Activity of some lysosomal enzymes in peripheral blood lymphocytes of patients with lung cancer. A cytochemical study

In 33 patients with lung cancer (6 women and 27 men, aged at average 61.2 years) the activity and intracellular localization of acid phosphatase, beta-glucuronidase and N-acetyl-beta-glucosaminidase in peripheral blood lymphocytes were determined by means of semiquantitative cytochemical methods. In comparison to the control group of healthy subjects, the patients with lung cancer showed increased counts of acid phosphatase-positive lymphocytes with granular-diffuse cytochemical reaction, increased counts of beta-glucuronidase-positive lymphocytes with solely granular type of reaction and increased numbers of N-acetyl-beta-glucosaminidase-positive cells showing the granular, granular-diffuse and diffuse type of reaction. The total count of beta-glucuronidase-positive and N-acetyl-beta-glucosaminidase-positive lymphocytes was significantly elevated in these patients. The authors discuss the significance of their observations for evaluating lymphocyte response in patients with lung cancer.     

Effect of radiotherapy on the neutrophil and the lymphocyte enzymatic equipment and serum immunoglobulins in patients with cancer of the larynx.

In 30 patients with cancer of the larynx, aged 40 to 70 years, treated by radiotherapy 6 to 9 years ago the decreased activity of neutrophil beta-glucuronidase and N-acetyl-beta-glucosaminidase accompanied by a decrease of absolute count of enzyme-positive cells was noted. Numbers of acid phosphatase-positive neutrophils were also decreased. Moderate increase of the neutrophil alkaline phosphatase activity and of numbers of enzyme-positive cells was another observed feature. The main finding in lymphocytes of the patients consisted in the appearance of cells exhibiting diffusion of lysosomal enzymes, especially of beta-glucuronidase, N-acetyl-beta-glucosaminidase and to a lesser degree of acid phosphatase into the cell cytoplasm. Moderate increase of immunoglobulin level, especially that of IgA, reflected probably the immunologic mobilization of patients.     

The lymphocyte cytochemical equipment and serum immunoglobulins in patients with precancerous states of the larynx.

In 24 men with precancerous states of the larynx, i.e. leukoplakia, papillomas and pachydermia, the peripheral blood lymphocytes were cytochemically stained for N-acetyl-beta-glucosaminidase, beta-glucuronidase, acid phosphatase and glycogen (PAS reaction). The results were expressed in terms of the absolute counts of enzyme- (or compound-) positive cells. The serum immunoglobulin IgG, IgA and IgM levels were also determined by Mancini's method. The results obtained were compared with those in 20 healthy men aged 20 to 30 years. It was found that the patients exhibited elevated numbers of N-acetyl-beta-glucosaminidase- and beta-glucuronidase-positive lymphocytes. A characteristic feature was an increase in the absolute counts of lymphocytes with diffuse and granular-diffuse types of cytochemical reaction for all enzymes studied. The number of cells with the granular type of enzymatic reaction (intact enzyme-positive lysosomes) was significantly diminished. These cytochemical alterations were accompanied by a significant increase in the serum IgA level. These results are discussed with reference to the lymphoid system response to tissues with precancerous lesions of the larynx.     

Intracellular enzymatic response of lymphocytes and neutrophils in patients with cancer of the larynx.

In 20 men, aged 35 to 55 years, with untreated cancer of the larynx activity of lysosomal acid phosphatase (AP), beta-glucuronidase (GR) and N-acetyl-beta-glucosaminidase was determined cytochemically in peripheral blood lymphocytes and neutrophils by means of Barka and Anderson, Hayashi et al. and Hayashi's method, respectively; the results obtained were compared with those in 20 healthy men aged 20 to 30 years. Total count of GR-positive lymphocytes was higher in the patients than in normal persons. Total counts of AP-, GR-, and GS-positive lymphocytes with not disrupted enzyme-positive lysosomal granules within the cell cytoplasm were significantly lower and total counts of cells exhibiting the disruption of lysosomal granules and the diffuse type of cytochemical reaction were significantly higher in the patients when compared with the control group. The response of neutrophils consisted of a significant elevation in numbers of AP-, and GS-positive cells; overall score of enzyme activity studied in neutrophils was not altered in the patients. The authors disucss the significance of their observations in the light of data on participation of lymphocytic and neutrophilic lysosomal apparatus in the immunological response against tumour specific antigen in patients with cancer.     

