Quantification of the degree of biotinylation of proteins using proteinase K digestion and competition ELISA

A multiplex PCR assay was evaluated for diagnosis of diarrheagenic Escherichia coli in stool samples of patients with diarrhoea submitted to a diagnostic microbiology laboratory. Two procedures of DNA template preparationproteinase K buffer method and the boiling method were evaluated to examine isolates of E. coli from 150 selected diarrhoeal cases. By proteinase K buffer method, 119 strains (79.3%) of E. coli were characterized to various categories by their genes that included 55.5% enteroaggregative E. coli (EAEC), 18.5% enterotoxigenic E. coli (ETEC), 1.7% enteropathogenic E. coli (EPEC), and 0.8% Shiga toxin-producing E. coli (STEC). Although boiling method was less time consuming (<24 hrs) and less costly (<8.0 US $/ per test) but was less efficient in typing E. coli compared to proteinase K method (41.3% vs. 79.3% ; p<0.001). The sensitivity and specificity of boiling method compared to proteinase K method was 48.7% and 87.1% while the positive and negative predictive value was 93.5% and 30.7%, respectively. The majority of pathogenic E. coli were detected in children (78.0%) under five years age with 53.3% under one year, and 68.7% of the children were male. Children under 5 years age were frequently infected with EAEC (71.6%) compared to ETEC (24.3%), EPEC (2.7%) and STEC (1.4%). The multiplex PCR assay could be effectively used as a rapid diagnostic tool for characterization of diarrheagenic E. coli using a single reaction tube in the clinical laboratory setting.Bangladesh J Med Microbiol 2007; 01 (02): 38-42

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AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

FLORA AND FAUNA ◽

10.33451/florafauna.v23i1pp25-36 ◽

2017 ◽

Vol 23 (1) ◽

Author(s):

N.NANDHA KUMAR ◽

K. SOURIANATHA SUNDARAM ◽

D. SUDHAKAR ◽

K.K. KUMAR

Keyword(s):

Polymerase Chain Reaction ◽

Quantitative Polymerase Chain Reaction ◽

Proteinase K ◽

Chain Reaction ◽

Banana Plant ◽

High Molecular Weight Dna ◽

Musa Spp ◽

Leaf Tissues ◽

Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

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Crystal structure of calcium-free proteinase K at 1.5-A resolution.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)31626-5 ◽

1994 ◽

Vol 269 (37) ◽

pp. 23108-23111

Author(s):

A. Müller ◽

W. Hinrichs ◽

W.M. Wolf ◽

W. Saenger

Keyword(s):

Crystal Structure ◽

Proteinase K

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Dementia with Lewy bodies—associated ß-synuclein mutations V70M and P123H cause mutation-specific neuropathological lesions

Human Molecular Genetics ◽

10.1093/hmg/ddab036 ◽

2021 ◽

Author(s):

Maryna Psol ◽

Sofia Guerin Darvas ◽

Kristian Leite ◽

Sameehan U Mahajani ◽

Mathias Bähr ◽

...

Keyword(s):

Neuronal Network ◽

Dementia With Lewy Bodies ◽

Lewy Bodies ◽

Sporadic Case ◽

Network Activity ◽

Proteinase K ◽

Neuronal Network Activity ◽

Distinct Disease

Abstract ß-Synuclein (ß-Syn) has long been considered to be an attenuator for the neuropathological effects caused by the Parkinson’s disease-related α-Synuclein (α-Syn) protein. However, recent studies demonstrated that overabundant ß-Syn can form aggregates and induce neurodegeneration in CNS neurons in vitro and in vivo, albeit at a slower pace as compared to α-Syn. Here we demonstrate that ß-Syn mutants V70M, detected in a sporadic case of Dementia with Lewy Bodies (DLB), and P123H, detected in a familial case of DLB, robustly aggravate the neurotoxic potential of ß-Syn. Intriguingly, the two mutations trigger mutually exclusive pathways. ß-Syn V70M enhances morphological mitochondrial deterioration and degeneration of dopaminergic and non-dopaminergic neurons, but has no influence on neuronal network activity. Conversely, ß-Syn P123H silences neuronal network activity, but does not aggravate neurodegeneration. ß-Syn WT, V70M and P123H formed proteinase K (PK) resistant intracellular fibrils within neurons, albeit with less stable C-termini as compared to α-Syn. Under cell free conditions, ß-Syn V70M demonstrated a much slower pace of fibril formation as compared to WT ß-Syn, and P123H fibrils present with a unique phenotype characterized by large numbers of short, truncated fibrils. Thus, it is possible that V70M and P123H cause structural alterations in ß-Syn, that are linked to their distinct neuropathological profiles. The extent of the lesions caused by these neuropathological profiles is almost identical to that of overabundant α-Syn, and thus likely to be directly involved into etiology of DLB. Over all, this study provides insights into distinct disease mechanisms caused by mutations of ß-Syn.

