Self-assembling Shell Proteins PduA and PduJ have Essential and Redundant Roles in Bacterial Microcompartment Assembly

Protein self-assembly is a common and essential biological phenomenon, and bacterial microcompartments present a promising model system to study this process. Bacterial microcompartments are large, protein-based organelles which natively carry out processes important for carbon fixation in cyanobacteria and the survival of enteric bacteria. These structures are increasingly popular with biological engineers due to their potential utility as nanobioreactors or drug delivery vehicles. However, the limited understanding of the assembly mechanism of these bacterial microcompartments hinders efforts to repurpose them for non-native functions. Here, we comprehensively investigate proteins involved in the assembly of the 1,2-propanediol utilization bacterial microcompartment from Salmonella enterica serovar Typhimurium LT2, one of the most widely studied microcompartment systems. We first demonstrate that two shell proteins, PduA and PduJ, have a high propensity for self-assembly upon overexpression, and we provide a novel method for self-assembly quantification. Using genomic knock-outs and knock-ins, we systematically show that these two proteins play an essential and redundant role in bacterial microcompartment assembly that cannot be compensated by other shell proteins. At least one of the two proteins PduA and PduJ must be present for the bacterial microcompartment shell to assemble. We also demonstrate that assembly-deficient variants of these proteins are unable to rescue microcompartment formation, highlighting the importance of this assembly property. Our work provides insight into the assembly mechanism of these bacterial organelles and will aid downstream engineering efforts.     

Starch content of etiolated radish (Raphanus sativus L.) hypocotyls as affected by chloramphenicol and external sucrose

Abstract

Starch was enzymatically assayed in the hypocotyls of radish (Raphanus sativus L.) seedlings grown in water or chloramphenicol (CAP) 1 × 10^4. CAP inhibits starch formation and its effect is related to the concentration. Both CAP- and water-grown hypocotyls are able to accumulate starch when sucrose is supplied in the medium, thus suggesting that the damage caused by CAP to the amyloplast is not irreversible. Apical segments of both water- and CAP- grown hypocotyls accumulate starch upon incubation in sucrose solutions while basal segments are unable to accumulate starch even in the presence of sucrose. The authors suggest that the basal segments are unable to perform sucrose uptake or that the amyloplast is incapable to starch synthesis. In any case the inability of the basal segment to perform sucrose uptake is independent of CAP. These findings confirm that the radish hypocotyls is not homogeneous along its axes.     

Critical role of bioanalytical strategies in investigation of clinical PK observations,a Phase I case study

RG7652 is a human immunoglobulin 1 (IgG1) monoclonal antibody (mAb) targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) and is designed for the treatment of hypercholesterolemia. A target-binding enzyme-linked immunosorbent assay (ELISA) was developed to measure RG7652 levels in human serum in a Phase I study. Although target-binding assay formats are generally used to quantify free therapeutic, the actual therapeutic species being measured are affected by assay conditions, such as sample dilution and incubation time, and levels of soluble target in the samples. Therefore, in the presence of high concentrations of circulating target, the choice of reagents and assay conditions can have a significant effect on the observed pharmacokinetic (PK) profiles. Phase I RG7652 PK analysis using the ELISA data resulted in a nonlinear dose normalized exposure. An investigation was conducted to characterize the ELISA to determine whether the assay format and reagents may have contributed to the PK observation. In addition, to confirm the ELISA results, a second orthogonal method, liquid chromatography tandem mass spectrometry (LC-MS/MS) using a signature peptide as surrogate, was developed and implemented. A subset of PK samples, randomly selected from half of the subjects in the 6 single ascending dose (SAD) cohorts in the Phase I clinical study, was analyzed with the LC-MS/MS assay, and the data were found to be comparable to the ELISA data. This paper illustrates the importance of reagent characterization, as well as the benefits of using an orthogonal approach to eliminate bioanalytical contributions when encountering unexpected observations.     

Evaluation of oxidative DNA damage and antioxidant defense in patients with nasal polyps

Abstract

Objectives

The presence of inflammatory cells indicates the development of epithelial cell injury in nasal polyposis (NP) and the potential for production of high levels of reactive oxygen and nitrogen species. The aim of our study was to clarify the role of oxidative stress and antioxidant status in the deterioration accompanying NP.

Methods

Twenty patients (11 men) aged 47.2 ± 17.0 years with nasal polyps were included in the study. Twenty healthy subjects (7 men) aged 48.2 ± 15.3 years formed the control group. The erythrocyte activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and plasma nitric oxide (NO) concentrations were measured. An alkaline comet assay was used to determine the extent of blood lymphocyte DNA damage of oxidized purines as glicosylo-formamidoglicosylase (Fpg) sites, and oxidized pyrimidines as endonuclease III (Nth) sites.

