Changes in the cytoskeleton of 3T3 fibroblasts induced by the phosphatase inhibitor,calyculin-A

Summary Addition of the protein phosphatase inhibitor, calyculin-A, to 3T3 fibroblasts causes a marked change in cell morphology. Initially the cells become rounded, develop surface blebs and then detach from the substratum. In the detached cells an unusual ball-like structure is observed. This study focuses on the cytoskeleton during these calyculin-A-induced morphological changes. Stress fibres disappear as the cells begin to round and aggregates of actin are formed towards the apical surface of the cell. These aggregates condense, in the detached cells, to form the ball structure of approximately 3 m diameter. Between the ball and the nucleus are cables of intermediate filaments that appear to be attached to the surface of the ball and to the nuclear lamina. Using a procedure designed for the isolation of nuclei the nucleus-ball complex can be obtained. Analysis of the nucleus-ball preparation by immunofluorescence and electron microscopy demonstrate that the ball contains actin and that intermediate filaments are located between the ball and the nucleus. In this preparation, the intermediate filaments also appear to attach to the surfaces of the ball and the nucleus. Electrophoretic analysis of the nucleus-ball preparation indicates that, in addition to actin, a major component of the ball is myosin. It is suggested that the formation of the ball is caused by an actin-myosin-based contractile process, initiated by the phosphorylation of myosin. The aggregation of the actomyosin draws together the intermediate filaments into the area between the ball and nucleus. This hypothesis requires that vimentin binds both to the nucleus and to some component of the ball.     

Frequent cellular senescence in small bile ducts in primary biliary cirrhosis: a possible role in bile duct loss

The pathogenesis of progressive bile duct loss in primary biliary cirrhosis remains unclear. In this study, the involvement of cellular senescence of biliary epithelial cells was examined in liver tissue samples from patients with primary biliary cirrhosis (n = 33), and compared with control diseased and normal livers (n = 83). In addition, cellular senescence was induced by oxidative stress in cultured mouse biliary epithelial cells. Biliary epithelial cells in small bile ducts in primary biliary cirrhosis, especially those in patients presenting with chronic non-suppurative cholangitis, frequently expressed senescence-associated beta-galactosidase, and senescence-associated p16(INK4) and p21(WAF1/CIP). In contrast, senescence-associated markers were rarely expressed in small bile ducts in control livers. The infiltration of myeloperoxidase-positive inflammatory cells into biliary epithelial cell layers was closely associated with the cellular senescence of biliary epithelial cells in early-stage PBC. Cellular senescence of cultured mouse biliary epithelial cells was induced by treatment with H2O2 via the p38MAPK-dependent pathway and nitric oxide-augmented H2O2-induced cellular senescence. Oxidative stress- and nitric oxide-mediated cellular senescence may be involved in bile duct lesions, which are followed by progressive bile duct loss in primary biliary cirrhosis.     

Effect of basic amino acids on susceptibility to carbapenems in clinical Pseudomonas aeruginosa isolates

We evaluated effects of medium composition, including basic amino acid content and pH, on susceptibility to carbapenems such as imipenem, panipenem and meropenem, in clinical isolates of Pseudomonas aeruginosa. Susceptibility to carbapenems was reduced by basic amino acids in the medium, while susceptibilities to ceftazidime and aztreonam were not. Among carbapenems, susceptibility to panipenem was most sharply reduced by addition of basic amino acids to 1:16 Mueller-Hinton agar (MHA). In 174 of 175 clinical isolates, MICs for carbapenems were affected to different degrees by medium composition. One isolate, in which MICs for carbapenems did not differ between MHA and 1:16 MHA, showed reduced production of porin (OprD). Our results suggest that susceptibility to individual carbapenems, especially panipenem, is difficult to evaluate based on MICs for other carbapenems determined on MHA. For a better prediction of antibiotic efficacy, it may be important to evaluate the susceptibility for each carbapenem individually.     

