Françoise Landon

Origin of the beta cells of the islets of Langerhans is further questioned by the expression of neuronal intermediate filament proteins, peripherin and NF-L, in the rat insulinoma RIN5F cell line

Intermediate filament proteins of the rat insulinoma RIN5F cell line were characterized. Two-dimensional gel analysis followed by immunostaining of proteins demonstrated that these cells express both peripherin and the low-molecular-mass neurofilament protein (NF-L); this was confirmed for peripherin by immunohistochemistry, peptide analysis and Northern blot. No expression of these proteins could be detected with these same methods either in the adult pancreas or in the tumor at the origin of the cell line, although such expression was apparent on sections of rat pancreas at embryonal day 16. These results were compared to those obtained on the rat pheochromocytoma PC12 cell line: expression in the adrenal medulla of the embryo, no expression either in the adult tissue or in the tumor, but solely in the derived cell line. The expression of neuronal intermediate filament proteins in the rat insulinoma RIN5F cell line is discussed in relation to its similarity in the rat pheochromocytoma PC12 cell line, and its meaning as to the developmental cell lineage; an ectodermal origin is suggested for the pancreatic islet cells.

Isolation and physico-chemical properties of blood platelet α-actinin

Abstract A procedure for the isolation of α-actinin from human blood platelets is described. Typical yields were 10–13 mg from 48 g of frozen platelets. The purified platelet α-actinin has many physico-chemical properties (molecular weight in native state, molecular weight in denaturing conditions, Stokes radius, ellipticities at 208 and 221 nm) similar to those of muscle α-actinins. However, in contrast to muscle α-actinins, it is composed of isoforms containing subunits of slightly different molecular weights and its effect on actin gelation is calcium-sensitive. These two characteristics are common to other known non-muscle α-actinins.

Phosphorylation of peripherin, an intermediate filament protein, in mouse neuroblastoma nie 115 cell line and in sympathetic neurons

Peripherin, an intermediate filament protein, described recently, is expressed in well defined neuronal populations. We studied the phosphorylation, in vivo, of this protein in mouse neuroblastoma NIE 115 cell line and in sympathetic neurons labelled with [32P]-orthophosphate. The autoradiograms of proteins separated on two-dimensional polyacrylamide gels were compared with the Coomassie-blue stainings. The results show that peripherin occurs as a mixture of phosphorylated and non-phosphorylated isoforms, and that these forms coexist in both differentiated and non-differentiated cells. We demonstrate by cleavage at the unique tryptophan residue, a characteristic shared by most other intermediate filament proteins (IFP), that the phosphorylation sites are located on the amino-terminal half of peripherin as it is for vimentin and desmin. These results are discussed in relation to the organization of the filamentous network constituted by peripherin.

Structure of the mouse gene encoding peripherin: a neuronal intermediate filament protein

Summary— The gene encoding mouse peripherin, a neuronal intermediate filament protein, has been cloned. Its sequence, through 1021 nucleotides composing the 5′‐flanking region, nine exons, eight introns and 547 nucleotides of the 3′‐flanking region, as well as its transcription initiation site have been determined. The amino acid coding sequence differs from that of the rat peripherin gene. The mouse gene has an additional histidine near the N‐terminal end, and shows three conservative and two non‐conservation changes. The promoter sequence, containing the binding sites for transcription factors as well as other sequences is homologous to promoter regions of other type III intermediate filament protein genes and other neuronal‐specific genes.

Crystallization of native striated-muscle actin

Since the amino acid sequence of skeletal-muscle actin has already been established [ 1 ] the next step in the study of actin conformation should be an X-ray diffraction analysis, to elucidate its three-dimensional structure. So far, however, the numerous difficulties involving the crystallization of actin have not been overcome. The crystallization of proteins is most commonly obtained from ammonium sulfate but the addition of this salt increases the ionic strength of the medium and brings actin to its fibrous form. As the fibrous form of actin cannot crystallize, investigations have been carried out concerning means to prevent polymerization. For skeletal-muscle actin, various chemical modifications of one amino acid residue were performed [2-41. The inhibition of polymerization has also been effected by the addition of another component [S] and in this way crystals of a threecomponent complex could be obtained with a nonmuscle actin [6]. Adopting a different approach, we have attempted to crystallize non-modified muscle actin by using, as crystallization reagent, polyethylene glycol, which does not increase the ionic strength of the actin solution.

Chromosomal localisation of the mouse and human peripherin genes

Using a mouse cDNA probe encoding for the major part of peripherin, a type III intermediate filament protein, we have assigned, by in situ hybridization, the mouse and human peripherin genes, Prph, to the E-F region of chromosome 15 and to the q12-q13 region of chromosome 12, respectively. These regions are known as homologous chromosomal segments containing other intermediate filament genes (keratins) and also other genes which could be co-ordinately regulated.

Susceptibility of platelet α-actinin to a Ca2+-activated neutral protease

Purified α-actinin from human platelets was digested with Ca2+-activated protease from muscle. The a subunit (Mr = 100 kDa) was degraded into a unique polypeptide b of slightly lower molecular mass. In fresh platelets, only the a subunit was detected by immunoblotting techniques, while in out-dated platelets, both a and b polypeptides were present. Since a similar conversion of a to b occurs in vitro as in whole platelets, it can be assumed that, in platelets, α-actinin is cleaved by the endogenous Ca2+-activated protease.

Comparison of the properties of two kinds of preparations of human blood platelet actin with sarcomeric actin

A new procedure of purification of actin from human blood platelets was used. This method starting from acetone powder of whole platelets gives a much higher yield than the one previously described (actin I) (Landon et al. (1977) Eur. J. Biochem., 81, 571-577). This actin II preparation has the same reduced viscosity as skeletal muscle actin, while the reduced viscosity of actin I preparation is about 1/10 of this value. Moreover actin I has the form of very short filaments as shown by electron microscopy. After an extra step of purification actin I, when polymerized, acquired a high reduced viscosity. We confirmed that platelet and sarcomeric actins are similar in their polymerization properties and their ability to activate muscular myosin. A circular dichroism study showed that the overall conformation of both actins are similar, but the environment of their aromatic chromophores is different.