Analysis of the roles of cysteine proteinases of Leishmania mexicana in the host–parasite interaction

Parasitology ◽  
2000 ◽  
Vol 121 (4) ◽  
pp. 367-377 ◽  
Author(s):  
M. J. FRAME ◽  
J. C. MOTTRAM ◽  
G. H. COOMBS
Keyword(s):  
Time Course ◽  
Cysteine Proteinase ◽  
Mammalian Host ◽  
Leishmania Mexicana ◽  
Wild Type ◽  
Large Numbers ◽  
Host Parasite

Promastigotes of Leishmania mexicana mutants lacking the multicopy CPB cysteine proteinase genes (ΔCPB) are markedly less able than wild-type parasites to infect macrophages in vitro. ΔCPB promastigotes invade macrophages in large numbers but are unable to survive in the majority of the cells. In contrast, ΔCPB amastigotes invade and survive within macrophages in vitro. This extreme in vitro stage-specific difference was not mimicked in vivo; both promastigotes and amastigotes of ΔCPB produced lesions in BALB/c mice, but in each case the lesions grew considerably more slowly than those caused by wild-type parasites and only small lesions resulted. Inhibition of CPB in situ using cell-permeant peptidyldiazomethylketones had no measurable effect on parasite growth or differentiation axenically in vitro. In contrast, N-benzoyloxycarbonyl-phe-ala-diazomethylketone reduced the infectivity of wild-type parasites to macrophages by 80%. Time-course experiments demonstrated that application of the inhibitor caused effects not seen with ΔCPB, suggesting that CPB may not be the prime target of this inhibitor. The data show that the CPB genes of L. mexicana encode enzymes that have important roles in intracellular survival of the parasite and more generally in its interaction with its mammalian host.

scholarly journals Use of Recombinase-BasedIn VivoExpression Technology To Characterize Enterococcus faecalis Gene Expression during Infection IdentifiesIn Vivo-Expressed Antisense RNAs and Implicates the Protease Eep in Pathogenesis

2011 ◽  
Vol 80 (2) ◽  
pp. 539-549 ◽  
Author(s):  
Kristi L. Frank ◽  
Aaron M. T. Barnes ◽  
Suzanne M. Grindle ◽  
Dawn A. Manias ◽  
Patrick M. Schlievert ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Rabbit Model ◽  
Microscopic Analysis ◽  
Mammalian Host ◽  
Wild Type ◽  
Antisense Transcripts ◽  
Content Type ◽  
Antisense Rnas

ABSTRACTEnterococcus faecalisis a member of the mammalian gastrointestinal microflora that has become a leading cause of nosocomial infections over the past several decades.E. faecalismust be able to adapt its physiology based on its surroundings in order to thrive in a mammalian host as both a commensal and a pathogen. We employed recombinase-basedin vivoexpression technology (RIVET) to identify promoters on theE. faecalisOG1RF chromosome that were specifically activated during the course of infection in a rabbit subdermal abscess model. The RIVET screen identified 249 putativein vivo-activated loci, over one-third of which are predicted to generate antisense transcripts. Three predicted antisense transcripts were detected inin vitro- andin vivo-grown cells, providing the first evidence ofin vivo-expressed antisense RNAs inE. faecalis. Deletions in thein vivo-activated genes that encode glutamate 5-kinase (proB[EF0038]), the transcriptional regulator EbrA (ebrA[EF1809]), and the membrane metalloprotease Eep (eep[EF2380]) did not hinder biofilm formation inin vitroassays. In a rabbit model of endocarditis, the ΔebrAstrain was fully virulent, the ΔproBstrain was slightly attenuated, and the Δeepstrain was severely attenuated. The Δeepvirulence defect could be complemented by the expression of the wild-type gene intrans. Microscopic analysis of early Δeepbiofilms revealed an abundance of small cellular aggregates that were not observed in wild-type biofilms. This work illustrates the use of a RIVET screen to provide information about the temporal activation of genes during infection, resulting in the identification and confirmation of a new virulence determinant in an important pathogen.

scholarly journals ATP keeps exocytosis sites in a primed state but is not required for membrane fusion: an analysis with Paramecium cells in vivo and in vitro.

