Crayfish exhibit innate immune responses via hemocytes and their products. There are 3 hemocyte populations: hyaline cells, granular cells, and semigranular cells. Hemocytes from laboratory housed, untreated crayfish (normal crayfish) have been quantified on the basis of cell type, cell size, and cell granularity using Flow Cytometry. These data present the first overall picture of normal hemocytes from Red Swamp Crayfish with regard to cell type, cell size, and cell granularity and will serve as a baseline for all future studies in our lab. Experiments using crayfish injected with Pseudomonas aeruginosa, Staphylococcus aureus, or crayfish saline alone showed significant and consistent changes in cell type in cells from crayfish injected with bacteria with a decrease in hyaline cells and an increase in granular cells. This effect was greater in crayfish injected with Gram - bacteria. In addition, crayfish injected with Pseudomonas aeruginosa showed a significant difference in Granular cell size with a shift to larger cells and a significant decrease in granularity in the Granular cell population. Cells from crayfish treated with Staphylococcus aureus did not show these changes.

In this paper, old as well as new technological findings to decrease premature infant mortality are reviewed. This paper discusses fetal development throughout pregnancy from conception to full-term status as well as fetal lung development specifically from conception until full-term status. Several ideas to rapidly develop and mature fetal lungs are discussed such as mothers ingesting artificial surfactant supplements, either independently or coupled with antenatal corticosteroids, as well as intra-amniotic instillation prior to 28 weeks gestational. Drawbacks regarding these two are mentioned as well such as the fetus’s lungs not being mature enough to use the artificial surfactant leading into the idea of researching ways to rapidly develop fetal lungs, either week-by-week or stage-by-stage. Lastly, if the baby is born pre-maturely and is severely underdeveloped, research is currently being done on an artificial womb that the baby can be placed in to simulate a uterus where the fetus can develop on a normal timeline as he or she would in the mother’s womb.

Cigarette smoking is a common and lethal worldwide habit, with considerable mortality stemming from its deleterious effects on heart function. While current theories posit altered blood lipids and fibrinogen metabolism as likely mediators, none have explored the role of the sphingolipid ceramide in exacerbating heart function with smoke exposure. Ceramide production is a consequence of cigarette smoke in the lung, and considering ceramide's harmful effects on mitochondrial function, we sought to elucidate the role of ceramide in mediating smoke-induced altered heart mitochondrial respiration. Lung cells were exposed to cigarette smoke extract and heart cells were exposed to the lung-cell conditioned medium. Adult male mice were exposed sidestream cigarette smoke for 8 weeks with dietary intervention and ceramide inhibition. Ceramides and heart cell or myocardial mitochondrial respiration were determined. Lung cell cultures revealed a robust response to cigarette smoke extract in both production and secretion of ceramides. Heart cells incubated with lung-cell conditioned medium revealed a pronounced inhibition of myocardial mitochondrial respiration, though this effect was mitigated with ceramide inhibition via myriocin. In vivo, heart ceramides increased roughly 600% in adult mice with long-term sidestream cigarette smoke exposure. This resulted in a significant ceramide-dependent reduction in left myocardial mitochondrial respiration, as heart mitochondria from the mice exposed to both smoke and myriocin injections respired normally. These results suggest ceramide to be an important mediator of altered myocardial mitochondrial function with cigarette smoke exposure. Thus, anti-ceramide therapies might be considered in the future to protect heart mitochondrial function with smoke exposure.

In vertebrate embryos, the ophthalmic trigeminal (opV) placode contributes sensory neurons to the trigeminal ganglion during development of the peripheral nervous system. FGFR4 is expressed transiently in the trigeminal placode as cells undergo an epithelial-mesenchymal transition and begin to migrate toward the condensing ganglion. Because of the restricted spatiotemporal expression of FGFR4 in the opV placode, it was hypothesized that FGFR4 is necessary for the process of epithelial-mesenchymal transition in the opV placode. Using electroporation, an FGFR4 inhibitory gene construct was introduced into 6—10 somite stage chick embryos. This secreted inhibitory form of the FGFR4 gene blocked endogenous FGFR4 signaling, which resulted in trigeminal placode cells remaining in the ectoderm that would have normally begun migration into the mesenchyme. These results show that FGFR4 is involved in trigeminal placode cell delamination.

