Intestinal-type Alkaline Phosphatase Research Articles

Application and discoveries of metabolomics and proteomics in the study of female infertility.

Female infertility is defined as the absence of clinical pregnancy after 12 months of regular unprotected sexual intercourse. This study employed metabolomics and proteomics approaches to investigate the relationship between metabolites and proteins and female infertility. The study used metabolomics and proteomics data from the UK Biobank to identify metabolites and proteins linked to infertility. The results showed that GRAM domain-containing protein 1C and metabolites fibrinogen cleavage peptides ADpSGEGDFXAEGGGVR and 3-Hydroxybutyrate had a positive correlation with infertility, whereas proteins such as Interleukin-3 receptor subunit alpha, Thrombospondin type-1 domain-containing protein 1, Intestinal-type alkaline phosphatase, and platelet and endothelial cell adhesion molecule 1 exhibited a negative correlation. These findings provide new clues and targets for infertility diagnosis and treatment. However, further research is required to validate these results and gain a deeper understanding of the specific roles of these metabolites and proteins in infertility pathogenesis. In conclusion, metabolomics and proteomics techniques have significant application value in the study of infertility, allowing for a better understanding of the biological mechanisms underlying infertility and providing new insights and strategies for its diagnosis and treatment. These research findings provide a crucial biological mechanistic basis for early infertility screening, prevention, and treatment.

Active-phase Plasma Alkaline Phosphatase Isozyme Activity Is a Sensitive Biomarker for Excessive Fructose Intake.

Excessive fructose intake reportedly leads to the development of nonalcoholic fatty liver disease (NAFLD). In our previous study, we reported that plasma activities of alkaline phosphatase (ALP) isozymes were markedly changed in rats with excessive fructose intake-induced hepatomegaly. In this study, we examined ALP isozyme activity prior to the occurrence of hepatomegaly, and investigated the effect of the timing of sample collection, to explore its potential as a biomarker. After 1-week intake of a 63% high-fructose diet (HFrD), blood samples were collected from male rats during sleep or active phases to analyze biochemical parameters. Body and liver weights were similar between the HFrD and control diet groups, indicating that hepatomegaly due to excessive fructose intake had not occurred. The triglyceride levels and glutamate dehydrogenase (GLDH) activity were significantly elevated to similar degrees at both time points. HFrD intake significantly increased liver-type ALP (L-ALP) activity, stimulating it by 12.7% at the sleep phase and by 124.3% at the active phase. HFrD consumption also significantly decreased intestinal-type ALP (I-ALP) at the active phase, but only showed a decreasing trend during the sleep phase. Measurements of plasma ALP isozyme and GLDH activity, and triglyceride levels are effective early biomarkers of impending NAFLD caused by excessive fructose intake. L-ALP and I-ALP activities during the active phase are particularly sensitive for detection of excessive fructose intake before the occurrence of NAFLD.

Novel Biomarker Establishment for Evaluation of Excessive Fructose Consumption Using a Rat Model.

The habitual consumption of excessive fructose is associated with the onset and progression of lifestyle-related diseases, such as nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the physiological changes observed when consuming a diet containing excessive fructose on the viewpoints of hepatotoxicity biological markers using a rat model and explored the biomarker candidates that could detect the effects of excessive fructose intake at an early stage. Male rats were fed 63% high fructose diet (HFrD) ad libitum and their blood samples were collected before and at 1, 2, 3, and 4 weeks after allocation. The plasma biochemical parameters, hepatotoxic enzyme activities including alkaline phosphatase (ALP) isozymes were analyzed. HFrD consumption for 4-weeks created NAFLD-like symptoms, including elevated plasma lipid parameters and hepatotoxicity markers, as well as fat accumulation in the liver compared with rats consuming a control diet. Alanine aminotransferase (ALT) and glutamate dehydrogenase (GLDH) were increased from the 3rd and 2nd weeks, respectively, but no changes were observed on ALP activity. However, the daily consumption of the HFrD increased the plasma activities of liver-type ALP isozyme, and decreased plasma small intestinal-type ALP isozyme soon after the start of feeding. ALP isozyme analysis in combination with GLDH and ALT activities in the plasma samples could be a useful tool to detect the physiological changes induced by excessive fructose intake at an early stage of the development of NAFLD.