1986 ◽  
Vol 103 (4) ◽  
pp. 1279-1288 ◽  
Author(s):  
J Vilmart-Seuwen ◽  
H Kersken ◽  
R Stürzl ◽  
H Plattner
Keyword(s):  
Membrane Fusion ◽  
Time Course ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Primed State ◽  
Different Strains ◽  
Exogenous Trigger ◽  
Free Media

We have tried to specify a widespread hypothesis on the requirement of ATP for exocytosis (membrane fusion). With Paramecium tetraurelia cells, synchronously (approximately 1 s) exocytosing trichocysts, ATP pools have been measured in different strains, including wild type cells, "non-discharge" (nd), "trichless" (tl), and other mutations. The occurrence of a considerable and rapid ATP consumption also in nd and tl mutations as well as its time course (with a maximum 3-5 s after exocytosis) in exocytosis-competent strains does not match the actual extent of exocytosis performance. However, from in vivo as well as from in vitro experiments, we came to the conclusion that ATP might be required to keep the system in a primed state and its removal might facilitate membrane fusion. (For the study of exocytosis in vitro we have developed a new system, consisting of isolated cortices). In vivo as well as in vitro exocytosis is inhibited by increased levels of ATP or by a nonhydrolyzable ATP analogue. In vitro exocytosis is facilitated in ATP-free media. In vivo-microinjected ATP retards exocytosis in response to chemical triggers, whereas microinjected apyrase triggers exocytosis without exogenous trigger. Experiments with this system also largely exclude any overlaps with other processes that normally accompany exocytosis. Our data also explain why it was frequently assumed that ATP would be required for exocytosis. We conclude that membrane fusion during exocytosis does not require the presence of ATP; the occurrence of membrane fusion might involve the elimination of ATP from primed fusogenic sites; most of the ATP consumption measured in the course of exocytosis may be due to other effects, probably to recovery phenomena.

Cabergoline Stimulates Human Endometrial Stromal Cell Decidualization and Reverses Effects of Interleukin-1β in vitro

2021 ◽  
Author(s):  
Yu Jie ◽  
Sarah L Berga ◽  
Qingying Meng ◽  
Mingjing Xia ◽  
Trudy A Kohout ◽  
...  
Keyword(s):  
Connexin 43 ◽  
Time Course ◽  
Sympathetic Innervation ◽  
Endometrial Stromal Cells ◽  
Catecholaminergic Neurons ◽  
Mrna And Protein Expression

Abstract Context Human embryonic implantation is regulated by neuroendocrine hormones, ovarian steroids, growth factors and cytokines. Sympathetic innervation of the uterus also may play a role. Objective We tested the hypothesis that cabergoline (Cb), an agonist of type 2 dopamine receptors (DRD2), could influence endometrial decidualization in vitro. Design Immunohistochemistry confirmed the presence of catecholaminergic neurons in human uterine tissue. DRD2 mRNA and protein expression in endometrial tissue and cells were validated by quantitative RT-PCR, cDNA microarrays, RNA sequencing and Western blotting. Isolated human endometrial stromal cells (ESC) were subjected to dose-response and time-course experiments in the absence or presence of decidualizing hormones (10 nM estradiol, 100 nM progesterone and 0,5 mM dibutyryl cAMP). In some cases, interleukin (IL)-1β (0.1 nM) was used as an inflammatory stimulus. Well-characterized in vitro biomarkers were quantified. Results DRD2 were maximally expressed in vivo in the mid-secretory phase of the cycle and upregulated in ESC in response to decidualizing hormones, as were classical (eg, prolactin) and emerging (eg, VEGF and connexin 43) differentiation biomarkers. Cabergoline treatment more than doubled decidual biomarker expression, whereas risperidone, a dopamine receptor antagonist, inhibited ESC differentiation by >50%. Cabergoline induced characteristic decidual morphology changes and blocked detrimental effects of IL-1β on decidual cytology. Conclusion Our results support the hypothesis that dopaminergic neurons modulate decidualization in situ. We postulate that dopamine agonists, like Cb, could be developed as therapeutic agents to enhance implantation in couples with inflammation-associated infertility.

scholarly journals Axenic cultivation and characterization ofLeishmania mexicanaamastigote-like forms

Parasitology ◽  
1992 ◽  
Vol 105 (2) ◽  
pp. 193-202 ◽  
Author(s):  
P. A. Bates ◽  
C. D. Robertson ◽  
L. Tetley ◽  
G. H. Coombs
Keyword(s):  
Cysteine Proteinase ◽  
Calf Serum ◽  
Foetal Calf Serum ◽  
Leishmania Mexicana ◽  
Sds Page ◽  
Cysteine Proteinase Activity ◽  
Axenic Cultivation

SUMMARYA new method is described which has made possible the long-term axenic cultivation ofLeishmania mexicanaamastigotelike forms in Schneider'sDrosophilamedium supplemented with 2% (v/v) foetal calf serum. Unlike previous methods, it utilizes direct culture of parasites obtained from the lesions of infected animals rather than adaptation of promastigotesin vitro. Ultrastructural (possession of megasomes), biochemical (cysteine proteinase activity and gelatin SDS-PAGE banding pattern) and infectivity (in vivo) data are presented which show the close similarity of the cultured forms to lesion amastigotes. The axenically cultured forms grew optimally at a temperature of 32–33 °C, providing further evidence for their amastigote nature. It was found that adjustment of the pH of the growth medium to 5·4 was required in order to retain the amastigote morphology of the cultured parasites. This supports the notion that leishmanial amastigotes are acidophiles.

