Higher Electrophoretic Mobility Research Articles

Structure and Functions of HMGB3 Protein.

HMGB3 protein belongs to the group of HMGB proteins from the superfamily of nuclear proteins with high electrophoretic mobility. HMGB proteins play an active part in almost all cellular processes associated with DNA-repair, replication, recombination, and transcription-and, additionally, can act as cytokines during infectious processes, inflammatory responses, and injuries. Although the structure and functions of HMGB1 and HMGB2 proteins have been intensively studied for decades, very little attention has been paid to HMGB3 until recently. In this review, we summarize the currently available data on the molecular structure, post-translational modifications, and biological functions of HMGB3, as well as the possible role of the ubiquitin-proteasome system-dependent HMGB3 degradation in tumor development.

Scaling the electrophoretic separation of rapeseed proteins and oleosomes

Gentle extraction of ingredients from raw materials is essential for high-quality food ingredients and can lead to reducing the use of water, chemicals, and energy in the extraction. For example, a simple aqueous extraction can yield a mixture of oil, in the form of a natural oil-in-water oleosome emulsion, and proteins. The oleosomes and proteins can then be further separated in a next step. We explored a continuous counter-current electrophoretic process that separates oleosomes and proteins based on their electrophoretic mobility by balancing an electric field with an opposing solvent flow. The separation is accomplished through the retention of the component with the higher electrophoretic mobility, the oleosomes, and the passage of the proteins, having lower mobility. The fluxes of oleosomes and proteins from rapeseed, after aqueous extraction, were analyzed as a function of the electric field (0–75 Vcm−1) and 1.2 ± 0.1 mLmin-1 solvent flow rate. At 50 Vcm−1, the permeation flux of proteins was 10-fold higher than that of oleosomes, as shown by the selectivity increasing to 9.84 from 1.90 at 25 Vcm−1. The difference in their flux promises to become more pronounced under an increasing treatment duration, but two main technical limitations, electrolysis-based pH alteration and membrane fouling, restrict further separation. We expect the listed challenges can be mitigated with the addition of electrode rinse chambers and the use of larger pore size membranes.

A novel allelic donkey β-lactoglobulin I protein isoform generated by a non-AUG translation initiation codon is associated with a nonsynonymous SNP

β-Lactoglobulin I (β-LG I) is one of the most important whey proteins in donkey milk. However, to our knowledge, there has been no study focusing on the full nucleotide sequences of this gene (BLG I). Current investigation of donkey BLG I gene is very limited with only 2 variants (A and B) characterized so far at the protein level. Recently, a new β-LG I variant, with a significantly higher mass (+1,915 Da) than known variants has been detected. In this study, we report the whole nucleotide sequence of the BLG I gene from 2 donkeys, whose milk samples are characterized by the β-LG I SDS-PAGE band with a normal electrophoretic mobility (18,514.25 Da, β-LG I B1 form) the first, and by the presence of a unique β-LG I band with a higher electrophoretic mobility (20,428.5 Da, β-LG I D form) the latter. A high genetic variability was found all over the 2 sequenced BLG I alleles. In particular, 16 polymorphic sites were found in introns, one in the 5' flanking region, 3 SNPs in the 5' untranslated region and one SNP in the coding region (g.458G > A) located at the 40th nucleotide of exon 2 and responsible for the AA substitutions p.Asp28 > Asn in the mature protein. Two SNPs (g.920-922CAC > TGT and g.1871G/A) were genotyped in 93 donkeys of 2 Italian breeds (60 Ragusana and 33 Amiatina, respectively) and the overall frequencies of g.920-922CAC and g.1871A were 0.3065 and 0.043, respectively. Only the rare allele g.1871A was observed to be associated with the slower migrating β-LG I. Considering this genetic diversity and those found in the database, it was possible to deduce at least 5 different alleles (BLG I A, B, B1, C, D) responsible for 4 potential β-LG I translations. Among these alleles, B1 and D are those characterized in the present research, with the D allele of real novel identification. Haplotype data analysis suggests an evolutionary pathway of donkey BLG I gene and a possible phylogenetic map is proposed. Analyses of mRNA secondary structure showed relevant changes in the structures, as consequence of the g.1871G > A polymorphism, that might be responsible for the recognition of an alternative initiation site providing an additional signal peptide. The extension of 19 AA sequence to the mature protein, corresponding to the canonical signal peptide with an additional alanine residue, is sufficient to provide the observed molecular weight of the slower migrating β-LG I encoded by the BLG I D allele.