Characterization and Structure of Alternatively Spliced Transcript Variant of Human Intestinal Alkaline Phosphatase (ALPI) Gene.

Intestinal-type alkaline phosphatase (IAP) is expressed at a high concentration in the brush border membrane of intestinal epithelial cells and is known to be a gut mucosal defense factor. In humans, a single gene (ALPI) for IAP has been isolated, and its transcription produces two kinds of alternatively spliced mRNAs (aAug10 and bAug10). Recently, we discovered that vitamin D up-regulated the expression of both types of human IAP alternative splicing variants in Caco-2 cells. However, the functional difference of protein encoded by the mRNA variants has remained elusive. In the present study, we aimed to provide further insight into the characterization and structure of IAP isoforms. To analyze the protein translated from the ALPI gene, we constructed two kinds of cDNA expression plasmids (aAug10 and bAug10), and the transfected cells were homogenized and assayed for alkaline phosphatase (ALP) activity. We also designed the homology-modeled 3D structures of the protein encoded by the mRNA variants (ALPI-aAug10 and ALPI-bAug10). The levels of ALP activity of COS-1 cells transfected with the aAug10 plasmid were increased significantly, while cells transfected with the bAug10 plasmid had undetectable ALP activity. The homology-modeled 3D structures revealed that the variant bAug10 lacks the central N-terminal α-helix and residue corresponding to Asp-42 of ALPI-aAug10 near the active site. This is the first report on the characterization and structure of alternatively spliced transcript variants of the human ALPI gene. Further studies on the regulation of aAug10 and/or bAug10 mRNA expression may identify novel physiological functions of IAP.

Effects of First Feeding Regime on Gene Expression and Enzyme Activity in Pikeperch (Sander lucioperca) Larvae

The present study investigates the effects of different feeding regimes with rotifers (Brachionus plicatilis) and Artemia salina on the gene expression and digestive enzymes in pikeperch (Sander lucioperca) larvae at 17 days post-hatch (DPH) over a period of 13 days. Five experimental feeding protocols were performed in four replicates. At 4 DPH, the larvae (total length= 5.62 ± 0.03 mm, body weight = 0.66 ± 0.16 mg) were divided into five experimental groups (2-L tanks) at initial density of 100 larvae per liter. Light intensity on the water surface was 90-100 lux and photoperiod was set at 13L: 11D (07:00 to 20:00 h). Water temperature, pH, and dissolved oxygen (DO) were measured before each feeding and the values were 17.8 ± 0.17°C, 7.3 ± 0.04 and 88.5 ± 2.53%. The fish larvae at 5 days post-hatch (DPH), were initially fed with rotifers (Brachionus plicatilis) for 3 days and from 8 to 17 DPH were fed with rotifers/Artemia for different time periods as follows: (A) only rotifers; (B) 8–13 DPH rotifers/14–17 DPH Artemia; (C) 8–10 DPH rotifers/11–17 DPH Artemia; (D) only Artemia; (E) a combination of rotifers and Artemia. Frozen paste of algae was added to the larval tanks twice a day (2 x 300,000 cells/mL). Rotifers and Artemia were provided as live feed to larvae three times a day with residual counts prior to each feeding. Feeding densities were steadily increased based on residual counts, performed prior to each feeding. The expression of genes related to intestinal development and maturation (aminopeptidase N, anpep; leucine aminopeptidase 3, lap3; intestinal-type alkaline phosphatase, alpi), together with key pancreatic digestive proenzymes (trypsinogen 1, try1; chymotrypsinogen b, ctrb; carboxyl ester lipase precursor, cel; phospholipase a2, pla2g1b; pancreatic alpha amylase, amy2a), were assessed. Additionally, the activity of six enzymes (trypsin, lipase, alkaline phosphatase, amino peptidase, amylase, and chymotrypsin) were determined. The highest expression of two genes related to intestine (lap3; anpep) were observed in the fish fed a combination of rotifers and Artemia from 8 DPH (Group E). The expression of amy2a, ctrb, pla2g1b, try1 was significantly lower in larvae fed rotifers until 14 DPH and replaced by Artemia afterwards (Group B). The specific activity of brush border membrane enzymes (alkaline phosphatase and aminopeptidase N) increased with combination of rotifers and Artemia in larval diet (Group E), indicating a more efficient functionality of digestive structures. The groups fed only with rotifers till 17 DPH (Group A) (38 ± 4.07%) and larvae fed with rotifers till 14 DPH followed by feeding with Artemia till 17 DPH (Group B) (36 ± 5.25%) showed significantly (P<0.05) lower survival rates than the other groups (54-67%). The group fed only with rotifers (Group A) showed significantly lower specific growth rate (SGR) than the other groups, and the highest SGR was found in the group fed with combination of rotifers and Artemia after 3 day rotifer feeding (Group E). The highest standard length (8.32 ± 0.48 mm) was obtained by combined feeding of rotifers and Artemia after 3 day of initial rotifer feeding. Combination of rotifers and Artemia from 8 DPH (Group E) could be considered a more appropriate diet for first feeding pikeperch larvae compared with later introduction of Artemia, as indicated by the higher expression of genes and activities of digestive enzymes. Our findings provide new insight into the effect of temporal sequence of rotifers and Artemia on the expression of genes and activities of digestive enzymes in pikeperch larvae.

