Effects of assisted hatching method and age on implantation rates of IVF and ICSI.

The objective of this study was to investigate whether a change in assisted hatching (AH) technique from total to partial penetration of the zona pellucida improved the outcome of IVF and intracytoplasmic sperm injection cycles where AH was indicated. This was an observational study conducted from the beginning of January 2000 to the end of April 2005. Total AH was performed in 312 cycles, while partial AH was performed in 592 cycles. In women of all ages, implantation, clinical pregnancy and live birth rates were higher in the partial AH group than in the total AH group (12.6 versus 7.2%, P = 0.0001; 22.3 versus 15.7%, P = 0.02; 18.2 versus 12.5%, P = 0.03 respectively). The benefit of partial AH was most marked in women under 38 years old (i.e. the recurrent implantation failure group). The authors conclude that partial AH is associated with higher implantation and pregnancy rates than total AH, especially in women under 38 years old who suffer from recurrent implantation failure.

Reduction of nitrogenase Fe protein from Azotobacter vinelandii by dithionite: quantitative and qualitative effects of nucleotides, temperature, pH and reaction buffer.

Oxidized Fe protein from Azotobacter vinelandii (Av2(0)) was reduced by dithionite (DT) in the absence and presence of nucleotides, over the temperature range 10-40 degrees C, over the pH range 7-8, and in various buffers--inorganic phosphate, TES, HEPES, and Tris. The reduction of each species of Fe protein--Av2(0), Av2(0)(MgATP)2, and Av2(0)(MgADP)2--was resolved into at least three exponential phases, with relative amplitudes of each phase varying over the range of experimental conditions, suggesting a dynamic population shift of kinetically distinct species. The rapid phase of Av2(0) reduction predominated at low temperature and pH, and in Tris buffer; rapid Av2(0)(MgATP)2 reduction was favored at high temperature and pH, and in phosphate buffer; and Av2(0)(MgADP)2 reduction was favored under more physiologically relevant conditions of 20 degrees C, pH 7.5, and in phosphate buffer. The rates of reduction of Fe protein species did not change with buffer, but temperature and pH do have an effect on the rates. With the appropriate constants, an empirically derived equation estimates the rate of Fe protein reduction at any temperature and pH within the limits 10-40 degrees C and pH 7-8, for a given species of Fe protein, and a given phase of the reaction. At 23.0 degrees C and pH 7.4, the rate of the dominant phase of Av2(0) reduction is 1.9 x 10(8) M(-1) s(-1). Under the same conditions, the rates of the two dominant phases of Av2(0)(MgATP)2 reduction are 1.2 x 10(6) and 1.5 x10 (5) M(-1) s(-1); and the rate of the dominant phase of Av2(0)(MgADP)2 reduction is 3.5 x 10(6) in M(-1) s(-1). Thermodynamic activation parameters for each phase of reduction were calculated. No breaks in the Arrhenius plots for any Fe protein species were observed.

Prenatal cocaine exposure alters potassium-evoked dopamine release dynamics in rat striatum.

The emerging profile for the effects of prenatal cocaine exposure presents two prominent features in the exposed offspring: cognitive/attention deficits and an age-associated trend toward motor/tone abnormalities up to 2 years of age. One candidate mechanism underlying these clinical features is long-lasting alterations to dopamine (DA) neuron function. However, the impact of prenatal cocaine exposure on DA release in dopaminergic terminal fields in vivo in mature offspring is poorly understood. Long-Evans female rats were implanted with an i.v. access port, bred, and given saline or cocaine-HCl (3 mg/kg/ml) for gestational days (GD) 8-14 (1x/day), GD 15-21 (2x/day), or GD 8-21 (1x/day-GD 8-14, 2x/day-GD 15-21). Using in vivo high-speed chronoamperometric recordings, potassium-stimulated DA release was measured in striatum of anesthetized male offspring 90-150 days after birth. There was a trend toward increased potassium-evoked DA signal amplitudes in offspring exposed to cocaine at any time period examined. In offspring exposed to cocaine during GD 8-21 and GD 15-21, but not at GD 8-14, there were significant decreases in the clearance capacity of the potassium-evoked DA signal compared with control offspring. The time required to clear 80% of the evoked DA signal (T(80)) in striatum for DA was significantly prolonged (approximately 150% of control) and this effect was further increased in the mean-evoked DA concentration range for these two groups. We also measured total dopamine transporter (DAT) and tyrosine hydroxylase protein levels in these offspring by blot immunolabeling and found a small, but significant, decrease in DAT protein in striatum from offspring exposed at GD 8-21 and GD 15-21. Collectively, these data demonstrate that prenatal cocaine exposure during dopamine neuron neurogenesis has long-lasting effects on DA neuron function lasting into early adulthood which may be related in part to steady state DAT protein levels. These molecular events may be associated with established cognitive deficits and perhaps the trends seen in altered motor behavior.

Duplication and extension of the Thorneley and Lowe kinetic model for Klebsiella pneumoniae nitrogenase catalysis using a MATHEMATICA software platform.

