Importance of renal mitochondria in the reduction of TEMPOL,a nitroxide radical

Spin probing methods using an electron spin resonance (ESR) spectrometer are used extensively and bring us a lot of information about in vivo redox mechanisms. However, the in vivo reducing mechanisms of exogenous nitroxide radicals, which serve as typical spin probing reagents are not clear. To clarify this, we examined the sequential kinetics of a spin probe, 4-hydroxy 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) in the in vivo organs, tissue homogenates and subcellular fractions of kidney and liver using an in vivo and X-band ESR spectrometers. As a parameter of reducing activity, we calculated the half-life of TEMPOL from the decay curve of ESR signal intensity. The half-life of TEMPOL in the whole organs and homogenates of the kidney was significantly shorter than that of the liver, this indicates that the kidney has more reducing activity against TEMPOL as compared to the liver. Subcellular fractional studies revealed that this reducing activity of the kidney mainly exists in the mitochondria. Contrarily, in addition to reduction in the mitochondria, TEMPOL in the liver was reduced by the microsome and cytosol.     

Reaction kinetics of a two-photon excitation microparticle based immunoassay--from modelling to practice

Real time observation of reaction kinetics is one of the key features of the newly developed microparticle based two-photon excitation fluorescence immunoassay system (TPX). By observing binding reactions at the surface of individual microparticles during the incubation of an assay, the binding constants of an assay become apparent. This paper describes the use of the new system in quantifying the reaction parameters of human thyroid stimulating hormone (hTSH) assay. A mechanistic reaction model for the assay is presented. The reaction model is further shown to precisely predict the behaviour of the assay kinetics over a wide range of analyte concentrations.     

Frequency response functions and information capacities of paired spider mechanoreceptor neurons

 Pseudorandom white-noise stimulation followed by direct spectral estimation was used to obtain linear frequency response and coherence functions from paired, but dynamically different, spider mechanosensory neurons. The dynamic properties of the two neuron types were similar with either mechanical or electrical stimulation, showing that action potential encoding dominates the dynamics. Phase-lag data indicated that action potential initiation occurs more rapidly during mechanical stimulation, probably in the distal sensory dendrites. Total information capacity, calculated from coherence, as well as information per action potential, were both similar in the two types of neurons, and similar to the few available estimates from other spiking neurons. However, information capacity and information per action potential both depended strongly on neuronal firing rate, which has not been reported before. Received: 7 August 2000 / Accepted in revised form: 5 April 2001     

Concerted Evolution of a Highly Repetitive DNA Family in Eptatretidae (Cyclostomata, Agnatha) Implies Specifically Differential Homogenization and Amplification Events in Their Germ Cells

In eight hagfish species, it is known that chromosome elimination occurs during early embryogenesis, and some highly repetitive DNA families, restricted to germ cells, have been isolated. One of these families, ``EEEo2,' has been isolated as DNA fragments by restriction enzyme analyses from Eptatretus okinoseanus and E. cirrhatus. In this study, EEEo2 sequences were isolated from germline DNA in E. burgeri, Paramyxine sheni, and P. atami using PCR methods. Sequence analysis revealed that these sequences are intraspecifically homogeneous, except in E. burgeri, and are interspecifically conserved with heterogeneity. The intraspecific sequence variability tends to decrease as the copy number increases. These results indicate that EEEo2 has evolved in a concerted manner. Moreover, an ancestral repeating motif consisting of triplicate subrepeats was deduced. These results suggest that EEEo2 arose as an initial amplification of this subrepeat and has evolved by saltatory replication. Phylogenetic analyses suggested the possibility that EEEo2 in E. okinoseanus and E. cirrhatus has been subjected to strong homogenizing forces for concerted evolution, whereas the force is weak in E. burgeri. In addition, EEEo2 in P. sheni and P. atami appear to have been incompletely subjected to these forces. Chromosomal in situ hybridization experiments revealed that EEEo2 sequences were located along almost their entire length of several heterochromatic chromosomes that are restricted to germ cells. These chromosomes are disposed to form a secondary association during the first meiotic metaphases, except in P. sheni. This chromosomal distribution may promote a concerted mode of sequence evolution in both nonhomologous chromosomes and homologous chromosomes and reflect the differential driving forces between species. Received: 17 April 1999 / Accepted: 10 September 1999     

