scholarly journals Dolichyl-Phosphate-Glucose Is Used To Make O-Glycans on Glycoproteins of Trichomonas vaginalis

2008 ◽  
Vol 7 (8) ◽  
pp. 1344-1351 ◽  
Author(s):  
Kariona A. Grabińska ◽  
Sudip K. Ghosh ◽  
Ziqiang Guan ◽  
Jike Cui ◽  
Christian R. H. Raetz ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Trichomonas Vaginalis ◽  
Gene Products ◽  
Gene Duplications ◽  
Gene Encoding ◽  
Dolichyl Phosphate ◽  
Genes Encoding ◽  
Carboxypeptidase N ◽  
Glycosylation Defect

ABSTRACT Trichomonas vaginalis, the protist that causes vaginal itching, has a huge genome with numerous gene duplications. Recently we found that Trichomonas has numerous genes encoding putative dolichyl-phosphate-glucose (Dol-P-Glc) synthases (encoded by ALG5 genes) despite the fact that Trichomonas lacks the glycosyltransferases (encoded by ALG6, ALG8, and ALG10 genes) that use Dol-P-Glc to glucosylate dolichyl-PP-linked glycans. In addition, Trichomonas does not have a canonical DPM1 gene, encoding a dolichyl-P-mannose (Dol-P-Man) synthase. Here we show Trichomonas membranes have roughly 300 times the Dol-P-Glc synthase activity of Saccharomyces cerevisiae membranes and about one-fifth the Dol-P-Man synthase activity of Saccharomyces membranes. Endogenous Dol-P-hexoses of Trichomonas are relatively abundant and contain 16 isoprene units. Five paralogous Trichomonas ALG5 gene products have Dol-P-Glc synthase activity when expressed as recombinant proteins, and these Trichomonas Alg5s correct a carboxypeptidase N glycosylation defect in a Saccharomyces alg5 mutant in vivo. A recombinant Trichomonas Dpm1, which is deeply divergent in its sequence, has Dol-P-Man synthase activity. When radiolabeled Dol-P-Glc is incubated with Trichomonas membranes, Glc is incorporated into reducing and nonreducing sugars of O-glycans of endogenous glycoproteins. To our knowledge, this is the first demonstration of Dol-P-Glc as a sugar donor for O-glycans on glycoproteins.

scholarly journals PhhB, a Pseudomonas aeruginosa Homolog of Mammalian Pterin 4a-Carbinolamine Dehydratase/DCoH, Does Not Regulate Expression of Phenylalanine Hydroxylase at the Transcriptional Level

1999 ◽  
Vol 181 (9) ◽  
pp. 2789-2796 ◽  
Author(s):  
Jian Song ◽  
Tianhui Xia ◽  
Roy A. Jensen
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Phenylalanine Hydroxylase ◽  
Transcriptional Level ◽  
Gene Encoding ◽  
Catalytic Function ◽  
Genes Encoding ◽  
Catalytic Role ◽  
Regulatory Effects

ABSTRACT Pterin 4a-carbinolamine dehydratase is bifunctional in mammals. In addition to playing a catalytic role in pterin recycling in the cytoplasm, it plays a regulatory role in the nucleus, where it acts as a dimerization-cofactor component (called DCoH) for the transcriptional activator HNF-1α. A thus far unique operon in Pseudomonas aeruginosa contains a gene encoding a homolog (PhhB) of the regulatory dehydratase, together with genes encoding phenylalanine hydroxylase (PhhA) and aromatic aminotransferase (PhhC). Using complementation of tyrosine auxotrophy in Escherichia colias a functional test, we have found that the in vivo function of PhhA requires PhhB. Strikingly, mammalian DCoH was an effective substitute for PhhB, and either one was effective in trans. Surprisingly, the required presence of PhhB for complementation did not reflect a critical positive regulatory effect of phhB onphhA expression. Rather, in the absence of PhhB, PhhA was found to be extremely toxic in E. coli, probably due to the nonenzymatic formation of 7-biopterin or a similar derivative. However, bacterial PhhB does appear to exert modest regulatory effects in addition to having a catalytic function. PhhB enhances the level of PhhA two- to threefold, as was demonstrated by gene inactivation ofphhB in P. aeruginosa and by comparison of the levels of expression of PhhA in the presence and absence of PhhB inEscherichia coli. Experiments using constructs having transcriptional and translational fusions with a lacZreporter indicated that PhhB activates PhhA at the posttranscriptional level. Regulation of PhhA and PhhB is semicoordinate; both PhhA and PhhB are induced coordinately in the presence of eitherl-tyrosine or l-phenylalanine, but PhhB exhibits a significant basal level of activity that is lacking for PhhA. Immunoprecipitation and affinity chromatography showed that PhhA and PhhB form a protein-protein complex.

