Proteomic characterization and cytotoxic potential of proteins from Cuscuta (Cuscuta epithymum (L.) crude herbal product against MCF-7 human breast cancer cell line.

BACKGROUND: The burden of breast cancer, the second leading cause of death worldwide, is increasing at an alarming rate. Cuscuta, used in traditional medicine for different ailments, including cancer, is known for containing phytochemicals that exhibit anticancer activity; however, the bioactivities of proteins from this plant remain unexplored. This study aimed to screen the cytotoxic potential of proteins from the crude herbal product of Cuscuta epithymum(L.) (CE) harvested from the host plants Alhagi maurorum and Medicago sativa. METHODS: The proteins from CE were extracted using a salting-out method, followed by fractionation with a gel filtration chromatography column. Gel-free shotgun proteomics was subsequently performed for protein characterization. The viability assay using MTT was applied to deduce the cytotoxic potential of proteins against MCF-7 breast cancer cells, with further exploration of the effect of treatment on the expression of the apoptotic mediator BCL2-associated X protein (BAX) and B-cell lymphoma protein 2 (BCL-2) proteins, using western blotting to strengthen the findings from the in vitro viability assay. RESULTS: The crude proteins (CP) of CE were separated into four protein peaks (P1, P2, P3, and P4) by gel filtration chromatography. The evaluation of potency showed a dose-dependent decline in the MCF-7 cell line after CP, P1, P2, and P3 treatment with the respective IC50 values of 33.8, 43.1, 34.5, and 28.6 µg/ml. The percent viability of the cells decreased significantly upon treatment with 50 µg/ml CP, P1, P2, and P3 (P < 0.001). Western-blot analysis revealed upregulation of proapoptotic protein BAX in the cells treated with CP, P3 (P < 0.01), and P2 (P < 0.05); however, the antiapoptotic protein, BCL-2 was downregulated in the cells treated with CP and P3 (P < 0.01), but no significant change was detected in P2 treated cells. The observed cytotoxic effects of proteins in the CP, P1, P2, and P3 from the in vitro viability assay and western blot depicted the bioactivity potential of CE proteins. The database search revealed the identities of functionally important proteins, including nonspecific lipid transfer protein, superoxide dismutase, carboxypeptidase, RNase H domain containing protein, and polyribonucleotide nucleotidyltransferase, which have been previously reported from other plants to exhibit anticancer activity. CONCLUSION: This study indicated the cytotoxic activity of Cuscuta proteins against breast cancer MCF-7 cells and will be utilized for future investigations on the mechanistic effect of active proteins. The survey of CE proteins provided substantial data to encourage further exploration of biological activities exhibited by proteins in Cuscuta.

Isolation, Virtual Screening, and Evaluation of Hazelnut-Derived Immunoactive Peptides for the Inhibition of SARS-CoV-2 Main Protease.

The aim of this study is to validate the activity of hazelnut (Corylus avellana L.)-derived immunoactive peptides inhibiting the main protease (Mpro) of SARS-CoV-2 and further unveil their interaction mechanism using in vitro assays, molecular dynamics (MD) simulations, and binding free energy calculations. In general, the enzymatic hydrolysis components, especially molecular weight < 3 kDa, possess good immune activity as measured by the proliferation ability of mouse splenic lymphocytes and phagocytic activity of mouse peritoneal macrophages. Over 866 unique peptide sequences were isolated, purified, and then identified by nanohigh-performance liquid chromatography/tandem mass spectrometry (NANO-HPLC-MS/MS) from hazelnut protein hydrolysates, but Trp-Trp-Asn-Leu-Asn (WWNLN) and Trp-Ala-Val-Leu-Lys (WAVLK) in particular are found to increase the cell viability and phagocytic capacity of RAW264.7 macrophages as well as promote the secretion of the cytokines nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß). Fluorescence resonance energy transfer assay elucidated that WWNLN and WAVLK exhibit excellent inhibitory potency against Mpro, with IC50 values of 6.695 and 16.750 µM, respectively. Classical all-atom MD simulations show that hydrogen bonds play a pivotal role in stabilizing the complex conformation and protein-peptide interaction. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) calculation indicates that WWNLN has a lower binding free energy with Mpro than WAVLK. Furthermore, adsorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions illustrate favorable drug-likeness and pharmacokinetic properties of WWNLN compared to WAVLK. This study provides a new understanding of the immunomodulatory activity of hazelnut hydrolysates and sheds light on peptide inhibitors targeting Mpro.

