Extracellular potassium controls responsiveness of the noradrenergic cAMP system in the rat retina to fluphenazine

The effects of fluphenazine (FLU) on the noradrenaline (NA) induced cAMP-synthesis in intact rat retinae were studied as a function of extracellular K⁺- and Ca²⁺-ions. Thus NA-induced cAMP levels were measured after incubating intact rat retinae with 50 μM NA in the presence or absence of FLU and in the presence of 1 or 10 mM theophylline. Results were: (1) Experimental condition a: standard NA-responses were measured after incubating retinae at 0.75 mM Ca²⁺, at 10 mM theophylline, at 10 μM FLU and at 2 and 0 mM K⁺. FLU does not affect the NA-response at 2 mM K⁺ significantly; however, it inhibits the NA-response at 0 mM K⁺ in this condition. (2) Experimental condition b: NA-responses were measured after incubating retinae at 0.125 mM Ca²⁺, 10 mM theophylline, 10 μM FLU and at 2 and 0 mM K⁺. At 2 mM K⁺ FLU replaces a Ca²⁺ function probably connected with the synthesis part of the NA-cAMP system and NA-responses in this low Ca²⁺ condition are consequently enhanced by FLU; however, FLU inhibits the NA-response at 0 mM K⁺ in this condition. (3) Experimental condition c: NA-responses were measured after incubating retinae at 0.75 mM Ca²⁺, 1 mM theophylline, 10 μM FLU and at 2 and 0 mM K⁺. At 2 mM K⁺ FLU enhances the NA-response by further inhibition of the degradation part of the NA-cAMP system; FLU inhibits the NA-response at 0 mM K⁺ in this condition. (4) The inhibitions of the NA-responses by FLU at 0 mM K⁺ in all three conditions a, b and c showed an apparent K_m of 1 μM. (5) Low concentrations of K⁺ (0.4–0.8 mM) maintain the property of FLU to enhance the NA-responses at condition b (0.125 mM Ca²⁺) and at condition c (1 mM theophylline). Results suggest that the activation of NA-receptor coupled adenylate cyclases (NA-AC-ases) by NA, resulting in activation of phosphodiesterase activity by the NA-elevated cAMP-levels, is sustained by (a) membraneous factor(s) connected to the NA-receptor. This (these) factor(s) is (are) switched off in the absence of K⁺. Evidence has been presented, that Ca²⁺ and FLU do not have access to this intramembraneous factor-enzyme activating moiety of the NA-cAMP system at 0 mM K⁺. Between 0.4 and 0.8 mM K⁺ the factor-enzyme-NA-receptor complex is still intact.     

Physiological changes in GRK2 regulate CCL2-induced signaling to ERK1/2 and Akt but not to MEK1/2 and calcium

G protein-coupled receptor (GPCR) kinase 2 (GRK2) regulates G protein-coupled receptor signaling via agonist-induced receptor phosphorylation and desensitization. GRK2 can also modulate cellular activation by interacting with downstream signaling molecules. The intracellular GRK2 level changes during inflammatory conditions. We investigated how IL-1β-induced changes in endogenous GRK2 expression influence chemokine receptor signaling in primary astrocytes. Culturing astrocytes with IL-1β for 24 h induced a 2–3-fold increase in GRK2 and decreased C–C chemokine ligand 2 (CCL2)-induced ERK1/2 activation. Conversely, the 45% decrease in GRK2 expression in astrocytes from GRK2+/− animals resulted in a more pronounced CCL2-induced ERK1/2 phosphorylation. Increased GRK2 inhibited CCL2-induced Akt phosphorylation at Thr308 and Ser473 as well as pPDK-1 translocation. In contrast, altered GRK2 levels did not change the CCL2-induced increase in intracellular calcium or MEK1/2 phosphorylation. These data suggest that altered GRK2 expression modulates chemokine signaling downstream of the receptor. We found that GRK2 kinase activity was not required to decrease chemokine-induced ERK1/2 phosphorylation, whereas regulation of CCL2-induced Akt phosphorylation did require an active GRK2 kinase domain. Collectively, these data suggest that changes in endogenous GRK2 expression in primary astrocytes regulate chemokine receptor signaling to ERK1/2 and to PDK-1-Akt downstream of receptor coupling via kinase-dependent and kinase-independent mechanisms, respectively.     

