TGF-beta1 in Aplysia: role in long-term changes in the excitability of sensory neurons and distribution of TbetaR-II-like immunoreactivity.

Exogenous recombinant human transforming growth factor beta-1 (TGF-beta1) induced long-term facilitation of Aplysia sensory-motor synapses. In addition, 5-HT-induced facilitation was blocked by application of a soluble fragment of the extracellular portion of the TGF-beta1 type II receptor (TbetaR-II), which presumably acted by scavenging an endogenous TGF-beta1-like molecule. Because TbetaR-II is essential for transmembrane signaling by TGF-beta, we sought to determine whether Aplysia tissues contained TbetaR-II and specifically, whether neurons expressed the receptor. Western blot analysis of Aplysia tissue extracts demonstrated the presence of a TbetaR-II-immunoreactive protein in several tissue types. The expression and distribution of TbetaR-II-immunoreactive proteins in the central nervous system was examined by immunohistochemistry to elucidate sites that may be responsive to TGF-beta1 and thus may play a role in synaptic plasticity. Sensory neurons in the ventral-caudal cluster of the pleural ganglion were immunoreactive for TbetaR-II, as well as many neurons in the pedal, abdominal, buccal, and cerebral ganglia. Sensory neurons cultured in isolation and cocultured sensory and motor neurons were also immunoreactive. TGF-beta1 affected the biophysical properties of cultured sensory neurons, inducing an increase of excitability that persisted for at least 48 hr. Furthermore, exposure to TGF-beta1 resulted in a reduction in the firing threshold of sensory neurons. These results provide further support for the hypothesis that TGF-beta1 plays a role in long-term synaptic plasticity in Aplysia.

Study of polytopic membrane protein topological organization as a function of membrane lipid composition.

A protocol is described using lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by the substituted-cysteine accessibility method as applied to transmembrane domains (SCAM). SCAM is adapted to follow changes in membrane protein topology as a function of changes in membrane lipid composition. The strategy described can be adapted to any membrane system.

Elevated albumin in retinas of monkeys with experimental glaucoma.

PURPOSE: To establish the identity of a prominent protein, approximately 70 kDa, that is markedly increased in the retina of monkeys with experimental glaucoma compared with the fellow control retina, the relationship to glaucoma severity, and its localization in the retina. METHODS: Retinal extracts were subjected to 2-D gel electrophoresis to identify differentially expressed proteins. Purified peptides from the abundant 70 kDa protein were analyzed and identified by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) separation, and collision-induced dissociation sequencing. Protein identity was performed on MASCOT (Matrix Science, Boston, MA) and confirmed by Western blot. The relationship between the increase in this protein and glaucoma severity was investigated by regression analyses. Protein localization in retina was evaluated by immunohistochemistry with confocal imaging. RESULTS: The abundant protein was identified as Macaca mulatta serum albumin precursor (67 kDa) from eight non-overlapping proteolytic fragments, and the identity was confirmed by Western blot. The average increase in retinal albumin content was 2.3 fold (P = 0.015). In glaucoma eyes, albumin was localized to some neurons of the inner nuclear layer, in the inner plexiform layer, and along the vitreal surface, but it was only found in blood vessels in control retinas. CONCLUSIONS: Albumin is the abundant protein found in the glaucomatous monkey retinas. The increased albumin is primarily localized to the inner retina where oxidative damage associated with experimental glaucoma is known to be prominent. Since albumin is a major antioxidant, the increase of albumin in the retinas of eyes with experimental glaucoma may serve to protect the retina against oxidative damage.

Heteroduplex analysis of VDJ amplified segments from rearranged IgH genes for clonality assessments in B-cell non-Hodgkin's lymphoma. A comparison between different strategies.

