Two New Subspecies of Herpetomonas muscarum (Leidy, 1856) Kent, 1880

SYNOPSIS. Herpetomonas muscarum muscarum n. subsp. was isolated from Musca domestica L. In culture at 20 C it assumed the opisthomastigote (up to 15%), double-flagellate and flagellate promastigote forms. At 30 C or with 4% urea added to cultures at 20 C, the proportion of opisthomastigotes was greater (up to 40%). In experimentally infected flies only transient infections, which included both opisthomastigotes and promastigotes, occurred. The promastigotes were 15–30 μ long and the kinetoplast was small and subspherical or transversely elongate. H. muscarum ingenoplastis n. subsp. was isolated from Phormia regina (Meigen). In culture at 20 C almost all individuals were double-flagellate promastigotes 20–40 μ long and less than 1% were opisthomastigotes. At 30 C or with added urea there was no increase in the proportion of opisthomastigotes and the cultures were not vigorous. In experimentally infected flies opisthomastigotes were 5–39% of the population depending on the part of the gut sampled. In all stages the kinetoplast was large (1.5–2.5 μ long) and tear-drop-shaped with the point directed posteriorly.
In artificially mixed cultures of H. m. muscarum and H. m. ingenoplastis the former predominated after a short time and eventually survived alone. A mixed culture that was about 98% H. m. muscarum was fed to Phormia regina and produced heavy pure infections of H. m. ingenoplastis , which lasted for 22 days with no indication of decline. No evidence of cyst formation was found in either subspecies.     

Changes in Cell Surface Anionogenic Groups Induced by Propranolol in Herpetomonas muscarum muscarum

The effects of propranolol (10⁻³ mM) on the surface anionic groups of Herpetomonas muscarum muscarum were analysed by cell electrophoresis, by ultrastructural cytochemistry and by identification of sialic acids using paper chromatography. Differentiation of H. muscarum muscarum induced by propranolol treatment caused a significant increase in the net negative surface charge. Binding of cationized ferritin (CF) and colloidal iron hydroxide particles was observed at the cell surface of both untreated and propranolol-treated cells. In cells incubated in the presence of the drug the CF particles were distributed in all membrane regions. However, there were small areas where the particles were absent. In H. muscarum muscarum exposed to propranolol the density of residues of sialic acid per cell was higher, and the agglutinating activity with Sendai virus was more intense. However, the pattern of sialic acid, characterized by the presence of N-acetylneuraminic acid derivative, was not modified upon cell interaction with the drug. Treatment of both control and propranolol-treated protozoa with neuraminidase significantly reduced the surface charge. These findings suggest that sialic acid residues are the major anionogenic groups exposed on the surface of H. muscarum muscarum .     

Effect of Platelet-Activating Factor on the Process of Cellular Differentiation of Herpetomonas muscarum muscarum

ABSTRACT. The effects of platelet-activating factor (PAF), at doses ranging from 10⁻⁶ M to 10⁻¹⁰ M, on cell growth and on cell differentiation of Herpetomonas muscarum muscarum were investigated. Cell differentiation was evaluated by both light and electron microscopy. At the concentrations used, PAF did not interfere with the protozoan growth. However, parasites grown in the presence of PAF (10⁻⁶ M) were significantly more differentiated than those grown in the absence of PAF, since the first day of culture. On the first two days of culture, PAF doses ranging from 10⁻¹⁰ M to 10⁻⁷ M, did not significantly interfere with the differentiation of these parasites, although after the third day of culture, all PAF doses used significantly increased the protozoan differentiation. Specific PAF receptor antagonists totally abrogated (WEB 2086 and WEB 2170)or significantly decreased (BN 52021) PAF effect on cell differentiation. These findings indicate PAF triggers the process of cell differentiation in Herpetomonas muscarum muscarum and suggest these parasites have receptors for PAF.     

