Furanocoumarins from Ruta chalepensis with Amebicide Activity.

Entamoeba histolytica (protozoan; family Endomoebidae) is the cause of amoebiasis, a disease related to high morbidity and mortality. Nowadays, this illness is considered a significant public health issue in developing countries. In addition, parasite resistance to conventional medicinal treatment has increased in recent years. Traditional medicine around the world represents a valuable source of alternative treatment for many parasite diseases. In a previous paper, we communicated about the antiprotozoal activity in vitro of the methanolic (MeOH) extract of Ruta chalepensis (Rutaceae) against E. histolytica. The plant is extensively employed in Mexican traditional medicine. The following workup of the MeOH extract of R. chalepensis afforded the furocoumarins rutamarin (1) and chalepin (2), which showed high antiprotozoal activity on Entamoeba histolytica trophozoites employing in vitro tests (IC50 values of 6.52 and 28.95 µg/mL, respectively). Therefore, we offer a full scientific report about the bioguided isolation and the amebicide activity of chalepin and rutamarin.

LipF increases rifampicin and streptomycin sensitivity in a Mycobacterium tuberculosis surrogate.

BACKGROUND: Mortality due to tuberculosis (TB) has increased due to the development of drug resistance, the mechanisms of which have not been fully elucidated. Our research group identified a low expression of lipF gene in Mycobacterium tuberculosis clinical isolates with drug resistance. The aim of this work was to evaluate the effect of lipase F (LipF) expression on mycobacterial drug resistance. RESULTS: The effects of expressing lipF from Mycobacterium tuberculosis in Mycobacterium smegmatis on resistance to antituberculosis drugs were determined with resazurin microtiter assay plate and growth kinetics. Functionality of ectopic LipF was confirmed. LipF expression reduced the rifampicin (RIF) and streptomycin (STR) minimum inhibitory concentration (MIC) from 3.12 µg/mL to 1.6 µg/mL and 0.25 µg/mL to 0.06 µg/mL respectively, moreover a reduced M. smegmatis growth in presence of RIF and STR compared with that of a control strain without LipF expression (p < 0.05 and p < 0.01) was shown. CONCLUSIONS: LipF expression was associated with increased RIF and STR sensitivity in mycobacteria. Reduced LipF expression may contribute to the development of RIF and STR resistance in Mycobacterium species. Our findings provide information pertinent to understanding mycobacterial drug resistance mechanisms.

Antiprotozoal Activity against Entamoeba histolytica of Flavonoids Isolated from Lippia graveolens Kunth.

Amebiasis caused by Entamoeba histolytica is nowadays a serious public health problem worldwide, especially in developing countries. Annually, up to 100,000 deaths occur across the world. Due to the resistance that pathogenic protozoa exhibit against commercial antiprotozoal drugs, a growing emphasis has been placed on plants used in traditional medicine to discover new antiparasitics. Previously, we reported the in vitro antiamoebic activity of a methanolic extract of Lippia graveolens Kunth (Mexican oregano). In this study, we outline the isolation and structure elucidation of antiamoebic compounds occurring in this plant. The subsequent work-up of this methanol extract by bioguided isolation using several chromatographic techniques yielded the flavonoids pinocembrin (1), sakuranetin (2), cirsimaritin (3), and naringenin (4). Structural elucidation of the isolated compounds was achieved by spectroscopic/spectrometric analyses and comparing literature data. These compounds revealed significant antiprotozoal activity against E. histolytica trophozoites using in vitro tests, showing a 50% inhibitory concentration (IC50) ranging from 28 to 154 µg/mL. Amebicide activity of sakuranetin and cirsimaritin is reported for the first time in this study. These research data may help to corroborate the use of this plant in traditional Mexican medicine for the treatment of dyspepsia.

In Vitro Study of Antiamoebic Activity of Methanol Extracts of Argemone mexicana on Trophozoites of Entamoeba histolytica HM1-IMSS.

