scholarly journals A Modified Shuttle Plasmid Facilitates Expression of a Flavin Mononucleotide-Based Fluorescent Protein in Treponema denticola ATCC 35405

2015 ◽  
Vol 81 (18) ◽  
pp. 6496-6504 ◽  
Author(s):  
Valentina Godovikova ◽  
M. Paula Goetting-Minesky ◽  
Jae M. Shin ◽  
Yvonne L. Kapila ◽  
Alexander H. Rickard ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Transformation Efficiency ◽  
Molecular Genetic ◽  
Methylation Status ◽  
Molecular Genetic Analysis ◽  
Fluorescence Labeling ◽  
Flavin Mononucleotide ◽  
Treponema Denticola ◽  
Content Type ◽  
Shuttle Plasmid

ABSTRACTOral pathogens, includingTreponema denticola, initiate the dysregulation of tissue homeostasis that characterizes periodontitis. However, progress of research on the roles ofT. denticolain microbe-host interactions and signaling, microbial communities, microbial physiology, and molecular evolution has been hampered by limitations in genetic methodologies. This is typified by an extremely low transformation efficiency and inability to transform the most widely studiedT. denticolastrain with shuttle plasmids. Previous studies have suggested that robust restriction-modification (R-M) systems inT. denticolacontributed to these problems. To facilitate further molecular genetic analysis ofT. denticolabehavior, we optimized existing protocols such that shuttle plasmid transformation efficiency was increased by >100-fold over prior reports. Here, we report routine transformation ofT. denticolaATCC 35405 with shuttle plasmids, independently of both plasmid methylation status and activity of the type II restriction endonuclease encoded by TDE0911. To validate the utility of this methodological advance, we demonstrated expression and activity inT. denticolaof a flavin mononucleotide-based fluorescent protein (FbFP) that is active under anoxic conditions. Addition of routine plasmid-based fluorescence labeling to theTreponematoolset will enable more-rigorous and -detailed studies of the behavior of this organism.

scholarly journals Nonhost Resistance in Arabidopsis-Colletotrichum Interactions Acts at the Cell Periphery and Requires Actin Filament Function

2006 ◽  
Vol 19 (3) ◽  
pp. 270-279 ◽  
Author(s):  
Chiyumi Shimada ◽  
Volker Lipka ◽  
Richard O'Connell ◽  
Tetsuro Okuno ◽  
Paul Schulze-Lefert ◽  
...  
Keyword(s):  
Actin Filament ◽  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Fluorescent Protein ◽  
Molecular Genetic ◽  
Molecular Genetic Analysis ◽  
Nonhost Resistance ◽  
Cell Periphery ◽  
Colletotrichum Species ◽  
Fungal Plant Pathogens

Pathogenesis of nonadapted fungal pathogens is often terminated coincident with their attempted penetration into epidermal cells of nonhost plants. The genus Colletotrichum represents an economically important group of fungal plant pathogens that are amenable to molecular genetic analysis. Here, we investigated interactions between Arabidopsis and Colletotrichum to gain insights in plant and pathogen processes activating nonhost resistance responses. Three tested nonadapted Colletotrichum species differentiated melanized appressoria on Arabidopsis leaves but failed to form intracellular hyphae. Plant cells responded to Colletotrichum invasion attempts by the formation of PMR4/GSL5-dependent papillary callose. Appressorium differentiation and melanization were insufficient to trigger this localized plant cell response, but analysis of nonpathogenic C. lagenarium mutants implicates penetration-peg formation as the inductive cue. We show that Arabidopsis PEN1 syntaxin controls timely accumulation of papillary callose but is functionally dispensable for effective preinvasion (penetration) resistance in nonhost interactions. Consistent with this observation, green fluorescent protein-tagged PEN1 did not accumulate at sites of attempted penetration by either adapted or nonadapted Colletotrichum species, in contrast to the pronounced focal accumulations of PEN1 associated with entry of powdery mildews. We observed extensive reorganization of actin microfilaments leading to polar orientation of large actin bundles towards appressorial contact sites in interactions with the nonadapted Colletotrichum species. Pharmacological inhibition of actin filament function indicates a functional contribution of the actin cytoskeleton for both preinvasion resistance and papillary callose formation. Interestingly, the incidence of papilla formation at entry sites was greatly reduced in interactions with C. higginsianum isolates, indicating that this adapted pathogen may suppress preinvasion resistance at the cell periphery.

