Basic processes of locomotor coordination in the rock lobster

Rock lobsters are able to perform long and stereotyped stepping sequences above a motor driven treadmill. Forward walking samples are estimated by mean of statistical methods to draw out the basic rules involved in the locomotor behaviour (Fig. 1).

- The spatial and temporal parameters defined in a single propulsive leg are either invariable in respect to the imposed speed, as the mean step length (L), the return stroke duration (Tr) and the pause times (T's, T'r), or speed dependent as the power stroke duration (Ts) and the whole period (Figs. 2 and 3).

- The interleg phase coupling is strong and stable in the ipsilateral rear pairs (4–5), these legs acting most of the time in absolute coordination (1:1) or in harmonic ratio (2:1). In the contralateral pairs (R4-L4, R5-L5) the legs roughly operate in antiphase, but the relationship appears much weaker and variable, with frequent episodes of relative coordination (Fig. 4).

- The time intervals between the ground contact of any leg and the swing initiation in the nearest ones appear somewhat constant and could be closely related to the mechanism of stepping synchronization. The “5 on - 4 off” delay, very stable and always positive, suggests that the rear legs could exert a predominant influence upon the rhythmical movements of the next anterior ipsilateral appendages (Fig. 5).

- To test the contralateral relationships, the treadmill belts can be decoupled in order to impose different walking speeds on each side. Such a conflicting stimulus reveals that:

1. The relative hierarchy always observed between the ipsilateral legs can be artificially created between the two sides (Fig. 6).
2. The driving influence of a given leg is closely linked to the intensity of EMG's discharges in its power stroke muscles.
3. The contralateral appendages are able to walk in absolute coordination despite a large speed difference between the two sides (up to 4 cm/s). Under such a constraint, the walking legs alter its invariable parameters (L and Tr) to reach a common step period and steadily maintain the alternating pattern (Figs. 6 and 7).

     

Response characteristics of wide-field non-habituating non-directional motion detecting neurons in the optic lobe of the locust,Locusta migratoria

1. Extracellular recordings from wide-field nonhabituating non-directional (ND) motion detecting neurons in the second optic chiasma of the locust Locusta migratoria are presented. The responses to various types of stepwise moving spot and bar stimuli were monitored (Fig. 1)
2. Stepwise motion in all directions elicited bursts of spikes. The response is inhibited at stimulus velocities above 5°/s. At velocities above 10°/s the ND neurons are slightly more sensitive to motion in the horizontal direction than to motion in the vertical direction (Fig. 2). The ND cells have a preference for small moving stimuli (Fig. 3).
3. The motion response has two peaks. The latency of the second peak depends on stimulus size and stimulus velocity. Increasing the height from 0.1 to 23.5° of a 5°/s moving bar results in a lowering of this latency time from 176 to 130 ms (Fig. 4). When the velocity from a single 0.1° spot is increased from 1 to 16°/s, the latency decreases from 282 to 180 ms (Figs. 5–6).
4. A change-of-direction sensitivity is displayed. Stepwise motion in one particular direction produces a continuous burst of spike discharges. Reversal or change in direction leads to an inhibition of the response (Fig. 7).
5. It shows that non-directional motion perception of the wide-field ND cells can simply be explained by combining self-and lateral inhibition.

     

Physiological changes in central neuronal pathways contributing to the generation of a reflex reversal

1. In the stick insect Carausius morosus the properties of the neuronal network governing the femur-tibia joint depend on the behavioral state of the animal. In the inactive animal flexion of the femur-tibia joint results in the generation of a resistance reflex, while in the active animal the same stimulus induces the so-called active reaction, the first part of which is a reflex reversal.
2. Recordings from motoneurons innervating the extensor tibiae muscle indicated that their time course of activity during the active reaction is due to inputs from intercalated pathways. We therefore investigated the role of identified nonspiking interneurons that transmit sensory information from the chordotonal organ onto the extensor motoneurons in the inactive animal. We can show that (i) the nonspiking interneurons received altered inputs whereas (ii) they provided qualitatively the same synaptic drive onto leg motoneurons.
3. From our results it is clear that (i) neuronal pathways contributing to the generation of the resistance reflex are also involved in the generation of the reflex reversal in the same control loop, (ii) thereby adopting the same principle of information processing (parliamentary principle), because both, supporting and opposing pathways contribute to the generation of the motor output.

