Studies on the phospholipid composition of pathogenic cell membranes of Mycoplasma hyopneumoniae

The membrane phospholipid and fatty acid compositions of Mycoplasma hyopneumoniae, a pathogen of porcine enzootic pneumoniae isolated in China, was studied by thin-layer chromatography and gas chromatography. The results showed that membrane phospholipids consisted predominantly of diphosphatidylglycerol. The percentage of C16 - C18 fatty acids comprised 79% of the total fatty acids, of which oleic acid as well as palmitic acid are the major fatty acids. Some differences were shown in fatty acid composition as compared with membranes of other species of Mycoplasma.     

Myosin thick filaments from adult rabbit skeletal muscles

Myosin subfragment 1 (S1) forms dimers in the presence of Mg(2+) or MgADP or MgATP. The entire myosin molecule forms head-head dimers in the presence of MgATP. The angle between the two subunits in the S1 dimer is 95 degrees. Assuming that the length of the globular part of S1 is approximately 12 nm and that the S1/S2 joint (lever arm approximately 7 nm) is clearly bent, the cylinder tangent to this dimer should have a diameter of approximately 18 nm, close to the approximately 16-20 nm suggested by many studies for the diameter of thick filaments in situ. These conclusions led us to re-examine our previous model, according to which two heads from two opposite myosin molecules are inserted into the filament core and interact as dimers. We studied synthetic filaments by electron microscopy, enzyme activity assays, controlled digestion and filament-filament interaction analysis. Synthetic filaments formed by rapid dilution in the presence of 1 mM EDTA at room temperature ( approximately 22 degrees C) had all their myosin heads outside the backbone. These filaments are called superfilaments (SF). Synthetic filaments formed by slow dilution, in the presence of either 2 mM Mg(2+) or 0.5 mM MgATP and at low temperature ( approximately 0 degrees C) had one myosin head outside the backbone and one head inside. These filaments are called filaments (F). Synthetic filaments formed by slow dilution, in the presence of 4 mM MgATP at low temperature ( approximately 0 degrees C) had most of their heads inserted in the filament core. These filaments are called antifilaments (AF). These experimental results provide important new information about myosin synthetic filaments. In particular, we found that myosin heads were involved in filament assembly and that filament-filament interactions can occur via the external heads. Native filaments (NF) from rabbit psoas muscle were also studied by enzyme assays. Their structure depended on the age of the rabbit. NF from 4-month-old rabbits were three-stranded, i.e. six myosin heads per crown, two of which were inside the core and four outside. NF from 18-month-old rabbits were two-stranded (similar to F).     

Vibrational spectroscopic study of glutathione complexation in aqueous solutions

A spectroscopic study of glutathione (GSH) and glutathione disulfide (GSSG) has been performed using Fourier-transformed infrared absorption and Raman scattering in order to pinpoint the sites of complexation of these two species with water and particularly with H2O2. Molecules of GSH and GSSG were studied in KBr pellets, and in aqueous solutions of H2O, D2O, and H2O with H2O2 (1 mol L(-1)) to characterize the specific influence of the solvent molecules. A time-resolved Raman study was performed for GSH/H2O2, in aqueous solution at 1:1 molar ratio in order to observe the formation of GSSG and to discuss the mechanism of this redox reaction.     

Development of pig embryos by nuclear transfer of cultured fibroblast cells

Pig fibroblast cells were transferred to enucleated oocytes by micromanipulation and electrofusion. The donor cells used for nuclear transfer were synchronized in presumptive G0 by serum starvation. In the first experiment, nuclear transfer was performed with fibroblasts that had either a smooth or a rough surface. A significant difference (p < 0.05) in the percentage of chromosome condensation (39.5%, 15/38 and 16.6%, 5/30) and nuclear formation (36.8%, 14/38 and 16.3%, 8/49) was found between the reconstructed embryos derived from the cells with smooth surface and with rough surfaces, respectively. The percentage of chromosome condensation (42.5%, 17/40 and 19.6%, 11/56) and nuclear formation (38.3%, 23/60 and 18.8%, 9/48) were higher (p < 0.05) in reconstructed embryos derived from small (15 microm) donor cells compared to large donor cells (20 microm), respectively. The percentage nuclei at 3 different time points (3, 6, and 9 hours in culture medium) was higher (p = 0.003) in the reconstructed embryos activated by thimerosal and dithiothreitol (20%, 36%, and 41.3%) compared to those without activation treatment (0%, 11.8%, and 22.2%). In addition, there was an increased percentage with nuclei as the time in culture increased from 3 to 9 hours (p = 0.029). The percentages of chromosome condensation (34.6%; 9/26) and nuclear formation (33.3%; 9/27) in nuclear transfer embryos were similar. The rate of blastocysts/morulae development (14.0%; 6/43) was low. However, 2 cavitated embryos (presumptive blastocysts) with 14 and 11 nuclei and 1 morula with 8 nuclei were obtained. This together with the above evidence indicate that the nuclei from pig fibroblast cells can be partially reprogrammed, which suggests that transfer of nuclei from fibroblast cells to in vitro matured oocytes resulting in production of identical or genetically altered pigs may be possible.     

