-
European Journal of Immunology Apr 2021HAMLET is a protein-lipid complex with a specific and broad bactericidal and tumoricidal activity, that lacks cytotoxic activity against healthy cells. In this study, we...
HAMLET is a protein-lipid complex with a specific and broad bactericidal and tumoricidal activity, that lacks cytotoxic activity against healthy cells. In this study, we show that HAMLET also has general immune-stimulatory effects on primary human monocyte-derived dendritic cells and macrophages (Mo-DC and Mo-M) and murine RAW264.7 macrophages. HAMLET, but not its components alpha-lactalbumin or oleic acid, induces mature CD14 CD83 Mo-DC and M1-like CD14 CD86 Mo-M surface phenotypes. Concomitantly, inflammatory mediators, including IL-2, IL-6, IL-10, IL-12 and MIP-1α, were released in the supernatant of HAMLET-stimulated cells, indicating a mainly pro-inflammatory phenotype. The HAMLET-induced phenotype was mediated by calcium, NFκB and p38 MAPK signaling in Mo-DCs and calcium, NFκB and ERK signaling in Mo-M as inhibitors of these pathways almost completely blocked the induction of mature Mo-DCs and M1-like Mo-M. Compared to unstimulated Mo-DCs, HAMLET-stimulated Mo-DCs were more potent in inducing T cell proliferation and HAMLET-stimulated macrophages were more efficient in phagocytosis of Streptococcus pneumoniae in vitro. This indicates a functionally activated phenotype of HAMLET-stimulated DCs and macrophages. Combined, we propose that HAMLET has a two-fold anti-bacterial activity; one inducing direct cytotoxic activity, the other indirectly mediating elimination of bacteria by activation of immune cells of the myeloid lineage.
Topics: Animals; Cell Survival; Cells, Cultured; Cytokines; Dendritic Cells; Humans; Inflammation Mediators; Lactalbumin; Macrophages; Mice; Monocytes; Myeloid Cells; NF-kappa B; Oleic Acids; Phagocytosis; Phenotype; RAW 264.7 Cells; Signal Transduction; p38 Mitogen-Activated Protein Kinases
PubMed: 33348422
DOI: 10.1002/eji.202048813 -
Biochemistry and Biophysics Reports Mar 2022Evolutionarily homologous proteins bovine α-lactoalbumin (αLA) and hen egg-white lysozyme (HEL) are very similar in primary, secondary and tertiary structures...
Sensitive and resistant of the homologous disulfide-bridged proteins α-lactalbumin and lysozyme to attack of hydrogen-atoms, dithiothreitol and trifluoroacetic acid, examined by matrix-assisted laser desorption/ionization mass spectrometry.
BACKGROUND
Evolutionarily homologous proteins bovine α-lactoalbumin (αLA) and hen egg-white lysozyme (HEL) are very similar in primary, secondary and tertiary structures involving the location of disulfide-bridges (S-S), and are resistant to the action of hydrolytic enzymes and reagents. It is of interest to examine and compare the difference in backbone cleavage characteristics, by using reductive and hydrolytic reagents.
METHODS
In-source decay (ISD) combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), reductive treatment of αLA and HEL with dithiothreitol (DTT) and acid hydrolysis with trifluoroacetic acid (TFA) were employed to examine the difference in the backbone cleavage characteristics of αLA and HEL.
RESULTS
The treatment of αLA and HEL with DTT/AcOHNH resulted in similar cleavage behaviors of the backbone N-Cα and S-S bonds, i.e., the enhancements of the intensity and range of sequence-reflected fragment ions were very similar. However, the treatment of αLA with DTT/TFA resulted in unexpected residue-specific degradation at the peptide bond of the Asp-Xxx, Xxx-Ser/Thr, Gln-Xxx, Xxx-Gly and Gly-Xxx residues, while HEL did not occur such degradation.
CONCLUSIONS
The results obtained above indicate that acidic αLA is very sensitive to acidic additive such as TFA, while basic HEL is resistance to acidic additives.
