Chloroplast magnesium transporters play essential but differential roles in maintaining magnesium homeostasis.

Magnesium (Mg2+) is essential for photosynthesis in the chloroplasts of land plants and algae. Being the central ion of chlorophyll, cofactor and activator of many photosynthetic enzymes including RuBisCO, magnesium-deficient plants may suffer from leaf chlorosis symptoms and retarded growth. Therefore, the chloroplast Mg2+ concentration is tightly controlled by magnesium transport proteins. Recently, three different transporters from two distinct families have been identified in the chloroplast inner envelope of the model plant Arabidopsis thaliana: MGT10, MGR8, and MGR9. Here, we assess the individual roles of these three proteins in maintaining chloroplast Mg2+ homeostasis and regulating photosynthesis, and if their role is conserved in the model green alga Chlamydomonas reinhardtii. Phylogenetic analysis and heterologous expression revealed that the CorC-like MGR8 and MGR9 transport Mg2+ by a different mechanism than the CorA-like MGT10. MGR8 and MGT10 genes are highest expressed in leaves, indicating a function in chloroplast Mg2+ transport. MGR9 is important for chloroplast function and plant adaptation in conditions of deficiency or excess of Mg2+. Transmission electron microscopy indicated that MGT10 plays a differential role in thylakoid stacking than MGR8 and MGR9. Furthermore, we report that MGR8, MGR9, and MGT10 are involved in building up the pH gradient across the thylakoid membrane and activating photoprotection in conditions of excess light, however the mechanism has not been resolved yet. While there are no chloroplast MGR-like transporters in Chlamydomonas, we show that MRS4 is a homolog of MGT10, that is required for photosynthesis and cell growth. Taken together, our findings reveal that the studied Mg2+ transporters play essential but differential roles in maintaining chloroplast Mg2+ homeostasis.

Functional analysis of whether the glycine residue of the GMN motif of the Arabidopsis MRS2/MGT/CorA-type Mg2+ channel protein AtMRS2-11 is critical for Mg2+ transport activity.

Magnesium (Mg2+) plays a critical role in many physiological processes. The AtMRS2/MGT family, which contains nine Arabidopsis genes (and two pseudogenes), belongs to a eukaryotic subset of the CorA superfamily of divalent cation transporters. AtMRS2-11/MGT10 possesses the signature GlyMetAsn sequence (the GMN motif) conserved in the CorA superfamily; however, little is known about the role of the GMN motif in AtMRS2. Direct measurement using the fluorescent dye mag-fura-2 revealed that reconstituted AtMRS2-11 mediated rapid Mg2+ uptake into proteoliposomes at extraliposomal Mg2+ concentrations of 10 and 20 mM. Mutations in the GMN motif, G417 to A, S or V, did not show a significant change in Mg2+ uptake relative to the wild-type protein. The G417W mutant exhibited a significant increase in Mg2+ uptake. The functional complementation assay in Escherichia coli strain TM2 showed that E. coli cells expressing AtMRS2-11 with mutations in G of the GMN motif did not grow in LB medium without Mg2+ supplementation, while growth was observed in LB medium supplemented with 0.5 mM Mg2+; no difference was observed between the growth of TM2 cells expressing the AtMRS2-11 G417W mutant and that of cells expressing wild-type AtMRS2-11. These results suggested that the Mg2+ transport activity of the AtMRS2-11 GMN-motif mutants was low at low physiological Mg2+ concentrations; thus, the Gly residue is critical for Mg2+ transport, and the Mg2+ transport activity of the GMN-motif mutants was increased at high Mg2+ concentrations.

Modulatory mechanisms of sliding of nine outer doublet microtubules for generating planar and half-helical flagellar waves.

It has been shown that sperm flagellar motility is generated and modulated by metachronal sliding and two types of synchronous sliding of the outer doublet microtubules. Metachronal sliding propagates around the axoneme circumferentially from one doublet to another along the flagellum, whereas the two types of synchronous sliding occur synchronously throughout an extended region along the doublet microtubules. Oscillatory synchronous sliding occurs between most pairs of the nine doublet microtubules, whereas non-oscillatory synchronous sliding occurs between a specific pair of the nine doublet microtubules. These types of sliding coexist in the flagellum and create beat cycles of flagellar movement. The circumferential propagation of active sliding around the nine doublet microtubules in the metachronal sliding suggests that it is easier for a flagellum to produce helical waves than planar waves. Most sperm flagellar movements are planar to a certain extent. Therefore, mechanisms that modulate the helical waves into planar waves may be present. Structures such as the central pair microtubules in 9 + 2 sperm flagella and the fusion of fibrous-sheath and 3-,8-doublet microtubules in mammalian sperm flagella partition the nine outer doublet microtubules into two groups. Accordingly, the sliding between these two groups generates planar flagellar waves. A similar effect is caused by the sliding between a specific pair of the nine doublet microtubules of the non-oscillatory synchronous sliding, occurring in a Ca2+ concentration-dependent manner. These hard- and soft-wired systems produce the nearly planar flagellar waves required for the efficient propulsion of spermatozoa.