Infections caused by parasites in humans represent one of the main public health concerns. Amoebiasis, a parasitic infection caused by Entamoeba histolytica (E. histolytica), is considered endemic in Mexico, where Argemone mexicana (A. mexicana) has been used in traditional medicine to treat intestinal parasitic diseases. The objective of this work was to evaluate the potential biological activity of A. mexicana on E. histolytica. For this purpose, a methanolic extract was prepared from A. mexicana leaves, and a differential fractionation was carried out with solvents of different polarities. The inhibitory capacities of the extract and its fractions were evaluated in vitro using HM1-IMSS, a strain of Entamoeba histolytica. A. mexicana extract was found to have a growth-inhibiting activity for E. histolytica, showing IC50 = 78.39 µg/mL. The extract was characterized phytochemically, and the methanolic extract fractions were analyzed by liquid chromatography (HPLC) and mass spectrometry (MS). Berberine and jatrorrhizine were present in the active fractions, and these compounds may be responsible for the antiparasitic activity. The identification of amoebicidal activity of A. mexicana on E. histolytica gives support to the traditional use. Further studies with berberine and jatrorrhizine will be carried out to understand the mechanism involved.

meso-Dihydroguaiaretic acid derivatives with antibacterial and antimycobacterial activity.

Thirty-three meso-dihydroguaiaretic acid (meso-DGA) derivatives bearing esters, ethers, and amino-ethers were synthesized. All derivatives were tested against twelve drug-resistant clinical isolates of Gram-positive and Gram-negative bacteria, including sensitive (H37Rv) and multidrug-resistant Mycobacterium tuberculosis strains. Among the tested compounds, four esters (7, 11, 13, and 17), one ether (23), and three amino-ethers (30, 31, and 33) exhibited moderate activity against methicillin-resistant Staphylococcus aureus, whereas 30 and 31 showed better results than levofloxacin against vancomycin-resistant Enterococcus faecium. Additionally, nineteen meso-DGA derivatives displayed moderate to potent activity against M. tuberculosis H37Rv with minimum inhibitory concentration (MIC) values ranging from 3.125 to 50µg/mL. Seven meso-DGA derivatives bearing amino-ethers (26-31 and 33) exhibited the lowest MICs against M. tuberculosis H37Rv and G122 strains, with 31 being as potent as ethambutol (MICs of 3.125 and 6.25µg/mL). The presence of positively charged group precursors possessing steric and hydrophobic features (e.g. N-ethylpiperidine moieties in meso-31) resulted essential to significantly increase the antimycobacterial properties of parent meso-DGA as supported by the R-group pharmacophoric and field-based QSAR analyses. To investigate the safety profile of the antimycobacterial compounds, cytotoxicity on Vero cells was determined. The amino-ether 31 exhibited a selectivity index value of 23, which indicate it was more toxic to M. tuberculosis than to mammalian cells. Therefore, 31 can be considered as a promising antitubercular agent for further studies.

An acidic sphingomyelinase Type C activity from Mycobacterium tuberculosis.

Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Sphingolipids are recognized as diverse and dynamic regulators of a multitude of cellular processes mediating cell cycle control, differentiation, stress response, cell migration, adhesion, and apoptosis. Bacterial SMases are virulence factors for several species of pathogens. Whole cell extracts of Mycobacterium tuberculosis strains H37Rv and CDC1551 were assayed using [N-methyl-(14)C]-sphingomyelin as substrate. Acidic Zn(2+)-dependent SMase activity was identified in both strains. Peak SMase activity was observed at pH 5.5. Interestingly, overall SMase activity levels from CDC1551 extracts are approximately 1/3 of those of H37Rv. The presence of exogenous SMase produced by M. tuberculosis during infection may interfere with the normal host inflammatory response thus allowing the establishment of infection and disease development. This Type C activity is different from previously identified M. tuberculosis SMases. Defining the biochemical characteristics of M. tuberculosis SMases helps to elucidate the roles that these enzymes play during infection and disease.

Naphthoquinones isolated from Diospyros anisandra exhibit potent activity against pan-resistant first-line drugs Mycobacterium tuberculosis strains.

