大豆异黄酮对去卵巢大鼠骨密度及骨代谢影响的实验研究

目的 探讨大豆异黄酮对去卵巢大鼠骨丢失的防治作用及其作用机理。方法 选用卵巢切除大鼠所诱发的骨质疏松模型,给与大豆异黄酮治疗。三个月后测定大鼠骨密度及骨代谢相关生化指标。结果 大豆异黄酮可提高卵巢切除大鼠的骨密度及血清雌激素水平,降低尿钙（Ca),尿磷（P）及尿羟脯氨酸（HOP）的排泄,同时降低血清总碱性磷酸酶（ALP）,骨碱性磷酸酶（BALP）,及抗酒石酸酸性磷酸酶（TRACP）的活性,还可使血清骨钙素（BGP）的浓度降低,促使卵巢切除大鼠子宫的相对重量增加,其作用与剂量相关。结论 小剂量大豆异黄酮有类似雌激素样作用,可有效防治卵巢切除大鼠的骨量丢失,其作用机制可能是通过降低骨转换率实现的。     

急性运动对大鼠骨骼肌中丙二醛和血清肌酸激酶的影响

研究一次急性力竭运动对大鼠骨骼肌红肌、白肌中脂质过氧化水平的影响及血清酶的变化情况。发现运动组大鼠红肌中丙二醛（ＭＤＡ）含量和碱性磷酸酶（ＡＫＰ）活性比安静组显著升高;白肌中ＭＤＡ含量、超氧化物歧化酶（ＳＯＤ）活性和ＡＫＰ活性有升高的趋势;血清肌酸激酶（ＣＫ）活性运动组比安静组有显著升高;但肌肉中ＭＤＡ含量与血清ＣＫ活性之间无显著相关。因此,ＭＤＡ增多可能并不是血清ＣＫ活性升高的主要原因。     

丹皮酚对过氧亚硝基阴离子致大鼠成骨细胞分化抑制的影响

探讨丹皮酚（Pae）拮抗过氧亚硝基阴离子（ONOO^-）对体外培养大鼠成骨细胞分化的影响。用改良的组织块法分离培养新生大鼠颅骨成骨细胞,采用淬灭流动反应方法体外制备ONOO^-,以不同终浓度加入成骨细胞培养体系,在作用不同时间后,用对硝基苯二钠动力学（PNPP）法检测细胞内碱性磷酸酶（ALP）的活性,用Lowry法测定蛋白含量,并以不同终浓度Pae消除ONOO^-（1000μmoL/L）对成骨细胞分化的影响。结果显示,不同浓度的ONOO^-（50-1000μmoL/L）均能抑制碱性磷酸酶的活性,影响分化;高浓度的丹皮酚（10^-3-10^-6mol/L）能消除ONOO^-（1000μmoL/L）对碱性磷酸酶活性的抑制,拮抗ONOO^-抑制成骨细胞分化的作用。     

不同程度老年骨质疏松患者血清BALP、OPG/PYR比值变化及其与骨密度、骨折发生的相关性分析

摘要 目的：分析不同程度老年骨质疏松患者血清骨特异性碱性磷酸酶(BALP)、骨保护素(OPG)/吡啶啉(PYR)比值变化及其与骨密度、骨折发生的相关性。方法：选择我院自2020年11月至2023年7月接诊的70例老年骨质疏松患者作为观察组,其中轻-中度骨质疏松44例、重度骨质疏松26例;另选70例老年非骨质疏松者作为对照组。检测所有受试者血清BALP、OPG/PYR比值、腰椎、股骨颈和髋部的骨密度(BMD),分析BALP、OPG/PYR比值与不同骨骼部位BMD的关系,使用受试者工作特征(ROC)曲线分析BALP、OPG/PYR比值对老年骨质疏松骨折的预测效能。结果：观察组血清BALP水平高于对照组,OPG/PYR比值小于对照组（P＜0.05）;重度骨质疏松组血清BALP水平高于轻-中度骨质疏松组,OPG/PYR比值小于轻-中度骨质疏松组（P＜0.05）;观察组腰椎、股骨颈及髋部的BMD均小于对照组（P＜0.05）;经Pearson相关性分析,腰椎、股骨颈及髋部的BMD与血清BALP水平呈负相关（P＜0.05）,与OPG/PYR比值呈正相关（P＜0.05）;经ROC曲线分析,血清BALP联合OPG/PYR比值预测老年骨质疏松骨折的AUC为0.890。结论：老年骨质疏松患者血清BALP水平升高、OPG/PYR比值减小,与病情严重程度及骨密度有关,联合预测骨折的效能较好,值得进一步研究应用。     