Focal hyperperfusion in a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes

2001 ◽  
Vol 94 (1) ◽  
pp. 133-136 ◽  
Author(s):  
Kenichi Amagasaki ◽  
Tsuneo Shimizu ◽  
Yoko Suzuki ◽  
Toshiyuki Kakizawa
Keyword(s):  
Lactic Acidosis ◽  
Genetic Analysis ◽  
Mitochondrial Myopathy ◽  
Molecular Genetic ◽  
Molecular Genetic Analysis ◽  
Metabolic Dysfunction ◽  
Nucleotide Pair ◽  
Content Type ◽  
Typical Point ◽  
Fine Print

✓ 28-year-old woman presented with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS). The diagnosis was based on the results of molecular genetic analysis, which indicated a typical point mutation at the nucleotide pair 3243. Xenon computerized tomography scans obtained during the strokelike episodes revealed the lesion responsible for the symptoms to be an area of focal hyperperfusion, and scans obtained after the episodes revealed an area of hypoperfusion. Pathogenesis of the strokelike episodes appears to be metabolic dysfunction, although the involvement of a vascular event cannot be excluded.

scholarly journals Actinoplanes Swims into the Molecular Age

2017 ◽  
Vol 199 (12) ◽  
Author(s):  
Mark J. Buttner
Keyword(s):  
Life Cycle ◽  
Genetic Analysis ◽  
Molecular Genetic ◽  
Molecular Genetic Analysis ◽  
Survival Strategy ◽  
Evolutionary Perspective ◽  
Content Type ◽  
Link Type

ABSTRACT The survival strategy of Actinoplanes is fascinating from an evolutionary perspective, combining a short motile phase in an otherwise nonmotile, filamentous life cycle and the somewhat paradoxical concept of spores—normally thought of as a resting stage—that swim. In the first paper to report a molecular genetic analysis of development in Actinoplanes, the authors identify a key regulator of the entry into development (Y. Mouri, K. Konishi, A. Fujita, T. Tezuka, Y. Ohnishi, J Bacteriol 199:e00840-16, 2017, https://doi.org/10.1128/JB.00840-16 ).

scholarly journals Golgi Structure Correlates with Transitional Endoplasmic Reticulum Organization in Pichia pastoris and Saccharomyces cerevisiae

1999 ◽  
Vol 145 (1) ◽  
pp. 69-81 ◽  
Author(s):  
Olivia W. Rossanese ◽  
Jon Soderholm ◽  
Brooke J. Bevis ◽  
Irina B. Sears ◽  
James O'Connor ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pichia Pastoris ◽  
Fluorescent Protein ◽  
Molecular Genetic ◽  
Molecular Genetic Analysis ◽  
Coat Proteins ◽  
Transport Vesicles ◽  
Copii Vesicle ◽  
Copii Vesicles ◽  
Green Fluorescent

Golgi stacks are often located near sites of “transitional ER” (tER), where COPII transport vesicles are produced. This juxtaposition may indicate that Golgi cisternae form at tER sites. To explore this idea, we examined two budding yeasts: Pichia pastoris, which has coherent Golgi stacks, and Saccharomyces cerevisiae, which has a dispersed Golgi. tER structures in the two yeasts were visualized using fusions between green fluorescent protein and COPII coat proteins. We also determined the localization of Sec12p, an ER membrane protein that initiates the COPII vesicle assembly pathway. In P. pastoris, Golgi stacks are adjacent to discrete tER sites that contain COPII coat proteins as well as Sec12p. This arrangement of the tER-Golgi system is independent of microtubules. In S. cerevisiae, COPII vesicles appear to be present throughout the cytoplasm and Sec12p is distributed throughout the ER, indicating that COPII vesicles bud from the entire ER network. We propose that P. pastoris has discrete tER sites and therefore generates coherent Golgi stacks, whereas S. cerevisiae has a delocalized tER and therefore generates a dispersed Golgi. These findings open the way for a molecular genetic analysis of tER sites.

scholarly journals Genome Sequence of the Oleaginous Red Yeast Rhodosporidium toruloides MTCC 457

2012 ◽  
Vol 11 (8) ◽  
pp. 1083-1084 ◽  
Author(s):  
Shailesh Kumar ◽  
Hariom Kushwaha ◽  
Anand Kumar Bachhawat ◽  
Gajendra Pal Singh Raghava ◽  
Kaliannan Ganesan
Keyword(s):  
Genome Sequence ◽  
De Novo ◽  
Molecular Genetic ◽  
Draft Genome ◽  
Molecular Genetic Analysis ◽  
Rhodosporidium Toruloides ◽  
Biofuel Production ◽  
Content Type ◽  
Oleaginous Fungi ◽  
Red Yeast

ABSTRACTWe report thede novoassembled 20.05-Mb draft genome of the red yeastRhodosporidium toruloidesMTCC 457, predicted to encode 5,993 proteins, 4 rRNAs, and 125 tRNAs. Proteins known to be unique to oleaginous fungi are present among the predicted proteins. The genome sequence will be valuable for molecular genetic analysis and manipulation of lipid accumulation in this yeast and for developing it as a potential host for biofuel production.

