Calnexin Delta 185-520 partially reverses the misprocessing of the Delta F508 cystic fibrosis transmembrane conductance regulator

We analyzed the interrelation between the efficiency of a gene expression and the nucleotide composition of all protein-coding sequences in 38 unicellular organisms whose complete genomic sequences are known. These organisms comprise 37 prokaryotic (29 eubacteria and eight archaebacteria) and one eukaryotic (yeast) species. We demonstrated that frequency analysis of gene codon composition fails to reflect adequately the gene expression efficiency of all these organisms. We constructed a measure, the elongation efficiency index, that considers simultaneously the information on codon frequencies and the degree of mRNA local self-complementarity. This measure recognizes the ribosome-coding genes as highly expressed in all the unicellular organisms studied. According to our analysis, these species fall into five groups differentiated by the process that makes the key contribution to the elongation rate.     

Chick bone collagenase inhibitor and latency of collagenase

Collagenase and collagenase inhibitor were isolated from the culture fluid of embryonic chick bone. The inhibitor, separated as a high molecular weight aggregate (160,000–200,000 daltons) during gel filtration in 1M NaCl, dissociated in 6M urea to species of approx 25,000 daltons. The inhibition of collagenase activity by the addition of inhibitor was not reversed by the addition of trypsin or p-aminophenylmercuric acetate. However, isolated inhibitor alone was inactivated by treatment with either trypsin or p-aminophenylmercuric acetate. The results suggest that the latent form of chick bone collagenase is a proenzyme which converts into an active form without a detectable change in molecular weight and that this occurs after the inactivation of collagenase inhibitor.     

Frequency distribution of axon diameters in the left aortic nerve of the rat raised in space--Neurolab Program.

Introduction: To study development of the aortic baroreflex mechanisms under conditions of microgravity, we carried out the various experiments in the neonate rats aged 25 days raised in microgravity for 16 days (flight: FLT group) in Neurolab program (STS-90, space shuttle Columbia, launch date: Apr 17, '98. Some results of the experiments were already reported. The results of histological examination of the aortic nerve which is the afferent of the aortic baroreflex showed that the number of unmyelinated fibers in FLT was significantly less in than those in two control groups and there was no difference between FLT and each control group in the analysis of myelin. In the present paper, the frequency distribution of axon diameters of the left aortic nerves in FLT was compared to that in two ground control groups to examine the growth of the aortic nerve fibers in space. Methods: After breeding Sprague-Dawley rats for 16 days in the shuttle in space and in the animal center in the Kennedy space center, a total of 43 deeply anesthetized rats were perfused with 1% parahormaldehyde and 1% glutaraldehyde or 4% parahormaldehyde solution buffered at pH7.4 with 0.12 M phosphate solution. Concerning the control groups, one group was the asynchronous ground control (AGC) group in which the rats were housed in the same cages as those on the shuttle, and the other was the vivarium ground control (VIV) group in which the rats were housed in commercial cages. The cervical region of the left aortic nerve which is a branch of the vagus was cut off and stored in the same fixative as that used for perfusion, and postfixed in the solution of 1% OsO4, for 2 hours within 24 hours after the perfusion. The fixed specimens were embedded in epoxy resin blocks by the usual method for electron microscopy following dehydration. Electron microscopic montages of transverse sections of these nerve trunks were made from the five left aortic nerves in each group. The magnification of the montages was approximately 13400 times. The long and short axes (a and b) of the nerve fibers and the myelin thickness (T) were measured with a caliper and the axon diameters (R were calculated by following formula: R2=[(a-2T)2+(b-2T)2]/2.     

