Interfacial properties of acidified skim milk.

The purpose of this study is to investigate the tension properties and dilatational viscoelastic modulus of various skim milk proteins (whole milk, EDTA-treated milk, beta-casein, and beta-lactoglobulin) at an oil/water interface at 20 degrees C. Measurements are performed using a dynamic drop tensiometer for 15,000 s. The aqueous bulk phase is a skim milk simulated ultrafiltrate containing 11 x 10(-3) g L(-1) milk protein. At pH 6.7, beta-casein appears as the best to decrease the interfacial tension, whereas beta-lactoglobulin leads to the highest interfacial viscoelastic modulus value. Whole milk was almost as surface-active as individual beta-casein in terms of the final (steady-state) lowering of the interfacial tension, but the rate of tension lowering was smaller. EDTA treatment improved the rate of tension lowering of whole milk. The acidification of milk, from previous measurements, would lead to the enhancement of surface activity. At t=15,000 s, the order of effectiveness is pH 4.3 > pH 5.3 = pH 5.6 > pH 6.7 whole milk, suggesting that pH 4.3 whole milk is the best surface active. As compared to pH 6.7 whole milk, the use of pH 5.3 and pH 5.6 milk as surface active would result in the use of milk containing more free beta-casein born of pH-dissociated casein micelles.

[Isolated muscular sarcoidosis mimicking a tumoral lesion]. / Manifestazione isolata di sarcoidosi muscolare simulante una lesione tumorale.

We report a case of a woman with a palpable painful nodule on her left leg. MR and CT showed a lesion that could be described as a neoplasm. Excisonal biopsy revealed a noncaseating granuloma. The woman presented the nodular type of muscular isolated sarcoidosis. Further the disease involved the lungs; this confirmed the accurate diagnosis of sarcoidosis. Sarcoidosis is a chronic, multisystem granulomatous disease of unknown etiology. Muscle involvement is frequent, but often asymptomatic. There are three forms of muscular sarcoidosis: only the nodular type can be recognized by technical imaging. MR and ultrasound are the best methods to attempt the diagnosis of nodular muscular sarcoidosis; nevertheless, the lesion must have a standardized behaviour because it can mimic a malignant neoform. In this case, biopsy is the only tool to identify the disease.

[Polysialylated NCAM in CSF, a marker for invasive medulloblastoma]. / La NCAM polysialylée du LCR, un marqueur des médulloblastomes invasifs.

We described a double-site enzyme-linked immunosorbent assay (ELISA) to measure polysialic acid neural cell adhesion molecule (PSA-NCAM) level in CSF. Immunocapture of PSA-bearing molecules is first effected by means of a monoclonal antibody (anti-MenB), directed against sialic acid polymers and adsorbed into plastic wells. Linked PSA-NCAM is then revealed by means of a second antibody, directed against an aminoacid sequence of NCAM and labelled with peroxydase. The lowest amount of PSA-NCAM detectable was estimated to be 0.11 microgram/l. This value was considered as the threshold for positivity. PSA-NCAM level was measured using this method in CSF from 29 patients with medulloblastoma. CSF had been collected at different times following tumor excision and stored at--80 degrees C. At the same times, cytological examination in CSF (medulloblastoma metastatic cells) and craniospinal imaging (tomographic scan or MRI) had been performed. PSA-NCAM was never detected in control CSF. For patients in remission, beyond the post-operative period of 1 or 2 months, 18 on 21 exhibited a PSA-NCAM level below the threshold value. For refractory patients, so classified according to the positivity of cytology and/or imaging, whatever the time after the tumor excision, PSA-NCAM was always positive (23/23), while either cytology or imaging were positive less frequently (16/23 for both). For relapses, PSA-NCAM was more frequently positive (6/7) than cytology and imaging (1/7 and 5/7, respectively). We concluded that PSA-NCAM positivity in CSF may be a reliable marker to detect the invasive or metastatic feature of medulloblastoma.

Stimulation of phosphatidylethanolamine synthesis during the last stages of the G1 phase in concanavalin A-activated human peripheral lymphocytes.

