Sperm volume regulation: maturational changes in fertile and infertile transgenic mice and association with kinematics and tail angulation

Laser light scatter analyzed by flow cytometry was used to monitor the volume of viable maturing murine spermatozoa. Upon release, dispersion, and dilution, epididymal sperm from fertile heterozygous c-ros knockout mice were smallest in the cauda region and largest in the corpus region. Cauda sperm from both infertile homozygous c-ros knockout and GPX5-Tag2 transgenic mice were abnormally large. When incubated, corpus and cauda sperm from normal mice became slightly enlarged and later returned to a smaller size. This suggests an immediate swelling due to high intracellular osmolality, which triggers a regulatory volume decrease (RVD) that results in a net volume reduction. Normal caput sperm increased in size continuously and became larger than the more mature sperm, indicating a lack of RVD. The ion-channel blocker quinine induced dose-dependent size increases in normal cauda sperm but not in caput sperm. Dose-dependent quinine action on mature sperm also included induction of tail angulation, and suppression of straight-line velocity and linearity. The kinematic effects were more sensitive, with a quicker onset, but they diminished with time in contrast to tail angulation, which intensified. These results suggest that kinematic changes are an early phenomenon of swelling, which gradually accumulates at the cytoplasmic droplet to cause flagellar angulation. Disruption of the epididymal maturation of sperm volume regulation capacity would hinder the transport of sperm in the female tract, and may thereby explain infertility under certain conditions, but may also provide a novel approach to male contraception.     

Role of the Na(+)-Ca(2+) exchanger as an alternative trigger of CICR in mammalian cardiac myocytes

Ca(2+) influx through the L-type Ca(2+) channels is the primary pathway for triggering the Ca(2+) release from the sarcoplasmic reticulum (SR). However, several observations have shown that Ca(2+) influx via the reverse mode of the Na(+)-Ca(2+) exchanger current (I(Na-Ca)) could also trigger the Ca(2+) release. The aim of the present study was to quantitate the role of this alternative pathway of Ca(2+) influx using a mathematical model. In our model 20% of the fast sodium channels and the Na(+)-Ca(2+) exchanger molecules are located in the restricted subspace between the sarcolemma and the SR where triggering of the calcium-induced calcium release (CICR) takes place. After determining the strengths of the alternative triggers with simulated voltage-clamps in varied membrane voltages and resting [Na](i) values, we studied the CICR in simulated action potentials, where fast sodium channel current contributes [Na](i) of the subspace. In low initial [Na](i) the Ca(2+) influx via the L-type Ca(2+) channels is the major trigger for Ca(2+) release from the SR, and the Ca(2+) influx via the reverse mode of the Na(+)-Ca(2+) exchanger cannot trigger the CICR. However, depending on the initial [Na](i), the contribution of the Ca(2+) entry via the exchanger may account for 25% (at [Na](i) = 10 mM) to nearly 100% ([Na](i) = 30 mM) of the trigger Ca(2+). The shift of the main trigger from L-type calcium channels to the exchanger reduced the delay between the action potential upstroke and the intracellular calcium transient. This may contribute to the function of the myocyte in physiological situations where [Na](i) is elevated. These main results remain the same when using different estimates for the most crucial parameters in the modeling or different models for the exchanger.     

Proton and electron pathways in the bacterial nitric oxide reductase

Electron- and proton-transfer reactions in bacterial nitric oxide reductase (NOR) have been investigated by optical spectroscopy and electrometry. In liposomes, NOR does not show any generation of an electric potential during steady-state turnover. This electroneutrality implies that protons are taken up from the same side of the membrane as electrons during catalysis. Intramolecular electron redistribution after photolysis of the partially reduced CO-bound enzyme shows that the electron transfer in NOR has the same pathway as in the heme-copper oxidases. The electron is transferred from the acceptor site, heme c, via a low-spin heme b to the binuclear active site (heme b3/FeB). The electron-transfer rate between hemes c and b is (3 +/- 2) x 10(4) s(-1). The rate of electron transfer between hemes b and b3 is too fast to be resolved (>10(6) s(-1)). Only electron transfer between heme c and heme b is coupled to the generation of an electric potential. This implies that the topology of redox centers in NOR is comparable to that in the heme-copper cytochrome oxidases. The optical and electrometric measurements allow identification of the intermediate states formed during turnover of the fully reduced enzyme, as well as the associated proton and electron movement linked to the NO reduction. The first phase (k = 5 x 10(5) s(-1)) is electrically silent, and characterized by the disappearance of absorbance at 433 nm and the appearance of a broad peak at 410 nm. We assign this phase to the formation of a ferrous NO adduct of heme b3. NO binding is followed by a charge separation phase (k = 2.2 x 10(5) s(-1)). We suggest that the formation of this intermediate that is not linked to significant optical changes involves movement of charged side chains near the active site. The next step creates a negative potential with a rate constant of approximately 3 x 10(4) s(-1) and a weak optical signature. This is followed by an electrically silent phase with a rate constant of 5 x 10(3) s(-1) leading to the last intermediate of the first turnover (a rate constant of approximately 10(3) s(-1)). The fully reduced enzyme has four electrons, enough for two complete catalytic cycles. However, the protons for the second turnover must be taken from the bulk, resulting in the generation of a positive potential in two steps. The optical measurements also verify two phases in the oxidation of low-spin hemes. Based on these results, we present mechanistic models of NO reduction by NOR. The results can be explained with a trans mechanism rather than a cis model involving FeB. Additionally, the data open up the possibility that NOR employs a P450-type mechanism in which only heme b3 functions as the NO binding site during turnover.     