     

Comparison between the movement detection systems underlying the optomotor and the landing response in the housefly

Flies evaluate movement within their visual field in order to control the course of flight and to elicit landing manoeuvres. Although the motor output of the two types of responses is quite different, both systems can be compared with respect to the underlying movement detection systems. For a quantitative comparison, both responses were measured during tethered flight under identical conditions. The stimulus was a sinusoidal periodic pattern of vertical stripes presented bilaterally in the fronto-lateral eye region of the fly. To release the landing response, the pattern was moved on either side from front to back. The latency of the response depends on the stimulus conditions and was measured by means of an infrared light-beam that was interrupted whenever the fly lifted its forelegs to assume a preprogrammed landing posture (Borst and Bahde 1986). As an optomotor stimulus the pattern moved on one side from front to back and on the other side in the opposite direction. The induced turning tendency was measured by a torque meter (Götz 1964). The response values which will be compared are the inverse latencies of the landing response and the amplitude of the yaw torque.

1. Optomotor course-control is more sensitive to pattern movement at small spatial wavelengths (10° and 20°) than the landing response (Fig. 1a and b). This suggests that elementary movement detectors (EMDs, Buchner 1976) with large detection base (the distance between interacting visual elements) contribute more strongly to the landing than to the optomotor system.
2. The optimum contrast frequencies of the different responses obtained at a comparatively high pattern contrast of about 0.6 was found to be between 1 and 10 Hz for the optomotor response, and around 20 Hz for the landing response (Fig. 2a and b). This discrepancy can be explained by the fact that the optomotor response was tested under stationary conditions (several seconds of stimulation) while for the landing response transient response characteristics of the movement detectors have to be taken into account (landing occurs under these conditions within less than 100 ms after onset of the movement stimulus). To test the landing system under more stationary conditions, the pattern contrast had to be reduced to low values. This led to latencies of several seconds. Then the optimum of the landing response is around 4 Hz. This is in the optimum range of the optomotor course-control response. The result suggests the same filter time constants for the movement detectors of both systems.
3. The dependence of both responses on the position and the size of the pattern was examined. The landing response has its optimum sensitivity more ventrally than the optomotor response (Fig. 3a and b). Both response amplitudes increase with the size of the pattern in a similar progression (Fig. 3c and d).

In first approximation, the present results are compatible with the assumption of a common set of movement detectors for both the optomotor course-control and the landing system. Movement detectors with different sampling bases and at different positions in the visual field seem to contribute with different gain to both responses. Accordingly, the control systems underlying both behaviors are likely to be independent already at the level of spatial integration of the detector output.     

Frequency as a releaser in the courtship song of two crickets,Gryllus bimaculatus (de Geer) and Teleogryllus oceanicus: a neuroethological analysis

1. The courtship behavior of male field crickets, Gryllus bimaculatus (De Geer) and Teleogryllus oceanicus, is a complex, multimodal behavioral act that involves acoustic signals (a courtship song; Fig. 1A,B). The dominant frequency is 4.5 kHz for T. oceanicus song (Fig. 1A) and 13.5 kHz for G. bimaculatus (Fig. IB).
2. When courting males are deprived of their courtship song by wing amputation, their courtship success declines markedly but is restored when courting is accompanied by tape-recordings of their courtship songs or a synthetic courtship song with only the dominant frequency of the natural song; other naturally occurring frequency components are ineffective for restoring mating success (Figs. 4, 5).
3. It has been suggested that an identified auditory interneuron, AN2, plays a critical role in courtship success. Chronic recordings of AN2 in an intact, tethered female show that AN2's response to the natural courtship song and synthesized songs at 4.5 and 13.5 kHz is similar in T. oceanicus. By contrast, in G. bimaculatus, AN2's response to the natural courtship song and synthesized song at 13.5 kHz, but not at 4.5 kHz, is similar (Figs. 2,3).
4. In behavioral experiments, playback of a 30 kHz synthetic courtship song in G. bimaculatus does not restore courtship success, yet this same stimulus elicits as strong a response from AN2 as does the normal courtship song (Fig. 6). Thus, contrary to earlier work by others, we conclude AN2 is not, by itself, a critical neural link in the courtship behavior of these two species of crickets.

