Expression in non-lens tissues of an enzyme activity related to the ‘lens-specific’ protein, delta crystallin

Development ◽  
1991 ◽  
Vol 111 (1) ◽  
pp. 181-190
Author(s):  
D.I. de Pomerai ◽  
W.K. Ip ◽  
M. McLaughlin ◽  
K.C. Perry
Keyword(s):  
Ectopic Expression ◽  
Molecular Size ◽  
Protein Band ◽  
Major Constituent ◽  
Lens Protein ◽  
Related Protein ◽  
Structural Protein ◽  
Cycle Enzyme ◽  
Published Evidence

When chick embryo neutral retina (NR) cells are cultured for long periods in vitro, they undergo extensive transdifferentiation into lens and express the lens protein, delta crystallin. We now demonstrate that this process is accompanied by a change in the chromatin conformation of the delta-gene locus from DNAase1-resistant to DNAase1-sensitive in the nuclei of most cells. Transcripts hybridising to a delta probe are also much more prevalent among the in vitro transcription products from lens or transdifferentiated NR culture nuclei, as compared to nuclei from fresh NR tissue. Published evidence indicates that the chick delta 1 crystallin gene encodes the major structural protein of embryonic lens fibres, whereas the closely related delta 2 gene may encode the urea-cycle enzyme argininosuccinate lyase (ASL). Our present data lends further support to this view. Both immunodetectable delta-related protein(s) and ASL activity are present in fresh embryonic NR tissue, as well as in mouse and Rana liver, and in Rana lens. Our polyclonal anti-delta antibody also cross-reacts with a major constituent of commercial bovine ASL, of the same molecular size as chick delta crystallin. Immunoselection studies suggest that the ASL activity in chick embryonic NR is conferred mainly by the delta-related protein band. So-called ‘ectopic’ expression of delta crystallin in embryonic NR (and other tissues) may thus involve the delta 2/ASL gene, and could reflect some metabolic requirement for ASL activity.

Electrogenic 2 Na/1 H antiport in crustacean epithelium is inhibited by a monoclonal antibody

1993 ◽  
Vol 264 (4) ◽  
pp. R804-R810
Author(s):  
H. Gert de Couet ◽  
L. Busquets-Turner ◽  
A. Gresham ◽  
G. A. Ahearn
Keyword(s):  
Brush Border ◽  
Membrane Vesicles ◽  
Structural Similarity ◽  
Protein Band ◽  
Monovalent Cation ◽  
Cation Binding ◽  
Western Blots ◽  
System A ◽  
Published Evidence

We have previously published evidence that suggests that Na/H exchange in crustacean and echinoderm epithelia occurs by an electrogenic antiporter protein with two external cation binding sites that accommodate Na, amiloride, or Ca and display a 2:1 monovalent cation antiport stoichiometry. The present study is an initial investigation into the molecular biology of this invertebrate electrogenic exchanger to ascertain its structural similarity to the analogous vertebrate electroneutral antiport system. A panel of monoclonal antibodies was prepared against components of lobster hepatopancreatic epithelial brush-border membranes and assayed immunohistochemically and by Western blotting. The antibodies were tested further in functional assays for their ability to interfere with electrogenic 2 Na/1 H antiport in isolated hepatopancreatic brush-border membrane vesicles. One cell line was identified producing an antibody that significantly inhibited the electrogenic exchange of cations by these membrane preparations and recognized a single protein band on Western blots of hepatopancreas, antennal gland, and gill epithelia corresponding to a molecular mass of 185 kDa. The existence of such an antibody probe may facilitate the purification of the electrogenic antiporter under denaturing conditions, in in vitro expression systems, or in prokaryotic expression libraries.

scholarly journals Adipocyte differentiation-related protein is secreted into milk as a constituent of milk lipid globule membrane

