Quaternary structure controls ligand dynamics in soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is the mammalian endogenous nitric oxide (NO) receptor. The mechanisms of activation and deactivation of this heterodimeric enzyme are unknown. For deciphering them, functional domains can be overexpressed. We have probed the dynamics of the diatomic ligands NO and CO within the isolated heme domain β(1)(190) of human sGC by piconanosecond absorption spectroscopy. After photo-excitation of nitrosylated sGC, only NO geminate rebinding occurs in 7.5 ps. In β(1)(190), both photo-dissociation of 5c-NO and photo-oxidation occur, contrary to sGC, followed by NO rebinding (7 ps) and back-reduction (230 ps and 2 ns). In full-length sGC, CO geminate rebinding to the heme does not occur. In contrast, CO geminately rebinds to β(1)(190) with fast multiphasic process (35, 171, and 18 ns). We measured the bimolecular association rates k(on) = 0.075 ± 0.01 × 10(6) M(-1) · S(-1) for sGC and 0.83 ± 0.1 × 10(6) M(-1) · S(-1) for β(1)(190). These different dynamics reflect conformational changes and less proximal constraints in the isolated heme domain with respect to the dimeric native sGC. We concluded that the α-subunit and the β(1)(191-619) domain exert structural strains on the heme domain. These strains are likely involved in the transmission of the energy and relaxation toward the activated state after Fe(2+)-His bond breaking. This also reveals the heme domain plasticity modulated by the associated domains and subunit.     

Hsp70 does not mediate the hyperthermia-associated hyporesponsiveness to angiotensin in isolated rat portal veins

In rat portal veins (RPV) isolated from septic rats, we previously showed that the contractile response to angiotensin II (AT(II)) was significantly decreased and that the vascular failure was correlated with the severity of the disease. We hypothesized that hyperthermia might be one of the factors responsible for the vascular failure. Moreover, hyperthermia should concomitantly increase heat shock proteins (Hsps) expression. We then compared the vascular contractility and the heat shock protein 70 (Hsp70) expression in RPV incubated at 37 degrees C and 39.5 degrees C and sought for a relationship between both events. In our experimental model, hyperthermia increased the Hsp70 expression and decreased the contractile response to AT(II). Incorporation of the Hsp70 antisense oligonucleotide in RPV blocked the increase in Hsp70 expression but had no consequence on the contractile response to AT(II). In conclusion, hyperthermia increases Hsp70 expression but does not mediate the decreased response to AT(II). Hsp70 overexpression has no effect on the actin-myosin interaction in vascular smooth muscle.     

Direct evidence of lower viral replication rates in vivo in human immunodeficiency virus type 2 (HIV-2) infection than in HIV-1 infection

Studies have shown that human immunodeficiency virus type 2 (HIV-2) is less pathogenic than HIV-1, with a lower rate of disease progression. Similarly, plasma viral loads are lower in HIV-2 infection, suggesting that HIV-2 replication is restricted in vivo in comparison to that of HIV-1. However, to date, in vivo studies characterizing replication intermediates in the viral life cycle of HIV-2 have been limited. In order to test the hypothesis that HIV-2 has a lower replication rate in vivo than HIV-1 does, we quantified total viral DNA, integrated proviral DNA, cell-associated viral mRNA, and plasma viral loads in peripheral blood samples from groups of therapy-na?ve HIV-1-infected (n = 21) and HIV-2-infected (n = 18) individuals from Dakar, Senegal, with CD4(+) T-cell counts of >200/microl. Consistent with our previous findings, total viral DNA loads were similar between HIV-1 and HIV-2 and plasma viral loads were higher among HIV-1-infected individuals. Proportions of DNA in the integrated form were also similar between these viruses. In contrast, levels of viral mRNA were lower in HIV-2 infection. Our study indicates that HIV-2 is able to establish a stable, integrated proviral infection in vivo, but that accumulation of viral mRNA is attenuated in HIV-2 infection relative to that in HIV-1 infection. The differences in viral mRNA are consistent with the differences in plasma viral loads between HIV-1 and HIV-2 and suggest that lower plasma viral loads, and possibly the attenuated pathogenesis of HIV-2, can be explained by lower rates of viral replication in vivo.     

