Alpha-naphthyl Butyrate Research Articles

Morphologic and Cytochemical Characteristics of the Blood Cells of the Yellow-Bellied Slider (Trachemys scripta scripta).

The increasing prevalence of yellow-bellied sliders (Trachemys scripta scripta) as pets in the European Union and also its utilization as animal models for experimental purposes makes crucial an accurate classification of their blood cells. The aim of this work was to provide a morphologic classification based on the cytochemical characteristics of the blood cells of 15 yellow-bellied sliders. Cytochemical stains included benzidine peroxidase, chloroacetate esterase, alpha-naphthyl butyrate esterase (with and without sodium fluoride), acid phosphatase (with and without tartaric acid), Sudan black B, periodic acid-Schiff and toluidine blue. Nuclear and cellular dimensions were also measured based on quick Romanowsky-type stained smears. Besides erythrocytes and thrombocytes, five types of white blood cells were identified: heterophils, eosinophils, basophils, lymphocytes and monocytes. The cytochemical patterns of heterophils, eosinophils and basophils were unique compared to those described for other chelonians. This paper provides a useful guideline for clinical settings and further haematological studies of this species.

C5 variant is associated with decreased risk of coronary artery disease in outpatients with severe hypercholinesterasemia.

Although the C5 variant of cholinesterase is known to be a cause of hypercholinesterasemia, the pathophysiological significance of the C5 variant and the C5 variant-related hypercholinesterasemia in cardiovascular diseases remain unclear. The present study aimed to clarify the pathophysiological significance of the C5 variant as a risk or protective factor for coronary artery disease (CAD) in patients with severe hypercholinesterasemia. Severe hypercholinesterasemia was defined as serum cholinesterase (ChE) activity >= 450 IU/L (>= 2.0 SD). We screened 11,648 consecutive outpatients between 2005 and 2011 at Toho University, Sakura Medical Center. In patients with severe hypercholinesterasemia, phenotyping of the C5 variant was conducted using polyacrylamide gel electrophoresis and alpha-naphthyl butyrate staining. 157 subjects (1.4% of 11,648 outpatients screened) were diagnosed with severe hypercholinesterasemia (mean serum ChE activity 574 ± 109 IU/L), and the frequency of the C5 variant was 45.2%. Subjects with the C5 variant had higher age, lower body mass index, milder dyslipidemia and liver dysfunction, and lower rates of hypertension and CAD compared with subjects without the C5 variant. Multivariate logistic regression model demonstrated that the presence of C5 variant independently lowered the risk of CAD, with odds ratio 0.071 (95% confidence interval (CI) 0.007 - 0.763, p = 0.029). The prevalence of the C5 variant was relatively high, and the C5 variant is associated with decreased risk of CAD in outpatients with severe hypercholinesterasemia.

Focal Positivity for Non-Specific Esterase in AML: A Surrogate Marker to Predict AML with t(8;21)/RUNX1-RUNX1T1

