Establishment of a protocol for obtention of neuronal stem cells lineages from the dog olfactory epithelium

A morphological and cell culture study from nasal mucosa of dogs was performed in order to establish a protocol to obtain a cell population committed to neuronal lineage, as a proposal for the treatment of traumatic and degenerative lesions in these animals, so that in the future these results could be applied to the human species. Twelve mongrel dogs of 60-day aged pregnancy were collected from urban pound dogs in São Paulo. Tissue from cribriform ethmoidal lamina of the fetuses was collected at necropsy under sterile conditions around 1h to 2h postmortem by uterine sections and sections from the fetal regions described above. Isolated cells of this tissue were added in DMEM/F-12 medium under standard conditions of incubation (5% CO², >37ºC). Cell culture based on isolated cells from biopsies of the olfactory epithelium showed rapid growth when cultured for 24 hours, showing phase-bright sphere cells found floating around the fragments, attached on culture flasks. After 20 days, a specific type of cells, predominantly ellipsoids or fusiform cells was characterized in vitro. The indirect immunofluorescence examination showed cells expressing markers of neuronal precursors (GFAP, neurofilament, oligodendrocyte, and III â-tubulin). The cell proliferation index showed Ki67 immunostaining with a trend to label cell groups throughout the apical region, while PCNA immunostaining label predominantly cell groups lying above the basal lamina. The transmission electron microscopy from the olfactory epithelium of dogs revealed cells with electron-dense cytoplasm and preserving the same distribution as those of positive cell staining for PCNA. Metabolic activity was confirmed by presence of euchromatin in the greatest part of cells. All these aspects give subsidies to support the hypothesis about resident progenitor cells among the basal cells of the olfactory epithelium, committed to renewal of these cell populations, especially neurons.

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Which cytochrome P450 metabolizes phenazepam? Step by step in silico, in vitro, and in vivo studies

Drug Metabolism and Personalized Therapy ◽

10.1515/dmpt-2017-0036 ◽

2018 ◽

Vol 33 (2) ◽

pp. 65-73 ◽

Cited By ~ 6

Author(s):

Dmitriy V. Ivashchenko ◽

Anastasia V. Rudik ◽

Andrey A. Poloznikov ◽

Sergey V. Nikulin ◽

Valeriy V. Smirnov ◽

...

Keyword(s):

Cell Culture ◽

Cytochrome P450 ◽

In Silico ◽

In Vivo Studies ◽

Cyp3a Activity ◽

Cell Culture Study ◽

Study Results ◽

Three Stages

Abstract Background: Phenazepam (bromdihydrochlorphenylbenzodiazepine) is the original Russian benzodiazepine tranquilizer belonging to 1,4-benzodiazepines. There is still limited knowledge about phenazepam’s metabolic liver pathways and other pharmacokinetic features. Methods: To determine phenazepam’s metabolic pathways, the study was divided into three stages: in silico modeling, in vitro experiment (cell culture study), and in vivo confirmation. In silico modeling was performed on the specialized software PASS and GUSAR to evaluate phenazepam molecule affinity to different cytochromes. The in vitro study was performed using a hepatocytes’ cell culture, cultivated in a microbioreactor to produce cytochrome P450 isoenzymes. The culture medium contained specific cytochrome P450 isoforms inhibitors and substrates (for CYP2C9, CYP3A4, CYP2C19, and CYP2B6) to determine the cytochrome that was responsible for phenazepam’s metabolism. We also measured CYP3A activity using the 6-betahydroxycortisol/cortisol ratio in patients. Results: According to in silico and in vitro analysis results, the most probable metabolizer of phenazepam is CYP3A4. By the in vivo study results, CYP3A activity decreased sufficiently (from 3.8 [95% CI: 2.94–4.65] to 2.79 [95% CI: 2.02–3.55], p=0.017) between the start and finish of treatment in patients who were prescribed just phenazepam. Conclusions: Experimental in silico and in vivo studies confirmed that the original Russian benzodiazepine phenazepam was the substrate of CYP3A4 isoenzyme.

