Effect of the phosphorylation structure in casein phosphopeptides on the proliferation, differentiation, and mineralization of osteoblasts and its mechanism.

Our previous studies have shown that highly phosphorylated casein phosphopeptides (residues 1-25) P5 could efficiently bind calcium and promote intestinal calcium absorption, and enhanced bone development in rats. The purpose of this study was to investigate the effect of the phosphorylation structure in P5 on the proliferation, differentiation, and mineralization of osteoblasts (MC3T3-E1) and its mechanism. P5 was obtained by high-performance liquid chromatography (HPLC) and non-phosphorylated peptide P5-0 was obtained by chemical synthesis. Compared with the control group, the proliferation rate of MC3T3-E1 cells treated by P5 was 1.10 times that of P5-0 at 200 µg mL-1. P5 caused the cell cycle retention of MC3T3-E1 cells in the G2/M phase, while P5-0 had no significant difference in the G2/M phase. MC3T3-E1 cells incubated with P5 showed stronger alkaline phosphatase (ALP) activity than with P5-0, suggesting a tendency to promote cellular differentiation. Compared to the P5-0 treatment group, the P5 treatment group at concentrations of 10 µg mL-1 showed significant differences in the mineralization rates (p < 0.05). P5 significantly upregulated the expressions of Runx2, ALP, ColIα1, and OCN compared with the control group (p < 0.05). In addition, in silico molecular docking showed that the binding force of the P5-EGFR complex was stronger than that of the P5-0-EGFR complex, which was significantly related to the phosphorylation structure in P5 and might be an important reason for osteoblast proliferation. In conclusion, the phosphorylation structure and amino acid composition in P5 stimulated the osteogenic activity of MC3T3-E1 cells, and could be expected to be a functional food for the prevention of osteoporosis.

Phosphorylated Peptide Derived from the Myosin Phosphatase Target Subunit Is a Novel Inhibitor of Protein Phosphatase-1.

Identification of specific protein phosphatase-1 (PP1) inhibitors is of special importance regarding the study of its cellular functions and may have therapeutic values in diseases coupled to signaling processes. In this study, we prove that a phosphorylated peptide of the inhibitory region of myosin phosphatase (MP) target subunit (MYPT1), R690QSRRS(pT696)QGVTL701 (P-Thr696-MYPT1690-701), interacts with and inhibits the PP1 catalytic subunit (PP1c, IC50 = 3.84 µM) and the MP holoenzyme (Flag-MYPT1-PP1c, IC50 = 3.84 µM). Saturation transfer difference NMR measurements established binding of hydrophobic and basic regions of P-Thr696-MYPT1690-701 to PP1c, suggesting interactions with the hydrophobic and acidic substrate binding grooves. P-Thr696-MYPT1690-701 was dephosphorylated by PP1c slowly (t1/2 = 81.6-87.9 min), which was further impeded (t1/2 = 103 min) in the presence of the phosphorylated 20 kDa myosin light chain (P-MLC20). In contrast, P-Thr696-MYPT1690-701 (10-500 µM) slowed down the dephosphorylation of P-MLC20 (t1/2 = 1.69 min) significantly (t1/2 = 2.49-10.06 min). These data are compatible with an unfair competition mechanism between the inhibitory phosphopeptide and the phosphosubstrate. Docking simulations of the PP1c-P-MYPT1690-701 complexes with phosphothreonine (PP1c-P-Thr696-MYPT1690-701) or phosphoserine (PP1c-P-Ser696-MYPT1690-701) suggested their distinct poses on the surface of PP1c. In addition, the arrangements and distances of the surrounding coordinating residues of PP1c around the phosphothreonine or phosphoserine at the active site were distinct, which may account for their different hydrolysis rate. It is presumed that P-Thr696-MYPT1690-701 binds tightly at the active center but the phosphoester hydrolysis is less preferable compared to P-Ser696-MYPT1690-701 or phosphoserine substrates. Moreover, the inhibitory phosphopeptide may serve as a template to synthesize cell permeable PP1-specific peptide inhibitors.

Salt-induced phosphoproteomic changes in the subfornical organ in rats with chronic kidney disease.

