Difference in the effects of acetazolamide and ammonium chloride acidosis on ventilatory responses to CO2 and hypoxia in humans

The effects of acetazolamide, a potent carbonic anhydrase inhibitor, and ammonium chloride (NH4Cl) on arterial blood gas tension, resting ventilation, and ventilatory responses to CO2 (HCVR) and hypoxia (HVR) were studied in healthy male subjects. Both drugs induced chronic metabolic acidosis with the reduction in plasma bicarbonate by a mean of 7.0 +/- 2.0 (S.D.) mM after acetazolamide and by 5.6 +/- 1.8 mM after NH4Cl. The ratio in the decrement of PaCO2 to that of plasma bicarbonate (delta PaCO2/delta [HCO3-]) was 1.51 in the former and 0.98 in the latter. Both drugs increased inspiratory minute ventilation (VI) predominantly due to increased tidal volume (VT) with acetazolamide and to increased respiratory frequency (f) with NH4Cl. In HCVR, the increments in CO2- ventilation slope and in ventilation at PETCO2 60 mmHg after drug administration were 0.77 +/- 0.51 l X min-1 X mmHg-1 and 20.0 +/- 11.2 l/min with acetazolamide and 0.59 +/- 0.40 l X min-1 X mmHg-1 and 8.0 +/- 2.8 l/min with NH4Cl, respectively. On the other hand, HVR both in terms of delta VI/delta SaO2 slope and of ventilation at SaO2 75% significantly increased after NH4Cl but not after acetazolamide administration. Thus, augmented VT and HCVR in the acetazolamide group and increased f and HVR in the NH4Cl group suggested that the central chemosensitive mechanism in the former and the peripheral chemosensitive mechanism in the latter may predominantly be responsible for the elevated ventilatory activities.     

Attachment of growth cones on substrate observed by multi-mode light microscopy

Evanescent light illumination was introduced into a multi-mode microscope to construct a new type of total internal reflection fluorescence microscope (TIRFM). This microscope, capable of TIRFM, high resolution video-enhanced differential interference contrast (DIC), epifluorescence, interference reflection (IR) imaging, was combined with an image acquisition system for time-lapse microscopy. Neuronal growth cones of a rat hippocampal neuron were stained with membrane labeling fluorescence dyes (DiI or octadecyl rhodamine B). Dynamic changes of the cell substrate contact of the neuronal growth cone were observed using the multi-imaging capacities of this system. When growth cone regions were stimulated by pressure ejection of a high potassium solution, TIRFM intensity at the basal membrane of the growth cone increased, suggesting that basal membrane of growth cone approaches the glass substrate when excited. The approach of the ventral membrane to the substrate during excitatory stimulation was also observed with IR microscope. The functional importance of cell/substrate contact in growth cones is discussed.     

Observation of cavitation in a mechanical heart valve in a total artificial heart

Recently, cavitation on the surface of mechanical heart valves has been studied as a cause of fractures occurring in implanted mechanical heart valves. The cause of cavitation in mechanical heart valves was investigated using the 25 mm Medtronic Hall valve and the 23 mm Omnicarbon valve. Closing of these valves in the mitral position was simulated in an electrohydraulic totally artificial heart. Tests were conducted under physiologic pressures at heart rates from 60 to 100 beats per minute with cardiac outputs from 4.8 to 7.7 L/min. The disk closing motion was measured by a laser displacement sensor. A high-speed video camera was used to observe the cavitation bubbles in the mechanical heart valves. The maximum closing velocity of the Omnicarbon valve was faster than that of the Medtronic Hall valve. In both valves, the closing velocity of the leaflet, used as the cavitation threshold, was approximately 1.3-1.5 m/s. In the case of the Medtronic Hall valve, cavitation bubbles were generated by the squeeze flow and by the effects of the venturi and the water hammer. With the Omnicarbon valve, the cavitation bubbles were generated by the squeeze flow and the water hammer. The mechanism leading to the development of cavitation bubbles depended on the valve closing velocity and the valve stop geometry. Most of the cavitation bubbles were observed around the valve stop and were generated by the squeeze flow.     

Involvement of Rho-kinase in inflammatory and neuropathic pain through phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS)