A monoclonal antibody-based enzyme-linked immunosorbent assay of aflatoxin B1 in peanut products

An improved enzyme-linked immunosorbent assay (ELISA) combined with monoclonal antibody (MAb) and one-step extraction method was established for the estimation of aflatoxin B₁ (AFB₁) in a peanut product. AFB₁ was converted to AFB₁-oxime, and then conjugated with bovine serum albumin (BSA). Spleen cells from mice immunized with AFB₁-BSA conjugates were fused with myeloma cells. After double selection with AFB₁-ovalbumin (OVA) and carbodiimide-modified OVA, five stable hybridoma cells secreting anti-AFB₁ MAbs (AF1, AF 2, AF 3, AF 4, and AF5) were cloned. Using these anti-AFB₁ MAbs, we developed the indirect competitive ELISA (cELISA) with alkaline phosphatase (ALP) — labeled sheep anti — mouse IgG as marker and the direct cELISA with AFBi-oxime horseradish peroxidase (POD) as marker. The minimum detectable limits of the indirect cELISA with AF 1, 2, 3, 4, and 5 were 5, 5, 5, 5, and 50 pg of standard AFB₁ per assay, respectively, and those of the direct cELISA with AF 1, 3, 4, and 5 were 2.5, 5, 25, and 100 pg of standard AFB₁ per assay, respectively. The cross reactivity of each toxins with these MAbs in the indirect cELISA was as follows: (a) AF 1 and AF 2 were reactive with AFB₂ as well as AFB₁, weakly with AFG₂ > AFG₁ > aflatoxicol II (COL II) > aflatoxicol I (COL I) and less weakly with other aflatoxins; (b) AF 3 and AF 4 were reactive with COL II as well as AFB₁, weakly with COLI > AFQ₁ and less weakly with others; (c) AF 5 was AFQ₁ as well as AFB₁ weakly with COL II > AFG₂ > COL I and less weakly with others. The 60% aqueous methanol extracts of oil-roasted blanched peanuts (“butter peanut”), naturally contaminated with AFB₁, were assayed by the direct cELISA without further purification. The direct cELISA with the most sensitive MAb AF 1 was allowed to determine 1 ng of AFB₁ per g samples.     

Asymmetric arrangement of auxiliary subunits of skeletal muscle voltage-gated l-type Ca(2+) channel

Highly purified L-type Ca(2+) channel complexes containing all five subunits (alpha(1), alpha(2), beta, gamma, and delta) and complexes of alpha(1)-beta subunits were obtained from skeletal muscle triad membranes by three-step purification and by 1% Triton X-100 treatment, respectively. Their structures and the subunit arrangements were analyzed by electron microscopy. Projection images of negatively stained Ca(2+) channels and alpha(1)-beta complexes were aligned, classified and averaged. The alpha(1)-beta complex showed a hollow trapezoid shape of 12 nm height. In top view, four asymmetric domains surrounded a central depression predicted to form the channel pore. The complete Ca(2+) channel complex exhibited the cylindrical shape of 20 nm in height binding a spherical domain on one edge. Further image analysis of higher complexes of the Ca(2+) channel using a monoclonal antibody against the beta subunit showed that the alpha(1)-beta complex forms the non-decorated side of the cylinder, which can traverse the membrane from outside the cell to the cytoplasm. Based on these results, we propose that the Ca(2+) channel exhibits an asymmetric arrangement of auxiliary subunits.     

Application of an enzyme-linked immunosorbent assay for screening of T-2 toxin-producing Fusarium spp

Culture filtrates of Fusarium species were subjected without clean-up procedures to an indirect competitive enzyme-linked immunosorbent assay with anti-T-2 toxin monoclonal antibody. Fusarium sporotrichioides, F. poae, F. tricinctum, and F. culmorum strains were positive for T-2 toxin, with a minimum detection limit of 5 pg per assay (100 pg/ml of culture filtrate), and the assay data correlated well with the gas-liquid chromatographic data.     

Properties of colony-stimulating factors produced by macrophage cell lines and hybrid cells

