A mutant small heat shock protein with increased thylakoid association provides an elevated resistance against UV-B damage in synechocystis 6803

Besides acting as molecular chaperones, the amphitropic small heat shock proteins (sHsps) are suggested to play an additional role in membrane quality control. We investigated sHsp membrane function in the model cyanobacterium Synechocystis sp. PPC 6803 using mutants of the single sHsp from this organism, Hsp17. We examined mutants in the N-terminal arm, L9P and Q16R, for altered interaction with thylakoid and lipid membranes and examined the effects of these mutations on thylakoid functions. These mutants are unusual in that they retain their oligomeric state and chaperone activity in vitro but fail to confer thermotolerance in vivo. We found that both mutant proteins had dramatically altered membrane/lipid interaction properties. Whereas L9P showed strongly reduced binding to thylakoid and model membranes, Q16R was almost exclusively membrane-associated, properties that may be the cause of reduced heat tolerance of cells carrying these mutations. Among the lipid classes tested, Q16R displayed the highest interaction with negatively charged SQDG. In Q16R cells a specific alteration of the thylakoid-embedded Photosystem II (PSII) complex was observed. Namely, the binding of plastoquinone and quinone analogue acceptors to the Q(B) site was modified. In addition, the presence of Q16R dramatically reduced UV-B damage of PSII activity because of enhanced PSII repair. We suggest these effects occur at least partly because of increased interaction of Q16R with SQDG in the PSII complex. Our findings further support the model that membrane association is a functional property of sHsps and suggest sHsps as a possible biotechnological tool to enhance UV protection of photosynthetic organisms.     

The Influence of Clinical Information in the Histopathologic Diagnosis of Melanocytic Skin Neoplasms

Background

We tested the relevance of clinical information in the histopathologic evaluation of melanocytic skin neoplasm (MSN).

Methods

Histopathologic specimens from 99 clinically atypical MSN were circulated among ten histopathologists; each case had clinical information available in a database with a five-step procedure (no information; age/sex/location; clinical diagnosis; clinical image; dermoscopic image); each step had a histopathologic diagnosis (D1 through D5); each diagnostic step had a level of diagnostic confidence (LDC) ranging from 1 (no diagnostic certainty) to 5 (absolute diagnostic certainty). The comparison of the LDC was employed with an analysis of variance (ANOVA) for repeated measures.

Findings

In D1 (no information), 36/99 cases (36.3%) had unanimous diagnosis; in D5 (full information available), 51/99 cases (51.5%) had unanimous diagnosis (p for difference between proportions <0.001). The observer agreement expressed as kappa increased significantly from D1 to D5. The mean LDC linearly increased for each observer from D1 through D5 (p for linear trend <0.001). On average, each histopathologist changed his initial diagnosis in 7 cases (range: 2–23). Most diagnostic changes were in D2 (age/sex/location).

Interpretation

The histopathologic criteria for the diagnosis of MSN can work as such, but the final histopathologic diagnosis is a clinically-aided interpretation. Clinical data sometimes reverse the initial histopathologic evaluation.     

Use of a pooled transposon mutation grid to demonstrate roles in disease development for Erwinia carotovora subsp. atroseptica putative type III secreted effector (DspE/A) and helper (HrpN) proteins

Soft rot Erwinia spp., like other closely related plant pathogens, possess a type III secretion system (TTSS) (encoded by the hrp gene cluster) implicated in disease development. We report the sequence of the entire hrp gene cluster and adjacent dsp genes in Erwinia carotovora subsp. atroseptica SCRI1039. The cluster is similar in content and structural organization to that in E. amylovora. However, eight putative genes of unknown function located within the E. carotovora subsp. atroseptica cluster do not have homologues in the E. amylovora cluster. An arrayed set of Tn5 insertional mutants (mutation grid) was constructed and pooled to allow rapid isolation of mutants for any given gene by polymerase chain reaction screening. This novel approach was used to obtain mutations in two structural genes (hrcC and hrcV), the effector gene dspE/A, and the helper gene hrpN. An improved pathogenicity assay revealed that these mutations led to significantly reduced virulence, showing that both the putative E. carotovora subsp. atroseptica TTSS-delivered effector and helper proteins are required for potato infection.     

