Electron tomography of insect flight muscle in rigor and AMPPNP at 23 degrees C

Treatment of rigor fibers of insect flight muscle (IFM) with AMPPNP at 23 degrees C causes a 70% drop in tension with little change in stiffness. In order to visualize the changes in crossbridge conformation and distribution that give rise to the mechanical response, we have produced three-dimensional reconstructions by tomography of both rigor and AMPPNP-treated muscle that do not average the repeating motifs of crossbridges, and thereby retain information on variability of crossbridge structure and distribution. Tomograms can be averaged when display of only the regular features is wanted. Tomograms of rigor IFM show double-headed lead and single-headed rear crossbridges. Tomograms of IFM treated with AMPPNP at 23 degrees C reveal many double-headed and some single-headed "lead" bridges but few crossbridges corresponding to the rear bridges of rigor. Instead, new non-rigor forms of variably angled crossbridges are found bound to actin sites not labeled with myosin heads in rigor. This indicates that the rear bridges of rigor have redistributed during the transition from rigor to the AMPPNP state, which could explain the maintenance of rigor stiffness despite the loss of tension. Comparison of in situ crossbridges in tomograms of rigor with atomic model of acto-S1, the complex formed by myosin subfragment 1 and actin, reveals that the regulatory domain of S1 would require significant bending and realignment to fit into both types of rigor crossbridges. The modifications are particularly significant for the rear bridges and suggest that differential strain in the regulatory domain of rear bridges may be the basis for their detachment and redistribution upon binding AMPPNP. Similar comparison using lead-type crossbridges in AMPPNP reveals departures from the rigor acto-S1 atomic model that include azimuthal straightening and a slight M-ward bending in the regulatory domain. Both the motor and regulatory domains of the new non-rigor crossbridges differ from those in the atomic model of acto-S1. A new crossbridge motif identified in AMPPNP-treated muscle consists of paired rigor-like and non-rigor crossbridges and suggests possible transitions in the myosin working stroke.     

Effects of calcium and nucleotides on the structure of insect flight muscle thin filaments

Using the original McCulloch-Pitts notion of simple on and off spike coding in lieu of rate coding, an Anderson-Kohonen artificial neural network (ANN) associative memory model was ported to a neuronal network with Hodgkin-Huxley dynamics. In the ANN, the use of 0/1 (no-spike/spike) units introduced a cross-talk term that had to be compensated by introducing balanced feedforward inhibition. The resulting ANN showed good capacity and fair selectivity (rejection of unknown input vectors). Translation to the Hodgkin-Huxley model resulted in a network that was functional but not at all robust. Evaluation of the weaknesses of this network revealed that it functioned far better using spike timing, rather than spike occurrence, as the code. The algorithm requires a novel learning algorithm for feedforward inhibition that could be sought physiologically.     

Crystal structure of elastase-substrate complex at -- 55 degrees C

The structure of a specific acyl-enzyme intermediate in the elastase-catalysed hydrolysis of N-carbobenzoxy-L-alanyl-p-nitrophenol ester has been determined by X-ray diffraction at 3.5 A resolution. The acyl-enzyme was stabilised by cooling the crystal to --55 degrees C during substrate addition and data collection.     

Stability of thiopental sodium and propofol in polypropylene syringes at 23 and 4 degrees C

The stability of thiopental sodium and propofol in an admixture stored in polypropylene syringes at room temperature and under refrigeration was studied. Propofol injection 10 mg/ mL and thiopental sodium 25 mg/mL were mixed to final concentrations of 5 and 12.5 mg/mL, respectively. The admixture was put into 60-mL polypropylene syringes, and two syringes were stored at 23 degrees C and two at 4 degrees C. For solutions stored at 23 degrees C, samples were taken at 0, 4, 8, 24, 48, 72, 120, 168, 216, 240, and 264 hours, and for samples stored at 4 degrees C, samples were taken at 0, 4, 8, 24, 48, 72, 120, 168, 216, and 312 hours. Drug concentrations were determined by high-performance liquid chromatography. Thiopental sodium and propofol retained > 90% of their initial concentrations for up to 312 hours at 4 degrees C. At 23 degrees C, > 90% of the initial concentration was retained by propofol for up to 120 hours and by thiopental sodium for up to 240 hours. No visual changes or significant change in pH occurred in any sample. When mixed and stored in polypropylene syringes, propofol 5 mg/mL and thiopental sodium 12.5 mg/mL were stable for up to 312 hours at 4 degrees C and for up to 120 hours at 23 degrees C.     

