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McGuire Dotson posted an update 6 months, 1 week ago
Human apurinic/apyrimidinic endonuclease APE1 is one of participants of the DNA base excision repair pathway. APE1 processes AP-sites and many other types of DNA damage via hydrolysis of the phosphodiester bond on the 5′ side of the lesion. APE1 also acts as an endoribonuclease, i.e., can cleave undamaged RNA.
Using pre-steady-state kinetic analysis we examined the role of certain catalytically important amino acids in APE1 enzymatic pathway and described their involvement in the mechanism of the target nucleotide recognition.
Comparative analysis of the cleavage efficiency of damaged DNAs containing an abasic site, 5,6-dihydrouridine, or α-anomer of adenosine as well as 3′-5′-exonuclease degradation of undamaged DNA and endonuclease hydrolysis of RNA substrates by mutant APE1 enzymes containing a substitution of an active-site amino acid residue (D210N, N212A, T268D, M270A, or D308A) was performed. Detailed pre-steady-state kinetics of conformational changes of the enzyme and of DNA substrate molecules during recognition and cleavage of the abasic site were studied.
It was revealed that substitution T268D significantly disturbed initial DNA binding, whereas Asn212 is critical for the DNA-bending stage and catalysis. Substitution D210N increased the binding efficacy and blocked the catalytic reaction, but D308A decreased the binding efficacy owing to disruption of Mg
coordination. Finally, the substitution of Met270 also destabilized the enzyme-substrate complex but did not affect the catalytic reaction.
It was found that the tested substitutions of the active-site amino acid residues affected different stages of the complex formation process as well as the catalytic reaction.
It was found that the tested substitutions of the active-site amino acid residues affected different stages of the complex formation process as well as the catalytic reaction.
In the endoplasmic reticulum (ER), folding of glycoproteins is assisted by a combined action of enzymes and chaperones that leads them to biologically functional structures. In this system, UDP-glucoseglycoprotein glucosyltransferase 1 (UGGT1) plays an essential role as the “folding sensor” by virtue of its ability to discriminate folding states of client glycoproteins. However, besides its transferase activity, whether UGGT1 possesses any chaperone activity that facilitates protein folding is yet to be addressed.
We prepared oligomannose-type glycan modified RNase (M9GN2-RNase) by chemoenzymatic means using M9GN-oxazoline and glycan truncated RNase B and analyzed the effect of human UGGT1 (HUGT1) for refolding of the denatured M9GN2-RNase. Refolding was evaluated based on the RNase activity which was measured by the cleavage of the RNA substrate.
HUGT1 slightly accelerated the folding of M9GN2-RNase and non-glycosylated RNase A as the same extent. However, HUGT1 remarkably accelerated the folding of M9GN2-RNase in the presence of UDP-Glc. DibutyrylcAMP In contrast, neither UDP nor UDP-Gal was effective in enhancing the folding. Additionally, an HUGT1 mutant which lacks the glucosyltransferase activity did not accelerate the protein folding of M9GN2-RNase.
HUGT1has the ability to promote the refolding of denatured protein and the effect would be enhanced when HUGT1 tightly interacts with the client protein via glycan recognition.
Our study provides a possibility that HUGT1 play a role not only in sensing the misfolded glycoprotein but also in promoting folding of glycoproteins in the endoplasmic reticulum glycoprotein quality control.
Our study provides a possibility that HUGT1 play a role not only in sensing the misfolded glycoprotein but also in promoting folding of glycoproteins in the endoplasmic reticulum glycoprotein quality control.
To describe our technique of simultaneous hand-assisted laparoscopic bilateral native nephrectomy (BNN) and kidney transplantation (KT) in patients with autosomal dominant polycystic kidney disease and present our experience.
We retrospectively reviewed a cohort of adult ESRD patients with symptomatic autosomal dominant polycystic kidney disease who underwent a hand-assisted laparoscopic BNN at the time of KT. We reviewed patients’ and donor characteristics, and perioperative and postoperative outcomes.
A total of 52 patients underwent hand-assisted laparoscopic BNN at the time of KT from January 2014 to October 2019. The median age of the recipients was 53.4 years, 57.7% were males, and the median body mass index was 29.0 kg/m
. All but one received a kidney from a living donor and the majority (86.5%) were pre-emptive. One patient required a small bowel resection due to an intraoperative small bowel injury. There was no solid organ injury during the procedure. All patients showed immediate allograft function and a steady decline in serum creatinine. The median decline in the creatinine and hemoglobin on day 1 was 1.2 mg/dL (inter quartile range 0.6-2.3) and 2.2 g/dL (inter quartile range 1.4-3.0), respectively.
Simultaneous hand-assisted laparoscopic bilateral nephrectomy with KT through a modified Gibson incision is feasible and safe in the hands of an experienced laparoscopic surgeon without compromising allograft function.
Simultaneous hand-assisted laparoscopic bilateral nephrectomy with KT through a modified Gibson incision is feasible and safe in the hands of an experienced laparoscopic surgeon without compromising allograft function.Autism spectrum disorders (ASDs) are a group of heterogenous neurodevelopmental disorders affecting 1 in 59 children. Syndromic ASDs are commonly associated with chromosomal rearrangements or dosage imbalance involving a single gene. Many of these genes are dosage-sensitive and regulate transcription, protein homeostasis, and synaptic function in the brain. Despite vastly different molecular perturbations, syndromic ASDs share core symptoms including social dysfunction and repetitive behavior. However, each ASD subtype has a unique pathogenic mechanism and combination of comorbidities that require individual attention. We have learned a great deal about how these dosage-sensitive genes control brain development and behaviors from genetically-engineered mice. Here we describe the clinical features of eight monogenic neurodevelopmental disorders caused by dosage imbalance of four genes, as well as recent advances in using genetic mouse models to understand their pathogenic mechanisms and develop intervention strategies.
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