BACKGROUND AND OBJECTIVES: The recent emergence of multidrug-resistant (MDR), extensively drug-resistant (XDR), and totally drug-resistant (TDR) Mycobacterium tuberculosis (MTB) strains have further complicated the control of tuberculosis (TB). There is an urgent need of new molecules candidates to be developed as novel, active, and less toxic anti-tuberculosis (anti-TB) drugs. Medicinal plants have been an excellent source of leads for the development of drugs, particularly as anti-infective agents. In previous studies, the non-polar extract of Diospyros anisandra showed potent anti-TB activity, and three monomeric and five dimeric naphthoquinones have been obtained. In this study, we performed bioguided chemical fractionation and the isolation of eight naphthoquinones from D. anisandra and their evaluation of anti-TB and cytotoxic activities against mammalian cells. METHODS: The n-hexane crude extract from the stem bark of the plant was obtained by maceration and liquid-liquid fractionation. The isolation of naphthoquinones was carried out by chromatographic methods and identified by gas chromatography and mass spectroscopy data analysis. Anti-TB activity was evaluated against two strains of MTB (H37Rv) susceptible to all five first-line anti-TB drugs and a clinical isolate that is resistant to these medications (pan-resistant, CIBIN 99) by measuring the minimal inhibitory concentration (MIC). Cytotoxicity of naphthoquinones was estimated against two mammalian cells, Vero line and primary cultures of human peripheral blood mononuclear (PBMC) cells, and their selectivity index (SI) was determined. RESULTS: Plumbagin and its dimers maritinone and 3,3'-biplumbagin showed the strongest activity against both MTB strains (MIC = 1.56-3.33 µg/mL). The bioactivity of maritinone and 3,3'-biplumbagin were 32 times more potent than rifampicin against the pan-resistant strain, and both dimers showed to be non-toxic against PBMC and Vero cells. The SI of maritinone and 3,3'-biplumbagin on Vero cells was 74.34 and 194.11 against sensitive and pan-resistant MTB strains, respectively. CONCLUSION: Maritinone and 3,3'-biplumbagin possess a very interesting potential for development as new drugs against M. tuberculosis, mainly resistant profile strains.

Antiprotozoal activity against Entamoeba histolytica of plants used in northeast Mexican traditional medicine. Bioactive compounds from Lippia graveolens and Ruta chalepensis.

Amoebiasis caused by Entamoeba histolytica is associated with high morbidity and mortality is becoming a major public health problem worldwide, especially in developing countries. Because of the side-effects and the resistance that pathogenic protozoa build against the standard antiparasitic drugs, e.g., metronidazole, much recent attention has been paid to plants used in traditional medicine around the world in order to find new antiprotozoal agents. We collected 32 plants used in Northeast Mexican traditional medicine and the methanolic extracts of these species were screened for antiprotozoal activity against E. histolytica trophozoites using in vitro tests. Only 18 extracts showed a significant inhibiting activity and among them six plant extracts showed more than 80% growth inhibition against E. histolytica at a concentration of 150 µg/mL and the IC50 values of these extracts were determined. Lippia graveolens Kunth and Ruta chalepensis Pers. showed the more significant antiprotozoal activity (91.54% and 90.50% growth inhibition at a concentration of 150 µg/mL with IC50 values of 59.14 and 60.07 µg/mL, respectively). Bioassay-guided fractionation of the methanolic extracts from these two plants afforded carvacrol (1) and chalepensin (2), respectively, as bioactive compounds with antiprotozoal activity.

Prevalence of obesity, diabetes and hypertension in immigrant populations in northeastern Mexico.

Introduction: Hispanic immigrants are a fast-growing population in the United States of America (USA) that disproportionately suffer from chronic diseases. Despite the increasing prevalence of obesity in Latin-American countries, only a few studies have examined the onset of chronic diseases in Mexican and Central American migrants in Mexico. Objective: The objective of this study is to determine the prevalence of obesity, diabetes, and hypertension in Central American immigrants who are in the process of traveling through northeastern Mexico to the United States. Methods: An observational, descriptive, cross-sectional study was conducted among migrants, mostly Central Americans. Migrants who agreed to participate in the study were interviewed face-to-face by researchers to obtain their sociodemographic data. To obtain the prevalence, many health indicators related to obesity, diabetes, and hypertension, including weight, height, fasting glucose, and blood pressure, were measured. Results: In total, 520 migrants were interviewed; sociodemographic data indicated that most participants were men (76%), from Honduras (72.6%), single (61.2%), and have elementary level of education (48.6%). The somatometric evaluation revealed that 28.9% were diagnosed as overweight, 10.7% with obesity, and 3.3% with malnutrition. Of less prevalence, 8.8% were detected with hypertension and 4.6% had fasting hyperglycemia. The mean participant age was 29.11 ± 10.00 years. For each participant, the average weight was 66.72 ± 13.09 kg; the average height was 1.64 ± 0.08 m; the average body mass index (BMI) was 24.59 ± 4.32; the mean systolic and diastolic pressures were 116.26 ± 15.13 and 74 ± 9.65, respectively; and the average glycemia was 100.97 ± 21.99. El Salvador showed the highest proportion of people with diabetes (14.7%). Women who participated in this study had a higher proportion of obesity (23.4%, p = 0.02) and overweight (36.2%) than men (8.4 and 29.2%, respectively). People from Mexico, Nicaragua, and Honduras reported a high prevalence of overweight participants (63.6, 47.4, and 30.7%, respectively), while people from El Salvador and Nicaragua had a high prevalence of obese participants (23.5 and 21.1%, respectively). Conclusion: We found significant differences in the rates of obesity, diabetes, and hypertension between groups of Central American migrants and their place of origin, age, educational level, and gender. Our findings highlight the importance of exploring differences within groups of Central American migrants traveling through northeastern Mexico to the United States, which may explain several health indicators.

