Simulated Microgravity Induces the Proliferative Inhibition and Morphological Changes in Porcine Granulosa Cells

Astronauts are always faced with serious health problems during prolonged spaceflights. Previous studies have shown that weightlessness significantly affects the physiological function of female astronauts, including a change in reproductive hormones and ovarian cells, such as granulosa and theca cells. However, the effects of microgravity on these cells have not been well characterized, especially in granulosa cells. This study aimed to investigate the effects of simulated microgravity (SMG) on the proliferation and morphology of porcine granulosa cells (pGCs). pGC proliferation from the SMG group was inhibited, demonstrated by the reduced O.D. value and cell density in the WST-1 assay and cell number counting. SMG-induced pGCs exhibited an increased ratio of cells in the G0/G1 phase and a decreased ratio of cells in the S and G2/M phase. Western blot analysis indicated a down-regulation of cyclin D1, cyclin-dependent kinase 4 (cdk4), and cyclin-dependent kinase 6 (cdk6), leading to the prevention of the G1-S transition and inducing the arrest phase. pGCs under the SMG condition showed an increase in nuclear area. This caused a reduction in nuclear shape value in pGCs under the SMG condition. SMG-induced pGCs exhibited different morphologies, including fibroblast-like shape, rhomboid shape, and pebble-like shape. These results revealed that SMG inhibited proliferation and induced morphological changes in pGCs.     

Identification of highly selective type II kinase inhibitors with chiral peptidomimetic tails

Identification of highly selective type II kinase inhibitors is described. Two different chiral peptidomimetic scaffolds were introduced on the tail region of non-selective type II kinase inhibitor GNF-7 to enhance the selectivity. Kinome-wide selectivity profiling analysis showed that type II kinase inhibitor 7a potently inhibited Lck kinase with great selectivity (IC₅₀ of 23.0 nM). It was found that 7a and its derivatives possessed high selectivity for Lck over even structurally conserved all Src family kinases. We also observed that 7a inhibited Lck activation in Jurkat T cells. Moreover, 7a was found to alleviate clinical symptoms in DSS-induced colitis mice. This study provides a novel insight into the design of selective type II kinase inhibitors by adopting chiral peptidomimetic moieties on the tail region.     

Ribosomal DNA Intergenic Spacer of the Swimming Crab, Charybdis japonica

We have determined the full sequence of the ribosomal DNA intergenic spacer (IGS) of the swimming crab, Charybdis japonica, by long PCR for the first time in crustacean decapods. The IGS is 5376 bp long and contains two nonrepetitive regions separated by one long repetitive region, which is composed mainly of four subrepeats (subrepeats I, II, III, and IV). Subrepeat I contains nine copies of a 60-bp repeat unit, in which two similar repeat types (60 bp-a and 60 bp-b) occur alternatively. Subrepeat II consists of nine successive repeat units with a consensus sequence length of 142 bp. Subrepeat III consists of seven copies of another 60-bp repeat unit (60 bp-c) whose sequence is complementary to that of subrepeat I. Immediately downstream of subrepeat III is subrepeat IV, consisting of three copies of a 391-bp repeat unit. Based on comparative analysis among the subrepeats and repeat units, a possible evolutionary process responsible for the formation of the repetitive region is inferred, which involves the duplication of a 60-bp subrepeat unit (60 bp-c) as a prototype. Received: 13 April 1999 / Accepted: 2 August 1999     

Urea biosensor based on PANi(urease)-Nafion/Au composite electrode

The polyaniline (PANi)-Nafion composite film was prepared onto the ceramic plate by the cyclic voltammetry (CV) method with the various cycle numbers. When the PANi-Nafion/Au/ceramic plate with the preparing cycle number of 5 was as working electrode, the cathodic peak current was achieved as 84.0 microA in 60 mg dl(-1) NH4Cl buffer solution. On the other hand, the small cathodic peak currents for buffer solution in the presence of 60 mg dl(-1) LiOH, NaCl and KCl, respectively, were found with the same composite electrode as working electrode. The cathodic peak current decreased from 84.0 to 16.3 microA in the 60 mg dl(-1) NH4Cl buffer solution when the cycle number for preparing PANi-Nafion/Au/ceramic plate composite electrode with the CV method increased from 5 to 15. The enzyme of urease was immobilized onto the PANi-Nafion/Au/ceramic plate composite film by the electrochemical immobilization and the casting methods and used as sensing electrode to detect the concentration of urea in the buffer solution. The sensitivity of composite electrode immobilized with the casting method was greater than that of electrochemical immobilization method. The sensitivity and the detecting limit of the urea sensor were found to be 0.7 and 5.27 microA (mg dl(-1))(-1)cm(-2), as well as 6 and 0.3 mg dl(-1), respectively, when urease was immobilized by glutaraldehyde (GA) cross-linker and Nafion network, respectively.     

Role of primary sensorimotor cortex and supplementary motor area in volitional swallowing: a movement-related cortical potential study

We investigated the role of the cerebral cortex, particularly the face/tongue area of the primary sensorimotor (SMI) cortex (face/tongue) and supplementary motor area (SMA), in volitional swallowing by recording movement-related cortical potentials (MRCPs). MRCPs with swallowing and tongue protrusion were recorded from scalp electrodes in eight normal right-handed subjects and from implanted subdural electrodes in six epilepsy patients. The experiment by scalp EEG in normal subjects revealed that premovement Bereitschaftspotentials (BP) activity for swallowing was largest at the vertex and lateralized to either hemisphere in the central area. The experiment by epicortical EEG in patients confirmed that face/tongue SMI and SMA were commonly involved in swallowing and tongue protrusion with overlapping distribution and interindividual variability. BP amplitude showed no difference between swallowing and tongue movements, either at face/tongue SMI or at SMA, whereas postmovement potential (PMP) was significantly larger in tongue protrusion than in swallowing only at face/tongue SMI. BP occurred earlier in swallowing than in tongue protrusion. Comparison between face/tongue SMI and SMA did not show any difference with regard to BP and PMP amplitude or BP onset time in either task. The preparatory role of the cerebral cortex in swallowing was similar to that in tongue movement, except for earlier activation in swallowing. Postmovement processing of swallowing was lesser than that of tongue movement in face/tongue SMI; probably suggesting that the cerebral cortex does not play a significant role in postmovement processing of swallowing. SMA plays a supplementary role to face/tongue SMI both in swallowing and tongue movements.     

Interaction of Leishmania (L.) chagasi with the Vero cell line

The Vero cell line, a non-phagocytic cell, has supported the intracellular mechanism of Leishmania (L.) chagasi. This strain (MHOM/BR/501/MS00) was isolated from a human case of visceral leishmaniasis in Mato Grosso do Sul, Brazil and cultivated in Schneider's Drosophila medium with 20% of heat inactivated fetal calf serum. It was allowed to infect the Vero cells at a ratio of 10 to 20 promastigotes per cell. Within six hours of incubation, promastigote forms were found attached to Vero cells without any particular orientation. The number of amastigotes per cell increased during the incubation period. Results showed that promastigotes of L. (L.) chagasi could interact, transform to amastigote forms and multiply in non-phagocytic cells, demonstrating a new model to study the intracellular cycle of this protozoan.