Gland mRNA Research Articles

Identification and characterization of calcium binding protein, spermatid-associated 1 (CABS1)# in selected human tissues and fluids.

Calcium binding protein, spermatid associated 1 (CABS1) is a protein most widely studied in spermatogenesis. However, mRNA for CABS1 has been found in numerous tissues, albeit with little information about the protein. Previously, we identified CABS1 mRNA and protein in human salivary glands and provided evidence that in humans CABS1 contains a heptapeptide near its carboxyl terminus that has anti-inflammatory activities. Moreover, levels of an immunoreactive form of CABS1 were elevated in psychological stress. To more fully characterize human CABS1 we developed additional polyclonal and monoclonal antibodies to different sections of the protein and used these antibodies to characterize CABS1 in an overexpression cell lysate, human salivary glands, saliva, serum and testes using western blot, immunohistochemistry and bioinformatics approaches exploiting the Gene Expression Omnibus (GEO) database. CABS1 appears to have multiple molecular weight forms, consistent with its recognition as a structurally disordered protein, a protein with structural plasticity. Interestingly, in human testes, its cellular distribution differs from that in rodents and pigs, and includes Leydig cells, primary spermatogonia, Sertoli cells and developing spermatocytes and spermatids, Geodata suggests that CABS1 is much more widely distributed than previously recognized, including in the urogenital, gastrointestinal and respiratory tracts, as well as in the nervous system, immune system and other tissues. Much remains to be learned about this intriguing protein.

Combined exposure of beta-cypermethrin and emamectin benzoate interferes with the HPO axis through oxidative stress, causing an imbalance of hormone homeostasis in female rats

The impact of pesticides on reproductive health has been increasingly recognized. β-cypermethrin (β-CYP) and emamectin benzoate (EMB) are commonly used with agricultural workers. There are few published studies on the effects of combined poisoning of these two pesticides on the reproductive system. This study investigated the toxic effects and mechanism of β-CYP and EMB on the reproductive system of female rats based on the hypothalamic-pituitary-ovarian (HPO) axis. The hypothalamic GnRH content tended to decrease, and Kiss-1 and GPR-54 mRNA and protein expression tended to increase in exposed rats. FSH content was elevated for the pituitary gland, and Kiss-1 and GPR-54 mRNA and protein expression were enhanced in all experimental groups compared with the control group. E2 content in rat ovaries and ERα mRNA and protein expression were reduced by β-CYP and EMB. Furthermore, there were interactive effects of β-CYP and EMB on FSH and E2 release, pituitary GPR-54 mRNA and protein, and ovarian ERα mRNA expression. To investigate causes of damage, oxidative damage indicators were tested and showed that exposure to β-CYP and EMB decreased GSH-Px and SOD activities in the HPO axis, increased MDA levels in the hypothalamus and ovary together with LDH activities in the HPO axis, with an interaction effect on GSH-Px and SOD activities in the hypothalamus and pituitary gland as well as on MDA in the ovary. The above results support the screening of sensitive molecular biomarkers and evaluation of the adverse effects of pesticide exposure in greenhouse operations on reproductive health.

Dapagliflozin downregulates G protein-coupled receptor Kinase-2 and upregulates regulator of g protein signaling-4 in adrenals to exert sympatholysis in heart failure

Abstract Dapagliflozin is a sodium-glucose co-transporter (SGLT)-2 inhibitor with beneficial cardiovascular effects independent of its anti-diabetic actions. Among these is sympatholysis, i.e., norepinephrine (NE) lowering which is beneficial in chronic human heart failure (HF). Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and sympathetic nervous system (SNS) hyperactivity in HF due to dysregulation of adrenal sympatho-inhibitory α2-adrenergic receptors (ARs) [1]. On the other hand, we recently reported that signaling by neuronal free fatty acid receptor (FFAR)-3, which stimulates NE release from sympathetic neurons, is blocked by Regulator of G protein Signaling (RGS)-4 [2,3]. We hypothesized herein that dapagliflozin may lower SNS hyperactivity in the adrenal gland by improving α2AR, and antagonizing FFAR3 signaling in adrenal chromaffin cells. We used the rat pheochromocytoma PC12 cell line expressing human α2A-AR, as well as freshly isolated adrenal glands from rats treated with dapagliflozin in vivo. Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217+67 pg/ml, n=6), compared to control, vehicle-treated rats (363+77 pg/ml, n=6, p<.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and tyrosine hydroxylase (TH) mRNA and protein levels in dapagliflozin-treated rat adrenals vs. vehicle-treated animal-derived glands. In contrast, RGS4 mRNA levels were increased in dapagliflozin-treated adrenals. Adrenal α2AR density was higher in dapagliflozin- vs. vehicle-treated rats (51.3+7.3 vs. 26.1+8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p<.05). These results (i.e., GRK2 & TH downregulation with RGS4 upregulation) were completely recapitulated in PC12 cells in culture, treated with 5 microM dapagliflozin (or vehicle) for 24 hours. Importantly, α2AR-dependent G protein-mediated signaling towards inhibition of CA secretion was markedly enhanced, whereas FFAR3-dependent phospholipase C/calcium signaling was reduced, at 24 hrs post-dapagliflozin treatment of PC12 cells. GRK2 overexpression partially reversed dapagliflozin`s effect on cholinergic-induced CA secretion. In conclusion, dapagliflozin exerts a sympatholytic action in the adrenal medulla via a) downregulation of TH, which reduces CA biosynthesis, and GRK2, which reduces α2AR desensitization; and b) upregulation of RGS4 to block FFAR3-stimulated CA secretion (Figure).Figure

