Cloning and sequencing of the dnaK region of Streptomyces coelicolor A3(2)
Abstract The dnaK homologue of Streptomyces coelicolor A3(2) strain M145 has been cloned and sequenced. Nucleotide sequence analysis of a 2.5-kb region revealed an open reading frame (ORF) encoding a predicted DnaK protein of 618 amino acids (Mr = 66 274). The dnaK coding sequence displays extreme codon bias and shows a strong preference for CGY and GGY, for Arg and Gly codons, respectively. The predicted DnaK sequence has a high Lys:Arg ratio which is not typical of streptomycete proteins. The region immediately downstream from dnaK contains an ORF for a GrpE-like protein; the predicted start codon of grpE overlaps the last two codons of dnaK, indicating that the two genes are translationa…
Agarose/κ-carrageenan-based hydrogel film enriched with natural plant extracts for the treatment of cutaneous wounds.
Abstract Hydrogels for complex and chronic wound dressings must be conformable, absorb and retain wound exudates and maintain hydration. They can incorporate and release bioactive molecules that can accelerate the healing process. Wound dressings have to be in contact with the wound and epidermis, even for long periods, without causing adverse effects. Hydrogel dressing formulations based on biopolymers derived from terrestrial or marine flora can be relatively inexpensive and well tolerated. In the present article hydrogel films composed by agarose (1.0 wt%), κ-carrageenan at three different concentrations (0.5, 1.0 and 1.5 wt%) and glycerol (3.0 wt%) were prepared without recourse to cros…
Poly(vinyl alcohol)/κ-Carrageenan-based hydrogels enriched with the adhesive mussel protein Pvfp5β as 3D cell culture scaffold for tissue engineering applications
Many marine organisms such as sandcastle worms, barnacles and mussels, produce natural adhesives to attach to wet surfaces in aqueous tidal environments. In mussels, the adhesion is possible through the secretion of a protein-based water-resistant glue, composed of a mixture of proteins called mussel adhesive proteins (MAPs) or mussel foot proteins (mfps), that allow anchoring to almost any kind of surface in wet conditions [1]. The proteins confined to adhesive plaques are mfp-2, -3, -4, -5, and -6. All these proteins contain an atypically high concentration of the catecholic amino acid 3,4- dihydroxy-l-phenylalanine (DOPA), obtained by the post-translational enzymatic hydroxylation of tyr…
Can Be miR-126-3p a Biomarker of Premature Aging? An Ex Vivo and In Vitro Study in Fabry Disease
Fabry disease (FD) is a lysosomal storage disorder (LSD) characterized by lysosomal accumulation of glycosphingolipids in a wide variety of cytotypes, including endothelial cells (ECs). FD patients experience a significantly reduced life expectancy compared to the general population
Recombinant mussel protein Pvfp5β enhances cell adhesion of poly(vinyl alcohol)/k-carrageenan hydrogel scaffolds
Polymeric hydrogels are increasingly being considered as a scaffold for tissue engineering because they show similarity to the extracellular matrix (ECM) of many tissues. To control various cellular processes, hydrogels are often functionalized or loaded with various bioactive molecules such as: specific ligands for adhesion receptors, growth factors, hormones, enzymes, and other natural or synthetic regulators of cellular behavior [1]. Cell adhesion is essential for cell communication and regulation of the cell cycle and is therefore of vital importance in tissue engineering. Biomimetic approaches have been investigated to facilitate cell-scaffold adhesion interactions. In particular, the …
Conserved alternative splicing in the 5'-untranslated region of the muscle-specific enolase gene. Primary structure of mRNAs, expression and influence of secondary structure on the translation efficiency.