<Emphasis Type="Italic">Brassica napus</Emphasis> lines with rearranged <Emphasis Type="Italic">Arabidopsis</Emphasis> mitochondria display CMS and a range of developmental aberrations

Numerous Brassica napus (+) Arabidopsis thaliana somatic hybrids were screened for male sterility and aberrant flower phenotypes. Nine hybrids were selected and backcrossed recurrently to B. napus. The resulting lines displayed stable maternal inheritance of flower phenotypes. Nuclear and organellar genomes were characterized molecularly using RFLP analysis. No DNA from A. thaliana was found in the nuclear genome after six back-crosses, whilst the mitochondrial genomes contained rearranged DNA from both A. thaliana and B. napus. Each line tested had a unique RFLP pattern of the mitochondrial DNA (mtDNA) that remained unchanged between the BC(3) and BC(6) generation. The plastid genomes consisted of B. napus DNA. Five lines of the BC(5) generation were subjected to more comprehensive investigations of growth, morphology and fertility. On the basis of these investigations, the five CMS lines could be assigned to two groups, one represented by three lines displaying reduced vegetative development, complete male sterility, and homeotic conversions of stamens into feminized structures. The second group, represented by the other two lines, were not completely male-sterile but still displayed severely affected flower morphologies. These two lines did not display any reduction in vegetative development. For both groups only stamens and petals suffered from the morphological and functional aberrations, while the sepals and pistils displayed normal morphology. All plants were fully female-fertile. Different rearrangements of the mitochondrial genome disturbed nuclear-mitochondrial interactions and led to various types of aberrant growth and flower development. The existence of numerous CMS lines with different mitochondrial patterns involving a species with a sequenced genome offers new opportunities to investigate the genetic regulation of CMS and its associated developmental perturbations.     

Reaction kinetics of a two-photon excitation microparticle based immunoassay--from modelling to practice

Real time observation of reaction kinetics is one of the key features of the newly developed microparticle based two-photon excitation fluorescence immunoassay system (TPX). By observing binding reactions at the surface of individual microparticles during the incubation of an assay, the binding constants of an assay become apparent. This paper describes the use of the new system in quantifying the reaction parameters of human thyroid stimulating hormone (hTSH) assay. A mechanistic reaction model for the assay is presented. The reaction model is further shown to precisely predict the behaviour of the assay kinetics over a wide range of analyte concentrations.     

Flexibility and viscometric studies of globular proteins ovalbumin and ovotransferrin

The ultrasonic velocity, density and viscosity of two egg proteins, ovalbumin and ovotransferrin in phosphate buffer have been studied at the physiological pH values. The thermodynamic functions for unfolding, ellipticity, surface amino acid residues and compressibility have been obtained for thermal and chemical denaturation in these food proteins. The computed values of Huggin's constant and shape factor, at a fixed ionic strength 0.16 M are found to be in agreement with the reported values for globular proteins. The slow increase in free-energy of unfolding with temperature at a fixed pH 7 suggests uncoiling and in turn, disappearance of biological activity. It has been observed that the effects of temperature and chemical denaturant on the native protein may give rise to different conformational states. In the presence of urea and sodium dodecyl sulphate (SDS), the proteins gave the excessively denatured states at 25 degrees C and pH 7, in comparison to the thermal denatured state. The positive values of partial adiabatic compressibility (see symbol in text) beta s over the temperature range 45-75 degrees C suggest the possibility of large internal flexibility in ovotransferrin than in ovalbumin.     

Cold acclimation increases basal heart rate but decreases its thermal tolerance in rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss, Walbaum) were acclimated to 4 degrees C and 17 degrees C for more than 4 weeks and heart rate was determined in the absence and presence of adrenaline to see how thermal adaptation influences basal heart rate and its beta-adrenergic control in a eurythermal fish species. The basal heart rate in vitro was higher in cold-acclimated than warm-acclimated rainbow trout at temperatures below 17 degrees C. On the other hand, adaptation to cold decreased thermal tolerance of heart rate so that the maximal heart rates were achieved at 17 degrees C (75 +/- 4 bpm) and 24 degrees C (88 +/- 2 bpm) in cold-acclimated and warm-acclimated trout, respectively. Beta-adrenergic response of the heart was enhanced by cold-adaptation, since adrenaline (100 nmol l(-1)) caused stronger stimulation of heart rate in cold-acclimated (29 +/- 14%) than in warm-acclimated fish (10 +/- 1%; P = 0.03). Furthermore, adrenaline strongly opposed the temperature-dependent deterioration of force production in cold-acclimated trout but not in warm-acclimated trout. The results indicate that adaptation to cold increases basal heart rate but decreases its thermal tolerance in rainbow trout. Cold acclimation up-regulates the beta-adrenergic system, and beta-adrenoceptor activation seems to provide cardioprotection against high temperatures in the cold-adapted rainbow trout.     

Xylitol purification by cross-linked glucose isomerase crystals

Xylitol is specifically bound by active, cross-linked glucose isomerase crystals (CLGI). CLGI can be used to purify xylitol or concentrate it from dilute and impure solutions. Bound xylitol can be eluted from CLGI by Ca2 and the material reactivated by Mg2. The binding capacity is 1 mg xylitol per 525 mg CLGI which equals one molecule per active center. CLGI can further be used to purify xylitol and sorbitol from impure mixtures of arabinitol, mannitol, ribitol and monosaccharides. © Rapid Science Ltd. 1998