GENERAL SIGNIFICANCE
The study demonstrates the sensitive and resistant of evolutionary homologous proteins αLA and HEL to the acid hydrolysis and these characters come from acidic and basic nature of the proteins.
PubMed: 35111980
DOI: 10.1016/j.bbrep.2022.101212 -
Molecules (Basel, Switzerland) Jan 2021In the last few decades, development of novel experimental techniques, such as new types of disulfide (SS)-forming reagents and genetic and chemical technologies for... (Review)
Review
In the last few decades, development of novel experimental techniques, such as new types of disulfide (SS)-forming reagents and genetic and chemical technologies for synthesizing designed artificial proteins, is opening a new realm of the oxidative folding study where peptides and proteins can be folded under physiologically more relevant conditions. In this review, after a brief overview of the historical and physicochemical background of oxidative protein folding study, recently revealed folding pathways of several representative peptides and proteins are summarized, including those having two, three, or four SS bonds in the native state, as well as those with odd Cys residues or consisting of two peptide chains. Comparison of the updated pathways with those reported in the early years has revealed the flexible nature of the protein folding pathways. The significantly different pathways characterized for hen-egg white lysozyme and bovine milk α-lactalbumin, which belong to the same protein superfamily, suggest that the information of protein folding pathways, not only the native folded structure, is encoded in the amino acid sequence. The application of the flexible pathways of peptides and proteins to the engineering of folded three-dimensional structures is an interesting and important issue in the new realm of the current oxidative protein folding study.
Topics: Animals; Cattle; Cysteine; Disulfides; Lactalbumin; Muramidase; Oxidation-Reduction; Peptides; Protein Conformation; Protein Folding; Proteins
PubMed: 33401729
DOI: 10.3390/molecules26010195 -
ACS Omega Oct 2020Protein aggregation is among the most challenging new frontiers in protein chemistry as well as in molecular medicine and has direct implications in protein misfolding....
Protein aggregation is among the most challenging new frontiers in protein chemistry as well as in molecular medicine and has direct implications in protein misfolding. This study investigated the role of sugar molecules (glucose, fructose, sucrose, and the mixture of glucose and fructose) in protecting the structural integrity of α-lactalbumin (α-LA) against aggregation. The research focused here is the inhibitory capabilities of sugars against α-LA fibril formation investigated employing diverse multispectroscopic and microscopic techniques. The aggregation was induced in α-LA thermally with a change in concentration. UV-vis spectroscopy, ThT binding assay, Trp fluorescence, Rayleigh scattering, and turbidity assay depicted synchronized results. Further, transmission electron microscopy (TEM) complemented that a mixture of glucose and fructose was the best inhibitor of α-LA fibril formation. Inhibition of α-LA aggregation by sugar osmolytes is attributed to the formation of hydrogen bonds between these osmolytes, as evidenced by the molecular docking results. This hydrogen bonding is a key player that prevents aggregation in α-LA in the presence of sugar osmolytes. This study provides an insight into the ability of naturally occurring sugar osmolytes to inhibit fibril formation and can serve as a platform to treat protein misfolding and aggregation-oriented disorders.
PubMed: 33111013
DOI: 10.1021/acsomega.0c04062 -
Theranostics 2021Most contemporary cancer therapeutic paradigms involve initial imaging as a treatment roadmap, followed by the active engagement of surgical operations. Current...
