Protein profiling of rat cerebella during development

Protein profiles of developing rat cerebella were analyzed by means of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). The analysis of adult rat cerebellum gave rise to a protein map comprising approximately 3000 spots detectable by silver staining following high resolution 2-DE with a pH range of 3-10 and a mass range of 8-100 kDa. To obtain landmarks for comparison of developmental profiles of cerebellar proteins, 100 spots were subjected to peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and 67 spots were assigned on the map. Analysis of profiles of the developing cerebella revealed significant changes in the expression of proteins during development. In most cases the expression levels of proteins increased as the cerebellum matured, while the expression of 42 spots appeared specific or remarkably abundant in the immature cerebellum. Peptide mass fingerprinting of these spots allowed us to identify 29 proteins, which include, in addition to proteins of unknown function, many proteins known to have roles in the development of the central nervous system. These results suggest that the proteomic approach is valuable for mass identification of proteins involved in cerebellar morphogenesis.     

Diverse redox-active molecules bearing O-, S-, or Se-terminated tethers for attachment to silicon in studies of molecular information storage

A molecular approach to information storage employs redox-active molecules tethered to an electroactive surface. Attachment of the molecules to electroactive surfaces requires control over the nature of the tether (linker and surface attachment group). We have synthesized a collection of redox-active molecules bearing different linkers and surface anchor groups in free or protected form (hydroxy, mercapto, S-acetylthio, and Se-acetylseleno) for attachment to surfaces such as silicon, germanium, and gold. The molecules exhibit a number of cationic oxidation states, including one (ferrocene), two [zinc(II)porphyrin], three [cobalt(II)porphyrin], or four (lanthanide triple-decker sandwich compound). Electrochemical studies of monolayers of a variety of the redox-active molecules attached to Si(100) electrodes indicate that molecules exhibit a regular mode of attachment (via a Si-X bond, X = O, S, or Se), relatively homogeneous surface organization, and robust reversible electrochemical behavior. The acetyl protecting group undergoes cleavage during the surface deposition process, enabling attachment to silicon via thio or seleno groups without handling free thiols or selenols.     

Quantitative analysis of relative protein contents by Western blotting: comparison of three members of the dystrophin-glycoprotein complex in slow and fast rat skeletal muscle

We have developed a method for accurate quantitative analysis and statistical comparison of the relative contents of the dystrophin-glycoprotein complex (DGC) in skeletal muscle. This method was applied to compare DGC contents in slow (soleus) and in fast (extensor digitorum longus, EDL) rat skeletal muscles. The quantitative analysis combines a modified bicinchoninic acid (BCA) assay with Western blotting and enhanced chemiluminescence (ECL). This combination allows the use of high levels of detergents and reducing reagents essential for extracting DGC. In addition, the evaluation of the total amount of proteins in each sample makes it possible to have a reference and to accurately compare relative protein levels without using a specific standard. With a large gradient gel, we could concomitantly compare two groups (n = 9) and quantify all protein contents differing highly in their molecular masses (from 35 kDa to 427 kDa). Each experiment was triplicated and normalized; the two muscles were compared using the Mann-Whitney test (P<0.001) to establish their protein content. The DGC relative levels for the slow muscle soleus and the fast muscle EDL differed significantly: dystrophin, beta-dystroglycan, and gamma-sarcoglycan levels were 130%, 110% and 120% higher in the soleus, respectively. The differences observed in the expression level of cytoskeletal associated protein (dystrophin) and transmembranous anchorage components may correspond to a physiological response of the muscle fibers to duration, magnitude, and frequency of the imposed mechanical loading.     

On the equivalence between the energy and virial routes to the equation of state of hard-sphere fluids

The energy route to the equation of state of hard-sphere fluids is ill defined since the internal energy is just that of an ideal gas, and thus it is independent of density. It is shown that this ambiguity can be avoided by considering a square-shoulder interaction and taking the limit of vanishing shoulder width. The resulting hard-sphere equation of state coincides exactly with the one obtained through the virial route. Therefore, the energy and virial routes to the equation of state of hard-sphere fluids can be considered as equivalent.     