Abstract 2534Introduction Cytomorphological evaluation of bone marrow remains a fast and easy accomplished tool in patients with suspected acute myeloid leukemia (AML). Some cytogenetic aberrations can be predicted by specific morphological features like abnormal eosinophils associated with inv(16)/t(16;16) and faggot cells with t(15;17). But implementations of cytogenetic and molecular tests nearly diminished the interest in studying cytomorphological features that might be associated with chromosomal aberrations. Common morphological characteristics often found in AML associated with t(8;21) are large blasts with abundant basophilic cytoplasm, containing numerous azurophilic granules, perinuclear clearing or hofs and single long and sharp Auer rods. But not all AML with t(8;21) show these characteristic signs. During our cytomorphological review for patients with AML within the German prospective multicenter randomized AML96 and AML2003 trials we observed that a focal non-specific esterase stain for blasts in t(8;21) appears to be another specific feature for this subentity. With this study we tried to analyze its predictive value for this chromosomal aberration. Materials and methodsBone marrow (BM) aspirates and peripheral blood smears of 156 eligible patients from the Study Alliance Leukemia (SAL) AML96 and AML2003 trials were investigated by conventional cytochemical methods (May-Giemsa, myeoloperoxidase, alpha-naphthyl butyrate esterase and iron staining). AML classification was performed according to FAB and WHO 2001. Diagnosis of t(8;21)/RUNX1-RUNX1T1 was made by FISH, chromosomal banding and RT-PCR analysis. Bone marrow samples from patients with AML associated with t(8;21)/RUNX1-RUNX1T1 were evaluated and reviewed by an independent and experienced morphologist. Blast morphology was evaluated for granulation, perinuclear clearing or hofs, focal positivity for non-specific esterase (NSE) and the presence of Auer rods, pseudo-Pelger neutrophils and eosinophils. Besides that we investigated its correlation for aberrant expression of CD19 and CD56 in immunophenotyping and the KIT exon-17 mutation. All statistical calculations were performed using the software program PASW statistics 17 (SPSS, Chicago, IL, USA) and R 2.10.1 (The R Foundation for Statistical Computing, 2010). ResultsOf 156 eligible patients, 72 did not have adequate bone marrow samples to assess the amount of blasts with NSE positivity; therefore, the final study cohort consisted of data sets from 84 patients. The median age was 44 years (range, 19–85 years). The distribution of gender was 39 women (46%) and 45 men (54%). The majority of cases (81%) were subclassified in FAB M2. Cytomorphology showed a focal NSE positivity in 82 patients (98% of cases). Chromosome banding analyses were performed in all cases and showed t(8;21) in all cases. Forty-three patients demonstrated additional abnormalities with 29 (35%) cases showing single additional aberrations and 14 (17%) cases having two or more additional aberrations. Focal positivity of non-specific-esterase stain was found in 25% in 11 (13%), 50% in 29 (35%), 75% in 22 (26%) and 100% in 20 (24%) of bone marrow blasts. The presence of focal NSE-positivity demonstrates a significant association with the cytogenetic aberration t(8;21) (p < 0.001). Focal NSE-positivity was present even in the absence of further morphological features associated with t(8;21). No correlation between focal NSE stain and the aberrant expression CD19 and CD56 and KIT-Mutation could be seen. In AML other than t(8;21) no focal positivity for NSE could be seen (n=100). ConclusionOur investigations could prove that focal positivity for NSE is a reliable surrogate marker to predict the chromosomal aberration t(8;21). Disclosures:No relevant conflicts of interest to declare.

Relationship of Insecticide Tolerance to Esterase Enzyme Activity in &lt;I&gt;Aphis pomi&lt;/I&gt; and &lt;I&gt;Aphis spiraecola&lt;/I&gt; (Hemiptera: Aphididae)

Green apple aphid, Aphis pomi De Geer, and Aphis spiraecola Patch (both Hemiptera: Aphididae), are sympatric aphid species that are pests of apples (Malus spp.) and other crops. A. spiraecola has been shown to be significantly more tolerant to several insecticides compared with A. pomi. To establish the mechanisms contributing to this difference in insecticide response, clones of both species were collected from British Columbia, Canada, and Washington state. Dose-response bioassays were conducted to determine relative tolerances to the insecticides pirimicarb, dimethoate, and imidacloprid; these results have been reported previously. Samples of adult aphids from each clone were assayed for the activity of esterase enzymes often involved in the detoxification of insecticides. A. spiraecola had higher esterase activity compared with A. pomi; this was apparent for two model substrates, alpha-naphthyl acetate (alpha-NA) and alpha-naphthyl butyrate (alpha-NB). Aphid clones of both species collected from Washington had higher esterase activity than clones collected from British Columbia. Clones from both species and locations hydrolyzed alpha-NA to a greater extent than alpha-NB. Esterase activity measured with both substrates was significantly positively correlated with the relative response to pirimicarb and dimethoate; a significant positive correlation also was found for hydrolysis of alpha-NB and imidacloprid. The apparent involvement of esterases in the differential response of A. pomi and A. spiraecola to insecticides indicated that the choice of control chemicals for A. spiraecola should not involve chemistries that are metabolized predominantly by esterases.

