Unexpected and coordinated expression of Spi-1, Fli-1, and megakaryocytic genes in four Epo-dependent cell lines established from transgenic mice displaying erythroid-specific expression of a thermosensitive SV40 T antigen.

Most erythroleukemic cell lines established in vitro coexpress erythrocytic and megakaryocytic markers that often are associated with expression of Spi-1 and/or Fli-1 transcription factors known as transactivators of megakaryocyte-specific promoters. In the present study, we examined the possibility of establishing new cell lines keeping strictly erythroid-specific properties in vitro through the targeted and conditional immortalization of erythrocytic progenitors. For that purpose, we established several lines of transgenic mice displaying erythroid-specific expression of a thermosensitive SV40 T antigen. As expected, these transgenic mice developed splenomegaly due to the massive amplification of Ter 119 positive erythroid nucleated cells expressing T antigen. Despite this drastic effect in vivo, the in vitro immortalization of erythropoietin-dependent erythroid progenitors unexpectedly occurred at low frequency, and all four cell lines established expressed both erythrocytic (globins) and megakaryocytic markers (glycoprotein IIb, platelet factor 4) as well as Spi-1 and Fli-1 transcripts at permissive temperature. Switching the cells to the nonpermissive temperature led to a marked increase in globin gene expression and concomitant decrease in expression of Spi-1, Fli-1, and megakaryocytic genes in an erythropoietin-dependent manner. Interestingly, enhanced expression of Spi-1 and Fli-1 genes already was detected in the Ter 119 positive cell population of transgenic mice spleen in vivo. However, like normal Ter 119 erythroid cells, these Ter 119 positive cells from transgenic mice still expressed high levels of beta-globin and very low or undetectable glycoprotein IIb and platelet factor 4 megakaryocytic transcripts. Taken together, these data indicate that the unexpected expression of megakaryocytic genes is a specific property of immortalized cells that cannot be explained only by enhanced expression of Spi-1 and/or Fli-1 genes.

Biochemical properties of protease resistant prion protein PrPsc in natural sheep scrapie.

Prions are infectious agents involved in neurodegenerative diseases, such as scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cows and Creutzfeldt-Jakob disease (CJD) in humans. These pathogens are characterized by unusual properties, and, in particular, by their strong resistance to common procedures of disinfection used against conventional microorganisms. A major component of highly infectious fractions is a proteinase K-resistant prion protein PrPsc (PrP-res), the normal host prion protein PrPc being sensitive to PK (PrP-sen). We used a biochemical approach to further characterize PrPsc protein in natural sheep scrapie. Western blot analyses using rabbit antiserum that recognized both normal and pathologic sheep prion proteins, were undertaken to study the biochemical behaviour of PrPsc extracted from brains of sheep naturally infected with scrapie after protease digestion and under denaturing conditions. Increasing concentrations of urea (1-7 M) or GdnSCN (0.25-3 M) and different pH from 2 to 11 were tested for their effects on protease resistance of PrPsc. Alkaline pH (pH = 10) and high concentrations of urea (> 3 M) and GdnSCN (> 0.75 M) greatly decreased the protease resistance of the prion protein. Identical experiments carried out on three different sheep from the same flock gave similar results. The biochemical behaviour of PrPsc under denaturing conditions and in the presence of proteinase K could thus provide a biochemical means for further characterization of different natural scrapie isolates.

Two distinct variants of erythrocyte spectrin beta IV domain.

We report two distinct variants affecting the beta IV domain of erythrocyte spectrin, designated spectrin Saint-Chamond and spectrin Tlemcen. They were discovered in a French family and an Algerian individual, respectively. They appeared clinically and morphologically asymptomatic in the heterozygous state. In two-dimensional maps of spectrin partial digests, both mutants were manifested by cathodic shifts (with no change of the molecular weights) of the peptides that cover the N-terminal region of spectrin beta IV domain. The relevance of the abnormal peptides to the beta IV domain was established by quantitative analysis and by Western blotting using anti-beta IV domain-specific antibodies. These two variants are thus far the most distal variants of spectrin to be defined on an unequivocal structural basis.

[Optical and scanning electron microscopic study of the structural development of the cecum and colon walls in the rabbit (Oryctolagus cuniculus L.) during the perinatal period]. / Etude en microscopie optique et electronique a balayage de l'evolution structurale des parois du caecum et du colon, durant la periode perinatale, chez le lapin (Oryctolagus cuniculus L.).

