A unique cell type in the lung--the Clara cell (the non-ciliated bronchiolar epithelial cell).

The non-ciliated bronchiolar epithelial cells (the Clara cells) are found most frequently in the distal conducting airways, but they are found throughout the tracheobronchial tree of different mammalian species. According to recent data, the main functions of the Clara cells can summarized as (1), the secretion of certain components of the extracellular bronchiolar lining layer (2), metabolism and detoxification of xenobiotics and other toxic compound (3) and participation in the renewal process of the bronchiolar epithelium. The main goal of this paper is to collect and discuss some of the general features of Clara cells from a functional-morphological point of view, and their possible role in pathological alterations of the lung especially in the pathogenesis of lung tumours originated from Clara cells.

Nucleolar activity after 3-methylcholanthrene treatment of rat liver cells studied by silver staining procedure.

The influence of a single dose of 3-methylcholanthrene (3-MC) was studied in nucleoli of young rat liver cells by means of conventional and ultracytochemical methods. The nucleolar activity was stimulated in our experimental conditions: the appearance of the fibrillar centres in the liver cell nucleoli as well as the silver staining protein content of the fibrillar centres and the dense fibrillar component were increased by 3-MC. The results suggest that the activity of ribosomal genes was increased following 3-MC treatment.

Isolation and characterization of nuclear hnRNP complexes from Drosophila melanogaster tissue culture cells.

Fractionation on sucrose gradients of nuclear described extracts prepared from cultured Drosophila melanogaster cells by sonication of the nuclei in the presence of rat liver cytosol RNAase inhibitor revealed a complex polysome-like pattern of nuclear ribonucleoprotein complexes. The bulk of these heterogeneous ribonucleoprotein (hnRNP) complexes sedimented in the 30S to 80S zone of the sucrose gradient. According to biochemical and morphological data, the monomer particle proved to be the 45S hnRNP and its average diameter was found by electron microscopy to be 24-26 nm. The buoyant density of both the mono and polyparticles was about 1.4 g/cm3, with a slight degree of heterogeneity. The proteins from different zones of the sucrose gradient were composed primarily of similar polypeptides of 47 000, 56 000, 64 000, 96 000 and 130 000 daltons. Complete dissociation of nuclear hnRNP complexes was observed by resedimentation of the particles in the presence of 0.7 M NaCl or 4 M urea. RNAase A digestion (0.1 microgram/ml at 0 degree C for 10 min) resulted in the solubilization of part of the hnRNP and aggregation of some particles. The bulk of the RNA isolated from the different sized hnRNP complexes sedimented in the 7 to 11S region in the sucrose gradient. The large hnRNP complexes contained hnRNA strands larger than 15S, up to 28S. The base composition of the RNA from the 45S monoparticles proved to be AU type: A + U/G + C = 1.7. The RNAs from the 60-75S and 90- 100S polyparticles were also AU type, with an A + U/G + C ratio of 1.46 and 1.21, respectively. The hnRNP complexes exhibited marked heterogeneity in the electron microscope. Our biochemical and morphological observation point to a nonrandom organization of hnRNP particles in Drosophila melanogaster nuclei.

HnRNP particles from Drosophila melanogaster cells.

The isolation and characterization of HnRNP from cultured Drosophila melanogaster cells is described. HnRNP particles were extracted from the purified nuclei of sonication in the presence of rat liver cytosol RNAse inhibitor. The nuclear extract was centrifuged on a 15-30% sucrose gradient. The main part of the heterogeneous HnRNP material was localized in the 30 to 80S region of the sucrose gradient. According to the results of re-sedimentation studies the monomer particle was 45S. The buoyant density of HnRNP particles from different regions of the sucrose gradient were equal to approximately 1.4. The protein composition of the particles was analyzed by urea-SDS-polyacrylamide gel electrophoresis. There are five main and a few minor bands. Only the main polypeptides have a slightly higher molecular weight than those of the major polypeptides of 30S subparticles from rat liver nuclei. According to electron = microscopic studies the particles are heterogeneous and the average diameter was found to be 24-26 nm both on the basis of negative contrast and platinum-palladium shadowed pictures.

Structural characterization of polysomal poly(A)-protein particles in rat liver.