Molecular cloning and functional analysis of HnSaratin from Hirudo nipponia

In order to finish a bloodmeal successfully, hematophagous organisms often stored a variety of anticoagulant proteins in their salivary glands, such as proteins that inhibit platelet aggregation. When they ingest a bloodmeal, these proteins are injected into the host to prevent the blood from clotting. As one of the origins of leeches used in traditional Chinese medicine, H. nipponia was proved to be clinically effective in treatment of cardiovascular and cerebrovascular diseases. This study cloned the sequence of HnSaratin cDNA derived from salivary glands of H. nipponia. The sequence contains an open reading frame of 387 bp, encoding a protein of 128 amino acids containing a signal peptide of 21 amino acids. After removal of the signal peptide, the molecular mass of mature HnSaratin was 12.37 kDa, with a theoretical isoelectric point (pI) of 3.89. The N-terminal of mature HnSaratin was folded into a globular structure, in which 3 disulfide bonds, a ββαβββ topology and 2 Glu residues that binds collagenous Lys2 were located, and the C-terminal formed a flexible region. The fusion HnSaratin protein was obtained by a prokaryotic expression system. The protein showed anti-platelet aggregation activity, and was observed to prevent blood clotting in rats. The significant high expression of HnSaratin mRNA in salivary glands was induced by bloodmeal ingestion of H. nipponia. Briefly, our work provides theoretical basis for further development and utilization of H. nipponia.

Abstract 11977: Dapagliflozin Downregulates Adrenal G Protein-Coupled Receptor-Kinase-2 to Exert Sympatholysis in Heart Failure

Introduction: Dapagliflozin is a sodium-glucose co-transporter (SGLT)-2 inhibitor exerting several cardiorenal protective effects independently of blood glucose regulation. Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and sympathetic nervous system (SNS) hyperactivity, especially in heart failure (HF). GRK2 severely dysregulates adrenal sympatho-inhibitory α 2 -adrenergic receptors (ARs), leading to unchecked, chronically elevated CA secretion. Hypothesis: SGLT2 inhibition with dapagliflozin lowers SNS hyperactivity in part by opposing adrenal GRK2 actions. Methods: We used the rat pheochromocytoma PC12 cell line expressing human α 2A AR, as well as freshly isolated adrenal glands from rats treated with dapagliflozin in vivo. Results: Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217+67 pg/ml, n=6), compared to control, vehicle-treated rats (363+77 pg/ml, n=6, p<.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and tyrosine hydroxylase (TH) mRNA and protein levels in dapagliflozin-treated rat adrenals vs. vehicle-treated animal-derived glands, indicating reduced adrenal CA synthesis and secretion. Finally, adrenal α 2 AR density was higher in dapagliflozin- vs. vehicle-treated rats(51.3+7.3 vs. 26.1+8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p<.05). GRK2 and TH downregulation were also observed in PC12 cells treated with 5 μM dapagliflozin for 24 hours. Importantly, α 2 AR-dependent inhibition of CA secretion was markedly enhanced 24 hrs post-dapagliflozin application. This was due to reduced GRK2-dependent receptor desensitization induced by dapagliflozin, since this effect was abolished in PC12 cells overexpressing GRK2. Conclusions: Dapagliflozin exerts a sympatholytic action in the adrenal medulla via downregulation of both TH, thus reducing CA biosynthesis, and GRK2, thus reducing α 2 AR desensitization to lower CA secretion. This adrenal-mediated sympatholysis may be part of the increasingly apparent cardiovascular benefits of SGLT2 inhibitors in human HF irrespective of diabetic status.

Sulfur Amino Acid Metabolism and the Role of Endogenous Cystathionine-γ-lyase/H2S in Holstein Cows with Clinical Mastitis.

