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.

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.

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.

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.

Anti-fatigue effects of enzyme-hydrolyzed okara in C2C12 myotubes and Sprague-Dawley rats.

Okara is a by-product of tofu or soymilk production processes. The disposal of huge quantities of okara is a significant issue. Based on previous reports, protein hydrolysis can release excess free amino acids and small peptides from okara and exhibit anti-fatigue function. We aimed to investigate the anti-fatigue effect of okara protein hydrolysate (OPH) in vitro and in vivo. In the first phase, we treated C2C12 myotubes with different processed OPHs to detect mitochondrial functions. The results revealed that OPH hydrolyzed with alcalase containing 2% E/S for 2 h increased the mitochondrial mRNA level (cytochrome b and cytochrome c oxidase I) and enzyme activity (citrate synthase and cytochrome c oxidase) most efficiently. In the second phase, we conducted animal studies to assess the anti-fatigue function of OPH. After acclimatization, 8 week-old male Sprague-Dawley (SD) rats were randomly classified into four groups: (1) control group, (2) 1X-OPH, (3) 2X-OPH, and (4) 5X-OPH (8 rats per group, treated for 28 days). The results indicated that the intake of OPH for 28 days increased the exhaustive swimming time of rats and lowered the increment of the lactate ratio, as well as the activity of lactate dehydrogenase and creatine kinase. These results indicated that OPH improves exercise performance and anti-fatigue function in male SD rats. Therefore, OPH could be a potential health supplement for anti-fatigue function.

Quorum Quenching-Guided Inhibition of Mixed Bacterial Biofilms and Virulence Properties by Protein Derived From Leaves of Carissa carandas.

The inhibition/degradation potential of Carissa carandas proteinaceous leaf extract against mixed bacterial biofilm of Staphylococcus aureus MTCC 96, Escherichia coli MTCC 1304, Pseudomonas aeruginosa MTCC 741, and Klebsiella pneumoniae MTCC 109, responsible for nosocomial infections, was evaluated. Distinct inhibition/degradation of mixed bacterial biofilm by the proteinaceous leaf extract of C. carandas was observed under a microscope, and it was found to be 80%. For mono-species biofilm, the maximum degradation of 70% was observed against S. aureus biofilm. The efficiency of aqueous plant extracts to inhibit the mono-species biofilm was observed in terms of minimum inhibitory concentration (MIC), and the best was found against P. aeruginosa (12.5 µg/ml). The presence of flavonoids, phenols, and tannins in the phytochemical analysis of the plant extract suggests the main reason for the antibiofilm property of C. carandas. From the aqueous extract, protein fraction was precipitated using 70% ammonium sulfate and dialyzed. This fraction was purified by ion-exchange chromatography and found to be stable and active at 10°C (pH 7). The purified fraction showed less than 40% cytotoxicity, which suggests that it can be explored for therapeutic purposes after in-depth testing. In order to investigate the mechanistic action of the biofilm inhibition, the plant protein was tested against Chromobacterium violaceum CV026, and its inhibitory effect confirmed its quorum quenching nature. Based on these experimental analyses, it can be speculated that the isolated plant protein might influence the signaling molecule that leads to the inhibition effect of the mixed bacterial biofilm. Further experimental studies are warranted to validate our current findings.

The acyclotide ribe 31 from Rinorea bengalensis has selective cytotoxicity and potent insecticidal properties in Drosophila.

Cyclotides and acyclic versions of cyclotides (acyclotides) are peptides involved in plant defense. These peptides contain a cystine knot motif formed by three interlocked disulfide bonds, with the main difference between the two classes being the presence or absence of a cyclic backbone, respectively. The insecticidal activity of cyclotides is well documented, but no study to date explores the insecticidal activity of acyclotides. Here, we present the first in vivo evaluation of the insecticidal activity of acyclotides from Rinorea bengalensis on the vinegar fly Drosophila melanogaster. Of a group of structurally comparable acyclotides, ribe 31 showed the most potent toxicity when fed to D. melanogaster. We screened a range of acyclotides and cyclotides and found their toxicity toward human red blood cells was substantially lower than toward insect cells, highlighting their selectivity and potential for use as bioinsecticides. Our confocal microscopy experiments indicated their cytotoxicity is likely mediated via membrane disruption. Furthermore, our surface plasmon resonance studies suggested ribe 31 preferentially binds to membranes containing phospholipids with phosphatidyl-ethanolamine headgroups. Despite having an acyclic backbone, we determined the three-dimensional NMR solution structure of ribe 31 is similar to that of cyclotides. In summary, our results suggest that, with further optimization, ribe 31 could have applications as an insecticide due to its potent in vivo activity against D. melanogaster. More broadly, this work advances the field by demonstrating that acyclotides are more common than previously thought, have potent insecticidal activity, and have the advantage of potentially being more easily manufactured than cyclotides.

