Interactions of the RAD7 and RAD23 excision repair genes of Saccharomyces cerevisiae with DNA repair genes in different epistasis groups

Summary The RAD7 and RAD23 genes of S. cerevisiae affect the efficiency of excision repair of UV-damaged DNA. We have examined the UV survival of strains carrying the rad7 or rad23 deletion mutation in combination with deletion mutations in genes affecting different DNA repair pathways. As expected, the rad7 and rad23 mutations interact epistatically with the excision repair defective rad1 mutation, and synergistically with the rad6 and rad52 mutations that affect the postreplication repair and recombinational repair pathways, respectively. However, the rad7rad6 and the rad23rad6 mutants exhibit the same level of UV sensitivity as the radlrad6 mutant. This observation is of interest since, in contrast to the rad7 or the rad23 mutations, the rad1 mutant is very UV sensitive and highly excision defective. This observation suggests that RAD6 and RAD7 and RAD23 genes compete for the same substrate during DNA repair.     

Ultrasonography gives directly but noninvasively elastic characteristic of human tendon in vivo

To obtain an insight into tendon elasticity during human movement, a real-time ultrasonography was applied to the contracting tibialis anterior muscle. The insertion point of fascicles onto the aponeurosis was clearly visualized, and its position relative to a fixed marker on the skin moved proximally (1) according to the increasing dorsiflexion force (F) with a fixed ankle joint. Notably, the 1 – F relationship in the tendon was found to be quadratic in nature (F = c1²; c=1.48 2.24, r=0.985 0.992, n=9) as has been reported in the isolated tendon, although the F – 1 curves were slightly underestimated in comparison with the stiffness constant estimated from tendon architecture. This underestimation might be caused by changes in the height of the foot arch with the application of force.     

Influence of muscle fibre type and pedal rate on the V̇O2-work rate slope during ramp exercise

We hypothesised that the ratio between the increase in oxygen uptake and the increase in work rate (O₂/WR) during ramp cycle exercise would be significantly related to the percentage type II muscle fibres at work rates above the gas exchange threshold (GET) where type II fibres are presumed to be active. We further hypothesised that ramp exercise at higher pedal rates, which would be expected to increase the proportional contribution of type II fibres to the total power delivered, would increase the O₂/WR slope at work rates above the GET. Fourteen healthy subjects [four female; mean (SD): age 25 (3) years, body mass 74.3 (15.1) kg] performed a ramp exercise test to exhaustion (25 W min^–1) at a pedal rate of 75 rev min^–1, and consented to a muscle biopsy of the vastus lateralis. Eleven of the subjects also performed two further ramp tests at pedal rates of 35 and 115 rev min^–1. The O₂/WR slope for exercise <GET (S ₁) was significantly correlated with O₂ peak in ml kg^–1 min^–1 (r=0.60; P<0.05), whereas the O₂/WR slope for exercise >GET (S ₂) was significantly correlated to percentage type II fibres (r=0.54; P=0.05). The ratio between the O₂/WR slopes for exercise above and below the GET (S ₂/S ₁) was significantly greater at the pedal rate of 115 rev min^–1 [1.22 (0.09)] compared to pedal rates of 35 rev min^–1 [0.96 (0.02)] and 75 rev min^–1 [1.09 (0.05), (P<0.05)]. The greater increase in S ₂ relative to S ₁ in subjects (1) with a high percentage type II fibres, and (2) at a high pedal rate, suggests that a greater recruitment of type II fibres contributes in some manner to the xs O₂ observed during ramp exercise.     

Mating-type suppression of the DNA-repair defect of the yeast rad6Δ mutation requires the activity of genes in the RAD52 epistasis group

The RAD6 gene of Saccharomyces cerevisiae is required for post-replication repair of UV-damaged DNA, UV mutagenesis, and sporulation. Here, we show that the radiation sensitivity of a MAT a rad6 strain can be suppressed by the MAT2 gene carried on a multicopy plasmid. The a1-2 suppression is specific to the RAD6 pathway, as mutations in genes required for nucleotide excision repair or for recombinational repair do not show such mating-type suppression. The a1-2 suppression of the rad6 mutation requires the activity of the RAD52 group of genes, suggesting that suppression occurs by channelling of post-replication gaps present in the rad6 mutant into the RAD52 recombinational repair pathway. The a1-2 repressor could mediate this suppression via an enhancement in the expression, or the activity, of recombination genes.     

