Detrimental Effects of Microgravity on Mouse Preimplantation Development In Vitro

Sustaining life beyond Earth either on space stations or on other planets will require a clear understanding of how the space environment affects key phases of mammalian reproduction. However, because of the difficulty of doing such experiments in mammals, most studies of reproduction in space have been carried out with other taxa, such as sea urchins, fish, amphibians or birds. Here, we studied the possibility of mammalian fertilization and preimplantation development under microgravity (µG) conditions using a three-dimensional (3D) clinostat, which faithfully simulates 10^–3 G using 3D rotation. Fertilization occurred normally in vitro under µG. However, although we obtained 75 healthy offspring from µG-fertilized and -cultured embryos after transfer to recipient females, the birth rate was lower than among the 1G controls. Immunostaining demonstrated that in vitro culture under µG caused slower development and fewer trophectoderm cells than in 1G controls but did not affect polarization of the blastocyst. These results suggest for the first time that fertilization can occur normally under µG environment in a mammal, but normal preimplantation embryo development might require 1G.     

Proteomic analysis of laser-microdissected paraffin-embedded tissues: (2) MRM assay for stage-related proteins upon non-metastatic lung adenocarcinoma

A preceding paper suggested 81 candidates of stage-specifically expressed proteins for either stage IA or IIIA by global shotgun proteomics and spectral counting. Six proteins, a subset of these proteins, were chosen for a further verification study since they are potentially soluble and/or secretory, which nature is convenient for detecting them in blood in clinical practice. The multiple-reaction monitoring (MRM) quantitative analysis suggested that napsin-A and anterior gradient protein 2 homolog (hAG-2) out of the 6 candidates would be useful for determining stage IA or IIIA and are related to metastasis. In the study we noted that stage IIIA patients with better outcome showed napsin-A profiles similar to that of stage IA patients. We therefore examined 14 additional patients for analysis, which contained the IA-stage patients of poorer outcome and the IIIA-stage patients of better outcome. The MRM analysis of napsin-A for all patients suggests that napsin-A contents correlate with better outcome in stage IA. This and discovery studies demonstrate that direct isolation of tumor cells alone by laser microdissection (LMD) greatly reduces complexity on comprehensive analyses, and that MRM mass spectrometry using the endogenous internal standard is a feasible technology for quantitative verification of target proteins in formalin-fixed paraffin embedded (FFPE) tissues.     

(−)‐Epigallocatechin‐3‐gallate inhibits human angiotensin‐converting enzyme activity through an autoxidation‐dependent mechanism

We investigated the molecular mechanisms involved in the angiotensin‐converting enzyme (ACE) inhibition by (?)‐epigallocatechin‐3‐gallate (EGCg), a major tea catechin. EGCg inhibited both the ACE activity in the lysate of human colorectal cancer cells and human recombinant ACE (rh‐ACE) in a dose‐dependent manner. Co‐incubation with zinc sulfate showed no influence on the rh‐ACE inhibition by EGCg, whereas it completely counteracted the inhibitory effect of ethylenediaminetetraacetic acid, a chelating‐type ACE inhibitor. Although hydrogen peroxide was produced by the autoxidation of EGCg, hydrogen peroxide itself had little effect on the ACE activity. Conversely, the co‐incubation of EGCg with borate or ascorbic acid significantly diminished the EGCg inhibition. A redox‐cycling staining experiment revealed that rh‐ACE was covalently modified by EGCg. A Lineweaver–Burk plot analysis indicated that EGCg inhibited the ACE activity in a non‐competitive manner. These results suggested that EGCg might allosterically inhibit the ACE activity through oxidative conversion into an electrophilic quinone.     

Mass balance analysis of contaminated heparin product

A quantitative analysis of a recalled contaminated lot of heparin sodium injection U.S. Pharmacopeia (USP) was undertaken in response to the controversy regarding the exact nature of the contaminant involved in the heparin (HP) crisis. A mass balance analysis of the formulated drug product was performed. After freeze-drying, a 1-ml vial for injection afforded 54.8 ± 0.3 mg of dry solids. The excipients, sodium chloride and residual benzyl alcohol, accounted for 11.4 ± 0.5 and 0.9 ± 0.5 mg, respectively. Active pharmaceutical ingredient (API) represented 41.5 ± 1.0 mg, corresponding to 75.7 wt% of dry mass. Exhaustive treatment of API with specific enzymes, heparin lyases, and/or chondroitin lyases was used to close mass balance. HP represented 30.5 ± 0.5 mg, corresponding to 73.5 wt% of the API. Dermatan sulfate (DS) impurity represented 1.7 ± 0.3 mg, corresponding to 4.1 wt% of API. Contaminant, representing 9.3 ± 0.1 mg corresponding to 22.4 wt% of API, was found in the contaminated formulated drug product. The recovery of contaminant was close to quantitative (95.6–100 wt%). A single contaminant was unambiguously identified as oversulfated chondroitin sulfate (OSCS).     

Carbamoyl-PROXYL-enhanced MRI detects very small disruptions in brain vascular permeability induced by dietary cholesterol

Background

Gd-DTPA-enhanced magnetic resonance imaging (MRI) is a conventional method for non-invasive investigation of blood-brain-barrier (BBB) permeability in animal models. It allows the visualization of serious injury to the BBB. We developed a novel approach for detecting very small disruptions in BBB permeability induced by dietary cholesterol by using carbamoyl-PROXYL (CMP) as an MRI contrast probe.

Methods

Mice were separated into two groups: normal diet (ND-mice) and high cholesterol diet (CD-mice). MRI-signal dynamics, plasma cholesterol, matrix metalloproteinase (MMP-9, MMP-2), and the white blood cell profile were analyzed. For the MRI analysis, two regions-of-interest (ROI) were selected: brain (ROI-1) and surrounding area (ROI-2).

Results

In the ROI-2 of ND-mice, CMP- or Gd-enhanced MRI-signal followed typical kinetics with a half-life of signal decay (τ_1/2) ~ 8 or ~ 15 min, respectively. In CD-mice, the MRI-signal increased continuously without decay.In the ROI-1 of ND- and CD-mice, MRI-signal enhancement was not detected by Gd-DTPA. In the ROI-1 of ND-mice, CMP-induced MRI-signal enhancement was negligible, while in CD-mice, it was significant (τ_1/2 > 15 min).Hypercholesterolemia increased the plasma levels of MMP-9 and neutrophils.

Conclusions

Hypercholesterolemia increases vascular permeability, which is mediated by MMP-9 and neutrophils.

General significance

Even very small disruptions in brain vascular permeability could be detected by CMP-enhanced MRI but not by Gd-DTPA-enhanced MRI.     

Phosphoinositide binding by par-3 involved in par-3 localization

Electrostatic interactions between lipids and proteins control many cellular events. We found that phospholipids, including phosphatidylinositol 3-phosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidylinositol 3,4,5-triphosphate, bound to the C-terminal coiled-coil region of par-3 at conserved, basic residues. We identified K1013 and K1014 as the phosphoinositide binding site, because the K1013E/K1014E mutation of rat par-3 abolished its lipid binding. Importantly, the K1013E/K1014E par-3 mutant exhibited significantly weaker localization at the cell-cell junctions than the wild-type par-3. Fluorescence recovery after photo-bleaching analyses confirmed the faster turnover of mutant par-3 at cell-cell junctions. The treatment of cells with an inhibitor of phosphatidylinositol 3-kinases partially increased the turnover of par-3. These data suggested that the putative phospholipid binding by par-3 is important for its localization at cell-cell junctions.