Bloodmeal digestion in the midgut of Phlebotomus papatasi and Phlebotomus langeroni

Abstract. Bloodmeal digestion in midguts of the sandflies Phlebotomus papatasi and Phlebotomus langeroni (Diptera: Psychodidae) was investigated in optimized assays to detect general protease, trypsin and aminopeptidase activities using synthetic substrates. Optimal activity occurred at pH 8-9 for all enzymes examined in both species. Protease activity peaked at 24-34h post human bloodmeal in midguts of P.papatasi and 34-48h in P'.langeroni; all endo- and exoprotease activities were completed by 50 h in P.papatasi compared to 72 h in P. langeroni. Hydrolysis of two chymotrypsin substrates was <2% of trypsin activity in both species. Aminopeptidase activity was associated mainly with the midgut wall, whereas trypsin activity was confined to the midgut lumen. A feature of digestion in P.langeroni was the high level of aminopeptidase recorded within 10h of the bloodmeal.     

Functional morphology of the midgut of a sandfly as compared to other hematophagous nematocera

The midgut epithelium of female Lutzomyia longipalpis was investigated by means of electron microscopic morphometry before and during blood digestion. Ultrastructure and cytological changes of the stomach cells upon blood feeding were generally similar to the ones described for Phlebotomus longipes (Gemetchu, 1974) and for mosquitoes (Hecker, 1977), In addition, the quantitative composition of the cells resembled the one of mosquitoes in many respects. Despite some morphological differences in the functional gut cytology, it can be admitted that, in general, digestive processes may run similarly in the midguts of sandflies and mosquitoes.     

Effect of Ditylenchus dipsaci and Pratylenchus penetrans on Verticillium Wilt of Alfalfa

Verticillium albo-atrum wilt symptoms appeared faster and were significantly more severe in the presence of Ditylenchus dipsaci in Vernal, a wilt-susceptible cultivar, than in Marls Kabul, a wilt-resistant cultivar. Winter kill in the field was not affected by the nematode during the first winter, but 50% of plants were killed in the second winter. Forage yield from nematode-infected plants was significantly reduced the second year. Interaction with V. albo-atrum did not significantly reduce forage yields below that of D. dipsaci alone. Pratylenchus penetrans did not increase the severity of wilt symptoms in the presence of V. albo-atrum, nor did it affect forage yield in the greenhouse. It did, however, reduce alfalfa yields in presence of V. albo-atrum under field conditions. D. dipsaci and P. penetrans reproduced faster in Vernal than in Maris Kabul when the fungus was present.     

Effect of Planting Date on Population Densities of Hoplolaimus columbus and Yield of Soybean

During the 1991 and 1992 soybean growing seasons, field plots were established in South Carolina to study the effect of planting date on at-planting nematode densities and subsequent yield losses caused by Hoplolaimus columbus. The susceptible and intolerant soybean cv. Braxton was planted on five dates from to May to 28 June in 1991 and from 12 May to 28 June in 1992. Nematodes were recovered from soil samples collected before nematicide treatment with 1,3-D (Pi), at 6 weeks after planting (Pm), and at harvest (Pf). Initial nematode population densities did not differ among the five dates of planting in either year. The increase in numbers of nematodes from planting to 6 weeks after planting (Pm/Pi) and from planting to harvest (Pf/Pi) were not different among the five planting dates in either year. Root samples also were collected at 6 weeks after planting and at harvest, but planting date did not affect the number of nematodes extracted from roots on any sample date in either year. Altering planting dates between early May and late June was not effective in preventing yield suppression due to H. columbus.     