Simple SummaryEndogenous hydrogen sulfide (H2S) has been implicated in many physiological and pathological processes, particularly in inflammatory responses and adaptive immunity. In this study, we identified the candidate differentially expressed proteins (DEPs) associated with H2S metabolism in Holstein cows with clinical mastitis (CM). The results revealed 17 DEPs included in 44 Gene Ontology (GO) terms and five Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to sulfide metabolism and indicated the important role of cystathionine-γ-lyase (CTH)/H2S in CM. Our findings can support research into the function and regulatory mechanism of CTH/H2S in Holstein cows and provide a basis for the prevention and treatment of CM.H2S plays an important role in various inflammatory diseases. However, the role of H2S and synthetic enzymes in Holstein cows with CM is unknown. The aim of this study was to identify DEPs associated with sulfide metabolism and further investigate their roles in dairy cows with CM. From 3739 DEPs generated by data-independent acquisition proteomics, we identified a total of 17 DEPs included in 44 GO terms and five KEGG pathways related to sulfide metabolism, including CTH and cystathionine-β-synthase (CBS). Immunohistochemical and immunofluorescence staining results showed that CTH and CBS proteins were present mainly in the cytoplasm of mammary epithelial cells. Endogenous H2S production in the serum of the CM group was significantly lower than that of the healthy Holstein cows. CTH and CBS mRNA and protein levels in the mammary glands of the CM group were significantly downregulated compared to those of the healthy group. These results indicate that CTH and H2S were correlated with the occurrence and development of CM in Holstein cows, which provides important insights into the function and regulatory mechanism of CTH/H2S in Holstein cows.

Serum CCL28 as a biomarker for diagnosis and evaluation of Sjögren’s syndrome

Objective The aim of this study was to explore the significance of serum CCL28 in Sjögren’s syndrome (SS) diagnosis and evaluation. Method The expression of CCL28 mRNA in salivary glands of SS patients from the GEO database was analysed. Serum levels of CCL28 of SS patients, rheumatoid arthritis (RA) patients, systemic lupus erythematosus (SLE) patients, and healthy controls (HCs) were measured by enzyme-linked immunosorbent assay. The serum immunoglobulin A (IgA) levels and the focus score of labial salivary gland (LSG) in patients with SS were also measured, and the correlation between serum IgA levels and serum CCL28 was explored. In addition, the level of serum CCL28 was compared between two subsets of SS patients who were classified by clinical symptoms and laboratory tests. Results SS patients displayed decreased expression of CCL28 mRNA in salivary glands, accompanying more severe pathological injury. Serum levels of CCL28 in both primary and secondary SS patients were significantly lower than those in the HC group, whereas no significant differences were observed between RA patients or SLE patients and HCs. Compared with RA and SLE patients alone, serum levels of CCL28 were dramatically lower in patients with SS secondary to RA or SLE. No remarkable correlation between serum IgA and CCL28 levels was observed, while the focus score of LSG negatively correlated with serum CCL28 levels. Serum levels of CCL28 were lower in SS patients who had dental caries and thrombocytopenia. Conclusion Serum CCL28 is a useful biomarker in the diagnosis and evaluation of SS.

Dapagliflozin Exerts Adrenal Sympatholysis via G protein-Coupled Receptor Kinase-2 & Tyrosine Hydroxylase Downregulation

Abstract The sodium-glucose co-transporter (SGLT)-2 inhibitor dapagliflozin was recently reported to reduce renal tyrosine hydroxylase (TH) and norepinephrine levels, lowering blood pressure and preventing endothelial dysfunction, in a murine model of neurogenic hypertension. This suggested that dapagliflozin may combat sympathetic nervous system (SNS) hyperactivity, which is known to accompany and aggravate chronic heart failure (CHF). Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and SNS hyperactivity, especially in CHF. GRK2 severely dysregulates adrenal sympatho-inhibitory α 2-adrenergic receptors (ARs), leading to unchecked, chronically elevated CA secretion. Therefore, we hypothesized herein that SGLT2 inhibition with dapagliflozin may lower SNS hyperactivity in the adrenal gland by antagonizing GRK2 actions on α 2ARs in adrenal chromaffin cells. We used the rat pheochromocytoma PC12 cell line expressing human α 2AAR, as well as freshly isolated adrenal glands from adult male Sprague-Dawley rats treated with dapagliflozin in vivo. We measured circulating norepinephrine (NE) in vivo via RIA, GRK2 & TH expression levels via real-time PCR and immunoblotting, adrenal α 2AR density (Bmax) via saturation radioligand binding with [methyl-3H]-rauwolscine, and G protein activation via the GTPγS assay. Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217+67 pg/ml, n=6), compared to control, vehicle-treated rats (363+77 pg/ml, n=6, p<.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and TH mRNA and protein levels in dapagliflozin-treated rat adrenals vs. vehicle-treated animal-derived glands, indicating reduced adrenal CA synthesis and secretion. Finally, adrenal α 2AR density was higher in dapagliflozin- vs. vehicle-treated rats (51.3+7.3 vs. 26.1+8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p<.05). These results (i.e. GRK2 and TH downregulation) were completely recapitulated in PC12 α 2AAR-expressing cells in culture, treated with 5 μM dapagliflozin (or vehicle) for 24 hours. Importantly, α 2AR-dependent G protein-mediated signaling towards inhibition of CA secretion was markedly enhanced at 24 hrs post-dapagliflozin application in PC12 cells. This was the result of reduced GRK2-dependent receptor desensitization, since dapagliflozin lacked this effect in cells co-transfected with a GRK2-encoding adenovirus to acutely overexpress GRK2. In conclusion, dapagliflozin exerts a sympatholytic action in the adrenal medulla via downregulation of both TH, which reduces CA biosynthesis, and GRK2, which reduces α 2AR desensitization in favor of enhanced inhibition of CA secretion.