     

Multisensory control of escape in the cockroach Periplaneta americana

1. All giant interneurons (GIs) were ablated from the nerve cord of cockroaches by electrocautery, and escape behavior was analyzed with high-speed videography. Animals with ablations retained the ability to produce wind-triggered escape, although response latency was increased (Table 1, Fig. 4). Subsequent lesions suggested that these non-GI responses depended in part on receptors associated with the antennae.
2. Antennal and cereal systems were compared by analyzing escape responses after amputating either cerci or antennae. With standard wind stimuli (high peak velocity) animals responded after either lesion. With lower intensity winds, animals lost their ability to respond after cereal removal (Fig. 6).
3. Removal of antennae did not cause significant changes in behavioral latency, but in the absence of cerci, animals responded at longer latencies than normal (Fig. 7).
4. The cercal-to-GI system can mediate short latency responses to high or low intensity winds, while the antennal system is responsive to high intensity winds only and operates at relatively longer latencies. These conclusions drawn from lesioned animals were confirmed in intact animals with restricted wind targeting the cerci or antennae only (Fig. 9).
5. The antennae do not represent a primary wind-sensory system, but may have a direct mechanosensory role in escape.

     

Improvements of the membrane filter method for DNA:rRNA hybridization

We describe and recommend the following improvements of DNA:rRNA membrane filter hybridization methods. One of our aims was to avoid DNA release from filter discs during hybridization.

1. Our hybridization conditions are 2 SSC in aq. dest., with 20% formamide, 50 C, overnight for 16 hr.
2. Duplexing is over in 8–10 hr.
3. Formamide has to be very pure (O.D.≤0.2/cm light path at 270 nm).
4. RNAase treatment: 250 μg/5 ml 2 SSC/filter at 37 C for 1 hr.
5. Our conditions for stepwise thermal denaturation are: 5°C steps from 50C to 90C in 1.5 SSC in 20% formamide.
6. Single-stranded DNA, fixed on membrane filters, and stored in vacuo at 4C, can be used reliably for hybridization for up to 20 months.
7. Concentrated DNA in 0.1 SSC, quick-frozen at ?50 C and stored at ?90 C for up to 2 years can be used for hybridization without much change.
8. A CsCl gradient purification step yields much purer DNA, but increases the release of DNA from filters by about 20%. Filters with 20% more DNA is a compensation.
9. rRNA can be stored for 20 months in SSC or 2 SSC at ?12C without changing the hybridization results.

     

Context-dependent stuttering

Bei einer Untersuchung des Stotterns in der deutschen Umgangssprache machten wir folgende Beobachtungen:

1. Das gestotterte Phonem wird häufig von einem identischen Phonem begleitet (definiert als das ?induzierende Phonem“) welches sowohl vor wie nach dem gestotterten Phonem auftreten kann.
2. Gewöhnlich folgte das induzierende Phonem dem gestotterten Phonem.
3. Der Abstand zwischen induzierendem und gestottertem Phonem war geringer als bei Zufälligkeit zu erwarten.
4. Induzierende und gestotterte Phoneme befanden sich gewöhnlich in identischen Silbenpositionen.
5. Gestotterte Phoneme traten in der Regel bei betonten Silben auf.

Um diese Beobachtungen zu erklären erschienen uns drei Annahmen erforderlich:

1. Sprach-Output ist hierarchisch bestimmt. Silben und Phoneme sind Glieder in dieser Hierarchie.
2. Unterschwellige Erregbarkeit ist in dieser Hierarchie stärker bei betonten als bei unbetonten motorischen Programmen.
3. Ähnliche Programme (sowohl auf Silbenals auch Phonemniveau) inhibieren einander.

Diese Annahme gibt zugleich eine mögliche Erklärung für Blockierung und Längung — Phänomene, die ebenfalls in der Sprache von Stotterern auftreten. Unsere Beobachtungen bieten also eine mögliche Lösung für das Rätsel des Stotterns.     

