Study on the interaction mechanism of whey protein isolate with phosphatidylcholine: By multispectral methods and molecular docking.

The elucidation of the interaction mechanism between phospholipids and milk proteins within emulsions is pivotal for comprehending the properties of infant formula fat globules. In this study, multispectral methods and molecular docking were employed to explore the relationship between phosphatidylcholine (PC) and whey protein isolate (WPI). Observations indicate that the binding constant, alongside thermodynamic parameters, diminishes as temperature ascends, hinting at a predominantly static quenching mechanism. Predominantly, van der Waals forces and hydrogen bonds constitute the core interactions between WPI and PC. This assertion is further substantiated by Fourier transform infrared spectroscopy, which verifies PC's influence on WPI's secondary structure. A detailed assessment of thermodynamic parameters coupled with molecular docking reveals that PC predominantly adheres to specific sites within α-lactalbumin, ß-lactoglobulin, and bovine serum albumin, propelled by a synergy of hydrophobic interactions, hydrogen bonding, and van der Waals forces, with binding energies noted at -5.59, -6.71, and -7.85 kcal/mol, respectively. An increment in PC concentration is observed to amplify the emulsification properties of WPI whilst concurrently diminishing the zeta potential. This study establishes a theoretical foundation for applying the PC-WPI interaction mechanism in food.

In vitro gastrointestinal digestion of cow's and sheep's dairy products: Impact of species and structure.

Sheep's milk (SM) is known to differ from cow's milk (CM) in nutritional composition and physicochemical properties, which may lead to different digestion behaviours. This work aimed to investigate the impact of the species (cow vs sheep) and the structure (milk vs yogurt) on the digestion of dairy products. Using an in vitro static gastrointestinal digestion model, CM, SM, cow's milk yogurt (CY) and sheep's milk yogurt (SY) were compared on particle size evolution, microscopic observations, degree of lipolysis, degree of proteolysis, specific protein degradation and calcium bioaccessibility. Species and structure affected particle size evolution during the gastric phase resulting in smaller particles for yogurts compared to milks as well as for CM products compared to SM products. Species impacted lipid composition and lipolysis, with SM products presenting higher short/medium-chain fatty acids content and higher intestinal degree of lipolysis. Proteolysis was influenced by structure, with milks showing higher intestinal degree of proteolysis compared to yogurts. Caseins were digested faster in CM, ⍺-lactalbumin was digested faster in SM despite its higher concentration, and during gastric digestion ß-lactoglobulin was more degraded in CM products compared to SM products and more in yogurts compared to milks. Lastly, SM products released more bioaccessible calcium than CM products. In conclusion, species (cow vs sheep) impacted more the digestion compared to the structure (milk vs yogurt). In fact, SM was different from CM mainly due to a denser protein network that might slow down the accessibility of the enzyme to its substrate which induce a delay of gastric disaggregation and thus lead to slower the digestion of the nutrients.

The BLV-miRNAs pathway of BLV inhibits the expression of Lactoferrin, Lactoperoxidase, Alpha-lactalbumin and Beta-lactoglobulin proteins.

Bovine leukemia virus (BLV) is a widespread virus that decreases milk production and quality in dairy cows. As crucial components of BLV, BLV-encoded microRNAs (BLV-miRNAs) affect BLV replication and may impact the synthesis of Lactoferrin (LTF), Lactoperoxidase (LPO), Alpha-lactalbumin (alpha-LA), and Beta-lactoglobulin (beta-LG). In this study, we investigated the targeting relationship between BLV-miRNAs and LTF, LPO, alpha-LA, and beta-LG in cow's milk. Additionally, we investigated the possible mechanisms by which BLV reduces milk quality. The results showed that cow's milk had significantly lower levels of LTF, LPO, and alpha-LA proteins in BLV-positive cows than in BLV-negative cows. BLV-△miRNAs (miRNA-deleted BLV) enhanced the reduction of LPO, alpha-LA, and beta-LG protein levels caused by BLV infection. Multiple BLV-miRNAs have binding sites with LTF and LPO mRNA; however, only BLV-miR-B1-5 P has a targeting relationship with LPO mRNA. The results revealed that BLV-miR-B1-5 P inhibits LPO protein expression by targeting LPO mRNA. However, BLV does not directly regulate the expression of LTF, alpha-LA, or beta-LG proteins through BLV-miRNAs.

