Protein Content In Supernatant Research Articles

Menstrual fluid endometrial stem/progenitor cell and supernatant protein content: cyclical variation and indicative range.

Does natural variation exist in the endometrial stem/progenitor cell and protein composition of menstrual fluid across menstrual cycles in women? Limited variation exists in the percentage of some endometrial stem/progenitor cell types and abundance of selected proteins in menstrual fluid within and between a cohort of women. Menstrual fluid is a readily available biofluid that can represent the endometrial environment, containing endometrial stem/progenitor cells and protein factors. It is unknown whether there is natural variation in the cellular and protein content across menstrual cycles of individual women, which has significant implications for the use of menstrual fluid in research and clinical applications. Menstrual fluid was collected from 11 non-pregnant females with regular menstrual cycles. Participants had not used hormonal medications in the previous 3 months. Participants collected menstrual fluid samples from up to five cycles using a silicone menstrual cup worn on Day 2 of menstrual bleeding. Menstrual fluid samples were centrifuged to separate soluble proteins and cells. Cells were depleted of red blood cells and CD45+ leucocytes. Menstrual fluid-derived endometrial stem/progenitor cells were characterized using multicolour flow cytometry including markers for endometrial stem/progenitor cells N-cadherin (NCAD) and stage-specific embryonic antigen-1 (SSEA-1) (for endometrial epithelial progenitor cells; eEPC), and sushi domain containing-2 (SUSD2) (for endometrial mesenchymal stem cells; eMSC). The clonogenicity of menstrual fluid-derived endometrial cells was assessed using colony forming unit assays. Menstrual fluid supernatant was analyzed using a custom magnetic Luminex assay. Endometrial stem/progenitor cells are shed in menstrual fluid and demonstrate clonogenic properties. The intraparticipant agreement for SUSD2+ menstrual fluid-derived eMSC (MF-eMSC), SSEA-1+ and NCAD+SSEA-1+ MF-eEPC, and stromal clonogenicity were moderate-good (intraclass correlation; ICC: 0.75, 0.56, 0.54 and 0.52, respectively), indicating limited variability across menstrual cycles. Endometrial inflammatory and repair proteins were detectable in menstrual fluid supernatant, with five of eight (63%) factors demonstrating moderate intraparticipant agreement (secretory leukocyte protein inhibitor (SLPI), lipocalin-2 (NGAL), lactoferrin, follistatin-like 1 (FSTL1), human epididymis protein-4 (HE4); ICC ranges: 0.57-0.69). Interparticipant variation was limited for healthy participants, with the exception of key outliers of which some had self-reported menstrual pathologies. N/A. There are no OMICS or other data sets relevant to this study. The main limitations to this research relate to the difficulty of obtaining menstrual fluid samples across multiple menstrual cycles in a consistent manner. Several participants could only donate across <3 cycles and the duration of wearing the menstrual cup varied between 4 and 6 h within and between women. Due to the limited sample size used in this study, wider studies involving multiple consecutive menstrual cycles and a larger cohort of women will be required to fully determine the normal range of endometrial stem/progenitor cell and supernatant protein content of menstrual fluid. Possibility for selection bias and true representation of the population of women should also be considered. Menstrual fluid is a reliable source of endometrial stem/progenitor cells and related endometrial proteins with diagnostic potential. The present study indicates that a single menstrual sample may be sufficient in characterizing a variety of cellular and protein parameters across women's menstrual cycles. The results also demonstrate the potential of menstrual fluid for identifying endometrial and menstrual abnormalities in both research and clinical settings as a non-invasive method for assessing endometrial health. This study was supported by grants from the Australian National Health and Medical Research Council to C.E.G. (Senior Research Fellowship 1024298 and Investigator Fellowship 1173882) and to J.E. (project grant 1047756), the Monash IVF Research Foundation to C.E.G. and the Victorian Government's Operational Infrastructure Support Program. K.A.W., M.L.D.-T., S.G.S. and J.E. declare no conflicts of interest. C.E.G. reports grants from NHMRC, during the conduct of the study; grants from EndoFound USA, grants from Ferring Research Innovation, grants from United States Department of Defence, grants from Clue-Utopia Research Foundation, outside the submitted work. CEF reports grants from EndoFound USA, grants from Clue-Utopia Research Foundation, outside the submitted work.