Most contemporary cancer therapeutic paradigms involve initial imaging as a treatment roadmap, followed by the active engagement of surgical operations. Current approved intraoperative contrast agents exemplified by indocyanine green (ICG) have a few drawbacks including the inability of pre-surgical localization. Alternative near-infrared (NIR) dyes including IRDye800cw are being explored in advanced clinical trials but often encounter low chemical yields and complex purifications owing to the asymmetric synthesis. A single contrast agent with ease of synthesis that works in multiple cancer types and simultaneously allows presurgical imaging, intraoperative deep-tissue three-dimensional visualization, and high-speed microscopic visualization of tumor margins spatiotemporally complementary modalities would be beneficial. Due to the lack of commercial availability and the absence of detailed synthesis and characterization, we proposed a facile and scalable synthesis pathway for the symmetric NIR water-soluble heptamethine sulfoindocyanine IRDye78. The synthesis can be accomplished in four steps from commercially-available building blocks. Its symmetric resonant structure avoided asymmetric synthesis problems while still preserving the benefits of analogous IRDye800cw with commensurable optical properties. Next, we introduced a low-molecular-weight protein alpha-lactalbumin (α-LA) as the carrier that effectively modulates the hepatic clearance of IRDye78 into the preferred renal excretion pathway. We further implemented Zr radiolabeling onto the protein scaffold for positron emission tomography (PET). The multimodal imaging capability of the fluorophore-protein complex was validated in breast cancer and glioblastoma. The scalable synthesis resulted in high chemical yields, typically 95% yield in the final step of the chloro dye. Chemical structures of intermediates and the final fluorophore were confirmed. Asymmetric IRDye78 exhibited comparable optical features as symmetric IRDye800cw. Its well-balanced quantum yield affords concurrent dual fluorescence and optoacoustic contrast without self-quenching nor concentration-dependent absorption. The NHS ester functionality modulates efficient covalent coupling to reactive side-chain amines to the protein carrier, along with desferrioxamine (DFO) for stable radiolabeling of Zr. The fluorophore-protein complex advantageously shifted the biodistribution and can be effectively cleared through the urinary pathway. The agent accumulates in tumors and enables triple-modal visualization in mouse xenograft models of both breast and brain cancers. This study described in detail a generalized strategic modulation of clearance routes towards the favorable renal clearance, the introduction of α-LA. IRDye78 as a feasible alternative of IRDye800cw currently in clinical phases was proposed with a facile synthesis and fully characterized for the first time. This fluorophore-protein complex with stable radiolabeling should have great potential for clinical translation where it could enable an elegant workflow from preoperative planning to intraoperative deep tissue and high-resolution image-guided resection.
Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Female; Fluorescence; Fluorescent Dyes; Glioblastoma; Humans; Indocyanine Green; Indoles; Lactalbumin; Mice; Mice, Inbred C57BL; Optical Imaging; Positron-Emission Tomography; Spectroscopy, Near-Infrared; Tissue Distribution; Tomography, X-Ray Computed
PubMed: 33456558
DOI: 10.7150/thno.54928 -
Frontiers in Nutrition 2022Observational studies suggest differences between breast-fed and formula-fed infants in developmental myelination, a key brain process for learning. The study aims to...
BACKGROUND AND OBJECTIVES
Observational studies suggest differences between breast-fed and formula-fed infants in developmental myelination, a key brain process for learning. The study aims to investigate the efficacy of a blend of docosahexaenoic acid (DHA), arachidonic acid (ARA), iron, vitamin B12, folic acid, and sphingomyelin (SM) from a uniquely processed whey protein concentrate enriched in alpha-lactalbumin and phospholipids compared with a control formulation on myelination, cognitive, and behavioral development in the first 6 months of life.
METHODS
These are 6-month results from an ongoing two-center, randomized controlled trial with a 12-month intervention period (completed for all participants). In this study, full term, neurotypical infants of both sexes ( = 81) were randomized into investigational ( = 42) or control groups ( = 39). In addition, non-randomized breast-fed children ( = 108) serve as a natural reference group. Main outcomes are myelination (MRI), cognitive (Bayley Scales of Infant and Toddler Development, 3rd edition [Bayley-III]), social-emotional development (Ages and Stages Questionnaires: Social-Emotional, 2nd edition [ASQ:SE-2]), sleep (Brief Infant Sleep Questionnaire [BISQ]), and safety (growth and adverse events [AEs]).