scholarly journals Mls is not a single gene, allelic system. Different stimulatory Mls determinants are the products of at least two nonallelic, unlinked genes.

1987 ◽  
Vol 166 (4) ◽  
pp. 1150-1155 ◽  
Author(s):  
R Abe ◽  
J J Ryan ◽  
R J Hodes
Keyword(s):  
T Cells ◽  
Single Gene ◽  
Surface Structures ◽  
Chromosome 1 ◽  
Gene Products ◽  
Spleen Cells ◽  
Gene Encoding ◽  
Cell Surface Structures ◽  
Genes Encoding ◽  
Polymorphic Cell

Mls determinants share with MHC products the unique property of stimulating T cells at extraordinarily high precursor frequencies. The Mls system was originally described as a single locus on chromosome 1, with four alleles, Mlsa, Mlsb, Mlsc, and Mlsd, that encode polymorphic cell surface structures. However, the fundamental issues of polymorphism and allelism in the Mls system remain controversial. To clarify these questions, a formal segregation analysis of the genes encoding Mlsa and Mlsc determinants was carried out by testing the capacity of spleen cells from progeny of (Mlsa X Mlsc)F1 X Mlsb breedings to stimulate responses by unprimed T cells and by Mlsa- and Mlsc-specific cloned T cells. The results of this analysis indicated that the gene encoding Mlsa determinants is neither allelic to nor linked to the gene encoding Mlsc determinants. Together with previous findings, these results also suggest that another strongly stimulatory type, Mlsd, in fact results from the independent expression of unlinked Mlsa and Mlsc gene products. Based on these observations, it is concluded that, contrary to conventional concepts, the stimulatory phenotypes designated as Mlsa, Mlsc, and Mlsd can be accounted for by the independent expression of the products of at least two unlinked gene loci.

scholarly journals Effects of Cordycepin in Cordyceps militaris during Its Infection to Silkworm Larvae

2021 ◽  
Vol 9 (4) ◽  
pp. 681
Author(s):  
Tatsuya Kato ◽  
Konomi Nishimura ◽  
Ahmad Suparmin ◽  
Kazuho Ikeo ◽  
Enoch Y. Park
Keyword(s):  
Cordyceps Militaris ◽  
Silkworm Larvae ◽  
Gene Encoding ◽  
Biosynthesis Gene Cluster ◽  
Expression Of Genes ◽  
Cuticular Proteins ◽  
Genes Encoding ◽  
Fat Bodies ◽  
In Vivo Growth

Cordyceps militaris produces cordycepin, a secondary metabolite that exhibits numerous bioactive properties. However, cordycepin pharmacology in vivo is not yet understood. In this study, the roles of cordycepin in C. militaris during its infection were investigated. After the injection of conidia, C. militaris NBRC100741 killed silkworm larvae more rapidly than NBRC103752. At 96 and 120 h, Cmcns genes (Cmcns1–4), which are part of the cordycepin biosynthesis gene cluster, were expressed in fat bodies and cuticles. Thus, cordycepin may be produced in the infection of silkworm larvae. Further, cordycepin enhanced pathogenicity toward silkworm larvae of Metarhizium anisopliae and Beauveria bassiana, that are also entomopathogenic fungi and do not produce cordycepin. In addition, by RNA-seq analysis, the increased expression of the gene encoding a lipoprotein 30K-8 (Bmlp20, KWMTBOMO11934) and decreased expression of genes encoding cuticular proteins (KWMTBOMO13140, KWMTBOMO13167) and a serine protease inhibitor (serpin29, KWMTBOMO08927) were observed when cordycepin was injected into silkworm larvae. This result suggests that cordycepin may aid the in vivo growth of C. militaris in silkworm larvae by the influence of the expression of some genes in silkworm larvae.

scholarly journals The regulation by iron of the synthesis of adhesins and cytoadherence levels in the protozoan Trichomonas vaginalis.

