The effect of internal biliary drainage on bile pigment accumulation and acid phosphatase activity in human liver during obstructive jaundice.

The effect of internal drainage after biliary obstruction due to primary cholangiocarcinoma has been studied in seven human liver biopsies with respect to bile pigment accumulation and acid phosphatase activity. Enzyme activity was demonstrated at the light-microscopic level in unfixed cryostat sections using an incubation medium containing naphthol AS-BI phosphate as substrate, and hexazotised pararosaniline as simultaneous coupling agent, and at the ultrastructural level in fixed tissue blocks and chopped tissue sections using sodium beta-glycerophosphate as substrate and lead or cerium ions as capture reagent. Large amounts of bilirubin were found in cryostat sections of non-drained cholestatic livers, especially in pericentral areas. At these sites a high acid phosphatase activity was found. At the ultrastructural level, acid phosphatase activity was found only in the lysosomal compartment--possibly due to the procedure necessary for tissue processing. After internal biliary drainage, the amount of bilirubin diminished, with a concomitant decrease in acid phosphatase activity. The co-localization of accumulations of bile compounds and acid phosphatase activity indicates that lysosomes play a role in the breakdown of bile compounds.     

Multiple forms of acid phosphatase activity in Gaucher's disease.

Although the primary genetic defect in all individuals with Gaucher's disease is a deficiency in glucocerebrosidase activity, the finding of marked elevations in splenic and serum acid phosphatase activity is almost as consistent a finding. Gaucher spleen and serum contain at least two forms of acid phosphatase that can be readily separated by chromatography on columns containing the cation exchange resin Sulphopropyl Sephadex. The major species of acid phosphatase (designated SP-I) contained in Triton X-100 (1% v/v) extracts of Gaucher spleen accounts for 65%--95% of the total activity and has the following properties: (1) it does not bind to the cation exchange column; (2) it exhibitis a pH optimum of 4.5--5.0; (3) it is inhibited by sodium fluoride (15 mM), L(+)-tartaric acid (20 mM), and beta-mercaptoethanol (2.1 M), and (4) it is resistant to inhibition by sodium dithionite (10 mM). The minor acid phosphatase activity (designated SP-II) present in extracts of Gaucher spleen has properties similar to those of the major species of acid phosphatase activity contained in serum from patients with Gaucher's disease: (1) it binds firmly to cation exchange columns (eluted by 0.5 M sodium chloride); (2) it exhibits a pH optimum of 5.0--6.0; (3) it is inhibited by sodium fluoride and sodium dithionite; and (4) it is resistant to inhibition by beta-mercaptoethanol (2.1 M) and L(+)-tartaric acid (20 mM). In addition, a second form of acid phosphatase that is tartrate resistant was found to be elevated in Gaucher serum. This form of serum acid phosphatase did not bind to Sulphopropyl Sephadex, was found to be significantly resistant to beta-mercaptoethanol (2.1 M), and was only partially inhibited by sodium dithionite (10 mM). The findings reported here indicate that at least three distinct forms of acid phosphatase activity are elevated in Gaucher's disease. Furthermore, the minor acid phosphatase activity contained in spleen homogenates has properties very similar to those of the major acid phosphatase activity observed to be present in serum of patients with Gaucher's disease. These data indicate that simple spleen spillage cannot account for the increased levels of serum acid phosphatase in patients with Gaucher's disease.     

Studies on the mechanism of cellular death—I. Enzymatic changes during early and late cardiac necroses

A battery of 15 different histoenzymological tests were employed to study the enzymatic changes which occur during the death of dog myocardial cells induced by local surgically induced cardiac ischemia and in autopsy specimens of infarcted human myocardium. The sequence of events was observed at intervals from 1 to 480 hr.

Succinoxidase activity was apparent both in the longitudinal and transverse axes of the myofibers. In the early necrotic phases, the network arrangement was disorganized and discrete mitochondria were observed at the edge of the infarcts. In later stages these mitochondria were associated with fat droplets.

Esterase activity was located on the same particulate elements as succinoxidase but no interconnecting network was evident. In older infarcts these particles increased in size and occurred both within and at the margin of the infarcts. Esterase activities persisted for a longer period of time than did the DPN diaphorase or succinoxidase activity.

