JoséLuis Avila

Studies on human polymorphonuclear leukocyte enzymes. I. Assay of acid hydrolases and other enzymes.

Abstract Eight acid hydrolases, peroxidase (EC 1.11.1.7), cyanide-insensitive NADH oxidase and alkaline phosphatase (EC 3.1.3.1.) were demonstrated in homogenates of human polymorphonuclear leukocytes. The main kinetic characters of these enzymes have been studied and methods for their quantitative assay have been developed. The activities present in human leukocytes are given and compared with those found in rabbit heterophil leukocytes. Homogenates were separated by differential centrifugation into two particulate fractions and a soluble fraction. The influence of the homogenization medium on the distribution of protein, DNA, cyanide-insensitive NADII oxidase and acid β-glycerophosphatase in these fractions was studied.

Studies on human polymorphonuclear leukocyte enzymes. II. Comparative study of the physical properties of primary and specific granules

1. 1. Seven distinct acid hydrolase activities present in cytoplasmic extracts from human polymorphonuclear leukocytes occur in latent form to the extent of 14–30% of their total activity, depending on the enzyme. This latency can be decreased or suppressed by exposure to Triton X-100 or to media of low osmotic pressure, by treatment in a Waring blender, by very acid pH and by freezing and thawing, but not by increasing the substrate concentration in the assay medium up to 16-fold the Michaelis constant of the enzymes. In preparations subjected to graded activating treatments, acid hydrolases are released in closely parallel fashion, suggesting that they are associated with particles possessing similar properties 2. 2. Acid phenylphosphatase and cyanide-insensitive NADH oxidase exhibited no latency under the conditions of the present experiments. It is concluded that, with the exception of acid phenylphosphatase activity, the acid hydrolases studied are associated with lysosome-like particles 3. 3. Alkaline phosphatase (EC 3.1.3.1) is also partly (44%) latent in cytoplasmic extracts of human polymorphonuclear leukocytes. Latent alkaline phosphatase can be released by some of the treatments that suppress the latency of the lysosomal enzymes, but differs from the latter by a complete insensitivity to exposure to media of low osmotic pressure and by an absolute requirement of an ionic media in order to maintain its latency. It is concluded from these results that the alkaline phosphatase-containing granules have some physical properties different from lysosomes.

Action of pyrazolopyrimidine derivatives on American Leishmania spp. promastigotes

The capacity of 54 different pyrazolo-(3,4-d)- or -(4,3-d)-pyrimidine derivatives to inhibit American Leishmania promastigote multiplication was evaluated. Among pyrazolo-(3,4-d)-pyrimidines, eight derivatives showed leishmanistatic activity, 4-aminopyrazolo-(3,4-d)-pyrimidine (APP) being the most active, about eight-fold more than 4-hydroxy-pyrazolo-(3,4-d)-pyrimidine (HPP). 7-Hydroxy-3-beta-D-ribofuranosylpyrazolo-(4,3-d)-pyrimidine (FoB) was as active as 7-amino-3-beta-D-ribofuranosylpyrazolo-(4,3-d)-pyrimidine (FoA), a situation different to that found for pyrazolo-(3,4-d)-pyrimidines. Furthermore, different chemical modifications in formycin structure did not modify inhibitory effects. It can be concluded that regarding American Leishmania the chemical analogy to hypoxanthine or inosine of pyrazolo-(3,4-d)- and pyrazolo-(4,3-d)-pyrimidine, respectively, is not absolutely critical, as different modifications on the heterocyclic ring did not abolish the inhibitory activity of these compounds.

