Tannin-controlled micelles and fibrils of κ-casein.

Effects of green tea tannin epigallocatechin-gallate (EGCG) on thermal-stress-induced amyloid fibril formation of reduced carboxymethylated bovine milk protein κ-casein were studied by dynamical light scattering and small angle X-ray scattering (SAXS). Two populations of aggregates, micelles, and fibrils dominated the time evolution of light scattering intensity and of effective hydrodynamic diameter. SAXS experiments allowed us to resolve micelles and fibrils so that the time dependence of the scattering profile revealed the structural evolution of the two populations. The low-Q scattering intensity prior to an expected increase in time due to fibril growth shows an intriguing rapid decrease, which is interpreted as the release of monomers from micelles. This phenomenon, observed both in the absence and in the presence of EGCG, indicates that under thermal stress free conditions, native monomers are converted to amyloid-prone monomers that do not form micelles. The consumption of free native monomers results in a release of native monomers from micelles because only native proteins participate in micelle-monomer (quasi)equilibrium. This release is reversible, indicating also that native-to-amyloid-prone monomer conversion is reversible as well. We show that EGCG does not bind to protein in fibrils, neither does it affect/prevent the proamyloid conversion of monomers. EGCG hinders the addition of monomers to growing fibrils. These facts allowed us to propose the kinetics model for EGCG-controlled amyloid aggregation of micellar proteins. Therein, we introduced the growth-rate inhibition function, which quantitatively accounts for the effect of EGCG on the fibril growth at any degree of thermal stress.

Inhibition of arginase via jugular infusion of Nω-hydroxy-nor-l-arginine inhibits casein synthesis in lactating dairy cows.

A previous in vitro study revealed that Arg elicits positive effects on casein synthesis through alterations of the Arg-ornithine pathway in bovine mammary epithelial cells. The main purpose of this work was to determine the effects of arginase inhibition using Nω-hydroxy-nor-l-arginine (nor-NOHA) on milk protein synthesis in vivo. Six healthy Chinese Holstein cows with similar body weight (550.0 ± 20 kg; means ± standard deviation), parity (4), body condition score (3.0), milk yield (21.0 ± 1.0 kg), and days in milk (80 ± 2) were selected and randomly assigned to 3 treatments in a replicated 3 × 3 Latin square design with 22 d for each period (7 d for infusion and 15 d for washout). The treatments were (1) control: saline infusion; (2) nor-NOHA: infusion of 125 mg/L of nor-NOHA; (3) nor-NOHA + Arg: infusion of 125 mg/L of nor-NOHA with 9.42 g/L of Arg. The activity of enzymes related to Arg metabolism, milk protein synthesis, and expression of AA transporters was determined. The infusion of nor-NOHA decreased the activity of arginase but had no effect on the activity of ornithine decarboxylase and nitric oxide synthase in serum, and these responses were the same at the gene expression level in mammary gland. In addition, the infusion of nor-NOHA also reduced protein and fat synthesis in milk but had no effect on milk yield. When Arg was infused with nor-NOHA, the activity of total arginase, ornithine decarboxylase, and nitric oxide synthase, and the concentration of casein, protein, and fat in milk did not change compared with the nor-NOHA group, but the milk protein yield, the expression of some Arg transporters (SLC7A5 and SLC7A8), and milk yield increased. Overall, results verified previous in vitro findings indicating that synthesis of casein protein is closely regulated by the Arg-ornithine pathway in bovine mammary gland.

Carboxymethylated-kappa-casein: a convenient tool for the identification of polyphenolic inhibitors of amyloid fibril formation.

Reduced and carboxymethylated-kappa-casein (RCM-kappa-CN) is a milk-derived amyloidogenic protein that readily undergoes nucleation-dependent aggregation and amyloid fibril formation via a similar pathway to disease-specific amyloidogenic peptides like amyloid beta (Abeta), which is associated with Alzheimer's disease. In this study, a series of flavonoids, many known to be inhibitors of Abeta fibril formation, were screened for their ability to inhibit RCM-kappa-CN fibrilisation, and the results were compared with literature data on Abeta inhibition. Flavonoids that had a high degree of hydroxylation and molecular planarity gave good inhibition of RCM-kappa-CN fibril formation. IC(50) values were between 10- and 200-fold higher with RCM-kappa-CN than literature results for Abeta fibril inhibition, however, with few exceptions, they showed a similar trend in potency. The convenience and reproducibility of the RCM-kappa-CN assay make it an economic alternative first screen for Abeta inhibitory activity, especially for use with large compound libraries.

