A naturally occurring mutation in the SLC21A6 gene causing impaired membrane localization of the hepatocyte uptake transporter

The organic anion transporter SLC21A6 (also known as OATP2, OATP-C, or LST-1) is involved in the hepatocellular uptake of a variety of endogenous and xenobiotic substances and drugs. We analyzed 81 human liver samples by immunoblotting and found one with a strongly reduced amount of SLC21A6 protein suggesting mutations in the SLC21A6 gene. The SLC21A6 cDNA from this sample contained five base pair changes in one allele; three of the mutations resulted in amino acid substitutions designated SLC21A6-N130D, SLC21A6-P155T, and SLC21A6-L193R. The former two were polymorphisms (SLC21A6*1b and SLC21A6*4), whereas SLC21A6-L193R represents the first naturally occurring mutation identified in one allele of the SLC21A6 gene, which affects protein maturation and organic anion transport. We introduced each of the mutations into the SLC21A6 cDNA and established stably transfected MDCKII cells expressing the respective mutant SLC21A6 protein. Immunofluorescence microscopy and uptake measurements were used to study localization and transport properties of the mutated proteins. Both proteins carrying the polymorphisms were sorted to the lateral membrane like wild-type SLC21A6, but their transport properties for the substrates cholyltaurine and 17beta-glucuronosyl estradiol were altered. Importantly, most of the mutant protein SLC21A6-L193R was retained intracellularly, and this single amino acid exchange abolished transport function.     

First detection of the microsporidium Enterocytozoon bieneusi in non-mammalian hosts (chickens)

Faecal samples taken from eight underweight, approximately 5-week-old broiler chickens in a poultry abattoir were investigated for microsporidial infections by light microscopy, electron microscopy, and PCR. In two of six chickens, which were suspected of being infected with microsporidia by light microscopy, Enterocytozoon bieneusi (genotype 'J') was detected by PCR and DNA sequencing, and in one of the two PCR-positive samples by extensive electron microscopy. This is the first time that E. bieneusi has been detected in chickens, i.e. in a non-mammalian species.     

Genetic analysis of functions involved in the late stages of biofilm development in Burkholderia cepacia H111

Burkholderia cepacia and Pseudomonas aeruginosa often co-exist as mixed biofilms in the lungs of patients suffering from cystic fibrosis (CF). Here, we report the isolation of 13 random mini-Tn5 insertion mutants of B. cepacia H111 that are defective in biofilm formation on a polystyrene surface. We show that the screening procedure used in this study is biased towards mutants defective in the late stages of biofilm development. A detailed quantitative analysis of the biofilm structures formed by wild-type and mutant strains revealed that the isolated mutants are impaired in their abilities to develop a typical three-dimensional biofilm structure. Molecular investigations showed that the genes required for biofilm maturation fall into several classes: (i). genes encoding for surface proteins; (ii). genes involved in the biogenesis and maintenance of an integral outer membrane; and (iii). genes encoding regulatory factors. It is shown that three of the regulatory mutants produce greatly reduced amounts of N-octanoylhomoserine lactone (C8-HSL). This compound serves as the major signal molecule of the cep quorum-sensing system. As this density-dependent regulatory system is involved in the regulation of biofilm maturation, we investigated the interplay between the three regulatory genes and the quorum-sensing cascade. The results of these investigations show that the identified genes encode for regulatory elements that are positioned upstream of the cep system, indicating that the quorum-sensing system of B. cepacia is a major checkpoint for biofilm formation.     

Biphasic oxygen kinetics of cellular respiration and linear oxygen dependence of antimycin A inhibited oxygen consumption

Oxygen kinetics in fibroblasts was biphasic. This was quantitatively explained by a major mitochondrial hyperbolic component in the low-oxygen range and a linear increase of rotenone-and antimycin A-inhibited oxygen consumption in the high-oxygen range. This suggests an increased production of reactive oxygen species and oxidative stress at elevated, air-level oxygen concentrations. The high oxygen affinity of mitochondrial respiration provides the basis for the maintenance of a high aerobic scope at physiological low-oxygen levels, whereas further pronounced depression of oxygen pressure induces energetic stress under hypoxia.