A sampling optimization analysis of soil‐bugs diversity (Crustacea,Isopoda, Oniscidea)

Biological diversity analysis is among the most informative approaches to describe communities and regional species compositions. Soil ecosystems include large numbers of invertebrates, among which soil bugs (Crustacea, Isopoda, Oniscidea) play significant ecological roles. The aim of this study was to provide advices to optimize the sampling effort, to efficiently monitor the diversity of this taxon, to analyze its seasonal patterns of species composition, and ultimately to understand better the coexistence of so many species over a relatively small area. Terrestrial isopods were collected at the Natural Reserve “Saline di Trapani e Paceco” (Italy), using pitfall traps monthly monitored over 2 years. We analyzed parameters of α‐ and β‐diversity and calculated a number of indexes and measures to disentangle diversity patterns. We also used various approaches to analyze changes in biodiversity over time, such as distributions of species abundances and accumulation and rarefaction curves. As concerns species richness and total abundance of individuals, spring resulted the best season to monitor Isopoda, to reduce sampling efforts, and to save resources without losing information, while in both years abundances were maximum between summer and autumn. This suggests that evaluations of β‐diversity are maximized if samples are first collected during the spring and then between summer and autumn. Sampling during these coupled seasons allows to collect a number of species close to the γ‐diversity (24 species) of the area. Finally, our results show that seasonal shifts in community composition (i.e., dynamic fluctuations in species abundances during the four seasons) may minimize competitive interactions, contribute to stabilize total abundances, and allow the coexistence of phylogenetically close species within the ecosystem.     

Cytoskeleton-associated, carbohydrate-metabolizing enzymes in maize identified by yeast two-hybrid screening

We have used yeast two-hybrid screens and biochemical methods to identify glycolytic enzymes that interact with subcellular structures in hypoxic maize seedlings. As binding domain-bait fusion constructs, we have cloned actin, cytosolic aldolase, the three sucrose synthase (SUS) isoforms SUS1, SUS3, and SH1 as well as the SNF1-related protein kinase into yeast and identified cytosolic isoforms of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), enolase, tubulin, and mitochondrial porin voltage-dependent anion channel protein (VDAC) as well as protein kinases and proteins involved in ubiquitinylation and proteasome-linked degradation as interacting activation domain-prey clones. The results were further confirmed using overlay blots (VDAC) as well as co-polymerization and co-precipitation assays (tubulin and actin). Some results were obtained that support the idea of metabolite and modification effects on the association, namely guanosine triphosphate (GTP)/MgCl₂ was necessary for the binding of enolase to actin. GAPDH is inactivated upon association with tubulin but then serves to stabilize the microtubules. The findings support the idea of the dynamic formation of locally associated complexes of enzymes involved in sucrose breakdown and glycolysis in plant cells depending on their metabolic state.     

Identification of a distinct side population of cancer cells in the Cal-51 human breast carcinoma cell line

“Side population” (SP) cells, which pump out the fluorescent dye H33342 via the ABCG2 transporter, define a putative stem/progenitor cell population in the mammary gland. Breast cancer SP cells recently isolated from the MCF-7 cell line possess similar properties and may represent stem cell-like cancer cells. This study extends SP cell analysis to a broad panel of human breast cancer cell lines and investigates the expression of differentiation-associated markers in isolated cancer SP cells. Expression of ABCG2 was determined in 16 breast cancer cell lines by quantitative RT-PCR, Western blotting and immunohistochemistry. Subsequently, all cell lines were screened for the presence of SP cells. Human breast cancer cell lines commonly express ABCG2. ABCG2-immunoreactivity was clearly restricted to rare cancer cells in several cell lines including Cal-51. Analysis of H33342-labeled Cal-51 cells revealed a small fraction of putative SP cells accounting for one percent of all cells. The genuine nature of Cal-51 SP cells was unambiguously verified by demonstrating a 30-fold increased ABCG2-expression in isolated Cal-51 SP cells. During in vitro expansion, Cal-51 SP cells generated heterologous non-SP (NSP) cells and ABCG2-expression declined dramatically. In contrast, NSP cells failed to sustain proliferation. Freshly isolated Cal-51 SP cells also exhibited increased expression of Muc1 and CALLA. Noteworthy, non-malignant mammary epithelial SP cells lack these differentiation markers, highlighting fundamental differences between non-malignant and breast cancer-derived SP cells. In summary, we established Cal-51 SP cells as a novel in vitro model to study differential gene expression in breast cancer-derived SP and NSP cells.     

