The use of bifunctional biotinyl compounds to determine the arrangement of subunits in avidin

A series of bisbiotinyl diamines was synthesized with between 9 and 25 bonds between the carboxyl groups of the two biotin residues. It was found that only one of the two biotin residues could combine with avidin when there were fewer than 12 bonds between the biotin residues. Compounds with longer chains behaved in a bifunctional manner and gave rise to linear polymers of avidin, which were characterized by electron microscopy and by gel filtration. The polymers formed with the shorter-chain reagents (12, 13 or 14 bonds) were relatively unstable and could be depolymerized by weakly bound analogues of biotin. The polymers of longer-chain reagents were not depolymerized under these conditions and were only slowly affected by added biotin. When the chain length of the reagent reached 23 bonds the polymers became much shorter, suggesting that the reagent was now able to link two subunits within the same avidin molecule. From the morphology of the polymers it could be concluded that the four subunits of the avidin molecules were arranged with 222 symmetry and that they were grouped in two pairs at opposite ends of the short axis of the molecule whose dimensions were 55Ax55Ax41A.     

Cotargeting Polo-Like Kinase 1 and the Wnt/β-Catenin Signaling Pathway in Castration-Resistant Prostate Cancer

The Wnt/β-catenin signaling pathway has been identified as one of the predominantly upregulated pathways in castration-resistant prostate cancer (CRPC). However, whether targeting the β-catenin pathway will prove effective as a CRPC treatment remains unknown. Polo-like kinase 1 (Plk1) is a critical regulator in many cell cycle events, and its level is significantly elevated upon castration of mice carrying xenograft prostate tumors. Indeed, inhibition of Plk1 has been shown to inhibit tumor growth in several in vivo studies. Here, we show that Plk1 is a negative regulator of Wnt/β-catenin signaling. Plk1 inhibition or depletion enhances the level of cytosolic and nuclear β-catenin in human prostate cancer cells. Furthermore, inhibition of Wnt/β-catenin signaling significantly potentiates the antineoplastic activity of the Plk1 inhibitor BI2536 in both cultured prostate cancer cells and CRPC xenograft tumors. Mechanistically, axin2, a negative regulator of the β-catenin pathway, serves as a substrate of Plk1, and Plk1 phosphorylation of axin2 facilitates the degradation of β-catenin by enhancing binding between glycogen synthase kinase 3β (GSK3β) and β-catenin. Plk1-phosphorylated axin2 also exhibits resistance to Cdc20-mediated degradation. Overall, this study identifies a novel Plk1-Wnt signaling axis in prostate cancer, offering a promising new therapeutic option to treat CRPC.     

Fibroblast growth factor and transforming growth factor beta repress transcription of the myogenic regulatory gene MyoD1.

In this report, we demonstrate that myogenic cultures inhibited from differentiating by treatment with fibroblast growth factor or transforming growth factor beta show reduced levels of MyoD1 mRNA. Although this repression may contribute to the inhibition of myogenesis by growth factors, additional regulatory pathways must be affected, since inhibition still occurs in cultures engineered to constitutively express MyoD1 mRNA.     

A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers

A set of mapping markers have been designed for Arabidopsis thaliana that correspond to DNA fragments amplifed by the polymerase chain reaction (PCR). The ecotype of origin of these amplified fragments can be determined by cleavage with a restriction endo-nuclease. Specifically, 18 sets of PCR primers were synthesized, each of which amplifies a single mapped DNA sequence from the Columbia and Landsberg erecta ecotypes. Also identifed was at least one restriction endonuclease for each of these PCR products that generates ecotype-specific digestion patterns. Using these co-dominant cleaved amplified polymorphic sequences (CAPS), an Arabidopsis gene can be unambiguously mapped to one of the 10 Arabidopsis chromosome arms in a single cross using a limited number of F₂ progeny.     

Glutamine synthetase in Lupinus luteus. Identification and preliminary characterization of nodule-specific cDNA clone

Two glutamine synthetase (GS) cDNA clones from L. luteus were identified and characterized. The nucleotide sequence analysis proved that they represent highly homologous but distinct mRNA species. Northern blot hybridization revealed that pc LINGS encodes the nodule-specific subunit of the GS while pcLIGS1 represents the nonspecific one present in nodule tissue as well as in uninfected roots.