Changes of Gap and Tight Junctions during Differentiation of Human Nasal Epithelial Cells Using Primary Human Nasal Epithelial Cells and Primary Human Nasal Fibroblast Cells in a Noncontact Coculture System

The epithelium of upper respiratory tissues such as nasal mucosa forms a continuous barrier to a wide variety of exogenous antigens. The epithelial barrier function is regulated in large part by the intercellular junctions, referred to as gap and tight junctions. However, changes of gap and tight junctions during differentiation of human nasal epithelial (HNE) cells are still unclear. In the present study, to investigate changes of gap and tight junctions during differentiation of HNE cells in vitro, we used primary human HNE cells cocultured with primary human nasal fibroblast (HNF) cells in a noncontact system. In HNE cells cocultured with HNF cells for 2 weeks, numerous elongated cilia-like structures were observed compared to those without HNF cells. In the coculture, downregulation of Cx26 and upregulation of Cx30.3 and Cx31 were observed together with extensive gap junctional intercellular communication. Furthermore, expression of the tight junction proteins claudin-1, claudin-4, occludin and ZO-2 was increased. These results suggest that switching in expression of connexins and induction of tight junction proteins may be closely associated with differentiation of HNE cells in vitro and that differentiation of HNE cells requires unknown soluble factors secreted from HNF cells.     

Metabolic engineering of Escherichia coli for 1-butanol production

Compared to ethanol, butanol offers many advantages as a substitute for gasoline because of higher energy content and higher hydrophobicity. Typically, 1-butanol is produced by Clostridium in a mixed-product fermentation. To facilitate strain improvement for specificity and productivity, we engineered a synthetic pathway in Escherichia coli and demonstrated the production of 1-butanol from this non-native user-friendly host. Alternative genes and competing pathway deletions were evaluated for 1-butanol production. Results show promise for using E. coli for 1-butanol production.     

Clinical value of plasma pentraxin 3 levels for predicting cardiac troponin elevation after percutaneous coronary intervention

Aims

Post-procedural myocardial necrosis manifested by elevated cardiac troponin T (cTnT) often complicates percutaneous coronary intervention (PCI). Plasma pentraxin 3 (PTX3) levels are increased in patients with arterial inflammation and especially unstable angina pectoris (UAP). This study tested whether plasma PTX3 levels can predict post-PCI cTnT elevation.

Main methods

We evaluated 94 consecutive patients with AP and normal pre-PCI cTnT levels who underwent PCI. Pre-PCI virtual histology-intravascular ultrasound was performed to assess culprit plaque composition. Plasma PTX3 and serum hs-CRP levels were measured pre-PCI. Patients were divided into 2 groups according to presence (Group I, n = 34) or absence (Group II, n = 60) of post-PCI cTnT elevation > 3 × the upper limit of normal at 24 h after PCI.

Key findings

Plasma PTX3 (4.06 ± 2.05 ng/ml vs 2.17 ± 1.02 ng/ml, p < 0.001), serum hs-CRP levels (0.25 ± 0.03 vs 0.16 ± 0.03 mg/dl, p = 0.048), plaque burden (80.9 ± 5.3 vs 75.4 ± 10.6%, p = 0.047), presence of positive remodeling (59 vs 25%, p = 0.034), and percent necrotic core area (19.0 ± 7.4 vs 14.0 ± 5.9%, p = 0.046) were significantly higher in Group I than in Group II. Receiver-operating characteristic curve analysis showed that with a best cut-off value of 2.83 ng/ml, plasma PTX3 level (AUC 0.823) predicted post-PCI cardiac TnT elevation better than did serum hs-CRP level (AUC 0.618). Multiple logistic regression analysis showed that plasma PTX3 level was the most independent predictor of post-PCI cardiac cTnT elevation (OR: 2.65; 95% CI: 1.56–10.1; p = 0.003).

Significance

Plasma PTX3 level may be a useful marker for predicting post-PCI cardiac cTnT elevation, which is associated with inflammatory status of culprit lesions.     

Difference in nucleocytoplasmic shuttling sequences of rat and human constitutive active/androstane receptor

Fluorescence recovery after photobleaching (FRAP) in spontaneous multinuclear cells shows that both rat and human constitutive active/androstane receptors (CARs) are shuttling proteins with both nuclear localization signals (NLSs) and nuclear export signals (NESs). We previously identified two NLSs in rat CAR: NLS1 in the hinge region (residues 100-108) and NLS2 in the ligand-binding domain (residues 111-320). In the present study, we compared the intracellular localization signals between rat and human CARs. There was a marked difference in their intracellular localization in COS-7 cells because, unlike rat CAR, human CAR does not contain NLS1 due to an amino acid change at position 106. A CRM1-dependent leucine-rich NES, which is sensitive to an inhibitory effect of leptomycin B, was found in the cytoplasmic retention region previously identified within the ligand-binding domain of rat CAR (residues 220-258). We found that human CAR instead has a NES in the ligand-binding domain between residues 170 and 220. Also, we detected CRM1-independent C-terminal NESs between residues 317-358 of rat and human CARs. Removal of NLS1 by N-terminal truncation and mutation of xenochemical response signal caused rat CAR to localize in the cytoplasm of COS-7 cells, which we suspect is due to the masking of NLS2.     

