Direct dynamin-actin interactions regulate the actin cytoskeleton

The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs.     

Using network dynamic fMRI for detection of epileptogenic foci

Background

Epilepsy is one of the most prevalent neurological disorders. It remains medically intractable for about one-third of patients with focal epilepsy, for whom precise localization of the epileptogenic zone responsible for seizure initiation may be critical for successful surgery. Existing fMRI literature points to widespread network disturbances in functional connectivity. Per previous scalp and intracranial EEG studies and consistent with excessive local synchronization during interictal discharges, we hypothesized that, relative to same regions in healthy controls, epileptogenic foci would exhibit less chaotic dynamics, identifiable via entropic analyses of resting state fMRI time series.

Methods

In order to first validate this hypothesis on a cohort of patients with known ground truth, here we test individuals with well-defined epileptogenic foci (left mesial temporal lobe epilepsy). We analyzed voxel-wise resting-state fMRI time-series using the autocorrelation function (ACF), an entropic measure of regulation and feedback, and performed follow-up seed-to-voxel functional connectivity analysis. Disruptions in connectivity of the region exhibiting abnormal dynamics were examined in relation to duration of epilepsy and patients’ cognitive performance using a delayed verbal memory recall task.

Results

ACF analysis revealed constrained (less chaotic) functional dynamics in left temporal lobe epilepsy patients, primarily localized to ipsilateral temporal pole, proximal to presumed focal points. Autocorrelation decay rates differentiated, with 100 % accuracy, between patients and healthy controls on a subject-by-subject basis within a leave-one-subject out classification framework. Regions identified via ACF analysis formed a less efficient network in patients, as compared to controls. Constrained dynamics were linked with locally increased and long-range decreased connectivity that, in turn, correlated significantly with impaired memory (local left temporal connectivity) and epilepsy duration (left temporal – posterior cingulate cortex connectivity).

Conclusions

Our current results suggest that data driven functional MRI methods that target network dynamics hold promise in providing clinically valuable tools for identification of epileptic regions.

     