1996 ◽  
Vol 320 (3) ◽  
pp. 1025-1030 ◽  
Author(s):  
Hans W. HEID ◽  
Martina SCHNÖLZER ◽  
Thomas W. KEENAN
Keyword(s):  
Adipocyte Differentiation ◽  
Sequence Similarity ◽  
Surface Membrane ◽  
Major Constituent ◽  
Milk Lipid ◽  
Related Protein ◽  
Membrane Material ◽  
Lipid Globule ◽  
Lactating Mammary Gland

Milk lipid globules from humans, cows and rats contained a protein identified as adipocyte differentiation-related protein (ADRP) associated with the globule surface membrane material. This protein, previously believed to be specific to adipocytes, was a major constituent of the globule surface and was present in a detergent-insoluble complex that contained stoichiometric amounts of butyrophilin and xanthine oxidase. Identification of ADRP was by sequence similarity of tryptic peptides from cow and human proteins with the sequence inferred from the cDNA for mouse ADRP. The putative ADRP of lipid globules from cow, human and rat milk was recognized specifically by antisera raised against a peptide synthesized to duplicate the N-terminal 26 residues of the mouse protein. In homogenates of lactating mammary gland, ADRP was found only in endoplasmic reticulum and in lipid droplet fractions. ADRP was modified, apparently post-translationally, and one modification apparently was acylation, primarily with C14, C16 and C18 fatty acids. Two isoelectric variants of ADRP were present in cow globule membrane material. In vitro, ADRP served as a substrate for protein kinases associated with milk lipid globule membrane, but this protein did not seem to become phosphorylated intracellularly.

scholarly journals Development and Characterization of Novel Rat Anti-mERβ Sera

Endocrinology ◽  
2016 ◽  
Vol 157 (7) ◽  
pp. 2844-2852 ◽  
Author(s):  
Horacio J. Novaira ◽  
J. B. Graceli ◽  
S. Capellino ◽  
A. Schoeffield ◽  
G. E. Hoffman ◽  
...  
Keyword(s):  
Physiological Role ◽  
Molecular Size ◽  
Keyhole Limpet Hemocyanin ◽  
Protein Band ◽  
Sprague Dawley ◽  
Amino Acid Peptide ◽  
Nuclear Extracts ◽  
Protein Partners ◽  
Mouse Tissues

Estrogens regulate normal sexual and reproductive development in females. Their actions are mediated mainly by estrogen receptor (ER)α and ERβ. Understanding the function of ERs necessitates knowing their cellular location and protein partners, which, in turn, requires reliable and specific antibodies. Several antibodies are available for ERα; however, discrepancies in immunoreactivity have been reported for ERβ. Here, we have developed antisera for mouse ERβ (mERβ) using a specific C-terminal 18-amino acid peptide conjugated to mariculture keyhole limpet hemocyanin. Sprague Dawley rats were immunized, and the resulting antisera were characterized by Western blot analysis of nuclear extracts from tissues of wild-type (WT) mice, and mice genetically modified to lack either ERα (CERαKO) or ERβ (CERβKO). An approximately 56-kDa protein was detected in the hypothalamus, uterus, ovary, mammary gland, testes, and epididymis of WT mice, consistent with the predicted molecular size of ERβ. In addition, the same protein band was identified in in vitro synthesized mERβ protein and in the mammary glands of CERαKO mice. The approximately 56-kDa protein was not observed in in vitro synthesized mERα protein or in any tissue examined in the CERβKO mice. Immunohistochemistry using the antisera revealed ERβ staining in the granulosa cells of WT ovaries and in the mediobasal hypothalamus, paraventricular nucleus, and cerebral cortex in the WT adult mouse brain. These data suggest that the novel rat anti-mERβ sera are specific to ERβ to allow investigators to explore to cellular and physiological role of ERβ in the brain and other mouse tissues.