Regulation of STAT4 by ETS1 in Sjögren syndrome pathogenesis

<h3>Objectives</h3> Primary Sjögren syndrome (pSS) is a chronic autoimmune disease mainly affecting women over the age of 40, causing dry mouth and eyes. The etiology of pSS is complex and includes the overexpression of inflammatory cytokines from affected salivary gland epithelial cells (SGECs). Our group has previously identified candidate biomarkers overexpressed in salivary gland tissues of patients with pSS. We showed that ETS proto-oncogene 1 (ETS1) had higher mRNA and protein expression in pSS salivary glands that correlated with higher levels of lymphocytic infiltration. It has also been shown that the signal transducer and activator of transcription 4 (STAT4) gene contains pSS susceptibility polymorphisms and is a significant mediator of cytokine (ie, interferon, tumor necrosis factor-α) production. Additionally, in previous studies unrelated to pSS, ETS1 was shown to regulate STAT4 expression at the transcription level. Thus, we hypothesized that ETS1 plays a significant role in regulating STAT4 transcription within SGECs of patients with pSS. Our objectives were as follows: (1) to determine whether downregulation of ETS1 mRNA by siRNA knockdown results in decreased mRNA and protein expression of STAT4 within the salivary gland cell line A253 and (2) to demonstrate the regulation of STAT4 mRNA expression by ETS1 siRNA knockdown in cultured SGECs from control patients with pSS and non-pSS sicca. <h3>Methods</h3> The salivary gland cell line, A253, was transfected with siRNA targeting ETS1 or a scrambled siRNA control. After 48 hours, mRNA and protein were isolated. cDNA synthesis was performed using 1 µg of mRNA. Expression of ETS1 and STAT4 mRNA was quantified by quantitative reverse transcription–polymerase chain reaction using the delta cycle threshold method to determine the relative fold changes normalized to glyceraldehyde 3-phosphate dehydrogenase in triplicate experiments. In addition, downregulation of STAT4 protein by ETS1 siRNA was determined by Western blot analysis and semiquantified by densitometry using cofilin as the loading control. SGECs were cultured from labial salivary gland biopsies of 2 patients with pSS (focus score [FS] ≥1) and 4 patients with non-pSS sicca (FS = 0, n = 2; 0 < FS < 1; n = 2) and transfected with siRNA targeting ETS1 or a scrambled siRNA control. After 48 hours, mRNA was isolated for cDNA synthesis and used to determine the mRNA expression of ETS1 and STAT4 as described above. <h3>Results</h3> In A253 cells, STAT4 mRNA expression was changed by −2.15-fold, on average, when treated with siRNA targeting ETS1. STAT4 protein expression was measured by Western blot analysis 48 hours post-ETS1 siRNA knockdown showing an average −1.25-fold change in A253 total cell lysate. SGECs cultured from patients with lymphocytic infiltration (ie, FS >0) showed a significantly higher expression of STAT4 (<i>P</i> = .0485) than patients with non-pSS sicca with FS = 0. However, we did not find a significant change in STAT4 mRNA expression (average, +1.14-fold) in cultured SGECs treated with siRNA targeting ETS1. <h3>Conclusions</h3> STAT4 is overexpressed by cultured SGECs in patients with pSS and those without pSS with focal lymphocytic infiltrates. STAT4 overexpression in SGECs of patients with pSS and those without pSS could be a result of inflammatory cytokine exposure by neighboring cells or effects of infiltrating lymphocyte exposure. Thus, our results suggest a separate regulatory mechanism of STAT4 in pSS bypassing ETS1 regulation, which might be due to pathogenic pathways involving other transcriptional regulation.