Thermodynamic Characterization of the Ca2+-Dependent Interaction Between SOUL and ALG-2.

SOUL, a heme-binding protein-2 (HEBP-2), interacts with apoptosis-linked gene 2 protein (ALG-2) in a Ca2+-dependent manner. To investigate the properties of the interaction of SOUL with ALG-2, we generated several mutants of SOUL and ALG-2 and analyzed the recombinant proteins using pulldown assay and isothermal titration calorimetry. The interaction between SOUL and ALG-2 (delta3-23ALG-2) was an exothermic reaction, with 1:1 stoichiometry and high affinity (Kd = 32.4 nM) in the presence of Ca2+. The heat capacity change (ΔCp) of the reaction showed a large negative value (-390 cal/K·mol), which suggested the burial of a significant nonpolar surface area or disruption of a hydrogen bond network that was induced by the interaction (or both). One-point mutation of SOUL Phe100 or ALG-2 Trp57 resulted in complete loss of heat change, supporting the essential roles of these residues for the interaction. Nevertheless, a truncated mutant of SOUL1-143 that deleted the domain required for the interaction with ALG-2 Trp57 still showed 1:1 binding to ALG-2 with an endothermic reaction. These results provide a better understanding of the target recognition mechanism and conformational change of SOUL in the interaction with ALG-2.

Male reproductive health statement (XIIIth international symposium on Spermatology, may 9th-12th 2018, Stockholm, Sweden.

On the occasion of the XIIIth International Symposium on Spermatology held from 9 to 13 May 2018 in Stockholm (Sweden), participants (guest speakers and audience) collectively felt the need to make a public statement on the general issue of male reproductive health. Our intention is to raise awareness of what we believe is a neglected area of research despite alarming situations around the world. The disclosure strategy desired by the co-authors is to bring it to the attention of the greatest number partly by considering co-publication in the various periodicals dealing with Reproductive Biology and Andrology. BaCA's editorial office accepted this mission and found it natural that our periodical, the official journal of the French Andrology Society (SALF), should carry this message.

A l'occasion du XIII eme Symposium international sur la Spermatologie qui s'est. tenu du 9 au 13 Mai 2018 à Stockholm (Suède), les participants (orateurs invités et l'auditoire) ont ressenti collectivement le besoin de faire une déclaration publique sur la question générale de la santé reproductive masculine. Notre intention est. de mieux faire connaître ce que nous pensons être un domaine de recherche négligé malgré des situations alarmantes dans le monde entier. La stratégie de divulgation souhaitée par les co-auteurs est. de le porter à l'attention du plus grand nombre en envisageant pour partie une co-publication dans les différents périodiques traitant de Reproduction et d'Andrologie. Le bureau éditorial de BaCA, a accepté cette mission et a trouvé naturel que notre périodique, journal officiel de la Société d'Andrologie en Langue Française (SALF) porte ce message.

The homologous Arabidopsis MRS2/MGT/CorA-type Mg2+ channels, AtMRS2-10 and AtMRS2-1 exhibit different aluminum transport activity.

