Photosynthetic compensation by the reproductive structures in the spring ephemeral Gagea lutea

Growth and reproduction of spring ephemerals inhabiting deciduous forests progress simultaneously during a short period from snowmelt to canopy closure. To clarify the mechanism to mitigate the cost of reproduction, contributions of foliar and non-foliar photosynthetic products to seed production were examined in a spring ephemeral Gagea lutea. Leaf growth, foliar and non-foliar photosynthetic activities, and total assimilated products were compared among reproductive-intact, floral bud-removal, and vegetative plants. Translocation of current photosynthetic products to individual organs was quantified by ¹³CO₂-trace experiment. Bulb growth was compared between hand-pollination and floral bud-removal treatments. Finally, seed set was compared between intact, leaf-clipping, and bract-clipping treatments. Fruit-forming plants retained leaves longer than vegetative and floral bud-removal plants, but the assimilative contribution of extended leaf longevity was negligible. Carbon supply by bract photosynthesis was large enough for fruit development, while carbon supply by fruit photosynthesis was offset by the high respiration loss. Foliar photosynthetic products were largely transported to bulbs, while translocation to reproductive functions was negligible. Because the floral bud-removal increased the bulb growth, lack of reproduction could lead to more storage. The leaf-clipping had no effect on seed production, while the bract-clipping significantly reduced the seed production. Therefore, current photosynthesis of leafy bracts might be a major carbon source for fruit development. This self-compensative mechanism of reproductive structure enables the continuous reproductive activity in this species.     

Lipid‐Labeling Facilitates a Novel Magnetic Isolation Procedure to Characterize Pathogen‐Containing Phagosomes

Here we describe a novel approach for the isolation and biochemical characterization of pathogen‐containing compartments from primary cells: We developed a lipid‐based procedure to magnetically label the surface of bacteria and visualized the label by scanning and transmission electron microscopy (SEM, TEM). We performed infection experiments with magnetically labeled Mycobacterium avium, M. tuberculosis and Listeria monocytogenes and isolated magnetic bacteria‐containing phagosomes using a strong magnetic field in a novel free‐flow system. Magnetic labeling of M. tuberculosis did not affect the virulence characteristics of the bacteria during infection experiments addressing host cell activation, phagosome maturation delay and replication in macrophages in vitro. Biochemical analyses of the magnetic phagosome‐containing fractions provided evidence of an enhanced presence of bacterial antigens and a differential distribution of proteins involved in the endocytic pathway over time as well as cytokine‐dependent changes in the phagosomal protein composition. The newly developed method represents a useful approach to characterize and compare pathogen‐containing compartments, in order to identify microbial and host cell targets for novel anti‐infective strategies.     

Genetics and prevention: a policy in the making

Abstract

This article explores the processes through which the advances of genetic research are incorporated into public health care in Denmark. Drawing on ethnographic fieldwork in cancer genetic counselling, the implementation of new medical advances is investigated by following the establishment of a policy on informing relatives at risk of hereditary cancer. This case material provides the occasion to examine how policies are shaped in a governmental process through which different actors seek to establish a common goal for a specific health practice. The struggle to define such a goal implies a struggle to define where to draw the line between health and disease and what makes up a healthy person in the context of genetic knowledge. The authors argue that in the process of establishing a policy in the field of cancer genetics the imperative of prevention comes to provide the framework within which an ethics of rights and responsibilities is constituted and the target group of cancer genetic counselling defined. This ethics is not determined by or inherent in genetic technology itself, but constituted in a social process and therefore negotiated within pre-existing frameworks of understanding in professional practice.     