OBJECTIVES: Subfornical organ (SFO) is vital in chronic kidney disease (CKD) progression caused by high salt levels. The current study investigated the effects of high salt on phosphoproteomic changes in SFO in CKD rats. METHODS: 5/6 nephrectomized rats were fed a normal-salt diet (0.4%) (NC group) or a high-salt diet (4%) (HC group) for three weeks, while sham-operated rats were fed a normal-salt diet (0.4%) (NS group). For phosphoproteomic analysis of SFO in different groups, TiO2 enrichment, isobaric tags for relative and absolute quantification (iTRAQ) labeling, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used. RESULTS: There were 6808 distinct phosphopeptides found, which corresponded to 2661 phosphoproteins. NC group had 168 upregulated and 250 downregulated phosphopeptides compared to NS group. Comparison to NC group, HC group had 154 upregulated and 124 downregulated phosphopeptides. Growth associated protein 43 (GAP43) and heat shock protein 27 (Hsp27) were significantly upregulated phosphoproteins and may protect against high-salt damage. Differential phosphoproteins with tight functional connection were synapse proteins and microtubule-associated proteins, implying that high-salt diet disrupted brain's structure and function. Furthermore, differential phosphoproteins in HC/NC comparison group were annotated to participate in GABAergic synapse signaling pathway and aldosterone synthesis and secretion, which attenuated inhibitory neurotransmitter effects and increased sympathetic nerve activity (SNA). DISCUSSION: This large scale phosphoproteomic profiling of SFO sheds light on how salt aggravates CKD via the central nervous system.

Osteopontin phosphopeptide mitigates calcium oxalate stone formation in a Drosophila melanogaster model.

Kidney stone disease affects nearly one in ten individuals and places a significant economic strain on global healthcare systems. Despite the high frequency of stones within the population, effective preventative strategies are lacking and disease prevalence continues to rise. Osteopontin (OPN) is a urinary protein that can inhibit the formation of renal calculi in vitro. However, the efficacy of OPN in vivo has yet to be determined. Using an established Drosophila melanogaster model of calcium oxalate urolithiasis, we demonstrated that a 16-residue synthetic OPN phosphopeptide effectively reduced stone burden in vivo. Oral supplementation with this peptide altered crystal morphology of calcium oxalate monohydrate (COM) in a similar manner to previous in vitro studies, and the presence of the OPN phosphopeptide during COM formation and adhesion significantly reduced crystal attachment to mammalian kidney cells. Altogether, this study is the first to show that an OPN phosphopeptide can directly mitigate calcium oxalate urolithiasis formation in vivo by modulating crystal morphology. These findings suggest that OPN supplementation is a promising therapeutic approach and may be clinically useful in the management of urolithiasis in humans.

The Therapeutic Effect of Phosphopeptide P140 Attenuates Inflammation Induced by Uric Acid Crystals in Gout Arthritis Mouse Model.

Gout is a painful form of inflammatory arthritis characterized by the deposition of monosodium urate (MSU) crystals in the joints. The aim of this study was to investigate the effect of peptide P140 on the inflammatory responses in crystal-induced mouse models of gout and cell models including MSU-treated human cells. Injection of MSU crystals into the knee joint of mice induced neutrophil influx and inflammatory hypernociception. Injection of MSU crystals subcutaneously into the hind paw induced edema and increased pro-inflammatory cytokines levels. Treatment with P140 effectively reduced hypernociception, the neutrophil influx, and pro-inflammatory cytokine levels in these experimental models. Furthermore, P140 modulated neutrophils chemotaxis in vitro and increased apoptosis pathways through augmented caspase 3 activity and reduced NFκB phosphorylation. Moreover, P140 increased the production of the pro-resolving mediator annexin A1 and decreased the expression of the autophagy-related ATG5-ATG12 complex and HSPA8 chaperone protein. Overall, these findings suggest that P140 exerts a significant beneficial effect in a neutrophilic inflammation observed in the model of gout that can be of special interest in the design of new therapeutic strategies.

Synthesis and characterization of nano bioactive glass for improving enamel remineralization ability of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP).