Efficacy of maturase K and rpL20 protein extracted from C. procera leaves on Anophelesstephensi.

The present work is carried out to protein isolation, purification, and characterization from leaves, stem, and seed of C. procera and to evaluate the larvicidal potential on Anopheles stephensi. The whole protein was isolated using protein extraction buffer and precipitated by ammonium sulphate and larvicidal active protein was purified by the column chromatography. The homogeneity of larvicidal protein was confirmed by the SDS-PAGE. The identification of protein was done by the HPLC and LC-MS/ESI-MS. The crude protein from leaves showed 100% mortality of 3rd instar larvae of An. stephensi at the concentration of 5.5 mg/ml after 24 h of exposure. The crude protein from stem showed 25% mortality and no mortality observed was observed in seed protein. The leaves crude protein was further purified by ion exchange chromatography and eluted fractions were tested for larvicidal potential. The purified single protein fractions L2 and L3 from C. procera leaves showed 100% mortality at concentration of 0.06 mg/ml. The homogeneity of purified protein was confirmed by SDS-PAGE and two bands of 26 kDa and 15 kDa protein were observed. The peptide sequence "R.SQMLENSFLIENVMKR.L" was identified in the trypsin digested homogenous protein fraction L2 and "R.DRGSQKR.N" peptide sequence in L3 fraction by LC-MS/ESI-MS. The CprL2 peptide showed the sequence similarity with the protein maturase K and CprL3 peptide showed the sequence similarity with ribosomal protein L20 of C. procera. The conserved functional domain was also identified in both the CprL2 and CprL3 peptide. The identified proteins showed strong larvicidal efficacy at very low concentration. The identified proteins are novel and natural larvicidal agents against An. stephensi and hence can be used to control the malaria.

The antioxidant peptides from walnut protein hydrolysates and their protective activity against alcoholic injury.

In this study, walnut protein was hydrolyzed, separated by ultrafiltration, purified by RP-HPLC, identified by LC-MS/MS, and screened by molecular docking to finally obtain three novel antioxidant peptides HGEPGQQQR (1189.584 Da), VAPFPEVFGK (1089.586 Da) and HNVADPQR (949.473 Da). These three peptides exhibited excellent cellular antioxidant activity (CAA) with EC50 values of 0.0120 mg mL-1, 0.0068 mg mL-1, and 0.0069 mg mL-1, respectively, which were superior to that of the positive control GSH (EC50: 0.0122 mg mL-1). In the ethanol injury model, three antioxidant peptides enhanced the survival of cells treated with ethanol from 47.36% to 62.69%, 57.06% and 71.64%, respectively. Molecular docking results showed that the three antioxidant peptides could effectively bind to Keap1, CYP2E1 and TLR4 proteins. These results suggested that walnut-derived antioxidant peptides could be potential antioxidants and hepatoprotective agents for application in functional foods.

Antifungal Activity of Ribosome-Inactivating Proteins.

The control of crop diseases caused by fungi remains a major problem and there is a need to find effective fungicides that are environmentally friendly. Plants are an excellent source for this purpose because they have developed defense mechanisms to cope with fungal infections. Among the plant proteins that play a role in defense are ribosome-inactivating proteins (RIPs), enzymes obtained mainly from angiosperms that, in addition to inactivating ribosomes, have been studied as antiviral, fungicidal, and insecticidal proteins. In this review, we summarize and discuss the potential use of RIPs (and other proteins with similar activity) as antifungal agents, with special emphasis on RIP/fungus specificity, possible mechanisms of antifungal action, and the use of RIP genes to obtain fungus-resistant transgenic plants. It also highlights the fact that these proteins also have antiviral and insecticidal activity, which makes them very versatile tools for crop protection.