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Urine lipoarabinomannan in HIV uninfected, smear negative, symptomatic TB patients: effective sample pretreatment for a sensitive immunoassay and mass spectrometry

Scientific Reports ◽

10.1038/s41598-021-82445-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Anita G. Amin ◽

Prithwiraj De ◽

Barbara Graham ◽

Roger I. Calderon ◽

Molly F. Franke ◽

...

Keyword(s):

Limit Of Detection ◽

Sample Pretreatment ◽

Positive Association ◽

Proteinase K ◽

Clinical Samples ◽

Sputum Smear ◽

Smear Negative ◽

Tuberculostearic Acid ◽

Negative Sputum ◽

Adult Culture

AbstractOur study sought to determine whether urine lipoarabinomannan (LAM) could be validated in a sample cohort that consisted mainly of HIV uninfected individuals that presented with tuberculosis symptoms. We evaluated two tests developed in our laboratory, and used them on clinical samples from Lima, Peru where incidence of HIV is low. ELISA analysis was performed on 160 samples (from 140 adult culture-confirmed TB cases and 20 symptomatic TB-negative child controls) using 100 μL of urine after pretreatment with Proteinase K. Two different mouse monoclonal antibodies-CS35 and CHCS9-08 were used individually for capture of urine LAM. Among cases, optical density (OD450) values had a positive association with higher bacillary loads. The 20 controls had negative values (below the limit of detection). The assay correctly identified all samples (97–100% accuracy confidence interval). For an alternate validation of the ELISA results, we analyzed all 160 urine samples using an antibody independent chemoanalytical approach. Samples were called positive only when LAM surrogates—tuberculostearic acid (TBSA) and d-arabinose (d-ara)—were found to be present in similar amounts. All TB cases, including the 40 with a negative sputum smear had LAM in detectable quantities in urine. None of the controls had detectable amounts of LAM. Our study shows that urinary LAM detection is feasible in HIV uninfected, smear negative TB patients.

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Synthesis, Properties, and Biodegradability of Thermoplastic Elastomers Made from 2-Methyl-1,3-propanediol, Glutaric Acid and Lactide

Life ◽

10.3390/life11010043 ◽

2021 ◽

Vol 11 (1) ◽

pp. 43

Author(s):

Lamya Zahir ◽

Takumitsu Kida ◽

Ryo Tanaka ◽

Yuushou Nakayama ◽

Takeshi Shiono ◽

...

Keyword(s):

Mechanical Properties ◽

Glass Transition ◽

Transition Temperature ◽

Glass Transition Temperature ◽

Glutaric Acid ◽

Triblock Copolymers ◽

Tensile Tests ◽

Thermoplastic Elastomers ◽

Proteinase K ◽

Synthesis Properties

An innovative type of biodegradable thermoplastic elastomers with improved mechanical properties from very common and potentially renewable sources, poly(L-lactide)-b-poly(2-methyl-1,3-propylene glutarate)-b-poly(L-lactide) (PLA-b-PMPG-b-PLA)s, has been developed for the first time. PLA-b-PMPG-b-PLAs were synthesized by polycondensation of 2-methyl-1,3-propanediol and glutaric acid and successive ring-opening polymerization of L-lactide, where PMPG is an amorphous central block with low glass transition temperature and PLA is hard semicrystalline terminal blocks. The copolymers showed glass transition temperature at lower than −40 °C and melting temperature at 130–152 °C. The tensile tests of these copolymers were also performed to evaluate their mechanical properties. The degradation of the copolymers and PMPG by enzymes proteinase K and lipase PS were investigated. Microbial biodegradation in seawater was also performed at 27 °C. The triblock copolymers and PMPG homopolymer were found to show 9–15% biodegradation within 28 days, representing their relatively high biodegradability in seawater. The macromolecular structure of the triblock copolymers of PLA and PMPG can be controlled to tune their mechanical and biodegradation properties, demonstrating their potential use in various applications.