藏红花提取液对去卵巢大鼠骨密度及骨代谢生化指标的影响

目的：研究中药藏红花提取液对去卵巢大鼠股骨骨密度及血清骨代谢生化指标的影响。方法：选用48只4月龄SD雌性大鼠,随机分为6组：假手术组、模型组、戊酸雌二醇组、藏红花低、中、高剂量组。术后4周各组分别给予相应制剂灌胃,术后12周处死,分别测定股骨骨密度、子宫指数、雌二醇、血钙、血磷、碱性磷酸酶。结果：与模型组相比,藏红花各剂量组股骨骨密度明显升高（p〈0.01）,雌二醇测定值升高（p〈0.01）,碱性磷酸酶显著降低（p〈0.01）,血钙及血磷无统计学差异（p〉0.05）;与戊酸雌二醇组比较,藏红花各剂量组子宫指数显著降低（p〈0.01）。结论：藏红花提取液有助于抑制去卵巢大鼠骨量的丢失,改善骨代谢,对骨质疏松症具有防治作用。     

益生菌补充剂治疗绝经后骨质疏松症或骨量减少的系统评价

目的 系统评价益生菌补充剂对绝经后骨质疏松症（PMOP）或骨量减少的有效性及安全性。方法 选取8个公共数据库,检索自建库至2022年9月,无语言限制,使用随机对照方法,评估补充益生菌补充剂对腰椎、股骨颈、总髋关节骨密度（BMD)、T值（T-value）、胶原降解产物（CTX）、I型前胶原氨基端前肽（PINP）、骨碱性磷酸酶（BALP）、骨钙素（BGP）水平和不良反应率的影响。结果 纳入了8篇RCT文献,647名参与者,治疗组有325名,对照组322名。结果显示益生菌组在提高PMOP和绝经后骨量减少患者的腰椎BMD、股骨颈BMD、T值、BGP和降低绝经后妇女CTX、BALP等方面显著优于对照组（P<0.05）。亚组分析结果显示益生菌治疗PMOP患者后显著提高腰椎BMD、股骨颈BMD、全髋关节BMD、T值和降低CTX、BALP优于对照组（P<0.05）。结论 益生菌干预PMOP和骨量减少患者后显著提高骨密度及降低骨代谢,对PMOP患者的作用更显著。     

凝集素亲和层析研究人血清肝型碱性磷酸酶的糖链结构

用麦胚凝集素将人血清肝型和骨型碱性磷酸酶分离，再将肝型ＡＬＰ通过蔓陀萝凝集素亲和层析柱作醋酸梯度洗脱，发现正常人、急性肝炎、慢性肝炎、肝硬化和原发性肝癌各组各自的混合血清ＡＬＰ的层析行为有明显差异。正常人只有不结合，而各种良性肝病出现数目不同（２－３个）的弱结合组分，肝癌则还出现３个强结合组分，各组分ＡＬＰ在不同肝病血清中的出峰时间基本恒定。这种ＡＬＰ洗脱峰的不均一性在单个病人血清中依然存在，是Ａ     