An elucidative study of the anti-biofilm effect of selenium nanoparticles (SeNPs) on selected biofilm producing pathogenic bacteria: A disintegrating effect of SeNPs on bacteria

In the present study, the antibacterial and anti-biofilm activity of selenium nanoparticles (SeNPs) produced by Bacillus subtilis BSN313 was investigated. Agar diffusion and microdilution methods did not reveal significant antibacterial activity of SeNPs against P. aeruginosa ATCC 9027, S. typhi ATCC 14028, and S. aureus ATCC 25923 due to the larger size of SeNPs and higher electrophoretic mobility, zeta potential, and/or poor diffusion properties in agar solids. Therefore, the agar overlay method was introduced, and significant antibacterial activity was observed with this method at concentrations (SeNPs) of 200 µg/mL, 150 µg/mL, and 150 µg/mL against P. aeruginosa ATCC 9027, S. typhi ATCC 14028, and S. aureus ATCC 25923, respectively. Atomic force spectroscopy (AFM) results, elucidated the disintegrating effect of SeNPs against Gram-negative (P. aeruginosa ATCC 9027) and Gram-positive (S. aureus ATCC 25923) pathogens. The disintegrating effect of SeNPs was caused by the destruction of the bacterial cell (biofilm producing) by rupturing the cell membrane. The hydrophobicity and zeta potential of the planktonic and biofilm populations were measured. Higher values of hydrophobicity and zeta potential heterogeneity were found for the biofilm population than for the planktonic population. The SeNPs showed significant anti-biofilm activity against biofilm-producing pathogenic strains. The ability of SeNPs to disperse biofilms was greatest at a concentration of 700 µg/mL within 15 min against P. aeruginosa ATCC 9027 (85.7%), S. typhi ATCC 14028 (89.6%), and S. aureus ATCC 25923 (78.3%). While the positive control (ascorbic acid, 32 µg/mL) showed an average dispersion of 95.1% against the biofilms of the strains studied within 90 min. Based on these results, we propose SeNPs as suitable candidates for the treatment of biofilm-producing pathogens.

Application of High Potential Electrophoretic Particles Modified with High Ionization Mono Ionic Liquid for Electrophoretic Displays

The electrophoretic display (EPD) has attracted widespread attention due to its great visual perception, energy-saving, portability, and bistability. However, the EPD still has many problems in response time, colorization, etc., which limits its practical application. In this paper, novel blue electrophoretic particles were prepared with copper (II) phthalocyanine and high ionization 1-butyl-1-methyl piperidinium bromide mono ionic liquid. It was shown that electrophoretic particles dispersed in a non-polar tetrachloroethylene medium had high Zeta potential and electrophoretic mobility. At the same time, electrophoretic particles showed better dispersion stability. Finally, the prepared blue electrophoretic particles and white titanium dioxide particles were compounded to prepare blue and white dual-color electrophoretic dispersion. An EPD cell was made to test its performance. The results showed that the prepared blue and white dual-color electrophoretic dispersion could realize a reversible response. Piperidine mono ionic liquid increased the surface potential of copper (II) phthalocyanine from +30.50 mV to +60.27 mV, enhancing it by 97.61%. Therefore, we believed that modifying particles with high ionization mono ionic liquid had great applicability to the modification of electrophoretic particles, and blue particles prepared with piperidine mono ionic liquid as a charge control agent (CCA) were excellent candidates for EPDs.

High Mobility Group Box 1: Biological Functions and Relevance in Oxidative Stress Related Chronic Diseases.

