The extreme Beringian/Atlantic disjunction in Saxifraga rivularis (Saxifragaceae) has formed at least twice

Aim The oceanic Saxifraga rivularis L. presents one of the most extreme disjunctions known in the arctic flora: it has a small amphi‐Beringian range and a larger amphi‐Atlantic one. It was recently suggested to have had a single allopolyploid origin in Beringia at least one glacial cycle ago, followed by gradual expansion in a more humid period and differentiation into two allopatric subspecies (the Atlantic ssp. rivularis and the Beringian ssp. arctolitoralis). Here we explore the history of its extreme disjunction. Location The amphi‐Beringian and northern amphi‐Atlantic regions. Methods We obtained amplified fragment length polymorphisms (AFLPs) and chloroplast DNA sequences from 36 populations (287 individuals) and 13 populations (15 individuals), respectively. The data were analysed using principal coordinates analyses, Bayesian clustering methods, and analyses of molecular variance. Results Two distinctly divergent AFLP groups were observed, corresponding to the two described subspecies, but, surprisingly, four of the West Atlantic populations belonged to the supposedly Beringian endemic ssp. arctolitoralis. This was confirmed by re‐examination of their morphological characteristics. The overall AFLP diversity in the species was low (26.4% polymorphic markers), and there was no variation in the five investigated chloroplast DNA (cpDNA) regions. There was little geographic structuring of the AFLP diversity within each subspecies, even across the extreme disjunction in ssp. arctolitoralis, across the Bering Sea, and across the Atlantic Ocean, except that most plants from the arctic Svalbard archipelago formed a separate genetic group with relatively high diversity. Main conclusions The extreme disjunction in S. rivularis has evidently formed at least twice. The first expansion from Beringia was followed by allopatric differentiation into one Beringian and one Atlantic subspecies, which are distinctly divergent at AFLP loci but still harbour identical cpDNA haplotypes, suggesting that the expansion was quite recent but before the last glaciation. The next expansion from Beringia probably occurred by means of several long‐distance dispersals in the current interglacial, resulting in the colonization of the western Atlantic region by ssp. arctolitoralis. The poor geographic structuring within each subspecies suggests frequent long‐distance dispersals from two main Weichselian refugia, one Beringian and one western‐central European, but it is possible that the genetic group in Svalbard originates from an additional refugium.     

Symptomatic relief precedes improvement of myocardial blood flow in patients under spinal cord stimulation

Background

Spinal cord electrical stimulation (SCS) has shown to be a treatment option for patients suffering from angina pectoris CCS III-IV although being on optimal medication and not suitable for conventional treatment strategies, e.g. CABG or PTCA. Although many studies demonstrated a clear symptomatic relief under SCS therapy, there are only a few short-term studies that investigated alterations in cardiac ischemia. Therefore doubts remain whether SCS has a direct effect on myocardial perfusion.

Methods

A prospective study to investigate the short- and long-term effect of spinal cord stimulation (SCS) on myocardial ischemia in patients with refractory angina pectoris and coronary multivessel disease was designed. Myocardial ischemia was measured by MIBI-SPECT scintigraphy 3 months and 12 months after the beginning of neurostimulation. To further examine the relation between cardiac perfusion and functional status of the patients we measured exercise capacity (bicycle ergometry and 6-minute walk test), symptoms and quality of life (Seattle Angina Questionnaire [SAQ]), as well.

Results

31 patients (65 ± 11 SEM years; 25 male, 6 female) were included into the study. The average consumption of short acting nitrates (SAN) decreased rapidly from 12 ± 1.6 times to 3 ± 1 times per week. The walking distance and the maximum workload increased from 143 ± 22 to 225 ± 24 meters and 68 ± 7 to 96 ± 12 watt after 3 months. Quality of life increased (SAQ) significantly after 3 month compared to baseline, as well. No further improvement was observed after one year of treament. Despite the symptomatic relief and the improvement in maximal workload computer based analysis (Emory Cardiac Toolbox) of the MIBI-SPECT studies after 3 months of treatment did not show significant alterations of myocardial ischemia compared to baseline (16 patients idem, 7 with increase and 6 with decrease of ischemia, 2 patients dropped out during initial test phase). Interestingly, in the long-term follow up after one year 16 patients (of 27 who completed the one year follow up) showed a clear decrease of myocardial ischemia and only one patient still had an increase of ischemia compared to baseline.

