Study of the mechanism of vanadate inhibition of the dynein cross-bridge cycle in sea urchin sperm flagella

The effect of vanadate on the ATP-induced disruption of trypsin-treated axonemes and the ATP-induced straightening of rigor wave preparations of sea urchin sperm was investigated. Addition of ATP to a suspension of trypsin-treated axonemes results in a rapid decrease in turbidity (optical density measured at 350 nm) concomitant with the disruption of the axonemes by sliding between microtubules to form tangles of connected doublet microtubules (Summers and Gibbons, 1971; Sale and Satir, 1977). For axonemes digested to approximately 93 percent of their initial turbidity, 5 {muM} vanadate completely inhibits the ATP-induced decrease in turbidity and the axonemes maintain their structural integrity. However, with axonemes digested to approximately 80 percent of their initial turbidity, vanadate fails to inhibit the ATP-induced decrease in turbidity and the ATP-induced structural disruption of axonemes, even when the vanadate concentration is raised as high as 100 μm. For such axonemes digested to 80 percent of their initial turbidity, the form of ATP-induced structural changes, in the presence of 25 μM vanadate, was observed by dark-field light microscopy and revealed that the axonemes become disrupted into curved, isolated doublet microtubules, small groups of doublet microtubules, and “banana peel” structures in which tubules have peeled back from the axoneme. Addition of 5 μM ATP to rigor wave sperm, which were prepared by abrupt removal of ATP from reactivated sperm, causes straightening of the rigor waves within 1 min, and addition of more than 10 μM ATP causes resumption of flagellar beating. Addition of 40 μM vanadate to the rigor wave sperm does not inhibit straightening of the rigor waves of 2 μM-1 mM ATP, although oscillatory beating is completely inhibited. These results suggest that vanadate inhibits the mechanochemical cycle of dyein at a step subsequent to the MgATP(2-)-induced release of the bridged dynein arms.     

Structure of Neuronal Impulse Trains of the Rat Inferior Olive under Conditions of Long-Lasting Vibrational Influence

In acute experiments on albino rats anesthetized with Nembutal (40 mg/kg, i.p.), we recorded the background impulse activity (BIA) generated by neurons of the inferior olive in the norm and after 5-, 10-, and 15-daylong vibrational influence (60 Hz, 2 h, daily). We characterized the distributions of neurons according to the regularity of impulse successions, their dynamics, and pattern of histograms of interspike intervals (ISIs); we also calculated the mean frequency of impulsation and the coefficient of variation of ISIs. It was demonstrated that the most significant shifts of the characteristics of BIA generated by neurons of the inferior olive were formed within the first 10 days of the vibrational influence. These shifts were observed mainly in the mean discharge frequency (increased within the initial period) and, to a lesser extent, in the intrinsic structure of impulse trains. Such shifts in the background activity of the inferior olive caused by long-lasting vibrational influence result, perhaps, from intensification of the influences of excitatory cerebellar/mesodiencephalic inputs to olivary neurons within the early periods of action of the above factor and prevalence of GABAergic influences within the later periods. It seems possible that, under such conditions, the characteristics of electrical synapses of the olivary neurons are also subjected to modification. Neirofiziologiya/Neurophysiology, Vol. 40, No. 4, pp. 340–347, July–August, 2008.     

Effects of Immobilization Stress on the Background Impulse Activity of Locus Coeruleus Neurons

We studied in rats changes in the impulse background activity (BA) of locus coeruleus (LC) neurons after short- and long-term immobilization stress; distributions of LC neurons by the level of regularity of their BA, dynamics of spike trains, and pattern of histograms of interspike intervals (ISI) were taken into account. We also calculated the means of the main BA statistical indices. Both short- and long-lasting immobilizations resulted in drops in the mean frequency of background discharges of LC neurons to about half of the initial value. Two-hour-long immobilization evoked statistically significant shifts in the distribution of LC neurons by the level of regularity of their BA, while after longer (15 h) immobilization this distribution nearly returned to the initial pattern. Short-lasting immobilization exerted no significant effect on the dynamic characteristics of BA; statistically significant changes in this respect developed only after longer stress. After 15-h-long immobilization, we also observed a noticeable increase in the number of neurons with polymodal ISI distributions. Therefore, stress results in significant modifications of the temporal parameters of the BA of LC neurons; characteristics of the BA of these neurons should be considered neuronal correlates of the stress state.     

One of the Pathways of Transmission of Afferent Activity to the Locus Coeruleus Under Conditions of Immobilization Stress

An electrophysiological proof has been obtained for the morphological data on the existence of afferent inputs from the medullary nucl. praepositus hypoglossi (NPH) to the brainstem locus coeruleus. The crucial role of the NPH in the formation of background spike activity of neurons of the locus coeruleus under conditions of short-term immobilization stress has been demonstrated. Neirofiziologiya/Neurophysiology, Vol. 37, No. 2, pp. 151–156, March–April, 2005.     

Preservation of native properties of mitochondria in rat liver homogenate

A protocol is developed for preparation of concentrated rat liver homogenate preserving assemblies of mitochondria in isotonic KCl under 0 and 15 degrees C. Assemblies preserve ability for self-organization during storage in homogenate. All key energy functions of mitochondria can be investigated in such a homogenate. Oxidative phosphorylation and membrane potential are stable for 5-7 h and can be still observed on the next day. Substrate-level phosphorylation is better pronounced for mitochondria in KCl than in sucrose medium while Ca2+ capacity is greater and lipid peroxidation is much lower. Sucrose addition impairs these functions. The rate of phosphorylating respiration is lower in large assemblies and higher in small. Transition from large to small assemblies corresponds to the transition from quiescent state of animal to adrenaline induced active state. The proposed method is particularly convenient for clinical investigations with small bioptates.     

Thoracic and Abdominal Respiration during the Reproduction of Fixed Respiration Volumes under Chemoreceptor Stimulation

Thoracic and abdominal components of the respiratory system during the reproduction without external feedback of fixed respiration volumes 1V _T, 0.5V _T, and 2V _T were studied in 13 young males. It was found that, during the reproduction of such respiration volumes without visual control, the thoracic and abdominal components of errors do not differ significantly. When the chemoreceptor stimulation increases (during the progressing hypoxia and hypercapnia), the accuracy of the reproduction decreases, mostly because of involuntary overestimation of the abdominal components of the respiration volume.