Ensemble noise and current relaxation analysis of K+ current in single isolated salivary acinar cells from rat

The K⁺ channel in rat parotid gland acinar cells were investigated by ensemble current noise analysis in single isolated cells employing the giga-seal whole cell current recording mode. Sets of 20–40 identical de- and hyperpolarization voltage steps were applied and the resultant current records were processed by computer to obtain the mean and the variance of the current. The time-course of the mean current could be fitted by the sum of two exponentials, suggesting a 3-state model. The simplest plausible hypothesis is a model with one open and two closed states. Assuming this model, the relationship between the variance (₂) and the mean current (I) could be fitted by the function ₂/I=i–I/N. The estimated single channeli/V-relations were similar to those taken from single channel current recordings, and the size of the population of channels per cell (N) was 76±26 (n=12). The validity of the model was tested by a successful simulation of the time-course of the variance.     

An ambulatory respirometer (Hotmate) as a tool for screening of sleep apnea syndrome

The gold standard diagnostic method for sleep apnea syndrome(SAS) is overnight polysomnography(PSG), but is costly in terms of time and money. We studied the usefulness of a 24-hour ambulatory respirometer equipped with oximeter(Hotmate) for screening of SAS. Seventy-six cases of suspected SAS were enrolled(68 males and 8 females, mean age 51). The correlation between data from Hotmate and PSG was evaluated in 24 cases who underwent both of the tests for the final diagnosis of SAS. There was a good correlation between the two parameters of the data obtained by Hotmate(H) (H-apnea index(AI) vs H-desaturation index(DI)). Among 24 cases who underwent both Hotmate and PSG, there was a good correlation between the data from PSG and Hotmate(PSG-AI vs H-AI: r = 0.80, p < 0.001). Both sensitivity and specificity were highest when screening criteria of H-DI > 15 was utilized(sensitivity = 91.7%, specificity = 66.7%). Our findings suggest that the respiromonitor with oximeter is useful for the screening the patients with SAS.     

Bradykinin Stimulates Type II Alveolar Cells to Release Neutrophil and Monocyte Chemotactic Activity and Inflammatory Cytokines

In the present study, we evaluated the potential of bradykinin (BK) to induce the release of neutrophil and monocyte chemotactic activity (NCA and MCA) and cytokines from an alveolar type II epithelial cell line, A549 cells. BK stimulated A549 cells to release NCA and MCA in a dose- and time-dependent manner (P < 0.001). Checkerboard analysis revealed that both NCA and MCA involved chemotactic and chemokinetic activity. Molecular sieve column chromatography showed three molecular weight masses (near 19 kd, 8 kd, and 400 d) for NCA and several molecular weight peaks (near 66 kd, 25 kd, 19 kd, 16 kd, and 400 d) for MCA. The release of NCA and MCA was inhibited by cycloheximide and lipoxygenase inhibitors (P < 0.01). The NCA and MCA were inhibited by leukotriene B4 (LTB4) receptor antagonist (P < 0.01), and the concentration of LTB4 was high enough for NCA and MCA. Antibodies to interleukin (IL)-8 and granulocyte colony-stimulating factor (G-CSF) attenuated NCA (P < 0.01), and antibodies to monocyte chemotactic protein-1 (MCP-1), G-CSF, and transforming growth factor (TGF)-β attenuated MCA (P < 0.01). The levels of IL-8, G-CSF, MCP-1, and TGF-β increased time dependently (P < 0.01). BK also stimulated the release of ILeukin-6 from A549 cells (P < 0.001). The receptors responsible for the release of NCA, MCA, and individual chemokines involved both BKB1 and BKB2 receptors. These data suggest that BK may stimulate alveolar type II pneumocytes to release inflammatory cytokines, which then may modulate the lung inflammation.     

Neuroexcitatory actions of Tamiflu and its carboxylate metabolite

Oseltamivir (Tamiflu) is now being stockpiled by several governments as a first line treatment for an anticipated outbreak of avian influenza caused by H5N1. However, abnormal behaviors and death associated with the use of Tamiflu have developed into a major issue in Japan where Tamiflu is often prescribed for seasonal influenza. Thus, it is critical to determine neuropsychiatric effects of oseltamivir and to establish methods for safe administration. Using juvenile rats and rat hippocampal slices, we investigated whether oseltamivir has adverse effects on the central nervous system. Systemic injection of oseltamivir (50 mg/kg i.p.) produced no change in behavior within 2 h. However, prior injection of oseltamivir significantly altered the duration of loss of lightning reflex following ethanol injection (3.3 g/kg, i.p.). Ethanol injection in the presence of oseltamivir also resulted in enhanced hypothermia. In the CA1 region of hippocampal slices, oseltamivir (100 μM) induced paired-pulse facilitation in population spikes without changes in excitatory postsynaptic potentials. Similarly, 3 μM oseltamivir carboxylate, the active metabolite of oseltamivir, facilitated neuronal firing, though the facilitation did not involve GABAergic disinhibition. Moreover, oseltamivir carboxylate produced further facilitation following administration of 60 mM ethanol. These findings indicate that oseltamivir has effects on the central nervous system, especially when combined with other agents.     

