High Molecular Weight Bands Research Articles

Selective Binding of Glutathione Conjugates of Fatty Acid Derivatives by Plant Glutathione Transferases

Proteomic studies with Arabidopsis thaliana have revealed that the plant-specific Tau (U) class glutathione transferases (GSTs) are selectively retained by S-hexylglutathione affinity supports. Overexpression of members of the Arabidopsis GST superfamily in Escherichia coli showed that 25 of the complement of 28 GSTUs caused the aberrant accumulation of acylated glutathione thioesters in vivo, a perturbation that was not observed with other GST classes. Each GSTU caused a specific group of fatty acyl derivatives to accumulate, which varied in chain length (C(6) to C(18)), additional oxygen content (0 or 1), and desaturation (0 or 1). Thioesters bound tightly to recombinant GSTs (K(d) approximately 1 microm), explaining their accumulation. Transient expression of GSTUs in Nicotiana benthamiana followed by recovery by Strep-tag affinity chromatography allowed the respective plant ligands to be extracted and characterized. Again, each GST showed a distinct profile of recovered metabolites, notably glutathionylated oxophytodienoic acid and related oxygenated fatty acids. Similarly, the expression of the major Tau protein GSTU19 in the endogenous host Arabidopsis led to the selective binding of the glutathionylated oxophytodienoic acid-glutathione conjugate, with the enzyme able to catalyze the conjugation reaction. Additional ligands identified in planta included other fatty acid derivatives including divinyl ethers and glutathionylated chlorogenic acid. The strong and specific retention of various oxygenated fatty acids by each GSTU and the conservation in binding observed in the different hosts suggest that these proteins have selective roles in binding and conjugating these unstable metabolites in vivo.

Calmodulin Activation Limits the Rate of KCNQ2 K+ Channel Exit from the Endoplasmic Reticulum

The potential regulation of protein trafficking by calmodulin (CaM) is a novel concept that remains to be substantiated. We proposed that KCNQ2 K+ channel trafficking is regulated by CaM binding to the C-terminal A and B helices. Here we show that the L339R mutation in helix A, which is linked to human benign neonatal convulsions, perturbs CaM binding to KCNQ2 channels and prevents their correct trafficking to the plasma membrane. We used glutathione S-transferase fused to helices A and B to examine the impact of this and other mutations in helix A (I340A, I340E, A343D, and R353G) on the interaction with CaM. The process appears to require at least two steps; the first involves the transient association of CaM with KCNQ2, and in the second, the complex adopts an "active" conformation that is more stable and is that which confers the capacity to exit the endoplasmic reticulum. Significantly, the mutations that we have analyzed mainly affect the stability of the active configuration of the complex, whereas Ca2+ alone appears to affect the initial binding step. The spectrum of responses from this collection of mutants revealed a strong correlation between adopting the active conformation and channel trafficking in mammalian cells. These data are entirely consistent with the concept that CaM bound to KCNQ2 acts as a Ca2+ sensor, conferring Ca2+ dependence to the trafficking of the channel to the plasma membrane and fully explaining the requirement of CaM binding for KCNQ2 function.

Mechanical Constraints on Hin Subunit Rotation Imposed by the Fis/Enhancer System and DNA Supercoiling during Site-Specific Recombination

Hin, a member of the serine family of site-specific recombinases, regulates gene expression by inverting a DNA segment. DNA inversion requires assembly of an invertasome complex in which a recombinational enhancer DNA segment bound by the Fis protein associates with the Hin synaptic complex at the base of a supercoiled DNA branch. Each of the four Hin subunits becomes covalently joined to the cleaved DNA ends, and DNA exchange occurs by translocation of a Hin subunit pair within the tetramer. We show here that, although the Hin tetramer forms a bidirectional molecular swivel, the Fis/enhancer system determines both the direction and number of subunit rotations. The chirality of supercoiling directs rotational direction, and the short DNA loop stabilized by Fis-Hin contacts limit rotational processivity, thereby ensuring that the DNA strands religate in the recombinant configuration. We identify multiple rotational conformers that are formed under different supercoiling and solution conditions.