Cytochrome oxidase activity persisted much longer than did either succinoxidase or DPN diaphorase activity.

Acid phosphatase activity using a standard azo dye coupling technique was also particulate and increased during the early stages of an infarct. The particulate acid phosphatase activity was replaced by a smooth, homogeneous staining of the cytoplasm at about 24 hr. Acid phosphatase activity revealed by ASBI as substrate was localized in the lipofuscin inclusions.

Leucine aminopeptidase activity was undetectable in early stages of cell death but reached very high persistent levels in the most advanced infarcts.

ATPase activity was localized at the blood vessels as well as myocardial tissue elements. The activity increased from 4 to 64 hr after occlusion and it disappeared thereafter. ADPase and alkaline phosphatase activities demonstrated a disruption of the blood vessels in the moderately advanced infarcts. ADPase tests revealed a greater number of vascular elements than did the alkaline phosphatase methods.

Leukocyte infiltration was observed at about 24 hr. Cells having positive leucine aminopeptidase activity as well as having acid phosphatase activity by the ASBI method were observed after 24 hr of infarction. Histiocytes, rich in acid phosphatase and esterase activities, appeared at about 144 hr and persisted thereafter.

Human material revealed a comparable sequence of histoenzymatic events.     

Age-related changes of activity of acid phosphatase in PHA-stimulated lymphocytes

Peripheral blood lymphocytes of 70-year-old individuals as well as spleen cells of 18-month-old Balb/c mice were characterized by diminished activity of acid phosphatase in relation to the activity of that enzyme in cells from young subjects. Simultaneously performed histochemical tests revealed that aging process in both species examined was accompanied by a reduction of the number of cells, disclosing the activity of acid phosphatase. Age-related differences with regard to the level of acid phosphatase became more pronounced after stimulation of cells with PHA. The decrease of acid phosphatase activity during aging is discussed in relation to the function of lymphocytes.     

Phosphorylation and inactivation of protein phosphatase 1 by cyclin-dependent kinases.

Protein phosphatase 1 and protein phosphatase 2A contain potential phosphorylation sites for cyclin-dependent kinases. In the present study we found that rabbit skeletal muscle protein phosphatase 1, as well as recombinant protein phosphatase 1 alpha and protein phosphatase 1 gamma 1, but not protein phosphatase 2A, was phosphorylated and inhibited by cdc2/cyclin A and cdc2/cyclin B. Phosphopeptide mapping and phospho amino acid analysis suggested that the phosphorylation site was located at a C-terminal threonine. Neither cdc2/cyclin A nor cdc2/cyclin B phosphorylated an active form of protein phosphatase 1 alpha in which Thr-320 had been mutated to alanine, indicating that the phosphorylation occurred at this threonine residue. Furthermore, protein phosphatase 1, but not protein phosphatase 2A, activity was found to change during the cell cycle of human MG-63 osteosarcoma cells. The observed oscillations in protein phosphatase 1 activity during the cell cycle may be due, at least in part, to phosphorylation of protein phosphatase 1 by cyclin-dependent kinases. Together, the results suggest a mechanism for direct regulation of protein phosphatase 1 activity.     

马尔尼菲青霉酸性磷酸酶在宿主氧化杀伤中的作用

目的 探讨马尔尼菲青霉(Penicillium marneffei, PM)酸性磷酸酶(Acid phosphatase,ACP)在宿主氧化杀伤中的作用。方法 在培养基中加入不同浓度H₂O₂模拟氧化应激环境,于25 ℃和37 ℃震荡培养后检测上清液中ACP的活性。通过用特定培养基诱导酶分泌、灭活、抑制剂处理的方法获得不同酶活性的PM分生孢子,建立分生孢子与巨噬细胞共培养模型;利用H₂DCFDA标记联合流式细胞术检测巨噬细胞ROS的释放量,CFU及透射电镜观察巨噬细胞对PM的杀伤情况。结果 PM菌丝相及酵母相均能分泌ACP,加入H₂O₂后两相ACP活性均提高;诱导PM分泌酸性磷酸酶,巨噬细胞ROS释放量及对菌体杀伤能力被抑制;将酶抑制后,巨噬细胞ROS释放量增加,对菌体杀伤能力增强。结论 PM在氧化应激条件下能通过分泌ACP抑制巨噬细胞ROS产生,从而抵御宿主的氧化杀伤作用。     

