Linear Time Course Research Articles

Influence of lesion level on the cardioventilatory adaptations in paraplegic wheelchair athletes during muscular exercise.

To characterize the influence of neurological lesion level on the cardiorespiratory and ventilatory responses of two groups of paraplegic athletes during incremental exercise on a treadmill and in the usual conditions for wheelchair exercise. Cardioventilatory responses evaluated in two groups of paraplegic wheelchair sportsmen designated as high paraplegic athletes (HPA) and low paraplegic athletes (LPA). After 2 min of data collection at rest and 3 min of warm-up at 4 km x h(-1), treadmill speed was increased by 1 km x h(-1) every minute until exhaustion. During this test, ventilation and its components, as well as respiratory exchanges, were measured breath by breath (C.P.X. Medical Graphics) every minute by taking the mean of the last 20 s of each increment. Spirometric values presented no significant differences between groups. At rest, no significant difference was observed between the two groups for all cardiorespiratory and ventilatory values obtained during the treadmill test. At submaximal exercise, all variables increased with the augmentation in workload. With the exception of R, there were no significant differences in the classic cardiorespiratory parameters (VO2, VCO2, HR, VE) between the two groups of paraplegics. For the ventilatory parameters, we observed significant differences between the two groups, with values of f and It/Trf significantly higher (0.01<P<0.001) and values of Trf and Vt significantly lower (0.01<P<0.001) for HPA versus LPA. We observed changes in breathing pattern, ie, in f, Vt, Trc and It/Trc, were significantly different between groups, with significantly higher values of f and It/Trc for HPA. We noted a ventilatory disturbance which was manifested by values of breathing frequency and tidal volume during exercise that were significantly different between groups. During maximal exercise, we observed no significant differences between the two groups concerning cardiorespiratory and ventilatory values. Despite the absence of significant differences, the more linear time course of the ensemble of HPA flows, the achievement of a greater number of work loads, and the higher maximal values indicate a better capacity for adaptation to exercise in the group of lower thoracic paraplegics. These results raise questions about the influence of neurological level and further research is needed to define with more precision the capacities of readaptation of the different cardiovascular and respiratory functions, as well as the training methods best adapted to the optimization of physical capacities.

Leukotriene A synthase activity of purified mouse skin arachidonate 8-lipoxygenase expressed in Escherichia coli

Mouse skin 8-lipoxygenase was expressed in COS-7 cells by transient transfection of its cDNA in pEF-BOS carrying an elongation factor-1α promoter. When crude extract of the transfected COS-7 cells was incubated with arachidonic acid, 8-hydroxy-5,9,11,14-eicosatetraenoic acid was produced as assessed by reverse- and straight-phase high performance liquid chromatographies. The recombinant enzyme also reacted on α-linolenic and docosahexaenoic acids at almost the same rate as that with arachidonic acid. Eicosapentaenoic and γ-linolenic acids were also oxygenated at 43% and 56% reaction rates of arachidonic acid, respectively. In contrast, linoleic acid was a poor substrate for this enzyme. The 8-lipoxygenase reaction with these fatty acids proceeded almost linearly for 40 min. The 8-lipoxygenase was also expressed in an Escherichia coli system using pQE-32 carrying six histidine residues at N-terminal of the enzyme. The expressed enzyme was purified over 380-fold giving a specific activity of approximately 0.2 μmol/45 min per mg protein by nickel–nitrilotriacetate affinity chromatography. The enzymatic properties of the purified 8-lipoxygenase were essentially the same as those of the enzyme expressed in COS-7 cells. When the purified 8-lipoxygenase was incubated with 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid, two epimers of 6- trans-leukotriene B 4, degradation products of unstable leukotriene A 4, were observed upon high performance liquid chromatography. Thus, the 8-lipoxygenase catalyzed synthesis of leukotriene A 4 from 5-hydroperoxy fatty acid. Reaction rate of the leukotriene A synthase was approximately 7% of arachidonate 8-lipoxygenation. In contrast to the linear time course of 8-lipoxygenase reaction with arachidonic acid, leukotriene A synthase activity leveled off within 10 min, indicating suicide inactivation.