Associations between Genetically Predicted Circulating Protein Concentrations and Endometrial Cancer Risk.

Simple SummaryEndometrial cancer is the leading female reproductive tract cancer in developed countries. Discovering new biomarkers is critical for understanding the etiology this cancer and identifying women with a higher risk of this cancer from the general population. Several blood protein biomarkers have been linked to endometrial cancer in previous studies, but these studies have assessed only a limited number of biomarkers usually among a small number of participants. The current study aimed at identifying novel circulating protein biomarkers of endometrial cancer by using the largest available dataset to date. Our finding suggested nine proteins to be associated with endometrial cancer risk, and five of the identified associations showed suggestive associations with risk of non-endometrioid EC, a much more lethal subtype. If validated by additional studies, our findings may contribute to understanding the pathogenesis of endometrial tumor development and facilitating the risk assessment of endometrial cancer.Endometrial cancer (EC) is the leading female reproductive tract malignancy in developed countries. Currently, genome-wide association studies (GWAS) have identified 17 risk loci for EC. To identify novel EC-associated proteins, we used previously reported protein quantitative trait loci for 1434 plasma proteins as instruments to evaluate associations between genetically predicted circulating protein concentrations and EC risk. We studied 12,906 cases and 108,979 controls of European descent included in the Endometrial Cancer Association Consortium, the Epidemiology of Endometrial Cancer Consortium, and the UK Biobank. We observed associations between genetically predicted concentrations of nine proteins and EC risk at a false discovery rate of <0.05 (p-values range from 1.14 × 10−10 to 3.04 × 10−4). Except for vascular cell adhesion protein 1, all other identified proteins were independent from known EC risk variants identified in EC GWAS. The respective odds ratios (95% confidence intervals) per one standard deviation increase in genetically predicted circulating protein concentrations were 1.21 (1.13, 1.30) for DNA repair protein RAD51 homolog 4, 1.27 (1.14, 1.42) for desmoglein-2, 1.14 (1.07, 1.22) for MHC class I polypeptide-related sequence B, 1.05 (1.02, 1.08) for histo-blood group ABO system transferase, 0.77 (0.68, 0.89) for intestinal-type alkaline phosphatase, 0.82 (0.74, 0.91) for carbohydrate sulfotransferase 15, 1.07 (1.03, 1.11) for D-glucuronyl C5-epimerase, and 1.07 (1.03, 1.10) for CD209 antigen. In conclusion, we identified nine potential EC-associated proteins. If validated by additional studies, our findings may contribute to understanding the pathogenesis of endometrial tumor development and identifying women at high risk of EC along with other EC risk factors and biomarkers.