Induction of virulence factors, apoptosis, and cytokines in precision-cut hamster liver slices infected with Entamoeba histolytica.

Precision-cut liver slices (PCLS) are mainly used to evaluate hepatotoxicity and metabolism of chemicals, as well as to study mechanisms of liver damage and repair. However, recently they have been used as a system to study amoebic infections. The aim of this study was to validate this model as an alternative for experimental amoebic liver absess (ALA) in animals. To do this, the PCLS was analyzed for the expression of amoebapore and cysteine proteinases 1 and 5, three of the most studied virulence factors of Entamoeba histolytica, as well as the induction of apoptosis and cytokines production in response to the ex vivo infection. PCHLS were prepared with the Brendel-Vitron tissue slicer and then, infected with 200,000 trophozoites of E. histolytica. Samples were taken at 0, 6, 12, 18, and 24 h and compared to control non-infected slices. Morphological studies were performed in order to verify the infection; while apoptosis was studied by TUNEL and PAS techniques. The expression of cysteine proteinases (1 and 5), and amoebapore, was analyzed by real-time PCR. By using ELISA assays, the production of cytokines was also studied. PCHLS were found to be a reproducible infection system, and E. histolytica caused the expression of cysteine proteinases and amoebapore in infected slices. At the same time, trophozoites induce release of cytokines and apoptotic death of the hepatocytes close to them. PCHLS represent a new and suitable alternative model to study the pathogenesis of hepatic amoebiasis.

Antigiardial Activity of Foeniculum vulgare Hexane Extract and Some of Its Constituents.

Foeniculum vulgare is used for the treatment of diarrhea in Mexican traditional medicine. Hexane extract showed 94 % inhibition of Giardia duodenalis trophozoites at 300 µg/mL. Therefore, 20 constituents of hexane extract were evaluated to determine their antigiardial activity. Interestingly, six compounds showed good activity toward the parasite. These compounds were (1R,4S) (+)-Camphene (61%), (R)(-)-Carvone (66%), estragole (49%), p-anisaldehyde (67%), 1,3-benzenediol (56%), and trans, trans-2,4-undecadienal (97%). The aldehyde trans, trans-2,4-undecadienal was the most active compound with an IC50 value of 72.11 µg/mL against G. duodenalis trophozoites. This aldehyde was less toxic (IC50 588.8 µg/mL) than positive control metronidazole (IC50 83.5 µg/mL) against Vero cells. The above results could support the use of F. vulgare in Mexican traditional medicine.

Latent tuberculosis infection in medical students in the Northeast of Mexico.

BACKGROUND: Medical students are considered to be personnel with a high level of risk for developing latent tuberculosis infection (LTBI). One possible reason is lack of knowledge about the transmission, prevention, and biosafety standards for tuberculosis disease. OBJECTIVE: This research aimed to determine the rate of LTBI among medical students studying in a private School of Medicine in Monterrey, Mexico. METHODS: In this cross-sectional study, we obtained blood samples from 174 medical students. LTBI was diagnosed using the QuantiFERON®-TB Gold Plus test. The prevalence of LTBI was compared with the socio-demographic data of the students and their level of knowledge and use of personal protective equipment (PPE). RESULTS: The proportion of LTBI in the students was 20.6%. Medical students in their first few years of medical school had a lower prevalence of LTBI than students in their final years of medical school. Additionally, students with a low level of knowledge on LTBI and low use of proper PPE had a higher prevalence of LTBI. CONCLUSIONS: In a School of Medicine in Monterrey, Mexico, the proportion of medical students with LTBI was low but the proportion increased in advanced students. Students who demonstrated adequate knowledge and use of respiratory protective masks had lower prevalence rates for LTBI.

D-Lactate and intestinal fatty acid-binding protein are elevated in serum in patients with acute ischemic stroke.