BACKGROUND AND OBJECTIVE: The main difficulty of PCR-based clonality studies for B-cell lymphoproliferative disorders (B-LPD) is discrimination between monoclonal and polyclonal PCR products, especially when there is a high background of polyclonal B cells in the tumor sample. Actually, PCR-based methods for clonality assessment require additional analysis of the PCR products in order to discern between monoclonal and polyclonal samples. Heteroduplex analysis represents an attractive approach since it is easy to perform and avoids the use of radioactive substrates or expensive equipment. DESIGN AND METHODS: We studied the sensitivity and specificity of heteroduplex PCR analysis for monoclonal detection in samples from 90 B-cell non Hodgkin's lymphoma (B-NHL) patients and in 28 individuals without neoplastic B-cell disorders (negative controls). Furthermore, in 42 B-NHL and in the same 28 negative controls, we compared heteroduplex analysis vs the classical PCR technique. We also compared ethidium bromide (EtBr) vs. silver nitrate (AgNO(3)) staining as well as agarose vs. polyacrylamide gel electrophoresis (PAGE). RESULTS: Using two pair consensus primers sited at VH (FR3 and FR2) and at JH, 91% of B-NHL samples displayed monoclonal products after heteroduplex PCR analysis using PAGE and AgNO(3) staining. Moreover, no polyclonal sample showed a monoclonal PCR product. By contrast, false positive results were obtained when using agarose (5/28) and PAGE without heteroduplex analysis: 2/28 and 8/28 with EtBr and AgNO(3) staining, respectively. In addition, false negative results only appeared with EtBr staining: 13/42 in agarose, 4/42 in PAGE without heteroduplex analysis and 7/42 in PAGE after heteroduplex analysis. INTERPRETATION AND CONCLUSIONS: We conclude that AgNO(3) stained PAGE after heteroduplex analysis is the most suitable strategy for detecting monoclonal rearrangements in B-NHL samples because it does not produce false-positive results and the risk of false-negative results is very low.

On-line hyphenation of capillary isoelectric focusing and capillary gel electrophoresis by a dialysis interface

An on-line two-dimensional (2D) capillary electrophoresis (CE) system consisting of capillary isoelectric focusing (CIEF) and capillary gel electrophoresis (CGE) was introduced. To validate this 2D system, a dialysis interface was developed by mounting a hollow fiber on a methacrylate resin plate to hyphenate the two CE modes. The two dimensions of capillary shared a cathode fixated into a reservoir in the methacrylate plate; thus, with three electrodes and only one high-voltage source, a 2D CE framework was successfully established. A practical 2D CIEF-CGE experiment was carried out to deal with a target protein, hemoglobin (Hb). After the Hb variants with different isoelectric points (pIs) were focused in various bands in the first-dimension capillary, they were chemically mobilized one after another and fed to the second-dimension capillary for further separation in polyacrylamide gel. During this procedure, a single CIEF band was separated into several peaks due to different molecular weights. The resulting electrophoregrarn is quite different from that of either CIEF or CGE; therefore, more information about the studied Hb sample can be obtained.

Acute phase protein profile in agoutis (Dasyprocta azarae; Lichtenstein, 1823) in captivity, determined by sodium dodecyl sulfate polyacrilamyde gel electrophoresis

The acute phase response refers to a nonspecific and complex systemic reaction of the organism that occurs shortly after any tissue injury. The acute phase response is considered a part of the innate host defense system, which is responsible for the survival of the host during the critical early stages of attack, and in evolutionary terms, it precedes the acquired immune response. The purpose of this study was to determine serum protein concentrations, including the acute phase protein profile in agoutis (Dasyprocta azarae) in captivity, by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Blood samples from 11 adult healthy animals (nine females and two males) were obtained. The serum proteinogram had 21 proteins with molecular weights ranging from 15 to 240 kD. The acute phase proteins identified were: ceruloplasmin, transferrin, albumin, haptoglobin, α-1-acid glycoprotein, and hemoglobin. IgA, IgG heavy and light chains, and nonnominal identified proteins of 240, 210, 140, 98, 78, 48, 35, 31, 23, and 15 kD were also identified. The determination of the acute phase protein concentrations is a useful method for the early detection of subclinical disease or changes in the healthy animal, with predictive information on the development of disease in the future. It is possible to standardize the reference values of the serum protein profile of agoutis, which can be used for diagnosis and prognosis, treatment and clinical follow-up of nutritional disorders, and immune-mediated inflammatory diseases that may affect these animals. © 2012 Springer-Verlag London Limited.

