Nomarski interference microscopy: use of combined contrast uranyl acetate-PTA for in vitro observation of monolayer cultures

In this paper is reported a technique for the fixation and staining of cellular monolayers in vitro for observation by the interferential microscope according to Nomarski. This method includes the use of glutaraldehyde at 2.5% in phosphate buffer 0.1 M pH 7.5 and the sequential use of Uranil acetate (0.9% in absolute alcohol) and phosphtungstic acid (1% in absolute alcohol). This study has been conducted on 3 cellular lines: PC12 (rat pheocromocytoma), R.P.C. (rat pineal cells), primary culture obtained from a human carcinoma of the uterus.     

Functional effects of a monoclonal antibody directed against a distinct epitope on 4F2 molecular complex in human peripheral blood mononuclear cell activation.

The 4F2 antigenic complex is expressed on most human cell lines in culture, on monocytes and activated lymphocytes, but not on resting T and B lymphocytes. Monoclonal antibody (mAb) CB43 recognizes an epitope of the 4F2 heterodimer either located on the light chain or dependent on the conformation of the molecule. The binding of CB43 mAb to peripheral blood mononuclear cells (PBMC) induced a dose-dependent comitogenic effect in the presence of submitogenic concentrations of anti-CD3 mAb. Significant amounts of interleukin (IL)-1 beta but not IL-2 or interferon-gamma were released in the supernatant. Pretreatment of monocytes with CB43 mAb increased the phytohemagglutinin-induced T lymphocyte proliferation. However, CB43 mAb did not exert agonistic effects on activated T lymphocytes. Depletion of CB43+ cells from PBMC decreased the proliferation and generation of cytotoxic effector cells induced by a mannoprotein (MP) derived from Candida albicans cell wall but not by recombinant IL-2. Furthermore, depletion of CB43+ cells from PBMC preactivated with MP or rIL-2 led to a significant decrease in their cytotoxic activity. CB43 mAb did not inhibit the growth of cell lines nor the proliferation of T cells. Thus CB43 mAb identifies a distinct functional epitope on the 4F2 molecular complex and might be useful in further studying the role of this molecule in cellular activation.     

Reversed-phase high-performance liquid chromatography separation of dimethylaminoazobenzene sulfonyl- and dimethylaminoazobenzene thiohydantoin-amino acid derivatives for amino acid analysis and microsequencing studies at the picomole level

A simple and fast reversed-phase high-performance liquid chromatographic method has been developed for the complete separation of 35 dimethylaminoazobenzene sulfonyl (DABS)-amino acids and by-products. This method allows simultaneous determination of primary and secondary amino acids which can be present in protein and peptide hydrolysates and also detects the presence of cysteic acid, S-sulfocysteine, hydroxyproline, taurine, norleucine, cystine, and delta-hydroxylysine. The precolumn derivatization of amino acids with dimethylaminoazobenzene sulfonyl chloride (DABS-Cl) is simple and quick (10 min at 70 degrees C) and allows the complete reaction of primary and secondary amino acids. The separation of the compounds under investigation is achieved in 25 min using a reversed-phase 3-microns Supelcosil LC-18 column at room temperature. The versatility of the proposed method is documented by amino acid determination on protein samples obtained using different hydrolysis techniques (HCl, methane-sulfonic acid, and NaOH), with attention given to the detection of tryptophan in protein samples with high sugar concentration. Furthermore, we have reported the experimental conditions necessary to apply this method to the amino acid analysis of very low amount of proteins (1 to 5 micrograms) electroeluted from a stained band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The stability of DABS-derivatives, the short time of analysis, the high reproducibility and sensitivity of the system, and the complete resolution of all compounds of interest make this method suitable for routine analysis. Furthermore, we have also developed a fast reversed-phase high-performance liquid chromatographic method for the complete separation of dimethylaminoazobenzene thiohydantoin (DABTH)-amino acids. The separation of the compounds under investigation is obtained, at room temperature, in less than 18 min using a reversed-phase Supelcosil LC-18 DB column, 3-micron particles, and also allows the complete separation of DABTH-Ile, DABTH-Leu, and DABTH-Norleu. The short time of analysis, together with the high reproducibility of the system and its sensitivity at picomole levels, make this method very suitable for the identification of DABTH-amino acids released during microsequencing studies of proteins and peptides with the dimethylaminoazobenzene isothiocyanate reagent. In addition, we have shown that it is possible to obtain complete separation of DABTH-amino acids also under isocratic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Head and Neck Veins of the Mouse. A Magnetic Resonance,Micro Computed Tomography and High Frequency Color Doppler Ultrasound Study