Differential androgen modulation of acid phosphatase isozymes in primary cultures of rat ventral prostate epithelial and stromal cells

The influence of androgen on prostate differentiated cell function was investigated using primary cultures of rat ventral prostate epithelial and stromal cells developed from sexually immature animals (21 days of age). As a biochemical marker of androgen action, total acid phosphatase activity, which comprises both the secretory and lysosomal isoforms, was measured. Testosterone increased total acid phosphatase activity approximately 2-fold in epithelial cell cultures. This increase occurred only after the cessation of cell proliferation (i.e. upon reaching a confluent monolayer). In contrast, stromal cells showed no significant change in total acid phosphatase activity in response to androgen. Polyacrylamide gel isoelectric focusing of total acid phosphatase activity from epithelial and stromal cell extracts revealed that secretory acid phosphatase activity was localized exclusively in the epithelial cells while lysosomal acid phosphatase activity was present in both cell types. Furthermore, the androgen-induced increases in epithelial total acid phosphatase activity were found to result from increases in the secretory isoform.     

An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid phosphatases tested from potato, wheat germ, milk, and bovine prostate did not show this degree of specificity. The plasma membrane activity also dephosphorylated phosphotyrosine histone at a much greater rate than did the other acid phosphatases. pH profiles for free O-phosphotyrosine and phosphotyrosine histone showed a shift toward physiological pH, indicating possible physiological significance. Phosphotyrosine histone dephosphorylation activity was nearly 10 times greater than that seen for phosphoserine histone dephosphorylation, and Km values were much lower for phosphotyrosine histone dephosphorylation (0.5 microM vs. 10 microM). Fluoride and zinc significantly inhibited phosphoserine histone dephosphorylation. Vanadate, on the other hand, was a potent inhibitor of phosphotyrosine histone dephosphorylation (50% inhibition at 0.5 microM) but not of phosphoserine histone. ATP stimulated phosphotyrosine histone dephosphorylation (160-250%) but inhibited phosphoserine histone dephosphorylation (95%). These results suggest the existence of a highly specific phosphotyrosine protein phosphatase activity associated with the plasma membrane of human astrocytoma.     

Distribution of acid phosphatase activity in the larval stages of Wuchereria bancrofti, Brugia malayi, B. pahangi and Dirofilaria immitis in the mosquito.

The histochemical distribution of acid phosphatase in microfilariae and in the larval stages of four mosquito-borne filariae: Wuchereria bancrofti, Brugia malayi, B. pahangi and Dirofilaria immitis was studied using naphthol AS-TR-hexazonium technique and light microscopy. Accurate differentiation between microfilariae of the four species could be made on the basis of their patterns of acid phosphatase activity. In contrast to microfilariae in the blood, the larval stages in the mosquito exhibited different patterns of acid phosphatase activity which were characteristic for each developmental stage. In the first-stage larva, maximum acid phosphatase activity was found in the anal vesicle, the growing anal membrane (anal plug), buccal cavity, forming intestine and rectum. In the second-stage larva, acid phosphatase activity was present throughout the alimentary canal, particularly in the section of the intestine and rectum. In the infective third-stage larva, the whole body stained densely red. The reaction for acid phosphatase in the excretory cell complex of W. bancrofti and of both species of Brugia gradually decreased in intensity and disappeared completely towards the end of the first-larval stage, whereas in D. immitis a strong reaction in this area persisted throughout the larval life in the mosquito. The presence or absence of enzymic activity in the excretory cell complex and in the Mundgebilde (amphids) of the developing larvae can be used as an adjunctive diagnostic method.     