A High-Resolution Genetic Map of Mouse Chromosome 5 Encompassing the Reeler (rl) Locus

Using interspecific crosses between BALB/c and Mus spretus (SEG) mice, the murine reeler (rl) gene was mapped to the proximal region of chromosome 5 between the hepatocyte growth factor gene (Hgf) and the D5Mit66 microsatellite. The following order was defined: (centromere)-Cchl2a/Hgf-D5Mit1-D5Nam1/D5Nam2 - rl/D5Mit61 - D5Mit72 - Xmv45 - Htr5a - Peplb - D5Nam3-D5Mit66. Estimated distances between reeler and the nearest flanking markers D5Nam1 and D5Mit72 are 1.5 and 1.0 cM, respectively (95% confidence level), suggesting that the region could be physically mapped using a manageable number of YAC clones.     

A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands

Helicases, enzymes that unwind DNA or RNA structure, are present in the cell nucleus and in the mitochondrion. Although the majority of the helicases unwind DNA or RNA duplexes, some of these proteins are known to resolve unusual structures such as G-quadruplexes (G4) in vitro. G4 may form stable barrier to the progression of molecular motors tracking on DNA. Monitoring G4 unwinding by these enzymes may reveal the mechanisms of the enzymes and provides information about the stability of these structures. In the experiments presented herein, we developed a reliable, inexpensive and rapid fluorescence-based technique to monitor the activity of G4 helicases in real time in a 96-well plate format. This system was used to screen a series of G4 structures and G4 binders for their effect on the Pif1 enzyme, a 5′ to 3′ DNA helicase. This simple assay should be adaptable to analysis of other helicases and G4 structures.     

Determination of the G + C content of twoSyntrophus buswellii strains by ultracentrifugation techniques

An anaerobic syntrophic bacterial culture degrading benzoate was isolated from a river sediment. The syntrophic organism was grown in coculture in the presence of a hydrogenotrophic strain,Desulfovibrio fructosovorans orMethanospirillum hungatei. The G+C content of the syntrophic benzoate degrader determined by density gradient ultracentrifugation was similar to that ofSyntrophus buswellii (54.3%). A method ensuring the G+C% determination of syntrophic bacteria is presented.     

Regulation of a COPII component by cytosolic O-glycosylation during mitosis

Endoplasmic reticulum (ER)-to-Golgi transport is blocked in mammalian cells during mitosis; however, the mechanism underlying this blockade remains unknown. Since COPII proteins are involved in this transport pathway, we investigated at the biochemical level post-translational modifications of COPII components during the course of mitosis that could be linked to inhibition of ER-to-Golgi transport. By comparing biochemical properties of cytosolic COPII components during interphase and mitosis, we found that Sec24p isoforms underwent post-translational modifications resulting in an increase in their apparent molecular weight. No such modification was observed for the other COPII components Sec23p, Sec13p, Sec31p or Sar1p. Analyzing in more details Sec24p isoforms in interphase and mitotic conditions, we found that the interphase form of Sec24p was O-N-acetylglucosamine modified, a feature lost upon entering into mitosis. This mitotic deglycosylation was coupled to Sec24p phosphorylation, a feature likely responsible for the increase in apparent molecular weight of these molecules. These modifications correlated with an alteration in the membrane binding properties of Sec24p. These data suggest that when entering into mitosis, the COPII component Sec24p is simultaneously deglycosylated and phosphorylated, a process which may contribute to the observed mitotic ER-to-Golgi traffic block.     