Expression Profiles of CircRNA and mRNA in Lacrimal Glands of AQP5-/- Mice With Primary Dry Eye.

Purpose: This work aimed to identify differentially expressed circular RNAs (circRNAs) and elucidate their potential function in aquaporin 5 (AQP5) knockout (AQP5–/–) mice with the primary dry eye phenotype.Methods: A slit lamp examination was performed on AQP5–/– mice to assess corneal epithelial defects using fluorescein sodium staining. Hematoxylin–eosin staining and transmission electron microscopy analysis were performed to identify structural changes in lacrimal gland epithelial cells due to AQP5 deficiency. The expression profiles of circRNA and messenger RNA (mRNA) were determined by a microarray analysis. The selected circRNA was verified by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to predict the biological functions and the potential pathways of parental genes involved in lacrimal gland epithelial cell changes. According to the bioinformatics analysis of identified circRNAs, we predicted a circRNA–miRNA–mRNA network of phagosomes.Results: The AQP5–/– mice spontaneously exhibit dry eye symptoms, wherein the AQP5 deficiency changes the structure of lacrimal gland epithelial cells. The analysis revealed that, compared to AQP5+/+ mice, 30 circRNAs in the lacrimal glands of AQP5–/– mice were differentially expressed (fold change ≥ 2.0, p < 0.05). Nine upregulated circRNAs were identified using qRT-PCR, and nine upregulated validated circRNAs, 40 altered microRNAs (miRNAs), and nine upregulated mRNAs were identified through a network analysis. The KEGG analysis showed that these nine target genes were expressed in phagosomes.Conclusion: The AQP5–/– mice have primary and stable dry eye phenotypes from birth. We identified differently expressed circRNAs in the lacrimal glands of AQP5–/– and AQP5+/+ mice, predicting a circRNA–miRNA–mRNA network of phagosomes. CircRNA likely plays an important role in lacrimal gland epithelial cell pathogenesis. Therefore, it is reasonable to use circRNA as a potential therapeutic agent for the treatment of dry eyes.

Effects of 17β-Estradiol on growth-related genes expression in female and male spotted scat (Scatophagus argus).

Growth hormone (GH) is the most important endocrine factor to regulate somatic growth. Spotted scat (Scatophagus argus) is a famous marine aquaculture species in China with a typical sexual growth dimorphism in which females grow faster and larger than males. In this study, gh messenger RNA (gh mRNA) and GH protein expression were examined in the pituitary glands of female and male spotted scat. Based on qPCR analysis, gh mRNA was mainly expressed in the pituitary gland, and weakly in the gonads and hypothalamus. Furthermore, gh mRNA expression in the pituitary gland was significantly higher in females at stages II-IV than in males at stages III-V. In addition, gh mRNA was highly expressed in the ovary and testis during mature development stages. In this study, spotted scat GH polyclonal antibody was produced. Western blot analysis showed that the molecular weight of spotted scat GH was about 21 KDa. Immunohistochemistry (IHC) in pituitary glands showed that GH was mainly expressed in the proximal pars distal (PPD) and a few cells were distributed in the rostral pairs distal (RPD). After injecting 17β-Estradiol (E2) in vivo, gh mRNA expression was significantly up-regulated in the pituitary gland, whereas igf1 and ghr1 mRNA levels were down-regulated in the liver, which might regulate gh mRNA expression in the pituitary gland. These results provide valuable insight into the molecular mechanisms of E2 regulating gh expression in spotted scat.

Molecular characterization of Antheraea mylitta arylphorin gene and its encoded protein

Antheraea mylitta arylphorin protein was extracted from the silk gland of fifth instar larvae and purified by ammonium sulphate precipitation, ion-exchange, and gel filtration chromatography. The N-terminal sequencing of ten amino acids (NH2-SVVHPPHHEV-COOH) showed similarity with Antheraea pernyi arylphorin. Based on N-terminal and C-terminal A. pernyi arylphorin sequences, primers were designed, and A. mylitta arylphorin cDNA was cloned by RT-PCR from silk gland mRNA. Sequencing of complete cDNA including 25 nucleotides at 5′ UTR (obtained by 5′ RACE) showed that it consisted of an ORF of 2115 nucleotides which could encode a protein of 704 amino acids (predominantly aromatic residues) having molecular weight 83 kDa. Homology modelling was done using A. pernyi arylphorin as a template. Cloned arylphorin cDNA was expressed in E. coli and recombinant His-tagged protein was purified by Ni-NTA affinity chromatography. Analysis of tissue-specific expression of arylphorin by real-time PCR showed maximum expression in the fat body followed by silk gland and integument. 5’ flanking region (759 bp) of arylphorin gene was amplified by inverse PCR and the full length gene (5359 nucleotides) containing five exons and four introns was cloned from the A. mylitta genomic DNA and sequenced. Polyclonal antibody was raised against purified arylphorin and more native arylphorin protein (500 kDa) was purified from the fat body by antibody affinity chromatography. Study of mitogenic effect of native and chymotrypsin hydrolysate of arylphorin on different insect cell lines showed that arylphorin could be used as serum substitute for in vitro cultivation of insect cells.