We report here the isolation and characterization of cDNAs covering the 5'-end region of mouse and rat mRNAs that encode the beta or muscle-specific isoform of the glycolytic enzyme enolase. As previously determined for humans, two classes of beta-enolase transcripts with distinct sequences in their 5'-untranslated regions are present in both mouse and rat muscles. A mechanism of alternative splicing, conserved from mouse to man, generates the two forms of mRNA. Secondary-structure predictions indicated that, in all cases, a more stable secondary structure could exist in the 5' end of the message with the longer leader. In vitro transcripts containing defined human or mouse 5'-untranslated …
Recombinant mussel protein Pvfp-5β: A potential tissue bioadhesive
During their lifecycle, many marine organisms rely on natural adhesives to attach to wet surfaces for movement and self-defence in aqueous tidal environments. Adhesive proteins from mussels are biocompatible and elicit only minimal immune responses in humans. Therefore these proteins have received increased attention for their potential applications in medicine, biomaterials and biotechnology. The Asian green mussel Perna viridis secretes several byssal plaque proteins, molecules that help anchor the mussel to surfaces. Among these proteins, protein-5β (Pvfp-5β) initiates interactions with the substrate, displacing interfacial water molecules before binding to the surface. Here, we establis…
miR-126-3p and miR-21-5p as Hallmarks of Bio-Positive Ageing; Correlation Analysis and Machine Learning Prediction in Young to Ultra-Centenarian Sicilian Population
Human ageing can be characterized by a profile of circulating microRNAs (miRNAs), which are potentially predictors of biological age. They can be used as a biomarker of risk for age-related inflammatory outcomes, and senescent endothelial cells (ECs) have emerged as a possible source of circulating miRNAs. In this paper, a panel of four circulating miRNAs including miR-146a-5p, miR-126-3p, miR-21-5p, and miR-181a-5p, involved in several pathways related to inflammation, and ECs senescence that seem to be characteristic of the healthy ageing phenotype. The circulating levels of these miRNAs were determined in 78 healthy subjects aged between 22 to 111 years. Contextually, extracellular miR-1…
Recombinant mussel protein Pvfp5β enhances cell adhesion of poly(vinyl alcohol)/k-carrageenan hydrogel scaffolds
Polymeric hydrogels are increasingly considered as scaffolds for tissue engineering due to their extraordinary resemblance with the extracellular matrix (ECM) of many tissues. As cell adhesion is a key factor in regulating important cell functions, hydrogel scaffolds are often functionalized or loaded with a variety of bioactive molecules that can promote adhesion. Interesting biomimetic approaches exploit the properties of mussel-inspired recombinant adhesive proteins. In this work, we prepared hydrogel scaffolds with a 50%w mixture of k-carrageenan (kC) and polyvinyl alcohol (PVA), by a two-step physical gelation process, and we coated them with Perna viridis foot protein-5 beta (Pvfp5 be…
Conserved Structure and Promoter Sequence Similarity in the Mouse and Human Genes Encoding the Zinc Finger Factor BERF-1/BFCOL1/ZBP-89
Abstract We have characterized the genomic structure of the mouse Zfp148 gene encoding Beta-Enolase Repressor Factor-1 (BERF-1), a Kruppel-like zinc finger protein involved in the transcriptional regulation of several genes, which is also termed ZBP-89, BFCOL1. The cloned Zfp148 gene spans 110 kb of genomic DNA encompassing the 5′-end region, 9 exons, 8 introns, and the 3′-untranslated region. The promoter region displays the typical features of a housekeeping gene: a high G+C content and the absence of canonical TATA and CAAT boxes consistent with the multiple transcription initiation sites determined by primary extension analysis. Computer-assisted search in the human genome database allo…
The molecular anatomy of human Hsp60 and its effects on Amyloid-β peptide
Heat Shock Protein 60 (HSP60) is ubiquitous and highly conserved, being present in eukaryotes and prokaryotes, including pathogens. This chaperonin is typically considered a mitochondrial protein but it is also found in other intracellular sites, extracellularly and in circulation. HSP60 is an indispensable component of the Chaperoning System and plays a key role in protein quality control, preventing off-pathway folding events and refolding misfolded proteins. This makes HSP60 a putative therapeutic agent for neurodegenerative diseases associated with aggregation of misfolded proteins, for example, Alzheimer’s Disease. We produced and purified recombinant human HSP60 and investigated the e…
Temperature-induced self-assembly of degalactosylated xyloglucan at low concentration
Xyloglucan is a natural polysaccharide having a cellulose-like backbone and hydroxyl groups-rich side-chains. In its native form the polymer is water-soluble and forms gel only in presence of selected co-solutes. When a given fraction of galactosyl residues are removed by enzymatic reaction, the polymer acquires the ability to form a gel in aqueous solution at physiological temperatures, a property of great interest for biomedical/pharmaceutical applications. This work presents data on the effect of a temperature increase on degalactosylated xyloglucan dispersed in water at concentration low enough not to run into macroscopic gelation. Results obtained over a wide interval of length scales …
Investigation on a MMACHC mutant from cblC disease: The c.394C>T variant
The cblC disease is an inborn disorder of the vitamin B12 (cobalamin, Cbl) metabolism characterized by methylmalonic aciduria and homocystinuria. The clinical consequences of this disease are devastating and, even when early treated with current therapies, the affected children manifest symptoms involving vision, growth, and learning. The illness is caused by mutations in the gene codifying for MMACHC, a 282aa protein that transports and transforms the different Cbl forms. Here we present data on the structural properties of the truncated protein p.R132X resulting from the c.394C > T mutation that, along with c.271dupA and c.331C > T, is among the most common mutations in cblC. Althou…
Hsp60, amateur chaperone in amyloid-beta fibrillogenesis
BACKGROUND: Molecular chaperones are a very special class of proteins that play essential roles in many cellular processes like folding, targeting and transport of proteins. Moreover, recent evidence indicates that chaperones can act as potentially strong suppressor agents in Alzheimer's disease (AD). Indeed, in vitro experiments demonstrate that several chaperones are able to significantly slow down or suppress aggregation of Aβ peptide and in vivo studies reveal that treatment with specific chaperones or their overexpression can ameliorate some distinct pathological signs characterizing AD. METHODS: Here we investigate using a biophysical approach (fluorescence, circular dichroism (CD), t…
ENO1 gene product binds to the c-myc promoter and acts as a transcriptional repressor: relationship with Myc promoter-binding protein 1 (MBP-1).
