Loss Of Alkaline Phosphatase Activity Research Articles

The Myb-like transcription factor phosphorus starvation response (PtPSR) controls conditional P acquisition and remodelling in marine microalgae.

Phosphorus (P) is one of the limiting macronutrients for algal growth in marine environments. Microalgae have developed adaptation mechanisms to P limitation that involve remodelling of internal phosphate resources and accumulation of lipids. Here, we used in silico analyses to identify the P-stress regulator PtPSR (Phaeodactylum tricornutum phosphorus starvation response) in the diatom P. tricornutum. ptpsr mutant lines were generated using gene editing and characterised by various molecular, genetics and biochemical tools. PtPSR belongs to a clade of Myb transcription factors that are conserved in stramenopiles and distantly related to plant P-stress regulators. PtPSR bound specifically to a conserved cis-regulatory element found in the regulatory region of P-stress-induced genes. ptpsr knockout mutants showed reduction in cell growth under P limitation. P-stress responses were impaired in ptpsr mutants compared with wild-type, including reduced induction of P-stress response genes, near to complete loss of alkaline phosphatase activity and reduced phospholipid degradation. We conclude that PtPSR is a key transcription factor influencing P scavenging, phospholipid remodelling and cell growth in adaptation to P stress in diatoms.

Unique and redundant roles of SOX2 and SOX17 in regulating the germ cell tumor fate.

Embryonal carcinomas (ECs) and seminomas are testicular germ cell tumors. ECs display expression of SOX2, while seminomas display expression of SOX17. In somatic differentiation, SOX17 drives endodermal cell fate. However, seminomas lack expression of endoderm markers, but show features of pluripotency. Here, we use chromatin immunoprecipitation sequencing to report and compare the binding pattern of SOX17 in seminoma-like TCam-2 cells to SOX17 in somatic cells and SOX2 in EC-like 2102EP cells. In seminoma-like cells, SOX17 was detected at canonical (SOX2/OCT4), compressed (SOX17/OCT4) and noncomposite SOX motifs. SOX17 regulates TFAP2C, PRDM1 and PRDM14, thereby maintaining latent pluripotency and suppressing somatic differentiation. In contrast, in somatic cells canonical motifs are rarely bound by SOX17. In sum, only 12% of SOX17-binding sites overlap in seminoma-like and somatic cells. This illustrates that binding site choice is highly dynamic and cell type specific. Deletion of SOX17 in seminoma-like cells resulted in loss of pluripotency, marked by a reduction of OCT4 protein level and loss of alkaline phosphatase activity. Furthermore, we found that in EC-like cells SOX2 regulates pluripotency-associated genes, most likely by partnering with OCT4. In conclusion, SOX17 (in seminomas) functionally replaces SOX2 (in ECs) to maintain expression of the pluripotency cluster.

Function of the N-terminal region of the phosphate-sensing histidine kinase, SphS, in Synechocystis sp. PCC 6803

In Synechocystis sp. PCC 6803 the histidine kinase SphS (sll0337) is involved in transcriptional activation of the phosphate (Pi)-acquisition system which includes alkaline phosphatase (AP). The N-terminal region of SphS contains both a hydrophobic region and a Per-Arnt-Sim (PAS) domain. The C-terminal region has a highly conserved transmitter domain. Immunological localization studies on heterologously expressed SphS in Escherichia coli indicate that the hydrophobic region is important for membrane localization. In order to evaluate the function of the N-terminal region of SphS, deletion mutants under the control of the native promoter were analysed for in vivo AP activity. Deletion of the N-terminal hydrophobic region resulted in loss of AP activity under both Pi-deficient and Pi-sufficient conditions. Substitution of the hydrophobic region of SphS with that from the Ni2+-sensing histidine kinase, NrsS, resulted in the same induction characteristics as SphS. Deletion of the PAS domain resulted in the constitutive induction of AP activity regardless of Pi availability. To characterize the PAS domain in more in detail, four amino acid residues conserved in the PAS domain were substituted with Ala. Among the mutants R121A constitutively expressed AP activity, suggesting that R121 is important for the function of the PAS domain. Our observations indicated that the presence of a transmembrane helix in the N-terminal region of SphS is critical for activity and that the PAS domain is involved in perception of Pi availability.