Phospholipid synthesis was investigated in concanavalin A-activated human peripheral lymphocytes up until 72 h following cell activation, i.e., during the G1 and S phases of the cell cycle. Using [32P]phosphate pulse experiments (5 h), striking differences were observed between phosphatidylethanolamine (PE) and phosphatidylcholine (PC) synthesis. Both the incorporation of [32P]phosphate into PE and the PE/PC incorporation ratio were greatly enhanced, 16-fold and 8-fold, respectively, after 48 h of incubation with the mitogen. This increase in PE synthesis was still observed when cell entry into the S phase was inhibited by an excess of concanavalin A; thereby it must be related to the late stages of the G1 phase. The stimulation of the incorporation into PE was the same for both [14C]ethanolamine and [32P]phosphate, therefore suggesting the involvement of the phosphoethanolamine synthesis pathway. Kinetics of continuous incorporation of [32P]phosphate into PE and PC indicated that the PE/PC net synthesis ratio was enhanced in activated cells, which corresponds to PE enrichment in lymphocyte membranes. The stimulation of PE synthesis in late G1 may be of importance for cell progression through the cell cycle by changing the membrane physical properties. Furthermore, it may serve as a test for checking lymphocyte reactivity to mitogens.

A monoclonal antibody against Meningococcus group B polysaccharides used to immunocapture and quantify polysialylated NCAM in tissues and biological fluids.

Polysialylated isoforms of neural cell adhesion molecule (PSA-NCAM) are transiently expressed in many tissues during development and in discrete areas of the adult central nervous system. In pathological situations, they are expressed by poorly differentiated tumor cells of neuroectodermal origin and by regenerating muscle. An ELISA is introduced here to estimate the relative concentrations of PSA-NCAM expressed by tissues or released into biological fluids. In this double-sandwich assay, an anti-PSA antibody (anti-MenB) was adsorbed onto plastic plates and permitted the immunocapture of PSA-bearing molecules. It is demonstrated that these molecules are major NCAM. The second antibody was directed against an amino acid sequence shared by NCAM isoforms in several species. The standard curves were established using Nonidet P40 extracts of human or mouse embryonic brain known to be rich in PSA-NCAM. The sensitivity of the assay allows for quantitation of PSA-NCAM in muscle during regeneration and in small samples of cerebrospinal fluid from patients with medulloblastoma metastasis.

Expression of NCAM and its polysialylated isoforms during mdx mouse muscle regeneration and in vitro myogenesis.

In order to understand the mechanism of the muscular regenerative process which occurs in mdx mice, the expression of neural cell adhesion molecule (NCAM) isoforms and their polysialylated (PSA) derivatives were studied during the postnatal development of normal and mdx mice in relation to the stage of the regeneration of muscle fibres, in the quadriceps. NCAM expression was also examined during in vitro differentiation of satellite cells isolated from both mdx and normal muscles. The immunohistochemical and biochemical analyses were done using antibodies for the different isoforms. The data presented here suggest that before the onset of necrosis and regeneration, the expression of NCAM isoforms in the quadriceps of mdx mice was similar to normal mice. Later, NCAM and PSA-NCAM expression in mdx mice increased and was related to the muscular regenerative process, and the overall level of NCAM expression can be considered as a good index of muscle regeneration. Young regenerative fibres expressed NCAM and PSA-NCAM, while mature regenerative fibres, in which myonuclei remained centrally located, did not express either NCAM or the PSA isoforms. Therefore, in terms of NCAM expression, the fibres in mdx muscle with centrally located nuclei appeared similar to mature fibres found in normal adult muscle. A major form of 145 kDa and a minor form of 115 kDa were detected in mdx regenerative muscle. The 145 kDa NCAM was sialylated, as demonstrated by its sensibility to exoneuraminidase which generates a desialoform of 125 kDa, but not polysialylated since it was not recognized by the anti-MenB antibody, specific for PSA-NCAM. In contrast, the molecular forms of NCAM migrating as a broad band from 160 kDa to 220 kDa were identified as PSA-NCAM. The comparison of in vitro differentiation of normal and mdx satellite cells showed that the expression of NCAM isoforms by mdx cells was similar to that expressed by normal cells. Both our in vivo and in vitro data concerning NCAM expression show that regeneration in mdx mice does not differ from that observed in other necrotic diseases. In other words, NCAM is unlikely to be a dystrophin-associated molecule since lack of dystrophin does not affect its expression.