In the early 1970s, a group of non-histone nuclear proteins with high electrophoretic mobility was discovered and named high-mobility group (HMG) proteins. High-mobility group box 1 (HMGB1) is the most studied HMG protein that detects and coordinates cellular stress response. The biological function of HMGB1 depends on its subcellular localization and expression. It plays a critical role in the nucleus and cytoplasm as DNA chaperone, chromosome gatekeeper, autophagy maintainer, and protector from apoptotic cell death. HMGB1 also functions as an extracellular alarmin acting as a damage-associated molecular pattern molecule (DAMP). Recent findings describe HMGB1 as a sophisticated signal of danger, with a pleiotropic function, which is useful as a clinical biomarker for several disorders. HMGB1 has emerged as a mediator in acute and chronic inflammation. Furthermore, HMGB1 targeting can induce beneficial effects on oxidative stress related diseases. This review focus on HMGB1 redox status, localization, mechanisms of release, binding with receptors, and its activities in different oxidative stress-related chronic diseases. Since a growing number of reports show the key role of HMGB1 in socially relevant pathological conditions, to our knowledge, for the first time, here we analyze the scientific literature, evaluating the number of publications focusing on HMGB1 in humans and animal models, per year, from 2006 to 2021 and the number of records published, yearly, per disease and category (studies on humans and animal models).

Self-assembly of bovine serum albumin (BSA)-dextran bio-nanoconjugate: structural, antioxidant and in vitro wound healing studies.

Glycation of proteins is often considered as a method to improve their functional properties for promising applications in wound healing. Furthermore, a marked increase in percentage of radical scavenging activity of the conjugates makes it an effective antioxidant synthetic strategy. A simple conjugation process was employed to develop bovine serum albumin–dextran conjugates (BSA–dextran) using Maillard reaction. Higher electrophoretic mobility and surface charge in the prepared conjugates was observed in native PAGE electrophoresis and zeta potential. Moreover, the fluorescence, FTIR and Raman analysis of the BSA–dextran conjugates shows significant shift in the fluorescence and wavelength as a consequence of conjugate formation. In vitro wound healing assay showed increased cell proliferation and migration effect. These finding suggests that BSA–dextran conjugate could open up a new practical way for exploration in the area of wound healing.

Electrophoretic Deposition of Nanoporous Oxide Coatings from Concentrated CuO Nanoparticle Dispersions.

Electrophoretic deposition (EPD) of nanoporous oxide coatings is an interesting research avenue owing to the experimental simplicity and broad scope of applications and materials. In this study, the properties of concentrated (up to 5000 mg/L), nonaqueous CuO nanoparticle (NP) dispersions were tailored to produce micrometer-thick, nanoporous CuO films by EPD. In particular, we performed a systematic investigation of the electrophoretic mobilities and size distributions of dispersed CuO aggregates and developing agglomerates in different organic solvents for concentrations ranging from 50 to 5000 mg/L with and without surfactant addition. Time-resolved dynamic light scattering analyses showed that aggregate mobilities and agglomeration rates decrease with increasing hydrocarbon chain length of the organic solvent (from ethanol to hexanol) and thus with increasing viscosity. The highest electrophoretic mobility was obtained for CuO NP aggregates and agglomerates dispersed in ethanol as a solvent. However, the addition of ≥0.5 wt % acetylacetone as a surfactant is required to stabilize these dispersions for subsequent EPD and at the same time introduce a net attractive (electrostatic) interaction between neighboring agglomerates on the substrate to promote layer formation during the EPD step. The produced micrometer-thick nanoporous CuO coatings can serve as high surface area nanostructured materials or nanoporous scaffolds in catalysis, combustion, propellants, and nanojoining.

THE ACTIVITY AND ISOFORMS OF SUPEROXIDE DISMUTASE IN TISUES OF REPRODUCTIVE ORGANS OF RATS

The aсtivity of superoxide dismutase, the content of its isoforms in testis, epididimis, and in epididimal spermatozoa of pubescent rat males were studied. The experiments were performed on adult male rats (age 5 - 6 months). After decapitation, the testes and the epididimis were taken, from which the spermatozoa were washed with 0.9 % sodium chloride solution. The total protein and activity of superoxide dismutase (SOD) were determined in the supernatant and the sperm suspension. SOD isoforms were detected after electrophoresis in 10 % polyacrylamide gel by specific staining of gel plates with nitrosine tetrazolium. The highest activity was found to be in the tissue of the testes (18.2 ± 2.59 IU/mg of protein), less on 34.6% in the epididymis and the lowest on 46.7% in epididymal sperm. Electrophoresis in 10 % polyacrylamide gel and specific staining in the tissues of testis, epididymis, and epididymal sperm of rats revealed five major SOD isoforms, which, depending on the speed of movement in PAAG, were labeled, from maximum to least mobile, such as S1, S2, S3, S4 and S5. The specifity of testicular tissue is the presence of two minor isoforms (S1a- and S1b-isoforms), which are characterized by high electrophoretic mobility and lower intensity of manifestation, and the color disappears over time, unlike other proteins of the enzyme. It was found that the SOD isoenzyme spectrum is characterized by tissue specificity and depended on the physiological and functional features of the tissue of the reproductive organs of males and spermatozoa. In the testis tissue, S2 isoform was in the largest amount– 36.7 ± 1.91%, less and almost the same content of S1-, S3- and S4-isoforms (18.0 ± 3.27, 19.3 ± 3.98 and 15.7 ± 2.15%) and the least – S5-, S1a- and S1b-isoform (6.2 ± 1.20%; 2.2 ± 0.03 and 1.9 ± 0.01%). In the epididimis tissue, the content of the S3-isoform was higher on 17.9% and S2- and S1- respectively on 7.9% and 4.9%, respectively, compared to the testis. SOD isoenzymes of epididymal spermatozoa are characterized by a high content of S2 isoforms, which is more on 11.2% than in the testis, and the content of S1 isoforms, by contrast, is lower on 10.0%. The established differences in the content and activity of individual isoforms of the enzyme indicate the existence of tissue specificity of SOD proteins, due to the functional characteristics of the studied tissues. Optimal protection of sperm from O2• - in the process of their differentiation and maturation is ensured by the activity of different SOD isoforms, confirming the established differences in the isoenzyme spectrum.

Low-level laser therapy as a modifier of erythrocytes morphokinetic parameters in hyperadrenalinemia.

Low-level laser therapy (LLLT) is widely used in clinical practice for treatment of various pathologies. It is assumed that LLLT impact on microcirculation is among the mechanisms underlying its therapeutic effect. The microcirculation disorder is observed in the pathogenesis of any inflammatory process and is significantly influenced by red blood cells (RBCs). On this point, studying the RBCs morphology under the influence of LLLT on alterated organism is of scientific interest and practical importance. The aim of the present study was to analyze the LLLT effect on morphokinetic parameters of RBCs in hyperadrenalinemia. The LLLT effect was analyzed on rats intraperitoneally injected with adrenaline hydrochloride solution (0.1mg/kg). As the comparison groups, the effects of LLLT, adrenaline, or saline injection as well as the parameters of intact animals were studied. LLLT was applied on the occipital region of rats for 10min. The light irradiation with pulse frequency 415Hz at 890nm wavelength and average power density in the plane of the output window at 193μW/cm2 was used. The dynamics of morphological characteristics of RBCs was studied by phase interference microscopy; the RBC electrophoretic mobility was tested by microelectrophoresis technique; photometric analyses of the RBCs amount, hemoglobin content, and osmotic fragility were performed. The adrenaline injection resulted in a significant increase in the amount of RBC pathological forms and a decrease in discocytes and normocytes by more than 50%. An increase in the optical density of RBC phase portraits, a decline in osmotic resistance, and electronegativity of RBC membranes and a reduction of their number in peripheral blood were also registered. The revealed effects persisted for 1week after the adrenaline administration. LLLT did not significantly impact on the RBC parameters 1h after adrenaline injection. However, a day later, LLLT reduced the severity of the adrenaline effect on RBSs, which was manifested in a decreased amount of the pathological forms of RBCs, restored RBC phase portraits, higher electrophoretic mobility and osmotic resistance, and RBSs amount in peripheral blood restored up to the level of intact animals. We suppose that the mechanism of LLLT action is realized both at cellular level through the laser radiation effect on RBC membranes, and at systemic level through the activation of stress-realizing systems of the organism with subsequent limitation of inflammatory response.

