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Hu11B6 is a monoclonal antibody that internalizes in cells expressing androgen receptor (AR)-regulated prostate-specific enzyme human kallikrein-related peptidase 2 (hK2; KLK2). In multiple rodent models, Actinium-225-labeled hu11B6-IgG1 ([225Ac]hu11B6-IgG1) has shown promising treatment efficacy. In the present study, we investigated options to enhance and optimize [225Ac]hu11B6 treatment. First, we evaluated the possibility of exploiting IgG3, the IgG subclass with superior activation of complement and ability to mediate FC-γ-receptor binding, for immunotherapeutically enhanced hK2 targeted α-radioimmunotherapy. Second, we compared the therapeutic efficacy of a single high activity vs. fractionated activity. Finally, we used RNA sequencing to analyze the genomic signatures of prostate cancer that progressed after targeted α-therapy. [225Ac]hu11B6-IgG3 was a functionally enhanced alternative to [225Ac]hu11B6-IgG1 but offered no improvement of therapeutic efficacy. Progression-free survival was slightly increased with a single high activity compared to fractionated activity. Tumor-free animals succumbing after treatment revealed no evidence of treatment-associated toxicity. In addition to up-regulation of canonical aggressive prostate cancer genes, such as MMP7, ETV1, NTS, and SCHLAP1, we also noted a significant decrease in both KLK3 (prostate-specific antigen ) and FOLH1 (prostate-specific membrane antigen) but not in AR and KLK2, demonstrating efficacy of sequential [225Ac]hu11B6 in a mouse model.

Experiments were performed to investigate the physiological dynamics of metabolic pathways in the GntII system genes of Escherichia coli through a biochemical analysis of the pathway enzyme gluconate and its accompanying intermediates and growth experiments involving mutants with lesions in particular sites in the pathway. DNA sequence analysis and nuclear magnetic resonance studies were performed on the mutants. Findings suggested that the GntII system genes encoded a pathway for catabolism of L-idonate in which D-gluconate was an intermediate.

Gluconate metabolism in Escherichia coli is under the control of a group of genes under two main systems. The transporter genes which belong to the GntI system are negatively regulated by the gene GntR. The product of this regulatory gene contains a helix turn helix motif which is similar to that of the lac operon. GntR negatively regulates the gluconate regulon gene by binding to the operator sequence.

Microbiological research using radioactive fructose shows that fructose incorporation by the gram-negative, obligately fermentative bacterium Zymomonas mobilis CP4 is reduced in the presence of glucose. However, the glucose-reducing effect is far less in vivo than in vitro. Evidence suggests that a single facilitated diffusion transport system is used in glucose and fructose uptake. Z. mobilis may accumulate fructose in the presence of high glucose concentrations so that it can produce sorbitol.

Single-copy gnt::lacZ fusion and Northern hybridization analysis were utilized to characterize the expression of the Escherichia coli gntT gene and determine the biochemical function of its protein product. Northern hybridization analysis of the Escherichia coli gntT gene indicated the presence of properties that were characteristic of integral membrane transport proteins. Furthermore, gntT gene expression was enhanced during the logarithmic phase indicating the role of GntT in promoting microbial growth.

A transcriptional factor called glutamate uptake regulatory protein (Grp) has been identified in Zymomonas mobilis. The protein increased glutamate uptake in E. coli but inhibited the secondary H+

Structural and functional analysis of the gluABCD DNA fragment of gram-positive Corynebacterium glutamicum reveals that the glutamate uptake system of the bacterium depends on binding proteins and that the system contains all the structures for control and transport on the DNA fragment analyzed. The gluA, gluB, gluC and gluD gene form an operon, as indicated by the terminator structure present downstream of gluD. Kinetic analysis with globomycin-grown cells indicates that the tethering of C-glutamicin binding protein is essential for proper functioning.

Estrogen receptor (ER) alpha and beta are ligand-activated nuclear transcription factors that mediate the effects of the steroid hormone 17beta-estradiol. Tissue-selective ER modulators have been developed for the treatment of a variety of diseases, including osteoporosis and hormone-dependent breast cancer. Second- and third-generation selective ER modulators are in development, with the goal of reducing toxicity and improving tissue-selective efficacy. Novel tissue-selective and ERsubtype specific ligands may have the potential of providing a new paradigm for maintaining the health of women. The traditional cell-based screening assays for nuclear receptors require 16-18 h of incubation, which limits the assay miniaturization for ultra-high-throughput screening. We have developed a new cell-based ERalpha transactivation assay for the screening of ERalpha-specific antagonists with only 4 h of incubation time. The assay was optimized and used for a fully automated ultrahigh-throughput screen in 3,456-well nanoplate format. The screening throughput was 250,000-300,000 compounds per day, and a number of valuable leads were identified.

Estrogen action is mediated via two estrogen receptor (ER) subtypes, ERalpha and ERbeta. Selective ER modulators with balanced high affinity for ERalpha and ERbeta have been developed as therapeutics for the treatment of a variety of diseases, including hormone-responsive breast cancer and osteoporosis. Recent data based primarily on the evaluation of ER-knockout mice have revealed that ERalpha and ERbeta may regulate separate and distinct biological processes. The identification of ERbeta specific ligands could further enhance our understanding of ERbeta biology. In addition, compounds targeting ERbeta may prove useful as therapeutic agents with activity profiles distinguishable from that of estradiol. To discover novel selective ligands for ERbeta, we developed and characterized a cell-based Gal4-ERbeta beta-lactamase reporter gene assay (GERTA) in CHO cells for the ligand-induced activation of the human ERbeta. The sensitivity and selectivity of this assay were found to be comparable to those of an ER ligand-binding assay. The assay was optimized for screening in an ultra high throughput 3456-well nanoplate format and was successfully used to screen a large compound collection for ERbeta agonists. Compounds identified in a primary screen were tested in an in vitro ligand-binding assay to characterize further the selectivity and potency for ERbeta.