scholarly journals Use of the fluorochrome 4'6-diamidino-2-phenylindole in genetic and developmental studies of chloroplast DNA.

1979 ◽  
Vol 82 (1) ◽  
pp. 299-305 ◽  
Author(s):  
A W Coleman
Keyword(s):  
Chloroplast Dna ◽  
Marine Algae ◽  
Developmental Changes ◽  
Developmental Studies ◽  
Wild Type ◽  
Peripheral Ring ◽  
Structural Plan ◽  
Chromophyte Algae ◽  
Dapi Fluorescence

Use of the DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) makes it possible to examine in situ the structure of chloroplast DNA (chDNA) with the fluorescence microscope. This simplifies the study of genetic and developmental changes in chloroplast DNA. Three examples are presented. (a) Wild-type Euglena gracilis B contains several chloroplast DNA nucleoids per chloroplast. A yellow mutant lacking functional chloroplasts is similar, but such nucleoids are absent in an aplastidic mutant strain known from biochemical studies to have lost its chDNA. (b) In vegetative cells of the giant-celled marine algae Acetabularia and Batophora, only about a quarter of the chloroplasts have even one discernible chloroplast DNA particle, and such particles vary in size, showing a 30-fold variation in the amount of DNA-bound DAPI fluorescence detected per chloroplast. By contrast, 98% of chloroplasts in developing Acetabularia cysts contain chDNA, with as many as nine nucleoids per chloroplast. (c) DAPI-stained chloroplasts of chromophyte algae display the peripheral ring of DNA expected from electron microscope studies. However, these rings are not uniform in thickness, but are necklace-like, with the appearance of beads on a string. Since the multiple nucleoids in plastids of chlorophyte algae also appear to be interconnected throughout the chloroplast, a common structural plan may underlie chDNA morphology in both groups of algae.

scholarly journals Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
Christine Landlinger ◽  
Lenka Tisakova ◽  
Vera Oberbauer ◽  
Timo Schwebs ◽  
Abbas Muhammad ◽  
...  
Keyword(s):  
Bacterial Vaginosis ◽  
Bactericidal Activity ◽  
High Selectivity ◽  
Ex Vivo ◽  
Vaginal Microbiome ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Promising Alternative ◽  
First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

scholarly journals Radixin deficiency causes deafness associated with progressive degeneration of cochlear stereocilia

2004 ◽  
Vol 166 (4) ◽  
pp. 559-570 ◽  
Author(s):  
Shin-ichiro Kitajiri ◽  
Kanehisa Fukumoto ◽  
Masaki Hata ◽  
Hiroyuki Sasaki ◽  
Tatsuya Katsuno ◽  
...  
Keyword(s):  
Hair Cells ◽  
Plasma Membranes ◽  
Wild Type ◽  
Cross Link ◽  
Erm Proteins ◽  
Hearing Ability ◽  
Adult Mice ◽  
Progressive Degeneration ◽  
Sensory Hair Cells

Ezrin/radixin/moesin (ERM) proteins cross-link actin filaments to plasma membranes to integrate the function of cortical layers, especially microvilli. We found that in cochlear and vestibular sensory hair cells of adult wild-type mice, radixin was specifically enriched in stereocilia, specially developed giant microvilli, and that radixin-deficient (Rdx−/−) adult mice exhibited deafness but no obvious vestibular dysfunction. Before the age of hearing onset (∼2 wk), in the cochlea and vestibule of Rdx−/− mice, stereocilia developed normally in which ezrin was concentrated. As these Rdx−/− mice grew, ezrin-based cochlear stereocilia progressively degenerated, causing deafness, whereas ezrin-based vestibular stereocilia were maintained normally in adult Rdx−/− mice. Thus, we concluded that radixin is indispensable for the hearing ability in mice through the maintenance of cochlear stereocilia, once developed. In Rdx−/− mice, ezrin appeared to compensate for radixin deficiency in terms of the development of cochlear stereocilia and the development/maintenance of vestibular stereocilia. These findings indicated the existence of complicate functional redundancy in situ among ERM proteins.

