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Serodiagnostic methods for diagnosing larval toxocariasis

Noordin R, Yunus MH, Tan Farrizam SN, Arifin N

Adv Parasitol, 2020;109:131-152.
PMID: 32381194 DOI: 10.1016/bs.apar.2020.01.003

Toxocariasis is a human infection primarily caused by larvae of Toxocara canis from dogs, and also by T. cati from cats. Children have a more significant risk of acquiring the infection due to their closer contact with pets, and greater chances of ingesting soil. Diagnosis of toxocariasis is based on clinical, epidemiological, and serological data. Indirect IgG ELISA is a widely used serodiagnostic method for toxocariasis, with native T. canis TES most commonly used as the antigen. Western blots, using the same antigen, can be used to confirm positive ELISA findings to reduce false-positive results. Improvements in Toxocara serodiagnosis include the use of recombinant TES antigens, simpler and more rapid assay formats, and IgG4 subclass detection. Also, incorporation of recombinant T. cati TES protein increases the diagnostic sensitivity. Development of antigen detection tests using polyclonal and monoclonal antibodies, nanobodies, or aptamers can complement the antibody detection assays, and enhance the effectiveness of the serodiagnosis.

Matched MeSH terms: Blotting, Western/methods
Heavy metals content in paraphilometroides nemipteri from south China sea may influence level of glutathione and p38 protein expression

Sains Malaysiana, 2018;47:1393-1400.

Numerous studies have shown that parasites potentially become bio-accumulators for heavy metals. The heavy metals
content in parasite-infected fish was reported to be lower compared to the parasite non-infected fish. Evaluation of heavy
metal content in Nemipterus peronii and Paraphilometroides nemipteri was performed using ICP-OES. Our result has
shown that arsenic was the most abundance heavy metal content in muscle N. peronii and P. nemipteri, suggesting that
the parasite has the ability to accumulate heavy metals. Heavy metals were reported to induce oxidative stress where
glutathione and p38 protein may be involved. Thus, expression of the p38 protein was determined using western blot
technique and glutathione content was measured fluorometrically. The p38 expression in P. nemipteri of Pulau Kambing
was higher compared to P. nemipteri of Besut has shown that the parasite may exposed to stress. Glutathione content
showed no significant changes due to detoxification mechanism occurred in the parasite. In this study, we could conclude
that P. nemipteri could be a bio-accumulator, whereas p38 protein and glutathione as indicator of stress level in the
parasite that exposed to the heavy metals.

Matched MeSH terms: Blotting, Western
Transient expression of an immunogenic envelope attachment glycoprotein of nipah virus in nicotiana benthamiana

Rofina Yasmin Othman, Jennifer Ann Harikrishna, Gan KS, Tan CS

Sains Malaysiana, 2018;47:499-509.

The Nipah virus is highly virulent to swine and humans. The envelope attachment glycoprotein (G) of Nipah virus
plays a key role in viral entry and induction of neutralizing antibody in mammalian hosts, thus is considered a good
candidate for vaccine development. Plant transient expression systems are gaining recognition as a viable alternative
for the production of vaccine antigens. In this study, we expressed the Nipah virus G protein heterologously in Nicotiana
benthamiana using an agroinfiltration approach. The highest expression of recombinant G protein in N. benthamiana at
RNA and protein levels was detected on day 9 post-infiltration. Western blot analysis demonstrated that the purified G
protein reacted specifically with rabbit anti-Nipah Virus serum, indicating its potential for vaccine use.

Matched MeSH terms: Blotting, Western
Fulltext Recombinant dense granular protein (GRA5) for detection of human toxoplasmosis by Western blot

Ching XT, Lau YL, Fong MY, Nissapatorn V, Andiappan H

Biomed Res Int, 2014;2014:690529.
PMID: 24987700 DOI: 10.1155/2014/690529

Toxoplasma gondii infects all warm-blooded animals, including humans, causing serious public health problems and great economic loss for the food industry. Commonly used serological tests require costly and hazardous preparation of whole Toxoplasma lysate antigens from tachyzoites. Here, we have evaluated an alternative method for antigen production, which involved a prokaryotic expression system. Specifically, we expressed T. gondii dense granular protein-5 (GRA5) in Escherichia coli and isolated it by affinity purification. The serodiagnostic potential of the purified recombinant GRA5 (rGRA5) was tested through Western blot analysis against 212 human patient serum samples. We found that rGRA5 protein was 100% specific for analysis of toxoplasmosis-negative human sera. Also, rGRA5 was able to detect acute and chronic T. gondii infections (sensitivities of 46.8% and 61.2%, resp.).