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. TraDΔN130 stimulated the TraI DNA transesterase activity apparently via interactions on both the protein and the DNA levels. TraM, a protein interaction partner of TraD, also increased DNA transesterase activity in vitro. The mechanism may involve altered DNA conformation as TraM induced underwinding of oriT plasmid DNA in vivo (ΔL_k = −4). Permanganate mapping of the positions of duplex melting due to relaxosome assembly with TraDΔN130 on supercoiled DNA in vitro confirmed localized unwinding at nic but ruled out formation of an open complex compatible with initiation of the TraI helicase activity. These data link relaxosome regulation to the T4CP and support the model that a committed step in the initiation of DNA export requires activation of TraI helicase loading or catalysis.Type IV secretion systems (T4SS) in gram-negative bacteria mediate translocation of macromolecules out of the bacterial cell (14). The transmission of effector proteins and DNA into plant cells or other bacteria via cell-cell contact is one example of their function, and conjugation systems as well as the transferred DNA (T-DNA) delivery system of the phytopathogen Agrobacterium tumefaciens are prototypical of the T4SS family. Macromolecular translocation is achieved by a membrane-spanning protein machinery comprised of 12 gene products, VirB1 to VirB11 and an associated factor known as the coupling protein (VirD4) (66). The T4SS-associated coupling protein (T4CP) performs a crucial function in recognition of appropriate secretion substrates and governing entry of those molecules to the translocation pathway (7, 8, 10, 30, 41). In conjugation systems substrate recognition is applied to the relaxosome, a nucleoprotein complex of DNA transfer initiator proteins assembled specifically at the plasmid origin of transfer (oriT). In current models, initiation of the reactions that provide the single strand of plasmid (T-strand) DNA for secretion to recipient bacteria is expected to resemble the initiation of chromosomal replication (for reviews, see references 18, 54, and 81). Controlled opening of the DNA duplex is required to permit entry of the DNA processing machinery. The task of remodeling the conjugative oriT is generally ascribed to two or three relaxosome auxiliary factors, of host and plasmid origin, which occupy specific DNA binding sites at this locus. Intrinsic to the relaxosome is also a site- and strand-specific DNA transesterase activity that breaks the phosphodiester backbone at nic (5). Upon cleavage, the transesterase enzyme (also called relaxase) forms a reversible phosphotyrosyl linkage to the 5′ end of the DNA. Duplex unwinding initiating from this site produces the single-stranded T strand to be exported. A wealth of information is available supporting the importance of DNA sequence recognition and binding by relaxosome components at oriT to the transesterase reaction in vitro and for effective conjugative transfer (for reviews, see references 18, 54, and 81). On the other hand, the mechanisms controlling release of the 3′-OH generated at nic and the subsequent DNA unwinding stage remain obscure.Equally little is known about the process of nucleoprotein uptake by the transport channel. DNA-independent translocation of the relaxases TrwC (R388), MobA (RSF1010), and VirD2 (Ti plasmid) has been demonstrated; thus, current models propose that the relaxase component of the protein-DNA adduct is the substrate actively secreted by the transport system after interaction with the T4CP (42, 66). Cotransport of the covalently linked single-stranded T strand occurs concurrently (42). The mechanisms underlying relaxosome recognition by T4CPs are not understood. Direct interactions have been observed biochemically between the RP4 TraG protein and relaxase proteins of the cognate plasmid (65) and heterologous relaxosomes that it mobilizes (73, 76). TrwB of R388 interacts in vitro with relaxase TrwC and an auxiliary component, TrwA (44). TraD proteins of plasmid R1 and F are known to interact with the auxiliary relaxosome protein TraM (20) via a cluster of C-terminal amino acids (3, 62). Extensive mutagenic analyses (45) plus recent three-dimensional structural data for a complex of the TraM tetramerization domain and the C-terminal tail of TraD (46) have provided more detailed models for the intermolecular contacts involved in recognition.Application of the Cre recombinase assay for translocation of conjugative relaxases as well as effector proteins to eukaryotic cells is currently the most promising approach to elucidate protein motifs recognized by T4CPs (56, 68, 78, 79). Despite that progress, the nature of the interactions between a T4CP and its target protein that initiate secretion and the mechanisms controlling this step remain obscure. In contrast to systems dedicated specifically to effector protein translocation, conjugation systems mobilize nucleoprotein complexes that additionally exhibit catalytic activities, which can be readily monitored. These models are therefore particularly well suited to investigate aspects of regulation occurring at the physical interface of a T4CP and its secretion substrate. For this purpose the MOB_F family of DNA-mobilizing systems is additionally advantageous, since DNA processing within this family features the fusion of a dedicated conjugative helicase to the DNA transesterase enzyme within a single bifunctional protein. The TraI protein of F-like plasmids, originally described as Escherichia coli DNA helicase I (1, 2, 23), and the related TrwC protein of plasmid R388 (25) are well characterized (reviewed in reference 18). Early work by Llosa et al. revealed a complex domain arrangement for TrwC (43). Similar analyses with TraI identified nonoverlapping transesterase and helicase domains (6, 77), while the remaining intermediate and C-terminal regions of the protein additionally provide functions essential to effective conjugative transfer (49, 71). The ability to physically separate the catalytic domains of TraI and TrwC has facilitated a detailed biochemical characterization of their DNA transesterase, ATPase, and DNA-unwinding reactions. Nonetheless, failure of the physically disjointed polypeptides to complement efficient conjugative transfer when coexpressed indicates a role(s) for these proteins in the strand transfer process that goes beyond the need for their dual catalytic activities (43, 50). The assignment of additional functional properties to regions within TraI is a focus of current investigation (16, 29, 49).In all systems studied thus far, conditions used to reconstitute relaxosomes on a supercoiled oriT plasmid have not supported the initiation steps necessary to enable duplex unwinding by a conjugative helicase. The question remains open whether additional protein components are required and/or whether the pathway of initiation is subject to specific repression. In the present study, we applied the IncFII plasmid R1 paradigm to investigate the potential for interaction between purified components of the relaxosome and its cognate T4CP, TraD, to exert regulatory effects on relaxosome activities in vitro. In this and in the accompanying report (72), we present evidence for wide-ranging stimulatory effects of the cytoplasmic domain of TraD protein and its interaction partner TraM on multiple aspects of relaxosome function.     

Examining the presence of ABO(H) antigens of blood types in the saliva of patients with oral cancer

Number of researches dealing with the influence of the ABO blood group antigens on the development of the oral cancer have hypothesized that people who do not secrete these substances in the saliva are more prone to suffer from this disease. The objective of this research is to examine this hypothesis. In total 114 subjects were examined, half of which suffered from oral cancer, while the other half was the healthy control group. All examinees were subjected to clinical examinations and the experimental group to pathohistological examination. An analysis of the secretor status was carried out using the Wiener agglutination test. The experimental group consisted of 78.95% of secretors, while the control group consisted of 82.46% of secretors. This difference is not statistically significant. The starting hypothesis that non-secretors are more prone to the development of oral cancer was not confirmed.     

Regulated expression of endothelial lipase by porcine brain capillary endothelial cells constituting the blood-brain barrier

Normal neurological function depends on a constant supply of polyunsaturated fatty acids to the brain. A considerable proportion of essential fatty acids originates from lipoprotein-associated lipids that undergo uptake and/or catabolism at the blood-brain barrier (BBB). This study aimed at identifying expression and regulation of endothelial lipase (EL) in brain capillary endothelial cells (BCEC), major constituents of the BBB. Our results revealed that BCEC are capable of EL synthesis and secretion. Overexpression of EL resulted in enhanced hydrolysis of extracellular high-density lipoprotein (HDL)-associated sn-2-labeled [(14)C]20 : 4 phosphatidylcholine. [(14)C]20 : 4 was recovered in cellular lipids, indicating re-uptake and intracellular re-esterification. To investigate local regulation of EL in the cerebrovasculature, BCEC were cultured in the presence of peroxisome-proliferator activated receptor (PPAR)- and liver X receptor (LXR)-agonists, known to regulate HDL levels. These experiments revealed that 24(S)OH-cholesterol (a LXR agonist), bezafibrate (a PPARalpha agonist), or pioglitazone (a PPARgamma agonist) resulted in down-regulation of EL mRNA and protein levels. Our findings implicate that EL could generate fatty acids at the BBB for transport to deeper regions of the brain as building blocks for membrane phospholipids. In addition PPAR and LXR agonists appear to contribute to HDL homeostasis at the BBB by regulating EL expression.     

