Methylamine dehydrogenase from the obligate methylotroph Methylomonas methylovora

An obligate methyltroph Methylomonas methylovora oxidized methylamine, formaldehyde, and formate. Enzymes oxidizing these substrates were detected in a cell-free system. Phenazine methosulfate-linked methylamine dehydrogenese was purified 21-fold. The enzyme had optimum activity at pH 7.5 and was stable at 60 °C for 5 min. The enzyme activity was inhibited by parachloromercuric benzoate, isonicotinic acid hydrazide, mercuric chloride, and sodium borate.

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Synthesis of β-glucan by cell-free extracts of Aureobasidium pullulans

Canadian Journal of Microbiology ◽

10.1139/m87-021 ◽

1987 ◽

Vol 33 (2) ◽

pp. 123-127 ◽

Cited By ~ 6

Author(s):

Malcolm A. J. Finkelman ◽

Alex Vardanis

Keyword(s):

Enzyme Activity ◽

Acetic Acid ◽

Bovine Serum Albumin ◽

Aureobasidium Pullulans ◽

Enzyme Preparation ◽

Glucanase Activity ◽

Free System ◽

Enzyme Preparations ◽

Partial Activation ◽

A Cell

A cell-free system catalysing the synthesis of β-glucan from uridine-diphosphoglucose was prepared from Aureobasidium pullulans. The activity was stable in the presence of 1 M sucrose and 10 mM MgSO4. The polymer produced was insoluble in H2O or acetic acid (0.5 M) and soluble in NaOH (0.5 M). Several enzyme preparations containing β-glucanase activity degraded the polymer to various extents. Synthesis of the polymer was enhanced by the presence of cellobiose and bovine serum albumin, but not by NaF or ATP. A Lineweaver–Burke plot of enzyme activity versus substrate concentration revealed biphasic kinetics. The enzyme preparation was subject to partial activation by trypsin and chymotrypsin.

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Cinnamate-4-Hydroxyiase in Polyporus hispidus

Canadian Journal of Biochemistry ◽

10.1139/o73-090 ◽

1973 ◽

Vol 51 (6) ◽

pp. 731-734 ◽

Cited By ~ 6

Author(s):

C. P. Vance ◽

A. M. D. Nambudiri ◽

G. H. N. Towers

Keyword(s):

Enzyme Activity ◽

Enzymatic Activity ◽

Microsomal Fraction ◽

Mitochondrial Fraction ◽

Maximum Activity ◽

Coumaric Acid ◽

Free System ◽

Optimum Ph ◽

A Cell ◽

Plant Enzyme

A cell-free system capable of hydroxylating cinnamic acid to p-coumaric acid has been isolated from 10-day-oid cultures of Polyporus hispidus. The enzyme requires NADPH and FAD for maximum activity. Enzyme activity appears to be localized in the microsomal fraction with slight activity occurring in the mitochondrial fraction. The optimum pH for enzymatic activity is 7.5. This is the first report on any properties of this enzyme in a fungus. The enzyme differs from the known plant enzyme in its FAD requirement.

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Evidence for the role of vitamin B-6 as a cofactor of lysyl oxidase

Biochemical Journal ◽

10.1042/bj1670463 ◽

1977 ◽

Vol 167 (2) ◽

pp. 463-467 ◽

Cited By ~ 32

Author(s):

J C Murray ◽

C I Levene

Keyword(s):

Enzyme Activity ◽

Isonicotinic Acid ◽

Lysyl Oxidase ◽

Acid Hydrazide ◽

Single Peak ◽

Partial Purification ◽

Vitamin B ◽

Epiphysial Cartilage

At 24 h after injection of 16-day chick embryos with [C-3H]pyridoxine hydrochloride, some of this label appears in the epiphysial cartilage. Over 35% of this radioactivity appears in the form of [G-3H]pyridoxal and a further 30% as other vitamin B-6 compounds. Partial purification of lysyl oxidase from the labelled epiphysial cartilage reveals a single peak of radioactivity coinciding with a single peak of enzyme activity. On dialysis against phosphate-buffered saline, 75% of this radioactivity is found to be non-diffusible. After incubation with isonicotinic acid hydrazide, a carbonyl reagent that appears to inhibit lysyl oxidase both in vivo and in vitro, a further 70% of the radioactivity is lost, with a roughly corresponding loss of enzyme activity. It is suggested that a form of vitamin B-6 is required as a cofactor of lysyl oxidase, and that this may have important implications in terms of connective-tissue metabolism.

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Asoprisnil, a Selective Progesterone Receptor Modulator (SPRM), Inhibits Melanosome Export in B16F10 Cells and HEMn-DP Melanocytes

Molecules ◽

10.3390/molecules25163581 ◽

2020 ◽

Vol 25 (16) ◽

pp. 3581

Author(s):

Shilpi Goenka ◽

Sanford R. Simon

Keyword(s):

