Test for analysing nerve conduction velocity]

Recently, many attempts have been made to measure the difference in velocity between the fastest and slowest fibers in a nerve trunk or to estimate the nerve fiber conduction velocity distribution in a nerve bundle using several different methods, such as collision technique (Hopf 1963) and computer analysis of the compound action potentials (Cummins et al. 1979; Barker et al. 1979). For the computer analysis, however, some assumptions in regard to the quantitative relationship among conduction velocity, single fiber action potential and fiber diameter are necessary, and there has been little agreement about them. There is also a problem about the relationship between conduction velocity and refractory period in Hopf's technique. Using a collision technique with a method of 3-point stimulation, Gilliat et al. (1976) now suggested that surface recording was unsatisfactory for measuring the velocity of the slow-conducting nerve fibers. With this method, however, we had a preliminary experiment to analyze conduction velocities of so-called A fibers in the bullfrog's sciatic-peroneal nerve using fluid electrode, and they were divided into 3 groups (Nakanishi et al. 1986). These findings were in good agreement with those obtained by Erlanger and Gasser (1937) using monophasic recording. Therefore, clinical measurement of the nerve conduction velocities with a method of this collision technique was performed using surface recording.     

Changes in HRCT findings in patients with respiratory bronchiolitis-associated interstitial lung disease after smoking cessation.

High-resolution computed tomography (HRCT) findings in patients with respiratory bronchiolitis-associated interstitial lung disease (RB-ILD) are varied and nonspecific. There is no known report of changes in HRCT findings and respiratory function test results for RB-ILD patients following the cessation of smoking. Five patients with RB-ILD, confirmed by surgical lung biopsy, were retrospectively studied. Each stopped cigarette smoking and did not receive corticosteroid therapy after diagnosis. The clinical symptoms, respiratory function test results and HRCT findings obtained at the final observation were compared with those from the time of diagnosis. Ground-glass opacity and centrilobular nodules corresponding to pathological respiratory bronchiolitis, as well as intralobular fine linear-reticular opacity corresponding to fibrosis involving the subpleural alveolar septa, showed computed tomography-pathological correlations. Both clinical symptoms and the diffusing capacity of the lungs for carbon monoxide improved significantly following smoking cessation, as did ground-glass opacity and centrilobular nodules seen during the initial HRCT examination. Centrilobular nodules and ground-glass opacity, which are the main features of high-resolution computed tomography of respiratory bronchiolitis-associated interstitial lung disease patients and represent pathological respiratory bronchiolitis, can be improved by smoking cessation. The diffusing capacity of the lung for carbon monoxide in respiratory function tests can be also improved.     

A primary lung carcinoma producing alpha-fetoprotein, carcinoembryonic antigen, and human chorionic gonadotropin. Immunohistochemical and biochemical studies

This article documents a patient with lung carcinoma that produced three oncofetal antigens including alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and human chorionic gonadotropin (hCG). Serum AFP, CEA, and hCG-beta-subunit were extremely high--118,000 ng/ml, 133 ng/ml and 0.9 ng/ml, respectively. Immunohistochemical staining of these tumor markers revealed that these proteins were present in different cells. The pattern of lectin affinity electrophoresis of AFP resembled that of hepatocellular carcinoma. Also investigated was the reactivity of serum CEA to monoclonal antibodies against peptide or sugar moieties. Serum CEA values measured by antipeptide monoclonal antibodies were higher than those measured by antisugar monoclonal antibodies. The demonstration of AFP, CEA, and hCG in different tumor cells suggests that three genomes were not reactivated together in a cell, and the lung carcinoma probably consisted of at least three clones of cancer cells with different phenotypes.     

Perforin expression in lymphocytes infiltrated to human colorectal cancer

Perforin (PFP) is a cytotoxic protein released from killer cells. PFP immunoreactivity in human peripheral blood lymphocytes (PBL) and tumour infiltrating lymphocytes (TIL) was investigated immunocytochemically with the aid of an anti-PFP monoclonal antibody. PFP was detected in the cytoplasm of 10% of PBL. We performed a double staining of PFP+ cells with Leu11b/CD16, Leu2a/CD8, or Leu3a/CD4 and showed that PFP was produced by 9% of CD8+ cells and 18% of CD16+ cells but not by CD4+ cells. In 28 colorectal cancer tissues, PFP immunoreactivity was observed in the lymphocytes infiltrating to the tumour stroma. The PFP+ cells were most numerous in Dukes A and decreased in number according to the progression of tumours. The PFP+ cells in TIL exhibited the same phenotypes as those in PBL but the PFP+ cells were more numerous in CD8+ cells than in CD16+ cells at all stages. This study represents the first evidence that PFP is mainly secreted from CD8+ cells in tumour tissues. It is hypothesised that the decrease in the number of PFP+ cells in accordance with tumour progression may reflect the suppression of the hosts local immunity.     

Phosphorylation and activation of tyrosine hydroxylase in PC18 cells: a cell line derived from rat pheochromocytoma PC12 cells.

