Comparative inotropic effect of calcium, endothelin-1, and forskolin on ferret papillary muscles during acidosis

Summary— Acidosis affects multiple steps in the excitation-contraction coupling pathway of myocardium, producing decreased calcium sensitivity of myofibrils and modification of the function of the sarcoplasmic reticulum. Our aim was to evaluate the effectiveness of three different classes of inotropic agents under acidotic conditions: 1) forskolin, an adenylate cyclase activator that enhances cellular cyclic AMP concentrations, 2) elevated extracellular Ca²⁺ and 3) endothelin-1, an activator of the inositol triphosphate, diacylglycerol pathway. Ferret papillary muscles were mounted in organ baths containing normal physiological solution (pH = 7.4). After baseline tension was measured, the muscles were bathed in an acidotic solution (pH = 6.98) that decreased tension to 40% of the control; subsequently, the muscles were washed with normal physiological solution until they returned to baseline. Each inotropic agent was added to the bathing solution in a concentration sufficient to increase tension by 40% above the baseline. Then the solution was made acidotic (pH = 6.98) in the continuous presence of that concentration of inotropic agent and the resultant steady-state developed tension measured. The increases in tension induced by each inotropic agent at normal pH were adjusted to be similar; in contrast, the response to each drug in acidosis was significantly different. Under acidotic conditions, endothelin-1 was the most effective inotropic agent in restoring the depressed developed tension. This was possibly due to enhancement of the myofilament sensitivity to Ca²⁺, which was more effective than increasing [Ca²⁺]_i through elevating extracellular Ca²⁺ or the addition of forskolin which increased [Ca²⁺]_i but desensitized the myofilaments to Ca²⁺.     

Effects of calcium in continuous cardioplegia on myocardial protection

The effects of calcium (Ca) on a hyperkalemic cardioplegic solution for continuous cardioplegia were examined in an isolated perfused working rat heart model. The coronary arteries were perfused with a modified Krebs-Henseleit bicarbonate buffer (K-H) solution, containing various concentrations of Ca(0.1, 0.6, 1.2, and 2.5 mmol/l) and a high concentration of potassium (20 mmol/l), for 180 min, after which cardiac arrest was induced at 37°C for 180 min. Cardiac function and creatine kinase (CK) were measured. In the control group, K-H solution was infused in place of the cardioplegic solution, and cardiac arrest was not induced. No significant differences were observed between the groups infused with the K-H solution containing Ca concentrations of 0.6, 1.2, and 2.5 mmol/l in the percent recovery of aortic flow (82.1±2.9%, 80.6±2.0%, and 71.5±3.7% (mean±SEM) respectively) or in the recovery of other indices of cardiac function, or in CK leakage. There were also no significant differences in the recovery of cardiac function and CK leakage between these groups and the control group. In the Ca 0.1 mmol/l group, however, the characteristic Ca paradox was observed. These findings suggest that if the Ca concentration in a cardioplegic solution is higher than 0.6 mmol/l during continuous cardioplegia, excellent cardioprotective effects will be achieved.     

A comparison of three bioelectrical impedance analyses for predicting lean body mass in a population with a large difference in muscularity

This study tested the hypothesis that, as compared to whole-body bioelectrical impedance (BI) analysis, segmental BI analysis can estimate lean body mass (LBM) more accurately in a population with a large difference in muscularity. In addition to whole-body BI, which determines impedance (Z) between the wrist and ankle, two segmental BI analyses which determine the Z value of every body segment in each of (1) the arms, legs and trunk (distal BI) and (2) the upper arms, upper legs and trunk (proximal BI) were applied to a group of 125 male athletes and 75 non-athletes. The subjects were divided into validation and cross-validation groups. Simple and multiple regression analyses were applied to (length)²/Z (BI index) values for the whole-body and each body segment, to develop the prediction equations of LBM measured using air-displacement plethysmography. In the validation group, the SE of estimation was similar in the whole-body (3.4 kg, 5.4%), distal (3.4 kg, 5.5%) and proximal BI (3.3 kg, 5.2%) analyses. However, the whole-body and distal BI analyses produced systematical errors in the estimates of LBM. Moreover, the residuals in the two methods significantly (P<0.05) correlated with the ratios of BI indices of the upper arms and upper legs to those of the arms and legs, respectively, calculated as variables approximating the relative development of lean tissues at the proximal area of limbs. On the other hand, the proximal BI analysis was validated and cross-validated. Thus, the accuracy of estimating LBM was similar in the whole-body and the two segmental BI analyses. However, the prediction equations derived from the use of the whole-body BI index and a combination of the arms, legs and trunk BI indices produced a systematical error relating to the difference between the limb segments in lean tissue development.     