Magnesium-dependent ecto-ATP diphosphohydrolase activity in Herpetomonas muscarum muscarum

In the present work we characterized the ecto-ATP diphosphohydrolase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum. This parasite hydrolyzed ATP at a rate of 15.52 nmol Pi/mg protein/min and this activity reached a maximum at pH 7.5. Classical inhibitors of acid phosphatases, such as sodium orthovanadate (NaVO3), sodium fluoride (NaF), and ammonium molybdate presented no effect on this activity. MgCl2, ZnCl2, and MnCl2 stimulated the ATP hydrolysis by H. m. muscarum. The ecto-ATPase activity was insensitive to oligomycin and sodium azide, two inhibitors of mitochondrial Mg-ATPase, bafilomycin A1, a V-ATPase inhibitor, ouabain, a Na(+)+K+-ATPase inhibitor and to levamizole, an inhibitor of alkaline phosphatase. An extracellular impermeant inhibitor 4,4'-diisothiocyanostylbene 2',2'-disulfonic acid (DIDS) and a inhibitor of some ecto-ATPases, suramin, which is also a competitive antagonist of P2-purinergic receptors, promoted a great inhibition on the ATP hydrolysis. This enzyme is able to hydrolysis ATP, ADP, UTP, and UDP, but not GTP, GDP, CTP, or CDP. ADP inhibited the enzymatic activity in a concentration dependent manner, reaching 70% inhibition.     

Changes in cell surface anionogenic groups induced by propranolol in Herpetomonas muscarum muscarum

The effects of propranolol (10(-3) mM) on the surface anionic groups of Herpetomonas muscarum muscarum were analysed by cell electrophoresis, by ultrastructural cytochemistry and by identification of sialic acids using paper chromatography. Differentiation of H. muscarum muscarum induced by propranolol treatment caused a significant increase in the net negative surface charge. Binding of cationized ferritin (CF) and colloidal iron hydroxide particles was observed at the cell surface of both untreated and propranolol-treated cells. In cells incubated in the presence of the drug the CF particles were distributed in all membrane regions. However, there were small areas where the particles were absent. In H. muscarum muscarum exposed to propranolol the density of residues of sialic acid per cell was higher, and the agglutinating activity with Sendai virus was more intense. However, the pattern of sialic acid, characterized by the presence of N-acetylneuraminic acid derivative, was not modified upon cell interaction with the drug. Treatment of both control and propranolol-treated protozoa with neuraminidase significantly reduced the surface charge. These findings suggest that sialic acid residues are the major anionogenic groups exposed on the surface of H. muscarum muscarum.     

Platelet-activating factor (PAF) activates casein kinase 2 in the protozoan parasite Herpetomonas muscarum muscarum

Herpetomonas muscarum muscarum is a flagellate parasite of the family Trypanosomatidae, whose cell differentiation can be triggered by the lipid mediator, PAF. In this study we demonstrate for the first time that PAF effect relies on the activation of casein kinase 2 (CK2). The classical antagonist of PAF receptor, WEB 2086, abrogated PAF-enhanced CK2 activity. CK2 activation by PAF was also inhibited when parasite extracts were assayed in the presence of modulators of PKC, MAPK, and both Ser/Thr and Tyr phosphatases. Finally, a cell permeable inhibitor of CK2 (DRB) suppressed PAF-induced cell differentiation in a dose-dependent manner.     