Carboxylesterase activity, cDNA sequence, and gene expression in malathion susceptible and resistant strains of the cotton aphid, Aphis gossypii

Levels of insecticide resistance, carboxylesterase activity, carboxylesterase expression, and the cDNA sequence of carboxylesterase gene were investigated in malathion resistant and susceptible strains of cotton aphids, Aphis gossypii (Glover). The resistant strain (MRR) exhibited 80.6-fold resistance to malathion compared to the susceptible strain (MSS) in cotton aphids. Five substrates, alpha-naphthyl acetate (alpha-NA), beta-naphthyl acetate (beta-NA), alpha-naphthyl propionate (alpha-NPr), alpha-naphthyl butyrate (alpha-NB), alpha-naphthyl caprylate (alpha-NC) and S-methyl thiobutyrate (S-MTB) were used to determine carboxylesterase activity in MRR and MSS strains of cotton aphids. Carboxylesterase activity was significantly higher in MRR strain than in MSS strain, 3.7-fold for alpha-NA, 3.0-fold for beta-NA, 2.0-fold for alpha-NPr, 2.9-fold for alpha-NB and 1.6-fold for alpha-NC, While for S-MTB, there was nearly no difference between the two strains. Two site mutations (K14Q and N354D) with high frequency were also found by sequence analysis in the MRR strain, compared with the MSS strain. The levels of gene expression for carboxylesterase of both MRR and MSS strains were determined by real-time quantitative PCRs. Compared with the MSS strain, the relative transcription levels and gene copy numbers of the carboxylesterase were 1.99- and 4.42-fold in the MRR strain, respectively. These results indicated that the increased expression of the carboxylesterase resulted from the increased transcription levels of carboxylesterase mRNA and gene copy numbers and combined with the site mutants might play role in cotton aphid resistance to malathion.

Esterase isoenzymes and insecticide resistance in Frankliniella occidentalis populations from the south‐east region of Spain

Frankliniella occidentalis (Pergande) is among the most important crop pests in the south-east region of Spain; its increasing resistance to insecticides constitutes a serious problem, and understanding the mechanisms involved is therefore of great interest. To this end, F. occidentalis populations, collected from the field at different locations in south-east Spain, were studied in terms of total esterase activity and esterase isoenzyme pattern. Individual thrips extracts were analysed by native polyacrylamide gel electrophoresis (PAGE) and stained for esterase activity with the model substrate alpha-naphthyl acetate. Significant correlations were found between resistance to the insecticides acrinathrin and methiocarb and the presence of a group of three intensely stained bands, named Triplet A. For each individual thrips extract, total esterase activity towards the substrates alpha-naphthyl acetate and alpha-naphthyl butyrate was also measured in a microplate reader. Insects possessing Triplet A showed a significantly higher alpha-naphthyl acetate specific activity and alpha-naphthyl acetate/alpha-naphthyl butyrate activity ratio. This observation allowed a reliable classification of susceptible or resistant insects either by PAGE analysis or by total esterase activity determination. The PAGE and microplate assays described can be used as a monitoring technique for detecting acrinathrin- and methiocarb-resistant individuals among F. occidentalis field populations.

Effects of the Homeodomain Gene Pitx2 on Self-Renewal of Hematopoietic Stem Cells.