Change in acid phosphatase and N-acetyl-beta-glucosaminidase activity in mouse lymphocytes induced by concanavalin A

The treatment with concanavalin A (5 micrograms/ml) of mouse lymphocytes containing 70-72% of T cells entails an increase in the activity of acid phosphatase and a decrease in the activity of N-acetyl-beta-glucosaminidase. These changes were detectable 15 h after lymphocyte incubation with Con A. After 24 h of incubation acid phosphatase activity rose 2-fold whereas that of N-acetyl-beta-glucosaminidase dropped 45-50%. Possible mechanisms of these changes are discussed.     

Subcellular fractionation of epithelial cells from toad urinary bladder. 1. Assay of marker enzymes and differential centrifugation

We have undertaken the analytical fractionation of epithelial cells from toad urinary bladder, a tissue extensively used to study epithelial transport of ions and water. In an attempt to establish markers for the main subcellular organelles, a number of enzymes were assayed in cell homogenates. The nearly ubiquitous plasma membrane marker 5'-nucleotidase, and the transferases that donate N-acetylglucosaminyl, galactosyl, and sialyl residues to glycoproteins and glycolipids in the Golgi complex were not detectable. Glucose-6-phosphatase activity was low in relation to that of nonspecific phosphatases and, therefore, not suitable for identifying the endoplasmic reticulum. Like the cytosolic enzyme lactate, dehydrogenase, catalase was essentially found in the high-speed supernatant, with a noteworthy part of aminopeptidase (substrate, leucyl-beta-naphthylamide) and NAD glycohydrolase. Other enzymes, including cytochrome c oxidase, acid phosphatase, acid N-acetyl-beta-glucosaminidase, alkaline phosphatase, alkaline phosphodiesterase I, nucleoside diphosphatase (substrate ADP), oligomycin-resistant Mg++-ATPase, and mannosyltransferase (acceptor, dolichylphosphate) were fairly active and largely sedimentable. After differential centrifugation, cytochrome oxidase, acid phosphatase, and acid N-acetyl-beta-glucosaminidase were typically associated with the large granule fraction, whereas the other sedimentable enzymes exhibited a broad distribution profile overlapping the nuclear, large granule, and microsome fractions. Their behavior in density equilibrium centrifugation is examined in a companion paper.     

The influence of tumor growth on natural cytotoxicity and activity of some lysosomal enzymes of human effector cells and the C3HA mouse splenocytes

In the course of malignant growth processes in patients with lung cancer, a decrease of natural cytotoxic activity of peripheral blood lymphocytes was observed. This process was accompanied by changes of activities of two lysosomal enzymes, arylsulfatase and acid phosphatase, suggesting participation of these enzymes in manifestation of effector functions of lymphocytes in cancer patients. The level of activity of granular enzyme, beta-glucuronidase, remained unchanged at all stages of disease. A study of natural killer activity of C3HA mice splenocytes after inoculation of transplantable hepatoma 22-a cells revealed a relative stability of the level of their cytotoxicity, and of the activities of lysosomal enzymes--arylsulfatase, acid phosphatase, alpha-mannosidase, acid lipase, N-acetyl-beta-D-glucosidase, and beta-galactosidase, beginning from the 3rd day after hepatoma implantation.     

