Alkaline Phosphatase Protects Lipopolysaccharide-Induced Early Pregnancy Defects in Mice

Excessive cytokine inflammatory response due to chronic or superphysiological level of microbial infection during pregnancy leads to pregnancy complications such as early pregnancy defects/loss and preterm birth. Bacterial toxin lipopolysaccharide (LPS), long recognized as a potent proinflammatory mediator, has been identified as a risk factor for pregnancy complications. Alkaline phosphatase (AP) isozymes have been shown to detoxify LPS by dephosphorylation. In this study, we examined the role of alkaline phosphatase (AP) in mitigating LPS-induced early pregnancy complications in mice. We found that 1) the uterus prior to implantation and implantation sites following embryo implantation produce LPS recognition and dephosphorylation molecules TLR4 and tissue non-specific AP (TNAP) isozyme, respectively; 2) uterine TNAP isozyme dephosphorylates LPS at its sites of production; 3) while LPS administration following embryo implantation elicits proinflammatory cytokine mRNA levels at the embryo implantation sites (EISs) and causes early pregnancy loss, dephosphorylated LPS neither triggers proinflammatory cytokine mRNA levels at the EISs nor induces pregnancy complications; 4) AP isozyme supplementation to accelerate LPS detoxification attenuates LPS-induced pregnancy complications following embryo implantation. These findings suggest that a LPS dephosphorylation strategy using AP isozyme may have a unique therapeutic potential to mitigate LPS- or Gram-negative bacteria-induced pregnancy complications in at-risk women.     

Distribution of Alkaline Phosphatase Within the Periplasmic Space of Gram-Negative Bacteria

Both reaction-product localization and ferritin-coupled antibody studies have shown that alkaline phosphatase is evenly distributed throughout the peri-plasmic space of Escherichia coli and a marine pseudomonad. This space is not locally enlarged except in cases where plasmolysis has occurred.     

On the Activity of Alkaline Phosphatase in Intestinal Mucosa from Casein-fed Rats

A novel optical activity of lutein was studied in dodecyltrimethylammonium bromide (DTAB) solution by the measurement of circular dichroism and absorbance. The surfactant was found to bring about the circular dichroism activity of the lutein below the critical micelle concentration (CMC) in a different way from that by sodium dodecyl sulfate (SDS). This phenomenon was interpreted by the card-pack model of the lutein aggregate in which lutein molecule was slightly shifted each other. The above optical activity abruptly became strong just before the CMC of DTAB. This seems to correspond to the transition from the polymeric aggregate of the lutein to the oligomeric one. Such an optical activity disappeared beyond the CMC on the incorporation of the lutein molecules into the surfactant micelles. The molar binding ratios of DTAB to the lutein were determined to be 130 to 210 on the basis of the lutein concentration dependence of the DTAB concentration showing the arbitrary ellipticity. These ratios were clearly larger than those for SDS. On the other hand, filtration measurement showed that the size of the lutein-DTAB complex was larger than 2 μm in diameter. These phenomena were discussed assuming the possible model of the aggregate as a comparative study of the anionic and cationic surfactants causing the novel optical activity of this aggregate.     

Supernatant from Cultured Intestinal Cells Inhibits the Growth of Gram-Negative Bacteria

Bacterial growth chambers of Transwell units bearing intestinal epithelial monolayers (C2BBe) was consistently observed to be stagnant during the course of transmigration studies with Salmonella typhi. This limitation could not be explained by varying the bacterial load in the inoculum. Conditioned media produced by cultured C2BBe cells would not support bacterial growth. Growth support of the media was restored by heating to 95 C for 10 min. C2BBe conditioned media had bacteriostatic activity for a large variety of gram-negative, enteropathogenic bacteria but had no effect on gram-positive bacteria.     

Influence of Indigenous Microbiota on Amount of Protein and Activities of Alkaline Phosphatase and Disaccharidases in Extracts of Intestinal Mucosa in Mice

The protein content and the activities of alkaline phosphatase, maltase, and sucrase were measured at 0800, 1000, 1200, 1400, and 1600 in saline extracts of the proximal small bowels of germfree and of ex-germfree mice colonized with an indigenous microbiota. In extracts prepared from germfree mice, the total activities of all of the enzymes were relatively constant throughout the sampling period. Likewise, the total activity of alkaline phosphatase in extracts prepared from associated mice varied little as a function of time. By contrast, the total activities of maltase and sucrase in the extracts from these latter animals varied significantly from sample to sample. The total activity levels in extracts from germfree mice were approximately twofold greater than the levels in extracts from associated mice. The specific activities of alkaline phosphatase and sucrase did not vary from sample to sample in extracts prepared from either type of mouse. In contrast, the specific activity of maltase in extracts prepared from both germfree and associated mice differed significantly from sample to sample. The specific activities of all three enzymes were greater in extracts from germfree animals than in those from associated animals. The protein content of extracts prepared from germfree mice also was greater than that of extracts prepared from associated animals at every sampling time. The amount of protein extractable from the mucosa of the small bowels of the former animals varied significantly at different sampling times during the day, whereas the amount of protein extractable from the tracts of associated animals remained relatively constant throughout the day. The indigenous microbiota apparently stabilizes in some way the amount of protein extractable from the mucosa of the mouse small bowel.     