Pitx2, a homeodomain gene preferentially expressed in murine hematopoietic stem/progenitor cells, is also a downstream target of genes important for hematopoiesis such as MLL and Wnt/Dvl/β-Catenin. We have previously reported that Pitx2 null hematopoietic stem cells (HSCs) can contribute to multi-lineage hematopoiesis under physiologic conditions. We have now carried out serial bone marrow transplantation experiments and demonstrated that after the 3rd round of serial transplantation, Pitx2 null cells reconstituted only 28.6% of the recipient hematopoietic cells as compared to 60% in the case of wild type cells (P<0.001). There were no Pitx2 null donor-derived cells in recipient mice after the 4th round of transplantation, whereas donor-derived chimerism was 57% with wild type cells (P<0.001), and 26% with Pitx2 +/− cells (P<0.001). Therefore, Pitx2 null HSCs have decreased self renewal capacity. To further study the function of Pitx2 in HSC, we constitutively overexpressed the Pitx2 gene in murine bone marrow cells following transduction using a MSCV/IRES/GFP retroviral vector, and analyzed the effects on hematopoiesis in vitro and in vivo. Bone marrow cells overexpressing Pitx2 were isolated on the basis of their GFP expression and analyzed for their colony forming ability in vitro. Retrovirally transduced bone marrow cells were also transplanted into lethally irradiated mice, and the transplanted mice were observed for long-term reconstitution. Colony-forming unit assays showed that Pitx2 overexpressing bone marrow cells, compared to control cells transduced with vector only, had increased numbers of GM colony forming units and reduced numbers of megakaryocytic colony forming units. Pitx2-overexpressing cells continued to form GM colonies after more than eight serial replatings. When these cells were cultured in liquid medium containing SCF, IL-3 and IL-6, they gave rise to cells that stained positively either for alpha naphthyl butyrate, indicating monocytic differentiation, or for peroxidase, indicating neutrophilic differentiation. The ability of these GM-colony forming cells to cause leukemia is currently under investigation. Long-term reconstitution of hematopoiesis in mice by Pitx2 over-expressing HSCs was demonstrated by identifying GFP positive multi-lineage peripheral blood cells four months following transplantation. One of these mice manifested leukemia at this time, as evidenced by a markedly elevated WBC count and other hematologic abnormalities. The leukemic WBCs had very high levels of GFP and Pitx2 expression and were shown to contain two retroviral integration sites, neither of which involved a known oncogene or overexpression of the gene at the integration site. Immunophenotyping by flow cytometry demonstrated that the majority of the leukemic cells were c-kit positive and expressed the megakaryocytic marker CD41, as well as the common myeloid progenitor marker, CD16/32. Some of the cells expressed the erythroid marker Ter119. The leukemic cells did not express any lymphoid markers, including CD3ε, B220, CD19, and IL7R3. This Pitx2-overexpression-associated leukemia was transplantable. Experiments are under way to characterize the leukemia initiating cells. Taken together, our results provide evidence that the homeodomain gene Pitx2 plays a role in the self-renewal of hematopoietic stem/progenitor cells.

Morphologic and cytochemical characteristics of blood cells of juvenile loggerhead sea turtles ( Caretta caretta)

A morphologic classification based on the cytochemical characteristics of blood cells of 35 juvenile loggerhead sea turtles ( Caretta caretta) is described. Cytochemical stains included benzidine peroxidase, chloroacetate esterase, alpha-naphthyl butyrate esterase (with and without sodium fluoride), acid phosphatase (with and without tartaric acid), Sudan black B, periodic acid-Schiff, and toluidine blue. The morphologic characteristics of erythrocytes were similar to those reported in green turtles. Six types of white blood cells were identified: heterophils, eosinophils, basophils, lymphocytes, monocytes and thrombocytes. Except for the basophils, the rest of the white blood cells from loggerhead turtles had different cytochemical characteristics compared to blood cells from other sea turtle species. The leukocyte differential count was different from that reported for other sea turtle species. Heterophils were the most numerous leukocytes from these loggerhead turtles, followed by lymphocytes, eosinophils, monocytes and basophils. This paper provides a morphologic classification of blood cells of loggerhead sea turtles that is useful for veterinary surgeons involved in sea turtle conservation.

Polymorphisms in a Carboxylesterase Gene Between Organophosphate-Resistant and -Susceptible &lt;I&gt;Aphis gossypii&lt;/I&gt; (Homoptera: Aphididae)

Resistance to omethoate was suppressible by the hydrolytic enzyme inhibitor SSS-tributyl phosphorotrithioate in a laboratory-selected resistant cotton aphid, Aphis gossypii Glover, strain, suggesting the involvement of hydrolytic enzymes in the detoxification process. The kinetic properties of carboxylesterases from both resistant and susceptible cotton aphids were characterized by four acyl ester substrates: alpha-naphthyl acetate (alpha-NA), alpha-naphthyl butyrate (alpha-NB), alpha-naphthyl phosphate (alpha-NP), and beta-naphthyl phosphate (beta-NP). No significant differences of carboxylesterase activity were found between resistant and susceptible strains by using either alpha-NP or beta-NP as substrates. In contrast, the susceptible A. gossypii exhibited significantly higher activity compared with resistant aphids with either alpha-NA or alpha-NB as substrates. To understand the molecular basis of this esterase-mediated resistance, carboxylesterase genes from both strains were cloned. Two genes share 99.4% identity at the nucleic acid level and 99.2% identity at the amino acid level. The full length of the cDNA opening reading frame is 1581 bp, encoding 526 amino acids. Four amino acid substitutions, Thr210 --> Met210, Asn294 --> Lys294, Gly408 --> Asp408, and Ser441 --> Phe441, were identified in the resistant strain. Probing of Southern blots with the 0.5 kb esterase fragment showed the same banding patterns and intensities with genomic DNA extracts from both resistant and susceptible A. gossypii. Furthermore, the MspI and HpaII fragments are the same in both strains, indicating there is no methylation of sequences detected by the probe. The combined results suggest that the structural gene substitution is likely the molecular basis of the organophosphate resistance in this laboratory-selected cotton aphid strain.