Molecular assessment of phosphorus and iron physiology in Trichodesmium populations from the western Central and western South Atlantic

Trichodesmium is capable of responding to low phosphorus (P) and low iron (Fe) concentrations through the induction of alkaline phosphatase (AP) activity (an enzyme that hydrolyzes inorganic phosphate from phosphomonoesters) and the IdiA protein (an iron deficiency protein that putatively binds iron), respectively. We monitored AP activity and IdiA expression as molecular diagnostics of P and Fe stress in Trichodesmium field samples on a transect from the western central to the western South Atlantic and compared the expression of these markers with physical, chemical, and N2 fixation measurements. Trichodesmium AP activity was detected at many stations, whereas IdiA expression was not. Incubation additions of inorganic P at one station resulted in a loss of AP activity and an increase in N2 fixation. Our data suggest that P, and not Fe, is the constraining factor limiting Trichodesmium N2 fixation along most of the transect. These results support previous modeling efforts predicting that diazotrophs in this region are P‐stressed and further validate the use of molecular diagnostics of nutritional physiology, in concert with more traditional approaches, for identifying constraints on marine N2 fixation.

Phosphatidylinositol-dependent bond between alkaline phosphatase and collagen fibers in the periodontal ligament of rat molars.

Alkaline phosphatase (ALP) is anchored to the outer leaflet of the lipid bilayer via phosphatidylinositol (PI) and ALP activity has been localized in the plasma membrane of numerous tissues. In the periodontal ligament ALP activity is found in the collagen fibers in addition to the plasma membrane of the osteoblasts and fibroblasts. In this study, we examined the distribution of ALP activity in the periodontal ligament of rat molars and also examined whether the bond between ALP and collagen fibers is dependent on PI by using phosphatidylinositol-specific phospholipase C (PI-PLC). ALP activity was distributed in the periodontal ligament. The activity mirrored the distribution of collagen fibers in the periodontal ligament. Cytochemical analysis also demonstrated that ALP activity was located not only in the plasma membrane of fibroblasts, but also in the collagen fiber bundles and fibrils in the periodontal ligament. After treatment with PI-PLC, the loss of ALP activity in the periodontal ligament was observed histochemically, and the loss of ALP activity in the fibroblasts as well as in the collagen fiber bundles and fibrils was observed cytochemically. These results strongly indicate that the bond between ALP and the collagen fibers is also dependent on PI.

Characterization of ectoenzyme activity and phosphate-regulated proteins in the coccolithophorid Emiliania huxleyi

Three phosphate-regulated proteins in the coccolithophorid Emiliania huxleyi were detected by the biotinylation of cell-surface proteins. Two of these phosphate-regulated proteins have reduced denatured molecular weights near 110 000 Da (118 078 and 110 541, respectively), while the third, and most abundant, is 69 087 Da. Induction of the three proteins and the common marker of phosphate stress, alkaline phosphatase activity, occur in the presence of <0.25M inorganic phosphate in batch culture. Phosphate-regulated proteins and enzyme activity differed among E. huxleyi strains. Alkaline phosphatase is an enzyme commonly induced by phytoplankton in response to phosphate stress in order for cells to scavenge inorganic phosphate from organic sources. In E. huxleyi, this enzyme activity and the phosphate-regulated proteins are rapidly lost when phosphate is added back to phosphate-stressed cultures. This contrasts with the slower loss of alkaline phosphatase activity in the dinoXagellate Prorocentrum minimum. The presence of the three phosphateregulated proteins and enzyme activity appear to differ somewhat among E. huxleyi strains. Based on these differences betweenstrains, kinetic data, growth experimentsand enzyme activities,the69087 Da protein may be a phosphatase with a high speciWcity for 59-nucleotides.

Maturation-dependent thiol loss increases chondrocyte susceptibility to apoptosis.

The major aim of the current investigation was to evaluate the role of thiols during chondrocyte maturation and apoptosis. Using a thiol-sensitive fluorescent probe, we found that in chick growth plate chondrocytes, hypertrophy is accompanied by a decrease in the glutathione content. In this study, we show that the maturation-dependent loss of thiol, although not causing death of maturing chondrocytes, drastically increases susceptibility to apoptosis by oxidative and nitrosoactive stress. To investigate how the loss of thiol content in cultured chondrocytes affects the expression of the hypertrophic phenotype, we chemically manipulated intracellular thiol levels and analyzed the expression of important maturation markers. We found that thiol depletion causes a decrease in the expression of osteopontin, type X and type II collagen and a significant loss of alkaline phosphatase activity, suggesting that the expression of the hypertrophic phenotype is tightly regulated by redox levels in chondrocytes. Furthermore, severe thiol depletion profoundly affected cell survival under oxidative and nitrosoactive stress. It was concluded that the loss of thiol reserve is not only linked to the expression of the hypertrophic phenotype but also influenced chondrocyte survival, linking chondrocyte maturation and the activation of the apoptotic pathway.