Spontaneous tension oscillation in skinned bovine cardiac muscle

 Skinned fibres from bovine ventricles exhibited spontaneous tension oscillations when MgADP and inorganic phosphate (Pi) were added to the solution bathing fibres in the relaxed state (ADP-SPOC). A similar type of oscillation was observed at intermediate concentrations of free Ca²⁺ in the absence of MgADP and Pi (Ca-SPOC). To investigate the correlation between ADP-SPOC and Ca-SPOC, we constructed two-dimensional state diagrams of cardiac muscle using different concentrations of Pi (0–20 mM) and free Ca²⁺ [pCa=around 5 (+Ca²⁺), pCa=5.15–6.9 and +EGTA (–Ca²⁺)], with varying concentrations of MgADP (0–10 mM), with 2 mM MgATP and 2 mM free Mg²⁺ maintaining ionic strength at 0.15±0.01 M, pH 7.0, 25 °C. The three-dimensional (pCa-Pi-MgADP) state diagram thus obtained was divided into three regions, i.e. the contraction region in which tension oscillation was undetectable, the spontaneous tension oscillation (SPOC) region and the relaxation region. We found that the regions of ADP-SPOC and Ca-SPOC were continuously connected by a single oscillation region sandwiched between the contraction and relaxation regions. The state diagram, which encompasses physiological conditions, shows that the probability of SPOC is higher in cardiac muscle than in skeletal muscle. From these results, we suggest that, despite distinct ionic conditions, the molecular state of cross-bridges during SPOC is common to both ADP-SPOC and Ca-SPOC. Received 19 February 1996 / Received after revision: 16 July 1996 / Accepted: 14 August 1996     

Oxatomide inhibits the release of chemical mediators from human lung tissues and from granulocytes

The effects of oxatomide on the release of histamine and leukotriene C4 (LTC4) from human lung fragments and granulocytes were examined and the findings compared with the effects of the antiallergic drugs ketotifen, azelastine and tranilast. Oxatomide inhibited the release of both histamine and LTC4 from human lung fragments in cases of passive sensitization with human IgE myeloma serum upon anti-epsilon stimulation. IC50 values for the release of LTC4 from human lung fragments were as follows: oxatomide, 2.35 x 10(-5) M; azelastine, 27.2 x 10(-5) M; ketotifen, 52.1 x 10(-5) M; tranilast, 62.9 x 10(-5) M. Oxatomide also inhibited the release of both histamine and LTC4 from human mixed leukocytes stimulated by the calcium ionophore A23187. IC50 values for the release of LTC4 from human mixed leukocytes were as follows: oxatomide, 1.67 x 10(-5) M; azelastine, 3.65 x 10(-5) M; ketotifen, 12.2 x 10(-5) M; tranilast, 15.1 x 10(-5) M. The effects of oxatomide on the release of LTC4 from purified human neutrophils and eosinophils were also given attention. Oxatomide inhibited the release of LTC4 from eosinophils more effectively than from neutrophils and mixed leukocytes. As there is evidence that eosinophils play an important role in the development of late asthmatic responses and/or in the aggravation of asthma, oxatomide is expected to be an effective treatment for such conditions.     

Three-dimensional ultrasonography in the first trimester of human pregnancy

Our purpose was to visualize normal embryonal and fetal surface anatomical structures in the first trimester of human pregnancy by use of three-dimensional ultrasonography with a specially developed abdominal three-dimensional transducer. Four embryos and 31 fetuses of 8-13 weeks gestation were studied with a specially-developed abdominal three-dimensional transducer (3.5 MHz). This imaging system can provide conventional two-dimensional ultrasonography images and can also generate, within seconds, high-quality three-dimensional images in the surface and transparent mode with no need for an external workstation. The percentage of surface anatomical structures visualized at each gestational age interval using two-dimensional and three-dimensional ultrasonography is presented. Head and trunk were depicted in all cases. The number and the clarity of visualization of face, upper and lower extremities, hand, and foot increased with advancing gestation. The free loop of the umbilical cord was depicted in most cases. The number of depictions of abdominal cord insertion, midgut herniation, and yolk sac decreased with the increase of gestation. Genitals could not be identified in the first trimester. The ability to view some surface anatomical structures (face, hand, and foot) was better with three-dimensional ultrasonography than with two-dimensional ultrasonography. Three-dimensional ultrasonography provides a novel means for visualization of surface anatomical structures of the embryo and early fetus. These results suggest that three-dimensional ultrasonography can become an important modality in future embryological and early fetal research and in detection of embryonic and fetal developmental disorders in the first trimester of pregnancy.      

