Raman Characterization of Fungal DHN and DOPA Melanin Biosynthesis Pathways

Fungal melanins represent a resource for important breakthroughs in industry and medicine, but the characterization of their composition, synthesis, and structure is not well understood. Raman spectroscopy is a powerful tool for the elucidation of molecular composition and structure. In this work, we characterize the Raman spectra of wild-type Aspergillus fumigatus and Cryptococcus neoformans and their melanin biosynthetic mutants and provide a rough “map” of the DHN (A. fumigatus) and DOPA (C. neoformans) melanin biosynthetic pathways. We compare this map to the Raman spectral data of Aspergillus nidulans wild-type and melanin biosynthetic mutants obtained from a previous study. We find that the fully polymerized A. nidulans melanin cannot be classified according to the DOPA pathway; nor can it be solely classified according to the DHN pathway, consistent with mutational analysis and chemical inhibition studies. Our approach points the way forward for an increased understanding of, and methodology for, investigating fungal melanins.

Download Full-text

Isolation and Characterization of Mutations in theEscherichia coli Regulatory Protein XapR

Journal of Bacteriology ◽

10.1128/jb.181.14.4397-4403.1999 ◽

1999 ◽

Vol 181 (14) ◽

pp. 4397-4403 ◽

Cited By ~ 22

Author(s):

Casper Jørgensen ◽

Gert Dandanell

Keyword(s):

Amino Acids ◽

Purine Nucleoside Phosphorylase ◽

Mutational Analysis ◽

Regulatory Protein ◽

Three Dimensional ◽

Dimensional Structure ◽

Wild Type ◽

Isolation And Characterization ◽

In The Wild

ABSTRACT In this work, the LysR-type protein XapR has been subjected to a mutational analysis. XapR regulates the expression of xanthosine phosphorylase (XapA), a purine nucleoside phosphorylase inEscherichia coli. In the wild type, full expression of XapA requires both a functional XapR protein and the inducer xanthosine. Here we show that deoxyinosine can also function as an inducer in the wild type, although not to the same extent as xanthosine. We have isolated and characterized in detail the mutants that can be induced by other nucleosides as well as xanthosine. Sequencing of the mutants has revealed that two regions in XapR are important for correct interactions between the inducer and XapR. One region is defined by amino acids 104 and 132, and the other region, containing most of the isolated mutations, is found between amino acids 203 and 210. These regions, when modelled into the three-dimensional structure of CysB from Klebsiella aerogenes, are placed close together and are most probably directly involved in binding the inducer xanthosine.

Download Full-text

Mutational analysis of the Drosophila miniature-dusky (m-dy) locus: effects on cell size and circadian rhythms.

Genetics ◽

10.1093/genetics/128.3.571 ◽

1991 ◽

Vol 128 (3) ◽

pp. 571-582

Author(s):

L M Newby ◽

L White ◽

S M DiBartolomeis ◽

B J Walker ◽

H B Dowse ◽

...

Keyword(s):

Cell Size ◽

Mutational Analysis ◽

Genetic Characterization ◽

Wing Development ◽

Direct Visualization ◽

Wild Type ◽

Genetic Studies ◽

Single Complex ◽

And Behavior

Abstract A mutational analysis has been performed to explore the function of the Drosophila melanogaster miniature-dusky (m-dy) locus. Mutations at this locus affect wing development, fertility and behavior. The genetic characterization of 13 different mutations suggests that m and dy variants are alleles of a single complex gene. All of these mutations alter wing size, apparently by reducing the volume of individual epidermal cells of the developing wing. In m mutants, epidermal cell boundaries persist in the mature wing, whereas they normally degenerate 1-2 hr after eclosion in wild-type or dy flies. This has permitted the direct visualization of cell size differences among several m mutants. Mutations at the m-dy locus also affect behavioral processes. Three out of nine dy alleles (dyn1, dyn3 and dyn4) lengthen the circadian period of the activity and eclosion rhythms by approximately 1.5 hr. In contrast, m mutants have normal circadian periods, but an abnormally large percentage of individuals express aperiodic bouts of activity. These behavior genetic studies also indicate that an existing "rhythm" mutation known as Andante is an allele of the m-dy locus. The differential effects of certain m-dy mutations on wing and behavioral phenotypes suggest that separable domains of function exist within this locus.