Experimental studies suggest that the intestinal barrier is affected in ischemic stroke. D-Lactate and intestinal fatty acid-binding protein (IFABP) are markers of intestinal mucosa integrity and barrier function. Our purpose was to evaluate the serum concentrations of these markers in patients with acute ischemic stroke (AIS). We included patients with AIS and used healthy subjects as controls. Clinical, demographic and outcome measures were recorded. Blood was drawn within 24 h of symptom onset. Serum concentrations of D-Lactate and IFABP were determined using commercially available colorimetric and ELISA kits, respectively. We included a total of 61 patients (median age of 64 years). The majority of patients were male (57.4%). The most common cause of stroke was atherosclerosis (34.4%), followed by small-vessel disease and cardioembolic (32.7% each). Mean admission NIHSS score was 8. Median IFABP and D-Lactate concentrations were significantly higher in patients than in controls. Concentrations were not associated with stroke severity or 3-month outcome. Patients with large-artery atherosclerosis and cardioembolic etiology had higher D-Lactate values than patients with small-vessel disease. D-Lactate and IFABP were significantly elevated in patients with AIS. This suggests that there is disruption of the intestinal barrier in patients with AIS.

Risk Factors for SARS-CoV-2 Infection, Pneumonia, Intubation, and Death in Northeast Mexico.

Despite the social distancing and mobility restriction measures implemented for susceptible people around the world, infections and deaths due to COVID-19 continued to increase, even more so in the first months of 2021 in Mexico. Thus, it is necessary to find risk groups that can benefit from more aggressive preventive measures in a high-density population. This is a case-control study of suspected COVID-19 patients from Nuevo León, Mexico. Cases were: (1) COVID-19-positive patients and COVID-19-positive patients who (2) developed pneumonia, (3) were intubated and (4) died. Controls were: (1) COVID-19-negative patients, (2) COVID-19-positive patients without pneumonia, (3) non-intubated COVID-19-positive patients and (4) surviving COVID-19-positive patients. ≥ 18 years of age, not pregnant, were included. The pre-existing conditions analysed as risk factors were age (years), sex (male), diabetes mellitus, hypertension, chronic obstructive pulmonary disease, asthma, immunosuppression, obesity, cardiovascular disease, chronic kidney disease and smoking. The Mann-Whitney U tests, Chi square and binary logistic regression were used. A total of 56,715 suspected patients were analysed in Nuevo León, México, with 62.6% being positive for COVID-19 and, of those infected, 14% developed pneumonia, 2.9% were intubated and 8.1% died. The mean age of those infected was 44.7 years, while of those complicated it was around 60 years. Older age, male sex, diabetes, hypertension, and obesity were risk factors for infection, complications, and death from COVID-19. This study highlights the importance of timely recognition of the population exposed to pre-existing conditions to prioritise preventive measures against the virus.

Acellular fraction from malignant effusions has cytotoxicity in breast cancer cells.

Malignant ascites (MA) and malignant pleural effusion (MPE) are frequently developed in patients with metastatic cancer; however, the biological properties of these fluids have not been clarified. The present study explored the biological role of a low molecular fraction derived from malignant effusions on the activation of peripheral blood mononuclear cells and on the proliferation of breast cancer cells and fibroblast 55x cells. A <10-kDa fraction from effusions of 41 oncological patients and 34 individuals without cancer was purified, and its potential role in inhibiting nitric oxide (NO) production on lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells was explored, as well as its cytotoxicity on MCF-7 breast cancer cells and fibroblast 55x cells. A significant decrease in NO production was observed in the <10-kDa fraction from malignant effusions. In addition, the acellular fraction from MA decreased the viability of breast cancer cells without affecting human fibroblasts. These data support the presence of low molecular weight molecules in malignant samples with a specific role in inhibiting the defense mechanisms of peripheral blood mononuclear cells and decreasing the viability of breast cancer cells in vitro.

Precision-cut hamster liver slices as an ex vivo model to study amoebic liver abscess.

Entamoeba histolytica is the etiological agent of amoebiasis, the second cause of global morbidity and mortality due to parasitic diseases in humans. In approximately 1% of the cases, amoebas penetrate the intestinal mucosa and spread to other organs, producing extra-intestinal lesions, among which amoebic liver abscess (ALA) is the most common. To study ALA, in vivo and in vitro models are used. However, animal models may pose ethical issues, and are time-consuming and costly; and cell cultures represent isolated cellular lineages. The present study reports the infection of precision-cut hamster liver slices with Entamoeba histolytica trophozoites. The infection time-course, including tissue damage, parallels findings previously reported in the animal model. At the same time amoebic virulence factors were detected in the infected slices. This new model to study ALA is simple and reproducible, and employs less than 1/3 of the hamsters required for in vivo analyses.