Phosphate dilution lowers net phosphatidyl choline synthesis in lymphocytes.

The rate of net phosphatidyl choline (PC) synthesis in lymphocytes was evaluated, under appropriate conditions, in terms of [32P]phosphate incorporation. Phosphate-diluted and normal media were compared, with the cells being stimulated by concanavalin A or not. In either case, incubation in phosphate-diluted medium lowered net PC synthesis. In concanavalin A-stimulated cells, phosphate dilution also abolished the stimulation effect on net PC synthesis in G1. Nevertheless, these changes did not inhibit blastogenic transformation of cells.

Effect of AY 9944 and chlorpromazine on Concanavalin A-induced stimulation of human lymphocytes.

Amphiphilic molecules AY 9944 and chlorpromazine (CPZ) inhibited DNA synthesis in Concanavalin A-stimulated lymphocytes in a dose-dependent manner. While AY 9944 strongly decreased 7-dehydrocholesterol conversion to cholesterol, CPZ did not significantly affect this reaction. Moreover, the inhibitory effect of AY 9944 and CPZ on DNA synthesis took place in the presence of cholesterol in the culture medium. These findings suggest that the mechanism of inhibition of DNA synthesis by AY 9944 or CPZ is not related to endogenous cholesterol synthesis or exogenous cholesterol supply. Results are discussed in relation to the amphiphilic properties of AY 9944 and CPZ and to the interaction of these drugs with membranes or other intracellular targets such as calmodulin.

Changes in ganglioside metabolism during in vitro differentiation of quail embryo myoblasts.

The metabolism of gangliosides was studied during the in vitro differentiation of both normal quail myoblasts and myoblasts which have been transformed by a temperature-sensitive mutant of Rous sarcoma virus (RSV). These transformed cells can be maintained undifferentiated if incubated at 35 degrees C, but they will differentiate when shifted to 41 degrees C. (D. Montarras and M. Y. Fiszman (1983) J. Biol. Chem. 258, 3882-3888). The analysis of [14C]Glucosamine-labeled gangliosides by two-dimensional thin-layer chromatography reveals variations in the metabolism of the gangliosides during the process of differentiation. During the formation of myotubes, it was observed that the accumulation of GD1a is reduced, while the accumulation of GD3 is increased. Therefore, this results in the variation of the ratio GD3/GD1a which increases from 1.8 to 25 in the case of clones of transformed myoblasts, and from 0.5 to 1.7 in the case of uninfected myoblasts. These variations which have been observed seem to be specific of the myogenic differentiation since they cannot be reproduced when differentiation is inhibited by BUdR treatment or when fibroblasts reach confluency and are blocked in the G1 phase of cell cycle. Furthermore, the transformed myoblasts in vitro are shown to be a good model system since their gangliosides composition is very similar to that of muscle cells in vivo.

Phosphate depletion decrease mitogen-mediated stimulation of phospholipid synthesis in human peripheral lymphocytes.

Concanavalin A-mediated stimulation of 32P-phosphate incorporation into phospholipids of human peripheral lymphocytes is comparatively studied in normal and phosphate-depleted media. In the phosphate-depleted medium, 2 hours after the start of cell activation, the stimulation sharply decreases for phosphatidylinositol (6.5-fold) and for phosphatidylcholine (in the latter case, the stimulation is even replaced by a slight inhibition of the incorporation). These results must be related to the rate-limiting effect of inorganic phosphate on ATP formation and thus on phospholipid synthesis, an effect which may be particularly pronounced when there is both phosphate depletion and cell activation.