Hormones and liver mitochondria: Influence of growth hormone,thyroxine, testosterone,and insulin on thermotropic effects of respiration and fatty acid composition of membranes

The effects of hypophysectomy and subsequent administration of growth hormone, thyroxine, insulin, and testosterone were examined in rat liver for the relationship between the thermotropic effects on State 3 respiration (ADP induced) and fatty acid composition of the phospholipid fraction of intact mitochondria as well as of inner membrane vesicles. The Arrhenius profile for energy-linked (succinate) State 3 respiration of mitochondria from hypophysectomized rats lacked the discontinuity at 23.5 °C seen with mitochondria from normal rats. After injections of the hormones the discontinuity representing the transition temperature from gel to liquid crystalline state of lipids occurred at different temperatures: 18.5 °C for growth hormone, 26.0 °C for thyroxine, 19.5 °C for growth hormone + thyroxine, 27.6 °C for insulin, and 25.3 °C for testosterone. The energy of activation between 37.5 and 23.5 °C was 1.9 times greater for hypophysectomy than for controls. Growth hormone was the most effective in restoring the energy of activation to normal, above as well as below transition temperature. The effect of thyroxine appears to be due to a larger stimulation of the State 4 respiration than that of growth hormone, insulin, or testosterone, especially at higher temperatures. Phospholipids extracted from intact mitochondria or inner membrane vesicles of hypophysectomized rats contained less arachidonic acid (20:4) and more linoleic acid (18:2) than those of normal rats. In addition, the contents of some of the minor fatty acids were also changed. Calculated unsaturation index showed an 18.8 and 14.9% depletion in unsaturation in whole mitochondria and inner membranes, respectively. Among the different hormones used to treat the hypophysectomized rats, growth hormone was the most effective in restoring the transition temperature and fatty acid composition to normal levels and increasing the gain in body weight. Although the other hormones increased total unsaturation index to some extent, some of the individual fatty acids were affected differently. Good correlation exists between the unsaturation index of mitochondrial fatty acids and transition temperature of State 3 respiration. These results strongly suggest a role for the hormones, particularly growth hormone, in the control of mitochondrial membrane fluidity of hypophysectomized rat liver, through fatty acid composition of phospholipids.     

Simultaneous RP‐HPLC‐DAD Separation,and Determination of Flavonoids and Phenolic Acids in Plantago L. Species

A rapid reversed‐phase (RP) high‐performance liquid chromatography method was developed and applied for simultaneous separation, and determination of flavonoids and phenolic acids in eight Plantago L. taxa (P. altissima L., P. argentea Chaix , P. coronopus L., P. holosteum Scop . ssp. depauperata Pilger , P. holosteum ssp. holosteum, P. holosteum ssp. scopulorum (Degen) Horvati? , P. lagopus L., and P. maritima L.) growing in Croatia. Chromatographic separation was carried out on Zorbax Eclipse XDB‐C18 using gradient elution with a H₂O (pH 2.5, adjusted with CF₃COOH) and MeCN mixture at 30°. The contents of analyzed phenolic compounds (% of the dry weight of the leaves, dw) varied among examined species: rutin (max. 0.024%, P. argentea), hyperoside (max. 0.020%, P. lagopus), quercitrin (max. 0.013%, P. holosteum ssp. holosteum), quercetin (max. 0.028%, P. holosteum ssp. scopulorum), chlorogenic acid (max. 0.115%, P. lagopus), and caffeic acid (max. 0.046%, P. coronopus). Isoquercitrin was detected only in P. argentea (0.020%), while isochlorogenic acid content was below limit of quantification in all investigated species. Multivariate analyses (UPGMA and PCA) showed significant differences in contents of investigated polyphenolic compounds between different Plantago taxa. Accordingly, investigated substances might be employed as chemotaxonomic markers in the study of the complex genus Plantago.     