Diclofenac Mediated Demodulation of Alkaline Phosphatase and Renal Cortical Damage in Experimental Albino Mice

Diclofenac sodium is known to interfere with renal physiology by inhibiting prostaglandins. Previous studies indicate that various nephrotoxins damage proximal renal tubules by altering alkaline phosphatase (APase) activity. APase has been reported to be a function related marker in renal proximal tubular epithelia where it is highly expressed. Present investigation deals with toxicity caused in mice kidney at histological and biochemical levels after diclofenac administration. Diclofenac toxicity was assessed by localizing APase in kidney histochemically and biochemically. Intramuscular diclofenac administration (10 mg/kg/body wt) for 30 days exhibited substantial degeneration in kidney. A marked change in APase activity was observed in histochemical and biochemical studies. A change was noticed in specific activity of APase at different periods of diclofenac treatment. Decrease in specific activity of APase after 10 days (18.41 %) and 30 days (55.3 %) of diclofenac exposure was observed. However, an insignificant hike in APase was observed after 20 days of drug therapy. Similar trends in APase activity were evidenced by the electrophoretic analysis. Histological and ultrastructural observations also corroborated above mentioned findings. Present investigation gives an insight into probable mechanism of renal pathology caused by diclofenac administration in mice.     

铅对鲫鱼碱性磷酸酶和酸性磷酸酶活性的影响

在实验条件下,将鲫鱼置于1、10、40 mg/L的Pb2 溶液中静态染毒,分别在24 h、48 h、96 h、144 h测定鳃、肝、胰、肾、脑组织的碱性磷酸酶(AKP)和酸性磷酸酶(ACP)活性.结果 表明,鲫鱼鳃、肝、胰、肾、脑组织中碱性磷酸酶和酸性磷酸酶的活性随着铅浓度的升高和染毒时间的延长均呈下降趋势,其中以肾脏和脑下降最明显.     

Morphine Induces Bacterial Translocation in Mice by Compromising Intestinal Barrier Function in a TLR-Dependent Manner

Opiates are among the most prescribed drugs for pain management. However, morphine use or abuse results in significant gut bacterial translocation and predisposes patients to serious infections with gut origin. The mechanism underlying this defect is still unknown. In this report, we investigated the mechanisms underlying compromised gut immune function and bacterial translocation following morphine treatment. We demonstrate significant bacterial translocation to mesenteric lymph node (MLN) and liver following morphine treatment in wild-type (WT) animals that was dramatically and significantly attenuated in Toll-like receptor (TLR2 and 4) knockout mice. We further observed significant disruption of tight junction protein organization only in the ileum but not in the colon of morphine treated WT animals. Inhibition of myosin light chain kinase (MLCK) blocked the effects of both morphine and TLR ligands, suggesting the role of MLCK in tight junction modulation by TLR. This study conclusively demonstrates that morphine induced gut epithelial barrier dysfunction and subsequent bacteria translocation are mediated by TLR signaling and thus TLRs can be exploited as potential therapeutic targets for alleviating infections and even sepsis in morphine-using or abusing populations.     

Mechanism of Action of Nalidixic Acid on Conjugating Bacteria

When nalidixic acid, a specific and effective inhibitor of cellular deoxyribonucleic acid synthesis, is added to conjugating bacteria at any time during mating, it stops genetic transfer provided the donor bacterium is sensitive to the drug. When this inhibition is released by the removal of the nalidixic acid, transfer does not resume at the point on the chromosome where it was stopped, but begins again at the transfer origin. Curves relating the effects of various low doses of nalidixic acid to the frequency of recombination reveal that several "hits" are necessary to inhibit recombination for early markers. The number of required "hits" decreases as the distance of the marker from the transfer origin increases. Transfer between drug-resistant cells may also be inhibited by nalidixic acid. The effect of high drug doses on matings between resistant cells is similar to that of low drug doses on matings with a sensitive male.     

耐热碱性磷酸酶突变体耐热性变化机理研究

从耐热碱性磷酸酶（TAP）200多个随机突变体的克隆库中选出耐热性明显下降的4株突变体,进行全序列及表达产物的最高耐受温度和最适反应温度测定和酶分子高级结构的模拟,分析突变位点、高级结构和耐热性表现三者的关系,探讨引起耐热性变化的机理。结构模拟显示所有突变位点都仅能引起细微的、局部的结构变化,除T320→I外都未直接触及酶的活性中心;结构上的细微改变虽然对最适反应温度影响不明显,但却使最高耐受温度降低了10℃左右;T320→I靠近酶的活性中心,尽管未能引起结构的较大变化,但却使最高耐受温度和最适反应温度同时显著降低。可见,多数点突变对高级结构的影响都不剧烈,但对耐热性尤其是最高耐受温度的影响却比较明显,一般地,在非活性区的突变通常只能引起最高耐受温度的降低,靠近活性区的突变则能同时引起最适反应温度和最高耐受温度的降低。     