Energetics of syntrophic fatty acid oxidation

Abstract: Fatty acids are key intermediates in methanogenic degradation of organic matter in sediments as well as in anaerobic reactors. Conversion of butyrate or propionate to acetate, (CO₂), and hydrogen is endergonic under standard conditions, and becomes possible only at low hydrogen concentrations (10^-4-10^-5 bar). A model of energy sharing between fermenting and methanogenic bacteria attributes a maximum amount of about 20 kJ per mol reaction to each partner in this syntrophic cooperation system. This amount corresponds to synthesis of only a fraction (one-third) of an ATP to be synthesized per reaction. Recent studies on the biochemistry of syntrophic fatty acid-oxidizing bacteria have revealed that hydrogen release from butyrate by these bacteria is inhibited by a protonophore or the ATPase inhibitor DCCD ( N , N '-dicyclohexyl carbodiimide), indicating that a reversed electron transport step is involved in butyrate or propionate oxidation. Hydrogenase, butyryl-CoA dehydrogenase, and succinate dehydrogenase acitivities were found to be partially associated with the cytoplasmic membrane fraction. Also glycolic acid is degraded to methane and CO₂ by a defined syntrophic coculture. Here the most difficult step for hydrogen release is the glycolate dehydrogenase reaction ( E '₀=−92 mV). Glycolate dehydrogenase, hydrogenase, and ATPase were found to be membrane-bound enzymes. Membrane vesicles produced hydrogen from glycolate only in the presence of ATP; protonophores and DCCD inhibited this hydrogen release. This system provides a suitable model to study reversed electron transport in interspecies hydrogen transfer between fermenting and methanogenic bacteria in methanogenic biomass degradation.     

Purine phosphoribosyltransferases of Leishmania mexicana mexicana and other flagellate protozoa

Amastigotes and cultured promastigotes of Leishmania mexicana mexicana and L. m. amazonensis, cultured promastigotes of L. donovani and L. tarentolae, and the culture forms of Crithidia fasciculata, Herpetomonas muscarum muscarum and H. m. ingenoplastis all possessed four phosphoribosyltransferase (PRTase) activities: adenine PRTase, hypoxanthine PRTase, guanine PRTase and xanthine PRTase. The enzymes of L. m. mexicana required divalent cations for activity; Mn2+ or Co2+ produced maximal activity in most cases. Hypoxanthine PRTase, guanine PRTase and xanthine PRTase from all organisms were sedimentable in part, suggesting that they may occur within glycosomes. The enzymes of L. m. mexicana cultured promastigotes were inhibited by a range of purine analogues.     

End products of metabolism in the anaerobic ciliate Trimyema compressum

Abstract The products of anaerobic and micro-aerobic (0.8% O₂) metabolism of the sapropelic ciliate Trimyema compressum strain N were studied. Under anaerobic conditions ethanol was formed in large amounts representing 44% of the total carbon excreted. Acetate, lactate, formate, CO₂ and H₂ were minor products, while succinate was formed in hardly detectable amounts. Under micro-aerobic conditions O₂ was consumed, CO₂ and formate were produced as major end products and no H₂, ethanol and succinate were formed.     

Platelet-Activating Factor Modulates a Secreted Phosphatase Activity of the Trypanosomatid Parasite Herpetomonas muscarum muscarum

In the present work we characterized the secreted phosphatase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum. This housefly parasite hydrolyzed p-nitrophenylphosphate at a rate of 10.26 nmol Pi/mg protein/min. Classical inhibitors of acid phosphatases, such as sodium orthovanadate (N_aVO₃), sodium fluoride (NaF), and ammonium molybdate promoted a decrease in this phosphatase activity. When the parasites were assayed in the presence of sodium tartrate, an inhibitor of Leishmania spp-secreted acid phosphatases, this activity was drastically diminished. Cytochemical analysis showed the localization of this enzyme on the external surface and in the flagellar pocket of these parasites. Sodium tartrate inhibited this reaction, confirming the biochemical data. Platelet-activating factor (PAF) inhibited the phosphatase activity determined in the supernatant of living H. m. muscarum. Received: 2 February 2001 / Accepted: 5 March 2001     

Varying Kinetoplast Ultrastructure in Two Subspecies of Herpetomonas muscarum (Leidy)*

SYNOPSIS. The kinetoplast nucleoid of Herpetomonas muscarum muscarum is a disk-shaped mass of filaments with sharply delimited anterior and posterior margins. The kinetoplast nucleoid of Herpetomonas muscarum ingenoplastis is elongate, tapered, and made up of long, helically arranged filaments. Possible explanations of these structures on the basis of work by others on isolated kinetoplast DNA are given. The double flagellum of Herpetomonas is seen by electron microscope to be two complete flagella in close contact but with no visible connection to each other.     