PSVIII-12 Comparative characterization of intestinal alkaline phosphatase kinetics in young piglets and human Caco-2 cells

Abstract This study reports the kinetics of young porcine jejunal alkaline phosphatase (IAP) towards the dephosphorylation of ATP, lipopolysaccharides (LPS) and p-nitrophenyl phosphate (pNPP) at the physiological conditions (pH = 7.4; and at 37 °C) in comparison with the IAP from human Caco-2 cells. The 10-day suckling young porcine jejunal IAP displayed the Km values of 1.26±0.50 mM, 1.35±0.64 mg/mL and 0.290±0.072 mM for the hydrolyses of ATP, LPS and pNPP, respectively; while the respective Km values were 0.030±0.007 mM, 0.66±0.22 mg/mL and 0.033±0.006 mM for the IAP in the Caco-2 cells towards the same set of substrates. In comparison, the Km values of the young porcine jejunal IAP were 2–40 times higher than those of IAP from the human Caco-2 cells. In addition, Pearson correlation analyses showed tight positive correlations (P &amp;lt; 0.001) between IAP activities towards pNPP, ATP and LPS in the piglet and the Caco-2 cells, suggesting that the IAP activity towards pNPP could be used to predict the IAP activities on the physiological substrates of ATP and LPS. Lastly, four AP genes were identified in the genome of Sus scrofa. Three of these porcine AP genes are annotated as intestinal-type alkaline phosphatase (IAP) genes and clustered at the distal end of chromosome 15, namely IAPX1, IAPX2, and IAPX3. The genomic context of APs in the pig genome is highly similar to those in the human genome. We predict that pig IAPX3 (XP_003133777.1) is likely an IAP gene for pigs. Further comparisons in post-translational modification and protein 3-D structure modelling were performed, indicating that the observed differences in kinetic affinity between the young porcine jejunal IAP and the human Caco-2 cell IAP in hydrolyzing ATP, LPS and pNPP might relate to their differences in the coding sequences of the IAP protein, and/or the IAP protein N-glycosylation.

1-alpha,25-Dihydroxyvitamin D3 up-regulates the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells and may be an important regulator of their expression in gut homeostasis

Vitamin D insufficiency is associated with a greater risk of osteoporosis and also influences skeletal muscle functions, differentiation, and development. The principal function of vitamin D in calcium homeostasis is to increase the absorption of calcium from the intestine, and the level of alkaline phosphatase (ALP) activity, a differentiation marker for intestinal epithelial cells, is regulated by vitamin D. Intestinal-type ALP is expressed at a high concentration in the brush border membrane of intestinal epithelial cells, and is known to be affected by several kinds of nutrients. Recent reviews have highlighted the importance of intestinal-type ALP in gut homeostasis. Intestinal-type ALP controls bacterial endotoxin-induced inflammation by dephosphorylating lipopolysaccharide and is a gut mucosal defense factor. In this study, we investigated the influence of vitamin D on the expression of 2 types of alternative mRNA variants encoding the human alkaline phosphatase, intestinal (ALPI) gene in human Caco-2 cells as an in vitro model of the small intestinal epithelium. After treatment with 1-alpha,25-dihydroxyvitamin D3, the biologically active form of vitamin D3, there were significant increases in the ALP activities of Caco-2 cells. Inhibitor and thermal inactivation experiments showed that the increased ALP had properties of intestinal-type ALP. Reverse transcription-polymerase chain reaction analysis revealed that expression of the 2 types of alternative mRNA variants from the ALPI gene was markedly enhanced by vitamin D in Caco-2 cells. In conclusion, these findings agree with the hypothesis: vitamin D up-regulated the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells; vitamin D may be an important regulator of ALPI gene expression in gut homeostasis.