The Thorneley and Lowe kinetic model for nitrogenase catalysis was developed in the early to mid 1980s, and has been of value in accounting for many aspects of nitrogenase catalysis. It has also been of value by providing a model for predicting new catalytic behavior. Since its original publication, new results have been obtained and have been successfully incorporated into the model. However, the computer program used for nitrogenase simulations has not been generally available. Using kinetic schemes and assumptions previously outlined by Thorneley and Lowe, we report attempts to duplicate the original T&L kinetic simulation for Klebsiella pneumoniae nitrogenase catalysis using an updated simulation based on the MATHEMATICA programming format, which makes it more user-friendly and more readily available. Comparisons of our simulations with the original T&L simulations are generally in agreement, but in some cases serious discrepancy is observed. Possible reasons for the differences are discussed. In addition to duplicating the original T&L model, we report effects of updating it by including information that has come to light subsequent to its original publication.

Analysis of steady state Fe and MoFe protein interactions during nitrogenase catalysis.

Steady state kinetic measurements are reported for nitrogenase from Azotobacter vinelandii (Av) and Clostridium pasteurianum (Cp) under a variety of conditions, using dithionite as reductant. The specific activities of Av1 and Cp1 are determined as functions of Av2:Av1 and Cp2:Cp1, respectively, at component protein ratios from 0.4 to 50, and conform to a simple hyperbolic rate law for the interaction of Av2 with Av1 and Cp2 with Cp1. The specific activities of Av2 and Cp2 are also measured as a function of increasing Av1 and Cp1 concentrations, producing 'MoFe inhibition' at large MoFe:Fe ratios. When the rate of product formation under MoFe inhibited conditions is re-plotted as increasing Av2:Av1 or Cp2:Cp1 ratios, sigmoidal kinetic behavior is observed, suggesting that the rate constants in the Thorneley and Lowe (T&L) model are more dependent upon the oxidation level of MoFe protein than previously suspected [R.N.F. Thorneley, D.J. Lowe, Biochem. J. 224 (1984) 887-894], at least when applied to Av and Cp. Calculation of Hill coefficients gave values of 1.7-1.9, suggesting a highly cooperative Fe-MoFe protein interaction in both Av and Cp nitrogenase catalysis. The T&L model lacks analytical solutions [R.N.F. Thorneley, D.J. Lowe, Biochem. J. 215 (1983) 393-404], so the ease of its application to experimental data is limited. To facilitate the study of steady state kinetic data for H(2) evolution, analytical equations are derived from a different mechanism for nitrogenase activity, similar to that of Bergersen and Turner [Biochem. J. 131 (1973) 61-75]. This alternative cooperative model assumes that two Fe proteins interact with one MoFe protein active site. The derived rate laws for this mechanism were fitted to the observed sigmoidal behavior for low Fe:MoFe ratios (<0.4), as well as to the commonly observed hyperbolic behavior for high values of Fe:MoFe for both Av and Cp.

Mechanistic interpretation of the dilution effect for Azotobacter vinelandii and Clostridium pasteurianum nitrogenase catalysis.

Nitrogenase activity for Clostridium pasteurianum (Cp) at a Cp2:Cp1 ratio of 1.0 and Azotobacter vinelandii (Av) at Av2:Av1 protein ratios (R) of 1, 4 and 10 is determined as a function of increasing MoFe protein concentration from 0.01 to 5 microM. The rates of ethylene and hydrogen evolution for these ratios and concentrations were measured to determine the effect of extreme dilution on nitrogenase activity. The experimental results show three distinct types of kinetic behavior: (1) a finite intercept along the concentration axis (approximately 0.05 microM MoFe); (2) a non-linear increase in the rate of product formation with increasing protein concentration (approximately 0.2 microM MoFe) and (3) a limiting linear rate of product formation at high protein concentrations (>0.4 microM MoFe). The data are fitted using the following rate equation derived from a mechanism for which two Fe proteins interact cooperatively with a single half of the MoFe protein. (see equation) The equation predicts that the cubic dependence in MoFe protein gives rise to the non-linear rate of product formation (the dilution effect) at very low MoFe protein concentrations. The equation also predicts that the rate will vary linearly at high MoFe protein concentrations with increasing MoFe protein concentration. That these limiting predictions are in accord with the experimental results suggests that either two Fe proteins interact cooperatively with a single half of the MoFe protein, or that the rate constants in the Thorneley and Lowe model are more dependent upon the redox state of MoFe protein than previously suspected [R.N. Thornley and D. J. Lowe, Biochem. J. 224 (1984) 887-894]. Previous Klebsiella pneumoniae and Azotobacter chroococcum dilution results were reanalyzed using the above equation. Results from all of these nitrogenases are consistent and suggest that cooperativity is a fundamental kinetic aspect of nitrogenase catalysis.

Hospice volunteer training: making the experience more meaningful.

Each volunteer coordinator continues to make changes and adjustments in the hospice training classes--some changes arise from circumstances such as staff availability, and some are conscious choices to try new ways of structuring the learning environment. As we shape and refine the experience that we provide for our new volunteers, let us make a special effort to create one that offers not only a comprehensive overview of hospice goals, philosophy, and end-of-life issues, but also a personally challenging and enriching team interaction.

