Presence Of Protease Inhibitors Research Articles

Activated CTHRC1 promotes glycolysis in endothelial cells: Implications for metabolism and angiogenesis

CTHRC1 is transiently expressed by activated fibroblasts during tissue repair and in certain cancers, and CTHRC1 derived from osteocytes is detectable in circulation. Because its biological activity is poorly understood, we investigated whether the N terminus of CTHRC1 encodes a propeptide requiring cleavage to become activated. The effects of full-length versus cleaved recombinant CTHRC1 on endothelial cell metabolism and gene expression were examined in vitro. Respirometry was performed on Cthrc1 null and wildtype mice to obtain evidence for biological activity of CTHRC1 in vivo. Cleavage of the propeptide observed in vitro was attenuated in the presence of protease inhibitors, and cleaved CTHRC1 significantly promoted glycolysis whereas full-length CTHRC1 was less effective. The respiratory exchange ratio was significantly higher in wildtype mice compared to Cthrc1 null mice, supporting the findings of CTHRC1 promoting glycolysis in vivo. Key enzymes involved in glycolysis were significantly upregulated in endothelial cells in response to treatment with CTHRC1. In healthy human subjects, 58% of the cohort had detectable levels of circulating full-length CTHRC1, whereas all subjects with undetectable levels of full-length CTHRC1 (with one exception) had measurable levels of truncated CTHRC1 (88 pg/ml to >400 ng/ml). Our findings support a concept where CTHRC1 induction in activated fibroblasts at sites of ischemia such as tissue injury or cancer functions to increase glycolysis for ATP production under hypoxic conditions, thereby promoting cell survival and tissue repair. By promoting glycolysis under normoxic conditions, CTHRC1 may also be a contributor to the Warburg effect characteristically observed in many cancers.

The dual role of eppin in immunity and biomineralization during nacreous layer formation in mollusks

The presence of protease inhibitors in a carbonated shell is intriguing and opens questions about their potential role in shell biomineralization.

Antileishmanial effects of Crotalaria spectabilis Roth aqueous extracts on Leishmania amazonensis.

Fifteen polar extracts from leaf, seed, pod, stem, flower and root of Crotalaria spectabilis were prepared using aqueous systems, based on the principles of green chemistry, and showed different protease inhibitor (PI) activities on trypsin, papain, pepsin and the extracellular L. amazonensis serine protease (LSPIII). The most pronounced inhibitory effect on LSPIII was observed in leaf (CS-P), root, stem, flower (CS-FPVPP) and pod (CS-VA) extracts. Crotalaria extracts exhibited low cytotoxicity on macrophages; however, they decreased the viability of L. amazonensis promastigotes and amastigotes, as observed in leaf (CS-AE, CS-P, CS-T and CS-PVPP), seed (CS-ST), flower and root (CS-RA) extracts. CS-P was chosen to study PI and secondary metabolites and a 10-12 kDa protein, analyzed by mass spectrometry, was identified as a serine PI homologous with papaya latex serine PI. Glycosylated flavonoids, such as quercetins, vitexin and tricin were the major secondary metabolites of CS-P. The presence of PIs in C. spectabilis is a new finding, especially in other organs than seeds since PIs have been reported only in seed legumes. Besides, this is the first report of antileishmanial activity of C. spectabilis extracts and the identification of serine polypeptide PI and glycosylated flavonoids from leaf.

P323 Virulence not required? Albumin promotes pathogenicity of (non)-damaging Candida strains

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMThe pathogenicity of the dimorphic yeast Candida albicans is associated with filamentation, adhesion, invasion, and production of the toxin Candidalysin. However, there are certain clinical isolates and other Candida spp., that cause infection independent of filamentation or the production of Candidalysin. Consequently, these strains and species are often non-damaging in vitro, this does not correlate with their potential to cause infection in patients. We hypothesize that specific host factors, which trigger pathogenicity, are absent in in vitro models, and thereby not reflecting the situation in the host. To determine the impact of albumin, the most abundant protein in the human body, vaginal epithelial cells were infected with different C. albicans strains and Candida species. Interestingly, after prolonged infection (45 h) albumin increased the damage potential, even in otherwise non-damaging and non-filamentous strains. This included deletion mutants deficient in filamentation, als3 adhesin/invasin, thigmotropism, or Candidalysin production. Yet, the increased damage was likely not solely an effect of increased growth and nutrient competition between the fungus and epithelial cells. Reduced damage in presence of protease inhibitors and albumin hint toward the role of proteases in the utilization of albumin. Albumin enhanced C. albicans metabolism, by stimulating the utilization of various nitrogen sources. This metabolic adaption could explain the advantage and enhanced growth as a strain and species-independent feature. Our data suggest that common host factors can impact C. albicans to cause damage independent of adhesion, invasion, filamentation, and toxin production. Possibly, also other host-derived factors can drive the pathogenic potential of fungi through unresolved mechanisms.