Site-specific glycosylation of SARS-CoV-2: Big challenges in mass spectrometry analysis

Glycosylation of viral proteins is required for the progeny formation and infectivity of virtually all viruses. It is increasingly clear that distinct glycans also play pivotal roles in the virus's ability to shield and evade the host's immune system. Recently, there has been a great advancement in structural identification and quantitation of viral glycosylation, especially spike proteins. Given the ongoing pandemic and the high demand for structure analysis of SARS-CoV-2 densely glycosylated spike protein, mass spectrometry methodologies have been employed to accurately determine glycosylation patterns. There are still many challenges in the determination of site-specific glycosylation of SARS-CoV-2 viral spike protein. This is compounded by some conflicting results regarding glycan site occupancy and glycan structural characterization. These are probably due to differences in the expression systems, form of expressed spike glycoprotein, MS methodologies, and analysis software. In this review, we recap the glycosylation of spike protein and compare among various studies. Also, we describe the most recent advancements in glycosylation analysis in greater detail and we explain some misinterpretation of previously observed data in recent publications. Our study provides a comprehensive view of the spike protein glycosylation and highlights the importance of consistent glycosylation determination.     

Interstitial Cajal-like cells of human Fallopian tube express estrogen and progesterone receptors

Cells of the female reproductive tract are subject to hormonal control via sex steroid genomic receptors expressed at nuclear level. We previously showed that interstitial Cajal-like cells (ICLC) of human myometrium expressed estrogen and progesterone receptors (ER/PR). Our aim, based on these results, was to see if ER and/or PR could be found also in tubal ICLC. Indeed, we present here immunohistochemical evidence that ICLC of human Fallopian tube (isthmic region) have such receptors. Stromal ICLC, as well as ICLC among smooth muscle layers, were identified in tissue sections by their morphological features (e.g. several very long, moniliform, prolongations of cell body) as well as by c-kit positivity, vital staining with methylene blue or silver impregnation. Additional evidence was provided by sequential staining for c-kit and for PR on the same cell, by ‘sandwich method’. In vitro, the 4th passage cell cultures from Fallopian tube muscularis exhibiting ICLC morphology showed the presence of ER-alpha and/or PR-A by immunofluorescence. In conclusion, our data suggest that ICLC could function as steroid sensors, and might be implicated in Fallopian tube motility (via gap junctions or juxta- and/or paracrine mechanisms).     

Protein profiling of human lung telocytes and microvascular endothelial cells using iTRAQ quantitative proteomics

Telocytes (TCs) are described as a particular type of cells of the interstitial space ( www.telocytes.com ). Their main characteristics are the very long telopodes with alternating podoms and podomers. Recently, we performed a comparative proteomic analysis of human lung TCs with fibroblasts, demonstrating that TCs are clearly a distinct cell type. Therefore, the present study aims to reinforce this idea by comparing lung TCs with endothelial cells (ECs), since TCs and ECs share immunopositivity for CD34. We applied isobaric tag for relative and absolute quantification (iTRAQ) combined with automated 2‐D nano‐ESI LC‐MS/MS to analyse proteins extracted from TCs and ECs in primary cell cultures. In total, 1609 proteins were identified in cell cultures. 98 proteins (the 5th day), and 82 proteins (10th day) were confidently quantified (screened by two‐sample t‐test, P < 0.05) as up‐ or down‐regulated (fold change >2). We found that in TCs there are 38 up‐regulated proteins at the 5th day and 26 up‐regulated proteins at the 10th day. Bioinformatics analysis using Panther revealed that the 38 proteins associated with TCs represented cellular functions such as intercellular communication (via vesicle mediated transport) and structure morphogenesis, being mainly cytoskeletal proteins and oxidoreductases. In addition, we found 60 up‐regulated proteins in ECs e.g.: cell surface glycoprotein MUC18 (15.54‐fold) and von Willebrand factor (5.74‐fold). The 26 up‐regulated proteins in TCs at 10th day, were also analysed and confirmed the same major cellular functions, while the 56 down‐regulated proteins confirmed again their specificity for ECs. In conclusion, we report here the first extensive comparison of proteins from TCs and ECs using a quantitative proteomics approach. Our data show that TCs are completely different from ECs. Protein expression profile showed that TCs play specific roles in intercellular communication and intercellular signalling. Moreover, they might inhibit the oxidative stress and cellular ageing and may have pro‐proliferative effects through the inhibition of apoptosis. The group of proteins identified in this study needs to be explored further for the role in pathogenesis of lung disease.     

