Ca2+ -dependent interaction of S100A1 with F1-ATPase leads to an increased ATP content in cardiomyocytes

S100A1, a Ca(2+)-sensing protein of the EF-hand family that is expressed predominantly in cardiac muscle, plays a pivotal role in cardiac contractility in vitro and in vivo. It has recently been demonstrated that by restoring Ca(2+) homeostasis, S100A1 was able to rescue contractile dysfunction in failing rat hearts. Myocardial contractility is regulated not only by Ca(2+) homeostasis but also by energy metabolism, in particular the production of ATP. Here, we report a novel interaction of S100A1 with mitochondrial F(1)-ATPase, which affects F(1)-ATPase activity and cellular ATP production. In particular, cardiomyocytes that overexpress S100A1 exhibited a higher ATP content than control cells, whereas knockdown of S100A1 expression decreased ATP levels. In pull-down experiments, we identified the alpha- and beta-chain of F(1)-ATPase to interact with S100A1 in a Ca(2+)-dependent manner. The interaction was confirmed by colocalization studies of S100A1 and F(1)-ATPase and the analysis of the S100A1-F(1)-ATPase complex by gel filtration chromatography. The functional impact of this association is highlighted by an S100A1-mediated increase of F(1)-ATPase activity. Consistently, ATP synthase activity is reduced in cardiomyocytes from S100A1 knockout mice. Our data indicate that S100A1 might play a key role in cardiac energy metabolism.     

Multilayering and loss of apical polarity in MDCK cells transformed with viral K-ras

The effects of viral Kirsten ras oncogene expression on the polarized phenotype of MDCK cells were investigated. Stable transformed MDCK cell lines expressing the v-K-ras oncogene were generated via infection with a helper-independent retroviral vector construct. When grown on plastic substrata, transformed cells formed continuous monolayers with epithelial-like morphology. However, on permeable filter supports where normal cells form highly polarized monolayers, transformed MDCK cells detached from the substratum and developed multilayers. Morphological analysis of the multilayers revealed that oncogene expression perturbed the polarized organization of MDCK cells such that the transformed cells lacked an apical--basal axis around which the cytoplasm is normally organized. Evidence for selective disruption of apical membrane polarity was provided by immunolocalization of membrane proteins; a normally apical 114-kD protein was randomly distributed on the cell surface in the transformed cell line, whereas normally basolateral proteins remained exclusively localized to areas of cell contact and did not appear on the free cell surface. The discrete distribution of the tight junction-associated ZO-1 protein as well as transepithelial resistance and flux measurements suggested that tight junctions were also assembled. These findings indicate that v-K-ras transformation alters cell-substratum and cell-cell interactions in MDCK cells. Furthermore, v-K-ras expression perturbs apical polarization but does not interfere with the development of a basolateral domain, suggesting that apical and basolateral polarity in epithelial cells may be regulated independently.     

Peptides isolated from human liver with specific inhibitory effects on reassociation/reactivation of in vitro dissociated lactic dehydrogenase (LDH-M4 and −H4) isozymes

Two different peptides have been purified from human liver, similar to those previously reported (Schoenenberger, G.A., and Wacker, W.E.C. (1966) Biochemistry 5, 1375–1379) to be present in human urine, which may serve as metabolic regulators of lactate dehydrogenase (EC 1.1 1.27) isoenzymes (LDH-M₄ = muscle type; LDH-H₄ = heart type). By trichloroacetic acid precipitation, ultrafiltration, Sephadex G-25 and Bio-Gel P-2 columns, affinity chromatography on immobilized LDH-isozymes and HPLC two peptides which differed with respect to molecular weight, retention on the affinity columns and amino acid composition were isolated. No effect was observed when native, tetrameric lactate dehydrogenase was incubated with these peptides. However, when lactate dehydrogenase was dissociated to monomers at low pH and allowed to reassociate by adjusting the pH to 7.5 complete inhibition of the reactivation occurred when the inhibitors were incubated together with respective reassociating monomeric isozymes. The two peptides showed no cross-specificity, i.e. each peptide exhibited inhibitory activity only on one of the two isozymes LDH-M₄ or LDH-H₄. From the amino acid analyses, gel-filtration and PAGE + SDS, molecular weight of 1800 for the M₄ and ≈2700 for the H₄ inhibitor were calculated. An apparent K_i of ≈3 × 10^?5 mM for the H₄ and ≈7 × 10^?5 mM for the H₄ inhibitor was estimated. The interaction of the inhibitors with the enzyme system showed strong cooperativity with Hill coefficients of 2.9 (LDH-M₄-specific) and 2.4 (LDH-H₄-specific). Mathematical modelling of the reassociation and reactivation of lactate dehydrogenase and its specific inhibition by the peptides led to the conclusion that the peptides reacts with monomers, dimers or a transition state during the tetramerisation process. k₁ for the dimerisation step of M₄ = 2.0 × 10⁵ M^?1 · s^?1 and of H₄ = 8.2 × 10⁴ M^?1 · s^?1; k₂ for the tetramerisation step of M₄ = 2.8 × 10⁵ M^?1 · s^?1 and of H₄ = 1.2 × 10⁵ · M^?1 · s^?1, were calculated, the second step still being the faster one.     