Clonal expansion of multiphenotypic Epstein-Barr virus-infected lymphocytes in chronic active Epstein-Barr virus infection

Chronic active Epstein-Barr virus (EBV) infection has been recognized as clonal non-neoplastic lymphoproliferative diseases. However, some reports of cases with a multiphenotypic expansion of EBV-infected lymphocytes give rise to questions of how EBV infects multiphenotypic lymphocytes and whether chronic active EBV infection is a truly monoclonal lymphoproliferative disease. We report two patients with chronic active EBV infection who showed expansion of multiphenotypic EBV-infected lymphocytes. EBV DNA was detected in CD4+ and CD8+ T cells and in B cells from pleural fluid of one patient and in T and B cells from a cervical lymph node of the other patient by polymerase chain reaction (PCR). Although real-time PCR showed that there were equally high loads of EBV genomes in CD4+ and CD8+ T cells from the pleural fluid, Southern blot hybridization with terminal repeats of the EBV genome showed a single band of the same molecular weight in three tissue samples from the patient. The results indicated biphenotypic expansions of CD4+ and CD8+ T cells infected with the same clone of EBV. Furthermore, bisulfite PCR analysis showed hypermethylated status in the Cp region in the two patients regardless of their cell populations. There has been a discrepancy between clonality and expansion of multiphenotypic EBV-infected lymphocytes. We speculate that lymphoid progenitor cells that have not differentiated into T and B cell progenitors are infected with EBV, resulting in clonal expansion of EBV-infected multiphenotypic cells.     

Argyrophil cell carcinoma (apudoma) of the esophagus

Summary In a series of 79 cases of primary esophageal carcinoma resected at The Center for Adult Diseases, Osaka, there were six tumors with specific histopathologic features valid for the diagnosis of argyrophil cell carcinoma. Of the 6 tumors, 3 were studied electron microscopically and assay for ACTH content was performed on 4 tumors.Clinically, the ages of the 6 patients ranged from 56 to 71 years; two were women and four men. Four of the 6 patients died with widespread tumor recurrences within 9 months of operation.Microscopically, the 6 tumors were composed largely or almost entirely of small, spindle-shaped cells resembling those of oat cell carcinoma of the lung, and were characterized by the arrangement of tumor cells in solid sheets or anastomosing cords, the presence of argyrophil tumor cells, and the deposits of amyloid. Electron microscopically, the three tumors contained neurosecretory-type granules. Using bioassay or radioimmunoassay ACTH activity in the tumor tissues was detected in 3 out of the 4 tumors determined.From the light and electron microscopic characteristics and the assay evidence, it seems reasonable to conclude that the 6 tumors are endocrine polypeptide producing tumors (apudomas) that arise from argyrophil cells normally found among the basal cells of the esophageal mucosa, and that they represent a distinct histopathologic entity clearly distinguishable from other types of esophageal carcinomas.Supported in part by a Grant-in Aid for Cancer Research from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare, JapanThe authors are grateful to Prof. H. Imura and Dr. Y. Hirate, Department of Internal Medicine, Kobe University School of Medicine for their interest and performing the assays for ACTH on the tumor tissues.     

