Multipoint linkage analysis of the short arm of the human X chromosome in families with X-linked muscular dystrophy

Summary Sixteen three generation families from the West of Scotland with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) have been studied using the Xg blood group and seven cloned DNA sequences which recognise DNA polymorphisms on the short arm of the X chromosome (Xp). Linkage has been established between DMD and probe 754 with a maximum lod score () of 4.47 at a recombination fraction ( ) of 0.04. DMD has also been linked to probe 99-6 (=3.75, =0.03). Combining the data in this study with that of previously published work has established linkage between DMD and L1.28 (=4.42, =0.17) and altered the linkage estimate between BMD and L1.28 (=3.50, =0.22).An approximate order for the loci has been deduced by the study of recombinant chromosomes in phase known families informative for three or more loci. The proposed order is centromere-L1.28-754-DMD/BMD-99-6-D2-782-Xg. These results conclusively map both DMD and BMD to the central region of Xp and add weight to the original suggestion that they may be allelic.     

Estimation of surface charges in some biological membranes

Summary The resting membrane potential data existing in the literature for the giant axon of the squid, frog muscle and barnacle muscle have been analyzed from the standpoint of the theory of membrane potential due to Kobatake and co-workers. The average values derived for the effective charge density (where is a constant, , and represents the fraction of counterions that are free, and is the stoichiometric charge density in the membrane) present on the different biomembranes existing in their normal ionic environment are 0.3, 0.325 and 0.17 M for the squid axon, frog and barnacle muscles, respectively. On the assumption that the values of are 0.4 and 0.2 for nerve and muscle membranes, respectively, values of 0.75, 1.62 and 0.85 M have been derived for the stoichiometric charge density present in the respective biological membranes. These correspond to 1 negative charge per 222, 103 and 195 Å of the membrane area of the squid axon, frog and barnacle muscles, respectively.     

A method for determining the maximal steady state of blood lactate concentration from two levels of submaximal exercise

The aim of this study was to estimate the characteristic exercise intensity _CL which produces the maximal steady state of blood lactate concentration (MLSS) from submaximal intensities of 20 min carried out on the same day and separated by 40 min. Ten fit male adults [maximal oxygen uptake _max 62 (SD 7) ml · min^–1 · kg^–1] exercisOed for two 30-min periods on a cycle ergometer at 67% (test 1.1) and 82% of _max (test 1.2) separated by 40 min. They exercised 4 days later for 30 min at 82% of _max without prior exercise (test 2). Blood lactate was collected for determination of lactic acid concentration every 5 min and heart rate and O₂ uptake were measured every 30 s. There were no significant differences at the 5th, 10th, 15th, 20th, 25th, or 30th min between , lactacidaemia, and heart rate during tests 1.2 and 2. Moreover, we compared the exercise intensities _CL which produced the MLSS obtained during tests 1.1 and 1.2 or during tests 1.1 and 2 calculated from differential values of lactic acid blood concentration ([1a^–]_b) between the 30th and the 5th min or between the 20th and the 5th min. There was no significant difference between the different values of _CL [68 (SD 9), 71 (SD 7), 73 (SD 6),71 (SD 11) % of _max (ANOVA test,P<0.05). Four subjects ran for 60 min at their _CL determined from periods performed on the same day (test 1.1 and 1.2) and the difference between the [la^–]_b at 5 min and at 20 min ( ([la^–]_b)) was computed. The [la^–]_b remained constant during exercise and ranged from 2.2 to 6.7 mmol · l^–1 [mean value equal to 3.9 (SD 1) mmol · l^–1]. These data suggest that the _CL protocol did not overestimate the exercise intensity corresponding to the maximal fractional utilization of _max at MLSS. For half of the subjects the _CL was very close to the higher stage (82% of _max where an accumulation of lactate in the blood with time was observed. It can be hypothesized that _CL was very close to the real MLSS considering the level of accuracy of [la^–]_b measurement. This study showed that exercise at only two intensities, performed at 65% and 80% of _max and separated by 40 min of complete rest, can be used to determine the intensity yielding a steady state of [la^–1]_b near the real MLSS workload value.     