Effects of priming exercise intensity on the dynamic linearity of the pulmonary V̇O2 response during heavy exercise

Prior heavy-intensity exercise facilitates the pulmonary oxygen uptake (O₂) response during subsequent exercise, such that its kinetics returns towards first-order. To better understand this priming phenomenon, we investigated the effect of priming exercise, over a range of intensities, on the O₂ response to heavy-intensity cycle ergometry at a work rate of 50% [halfway between lactate threshold (LT) and O_2max]. Eight subjects performed two consecutive 6-min bouts separated by 6 min at 20 W. The first bout was each of: no warm-up control (CON), sub-lactate threshold (LT) at 80% of LT, and three supra-LT conditions (20%, 40%, and 60%). The O₂ response during the subsequent bout was evaluated using the effective time constant (), and the O₂ difference between minutes 3 and 6 (O_2(6–3)). The goodness-of-fit, indicative of first-order kinetics, was determined by the residual profile, and the mean square of errors (MSEr). The heart rate and blood lactate concentration ([La]r) just prior to the second bout were also measured. Compared with CON, and O_2(6–3) were significantly reduced following all supra-LT priming bouts, while the goodness-of-fit was significantly improved following 40% exercise. O_2(6–3) and [La]r were negatively correlated (P<0.05), unlike HR. In conclusion, prior exercise just above, but not below, LT facilitated the O₂ response in a threshold-like manner. Supra-LT priming exercise influenced the O₂ response allowing it to return to within as little as 12% from first-order (compared to ~50% in CON). The associated increases in circulating lactate and/or related factors seem to be centrally involved in this phenomenon.     

Indices for the role played by regional flow resistance in blood pressure reflex

Summary An index, = (r/r)/(R/R), is proposed for assessment of the relative degree of participation of a regional flow resistance in a blood pressure reflex, wherer andR are regional and total peripheral resistances respectively and 's refer to their reflexive changes. For actual computation, =I (iP – Pi)/i(IP – PI), whereI=cardiac output,i=regional flow rate,P=mean arterial pressure. When the change in cardiac output is neglegible, the above equation is simplified as =1 – Pi/iP. Another index, = g/G = (Pi – iP)/(PI – IP), is introduced for the degree of contribution of a region to a reflex, whereg andG are regional and total conductances.i = 1. Some examples of application of the indices are presented.Supported in part by a grant from the Ministry of Education of Japan.     

Organization of assembly factors Cbp3p and Cbp4p and their effect on bc 1 complex assembly in Saccharomyces cerevisiae

The bc₁ complex (complex III) of Saccharomyces cerevisae is composed of ten subunits that are assembled in the inner mitochondrial membrane. Cbp3p and Cbp4p are two mitochondrial proteins which are postulated to act as chaperones in bc₁ complex formation. Here, we show by blue native PAGE that cbp3 and cbp4 mutants are disturbed in complex III assembly and accumulate intermediate-sized forms of the complex. Moreover, deletion of CBP3 interferes with the formation of complex III/IV supracomplexes. Our studies show that Cbp3p and Cbp4p interact and are present in high-molecular-weight complexes, some of which might represent intermediates of complex III assembly. Overexpression of Cbp4p cannot substitute for the function of Cbp3p, but high-level expression of Cbp3p can partially compensate for the lack of Cbp4p. The finding that mitochondria of cbp3 and cbp4 mutants exhibit a wild-type lipid composition favors the idea that Cbp3p and Cbp4p are specific assembly factors for complex III rather than components of the mitochondrial lipid metabolism.     

