Transcriptional inactivation of p53 by deletions and single amino acid changes in mouse mot-1 protein

Mouse mortalin proteins, mot-1 and mot-2, differ by only two amino acid residues in their C-terminus. In previous studies we showed that they differ in their subcellular distributions and interactions with the tumor suppressor protein, p53. By using mot-1 deletion mutants and amino acid substitution constructs, we report here that inability of mot-1 to affect p53 activity in vivo is dependent on the presence of both of the unique mot-1 amino acids and all three of the predicted hsp70, EF hand, and leucine zipper motif regions. The two proteins and their single amino acid mutants showed different mobilities on SDS-polyacrylamide gel presenting an evidence for their different secondary structures. Taken together, the data suggest that each of the two differing amino acids between mot-1 and mot-2 is an important determinant of their secondary structures and in vivo activities.     

Quantitative determination of disopyramide, verapamil and flecainide enantiomers in rat plasma and tissues by high-performance liquid chromatography

Enantiomers of disopyramide (DP), flecainide (FLC) and verapamil (VP) were extracted from rat plasma and tissues (brain, lung, heart, liver, kidney and muscle), followed by quantitative determination using enantioselective high-performance liquid chromatography with chiral stationary-phase columns. The recoveries of S-(+)- and R-(−)-DP from tissues were higher than 69%, and the within- and between-day coefficients of variation were very low (0.5 – 5.7%). The lower limits of detection in each tissue were less than 289 ng/g tissue. The recoveries of S-(+)- and R-(−)-FLC from tissues were higher than 88%, and the within- and between-day coefficients of variation were 1.2–6.0%. The lower limits of detection in each tissue were less than 37 ng/g tissue. The recoveries of S-(−)- and R-(+)-VP from tissues were higher than 80%, and the within- and between-day coefficients of variation were 0.5–6.2%. The lower limits of detection in each tissue were less than 51 ng/g tissue. The analytical methods established in this study will be suitable for determining the concentrations of the enantiomers of these anti-arrhythmic agents in rat plasma and tissues.     

Raman and Autofluorescence Spectrum Dynamics along the HRG-Induced Differentiation Pathway of MCF-7 Cells

Cellular differentiation proceeds along complicated pathways, even when it is induced by extracellular signaling molecules. One of the major reasons for this complexity is the highly multidimensional internal dynamics of cells, which sometimes causes apparently stochastic responses in individual cells to extracellular stimuli. Therefore, to understand cell differentiation, it is necessary to monitor the internal dynamics of cells at single-cell resolution. Here, we used a Raman and autofluorescence spectrum analysis of single cells to detect dynamic changes in intracellular molecular components. MCF-7 cells are a human cancer-derived cell line that can be induced to differentiate into mammary-gland-like cells with the addition of heregulin (HRG) to the culture medium. We measured the spectra in the cytoplasm of MCF-7 cells during 12 days of HRG stimulation. The Raman scattering spectrum, which was the major component of the signal, changed with time. A multicomponent analysis of the Raman spectrum revealed that the dynamics of the major components of the intracellular molecules, including proteins and lipids, changed cyclically along the differentiation pathway. The background autofluorescence signals of Raman scattering also provided information about the differentiation process. Using the total information from the Raman and autofluorescence spectra, we were able to visualize the pathway of cell differentiation in the multicomponent phase space.     

Induction of antibacterial activity in larvae of the blowfly Lucilia sericata by an infected environment

Maggot debridement therapy (MDT) is a method for the treatment of intractable, infected and necrotic wounds. In MDT, sterile larvae of Lucilia sericata Meigen (Diptera: Calliphoridae) are applied to infected wounds, where they exert antibacterial effects. Once the larvae are placed in the wound, they are no longer germ-free. This study analysed the influence of infected environments on larval antibacterial activities. Sterile larvae were mixed in a test tube containing a bacterial suspension of Staphylococcus aureus or Pseudomonas aeruginosa, transferred to liver puree agar, and incubated at 25 °C for set periods. To collect the larval extracts, the incubated larvae were transferred to a test tube containing phosphate buffered saline (PBS), cut into multiple pieces with scissors, and centrifuged. The supernatant was used to test antibacterial activities. The results showed that infected larvae had better antibacterial capacities than sterile larvae. Antibacterial activities were induced by pretreatment with a single bacterial species, S. aureus or P. aeruginosa, within 24 h and 12 h, respectively, and disappeared after 36 h. The activities were effective against S. aureus, but not against P. aeruginosa. This natural infection model is very similar to the clinical wound context in MDT and will be a powerful tool with which to study the antibacterial activities of L. sericata larvae in MDT.     

