Determination of kinetic parameters of enantiomerization of benzothiadiazines by DCXplorer

Benzothiadiazines differently substituted at the sulfonamidic nitrogen atom, at the stereogenic carbon atom and at the anilinic nitrogen atom have been synthesized and fully characterized. Enantioseparation of these compounds has revealed rapid on‐column enantiomerization. The recently developed software DCXplorer has been successfully applied to calculate enantiomerization kinetic parameters. Enantiomerization barriers of 3‐phenyl substituted benzothiadiazines, calculated in this work, have indicated a higher enantiomerization rate suggesting that the aromatic substituent exerts a strong effect on the enantiomerization process. Methyl substitution on N² position led to higher free energy barriers of enantiomerization, suggesting negative influence of methyl in the N² position on enantiomerization kinetics. However, methylation on N⁴ position increases the enantiomerization rates significantly. The results obtained have been employed to postulate an enantiomerization mechanism for chiral benzothiadiazine type compounds. Chirality 2010. © 2010 Wiley‐Liss, Inc.     

Leucine-rich repeat kinase 2 induces α-synuclein expression via the extracellular signal-regulated kinase pathway

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of autosomal-dominant Parkinson's disease (PD). The second known autosomal-dominant PD gene (SNCA) encodes α-synuclein, which is deposited in Lewy bodies, the neuropathological hallmark of PD. LRRK2 contains a kinase domain with homology to mitogen-activated protein kinase kinase kinases (MAPKKKs) and its activity has been suggested to be a key factor in LRRK2-associated PD. Here we investigated the role of LRRK2 in signal transduction pathways to identify putative PD-relevant downstream targets. Over-expression of wild-type [wt]LRRK2 in human embryonic kidney HEK293 cells selectively activated the extracellular signal-regulated kinase (ERK) module. PD-associated mutants G2019S and R1441C, but not kinase-dead LRRK2, induced ERK phosphorylation to the same extent as [wt]LRRK2, indicating that this effect is kinase-dependent. However, ERK activation by mutant R1441C and G2019S was significantly slower than that for [wt]LRRK2, despite similar levels of expression. Furthermore, induction of the ERK module by LRRK2 was associated to a small but significant induction of SNCA, which was suppressed by treatment with the selective MAPK/ERK kinase inhibitor U0126. This pathway linking the two dominant PD genes LRRK2 and SNCA may offer an interesting target for drug therapy in both familial and sporadic disease.     

Strain-dependent expression of metabolic proteins in the mouse hippocampus

Individual mouse strains differ significantly in terms of behavior and cognitive function. Strain-specific variation of metabolic protein levels in the hippocampus among various commonly used mouse strains, however, has not been investigated yet. A proteomic approach based on two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry [high capacity ion trap (HCT)] has been chosen to address this question by determining strain-dependent levels of metabolic proteins in hippocampal tissue of four inbred and one outbred mouse strain. Statistical analysis of protein spots on 2-DE gels of the individual strains (n = 10) revealed significant strain-dependent differences in densities of 39 spots. Subsequent HCT analysis led to the identification of 22 different metabolic proteins presenting with differential protein levels among the five mouse strains investigated. Among those are proteins concerned with the metabolism of amino acid, nucleic acid, carbohydrate and energy. Moreover, proteins known to play a pivotal role in the processes of learning and memory, such as calcium/calmodulin-dependent protein kinase type II alpha chain, were found to present with significant inter-strain variability, which is also in agreement with our previous reports. Strain-specific protein levels of metabolic proteins in the mouse hippocampus may provide some insight into the molecular underpinnings and genetic determination of strain-dependent neuronal function. Furthermore, data presented herein emphasize the significance of the genetic background for the analysis of metabolic pathways in the hippocampus in wild-type mice as well as in gene-targeting experiments.     

PavB is a surface‐exposed adhesin of Streptococcus pneumoniae contributing to nasopharyngeal colonization and airways infections