Juxtaposition of two heterotypic monolayer cell cultures of chick limb buds

In chick limb buds, mesenchymal cells of the progress zone (PZ-cells) at different developmental stages segregate one from the other in mixed cell cultures, suggesting they have different cell affinity. In order to learn the possible roles of such differences in the cells, two heterotypic leg PZ-cell populations (cells from stages 25/26 and 20/21) in vitro were juxtaposed to allow them to form the boundary. A method with double cylindrical columns was used to make adjoining monolayer cell cultures. It was shown that heterotypic juxtaposition produced two chondrogenic patterns along the boundary: aggregates of chondrocytes formed by stage 20/21 PZ-cells and a chondrocyte-free band formed by those at stage 25/26. Juxtaposition of PZ-cells and proximal cells also formed these patterns, while that between cells from anterior and posterior PZ formed indistinct patterns along the boundary. Homotypic PZ-cell juxtaposition did not produce these patterns. The results suggest that different cell affinity has a role in the segmentation of cartilage patterns at a point along the proximodistal axis, as well as a role in retaining cells in one area so as not to be recruited to other condensation areas.     

Fear Conditioning Induced by Interpersonal Conflicts in Healthy Individuals

Psychophysiological markers have been focused to investigate the psychopathology of psychiatric disorders and personality subtypes. In order to understand neurobiological mechanisms underlying these conditions, fear-conditioning model has been widely used. However, simple aversive stimuli are too simplistic to understand mechanisms because most patients with psychiatric disorders are affected by social stressors. The objective of this study was to test the feasibility of a newly-designed conditioning experiment using a stimulus to cause interpersonal conflicts and examine associations between personality traits and response to that stimulus. Twenty-nine healthy individuals underwent the fear conditioning and extinction experiments in response to three types of stimuli: a simple aversive sound, disgusting pictures, and pictures of an actors’ face with unpleasant verbal messages that were designed to cause interpersonal conflicts. Conditioned response was quantified by the skin conductance response (SCR). Correlations between the SCR changes, and personality traits measured by the Zanarini Rating Scale for Borderline Personality Disorder (ZAN-BPD) and Revised NEO Personality Inventory were explored. The interpersonal conflict stimulus resulted in successful conditioning, which was subsequently extinguished, in a similar manner as the other two stimuli. Moreover, a greater degree of conditioned response to the interpersonal conflict stimulus correlated with a higher ZAN-BPD total score. Fear conditioning and extinction can be successfully achieved, using interpersonal conflicts as a stimulus. Given that conditioned fear caused by the interpersonal conflicts is likely associated with borderline personality traits, this paradigm could contribute to further understanding of underlying mechanisms of interpersonal fear implicated in borderline personality disorder.     

Cerebral Hemodynamic Changes of Mild Traumatic Brain Injury at the Acute Stage

Mild traumatic brain injury (mTBI) is a significant public health care burden in the United States. However, we lack a detailed understanding of the pathophysiology following mTBI and its relation to symptoms and recovery. With advanced magnetic resonance imaging (MRI), we can investigate brain perfusion and oxygenation in regions known to be implicated in symptoms, including cortical gray matter and subcortical structures. In this study, we assessed 14 mTBI patients and 18 controls with susceptibility weighted imaging and mapping (SWIM) for blood oxygenation quantification. In addition to SWIM, 7 patients and 12 controls had cerebral perfusion measured with arterial spin labeling (ASL). We found increases in regional cerebral blood flow (CBF) in the left striatum, and in frontal and occipital lobes in patients as compared to controls (p = 0.01, 0.03, 0.03 respectively). We also found decreases in venous susceptibility, indicating increases in venous oxygenation, in the left thalamostriate vein and right basal vein of Rosenthal (p = 0.04 in both). mTBI patients had significantly lower delayed recall scores on the standardized assessment of concussion, but neither susceptibility nor CBF measures were found to correlate with symptoms as assessed by neuropsychological testing. The increased CBF combined with increased venous oxygenation suggests an increase in cerebral blood flow that exceeds the oxygen demand of the tissue, in contrast to the regional hypoxia seen in more severe TBI. This may represent a neuroprotective response following mTBI, which warrants further investigation.     

