Investigation of α-naphthyl acetate esterase and acid phosphatase in the peripheral blood leukocytes of greyhounds

The purpose of our study was to determine the percentages of α-naphthyl acetate esterase (ANAE)- and acid phosphatase (ACP)-positive peripheral blood lymphocytes (PBL), the presence of the ANAE and ACP enzymes in leukocytes, and the proportion of PBL in greyhounds. Peripheral blood samples were collected from the cephalic antebrachial vein of 14 (7 animals of each sex) healthy 1-2-year-old greyhounds. Mean percentages of ANAE-positive PBL were found to be 73.29 ± 0.95% in female and 74.29 ± 2.21% in male dogs. The difference between mean values of the genders was not statistically significant. The ACP values were 36.00 ± 2.94% for females and 33.57 ± 2.15% for males. No significant differences were found with regard to gender. For both enzymes, although monocytes and eosinophilic granulocytes displayed a positive reaction, neutrophils gave negative reactions. The proportion of PBL was 36.29 ± 5.31% and 33.00 ± 2.38 % in female and male dogs, respectively. The differences were not significant.     

Investigation of α-naphthyl acetate esterase and acid phosphatase in the peripheral blood leukocytes of greyhounds

The purpose of our study was to determine the percentages of α-naphthyl acetate esterase (ANAE)- and acid phosphatase (ACP)-positive peripheral blood lymphocytes (PBL), the presence of the ANAE and ACP enzymes in leukocytes, and the proportion of PBL in greyhounds. Peripheral blood samples were collected from the cephalic antebrachial vein of 14 (7 animals of each sex) healthy 1-2-year-old greyhounds. Mean percentages of ANAE-positive PBL were found to be 73.29 ± 0.95% in female and 74.29 ± 2.21% in male dogs. The difference between mean values of the genders was not statistically significant. The ACP values were 36.00 ± 2.94% for females and 33.57 ± 2.15% for males. No significant differences were found with regard to gender. For both enzymes, although monocytes and eosinophilic granulocytes displayed a positive reaction, neutrophils gave negative reactions. The proportion of PBL was 36.29 ± 5.31% and 33.00 ± 2.38 % in female and male dogs, respectively. The differences were not significant.     

Determination of acid α-naphthyl acetate esterase enzyme activity in peripheral blood leukocytes of gazelles (Gazella subgutturosa)

We examined gazelle peripheral blood leucocytes using the α-Naphthyl acetate esterase (ANAE) staining technique (pH 5.8). Our purpose was to determine the percentage of ANAE positive lymphocytes. The proportion of ANAE positive T-lymphocytes was 72%. T-lymphocytes showed an ANAE positive reaction, but eosinophilic granulocytes and monocytes also showed a positive reaction. By contrast, no reaction was detected in B-lymphocytes, neutrophil granulocytes or platelets. The reaction observed in T-lymphocytes was a red-brown coloration, usually 1–2 granules, but enough granules to fill the cytoplasm were detected rarely. As a result of ANAE enzyme staining, we concluded that the staining technique can be used as a cytochemical marker for gazelle T-lymphocytes.     

SH groups in the α-naphthyl acetate esterase in the thyroid of the guinea-pig

Summary The -naphthyl acetate esterase in both group I and group II thyroid cells is shown to contain SH groups since there is a decline in activity in both cell groups when certain sulfhydryl reagents [DTNB; 5,5-Dithiobis-(2-nitrobenzoic acid)-AgNO₃-Mersalyl-PCMB (parachloro mercuribenzoate)+urea] are added to the incubation media. Thus the inhibition is by far the greatest in group I cells, which also show the greatest activity after incubation in conventional media, when long fixation and storage times are used. In all cases the inhibiting effect was complete or almost completely reversed if cysteine was added to the incubation media in equivalent concentrations to the SH blocker. There were great differences among the sulfhydryl reagents used in their ability to bring about enzyme inhibition. The alkylating agents NEM (N-ethylmaleimide) and iodoacetamide had no or little effect while PCMB could only inhibit the activity of the -naphthylacetate esterase if the enzyme was denaturated with 5 m urea. The maximal inhibitory effect of PCMB was only obtained when NaCl was added to the incubation media. The most effective inhibitor was AgNO₃.     

