100-kDa Band Research Articles

Evidence of γ-secretase complex involved in the regulation of intramembrane proteolysis in Entamoeba histolytica

Presenilins (PSNs) are multifunctional membrane proteins involved in signal transduction, lysosomal acidification, and certain physiological processes related to mitochondria. The aspartic protease activity of PSN and the formation of a γ-secretase complex with other subunits such as nicastrin (NCT) are required for the biological functions. Although PSN is widely conserved in eukaryotes, most studies on PSN were conducted in metazoans. Homologous genes for PSN and NCT (EhPSN and EhNCT, respectively) are encoded in the genome of Entamoeba histolytica, however, their functions remain unknown. In this study, we showed that EhPSN and EhNCT form a complex on the cell membrane, demonstrating that the parasite possesses γ-secretase. The predicted structure of EhPSN was similar to the human homolog, demonstrated by the crystal structure, and phylogenetic analysis indicated good conservation between EhPSN and human PSN, supporting the premise that EhPSN functions as a subunit of γ-secretase. By contrast, EhNCT appears to have undergone remarkable structural changes during its evolution. Blue native-polyacrylamide gel electrophoresis combined with western blotting indicated that a 150-kDa single band contains both EhPSN (estimated molecular size: 47-kDa) and EhNCT (64-kDa), suggesting that the complex also contains other unknown components or post-translational modifications. Coimmunoprecipitation from amebic lysates also confirmed that EhPSN and EhNCT formed a complex. Indirect immunofluorescence analysis revealed that the complex localized to the plasma membrane. Moreover, EhPSN exhibited protease activity, which was suppressed by a γ-secretase inhibitor. This is the first report of a γ-secretase complex in protozoan parasites.

Production and characterization of immunoglobulin G anti-rLipL32 antibody as a biomarker for the diagnosis of leptospirosis.

Microscopic agglutination test (MAT) for the diagnosis of leptospirosis requires live cultures and is serovar-specific, while polymerase chain reaction (PCR) requires expensive equipment and sample preparation. The rLipL32 protein is conserved and can be used for the production of immunoglobulin G (IgG) anti-rLipL32 antibody, which can be used as a biomarker for leptospirosis diagnosis. This study aimed to produce and characterize an IgG anti-rLipL32 antibody as a biomarker for leptospirosis diagnosis. Escherichia coli rLipL32 was cultured and analyzed by PCR and sequencing. Cultures were used for rLipL32 protein expression and purification and the rLipL32 protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The rLipL32 protein was used to produce anti-rLipL32 serum and was analyzed by enzyme-linked immunosorbent assay (ELISA). Serum was purified to obtain IgG anti-rLipL32 antibody and characterized by SDS-PAGE and western blotting. PCR was able to amplify the LipL32 gene from E. coli rLipL32, and sequencing analysis showed 99.19% similarity with pathogenic Leptospira. SDS-PAGE analysis showed a 32-kDa band. ELISA results showed an increase in OD in anti-rLipL32 serum compared to preimmune serum. Western blotting results showed that the IgG anti-rLipL32 antibody was able to bind and cross-reacts with pathogenic Leptospira serovar but not with E. coli or Staphylococcus aureus. IgG anti-rLipL32 antibody has high specificity and sensitivity against Leptospira pathogens. These findings suggest that IgG anti-rLipL32 antibody is a promising biomarker for the diagnosis of leptospirosis.

Heterologous expression and purification of the phage lysin-like bacteriocin LysL from Lactococcus lactis LAC460.

