lung cancer cells on GEM treatment preceded the nuclear accumulation of NF-κB and HIF-1α and suppression of ROS diminished these effects. ERK1/2 and Akt activation mediated the effect of ROS on NF-κB and HIF-1α and their pharmacological inhibition suppressed GEM-induced PTX3. Our study findings reinforced the role regarding PTX3 signaling in GEM-induced resistance and pointed toward an unintended and undesired effect of chemotherapy and to get an active regimen; the synergy was associated with NF-κB downregulation in lung cancer.