Magnesium (Mg2+) plays a critical role in many physiological processes. The AtMRS2/MGT family, which consists of nine Arabidopsis genes (and two pseudo-genes) belongs to a eukaryotic subset of the CorA superfamily of divalent cation transporters. AtMRS2-10 and AtMRS2-1 possess the signature GlyMetAsn sequence conserved in the CorA superfamily; however, they have low sequence conservation with CorA. Direct measurement using the fluorescent dye mag-fura-2 revealed that reconstituted AtMRS2-10 and AtMRS2-1 mediated rapid Mg2+ uptake into proteoliposomes. The rapid Mg2+ uptake through AtMRS2-10 was inhibited by aluminum. An assay using the Al-sensitive dye morin indicated Al uptake into the proteoliposomes through AtMRS2-10. AtMRS2-10 also exhibited Ni2+ transport activity but almost no Co2+ transport activity. The rapid Mg2+ uptake through AtMRS2-1 was not inhibited by aluminum. Al uptake into the proteoliposomes through AtMRS2-1 was not observed. The functional complementation assay in Escherichia coli strain TM2 showed that AtMRS2-1 was capable of mediating Mg2+ uptake. Heterologous expression using the E. coli mutant cells also showed that the E. coli cells expressing AtMRS2-1 was more resistant to aluminum than the E. coli cells expressing AtMRS2-10. The results suggested that AtMRS2-10 transported Al into the E. coli cells, and then the transported Al inhibited the growth of E. coli. AtMRS2-1 has been localized to the Arabidopsis tonoplast, indicating that AtMRS2-1 is exposed to much higher concentration of aluminum than AtMRS2-10. Under the conditions, it may be required that the Mg2+ transport of AtMRS2-1 is insensitive to Al inhibition, and AtMRS2-1 is impermeable to Al.

Regulatory mechanisms of sperm flagellar motility by metachronal and synchronous sliding of doublet microtubules.

STUDY QUESTION: What is the role of metachronal and synchronous sliding in sperm flagellar motility? SUMMARY ANSWER: Both metachronal and oscillatory synchronous sliding are essential for sperm flagellar motility, while the change in mode of synchronous sliding between the non-oscillatory synchronous sliding of a specific pair of the doublet microtubules and the oscillatory synchronous sliding between most pairs of doublet microtubules modulates the sperm flagellar motility. WHAT IS KNOWN ALREADY: Metachronal and synchronous sliding of doublet microtubules are involved in sperm flagellar motility and regulation of these sliding movements controls flagellar bend formation. STUDY DESIGN, SIZE, DURATION: To study the regulatory mechanisms of metachronal and synchronous sliding in flagellar movement of golden hamster spermatozoa, changes in these sliding movements during hyperactivation were examined by measuring the angle of the tangent to the flagellar shaft with reference to the central axis of the sperm head (the shear angle) along the flagellum. Golden hamster spermatozoa were obtained from the caudal epididymis of five sexually mature golden hamsters. Results from three experiments were averaged. The number of spermatozoa analyzed is 15 activated sperm, 22 hyperactivated sperm and 20 acrosome-reacted sperm. PARTICIPANTS/MATERIALS, SETTING, METHODS: For detailed field-by-field analysis, an individual flagellar image was tracked automatically using the Autotrace module of image analysis software. The coordinate values of the flagellar shaft were used to calculate the shear angle, which is proportional to the amount of microtubule sliding at any given position along the flagellum. The maximum shear angles of metachronal and synchronous sliding were obtained from the mean shear angles between the maximum shear angles of pro-hook bends and the absolute values of the minimum shear angles of anti-hook bends, which represent the amplitude of a set of successive shear angle curves, with 3-12 shear curves covering one beat cycle of sperm flagellar movement. Asymmetry of flagellar waves was expressed by the mean shear angle between the maximum shear angle of pro-hook bends and the minimum shear angle of anti-hook bends at 100 µm from the head-midpiece junction. MAIN RESULTS AND THE ROLE OF CHANCE: The asymmetrical flagellar movements observed in the activated (non-hyperactivated) and hyperactivated spermatozoa were characterized by the non-oscillatory synchronous sliding of a specific pair of the doublets; the large asymmetrical flagellar movement in the hyperactivated spermatozoa was generated by the large non-oscillatory synchronous sliding. Both the metachronal and synchronous sliding increased during the hyperactivation; however, the large symmetrical flagellar movement of the acrosome-reacted spermatozoa was characterized by the oscillatory synchronous sliding between most pairs of doublets. These results demonstrated that the metachronal and synchronous sliding are involved in generation and modulation of sperm flagellar motility; however, two types of synchronous sliding, non-oscillatory and oscillatory sliding, modulate the sperm flagellar motility by enhancing the sliding of a specific pair of the doublets or the sliding between most pairs of the doublets. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: This is an indirect study of the metachronal and synchronous sliding of doublet microtubules. Studies based on the direct observation of behavior of dynein are needed to clarify the sliding microtubule theory of flagellar movement of spermatozoa. WIDER IMPLICATIONS OF THE FINDINGS: Both the metachronal and oscillatory synchronous sliding of doublet microtubule generate and modulate sperm flagellar motility, while the change in mode of synchronous sliding between the non-oscillatory synchronous sliding and oscillatory synchronous sliding modulates the sperm flagellar motility. The coordination between these sliding leads to various types of flagellar and ciliary motility, including the asymmetrical beating in flagellar and ciliary movement and planar or helical beating in sea urchin spermatozoa. Moreover, the finding that the metachronal sliding and two types of synchronous sliding generate and modulate the flagellar motility will open a new avenue for quantitative analysis of flagellar and ciliary motility. STUDY FUNDING AND COMPETING INTEREST(S): The authors have no conflict of interest and no funding to declare.