Higher-order structures of chromatin in solution.

Neutron scatter studies have been made on gently prepared chicken erythrocyte chromatin over a range of ionic strength. At low ionic strength the mass per unit length of the '10 nm nucleofilament corresponds to one nucleosome per 8--12 nm and a DNA packing ratio of between 6 and 9. From the contrast dependence of the cross-section radius of gyration of the nucleofilament the following parameters have been obtained; RgDNA' the cross-section radius of gyration (Rg) when DNA dominates the scatter; RgP, the cross-section Rg when protein dominates the scatter; Rc, the cross-section Rg at infinite contrast and alpha, the constant which describes the dependence of the cross-section Rg on contrast variation. From our understanding of the structure of the core particle, various arrangement of core particles in the nucleofilament have been tested. In models consistent with the above parameters the core particles are arranged edge-to-edge or with the faces of the core particles inclined to within 20 degrees to the axis of the nucleofilament. With increase of ionic strength the transition to the second-order chromatin structure has been followed. This gave the interesting result that above 20 microM NaCL or 0.4 mM MgCL2 the cross-section Rg increases abruptly to about 9 nm with a packing ratio of 0.2 nucleosome/mn and with further increase of ionic strength the Rg increases to 9.5 nm while the packing ratio increases threefold to 0.6 nucleosome/nm. This suggests a family of supercoils of nucleosomes which contract with increasing ionic strength. In its most contracted form the diameter of the hydrated supercoil has been found from the radial distribution function to be 34 nm. Models for the arrangements of core particles in the 34-nm supercoil are discussed.     

Free DNA stretches in histone H1-depleted chromatin and their possible relation to chromomere structure

Oligomers of chromatin subunits (oligonucleosomes) were prepared by a mild digestion of chromatin with staphylococcal nuclease followed by a purification of a high molecular weight material (hexanucleosomes and larger DNP particles) by gel chromatography. The main finding is that a mild removal of histone H1 from the oligonucleosome preparation by treatment with tRNA in the absence of any significant hydrodynamic shearing leads to the formation of free DNA molecules which constitute 5–6% of the total oligonucleosomal DNA.The size of nucleosome-free DNA stretches in H1-depleted hydrodynamically sheared chromatin is about 6000 base pairs and their content is apparently 10–12% of the total DNA. These and related findings are discussed in terms of the previously proposed asymmetric hairpin model of DNA packing in chromatin [1–4]. Different kinds of the asymmetric hairpin are considered and ambiguities in interpretations of experimental data are pointed out.     

A sensitive radioimmuno assay for thymine dimers

Summary A sensitive radioimmuno assay (RIA) method for detection of the UV photoproduct, thymine dimers has been developed. The limit of detection of this method is 6×10^-14 mol or 15 pg thymine dimer. It is highly specific: A structurally similar compound such as uridine dimer interferes with the detection of thymine dimers only when it is 53,000-fold or more in molar excess. Since this RIA method does not require the use of labeled DNA, it represents a considerable improvement for repair studies with radiation-sensitive cells.     

Relationship between the octanol-water partition coefficient of tertiary amines and their effect of ‘selective’ uncoupling of photophosphorylation

