Antitumor activity of paclitaxel and epirubicin in human breast carcinoma, R-27

The antitumor effect of paclitaxel, epirubicin, and both in combination was tested using R-27, an estrogen receptor-positive human breast carcinoma. In anin vivo study using nude mice both drugs showed an additive effect, whereas they showed a supraadditive pattern inin vitro MTT assay. The combination of paclitaxel and epirubicin may enhance the antitumor effect on breast carcinoma.     

Ni–Fe–La(Sr)Fe(Mn)O3 as a New Active Cermet Cathode for Intermediate‐Temperature CO2 Electrolysis Using a LaGaO3‐Based Electrolyte

Various additives to Ni–Fe systems are studied as cermet cathodes for CO₂ electrolysis (973–1173 K) using a La_0.9Sr_0.1Ga_0.8Mg_0.2O₃ (LSGM) electrolyte, which is one of the most promising oxide‐ion conductors for intermediate‐temperature solid‐oxide electrolysis cells in terms of ionic‐transport number and conductivity. It is found that Ni–Fe–La_0.6Sr_0.4Fe_0.8Mn_0.2O₃ (Ni–Fe–LSFM) exhibits a remarkable performance with a current density of 2.32 A cm^?2 at 1.6 V and 1073 K. The cathodic overpotential is significantly decreased by mixing the LSFM powder with Ni–Fe, which is related to the increase in the number of reaction sites for CO₂ reduction. For Ni–Fe–LSFM, much smaller particles (<200 nm) are sustained under CO₂ electrolysis conditions at high temperatures than for Ni–Fe. X‐ray diffraction analysis suggests that the main phases of Ni–Fe–LSFM are Ni and LaFeO₃; thus, the oxide phase of LaFeO₃ is also maintained during CO₂ electrolysis. Analysis of the gaseous products indicates that only CO is formed, and the rate of CO formation agrees well with that of a four‐electron reduction process, suggesting that the reduction of CO₂ to CO proceeds selectively. It is also confirmed that almost no coke is deposited on the Ni–Fe–LSFM cathode after CO₂ electrolysis.     

Structural basis of the collagen-binding mode of discoidin domain receptor 2

Discoidin domain receptor (DDR) is a cell-surface receptor tyrosine kinase activated by the binding of its discoidin (DS) domain to fibrillar collagen. Here, we have determined the NMR structure of the DS domain in DDR2 (DDR2-DS domain), and identified the binding site to fibrillar collagen by transferred cross-saturation experiments. The DDR2-DS domain structure adopts a distorted jellyroll fold, consisting of eight beta-strands. The collagen-binding site is formed at the interloop trench, consisting of charged residues surrounded by hydrophobic residues. The surface profile of the collagen-binding site suggests that the DDR2-DS domain recognizes specific sites on fibrillar collagen. This study provides a molecular basis for the collagen-binding mode of the DDR2-DS domain.     

Prolonged transient acidosis during early reperfusion contributes to the cardioprotective effects of postconditioning

We have previously reported that the prolonged transient acidosis during early reperfusion mediates the cardioprotective effects in canine hearts. Recently, postconditioning has been shown to be one of the novel strategies to mediate cardioprotection. We tested the contribution of the prolonged transient acidosis to the cardioprotection of postconditioning. Open-chest anesthetized dogs subjected to 90-min occlusion of the left anterior descending coronary artery and 6-h reperfusion were divided into four groups: 1) control group; no intervention after reperfusion (n = 6); 2) postconditioning (Postcon) group; four cycles of 1-min reperfusion and 1-min reocclusion (n = 7); 3) Postcon + sodium bicarbonate (NaHCO(3)) group; four cycles of 1-min reperfusion and 1-min reocclusion with the administration of NaHCO(3) (n = 8); and 4) NaHCO(3) group; administration of NaHCO(3) without postconditioning (n = 6). Infarct size, the area at risk (AAR), collateral blood flow during ischemia, and pH in coronary venous blood were measured. The phosphorylation of Akt and extracellular signal-regulated kinase (ERK) in ischemic myocardium was assessed by Western blot analysis. Systemic hemodynamic parameters, AAR, and collateral blood flow were not different among the four groups. Postconditioning induced prolonged transient acidosis during the early reperfusion phase. Administration of NaHCO(3) completely abolished the infarct size-limiting effects of postconditioning. Furthermore, the phosphorylation of Akt and ERK in ischemic myocardium induced by postconditioning was also blunted by the cotreatment of NaHCO(3). In conclusion, postconditioning mediates its cardioprotective effects possibly via prolonged transient acidosis during the early reperfusion phase with the activation of Akt and ERK.     