Self-Sustained Oscillatory Sliding Movement of Doublet Microtubules and Flagellar Bend Formation.

It is well established that the basis for flagellar and ciliary movements is ATP-dependent sliding between adjacent doublet microtubules. However, the mechanism for converting microtubule sliding into flagellar and ciliary movements has long remained unresolved. The author has developed new sperm models that use bull spermatozoa divested of their plasma membrane and midpiece mitochondrial sheath by Triton X-100 and dithiothreitol. These models enable the observation of both the oscillatory sliding movement of activated doublet microtubules and flagellar bend formation in the presence of ATP. A long fiber of doublet microtubules extruded by synchronous sliding of the sperm flagella and a short fiber of doublet microtubules extruded by metachronal sliding exhibited spontaneous oscillatory movements and constructed a one beat cycle of flagellar bending by alternately actuating. The small sliding displacement generated by metachronal sliding formed helical bends, whereas the large displacement by synchronous sliding formed planar bends. Therefore, the resultant waveform is a half-funnel shape, which is similar to ciliary movements.

Magnesium uptake of Arabidopsis transporters, AtMRS2-10 and AtMRS2-11, expressed in Escherichia coli mutants: Complementation and growth inhibition by aluminum.

Magnesium (Mg2+) plays a critical role in many physiological processes. Mg2+ transport systems in Salmonella have been well documented, but those in Escherichia coli have not been fully elucidated. We examined the effects of corA, mgtA, yhiD and corC gene deletion on Mg2+ transport in E. coli. We obtained every combination of double, triple and quadruple mutants. The corA and mgtA double mutant required addition of 10 mM Mg2+ to Luria-Bertani (LB) medium for growth, and the corA, mgtA and yhiD triple mutant TM2 required a higher Mg2+ concentration. The Mg2+ requirement of the quadruple mutant was similar to that of TM2. The results demonstrated that either CorA or MgtA is necessary for normal E. coli growth in LB medium and that YhiD plays a role in Mg2+ transport under high Mg2+ growth conditions in E. coli. The Arabidopsis Mg2+ transporters, AtMRS2-10 and AtMRS2-11, were heterologously expressed in TM2 cells. TM2 cells expressing AtMRS2-10 and AtMRS2-11 could grow in LB medium that had been supplemented with 1 mM Mg2+ and without Mg2+ supplementation, respectively, and cell growth was inhibited by 2 mM AlCl3. The results indicated that the growth of TM2 expressing AtMRS2-10 and AtMRS2-11 reflected these AtMRS2 function for Mg2+ and aluminum. The E. coli TM2 cells are useful for functional analysis of Arabidopsis MRS2 proteins.

Ca2+ and cAMP regulations of microtubule sliding in hyperactivated motility of bull spermatozoa.

To reach and fertilize the egg, mammalian spermatozoa change their flagellar movement in the female reproductive tract, named hyperactivation. The biochemical analyses of the hyperactivated movement using demembranated spermatozoa defined the factors inducing this peculiar movement; namely, large asymmetrical flagellar movement observed in the early stage of the hyperactivation was induced with a high Ca(2+) concentration while large symmetrical flagellar movement in the late stage of the hyperactivation was generated with low Ca(2+) and high cAMP concentrations. Under these conditions, the microtubule sliding of bull sperm flagella was investigated by disintegrating the sperm flagella with MgATP(2-) after extracting their plasma membrane and mitochondria. The large asymmetrical flagellar movement was caused by a long sliding displacement of a fiber of the doublet microtubules. On the other hand, the large symmetrical flagellar movement was generated by a large amount of microtubule sliding by many doublet microtubules.

Changes in pH and NADPH regulate the DNA binding activity of neuronal PAS domain protein 2, a mammalian circadian transcription factor.