自然発症糖尿病WBN/Kobラットにおけるアルカリホスファターゼについて

The male WBN/Kob rat is an animal model of chronic pancreatitis and spontaneous diabetes. Here, we performed a kinetic study of alkaline phosphatase (ALP) in WBN/Kob rats. The level of serum ALP activities in the WBN/Kob rats was three times higher than that of the control group (p<0.001). Specific ALP activities in bone tissues or the ileum increased significantly (p<0.01 and p<0.05, respectively). By 4-20% gradient polyacrylamide gel electrophoresis, serum ALP isozymes were separated into two bands. One was heat-unstable, showing the same mobility as an ALP preparation from bone tissue, and the other was heat-stable. We considered these isozymes to be bone-type and intestinal-type ALPs, respectively, and both of them were enhanced in the WBN/Kob rats. These results demonstrate the enzyme kinetics of ALP isozymes in WBN/Kob rats, and further studies will be necessary to reveal the association with diabetes.

Menaquinone-4 (vitamin K2) up-regulates expression of human intestinal alkaline phosphatase in Caco-2 cells

Alkaline phosphatase (ALP) hydrolyzes several monophosphate esters into inorganic acid and alcohol. In humans, 4 kinds of ALP isozymes have been identified: tissue-nonspecific ALP, intestinal ALP, placental ALP, and germ cell ALP. Intestinal ALP is expressed at a high concentration in the brush border membrane of intestinal epithelial cells and is known to be affected by several kinds of nutrients, such as lipids, but the physiological function of intestinal ALP has remained elusive. Vitamin K is an essential cofactor for the posttranslational carboxylation of glutamate residues into γ-carboxy glutamate. Menaquinone-4 (MK-4) with 4 isoprene units, vitamin K2, has been shown to induce bone-type ALP activity and osteoblastogenesis in human bone marrow cells. In this study, we investigated the effects of MK-4 on the level of ALP activity and expression of ALP messenger RNA in the human colon carcinoma cell line Caco-2, which is known to differentiate into small intestinal epithelial cells in vitro. After treatment with MK-4, there were significant increases in the ALP activities of Caco-2 cells. Inhibitor and thermal inactivation experiments demonstrated that the increased ALP had properties of intestinal-type ALP. Semiquantitative reverse transcription–polymerase chain reaction analysis revealed that expressions of human intestinal ALP and sucrase-isomaltase, which are intestinal differentiation markers, were highly enhanced in Caco-2 cells by MK-4. This is the first report concerning ALP messenger RNA expression induced by vitamin K2 in Caco-2 cells. Further studies on the physiological functions of human intestinal ALP will provide useful data on the novel effects of vitamin K.

Rifaximin-Mediated Changes to the Epithelial Cell Proteome: 2-D Gel Analysis

Rifaximin is a semi-synthetic rifamycin derivative that is used to treat different conditions including bacterial diarrhea and hepatic encephalopathy. Rifaximin is of particular interest because it is poorly adsorbed in the intestines and has minimal effect on colonic microflora. We previously demonstrated that rifaximin affected epithelial cell physiology by altering infectivity by enteric pathogens and baseline inflammation suggesting that rifaximin conferred cytoprotection against colonization and infection. Effects of rifaximin on epithelial cells were further examined by comparing the protein expression profile of cells pretreated with rifaximin, rifampin (control antibiotic), or media (untreated). Two-dimensional (2-D) gel electrophoresis identified 36 protein spots that were up- or down-regulated by over 1.7-fold in rifaximin treated cells compared to controls. 15 of these spots were down-regulated, including annexin A5, intestinal-type alkaline phosphatase, histone H4, and histone-binding protein RbbP4. 21 spots were up-regulated, including heat shock protein (HSP) 90α and fascin. Many of the identified proteins are associated with cell structure and cytoskeleton, transcription and translation, and cellular metabolism. These data suggested that in addition to its antimicrobial properties, rifaximin may alter host cell physiology that provides cytoprotective effects against bacterial pathogens.

