Etoposide-resistant HT-29 human colon carcinoma cells during glucose deprivation are sensitive to piericidin A, a GRP78 down-regulator

Glucose deprivation, a pathophysiological cell condition, causes up-regulation of GRP78 and induction of etoposide resistance in human cancer cells. The induction of drug resistance can be partly explained by the fact that GRP78 can block activation of caspase-7 induced by treatment with etoposide. Therefore, downregulating GRP78 expression may be a novel strategy anticancer drug development. Based on that premise, we established a screening program for anticancer agents that exhibit preferential cytotoxic activity for etoposide-resistant cancer cells under glucose-deprived conditions. We recently isolated an active compound, AR-054, from the culture broth of Streptomyces sp., which prevents stress-induced etoposide resistance in vitro. AR-054 was identified as piericidin A, a prototypical compound, by ESI-MS analysis and various NMR spectroscopic methods. Here, we showed that piericidin A suppressed the accumulation of GRP78 protein and was also highly toxic to etoposide-resistant HT-29 cells, with IC50 values for colony formation of 6.4 and 7.7 nM under 2-deoxyglucose supplemented and glucose-deprived conditions, respectively. Interestingly, piericidin A had no effect under normal growth conditions. Therefore, we suggest that piericidin A prevents up-regulation of GRP78, and exhibits cytotoxicity in glucose-deprived HT-29 cells that are resistant to etoposide.     

Synthesis and bradykinin inhibitory activity of novel non-peptide compounds,and evaluation of in vivo analgesic activity

A series of novel non-peptide diamide compounds was synthesized and evaluated as antibradykinin agents by utilizing guinea-pig ileum smooth muscle. Among the final compounds, (Z)-4-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-4-oxo-N-(4-phenylbutan-2-yl)but-2-enamide showed most favorable bradykinin inhibitory activity and demonstrated analgesic efficacies in the rat models of inflammatory and neuropathic pain.     

Discovery of aryl-biphenyl-2-ylmethylpiperazines as novel scaffolds for 5-HT7 ligands and role of the aromatic substituents in binding to the target receptor

It has been reported that 5-HT₇ receptors are promising targets of depression and neuropathic pain. 5-HT₇ receptor antagonists have exhibited antidepressant-like profiles, while agonists have represented potential therapeutics for pain. In the course of our ongoing efforts to discover novel 5-HT₇ modulators, we designed an arylpiperazine scaffold with a substituted biphenyl-2-ylmethyl group. A series of biphenyl-2-yl-arylpiperazinylmethanes were then prepared, which showed a broad spectrum of binding affinities to the 5-HT₇ receptor depending upon the substituents attached to the biphenyl and aryl functionalities. Among those synthesized compounds, the compounds 1–24 and 1–26 showed the best binding affinities to the 5-HT₇ receptor with K_i values of 43.0 and 46.0 nM, respectively. Structure–activity relationship study in conjunction with molecular docking study proposed that the 5-HT₇ receptor might have two distinctive hydrophobic binding sites, one specific for aromatic 2-OCH₃ substituents within the arylpiperazine and the other for biphenyl methoxy group.     

Human Embryonic Stem Cell Technology: Large Scale Cell Amplification and Differentiation

Embryonic stem cells (ESC) hold the promise of overcoming many diseases as potential sources of, for example, dopaminergic neural cells for Parkinson’s Disease to pancreatic islets to relieve diabetic patients of their daily insulin injections. While an embryo has the innate capacity to develop fully functional differentiated tissues; biologists are finding that it is much more complex to derive singular, pure populations of primary cells from the highly versatile ESC from this embryonic parent. Thus, a substantial investment in developing the technologies to expand and differentiate these cells is required in the next decade to move this promise into reality. In this review we document the current standard assays for characterising human ESC (hESC), the status of ‘defined’ feeder-free culture conditions for undifferentiated hESC growth, examine the quality controls that will be required to be established for monitoring their growth, review current methods for expansion and differentiation, and speculate on the possible routes of scaling up the differentiation of hESC to therapeutic quantities.     

