Multi-layered molecular mechanisms of polypeptide holding, unfolding and disaggregation by HSP70/HSP110 chaperones.

Members of the HSP70/HSP110 family (HSP70s) form a central hub of the chaperone network controlling all aspects of proteostasis in bacteria and the ATP-containing compartments of eukaryotic cells. The heat-inducible form HSP70 (HSPA1A) and its major cognates, cytosolic HSC70 (HSPA8), endoplasmic reticulum BIP (HSPA5), mitochondrial mHSP70 (HSPA9) and related HSP110s (HSPHs), contribute about 3% of the total protein mass of human cells. The HSP70s carry out a plethora of housekeeping cellular functions, such as assisting proper de novo folding, assembly and disassembly of protein complexes, pulling polypeptides out of the ribosome and across membrane pores, activating and inactivating signaling proteins and controlling their degradation. The HSP70s can induce structural changes in alternatively folded protein conformers, such as clathrin cages, hormone receptors and transcription factors, thereby regulating vesicular trafficking, hormone signaling and cell differentiation in development and cancer. To carry so diverse cellular housekeeping and stress-related functions, the HSP70s act as ATP-fuelled unfolding nanomachines capable of switching polypeptides between different folded states. During stress, the HSP70s can bind (hold) and prevent the aggregation of misfolding proteins and thereafter act alone or in collaboration with other unfolding chaperones to solubilize protein aggregates. Here, we discuss the common ATP-dependent mechanisms of holding, unfolding-by-clamping and unfolding-by-entropic pulling, by which the HSP70s can apparently convert various alternatively folded and misfolded polypeptides into differently active conformers. Understanding how HSP70s can prevent the formation of cytotoxic protein aggregates, pull, unfold, and solubilize them into harmless species is central to the design of therapies against protein conformational diseases

Pengaruh pemberian ekstrak etanol daun sirsak (Annona muricata L) terhadap kadar Superoksida Dismutase (SOD) dan Malondialdehide (MDA) hepar mencit betina (Mus musculus) yang diinduksi 7,12-dimetilbenz (α) antrasen secara in vivo

INDONESIA: Adanya zat radikal bebas yang berlebihan menyebabkan terjadinya stress oksidatif. Tingginya stress oksidatif, menyebabkan peningkatan aktivitas radikal bebas dalam tubuh dan menurunkan aktivitas antioksidan enzimatis. Aktivitas radikal bebas tersebut hanya dapat dihambat oleh adanya antioksidan. Salah satu tanaman yang telah terbukti mengandung antioksidan adalah daun sirsak (Annona muricata L.). Kandungan daun sirsak yang bersifat sebagai antioksidan adalah flavonoid. Penelitian ini bertujuan untuk mengetahui pengaruh pemberian ekstrak etanol daun sirsak (Annona muricata L.) terhadap kadar Superoksida Dismutase (SOD) dan Malondialdehide (MDA) hepar mencit betina yang diinduksi 7,12- dimetilbenz (α) antrasen secara in vivo. Penelitian ini menggunakan Rancangan Acak Lengkap (RAL) dengan enam perlakuan dan empat kali ulangan. Perlakuan yang digunakan adalah pemberian ekstrak etanol daun sirsak (Annona muricata L.) dengan empat dosis, yaitu dosis I (100 mg/kg BB/oral/hari), dosis II (150 mg/kg BB/oral/hari), dosis III (200 mg/kg BB/oral/hari) dan dosis IV (250 mg/kg BB/oral/hari). Untuk mengukur aktivitas antioksidan enzimatis, parameter yang digunakan adalah kadar Superoksida Dismutase (SOD). Untuk mengukur aktivitas radikal bebas, parameter yang digunakan adalah kadar Malondialdehide (MDA). Data yang didapat dianalisis menggunakan ANOVA dengan taraf signifikansi 1%. Hasil penelitian menunjukkan bahwa pemberian ekstrak etanol daun sirsak (Annona muricata L) berpengaruh terhadap peningkatan kadar SOD dan penurunan kadar MDA pada hepar mencit betina (Mus musculus) yang diinduksi 7,12-dimetilbenz (α) antrasen (p < 0,05). Dari keempat dosis ekstrak etanol daun sirsak yang diberikan, dosis 3 (200 mg/kg BB/oral/hari) merupakan dosis yang paling efektif dalam meningkatkan kadar SOD(99,6 U/ml) hingga mendekati kadar normal (135,4 U/ml) dan mampu menurunkan kadar MDA (16,607 nmol/ml) hingga mendekati kadar normal (8,25 nmol/ml). ENGLISH: The presence of excessive free radicals that cause oxidative stress. The high oxidative stress, causing an increase in free radical activity in the body and lower the antioxidant enzymatic activity. Free radical activity can only be inhibited by the presence of antioxidants. One of the plants that have been shown to contain antioxidants is the leaves of Annona muricata L. The content that is Annona muricata leaves as antioxidants are flavonoids. This study aimed to determine the effect of ethanol extract of leaves of Annona muricata L. on levels of Superoxide Dismutase (SOD) and Malondialdehyde (MDA) female mice liver induced by 7,12 - dimetilbenz (α) antrasen in vivo. This research used Completely Randomized Design (CRD) with six treatments and four replications. The treatment used is ethanol extract of leaves of Annona muricata L. with four doses, the first dose (100 mg / kg / oral / day), II dose (150 mg / kg / oral / day), doses III (200 mg / kg / oral / day) and IV dose (250 mg / kg / oral / day). To measure the enzymatic antioxidant activity, the parameters used are the levels of superoxide dismutase (SOD). To measure the activity of free radicals, the parameters used are the levels of malondialdehyde (MDA). The data were analyzed using ANOVA with a significance level of 1%. The results showed that the ethanol extract of leaves of Annona muricata L. effect on increased levels of SOD and decreased MDA levels in the liver of female mice (Mus musculus) induced by 7.12-dimetilbenz (α)antrasen (p < 0,05). Four doses of ethanol extract of leaves of Annona muricat is given, 3 doses (200 mg / kg / oral / day) is the most effective dose in increasing levels of SOD (99.6 U / ml) to near normal levels (135.4 U / ml) and is able to reduce levels of MDA (16.607 nmol / ml) to near normal levels (8.25 nmol / ml)