Heat storage and body temperature during cooling and rewarming

Summary During calorimetric experiments with forced cooling and rewarming, changes in rectal temperature (T _re) and mean skin temperature ( _sk) allowed calculations of Burton's (1935) weighting coefficient a, which relates body temperature change to change in mean body temperature ( _b). Calculating _b from change in body heat content (H _b), which was determined from direct and indirect calorimetry, included individualized values for body specific heat based on body fat content. In five different cooling procedures there were two with cooling by exposure to cold water and three with cooling in a tubing suit; two of the procedures included mild exercise. The H _b ranged from –335 to –1600 kJ; rewarming restored body heat content. The mean (SEM) value of a in 119 determinations was 0.75 (0.01). This small variability in the coefficient probably came from the large values of H _b and from the use of maximal changes in _sk andT _re, including afterdrop. Change inT _re by itself correlated with _b, but with much variability. In forced body cooling and rewarming, 0.75(T _re) + 0.25 ( _sk) gives an accurate estimate of _b, hence change in body heat storage.     

Quantitative and sustained suppression of renal sympathetic nerve activity by left atrial distension in conscious dogs

In order to investigate the interrelations between left atrial pressure (Pla) and renal sympathetic nerve activity (RSNA) and heart rate (HR), Pla was increased by a balloon both in conscious sham-operated and cardiac-denervated dogs. RSNA was decreased and HR was increased with increasing Pla in sham-operated dogs. The reflex changes in RSNA and HR induced by the stimulation of atrial receptors persisted for at least 15 min. There was a consistent relationship between Pla and RSNA which could be described by a single exponential equation: % RSNA = 80.0(1–e^{–0.395 Pla}), or by a linear equation when Pla was less than 6 mmHg; %RSNA=-13.5Pla–2.25. The relationship between Pla and HR can be described by a single exponential equation: HR = 86.5(1–e^{–0.125 Pla}). A significant linear relationship between RSNA and HR was obtained during a graded left atrial distention (%RSNA=-1.08HR–17.7). In cardiac-denervated dogs, RSNA tended to increase and HR remained constant during the left atrial distention. These results indicate that left atrial receptors regulate RSNA and HR in both a quantitative and sustained manner in conscious sham-operated dogs.     

Novel adenoviral vectors coding for GFP-tagged wtCFTR and ΔF508-CFTR: characterization of expression and electrophysiological properties in A549 cells

E1/E3-deleted adenoviral vectors expressing an N-terminal green fluorescent protein (GFP) reporter gene fused to either wtCFTR (H5.040CMVEGFP-wtCFTR) or F508-CFTR (H5.040CMVEGFP-F508CFTR) were generated. To characterize the expression and activity, A549 cells were infected with vectors expressing GFP-tagged and non-tagged forms of CFTR and F508CFTR. CFTR activity was assayed in cell-attached and excised patches. For H5.040CMVEGFP-wtCFTR, forskolin-dependent outward current was observed in cell-attached patches from 56 of 67 GFP-positive cells. Single-channel conductances, open probability, mean open and mean closed time values for GFP-CFTR and CFTR were not significantly different. After excision, GFP-CFTR activity required ATP and exhibited a linear I-V relationship. For H5.040CMVEGFP-F508CFTR, media were supplemented with 5 mM butyrate 16 h after infection. Forskolin-dependent outward current was observed in cell-attached patches from 21 of 30 butyrate-treated GFP-positive cells and 0 of 8 GFP-positive cells without butyrate. Single-channel conductances, open probability, mean open and mean closed time values for GFP-F508CFTR and F508CFTR were not significantly different. However, the increase in open probability with genistein was significantly smaller for GFP-F508CFTR than for F508CFTR. In excised patches, GFP-F508CFTR activity required ATP and exhibited a linear I-V relationship. Despite the consistent detection of GFP-CFTR and GFP-F508CFTR channels in the plasma membrane by patch clamping, GFP fluorescence was observed only in intracellular regions and was not altered by butyrate. The data show that high levels of functional GFP-tagged CFTR channels can be expressed with these adenoviral vector constructs.     