Developmental changes in dopamine levels in larvae of the fly Chymomyza costata: comparison between wild-type and mutant-nondiapause strains

1998 ◽  
Vol 44 (7-8) ◽  
pp. 605-614 ◽  
Author(s):  
Vladimir Kostal ◽  
Hirofumi Noguchi ◽  
Kimio Shimada ◽  
Yoichi Hayakawa
Keyword(s):  
Developmental Changes ◽  
Wild Type

Developmental changes in the expression of growth-associated protein-43 mRNA in the monkey thalamus: Northern blot and in situ hybridization studies

Neuroscience ◽  
2005 ◽  
Vol 136 (2) ◽  
pp. 497-507 ◽  
Author(s):  
Y. Murata ◽  
N. Higo ◽  
T. Oishi ◽  
A. Yamashita ◽  
K. Matsuda ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Northern Blot ◽  
Developmental Changes

Changes in glucocorticoid receptor mRNA in the developing ovine pituitary and the effects of exogenous cortisol

1995 ◽  
Vol 144 (3) ◽  
pp. 483-490 ◽  
Author(s):  
S G Matthews ◽  
K Yang ◽  
J R G Challis
Keyword(s):  
Glucocorticoid Receptor ◽  
Late Gestation ◽  
Developmental Changes ◽  
Pars Intermedia ◽  
Pars Distalis ◽  
Short Term ◽  
Neonatal Life ◽  
Inferior Aspect ◽  
Glucocorticoid Receptor Mrna

Abstract Developmental changes in pituitary glucocorticoid receptor (GR) mRNA were examined during gestation and early neonatal life using in situ hybridization. Pituitaries were harvested from sheep fetuses at days 60–80, 100–120, 130–135, 140–142 and term, and from lambs of days 0–7 and 30–60, and adults. GR mRNA was present in the pars distalis by day 60, levels increased through gestation, and there was a redistribution of GR mRNA, resulting in a relatively greater abundance at the base of the pars distalis. At term, there was a significant (P<0·05 compared with the day 140–142 fetuses) elevation of GR mRNA, which was maintained in the newborn lamb, reaching highest levels at days 30–60 of neonatal life. GR mRNA was undetectable in the pars intermedia until day 120, but subsequently increased to high levels at term. Interestingly, the expression of GR mRNA in the pars intermedia dropped precipitously in the newborn (P<0·05 compared with term), though levels recovered in the older lambs and adults. The regional and cellular distribution of GR mRNA correlated closely with the presence of immuno-reactive GR (irGR) in the pituitary; the majority of irGR was present in the nuclei. Intrafetal infusion of cortisol (12 h; 5 μg/min) in late gestation (day 135) had no effect on GR mRNA expression in either the pars distalis or pars intermedia. These results indicated that, in the fetal pituitary, (1) the GR gene is expressed in both the pars distalis and pars intermedia, (2) levels of GR mRNA in the fetal pituitary correlated with the distribution of nuclear irGR, (3) GR mRNA is present at higher levels in the inferior aspect of the pars distalis, its abundance increases immediately prior to parturition and is maintained in the newborn, and (4) cortisol infusion for 12 h does not affect GR mRNA in either region of the pituitary, suggesting that, in the short term, glucocorticoids do not directly regulate GR synthesis. Journal of Endocrinology (1995) 144, 483–490

scholarly journals Prohormone convertases 1/3 and 2 together orchestrate the site-specific cleavages of progastrin to release gastrin-34 and gastrin-17

2008 ◽  
Vol 415 (1) ◽  
pp. 35-43 ◽  
Author(s):  
Jens F. Rehfeld ◽  
Xiaorong Zhu ◽  
Christina Norrbom ◽  
Jens R. Bundgaard ◽  
Anders H. Johnsen ◽  
...  
Keyword(s):  
Structural Factors ◽  
Cleavage Sites ◽  
Wild Type ◽  
Prohormone Convertases ◽  
Site Specific ◽  
G Cells ◽  
Processing Site ◽  
Vitro Effect

