Identification of CRAM, a novel unc-33 gene family protein that associates with CRMP3 and protein-tyrosine kinase(s) in the developing rat brain

Four members of collapsin response mediator proteins (CRMPs) are thought to be involved in the semaphorin-induced growth cone collapse during neural development. Here we report the identification of a novel CRMP3-associated protein, designated CRAM for CRMP3-associated molecule, that belongs to the unc-33 gene family. The deduced amino acid sequence reveals that the CRAM gene encodes a protein of 563 amino acids, shows 57% identity with dihydropyrimidinase, and shows 50-51% identity with CRMPs. CRAM appears to form a large complex composed of CRMP3 and other unidentified proteins in vivo. Indeed, CRAM physically associates with CRMP3 when co-expressed in COS-7 cells. The expression of CRAM is brain-specific, is high in fetal and neonatal rat brain, and decreases to very low levels in adult brain. Moreover, CRAM expression is up-regulated during neuronal differentiation of embryonal carcinoma P19 and PC12 cells. Finally, immunoprecipitation analysis of rat brain extracts shows that CRAM is co-immunoprecipitated with proteins that contain protein-tyrosine kinase activity. Taken together, our results suggest that CRAM, which interacts with CRMP3 and protein-tyrosine kinase(s), is a new member of an emerging family of molecules that potentially mediate signals involved in the guidance and outgrowth of axons.     

Phosphorylation of the GTS1 gene product of the yeast Saccharomyces cerevisiae and its effect on heat tolerance and flocculation

The GTS1 gene from the yeast Saccharomyces cerevisiae showed pleiotropic effects on yeast phenotypes, including an increase of heat tolerance in stationary-phase cells and an induction of flocculation. Here, we found that the GTS1 product, Gts1p, was partially phosphorylated at some serine residue(s) in cells grown on glucose. Studies using mutants of protein kinase A (PKA) and CDC25, the Ras-GTP exchange activator, showed that PKA positively regulated the phosphorylation level of Gts1p. Overexpression of Gts1p in a mutant with attenuated PKA activity did not show any increase of heat tolerance and partially decreased flocculation inducibility, suggesting that phosphorylation of Gts1p is required for induction of these phenomena.     

Functional association between nicotinic acetylcholine receptor and sarcomeric proteins via actin and desmin filaments

By affinity chromatography utilizing alpha-cobrotoxin from digitonin-solubilized fractions of rabbit skeletal muscle, we found that many proteins are associated with the nicotinic acetylcholine receptor (AChR). In addition to the proteins we previously reported to bind to AChR (including dystrophin-dystrophin-associated protein (DAP) complex, utrophin, rapsyn, and actin; Mitsui et al. [1996] Biochem. Biophys. Res. Commun.224:802-807), alpha-actinin, desmin, myosin, tropomyosin, troponin T, and titin are also identified to be associated with AChR. Alkaline treatment or Triton X-100 solubilization released dystrophin-DAP complex, utrophin, and rapsyn from the AChR fraction, while actin and desmin remained associated. These findings demonstrate that AChR is supported primarily by a submembranous organization of actin and desmin filaments, and is linked to sarcomeric proteins via these filaments. To further investigate whether the association has any functional role, we studied the effect of acetylcoline on ATPase activity of the AChR fraction. Acetylcholine (0.5-4 microM) significantly activated Mg(2+)-ATPase activity of digitonin-solubilized AChR fraction (P < 0.05). Furthermore, we found that desmin as well as actin activated myosin Mg(2+)-ATPase activity. From these findings, it is suggested that desmin and actin form a submembranous organization in the postsynaptic region, and function as mediators of excitation of AChR to the sarcomeric contraction system.     

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.     

Phylogenetic relationships among strains of the entomopathogenic fungus Beauveria bassiana (Hypocreales: Clavicipitaceae) isolated from Japan

The fungus Beauveria bassiana (Balsamo) Vuillemin has previously been classified using morphological characteristics, but morphology cannot reveal the phylogenetic relationships among conventionally classified strains. High levels of homology have been found in gene sequences among various B. bassiana strains, complicating the determination of their evolutionary relationships. To elucidate phylogenetic relationships among conventionally known Beauveria species, we analyzed 57 major strains of B. bassiana and 3 strains of B. brongniartii (Saccardo) Petch isolated from Japan by analysis of internal transcribed spacer (ITS) sequences and genome profiling (GP) based on temperature gradient gel electrophoresis of random PCR products. The ITS sequence analysis placed the 57 conventional B. bassiana strains into two clusters, B. bassiana and Beauveria pseudobassiana Rehner et Humber. In contrast, GP analysis produced five clusters of B. bassiana strains that included B. pseudobassiana clusters. These results suggested that GP was more accurate than ITS sequence analysis for determining phylogenetic relationships within B. bassiana. In addition, our findings suggested that conventional strains of B. bassiana isolated from Japan include both B. bassiana and B. pseudobassiana groups.     

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.