Validity of NIR spectroscopy for quantitatively measuring muscle oxidative metabolic rate in exercise

The purpose of this studywas to examine the validity of the quantitative measurement of muscleoxidative metabolism in exercise by near-infrared continuous-wavespectroscopy (NIRcws). Twelve male subjects performed two bouts ofdynamic handgrip exercise, once for the NIRcws measurement and once forthe ³¹P-magnetic resonance spectroscopy (MRS) measurementas a standard measure. The resting muscle metabolic rate (RMRmus) wasindependently measured by ³¹P-MRS during 15 min of arterialocclusion at rest. During the first exercise bout, the quantitativevalue of muscle oxidative metabolic rate at 30 s postexercisewas evaluated from the ratio of the rate of oxyhemoglobin/myoglobindecline measured by NIRcws during arterial occlusion 30 s afterexercise and the rate at rest. Therefore, the absolute values of muscleoxidative metabolic rate at 30 s after exercise[O_2NIR(30)] wascalculated from this ratio multiplied by RMRmus. During the secondexercise bout, creatine phosphate (PCr) resynthesis rate was measuredby ³¹P-MRS at 30 s postexercise[Q₍₃₀₎] under the same conditions but without arterial occlusion postexercise. To determine the validity ofNIRcws, O_2NIR(30) wascompared with Q₍₃₀₎. There was a significant correlation betweenO_2NIR(30), which rangedbetween 0.018 and 0.187 mM ATP/s, and Q₍₃₀₎,which ranged between 0.041 and 0.209 mM ATP/s (r = 0.965, P < 0.001). This result supports theapplication of NIRcws to quantitatively evaluate muscle oxidativemetabolic rate in exercise.

     

High-throughput single-nucleotide polymorphism genotyping by fluorescent competitive allele-specific polymerase chain reaction (SNiPTag)

Single nucleotide polymorphisms (SNPs) are becoming widely recognized as the new currency for gene mapping as increasing numbers are discovered. Here we outline a method for their rapid analysis based on an allele-specific polymerase chain reaction (PCR) which employs a competitive approach, whereby both allele-specific primers are present in the same reaction and carry different fluorescent labels. This procedure is simple and amenable to high-throughput genotyping using conventional automated sequencing equipment, and no post-PCR modifications are required. Verification of the procedure was carried out by comparison of results derived by this method with those from restriction enzyme digestion of the ALDH2 exon 12 functional polymorphism (Glu-487-Lys) in 109 individuals. Additionally, we have examined all combinations of nucleotide substitutions and shown them to be differentiated by this method. As proof of concept, several assays were combined and loaded on a single gel lane/capillary to substantially improve throughput. This was made possible by designing the PCR products to be of different lengths and no interference was observed between products differing in size by only six nucleotides. We outline a number of test assays for well-characterized SNPs in human candidate genes for behavioral disorders.     

Affinities between Asian non-human Schistosoma species,the S. indicum group,and the African human schistosomes

Schistosoma species have traditionally been arranged in groups based on egg morphology, geographical origins, and the genus or family of snail intermediate host. One of these groups is the 'S. indicum group' comprising species from Asia that use pulmonate snails as intermediate hosts. DNA sequences were obtained from the four members of this group (S. indicum, S. spindale, S. nasale and S. incognitum) to provide information concerning their phylogenetic relationships with other Asian and African species and species groups. The sequences came from the second internal transcribed spacer (ITS2) of the ribosomal gene repeat, part of the 28S ribosomal RNA gene (28S), and part of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene. Tree analyses using both distance and parsimony methods showed the S. indicum group not to be monophyletic. Schistosoma indicum, S. spindale and S. nasale were clustered among African schistosomes, while S. incognitum was placed as sister to the African species (using ITS2 and 28S nucleotide sequences and CO1 amino acid sequences), or as sister to all other species of Schistosoma (CO1 nucleotide sequences). Based on the present molecular data, a scenario for the evolution of the S. indicum group is discussed.     

Transgenic mouse model of tauopathies with glial pathology and nervous system degeneration

Frontotemporal dementias (FTDs), including corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP), are neurodegenerative tauopathies characterized by widespread CNS neuronal and glial tau pathologies, but there are no tau transgenic (Tg) mice that model neurodegeneration with glia tau lesions. Thus, we generated Tg mice overexpressing human tau in neurons and glia. No neuronal tau aggregates were detected, but old mice developed Thioflavin S- and Gallyas-positive glial tau pathology resembling CBD astrocytic plaques. Tau-immunoreactive and Gallyas-positive oligodendroglial coiled bodies (similar to CBD and PSP), glial degeneration, and motor deficits were associated with age-dependent accumulations of insoluble hyperphosphorylated human tau and tau immunopositive filaments in degenerating glial cells. Thus, tau-positive glial lesions similar to human FTDs occur in these Tg mice, and these pathologies are linked to glial and axonal degeneration.     