Cellular synthesis of peptide hormones requires PCs (prohormone convertases) for the endoproteolysis of prohormones. Antral G-cells synthesize the most gastrin and express PC1/3, 2 and 5/6 in the rat and human. But the cleavage sites in progastrin for each PC have not been determined. Therefore, in the present study, we measured the concentrations of progastrin, processing intermediates and α-amidated gastrins in antral extracts from PC1/3-null mice and compared the results with those in mice lacking PC2 and wild-type controls. The expression of PCs was examined by immunocytochemistry and in situ hybridization of mouse G-cells. Finally, the in vitro effect of recombinant PC5/6 on progastrin and progastrin fragments containing the relevant dibasic cleavage sites was also examined. The results showed that mouse G-cells express PC1/3, 2 and 5/6. The concentration of progastrin in PC1/3-null mice was elevated 3-fold. Chromatography showed that cleavage of the Arg36Arg37 and Arg73Arg74 sites were grossly decreased. Accordingly, the concentrations of progastrin products were markedly reduced, α-amidated gastrins (-34 and -17) being 25% of normal. Lack of PC1/3 was without effect on the third dibasic site (Lys53Lys54), which is the only processing site for PC2. Recombinant PC5/6 did not cleave any of the dibasic processing sites in progastrin and fragments containing the relevant dibasic processing sites. The complementary cleavages of PC1/3 and 2, however, suffice to explain most of the normal endoproteolysis of progastrin. Moreover, the results show that PCs react differently to the same dibasic sequences, suggesting that additional structural factors modulate the substrate specificity.

scholarly journals The microRNA miR-18a Links Proliferation and Inflammation During Photoreceptor Regeneration in the Injured Zebrafish Retina

2021 ◽  
Author(s):  
Evin Magner ◽  
Pamela Sandoval-Sanchez ◽  
Peter F Hitchcock ◽  
Scott M Taylor
Keyword(s):  
Muller Glia ◽  
Müller Glia ◽  
Wild Type ◽  
Post Injury ◽  
Photoreceptor Layer ◽  
Brdu Labeling ◽  
Key Aspects ◽  
The Brain ◽  
Embryonic Retina

Abstract In mammals, photoreceptor loss causes permanent blindness, but in zebrafish (Danio rerio), Müller glia function as intrinsic stem cells, producing progenitor cells that regenerate photoreceptors and restore vision. MicroRNAs (miRNAs) critically regulate neurogenesis in the brain and retina, but the roles of miRNAs in injury-induced neuronal regeneration are largely unknown. The miRNA miR-18a regulates photoreceptor differentiation in the embryonic retina. The purpose of the current study was to determine the function of miR-18a during injury-induced photoreceptor regeneration. RT-qPCR, in-situ hybridization (ISH) and immunohistochemistry (IHC) showed that miR-18a expression increases throughout the retina by 1-day post-injury (dpi) and continues to increase through 5 dpi. Bromodeoxyuridine (BrdU) labeling showed that at 7 and 10 dpi, when regenerated photoreceptors are normally differentiating, there are more proliferating Müller glia-derived progenitors in homozygous miR-18a mutant (miR-18ami5012) retinas compared with wild type (WT), indicating that miR-18a negatively regulates injury-induced proliferation. At 7 and 10 dpi, miR-18ami5012 retinas have fewer mature photoreceptors than WT, but there is no difference at 14 dpi, revealing that photoreceptor regeneration is delayed. BrdU labeling showed that the excess progenitors in miR-18ami5012 retinas migrate to other retinal layers besides the photoreceptor layer. Inflammation is critical for photoreceptor regeneration and RT-qPCR showed that, in the absence of miR-18a, inflammation is prolonged. Suppressing inflammation with dexamethasone rescues the miR-18ami5012 phenotype. Together, these data show that during injury-induced photoreceptor regeneration, miR-18a regulates proliferation and photoreceptor regeneration by regulating key aspects of the inflammatory response during photoreceptor regeneration in zebrafish.

scholarly journals Imaging Sterols and Oxysterols in Mouse Brain Reveals Distinct Spatial Cholesterol Metabolism

2018 ◽  
Author(s):  
Eylan Yutuc ◽  
Roberto Angelini ◽  
Mark Baumert ◽  
Natalia Mast ◽  
Irina Pikuleva ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Mouse Brain ◽  
Brain Function ◽  
Mass Spectrometry Imaging ◽  
Cholesterol Metabolism ◽  
Biologically Active ◽  
Wild Type ◽  
Tissue Slices ◽  
The Brain

