[Effect of prenatal stressful life event exposure on child emotional and behavioral problem at age 2-6 years].

Objective: To investigate the influence of prenatal stressful life event (SLE) exposure on child emotional and behavioral problem at age 2-6 years and identify the most risk exposure period. Methods: A total of 2 524 mother-child pairs were selected from Shanghai Maternal-Child Pairs Cohort based on pregnant women form 2016 to 2018 in Shanghai. Prenatal SLE exposure was assessed by Life Events Scale for Pregnant Women Questionnaire during the first and third trimester of pregnancy. Child emotional and behavioral problem was evaluated by Strengths and Difficulties Questionnaire at age 2-6 years. Multivariate binary logistic regression model and generalized estimating equation were conducted to quantify the association between prenatal SLE exposure and child emotional and behavioral problem at age 2-6 years, and identify the pregnancy period with strongest adverse effect. Results: The 2 524 mother-child pairs were divided into 4 groups: group with consistent low exposure to SLE (61.8%), group with high exposure to SLE in the first trimester (13.2%), group with high exposure to SLE in the third trimester (13.2%) and group with consistent high exposure to SLE (11.8%). The detection rates of emotional problem, hyperactivity, peer interaction problem and total difficulty score in children aged 3-6 years were highest in the group with consistent high exposure to SLE. Generalized estimating equation analysis showed that after controlling the confounding factors, compared with the consistent low exposure group, the children in the group with high exposure to SLE in the first trimester had significant increased risk for conduct problem at age 2-6 years (aOR=1.41, 95%CI:1.07-1.87). The children in the group with consistent high exposure to SLE were at increased risk for emotional problem, peer interaction problem, and high total difficulty score with the aOR of 1.41 (95%CI: 1.09-1.83), 1.46 (95%CI: 1.15-1.86) and 1.51(95%CI: 1.17-1.93). Conclusion: These findings indicated that prenatal exposure to SLE have adverse effect on child emotional and behavioral problem at age 2-6 years, especially the exposure in the first trimester.

The chaperone activity of trigger factor is distinct from its isomerase activity during co-expression with adenylate kinase in Escherichia coli.

To investigate the molecular chaperone function of trigger factor (TF) and its relationship with isomerase activity in vivo, the assisted folding of adenylate kinase (AK) by TF in Escherichia coli was examined by measuring the amounts of soluble AK produced during co-expression. When the mutant of chicken AK, P17G, is expressed in plasmid pBVAK, 95% of the protein is found in inclusion bodies. Co-expression of AK with TF was achieved using a plasmid pBVAT that allowed expression of TF and AK in the same plasmid under separate control. Co-expression with TF resulted in an increase in the amount of soluble AK, with a higher increase when TF was expressed at higher levels in the cell. Co-expression of AK with the two TF mutants, Y221G and F233Y, in which peptidyl-prolyl cis/trans isomerase activity was 1% of wild-type, gave the same results as wild-type TF. This provides in vivo evidence that the molecular chaperone activity of TF is distinct from its isomerase activity.

Conformational adjustments of SNase R and its N-terminal fragments probed by monoclonal antibodies.

Two monoclonal antibodies specific for staphylococcal nuclease R (SNase R) (McAb2C9 and McAb1B8) were prepared and used to probe protein folding during peptide elongation, by measuring antibody binding to seven N-terminal fragments (SNR141, SNR135, SNR121, SNR110, SNR102, SNR79 and SNR52) of SNase R. Comparative studies of the conformations of the N-terminal fragments have shown that all seven fragments of SNase R have a certain amount of residual structure, indicating that folding may occur during elongation of the nascent peptide chain. We show that the binding abilities of the intact enzyme and its seven fragments to the monoclonal antibodies are not simply proportional to the length of the peptide chain, suggesting that there may be continuous conformational adjustment in the nascent peptide chain as new C-terminal amino acids are added. A folding intermediate close in structure to the native state but with structural features in common with SNR121 is highly populated in 0.6 M GuHCl, and is also formed transiently during folding.