Impaired aerobic glycolysis in muscle phosphofructokinase deficiency results in biphasic post-exercise phosphocreatine recovery in 31P magnetic resonance spectroscopy

Using 31P magnetic resonance spectroscopy, energy metabolism in calf muscles of two patients with biochemically and genetically proven muscular phosphofructokinase deficiency, and an asymptomatic heterozygote was monitored during isometric foot plantarflexion performed under aerobic and anaerobic conditions and in the aerobic recovery phases. In the heterozygote only a moderate alteration from normal was found in terms of an elevated ATP demand during exercise. In the homozygote, hexose phosphates, indicated as phosphomonoesters, increased dramatically during contraction. Phosphomonoester accumulation resulted in consumption of free inorganic phosphate (P i). During ischemic exercise the absence of glycolytic ATP formation resulted in a linear time course of phosphocreatine breakdown and a moderate alkalinization. During the recovery, phosphocreatine resynthesis showed a biphasic time course, indicating that mitochondrial function itself was not directly affected. At first glance, the early depletion of P i below initial resting levels and the rate of phosphate splitting from sugar phosphates seemed to become the limiting factor for the rate of the oxidative phosphorylation and creatine kinase reaction. However, the actual concentrations of P i and ADP estimated at the onset of delay were too high to exclusively explain the dramatic delay in PCr resynthesis. For this reason, a reduced turnover of the citric acid cycle was assumed, which was caused by the complete absence of glycolysis in PFK deficiency patients. Furthermore, results from PFK deficiency patients were compared with previous findings from myophosphorylase deficiency patients in the literature.

METHODOLOGICAL ASPECTS OF THE CONCONI-TEST

1860 The aim of the study was to test protocol variations on the heart rate performance curve (HRPC) and the heart rate turn point (HRTP) according to Conconi et al. (Int J. Sports Med. 1996, 17: 509-519). Respiratory gas exchange variables were used to define the 3 phases of energy supply(I,II,III). 18 healthy young male subjects performed 4 tests (T1-T4). T1: uniform speed (S) increments every 60 s. Subjects were than randomized for the next tests. T2: uniform S increments every 20 s; T3: like T2, in the second half of phase III greater acceleration in S. T4: like T2, at the begin of phase III greater acceleration in S. No differences were found in the degree of the deflection of the HRPC expressed as factor kHR between T1(0.228±0.225) and T2 (0.248±0.231) but an increase was found in T3 (0.533±0.248) and T4 (0.770±0.258). 11 subjects showed a deflection in the HRPC in all tests, and no significant difference could be found for S, HR and VO2 at the HRTP. HRTP was not found in 7 subjects in T1 and T2, due to linearity of the HRPC. In T3 and T4 subjects showed a deflection of HRPC resulting from the protocol. A HRTP was found to be dependent on the start of the acceleration in S. In cases with a linear time course in the HRPC in T1 and T2, in T3 a HRTP was found at 15.6 km/h and in T4 at 13.6 km/h, respectively. The Conconi test protocol with an accelerated increase in S in the final phase of the test has a major influence on the occurrence of the HRTP in cases of near linear HRPC.

In Vitro Regulation of FcεRIα Expression on Human Basophils by IgE Antibody

AbstractIn vivo studies suggested the possibility of an IgE-dependent regulation of high-affinity (FcεRI) IgE receptor expression on basophils. The current studies extend these observations to in vitro cultures of human basophils. Incubation of basophils for 3 to 4 weeks resulted in a slow dissociation of IgE antibody, during which time FcεRI expression decreased, as measured by flow cytometry using the anti-FcεRIα monoclonal antibody, 22E7, or by measuring FcεRIα mass by Western blotting of whole-cell lysates. Culture of basophils with IgE resulted in upregulation of FcεRIα expression by both flow cytometry and Western blotting of whole-cell lysates. Upregulation followed a linear time course during 2 weeks of culture. The relative increase in FcεRIα density depended on the starting density; with starting densities of FcεRIα of 10,000 to 170,000 per basophil, the upregulation varied 20- to 1.1-fold, respectively. Upregulation occurred in high-purity basophils, was not influenced by IgG at concentrations up to 1 mg/mL, and was inhibited by dimeric IgE. Heat-inactivated IgE was less effective and the monoclonal antibody CGP51901 that prevents IgE binding to FcεRIα blocked the ability of IgE to induce upregulation. The dose-response curve for IgE-induced upregulation had an effective concentration50 of 230 ng/mL. Although the receptor through which IgE induces this upregulation is not yet known, several characteristics suggest that the upregulation is mediated by IgE interacting through FcεRIα itself.