Ontogeny of Na+/D-glucose cotransport in guinea-pig jejunal vesicles: only one system is involved at both 20 degrees C and 35 degrees C

The first three-dimensional structure of a DNA methyltransferase is presented. The crystal structure of the DNA (cytosine-5)-methyltransferase, M.HhaI (recognition sequence: GCGC), complexed with S-adenosyl-L-methionine has been determined and refined at 2.5 A resolution. The core of the structure is dominated by sequence motifs conserved among all DNA (cytosine-5)-methyltransferases, and these are responsible for cofactor binding and methyltransferase function.     

Effects of CO2-induced acidification on the fatigue resistance of single mouse muscle fibers at 28 degrees C

The role of reduced muscle pH in the development of skeletal muscle fatigue is unclear. This study investigated the effects of lowering skeletal muscle intracellular pH by exposure to 30% CO2 on the number of isometric tetani needed to induce significant fatigue. Isolated single mouse muscle fibers were stimulated repetitively at intervals of 4-2.5 s by using 80-Hz, 400-ms tetani at 28 degrees C in Tyrode solution bubbled with either 5 or 30% CO2. Stimulation continued until tetanic force had fallen to 40% of the initial value. Exposure to 30% CO2 caused a significant fall in intracellular pH of approximately 0.3 pH unit but did not cause any significant changes in initial peak tetanic force. During the course of repetitive stimulation, intracellular pH fell by approximately 0.3 pH unit in both normal and acidified fibers. The number of tetani needed to reduce force to 40% of the initial value was not significantly different in 5 and 30% CO2 Tyrode. The sole effect of acidosis was to reduce the rate of relaxation of force, especially in fatigued fibers. It is concluded that, at 28 degrees C, acidosis per se does not accelerate the development of fatigue during repeated tetanic stimulation of isolated mouse skeletal muscle fibers.     

Hydrolysis of CaHPO4 in sodium fluoride solutions at 37.4 degrees C

A possible chemical process occurring during caries reversal is conversion of acidic calcium phosphates to apatite. The role of fluoride in this process is of particular interest. The effects of fluoride on the rate of CaHPO4 hydrolysis at 37.4 degrees C were studied by a multimethod analysis involving X-ray diffraction, analyses of variations in solution chemistry, and observation of microstructural evolution. Hydrolysis in low NaF concentrations results in the formation of discrete fluorapatite crystals on the surfaces of the CaHPO4 crystallites. At CaHPO4/NaF molar ratios from approximately 9:1 to 10:2, fluorapatite formed in approximately 3 h as the only crystalline product and complete hydrolysis of CaHPO4 occurred; a pH value as low as 2.4 was attained with solution species being predominantly sodium phosphate. At NaF concentrations beyond those which result in pH minima, fluorapatite and CaF2 are the crystalline products. At 0.6 M NaF, pseudomorphs composed of fluorapatite and CaF2 crystals form without developing morphologies characteristic of individual fluorapatite and CaF2 crystals. CaHPO2 can hydrolyze completely to fluorapatite and CaF2 within a few hours depending on NaF concentration and liquid-to-solids ratio.     

Comparison of responses of men to immersion in circulating water at 40.0 and 41.5 degrees C

BACKGROUND: The recommended maximum water temperature for public hot tubs has been set at 40.0 degrees C, but no research has been published on human immersion in hot water at higher temperatures. HYPOTHESIS: We hypothesized that thermoregulatory and cardiovascular responses at two water temperatures would be proportional to the water:blood temperature gradients. METHODS: Six healthy men were immersed for 21 min in circulating hot water at 40.0 and 41.5 degrees C in separate trials in random order 1-3 wk apart. Measurements included heart rate, systolic BP, esophageal, rectal, and non-immersed skin temperatures, sweat rate, and perceived comfort. RESULTS: The rise in all body temperatures, sweat rate, and heart rate were significantly greater in the 41.5 vs. 40.0 degrees C water. Peak esophageal temperatures were 38.3 +/- 0.2 degrees C vs. 37.8 +/- 0.03 degrees C, peak sweat rates were 0.48 +/- 0.05 vs. 0.32 +/- 0.03 kg x m(-2) x h(-1), and peak heart rates were 123 +/- 7 vs. 108 +/- 5 bpm, respectively. Systolic BPs followed different patterns of response in each trial, whereas diastolic pressures were not different between trials. Comfort at each level of immersion was reduced during the 41.5 degrees C trial compared with the 40.0 degrees C in excess of that predicted by difference in esophageal temperature between the trials. CONCLUSIONS: These results suggest that risks of hyperthermia or adverse cardiovascular effects in hot tubs may not be greater in water above 40.0 degrees C unless perceptual judgment is impaired. Hypotension when standing to exit the tub occurred in both trials and may represent a potential hazard to hot tub use.     

