Control of germline stem cell division frequency--a novel, developmentally regulated role for epidermal growth factor signaling

Exploring adult stem cell dynamics in normal and disease states is crucial to both better understanding their in vivo role and better realizing their therapeutic potential. Here we address the division frequency of Germline Stem Cells (GSCs) in testes of Drosophila melanogaster. We show that GSC division frequency is under genetic control of the highly conserved Epidermal Growth Factor (EGF) signaling pathway. When EGF signaling was attenuated, we detected a two-fold increase in the percentage of GSCs in mitotic division compared to GSCs in control animals. Ex vivo and in vivo experiments using a marker for cells in S-phase of the cell cycle showed that the GSCs in EGF mutant testes divide faster than GSCs in control testes. The increased mitotic activity of GSCs in EGF mutants was rescued by restoring EGF signaling in the GSCs, and reproduced in testes from animals with soma-depleted EGF-Receptor (EGFR). Interestingly, EGF attenuation specifically increased the GSC division frequency in adult testes, but not in larval testes. Furthermore, GSCs in testes with tumors resulting from the perturbation of other conserved signaling pathways divided at normal frequencies. We conclude that EGF signaling from the GSCs to the CySCs normally regulates GSC division frequency. The EGF signaling pathway is bifurcated and acts differently in adult compared to larval testes. In addition, regulation of GSC division frequency is a specific role for EGF signaling as it is not affected in all tumor models. These data advance our understanding concerning stem cell dynamics in normal tissues and in a tumor model.     

Properdin deficiency in murine models of nonseptic shock

Hereditary properdin deficiency is linked to susceptibility to meningococcal disease (Neisseria meningitidis serotypes Y and W-135) with high mortality. Its relative contribution toward the outcome of nonseptic shock has not been investigated. Using properdin-deficient C57BL/6 mice and their littermates, this study examines their survival of zymosan-induced and LPS-induced shock. Properdin-deficient mice were more resistant to zymosan shock compared with wild-type mice, which showed greater impairment of end-organ function 24 h after zymosan injection, higher TNF-alpha production by alveolar and peritoneal macrophages, higher TNF-alpha, and, inversely, lower IL-10 levels in peritoneal lavage and circulation and higher plasma C5a levels. Properdin-deficient mice showed significantly higher mortality in LPS shock, elevated TNF-alpha, and, inversely, reduced IL-10 production by peritoneal macrophages as well as lower plasma C5a levels compared with wild-type littermates. NO production by peritoneal macrophages and plasma alpha1-antitrypsin levels at 24 h after the injection of LPS or zymosan were decreased in properdin-deficient mice in both models, and fewer histopathologic changes in liver were observed in properdin-deficient animals. This study provides evidence that properdin deficiency attenuates zymosan-induced shock and exacerbates LPS-induced shock.     

Optimizing DHA levels in piglets by lowering the linoleic acid to alpha-linolenic acid ratio

We examined the effect of altering the linoleic acid (LA, 18:2n-6) to alpha-linolenic acid (ALA, 18:3n-3) ratio in the dietary fats of 3 day old piglets fed formula for 3 weeks. The LA-ALA ratios of the experimental formulas were 0.5:1, 1:1, 2:1, 4:1, and 10:1. The level of LA was held constant at 13% of total fats while the level of ALA varied from 1.3% (10:1 group) to 26.8% (0.5:1 group). Incorporation of the n-3 long chain PUFA EPA and 22:5n-3 into erythrocytes, plasma, liver, and brain tissues was linearly related to dietary ALA. Conversely, incorporation of DHA into all tissues was related to dietary ALA in a curvilinear manner, with the maximum incorporation of DHA appearing to be between the LA-ALA ratios of 4:1 and 2:1. Feeding LA-ALA ratios of 10:1 and 0.5:1 resulted in lower and similar proportions of DHA in tissues despite the very different levels of dietary ALA (1.3 vs. 26.8% of total fats, respectively). These results are relevant to term infant studies in that they confirm our earlier findings of the positive effect on DHA status by lowering the LA-ALA ratio from 10:1 to 3:1 or 4:1, and they predict that ratios of LA-ALA below 4:1 would have little further beneficial effect on DHA status.     