A series of tertiary amines was investigated for effects on the transmembrane proton potential difference ( H), on photophosphorylation and on electron-flux control related to the intrathylakoid proton potential ( H_I), using isolated chloroplasts ofSpinacia oleracea L. As indicated by 9-aminoacridine fluorescence and [14C]methylamine uptake, all amines studied inhibited a build-up of H and, in parallel, ATP synthesis. Even when H was low, strong H₁-dependent electron-flux control was observed under the influence of tertiary amines. The strength of flux control in the presence of low H and the effectiveness of inhibition of ATP synthesis linearly increased with the lipophilicity of the amines. The most effective of the amines tested caused 50% inhibition of ATP synthesis at a concentration of 6 M, which is about 1000-fold lower than the concentration required for inhibition by methylamine. The data presented indicate the existence of two proton domains in the thylakoid vesicles, one of them feeding the ATP-synthase, the other the sites of pH-dependent electron-flux control. It is concluded that tertiary amines develop their action in a lipophilic domain of the thylakoid membrane, in the vicinity of the ATP-synthase complex. A mechanism for selective uncoupling and for the maintenance of H_I-dependent electron flux control in the presence of low H is discussed.Abbreviations and symbols coefficient for pH-dependent electron flux control - 9-AA 9-aminoacridine - Chl chlorophyll - I₅₀ amine concentration producing 50% inhibition of ATP-synthesis - J_e flux of photosynthetic electron transport - k _H apparent rate constant for proton efflux - H₁ proton potential in the thylakoid lumen - H₁ transthylakoid proton potential difference - p partition coefficient - q _AA coefficient for 9-aminoacridine fluorescence quenching - PS photosystem - Q quantum flux of photosynthetically active light Dedicated to Professor Wilhelm Simonis, on the occasion of his 80th birthday     

Minimal Functional Unit of Lactate Dehydrogenase

The tetrameric heart isozyme of lactate dehydrogenase (H₄) is modified by p-chloromercuribenzoate (PCMB) to produce the inactive tetramer and then hybridized with native tetrameric muscle isozyme (M₄). The hybrid mixture was isolated by polyacrylamide gel electrophoresis (PAGE) and then stained for enzyme activity and with Coomassie brilliant blue. Only three bands were found on the gels in either case. The hybrid enzymes as isolated by PAGE have half the specific activity of the native muscle enzyme. The electrophoresis properties of HM₃ are very similar to those of HM₃, while the electrophoresis properties of are very similar to those of H₂M₂. The above results strongly suggest that the tetramer having enzymatic activity contains at least two native subunits, and the di-subunit in the tetrameric enzyme is the minimal functional unit.     

Cardiovascular responses to facial cooling during low and moderate intensity exercise

To investigate the hypothesis that facial cooling (FC) exerts a greater influence on the cardiovascular system at lower versus higher levels of exercise, this study examined the effect of facial cooling [mean (SE): 0 (2)°C at 0.8 m·s^–1 wind velocity] during 30 min low [35% maximum oxygen consumption ( O_2max)] and moderate (70% O_2max) levels of cycle ergometry in the supine position. Five male subjects were assigned in random order to four exercise conditions: (1) FC at 35% O_2max(FC35), (2) no cooling (NFC35), (3) FC at 70% O_2max(FC70), and (4) no cooling (NFC70). Heart rate (f _c), stroke volume (V _s), and cardiac output ( _c) were measured at rest and every 10 min of exercise using impedance cardiography. During FC35, the change in f _c [mean (SE)] was significantly lower (P < 0.05) than NFC35 at 10 [22 (5) vs 31 (3) beats· min^–1], 20 [29 (6) vs 35 (3) beats·min^–1], and 30 [29 (5) vs 38 (4) beats·min^–1] min. No differences in f _c were observed between FC70 and NFC70. Furthermore, FC had no effect on V _s or _cat either exercise intensity. However, when comparing the FC70 and NFC70 conditions, there was a significant main effect (P<0.05) in mean arterial pressure (P _a) response with cooling despite the fact that neither V _s or _cwere different from the NFC70 control. The increase (P < 0.05) in the estimated change in systemic vascular resistance ( _a· _c ^–1) could partly explain the relative rise in _aat FC70. No pressor effect of cooling was observed at 35% O_2max. The results suggest that the FC condition promotes exercise bradycardia at low levels of exercise and exerts a greater pressor response during moderate exercise.     

