Involvement of A1 adenosine receptors in the acquisition of fertilizing capacity

Ejaculated mammalian spermatozoa acquire competence to fertilize oocytes by a two-step process: capacitation followed by acrosome reaction. The biochemical and biophysical modifications occurring in vivo in the female reproductive tract can be reproduced in vitro, and previous studies have suggested a capacitative role for adenosine A(1) receptor (A(1)R). Mice with a targeted disruption of the Adora 1 gene (A(1)R-/- mice) provide a useful model for better understanding the role of the A(1)R in fertility. Murine spermatozoa express A(1)R in the head, neck, midpiece region, and tail. The number of capacitated spermatozoa incubated in human tubal fluid was significantly reduced in A(1)R-/- compared with A(1)R+/+ and A(1)R+/- spermatozoa. The difference between A(1) R+/+ and A(1)R-/- mouse spermatozoa was mainly in the time necessary to reach the maximum percentage of capacitation. A(1)R+/+ murine sperm obtained the full state of capacitation within 90 minutes whereas A(1)R-/- sperm required 240 minutes. Caffeine, a known antagonist of A(1) and A(2A) adenosine receptors, lowered the number of capacitated sperm and affected the time of capacitation in a dose-dependent manner, mimicking the effects of the lack of A(1) receptors. Although number, motility, and viability of A(1)R-/- murine sperm was not significantly different from A(1)R+/+ mouse spermatozoa, a significant reduction of the number of pups produced by A(1)R-/- male mice suggests that A(1) receptors must be fully operative to accomplish the optimal degree of capacitation and thereby fertilization.     

Serum concentration and peritoneal transfer of aluminum during treatment by continuous ambulatory peritoneal dialysis

The evolution of the aluminum (A1) serum levels during a 2-year follow-up and the peritoneal transfer of A1 were studied in 22 patients treated by continuous ambulatory peritoneal dialysis (CAPD), using a dialysate with a very low A1 concentration (r = 0.25 - 0.30 mumoles/liter). Patients were divided in three groups. A transfer of A1 from the patient to the dialysate was observed in all patients. In group 1, patients exclusively treated by CAPD and who have never received aluminum-containing phosphate binders (ACPB), mean level (+/- SD) of serum A1 stabilized within a safe range (0.60 +/- 0.28 mumoles/liter). In group 2 the oral administration of ACPB in patients exclusively treated by CAPD induced a slow and progressive increase of A1 serum concentration despite the increase of the A1 excretion through the peritoneal route. In group 3, patients previously treated by hemodialysis and receiving ACPB, the high serum A1 levels observed before treatment by CAPD decreased rapidly on CAPD. A1 removal through the peritoneum was higher in group 3 than in group 2 despite serum A1 levels not statistically different in both groups. A1 removal through the peritoneum is mainly influenced by serum and dialysate A1 concentration. A1 body stores could play a role in the transfer of A1 through the peritoneum. Three cases of A1 poisoning due to the accidental use of a dialysate with a high A1 content are reported.     

Identification of COL4A5 defects in Alport's syndrome by immunohistochemistry of skin

BACKGROUND: The COL4A3-COL4A4-COL4A5 network in the glomerular basement membrane is affected in the inherited renal disorder Alport's syndrome (AS). Approximately 85% of the AS patients are expected to carry a mutation in the X-chromosomal COL4A5 gene and 15% in the autosomal COL4A3 and COL4A4 genes. The COL4A5 chain is also present in the epidermal basement membrane (EBM). It is predicted that approximately 70% of the COL4A5 mutations prevent incorporation of this chain in basement membranes. METHODS: We investigated whether or not COL4A5 defects could be detected by immunohistochemical analysis of the EBM. Punch skin biopsies were obtained from 22 patients out of 17 families and two biopsy specimens from healthy males were used as controls. RESULTS: In four cases with the COL4A5 frameshift or missense mutations, the COL4A5 chain was either lacking from the EBM (male) or showed a focally negative pattern (female). In three other patients with a COL4A5 missense mutation, a COL4A3 and a COL4A4 mutation, respectively, the COL4A5 staining was normal. A (focally) negative EBM-COL4A5 staining was found in three patients of six families with a diagnosis of AS and in one family of a group of four families with possible AS. CONCLUSIONS: The (focal) absence of COL4A5 in the EBM of skin biopsy specimens can be used for fast identification of COL4A5 defects. Combined with polymorphic COL4A5 markers, both postnatal and prenatal DNA diagnosis are possible in the family of the patient.     