Expression of intestinal-type alkaline phosphatase mRNA in liver of Akp3 knockout mice

Previously, we discovered that the rat liver showed enhanced expression of intestinal-type alkaline phosphatase (ALP) on high fat-feeding. Here we tested the hypothesis that intestinal-type ALP (Akp3 or Akp6) might also be regulated in the liver of Akp3-/- mice upon high fat-feeding. C57Bl/6J male Akp3-/-, Akp3+/-, or wild-type mice were fed a normal control or high-fat diet and the effects on intestinal-type ALP mRNA in liver were investigated. We found that Akp3 mRNA is only expressed in the intestine and not in the liver, while Akp6 mRNA is expressed in both the upper intestine and liver of wild-type mice. The intensity of Akp6 mRNA expression in the liver was not enhanced in the Akp3-/- mice compared with the wild-type mice fed on a high-fat diet. The nucleotide sequence of the PCR product of Akp6 from the liver was identified as being the same as that of Akp6 in the intestine. This is the first report concerning IAP mRNA expression in the mouse liver, but further studies will be needed to determine if this ectopic expression of intestinal-type ALP is associated with any unique function.

Non-cloning Synthesis of cRNA Probe and Its Application to In Situ Hybridization Analysis of Formalin-fixed Paraffin Embedded Tissue

Intestinal type alkaline phosphatase (IALP) mRNA-specific cRNA probe was synthesized by non-cloning method for in situ hybridization (ISH) analysis. Total RNA was extracted from small intestine of male F344 rat. Reverse transcription-polymerase chain reaction (RT-PCR) was performed for IALP gene. T7 promoter adapter was directly added to IALP cDNA-specific PCR product by ligation reaction. IALP cDNA accompanied by T7 promoter adapter was then amplified by PCR using IALP gene-specific primer in combination with T7 promoter adapter primer sequence and used as a template of cRNA probe. Digoxigenin-labeled cRNA was synthesized by in vitro transcription method using RNA polimerase. ISH was performed using formalin-fixed, paraffin embedded small intestine tissue. As a result, the cytoplasm of mucosal epithelial cells was successfully stained with this cRNA probe. The present procedure is quite simple and a rapid way to prepare the cRNA probe and would be very useful for ISH analysis of formalin-fixed paraffin embedded tissue sample.

Down-regulation of intestinal-type alkaline phosphatase in the tumor vasculature and stroma provides a strong basis for explaining amifostine selectivity

Strong clinical and experimental evidence indicates that amifostine confers cytoprotection in normal, but not in tumor, tissues. However, the mechanism of such selective action is poorly understood. Intestinal-type alkaline phosphatase (IAP) is a major isoenzyme involved in the hydrolysis of amifostine (WR-2721) to its active thiol form WR-I065. Could differences in IAP expression between normal and malignant tissues account for amifostine's selectivity? Paraffin-embedded material from normal breast, lung, colon, and head and neck tissues, together with their malignant counterparts, were retrieved and stained immunohistochemically for human IAP (antibody 7324, Abcam, Cambridge, UK) and endothelial cell CD31 antigen. Normal tissues (epithelium, fibroblasts, and vessels) consistently displayed strong nuclear and cytoplasmic IAP reactivity. The vascular density (number of positive vessels per x 200 optical field), whether assessed in anti-IAP or anti-CD31 stained sections, was very similar, indicating a strong IAP content for the entire normal vasculature. Therefore, amifostine hydrolysis is ensured in normal tissues and may occur at both vascular and interstitial levels. By contrast, 60% of the tumors analyzed showed a loss of IAP expression in both epithelial cells and stroma, and only 10% to 15% of them demonstrated nuclear/cytoplasmic reactivity, which was confined to the epithelial cells. Similarly, the percentage of tumor vessels exhibiting some IAP reactivity was very low (6% to 17%). This dramatic loss of IAP expression from tumor stroma/vasculature may form a strong basis for explaining amifostine selectivity. In contrast, the abundance of IAP expression in normal tissues, stromal and vascular, ensures an intense hydrolysis of WR-2721 and rapid intracellular accumulation of WR-1065.