Molecular docking studies and anti-snake venom metalloproteinase activity of Thai mango seed kernel extract.

Snakebite envenomations cause severe local tissue necrosis and the venom metalloproteinases are thought to be the key toxins involved. In this study, the ethanolic extract from seed kernels of Thai mango (Mangifera indica L. cv. 'Fahlun') (Anacardiaceae) and its major phenolic principle (pentagalloylglucopyranose) exhibited potent and dose-dependent inhibitory effects on the caseinolytic and fibrinogenolytic activities of Malayan pit viper and Thai cobra venoms in in vitro tests. molecular docking studies revealed that the binding orientations of the phenolic principles were in the binding pockets of snake venom metalloproteinases (SVMPs). The phenolic principles could form hydrogen bonds with the three histidine residues in the conserved zinc-binding motif and could chelate the Zn(2+) atom of the SVMPs, which could potentially result in inhibition of the venom enzymatic activities and thereby inhibit tissue necrosis.

Trimethylamine N-oxide abolishes the chaperone activity of α-casein: an intrinsically disordered protein.

Osmolytes (small molecules that help in circumventing stresses) are known to promote protein folding and prevent aggregation in the case of globular proteins. However, the effect of such osmolytes on the structure and function of intrinsically disordered proteins (IDPs) has not been clearly understood. Here we have investigated the effect of methylamine osmolytes on α-casein (an IDP present in mammalian milk) and discovered that TMAO (Trimethylamine-N-oxide) but not other methylamines renders α-casein functionless. We observed that the loss of chaperone activity of α-casein in presence of TMAO was due to the induction of an unstable aggregation-prone intermediate. The results indicate that different osmolytes may have different structural and functional consequences on IDPs, and therefore might have clinical implications for a large number of human diseases (e.g., amyloidosis, cancer, diabetes, and neurodegeneration) where IDPs are involved.

Characterization and study of a kappa-casein-like chymosin-sensitive linkage.

The present report is dealing with the identification, in various unrelated proteins, of protein fragments sharing local sequence and structure similarities with the chymosin-sensitive linkage surrounding the Phe-Met/Ile bond of kappa-caseins. In all these proteins, this linkage is observed within an exposed beta-strand-like structure, as also predicted for kappa-caseins. The structure of one of these fragments, included in glutamine synthetase, particularly superimposes well with the conformation observed for a chymosin inhibitor (CP-113972) within the complex it forms with chymosin and can be similarly accommodated by specificity pockets within the enzyme substrate binding cleft. The effect of the enzyme activity of chymosin was thus tested on glutamine synthetase. Chymosin cut the latter at the Phe-Met linkage, suggesting that this system may locally resemble the kappa-casein/chymosin complex.

Isolation and characterization of DM40 and DM43, two snake venom metalloproteinase inhibitors from Didelphis marsupialis serum.

From Didelphis marsupialis serum, two antihemorrhagic proteins were isolated by DEAE-Sephacel, Phenyl-Sepharose and Superdex 200 and characterized. Their masses by mass spectrometry were 40318 AMU for DM40 and 42373 and 43010 AMU for DM43, indicating the presence of isoforms for the last. Molecular masses of 44.8 and 47.3 were obtained by SDS-PAGE, respectively for DM40 and DM43. Both inhibitors showed isoelectric points lower than 3.5 and glycosylation percentages varying from 20.5 to 29.0%, as estimated by chemical deglycosylation and amino acid analysis. N-terminal sequences of the first 17 residues of DM40 and DM43 were identical except for the exchange of R9 for P9. Both were homologous to oprin, a similar inhibitor from Didelphis virginiana serum. No evidence of complex formation between DM40 and DM43 was observed either by native PAGE or gel filtration chromatography. In addition to the antihemorrhagic activity, DM40 and DM43 inhibited the hydrolysis of casein, fibrinogen and fibronectin by Bothrops jararaca venom. DM43 also showed antilethal, antiedematogenic and antihyperalgesic activities. None of the inhibitors showed enzymatic activity on casein. Both proteins formed stable complexes with jararhagin and inhibited its hemorrhagic effect as well as the enzymatic activity of this toxin on fluorogenic substrate.

Inhibitory effect of fluvoxamine on ß-casein expression via a serotonin-independent mechanism in human mammary epithelial cells.