In Vitro Regulation of FcRIα Expression on Human Basophils by IgE Antibody

In vivo studies suggested the possibility of an IgE-dependent regulation of high-affinity (FcepsilonRI) IgE receptor expression on basophils. The current studies extend these observations to in vitro cultures of human basophils. Incubation of basophils for 3 to 4 weeks resulted in a slow dissociation of IgE antibody, during which time FcepsilonRI expression decreased, as measured by flow cytometry using the anti-FcepsilonRIalpha monoclonal antibody, 22E7, or by measuring FcRIalpha mass by Western blotting of whole-cell lysates. Culture of basophils with IgE resulted in upregulation of FcepsilonRIalpha expression by both flow cytometry and Western blotting of whole-cell lysates. Upregulation followed a linear time course during 2 weeks of culture. The relative increase in FcepsilonRIalpha density depended on the starting density; with starting densities of FcepsilonRIalpha of 10,000 to 170,000 per basophil, the upregulation varied 20- to 1.1-fold, respectively. Upregulation occurred in high-purity basophils, was not influenced by IgG at concentrations up to 1 mg/mL, and was inhibited by dimeric IgE. Heat-inactivated IgE was less effective and the monoclonal antibody CGP51901 that prevents IgE binding to FcepsilonRIalpha blocked the ability of IgE to induce upregulation. The dose-response curve for IgE-induced upregulation had an effective concentration50 of 230 ng/mL. Although the receptor through which IgE induces this upregulation is not yet known, several characteristics suggest that the upregulation is mediated by IgE interacting through FcepsilonRIalpha itself.

Evidence for P2-purinoceptor-mediated uptake of Ca2+ across a fish (Oreochromis mossambicus) intestinal brush border membrane.

We have studied the effect of ATP on Ca2+ uptake in intestinal brush border membrane vesicles (BBMVs) of the teleost tilapia (Oreochromis mossambicus). ATP stimulated Ca2+ uptake 12-fold over the control, with a linear time course. Ionomycin and detergent treatment did not reduce BBMVs' Ca2+ content, indicating the binding of Ca2+ to a membrane component. A rank order of ATP > ADP > AMP was established for the stimulation of Ca2+ uptake. Adenosine, vanadate, adenosine 5'-[alpha, beta-methylene]triphosphate (a P2x purinoceptor agonist) and adenosine 5'-[gamma-thio]triphosphate (a P-type ATPase inhibitor) were without effect. 2-Methylthioadenosine 5'-triphosphate, a P2y purinoceptor agonist, mimicked the stimulation by ATP. As judged from a kinetic comparison, ATP hydrolysis and the stimulation by ATP of Ca2+ uptake were not compatible. The P2 purinoceptor antagonist suramin and the P2y purinoceptor antagonist Reactive Blue-2 inhibited the Ca2+ uptake stimulated by 1 mM ATP (IC50 0.17 mM and 58 microM respectively). We conclude that ATP-stimulated Ca2+ uptake in tilapia intestine is dissociated from ATPase activity and is mediated through a P2 purinoceptor.

An electrophysiological study of the mechanism of fatigue in multiple sclerosis.