Matched MeSH terms: Blotting, Western/methods*
Fulltext Evaluation of Pichia pastoris-expressed recombinant rhoptry protein 2 of Toxoplasma gondii for its application in diagnosis of toxoplasmosis

Chang PY, Fong MY, Nissapatorn V, Lau YL

Am J Trop Med Hyg, 2011 Sep;85(3):485-9.
PMID: 21896809 DOI: 10.4269/ajtmh.2011.11-0351

Rhoptry protein 2 (ROP2) of Toxoplasma gondii is a rhoptry-secreted protein that plays a critical role in parasitophorous vacuole membrane formation during invasion. In previous studies, ROP2 has been shown to be efficient in triggering humoral and cell-mediated responses. High immunogenicity of ROP2 makes it a potential candidate for diagnosis and vaccination against toxoplasmosis. In this study, the ROP2 gene was cloned into pPICZα A expression vector and extracellularly expressed in the yeast Pichia pastoris, which has numerous advantages over other expression systems for eukaryotic proteins expression. The effectiveness of the secreted recombinant ROP2 as a diagnosis agent was assessed by Western Blot with 200 human serum samples. Recombinant ROP2 reacted with toxoplasmosis-positive human serum samples and yielded an overall sensitivity of 90% and specificity of 95%. However, recombinant ROP2 is a better marker for detection of IgG (91.7%) rather than IgM (80%).

Matched MeSH terms: Blotting, Western/methods
Seroprevalence of sarcocystosis in the local communities of Pangkor and Tioman Islands using recombinant surface antigens 3 (rSAG3) of Sarcocystis falcatula

Tengku-Idris TIN, Fong MY, Lau YL

Trop Med Int Health, 2018 12;23(12):1374-1383.
PMID: 30286271 DOI: 10.1111/tmi.13160

OBJECTIVE: To investigate the seroprevalence of Sarcocystosis in the local communities of Pangkor and Tioman islands, Malaysia, by using antigenic recombinant surface antigens 2 and 3 from Sarcocystis falcatula (rSfSAG2 and rSfSAG3) as the target proteins via Western blot and ELISA assays.
METHODS: SfSAG2 and SfSAG3 genes were isolated from S. falcatula and expressed in Escherichia coli expression system. A total of 348 serum samples [volunteers from both islands (n = 100), non-Sarcocystis parasitic infections patients (n = 50) and healthy donors (n = 100)] were collected and tested with purified SfSAGs in Western blot and ELISA assays to measure the seroprevalence of human sarcocystosis.
RESULTS: None of the sera in this study reacted with rSfSAG2 by Western blot and ELISA. For rSfSAG3, relatively high prevalence of sarcocystosis was observed in Tioman Island (75.5%) than in Pangkor Island (34%) by Western blot. In ELISA, the different prevalence rate was observed between Tioman Island (43.8%) and Pangkor Island (37%). The prevalence rate in other parasitic infections (amoebiasis, cysticercosis, filariasis, malaria, toxocariasis and toxoplasmosis) was 30% by Western blot and 26% by ELISA. Only 8% (by Western blot) and 10% (by ELISA) of healthy donors showed reactivity towards rSfSAG3.
CONCLUSION: This is the first study reporting a seroprevalence of sarcocystosis in Pangkor and Tioman Islands, Malaysia. The combination of Western blot and ELISA is suitable to be used for serodiagnosis of sarcocystosis. With further evaluations, SfSAG3 can potentially be used to confirm infection, asymptomatic screening, surveillance and epidemiological studies.