Down-regulation of intestinal-type alkaline phosphatase in the tumor vasculature and stroma provides a strong basis for explaining amifostine selectivity

Strong clinical and experimental evidence indicates that amifostine confers cytoprotection in normal, but not in tumor, tissues. However, the mechanism of such selective action is poorly understood. Intestinal-type alkaline phosphatase (IAP) is a major isoenzyme involved in the hydrolysis of amifostine (WR-2721) to its active thiol form WR-I065. Could differences in IAP expression between normal and malignant tissues account for amifostine's selectivity? Paraffin-embedded material from normal breast, lung, colon, and head and neck tissues, together with their malignant counterparts, were retrieved and stained immunohistochemically for human IAP (antibody 7324, Abcam, Cambridge, UK) and endothelial cell CD31 antigen. Normal tissues (epithelium, fibroblasts, and vessels) consistently displayed strong nuclear and cytoplasmic IAP reactivity. The vascular density (number of positive vessels per × 200 optical field), whether assessed in anti-IAP or anti-CD31 stained sections, was very similar, indicating a strong IAP content for the entire normal vasculature. Therefore, amifostine hydrolysis is ensured in normal tissues and may occur at both vascular and interstitial levels. By contrast, 60% of the tumors analyzed showed a loss of IAP expression in both epithelial cells and stroma, and only 10% to 15% of them demonstrated nuclear/cytoplasmic reactivity, which was confined to the epithelial cells. Similarly, the percentage of tumor vessels exhibiting some IAP reactivity was very low (6% to 17%). This dramatic loss of IAP expression from tumor stroma/vasculature may form a strong basis for explaining amifostine selectivity. In contrast, the abundance of IAP expression in normal tissues, stromal and vascular, ensures an intense hydrolysis of WR-2721 and rapid intracellular accumulation of WR-1065. Semin Oncol 29 (suppl 19):14-21. Copyright 2002, Elsevier Science (USA). All rights reserved.

Heat shock induces intestinal-type alkaline phosphatase in rat IEC-18 cells.

We demonstrate a previously unknown regulation for intestinal-type alkaline phosphatase (IAP) as a heat shock protein (HSP). Heat shock to rat intestinal epithelial cells (IEC)-18 at 43 degrees C induced the expression of IAP-I and HSP72 mRNAs time dependently (<60 min) but did not induce expression of IAP-II, tissue nonspecific-type alkaline phosphatase (TNAP), or HSP90 as determined by the RT-PCR method. To confirm the identity of the IAP-I gene, we sequenced the amplification product of IAP-I and found the gene to have 99% homology with the sequence of the IAP-I gene in rat intestine. Under the subculture conditions used, no IAP protein was detected in IEC-18 cells, but it became detectable as a 62-kDa band on a Western blot after heat shock. IAP-I was also induced by sodium arsenite, which generates reactive oxygen species and is an inducer of members of the HSP family. Glutathione suppressed activating protein-1 and cAMP response element-binding protein activation caused by heat shock but did not suppress the expression of IAP-I. These results suggest that cellular stress induces the elevation of IAP-I mRNA and protein synthesis. IAP-I may play an important role as a dephosphorylating enzyme under stress conditions.