Modulation of enzymatic activities during spontaneous and induced differentiation in a human pancreatic adenocarcinoma cell line CAPAN-1

Different enzymatic activities were studied in the human pancreatic cancer cell line CAPAN-1 in order to analyze their relation to differentiation. Alkaline phosphatase (Alk Ph), acid phosphatase, aminopeptidase, dipeptidyl peptidase IV, acid and neutral alpha-glucosidases, and acid beta-galactosidase were present. Especially alkaline phosphatase, which we have found to be of the placental type isoenzyme, is being highly expressed. Spontaneous cell differentiation at confluence as well as differentiating agents: sodium butyrate and DMSO, modulated the levels of three enzymes: Alk. Ph., aminopeptidase, and acid alpha-glucosidase. The exposure of the cells to the differentiating agents amplified the modulations occurring during the spontaneous differentiation. Aminopeptidase and acid alpha-glucosidase were found to be induced by differentiation. Alk Ph specific activity was significantly increased by the spontaneous and the butyrate-induced differentiations; whereas DMSO exerted an opposite effect, probably related to its biphasic action on cell proliferation.     

Changes of enzyme activities recognized in lymphocytes from patients with carcinoma of the gastrointestinal tract

Adenosine triphosphatase (ATPase) activity and acid phosphatase activity in lymphocytes from patients with carcinoma of the gastrointestinal tract were determined in order to investigate whether or not changes in these enzyme activities has any relation to the immune reactivity of carcinoma-bearing patients. In patients with a performance status of more than 60%, the mean value of the total ATPase activity in lymphocytes differed little from that in controls, but the mean value of the oligomycin-sensitive ATPase activity decreased as compared to that in the controls. On the other hand, the mean values of the activities of both free and total acid phosphatase in lymphocytes increased as compared to those in controls. The mean values of the activities of both ATPase and acid phosphatase in lymphocytes from patients whose performance status was less than 50% decreased as compared to those from controls and patients whose performance status was more than 60%. The change of the activities of both ATPase and acid phosphatase in lymphocytes has relation to that of the immunological parameters of the patients with carcinoma of the gastrointestinal tract. These results indicate that both ATPase and acid phosphatase in lymphocytes may play an important role in the immune mechanism.     

A preliminary study of elevated alkaline phosphatase and cathepsin in bronchial aspirates of patients with lung cancer and bronchitis.

The measurement of acid and alkaline phosphatase and cathepsin activities in bronchial aspirates obtained through bronchoscopy from a series of 75 patients suggests a procedure that may have value as a routine diagnostic examination. Using this approach, seven patients with neoplasms in the lung showed elevated levels of alkaline phosphatase and cathepsin in bronchial aspirates without elevation in acid phosphatase activity.     

Histochemical study of enzyme activity of the exocrine and endocrine pancreas at different glucocorticoid levels in animal body

A comparative study was made of the activity of nonspecific phosphatases and lactate dehydrogenases, succinate and NADH in exocrine and endocrine pancreas in excess and deficiency of body glucocorticoids. SDH, NADH-DH and LDH activity in pancreatic acinar cells was shown to be higher than that in the endocrine epithelium but significantly lower than the activity of nonspecific phosphatases, acid phosphatase being the predominant enzyme of B-insulocytes and alkaline phosphatase the predominant enzyme of A-insulocytes. A stimulating effect of hydrocortisone physiological doses on pancreatic secretory activity was accompanied by the enhanced activity of nonspecific phosphatases and enzymes of the Krebs cycle in the exocrine epithelium and acid phosphatase, NADH-DH in B-insulocytes. Large hydrocortisone doses as well as hormonal balance deficiency resulted in a decrease in the energy potential of acinar and endocrine cells.     

Purification and properties of branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney.

Branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase was purified about 8000-fold from extracts of bovine kidney mitochondria. The highly purified phosphatase exhibited a molecular weight of approximately 460,000, as estimated by gel-permeation chromatography. Another form of the phosphatase, with an apparent molecular weight of approximately 230,000, was also detected under conditions of high dilution. In contrast to pyruvate dehydrogenase phosphatase, BCKDH phosphatase was active in the absence of divalent cations. BCKDH phosphatase was inactive toward 32P-labeled phosphorylase a, but exhibited approximately 10% maximal activity with 32P-labeled pyruvate dehydrogenase complex. BCKDH phosphatase activity was inhibited by GTP, GDP, ATP, ADP, UTP, UDP, CTP, and CDP. Half-maximal inhibition occurred at about 60, 200, 200, 400, 100, 250, 250, and 400 microM, respectively. These inhibitions were reversed completely by 2 mM Mg2+. GTP was replaceable by guanosine 5'-(beta, gamma-imido)triphosphate. GMP, AMP, UMP, CMP, NAD, and NADH showed little effect, if any, on BCKDH phosphatase activity at concentrations up to 1 mM. Heparin showed half-maximal inhibition at 2 micrograms/ml. This inhibition was only partially (30%) reversed by 2 mM Mg2+. CoA and various acyl-CoA compounds exhibited half-maximal inhibition at 150-300 microM. These inhibitions were not reversed by 2 mM Mg2+. BCKDH phosphatase activity was stimulated 1.5- to 3-fold by protamine, poly(L-lysine), and poly(L-arginine) at 3.6 micrograms/ml.     

The enzymatic activities and NBT reduction test of granulocytes in untreated and dialysed uraemic patients.

It was found that neutrophils in untreated uraemic patients as well as in subjects on regular dialysis treatment displayed higher activity of acid phosphatase, alkaline phosphatase and peroxidase. Spontaneous reduction of nitro blue tetrazolium (NBT) by granulocytes was also higher in both groups in comparison to controls. Stimulation with latex particles gave similar results of NBT reduction in investigated patients and controls. Lymphocytes also showed an increase in acid phosphatase activity if compared to healthy persons. It seems possible that granulocytes which take part in unspecific defense mechanisms are more active in uraemic patients due perhaps to subclinical infections.     

Iodinated particles in the rat thyroid. II. Lysosomal characteristics.

In a previous study (Simon et al. 1971) a procedure for the preparation and separation of iodinated particles was described in the rat. The present paper deals with further investigations on the nature of these particles. Acid phosphatase and iodine are conjointly sedimentable and display a latency that is unmasked on dilution in a hypo-osmotic medium and under acidification to pH 5.0. These properties together with the sensitivity to Triton X-100 are best accounted for by assuming that iodinated particles of the thyroid gland are lysosomes. Part of the particulate iodine is soluble in n-butanol (BEI fraction). The existence of this BEI fraction demonstrates that hydrolysis of thyroglobulin occurs within the particles which thus exhibit an acid protease activity. Both the sedimentable iodine pool and acid phosphatase are increased under TSH stimulation and decreased after thyroxine treatment. In addition, the general activity of the iodinated particles is dependent on the daily iodine intake as shown by the variation of their iodine pool, acid phosphatase activity and BEI fraction with the iodine diet. It is concluded that iodinated particles of the thyroid gland are secondary lysosomes which participate in iodine secretion under TSH control. By in vitro treatment with destabilizing media or after in vivo treatment with thyroxine, iodinated particles exhibit a parallel loss of iodine and acid phosphatase. After a short-term TSH treatment in vivo, their iodine pool is more increased than their acid phosphatase activity. It is concluded that, at least in the normal rat thyroid, iodinated particles are essentially secondary lysosomes; true colloid droplets actually accumulate only after sufficient TSH stimulation.     

A single domain of human prostatic acid phosphatase shows antibody-mediated restoration of catalytic activity.

By limited proteolysis with mouse submaxillaris protease, human prostatic acid phosphatase (EC 3.1.3.2) was cleaved into three fragments, Sp1, Sp2, and Sp3, which individually had no enzymatic activity. One of the fragments, Sp3, regained enzymatic activity after interaction with rabbit antibody to prostatic acid phosphatase. The Sp3 fragment was purified and characterized as to its molecular weight, amino acid composition, and carbohydrate content. The Sp3 fragment behaved like the parent molecule in L(+)-tartrate affinity and in trapping of a phosphoryl intermediate. The same Sp3 fragment also bears the most prominent antigenic determinants. This evidence suggest that Sp3 is the enzymatically active domain of prostatic acid phosphatase.     