Evaluation of the in vitro protein bioaccessibility of several microalgae and cyanobacteria as potential dietary ingredients in gilthead seabream (Sparus aurata) juveniles

This work addresses the evaluation in vitro of different marine (Dunaliella salina REC-0214B and Microchloropsis gaditana REC-0251B) and freshwater (Anabaena sp. BEA-0300B, Arthrospira platensis BEA-0007B, Chlorella vulgaris BEA-0753B, Spirogyra sp. BEA-0666B) microalgae and cyanobacteria as potential dietary ingredients in aquafeeds. For this purpose, total protein content, amino acid composition, and the presence of protease inhibitors were evaluated. In addition, protein bioaccessibility was estimated using a species-specific in vitro assay using Sparus aurata digestive enzymes. Overall, all the microalgae showed high protein content ranging from 25 to 61%, and a balanced essential to non-essential amino acid content (from 0.81 to 0.95). The inhibition assay confirmed the absence of protease inhibitors whatever the microalgae considered. Finally, the in vitro assays showed differences in the degree of protein hydrolysis with values for the coefficient of protein degradation ranging from 49.4% in Spirogyra sp. to 85.5% in D. salina. Similarly, the total amount of free amino acids released from the microalgal biomass (from 12.8 to 20.8 g L-leucine equivalents (100 g protein)−1), as well as their qualitative amino acids profile varied among the different species, although the profile can be considered as well balanced in all cases. In conclusion, the results obtained revealed that, even if significant differences were observed among species in terms of their susceptibility to be hydrolysed by S. aurata digestive enzymes, all the microalgae and cyanobacteria evaluated presented an adequate protein content and a balanced amino acid profile.

Presence of Protease Inhibitor 9 and Granzyme B in Healthy and Pathological Human Corneas.

Simple SummaryDetailed knowledge of the structure and properties of the human cornea is a prerequisite not only for the treatment of various corneal diseases but also for successful corneal transplantation and its long-term survival after grafting. Using various cell and molecular biology approaches, we found in cornea the protease inhibitor 9. This protein, known to be present in other human tissues but not yet reported in cornea, is directly involved in the immune response after transplantation. Together with its inhibitor (granzyme B), we localized this protein, especially in the superficial and inner cornea layers. This localization indicates that protease inhibitor 9 protein may be involved in protecting the cornea from external damage, but also in protection against immune cells inducing corneal graft rejection. Furthermore, we have shown on pathological corneal samples from corneal melting and herpes virus keratitis that the increased expression of both proteins is linked to these diseases. These experiments and their results represent an important contribution to the basic research of cornea biological properties with direct overlap into clinical practice.The aim of this study was to find out whether protease inhibitor 9 (PI-9) and granzyme B (GrB) molecules that contribute to immune response and the immunological privilege of various tissues are expressed in healthy and pathological human corneas. Using cryosections, cell imprints of control corneoscleral discs, we showed that PI-9 was expressed particularly in the endothelium, the superficial and suprabasal epithelium of healthy corneas, limbus, and conjunctiva. GrB was localized in healthy corneal and conjunctival epithelium, while the endothelium showed weak immunostaining. The expression of PI-6 and GrB was confirmed by qRT-PCR. Increased expression levels of the PI-9 and GrB genes were determined when the corneas were cultured with proinflammatory cytokines. Fluorescent and enzymatic immunohistochemistry of pathological corneal explants (corneal melting and herpes virus keratitis) showed pronounced PI-9, GrB, human leucocyte antigen (HLA)-DR, and leukocyte-common antigen (CD45) signals localized in multicellular stromal infiltrates and inflammatory cells scattered in the corneal stroma. We conclude that increased expression of the PI-9 and GrB proteins under pathological conditions and their upregulation in an inflammatory environment indicate their participation in immune response of the cornea during the inflammatory process.

