Dopamine was injected intravenously (9 μg/kg) or intraperitoneally (15 μg/kg) to Wistar rats (3-4 months, 300-400 g). Hepatocytes were isolated 40 min after dopamine injection. Dense cultures were ...maintained on collagen-coated glasses. By the 5th hour, the circaholarian rhythm of protein synthesis in hepatocytes cultures was absent in the dopamine group, but was present in cultures from animals receiving physiological saline (NaCl). The rhythm-disorganizing effect of dopamine was reversible. The rhythm was observed in cultures of hepatocytes isolated 1 day after dopamine treatment. The effect of dopamine was abolished by melatonin. The protein synthesis rhythm was revealed in 5-h cultures of hepatocytes from rats receiving melatonin (32 ng/kg) 40 min after intraperitoneal injection of dopamine. The results of our
in vitro
experiments with addition of dopamine into the medium of cultured hepatocytes 1 suggest that dopamine
in vivo
produces a direct effect on liver cells. The observed changes are discussed taking into account the biochemical mechanisms for a direct cell–cell interaction and previously unknown properties of catecholamines.
Melatonin injected intraperitoneally into rat synchronizes the ultradian rhythm of protein synthesis after 100 min in primary hepatocyte cultures isolated from this rat, which are studied after 1 or ...2 days. The effective synchronization concentrations of melatonin—0.01-0.02 μg per kg of rat weight—are three orders lower than melatonin doses used in clinical practice in human treatment.
Two main gangliosides (G-1 and G-2) were isolated from eggs and embryos S. intermedius. They contain glucose, N-glucolylneuraminic acids, phytosphyngosine, fatty acids and alpha-hydroxy fatty acids. ...Molar ratios and sequence of these components are the same for both gangliosides, but G-2 contains sulphate residue which is attached to the terminal neuraminic acid. To obtain specific antisera rabbits were immunized by G-1 or G-2, which were mixed with bovine serum albumin and Freund's adjuvant. Both gangliosides possessed electrophoretic and antigenic heterogeneity. G-1 and G-2 gangliosides have common and individual antigenic determinants. Glucosylceramide of gangliosides is immunologically inactive. Individual antigenic specificity of the gangliosides depends on the presence of N-glycolylneuraminic acid (G-1) and SO3H-group (G-2). Egg gangliosides were demonstrated by immunofluorescence throughout the cell surface. After fertilization of immunofluorescent label was concentrated on one pole of the embryo only. During the development of specific fluorescence was again uniformly distributed at the blastomer surface. The most intense fluorescence was observed in the junction areas of the blastomers.
From eggs and embryos of the sea urchin Strongylocentrotus intermedius two gangliosides, provisionally named G-1 and G-2, were isolated in the pure state. Both gangliosides contained glucose, ...N-glycoloylneuraminic acid and sphingosines in a 2:2:1 ratio; G-2 contained also a sulfate group, and yielded G-1 on desulfation. By periodate oxidation/borohydride reduction, permethylation analysis, neuraminidase degradation, analysis of the aldohexitol acetates and mass-spectrometry G-1 and G-2 were shown to have hitherto unknown structures: G-1 was identified as N-glycoloylneuraminosyl-(alpha 2 leads to 6)-glucosyl-(1 leads to 8)-N-glycoloylneuraminosyl-(2 leads to 6)-glucosyl-(1 leads to 1)-ceramide, and G-2 as sulfated G-1, carrying a sulfate ester group at C-8 of the terminal sialic acid. Antisera against the two gangliosides were prepared in rabbits by immunization with ganglioside G-1 or G-2. The specificity of the antisera was revealed by immunoelectrophoresis and immunodiffusion. The antisera did not react with bovine-brain and rat-liver gangliosides, with glucosylceramide and with various hydrolytic fragments of G-1 and G-2. The surface localization of the gangliosides in embryos incubated at different cell densities was studied by immunofluorescence microscopy. The intensity of the immunofluorescence was found to increase with decreasing cell density, indicating a different surface organization in sparse and dense embryos. In the sparse embryos immunofluorescence was seen mainly in the contact regions between the blastomers.
Ultradian protein synthesis rhythm was used as a marker of cell cooperation in synchronous dense and non-synchronous sparse hepatocyte cultures. Phenylephrine (2 μM, 2 min), an
α
1-adrenoreceptor ...agonist, which exerts Ca
2+
cytelevation from intracellular stores, affected protein synthesis rhythm in sparse cultures, i.e. initiated cooperative activity of the cells. The same effect was produced by 2,5-di(tertiary-butyl)-1,4-benzohydroquinone (10 μM, 2 min), which increases Ca
2+
cytby a non-receptor pathway. Pretreatment of dense cultures with the intracellular calcium chelator, 1,2-bis (2-aminophenoxy) ethane-
N,
N,
N′,
N′- tetraacetic acid (acetoxymethyl) ester (BAPTA-AM) at 10–20 μM for, 30–60 min resulted in loss of the rhythm of protein synthesis, i.e. loss of cooperative activity between the cells. The medium conditioned by control dense cultures initiated rhythm in sparse cultures, whereas the conditioned medium of cultures pretreated with BAPTA-AM did not. Ca
2+
cytincrease is known to occur with monosialoganglioside GM1 treatment. By ELISA estimation, the GM1 content in 3 h conditioned medium was similar in control dense cultures to that in cultures pretreated with BAPTA-AM. Bearing in mind data on the Ca
2+-dependence of vesicle formation and shedding, the conditioned medium was separated by 150,000
g centrifugation to supernatant containing monomers and micelles, and a pellet containing vesicular form of gangliosides. Only the latter initiated cooperative activity of the cells of sparse cultures. These cultures were also synchronized by GM1-containing liposomes at lower concentrations than added free GM1, 0.0003 and 0.06 μM respectively. Thus, GM1 and calcium are both involved in cell–cell synchronization. Activation of gangliosides, including GM1 and elevation of Ca
2+
cyt,is known to lead to changes of protein kinase activity and protein phosphorylation resulting in modulation of oscillations in protein metabolism.