Circadian rhythm in hypothalamic leptin receptor (Ob-Rb) mRNA expressions and cerebrospinal fluid and circulating glucose and leptin levels in lactating rats

In lactating animals, the food consumption increases several-fold for milk supply to the pups. The present study was conducted to clarify the relationship between the hyperphagia during lactation and hypothalamic leptin receptor (Ob-Rb) mRNA expression, cerebrospinal fluid (CSF) and circulating leptin and glucose levels. Food intakes significantly higher in lactation than in non-lactation at all time points (3 points: light phase, 4 points: dark phase) of the day. However, the expression of the hypothalamic Ob-Rb mRNA showed similar circadian rhythms in both the non-lactation and lactation, with only slight differences between the two groups. CSF leptin and glucose levels were constant throughout the day in both non-lactation and lactation, and there was almost no difference between the two groups at each time point. Circulating leptin and glucose levels showed circadian rhythms only in the non-lactating period, and were lower in lactation than in non-lactation, especially in the dark phase. In conclusion, the present study provides evidence that Ob-Rb mRNA expression fluctuates in the lactation period as well as in the non-lactation period, suggesting that the expression profile of whole hypothalamic Ob-Rb may not contribute to the difference in food consumption between lactation and non-lactation, and that chronic decrease in blood glucose levels may be associated with the increase in food consumption during lactation.     

Catalytic antibody light chain capable of cleaving a chemokine receptor CCR-5 peptide with a high reaction rate constant

A monoclonal antibody (MAb), ECL2B-2, was obtained by immunizing a peptide possessing a part of a sequence of a chemokine receptor, CCR-5, which is present as a membrane protein on the macrophage surface, and which plays an important role in human immunodeficiency virus (HIV) infection. From the DNA and the deduced amino acid sequences of the light and heavy chains of ECL2B-2 MAb, molecular modeling was conducted to calculate the steric conformation of the antibody. Modeling suggested that the structure of ECL2B-2 could possess one or two catalytic triad(s), composed of Asp(1), Ser(27a) (or Ser(27e)), and His(93) (or His(27d)), in the light chain of ECL2B-2. The three amino acid residues, Asp(1), Ser(27a), and His(93), are identical to those of catalytic antibody light chains such as VIPase and i41SL1-2. The light chain of ECL2B-2 MAb degraded the antigenic peptide CCR-5 within about 100 h. Surprisingly, the light chain had a very high catalytic reaction rate constant (k(cat)) of 2.23 min(-1), which is greater by factors of tens to hundreds than those of natural catalytic antibodies obtained previously. The heavy chain of ECL2B-2 MAb, which has no catalytic triad because of a lack of His residue, did not degrade the CCR-5 peptide.     

Efficient and Rapid Induction of Human iPSCs/ESCs into Nephrogenic Intermediate Mesoderm Using Small Molecule-Based Differentiation Methods

The first step in developing regenerative medicine approaches to treat renal diseases using pluripotent stem cells must be the generation of intermediate mesoderm (IM), an embryonic germ layer that gives rise to kidneys. In order to achieve this goal, establishing an efficient, stable and low-cost method for differentiating IM cells using small molecules is required. In this study, we identified two retinoids, AM580 and TTNPB, as potent IM inducers by high-throughput chemical screening, and established rapid (five days) and efficient (80% induction rate) IM differentiation from human iPSCs using only two small molecules: a Wnt pathway activator, CHIR99021, combined with either AM580 or TTNPB. The resulting human IM cells showed the ability to differentiate into multiple cell types that constitute adult kidneys, and to form renal tubule-like structures. These small molecule differentiation methods can bypass the mesendoderm step, directly inducing IM cells by activating Wnt, retinoic acid (RA), and bone morphogenetic protein (BMP) pathways. Such methods are powerful tools for studying kidney development and may potentially provide cell sources to generate renal lineage cells for regenerative therapy.     

Genetic structure and reproduction dynamics of Salix reinii during primary succession on Mount Fuji,as revealed by nuclear and chloroplast microsatellite analysis

The early stage of volcanic desert succession is underway on the southeastern slope of Mount Fuji. We used markers of nuclear microsatellites (simple sequence repeats; SSR) and chloroplast microsatellites (cpSSR) to investigate the population genetic structure and reproduction dynamics of Salix reinii, one of the dominant pioneer shrubs in this area. The number of S. reinii genets in a patch and the area of the largest genet within the patch increased with patch area, suggesting that both clonal growth and seedling recruitment are involved in the reproduction dynamics of S. reinii. Five polymorphic cpSSR markers were developed for S. reinii by sequencing the noncoding regions between universal sequences in the chloroplast genome. Nineteen different cpSSR haplotypes were identified, indicating that S. reinii pioneer genets were created by the long-distance dispersal of seeds originating from different mother genets around the study site, where all vegetation was destroyed during the last eruption. Furthermore, the clustered distributions of different haplotypes within each patch or plot suggested that newly colonized genets tended to be generated from seeds dispersed near the initially established mother genets. These results revealed that the establishment of the S. reinii population on the southeastern slope of Mount Fuji involved two sequential modes of seed dispersal: long-distance dispersal followed by short-distance dispersal.     

Interaction between trehalose and alkaline-earth metal ions

We investigated the interaction between trehalose and alkaline-earth metal ions. The nuclear relaxation times of carbon atoms of trehalose were shortened by addition of the alkaline-earth chloride salts, MgCl2, CaCl2, and SrCl2, indicating that trehalose formed metal-complexes with the alkaline-earth metal chlorides. From the data of the 1H-1H coupling constants of trehalose in the presence of the alkaline-earth chlorides, it appeared that trehalose formed complexes with MgCl2, and CaCl2 at the various complexing sites: Mg2+ was coordinated to O-4 and O-4' of trehalose, and Ca2+ to O-2 and O-3. We succeeded in the preparation of two types of crystals of the trehalose/CaCl2. One was a crystal consisting of trehalose, CaCl2, and water in a ratio of 1:1:1. The other was an anhydrous crystal containing trehalose and CaCl2 in a ratio of 1:2. Several applications of the complexing between trehalose and the metal ions for food processing are proposed.