OBJECTIVE: Nanomaterials with superior properties such as high surface area over volume ratio are widely used in dentistry and medicine. This in vitro study was performed to synthesize and characterize nano bioactive glass (nBG) and to evaluate the effect of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) containing nBG (CPP-ACP@nBG) on enamel remineralization by its application to pH-cycled, synthetically demineralized enamel surfaces. MATERIALS AND METHODS: nBG particles were prepared by sol-gel method. X-ray diffraction pattern (XRD), Fourier-transform infrared spectroscopy (FTIR) and transmittance electron microscopy (TEM) were used for nBG characterization. Synthetic CPP-ACP paste was prepared and nBG particles were added to it. To evaluate the degree of remineralization, 32 healthy human premolars were selected. The samples were randomly divided into 4 groups as: Group 1: Commercial CPP-ACFP (MI paste plus), Group 2: Synthetic casein phosphopeptide-amorphous calcium phosphate containing fluoride (CPP-ACP@F), Group 3: Synthetic CPP/ACP containing nBG (CPP-ACP@nBG), and Group 4: Control (received no treatment). The pastes were then applied on the tooth surfaces for 28 days. The Vickers microhardness of enamel surfaces was evaluated, and enamel surface morphology was assessed using scanning electron microscopy (SEM). RESULTS: X-Ray diffraction pattern (XRD) of the synthesized nBG show its crystalline nature with the Larnite crystalline mode. Transmittance electron microscope (TEM) microimage of the synthesized nBG shows its formation as less that 100 nm spherical nanoparticle with partial agglomeration. Fourier transform infrared spectroscopy (FTIR) confirm the success formation of nBG with high purity. The results of this study showed that microhardness of the experimental groups was significantly higher than the control group (p ≥ 0.05). SEM images showed a layer of hydroxyapatite in the CPP-ACP@nBG, synthetic and commercial CPP-ACP@F remineralized groups. CONCLUSION: The results of this study demonstrated that CPP-ACP@F and CPP-ACP@nBG remineralize the surface of the demineralized enamel. Microhardness of the remineralized enamel in the CPP-ACP@nBG group was higher than synthetic and commercial CPP-ACP@F groups.

Influence of two remineralizing agents on bleached enamel surface morphology and mineral composition - An In Vitro study.

Aim: To investigate the effects of bleaching with 35% hydrogen peroxide on the structure of tooth enamel and the role of two remineralizing agents for their potential to remineralize any damaged regions of enamel. Materials and Methods: Freshly extracted 32 mature permanent central incisors were selected and sectioned at the level of the cemento-enamel junction. The teeth were divided into four groups consisting of eight teeth each: No bleaching (control) [Group 1], bleaching with 35% hydrogen peroxide [Group 2], bleaching with 35% hydrogen peroxide followed by application of casein phosphopeptide amorphous calcium phosphate fluoride paste [Group 3], and bleaching with 35% hydrogen peroxide followed by application of xylitol-coated calcium phosphate fluoride varnish [Group 4]. The enamel surfaces were analyzed under the scanning electron microscope and quantitative energy dispersive X-ray analysis. Results: Results were statistically analyzed by one-way analysis of variance and Tukey's posthoc test. Group 2 revealed changes in enamel surface morphology and a statistically significant decrease in mineral content. Groups 3 and 4 showed statistically significant remineralization potential. Intergroup comparison showed that samples in Group 4 had a higher mineral content compared to Group 3. Conclusions: The application of the tested remineralizing agents following bleaching was effective in repairing the enamel surface morphology with higher efficacy for the fluoride varnish product. Since bleaching regimes with high concentrations of hydrogen peroxide adversely affect the enamel surface, these findings can translate to clinical practice to reduce the long-term damaging effects of tooth bleaching.

Evaluation of the changes in physical properties and mineral content of enamel exposed to radiation after treating with remineralization agent.

OBJECTIVE: The aim of this study was to investigate the effect of different remineralization agents on the physical properties and elemental content of enamel exposed to radiation. MATERIAL AND METHOD: The enamel surfaces of impacted third molar teeth were prepared, and six study groups were created (n = 6). Next, 60 Gy radiation was applied to each group. Between applications, each group except for the control group was treated with a different remineralization agent (sodium fluoride, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), casein phosphopeptide amorphous calcium phosphate with fluorite (CPP-ACFP), bioactive glass, or chitosan). The results were evaluated in terms of pre- and post-radiation values and the difference between the two. The paired-samples t test and analysis of variance test were used in the analysis of normally distributed hardness and roughness values, while Wilcoxon's signed ranks test, and the Kruskal Wallis and Mann-Whitney U tests were used in the analysis of elemental content without normal distribution. RESULTS: A statistically significant decrease was observed in microhardness measurements in all groups. Intragroup evaluation revealed a statistically significant difference between the NaF and bioactive glass groups (p < 0.05). No significant difference was observed between the groups' roughness measurements (p < 0.05). Intergroup evaluation of surface roughness revealed a significant difference in the CPP-ACFP and chitosan groups (p < 0.05). Pre- and post-radiation oxygen, magnesium, and potassium levels and Ca/P ratios also differed significantly (p < 0.05). CONCLUSION: Radiation caused a statistically significant difference in the microhardness and elemental content of enamel. However, no significant difference was observed in enamel roughness. The applied remineralizing agents have a partial ameliorating effect on the adverse impacts of radiation. CLINICAL RELEVANCE: Radiation causes changes in the mechanical properties and elemental content of tooth enamel. Remineralizing agent application is a promising option in reducing the adverse effects of irradiation.