Differential Effect of Extracellular Acidosis on the Release and Dispersal of Soluble and Membrane Proteins Secreted from the Weibel-Palade Body

Proteins secreted from Weibel-Palade bodies (WPBs) play important roles in regulating inflammatory and hemostatic responses. Inflammation is associated with the extracellular acidification of tissues and blood, conditions that can alter the behavior of secreted proteins. The effect of extracellular pH (pH_o) on the release of von Willebrand factor (VWF), the VWF-propolypeptide (Proregion), interleukin-8, eotaxin-3, P-selectin, and CD63 from WPBs was investigated using biochemical approaches and by direct optical analysis of individual WPB fusion events in human endothelial cells expressing green or red fluorescent fusions of these different cargo proteins. Between pH_o 7.4 and 7.0, ionomycin-evoked WPB exocytosis was characterized by the adhesion of VWF to the cell surface and the formation of long filamentous strands. The rapid dispersal of Proregion, interleukin-8, and eotaxin-3 into solution, and of P-selectin and CD63 into the plasma membrane, was unaltered over this pH_o range. At pH_o 6.8 or lower, Proregion remained associated with VWF, in many cases WPB failed to collapse fully and VWF failed to form filamentous strands. At pH_o 6.5 dispersal of interleukin-8, eotaxin-3, and the membrane protein CD63 remained unaltered compared with that at pH_o 7.4; however, P-selectin dispersal into the plasma membrane was significantly slowed. Thus, extracellular acidification to levels of pH_o 6.8 or lower significantly alters the behavior of secreted VWF, Proregion, and P-selectin while rapid release of the small pro-inflammatory mediators IL-8 and eotaxin-3 is essentially unaltered. Together, these data suggest that WPB exocytosis during extracellular acidosis may favor the control of inflammatory processes.Local acidosis is associated with inflammation and ischemia and can have significant effects on the normal function of cells, tissues, cellular, and blood components, in particularly those associated with the immune, vascular, and hemostatic systems (1-8). Endothelial cells regulate inflammatory, vascular and hemostatic responses through the secretion of a wide range of bioactive molecules from specialized secretory organelles, the Weibel-Palade bodies (WPBs).³The major WPB core proteins are von Willebrand factor (VWF) and the VWF-propolypeptide (Proregion). VWF is synthesized as a pre-proprotein comprising an N-terminal signal peptide (pre-), and several distinct repeating structural domains (termed A, B, C, and D) arranged as D1-D2-D′-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK (9). During translation, the signal peptide is removed to yield proVWF, which then undergoes disulfide-linked dimerization to produce proVWF dimers (10). The Proregion domains (D1-D2) are cleaved from the main peptide in the Golgi apparatus, and further disulfide bond formation produces VWF multimers. The two resulting proteins, VWF and Proregion are co-packaged into the WPB where they noncovalently associate to form ordered tubules in a pH- and Ca²⁺-dependent fashion (11). Other physiologically important WPB proteins include P-selectin, interleukin-8 (IL-8), eotaxin-3, osteoprotegerin, and angiopoietin-2, (reviewed in Ref. 12).At physiological extracellular pH (pH_o 7.4) the majority of agonist-evoked WPB fusion events (∼75-90%) result in complete exocytosis and release of the stored molecules (13, 14). Secreted VWF adheres to the cell surface and can form long filamentous strands, particularly under flow conditions, that are essential for the efficient capture of platelets from solution both in vitro and in vivo (e.g. Ref. 15). Imaging individual WPB exocytotic events in live endothelial cells expressing fluorescent WPB cargo molecules has shown that Proregion, along with IL-8, disperse quickly into solution, while P-selectin rapidly diffuses into the plasma membrane at sites of fusion (14, 16).Under acidic conditions, however, the behavior of VWF and Proregion, are reported to change. At pH_o 6.4 or lower Proregion remains associated with VWF, either at the cell surface after stimulated exocytosis of WPBs (17) or in the cell following removal of the WPB membrane by detergent treatment (15). In addition, the unfurling of VWF to form long filamentous strands is attenuated (15, 17), a situation that impairs its capacity to efficiently support platelet adhesion (15). Retention of Proregion at the cell surface has led to speculation that it might play some biological role at such sites (17). In vitro Proregion is a ligand for the integrins α4β1 (VLA-4) (18) and α9β1 (VLA-9) (19) present on monocytes, leukocytes, eosinophils (VLA-4), and neutrophils (VLA-9). Thus it is possible that at sites of local acidosis produced during inflammation or ischemia, Proregion could play a role, along with other secreted factors, in regulating inflammatory responses. The influence of extracellular acidification on the release of these other secreted factors (e.g. IL-8, eotaxin-3, and P-selectin) is not known.pH_o values of 6.4 or lower represent extreme conditions of acidosis, with decreases in pH_o between 7.2 and 6.5 more typically reported during inflammation or ischemia (7, 20-26). The aim of this study was to determine the influence of extracellular acidification in this range (pH_o 7.4-6.5) on the release of a variety of soluble and membrane proteins of the WPB involved in coagulant and inflammatory responses. Specifically, we define more precisely the value of pH_o at which VWF fails to form filamentous strands and remains associated with Proregion at the cell surface, demonstrate the rapid reversibility of this pH-dependent association at individual sites of WPB fusion, and examine in detail the influence of extracellular acidification on the release and dispersal of fluorescent fusion proteins of the soluble mediators IL-8, eotaxin-3, and the membrane proteins P-selectin and CD63 from individual WPBs.The data show that the external conditions into which WPB deliver their cargo has significant effects on the dispersal and behavior of some but not other secreted molecules. Under acidic conditions, these changes could lead to a subtle shift from a coagulant to a more inflammatory phenotype. The data also highlight a more general problem of interpreting biochemical data of soluble secreted proteins in terms of underlying secretory granule exocytosis.     