鹿瓜多肽注射液与补肾壮骨胶囊对膝关节骨性关节炎患者血清BALP、BGP、TRACP-5b及CTX-I水平的影响

目的:研究鹿瓜多肽注射液联合补肾壮骨胶囊对膝关节骨性关节炎的临床疗效及对血清骨特异性碱性磷酸酶(BALP)、骨钙素(BGP)、抗酒石酸盐酸性磷酸酶异构体5b(TRACP-5b)、I型胶原C端肽(CTX-I)水平的影响。方法:选取2015年4月至2016年3月我院收治的84例膝关节骨性关节炎患者,根据患者入院顺序分为观察组和对照组,42例每组。对照组使用鹿瓜多肽注射液,观察组在此基础上加以补肾壮骨胶囊。比较两组患者临床疗效,治疗前后血清BALP、BGP、TRACP-5b、CTX-I水平、视觉模拟评分法(VAS)的变化。结果:治疗后,观察组临床总有效率显著高于对照组[90.48%(38/42)比64.29%(27/42)](P0.05);两组患者血清BALP、BGP水平均较治疗前显著升高(P0.05),血清TRACP-5b、CTX-I水平、VAS评分均较治疗前显著降低(P0.05),且观察组的血清BALP、BGP水平显著高于对照组(P0.05),血清TRACP-5b、CTX-I水平、VAS评分明显低于对照组(P0.05)。结论:鹿瓜多肽注射液联合补肾壮骨胶囊治疗膝关节骨性关节炎的临床疗效明显优于鹿瓜多肽注射液单药治疗,其能显著升高患者的血清BALP、BGP水平,降低血清RACP-5b、CTX-I水平,改善患者骨代谢。     

碱性磷酸酶在文昌鱼胚胎和幼体中表达图式的研究(英文)

研究组织特异性酶在胚胎发育中的表达图式对于研究细胞、组织和器官的分化具有重要意义。本文以 ＢＣＩＰ为底物研究了碱性磷酸酶在文昌鱼胚胎和幼体中的表达图式。在囊胚、原肠胚和神经胚早期（１２～１３小时）未检测到碱性磷酸酶的特异性表达;到神经胚中期（１５小时,约６个体节）,碱性磷酸酶在胚胎每一体节的中部开始表达,在胚胎中形成３～６个条纹（Ｆｉｇ．１）;在１８小时神经胚（９～１０体节）中,碱性磷酸酶在肌节中表达（Ｆｉｇ．２）;到２４小时在后部内胚层中也开始表达（Ｆｉｇ．３）;在３６～４８小时幼体中,碱性磷酸酶在消化道中强烈表达,但在咽鳃区不表达,同时在肌节中仍有弱的表达（Ｆｉｇ．４）。研究表明碱性磷酸酶可能在肌节的发育过程中具有一定作用。碱性磷酸酶在消化道中的表达可能与这一时期消化道开始行使功能有关。另外,Ｌ－苯丙氨酸只抑制３６、４８小时文昌鱼肠碱性磷酸酶活性,不抑制其体节肠碱性磷酸酶活性（Ｆｉｇ．５）、表明文昌鱼至少存在两种碱性磷酸酶：在消化道中表达的是一种肠型的,与脊椎动物碱性磷酸酶可能为同一类型;另一种主要在肌节中表达,可能是组织非特异性的破性磷酸酶。以上结果说明文昌鱼早期发育中碱性磷酸酶的表达与脊椎动物相似,具     

High xylosyltransferase activity in children and during mineralization of osteoblast-like SAOS-2 cells

Skeletal growth and tissue remodelling processes are characterized by an elevated collagen and proteoglycan biosynthesis. The xylosyltransferases I and II are the rate-limiting step enzymes in proteoglycan biosynthesis and serum xylosyltransferase (XT) activity has been shown to be a biomarker for the actual proteoglycan biosynthesis rate. Here, XT, alkaline phosphatase (ALP), bone ALP (BALP) activities were measured in 133 juvenile Caucasians. Serum XT activities in juveniles were elevated and significantly correlated with ALP and BALP. In an osteoblast-like cell model using SAOS-2 cells mineralization and bone nodule formation were induced and XT-I, XT-II and ALP were monitored. Induction of mineralization in SAOS-2 cells resulted in a long-term increase of XT-I mRNA and enzyme activity, which could be paralleled with elevated ALP activity. In addition, HGH and IGF-I treatment of SAOS-2 cells led to an increased expression of XT-I and ALP. These results point to skeletal growth and tissue remodeling as a cause of the high XT activity in children.     