Characterization of a Novel Protease Inhibitor from the Edible Mushroom Agaricus bisporus.

Protease inhibitors inhibit the activity of protease enzymes; hence, they are essentially involved in the regulation of the metabolic processes involving protease enzymes and the protection of the host organism against external damage due to proteases. These inhibitors are abundantly present in all living organisms but have not been much reported in mushrooms. Mushrooms are one of the major food components of humans, with delicious taste and high nutritional value. Mushrooms also have therapeutic and economic significance. The edible mushrooms with medicinal properties are much in commercial demand. To date, the presence of protease inhibitors has not been reported much in edible mushrooms. The present study reports the characterization of a protease inhibitor isolated from the common white button mushroom Agaricus bisporus. The objective of the present study is to characterize the novel protease inhibitor from Agaricus bisporus in order to determine its nature and activity under varying environmental conditions. The protease inhibitor was characterized through SDS PAGE, gel filtration chromatography, and de novo sequencing in order to determine its molecular mass and sequence, respectively. The optimum pH, temperature, and thermal stability were studied to determine the optimum working range of the protease inhibitor. The protease inhibitory activity (%) was determined in the presence of metal ions, surfactants, oxidizing agents, and reducing agents. The kinetic parameters and the type of inhibition exhibited by the protease inhibitor were determined using casein and trypsin protease enzyme. The protease inhibitor was found to be a low molecular mass compound of 25 kDa. The de novo sequencing matched the inhibitor against a 227 amino acid containing peptide molecular mass of 24.6 kDa molecular mass. The protease inhibitory activity (%) was found highest at pH 7.0 and temperature 50 °C, and the inhibitor was stable from pH 4.0-9.0 and temperature 30-80 °C. In the presence of metal ions, the residual protease inhibitory activity (%) enhanced in the presence of Na+, Mg2+, and Fe3+. The residual activity increased in the presence of the surfactant SDS slightly in comparison to control, while it decreased in the case of Triton-X and Tween 20. The presence of oxidizing agents, hydrogen peroxide and dimethyl sulfoxide decreased the residual inhibitory activity. The protease inhibitor's activity was unaffected by the reducing agents, dithiothreitol and β-mercaptoethanol, at up to 2mM concentration, but it decreased at higher concentrations. The inhibitor exhibited uncompetitive inhibition against trypsin with an inhibitory constant of 166 nM, indicating a strong affinity towards the protease, with a half-life of 93.90 minutes at 37 °C. Protease inhibitors isolated from mushrooms are generally small in size, more stable, and tolerant towards varying external conditions. The protease inhibitor isolated from Agaricus bisporus also exhibited similar characteristics.

Leishmania tarentolae novel responses to Bi3+-doped strontium aluminum oxyfluorides

Novel therapeutics for the treatment of leishmaniasis are of interest as the disease not only is becoming more prevalent, but drug resistance is increasing in certain regions of the world. Reported here is the use of Bi3+-doped strontium aluminum oxyfluoride phosphors and protease inhibitors to test in vitro inhibitory activity against cultured promastigote Leishmania tarentolae and effects on L. tarentolae secreted acid phosphatase (SAP) activity. Cell viability did not significantly decrease in the presence of 50 μM anti-perovskite compounds, implying limited cytotoxicity. Yet SAP activity did increase in the cell free preparations with time in the presence of strontium compounds. Of interest was the observation that cell free SAP activity did not increase in the presence of protease inhibitors with or without added strontium compounds. Since secreted proteases may play a role in the maturation of Leishmania SAP and thus be involved with parasite-host infection establishment, this is in further need of evaluation. Nitric oxide production on day 4 post-addition of the strontium compounds was evaluated and showed an approximately 50% decrease in NO production in the presence of two test compounds relative to DMSO control cells. This is the first report of anti-perovskite compound inhibition of NO production by Leishmania.

