Identification of the 190 kD microtubule-associated protein in cultured fibroblasts and its association with interphase and mitotic microtubules

We previously investigated the biochemical characteristics of microtubule-associated proteins (MAPs) of the adrenal medulla and adrenal cortex and found that they contain a new kind of MAP with a molecular weight of 190,000 (190 kD MAP) as a major species (Kotani, S., H. Murofushi, S. Maekawa, C. Sato, and H. Sakai. Eur. J. Biochem. 156, 23-29, 1986). We now have used an affinity purified anti-(190 kD MAP) antibody and show by indirect immunofluorescent microscopy the association of this MAP with microtubules in situ in TIG-3 cells (human embryonic lung fibroblasts). The 190 kD MAP was present along the interphase and mitotic microtubules, and there was no marked difference between the staining pattern with anti-tubulin and that with anti-(190 kD MAP) antibodies, evidence that the localization of 190 kD MAP is not restricted to the subset of microtubules. We also isolated MAPs from TIG-3 cells and identified their 190 kD MAP as a major heat-stable component. Several other unidentified polypeptides were recovered in the MAP fraction specifically.     

Morphological pathway of flagellar assembly in Salmonella typhimurium.

The process of flagellar assembly was investigated in Salmonella typhimurium. Seven types of flagellar precursors produced by various flagellar mutants were purified by CsCl density gradient protocol. They were characterized morphologically by electron microscopy, and biochemically by two-dimensional gel electrophoresis. The MS ring is formed in the absence of any other flagellar components, including the switch complex and the putative export apparatus. Four proteins previously identified as rod components, FlgB, FlgC, FlgF, FlgG, and another protein, FliE, assemble co-operatively into a stable structure. The hook is formed in two distinct steps; formation of its proximal part and elongation. Proximal part formation occurs, but elongation does not occur, in the absence of the LP ring. FlgD is necessary for hook formation, but not for LP-ring formation. A revised pathway of flagellar assembly is proposed based on these and other results.     

Precise identification of gene products in hearts after in vivo gene transfection, using Sendai virus-coated proteoliposomes.

Both efficient gene transfer and the exact identification of gene product are required for gene therapy. Gene transfection of green fluorescence protein (GFP) might be useful for the reporter. After in vivo cotransfection of GFP and beta-galactosidase (beta-Gal) genes in Sendai virus-coated proteoliposomes to rat hearts, we compared the sensitivity and specificity of three methods: GFP detection, histochemical staining (HC) of beta-Gal activity, and immunostaining (IS) of the beta-Gal protein. Fluorescence microscopy and double staining of HC and IS revealed that both GFP and IS were equally sensitive and fourfold superior to HC at the peak of gene expression. However, different from skeletal muscle, the GFP of transfected cardiomyocytes showed two demerits: the fluorescence quenching due to the intense staining of beta-Gal activity, and nonspecific autofluorescence from myocardium. Thus, specific IS would be so far the most reliable to identify the gene product in heart.     

Suppression of PPARγ through MKRN1-mediated ubiquitination and degradation prevents adipocyte differentiation

The central regulator of adipogenesis, PPARγ, is a nuclear receptor that is linked to obesity and metabolic diseases. Here we report that MKRN1 is an E3 ligase of PPARγ that induces its ubiquitination, followed by proteasome-dependent degradation. Furthermore, we identified two lysine sites at 184 and 185 that appear to be targeted for ubiquitination by MKRN1. Stable overexpression of MKRN1 reduced PPARγ protein levels and suppressed adipocyte differentiation in 3T3-L1 and C3H10T1/2 cells. In contrast, MKRN1 depletion stimulated adipocyte differentiation in these cells. Finally, MKRN1 knockout MEFs showed an increased capacity for adipocyte differentiation compared with wild-type MEFs, with a concomitant increase of PPARγ and adipogenic markers. Together, these data indicate that MKRN1 is an elusive PPARγ E3 ligase that targets PPARγ for proteasomal degradation by ubiquitin-dependent pathways, and further depict MKRN1 as a novel target for diseases involving PPARγ.     

