Physiological and Biochemical Parameters of Common Duckweed Lemna minor after the Exposure to Tetracycline and the Recovery from This Stress

In this study, the ability of Lemna minor L. to recover to normal growth, after being degraded in a tetracycline-containing medium, was extensively investigated. The plants were exposed to tetracycline (TC) at concentrations of 1, 2.5, and 10 mM. Subsequently, their physiological status was analysed against the following criteria: rate of plant growth; free radical accumulation; antioxidant enzyme activity; chlorophyll content; HSP70 protein content; cell membrane permeability, and mitochondrial activity. The study showed that duckweed can considerably recover from the damage caused by antibiotics, within a week of cessation of stress. Of the plant properties analysed, mitochondrial activity was the most sensitive to antibiotic-induced disturbances. After transferring the plants to a tetracycline-free medium, all plant parameters improved significantly, except for the mitochondrial activity in the plants grown on the medium containing the highest dose of tetracycline. In the plants treated with this antibiotic at the concentration of 10 mM, the proportion of dead mitochondria increased and was as high as 93% after one week from the beginning of the recovery phase, even after the transfer to the tetracycline-free medium.

A novel multifunctional role for Hsp70 in binding post-translational modifications on clients

Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and inability of current technologies to distinguish direct vs bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the budding yeast Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multi-point interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors which can be used to probe chaperone function in cells, we have also identified a suite of PTM-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically-important PTMs on client proteins.

Identification and Characterization of MortaparibPlus—A Novel Triazole Derivative That Targets Mortalin-p53 Interaction and Inhibits Cancer-Cell Proliferation by Wild-Type p53-Dependent and -Independent Mechanisms

p53 has an essential role in suppressing the carcinogenesis process by inducing cell cycle arrest/apoptosis/senescence. Mortalin/GRP75 is a member of the Hsp70 protein family that binds to p53 causing its sequestration in the cell cytoplasm. Hence, p53 cannot translocate to the nucleus to execute its canonical tumour suppression function as a transcription factor. Abrogation of mortalin-p53 interaction and subsequent reactivation of p53’s tumour suppression function has been anticipated as a possible approach in developing a novel cancer therapeutic drug candidate. A chemical library was screened in a high-content screening system to identify potential mortalin-p53 interaction disruptors. By four rounds of visual assays for mortalin and p53, we identified a novel synthetic small-molecule triazole derivative (4-[(1E)-2-(2-phenylindol-3-yl)-1-azavinyl]-1,2,4-triazole, henceforth named MortaparibPlus). Its activities were validated using multiple bioinformatics and experimental approaches in colorectal cancer cells possessing either wild-type (HCT116) or mutant (DLD-1) p53. Bioinformatics and computational analyses predicted the ability of MortaparibPlus to competitively prevent the interaction of mortalin with p53 as it interacted with the p53 binding site of mortalin. Immunoprecipitation analyses demonstrated the abrogation of mortalin-p53 complex formation in MortaparibPlus-treated cells that showed growth arrest and apoptosis mediated by activation of p21WAF1, or BAX and PUMA signalling, respectively. Furthermore, we demonstrate that MortaparibPlus-induced cytotoxicity to cancer cells is mediated by multiple mechanisms that included the inhibition of PARP1, up-regulation of p73, and also the down-regulation of mortalin and CARF proteins that play critical roles in carcinogenesis. MortaparibPlus is a novel multimodal candidate anticancer drug that warrants further experimental and clinical attention.

Scutellaria barbata flavonoids improve the composited Aβ-induced abnormal changes of glial cells in rats’ brain

Aim: It has been reported that glial cells are involved in Alzheimer’s disease (AD). According to our previous research, Scutellaria barbata flavonoids (SBFs) can protect the neuronal disorder and memory impairment for AD-like rats, while the effect of SBFs on the glial cells disorder in AD-like rats has been less well studied. The effects of SBFs on astrocytes(ASs), microglial cells (MGs) and oligodendrocytes (Ols), as well as heat shock proteins 70 (Hsp70) and apolipoprotein E (ApoE) were investigated in the present study. Methods: The successful model rats, screened by Morris water maze, were daily orally administrated with 35, 70 and 140 mg/kg SBFs for 36 d. The numbers of brain’s astrocytes (ASs), microglial cells (MGs) and oligodendrocytes (Ols) were examined by immunohistochemistry. The cortical glial fibrillary acidic protein (GFAP), leukocyte common antigen (LCA) (CD45), Claudin 11 and heat shock proteins 70 (Hsp70) protein expression were assayed by Western blotting, and apolipoprotein E (ApoE) mRNA expression was analyzed by real-time quantitative polymerase chain reaction (qPCR). Results: Compared with the sham-operated group, the numbers of ASs and MGs in the brain were significantly increased in the model group (P<0.05, P<0.01), and accompanied with increases of GFAP, CD45 and Hsp70 protein and ApoE mRNA expression (P<0.05, P<0.01). Both Ols number and Claudin 11 protein expression decreased in the brain in the model group (P<0.05, P<<.01). However, the above abnormal changes induced by composited Aβ were differently reversed by treatment of SBFs at three doses of 35, 70 and 140 mg/kg (P<0.05, P<0.01). Conclusions: SBFs can dramatically improve the abnormal changes of glial cells in rats’ brain induced by composited Aβ, which may be a helpful treatment of neurodegenerative diseases.