Leishmania species: Comparative ultrastructure of experimental nodules and diffuse human cutaneous lesions in American leishmaniases

Experimental nodules of American leishmaniases were obtained by inoculating 0.1-1 x 10(5) amastigotes into the dorsum of the hindpaws of golden hamsters and of C57Bl/6J mice. The amastigotes were obtained by biopsy of lesions in six human cases of cutaneous leishmaniases and were serially maintained in golden hamsters and in a fetal calf serum-containing medium. Human nodules were obtained by biopsy from several patients with diffuse cutaneous leishmaniases, always prior to treatment. Within the same host species, no ultrastructural differences were seen in the tissue response to isolates of Leishmania mexicana, L. brasiliensis, or L. garnhami, nor were there differences between the host species in response to a particular isolate of the genus Leishmania. The typical inflammatory response was a macrophage granuloma with abundant polymorphonuclear neutrophils, some eosinophils, and plasma cells. Simple human cutaneous leishmanial lesions, as well as experimental nodules in regression, show many fibroblasts, much collagen fiber, but very few parasites. In typical lesions, parasites occurred within macrophage phagolysosomes, within distended lacunar cells, and in the intercellular spaces. Leishmaniae strongly adhered to parasitophorous vacuoles by a site of their plasma membrane directly opposite the flagellum, and the host cell cytoplasm close to the adherence site became highly vacuolated. In most cases the intra- and extracellular parasites show normal morphology, which suggest the inability of phagocytic cells to attack them.

Leukocytic enzyme differences between the clinical forms of malnutrition

Abstract The enzymatic activities of peripheral polymorphonuclear leukocytes obtained by dextran sedimentation from 15 children with both clinical forms of malnutrition were compared to the activity found in normal children. No differences were detected in peroxidase and lysozyme activities between control subjects and children affected with marasmus or kwashiorkor. However, nitro-blue tetrazolium reduction was found increased in marasmus (P

Differences in allopurinol and 4-aminopyrazolo(3,4-d) pyrimidine metabolism in drug-sensitive and insensitive strains of Trypanosoma cruzi

Most freshly isolated Trypanosoma cruzi blood trypomastigotes were insensitive to allopurinol (HPP) and 4-aminopyrazolo(3,4-d)pyrimidine (APP). Strains EP and Ya were, however, strongly inhibited by both drugs while strains DS and A-35 were HPP-insensitive but APP-sensitive. In contrast, epimastigotes resulting from one in vitro passage of all eleven T. cruzi strains were highly sensitive to both drugs. While hypoxanthine/guanine and adenine phosphoribosyltransferase and succino-AMP synthetase activities were similar in trypomastigotes of sensitive and insensitive T. cruzi strains, the uptake and metabolism of [14C]HPP and [14C]APP was significantly slower in T. cruzi trypomastigotes of insensitive strains than in sensitive strains. The results suggest the importance of drug uptake rates in determining the pyrazolopyrimidine sensitivity of different T. cruzi strains.

Studies on human polymorphonuclear leukocyte enzymes IV. Intracellular distribution and properties of α-l-fucosidase

Abstract 1. 1. The location and characteristics of the α- l -fucosidase (EC 3.2.1.-) activity in human polymorphonuclear leukocytes were studied. 2. 2. There was a fairly strong α- l -fucosidase activity in human leukocytes. The pH-activity curve showed a broad peak with highest activity between pH 5.4 and 5.6, using p- nitrophenyl -α- l -fucoside as substrate. The apparent Km and V were 0.168 mM and 149 nmoles/mg protein per h. The temperature optimum was 45 °C. 3. 3. The enzyme was stable during preincubation at 37 °C in 0.1 M acetate buffer pH 5.5, up to 2 h, and was also extremely sensitive to thiol-blocking agents. 4. 4. Despite the use of several different biochemical approaches (gel filtration, thermostability, pH stability and subcellular fractionation) no clear evidence for the presence of several forms of α- l -fucosidase was obtained. 5. 5. In cytoplasmic extracts, about 30% of the activity of α- l -fucosidase is latent, and is unmasked by a number of treatments (detergents, sonication, low osmotic pressure, Waring-Blendor) that unmask other lysosomal enzymes. On the other hand, the distribution pattern of α- l -fucosidase in subcellular fractions of polymorphonuclear leukocytes is closely similar to that reported for other acid hydrolases of this tissue. These results support the concept of an association of α- l -fucosidase with primary granules.