Effect of adding salt during the diafiltration step of milk protein concentrate powder manufacture on mineral and soluble protein composition

Milk protein concentrate (MPC) powders offer a great potential for use in an array of food applications because of their nutritional and functional values. However, MPC powders with protein content ≥80% (MPC80) exhibit poor solubility and hence restrict their potential use. The objective of this study was to determine the impact of adding salt during the diafiltration stage of MPC80 manufacture on solubility, turbidity, and to compare minerals and protein content of supernatants of ultracentrifuged samples with control sample. Three types of samples were produced: MPC80-C (control) with or without salt treatment, MPC80-Na (150 mM NaCl), and MPC80-K (150 mM KCl). Lower solubility was observed in MPC80-C (53%) as compared to MPC80-Na or MPC80-K (100%). Higher turbidity was observed in MPC80-C (530 NTU) and lower turbidity was observed in samples of MPC80-Na (128 NTU) and MPC80-K (131 NTU). Furthermore, lower protein and calcium contents were observed in supernatants of ultracentrifuged samples of MPC80-C (2.3%; 0.35 mg.mL−1) as compared to MPC80-Na (3.8%; 0.63mg.mL−1) and MPC80-K (3.7%; 0.67 mg.mL−1). The opposite trend was found in reconstituted samples (5% TS). Our results showed that the addition of salt impacted the distribution of minerals and proteins in colloidal and soluble phases of MPC80-Na and MPC80-K. The results from this work will contribute to our understanding of the role that mineral-induced changes (depletion or addition) play in the functionality of MPC80.

Inhibition of airway mucous hypersecretion by azithromycin through matrix metalloproteinase 9

To investigate the mechanism of azithromycin (AZT) inhibiting the airway mucous hypersecretion through matrix metalloproteinase 9 (MMP9). After culturing, the airway epithelial cells of line HBE16 were stimulated with neutrophil elastase (NE) and pretreated with AZT and epidermal growth factor receptor (EGFR) antagonist BIBX1522. Then the cells were divided into 4 groups: control group, NE-stimulated group, AZT-pretreated & NE-stimulated group and BIBX1522-pretreated & NE-stimulated group. The mucin (MUC)5AC mRNA and MMP9 mRNA levels were analyzed by RT-PCR (reverse transcription-polymerase chain reaction). And the MUC5AC protein content in supernatant was detected by ELISA (enzyme-linked immunosorbent assay). Gelatin zymogrphy was employed to assay the MMP9 activity. Western blot was used to detect the protein levels of MMP9, prozymogen MMP9 (pro-MMP9), tissue inhibitors of metalloproteinases 1 (TIMP-1), phosphorylated EGFR (p-EGFR) and phosphorylated external-signal regulated kinase (p-ERK). As compared with the control group, the levels of MUC5AC and MMP9 gene transcription (0.83 ± 0.17, 0.79 ± 0.16) and protein expression (0.84 ± 0.15, 0.88 ± 0.16) in the NE stimulated group were obviously higher than those of the control group (all P < 0.01). And the activity of MMP9 increased (392.33 ± 18.33, P < 0.01) while the protein level of pro-MMP9 decreased (0.17 ± 0.10, P < 0.01). But the expression of TIMP-1 showed no significant change. The protein expressions of p-EGFR and p-ERK increased (0.86 ± 0.23, 0.85 ± 0.22, both P < 0.01); as compared with the NE-stimulated group, there was a reduction of MUC5AC and MMP9 gene transcription (0.36 ± 0.15, 0.41 ± 0.09, both P < 0.01) and protein expression levels (0.30 ± 0.08, 0.37 ± 0.14, both P < 0.01) in the AZT-pretreated and NE-stimulated group. The MMP9 activity decreased (295.33 ± 14.54, P < 0.01), the protein levels of pro-MMP9 and TIMP-1 increased (0.46 ± 0.14, 0.67 ± 0.17, both P < 0.05), p-ERK protein level decreased (0.40 ± 0.19, P < 0.01) while the expression of p-EGFR showed no significant decline. As compared with the NE-stimulated group, except for the expressions of MUC5AC mRNA (0.37 ± 0.14), MMP9 mRNA (0.37 ± 0.13), p-EGFR (0.36 ± 0.13) and p-ERK (0.37 ± 0.18) decreasing (all P < 0.01) in BIBX1522-pretreated and NE-stimulated group, the other results had no obvious change. AZT may suppress the activity and production of MMP9 in HBE16 cells so as to inhibit the airway mucous hypersecretion.