Cytotoxic Cyclotides from Anchietea pyrifolia, a South American Plant Species.

Cyclotides are mini-proteins with potent bioactivities and outstanding potential for agricultural and pharmaceutical applications. More than 450 different plant cyclotides have been isolated from six angiosperm families. In Brazil, studies involving this class of natural products are still scarce, despite its rich floristic diversity. Herein were investigated the cyclotides from Anchietea pyrifolia roots, a South American medicinal plant from the family Violaceae. Fourteen putative cyclotides were annotated by LC-MS. Among these, three new bracelet cyclotides, anpy A-C, and the known cycloviolacins O4 (cyO4) and O17 (cyO17) were sequenced through a combination of chemical and enzymatic reactions followed by MALDI-MS/MS analysis. Their cytotoxic activity was evaluated by a cytotoxicity assay against three human cancer cell lines (colorectal carcinoma cells: HCT 116 and HCT 116 TP53-/- and breast adenocarcinoma, MCF 7). For all assays, the IC50 values of isolated compounds ranged between 0.8 and 7.3 µM. CyO17 was the most potent cyclotide for the colorectal cancer cell lines (IC50, 0.8 and 1.2 µM). Furthermore, the hemolytic activity of anpy A and B, cyO4, and cyO17 was assessed, and the cycloviolacins were the least hemolytic (HD50 > 156 µM). This work sheds light on the cytotoxic effects of the anpy cyclotides against cancer cells. Moreover, this study expands the number of cyclotides obtained to date from Brazilian plant biodiversity and adds one more genus containing these molecules to the list of the Violaceae family.

The Activity of Chelidonium majus L. Latex and Its Components on HPV Reveal Insights into the Antiviral Molecular Mechanism.

Yellow-orange latex of Chelidonium majus L. has been used in folk medicine as a therapeutic agent against warts and other visible symptoms of human papillomavirus (HPV) infections for centuries. The observed antiviral and antitumor properties of C. majus latex are often attributed to alkaloids contained therein, but recent studies indicate that latex proteins may also play an important role in its pharmacological activities. Therefore, the aim of the study was to investigate the effect of the crude C. majus latex and its protein and alkaloid-rich fractions on different stages of the HPV replication cycle. The results showed that the latex components, such as alkaloids and proteins, decrease HPV infectivity and inhibit the expression of viral oncogenes (E6, E7) on mRNA and protein levels. However, the crude latex and its fractions do not affect the stability of structural proteins in HPV pseudovirions and they do not inhibit the virus from attaching to the cell surface. In addition, the protein fraction causes increased TNFα secretion, which may indicate the induction of an inflammatory response. These findings indicate that the antiviral properties of C. majus latex arise both from alkaloids and proteins contained therein, acting on different stages of the viral replication cycle.

Sapovaccarin-S1 and -S2, Two Type I RIP Isoforms from the Seeds of Saponaria vaccaria L.

Type I ribosome-inactivating proteins (RIPs) are plant toxins that inhibit protein synthesis by exerting rRNA N-glycosylase activity (EC 3.2.2.22). Due to the lack of a cell-binding domain, type I RIPs are not target cell-specific. However once linked to antibodies, so called immunotoxins, they are promising candidates for targeted anti-cancer therapy. In this study, sapovaccarin-S1 and -S2, two newly identified type I RIP isoforms differing in only one amino acid, were isolated from the seeds of Saponaria vaccaria L. Sapovaccarin-S1 and -S2 were purified using ammonium sulfate precipitation and subsequent cation exchange chromatography. The determined molecular masses of 28,763 Da and 28,793 Da are in the mass range typical for type I RIPs and the identified amino acid sequences are homologous to known type I RIPs such as dianthin 30 and saporin-S6 (79% sequence identity each). Sapovaccarin-S1 and -S2 showed adenine-releasing activity and induced cell death in Huh-7 cells. In comparison to other type I RIPs, sapovaccarin-S1 and -S2 exhibited a higher thermostability as shown by nano-differential scanning calorimetry. These results suggest that sapovaccarin-S1 and -S2 would be optimal candidates for targeted anti-cancer therapy.