Modulating Whey Proteins Antigenicity with Lactobacillus delbrueckii subsp. bulgaricus DLPU F-36 Metabolites: Insights from Spectroscopic and Molecular Docking Studies.

Numerous studies have highlighted the potential of Lactic acid bacteria (LAB) fermentation of whey proteins for alleviating allergies. Nonetheless, the impact of LAB-derived metabolites on whey proteins antigenicity during fermentation remains uncertain. Our objective was to elucidate the impact of small molecular metabolites on the antigenicity of α-lactalbumin (α-LA) and ß-lactoglobulin (ß-LG). Through metabolomic analysis, we picked 13 bioactive small molecule metabolites from Lactobacillus delbrueckii subsp. bulgaricus DLPU F-36 for coincubation with α-LA and ß-LG, respectively. The outcomes revealed that valine, arginine, benzoic acid, 2-keto butyric acid, and glutaric acid significantly diminished the sensitization potential of α-LA and ß-LG, respectively. Moreover, chromatographic analyses unveiled the varying influence of small molecular metabolites on the structure of α-LA and ß-LG, respectively. Notably, molecular docking underscored that the primary active sites of α-LA and ß-LG involved in protein binding to IgE antibodies aligned with the interaction sites of small molecular metabolites. In essence, LAB-produced metabolites wield a substantial influence on the antigenic properties of whey proteins.

Milk: A Natural Guardian for the Gut Barrier.

The gut barrier plays an important role in health maintenance by preventing the invasion of dietary pathogens and toxins. Disruption of the gut barrier can cause severe intestinal inflammation. As a natural source, milk is enriched with many active constituents that contribute to numerous beneficial functions, including immune regulation. These components collectively serve as a shield for the gut barrier, protecting against various threats such as biological, chemical, mechanical, and immunological threats. This comprehensive review delves into the active ingredients in milk, encompassing casein, α-lactalbumin, ß-lactoglobulin, lactoferrin, the milk fat globular membrane, lactose, transforming growth factor, and glycopeptides. The primary focus is to elucidate their impact on the integrity and function of the gut barrier. Furthermore, the implications of different processing methods of dairy products on the gut barrier protection are discussed. In conclusion, this study aimed to underscore the vital role of milk and dairy products in sustaining gut barrier health, potentially contributing to broader perspectives in nutritional sciences and public health.

Whey proteins as multifunctional food materials: Recent advancements in hydrolysis, separation, and peptidomimetic approaches.