Morphologic characterization of specific granules in Greyhound eosinophils

"Vacuolated" eosinophils (ie, eosinophils with empty, nonstaining granules) have been described previously in normal Greyhounds. However, to our knowledge, detailed studies of granules in vacuolated and normal eosinophils in this breed have not been performed. The objective of this prospective study was to characterize some of the morphologic, ultrastructural, and cytochemical staining features of specific (primary) granules in both normal and vacuolated eosinophils in Greyhound blood. Morphologic features of eosinophils in Wright's- and Diff-Quik-stained peripheral blood smears from 49 Greyhounds were compared with 200 blood smears from non-Greyhound dogs. Transmission electron microscopy was done on blood from 3 Greyhounds with vacuolated eosinophils and 3 with normal eosinophil granules. Blood smears from 4 of these dogs also were stained cytochemically with alkaline phosphatase (AP), chloracetate esterase (CAE), and alpha naphthyl butyrate esterase (ANBE). The morphologic features and tinctorial properties of vacuolated and normal eosinophils were compared. Twenty-six Greyhounds (53%) had vacuolated eosinophils and 23 (47%) had normal granulated eosinophils in smears stained with Wright's stain. Only 1% of eosinophils were vacuolated in non-Greyhound dogs. Twenty of the 23 (85%) Greyhounds with normal granulated eosinophils on Wright's-stained smears had vacuolated eosinophils in smears stained with Diff-Quik. Ultrastructurally, no morphologic differences were observed between granules of vacuolated and normal eosinophils. Both vacuolated and normal eosinophils in Greyhounds were positive for AP and negative for CAE and ANBE, as expected for normal dogs. Vacuolated eosinophils in Greyhounds likely reflect, at least in part, differential staining properties of the specific granules with different hematologic stains. Ultrastuctural and cytochemical features of eosinophil granules were similar in normal and vacuolated eosinophils from Greyhounds.

Hematologic reference intervals for koi (Cyprinus carpio), including blood cell morphology, cytochemistry, and ultrastructure.

Hematologic data are used routinely in the health care of humans and domestic mammals. Similar data for fish are largely fragmentary or have not been collected. The primary purpose of this study was to determine hematologic reference intervals for koi, an ornamental strain of the common carp (Cyprinus carpio). Secondarily, the morphology, cytochemical reactions, and ultrastructure of koi blood cells were characterized. A CBC was performed manually on heparin-anticoagulated blood specimens using Natt and Herrick's diluent and a Neubauer-ruled hemacytometer. Leukocyte differential counts were done on Wright-Leishman- and Diff-Quik-stained blood smears. Cytochemical reactions of koi leukocytes were determined using commercial kits. Transmission electron microscopy was performed to characterize the ultrastructural features of koi blood cells. Hematologic reference intervals were established for healthy koi for PCV (30-34%), hemoglobin concentration (6.3-7.6 g/dL), RBC count (1.7-1.9 X 10(6)/ microL), WBC count (19.8-28.1 X 10(3)/ microL), RBC indices, and differential leukocyte counts. Lymphocytes were the predominant leukocyte (accounting for up to 80% of all leukocytes), whereas eosinophils were rare. Basophils were positive with PAS staining. Naphthol AS-D chloroacetate esterase activity was observed only in eosinophils. alpha-Naphthyl butyrate esterase and beta-glucuronidase activities were positive in monocytes. Some lymphocytes were reactive for alpha-naphthyl butyrate esterase and acid phosphatase activity. Ultrastructurally, leukocytes, erythrocytes, and thrombocytes were identified on the basis of cytoplasmic organelles and granule appearance. Hematologic reference intervals and knowledge of the cytochemical reactions and ultrastructural characteristics of koi leukocytes will help standardize hematologic studies in this species.

Effect of chlorpromazine on human and murine intracellular carboxylesterases.