The genomic analysis of Streptococcus pneumoniae strains identified the Pneumococcal adherence and virulence factor B (PavB), whose repetitive sequences, designated Streptococcal Surface REpeats (SSURE), interact with human fibronectin. Here, we showed the gene in all tested pneumococci and identified that the observed differences in the molecular mass of PavB rely on the number of repeats, ranging from five to nine SSURE. PavB interacted with fibronectin and plasminogen in a dose‐dependent manner as shown by using various SSURE peptides. In addition, we identified PavB as colonization factor. Mice infected intranasally with ΔpavB pneumococci showed significantly increased survival times compared with wild‐type bacteria. Importantly, the pavB‐mutant showed a delay in transmigration to the lungs as observed in real‐time using bioluminescent pneumococci and decreased colonization rates in a nasopharyngeal carriage model. In co‐infection experiments the wild‐type out‐competed the pavB‐mutant and infections of epithelial cells demonstrated that PavB contributes to adherence to host cell. Blocking experiments suggested a function of PavB as adhesin, which was confirmed by direct binding of SSURE peptides to host cells. Finally, PavB may represent a new vaccine candidate as SSURE peptides reacted with human sera. Taken together, PavB is a surface‐exposed adhesin, which contributes to pneumococcal colonization and infections of the respiratory airways.     

Leptotene/zygotene chromosome movement via the SUN/KASH protein bridge in Caenorhabditis elegans

The Caenorhabditis elegans inner nuclear envelope protein matefin/SUN-1 plays a conserved, pivotal role in the process of genome haploidization. CHK-2-dependent phosphorylation of SUN-1 regulates homologous chromosome pairing and interhomolog recombination in Caenorhabditis elegans. Using time-lapse microscopy, we characterized the movement of matefin/SUN-1::GFP aggregates (the equivalent of chromosomal attachment plaques) and showed that the dynamics of matefin/SUN-1 aggregates remained unchanged throughout leptonene/zygotene, despite the progression of pairing. Movement of SUN-1 aggregates correlated with chromatin polarization. We also analyzed the requirements for the formation of movement-competent matefin/SUN-1 aggregates in the context of chromosome structure and found that chromosome axes were required to produce wild-type numbers of attachment plaques. Abrogation of synapsis led to a deceleration of SUN-1 aggregate movement. Analysis of matefin/SUN-1 in a double-strand break deficient mutant revealed that repair intermediates influenced matefin/SUN-1 aggregate dynamics. Investigation of movement in meiotic regulator mutants substantiated that proper orchestration of the meiotic program and effective repair of DNA double-strand breaks were necessary for the wild-type behavior of matefin/SUN-1 aggregates.     

Endogenous NO levels regulate nodule functioning: Potential role of cGMP in nodule functioning?

Nitric oxide is a small gaseous signaling molecule which functions in the regulation of plant development and responses to biotic and abiotic stresses. Recently, we have shown that nitric oxide is required for development of functional nodules. Here, we show that inhibition of nitric oxide synthase enzymatic activity (using N_ω-nitro-L-arginine) reduces nitric oxide content in soybean root nodules and this is coupled by reduction of endogenous cyclic guanosine monophosphate content in the nodules. We postulate that the regulation of soybean nodule development by nitric oxide is transduced via cyclic guanosine monophosphate through activation of nitric oxide-responsive soluble guanylate cyclase. Furthermore, we hypothesize that this signaling cascade is mediated via modulation of the activities of antioxidant metabolic pathways.Key words: cyclic guanosine monophosphate, nitric oxide synthase, nitric oxide, nitrogen fixation, nodulation efficiency, nodule functioning, reactive oxygen species     

Pneumolysin activates the NLRP3 inflammasome and promotes proinflammatory cytokines independently of TLR4

Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1β, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1β plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1β secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system.     

Plus- and Minus-End Directed Microtubule Motors Bind Simultaneously to Herpes Simplex Virus Capsids Using Different Inner Tegument Structures

Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1) show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during cell entry or to cytoplasmic membranes for envelopment during assembly.     

Volatile metabolic profiles of cell suspension cultures of Lavandula vera,Nicotiana tabacum and Helianthus annuus,cultivated under different regimes

Cell suspension cultures of Lavandula vera (Lamiaceae), Nicotiana tabacum (Solanaceae), and Helianthus annuus (Asteraceae) were cultivated in three different ways: in shake flasks both as free suspensions and in two‐phase systems (in the presence of Amberlite XAD‐4 resin as a second phase), as well as in 3‐L stirred tank reactor, and their volatile metabolic profiles were studied using GC‐MS. A number of compounds, some of them having allelochemical and biological activities, were identified in all the three cell suspension cultures under study. Also the presence of some compounds, unusual for the intact plants, was observed. It was found that the cultivation mode strongly influences the production and the transport (secretion into the culture medium) of the low‐molecular‐mass volatile metabolites. Principal component analyses of 12 common hydrocarbons showed discrimination between the different cultivation modes (shake flasks and two‐phase systems cultivation) by first principal component (PC1) and second principal component (PC2).