Aging and lymphocyte lysosomes in man

It has been stated in 120 healthy subjects of varying age that last three decades of human life that is between 60 and 90 year of life, the dramatic decrease in numbers of peripheral blood lymphocytes having intact lysosomes containing acid phosphatase, beta-glucuronidase and N-acetyl-beta-glucosaminidase occurs. In contrast, the first five decades of life are characterized by relative stability of enzyme-positive lysosomal apparatus of lymphocytes. Above studies have been performed using semiquantitative cytochemical methods. The authors discuss their results with regard to similar phenomenon of lysosomes disintegration in lymphocytes in patients with malignancies.     

Acid and alkaline phosphatases activities in vascular smooth muscle: species differences and subcellular distribution

1. Acid and alkaline phosphatase activities were studied in rat and dog aortic muscle using p-nitrophenyl phosphate (p-NPP) as the substrate. Alkaline phosphatase activity was quite comparable to acid phosphatase activity in rat aortic microsomes as well as further purified plasma membranes, but considerably lower than acid phosphatase activity in dog aortic membranes. 2. Subcellular distribution of acid and alkaline phosphatase activities in these vascular muscles indicated that alkaline phosphatases and a large portion of acid phosphatase activities were primarily associated with plasma membranes and the distribution of acid phosphatase showed little resemblance to that of N-acetyl-beta-glucosaminidase, a lysosomal marker enzyme. 3. The rat aortic plasmalemmal acid and alkaline phosphatase activities responded very differently to magnesium, fluoride, vanadate and EDTA. The alkaline phosphatase was more susceptible to heat inactivation than acid phosphatase. 4. These results suggest that these two phosphatases are likely to be two different enzymes in the smooth muscle plasma membranes. The implication of the present findings is discussed in relation to the alteration of these phosphatases in hypertensive vascular diseases.     

The effects of chlorhexidine on the maximum specific growth rate, biomass and hydrolytic enzyme production of Bacteroides gingivalis grown in continuous culture

Bacteroides gingivalis was grown in continuous culture in the presence of chlorhexidine. Maximum specific growth rates and biomass levels initially increased but then decreased as the chlorhexidine level increased from 0 to 30 micrograms/ml. Total inhibition of growth occurred when the chlorhexidine concentration reached 60 micrograms/ml. The steady-state levels of cell-bound, extracellular vesicle and extracellular soluble enzymes, trypsin-like protease, alkaline phosphatase and N-acetyl-beta-glucosaminidase were measured. With increasing sub-lethal concentrations of chlorhexidine, levels of alkaline phosphatase increased noticeably in all three fractions of culture, whilst cell-bound and extracellular vesicle levels of N-acetyl-beta-glucosaminidase remained approximately constant. Extracellular soluble levels of alkaline phosphatase and N-acetyl-beta-glucosaminidase increased with increasing levels of chlorhexidine. The levels of trypsin-like protease decreased significantly in all fractions of the culture when cells were grown in the presence of chlorhexidine. Thus, chlorhexidine has a differential effect on the production of B. gingivalis hydrolytic enzymes.     

Subcellular fractionation of epithelial cells from toad urinary bladder. 2. Isopycnic centrifugation and effect of density perturbants