Plant biomechanics and resilience to environmental changes are controlled by specific lignin chemistries in each vascular cell type and morphotype

The biopolymer lignin is deposited in the cell walls of vascular cells and is essential for long-distance water conduction and structural support in plants. Different vascular cell types contain distinct and conserved lignin chemistries, each with specific aromatic and aliphatic substitutions. Yet, the biological role of this conserved and specific lignin chemistry in each cell type remains unclear. Here, we investigated the roles of this lignin biochemical specificity for cellular functions by producing single cell analyses for three cell morphotypes of tracheary elements, which all allow sap conduction but differ in their morphology. We determined that specific lignin chemistries accumulate in each cell type. Moreover, lignin accumulated dynamically, increasing in quantity and changing in composition, to alter the cell wall biomechanics during cell maturation. For similar aromatic substitutions, residues with alcohol aliphatic functions increased stiffness whereas aldehydes increased flexibility of the cell wall. Modifying this lignin biochemical specificity and the sequence of its formation impaired the cell wall biomechanics of each morphotype and consequently hindered sap conduction and drought recovery. Together, our results demonstrate that each sap-conducting vascular cell type distinctly controls their lignin biochemistry to adjust their biomechanics and hydraulic properties to face developmental and environmental constraints.

During the development of each vascular cell, specific lignin chemistries control their biomechanics and water conduction properties to face environmental changes.

IN A NUTSHELL Background: Lignin comprises multiple cell wall–localized aromatic polymers that are essential for vascular plants to conduct water and strengthen their organs. It has long been thought that lignin was randomly and nonspecifically assembled to provide mechanical strengthening and waterproofing to cells by filling-up the empty spaces in the cell walls. However, the different cell types and morphotypes forming the different sap-conducting pipes and their cell wall layers (inner vs. outer layer) exhibit specific lignin chemistries that are conserved among plant species. We, therefore, investigated the function of these specific lignin chemistries at the cell and cell wall layer levels for the different sap-conducting pipes in plants. Question: What is the function of a specific lignin chemistry for the different plant sap-conducting pipe cells? Can changes in the lignin chemistry of sap-conducting cells affect their hydraulic capacity when facing environmental conditions such as drought? Findings: We answered these questions by changing lignin levels and composition, using drugs to block lignin formation, and/or genetic engineering to switch off genes, in three complementary systems: (1) isolated cells grown in test tubes that we can trigger to become sap conduits, (2) annual plants, and (3) hardwood trees. We show that lignin chemistry is specific to each cell morphotype and changes during cell maturation, modifying the amount of lignin, the chemical composition of lignin units, and the position of these units in the longer polymer. These specific lignin chemistries are required for the proper function of each cell morphotype to properly conduct the sap and strengthen plant organs. Modifying the amount, the composition, and the time when specific units with distinct chemistry are incorporated in lignin of each cell morphotype has dramatic effects, causing defects in sap conduit hydraulic and biomechanical properties. The ratio between the different chemical units of lignin needs to be fine-tuned to adjust plant sap conduction and mechanical strengthening. Thus, changes in the proportion of lignin units with distinct chemistries confer different hydraulic and mechanical properties enabling plants to better resist and/or recover from drought. We also revealed that increases in the proportion of lignin units with aldehyde modulate plant pipe hydraulic and mechanical properties. Next steps: We are now working to identify and understand the molecular mechanisms that control the formation of specific lignin chemistries in distinct sites and times during the development of the different cell wall layers in each cell type and morphotype.     

Synthesis of piperazine derivatives and evaluation of their antihistamine and antibradykinin effects.

Piperazine derivatives were prepared as histamine antagonists. Some of the synthesized compounds showed dual antagonistic activity against bradykinin as well as histamine.     

Total synthesis and biological evaluation of methylgerambullone

First total synthesis of methylgerambullone (MGB, 1) isolated from Glycosmis angustifolia was completed via a convergent route. The effect of MGB on the contractile responses of the isolated guinea-pig ileum induced by acetylcholine was investigated. As a result, it showed a potent relaxation rate (78.66 ± 4.30% at 100 mg/L) in a concentration-dependent manner on longitudinal smooth muscle contraction of isolated guinea-pig ileum induced by 1 μM acetylcholine.     