Effect of different surface treatments on the microhardness and colour change of artificial enamel lesions.

BACKGROUND: To investigate the effect of three different surface treatments on the microhardness and colour change of artificial enamel lesions. MATERIALS AND METHODS: One hundred bovine teeth were randomly assigned into four groups. Artificial enamel lesions were created using demineralizing solution for all groups except the sound enamel group. Different surface treatments were then performed G1: resin-infiltrant; G2: Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP); G3: artificial saliva; G4: Sound Enamel. Each group was subdivided into three subgroups, where each subgroup was subjected to a different testing method. Subgroup 1: surface microhardness; subgroup 2: cross-sectional microhardness; subgroup 3: colour measurement. Statistical analysis was performed by ANOVA, followed by Tukey's post hoc test. RESULTS: Sound enamel group recorded the highest surface and cross-sectional microhardness results. No significant difference was found between the resin-infiltrant group and CPP-ACP regarding surface and cross-sectional microhardness at different lesion depths. Resin-infiltrant group showed the least colour change (∆E) results compared to the other groups. CONCLUSION: Resin-infiltrant can effectively enhance surface microhardness and enamel resistance to demineralization, additionally, reduces the staining susceptibility of white spot lesions (WSLs) after treatment. CPP-ACP application for 4 weeks seems to improve surface microhardness; however, has a limited effect in resisting staining of WSLs after treatment. © 2022 Australian Dental Association.

Biomimetic calcium-inactivated ion/molecular channel.

A phosphopeptide-modified nanochannel was prepared based on a conical polymeric nanopore. It shows a reversible Ca2+-induced inactivation effect toward the ion flow and molecular transport, resulting from Ca2+ binding-caused surface charge neutralization and hydrophilicity reduction, and Ca2+ removal by the competitive binding.

MHC Phosphopeptides: Promising Targets for Immunotherapy of Cancer and Other Chronic Diseases.

Major histocompatibility complex-associated peptides have been considered as potential immunotherapeutic targets for many years. MHC class I phosphopeptides result from dysregulated cell signaling pathways that are common across cancers and both viral and bacterial infections. These antigens are recognized by central memory T cells from healthy donors, indicating that they are considered antigenic by the immune system and that they are presented across different individuals and diseases. Based on these responses and the similar dysregulation, phosphorylated antigens are promising candidates for prevention or treatment of different cancers as well as a number of other chronic diseases.

Effect of fluoride, casein phosphopeptide-amorphous calcium phosphate and sodium trimetaphosphate combination treatment on the remineralization of caries lesions: An in vitro study.

OBJECTIVE: To evaluate the effects of combination of treatments with fluoridated toothpastes supplemented with sodium trimetaphosphate (TMP) and casein phosphopeptide-amorphous calcium phosphate (MI Paste Plus®), on the remineralization of dental enamel. DESIGN: Enamel blocks with artificial caries were randomly allocated into six groups (n = 12), according to the toothpastes: 1) without F-TMP-MI Paste Plus® (Placebo); 2) 1100 ppm F (1100 F), 3) MI Paste Plus®, 4) 1100 F + MI Paste Plus® (1100 F-MI Paste Plus®), 5) 1100 F + 3% TMP (1100 F-TMP) and 6) 1100 F-TMP + MI Paste Plus® (1100 F-TMP-MI Paste Plus®). Blocks were treated 2×/day with slurries of toothpastes (1 min). Furthermore, groups 4 and 6 received the application of MI Paste Plus® for 3 min. After pH cycling, the percentage of surface hardness recovery (%SHR); integrated loss of subsurface hardness (ΔKHN); profile analysis and lesion depth subsurface through polarized light microscopy (PLM), confocal laser scanning microscopy (LSCM), scanning electron microscopy (SEM), fluoride (F), calcium (Ca), phosphorus (P) concentrations in the enamel were determined. The data were analyzed by ANOVA (1-criterion) and Student-Newman-Keuls test (p < 0.001). RESULTS: 1100 F-TMP-MI Paste Plus® group showed the best results of %SHR, ΔKHN and PLM (p < 0.001). F concentration was similar between the 1100 F, 1100 F-MI Paste Plus®, and 1100 F-TMP-MI Paste Plus® groups (p > 0.001). 1100 F-TMP-MI Paste Plus® group showed the highest concentration of Ca and P in the enamel (p < 0.001). CONCLUSION: The association of 1100 F-TMP and MI Paste Plus® led to a significant increase in the remineralization of initial carious lesions.