The wild-type Lactococcus lactis strain LAC460 produces two bacteriocin-like phage lysins, LysL and LysP. This study aimed to produce and secrete LysL in various heterologous hosts and an in vitro cell-free expression system for further functional studies. Initially, the lysL gene from L. lactis LAC460 was cloned into Lactococcus cremoris NZ9000 and L. lactis N8 strains, with and without the usp45 signal sequence (SSusp45), under a nisin-inducible promoter. Active LysL was primarily produced intracellularly in recombinant L. lactis N8, with some secretion into the supernatant. Recombinant L. cremoris NZ9000 lysed upon nisin induction, indicating successful lysL expression. However, fusion with Usp45 signal peptide (SPUsp45-LysL) weakened LysL activity, likely due to incomplete signal peptide cleavage during secretion. Active LysL was also produced in vitro, and analysed in SDS-PAGE, giving a 42-kDa band. However, the yield of LysL protein was still low when produced from recombinant lactococci or by in vitro expression system. Therefore, His-tagged LysL was produced in Escherichia coli BL21(DE3). Western blot confirmed the intracellular production of about 44-kDa His-tagged LysL in E. coli. His-tagged active LysL was then purified by Ni-NTA affinity chromatography yielding sufficient 4.34mg of protein to be used in future functional studies.

Construing recombinant ZFP160 from Aspergillus terreus as pterin deaminase enzyme.

Pterin deaminase stands as a metalloenzyme and exhibits both antitumor and anticancer activities. Therefore, this study aimed to explore the molecular function of zinc finger protein-160 (zfp160) from Aspergillus terreus with its enzyme mechanism in detail. Subsequently, preliminary molecular docking studies on zfp160 from A. terreus were done. Next, the cloning and expression of zfp160 protein were carried out. Following, protein expression was induced and purified through nickel NTA column with imidazole gradient elution. Through the Mascot search engine tool, the expressed protein of MALDI-TOF was confirmed by 32kDa bands of SDS-PAGE. Furthermore, its enzymatic characterization and biochemical categorization were also explored. The optimum conditions for enzyme were determined to be pH 8, temperature 35°C, km 50μm with folic acid as substrate, and Vmax of 24.16 (IU/mL). Further, in silico analysis tried to explore the interactions and binding affinity of various substrates to the modeled pterin deaminase from A. terreus. Our results revealed the binding mode of different substrate molecules with pterin deaminase using the approximate scoring functions that possibly correlate with actual experimental binding affinities. Following this, molecular dynamic simulations provided the in-depth knowledge on deciphering functional mechanisms of pterin deaminase over other drugs.

PRODUCTION OF ANTI-LACTOFERRIN ANTIBODIES AND THEIR APPLICATION IN ANALYSIS OF THE TEAR FLUID IN HEALTH AND CORNEAL INJURIES

Lactoferrin is a ubiquitous and multifunctional protein, which has antimicrobial and immunomodulatory activities. Lactoferrin plays an important role in the maintenance of ocular health. The aim of the study was to produce polyclonal antibodies against human lactoferrin in order to apply them in evaluation of lactoferrin levels in tear fluid collected from healthy eye and after corneal injury. Materials and methods. Affine chromatography on Protein A-sepharose was applied in order to isolate immunoglobulin G (IgG) fraction from the blood serum of lactoferrin-immunized rabbits. Each step of protein purification was monitored by denaturing gel electrophoresis (SDS-PAGE). Target antigen recognition by produced antibodies was established by western blot analysis with the use of diluted IgG fraction. Lactoferrin levels in the tear fluids collected from healthy individuals (n = 4) and patients with non-penetrating corneal injures (n = 6) were determined immunochemically with the use of purified antibodies. The results of western blot of lactoferrin levels in the tear fluids of healthy individuals and patients with corneal wounds were analysed using Mann-Whitney U-test. The difference between group mean values was considered significant at P<0.05. Results. Using affine chromatography on Protein A-sepharose, antibodies against human lactoferrin were purified as IgG fraction from blood serum of lactoferrin-immunized rabbits. Western blot analysis showed that obtained antibodies recognize the antigen as a 75-kDa band, which corresponds to the intact human lactoferrin polypeptide. The same major polypeptide band was visualized by western blot with enhanced chemiluminescence detection in the tear fluid samples. Densitometry analysis of 75-kDa lactoferrin band showed 3.2-fold decrease in lactoferrin level in the tear fluid samples obtained from patients with non-penetrating corneal traumas as compared with samples collected from healthy persons (P<0.05). Besides, tear fluid of patients with injured corneas contained large amounts of truncated lactoferrin immunoreactive polypeptides as well as high molecular weight bands, which could correspond to lactoferrin complexes with other proteins occurring during inflammation. Conclusions. According to our data, obtained anti-lactoferrin antibodies can be used as a valuable tool for development of advanced tests and procedures for diagnostics of eye diseases associated with the corneal lesions. Reduced lactoferrin concentration might represent a potential prognostic biomarker for diagnosis of ocular diseases including non-penetrating corneal injuries in a simple and non-invasive way.

