Monocarboxylate Transporter 8 Modulates the Viability and Invasive Capacity of Human Placental Cells and Fetoplacental Growth in Mice

Monocarboxylate transporter 8 (MCT8) is a well-established thyroid hormone (TH) transporter. In humans, MCT8 mutations result in changes in circulating TH concentrations and X-linked severe global neurodevelopmental delay. MCT8 is expressed in the human placenta throughout gestation, with increased expression in trophoblast cells from growth-restricted pregnancies. We postulate that MCT8 plays an important role in placental development and transplacental TH transport. We investigated the effect of altering MCT8 expression in human trophoblast in vitro and in a Mct8 knockout mouse model. Silencing of endogenous MCT8 reduced T3 uptake into human extravillous trophoblast-like cells (SGHPL-4; 40%, P<0.05) and primary cytotrophoblast (15%, P<0.05). MCT8 over-expression transiently increased T3 uptake (SGHPL-4∶30%, P<0.05; cytotrophoblast: 15%, P<0.05). Silencing MCT8 did not significantly affect SGHPL-4 invasion, but with MCT8 over-expression T3 treatment promoted invasion compared with no T3 (3.3-fold; P<0.05). Furthermore, MCT8 silencing increased cytotrophoblast viability (∼20%, P<0.05) and MCT8 over-expression reduced cytotrophoblast viability independently of T3 (∼20%, P<0.05). In vivo, Mct8 knockout reduced fetal:placental weight ratios compared with wild-type controls at gestational day 18 (25%, P<0.05) but absolute fetal and placental weights were not significantly different. The volume fraction of the labyrinthine zone of the placenta, which facilitates maternal-fetal exchange, was reduced in Mct8 knockout placentae (10%, P<0.05). However, there was no effect on mouse placental cell proliferation in vivo. We conclude that MCT8 makes a significant contribution to T3 uptake into human trophoblast cells and has a role in modulating human trophoblast cell invasion and viability. In mice, Mct8 knockout has subtle effects upon fetoplacental growth and does not significantly affect placental cell viability probably due to compensatory mechanisms in vivo.     

The influence of insulin administration after weaning the first litter on ovulation rate and embryo survival in sows

Primiparous crossbred sows (n = 43), lactating for an average of 21.1 +/- 0.1 d and weaning 8.7 +/- 0.1 pigs, were used to evaluate the influence of insulin on ovulation rate and embryo survival. The sows were maintained on 2.3 kg/head/d of a 14% protein gestation diet during pregnancy, fed ad libitum during lactation, given 2.7 kg/head/d from weaning until re-breeding and fed 2.3 kg/head/d after mating. Beginning the day after weaning (Day 0) sows were treated with 0.4 IU/kg body weight (BW) insulin (n = 21) or were administered an equivalent volume of saline (n = 22) for 4 d. Beginning on Day 3 and continuing until Day 14 after weaning, the sows were checked for estrus twice daily and were artificially inseminated using pooled semen from 2 fertile boars. At slaughter (days 30 to 40 of gestation), ovaries and uteri were collected, and the ovulation rate, embryo number and viability, and uterine weight and length were evaluated and recorded. Use of insulin decreased the average interval from weaning to estrus compared with saline by increasing percentage in estrus by Day 14 after weaning (5.0 +/- 0.57 vs 6.9 +/- 0.56 d, respectively; P < 0.03). Ovulation rate, number of embryos, embryo survival, and average uterine length and weight were not influenced by insulin treatment. Overall, insulin affected reproductive efficiency in primiparous sows by increasing the percentage of sows in estrus.     

Hepatocyte growth factor induced human trophoblast motility involves phosphatidylinositol-3-kinase,mitogen-activated protein kinase,and inducible nitric oxide synthase

Hepatocyte growth factor (HGF) increases human trophoblast motility and invasion, an effect which is abrogated when inducible nitric oxide synthase (iNOS) is inhibited. In this study we have investigated the pathways involved in the regulation of trophoblast motility. Both basal and HGF-stimulated motility of the extravillous trophoblast cell line, SGHPL-4, were inhibited in a dose-dependent manner by the phosphatidylinositol-3-kinase (PI3-kinase) inhibitor, LY294002. HGF-stimulated iNOS expression was also inhibited by LY294002 and direct activation of PI3-kinase, using the peptide 740Y-P, led to an increase in iNOS expression and cell motility. Pretreatment with rapamycin, which acts at a point distal to PI3-kinase activation, also inhibited basal and HGF-stimulated motility. Inhibition of the p42/p44 mitogen activated protein kinase (MAPK) pathway but not the p38 MAPK pathway had significant inhibitory effects on HGF-stimulated but not basal trophoblast motility. Inhibition of p42/p44 MAPK also inhibited HGF-induced iNOS expression. This data demonstrate that the PI3-kinase signaling pathway is involved in basal trophoblast motility and that both MAPK and PI3-kinase signaling pathways are important in HGF-stimulated motility and iNOS expression.     

High-fidelity discrete modeling of the HPA axis: a study of regulatory plasticity in biology

Background

The hypothalamic-pituitary-adrenal (HPA) axis is a central regulator of stress response and its dysfunction has been associated with a broad range of complex illnesses including Gulf War Illness (GWI) and Chronic Fatigue Syndrome (CFS). Though classical mathematical approaches have been used to model HPA function in isolation, its broad regulatory interactions with immune and central nervous function are such that the biological fidelity of simulations is undermined by the limited availability of reliable parameter estimates.