The oxidative response of rabbit peritoneal neutrophils to leishmanias and other trypanosomatids

Luminometry has been used to measure the respiratory burst of rabbit peritoneal neutrophils that is elicited by different forms and species of Leishmania and Herpetomonas. Mid-log phase and metacyclic promastigotes of L. major evoked large responses; that due to metacyclics was lower and slower, but they also bound in smaller numbers than mid-log phase cells. Promastigotes of L. mexicana mexicana also stimulated a large respiratory burst whereas amastigotes elicited little or none. Leishmania donovani promastigotes and culture forms of H. muscarum muscarum and H. m. ingenoplastis all evoked large responses by neutrophils. There was, however, very little response to L. mexicana mexicana promastigotes, L. donovani promastigotes or H. muscarum muscarum when they were added in large numbers. This 'inhibition' was not apparent with L. major.     

Biological effects of lithium chloride on Herpetomonas muscarum muscarum and Blastocrithidia culicis (kinetoplastida: trypanosomatidae).

Lithium is widely used in medicine as an antidepressive drug and for myelosuppression attenuation during chemotherapy. In spite of abundant literature, questions on the biological action of lithium ions are far from being answered. We have here examined the effects of lithium (10-200 mM) on culture forms of the trypanosomatid protozoa Herpetornonas muscarum muscarum and Blastocrithidia culicis. Incubation of these parasites with LiCl inhibited cell growth in a concentration-dependent manner, but growth could be restored when the drug was removed from the medium. Furthermore, Li+ induced cell differentiation in H. m. muscarum. Light microscopy examination of cell viability, using erythrosin B staining, showed that all treated parasites remained viable with all drug concentrations used. Ultrastructural analysis by transmission electron microscopy showed that the cells presented no signs of degeneration. However, in H. m. muscarum the nuclei lost their peripheral heterochromatin and appeared filled with a homogeneous matrix, whereas in B. culicis an increased amount of lipid droplets was present in the cytoplasm. Our data show that LiCl treatment arrested the cell division process, stimulated cell differentiation, and affected the metabolism of these parasites.     

A comparative study of the purine- and pyrimidine-metabolising enzymes of a range of trypanosomatids

A range of trypanosomatids (amastigotes and cultured promastigotes of Leishmania mexicana mexicana, cultured promastigotes of L. m. amazonensis, L. donovani and L. tarentolae, culture forms of Crithidia fasciculata, Herpetomonas muscarum muscarum and H. m. ingenoplastis and procyclic trypomastigotes of Trypanosoma brucei brucei) have been surveyed for the presence of purine- and pyrimidine-metabolising enzymes. Several common features were observed, including the presence of nucleosidases, catabolic phosphorylases, phosphoribosyltransferases, kinases and cytidine deaminase and the apparent absence of AMP deaminase, anabolic purine phosphorylase and cytosine deaminase. Significant differences between species were discovered, notably in adenine and adenosine metabolism. Nucleoside phosphotransferase active on inosine was detected in insect trypanosomatids but not in L. m. mexicana.     

Metabolism of position-labelled glucose in anoxic methanogenic paddy soil and lake sediment