Entamoeba histolytica: soluble and membrane-associated neutral sphingomyelinase-C and other unidentified esterase activity.

Sphingomyelinase (SMase) activity was measured in Entamoeba histolytica particulate and soluble subcellular fractions. The effects on SMase of incubation time, total protein concentration, pH, and several divalent cations were determined. SMase-C and other unidentified esterase activity were detected in soluble and particulate fractions. SMase-C was 94.5-96.0% higher than the unidentified esterase activity. Soluble and insoluble SMase-C specific activities increased with protein dose and incubation time. Soluble and insoluble SMase-C activities were maximum at pH 7.5 and were dependent on Mg(2+), Mn(2+), or Co(2+), and inhibited by Zn(2+), Hg(2+), Ca(2+), and EDTA. SMase-C was active in the pH range of 3-10 and its maximum activity was at pH 7.5. The soluble and insoluble SMases have remarkably similar physicochemical properties, strongly suggesting that E. histolytica has just one isoform of neutral SMase-C that had not been described before and might be essential for E. histolytica metabolism or virulence.

Antimycobacterial neolignans isolated from Aristolochia taliscana.

Tuberculosis (TB - Mycobacterium tuberculosis) is an ancient infectious disease that has appeared once again as a serious worldwide health problem and now comprises the second leading cause of death resulting from a single infection. The prevalence of multidrug resistance (MDR) TB is increasing and therapeutic options for treatment are not always accessible; in fact, some patients do not respond to the available drugs. Therefore, there is an urgent need to develop novel anti-TB agents. The aim of the present study was to screen extracts of Aristolochia taliscana, a plant used in traditional Mexican medicine to treat cough and snake bites, for antimycobacterial activity. The hexanic extract of A. taliscana was tested by microdilution alamar blue assay against Mycobacterium strains and bioguided fractionation led to the isolation of the neolignans licarin A, licarin B and eupomatenoid-7, all of which had antimycobacterial activity. Licarin A was the most active compound, with minimum inhibitory concentrations of 3.12-12.5 microg/mL against the following M. tuberculosis strains: H37Rv, four mono-resistant H37Rv variants and 12 clinical MDR isolates, as well as against five non-tuberculous mycobacteria (NTM) strains. In conclusion, licarin A represents a potentially active anti-TB agent to treat MDR M. tuberculosis and NTM strains.

Entamoeba invadens: influence of 60 Hz magnetic fields on growth and differentiation.

Exposure to extremely low-frequency (ELF) electromagnetic fields appears to result in a number of important biological changes. In the present study, we evaluated the effects of 60 Hz sinusoidal magnetic fields (MF) at magnetic flux densities of 1.0, 1.5 and 2.0 mT on growth and differentiation of the protozoan Entamoeba invadens. We demonstrated an inhibitory growth effect when trophozoite cultures were exposed to 1.5 and 2.0 mT. Furthermore, we found that there was not a synergistic effect in cultures co-exposed to MF and Metronidazole, a cytotoxic drug against amoebic cells. In addition, MF exposure inhibited the encystation process of E. invadens.

Activity of intracellular phospholipase A1 and A2 in Giardia lamblia.

Neither phospholipase A1 (PLA A1) nor phospholipase A2 (PLA A2), nor their respective genes, have been identified in Giardia lamblia, even though they are essential for lipid metabolism in this parasite. A method to identify, isolate, and characterize these enzymes is needed. The activities of PLA A1 and PLA A2 were analyzed in a total extract (TE) and in vesicular (P30) and soluble (S30) subcellular fractions of G. lamblia trophozoites; the effects of several chemical and physicochemical factors on their activities were investigated. The assays were performed using substrate labeled with 14C, and the mass of the 14C-product was quantified. PLA A1 and PLA A2 activity was present in the TE and the P30 and S30 fractions, and it was dependent on pH and the concentrations of protein and Ca2+. In all trophozoite preparations, PLA A1 and PLA A2 activities were inhibited by ethylenediaminetetraacetic acid and Rosenthal's inhibitor. These results suggest that G. lamblia possesses several PLA A1 and PLA A2 isoforms that may be soluble or associated with membranes. In addition to participating in G. lamblia phospholipid metabolism, PLA A1 and PLA A2 could play important roles in the cytopathogenicity of this parasite.