SDS-polyacrylamide Gradient Gel Electrophoresis Research Articles

Sustained small and intermediate size proteins expression in phorbol 12-myristate 13-acetate/ionomycine prolonged stimulated human fibroblasts

ObjectiveTo compare the protein profile of culture supernatants in stimulated and unstimulated human fibroblasts to find some proteins indicating the presence of fibroblasts and their activation status. MethodsDermal fibroblasts were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycine for 72 h. MTT assay was done to determine cell viability and A/E fluorescent staining was used to evaluate the cell death pattern. Protein analysis was performed by gradient SDS polyacrylamide gel electrophoresis 8%–16%. ResultsThe supernatant of 24 h cultured both stimulated and unstimulated fibroblasts showed two bands in SDS-PAGE analysis with relative molecular weights of 8.59 and 78.8 kDa. These bands density was decreased during the next 48 h in unstimulated cells while their expression was continued in PMA or PMA/ionomycine stimulated cells and a new 85.3 kDa band was appeared in unstimulated and 72 h PMA stimulated cells. Moreover, we found another seven small size (10–19.5 kDa) proteins in supernatants of 48 h and 72 h unstimulated but not in PMA or PMA/Ionomycine stimulated fibroblasts. Most of these proteins expression were down regulated following fibroblast activation. This down-regulation is consistent with our finding that PMA or PMA/ionomycine stimulated cells exhibited a significant level of apoptosis cell death. ConclusionsHuman fibroblasts produce some small to intermediate sized proteins with specific SDS-PAGE profile upon cell activation. Most of these proteins can be excreted in urine and can be immunogen theoretically so this data provided a reliable clue for fibrosis biomarker screening based on designation of an appropriated immunoassay.

Proteome profiling of lipopolysaccharide induced L6 rat skeletal muscle cells response to flavonoids from Scutellaria baicalensis Georgi.

BackgroundScutellaria baicalensis Georgi is a commonly used medicinal herb in several Asian countries like Korea, China and Japan for thousands of years. It has been reported to have various medicinal properties such as anti-microbial, anti-inflammatory and anti-cancer effects. However, the anti-inflammatory mechanism of S. baicalensis G at proteome level has not yet been reported. Hence, we performed a proteome analysis to study differentially expressed proteins and its anti-inflammatory role in lipopolysaccharide (LPS) stimulated L6 skeletal muscle cells response to flavonoids isolated from S. baicalensis G.MethodsFor that, 150 μg of proteins from the L6 cells of the control (Vehicle only), LPS treated and flavonoid treated groups were separated using 18 cm, pH 4–7 IPG strips in the first dimension and resolved by 12% linear gradient SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The silver stained gels were analyzed by using progenesis SameSpots software and twenty six differentially expressed protein spots (≥2 fold, p < 0.05) were selected for matrix assisted laser desorption ionization- time of flight mass spectroscopy/mass spectrometry (MALDI-TOF/MS) analysis. Also, the expression of COX-2, iNOS and Annexin A2 proteins were analyzed by western blot.ResultsTotally, 12 differentially expressed proteins were successfully identified by MALDI-TOF/MS and database searching, that’s involved in inflammatory responses such vimentin, T-box transcription factor TBX3, annexin A1, annexin A2 and annexin A5. In addition, flavonoids inhibited the expression of COX-2, iNOS and Annexin A2 proteins in LPS-stimulated L6 skeletal muscle cells.ConclusionsThe findings revealed that the flavonoids from S. baicalensis G. directly protect the LPS stimulated inflammation process in L6 cells and, would be helpful to study further the muscle cell inflammatory mechanism. This is the first proteome study provide the anti-inflammatory mechanism of flavonoids from S. baicalensis G. in LPS stimulated L6 skeletal muscle cells.