Effects of Aged Garlic Extract on Left Ventricular Diastolic Function and Fibrosis in a Rat Hypertension Model

Daily consumption of garlic is known to lower the risk of hypertension and ischemic heart disease. In this study, we examined whether aged garlic extract (AGE) prevents hypertension and the progression of compensated left ventricular (LV) hypertrophy in Dahl salt-sensitive (DS) rats. DS rats were randomly divided into three groups: those fed an 8% NaCl diet until 18 weeks of age (8% NaCl group), those additionally treated with AGE (8% NaCl + AGE group), and control rats maintained on a diet containing 0.3% NaCl until 18 weeks of age (0.3% NaCl group). AGE was administered orally by gastric gavage once a day until 18 weeks of age. LV mass was significantly higher in the 8% NaCl + AGE group than in the 0.3% NaCl group at 18 weeks of age, but significantly lower in the 8% NaCl + AGE group than in the 8% NaCl group. No significant differences were observed in systolic blood pressure (SBP) between the 8% NaCl and 8% NaCl + AGE groups at 12 and 18 weeks of age. LV end-diastolic pressure and pressure half-time at 12 and 18 weeks of age were significantly lower in the 8% NaCl + AGE group compared with the 8% NaCl group. AGE significantly reduced LV interstitial fibrosis at 12 and 18 weeks of age. Chronic AGE intake attenuated LV diastolic dysfunction and fibrosis without significantly decreasing SBP in hypertensive DS rats.     

Effects of Selected Insecticides and Nematicides on the In Vitro Development of the Entomogenous Nematode Neoaplectana carpocapsae

The effects of organophosphates (mevinphos, phenamiphos, trichlorfon), carbamates (carbofuran, methomyl, oxamyl), a formamidine (chlordimeform), a synthetic pyrethroid (fenvalerate), a chlorinated hydrocarbon (methoxychlor). and an insect growth regulator (diflubenzuron) on in vitro development and reproduction of Neoaplectana carflocapsae were tested by incorporating each chemical into a nematode rearing medium. Organophosphates and carbamates adversely affected development and reproduction at concentrations ≥ 0.1 mg/ml. Phenamiphos was the most toxic, with no nematode reproduction at 0.01 mg/ml. Inoculated infective juveniles developed to adults with some of the organophosphates and carbamates, but limited or no reproduction occurred. Chlordimeform inhibited development at 1.0 mg/ml, while diflubenzuron, fenvalerate, and methoxychlor did not significantly (P > 0.05) reduced reproduction at 1.0 mg/ml. The organophosphate and carbamate nematicides in use for control of plant-parasitic nematodes may be toxic to N. carpocapsae in the soil.     