Colony-stimulating factors (CSFs) produced by two simian virus 40(SV40) transformed macrophage cell lines (BAM1 and BAM3), and three hybrids (HM3-11, HM3-12, and HM3-14) derived from fusion between BAM3 and a Chinese hamster cell line (hs222-16) were examined. HM3-11 and HM3-14 produce two molecular species of CSF, which are not found in the conditioned media from cultures of BAM1 and BAM3 or lipopolysaccharide (LPS), phorbolmyristate-acetate (PMA), and zymosan-stimulated BAM3. HM3-12, which is classified into another group in terms of CSF secretion, does not produce these two CSFs. On the basis of various criteria, one of these CSF species (peak 1-CSF) was characterized as a macrophage-colony-stimulating factor (M-CSF). The other CSF (peak 2-CSF) induced a group of bone marrow cells in granulocytes and macrophages as well as growth of a mast cell line, IC2. This CSF has an apparent molecular weight of 18,000, estimated by SDS-polyacrylamide gel electrophoresis. Unlike interleukin 3 (IL3) from WEHI-3 cells, the growth factor activity of peak 2-CSF binds to DEAE-Sephacel. Thus, peak 2-CSF is similar to a granulocyte-macrophage colony-stimulating factor (GM-CSF) rather than to IL3. The anti L cell CSF serum does not inhibit the CSF activity in Chinese hamster fibroblast conditioned medium, and the IC2 cells do not respond to Chinese hamster lung conditioned medium (CHLCM), suggesting that peak 1- and peak 2-CSF are of mouse origin.     

MEK and Cdc2 kinase are sequentially required for Golgi disassembly in MDCK cells by the mitotic Xenopus extracts

At the onset of mitosis, the Golgi apparatus, which consists of several cisternae, disperses throughout the cell to be partitioned into daughter cells. The molecular mechanisms of this process are now beginning to be understood. To investigate the biochemical requirements and kinetics of mitotic Golgi membrane dynamics in polarized cells, we have reconstituted the disassembly of the Golgi apparatus by introducing Xenopus egg extracts into permeabilized Mardin-Darby canine kidney (MDCK) cells. We used green fluorescence protein (GFP)-tagged galactosyltransferase-expressing MDCK cells to analyze the morphological changes of the Golgi membrane in the semi-intact system. Analyses by fluorescence and electron microscopies showed that the Golgi disassembly can be dissected into two elementary processes morphologically. In the first process, the perinuclear Golgi stacks break into punctate structures, intermediates, which are comprised of mini-stacks of cisternae associating with apical microtubule networks. In the second process, the structures fragment more thoroughly or substantially relocate to the ER. Our analyses further showed that cdc2 kinase and mitogen-activated protein kinase kinase (MAPKK = MEK) are differently involved in these two processes: the first process is mainly regulated by MEK and the second mainly by cdc2.     

Schistosoma mansoni and alpha-galactosylceramide: prophylactic effect of Th1 Immune suppression in a mouse model of Graves' hyperthyroidism

Graves' hyperthyroidism, an organ-specific autoimmune disease mediated by stimulatory thyrotropin receptor (TSHR) autoantibodies, has been considered a Th2-dominant disease. However, recent data with mouse Graves' models are conflicting. For example, we recently demonstrated that injection of BALB/c mice with adenovirus coding the TSHR induced Graves' hyperthyroidism characterized by mixed Th1 and Th2 immune responses against the TSHR, and that transient coexpression of the Th2 cytokine IL-4 by adenovirus skewed Ag-specific immune response toward Th2 and suppressed disease induction. To gain further insight into the relationship between immune polarization and Graves' disease, we evaluated the effect of Th2 immune polarization by helminth Schistosoma mansoni infection and alpha-galactosylceramide (alpha-GalCer), both known to bias the systemic immune response to Th2, on Graves' disease. S. mansoni infection first induced mixed Th1 and Th2 immune responses to soluble worm Ags, followed by a Th2 response to soluble egg Ags. Prior infection with S. mansoni suppressed the Th1-type anti-TSHR immune response, as demonstrated by impaired Ag-specific IFN-gamma secretion of splenocytes and decreased titers of IgG2a subclass anti-TSHR Abs, and also prevented disease development. Similarly, alpha-GalCer suppressed Ag-specific splenocyte secretion of IFN-gamma and prevented disease induction. However, once the anti-TSHR immune response was fully induced, S. mansoni or alpha-GalCer was ineffective in curing disease. These data support the Th1 theory in Graves' disease and indicate that suppression of the Th1-type immune response at the time of Ag priming may be crucial for inhibiting the pathogenic anti-TSHR immune response.