1991 ◽  
Vol 174 (2) ◽  
pp. 311-318 ◽  
Author(s):  
M W Lehker ◽  
R Arroyo ◽  
J F Alderete
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Trichomonas Vaginalis ◽  
Actinomycin D ◽  
Cultivation Medium ◽  
Genes Encoding ◽  
Transcription Data ◽  
Alpha Amanitin ◽  
Adherence Property

Levels of adherence of Trichomonas vaginalis to epithelial cells was found to be modulated by iron. Cytoadherence values were greater than or equal to twofold higher for trichomonads grown in a complex cultivation medium supplemented with iron. This increase in adherence levels was specifically mediated by iron; parasites cultured in a low-iron medium in the presence of salts other than iron were unresponsive to changes in adherence levels. Expression of the higher adherence property, by parasites grown first in low-iron medium followed by supplementation with iron, was a function of time, and the extent of cytoadherence was proportional to the concentration of iron added to the medium. Lactoferrin, an important iron source for trichomonads at the site of infection, elevated adherence of the parasite to epithelial cells, demonstrating the likely in vivo modulation of adherence by iron. The alteration of levels of adherence caused by iron was determined to be a reflection of gene expression of previously characterized trichomonad adhesins. Parasites grown under iron-replete conditions had higher quantities of surface-exposed adhesins, and this was a result of increased synthesis of adhesins. Actinomycin D and alpha-amanitin prevented expression of adhesin molecules, which resulted in decreased cytoadherence, showing that adhesin synthesis was dependent on gene transcription. Data indicated that genes encoding the four trichomonad adhesins are coordinately regulated by iron.

scholarly journals An inhibitor of yeast cyclin-dependent protein kinase plays an important role in ensuring the genomic integrity of daughter cells.

1994 ◽  
Vol 14 (5) ◽  
pp. 3320-3328 ◽  
Author(s):  
T T Nugroho ◽  
M D Mendenhall
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Activity ◽  
Gene Products ◽  
Dependent Protein Kinase ◽  
Gene Encoding ◽  
Daughter Cells ◽  
G2 Checkpoint ◽  
Dependent Protein ◽  
Segregation Of Chromosomes

The gene encoding a 40-kDa protein, previously studied as a substrate and inhibitor of the yeast cyclin-dependent protein kinase, Cdc28, has been cloned. The DNA sequence reveals that p40 is a highly charged protein of 32,187 Da with no significant homology to other proteins. Overexpression of the gene encoding p40, SIC1, produces cells with an elongated but morphology similar to that of cells with depleted levels of the CLB gene products, suggesting that p40 acts as an inhibitor of Cdc28-Clb complexes in vivo. A SIC1 deletion is viable and has highly increased frequencies of broken and lost chromosomes. The deletion strain segregates out many dead cells that are primarily arrested at the G2 checkpoint in an asymmetric fashion. Only daughters and young mothers display the lethal defect, while experienced mothers appear to grow normally. These results suggest that negative regulation of Cdc28 protein kinase activity by p40 is important for faithful segregation of chromosomes to daughter cells.

scholarly journals Translational Paradigms in Cerebrovascular Gene Transfer

2003 ◽  
Vol 23 (11) ◽  
pp. 1251-1262 ◽  
Author(s):  
Vini G Khurana ◽  
Fredric B Meyer
Keyword(s):  
Gene Transfer ◽  
Blood Vessels ◽  
Ex Vivo ◽  
Present Report ◽  
Cerebral Arteries ◽  
Experimental Studies ◽  
Gene Encoding ◽  
Expression Of Genes ◽  
Genes Encoding

