[The possible role of the inner mitochondrial membrane in regulating oxidative phosphorylation in cells in vivo]. / O vozmozhnoi roli vneshnei membrany mitokhondrii v reguliatsii okislitel'nogo fosphorilirovaniia v kletkakh in vivo.

It has been shown for the first time that the outer mitochondrial membrane has a low permeability for ADP and can control its diffusion into cells in vivo. Respiration of saponin-skinned cardiac and skeletal muscle fibers is maximally stimulated by millimolar concentrations of external ADP. The apparent Km values for ADP are equal to 297 +/- 35 and 334 +/- 54 microM, respectively. After complete extraction of myosin with 0.8 M KCl, which fully preserves the intact structure of the mitochondria, the apparent Km values for exogenously added ADP does not change. However, disruption of the outer mitochondrial membrane by osmotic shock (treatment with 40 mOsM KCl) causes a reduction of the apparent Km value down to 32.3 +/- 5.0 microM of ADP. The apparent Km for ADP in isolated heart mitochondria is 17.6 +/- 1.0 microM. It is concluded that there exists an intracellular factor in the cells in vivo which controls the outer mitochondrial membrane and notably decreases its permeability for ADP. After isolation of mitochondria this factor is lost. When mitochondrial creatine kinase is activated, weak intracellular fluxes of ADP passing through the outer mitochondrial membrane in the skinned fibers are amplified manifold due to the tight functional coupling between mitochondrial creatine kinase and the oxidative phosphorylation system. This coupling is considered to be the central mechanism in the control of cell respiration.

Retarded diffusion of ADP in cardiomyocytes: possible role of mitochondrial outer membrane and creatine kinase in cellular regulation of oxidative phosphorylation.

Possible reasons for retarded intracellular diffusion of ADP were investigated. The isolated skinned cardiac fibers were used to study apparent kinetic parameters for externally added ADP in control of mitochondrial respiration. Participation of myosin-ATPase in binding of ADP within cells as it was supposed earlier (Saks, V.A., Belikova, Yu.O. and Kuznetsov, A.V. (1991) Biochim. Biophys. Acta 1074, 302-311) was completely excluded, since myosin-deprived skinned cardiac fibers ('ghosts') displayed the same kinetic parameters as intact ones (Kmapp for ADP about 300 microM). Significantly lower apparent Km values were obtained for fibers with osmotically disrupted outer mitochondrial membrane (25-35 microM), which was close to that observed for isolated heart mitochondria. The data obtained are in favor of limitation of ADP movement via anion-selective low-conductance porine channels in the outer membrane of mitochondria. It is proposed that the permeability of this membrane is controlled by some unknown intracellular factor(s). In the presence of saturating concentrations of creatine (25 mM) the apparent Km for ADP significantly decreases due to coupling of creatine kinase and oxidative phosphorylation reactions in mitochondria. This coupling is not observed in KCl medium in which mitochondrial creatine kinase is detached from the membrane. It is concluded that in the cells in-vivo ADP movement between cytoplasm and intramitochondrial space is controlled by low-conductivity anion channels in the outer membrane. Thus, the mitochondrial creatine kinase reaction coupled to the adenine nucleotide translocase is an important mechanism in control of oxidative phosphorylation in vivo due to its ability to manifold amplify these very weak ADP signals from cytoplasm.

[Damage to human endothelial cells by cholestane-3 beta,5 alpha,6 beta-triol and the protective effects of preparations that raise the intracellular level of cAMP]. / Povrezhdenie éndotelial'nykh kletok cheloveka kholestan-3 beta,5 al'fa,6 beta-triolom i zashchitnye éffekty preparatov, povyshaiushchikh vnutrikletochnyi uroven' tsAMF.

Effect of drugs, which are able to elevate the intracellular level of cAMP, on resistance of human umbilical vein endothelial cells (HUVEC) to cholestane-3 beta,5 alpha, 6 beta-triol (Triol)-induced injury was studied. Triol at a concentration of 62 microM caused death of 50% of cells after a 24 hour incubation. Addition of forskolin (10 microM), methylisobutylxantine (100 microM), or 8-Br-cAMP (100 microM) into the incubation medium prevented injury of HUVEC under these conditions. These findings indicate that endothelial resistance to the injury can be regulated by the adenylate cyclase system. A comparative study on Triol-induced injury of adult human aortic endothelial cells isolated separately from zones of low (LP) and high (HP) probability of atherosclerosis was also performed. In 7 cases endothelial cells isolated from the LP zones were more resistant to Triol-induced injury, in 2 cases the differences were not significant. The development of atherosclerotic lesion in HP zones is likely to be associated with a higher sensitivity of endothelial cells from these zones to different injuring agents.

[The structure of the centriolar apparatus of the endothelial cells in the embryonic and definitive human aorta]. / Struktura tsentrioliarnogo apparata éndotelial'nykh kletok v émbrional'noi i definitivnoi aorte cheloveka.

The fine structure of the centriolar system was studied on serial sections of 90 endothelial cells of human aorta (50 to 60 years) in regions without atherosclerotic platelets and with fibrous and atheromatous platelets and of 30 endothelial cells of human embryonic aorta (22-24 weeks). The vast majority (95%) of endothelial cells of the atheromatous platelets were shown to have a primary cilium over 1 micron long which gives on the basal surface in all the cells. In the regions without platelets and with fibrous platelets a cilium was observed in about 20% of cells and it gives in the vessel lumen. Endothelial cells with a cilium fully embedded in the cytoplasm and with abnormal cilium structure were found in the embryonic aorta. A suggestion is put forward that cilia of the endothelial cells of embryonic aorta and those of adult aorta differ by the mechanism of their formation and can have different functions.

An ultrastructural study of centriolar complexes in adult and embryonic human aortic endothelial cells.

Ultrastructural organization of centriolar complexes in 90 adult human aortic endothelial cells from uninvolved areas, fibrous and atheromatous plaques and 30 endothelial cells from human embryonic aorta were studied using serial sections. Primary cilia protruding from the abluminal cell surface were found on 28 of 30 endothelial cells from atheromatous plaques. Only five of 30 cells from either fibrous plaques or uninvolved areas developed primary cilia protruding to the lumen. Impaired primary cilia entirely immersed into the cytoplasm were found in embryonic endothelial cells. It was speculated that both the modes of formation and the functions of endothelial cilia in embryonic and adult aortas are different.

Primary culture of endothelial cells from atherosclerotic human aorta. Part 1. Identification, morphological and ultrastructural characteristics of two endothelial cell subpopulations.

Endothelial cells (EC) were harvested by 0.1% collagenase treatment for adult human thoracic aortas obtained 1-3 h after sudden death. At least 35-70% of EC were removed from the intimal surface of aorta, 90-95% of them being viable. Plating efficiency was 70-80%. Monolayer formation was achieved at a seeding density of 5-8 X 10(2) cells/mm2. The cells were identified as endothelium by the presence of Factor VIII antigen, Weigel-Palade bodies and typically endothelial morphology at confluence. Unlike endothelial cultures derived from human umbilical veins and infant aortas, primary cultures obtained from human adult aortas contain multinuclear EC with Factor VIII antigen and Weibel-Palade bodies. The number of multinuclear EC in cultures isolated from aortas affected by atherosclerosis was 2-fold higher (P less than 0.05) than in cultures obtained from grossly normal aortas taken from donors of the same age. EC with numerous lipid inclusions revealed by oil-red-O staining were present in all the EC primary cultures derived from aortas affected by atherosclerosis. No oil-red-O-positive cells were detected among the EC cultured from infant aorta, aorta of young donors, and umbilical vein. An electron microscopic examination of EC from atherosclerotic aorta in culture and in situ failed to reveal any ultrastructural peculiarities distinguishing multinuclear EC from the mononuclear EC.

[Presence of an insulinlike protein in the submandibular salivary glands of man and agricultural animals]. / K voprosu o nalichii insulinpodobnogo belka v podnizhnecheliustnykh sliunnykh zhelezakh cheloveka i sel'skokhoziaistvennykh zhivotnykh.

An insulinlike protein (ILP) has been extracted from the submaxillary glands of men, bulls and boars. Its electrophoretic mobility is similar to that of the cristalline insulin. In addition, electrophoregrams of bulls and boars show that levels of the main zones of ILP and the insulin extracted from the pancreas by the same method coinside. Immunological properties of ILP and those of insulin are identical. The immuno-fluorescence method has detected ILP in striated ducts of the salivary glands. Nonspecific luminescence in these ducts is revealed around the human gland nuclei and is likely to be caused by the presence of lysosomes. It is not yet clear what microstructures are responsible for LIP localization in salivary ducts.

[Differential reaction of polytene chromosome segments to high doses of actinomycin D]. / Differentsial'naia uchastkov politennykh khromosom na deistvie vysokikh doz aktinomitsina D.

The inhibition of RNA synthesis by a high dose of actinomycin D (10 and 50 mg/ml) in Chironomus thummi polytene chromosomes was followed by drastic changes in the ultrastructure of puffs and some bands: 1) in some puffs, the number of puff granules was sharply reduced, and such puffs were collapsed, 2) in other puffs, the number of puff particles did not decrease but puff granules were substituted by thin fibrils. These puffs remained in an decondensed state and even increased their size, 3) in some puffs, large droplets or thick fibrils appeared never observed in these puffs in normal conditions, 4) some bands are decondensed like the puffs, 5) band segregation (particularly in centromere regions) into two substances with low and high electron densities has been described for the first time. A low electron dense substance was Bernhard--positive and contained RNA. The phenomenon of band segregation has been compared with nucleolar segregation. The results are discussed in relation to the ultrastructural organization of the transcription complex and puff products in polytene chromosomes.

[Ultrastructure of the human submandibular salivary glands]. / Ul'trastruktura podnizhnecheliustnykh sliunnykh zhelez cheloveka.

By means of electron microscopy cells in the human submandibular glands were studied. It was demonstrated that in acini two types of glandular cells were present: mucosal and seromucosal. In the latter, secretory granules are descrete with electron opaque cores in most of them. Mucocytes are filled with an electron transparent secrete; secretory granules often confluent and their membranes rupture. The acini are surrounded with myoepithelial cells. Intercalated ducts consist of cells with moderately electron opaque granules. In some granules there are dense bodies excentrically situated. In these cells there occur lipid inclusions. Striated ducts are composed of basal (electron transparent) and high cylindric (light and dark) cells. The cylindrical cells have a large amount of mitochondria, deep folds in their basal plasmolemma protruding into cytoplasma. Most of the cells in these parts contain small apically accumulated secretory granules with a dense matrix and separate larger ones scattered in the cell. It is possible to suggest that some secretory granules of ductal or, perhaps, acinar origin contain hormonal products.

Electron microscopic studies on the polytene chromosomes of Chironomus thummi salivary glands. I. Ultrastructural mapping.

The method of ultrathin sections of unsquashed salivary gland polytene chromosomes of Ch. thummi was applied to their ultrastrual mapping. There was a good agreement between electron micrographs and Hägele's light microscopic map (1970) with respect to the pattern and number of bands. 94% of bands were identified in larval and prepupal chromosomes. In Ch. thummi, band thickness varied from 0.05-0.5 mum. Most characteristic were 0.2-0.3 mum bands. Morphologically, bands were classified as: continuous (frequently with holes and gaps), discrete, dotted and continuous-discrete, discrete-dotted. Band morphology is related to band size, such that smaller bands, as a rule, were also dotted. Bands beginning to puff likewise became dotted. Interbands in unsquashed chromosome sections were from 0.05-0.15 mum. The smallest interbands contained only fibrils, in the larger interbands few granules could be observed. This makes interbands distinguishable from a typical puff with many such granules.

[Ultrastructural organization of polytene chromosomes of Chironomus thummi salivary glands]. / Ul'trastrukturnaia organizatsiia politennykh khromosom sliunnykh zhelez Chironomus thummi

An electronmicroscopical mapping of a number of regions of the polytene chromosomes of Ch. thummi salivary glands (3rd chromosome, right arm of the 1st chromosome, centromere regions, puffs 1-A2e, 1-A3ij, III-A5c and others) was done by the method of oriented ultrastructural sections of the unsquashed polytene chromosomes. The banding pattern on the electron micrograph was similar to the observed with the light microscope. The difference was that some doublets appeared as single cavity-containing bands with the double structure only in short regions under the electron microscope. It was also difficult to distinguish single bands in those regions where heavy adjacent bands were connected by dens, protrusions and anastomoses. These connections were most pronounced in the regions of the centromerers which had "spongy" appearance on the electron micrographs. These pictures may be connected with small interbands between heavy bands. Thin bands and some broad bands were frequently dotted. The puffs examined contained mainly RNP granules 200-400 A in diameter and RNP fibrils; BR-1 and BR-2 contained granules 500 A, RNP fibrils and smaller granules (200-400 A). BR and puffs were characterized by loop-like structures composed of granules arranged along the central DNP fibril. Only fibrils were presented in small interbands (0.05 mk), while larger interbands could include a small number of granules similar to those observed in puffs. It was found that centromere, telomeres and some heavy bands formed characteristic contacts with the nuclear membrane.