DNA double-strand break and chromosomal rejoining defects with misrejoining in Nijmegen breakage syndrome cells

NBS1-deficient cells exhibit pronounced radiosensitivity and defects in chromosome integrity after ionizing radiation (IR) exposure, yet show only a minor defect in DNA double-strand break (DSB) rejoining, leaving an as yet unresolved enigma as to the nature of the radiosensitivity of these cells. To further investigate the relationship between radiosensitivity, DSB repair, and chromosome stability, we have compared cytological and molecular assays of DSB misrejoining and repair in NBS1-defective, wild type, and NBS1-complemented cells after IR damage. Our findings suggest a subtle defect in overall DSB rejoining in NBS1-defective cells and uniquely also reveal reduced ability of NBS1-defective cells to rejoin correct ends of DSBs. In agreement with published results, one of two different NBS1-defective cell lines showed a slight defect in overall rejoining of DSBs compared to its complemented counterpart, whereas another NBS line did not show any difference from wild type cells. Significant defects in the correct rejoining of DSBs compared to their respective controls were observed for both NBS1-defective lines. The defect in DSB rejoining and the increased misrejoining detected at the molecular level were also reflected in higher levels of fragments and translocations, respectively, at the chromosomal level. This work provides both molecular and cytological evidence that NBS1-deficient cells have defects in DSB processing and reveals that these molecular events can be manifest cytologically.     

Organization of the <Emphasis Type="Italic">MASP2</Emphasis> locus and its expression profile in mouse and rat

The identification of vertebral fracture in osteoporosis is based mainly on the identification of abnormal variation in vertebral shape, but this can be misleading in the presence of a non-fracture deformity or normal variant of vertebral shape. Qualitative identification of vertebral fracture (Qual) is influenced by the subjectivity of the approach, and although more objective, the semiquantitative method (SQ) can be difficult to apply. In addition, there has been little independent evaluation of SQ in relation to other approaches. We aimed to evaluate a new algorithm-based approach for the qualitative identification of vertebral fracture (ABQ) and to compare it with SQ and Qual. Two radiologists reported spinal radiographs for 372 postmenopausal women using Qual (reader 1), and SQ and ABQ (reader 2). Non-fracture deformities and normal variants were also reported using Qual and ABQ. The prevalence of vertebral fracture by subjects was higher for SQ (24%) than for Qual (11%) and ABQ (7%). Agreement was poor between SQ and the other methods, and moderate between Qual and ABQ. Twenty-two women with vertebral fracture were agreed by all three methods, similar to the total identified by ABQ (25 women). Seventeen women diagnosed with fracture by Qual, had non-fracture deformity or normal variant (but no fracture) according to ABQ. Of the women with SQ fractures, 53% and 70% were identified negative for fracture but positive for non-fracture deformity or normal variant by ABQ and Qual. The main sources of discrepancy between SQ and the other methods were Scheuermanns disease, normal variation, and degenerative change accompanied by short anterior vertebral height. For all methods, bone mineral density (BMD) and BMD Z-scores were lower in women with vertebral fractures than in those with no fractures. Bone mineral density and BMD Z-scores were also lower at the lumbar spine and total body in women with vertebral fractures according to Qual and ABQ than they were for SQ, and were lower in women with SQ fractures agreed by Qual and ABQ, compared with those diagnosed negative for fracture by Qual and ABQ (p<0.01). We conclude that poor agreement between methods arises mainly from difficulties in differentiating true fracture from non-fracture deformity. Our new approach attempts to address this problem but requires further testing in a larger study population.     

Analysis of the Arabidopsis O-acetylserine(thiol)lyase gene family demonstrates compartment-specific differences in the regulation of cysteine synthesis

Cys synthesis in plants takes place in plastids, cytosol, and mitochondria. Why Cys synthesis is required in all compartments with autonomous protein biosynthesis and whether Cys is exchanged between them has remained enigmatic. This question was addressed using Arabidopsis thaliana T-DNA insertion lines deficient in the final step of Cys biosynthesis catalyzed by the enzyme O-acetylserine(thiol)lyase (OAS-TL). Null alleles of oastlA or oastlB alone showed that cytosolic OAS-TL A and plastid OAS-TL B were completely dispensable, although together they contributed 95% of total OAS-TL activity. An oastlAB double mutant, relying solely on mitochondrial OAS-TL C for Cys synthesis, showed 25% growth retardation. Although OAS-TL C alone was sufficient for full development, oastlC plants also showed retarded growth. Targeted affinity purification identified the major OAS-TL-like proteins. Two-dimensional gel electrophoresis and mass spectrometry showed no compensatory changes of OAS-TL isoforms in the four mutants. Steady state concentrations of Cys and glutathione and pulse-chase labeling with [35S]sulfate indicated strong perturbation of primary sulfur metabolism. These data demonstrate that Cys and also sulfide must be sufficiently exchangeable between cytosol and organelles. Despite partial redundancy, the mitochondria and not the plastids play the most important role for Cys synthesis in Arabidopsis.     