Roles of Protein-disulfide Isomerase-mediated Disulfide Bond Formation of Yeast Mnl1p in Endoplasmic Reticulum-associated Degradation

The endoplasmic reticulum (ER) has a strict protein quality control system. Misfolded proteins generated in the ER are degraded by the ER-associated degradation (ERAD). Yeast Mnl1p consists of an N-terminal mannosidase homology domain and a less conserved C-terminal domain and facilitates the ERAD of glycoproteins. We found that Mnl1p is an ER luminal protein with a cleavable signal sequence and stably interacts with a protein-disulfide isomerase (PDI). Analyses of a series of Mnl1p mutants revealed that interactions between the C-terminal domain of Mnl1p and PDI, which include an intermolecular disulfide bond, are essential for subsequent introduction of a disulfide bond into the mannosidase homology domain of Mnl1p by PDI. This disulfide bond is essential for the ERAD activity of Mnl1p and in turn stabilizes the prolonged association of PDI with Mnl1p. Close interdependence between Mnl1p and PDI suggests that these two proteins form a functional unit in the ERAD pathway.

Seed protein polymorphism in nine species of Jute (Corchorus, Family: Tiliaceae)

Distinct polymorphism in electrophoretic banding patterns of seed protein following SDSPAGE was noted in nine jute (Corchorus; Family: Tiliaceae) species and led to the detection of 52 polypeptide bands(cultivated members: C. olitorius L.-42, C. capsularis L.-40; wild species: C. fascicularis Lamk.- 23, C. aestuans L.- 28, C. pseudoolitorius I. and Z.- 34, C. pseudocapsularis L. and C. tridens - 30, C. trilocularis L.- , 26 and C. urticaefolius W. and A.- 35) with molecular weight ranging between 13.0kD to 122.5 kD. Polypeptide bands were mostly medium (25.0 kD to 49.9 kD: 8 to 17 bands) to low (<25.0 kD: 6 to 11 bands) molecular weights but very high (> 80.0 kD: 1-6 bands) and high (50.0 kD to 80.0 kD: 5-11 bands) molecular weight bands were also noted. Band number 3 (97.5 kD), 8 (76.0 kD), 15 (57.5 kD), 20 (47.0 kD), 29 (35.0 kD) and 35 (28.0 kD) were common band in all the species. Species specific polypeptide bands were detected( no. 17-53.5 kD, C. olitorius; 36-27.0 kD, C. capsularis; 30-34.0 kD, C. fascicularis; 33-30.3 kD, C. aestuans; 22 and 45-41.0 kD and 20.0 kD respectively, C. pseudocapsularis; 7 and 42 -79.0 kD and 22.0 kD respectively, C. tridens; 39-24.0 kD, C. trilocularis and 43 -21.0 kD, C. urticaefolius). The clustering (Hierarchical cluster analysis; based on proximity matrix) of the species on un-weighted pair group method with arithmetic mean algorithm (UPGMA) analysis was made and 3 prominent clusters were noted (Cluster1: C. trilocularis and C. urticaefolius; cluster 2: C. fascicularis, C. tridens and C. pseudoolitorius and cluster 3: C. olitorius, C. capsularis and C. aestuans). Significance of the findings has been discussed. Keywords: Corchorus species, Seed protein polymorphism, SDS-PAGE, UPGMA analysis.

Seed protein polymorphism in nine species of Jute (Corchorus, Family: Tiliaceae)