Difference in the nutritional, in vitro, and functional characteristics of protein and fat isolates of two Indian chia (Salvia hispanica L) seed genotypes with variation in seed coat color.

This study aimed to determine the association between the seed coat color of two chia seed genotypes for their composition, protein content, amino acid, and fatty acid profiles. The optimal pH for protein isolation for both genotypes (BCPI and WCPI) was 10, based on protein purity and solubility. Fatty acid profiling indicated, overall, 18 different fatty acids higher in BCPI10 with linolenic acid domination (∼66%) followed by linoleic acid (∼19%) and oleic acid (∼6%), contributing PUFAs (∼86%). Optimized protein isolates, black (BCPI10) and white (WCPI10) chia, had shown purity, L*-value, solubility, and yields of 90.65%, 75.86%, 77.75%, 11.30%, and 90.00%, 77.83%, 76.07%, 10.69%, respectively. BCPI10 depicted higher EAA (33.19g/100 g N) and EEA indices (57.676%) compared to WCPI10 (32.14g/100 g N) and 56.360%, respectively.Amino acid profiling indicated higher, PER, TAA, TEAA, TNEAA, TAAA, TBA, acidic AA values for BCPI10, and higher leucine/isoleucine ratio for WCPI10 having leucine and sulfur amino acids as limiting amino acids. BCPI10 had higher sulfur-containing amino acid contents, as the main contributor to the albumin a water-soluble fraction, leading to its higher in vitro digestibility (71.97%) than WCPI10 (67.70%). Both isolates exhibited good WHC and OHC of 3.18, 2.39 and 3.00, 2.20, respectively. Both protein isolates had similar ∆Td (°C) values with some variation in FTIR spectrum from 1000 cm-1 to 1651cm-1 having more peak intensity for BCPI10. SDS-PAGE indicated bands at 150kDa, representing globulin and mild bands at 25-33kDa for glutelin and albumin. A significant (p<0.05) variation reported in this study for protein and lipid profiles of both genotype attributes to genetic differences between the seeds. PRACTICAL APPLICATION: Based on the nutritional profile, both chia seed isolates (black and white) are suitable for consumption with an edge for black seed when supplemented with their limiting amino acids. The high values of the functional properties and structural characteristics combined with high nutritional values make the chia protein isolate an excellent source of raw material for various food formulations. Fatty acid profile of the oils from the genotypes showed the presence of high amounts of unsaturated fatty acids, especially the PUFAs with more number of fatty acids in black chia seed. The excellent lipid profile of chia seed oil indicates the benefit of using chia seed oil as a source of essential fatty acids in the human diet for optimal health.

Evaluation of circulating insulin-like growth factor (IGF)-I and IGF-binding proteins as growth indices in rainbow trout (Oncorhynchus mykiss)