Herring egg phosphopeptides as calcium carriers for improving calcium absorption and bone microarchitecture in vivo.

Phosphorylation may enhance the functional properties of proteins/peptides. Herring egg phosphopeptides (HEPPs) have been found to be more effective than the non-phosphorylated variant in calcium-binding activities due to the introduced phosphate groups. However, whether HEPPs as calcium carriers will be superior to herring egg peptides (HEPs) in improving calcium bioavailability in vivo, for the equivalent calcium intake prerequisite, remains to be clarified. This study aimed to evaluate the effect of HEPPs-calcium complex and HEPs-calcium complex on calcium absorption and bioavailability in calcium-deficient mice. Results showed that the remarkably lower calcium absorption and bone calcium deposition induced by long-term calcium deficiency were accompanied by deterioration of the trabecular bone microarchitecture (P < 0.05). The HEPPs-Ca supplements significantly improved the apparent calcium absorption, increased the serum calcium level, decreased the alkaline phosphatase activity, strengthened the bone biomechanical property, and increased bone volume/tissue volume (BV/TV) and trabecular number (Tb·N) in calcium-deficient mice (P < 0.05), as determined by micro-computed tomography (micro-CT) assay. The effect of HEPPs-Ca on calcium absorption and bioavailability was comparable to that of CPPs-Ca, but better than that of HEPs-Ca and CaCO3. This study brings new insights into the potential of HEPPs as an alternative to CPPs for use in calcium supplements.

Characterization of Phosphopeptide Positional Isomers on the Transcriptional Co-activator TAZ.

The transcriptional co-activator with the PDZ binding motif (TAZ) is a critical regulator of numerous cellular processes such as cell differentiation, development, proliferation, and cell growth. Aberrant expression and activity of TAZ are also featured in many human malignancies. A hallmark of TAZ biology is its cytoplasmic retention mediated by 14-3-3 isoforms in response to phosphorylation of Ser89 by members of the LATS family of kinases. Following the observation that TAZ is a highly phosphorylated protein even when Ser89 is mutated, high-resolution mass spectrometry employing data-independent acquisition and ion mobility separation was conducted to elucidate additional TAZ phosphorylation sites that may play a role in regulating this critical transcriptional rheostat. Numerous phosphorylation sites on TAZ were identified, including several novel modifications. Of notable interest was the identification of positional phosphoisomers on a phosphopeptide containing Ser89. Optimized use of a so-called wideband enhancement acquisition technique yielded higher-quality fragmentation data that confirmed the detection of Ser93 as the positional phosphoisomer partner of Ser89 and identified diagnostic fragment ions for the phosphorylation events. Functional analysis indicated that Ser93 phosphorylation reduces the level of 14-3-3 association and increases the level of nuclear translocation, indicating this phosphorylation event attenuates the 14-3-3-mediated TAZ cytoplasmic retention mechanism. These findings suggest that the biological activities of TAZ are likely dynamically regulated by multisite phosphorylation.

In Vivo Remodeling of Altered Autophagy-Lysosomal Pathway by a Phosphopeptide in Lupus.