Neuronal PAS domain protein 2 (NPAS2) is a core clock transcription factor that forms a heterodimer with BMAL1 to bind the E-box in the promoter of clock genes and is regulated by various environmental stimuli such as heme, carbon monoxide, and NAD(P)H. In this study, we investigated the effects of pH and NADPH on the DNA binding activity of NPAS2. In an electrophoretic mobility shift (EMS) assay, the pH of the reaction mixture affected the DNA binding activity of the NPAS2/BMAL1 heterodimer but not that of the BMAL1/BMAL1 homodimer. A change in pH from 7.0 to 7.5 resulted in a 1.7-fold increase in activity in the absence of NADPH, and NADPH additively enhanced the activity up to 2.7-fold at pH 7.5. The experiments using truncated mutants revealed that N-terminal amino acids 1-61 of NPAS2 were sufficient to sense the change in both pH and NADPH. We further analyzed the kinetics of formation and DNA binding of the NPAS2/BMAL1 heterodimer at various pH values. In the absence of NADPH, a change in pH from 6.5 to 8.0 decreased the KD(app) value of the E-box from 125 to 22 nM, with an 8-fold increase in the maximal level of DNA binding for the NPAS2/BMAL1 heterodimer. The addition of NADPH resulted in a further decrease in KD(app) to 9 nM at pH 8.0. Furthermore, NPAS2-dependent transcriptional activity in a luciferase assay using NIH3T3 cells also increased with the pH of the culture medium. These results suggest that NPAS2 has a role as a pH and metabolite sensor in regulating circadian rhythms.

Effects of NAD(P)H and its derivatives on the DNA-binding activity of NPAS2, a mammalian circadian transcription factor.

NPAS2 is a transcription factor that regulates mammalian circadian rhythms. It has been suggested that NPAS2 DNA-binding activity is regulated by the intracellular redox state of NAD(P)H, although the mechanism remains unclear. To investigate the NAD(P)H interaction site of murine NPAS2, we performed electrophoretic mobility shift assays using several truncation mutants of the NPAS2 bHLH domain. Among the mutants, NPAS2 containing the N-terminal 61 residues formed a heterodimer with BMAL1 to bind DNA, and NAD(P)H enhanced the binding activity, while NAD(P)H inhibited the DNA-binding activity of the BMAL1 homodimer in a dose-dependent manner. NAD(P)H derivatives such as 2',5'-ADP, nicotinamide, nicotinic acid and nicotinic acid adenine dinucleotide (NAAD) did not affect the DNA-binding activity. Interestingly, NAD(P)(+), previously reported as an inhibitor, did not affect NPAS2 binding activity in the presence or absence of NAD(P)H in our system. These results suggest that NPAS2 DNA-binding activity is specifically enhanced by NAD(P)H independently of NAD(P)(+) and that the N-terminal 1-61 amino acids of NPAS2 are sufficient to sense NAD(P)H.

Regulations of microtubule sliding by Ca2+ and cAMP and their roles in forming flagellar waveforms.

The function of Ca(2+) and cAMP in extruding doublet microtubules from sea urchin sperm axoneme and generating flagellar waves was investigated in order to clarify the regulatory mechanism of microtubule sliding and the formation mechanism of beating patterns of cilia and flagella. Almost all potentially asymmetric spermatozoa that were demembranated with Triton in the absence of Ca(2+) and reactivated with MgATP(2-) (Gibbons, B.H. and Gibbons, I.R. (1980). J. Cell Biol., 84: 13-27), beat with planar waves closely resembling those of the intact spermatozoa, whereas potentially symmetric spermatozoa, in which axonemal calmodulin was removed by detergent extraction in the presence of millimolar Ca(2+) (Brokaw, C.J. and Nagayama, S.M. (1985). J. Cell Biol., 100: 1875-1883), beat with three-dimensional waves if they were reactivated with low MgATP(2-). At a high MgATP(2-), almost all demembranated spermatozoa beat with planar waves. cAMP enhanced the three-dimensionality of the flagellar waves at a low Ca(2+). These changes in the flagellar waves were caused by different regulations of the microtubule sliding by calcium, cAMP, and MgATP(2-).

Tubulin-dynein system in flagellar and ciliary movement.

Eukaryotic flagella and cilia have attracted the attention of many researchers over the last century, since they are highly arranged organelles and show sophisticated bending movements. Two important cytoskeletal and motor proteins, tubulin and dynein, were first found and described in flagella and cilia. Half a century has passed since the discovery of these two proteins, and much information has been accumulated on their molecular structures and their roles in the mechanism of microtubule sliding, as well as on the architecture, the mechanism of bending movement and the regulation and signal transduction in flagella and cilia. Historical background and the recent advance in this field are described.

Comparative analysis of movement characteristics of lancelet and fish spermatozoa having different morphologies.

The movement characteristics of the sperm and their flagella obtained from a lancelet and 35 species from almost all orders of fishes were examined using high-speed video microscopy. The aim was to clarify the relationship between the motility parameters of the spermatozoa having different morphologies and how these motility parameters affect the swimming speed of the spermatozoa. The motility parameters representing the flagellar waveform, the wavelength, and the amplitude were neither very different between the spermatozoa of the different species nor related to the swimming speed. In contrast, the beat frequency was remarkably changed in the different spermatozoa and was proportional to the swimming speed. The maximum shear angle of the flagellar wave, which is directly related to the maximum sliding displacement between the doublet microtubules, remained nearly constant while the beat frequency varied widely; therefore, the spermatozoa beat in the constant sliding displacement mode. An analysis of the relationship between swimming speed and flagellar length revealed that short flagella were at a disadvantage in developing swimming speed; however, so were extra-long flagella. The ratio of the swimming speed to the wave velocity calculated from the wavelength and the beat frequency depended on the distance from the glass surface. The swimming speeds calculated using the original resistive-force theory were greater than the measured values. To rationalize the measured values, the ratio between the normal and tangential drag coefficient in the resistive-force theory was corrected; namely, 1.99 at 1 µm and 1.63 at 3 µm from the glass surface.

Functional reconstitution and characterization of the Arabidopsis Mg(2+) transporter AtMRS2-10 in proteoliposomes.

Magnesium (Mg(2+)) plays critical role in many physiological processes. The mechanism of Mg(2+) transport has been well documented in bacteria; however, less is known about Mg(2+) transporters in eukaryotes. The AtMRS2 family, which consists of 10 Arabidopsis genes, belongs to a eukaryotic subset of the CorA superfamily proteins. Proteins in this superfamily have been identified by a universally conserved GlyMetAsn motif and have been characterized as Mg(2+) transporters. Some members of the AtMRS2 family, including AtMRS2-10, may complement bacterial mutants or yeast mutants that lack Mg(2+) transport capabilities. Here, we report the purification and functional reconstitution of AtMRS2-10 into liposomes. AtMRS2-10, which contains an N-terminal His-tag, was expressed in Escherichia coli and solubilized with sarcosyl. The purified AtMRS2-10 protein was reconstituted into liposomes. AtMRS2-10 was inserted into liposomes in a unidirectional orientation. Direct measurement of Mg(2+) uptake into proteoliposomes revealed that reconstituted AtMRS2-10 transported Mg(2+) without any accessory proteins. Mutation in the GMN motif, M400 to I, inactivated Mg(2+) uptake. The AtMRS2-10-mediated Mg(2+) influx was blocked by Co(III)hexamine, and was independent of the external pH from 5 to 9. The activity of AtMRS2-10 was inhibited by Co(2+) and Ni(2+); however, it was not inhibited by Ca(2+), Fe(2+), or Fe(3+). While these results indicate that AtMRS2-10 has similar properties to the bacterial CorA proteins, unlike bacterial CorA proteins, AtMRS2-10 was potently inhibited by Al(3+). These studies demonstrate the functional capability of the AtMRS2 proteins in proteoliposomes to study structure-function relationships.

Mechanical constraint converts planar waves into helices on tunicate and sea urchin sperm flagella.

The change in the flagellar waves of spermatozoa from a tunicate and sea urchins was examined using high-speed video microscopy to clarify the regulation of localized sliding between doublet microtubules in the axoneme. When the tunicate Ciona spermatozoa attached to a coverslip surface by their heads in seawater or they moved in seawater with increased viscosity, the planar waves of the sperm flagella were converted into left-handed helical waves. On the other hand, conversion of the planar waves into helical waves in the sea urchin Hemicentrotus spermatozoa was not seen in seawater with an increased viscosity as well as in ordinary seawater. However, the sea urchin Clypeaster spermatozoa showed the conversion, albeit infrequently, when they thrust their heads into seawater with an increased viscosity. The chirality of the helical waves of the Clypeaster spermatozoa was right-handed. When Ciona spermatozoa swam freely near a glass surface, they moved in relatively large circular paths (yawing motion). There was no difference in the proportion of spermatozoa yawing in either a clockwise or counterclockwise direction when viewed from above, which was also different from that of the sea urchin spermatozoa. These observations suggest that the planar waves generally observed on the sperm flagella are mechanically regulated, although their stability must depend on the Ca(2+) concentration in the cell. Furthermore, the chirality of the helical waves may be determined by the intracellular Ca(2+) concentration and changed by transmitting the localized active sliding between the doublet microtubules around the axoneme in an alternative direction.

