In this study a preliminary report is given of enzymatic studies of the perilymph from otosclerotic patients. Two methods were used. Firstly an enzymatic dissolution of a gelatin film coating was used as a screening method. This method showed hydrolytic activity in 75% and a remarkably close correlation to the evolution of the perceptive hearing loss. Secondly, to identify the different enzymes, Uriel & Avrameas' micro-electrophoretic method was used, and a high activity of especially acid phosphatase, collagenase and chymotrypsin was demonstrated. In this study there was also a striking correlation of hydrolytic activity to the development of the perceptive hearing loss. It is suggested that these enzymes play a major role in the progressive inner ear disease in otosclerosis.
AbstractNIH 3T3 cells cultured in suspension fail to express cyclin A and hence cannot enter S phase and divide. We show that loss of cell adhesion to substratum abrogates cyclin A gene expression by blocking its promoter activity through the E2F site that mediates its cell cycle regulation in adherent cells. In suspended cells, G0-speciflc E2F complexes remain bound to the cyclin A promoter. Overexpression of cyclin D1 restores cyclin A transcription in suspended cells and rescues them from cell cycle arrest. In suspended cells, cyclin D1 and cyclin E accumulate normally upon serum stimulation, but their associated kinases remain inactive; their substrates, pRb and p107, are not hyperphosphorylated. Concomitantly, the cyclin-dependent kinase inhibitor, p27KIP1, is stabilized. Ectopic expression of p27KIP1 blocks cyclin A promoter activity through its E2F binding site. These data suggest that the block to cyclin A transcription in nonadherent NIH 3T3 cells results from stabilization of p27KIP1 and subsequent inactivation of the specific E2F moiety required for its induction.
Parallel anatompathological and enzymatic research led to the discovery of the role played by histiocytes, actively engaged in the lytic process and containing numerous lysosomes, and thus to postulate a cellular and enzymatic concept of otospongiosis. The enzymatic experiments were continued by mico-electrophoresis and allowed the authors to confirm this enzymatic action. In a third step, specific micro-concentrations of trypsin and alpha-1 antitrypsin correlated with cochlear progression, and showed an identical sudden reversal of activity. It was deduced that, when the balance of trypsinlantitrypsin is upset, cochlear deterioration can occur. Confirmation of this concept was done by experimental cytologic findings by scanning electron microscopy. Alteration or destruction of hair cells were the proof of the toxicity of the trypsin action on the inner ear, chiefly on the outer hair cells. The upset of the equilibrium of trypsin and antitrypsin in otospongiotic microfoci and resulting spreading of enzymes from the microfoci into different parts of the cochlea, provokes the peculiar response of the otic capsule with various clinical expressions according to the localization of the impairment: a clinical, stapedial one, resulting in conductive hearing loss; a cochlear one, leading to a sensorineural deafness; a vestibular one, provoking dizziness or even vertigo. The relationship of this enzymatic concept with the disease is explained in detail and leads the authors to express genetic prospects and possibilities of treatment by enzymatic inhibitors.
Cyclin A is involved in the control of S phase and mitosis in mammalian cells. Expression of the cyclin A gene in nontransformed cells is characterized by repression of its promoter during the G1 phase of the cell cycle and its induction at S-phase entry. We show that this mode of regulation is mediated by the transcription factor E2F, which binds to a specific site in the cyclin A promoter. It differs from the prototype E2F site in nucleotide sequence and protein binding; it is bound by E2F complexes containing cyclin E and p107 but not pRB. Ectopic expression of cyclin D1 triggers premature activation of the cyclin A promoter by E2F, and this effect is blocked by the tumor suppressor protein p16INK4.
Abstract A procedure adapted from that described by Mitchison and Kirschner [Nature 312:232–237, 1984] was used to isolate centrosomes from human lymphoid cells. High yields of homogeneous centrosomes (60% of the theoretical total, assuming one centrosome per cell) were obtained. Centrosomes were isolated as pairs of centrioles, plus their associated pericentriolar material. Ultrastructural investigation revealed: 1) a link between both centrioles in a centrosome formed by the gathering in of a unique bundle of thin filaments surrounding each centriole; 2) a stereotypic organization of the pericentriolar material, including a rim of constant width at the proximal end of each centriole and a disc of nine satellite arms organized according to a ninefold symmetry at the distal end and; 3) an axial hub in the lumen of each centriole at the distal end surrounded by some ill‐defined material. The total protein content was 2 to 3 × 10 −2 pg per isolated centrosome, a figure that suggests that the preparations were close to homogeneity. The protein composition was complex but specific, showing proteins ranging from 180 to 300 kD, one prominent band at 130 kD, and a group of proteins between 50 and 65 kD. Actin was also present in centrosome preparations. Functional studies demonstrated that the isolated centrosomes were competent to nucleate microtubules in vitro from purified tubulin in conditions in which spontaneous assembly could not occur. They were also very effective at inducing cleavage when microinjected into unfertilized Xenopus eggs.