Download Full-text

Characterization of two‐dimensional materials from Raman spectral data

Journal of Raman Spectroscopy ◽

10.1002/jrs.5744 ◽

2019 ◽

Vol 51 (1) ◽

pp. 37-45

Author(s):

Preet Mishra ◽

Laxmi Narayan Tripathi

Keyword(s):

Spectral Data ◽

Two Dimensional ◽

Two Dimensional Materials ◽

Raman Spectral Data ◽

Raman Spectral

Download Full-text

Mutational Analysis of the [Het-s] Prion Analog of Podospora anserina: A Short N-Terminal Peptide Allows Prion Propagation

Genetics ◽

10.1093/genetics/153.4.1629 ◽

1999 ◽

Vol 153 (4) ◽

pp. 1629-1640 ◽

Cited By ~ 1

Author(s):

Virginie Coustou ◽

Carole Deleu ◽

Sven J Saupe ◽

Joël Bégueret

Keyword(s):

Amino Acid ◽

Mutational Analysis ◽

Podospora Anserina ◽

Wild Type ◽

Haploid Nucleus ◽

Abnormal Growth ◽

Prion Propagation ◽

Allele Specificity ◽

S Allele

Abstract The het-s locus is one of nine known het (heterokaryon incompatibility) loci of the fungus Podospora anserina. This locus exists as two wild-type alleles, het-s and het-S, which encode 289 amino acid proteins differing at 13 amino acid positions. The het-s and het-S alleles are incompatible as their coexpression in the same cytoplasm causes a characteristic cell death reaction. We have proposed that the HET-s protein is a prion analog. Strains of the het-s genotype exist in two phenotypic states, the neutral [Het-s*] and the active [Het-s] phenotype. The [Het-s] phenotype is infectious and is transmitted to [Het-s*] strains through cytoplasmic contact. het-s and het-S were associated in a single haploid nucleus to generate a self-incompatible strain that displays a restricted and abnormal growth. In the present article we report the molecular characterization of a collection of mutants that restore the ability of this self-incompatible strain to grow. We also describe the functional analysis of a series of deletion constructs and site-directed mutants. Together, these analyses define positions critical for reactivity and allele specificity. We show that a 112-amino-acid-long N-terminal peptide of HET-s retains [Het-s] activity. Moreover, expression of a mutant het-s allele truncated at position 26 is sufficient to allow propagation of the [Het-s] prion analog.

Download Full-text

LYT1 Protein Is Required for Efficient In Vitro Infection by Trypanosoma cruzi

Infection and Immunity ◽

10.1128/iai.69.6.3916-3923.2001 ◽

2001 ◽

Vol 69 (6) ◽

pp. 3916-3923 ◽

Cited By ~ 40

Author(s):

Rebeca Manning-Cela ◽

Arantxa Cortés ◽

Elena González-Rey ◽

Wesley C. Van Voorhis ◽

John Swindle ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Mutational Analysis ◽

Parasitophorous Vacuole ◽

Critical Role ◽

Host Cells ◽

Wild Type ◽

Stage Transition

ABSTRACT Trypanosoma cruzi invasion of host cells involves several discrete steps: attachment, parasite internalization mediated by recruitment and fusion of host cell lysosomes, and escape from the parasitophorous vacuole to liberate amastigotes to multiply freely in the cytosol. This report describes the initial characterization of theLYT1 gene and the demonstration that the gene product is involved in cell lysis and infectivity. Mutational analysis demonstrated that deletion of LYT1 resulted in attenuation of infection, which was associated with diminished hemolytic activity. Reintroduction of LYT1 restored infectivity in null mutants, confirming the critical role of LYT1 in infection. Additionally, in vitro stage transition experiments withLYT1-deficient lines showed that these parasites converted to extracellular amastigote-like cells and metacyclic trypomastigotes more rapidly than wild-type parasites, suggesting that the diminished infectivity was not a result of the LYT1 deficiency that affected the parasite's ability to complete the life cycle.

Download Full-text

Identification of toxic mold species through Raman spectroscopy of fungal conidia

PLoS ONE ◽

10.1371/journal.pone.0242361 ◽

2020 ◽

Vol 15 (11) ◽

pp. e0242361

Author(s):

Benjamin D. Strycker ◽

Zehua Han ◽

Zheng Duan ◽

Blake Commer ◽

Kai Wang ◽

...