Matched MeSH terms: Blotting, Western/methods
Fulltext A Toxoplasma gondii 10 kDa in vitro excretory secretory antigen reactive with human IgM and IgA antibodies

Saadatnia G, Ghaffarifar F, Khalilpour A, Amerizadeh A, Rahmah N

Trop Biomed, 2011 Dec;28(3):606-14.
PMID: 22433890 MyJurnal

Toxoplasmosis can cause serious disease in immunocompromised patients and to congenitally infected foetuses. Appropriate laboratory investigations in potential cases of acute Toxoplasma infection are important. Excretory secretory antigen (ESA) is immunogenic during both human and experimental infections, therefore is considered as a good candidate for investigation into new infection markers. In this study, ESA was prepared from in vitro cultures of Toxoplasma gondii to identify T. gondii ESA antigenic component(s) that is/are most reactive with serum samples from probable acute cases of toxoplasmosis. Serum samples were obtained from several categories of individuals with the following Toxoplasma serology: Group I: IgM+ IgG+ (low IgG avidity) or IgM+ IgG- from sera of patients who had clinical query of toxoplasmosis (n=35). Group II: IgM- IgG+ (high IgG avidity) from chronically infected individuals (n=30). Group III: normal/healthy individuals with anti-Toxoplasma IgMIgG- (n=20). Group IV: individuals with other infections who had anti-Toxoplasma IgM- IgG- (n=10). The ESA was subjected to SDS-PAGE, followed by Western blot analysis using the above sera and probed with peroxidase conjugated anti-human IgM and IgA antibodies. The blots were then developed using chemiluminescence substrate. The selected antigenic band was excised from the gel after two dimensional electrophoresis and sent for mass spectrometry analysis using MALDI TOF-TOF. The most promising antigenic band was a 10 kDa protein which showed sensitivity of 80% in both IgM and IgA blots, and specificity of 96.7% with sera from other infections and healthy controls. The two best identifications for the 10 kDa band were ubiquitin (ribosomal protein CEP52 fusion protein) and polyubiquitin.

Matched MeSH terms: Blotting, Western/methods
An assay for selection of sera with circulating Toxoplasma gondii antigens

Emelia O, Zeehaida M, Sulaiman O, Rohela M, Saadatnia G, Yeng C, et al.

J Immunoassay Immunochem, 2010;31(1):79-91.
PMID: 20391020 DOI: 10.1080/15321810903405134

We have developed an ELISA that employs monoclonal anti-Toxoplasma SAG1 (p30) as the capture antibody to detect T. gondii circulating antigens in patients' serum samples. Using serum spiked with Toxoplasma soluble and with SAG1 recombinant proteins, the detection limits were 31.25 ng/mL and 62.50 ng/mL, respectively. We obtained positive results in 28% (21/75) and 11% (23/206) of probable active and chronic toxoplasmosis serum samples, respectively. Western blot analysis on pooled antigen-positive serum samples showed antigenic bands of molecular weights 25 and 75 kDa from sera of probable active infection and five antigenic bands ranging in size from 26 to 33 kDa from chronic infection sera. This assay would be useful as an initial serum selection step in developing a Toxoplasma antigen detection test and for characterization studies.

Matched MeSH terms: Blotting, Western
Mimotopes of heat shock proteins of Salmonella enterica serovar Typhi identified from phage-displayed peptide library

Yuen HL, Shamala D, Thong KL

J Infect Dev Ctries, 2008 Aug 30;2(4):313-23.
PMID: 19741295

BACKGROUND: Heat shock proteins (HSPs) are known to be involved in the pathogenesis of Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever. The objective of this study was to apply a phage display library to identify mimotopes of two HSPs, HSP90 and DnaK in S. Typhi.
METHODOLOGY: A 12-mer random peptide library expressed on the surface of the filamentous phage, M13, was used to select the mimotopes of two S. Typhi heat shock proteins by biopanning with monoclonal antibodies (mAbs), DnaK and HSP90. The immunogenicity of the selected peptides was determined through binding affinity with polyclonal antibodies from pooled typhoid-confirmed patients' sera and purified HSPs mAb using Western blotting and ELISA.
RESULTS: Five rounds of biopanning resulted in enrichment of phage clones expressing the binding motifs TDxSTRP and FPSHYWLYPPPT, respectively. The selected peptides showed strong immunoreactivity with patients' sera. Thus, monoclonal antibodies against HSP and patient sera can select common mimotopes from the random peptide library.
CONCLUSION: These findings may provide fundamental information for further studies on diagnostic application or vaccine design against this aetiologic agent of typhoid fever.