Choriocapillaris degeneration and related pathologic changes in human diabetic eyes.

To measure the extent of choriocapillaris degeneration (CCD) in diabetic choroids and to study the association of CCD with choroidal neovascularization and pathologic changes in Bruch's membrane-like basal laminar deposits. Human choroids from 10 postmortem subjects (diabetic, 5 [group 1]; nondiabetic, 5 [group 2]) were incubated for the histochemical demonstration of alkaline phosphatase and nonspecific esterase activities, permitting analysis of the choroidal vasculature and polymorphonuclear leukocytes, respectively. The tissue was then flat embedded and sectioned for structural analysis. Areas of CCD were measured in the flat perspective by computer-assisted image analysis and verified in cross-sections of flat-embedded tissue. The CCD in choroids from subjects with diabetes (group 1) appeared in 2 patterns: diffuse (partial loss of alkaline phosphatase activity in a poorly defined area, ie, degeneration of some capillary segments) and focal (complete degeneration of choriocapillaris or loss of alkaline phosphatase activity in a relatively well-defined area). The mean+/-SD percentage of the choroid with focal CCD in group 1 was 5.08%+/-1.13% of the total choroidal area vs 1. 16%+/-0.35% in group 2 (P<.001). Focal CCD in group 1 was more prominent in the posterior pole than in the peripheral choroid. Choroidal neovascularization was associated with some areas of diffuse CCD in group 1. Pathologic changes in Bruch's membrane-like basal laminar deposits were often associated with CCD; the thickness of the deposits was greater in group 1 than in group 2 and greater in areas with focal CCD than in areas with diffuse or no CCD. The percentage of choroid with focal CCD in group 1 choroids was more than 4-fold greater than that in nondiabetic choroids. The presence of CCD was related to basal laminar deposits and, in some cases, to choroidal neovascularization.

Loss of Alkaline Phosphatase Activity in Meningiomas

Apart from defined histomorphologic features, increased Ki-67 indices and various numeric and structural chromosome aberrations, meningiomas of the intermediate (WHO grade II, atypical meningioma) and anaplastic type (WHO grade III) are cytogenetically distinguished from common-type meningiomas (WHO grade I) by frequent loss of the distal part of the short arm of one chromosome 1 (1p-), which formerly proved to be an independent predictor of shorter recurrence-free intervals. Histochemically, loss of alkaline phosphatase activity (ALPL, liver/bone/kidney type, EC 3.1.3.1) was another frequent, specific finding in meningiomas with signs of dedifferentiation. In a prospective study including 66 meningiomas, all common-type meningiomas except one case (18/19) were reactive for ALPL, whereas 75% (30/39) of intermediate type and all anaplastic meningiomas (8/8) showed loss of enzyme activity in large areas of the tumor. Exclusively, the ALPL negative phenotype was associated with 1p loss (15/19). Our data suggest that ALPL, which is coded as a single copy gene on chromosome 1p36.1-p34, is a useful marker enzyme for the loss of a putative regulatory (tumor suppressor) gene on chromosome 1p, or that ALPL itself represents a new tumor suppressor gene homozygously inactivated in meningiomas.

Fractionation response and repair kinetics of radiation-induced heart failure in the rat

Local heart irradiation with single or fractionated doses leads to heart failure after dose-dependent latency times. Clinical symptoms of heart failure are dyspnoea at rest, apathy and subcutaneous oedema. Animals autopsied when they presented with these symptoms, have a congested liver and occasional pleural effusions. The left ventricle is dilated, showing a reduction in wall thickness by 15–17% of control values. Histological examination reveals a focal degeneration and necrosis of about 23% of the total myocardial volume. Loss of alkaline phosphatase activity from myocardial capillaries, which is known to precede myocardial degeneration, involves 77% of the myocardium. These findings at the time of manifest heart failure are constant, independent on whether injury to the heart was inflicted by single-dose or fractionated irradiation or whether heart failure developed within a relatively short time after high total doses or within many months after low total doses. The latent time of heart failure therefore can be considered an appropriate endpoint for comparison of treatment groups. From experiments giving 1, 2, 4 or 10 dose fractions, a low alpha/beta ratio of 3.7 Gy (95% confidence interval 1.8–5.6 Gy) can be calculated. When the time interval between dose fractions is varied in a split-dose experiment, time intervals of up to 3 h do not increase the survival time significantly. This appears to indicate very slow repair of sublethal damage. On the other hand, it cannot be excluded that pathogenetic mechanisms independent of cell death in the renewing cell population contribute to this effect, making an interpretation of the alpha/beta ratio in terms of cell survival parameters of a defined target cell population difficult.

