Use of DNA Quantification To Measure Growth and Autolysis of Lactococcus and Propionibacterium spp. in Mixed Populations

Autolysis is self-degradation of the bacterial cell wall that results in the release of enzymes and DNA. Autolysis of starter bacteria, such as lactococci and propionibacteria, is essential for cheese ripening, but our understanding of this important process is limited. This is mainly because the current tools for measuring autolysis cannot readily be used for analysis of bacteria in mixed populations. We have now addressed this problem by species-specific detection and quantification of free DNA released during autolysis. This was done by use of 16S rRNA gene single-nucleotide extension probes in combination with competitive PCR. We analyzed pure and mixed populations of Lactococcus lactis subsp. lactis and three different species of Propionibacterium. Results showed that L. lactis subsp. lactis INF L2 autolyzed first, followed by Propionibacterium acidipropionici ATCC 4965, Propionibacterium freudenreichii ISU P59, and then Propionibacterium jensenii INF P303. We also investigated the autolytic effect of rennet (commonly used in cheese production). We found that the effect was highly strain specific, with all the strains responding differently. Finally, autolysis of L. lactis subsp. lactis INF L2 and P. freudenreichii ISU P59 was analyzed in a liquid cheese model. Autolysis was detected later in this cheese model system than in broth media. A challenge with DNA, however, is DNA degradation. We addressed this challenge by using a DNA degradation marker. We obtained a good correlation between the degradation of the marker and the target in a model experiment. We conclude that our DNA approach will be a valuable tool for use in future analyses and for understanding autolysis in mixed bacterial populations.     

Ultrastructure of tarsal sensilla and other integument structures of two Pseudocellus species (Ricinulei, Arachnida)

Ricinuleids are one of the least investigated groups of Arachnida. In particular, knowledge of their ultrastructure is poor. Observations of the distal tarsomeres of ricinuleids show differences in their shape and equipment of surface structures. Legs I and II are used by the Ricinulei to explore their surroundings with tentative movements. The tarsomeres of these legs show similarities in shape and surface structures that distinguish them from those of legs III and IV. In this study, 11 different structures of the tarsomere surfaces of two cave-dwelling species, Pseudocellus pearsei and P. boneti from México, were investigated for the first time with scanning and transmission electron microscopy and discussed regarding their possible function: 1) a single treelike ramifying seta resembles a no pore single-walled (np-sw) sensillum; 2) setae occurring in a small number and possessing a bipartite shaft represent terminal pore single-walled (tp-sw) sensilla. The surface of the proximal half of the shaft shows small branches. The distal half has a smooth surface; 3) long setae with conspicuous longitudinal lamellae show characteristics of chemoreceptive wall pore single-walled (wp-sw) sensilla; 4) frequent small wp-sw sensilla with flat and irregular lamellae; 5) very short wp-sw sensilla occurring solitary or in groups; 6) a few short setae with smooth surface correspond to wp-sw sensilla; 7) a single short clubbed seta articulating in a flat pit is considered to be a np-sw sensillum; 8) common long setae with a pointed tip show characteristics of mechanoreceptive np-sw sensilla; 9) ventral setae with adhesive and mechanosensory function are accompanied by multicellular "class III" glands; 10) slit organs with mechanoreceptive function; and 11) dome-like tubercles with no indication of sensorial function. Several of these sensilla form a sensory field on the dorsofrontal surface which is particularly pronounced on the distal tarsomeres of legs I and II.     

A low molecular weight agonist signals by binding to the transmembrane domain of thyroid-stimulating hormone receptor (TSHR) and luteinizing hormone/chorionic gonadotropin receptor (LHCGR)

Many cognate low molecular weight (LMW) agonists bind to seven transmembrane-spanning receptors within their transmembrane helices (TMHs). The thienopyrimidine org41841 was identified previously as an agonist for the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and suggested to bind within its TMHs because it did not compete for LH binding to the LHCGR ectodomain. Because of its high homology with LHCGR, we predicted that thyroid-stimulating hormone receptor (TSHR) might be activated by org41841 also. We show that org41841 is a partial agonist for TSHR but with lower potency than for LHCGR. Analysis of three-dimensional molecular models of TSHR and LHCGR predicted a binding pocket for org41841 in common clefts between TMHs 3, 4, 5, 6, and 7 and extracellular loop 2 in both receptors. Evidence for this binding pocket was obtained in signaling studies with chimeric receptors that exhibited improved responses to org41841. Furthermore, a key receptor-ligand interaction between the highly conserved negatively charged E3.37 and the amino group of org41841 predicted by docking of the ligand into the three-dimensional TSHR model was experimentally confirmed. These findings provide the first evidence that, in contrast to the ectodomain binding of cognate ligands, a LMW agonist can bind to and activate glycoprotein hormone receptors via interaction with their transmembrane domain.     

Significance of ectodomain cysteine boxes 2 and 3 for the activation mechanism of the thyroid-stimulating hormone receptor

Recently, we identified constitutively activating mutations at positions Asp-403, Glu-404, and Asn-406 in the third extracellular cysteine box (C-b3) of the thyroid-stimulating hormone receptor. We hypothesized that this region could act as a molecular interface between the extracellular and serpentine domain. In this study we present a model for properties of potential interaction partners for this region. Moreover, we show that Pro-400 and Pro-407 adjacent to this epitope are also important for stabilizing the partially active, basal conformation of the wild-type (WT) thyroid-stimulating hormone receptor. Furthermore, the mutation K291A in the second extracellular cysteine box (C-b2) was identified as a new constitutively activating mutation that releases the basal conformation of the WT receptor like the known tryptic cleavage in its close vicinity. Taken together, we provide an activation scenario at the C-b2/C-b3 unit. Three anchor fragments (anchors I-III) most likely constrain the basal conformation. The three anchor fragments are tightly packed. A disulfide bridge holds the C-b2/C-b3 portions in close positions. Independent of the type of conformational interference such as side chain modifications, tryptic cleavage, or hormone stimulation that act on the constrained C-b2/C-b3 WT conformation, it will always release one of the anchor fragments. Subsequently, this results in a conformational displacement of the C-b2/C-b3 portions relative to each other, inducing receptor activation.     

Selective role of intracellular chloride in the regulation of the intrinsic but not extrinsic pathway of apoptosis in Jurkat T-cells

Apoptosis is a genetic program for the removal of unwanted cells from an organism, which is distinct from necrosis by its characteristic volume loss or apoptotic volume decrease. This cell shrinkage is the result of ion redistribution that is crucial for both the activation and execution of apoptosis. Here we report that UV-C but not Fas ligand treatment results in a significant decrease in intracellular chloride that can be abolished by modulation of chloride flux using either the chloride channel inhibitor SITS or medium with a reduced chloride concentration. Accordingly, downstream events are diminished during UV-C-induced apoptosis following chloride flux modulation, whereas Fas ligand-induced apoptotic characteristics are not affected. Moreover, the activation of the mitogen-activated protein kinase signal transduction pathway early in the apoptotic signaling cascade was affected by chloride flux in Jurkat T-cells. Thus, an alteration of intracellular chloride plays an important role in the activation of signaling molecules upstream of the mitochondria, specifically impairing the intrinsic but not extrinsic apoptotic pathway.     

Methylene blue for malaria in Africa: results from a dose-finding study in combination with chloroquine

Background

Atovaquone is part of the antimalarial drug combination atovaquone-proguanil (Malarone^®) and inhibits the cytochrome bc₁ complex of the electron transport chain in Plasmodium spp. Molecular modelling showed that amino acid mutations are clustered around a putative atovaquone-binding site resulting in a reduced binding affinity of atovaquone for plasmodial cytochrome b, thus resulting in drug resistance.

Methods

The prevalence of cytochrome b point mutations possibly conferring atovaquone resistance in Plasmodium falciparum isolates in atovaquone treatment-naïve patient cohorts from Lambaréné, Gabon and from South Western Ethiopia was assessed.

Results

Four/40 (10%) mutant types (four different single polymorphisms, one leading to an amino acid change from M to I in a single case) in Gabonese isolates, but all 141/141 isolates were wild type in Ethiopia were found.

Conclusion

In the absence of drug pressure, spontaneous and possibly resistance-conferring mutations are rare.     

LC-MS/MS-analysis of sphingosine-1-phosphate and related compounds in plasma samples

Sphingosine-1-phosphate (S1P) and related compounds are important signaling molecules and are normal constituents of human plasma. So far, only a few methods exist for their determination specifically in plasma demanding radioactive agents, more or less time consuming extraction or derivatization procedures. Here, we describe a very simple, reliable, sensitive standard-addition method for the simultaneous determination of S1P, sphingosine (SPH), sphinganine (SAPH) and sphinganine-1-phosphate (SA1P) in human and rat plasma samples. After methanol precipitation of plasma samples the supernatants were directly assessed by liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). HPLC analysis was done under gradient conditions using a C18 reversed phase column. The lower limit of quantification (LLOQ) was <10.2, <4.6, <1.9 and 0.57ng/ml for S1P, SPH, SAPH and SA1P, respectively. Variations in accuracy and intraday and interday precision were <15% over the range of calibration. All analytes were normal constituents both in human and rat plasma although the SA1P concentrations in a few rat plasma samples were below the lower limit of quantification. This validated method is suitable to generate new pharmacological findings by monitoring plasma concentrations of S1P and related compounds especially when low amounts of plasma samples are present (e.g. plasma samples from rodents).