Enhancement of the antibody-dependent cellular cytotoxicity of human peripheral blood lymphocytes with interleukin-2 and interferon α

Antibody-dependent cellular cytotoxicity (ADCC) is regarded as an important mechanism by which monoclonal antibodies (mAb) can exert an antitumour effect in vivo. It may be possible, therefore, to enhance the therapeutic efficacy of mAb by cytokines that are able to enhance the ADCC of human CD3^–, CD56⁺, CD16⁺ natural killer (NK) cells. We investigated in vitro the effects of recombinant interferon (rIFN) and recombinant interleukin 2 (rIL-2), alone or in combination, on the ADCC of human peripheral blood NK cells. Both cytokines enhanced the ADCC of the human effector cells. rIFN induced a maximally increased ADCC after an exposure of human effector cells to 20 IU/ml for 15–30 min, while rIL-2 induced optimal ADCC after incubation of the cells for 2 days in 20–50 U/ml. We now show that activation of the NK cells with a combination of rIL-2and rIFN induced significantly higher levels of ADCC than either cytokine alone. The highest ADCC was induced if the cells were first exposed to rIL-2 before rIFN was added to the culture. Culture of NK cells in medium or rIL-2 decreased the expression of FcRIII (CD16), indicating that intensity of CD16 expression and level of ADCC are not directly correlated, although blocking experiments with a mAb directed against CD16 showed that this FcR was essential for ADCC of the human effector cells.Supported by a grant from the Dutch Cancer Society (grant NKI-84-14)     

Exploring the thermal stability and activity of α-chymotrypsin in the presence of spermine

Polyamines such as spermine can have interaction with protein. The aim of the present study was to investigate how spermine could influence the structure, thermal stability, and the activity of α-chymotrypsin. Kinetics, thermodynamics, molecular dynamics (MD), and docking simulations studies were conducted to investigate the effect of spermine on the activity and structure of α-Chymotrypsin (α-Chy) in 50 mM Tris–HCl buffer, with the pH 8, using different spectroscopic techniques as well as molecular docking and MD simulations. The stability and activity of α-Chy were increased in the presence of spermine. The results of the kinetic study showed that the activity of spermine was increased. Enzyme activation was accompanied by changes on the α-Chy conformation. Fluorescence intensity changes showed dynamic quenching during spermine binding. The fluorescence quenching of the α-Chy suggested the more polar location of Trp residues. Near-UV and Far-UV circular dichroism studies also demonstrated the transfer of Trp, Phe, and Tyr residues to a more flexible environment. The increase in the absorption of α-Chy in the presence of spermine was as a result of the formation of spermine–α-Chy complex. Molecular docking results revealed the presence of one binding site with a negative value for the Gibbs free energy of the binding of spermine to α-Chy. Docking study also revealed that van der Waals interactions and hydrogen bonds played a major role in stabilizing the complex.     

Uneven distribution of α-naphthyl acetate in tissues

Summary In the mouse ear in which esterases had been inactivated with warm formol the distribution of -naphthyl acetate was studied by an UV absorption technique. The substrate was found to concentrate in structures containing lipids. The physicochemical properties of substrates used in enzyme chemistry seem to be such that it might prove difficult to find any substrate that would distribute evenly in tissues. So far it does not seem to have been ruled out that this influences enzyme reactions by some mass law effect.

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Zusammenfassung Die Verteilung von -Naphthylazetat wurde mit Hilfe einer UV Absorptionstechnik in einem Mausohr untersucht, in dem die Esterasen mit heißem Formol inaktiviert worden waren. Das Substrat konzentrierte sich in lipoid-haltigen Strukturen. Die physikalisch-chemischen Eigenschaften histochemischer Enzym substratescheinen derartig zu sein, daß es schwer fallen dürfte, ein Substrat zu finden, das sich in den Geweben gleichmäßig verteilen würde. Bis aufs weitere scheint es durchaus nicht ausgeschlossen zu sein, daß dies Enzymreaktionen durch irgendein Massenwirkungsgesetz beeinflußt.

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Aided by a grant from Sigrid Jusélius Foundation.     