Assessment of markers of bone formation under controlled environmental factors and their correlation with serum minerals in adult sheep as a model for orthopaedic research

Eighteen healthy skeletally mature (3 years old) ewes, with an average weight of 45 kg, of the Portuguese Churra da Terra Quente breed were used to evaluate the normal values of total and bone-specific isoform of alkaline phosphatase serum activities (ALP and BALP, respectively) and serum osteocalcin (OC) and their correlation with the serum minerals - calcium (Ca), phosphorus (P), magnesium (Mg) and ionized calcium (Ca(2+)). The sheep were maintained under controlled environmental conditions (constant diurnal photoperiod cycle and identical husbandry and feeding) for six weeks before the collection of the blood samples. The measurement of the total ALP and serum minerals was performed with automated biochemistry analysers using the BioMérieux kits, the serum electrolyte Ca(2+) Diametrics Medical, Inc specific cassettes and the BALP and OC METRATM kits from QUIDEL Corporation. The mean +/- standard deviation values obtained were: total ALP 90.17 +/- 85.72 U/L, BALP 15.0 +/- 5.44 U/L, ratio BALP/ total ALP 29.28 +/- 24.22, OC 13.02 +/- 1.87 ng/mL, Ca 2.57 +/- 0.37 mmol/L, P 2.13 +/- 0.42 mmol/L, Mg 1.04 +/- 0.13 mmol/L, Ca(2+) 1.29 +/- 0.04 mmol/L. Significant correlations were observed between the total ALP and Ca (r = 0.5939; P = 0.05) and OC and Ca (r = 0.5706; P = 0.05). Reference to the serum values of bone turnover parameters in sheep could be of great value in research and could provide complementary non-invasive information on the bone healing process, particularly with regard to obtaining an early prognosis of fracture healing.     

Effect of cysteine,glutamate and glycine supplementation to in vitro fertilization medium during bovine early embryo development

Glutathione (GSH) is an antioxidant synthesized from three constitutive amino acids (CAA): cysteine (Cys), glycine (Gly) and glutamate (Glu). Glutathione plays an important role in oocyte maturation, fertilization and early embryo development. This study aimed to investigate the effect of Cys (0.6 mM), Gly (0.6 mM) and Glu (0.9 mM) supplementation during in vitro fertilization (IVF) of cattle oocytes. In a Pilot Experiment, de novo synthesis of GSH in bovine zygote was evaluated using a modified TALP medium prepared without MEM-essential and MEM-non-essential amino acids (mTALP): mTALP + CAA (constitutive amino acids); mTALP + CAA+5 mMBSO (buthionine sulfoximide); mTALP + Cys + Gly; mTALP + Cys + Glu and mTALP + Gly + Glu. This evidence led us to investigate the impact of CAA supplementation to TALP medium (with essential and non-essential amino acids) on zygote viability, lipid peroxidation, total intracellular GSH content (include reduced and oxidized form; GSH-GSSG), pronuclear formation in zygotes and subsequent embryo development. IVF media contained a) TALP; b) TALP + Cys + Gly + Glu (TALP + CAA); c) TALP + Cys + Gly; d) TALP + Cys + Glu; e) TALP + Gly + Glu, were used. Total GSH-GSSG concentration was increased in TALP, TALP + CAA, and TALP + Cys + Gly. The viability of zygote was similar among treatments. Lipid peroxidation was increased in zygote fertilized with TALP + Cys + Gly; TALP + Cys + Glu; TALP + Gly + Glu and TALP + CAA. The percentage of penetrated oocytes decreased in TALP + CAA and TALP + Cys + Gly. The cleavage rate was lower in TALP + CAA and TALP + Gly + Glu. The percentage of embryos developing to the blastocyst stage was lower in TALP + Cys + Glu and TALP + CAA. In conclusion, we have demonstrated the synthesis of GSH during IVF. However, Cys, Gly and Glu supplementation to TALP medium had negative effects on embryonic development.     