A New Approach for Quantitative Phosphoproteomic Dissection of Signaling Pathways Applied to T Cell Receptor Activation

Reversible protein phosphorylation plays a pivotal role in the regulation of cellular signaling pathways. Current approaches in phosphoproteomics focus on analysis of the global phosphoproteome in a single cellular state or of receptor stimulation time course experiments, often with a restricted number of time points. Although these studies have provided some insights into newly discovered phosphorylation sites that may be involved in pathways, they alone do not provide enough information to make precise predictions of the placement of individual phosphorylation events within a signaling pathway. Protein disruption and site-directed mutagenesis are essential to clearly define the precise biological roles of the hundreds of newly discovered phosphorylation sites uncovered in modern proteomics experiments. We have combined genetic analysis with quantitative proteomic methods and recently developed visual analysis tools to dissect the tyrosine phosphoproteome of isogenic Zap-70 tyrosine kinase null and reconstituted Jurkat T cells. In our approach, label-free quantitation using normalization to copurified phosphopeptide standards is applied to assemble high density temporal data within a single cell type, either Zap-70 null or reconstituted cells, providing a list of candidate phosphorylation sites that change in abundance after T cell stimulation. Stable isotopic labeling of amino acids in cell culture (SILAC) ratios are then used to compare Zap-70 null and reconstituted cells across a time course of receptor stimulation, providing direct information about the placement of newly observed phosphorylation sites relative to Zap-70. These methods are adaptable to any cell culture signaling system in which isogenic wild type and mutant cells have been or can be derived using any available phosphopeptide enrichment strategy.

Mass spectrometric determination of apolipoprotein molecular stoichiometry in reconstituted high density lipoprotein particles

Plasma HDL-cholesterol and apolipoprotein A-I (apoA-I) levels are strongly inversely associated with cardiovascular disease. However, the structure and protein composition of HDL particles is complex, as native and synthetic discoidal and spherical HDL particles can have from two to five apoA-I molecules per particle. To fully understand structure-function relationships of HDL, a method is required that is capable of directly determining the number of apolipoprotein molecules in heterogeneous HDL particles. Chemical cross-linking followed by SDS polyacrylamide gradient gel electrophoresis has been previously used to determine apolipoprotein stoichiometry in HDL particles. However, this method yields ambiguous results due to effects of cross-linking on protein conformation and, subsequently, its migration pattern on the gel. Here, we describe a new method based on cross-linking chemistry followed by MALDI mass spectrometry that determines the absolute mass of the cross-linked complex, thereby correctly determining the number of apolipoprotein molecules in a given HDL particle. Using well-defined, homogeneous, reconstituted apoA-I-containing HDL, apoA-IV-containing HDL, as well as apoA-I/apoA-II-containing HDL, we have validated this method. The method has the capability to determine the molecular ratio and molecular composition of apolipoprotein molecules in complex reconstituted HDL particles.

Enrichment of C-Terminal Fragments in TAR DNA-Binding Protein-43 Cytoplasmic Inclusions in Brain but not in Spinal Cord of Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis

TAR DNA-binding protein (TDP-43) has been recently described as a major pathological protein in both frontotemporal dementia with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis. However, little is known about the relative abundance and distribution of different pathological TDP-43 species, which include hyperphosphorylated, ubiquitinated, and N-terminally cleaved TDP-43. Here, we developed novel N-terminal (N-t) and C-terminal (C-t)-specific TDP-43 antibodies and performed biochemical and immunohistochemical studies to analyze cortical, hippocampal, and spinal cord tissue from frontotemporal dementia with ubiquitin-positive inclusions and amyotrophic lateral sclerosis cases. C-t-specific TDP-43 antibodies revealed similar abundance, morphology, and distribution of dystrophic neurites and neuronal cytoplasmic inclusions in cortex and hippocampus compared with previously described pan-TDP-43 antibodies. By contrast, N-t-specific TDP-43 antibodies only detected a small subset of these lesions. Biochemical studies confirmed the presence of C-t TDP-43 fragments but not extreme N-t fragments. Surprisingly, immunohistochemical analysis of inclusions in spinal cord motor neurons in both diseases showed that they are N-t and C-t positive. TDP-43 inclusions in Alzheimer's disease brains also were examined, and similar enrichment in C-t TDP-43 fragments was observed in cortex and hippocampus. These results show that the composition of the inclusions in brain versus spinal cord tissues differ, with an increased representation of C-t TDP-43 fragments in cortical and hippocampal regions. Therefore, regionally different pathogenic processes may underlie the development of abnormal TDP-43 proteinopathies.