Abstract Turnover times of radioactive glucose were shorter in paddy soil (4–16 min) than in Lake Constance sediment (18–62 min). In the paddy soil, 65–75% of the radioactive glucose was converted to soluble metabolites. In the sediment, only about 25% of the radioactive glucose was converted to soluble metabolites, the rest to particulate material. In anoxic paddy soil, the degradation pattern of position-labelled glucose was largely consistent with glucose degradation via the Embden-Meyerhof-Parnas (EMP) pathway followed by methanogenic acetate cleavage: CO₂ mainly originated from C-3,4, whereas CH₄ mainly originated from C-1 and C-6 of glucose. Acetate-carbon originated from C-1, C-2 and C-6 rather than from C-3,4 of glucose. In both paddy soil and Lake Constance sediment acetate and CO₂ were the most important early metabolites of radioactive glucose. Other early products included propionate, ethanol/butyrate, succinate, and lactate, but accounted each for less than 1–8% of the glucose utilized. The labelling of propionate by [3,4-¹⁴C]glucose suggests that it was mainly produced from glucose or lactate rather than from ethanol. Isopropanol and caproate were also detectable in paddy soil, but were not produced from radioactive glucose. Chloroform inhibited methanogenesis, inhibited the further degradation of radioactive acetate and resulted in the accumulation of H₂, however, did not inhibit glucose degradation. Since acetate was the main soluble fermentation product of glucose and was produced at a relatively high molar acetate: CO₂ ratio (2.5:1), homoacetogenesis appeared to be the most important glucose fermentation pathway.     

The ecology and taxonomy of anaerobic halophilic eubacteria

Abstract A number of obligately anaerobic chemoorganotrophic moderately halophilic bacteria have been isolated from the bottom sediments of the Dead Sea and the Great Salt Lake, Utah: (1) Halobacteroides halobius , a long motile rod from the Dead Sea, fermenting sugars to ethanol, acetate, H₂ and CO₂; (2) Clostridium lortetii , a rod-shaped bacterium from the Dead Sea, producing endospores with attached gas vacuoles; (3) a spore-forming motile rod-shaped bacterium, fermenting sugars, isolated from the Dead Sea; (4) Haloanaerobium praevalens , isolated from the Great Salt Lake, fermenting carbohydrates, peptides, amino acids and pectin to acetate, propionate, butyrate, H₂ and CO₂.
Analysis of their 16S rRNA shows that these organisms are related to each other, but unrelated to any of the other subgroups of the eubacterial kingdom, to which they belong.
Ha. praevalens and Hb. halobius regulate their internal osmotic pressure by the accumulation of salt (Na⁺, K⁺, Cl⁻) rather than by organic osmotic solutes.     

Growth and end-product formation in fermenter cultures of Brochothrix thermosphacta ATCC 11509T and two psychrotrophic Lactobacillus spp. in different gaseous atmospheres

The effects of different gaseous atmospheres were determined on the maximum specific growth rate (μ_max) and end-product formation by Brochothrix thermosphacta ATCC 11509^T, Lactobacillus viridescens SMRICC 174 and Lactobacillus sp. SMRICC 173 (homofermentative). The highest μ_max-values for Lact. viridescens (0.47/h) and Broc. thermosphacta (0.49/h) were obtained in air. Under anaerobic conditions μ_max was reduced, an atmosphere containing CO₂ alone giving the greatest reduction. Lactobacillus sp. 173 did not grow in air or N₂. Aerobic growth was obtained by adding peroxidase while anaerobic growth occurred in the presence of 5–20% CO₂. Carbon dioxide alone reduced the growth rate. All test organisms produced mainly lactic acid anaerobically. Lactobacillus viridescens also produced ethanol while Broc. thermosphacta produced small amounts of ethanol and formic acid. With O₂ present, the number of end-products increased for all organisms. Lactobacillus sp. 173 produced small amounts of acetic acid and acetoin together with lactic acid. Oxygen induced acetic acid production in Lact. viridescens and Broc. thermosphacta . Aerobically, Broc. thermosphacta also produced a large amount of acetoin and smaller amounts of 2,3-butanediol, iso -valeric acid and iso -butyric acid. The production of lactic acid by Broc. thermosphacta was completely prevented under strictly aerobic conditions. All test organisms consumed O₂ during aerobic growth. Hydrogen peroxide was produced by Lact. viridescens and Lactobacillus sp. 173.     