Minimal Acute Rejection after Lung Transplantation: A Risk for Bronchiolitis Obliterans Syndrome

Bronchiolitis obliterans syndrome (BOS) is a major cause of lung allograft dysfunction. Although previous studies have identified mild to severe rejection (grade>or=A2) as a risk factor for BOS, the role of minimal rejection (grade A1) remains unclear. To determine if A1 rejection by itself is a risk factor for BOS, we performed a retrospective cohort study on 228 adult lung transplant recipients over a 7-year period. Cohorts were defined by their most severe rejection episode (none, A1 only, and >or=A2) and analyzed for the subsequent development and progression of BOS using univariate and multivariate time-dependent Cox regression analysis. In the univariate model, the occurrence of isolated minimal rejection was a risk factor for all stages of BOS. Similarly, multivariate models that included HLA mismatch, cytomegalovirus pneumonitis, community acquired viral infection, underlying disease and type of transplant demonstrated that A1 rejection was a distinct risk factor for BOS. Furthermore, the associated risk with A1 rejection was slightly greater than the risk from >or=A2 and treatment of A1 rejection decreased the risk for subsequent BOS stage 1. We conclude that minimal rejection is associated with an increased risk for BOS development and progression that is comparable to A2 rejection.     

不同剂量布托啡诺对胃癌根治术后患者静脉自控镇痛效能的比较

目的观察不同剂量的布托啡诺对胃癌根治术后患者静脉自控镇痛效能的比较。方法选择行胃癌根治的患者232例,ASA为Ⅰ～Ⅱ级。将232例患者随机分为布托啡诺静脉自控镇痛组和对照组,每组均为29例。其中布托啡诺组分为6个小组,分别是布托啡诺浓度为0.004%、0.005%、0.006%、0.007%、0.008%和0.009%的A1、A2、A3、A4、A5和A6组。对照组分为2个小组,分别是吗啡浓度为0.025%的B1组以及传统方法治疗B2组。在胃癌根治术后1d观察患者情况,详细记录患者的舒适度评分、镇静度评分、视觉模拟评分以及患者的不良反应与PCIA的总按压频率次数和实际用药情况。结果①A3～A6组以及B1组的VAS评分明显低于B2和A1、A2组,而BCS的评分要高于B2和A1、A2组（P〈0.05）。②A3～A6组的PCIA总压次数明显低于A1、A2组,然而用药量远远高于A1、A2两组（P〈0.05）。③B1组患者腹胀、恶心、呕吐等不良症状发生频率远远高于A1～A6以及B2组患者（P〈0.05）。A6组患者的嗜睡程度最高。A1、A2组需要其他镇痛药物辅助,镇痛效果才能达到A3～A6组。结论布托啡诺的浓度为0.006%、0.007%时,以0.0022～0.0026mg/（kg.h）的速率给药,负荷剂量为0.005mg/（kg.h）的静脉PCIA会获得最佳的镇痛疗效,并且不良反应的发生率极低,可以应用于相关临床疾病的治疗中。     

Effects of Weekly Administrations of Alendronate+Clodronate on Young Mouse Tibia: Localized Action at the Proximal Growth Plate