Loss of alkaline phosphatase activity in duodenal mucosa: A marker for precursors of gastric metaplasia?

Biopsy specimens from duodenal mucosa in 34 patients with upper gastrointestinal symptoms and endoscopically abnormal mucosa (including duodenitis, active duodenal ulcer, and healed duodenal ulcer) and in 9 patients with histologically normal mucosa were examined histologically for gastric metaplasia and endogenous alkaline phosphatase (AP) activity. Using haematoxylin and eosin (H&E) and periodic acid-Schiff (PAS), we found gastric metaplasia in 91.2 per cent (31 out of 34) of patients with altered duodenal mucosa and in 33.3 per cent (3 out of 9) of patients with histologically normal, non-inflamed duodenal mucosa (P less than 0.001). To characterize gastric metaplasia further, histochemical methods for AP activity were applied to duodenal mucosa specimens. No AP activity was detected in complete metaplastic cells, but focal or diffuse loss of AP activity was frequently shown in otherwise normal appearing enterocytes next to metaplastic cell groups. Focal loss of AP activity was also detected in seven out of nine healthy controls (= 77.8 per cent) which appeared normal when stained with H&E. Our results suggest that the loss of AP activity in enterocytes may be an early marker of developing gastric metaplasia or at least a morphological manifestation of cell damage.

Morphological and Functional Changes in the Rat Heart after X Irradiation: Strain Differences

The hearts of mature male rats of the Wistar and Sprague-Dawley strains were locally irradiated with single doses of 17.5 and 20.0 Gy of X rays, respectively. These two dose levels had previously been shown to result in a comparable latent period between irradiation and the death of rats of these two strains from cardiac failure. Morphological changes in the myocardium and modifications in cardiac function were assessed in the animals at 28, 70, and 100 days after irradiation. The first radiation-induced change which was observed in the myocardium was a rapid decline in capillary density and a loss of alkaline phosphatase activity by the capillary endothelial cells. The capillary density was reduced to approximately 50% of that of unirradiated control values at 28 days and to approximately 40% of the control values between 70 and 100 days after irradiation. The loss of enzyme activity was also detected at 28 days. Examination of histological sections showed an increase by 70 days in the areas with negative enzyme activity up to approximately 70% of the myocardium. The reduction in capillary density and the loss of enzyme activity occurred before any marked pathological changes were seen in the myocardium. The pathological lesions seen in the myocardium at 100 days after irradiation were qualitatively and quantitatively the same in the two strains of rat. Measurements of cardiac output in Sprague-Dawley rats showed a gradual decline in output after irradiation; however, measurements in Wistar rats showed a progressive increase in cardiac output over the same period of time. It was shown by rubidium extraction that there was an increase in the percentage of the total cardiac output distributed to the ventricular muscle of Sprague-Dawley rats, while similar measurements in Wistar rats showed no significant change. In spite of the marked strain differences observed in cardiac output and rubidium extraction, blood perfusion per gram of ventricular muscle was apparently not modified in both strains of rat after irradiation. These findings indicated that the correlation between morphological effects after irradiation and the functional expression of damage is highly complex.

