Cell cycle-regulated inactivation of endothelial NO synthase through NOSIP-dependent targeting to the cytoskeleton

Nitric oxide (NO) plays a key role in vascular function, cell proliferation, and apoptosis. Proper subcellular localization of endothelial NO synthase (eNOS) is crucial for its activity; however, the role of eNOS trafficking for NO biosynthesis remains to be defined. Overexpression of NOS-interacting protein (NOSIP) induces translocation of eNOS from the plasma membrane to intracellular compartments, thereby impairing NO production. Here we report that endogenous NOSIP reduces the enzymatic capacity of eNOS, specifically in the G(2) phase of the cell cycle by targeting eNOS to the actin cytoskeleton. This regulation is critically dependent on the nucleocytoplasmic shuttling of NOSIP and its cytoplasmic accumulation in the G(2) phase. The predominant nuclear localization of NOSIP depends on a bipartite nuclear localization sequence (NLS) mediating interaction with importin alpha. Mutational destruction of the NLS abolishes nuclear import and interaction with importin alpha. Nuclear export is insensitive to leptomycin B and hence different from the CRM1-dependent default mechanism. Inhibition of NOSIP expression by RNA interference completely abolishes G(2)-specific cytoskeletal association and inhibition of eNOS. These findings describe a novel cell cycle-dependent modulation of endogenous NO levels that are critical to the cell cycle-related actions of NO such as apoptosis or cell proliferation.     

Rapid fluctuations in atrial fibrillatory electrophysiology detected during controlled respiration

Heart rate during sinus rhythm is modulated through the autonomic nervous system, which generates short-term oscillations. The high-frequency components in these oscillations are associated with respiration, causing sinus arrhythmia, mediated by the parasympathetic nervous system. In this study, we evaluated whether slow, controlled respiration causes cyclic fluctuations in the frequency of the fibrillating atria. Eight patients (four women; median age 63 yr, range 53-68 yr) with chronic atrial fibrillation (AF) and third-degree atrioventricular block treated by permanent pacemaker were studied. ECG was recorded during baseline rest, during 0.125-Hz frequency controlled respiration, and finally during controlled respiration after full vagal blockade. We calculated fibrillatory frequency using frequency analysis of the fibrillatory ECG for overlapping 2.5-s segments; spectral analysis of the resulting frequency trend was performed to determine the spectrum of variations of fibrillatory frequency. Normalized spectral power at respiration frequency increased significantly during controlled respiration from 1.4 (0.76-2.0) (median and range) at baseline to 2.7 (1.2-5.8) (P = 0.01). After vagal blockade, the power at respiration frequency decreased to 1.2 (0.23-2.8) (P = 0.01). Controlled respiration causes cyclic fluctuations in the AF frequency in patients with long-duration AF. This phenomenon seems to be related to parasympathetic modulations of the AF refractory period.     

A metamemory perspective on odor naming and identification

A metacognitive perspective is utilized to elucidate why it is so difficult to name common odors and what characterizes the subjective knowledge people have about their actual odor knowledge. Odor-naming failures are often accompanied by strong feelings of knowing (FOK) or feelings of imminent retrieval of what it is that smells. The paper's two experiments investigate FOK judgements and tip of the tongue (TOT) experiences for odor and person names. The data indicate that our inability to correctly name odors are typically not due to the often proposed uniquely poor association between odors and their proper names, but rather due to failures to identify the odors, that is, failures to know 'what it is'. It was also found that (i) TOT experiences are very unusual for odor names and more so than for person names; (ii) FOK judgements about odor names are significantly less predictive of later retrieval than equivalent judgements about names of persons; (iii) FOK judgements were highly correlated with the familiarity of the cue (odor or picture of famous person), rendering some support for the idea that FOK judgements are based on the perceived familiarity of the cue triggering the FOK; and (iv) the idea that FOK judgements are based on the amount of available information about the sought-for memory (accessibility theory) was also supported.     

Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization

Direct cultivation-independent sequence retrieval of unidentified bacteria from histological tissue sections has been limited by the difficulty of selectively isolating specific bacteria from a complex environment. Here, a new DNA isolation approach is presented for prokaryotic cells. By this method, a potentially pathogenic strain of the genus Brachyspira from formalin-fixed human colonic biopsies were visualized by fluorescence in situ hybridization (FISH) with a 16S rRNA-targeting oligonucleotide probe, followed by laser capture microdissection (LCM) of the targeted cells. Direct 16S rRNA gene PCR was performed from the dissected microcolonies, and the subsequent DNA sequence analysis identified the dissected bacterial cells as belonging to the Brachyspira aalborgi cluster 1. The advantage of this technique is the ability to combine the histological recognition of the specific bacteria within the tissue with molecular analysis of 16S rRNA gene or other genes of interest. This method is widely applicable for the identification of noncultivable bacteria and their gene pool from formalin-fixed paraffin-embedded tissue samples.     

Brevican-deficient mice display impaired hippocampal CA1 long-term potentiation but show no obvious deficits in learning and memory

Brevican is a brain-specific proteoglycan which is found in specialized extracellular matrix structures called perineuronal nets. Brevican increases the invasiveness of glioma cells in vivo and has been suggested to play a role in central nervous system fiber tract development. To study the role of brevican in the development and function of the brain, we generated mice lacking a functional brevican gene. These mice are viable and fertile and have a normal life span. Brain anatomy was normal, although alterations in the expression of neurocan were detected. Perineuronal nets formed but appeared to be less prominent in mutant than in wild-type mice. Brevican-deficient mice showed significant deficits in the maintenance of hippocampal long-term potentiation (LTP). However, no obvious impairment of excitatory and inhibitory synaptic transmission was found, suggesting a complex cause for the LTP defect. Detailed behavioral analysis revealed no statistically significant deficits in learning and memory. These data indicate that brevican is not crucial for brain development but has restricted structural and functional roles.     

Proteomics of Synechocystis sp. strain PCC 6803: identification of plasma membrane proteins

Cyanobacteria are unique prokaryotes since they in addition to outer and plasma membranes contain the photosynthetic membranes (thylakoids). The plasma membranes of Synechocystis 6803, which can be completely purified by density centrifugation and polymer two-phase partitioning, have been found to be more complex than previously anticipated, i.e. they appear to be essential for assembly of the two photosystems. A proteomic approach for the characterization of cyanobacterial plasma membranes using two-dimensional gel electrophoresis and mass spectrometry analysis revealed a total of 57 different membrane proteins of which 17 are integral membrane spanning proteins. Among the 40 peripheral proteins 20 are located on the periplasmic side of the membrane, while 20 are on the cytoplasmic side. Among the proteins identified are subunits of the two photosystems as well as Vipp1, which has been suggested to be involved in vesicular transport between plasma and thylakoid membranes and is thus relevant to the possibility that plasma membranes are the initial site for photosystem biogenesis. Four subunits of the Pilus complex responsible for cell motility were also identified as well as several subunits of the TolC and TonB transport systems. Several periplasmic and ATP-binding proteins of ATP-binding cassette transporters were also identified as were two subunits of the F(0) membrane part of the ATP synthase.     

17S,20S-Methanofusidic acid,a new potent semi-synthetic fusidane antibiotic

A novel fusidic acid type antibiotic having the side chain linked to the tetracyclic ring system via a spiro-cyclopropane system is described. 17S,20S-Methanofusidic acid is obtained by an efficient synthetic route including cyclopropanation of the Δ17(20) bond with attack solely from the least hindered -face. The spiro-cyclopropane system orients the side chain into a bioactive conformational space. The new 17S,20S-methanofusidic acid exerts antibacterial activity against several Gram-positive species with potency essentially equal to natural fusidic acid.     