Selective serotonin reuptake inhibitors (SSRIs) are widely used as a first-line therapy in postpartum depression. The objective of this study was to determine the mechanism underlying the inhibitory effects of the SSRI, fluvoxamine, on ß-casein expression, an indicator of lactation, in MCF-12A human mammary epithelial cells. Expression levels of serotonin (5-hydroxytryptamine; 5-HT) transporter, an SSRI target protein, and tryptophan hydroxylase 1, a rate-limiting enzyme in 5-HT biosynthesis, were increased in MCF-12A cells by prolactin treatment. Treatment with 1 µM fluvoxamine for 72 h significantly decreased protein levels of ß-casein and phosphorylated signal transducer and activator transcription 5 (pSTAT5). Extracellular 5-HT levels were significantly increased after exposure to 1 µM fluvoxamine, in comparison with those of untreated and vehicle-treated cells; however, extracellular 5-HT had little effect on the decrease in ß-casein expression. Expression of glucose-related protein 78/binding immunoglobulin protein, a regulator of endoplasmic reticulum (ER) stress, was significantly increased after treatment with 1 µM fluvoxamine for 48 h. Exposure to tunicamycin, an inducer of ER stress, also decreased expression of ß-casein and pSTAT5 in a manner similar to fluvoxamine. Our results indicate that fluvoxamine suppresses ß-casein expression in MCF-12A cells via inhibition of STAT5 phosphorylation caused by induction of ER stress. Further studies are required to confirm the effect of fluvoxamine on the function of mammary epithelial cells.

The liver-enriched inhibitory protein isoform of CCAAT/enhancer-binding protein beta, but not nuclear factor-kappaB, mediates the transcriptional inhibition of beta-casein by tumor necrosis factor-alpha.

TNF-alpha is a physiological regulator of mammary gland development that stimulates the growth of both normal and malignant mammary epithelial cells in primary culture and inhibits functional differentiation. To understand how TNF exerts its effects, the current study examined the mechanism by which TNF down-regulates expression of the beta-casein and whey acidic protein (WAP) genes. TNF treatment markedly decreased activity of the beta-casein and WAP promoters in transiently transfected HC11 mammary epithelial cells. Overexpression of the nuclear factor-kappaB (NFkappaB) p50 and/or p65 proteins increased the transcriptional activity of the beta-casein and WAP promoters in HC11 cells, suggesting that the inhibitory effect of TNF on transcription of these genes is not mediated by NFkappaB. This was further confirmed in experiments in which an NFkappaB super-repressor was overexpressed, and by deletion of an NFkappaB binding site in the beta-casein promoter. In contrast, we found that TNF induced both nuclear expression and the DNA-binding activity of liver-enriched inhibitory protein (LIP) isoform of CCAAT/enhancer-binding protein beta. Moreover, cotransfection of LIP and beta-casein expression vectors showed that LIP suppressed the transcriptional activity of the beta-casein promoter. Together, these results suggest that LIP plays a critical role in mediating TNF-induced down-regulation of the beta-casein gene.

Impairment of proteasome function upon UVA- and UVB-irradiation of human keratinocytes.

The major environmental influence for epidermal cells is sun exposure and the harmful effect of UV radiation on skin is related to the generation of reactive oxygen species that are altering cellular components including proteins. It is now well established that the proteasome is responsible for the degradation of oxidized proteins. Therefore, the effects of UV-irradiation on proteasome have been investigated in human keratinocyte cultures. Human keratinocytes were irradiated with 10 J/cm(2) of UVA and 0.05 J/cm(2) of UVB and proteasome peptidase activities were measured in cell lysates using fluorogenic peptides. All three peptidase activities were decreased as early as 1 h and up to 24 h after irradiation of the cells. Increased levels of oxidized and ubiquitinated proteins as well as proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal were also observed in irradiated cells. However, immunopurified 20S proteasome exhibited no difference in both peptidase specific activities and 2D gel pattern of subunits in irradiated cells, ruling out the possibility that the 20S proteasome could be a target for the UV-induced damage. Finally, extracts from irradiated keratinocytes were able to inhibit degradation by the proteasome, demonstrating the presence of endogeneous inhibitors, including 4-hydroxy-2-nonenal modified proteins, generated upon UV-irradiation.

Effect of beta-casomorphin on neonatal sleep in rats.

The effects of bovine beta-casomorphin(1-7) (Tyr-Pro-Phe-Pro-Gly-Pro-Ile) on neonatal sleep in rats were studied. The pups received intraperitoneal injections of beta-casomorphin(1-7) (1 mg, 5 mg, 10 mg, 50 mg, or 100 mg/kg) or a corresponding volume of sodium chloride. In any of the doses used, beta-casomorphin(1-7) had no effect on waking. Only 100 mg/kg caused significant changes in sleep: the percentage of quiet state of the total recording time (TRT) increased and the percentage of active sleep decreased. Beta-casomorphin(1-7) did not cause significant respiratory depression. Naloxone pretreatment (1 mg/kg IP) reversed the effects of beta-casomorphin(1-7) on sleep, a finding which suggests that opiate mu-receptors are involved in mediating the sleep effects of beta-casomorphin.