TickSialoFam (TSFam): A Database That Helps to Classify Tick Salivary Proteins, a Review on Tick Salivary Protein Function and Evolution, With Considerations on the Tick Sialome Switching Phenomenon.

Tick saliva contains a complex mixture of peptides and non-peptides that counteract their hosts' hemostasis, immunity, and tissue-repair reactions. Recent transcriptomic studies have revealed over one thousand different transcripts coding for secreted polypeptides in a single tick species. Not only do these gene products belong to many expanded families, such as the lipocalins, metalloproteases, Antigen-5, cystatins, and apyrases, but also families that are found exclusively in ticks, such as the evasins, Isac, DAP36, and many others. Phylogenetic analysis of the deduced protein sequences indicate that the salivary genes exhibit an increased rate of evolution due to a lower evolutionary constraint and/or positive selection, allowing for a large diversity of tick salivary proteins. Thus, for each new tick species that has its salivary transcriptome sequenced and assembled, a formidable task of annotation of these transcripts awaits. Currently, as of November 2019, there are over 287 thousand coding sequences deposited at the National Center for Biotechnology Information (NCBI) that are derived from tick salivary gland mRNA. Here, from these 287 thousand sequences we identified 45,264 potential secretory proteins which possess a signal peptide and no transmembrane domains on the mature peptide. By using the psiblast tools, position-specific matrices were constructed and assembled into the TickSialoFam (TSF) database. The TSF is a rpsblastable database that can help with the annotation of tick sialotranscriptomes. The TSA database identified 136 tick salivary secreted protein families, as well as 80 families of endosomal-related products, mostly having a protein modification function. As the number of sequences increases, and new annotation details become available, new releases of the TSF database may become available.

Severe chronic kidney disease environment reduced calcium-sensing receptor expression in parathyroid glands of adenine-induced rats even without high phosphorus diet

BackgroundChronic kidney disease (CKD) disrupts mineral homeostasis and its main underlying cause is secondary hyperparathyroidism (SHPT). We previously reported that calcium-sensing receptor (CaSR) mRNA and protein expression in parathyroid glands (PTGs) significantly decreased in a CKD rat model induced by a 5/6 nephrectomy that were fed a high phosphorus diet. However, there was a significant difference in the severity of CKD between high phosphorus and adequate phosphorus diet groups. Thus, it was unclear whether CKD environment or the high phosphorus diet influenced CaSR expression, and the underlying mechanism remains largely unknown.MethodsCKD was induced in rats with 0.75% adenine-containing diet. CKD and control rats were maintained for 5 days and 2 weeks on diets with 0.7% or 1.3% phosphorus. For gene expression analysis, quantitative real-time polymerase chain reaction was performed with TaqMan probes. Protein expression was analyzed by immunohistochemistry.ResultsPTG CaSR expression significantly decreased in the presence of a severe CKD environment, even without the high phosphate load. Ki67 expressing cells in PTGs were significantly higher only in the CKD rats fed a high phosphorus diet. Furthermore, among the many genes that could affect CaSR expression, only vitamin D receptor (VDR) and glial cells missing 2 (Gcm2) showed significant changes. Moreover, Gcm2 was significantly reduced at an early stage without significant changes in serum calcium, phosphorus and 1,25(OH)2 vitamin D, and there was no significant reduction in CaSR and VDR expressions. Then, significantly elevated Ki67-positive cell numbers were also only observed in the early CKD PTGs with high-phosphorus diets.ConclusionsOur data suggest that the cause of the decreased PTG CaSR expression is the reduction in VDR and Gcm2 expression; Gcm2 may play a role in the onset and progression of SHPT.

The Presence and Distribution of TRPM7 in the Canine Mammary Glands.