Distinct polymorphism in electrophoretic banding patterns of seed protein following SDS-PAGE was noted in nine jute ( Corchorus ; Family: Tiliaceae) species and led to the detection of 52 polypeptide bands(cultivated members: C. olitorius L.-42, C. capsularis L.-40; wild species: C. fascicularis Lamk.- 23, C. aestuans L.- 28, C. pseudoolitorius I. and Z.- 34, C. pseudocapsularis L. and C. tridens - 30, C. trilocularis L.- , 26 and C. urticaefolius W. and A.- 35) with molecular weight ranging between 13.0kD to 122.5 kD. Polypeptide bands were mostly medium (25.0 kD to 49.9 kD: 8 to 17 bands) to low ( 80.0 kD: 1-6 bands) and high (50.0 kD to 80.0 kD: 5-11 bands) molecular weight bands were also noted. Band number 3 (97.5 kD), 8 (76.0 kD), 15 (57.5 kD), 20 (47.0 kD), 29 (35.0 kD) and 35 (28.0 kD) were common band in all the species. Species specific polypeptide bands were detected( no. 17-53.5 kD, C. olitorius ; 36-27.0 kD, C. capsularis ; 30-34.0 kD, C. fascicularis ; 33-30.3 kD, C. aestuans ; 22 and 45-41.0 kD and 20.0 kD respectively, C. pseudocapsularis ; 7 and 42 -79.0 kD and 22.0 kD respectively, C. tridens ; 39-24.0 kD, C. trilocularis and 43 -21.0 kD, C. urticaefolius ). The clustering (Hierarchical cluster analysis; based on proximity matrix) of the species on un-weighted pair group method with arithmetic mean algorithm (UPGMA) analysis was made and 3 prominent clusters were noted (Cluster1: C. trilocularis and C. urticaefolius ; cluster 2: C. fascicularis, C. tridens and C. pseudoolitorius and cluster 3: C. olitorius, C. capsularis and C. aestuans ). Significance of the findings has been discussed.

Biosynthesis of Prion Protein Nucleocytoplasmic Isoforms by Alternative Initiation of Translation

The cellular prion protein PrP(C) is synthesized as a family of four distinct forms. Of these, (Cyt)PrP is a minor member that segregates outside of the secretory route and can generate cytotoxic forms. Using signal sequence mutants, we found that (Cyt)PrP is translated from a downstream AUG (coding for Met-8 in human PrP or Met-15 in Syrian hamster PrP). Shortening of the signal sequence dictated the spillage of this isoform into the cytosol, from where it accessed the nucleus or formed insoluble cytosolic aggregates if the proteasome is inhibited. The PrP isoform isolated from the nuclear fractions of cell and brain homogenates was partially SUMO-1-conjugated. Expression of HaPrP(M15) in cells caused an antiproliferative phenotype due to a cell cycle arrest at the G(0)/G(1) phase. The identification of this PrP isoform and its properties provides novel insight into PrP(C) physiological and pathological functions.

Molecular properties of the partially SDS-resistant lectin from the albumen gland of Helix pomatia and demonstration of lectin-related molecules on the surface of H. pomatia haemocytes

Lectins (agglutinins) are components of the immunobiological recognition system of vertebrates and invertebrates. The present study focused on the molecular properties of the agglutinin from the albumen gland of Helix pomatia (HPA) and on the occurrence of lectin-related molecules on the surface of H. pomatia haemocytes. According to the current model (Hammarstrom et al., 1972, Scandinavian Journal of Immunology, 1: 259-301), the hexameric HPA of about 79 kDa is composed of three non-covalently associated dimers (26 kDa), each consisting of two disulphide-bridged 13 kDa monomers. However, on native-gradient polyacrylamide gel electrophoresis (PAGE), we obtained high molecular weight bands representing lectin polymers. The stepwise dissociation of these was achieved by incubation with SDS at temperatures from 20 to 408C (1 h) and at 1008C (10 min). The results obtained on SDS-PAGE included the occurrence of partially SDS-resistant hexamers of about 66 kDa, of two dimer bands of 22 and 19 kDa, and of two minor heteromonomer fractions. Complete dissociation into heteromonomers of 13 and 11 kDa was achieved by boiling the lectin (10 min) with SDS under reducing conditions. For native lectin molecules, both monomers occurred as disulphide-linked homodimers. Monomers or dimers electroeluted from an SDS -gel, reassociated to SDS-resistant oligomers upon re-electrophoresis. Finally, molecules antigenetically related to the lectin were extracted from the membrane of H. pomatia haemocytes. Anti-HPA antibodies recognized peptides with an apparent molecular weight of about 30 and 56 kDa, which were shown to represent cell-surface molecules.