Alendronate (A), a typical aminobisphosphonate (aminoBP), has a strong bone-resorption-inhibitory activity (BRIA). However, like other aminoBPs it has inflammatory side effects. In contrast, the BRIA of clodronate (C), a non-aminoBP, is much weaker, and in animal experiments it suppresses aminoBP-induced inflammatory reactions. In the present study, we examined the effects of weekly administrations of A (1.6 µmol/kg) + C (160 µmol/kg) on the tibias in young mice and compared them to those induced by A or C alone. Radiophotography showed that A increased bone density at a selective site in the tibia. Indeed, one week after the final injection of A (given alone), clear sclerotic lines (tentatively called BP-lines) were visible at sites corresponding to the location of the growth plate at the time of the each injection. C also produced BP-lines, although they were weaker than those produced by A. Combined administration of A and C produced similar BP-lines as seen in mice given A alone. These results together with other physicochemical effects of A on the tibia suggest that (1) each injection of A and C inhibits bone resorption selectively and transiently at the tibial growth plate in young mice, although minor effects on other sites cannot be excluded, and (2) the combination of A and C keeps still a strong BRIA. Our findings may suggest a strategy for the prevention or reduction of some inflammatory side effects of A or other aminoBPs.     

Using rotational thromboelastometry clot firmness at 5 minutes (ROTEM® EXTEM A5) to predict massive transfusion and in‐hospital mortality in trauma: a retrospective analysis of 1146 patients

Viscoelastic assays such as TEG ^® and ROTEM ^® are increasingly used to guide transfusion of blood products. The EXTEM assay maximum clot firmness (MCF) is a ROTEM measure available after 25–29 min used to guide early decisions. EXTEM A10, the clot firmness at 10 min, is an accepted early surrogate, but investigators differ on whether A5, the clot firmness at 5 min, is acceptable. We re‐examined this in a retrospective observational analysis of 1146 trauma patients in one centre who had ROTEM data recorded. A5 and A10 both correlated well with maximum clot firmness, with Pearson coefficients of r = 0.92 and r = 0.96, respectively. The correlations of A5, A10 and maximum clot firmness with requirement for massive transfusion were all similarly high, with c‐stats of 0.87, 0.89 and 0.90, respectively. The correlations with mortality were also similar but weaker, with c‐stats of 0.67, 0.69 and 0.69, respectively. Using a previously validated cut‐off of A5 < 35 mm to predict massive transfusion gave a sensitivity of 95%, specificity 83%, positive predictive value 9.3% and negative predictive value 100%. Using a value of A5 < 29 mm, for a pragmatic positive predictive value of 20%, gave a sensitivity of 67%, specificity 95% and negative predictive value 99%. Whether aiming for a high sensitivity or a strong predictive value, A5 was non‐inferior to A10 and actually missed fewer cases needing massive transfusion. A5 has similar utility to both A10 and maximum clot firmness as an early measure of clot firmness, and a low A5 value is strongly predictive of the need for massive transfusion.     

腺苷A2A受体激活缺血后处理保护皮瓣作用研究

目的：缺血后处理需要反复夹闭血管蒂,可能对血管造成机械性损伤。药物缺血后处理成为缺血后处理的发展方向。腺苷A2A是调控皮瓣炎症的关键靶点,进行相关研究进一步探究腺苷A2A激活缺血后处理对皮瓣有否保护作用。方法：健康成年新西兰大白兔,分为3组。A组为假手术组;B组为缺血再灌注损伤组;C组,再灌注前5min注射腺苷A2A激活剂。分别进行皮瓣存活率检测和ELISA检测TNF以及IL-6。结果：腺苷A2A激活剂组皮瓣存活率高,炎症因子检测活性低于缺血再灌注损伤组。结论：腺苷A2A激活剂缺血后处理可以抑制炎症因子,具有保护皮瓣作用。运用腺苷A2A受体激活缺血后处理可能成为保护皮瓣的新措施。     

Glomerular activin A overexpression is linked to fibrosis in anti-Thy1 glomerulonephritis.

