Soritesidine, a Novel Proteinous Toxin from the Okinawan Marine Sponge Spongosorites sp.

A novel protein, soritesidine (SOR) with potent toxicity was isolated from the marine sponge Spongosorites sp. SOR exhibited wide range of toxicities over various organisms and cells including brine shrimp (Artemia salina) larvae, sea hare (Aplysia kurodai) eggs, mice, and cultured mammalian cells. Toxicities of SOR were extraordinary potent. It killed mice at 5 ng/mouse after intracerebroventricular (i.c.v.) injection, and brine shrimp and at 0.34 µg/mL. Cytotoxicity for cultured mammalian cancer cell lines against HeLa and L1210 cells were determined to be 0.062 and 12.11 ng/mL, respectively. The SOR-containing fraction cleaved plasmid DNA in a metal ion dependent manner showing genotoxicity of SOR. Purified SOR exhibited molecular weight of 108.7 kDa in MALDI-TOF MS data and isoelectric point of approximately 4.5. N-terminal amino acid sequence up to the 25th residue was determined by Edman degradation. Internal amino acid sequences for fifteen peptides isolated from the enzyme digest of SOR were also determined. None of those amino acid sequences showed similarity to existing proteins, suggesting that SOR is a new proteinous toxin.

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Application of the Innovative and Non-Invasive Technique, Molecular Music Therapy (MMT), Bio-Frequency Therapy, for the Treatment of a Wide Range of Disorders and Pathologies, with Consequent Verification of Molecular Parameters by Using Bi-Digital O-Ring Test (BDORT)

Acupuncture & Electro-Therapeutics Research ◽

10.3727/036012920x15779969212928 ◽

2020 ◽

Vol 44 (3) ◽

pp. 177-189

Author(s):

Momir Dunjic ◽

Stefano Turini ◽

Dejan Krstic ◽

Katarina Dunjic ◽

Marija Dunjic ◽

...

Keyword(s):

Amino Acid ◽

Music Therapy ◽

Amino Acid Sequences ◽

Sirtuin 1 ◽

Ring Test ◽

Invasive Technique ◽

Specific Gene ◽

Radiofrequency Therapy ◽

Wide Range ◽

Clinical Pictures

Radiofrequency therapy is an unconventional method, already applied for some time, with numerous results in numerous clinical pictures. Our group has developed a software, later called SONGENPROT-SOLARIS, capable of directly converting nucleotide sequences (DNA and/or RNA) and amino acid sequences (polypeptides and proteins) into musical sequences, based on mathematic matrices, designed by the French physicist and musician Joel Sternheimer, which allows to associate a musical note with a nucleotide or an amino acid. Innovation in our software is that, in the algorithm that defines it, a variant is directly implemented that allows the reproduction of sounds, phase-shifted by 30 Hz, between one ear and another reproducing the phenomenon of Binaural Tones, capable of induce a specific brain activity and also the release of particles called solitons. Thanks to this software we have developed a technique called MMT (Molecular Music Therapy) and currently, we are in the phase of applying the technique on a cohort of 91 patients, with a high spectrum of clinical pictures, examining the same, using the technique Bi-Digital-ORing-Test (BDORT), before and after treatment with MMT. Aim of project is to stimulate the expression of a specific gene (the same genetic sequence that the patient listens to, translated into music), only through the use of sound sequences. We have concentrated our attention on three main molecules: Sirtuin-1, Telomers and TP-53. The results obtained with BDORT, after treatment with MMT, showed a significant increase in the values of the three molecules, on all the examined patients, demonstrating the operative efficacy of the technique and the its applicability to numerous diseases. In order to confirm the data obtained by BDORT, we propose, with the help of an accredited laboratory, to perform epigenetic tests on the three parameters listed above, paving the way to understanding how frequencies can influence gene expression.

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Identification, characterization and cloning of myr 1, a mammalian myosin-I.