Sphingosine kinase regulates oxidized low density lipoprotein-mediated calcium oscillations and macrophage survival

We recently reported that oxidized LDL (oxLDL) induces an oscillatory increase in intracellular calcium ([Ca(2+)](i)) levels in macrophages. Furthermore, we have shown that these [Ca(2+)](i) oscillations mediate oxLDL's ability to inhibit macrophage apoptosis in response to growth factor deprivation. However, the signal transduction pathways by which oxLDL induces [Ca(2+)](i) oscillations have not been elucidated. In this study, we show that these oscillations are mediated in part by intracellular mechanisms, as depleting extracellular Ca(2+) did not completely abolish the effect. Inhibiting sarco-endoplasmic reticulum ATPase (SERCA) completely blocked [Ca(2+)](i) oscillations, suggesting a role for Ca(2+) reuptake by the ER. The addition of oxLDL resulted in an almost immediate activation of sphingosine kinase (SK), which can increase sphingosine-1-phosphate (S1P) levels by phosphorylating sphingosine. Moreover, S1P was shown to be as effective as oxLDL in blocking macrophage apoptosis and producing [Ca(2+)](i) oscillations. This suggests that the mechanism in which oxLDL generates [Ca(2+)](i) oscillations may be 1) activation of SK, 2) SK-mediated increase in S1P levels, 3) S1P-mediated Ca(2+) release from intracellular stores, and 4) SERCA-mediated Ca(2+) reuptake back into the ER.

The influence of dilute acid pretreatment conditions on the enzymatic saccharification of Erica spp. for bioethanol production

Production of bioethanol from biomass is one way to reduce both consumption and depletion of fossil fuels and environmental pollution. The conversion of lignocellulosic biomass-to-ethanol comprehends three major steps: thermochemical pretreatment, enzymatic saccharification and fermentation. The aim of this work is to study the influence of dilute acid pretreatment conditions in sugars removal and in enzymatic saccharification of Erica spp. for bioethanol production as well as the influence of poly(ethylene)glycol addition to the enzymatic saccharification step. Pretreatment of Erica spp. with dilute sulfuric acid was studied at different conditions of reaction time, temperature and acid concentration. The influence of supplementation of enzymatic saccharification with poly(ethylene)glycol 4000 and concentration of polymer were tested, using as response the release of glucose and the adsorption of cellulase to biomass by protein content in supernatant and by SDS-PAGE. The addition of PEG 4000 affects the adsorption of cellulase and influences the yield of enzymatic saccharification, leading to an improvement of 13–74% depending on the pretreatment applied to biomass. A maximum of 440 mg glucose/g dry biomass was obtained by enzymatic saccharification when pretreated Erica spp. at 180 °C with 2.75% of sulfuric acid for 75 min, supplemented with 0.25 g/g dry biomass of PEG 4000, was used. The observation of biomass structure by scanning electron microscopy after pretreatment reveals visible breaks in the structure, which could be related to the improvement of enzymatic process.

Dependence of Leucine-rich Repeat Kinase 2 (LRRK2) Kinase Activity on Dimerization

Dominant missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known genetic cause of Parkinson disease. LRRK2 encodes a serine/threonine protein kinase, and pathogenic mutations may increase kinase activity. Intrinsic GTP binding in the GTPase domain may govern kinase activity through an internal signal transduction cascade. As with many protein kinases, LRRK2 self-interacts through mechanisms that may regulate enzymatic activity. We find that the disruption of either GTPase or kinase activity enhances the formation of high molecular weight oligomers and prevents the formation of LRRK2 dimer structures. In addition, brief application of the broad spectrum kinase inhibitor staurosporine ablates LRRK2 dimers and promotes LRRK2 high molecular weight oligomers. LRRK2 interactions with other proteins in cell lines are kinase-independent and include chaperones and cell cytoskeleton components, suggesting that LRRK2 self-assembly principally dictates complex size. To further explore the mechanics of kinase activation, we separate soluble LRRK2 protein that encodes the pathogenic G2019S mutation into high molecular weight oligomers, dimers, and monomers and find that kinase activity resides with dimeric LRRK2. Some PD-associated mutations that increase kinase activity in vitro significantly increase the proportion of dimer structures relative to total LRRK2 protein, providing additional insight into how pathogenic mutations may alter normal enzymatic regulation. Targeting and tracking LRRK2 dimerization may provide a clear way to observe LRRK2 kinase activity in living cells, and disruption of dimeric LRRK2 through kinase inhibition or other means may attenuate pathogenic increases in LRRK2 enzymatic output.