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Microtubules facilitate the stimulated secretion of beta-hexosaminidase in lacrimal acinar cells

Journal of Cell Science ◽

10.1242/jcs.111.9.1267 ◽

1998 ◽

Vol 111 (9) ◽

pp. 1267-1276 ◽

Cited By ~ 3

Author(s):

S.R. da Costa ◽

F.A. Yarber ◽

L. Zhang ◽

M. Sonee ◽

S.F. Hamm-Alvarez

Keyword(s):

Confocal Microscopy ◽

Cytochalasin D ◽

Apical Plasma Membrane ◽

Secretory Proteins ◽

Apical Region ◽

Cell Periphery ◽

Isolated Cells ◽

Microtubule Array ◽

Lacrimal Acinar Cells

Stimulation of lacrimal acini with secretagogues such as carbachol initiates movement and fusion of acinar secretory vesicles with the apical plasma membrane, resulting in release of protein into the nascent tear fluid. Using rabbit lacrimal acini reconstituted in vitro from isolated cells, we have investigated the organization of the apical cytoskeleton and its role in stimulated secretion. Confocal microscopy revealed a microtubule array emanating from the apical region of the acini; the apical region was also enriched in microfilaments and (gamma)-tubulin. Cytokeratin-based intermediate filaments were apically concentrated, and also detected at the cell periphery. Neither confocal microscopy nor biochemical analysis revealed any reorganization of lumenal microfilaments or microtubules which might accompany carbachol-stimulated release of secretory proteins. However, major changes in the acinar microtubule array induced by taxol or nocodazole were correlated with inhibition of carbachol-dependent release of the secreted protein, beta-hexosaminidase. Major changes in lumenal microfilaments induced by jasplakinolide or cytochalasin D did not inhibit the carbachol-dependent release of beta-hexosaminidase; rather, release of beta-hexosaminidase from jasplakinolide- or cytochalasin D-treated carbachol-stimulated acini was markedly increased relative to the release from untreated stimulated acini. Our findings demonstrate that microtubules play a major role in stimulated lacrimal secretion, and suggest a contributory role for microfilaments.

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Endogenous pH 6.0 β-Galactosidase Activity Is Linked to Neuronal Differentiation in the Olfactory Epithelium

Cells ◽

10.3390/cells11020298 ◽

2022 ◽

Vol 11 (2) ◽

pp. 298

Author(s):

José Antonio de Mera-Rodríguez ◽

Guadalupe Álvarez-Hernán ◽

Yolanda Gañán ◽

Ana Santos-Almeida ◽

Gervasio Martín-Partido ◽

...

Keyword(s):

Enzymatic Activity ◽

Neuronal Differentiation ◽

Olfactory Epithelium ◽

Cellular Senescence ◽

Time Windows ◽

Lectin Histochemistry ◽

Apical Region ◽

Basal Region ◽

Basal Cells ◽

Histochemical Detection

The histochemical detection of β-galactosidase enzymatic activity at pH 6.0 (β-gal-pH6) is a widely used biomarker of cellular senescence in aging tissues. This histochemical assay also detects the presence of programmed cell senescence during specific time windows in degenerating structures of vertebrate embryos. However, it has recently been shown that this enzymatic activity is also enhanced in subpopulations of differentiating neurons in the developing central nervous system in vertebrates. The present study addressed the histochemical detection of β-gal-pH6 enzymatic activity in the developing postnatal olfactory epithelium in the mouse. This activity was detected in the intermediate layer of the olfactory epithelium. As development progressed, the band of β-gal-pH6 labeling in this layer increased in width. Immunohistochemistry and lectin histochemistry showed the β-gal-pH6 staining to be strongly correlated with the immunolabeling of the olfactory marker protein (OMP) that identifies mature olfactory sensory neurons. The cell somata of a subpopulation of differentiated olfactory neurons that were recognized with the Dolichos biflorus agglutinin (DBA) were always located inside this band of β-gal-pH6 staining. However, the β-gal-pH6 histochemical signal was always absent from the apical region where the cytokeratin-8 positive supporting cells were located. Furthermore, no β-gal-pH6 staining was found in the basal region of the olfactory epithelium where PCNA/pHisH3 immunoreactive proliferating progenitor cells, GAP43 positive immature neurons, and cytokeratin-5 positive horizontal basal cells were located. Therefore, β-gal-pH6 seems to be linked to neuronal differentiation and cannot be regarded as a biomarker of cellular senescence during olfactory epithelium development in mice.

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Characterization of the intrinsic properties of the anterior cruciate and medial collateral ligament cells: An in vitro cell culture study

Journal of Orthopaedic Research® ◽

10.1002/jor.1100100402 ◽

1992 ◽

Vol 10 (4) ◽

pp. 465-475 ◽

Cited By ~ 158

Author(s):

Chandrasekharam N. Nagineni ◽

David Amiel ◽

Melvin H. Green ◽

Matthew Berchuck ◽

Wayne H. Akeson

Keyword(s):

Cell Culture ◽

Medial Collateral Ligament ◽

Intrinsic Properties ◽

Collateral Ligament ◽

Culture Study ◽

Cell Culture Study ◽

In Vitro Cell Culture ◽

Anterior Cruciate

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Cytotoxic and Enzyme-Inducing Effects of Rodent Diets and Cage Bedding Materials: Evaluation by a Cell Culture Study