Matched MeSH terms: Blotting, Western
SDS-PAGE electrophoretic property of human chorionic gonadotropin (hCG) and its beta-subunit

Gam LH, Latiff A

Int. J. Biol. Sci., 2005;1(3):103-9.
PMID: 16094462

The microheterogeneity property of hCG with regards to its sialic acid contents resulted in variable mobility of the glycoprotein in SDS-PAGE. The intact hCG molecule is composed of two dissimilar subunits, namely alpha- and beta-subunits. The identification of hCG bands in SDS-PAGE was accomplished by the immunoblotting experiment, whereby the antibody directed toward the specific region of beta-subunit of hCG was used. The data shows that the different mobility of intact hCG was attributed to the different degree of desialylation of the glycoprotein. Nevertheless, unlike the intact hCG, the mobility of its beta-subunit was not affected by its variety sialic acid content. This characteristic of beta-hCG is beneficial when semi-quantification of total hCG is required. Quantification of hCG using the HPLC-reversed phase C18 analytical column is not possible as the glycoprotein was eluted in multiple fractions at different retention times. The identification of denatured hCG (HPLC eluted fractions) was carried out by immunoblotting experiment whilst immunoassay technique failed to detect its presence in any fraction.

Matched MeSH terms: Blotting, Western
Cloning and expression of Vibrio cholerae virulence gene, accessory cholera enterotoxin (ace)

Somarny WM, Mariana NS, Rozita R, Raha AR

Southeast Asian J. Trop. Med. Public Health, 2004 Dec;35(4):856-62.
PMID: 15916081

The cholera enterotoxin (CT) has been considered a major virulence factor of Vibrio cholerae. The accessory cholera enterotoxin (ace) gene is the third gene of V. cholerae virulence cassette. The gene coding for the Ace toxin was amplified from V. cholerae isolates producing a single band of 314 bp. The presence of ace gene was confirmed by hybridization as well as by sequencing. The gene was successfully expressed in Escherichia coli (LMG194) using expression, pBAD/Thio-TOPO vector. Optimal conditions for expression included choice of host strain, temperature used for culturing, and concentration of antibiotic and arabinose inducer. The Ace protein was obtained from the cell supernatant as a fusion protein with a molecular mass 34 kDa which was detected using an anti V5-HRP epitope tagged antibody.

Matched MeSH terms: Blotting, Western
Fulltext In depth analysis of the Sox4 gene locus that consists of sense and natural antisense transcripts

Ling KH, Brautigan PJ, Moore S, Fraser R, Leong MP, Leong JW, et al.

Data Brief, 2016 Jun;7:282-90.
PMID: 26958646 DOI: 10.1016/j.dib.2016.01.045

SRY (Sex Determining Region Y)-Box 4 or Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts [1], [2]. Here we provide accompanying data on various analyses performed and described in Ling et al. [2]. The data include a detail description of various features found at Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH), Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR), gain-of-function and in situ hybridization (ISH) experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1.

Matched MeSH terms: Blotting, Western
Fulltext Effect of glycerol feed in methanol induction phase for Hepatitis B surface antigen expression in Pichia pastoris strain KM71

Morvarid, A.R., Zeenathul, N.A., Tam, Y.J., Zuridah, H., Mohd-Azmi, M.L., Azizon, B.O.

Pertanika Journal of Science & Technology, 2012;20(1):31-42.
MyJurnal

This study describes expression of HBs Ag in methylotrophic yeast, Pichia Pastoris under alcohol oxidase promoter. A single copy number of HBs Ag gene was transformed into pichia strain of KM 71, a Muts type, by using pA0815 pichia expression vector. The recombinant was cultivated in a shake flask either using methanol or a mixed feed of glycerol -methanol for induction. The HBs Ag gene integrity was justified using direct PCR method. The expressed products in the soluble cell extracts were analyzed by Western blot, SDS page, Bradford assay and ELISA tests. The recombinant HBs Ag was expressed successfully in Pichia pastoris strain KM71 at a high level of HBs Ag protein expression. Thus, an addition of glycerol in the ratio of glycerol per methanol 1/1 (g g-1) consistently produced 2-fold increment in both biomass accumulation and HBs Ag productivity.