Kinesin-1-mediated capsid disassembly and disruption of the nuclear pore complex promote virus infection

Many viruses deliver their genomes into the host cell nucleus for replication. However, the size restrictions of the nuclear pore complex (NPC), which regulates the passage of proteins, nucleic acids, and solutes through the nuclear envelope, require virus capsid uncoating before viral DNA can access the nucleus. We report a microtubule motor kinesin-1-mediated and NPC-supported mechanism of adenovirus uncoating. The capsid binds to the NPC filament protein Nup214 and kinesin-1 light-chain Klc1/2. The nucleoporin Nup358, which is bound to Nup214/Nup88, interacts with the kinesin-1 heavy-chain Kif5c to indirectly link the capsid to the kinesin motor. Kinesin-1 disrupts capsids docked at Nup214, which compromises the NPC and dislocates nucleoporins and capsid fragments into the cytoplasm. NPC disruption increases nuclear envelope permeability as indicated by the nuclear influx of large cytoplasmic?dextran polymers. Thus, kinesin-1 uncoats viral DNA?and compromises NPC integrity, allowing viral genomes nuclear access to promote infection.     

Neuronal profilin isoforms are addressed by different signalling pathways

Profilins are prominent regulators of actin dynamics. While most mammalian cells express only one profilin, two isoforms, PFN1 and PFN2a are present in the CNS. To challenge the hypothesis that the expression of two profilin isoforms is linked to the complex shape of neurons and to the activity-dependent structural plasticity, we analysed how PFN1 and PFN2a respond to changes of neuronal activity. Simultaneous labelling of rodent embryonic neurons with isoform-specific monoclonal antibodies revealed both isoforms in the same synapse. Immunoelectron microscopy on brain sections demonstrated both profilins in synapses of the mature rodent cortex, hippocampus and cerebellum. Both isoforms were significantly more abundant in postsynaptic than in presynaptic structures. Immunofluorescence showed PFN2a associated with gephyrin clusters of the postsynaptic active zone in inhibitory synapses of embryonic neurons. When cultures were stimulated in order to change their activity level, active synapses that were identified by the uptake of synaptotagmin antibodies, displayed significantly higher amounts of both isoforms than non-stimulated controls. Specific inhibition of NMDA receptors by the antagonist APV in cultured rat hippocampal neurons resulted in a decrease of PFN2a but left PFN1 unaffected. Stimulation by the brain derived neurotrophic factor (BDNF), on the other hand, led to a significant increase in both synaptic PFN1 and PFN2a. Analogous results were obtained for neuronal nuclei: both isoforms were localized in the same nucleus, and their levels rose significantly in response to KCl stimulation, whereas BDNF caused here a higher increase in PFN1 than in PFN2a. Our results strongly support the notion of an isoform specific role for profilins as regulators of actin dynamics in different signalling pathways, in excitatory as well as in inhibitory synapses. Furthermore, they suggest a functional role for both profilins in neuronal nuclei.     

Relative microelastic mapping of living cells by atomic force microscopy.

The spatial and temporal changes of the mechanical properties of living cells reflect complex underlying physiological processes. Following these changes should provide valuable insight into the biological importance of cellular mechanics and their regulation. The tip of an atomic force microscope (AFM) can be used to indent soft samples, and the force versus indentation measurement provides information about the local viscoelasticity. By collecting force-distance curves on a time scale where viscous contributions are small, the forces measured are dominated by the elastic properties of the sample. We have developed an experimental approach, using atomic force microscopy, called force integration to equal limits (FIEL) mapping, to produce robust, internally quantitative maps of relative elasticity. FIEL mapping has the advantage of essentially being independent of the tip-sample contact point and the cantilever spring constant. FIEL maps of living Madine-Darby canine kidney (MDCK) cells show that elasticity is uncoupled from topography and reveal a number of unexpected features. These results present a mode of high-resolution visualization in which the contrast is based on the mechanical properties of the sample.     