Conclusion

Thus, spinal cord stimulation not only relieves symptoms, but reduces myocardial ischemia as well. However, since improvement in symptoms and exercise capacity starts much earlier, decreased myocardial ischemia might not be a direct effect of neurostimulation but rather be due to a better coronary collateralisation because of an enhanced physical activity of the patients.     

Characterization of a novel chemokine-containing storage granule in endothelial cells: evidence for preferential exocytosis mediated by protein kinase A and diacylglycerol

We have recently shown that several proinflammatory chemokines can be stored in secretory granules of endothelial cells (ECs). Subsequent regulated exocytosis of such chemokines may then enable rapid recruitment of leukocytes to inflammatory sites. Although IL-8/CXCL8 and eotaxin-3/CCL26 are sorted to the rod-shaped Weibel-Palade body (WPB), we found that GROalpha/CXCL1 and MCP-1/CCL2 reside in small granules that, similarly to the WPB, respond to secretagogue stimuli. In the present study, we report that GROalpha and MCP-1 colocalized in 50- to 100-nm granules, which occur throughout the cytoplasm and at the cell cortex. Immunofluorescence confocal microscopy revealed no colocalization with multimerin or tissue plasminogen activator, i.e., proteins that are released from small granules of ECs by regulated exocytosis. Moreover, the GROalpha/MCP-1-containing granules were Rab27-negative, contrasting the Rab27-positive, WPB. The secretagogues PMA, histamine, and forskolin triggered distinct dose and time-dependent responses of GROalpha release. Furthermore, GROalpha release was more sensitive than IL-8 release to inhibitors and activators of PKA and PKC but not to an activator of Epac, a cAMP-regulated GTPase exchange factor, indicating that GROalpha release is regulated by molecular adaptors different from those regulating exocytosis of the WPB. On the basis of these findings, we designated the GROalpha/MCP-1-containing compartment the type 2 granule of regulated secretion in ECs, considering the WPB the type 1 compartment. In conclusion, we propose that the GROalpha/MCP-1-containing type 2 granule shows preferential responsiveness to important mediators of EC activation, pointing to the existence of selective agonists that would allow differential release of selected chemokines.     

The susceptibility of grayling (Thymallus thymallus) to experimental infections with the monogenean Gyrodactylus salaris

The pathogenic monogenean Gyrodactylus salaris infecting Atlantic salmon (Salmo salar) is found to attach and reproduce under laboratory conditions on several species in the subfamily Salmoninae other than the Atlantic salmon. The gyrodactylid species Gyrodactylus thymalli infecting grayling (Thymallus thymallus) in another subfamily, Thymallinae, is previously said to be very similar to G. salaris based on morphometry and genetical analysis which prompted the present laboratory experiments to test the susceptibility and resistance of grayling to G. salaris. All 0+ and 1+ grayling became infected with G. salaris during the experimental infection procedure. However, both innate resistant and susceptible grayling were found. In susceptible individually isolated fish, parasite reproduction lasted for more than 35 days. Parasite reproduction also occurred among grouped grayling as judged from the duration of infection of more than 50 days. However, grayling susceptibility as judged from G. salaris reproduction, was very limited. Hence, the results indicate significant biological differences between the function of Atlantic salmon and grayling as host for G. salaris. The grayling is interpreted as unable to sustain G. salaris in nature which implies that G. thymalli is not conspecific with G. salaris. However, G. salaris dispersal by grayling cannot be excluded.     

A-like cells in the rat stomach contain ghrelin and do not operate under gastrin control

