H2O2 enhances Ca2+ release from osteoblast internal stores

The physiological activity of osteoblasts is known to be closely related to increased intracellular Ca2+ activity ([Ca2+]i) in osteoblasts. The cellular regulation of [Ca2+]i in osteoblasts is mediated by Ca2+ movements associated with Ca2+ release from intracellular Ca2+ stores, and transmembrane Ca2+ influx via Na+-Ca2+ exchanger, and Ca2+ ATPase. Reactive oxygen species, such as H2O2, play an important role in the regulation of cellular functions, and act as signaling molecules or toxins in cells. In this study, we investigated the effects of H2O2 on cellular Ca2+ regulation in osteoblasts by measuring intracellular Ca2+ activities using cellular calcium imaging techniques. Osteoblasts were isolated from the femurs and tibias of neonatal rats, and cultured for 7 days. The cultured osteoblasts were loaded with a Ca2+-sensitive fluorescent dye, Fura-2, and fluorescence images were monitored using a cooled CCD camera, and subsequently analyzed using image analyzing software. The results obtained are as follows: (1) The osteoblasts with lower basal Ca2+ activities yielded a transient Ca2+ increase, a Ca2+ spike, while osteoblasts with higher basal Ca2+ activities showed a continuous increase in [Ca2+]i leading to cell death. (2) Ca2+ spikes, generated after removing Na+ from superfusing solutions, were blocked by H2O2 and this was followed by a sustained increase in Ca2+ activity. (3) ATP- induced Ca2+ spikes were inhibited by pretreating with H2O2 and this was followed by a continuous increase of [Ca2+]i. When cells were pretreated with the exogenous nitric oxide (NO) donor S-Nitroso-N-acetylpenicilance (SNAP, 50 microM), treatments of ATP (1 mM) induced a Ca2+ spike-like increase, but [Ca2+]i did not return to the basal level. (4) The expression of inositol- 1,4,5-triphosphate receptor (IP3R) was enhanced by H2O2. Our results suggest that H2O2 modulates intracellular Ca2+ activity in osteoblasts by increasing Ca2+ release from the intracellular Ca2+ stores.     

Cell signaling in aging and apoptosis

Alterations in apoptotic potential, due to perturbations in cell signaling cascades, could underlie age-related organ-specific cellular degeneration and death. While increased apoptosis could lead to cell loss, as in neuronal degeneration, loss of apoptosis competence might well result in the loss of phenotypic fidelity of somatic cells, which could explain to some extent, the age-related increase in cancer incidence. Results from our laboratory indicate that after subjecting young and old rats to genotoxic stress in the form of methyl methanesulfonate (MMS), an apoptotic response is quickly mounted in the liver of the young animals but virtually absent in the same organ of old animals (Nature Med. 8 (2002) 3). To address the possible molecular signaling defect(s) responsible for the age-related dysfunction of apoptosis in response to MMS, mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases (ERKs), c-Jun NH(2)-terminal kinases (JNKs) and p38 MAPKs, were evaluated in the liver of young and old rats after MMS treatment. The results demonstrated distinct age-specific patterns of MMS-induced MAPKs activation, suggesting that the balance between cell survival and apoptosis after genotoxic stress may be impaired during aging. These results are discussed in terms of the relative importance in aging of biological redundancy, a concept put forward by the late Bernard Strehler, and cellular fidelity.     

The segmented regional volumes of the cerebrum and cerebellum in boys with Tourette syndrome

Neuropathological deficits are an etiological factor in Tourette syndrome (TS), and implicate a network linking the basal ganglia and the cerebrum, not a particular single brain region. In this study, the volumes of 20 cerebral and cerebellar regions and their symmetries were measured in normal boys and TS boys by brain magnetic resonance imaging. Brain magnetic resonance images were obtained prospectively in 19 boys with TS and 17 age-matched normal control boys. Cerebral and cerebellar regions were segmented to gray and white fractions using algorithm for semi-automated fuzzy tissue segmentation. The frontal, parietal, temporal, and the occipital lobes and the cerebellum were defined using the semiautomated Talairach atlas-based parcellation method. Boys with TS had smaller total brain volumes than control subjects. In the gray matter, although the smaller brain volume was taken into account, TS boys had a smaller right frontal lobe and a larger left frontal lobe and increased normal asymmetry (left>right). In addition, TS boys had more frontal lobe white matter. There were no significant differences in regions of interest of the parietal, temporal, or the occipital lobes or the cerebellum. These findings suggest that boys with TS may have neuropathological abnormalities in the gray and the white matter of the frontal lobe.     