Selective phonotaxis to advertisement calls in the gray treefrog Hyla versicolor: behavioral experiments and neurophysiological correlates

1. The significance of particular acoustic properties of advertisement calls for selective phonotaxis by the gray treefrog, Hyla versicolor (= HV), was studied behaviorally and neurophysiologically. Most stimuli were played back at 85 dB SPL, a level typically measured at 1–2 m from a calling male.
2. Females preferred stimuli with conspecific pulse shapes at 20° and 24°C, but not at 16°C. Tests with normal and time-reversed pulses indicated the preferences were not influenced by the minor differences in the long-term spectra of pulses of different shape.
3. Pulse shape and rate had synergistic or antagonistic effects on female preferences depending on whether the values of one or both of these properties in alternative stimuli were typical of those in HV or heterospecific (H. chrysoscelis = HC) calls.
4. More auditory neurons in the torus semicircularis were temporally selective to synthetic calls (90%) than to sinusoidally AM tones and noise (< 70%).
5. Band-pass neurons were tuned to AM rates of 15–60 Hz. Neurons were more likely to be tuned to HV AM rates ( < 40 Hz) when stimuli had pulses with HV rather than HC shapes.
6. Sharp temporal tuning was uncommon and found only in neurons with band-pass or low-pass characteristics.
7. Many neurons differed significantly in response to HV and HC stimulus sets. Maximum spike rate was more often elicited by an HV stimulus (74%) than by an HC stimulus (24%).
8. Differences in spike rates elicited by HV and HC stimuli were attributable to combinations of differences in the rise times and shapes of the pulses.

     

Morphological and physiological properties of pheromone-triggered flipflopping descending interneurons of the male silkworm moth,Bombyx mori

1. The morphology of descending interneurons (DNs) which have arborizations in the lateral accessory lobe (LAL) of the protocerebrum, the higher order olfactory center, and have an axon in the ventral nerve cord (VNC), were characterized in the male silkworm moth, Bombyx mori.
2. Two clusters (group I, group II) of DNs which have arborizations mainly in the LALs were morphologically characterized. The axons of these DNs are restricted to the dorsal part of the each connective (Figs. 1–5).
3. Pheromonal responses of the group I and group II DNs were characterized. Flipflopping activity patterns, which have two distinct firing frequencies (high and low) in response to sequential pheromonal stimulation, were usually recorded (Figs.6–10).
4. Two types of flipflopping activity patterns were classified into those that had an antiphasic relationship (called the ‘FF’ type) between the left and right connectives and those with a synchronized relationship (‘ff’ type) (Figs. 8–12). We propose that some group II DNs show ‘FF’ flipflopping activity patterns (Fig. 10).
5. A state transition was usually elicited by less than 10 ng bombykol, the principal pheromone component. Extra impulses were elicited during constant light stimulation (Fig. 9).
6. Our results suggest that the LAL olfactory pathways might be important for producing flipflopping activity patterns (Fig. 11).

     

Polarization sensitivity in crayfish lamina monopolar neurons

1. Polarization sensitivity (PS) was examined in photoreceptors and lamina monopolar cells (LMCs) in two species of crayfish, Procambarus clarkii and Pacifasticus leniusculus. The measurements were made with intracellular recordings and broad field illumination.
2. PS is about 40% greater in Pacifasticus than in Procambarus (Table 1). In both species the LMC stationary PS profiles (estimated with flashes) are similar to those of receptors (Figs. 1 and 2). Both receptor and LMC sensitivity profiles are well described by cos² θ functions (Fig. 3). PS was observed in all receptors and 78% of LMCs.
3. When stimulated with a rotating polarizer, receptors and LMCs exhibit membrane potential modulation with phase predicted by the stationary PS profile (Fig. 5). In photoreceptors, the polarization-elicited percent modulation falls off steeply as intensity increases. The LMC modulation is stronger than that in receptors and relatively insensitive to the mean intensity (Figs. 6 to 8). For low intensities the LMC modulation is 100%. The LMC dynamic behavior is consistent with either an opponency mechanism or strong but polarization-insensitive lateral inhibition.
4. Receptors and LMCs exhibit steady-state differential sensitivity to stationary e-vector orientation (Fig. 9).
5. About 10% of the LMC neurons exhibit PS maxima separated by 90°. These results imply a nonlinear summation of signals from orthogonal receptor channels (Fig. 10).