Circulating insulin-like growth factor (IGF)-I has been proposed as a growth index in several teleosts, including salmonids, and its level in circulation is stabilized by multiple IGF-binding proteins (IGFBPs). Three IGFBPs, IGFBP-2b, -1a, and -1b, are consistently detected in salmonid blood and are suggested to be indices of positive or negative growth, although their applicability to rainbow trout (Oncorhynchus mykiss) is unclear. The present study examined the usefulness of IGFBPs along with IGF-I as a physiological indicator of growth rate in rainbow trout through a rearing experiment. Two groups of underyearling rainbow trout were pit-tagged and either fed or fasted for 33 days. A third group was fasted for 22 days, followed by refeeding for 11 days. Serum IGF-I levels were reduced after fasting for 22 days, but refeeding did not retore its levels to those of the fed control. Nevertheless, there was a positive relationship between serum IGF-I levels and individual growth rates over 33 days of experimentation, confirming its validity as a growth index. Ligand blotting using labeled human IGF-I revealed two IGFBP bands at 43 and 32 kDa, which corresponded to IGFBP-2b and an unidentified form, respectively. In contrast, bands corresponding to IGFBP-1a and -1b, which usually increase after fasting, were hardly detected, even in the fasted fish. The responses of circulating IGFBP-2b to fasting and refeeding were similar to those of circulating IGF-I and positively correlated with growth rate and IGF-I levels. The intensity of the serum 32-kDa IGFBP band was higher in constantly fed fish than in the fasted fish; however, its correlation with growth rate was weaker than those of IGF-I and IGFBP-2b. The present study shows that IGF-I and IGFBP-2b can be used as growth indices for rainbow trout. In contrast, circulating IGFBP-1a and -1b may not serve as negative growth indices in rainbow trout under regular aquaculture conditions because they are rarely detected by ligand blotting or respond to fasting/refeeding.

Expression and production of protoscolex recombinant P29 protein and its serological evaluation for diagnosis of human hydatidosis.

Cystic Echinococcosis/Hydatidosis considered as one of the most important parasitic diseases in humans and animals across the world. The goal of the present study was to determine a native antigen with an acceptable sensitivity and specificity to be used in the human hydatid cyst diagnostic methods. In the present study, recombinant P29 antigen was used to detect the antibodies in the serum of patients with hydatid cysts of the liver. In fact, purified recombinant P29 protein is used as an antigen in ELISA. In order to evaluate the recombinant P29 protein for diagnostic ELISA, 25 serums obtained from people harboring the hydatid cysts were tested. The result of the gene expression on a 12% SDS-PAGE gel showed a band with a length of 28 KD. Also, 28KDa band was observed through the reaction of recombinant P29 protein with Anti T7-tag monoclonal antibody in the western blotting method. This protein showed satisfactory results in detecting the hydatid cyst antibodies in the serum of patients having hydatid cysts. Twenty two of 25 hydatidosis serums positively reacted in ELISA using with P29 protein, indicating in 92% of ELISA sensitivity, 95% of specificity, 95.83% of positive predictive value, and 90.42% of negative predictive value for recombinant P29 protein. Whereas the produced recombinant protein P29 showed promising results in the diagnosis of hydatidosis but of more research needs to be done to reach a more accurate conclusion.

Freezing-thawing induces deprotamination, cryocapacitation-associated changes; DNA fragmentation; and reduced progesterone sensitivity in buck spermatozoa

In the present study, there was evaluation of cryocapacitation-associated changes, apoptotic-like changes, deprotamination, total antioxidant capacity (TAC), and in vitro sperm functional attributes in Barbari bucks after freezing-thawing. The correlation between deprotamination and sperm functional characteristics was established. Using immunoblotting procedures, there was detection of the presence of a single 28-kDa protein band corresponding to protamine-1. The localization in the head region of the spermatozoa was further validated by an immunofluorescence test. Capacitated (B-) and acrosome-reacted (AR-) pattern spermatozoa, spermatozoa with the externalization of phosphatidylserine and a relatively lesser mitochondrial transmembrane potential, and deprotamination and DNA fragmentation was greater (P < 0.05) after freezing-thawing and indicated there were cryocapacitation- and apoptotic-like changes, respectively. Furthermore, the detection of phosphorylation of tyrosine-containing proteins with use of immunoblotting and immunofluorescence procedures confirmed there were cryocapacitation-like changes in the buck spermatozoa after freezing-thawing. Total antioxidant capacity (TAC), in vitro thermal resistance response, Vanguard distance, progesterone sensitivity, and in vitro capacitation response were less (P < 0.05) in the spermatozoa after freezing-thawing compared with spermatozoa after initial dilution and equilibration. Deprotamination (chromomycin A3-positive cells, CMA3+) and DNA fragmentation (TUNEL+ve) were positively correlated with B- and AR-pattern spermatozoa, while other values for other variables were negatively correlated. In conclusion, the results of this study indicated there was protamine-1 in buck spermatozoa and after freezing-thawing there was a loss of protamine-1 combined with cryocapacitation-associated changes and apoptotic-like changes in buck spermatozoa. Spermatozoa deprotamination might be attributed to increased DNA fragmentation, resulting in compromised fertilizing capacity of buck spermatozoa.