Results

A significant increase of NO (P < 0.05) and non-significant decreases of SOD (P > 0.05), CAT (P > 0.05), and GPx (P > 0.05) were seen in NP patients compared to healthy controls. The level of blood lymphocyte oxidative DNA damage in NP patients was significantly higher compared to the control group (P = 0.01).

Discussion

The blood lymphocyte DNA damage level increased in patients with NP. Elevated DNA damage may be related to overproduction of reactive oxygen and nitrogen species and/or decreased antioxidant protection.     

Coming soon to a journal near you—The updated guidelines for the use and interpretation of assays for monitoring autophagy

In 2008 we published the first guidelines paper for monitoring autophagy and interpreting the data resulting from the various assays used in our field. The guidelines paper was substantially expanded and updated in 2012. Based on the number of citations, and on comments from many users, I think it is accurate to say that the guidelines have been very useful for many researchers. Because the field continues to undergo rapid development, it is necessary to update the guidelines once again.     

Comparison Between Different Assays for Superoxide Dismutase-Like Activity

The direct and indirect methods for assaying the superoxide dismutase activity of a compound are compared. With the use of a direct method. the mechanism of the catalysis of O₂-dismutation by the tested compound can be determined. while with the indirect method it cannot. and this may lead to misinterpretation of the results. Assuming that the catalysis occurs via the ‘ping-pong’ mechanism, both the direct and indirect methods are limited to the determination of values of k_cat ≥ 10⁵M^?1s^?1 and k_cat ≥ 3 × 10⁶M^?1s^?1. respectively. Moreover, many side reactions may occur with the indirect method which may interfere with the measurements. Nevertheless. the indirect method approximates better the in vivo conditions than the direct method, and a tested compound that has high SOD activity using a direct method and low SOD activity using an indirect method. will most probably be a poor SOD mimic in vivo.     

Synthesis,anticancer assessment on human breast,liver and colon carcinoma cell lines and molecular modeling study using novel pyrazolo[4,3-c]pyridine derivatives

The key intermediate 3-aminopyrazolo[4,3-c]pyridine-4,6-dione (2) is considered as a precursor for some novel pyrazolo[4,3-c]pyridines 4a-c, arylhydrazopyrazolo[4,3-c]pyridines 8a-e, pyrazolo[4,5,1-ij][1,6]naphthyridines 11a-e and pyrido[4′,3′:3,4]pyrazolo[1,5-a]-pyrimidines 15a-d through Knovenegal condensation, coupling reaction and Michael addition. Some of the newly synthesized pyrazolo[4,3-c]pyridine derivatives were investigated for anticancer activity. The results of the cytotoxic activity revealed that compound 6b was the most active compound against the breast and liver carcinoma cell lines which gives IC₅₀ values of 1.937 and 3.695 µg/mL, respectively compared to reference drug (doxorubicin) with IC₅₀ values of 2.527 and 4.749 µg/ml, respectively. Moreover, compound 6c was potent compound against the colon carcinoma cell line which gives the value of IC₅₀ = 2.914 µg/ml compared to doxorubicin with IC₅₀ value of 3.641 µg/ml. Some selected of the novel synthesized compounds were docked inside the active site of ERK2 enzyme and were found display a suitable binding with the active site amino acids according to their bond lengths, angles and conformational energy.     

Synthesis of an insulin analogue embodying a strongly fluorescent moiety, [19-Tryptophan-A]insulin

As part of our aim to study the conformation of insulin in solution by time-resolved fluorescence spectroscopy, we have synthesized the analogue [19-Tryptophan-A]insulin. In this compound, the tyrosine residue at position 19 of the A-chain of insulin, one of the most strongly conserved residues in insulins from various species, is substituted with the strongly fluorescent tryptophan residue. [19-Tryptophan-A]insulin displays 4.1±1.9% of the potency of natural insulin in binding to the insulin receptor from rat liver plasma membranes, 5.0±2.3% in stimulating lipogenesis in rat adipocytes, and 75.7±4% of the potency of insulin in radioimmunoassay. In connection with our previous work, these data indicate that an aromatic side chain at position A¹⁹ of insulin seems necessary but not sufficient for high biological activity. We further conclude that in regard to the immunogenic determinants of insulin, tryptophan in position A¹⁹ is an essentially neutral substitution for tyrosine in that position, in sharp contrast to the situation with regard to biological activity.