Matched MeSH terms: Blotting, Western
Fulltext Expression of recombinant E1 Glycoprotein of Chikungunya virus in Baculovirus expression system

Sum, Magdline Sia Henry, Andrew, Anna, Maling, Milda Aren

Journal of Biochemistry, Microbiology and Biotechnology, 2015;3(1):26-29.
MyJurnal

Chikungunya is an acute febrile illness caused by chikungunya virus (CHIKV). In this study, the envelope E1 gene of CHIKV was cloned and expressed in a baculovirus system. The recombinant E1 protein with N-term 6-His residues protein was successfully expressed and purified as confirmed by SDS-PAGE and western blot analysis. The seroreactivity of the recombinant protein was evaluated in immunoassay for anti-CHIKV IgM and IgG antibodies. The recombinant antigen showed 69% sensitivity and 100% specificity for anti-CHIKV IgG by dot blot assay. Detection of anti-CHIKV IgM by dot assay showed 79% sensitivity and 100% specificity. No cross reactivity of the antigen was observed with anti-dengue virus serum samples. The results strongly support that the recombinant E1 protein has potential to be used as diagnostic antigen. The used of the antigen in a dot blot assay gives an advantage for laboratory detection without the need of any specialised equipment.

Matched MeSH terms: Blotting, Western
Fulltext Identification and Preliminary Evaluation of a Novel Recombinant Protein for Serodiagnosis of Strongyloidiasis

Arifin N, Yunus MH, Nolan TJ, Lok JB, Noordin R

Am J Trop Med Hyg, 2018 04;98(4):1165-1170.
PMID: 29436335 DOI: 10.4269/ajtmh.17-0697

Strongyloides stercoralis is a human parasite that can cause a long-term infection. In immunosuppressed patients, strongyloidiasis may be fatal when there is overwhelming autoinfection resulting in the migration of large numbers of larvae through many organs. Definitive diagnosis is still a challenge, and a combination of symptoms, microscopic identification, and serology test results are often used to arrive at a clinical decision. However, intermittent larval excretion, low parasite burden, and occult infections are challenges with parasitological diagnosis of infection with S. stercoralis. Meanwhile, serologic tests using immunoglobulin G and parasite antigen extract have problems of cross-reactivity with other helminthic infections. Recombinant antigen-based serodiagnosis is a good alternative to overcome the laboratory diagnostic issues. Herein, we report on the isolation of cDNA clone encoding an antigen of potential diagnostic value identified from immunoscreening of a S. stercoralis cDNA library. The translated protein had highest similarity to Strongyloides ratti immunoglobulin-binding protein 1. The recombinant antigen produced, rSs1a, was assessed using western blot and enzyme-linked immunosorbent assay. The latter showed 96% diagnostic sensitivity and 93% specificity; thus, rSs1a has good potential for use in serodiagnosis of human strongyloidiasis.

Matched MeSH terms: Blotting, Western
Fulltext Can We Use 2,3,5-Triphenyltetrazolium Chloride-Stained Brain Slices for Other Purposes? The Application of Western Blotting

Sanchez-Bezanilla S, Nilsson M, Walker FR, Ong LK

Front Mol Neurosci, 2019;12:181.
PMID: 31417355 DOI: 10.3389/fnmol.2019.00181

2,3,5-Triphenyltetrazolium chloride (TTC) staining is a commonly used method to determine the volume of the cerebral infarction in experimental stroke models. The TTC staining protocol is considered to interfere with downstream analyses, and it is unclear whether TTC-stained brain samples can be used for biochemistry analyses. However, there is evidence indicating that, with proper optimization and handling, TTC-stained brains may remain viable for protein analyses. In the present study, we aimed to rigorously assess whether TTC can reliably be used for western blotting of various markers. In this study, brain samples obtained from C57BL/6 male mice were treated with TTC (TTC+) or left untreated (TTC-) at 1 week after photothrombotic occlusion or sham surgery. Brain regions were dissected into infarct, thalamus, and hippocampus, and proteins were extracted by using radioimmunoprecipitation assay buffer. Protein levels of apoptosis, autophagy, neuronal, glial, vascular, and neurodegenerative-related markers were analyzed by western blotting. Our results showed that TTC+ brains display similar relative changes in most of the markers compared with TTC- brains. In addition, we validated that these analyses can be performed in the infarct as well as other brain regions such as the thalamus and hippocampus. Our findings demonstrate that TTC+ brains are reliable for protein analyses using western blotting. Widespread adoption of this approach will be key to lowering the number of animals used while maximizing data.