Itolizumab-induced antigenic modulation of CD6 inhibits T cell activity

Abstract CD6 is an immunomodulatory receptor on T cells that promotes immune synapse formation, T-cell activation, and cell migration into tissues by binding activated leukocyte cell adhesion molecule (ALCAM). Excessive activation through the CD6-ALCAM pathway has been implicated in the pathogenesis of multiple autoimmune and inflammatory diseases. Hence, the ability to modulate the level of activation is beneficial to disease resolution. Itolizumab is a humanized anti-CD6 monoclonal antibody that is specific for the membrane distal domain 1 of CD6. Previously, itolizumab was believed to sterically hinder the CD6-ALCAM interaction, thereby blocking T-cell co-stimulation. Here we describe antigenic modulation as an additional mechanism whereby binding of itolizumab to CD6 induces proteolytic cleavage of the extracellular portion of CD6. Upon treatment with itolizumab, surface levels of CD6 decreased in a dose- and time-dependent manner, as monitored by flow cytometry using a noncompetitive anti-CD6 monoclonal antibody. This loss was inhibited in the presence of protease inhibitors and the decrease in surface levels of CD6 was accompanied by an increase in levels of soluble CD6 in the supernatant. Furthermore, we assessed the effect of surface levels of CD6 on the response of cells to T cell stimulation in the presence of ALCAM. CD6low cells showed reduced T-cell activity compared to CD6highcells as measured by cell surface activation makers, including CD25, CD69 and PD-1, and cytokine production. These findings demonstrate that CD6 is an important regulator of T-cell activity and that modulating surface levels of CD6 is an effective method for fine-tuning the activity of T-cells.

LncRNA MIR503HG inhibits cell proliferation and promotes apoptosis in TNBC cells via the miR-224-5p/HOXA9 axis

Triple-negative breast cancer (TNBC) is a highly invasive subtype of breast cancer. This study investigated the molecular mechanism and influences of MIR503HG, miR-224-5p, and homeobox A9 (HOXA9) on TNBC cell growth and migration. Dual-luciferase reporter gene and RNA immunoprecipitation were performed to examine the regulation of MIR503HG, miR-224-5p, and HOXA9. Cell proliferation, apoptosis, migration, and invasion were evaluated by colony formation, flow cytometry, and Transwell assays. Finally, nude mice were employed to investigate the influence of MIR503HG on TNBC tumor growth. HOXA9 protein levels were detected by immunohistochemical staining. MIR503HG and HOXA9 expression were reduced in TNBC, while miR-224-5p was increased. Overexpression of MIR503HG or HOXA9 reduced the cell migration ability and proliferation and promoted apoptosis, and knockdown of MIR503HG or overexpression of miR-224-5p exhibited the opposite effects. Furthermore, MIR503HG promoted HOXA9 expression by inhibiting miR-224-5p. Overexpression of miR-224-5p reversed the effects of MIR503HG overexpression on TNBC cells, while overexpression of HOXA9 reversed the effect of MIR503HG knockdown. Additionally, an in vivo study proved that MIR503HG inhibited TNBC tumor growth via the miR-224-5p/HOXA9 axis. MIR503HG inhibited cell proliferation and promoted the apoptosis of TNBC cells via the miR-224-5p/HOXA9 axis, which may function as a novel target for the treatment of TNBC.

The distinct N-terminomes of Bothrops jararaca newborn and adult venoms

Using approaches of transcriptomics and proteomics we have shown that the phenotype of Bothrops jararaca venom undergoes a significant rearrangement upon neonate to adult transition. Most regulatory processes in biology are intrinsically related to modifications of protein structure, function, and abundance. However, it is unclear to which extent intrinsic proteolysis affects toxins and snake venom phenotypes upon ontogenesis. Here we assessed the natural N-terminome of Bothrops jararaca newborn and adult venoms and explored the degree of N-terminal protein truncation in ontogenetic-based proteome variation. To this end we applied the Terminal Amine Isotopic Labeling of Substrates (TAILS) technology to characterize venom collected in the presence of proteinase inhibitors. We identified natural N-terminal sequences in the newborn (71) and adult (84) venoms, from which only 37 were common to both. However, truncated toxins were found in higher number in the newborn (212) than in the adult (140) venom. Moreover, sequences N-terminally blocked by pyroglutamic acid were identified in the newborn (55) and adult (49) venoms. Most toxin classes identified by their natural N-terminal sequences showed a similar number of unique peptides in the newborn and adult venoms, however, those of serine proteinases and C-type lectins were more abundant in the adult venom. Truncated sequences from at least ten toxin classes were detected, however the catalytic and cysteine-rich domains of metalloproteinases were the most prone to proteolysis, mainly in the newborn venom. Our results underscore the pervasiveness of truncations in most toxin classes and highlight variable post-translational events in newborn and adult venoms.