Gene expression analysis of ischaemia and reperfusion in human microsurgical free muscle tissue transfer

The aim of this study was to analyse various gene expression profiles of muscle tissue during normoxia, ischaemia and after reperfusion in human muscle free flaps, to gain an understanding of the occurring regulatory, inflammatory and apoptotic processes on a cellular and molecular basis. Eleven Caucasian patients with soft tissue defects needing coverage with microsurgical free muscle flaps were included in this study. In all patients, the muscle samples were taken from free myocutaneous flaps. The first sample was taken before induction of ischaemia in normoxia (I), another one after ischaemia (II), and the last one was taken after reperfusion (III). The samples were analysed using DNA‐microarray, real‐time‐quantitative‐PCR and immunohistochemistry. DNA‐microarray analysis detected multiple, differentially regulated genes when comparing the different groups (I–III) with statistical significance. Comparing ischaemia (II) versus normoxia (I) educed 13 genes and comparing reperfusion (III) versus ischaemia (II) educed 19 genes. The comparison of reperfusion (III) versus normoxia (I) yielded 100 differentially regulated genes. Real‐time‐quantitative‐PCR confirmed the results of the DNA‐microarrays for a subset of four genes (CASP8, IL8, PLAUR and S100A8). This study shows that ischaemia and reperfusion induces alterations on the gene expression level in human muscle free flaps. Data may suggest that the four genes CASP8, IL8, PLAUR and S100A8 are of great importance in this context. We could not confirm the DNA‐microarry and real‐time‐quantitative‐PCR results on the protein level. Finally, these findings correspond with the surgeon’s clinical experience that the accepted times of ischaemia, generally up to 90 min., are not sufficient to induce pathophysiological processes, which can ultimately lead to flap loss. When inflammatory and apoptotic proteins are expressed at high levels, flap damage might occur and flap loss is likely. The sole expression on mRNA level might explain why flap loss is unlikely.     

Human placental transthyretin in fetal growth restriction in combination with preeclampsia and the HELLP syndrome

Fetal growth restriction is a serious, still poorly understood pregnancy-related pathology often associated with preeclampsia. Recent studies speculate on the role of human transthyretin, a carrier protein for thyroxin and retinol binding protein, in the etiology of both pregnancy pathologies. Objective was to investigate the localization and abundance of transthyretin (TTR) in placentas of pregnancies suffering from fetal growth restriction with and without preeclampsia and HELLP. This was a retrospective case control study on human paraffin-embedded placentas from pregnancies with a gestational age at delivery between the 24th and 34th week of gestation. 16 placentas were included in this study, 11 cases and 5 from normotensive pregnancies as controls. Cases were divided into three groups: four from early onset idiopathic intrauterine growth restriction (IUGR), four from early-onset severe preeclampsia (PE), and three from early-onset IUGR with preeclampsia plus HELLP syndrome. Distribution and abundance of TTR were investigated by means of immunohistochemistry. Semi quantitative analysis of TTR staining of placental sections revealed that TTR was mostly expressed in the villous trophoblast covering placental villi. Only weak staining of TTR in villous stroma could be detected. The comparison of placentas revealed that in pure IUGR and severe PE there is a much stronger TTR reactivity compared to controls and cases with IUGR?+?PE?+?HELLP. Concluding, the study showed that TTR is dysregulated in cases of IUGR and severe early onset preeclampsia. Interestingly, TTR expression is not affected in cases with HELLP syndrome that reveal the same staining intensities as age-matched controls.     

Twisted gastrulation modulates bone morphogenetic protein-induced collagen II and X expression in chondrocytes in vitro and in vivo

Twisted gastrulation (TSG) is an extracellular modulator of bone morphogenetic protein (BMP) activity and regulates dorsoventral axis formation in early Drosophila and Xenopus development. Studies on tsg-deficient mice also indicated a role of this protein in skeletal growth, but the mechanism of TSG activity in this process has not yet been investigated. Here we show for the first time by in situ hybridization and immunohistochemistry that TSG is strongly expressed in bovine and mouse growth plate cartilage as well as in fetal ribs, vertebral cartilage, and cartilage anlagen of the skull. Furthermore we provide evidence that TSG is directly involved in BMP-regulated chondrocyte differentiation and maturation. In vitro, TSG impaired the dose-dependent BMP-2 stimulation of collagen II and X expression in cultures of MC615 chondrocytes and primary mouse chondrocytes. In the presence of chordin, a BMP antagonist, the inhibitory effect of TSG was further enhanced. TSG also inhibited BMP-2-stimulated phosphorylation of Smad factors in chondrocytes, confirming the role of TSG as a modulator of BMP signaling. For analysis of TSG functions in cartilage development in vivo, the gene was overexpressed in transgenic mice under the control of the cartilage-specific Col2a1 promoter. As a result, Col10a1 expression was significantly reduced in the growth plates of transgenic embryos and newborns in comparison with wild type littermates as shown by in situ hybridization and by real time PCR analysis. The data suggest that TSG is an important modulator of BMP-regulated cartilage development and chondrocyte differentiation.