Real-Time Tracking of BODIPY-C12 Long-Chain Fatty Acid in Human Term Placenta Reveals Unique Lipid Dynamics in Cytotrophoblast Cells

While the human placenta must provide selected long-chain fatty acids to support the developing fetal brain, little is known about the mechanisms underlying the transport process. We tracked the movement of the fluorescently labeled long-chain fatty acid analogue, BODIPY-C₁₂, across the cell layers of living explants of human term placenta. Although all layers took up the fatty acid, rapid esterification of long-chain fatty acids and incorporation into lipid droplets was exclusive to the inner layer cytotrophoblast cells rather than the expected outer syncytiotrophoblast layer. Cytotrophoblast is a progenitor cell layer previously relegated to a repair role. As isolated cytotrophoblasts differentiated into syncytialized cells in culture, they weakened their lipid processing capacity. Syncytializing cells suppress previously active genes that regulate fatty-acid uptake (SLC27A2/FATP2, FABP4, ACSL5) and lipid metabolism (GPAT3, LPCAT3). We speculate that cytotrophoblast performs a previously unrecognized role in regulating placental fatty acid uptake and metabolism.     

The demonstration of arylsulfatases with 4-nitro-1,2-benzenediol mono(hydrogen sulfate) by the formation of osmium blacks at the sites of copper capture.

A new method is described for the direct cytochemical demonstration of lysosomal arylsulfatases utilizing a synthetic substrate, 4-nitro-1,2-benzenediol mono(hydrogen sulfate), and a copper capture reaction. A small amount of Hatchett's brown (cupric ferrocyanide, Cu2Fe(CN)6-7 H2O) formed at the subcellular sites of copper capture is then utilized as a heterogeneous catalyst to effect the oxidative polymerization of 3,3'-diaminobenzidine which results in the formation of an insoluble, highly colored osmiophilic indamine polymer at the sites of enzymatic activity. The reaction product even at this stage prior to osmication is highly visible. It is readily seen with a light microscope in 50 mum sections of fixed tissues prepared with a mechanical chopper or in 10 micron cryostat sections treated for arylsulfatase activity. Upon osmication, an electron-opaque osmium black is formed which is much less soluble than the products of either the lead or barium capture reactions currently used for the demonstration of arylsulfatase with the electron microscope. The selection of areas of plastic-embedded tissues for ultrathin sectioning is facilitated by the ready visibility of these osmium black end products on 1-2 mum plastic sections which can be studied with the light microscope. This method gives permanent specimens demonstrating arylsulfatases A or B in lysosomes and autophagic vacuoles. In addition, enzyme activity is seen occasionally in the Golgi region or lamellae of certain cells believed to be elaborating sulfated products. In these instances, it may be demonstrating sulfotransferase activity.     

Interaction of Nectarin 4 with a fungal protein triggers a microbial surveillance and defense mechanism in nectar

Understanding the biochemical mechanisms by which plants respond to microbial infection is a fundamental goal of plant science. Extracellular dermal glycoproteins (EDGPs) are widely expressed in plant tissues and have been implicated in plant defense responses. Although EDGPs are known to interact with fungal proteins, the downstream effects of these interactions are poorly understood. To gain insight into these phenomena, we used tobacco floral nectar as a model system to identify a mechanism by which the EDGP known as Nectarin IV (NEC4) functions as pathogen surveillance molecule. Our data demonstrates that the interaction of NEC4 with a fungal endoglucanase (XEG) promotes the catalytic activity of Nectarin V (NEC5), which catalyzes the conversion of glucose and molecular oxygen to gluconic acid and H(2)O(2). Significantly enhanced NEC5 activity was observed when XEG was added to nectar or nectarin solutions that contain NEC4. This response was also observed when the purified NEC4:XEG complex was added to NEC4-depleted nectarin solutions, which did not respond to XEG alone. These results indicate that formation of the NEC4:XEG complex is a key step leading to induction of NEC5 activity in floral nectar, resulting in an increase in concentrations of reactive oxygen species (ROS), which are known to inhibit microbial growth directly and activate signal transduction pathways that induce innate immunity responses in the plant.     

The intrauterine origins of Hodgkin's lymphoma

Background: Both small and large body size at birth are now known to predict a range of chronic disorders in adult life, including certain cancers. These associations are thought to reflect “fetal programming.” This may lead to impairment of a small number of key systems including the immune system. Hodgkin's lymphoma is a disease of the immune system. We have therefore examined the association between Hodgkin's lymphoma and early development. Our hypothesis was that the disease would be associated with markers of poor fetal growth, specifically small body size or small placental size at birth. Methods: Using the Finnish Cancer Registry we identified patients with Hodgkin's disease in a cohort of 20,431 people born in Helsinki during 1924–44. Each person has a detailed birth record. Results: There were 12 patients with Hodgkin's disease, giving an incidence comparable to international rates. The disease was associated with prolonged gestation. For every additional week of gestation the hazard ratio was 1.37 (95% CI 1.00–1.87; p = 0.05). The disease was also associated with a short placental surface. After allowing for gestation, for each centimetre increase in surface length, the hazard ratio was 0.70 (0.53–0.92; p = 0.01). The disease was not associated with birth weight or length or maternal body size. Conclusions: We have shown that Hodgkin's lymphoma is associated with prolonged gestation and a short length of the placental surface. We speculate that Hodgkin's lymphoma is initiated by two events in fetal life. One, which is an immune event, is associated with prolonged gestation, while the other is associated with growth faltering.     

