Caudatonigral projections under normal conditions and after an MPTP-induced lesion of the cat nigrostriatal system: Quantitative ultrastructural analysis

Ultrastructural and morphometric studies of caudatonigral synapses located on the nigrothalamic neurons were carried out on intact and N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated adult cats. Three types of synapses with different ultrastructural features were found. Morphometric analysis showed that 11.3% of analyzed junctions were caudatonigral synapses; 5.9% and 5.4% of them were located on the somata of nigrothalamic neurons and on their dendrites respectively. Among axo-somatic synapses, the caudatonigral ones amounted to 11.9%: 7.6% were type-I synapses and 4.3% belonged to type-III synapses. Both types had symmetrical contacts and could be considered inhibitory. Caudatonigral axodendritic synapses amounted to 10.6%: 3.2% were of type I; 4.2%, of type III; and 3.2%, of type II synapses with asymmetric contacts. The labelled type-II synapses were found exclusively on the nigrothalamic dendrites. The ultrastructural changes and the statistically significant decrease in the size of caudate axon terminals following the MPTP treatment took place only in the type I exosomatic synapses. It is suggested that the development of motor disorders in the cat after experimentally induced striatal dopamine insufficiency is due to the decrease in the efficacy of caudatonigral influences, which causes disinhibition of GABA-ergic inhibitory nigrothalamic neurons, and to enhanced influences of the latter on the cells of the motor thalamic nuclei.Neirofiziologiya/Neurophysiology, Vol. 26, No. 2, pp. 150–156, March–April, 1994.     

Quantitative cleavage of the N-glycosidic bond under the normal conditions of methanolysis used for the analysis of glycoprotein monosaccharides

The most common method used for the liberation of monosaccharides from glycoprotein N-glycans involves anhydrous methanolysis because it liberates almost quantitatively monosaccharides as O-methylglycosides, which are resistant to further degradation. However, it is generally assumed that this method does not cleave quantitatively the N-glycosidic bonds. This paper demonstrates that classical methanolysis conditions quantitatively cleave the N-glycosidic bond (96%), liberating glucosamine (and not its O-methylglycosides) and other minor reaction products which were identified. Because other N-acetyl-d-glucosamine (GlcNAc) residues are quantitatively liberated as the O-methylglycosides of glucosamine, the GlcNAc residue involved in the N-glycosidic bond is separated from the others using gas chromatography of heptafluorobutyrate derivatives.     

Quantitative analysis of benign paroxysmal positional vertigo fatigue under canalithiasis conditions

In our daily life, small flows in the semicircular canals (SCCs) of the inner ear displace a sensory structure called the cupula which mediates the transduction of head angular velocities to afferent signals. We consider a dysfunction of the SCCs known as canalithiasis. Under this condition, small debris particles disturb the flow in the SCCs and can cause benign paroxysmal positional vertigo (BPPV), arguably the most common form of vertigo in humans. The diagnosis of BPPV is mainly based on the analysis of typical eye movements (positional nystagmus) following provocative head maneuvers that are known to lead to vertigo in BPPV patients. These eye movements are triggered by the vestibulo-ocular reflex, and their velocity provides an indirect measurement of the cupula displacement. An attenuation of the vertigo and the nystagmus is often observed when the provocative maneuver is repeated. This attenuation is known as BPPV fatigue. It was not quantitatively described so far, and the mechanisms causing it remain unknown. We quantify fatigue by eye velocity measurements and propose a fluid dynamic interpretation of our results based on a computational model for the fluid–particle dynamics of a SCC with canalithiasis. Our model suggests that the particles may not go back to their initial position after a first head maneuver such that a second head maneuver leads to different particle trajectories causing smaller cupula displacements.     

