20-Hydroxyecdysone-induced differentiation and apoptosis in the Drosophila cell line, l(2)mbn

20-Hydroxyecdysone has an inhibitory effect on the proliferation of l(2)mbn cells, causes vacuolization and fragmentation of cells, and promotes a strong phagocytotic activity. From several lines of evidence, it can be concluded that 20-hydroxyecdysone induces apoptosis. Long-term video observations following the fate of individual cells, scanning and transmission electron microscopy reveal the typical characteristics of apoptosis: sequestration of small cellular protuberances or larger parts of the cell with nuclear fragments (apoptotic bodies), chromatin condensation, condensation and vesiculation of cytoplasm, whereas the mitochondria retain their normal appearance. The induction of apoptosis by 20-hydroxyecdysone was confirmed by the TUNEL reaction and quantitatively determined by a method based on this reaction. Onset of apoptosis precedes phagocytotic activity. JH III alone has no clear-cut effect on l(2)mbn cells. In double treatments, the inhibitory effect of 20-hydroxyecdysone on cell proliferation is significantly reduced by the addition of JH III. Whether or not JH III also reduces apoptotic activity is not yet clear. It is shown that the l(2)mbn cell line is an advantageous model system for the exploration of steroid-induced apoptosis.     

GA4 Does Not Require Conversion into GA1 to Delay Senescence of Alstroemeria hybrida Leaves

The biological activity and metabolism of applied GA₁ and GA₄ were studied in leaves of alstroemeria (Alstroemeria hybrida). It appeared that GA₄ was 2 orders of magnitude more active in delaying leaf senescence than GA₁. GA₃-13-OMe, a GA analog that cannot be hydroxylated on the 13-C position, also retarded chlorophyll loss, although less efficiently. Tritiated and deuterated GA₁, GA₄, and GA₉ were applied to leaves, and their metabolites were analyzed. According to high performance liquid chromatography and gas chromatography-mass spectrometry analyses, GA₉ was converted into GA₄ and GA₃₄, and GA₄ was converted into GA₃₄ and more polar components. No evidence was found for the conversion of both GA₉ and GA₄ into GA₁, even at the relatively high concentrations that were taken up by the leaf. The results strongly suggest that GA₄ is recognized directly by a receptor involved in regulation of leaf senescence in alstroemeria. Received November 24, 1997; accepted February 17, 1998     

Thermomorphogenic and Photomorphogenic Control of Stem Elongation in Fuchsia Is Not Mediated by Changes in Responsiveness to Gibberellins

Stem elongation in Fuchsia × hybrida was influenced by cultivation at different day and night temperatures or in different light qualities. Internode elongation of plants grown at a day (25°C) to night (15°C) temperature difference (DIF+10) in white light was almost twofold that of plants grown at the opposite temperature regime (DIF−10). Orange light resulted in a threefold stimulation of internode elongation compared with white light DIF−10. Surprisingly, internode elongation in orange light was similar for plants grown at DIF−10 and DIF+10. Flower development was accelerated at DIF−10 compared with DIF+10 in both white and orange light. To examine whether the effects of DIF and light quality on shoot elongation were related to changes in gibberellin metabolism or plant sensitivity to gibberellins (GAs), the stem elongation responses of paclobutrazol-treated plants to applied gibberellins were determined. In the absence of applied gibberellins paclobutrazol (>0.32 μmol plant⁻¹) strongly retarded shoot elongation. This inhibition was nullified by the application of about 10–32 nmol of GA₁, GA₄, GA₉, GA₁₅, GA₁₉, GA₂₀, GA₂₄, or GA₄₄. The results are discussed in relation to possible effects of DIF and light quality on endogenous gibberellin levels and gibberellin sensitivity of fuchsia and their effects on stem elongation. Received October 4, 1997; accepted December 17, 1997     

From Africa via Europe to South America: migrational route of a species‐rich genus of Neotropical lowland rain forest trees (Guatteria,Annonaceae)