Salivary HSP70 and Progesterone are Upregulated in Preeclamptic Antenatal Mothers

Introduction and Aim: Most of the pregnancies are complicated with complexities due to the environment, psychological, physiological circumstances. It is crucial to diagnose the pregnant women for complications during the earlier stages of pregnancy. Most importantly, the ability to monitor health status, disease onset, and the progression and treatment outcome through non-invasive means is a highly desirable goal in healthcare management. In that, the saliva represents a suitable, potential, and alternative biological filtrate/diagnostic fluid/medium for exploring the surveillance of health and disease. Also, it offers further opportunities as a pool containing a vast repertoire of specific, biologically-active peptides and proteins. Since hypertension represents the major risk contributing to diverse pregnancy complications; the present study is aimed to quantify the level of HSP70 and Progesterone in the saliva of normotensive and preeclamptic antenatal mothers. Materials and Methods: A saliva sample was collected from normotensive and preeclamptic antenatal mothers (n=10 each group) by passive drool method. Progesterone and HSP70 were quantified using ELISA kits. Results: The present study shows an increase in the expression of HSP70 and Progesterone in the salivary sample of preeclamptic pregnant women. Conclusion: Results with a higher value of HSP70 protein expression suggests that salivary HSP70 may serve as a novel and valuable marker in diagnosing pregnancy complicated with hypertensive disorders and salivary progesterone may be used as an indicator for imminent delivery in pregnant women.

Comparative Homology Modeling and Physicochemical Characterization of Cyprinus carpio Hsp70 Protein

Background: Expression of the heat shock proteins (Hsp) is responsible for the protection from adverse climatic changes particularly heat stress in Common carp (Cyprinus carpio). Although, with advancement of molecular techniques, Hsp70 protein has been isolated but this protein needs to be characterized by both physicochemically and structurally for the functional annotation of fish genome. So this current study was undertaken with aim of generation of various protein models and also for thorough physiochemical characterization of this protein.Methods: In this study, Hsp70 protein of common carp was characterized by both physiochemical and structurally through insilco platform and as the crystal structure of this protein is not available, protein models were created though homology modelling upon Modeller version 9.21, Phyre2 and Swiss-model and then the generated predicted models were evaluated through Rampage, Errat, Verify 3D, ProQ and ProSA analysis.Result: Our investigation showed that this protein is very stable, hydrophilic with no disulphide bonds and the protein models which were generated from this study, are of good quality with z value of - 9.58, -9.48 and -10.93 and quality factor of 82.56, 59.43 and 95.27 respectively. So this study was concluded that the generated Hsp70 protein models would provide an avenue for the other researchers for development of high-throughput gene function assignment in fish.

A novel assay provides insight into tRNAPhe retrograde nuclear import and re-export in S. cerevisiae

In eukaryotes, tRNAs are transcribed in the nucleus and subsequently exported to the cytoplasm where they serve as essential adaptor molecules in translation. However, tRNAs can be returned to the nucleus by the evolutionarily conserved process called tRNA retrograde nuclear import, before relocalization back to the cytoplasm via a nuclear re-export step. Several important functions of these latter two trafficking events have been identified, yet the pathways are largely unknown. Therefore, we developed an assay in Saccharomyces cerevisiae to identify proteins mediating tRNA retrograde nuclear import and re-export using the unique wybutosine modification of mature tRNAPhe. Our hydrochloric acid/aniline assay revealed that the karyopherin Mtr10 mediates retrograde import of tRNAPhe, constitutively and in response to amino acid deprivation, whereas the Hsp70 protein Ssa2 mediates import specifically in the latter. Furthermore, tRNAPhe is re-exported by Crm1 and Mex67, but not by the canonical tRNA exporters Los1 or Msn5. These findings indicate that the re-export process occurs in a tRNA family-specific manner. Together, this assay provides insights into the pathways for tRNAPhe retrograde import and re-export and is a tool that can be used on a genome-wide level to identify additional gene products involved in these tRNA trafficking events.