Heterogeneity of acid phosphatase activity in human polymorphonuclear leukocytes

Abstract Evidence is presented indicating the existence of two acid phosphatase activities in human polymorphonuclear leukocytes. There is an acid phosphatase activity, acting preferentially on β-glycerophosphate. This enzyme has a typical lysosomal localization ( p = 1.24) and is strongly inhibited by 1.5 mM (+)-tartrate and 15 mM fluoride, being insensitive to 45 mM alloxan and 0.3 M citrate. The second acid phosphatase acts preferentially on phenyl phosphate and is bound to structures of low density ( p = 1.18). It is strongly activated by 0.3 M citrate and dl -tartrate, and insensitive to 15 mM fluoride. It is also extremely sensitive to 45 mM alloxan and to thiol-blocking agents, and to denaturation by preincubation under varying conditions. It was not possible to demonstrate any difference in K m values, pH effect or response to different substances or ions between the β-glycerophospliatase or phenylphosphatase localized in the granular or supernatant fractions. It is concluded that the soluble acid phosphatase activities are similar to that of the granular fraction and may be derived from ruptured lysosomes or solubilization from other subcellular structures.

Correlation of sinefungin susceptibility and drug-affinity for protein carboxymethyltransferase activity in American Leishmania species

Among promastigotes of 22 different American Leishmania strains, a 5000-fold variation in sinefungin susceptibility was found, apparently independent of their taxonomic classification, although L. mexicana strains did tend to be more resistant than L. braziliensis. Protein carboxymethyltransferase (EC 2.1.1.24) and glycine N-methyltransferase (EC 2.1.1.20) activities were not substantially different in sinefungin-susceptible and -resistant American Leishmania strains. However, when [methyl-3H]methionine incorporation into total protein or gamma-glutamyl residues of leishmanial proteins was carried out in the presence or absence of sinefungin, protein carboxymethylating activity was significantly inhibited only in sinefungin-susceptible Leishmania strains. Furthermore, when protein carboxymethyltransferase activity was purified from several leishmanial strains to a state of electrophoretic homogeneity (sp. act. = 240 nmol h-1 (mg protein)-1), the enzyme from the resistant cells showed a higher inhibition constant (mean Ki 55 microM against 2 microM in susceptible cells) for sinefungin. This 28-times stronger affinity of the susceptible cell enzyme towards sinefungin despite normal protein carboxymethyltransferase specific activity seems to be a key element of the resistance mechanism of certain American Leishmania strains.

Biological action of pyrazolopyrimidine derivatives against Trypanosoma cruzi. Studies in vitro and in vivo

The capacity of 54 different pyrazolo(3,4-d) or (4,3-d)pyrimidine derivatives to inhibit Trypanosoma cruzi epimastigote and trypomastigote multiplication, and for some of them its chemotherapeutic activity, was evaluated. Six pyrazolo(3,4-d)pyrimidines showed inhibitory activity against epimastigote forms, 4-aminopyrazolo(3,4-d)pyrimidine being the most active, 5-fold more so than 4-hydroxypyrazolo(3,4-d)-pyrimidine. Neither compound was active against freshly isolated trypomastigotes, suggesting biochemical differences between culture and bloodstream forms of T. cruzi. On both epimastigote and trypomastigote forms, 7-amino-3-beta-D-ribofuranosylpyrazolo-(4,3-d)pyrimidine (FoA) was about 2-fold more active than 7-hydroxy-3-beta-D-ribofuranosylpyrazolo-(4,3-d)pyrimidine (FoB); however, when tested on T. cruzi-infected mice, only FoB exhibited significant chemotherapeutic activity. Previous results suggest that, except for FoB and FoA: (a) pyrazolopyrimidine insensitivity is trypomastigote-specific and (b) drug-insensitivity is lost when trypomastigotes transform into epimastigotes and vice versa.