An autoinhibitory control element defines calcium-regulated isoforms of nitric oxide synthase

Nitric oxide synthases (NOSs) are classified functionally, based on whether calmodulin binding is Ca2+-dependent (cNOS) or Ca2+-independent (iNOS). This key dichotomy has not been defined at the molecular level. Here we show that cNOS isoforms contain a unique polypeptide insert in their FMN binding domains which is not shared with iNOS or other related flavoproteins. Previously identified autoinhibitory domains in calmodulin-regulated enzymes raise the possibility that the polypeptide insert is the autoinhibitory domain of cNOSs. Consistent with this possibility, three-dimensional molecular modeling suggested that the insert originates from a site immediately adjacent to the calmodulin binding sequence. Synthetic peptides derived from the 45-amino acid insert of endothelial NOS were found to potently inhibit binding of calmodulin and activation of cNOS isoforms. This inhibition was associated with peptide binding to NOS, rather than free calmodulin, and inhibition could be reversed by increasing calmodulin concentration. In contrast, insert-derived peptides did not interfere with the arginine site of cNOS, as assessed from [3H]NG-nitro-L-arginine binding, nor did they potently effect iNOS activity. Limited proteolysis studies showed that calmodulin's ability to gate electron flow through cNOSs is associated with displacement of the insert polypeptide; this is the first specific calmodulin-induced change in NOS conformation to be identified. Together, our findings strongly suggest that the insert is an autoinhibitory control element, docking with a site on cNOSs which impedes calmodulin binding and enzymatic activation. The autoinhibitory control element molecularly defines cNOSs and offers a unique target for developing novel NOS activators and inhibitors

Nitric oxide and cyclic nucleotides: Their roles in junction dynamics and spermatogenesis

Spermatogenesis is a highly complicated process in which functional spermatozoa (haploid, 1n) are generated from primitive mitotic spermatogonia (diploid, 2n). This process involves the differentiation and transformation of several types of germ cells as spermatocytes and spermatids undergo meiosis and differentiation. Due to its sophistication and complexity, testis possesses intrinsic mechanisms to modulate and regulate different stages of germ cell development under the intimate and indirect cooperation with Sertoli and Leydig cells, respectively. Furthermore, developing germ cells must translocate from the basal to the apical (adluminal) compartment of the seminiferous epithelium. Thus, extensive junction restructuring must occur to assist germ cell movement. Within the seminiferous tubules, three principal types of junctions are found namely anchoring junctions, tight junctions, and gap junctions. Other less studied junctions are desmosome-like junctions and hemidesmosome junctions. With these varieties of junction types, testes are using different regulators to monitor junction turnover. Among the uncountable junction modulators, nitric oxide (NO) is a prominent candidate due to its versatility and extensive downstream network. NO is synthesized by nitric oxide synthase (NOS). Three traditional NOS, specified as endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS), and one testis-specific nNOS (TnNOS) are found in the testis. For these, eNOS and iNOS were recently shown to have putative junction regulation properties. More important, these two NOSs likely rely on the downstream soluble guanylyl cyclase/cGMP/protein kinase G signaling pathway to regulate the structural components at the tight junctions and adherens junctions in the testes. Apart from the involvement in junction regulation, NOS/NO also participates in controlling the levels of cytokines and hormones in the testes. On the other hand, NO is playing a unique role in modulating germ cell viability and development, and indirectly acting on some aspects of male infertility and testicular pathological conditions. Thus, NOS/NO bears an irreplaceable role in maintaining the homeostasis of the microenvironment in the seminiferous epithelium via its different downstream signaling pathways

On the origin and evolution of the material in 67P/Churyumov-Gerasimenko

International audiencePrimitive objects like comets hold important information on the material that formed our solar system. Several comets have been visited by spacecraft and many more have been observed through Earth- and space-based telescopes. Still our understanding remains limited. Molecular abundances in comets have been shown to be similar to interstellar ices and thus indicate that common processes and conditions were involved in their formation. The samples returned by the Stardust mission to comet Wild 2 showed that the bulk refractory material was processed by high temperatures in the vicinity of the early sun. The recent Rosetta mission acquired a wealth of new data on the composition of comet 67P/Churyumov-Gerasimenko (hereafter 67P/C-G) and complemented earlier observations of other comets. The isotopic, elemental, and molecular abundances of the volatile, semi-volatile, and refractory phases brought many new insights into the origin and processing of the incorporated material. The emerging picture after Rosetta is that at least part of the volatile material was formed before the solar system and that cometary nuclei agglomerated over a wide range of heliocentric distances, different from where they are found today. Deviations from bulk solar system abundances indicate that the material was not fully homogenized at the location of comet formation, despite the radial mixing implied by the Stardust results. Post-formation evolution of the material might play an important role, which further complicates the picture. This paper discusses these major findings of the Rosetta mission with respect to the origin of the material and puts them in the context of what we know from other comets and solar system objects

La Galerie / The Gallery

The catalogue for the 1973 exhibition of Canadian works produced by the Still Photography Division of the NFB includes six biographical profiles and five artists statements

In Beam Tests of Implanted Helium Targets

Targets consisting of 3,4He implanted into thin aluminum foils (approximately 100, 200 or 600 ug/cm^2) were prepared using intense (a few uA) helium beams at low energy (approximately 20, 40 or 100 keV). Uniformity of the implantation was achieved by a beam raster across a 12 mm diameter tantalum collimator at the rates of 0.1 Hz in the vertical direction and 1 Hz in the horizontal direction. Helium implantation into the very thin (approximately 80-100 ug/cm^2) aluminum foils failed to produce useful targets (with only approximately 10% of the helium retained) due to an under estimation of the range by the code SRIM. The range of low energy helium in aluminum predicted by Northcliffe and Shilling and the NIST online tabulation are observed on the other hand to over estimate the range of low energy helium ions in aluminum. An attempt to increase the amount of helium by implanting a second deeper layer was also carried out, but it did not significantly increase the helium content beyond the blistering limit (approximately 6 x 10^17 helium/cm^2). The implanted targets were bombarded with moderately intense 4He and 16O beams of 50-100 particle nA . Rutherford Back Scattering of 1.0 and 2.5 MeV proton beams and recoil helium from 15.0 MeV oxygen beams were used to study the helium content and profile before, during and after bombardments. We observed the helium content and profile to be very stable even after a prolonged bombardment (up to two days) with moderately intense beams of 16O or 4He. Helium implanted into thin (aluminum) foils is a good choice for thin helium targets needed, for example, for a measurement of the 3he(a,g)7Be reaction and the associated S34 astrophysical cross section factor (S-factor). Comment: Submitted to the New Online Journal of Instrumentation, JINST. Work Supported by USDOE Grant Nos: DE-FG02-94ER40870, DE-FG02-91ER40609, DE-FG02-97ER41033, and DE-FG02-97ER4104

β\beta-decay of $^{22}O

NESTERA mass-separated $^{12}$C$^{22}$O molecular ion beam from the ISOLDE facility was used to study the decay of neutron-rich $^{22}$O. The experimental results were compared with the results from an earlier experiment and predictions by shell-model calculations using various effective interactions. The mechanism leading to the vanishing decay strength to the first 1$^+$ level of the $^{22}$F nucleus, predicted with the USD effective interaction but not supported by the experimental data, is analysed