SAXS Analysis and Characterization of Anticancer Activity of PNP-UDP Family Protein from Putranjiva roxburghii.

A class of plant defense and storage proteins, including Putranjiva roxburghii PNP protein (PRpnp), belongs to PNP-UDP family. The PRpnp and related plant proteins contain a disrupted PNP-UDP domain as revealed in previous studies. In PRpnp, the insert disrupting the domain contains the trypsin inhibitory site. In the present work, we analyzed native PRpnp (nPRpnp) complex formation with trypsin and inosine using SAXS experiments and established its dual functionality. Results indicated a relatively compact nPRpnp:Inosine structure, whereas trypsin complex showed conformational changes/flexibility. nPRpnp also exhibited a strong anti-cancer activity toward breast cancer (MCF-7), prostate cancer (DU-145) and hepatocellular carcinoma (HepG2) cell lines. MCF-7 and DU-145 were more sensitive to nPRpnp treatment as compared to HepG2. However, nPRpnp treatment showed no effect on the viability of HEK293 cells indicating that nPRpnp is specific for targeting the viability of only cancer cells. Further, acridine orange, DAPI and DNA fragmentation studies showed that cytotoxic effect of nPRpnp is mediated through induction of apoptosis as evident from the apoptosis-associated morphological changes and nuclear fragmentation observed after PRpnp treatment of cancer cells. These results suggest that PRpnp has the potential to be used as an anticancer agent. This is first report of anticancer activity as well as SAXS-based analysis for a PNP enzyme with trypsin inhibitory activity.

A Novel Antiviral Protein Derived from Oenanthe javanica: Type I Interferon-Dependent Antiviral Signaling and Its Pharmacological Potential.

Pathogenesis-related (PR) proteins produced in plants play a crucial role in self-defense against microbial attacks. Previously, we have identified a novel PR-1-like protein (OPRP) from Oenanthe javanica and examined its pharmacologic relevance and cell signaling in mammalian cells. Purified full-length OPRP protein significantly increased toll-like receptor 4 (TLR4)-dependent expression levels of genes such as inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and CD80. We also found that small peptides (OPRP2 and OPRP3) designed from OPRP remarkably upregulated myxovirus resistance (Mx1), 2'-5' oligoadenylate sythetase (OAS), and interferon (IFN) α/ß genes in mouse splenocytes as well as human epithelial cells. Notably, OPRP protein distinctively activated STAT1 phosphorylation and ISGF-3γ. Interestingly, OPRP2 and OPRP3 were internalized to the cytoplasm and triggered dimerization of STAT1/STAT2, followed by upregulation of type I IFN-dependent antiviral cytokines. Moreover, OPRP1 successfully inhibited viral (Pseudo SARS-CoV-2) entry into host cells. Taken together, we conclude that OPRP and its small peptides (OPRP1 to 3) present a new therapeutic intervention for modulating innate immune activity through type I IFN-dependent antiviral signaling and a new therapeutic approach that drives an antiviral state in non-immune cells by producing antiviral cytokines.

Successful Pre-Clinical Management of Irinotecan-Debilitated Animals: A Protein- Based Accessory Phytomedicine.

BACKGROUND: Calotropis procera is a laticiferous plant (Apocynaceae) found in tropical regions all over the world. The ultrastructural characteristics of laticifers, their restricted distribution among different taxonomic groups, and in some species in each clade, as peptidases from latex, make them very attractive for biological analysis. OBJECTIVE: The study aims to investigate the effects of LP-PII-IAA (laticifer protein (LP) sub-fraction II (PII) of C. procera presenting an iodoacetamide-inhibited cysteine proteinase activity) on irinotecan-induced intestinal mucositis, a serious adverse effect of this medicine for the treatment of cancer. METHODS: LP-PII-IAA is composed of closely related isoforms (90%) of peptidases derived from catalysis and an osmotin protein (5%). Animals receiving co-administration of LP-PII-IAA presented a significant decrease in mortality, absence of diarrhea, histological preservation, and normalization of intestinal functions. RESULTS: Clinical homeostasis was accompanied by a reduction in MPO activity and declined levels of IL-1ß, IL-6 and KC, while the IL-10 level increased in LP-PII-IAA-treated animals. COX-2 and NF-kB immunostaining was reduced and the levels of oxidative markers (GSH, MDA) were normalized in animals that received LP-PII-IAA. CONCLUSION: We suggest that peptidases from the latex of Calotropis procera were instrumental in the suppression of the adverse clinical and physiological effects of irinotecan.