Delta Sleep-Inducing Peptide Modulates the Stimulation of Rat Pineal N-Acetyltransferase Activity by Involving the α1 -Adrenergic Receptor

Delta sleep-inducing peptide (DSIP) has been isolated and characterized by its capacity to enhance delta sleep in rabbits. Up to now, sleep was the main target of DSIP research, but different extra-sleep effects of the peptide have been reported as well. Several mechanisms of action have been proposed, though no convincing evidence for any of them has been obtained so far. We recently detected that DSIP reduced the nocturnal increase of N-acetyltransferase (NAT) activity in rat pineal in a dose-dependent manner. The activity of this enzyme is known to be induced by adrenergic agonists and several studies have suggested that stimulation of alpha 1-adrenergic receptors potentiates the "basic" effect of beta-receptors. DSIP in the range between 20 and 300 nM significantly enhanced NAT activity induced by 10(-6) M norepinephrine in vitro, and a similar effect was observed with 2nMP-DSIP, a phosphorylated analog. Incubation with prazosin eliminated the enhancement, whereas propranolol reduced norepinephrine stimulation that was still increased by P-DSIP and probably DSIP. It was concluded that the sleep-peptide and its analog modulate the alpha 1-adrenergic receptor of rat pineal in its response to adrenergic agonists. The same mechanism may also be responsible for other biological activities of DSIP such as sleep-induction and stress-tolerance.     

Separation of tryptophan from DSIP, a Trp-nonapeptide, by adsorption to aluminum oxide

Delta sleep-inducing peptide (DSIP) has been found to induce sleep as well as extra-sleep effects. Although the presence of endogenous DSIP-like material has been demonstrated, the metabolic fate of injected DSIP has not been clarified so far. A major obstacle in monitoring degradation of DSIP has been the lack of an easy method to separate DSIP from tryptophan (Trp). Cleavage of the N-terminal Trp apparently represents the first and most important step in the metabolism of the peptide. Adsorption to aluminum oxide has been found to separate the two compounds and optimal conditions for the separation are described. Quantitative determination of the degradation of DSIP in plasma or serum is now rapidly achieved. The method should help to advance metabolic studies of DSIP. Other applications such as extraction of DSIP from solutions are also possible.     

Resting and post bronchial challenge testing carbon dioxide partial pressure in individuals with and without asthma

Objective

There is conflicting evidence about resting carbon dioxide levels in asthmatic individuals. We wanted to determine if transcutaneously measured carbon dioxide levels prior and during bronchial provocation testing differ according to asthma status reflecting dysfunctional breathing.

Methods

We investigated active firefighters and policemen by means of a validated questionnaire on respiratory symptoms, spirometry, bronchial challenge testing with methacholine (MCT) and measurement of transcutaneous blood carbon dioxide partial pressure (PtcCO₂) at rest prior performing spirometry, one minute and five minutes after termination of MCT. A respiratory physician blinded to the PtcCO₂ results assigned a diagnosis of asthma after reviewing the available study data and the files of the workers medical screening program.

Results

The study sample consisted of 128 male and 10 female individuals. Fifteen individuals (11%) had physician-diagnosed asthma. There was no clinically important difference in median PtcCO₂ at rest, one and five minutes after recovery from MCT in asthmatics compared to non-asthmatics (35.6 vs 35.7 mmHg, p = 0.466; 34.7 vs 33.4 mmHg, p = 0.245 and 37.4 vs 36.4 mmHg, p = 0.732). The median drop in PtcCO₂ during MCT and the increase after MCT was lower in asthmatics compared to non-asthmatics (0.1 vs 3.2 mmHg, p = 0.014 and 1.9 vs 2.9 mmHg, p = 0.025).

Conclusions

PtcCO₂ levels at rest prior and during recovery after MCT do not differ in individuals with or without physician diagnosed asthma. The fall and subsequent increase in PtcCO₂ levels are higher in non-asthmatics than in asthmatics and seems to be related with increased number of respiratory maneuvers during MCT.