The Myc promoter-binding protein-1 (MBP-1) is a 37-38 kDa protein that binds to the c-myc P2 promoter and negatively regulates transcription of the protooncogene. MBP-1 cDNA shares 97% similarity with the cDNA encoding the glycolytic enzyme alpha-enolase and both genes have been mapped to the same region of human chromosome 1, suggesting the hypothesis that the two proteins might be encoded by the same gene. We show here data indicating that a 37 kDa protein is alternatively translated from the full-length alpha-enolase mRNA. This shorter form of alpha-enolase is able to bind the MBP-1 consensus sequence and to downregulate expression of a luciferase reporter gene under the control of the c…
Oligomeric State and Holding Activity of Hsp60
Similar to its bacterial homolog GroEL, Hsp60 in oligomeric conformation is known to work as a folding machine, with the assistance of co-chaperonin Hsp10 and ATP. However, recent results have evidenced that Hsp60 can stabilize aggregation-prone molecules in the absence of Hsp10 and ATP by a different, “holding-like” mechanism. Here, we investigated the relationship between the oligomeric conformation of Hsp60 and its ability to inhibit fibrillization of the Ab40 peptide. The monomeric or tetradecameric form of the protein was isolated, and its effect on beta-amyloid aggregation was separately tested. The structural stability of the two forms of Hsp60 was also investigated using differentia…
Solution structure of recombinant Pvfp-5β reveals insights into mussel adhesion
Solution structure of byssal plaque protein Pvfp-5 beta secreted by the Asian green mussel Perna viridis gives molecular insight into mussel adhesion on wet surfaces.Some marine organisms can resist to aqueous tidal environments and adhere tightly on wet surface. This behavior has raised increasing attention for potential applications in medicine, biomaterials, and tissue engineering. In mussels, adhesive forces to the rock are the resultant of proteinic fibrous formations called byssus. We present the solution structure of Pvfp-5 beta, one of the three byssal plaque proteins secreted by the Asian green mussel Perna viridis, and the component responsible for initiating interactions with the…
Water Extract of Cryphaea heteromalla (Hedw.) D. Mohr Bryophyte as a Natural Powerful Source of Biologically Active Compounds
Bryophytes comprise of the mosses, liverworts, and hornworts. Cryphaea heteromalla, (Hedw.) D. Mohr, is a non-vascular lower plant belonging to mosses group. To the date, the most chemically characterized species belong to the liverworts, while only 3.2% and 8.8% of the species belonging to the mosses and hornworts, respectively, have been investigated. In this work, we present Folin–Ciocalteu and oxygen radical absorbance capacity (ORAC) data related to crude extracts of C. heteromalla obtained by three di erent extraction solvents: pure water (WT), methanol:water (80:20 v/v) (MET), and ethanol:water (80:20 v/v) (ETH). The water extract proved to be the best solvent showing the highest con…
Expression of vesicle-associated membrane-protein-associated protein B cleavage products in peripheral blood leukocytes and cerebrospinal fluid of patients with sporadic amyotrophic lateral sclerosis
Background and purpose Vesicle-associated membrane-protein-associated protein B (VAPB) is an endoplasmic reticulum (ER) resident protein participating in ER function, vesicle trafficking, calcium homeostasis and lipid transport. Its N-terminal domain, named MSP, is cleaved and secreted, serving as an extracellular ligand. VAPB mutations are linked to autosomal-dominant motor neuron diseases, including amyotrophic lateral sclerosis (ALS) type 8. An altered VAPB function is also suspected in sporadic ALS (SALS). Methods The expression pattern of VAPB cleavage and secreted products in the peripheral blood leukocytes (PBL) and cerebrospinal fluid (CSF) of SALS patients and neurological controls…
Negative Regulation of β Enolase Gene Transcription in Embryonic Muscle Is Dependent upon a Zinc Finger Factor That Binds to the G-rich Box within the Muscle-specific Enhancer
We have previously identified a muscle-specific enhancer within the first intron of the human beta enolase gene. Present in this enhancer are an A/T-rich box that binds MEF-2 protein(s) and a G-rich box (AGTGGGGGAGGGGGCTGCG) that interacts with ubiquitously expressed factors. Both elements are required for tissue-specific expression of the gene in skeletal muscle cells. Here, we report the identification and characterization of a Kruppel-like zinc finger protein, termed beta enolase repressor factor 1, that binds in a sequence-specific manner to the G-rich box and functions as a repressor of the beta enolase gene transcription in transient transfection assays. Using fusion polypeptides of b…
A breakdown in macromolecular synthesis preceding differentiation in Streptomyces coelicolor A3(2)
Summary: A transitory cessation of growth was recorded in Streptomyces coelicolor A3(2) at the end of vegetative mycelium formation on solid medium. In the same phase a striking reduction in protein and nucleic acid synthesis was detected. Growth and macromolecular synthesis resumed, nearly reaching the original values, when morphological differentiation occurred. It is concluded that a physiological stress occurs within the bacterial population just before the onset of the morphological differentiation.