Fatigue is a common and disabling symptom in multiple sclerosis but is poorly understood. We investigated 'physiological' fatigue in 21 patients with multiple sclerosis who complained of disabling fatigue by measuring the decline in strength during a 45 s maximal contraction of the adductor pollicis muscle. The results were compared with those from a control group of 19 healthy subjects. The strength of control subjects declined by approximately 20% during the contraction; twitch interpolation showed central drive remained almost maximal throughout, and therefore that the fatigue was peripheral in origin. Patients had normal baseline strength, but developed greater fatigue (approximately 45%), which was central in origin. In both cases, the decline in strength followed a roughly linear time course suggesting that the patients, like the normals, were trying to maintain a maximum voluntary effort. Evidence for frequency-dependent conduction block (FDCB) in the patients' central motor pathways was sought by measuring the EMG responses to single and paired transcranial magnetic stimuli. Fatigue had no effect on the latency or size of EMG responses to transcranial magnetic stimulation, suggesting that FDCB was unlikely to have occurred. This was supported by measurements of the maximum speed of voluntary muscle contraction; although the patients showed relatively slow speeds before exercise, the decline in speed after fatigue was no greater than in normal subjects. We conclude that excessive 'physiological' fatigue contributes to the symptom of fatigue in multiple sclerosis and is central in origin. However, since the degree of exercise-induced fatigue did not correlate with the baseline complaint of fatigue, other factors must also be operating to produce the full range of clinical symptoms. We found no conclusive evidence that central fatigue is related to increased dysfunction in the primary central motor pathways and no evidence that FDCB is the pathophysiological mechanism. We postulate that central fatigue in multiple sclerosis is due to impaired drive to the primary motor cortex and several lines of evidence strongly suggest that this is not due to a lack of motivation.

Application of magnetic resonance neurography in the evaluation of patients with peripheral nerve pathology.

Currently, diagnosis and management of disorders involving nerves are generally undertaken without images of the nerves themselves. The authors evaluated whether direct nerve images obtained using the new technique of magnetic resonance (MR) neurography could be used to make clinically important diagnostic distinctions that cannot be readily accomplished using existing methods. The authors obtained T2-weighted fast spin-echo fat-suppressed (chemical shift selection or inversion recovery) and T1-weighted images with planes parallel or transverse to the long axis of nerves using standard or phased-array coils in healthy volunteers and referred patients in 242 sessions. Longitudinal and cross-sectional fascicular images readily distinguished perineural from intraneural masses, thus predicting both resectability and requirement for intraoperative electrophysiological monitoring. Fascicle pattern and longitudinal anatomy firmly identified nerves and thus improved the safety of image-guided procedures. In severe trauma, MR neurography identified nerve discontinuity at the fascicular level preoperatively, thus verifying the need for surgical repair. Direct images readily demonstrated increased diameter in injured nerves and showed the linear extent and time course of image hyperintensity associated with nerve injury. These findings confirm and precisely localize focal nerve compressions, thus avoiding some exploratory surgery and allowing for smaller targeted exposures when surgery is indicated. Direct nerve imaging can demonstrate nerve continuity, distinguish intraneural from perineural masses, and localize nerve compressions prior to surgical exploration. Magnetic resonance neurography can add clinically useful diagnostic information in many situations in which physical examinations, electrodiagnostic tests, and existing image techniques are inconclusive.

Electroresponsive properties and membrane potential trajectories of three types of inspiratory neurons in the newborn mouse brain stem in vitro.