The effectiveness of the control of pH in the extracellular fluid of the brain by the respiratory control system

Summary The effectiveness of the respiratory control system as a regulator of the pH in the extracellular fluid of the brain is defined by pH_{ECF op}/pH_{ECF cl} where pH_{ECF op} means the primary or open loop shift and pH_{ECF cl} the final or closed loop shift of brain extracellular fluid pH. The analysis of a steady state model described in a preceding paper (Loeschcke, 1973) allows, in the limits of the suppositions and simplifications, to calculate the effectiveness of the feedback regulator in the cases of increased metabolism, metabolic acidosis-alkalosis and inhalation of CO₂. The effectiveness is diminished if CO₂ production is increased, it drops in metabolic acidosis and rises in metabolic alkalosis and it drops steeply if CO₂ is inhaled. The effectiveness of this control system depends on the controlling action of the controlling element (the ventilation) rather than on varying sensitivity of the sensing element. The controlling effect is defined asC=–d P _CO2/d V _A orC=d pH_ECF/d V _A.Im Rahmen des Programms des Sonderforschungsbereiches 114 (Bionach) der Deutschen Forschungsgemeinschaft.     

Mechanical step variability during treadmill running

The present study was designed to study intra-individual step variability measured both on vertical displacement of the body (Z) and on step time (t) parameters by means of a kinematic arm and during treadmill running. A group of 17 subjects ran successively at 60%, 80%, 100% and 140% of their maximal aerobic velocity (v _amax). The total number of steps analysed was 6116. The absolute Z step variability (Z) ranged between 5 mm and 21 mm while the absolute t variability (t) ranged between 6 ms and 40 ms. Step variabilities were due to step asymmetry (from 38.5% to 48.5% of the step variability) and to stride variability. For submaximal velocities (60%, 80%, and 100%v _amax) both t and Z were independent of velocity or body dimensions whereas differences between subjects were significant (P < 0.01) for Z. On the other hand, variabilities were significantly increased when velocity was changed from submaximal to the 140%v _amax level. Furthermore, at submaximal levels Z was linked to the subject's energy cost of running (P < 0.05). Therefore, the intra-individual step variability should not be neglected in future studies on mechanical efficiency of running and it is suggested that, to obtain a good accuracy (better than 1%,P < 0.05) on mean value and variability of the mechanical parameters, measurements should be performed on at least 32–64 consecutive steps, which corresponds to about 15 to 20 s of running.     

Cupula displacement,hair bundle deflection,and physiological responses in the transparent semicircular canal of young eel

The transparent labyrinth of young eels (Anguilla anguilla L.) was used in toto for studying the configuration of cupula displacement, deflection of the hair bundle, and correlated changes in transepithelial voltage (TEV) and nerve activity (NA) in the semicircular canal. Microcapillaries were introduced into the canal through holes produced by a microthermocauter. Mechanical stimulation was applied either by injection of fluid into the ampulla or by electromagnetically displacing ferrofluid as a piston within the canal. Motion of individual kinocilia, stained cupulae or the ferrofluid piston was analysed by double-exposed microphotographs, photodiodes, or a video-system. The three-dimensional cupula displacement configuration was found to be piston-to diaphragm-like. Hair bundles at different sites on the crista exhibit differences in amplitude and time course of deflection. The transfer factor between shifts of the canal fluid and the tips of the kinocilia is 0.4–0.6. Displacements in opposite directions induce TEV and NA of opposite sign. Various tests confirmed TEV to reflect receptor potential responses. Nerve activity adapts to a tonic response with a time constant of 6.4s. No similar adaptation occurred in TEV. Stimulus-response curves of TEV- and NA-responses are similar and signoid in shape with saturation at ciliary deflections of roughly + 6° and –3°.     