Conformational features of a truncated staphylococcal nuclease R (SNR135) and their implications for catalysis.

Conformational features of a truncated (14 amino acid residues deleted from the C-terminus) staphylococcal nuclease R (SNR135) and the ternary complex of SNR135-Ca2+-pdTp were studied using circular dichroism (CD) spectra and 1-anilinonaphthalene-8-sulfonate (ANS)-binding fluorescence spectra under different conditions. Kinetic parameters such as KDNAM, KDNAS, KCaM, KCaA, and KpdTpd of SNR135 were also determined. The results show that SNR135 contains some residual secondary structure and some tertiary structure elements as indicated by far-UV and near-UV CD spectra and that it has the ability to fold into a native-like state in the presence of pdTp and Ca2+, but there are obvious differences both in secondary structure and in tertiary structure between the SNR135-Ca2+-pdTp complex and SNase R. The unfolding curves in Gdn-HCl show that the stability of the native-like conformation of the SNR135-Ca2+-pdTp complex is much less than that of SNase R though the ligand (Ca2+, pdTp) binding increases the stability of the SNR135-Ca2+-pdTp complex to some extent. Comparison of the kinetic parameters of SNR135 with those of the full-length nuclease shows that both SNR135 and SNase R have the same value of KpdTpd and very similar values of KCaM and KCaA, but SNR135 has larger values of KDNAM and KDNAS than SNase R. Such results indicate that the C-terminal deletion for SNR135 does not greatly affect the ligand (Ca2+, pdTp) binding and decreases the binding affinity of the DNA substrate to the nuclease, implying that the amino acid residues at the ligand binding sites in SNR135 are probably arranged in a similar topology to those in SNase R and that effective binding of the DNA substrate to the enzyme needs the conformational integrity of the entire enzyme molecule. Furthermore, it is suggested that the binding sites of pdTp and DNA substrate may overlap but are not exactly the same. This paper also provides evidence obtained by monitoring ANS-binding fluorescence that the partially unfolded conformation of SNR135 is not in the molten globule state.

Free luciferase may acquire a more favorable conformation than ribosome-associated luciferase for its activity expression.

A variant of firefly luciferase in which the C-terminal end was extended with 44 amino acid residues served as a model protein in this study. After transcription and translation in vitro, the enzyme activity was measured when still attached to the ribosome and when released from the ribosome by incubation with RNase A or puromycin. It was found that the C-terminally extended luciferase already had activity when linked to the ribosome, but its activity was greatly increased when released from the ribosome. These results indicate that the luciferase is folded during synthesis on the ribosome; however, some conformational adjustments occur after its release from the ribosome which are required for the full expression of its enzymatic activity.

Soluble expression in Escherichia coli, one-step purification, and characterization of Chinese hamster dihydrofolate reductase.

Chinese hamster dihydrofolate reductase (ch-DHFR) was overexpressed in Escherichia coli DH5 alpha under the transcriptional control of PRPL promoters regulated by temperature-sensitive repressors. The desired recombinant product is soluble and constitutes about 30% of the total soluble proteins of the bacterial cell. With repeated cycles of freezing and thawing as a first step, the purification of the recombinant ch-DHFR to homogeneity requires only one further step, gel filtration on a Sephadex G-75 column with 85-90% enzyme recovery, two to three times higher than that obtained with the commonly used affinity chromatography on a methotrexate-Sepharose column. The purified enzyme migrates as a single protein band on SDS-polyacrylamide gel electrophoresis with approximate mass of 23 kDa, in accord with that calculated from the DNA sequence. The initiation methionine residue at the N-terminus of the enzyme is completely removed by E. coli methionine aminopeptidase as judged by amino-terminal analysis. The steady-state kinetic parameters, dissociation constants for binary complexes of dihydrofolate, NADPH, and methotrexate with ch-DHFR, and the inhibitor constant of methotrexate have also been determined. The enzyme is activated about 4-fold in 3 M urea and about 2.5-fold in 0.5 M guanidine hydrochloride.