1. The electrophysiological properties of inspiratory neurons were studied in a rhythmically active thick-slice preparation of the newborn mouse brain stem maintained in vitro. Whole cell patch recordings were performed from 60 inspiratory neurons within the rostral ventrolateral part of the slice with the aim of extending the classification of inspiratory neurons to include analysis of active membrane properties. 2. The slice generated a regular rhythmic motor output recorded as burst of action potentials on a XII nerve root with a peak to peak time of 11.5 +/- 3.4 s and a duration of 483 +/- 54 ms (means +/- SD, n = 50). Based on the electroresponsive properties and membrane potential trajectories throughout the respiratory cycle, three types of inspiratory neurons could be distinguished. 3. Type-1 neurons were spiking in the interval between the inspiratory potentials (n = 9) or silent with a resting membrane potential of -48.6 +/- 10.1 mV and an input resistance of 306 +/- 130 M omega (n = 15). The spike activity between the inspiratory potentials was burst-like with spikes riding on top of an underlying depolarization (n = 11) or regular with no evidence of bursting (n = 12). Hyperpolarization of the neurons below threshold for spike initiation did not reveal any underlying phasic synaptic activity, that could explain the bursting behavior. 4. Type-1 neurons showed delayed excitation after hyperpolarizing square current pulses or when the neurons were depolarized from a hyperpolarized level. This membrane behavior resembles the response seen in other CNS neurons expressing an IA. The response to 1-s long depolarizing pulses with a large current strength showed signs of activation of an active depolarizing membrane response leading to a transient reduction in the spike amplitude. The relationship between the membrane potential and the amplitude of square current pulses (Vm-I) showed a small upward rectification below -70 mV, and spike adaptation throughout a 1-s pulse had a largely linear time course. 5. Type-1 neurons depolarized and started to fire spikes 398 +/- 102 ms (n = 20) before the upstroke of the integrated XII nerve discharge. The inspiratory potential was followed by fast hyperpolarization, a short fast-repolarizing phase (1,040 +/- 102 ms, n = 5) and a longer slow-repolarizing phase (lasting until the next inspiratory discharge). 6. Type-2 neurons were spiking in the interval between the inspiratory potentials with no evidence of bursting behavior and had an input resistance of 296 +/- 212 M omega (n = 26). The response to hyperpolarizing pulses revealed an initial sag and postinhibitory rebound depolarization. This membrane behavior resembles the response seen in other CNS neurons expressing an Ih. The Vm-I relationship was linear at depolarized potentials and showed a marked upward rectification below -60 mV. Spike trains elicited by 1-s long pulses showed a pronounced early and late adaptation. 7. Type-2 neurons depolarized and started to fire spikes 171 +/- 87 ms (n = 23) before the upstroke of the integrated XII nerve discharge. The inspiratory potential had a variable amplitude from cell to cell and was followed by a short hyperpolarization in the cells displaying a large amplitude inspiratory potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Microbial formation of dimethyl sulfide in anoxic sphagnum peat

Peat bogs dominated by Sphagnum spp. have relatively high areal rates of dimethyl sulfide (DMS) emission to the atmosphere. DMS was produced in anoxic slurries of Sphagnum peat with a linear time course and with an average rate of 40.4 (range, 22.0 to 68.6) nmol per liter of slurry (middot) day(sup-1) observed in nine batches of slurry. Methanethiol (MeSH) was produced at roughly similar rates over the typical 4- to 8-day incubations. DMS and MeSH production in these acidic (pH 4.2 to 4.6) peats were biological, as they were stopped completely by autoclaving and inhibited strongly by addition of antibiotics and 500 (mu)M chloroform. Endogenous DMS production may be due to the degradation of S-methyl-methionine, dimethyl sulfoxide, or methoxyaromatic compounds (e.g., syringic acid), each of which stimulated DMS formation when added at 5 to 10 (mu)M concentrations. However, on the basis of the high rates of thiol (MeSH and ethanethiol) methylation activity that we observed and the availability of endogenous MeSH, we suggest that methylation of MeSH is the major pathway leading to DMS formation in anaerobic peat. Solid-phase adsorption of MeSH plays a key role in its availability for biomethylation reactions. Additions of acetate (1.5 mM) or compounds which could cause acetate to accumulate (e.g., glucose, alanine, and 2-bromoethanesulfonate) suppressed DMS formation. It is likely that acetogenic bacteria are involved in DMS formation, but our data are insufficient to allow firm conclusions about the metabolic pathways or organisms involved. Our observations are the first which point to the methylation of MeSH as the major mechanism for endogenous DMS production in any environment. The rates of net DMS production observed are sufficient to explain the relatively high fluxes of DMS emitted to the atmosphere from Sphagnum sp.-dominated wetlands.

Phosphatidylinositol transfer protein dictates the rate of inositol trisphosphate production by promoting the synthesis of PIP2.