The serine protease HtrA plays a key role in heat-induced dispersal of pneumococcal biofilms

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx by forming multicellular biofilms. Due to the high level of asymptomatic carriage, transition to infections, such as otitis media, pneumonia, sepsis, and meningitis, occurs often enough that the pneumococcus remains a major cause of disease and death globally. Virus infection and virus-induced responses, such as increased temperature (fever), trigger release of virulent bacteria from colonizing biofilms. The exact mechanisms involved in pneumococcal egress during biofilm dispersal remain unknown, although we hypothesize that disruption of the biofilm matrix encasing the bacteria is necessary. Here, we utilized established in vitro biofilm dispersal models to investigate the involvement of proteases in bacterial egress from pneumococcal biofilms. We demonstrate the importance of protease activity, both through increased bacterial release following addition of proteases and reduced heat-induced biofilm dispersal in the presence of protease inhibitors. We identify a key role for the surface-exposed serine protease HtrA, but not PrtA, in heat-induced biofilm dispersal. Bacterial release from htrA-negative biofilms was significantly reduced compared to wild-type isogenic strains but was restored and increased above wild-type levels following addition of recombinant HtrA. Understanding the specific mechanisms involved in bacterial egress may provide novel targets for future strategies aimed to specifically interfere with disease progression without disturbing nasopharyngeal biofilm colonization.

Time-dependent aggregation of casein micelle concentrates

This research focused on understanding physical and chemical changes occurring to concentrated milk protein suspensions as a function of time. Skim milk (untreated and heat treated at 90°C for 10 min) was concentrated at 6 times the original volume using osmotic stressing, a noninvasive concentration method, maintaining the serum composition as close as possible to that of native milk. A protease inhibitor cocktail, with broad specificity for the inhibition of serine, cysteine, aspartic proteases, and aminopeptidases, was added in selected samples. Within 9 d of storage at 4°C, the apparent viscosity increased markedly for both unheated and heated concentrated milk, but not for those in the presence of protease inhibitors. However, only unheated milk showed a significant increase in the apparent diameter of the casein micelles. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry measurements indicated a significantly lower extent of proteolysis in heated than in unheated samples. The microstructure of the aggregates was observed using field emission scanning electron microscopy, and unheated samples clearly showed aggregation of casein micelles with storage time. In heated samples, aggregation was instead triggered by heat-induced protein-protein interactions.

NNRTI-induced HIV-1 protease-mediated cytotoxicity induces rapid death of CD4 T cells during productive infection and latency reversal

BackgroundCurrent efforts towards HIV-1 eradication focus on the reactivation and elimination of the latent viral reservoir, so-called shock and kill therapy. However, work from several groups indicates that infected cell death following virus reactivation is not guaranteed. Thus, it is imperative to develop strategies to foster specific elimination of cells carrying integrated proviruses. It has been shown that some non-nucleoside reverse transcriptase inhibitors (NNRTIs) including efavirenz can induce premature HIV-1 GagPol dimerization in productively infected cells, resulting in intracellular HIV-1 Protease (PR) activation and a reduction in HIV-1 expressing cells.ResultsHere, we document that NNRTI-induced PR activation triggers apoptotic death of productively infected resting or activated T cells in as little as 2 h via caspase-dependent and independent pathways. Rilpivirine, efavirenz and etravirine were the most potent NNRTIs, whereas nevirapine had almost no effect. NNRTI-induced cell killing was prevented by inhibitors of HIV-1 Protease (PR) activity including indinavir and nelfinavir. HIV-1 transmitter founder viruses induced cell killing similarly to lab-adapted HIV-1 except when NNRTI resistance conferring mutations were present in reverse transcriptase. Mutations in PR that confer PR inhibitor (PI) resistance restore NNRTI-induced killing in the presence of PI. Finally, we show that NNRTIs can rapidly eliminate cells in which latent viruses are stimulated to active expression.ConclusionsThis work supports the notion that select NNRTIs might help promote the elimination of HIV-1 producing cells as an adjuvant during shock and kill therapy.

