Radioimmunoassay of chenodeoxycholic acid-3-sulfate in urine

A sensitive and specific radioimmunoassay (RIA) for the measurement of urinary total chenodeoxycholic acid-3-sulfate (SCDCA) was developed and the accuracy was confirmed. SCDCA bound to bovine serum albumin as the antigen and emulsified with Freund's complete adjuvant was injected into rabbits. The antiserum obtained was capable of binding 75% of [11,12-3H]SCDCA at 1:1000 dilution. The percentage of bound radioactivity decreased linearly with logarithmic increases in unlabeled SCDCA, from 8 to 200 pmol/ml. The antiserum showed an extremely high specificity for SCDCA (free and conjugated), and the values determined by RIA indicated a close correlation with those found by gas-liquid chromatography. The daily urinary SCDCA level was determined using SCDCA-RIA in 12 disease-free humans and 74 patients with chronic liver diseases. In the normal subjects the daily urinary SCDCA level was 0.74 +/- 0.83 mg/day and increased levels were evident in all groups with chronic liver diseases. The daily urinary SCDCA level corresponds closely with the extent of hepatic dysfunction.     

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.     

Comparative studies of phosphoenolpyruvate carboxylase from c(3) and c(4) plants

Phosphoenolpyruvate carboxylase (PEPC) from several C₃ plants was compared to maize PEPC by immunoblotting using an antibody against maize PEPC and by peptide mapping. In C₃ gramineous plants, PEPCs of slightly different monomeric sizes were detected as two bands for wheat and barley leaves, as three bands for etiolated maize leaves and as four bands for rice leaves by SDS-polyacrylamide gel electrophoresis and immunoblotting, whereas only one PEPC band was detected for maize leaves, a C₄ plant, or tobacco leaves, a dicotyledonous C₃ plant. The peptide fragment patterns of the lower molecular weight PEPC (major band in immunoblotting) in wheat leaves was similar to that of maize PEPC in peptide mapping by protein staining or by immunological detection, but the upper one (minor band) had a different pattern from the lower one in peptide mapping by immunological detection and few peptide fragments from this were recognized by the anti-(maize) PEPC antibody. These results suggest that there are multiple forms of PEPC subunits in the gramineous plants tested, and the major PEPC has a primary structure similar to that of maize PEPC. To obtain information about the expression of PEPCs in C₃ plants, changes in the amount of PEPC protein were investigated during the greening of rice and wheat seedlings. Judging from the regulation by light, there were two types of PEPCs in greening rice seedlings, one induced by light and the other reduced by it. Greening wheat seedlings also show a PEPC band induced by light. These findings indicate that some PEPCs in C₃ gramineous plants not only have structures similar to that of maize PEPC, but also are regulated by light in a similar manner.     

Nucleotide sequence of the PR-1 gene of Nicotiana tabacum

A gene encoding one of the pathogenesis-related proteins, PR1a, and two related pseudogenes were isolated from Nicotiana tabacum. The cloned PR1a gene (pPR-gamma) and one of the pseudogenes (pPR-alpha) were sequenced and found to have similar structures. The sequence of pPR-gamma was quite similar to that of the cDNA clone of PR1a. The plasmid pPR-gamma did not contain an intron and had a typical promoter sequence in the 5'-flanking region.