Obesity--new threat to Croatian longevity

The aim of this study was to examine the association of weight gain and life expectancy at birth in Croatia. Mean body mass index was based on the data from the Croatian Adult Health Survey 2003. Birth rate and mortality data needed for life expectancy calculation were supplied by the Central Bureau of Statistics. The results suggest that the increase in mean body mass index value (1.31 kg m(-2) for women and 1.41 kg m(-2) for men) will shorten life expectancy at birth for one year. Obesity, if unchecked, might have a negative effect on life expectancy in Croatia. Despite widespread knowledge about how to reduce the severity of the problem, observed trends in obesity in Croatia continue to worsen. These trends threaten to diminish the health and life expectancy of current and future generations.     

B-type natriuretic peptide predicts new-onset atrial fibrillation in patients with ST-segment elevation myocardial infarction treated by primary percutaneous coronary intervention

The predictive value of B-type natriuretic peptide (BNP) with respect to the occurrence of new-onset atrial fibrillation (AF) in patients with ST-segment elevation myocardial infarction (STEMI) treated by primary percutaneous coronary intervention (PCI) is unknown. The aim of this study was to evaluate whether BNP has a predictive value for the occurrence of new-onset AF in patients with STEMI treated by primary PCI. In 180 patients with STEMI treated by primary PCI, BNP concentrations were measured 24h after chest pain onset. The Receiver Operating Characteristic analysis was performed to identify the most useful BNP cut-off level for the prediction of AF. The patients were divided into the two groups according to calculated cut-off level: high BNP group (BNP≥720 pg/mL, n=33) and low BNP group (BNP<720 pg/mL, n=147). The incidence of AF was 5.0%, and occurred more frequently in high BNP group (7/33, 21.2%) than in low BNP group (2/147, 1.4%), (p<0.001). Patients with high BNP were older (p=0.017), had more often anterior wall infarction (p=0.015), higher Killip class on admission (p=0.038), higher peak troponin I (p=0.002), lower left ventricular ejection fraction (p=0.029) than patients with low BNP. After multivariate adjustment, BNP was an independent predictor of AF (OR 3.70, 95% CI 1.40-9.77, p=0.008). BNP independently predicts the occurrence of new-onset AF in STEMI patients treated by primary PCI.     

Life‐history strategy,with ctenidial and pallial larval brooding,of the troglodytic ‘living fossil’ Congeria kusceri (Bivalvia: Dreissenidae) from the subterranean Dinaric Alpine karst of Croatia

Among freshwater bivalves, the brooding of embryos and larvae within the maternal ctenidia is well known. Exceptions to this generalization are the non‐brooding freshwater and estuarine species of Dreissena and Mytilopsis, respectively. It was reported that the freshwater troglodytic cousin, Congeria kusceri Bole, 1962, of these dreissenids does not brood either. It is herein demonstrated that C. kusceri undergoes one reproductive cycle each year. Sexes are separate, with an early male and later female bias. A small percentage (2.14%) of individuals is hermaphroditic. The gonads mature over summer from May to November. Spawning commences in September, when females release mature oocytes into their ctenidia and inhale sperm from mature males. Here the oocytes are fertilized, and develop within interfilamentary marsupia. Ctenidial tissues glandularize, and may provide a source of maternal nutrition for the embryos. At the late prodissoconch‐1 or prodissoconch‐2 stage (PR2, ~220 μm), larvae are released into the infrabranchial chamber via a birth channel along the outer edge of the ventral marginal food groove of both inner demibranchs. Here, they are brooded further in mantle pouches located beneath the inhalant siphon. Subsequently, after the PR2 stage (nepioconch/dissoconch), they are released from the inhalant siphon and assume an independent life as crawling juveniles. Such juveniles may be found amongst clusters of adults. Not only is C. kusceri unique amongst the Dreissenidae in possessing the capacity to brood internally fertilized ova, but it is also exceptional amongst the Bivalvia in possessing the described methods of brooding and birth. Explanations for both lie in its troglodytic lifestyle, decadal length longevity and habitat: that of byssal attachment to the hard surfaces of underground freshwater rivers, caves, pits, and sinkholes in the Tethyan arc of the Dinaric karst. Internal fertilization of a few large yolky eggs, lecithotrophic larvae, ctenidial brooding, and secondary pallial parental care represent relatively recent, Late Miocene, evolutionary adaptations from a Tethyan lentic ancestor.