Freilebende Nematoden als Nahrung der SandgarneleCrangon crangon

Zusammenfassung Sandgarnelen (Crangon crangon) verschiedener Größenklassen konnten unter Laboratoriumsbedingungen mit freilebenden Nematoden als einzige Nahrung bis zu 210 Tagen am Leben erhalten werden. Für diese Experimente wurde der saprobe NematodePanagrellus redivivus benutzt, der etwa gleichgroß ist wie marine freilebende Nematoden (0,0003–0,0015 mg Trockengewicht), und der ebensogern wie diese von den Garnelen als Nahrung angenommen wird. Die Garnelen können bis zu 5 Nematoden pro Minute aus dem Lückensystem des Sandes herausfangen, doch halten sie dieses Tempo nur etwa 1/2 Std durch und legen nach der Erbeutung von etwa 300 Nematoden (0,2 mg Trockengewicht) eine Freßpause ein. Deshalb wird sehr viel weniger Nahrung aufgenommen als bei der Erbeutung von Makrofauna-Vertretern, denn ein 1,5 mg schweres Stück des PolychaetenLanice füllt den Magen. Die größte Nahrungsaufnahme bei Nematodenernährung war bei 20 mm langenCrangon nur 1,5 mg am Tag. Hierdurch wird verständlich, daß bei den Experimenten nur ein sehr geringer Zuwachs beobachtet wurde (1 mm in 25–74 Tagen) oder ein Wachstumsstillstand eintrat.Ohne Nahrung sterbenCrangon unter den Versuchsbedingungen in 27–47 Tagen ab. Auch wenn es sicher ist, daß insbesondere größere Garnelen nicht gut mit einer ausschließlichen Nematoden-Ernährung gedeihen können, so ist doch nicht von der Hand zu weisen, daß Meiofauna-Populationen in Ermangelung besserer NahrungsquellenCrangon auch in freier Natur vor dem Verhungern bewahren können.

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Free-living nematodes as food of the sand shrimp, Crangon crangonExperiments on the role of meiofauna as food of the marine macrobenthos

Summary Under laboratory conditions,Crangon crangon of different size classes survived periods of up to 210 days if fed exclusively with free-living nematodes. Experiments were performed with the saprobic nematode speciesPanagrellus redivivus which is of similar size (0.0003–0.0015 mg dry weight) as marine freeliving species and which is in the same way accepted as food by the shrimps. The shrimps are able to catch nematodes out of a sandy substratum at a rate of 5 nematodes/min, but they do so for only about 30 minutes, stopping after ingesting approximately 0.2 mg dry weight of nematodes. Thus the food intake is much less than with macrofauna prey, e.g. with the polychaeteLanice of which a shrimp may ingest 1.5 mg at one sitting. 20 mm long specimens ofCrangon feeding on nematodes had a maximum food intake of 1.5 mg dry weight per day only which explains why growth was insignificant (1 mm in 25–74 days) or non-existent.Shrimps left without food died within 27–47 days. Even if these animals, especially the larger ones, could not prosper on an exclusive diet of nematodes, in the absence of better food natural meiofauna populations may preventCrangon crangon from starving to death.

     

Effects of Murine and Human Bone Marrow-Derived Mesenchymal Stem Cells on Cuprizone Induced Demyelination

For the treatment of patients with multiple sclerosis there are no regenerative approaches to enhance remyelination. Mesenchymal stem cells (MSC) have been proposed to exert such regenerative functions. Intravenous administration of human MSC reduced the clinical severity of experimental autoimmune encephalomyelitis (EAE), an animal model mimicking some aspects of multiple sclerosis. However, it is not clear if this effect was achieved by systemic immunomodulation or if there is an active neuroregeneration in the central nervous system (CNS). In order to investigate remyelination and regeneration in the CNS we analysed the effects of intravenously and intranasally applied murine and human bone marrow-derived MSC on cuprizone induced demyelination, a toxic animal model which allows analysis of remyelination without the influence of the peripheral immune system. In contrast to EAE no effects of MSC on de- and remyelination and glial cell reactions were found. In addition, neither murine nor human MSC entered the lesions in the CNS in this toxic model. In conclusion, MSC are not directed into CNS lesions in the cuprizone model where the blood-brain-barrier is intact and thus cannot provide support for regenerative processes.     