Expression and Purification of Recombinant Bowman-Birk Trypsin Inhibitor from Foxtail Millet Bran and Its Anticolorectal Cancer Effect In Vitro and In Vivo.

Trypsin inhibitors derived from plants have various pharmacological activities and promising clinical applications. In our previous study, a Bowman-Birk-type major trypsin inhibitor from foxtail millet bran (FMB-BBTI) was extracted with antiatherosclerotic activity. Currently, we found that FMB-BBTI possesses a prominent anticolorectal cancer (anti-CRC) activity. Further, a recombinant FMB-BBTI (rFMB-BBTI) was successfully expressed in a soluble manner in host strain Escherichia coli. BL21 (DE3) was induced by isopropyl-ß-d-thiogalactoside (0.1 mM) at 37 °C for 3.5 h by the pET28a vector system. Fortunately, a purity greater than 93% of rFMB-BBTI with anti-CRC activity was purified by nickel-nitrilotriacetic acid affinity chromatography. Subsequently, we found that rFMB-BBTI displays a strikingly anti-CRC effect, characterized by the inhibition of cell proliferation and clone formation ability, cell cycle arrest at the G2/M phase, and induction of cell apoptosis. It is interesting that the rFMB-BBTI treatment had no obvious effect on normal colorectal cells in the same concentration range. Importantly, the anti-CRC activity of rFMB-BBTI was further confirmed in the xenografted nude mice model. Taken together, our study highlights the anti-CRC activity of rFMB-BBTI in vitro and in vivo, uncovering the clinical potential of rFMB-BBTI as a targeted agent for CRC in the future.

Litchi pulp-derived gamma-aminobutyric acid (GABA) extract counteracts liver inflammation induced by litchi thaumatin-like protein.

Gamma-aminobutyric acid (GABA) is the predominant amino acid in litchi pulp, known for its neuroregulatory effects and anti-inflammatory properties. Although previous research has highlighted the pro-inflammatory characteristics of litchi thaumatin-like protein (LcTLP), interplay between GABA and LcTLP in relation to inflammation remains unclear. This study aims to explore the hepatoprotective effects of the litchi pulp-derived GABA extract (LGE) against LcTLP-induced liver inflammation in mice and LO2 cells. In vivo experiments demonstrated that LGE significantly reduced the levels of aspartate transaminase and alanine transaminase, and protected the liver against infiltration of CD4+ and CD8+ T cells and histological injury induced by LcTLP. Pro-inflammatory cytokines including interleukin-6, interleukin-1ß, and tumor necrosis factor-α were also diminished by LGE. The LGE appeared to modulate the mitogen-activated protein kinase (MAPK) signaling pathway to exert its anti-inflammatory effects, as evidenced by a reduction of 47%, 35%, and 31% in phosphorylated p38, JNK, and ERK expressions, respectively, in the liver of the high-dose LGE group. Additionally, LGE effectively improved the translocation of gut microbiota by modulating its microbiological composition and abundance. In vitro studies have shown that LGE effectively counteracts the increase in reactive oxygen species, calcium ions, and pro-inflammatory cytokines induced by LcTLP. These findings may offer new perspectives on the health benefits and safety of litchi consumption.

A novel angiotensin I-converting enzyme inhibitory peptide from walnut (Juglans sigillata) protein hydrolysates and its evaluation in Ang II-induced HUVECs and hypertensive rats.