Identification, cloning, expression and functional characterization of an astacin-like metalloprotease toxin from Loxosceles intermedia (brown spider) venom

Injuries caused by brown spiders (Loxosceles genus) are associated with dermonecrotic lesions with gravitational spreading and systemic manifestations. The venom has a complex composition containing many different toxins, of which metalloproteases have been described in many different species of this genus. These toxins may degrade extracellular matrix constituents acting as a spreading factor. By using a cDNA library from an Loxosceles intermedia venom gland, we cloned and expressed a 900 bp cDNA, which encoded a signal peptide and a propeptide, which corresponded to a 30 kDa metalloprotease, now named LALP (Loxosceles astacin-like protease). Recombinant LALP was refolded and used to produce a polyclonal antiserum, which showed cross-reactivity with a 29 kDa native venom protein. CD analysis provided evidence that the recombinant LALP toxin was folded correctly, was still in a native conformation and had not aggregated. LALP addition to endothelial cell cultures resulted in de-adhesion of the cells, and also in the degradation of fibronectin and fibrinogen (this could be inhibited by the presence of the bivalent chelator 1,10-phenanthroline) and of gelatin in vitro. Sequence comparison (nucleotide and deduced amino acid), phylogenetic analysis and analysis of the functional recombinant toxin revealed that LALP is related in both structure and function to the astacin family of metalloproteases. This suggests that an astacin-like toxin is present in a animal venom secretion and indicates that recombinant LALP will be a useful tool for future structural and functional studies on venom and the astacin family.     

Astacin-like metalloproteases are a gene family of toxins present in the venom of different species of the brown spider (genus Loxosceles)

Brown spiders have a worldwide distribution, and their venom has a complex composition containing many different molecules. Herein, we report the existence of a family of astacin-like metalloprotease toxins in Loxosceles intermedia venom, as well as in the venom of different species of Loxosceles. Using a cDNA library from the L. intermedia venom gland, we cloned two novel cDNAs encoding astacin-like metalloprotease toxins, LALP2 and LALP3. Using an anti-serum against the previously described astacin-like toxin in L. intermedia venom (LALP1), we detected the presence of immunologically-related toxins in the venoms of L. intermedia, Loxosceles laeta, and Loxosceles gaucho. Zymographic experiments showed gelatinolytic activity of crude venoms of L. intermedia, L. laeta, and L. gaucho (which could be inhibited by the divalent metal chelator 1,10-phenanthroline) at electrophoretic mobilities identical to those reported for immunological cross-reactivity. Moreover, mRNAs extracted from L. laeta and L. gaucho venom glands were screened for astacin-like metalloproteases, and cDNAs obtained using LALP1-specific primers were sequenced, and their deduced amino acid sequences confirmed they were members of the astacin family with the family signatures (HEXXHXXGXXHE and MXY), LALP4 and LALP5, respectively. Sequence comparison of deduced amino acid sequences revealed that LALP2, LALP3, LALP4, and LALP5 are related to the astacin family. This study identified the existence of gene family of astacin-like toxins in the venoms of brown spiders and raises the possibility that these molecules are involved in the deleterious effects triggered by the venom.     