Identification of Biomarkers for Diagnosis and Prognosis of Congenital and Acute Toxoplasmosis.

The diagnosis of congenital toxoplasmosis can be inconclusive in many cases. Despite the several serological tests developed, the literature on biomarkers that can assist in the diagnosis of congenital an acute toxoplasmosis is limited. The objective of this study was to analyze the immunoreactive profile of Toxoplasma gondii protein bands with the potential to be biomarkers for diagnosis and prognosis of congenital and acute toxoplasmosis. Peripheral blood samples from women of childbearing age and/or pregnant women diagnosed with acquired toxoplasmosis as well as from congenitally infected children were selected and submitted to immunoblotting for analysis of the immunoreactive bands profile by immunoglobulin G (IgG) antibodies. When comparing the immunoreactive bands profile for antibodies present in samples from different groups and subgroups, the 150, 18.5, and 16.96-kDa bands were more immunoreactive with the antibodies present in serum samples from the acquired infection group. The 343, 189, 150, 75, and 42-kDa bands showed more chance to be detected by the symptomatic congenital infection subgroup samples, while the 61, 50, and 16.96-kDa bands were significantly immunoreactive with the acute infection subgroup samples. The identification of these potential biomarkers can assist in early diagnosis and treatment of congenital toxoplasmosis.

Evaluation of the Toxicity of Supernatant Cultures and Spore-Crystal Mixtures of Bacillus thuringiensis Strains Isolated from Algeria.

Bacillus thuringiensis (Bt) is the most used technology for biological control of insect pathogens worldwide. In order to select new Bt candidates challenging the emergence of insect's resistance, a mass bioassay and molecular screening was performed on an autochthonous collection. Toxicity assays against neonate larvae of three lepidopteran species (Mamestra brassicae, Grapholita molesta, and Spodoptera exigua) were conducted using spore-crystal mixtures and supernatant cultures of 49 Bt isolates harboring at least one gene coding for a lepidopteran-specific insecticidal protein. A threshold of 30% of "functional mortality" was used to discriminate between "nontoxic" and "toxic" isolates. The toxicity of many Bt isolates competed with that of Btk-HD1. However, only three of them (Bl4NA, Bl5NA, and Bl9NA) showed high toxicity in both spore-crystal mixtures and supernatant cultures against the three lepidopteran species. The Bt isolates Bl4NA and Bl9NA express a protein of 130kDa whereas the Bt isolate Bl5NA expresses a protein of 65-70kDa. The LC-MS/MS results indicate that the major peptides in the 130kDa band of Bl9NA were Cry1Da, Cry1Ca, Cry1Ab, and Cry1Aa, and those in the 70kDa band of Bl5NA were Cry1Aa and Cry1Ca. The evaluation of the protein content of the supernatants by comparison to Btk-HD1 indicates the overproduction of Vip3 proteins in these strains (most likely Vip3Aa in Bl4NA and Bl9NA and Vip3Ca in Bl5NA). In addition, these three Bt strains do not produce β-exotoxins. Based on our results, the three selected strains could be considered promising candidates to be used in insect pest control.

Anti-stress phytohormones impact on proteome profile of green gram (Vigna radiata) under salt toxicity

Anti-stress phytohormones impact on proteome profile of green gram (Vigna radiata) under salt toxicity

Autoimmune inner ear disease patient-associated 28-kDa proinflammatory IL-1β fragment results from caspase-7-mediated cleavage in vitro.