Aim Several recent studies have suggested that a substantial portion of today’s plant diversity in the Neotropics has resulted from the dispersal of taxa into that region rather than by vicariance. In general, three routes have been documented for the dispersal of taxa onto the South American continent: (1) via the North Atlantic Land Bridge, (2) via the Bering Land Bridge, or (3) from Africa directly onto the continent. Here a species‐rich genus of Neotropical lowland rain forest trees (Guatteria, Annonaceae) is used as a model to investigate these three hypotheses. Location The Neotropics. Methods The phylogenetic relationships within the long‐branch clade of Annonaceae were reconstructed (using maximum parsimony, maximum likelihood and Bayesian inference) in order to gain insight in the phylogenetic position of Guatteria. Furthermore, Bayesian molecular dating and Bayesian dispersal–vicariance (Bayes‐DIVA) analyses were undertaken. Results Most of the relationships within the long‐branch clade of Annonaceae were reconstructed and had high support. However, the relationship between the Duguetia clade, the Xylopia–Artabotrys clade and Guatteria remained unclear. The stem node age estimate of Guatteria ranged between 49.2 and 51.3 Ma, whereas the crown node age estimate ranged between 11.4 and 17.8 Ma. For the ancestral area of Guatteria and its sister group, the area North America–Africa was reconstructed in 99% of 10,000 DIVA analyses, while South America–North America was found just 1% of the time. Main conclusions The estimated stem to crown node ages of Guatteria in combination with the Bayes‐DIVA analyses imply a scenario congruent with an African origin followed by dispersal across the North Atlantic Land Bridge in the early to middle Eocene and further dispersal into North and Central America (and ultimately South America) in the Miocene. The phylogenetically and morphologically isolated position of the genus is probably due to extinction of the North American and European stem lineages in the Tertiary.     

Evaluation of 111In-Labelled Exendin-4 Derivatives Containing Different Meprin β-Specific Cleavable Linkers

Background

Cleavable linkers, which are specifically cleaved by defined conditions or enzymes, are powerful tools that can be used for various purposes. Amongst other things, they have been successfully used to deliver toxic payloads as prodrugs into target tissues. In this work novel linker sequences targeting meprin β, a metalloprotease expressed in the kidney brush-border membrane, were designed and included in the sequence of three radiolabelled exendin-4 derivatives. As radiolabelled exendin-4 derivatives strongly accumulate in the kidneys, we hypothesised that specific cleavage of the radiolabelled moiety at the kidney brush-border membrane would allow easier excretion of the activity into the urine and therefore improve the pharmacological properties of the peptide.

Results

The insertion of a cleavable linker did not negatively influence the in vitro properties of the peptides. They showed a good affinity to the GLP-1 receptor expressed in CHL cells, a high internalisation and sufficiently high stability in fresh human blood plasma. In vitro digestion with recombinant meprin β rapidly metabolised the corresponding linker sequences. After 60 min the majority of the corresponding peptides were digested and at the same time the anticipated fragments were formed. The peptides were also quickly metabolised in CD1 nu/nu mouse kidney homogenates. Immunofluorescence staining of meprin β in kidney sections confirmed the expression of the protease in the kidney brush-border membrane. Biodistribution in GLP-1 receptor positive tumour-xenograft bearing mice revealed high specific uptake of the ¹¹¹In-labelled tracers in receptor positive tissue. Accumulation in the kidneys, however, was still high and comparable to the lead compound ¹¹¹In-Ex4NOD40.

Conclusion

In conclusion, we show that the concept of cleavable linkers specific for meprin β is feasible, as the peptides are rapidly cleaved by the enzyme while retaining their biological properties.     

Left ventricular assist device for end-stage heart failure: results of the first LVAD destination program in the Netherlands

PurposeMechanical circulatory support with a continuous-flow left ventricular assist device (LVAD) may be a valuable treatment in end-stage heart failure patients for an extended period of time. The purpose of this study was to evaluate the safety and efficacy of implantation of a continuous-flow LVAD in end-stage heart failure patients within the first destination program in the Netherlands.MethodsA third-generation LVAD was implanted in 16 heart failure patients (age 61 ± 8; 81 % male; left ventricular ejection fraction 20 ± 6 %) as destination therapy. All patients were ineligible for heart transplant. At baseline, 3 and 6 months, New York Heart Association (NYHA) functional class, quality-of-life and exercise capacity were assessed. Clinical adverse events were registered.ResultsSurvival at 30 days and 6 months was 88 and 75 %, respectively. In the postoperative phase, 6 (38 %) patients required continuous veno-venous haemofiltration for renal failure and 2 (13 %) patients required extracorporeal membrane oxygenation because of severe right ventricular failure. During follow-up, NYHA functional class and quality-of-life improved from 3.7 ± 0.1 to 2.3 ± 0.1 and 57 ± 5 to 23 ± 3 at 6 months (P < 0.001), respectively. The 6 min walking distance improved from 168 ± 42 m to 291 ± 29 m at 6 months (P = 0.001).ConclusionContinuous-flow LVAD therapy is a promising treatment for patients with end-stage heart failure ineligible for heart transplant.     