Self-Organization in Primates: Understanding the Rules Underlying Collective Movements

Patterns of collective movements, such as the distribution of leadership and the organization of individuals, may be either homogeneously (no leader, no specific order), or heterogeneously (1 or several leaders, and a highly stable order) distributed. Members of a group need to synchronize their activities and coordinate their movements, despite the fact that they differ in physiological or morphological traits. The degree of difference in these traits may affect their decision-making strategy. We demonstrate how a theoretical model based on a variation of a simple mimetic rule, i.e., an amplification process, can result in each of the various collective movement patterns and decision-making strategies observed in primates and other species. We consider cases in which 1) the needs of different individuals are identical and social relationships are equivalent between group members, 2) the needs of individuals are different and social relationships are equivalent, and 3) the needs of individuals are different and social relationships are different. Finally, 4) we assess how the synergy between 2 mimetism rules, specifically the probability of joining a movement and that of canceling an initiation, allows group members to stay synchronized and cohesive. Our models suggest that similar self-organized processes have been selected as reliable and well-adapted means for optimal collective decisions across species, despite differences in their biological and social characteristics.     

Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, IX, and XII with N-hydroxysulfamides--a new zinc-binding function in the design of inhibitors

A small library of N-hydroxysulfamides was synthesized by an original approach in order to investigate whether this zinc-binding function is efficient for the design of inhibitors targeting the cytosolic (hCA I and II) and transmembrane, tumor-associated (hCA IX and XII) isozymes of carbonic anhydrase (CA, EC 4.2.1.1). The parent derivative, N-hydroxysulfamide was a more potent inhibitor as compared to sulfamide or sulfamic acid against all isozymes, with inhibition constants in the range of 473 nM-4.05 microM. Its substituted n-decyl-, n-dodecyl-, benzyl-, and biphenylmethyl-derivatives were less inhibitory against hCA I (K(I)s in the range of 5.8-8.2 microM) but more inhibitory against hCA II (K(I)s in the range of 50.5-473 nM). The same situation was true for the tumor-associated isozymes, with K(I)s in the range of 353-790 nM against hCA IX and 372-874 nM against hCA XII. Some sulfamides/sulfamates possessing similar substitution patterns have also been investigated for the inhibition of these isozymes, being shown that in some particular cases sulfamides are more efficient inhibitors as compared to the corresponding sulfamates. Potent CA inhibitors targeting the cytosolic or tumor-associated CA isozymes can thus be designed from various classes of sulfonamides, sulfamides, or sulfamates and their derivatives, considering the extensive interactions in which the inhibitor and the enzyme active site are engaged, based on recent X-ray crystallographic data.     

Role of membrane potential in vasomotion of isolated pressurized rat arteries

Vasomotion, the phenomenon of vessel diameter oscillation, regulates blood flow and resistance. The main parameters implicated in vasomotion are particularly the membrane potential and the cytosolic free calcium in smooth muscle cells. In this study, these parameters were measured in rat perfused-pressurized mesenteric artery segments. The application of norepinephrine (NE) caused rhythmic diameter contractions and membrane potential oscillations (amplitude; 5.3 +/- 0.3 mV, frequency; 0.09 +/- 0.01 Hz). Verapamil (1 microM) abolished this vasomotion. During vasomotion, 10(-5) M ouabain (Na(+)-K(+) ATPase inhibitor) decreased the amplitude of the electrical oscillations but not their frequency (amplitude; 3.7 +/- 0.3 mV, frequency; 0.08 +/- 0.002 Hz). Although a high concentration of ouabain (10(-3) M) (which exhibits non-specific effects) abolished both electrical membrane potential oscillations and vasomotion, we conclude that the Na+-K+ ATPase could not be implicated in the generation of the membrane potential oscillations. We conclude that in rat perfused-pressurized mesenteric artery, the slow wave membrane type of potential oscillation by rhythmically gating voltage-dependent calcium channels, is responsible for the oscillation of intracellular calcium and thus vasomotion.