This study aims to seek angiotensin-I-converting enzyme inhibitory (ACEi) peptides from walnut using different enzymatic hydrolysis, and further to validate the potent ACEi peptides identified and screened via peptidomics and in silico analysis against hypertension in spontaneously hypertensive rats (SHRs). Results showed that walnut protein hydrolysate (WPH) prepared by combination of alcalase and simulated gastrointestinal digestion exhibited high ACEi activity. WPH was separated via Sephadex-G25, and four peptides were identified, screened and verified based on their PeptideRanker score, structural characteristic and ACE inhibition. Interestingly, FDWLR showed the highest ACEi activity with IC50 value of 8.02 µg/mL, which might be related to its close affinity with ACE observed in molecular docking. Subsequently, high absorption and non-toxicity of FDWLR was predicted via in silico absorption, distribution, metabolism, excretion and toxicity. Furthermore, FDWLR exhibited positively vasoregulation in Ang II-induced human umbilical vein endothelial cells, and great blood pressure lowering effect in SHRs.

A Novel Trypsin Kunitz-Type Inhibitor from Cajanus cajan Leaves and Its Inhibitory Activity on New Cancer Serine Proteases and Its Effect on Tumor Cell Growth.

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 µM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and ß-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

Discovery of Transfer Factors in Plant-Derived Proteins and an In Vitro Assessment of Their Immunological Activities.

Repeated exposure to pathogens leads to evolutionary selection of adaptive traits. Many species transfer immunological memory to their offspring to counteract future immune challenges. Transfer factors such as those found in the colostrum are among the many mechanisms where transfer of immunologic memory from one generation to the next can be achieved for an enhanced immune response. Here, a library of 100 plants with high protein contents was screened to find plant-based proteins that behave like a transfer factor moiety to boost human immunity. Aqueous extracts from candidate plants were tested in a human peripheral blood mononuclear cell (PBMC) cytotoxicity assay using human cancerous lymphoblast cells-with K562 cells as a target and natural killer cells as an effector. Plant extracts that caused PBMCs to exhibit enhanced killing beyond the capability of the colostrum-based transfer factor were considered hits. Primary screening yielded an 11% hit rate. The protein contents of these hits were tested via a Bradford assay and Coomassie-stained SDS-PAGE, where three extracts were confirmed to have high protein contents. Plants with high protein contents underwent C18 column fractionation using methanol gradients followed by membrane ultrafiltration to isolate protein fractions with molecular weights of <3 kDa, 3-30 kDa, and >30 kDa. It was found that the 3-30 kDa and >30 kDa fractions had high activity in the PBMC cytotoxicity assay. The 3-30 kDa ultrafiltrates from the top two hits, seeds from Raphanus sativus and Brassica juncea, were then selected for protein identification by mass spectrometry. The majority of the proteins in the fractions were found to be seed storage proteins, with a low abundance of proteins involved in plant defense and stress response. These findings suggest that Raphanus sativus or Brassica juncea extracts could be considered for further characterization and immune functional exploration with a possibility of supplemental use to bolster recipients' immune response.

High Plant Protein Diet Ameliorated Hepatic Lipid Accumulation Through the Modulation of Gut Microbiota.

SCOPE: Substituting plant protein for animal protein has emerged as a promising strategy for managing atherogenic lipids. However, the impact of long-term intake of a high plant protein diet (HPD) on hepatic lipid disorder remains unclear. METHODS AND RESULTS: Eight-week-old apolipoprotein E deficient (apoE-/- ) mice are fed with either a normal protein diet (NCD) or HPD for 12 weeks. HPD intervention results in decreased body weight accompanied by increased energy expenditure, with no significant effect on glycemic control. Long-term intake of HPD improves the serum and hepatic lipid and cholesterol accumulation by suppressing hepatic squalene epoxidase (SQLE) expression, a key enzyme in cholesterol biosynthesis. Integrated analysis of 16S rDNA sequencing and metabolomics profiling reveals that HPD intervention increases the abundance of the Lachnospiraece family and serum levels of 12,13-DiHOME. Furthermore, in vivo studies demonstrate that 12,13-DiHOME significantly inhibits lipid accumulation, as well as SQLE expression induced by oleic acid in HepG2 cells. CONCLUSION: Diet rich in plant protein diet alleviates hyperlipidemia via increased microbial production of 12,13-DiHOME.