Protective effect of a protease inhibitor from Agaricus bisporus on Saccharomyces cerevisiae cells against oxidative stress

Protease inhibitors are known to resist damage to host organisms against external threats, hence form a part of their defense system. This property of protease inhibitors was studied on protecting oxidatively stressed Saccharomyces cerevisiae yeast cells. The protease inhibitor was extracted from Agaricus bisporus, an edible mushroom. The inhibitor showed the presence of antioxidant activity as the purified inhibitor fraction gave an IC50 value of 45.13 ± 0.88 µg/mL and 33.30 ± 1.5 µg/mL when checked, respectively, by 2, 2-diphenyl-1-picrylhydrazyl, DPPH and 2, 2′-azo-bis(3-ethylbenzthiazoline-6- sulfonic acid), ABTS•+ scavenging activity. The yeast cells’ survival rate (%), was determined through 3-(4, 5-dimethylthiazol-2-yl) - 2, 5-diphenyltetrazolium bromide, MTT assay, and it was found that in the presence of 2 mM H2O2 cell survival decreased to 26.33%, whereas when the experiment was conducted in the presence of protease inhibitor and 2 mM H2O2 cell survival percentage rose to 74%. The protease inhibitor’s effect on the oxidatively stressed yeast cells was further studied by using Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and Confocal Microscopy to understand the morphological changes. The viable and non-viable cell populations were quantified using Fluorescence Assorted Cell Sorting (FACS) using propidium iodide, PI, 4′, 6-diamidino-2-phenylindole, DAPI and 2′, 7′-dichlorofluorescein, DCF dyes.

The protease-based compensatory mechanism to minimize the effect of dietary Soybean Trypsin Inhibitor in Litopenaeus vannamei

Soybean meal is a recurrent ingredient in shrimp feed formulations; although economically convenient, the downside is the antinutritional molecules often present in its active forms. Recently, a midgut gland proteolytic activity adjustment to compensate the effect of increasing concentrations of soybean trypsin inhibitor (SBTI) delivered in shrimp feed was found in Litopenaeus vannamei; which prompted us to inquire on the transcriptional response of shrimp digestive peptidases against exogenous peptidase inhibitors and connect the results for a broader comprehension of the compensatory mechanism triggered by ingestion of SBTI. Our results indicate that such response involves at least two stages: in the first hours, a drastic increase in transcription of trypsin, an inhibitor sensitive protease was observed followed by a gradual increase in transcription of putatively less-sensitive protease chymotrypsin, and a non-sensitive metalloprotease was observed. At 23 h a second doses of SBTI was administered and at 24 h, trypsin transcripts returned to basal levels, whereas the transcription of genes encoding inhibitor-insensitive peptidases increased. Taken together, this paper is the first comprehensive study of the compensatory mechanisms to exogenous protease inhibitors in Penaeidae. Specific activity and gene expression of digestive proteases observe the same pattern, indicating a phenotypic plasticity in the digestive system of L. vannamei as an adaptive response to compensate the protein digestion capacity when some of the peptidase activities are reduced by the presence of protease inhibitors.

Pregnancy History Influences the Level of Autophagy in Peripheral Blood Mononuclear Cells From Pregnant Women.

Maternal immune responses are altered during pregnancy and differ between nulliparous and multiparous women. The influence of a prior gestation on autophagy in peripheral blood mononuclear cells (PBMCs) from pregnant women has not been determined and is the subject of this investigation. Peripheral blood mononuclear cells were isolated from 212 pregnant women and immediately lysed in the presence of protease inhibitors, and the extent of autophagy was determined by quantitation of the concentration of p62 (sequestosome-1) in the lysates by enzyme-linked immunosorbent assay (ELISA). In PBMCs, the p62 level is inversely related to the extent of autophagy. The level of the stress-inducible 70-kDa heat shock protein (hsp70), an inhibitor of autophagy, was also measured in the lysates by ELISA. Data were analyzed by the Spearman rank correlation, Mann-Whitney U test, or Kruskal-Wallis test, as appropriate. The p62 concentration in PBMCs increased (autophagy decreased) with the number of previous live ( P = .0322), preterm ( P = .0143), or term ( P = .0418) deliveries. The p62 level was lower (autophagy higher) in women with a prior spontaneous pregnancy loss but no deliveries as compared to women with their first conception ( P = .0087). The intracellular hsp70 concentration correlated with the p62 level ( P < .0001). Multiparity is associated with a reduced level of autophagy in PBMCs. Dysregulated autophagy might be one mechanism leading to spontaneous abortion in nulliparous women.

