Missense mutations in SGLT1 cause glucose-galactose malabsorption by trafficking defects

Glucose-galactose malabsorption (GGM) is an autosomal recessive disorder caused by defects in the Na+/glucose cotransporter (SGLT1). Neonates present with severe diarrhea while on any diet containing glucose and/or galactose [1]. This study focuses on a patient of Swiss and Dominican descent. All 15 exons of SGLT1 were screened using single stranded conformational polymorphism analyses, and aberrant PCR products were sequenced. Two missense mutations, Gly318Arg and Ala468Val, were identified. SGLT1 mutants were expressed in Xenopus laevis oocytes for radiotracer uptake, electrophysiological experiments, and Western blotting. Uptakes of [14C]alpha-methyl-d-glucoside by the mutants were 5% or less than that of wild-type. Two-electrode voltage-clamp experiments confirmed the transport defects, as no noticeable sugar-induced current could be elicited from either mutant [2]. Western blots of cell protein showed levels of each SGLT1 mutant protein comparable to that of wild-type, and that both were core-glycosylated. Presteady-state current measurements indicated an absence of SGLT1 in the plasma membrane. We suggest that the compound heterozygote missense mutations G318R and A468V lead to GGM in this patient by defective trafficking of mutant proteins from the endoplasmic reticulum to the plasma membrane.     

Characterization of the carp myosin heavy chain multigene family

We isolated partial coding sequences for 29 carp myosin heavy chain genes (MyoHCs) and determined the nucleotide sequences around the region encoding the loop 2 of the myosin molecule. The predicted amino acid sequences from the isolated genes all showed very high similarity to those of skeletal and cardiac muscles from higher vertebrates, but not to those of smooth and non-muscle counterparts. Among all clones isolated, carp MyoHC10, MyoHCI-1-3 and MyoHC30 showed exon-nucleotide sequences identical to those of cDNAs encoding the loop 2 region of the 10 degrees C-, intermediate- and 30 degrees C-type fast skeletal isoforms [Hirayama and Watabe, Euro. J. Biochem. 246 (1997) 380-387]. The loop 2 of 28 types of carp MyoHCs was encoded by two exons separated by an intron corresponding to that of the 16th in higher vertebrate MyoHCs, whilst this intron was not found in carp MyoHC30. Although carp MyoHC30 had a gene organization different from those of higher vertebrates and other carp MyoHCs, its predicted amino acid sequence for loop 2 showed the highest homology to those of higher vertebrates among carp MyoHCs. In the 28 carp MyoHCs containing the intron, a combination of different nucleotide sequences for the two resulted in 14 distinct series for the combined coding sequence. These different nucleotide sequences encoded nine distinct amino acid sequences. Phylogenetic analysis for the present loop 2 and light meromyosin previously reported for carp MyoHCs [Imai et al., J. Exp. Biol. 200 (1997) 27-34] revealed that carp MyoHCs have recently diverged and are more closely related to each other than to MyoHCs from other species.     

Stable isotope labeling of Arabidopsis thaliana for an NMR-based metabolomics approach

Nuclear magnetic resonance (NMR) will become a key technology in plant metabolomics with the use of stable isotope labeling and advanced hetero-nuclear NMR methodologies. To demonstrate the power of this approach, we performed multi-dimensional hetero-nuclear NMR analysis of metabolic movement of carbon and nitrogen nuclei in Arabidopsis thaliana. First, distinct ethanol-stress response was investigated using (13)C-labeled wild type and an ethanol-hypersensitive mutant plants. Furthermore, we followed nitrogen fluxes in (15)N-labeled seeds during the initiation of germination in vivo. The future role of stable isotope-labeling combined with advanced hetero-nuclear NMR in plant metabolomics is discussed.     

Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The product of LAMP is detected by the turbidity of the reaction mixture without electrophoresis. The objective of this study was to develop a rapid sexing method for bovine preimplantation embryos using LAMP. The first experiment was conducted to optimize the DNA extraction method for LAMP-based embryo sexing. The DNA of single blastomeres was extracted using three methods: heat, NaOH, and proteinase K-Tween 20 (PK-TW) treatments. Sexing was performed with two LAMP reactions, male-specific and male-female common reaction, after DNA extraction. The rates of correct determination of sex were 88.9-94.4%, with no difference among methods. The sensitivity and accuracy of LAMP-based embryo sexing were evaluated in the next experiment. The proportion of samples in which the sex was correctly determined was 75-100% for one to five biopsied cells. Lastly, in vivo-derived embryos were examined to verify the usefulness of LAMP-based embryo sexing, and some of these fresh, sexed embryos were transferred into recipient animals. The time needed for sexing was <1 h. The pregnancy rate was 57.4% and all calves born were of the predicted sex (12 male and 21 female). Therefore, LAMP-based embryo sexing accurately determined gender and is suitable for field application.     