Segregated regulatory CD39+CD4+ T cell function: TGF-β-producing Foxp3- and IL-10-producing Foxp3+ cells are interdependent for protection against collagen-induced arthritis

Oral immunization with a Salmonella vaccine vector expressing enterotoxigenic Escherichia coli colonization factor Ag I (CFA/I) can protect against collagen-induced arthritis (CIA) by dampening IL-17 and IFN-γ via enhanced IL-4, IL-10, and TGF-β. To identify the responsible regulatory CD4(+) T cells making the host refractory to CIA, Salmonella-CFA/I induced CD39(+)CD4(+) T cells with enhanced apyrase activity relative to Salmonella vector-immunized mice. Adoptive transfer of vaccine-induced CD39(+)CD4(+) T cells into CIA mice conferred complete protection, whereas CD39(-)CD4(+) T cells did not. Subsequent analysis of vaccinated Foxp3-GFP mice revealed the CD39(+) T cells were composed of Foxp3-GFP(-) and Foxp3-GFP(+) subpopulations. Although each adoptively transferred Salmonella-CFA/I-induced Foxp3(-) and Foxp3(+)CD39(+)CD4(+) T cells could protect against CIA, each subset was not as efficacious as total CD39(+)CD4(+) T cells, suggesting their interdependence for optimal protection. Cytokine analysis revealed Foxp3(-) CD39(+)CD4(+) T cells produced TGF-β, and Foxp3(+)CD39(+)CD4(+) T cells produced IL-10, showing a segregation of function. Moreover, donor Foxp3-GFP(-) CD4(+) T cells converted to Foxp3-GFP(+) CD39(+)CD4(+) T cells in the recipients, showing plasticity of these regulatory T cells. TGF-β was found to be essential for protection because in vivo TGF-β neutralization reversed activation of CREB and reduced the development of CD39(+)CD4(+) T cells. Thus, CD39 apyrase-expressing CD4(+) T cells stimulated by Salmonella-CFA/I are composed of TGF-β-producing Foxp3(-) CD39(+)CD4(+) T cells and support the stimulation of IL-10-producing Foxp3(+) CD39(+)CD4(+) T cells.     

Stuttered swallowing: Electric stimulation of the right insula interferes with water swallowing. A case report

Background  

Various functional resonance imaging, magnetoencephalographic and lesion studies suggest the involvement of the insular cortex in the control of swallowing. However, the exact location of insular activation during swallowing and its functional significance remain unclear.     

Vulnerabilities in Yersinia pestis caf operon are unveiled by a Salmonella vector

During infection, Yersinia pestis uses its F1 capsule to enhance survival and cause virulence to mammalian host. Since F1 is produced in large quantities and secreted into the host tissues, it also serves as a major immune target. To hold this detrimental effect under proper control, Y. pestis expresses the caf operon (encoding the F1 capsule) in a temperature-dependent manner. However, additional properties of the caf operon limit its expression. By overexpressing the caf operon in wild-type Salmonella enterica serovar Typhimurium under a potent promoter, virulence of Salmonella was greatly attenuated both in vitro and in vivo. In contrast, expression of the caf operon under the regulation of its native promoter exhibited negligible impairment of Salmonellae virulence. In-depth investigation revealed all individual genes in the caf operon attenuated Salmonella when overexpressed. The deleterious effects of caf operon and the caf individual genes were further confirmed when they were overexpressed in Y. pestis KIM6+. This study suggests that by using a weak inducible promoter, the detrimental effects of the caf operon are minimally manifested in Y. pestis. Thus, through tight regulation of the caf operon, Y. pestis precisely balances its capsular anti-phagocytic properties with the detrimental effects of caf during interaction with mammalian host.     

Thyroid hormone drives fetal cardiomyocyte maturation

Tri-iodo-l-thyronine (T(3)) suppresses the proliferation of near-term serum-stimulated fetal ovine cardiomyocytes in vitro. Thus, we hypothesized that T(3) is a major stimulant of cardiomyocyte maturation in vivo. We studied 3 groups of sheep fetuses on gestational days 125-130 (term ～145 d): a T(3)-infusion group, to mimic fetal term levels (plasma T(3) levels increased from ～0.1 to ～1.0 ng/ml; t(1/2)～24 h); a thyroidectomized group, to produce low thyroid hormone levels; and a vehicle-infusion group, to serve as intact controls. At 130 d of gestation, sections of left ventricular freewall were harvested, and the remaining myocardium was enzymatically dissociated. Proteins involved in cell cycle regulation (p21, cyclin D1), proliferation (ERK), and hypertrophy (mTOR) were measured in left ventricular tissue. Evidence that elevated T(3) augmented the maturation rate of cardiomyocytes included 14% increased width, 31% increase in binucleation, 39% reduction in proliferation, 150% reduction in cyclin D1 protein, and 500% increase in p21 protein. Increased expression of phospho-mTOR, ANP, and SERCA2a also suggests that T(3) promotes maturation and hypertrophy of fetal cardiomyocytes. Thyroidectomized fetuses had reduced cell cycle activity and binucleation. These findings support the hypothesis that T(3) is a prime driver of prenatal cardiomyocyte maturation.