Optimization of methodology for production of CD25/CD71 allodepleted donor T cells for clinical use

Background aimsImmunotherapy with allodepleted donor T cells improves immunity after T cell-depleted hematopoietic stem cell transplantation. We developed a methodology for selective depletion of alloreactive T cells after activation with host antigen-presenting cells by targeting T cells up-regulating CD25 and CD71. Combined depletion of these cells yields a pool of allodepleted donor T cells with antiviral properties with minimal capacity to cause graft-versus-host disease.MethodsMature dendritic cells were irradiated and used to stimulate donor peripheral blood mononuclear cells for 4 days. The co-culture was stained with anti-CD71-biotin followed by CliniMACS CD25 and Anti-Biotin Reagents (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany) before depletion on the CliniMACS Plus (Miltenyi Biotec GmbH). Residual alloreactivity was tested by flow cytometry, a secondary mixed lymphocyte reaction and limiting dilution analysis, and specific anti-viral immunity with pentamer staining. The large-scale protocol was tested under current good manufacturing practice conditions in five donor-recipient pairs of human leukocyte antigen-matched volunteer donors.ResultsWe developed a closed-system methodology using cell differentiation bags for cell culture and the COBE2991 Cell Processor (CaridianBCT, Lakewood, CO, USA). We also validated an anti-CD71-biotin generated for ex vivo clinical use. In five large-scale runs, the depleted fraction demonstrated excellent viability (99.9%), minimal residual expression of CD3/CD25 and CD3/CD71 (<0.2%) and passed tests for Mycoplasma, endotoxin, bacterial and fungal sterility. In secondary mixed lymphocyte reaction assays, the median response to host after allodepletion was 0%, whereas responses to third-party peripheral blood mononuclear cells were preserved (median, 105%; range 37%–350%). Limiting dilution analysis assays also demonstrated a reduction in response to host (median, ?1.11 log) with preservation of third-party responses, and testing with human leukocyte antigen-restricted pentamers showed that populations of Epstein-Barr virus-specific and cytomegalovirus-specific CD8⁺ T cells were retained after depletion.ConclusionsWe optimized a protocol for the combined immunomagnetic depletion of alloreactive CD25/CD71 T cells under current good manufacturing practice conditions and tested the efficacy in five donor-recipient pairs.     

Prevalence of mandibular osteomyelitis in roe deer (Capreolus capreolus) in Slovenia

Mandibular osteomyelitis in free-ranging cervids is a rare, but eventually fatal, disease. We examined 41,895 defleshed mandibles of roe deer collected throughout Slovenia in 2007. Mandibles from 14,679 fawns had no signs of osteomyelitis, and were excluded from further analysis. Of the remaining 27,216 specimens, chronic osteomyelitis ("lumpy jaw") was found in 113 mandibles (4.2%; 7.0% of adults). The majority of cases were observed from the Mediterranean and subalpine regions, near larger cities and thermal power plants. There was no statistically significant correlation between severity of the mandibular osteomyelitis and body weight. Females were more frequently affected than males. Coarse and abrasive food, and to some extent dental fluorosis, are the most probable triggers for development of lesions.     

Angiotensin II and norepinephrine activate specific calcineurin-dependent NFAT transcription factor isoforms in cardiomyocytes

Norepinephrine (NE) and angiotensin II (ANG II) are primary effectors of the sympathetic adrenergic and the renin-angiotensin-aldosterone systems, mediating hypertrophic, apoptotic, and fibrotic events in the myocardium. As NE and ANG II have been shown to affect intracellular calcium in cardiomyocytes, we hypothesized that they activate the calcium-sensitive, prohypertrophic calcineurin-nuclear factor of activated T-cell (NFATc) signaling pathway. More specifically, we have investigated isoform-specific activation of NFAT in NE- and ANG II-stimulated cardiomyocytes, as it is likely that each of the four calcineurin-dependent isoforms, c1-c4, play specific roles. We have stimulated neonatal ventriculocytes from C57/B6 and NFAT-luciferase reporter mice with ANG II or NE and quantified NFAT activity by luciferase activity and phospho-immunoblotting. ANG II and NE increased calcineurin-dependent NFAT activity 2.4- and 1.9-fold, measured as luciferase activity after 24 h of stimulation, and induced protein synthesis, measured by radioactive leucine incorporation after 24 and 72 h. To optimize measurements of NFAT isoforms, we examined the specificity of NFAT antibodies on peptide arrays and by immunoblotting with designed blocking peptides. Western analyses showed that both effectors activate NFATc1 and c4, while NFATc2 activity was regulated by NE only, as measured by phospho-NFAT levels. Neither ANG II nor NE activated NFATc3. As today's main therapies for heart failure aim at antagonizing the adrenergic and renin-angiotensin-aldosterone systems, understanding their intracellular actions is of importance, and our data, through validating a method for measuring myocardial NFATs, indicate that ANG II and NE activate specific NFATc isoforms in cardiomyocytes.     