Ultrastructural localization of α-galactose-containing glycoconjugates in the rat vomeronasal organ

Binding sites of Griffonia simplicifolia I-B₄ isolectin (GS-I-B₄), which recognizes terminal α-galactose residues of glycoconjugates, were examined in the juxtaluminal region of the rat vomeronasal sensory epithelium and its associated glands of the vomeronasal organ, using a lectin cytochemical technique. Lowicryl K4M-embedded ultra-thin sections, which were treated successively with biotinylated GS-I-B₄ and streptavidin-conjugated 10 nm colloidal gold particles, were observed under a transmission electron microscope. Colloidal gold particles, which reflect the presence of terminal α-galactose-containing glycoconjugates, were present in vomeronasal receptor neurons in the sensory epithelium and secretory granules of acinar cells of associated glands of the epithelium. Quantitative analysis demonstrated that the density of colloidal gold particles associated with sensory cell microvilli that projected from dendritic endings of vomeronasal neurons was considerably higher than that of microvilli that projected from neighboring sustentacular cells. The same was true for the apical cytoplasms of these cells just below the microvilli. These results suggest that of the sensory microvilli and dendritic endings contained a much larger amount of the α-galactose-containing glycoconjugates, compared with those in sustentacular microvilli. Further, biochemical analyses demonstrated several vomeronasal organ-specific glycoproteins with terminal α-galactose.     

Summer and autumn activity patterns in the Vicuña

Airborne input of minerals essential for plant growth is significantly more important for the Amazonian rain forest than elsewhere. Annual rates have been calculated to an average of 26.9 kg/ha/yr for phosphate phosphorus and 12.6 kg/ha/yr for potassium. Other nutrients show a similar amount. If these values are representative for Amazonia as a whole, the total amount of nutrient influx via the air (dry input and precipitation) attains a magnitude, which may be well above the level for assuming a local South American source of origin. Phosphate and potassium alone give an average of 15 to 25 million tons per year. It is concluded, therefore, that dust from the Sahara carried by the trade winds provides the bulk of this mineral input, thus influencing the stability and productivity of the Amazonian rain forest. If this conclusion holds true, the pleistocene forest dynamics in Amazonia should have been influenced not only by changes in the amount of rain fall, but also by the transatlantic transport of nutrients.     

Ultrastructural localization of Interleukin 1 in human peripheral blood monocytes; evidence for IL-1β in mitochondria

Summary The pathway of interleukin 1 (IL-1) secretion from the cell remains unclear. IL-1β is the major form produced by human monocytes, and is synthesized as a precursor of 35kDa which is processed to the extracellular biologically active 17kDa form. We have examined the intracellular localization of IL-1β in lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes, by immunocytochemistry and immunoprecipitation of subcellular fractions. LPS treatment slightly damaged the cells. Unstimulated cells showed very little immunolabelling. In contrast, there was heavy immunolabelling on LPS stimulated cells. Immunolabelling occured within the cytoplasm, nucleus and mitochondria. There was no immunolabelling on the membranous secretory organelles and the plasma membrane. Blebs of cytoplasm budding from the cell surface were immunolabelled, suggesting an alternative route of secretion of IL-1β from the cell. Immunoprecipitation studies confirmed these results.     

Interleukin-15 effectively potentiates the in vitro tumor-specific activity and proliferation of peripheral blood γδT cells isolated from glioblastoma patients

γδT cells play a regulatory role in both primary and metastatic tumor growth in humans. The mechanisms responsible for the activation and proliferation of circulating γδT cells should be fully understood prior to their adoptive transfer to cancer patients. We have examined in vitro functional effects of interleukin-15 (IL-15) on highly purified γδT cells isolated from glioblastoma patients. γδT cells constitutively express the heterotrimeric IL-2 receptor (IL-2R) αβγ, but the levels of IL-2Rβ or γ expression were not increased by incubation with saturating amounts of IL-15. IL-15 was shown to induce a maximal γδT cell proliferation, although at much higher concentrations (at least 2000 U/ml) than IL-2 (100 U/ml). Submaximal concentrations of IL-15 plus low concentrations of IL-2 produced an additive proliferative response. In contrast to the IL-2-induced response, this activity was completely or partially abrogated by anti-IL-2Rβ, or anti-IL-2Rγ antibodies, but not by anti-IL-2Rα antibodies. Incubation of γδT cells in the presence of IL-15 resulted not only in the appearance of NK and LAK activity, but also in specific autologous tumor cell killing activity, an additive effect being seen with IL-15 and IL-2. This IL-15-induced tumor-specific activity could be significantly blocked by anti-IL-2Rγ and anti-IL-2R-β mAb, but not by anti-IL-2Rα mAb. Thus, in contrast to IL-2, IL-15 activates tumor-specific γδT cells through the components of IL-2Rβ and IL-2Rγ, but not IL-2Rα. These enhanced in vitro tumor-specific and proliferative responses of γδT cells seen with IL-15 suggest a rational adjuvant imunotherapeutic use of γδT cells in cancer patients. Received: 23 January 1998 / Accepted: 20 May 1998     