The Journal of Cell Biology ◽

10.1083/jcb.120.6.1393 ◽

1993 ◽

Vol 120 (6) ◽

pp. 1393-1403 ◽

Cited By ~ 71

Author(s):

C Ruppert ◽

R Kroschewski ◽

M Bähler

Keyword(s):

Amino Acid ◽

Light Chain ◽

Brush Border ◽

Binding Sites ◽

Neuronal Development ◽

Amino Acid Sequences ◽

Dependent Manner ◽

Tail Domain ◽

Myosin I ◽

Splice Forms

We have identified, characterized and cloned a novel mammalian myosin-I motor-molecule, called myr 1 (myosin-I from rat). Myr 1 exists in three alternative splice forms: myr 1a, myr 1b, and myr 1c. These splice forms differ in their numbers of putative calmodulin/light chain binding sites. Myr 1a-c were selectively released by ATP, bound in a nucleotide-dependent manner to F-actin and exhibited amino acid sequences characteristic of myosin-I motor domains. In addition to the motor domain, they contained a regulatory domain with up to six putative calmodulin/light chain binding sites and a tail domain. The tail domain exhibited 47% amino acid sequence identity to the brush border myosin-I tail domain, demonstrating that myr 1 is related to the only other mammalian myosin-I motor molecule that has been characterized so far. In contrast to brush border myosin-I which is expressed in mature enterocytes, myr 1 splice forms were differentially expressed in all tested tissues. Therefore, myr 1 is the first mammalian myosin-I motor molecule with a widespread tissue distribution in neonatal and adult tissues. The myr 1a splice form was preferentially expressed in neuronal tissues. Its expression was developmentally regulated during rat forebrain ontogeny and subcellular fractionation revealed an enrichment in purified growth cone particles, data consistent with a role for myr 1a in neuronal development.

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In vitro and In Silico Approach For Characterization of Antimicrobial Peptide From Probiotics Against Staphylococcus Aureus and Escherichia Coli

10.21203/rs.3.rs-366314/v2 ◽

2021 ◽

Author(s):

Amrutha Bindu ◽

Lakshmi Devi

Keyword(s):

Amino Acid ◽

Antimicrobial Peptide ◽

Lactobacillus Plantarum ◽

Exchange Chromatography ◽

Amino Acid Sequences ◽

Proteinase K ◽

Kinetic Assay ◽

Wide Range

Abstract The focus of present study was to characterize antimicrobial peptide produced by probiotic cultures, Enterococcus durans DB-1aa (MCC4243), Lactobacillus plantarum Cu2-PM7 (MCC4246) and Lactobacillus fermentum Cu3-PM8 (MCC4233) against Staphylococus aureus and E. coli. The growth kinetic assay revealed 24 h of incubation to be optimum for bacteriocin production. The partially purified compound after ion-exchange chromatography was found to be thermoresistant and stable under wide range of pH. The compound was sensitive to proteinase-K, but resistant to trypsin, a-amylase and lipase. The apparent molecular weight of bacteriocin from MCC4243 and MCC4246 was found to be 3.5 KDa. Translated partial amino acid sequence of plnA gene in MCC4246 displayed 48 amino acid sequences showing 100% similarity with plantaricin A of Lactobacillus plantarum (WP_0036419). The sequence revealed 7 β sheets, 6 α sheets, 6 predicted coils and 9 predicted turns. The functions on cytoplasm show 10.82 isoelectric point and 48.6% hydrophobicity. The molecular approach of using Geneious Prime software and protein prediction data base for characterization of bacteriocin is novel and predicts “KSSAYSLQMGATAIKQVKKLFKKWGW” as peptide responsible for antimicrobial activity. The study provides information about broad spectrum bacteriocin in native probiotic culture and paves a way towards its application in functional foods as biopreservative agents.