Optimization of dynamic pH barrage junction focusing for weakly alkaline or zwitterionic analytes in capillary electrophoresis

Dynamic pH junction focusing prior to electrophoretic separation has been widely used for online pre-concentration of biologically important analytes, which are mostly weakly alkaline/acidic or zwitterionic species such as neurotransmitters, peptides, and proteins. A pH junction is formed when background electrolytes with different pH values are injected sequentially into the separation column of a capillary electrophoresis (CE) system. Unlike the traditional dynamic pH junction configuration with analyte molecules located in a different chemical environment to the separation background electrolyte (BGE), the pH barrage junction has a separate high pH (or low pH) region containing no analyte. Based on Simul 5 Complex simulations and experimental verification with three series of electrolyte combinations, four basic principles for pH barrage junction focusing were identified for its optimization. First, the peak shape after focusing is slightly asymmetric, but this has negligible influence on the analysis result. Second, longer length of the barrage segment is needed for complete focusing with lower concentration of the buffering species. Third, this technique is more advantageous for analytes with relatively high electrophoretic mobility in a capillary without electroosmotic flow. Fourth, provided the analyte region and pH junction buffering species are separated, this quantitative technique is compatible with both optical and mass spectrometric detection.

Surface properties of Enterococcus faecalis cells isolated from chicken hearts determine their low ability to form biofilms

Enterococcus faecalis is one of the most significant bacterial pathogens associated with the first-week mortality of chickens. Here, the surface properties of bacterial cells and the selected virulence factors of E. faecalis strains isolated from the hearts of clinically healthy broiler chickens were studied. Investigations were carried out on live and autoclaved cells. E. faecalis (ATCC 29212) was used as a reference strain. The bacterial cells revealed different haemolytic activities. Their surface free energy was dominated by the hydrophobic component. The cell walls of the bird isolates showed slightly weaker acidic characteristics than those of E. faecalis (ATCC 29212). Moreover, the bacterial cells from the chicken hearts showed higher electrophoretic mobility and surface electrical charge than the reference strain, and consequently demonstrated a low ability to form biofilms.

The Involvement of Arginase and Nitric Oxide Synthase in Breast Cancer Development: Arginase and NO Synthase as Therapeutic Targets in Cancer.

It is well established that, during development of malignancies, metabolic changes occur, including alterations of enzyme activities and isoenzyme expression. Arginase and nitric oxide (NO) synthase (NOS) are two of those enzymes considered to be involved in tumorigenesis. The goal of this article was to study the involvement of arginase and NOS in the development of different stages of breast cancer. Our results have shown that human serum arginase activity and NO (resp., and NOS activity) and polyamines quantities increased in parallel with cancer stage progression and decreased after neoadjuvant chemotherapy. For breast cancer, the only isoenzyme of arginase expressed in serum before and after chemotherapy was in a cationic form. The data of Lineweaver-Burk plot with a Km value of 2 mM was calculated, which is characteristic for human liver type isoform of arginase. During electrophoresis at pH 8.9, the enzyme exhibited high electrophoretic mobility and was detected near the anode. The presented results demonstrated that arginase in human serum with breast cancer and after chemotherapy is not polymorphic. We suggest that arginase and NOS inhibition has antitumor effects on cancer development, as it can inhibit polyamines and NO levels, a precursor of cancer cell proliferation, metastasis, and tumor angiogenesis.