Induction of interferon-inducible protein-10 and monokine induced by interferon-gamma from human endothelial cells infected with Influenza A virus

Primary human umbilical vein endothelial cells (HUVECs) were infected with Influenza virus A/Aichi/2/68 (H3N2) in order to determine the role of endothelial cells in mediating inflammation induced upon virus infection. Structural proteins of the virus and mRNA of the M2 protein were detected in the infected cells, indicating that virus infection had occurred in HUVECs. The Influenza A virus-infected HUVECs showed elevated levels of gene expression of interferon (IFN)-inducible protein (IP)-10 and monokine induced by IFN-gamma (Mig), while heat-, formalin- and diethyl ether-inactivated viruses did not enhance the IP-10 and Mig gene expression. The results thus indicate that infection of live Influenza A virus is responsible for elevation of IP-10 and Mig gene expression. The elevation of IP-10 and Mig gene expression in infected HUVECs was not accompanied by the elevation of IFN-gamma gene expression, indicating that the elevation of IP-10 and Mig gene expression was independent of the IFN-gamma pathway.     

Arpp,a new homolog of carp,is preferentially expressed in type 1 skeletal muscle fibers and is markedly induced by denervation

In this study, we isolated and characterized a murine counterpart of the human Arpp (hArpp) gene. Sequence analysis revealed that the murine Arpp (mArpp) gene is almost identical to the Ankrd2 gene, which has recently been isolated as a mouse gene induced in stretched skeletal muscle. The mArpp gene encodes a protein of 332 amino acids that contains four well-conserved ankyrin-repeat domains in the central portion of the protein. The amino acid sequence of mArpp protein (mArpp) is highly homologous to that of mouse cardiac-restricted ankyrin-repeat protein (Carp), which is proposed to be a putative genetic marker for cardiac hypertrophy. Immunohistochemical analysis revealed that mArpp is preferentially expressed in type 1 skeletal muscle fibers, and that mArpp is localized in both the nucleus and the sarcomeric I-band of muscle fibers, suggesting that Arpp may function as a nuclear and sarcomeric protein. Furthermore, mArpp was also expressed in neurons of the cerebellum and cerebrum, the islets of Langerhans in the pancreas, and the esophageal epithelium, suggesting that mArpp may play a functional physiologic role in brain, pancreas, and esophagus as well as in type 1 muscle fibers. Interestingly, although mArpp was localized in both nucleus and cytoplasm in neurons, its localization was restricted to nucleus in pancreas and esophagus, suggesting that intracellular localization of mArpp is regulated in a tissue-specific manner. Furthermore, we found that mArpp- and Carp-expression in skeletal muscle were markedly up-regulated after denervation. Although the elevated expression level of Carp was kept only for two weeks after denervation, that of Arpp was kept at least for 4 weeks, suggesting that mArpp and Carp may play distinct functional roles in denervated skeletal muscle.     

Wnt signaling plays an essential role in neuronal specification of the dorsal spinal cord

In the developing spinal cord, signals from the roof plate are required for the development of three classes of dorsal interneuron: D1, D2, and D3, listed from dorsal to ventral. Here, we demonstrate that absence of Wnt1 and Wnt3a, normally expressed in the roof plate, leads to diminished development of D1 and D2 neurons and a compensatory increase in D3 neuron populations. This occurs without significantly altered expression of BMP and related genes in the roof plate. Moreover, Wnt3a protein induces expression of D1 and D2 markers in the isolated medial region of the chick neural plate, and Noggin does not interfere with this induction. Thus, Wnt signaling plays a critical role in the specification of cell types for dorsal interneurons.     

Detection methods of possible prion contaminants in collagen and gelatin

Summary.  We describe methods for the preparation of collagen and gelatin samples to detect possible prion contaminants using Western blotting of a major component of prions, PrP^Sc. A commercially available collagen solution containing 2% athero-collagen was spiked with rodent adapted scrapie prion and used as the prion-contaminating collagen. The methods developed center on the enzymatic reduction of the collagen solution viscosity with protease treatments and on the concentration of the prion from the protease-digests with polyethylene glycol-#6000 and NaCl. Recovery of the spiked prion as a partially protease-resistant core fragment of PrP^Sc fluctuated from 30% to 46% of the input amount. Received June 19, 1998 Accepted August 5, 1998