Affinity transfer to a human protein by CDR3 grafting of camelid VHH

VHH is the binding domain of the IgG heavy chain. Some VHHs have an extremely long CDR3 that contributes to antigen binding. We studied the antigen binding ability of CDR3 by grafting a CDR3 from an antigen-binding VHH onto a nonbinding VHH. cAb-CA05-(1RI8), the CDR3-grafted VHH, had an antigen-binding ability. To find a human scaffold protein acceptable for VHH CDR3 grafting, we focused on the conserved structure of VHH, especially the N-terminal and C-terminal amino acid residues of the CDR3 loop and the Cys residue of CDR1. Human origin protein structures with the same orientation were searched in PDB and ubiquitin was selected. Ubi-(1RI8), the CDR3-grafted ubiquitin, had antigen-binding ability, though the affinity was relatively low compared to cAb-CA05-(1RI8). The thermodynamic parameters of Ubi-(1RI8) binding to HEWL were different from cAb-CA05-(1RI8). Hydrogen-deuterium exchange experiments showed decreased stability around the CDR3 grafting region of Ubi-(1RI8), which might explain the decreased antigen-binding ability and the differences in thermodynamic properties. We concluded that the orientation of the CDR3 sequence of Ubi-(1RI8) could not be reconstructed correctly.     

Rapid identification of protein-protein interfaces for the construction of a complex model based on multiple unassigned signals by using time-sharing NMR measurements

Protein-protein interactions are necessary for various cellular processes, and therefore, information related to protein-protein interactions and structural information of complexes is invaluable. To identify protein-protein interfaces using NMR, resonance assignments are generally necessary to analyze the data; however, they are time consuming to collect, especially for large proteins. In this paper, we present a rapid, effective, and unbiased approach for the identification of a protein-protein interface without resonance assignments. This approach requires only a single set of 2D titration experiments of a single protein sample, labeled with a unique combination of an (15)N-labeled amino acid and several amino acids (13)C-labeled on specific atoms. To rapidly obtain high resolution data, we applied a new pulse sequence for time-shared NMR measurements that allowed simultaneous detection of a ω(1)-TROSY-type backbone (1)H-(15)N and aromatic (1)H-(13)C shift correlations together with single quantum methyl (1)H-(13)C shift correlations. We developed a structure-based computational approach, that uses our experimental data to search the protein surfaces in an unbiased manner to identify the residues involved in the protein-protein interface. Finally, we demonstrated that the obtained information of the molecular interface could be directly leveraged to support protein-protein docking studies. Such rapid construction of a complex model provides valuable information and enables more efficient biochemical characterization of a protein-protein complex, for instance, as the first step in structure-guided drug development.     

In vivo tracking of transplanted mononuclear cells using manganese-enhanced magnetic resonance imaging (MEMRI)

Background

Transplantation of mononuclear cells (MNCs) has previously been tested as a method to induce therapeutic angiogenesis to treat limb ischemia in clinical trials. Non-invasive high resolution imaging is required to track the cells and evaluate clinical relevance after cell transplantation. The hypothesis that MRI can provide in vivo detection and long-term observation of MNCs labeled with manganese contrast-agent was investigated in ischemic rat legs.