Adeno-associated Virus Type 5 Reduces Learning Deficits and Restores Glutamate Receptor Subunit Levels in MPS VII Mice CNS

A major challenge in treating lysosomal storage diseases with enzyme therapy is correcting symptoms in the central nervous system (CNS). This study used a murine model of mucopolysaccharidosis type VII (MPS VII) to test whether pathological and functional CNS defects could be corrected by expressing beta-glucuronidase via bilateral intrastriatal injection of adeno-associated virus type 5 (AAV5betagluc) vectors. After injecting AAV5betagluc, different brain regions expressed active beta-glucuronidase, which corrected lysosomal storage defects. Compared to age-matched littermates, adult MPS VII mice were impaired in spatial learning and memory, as measured by the repeated acquisition and performance chamber (RAPC) assay. AAV5betagluc-treated MPS VII mice improved significantly in the RAPC assay, relative to saline-injected littermates. Moreover, our studies reveal that cognitive changes in MPS VII mice correlate with decreased N-methyl-d-aspartate and alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor expression. Importantly, AAV5betagluc delivery restored glutamate receptor levels. Together, these data demonstrate that AAV5 vectors deliver a therapeutically effective beta-glucuronidase gene to the CNS and further suggest a possible mechanism underlying spatial learning defects in MPS VII mice.

Neprilysin-sensitive Synapse-associated Amyloid-β Peptide Oligomers Impair Neuronal Plasticity and Cognitive Function

Neprilysin-sensitive Synapse-associated Amyloid-β Peptide Oligomers Impair Neuronal Plasticity and Cognitive Function

Adaptation of tobacco budworm Heliothis virescens to proteinase inhibitors may be mediated by the synthesis of new proteinases

The tobacco budworm Heliothis virescens is adapted to feed on tobacco leaves that have proteinase protein inhibitors (PIs). To study this adaptation, the midgut proteinases of Heliothis virescens larvae reared on artificial PI-free diet and on tobacco leaves were compared using ion exchange chromatography, hydrophobic chromatography, gel filtration and polyacrylamide gel electrophoresis at different conditions. SDS polyacrylamide-gradient gel electrophoresis (SDS-PAGE) and kinetic studies shown that leaf-fed larvae have a chymotrypsin ( M r 26 000) and four trypsins (T1–T4) with the following properties: T1, K m 0.3 μM, M r 70 000 ; T2, K m 0.4 μM, M r 67 000; T3, K m 2.4 μM, M r 29 000; T4, K m 15 μM, M r 17 000. Diet-fed larvae have a chymotrypsin ( M r 26 000) and a major trypsin ( K m 2.9 μM, M r 29 000). Native PAGE at different gel concentrations showed that in these conditions, only T1 and T2 occur in leaf-fed larvae, whereas gel filtration in the absence and presence of SDS revealed that T1 and T2 might arise by polymerization of T3 and T4, respectively. The data suggest that, in the presence of PI-containing food, H. virescens larvae express new trypsin molecules that form oligomers and are apparently less affected by PIs because of tighter binding to the substrate (lower K m values) and a putative decreased affinity for PIs.

Genetic Evaluation of proteins with High Resolution Two Dimensional (2D) Gel Electrophoresis

High resolution two dimensional gel electrophoresis with the combination of isoelectric focusing (IEF) and density gradient SDS polyacrylamide gel electrophoresis (DGSDS-PAGE) is a highly sensitive...

Oral mucosal permeability and stability of transforming growth factor beta-3 in vitro.

To investigate the permeability and localization of topically applied 125I-TGF-beta3 in porcine floor-of-mouth mucosa as a function of concentration and exposure. The 125I-TGF-beta3 diluted in three different vehicles was applied to the tissue samples mounted in perfusion cells maintained at 37 degrees C. Flux and Kp values were calculated from the perfusate collected over a 24 hour period. The quantity of 125I-TGF-beta3 present in the tissue was determined by horizontal sectioning and subsequent counting. The stability of 125I-TGF-beta3 in saliva and in the tissue was analyzed by SDS polyacrylamide gradient gel electrophoresis. 125I-TGF-beta3 was relatively stable in saliva and in the epithelium; approximately 50% of the total counts in the deeper epithelium were resident in the 25kDa TGF-beta3 homodimer. A steady-state flux was reached approximately 6 hours post application and Kp value was 4.0+/-0.6 x 10(-6) (mean +/- sem). Penetration of 125I-TGF-beta3 to the basal cell layer was concentration dependent but reached nanomolar concentrations even after extensive surface rinsing, representing over one-thousand fold the IC50 for epithelial cell cycle arrest. The data suggest that topical application of TGF-beta3 to the oral mucosa in an appropriate vehicle can provide effective therapeutic delivery to the tissue.