Genotype,serotype, and phylogenetic characterization of the complete genome sequence of hepatitis B virus isolates from Malawian chronic carriers of the virus

Hepatitis B virus (HBV) genotypes have distinct geographical distribution. HBV sequences among hepatitis B carriers in Malawi have not been evaluated thus far. HBsAg serotype and genotype of HBV was determined in 20 serum samples from Malawian chronic HBV carriers, and two complete genomes and 13 entire pre-S2/S genes were sequenced directly. Genotype A HBV isolates were found in all of the samples, and serotype with adw2 and ayw2 were detected in three and 17 samples, respectively. In phylogenetic analyses, two complete genomes were classified into a subgroup A' that was described previously in South African isolates of the virus, and were separated from HBV isolates in Western countries with nucleotide differences ranging from 4.1-6.2%. The separation of subgroup A' was also evident in the tree topology of the entire pre-S1/S2, X and precore/core region, but not evident in the small-S region. The nucleotide divergences in subgroup A' were higher than those among genotype A without subgroup A' in the complete genomes as well as each of four open reading frames. All of the 13 pre-S2/S sequences were classified into the subgroup A', and clustered with known HBV isolates with ayw2 in carriers from South Africa and Zimbabwe. Three amino acids in the pre-S2/S gene were characteristic of subgroup A' with ayw2. In conclusion, unique HBV isolates of subgroup A' with ayw2 are prevalent in Malawi, and subgroup A' with a relatively higher nucleotide diversity may be a HBV isolate characteristic of the indigenous population of some African countries.     

The influence of social environmental condition on the production of stress-induced 22 kHz calls in adult male Wistar rats

Adult rats emit 22 kHz ultrasonic vocalizations (USVs) in response to aversive stimuli, and these sounds are suggested to have communicative information among conspecifics. It is conceivable that social environment during development of rats has relevance to the emission of 22 kHz USVs. To examine the effects of social environment after weaning on production of stress-induced USVs, we compared the amount of emission of USVs among three groups of rats reared under different conditions after weaning. One group of rats was housed individually, and the other two groups were housed in pairs, in which social hierarchy of the pair was determined by social dominance-subordination relationships. The USVs were induced by acute mild somatic stimuli on the back and neck. Individually reared rats emitted much fewer USVs than pair-reared rats. In addition, socially subordinate rats emitted more USVs compared with socially dominant ones. These results suggest that not only social interaction but also the status in social hierarchy may play an important role in the process of the development of USVs induced by somatic stimuli.     

Leukocytic fragments in blood smears

We retrospectively reviewed blood smears from patients with septic shock and/or disseminated intravascular coagulation (DIC) in order to determine whether the appearance of leukocytic fragments on blood smears has any clinical significance. Leukocytic fragments were seen in six of twelve cases with septic shock but in none of three cases of DIC without sepsis. All six cases with leukocytic fragments also showed fragmented erythrocytes. The fact that five of these six cases died and that four were dead within two days after the leukocytic fragments were noted may indicate that this appearance heralds a severe and terminal condition of sepsis. Although the exact reason for the appearance of these fragments is unclear, it seems to be related either to mechanical shearing through microangiopathic fibrin strands, which may also cause erythrocytic fragments, or to a complex alteration in the cell membrane due to bacteria, endotoxin, or other factors. We would like to stress that the presence of leukocytic fragments on blood smears provides an important clue to the patient's prognosis.