Acute effect of ethanol on firing patterns of Purkinje cells in the rat cerebellar slice preparation

This study examined the acute effects of ethanol (EtOH) on the firing patterns of Purkinje cells (PCs) using an intracellular recording in slice preparation of rat cerebellum. The experiments were performed in sagittal cerebellar slices (400 micrometer) of adult Sprague-Dawley rats (80-100g). Ethanol was applied by a bath superfusion with a known concentration expressed as the percentage of solution by volume (v/v) at 0.1, 0.5, 1, 2, and 4%. The result of the Chi-square test illustrated that the firing patterns were altered significantly after EtOH (p=0.007). However, the firing patterns that were altered by EtOH application were not affected by EtOH concentration (p= 0.1296). Among the 54 PCs tested, 30 PCs did not display any spontaneous firing activity and 24 PCs displayed spontaneous spike activity, either spiking in the simple manner (n=14) or cyclicly oscillating (n=10). In the presence of EtOH, 31 PCs were quiet, 22 PCs exhibited simple spiking activity and 1 PC continued to oscillate. Most PCs that displayed spontaneous activity before EtOH application progressively slowed their spike activity after EtOH superfusion. Especially, it was evident that 9 out of 10 oscillating PCs stopped their regular cyclic activity. In addition, 9 out of 14 PCs that displayed simple spike activity ceased to fire after EtOH application. Eleven out of 30 quiet PCs began to fire irregularly after EtOH application and this phenomenon usually occurred with membrane depolarization. EtOH induced spontaneous activity in 36.7% (11/30) of the quiescent PCs. In conclusion, there was differential EtOH sensitivity in the vitro slice preparation. EtOH depressed the endogenously generated spontaneous activity, especially the oscillatory firing activity. In contrast, the silent PCs were excited after EtOH application. Since this differential sensitivity persists in the presence of tetrodotoxin (TTX), it is suggested that this differential sensitivity is peculiar to the PCs.     

Applications of the pulsatile flow versatile ECLS: in vivo studies

INTRODUCTION: T-PLS (Twin-Pulse Life Support) is the first commercial pulsatile ECLS (Extra Corporeal Life Support) device (1). The dual sac structure of T-PLS can effectively reduce high membrane oxygenator inlet pressure and hemolysis. To verify both the use of T-PLS for ECLS and the advantages of T-PLS, we tested various models. METHOD AND RESULTS: In the partial CPB (cardio pulmonary bypass) model (swine), T-PLS (N = 6), and Biopump (N = 2), a single pulsatile pump (N = 2), were compared. In the case of single pulsatile flow, during pump systole, pressure increased to 700 - 800 mmHg at the inlet port of the membrane oxygenator. fHb, a hemolysis measurement value, was about 80 mg/dL at 3 hours. On the contrary, because of T-PLS's dual sac system, the pressure of T-PLS had a maximum value of about 250 mmHg and fHb was similar to that of the commercial centrifugal pumps. In the total CPB model (bovine, N = 6), the heart was stopped via cardioplegia (Kcl). T-PLS flow was maintained at 3.0-4.5 L/min. T-PLS functioned like a natural heart, having a pulse pressure of 26-43 mmHg and a pulse rate of 40-60 bpm (beats per minute). In the emergency case model (canine, N = 6), T-PLS was started 10 minutes after cardiac arrest from electronic shock. In spite of cardiac arrest for a period of 40 minutes, the heart was recovered after defibrillation. In the ARDS (Acute Respiratory Distress Syndrome) model (canine, N = 6), minimal ventilator parameters were set: tidal volume 130 ml, respiration rate = bpm, FiO2 = 10%. Three hours after starting T-PLS, PO2 of the carotid artery blood (after 2 hours: 195 +/- 89.4; after 3 hours: 258 +/- 99.3 mmHg) was above half the value of the femoral artery but was within normal range. CONCLUSION: It is suggested that a portable pulsatile ECLS like T-PLS may be used as a CPB device and as an alternative CPR (cardiopulmonary resuscitation) device in the case of cardiac arrest. Due to the pulsatile flow, oxygenated blood is delivered to the patient without overloading the ARDS patients heart.     

Microcystic adenomas of the pancreas. Report of three cases with two of multicentric origin

Three cases of microcystic adenomas of the pancreas with special reference to multicentric origin are described. The gross features and light microscopic findings were consistent with those described as being microcystic adenomas, but in two cases the gross examination and gelatin-embedded giant slices revealed multiple, isolated development of tumors ranging from submacroscopic nodules to tumors 4.5 cm in diameter. The larger tumors often showed ragged margins with small satellite nodules around the masses. A central fibrolamellar stellate core with centrifugally radiating septation was found in most of the tumor masses, even in the smaller ones. Ultrastructural and immunohistochemical findings revealed a single row of glycogen-rich epithelial cells, but participation of myoepithelial cells was not confirmed. Instead, vimentin-positive cells (pericytes) within the interstitial space incorporated closely with the basal lamina of the cyst wall. This study suggests that a small percentage of microcystic adenomas of the pancreas develop in multiple tumors, and some appear as a single tumor by their confluence.     