Whey protein derived bioactives, including α-lactalbumin, ß-lactoglobulin, bovine serum albumin, lactoferrin, transferrin, and proteose-peptones, have exhibited wide ranges of functional, biological and therapeutic properties varying from anticancer, antihypertensive, and antimicrobial effects. In addition, their functional properties involve gelling, emulsifying, and foaming abilities. For these reasons, this review article is framed to understand the relationship existed in between those compound levels and structures with their main functional, biological, and therapeutic properties exhibited either in vitro or in vivo. The impacts of hydrolysis mechanism and separation techniques in enhancing those properties are likewise discussed. Furthermore, special emphasize is given to multifunctional effects of whey derived bioactives and their future trends in ameliorating further food, pharmaceutical, and nutraceutical products. The underlying mechanism effects of those properties are still remained unclear in terms of activity levels, efficacy, and targeted effectiveness. For these reasons, some important models linking to functional properties, thermal properties and cell circumstances are established. Moreover, the coexistence of radical trapping groups, chelating groups, sulfhydryl groups, inhibitory groups, and peptide bonds seemed to be the key elements in triggering those functions and properties. Practical Application: Whey proteins are the byproducts of cheese processing and usually the exploitation of these food waste products has increasingly getting acceptance in many countries, especially European countries. Whey proteins share comparable nutritive values to milk products, particularly on their richness on important proteins that can serve immune protection, structural, and energetic roles. The nutritive profile of whey proteins shows diverse type of bioactive molecules like α-lactalbumin, ß-lactoglobulin, lactoferrin, transferrin, immunoglobulin, and proteose peptones with wide biological importance to the living system, such as in maintaining immunological, neuronal, and signaling roles. The diversification of proteins of whey products prompted scientists to exploit the real mechanisms behind of their biological and therapeutic effects, especially in declining the risk of cancer, tumor, and further complications like diabetes type 2 and hypertension risk effects. For these reasons, profiling these types of proteins using different proteomic and peptidomic approaches helps in determining their biological and therapeutic targets along with their release into gastrointestinal tract conditions and their bioavailabilities into portal circulation, tissue, and organs. The wide applicability of those protein fractions and their derivative bioactive products showed significant impacts in the field of emulsion and double emulsion stabilization by playing roles as emulsifying, surfactant, stabilizing, and foaming agents. Their amphoteric properties helped them to act as excellent encapsulating agents, particularly as vehicle for delivering important vitamins and bioactive compounds. The presence of ferric elements increased their transportation to several metal-ions in the same time increased their scavenging effects to metal-transition and peroxidation of lipids. Their richness with almost essential and nonessential amino acids makes them as selective microbial starters, in addition their richness in sulfhydryl amino acids allowed them to act a cross-linker in conjugating further biomolecules. For instance, conjugating gold-nanoparticles and fluorescent materials in targeting diseases like cancer and tumors in vivo is considered the cutting-edges strategies for these versatile molecules due to their active diffusion across-cell membrane and the presence of specific transporters to these therapeutic molecules.

Diet Plus Inositols, α-Lactalbumin and Gymnema sylvestre: The Successful Combo to Restore Body Weight and Metabolic Profile in Obese and Dysmetabolic Patients.

The primary control of dysmetabolic patients is extremely challenging worldwide, with inadequate dietary habits and sporadic physical activity among the key risk factors for metabolic syndrome onset. Nowadays, there is no exclusive treatment for this condition, and considering that preventive measures usually fail, new therapeutic approaches need to be proposed and investigated. This present pilot study compared the effects of diet alone and in association with a combination of myo-inositol and d-chiro-inositol in their 40:1 ratio, α-lactalbumin, and Gymnema sylvestre on different metabolic parameters in obese dysmetabolic patients. To this purpose, 37 patients with BMI between 30 and 40 and fasting blood glucose between 100 and 125 mg/dL were divided into two groups: (i) the control group followed a hypocaloric Mediterranean diet, (ii) while the study group was also supplemented with a daily dosage of two sachets, each one containing 1950 mg myo-inositol, 50 mg d-chiro-inositol, 50 mg α-lactalbumin, and 250 mg Gymnema Sylvestre. After a 6-month treatment, all parameters improved in both groups. Nevertheless, the treated group experienced a greater improvement, especially concerning the variation from the baseline of HOMA index, triglycerides, BMI, body weight, and waist circumference. These findings support the supplementation with myo-inositol and d-chiro-inositol in the 40:1 ratio, α-lactalbumin, and Gymnema sylvestre as a therapeutical strategy to potentiate the beneficial effects induced via dietary programs in dysmetabolic patients.

α-Lactalbumin Peptide Asp-Gln-Trp Ameliorates Hepatic Steatosis and Oxidative Stress in Free Fatty Acids-Treated HepG2 Cells and High-Fat Diet-Induced NAFLD Mice by Activating the PPARα Pathway.