Gene transfer involves the use of an engineered biologic vehicle known as a vector to introduce a gene encoding a protein of interest into a particular tissue. In diseases with known defects at a genetic level, gene transfer offers a potential means of restoring a normal molecular environment via vector-mediated entry (transduction) and expression of genes encoding potentially therapeutic proteins selectively in diseased tissues. The technology of gene transfer therefore underlies the concept of gene therapy and falls under the umbrella of the current genomics revolution. Particularly since 1995, numerous attempts have been made to introduce genes into intracranial blood vessels to demonstrate and characterize viable transduction. More recently, in attempting to translate cerebrovascular gene transfer technology closer to the clinical arena, successful transductions of normal human cerebral arteries ex vivo and diseased animal cerebral arteries in vivo have been reported using vasomodulatory vectors. Considering the emerging importance of gene-based strategies for the treatment of the spectrum of human disease, the goals of the present report are to overview the fundamentals of gene transfer and review experimental studies germane to the clinical translation of a technology that can facilitate genetic modification of cerebral blood vessels.

scholarly journals The katX Gene, Which Codes for the Catalase in Spores of Bacillus subtilis, Is a Forespore-Specific Gene Controlled by ςF, and KatX Is Essential for Hydrogen Peroxide Resistance of the Germinating Spore

1998 ◽  
Vol 180 (8) ◽  
pp. 2057-2062 ◽  
Author(s):  
Irina Bagyan ◽  
Lilliam Casillas-Martinez ◽  
Peter Setlow
Keyword(s):  
Hydrogen Peroxide ◽  
Bacillus Subtilis ◽  
Mature Spore ◽  
Specific Gene ◽  
Gene Encoding ◽  
Major Function ◽  
Gene Coding ◽  
Genes Encoding ◽  
Dormant Spore

ABSTRACT Previous work has shown that the katX gene encodes the major catalase in dormant spores of Bacillus subtilis but that this enzyme has no role in dormant spore resistance to hydrogen peroxide. Expression of a katX-lacZ fusion began at approximately h 2 of sporulation, and >75% of thekatX-driven β-galactosidase was packaged into the mature spore. A mutation in the gene coding for the sporulation-specific RNA polymerase sigma factor ςF abolishedkatX-lacZ expression, while mutations in genes encoding ςE, ςG, and ςK did not. Induction of ςF synthesis in vegetative cells also resulted in katX-lacZ expression, while induction of ςG expression did not; the katX-lacZ fusion was also not induced by hydrogen peroxide. Upstream of the in vivokatX transcription start site there are sequences with good homology to those upstream of known ςF-dependent start sites. These data indicate that katX is an additional member of the forespore-specific ςF regulon. A mutant in the katA gene, encoding the major catalase in growing cells, was sensitive to hydrogen peroxide during sporulation, while akatX mutant was not. However, outgrowth of katXspores, but not katA spores, was sensitive to hydrogen peroxide. Consequently, a major function for KatX is to protect germinating spores from hydrogen peroxide.

scholarly journals Genetic and Physiological Characterization of Fructose-1,6-Bisphosphate Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase in the Crabtree-Negative Yeast Kluyveromyces lactis

2022 ◽  
Vol 23 (2) ◽  
pp. 772
Author(s):  
Rosaura Rodicio ◽  
Hans-Peter Schmitz ◽  
Jürgen J. Heinisch
Keyword(s):  
Kluyveromyces Lactis ◽  
Regulation Of Gene Expression ◽  
Carbon Sources ◽  
Pentose Phosphate ◽  
Gene Encoding ◽  
Physiological Characterization ◽  
Genes Encoding ◽  
Fructose 1,6 Bisphosphate

The milk yeast Kluyveromyces lactis degrades glucose through glycolysis and the pentose phosphate pathway and follows a mainly respiratory metabolism. Here, we investigated the role of two reactions which are required for the final steps of glucose degradation from both pathways, as well as for gluconeogenesis, namely fructose-1,6-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In silico analyses identified one gene encoding the former (KlFBA1), and three genes encoding isoforms of the latter (KlTDH1, KlTDH2, KlGDP1). Phenotypic analyses were performed by deleting the genes from the haploid K. lactis genome. While Klfba1 deletions lacked detectable FBA activity, they still grew poorly on glucose. To investigate the in vivo importance of the GAPDH isoforms, different mutant combinations were analyzed for their growth behavior and enzymatic activity. KlTdh2 represented the major glycolytic GAPDH isoform, as its lack caused a slower growth on glucose. Cells lacking both KlTdh1 and KlTdh2 failed to grow on glucose but were still able to use ethanol as sole carbon sources, indicating that KlGdp1 is sufficient to promote gluconeogenesis. Life-cell fluorescence microscopy revealed that KlTdh2 accumulated in the nucleus upon exposure to oxidative stress, suggesting a moonlighting function of this isoform in the regulation of gene expression. Heterologous complementation of the Klfba1 deletion by the human ALDOA gene renders K. lactis a promising host for heterologous expression of human disease alleles and/or a screening system for specific drugs.