Effect of glucocorticoids on alkaline phosphatase, alanine aminotransferase, and gamma-glutamyltransferase in cultured dog hepatocytes

Glucocorticoid(GC)-induced hepatopathy in the dog is characterized by abnormal liver morphology and increases in serum alanine aminotransferase (ALT), gamma-glutamyltransferase (GGT), and the liver alkaline phosphatase isoenzyme (LALP) and by the appearance of an unusual isoenzyme of alkaline phosphatase known as the corticosteroid-induced alkaline phosphatase isoenzyme (CALP). It has not been shown whether the increases in serum ALT, GGT, and LALP are as a result of an increase in production of these enzymes or as a result of the GC-induced hepatocellular swelling and possible membrane alterations. Also, it has been assumed that the mechanism of production of CALP is via GC-induced gene derepression and de novo protein synthesis; however, this hypothesis has not been directly tested. Using isolated dog hepatocytes maintained in a confluent monolayer culture in the presence and absence of GC or cyclic AMP, no statistical increase in serum ALT, GGT, or LALP was observed. A combination of GC and cyclic AMP also caused no statistical increase in ALT and GGT; however, we demonstrate that these conditions clearly stimulated the de novo synthesis of LALP. These conditions do not induce the synthesis of CALP as determined by a sensitive immunoassay. The data obtained using this in vitro model suggest that the primary mechanism(s) of the in vivo increase of serum ALT and GGT in GC treated dogs may be other than that of de novo protein synthesis. Likewise, in vitro production of CALP may be a mechanism more complex than the conditions tested in this study.     

Activity and stability of alkaline phosphatase (ALP) immobilized onto magnetic nanoparticles (Fe3O4)

Direct binding of alkaline phosphatase (ALP) on magnetic nanoparticles (Fe(3)O(4)) in the presence of carbodiimide (CDI) using two different methods is described. The activity and stability of immobilized ALP with both shaking and sonication method were compared. The results indicated the ALP binding efficiency to be in the range of 80-100% with both the immobilization techniques. The activities retained were in the range of 20-38% with shaking method and 30-43% with sonication method, respectively. The activities of the immobilized ALP preparations were found to be stable compared to the free (unbound) ALP for at least 16-week storage period. Moreover, ALP immobilized on magnetic nanoparticles was successfully used for dephosphorylation of plasmid DNA before it was used for ligation reaction. The use of immobilized ALP for plasmid dephosphorylation allows easy manipulation, reduces the procedural time, and also avoids exposure of reaction mixture to high temperature.     

Molecular cloning and characterization of a novel mammalian endo-apyrase (LALP1)

Here we describe the cloning, localization, and characterization of a novel mammalian endo-apyrase (LALP1) in human and mouse. The predicted human LALP1 gene encodes a 604-amino acid protein, whereas the mouse Lalp1 gene encodes a 606-amino acid protein. The human and mouse genes have 88% amino acid sequence identity. These genes share considerable homologies with hLALP70, a recently discovered mammalian lysosomal endo-apyrase. The human LALP1 gene resides on chromosome 10q23-q24 and contains 12 exons and 11 introns covering a genomic region of approximately 46 kilobase pairs. The subcellular localization and enzymatic activity of LALP1 indicated that LALP1 is indeed an endo-apyrase with substrate preference for nucleoside triphosphates UTP, GTP, and CTP.     

Rapid and highly sensitive electrochemical determination of alkaline phosphatase using a composite tyrosinase biosensor

The use of an amperometric graphite-Teflon composite tyrosinase biosensor for the rapid monitoring of alkaline phosphatase (ALP), with no need of an incubation step and using phenyl phosphate as the substrate, is reported. Phenol generated by the action of ALP is monitored at the tyrosinase composite electrode through the electrochemical reduction of the o-quinone produced to catechol, which produces a cycle between the tyrosinase substrate and the electroactive product, giving rise to the amplification of the biosensor response and to the sensitive detection of ALP. The current was measured at -0.10 V 5 min after the addition of ALP. As a compromise between high ALP activity and high sensitivity for the detection of phenol, a pH of 8.5 was chosen. The substrate concentration was also optimized. A linear calibration plot was obtained for ALP between 2.0 x 10(-13) and 2.5 x 10(-11), with a detection limit of 6.7 x 10(-14) M. Different types of milk were analyzed with good results, using an extremely simple and rapid procedure.