Characterization of oocyte retrieval cycles with empty zona pellucida.

Purpose: To identify the factors that characterize cycles with empty zona pellucida (EZP). Methods: Thirty-six oocyte retrieval cycles from which EZP were collected and another 36 cycles from which no EZP was collected were compared. The patients were divided into three groups: those with no EZP collected during any cycle, those with EZP collected during all cycles, and those experiencing cycles both with and without EZP. Results: The mean number of oocytes collected per cycle was higher in the cycles with EZP than without EZP. The fertilization rate of the collected oocytes and the rate of good embryo formation were significantly lower in the cycles with EZP. No significant difference was observed between the three groups in terms of age, number of oocytes collected, or hormone levels before and after the oocyte retrieval. The fertilization and pregnancy rates were highest in the patients with no EZP being collected during any cycle, followed by those experiencing cycles both with and without EZP, and then by those with EZP collected during all cycles. Conclusion: The observation of lower fertilization, poor embryo formation, and a low pregnancy rate in the patients with EZP suggests the poor quality of oocytes that were collected with EZP in the same cycle.

Structure-activity relationship study of BACE1 inhibitors possessing a chelidonic or 2,6-pyridinedicarboxylic scaffold at the P(2) position.

We have previously reported potent substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. While these inhibitors exhibited potent activities in enzymatic and cellular assays (KMI-429 in particular inhibited Aß production in vivo), these inhibitors contained some natural amino acids that seemed to be required to improve enzymatic stability in vivo and permeability across the blood-brain barrier, so as to be practical drug. Recently, we synthesized non-peptidic and small-sized BACE1 inhibitors possessing a heterocyclic scaffold at the P2 position. Herein we report the SAR study of BACE1 inhibitors possessing this heterocyclic scaffold, a chelidonic or 2,6-pyridinedicarboxylic moiety.

[A case of an advanced gastric cancer patient on hemodialysis achieving long-term progression-free survival after CPT-11+CDDP therapy].

A 59-year-old male with chronic kidney disease was diagnosed as having advanced gastric cancer(cT2N1P0H1M0), and CPT-11+CDDP therapy was started for him simultaneously with hemodialysis(HD). Serum CDDP concentrations were measured in the 1st course, and free-platinum(f-Pt)showing the anti-tumor effect was found to be eliminated by HD. Serum f-Pt levels, however, re-elevated until 24 hours after HD completion. Serum concentrations measured in the 15th course showed that f-Pt levels became higher than those observed in the 1st course, suggesting that CDDP was not completely removed by HD. Medical treatment was continued until the liver metastases were judged to be a progression disease at completion of the 18th course. When CDDP was administered to patients on HD, it was necessary to pay attention to various CDDP serum concentrations, and to tailor the dose to a tolerable level in each patient. Such an individual therapy might enable CPT-11+CDDP therapy to be one of the medical treatments of choice for advanced gastric cancer patients on HD.

Significance of interactions of BACE1-Arg235 with its ligands and design of BACE1 inhibitors with P2 pyridine scaffold.

Recently, we reported potent substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. Because these inhibitors contained some natural amino acids, we would need to improve their enzymatic stability in vivo and permeability across the blood-brain barrier, so that they become practically useful. Subsequently, non-peptidic and small-sized BACE1 inhibitors possessing a heterocyclic scaffold, 2,6-pyridenedicarboxylic, chelidamic or chelidonic moiety, at the P(2) position were reported. These inhibitors were designed based on the conformer of docked inhibitor in BACE1. In this study, we discuss the role and significance of interactions between Arg235 of BACE1 and its inhibitor in BACE1 inhibitory mechanism. Moreover, we designed more potent small-sized BACE1 inhibitors with a 2,6-pyridinedicarboxylic scaffold at the P(2) position, that were optimized for the interactions with Arg235 of BACE1.

Structural characterization of multibranched oligosaccharides from seal milk by a combination of off-line high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and sequential exoglycosidase digestion.

A complex mixture of diverse oligosaccharides related to the carbohydrates in glycoconjugates involved in various biological events is found in animal milk/colostrum and has been challenging targets for separation and structural studies. In the current study, we isolated oligosaccharides having high molecular masses (MW approximately 3800) from the milk samples of bearded and hooded seals and analyzed their structures by off-line normal-phase-high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight (NP-HPLC-MALDI-TOF) mass spectrometry (MS) by combination with sequential exoglycosidase digestion. Initially, a mixture of oligosaccharides from the seal milk was reductively aminated with 2-aminobenzoic acid and analyzed by a combination of HPLC and MALDI-TOF MS. From MS data, these oligosaccharides contained different numbers of lactosamine units attached to the nonreducing lactose (Galbeta1-4Glc) and fucose residue. The isolated oligosaccharides were sequentially digested with exoglycosidases and characterized by MALDI-TOF MS. The data revealed that oligosaccharides from both seal species were composed from lacto-N-neohexaose (LNnH, Galbeta1-4GlcNAcbeta1-6[Galbeta1-4GlcNAcbeta1-3]Galbeta1-4Glc) as the common core structure, and most of them contained Fucalpha1-2 residues at the nonreducing ends. Furthermore, the oligosaccharides from both samples contained multibranched oligosaccharides having two Galbeta1-4GlcNAc (N-acetyllactosamine, LacNAc) residues on the Galbeta1-4GlcNAcbeta1-3 branch or both branches of LNnH. Elongation of the chains was observed at 3-OH positions of Gal residues, but most of the internal Gal residues were also substituted with an N-acetyllactosamine at the 6-OH position.