Arthritic manifestations of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is commonly associated with arthritic manifestations. They are divided into three clinical categories; peripheral arthritis, spondylitis, and sacroiliitis. To evaluate the incidence of arthritis associated with IBD in Korea, we retrospectively reviewed one hundred and twenty-nine patients with IBD, 77 with ulcerative colitis (UC) and 52 with Crohn''s disease (CD). Arthritis occurred in twenty-two patients (17.1%); 15 with UC(19.6%), 7 with CD (13.5%). Patients with arthritis had more active inflammations and all were seronegative except one patient. Peripheral arthritis was found in twenty patients (15.5%) and more common in UC (19.6%) than in CD (9.6%). Joint involvements tended to be monoarticular or pauciarticular, and most frequently developed in the knee and ankle. Spondylitis was diagnosed in one patient (1.6%) who showed HLA B27 positivity. Radiographic sacroiliitis was observed in eight patients (6.2%) who revealed HLA B27 negativity. Both peripheral arthritis and sacroiliitis were found in six patients (4.6%). In CD, arthritis occurred in 20% of the patients with colonic involvement but in none of the patients without colonic involvement. In conclusion, arthritis was frequent in patients with IBD. Peripheral arthritis was more common in patients with UC than CD. All the patients with CD and arthritis had colonic involvement.     

Thyroglobulin synthesis of oxyphilic cells in various types of neoplastic and autoimmune thyroid diseases.

To determine the content of thyroglobulin in oxyphilic cells of the thyroid, which have been considered as non-thyroglobulin producing cells, the degree of stainability of the various oxyphilic cells for thyroglobulin was compared with that of non-oxyphilic follicular cells in either same or different lesion. A total of 13 oxyphilic lesions, including three follicular adenomas containing oxyphilic cell nodules, four pure oxyphilic cell adenomas, and six Hashimoto's thyroiditis were compared with 16 of non-oxyphilic lesions such as, seven follicular adenomas, four chronic lymphocytic thyroiditis, and five Graves' disease. Many oxyphilic cells stained positively for thyroglobulin regardless of their morphologic variation, but less intensely than the usual follicular cells in follicular adenomas, chronic lymphocytic thyroiditis, and Graves' disease. The stainability of oxyphilic cells for thyroglogulin did not show any significant correlation with morphologic features, whereas in follicular adenomas, the non-oxyphilic follicular cells forming microfollicles stained less strongly for thyroglobulin than the same cells lining large mature follicles in a reproducible way. With above findings, we concluded that oxyphilic cells maintain the functional activity in terms of thyroglobulin synthesis, although the content of the thyroglobulin is less than that of non-oxyphilic colloid forming follicular cells.     

Differential expression of hypoxia inducible factor-1 alpha and tumor cell proliferation between squamous cell carcinomas and adenocarcinomas among operable non-small cell lung carcinomas

This study aimed to evaluate whether the elevated level of hypoxia-inducible factor-1alpha (HIF-1alpha) correlated with histologic types, angiogenesis, tumor cell proliferation, and clinical parameters in common non-small cell lung carcinomas (NSCLCs). We performed immunohistochemical stains using paraffin-embedded tissue blocks from 84 cases of operable NSCLC [No. of squamous cell carcinoma (SCC), 45; No. of adenocarcinoma (AC), 39]. HIF-1alpha expression was related with histologic types (66.7% in SCCs vs 20.5% in ACs, p<0.001), but not with lymph node status, tumor stage, vascular endothelial growth factor expression, microvessel density (MVD), and proliferating cell nuclear antigen (PCNA) index (p>0.05, respectively). As for the histologic types, MVD and PCNA index were significantly higher in SCCs than in ACs (p=0.009 and p=0.016, respectively). Among HIF-1alpha positive carcinomas, MVD was significantly higher in HIF-1alpha positive SCCs than in HIF-1alpha positive ACs (p=0.023). The overall survival curves were not associated with HIF-1alpha expression or any other histologic parameters (p>0.05). These findings suggest that HIF-1alpha expression in NSCLCs may play a differential role according to histologic types, but its prognostic significance is indeterminate.     

Hepatic and small bowel mucormycosis after chemotherapy in a patient with acute lymphocytic leukemia

Mucormycosis is a rare but invasive opportunistic fungal infection with increased frequency during chemotherapy-induced neutropenia. The clinical infections due to Mucor include rhinocerebral, pulmonary, cutaneous, gastrointestinal and disseminated diseases. The first two are the most common diseases and all entities are associated with a high mortality rate. Still hepatic involvement of Mucor is rarely reported. We experienced a case of hepatic and small bowel mucormycosis in a 56-year-old woman after induction chemotherapy for B-cell acute lymphocytic leukemia. Initial symptoms were a high fever unresponsive to broad spectrum antibiotics and pain in the left lower abdominal quadrant. It was followed by septic shock, deterioration of icterus and progressively elevated transaminase. An abdominal CT demonstrated multiple hypodense lesions with distinct margins in both lobes of liver and pericolic infiltration at small bowel and ascending colon. Diagnosis was confirmed by biopsy of the liver. The histopathology of the liver showed hyphae with the right-angle branching, typical of mucormycosis. The patient was managed with amphotericin B and operative correction of the perforated part of the small bowel was performed. However, the patient expired due to progressive hepatic failure despite corrective surgery and long-term amphotericin B therapy.