Breathing pattern and growth: comparative aspects

Summary The resting oxygen consumption and breathing pattern of nine newborn and adult species (ranging in body size from mouse to human) have been compared on the basis of data collected from the literature. Minute ventilation is similarly linked to at both ages, the percent of extracted as O₂ about 2.2. Tidal volume/kg is an interspecies constant in newborns and adults, approximately 8 ml/kg. Breathing frequency decreases with the increase in size in a different way at the two ages: large species have newborns breathing at rates 2–3 times above the corresponding adults' values, while in the small species newborns and adults breathe at almost the same rate. Therefore the newborns of the smallest species have both and below the expected values, implying a greater inability to cope with the external demands than newborns of larger species. Several considerations indicate that in the smallest newborns the mechanical properties of the respiratory system could be a constraint to resting ventilations larger than observed. It is therefore possible that their low is the cause, and not the effect, of the relatively small .     

Spondyloepiphyseal dysplasia tarda: linkage with genetic markers from the distal short arm of the X chromosome

Summary A linkage study of six families with spondyloepiphyseal dysplasia tarda (SEDL) has been performed. A linkage to site DXS41 ( =0.08; =3.07) and DXS92 ( =0.05; =2.95) has been established. We propose, that the SEDL locus lies on the distal part of the short arm of the X chromosome.     

Construction of population growth equations in the presence of viability constraints

A mathematical method based on the G-projection of differential inclusions is used to construct dynamical models of population biology. We suppose that the system under study, not being limited by resources, may be described by a control system

Optimal use of restriction enzymes in the analysis of human DNA polymorphism

Summary Numerous biological factors have an effect upon the probability of the presence of a theoretical restriction site at a given point, ( ), in a DNA molecule, where n represents the number of base pairs composing the site. Indeed, it is a fact that the ratio does not usually equal unity. In addition, the sequence of the nucleotides appears to change haphazardly, with certain dinucleotides present more often than others. When considering , it is observed that the influence of this first parameter is very important for sites with either four or six base pairs. The balance of the average frequency of each site according to the preponderance of certain dinucleotides and the G+C content has been the subject of an involved study which makes use of a certain number of restriction enzymes which are commercially available. The important frequency variations of different sites are due primarily to the presence of one or two GC doublets which go as far as reducing the frequency by a factor of 3.     

Structure of the chromatin with long linker DNA

The method of velocity sedimentation have been used to investigate ionic-strength-induced compaction of sea urchin sperm chromatin characterized by extremely long linker DNA (100 b.p.). The dependence of sedimentation coefficients of oligonucleosomes on the number of nucleosomes in the chain have been studied in the range of ionic strength from 0.005 to 0.085. Analysis of these data indicates that such structural parameters of sea urchin sperm chromatin fibre as the diameter of the chain and the length of the chain per nucleosome are quite similar to those of chromatin with shorter linker DNA, but the DNA packing ratio is higher. The structure of sea urchin sperm oligonucleosomes agrees well with the model of three-dimensional zig-zag-shaped chain with linker DNA forming a loop. The possible role of alpha-helical regions of the C-terminal domain of sea urchin sperm histone H1 in the long linker DNA folding is discussed.     

Effect of exposure to oxygen at 101 and 150 kPa on the cerebral circulation and oxygen supply in conscious rats

Hyperbaric oxygen at pressures of 300 to 500 kPa has been shown to induce changed distribution of cerebral blood flow ( _CBF) in rats, in places reducing the supply of the supplementary O₂. Thus, in the present study, the effect of hyperoxia at 101 (group 1, n = 9) and 150 (group 2, n = 9) kPa OZ on cerebral blood flow distribution and central haemodynamics was tested in conscious, habituated rats. During the control period the systolic arterial pressure (BP_s), heart rate (f _c), breathing frequency (f _b), cardiac output ( _c), arterial acid-base chemistry and glucose, as well as _CBF distribution (r _CBF) were similar in the two groups of animals. During O₂ exposure, the acid-base chemistry remained unchanged. The haemoglobin decreased in group 2, but remained unchanged in group 1. The f _c decreased rapidly in both groups during the change in gas composition, after which f _c remained constant both in group 1 and in group 2, for whom pressure was increased. The _c and f _b decreased and BP_s increased similarly in the two groups. Total _CBF and r _CBF decreased to the same extent in both groups, and the r _CBF changes were equally scattered. In group 1, breathing of pure O₂ did not increase the O₂ supply to any cerebral region except to the thalamus and colliculi after 60 min, whereas the O₂ supply to the hypothalamus decreased and remained low. In group 2, the O₂ supply was unchanged compared to the control period in all regions. These findings agree with previous observations during exposures to higher O₂ pressures. In air after O₂ exposure the acid-base chemistry remained normal. The f _c and f _b increased to higher levels than during the control period. The BP_s remained high. The brain blood flows were increased, inducing elevated O₂ supply to several brain regions compared to the control period. In conclusion, O₂ supply to the central nervous system was found to be in the main unchanged during breathing of O₂ at 101 kPa and 150 kPa.     