Purification and partial characterisation of a fish lethal extracellular protease from Vibrio pelagius.

The purification, characterisation and lethal effect of an extracellular protease present in the extracellular products (ECPs) of a pathogenic Vibrio pelagius (7P) strain are described. The extracellular protease was purified by size-exclusion high-performance liquid chromatography and characterised by enzymatic assays. The lethal effect was evaluated by injection into fish. The native protease had a molecular mass of 39 kDa, was active on casein and L-leucyl-beta-naphthylamide (LNA) and its metalloprotease nature was shown by the LNA inhibition profile. Kinetic studies on the hydrolysis of casein and LNA confirmed a competitive inhibition of one substrate with respect to the other. The temperature assays showed that both aminopeptidolytic and caseinolytic activities were labile at 70 degrees C for 3 min. The N-terminal amino acid sequence of 7P protease revealed a high degree of homology with other metalloproteases of Vibrio species that are implicated in virulence. The purified 7P protease showed an LD(50) of 1.77 microgprotein/g fish for turbot. The quick lethal effect (<24h) and the macroscopic damage (external haemorrhagic areas, principally on fins and mouth, petechial haemorrhages in internal organs, but with no external or internal apparent necrotic areas) detected in the host were similar to those obtained by injection of total ECP and live cells of 7P strain. An extracellular protease with endopeptidolytic and exopeptidolytic activities, responsible for the lethal effect of ECP and clinical signs of vibriosis in turbot was purified from a pathogenic V. pelagius (7P) strain.

Inhibitory activity of YKL-40 in mammary epithelial cell differentiation and polarization induced by lactogenic hormones: a role in mammary tissue involution.

We previously reported that a secreted glycoprotein YKL-40 acts as an angiogenic factor to promote breast cancer angiogenesis. However, its functional role in normal mammary gland development is poorly understood. Here we investigated its biophysiological activity in mammary epithelial development and mammary tissue morphogenesis. YKL-40 was expressed exclusively by ductal epithelial cells of parous and non-parous mammary tissue, but was dramatically up-regulated at the beginning of involution. To mimic ductal development and explore activity of elevated YKL-40 during mammary tissue regression in vivo, we grew a mammary epithelial cell line 76N MECs in a 3-D Matrigel system in the presence of lactogenic hormones including prolactin, hydrocortisone, and insulin. Treatment of 76N MECs with recombinant YKL-40 significantly inhibited acinar formation, luminal polarization, and secretion. YKL-40 also suppressed expression of E-cadherin but increased MMP-9 and cell motility, the crucial mechanisms that mediate mammary tissue remodeling during involution. In addition, engineering of 76N MECs with YKL-40 gene to express ectopic YKL-40 recapitulated the same activities as recombinant YKL-40 in the inhibition of cell differentiation. These results suggest that YKL-40-mediated inhibition of cell differentiation and polarization in the presence of lactogenic hormones may represent its important function during mammary tissue involution. Identification of this biophysiological property will enhance our understanding of its pathologic role in the later stage of breast cancer that is developed from poorly differentiated and highly invasive cells.

Presence of a lethal protease in the extracellular products of Vibrio splendidus-Vibrio lentus related strains.

The presence of a lethal extracellular 39-kDa protease, a virulence determinant of a Listonella pelagia strain which produces vibriosis in turbot, was determined in the extracellular products (ECP) of 33 Vibrionaceae strains. Both immunological and enzymatic techniques distinguished this specific protease from other Vibrionaceae proteins. It was detected in 15% (5/33) of the ECPs assayed belonging to strains of the Vibrio splendidus-V. lentus related group isolated in Galician aquaculture systems (NW Spain). As these strains were associated with diseased octopus and cultured turbot, were able to colonize the internal organs of fish and produced a lethal ECP for fish, they are a potential risk for the health of reared aquatic organisms.

Colchicine inhibition of the first phase of amyloid synthesis in experimental animals.

Colchicine was found to inhibit the first phase of casein-induced synthesis of murine amyloid. When mice were treated with colchicine during the first 7 days of an amyloid induction regimen or when colchicine was given to the donor mice in a transfer model, the amyloidogenic stimulus of casein was blocked completely. Amyloid synthesis was however, not interrupted by the administration of colchicine during the last 7 days of the casein regimen nor by colchicine treatment of recipient mice in a transfer model.