Immunohistochemical characteristics of submucosal Dogiel type II neurons in rat colon

Secretory and motility reflexes are evoked by physiological stimuli in the isolated rat distal colon, which is therefore expected to contain intrinsic primary afferent (sensory) neurons. Dogiel type II neurons (putative intrinsic primary afferent neurons) exhibit several long processes emerging from large oval or round cell bodies. This study has examined the immunohistochemical characteristics of type II neurons in the submucosal plexus of rat distal colons by using whole-mount preparations. Neuronal cell bodies positive for both substance P (SP) and calretinin have been observed in colchicine-treated rats. Neurofilament 200 immunostaining has confirmed the type II morphology of SP-positive neurons. Moreover, all submucosal type II neurons identified by neurofilament 200 immunoreactivity are positive for calretinin. Calcitonin gene-related peptide (CGRP)-positive neurons in the submucosal plexus are distinct from type II neurons because they are negative for calretinin and have smaller cell bodies than the SP-positive submucosal type II neurons. Most (73%) of the submucosal neurons including type II neurons exhibit immunoreactivity for the neurokinin-1 receptor (NK1R), a receptor for SP, on the surface of cell bodies. Immunoreactivity for the EP3 receptor (EP3R), a receptor for prostaglandin E2, has been detected in 51% of submucosal neurons including type II neurons. Thus, submucosal type II neurons in the rat distal colon are immunopositive for SP/calretinin but immunonegative for CGRP. SP released from submucosal type II neurons probably acts via NK1Rs on type II and non-type II submucosal neurons to mediate intrinsic reflexes. EP3R-positive submucosal type II neurons may be potential targets of prostaglandin E2.     

Mesenchymal stem cells cultured under hypoxia escape from senescence via down-regulation of p16 and extracellular signal regulated kinase

Hypoxia has been considered to affect the properties of tissue stem cells including mesenchymal stem cells (MSCs). Effects of long periods of exposure to hypoxia on human MSCs, however, have not been clearly demonstrated. MSCs cultured under normoxic conditions (20% pO₂) ceased to proliferate after 15-25 population doublings, while MSCs cultured under hypoxic conditions (1% pO₂) retained the ability to proliferate with an additional 8-20 population doublings. Most of the MSCs cultured under normoxic conditions were in a senescent state after 100 days, while few senescent cells were found in the hypoxic culture, which was associated with a down-regulation of p16 gene expression. MSCs cultured for 100 days under hypoxic conditions were superior to those cultured under normoxic conditions in the ability to differentiate into the chondro- and adipogenic, but not osteogenic, lineage. Among the molecules related to mitogen-activated protein kinase (MAPK) signaling pathways, extracellular signal regulated kinase (ERK) was significantly down-regulated by hypoxia, which helped to inhibit the up-regulation of p16 gene expression. Therefore, the hypoxic culture retained MSCs in an undifferentiated and senescence-free state through the down-regulation of p16 and ERK.     