Quantitative analysis of chromatin structure by circular dichroism

Quantitative analysis of the circular dichroism of nucleohistones and protein-free DNA was carried out in order to determine the structure and the role of the linker region DNA in chromatin, in terms of the conformational change of chromatin as a function of the ionic strength. It is shown clearly that the circular dichroism of Hl-depleted chromatin isolated from calf thymus is determined only by the ratio of the core region to the linker region and demonstrated by the linear combination of the spectrum of protein-free DNA and that of the nucleosome core in 5 mm-Tris · HCl, 1 mm-EDTA (pH 7.8). The calculated spectrum for the linker region in the H1-depleted chromatin was in good agreement with that of protein-free DNA. From the difference spectra between nucleohistones and protein-free DNA, it is suggested that the chromatin has an additional winding of DNA other than 146 base-pairs of DNA around the histone core. By decreasing the ionic strength to values lower than 5 mm-Tris · HCl, 1 mm-EDTA, the ellipticity of H1-depleted chromatin increased greatly between 250 nm and 300 nm while the increase was small in the case of chromatin and the nucleosome core. Nucleosomes with linker region DNA but without histone H1 also show great increase in ellipticity in this range of wavelengths as the ionic strength is decreased. Therefore, the linker region in H1-depleted chromatin plays an important role in the conformational changes brought about by changes in the ionic strength, and the conformational changes caused in the DNA of chromatin by decreasing the ionic strength are suppressed by the presence of histone H1.     

Ionol (BHT) produces superoxide anion

In aqueous medium etiolated wheat seedlings release superoxide anion ( ). Interaction of a synthetic antioxidant, butylated hydroxytoluene (BHT, ionol), with oxygen in the aqueous medium is accompanied by formation. This suggests that under certain conditions BHT behaves as a prooxidant. A natural antioxidant, superoxide dismutase (SOD), and also a wound healing preparation, emulsified denatured placenta (EDP), do not exhibit the prooxidant properties. In contrast to BHT, they reduce production by the etiolated wheat seedling system.     

The generation of the proton electrochemical potential and its role in energy transduction

Summary The evidence that all energy transducing membranes can generate a proton electrochemical potential difference, _H, across the membrane and that this potential can be used to transfer energy among energy transducing units and to generate ATP, has increased the interest for the view that _H plays an obligatory role in energy transduction and ATP synthesis. In the present article we shall concentrate on two experimental questions related with the generation and role of _H: (a) the charge/site ratio; (b) the relation between the proton electrochemical potential on one side and the cation electrochemical potential, the phosphate potential and the redox potential on the other. We shall then discuss the view that energy transduction corresponds to a molecular energy machine rather than to a fuel cell.     

Genetic mapping of four DNA markers (DXS16, DXS43, DXS85, and DXS143) from the p22 region of the human X chromosome

Summary Linkage analysis of four polymorphic anonymous DNA markers from the Xp22 region was performed using families from the Centre d'Etude du Polymorphisme Humain. The loci DXS43 (pD2) and DXS16 (pXUT23) were found to be tightly linked ( = 0.02 at = 14.96) and proximal to both DXS85 (782) and DXS143 (dic56). Multipoint linkage analysis suggests the order: