Detection of oligonucleotide hybridization at femtomolar level and sequence-specific gene analysis of the Arabidopsis thaliana leaf extract with an ultrasensitive surface plasmon resonance spectrometer

A flow-injection (FI) device is combined, through the use of a low-volume (4 µl) flow cell, with an ultrasensitive surface plasmon resonance (SPR) spectrometer equipped with a bi-cell photodiode detector. The application of this novel FI–SPR device for sequence-specific ultratrace analysis of oligodeoxynucleotides (ODNs) and polydeoxynucleotides was demonstrated. Self-assembled monolayers of ODN probes are tethered onto Au films with a mercaptohexyl group at the 3′ ends. The FI–SPR provides a detection level (≤54 fM) 2–3 orders of magnitude lower than other SPR devices and compares well with several ultrasensitive detection methods for labeled DNA targets (e.g. fluorophore-tagged and radiolabeled DNA samples). The technique is also highly selective, since a 47mer ODN target with a single-base mismatch yielded a much smaller SPR signal, and a specific interaction was detected when the complementary target was present at 0.001% of the total DNA. The FI–SPR was extended to the measurement of two individual genes in a cDNA mixture transcribed from an Arabidopsis thaliana leaf mRNA pool. The greatly enhanced sensitivity not only obviates the necessity of DNA labeling, but also significantly reduces sample consumption, allowing direct quantification of low abundance mRNAs in cellular samples without amplification.     

Direct measurement of local and global contributions in the binding of coformycin to bovine adenosine deaminase

A general method is outlined that determines quantitatively the extent to which tight ligand binding to an enzyme active site is facilitated by the adoption of a stabler macromolecular conformation in the complex. The method therefore rejects the general assumption that competitive inhibitor binding to enzyme active sites involves only local (active site) interactions. The procedure involves comparing the unfolding transition state free energies of the free and complexed enzyme from physiological conditions. For the interaction of the transition state analog coformycin with bovine adenosine deaminase we observed that the binding free energy by the physiological enzyme was approximately 92% due to the assumption of a stabler enzyme conformation in the complex. The significance of these findings in terms of general enzyme catalysis is discussed.     

Effect of hyperhomocysteinemia on the protein kinase DYRK1A in liver of mice

Hyperhomocysteinemia due to cystathionine beta synthase (CBS)-deficiency confers diverse clinical manifestations, notably liver diseases. Even if hyperhomocysteinemia in liver of CBS-deficient mice, a murine model of hyperhomocysteinemia, promotes mitochondrial oxidative stress and pro-apoptotic signals, protective signals may counteract these pro-apoptotic signals, leading to chronic inflammation. As DYRK1A, a serine/threonine kinase, has been described as a candidate antiapoptotic factor, we have analyzed the expression of DYRK1A in liver of CBS-deficient mice. We found that DYRK1A protein level was reduced in liver of CBS-deficient mice, which was not observed at the gene expression level. Moreover, the use of primary hepatocytes/Kupffer cells co-culture showed that degradation of DYRK1A induced by hyperhomocysteinemia requires calpain activation. Our results demonstrate a deleterious effect of hyperhomocysteinemia on DYRK1A protein expression, and emphasize the role of hyperhomocysteinemia on calpain activation.     

State of the APC/C: organization, function, and structure

The ubiquitin-proteasome protein degradation system is involved in many essential cellular processes including cell cycle regulation, cell differentiation, and the unfolded protein response. The anaphase-promoting complex/cyclosome (APC/C), an evolutionarily conserved E3 ubiquitin ligase, was discovered 15 years ago because of its pivotal role in cyclin degradation and mitotic progression. Since then, we have learned that the APC/C is a very large, complex E3 ligase composed of 13 subunits, yielding a molecular machine of approximately 1 MDa. The intricate regulation of the APC/C is mediated by the Cdc20 family of activators, pseudosubstrate inhibitors, protein kinases and phosphatases and the spindle assembly checkpoint. The large size, complexity, and dynamic nature of the APC/C represent significant obstacles toward high-resolution structural techniques; however, over the last decade, there have been a number of lower resolution APC/C structures determined using single particle electron microscopy. These structures, when combined with data generated from numerous genetic and biochemical studies, have begun to shed light on how APC/C activity is regulated. Here, we discuss the most recent developments in the APC/C field concerning structure, substrate recognition, and catalysis.     

A role for septins in the interaction between the Listeria monocytogenes INVASION PROTEIN InlB and the Met receptor

Septins are conserved GTPases that form filaments and are required for cell division. During interphase, septin filaments associate with cellular membrane and cytoskeleton networks, yet the functional significance of these associations have, to our knowledge, remained unknown. We recently discovered that different septins, SEPT2 and SEPT11, regulate the InlB-mediated entry of Listeria monocytogenes into host cells. Here we address the role of SEPT2 and SEPT11 in the InlB-Met interactions underlying Listeria invasion to explore how septins modulate surface receptor function. We observed that differences in InlB-mediated Listeria entry correlated with differences in Met surface expression caused by septin depletion. Using atomic force microscopy on living cells, we show that septin depletion significantly reduced the unbinding force of InlB-Met interaction and the viscosity of membrane tethers at locations where the InlB-Met interaction occurs. Strikingly, the same order of difference was observed for cells in which the actin cytoskeleton was disrupted. Consistent with a proposed role of septins in association with the actin cytoskeleton, we show that cell elasticity is decreased upon septin or actin inactivation. Septins are therefore likely to participate in anchorage of the Met receptor to the actin cytoskeleton, and represent a critical determinant in surface receptor function.     