Effects of a Peptide Derived from the Primary Sequence of a Kallikrein Inhibitor Isolated from Bauhinia bauhinioides (pep-BbKI) in an Asthma-COPD Overlap (ACO) Model.

(1) There are several patients with asthma-COPD overlap (ACO). A peptide derived from the primary sequence of a kallikrein inhibitor isolated from Bauhinia bauhinioides (pep-BbKI) has potent anti-inflammatory and antioxidant effects. Purpose: To investigate the effects of pep-BbKI treatment in an ACO model and compare them with those of corticosteroids. (2) BALB/c mice were divided into groups: SAL (saline), OVA (ovalbumin), ELA (elastase), ACO (ovalbumin + elastase), ACO-pep-BbKI (treated with inhibitor), ACO-DX (dexamethasone treatment), ACO-DX-pep-BbKI (both treatments), and SAL-pep-BbKI (saline group treated with inhibitor). We evaluated: hyperresponsiveness to methacholine, bronchoalveolar lavage fluid (BALF), exhaled nitric oxide (eNO), IL-1ß, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IFN-γ, TNF-α, MMP-9, MMP-12, TGF-ß, collagen fibers, iNOS, eNO, linear mean intercept (Lm), and NF-κB in airways (AW) and alveolar septa (AS). (3) ACO-pep-BbKI reversed ACO alterations and was similar to SAL in all mechanical parameters, Lm, neutrophils, IL-5, IL-10, IL-17, IFN-γ, TNF-α, MMP-12 (AW), collagen fibers, iNOS (AW), and eNO (p > 0.05). ACO-DX reversed ACO alterations and was similar to SAL in all mechanical parameters, Lm, total cells and differentials, IL-1ß(AS), IL-5 (AS), IL-6 (AS), IL-10 (AS), IL-13 (AS), IFN-γ, MMP-12 (AS), TGF-ß (AS), collagen fibers (AW), iNOS, and eNO (p > 0.05). SAL was similar to SAL-pep-BbKI for all comparisons (p > 0.05). (4) Pep-BbKI was similar to dexamethasone in reducing the majority of alterations of this ACO model.

Muscle Protein Synthesis with a Hybrid Dairy and Plant-Based Protein Blend (P4) Is Equal to Whey Protein in a Murine Ageing Model after Fasting.

P4, a specific combination of dairy proteins (whey and casein) and plant-based protein isolates (pea and soy), has been shown to provide a more balanced amino acid (AA) profile than its single constituent proteins; however, less is known about how this translates to muscle protein synthesis (MPS). The aim of this study was to investigate the effect of P4 compared to whey or casein against fasted control on MPS. C57BL/6J mice, aged 25 months, were fasted overnight, followed by oral gavage of either whey, P4, casein, or water as a fasted control. Thirty minutes after ingestion, puromycin (0.04 µmol∙g-1 bodyweight) was subcutaneously injected; 30-min thereafter, mice were sacrificed. MPS was measured by the SUnSET method, and signalling proteins were determined in the left-tibialis anterior (TA) muscle by the WES technique. AA composition was determined in plasma and right-TA muscle. Dried blood spots (DBS) were analysed for postprandial AA dynamics at 10, 20, 45, 60 min. MPS was 1.6-fold increased with whey (p = 0.006) and 1.5-fold with P4 compared to fasted (p = 0.008), while no change was seen with casein. This was confirmed by a significant increase of phosphorylated/total ratio of 4E-BP1 for both whey (p = 0.012) and P4 (p = 0.001). No changes were observed in p70S6K and mTOR phosphorylation/total ratio with whey or P4. Intramuscular leucine levels were lower for P4 (0.71 µmol∙g dry weight-1) compared to whey (0.97 µmol∙g dry weight-1) (p = 0.0007). Ten minutes postprandial, DBS showed significantly increased blood AA levels of BCAAs, histidine, lysine, threonine, arginine, and tyrosine for P4 versus fasted. In conclusion, a hybrid mix of dairy and plant-based proteins (P4) resulted in a MPS response that was similar to whey protein in aged mice after fasting. This suggests that other anabolic triggers beyond leucine or the well-balanced amino acid profile and bioavailability of the blend benefit stimulation of MPS.

