5a-Carba-β-D-glucopyranose derivatives as novel sodium-dependent glucose cotransporter 2 (SGLT2) inhibitors for the treatment of type 2 diabetes

5a-Carba-β-D-glucopyranose derivatives were synthesized and identified as novel SGLT2-selective inhibitors. These inhibitors exhibited potent SGLT2 inhibition with high selectivity over SGLT1. Among the tested compounds, 6f indicated the most potent hSGLT2 inhibition and the highest selectivity over hSGLT1. Moreover, the pharmacokinetics data also showed that 6h, which had the same aglycon structure as sergliflozin-active (3-active), had a threefold longer half-life time (T(1/2)) than sergliflozin (3) with a high distribution volume in db/db mice. Subsequently, 6h lowered blood glucose levels as much as 3 and showed longer hypoglycemic action than 3 in db/db mice.     

Whole cell segmentation in solid tissue sections.

BACKGROUND: Understanding the cellular and molecular basis of tissue development and function requires analysis of individual cells while in their tissue context. METHODS: We developed software to find the optimum border around each cell (segmentation) from two-dimensional microscopic images of intact tissue. Samples were labeled with a fluorescent cell surface marker so that cell borders were brighter than elsewhere. The optimum border around each cell was defined as the border with an average intensity per unit length greater that any other possible border around that cell, and was calculated using the gray-weighted distance transform. Algorithm initiation requiring the user to mark two points per cell, one approximately in the center and the other on the border, ensured virtually 100% correct segmentation. Thereafter segmentation was automatic. RESULTS: The method was highly robust, because intermittent labeling of the cell borders, diffuse borders, and spurious signals away from the border do not significantly affect the optimum path. Computer-generated cells with increasing levels of added noise showed that the approach was accurate provided the cell could be detected visually. CONCLUSIONS: We have developed a highly robust algorithm for segmenting images of surface-labeled cells, enabling accurate and quantitative analysis of individual cells in tissue.     

Application of a new probabilistic model for recognizing complex patterns in glycans

MOTIVATION: The study of carbohydrate sugar chains, or glycans, has been one of slow progress mainly due to the difficulty in establishing standard methods for analyzing their structures and biosynthesis. Glycans are generally tree structures that are more complex than linear DNA or protein sequences, and evidence shows that patterns in glycans may be present that spread across siblings and into further regions that are not limited by the edges in the actual tree structure itself. Current models were not able to capture such patterns. RESULTS: We have applied a new probabilistic model, called probabilistic sibling-dependent tree Markov model (PSTMM), which is able to inherently capture such complex patterns of glycans. Not only is the ability to capture such patterns important in itself, but this also implies that PSTMM is capable of performing multiple tree structure alignments efficiently. We prove through experimentation on actual glycan data that this new model is extremely useful for gaining insight into the hidden, complex patterns of glycans, which are so crucial for the development and functioning of higher level organisms. Furthermore, we also show that this model can be additionally utilized as an innovative approach to multiple tree alignment, which has not been applied to glycan chains before. This extension on the usage of PSTMM may be a major step forward for not only the structural analysis of glycans, but it may consequently prove useful for discovering clues into their function.     

Acceleration of matrix metalloproteinase-1 production and activation of platelet-derived growth factor receptor beta in human coronary smooth muscle cells by oxidized LDL and 4-hydroxynonenal

Increases in matrix metalloproteinases (MMPs) at atherosclerotic lesions are involved in the migration of smooth muscle cells (SMCs) into the intima and to the rupture of plaques, being implicated in the progression of atherosclerosis. The present study examined the mechanisms underlying the production of MMP-1, interstitial collagenase-1, induced by oxidized low-density lipoprotein (oxLDL) and 4-hydroxynonenal (4-HNE), factors proposed to play a pivotal role in atherogenesis, in human coronary SMCs. oxLDL promoted the production of MMP-1 with the preceding phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Immunoprecipitation of platelet-derived growth factor receptor beta (PDGFR-beta) revealed that oxLDL induced tyrosine phosphorylation of the receptor. Inhibition of the activation of PDGFR-beta and ERK1/2 resulted in a suppression of the production of MMP-1. Consistently, 4-HNE also elicited the production of MMP-1 with the preceding phosphorylation of PDGFR-beta and ERK1/2. The 4-HNE-induced production of MMP-1 was prevented when the activation of PDGFR-beta and ERK1/2 was inhibited. The present results suggest that the activation of PDGFR-beta and ERK1/2 is involved in the production of MMP-1 in oxLDL- and 4-HNE-stimulated human coronary SMCs.     

Production of human erythropoietin by chimeric chickens

The use of transgenic avian allows cost effective and safe production of pharmaceutical proteins. Here, we report the successful production of chimeric chickens expressing human erythropoietin (hEpo) using a high-titer retroviral vector. The hEpo expressed by transgenic hens accumulated abundantly in egg white and had N- and O-linked carbohydrates. While attachment of terminal sialic acid and galactose was incomplete, portions of N- and O-linked carbohydrates were present. In vitro biological activity of egg white-hEpo was comparable to that produced by recombinant CHO cells.     

Two forms of secreted and thermostable luciferases from the marine copepod crustacean, Metridia pacifica

We cloned two forms of the secreted and thermostable luciferase genes, MpLuc1 and MpLuc2, from the marine copepod, Metridia pacifica. The 840-bp MpLuc1 cDNA comprised a 630-bp open reading frame encoding a 210-amino acid polypeptide (22.7 kDa). MpLuc1 had the closest homology with Metridia longa luciferase. The 753-bp MpLuc2 cDNA consisted of a 567-bp open reading frame (20.3 kDa), and it had the closest homology with Gaussia princeps luciferase. Single-specimen genomic PCR confirmed the presence of two luciferase genes in M. pacifica, and single-specimen RT-PCR revealed that both luciferase mRNAs were expressed. Both MpLuc1 and MpLuc2 (MpLucs) specifically reacted with the substrate coelenterazine producing identical bioluminescent spectra (lambdamax, 485 nm), but with different kinetics. Adding salt such as MgCl2 and CaCl2 to the reaction mixture significantly enhanced MpLuc1 and MpLuc2 activities. Wild-type MpLucs were remarkably thermostable; MpLuc1 retained about 60% of the original activity even after incubation at 90 degrees C for 30 min. MpLucs expressed in NIH-3T3 and HeLa cells were largely secreted into the culture medium. Continuous monitoring of secreted MpLuc1 driven by the c-fos promoter demonstrated the potential usefulness of MpLuc1 in nondisruptive reporter assays.     

The reactive site of eggplant trypsin inhibitor.

The reactive site peptide bond of the eggplant inhibitor against trypsin [EC 3.4.21.4] was identified by chemical modifications with 1,2-cyclohexanedione, 2,4,6-trinitrobenzenesulfonic acid, acetic anhydride and glyoxal, and by sequential treatments with trypsin and carboxypeptidase B [EC 3.4.12.3]. The inhibitor was significantly inactivated by chemical modifications of arginine residues, but was not affected by lysine modifications. Free arginine was released from the trypsin-modified inhibitor by carboxypeptidase B digestion, accompanied by a marked loss of inhibitory activity. A serine residue was newly exposed at the N-terminal amino acid of the inhibitor after modification with trypsin. The reactive site of the inhibitor against trypsin was concluded to be an arginylseryl bond. The inhibitor was completely inactivated by full reduction of its disulfide bonds.     

'Crystal lattice engineering,' an approach to engineer protein crystal contacts by creating intermolecular symmetry: crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines

A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild-type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X-ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L- and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L-RNase 1) was also evaluated. N4L-RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.