The study, through optical and scanning microscopy, of various developmental stages, from the 24th day of gestation to the 13th day after birth, shows a quick development of the structures of the caecal and colic mucosal layers to the adult forms, from the very first week of postnatal life. The distal part of the colon is full-grown before the other parts of the alimentary canal, and is particularly rich in mucous cells. The caecum is the place where an early lymphoid defense is provided, from the 30th day of gestation.

[In vitro study of the lipase activity of bacterial strains isolated from the rabbit cecum on C14-labeled triolein]. / Etude in vitro de l'activité lipasique de souches bactériennes isolées de caecum de lapins sur la trioléine marquée au 14C.

The lipase activity of bacterial species isolated from the caecum of conventional rabbits was studied by means of two groups of tests: (1) bacteriologic isolation and an identification of nine aerobic and anaerobic clones and (2) incubation of each of these clones in the appropriate medium in the presence of labelled triolein (oleyl 1-14C), a biochemical analysis of the radioactivity of the fractions obtained. The main results can be expressed in the following way: maximum hydrolysis of glycerides is achieved by two species: Bacillus licheniformis and Clostridium sporogenes. The reaction is proportional to the incubation time for incubations of less than 8 h. The release of fatty acids is concomitant to the synthesis of phospholipids and of fatty acid methyl esters.

GM3 and GD3 are the major gangliosides of rat fetal intestine.

Gangliosides were extracted from intestine of rat fetuses on each day between 17 days of gestation and 1 day after birth. They were purified from the lipid extract by DEAE-Sephadex chromatography. Two major gangliosides are found at the earliest time: GD3 and GM3. Their contributions to the ganglioside content are, respectively, 48.2 and 33.7%. Between 19 days of gestation and birth, a 3.5-fold increase of the total ganglioside concentration is observed which is due to sharp rise of the GM3 concentration. In GD3 and GM3 of rat fetal intestine, sialic acid is exclusively N-acetylneuraminic acid and fatty acids are all non-hydroxylated.

[Tolerance and metabolic results of long-term administration of a mixture of saturated triglycerides by recto-colic route in rabbits]. / Tolérance et conséquences métaboliques résultant de l'administration chronique de mélange de triglycérides saturés par voie recto-colique chez le lapin.

Rabbit's and Dog's colonic absorption of triglycerides is shown in previous works. Further, we are studying the possibility to use the rectal route for nutritive substances. This way remains now poorly used and an exceptional therapeutic. However, bringing triglycerides in adequate state would be an energetic, supply, all the more as it is very difficult to provide potent caloric source in parenteral nutrition. This is why we investigate, on the Rabbit, the issues of chronic administration of glycerides by rectal route, on caloric balance and on a biochemical view to seek for in lipid composition of tissues the print of lipids administrated by transanal way. The animals are individually housed in metabolism units feeding a standard diet and drinking ad libitum. The treated subjects are given by transanal way, twice a day, for eight weeds, a sum of 1 g/kg of medium chain triglycerides. The metabolic balance-sheet is daily drawn. The clinic balance-sheet is set on the end, by biological controls to explore the hepatic and renal function (prothrombine ratio, transaminases (SGOT, SGPT), ornithine, carbamyl transferase (OCT), urea, total proteins ratio and electrolyte imbalance. We measure also the concentration of total lipids plasma and tissue (liver, kidney, heart, lungs, perirenal adipose tissue). The triglyceride composition and fatty acid composition of different lipids fractions of control and treated subjects are analyzed by gas-liquid chromatography. The animals support perfectly, without damage, a chronic and massive (1 g/kg/each day) administration of medium chain triglycerides by rectal route. The growth of treated subjects is normal. On the opposite, they adjust their alimentary consumption with the caloric charge of the diet, this is the reason why the reduction is about 12p. 100 with respect to the control animals. The coli-rectal administration of saturated glycerides produces at middle end only qualitative variations of lipids extracts. They essentially affect the triglyceride and fatty acid fraction of the tissue lipids, they become intermediate between that of control animals and the mixture administered by the transanal way. These results prove the integration of the glycerides in the metabolic pathway. They show the problem of the colonic absorption for the liposoluble substances. They authorize researches in view of a possible caloric assistance by this way.