Matched MeSH terms: Blotting, Western
Chronic Cold-Water-Induced Hypothermia Impairs Memory Retrieval and Nepeta menthoides as a Traditional "Hot" Herb Reverses the Impairment

Ahmadian-Attar MM, Ahmadiani A, Kamalinejad M, Dargahi L, Mosaddegh M

Iran J Pharm Res, 2014;13(Suppl):185-93.
PMID: 24711845

Iranian Traditional Medicine (ITM) describes a kind of dementia with similar signs and symptoms of Alzheimer's disease (AD). It explains the pathology of dementia with cold intemperament of the brain, which means that the brain is colder than its healthy form. ITM strategy for treatment of dementia is to heat the brain up by medical "hot" herbs. Nepeta menthoides (NM) is one of these "hot" herbs. To evaluate the veracity of ITM concept about dementia and its treatment, we first try to examine if coldness of brain can make memory impairment. If so, can NM reverse memory impairment? Rats in cold-water-induced hypothermic (CWH) groups were immersed up to the neck in 3.5 °C water, for 5 min during 14 consecutive days. As a control, rats were forced to swim in warm water at the same conditions. To eliminate the impact of forced swimming stress, a group of intact rats was also added. After last swimming in day 14, some groups received drug (100 or 500 mg/ Kg aqueous extract of NM) or vehicle via i.p. injection. Learning and memory were assessed by Morris water maze, and tau hyperphosphorylation was measured by western blotting. The results showed that CWH impairs learning and memory and induces tau hyperphosphorylation. 100 mg/Kg of NM reversed memory impairment as well as tau hyperphosphorylation. ITM theory about the relationship between brain hypothermia and dementia is in accordance with our findings.

Matched MeSH terms: Blotting, Western
Fulltext Pharmacokinetic evaluation, molecular docking and in vitro biological evaluation of 1, 3, 4-oxadiazole derivatives as potent antioxidants and STAT3 inhibitors

Khanam R, Hejazi II, Shahabuddin S, Bhat AR, Athar F

J Pharm Anal, 2019 Apr;9(2):133-141.
PMID: 31011470 DOI: 10.1016/j.jpha.2018.12.002

1, 3, 4-Oxadiazole derivatives (4a-5f) were previously synthesized to investigate their anticancer properties. However, studies relating to their antioxidant potential and signal transducer and activator of transcription (STAT) inhibition have not been performed. We investigated previously synthesized 1, 3, 4-oxadiazole derivatives (4a-5f) for various radical scavenging properties using several in vitro antioxidant assays and also for direct inhibition of STAT3 through molecular docking. The data obtained from various antioxidant assays such as 2, 2,-diphenyl-1-picrylhydrazyl radical (DPPH), nitric oxide, hydrogen peroxide, and superoxide anion radical revealed that among all the derivatives, compound 5e displayed high antioxidant activities than the standard antioxidant L-ascorbic acid. Additionally, the total reduction assay and antioxidant capacity assay further confirmed the antioxidant potential of compound 5e. Furthermore, the molecular docking studies performed for all derivatives along with the standard inhibitor STX-0119 showed that binding energy released in direct binding with the SH2 domain of STAT3 was the highest for compound 5e (-9.91kcal/mol). Through virtual screening, compound 5e was found to exhibit optimum competency in inhibiting STAT3 activity. Compound 5e decreased the activation of STAT3 as observed with Western blot. In brief, compound 5e was identified as a potent antioxidant agent and STAT3 inhibitor and effective agent for cancer treatment.