SCOPE: Dietary intervention has emerged as a promising strategy for the management of nonalcoholic fatty liver disease (NAFLD). The aim of this study is to investigate the ameliorative effects of the α-lactalbumin peptide Asp-Gln-Trp (DQW) against NAFLD and the underlying mechanism. METHODS AND RESULTS: The models of lipid metabolism disorders are established both in HepG2 cells and in C57BL/6J mice. The results demonstrate that DQW activates peroxisome proliferator-activated receptor α (PPARα) and subsequently ameliorates lipid deposition and oxidative stress in vitro. Interestingly, GW6471 markedly attenuates the modulatory effects of DQW on the PPARα pathway in HepG2 cells. Moreover, results of in vivo experiments indicate that DQW alleviates body weight gain, dyslipidemia, hepatic steatosis, and oxidative stress in high-fat-diet (HFD)-induced NAFLD mice. At the molecular level, DQW activates PPARα, subsequently enhances fatty acid ß-oxidation, and reduces lipogenesis, thereby ameliorating hepatic steatosis. Meanwhile, DQW may ameliorate liver injury and oxidative stress via activating the PPARα/nuclear-factor erythroid 2 (Nrf2)/heme-oxygenase 1 (HO-1) pathway. CONCLUSION: Those results indicate that α-lactalbumin peptide DQW may be an effective dietary supplement for alleviating NAFLD by alleviating lipid deposition and oxidative stress.

Tandem mass tag-based quantitative proteomics analysis reveals the effects of the α-lactalbumin peptides GINY and DQW on lipid deposition and oxidative stress in HepG2 cells.

The objective of this study was to investigate the mechanism by which the α-lactalbumin peptides Gly-Ile-Asn-Tyr (GINY) and Asp-Gln-Trp (DQW) ameliorate free fatty acid-induced lipid deposition in HepG2 cells. The results show that GINY and DQW reduced triglyceride, total cholesterol, and free fatty acid levels significantly in free fatty acid-treated HepG2 cells. Based on proteomic analysis, GINY and DQW alleviated lipid deposition and oxidative stress mainly through the peroxisome proliferator-activated receptor (PPAR) pathway, fatty acid metabolism, oxidative phosphorylation, and response to oxidative stress. In vitro experiments confirmed that GINY and DQW upregulated the mRNA and protein expression of fatty acid ß-oxidation-related and oxidative stress-related genes, and downregulated the mRNA and protein expression of lipogenesis-related genes by activating peroxisome proliferator-activated receptor α (PPARα). Meanwhile, GINY and DQW reduced free fatty acid-induced lipid droplet accumulation and reactive oxygen species generation, and enhanced the mitochondrial membrane potential and ATP levels. Furthermore, GINY and DQW enhanced carnitine palmitoyl-transferase 1a (CPT-1a) and superoxide dismutase activities, and diminished acetyl-coenzyme A carboxylase 1 (ACC1) and fatty acid synthase (FASN) activities in a PPARα-dependent manner. Interestingly, GW6471 (a PPARα inhibitor) weakened the effects of GINY and DQW on the PPARα pathway. Hence, our findings suggest that GINY and DQW have the potential to alleviate nonalcoholic fatty liver disease by activating the PPARα pathway.

The intracellular fate and transport mechanism of shape, size and rigidity varied nanocarriers for understanding their oral delivery efficiency.

Nanocarriers have become an effective strategy to overcome epithelial absorption barriers. During the absorption process, the endocytosis mechanisms, cell internalization pathways, and transport efficiency of nanocarriers are greatly impacted by their physical properties. To understand the relationship between physical properties of nanocarriers and their abilities overcoming multiple absorption barriers, nanocarriers with variable physical properties were prepared via self-assembly of hydrolyzed α-lactalbumin peptide fragments. The impacts of size, shape, and rigidity of nanocarriers on epithelial cells endocytosis mechanisms, internalization pathways, transport efficiency, and bioavailability were studied systematically. The results showed that nanospheres were mainly internalized via clathrin-mediated endocytosis, which was then locked in lysosomes and degraded enzymatically in cytoplasm. While macropinocytosis was the primary pathway of nanotubes and transported to the endoplasmic reticulum and Golgi apparatus, resulting in a high drug concentration and sustained release in cytoplasm. Besides, nanotubes can overcome the multi-drug resistance by inhibiting the P-glycoprotein efflux. Furthermore, nanotubes can open intercellular tight-junctions instantaneously and reversibly, which promotes transport into blood circulation. The aqueous solubility of hydrophobic bioactive mangiferin (Mgf) was improved by nanocarriers. Most importantly, the bioavailability of Mgf was the highest for cross-linked short nanotube (CSNT) which outperformed free Mgf and other formulations by in vivo pharmacokinetic studies. Finally, Mgf-loaded CSNT showed an excellent therapeutic efficiency in vivo for the intervention of streptozotocin-induced diabetes. These results indicate that cross-linked α-lactalbumin nanotubes could be an effective nanocarrier delivery system for improving the epithelium cellular absorption and bioavailability of hydrophobic bioactive compounds.