Large-volume sample stacking for the analysis of low molecular mass amines in steam water by CE using novel highly absorbing probe for indirect UV detection

A rapid, simple, sensitive and ultrafast capillary electrophoretic method – large-volume sample stacking (LVSS) without a polarity switching step has been developed in this study for determining ammonia and 9 amines, including ethanolamine, cyclohexylamine, hydrazine, morpholine, triethanolamine, and dimethylamine in steam water and their degradation products, such as methylamine, ethylamine, diethanolamine, and ammonia. The background electrolyte (BGE) containing acridine as a probe was firstly used for indirect UV detection. Acridine was shown to be very beneficial for the determination of low molecular mass amines due to its high electrophoretic mobility and at the same time high molar absorption coefficient (ε252) of 1.7∗105. No laborious sample preparation was needed. Separation and detection was achieved in about 8.5min for BGE containing acridine per sample. Limits of detection (LOD) using acridine were as low as 0.003mg/L. The calibration plots showed good linearity over the concentration range from 0.01 to 1mg/L for ammonia and the amines of interest for nuclear and thermal power plants. The proposed method was successfully applied to the determination of ethanolamine and cyclohexylamine in steam water samples obtained from thermal power plant and the analytical results were in good agreement with those obtained by reference ion chromatography (IC) method.

Characterization of conformers and dimers of antithrombin by capillary electrophoresis-quadrupole-time-of-flight mass spectrometry

Antithrombin (AT) is a plasma glycoprotein which possesses anticoagulant and anti-inflammatory properties. AT exhibits various forms, among which are native, latent and heterodimeric ones. We studied the potential of capillary electrophoresis-mass spectrometry (CE-MS) using a sheath liquid interface, electrospray ionization (ESI), and a quadrupole-time-of-flight (Q-TOF) mass spectrometer to separate and quantify the different AT forms. For CE separation, a neutral polyvinyl alcohol (PVA) coated capillary was employed. The protein conformation was preserved by using a background electrolyte (BGE) at physiological pH. A sheath liquid of isopropanol-water 50:50 (v/v) with 14 mM ammonium acetate delivered at a flow rate of 120 μL h−1 resulted in optimal signal intensities. Each AT form exhibited a specific mass spectrum, allowing unambiguous distinction. Several co-injection experiments proved that latent AT had a higher electrophoretic mobility (μep) than native AT, and that these conformers could associate to form a heterodimer during the CE analysis. The developed CE-MS method enabled the detection and quantitation of latent and heterodimeric forms in a commercial AT preparation stored at room temperature for three weeks.

Effects of different irradiance levels on peroxidase activities in Quercus castaneifolia C.A. Mey. seedlings from different provenances

To understand the function of peroxidase (POD) in relation to a light gradient, changes in POD activity were studied in five different provenances of chestnut oak seedlings. An experiment was conducted in controlled conditions and six different irradiances (10, 20, 30, 50, 60, and 70% of full light) were considered. According to the results, POD activity was strongly related to irradiance and showed a decreasing response to light, with the largest changes at low light (10 to 30%) and a levelling-off at high light (50-70%). Five provenances were also significantly distinguished at 10-30% of full light. The gel analysis showed that there were several different bands between irradiances from 10 to 70% regardless of provenances. One isoenzyme with the highest electrophoretic mobility was significantly increased in response to reduced light and slightly decreased at high irradiance. Other isoenzymes were significantly increased at high irradiance, suggesting that these isoenzymes are most likely involved in response to light stress and leaf adaptation to high irradiance.

Screening of Small Intact Proteins by Capillary Electrophoresis Electrospray Ionization-Mass Spectrometry (CE-ESI-MS).

Capillary electrophoresis (CE) has been shown to be a suitable separation technique for complex samples. Combined with electrospray ionization-mass spectrometry (ESI-MS), it is a powerful tool offering the opportunity of high selectivity and sensitivity combined with the possibility to identify and characterize intact proteins. In this protocol, we demonstrate a screening method for intact proteins based on capillary zone electrophoresis (CZE) separation coupled with online mass spectrometric detection. In order to avoid protein-wall interactions, a neutral coated capillary is used to create a universal method for proteins with both low and high electrophoretic mobilities. In addition, we show the successful validation and application of this screening method for a set of eight standard proteins and the glycoprotein erythropoietin.