AbstractDysregulated cholesterol metabolism is implicated in a number of neurological disorders. Many sterols, including cholesterol and its precursors and metabolites, are biologically active and important for proper brain function. However, spatial cholesterol metabolism in brain and the resulting sterol distributions are poorly defined. To better understand cholesterol metabolism in situ across the complex functional regions of brain, we have developed on-tissue enzyme-assisted derivatisation in combination with micro-liquid-extraction for surface analysis and liquid chromatography - mass spectrometry to image sterols in tissue slices (10 µm) of mouse brain. The method provides sterolomic analysis at 400 µm spot diameter with a limit of quantification of 0.01 ng/mm2. It overcomes the limitations of previous mass spectrometry imaging techniques in analysis of low abundance and difficult to ionise sterol molecules, allowing isomer differentiation and structure identification. Here we demonstrate the spatial distribution and quantification of multiple sterols involved in cholesterol metabolic pathways in wild type and cholesterol 24S-hydroxylase knock-out mouse brain. The technology described provides a powerful tool for future studies of spatial cholesterol metabolism in healthy and diseased tissues.SignificanceThe brain is a remarkably complex organ and cholesterol homeostasis underpins brain function. It is known that cholesterol is not evenly distributed across different brain regions, however, the precise map of cholesterol metabolism in the brain remains unclear. If cholesterol metabolism is to be correlated with brain function it is essential to generate such a map. Here we describe an advanced mass spectrometry imaging platform to reveal spatial cholesterol metabolism in situ at 400 µm resolution on 10 µm tissue slices from mouse brain. We mapped, not only cholesterol, but also other biologically active sterols arising from cholesterol turnover in both wild type and mice lacking cholesterol 24-hydroxylase (Cyp46a1), the major cholesterol metabolising enzyme.

scholarly journals MNB1 gene is involved in regulating the iron-deficiency stress response in Arabidopsis thaliana

2021 ◽  
Author(s):  
Hui Song ◽  
Feng Chen ◽  
Xi Wu ◽  
Min Hu ◽  
Qingliu Geng ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Arabidopsis Thaliana ◽  
Normal Growth ◽  
Developmental Changes ◽  
Fe Deficiency ◽  
Oxygen Species ◽  
Mineral Element ◽  
Wild Type ◽  
Transcription Level ◽  
Fe Uptake

Abstract Abstract Iron (Fe) is an indispensable mineral element for normal growth of plants. Fe deficiency induces a complex series of responses in plants, involving physiological and developmental changes, to increase Fe uptake from soil. However, the molecular mechanism involved in plant Fe-deficiency is not well understood. Here, we found that the MNB1 gene is involved in modulating Fe-deficiency response in Arabidopsis thaliana . The expression of MNB1 was inhabited by Fe-deficiency stress. Knockout of MNB1 led to enhanced Fe accumulation and tolerance, whereas the MNB1-overexpressing plants were sensitive to Fe-deficiency stress. Lower H 2 O 2 concentrations in mnb1 mutant plants were examined under Fe deficiency circumstances compared to wild-type. On the contray, higher H 2 O 2 concentrations were found in MNB1-overexpressing plants, which was adversely linked with malondialdehyde (MDA) concentrations. Furthermore, in mnb1 mutants, the transcription level of the Fe-uptake and translocation genes, FIT , IRT1 , FRO2 , Z IF , FRD3 , NAS4 , PYE and MYB72 , were considerably elevated during Fe-deficiency stress, resulting in higher Fe accumulation. Together, our findings show that the MNB1 gene negatively controls the Fe-deficiency response in Arabidopsis via modulating reactive oxygen species (ROS) levels and the ROS-mediated signaling pathway, thereby affecting the expression of Fe-uptake and translocation genes.

scholarly journals Structural insights into flagellar stator–rotor interactions

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Yunjie Chang ◽  
Ki Hwan Moon ◽  
Xiaowei Zhao ◽  
Steven J Norris ◽  
MD A Motaleb ◽  
...  
Keyword(s):  
Conformational Change ◽  
High Speed ◽  
Electron Tomography ◽  
Deletion Mutants ◽  
Flagellar Motor ◽  
Wild Type ◽  
Cryo Electron Tomography ◽  
Flagellar Filament ◽  
Structural Insights

The bacterial flagellar motor is a molecular machine that can rotate the flagellar filament at high speed. The rotation is generated by the stator–rotor interaction, coupled with an ion flux through the torque-generating stator. Here we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirochete, Borrelia burgdorferi. By analyzing the motor structures of wild-type and stator-deletion mutants, we not only localized the stator complex in situ, but also revealed the stator–rotor interaction at an unprecedented detail. Importantly, the stator–rotor interaction induces a conformational change in the flagella C-ring. Given our observation that a non-motile mutant, in which proton flux is blocked, cannot generate the similar conformational change, we propose that the proton-driven torque is responsible for the conformational change required for flagellar rotation.

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