Anti-Hyperuricemic, Nephroprotective, and Gut Microbiota Regulative Effects of Separated Hydrolysate of α-Lactalbumin on Potassium Oxonate- and Hypoxanthine-Induced Hyperuricemic Mice.

SCOPE: This study aims to investigate the anti-hyperuricemic and nephroprotective effects and the potential mechanisms of the separated gastrointestinal hydrolysates of α-lactalbumin on hyperuricemic mice. METHODS AND RESULTS: The gastrointestinal hydrolysate of α-lactalbumin, the hydrolysate fraction with molecular weight (MW) < 3 kDa (LH-3k), and the fragments with smallest MW among LH-3K harvested through dextran gel chromatography (F5) are used. Hyperuricemia mice are induced via daily oral gavage of potassium oxonate and hypoxanthine. F5 displays the highest in vitro xanthine oxidase (XO) inhibition among all the fractions separated from LH-3k. Oral administration of F5 significantly reduces the levels of serum uric acid (UA), creatinine, and urea nitrogen. F5 treatment could ameliorate kidney injury through alleviating oxidative stress and inflammation. F5 alleviates hyperuricemia in mice by inhibiting hepatic XO activity and regulating the expression of renal urate transporters. Gut microbiota analysis illustrates that F5 administration increases the abundance of some SCFAs producers, and inhibits the growth of hyperuricemia and inflammation associated genera. LH-3k exhibits similar effects but does not show significance as those of the F5 fraction. CONCLUSION: The anti-hyperuricemia and nephroprotective functions of F5 are mediated by inhibiting hepatic XO activity, ameliorating oxidative stress and inflammation, regulating renal urate transporters, and modulating the gut microbiota in hyperuricemic mice.

Unveiling unstable non-acid incidence in Holstein cows fed with corn silage or sugarcane.

We aimed to evaluate the incidence of unstable non-acid milk (UNAM) in cows fed either sugarcane or corn silage. Second, we aimed to evaluate the effect of daily variation (d 1 to 4) and alcohol grades (72, 78, and 80%) on UNAM incidence. The experiment was conducted as a split-plot crossover design, with 2 periods and 2 roughage types (sugarcane or corn silage). Thirteen multiparous Holstein cows with an average of 281 ± 29 d in milk were randomly distributed into 2 diets. Individual blood (analysis of total proteins, albumin, urea, calcium, phosphorus, magnesium, iron, chloride, glucose, and lactate) and milk samples (analysis of protein, fat, lactose and total solids, somatic cell count, and characterization of the protein profile) were collected during the last 4 d of each period. For UNAM identification, the alcohol test was conducted in milk samples at 4°C; specifically, if the sample presented the formation of clots, this would be noted as positive for UNAM. In addition, the Dornic acidity analysis was performed in the same samples to evaluate the true milk acidity. The use of sugarcane and higher degrees of alcohol were associated with increased UNAM. We observed no daily variation in UNAM. Nevertheless, we found no roughage type effect on the variables most commonly associated with UNAM, such as changes in salts in the casein micelle and, consequently, the zeta potential and the κ-casein (CN) fraction. The Pearson correlation analysis showed that the zeta potential and the concentrations of αS2-CN, blood ionic calcium, lactate, and glucose increased as the incidence of UNAM increased, showing a positive correlation among these variables. In contrast, the concentrations of lactose, phosphorus, and potassium decreased as UNAM increased, presenting a negative correlation. This study brought important discoveries to unveil why cows manifest UNAM. For instance, higher alcohol grades and cows fed with sugarcane had increased the incidence of UNAM. Additionally, animals with a higher incidence of UNAM (sugarcane-fed cows) were related to increased ionic calcium and glucose and changes in milk protein profile, with lower levels of BSA, ß-CN, and α-lactalbumin and greater αS1-CN content, all of which were correlated with UNAM. Nonetheless, this trial also provides evidence for the need for further studies to better understand the physiological mechanisms that directly affect the stability of milk protein.