Efficient Differentiation of Steroidogenic and Germ-Like Cells from Epigenetically-Related iPSCs Derived from Ovarian Granulosa Cells

To explore restoration of ovarian function using epigenetically-related, induced pluripotent stem cells (iPSCs), we functionally evaluated the epigenetic memory of novel iPSC lines, derived from mouse and human ovarian granulosa cells (GCs) using c-Myc, Klf4, Sox2 and Oct4 retroviral vectors. The stem cell identity of the mouse and human GC-derived iPSCs (mGriPSCs, hGriPSCs) was verified by demonstrating embryonic stem cell (ESC) antigen expression using immunocytochemistry and RT-PCR analysis, as well as formation of embryoid bodies (EBs) and teratomas that are capable of differentiating into cells from all three germ layers. GriPSCs’ gene expression profiles associate more closely with those of ESCs than of the originating GCs as demonstrated by genome-wide analysis of mRNA and microRNA. A comparative analysis of EBs generated from three different mouse cell lines (mGriPSCs; fibroblast-derived iPSC, mFiPSCs; G4 embryonic stem cells, G4 mESCs) revealed that differentiated mGriPSC-EBs synthesize 10-fold more estradiol (E2) than either differentiated FiPSC- or mESC-EBs under identical culture conditions. By contrast, mESC-EBs primarily synthesize progesterone (P4) and FiPSC-EBs produce neither E2 nor P4. Differentiated mGriPSC-EBs also express ovarian markers (AMHR, FSHR, Cyp19a1, ER and Inha) as well as markers of early gametogenesis (Mvh, Dazl, Gdf9, Boule and Zp1) more frequently than EBs of the other cell lines. These results provide evidence of preferential homotypic differentiation of mGriPSCs into ovarian cell types. Collectively, our data support the hypothesis that generating iPSCs from the desired tissue type may prove advantageous due to the iPSCs’ epigenetic memory.     

Sex Differences in the Quality of Diabetes Care in the Netherlands (ZODIAC-45)

Objective

Our aim was to investigate whether trends in quality of diabetes care differ between sexes in the Netherlands from 1998 till 2013.

Research Design and Methods

In this prospective observational cohort study quality of care was measured using process and outcome measures in patients with type 2 diabetes in primary care. Trend and absolute differences between sexes were investigated for patients <75 years. Subgroup analyses were performed in patients ≥75 years. 10-year mortality risk was assessed with the Globorisk risk equation in patients without cardiovascular diseases <75 years.

Results

The number of patients increased from 2,644 in 1998 to 62,230 in 2013. In 1998, 51% of the men and 60% of the women <75 years had an HbA1c >53 mmol/mol; this decreased to approximately 29% in both sexes in 2013. Patients having a systolic blood pressure >140 mmHg decreased from 70% to 42%, and from 80% to 40% in men and women <75 years, respectively. In patients ≥75 years it decreased from 72% to 50% in men and 85% to 56% in women. Obesity increased in both sexes, whereas smoking in men and women declined in patients <75 years (men: 34% to 22%; women: 22% to 18%). The number of patients with a mortality risk >20% over 10 years decreased from 15% to 3% in men and from 18% to 3% in women.

Conclusions

Quality of diabetes care has improved considerably in the period 1998–2013 in both sexes. Possibly relevant trend differences between sexes were observed for HbA1c, systolic blood pressure, BMI and smoking. The predicted mortality risk decreased over time in both sexes. Except for BMI in both age groups and systolic blood pressure in patients ≥75 years, no evident poorer risk factor control in women compared to men was found at the end of the study period.