BACKGROUND: Activin A, a member of the transforming growth factor-beta (TGF-beta) superfamily of proteins, shares many biological features with the pro-fibrotic cytokine TGF-beta1, which is primarily responsible for the accumulation of extracellular matrix proteins in renal disease. This study was designed to identify regulators of activin A production in glomerular mesangial cells and test if activin A acts as a pro-fibrotic cytokine in mesangial cells. METHODS: The effect of inflammatory cytokines on activin A production and the effect of exogenous activin A on mediators of fibrosis were analysed in cultured rat mesangial cells. Expression of activin A and of established mediators of fibrosis was analysed in a rat model of glomerular fibrosis (anti-Thy1 glomerulonephritis). RESULTS: In cultured mesangial cells, interleukin-1 and basic fibroblast growth factor, both mediators of glomerular inflammatory injury, dose-dependently increased activin A expression. Incubation with activin A significantly stimulated TGF-beta1, PAI-1 and connective tissue growth factor RNA expression and increased production of extracellular matrix proteins in mesangial cells. In rats with anti-Thy1 glomerulonephritis, expression of glomerular activin A mRNA and protein paralled the expression of TGF-beta and other indices of fibrosis, showing little change from normal on day 1, a marked, 70-fold increase of activin protein production on day 6, and a subsequent decrease at day 12. Antifibrotic therapy with the angiotensin-converting enzyme inhibitor enalapril significantly reduced glomerular activin A production. CONCLUSION: Taken together, the results of this study link overexpression of activin A to glomerular matrix protein expansion in vivo and in vitro, suggesting that activin A acts as pro-fibrotic cytokine in renal disease.     

CSF biomarker and PIB-PET-derived beta-amyloid signature predicts metabolic, gray matter, and cognitive changes in nondemented subjects

Beta-amyloid (Aβ) is a histopathological hallmark of Alzheimer's disease dementia, but high levels of Aβ in the brain can also be found in a substantial proportion of nondemented subjects. Here we investigated which 2-year rate of brain and cognitive changes are present in nondemented subjects with high and low Aβ levels, as assessed with cerebrospinal fluid and molecular positron emission tomography (PET)-based biomarkers of Aβ. In subjects with mild cognitive impairment, increased brain Aβ levels were associated with significantly faster cognitive decline, progression of gray matter atrophy within temporal and parietal brain regions, and a trend for a faster decline in parietal Fludeoxyglucose (FDG)-PET metabolism. Changes in gray matter and FDG-PET mediated the association between Aβ and cognitive decline. In contrast, elderly cognitively healthy controls (HC) with high Aβ levels showed only a faster medial temporal lobe and precuneus volume decline compared with HC with low Aβ. In conclusion, the current results suggest not only that both functional and volumetric brain changes are associated with high Aβ years before the onset of dementia but also that HC with substantial Aβ levels show higher Aβ pathology resistance, lack other pathologies that condition neurotoxic effects of Aβ, or accumulated Aβ for a shorter time period.     

肝细胞癌中HBX和MATs基因启动子甲基化的关系

目的 观察肝细胞癌(HCC)中乙肝病毒X蛋白(HBx)、甲硫氨酸腺苷转移酶1A(MAT1A)和甲硫氨酸腺苷转移酶2A(MAT2A)的表达以及HBx对MAT1A、MAT2A基因启动子的甲基化状态的影响,探讨HCC中MATs发生转化的机制.方法 应用免疫组织化学法检测78例乙型肝炎病毒(HBV)阳性HCC组织中HBx、MAT1A、MAT2A的表达,并用甲基化特异性聚合酶链反应(MS-PCR)法检测上述组织中MAT1A、MAT2A的甲基化状态,并将上述检测结果进行相关分析.结果 免疫组织化学结果表明,在78例HCC组织中,HBx、MAT1A、MAT2A阳性表达分别为59例(75.64%)、17例(21.79%)和66例(84.62%);HBx表达和MAT1A表达为负相关(P＜0.05),和MAT2A表达为正相关(P＜0.05),MAT1A和MAT2A表达为负相关(P＜0.05);MS-PCR结果表明,78例HCC组织中,MAT1A甲基化占58例(74.36%),MAT2A甲基化为9例(11.54%),HBx表达和MAT1A启动子甲基化状态呈正相关(P＜0.01),和MAT2A启动子甲基化状态呈负相关(P＜0.01).结论 HBx同MAT1A、MAT2A的表达明确相关,它能通过某些途径引起MAT1A启动子高甲基化、MAT2A启动子低甲基化来改变MATs的表达状态.