α-Lactalbumin peptide Asp-Gln-Trp alleviates hepatic insulin resistance and modulates gut microbiota dysbiosis in high-fat diet-induced NAFLD mice.

The progression of nonalcoholic fatty liver disease (NAFLD) is closely related to insulin resistance and gut microbiota. Dietary interventions have emerged as effective palliative strategies for NAFLD. The present study investigated the potential mechanisms by which α-lactalbumin peptide Asp-Gln-Trp (DQW) ameliorated insulin resistance and gut microbiota dysbiosis in high-fat diet (HFD)-induced NAFLD mice. The results demonstrated that DQW treatment alleviated HFD-induced body weight gain, hepatic steatosis, insulin resistance, and dyslipidemia. DQW treatment also increased the ratio of Bacteroides to Firmicutes in the gut, reduced the relative abundance of pathogenic bacteria (such as Bacteroides, Blautia, and Alistipes) and enhanced the relative abundance of short-chain fatty acid (SCFA)-producing bacteria (such as Muribaculaceae, Lachnospiraceae_NK4A136_group, and Rikenellaceae_RC9_gut_group). DQW treatment promoted the production of SCFAs and subsequently improved intestinal barrier integrity and inflammation. Furthermore, the results of real-time quantitative PCR (qRT-PCR) and western blotting further proved that the effects of DQW on the attenuation of hepatic insulin resistance were mediated by the PPARα and IRS1/PI3K/AKT pathways. Taken together, these results indicated that DQW treatment could attenuate HFD-induced NAFLD and insulin resistance by modulating gut microbiota composition, enhancing the SCFA levels, and activating the PPARα and IRS1/PI3K/Akt pathways.

Endosperm-specific accumulation of human α-lactalbumin increases seed lysine content in maize.

KEY MESSAGE: This study demonstrated high expression and accumulation of human α-lactalbumin in transgenic maize, and significant improvement of lysine content in maize endosperm. As a high-yield crop, lack of lysine in endosperm storage protein is a major defect of maize (Zea mays L.). Specifically expression of foreign proteins is a potential way to improve lysine content in maize endosperm. Human α-lactalbumin is such a protein with high lysine content and high nutritional value. In this study, the codon-optimized human lactalbumin alpha (LALBA) gene was driven by maize endosperm-specific 27 kD γ-zein promoter, and transformed into maize. Five independent transgenic lines were obtained, and LALBA was highly expressed in endosperm in all these lines. Protein assay indicated that human α-lactalbumin was highly accumulated in maize endosperm. Immuno-localization assay indicated that human α-lactalbumin was mainly deposited into the protein body (PB). Protein interaction assay showed that human α-lactalbumin interacted with 16 kD γ-zein, which might lead to its deposition to the PBs. Amino acid analysis of two independent transgenic lines showed significant increase of lysine contents in transgenic endosperm, with 47.26% and 45.15% increase to their non-transgenic seeds, respectively. We obtained transgenic maize with endosperm-specific accumulation of human α-lactalbumin at high level and increased the lysine content in maize endosperm. This study demonstrated an effective way to improve the nutritional value of maize seeds.

The kinetic mechanism of cations induced protein nanotubes self-assembly and their application as delivery system.