Natriumtransport in den proximalen Tubuli und den Sammelrohren bei Variation der Natriumkonzentration im umgebenden Intterstitium

Zusammenfassung An Ratten wurden Mikropunktionsuntersuchungen am proximalen Tubulus und am Sammelrohr bei Durchblutung der peritubulären Capillaren bzw. Vasa recta sowie bei künstlicher Perfusion dieser Blutgefäße durchgeführt. In Abhängigkeit von der Höhe der interstitiellen Natriumkonzentration wurden der Nettonatriumtransport (_{Na iso}) bei gleicher Natriumkonzentration zu beiden Seiten der Tubuluswand und die Gleichgewichtskonzentrationsdifferenz (c _Na) bei fehlendem Nettovolumen- und Nettosubstanzfluß gemessen.Bei Variation der Natriumkonzentration im Gewebe durch Perfusion der peritubulären Capillaren mit 155 bzw. 300 mÄq/l Na änderten sich weder _{Na iso} noch c _Na für den proximalen Tubulus (_{Na iso} 8,4 bzw. 7,9·10^–5 Äq · mm^–2 · sec^–1; c _Na 24 bzw. 24 mÄq/l). Veränderung der Natriumkonzentration im Blut durch Infusion hypertoner NaCl Lösung oder Peritonealdialyse mit isotoner Mannitlösung führten zu prinzipiell gleichen Ergebnissen. Bei Perfusion der Vasa recta mit Lösungen, die 145 und 300 mÄq/l Natrium entheilten, blieben _{Na iso} wie c _Na über die Sammelrohrwand ebenfalls konstant (_{Na iso} 4,1 bzw. 4,1·10^–5 Äq · mm^–2 · sec^–1; c _Na 98 bzw. 104 mÄq/l). Proximale Tubuli und Sammelrohre verhalten sich demnach bei Variation der interstitiellen Natriumkonzentration gleich.Da die Wasserresorption aus den Sammelrohren von dem durch Gegenstrommultiplikation erzeugten Natriumkonzentrationsanstieg im Markinterstitium abhängt, die Natriumresorption aus den Sammelrohren aber wie die vorliegenden Befunde zeigen von eben dieser Natriumkonzentration unabhängig ist, ist dem Warmblüterorganismus die Möglichkeit gegeben, Natrium- und Wasserresorption unabhängig voneinander zu variieren. Die Natrium- und Wasserresorption aus den Sammelrohren werden jedoch beide durch den Gehalt der Sammelrohrflüssigkeit an permeablen Nichtelektrolyten, wie z. B. Harnstoff, beeinflußt.Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Single cell assay of exocytosis from pancreatic islet B cells

We have measured changes in membrane capacitance (C_m) in pancreatic B cells as a single cell assay of insulin secretion. Evidence that depolarization evoked C_m reflects exocytosis includes it's voltage, temperature and Ca²⁺ _o dependence. Decreases in C_m, presumably reflecting endocytosis, occur on a variable time scale. Two features that make B cells unique among excitable cells are the large magnitude of C_m, when normalized to Ca²⁺ current, and the rapid fatigue of the response for even minimal stimulation, perhaps due to the depletion of a readily releasable pool of vesicles.     

Influence of mechanical and metabolic strain on the oxygen consumption slow component during forward pulled running

The possible influence of increased eccentric mechanical work on the increase in oxygen uptake (O₂) after 3 min of running (O₂) was investigated through forward pulled running. Ten subjects ran at individually predetermined constant velocity on a treadmill, while being pulled forward. Ground reaction forces, expired gas and EMGs from leg muscles were collected after 3 min and at the end of the run. O₂ and mechanical work were then calculated. The amplitude of O₂ was 138 (139) ml·min^–1 [mean (SD)]. Increased ventilation explained only 8% of O₂. Stride frequency slightly decreased, inducing a similar decrease in internal work and total mechanical work (all P<0.01), while integrated EMG showed no modifications. It was concluded that O₂ does not come from either an increase in mechanical work production or an increase in muscular activity. O₂ could come from a lower muscle efficiency that could be due to a modification of fibre type recruitment.     