Methods and Findings

The Mn-labeled MNCs were evaluated using 7-tesla high-field magnetic resonance imaging (MRI). Intramuscular transplanted Mn-labeled MNCs were visualized with MRI for at least 7 and up to 21 days after transplantation in the ischemic leg. The distribution of Mn-labeled MNCs was similar to that of ¹¹¹In-labeled MNCs measured with single-photon emission computed tomography (SPECT) and DiI-dyed MNCs with fluorescence microscopy. In addition, at 1–2 days after transplantation the volume of the site injected with intact Mn-labeled MNCs was significantly larger than that injected with dead MNCs, although the dead Mn-labeled MNCs were also found for approximately 2 weeks in the ischemic legs. The area covered by CD31-positive cells (as a marker of capillary endothelial cells) in the intact Mn-MNCs implanted site at 43 days was significantly larger than that at a site implanted with dead Mn-MNCs.

Conclusions

The present Mn-enhanced MRI method enabled visualization of the transplanted area with a 150–175 µm in-plane spatial resolution and allowed the migration of labeled-MNCs to be observed for long periods in the same subject. After further optimization, MRI-based Mn-enhanced cell-tracking could be a useful technique for evaluation of cell therapy both in research and clinical applications.     

Spatial frequency-based analysis of mean red blood cell speed in single microvessels: investigation of microvascular perfusion in rat cerebral cortex

Background

Our previous study has shown that prenatal exposure to X-ray irradiation causes cerebral hypo-perfusion during the postnatal development of central nervous system (CNS). However, the source of the hypo-perfusion and its impact on the CNS development remains unclear. The present study developed an automatic analysis method to determine the mean red blood cell (RBC) speed through single microvessels imaged with two-photon microscopy in the cerebral cortex of rats prenatally exposed to X-ray irradiation (1.5 Gy).

Methodology/Principal Findings

We obtained a mean RBC speed (0.9±0.6 mm/sec) that ranged from 0.2 to 4.4 mm/sec from 121 vessels in the radiation-exposed rats, which was about 40% lower than that of normal rats that were not exposed. These results were then compared with the conventional method for monitoring microvascular perfusion using the arteriovenous transit time (AVTT) determined by tracking fluorescent markers. A significant increase in the AVTT was observed in the exposed rats (1.9±0.6 sec) as compared to the age-matched non-exposed rats (1.2±0.3 sec). The results indicate that parenchyma capillary blood velocity in the exposed rats was approximately 37% lower than in non-exposed rats.

Conclusions/Significance

The algorithm presented is simple and robust relative to monitoring individual RBC speeds, which is superior in terms of noise tolerance and computation time. The demonstrative results show that the method developed in this study for determining the mean RBC speed in the spatial frequency domain was consistent with the conventional transit time method.     

Characterization and Transcription of the Genes Involved in Butyrate Production in Butyrivibrio fibrisolvens TypeI and II Strains

The genes in Butyrivibrio fibrisolvens that encode the enzymes involved in butyrate production were sequenced. In a type I strain (ATCC 19171(T)), the genes coding for the enzymes that catalyze the conversion from acetyl-CoA to butyryl-CoA, thl (thiolase), crt (crotonase), hbd (beta-hydroxybutyryl-CoA dehydrogenase), bcd (butyryl-CoA dehydrogenase), etfB (electron transfer flavoprotein [ETF]-beta), and etfA (ETF-alpha), were found to be clustered and arranged in this order. A type II strain (ATCC 51255) had the same clustered genes with the same arrangement, except that crt was not present in the clustered genes. The deduced amino acid sequences of these enzymes did not greatly differ between the two strains, and even between B. fibrisolvens and clostridia. Amino acid identity appeared to be higher within the same type than between types I and II. The clustered genes were shown to be cotranscribed, and constitutively transcribed without being affected significantly by culture conditions.