The amphiphilic proteins can be used as building blocks (BBs) forming various self-assemblies. Understanding their self-assembly mechanism is important for designing novel nanomaterials. Herein, the BBs dimers were first prepared from carboxyl-abundant enzymolyzed α-lactalbumin (α-lac) at 50 °C. Then the unidentate coordination of Ca2+ between the BBs caused a ß-sheet stacking to further self-assemble into nanotubes (NTs). Compared with the traditional "one-pot" method, a step-wise new method was applied to study hydrolysis, aggregation and self-assembly processes separately. The α-lac was hydrolyzed into 11 kDa amphiphilic peptides independent of temperature while a BBs dimer was formed at 50 °C by hydrophobic interaction. Ca2+ induced a conformational change of BBs and promoted these BBs gradually aggregate into 10 strands of filaments, which twisted into helical ribbons by electrostatic repulsion. Ca2+ further induced the twisted helical ribbons closed into NTs driven by the reduction of line tension energy. Besides, the carboxyl-Ca2+ coordination dominated NTs elongation in the longitudinal direction and filaments aggregation in the lateral direction with the same binding stoichiometry of 1:1 respectively. Finally, NTs successfully encapsulated curcumin and improved the viscosity of liquid food. α-Lac NTs show a high potential as a delivery system for food applications.

Lipid bilayer composition as a determinant of cancer cell sensitivity to tumoricidal protein-lipid complexes.

Complexes formed by the alpha1 N-terminal peptide of alpha-lactalbumin and oleic acid (alpha1-oleate) interact with lipid bilayers. Plasma membrane perturbations trigger tumor cell death but normal differentiated cells are more resistant, and their plasma membranes are less strongly affected. This study examined membrane lipid composition as a determinant of tumor cell reactivity. Bladder cancer tissue showed a higher abundance of unsaturated lipids enriched in phosphatidylcholine, PC (36:4) and PC (38:4), and sphingomyelin, SM (36:1) than healthy bladder tissue, where saturated lipids predominated and the lipid extracts from bladder cancer tissue inhibited the tumoricidal effect of the complex more effectively than healthy tissue extracts. Furthermore, unsaturated PC in solution inhibited tumor cell death, and the complex interacted with giant unilamellar vesicles formed by PC, confirming the affinity of alpha1-oleate for fluid membranes enriched in PC. Quartz Crystal Microbalance with dissipation monitoring (QCM-D) detected a preference of the complex for the liquid-disordered phase, suggesting that the insertion into PC-based membranes and the resulting membrane perturbations are influenced by membrane lipid saturation. The results suggest that the membrane lipid composition is functionally important and that specific unsaturated membrane lipids may serve as "recognition motifs" for broad-spectrum tumoricidal molecules such as alpha1-oleate.

SIRT1 Mediates Neuroprotective and Neurorescue Effects of Camel α-Lactalbumin and Oleic Acid Complex on Rotenone-Induced Parkinson's Disease.

Parkinson's disease (PD) is the second most common devastating neurodegenerative disorder. Presently used therapies for PD have severe side effects and are limited to only temporary improvement. Therefore, a new therapeutic approach to treat PD urgently needs to be developed. α-Lactalbumin, the most abundant milk protein in camel milk, has been attributed to various medicinal properties. This study intended to investigate the neuroprotective efficacy of the camel α-lactalbumin and oleic acid (CLOA) complex. One mechanism postulated to underlie neuroprotection by the CLOA complex is the induction of silent information regulatory protein (SIRT1). SIRT1 is known to be involved in several pathological and physiological processes, and it has been suggested that SIRT1 plays a protective role in PD. Oxidative stress, inflammation, mitochondrial dysfunction, and apoptosis are involved in PD pathogenesis. Our results revealed that SIRT1 inhibits oxidative stress by maintaining HIF-1α in a deacetylated state. SIRT1 upregulates the expression of FOXO3a and HSF-1, thus inhibiting apoptosis and maintaining the homeostasis of cellular proteins. Increased SIRT1 expression reduces the levels of TNF-α, IL-6, and IL-8, which in turn inhibits neuroinflammation. In addition to SIRT1, the CLOA complex also enhances the expression of survivin and leptin and promotes the survival of neuroblastoma cells. Altogether, our results suggest that the CLOA complex might be a novel therapeutic molecule that could ameliorate neuronal cell damage in PD.