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Differentiating closely affiliated Dehalococcoides lineages by a novel genetic marker identified via computational pangenome analysis

Applied and Environmental Microbiology ◽

10.1128/aem.02181-21 ◽

2021 ◽

Author(s):

Siyan Zhao ◽

Chen Zhang ◽

Matthew J. Rogers ◽

Xuejie Zhao ◽

Jianzhong He

Keyword(s):

Amino Acid ◽

Microbial Communities ◽

Genetic Marker ◽

Genetic Markers ◽

Unknown Function ◽

Computational Approach ◽

Amino Acid Sequences ◽

Reductive Dehalogenation ◽

Wide Range

As a group, Dehalococcoides dehalogenate a wide range of organohalide pollutants but the range of organohalide compounds that can be utilized for reductive dehalogenation differs among the Dehalococcoides strains. Dehalococcoides lineages cannot be reliably disambiguated in mixed communities using typical phylogenetic markers, which often confounds bioremediation efforts. Here, we describe a computational approach to identify Dehalococcoides genetic markers with improved discriminatory resolution. Screening core genes from the Dehalococcoides pangenome for degree of similarity and frequency of 100% identity found a candidate genetic marker encoding a bacterial neuraminidase repeat (BNR)-containing protein of unknown function. This gene exhibits the fewest completely identical amino acid sequences and among the lowest average amino acid sequence identity in the core pangenome. Primers targeting BNR could effectively discriminate between 40 available BNR sequences ( in silico ) and 10 different Dehalococcoides isolates ( in vitro ). Amplicon sequencing of BNR fragments generated from 22 subsurface soil samples revealed a total of 109 amplicon sequence variants, suggesting a high diversity of Dehalococcoides distributed in environment. Therefore, the BNR gene can serve as an alternative genetic marker to differentiate strains of Dehalococcoides in complicated microbial communities. Importance The challenge of discriminating between phylogenetically similar but functionally distinct bacterial lineages is particularly relevant to the development of technologies seeking to exploit the metabolic or physiological characteristics of specific members of bacterial genera. A computational approach was developed to expedite screening of potential genetic markers among phylogenetically affiliated bacteria. Using this approach, a gene encoding a bacterial neuraminidase repeat (BNR)-containing protein of unknown function was selected and evaluated as a genetic marker to differentiate strains of Dehalococcoides , an environmentally relevant genus of bacteria whose members can transform and detoxify a range of halogenated organic solvents and persistent organic pollutants, in complex microbial communities to demonstrate the validity of the approach. Moreover, many apparently phylogenetically distinct, currently uncharacterized Dehalococcoides were detected in environmental samples derived from contaminated sites.

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Unevolved De Novo Proteins Have Innate Tendencies to Bind Transition Metals

Life ◽

10.3390/life9010008 ◽

2019 ◽

Vol 9 (1) ◽

pp. 8 ◽

Cited By ~ 4

Author(s):

Michael S. Wang ◽

Kenric J. Hoegler ◽

Michael H. Hecht

Keyword(s):

Amino Acid ◽

Transition Metals ◽

Metal Binding ◽

Combinatorial Library ◽

De Novo ◽

Protein Sequences ◽

Amino Acid Sequences ◽

Ancestral Sequences ◽

Wide Range ◽

Catalytic Functions

Life as we know it would not exist without the ability of protein sequences to bind metal ions. Transition metals, in particular, play essential roles in a wide range of structural and catalytic functions. The ubiquitous occurrence of metalloproteins in all organisms leads one to ask whether metal binding is an evolved trait that occurred only rarely in ancestral sequences, or alternatively, whether it is an innate property of amino acid sequences, occurring frequently in unevolved sequence space. To address this question, we studied 52 proteins from a combinatorial library of novel sequences designed to fold into 4-helix bundles. Although these sequences were neither designed nor evolved to bind metals, the majority of them have innate tendencies to bind the transition metals copper, cobalt, and zinc with high nanomolar to low-micromolar affinity.