Automated analysis and survival selection of anchorage-dependent cells under normal growth conditions

An instrument is described that can automatically analyze and select for a subpopulation of anchorage-dependent cells in tissue culture. Cells that label with fluorescently tagged antibodies or demonstrate structural variations are saved from exposure to a destructive high-intensity argon laser beam. The surviving population may then be cloned. The cell selection may occur in a tissue culture plate or in a microflow incubator which is designed to maintain a constant flow of media at 37 degrees C across cells growing on a glass coverslip. This incubator sits on an inverted microscope which focuses the laser beam to a diameter as small as 1 micron. A high-speed computer-controlled two-dimensional stage moves the cells past the beam for analysis, the results of which determine the fate of each cell: whether it is to be destroyed by radiant energy or selected for survival and subsequent proliferation. Another selection strategy performed by the instrument involves growing the cells on a thin, blackened polyester film which can be cut by the argon laser beam. Cells selected for cloning are then circumscribed. The heat of cutting welds the circumscribed film to a plastic coverslip surface or tissue culture chamber bottom. Nonselected cells may be removed by pulling the unattached polyester sheet from the attachment surface. The selected cells remain on polyester film disks welded to the plastic. Selections may be done automatically under computer control or manually by operator direction of stage movements. This instrument extends the art of automated cell selection and analysis to normal cell lines that must maintain cell-substratum contact (anchorage dependence) for differentiated cell function, e.g., neurons, fibroblasts, or kidney cells.     

Quantitative analysis of anti-apoptotic function of Akt in Akt1 and Akt2 double knock-out mouse embryonic fibroblast cells under normal and stressed conditions

The serine/threonine kinases Akt1/PKBalpha, Akt2/PKBbeta, and Akt3/PKBgamma have been implicated in preventing cells from undergoing apoptosis. Although several small molecule inhibitors of Akt have been reported to induce apoptosis in cancer cells, these inhibitors may have additional targets. In the current study, we used an Akt3 small interfering RNA (Akt3 siRNA) to analyze apoptosis induction in Akt1 and Akt2 double knock-out mouse embryonic fibroblast cells (MEF-Akt1,2-DKO). Our data indicated that Akt3 siRNA inhibited Akt3 protein expression in a dose-dependent manner. As a result, phosphorylation of Akt and its downstream targets, including FKHRL1 and GSK3alpha/beta, were reduced accordingly. The treatment also induced apoptosis in MEF-Akt1,2-DKO cells. However, apoptosis induction is significant only when more than 80% of Akt3 protein was depleted. Reintroducing Akt3 totally rescued Akt3-siRNA-induced apoptosis in MEF-Akt1,2-DKO cells. In addition, reintroducing Akt1 also inhibited apoptosis induced by Akt3 siRNA. Moreover, Akt3 siRNA potentiated different stress-induced apoptosis in MEF-Akt1,2-DKO cells at a lower dose when compared with what is required for apoptosis induction by itself. Our study suggests that only a small portion of Akt is active in wild-type MEF cells and a threshold of Akt inhibition is required to induce apoptosis by pure Akt inhibitors. In addition, our data indicate that cells under stress require more Akt for its survival.     

Ciliary activity under normal conditions and under viscous load

Ciliary metachronism and motility were examined optically in muco-ciliary tissue cultures from three different systems: a) frog's palate epithelium, b) frog's oesophagus, and c) human nasal polyps. In addition, lateral cilia of Mytilus edulis (water transporting cilia) were examined. It was revealed that the degree of synchronization between muco-ciliary systems is lower than that of water transporting cilia. There are no significant differences between different muco-ciliary systems, within the accuracy of our measurement although relatively large statistical ensembles were used. In addition the wavelength and wave direction of the metachronal wave was examined. All four systems exhibit similar wavelength. The metachronal parameters of muco-ciliary systems exhibit fluctuations (as was demonstrated by the degree of synchronization), however, the magnitude and repetitivity of these fluctuations, is dependent on the loading of the ciliary system. We have loaded the system by increasing the viscosity of the medium. Under viscous load the frequency of the beating decreased. The metachronal wavelength became longer and the metachronal coordination type more orthoplectic.     

Quantitative analysis of propofol in whole blood using capillary electrophoresis

We have developed a straightforward capillary electrophoresis method capable of quantifying clinically relevant propofol concentrations in whole blood from patients undergoing aortocoronary bypass grafting with cardiopulmonary bypass. The method utilizes 400 μL of whole blood and is capable of detecting propofol in the ng/mL range. Factors affecting reproducibility and reliability of analytical results for clinically relevant samples are discussed. The method was used to evaluate propofol concentrations in blood samples from 30 patients. The distribution in the whole blood concentration achieved in patients advocates the need for target-achieved monitoring techniques.     