mSEL-1L (Suppressor/enhancer Lin12-like) protein levels influence murine neural stem cell self-renewal and lineage commitment

Murine SEL-1L (mSEL-1L) is a key component of the endoplasmic reticulum-associated degradation pathway. It is essential during development as revealed by the multi-organ dysfunction and in uterus lethality occurring in homozygous mSEL-1L-deficient mice. Here we show that mSEL-1L is highly expressed in pluripotent embryonic stem cells and multipotent neural stem cells (NSCs) but silenced in all mature neural derivatives (i.e. astrocytes, oligodendrocytes, and neurons) by mmu-miR-183. NSCs derived from homozygous mSEL-1L-deficient embryos (mSEL-1L(-/-) NSCs) fail to proliferate in vitro, show a drastic reduction of the Notch effector HES-5, and reveal a significant down-modulation of the early neural progenitor markers PAX-6 and OLIG-2, when compared with the wild type (mSEL-1L(+/+) NSCs) counterpart. Furthermore, these cells are almost completely deprived of the neural marker Nestin, display a significant decrease of SOX-2 expression, and rapidly undergo premature astrocytic commitment and apoptosis. The data suggest severe self-renewal defects occurring in these cells probably mediated by misregulation of the Notch signaling. The results reported here denote mSEL-1L as a primitive marker with a possible involvement in the regulation of neural progenitor stemness maintenance and lineage determination.     

Behaviour of rainbow trout Oncorhynchus mykiss presented with a choice of normoxia and stepwise progressive hypoxia

The objective of this study was to identify behavioural adjustments leading to avoidance of hypoxia. Using the oxygen-sensitive species rainbow trout Oncorhynchus mykiss as a model, individual fish were recorded while moving freely between two sides of a test arena: one with normoxia and one with stepwise progressive hypoxia [80-30% dissolved oxygen (DO) air saturation]. The results demonstrated a gradual decrease in the total time spent in hypoxia starting at 80% DO air saturation. At this DO level, the avoidance of hypoxia could not be attributed to changes in spontaneous swimming speed, neither in normoxia nor in hypoxia. Reducing the DO level to 60% air saturation resulted in decreased spontaneous swimming speed in normoxia, yet the number of trips to the hypoxic side of the test arena remained unchanged. Moreover, data revealed increased average residence time per trip in normoxia at DO levels ≤60% air saturation and decreased average residence time per trip in hypoxia at DO levels ≤50% air saturation. Finally, the spontaneous swimming speed in hypoxia increased at DO levels ≤40% air saturation and the number of trips to hypoxia decreased at the 30% DO air saturation level. Thus, avoidance of the deepest hypoxia was connected with a reduced number of trips to hypoxia as well as decreased and increased spontaneous swimming speed in normoxia and hypoxia, respectively. Collectively, the data support the conclusions that the mechanistic basis for avoidance of hypoxia may (1) not involve changes in swimming speed during mild hypoxia and (2) depend on the severity of hypoxia.     

Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp

Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. However, substitutions within the fourth FAS1 (FAS1-4) domain (A546T, R555Q, and R555W) affected the overall stability of intact TGFBIp revealing the following stability ranking R555W>WT>R555Q>A546T. Significantly, the stability ranking of the isolated FAS1-4 domains mirrored the behavior of the intact protein. In addition, it was linked to the aggregation propensity as the least stable mutant (A546T) forms amyloid fibrils while the more stable variants generate non-amyloid amorphous deposits in vivo. Significantly, the data suggested that both an increase and a decrease in the stability of FAS1-4 may unleash a disease mechanism. In contrast, amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation.