Biomedical subjects
Publications and source records attributed to D L Xu.
Parvovirus B19 involvement was investigated in 30 children with severe aplastic anaemia. Active or recent parvovirus B19 infection, as shown by B19 DNA viraemia, positive B19 specific IgM antibodies, or both, was diagnosed in six patients. There were no other plausible causes. We suggest that parvovirus B19 infection might be associated with severe aplastic anaemia.
Naturally infected cases of swine mycobacteriosis were divided into two groups, localized infection (LI) and disseminated infection (DI). Lymphoproliferative response (LPR) was then examined to estimate their immunological states. Both control and LI groups showed strong response to Concanavalin A (Con A) and phytohemagglutinin (PHA) in the LPR, and lymphocytes recovered from the LI responded well to purified protein derived from M. avium (PPD). On the other hand, the DI group showed weak response to both Con A and PHA, despite their strong response to PPD stimulation. These data suggest that the low LPR to Con A and PHA observed in the DI groups was probably not due to the general unresponsiveness of T-cells.
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Nucleic acid sequence of complemental DNA open reading frame for Nramp-1 gene was compared among DBA/2 (Bcgr), C57BL/6 (Bcgs) and C57BL/6-Bcgr mice which was previously developed as M. avium-resistant mouse strain (Xu, et al. Veterinary Microbiology 50:73-79 (1996). Total RNA was isolated from various organs of DBA/2, C57BL/6 and C57BL/6-Bcgr. Nramp-1 cDNA was constructed from their mRNAs by gene amplification (PCR) technique and their open reading frame sequences were compared. The results clearly showed that our C57BL/6-Bcgr was almost identical with the DNA sequence of the DBA/2 mice. In contrast, C57BL/6-Bcgs mice differed only on the substitution of adenine for guanine of the nucleic acid at position 596. This corresponded to the site of amino acid substitution (glycine to asparate) at position 169 in predicted NRAMP which had been reported. The expression of Nramp-1 mRNA was more prominent in spleens and livers and there appeared to be no significant difference among the strains of mice.
The endotoxins from two recently-classified subspecies of Fusobacterium, namely F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, were compared. Chemical analysis of the isolated endotoxins revealed that they were clearly different. Distinct levels of polysaccharides were demonstrated. The endotoxins isolated were devoid of heptose and 3-deoxy-D-manno-octulosonate (KDO). The endotoxins of F. n. necrophorum and F. n. funduliforme contained lipid A in a ratio of 4:1 which may account for the variations in their virulence.
This study was to investigate cell cycle distribution of the vascular smooth muscle cells (VSMCs) and negative regulator of cell proliferation p27 expression caused by platelet derived growth factor BB (PDGF-BB), angiotensin II (Ang II) and arginine vasopressin (AVP). Deprived of fotal calf serum for 48 h, cultured VSMCs in quiescent condition were collected at different times after stimulation of Ang II, AVP and PDGF-BB. Cell cycle distribution and p27 expression were determined with a flow cytometer. The results showed that the protein content of VSMCs was significantly increased (43.6%) by Ang II as a result of hypertrophy, but Ang II did not lead to downregulation of p27. AVP could downregulate p27 slightly. PDGF could inhibit p27 expression significantly and cause VSMCs hyperplasia. These results suggest that the progression of VSMCs through G1 to S phase might be brought out by the inhibition of p27 during proliferation.
OBJECTIVE: To study the bone formation and osteogenesis after transplantation of human periosteal mesenchymal stem cells(PMSC). METHODS: Suspension of PMSC which obtained from cell culture of periosteal segments in vitro were injected into the backs of nude mice subcutaneously, and the fracture site of neck of femur in old person. RESULTS: Subdermal nodules were observed by naked eyes after 11 days of transplantation. 4 weeks later, their anatomic diameter reached 2-7 mm(averaged 3.6 mm). It was proved that the subdermal nodules were trabecular ball trapped with fibrous tissue. The nodules were investigated by human special apoB gene with PCR, and the test of anti-human-tissue precipitin reaction(AHTPR). The results of PCR and AHTPR were positive reaction. There were no subdermal nodules formed in the sites of injection of frozen-melted PMSC or culture medium. The new callus in the sites of fracture were tested by PCR test, and two kinds of apoB gene products were detected. CONCLUSION: The results indicated that the implanted PMSC could form new bone directly in nude mice, and the cells of donor and recipient all could form new bone.
An attempt was made to determine the receptor for the hemolysin of Fusobacterium necrophorum using horse erythrocyte or its membranes as target. The spectrum of erythrocyte sensitivity has indicated that horse, dog and mouse erythrocytes are highly sensitive whereas cattle, sheep, goat and chicken red blood cells are insensitive to this hemolysin. A high correlation between sensitivity and phosphatidylcholine content of the erythrocyte membranes was noted. Binding of hemolysin to horse erythrocyte membranes was reduced significantly by prior treatment of membranes with phospholipase A2 but not with phospholipase C. Pretreatment of erythrocyte membranes with pronase, proteinase K, trypsin or neuraminidase did not alter binding of hemolysin to the membranes, suggesting that protein or sialyl residues are not involved as receptors. Gas liquid chromatography analysis showed that the fatty acid profile from hydrolysis of bovine liver phosphatidylcholine by hemolysin and phospholipase A2 were similar. In conclusion, this report presents evidence that phosphatidylcholine may be acting as a possible receptor for the hemolysin of F. necrophorum.
Water retention is characteristic of pregnancy but the mechanism(s) of the altered water metabolism has yet to be elucidated. The collecting duct water channel, aquaporin 2 (AQP2), plays a pivotal role in the renal water regulation, and we hypothesized that AQP2 expression could be modified during pregnancy. Sprague-Dawley female rats were studied on days 7 (P7), 14 (P14), and 20 (P20) of pregnancy, and expression of AQP2 in papillae was examined. Nonpregnant (NP) littermates were used as controls. Plasma osmolalities were significantly lower in pregnant rats by day 7 of gestation (P7 283.8+/-1.82, P14 284.3+/-1.64, P < 0.001, P20 282. 4+/-1.32, P < 0.0001, vs. NP 291.8+/-1.06 mosmol/kgH2O). However, plasma vasopressin concentrations in pregnant rats were not significantly different than in nonpregnant rats (NP 1.03+/-0.14, P7 1.11+/-0.21, P14 1.15+/-0.21, P20 1.36+/-0.24 pg/ml, NS). The mRNA of AQP2 was increased early during pregnancy: AQP2/beta actin: P7 196+/-17.9, P14 200+/-6.8, and P20 208+/-15.5%, P < 0.005 vs. NP (100+/-11.1%). AQP2 protein was also increased during pregnancy: AQP2 protein: P7 269+/-10.0, P14 251+/-12.0, P < 0.0001, and P20 250+/-13.6%, P < 0.001 vs. NP (100+/-12.5%). The effect of V2 vasopressin receptor antagonist, OPC-31260, was then investigated. AQP2 mRNA was suppressed significantly by OPC-31260 administration to P14 rats (AQP2/beta actin: P14 with OPC-31260 39.6+/-1.7%, P < 0.001 vs. P14 with vehicle) and was decreased to the same level of expression as NP rats receiving OPC-31260. Similar findings were found with the analysis of AQP2 protein. The decreased plasma osmolality of P14 rats was not modified by OPC-31260. The results of the study indicate that upregulation of AQP2 contributes to the water retention in pregnancy through a V2 receptor-mediated effect. In addition to vasopressin, other factors may be involved in this upregulation.
Normalization of the increased vascular nitric oxide (NO) generation with low doses of NG-nitro-L-arginine methyl ester (L-NAME) corrects the hemodynamic abnormalities of cirrhotic rats with ascites. We have undertaken this study to investigate the effect of the normalization of vascular NO production, as estimated by aortic cyclic guanosine monophosphate (cGMP) concentration and endothelial nitric oxide synthase (eNOS) protein expression in the aorta and mesenteric artery, on sodium and water excretion. Rats with carbon tetrachloride-induced cirrhosis and ascites were investigated using balance studies. The cirrhotic rats were separated into two groups, one receiving 0.5 mg/kg per day of L-NAME (CIR-NAME) during 7 d, whereas the other group (CIR) was administrated the same volume of vehicle. Two other groups of rats were used as controls, one group treated with L-NAME and another group receiving the same volume of vehicle. Sodium and water excretion was measured on days 0 and 7. On day 8, blood samples were collected for electrolyte and hormone measurements, and aorta and mesenteric arteries were harvested for cGMP determination and nitric oxide synthase (NOS) immunoblotting. Aortic cGMP and eNOS protein expression in the aorta and mesenteric artery were increased in CIR as compared with CIR-NAME. Both cirrhotic groups had a similar decrease in sodium excretion on day 0 (0.7 versus 0.6 mmol per day, NS) and a positive sodium balance (+0.9 versus +1.2 mmol per day, NS). On day 7, CIR-NAME rats had an increase in sodium excretion as compared with the CIR rats (sodium excretion: 2.4 versus 0.7 mmol per day, P < 0.001) and a negative sodium balance (-0.5 versus +0.8 mmol per day, P < 0.001). The excretion of a water load was also increased after L-NAME administration (from 28+/-5% to 65+/-7, P < 0.05). Plasma renin activity, aldosterone and arginine vasopressin were also significantly decreased in the CIR-NAME, as compared with the CIR rats. The results thus indicate that normalization of aortic cGMP and eNOS protein expression in vascular tissue is associated with increased sodium and water excretion in cirrhotic rats with ascites.
The interactions between the hemolysin of Fusobacterium necrophorum subsp. necrophorum, erythrocytes and erythrocyte membranes were studied as an attempt to determine the initial characteristics leading to hemolysis. The spectrum of erythrocyte sensitivity indicated that horse, dog and mouse erythrocytes were highly sensitive whereas those of cattle, sheep, goat and chicken were insensitive to the hemolysin. Binding of hemolysin to horse and dog erythrocytes or their ghosts was more pronounced than to those of cattle and sheep as detected by a decrease of hemolytic activity from hemolysin preparations. The kinetics of hemolysis revealed that lysis is preceded by a prelytic phase characterized by binding of hemolysin to erythrocytes. Treatment of horse erythrocytes with hemolysin at various temperatures prior to incubation at 37 degrees C also revealed that this binding prelytic phase is temperature independent. This was followed by a temperature dependent lytic stage since erythrocytes pretreated with hemolysin and incubated at 4 degrees C showed no hemolysis. An inverse relation was found between erythrocyte concentration and hemolytic activity suggesting a multiple-hit mechanism of hemolysis.
Aquaporin-2 (AQP2) mediates vasopressin-regulated collecting duct water permeability. Chronic heart failure (CHF) is characterized by abnormal renal water retention. We hypothetized that upregulation of aquaporin-2 water channel could account for the water retention in CHF. Male rats underwent either a left coronary artery ligation, a model of CHF, or were sham operated. 31-33 d after surgery, mean arterial pressure (MAP) and cardiac output were measured in conscious animals, and the animals were killed 24 h later. Cardiac output (CO) and plasma osmolality were significantly decreased and plasma vasopressin increased in the CHF as compared to the sham-operated rats. Both mRNA and protein AQP2 were significantly increased in the kidneys of the CHF rats. The effect of oral administration of a nonpeptide V2 vasopressin receptor antagonist, OPC 31260, was therefore investigated. OPC 31260 induced a significant increase in diuresis, decrease in urinary osmolality, and rise in plasma osmolality in the OPC 31260-treated CHF rats as compared to untreated CHF rats. The mRNA and protein AQP2 were significantly diminished in both cortex and inner medulla of the treated CHF rats. In conclusion, an early upregulation of AQP2 is present in CHF rats and this upregulation is inhibited by the administration of a V2 receptor antagonist. The results indicate a major role for vasopressin in the upregulation of AQP2 water channels and water retention in experimental CHF in the rat.
To determine the role of the Bcg gene in the resistance and susceptibility of BCG-infected C57BL/6 (Bcg(s)) and its Bcg(r) congenic mice, the antigen presenting ability of spleen adherent cells and peritoneal macrophages, delayed type hypersensitivity (DTH) and lymphocyte blastogenic responses were investigated. The results obtained indicate that the DTH and lymphocyte blastogenic responses in Bcg(r) congenic mice were higher than in the Bcg(s) mice. Stimulation of spleen adherent cells with live Mycobacterium bovis BCG or PPD-BCG resulted in a higher antigen presenting ability in Bcg(r) than in Bcg(s) mice. However, comparatively low responses were associated with M. avium stimulation, with those in Bcg(r) being higher than in Bcg(s). I-A expression was also comparatively higher in Bcg(r) than in Bcg(s) mice. This study demonstrates that the Bcg gene seems to exhibit some effect on the antigen presenting ability of macrophages in immune responses of the congenic mice.
Bcg congenic mice were developed by using C57BL/6 and DBA/2 strains of mice as progenitors. They were obtained by introgressively backcrossing the Bcgr marker of DBA/2 onto C57BL/6. After twenty successive backcrossings, the heterozygous resistant mice were mated with each other to obtain homozygous mice as the Bcgr congenic mice. The results of immunogenic and genetic markers coupled with those of an mixed lymphocyte reaction, all confirmed that the newly developed mice were highly congenic. These congenic mice were found to be resistant to in vivo infections by Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium bovis BCG.
The stability and stabilization of the hemolytic activity of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme were monitored over a period of four weeks using culture supernatants. The hemolytic activity was completely lost after one week at room temperature and 37 degrees C. After a two-week storage at 4 degrees C and -80 degrees C only trace activity was detected with -80 degrees C being the better of the two conditions. The addition of cysteine monohydrochloride, bovine serum albumin or Tween 80 as stabilizers, however, led to the detection of a considerable amount of the hemolytic activity in the sample stored at 4 degrees C and - 80 degrees C throughout the period investigated. The hemolytic activity appeared to be more stable in the presence of Tween 80 at -80 degrees C. Cysteine monohydrochloride was found to crystallize at - 80 degrees C and was therefore ineffective as a stabilizer at this temperature. Hemoglobin was also ineffective as a stabilizer.
The effect of various agents as enhancers or inhibitors of hemolysin secretion by Fusobacterium necrophorum was investigated. The hemolysin secreted in phosphate buffered saline (PBS) alone was inactivated shortly after secretion. Tween-80 or albumin preserved the hemolytic activity in PBS in which cultures of F. necrophorum had been suspended. Hemoglobin was found to enhance hemolysin secretion. However, higher concentrations diminished secretion. Chloramphenicol, an inhibitor of protein synthesis, exhibited no effect on the hemolytic activity. However, the addition of sodium azide, an energy metabolic inhibitor, significantly reduced the hemolytic activity. Lower temperatures and pH above 9 and below 6 all yielded a low hemolytic activity. Cells suspended in Tween-80 prior to sonication yielded a substantial amount of extracellular hemolytic activity with low intracellular activity detected. However, cells suspended in PBS alone yielded a low extracellular activity but rather a high intracellular activity. The same spectrum of red blood cells of different species were found to be sensitive to both the extracellular and intracellular hemolysins.
Pregnancy is characterized by hemodynamic and body fluid alterations. Increased nitric oxide (NO) production has been suggested to play a role in the hemodynamic alterations of pregnancy and has also been reported to increase arginine vasopressin (AVP) release. We therefore hypothesized that gestation could increase both NO synthase (NOS) constitutive isoforms, neuronal NOS and endothelial NOS, and thereby contribute to the hyposmolality and peripheral arterial vasodilation of pregnancy, respectively. The present study was therefore undertaken to examine the constitutive NOS isoforms in aortas, mesenteric arteries, and hypothalami of pregnant rats on day 20 of gestation compared with age-matched nonpregnant rats. Plasma AVP was determined by radioimmunoassay and hypothalamic mRNA AVP by solution hybridization assay. Hypothalamic neuronal NOS was assessed by Northern blot and Western blot; endothelial NOS was assessed by Western blot in arteries and hypothalamus. The results demonstrated that 1) plasma AVP and hypothalamic AVP mRNA are increased in pregnant rats (n = 8), 2) neuronal NOS protein and mRNA are increased in hypothalamus of pregnant rats (n = 4), and 3) endothelial NOS expression, as assessed by Western blot analysis, is increased in both conductance (aorta) as well as resistance (mesenteric) arteries of pregnant rats (n = 4). We conclude that both of the constitutive NOS isoforms are increased in pregnant rats, suggesting that the peripheral arterial vasodilation and hyposmolality of pregnancy could be mediated by these isoforms.
Nitric oxide (NO) is postulated to mediate the peripheral arterial vasodilation in cirrhosis. However, it is not known which isoform of the nitric oxide synthase (NOS) is involved in the increased production of NO. This study was therefore undertaken to examine the expression of the NOS isoforms in arteries of cirrhotic rats compared with controls. Cirrhosis was induced by CCl4, and vessels were harvested for immunoblots using antibodies against inducible NOS (iNOS) and endothelial constitutive NOS (ecNOS). Endothelial cells were used as controls for ecNOS, and vascular smooth muscle cells treated with lipopolysaccharide or septic rats were used for iNOS controls. The results demonstrated an upregulation of ecNOS in both the aortas and mesenteric arteries of cirrhotic compared with control rats. Chronic inhibition of NOS decreased ecNOS in cirrhotic vessels. Although iNOS mRNA was found by reverse transcription-polymerase chain reaction in arteries of cirrhotic rats, iNOS protein was not detectable by immunoblotting compared with septic rats, suggesting a low vascular level of this isoform. In conclusion, the ecNOS seems to play a major role in the increased NO production in cirrhotic rats, whereas the role of iNOS remains elusive.