62 research outputs found
Protein tyrosine kinases in Schistosoma mansoni
The identification and description of signal transduction molecules and mechanisms are essential to elucidate Schistosoma mansoni host-parasite interactions and parasite biology. This mini review focuses on recent advancements in the study of signalling molecules and transduction mechanisms in S. mansoni, drawing special attention to the recently identified and characterised protein tyrosine kinases of S. mansoni.Fiocruz Centro de Pesquisas René RachouSanta Casa de Misericórdia de Belo Horizonte Programa de Pós-Graduação e PesquisaUniversidade Federal de São Paulo (UNIFESP), Escola Paulista de Medicina (EPM)UNIFESP, EPMSciEL
Mixoma cutâneo em um pintagol
Mixomas são tumores mesenquimais benignos incomuns em aves. Este trabalho objetiva descrever os achados clínico-patológicos de um caso de mixoma em um pintagol. A ave apresentou aumento de volume na região dorsal do terceiro dígito do membro pélvico esquerdo. Macroscopicamente, notou-se um nódulo de 0,9x0,5x0,4cm, macio, esbranquiçado, com áreas amareladas e enegrecidas na superfície de corte. A histopatologia revelou população monomórfica de células fusiformes, com baixo pleomorfismo, arranjadas em meio à matriz mixóide positiva para a coloração de azul alciano. Com base nos achados histopatológicos, foi firmado o diagnóstico de mixomaMyxomas are benign mesenchymal tumors rarely described in birds. This report describes the clinical and pathological findings in a case of myxoma in a pintagol (Sporagra magellanica X Serinus canaria). The animal had a nodule on the dorsal region of the third digit on the left hindlimb. Grossly, it was a 0.9×0.5×0.4cm, soft, white nodule, with black and yellow areas on the cut surface. Microscopically, a well-differentiated monomorphic population of spindle cells arranged in an abundant Alcian blue-positive myxoid matrix was observed. The diagnosis of myxoma was based on the microscopic findings
Serological screening of the Schistosoma mansoni adult worm proteome
BackgroundNew interventions tools are a priority for schistosomiasis control and elimination, as the disease is still highly prevalent. The identification of proteins associated with active infection and protective immune response may constitute the basis for the development of a successful vaccine and could also indicate new diagnostic candidates. In this context, post-genomic technologies have been progressing, resulting in a more rational discovery of new biomarkers of resistance and antigens for diagnosis.Methodology/Principal FindingsTwo-dimensional electrophoresed Schistosoma mansoni adult worm protein extracts were probed with pooled sera of infected and non-infected (naturally resistant) individuals from a S. mansoni endemic area. A total of 47 different immunoreactive proteins were identified by mass spectrometry. Although the different pooled sera shared most of the immunoreactive protein spots, nine protein spots reacted exclusively with the serum pool of infected individuals, which correspond to annexin, major egg antigen, troponin T, filamin, disulphide-isomerase ER-60 precursor, actin and reticulocalbin. One protein spot, corresponding to eukaryotic translation elongation factor, reacted exclusively with the pooled sera of non-infected individuals living in the endemic area. Western blotting of two selected recombinant proteins, major egg antigen and hemoglobinase, showed a similar recognition pattern of that of the native protein.Concluding/SignificanceUsing a serological proteome analysis, a group of antigens related to the different infection status of the endemic area residents was identified and may be related to susceptibility or resistance to infection
Use of humanised rat basophilic leukaemia cell line RS-ATL8 for the assessment of allergenicity of Schistosoma mansoni proteins.
BACKGROUND
Parasite-specific IgE is thought to correlate with protection against Schistosoma mansoni infection or re-infection. Only a few molecular targets of the IgE response in S. mansoni infection have been characterised. A better insight into the basic mechanisms of anti-parasite immunity could be gained from a genome-wide characterisation of such S. mansoni allergens. This would have repercussions on our understanding of allergy and the development of safe and efficacious vaccinations against helminthic parasites.
METHODOLOGY/PRINCIPAL FINDINGS
A complete medium- to high-throughput amenable workflow, including important quality controls, is described, which enables the rapid translation of S. mansoni proteins using wheat germ lysate and subsequent assessment of potential allergenicity with a humanised Rat Basophilic Leukemia (RBL) reporter cell line. Cell-free translation is completed within 90 minutes, generating sufficient amounts of parasitic protein for rapid screening of allergenicity without any need for purification. Antigenic integrity is demonstrated using Western Blotting. After overnight incubation with infected individuals' serum, the RS-ATL8 reporter cell line is challenged with the complete wheat germ translation mixture and Luciferase activity measured, reporting cellular activation by the suspected allergen. The suitability of this system for characterization of novel S. mansoni allergens is demonstrated using well characterised plant and parasitic allergens such as Par j 2, SmTAL-1 and the IgE binding factor IPSE/alpha-1, expressed in wheat germ lysates and/or E. coli. SmTAL-1, but not SmTAL2 (used as a negative control), was able to activate the basophil reporter cell line.
CONCLUSION/SIGNIFICANCE
This method offers an accessible way for assessment of potential allergenicity of anti-helminthic vaccine candidates and is suitable for medium- to high-throughput studies using infected individual sera. It is also suitable for the study of the basis of allergenicity of helminthic proteins
Avaliação do efeito do revestimento à base de quitosana na conservação pós-colheita do Umbu / Evaluation of the effect of chitosan-based coating on the post-harvest conservation of Umbu
Para manter a qualidade e estender o tempo de prateleira das frutas, as indústrias têm buscado novas técnicas de aprimoramento, como o uso de revestimentos comestíveis. A quitosana é um biopolímero promissor nessa área, por apresentar atividade antimicrobiana, características físico-químicas e biodegradáveis pertinentes para a utilização como revestimento comestível. O umbu é um fruto da região semiárida brasileira que tem importância econômica e nutricional contribuindo para a geração de renda da região Nordeste do Brasil. Neste estudo foi realizada a avaliação do efeito do revestimento comestível à base de quitosana sobre o tempo de prateleira dos umbus. Para tal, os frutos foram imersos numa solução de quitosana 1% (p/v) em ácido acético 1% (v/v) por 2 min à 25°C. Grupos com e sem revestimento foram estocados à 8 °C por 15 dias e as análises de perda de massa, acidez total titulável, pH, sólidos solúveis totais e parâmetros de cor (L e ºH) foram realizados. Os resultados revelaram que durante o período de armazenamento houve redução da perda de massa, aumento da acidez titulável e diminuição do pH nos frutos revestidos em comparação com o grupo sem revestimento (controle). Contudo, nenhuma diferença foi encontrada nas análises colorimétricas, nem nos sólidos solúveis totais. O estudo sugere que o revestimento de quitosana pode ser um candidato promissor para aumentar a vida de prateleira dos umbus.
Vaccination with a CD4+ and CD8+ T-cell epitopes-based recombinant chimeric protein derived from Leishmania infantum proteins confers protective immunity against visceral leishmaniasis.
Vaccination seems to be the best approach to control visceral leishmaniasis (VL). Resistance against infection is based on the development of a Th1 immune response characterized by the production of interferons-? (IFN-?), interleukin-12 (IL-12), granulocyte-macrophage-colony-stimulating factor (GM-CSF), and tumor necrosis factor-? (TNF-?), among others. A number of antigens have been tested as potential targets against the disease; few of them are able to stimulate human immune cells. In the present study, 1 prediction of MHC class I and II molecules-specific epitopes in the amino acid sequences of 3 Leishmania proteins: 1 hypothetical, prohibitin, and small glutamine-rich tetratricopeptide repeat-containing proteins, was performed using bioinformatics tools, and a T-cell epitopes-based recombinant chimeric protein was constructed, synthetized and purified to be evaluated in invitro and in vivo experiments. The purified protein was tested regarding its immunogenicity in peripheral blood mononuclear cells (PBMCs) from healthy subjects and VL patients, as well as to its immunogenicity and protective efficacy in a murine model against Leishmania infantum infection. Results showed a Th1 response based on high IFN-? and low IL-10 levels derived from in chimera-stimulated PBMCs in both healthy subjects and VL patients. In addition, chimera and/or saponin-immunized mice presented significantly lower parasite burden in distinct evaluated organs, when compared to the controls, besides higher levels of IFN-?, IL-2, IL-12, and GM-CSF, and an IgG2a isotype-based humoral response. In addition, the CD4+ and CD8+ T-cell subtypes contributed to IFN-? production in the protected animals. The results showed the immunogenicity in human cells and the protective efficacy against L. infantum in a murine model, and well indicate that this recombinant chimera can be considered as a promising strategy to be used against human disease
In vivo antileishmanial efficacy of a naphthoquinone derivate incorporated into a Pluronic? F127-based polymeric micelle system against Leishmania amazonensis infection.
New therapeutic strategies against leishmaniasis are desirable, since the treatment against disease presents
problems, such as the toxicity, high cost and/or parasite resistance. As consequence, new antileishmanial
compounds are necessary to be identified, as presenting high activity against Leishmania parasites, but low
toxicity in mammalian hosts. Flau-A is a naphthoquinone derivative recently showed to presents an in vitro
effective action against Leishmania amazonensis and L. infantum species. In the present work, the in vivo efficacy
of Flau-A, which was incorporated into a Poloxamer 407-based micelle system, was evaluated in a murine model
against L. amazonensis infection. Amphotericin B (AmB) and Ambisome? were used as controls. The animals were
infected and later treated with the compounds. Thirty days after the treatment, parasitological and immunological parameters were evaluated. Results showed that AmB, Ambisome?
, Flau-A or Flau-A/M-treated
animals presented significantly lower average lesion diameter and parasite burden in tissue and organs evaluated, when compared to the control (saline and micelle) groups. Flau-A or Flau-A/M-treated mice were those
presenting the most significant reductions in the parasite burden, when compared to the others. These animals
developed also a more polarized antileishmanial Th1 immune response, which was based on significantly higher
levels of IFN-?, IL-12, TNF-?, GM-CSF, and parasite-specific IgG2a isotype; associated with low levels of IL-4, IL10, and IgG1 antibody. The absence of toxicity was found in these animals, although mice receiving AmB have
showed high levels of renal and hepatic damage markers. In conclusion, results suggested that the Flau-A/M
compound may be considered as a possible therapeutic target to be evaluated against human leishmaniasis
Caracterização molecular de uma nova proteína tirosina quinase, SmFes, de Schistosoma mansoni.
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Previous issue date: 2005Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil.A identif
icação de moléculas sinalizadoras
e a elucidação
de processo
s de transdução
de sinal
de
Schistosoma
mansoni
são necessárias
para o entendi
mento da interação
hospedeiro-
parasito
e de proces
sos fisiológicos
do parasito.
Proteínas
tirosinas
quinase
(PTK
s) são
molécu
las importan
tes na comunica
ção intra e intercelu
lar, tendo
um papel
crucial
nos
mecan
ismos de transdução
de sinal.
Estas
proteínas
estão
envolvidas
nos processo
s de
desenvolvim
ento,
diferenciação
e comuni
cação
celular
entre
outros.
Uma
nova
PTKs foi
identificad
a em
S. mansoni
e nomeada,
SmFes.
SmFes
é um gene de cópia
única
e o seu
cDNA comple
to contém
uma ORF
de 3.780
pb. SmFes
exibe
característi
cas da família
Fes/Fps/Fer
das PTKs,
com assinaturas
de domín
io proteína
quinase,
SH2 e
coiled-
coil
característ
icos. SmFes
é o prime
iro gene da família Fes/Fp
s/Fer
identificado
em
S. mansoni.
Análise
filogenétic
a revelou
que SmFes
está relacionada
a proteínas
da família Fes/Fps/Fer,
agrupadas
mais próximas
às proteínas
ortólogas
de organismos
invertebrados.
O alinham
ento
entre
o cDNA de SmFes
e seqüências
genôm
icas depositadas
pelos
bancos
de dados
de
S.
mansoni
indicara
m a presença
de 17 introns,
alguns
demonstrados
experi
mentalmente.
Análises
parciais
de clones
de cDNA
indicara
m a inserção
de 9 pb na posição
3’ do exon 10,
formando
duas populações
de cDNA,
comprovadas
por RFLP.
A análise
da seqüência
genômic
a indicou
que existem
dois possíveis
sítios
de edição
do RNA na proximidad
e 5’ do
intron,
indicando
um evento
de edição
alternat
iva. A existência
de um alelo
contendo
uma
inserção
de 15 pb na seqüência
genômi
ca foi observada.
As amplif
icaçõ
es de diferentes
espécies
de
Schistosoma
com pares
de iniciador
es desenhados
para SmFes
indicara
m a
presença
de ortólogos
de SmFes
em várias
espécies
do gênero.
SmFes
é transcrita
em baixos
níveis
nas diversas
fases
de desenvolvi
mento do parasito,
com uma maior taxa de transcrição
em verm
es machos,
como
detectado
por PCR em tempo
real e
northern-
blot.
A expressão
da
proteína
SmFes
foi verificada
em esporocisto,
mirac
ídio, verme
adulto
e em nível
mais
elevado
em cercári
a. Uma proteína
recombinan
te de SmFes
foi expressa
para ser usada
em
experim
entos
de identif
icação de parceiros
de SmFes
em futuros
ensaios
de co-precipitaç
ão e
també
m em experim
entos
de duplo
híbrido.
Vários
possíveis
parceiros
de SmFes
foram
identificados
por análise
compu
tacion
al. A compre
ensão
de genes
envolvidos
nos
mecan
ismos de transdução
de sinal pode
fornecer
novas
estratégias
de desenvolvi
mento
de
drogas.The identifi
cation and elucida
tion of signal
transduction
molecules
and mechan
isms are
necessary
for the understanding
of the
Schistosoma
mansoni
host-parasite
interac
tion and of
physiologica
l process
of the parasite.
Protein
Tyrosine kinases
(PTKs)
are impor
tant
molecu
les for intra
and interce
llular comm
unication,
playing a crucial
role in signal
transduction
processes.
These
proteins
are known
to be involved
in developm
ental,
differentiat
ion and communication
processes
of cells,
among
others.
A novel
member of PTK
was identified
in
S. mansoni
and designated
SmFes.
SmFes
is a single
copy
gene and the
comple
te cDNA contains
a 3.780
bp ORF. SmFes
exhibits
the characteristi
c features
of
Fes/Fps/Fer
protein
tyrosine kinases
family, containing
three
coiled-
coil regions,
an SH2 and
a protein
tyrosine kinase catal
ytic doma
in signatures.
SmFes
is the first gene
from
Fes/Fps/Fer
family identified
in
S. mansoni.
Phylogene
tic analyses reveled
that SmFes
is more
closel
y
related
to invertebrate
proteins
orthologues
of the Fes/Fps/Fer
family. The assembly of the
SmFes
cDNA
and genomi
c sequences
indicated
the presence
of 17 introns,
some
experim
entally demonstra
ted. Analy
sis of partial
cDNA clones
indicated
the presence
of a 9
pb insertion
at the 3’ end of exon
10, producing
two different
populations
of cDNA. The
analysis of the genomi
c sequence
indicated
that there
are two possible
splice
sites at the 5’end
of the intron,
pointing
to an alternative
splicing
event
in the SmFes
pre-mRNA.
An allele
of
the SmFes
containing
a 15 pb insertion
was observed
in genomi
c sequence.
The amplification
of different
Schistosoma
species
with SmFes
specific
primers
indicat
ed that orthologues
of
SmFes
are present
in several
species
of the genus.
SmFes
is transcribed
at low levels
in the
different
develop
mental stages
of the parasite,
with higher
level
in adult
male worms
as
detected
by real time PCR and
northern-blot
. The expression
of SmFes protein
was verified
in
sporoc
yst, mirac
idium, adult
worm and in higher
levels
in cercariae.
A recombin
ant protein
was expressed
to be used in experi
ments to identif
y SmFes
partner
proteins
in future
pull-
down assay
s and also in two-h
ybrid systems. Various
possible
partners
or SmFes
were
identified
by computa
tional analy
sis. The comprehension
of signal
transduction
processes
could also
contribute
to the
identific
ation
of po
ssible
new targets
for drug
s
- …