Effect of hypochlorous acid solution on the eradication and prevention of Pseudomonas aeruginosa infection,serum biochemical variables,and cecum microbiota in rats

In this study, hypochlorous acid solution, a weak acid, provided as drinking water to rats, was evaluated for its ability to eradicate and prevent Pseudomonas aeruginosa infection, while monitoring its simultaneous effect on serum biochemical variables and microbiota in the rat cecum. The results suggest that the solution could not eliminate the bacteria in the experimentally infected rats; however, the administration of a 10-parts-per-million (ppm) hypochlorous acid solution as drinking water was effective in inhibiting horizontal spread of P. aeruginosa infection among cage mates. Additionally, exposure to hypochlorous solution did not have any effect on serum biochemical variables of the rat including levels of total cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin, total bilirubin, lipase, amylase, urea nitrogen, total protein, calcium (Ca), phosphorus (P), sodium (Na), chlorine (Cl), except for potassium (K) levels. The most frequently isolated bacteria in the rat cecum included species belonging to Bacteroidales, Lactobacillus, Clostridiales, Erysipelotrichaceae, Akkermansia, Coriobacteriales, and Firmicutes. The ratio of the terminal restriction fragment length polymorphism (T-RFLP) peaks did not differ across rats administered with 5 and 10 ppm weak acid solution as compared to the control group for any of the bacteria, except for Erysipelotrichaceae and Firmicutes, where the ratio of T-RFLP peaks was higher in the 5 ppm group for Erysipelotrichaceae and in the 10 ppm group for Firmicutes than that in the control group (P<0.01). The results suggest that the weak acid hypochlorous solution could not eradicate P. aeruginosa completely from rats. The solution was effective in preventing infection without affecting serum biochemical variables; however, some of bacterial microbiota may have changed due to administration of the solution.     

Effects of the Ionic Composition and Water Potential of Aqueous Solution on the Activity and Survival of Orrina phyllobia

The activity and survival of Orrina phyllobia fourth-stage juveniles (J4) were examined in aqueous solutions representing 96 combinations of eight predominant soil solution ions at total concentrations of 100, 200, and 1,000 meq/liter. Various water potentials were imposed by the addition of mannitol or polyethylene glycol to ionic solutions. Nematode longevity increased as water potential was decreased. Longevity was approximately doubled at a water potential of -23 × 10⁵ Pa and more than tripled at -60 × 10⁵ Pa. No combination oflons at 200 meq/liter was lethal after a 6-day exposure. Several ion combinations significantly increased longevity at -10 and -23 × 10⁵ Pa. Single cation Na⁺ solutions consistently inhibited activity and more than doubled nematode longevity.     

A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis

Developing a fast, inexpensive, and specific test that reflects the mutations presentin Mycobacterium tuberculosis isolates according to geographicregion is the main challenge for drug-resistant tuberculosis (TB) control. Theobjective of this study was to develop a molecular platform to make a rapid diagnosisof multidrug-resistant (MDR) and extensively drug-resistant TB based on singlenucleotide polymorphism (SNP) mutations present in therpoB,katG, inhA,ahpC, andgyrA genes from Colombian M. tuberculosisisolates. The amplification and sequencing of each target gene was performed. Captureoligonucleotides, which were tested before being used with isolates to assess theperformance, were designed for wild type and mutated codons, and the platform wasstandardised based on the reverse hybridisation principle. This method was tested onDNA samples extracted from clinical isolates from 160 Colombian patients who werepreviously phenotypically and genotypically characterised as having susceptible orMDR M. tuberculosis. For our method, the kappa index of thesequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625forrpoB, katG,inhA,ahpC, and gyrA,respectively. Sensitivity and specificity were ranked between 90-100% compared withthose of phenotypic drug susceptibility testing. Our assay helps to pave the way forimplementation locally and for specifically adapted methods that can simultaneouslydetect drug resistance mutations to first and second-line drugs within a fewhours.     