Role of the specifically targeted lysine residues in the glycation dependent loss of chaperone activity of αA- and αB-crystallins

Earlier studies have shown significant loss of chaperone activity in α-crystallin from diabetic lenses. In vitro glycation studies have suggested that glycation of α-crystallin could be the major cause of chaperone activity loss. The following lysine (K) residues in α-crystallin have been identified as the major glycation sites: K11, K78, and K166 in αA-crystallin and K90, K92, and K166 in αB-crystallin. The present study was aimed to assess the contribution of each of the above glycation site in the overall glycation and loss of chaperone activity by mutating them to threonine followed by in vitro glycation with fructose. Level of glycated protein (GP) was determined by phenylboronate affinity chromatography, advanced glycation end products (AGEs) by direct ELISA using anti-AGE polyclonal antibody, and chaperone activity by using alcohol dehydrogenase as the target protein. K11T, K78, and K166T mutants of αA showed 33, 17, and 27% decrease in GP and 32, 18, and 21% decrease in AGEs, respectively, as compared to αA-wt. Likewise, K90T, K92T, K90T/K92T, and K166T mutants of αB showed 18, 21, 29, and 12% decrease in GP and 22, 24, 32, and 16% decrease in AGEs, respectively. Chaperone activity also showed concomitant increase with decreasing glycation and AGEs formation. αA-K11T and αB-K90T/K92T mutants showed the largest decrease in glycation and increase in chaperone activity.     

Ultrastructural localisation of acid phosphatase,non-specific esterase and β-glucuronidase in the midgut epithelium of Tenebrio molitor,Schistocerca gregaria and Carausius morosus

Summary Acid phosphatase, non-specific esterase and -glucuronidase have been localised in the midgut epithelium of three species of insect using naphthol esters as substrates and triphenyl-p-amino-phenethyl lead as coupling salt. In all three species acid phosphatase and -glucuronidase appear to be confined to primary and secondary lysosomes. Non-specific esterase activity was demonstrated within membrane-enclosing bodies in all three species, associated with lipid droplets in T. molitor and C. morosus and with an unidentified intranuclear structure in C. morosus.     

Ultrastructural localization of corticotropin, β-lipotropin,and αand β-endorphin in the porcine anterior pituitary

Summary The peroxidase-antiperoxidase immunocytochemical technique was used to identify the ACTH/endorphin cells in the porcine pituitary at the ultrastructural level and to determine the precise subcellular localization of the pro-ACTH/endorphin fragments. The cells display different aspects: 1) large, regular shapes with numerous and large secretory granules; 2) small, irregular and angular shapes with small granules aligned along the periphery of the cell; and 3) intermediate forms. The presence of and -endorphin not only in the same cells but also in the same secretory granules that contain ACTH and -LPH clearly indicates that both the precursor or its fragments and the abovementioned peptides are stored in the same granules and released simultaneously by the corticotropic cells. The presence of FSH in some corticotropic cells is also discussed.Abbreviations used in this Article ACTH corticotropin - -MSH -melanotropin (ACTH I–I3) - CLIP corticotropin-like intermediate lobe peptide (ACTH 18–39) - -LPH -lipotropin - -MSH -melanotropin (-LPH 41–58); -endorphin (-LPH 61–91); -endorphin (-LPH 61–76)     

Direct observation of the interconversion of normal and toxic forms of α-synuclein

Here, we use single-molecule techniques to study the aggregation of α-synuclein, the protein whose misfolding and deposition is associated with Parkinson's disease. We identify a conformational change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads ultimately to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added soluble protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, hence acting as a source of cytotoxic species.     