Treatment of rat pheochromocytoma PC18 cells (a variant subclone of PC12 cells) with forskolin produced increased activity and phosphorylation of tyrosine hydroxylase. In contrast, treatment of the PC18 cells with 56 mM K+, A23187, phorbol-12-myristate-13-acetate (PMA) or phorbol-12,13-dibutyrate (PDB) did not affect the activity and only slightly increased the phosphorylation of tyrosine hydroxylase. None of the treatments except forskolin increased cyclic AMP levels in PC18 cells. Furthermore, 45Ca2+ uptake into PC18 cells was not affected by 56 mM K+, PDB or forskolin; however, A23187 increased 45Ca2+ uptake 4-fold over basal uptake. Nevertheless, no activation and little increase in phosphorylation of tyrosine hydroxylase was observed in PC18 cells treated with A23187. When tyrosine hydroxylase levels in PC18 cells were elevated by treatment with dexamethasone, activation of tyrosine hydroxylase by 56 mM K+, PDB or A23187 was still not observed. Both purified Ca2+/calmodulin-dependent protein kinase and cyclic AMP-dependent protein kinase catalyzed the phosphorylation of tyrosine hydroxylase purified from PC18 cells in vitro. Furthermore, crude cell extracts from PC12 cells and PC18 cells possessed Ca2+/calmodulin-dependent protein kinase activity that catalyzed the phosphorylation of purified tyrosine hydroxylase. These results suggest that tyrosine hydroxylase activity in PC18 cells is regulated by a cyclic AMP-dependent mechanism. However, due to a number of abnormalities the Ca(2+)-dependent mechanisms do not result in the activation of tyrosine hydroxylase and only slightly increase the phosphorylation of the enzyme in PC18 cells.     

Immunohistochemical localization of ryanodine receptors in mouse central nervous system.

The distribution of ryanodine receptor-like immunoreactivity in the mouse central nervous system was studied using two antibodies raised against synthetic peptides. These peptides represented a region conserved between the cardiac and skeletal muscle forms and a region specific to the cardiac form. Western blotting analysis and [3H]ryanodine binding analysis showed ryanodine receptors are expressed in all the brain regions. The activity was prominent in hippocampus and cerebral cortex. Immunohistochemical study demonstrated that the ryanodine receptors were localized unevenly in somata. Some apical and proximal dendrites in some cells were also labeled. In hippocampus pyramidal neurons in CA2-3 region were more labeled than CA1 region. Immunohistochemical distribution revealed by two antibodies was essentially the same but the fibers were more immunoreactive with the antibody raised against the cardiac muscle ryanodine form. The localization of ryanodine receptors was quite different from that of inositol 1,4,5-trisphosphate receptors.     

Reevaluation of optimum extraction condition for urinary organic acids using a stable isotope dilution technique]

We reevaluated the optimum conditions for analysis of 13 urinary organic acids using solvent extraction and GC/MS by the stable isotope dilution technique. The acids analyzed were uracil, and lactic, oxalic, 3-hydroxybutyric, succinic, fumaric, glutaric, adipic, pyroglutamic, 2-ketoglutaric, orotic, sebacic, and citric acids. Analytical recovery and accuracy for 13 organic acids ranged from 90 to 107% and from 1.6 to 13.7%, respectively. The optimal pH for most organic acids was 1-2, while that for oxalic and citric acids was 0.25, and that for 2-ketoglutaric and orotic acids was 0.5. The presence of urinary albumin decreased the extraction rates of organic acids, especially of orotic and citric acids; in slight albuminuria (0.5 g/l) the extraction recovery (the extraction rate with albumin/that without albumin) of orotic and citric acid was 38% and 67%, respectively, and in more marked albuminuria (5 g/l), 6% and 26%. Membrane pretreatment with Centricon-3 improved these extraction rates under the condition of albuminuria. Dehydration with the desiccant agent decreased urinary acid extraction rates, especially of uracil and orotic and citric acids. The extraction rates of these three organic acids was decreased in albuminuria and by the desiccant agent. Accurate quantitative analysis of urinary organic acids by the stable isotope dilution technique is necessary for routine examination conducted in clinical laboratories.     

KT5926, a potent and selective inhibitor of myosin light chain kinase

KT5926, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3 ,9, 10-tetrahydro-8,11-epoxy, 1H,8H, 11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde] trinden-1-one, was found to be a potent and selective inhibitor of myosin light chain kinase. The compound inhibited both Ca2+/calmodulin-dependent and -independent smooth muscle myosin light chain kinases to a similar extent. The inhibition was not affected by the concentration of calmodulin. Kinetic analyses showed that the mode of inhibition was of the competitive type with respect to ATP (Ki, 18 nM) and of the noncompetitive type with respect to myosin light chain (Ki, 12 nM). These results indicated that KT5926 directly interacted with the enzyme at the catalytic site. KT5926 also inhibited other protein kinases, but with relatively high Ki values; the values for protein kinase C, cAMP-dependent protein kinase, and cGMP-dependent protein kinase were 723, 1200, and 158 nM, respectively. Ca2(+)-ATPase, Na+/K(+)-ATPase, hexokinase, and 5'-nucleotidase were not inhibited by KT5926 at less than 10 microM. The effect of KT5926 on serotonin secretion and protein phosphorylation induced by platelet-activating factor or phorbol ester was examined in rabbit platelets. KT5926 inhibited the phosphorylation of a 20-kDa protein but had no effect on the phosphorylation of a 40-kDa protein, thereby indicating that the compound exerts its selective inhibition of myosin light chain kinase in intact cells. The compound inhibited serotonin secretion induced by platelet-activating factor, but its potency was significantly less than that of K-252a, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9, 10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b, 11a-triazadibenzo[a,g]cycloocta [cde]trinden-1-one, which inhibited the phosphorylation of both the 20-kDa protein and the 40-kDa protein. Phorbol ester-induced secretion was not suppressed by KT5926. These results provide the evidence that both the 20-kDa protein phosphorylation by myosin light chain kinase and the 40-kDa protein phosphorylation by protein kinase C substantially contribute to the secretion response in platelets.