Unfolding and refolding of the N-terminal fragments of staphylococcal nuclease R in guanidine hydrochloride.

The conformational and activity changes of a family of peptide fragments of staphylococcal nuclease R, which extend from residues -6 to 102, -6 to 110, -6 to 121, -6 to 135, and -6 to 141, during unfolding and refolding in different concentrations of guanidine hydrochloride have been studied. The studies indicate that the conformational stability in guanidine hydrochloride solution of the N-terminal fragment increases with increasing chain length, and that interaction and recognition between amino acid residues which are related to formation of the native conformation also increase with growth of the peptide chain, but such interaction becomes effective only when the polypeptide chain reaches a certain length. The changes in conformation and catalytic activity of the N-terminal fragments during unfolding and refolding demonstrate that conformational adjustments are necessary during chain elongation to generate the native conformation of a biologically active protein.

Reversible thermal denaturation of staphylococcal nuclease: a Fourier transformed infrared spectrum study.

The secondary structures of staphylococcal nuclease (SNase) have been assigned and semiquantitatively estimated from the deconvoluted Fourier transformed infrared (FTIR) spectrum. The changes in the secondary structures accompanying unfolding and refolding of SNase during reversible thermal denaturation up to 70 degrees C are followed by FTIR measurements. Only slight perturbation was observed up to 35 degrees C. The unfolding transition temperatures of beta-structure and alpha-helix are almost the same at 48.0-48.5 degrees C. During refolding the formation of the beta-structure follows the same pathway but that of the alpha-helix does not, although it recovers its original content almost completely. The final thermally denatured state at high temperature (60-70 degrees C) contains nonrandom structures in the complicated interaction, energetically resembling many kinds of structures. The occurrence of local conformational change before the final cooperative transition to the unfolded state during thermal denaturation as judged by FTIR spectra indicates that the unfolding and refolding of SNase may not follow the typical two-state model.

Comparative studies of the conformation of the N-terminal fragments of staphylococcal nuclease R in solution.

In order to elucidate the folding of nascent peptide, five peptide fragments of staphylococcal nuclease R starting from N-terminal end and of different chain lengths are made by deletion of 47, 39, 28, 14 and 8 amino-acid residues from its C-terminal end, respectively. Changes in conformation of the N-terminal fragments have been compared by using Fourier-transform infrared spectra, far-ultraviolet circular dichroism spectra and analysis of surface hydrophobicity. The experiments indicate that all the five fragments have certain amounts of residual structure; in general, with increasing the peptide chain, the contents of secondary structure and the enzyme's activity of the peptide increase, and the exposed hydrophobic side chains brought about by the deletion of C-terminal residues are gradually buried in the interior of the nuclease. However, the ordered secondary structures do not always increase with increasing the peptide chain. Further growth of the length of the peptide chain could have an important effect on the conformation of the peptide fragment already synthesized, suggesting some structural adjustments should be necessary in order for the newly synthesized polypeptide to attain its final native conformation. These results support Tsou's nascent peptide folding hypothesis (Tsou, C.-L. (1988) Biochemistry 27, 1809-1812).

A method for the preparation of size marker for synthetic oligonucleotides.

Terminal deoxynucleotidyltransferase was used for the addition of [alpha-32P]dCTP to the 3'-OH termini of oligo(dT)12-18. A collection of oligonucleotides with chain lengths ranging continuously from 13-mer to over 100-mer was generated. The reaction mixture was then mixed with oligo(dT)12-18 labeled with [gamma-32P]ATP by T4 polynucleotide kinase. A sequence ladder with the bottom base as 12-mer was then formed. These oligonucleotides served as size marker for the purification and identification of oligonucleotides on polyacrylamide gel.