Trait anxiety,submaximal physical exercise and blood androgens

Summary This study evaluates the relationship between trait anxiety and both androgen and gonadotrophic hormone levels at rest and during severe physical exercise. Twelve volunteers were selected among 160 untrained male collegial students and classified as anxious (N=6) or non-anxious (N=6) subjects according to their scores on three trait-anxiety tests (STAI, IPAT, 16 PF). Serum ⁴-androgen (testosterone and ⁴-androstenedione), ⁴-androgen (DHEA and DHEA-SO₄) and gonadotrophin (LH and FSH) concentrations were measured by radioimmunoassay before, during and after 20 minutes of intensive bicycle exercise (80% of maximal heart rate). Results indicate significantly lower serum ⁴-androgens in anxious subjects before exercise. However, for each subject and irrespective of his anxiety level, all measured serum androgen concentrations increased significantly during exercise, although ⁴-androstene-dione remained lower in anxious subjects than in non-anxious ones. Serum LH concentrations (but not FSH) were signicantly higher in anxious subjects throughout the observation periods. However, exercise induced in each subject a significant decrease in the serum level of both gonadotrophic hormones. The results suggest that trait anxiety level may constitute an important factor that affects both pre-exercise and exercise serum androgen concentrations in untrained subjects.     

Klinische,morphologische und biochemische Untersuchungen bei einem androgenbildenden Hodentumor

Zusammenfassung Bei einem Knaben mit einer Pseudopubertas praecox wurde ein androgenproduzierender Hodentumor entfernt. Präoperativ war die Harnausscheidung der gesamten 17-Ketosteroide, von Androsteron und Ätiocholanolon, der 11-Oxy-17-Ketosteroide, insbesondere von 11-Hydroxy-androsteron, von Pregnandiol, Pregnantriol sowie der Oestrogene abnorm erhöht und fiel nach der Operation auf Normwerte ab. Nach Inkubation von Glucose allein mit Tumorschnitten wurden 11-Hydroxy- ⁴-androstendion und ⁴-Androstendion gefunden, nach Inkubation von ⁴-Androstendion: 11-Hydroxy- ⁴-androstendion, Testoseron und Adrenosteron, jedoch keine Oestrogene, nach Inkubation von Desoxycorticosteron: Corticosteron und 17-Hydroxy-desoxy-corticosteron. Im V. spermatica-Blut wurden große Mengen ⁴-Androstendion, ferner 17-Hydroxy-progesteron, 11-Hydroxy- ⁴-androstendion und Testosteron nachgewiesen.Die biochemischen und morphologischen Gesichtspunkte sowie die klinischen Auswirkungen des Tumors werden diskutiert. Morphologisch hatte der Tumor den Aspekt eines Leydigzell-Adenoms. In Übereinstimmung hiermit wurde eine hohe Sekretion von Androgenen festgestellt. Durch den Nachweis einer 11-Hydroxylase-Aktivität zeigte der Tumor jedoch gleichzeitig eine Eigenschaft, die sonst nur NNR-Zellen zukommt. Der Tumor wird vermutungsweise von atypischen Leydigzellen abgeleitet.Diese Art von Leydigzell-Adenom wird der beidseitigen tumorartigen Hodenvergrößerung, die gelegentlich beim angeb. adrenogenitalen Syndrom beobachtet wird, gegenübergestellt. Die gemeinsamen und unterschiedlichen Eigenschaften der beiden Wucherungen werden besprochen.