Abstract：

Objective To investigate the effect of hepatitis B virus x-protein (HBx) on the expression of MATs, the methylation status of methionine adenosyltransferases (MATs) ' promoters in hepatocellular carcinoma ( HCC), and the possible mechanism of MATs' change in HCC. Methods The expression of HBx, MAT1 A and MAT2A was detected in 78 HBV-positive HCC samples by IHC method and their correlation was analyzed. The methylation status of the promoters of MAT1A and MAT2A was examined. Results In 78 HCC tumor specimens, the positive expression rate of HBx, MAT1A and MAT2A was 75.64% (59 cases), 21.79% ( 17 cases) and 84. 62% (66 cases). There was a negative correlation between the expression of HBx and MAT1A (P ＜0. 05), a positive correlation between HBx and MAT2A ( P ＜ 0. 05), a negative correlation between MAT1A and MAT2A ( P ＜ 0. 05 ). MS-PCR revealed that there were 58 cases of MAT1A promoter' hypomethylation (74. 36% ), 9 cases of MAT2A promoter' hypomethylation ( 11.54% ). The positive expression of HBx was positively related with promoter' hypomethylation of MAT1A (P ＜ 0. 01 ), but negatively with promoter' hypomethylation of MAT2A ( P ＜ 0. 01 ).Conclusion The expression of HBx has relationship to the expression MAT1A and MAT2A in HCC tumor tissues, which is contributed to the fact that HBx can change the methylation status of MATs' promoters by some unknown pathways.     

Multiple functions of the von Willebrand Factor A domain in matrilins: secretion, assembly, and proteolysis

The von Willebrand Factor A (vWF A) domain is one of the most widely distributed structural modules in cell-matrix adhesive molecules such as intergrins and extracellular matrix proteins. Mutations in the vWF A domain of matrilin-3 cause multiple epiphyseal dysplasia (MED), however the pathological mechanism remains to be determined. Previously we showed that the vWF A domain in matrilin-1 mediates formation of a filamentous matrix network through metal-ion dependent adhesion sites in the domain. Here we show two new functions of the vWF A domain in cartilage-specific matrilins (1 and 3). First, vWF A domain regulates oligomerization of matrilins. Insertion of a vWF A domain into matrilin-3 converts the formation of a mixture of matrilin-3 tetramer, trimer, and dimer into a tetramer only, while deletion of a vWF A domain from matrilin-1 converts the formation of the native matrilin-1 trimer into a mixture of trimer and dimer. Second, the vWF A domain protects matrilin-1 from proteolysis. We identified a latent proteolytic site next to the vWF A2 domain in matrilin-1, which is sensitive to the inhibitors of matrix proteases. Deletion of the abutting vWF A domain results in degradation of matrilin-1, presumably by exposing the adjacent proteolytic site. In addition, we also confirmed the vWF A domain is vital for the secretion of matrilin-3. Secretion of the mutant matrilin-3 harbouring a point mutation within the vWF A domain, as occurred in MED patients, is markedly reduced and delayed, resulting from intracellular retention of the mutant matrilin-3. Taken together, our data suggest that different mutations/deletions of the vWF A domain in matrilins may lead to distinct pathological mechanisms due to the multiple functions of the vWF A domain.     