Skeletal muscle contraction results in the phosphorylation and activation of the AMP-activated protein kinase (AMPK) by an upstream kinase, AMPKK. The LKB1-STRAD-MO25 complex is the major AMPKK in skeletal muscle; however, LKB1-STRAD-MO25 activity is not increased by muscle contraction. This relationship suggests that phosphorylation of AMPK by LKB1-STRAD-MO25 during skeletal muscle contraction may be regulated by allosteric mechanisms. In this study we tested an array of metabolites including glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose 1,6-bisphosphate (F1,6-P2), 3-phosphoglycerate (3PG), glucose-1-phosphate (G1P), glucose-1,6-bisphosphate (G1,6-P2), adenosine diphosphate (ADP), carnitine (Carn), acetyl-carnitine (Acarn), inosine monophosphate (IMP), inosine, and ammonia for allosteric regulation. We found that 3PG stimulated LKB1-STRAD-MO25 activity and allowed for increased AMPK phosphorylation. 3PG did not stimulate LKB1-STRAD-MO25 activity toward the peptide substrate LKB1tide. These results have identified 3PG as an AMPK-specific regulator of AMPK phosphorylation and activation by LKB1-STRAD-MO25.

The LKB1/STRAD/MO25 complex (LSMK) has been identified as the major upstream kinase for AMP-activated protein kinase (AMPK). PKA phosphorylates LKB1 at the Ser428 residue in humans and Ser431 residue in mice. We investigated PKA as an upstream kinase for LSMK. LKB1 that had been incubated with PKA prior to incubation with AMPK experienced up to a 51% increase in AMPK Kinase activity compared to LKB1 alone (p < 0.05). When blocked with a PKA Inhibitor, the kinase effect of PKA on LKB1 was eliminated. Rat epitrochlearis muscle tissue incubated with epinephrine experienced no increase in AMPK activity compared with controls indicating that epinephrine does not cause AMPK activity in this type of tissue. In conclusion, phosphorylation by PKA can increase the AMPKK activity of LKB1-STRAD-MO25 in vitro. Because LKB1 has been found to be constitutively active, it is postulated that phosphorylation by PKA may act to enhance LKB1-AMPK interaction and thus achieve its effect.

Exposure of human erythrocytes to elevated intracellular calcium causes alterations in cell shape and stimulates shedding of the cell membrane in the form of microvesicles. We hypothesized that both the shape transition and microvesiculation are influenced by microscopic membrane physical properties such as lipid order. To test this hypothesis, membrane properties were manipulated by varying the experimental temperature, membrane cholesterol content, and the internal ionic environment. Changes in membrane order were assessed using steady-state fluorescence spectroscopy with an environment-sensitive probe, laurdan. Our observations led us to the following conclusions: 1) the modest temperature dependence of membrane structure observed with laurdan is shifted to lower temperatures and becomes more cooperative upon removal of membrane cholesterol, 2) the calcium-induced shape change observed in erythrocytes requires a decrease in membrane order, 3) the influence of membrane order is not limited to shape transitions induced only by calcium, and 4) decreased order is also a permissive factor for microvesicle shedding. Our data suggest that while the mechansims that regulate the shape transition and the release of microvesicles are different, they both require a state of membrane disorder.