LOX-1 augments oxLDL uptake by lysoPC-stimulated murine macrophages but is not required for oxLDL clearance from plasma

Oxidized LDL (oxLDL) promotes lipid accumulation as well as growth and survival signaling in macrophages. OxLDL uptake is mainly due to scavenger receptors SR-AI/II and CD36. However, other scavenger receptors such as lectin-like oxLDL receptor-1 (LOX-1) may also play a role. We used mice with targeted inactivation of the LOX-1 gene to define the role of this receptor in the uptake of oxLDL and in activation of survival pathways. There was no difference in uptake or degradation of 125I-oxLDL in unstimulated macrophages from wild-type and LOX-1 knockout mice and no difference in the rate of clearance of oxLDL from plasma in vivo. However, when expression of LOX-1 was induced with lysophosphatidylcholine, oxLDL uptake and degradation increased 2-fold in wild-type macrophages but did not change in LOX-1 knockout macrophages. Macrophages lacking LOX-1 showed the same stimulation of PKB phosphorylation and enhancement of survival by oxLDL as wild-type cells. These data show that LOX-1 does not alter the uptake of oxLDL in unstimulated macrophages and is not essential for the pro-survival effect of oxLDL in these cells. However, LOX-1 expression is highly inducible by lysophosphatidylcholine and pro-inflammatory cytokines, and if that occurred in macrophages within atheromas, LOX-1 could substantially increase oxLDL uptake by lesion macrophages.

High density fermentation and activity of a recombinant lumbrokinase (PI239) from Pichia pastoris

A system for the expression of recombinant lumbrokinase (rPI239) was developed in the yeast Pichia pastoris. A total supernatant protein content of 0.174 g/L of high density fermentation broth was obtained. The rPI239 exhibited in vitro fibrinolytic activity. The in vivo activity of rPI239 was measured by prothrombin time, kaolin part thrombin time, thrombin time, and fibrinolytic activity. This work presents the high-density fermentation of rPI239 from P. pastoris and shows that the recombinant protein has similar fibrinolytic activity both in vivo and in vitro.

Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference.

RNA interference (RNAi) is an ancient antiviral response that processes dsRNA and associates it into a nuclease complex that identifies RNA with sequence homology and specifically cleaves it. We demonstrate that RNAi mediated by 21-bp dsRNA specifically inhibits HIV-1 infection of permanent cell lines and primary CD4(+) T cells. Inhibition of HIV replication was measured by p24 Gag protein content in supernatant, Northern blot analysis, and DNA PCR for products of reverse transcription. The inhibition occurred at two points in the viral life cycle, after fusion and before reverse transcription and during transcription of viral RNA from integrated provirus. Treatment of HIV-infected activated CD4(+) T cells with a fluorine-derivatized siRNA that is resistant to RNase A yielded similar inhibition of HIV infection. In addition, the derivatized siRNA could be delivered without lipofectin complexing and in the presence of serum. The identification of RNAi activity against HIV-1 presents a new approach to study viral infections and a proof of concept of RNAi antiviral activity in mammalian cells.