scholarly journals THE ACTION POTENTIAL OF SPINAL AXONS IN VITRO

1954 ◽  
Vol 37 (4) ◽  
pp. 505-538 ◽  
Author(s):  
Donald O. Rudin ◽  
George Eisenman
Keyword(s):  
Action Potential ◽  
Peripheral Nerve ◽  
Time Course ◽  
Dorsal Column ◽  
Myelinated Axons ◽  
Spike Potential ◽  
Tissue Mass

Despite the trauma of dissection and special metabolic requirements, the physiological properties of funiculi of the mammalian spinal cord can be studied in vitro. They are adequately oxygenated by diffusion at 0.88 atm. pO2 and remain in a functionally normal state for over 12 hours. The internal consistency of several kinds of data presented in this and the foregoing papers (5, 38) serves to characterize certain properties of central myelinated axons whether excised or in situ. (1) Spinal tracts support a large spike potential in vitro whose form, duration, and velocity are comparable to those of alpha fibers in vitro and spinal tracts in vivo. (2) Properties consistent with a large L fraction are found in central axons whether excised or in situ. (3) Following conduction there has been identified post-spike supernormality with exponential time course (7.5 msecs. half-time) which is the result of activity intrinsic to parent fibers of dorsal columns. The supernormality is similar in form and magnitude both in excised and intact funiculi. (4) In excised funiculi the action potential of parent axons includes a large negative after-potential whose form and duration correspond satisfactorily with this supernormality. This potential appears not to result from activity arising in broken collaterals. (5) Central axons, excised or intact, fire spontaneously in the presence of citrate ion, and when synchronized by stimulation develop periodic oscillations at about 400 C.P.S. but show no such behavior in the presence of excess potassium ion. Certain characteristics peculiar to central axons indicate that they occupy an extreme position in the spectrum of properties encountered in conducting tissues. Dorsal column myelinated axons differ from their peripheral counterparts, even though they are parts of the same cell, in the following ways. The maintenance of the column spike potential is more critically dependent on CO2 and the entire tissue mass has a higher oxygen consumption. The negative after-potential is much larger and the positive after-potential, non-existent following a single volley, is more difficult to develop by repetitive stimulation. Unlike peripheral nerve, central axons are not incited to spontaneous activity by manipulation of certain constituents normally present in their environment. However, when induced by the application of citrate the resulting rhythmic behavior has twice the frequency of that in peripheral nerve. In general, the recovery process in central axons is more invariant than that in peripheral axons when they are subjected to similar changes in their artificial environments.

A quick, simple method for purifying Leishmania mexicana amastigotes in large numbers

Parasitology ◽  
1981 ◽  
Vol 82 (3) ◽  
pp. 345-355 ◽  
Author(s):  
D. T. Hart ◽  
K. Vickerman ◽  
G. H. Coombs
Keyword(s):  
Electron Microscopy ◽  
Buoyant Density ◽  
Light And Electron Microscopy ◽  
Host Cells ◽  
Leishmania Mexicana ◽  
Cell Debris ◽  
Simple Method ◽  
Large Numbers

SUMMARYA rapid method for the bulk isolation of purified Leishmania mexicana mexicana amastigotes from parasite-induced lesions in experimentally infected mice is described. The procedure includes purification steps based on differences in net cell charge, lysis susceptibility and buoyant density between parasite and host cells. Yields of up to 2 × 1010 untransformed amastigotes with minimal contamination with host cells and cell debris can be obtained. At least 90 % of the purified amastigotes are viable as judged by light and electron microscopy, the staining of their lysosomes with acridine orange, their ability to transform to promastigotes and their infectivity to macrophages in vivo and in vitro.

scholarly journals Analysis of Recombinant H7N9 Wild-Type and Mutant Viruses in Pigs Shows that the Q226L Mutation in HA Is Important for Transmission

2014 ◽  
Vol 88 (14) ◽  
pp. 8153-8165 ◽  
Author(s):  
Qinfang Liu ◽  
Bin Zhou ◽  
Wenjun Ma ◽  
Bhupinder Bawa ◽  
Jingjiao Ma ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Viral Population ◽  
Wild Type Virus ◽  
Mammalian Host ◽  
H7n9 Virus ◽  
Loss Of Function ◽  
Wild Type ◽  
Human Infections

ABSTRACTThe fact that there have been more than 300 human infections with a novel avian H7N9 virus in China indicates that this emerging strain has pandemic potential. Furthermore, many of the H7N9 viruses circulating in animal reservoirs contain putative mammalian signatures in the HA and PB2 genes that are believed to be important in the adaptation of other avian strains to humans. To date, the definitive roles of these mammalian-signature substitutions in transmission and pathogenesis of H7N9 viruses remain unclear. To address this we analyzed the biological characteristics, pathogenicity, and transmissibility of A/Anhui/1/2013 (H7N9) virus and variantsin vitroandin vivousing a synthetically created wild-type virus (rAnhui-WT) and two mutants (rAnhui-HA-226Q and rAnhui-PB2-627E). All three viruses replicated in lungs of intratracheally inoculated pigs, yet nasal shedding was limited. The rAnhui-WT and rAnhui-PB2-627E viruses were transmitted to contact animals. In contrast, the rAnhui-HA-226Q virus was not transmitted to sentinel pigs. Deep sequencing of viruses from the lungs of infected pigs identified substitutions arising in the viral population (e.g., PB2-T271A, PB2-D701N, HA-V195I, and PB2-E627K reversion) that may enhance viral replication in pigs. Collectively, the results demonstrate that critical mutations (i.e., HA-Q226L) enable the H7N9 viruses to be transmitted in a mammalian host and suggest that the myriad H7N9 genotypes circulating in avian species in China and closely related strains (e.g., H7N7) have the potential for further adaptation to human or other mammalian hosts (e.g., pigs), leading to strains capable of sustained human-to-human transmission.IMPORTANCEThe genomes of the zoonotic avian H7N9 viruses emerging in China have mutations in critical genes (PB2-E627K and HA-Q226L) that may be important in their pandemic potential. This study shows that (i) HA-226L of zoonotic H7N9 strains is critical for binding the α-2,6-linked receptor and enables transmission in pigs; (ii) wild-type A/Anhui/1/2013 (H7N9) shows modest replication, virulence, and transmissibility in pigs, suggesting that it is not well adapted to the mammalian host; and (iii) both wild-type and variant H7N9 viruses rapidly develop additional mammalian-signature mutations in pigs, indicating that they represent an important potential intermediate host. This is the first study analyzing the phenotypic effects of specific mutations within the HA and PB2 genes of the novel H7N9 viruses created by reverse genetics in an important mammalian host model. Finally, this study illustrates that loss-of-function mutations can be used to effectively identify residues critical to zoonosis/transmission.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Comparison of High Purity Factor IX Concentrates and a Prothrombin Complex Concentrate in a Canine Model of Thrombogenicity

1991 ◽  
Vol 66 (05) ◽  
pp. 609-613 ◽  
Author(s):  
I R MacGregor ◽  
J M Ferguson ◽  
L F McLaughlin ◽  
T Burnouf ◽  
C V Prowse
Keyword(s):  
High Purity ◽  
Time Course ◽  
Prothrombin Complex Concentrate ◽  
Degradation Products ◽  
Canine Model ◽  
Factor Ix ◽  
In Vitro Tests ◽  
Albumin Infusion

SummaryA non-stasis canine model of thrombogenicity has been used to evaluate batches of high purity factor IX concentrates from 4 manufacturers and a conventional prothrombin complex concentrate (PCC). Platelets, activated partial thromboplastin time (APTT), fibrinogen, fibrin(ogen) degradation products and fibrinopeptide A (FPA) were monitored before and after infusion of concentrate. Changes in FPA were found to be the most sensitive and reproducible indicator of thrombogenicity after infusion of batches of the PCC at doses of between 60 and 180 IU/kg, with a dose related delayed increase in FPA occurring. Total FPA generated after 100-120 IU/kg of 3 batches of PCC over the 3 h time course was 9-12 times that generated after albumin infusion. In contrast the amounts of FPA generated after 200 IU/kg of the 4 high purity factor IX products were in all cases similar to albumin infusion. It was noted that some batches of high purity concentrates had short NAPTTs indicating that current in vitro tests for potential thrombogenicity may be misleading in predicting the effects of these concentrates in vivo.

Evaluation of Structure-Controlled Silk Fibroin Coatings for Orthopedic Infection and In-Situ Osteogenesis: In Vitro and In Vivo

2020 ◽  
Author(s):  
Wenhao Zhou ◽  
Teng Zhang ◽  
Jianglong Yan ◽  
QiYao Li ◽  
Panpan Xiong ◽  
...  
Keyword(s):  
Silk Fibroin ◽  
Orthopedic Infection
Sign in / Sign up

Export Citation Format

Share Document