Incidence of microscopically detectable degenerative characteristics in skeletal muscle of Turkey

1. The incidence of microscopically detectable degenerative characteristics in 5 skeletal muscles (m. pectoralis thoracicus, m. supracoracoideus, m. biceps femoris, m. semitendinosus, m. femorotibialis medius) of turkeys was investigated. 2. Samples were obtained from 30 Large White turkey males 14, 16 and 18 weeks old. Hyaline degeneration, infiltration of mononuclear cells and necrotic fibres were observed. 3. Individual fibres varied greatly in size and muscle fibre nuclei were often shrunken and pyknotic. 4. Weak and/or uniform reaction for Ca++-ATPase and SDH in all types of muscle fibres and loss of alkaline phosphatase activity in cell membranes were noted. A positive reaction for acid phosphatase occurred in regions of perivascular infiltration and in necrotic muscle fibres. The majority of muscle fibres possessed high activity for phosphorylase a and b. 5. Based on the use of fluorescein alpha-bungarotoxin conjugate, motor end-plates appeared to be morphologically intact. Direct immunofluorescence with anti-chicken IgG showed positive reaction in muscle fibres undergoing necrosis and in the involved connective tissue. 6. Degenerative changes varied with age and were most marked in the oldest birds. 7. Because gross degenerative symptoms were absent from both the birds and the meat from them, the condition appears to be either different from or a precursor to the degenerative myopathy characterised by other authors.

Correlation between loss of alkaline phosphatase activity and accumulation of calcium during matrix vesicle-mediated mineralization.

Activity of the bone/liver/kidney isozyme of alkaline phosphatase (AP) is known to be critical for mineralization in developing bone, although its role is unclear. The work now reported explores changes in the activity of this Zn2+-containing enzyme that occur during Ca2+ accumulation by matrix vesicles (MV). A marked loss (up to 65-70%) in AP activity was found to accompany Ca2+ accumulation by MV. These two events were highly correlated, both temporally and quantitatively. Investigation into possible causes revealed that the decline in AP activity during Ca2+ uptake was not due to action of proteases but rather resulted from interaction with the developing mineral phase, loss of metal ions (Zn2+ and Mg2+) from the active site of the enzyme, and concomitant irreversible denaturation of the enzyme. Protease inhibitors did not protect AP from loss of activity during mineralization; in contrast, protease treatments, which progressively destroyed the ability of MV to accumulate Ca2+ actually reduced loss of AP activity. These findings clearly demonstrate that AP is present at the site of MV mineralization and that its catalytic activity is profoundly reduced by the mineralization process.

Mechanisms of triple stress‐mediated damage in stationary phase cells of Salmonella typhimurium exposed to succinate‐acidified hypochlorite system at 5° C

Exposure for 20 min of stationary phase cells of Salmonella typhimurium to a combined triple stress system (TSS) treatment comprising hypochlorite derived 5 ppm free available chlorine in solution acidified with 1% succinate (pH 2.5) and at a chill shock temperature of 5 degrees C resulted in symptoms of injury. Cells became sensitive to 40 micrograms/ml lysozyme, 50 micrograms/ml actinomycin D and 100 micrograms/ml ribonuclease B, to which control cells were resistant. Metabolic injury was indicated by reduction in colony forming ability of stressed cells on minimal salts glucose agar M9 medium. There was no detectable leakage loss of 260-280 nm-absorbing materials. This was also confirmed by assay of the cellular RNA material components. Loss of alkaline phosphatase activity was observed in the stressed cells. The intensity of induced cellular damage as measured by lysozyme sensitivity was greatest in the cells exposed to the complete TSS, followed by those stressed in 1% succinate at 5 degrees C, then 5 ppm chlorine at 5 degrees C and the singular chill shock stress at 5 degrees C, respectively. The magnitudes of cellular damage, however, were suggestive of synergistic interactions among the component stress factors of the TSS. The findings obtained indicated impairment of the structural integrity and functional capabilities of the permeability barriers and the inactivation of certain periplasmic enzymes. The resultant cumulative cellular damage from the TSS exposure may therefore enhance greater sensitivity of treated cells to subsequent stress factors.

Histochemical investigations on human bladder cancer (author's transl)

The histochemical enzyme activity of alkaline phosphatase, nonspecific esterase, 5-nucleotidase, beta-glucuronidase, glucose-6-phosphatase, succinate dehydrogenase, and glucose-6-phosphate dehydrogenase in human bladder cancer was investigated. Tumors of 84 patients, classified into grades I-III according to the WHO classification, were compared with 12 normal and 16 inflamed bladder epithelia. As a rule, loss of alkaline phosphatase activity and a decrease of nonspecific esterase activity was found in most of these tumors. The activity of beta-glucuronidase was decreased and compared with normal tissue, also the activity of 5-nucleotidase. The succinate dehydrogenase activity in tumor tissue was frequently increased, whereas glucose-6-phosphate dehydrogenase did not show any significant reaction.