RESULTS
The full analyses set comprises = 66 infants. Significant differences in myelin structure, volume, and rate of myelination were observed in favor of the investigational myelin blend at 3 and 6 months of life. Effects were demonstrated for whole brain myelin and for cerebellar, parietal, occipital, and temporal regions, known to be functionally involved in sensory, motor, and language skills. No statistically significant differences were found for early behavior and cognition scores.
CONCLUSIONS
This is the first study demonstrating the efficacy of a myelin nutrient blend in well-nourished, term infants on developmental myelination, which may be foundational for later cognitive and learning outcomes.
CLINICAL TRIAL REGISTRATION
ClinicalTrials.gov, identifier: NCT03111927.
PubMed: 35242798
DOI: 10.3389/fnut.2022.823893 -
Journal of Ovarian Research May 2018Myo-inositol (MI), successfully used in polycystic ovary syndrome (PCOS), was administered with α-LA to exploit its action of favouring the passage of other molecules... (Clinical Trial)
Clinical Trial
BACKGROUND
Myo-inositol (MI), successfully used in polycystic ovary syndrome (PCOS), was administered with α-LA to exploit its action of favouring the passage of other molecules through biological barriers, and also considering its anti-inflammatory effect.
METHODS
PCOS patients, according to the Rotterdam ESHRE-ASRM criteria, with anovulation and infertility > 1 year, were included in this open and prospective study. The preliminary phase was aimed at determining a set of MI-resistant PCOS patients. This treatment involved 2 g MI, taken twice per day by oral route, for three months. The Homeostasis Model Assessment (HOMA) index and MI plasma levels were measured. In the main phase, previously selected MI-resistant patients received the same daily amount of MI plus 50 mg α-LA twice a day, for a further three months. Ovulation was assessed using ultrasound examination on days 12, 14 and 20 of the cycle. The HOMA index, lipid, hormone and MI plasma levels were detected at baseline and at the end of this phase.
RESULTS
Thirty-seven anovulatory PCOS subjects were included in the study. Following MI treatment, 23 of the 37 women (62%) ovulated, while 14 (38%) were resistant and did not ovulate. In the latter group, MI plasma levels did not increase. These MI-resistant patients underwent treatment in the main phase of the study, receiving MI and α-LA. After this combined treatment, 12 (86%) of them ovulated. Their MI plasma levels were found to be significantly higher than at baseline; also, a hormone and lipid profile improvement was recorded.
CONCLUSION
The combination of MI with α-LA allowed us to obtain significant progress in the treatment of PCOS MI-resistant patients. Therefore, this new formulation was able to re-establish ovulation, greatly increasing the chances of desired pregnancy.
TRIAL REGISTRATION
Clinical trial registration number: NCT03422289 ( ClinicalTrials.gov registry).
Topics: Adult; Dietary Supplements; Drug Combinations; Drug Resistance; Female; Folic Acid; Humans; Inositol; Lactalbumin; Polycystic Ovary Syndrome; Pregnancy
PubMed: 29747700
DOI: 10.1186/s13048-018-0411-2 -
Food Chemistry Jun 2018This paper set out to differentiate the Maillard induced glycation reactivity of individual milk proteins using different saccharides under well-defined reaction...
This paper set out to differentiate the Maillard induced glycation reactivity of individual milk proteins using different saccharides under well-defined reaction conditions. α-Lactalbumin, β-lactoglobulin and β-casein were incubated with mono-, di- and trisaccharides in the dry state under standardised buffered conditions and glycation was expressed relative to the available reactive groups per protein (DG). Protein reactivity, described by the DG and initial speed of glycation (v), followed the same order for each protein-saccharide incubation: α-lactalbumin > β-lactoglobulin ≫ β-casein. Glycation of whey proteins by different monosaccharides was double that of β-casein. Differences in DG between whey proteins and β-casein decreased with increased saccharide size. A two-fold difference was found for glycation in the presence of the dimers lactose and maltose for β-casein but not for the whey proteins. The percentage of glycated lysines increased with increased lysine to protein size ratio.