Presynaptic alpha2-receptors regulate reverse Na+/Ca2+-exchange and transmitter release in Na+-loaded peripheral sympathetic nerves

Electrical depolarisation-(2 Hz, 1 ms)-induced [3H]noradrenaline ([3H]NA) release has been measured from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3 x 10(-5) M; corticosterone, 5 x 10(-5) M). Substitution of most of the external Na+ by Li+ (113 mM; [Na+]0: 25 mM) slightly potentiated the axonal stimulation-evoked release of [3H]NA in a tetrodotoxin (TTX, 10(-7) M) sensitive manner. The reverse Na+/Ca2+-exchange inhibitor KB-R7943 (3 x 10(-5) M) failed to inhibit the stimulation-evoked release of [3H]NA, but increased the resting outflow of neurotransmitter. The 'N-type' voltage-sensitive Ca2+-channel (VSCC) blocker omega-conotoxin (omega-CgTx) GVIA (10(-8) M) significantly and irreversibly inhibited the release of [3H]NA on stimulation (approximately 60-70%). The 'residual release' of NA was abolished either by TTX or by reducing external Ca2+ from 2.5 to 0.25 mM. The 'residual release' of NA was also blocked by the non-selective VSCC-blocker neomycin (3 x 10(-3) M). Correlation was obtained between the extent of VSCC-inhibition and the transmitter release-enhancing effect of presynaptic alpha2-receptor blocker yohimbine (3 x 10(-7) M). When the release of [3H]NA was blocked by omega-CgTx GVIA plus neomycin, yohimbine was ineffective. Inhibition of the Na+-pump by removal of K+ from the external medium increased both the resting and the axonal stimulation-evoked release of [3H]NA in the absence of functioning VSCCs (i.e., in the presence of neomycin and after omega-CgTx treatment). Under these conditions the stimulation-evoked release of NA was abolished either by TTX or by external Ca2+-removal (+1 mM EGTA). Similarly, external Li+ (113 mM) or the reverse Na+/Ca2+ exchange blocker KB-R7943 (3 x 10(-5) M) significantly inhibited the stimulation-induced transmitter release in 'K+-free' solution. KB-R7943 decreased the resting outflow of NA as well. Under conditions in which the Na+-pump was inhibited in the absence of functioning VSCCs, yohimbine (3 x 10(-7) M) further enhanced the release of neurotransmitter, while l-noradrenaline (l-NA, 10(-6) M), an agonist of presynaptic alpha2-receptors, inhibited it. The yohimbine-induced enhancement of NA-release was abolished by Li+-substitution and significantly inhibited by KB-R7943 application. It is concluded that after blockade of VSCCs brief depolarising pulses may reverse Na+/Ca2+-exchange and release neurotransmitter in Na+-loaded sympathetic nerves. Further, similar to that of VSCCs, the reverse Na+/Ca2+-exchange may also be regulated by presynaptic alpha2-receptors.     

Joint explorative analysis of neuroreceptor subsystems in the human brain: application to receptor-transporter correlation using PET data

Positron emission tomography (PET) has proved to be a highly successful technique in the qualitative and quantitative exploration of the human brain's neurotransmitter-receptor systems. In recent years, the number of PET radioligands, targeted to different neuroreceptor systems of the human brain, has increased considerably. This development paves the way for a simultaneous analysis of different receptor systems and subsystems in the same individual. The detailed exploration of the versatility of neuroreceptor systems requires novel technical approaches, capable of operating on huge parametric image datasets. An initial step of such explorative data processing and analysis should be the development of novel exploratory data-mining tools to gain insight into the "structure" of complex multi-individual, multi-receptor data sets. For practical reasons, a possible and feasible starting point of multi-receptor research can be the analysis of the pre- and post-synaptic binding sites of the same neurotransmitter. In the present study, we propose an unsupervised, unbiased data-mining tool for this task and demonstrate its usefulness by using quantitative receptor maps, obtained with positron emission tomography, from five healthy subjects on (pre-synaptic) serotonin transporters (5-HTT or SERT) and (post-synaptic) 5-HT(1A) receptors. Major components of the proposed technique include the projection of the input receptor maps to a feature space, the quasi-clustering and classification of projected data (neighbourhood formation), trans-individual analysis of neighbourhood properties (trajectory analysis), and the back-projection of the results of trajectory analysis to normal space (creation of multi-receptor maps). The resulting multi-receptor maps suggest that complex relationships and tendencies in the relationship between pre- and post-synaptic transporter-receptor systems can be revealed and classified by using this method. As an example, we demonstrate the regional correlation of the serotonin transporter-receptor systems. These parameter-specific multi-receptor maps can usefully guide the researchers in their endeavour to formulate models of multi-receptor interactions and changes in the human brain.     