The sandfly fauna (Diptera: Psychodidae: Phlebotominae) of the Parque Estadual da Serra da Tiririca,Rio de Janeiro, Brazil

Cutaneous leishmaniasis (CL) in the state of Rio de Janeiro is sporadic and canbe characterised as a peridomestic transmission that occurs in modified naturalenvironments. The aim of this work was to study the fauna and ecologicalcharacteristics of sandflies in an environmentally protected area (the StatePark of Serra da Tiririca) within the remnants of the Atlantic Forest in themunicipalities of Niterói and Maricá and their possible relationship withleishmaniasis. Captures were performed using light traps during the night once amonth for one year in both sylvatic environments and areas surrounding homesnear the park. A total of 1,037 sandflies were captured, belonging to ninegenera and 12 species: Evandromyia tupynambai (34.1%),Migonemyia migonei (20.6%), Brumptomyiacunhai (13.8%), Micropygomyia schreiberi (9.7%),Psathyromyia lanei (6.5%), Brumptomyianitzulescui (5.7%), Evandromyia edwardsi(5.4%), Nyssomyia intermedia (2.8%), Evandromyiacortelezzii (0.6%), Pintomyia bianchigalatiae(0.5%), Lutzomyia longipalpis (0.2%) and Sciopemyiamicrops (0.1%). Both Mg. migonei and Ny.intermedia may be acting as vectors of CL in this area.     

Effects of Soil Texture on the Interaction Between Rhizoctonia solani and Meloidogyne incognita on Cotton Seedlings

Soils containing 60, 75, and 90% coarse particles (sand plus coarse silt) were prepared by dilution of a field soil with 246μm (60-mesh) silica sand. As the coarse-particle content of the soils increased, the synergistic interaction between Meloidogyne incognita and Rhizoctonia solani on cotton seedlings increased. Increasing the coarse-particle content of the soil also increased damage from the nematode alone and slightly increased soreshin damage from R. solani alone.     

The Effect of Arthrobotrys conoides on Meloidogyne incognita Population Densities in Corn as Influenced by Temperature,Fungus Inoculum Density,and Time of Fungus Introduction in the Soil

In greenhouse experiments, the effect of Arthrobotrys conoides on Meloidogyne incognita population densities as affected by soil temperature, inoculum density, and green alfalfa was determined. The effect on M. incognita population densities was greater at a soil temperature of 25 C than at 18 or 32 C. Nematode control by A. conoides was most effective when the fungus was introduced into the soil 2 wk prior to nematode inoculation and planting of corn. Inoculum density of A. conoides was positively correlated with plant shoot weight (r = 0.81) and negatively correlated with numbers of Meloidogyne juveniles (r = -0.96), eggs (-0.89) and galls per gram of root (-0.91). A. conoides was not isolated from green alfalfa, but was isolated from alfalfa-amended soil to which no fungus had been added.     

Comparative study of behavioural tests in the SOD1G93A mouse model of amyotrophic lateral sclerosis

In preclinical trials, a sensitive functional test is required to detect changes in the motor behaviour of the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS). We evaluated changes in body weight and motor impairment in behavioural tests, such as the rotarod, the hanging-wire test and the treadmill, of transgenic and wild type mice. We found differences in detection of the onset of symptoms and progression of the disease between the different tests assessed. Moreover, the data showed significant gender differences in the motor behaviour of this mouse model. The rotarod and the hanging-wire test were more sensitive to detect early motor impairment. Moreover, the results suggested that the rotarod and hanging-wire became the most accurate tests rather than treadmill to characterise the ALS disease phenotype.     