Comparative proteomic analysis of human lung telocytes with fibroblasts

Telocytes (TCs) were recently described as interstitial cells with very long prolongations named telopodes (Tps; www.telocytes.com ). Establishing the TC proteome is a priority to show that TCs are a distinct type of cells. Therefore, we examined the molecular aspects of lung TCs by comparison with fibroblasts (FBs). Proteins extracted from primary cultures of these cells were analysed by automated 2‐dimensional nano‐electrospray ionization liquid chromatography tandem mass spectrometry (2D Nano‐ESI LC‐MS/MS). Differentially expressed proteins were screened by two‐sample t‐test (P < 0.05) and fold change (>2), based on the bioinformatics analysis. We identified hundreds of proteins up‐ or down‐regulated, respectively, in TCs as compared with FBs. TC proteins with known identities are localized in the cytoskeleton (87%) and plasma membrane (13%), while FB up‐regulated proteins are in the cytoskeleton (75%) and destined to extracellular matrix (25%). These identified proteins were classified into different categories based on their molecular functions and biological processes. While the proteins identified in TCs are mainly involved in catalytic activity (43%) and as structural molecular activity (25%), the proteins in FBs are involved in catalytic activity (24%) and in structural molecular activity, particularly synthesis of collagen and other extracellular matrix components (25%). Anyway, our data show that TCs are completely different from FBs. In conclusion, we report here the first extensive identification of proteins from TCs using a quantitative proteomics approach. Protein expression profile shows many up‐regulated proteins e.g. myosin‐14, periplakin, suggesting that TCs might play specific roles in mechanical sensing and mechanochemical conversion task, tissue homoeostasis and remodelling/renewal. Furthermore, up‐regulated proteins matching those found in extracellular vesicles emphasize TCs roles in intercellular signalling and stem cell niche modulation. The novel proteins identified in TCs will be an important resource for further proteomic research and it will possibly allow biomarker identification for TCs. It also creates the premises for understanding the pathogenesis of some lung diseases involving TCs.     

Spatial distribution and seasonal variation in fluoride enrichment in groundwater and its associated human health risk assessment in Telangana State,South India

Groundwater is a vital source of drinking water in Siddipet rural and urban regions of Central Telangana, South India and it is a major cause of fluoride toxicity in humans. The intake of elevated fluoride has a significant impact on human health, especially immediate problems that are seen in children's teeth. The primary aim of the study was to identify the seasonal variation in fluoride concentration and associated health risks in the residents of the study region. To assess the fluoride contamination in groundwater, a total of 158 samples were analyzed in two seasons. The mean concentrations of fluoride 1.26 mg/L and 2.21 mg/L were 1.46 and 2.8 times higher than the acceptable limit of 1.5 mg/L, before and after monsoon respectively. To estimate the human health risks due to the ingestion of elevated fluoride through drinking water, hazard quotient fluoride (HQ_Fluoride) was calculated using the United States Environmental Protection Agency method. HQ_Fluoride values were 0.44–2.44 and 0.89–4.67 for children, 0.36–2.00 and 0.73–3.82 for females, and 0.41–2.26 and 0.82–4.31 for males in pre- and post-monsoon seasons respectively, suggesting emphatically greater risk than the acceptable limits (HQ_Fluoride > 1), which generates health risks.