A rationale for the loss of growth control during experimental bladder carcinogenesis

A rationale is presented which provides an explanation for the loss of growth control which is associated with the early phase of experimentally-induced bladder cancer. Two early events which occur in the urothelium following exposure to carcinogen, the focal loss of alkaline phosphatase activity and the initiation of cell proliferation, are both proposed to be the result of a defect in the interaction between glucocorticoid hormone and urothelium. The possible causes for this defect are discussed in terms of a defect in, or an interference with, the glucocorticoid-receptor mechanism.

Changes in alkaline phosphatase activity in periosteal cells in healing fractures.

The quantitative changes in alkaline phosphatase activity in the periosteal cells close to the fracture in rat metatarsal bones has been measured during the first 5 days postfracture. This study has been made possible by two technological advances, firstly the development of cryostat microtomy for cutting unfixed, undemineralised bone, and secondly the use of scanning and integrating microdensitometry for quantifying the activity in each periosteal cell. The results showed a loss of alkaline phosphatase activity close to the fracture site, with activity rising to normal values 0.8-1.0 mm from the site. No alkaline phosphatase activity was found in the cells which proliferated from the periosteum. It is suggested that reduced glutathione could cause such inhibition.

Focal loss of alkaline phosphatase and increase of proliferation in preneoplastic areas of the rat urothelium after administration of n-butyl-n-(4-hydroxybutyl)-nitrosamine and n-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide.

Development of tumours of the urinarY bladder was studied in 59 Male and female Sprague-Dawley and Wistar rats with combined enzyme-histochemical and autoradiographic methods after oral application of n-butyl-n-(4-hydroxybutyl)-nitrosamine (BBN) and n-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT). as the first carcinogenic lesion detectable by light-microscopy a focal, sharply defined irreversible loss of alkaline phosphatase activity was consistently demonstrated in the urothelium, which appeared normal histologically and cytologically. In about 2/3 of the cases, NADH-diaphorase activity was markedly reduced in identical regions. The enzyme-deficient areas are to be considered as preneoplastic, because papillomas and carcinomas developed from them through different stages of hyperplasia. As a rule, these also were characterized by total loss of alkaline phosphatase activity and attenuation of the NADH-diaphorase in all parts or circumscribed areas. Autoradiographically 3H-thymidine-labelling index revealed a 43.2-fold (BBN) and 22.6-fold (FANFT) increase, respectively, in the enzyme-deficient areas, as compared with the surrounding emzyme-containing urothelium. After 54 hrs of continous labelling, there was a mean 3H-thymidine-labelling index of 54.9% in the enzyme-negative regions. The physiological mode of regeneration was no longer maintained in the areas of enzyme deficiency as there was an increased proliferation of suprabasal cells. Areas of papillomas that showed a marked attention of NADH-diaphorase had a 3H-thymidine-labelling index 4.5 (BBN) and 3.1 (FANFT) greater than the surrounding areas with preserved enzyme activity. Since loss of alkaline phosphatase activity occurs regulary and consistently after application of carcinogens with chemically different structures it appears to indicate the initial phase of tumor development in the urinary bladder of the rat.

Alkaline phosphatase activity in hyperplastic and neoplastic urinary bladder epithelium of mice fed 2-acetylaminofluorene.

Studies were made on the urinary bladder of 66 BALB/c mice of both sexes; 21 were fed a normal diet and 45, a diet containing 100-500 parts per million (ppm) of 2-acetylaminofluorene (2-AAF). Bladder epithelial hyperplasia was noted in 22 of 24 mice given 2-AAF for 7 months and in 20 of 21 given 2-AAF for 19 months. Transitional cell carcinomas were in 12 of 21 mice given 2-AAF for 19 months and in all except the 9 females given the low dose (100 ppm). Special stains revealed marked alkaline phosphatase activity in the bladder epithelium of untreated controls and slightly to moderately reduced activity in the hyperplastic epithelium, particularly the lower layers, of mice given 2-AAF for 7 months. Activity was markedly reduced and confined largely to the upper layers in mice treated for 19 months. The transitional cell carcinomas showed no activity except focally in a few tumor masses in 11 of 12 mice with tumors. Alkaline phosphatase activity was usually slightly to moderately increased in the sub-epithelial stroma after 7 months' treatment with 2-AAF and markedly increased after 19 months. Loss of alkaline phosphatase activity in the bladder epithelium may be a preneoplastic change.