Meta-analysis of heat- and chemically upregulated chaperone genes in plant and human cells

Molecular chaperones are central to cellular protein homeostasis. In mammals, protein misfolding diseases and aging cause inflammation and progressive tissue loss, in correlation with the accumulation of toxic protein aggregates and the defective expression of chaperone genes. Bacteria and non-diseased, non-aged eukaryotic cells effectively respond to heat shock by inducing the accumulation of heat-shock proteins (HSPs), many of which molecular chaperones involved in protein homeostasis, in reducing stress damages and promoting cellular recovery and thermotolerance. We performed a meta-analysis of published microarray data and compared expression profiles of HSP genes from mammalian and plant cells in response to heat or isothermal treatments with drugs. The differences and overlaps between HSP and chaperone genes were analyzed, and expression patterns were clustered and organized in a network. HSPs and chaperones only partly overlapped. Heat-shock induced a subset of chaperones primarily targeted to the cytoplasm and organelles but not to the endoplasmic reticulum, which organized into a network with a central core of Hsp90s, Hsp70s, and sHSPs. Heat was best mimicked by isothermal treatments with Hsp90 inhibitors, whereas less toxic drugs, some of which non-steroidal anti-inflammatory drugs, weakly expressed different subsets of Hsp chaperones. This type of analysis may uncover new HSP-inducing drugs to improve protein homeostasis in misfolding and aging disease

Enhancing the selective extracellular location of a recombinant E. coli domain antibody by management of fermentation conditions

The preparation of a recombinant protein using Escherichia coli often involves a challenging primary recovery sequence. This is due to the inability to secrete the protein to the extracellular space without a significant degree of cell lysis. This results in the release of nucleic acids, leading to a high viscosity, difficulty to clarify, broth and also to contamination with cell materials such as lipopolysaccharides and host cell proteins. In this paper, we present different fermentation strategies to facilitate the recovery of a V H domain antibody (13.1 kDa) by directing it selectively to the extracellular space and changing the balance between domain antibody to nucleic acid release. The manipulation of the cell growth rate in order to increase the outer cell membrane permeability gave a small ~1.5-fold improvement in released domain antibody to nucleic acid ratio without overall loss of yield. The introduction during fermentation of release agents such as EDTA gave no improvement in the ratio of released domain antibody to nucleic acid and a loss of overall productivity. The use of polyethyleneimine (PEI) during fermentation was with the aim to (a) permeabilise the outer bacterial membrane to release selectively domain antibody and (b) remove selectively by precipitation nucleic acids released during cell lysis. This strategy resulted in up to ~4-fold increase in the ratio of domain antibody to soluble nucleic acid with no reduction in domain antibody overall titre. In addition, a reduction in host cell protein contamination was achieved and there was no increase in endotoxin levels. Similar results were demonstrated with a range of other antibody products prepared in E. coli

A critically appraised topic (CAT) to compare the effects of single and multi-cat housing on physiological and behavioural measures of stress in domestic cats in confined environments

Background Domestic cats have evolved from solitary, asocial predators and whilst they may display social behaviours, they can still exist as solitary survivors. Over-population and relinquishment of pet cats are ubiquitous problems worldwide, and rehoming centres (also known as rescues/ shelters) aim to ameliorate this by holding cats in confinement for a variable period until a new home is found. The provision of optimal housing for large numbers of cats in close confinement, such as in rehoming centres, is therefore inherently difficult. Under these conditions there is the potential for individuals to develop signs of physical and psychological ill health, and thus experience compromised welfare. Available information regarding housing practices that maximise welfare currently provides conflicting results, and as a consequence there are no unanimous housing recommendations. The aim of this study was therefore to review the evidence on the impact of single housing compared to multi-cat housing on stress in confined cats, as measured by physiological and/or behavioural outcomes. The review was conducted using a Critically Appraised Topic (CAT) format. A systematic search of electronic databases (CAB Abstracts, Zoological Records and Medline) was carried out to identify peer-reviewed literature comparing single and multi-cat housing in confined environments. Results A total of 959 papers were initially identified, six of which met sufficient criteria based on their relevance to be included within this review. All of the studies had significant limitations in design and methodology, including a lack of information on how groups were assigned, inconsistent handling and enrichment provision between groups, and lack of information on the socialisation status of cats. Conclusions Whilst some studies suggested that single housing may be less stressful for cats, others suggested group housing was less stressful. Several other important factors were however identified as potential mediators of stress within the different housing systems, and recommendations based upon these findings are presente