     

Protease and Amylase in the digestive tract ofBarbus paludinosus

1. Protease and amylase activity in the digestive system ofBarbus paludinosus Peters (Pisces, Cyprinidae) has been investigated.
2. Chromatographic analysis showed seven amino acids to be present in both the anterior and posterior intestine. Only leucine, phenylalanine, valine, glycine and aspartic acid were positively identified.
3. In the anterior intestine chromatography revealed two sugars, but only one in the posterior intestine which was identified as glucose.
4. The pH of the intestinal fluid was found to be 5.8 and 7.8 for the fore and hind gut respectively, This correlates well with the enzyme pH optima found in in vitro experiments.
5. Protease and amylase activity was found throughout the digestive tract. Maximum proteolytic activity being present in the anterior intestine. Amylase activity is similar in both regions of the gut.
6. Correlation between the digestive enzymes and the fishes diet is briefly discussed.

     

Effect of external factors on phototaxis of Chlamydomonas reinhardtii

1. A fully automated phototaxis monitoring device is described for measuring photo-topatactic responses of flagellated organisms.
2. Photokinesis can be demonstrated in Chlamydomonas cells only after a dark period of about 72 hrs.
3. Pre-darkening of a few hours duration raises the phototactic disposition, whereas pre-illumination has no significant effect.
4. Circadian rhythms can be initiated by only one period of darkness or lower light intensity, whereas a period of higher intensity does not induce rhythms. The period length of the circadian rhythms is about 24 hrs.

     

Physiological and pharmacological properties of responses to GABA and ACh by abdominal motor neurons in Manduca sexta

1. GABA, ACh, and other agents were applied by pressure ejection to the neuropil of the third abdominal ganglion in the isolated nerve cord of Manduca sexta. Intersegmental muscle motor neurons with dendritic arborizations in the same hemiganglion were inhibited by GABA (Fig. 2) and excited by ACh (Fig. 5).
2. Picrotoxin was a potent antagonist of GABA (Fig. 4A). Bicuculline reduced GABA responses in some motor neurons (Fig. 4C), but had no effect on many other motor neurons. Curare reduced ACh responses (Fig. 6A). Bicuculline was an effective ACh antagonist in most motor neurons tested (Fig. 6B).
3. Motor neurons with dendrites across the ganglion from the ejection pipette exhibited different responses to GABA and ACh. Contralateral motor neurons often showed smaller, delayed hyperpolarizing GABA responses (Fig. 7). On two occasions, contralateral motor neurons had excitatory responses (Fig. 8). Contralateral motor neurons were hyperpolarized by ACh (Fig. 9). The inhibitory responses had only slightly longer latencies than ipsilateral excitatory ACh responses (Fig. 10A). The contralateral inhibitory ACh responses, but not the ipsilateral excitatory ACh responses, were eliminated by TTX (Fig. 10B).
4. A model, which includes inhibitory interneurons that cross the ganglionic midline to inhibit their contralateral homologs and motor neurons (Fig. 11), is proposed to account for contralateral responses to GABA and ACh and antagonistic patterns of activity of motor neurons during mechanosensory reflex responses.

     

Inhibition of carotenoid synthesis in Myxococcus fulvus (Myxobacterales)

1. The inhibitory effects of CPTA, nicotine, DPA, and San 6706 on carotenogenesis in Myxococcus fulvus were investigated.
2. The effects of CPTA, D-nicotine, and L-nicotine were very similar. The action of the drugs wasadditive. The cyclization was inhibited at low doses, the introduction of the hydroxyl group at C-1′ at higher doses. Lycopene accumulated at high drug concentration. The mode of action of the inhibitors is discussed.
3. In a carotenoid mutant of M. fulvus a stimulation of the “7,8-dehydrogenase” by CPTA was observed.
4. The specific carotenoid content of bacteria was increased by DPA due to an enhanced formation of phytoene. At low doses of DPA small amounts of an intermediate carotenoid glucoside ester, a 7,8-dihydro derivative, were detected.
5. DPA was taken up by the plasma membrane. Quantitative removal of DPA by washing was not possible.
6. San 6706 specifically and reversibly blocked the desaturation of phytoene.