Keyword(s):

Spectral Data ◽

Raman Spectra ◽

Network Models ◽

Excitation Wavelength ◽

Raman Signal ◽

Neural Network Models ◽

Species Classification ◽

Melanin Biosynthesis ◽

Raman Spectral Data ◽

Raman Spectral

We use a 785 nm shifted excitation Raman difference (SERDS) technique to measure the Raman spectra of the conidia of 10 mold species of especial toxicological, medical, and industrial importance, including Stachybotrys chartarum, Penicillium chrysogenum, Aspergillus fumigatus, Aspergillus flavus, Aspergillus oryzae, Aspergillus niger, and others. We find that both the pure Raman and fluorescence signals support the hypothesis that for an excitation wavelength of 785 nm the Raman signal originates from the melanin pigments bound within the cell wall of the conidium. In addition, the major features of the pure Raman spectra group into profiles that we hypothesize may be due to differences in the complex melanin biosynthesis pathways. We then combine the Raman spectral data with neural network models to predict species classification with an accuracy above 99%. Finally, the Raman spectral data of all species investigated is made freely available for download and use.

Download Full-text

Raman Spectroscopy and Mapping Analysis of Low-Dimensional Nanostructured Materials and Systems

10.5772/intechopen.99775 ◽

2021 ◽

Author(s):

Karthikeyan Krishnamoorthy ◽

Sang-Jae Kim

Keyword(s):

Raman Spectroscopy ◽

Stoichiometric Composition ◽

Electrical Energy ◽

Composition And Structure ◽

Nanostructured Polymer ◽

Mapping Analysis ◽

Low Dimensional ◽

Low Dimensional Materials ◽

Raman Spectral

This chapter describes the use of Raman spectroscopy and mapping analysis for the characterization of low dimensional nanostructures, including 2D sheets (graphene oxide, graphene sheets, MoS2, siloxene), and one-dimensional carbyne chains. The Raman mapping analysis and their application towards understanding the molecular level interactions in these low dimensional materials, nanostructured polymer composites, and nanopaints are also discussed. The stoichiometric composition and structure of these low dimensional materials were correlated with the Raman spectral and mapping analysis. Further, Raman spectroscopy for understanding or probing the mechanism of mechanical to electrical energy harvesting properties of carbyne films via the structural transformation from cumulene to polynne networks of carbyne is demonstrated.

Download Full-text

Mutational Analysis of VKOR.

Blood ◽

10.1182/blood.v108.11.1628.1628 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1628-1628

Author(s):

Da-Yun Jin ◽

Jian-Ke Tie ◽

Darrel W. Stafford

Keyword(s):

Vitamin K ◽

Disulfide Bond ◽

Active Sites ◽

Mutational Analysis ◽

Residual Activity ◽

Wild Type ◽

Vitamin K Epoxide Reductase ◽

The Us ◽

Epoxide Reductase ◽

Inhibition Studies

Abstract More than 21 million prescriptions for warfarin are written yearly in the US for Vitamin K epoxide reductase (VKOR), the target of warfarin The vitamin K epoxide reductase, VKOR, apparently uses cysteines, 132 and 135 as active sites. In addition to cysteines 132 and 135, cysteines 43 and 51 are conserved throughout evolution. Rost et al. have mutated each of the cysteines in VKOR and found that, in whole cell lysates, mutations in of C43 result in less than 20% of wild-type activity while mutation of C53 eliminates activity. We have repeated these experiments and mutated all of the cysteine residues in VKOR. Our results in microsomes are similar to the results of Rost et. al. (2005) Thromb. Haemost. 94, 780–786. However, when the mutated enzymes are purified we find that the activity of C43 has 30% residual activity while C51 has 60% residual activity. Mutation of both residues in the same molecule results in an enzyme that is similar to the C51 mutation. In addition, we find that a portion of purified VKOR has a disulfide bond between residues 43 and 51. This suggested that we might be able to remove the loop between C43 and C51 and retain activity. Indeed, the mutated enzyme with this loop removed also has substantial activity. Warfarin inhibition studies suggest that these mutations do not materially affect warfarin sensitivity. Our results stress the importance of utilizing purified enzyme for interpreting the results of mutations in VKOR.

Download Full-text

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649147 ◽

1974 ◽

Vol 31 (01) ◽

pp. 072-085 ◽

Cited By ~ 5

Author(s):

M Kopitar ◽

M Stegnar ◽

B Accetto ◽

D Lebez

Keyword(s):

Molecular Weight ◽

Plasminogen Activator ◽

Gel Electrophoresis ◽

Gel Filtration ◽

Ph Optimum ◽

Isolation And Characterization ◽

Ph Range ◽

Inhibition Studies ◽

Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

Download Full-text