Peptidomic Analysis and Antimicrobial Activity of Serum Peptide from Hevea brasiliensis Clone BPM24.

BACKGROUND: Hevea brasiliensis is severely affected by the fungal disease caused by Phytophthora spp. Significant loss of rubber yield is widespread and extensive use of chemical fungicides has resulted in health and environmental problems. OBJECTIVE: This work aims to extract and identify the latex serum peptides from a disease tolerant clone of H. brasiliensis, and study the inhibitory efficacy against pathogenic bacteria and fungi. METHODS: Serum peptides were extracted from H. brasiliensis BPM24 using mixed lysis solution. Low molecular weight peptides were screened and fractionated by solid-phase extraction and then identified by tandem mass spectrometry. Total and fractionated serum peptides were assayed for bacterial and fungal inhibition using broth microdilution and poisoned food methods. An inhibitory control study in the greenhouse was also performed using susceptible clones for pre and postinfection with Phytophthora spp. RESULTS: Forty-three serum peptide sequences were successfully identified. Thirty-four peptides matched with the proteins associated with plant defense response signaling, host resistance, and adverse environmental factors. The inhibitory study of total serum peptides demonstrated antibacterial and anti-fungal properties. The greenhouse study exhibited disease inhibitory efficacy of 60% for the treatment of Phytophthora spp. in post-infected plants and 80% for pre-treated samples. CONCLUSION: Latex serum peptides from disease tolerant H. brasiliensis revealed several proteins and peptides associated with plant defense and disease resistance. The peptides play a vital role for defense against bacteria and fungi pathogens, including Phytophthora spp. Enhanced disease protection can be obtained when the extracted peptides were applied to the susceptible plants before exposure to the fungi. These findings provided an insight and may pave the way for the development of biocontrol peptides from natural resources.

Streptavidin-Saporin: Converting Biotinylated Materials into Targeted Toxins.

Streptavidin-Saporin can be considered a type of 'secondary' targeted toxin. The scientific community has taken advantage of this conjugate in clever and fruitful ways using many kinds of biotinylated targeting agents to send saporin into a cell selected for elimination. Saporin is a ribosome-inactivating protein that causes inhibition of protein synthesis and cell death when delivered inside a cell. Streptavidin-Saporin, mixed with biotinylated molecules to cell surface markers, results in powerful conjugates that are used both in vitro and in vivo for behavior and disease research. Streptavidin-Saporin harnesses the 'Molecular Surgery' capability of saporin, creating a modular arsenal of targeted toxins used in applications ranging from the screening of potential therapeutics to behavioral studies and animal models. The reagent has become a well-published and validated resource in academia and industry. The ease of use and diverse functionality of Streptavidin-Saporin continues to have a significant impact on the life science industry.

Walnut-Derived Peptides Promote Autophagy via the Activation of AMPK/mTOR/ULK1 Pathway to Ameliorate Hyperglycemia in Type 2 Diabetic Mice.