Cytoplasmic granules obtained from toad urinary bladder epithelial cells were brought to buoyancy in a linear sucrose gradient. The gradient was loaded either with untreated cytoplasmic granules, or with granules treated with Na pyrophosphate (PPi), with digitonin, or with PPi and digitonin in succession. The following enzymes were assayed in the gradient subfractions: oligomycin-insensitive Mg++-ATPase, alkaline phosphodiesterase I, alkaline phosphatase, acid N-acetyl-beta-glucosaminidase, cytochrome oxidase, nucleoside diphosphatase (substrate, ADP), aminopeptidase (substrate, leucyl-beta-naphthylamide), and mannosyltransferase (acceptor, dolichylphosphate). Comparison of the density distributions of enzymes in untreated and treated preparations led to the characterization of 4 distinct subcellular entities. In agreement with the properties of mitochondria from other cell types, cytochrome oxidase buoys at 1.18 within a narrow density range and its behavior is not significantly altered by PPi or digitonin. Under all conditions, acid N-acetyl-beta-glucosaminidase is recovered over a broad density range in the lower part of the gradient and appears as a qualified lysosomal marker. Mg++-ATPase, alkaline phosphodiesterase I, and alkaline phosphatase belong to a group with the distinguishing features of a low equilibrium density in native cytoplasmic granules and a marked shift (+0.03 density units) after digitonin treatment. Such properties are typical of the plasma membranes. Part of the aminopeptidase activity probably also belongs to plasma membrane-derived elements. Minor differences between alkaline phosphatase and the other 2 members of that group make it possible that their distribution domains in the membrane do not overlap or coincide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Digestion in the soil predatory mite Pergamasus longicornis (Berlese) (Acari: Mesostigmata: Parasitidae)--detectable hydrolases

Nineteen hydrolytic enzymes were detected in individual adult Pergamasus longicornis (Berlese) mites--amylase, hide protease, alkali phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, phosphoamidase, alpha-galactosidase, beta-galactosidase, beta-glucuronidase, alpha-glucosidase, beta-glucosidase, N-acetyl-beta-glucosaminidase, alpha-mannosidase, and alpha-fucosidase. All but the phosphatases were detected for the first time. Tryptic and chymotryptic activity were consistently not demonstrable. Comparisons are made with saprophagous mites. No clear enzymic specialization for predation was found.     

The distribution of some hydrolases in glomeruli and tubular fragments prepared from rat kidney by zonal centrifugation

1. A collagenase digest of rat kidney cortex was separated into four bands by zonal centrifugation. 2. Two of these bands were shown by light-microscopy to contain glomeruli and tubular fragments, which were free from each other and well separated from other renal material. 3. Protein, N-acetyl-beta-glucosaminidase, 5'-nucleotidase, l-leucine beta-naphthylamidase, leucine aminopeptidase, acid phosphatase and alkaline phosphatase were assayed across the gradient. 4. The greater proportion of these enzyme activities was recovered in the tubular fragments and acid phosphatase was the only enzyme detected in significant amounts in the glomeruli. 5. Tubular fragments and glomeruli were sedimented and multiple forms of beta-naphthylamidase, N-acetyl-beta-glucosaminidase, acid phosphatase and alkaline phosphatase were investigated by starch-gel electrophoresis.     

Semiquantitative cytochemical determination of acid phosphatase, beta-glucuronidase, and beta-glucosaminidase activity in peripheral blood lymphocytes.

Activity of acid phosphatase, beta-glucuronidase, and beta-glucosaminidase in peripheral blood lymphocytes was determined cytochemically in 20 normal subjects, 10 male and 10 female, by the use of BARKA and ANDERSON's (1962), HAYASHI et al. (1964) and HAYASHI's (1965) methods, respectively. Results obtained were semiquantitatively according to subdivision of lymphocytes into enzyme-negative and enzyme-positive cells. Enzyme-positive lymphocytes were divided into cells with granular, mixed granular and diffuse enzymatic reaction type. In the first two types of cytochemical reaction a number of enzyme-positive lysosomal granules were counted and expressed in terms of both absolute count and percentage of circulating lymphocytes. Enzyme-positive lymphocytes represented 80.3%, 40.5% and 41.5% of the total lymphocyte count in regard to the presence of acid phosphatase, beta-glucuronidase, and beta-glucosaminidase, respectively.     