The phosphopeptide P140/Lupuzor, which improves the course of lupus disease in mice and patients, targets chaperone-mediated autophagy (CMA), a selective form of autophagy that is abnormally upregulated in lupus-prone MRL/lpr mice. Administered intravenously to diseased mice, P140 reduces the expression level of two major protein players of CMA, LAMP2A and HSPA8, and inhibits CMA in vitro in a cell line that stably expresses a CMA reporter. Here, we aimed to demonstrate that P140 also affects CMA in vivo and to unravel the precise cellular mechanism of how P140 interacts with the CMA process. MRL/lpr mice and CBA/J mice used as control received P140 or control peptides intravenously. Lysosome-enriched fractions of spleen or liver were prepared to examine lysosomal function. Highly purified lysosomes were further isolated and left to incubate with the CMA substrate to study at which cellular step P140 interacts with the CMA process. The data show that P140 effectively regulates CMA in vivo in MRL/lpr mice at the step of substrate lysosomal uptake and restores some alterations of defective lysosomes. For the first time, it is demonstrated that by occluding the intralysosome uptake of CMA substrates, a therapeutic molecule can attenuate excessive CMA activity in a pathological pro-inflammatory context and protect against hyperinflammation. This recovery effect of P140 on hyperactivated CMA is not only important for lupus therapy but potentially also for treating other (auto)inflammatory diseases, including neurologic and metabolic disorders, where CMA modulation would be highly beneficial.

Casein phosphopeptide combined with fluoride enhances the inhibitory effect on initial adhesion of Streptococcus mutans to the saliva-coated hydroxyapatite disc.

BACKGROUND: Recent preventive strategies for dental caries focus on targeting the mechanisms underlying biofilm formation, including the inhibition of bacterial adhesion. A promising approach to prevent bacterial adhesion is to modify the composition of acquired salivary pellicle. This in vitro study investigated the effect and possible underlying mechanism of pellicle modification by casein phosphopeptide (CPP) on Streptococcus mutans (S. mutans) initial adhesion, and the impact of fluoride on the efficacy of CPP. METHODS: The salivary pellicle-coated hydroxyapatite (s-HA) discs were treated with phosphate buffered saline (negative control), heat-inactivated 2.5% CPP (heat-inactivated CPP), 2.5% CPP (CPP) or 2.5% CPP supplemented with 900 ppm fluoride (CPP + F). After cultivation of S. mutans for 30 min and 2 h, the adherent bacteria were visualized by scanning electron microscopy (SEM) and quantitatively evaluated using the plate count method. Confocal laser scanning microscopy (CLSM) was used to evaluate the proportions of total and dead S. mutans. The concentrations of total, free, and bound calcium and fluoride in the CPP and fluoride-doped CPP solutions were determined. The water contact angle and zeta potential of s-HA with and without modification were measured. The data were statistically analyzed using one-way ANOVA followed by a Turkey post hoc multiple comparison test. RESULTS: Compared to the negative control group, the amount of adherent S. mutans significantly reduced in the CPP and CPP + F groups, and was lowest in the CPP + F group. CLSM analysis showed that there was no statistically significant difference in the proportion of dead S. mutans between the four groups. Water contact angle and zeta potential of s-HA surface significantly decreased in the CPP and CPP + F groups as compared to the negative control group, and both were lowest in the CPP + F group. CONCLUSIONS: Pellicle modification by CPP inhibited S. mutans initial adhesion to s-HA, possibly by reducing hydrophobicity and negative charge of the s-HA surface, and incorporating fluoride into CPP further enhanced the anti-adhesion effect.

A cell permeant phosphopeptide mimetic of Niban inhibits p38 MAPK and restores endothelial function after injury.

Vascular injury leads to membrane disruption, ATP release, and endothelial dysfunction. Increases in the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and decreases in the phosphorylation of Niban, a protein implicated in ER stress and apoptosis, are associated with vascular injury. A cell permeant phosphopeptide mimetic of Niban (NiPp) was generated. The effects of NiPp in restoring endothelial function were determined ex vivo using intact rat aortic tissue (RA) after pharmacological activation of p38 MAPK and also in multiple clinically relevant injury models. Anisomycin (Aniso) increased p38 MAPK phosphorylation and reduced endothelial-dependent relaxation in RA. Treatment with NiPp prevented Ansio-induced reduction in endothelial function and increases in p38 MAPK phosphorylation. NiPp treatment also restored endothelial function after stretch injury (subfailure stretch), treatment with acidic Normal Saline (NS), and P2X7R activation with 2'(3')-O-(4-Benzoylbenzoyl)adenosine 5'-triphosphate (BzATP). Aged, diseased, human saphenous vein (HSV) remnants obtained from patients undergoing coronary bypass surgical procedures have impaired endothelial function. Treatment of these HSV segments with NiPp improved endothelial-dependent relaxation. Kinome screening experiments indicated that NiPp inhibits p38 MAPK. These data demonstrate that p38 MAPK and Niban signaling have a role in endothelial function, particularly in response to injury. Niban may represent an endogenous regulator of p38 MAPK activation. The NiPp peptide may serve as an experimental tool to further elucidate p38 MAPK regulation and as a potential therapeutic for endothelial dysfunction.