Novel non-peptidic and small-sized BACE1 inhibitors.

Recently, we reported substrate-based beta-secretase (BACE1) inhibitors with a hydroxymethylcarbonyl (HMC) isostere as a substrate transition-state mimic. These inhibitors showed potent BACE1 inhibitory activities (approximately 1.2 nM IC(50)). In order to improve in vivo enzymatic stability and permeability across the blood-brain barrier, these penta-peptidic inhibitors would need to be further optimized. On the other hand, non-peptidic inhibitors possessing isophthalic residue at the P(2) position were reported from other research groups. We selected isophthalic-type aromatic residues at the P(2) position and an HMC isostere at the P(1) position as lead compounds. On the basis of the design approach focused on the conformer of docked inhibitor in BACE1, we found novel non-peptidic and small-sized BACE1 inhibitors possessing a 2,6-pyridinedicarboxylic, chelidamic or chelidonic residue at the P(2) position.

Electrochemical probe for on-chip type flow immunoassay: immunoglobulin G labeled with ferrocenecarboaldehyde.

Labeling of ferrocenecarboaldehyde (Fc-CHO) to immunoglobulin G (IgG) via formation of Schiff-base and its reduction was investigated for construction of an electrochemical probe for miniaturized amperometric flow immunoassay. Approximately eight molecules of Fc-CHO were labeled to IgG and the reversible redox property of ferrocene was observed. Labeling efficiency improved by over three times as compared to the conventional method using ferrocenemonocarboxylic acid (Fc-COOH). Also, binding affinity of IgG labeled with Fc-CHO to its antigen, IgE, was investigated by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance assay. IgG labeled with Fc-CHO that retained eight ferrocene moiety showed sufficient binding affinity to its antigen and the current response obtained in the flow electrochemical detection system increased by 14-fold as compared with IgG labeled with Fc-COOH when applying the potential of 390 mV vs. Ag/AgCl. The minimum detectable concentration of IgG labeled with Fc-CHO was 0.06 microM. IgG labeled with Fc-CHO demonstrate biochemical and electrochemical properties that are useful for electrochemical immunosensors.

Capturing of acidic macromolecules from biological samples using a temperature-responsive polymer modified with poly-l-lysine.

We synthesized a temperature-responsive polymer, N-(isopropylacrylamide)-methacrylic acid copolymer, to which poly-l-lysine was introduced. The synthesized polymer as well as the parent polymer showed reversible soluble-insoluble changes in response to temperature changes across the lower critical solution temperature at 32 degree C in an aqueous solution. We found that the polymer efficiently captured acidic bio-macromolecules such as RNA, glycosaminoglycans and mucin-type glycoproteins in biological samples, and the captured molecules were recovered using aqueous NaCl solutions at high concentration. The target acidic molecules thus obtained will be employed for further studies such as structural analysis after brief desalting procedure. The proposed method does not require any chromatographic separations, but only needs a small volume of an aqueous salt solution for releasing captured molecules. Overall procedures are quite easy and simple, and are completed at least within 1 h. We show a few examples for capturing RNA and glycosaminoglycans from cultured cells using the polymer.

Microfabricated on-chip-type electrochemical flow immunoassay system for the detection of histamine released in whole blood samples.

This paper describes an on-chip-type electrochemical flow immunoassay system with a multichanneled matrix column. The multichanneled matrix column was functionally coated with cation-exchange resin and used for separation of proteins. Antihistamine immunoglobulin G (IgG) antibody conjugated with ferrocenemonocarboxylic acid (Fc) was also prepared and used as a novel analytical reagent. Antibody-antigen complexes were separated from free Fc-conjugated IgG antibody (Fc-IgG) on the basis of differences in isoelectric point (pI) using the multichanneled matrix column coated with cation-exchange resin. The assay yields a good relationship between current and histamine concentration in the range of 200-2000 ng/mL. This simple technique enables the assay of histamine released in whole blood within 2 min. Furthermore, a good correlation was found between the response of the electrochemical immunoassay described in this paper and the conventional RIA (radioimmunoassay). This on-chip-type electrochemical flow immunoassay requires only minute quantities of whole blood samples and generates highly reproducible results.