Mechanisms for lateral turns in lamprey in response to descending unilateral commands: a modeling study

 Straight locomotion in the lamprey is, at the segmental level, characterized by alternating bursts of motor activity with equal duration and spike frequency on the left and the right sides of the body. Lateral turns are characterized by three main changes in this pattern: (1) in the turn cycle, the spike frequency, burst duration, and burst proportion (burst duration/cycle duration) increase on the turning side; (2) the cycle duration increases in both the turn cycle and the succeeding cycle; and (3) in the cycle succeeding the turn cycle, the burst duration increases on the non-turning side (rebound). We investigated mechanisms for the generation of turns in single-segment models of the lamprey locomotor spinal network. Activation of crossing inhibitory neurons proved a sufficient mechanism to explain all three changes in the locomotor rhythm during a fictive turn. Increased activation of these cells inhibits the activity of the opposite side during the prolonged burst of the turn cycle, and slows down the locomotor rhythm. Secondly, this activation of the crossing inhibitory neurons is accompanied by an increased calcium influx into the cells. This gives a suppressed activity on the turning side and a contralateral rebound after the turn, through activation of calcium-dependent potassium channels. Received: 28 June 2000 / Accepted for publication: 10 May 2001     

Myogenic tone,reactivity, and forced dilatation: a three-phase model of in vitro arterial myogenic behavior

Myogenic behavior, prevalent in resistance arteries and arterioles, involves arterial constriction in response to intravascular pressure. This process is often studied in vitro by using cannulated, pressurized arterial segments from different regional circulations. We propose a comprehensive model for myogenicity that consists of three interrelated but dissociable phases: 1) the initial development of myogenic tone (MT), 2) myogenic reactivity to subsequent changes in pressure (MR), and 3) forced dilatation at high transmural pressures (FD). The three phases span the physiological range of transmural pressures (e.g., MT, 40-60 mmHg; MR, 60-140 mmHg; FD, >140 mmHg in cerebral arteries) and are characterized by distinct changes in cytosolic calcium ([Ca(2+)](i)), which do not parallel arterial diameter or wall tension, and therefore suggest the existence of additional regulatory mechanisms. Specifically, the development of MT is accompanied by a substantial (200%) elevation in [Ca(2+)](i) and a reduction in lumen diameter and wall tension, whereas MR is associated with relatively small [Ca(2+)](i) increments (<20% over the entire pressure range) despite considerable increases in wall tension and force production but little or no change in diameter. FD is characterized by a significant additional elevation in [Ca(2+)](i) (>50%), complete loss of force production, and a rapid increase in wall tension. The utility of this model is that it provides a framework for comparing myogenic behavior of vessels of different size and anatomic origin and for investigating the underlying cellular mechanisms that govern vascular smooth muscle mechanotransduction and contribute to the regulation of peripheral resistance.     

Vascular smooth muscle cell stress as a determinant of cerebral artery myogenic tone

Although the level of myogenic tone (MT) varies considerably from vessel to vessel, the regulatory mechanisms through which the actual diameter set point is determined are not known. We hypothesized that a unifying principle may be the equalization of active force at the contractile filament level, which would be reflected in a normalization of wall stress or, more specifically, media stress. Branched segments of rat cerebral arteries ranging from <50 microm to >200 microm in diameter were cannulated and held at 60 mmHg with the objectives of: 1) evaluating the relationship between arterial diameter and the extent of myogenic tone, 2) determining whether differences in MT correlate with changes in cytosolic calcium ([Ca(2+)](i)), and 3) testing the hypothesis that a normalization of wall or media stress occurs during the process of tone development. The level of MT increased significantly as vessel size decreased. At 60 mmHg, vascular smooth muscle [Ca(2+)](i) concentrations were similar in all vessels studied (averaging 230 +/- 9.2 nM) and not correlated with vessel size or the extent of tone. Wall tension increased with increasing arterial size, but wall stress and media stress were similar in large versus small arteries. Media stress, in particular, was quite uniform in all vessels studied. Both morphological and calcium data support the concept of equalization of media stress (and, hence, vascular smooth muscle cell stress and force) as an underlying mechanism in determining the level of tone present in any particular vessel. The equalization of active (vascular smooth muscle cell) stress may thus explain differences in MT observed in the different-sized vessels constituting the arterial network and provide a link between arterial structure and function, in both short- and long-term (hypertension) pressure adaptation.