Clinical use of chlorpromazine (CPZ), an antipsychotic drug, is limited due to its hepatotoxicity. CPZ is found to inhibit in vitro intracellular carboxylesterases (CE), such as alpha-naphthyl acetate esterase, naphthol AS-D chloroacetate esterase, and alpha-naphthyl butyrate esterase in polymorphonuclear neutrophils, hepatocytes, and neuronal brain cells from mice. CPZ inhibits CE of all these cell types, whereby the degree of the inhibition depends on the incubation time and CPZ concentration. The polymorphonuclear neutrophils are most sensitive to CPZ. Comparable results were obtained with polymorphonuclear neutrophils from mice and humans. Since leukocytes are much more available than hepatocytes or neuronal cells in humans, we assume that CE in peripheral blood leukocytes (neutrophils and monocytes) can be used as markers for indication of pending liver damage by CPZ.

Effect of tetrachloromethane on the immune system.

Acute poisoning with tetrachloromethane in a dose of 0.75 LD(50) suppressed humoral and cell immune reactions in Wistar rats. Immunotoxicity of tetrachloromethane is realized via initiation of lipid peroxidation and inhibition of acetylcholine esterase in T lymphocytes and alpha-naphthyl AS-acetate esterase and alpha-naphthyl butyrate esterase in splenocytes.

Monocytes with altered phenotypes in posttrauma patients.

Posttrauma patients show impaired immune responsiveness and increased susceptibility to infections. Although monocytes in these patients have been known to express decreased HLA-DR, induction of HLA-DR using interferon gamma failed to reduce susceptibility to infection, suggesting additional factors also may be involved in the impaired immune responsiveness. CD4 plays an integral role in most of the functions of HLA-DR. In newborn infants, who have impaired immune responsiveness, we found a concomitant reduction of CD4 on monocytes with decreased HLA-DR expression. Because monocytes in posttrauma patients have not been previously studied for morphology, coexpression of CD4 and HLA-DR, and activity of alpha-naphthyl butyrate esterase, the purpose of this study was to analyze these factors in this population. Monocyte morphology; expression of CD4, CD11b, CD13, CD16, and HLA-DR by 3-color flow cytometry; and analysis of alpha-naphthyl butyrate esterase activity by cytochemical staining were studied in 27 posttrauma patients and 20 control subjects. Monocytes in posttrauma patients showed significant differences in the following characteristics compared with controls: (1) increase of subsets displaying the phenotypes CD4-/CD14+/HLA-DR- and CD4-/CD14+/CD16-, (2) decrease in mean fluorescence intensity of CD4 and HLA-DR expression in monocytes that were positive for these markers, (3) decrease in alpha-naphthyl butyrate esterase activity, and (4) decreased amount of cytoplasm and cytoplasmic vacuoles.Conclusion.-Our study suggests that in posttrauma patients, as in newborns, there is a marked increase of monocytes with decreased expression of CD4 and HLA-DR, as well as decreased alpha-naphthyl butyrate esterase activity. Concomitant reduction in CD4 and HLA-DR expression on monocytes may contribute to impaired immune responsiveness in these patients.

The professional phagocytes of sea bass (Dicentrarchus labrax L.): cytochemical characterisation of neutrophils and macrophages in the normal and inflamed peritoneal cavity

In order to identify the phagocytic cells of sea bass, the peritoneal leucocyte population of fish injected intraperitoneally with Photobacterium damselae subspecies piscicida was studied by light microscopy using cytocentrifuge preparations stained by the Antonow technique for peroxidase detection. Among the leucocytes present in the peritoneal exudate of the infected fish (macrophages, neutrophils, eosinophilic granular cells, lymphocytes and thrombocytes), macrophages and neutrophils were the only phagocytic cells. Neutrophils were easily distinguished from macrophages in Antonow stained preparations by the pattern of peroxidase positivity. Using ultrastructural cytochemistry, neutrophils were found to have abundant cytoplasmic granules positive for peroxidase and arylsulphatase and were negative for alpha-naphthyl butyrate (ANB) esterase. In contrast, ANB esterase activity was detected in macrophages. These leucocytes were typically negative for peroxidase, but ocasionally, some macrophages with peroxidase or arylsulphatase-positive vacuoles were observed. Both phagocytes had cytoplasmic granules positive for acid phosphatase. Glycogen particles were found in the cytoplasm of the two phagocytic cells, but they were much more abundant in neutrophils. Macrophages were much more abundant than neutrophils in the peritoneal cavity of non-injected sea bass but early after the intraperitoneal injection of bacteria, the number of neutrophils increased quickly and extensively. Higher numbers of intraperitoneally injected bacteria were found inside macrophages as compared to neutrophils because macrophages strongly predominated in the peritoneal population at the time of injection. However, when the bacteria were injected into peritoneal cavities with high numbers of neutrophils (attracted by a previous injection of 12% casein), the percentage of neutrophils with phagocytosed bacteria increased, approaching that of infected macrophages. Taken together, these results show that in sea bass, as in many other organisms, in addition to macrophages, neutrophils are important phagocytic cells, the relative participation of each of the two phagocytes in defense mechanisms against infection depending on the opportunity to encounter the invading infectious agents.