Repair of extensive ionizing-radiation DNA damage at 95 degrees C in the hyperthermophilic archaeon Pyrococcus furiosus

We investigated the capacity of the hyperthermophile Pyrococcus furiosus for DNA repair by measuring survival at high levels of 60Co gamma-irradiation. The P. furiosus 2-Mb chromosome was fragmented into pieces ranging from 500 kb to shorter than 30 kb at a dose of 2,500 Gy and was fully restored upon incubation at 95 degrees C. We suggest that recombination repair could be an extremely active repair mechanism in P. furiosus and that it might be an important determinant of survival of hyperthermophiles at high temperatures.     

Serum concentrations of retinol, d-alpha-tocopherol and beta-carotene: effects of storage at -70 degrees C for five years

To investigate the effects of prolonged storage of serum samples at -70 degrees C on concentrations of micronutrients, we measured concentrations of retinol, d-alpha-tocopherol, and beta-carotene in serum samples drawn in 1986. We compared values we measured in 1991 to values we obtained in 1986, using the same analytical methods. The relative concentrations obtained in 1991 (mean +/- S.D.) were: retinol 99.7 +/- 12.6% (n = 23), d-alpha-tocopherol 100.7 +/- 6.4% (n = 19), and beta-carotene 103.4 +/- 13.7% (n = 28). Using these techniques of sample preparation and high-performance liquid chromatographic analysis, we found that the effects of storage of serum at -70 degrees C for five years appear insignificant in a small population of patients. However, we did identify clinically important changes in concentration (> 20% difference) in several individual subjects.     

An isothermal section at 550 °C in the Al-Rich corner of the Al-Fe-Mn-Si system

Phase equilibria in the Al corner of the Al-Fe-Mn-Si system at 550 °C have been explored. Twenty-six quaternary alloys were prepared and analyzed by microprobe analysis. Elemental powders were mixed under air and molten at 1000 °C under argon flux. These specimens received a heat treatment at 550 °C during 4 to 12 weeks and finally were water quenched. A four-phase region, where Al, Si,α-Al(Fe,Mn)Si, andβ-Al(Fe,Mn)Si are in equilibrium with each other, was found. The presence of a single-phaseα-Al(Fe,Mn)Si region, starting in the Al-Mn-Si subsystem and extending toward the Al-Fe-Si subsystem, could be confirmed. Some problems concerning the fit of the quaternary system with its ternary Al-Fe-Mn subsystem remain to be solved.     

The structure of cucurbitin: subunit symmetry and organization in situ

The low-resolution (2 nm) subunit symmetry of cucurbitin, the crystalline seed storage globulin of cucurbits, has been determined by X-ray diffraction. The wet crystals belong to the cubic space group F23 and there are 4 molecules per unit cell. The molecules therefore possess point-group symmetry 23 and contain 12 structural units which at this resolution are indistinguishable. On drying, the crystal lattice dimension shrinks from 13.6 nm to 12.4 nm with no apparent change in symmetry. Diffraction patterns of small crystals spun into a pellet, and sections of dry and wet native seed indicate that in situ the protein is organised in microcrystals of the same unit cell and symmetry. Edestin, the crystalline storage globulin from cannabis, and a crystalline globulin from tobacco seed both have the same crystal lattice as cucurbitin and, very likely, the same subunit symmetry.     

Identification of the high-virulence clone of group B streptococci in Mexican isolates by growth characteristics at 40 degrees C

Group B streptococci (GBS) colonizing the vagina and rectum of pregnant women cause invasive disease of the offspring in a small number of cases. The immune status of the host and differences in virulence among strains appear to be the main determinants for neonatal infection. A high-virulence clone (HVC) was proposed to cause much of the morbidity and mortality when a collection of GBS isolates was examined by multilocus enzyme electrophoresis. HVC isolates could be further distinguished by their inability to grow at 40 degrees C. This characteristic was used in the present study to examine a collection of 57 GBS isolates from Mexico City for the HVC. Three serotype III invasive strains were classified in the HVC. The other eleven invasive strains and all carrier isolates had growth curves unaffected at 40 degrees C. These results demonstrate the presence of the HVC in Mexico. Such a low prevalence could explain in part the low rate of GBS invasive neonatal disease in Mexico.