A protein tyrosine phosphatase inhibitor, sodium orthovanadate, causes parthenogenetic activation of pig oocytes via an increase in protein tyrosine kinase activity.

This study was conducted to determine whether a protein tyrosine kinase (PTK) activity is involved in the initiation of the events that occur at fertilization in pig oocytes. After maturation for 47 h, a 7-h treatment of oocytes with 1 mM sodium orthovanadate, which is an inhibitor of protein tyrosine phosphatase, caused more than 90% pronuclear formation, cortical granule exocytosis, and a decrease in mitogen-activated protein kinase activity. Immunoblotting with an antibody specific for phosphotyrosine showed at least three proteins whose phosphotyrosine contents were significantly increased upon treatment of oocytes with 1 mM sodium orthovanadate. Preincubation of pig oocytes with 50 microM tyrphostin 47, a specific PTK inhibitor, completely blocked the ability of sodium orthovanadate to trigger activation events. In addition, when oocytes were pretreated with the calcium-chelating agent BAPTA-AM, sodium orthovanadate-stimulated pronuclear formation was significantly (P < 0.01) reduced (94.0% vs. 43.1%). These results suggest that PTK may be involved in pig oocyte activation in a calcium-dependent manner and that the stimulation of tyrosine kinase is able to signal a series of intracellular changes that lead to the activation events associated with fertilization.     

低分子抑瘤物净化白血病细胞的实验研究及临床应用

胎儿肝脏中存在一类小分子量（分子量〈１０ｋＤ）的肿瘤抑制物,其在体外对ＨＬ－６０等多种白血病细胞系具有明显的选择性抑制作用,对急性白血病患者骨髓的白血病祖细胞也具有这种选择性抑制效果;分离纯化获得两种天然低分子抑瘤物（７－ＫＣ和７－β－ＨＣ）和一种外源性低分子抑瘤物（ＤＢＰ）,研究证实其抑制白血病细胞生长的作用机制是诱导白血病细胞凋亡;将其应用于白血病及恶性淋巴瘤自体骨髓移植时的体外净化,完成１２     

Paravascular pathways contribute to vasculitis and neuroinflammation after subarachnoid hemorrhage independently of glymphatic control

Subarachnoid hemorrhage (SAH) is a devastating disease with high mortality. The mechanisms underlying its pathological complications have not been fully identified. Here, we investigate the potential involvement of the glymphatic system in the neuropathology of SAH. We demonstrate that blood components rapidly enter the paravascular space following SAH and penetrate into the perivascular parenchyma throughout the brain, causing disastrous events such as cerebral vasospasm, delayed cerebral ischemia, microcirculation dysfunction and widespread perivascular neuroinflammation. Clearance of the paravascular pathway with tissue-type plasminogen activator ameliorates the behavioral deficits and alleviates histological injury of SAH. Interestingly, AQP4^−/− mice showed no improvements in neurological deficits and neuroinflammation at day 7 after SAH compared with WT control mice. In conclusion, our study proves that the paravascular pathway dynamically mediates the pathological complications following acute SAH independently of glymphatic control.Cerebral aneurysm rupture causes subarachnoid hemorrhage (SAH), which is associated with a high mortality due to its secondary complications, including hemorrhage, hydrocephalus and delayed cerebral ischemia (DCI).^{1, 2, 3} Therapeutic interventions against the secondary complications, especially DCI, are yet limited, as the pathological mechanism underlying that is not fully understood.^{2, 3, 4, 5, 6, 7} Current hypotheses of the development of the secondary complications mainly include cerebral vasospasm (CVS) and the microcirculation disturbance, as well as parenchymal arterial lesions, microthrombosis and neuroinflammation.^{1, 2, 4, 7, 8, 9}Previous studies have shown that the blockade of cerebral lymphatic drainage deteriorated the secondary cerebral ischemia after SAH, suggesting that the cerebral lymphatic drainage pathway could be involved in the pathological mechanism of SAH.^{10, 11} However, the central nervous system (CNS) was considered lack of a conventional lymphatic drainage system in the past. Recently, several studies have shown that the brain has in fact the proper lymphatic system, including sinus-associated lymphatic vessels and the glymphatic system (GS).^{12, 13, 14, 15} Sinus-associated lymphatic vessels express all of the molecular hallmarks of lymphatic endothelial cells, contain cerebrospinal fluid (CSF) and immune cells, and drain into the deep cervical lymph nodes.^{12, 13}There is a histologically defined space in the brain, the Virchow–Robin space, where the subarachnoid space meets the paravascular space (or perivascular space in somewhere, PVS).¹⁶ The GS is a specialized brain-wide anatomic structure locating at the PVS surrounding the brain vasculature, which is ensheathed with the astroglial endfeet and astroglial water channel aquaporin-4 (AQP4).^{14, 15} The GS facilitates the efficient lymphatic clearance of extracellular solutes and fluid in the brain through astroglial-mediated interstitial fluid bulk flow.¹⁴Impairment of GS involves neurological conditions including traumatic brain injuries,¹⁷ ischemic stroke¹⁸ and aged brain.¹⁹ Interestingly, brain imaging study with magnetic resonance imaging reported weakened GS perfusion following acute stroke or SAH.^{18, 20} However, little is known about whether the GS is involved in the secondary complications of SAH. Here, we examined the potential involvement of GS in SAH-associated pathology progression with in vivo two-photon microscopy and CLARITY technique.^{21, 22} Our data showed that subarachnoid blood flowed into the brain parenchyma rapidly through the PVS, causing CVS, vasculitis, widespread microinfraction and neuroinflammation in the animal model of SAH and SAH patients. Prevention of CVS with Fasudil²³ did not improve the neurological impairment nor alleviated the pathology, while the PVS clearance with tissue-type plasminogen activator (tPA) infusion improved the behavioral recovery and reduced neuroinflammation in the brain. Interestingly, AQP4^−/− mice showed no improvements in neurological deficits and neuroinflammation at day 7 after SAH compared with WT control mice. Our study therefore suggested that the paravacular pathway dynamically mediates the pathological complications following acute SAH independently of glymphatic control.     