Interleukin-1β (IL-1β) is a key proinflammatory cytokine involved in the progression of many autoinflammatory and autoimmune diseases, including autoimmune inner ear disease (AIED). IL-1β inhibition has been shown to result in clinical hearing improvement in a small cohort of corticosteroid-resistant patients with AIED. Canonical processing of pro-IL-1β by caspase-1 generates an active 17-kDa fragment, capable of instigating a proinflammatory microenvironment. However, in response to LPS, PBMCs from patients with AIED uniquely express a 28-kDa IL-1β fragment, as compared with PBMCs from control subjects. We synthesized and compared the biologic activity of the 28-kDa fragment to the 17-kDa IL-1β product and the pro-IL-1 31-kDa protein. The 28-kDa IL-1β fragment induces IL-6, TNF-α, and CCL3 in PBMCs. Uniquely, only caspase-7 treatment showed a dose- and time-dependent increase in 28-kDa band generation. Mass spectrometry confirmed the putative caspase-7 cleavage site of pro-IL-1β, which was used to generate the 28-kDa fragment used for PBMC stimulation studies. Collectively, these results provide insight into the function of a poorly understood, processed 28-kDa form of IL-1β in patients with AIED that is uniquely generated by caspase-7 and is capable of activating further downstream proinflammatory cytokines. Further investigation may provide novel pharmacologic targets for the treatment of this rare disease.

Impaired vascular endothelial growth factor expression and secretion during in vitro differentiation of human primary term cytotrophoblasts.

Vascular endothelial growth factor A (VEGF-A) is one of the main growth factors involved in placental vasculogenesis and angiogenesis, but its placental expression is still ambiguous. During in vitro cultures of primary term cytotrophoblasts, VEGF could not be detected in the supernatants by enzyme-linked immunosorbent assays (ELISA). One hypothesis is that VEGF is immediately and completely bound to its soluble receptor after secretion, and cannot be recognized by the antibodies used in the commercial ELISA kits. We decided to verify this hypothesis by measuring VEGF-A expression during in vitro cultures of primary term cytotrophoblasts. Term cytotrophoblasts were cultured under 21% and 2.5% O2 for 4days. VEGF-A transcripts were quantified by real-time polymerase chain reaction. The proteins from cell lysates and concentrated media were separated by polyacrylamide gel electrophoresis (PAGE) under denaturing and reducing conditions, and VEGF-A immunodetected by western blotting. VEGF mRNA expression did not increase during in vitro cell differentiation under 21% O2, but slightly increased under 2.5% O2 only at 24h. VEGF-A monomer was not detected in the cell lysates and in the concentrated supernatants, while a ~ 42KDa band corresponding to the precursor L-VEGF was detected in all the cellular extracts. Isolated term villous cytotrophoblasts produce the L-VEGF precursor but they do not secrete VEGF-A even under low-oxygen tension. The question remains about the origin of VEGF in pregnancy but also about the biological role of L-VEGF, which can represent a form of storage for rapid VEGF secretion when needed.

Cloning and Expression of EBI3 and p28 Subunits of Human Interleukin 27 in E. coli

Background: Interleukin-27 is a heterodimeric cytokine belonging to IL-12 and IL-23 families, secreted by Antigen Presenting Cells( APCs ) .The IL-27 is composed of 2 subunits: Epstein-Barr virus induced gene 3 (EBI3) and p28. IL-27 is an anti-inflammatory cytokine which has an inhibitory effect on Th17 population and suppress the IL-17 expression. It is suggested that IL-27 could be a potent drug candidate for treating auto immune diseases. Methods: The IL-27 subunit genes were constructed into plasmid vectors, they sub-cloned into pETDeut-1an expression vector in restriction sites of BamHI, SacI and NotI. Subsequently the induction was done by IPTG and the recombinant proteins were confirmed by SDS-PAGE and Western Blot analysis using anti His-tag antibody. Results: The 28kDa and 25kDa protein bands were observed on SDS-PAGE and finally confirmed by Western blot technique.

Defective Tmprss3-Associated Hair Cell Degeneration in Inner Ear Organoids.