Winter body condition of moose (Alces alces) in a declining population in northeastern Minnesota

Assessments of the condition of moose (Alces alces) may be particularly informative to understanding the dynamics of populations and other influential factors. During February-March 2003 to 2005, we assessed the nutritional condition of 79 moose (39 females, 40 males) in northeastern Minnesota by body condition scoring (BCS(F), scale of 0-10); 67 of these by were assessed by ultrasonographic measurements of rump fat (Maxfat), which was used to estimate ingesta-free body fat (IFBF) in all but two of the females. Scores of the BCS(F) were related (r(2)=0.34, P<0.0001) to Maxfat. Body condition scores were not affected by sex × capture-year, capture-year, or age-at-capture, but the mean body condition score of males (6.5 ± 0.2 [SE], n=40) was less (P ≤ 0.009) than that of females (7.4 ± 0.2, n=39). Overall, Maxfat ranged from 0.0 to 4.6 and 0.3 to 2.8 cm in females and males, respectively, and was unaffected by age-at-capture. There was a sex×capture-year effect (P=0.021) on Maxfat; mean values were stable for males during the winters of 2003 to 2005 but in females were lowest during 2003, consistent with the lowest pregnancy rates and lowest winter and spring survival compared to 2004 and 2005. Based on estimates of percent IFBF, late winter-early spring survival in 2003 of at least 11% of the collared animals assessed by Maxfat, 21% of the adult females, specifically, may have been seriously challenged directly by poor condition. Data from this study provide reference values and assessments of body condition of moose that will be an essential component of the additional, comprehensive research needed to better understand the influence of extrinsic and intrinsic factors on the performance of this viable, but declining, population. For future research, we will concentrate on developing a more-reliable BCS which would allow IFBF estimation once rump fat is depleted.     

Intranasal Administration of Human MSC for Ischemic Brain Injury in the Mouse: In Vitro and In Vivo Neuroregenerative Functions

Intranasal treatment with C57BL/6 MSCs reduces lesion volume and improves motor and cognitive behavior in the neonatal hypoxic-ischemic (HI) mouse model. In this study, we investigated the potential of human MSCs (hMSCs) to treat HI brain injury in the neonatal mouse. Assessing the regenerative capacity of hMSCs is crucial for translation of our knowledge to the clinic. We determined the neuroregenerative potential of hMSCs in vitro and in vivo by intranasal administration 10 d post-HI in neonatal mice. HI was induced in P9 mouse pups. 1×10⁶ or 2×10⁶ hMSCs were administered intranasally 10 d post-HI. Motor behavior and lesion volume were measured 28 d post-HI. The in vitro capacity of hMSCs to induce differentiation of mouse neural stem cell (mNSC) was determined using a transwell co-culture differentiation assay. To determine which chemotactic factors may play a role in mediating migration of MSCs to the lesion, we performed a PCR array on 84 chemotactic factors 10 days following sham-operation, and at 10 and 17 days post-HI. Our results show that 2×10⁶ hMSCs decrease lesion volume, improve motor behavior, and reduce scar formation and microglia activity. Moreover, we demonstrate that the differentiation assay reflects the neuroregenerative potential of hMSCs in vivo, as hMSCs induce mNSCs to differentiate into neurons in vitro. We also provide evidence that the chemotactic factor CXCL10 may play an important role in hMSC migration to the lesion site. This is suggested by our finding that CXCL10 is significantly upregulated at 10 days following HI, but not at 17 days after HI, a time when MSCs no longer reach the lesion when given intranasally. The results described in this work also tempt us to contemplate hMSCs not only as a potential treatment option for neonatal encephalopathy, but also for a plethora of degenerative and traumatic injuries of the nervous system.     

Excessive heart rate response to orthostatic stress in postural tachycardia syndrome is not caused by anxiety.

Postural tachycardia syndrome (POTS) is characterized by excessive increases in heart rate (HR) without hypotension during orthostasis. The relationship between the tachycardia and anxiety is uncertain. Therefore, we tested whether the HR response to orthostatic stress in POTS is primarily related to psychological factors. POTS patients (n = 14) and healthy controls (n = 10) underwent graded venous pooling with lower body negative pressure (LBNP) to -40 mmHg while wearing deflated antishock trousers. "Sham" venous pooling was performed by 1) trouser inflation to 5 mmHg during LBNP and 2) vacuum pump activation without LBNP. HR responses to mental stress were also measured in both groups, and a questionnaire was used to measure psychological parameters. During LBNP, HR in POTS patients increased 39 +/- 5 beats/min vs. 19 +/- 3 beats/min in control subjects at -40 mmHg (P < 0.01). LBNP with trouser inflation markedly blunted the HR responses in the patients (9 +/- 2 beats/min) and controls (2 +/- 1 beats/min), and there was no HR increase during vacuum application without LBNP in either group. HR responses during mental stress were not different in the patients and controls (18 +/- 2 vs. 19 +/- 1 beats/min; P > 0.6). Anxiety, somatic vigilance, and catastrophic cognitions were significantly higher in the patients (P < 0.05), but they were not related to the HR responses during LBNP or mental stress (P > 0.1). These results suggest that the HR response to orthostatic stress in POTS patients is not caused by anxiety but that it is a physiological response that maintains arterial pressure during venous pooling.     

The bovine corneal opacity and permeability test in routine ocular irritation testing and its improvement within the limits of OECD test guideline 437

Data on eye irritation are generally needed for the hazard identification of chemicals. As the Bovine Corneal Opacity and Permeability (BCOP) test has been accepted by many regulatory agencies for the identification of corrosive and severe ocular irritants since September 2009 (OECD Test Guideline 437, TG 437), we evaluated this alternative method for routine testing at BASF. We demonstrated our technical proficiency by testing the reference standards recommended in TG 437, and 21 additional materials with published BCOP and in vivo data. Our results matched the published in vitro data very well, but with some intentionally selected false negatives (FNs) and false positives (FPs), the concordance was 77% (24/31), with FN and FP rates of 20% (2/10) and 24% (5/21), respectively. In addition, we tested 21 in-house materials, demonstrating the utility of the BCOP assay for our own test material panel. Histopathological assessment of the corneas by light microscopy was also conducted, as this was suggested as a means of improving the identification of FNs. The histopathology corrected the classification of some FNs, but also increased the number of FPs. Parallel to the test method evaluation, we compared three new opacitometer models with the current standard device. We recommend the use of an opacitometer developed in our BASF laboratory, which has certified components and electronic data storage, resulting in what we consider to be excellent sensitivity, stability and reproducibility.     

Endometrial Stromal Cells of Women with Recurrent Miscarriage Fail to Discriminate between High- and Low-Quality Human Embryos

Background

The aetiology of recurrent miscarriage (RM) remains largely unexplained. Women with RM have a shorter time to pregnancy interval than normally fertile women, which may be due to more frequent implantation of non-viable embryos. We hypothesized that human endometrial stromal cells (H-EnSCs) of women with RM discriminate less effectively between high-and low-quality human embryos and migrate more readily towards trophoblast spheroids than H-EnSCs of normally fertile women.

Methodology/Principal Findings

Monolayers of decidualized H-EnSCs were generated from endometrial biopsies of 6 women with RM and 6 fertile controls. Cell-free migration zones were created and the effect of the presence of a high-quality (day 5 blastocyst, n = 13), a low-quality (day 5 blastocyst with three pronuclei or underdeveloped embryo, n = 12) or AC-1M88 trophoblast cell line spheroid on H-ESC migratory activity was analyzed after 18 hours. In the absence of a spheroid or embryo, migration of H-EnSCs from fertile or RM women was similar. In the presence of a low-quality embryo in the zone, the migration of H-EnSCs of control women was inhibited compared to the basal migration in the absence of an embryo (P<0.05) and compared to the migration in the presence of high-quality embryo (p<0.01). Interestingly, the migratory response H-EnSCs of women with RM did not differ between high- and low-quality embryos. Furthermore, in the presence of a spheroid their migration was enhanced compared to the H-EnSCs of controls (p<0.001).

Conclusions

H-EnSCs of fertile women discriminate between high- and low-quality embryos whereas H-EnSCs of women with RM fail to do so. H-EnSCs of RM women have a higher migratory response to trophoblast spheroids. Future studies will focus on the mechanisms by which low-quality embryos inhibit the migration of H-EnSCs and how this is deregulated in women with RM.