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Targeting motifs and functional parameters governing the assembly of connexins into gap junctions

Biochemical Journal ◽

10.1042/bj3490281 ◽

2000 ◽

Vol 349 (1) ◽

pp. 281-287 ◽

Cited By ~ 7

Author(s):

Patricia E. M. MARTIN ◽

James STEGGLES ◽

Claire WILSON ◽

Shoeb AHMAD ◽

W. Howard EVANS

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Gap Junctions ◽

Gap Junction ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Lucifer Yellow ◽

Amino Acid Sequences ◽

Amino Acid Residues ◽

Green Fluorescent

To study the assembly of gap junctions, connexin-green-fluorescent-protein (Cx-GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26- and Cx32-GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32-GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx-GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.

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Purification of two distinct types of phosphoinositide-specific phospholipase C from rat liver. Enzymological and structural studies

Biochemical Journal ◽

10.1042/bj2560453 ◽

1988 ◽

Vol 256 (2) ◽

pp. 453-459 ◽

Cited By ~ 21

Author(s):

O Nakanishi ◽

Y Homma ◽

H Kawasaki ◽

Y Emori ◽

K Suzuki ◽

...

Keyword(s):

Amino Acid ◽

Phospholipase C ◽

Rat Liver ◽

Peptide Mapping ◽

Polyacrylamide Gel Electrophoresis ◽

Liver Homogenate ◽

Amino Acid Sequences ◽

Dependent Manner ◽

Phosphatidylinositol 4 ◽

Hydrolysis Of

Two kinds of phosphoinositide-specific phospholipase C (PLC) were purified from rat liver by acid precipitation and several steps of column chromatography. About 50% of the activity could be precipitated when the pH of the liver homogenate was lowered to pH 4.7. The redissolved precipitate yielded two peaks, PLC I and PLC II, in an Affi-gel Blue column, and each was further purified to homogeneity by three sequential h.p.l.c. steps, which were different for the two enzymes. The purified PLC I and PLC II had estimated Mr values of 140,000 and 71,000 respectively on SDS/polyacrylamide-gel electrophoresis. Both enzymes hydrolysed phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in a Ca2+- and pH-dependent manner. PLC I was most active at 10 microM- and 0.1 mM-Ca2+ for hydrolysis of PI and PIP2 respectively, whereas PLC II showed the highest activity at 5 mM- and 10 microM-Ca2+ for that of PI and PIP2 respectively. The optimal pH of the two enzymes also differed with substrates or Ca2+ concentration, in the range pH 5.0-6.0. Hydrolysis of phosphoinositides by these enzymes was completely inhibited by Hg2+ and was affected by other bivalent cations. From data obtained by peptide mapping and partial amino acid sequencing, it was clarified that PLC I and PLC II had distinct structures. Moreover, partial amino acid sequences of three proteolytic fragments of PLC I completely coincided with those of PLC-148 [Stahl, Ferenz, Kelleher, Kriz & Knopf (1988) Nature (London) 332, 269-272].

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Identification of metal ion binding sites based on amino acid sequences

PLoS ONE ◽

10.1371/journal.pone.0183756 ◽

2017 ◽

Vol 12 (8) ◽

pp. e0183756 ◽

Cited By ~ 10

Author(s):

Xiaoyong Cao ◽

Xiuzhen Hu ◽

Xiaojin Zhang ◽

Sujuan Gao ◽

Changjiang Ding ◽

...

Keyword(s):

Amino Acid ◽

Binding Sites ◽

Metal Ion ◽

Amino Acid Sequences ◽

Ion Binding ◽

Metal Ion Binding ◽

Ion Binding Sites ◽

Metal Ion Binding Sites

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New 19-Residue Peptaibols from Trichoderma Clade Viride

Microorganisms ◽

10.3390/microorganisms6030085 ◽

2018 ◽

Vol 6 (3) ◽

pp. 85 ◽

Cited By ~ 10

Author(s):

Tamás Marik ◽

Chetna Tyagi ◽

Gordana Racić ◽

Dávid Rakk ◽

András Szekeres ◽

...