Selection of zinc fingers that bind single-stranded telomeric DNA in the G-quadruplex conformation

There is considerable interest in molecules that bind to telomeric DNA sequences and G-quadruplexes with specificity. Such molecules would be useful to test hypotheses for telomere length regulation, and may have therapeutic potential. The versatility and modular nature of the zinc finger motif makes it an ideal candidate for engineering G-quadruplex-binding proteins. Phage display technology has previously been widely used to screen libraries of zinc fingers for binding to novel duplex DNA sequences. In this study, a three-finger library has been screened for clones that bind to an oligonucleotide containing the human telomeric repeat sequence folded in the G-quadruplex conformation. The selected clones show a strong amino acid consensus, suggesting analogous modes of binding. Binding was found to be both sequence dependent and structure specific. This is the first example of an engineered protein that binds to G-quadruplex DNA, and represents a new type of binding interaction for a zinc finger protein.     

Remodeling of the glycosylation pathway in the methylotrophic yeast Hansenula polymorpha to produce human hybrid-type N-glycans

As a step forward to achieve the generation of human complex-type N-glycans in the methylotrophic yeast Hansenula polymorpha, we here report the modification of the yeast glycosylation pathway by heterologous expression of the human gene encoding β-1,2-N-acetylglucosaminyltransferase I (GnTI). For the optimal expression of human GnTI in the yeast Golgi compartment, the catalytic domain of the GnTI was fused to various N-terminal leader sequences derived from the yeast type II membrane proteins. The vectors containing GnTI fusion constructs were introduced into the H. polymorpha och1Δ single and och1Δalg3Δ double mutant strains expressing the ER-targeted Aspergillus saitoi α-1,2 mannosidase, respectively. Both of the glycoengineered Hpoch1Δ and Hpoch1ΔHpalg3Δ strains were shown to produce successfully the hybrid-type glycans with a monoantennary N-acetylglucosamine (GlcNAc₁Man₅GlcNAc₂ and GlcNAc₁Man₃GlcNAc₂, respectively) by N-glycan profile analysis of cell wall proteins. Furthermore, by comparative analysis of byproduct formation and the glycosylation site occupancy, we propose that the Hpoch1Δ strain would be more suitable than the Hpoch1ΔHpalg3Δ strain as a host for the production of recombinant proteins with humanized glycans.     

The kinetics and mechanism of shear inactivation of lipase from Candida cylindracea

Shearing experiments were conducted in a stirred tank reactor with 0.1% lipase solutions of Candida cylindracea. Inactivation of the lipase solutions were observed at various shear rates from 50 to 150 s(-1) after continuous shearing for ca. 30-240 min under optimal pH and temperature conditions. However, there was no shear stress denaturation of the lipase when it was subjected to shear stresses of 0.72-109.2 kg/m/s(2) and shear rate of 100 s(-1). In the presence of polypropylene glycol, the rate of denaturation of the lipase decreased by 93%. When the lipase solution was filled to the brim, the rate of denaturation of the lipase decreased by 97% compared to that when reactor was half-filled. The rate of denaturation of the lipase decreased by 61% when probes in the fermentor were removed. There was no significant difference in the rate of denaturation of the lipase under ambient conditions compared with that in the absence of oxygen, or in the absence of free metal ions. Recovery of lipase activity from the first hour of shearing was observed at a shear rate of 150 s(-1). The native lipase and the lipase which had recovered its activity showed similar pH profiles, temperature profiles, and activation energies. Temperature was found to have no effect in the rate of shear-induced denaturation of the lipase in the range 20 to 30 degrees C during shearing at 100 s (-1)and optimal pH. Above 30 degrees C, the rate of denaturation of the lipase increased drastically as a function of temperature. The significance of the findings in the de sign of reactor systems for hydrolysis or esterification of oils by lipase will be discussed.