Esterase-3 polymorphism in the sugarcane borer Diatraea saccharalis (Lepidoptera, Pyralidae)

The migration rate of esterases and their substrate specificity for 4-methylumbelliferyl esters (acetate, propionate, and butyrate) and alpha- and beta-naphthyl esters were analyzed in Diatraea saccharalis by starch gel electrophoresis. Substrate preference of esterases was observed with Est-2 and Est-8 isozymes showing substrate specificity for 4-methylumbelliferyl esters and Est-4 isozyme showing specificity for 4-methylumbelliferyl butyrate and alpha-naphthyl butyrate. Allele variation was detected at the Est-3 locus. Two alleles, Est-3F and Est-3S, were identified in pupae with fluorogenic and ester-naphthyl substrates. Chi-square analysis showed no differences between the observed genotypic frequencies and those expected on the basis of Hardy-Weinberg frequencies for the Est-3 locus (chi² = 2.4; p < 0.01). The negative value for the Wright's fixation index (F = -0.2096) calculated for the D. saccharalis population maintained under laboratory conditions indicates an excess of heterozygotes, however, the observed Hardy-Weinberg equilibrium indicates that in the laboratory the population of D. saccharalis behaved as if the moth were randomly mating in nature. The high level of heterozygosity at the Est-3 locus indicates also that this esterase may be a good genetic marker for studies of natural D. saccharalis populations.

CD4+CD56+CD68+hematopoietic tumor of probable plasmacytoid monocyte derivation with weak expression of cytoplasmic CD3.

Hematopoietic neoplasm coexpressing CD4 and CD56 includes a subset of acute myeloid leukemia with myelomonocytic differentiation, plasmacytoid monocyte tumor, and other immature hematopoietic neoplasms of undefined origin. Herein, we report a CD4+CD56+CD68+ hematopoietic tumor that was thought to be a tumor of plasmacytoid monocytes. This case is unique in the absence of accompanying myelomonocytic leukemia and the faint expression of cCD3 on the tumor cells. The patient was a 22-yr old man presented with multiple lymphadenopathy and an involvement of the bone marrow. Tumor cells were large and monomorphic with an angulated eosinophilic cytoplasm of moderate amount. Nuclei of most tumor cells were eccentric and round with one or two prominent nucleoli. Rough endoplasmic reticulum was prominent in electron microscopic examination. Tumor cells expressed CD4, CD7, CD10, CD45RB, CD56, CD68, and HLA-DR and were negative for CD1a, CD2, sCD3, CD5, CD13, CD14, CD20, CD33, CD34, CD43, CD45RA, TIA-1, S-100, and TdT. cCD3 was not detected in the immunostaining using paraffin tissue, but was faintly expressed in flow cytometry and immunostaining using a touch imprint slide. T-cell receptor gene rearrangement analysis and EBV in situ hybridization showed negative results. Cytochemically, myeloperoxidase, Sudan black B, and alpha naphthyl butyrate esterase were all negative.

Cell differentiation and apoptosis of monocytic and promyelocytic leukemia cells (U-937 and HL-60) by tryptanthrin, an active ingredient of Polygonum tinctorium Lour.