Simple SummaryMammary gland tumors are one of the major causes of mortality in dogs. It is, therefore, imperative to clarify the nature of this disease. Transient receptor potential melastatin-subfamily member 7 (TRPM7), a bifunctional ion channel found in human cells, plays a role in normal physiological processes such as cell development, survival, proliferation, differentiation, and migration. However, TRPM7 is also active in several types of cancers in humans, and blocking the expression of this pathway leads to a decrease in the proliferation, migration, and invasion of cancer cells. We have proved the presence of TRPM7 in healthy canine mammary tissues, which could help further studies that aim to establish the relationship between TRPM7 and its physiological and pathophysiological effects in the canine mammary glands and, possibly, tumors.The transient receptor potential melastatin-subfamily member 7 (TRPM7) cation channel is a bifunctional ion channel with intrinsic kinase activity and is ubiquitously expressed in the animal/human body. Accumulated knowledge of TRPM7 suggests that it plays an essential role in normal physiological processes, including the development, survival, proliferation, differentiation, and migration of cells. The aim of this study was to demonstrate the presence and expression patterns of TRPM7 in normal canine mammary glands using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry. Normal mammary gland tissue samples were obtained from five female beagle dogs. RT-PCR and sequencing of the amplified PCR products demonstrated the presence of TRPM7 mRNA in normal mammary glands, and the presence of TRPM7 protein was confirmed by Western blotting. Immunohistochemical investigations demonstrated the expression of TRPM7 in the apical membrane of acinar and ductal epithelial cells in the canine mammary glands. These results provide the first evidence of the presence and distribution of TRPM7 in the canine mammary gland and could help explain the physiological and pathological roles of TRPM7 in the canine mammary gland; however, additional studies are required to elucidate these roles.

Genome-wide analysis of mRNAs and lncRNAs in Mycoplasma bovis infected and non-infected bovine mammary gland tissues

Mycoplasma bovis (M. bovis) causes diseases such as arthritis, pneumonia, abortion, and mastitis, leading to great losses in the bovine dairy industries. RNA types such as messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) play significant roles in regulating the immune responses triggered by bacteria. The expression profiles of mRNA and lncRNA as they occur in bovine mammary gland tissues infected with M. bovis are still not well understood. To illuminate this issue, transcription analysis of mRNA and LncRNAs were conducted on the mammary gland tissues belonging to Holstein cattle infected and not infected with M. bovis. The analysis revealed 1310 differentially expressed mRNAs and 57 differentially expressed lncRNAs in the bovine mammary gland tissues infected and not infected with M. bovis. In addition, 392 novel lncRNAs were detected, 19 of which were differentially expressed. Gene ontology analysis reveals that differentially expressed mRNAs and lncRNAs play significant roles in such vital biological pathways as metabolic pathways, T-cell receptor signaling, TGF-beta signaling, pathways in cancer, PI3K-Akt signaling, NF-kappa B signaling, mTOR signaling, and apoptosis, including in the immune response to cancer. Based on our literature review, this study is the first genome-wide lncRNA research conducted on bovine mammary gland tissues infected with M. bovis. Our results provide bovine mammary gland lncRNA and mRNA resources to understand their roles in the regulation of the immune response against the agent M. bovis in bovine mammary gland tissues.

Serotonin regulates maternal calcium homeostasis during the perinatal period of sheep.

The goal of this experiment was to demonstrate the ability of an infusion of serotonin (5-HT; 5-hydroxytryptamine) precursors to increase 5-HT production during the transition from pregnancy to lactation and its effects on gene expression related to calcium (Ca) transporters in the mammary gland and bone resorption markers in the femur. Thirty pregnant Bamei mutton sheep were randomly assigned to 3 experimental groups. All groups received a daily intravenous infusion of saline (control group; n = 10), saline containing 0.178 mg of L-tryptophan/kg body weight (BW) (TRP group, n = 10) or 0.178 mg of 5-hydroxytryptophan/kg BW (5-HTP group, n = 10), beginning on day 7 of prepartum and continuing until delivery. Serum (pre- and postpartum), milk (postpartum), and femur and mammary gland tissue (day 9) were collected. Sheep infused with 5-HTP had a larger total serum Ca concentration on days 3, 6, 15, and 30 of lactation and total milk Ca concentration on days 3, 6, 12, and 15 of lactation compared with that of the control group. Sheep infused with 5-HTP and TRP increased blood and milk concentrations of 5-HT on days 3, 6, 9, and 30 of lactation and parathyroid hormone-related protein (PTHrP) on day 3 of prepartum and on days 3, 6, and 15 of lactation (P < 0.05). In addition, compared to that of the control group, the TRP or 5-HTP infusion upregulated PTHrP, a sodium/calcium exchanger, plasma membrane Ca2+ ATPase 2, secretory pathway Ca2+ ATPase 1, and calcium sensing receptor mRNA expression in mammary gland and receptor-activated nuclear factor kappa-B ligand mRNA expression in the femur, but had no effect on receptor-activated nuclear factor kappa-B and osteoprotegerin mRNA expression in the femur (P < 0.05). This suggests that 5-HT and PTHrP may be involved in regulating maternal Ca homeostasis during the transition from pregnancy to lactation in the sheep.