In vitro schistosomicidal activity of Usnea steineri extract and its major constituent (+)-usnic acid against Schistosoma mansoni

Planta Medica ◽  
2012 ◽  
Vol 78 (11) ◽  
Author(s):  
AIO Salloum ◽  
R Lucarini ◽  
MG Tozatti ◽  
J Medeiros ◽  
MLA Silva ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Usnic Acid ◽  
Major Constituent

Biological and In silico Studies on Synthetic Analogues of Tyrosine Betaine as Inhibitors of Neprilysin - A Drug Target for the Treatment of Heart Failure

2018 ◽  
Vol 24 (17) ◽  
pp. 1899-1904
Author(s):  
Daniel Fabio Kawano ◽  
Marcelo Rodrigues de Carvalho ◽  
Mauricio Ferreira Marcondes Machado ◽  
Adriana Karaoglanovic Carmona ◽  
Gilberto Ubida Leite Braga ◽  
...  
Keyword(s):  
Heart Failure ◽  
Secondary Metabolites ◽  
Structural Similarity ◽  
Structural Features ◽  
Major Constituent ◽  
Binding Modes ◽  
Starting Point ◽  
In Silico Studies ◽  
Nep Inhibition

Background: Fungal secondary metabolites are important sources for the discovery of new pharmaceuticals, as exemplified by penicillin, lovastatin and cyclosporine. Searching for secondary metabolites of the fungi Metarhizium spp., we previously identified tyrosine betaine as a major constituent. Methods: Because of the structural similarity with other inhibitors of neprilysin (NEP), an enzyme explored for the treatment of heart failure, we devised the synthesis of tyrosine betaine and three analogues to be subjected to in vitro NEP inhibition assays and to molecular modeling studies. Results: In spite of the similar binding modes with other NEP inhibitors, these compounds only displayed moderate inhibitory activities (IC50 ranging from 170.0 to 52.9 µM). However, they enclose structural features required to hinder passive blood brain barrier permeation (BBB). Conclusions: Tyrosine betaine remains as a starting point for the development of NEP inhibitors because of the low probability of BBB permeation and, consequently, of NEP inhibition at the Central Nervous System, which is associated to an increment in the Aβ levels and, accordingly, with a higher risk for the onset of Alzheimer's disease.

Chemical Composition and in-vitro antimicrobial screening of leaf essential oil of Plectranthus gerardianus Benth., and isolation of the major constituent of oil

2020 ◽  
Vol 10 ◽  
Author(s):  
Navadha Bhatt ◽  
Navabha Joshi ◽  
Kapil Ghai ◽  
Om Prakash
Keyword(s):  
Antimicrobial Activity ◽  
Essential Oil ◽  
Major Constituent ◽  
In Vitro Antimicrobial Activity ◽  
Gram Positive Bacteria ◽  
Medicinal Value ◽  
Leaf Essential Oil ◽  
Plant Families ◽  
Total Oil

Background: The Lamiaceae (Labiatae) is one of the most diverse and widespread plant families’ in terms of ethno medicine and its medicinal value is based on the volatile oils concentration. This family is important for flavour, fragrance and medicinal properties. Manyplants belonging to this family have indigenous value. Method: The essential oil of Plectranthus gerardianusBenth. (Lamiaceae), was analysed by GC and GC-MS analysis, while the major component was isolated and conformed by NMR spectroscopy. Result: The oil was found to be rich in oxygenated monoterpenes, which contribute around 62% of the total oil. The major components identified were fenchone (22.90%) and carvenone oxide (16.75%), besides other mono and sesquiterpenoids. The in-vitro antimicrobial activity of essential oil was tested against three gram negative bacteria viz. Pasteurellamultocida, Escherichia coli, and Salmonella enterica, two gram positive bacteria viz. Staphylococcus aureus and Bacillus subtilis and two fungi viz. Candida albicans and Aspergillusflavus. The antimicrobial activity of the oil was also compared to the antimicrobial activity of leaf essential oil of another Himalayan plant viz. Nepetacoerulescens. Conclusion: The oil showed in-vitro antimicrobial activity against all the microbial strains and can lessen the ever-growing demand of potentially hazardous antibiotics for treatment.