Isolation and Partial Characterization of an Antiviral Proteolytic Fraction from the Venom of Echis Carinatus Sochureki

The venom of the viper Echis carinatus sochureki suppresses the hemolytic activity of Sendai virus on human erythrocytes, when pre-incubated with the virions prior to their binding to cells. A fraction (C1), with an IC50 of 1.25 􀀁g/ml, was isolated from the venom. Fraction C1 possesses strong azocollase, azocaseinase and gelatinase activity. The proteolytic and anti-hemolytic potency of C1 depends on the period and temperature of incubation. Its antiviral activity is inhibited by Sodium-EDTA but not by PMSF. SDS PAGE of Sendai virus incubated with fraction C1 shows disappearance of several of the virion high molecular weight bands. We suggest that inhibition of the hemolytic activity of the virions is probably a result of the cleavage of viral surface proteins, such as the hemagglutinin-neuraminidase glycoprotein found on the virion envelope that mediates the absorption of the virus to cells.

CYTOLGICAL AND ISOZYMES STUDIES OF GARLIC CULTIVATED IN EGYPT.

Generally It has been accepted that the apomictic nature of nature of garlic clones could affect its cytological and genetical features as a result of accumulation of certain somatic mutations. Thus, the aim of this study is to examine some of the cytological (karyotyping, mitotic index and mitotic abnormalities) and isozyme characteristics of five garlic genotypes cultivated in Egypt (Baladi , Chinese , Chinese , 30-4 , Chinese 31-1and Chinese 31-3).Relative chromosome lengths and centromere position of these genotypes revealed insignificant difference in their karyotype formula (5 large , 2 medium from submetacentric pair and one large metacentric pair no. 5). The percentage of mitotic index of Baladi genotype (8.28%) was not very much different from that of Chinese (12.08%) whereas it was significantly lower than those of Chinese 30-4 (15.6%),Chinese 31-1(18.18%)and Chinese 31-3 (16. 8%). Means of total chromosomal abnormalities (including anuepliody, break and stickiness) were remarkably lower in genotypes Baladi (7.64%) and Chinese (9.38%)than those of Chinese 30-4 (11.94%), Chinese 31-3 (11.2%). Statistical analysis revealed that there was a high correlation (0.77) between the mitotic index and the percentage of mitotic chromosome abnormalities. Isozymes [Acid phosphatase, Glutamate Oxaloacetate Transaminase (GOT), Peroxidase and Esterase) banding patterns were qualitatively compared. The banding patterns of esterase (2bands) and GOT (one band) were similar in all five genotypes. As for Pyroxidase, the four genotypes of the Chinese origin had two bands, whereas only one band was shown by the genotype Baladi that was similar in molecular weight to small band of Chinese genotypes. All studies genotypes of acid phosphatase showed one band, which was of a low molecular weight in all except in Chinese genotype where it was a high molecular weight and Chinese 31-1 in which both low and high molecular weight bands were found.

Sialylation of β1 Integrins Blocks Cell Adhesion to Galectin-3 and Protects Cells against Galectin-3-induced Apoptosis

Sialylation of β1 Integrins Blocks Cell Adhesion to Galectin-3 and Protects Cells against Galectin-3-induced Apoptosis

Phosphorylation of the Human Retinoid X Receptor α at Serine 260 Impairs Coactivator(s) Recruitment and Induces Hormone Resistance to Multiple Ligands

The retinoid X receptor alpha (RXRalpha) is a member of the nuclear receptor superfamily that regulates transcription of target genes through heterodimerization with several partners, including peroxisome proliferator-activated receptor, retinoic acid receptor, thyroid receptor, and vitamin D receptor (VDR). We have shown previously that signaling through VDR.RXRalpha heterodimers was attenuated in ras-transformed keratinocytes due to phosphorylation of serine 260 of the RXRalpha via the activated Ras-Raf-MAPK cascade in these cells. In this study we demonstrate that phosphorylation at serine 260, a site located in the omega loop-AF-2 interacting domain of RXRalpha, inhibits signaling through several heterodimeric partners of the RXRalpha. The inhibition of signaling results in reduced transactivational response to ligand presentation and the reduced physiological response of growth inhibition not only of 1,25-dihydroxyvitamin D3 but also of retinoic acid receptor alpha ligands and LG1069 (an RXRalpha ligand). This partial resistance to ligands could be reversed by inhibition of MAPK activity or by overexpression of a non-phosphorylable RXRalpha mutant at serine 260 (RXRalpha Ser-260-->Ala). Importantly, phosphorylation of RXRalpha at serine 260 impaired the recruitment of DRIP205 and other coactivators to the VDR.RXRalpha complex. Chromatin immunoprecipitation and pulldown assays further demonstrated that coactivator recruitment to the VDR.RXR complex could be restored by treatment with a MAPK inhibitor. Our data suggest that phosphorylation at serine 260 plays a critical role in inducing hormone resistance of RXRalpha-mediated signaling likely through structural changes in the H1-H3 omega loop-AF2 coactivator(s) interacting domain.

CHARACTERIZATION OF APHID-TRANSMITTED VIRUS ASSOCIATED WITH BLACK RASPBERRY DECLINE IN OREGON

A serious disease of black raspberry (Rubus occidentalis) was observed in Oregon in the last decade. Plants showing mosaic symptoms declined rapidly and in many cases died over a period of several years. Double-stranded RNA extraction from symptomatic black raspberry revealed the presence of two high molecular weight bands, which were cloned and partially sequenced. Sequence analysis disclosed the presence of Black raspberry virus (BRV). A newly planted field of black raspberries in Oregon was studied to assess the rate of spread of BRV. The timing of BRV infection as it relates to aphid populations and flights was also determined. Testing of nearby vegetation identified several symptomless Rubus hosts of BRV. It was determined that BRV spreads rapidly with a low aphid threshold and was associated with symptoms of decline in black raspberries in Oregon.

The Motor Protein Prestin Is a Bullet-shaped Molecule with Inner Cavities

Prestin is a transmembrane motor protein localized at the outer hair cells (OHCs) of the mammalian inner ear. Voltage-dependent conformational changes in prestin generate changes in the length of OHCs. A loss of prestin function is reported to induce severe auditory deficiencies, suggesting prestin-dependent changes of OHC length may be at least a part of cochlear amplification. Here we expressed the recombinant FLAG-fused prestin proteins in Sf9 cells and purified to particles of a uniform size in EM. The square-shaped top view of purified prestin, the binding of multiple anti-FLAG antibodies to each prestin particle, the native-PAGE analysis, and the much larger molecular weight obtained from size exclusion chromatography than the estimation for the monomer all support that prestin is a tetramer (Zheng, J., Du, G. G., Anderson, C. T., Keller, J. P., Orem, A., Dallos, P., and Cheatham, M. (2006) J. Biol. Chem. 281, 19916-19924). From negatively stained prestin particles, the three-dimensional structure was reconstructed at 2 nm resolution assuming 4-fold symmetry. Prestin is shown to be a bullet-shaped particle with a large cytoplasmic domain. The surface representation demonstrates indentations on the molecule, and the slice images indicate the inner cavities of sparse densities. The dimensions, 77 x 77 x 115 A, are consistent with the previously reported sizes of motor proteins on the surface of OHCs.

High molecular weight protein detected in higher plant cells by antibodies against dynein is associated with vesicular organelles including Golgi apparatus

The cytoplasmic dynein is a multisubunit complex driving organelles along microtubules to their minus-end. We used antibodies against two functional domains (motor and microtubule-binding) of one of principal components of the complex--dynein heavy chain of slime mould Dictyostelium discoideum--to test root meristem cells of wheat Triticum aestivum. The antibodies reacted with a high molecular weight protein (> 500 kDa) in the total cell extract and the band recognized by the antibodies in plant extracts had a lower electrophoretic mobility than the high molecular weight band of mammalian dynein. Antibodies coupled to protein A-Sepharose precipitated the high molecular weight protein from the purified cell extracts. Immunocytochemical analysis demonstrated that the antigen recognized by antibodies against dynein heavy chains is associated with the vesicles whose localization depends on the cell cycle stage. The antigen-positive vesicles were localized to the perinuclear region in interphase and early prophase, to the spindle periphery and to spindle pole region during mitosis, and to the interzonal region in the period of fragmoplast and cell plate formation. Some antigen-positive vesicles also reacted with antibodies against Golgi protein markers. The obtained data indicate that higher plant cells contain a high molecular weight protein interacting with antibodies against the motor and microtubules-binding domains of Dictyostelium dynein heavy chain. The revealed antigen was associated with the vesicular structures in the cytoplasm including the Golgi apparatus.