Implantation of BDNF-producing packaging cells into brain

In order to invent a screening system to check in vivo gene function and the efficiency of gene transfer mediated by a retroviral vector system, we established a novel packaging cell, PacC6/A8, that is transplantable to rat brains. The packaging cell is based on the gene of the neuropatogenic retrovirus, A8-V. For expression in the brain, a vector that expresses brain-derived neurotrophic factor (BDNF) tagged by c-Myc-His6 (LxA/bdmh) was constructed. After transfection of LxA/bdmh to PacC6/A8, a cloned cell line, PacC6/A8/bmh, was established. PacC6/A8/bmh cells stably produced pseudotyped retroviruses carrying LxA/bdmh. For a control, a retroviral vector that bears the gene that codes enhanced green fluorescent protein (EGFP) tagged by C-Mic-His6 was also created and used for the establishment of PacC6/A8/gfmh cells that produce pseudotyped retroviruses carrying LxA/gfmh. PacC6/A8/bmh and PacC6/A8/gfmh cells were injected to the brain of newborn rats. A tumor was formed in all the rats injected that did not exhibit any symptoms until 3-4 weeks after the injection. A histological study of the injected rats revealed that the transferred BDNF gene was expressed in the brain of rats injected with PacC6/A8/bmh cells, but not in rats with PacC6/A8/gfmh cells. Interestingly, many activated microglia had migrated into the tumor induced by PacC6/A8/bmh cells, and expressed a high amount of BDNF.     

Characterization of binding and phosphorylation defects of erythrocyte insulin receptors in the type A syndrome of insulin resistance

The type A syndrome of insulin resistance and acanthosis nigricans is characterized by severe insulin resistance due to a cellular defect in insulin action. To better understand the molecular nature of this defect, we have investigated insulin binding to circulating monocytes, erythrocytes, and the Triton X-100-solubilized erythrocyte receptor, and insulin-stimulated receptor autophosphorylation using cells and receptor from three type A patients. Insulin binding in both circulating cells and the soluble extract of erythrocytes indicated a heterogeneity of defects. Patients A1 and A2 both presented a major decrease in tracer insulin binding to intact cells and soluble insulin receptor. Determination of stoichiometric binding parameters using a cooperative model indicated that in patient A1 this was due to a reduction in the number of receptors, whereas in patient A2 the affinity constant for binding was decreased. Patient A3 presented near-normal insulin binding to erythrocytes and normal binding in intact monocytes, solubilized erythrocyte receptors, and cultured fibroblasts. Affinity labeling of erythrocyte receptor from this patient revealed a normal alpha-subunit and also a normal relative distribution of the higher-molecular-weight, nonreduced oligomeric forms of the receptor. Receptor autophosphorylation was measured using the solubilized insulin receptor from erythrocytes. The maximal stimulated phosphorylation was reduced by 79%, 76%, and 52% in patients A1, A2, and A3, respectively, relative to the simultaneous control. In all three patients, the autophosphorylation was stimulated only 1.0-3.5 times the basal level compared with controls, in which the stimulation was 5.7-fold +/- 1.2 (mean +/- 1 SD, P less than 0.005). In addition, in patients A1 and A2 a decrease in basal phosphorylation was observed and in patient A2 there was a rightward shift of the dose-response curve for insulin stimulation. These data and the correlation of coupling of receptor phosphorylation with the fractional occupancy of the receptor measured in the same extract suggest that these patients exhibit three types of defects. In patient A1, there is a loss in receptor number manifested by a parallel decrease in insulin binding and receptor phosphorylation. In patient A2, there is an additional decrease in the affinity constant leading to a decrease in both binding and receptor phosphorylation with an almost linear coupling between receptor occupancy and receptor phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunoabsorption in an Extracorporeal Plasma Perfusion System: In Vitro Studies

A rechargeable plasma perfusion system developed to remove IgG immunoglobulins is described. Partially purified protein A was covalently bonded on two separate carriers, Sepharose 6MB and carboxyl acrylic beads. Plasma from a patient with IgG multiple myeloma was perfused through the Sepharose-protein. A column, and this resulted in a maximum removal of 177% of the estimated removal capacity of the column. Plasma from the same source was perfused through the acrylic bead-protein. A column and yielded an average of 220% of the estimated removal capacity of the column. In addition, plasma from a normal subject and from six patients with various autoimmune diseases was perfused through the Sepharose–protein A column, and varying percent removal was obtained. A detailed procedure for covalent coupling of purified protein A to carboxylated acrylic beads is also given.