Activity of acid phosphatase and succinate and dehydroorotate dehydrogenases during the interaction of allogeneic lymphocytes and target cells in tissue culture]

A study was made of the activity of acid phosphatase in interaction of lymphocytes and target cells; it was shown that in the lymphocytes its greatest increase occurred in the 1- and sometimes in the 3-hour cultures; sometimes the activity of the enzyme in the L-cells increased by the 3rd-6th hour. The initial activity of acid phosphatase was greater in the immune lymphocytes; later, on their addition to the L-cell culture, there was no significant difference in the changes in the activity of the enzyme in the immune and normal lymphocytes. In the L-cells the activity of acid phosphatase was lower on addition of normal lymphocytes in comparison with the immune ones. The activity of dehydrogenases in the lymphocytes increased by the 3rd hour of the incubation period and was greater in the immune lymphocytes than in the normal ones. The activity of the succininc- and dehydroorotic dehydrogenases altered practically at the same periods in the L-cells as in the lymphocytes. The intensification of the activity of the redox enzymes and of the acid phosphatase during the first hours of contact pointed to the rapid activation of the effector lymphocytes.     

Intracellular transport and degradation of 125I-tyramine cellobiose-labelled low-density lipoprotein endocytosed in vivo in rat liver cells studied by means of subcellular fractionation

The intracellular transport and degradation of in vivo endocytosed 125I-tyramine cellobiose-labelled low density lipoprotein (125I-TC-LDL) in rat liver cells were studied by means of subcellular fractionation in Nycodenz, sucrose and Percoll density gradients, as well as by means of analytical differential centrifugation. Initially, labelled LDL was located in endocytic vesicles of low densities. Subsequently, acid-soluble and acid-precipitable radioactivities were found in organelles with buoyant densities distinctly lower than that of the main peaks of the lysosomal marker enzymes acid phosphatase and N-acetyl-beta-glucosaminidase. These prelysosomal organelles may represent multivesicular bodies (MVBs). Finally, 6 h after injection and onwards, the acid-soluble radioactivity cosegregated completely with the two lysosomal marker enzymes, suggesting that the degradation products were in secondary lysosomes. The rate of intracellular processing of LDL was very slow compared to that of asialoglycoproteins, suggesting that LDL followed a unique intracellular pathway, that may be specific for this type of ligand.     

Studies on the nephrotoxicity of p-nitrophenylarsonic acid: changes in rat kidney and urinary enzyme activities following the administration of p-nitrophenylarsonic acid.

Histological studies showed that the administration of p-nitrophenylarsonic acid to rats resulted in renal tubular necrosis. The nephrotoxin was administered intraperitoneally and doses greater than 30 mg/kg were found to be fatal. The severity of the renal lesion depended on the amount of the nephrotoxin used. Elevated serum urea levels, urinary protein and volume were recorded over an 8-day period following the injection of the nephrotoxin. These changes were paralleled by an increase in the activity of lactate dehydrogenase, acid and alkaline phosphatase, N-acetyl-beta-glucosaminidase and beta-glucosidase in the urine. beta-Glycosidase activities increased in kidney homogenates, immediately after the injection of the nephrotoxin, but this eventually fell to well below the normal range. Subcellular fractions were prepared from sucrose homogenates by differential centrifugation and beta-glycosidases and cytochrome oxidase were used as enzyme markers. Only minor changes in the activity of cytochrome oxidase activity resulted from the administration of p-nitrophenylarsonic acid. One of the earliest indications of renal damage was a decrease in lysosomal latency. The activities of the lysosomal and soluble enzymes were elevated above normal during the first two days after the injection of p-nitrophenylarsonic acid, but they fell to values, significantly lower than normal, on the third day. The isoenzymic forms of beta-galactosidase, beta-glucosidase and N-acetyl-beta-glucosaminidase in normal and damaged kidneys were studied, using starch gel electrophoresis. The activities of both the lysosomal and the soluble forms of these enzymes decreased following the injection of the nephrotoxin, confirming the results obtained with whole homogenates. The relationship between the changes in renal enzyme activity and urinary enzyme excretion during the nephrotoxic process is discussed.     