Use of pulsed field gel electrophoresis to characterize BCR gene involvement in CML patients lacking M-BCR rearrangement.

We studied the pattern of BCR involvement in 52 patients with chronic myeloid leukemia by Southern blotting. Of 33 Philadelphia (Ph)-positive patients, 30 had evidence of M-BCR rearrangement, two cases were difficult to interpret, and one clearly lacked evidence of M-BCR rearrangement. Of 19 Ph-negative patients, nine showed M-BCR rearrangement, nine showed no rearrangement, and one result was uncertain. We selected for more detailed study eight patients (three Ph-positive and five Ph-negative). Two of the Ph-positive patients, whose Southern blots were difficult to interpret, had rearranged bands when the BCR gene was studied by pulsed field gel electrophoresis (PFGE). Results of PFGE studies and in situ hybridization to metaphase chromosomes in the third Ph-positive patient, whose DNA clearly lacked M-BCR rearrangement on Southern analysis, were consistent with a breakpoint on chromosome 22 located 3' of all known exons of the BCR gene. However, mRNA studied with the polymerase chain reaction showed evidence of a classical b2-a2 linkage. The findings in this patient may be explained by an unusual genomic breakpoint downstream of the BCR gene associated with long range splicing that excluded all of the 3' BCR exons. Of the five patients with Ph-negative M-BCR non-rearranged CML studied by PFGE for BCR gene rearrangement, none had evidence of rearranged bands. We conclude that PFGE is a valuable adjunct to standard molecular techniques for the study of atypical cases of CML. Occasional patients with Ph-positive CML have breakpoints outside M-BCR. The BCR gene is probably not involved in patients with Ph-negative, M-BCR non-rearranged CML.

Separating DNA with different topologies by atomic force microscopy in comparison with gel electrophoresis.

Atomic force microscopy, which is normally used for DNA imaging to gain qualitative results, can also be used for quantitative DNA research, at a single-molecular level. Here, we evaluate the performance of AFM imaging specifically for quantifying supercoiled and relaxed plasmid DNA fractions within a mixture, and compare the results with the bulk material analysis method, gel electrophoresis. The advantages and shortcomings of both methods are discussed in detail. Gel electrophoresis is a quick and well-established quantification method. However, it requires a large amount of DNA, and needs to be carefully calibrated for even slightly different experimental conditions for accurate quantification. AFM imaging is accurate, in that single DNA molecules in different conformations can be seen and counted. When used carefully with necessary correction, both methods provide consistent results. Thus, AFM imaging can be used for DNA quantification, as an alternative to gel electrophoresis.

Outer membrane protein profiles and multilocus enzyme electrophoresis analysis for differentiation of clinical isolates of Proteus mirabilis and Proteus vulgaris

Outer membrane protein (MP) profiles and multilocus enzyme electrophoresis (MEE) analysis were used as tools for differentiating clinical isolates of Proteus spp. Fourteen distinct MP profiles were established by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis in 54 clinical isolates of Proteus spp. (44 strains identified as P. mirabilis and 10 strains identified as P. vulgaris). Forty-one isolates of P. mirabilis and eight isolates of P. vulgaris were grouped within six and three MP profiles, respectively. The remaining P. mirabilis and P. vulgaris isolates had unique profiles. MEE analysis was used to further discriminate among the strains belonging to the same MP groups. Thirty-five distinct electrophoretic types (ETs) were identified among P. mirabilis isolates. The isolates of P. mirabilis from the four most common MP groups were subgrouped into 30 ETs. All of the P. vulgaris strains had unique ETs. The results suggest that upon biochemical classification of Proteus isolates as P. mirabilis or P. vulgaris, further differentiation among strains of the same species can be obtained by the initial determination of MP profiles followed by MEE analysis of strains with identical MPs.