Ginkgolic acid induces apoptosis and autophagy of endometrial carcinoma cells via inhibiting PI3K/Akt/mTOR pathway in vivo and in vitro

2021 ◽  
pp. 096032712110237
Author(s):  
L Zhou ◽  
S Li ◽  
J Sun
Keyword(s):  
Endometrial Cancer ◽  
B Cells ◽  
Western Blot ◽  
Signal Pathway ◽  
Mtor Pathway ◽  
Related Protein ◽  
Western Blot Assay ◽  
Ginkgolic Acid

Endometrial cancer (EC) is the fourth most common malignancy in women in developed countries. The prognosis of EC is extremely poor, and it is an important factor that contributes to the death of patients. Therefore, studying EC pathogenesis and therapeutic targets, and exploring effective drugs are the primary tasks to improve the prognosis of EC. In the present study, we aimed to explore the function of ginkgolic acid (GA) in EC cell apoptosis and autophagy through PI3K/Akt/mTOR signal pathway in vitro and in vivo. Firstly, MTT assay and clone formation assay were employed to analyze the Ishikawa and HEC-1-B cell viabilities and proliferation after treatment with GA. The results showed that GA inhibited endometrial cancer cell survival. Flow cytometry assay and western blot assay were applied to examine the apoptosis and apoptosis related protein Bcl-2, Bax, Cleaved caspase-3 expression levels of Ishikawa and HEC-1-B cells after treatment with GA. Next, we applied western blot assay to analyze the autophagy associated proteins LC3I, LC3II, p62 and Beclin-1 in GA treated Ishikawa and HEC-1-B cells. We found that GA promoted apoptosis and induced autophagy of endometrial cancer cells. Meanwhile, western blot assay was also used to determine the expression levels of the PI3K/Akt/mTOR signal pathway related protein and the results revealed that GA inhibited the activity of PI3K/Akt/mTOR pathway. Finally, we found that GA inhibited tumor growth in vivo through immunohistochemistry assay. In conclusion, GA induces apoptosis and autophagy of EC cells via inhibiting PI3K/Akt/mTOR pathway in vivo and vitro.

scholarly journals Nanoceria Prevents Glucose-Induced Protein Glycation in Eye Lens Cells

Nanomaterials ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1473
Author(s):  
Belal I. Hanafy ◽  
Gareth W. V. Cave ◽  
Yvonne Barnett ◽  
Barbara K. Pierscionek
Keyword(s):  
Protein Glycation ◽  
Human Lens ◽  
Lens Epithelial Cells ◽  
Oxide Nanoparticles ◽  
Lens Protein ◽  
Oxygen Species ◽  
Human Lens Epithelial Cells ◽  
Age Related ◽  
Age Related Changes

Cerium oxide nanoparticles (nanoceria) are generally known for their recyclable antioxidative properties making them an appealing biomaterial for protecting against physiological and pathological age-related changes that are caused by reactive oxygen species (ROS). Cataract is one such pathology that has been associated with oxidation and glycation of the lens proteins (crystallins) leading to aggregation and opacification. A novel coated nanoceria formulation has been previously shown to enter the human lens epithelial cells (HLECs) and protect them from oxidative stress induced by hydrogen peroxide (H2O2). In this work, the mechanism of nanoceria uptake in HLECs is studied and multiple anti-cataractogenic properties are assessed in vitro. Our results show that the nanoceria provide multiple beneficial actions to delay cataract progression by (1) acting as a catalase mimetic in cells with inhibited catalase, (2) improving reduced to oxidised glutathione ratio (GSH/GSSG) in HLECs, and (3) inhibiting the non-enzymatic glucose-induced glycation of the chaperone lens protein α-crystallin. Given the multifactorial nature of cataract progression, the varied actions of nanoceria render them promising candidates for potential non-surgical therapeutic treatment.

scholarly journals Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

2021 ◽  
Vol 30 ◽  
pp. 096368972097821
Author(s):  
Andrea Tenorio-Mina ◽  
Daniel Cortés ◽  
Joel Esquivel-Estudillo ◽  
Adolfo López-Ornelas ◽  
Alejandro Cabrera-Wrooman ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Neural Induction ◽  
Human Keratinocytes ◽  
Differentiation Potential ◽  
Neuronal Proteins ◽  
Green Fluorescent

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

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