Poly(A)-protein particles were prepared from rat liver polyribosomes, washed with 0.5 M KCl or unwashed, after digestion with pancreatic ribonuclease and ribonuclease T1 by two successive rounds of sucrose gradient centrifugation. The particles were sedimented in a range of 5--13 S with a peak at about 9 S. The KCl wash of polysomes had no effect on the sedimentation properties of the particles. The particles isolated in this manner were 99% resistant to further pancreatic ribonuclease treatment and contained about 96% adenylic acid. The length of the poly(A) molecules prepared from the poly(A)-protein particles showed a broad distribution of about 70--290 nucleotides with a peak around 130 nucleotides, as measured by polyacrylamide gel electrophoresis. In CsCl density gradient the poly(A)-protein particles banded in a density range of 1.30--1.42 g/cm3 with a peak at 1.36 g/cm3, which amounts to about 80% of the protein content. Sodium dodecyl sulfate/polyacrylamide and urea/sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated six polypeptides with molecular weights of 50 000, 54 000, 58 000, 63 000, 76 000 and 90 000 in the poly(A)-protein particles, but the main components were dependent on the method. The treatment of polysomes with KCl resulted in a loss of the 90 000-molecular-weight component. Amino acid analysis of the polypeptides bound to poly(A) revealed that they contained a relatively large amount of aspartic plus glutamic acid (21.6%) as well as hydrophobic amino acids (41.4%). Digestion of glutaraldehyde-fixed particles with ribonuclease T2 showed that about 50% of poly(A) was accessible to the enzyme, thus this part of poly(A) was located on the surface of the particles. In the electron micrographs the shadowed poly(A)-protein particles appeared in a globular, somewhat elongated form and were mostly 14-18 nm in diameter. On the basis of the results a model for the 'average' 9-S particles was constructed.

Studies on the structure of rat liver messenger ribonucleoprotein. II. Non-poly(A) RNase resistant fragments and electron microscopic appearance of mRNP.

Partially fragmented 12-21S rat liver messenger ribonucleoprotein (mRNP), labelled either with [3H]-orotic acid or [3H]-adenine was treated with 5 (micrograms/ml or 0.1 microgram/ml pancreatic ribonuclease (EC 3.1.27.5) and the resistant fragments were separated by sucrose gradient centrifugation. Two types of fragments were obtained. Digestion of mRNP with ribonuclease at a concentration of 5 micrograms/ml resulted in 9S poly(A)-protein particles of mRNP as evidenced by their characteristic sedimentation, electrophoretic mobility of the RNA moiety and protein composition. In contrast, ribonuclease at a concentration of 0.1 microgram/ml produced 2-5S pyrimidine labelled fragments carrying polynucleotide sequences consisting of approximately 16-50 residues. Two polypeptides of rat liver mRNP with molecular weights of 38 000 (P38) and 44 000 (P44) were found to be attached to these sequences. The data demonstrate that RNA sequences other than poly(A) interact with protein in mRNP. Electron microscopic studies revealed that the liberation of mRNP from the polysomes by EDTA changes its surface properties since EDTA releases the mRNP mainly in the form of globular particles. However, a small proportion of the mRNP remains in fibril-like form. Among the fibrils several blobs could be seen which were probably the proteins attached to the mRNA. From the available data a model for the structure of an "average" mRNP molecule is proposed.

Swelling of nuclei and decondensation of chromatin in a multinuclear model.

Rat liver nuclei were isolated in the presence of a nonionic detergent, and the soluble proteins were extracted from them using low-salt solution. The isolated nuclei were then embedded in a fibrin clot which was cut into 4 to 5 mm3 pieces and placed into an AMICON pressure cell in the presence and absence of the soluble nuclear proteins. When the soluble nuclear proteins were present and when their concentrations reached 40 mg/ml, the nuclei were significantly larger and their chromatin more decondensed than in the absence of added proteins. Our findings suggest that the soluble nuclear proteins play a role in the regulation of nuclear volume and the hydration of chromatin.

Effect of 3-methylcholanthrene on RNA polymerase and protein kinase activities and on the nuclear ultrastructure of rat liver.

The polycyclic aromatic hydrocarbon 3-methylcholanthrene (3-MC) is a well known inducer of the microsomal mixed function oxidase enzyme system in rat liver cells. It seems probable that the inductive action of 3-MC is realized, at least partly, at the transcriptional level of protein synthesis regulation. The present experiments indicated that in the liver of young rats there was a significant alteration in the activities of nucleolar as well as nucleoplasmic protein kinase and RNA polymerase enzymes during the first days of exposure to a single dose of 3-MC. The ultrastructural investigations showed that 3-MC treatment caused nucleolar hypertrophy and accumulation of lipid inclusions in the nucleoplasm.

Perichromatin granules in the liver nuclei after isolation of hnRNP (informofer) particles.

The influence of the extraction of hnRNP (informofer) particles on the ultrastructural appearance of the perichromatin granules was studied. The extraction of hnRNP particles was carried out by SAMARINA's procedure from the rat liver nuclear fraction. The extraction did not change the numbers and the general ultrastructural features of perichromatin granules. It is therefore unlikely that the perichromatin granules are the morphological representatives of storaged or transported hnRNA.

Electron microscopic observations on the poly(A)-RNP component of the pre-mRNA containing nuclear complexes.

The nuclear ribonucleoprotein particles containing polyadenylic acid were prepared from rat liver nuclear extract by treatment with RNase I. The sedimentation coefficient of the particles was approximately 14 S. These particles represent a component of the pre-mRNA containing ribonucleoprotein complexes of the nucleus. The submicroscopic morphology of the particles was examined in preparations pretreated by platinum-palladium shadowing and by various negative staining methods as well as in ultrathin sections. The 14 S particles are globular structures, 120-140 A in diameter. The particles are characterized by a strong tendency to aggregation. The results are discussed with respect to mRNA transport.