Phosphatidylinositol transfer protein (PI-TP), which has the ability to transfer phosphatidylinositol (PI) from one membrane compartment to another, is required in the inositol lipid signalling pathway through phospholipase C-beta (PLC-beta) that is regulated by GTP-binding protein(s) in response to extracellular signals. Here, we test the hypothesis that the principal role of PI-TP is to couple sites of lipid hydrolysis to sites of synthesis, and so to replenish depleted substrate for PLC-beta. We have designed an experimental protocol that takes advantage of the different rates of release of endogenous PI-TP and PLC-beta from HL60 cells permeabilized with streptolysin O. We have examined the kinetics of stimulated inositol lipid hydrolysis in cells depleted of PI-TP, but not of endogenous PLC-beta, in the presence and absence of exogenous PI-TP. Linear time-courses were observed in the absence of any added protein, and the rate was accelerated by PI-TP using either guanosine 5'[gamma-thio]-triphosphate (GTP gamma S) or the receptor-directed agonist fMetLeuPhe as activators. In addition, depletion from the cells of both PI-TP and PLC-beta isoforms by extended permeabilization (40 minutes) allowed us to control the levels of PLC-beta present in the cells. Once again, PI-TP increased the rates of reactions. To identify whether the role of PI-TP was to make available the substrate phosphatidylinositol bisphosphate (PIP2) for the PLC, we examined the synthesis of PIP2 in cells depleted of PI-TP. We found that PI-TP was essential for the synthesis of PIP2. The predicted function of PI-TP in inositol lipid signalling is the provision of substrate for PLC-beta from intracellular sites where PI is synthesized. We propose that PI-TP is in fact a co-factor in inositol lipid signalling and acts by interacting with the inositol lipid kinases. We hypothesize that the preferred substrate for PLC-beta is not the lipid that is resident in the membrane but that provided through PI-TP.

Biosorption of chromium to fungi

Eighteen fungal strains were isolated from water and soil samples and tested for their ability to enrich chromium. The microorganism with the highest enrichment capacity, a zygomycete (Mucor hiemalis MP/92/3/4), was chosen for detailed investigations. Some basic tests such as the pH-dependence, the kinetics of the enrichment and the metal selectivity were carried out with the two most frequent oxidation states of chromium, the trivalent cation (Cr3+) and the hexavalent anion (CrO42−). With Cr3+ the enrichment showed a saturation kinetic reaching 70% of the maximum capacity after about 30 min, whereas with CrO42− a linear time course with a much lower metal enrichment was observed. The highest level of enrichment for Cr3+ was observed at pH 5.5 (21.4 mg/g dry wt), and for CrO42− at pH 1 (4.3 mg/g dry wt). Investigations concerning the metal enrichment selectivity resulted in the following series of decreasing ion uptake: Cr3+ > Cu2+ > Pb2+ > Ag+ > Al3+ > Co2+ > Zn2+ > Ni2+ > Fe2+ > Mo5+ > Cd2+ > 2− > CrO42− > VO3−, calculated on a molar basis. Trivalent chromium caused a staining of the outer cell wall region in transmission electron microscopy. The localization of chromium in the stained outer layers of the cell wall could be verified by electron energy loss spectroscopy. The enrichment of Cr3+ by M. hiemalis seemed to be mainly a passive biosorption to the cell wall, whereas for the uptake of CrO42− intracellular accumulation as well as biosorption is possible.

Autoprocessing of the protease that degrades small, acid-soluble proteins of spores of Bacillus species is triggered by low pH, dehydration, and dipicolinic acid.