Malnutrition impairs alveolar fluid clearance in rat lungs

Inadequate nutrition complicates the clinical course of critically ill patients, and many of these patients develop pulmonary edema. However, little is known about the effect of malnutrition on the mechanisms that resolve alveolar edema. Therefore, we studied the mechanisms responsible for the decrease in alveolar fluid clearance in rats exposed to malnutrition. Rats were allowed access to water, but not to food, for 120 h. Then, the left and right lungs were isolated for the measurement of lung water volume and alveolar fluid clearance, respectively. The rate of alveolar fluid clearance was measured by the progressive increase in the concentration of Evans blue dye that was instilled into the distal air spaces with an isosmolar 5% albumin solution over 1 h. Malnutrition decreased alveolar fluid clearance by 38% compared with controls. Amiloride (10(-3) M) abolished alveolar fluid clearance in malnourished rats. Either refeeding for 120 h following nutritional deprivation for 120 h or an oral supply of sodium glutamate during nutritional deprivation for 120 h restored alveolar fluid clearance to 91 and 86% of normal, respectively. Dibutyryl-cGMP, a cyclic nucleotide-gated cation channel agonist, increased alveolar fluid clearance in malnourished rats supplied with sodium glutamate. Terbutaline, a beta(2)-adrenergic agonist, increased alveolar fluid clearance in rats under all conditions (control, malnutrition, refeeding, and glutamate-treated). These results indicate that malnutrition impairs primarily amiloride-insensitive and dibutyryl-cGMP-sensitive alveolar fluid clearance, but this effect is partially reversible by refeeding, treatment with sodium glutamate, or beta-adrenergic agonist therapy.     

Involvement of syntaxin 7 in human gastric epithelial cell vacuolation induced by the Helicobacter pylori-produced cytotoxin VacA

The Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. The formed vacuole is assumed to be a hybrid of late endosome and lysosome. To elucidate the molecular mechanism of VacA-induced vacuolation, we examined the participation of syntaxin 7 in the human gastric epithelial cell line AGS. Immunocytochemistry revealed that endogenous syntaxin 7 was localized to vacuoles induced by VacA. Northern and Western blotting demonstrated that VacA intoxication increased syntaxin 7 mRNA and protein expression, respectively, in a time-dependent manner. Transient transfection of dominant-negative mutant syntaxin 7, which lacks a carboxyl-terminal transmembrane domain, inhibited VacA-induced vacuolation. In contrast, transient transfection of wild-type syntaxin 7, dominant-negative mutant syntaxin 1a, or dominant-negative mutant syntaxin 4 did not alter VacA-induced vacuolation. Furthermore, under VacA treatment, neutral red dye uptake, a parameter of VacA-induced vacuolation, was inhibited in cells stably transfected with mutant syntaxin 7 but not in cells stably transfected with wild-type syntaxin 7, mutant syntaxin 1a, or mutant syntaxin 4. Sequential immunocytochemical observation confirmed that expression of mutant syntaxin 7 did not affect VacA attachment to or internalization into AGS cells. We suggest that syntaxin 7 is involved in the intracellular vacuolation induced by VacA.     

Levels and behavior of 2-phenylbenzotoriazole-type mutagens in the effluent of a sewage treatment plant

We previously reported on the isolation and structural determination of five 2-phenylbenzotriazole (PBTA)-type mutagens (PBTA-1, PBTA-2, PBTA-3, PBTA-4 and PBTA-6) in blue rayon/cotton adsorbed substances collected from surface waters at sites located downstream of sewage treatment plants. We also noted that PBTA-1 and PBTA-2 were discharged from sewage treatment plants and subsequently diluted or decomposed while moving down the Yodo River system. However, it has not been investigated whether they are commonly discharged from sewage treatment plants into rivers. The main purpose of this study was to make a comprehensive survey of levels and behavior of PBTA-type mutagens in effluents discharged from the sewage treatment plant located along the bank of the Uji River, one tributary of the Yodo River system. Water samples were collected at the outlet of the sewage treatment plant for 16 consecutive days in May 1999 and 11 consecutive days in December 1999. Organic constituents were obtained via sorption to blue rayon and subsequent methanol elution. Extract mutagenic activity was measured using Salmonella typhimurium YG1024 with metabolic activation. PBTA-type mutagens (PBTA-1, PBTA-2, PBTA-3, PBTA-4, PBTA-5 and PBTA-6) were quantified by HPLC with electrochemical detection, followed by HPLC purification on reverse-phase columns. The study showed that PBTA-2, PBTA-3, PBTA-4 and PBTA-6 were detected in most samples. The total contribution of these four PBTA-type mutagens to overall extract mutagenicity is on average 33% for the May 1999 sample and 58% for the December 1999 sample. The individual PBTA compounds that had the largest contribution to the overall mutagenicity were PBTA-3 and PBTA-4, accounting for 11 and 16% in May 1999, and 25 and 26% in December 1999. A further comparative study was done in December 1999 using the blue rayon hanging method and the results were similar to those obtained using the blue rayon column method. In conclusion, the present study showed that PBTA-2, PBTA-3, PBTA-4 and PBTA-6 were commonly discharged from a sewage treatment plant into the Uji River, and they accounted for a substantial portion of the effluent mutagenicity.     

The protozoa dinoflagellate Oxyrrhis marina contains selenoproteins and the relevant translation apparatus

In the phylogenetic tree, selenoproteins and the corresponding translation machinery are found in Archaea, Eubacteria, and animals, but not in fungi and higher plants. As very little is known about Protozoa, we searched for the presence of selenoproteins in the primitive dinoflagellate Oxyrrhis marina, belonging to the Protoctista kingdom. Four selenoproteins could be obtained from O. marina cells cultured in the presence of 75Se. Using O. marina or bovine liver cytosolic extracts, we could serylate and selenylate in vitro total O. marina tRNAs. Moreover, the existence of a tRNA(Sec) could be deduced from in vivo experiments. Lastly, an anti-serum against the specialized mammalian translation elongation factor mSelB reacted with a protein of 48-kDa molecular mass. Altogether, our data showed that O. marina contains selenoproteins and suggests that the corresponding translation machinery is related to that found in animals.