Method

Here we introduce and apply a generalized discrete formalism to recover multiple stable regulatory programs of the HPA axis using little more than connectivity between physiological components. This simple discrete model captures cyclic attractors such as the circadian rhythm by applying generic constraints to a minimal parameter set; this is distinct from Ordinary Differential Equation (ODE) models, which require broad and precise parameter sets. Parameter tuning is accomplished by decomposition of the overall regulatory network into isolated sub-networks that support cyclic attractors. Network behavior is simulated using a novel asynchronous updating scheme that enforces priority with memory within and between physiological compartments.

Results

Consistent with much more complex conventional models of the HPA axis, this parsimonious framework supports two cyclic attractors, governed by higher and lower levels of cortisol respectively. Importantly, results suggest that stress may remodel the stability landscape of this system, favoring migration from one stable circadian cycle to the other. Access to each regime is dependent on HPA axis tone, captured here by the tunable parameters of the multi-valued logic. Likewise, an idealized glucocorticoid receptor blocker alters the regulatory topology such that maintenance of persistently low cortisol levels is rendered unstable, favoring a return to normal circadian oscillation in both cortisol and glucocorticoid receptor expression.

Conclusion

These results emphasize the significance of regulatory connectivity alone and how regulatory plasticity may be explored using simple discrete logic and minimal data compared to conventional methods.

     

Vicinity analysis: a methodology for the identification of similar protein active sites

Vicinity analysis (VA) is a new methodology developed to identify similarities between protein binding sites based on their three-dimensional structure and the chemical similarity of matching residues. The major objective is to enable searching of the Protein Data Bank (PDB) for similar sub-pockets, especially in proteins from different structural and biochemical series. Inspection of the ligands bound in these pockets should allow ligand functionality to be identified, thus suggesting novel monomers for use in library synthesis. VA has been developed initially using the ATP binding site in kinases, an important class of protein targets involved in cell signalling and growth regulation. This paper defines the VA procedure and describes matches to the phosphate binding sub-pocket of cyclin-dependent protein kinase 2 that were found by searching a small test database that has also been used to parameterise the methodology.     

Proteins of Vesicular Stomatitis Virus and of Phenotypically Mixed Vesicular Stomatitis Virus-Simian Virus 5 Virions

The identity of the glycoprotein of vesicular stomatitis virus (VSV) as the spike protein has been confirmed by the removal of the spikes with a protease from Streptomyces griseus, leaving bullet-shaped particles bounded by a smooth membrane. This treatment removes the glycoprotein but does not affect the other virion proteins, apparently because they are protected from the enzyme by the lipids in the viral membrane. The proteins of phenotypically mixed, bullet-shaped virions produced by cells mixedly infected with VSV and the parainfluenza virus simian virus 5 (SV5) have been analyzed by polyacrylamide gel electrophoresis. These virions contain all the VSV proteins plus the two SV5 spike proteins, both of which are glycoproteins. The finding of the SV5 spike glycoproteins on virions with the typical morphology of VSV indicates that there is not a stringent requirement that only the VSV glycoprotein can be used to form the bullet-shaped virion. On the other hand, the SV5 nucleocapsid protein and the major non-spike protein of the SV5 envelope were not detected in the phenotypically mixed virions, and this suggests that a specific interaction between the VSV nucleocapsid and regions of the cell membrane which contain the nonglycosylated VSV envelope protein is necessary for assembly of the bullet-shaped virion.     

Theileria parva: significance of leukocytes for infecting cattle

Using an artificial feeding technique, infective particles of Theileria parva were harvested in bovine blood in capillary tubes from prefed female Rhipicephalus appendiculatus over a 2-hr period. Inoculations of this blood feed pool invariably resulted in the establishment of patent East Coast fever in autogeneic or syngeneic cattle, i.e., the blood donors or their monozygotic twins, but not in unrelated animals. Mechanical passage of 4 × 10⁶ macroschizont-infected lymphoid cells, harvested from a heifer with East Coast fever, successfully induced patent theileriosis in the donor's monozygotic twin but not in a susceptible allogeneic bovid. The significance of parasite-cell association and histocompatibility of infected cells is discussed in relation to natural and mechanical transmission of Theileria parva.     

Mosaicism of Podocyte Involvement Is Related to Podocyte Injury in Females with Fabry Disease

Background

Fabry disease. an X-linked deficiency of α-galactosidase A coded by the GLA gene, leads to intracellular globotriaosylceramide (GL-3) accumulation. Although less common than in males, chronic kidney disease, occurs in ∼15% of females. Recent studies highlight the importance of podocyte injury in Fabry nephropathy development and progression. We hypothesized that the greater the % of podocytes with active wild-type GLA gene (due to X-inactivation of the mutant copy) the less is the overall podocyte injury.

Methods

Kidney biopsies from 12 treatment-naive females with Fabry disease, ages 15 (8–63), median [range], years were studied by electron microscopy and compared with 4 treatment-naive male patients.

Results

In females, 51 (13–100)% of podocytes (PC) per glomerulus had no GL-3 inclusions, this consistent with a non-Fabry podocyte phenotype (NFPC). In PC with GL-3 inclusions [Fabry podocyte phenotype (FPC)], GL-3 volume density per podocyte was virtually identical in females and males, consistent with little or no cross-correction between FPC and NFPC. %NFPC per glomerulus (%NFPC/glom) correlated with age in females (r = 0.65, p = 0.02), suggesting a survival disadvantage for FPC over time. Age-adjusted %NFPC/glom was inversely related to foot process width (FPW) (r = −0.75, p = 0.007), an indicator of PC injury. GL-3 volume density in FPC in females correlated directly with FPW.

Conclusions

These findings support important relationships between podocyte mosaicism and podocyte injury in female Fabry patients. Kidney biopsy, by providing information about podocyte mosaicism, may help to stratify females with Fabry disease for kidney disease risk and to guide treatment decisions.