Rapid determination of aliphatic amines in water samples by pressure-assisted monolithic octadecylsilica capillary electrochromatography-mass spectrometry

A pressure-assisted capillary chromatography-mass spectrometry method based on the use of a monolithic octadecylsilica (ODS) capillary is proposed for the determination of aliphatic amines. A 25 mM citric acid buffer containing 10% methanol is used as running electrolyte. Separation is achieved by simultaneously applying a capillary electrophoresis (CE) voltage of 13 kV and an overimposed pressure of 8 bar. The use of pressure is required to ensure stable electrospray conditions. Analysis times are reduced by using a capillary column consisting of a 30 cm long monolithic silica capillary column bound with ODS and a fused-silica capillary column also 30 cm long. The proposed method was successfully applied to the determination of low-molecular-weight aliphatic amines in tap and river water. The analysis of real samples requires cleanup and preconcentration, which can be performed automatically by inserting a minicolumn in the replenishment system of the commercial instrument.     

Dynamic behaviour of river colloidal and dissolved organic matter through cross-flow ultrafiltration system

Through cross-flow filtration (CFF) with a 1-kDa regenerated cellulose Pellicon 2 module, the ultrafiltration characteristics of river organic matter from Longford Stream, UK, were investigated. The concentration of organic carbon (OC) in the retentate in the Longford Stream samples increased substantially with the concentration factor (cf), reaching approximately 40 mg/L at cf 15. The results of dissolved organic carbon (DOC) and colloidal organic carbon (COC) analysis, tracking the isolation of colloids from river waters, show that 2 mg/L of COC was present in those samples and good OC mass balance (77-101%) was achieved. Fluorescence measurements were carried out for the investigation of retentate and permeate behaviour of coloured dissolved organic materials (CDOM). The concentrations of CDOM in both the retentate and permeate increased with increasing cf, although CDOM were significantly more concentrated in the retentate. The permeation model expressing the correlation between log[CDOM] in the permeate and logcf was able to describe the permeation behaviour of CDOM in the river water with regression coefficients (r(2)) of 0.94 and 0.98. Dry weight analysis indicated that the levels of organic colloidal particles were from 49 to 71%, and between 29 and 51% of colloidal particles present were inorganic. COC as a percentage of DOC was found to be 10-16% for Longford Stream samples.     

Assessment of drinking water radioactivity content by liquid scintillation counting: set up of high sensitivity and emergency procedures

In our institute, different procedures have been developed to measure the radioactivity content of drinking water both in normal and in emergency situations, such as those arising from accidental and terrorist events. A single radiometric technique, namely low level liquid scintillation counting (LSC), has been used. In emergency situations a gross activity screening is carried out without any sample treatment by a single and quick liquid scintillation counting. Alpha and beta activities can be measured in more than one hundred samples per day with sensitivities of a few Bq/L. Higher sensitivity gross alpha and beta, uranium and radium measurements can be performed on water samples after specific sample treatments. The sequential method proposed is designed in such a way that the same water sample can be used in all the stages, with slight modifications. This sequential procedure was applied in a survey of the Lombardia district. At first tap waters of the 13 largest towns were examined, then a more detailed monitoring was carried out in the surroundings of Milano and Lodi towns. The high sensitivity method for the determination of uranium isotopes was used to check the presence of depleted uranium in Lake Garda. Reduced equipment requirements and relative readiness of radiochemical procedures make LSC an attractive technique which can also be applied by laboratories lacking specific radiochemistry facilities and experience.     

Emulsifying and foaming properties of amaranth seed protein isolates

The emulsifying and foaming properties of amaranth seed protein isolates prepared by wet extraction methods, such as isoelectric precipitation and dialysis, were investigated. The various isolates differ from each other in many ways. The isolate prepared by isoelectric precipitation mainly contains the globulin but not the albumin fraction and a considerable amount of polysaccharides, while the other isolate prepared by the dialysis method contains all the globulin and albumin fractions. The protein-polysaccharide complexes enhance emulsion stability due to steric repulsion effects. Measurements of the emulsion stability show that the studied protein isolates act as effective stabilizing agents. Foam expansion is dominated by the surface activity and availability of protein in the solution, while foam stability is determined by the properties of the interfacial layer. The results show that amaranth protein isolates act as an effective foaming agent. Both foaming properties intensified from the presence of protein-polysaccharide complexes.