Assessment of time patterns of activity and rest in full-shift recordings of trapezius muscle activity – Effects of the data processing procedure

The purpose of this paper was to compare the effects of different data reduction procedures on the values of variables characterizing the time pattern of trapezius muscle activity during full work shifts. Surface electromyography (EMG) of the right and left upper trapezius muscles were obtained from 40 young subjects in different occupations, mainly electricians, hairdressers and students. The target EMG variables were gap frequency, muscle rest, and the number and duration of episodes with sustained muscle activity (from 0.13 s to 30 min as minimum duration). These variables were derived from the EMG recordings using different Root Mean Square (RMS) windows (from 0.13 to 6.38 s), and discrimination levels between “activity” and “rest” (0.5%, 1% and 2% of maximal EMG).The results give basis for practical suggestions for EMG analyses of full work shifts. For most variables, a discrimination level of 0.5% EMG_max showed to be preferable. The time proportion of muscle rest and sustained muscle activity should, in general, be preferred over the corresponding frequency measures. Sustained muscle activity should be calculated using a RMS window between 1 and 3 s, and preferably be stated in terms of variables describing time proportions of activity. Uninterrupted activity episodes longer than 10 min proved not to be a useful variable due to limited occurrence in many work shifts.     

17-β estradiol in the acetylcholinesterase activity and lipid peroxidation in the brain and blood of ovariectomized adult and middle-aged rats

AimsTo investigate the 17-β estradiol in the acetylcholinesterase activity and lipid peroxidation in the brain and blood of ovariectomized rats of different ages.Main methodsAnimals were randomly assigned into three experimental groups of each age (n = 6). Control groups consisted of adult (sham-A) and middle-aged (sham-MA) female rats, ovariectomized adult (OVX-A) and middle-aged (OVX-MA) rats without estrogen therapy reposition, and ovariectomized adult (OVX + E2-A) and middle-aged (OVX + E2-MA) rats treated with 17-β estradiol for 30 days. After this period, AChE activity and lipid peroxidation were measured in the brain and blood.Key findingsThe AChE activity increased (p < 0.05) in striatum (ST) in OVX-A, OVX + E2-A and OVX-MA, and hippocampus (HP) in OVX-MA. The enzyme activity decreased (p < 0.05) in ST of OVX + E2-MA, and cerebral cortex (CC) in OVX + E2-A, OVX-MA and OVX + E2-MA. Blood AChE activity increased (p < 0.05) in OVX + E2-A and decreased (p < 0.05) in OVX-MA. Lymphocyte AChE activity increased (p < 0.05) in OVX-A and OVX + E2-A and decreased (p < 0.05) in OVX-MA. Lipid peroxidation increased (p < 0.05) in ST of OVX-A, CC of OVX-A and OVX-MA, HP of OVX-A, and cerebellum (CE) of OVX-A, OVX-MA, and OVX + E2-MA. Lipid peroxidation decreased (p < 0.05) in ST, CC and CE of OVX + E2-A, and ST and HP of OVX + E2-MA. Similar values of lipid peroxidation to control groups were found in ST and HP of OVX-MA, HP of OVX + E2-A and CC of OVX + E2-MA.Significance17-β estradiol is able to modulate the AChE activity and non-neuronal cholinergic response as well as to reduce lipid peroxidation. Its response is dependent on the age and brain structure analyzed.     

Ultrastructural localization of amyloid β/A4 protein precursor in the normal rat brain

The ultrastructural localization of amyloidβ/ A4 protein precursor (APP) was studied immunohistochemically in normal rat brains using antibodies against different portions of APP. In cerebral cortical neurons and Purkinje cells, APP reaction products were located in the cytoplasm and on cell surface membranes. Some Golgi apparatuses and rough endoplasmic reticulum also showed APP immunoreactivity on their membranes and some vesicles near the trans face of the Golgi apparatuses were stained. In the neuropil of the cerebral cortex and the cerebellar molecular layer, many cell processes, which surrounded synapses and were considered to be astrocytic, were APP-positive. Foot processes around capillaries and subpial astrocytic processes were also immuno-positive. At the ultrastructural level, APP-positive astrocytic processes were identified.