Controlling pore assembly of staphylococcal gamma-haemolysin by low temperature and by disulphide bond formation in double-cysteine LukF mutants

Staphylococcal LukF and Hlg2 are water-soluble monomers of gamma-haemolysin that assemble into oligomeric pores on the erythrocyte membranes. Here, we have created double-cysteine LukF mutants, in which single disulphide bonds connect either the prestem domain and the cap domain (V12C-T136C, Cap-Stem), or two beta-strands within the prestem domain (T117C-T136C, Stem-Stem) to control pore assembly of gamma-haemolysin at intermediate stages. The disulphide-trapped mutants were inactive in erythrocyte lysis, but gained full haemolytic activity if the disulphide bonds were reduced. The disulphide bonds blocked neither the membrane binding ability nor the intermediate prepore oligomerization, but efficiently inhibited the transition from prepores to pores. The prepores of Cap-Stem were dissociated into monomers in 1% SDS. In contrast, the prepores of Stem-Stem were stable in SDS and had ring-shaped structures similar to those of wild-type LukF, as observed by transmission electron microscopy. The transition of both mutants from prepores to pores could even be achieved by reducing disulphide bonds at low temperature (2 degrees C), whereas prepore oligomerization was effectively inhibited by low temperature. Finally, real-time transition of Stem-Stem from prepores to pores on ghost cells, visualized using a Ca2+-sensitive fluorescent indicator (Rhod2), was shown by the sequential appearance of fluorescence spots, indicating pore-opening events. Taken together, these data indicate that the prepores are legitimate intermediates during gamma-haemolysin pore assembly, and that conformational changes around residues 117 and 136 of the prestem domain are essential for pore formation, but not for membrane binding or prepore oligomerization. We propose a mechanism for gamma-haemolysin pore assembly based on the demonstrated intermediates.     

Efficient biallelic mutagenesis with Cre/loxP-mediated inter-chromosomal recombination

The isolation of mutant cells with phenotypes caused by random mutagenesis has been hampered in mammalian cells because there are two alleles per gene and the disruption of both alleles is extremely rare. We describe a method for the efficient biallelic mutagenesis in embryonic stem cells. loxP sites were introduced near the centromeric regions of a pair of chromosome 1s. A mutant neo gene was inserted at the distal part of one of the loxP sites so that biallelic mutants would be selected by high-dose G418. Expression of Cre induced the recombination between homologous chromosomes and led to an elevation in the number of biallelic mutants. This system will facilitate phenotype-driven gene function study in the mammalian system.     

High-level expression of naked DNA delivered to rat liver via tail vein injection

Background

High levels of foreign gene expression in mouse hepatocytes can be achieved by rapid tail vein injection of a large volume of a naked DNA solution, the ‘hydrodynamics‐based procedure’. Rats are more tolerant of the frequent phlebotomies required for monitoring blood parameters than mice, and thus are better for some biomedical research.

Methods

We tested this technique for the delivery of a therapeutic protein in normal rats, using a rat erythropoietin (Epo) expression plasmid vector, pCAGGS‐Epo.

Results

We obtained maximal Epo expression when the DNA solution was injected in a volume of 25 ml (approximately 100 ml/kg body weight) within 15 s. We observed a dose‐response relationship between serum Epo levels and the amount of injected DNA up to 800 µg. Using quantitative real‐time PCR, the vector‐derived Epo mRNA expression was mainly detected in the liver. When a lacZ expression plasmid was injected similarly, β‐galactosidase was exclusively detected in the liver, mainly in hepatocytes. Toxicity attributable to the technique was mild and transient, as assessed by histochemical analysis. Epo gene expression and erythropoiesis occurred with Epo gene transfer in a dose‐dependent manner, and persisted for at least 12 weeks, the last time point examined. Repeated administration of the plasmid DNA also effectively led to erythropoiesis.

Conclusions

These results demonstrate that gene transfer into the liver via rapid tail vein injection can easily be achieved in the rat, which is more than 10 times larger than the mouse, and has significant value for gene function analysis in rats. Copyright © 2002 John Wiley & Sons, Ltd.

     

Arachidonic acid converts the glutathione depletion-induced apoptosis to necrosis by promoting lipid peroxidation and reducing caspase-3 activity in rat glioma cells

Intracellular glutathione (GSH) depletion induced by buthionine sulfoximine (BSO) caused cell death that seemed to be apoptosis in C6 rat glioma cells. Arachidonic acid (AA) promoted BSO-induced cell death by accumulating reactive oxygen species (ROS) or hydroperoxides. AA inhibited caspase-3 activation and internucleosomal DNA fragmentation during the BSO-induced GSH depletion. Furthermore, AA reduced intracellular ATP content, induced dysfunction of mitochondrial membrane and enhanced 8-hydroxy-2'-deoxyguanosine (8-OH-dG) production. There was significant increase of 12-lipoxygenase activity in the presence of AA under the BSO-induced GSH depletion in C6 cells. These results suggest that AA promotes cell death by changing to necrosis from apoptosis through lipid peroxidation initiated by lipid hydroperoxides produced by 12-lipoxygenase under the GSH depletion in C6 cells. Some ROS such as hydroperoxide produced by unknown pathway make hydroxy radicals and induce 8-OH-dG formation in the cells. The conversion of apoptosis to necrosis may be a possible event under GSH depleted conditions.