Enzyme Activity ◽

Progesterone Receptor ◽

Human Keratinocytes ◽

Tyrosinase Activity ◽

Free System ◽

Cell Free System ◽

Receptor Modulator ◽

B16f10 Cells ◽

A Cell ◽

Tyrosinase Enzyme

Previous studies have reported that estrogen hormone promotes melanogenesis while progesterone inhibits it. A selective estrogen receptor modulator (SERM), tamoxifen, has been shown to promote melanogenesis; however, to date, there have been no reports on the effects of a selective progesterone receptor modulator (SPRM) on melanogenesis. In the present study, we hypothesized that asoprisnil (AP), a SPRM, inhibits melanogenesis. AP was tested for cytotoxicity to B16F10 mouse melanoma cells for screening the nontoxic concentrations using MTS cytotoxicity assay. Extracellular and intracellular melanin levels were estimated at nontoxic concentrations of AP. To evaluate the direct effect of AP on tyrosinase enzyme, tyrosinase activity and copper chelating activities were measured. Next, the effects of AP on melanogenesis were tested in normal human melanocytes, neonatal, darkly pigmented (HEMn-DP). Our results demonstrate that AP was nontoxic at a concentration range of 10–50 μM in B16F10 cells; AP at 50 μM significantly suppressed extracellular melanin levels comparable to kojic acid at 500 μM, with no significant effect on intracellular melanin levels. The mechanism of melanogenesis inhibition was studied to assess if AP downregulated tyrosinase activity in cell lysates or in a cell-free system. However, AP was found to increase intracellular tyrosinase activity without any effect on tyrosinase enzyme activity or copper chelating activity in a cell-free system, indicating that AP inhibits melanogenesis by mechanisms other than direct effects on tyrosinase enzyme activity. The capacity of AP to inhibit melanosome export was further validated in HEMn-DP cells; AP significantly suppressed dendricity at concentrations of 20 and 30 μM in the absence of effects on melanin synthesis or intracellular tyrosinase activity. In addition, AP was nontoxic to human keratinocytes (HaCaT) at these concentrations, validating its safety for topical use. Taken together, our preliminary results demonstrate that AP might be repurposed as a candidate therapeutic for treatment of hyperpigmentation disorders via a unique mechanism, which encompasses a selective inhibition of melanosome export.

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Combination of ribosome and phage display for fast selection of high affinity VHH antibody fragments

Bulletin of Russian State Medical University ◽

10.24075/brsmu.2019.002 ◽

2019 ◽

pp. 27-33

Author(s):

YuE Kravchenko ◽

SV Ivanov ◽

DS Kravchenko ◽

EI Frolova ◽

SP Chumakov

Keyword(s):

Phage Display ◽

Selection Procedure ◽

Free System ◽

Phage Displays ◽

High Affinity ◽

Binding Domains ◽

A Cell ◽

Vhh Antibodies ◽

Efficiency Of Selection ◽

Selection Of

Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. Selection in cell-free conditions using a ribosomal display is devoid from this drawback, however is highly sensitive to PCR artifacts and the RNase contamination. The aim of the study was to test the efficiency of a combination of both methods, including pre-selection in a cell-free system to enrich the source library, followed by cloning and final selection using phage display. This approach may eliminate the shortcomings of each method and increase the efficiency of selection. For selection, alpaca VHH antibody sequences suitable for building an immune library were used due to the lack of VL domains. Analysis of immune libraries from the genes of the VH3, VHH3 and VH4 families showed that the VHH antibodies share in the VH3 and VH4 gene groups is insignificant, and selection from the combined library is less effective than from the VHH3 family of sequences. We found that the combination of ribosomal and phage displays leads to a higher enrichment of high-affinity fragments and avoids the loss of the original diversity during cloning. The combined method allowed us to obtain a greater number of different high-affinity sequences, and all the tested VHH fragments were able to specifically recognize the target, including the total protein extracts of cell cultures.

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Hybrid Design of Isonicotinic Acid Hydrazide Derivatives: Machine Learning Studies, Synthesis and Biological Evaluation of their Antituberculosis Activity

Current Drug Discovery Technologies ◽

10.2174/1570163816666190411110331 ◽

2020 ◽

Vol 17 (3) ◽

pp. 365-375

Author(s):

Vasyl Kovalishyn ◽

Diana Hodyna ◽

Vitaliy O. Sinenko ◽

Volodymyr Blagodatny ◽

Ivan Semenyuta ◽

...

Keyword(s):

Machine Learning ◽

Isonicotinic Acid ◽

Acid Hydrazide ◽

Predictive Ability ◽

Antitubercular Activity ◽

Biological Evaluation ◽

Starting Point ◽

Binary Classifiers ◽

Synthesis And Biological Evaluation

Background: Tuberculosis (TB) is an infection disease caused by Mycobacterium tuberculosis (Mtb) bacteria. One of the main causes of mortality from TB is the problem of Mtb resistance to known drugs. Objective: The goal of this work is to identify potent small molecule anti-TB agents by machine learning, synthesis and biological evaluation. Methods: The On-line Chemical Database and Modeling Environment (OCHEM) was used to build predictive machine learning models. Seven compounds were synthesized and tested in vitro for their antitubercular activity against H37Rv and resistant Mtb strains. Results: A set of predictive models was built with OCHEM based on a set of previously synthesized isoniazid (INH) derivatives containing a thiazole core and tested against Mtb. The predictive ability of the models was tested by a 5-fold cross-validation, and resulted in balanced accuracies (BA) of 61–78% for the binary classifiers. Test set validation showed that the models could be instrumental in predicting anti- TB activity with a reasonable accuracy (with BA = 67–79 %) within the applicability domain. Seven designed compounds were synthesized and demonstrated activity against both the H37Rv and multidrugresistant (MDR) Mtb strains resistant to rifampicin and isoniazid. According to the acute toxicity evaluation in Daphnia magna neonates, six compounds were classified as moderately toxic (LD50 in the range of 10−100 mg/L) and one as practically harmless (LD50 in the range of 100−1000 mg/L). Conclusion: The newly identified compounds may represent a starting point for further development of therapies against Mtb. The developed models are available online at OCHEM http://ochem.eu/article/11 1066 and can be used to virtually screen for potential compounds with anti-TB activity.

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