Invalidation of TASK1 potassium channels disrupts adrenal gland zonation and mineralocorticoid homeostasis

TASK1 (KCNK3) and TASK3 (KCNK9) are two-pore domain potassium channels highly expressed in adrenal glands. TASK1/TASK3 heterodimers are believed to contribute to the background conductance whose inhibition by angiotensin II stimulates aldosterone secretion. We used task1-/- mice to analyze the role of this channel in adrenal gland function. Task1-/- exhibited severe hyperaldosteronism independent of salt intake, hypokalemia, and arterial 'low-renin' hypertension. The hyperaldosteronism was fully remediable by glucocorticoids. The aldosterone phenotype was caused by an adrenocortical zonation defect. Aldosterone synthase was absent in the outer cortex normally corresponding to the zona glomerulosa, but abundant in the reticulo-fasciculata zone. The impaired mineralocorticoid homeostasis and zonation were independent of the sex in young mice, but were restricted to females in adults. Patch-clamp experiments on adrenal cells suggest that task3 and other K+ channels compensate for the task1 absence. Adrenal zonation appears as a dynamic process that even can take place in adulthood. The striking changes in the adrenocortical architecture in task1-/- mice are the first demonstration of the causative role of a potassium channel in development/differentiation.

SorLA/LR11 Regulates Processing of Amyloid Precursor Protein via Interaction with Adaptors GGA and PACS-1

SorLA has been recognized as a novel sorting receptor that regulates trafficking and processing of the amyloid precursor protein (APP) and that represents a significant risk factor for sporadic Alzheimer disease. Here, we investigated the cellular mechanisms that control intracellular trafficking of sorLA and their relevance for APP processing. We demonstrate that sorLA acts as a retention factor for APP in trans-Golgi compartments/trans-Golgi network, preventing release of the precursor into regular processing pathways. Proper localization and activity of sorLA are dependent on functional interaction with GGA and PACS-1, adaptor proteins involved in protein transport to and from the trans-Golgi network. Aberrant targeting of sorLA to the recycling compartment or the plasma membrane causes faulty APP trafficking and imbalance in non-amyloidogenic and amyloidogenic processing fates. Thus, our findings identified altered routing of sorLA as a major cellular mechanism contributing to abnormal APP processing and enhanced amyloid beta-peptide formation.

Development of silverleaf assay, protein and nucleic acid-based diagnostic techniques for the quick and reliable detection and monitoring of biotype B of the whitefly, Bemisia tabaci (Gennadius)

The aim of this study was to develop and optimize silverleaf bioassay, esterase analysis and PCR-based techniques to distinguish quickly and reliably biotype B of the whitefly, Bemisia tabaci (Gennadius), from Indian indigenous biotypes. Zucchini and squash readily develop silverleaf symptoms upon feeding by the B biotype, but they are not readily available in Indian markets. A local pumpkin variety 'Big' was, therefore, used in silverleaf assay, which developed symptoms similar to those on zucchini and squash and can be used reliably to detect B biotype. Analysis of non-specific esterases of B and the indigenous biotypes indicated both quantitative and qualitative differences in esterase patterns. Two high molecular weight bands were unique to B biotype and they occurred in abundance. These esterases were used to develop quick and field-based novel detection methods for differentiating B from the indigenous biotypes. Development of these simple and cost-effective protocols has wider application as they can be potentially used to identify other agricultural pests. Mitochondrial cytochrome oxidase I gene sequences and randomly amplified polymorphic DNA (RAPD) polymorphisms, generated using the primer OpB11, were also found useful for detecting B. tabaci biotypes. A B biotype-specific RAPD band of 800 bp was sequenced, which was used to a develop sequence characterized amplified region (SCAR) marker. The SCAR marker involved the development of B biotype-specific primers that amplified 550 bp PCR products only from B biotype genomic DNA. Silverleaf assay, esterases, RAPDs or a SCAR marker were used in combination to analyse whitefly samples collected from selected locations in India, and it was found that any of these techniques can be used singly or in combination to detect B biotype reliably. The B biotype was found in southern parts of India but not in the north in 2004-06.