Effects of compressive force on the differentiation of pluripotent mesenchymal cells

The purpose of this study was to determine the effect of mechanical stress on the differentiation of the pluripotent mesenchymal cell line C2C12. C2C12 cells were cultured continuously under compressive force (0.25-2.0 g/cm(2)). After mechanical stress loading, the levels of expression of mRNAs and proteins for phenotype-specific markers of osteoblasts (Runx2, Msx2, Dlx5, Osterix, AJ18), chondroblasts (Sox5, Sox9), myoblasts (MyoD), and adipocytes (PPAR gamma) were measured by real-time polymerase chain reaction analysis and Western blot analysis, respectively. The expression of activated p38 mitogen-activated protein kinase (p38 MAPK) was measured by Western blotting and/or ELISA. Loading 0.5 g/cm(2) of compressive force significantly increased the expression levels of Runx2, Msx2, Dlx5, Osterix, Sox5, and Sox9. In contrast, the expression levels of AJ18, MyoD, and PPAR gamma were decreased by exposure to 0.5 g/cm(2) of compressive force. Loading 0.5 g/cm(2) of compressive force also induced the phosphorylation of p38 MAPK. SB203580, which is a specific inhibitor of p38 MAPK, inhibited the compressive force-induced phosphorylation of p38 MAPK and partially blocked compressive force-induced Runx2 mRNA expression. These results demonstrate that compressive force stimulation directs the differentiation pathway of C2C12 cells into the osteoblast and chondroblast lineage via activated phosphorylation of p38 MAPK.     

Loss of calcineurin homologous protein-1 in chicken B lymphoma DT40 cells destabilizes Na+/H+ exchanger isoform-1 protein

NHE1/SLC9A1 is a ubiquitous isoform of vertebrate Na⁺/H⁺ exchangers (NHEs) functioning in maintaining intracellular concentrations of Na⁺ and H⁺ ions. Calcineurin homologous protein-1 (CHP1) binds to the hydrophilic region of NHE1 and regulates NHE1 activity but reportedly does not play a role in translocating NHE1 from the endoplasmic reticulum to the plasma membrane. However, an antiport function of NHE1 requiring CHP1 remains to be clarified. Here we established CHP1-deficient chicken B lymphoma DT40 cells by gene targeting to address CHP1 function. CHP1-deficient cells showed extensive decreases in Na⁺/H⁺ activities in intact cells. Although NHE1 mRNA levels were not affected, NHE1 protein levels were significantly reduced not only in the plasma membrane but in whole cells. The expression of a CHP1 transgene in CHP1-deficient cells rescued NHE1 protein expression. Expression of mutant forms of CHP1 defective in Ca²⁺ binding or myristoylation also partially decreased NHE1 protein levels. Knockdown of CHP1 also caused a moderate decrease in NHE1 protein in HeLa cells. These data indicate that CHP1 primarily plays an essential role in stabilization of NHE1 for reaching of NHE1 to the plasma membrane and its exchange activity. membrane protein; transporter; antiporter; quality control; degradation     

Characterization of the Rv3378c gene product, a new diterpene synthase for producing tuberculosinol and (13R, S)-isotuberculosinol (nosyberkol), from the Mycobacterium tuberculosis H37Rv genome

The Rv3377c and Rv3378c genes from Mycobacterium tuberculosis are specifically found in the virulent Mycobacterium species, but not in the avirulent species. The Rv3378c-encoded enzyme produced tuberculosinol 2 (5(6), 13(14)-halimadiene-15-ol), 13R-5a and 13S-isotuberculosinol 5b (5(6), 14(15)-halimadiene-13-ol) as its enzymatic products from tuberculosinyl diphosphate 3, indicating that the Rv3378c enzyme catalyzed the nucleophilic addition of a water molecule after the release of a diphosphate moiety. The three enzymatic products 2, 5a, and 5b were produced irrespective of the N- and C-terminal His-tagged Rv3378c enzymes, and of the maltose-binding protein fusion enzyme; the product distribution ratio was identical between the enzymes as 1:1 for 2:5, and 1:3 for 5a:5b. The successful separation of 5a and 5b by a chiral HPLC column provided the first complete assignments of 1H- and 13C-NMR data for 5a and 5b. The enzymatic mechanism for producing 2, 5a, and 5b is proposed here, and the optimal catalytic conditions and kinetic parameters, in addition to the divalent metal effects, are described. Site-directed mutagenesis of Asp into Asn, targeted at the DDXXD motif, resulted in significantly decreased enzymatic activity.