The discovery of 3-(N-alkyl)aminopropylphosphonic acids as potent S1P receptor agonists

3-(N-Alkyl)aminopropylphosphonic acids are potent agonists of four of the five known sphingosine-1-phosphate (S1P) receptor subtypes.     

Quantitative microinjection of trehalose into mouse oocytes and zygotes,and its effect on development

Sugars such as trehalose are effectively used by various organisms as protective agents to undergo anhydrobiosis and cryobiosis. The objective of this study was first to establish a method for quantitative delivery of trehalose as a model sugar into oocytes, and then to evaluate its effect on development of mouse zygotes. To this end, a quantitative microinjection technique was developed using volumetric response of microdroplets suspended in dimethylpolysilaxene. To verify accuracy of this technique, both microdroplets and oocytes were microinjected with fluorophore-labeled dextran. Thereafter, injection volumes were calculated from fluorescence intensity, and volumetric responses of both microdroplets and oocytes. Comparison of calculated injection volumes revealed that this technique reflects microinjection into oocytes with pL-accuracy. The next series of experiments focused on toxicity of injection buffers (i.e., 10mM Tris and 15mM Hepes) and trehalose. Microinjection of Hepes and Tris buffer in the presence of 0.1M trehalose resulted in blastocyst rates of 86 and 72%, respectively, without a significant difference when compared to controls (86%). In subsequent experiments, Hepes was used as the injection buffer, and embryonic development of zygotes was studied as a function of intracellular trehalose concentrations. Microinjection of trehalose up to 0.15M resulted in development to blastocyst stage similar to controls (85 and 87%, respectively) while the blastocyst rate was significantly decreased (43%) in the presence of 0.20M intracellular trehalose. When transferred to foster mothers, trehalose-injected zygotes (0.1M) implanted and developed to day 16 fetuses similar to controls, healthy pups were born. The findings of this study suggest that trehalose at effective intracellular concentrations does not impair development of mouse zygotes.     

On the role of mechanosensitive mechanisms eliciting reactive hyperemia

We hypothesized that changes in hemodynamic forces such as pressure (P) and flow (F) contribute importantly to the development of reactive hyperemia. To exclude the effects of vivo factors, isolated rat skeletal muscle arterioles ( approximately 130 microm) were utilized. We found that changes in P or P + F following occlusions elicited reactive dilations (RD). The peak of RD (up to approximately 45 microm), but not the duration of RD, increased to changes in P (80 to 10, then back to 80 mmHg) as a function of the length of occlusions (30, 60, and 120 s). However, changes in P + F (80-10 -80 mmHg + 25-0-25 microl/min) increased both the peak and duration of RD (from approximately 25 to 90 s) with longer occlusions. When only P changed, inhibition of nitric oxide synthesis or endothelium removal (E-) reduced only the peak of RD, whereas when P + F were changed, both the peak and duration of RD became reduced. Inhibition of stretch-activated cation channels by gadolinium reduced the peak but enhanced the duration of RD (both to P or P + F) that was unaffected by N(G)-nitro-l-arginine methyl ester (l-NAME) or by E-. When only P changed, inhibition of tyrosine kinases by genistein reduced peak RD but did not affect the RD duration. However, when P + F changed, genistein reduced both the peak and the duration of RD, additional l-NAME reduced the peak RD, but did not affect the duration of RD. Thus in isolated arterioles an RD resembling the characteristics of reactive hyperemia can be generated that is elicited by deformation, stretch, pressure, and flow/shear stress-sensitive mechanisms and is, in part, mediated by nitric oxide.