Malignant transformation of a gangliocytoma/ganglioglioma into a glioblastoma multiforme: a molecular genetic analysis

2001 ◽  
Vol 95 (1) ◽  
pp. 138-142 ◽  
Author(s):  
Yutaka Hayashi ◽  
Masayuki Iwato ◽  
Mitsuhiro Hasegawa ◽  
Osamu Tachibana ◽  
Andreas von Deimling ◽  
...  
Keyword(s):  
Glioblastoma Multiforme ◽  
Molecular Analysis ◽  
Molecular Genetic ◽  
Growth Factor Receptor ◽  
Molecular Genetic Analysis ◽  
World Health ◽  
Recurrent Tumor ◽  
Content Type ◽  
Multinucleated Cells ◽  
Second Resection

✓ A gangliocytoma/ganglioglioma with no atypical or malignant features was subtotally resected from the right temporal lobe of a 16-year-old woman. A second resection was performed 8 years later to treat a locally recurrent lesion with increased cellularity that was diagnosed as a World Health Organization Grade II ganglioglioma on the basis of neuropathological examination. Molecular analysis of the recurrent tumor revealed a TP53 gene mutation, but no amplification of the epidermal growth factor receptor (EGFR) gene. Radiotherapy (60 Gy) was administered after the second resection. The patient returned 1 year later with a second focal recurrence. The specimen obtained during the third resection of tumor exhibited exclusively astrocytic differentiation, cellular pleomorphism with multinucleated cells, high mitotic activity, and endothelial proliferation. Therefore, the tumor was diagnosed to be a glioblastoma multiforme (GBM). Molecular analysis of tumor DNA from the second recurrent tumor demonstrated the presence of the TP53 mutation, which previously had been observed in the first recurrent tumor, but again no evidence of EGFR amplification. Findings demonstrate that the presence of TP53 mutation in progressed gangliogliomas should be interpreted as a progression-associated mutation rather than a consequence of treatment. This is the first report to indicate that the molecular pathways of gangliocytomas/gangliogliomas progressing to become GBMs may parallel those of diffuse astrocytomas progressing to become GBMs.

The hPMS2 exon 5 mutation and malignant glioma

1999 ◽  
Vol 90 (5) ◽  
pp. 946-950 ◽  
Author(s):  
Michael D. Taylor ◽  
James Perry ◽  
Magdalena C. Ƶlatescu ◽  
Anat O. Stemmer-Rachamimov ◽  
L. C. Ang ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Molecular Genetic ◽  
Genetic Defect ◽  
Malignant Gliomas ◽  
Molecular Genetic Analysis ◽  
Allelic Loss ◽  
Mismatch Repair Gene ◽  
Polymorphism Analysis ◽  
Repair Gene ◽  
Content Type

✓ Patients with Turcot syndrome (TS) are predisposed to colon tumors and primary brain tumors, typically glioblastomas or medulloblastomas. The authors describe a patient with TS featuring a known germline mutation of exon 5 of the hPMS2 mismatch repair gene who developed two metachronous glioblastomas, both with distinct oligodendroglial features. Molecular genetic analysis revealed allelic loss of chromosome 19q in the patient's second tumor but no allelic loss of chromosome 1p. Prominent microsatellite instability was also found in this tumor, consistent with a germline mismatch repair defect. Because this patient had an unusual underlying condition and his tumor had a unique histological appearance for TS, it was hypothesized that this genetic defect may predispose to malignant gliomas with oligodendroglial features. The authors therefore evaluated whether sporadic glioblastomas and oligodendrogliomas undergo mutations of this region of the hPMS2 gene. However, single-strand conformation polymorphism analysis of hPMS2 exon 5 failed to reveal mutations in 20 sporadic glioblastomas and 16 sporadic oligodendroglial gliomas. Thus, although it is possible that the germline hPMS2 exon 5 mutation may predispose to glioblastomas with an oligodendroglial component, the same genetic defect is not commonly involved in sporadic oligodendrogliomas or glioblastomas.