Metformin improves salivary gland inflammation and hypofunction in murine Sj\xf6gren\u2019s syndrome

BackgroundActivated T and B cells participate in the development and progression of Sjögren’s syndrome (SS). Metformin, a first-line anti-diabetic drug, exerts anti-inflammatory and immunomodulatory effects by activating AMPK. We investigated the therapeutic effect of metformin in non-obese diabetic (NOD)/ShiLtJ mice, an animal model of SS.MethodsMetformin or vehicle was administered orally to the mice for 9 weeks. The salivary flow rate was measured at 11, 13, 15, 17, and 20 weeks. Histological analysis of the salivary glands from vehicle- and metformin-treated mice was conducted. CD4+ T and B cell differentiation in the peripheral blood and/or spleen was determined by flow cytometry. Serum total IgG, IgG1, and IgG2a levels were determined by enzyme-linked immunosorbent assay.ResultsMetformin reduced salivary gland inflammation and restored the salivary flow rate. Moreover, metformin reduced the interleukin (IL)-6, tumor necrosis factor-α, IL-17 mRNA, and protein levels in the salivary glands. Metformin reduced the Th17 and Th1 cell populations and increased the regulatory T cell population in the peripheral blood and spleen and modulated the balance between Tfh and follicular regulatory T cells. In addition, metformin reduced B cell differentiation into germinal center B cells, decreased the serum immunoglobulin G level, and maintained the balance between IL-10- and IL-17-producing B cells.ConclusionMetformin suppresses effector T cells, induces regulatory T cells, and regulates B cell differentiation in an animal model of SS. In addition, metformin ameliorates salivary gland inflammation and hypofunction, suggesting that it has potential for the treatment of SS.

Effect of chronic corticosterone treatment on expression and distribution of serotonin 5-HT7 receptors in rat adrenal glands

Sensitized stress-induced corticosterone (CORT) secretion in chronically stressed rats involves 5-HT7 receptor activation. The effect of 14-day chronic CORT and vehicle (VEH) administration on 5-HT7 receptor expression in adrenal glands, adrenal 5-HT content, and adrenocorticotropic hormone and CORT secretion was analysed. On day 15, VEH- and CORT-treated animals were perfused or decapitated without stress exposure (0 min) or after 10 and 30 min of restraint for collection of trunk blood and tissues. 5-HT7 receptor-like immunoreactivity (5-HT7R-LI), 5-HT7 receptor protein, and mRNA levels were determined by immunohistochemistry, Western blot, and reverse transcription polymerase chain reaction assays, respectively; 5-HT levels and hormones were quantified using HPLC and ELISA kits, respectively. An undisturbed control group was included for most experimental comparisons. Chronic CORT strongly increased 5-HT7R-LI in the outer adrenal cortex, as well as 5-HT7 receptor protein and mRNA in whole adrenal glands; adrenal 5-HT content also increased in these animals. Decreased adrenocorticotropic hormone and CORT secretion at 30 min of restraint occurred in CORT-treated rats. The results support the notion that chronic stress-induced increase of adrenocortical 5-HT7 receptors and adrenal 5-HT content is a glucocorticoid-dependent phenomenon; the development of magnified stress-induced 5-HT7 receptor-mediated CORT responses in chronically stressed animals nevertheless likely involves additional mechanisms.

Effect of Monochromatic Light on Plasma and Mrna Levels of Luteinising Hormone, Follicle-Stimulating Hormone, and their Receptors in the Hypothalamic–Pituitary–Gonadal Axis of Pigeons

This study was conducted to evaluate the effect of monochromatic light on luteinising hormone (LH) and follicle-stimulating hormone (FSH) concentrations, LH and FSH mRNA levels, and mRNA levels of their receptors (LHR and FSHR) in Pigeons (Columba livia). A total of 60 paired adult White King pigeons were randomly divided into four groups; each group was exposed to violet light (VL), green light (GL), red light (RL), or white light for 16 h. Nine paired pigeons from each group were selected for analysis of FSH and LH concentrations and six paired pigeons from each group were selected for analysis of mRNA. The results showed that the concentration of LH in females was lower in the VL group (P&lt;0.05), and in males, it was lower in the VL and GL groups (P&lt;0.05). The concentration of FSH in females was significantly higher in the RL group than in the VL group (P&lt;0.05), whereas no significant difference was observed in males (P&gt;0.05). In females, the LH and FSH mRNA levels were relatively high in the pituitary gland, whereas LHR and FSHR mRNA levels were relatively high in the ovary. In males, the LH, LHR, FSH, and FSHR mRNA levels were high in the pituitary gland, whereas their respective mRNA levels were relatively low in the testis. These results suggest that RL has the potential to improve LH and FSH concentrations and mRNA levels, and indicates that RL could promote reproductive performance of pigeons.