Optimized fed-batch fermentation of L-β-hydroxy isobutyric acid by Yarrowia lipolytica

Yarrowia lipolytica KCCM50506, which transforms isobutyric acid to L-#-hydroxy isobutyric acid (L-#-HIBA), was screened. Chemostat cultures were carried out in jar fermentors at dilution rates of 0.02 h^у to 0.12 h^у. L-#-HIBA fermentation-regulating factors were determined to be specific growth rate, and concentrations of glucose and isobutyric acid in fermentor from analysis of steady-state data. The specific productivity of L-#-HIBA increased as the specific growth rate increased, apparently as a growth-associated type of product formation. A fed-batch culture was carried out under optimum conditions where the concentrations of glucose and isobutyric acid in the fermentor were maintained at 23 g l^у and 9 g l^у, respectively. The concentrations of cells and L-#-HIBA obtained at the end of fermentation were 20 g l^у and 49 g l^у, respectively, corresponding to 2.0 and 2.7 times more than concentrations in batch culture.     

Swabbing Often Fails to Detect Amphibian Chytridiomycosis under Conditions of Low Infection Load

The pathogenic chytrid fungus, Batrachochytrium dendrobatidis (denoted Bd), causes large-scale epizootics in naïve amphibian populations. Intervention strategies to rapidly respond to Bd incursions require sensitive and accurate diagnostic methods. Chytridiomycosis usually is assessed by quantitative polymerase chain reaction (qPCR) amplification of amphibian skin swabs. Results based on this method, however, sometimes yield inconsistent results on infection status and inaccurate scores of infection intensity. In Asia and other regions where amphibians typically bear low Bd loads, swab results are least reliable. We developed a Bd-sampling method that collects zoospores released by infected subjects into an aquatic medium. Bd DNA is extracted by filters and amplified by nested PCR. Using laboratory colonies and field populations of Bombina orientalis, we compare results with those obtained on the same subjects by qPCR of DNA extracted from swabs. Many subjects, despite being diagnosed as Bd-negative by conventional methods, released Bd zoospores into collection containers and thus must be considered infected. Infection loads determined from filtered water were at least 1000 times higher than those estimated from swabs. Subjects significantly varied in infection load, as they intermittently released zoospores, over a 5-day period. Thus, the method might be used to compare the infectivity of individuals and study the periodicity of zoospore release. Sampling methods based on water filtration can dramatically increase the capacity to accurately diagnose chytridiomycosis and contribute to a better understanding of the interactions between Bd and its hosts.     

VARIATION IN THE ULTRASTRUCTURE OF THE BIFLAGELLATE MOTILE CELLS OF SIX UNICELLULAR GENERA OF THE CHLAMYDOMONADALES AND CHLOROCOCCALES (CHLOROPHYCEAE), WITH EMPHASIS ON THE FLAGELLAR APPARATUS

The ultrastructural features of the biflagellate motile cells of six different species of the Chlorophyceae, namely Dunaliella lateralis (Polyblepharidaceae, Chlamydomonadales), Chlorococcum hypnosporum, Spongiochloris spongiosa, Protosiphon botryoides (Chlorococcaceae, Chlorococcales), Tetracystis aeria and Pseudotetracystis terrestris (Tetracystidaceae, Chlorococcales), were examined with an emphasis on the flagellar apparatus (FA). They have different vegetative characteristics, such as, being motile or nonmotile, variations in chloroplast morphology, possession of one or more nuclei, and reproductive features such as formation of tetrahedral tetrads, and naked or walled zoospores. Ultrastructural differences amongst reproductive cells of the six species include variations in cell surface structure, basal body to basal body angle, beamlike extensions of the distal fiber, extensive connections of the proximal sheath between basal bodies, two-membered rootlets, striated microtubule-associated components, two-membered rootlet-nucleus and/or mitochondria connections, X-membered rootlets, connections of rootlets and basal bodies, rhizoplasts and accessory basal bodies. All six species possess pyrenoids penetrated by thylakoid membranes, and the FA typical of the Chlorophyceae (sensu Mattox and Stewart, 1984). These six species should be divided into two groups. The first includes D. lateralis, C. hypnosporum, and T. aeria, in which accessory basal bodies are present, the basal body to basal body angle is relatively fixed, and a cell wall or surface coat is present. The second group includes Ps. terrestris, S. spongiosa, and Pr. botryoides, in which accessory basal bodies are absent, the basal body to basal body angle is variable and the zoospores are naked.