Long-term survival of a patient with giant cell glioblastoma

2001 ◽  
Vol 94 (4) ◽  
pp. 605-611 ◽  
Author(s):  
Michael Sabel ◽  
Julia Reifenberger ◽  
Ruthild G. Weber ◽  
Guido Reifenberger ◽  
Horst P. Schmitt
Keyword(s):  
Giant Cell ◽  
Tumor Cells ◽  
Postoperative Radiotherapy ◽  
Molecular Genetic ◽  
Tumor Resection ◽  
Molecular Genetic Analysis ◽  
Comparative Genomic ◽  
Term Survival ◽  
Content Type ◽  
Giant Cell Glioblastoma

✓ The authors report on a patient who had undergone resection of a left-sided temporal giant cell glioblastoma at the age of 69 years and who survived for more than 17 years. This man had not undergone postoperative radiotherapy or adjuvant chemotherapy. He died at the age of 86 years without clinical evidence of tumor recurrence. Histologically, the lesion was characterized by highly pleomorphic tumor cells (including bizarre multinucleated giant cells) with high mitotic activity, large necroses, and prominent mononuclear infiltration. A point mutation in the TP53 tumor suppressor gene (c.524G>A: R175H) and no epidermal growth factor receptor gene amplification were revealed on molecular genetic analysis. No diagnostic chromosomal imbalances were identified on comparative genomic hybridization, although the average ratio profile for chromosome 10 indicated loss of 10p15 in a subpopulation of tumor cells. This patient is exceptional because tumor resection, probably in conjunction with a marked antitumor immune response, apparently resulted in eradication of the lesion.

Diffuse leptomeningeal involvement by a ganglioglioma in a child

1992 ◽  
Vol 77 (2) ◽  
pp. 302-306 ◽  
Author(s):  
Margaret R. Wacker ◽  
Philip H. Cogen ◽  
Joan E. Etzell ◽  
Laleh Daneshvar ◽  
Richard L. Davis ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Dna Sequences ◽  
Tumor Tissue ◽  
Molecular Genetic ◽  
Molecular Genetic Analysis ◽  
Content Type ◽  
Leptomeningeal Involvement ◽  
Brain And Spinal Cord ◽  
The Brain ◽  
Fine Print

✓ Gangliogliomas are tumors composed of neuronal and glial elements that typically grow slowly by expansion only. This report describes a 20-month-old girl with a ganglioglioma that extensively involved the subarachnoid space; microscopic foci of tumor were found in the brain and spinal cord. Despite chemotherapy and radiation therapy, the child died 5 months after diagnosis. Molecular genetic analysis showed loss of chromosome 17p DNA sequences in the tumor tissue.

Use of fluorescence in situ hybridization to detect loss of chromosome 10 in astrocytomas

1995 ◽  
Vol 83 (2) ◽  
pp. 316-323 ◽  
Author(s):  
Stephen J. Dalrymple ◽  
John F. Herath ◽  
Steven R. Ritland ◽  
Cheryl A. Moertel ◽  
Robert B. Jenkins
Keyword(s):  
Molecular Genetic ◽  
Molecular Genetic Analysis ◽  
Fish Analysis ◽  
Chromosome 10 ◽  
Content Type ◽  
Valuable Adjunct ◽  
Length Polymorphisms ◽  
Grade Ii ◽  
Fine Print

✓ Models describing progression in the genetic derangement of glial tumors have shown loss of chromosome 10 to occur most frequently in high-grade lesions, suggesting that identification of this loss may be prognostically significant. Fluorescence in situ hybridization (FISH) analysis may be a valuable adjunct to histological grading if it can accurately detect this loss. In this paper the authors correlate results obtained from FISH, cytogenetic, molecular genetic, and flow cytometric analyses of a series of 39 brain specimens, including seven normal, two gliotic, and 30 neoplastic (one Grade II, one Grade III, and 28 Grade IV astrocytoma) specimens. Contiguous section of freshly resected surgical tissue were submitted for tissue culturing (karyotype) and touch preparation (FISH), snap-frozen (molecular genetic), or paraffin-embedded (histology and flow cytometry). Centromere-specific probes for chromosomes 10 and 12 were used for FISH analysis, and 19 restriction fragment length polymorphisms (two p-arm and 17 q-arm) and four microsatellite sequence polymorphisms (three p-arm and one q-arm) were used for molecular genetic analysis of chromosome 10. Findings showed FISH and loss of heterozygosity (LOH) analyses to be concordant in 33 of 38 specimens (sensitivity 94%, specificity 81%), with one specimen indeterminate on LOH analysis. Both FISH and LOH analyses were more sensitive at detecting chromosome 10 loss than conventional cytogenetic (karyotype) analysis. The authors conclude that FISH is a sensitive test for detecting chromosome 10 loss and ploidy in astrocytic tumors.

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