Anaerobic promotion of stem extension in Potamogeton pectinatus. Roles for carbon dioxide, acidification and hormones

Dark-grown shoots of tubers of the aquatic monocot Potamogön pectinatus L. elongated more strongly in anaerobic than aerobic solutions over 5 days. The response was located in the stem rather than the leaf. Anaerobic carbon dioxide (CO₂) production was similar to that in aerobic conditions. Approximately half the anaerobic stem extension was attributed to acidification of the submerging medium by respiratory CO₂. Sparging with an anaerobic gas mixture of nitrogen and hydrogen to remove dissolved CO₂ inhibited stem elongation and prevented acidification of the medium. Similarly, supplying CO₂ anaerobically promoted stem elongation while acidifying the medium. Carbon dioxide was also active on aerobic shoots. The effect of CO₂ on anaerobic stem extension could be mimicked with an acidic buffer. Anaerobic stem extension was inhibited by exogenous abscisic acid (ABA), while gibberellic acid and the gibberellin-biosynthesis inhibitor paclobutrazol proved inactive. Exogenous indole-3-acetic acid promoted stem extension in the absence of oxygen. A strong gravitropic response by anaerobic stems of P. pectinatus was inhibited by the auxin-efflux inhibitor naphthylphthalamic acid.     

Measurement of mercaptoacetate levels in anaerobic batch culture of colonic bacteria

Abstract Mercaptoacetate levels were measured by HPLC utilizing precolumn derivitisation with o -phthalaldehyde in bacteria suspensions incubated anaerobically in batch culture. Reproducibility of measurement had a coefficient of variation of 4.8% and the recovery was 98%. Suspensions of faecal bacteria were incubated under H₂/CO₂ or N₂/CO₂ (4:1 v/v) in anaerobic dilution solution, reduced with ascorbate, with either glucose, starch, dextran or dextran sulphate. Production of the short chain fatty acids (acetate, propionate and butyrate) and utilization of H₂ showed continuing microbial fermentation. Under these conditions mercaptoacetate was produced at variable rates between 0.06–12.34 μmol/g (dry weight) over 24 h. Incubations from 24 to 48 h revealed that mercaptoacetate was both produced and utilized. Endogenous mercaptoacetate production in the colon would assist in maintaining anaerobiosis in an environment exposed to variable amounts of oxygen.     

Gas exchange in intact isolated chloroplasts from Chlamydomonas reinhardtii during starch degradation in the dark

During starch degradation in intact isolated chloroplasts from Chlamydomonas reinhardtii gas exchange was studied with a mass spectrometer. Oxygen uptake by intact chloroplasts in the dark never exceeded 1.5% of the starch degradation rate [maximum 15 nmol O₂ (mg Chl)⁻¹ h⁻¹ consumed. 1 000 nmol glucose (mg Chl)⁻¹h⁻¹ degraded]. Evolution of CO₂ under aerobic conditions [9.8–28 nmol (mg Chl)⁻¹ h⁻¹] was stimulated by addition of 0.1–0.5 m M oxaloacetate [393–425 nmol CO₂ (mg Chl)⁻¹ h⁻¹]. Pyridoxal phosphate (5 m M ) inhibited starch degradation by more than 80%, but had no effect on O₂ uptake. Starch degradation rates and CO₂ evolution did not differ under acrobic and anaerobic conditions. Increasing P_i in the reaction medium from 0.5 m M to 5.0 m M stimulated starch degradation by 230 and 260% under aerobic and anaerobic conditions, respectively. A rapid autooxidation of reduced ferredoxin was observed in a reconstituted system consisting of purified Chlamydomonas ferredoxin, purified Chlamydomonas NADP-ferredoxin oxidoreductase (EC 1.6.7.1) and NADPH. Addition of isolated thylakoids from C. reinhardtii did not affect the rate of O₂ uptake. Our results clearly indicate the absence of any oxygen requirement during starch degradation in isolated chloroplasts.