Proteomic Analysis of DNA Synthesis on a Structured DNA Template in Human Cellular Extracts: Interplay Between NHEJ and Replication‐Associated Proteins

It is established that short inverted repeats trigger base substitution mutagenesis in human cells. However, how the replication machinery deals with structured DNA is unknown. It has been previously reported that in human cell‐free extracts, DNA primer extension using a structured single‐stranded template is transiently blocked at DNA hairpins. Here, the proteomic analysis of proteins bound to the DNA template is reported and evidence that the DNA‐PK complex (DNA‐PKcs and the Ku heterodimer) recognizes, and is activated by, structured single‐stranded DNA is provided. Hijacking the DNA‐PK complex by double‐stranded oligonucleotides results in a large removal of the pausing sites and an elevated DNA extension efficiency. Conversely, DNA‐PKcs inhibition results in its stabilization on the template, along with other proteins acting downstream in the Non‐Homologous End‐Joining (NHEJ) pathway, especially the XRCC4‐DNA ligase 4 complex and the cofactor PAXX. Retention of NHEJ factors to the DNA in the absence of DNA‐PKcs activity correlates with additional halts of primer extension, suggesting that these proteins hinder the progression of the DNA synthesis at these sites. Overall these results raise the possibility that, upon binding to hairpins formed onto ssDNA during fork progression, the DNA‐PK complex interferes with replication fork dynamics in vivo.     

Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas

One major unresolved question in the field of pancreas biology is whether ductal cells have the ability to generate insulin-producing β-cells. Conclusive examination of this question has been limited by the lack of appropriate tools to efficiently and specifically label ductal cells in vivo. We generated Sox9CreER(T2) mice, which, during adulthood, allow for labeling of an average of 70% of pancreatic ductal cells, including terminal duct/centroacinar cells. Fate-mapping studies of the Sox9(+) domain revealed endocrine and acinar cell neogenesis from Sox9(+) cells throughout embryogenesis. Very small numbers of non-β endocrine cells continue to arise from Sox9(+) cells in early postnatal life, but no endocrine or acinar cell neogenesis from Sox9(+) cells occurs during adulthood. In the adult pancreas, pancreatic injury by partial duct ligation (PDL) has been suggested to induce β-cell regeneration from a transient Ngn3(+) endocrine progenitor cell population. Here, we identify ductal cells as a cell of origin for PDL-induced Ngn3(+) cells, but fail to observe β-cell neogenesis from duct-derived cells. Therefore, although PDL leads to activation of Ngn3 expression in ducts, PDL does not induce appropriate cues to allow for completion of the entire β-cell neogenesis program. In conclusion, although endocrine cells arise from the Sox9(+) ductal domain throughout embryogenesis and the early postnatal period, Sox9(+) ductal cells of the adult pancreas no longer give rise to endocrine cells under both normal conditions and in response to PDL.     

Dyrk1A induces pancreatic β cell mass expansion and improves glucose tolerance

Type 2 diabetes is caused by a limited capacity of insulin-producing pancreatic β cells to increase their mass and function in response to insulin resistance. The signaling pathways that positively regulate functional β cell mass have not been fully elucidated. DYRK1A (also called minibrain/MNB) is a member of the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family. A significant amount of data implicates DYRK1A in brain growth and Down syndrome, and recent data indicate that Dyrk1A haploinsufficient mice have a low functional β cell mass. Here we ask whether Dyrk1A upregulation could be a way to increase functional β cell mass.

We used mice overexpressing Dyrk1A under the control of its own regulatory sequences (mBACTgDyrk1A). These mice exhibit decreased glucose levels and hyperinsulinemia in the fasting state. Improved glucose tolerance is observed in these mice as early as 4 weeks of age. Upregulation of Dyrk1A in β cells induces expansion of β cell mass through increased proliferation and cell size. Importantly, mBACTgDyrk1A mice are protected against high-fat-diet-induced β cell failure through increase in β cell mass and insulin sensitivity.

These studies show the crucial role of the DYRK1A pathway in the regulation of β cell mass and carbohydrate metabolism in vivo. Activating the DYRK1A pathway could thus represent an innovative way to increase functional β cell mass.     

Lipids and prostate cancer

The role of lipid metabolism has gained particular interest in prostate cancer research. A large body of literature has outlined the unique upregulation of de novo lipid synthesis in prostate cancer. Concordant with this lipogenic phenotype is a metabolic shift, in which cancer cells use alternative enzymes and pathways to facilitate the production of fatty acids. These newly synthesized lipids may support a number of cellular processes to promote cancer cell proliferation and survival. Hence, de novo lipogenesis is under intense investigation as a therapeutic target. Epidemiologic studies suggest dietary fat may also contribute to prostate cancer; however, whether dietary lipids and de novo synthesized lipids are differentially metabolized remains unclear. Here, we highlight the lipogenic nature of prostate cancer, especially the promotion of de novo lipid synthesis, and the significance of various dietary lipids in prostate cancer development and progression.