We studied dense 24-hour cultures of rat hepatocytes in serum-free medium on collagencoated slides. As before, a circahoralian rhythm of protein synthesis was observed in control cultures in a fresh ...medium. No rhythm was found after addition of 1-10 μM dopamine to the medium containing such cultures. The rhythm was observed after addition of 0.3 μM ganglioside to pretreated-dopamine cultures. Dopamine is likely to influence the conditioning of intercellular medium with gangliosides. Deficit of this endogenous synchronizing factor in the intercellular medium blocks self-organization of the protein synthesis rhythm. Thus, in contrast to previously studied norepinephrine and serotonin, as well as gangliosides, which organized the population rhythm of protein synthesis, dopamine disorganized the rhythm, impairing direct intercellular interactions.
Melatonin (5 nM) added to medium with primary hepatocyte cultures shifted the phase of circahoralian rhythm of protein synthesis and hence, can be a factor synchronizing fluctuations in protein ...synthesis and rhythm organizer in the hepatocyte population. Blockade of melatonin receptors with luzindole (20 nM) arrested rhythm organization of protein synthesis by melatonin. Prospects of studying biochemical mechanisms of protein synthesis rhythm organization with other drugs (calcium agonists, similarly to melatonin) are discussed.
Dopamine in the concentration 0.4 μg/mL abolishes protein synthesis rhythm in HaCaT keratinocytes and hepatocytes unlike noradrenaline or melatonin, which synchronize direct intercellular ...interactions and organize protein synthesis rhythm. Experiments with D2 dopamine receptors blocking agent metoclopramide (tserukal) in the concentration 2 μg/mL show that a disorganizing effect of dopamine is driven by the activation of D2 receptors, which block adenylyl cyclase and the efflux of calcium ions from internal depos according to the literature. It is shown that tserukal does not activate serotonin receptors in our experimental settings. Cellular interactionsâ recovery during or after dopamine action is carried out by melatonin in the concentration 0.001 μg/mL. A recommendation to inject melatonin before dopamine administration for different medical indications is discussed.
The effect of inhibition of proteasome activity on direct cell-cell interactions in primary hepatocyte cultures was studied. The circahoralian rhythm of protein synthesis was a marker of cell-cell ...communication. The addition of the proteasome inhibitor MG132 at doses of 10 or 20 μM to the medium with hepatocyte cultures for 19 h resulted in a significant reduction in the total pool of³H-leucine in cells. The incorporation of leucine into proteins changed slightly or negligibly, whereas the content of free labeled leucine in hepatocytes decreased. The rhythm of protein synthesis was distorted compared to the control. The rhythm was restored by external organizers, such as gangliosides and melatonin, as well as by enhancing the activity of protein kinases—the key factor in the organization of the rhythm of protein synthesis. A short-term (3-h) exposure to MG132 did not change the pool of leucine, but the rhythm of protein synthesis was also disturbed. Thus, protein catabolism affects cell-cell interactions organizing the rhythm of protein synthesis. Another factor of the downregulation of the rhythm of protein synthesis, the secretion of proteins from the hepatocytes in vivo, which was shown in vivo in many studies, was also revealed in our study when measuring the content of proteins stained with Coomassie Brilliant Blue G250 in the medium with hepatocyte cultures.
This report extends the genetic map of the common shrew (Sorex araneus) by use of a clone panel of shrew-Chinese hamster and shrew-mouse hybrid cells (Pack et al., 1995; Matyakhina et al., 1996). ...This set of hybrid clones made it possible to assign the shrew genes for isocitrate dehydrogenase 2 (IDH2), inorganic pyrophosphatase (PP), glutamicpyruvate transaminase (GPT), adenosine kinase (ADK), glucuronidase 2 (GUSB) and acid phosphatase 2 (ACP2) to chromosome ik; the genes for adenylate kinases 1 and 3 (AK1 and AK3) to chromosome af; the genes for glutamate-oxaloacetate transaminase 2 (GOT2), peptidase D (PEPD) and growth hormone (GH) to chromosome hn; the gene for phosphoglucomutase 2 (PGM2) to chromosome go, the gene for enolase 1 (ENO1) to chromosome ji, the gene for ornithine carbamoyl-transferase (OTC) to chromosome de, the gene for aminoacylase 1 (ACY1) to arm m (chromosome mp), the gene for glutamate-oxaloacetate transaminase 1 (GOT1) to arm q (chromosome qr). Thus, the genetic map of the common shrew now contains 33 genes and it is possible to compare the syntenic associations with other species.