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Summary An androgen producing tumour of the testis was removed from a boy with pseudopubertas praecox. Urinary excretion of total 17-oxosteroids, androsterone and aetiocholanolone, 11-oxy-17-oxosteroids (particularly 11-hydroxy-androsterone), pregnandiol, pregnantriol and oestrogens were abnormally high before operation, and had decreased to normal after operation. After incubation of tumour slices with glucose alone androst-4-en-3,17-dione and 11-hydroxyandrost-4-en-3,17-dione were found; after incubation with androst-4-en-3,17-dione: 11-hydroxyandrost-4-en-3,17-dione, testosterone and adrenosterone, but no oestrogens were found; after incubation with desoxycorticosterone: corticosterone and 17-hydroxycorticosterone. A large amount of androst-4-en-3,17-dione was found in Vena spermatica blood, as well as 17-hydroxyprogesterone, 11-hydroxyandrost-4-en-3,17-dione and testosterone.The biochemical and morphological aspects are discussed in relation to the clinical manifestations of the tumour. The tumour had the morphological appearance of a Leydig cell adenoma and also showed a high rate of androgen secretion. The finding of 11-hydroxylase activity in the tumour shows that it possessed a characteristic usually found only in adrenocortical cells. The tumour was conceivably derived from atypical Leydig cells.This type of Leydig cell adenoma is compared with the bilateral tumour-like enlargement of the testis, which is ocassionally observed in congenital adrenogenital syndrome. The common and contrasting characteristics of the two growths are discussed.

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Herrn Prof. Dr.E. Rehn zum 85. Geburtstag gewidmet.     

Isolation of the ERG2 gene,encoding sterol Δ8Δ→7 isomerase,from the rice blast fungus Magnaporthe grisea and its expression in the maize smut pathogen Ustilago maydis

The Magnaporthe grisea ERG2 gene, encoding ⁸⁷ sterol isomerase, was isolated from a genomic library by heterologous hybridization to a fragment of the Ustilago maydis ERG2 gene. The isolated gene contained a reading frame of 745 bp which encoded a protein of 221 amino acids. The coding region was interrupted by a single putative 79-bp-long intron. The deduced amino-acid sequence exhibited similarity to the ERG2 gene products of U. maydis and of Saccharomyces cerevisiae, particularly in the central region of the proteins. The NH₂-terminal of all three proteins contained a long stretch of amino acids that were strongly hydrophobic, suggesting that they may function by anchoring the protein to a membrane surface. The M. grisea ERG2 gene complemented a U. maydis deletion mutant in which the ERG2 gene had been removed using a one-step gene replacement procedure. The ⁸⁷ sterol isomerase produced by the M. grisea ERG2 gene exhibited a level of sensitivity to the sterol biosynthesis inhibitor, tridemorph, similar to that of the enzyme derived from the U. maydis ERG2 gene.     

End-plate currents evoked by paired stimuli in frog muscle fibres

Using voltage-clamp techniques spontaneously occuring miniature end-plate currents (mepc) and nerve-evoked end-plate currents (epc) were recorded in frog glycerol-treated or cut muscle preparations. Epcs were induced by pairs of stimuli (the delay of the 2nd stimulus, t being 6 ms–30 s; one pair was delivered every 60–90 s). The decay time constant of the epc (_epc) was longer, the larger its quantal content despite the presence of active acetylcholinesterase (AChE). After treatment with anticholinesterases (prostigmine or armin, an irreversible inhibitor) this increase in _epc became more pronounced. When AChE was fully active the decay of the 1st epc ₁ was slightly faster than the decay of the 2nd epc ₂ only when the interstimulus interval was rather short (t<20 ms). Following treatment with anticholinesterases this difference between ₂ and ₁ could be determined even when t was as long as 30 s. In anticholinesterase-treated preparations was found to be inversely proportional to log t: a 50% increase in the decay time-constant of the 2nd epc occurred with t=120 ms. During continuous stimulation (10 impulses/s) _epc increased from the 1st to the 5–6th responses, but then decreased in parallel with the fall in the epc amplidude. The phenomenon of postsynaptic potentiation we observed could be readily abolished when quantal content was decreased by the presence of magnesium ions, but it was relatively unaffected when the receptor density was decreased by -bungarotoxin (-BuTX).The possible existence is discussed of two kinds of repetitive binding of ACh molecules, first, to free cholinoreceptors (a process which could be inhibited by -BuTX) and, second, to a complex of the cholinoreceptor plus one molecule of ACh (a process which is less sensitive to -BuTX blocking action).