Dynamics of flagellar force generated by a hyperactivated spermatozoon.

The flagellar force generated by a hyperactivated monkey spermatozoon was evaluated using the resistive force theory applied to the activated (nonhyperactivated) and hyperactivated flagellar waves that were obtained using high-speed video microscopy and digital image processing in order to clarify the mechanism of sperm penetration through the zona pellucida. No difference in the maximum propulsive force, which was parallel to the longitudinal sperm head axis, was found between the activated and hyperactivated spermatozoa. The maximum transverse force (45 pN), which was perpendicular to the longitudinal sperm head axis, of the hyperactivated spermatozoon was ∼2.5 times its propulsive force. As the beat frequency of the flagellar beating remarkably decreased during the hyperactivation, the slowly oscillating transverse force (5 Hz) by the hyperactivated spermatozoon seems to be most effective for sperm penetration through the zona pellucida.

Thermodynamic analysis of interactions between cofactor and neuronal nitric oxide synthase.

The thermodynamics of cofactor binding to the isolated reductase domain (Red) of nNOS and its mutants have been studied by isothermal titration calorimetry. The NADP(+) and 2',5'-ADP binding stoichiometry to Red were both 1:1, consistent with a one-site kinetic model instead of a two-site model. The binding constant (K(D) = 71 nM) and the large heat capacity change (ΔC(p) = -440 cal mol(-1) K(-1)) for 2',5'-ADP were remarkably different from those for NADP(+) (1.7 µM and -140 cal mol(-1) K(-1), respectively). These results indicate that the nicotinamide moiety as well as the adenosine moiety has an important role in binding to nNOS. They also suggest that the thermodynamics of the conformational change in Red caused by cofactor binding are significantly different from the conformational changes that occur in cytochrome c reductase, in which the nicotinamide moiety of the cofactor is not essential for binding. Analysis of the deletion mutant of the autoinhibitory helix (RedΔ40) revealed that the deletion resulted in a decrease in the binding affinity of 2',5'-ADP with more unfavorable enthalpy gain. In the case of RedCaM, which contains a calmodulin (CaM) binding site, the presence of Ca(2+)/CaM caused a 6.7-fold increase in the binding affinity for 2',5'-ADP that was mostly due to the favorable entropy change. These results are consistent with a model in which Ca(2+)/CaM induces a conformational change in NOS to a flexible "open" form from a "closed" form that locked by cofactor binding, and this change facilitates the electron transfer required for catalysis.

Gymnemic acid interacts with mammalian glycerol-3-phosphate dehydrogenase.

In a previous study, we found interaction of gymnemic acid (GA) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis. We now examined interaction of GA with glycolytic and related enzymes. We found that (1) GA induced a band smearing of glycerol-3-phosphate dehydrogenase (G3PDH) as well as that of GAPDH in SDS-PAGE, (2) GA diminished the G3PDH band detected by an antibody to phosphoserine, and (3) GA inhibited the G3PDH activity. The GA-induced smearing of the G3PDH band was diminished by prior incubation of GA with gamma-cyclodextrin. GA gave no effects on the electrophoretic and phosphoserine bands of other glycolytic enzymes. NAD and NADH diminished the GA-induced smearing of the G3PDH and GAPDH bands in different concentration-dependent manner. Pretreatment of G3PDH with heated SDS-containing buffer or pretreatment with hydroxylamine diminished the GA-induced smearing of G3PDH. Deacylation of GA by alkaline hydrolysis diminished the smearing of G3PDH band, thereby indicating that the acyl moieties of GA were necessary for the GA-induced smearing of G3PDH. These results indicated the interaction of GA with G3PDH, an enzyme involved in glycerol metabolism. These studies suggest that GA may have some pharmacological activities including antidiabetic activity and lipid lowering effects via interaction with GAPDH and G3PDH.