Elektrophoretische Analyse humaner Uterussekretproteine: ein neuer Weg zur Funktionsdiagnostik des Endometriums

Objective: The protein composition of human uterine secretions was studied by SDS polyacrylamide gradient gel electrophoresis (SDS-PAGE). Material and methods: 278 secretion samples from 246 hormonally stimulated or unstimulated patients treated for infertility or cervical dysplasia were collected at different phases of the menstrual cycle by sampling transcervically uterine secretions. The proteins of the samples were studied by SDS-PAGE and by densitometric analysis of the electrophoretic protein patterns. Expression of certain proteins were evaluated, mathematically analysed and semiquantitatively described as a reproducible and comparable parameter. Results: The analysis reveals changing protein patterns during the menstrual cycle: An increasing number of small protein bands between 15 kDa and 60 kDa appears during the proliferative and the early and midsecretory phases. In the secretory phase the protein pattern is completed by three characteristic bands between 12.5 kDa and 21.0 kDa, known as histones H2 A, H2 B and H3. This results in a maximal expression of the protein pattern during the mid-secretory phase, the time of implantation. Analysis of the protein patterns in relation to infertility reveals weaker protein expression in patients suffering from tubal occlusion and idiopathic infertility in comparison to those apparently fertile. Conclusion: This study supports the results of an earlier study by Beier-Hellwig et al. (1989) where characteristic changes of the protein patterns during the menstrual cycle were evaluated by a visual and subjective analysis. The introduction of a semi-quantitative analysis of the protein patterns leads to a reproducible description of the fine tuning of the protein patterns during the cycle. This technique promises to be useful as a diagnostic tool for the analysis of endometrial function.

Purification and Characterization of Hepatic Aldehyde Oxidase in Male and Female Mice

To examine whether the hepatic aldehyde oxidases of male and female mice, which exhibit sex-related differences in benzaldehyde oxidation, are qualitatively different, we purified the enzymes from untreated male and femaleddymice and testosterone-pretreated female mice by sequential chromatography of benzamidine–Sepharose 6B and DEAE-5PW columns and characterized the enzymes. Purified enzymes from untreated male and female mice and from testosterone-treated females gave a single protein band atMr265K andMr138K on native and SDS–polyacrylamide gradient gel electrophoresis, respectively. The susceptibilities of the enzymes from both sexes to inhibitors such as menadione, norharman, quinacrine, estradiol, and SKF 525-A were also indistinguishable. However, theKmvalues for benzaldehyde,p-dimethylaminocinnamaldehyde, and 2-hydroxypyrimidine were two- to fourfold higher in the untreated female enzyme than in the untreated male enzyme, while the testosterone-induced female enzyme showedKmvalues similar to those of the male enzyme. These results indicate that the hepatic aldehyde oxidases of the sexes are quite similar with respect to molecular size and susceptibility to inhibitors, but their kinetic properties are somewhat different, suggesting the existence of microheterogeneity in sex differences. It also suggests that treatment of female mice with testosterone might induce the male-type enzyme.

アメマスＳａｌｖｅｌｉｎｕｓ ｌｅｕｃｏｍａｅｎｉｓビテロゲニンの卵黄蛋白質への特異的分解

Vitellogenin (Vg) was purified from white spotted-charr Salvelinus leucomaenis serum and biotinylated (b-Vg). The b-Vg was digested under mild acidic conditions by bovine cathepsin-D (BCD) or a water soluble fraction of charr eggs (WSF), Digests were analyzed by 5.0-22.5% gradient SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Westem blotting, and gel filtration on Superose 6. Westem blotting was done using: 1) streptavidin coupled to horseradish peroxidase (HRPO) to identify biotinylated products of b-Vg digestion, and 2) antisera against lipovitellin (a-Lv) or β-component (a-β') to immunologically relate digestion products to specific charr egg yolk proteins. With respect to antigenicity and apparent molecular weight, fragments resulting from digestion of b-Vg by WSF or BCD were almost indistinguishable from those found in charr egg yolk, except for phosvitin (Pv). Pv-related phosphorus in BCD digests eluted at a position corresponding to a molecular weight slightly greater than that of Pv in egg yolk. The present study is the first to demonstrate in vitro conversion of salmonid Vg to its related egg yolk proteins. The similar catalytic effect of WSF or BCD on in vitro generation of egg yolk proteins suggests that cathepsin-D, present in WSF or intact eggs, may play a key role in natural yolk formation in salmonids.