Chicken Intestinal Alkaline Phosphatase : I. The kinetics and thermodynamics of reversible inactivation II. Reactivation by zinc ions

Purified chicken intestinal alkaline phosphatase is active at pH 8 to 9, but becomes rapidly inactivated with change of pH to 6 or less. Also, a solution of the inactivated enzyme at pH 4.5 rapidly regains its activity at pH 8. In the range of pH 6 to 8 a solution of purified alkaline phosphatase consists of a mixture of active and inactive enzyme in equilibrium with each other. The rate of inactivation at lower pH and of reactivation at higher pH increases with increase in temperature. Also, the activity at equilibrium in the range of pH 6 to 8 increases with temperature so that a solution equilibrated at higher temperature loses part of its activity on cooling, and vice versa, a rise in temperature shifts the equilibrium toward higher activity. The kinetics of inactivation of the enzyme at lower pH and the reactivation at higher pH is that of a unimolecular reaction. The thermodynamic values for the heat and entropy of the reversible inactivation and reactivation of the enzyme are considerably lower than those observed for the reversible denaturation of proteins. The inactivated enzyme at pH 4 to 6 is rapidly reactivated on addition of Zn ions even at pH 4 to 6. However, zinc ions are unable to replace magnesium ions as cocatalysts for the enzymatic hydrolysis of organic phosphates by alkaline phosphatase.     

耐热碱性磷酸酯酶的功能结构域的定位

 为了确定耐热碱性磷酸酯酶 (TAPND2 7)发挥活性所必需的功能结构域 ,通过 PCR介导的诱变缺失 ,得到了 N端分别缺失 8、1 6、2 5个氨基酸的 3个缺失体 p TAPN8、p TAPN1 6和p TAPN2 5以及 C端分别缺失 1 0和 30个氨基酸的两个缺失体 p TAPC1 0和 p TAPC30 .经表达和活性测定 ,发现 p TAPN8和 TAPC1 0保持了较高的活性而其余 3个缺失体则失去酶活性 .据此 ,TAPND2 7的活性区域被定位在 8～ 465氨基酸之间 .在分离纯化的基础上测定了一些酶学性质 .发现 TAPN8和 TAPC1 0的比活没有大的改变 ,Tm 下降了 5.5℃ ;TAPN8的最适反应温度上升了1 0℃ .结果提示了 N端和 C端的这些氨基酸残基对热稳定性有一定的贡献 ,N端氨基酸残基还对酶的亲热性有贡献 .     

Mechanism of Action of an Imidopiperidine Inhibitor of Human Polynucleotide Kinase/Phosphatase

The small molecule, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo[3,4-b]pyridine-5,7(2H,4aH)-dione (A12B4C3), is a potent inhibitor of the phosphatase activity of human polynucleotide kinase/phosphatase (PNKP) in vitro. Kinetic analysis revealed that A12B4C3 acts as a noncompetitive inhibitor, and this was confirmed by fluorescence quenching, which showed that the inhibitor can form a ternary complex with PNKP and a DNA substrate, i.e. A12B4C3 does not prevent DNA from binding to the phosphatase DNA binding site. Conformational analysis using circular dichroism, UV difference spectroscopy, and fluorescence resonance energy transfer all indicate that A12B4C3 disrupts the secondary structure of PNKP. Investigation of the potential site of binding of A12B4C3 to PNKP using site-directed mutagenesis pointed to interaction between Trp⁴⁰² of PNKP and the inhibitor. Cellular studies revealed that A12B4C3 sensitizes A549 human lung cancer cells to the topoisomerase I poison, camptothecin, but not the topoisomerase II poison, etoposide, in a manner similar to small interfering RNA against PNKP. A12B4C3 also inhibits the repair of DNA single and double strand breaks following exposure of cells to ionizing radiation, but does not inhibit two other key strand-break repair enzymes, DNA polymerase β or DNA ligase III, providing additional evidence that PNKP is the cellular target of the inhibitor.     

耐热碱性磷酸酶功能结构域的进一步定位

为了进一步精确定位耐热碱性磷酸酶 (TAP)的功能结构域和建立酶功能结构域定位的有效方法 ,在对TAP进行序列相容性比对及二级结构统计预测的基础上 ,以二级结构单元的完整性为依据对其功能结构域进行定位 .TAP的功能结构域在 35～ 4 76氨基酸之间 ,记为TAPN3 4C2 5(TAP的N端去掉 34个氨基酸而C端去掉 2 5个氨基酸 ) ,比盛小禹等定位的少 15个氨基酸 .克隆、表达实验结果证明了TAPN3 4C2 5的酶学活性 ,其最适反应温度为 72℃ ,比TAPND2 7(N端去除了 2 7个信号肽氨基酸的TAP)上升了 7℃ ,而最高耐受温度为 83℃ ,比TAPND2 7下降了 16℃ .实验结果表明 ,以二级结构预测结果进行酶功能结构域定位是一种更为精确的方法 .