Alkaline phosphatases reduce toxicity of lipopolysaccharides in vivo and in vitro through dephosphorylation

Intestinal alkaline phosphatase (AP), as a host defense factor, was first investigated in vivo using rats orally exposed to lipopolysaccharide (LPS). After the oral administration of LPS to rats, serum LPS content was increased within 2 hr and then decreased to 6 hr. In contrast, when l-phenylalanine ( l-Phe), an inhibitor of intestinal-type AP isozymes, was simultaneously administered with LPS, serum LPS content significantly increased from 1 hr and the area under the concentration-time curve of serum LPS was augmented approximately 2-fold, suggesting that APs in the gastrointestinal tract reduced serum LPS content. In addition, LPS toxicity diminished by a treatment in vitro with intestinal APs, were recovered by the treatment in the co-presence of l-Phe. In the experiment using human aortic endothelial cells (HAECs), we observed that the cell viability decreased in a dose-dependent manner of LPS-exposure, and the LPS dose, exhibiting 50% viability of the cells, was 0.05 μg/ml. When the cells were exposed to LPS pretreated with 50 nIU/ml of intestinal AP at pH 10.0 and 8.0, the 50% viability was at 2.0 μg/ml of LPS. These results strongly suggest that the APs reduced the toxicity of LPS, as a host defense factor against LPS.

Expression of sucrase and intestinal-type alkaline phosphatase in colorectal carcinoms in rats treated with methylazoxymethanol acetate

In this study the small-intestine phenotype in rat colonic tumors was investigated in terms of sucrase and intestinal-type alkaline phosphatase (I-ALP) activity. F344 rats were given intraperitoneal injections of methylazoxymethanol acetate at a dose level of 25 mg/kg body weight once a week for 8 weeks and were killed 40 weeks after the first injection. Sucrase and I-ALP activities in proximal and distal colon adenocarcinomas were significantly higher than those in the normal colon epithelium. In the jejunum, by contrast, normal tissue had significantly higher levels than tumors. Immunohistochemical staining of I-ALP was also strong in striated cell borders of colon adenocarcinoma cells. These data suggest that, whereas absorptive cells of the small intestine lose their own traits with tumor development, colonocytes acquire phenotypic features of the small intestine. Intestinal enzymes associated with the striated-cell border, such as sucrase and I-ALP, may be useful markers for malignant phenotypic expression in colonocytes.

Increased expression of sucrase and intestinal-type alkaline phosphatase in human gastric carcinomas with progression.

The activities of sucrase, total alkaline phosphatase (total ALP) and intestinal‐type alkaline phosphatase (I‐ALP) were assayed in gastric carcinomas and in their surrounding mucosae from 57 patients with advanced cancers, and the localization of sucrase in 203 carcinomas, including 86 early cancers, was examined immunohistochemically using polyclonal anti‐sucrase antibody. All three enzymes were active in the 57 carcinomas as well as in their surrounding mucosae, but the levels were fairly low as compared to those in normal jejunum mucosa. A considerable part of the total ALP activity in tumor specimens was assumed to be due to I‐ALP itself. Increased sucrase and I‐ALP were found with greater depth of invasion by undifferentiated‐type carcinomas. The pattern of immunohistochemical localization of sucrase in the 203 carcinomas also clearly indicated increased expression with greater depth of invasion even in differentiated‐type carcinomas.

The tumor-derived fetal-intestinal alkaline phosphatase cDNA is identical in sequence to the adult intestinal alkaline phosphatase isozyme gene

The alkaline phosphatase (AP) of Caco-2 cells, a cell line derived from a human adenocarcinoma of the colon, is quite similar to fetal intestinal AP in its enzymatic properties. The nucleotide sequence of a cDNA encoding AP produced in Caco-2 cells was examined. The sequence was identical to one of the three sequences of adult intestinal AP reported previously. We further investigated the entire nucleotide sequence of cDNA of intestinal-type AP produced in cancer cell lines such as HuH-7 cells, FL-amnion cells, and HuG-1 cells. The sequence of these cell APs was identical to that of Caco-2 cell AP. These results indicate that cancer cells producing intestinal-type AP have the same nucleotide sequence as that of adult intestinal AP, and suggest that the differences in electrophoretic mobilities of these cell APs compared with adult intestinal AP may be due to post-translational modifications.