Cultured astrocytes express biglycan, a chondroitin/dermatan sulfate proteoglycan supporting the survival of neocortical neurons

Astrocyte-conditioned medium (ACM) supports the survival of rat E15 neocortical neurons. Using a microtiter assay for neuronal survival, we demonstrated that part of the survival activity is associated with a proteoglycan fraction obtained after two chromatographic steps: (1) preparative Q-Sepharose anion-exchange chromatography under non-denaturating conditions and (2) MonoQ chromatography in the presence of 8 M urea. Analytical SDS-polyacrylamide gradient gel electrophoresis of pooled active MonoQ-fractions (MQ-pool) revealed a broad proteoglycan band migrating with an apparent M r in the range of 150–400 kDa. Digestion of the MQ-pool with chondroitin-ABC-lyase yielded a major core protein of 50 kDa. In Western blots the high molecular weight (150–400 kDa) material as well as the 50 kDa core protein band were immunoreactive to chicken polyclonal antibodies raised against purified biglycan from rat meningeal fibroblasts. Northern blot analysis of total RNA prepared from highly enriched astrocyte cultures revealed a single 2.9 kb biglycan transcript. By using in situ hybridization we demonstrated that essentially all cells in these cultures expressed biglycan mRNA. Furthermore, highly purified biglycan from bovine cartilage was shown to markedly enhance survival of rat neocortical neurons. In conclusion, we have shown that astrocytes synthesize and release the small chondroitin/dermatan sulfate proteoglycan (CS/DSPG) biglycan, a molecule that was found to support survival of neocortical neurons in vitro.

Purification and Partial Characterization of a Lutein-binding Protein from the Midgut of the Silkworm Bombyx mori

A lutein-binding protein was purified from fifth instar larval midgut of Bombyx mori by a combination of ammonium sulfate fractionation and three chromatographic procedures, gel filtration, chromatofocusing, and anion exchange chromatography. The protein has a pI of 5.4 and an apparent molecular mass of 35,000 Da, as determined by a linear gradient SDS-polyacrylamide gel electrophoresis. The lutein-protein complex is water-soluble and more stable than the carotenoid or protein alone. The carotenoid moiety was identified by thin layer chromatography, light absorption spectroscopy, and high performance liquid chromatography as all-trans-lutein. Lutein is specifically and stoichiometrically bound to the protein, with a ratio of 3 mol of lutein per mol of protein. Binding of lutein (absorption maximum in hexane at 454 nm) to the apoprotein results in a marked red spectral shift of about 38 nm, giving rise to absorption maxima at 432, 462, and 492 nm in 20 mM Tris-HCl, pH 7.0. The lutein-protein complex is characterized by fine spectral structure indicating that lutein is in a relatively rigid environment. This protein is distributed in equal amounts throughout the midgut and in all developmental stages of the larval B. mori.

Influence of nutrient excess on intracellular proteins composition in relation to spiramycin production, in Streptomyces ambofaciens

The protein profile on media favouring or reducing spiramycin biosynthesis in S. ambofaciens, was examined using SDS-polyacrylamide gradient gel electrophoresis over a time course of 144 h. Changes in the pattern of protein synthesis were observed which varied according to the nutrient medium conditions and appeared to suggest the presence of protein subsets specific to the production phase, which were probably involved in the switch to secondary metabolism and the onset of antibiotic synthesis. Excess of ammonium, glycerol or phosphate provoked the persistence of a protein band during the whole culture period, which was detected only during growth phase in the control producer culture. In addition, the nutrient excess caused the suppression of spiramycin production and the absence of two protein bands which appeared only in production phase in the control producer culture.

Evidence That Meizothrombin Is an Intermediate Product in the Clotting of Whole Blood