Matched MeSH terms: Blotting, Western
Estrogen, progesterone, and genistein differentially regulate levels of expression of α-, β-, and γ-epithelial sodium channel (ENaC) and α-sodium potassium pump (Na⁺/K⁺-ATPase) in the uteri of sex steroid-deficient rats

Chinigarzadeh A, Muniandy S, Salleh N

Theriogenology, 2015 Oct 1;84(6):911-26.
PMID: 26154487 DOI: 10.1016/j.theriogenology.2015.05.029

Estrogen, progesterone, and genistein could induce changes in uterine fluid volume and Na(+) concentration. Progesterone upregulates expression of epithelial sodium channel (ENaC) and Na(+)/K(+)-ATPase which contributed toward these changes. However, effects of estrogen and genistein were unknown. This study therefore investigated changes in expression of these proteins in the uterus under estrogen, progesterone, and genistein influences to further understand mechanisms underlying sex steroids and phytoestrogen effects on uterine fluid Na(+) regulation. In this study, uteri of ovariectomized female rats receiving 7-day treatment with genistein (25, 50, and 100 mg/kg/day), estrogen (0.8 × 10(-4) mg/kg/day), or progesterone (4 mg/kg/day) were harvested, and expression levels of α-, β-, and γ-ENaC proteins and messenger RNAs (mRNAs) and α-Na(+)/K(+)-ATPase protein were determined by Western blotting (proteins) and real-time polymerase chain reaction (mRNA). Meanwhile, distribution of α-, β-, and γ-ENaC and α-Na(+)/K(+)-ATPase proteins in the uterus was identified by immunohistochemistry. Our findings indicated that expression of α-, β-, and γ-ENaC proteins and mRNAs and α-Na(+)/K(+)-ATPase protein were enhanced under progesterone influence. Lower expressions were noted under estrogen and genistein influences compared to progesterone. Under estrogen, progesterone, and genistein influences, α- and β-ENaC were distributed at apical membrane and γ-ENaC was distributed at apical and basolateral membranes of uterine luminal epithelia. Under progesterone influence, α-Na(+)/K(+)-ATPase was highly expressed at basolateral membrane. In conclusion, high expression of α-, β-, and γ-ENaC and α-Na(+)/K(+)-ATPase under progesterone influence would contribute toward increased uterine fluid Na(+) reabsorption, whereas lesser expression of these proteins under estrogen and genistein influences would contribute toward lower reabsorption of uterine fluid Na(+).

Matched MeSH terms: Blotting, Western
Fulltext Differential Regulation of LPS-Mediated VE-Cadherin Disruption in Human Endothelial Cells and the Underlying Signaling Pathways: A Mini Review

Chan YH, Harith HH, Israf DA, Tham CL

Front Cell Dev Biol, 2019;7:280.
PMID: 31970155 DOI: 10.3389/fcell.2019.00280

Endothelial cells lining the inner vascular wall form a monolayer that contributes to the selective permeability of endothelial barrier. This selective permeability is mainly regulated by an endothelium-specific adherens junctional protein, known as vascular endothelial-cadherin (VE-cadherin). In endothelial cells, the adherens junction comprises of VE-cadherin and its associated adhesion molecules such as p120, α-catenin, and β-catenin, in which α-catenin links cytoplasmic tails of VE-cadherin to actin cytoskeleton through β-catenin. Proinflammatory stimuli such as lipopolysaccharide (LPS) are capable of attenuating vascular integrity through the disruption of VE-cadherin adhesion in endothelial cells. To date, numerous studies demonstrated the disruption of adherens junction as a result of phosphorylation-mediated VE-cadherin disruption. However, the outcomes from these studies were inconsistent and non-conclusive as different cell fractions were used to examine the effect of LPS on the disruption of VE-cadherin. By using Western Blot, some studies utilized total protein lysate and reported decreased protein expression while some studies reported unchanged expression. Other studies which used membrane and cytosolic fractions of protein extract demonstrated decreased and increased VE-cadherin expression, respectively. Despite the irregularities, the results of immunofluorescence staining are consistent with the formation of intercellular gap. Besides that, the overall underlying disruptive mechanisms of VE-cadherin remain largely unknown. Therefore, this mini review will focus on different experiment approaches in terms of cell fractions used in different human endothelial cell studies, and relate these differences to the results obtained in Western blot and immunofluorescence staining in order to give some insights into the overall differential regulatory mechanisms of LPS-mediated VE-cadherin disruption and address the discrepancy in VE-cadherin expression.

Matched MeSH terms: Blotting, Western

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