Regulation of morphologicaland functional properties of astroglial cells by hydrogen peroxide

Effects of hydrogen peroxide on morphological characteristics, proliferation index, menadione-dependent lucigenin-enhanced chemiluminescence of C6 glioma cells were studied. It was established that H2O2 at 1 x 10(-8) - 5 x 10(-7) M concentrations acts as a regulator of morphological and functional properties of astrocytes by inducing their reactivation that is manifested as a cell body hypertrophy and an increase of proliferative activity and of menadione-dependent production of superoxide (O2- ). Cytodestructive action of hydrogen peroxide at a concentration higher than 1 microM on C6 glioma cells shows itself as a decrease of their proliferation index and the ability to generate O2- under menadione action. Using lipopolysaccharide B as a functional stimulator it has been shown that H2O2 modifies signaling pathways leading to the increase of mitotic activity of C6 glioma cells and decreases the yield of lucigenin-enhanced chemiluminescence of astrocytes under menadione action to the level of control values.     

Perturbation of cytochrome P450, generation of oxidative stress and induction of DNA damage in Cyprinus carpio exposed in situ to potable surface water

Epidemiological evidence suggests a link between consumption of chlorinated drinking water and various cancers. Chlorination of water rich in organic chemicals produces carcinogenic organochlorine by-products (OBPs) such as trihalomethanes and haloacetic acids. Since the discovery of the first OBP in the 1970s, there have been several investigations designed to determine the biological effects of single chemicals or small artificial OBP combinations. However, there is still insufficient information regarding the general biological response to these compounds, and further studies are still needed to evaluate their potential genotoxic effects. In the current study, we evaluated the effect of three drinking water disinfectants on the activity of cytochrome P450 (CYP)-linked metabolizing enzymes and on the generation of oxidative stress in the livers of male and female Cyprinus carpio fish (carp). The fish were exposed in situ for up 20 days to surface water obtained from the Trasmene lake in Italy. The water was treated with 1-2 mg/L of either sodium hypochlorite (NaClO) or chlorine dioxide (ClO2) as traditional disinfectants or with a relatively new disinfectant product, peracetic acid (PAA). Micronucleus (MN) frequencies in circulating erythrocytes from the fish were also analysed as a biomarker of genotoxic effect. In the CYP-linked enzyme assays, a significant induction (up to a 57-fold increase in the deethylation of ethoxyresorufin with PAA treatment) and a notable inactivation (up to almost a 90% loss in hydroxylation of p-nitrophenol with all disinfectants, and of testosterone 2beta-hydroxylation with NaClO) was observed in subcellular liver preparations from exposed fish. Using the electron paramagnetic resonance (EPR) spectroscopy radical-probe technique, we also observed that CYP-modulation was associated with the production of reactive oxygen species (ROS). In addition, we found a significant increase in MN frequency in circulating erythrocytes after 10 days of exposure of fish to water treated with ClO2, while a non-significant six-fold increase in MN frequency was observed with NaClO, but not with PAA. Our data suggest that the use of ClO2 and NaClO to disinfect drinking water could generate harmful OBP mixtures that are able to perturb CYP-mediated reactions, generate oxidative stress and induce genetic damage. These data may provide a mechanistic explanation for epidemiological studies linking consumption of chlorinated drinking water to increased risk of urinary, gastrointestinal and bladder cancers.