Study of the irreversible binding of Bacillus thuringiensis Cry1Aa to brush border membrane vesicles from Bombyx mori midgut

The binding of Bacillus thuringiensis δ-endotoxin to brush border membrane vesicles (BBMVs) from the target insect larval midgut comprises with not only a reversible but also an irreversible component. The irreversible binding of δ-endotoxin is thought to be a pathologically important factor. Here, we studied the irreversible binding of Cry1Aa to the BBMVs of Bombyx mori. The ¹²⁵I-labeled Cry1Aa bound to the solubilized brush border membrane (BBM) through rapid dissociation only, unlike the binding to BBMVs, indicating that the toxin bound to the solubilized BBM through only a reversible process. Low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the toxin bound irreversibly to BBMVs formed an oligomer of 220 kDa, whereas that bound reversibly to the solubilized BBM did not oligomeraize. When the ¹²⁵I-labeled Cry1Aa bound irreversibly to the BBMVs was digested by proteinase K, approximately 40% of the toxin observed to be resistant to proteinase K. The molecular mass of the toxin resistant to proteinase K was 60 kDa, suggesting that the irreversible binding comprise two forms. These results support the notion that the irreversible binding of the toxin to BBMVs is due to the insertion of the toxin into the lipid bilayers and oligomerization to form channels.     

Effect of<Emphasis Type="Italic">in vitro</Emphasis> digestion on fumonisin B<Subscript>1</Subscript> in corn flakes

Low levels of fumonisins have been found frequently in corn based breakfast cereals and can occur bound to protein and other matrix components.In vitro digestion of two samples of corn flakes was carried out under "fed conditions." Fumonisins were measured as o-phthaldialdehyde/mercaptoethanol derivatives by LC-fluorescence. One sample of corn flakes (FN12) had high concentrations of fumonisin B₁ (FB) (average 125 ng/g) and total bound FB₁, (TB FB₁) (average 92 ng/g) and the other (FN11) had a low level of free FB₁ (average 29 ng/g) and no detectable TB FB₁. After incubation of the samples with gastrointestinal tract solutions simulating saliva plus stomach and duodenal juices, chyme was analysed for FB₁, hydrolyzed FB₁ (HFB₁) and partially hydrolyzed fumonisin B₁ (PHFB₁). The bioaccessibility (percentage of FB₁ released from corn flakes into chyme) was 38-78% for incurred FB₁ in FN12, 8-54% for incurred plus spiked FB₁ in FN12, and 19-66% for incurred plus spiked FB₁ in FN11. HFB₁ and PHFB₁ were not detected. If free FB₁ was first extracted from sample FN12, no FB1 was detected in the chyme, indicating no contribution from TB FB₁. Concentrations were corrected for method recovery of FB₁ or, for bound FB₁, partial method recovery of HFB₁ Presented at the XIIth IUPAC International Symposium on Mycotoxins and Phycotoxins, Istanbul, Turkey, 21–25 May, 2007     

Effects of C-terminal amino acids truncation on enzyme properties of <Emphasis Type="Italic">Aeromonas caviae</Emphasis> D1 chitinase

C-Terminal truncation mutagenesis was used to explore the functional and structural significance of the C-terminal region of Aeromonas caviae D1 chitinase (AcD1ChiA). Comparative studies between the engineered full-length AcD1ChiA and the truncated mutant (AcD1ChiAK606) included initial rate kinetics, fluorescence and circular dichroism (CD) spectrometric properties, and substrate binding and hydrolysis abilities. The overall catalytic efficiency, k _cat/K _M, of AcD1ChiAK606 with the 4MU-(GlcNAc)₂ and the 4MU-(GlcNAc)₃ chitin substrates was 15–26% decreased. When compared with AcD1ChiA, the truncated mutant AcD1ChiAK606 maintained 80% relative substrate-binding ability and about 76% of the hydrolyzing efficiency against the insoluble α-chitin substrate. Both fluorescence and CD spectroscopy indicated that AcD1ChiAK606 retained the same conformation as AcD1ChiA. These results indicated that removal of the C-terminal 259 amino acid residues, including the putative chitin-binding motif and the A region (a motif of unknown function) of AcD1ChiA, did not seriously affect the enzyme structure integrity as well as activity. The present study provided evidences illustrating that the binding and hydrolyzing of insoluble chitin substrates by AcD1ChiA were not absolutely dependent on the putative C-terminal chitin-binding domain and the function-unknown A region.