The sequence-specific protease (termed GPR) that degrades small, acid-soluble proteins (SASP) during germination of spores of Bacillus species is synthesized during sporulation as an inactive precursor termed P46. Approximately 2 h later in sporulation, P46 is converted proteolytically to a smaller form, termed P41, which is active in vitro, but which does not act significantly on SASP in vivo until spore germination is initiated. While it appears likely that P46-->P41 conversion is an autoprocessing event, the mechanisms regulating P46-->P41 conversion in vivo are not clear. In this work we found that P46-->P41 conversion in vitro was stimulated tremendously in an allosteric manner by pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) plus Ca2+ but not by Ca2+ in combination with a variety of DPA analogs. The processing reaction stimulated by Ca(2+)-DPA was seen at pH 5.1 but not at pH 6.2 or 7, and under these conditions P46-->P41 conversion exhibited a linear time course and was a first-order reaction, indicative of an intramolecular autoprocessing reaction. At pH 5.1, P46-->P41 conversion was stimulated markedly by very high ionic strength. At pHs from 5.1 to 6.6, P46-->P41 conversion also occurred when P46 was dehydrated to approximately 54% relative humidity. This processing was stimulated markedly when dehydration was carried out in the presence of DPA and NaCl but was greatly decreased when dehydration was to 10, 33, or 75% relative humidity. Since previous work has shown that P(46)-->P(41) processing in vivo takes place (i) after a fall in forespore pH to 6.3 to 6.9 and approximately in parallel with (ii) DPA accumulation by the forespore and (iii) dehydration of the forespore, out new finings in vitro suggest that these three changes may synergistically trigger P(46)-->P(41) autoprocessing in the developing forespore. Presumably the conditions in vivo during this authoprocessing preclude significant attack of the P(41) generated on its SASP substrates.

Kinetic microphotometric evaluation of in situ hybridization for mRNA of slow myosin heavy chain in type I and C fibres of rabbit muscle

The present study was undertaken in order to test the possibility of microphotometric evaluation of in situ hybridizations. The histochemical detection of mRNA specific to the slow myosin heavy chain (HCI), in fibre cross sections of normal and transforming rabbit muscles with a digoxigenin-labelled complementary RNA (cRNA) probe was used as a model. Scanning densitometry of Northern blot hybridizations showed that the detection of cRNA/mRNA hybrids by a staining reaction catalysed by alkaline phosphatase coupled to an anti-digoxigenin antibody occurs in a concentration-dependent manner and follows a linear time course. These findings were the basis for elaborating a comparative microphotometric evaluation of in situ hybridization in tissue sections by measuring the reaction rate of the alkaline phosphatase-catalysed formazan production. Relative amounts of HCI mRNA were thus determined by comparing reaction rates instead of by single point microphotometry. This method was applied to studies on the distribution of HCI mRNA in different fibre types of normal rabbit muscles and and muscles undergoing fast-to-slow fibre transformation in response to low-frequency stimulation. The different fibre types were identified by histochemical staining for myofibrillar actomyosin ATPase (mATPase) in cross sections adjacent to the sections processed for in situ hybridization. On the average, type I fibres displayed 2.3-fold higher reaction rates than the mean value recorded for C fibres. According to the pronounced scattering of the values measured in single C fibres, these fibres represented a heterogeneous population in the transforming muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of ATP hydrolysis during the DNA helicase II-promoted unwinding of duplex DNA

The ATP hydrolysis activity of DNA helicase II from Escherichia coli was examined in the presence of linear single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA). In the presence of ssDNA, the ATP hydrolysis reaction followed a linear time course until the ATP was depleted. In the presence of dsDNA, in contrast, there was a kinetic lag before a linear phase of ATP hydrolysis was achieved. The nonlinear kinetics of the dsDNA-dependent ATP hydrolysis reaction could be modeled by a kinetic scheme in which helicase II undergoes a time-dependent transition from an ATPase-inactive to an ATPase-active form. Order of addition experiments indicated that this transition was not due to a rate-limiting association event between helicase II and any other component of the reaction. Instead, agarose gel assays showed that progressive unwinding of the dsDNA occurs during the same time period as the lag phase of the ATP hydrolysis reaction. No significant ATP hydrolysis was observed when the linear dsDNA was replaced with closed circular dsDNA, suggesting that the ATP hydrolysis reaction requires a dsDNA substrate that can be unwound to the complementary single strands. These results are consistent with a model in which the lag phase of the dsDNA-dependent ATP hydrolysis reaction corresponds to progressive unwinding of the dsDNA, with the ATP hydrolysis reaction arising from helicase II molecules that are bound to the separated single strands.