Synthesis and Antimicrobial Activity of Phosphonopeptide Derivatives Incorporating Single and Dual Inhibitors.

In diagnostic microbiology, culture media are widely used for detection of pathogenic bacteria. Such media employ various ingredients to optimize detection of specific pathogens such as chromogenic enzyme substrates and selective inhibitors to reduce the presence of commensal bacteria. Despite this, it is rarely possible to inhibit the growth of all commensal bacteria, and thus pathogens can be overgrown and remain undetected. One approach to attempt to remedy this is the use of "suicide substrates" that can target specific bacterial enzymes and selectively inhibit unwanted bacterial species. With the purpose of identifying novel selective inhibitors, six novel phosphonopeptide derivatives based on d/l-fosfalin and ß-chloro-l-alanine were synthesized and tested on 19 different strains of clinically relevant bacteria. Several compounds show potential as useful selective agents that could be exploited in the recovery of several bacterial pathogens including Salmonella, Pseudomonas aeruginosa, and Listeria.

Overexpression of amyA and glaA substantially increases glucoamylase activity in Aspergillus niger.

The purpose of this study was to obtain an engineered Aspergillus niger strain with high glucoamylase activity by overexpressing the glucoamylase gene glaA and α-amylase gene amyA in A. niger CICC2462. Three recombinant strains containing a single copy of amyA (1A), containing two copies of amyA (2A), and coexpressing amyA and glaA (AG), respectively, were constructed. The transcript levels of amyA in 1A and 2A were increased by 2.95 folds and 3.09 folds, respectively. The levels of amyA and glaA in AG were increased by 1.21 folds and 2.86 folds, but the maximum extracellular glucoamylase activities did not differ significantly. In addition, after 1% casein phosphopeptides (CPPs) was added to the fermentation medium, the maximum extracellular glucoamylase activities for strains 1A, 2A, and AG were 35,200, 37,300, and 40,710 U/ml, respectively, which were significantly higher than that of the parental strain CICC2462 (28,250 U/ml), while CPPs alone had no effect on the parental strain CICC2462. We demonstrate that overexpression of amyA and glaA substantially increases the expression and secretion of glucoamylase in A. niger, and CPPs effectively improves the yield of glucoamylase in recombinant A. niger strains overexpressing amyA and glaA. The newly developed strains and culture methods may have extensive industrial applications.

Enhanced Anti-Allergic Activity of Milk Casein Phosphopeptide by Additional Phosphorylation in Ovalbumin-Sensitized Mice.

The proteolytic digest of milk casein, known as casein phosphopeptide (CPP-III), exhibits diverse biological activities, including calcium absorption and antioxidant activities. We hypothesized that the additional phosphorylation of this peptide can enhance its immunomodulatory activity such as suppression of allergy-associated cytokine and antigen-specific immune response. This study was conducted to assess whether oral intake of additionally phosphorylated CPP-III (P-CPP) attenuates ovalbumin (OVA)-induced IgE-mediated allergic reactions because of the additional phosphate groups. Female BALB/c mice were intraperitoneally sensitized with OVA twice at intervals of 14 days and then orally fed native CPP-III (N-CPP), P-CPP, and dephosphorylated CPP-III (D-CPP) for 6 weeks. Next, the mice were orally challenged with 50 mg of OVA. Oral administration of P-CPP suppressed total and specific IgE levels in the serum. Mice fed P-CPP exhibited low levels of OVA-specific IgG1 and increased OVA-specific IgG2a. P-CPP also suppressed IL-4 production, while D-CPP showed similar a level compared to that of the control. Further, P-CPP increased the population of the T follicular helper (Tfh) cell in the spleen. These results suggest that additional phosphorylation of CPP can enhance the attenuation of allergen-specific IgE-modulated allergic reactions in a murine food allergy model.