Peptic and Tryptic Hydrolysis of Native and Heated Whey Protein to Reduce Its Antigenicity

This study examined the effects of enzymes on the production and antigenicity of native and heated whey protein concentrate (WPC) hydrolysates. Native and heated (10min at 100°C) WPC (2% protein solution) were incubated at 50°C for 30, 60, 90, and 120min with 0.1, 0.5, and 1% pepsin and then with 0.1, 0.5, and 1% trypsin on a protein-equivalent basis. A greater degree of hydrolysis was achieved and greater nonprotein nitrogen concentrations were obtained in heated WPC than in native WPC at all incubation times. Hydrolysis of WPC was increased with an increasing level of enzymes and higher incubation times. The highest hydrolysis (25.23%) was observed in heated WPC incubated with 1% pepsin and then with 1% trypsin for 120min. High molecular weight bands, such as BSA, were completely eliminated from sodium dodecyl sulfate-PAGE of both native and heated WPC hydrolysates produced with pepsin for the 30-min incubation. The α-lactalbumin in native WPC was slightly degraded when incubated with 0.1% pepsin and then with 0.1% trypsin; however, it was almost completely hydrolyzed within 60min of incubation with 0.5% pepsin and then with 0.5% trypsin. Incubation of native WPC with 1% pepsin and then with 1% trypsin for 30min completely removed the BSA and α-lactalbumin. The β-lactoglobulin in native WPC was not affected by the pepsin and trypsin treatments. The β-lactoglobulin in heated WPC was partially hydrolyzed by the 0.1 and 0.5% pepsin and trypsin treatments and was completely degraded by the 1% pepsin and trypsin treatment. Antigenicity reversibly mimicked the hydrolysis of WPC and the removal of β-lactoglobulin from hydrolysates. Antigenicity in heated and native WPC was reduced with an increasing level of enzymes. A low antigenic response was observed in heated WPC compared with native WPC. The lowest antigenicity was observed when heated WPC was incubated with 1% pepsin and then with 1% trypsin. These results suggested that incubation of heated WPC with 1% pepsin and then with 1% trypsin was the most effective for producing low-antigenic hydrolysates by WPC hydrolysis and obtaining low molecular weight small peptides. Further research is warranted to identify the low molecular weight small peptides in the WPC hydrolysates produced by pepsin and trypsin, which may enhance the use of whey.

Post-Transcriptional Regulation of Melanin Biosynthetic Enzymes by cAMP and Resveratrol in Human Melanocytes

Upregulation of microphthalmia-associated transcription factor (MITF) expression has been proposed to mediate melanogenesis stimulated by cAMP, whereas downregulation of MITF has been suggested to underlie the depigmentary effects of resveratrol, a promising chemotherapeutic found in red wine. We have assessed the contribution of MITF to pigmentation regulation by treating primary cultures of normal human melanocytes with the adenylate cyclase activator forskolin and/or resveratrol, then quantifying mRNA and protein levels for MITF, tyrosinase, tyrosinase-related protein-1, and dopachrome tautomerase (DCT). The inhibition of tyrosinase activity by resveratrol was not due to alterations in MITF, but instead was explained by both direct tyrosinase inhibition and a post-transcriptional effect that reduced the amount of fully processed tyrosinase. Glycosidase digestion revealed that the basis for the tyrosinase decrease was the retention of an immature form in the ER and subsequent loss of the mature, Golgi-processed enzyme. Elevation of intracellular cAMP by forskolin markedly increased protein levels for MITF, tyrosinase and DCT, however there was no concomitant increase in tyrosinase or DCT mRNA. This indicated that elevated levels of MITF were not sufficient to promote transcription of these melanogenic genes and that the increase in their protein abundance appeared to be predominantly mediated through post-transcriptional processing events.