Coagulation monitor based on serum migration through absorbent materials.

I describe a method to measure blood coagulation properties, based on the hypothesis that the distance the serum component of a clotted plasma sample moves through a suitable absorbent material should be proportional to the blood's ability to clot. A simple apparatus was constructed to test this principle, in which an absorbent strip contacts clotted plasma samples. At various times, the liquid migration distance into the strip was measured and correlated with clotting times determined by well-accepted procedures. Use of this device to test lyophilized normal and barium-absorbed plasmas, factor VII-deficient plasma, frozen normal plasmas, plasmas from patients undergoing oral anticoagulation, and saline-diluted plasmas (as for Quick Percent assays) showed that clotting times correlated with migration distances for all types of samples (r2 = 0.93-0.99). The device distinguishes between samples from normal and coumadin-treated subjects. The concept can be integrated into an inexpensive, point-of-care coagulation monitor.

Galvanic reactions between cerclage wires and Kuntscher nails.

Four patients are presented who had closed femoral shaft fractures and developed either extensive callus or osteomyelitis following open Kuntscher nailing and cerclage wiring of a butterfly fragment. Both the Kuntscher nail and cerclage wire are made of stainless steel but with different percentages of constituent elements. We looked at the galvanic reactions between the two and showed potential differences of up to 4.5 m.v. in wound drainage fluid. Potential differences in this range have been reported in other studies to be associated with extensive callus and infections associated with stainless steel anode corrosion.

Pediatric wheelchair athletics: sports injuries and prevention.

Two hundred and forty-seven competitors at the 1990 Junior National Wheelchair Games (USA) were surveyed for general information, training techniques, and injury patterns. Eighty-three athletes (34%) responded to the retrospective survey. Injuries were reported by 97% of those participating in track, 22% of the field participants, and 91% of the swimming competitors. A wide variety of injuries was reported ranging from minor bruises, blisters and abrasions to more serious problems (bladder infections, hyperthermia, and soft tissue injuries). While information-reporting biases may be inherent in this type of study, these data are significant in that they are the first obtained for this specific pediatric population via a systemic scientific approach and not from merely anecdotal information.

Proliferation of the surface-connected intracytoplasmic membranous network in skeletal muscle disease.

A surface-connected intracytoplasmic membranous (SCIM) network proliferates in skeletal muscle diseases and in myotubes grown in vitro. The authors observed frequent occurrence of "coated" microdomains in the form of budding vesicles in the proliferated components of this network and suspected a potential role the proliferated membranes might have in the endocytosis of molecules into myotubes undergoing repair or regeneration. Five-day-old myotubes in culture were incubated at 37 C and between 2 and 4 C with two tracers, Lucifer yellow and ferritin, both known to enter other types of cells via a fluid-phase endocytotic pathway. The differential penetration of Lucifer yellow at 37 C and below 2-4 C was examined by fluorescence microscopy and by electron microscopy. Lucifer yellow was rendered electron-opaque by photoreacting it with an intense light in the presence of DAB. Ferritin penetration at 37 C and between 2 and 4 C was compared and quantitated ultrastructurally. The authors found that endocytosis of the tracers into myotubes and eventually into lysosomes took place after the tracers had diffused into the lumen of the proliferated SCIM network. These processes were inhibited below 4 C. This finding, coupled with the presence of "coated" microdomains in the proliferated membranes, led us to suspect that the SCIM network may have a role in membrane turnover of metabolically active diseased muscle cells undergoing regeneration.

Monoclonal antibody specific for the T-tubule of skeletal muscle.

Monoclonal antibodies were raised against a triad-enriched (sarcoplasmic reticulum-T-tubule complex) microsomal membrane fraction of rabbit skeletal muscle. The avidin-biotin complex (ABC) immunoperoxidase staining method was used to screen hybrid colonies. Positive antibodies exhibited a granular doublet pattern at the A-I junction, consistent with the location of triads in rabbit muscle. One monoclonal antibody, M171, was further characterized by ultrastructural and immunoadsorption techniques. Postembedding electron immunocytochemistry was performed on tissue sections embedded in Lowicryl K4M. Goat anti-mouse immunoglobulin absorbed to 10 nm colloidal gold particles was used as an ultrastructural label. In these studies, M171 recognized an epitope at the triads and at periodic openings along the plasmalemma. Immunoadsorption on protein transfers of isolated sarcoplasmic reticulum, surface membrane (plasmalemma and T-tubule), and triad-enriched fractions showed that M171 reacts with a surface membrane component. Taken together, these studies suggest that M171 recognizes an epitope associated with the T-tubule at the triad and at the "mouth" of the T-system at the plasmalemma.

Infections in a hospital for patients with diseases of the skin.

Studies on infections in a hospital for diseases of the skin are described. Patients were shown to acquire staphylococci in the groin and on the chest at about the same rate as in the nose. In contrast to surgical wards, many staphylococci were resistant to tetracycline but sensitive to penicillin. Nevertheless, much of the epidemic spread of staphylococci was with typical surgical-ward strains rather than with phage group II strains which might be thought typical of skin diseases.