Autophagy flux plays a significant protective role in type 2 diabetes mellitus (T2DM). However, the mechanisms by which autophagy mediates insulin resistance (IR) to ameliorate T2DM remain unclear. This study explored the hypoglycemic effects and mechanisms of walnut-derived peptides (fraction 3-10 kDa and LP5) in streptozotocin and high-fat-diet-induced T2DM mice. Findings revealed that walnut-derived peptides reduced the levels of blood glucose and FINS and ameliorated IR and dyslipidemia. They also increased SOD and GSH-PX activities and inhibited the secretion of TNF-α, IL-6, and IL-1ß. Additionally, they increased the levels of ATP, COX, SDH, and MMP of liver mitochondria. Western blotting indicated that walnut-derived peptides up-regulated LC3-II/LC3-I and Beclin-1 expression, while they down-regulated p62 expression, which may be associated with the activation of the AMPK/mTOR/ULK1 pathway. Finally, the AMPK activator (AICAR) and inhibitor (Compound C) were used to verify that LP5 could activate autophagy through the AMPK/mTOR/ULK1 pathway in IR HepG2 cells.

Expression profiling of anticancer genes in colorectal cancer patients and their in vitro induction by riproximin, a ribosomal inactivating plant protein.

BACKGROUND: Ectopic expression of anticancer genes (ACGs) imposes antineoplastic effects on transformed cells. Clinically, reduced expression of these genes has been linked with poor prognosis, metastasis and chemo/radiotherapy resistance in cancers. Identifying expression pattern of ACGs is crucial to establish their prognostic and therapeutic relevance in colorectal cancer (CRC). In addition to the clinical perspective, naturally occurring compounds can be explored in parallel for inducing ACGs to achieve cancer cell-specific death. METHODOLOGY: Expression profiles of three ACGs (NOXA, PAR-4, TRAIL) were identified via real-time PCR in CRC clinical isolates. Time lapse-based expression modifications in ACGs were studied in a CRC liver metastasis animal model using microarray methodology. Effects of a purified plant protein (riproximin) on selected ACGs were identified in three primary and metastatic CRC cell lines by real-time PCR. Lastly, importance of the ACGs in a cellular environment was highlighted via bioinformatic analysis. RESULTS: ACGs (except NOXA) were persistently downregulated in clinical isolates when comparing the overall mean expression values with normal mucosa levels. In vivo studies showed a prominent inhibition of NOXA and PAR-4 genes in implanted CRC cells during rat liver colonization. TRAIL showed deviation from this theme while showing marked induction during the early period of liver colonization (days 3 and 6 after CRC cell implantation). Riproximin exhibited substantial potential of inducing ACGs at transcriptome levels in selected CRC cell lines. Bioinformatic analysis showed that vital molecular/functional aspects of a cell are associated with the presence of ACGs. CONCLUSION: ACGs are downregulated in primary and metastatic phase of CRC. Riproximin effectively induces ACGs in CRC cells and can be exploited for clinical investigations over time.

The heat-stable protein fraction from Opuntia ficus-indica seeds exhibits an enzyme protective effect against thermal denaturation and an antibacterial activity.

Desiccation tolerance in developing seeds occurs through several mechanisms among which, a common group of proteins named dehydrins has received considerable attention. So far, there is no information dealing with the accumulation of dehydrins in seeds of Opuntia ficus-indica. We have initiated here an extraction protocol based on two critical steps: heat and acid treatments, and the purity of this fraction was analyzed by FTIR spectroscopy. Western blot analysis of the heat-stable protein fraction (HSF) revealed two main bands of approximately 45 and 44 kDa, while three others of ∼40, 32, and 31 kDa were faintly visible, which were recognized by anti-dehydrin antibodies. This fraction exhibited a Cu2+ -dependent resistance to protease treatments. Next, we performed a series of assays to compare the functional properties of the HSF with those of the previously characterized wheat dehydrin (DHN-5). Antibacterial assays revealed that HSF exhibits only moderate antibacterial activities against gram-negative and gram-positive bacteria, with a minimum inhibition concentration ranging from 0.25 to 1 mg/ml. However, in vitro assays revealed that compared to DHN-5, HSF exhibits higher protective activities of the lactate dehydrogenase (LDH) when exposed to heat, freezing, and dehydration stresses. The protective role of HSF seems to be linked to its best ability to minimize protein aggregation.

Protein intake and bone mineral density: Cross-sectional relationship and longitudinal effects in older adults.

BACKGROUND: There are several mechanisms via which increased protein intake might maintain or improve bone mineral density (BMD), but current evidence for an association or effect is inconclusive. The objectives of this study were to investigate the association between dietary protein intake (total, plant and animal) with BMD (spine and total body) and the effects of protein supplementation on BMD. METHODS: Individual data from four trials that included either (pre-)frail, undernourished or healthy older adults (aged ≥65 years) were combined. Dietary intake was assessed with food records (2, 3 or 7 days) and BMD with dual-energy X-ray absorptiometry (DXA). Associations and effects were assessed by adjusted linear mixed models. RESULTS: A total of 1570 participants [57% women, median (inter-quartile range): age 71 (68-75) years] for which at least total protein intake and total body BMD were known were included in cross-sectional analyses. In fully adjusted models, total protein intake was associated with higher total body and spine BMD [beta (95% confidence interval): 0.0011 (0.0006-0.0015) and 0.0015 (0.0007-0.0023) g/cm2 , respectively]. Animal protein intake was associated with higher total body and spine BMD as well [0.0011 (0.0007-0.0016) and 0.0017 (0.0010-0.0024) g/cm2 , respectively]. Plant protein intake was associated with a lower total body and spine BMD [-0.0010 (-0.0020 to -0.0001) and -0.0019 (-0.0034 to -0.0004) g/cm2 , respectively]. Associations were similar between sexes. Participants with a high ratio of animal to plant protein intake had higher BMD. In participants with an adequate calcium intake and sufficient serum 25(OH)D concentrations, the association between total protein intake with total body and spine BMD became stronger. Likewise, the association between animal protein intake with total body BMD was stronger. In the longitudinal analyses, 340 participants [58% women, median (inter-quartile range): age 75 (70-81) years] were included. Interventions of 12 or 24 weeks with protein supplementation or protein supplementation combined with resistance exercise did not lead to significant improvements in BMD. CONCLUSIONS: An association between total and animal protein intake with higher BMD was found. In contrast, plant protein intake was associated with lower BMD. Research is warranted to further investigate the added value of dietary protein alongside calcium and vitamin D for BMD improvement, especially in osteopenic or osteoporotic individuals. Moreover, more research on the impact of a plant-based diet on bone health is needed.

Plant-Derived Proteins and Peptides as Potential Immunomodulators.

The immune response of humans may be modulated by certain biopeptides. The present study aimed to determine the immunomodulatory potential of plant-derived food proteins and hydrolysates obtained from these proteins via monocatalytic in silico hydrolysis (using ficin, stem bromelainm or pepsin (pH > 2)). The scope of this study included determinations of the profiles of select bioactivities of proteins before and after hydrolysis and computations of the frequency of occurrence of selected bioactive fragments in proteins (parameter A), frequency/relative frequency of the release of biopeptides (parameters AE, W) and the theoretical degree of hydrolysis (DHt), by means of the resources and programs available in the BIOPEP-UWM database. The immunomodulating (ImmD)/immunostimulating (ImmS) peptides deposited in the database were characterized as well (ProtParam tool). Among the analyzed proteins of cereals and legumes, the best precursors of ImmD immunopeptides (YG, YGG, GLF, TPRK) turned out to be rice and garden pea proteins, whereas the best precursors of ImmS peptides appeared to be buckwheat (GVM, GFL, EAE) and broad bean (LLY, EAE) proteins. The highest number of YG sequences was released by stem bromelain upon the simulated hydrolysis of rice proteins (AE = 0.0010-0.0820, W = 0.1994-1.0000, DHt = 45-82%). However, antibacterial peptides (IAK) were released by ficin only from rice, oat, and garden pea proteins (DHt = 41-46%). Biopeptides (YG, IAK) identified in protein hydrolysates are potential immunomodulators, nutraceuticals, and components of functional food that may modulate the activity of the human immune system. Stem bromelain and ficin are also active components that are primed to release peptide immunomodulators from plant-derived food proteins.