Tryptanthrin, a bioactive ingredient of Polygonum tinctorium Lour., is a member of the Indigo plant family and has potent cytocidal effects on various human leukemia cells in vitro. At low concentrations, tryptanthrin enhanced the expression of cell differentiation (CD) markers in human monocytic (U-937) and promyelocytic (HL-60) leukemia cells indicative of differentiation to monocytes/macrophages. Furthermore, nitroblue tetrazolium (NBT) reductive and alpha-naphthyl butyrate esterase (NBE) activities were markedly increased after treatment. Tryptanthrin was more potent than dimethyl sulfoxide (DMSO) at inducing U-937 cell differentiation into monocytes/macrophages. After treatment with higher concentrations of tryptanthrin for 24 h, cytoplasmic vacuolation and destruction of mitochondria were observed. The leukemia cells died via apoptosis 48 h after treatment. Cytoplasmic vacuolation and apoptotic changes correlated with the dysfunction of mitochondria. Electron microscopic observations revealed marked swelling and destruction of mitochondria after exposure of the leukemia cells to tryptanthrin. Exposure to tryptanthrin enhanced Fas-induced apoptosis and increased caspase-3 activity before induction of apoptosis. These results show that low concentrations of tryptanthrin can induce differentiation of leukemia cells but higher concentrations will kill leukemia cells through apoptosis, possibly through a caspase-3/Fas antigen pathway.

Decapitation Impacting Effect of Topically Applied Chlorpyrifos on Acetylcholinesterase and General Esterases in Susceptible and Resistant German Cockroaches (Dictyoptera: Blattellidae)

The effect of topically applied chlorpyrifos on acetylcholinesterase and other esterases in heads and decapitated bodies of CSMA and Crawford German cockroaches was examined with spectrophotometric enzyme assay and native polyacrylamide gel electrophoresis. The toxicity of chlorpyrifos was greatly reduced in decapitated CSMA male cockroaches with LD50 value 17.1-fold higher than that of normal CSMA cockroaches. Acetylcholinesterase activity from heads was significantly higher in the Crawford compared with the CSMA strain and did not change until 24 h after chlorpyrifos in vivo treatment in both strains. The p-nitrophenyl butyrate (NPB) esterase activities from both heads and decapitated bodies of the resistant Crawford strain were significantly greater than the susceptible CSMA strain. The p-NPB esterase activity was significantly inhibited by chlorpyrifos in vivo treatment, and total p-NPB esterase activity was significantly reduced in decapitated bodies compared with heads of both strains. Native polyacrylamide gel electrophoresis (PAGE) analysis of extracts solubilized with Triton X-100 from heads and decapitated bodies revealed five major esterase bands and an acetylcholinesterase (AChE) band with a high capability of hydrolyzing alpha-naphthyl butyrate and acetylthiocholine, respectively. In the heads of susceptible CSMA male cockroaches, the activity of mobile isozymes d1 and d2 was completely inhibited at 24 h after chlorpyrifos application, and isozyme e was partially inhibited. In contrast, isozymes c1 and c2 from the decapitated bodies of CSMA cockroaches were mostly affected at 24 h after the topical application of chlorpyrifos. The activities of acetylcholinesterase and esterase isozymes a and b from the decapitated body remained uninhibited in both strains. Inhibition of isozymes d1 and d2 seems to be more important in chlorpyrifos intoxication than acetylcholinesterase.

Characterization of Nasutitermes globiceps (Isoptera: Termitidae) esterases.

Esterases of Nasutitermes globiceps termites which occur on the Upper Paraná River floodplain (Brazil) were characterized. The electrophoretic pattern of the termite esterases Nasutitermes globiceps was obtained by starch gel electrophoresis. Six esterase activity zones were obtained and numbered, with esterase-1 being the most anodall one and esterase-6 the most cathodal one. Esterase-2 was detected only with substrates derived from the 4-methylumbelliferyl radical. The esterases of N. globiceps present wide substrate specificity, having been observed with substrates derived from alpha-naphthyl (acetate, propionate, and butyrate) and beta-naphthyl (acetate, butyrate) and from 4-methylumbelliferyl (acetate, propionate and butyrate). Esterase-6 is a caste-specific enzyme detected in soldiers. Only esterases 1, 3 and 5 were detected in nymphs. No genetic polymorphism has been detected thus far in the esterases of Nasutitermes globiceps. This study suggests that allozyme variation can be explored to understand Nasutitermes social structure.