scholarly journals Functional Characterisation of Banana (Musa spp.) 2-Oxoglutarate-Dependent Dioxygenases Involved in Flavonoid Biosynthesis

2021 ◽  
Vol 12 ◽  
Author(s):  
Mareike Busche ◽  
Christopher Acatay ◽  
Stefan Martens ◽  
Bernd Weisshaar ◽  
Ralf Stracke
Keyword(s):  
Recombinant Proteins ◽  
Nutritional Value ◽  
Flavonoid Biosynthesis ◽  
Loss Of Function ◽  
Structural Genes ◽  
In Planta ◽  
Anthocyanidin Synthase ◽  
Functional Characterisation ◽  
Genes Encoding

Bananas (Musa) are non-grass, monocotyledonous, perennial plants that are well known for their edible fruits. Their cultivation provides food security and employment opportunities in many countries. Banana fruits contain high levels of minerals and phytochemicals, including flavonoids, which are beneficial for human nutrition. To broaden the knowledge on flavonoid biosynthesis in this major crop plant, we aimed to identify and functionally characterise selected structural genes encoding 2-oxoglutarate-dependent dioxygenases, involved in the formation of the flavonoid aglycon. Musa candidates genes predicted to encode flavanone 3-hydroxylase (F3H), flavonol synthase (FLS) and anthocyanidin synthase (ANS) were assayed. Enzymatic functionalities of the recombinant proteins were confirmed in vivo using bioconversion assays. Moreover, transgenic analyses in corresponding Arabidopsis thaliana mutants showed that MusaF3H, MusaFLS and MusaANS were able to complement the respective loss-of-function phenotypes, thus verifying functionality of the enzymes in planta. Knowledge gained from this work provides a new aspect for further research towards genetic engineering of flavonoid biosynthesis in banana fruits to increase their antioxidant activity and nutritional value.

scholarly journals Functional characterisation of banana (Musa spp.) 2-oxoglutarate-dependent dioxygenases involved in flavonoid biosynthesis

2021 ◽  
Author(s):  
Mareike Busche ◽  
Christopher Acatay ◽  
Bernd Weisshaar ◽  
Ralf Stracke
Keyword(s):  
Recombinant Proteins ◽  
Nutritional Value ◽  
Flavonoid Biosynthesis ◽  
Loss Of Function ◽  
Structural Genes ◽  
In Planta ◽  
Anthocyanidin Synthase ◽  
Functional Characterisation ◽  
Genes Encoding

Bananas (Musa) are monocotyledonous, perennial plants that are well-known for their edible fruits. Their cultivation provides food security and employment opportunities in many countries. Banana fruits contain high levels of minerals and phytochemicals, including flavonoids, which are beneficial for human nutrition. To broaden the knowledge on flavonoid biosynthesis in this major crop plant, we aimed to identify and functionally characterise selected structural genes encoding 2-oxoglutarate-dependent dioxygenases, involved in the formation of the flavonoid aglycon. Musa candidates genes predicted to encode flavanone 3 hydroxylase (F3H), flavonol synthase (FLS) and anthocyanidin synthase (ANS) were assayed. Enzymatic functionalities of the recombinant proteins were confirmed in vivo using bioconversion assays. Moreover, transgenic analyses in corresponding Arabidopsis thaliana mutants showed that MusaF3H, MusaFLS and MusaANS were able to complement the respective loss-of-function phenotypes, thus verifying functionality of the enzymes in planta. Knowledge gained from this work provides a new aspect for further research towards genetic engineering of flavonoid biosynthesis in banana fruits to increase their antioxidant activity and nutritional value.

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