SummaryMutations in the gene encoding the type II transmembrane protease 3 (TMPRSS3) cause human hearing loss, although the underlying mechanisms that result in TMPRSS3-related hearing loss are still unclear. We combined the use of stem cell-derived inner ear organoids with single-cell RNA sequencing to investigate the role of TMPRSS3. Defective Tmprss3 leads to hair cell apoptosis without altering the development of hair cells and the formation of the mechanotransduction apparatus. Prior to degeneration, Tmprss3-KO hair cells demonstrate reduced numbers of BK channels and lower expressions of genes encoding calcium ion-binding proteins, suggesting a disruption in intracellular homeostasis. A proteolytically active TMPRSS3 was detected on cell membranes in addition to ER of cells in inner ear organoids. Our in vitro model recapitulated salient features of genetically associated inner ear abnormalities and will serve as a powerful tool for studying inner ear disorders.

A Vitellogenin Antibody in Honey Bees (Apis mellifera): Characterization and Application as Potential Biomarker for Insecticide Exposure.

The insect yolk precursor vitellogenin is a lipoglycoprotein synthesized and stored in the fat body and secreted into the hemolymph. In honey bees, vitellogenin displays crucial functions in hormone signaling, behavioral transition of nurse bees to foragers, stress resistance, and longevity in workers. Plant protection products such as neonicotinoids, pyrethroids, and organophosphates alter the transcriptional expression of vitellogenin. To assess plant protection product-induced alterations on the protein level, we developed a rabbit polyclonal vitellogenin antibody. After characterization, we assessed its specificity and vitellogenin levels in different tissues of worker bees. The vitellogenin antibody recognized full-length 180-kDa vitellogenin and the lighter fragment of 150 kDa in fat body, hemolymph, and brain. In hemolymph, a band of approximately 75 kDa was detected. Subsequent mass spectrometric analysis (liquid chromatography-mass spectrometry) confirmed the 180- and 150-kDa bands as vitellogenin. Subsequently, we evaluated vitellogenin expression in brain, fat body, and hemolymph on 24-h exposure of bees to 3 ng/bee to the neonicotinoid clothianidin. Full-length vitellogenin was upregulated 3-fold in the fat body, and the 150-kDa fragment was upregulated in the brain of exposed honey bees, whereas no alteration occurred in the hemolymph. Upregulation of the vitellogenin protein by the neonicotinoid clothianidin is in line with the previously shown induction of its transcript. We conclude that vitellogenin might serve as a potential biomarker for neonicotinoid and other pesticide exposure in bees. Environ Toxicol Chem 2019;00:1-10. © 2019 SETAC.

Cloning and Expression of Immunogenic Regions of EMA-1 Gene of Theileria equi From Infected Horses.

Diversity among the pathogenic strains of Theileria equi (T. equi), a major agent of equine piroplasmosis, can affect the appropriate detection of parasite and host immunization. Production of recombinant surface proteins from an infected horse in natural endemic area provides a reliable tool for immunodiagnosis of parasite. Regarding this, the present study was targeted toward the cloning, expression, and purification of the immunogenic regions of equine merozoite antigen 1 (EMA-1 gene), as one of the most important immunodominant surface proteins in T. equi, from naturally infected horses in Iran. The immunogenic region of EMA-1 gene was amplified using the blood of infected horses. EMA-1 gene was cloned into pET26b vector. Then, recombinant plasmids (pET 26b-EMA-1) were transformed into competent E. coli BL21 (DE3) cells. Cloning was confirmed by polymerase chain reaction (PCR), restriction enzyme assays, and DNA sequence analysis. The recombinant protein was expressed using isopropyl &amp;beta;-D-1-thiogalactopyranoside as an inducer, purified using nickle-nitrilotriacetic acid column, and then confirmed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and dot blot analysis utilizing Anti-His Tag antibody. Furthermore, the immunoreactivity of recombinant protein against the serum of the infected horses was evaluated using dot blot analysis. The PCR product analysis showed a 750-bp band belonging to immunogenic regions of EMA-1 gene. Sequence analysis revealed that cloned EMA-1 and protein had 94% and 97% homology to EMA-1 sequences submitted to GenBank from different countries, respectively. Restriction enzyme and sequence analyses confirmed the subcloning and correction of the orientation of inserted gene. The SDS-PAGE analysis confirmed the expression of EMA-1 protein with a 28-kDa band. The results of the dot blot analysis revealed that the horse serum containing antibody against T. equi could react with the purified recombinant protein. Purified EMA-1 protein can be used as a reliable tool for the future development of diagnostic tests or vaccines.