Antibiotic-loaded chitosan-Laponite films for local drug delivery by titanium implants: cell proliferation and drug release studies.

In this study, chitosan-Laponite nanocomposite coatings with bone regenerative potential and controlled drug-release capacity are prepared by electrophoretic deposition technique. The controlled release of a glycopeptide drug, i.e. vancomycin, is attained by the intercalation of the polymer and drug macromolecules into silicate galleries. Fourier-transform infrared spectrometry reveals electrostatic interactions between the charged structure of clay and the amine and hydroxyl groups of chitosan and vancomycin, leading to a complex positively-charged system with high electrophoretic mobility. By applying electric field the charged particles are deposited on the surface of titanium foils and uniform chitosan films containing 25-55wt% Laponite and 937-1655µg/cm(2) vancomycin are obtained. Nanocomposite films exhibit improved cell attachment with higher cell viability. Alkaline phosphatase assay reveals enhanced cell proliferation due the gradual dissolution of Laponite particles into the culture medium. In-vitro drug-release studies show lower release rate through a longer period for the nanocomposite compared to pristine chitosan.

Differential latency toward (–)-epicatechin and catechol mediated by avocado mesocarp polyphenol oxidase (PPO)

The polyphenol oxidase (PPO) metabolism of the endogenous substrate (–)-epicatechin has been correlated with browning in avocados and other fruit. Although total PPO activity declined during ripening in the avocado mesocarp, a selective retention of high electrophoretic mobility isoforms was observed during ripening. Avocado PPO was virtually inactive toward (–)-epicatechin without the addition of an activator such as sodium dodecyl sulfate. In contrast, PPO was active toward catechol, an exogenous substrate, in the absence of an activator. Low concentrations of the free fatty acids oleic acid, linoleic acid, and arachidonic acid were effective activators of (–)-epicatechin oxidation in vitro. The data suggest that these endogenous free fatty acids may play a role in the PPO-mediated browning of avocado fruit in vivo.

Activities of indigenous proteolytic enzymes in caprine milk of different somatic cell counts

Individual caprine milk with different somatic cell counts (SCC) were studied with the aim of investigating the percentage distribution of leukocyte cell types and the activities of indigenous proteolytic enzymes; proteolysis of casein was also studied in relation to cell type following recovery from milk. The experiment was conducted on 5 intensively managed dairy flocks of Garganica goats; on the basis of SCC, the experimental groups were denoted low (L-SCC; <700,000cells/mL), medium (M-SCC; from 701,000 to 1,500,000cells/mL), and high (H-SCC; >1,501,000cells/mL) SCC. Leukocyte distribution differed between groups; polymorphonuclear neutrophilic leukocytes were higher in M-SCC and H-SCC milk samples, the percentage macrophages was the highest in H-SCC, and levels of nonviable cells significantly decreased with increasing SCC. Activities of all the main proteolytic enzymes were affected by SCC; plasmin activity was the highest in H-SCC milk and the lowest in L-SCC, and elastase and cathepsin D activities were the highest in M-SCC. Somatic cell count influenced casein hydrolysis patterns, with less intact α- and β-casein in H-SCC milk. Higher levels of low electrophoretic mobility peptides were detected in sodium caseinate incubated with leukocytes isolated from L-SCC milk, independent of cell type, whereas among cells recovered from M-SCC milk, macrophages yielded the highest levels of low electrophoretic mobility peptides from sodium caseinate. The level of high electrophoretic mobility peptides was higher in sodium caseinate incubated with polymorphonuclear neutrophilic leukocytes and macrophages isolated from M-SCC, whereas the same fraction of peptides was always the highest, independent of leukocyte type, for cells recovered from H-SCC milk. In caprine milk, a level of 700,000cells/mL represented the threshold for changes in leukocyte distribution, which is presumably related to the immune status of the mammary gland. Differences in the profile of indigenous lysosomal proteolytic enzymes in caprine milk may influence the integrity of casein based on proteolysis patterns of sodium caseinate incubated with isolated and lysed leukocyte types.