Lymphocyte and neutrophil cytochemistry in the dynamics of different forms of diphtheria in adults]

The cytochemical study of lymphocytes and neutrophils isolated from fractionated blood of diphtheria patients and carriers has revealed that a decrease in the activity of lymphocyte succinic dehydrogenase and myeloperoxidase can be observed at all periods of the disease and in all its forms. A decrease in the activity of lymphocyte nonspecific esterase has been noted only in patients with toxic and subtoxic diphtheria and a decrease in the activity of neutrophil alkaline phosphatase, in carriers. The analysis of correlations between the parameters of five enzymes under study (lymphocyte succinic dehydrogenase, lymphocyte acid phosphatase, lymphocyte non-specific esterase, myeloperoxidase, neutrophil alkaline phosphatase) and enzymatic rosette parameters has been made. The analysis has revealed an essential increase in the number of correlations in comparison with donors, changes in the qualitative nature of these correlations and sometimes the reversion of the correlations. Carriers have shown the greatest number of correlations. By the end of the terms of observations no restoration of normal correlations has been observed.     

Acid and neutral hydrolases in Trypanosoma cruzi. Characterization and assay.

Twelve acid hydrolases, 4 near-neutral hydrolases, and alkaline phosphatase were demonstrated in 0.34 M sucrose homogenates of Trypanosoma cruzi strain Y: p-nitrophenylphosphatase and alpha-naphthylphosphatase, with optimum pH at approximately 6.0; alpha=ga;actpsodase. beta=ga;actpsodase. beta=g;icpsodase, N-acetyl-beta-glucosaminidase, cathepsin A and peptidase I and III, with optimum pH between 5.0 and 6.0; and arylsulfatase, cathepsin D, alpha-arabinase and alpha-mannosidase with optimum pH at approximately 4.0. alpha-Glucosidase, glucose-6-phosphatase and peptidase II had optimum pH at approximately 7.0. beta-Glycerophosphatase had a broad pH-activity curve from 4,0 to 7.4, with maximum activity at pH 7.0. The main kinetic characteristics of these enzymes and their quantitative assay methods were studied. No activity was detected for alpha-fucosidase, beta-xylosidase, beta-glucuronidase, elaidate esterase, acid lipase, and alkaline phosphodiesterase.     

The salubrious effect of tamaxifen on serum marker enzymes,glycoproteins, and lysosomal enzymes level in breast cancer women

Tumour markers correlate strongly with prognosis based on tumour burden and surgical resectability. If chemotherapy is extremely effective in certain stage of the disease, the sensitive marker may be of great use in monitoring disease response and drug treatment. Hence, this study was launched to evaluate the changes in tumour marker enzymes like lactate dehydrogenase (LDH), glumate oxaloacetate transaminase (SGOT), glutamate pyruvate transaminase (SGPT), alkaline phosphatase, and acid phosphatase in before and after 3 and 6 months tamoxifen treated breast cancer patients. In addition, the changes in serum glycoproteins viz., hexose, hexosamine, and sialic acid and lysosomal enzymes such as N-acetyl-beta-D-glucosaminidase, beta-D-galactosidase, and beta-D-glucuronidase were analysed in these patients. These values were compared with their age matched healthy control subjects. At 6 months evaluation, the tamoxifen treated postmenopausal breast cancer women showed a statistically significant decreased (p < 0.001, 0.05 respectively) levels of LDH, SGOT, SGPT, alkaline and acid phosphatases than their baseline values. Similarly, the levels of hexose, hexosamine, and sialic acid and N-acetyl-beta-D-glucosaminidase, beta-D-galactosidase, and beta-D-glucuronidase were decreased significantly (p < 0.001 ) in tamoxifen received postmenopausal women. The result of this study suggested that tamoxifen potentially retard the metastasis of breast cancer as well as the bone demineralisation in postmenopausal breast cancer women. Thus, tamoxifen may also have its antitumour activity through its beneficial effects on tumour marker enzymes and serum proteins in breast cancer women.