Ghrelin is a 28 a.a. gastric peptide, recently identified as a natural ligand of the growth hormone secretagogue receptor (orphan receptor distinct from the receptor for growth hormone releasing hormone). In the present study, radioimmunoassay demonstrated ghrelin-like material in the rat oxyntic mucosa with moderate amounts also in antrum and duodenum. Small amounts were found in the distal intestines and pancreas. Northern blot analysis revealed abundant ghrelin mRNA in the oxyntic mucosa. Immunocytochemistry demonstrated ghrelin-immunoreactivity in endocrine-like cells in the oxyntic mucosa. Such cells occurred in low numbers also in the antrum and duodenum. The rat oxyntic mucosa is rich in endocrine (chromogranin A/pancreastatin-immunoreactive) cells, such as the histamine-rich ECL cells (65-75% of the endocrine cells), the A-like cells (20-25%) and the D cells (somatostatin cells) (10%). The ghrelin-immunoreactive (IR) cells contained pancreastatin but differed from ECL cells and D cells by being devoid of histamine-forming enzyme (ECL cell constituent) and somatostatin (D cell constituent). Hence, ghrelin seems to occur in the A-like cells. The ghrelin-IR cells in the antrum were distinct from the gastrin cells, the serotonin-containing enterochromaffin cells and the D cells. Conceivably, ghrelin cells in the antrum and distally in the intestines also belong to the A-like cell population. The concentration of ghrelin in the circulation was lowered by about 80% following the surgical removal of the acid-producing part of the stomach in line with the view that the oxyntic mucosa is the major source of ghrelin. The serum ghrelin concentration was higher in fasted rats than in fed rats; it was reduced upon re-feeding and seemed unaffected by 1-week treatment with the proton pump inhibitor omeprazole, resulting in elevated serum gastrin concentration. Infusion of gastrin-17 for 2 days failed to raise the serum ghrelin concentration. Omeprazole treatment for 10 weeks raised the level of HDC mRNA but not that of ghrelin mRNA or somatostatin mRNA in the oxyntic mucosa. Hence, unlike the ECL cells, ghrelin-containing A-like cells do not seem to operate under gastrin control.     

Intestinal epithelial cell proliferation and migration in Atlantic salmon, Salmo salar L.: effects of temperature and inflammation

A 28-day feeding trial was carried out to characterise intestinal epithelial cell (IEC) turnover in Atlantic salmon (Salmo salar L.) post-smolts in seawater. Four groups of fish raised at two temperatures of 8°C or 12°C and fed two different diets were investigated. The diets included a reference maize gluten and fishmeal-based diet (FM) and an experimental enteropathy-causing diet containing 20% extracted soybean meal (SBM). IEC proliferation and migration were investigated by labelling cells with the in vivo proliferation marker 5-bromo-2′-deoxyuridine (BrdU). Proliferating cell nuclear antigen (PCNA) labelling was used as a control for identifying proliferating cells. Samples of the proximal (PI), mid (MI) and distal (DI) intestinal regions were collected at five time points (3 h–28 days) over the experimental period. Histologically, FM-fed fish had normal mucosa, whereas the SBM-fed fish developed DI enteropathy. Major zones of cell proliferation were observed in the mucosal fold bases for all intestinal regions. Over time, BrdU-labelled cells migrated up mucosal folds to the tips before being lost. Migration rates were dependent on intestinal region, temperature and diet. Highest migration rates were observed in the PI followed by the MI and DI for FM-fed fish. Diet and temperature barely affected migration in the PI and MI. Migration in the DI was most sensitive to diet and temperature, with both SBM and the higher water temperature increasing proliferation and migration rates. The slow IEC turnover in the DI might help to explain the sensitivity of this region to dietary SBM-induced enteropathy.     

The production and characterization of murine monoclonal antibodies to a DNA receptor on human leukocytes

Two murine mAb have been generated with a reactivity toward a 30,000 m.w. DNA binding protein found on the cell surface of human leukocytes; mAb 12A has an IgG1/k isotype, and mAb 24T has an IgG2b/k isotype. Both react with the DNA binding domain or adjacent region of the putative DNA receptor and inhibit the binding of [3H]DNA to PBMC at concentrations as low as 100 ng/ml. Stoichiometric studies indicate that both mAb react with monocytes and T cells with a kDa of 10(-7) M; about 0.5 x 10(6) molecules bind per cell at saturation. Flow cytometry indicated that 67% of lymphocytes and 98% of monocytes bore the DNA receptor. Dual labeling studies showed that 90% of B cells and 50% of T cells express the receptor; 50% of CD4+ T cells are receptor positive. Immunomatrices constructed with both mAb 12A and 24T allowed the receptor to be purified to a high degree of purity. A single protein of Mr 30,000 was readily observed after SDS-PAGE and silver staining of the gel; after electropheretic transfer of nitrocellulose this protein was shown to be a DNA binding molecule by use of a probe of biotin labeled DNA. These experiments provide further evidence to support the existence of a specific DNA receptor on human leukocytes; the availability of mAb to the receptor should be useful in its further characterization.     

Gene expression analysis uncovers novel hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

Hedgehog interacting protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis.