14 research outputs found
National Origin and Behavioural Problems of Toddlers: The Role of Family Risk Factors and Maternal Immigration Characteristics
In many societies the prevalence of behavioural problems in school-aged children varies by national origin. We examined the association between national origin and behavioural problems in 1½-year-old children. Data on maternal national origin and the Child Behavior Checklist for toddlers (n = 4943) from a population-based cohort in the Netherlands were used. Children from various non-Dutch backgrounds all had a significantly higher mean behavioural problem score. After adjustment for family risk factors, like family income and maternal psychopathology, the differences attenuated, but remained statistically significant. Non-Dutch mothers with immigration risk factors, such as older age at immigration or no good Dutch language skills, reported significantly more behavioural problems in their offspring. In conclusion, the present study indicated more behavioural problems in immigrant toddlers from various backgrounds. Researchers and policymakers aiming to tackle disparities in behavioural problems should take into account that risks associated with national origin are intertwined with unfavourable family and immigration characteristics
Use of a Chimeric Hsp70 to Enhance the Quality of Recombinant Plasmodium falciparum S-Adenosylmethionine Decarboxylase Protein Produced in Escherichia coli
S1 Fig. KPf and PfHsp70 do not co-purify with PfAdoMetDC. Western blot representing the
purification of PfAdoMetDC expressed in E. coli BL21 (DE3) Star cells rehosted with various chaperone
combinations. Lanes: U–PfAdoMetDC expressed in the absence of supplemented chaperones;
K–PfAdoMetDC co-expressed with supplemented DnaK; KPf–PfAdoMetDC expressed in
cells supplemented with KPf; Pf70 –PfAdoMetDC expressed in cells supplemented with PfHsp70;
K-EL–PfAdoMetDC expressed in cells supplemented with DnaK and GroEL-GroES; KP-EL–PfAdoMetDC
expressed in cells supplemented with KPf and GroEL-GroES; Pf70-EL–PfAdoMetDC
expressed in cells supplemented with PfHsp70 and GroEL-GroES; +C–positive consisting of purified
PfHsp70 protein.Western blot analysis of PfHsp70 (70 kDa) detected using α-PfHsp70 antibody.
Numbers to the left represent protein markers (Fermentas) in kDa.S2 Fig. Sequence alignment of PfHsp70 and E. coli DnaK. Sequence alignment of E. coli
DnaK (accession number: BAA01595.1) and PfHsp70 (accession number: PF08_0054) were
conducted using ClustalW and Boxshade. The following structural features are highlighted: the
highly conserved linker segment (black horizontal line) which separates the ATPase domain
from the peptide binding domain. Residues Y145, N147, D148, N170 and T173 in the ATPase
domain that interact with DnaJ as reviewed by Shonhai et al (8) are shown with black arrows.
Residues G400, D526 and G539 in the peptide binding domain of DnaK that are important for
interaction with DnaJ, and the aligned residues in PfHsp70 are shown as black arrows. Identical
residues are presented in white against a black background and similar residues are shown in
black against a grey background).S1 Table. E. coli strains and plasmids used in this study.S2 Table. Description of primers used towards generation of destination plasmids.S-adenosylmethionine decarboxylase (PfAdoMetDC) from Plasmodium falciparum is a prospective
antimalarial drug target. The production of recombinant PfAdoMetDC for biochemical
validation as a drug target is important. The production of PfAdoMetDC in Escherichia
coli has been reported to result in unsatisfactory yields and poor quality product. The coexpression
of recombinant proteins with molecular chaperones has been proposed as one
way to improve the production of the former in E. coli. E. coli heat shock proteins DnaK,
GroEL-GroES and DnaJ have previously been used to enhance production of some recombinant
proteins. However, the outcomes were inconsistent. An Hsp70 chimeric protein, KPf,
which is made up of the ATPase domain of E. coli DnaK and the substrate binding domain
of P. falciparum Hsp70 (PfHsp70) has been previously shown to exhibit chaperone function
when it was expressed in E. coli cells whose resident Hsp70 (DnaK) function was impaired.
We proposed that because of its domain constitution, KPf would most likely be recognised
by E. coli Hsp70 co-chaperones. Furthermore, because it possesses a substrate binding
domain of plasmodial origin, KPf would be primed to recognise recombinant PfAdoMetDC
expressed in E. coli. First, using site-directed mutagenesis, followed by complementation
assays, we established that KPf with a mutation in the hydrophobic residue located in its
substrate binding cavity was functionally compromised. We further co-expressed PfAdo-
MetDC with KPf, PfHsp70 and DnaK in E. coli cells either in the absence or presence of over-expressed GroEL-GroES chaperonin. The folded and functional status of the produced
PfAdoMetDC was assessed using limited proteolysis and enzyme assays. PfAdo-
MetDC co-expressed with KPf and PfHsp70 exhibited improved activity compared to
protein co-expressed with over-expressed DnaK. Our findings suggest that chimeric KPf may be an ideal Hsp70 co-expression partner for the production of recombinant plasmodial
proteins in E. coli.The
National Research Foundation for an equipment
grant (UID, 75464) awarded to AS. AS is a recipient
of a Georg Foster research fellowship awarded by the
Alexander von Humboldt Foundation of Germany.
XHM is a recipient of a National Research
Foundation (South Africa) scarce skills scholarship
and also received a grant from the University of Zululand Research Committee. AB is a recipient of a
postdoctoral fellowship awarded by the NRF.http://www.plosone.orgam2016BiochemistryUP Centre for Sustainable Malaria Control (UP CSMC