Mesenchymal stem cells from ischemic heart disease patients improve left ventricular function after acute myocardial infarction

Mesenchymal stem cells (MSCs) from healthy donors improve cardiac function in experimental acute myocardial infarction (AMI) models. However, little is known about the therapeutic capacity of human MSCs (hMSCs) from patients with ischemic heart disease (IHD). Therefore, the behavior of hMSCs from IHD patients in an immune-compromised mouse AMI model was studied. Enhanced green fluorescent protein-labeled hMSCs from IHD patients (hMSC group: 2 x 10(5) cells in 20 microl, n = 12) or vehicle only (medium group: n = 14) were injected into infarcted myocardium of NOD/scid mice. Sham-operated mice were used as the control (n = 10). Cardiac anatomy and function were serially assessed using 9.4-T magnetic resonance imaging (MRI); 2 wk after cell transplantation, immunohistological analysis was performed. At day 2, delayed-enhancement MRI showed no difference in myocardial infarction (MI) size between the hMSC and medium groups (33 +/- 2% vs. 36 +/- 2%; P = not significant). A comparable increase in left ventricular (LV) volume and decrease in ejection fraction (EF) was observed in both MI groups. However, at day 14, EF was higher in the hMSC than in the medium group (24 +/- 3% vs. 16 +/- 2%; P < 0.05). This was accompanied by increased vascularity and reduced thinning of the infarct scar. Engrafted hMSCs (4.1 +/- 0.3% of injected cells) expressed von Willebrand factor (16.9 +/- 2.7%) but no stringent cardiac or smooth muscle markers. hMSCs from patients with IHD engraft in infarcted mouse myocardium and preserve LV function 2 wk after AMI, potentially through an enhancement of scar vascularity and a reduction of wall thinning.     

Trypanosoma cruzi: involvement of glycoinositolphospholipids in the attachment to the luminal midgut surface of Rhodnius prolixus

Trypanosoma cruzi epimastigotes adhere in vivo to the luminal surface of their triatomid vector digestive tract by molecular mechanisms, as yet, unknown. Here, we show that the administration of 0.5 microM epimastigote major surface glycoinositolphospholipids (GIPLs) to the infected bloodmeal inhibits up to 90% parasite infection in Rhodnius prolixus. The parasite behavior was investigated in vitro using fragments of the insect midgut. The addition of GIPLs in concentration as low as 50-100 nM impaired 95% the attachment of epimastigotes. Previous treatment of GIPLs with trifluoroacetic acid to remove the terminal beta-galactofuranosyl residues reversed 50% the epimastigote in vitro attachment. The binding sites of purified GIPLs on the luminal surface of the posterior midgut were exposed by immunofluorescence microscopy. These observations indicate that GIPLs are one of the components involved in the adhesion of T. cruzi to the luminal insect midgut surface and possibly one of the determinants of parasite infection in the insect vector.     

Gonadoinhibitory effects of Neb-colloostatin and Neb-TMOF on ovarian development in the mealworm, Tenebrio molitor L

The gonadostatic action of the peptides Neb-colloostatin (SIVPLGLPVPIGPIVVGPR) and Neb-TMOF (NPTNLH) from Neobellieria bullata was studied in female mealworm Tenebrio molitor. Both peptides potently inhibit ovarian development and terminal oocyte maturation of mated females during their first reproductive cycle. Injection of 4 mug of Neb-colloostatin or Neb-TMOFNeb-TMOF reduced, at day 4 of the cycle, the size of the terminal oocytes to about half or one third of the normal size in saline-injected controls. In addition, follicular patency was arrested. The injections of Neb-colloostatin and Neb-TMOF also caused a delay to the first ovulation and oviposition as well as a reduction of the number of eggs by about 50% in the first 3 days of the oviposition period. At 4 days after adult emergence, none of the peptides had caused significant changes in protein concentration or composition of the haemolymph. However, both peptides reduced total protein content in ovaries and induced qualitative changes in ovarian protein patterns. Electrophoretic analyses indicated that Neb-colloostatin and Neb-TMOF caused a loss of two proteins (150, 180 kDa) and a drastic reduction of 4 others (39, 43, 47, 130 kDa), which are the most abundant ones in ovaries of control females. On the other hand, they increased the concentration of 2 other polypeptides (65, 70 kDa), which normally occur in insignificant quantities in ovaries. Our results indicate that both peptides have a very similar mode of action despite the differences in their amino acid sequence. They seem to interfere with vitellogenin production by the fat body as well as with vitellogen uptake by the oocytes through modification of patency.