     

Effects of in vivo ethanol administration on Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ uptake activity in synaptosomal membranes

1. Acute administration of ethanol (4 g/kg, i.p.) to mice inhibits the sequestration of calcium into endoplasmic reticulum-like organelles in synaptosomal membranes.
2. Ethanol administration inhibits both Ca²⁺-stimulated adenosine triphosphate hydrolysis and ATP-dependent calcium uptake in the vesicles at time of loss of righting reflex.
3. At recovery of righting reflex, the Ca²⁺-ATPase activity returns to normal levels, while the ATP-dependent uptake remains inhibited.
4. The effect of ethanol is specific for the sequestration (active transport) of calcium since calcium binding to synaptic membranes is not altered.
5. Alteration in mechanisms responsible for synaptosomal buffering of cytosolic Ca²⁺ levels by in vivo ethanol may contribute to altered transmitter release rates following ethanol adminstration.

     

Rigorous and extended application of information theory to the afferent visual system of the cat

1. Intracellular recording were obtained from P-cells of the LGN of the cat. The impulse trains of a single presynaptic retinal ganglion cell and the postsynaptic P-cell were separated by band-pass-filtering and subsequent amplitude discrimination.
2. The rates of information and transinformation for the visual channel from the eye to a ganglion cell and to the connected P-cell were calculated. Input signals to the channel were trains of light flashes of different rate, luminance and spatial distribution.
3. Transinformation was calculated without restrictive assumptions for the code.
4. The transient behaviour of the system in response to a flash was fully considered for information calculations. Additionally, it was ensured that the state of the (adaptive) channel was considered correctly.
5. Information theory was applied in an extended way. The time courses of information transfer were calculated for various flash stimuli and compared with each other.

     

Biogenesis of mitochondria 20 the effects of altered membrane lipid composition on mitochondrial oxidative phosphorylation inSaccharomyces cerevisiae

1. The lipid composition of mitochondria isolated from a fatty acid desaturase mutant ofSaccharomyces cerevisiae may be extensively manipulated by growing the organism on defined supplements of unsaturated fatty acid (UFA).
2. The fatty acid composition of the mitochondrial lipids closely follows that of the whole cells from which the mitochondria are isolated. UFA-depleted mitochondria contain normal levels of sterols, neutral lipids and total phospholipids, but have much lower levels of phosphatidyl inositides.
3. UFA-depleted mitochondria possess a full complement of cytochromes, oxidase both NAD-linked and flavoprotein-linked substrates at normal rates, and have levels of succinate and malate dehydrogenases similar to those of UFA-supplemented mitochondria. However, UFA-depletion has a marked effect on the ability of cytochromec to reactivate the NADH oxidase activity of cytochromec-depleted mitochondria.
4. The efficiency of oxidative phosphorylation decreases progressively with the UFA content of the mitochondria, and oxidative phosphorylation is completely lost in mitochondria containing approximately 20% UFA.
5. The incorporation of UFA into the lipids of UFA-depleted mitochondriain vivo results in a recoupling of oxidative phosphorylation. Recoupling is insensitive to both chloramphenicol and cycloheximide, indicating that all the proteins necessary for oxidative phosphorylation are present in UFA-depleted mitochondria, and that the less of oxidative phosphorylation is a purely lipid lesion.
6. ATPase activity is apparently unaffected by UFA-depletion, but³²P_i-ATP exchange activity is lost in mitochondria which have been extensively depleted in UFA.
7. Valinomycin stimulates the respiration of UFA-supplemented mitochondria in media containing potassium, but has no effect on the respiration of UFA-depleted mitochondria, suggesting that active transport of potassium is lost as a result of UFA-depletion.

     