Keyword(s):

Amino Acid ◽

Structural Investigation ◽

Amino Acid Sequences ◽

Amino Acid Residues ◽

Helix Formation ◽

Left Handed ◽

Wall Structures ◽

High Preference ◽

Wide Range ◽

Unusual Amino Acid

Trichoderma koningiopsis and T. gamsii belong to clade Viride of Trichoderma, the largest and most diverse group of this genus. They produce a wide range of bioactive secondary metabolites, including peptaibols with antibacterial, antifungal, and antiviral properties. The unusual amino acid residues of peptaibols, i.e., α-aminoisobutyric acid (Aib), isovaline (Iva), and the C-terminal 1,2-amino alcohol make them unique among peptides. In this study, the peptaibiomes of T. koningiopsis and T. gamsii were investigated by HPLC-ESI-MS. The examined strains appeared to produce 19-residue peptaibols, most of which are unknown from literature, but their amino acid sequences are similar to those of trikoningins, tricholongins, trichostrigocins, trichorzianins, and trichorzins. A new group of peptaibols detected in T. koningiopsis are described here under the name “Koningiopsin”. Trikoningin KA V, the closest peptaibol compound to the peptaibols produced by these two strains, was selected for structural investigation by short MD simulation, which revealed that many residues show high preference for left handed helix formation. The bioactivity of the peptaibol mixtures produced by T. koningiopsis and T. gamsii was tested on agar plates against bacteria, yeasts, and filamentous fungi. The results revealed characteristic differences in bioactivities towards the different groups of target microorganisms, which can be explained with the differences in their cell wall structures.

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Enzymic and structural characterization of nepenthesin, a unique member of a novel subfamily of aspartic proteinases

Biochemical Journal ◽

10.1042/bj20031575 ◽

2004 ◽

Vol 381 (1) ◽

pp. 295-306 ◽

Cited By ~ 80

Author(s):

Senarath B. P. ATHAUDA ◽

Koji MATSUMOTO ◽

Sanath RAJAPAKSHE ◽

Masayuki KURIBAYASHI ◽

Masaki KOJIMA ◽

...

Keyword(s):

Amino Acid ◽

Computer Modelling ◽

Structural Characteristics ◽

Amino Acid Sequences ◽

Cysteine Residues ◽

Aspartic Proteinases ◽

Protein Databases ◽

Wide Range ◽

First Time

Carnivorous plants are known to secrete acid proteinases to digest prey, mainly insects, for nitrogen uptake. In the present study, we have purified, for the first time, to homogeneity two acid proteinases (nepenthesins I and II) from the pitcher fluid of Nepenthes distillatoria (a pitcher-plant known locally as badura) and investigated their enzymic and structural characteristics. Both enzymes were optimally active at pH approx. 2.6 towards acid-denatured haemoglobin; the specificity of nepenthesin I towards oxidized insulin B chain appears to be similar, but slightly wider than those of other APs (aspartic proteinases). Among the enzymic properties, however, the most notable is their unusual stability: both enzymes were remarkably stable at or below 50 °C, especially nepenthesin I was extremely stable over a wide range of pH from 3 to 10 for over 30 days. This suggests an evolutionary adaptation of the enzymes to their specific habitat. We have also cloned the cDNAs and deduced the complete amino acid sequences of the precursors of nepenthesins I and II (437 and 438 residues respectively) from the pitcher tissue of N. gracilis. Although the corresponding mature enzymes (each 359 residues) are homologous with ordinary pepsin-type APs, both enzymes had a high content of cysteine residues (12 residues/molecule), which are assumed to form six unique disulphide bonds as suggested by computer modelling and are supposed to contribute towards the remarkable stability of nepenthesins. Moreover, the amino acid sequence identity of nepenthesins with ordinary APs, including plant vacuolar APs, is remarkably low (approx. 20%), and phylogenetic comparison shows that nepenthesins are distantly related to them to form a novel subfamily of APs with a high content of cysteine residues and a characteristic insertion, named ‘the nepenthesin-type AP-specific insertion’, that includes a large number of novel, orthologous plant APs emerging in the gene/protein databases.

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