Topics: Animals; Caseins; Cattle; Glycosylation; Lactalbumin; Lactoglobulins; Maillard Reaction; Milk Proteins; Monosaccharides; Trisaccharides; Whey Proteins
PubMed: 29478547
DOI: 10.1016/j.foodchem.2018.01.106 -
Breastfeeding Medicine : the Official... Apr 2023Human milk (HM) fortification has been recommended for the nutritional optimization of very low-birthweight infants. This study analyzed the bioactive components of HM... (Observational Study)
Observational Study
Exclusive Human Milk Diet for Extremely Premature Infants: A Novel Fortification Strategy That Enhances the Bioactive Properties of Fresh, Frozen, and Pasteurized Milk Specimens.
Human milk (HM) fortification has been recommended for the nutritional optimization of very low-birthweight infants. This study analyzed the bioactive components of HM and evaluated fortification choices that could accentuate or attenuate the concentration of such components, with special reference to human milk-derived fortifier (HMDF) offered to extremely premature infants as an exclusive human milk diet. An observational feasibility study analyzed the biochemical and immunochemical characteristics of mothers' own milk (MOM), both fresh and frozen, and pasteurized banked donor human milk (DHM), each supplemented with either HMDF or cow's milk-derived fortifier (CMDF). Gestation-specific specimens were analyzed for macronutrients, pH, total solids, antioxidant activity (AA), -lactalbumin, lactoferrin, lysozyme, and α- and -caseins. Data were analyzed for variance applying general linear model and Tukey's test for pairwise comparison. DHM exhibited significantly lower ( < 0.05) lactoferrin and α-lactalbumin concentrations than fresh and frozen MOM. HMDF reinstated lactoferrin and α-lactalbumin and exhibited higher protein, fat, and total solids ( < 0.05) in comparison to unfortified and CMDF-supplemented specimens. HMDF had the highest ( < 0.05) AA, suggesting the potential capability of HMDF to enhance oxidative scavenging. DHM, compared with MOM, has reduced bioactive properties, and CMDF conferred the least additional bioactive components. Reinstatement and further enhancement of bioactivity, which has been attenuated through pasteurization of DHM, is demonstrated through HMDF supplementation. Freshly expressed MOM fortified with HMDF and given , , and appears an optimal nutritional choice for extremely premature infants.
Topics: Infant, Newborn; Infant; Female; Animals; Cattle; Humans; Milk, Human; Infant, Extremely Premature; Lactalbumin; Lactoferrin; Breast Feeding; Diet
PubMed: 37071630
DOI: 10.1089/bfm.2022.0254 -
Heliyon Sep 2020High-value milk proteins, which can be obtained by optimized fractionation procedures, are ideal ingredients in many food applications. Thus, a simple and robust...
High-value milk proteins, which can be obtained by optimized fractionation procedures, are ideal ingredients in many food applications. Thus, a simple and robust analytical method is required for the identification and quantification of these individual milk proteins. Here, we present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) to simultaneously detect and measure target peptides of two major milk proteins, α-lactalbumin (α-LA) and β-casein (β-CN), in raw milk samples from 662 Danish Holstein cows. The MRM quantification of α-LA and β-CN was achieved with limit of detection (LOD) of 0.14 and 0.16 g/L, respectively and reproducibility of the assay <15%. By this newly established MRM-based method, the concentration of α-LA and β-CN in an individual cow's milk ranged from 0.5 to 1.9 (average 1.1) g/L, and from 7.5 to 23.4 (average 15) g/L, respectively. There was no significant effect of parity, whereas significantly increasing concentrations of α-LA and β-CN were observed through lactation (P < 0.001). This shows a considerable biological variation of these two ingredient milk proteins, providing potential varying outputs of fractionation in the dairy streams.
PubMed: 32995587
DOI: 10.1016/j.heliyon.2020.e04620