Effects of ISI and stimulus probability on event-related go/nogo potentials after somatosensory stimulation

The present study investigated the characteristics of the middle-latency negative potential of event-related potentials (ERPs) using somatosensory go/nogo tasks. We manipulated interstimulus interval (ISI) in Experiment 1 and stimulus probability in Experiment 2 and analyzed the subtracted difference waveform resulting from subtraction of the ERP evoked by the go stimulation from that evoked by the nogo stimulation. In Experiment 1, the peak latency of negativity became significantly longer as the ISI increased, but the peak amplitude was unchanged. The reaction time (RT) was longer with increasing ISI. In Experiment 2, manipulation of the stimulus probability yielded an increase in peak amplitude with decreasing probability of the nogo stimulus, but did not affect the latency. The RT increased as the probability of a nogo stimulus rose. Because manipulation of the ISI and stimulus probability elicited different brain activities, we hypothesized that manipulation of the ISI elicited a delay of the stimulus evaluation process including response inhibition, and that stimulus probability significantly affected the strength of the response inhibition process.     

Deoxycorticosterone-secreting adrenocortical carcinoma

A case of DOC-secreting adrenocortical carcinoma in a 66-year-old man is reported. He had hypertension, hypokalemia, suppressed PRA, and excessive serum levels of DOC. His serum aldosterone level was normal. The resected adrenal mass weighed 230 g. Histologically, the tumor was mainly composed of compact cells associated with necrosis and atypical mitoses. Invasion of venous structure, sinusoids, and capsule was also present. Immunohistochemically, P450 C21 (21 -hydroxylase) was positive in many tumor cells, and P450 C17(17 α-hydroxylase) was intensely positive in a relatively small number of tumor cells. The patient died 9 months after operation due to rupture of metastatic liver tumor.Endocr Pathol 4:165–168, 1993.     

IgG heavy chain allotypes (Gm) in autoimmune diseases.

Serum samples from 100 patients with myasthenia gravis, 322 with Graves' disease, 113 with Hashimoto's disease, 132 with systemic lupus erythematosus (SLE), 192 with insulin-dependent juvenile diabetes mellitus, 83 with Behçet's syndrome, 73 with psoriasis vulgaris, 258 with leprosy, 112 with Duchenne progressive muscular dystrophy and 343 non-related normal controls were studied for Gm allotypes. The incidence of Gm phenotypes with Gm(2) was significantly increased in patients with myasthenia gravis. Graves' disease, Hashimoto's disease, and high in SLE patients. The Gm1,2,21 haplotype was increased in patients with myasthenia gravis (chi 2 = 34 . 08, corrected P less than 0 . 001), Hashimoto's disease (chi 2 = 12 . 39, corrected P less than 0 . 05), Graves' disease (chi 2 = 8 . 65, corrected P less than 0 . 05), and SLE (chi 2 = 6 . 41, 0 . 1 greater than corrected P greater than 0 . 05). The total chi-square for the four different Gm haplotypes was significantly increased in patients with myasthenia gravis (chi 2 = 44 . 46, corrected P less than 0 . 001), SLE (chi 2 = 20 . 70, corrected P less than 0 . 005), Hashimoto's disease (chi 2 = 17 . 03, corrected P less than 0 . 025), and Graves' disease (chi 2 = 11 . 87, corrected P less than 0 . 025). Our data suggest the presence of Gm-associated pathogenic polygenes in certain autoimmune disorders.     

Elucidation of the protein kinase C-dependent apoptosis pathway in distinct subsets of T lymphocytes in MRL-lpr/lpr mice