Quantitative metabolome analysis using capillary electrophoresis mass spectrometry

A new approach for the comprehensive and quantitative analysis of charged metabolites by capillary electrophoresis mass spectrometry (CE-MS) is proposed. Metabolites are first separated by CE based on charge and size and then selectively detected using MS by monitoring over a large range of m/z values. This method enabled the determination of 352 metabolic standards and its utility was demonstrated in the analysis of 1692 metabolites from Bacillus subtilis extracts, revealing significant changes in metabolites during B. subtilis sporulation.     

Effects of antitussive drugs under normal and pathological conditions

The antitussive effect codeine and 1-propoxyphene have been studied in non-anaesthetized healthy cats and cats with respiratory tract inflammation elicited by undiluted croton oil. The drugs were administered intraperitoneally in doses of 10 and 20 mg/kg body weight. The antitussive effect was studied on the 4th day, after inflammation had set in. Cough induced in nonanaesthetized cats by mechanical irritation of the laryngopharyngeal and tracheobronchial areas was evaluated by changes of the lateral tracheal pressure. Experimentally induced inflammatory changes of the respiratory tract due to the antitussive activity of 1-porpoxyphene were significantly reduced, but that of codeine had not changed at all.     

Quantitative proteomic analysis of Salmonella enterica serovar Typhimurium under PhoP/PhoQ activation conditions

The PhoP/PhoQ two-component system plays a central regulatory role in the pathogenesis of Salmonella enterica serovar Typhimurium (S. Typhimurium), and it can be activated by low Mg(2+) concentrations and sublethal concentrations of cationic antimicrobial peptides (CAMP). Therefore, these two PhoP/PhoQ activation signals are considered as in vivo environmental cues sensed by S. Typhimurium for adaptation and survival. In this work, we conducted a SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative proteomic study to survey the proteomic changes of S. Typhimurium in response to low Mg(2+) concentrations or CAMP. We discovered that CAMP activated a portion of the PhoP/PhoQ regulatory network, whereas low Mg(2+) concentrations upregulated nearly all known members of this network, a number of previously unknown proteins, and some proteins regulated by IHF and RpoS. Systematic analysis following metabolic pathways revealed that low Mg(2+) concentrations selectively influenced proteins of certain metabolic functions while CAMP did not. Our study indicates that the low Mg(2+)-concentration condition may lead S. Typhimurium into a growth-control lifestyle, which provides new perspectives about Salmonella's adaptation to the host environment.     

Annulate lamellae in frog adenohypophysis under normal and experimental conditions

Cytoplasmic annulate lamellae have been observed in frog (Rana ridibunda) adenohypophysis pars distalis from normal spring animals and from others which were submitted to experimental conditions inducing selective activation of different cell types. Cell activation, because of either the normal active period in the frog cycle or the experimental treatments, seems to be correlated with the occurrence annulate lamellae. These annulate lamellae consist of a succession of two relatively parallel membranes interrupted periodically by discontinuities similar to nuclear pores. Sometimes they have been observed connected to endoplasmic reticulum.     

Multimeasurement analysis of the quantitative spatial organization of epicardial lymphatic capillaries under normal conditions

Principles of quantitative-spatial organization of the epicardial lymphatic capillaries have been studied in 26 normal dogs' hearts. Multimeasurable systemic models have been constructed to demonstrate how quantitative indices of metrical properties of the capillaries depend on the cardiac size. The possibility is discussed to apply the data obtained in the study of microcirculatory pathology of different genesis.     

Simulation of mechanoelectrical coupling in cardiomyocytes under normal and abnormal conditions

Mathematical models of the electromechanical function of cardiomyocytes and muscle duplexes, the simplest mechanically inhomogeneous myocardial systems, are developed. Using these models, the contribution of mechanoelectrical feedbacks to the contractive activity of the myocardium in normal and abnormal conditions is studied. In particular, the influence of the mechanical conditions of contraction on the shape and duration of the action potentials is reproduced and interpreted. In this context, different types of mechanical heterogeneity of the myocardium are analyzed. It is established that this heterogeneity can play a positive or negative role depending on the distribution of heterogeneous properties and on the order the elements of the system are activated. Using the same models, the contribution of mechanical factors to arrhythmogenesis under calcium overload of cardiomyocytes due to the weakening of the sodium-potassium pump is studied. Methods for correction of the contractive activity of cardiomyocytes in the case of such abnormalities are outlined.