To characterize the anatomy of the venous outflow of the mouse brain using different imaging techniques. Ten C57/black male mice (age range: 7-8 weeks) were imaged with high-frequency Ultrasound, Magnetic Resonance Angiography and ex-vivo Microcomputed tomography of the head and neck. Under general anesthesia, Ultrasound of neck veins was performed with a 20MHz transducer; head and neck Magnetic Resonance Angiography data were collected on 9.4T or 7T scanners, and ex-vivo Microcomputed tomography angiography was obtained by filling the vessels with a radiopaque inert silicone rubber compound. All procedures were approved by the local ethical committee. The dorsal intracranial venous system is quite similar in mice and humans. Instead, the mouse Internal Jugular Veins are tiny vessels receiving the sigmoid sinuses and tributaries from cerebellum, occipital lobe and midbrain, while the majority of the cerebral blood, i.e. from the olfactory bulbs and fronto-parietal lobes, is apparently drained through skull base connections into the External Jugular Vein. Three main intra-extracranial anastomoses, absent in humans, are: 1) the petrosquamous sinus, draining into the posterior facial vein, 2) the veins of the olfactory bulb, draining into the superficial temporal vein through a foramen of the frontal bone 3) the cavernous sinus, draining in the External Jugular Vein through a foramen of the sphenoid bone. The anatomical structure of the mouse cranial venous outflow as depicted by Ultrasound, Microcomputed tomography and Magnetic Resonance Angiography is different from humans, with multiple connections between intra- and extra- cranial veins.     

Increased expression of titin in mouse gastrocnemius muscle in response to an endurance-training program

Titin, a sarcomeric giant protein, plays crucial roles in muscle assembly, elasticity and stability. Little is known about titin adaptation to endurance exercise. We studied the effects of endurance training on titin expression in mouse gastrocnemius muscles (MGM). Sixty-three ten-week-old male Swiss mice were divided into seven groups. Four groups were composed of untrained control animals (C0, C15, C30, C45) instead the other three included mice trained for 15 (T15), 30 (T30) and 45 (T45) days by treadmill. The training protocol was mainly aerobic, characterized by moderate-intensity, rhythmic and continuous exercises. Titin expression was determined by immunohistochemistry on MGM sections. Results revealed a significant reduction in body weight of the T45 mice and a significant increase in titin expression (% titin immunoreactivity median [range] = 41.11 [20-60] vs. 30.00 [10-50]). It is postulated that the up-regulation of titin expression is an adaptative mechanism to increase muscle elasticity and stability in response to the high number of stretch-shorten cycles during endurance training. Such a mechanism may be important for minimizing muscle energy consumption and improving performance during running.     

Complement factor H binds to denatured rather than to native pentameric C-reactive protein

Binding of the complement regulatory protein, factor H, to C-reactive protein has been reported and implicated as the biological basis for association of the H402 polymorphic variant of factor H with macular degeneration. Published studies utilize solid-phase or fluid-phase binding assays to show that the factor H Y402 variant binds C-reactive protein more strongly than H402. Diminished binding of H402 variant to C-reactive protein in retinal drusen is posited to permit increased complement activation, driving inflammation and pathology. We used well validated native human C-reactive protein and pure factor H Y402H variants to test interactions. When factor H variants were incubated with C-reactive protein in the fluid phase at physiological concentrations, no association occurred. When C-reactive protein was immobilized on plastic, either non-specifically by adsorption in the presence of Ca(2+) to maintain its native fold and pentameric subunit assembly or by specific Ca(2+)-dependent binding to immobilized natural ligands, no specific binding of either factor H variant from the fluid phase was observed. In contrast, both factor H variants reproducibly bound to C-reactive protein immobilized in the absence of Ca(2+), conditions that destabilize the native fold and pentameric assembly. Both factor H variants strongly bound C-reactive protein that was denatured by heat treatment before immobilization, confirming interaction with denatured but not native C-reactive protein. We conclude that the reported binding of factor H to C-reactive protein results from denaturation of the C-reactive protein during immobilization. Differential binding to C-reactive protein, thus, does not explain association of the Y402H polymorphism with macular degeneration.