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a major in vivo substrate for protein kinase C in the brain and has been implicated in cellular processes associated with cytoskeletal restructuring such as synaptic trafficking and neurotransmitter release. A phosphorylation-site specific antibody against Ser159-phospho-MARCKS (pS159-Mar-Ab) revealed that MARCKS is phosphorylated at Ser159 by Rho-kinase and that its phosphorylation is inhibited by the Rho-kinase specific inhibitor H-1152. Since the function of MARCKS is regulated by phosphorylation at multiple sites, here we examined the involvement of Rho-kinase in relation to phosphorylation of MARCKS at Ser159 in inflammatory and neuropathic pain by H-1152. When intrathecally administered 10 min before s.c. injection of formalin, H-1152 at 10 and 100 ng attenuated the second-phase, but not the first-phase, pain-like behaviors in the formalin test. Neuropathic pain induced by selective L5 spinal nerve transection was also relieved by intrathecal injection of H-1152. Nitric oxide synthase activity visualized by NADPH diaphorase histochemistry increased in the superficial layer of the spinal cord 30 min after formalin injection and 7 days after nerve transection, which were blocked by H-1152. Phosphorylation of MARCKS at Ser159 was detected in the spinal cord by pS159-Mar-Ab and the level of phosphorylation increased in the superficial layer after nerve transection. In contrast, immunoreactivities of neuronal nitric oxide synthase and MARCKS did not change significantly in the spinal cord before and after nerve transection. Taken together, the present study demonstrates that Rho-kinase is involved in inflammatory pain and the maintenance of neuropathic pain through phosphorylation of MARCKS at Ser159.     

Novel mutations in TNFRSF1A in patients with typical tumor necrosis factor receptor-associated periodic syndrome and with systemic lupus erythematosus in Japanese

Molecular defects of TNFRSF1A was investigated in members of a family presenting with typical phenotypes of tumor necrosis factor receptor-associated periodic syndrome (TRAPS) and in patients with the autoimmune disorders, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Genomic DNA from the members of a family with typical TRAPS, as well as from 100 patients with SLE, 100 patients with RA and 100 healthy individuals, was studied for mutations in exons 2, 3 and 4 of the TNFRSF1A gene. All individuals were Japanese. Three novel missense mutations were identified in the TNFRSF1A. The C70G mutation was identified in family members with typical TRAPS, which was the second case in eastern Asian population. In addition, the T61I and R104Q mutations were each identified in 2 of the 100 SLE patients. The T61I mutation was identified in one of the 100 healthy individuals. No mutations were identified in the 100 RA patients. Functional analysis revealed that PMA-induced shedding of TNFRSF1A from PBMCs was impaired in a patient carrying T61I. A larger scale of study will clarify whether these two mutations, T61I and R104Q, are associated with chronic inflammatory disorders, such as SLE, or not.     

Total hematological analysis system as a screening test for hematological malignancy

Measurement of complete blood cell count and white blood cell differentiation is an essential laboratory test and the most important screening test for hematological malignancy. Recently, several automated blood cell analyzers have been developed to improve accuracy and precision. When flag messages generated in the presence of morphological abnormalities of the samples are displayed, manual revision is necessary. In our laboratory, the manual revision rate has been 35-40%. Therefore blood cell analyzers are useful in screening for abnormalities as well as greatly reducing expensive and time-consuming manual differential procedures. In addition, automated blood cell analyzers can provide several types of useful information including the leukocyte distribution scattergram. However, most such information is not utilized in the clinical field. In the future, a total hematological analysis system will be constructed so that all information provided by automated blood cell analyzer and by manual methods are available.     

Evaluation of the test method “activated sludge, respiration inhibition test” proposed by the OECD

The test method of "activated sludge, respiration inhibition test" proposed by the OECD was critically carried out and compared with other test methods. Investigation of test conditions showed that the moderate deviation from the test conditions defined by the OECD Test Guidelines did not have much effect on the result, and some modifications were proposed to improve the method. This method had a poor detection limit compared with the LC50 test with Oryzias latipes and EC50 of the growth inhibition test with Tetrahymena pyriformis. The susceptivity of the method was particularly poor for the chemicals which were highly toxic in the other two tests.     

Reductive debromination of (alpha-bromoiso-valeryl)urea by intestinal bacteria

The reductive debromination of the hypnotic (alpha-bromoiso-valeryl)urea to (3-methylbutyryl)urea by intestinal bacteria has been studied. The caecal contents of rats, mice, hamsters, guinea-pigs and rabbits had significant debrominating activity toward (alpha-bromoiso-valeryl)urea. The cell-free extract of intestinal bacteria from the caecal contents of rats had debrominating activity in the presence of both flavin mononucleotide (FMN) and NADH (or NADPH) under anaerobic conditions. Seven pure strains of intestinal bacteria were also tested and the highest activity was observed with Clostridium sporogenes. The cell-free extract of Clostridium sporogenes had debrominating activity in the presence of both FMN and NADH (or NADPH), and this activity was inhibited by sodium arsenite and potassium cyanide. The activity of the cell-free extract was also supported by the photochemically reduced form of FMN. The debromination in intestinal bacteria seems to proceed in two steps--reduction of flavins by bacterial flavin reductase(s) in the presence of NADPH or NADH, and then the reductive debromination of (alpha-bromoiso-valeryl)urea to (3-methylbutyryl)urea by bacterial dehalogenase(s) using the reduced flavins as an electron donor. These results indicate that intestinal bacteria play a role in the reductive debromination of (alpha-bromoiso-valeryl)urea to (3-methylbutyryl)urea in animals. The debromination is inhibited by oxygen and dependent on flavins.