Procyanidins from Cinnamomi Cortex promote proteasome-independent degradation of nuclear Nrf2 through phosphorylation of insulin-like growth factor-1 receptor in A549 cells

Many lines of evidence demonstrate that transcription factor nuclear factor-E2-related factor 2 (Nrf2) plays essential roles in cancer cell proliferation and resistance to chemotherapy, thereby indicating that suppression of abnormal Nrf2 activation is needed for a new therapeutic approach. Our previous studies reported that procyanidins prepared from Cinnamomi Cortex extract (CCE) have an ability to suppress cytoprotective enzymes and cell proliferation in human cancer cells with activated Nrf2. In the present study, we investigated the mechanism of CCE procyanidin-mediated antagonization of Nrf2. CCE procyanidin treatment rapidly reduced nuclear Nrf2 expression and phosphorylated insulin-like growth factor-1 receptor (IGF-1R) in A549 cells. Nrf2 protein expression in A549 cells with reduced IGF-1R expression and function was not affected by treatment with CCE procyanidins, which suggested that CCE procyanidins decreased Nrf2 through IGF-1R. Nrf2 suppression by CCE procyanidins was mitigated in the presence of protease inhibitors, not proteasome inhibitors. In addition, CCE procyanidin treatment led to enhancement of nuclear cysteine protease activity in A549 cells. Our findings suggest a novel mechanism by which CCE procyanidins can promote proteasome-independent degradation of nuclear Nrf2 through IGF-1R phosphorylation and cysteine protease activation.

Aflatoxin B1 degradation by culture and lysate of a Pontibacter specie

The presence of aflatoxin B1 (AFB1) along the food chain poses a significant threat, thus propelling the need for an effective approach to control it. This study was therefore, aimed at investigating AFB1 degradation of liquid cultures and lysates of an isolated Pontibacter sp. (VGF1). Liquid cultures, lysed bacterial cells in the absence (uninhibited lysates) and presence of protease inhibitors (protease inhibited lysates) were respectively incubated with AFB1 for 3, 6, 12, 24 and 48 h. AFB1 degradation was monitored during this period on high performance liquid chromatography (HPLC) and results obtained revealed that after 6 h of incubation, the protease inhibited (PI) lysates yielded a 65% AFB1 degradation, whereas after 12 h, no residual AFB1 was detected. Conversely, after 48 h of incubation, a significantly (p≤0.05) lower AFB1 degradation of 50 and 36% by the liquid culture and uninhibited lysate, respectively, were noted. It was further confirmed that the degradation mechanism was enzymatic. Data from cytotoxicity studies against human lymphocytes further demonstrated that extracts of biotransformed AFB1 were less toxic when compared to that of AFB1. Findings from this study have demonstrated an alternative approach for the decontamination and biocontrol of AFB1 in various agricultural commodities.

Mass Spectrometry Based Proteomics Study of Cisplatin-Induced DNA-Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells.

Platinum-based antitumor drugs such as 1,1,2,2-cis-diamminedichloroplatinum(II) (cisplatin), carboplatin, and oxaliplatin are currently used to treat nearly 50% of all cancer cases, and novel platinum based agents are under development. The antitumor effects of cisplatin and other platinum compounds are attributed to their ability to induce interstrand DNA-DNA cross-links, which are thought to inhibit tumor cell growth by blocking DNA replication and/or preventing transcription. However, platinum agents also induce significant numbers of unusually bulky and helix-distorting DNA-protein cross-links (DPCs), which are poorly characterized because of their unusual complexity. We and others have previously shown that DPCs block DNA replication and transcription and causes toxicity in human cells, potentially contributing to the biological effects of platinum agents. In the present work, we have undertaken a system-wide investigation of cisplatin-mediated DNA-protein cross-linking in human fibrosarcoma (HT1080) cells using mass spectrometry-based proteomics. DPCs were isolated from cisplatin-treated cells using a modified phenol/chloroform DNA extraction in the presence of protease inhibitors. Proteins were released from DNA strands and identified by mass spectrometry-based proteomics and immunological detection. Over 250 nuclear proteins captured on chromosomal DNA following treatment with cisplatin were identified, including high mobility group (HMG) proteins, histone proteins, and elongation factors. To reveal the exact molecular structures of cisplatin-mediated DPCs, isotope dilution HPLC-ESI+-MS/MS was employed to detect 1,1-cis-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2'-deoxyguanosine-7-yl)-platinum(II) (dG-Pt-Lys) conjugates between the N7 guanine of DNA and the ε-amino group of lysine. Our results demonstrate that therapeutic levels of cisplatin induce a wide range of DPC lesions, which likely contribute to both target and off target effects of this clinically important drug.