MRL-lpr mice are severely impaired in the Fas pathway of apoptosis induction. We here evaluate another pathway of apoptosis induction in MRL-lpr mice which is protein kinase C (PKC) dependent. Despite the defect of the Fas pathway, apoptosis developed during culture in vitro in splenic T lymphocytes from MRL-lpr mice more extensively than in T lymphocytes from MRL-+/+ mice. Apoptosis induction in the former cells was then found to be greatly promoted by PKC inhibitor H-7, and partially prevented by PKC activator phorbol 12-myristate 13-acetate (PMA). High sensitivity to H-7, but not to PKA inhibitor HA 1004, of these cells for apoptosis induction was confirmed by detailed time course and dose-dependency experiments of the drug effect. Population analysis showed that both CD4⁺ T lymphocytes and CD8⁺ T lymphocytes from MRL-lpr mice were highly sensitive to H-7, whereas CD8⁺ T lymphocytes, but not CD4⁺ T lymphocytes, from MRL-+/+ mice were susceptible to the reagent. Interestingly, B220⁺Thy-1⁺CD4^?CD8^? T lymphocytes from MRL-lpr mice were most sensitive to H-7 for apoptosis induction. Correspondingly, the membrane-translocated activated PKC-α level in splenic T lymphocytes from MRL-lpr was more extensively up-regulated by PMA than in splenic T lymphocytes from MRL-+/+. These results suggest that some signal consistently activates PKC in MRL-lpr T lymphocytes, and this event is needed for survival of these cells. On the other hand, CD4⁺CD8⁺ thymocytes were deleted by apoptosis in culture with PMA, whether these thymocytes were from MRL-lpr mice or MRL-+/+ mice. This finding suggested that the apoptosis induction pathway linked to PKC activation is intact in CD4⁺ CD8⁺ thymocytes from the Fas-defective MRL-lpr mice. We conclude from these results that the PKC-dependent signal pathways for either cell death or cell activation are intact or even accelerated in lpr mice, which could both compensate for the loss of the Fas pathway and promote the generation of autoreactive T lymphocytes.     

Genitourinary phenotype in XX patients with distal 9p monosomy

Although testicular development has been shown to be variably impaired in XY patients with distal 9p monosomy, ovarian and other genitourinary phenotype has poorly been studied in XX patients monosomic for the distal 9p region. Thus, we studied a 13-month-old infant with 46,XX,der(9)t(9;10)(p23;p13) (case 1) and an 11-year-old girl with 46,XX,der(9)t(9;16)(p23;q22) (case 2). Case 1 had primary hypogonadism (basal serum follicle stimulating hormone [FSH], 40.0 mIU/mL; leteinizing hormone [LH], 1.2 mIU/mL; estradiol [E2], <10 pg/mL), whereas case 2 had age-appropriate pubertal development (breast, Tanner stage 4; pubic hair, Tanner stage 3; menarche 11.7 years of age) and hormone values (FSH, 7.3 mIU/mL; LH, 6.7 mIU/mL; E2, 47 pg/mL). In addition, case 1 had hypoplastic labia majora, short distance between the vaginal orifice and the anus, and five renal cysts, and case 2 had anal atresia, short distance between the vaginal orifice and the anus, bilateral hydronephrosis of grade 3 with probable ureteropelvic junction stenosis, and renal dysfunction (serum creatinine, 1.52 mg/dL; urea nitrogen, 34.5mg/dL). Fluorescence in situ hybridization analysis for five regions and microsatellite analysis for 10 loci on 9p confirmed hemizygosity for the distal 9p region with the breakpoints between IFNA and D9S285 in case 1 and between D9S168 and D9S286 in case 2. The results, in conjunction with the previous data in XX patients with molecularly defined distal 9p monosomy, are consistent with the presence of a gene(s) involved in the development of indifferent gonad or subsequent ovarian differentiation in a approximately 11 Mb region distal to D9S168. In addition, it is possible that a gene(s) for anoperineal and renal development also maps distal to D9S168 and that for external genital development maps distal to D9S285 at the position approximately 16 Mb from the 9p telomere.     

Genotyping of Mycobacterium leprae on the basis of the polymorphism of TTC repeats for analysis of leprosy transmission

The polymorphism of TTC repeats in Mycobacterium leprae was examined using the bacilli obtained from residents in villages at North Maluku where M. leprae infections are highly endemic (as well as from patients at North Sulawesi of Indonesia) to elucidate the possible mode of leprosy transmission. TTC genotypes are stable for several generations of passages in nude mice footpads and, hence, are feasible for the genotyping of isolates and epidemiological analysis of leprosy transmission. It was found that bacilli with different TTC genotypes were distributed among residents at the same dwelling in villages in which leprosy is endemic and that some household contacts harbored bacilli with a different genotype from that harbored by the patient. Investigations of a father-and-son pair of patients indicated that infections of bacilli with 10 and 18 copies, respectively, had occurred. Genotypes of TTC repeats were found to differ between a son under treatment and two brothers. These results reveal the possibility that in addition to exposure via the presence of a leprosy patient with a multibacillary infection who was living with family members, there might have been some infectious sources to which the residents had been commonly exposed outside the dwellings. A limited discriminative capacity of the TTC polymorphism in the epidemiological analysis implies the need of searching other useful polymorphic loci for detailed subdivision of clinical isolates.