Analysis of opossum kidney NaPi-IIc sodium-dependent phosphate transporter to understand Pi handling in human kidney.

The role of Na+-dependent inorganic phosphate (Pi) transporters in the human kidney is not fully clarified. Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is caused by loss-of-function mutations in the IIc Na+-dependent Pi transporter (NPT2c/Npt2c/NaPi-IIc) gene. Another Na+-dependent type II transporter, (NPT2A/Npt2a/NaPi-IIa), is also important for renal Pi reabsorption in humans. In mice, Npt2c deletion does not lead to hypophosphatemia and rickets because Npt2a compensates for the impaired Pi reabsorption. To clarify the differences between mouse and human, we investigated the relation between NaPi-IIa and NaPi-IIc functions in opossum kidney (OK) cells. We cloned NaPi-IIc from OK cells and created opossum NaPi-IIc (oNaPi-IIc) antibodies. We used oNaPi-IIc small interference (si)RNA and investigated the role of NaPi-IIc in Pi transport in OK cells. We cloned opossum kidney NaPi-IIc cDNAs encoding 622 amino acid proteins (variant1) and examined their pH- and sodium-dependency. The antibodies reacted specifically with 75-kDa and 150-kDa protein bands, and the siRNA of NaPi-IIc markedly suppressed endogenous oNaPi-IIc in OK cells. Treatment with siRNA significantly suppressed the expression of NaPi-4 (NaPi-IIa) protein and mRNA. oNaPi-IIc siRNA also suppressed Na+/H+ exchanger regulatory factor 1 expression in OK cells. These findings suggest that NaPi-IIc is important for the expression of NaPi-IIa (NaPi-4) protein in OK cells. Suppression of Npt2c may downregulate Npt2a function in HHRH patients.

A-kinase anchor protein 4 precursor (pro-AKAP4) in human spermatozoa.

A-kinase anchor protein 4 (AKAP4) and its precursor pro-AKAP4 are two major proteins in spermatozoa of rodents and mammals. Although researchers have characterized the AKAP4 expression in various species, the protein's expression in humans has not been described in detail. The objective of this study was to characterize human pro-AKAP4 more precisely (notably the definition of its localization and expression levels in human spermatozoa and testes). pro-AKAP4 protein expression levels were assessed by Western blotting. The pro-AKAP4's localization in spermatozoa and testes was determined using immunofluorescence staining and immunogold electron microscopy. Furthermore, pro-AKAP4 protein expression levels were assessed in a series of 77 human semen samples, and associations with semen parameters were evaluated. Western blotting revealed a 100-kDa band in human sperm protein extracts. The pro-AKAP4 was immunolocalized in the fibrous sheath of the flagellum of ejaculated spermatozoa and in elongated spermatids in human testes. A Western blot analysis of 77 normozoospermic semen samples evidenced striking differences in pro-AKAP4 levels from one to another sample (median [interquartile range] integrated optical density=305 [49-1038]). No correlations were found for pro-AKAP4 levels on one hand and semen volume, sperm concentration, sperm count, sperm motility, or sperm morphology on the other (p>0.05 for all). However, pro-AKAP4 levels were positively correlated with motility after density gradient centrifugation of the semen (r=0.224, p=0.049). AKAP4 protein might be activated as an alternative pathway to rescue sperm motility. In human spermatozoa, pro-AKAP4 might therefore be a 'reservoir' of mature AKAP4. This study generated new knowledge about pro-AKAP4 in human semen, which may be of interest in the management of male infertility.