Predictors of osteoclast activity in patients with sickle cell disease

Background

Bone changes are common in sickle cell disease, but the pathogenesis is not fully understood. Tartrate-resistant acid phosphatase (TRACP) type 5b is produced by bone-resorbing osteoclasts. In other forms of hemolytic anemia, increased iron stores are associated with osteoporosis. We hypothesized that transfusional iron overload would be associated with increased osteoclast activity in patients with sickle cell disease.

Design and Methods

We examined tartrate-resistant acid phosphatase 5b concentrations in patients with sickle cell disease and normal controls of similar age and sex distribution at steady state. Serum tartrate-resistant acid phosphatase 5b concentration was measured using an immunocapture enzyme assay and plasma concentrations of other cytokines were assayed using the Bio-Plex suspension array system. Tricuspid regurgitation velocity, an indirect measure of systolic pulmonary artery pressure, was determined by echocardiography.

Results

Tartrate-resistant acid phosphatase 5b concentrations were higher in 58 adults with sickle cell disease than in 22 controls (medians of 4.4 versus 2.4 U/L, respectively; P=0.0001). Among the patients with sickle cell disease, tartrate-resistant acid phosphatase 5b independently correlated with blood urea nitrogen (standardized beta=0.40, P=0.003), interleukin-8 (standardized beta=0.30, P=0.020), and chemokine C-C motif ligand 5 (standardized beta=−0.28, P=0.031) concentrations, but not with serum ferritin concentration. Frequent blood transfusions (>10 units in life time) were not associated with higher tartrate-resistant acid phosphatase 5b levels in multivariate analysis. There were strong correlations among tartrate-resistant acid phosphatase 5b, alkaline phosphatase and tricuspid regurgitation velocity (r>0.35, P<0.001).

Conclusions

Patients with sickle cell disease have increased osteoclast activity as reflected by serum tartrate-resistant acid phosphatase 5b concentrations. Our results may support a potential role of inflammation rather than increased iron stores in stimulating osteoclast activity in sickle cell disease. The positive relationships among tartrate-resistant acid phosphatase 5b, alkaline phosphatase and tricuspid regurgitation velocity raise the possibility of a common pathway in the pulmonary and bone complications of sickle cell disease.     

Acid phosphatase activity of the brain in SHR-- the first report of enzyme histochemical studies of spontaneously hypertensive rat brain.

Changes in acid phosphatase activity in the cerebrovascular system and brain parenchyma in SHR were investigated histochemically. An increased activity of the enzyme was demonstrated in the SHR endothelial and medial smooth muscle cells of the cerebral arterial system as compared to the control. The pericytes of intraparenchymal blood vessels also showed an intensified enzyme activity. The enzyme activity increased with advancing age. In SHR brain parenchyma, the enzyme activity was decreased in the cortical nerve cells. Glial cells with the enzyme activity were increased in number and showed an intensified activity. Causative factors of changes in acid phosphatase activity in SHR cerebral arteries and parenchyma were discussed.     

Appearance of Hydrolase Rich Granules in Human Lymphocytes Induced by Phytohemagglutinin and Antigens

Purified human peripheral blood lymphocytes have been shown to developacid hydrolase-rich granules between 24 and 48 hours after stimulation byphytohemagglutinin, prior to mitosis. This increase has been measured biochemically as a net increase in total activity of the lysosomal enzymes: acid-glycerophosphatase, acid phenolpthalein phosphatase, and aryl sulfatase. Thesubcellular localization of acid hydrolytic enzyme activities has been investigated, and they have been shown to be concentrated in a large granulefraction of sucrose homogenates and to behave as if they were membrane-bounded, in that their activity could be released by lysolecithin. It has alsobeen demonstrated by histochemical technics that stimulation of lymphocytesby antigen (PPD) and by streptolysin S, as well as by phytohemagglutinin,produced an increase in acid phosphatase activity. Chloroquine, an inhibitorof the response to phytohemagglutinin, has also been shown to inhibit thedevelopment of acid phosphatase activity. These results are interpreted tosuggest that both specific and nonspecific stimulants of lymphocytes inducelysosome-like structures in premitotic cells.

Submitted on November 1, 1966 Accepted on February 17, 1967