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Acrylamide-encapsulated glucose oxidase inhibits breast cancer cell viability

Turkish Journal of Biochemistry ◽

10.1515/tjb-2020-0247 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Trëndelina Rrustemi ◽

Öykü Gönül Geyik ◽

Ali Burak Özkaya ◽

Taylan Kurtuluş Öztürk ◽

Zeynep Yüce ◽

...

Keyword(s):

Breast Cancer ◽

Glucose Oxidase ◽

Cancer Cells ◽

Half Life ◽

Neutralizing Antibodies ◽

Redox Homeostasis ◽

Proteinase K ◽

Dependent Manner ◽

Production Of Hydrogen ◽

Mcf 7

AbstractObjectivesCancer cells modulate metabolic pathways to ensure continuity of energy, macromolecules and redox- homeostasis. Although these vulnerabilities are often targeted individually, targeting all with an enzyme may prove a novel approach. However, therapeutic enzymes are prone to proteolytic degradation and neutralizing antibodies leading to a reduced half-life and effectiveness. We hypothesized that glucose oxidase (GOX) enzyme that catalyzes oxidation of glucose and production of hydrogen peroxide, may hit all these targets by depleting glucose; crippling anabolic pathways and producing reactive oxygen species (ROS); unbalancing redox homeostasis.MethodsWe encapsulated GOX in an acrylamide layer and then performed activity assays in denaturizing settings to determine protection provided by encapsulation. Afterwards, we tested the effects of encapsulated (enGOX) and free (fGOX) enzyme on MCF-7 breast cancer cells.ResultsGOX preserved 70% of its activity following encapsulation. When fGOX and enGOX treated with guanidinium chloride, fGOX lost approximately 72% of its activity, while enGOX only lost 30%. Both forms demonstrated remarkable resilience against degradation by proteinase K and inhibited viability of MCF-7 cells in an activity-dependent manner.ConclusionsEncapsulation provided protection to GOX against denaturation without reducing its activity, which would prolong half-life of the enzyme when administered intravenously.

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Proteinase K treatment in absence of RNA isolation classical procedures is a quick and cheaper alternative for SARS-CoV-2 molecular detection

Journal of Virological Methods ◽

10.1016/j.jviromet.2021.114131 ◽

2021 ◽

pp. 114131

Author(s):

L. Mallmann ◽

B.S. Hermann ◽

K. Schallenberger ◽

M. Demoliner ◽

A.K.A. Eisen ◽

...

Keyword(s):

Molecular Detection ◽

Rna Isolation ◽

Proteinase K ◽

Proteinase K Treatment

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T Cells Limit Accumulation of Aggregate Pathology Following Intrastriatal Injection of α-Synuclein Fibrils

Journal of Parkinson s Disease ◽

10.3233/jpd-202351 ◽

2021 ◽

pp. 1-19

Author(s):

Sonia George ◽

Trevor Tyson ◽

Nolwen L. Rey ◽

Rachael Sheridan ◽

Wouter Peelaerts ◽

...

Keyword(s):

T Cells ◽

Immune System ◽

B Cells ◽

Substantia Nigra ◽

Adaptive Immune System ◽

Proteinase K ◽

T And B Cells ◽

Adaptive Immune ◽

Immunocompromised Mice ◽

The Impact

Background: α-Synuclein (α-syn) is the predominant protein in Lewy-body inclusions, which are pathological hallmarks of α- synucleinopathies, such as Parkinson’s disease (PD) and multiple system atrophy (MSA). Other hallmarks include activation of microglia, elevation of pro-inflammatory cytokines, as well as the activation of T and B cells. These immune changes point towards a dysregulation of both the innate and the adaptive immune system. T cells have been shown to recognize epitopes derived from α-syn and altered populations of T cells have been found in PD and MSA patients, providing evidence that these cells can be key to the pathogenesis of the disease. Objective To study the role of the adaptive immune system with respect to α-syn pathology. Methods: We injected human α-syn preformed fibrils (PFFs) into the striatum of immunocompromised mice (NSG) and assessed accumulation of phosphorylated α-syn pathology, proteinase K-resistant α-syn pathology and microgliosis in the striatum, substantia nigra and frontal cortex. We also assessed the impact of adoptive transfer of naïve T and B cells into PFF-injected immunocompromised mice. Results: Compared to wildtype mice, NSG mice had an 8-fold increase in phosphorylated α-syn pathology in the substantia nigra. Reconstituting the T cell population decreased the accumulation of phosphorylated α-syn pathology and resulted in persistent microgliosis in the striatum when compared to non-transplanted mice. Conclusion: Our work provides evidence that T cells play a role in the pathogenesis of experimental α-synucleinopathy.

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