Longitudinal Study of the Decline in Renal Function in Healthy Subjects

Background

Chronic kidney disease is an important concern in preventive medicine, but the rate of decline in renal function in healthy population is not well defined. The purpose of this study was to determine reference values for the estimated glomerular filtration rate (eGFR) and rate of decline of eGFR in healthy subjects and to evaluate factors associated with this decline using a large cohort in Japan.

Methods

Retrospective cross-sectional and longitudinal studies were performed with healthy subjects aged ≥18 years old who received a medical checkup. Reference values for eGFR were obtained using a nonparametric method and those for decline of eGFR were calculated by mixed model analysis. Relationships of eGFR decline rate with baseline variables were examined using a linear least-squares method.

Results

In the cross-sectional study, reference values for eGFR were obtained by gender and age in 72,521 healthy subjects. The mean (±SD) eGFR was 83.7±14.7ml/min/1.73m². In the longitudinal study, reference values for eGFR decline rate were obtained by gender, age, and renal stage in 45,586 healthy subjects. In the same renal stage, there was little difference in the rate of decline regardless of age. The decline in eGFR depended on the renal stage and was strongly related to baseline eGFR, with a faster decline with a higher baseline eGFR and a slower decline with a lower baseline eGFR. The mean (±SD) eGFR decline rate was ‒1.07±0.42ml/min/1.73m²/year (‒1.29±0.41%/year) in subjects with a mean eGFR of 81.5±11.6ml/min/1.73m².

Conclusions

The present study clarified for the first time the reference values for the rate of eGFR decline stratified by gender, age, and renal stage in healthy subjects. The rate of eGFR decline depended mainly on baseline eGFR, but not on age, with a slower decline with a lower baseline eGFR.     

[18F]FDG Accumulation in Early Coronary Atherosclerotic Lesions in Pigs

Objective

Inflammation is an important contributor to atherosclerosis progression. A glucose analogue ¹⁸F-fluorodeoxyglucose ([¹⁸F]FDG) has been used to detect atherosclerotic inflammation. However, it is not known to what extent [¹⁸F]FDG is taken up in different stages of atherosclerosis. We aimed to study the uptake of [¹⁸F]FDG to various stages of coronary plaques in a pig model.

Methods

First, diabetes was caused by streptozotocin injections (50 mg/kg for 3 days) in farm pigs (n = 10). After 6 months on high-fat diet, pigs underwent dual-gated cardiac PET/CT to measure [¹⁸F]FDG uptake in coronary arteries. Coronary segments (n = 33) were harvested for ex vivo measurement of radioactivity and autoradiography (ARG).

Results

Intimal thickening was observed in 16 segments and atheroma type plaques in 10 segments. Compared with the normal vessel wall, ARG showed 1.7±0.7 times higher [¹⁸F]FDG accumulation in the intimal thickening and 4.1±2.3 times higher in the atheromas (P = 0.004 and P = 0.003, respectively). Ex vivo mean vessel-to-blood ratio was higher in segments with atheroma than those without atherosclerosis (2.6±1.2 vs. 1.3±0.7, P = 0.04). In vivo PET imaging showed the highest target-to-background ratio (TBR) of 2.7. However, maximum TBR was not significantly different in segments without atherosclerosis (1.1±0.5) and either intimal thickening (1.2±0.4, P = 1.0) or atheroma (1.6±0.6, P = 0.4).

Conclusions

We found increased uptake of [¹⁸F]FDG in coronary atherosclerotic lesions in a pig model. However, uptake in these early stage lesions was not detectable with in vivo PET imaging. Further studies are needed to clarify whether visible [¹⁸F]FDG uptake in coronary arteries represents more advanced, highly inflamed plaques.