Proteinograma sérico de cães sadios e com linfoma obtido por eletroforese em gel de poliacrilamida (SDS-PAGE)

Foram avaliadas amostras de soro sanguíneo de 10 cães sadios e de 12 com linfoma, utilizando-se a eletroforese em gel de poliacrilamida contendo dodecil sulfato de sódio. Houve diferença entre as médias dos teores de proteína total de cães sadios, 7,68g/dL±0,46 e de cães com linfoma, 7,93g/dL±2,49. As concentrações de IgA e IgG não foram diferentes entre os grupos. Os teores das proteínas de pesos moleculares 142000, 110000, 52000, 49000, 24000 e 18000 dáltons foram mais elevados em cães com linfoma. Os cães com linfoma apresentaram concentrações mais elevadas de ceruloplasmina, 43,95mg/dL±18,19, e haptoglobina, 554mg/dL±449,51, e menores de albumina, 2908mg/dL±476,67, em comparação aos cães sadios (ceruloplasmina: 3,42mg/dL±7,44; haptoglobina: 94,54mg/dL±59,50 e albumina: 4207mg/dL±206,18). Conclui-se que concentrações séricas mais elevadas de ceruloplasmina e haptoglobina e menores de albumina podem estar associadas ao linfoma em cães.

Influência do puerpério sobre o proteinograma sérico de caprinos da raça Saanen obtido por eletroforese em gel de poliacrilamida

Aiming to evaluate the puerperal influence on the proteinogram of Saanen goats, 108 samples of blood serum from 12 goats were collected, and the results were presented at nine times: just after parturition, 1, 3, 5, 7, 10, 15, 21 and 30 days after parturition. Total amount of serum proteins were determined by the biuret technique, and the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to the protein fractionation. In this last method, 17 protein bands were observed, from which molecular weights varied between 25 KDa and 275 KDa. In addition, it was possible to identify the following protein fractions: immunoglobulin A (180 KDa), ceruloplasmin (115 KDa), transferrin (79 KDa), albumin (65 KDa), heavy-chain immunoglobulin G (58 KDa), haptoglobin (45 KDa), acid glycoprotein (37 KDa) and light-chain immunoglobulin G (28 KDa). Another 9 nonidentified protein fractions presented, each molecular weights equal to 275 KDa, 140 KDa, 125 KDa, 103 KDa, 95 KDa, 41 KDa, 35 KDa, 30 Kda and 25 KDa. The results allow us to conclude that by the first week of puerperium, an improvement of acid glycoprotein occurs, whereas those others protein fractions do not suffer any puerperal influence.

Solubilization of proteins from human lymph node tissue and two-dimensional gel storage.

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT (dithiothreitol) and 0.2% carrier ampholytes; (b) 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.

Selênio em tilápia do Nilo utilizando eletroforese em gel e espectrometria atômica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Proteinograma do líquido sinovial de equinos hígidosobtido por eletroforese em gel de poliacrilamida

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Novel PCL-based honeycomb scaffolds as drug delivery systems for rhBMP-2

This study investigated a novel drug delivery system (DDS), consisting of polycaprolactone (PCL) or polycaprolactone 20% tricalcium phosphate (PCL-TCP) biodegradable scaffolds, fibrin Tisseel sealant and recombinant bone morphogenetic protein-2 (rhBMP-2) for bone regeneration. PCL and PCL-TCP-fibrin composites displayed a loading efficiency of 70% and 43%, respectively. Fluorescence and scanning electron microscopy revealed sparse clumps of rhBMP-2 particles, non-uniformly distributed on the rods’ surface of PCL-fibrin composites. In contrast, individual rhBMP-2 particles were evident and uniformly distributed on the rods’ surface of the PCL-TCP-fibrin composites. PCL-fibrin composites loaded with 10 and 20 μg/ml rhBMP-2 demonstrated a triphasic release profile as quantified by an enzyme-linked immunosorbent assay (ELISA). This consisted of burst releases at 2 h, and days 7 and 16. A biphasic release profile was observed for PCL-TCP-fibrin composites loaded with 10 μg/ml rhBMP-2, consisting of burst releases at 2 h and day 14. PCL-TCP-fibrin composites loaded with 20 μg/ml rhBMP-2 showed a tri-phasic release profile, consisting of burst releases at 2 h, and days 10 and 21. We conclude that the addition of TCP caused a delay in rhBMP-2 release. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline phosphatase assay verified the stability and bioactivity of eluted rhBMP-2 at all time points