Meizothrombin is an intermediate that is produced during the conversion of prothrombin to thrombin in systems composed of purified factor Xa and factor Va that are quantitatively assembled on an anionic phospholipid surface. The biological significance of this intermediate has recently been challenged by the apparent absence of meizothrombin during clotting of sodium citrate-anticoagulated plasma. We analyzed the formation of thrombin during coagulation of nonanticoagulated, unchilled, minimally manipulated whole blood in glass tubes. The process was stopped at 0, 3, 5, and 7 minutes by the addition of biotinylated peptidyl chloromethyl-ketone active-site labeling reagents. Plasma/serum was separated by centrifugation, and labeled species were extracted by immunoadsorption with a polyclonal anti-prothrombin antibody. The purified prothrombin-derived species were separated by SDS-polyacrylamide gradient gel electrophoresis and visualized on a chemiluminescent avidin blot. Meizothrombin appeared as an intermediate product of this reaction and persisted with some increase through the 7-minute time point. We also observed incorporation of the active-site label into a species of lower molecular weight consistent with the B1 chain of beta- and/or gamma-thrombin. These degraded forms of thrombin have not been previously demonstrated in a biologically relevant preparation. Our data clearly establish the generation of meizothrombin as an intermediate product of thrombin generation during whole-blood clotting. The data also represent the first experimental evidence for the generation of beta- and gamma-thrombin in a biologically relevant environment and time scale.

Identification, isolation and partial characterisation of a 14–15 kDa pigment binding protein complex of PS II from spinach

We described recently a new Chl a b binding polypeptide in PS II from spinach (Irrgang, K.-D., Bechtel, C., Vater, J. and Renger, G. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., ed.), Vol. I, pp. 375–378, Kluwer, Dordrecht). This 14–15 kDa chlorophyll a b binding polypeptide complex was isolated and purified from intrinsic photosystem II membrane polypeptides. About 3 Chl a and 1 Chl b were bound per protein molecule. A polyclonal antiserum was induced against this pigment-protein complex. SDS polyacrylamide gradient gel electrophoresis in combination with immunoblotting revealed reactivities with two polypeptides of very similar relative molecular masses of 14 and 15 kDa that are clearly identified in thylakoids, PS II membrane fragments, ISO and RSO thylakoids. Their N-termini were blocked by an as yet unidentified modifying group. Using analytical isoelectric focusing under denaturing conditions their isoelectric points were determined to be 5.2–5.3 and around 6.0–6.5. The isolated polypeptides of the pigment-protein complex tend to self-associate into oligomeric forms of about 66–70 kDa. Furthermore, under mild solubilisation conditions an oligomeric pigment-protein complex of 120 kDa was observed. This oligomer was shown to be heterogeneously composed of the 14–15 kDa proteins and at least another pigment-binding polypeptide with an M r of 22–24 kDa. The low molecular mass pigment-protein complex ( CP14–15) is proposed to act as an additional antenna complex within PS II.

A bacteria-digesting midgut-lysozyme from Musca domestica (diptera) larvae. Purification, properties and secretory mechanism

Two lysozymes were purified to homogeneity from heated acid extracts of Musca domestica larval midguts, using an S-Sepharose column, and a semi-preparative polyacrylamide gel electrophoresis (PAGE). The final yield was 60%. Lysozymes 1 and 2 display M r 22,000, determined by ultracentrifugation or electrophoresis in non-denaturing conditions, and M r 17,000, determined by SDS-polyacrylamide gradient gel electrophoresis. Isoelectric focusing showed the following pI values: lysozyme 1, 7.9; lysozyme 2, 8.2. Lysozyme 1 and 2 display identical kinetic properties, which include decrease in activity, displacement of the pH optimum toward acidic values and a K m increase as the ionic strength of the medium becomes higher. The lysozymes are resistant to a cathepsin D-like proteinase present in M. domestica midgut, and display a chitinase activity which is 6-fold higher than that of chicken lysozyme. Lysozyme immunolabeling revealed that lysozyme mainly occurs in secretory vesicles and at the outside surface of microvilli from M. domestica anterior midgut cells. The results showed that M. domestica lysozymes are similar to ruminant stomach lysozyme in being more active at acid pH values, when present in media with physiological ionic strengths, and in being resistant to and acid proteinase derived from the same animal as the lysozyme. Furthermore, the datta support the assertion that M. domestica midgut lysozyme is secreted by exocytosis, partly remaining adsorbed to the cell glycocalyx.

Electrophoretic protein profiles of strains of Ceratocystis fimbriata f. sp. platani

Soluble proteins extracted from the mycelium of strains of Ceratocystis fimbriata (Ell. et Halst) Davidson f. sp. platani Walter were separated on native and SDS-polyacrylamide gradient gel electrophoresis. The protein patterns were compared and the electrophoretic profiles of all the strains examined showed striking intraspecific similarities. The use or protein banding patterns as a diagnostic criterion to distinguish strains of C. fimbriata is discussed.