Fluid-phase endocytosis by rainbow trout head-kidney macrophages

Fluid-phase endocytosis in rainbow trout macrophages was studied using 125 I-poly (vinylpyrrolidone) (125I-PVP) as a marker. Uptake of 125I-PVP was measurable at 12° C using a high cell concentration (30-50 × 106 cell ml-1) and after extensive washing of the cells by centrifugation. The amount of extracellular fluid taken up after 30 min incubation with labelled PVP was estimated as 3.6±2.2 nl/106 cells, corresponding to a fluid uptake of approximately 1·4% of the cell volume per hour. Uptake of 125 I-PVP was temperature dependent and was not saturable after coincubation with excess unlabelled PVP. Fluid-phase endocytosis of labelled PVP did not follow a linear time course. This seemed to be caused by exocytosis of internalised 125I-PVP to the medium.

The effect of picrylsulphonic acid on In vitro conversion of cyanide to thiocyanate by 3-mercaptopyruvate sulphurtransferase and rhodanese

This study indicates that 3-mercaptopyruvate sulphurtransferase (MPST; EC 2.8.1.2) activity may serve as a useful in vitro indicator for the analysis of cyanide detoxification to thiocyanate. The time course and capacity of MPST to detoxify cyanide was equal to or exceeded that of rhodanese. Picrylsulphonic acid strongly inhibited purified rhodanese, but in the presence of mercaptopyruvate, it could increase the formation of thiocyanate catalysed by MPST. Formation of thiocyanate by MPST followed a linear time course and had a linear relation to enzyme level. However, substrate dependence did not produce linear Lineweaver-Burk plots when either mercaptopyruvate or cyanide concentration was varied. The differential effect of picrylsulphonic acid on the activities of these two enzymes was confirmed by using a crude kidney extract as the source of both enzymes. Picrylsulphonic acid may provide a useful scientific tool to examine which sulphurtransferase is most responsible for the detoxification of cyanide.

Ｄ‐酵素とホスホリラーゼのサブサイト親和力評価 サブサイト理論の２基質反応への適用

Disproportionating enzyme (D-enzyme, EC 2.4.1.25) is a transglycosylase and its reaction involves the participation of more than two molecules of a substrate; E+2×Gn→Gn-i+Gn+i. The HPLC analysis of digests of maltooligosaccharides (G3-G7) showed that maltose is not formed in any case. The products from all substrates except G4 are those resulting from maltosyl transfer as the predominant reaction. Glucan phosphorylase(EC 2.4.1.1) has a rapid equilibrium-random Bi Bi mechanism involving the two kinds of substrate; E+Gn+G1P→E+Gn-1+Pi. Purified G3 is of poor primer ability, and the time course of the reaction shows an accelerating curve. By incorporating a sufficient quantity of β-amylase in the digests, the true rates of the G3-primed reaction could be determined from the linear time courses to give the K4 value of 9.3 mM. Other kinetic parameters for a series of maltooligosaccharides (G4-G8) were also determined in both the synthetic and the phosphorolytic directions. The reaction mechanisms of both enzymes are more complicated than the hydrolytic reaction of amylases and do not obey the simple mechanism of Michaelis-Menten type. We attempted to apply the subsite theory to the two enzyme reactions to analyze the characteristics of their action patterns. The two enzymes were isolated from a β-amylase-deficient variety of sweet potato.

Chromophoric peptide substrates for the spectrophotometric assay of HIV-1 protease

Purified HIV-1 protease hydrolyzes H-Ser-Gln-Asn-Leu-Phe(NO 2)-Leu-Asp-Gly-NH 2 (Peptide 1) and acetyl-Arg-Lys-Ile-Leu-Phe(NO 2)-Leu-Asp-Gly-NH 2 (Peptide 2) between the ( p-nitro)phenylalanyl and leucyl residues. The cleavage of Peptides 1 and 2 resulted in a decrease in uv absorbance at 310 nm. The HIV-1 protease-catalyzed peptidolysis of Peptides 1 and 2 was characterized by a linear time course at substrate turnover of ≤20%. The solubilities of these substrates at pH 5.0 were sufficient to provide initial rate measurements over a concentration range of 0.05–0.5 mM. Steady-state kinetic data and inhibition constants using both spectrophotometric and high performance liquid chromatography (HPLC) analysis of the peptidolysis of these substrates resulted in comparable values.