Measurement of fibrinolytic components in human tissue

The fibrinolytic system is involved in the resolution of thrombi and in tissue repair. Quantitation of the activators and inhibitors of this system at tissue level is crucial to further characterise these processes. Hitherto, there have been difficulties in measuring the individual components of the plasmin system in human vascular and peritoneal tissue. The aim of this study was to develop a protocol allowing quantitation of activators and inhibitors of plasmin generation at the tissue level. Following a strict protocol in the processing of tissue, the efficiency of extraction in tissue homogenisates was compared using buffers containing acetic acid, 1% Triton X-100 and thiocyanate. The influence of different modalities of normalisation was investigated by normalising to wet weight, total protein content, protein content in supernatant or DNA. Using the protocol, all buffers extracted components of the plasmin system sufficiently for detection. The acetic acid buffer yielded the greatest amount of protein, and in extracting plasminogen activators was comparable to the thiocyanate buffer and significantly more efficient than the Triton buffer (p<0.05). The relationship between the individual components was unaltered by different means of normalisation. The protocol described, using an acetic acid buffer and normalising to wet weight, seems to be a simple and efficient technique for measuring components of the fibrinolytic system, at least in the tissues investigated.

Dose-related effects of Ampligen (poly(I).poly(C12U)), a mismatched double-stranded RNA, in a bleomycin-mouse model of pulmonary fibrosis.

The antifibrotic effect of the mismatched double-stranded RNA, Ampligen (poly(I).poly(C12U)), was evaluated in a bleomycin-mouse model of pulmonary fibrosis. Mice received a single intratracheal dose of bleomycin (0.125 U/mouse) or saline (50 microL) at the beginning of the experiment, followed by 5 or 6 intraperitoneal injections of Ampligen (1.0, 5.0, 10.0, 15.0, or 25.0 mg/kg) or saline at regular intervals for 2 weeks. Ampligen did not produce increased mortality or weight loss by itself. However, it produced varying degrees of mortality in combination with bleomycin. Five injections of 10 mg/kg Ampligen or three injections of 25 mg/kg Ampligen plus three injections of 10 mg/kg Ampligen in combination with bleomycin .produced significant reductions in lung collagen accumulation as indicated by lung hydroxyproline content compared to the bleomycin control group. Animals receiving bleomycin plus Ampligen at all dosages had significantly reduced prolyl hydroxylase activity compared to the bleomycin control group. Lipid peroxidation and bronchoalveolar lavage fluid (BALF)-supernatant protein content for the groups receiving bleomycin plus Ampligen were not reduced compared to the bleomycin control group. In the BALF-supernatant, the activity of acid phosphatase, a lysosomal enzyme produced by neutrophils, monocytes, and macrophages, was significantly decreased in the group receiving bleomycin plus 10 mg/kg Ampligen. Also, selected BALF differential immune cell counts were reduced in some of the groups receiving bleomycin plus Ampligen, but not in a consistent or dose-dependent manner. The results of this study indicate that Ampligen can significantly reduce the bleomycin-induced increased collagen accumulation and may be therapeutically useful in the management of lung fibrosis in humans.

Effects of an interferon inducer bropirimine on bleomycin-induced lung fibrosis in hamsters.

The antifibrotic effect of an interferon inducer, bropirimine (2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone, ABPP) was evaluated in bleomycin (BLM)-hamster model of lung fibrosis. ABPP is an orally active biological response modifier and has immunomodulatory, antiviral, and antineoplastic activities. The hamsters were randomized in four groups and treated with either bropirimine (100 mg/kg, intraperitoneally) suspended in carboxymethylcellulose (CMC, 10 mg/10 mg/ml) or CMC alone each day for sixteen days. After two days, the hamsters received either single bolus of BLM (7.5 U/5 ml/kg) or an equivalent volume of saline by the transoral endotracheal route. Groupings were assigned as: CMC + saline (CS), ABPP + saline (AS), CMC + BLM (CB) and ABPP + BLM (AB). Animals were sacrificed at fourteen days after intratracheal installation of either BLM or saline. Their lungs were lavaged and processed for morphometric and biochemical studies. ABPP had little effect in preventing BLM-induced weight loss and lung injury. ABPP was found to reduce the BLM-induced accumulation of collagen in the lung as measured by hydroxyproline content. The hamsters in AB group had significantly less collagen than the hamsters in CB group: 995 and 1157 micrograms hydroxyproline/lung, respectively. Administration of ABPP prevented the BLM-induced increase in the lung prolyl hydroxylase activity. The total number of monocytes and eosinophils recovered from the bronchoalveolar lavage fluid (BALF) of the AB group were significantly lower than that of the animals in CB group. However, the BALF supernatant protein content from animals in AB group (7.9 mg/lung) was significantly higher than that of CB group (4.5 mg/lung).(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental Protein Malnutrition Decreases Calcium-Binding Protein in Rat Intestinal Mucosa

Calcium-binding activity was measured in duodenal mucosal homogenates of rats 50 days after weaning onto a protein-deficient diet providing 3–4 g of protein per kilogram of body weight per day compared to control animals, who were fed an isocaloric diet providing 9–12 g of protein per kilogram of body weight per day. Calcium-binding activity was decreased (44% of control) further than can be explained by the decrease in intestinal mucosal weight (70% of control) or supernatant protein content (80% of control). The results suggest that the decrease in calcium-binding activity reflects decreased synthesis of the vitamin D-dependent calcium-binding protein (CaBP) as an adaptive response to the stunted growth associated with protein malnutrition.intestinal calcium-binding protein calcium absorption growth protein malnutrition

Differential Centrifugation of Peanut and Soybean Protein Concentrates as Influenced by Preparation Technique and Heat Treatment1

Abstract Protein extracts prepared from defatted Florunner peanuts and Cobb soybeans were dried by spray-, freeze- and drum-drying techniques. Differential centrifugation was performed on 1.0% protein dispersions at 2,000 and 40,000 x g for 20 min at 24°C and at 200,000 × g for 1 hr at 15°C Lowest supernatant protein content under all centrifugation conditions studied was observed in drum-dried protein preparations. Increased centrifugal force from 2,000 to 40,000 × g only slightly affected supernatant protein in freeze-dried and spray-dried oilseed preparations and did not affect drum-dried preparations. Heat treatment (70°C for 30 min) of the protein dispersions increased supernatant protein (2,000 and 40,000 × g) for spray-and drum-dried peanut, and for all soy protein preparations. Heat treatment only slightly affected ultracentrifuge supernatant protein. Sepharose 6B gel filtration indicated four major fractions in spray- and freeze-dried peanut preparations and three major fractions in spray- and freeze-dried soy protein preparations.

The effect of propoxur on rats fed diets differing in protein content

In 6-week-old Wistar rats fed for 30 and 93 days either 4.5 or 26% casein diets containing 0, 500, 1500, 4500, and 9000 ppm of propoxur several biological changes were found which were more pronounced when propoxur was added to low-protein diet for 93 days, particularly in concentrations of 4500 and 9000 ppm. The changes were as follows. (1) In the liver—decreased activity of aromatic amino acids aminotransferases (AAA) and β-glucuronidase (β-GR) as well as diminished protein content in supernatant; unchanged activity of aspartate aminotransferase (AspAT), glucosephosphate isomerase (PHI) and fructosediphosphate aldolase (ALD). (2) In the serum—decreased activity of acetylcholinesterase (AChE) as well as β-GR and increased PHI activity; unaltered activity of ALD and sorbitol dehydrogenase, (SDH). (3) In the brain—decreased activity of AChE, PHI, ALD, AAA, reduced protein content in supernatant; unchanged activity of AspAT and biogenic amines level. (4) Decreased rate of rat body weight gain on contaminated normoprotein diet and weight loss on contaminated low-protein diet. (5) Changes in the relative weight of certain internal organs; no detectable anatomical changes in the examined organs. The acute oral toxicity of propoxur was definitely enhanced by the previous feeding of low-protein diet: 1.7 times in males and 1.3 times in females after 30 days, and after 93 days 4.3 and 2.7 times, respectively.

Developmental Pattern of Hepatic Uridine Diphosphoglucuronosyl Transferase Activity in Growing Male Rats

Hepatic uridine diphospho(UDP)glucuronosyl transferase activity was measured in growing male rats to gain the developmental activity pattern of this enzyme catalyzing conjugation of xenobiotics and endogenous substrates. The activity was at a very low level at the age of 14 days showing a 10-fold increase to the age of 72 days whereafter a slight decrease of activity was observed. Simultaneously there was about 30% increase in the hepatic postmitochondrial supernatant protein content. The highly enhanced UDPglucuronosyl transferase activity during the growth may be caused by increased synthesis of enzyme protein or by changes in the micro-environment of the enzyme.