Experimental Milestones in the Discovery of Molecular Chaperones as Polypeptide Unfolding Enzymes.

Molecular chaperones control the cellular folding, assembly, unfolding, disassembly, translocation, activation, inactivation, disaggregation, and degradation of proteins. In 1989, groundbreaking experiments demonstrated that a purified chaperone can bind and prevent the aggregation of artificially unfolded polypeptides and use ATP to dissociate and convert them into native proteins. A decade later, other chaperones were shown to use ATP hydrolysis to unfold and solubilize stable protein aggregates, leading to their native refolding. Presently, the main conserved chaperone families Hsp70, Hsp104, Hsp90, Hsp60, and small heat-shock proteins (sHsps) apparently act as unfolding nanomachines capable of converting functional alternatively folded or toxic misfolded polypeptides into harmless protease-degradable or biologically active native proteins. Being unfoldases, the chaperones can proofread three-dimensional protein structures and thus control protein quality in the cell. Understanding the mechanisms of the cellular unfoldases is central to the design of new therapies against aging, degenerative protein conformational diseases, and specific cancers

Evaluation of options for harvest of a recombinant E. coli fermentation producing a domain antibody using ultra scale-down techniques and pilot-scale verification

Ultra scale-down (USD) methods operating at the millilitre scale were used to characterise full-scale processing of E. coli fermentation broths autolysed to different extents for release of a domain antibody. The focus was on the primary clarification stages involving continuous centrifugation followed by depth filtration. The performance of this sequence was predicted by USD studies to decrease significantly with increased extents of cell lysis. The use of polyethyleneimine (PEI) reagent was studied to treat the lysed cell broth by precipitation of soluble contaminants such as DNA and flocculation of cell debris material. The USD studies were used to predict the impact of this treatment on the performance and here it was found that the fermentation could be run to maximum productivity using an acceptable clarification process (e.g a centrifugation stage operating at 0.11 L per m(2) equivalent gravity settling area per h followed by a resultant required depth filter area of 0.07 m(2) per L supernatant). A range of USD predictions was verified at the pilot scale for centrifugation followed by depth filtration. This article is protected by copyright. All rights reserved

Infeksi Parasit Usus Pada Anak Sekolah Dasar Di Pesisir Pantai Kecamatan Wori Kabupaten Minahasa Utara

: Parasite infection is still an endemic disease that can be found in any place in Indonesiathat can cause problems in public health, especially for children in elementary school age. The purpose of this research is to describe the intestinal parasite infection in children at coastal area in Wori district, North Minahasa regency. The method of this research is descriptive survey. The subject of this research is student class I to VI of GMIM Budo and Kima Bajo elementary school in Wori district.The result of this research according to the finding of the stool examination: hookworm infection 4.7%, Entamoeba coli 39%, Chilomastix mesnili 3.1%, Blastocystis hominis 3.1%, Giardia lambia 3.9% and mixed infection 1.5 %

Intensitas Serangan Akibat Hama Pemakan Daun Setelah Aplikasi Ekstrak Daun Kirinyuh (Chromolaena Odorata) pada Tanaman Sawi (Brassica Juncea L.)

Penelitian ini bertujuan untuk mengevaluasi intensitas serangan akibat hama pemakan daun setelah aplikasi ekstrak daun kirinyuh pada tanaman sawi. Penelitian ini dilaksanakan di lahan percobaan di Kelurahan Lansot Kecamatan Tomohon Selatan Kota Tomohon pada bulan Desember 2018 – Februari 2019. Penelitian ini menggunakan rancangan acak lengkap (RAL) yang terdiri dari 4 perlakuan konsentrasi ekstrak daun kirinyuh yaitu K0 (kontrol) = 0 g/L air, K1 = 100 g/L air, K2 = 200 g/L air, dan K3 = 300 g/L air. Intensitas serangan diamati berdasarkan luas area daun yang dirusak oleh hama pemakan daun pada daun sawi setelah aplikasi ekstrak daun krinyuh. Hasil penelitian menunjukkan bahwa pemberian ekstrak daun kirinyuh berpengaruh nyata terhadap intensitas serangan pada tanaman sawi dan konsentrasi ekstrak daun kirinyuh yang dapat menekan serangan hama pemakan daun yaitu yang tertinggi pada perlakuan K3 (300 g/L air)