Efficient Bioproduction of Human Milk Alpha-Lactalbumin in Komagataella phaffii.

Alpha-lactalbumin (α-LA; the most abundant whey protein in human milk) contributes to infant development, providing bioactive peptides and essential amino acids. Here, Komagataella phaffii (K. phaffii) was selected as the production host. We found that the K. phaffii host X33 was suitable for expressing the target protein, yielding 5.2 mg·L-1 α-LA. Thereafter, several secretory signal peptides were applied to obtain a higher titer of α-LA. The strain with α-factor secretory signal peptide secreted the highest extracellular titer. Additionally, promoters AOX1, GAP, and GAP(m) were compared and applied. The strain with the promoter AOX1 produced the highest extracellular titer. In addition, coexpressing human protein disulfide isomerase A3 (hPDIA3) increased the titer by 27%. Human α-LA production by the strain X33-pPICZαA-hLALBA-hPDIA3 reached 56.3 mg·L-1 in a 3 L bioreactor. This is the first report of successful secretory human α-LA expression in K. phaffii and lays foundations for the simulation of human milk for infant formulas and further development of bioengineered milk.

Purification and Handling of the Chaperonin GroEL.

GroEL is an important model molecular chaperone. Despite being extensively studied, several critical aspects of its functionality are still in dispute due partly to difficulties in obtaining protein samples of consistent purity. Here I describe an easy-to-carry-out purification protocol that can reliably produce highly purified and fully functional GroEL protein in large quantities. The method takes advantage of the remarkable stability of the GroEL tetradecamer in 45% acetone which efficiently extracts and removes tightly bound substrate proteins that cannot be separated from GroEL by the usual chromatographic methods. The efficiency of the purification method can be assessed by the amount of residual tryptophan fluorescence associated with the purified GroEL sample. The functionality of the thus obtained GroEL sample is demonstrated by measuring its ATPase turnover both in the presence and absence of the GroEL model substrate protein α-lactalbumin.

Evolution of milk oligosaccharides: Origin and selectivity of the ratio of milk oligosaccharides to lactose among mammals.

BACKGROUND: The carbohydrate fraction of mammalian milk is constituted of lactose and oligosaccharides, most of which contain a lactose unit at their reducing ends. Although lactose is the predominant saccharide in the milk of most eutherians, oligosaccharides significantly predominate over lactose in the milk of monotremes and marsupials. SCOPE OF REVIEW: This review describes the most likely process by which lactose and milk oligosaccharides were acquired during the evolution of mammals and the mechanisms by which these saccharides are digested and absorbed by the suckling neonates. MAJOR CONCLUSIONS: During the evolution of mammals, c-type lysozyme evolved to α-lactalbumin. This permitted the biosynthesis of lactose by modulating the substrate specificity of ß4galactosyltransferase 1, thus enabling the concomitant biosynthesis of milk oligosaccharides through the activities of several glycosyltransferases using lactose as an acceptor. In most eutherian mammals the digestion of lactose to glucose and galactose is achieved through the action of intestinal lactase (ß-galactosidase), which is located within the small intestinal brush border. This enzyme, however, is absent in neonatal monotremes and macropod marsupials. It has therefore been proposed that in these species the absorption of milk oligosaccharides is achieved by pinocytosis or endocytosis, after which digestion occurs through the actions of several lysosomal acid glycosidases. This process would enable the milk oligosaccharides of monotremes and marsupials to be utilized as a significant energy source for the suckling neonates. GENERAL SIGNIFICANCE: The evolution and significance of milk oligosaccharides is discussed in relation to the evolution of mammals.