Fumarate reduction inProteus mirabilis

1. Proteus mirabilis formed fumarate reductase under anaerobic growth conditions. The formation of this reductase was repressed under conditions of growth during which electron transport to oxygen or to nitrate is possible. In two of three tested chlorateresistant mutant strains of the wild type, fumarate reductase appeared to be affected.
2. Cytoplasmic membrane suspensions isolated from anaerobically grownP. mirabilis oxidized formate and NADH with oxygen and with fumarate, too.
3. Spectral investigation of the cytoplasmic membrane preparation revealed the presence of (probably at least two types of) cytochromeb, cytochromea ₁ and cytochromed. Cytochromeb was reduced by NADH as well as by formate to approximately 80%.
4. 2-n-Heptyl-4-hydroxyquinoline-N-oxide and antimycin A inhibited oxidation of both formate and NADH by oxygen and fumarate. Both inhibitors increased the level of the formate/oxygen steady state and the formate/fumarate steady state.
5. The site of inhibition of the respiratory activity by both HQNO and antimycin A was located at the oxidation side of cytochromeb.
6. The effect of ultraviolet-irradiation of cytoplasmic membrane suspensions on oxidation/reduction phenomena suggested that the role of menaquinone is more exclusive in the formate/fumarate pathway than in the electron transport route to oxygen.
7. Finally, the conclusion has been drawn that the preferential route for electron transport from formate and from NADH to fumarate (and to oxygen) includes cytochromeb as a directly involved carrier. A hypothetical scheme for the electron transport in anaerobically grownP. mirabilis is presented.

     

A study on the mechanism of energy coupling in the redox chain

The present study demonstrates that the mitochondrial respiratory chain includes not three but four energy coupling sites, the fourth site being localized at the NADPH→NAD⁺ step.

1. The NADPH→NAD⁺-directed transhydrogenase reaction in sonicated beef heart submitochondrial particles energizes the particle membrane as judged by two membrane potential probes, i.e. uptake of a penetrating anion, phenyldicarbaundecaborane (PCB^?), and enhancement of anilinonaphthalene sulfonate (ANS^?) fluorescence.
2. The reverse reaction (NADH→NADP⁺) is accompanied by the oppositely directed anion movement, i.e. PCB^? efflux.
3. Being insensitive to rotenone, antimycin, cyanide, and oligomycin, both the influx and efflux of PCB^? coupled with transhydrogenase reaction can be prevented or reversed by uncouplers.
4. Equalization of concentrations of the transhydrogenase substrates and products also prevents (or reverses) the PCB^? influx coupled with oxidation of NADPH by NAD⁺, as well as the PCB^? efflux coupled with reduction of NADP⁺ by NADH.
5. The transhydrogenase-linked PCB^? uptake depends linearly on the energy yield of the oxidation reaction calculated according to formula $$\Delta G = RTln\frac{{[NADPH] x [NAD^ + ]}}{{[NADP^ + ] x [NADH]^ \cdot }}$$ No threshold value of Δ was found. Measurable PCB^? transport was still observed at Δ≤0.5 kcal/mole NADPH oxidized.
6. Partial uncoupling of transhydrogenase reaction and PCB^? transport, induced by low concentrations ofp-trifluoromethoxycarbonylcyanide phenylhydrazone (FCCP), dinitrophenol, or by removing coupling factor F₁, results in the decrease of the slope of the straight line showing the PCB^? uptake as a function of Δ. Oligomycin improves the coupling in F₁-deprived particles, the slope being increased. Rutamycin, dicyclohexylcarbodiimide (DCCD) and reconstitution of particles with F₁, also increase the coupling.
7. In phosphorylating particles oxidizing succinate by O₂, both the energy-dependent NADH→NADP⁺ hydrogen transfer and PCB^? influx possess equal sensitivity to FCCP, which is lower than the sensitivity of oxidative phosphorylation. Similarly, the decrease in the succinate oxidation rate induced by malonate arrests first phosphorylation and then under higher malonate concentration, PCB^? influx. The rate of NADPH→NAD⁺ transhydrogenase reaction was found to be lower than the threshold value of rate of succinate oxidation, still coupled with phosphorylation. Respectively, the values of PCB^? uptake under transhydrogenase reaction are lower than those inherent in phosphorylating oxidation of succinate.

The conclusion is made that the NADPH→NAD⁺-directed transhydrogenase reaction generates the membrane potential of the same polarity as respiration and ATP hydrolysis but of a lower magnitude (“plus” inside particles; the forward hydrogen transfer). The NADH→NADP⁺-directed transhydrogenase reaction forms the membrane potential of the opposite polarity (“minus” inside particles; the reverse hydrogen transfer). Under conditions used, the transhydrogenase-produced membrane potential proves to be too low to support ATP synthesis (and, most probably, the synthesis of any other high-energy compound) maintaining, nevertheless, some electrophoretic ion fluxes. A conclusion is made that transhydrogenase forms a membrane potential with no high-energy intermediates involved.