Alternatives to Laboratory Animals ◽

10.1177/026119299001700309 ◽

1990 ◽

Vol 17 (3) ◽

pp. 182-187

Author(s):

Riitta Törrönen ◽

Kai Pelkonen ◽

Sirpa Karenlampi

Keyword(s):

Cell Culture ◽

Hepatoma Cell ◽

Screening Method ◽

Laboratory Animals ◽

Hepatoma Cell Line ◽

Cell Culture Study ◽

A Cell ◽

Materials Used ◽

Bedding Materials

Several rodent diets and wood-based materials used as cage bedding for laboratory animals were studied for their cytotoxic and cytochrome P450-inducing properties by an in vitro method. The cell culture system using a mouse hepatoma cell line (Hepa-1) proved to be a convenient and sensitive screening method, and considerable differences among both the feeds and the bedding materials were detected.

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Effect of Breast Milk Calcium and Fluidity on Breast Cancer Cells: An In Vitro Cell Culture Study

Breastfeeding Medicine ◽

10.1089/bfm.2016.0048 ◽

2016 ◽

Vol 11 (9) ◽

pp. 474-478 ◽

Cited By ~ 2

Author(s):

Recep Bayram ◽

Muhsine Zeynep Yavuz ◽

Bedri Selim Benek ◽

Ayşenur Aydoğar Bozkurt ◽

Ali Ucbek ◽

...

Keyword(s):

Breast Cancer ◽

Cell Culture ◽

Breast Milk ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Culture Study ◽

Cell Culture Study ◽

In Vitro Cell Culture

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The characteristics of intracellular Ca2+ signals in vivo necessitate a new model for salivary fluid secretion

10.1101/2020.12.30.424864 ◽

2021 ◽

Author(s):

Takahiro Takano ◽

Amanda M. Wahl ◽

Kai-Ting Huang ◽

John Rugis ◽

James Sneyd ◽

...

Keyword(s):

Mathematical Model ◽

Fluid Secretion ◽

Salivary Secretion ◽

Apical Region ◽

Isolated Cells ◽

Marked Contrast ◽

K Channels ◽

Stimulation Of

AbstractSalivary fluid secretion involves an intricate choreography to result in the trans-epithelial movement of NaCl and water into the acinus lumen. Current models are based on experimental observations in enzymatically isolated cells where the Ca2+ signal invariably propagates globally and thus appears ideally suited to activate spatially separated Cl and K channels. We monitored Ca2+ signals and salivary secretion in live mice expressing GCamp6F, following stimulation of the nerves innervating the submandibular gland. Consistent with in vitro studies, Ca2+ signals were initiated in the apical endoplasmic reticulum. In marked contrast to in vitro data, highly localized trains of Ca2+ transients that failed to propagate from the apical region were observed. Following stimuli optimum for secretion, large apical-basal gradients were elicited. Given this incompatibility to the previous model, a new mathematical model was constructed to explain how salivary secretion can be efficiently stimulated by apically localized Ca2+ signals.

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Highly Localized intracellular Ca2+ signals promote optimal salivary gland fluid secretion

eLife ◽

10.7554/elife.66170 ◽

2021 ◽

Vol 10 ◽

Author(s):

Takahiro Takano ◽

Amanda Wahl ◽

Kai-Ting Huang ◽

Takanori Narita ◽

John Rugis ◽

...

Keyword(s):

Fluid Secretion ◽

Salivary Secretion ◽

Membrane Transporters ◽

Apical Region ◽

Isolated Cells ◽

Marked Contrast ◽

K Channels ◽

Basolateral Plasma Membrane ◽

Stimulation Of

Salivary fluid secretion involves an intricate choreography of membrane transporters to result in the trans-epithelial movement of NaCl and water into the acinus lumen. Current models are largely based on experimental observations in enzymatically isolated cells where the Ca2+ signal invariably propagates globally and thus appears ideally suited to activate spatially separated Cl and K channels, present on the apical and basolateral plasma membrane, respectively. We monitored Ca2+ signals and salivary secretion in live mice expressing GCamp6F, following stimulation of the nerves innervating the submandibular gland. Consistent with in vitro studies, Ca2+ signals were initiated in the apical endoplasmic reticulum. In marked contrast to in vitro data, highly localized trains of Ca2+ transients that failed to fully propagate from the apical region were observed. Following stimuli optimum for secretion, large apical-basal gradients were elicited. A new mathematical model, incorporating these data was constructed to probe how salivary secretion can be optimally stimulated by apical Ca2+ signals.

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