Activation of AMP-activated protein kinase (AMPK) results in the upregulation of several intracellular systems which help to prepare a cell for a high energy challenge. The magnitude of the AMPK response to a 10 min bout of exercise has been found to decrease in red quadriceps (RQ) following training, while putative AMPK roles seem to be maintained; specifically, the biogenesis of mitochondria and higher levels of hexokinase II and glucose transporter 4 (GLUT4). If the AMPK response to exercise is responsible in part for these adaptations, how can they be maintained if the AMPK response is attenuated? The purpose of this study was to determine whether phosphorylation of AMPK in RQ increases during 2-hr training bouts after rats have trained for 8 wks. Male Sprague-Dawley rats ran up to 30 m/min up a 15% grade, 2 hr/day for 8 wks. On the final bout of exercise, trained rats ran for 0 (TRC), 30 (TR1), or 120 min (TR2) up a 15% grade at 30 m/min. Red quadriceps (RQ), soleus, and white quadriceps (WQ) were immediately collected and frozen for analysis. Citrate synthase activity increased in RQ (79 ± 3 vs. 37 ± 4 µmol/g/min) and soleus (64 ± 4 vs. 35 ± 2 µmol/g/min) but not in WQ compared to non-trained controls. In trained rats, maximal increases in T-172 phosphorylation of AMPK occurred after 30 min of exercise (relative values = 1.29 ± 0.06 vs. 1.00 ± 0.06). AMPK phosphorylation did not change significantly in trained rats that ran for 2 hrs (1.31 ± 0.09) compared to rats that ran for 30 min. Similarly, maximal increases in AMPK activity in trained rats occurred after 30 min of exercise (pmoles/min/mg = 2.67 ± .05 vs. 1.09 ± .41) and AMPK activity did not change significantly in trained rats that ran for 2 hrs (2.79 ± .17) compared to rats that ran for 30 min. Previous studies demonstrated a 2−3 fold increase in AMPK activity in non-trained rats after 30 min of exercise at lower work rates. These results demonstrate that the AMPK response to exercise is attenuated even after two-hr bouts of exercise. This implies that the increase in mitochondrial oxidative enzymes, GLUT4, and hexokinase II may be maintained by signals other than the AMPK signaling system. The CREB signaling pathway is one such system. Western analysis of phospho-CREB (Ser133) showed a statistically significant increase in phospho-CREB content in trained rats relative to control. No change in phospho-CREB protein expression was observed between TRC, TR1, and TR2 rats. Significant increases of muscle phospho-CREB content in TRC relative to untrained rats suggest that CREB remains phosphorylated in trained rats even after 24 hrs of rest. Accordingly, chronically increased phospho-CREB in muscle of trained rats relative to controls may explain in part how increased levels of mitochondria are maintained in the face of reduced AMPK response. Alternatively, the attenuated AMPK response may still be above the threshold required for inducing adaptations to endurance training.

Equol is known to be a selective androgen modulator and has the ability to bind and inhibit 5-alpha dihydrotestosterone (5α-DHT). Equol is also a selective estrogen receptor modulator and is able to bind beta estrogen receptors with high affinity. As estrogen receptors are found in the hypothalamus, pituitary, and gonads, prenatally administered equol could affect the morphological and reproductive development of offspring. To test this hypothesis, during gestational days 14 to 20, forty-two pregnant Long-Evans rats were given one of six treatments: 1) no treatment, 2) injection with dimethyl sulfoxide (DMSO), 3) injection with 10 mg/kg equol, 4) injection with 21.0 mg/kg equol, 5) injection with 63.0 mg/kg equol, or 6) injection with 90.0 mg/kg flutamide. At birth the pups were weighed, anogenital distance measured, and sex was determined. Some of the animals were sacrificed and trunk blood collected from both the mothers and pups. Serum levels of phytoestrogens, estradiol, testosterone, and 5α-DHT levels were determined. Some pups were allowed to grow up to day 29 and were tested on the forced-swim test with the parameters of time mobile, time immobile, swim distance, and average speed measured. The flutamide treated pups had the lowest anogenital distance. The low equol dose animals had the largest anogenital distance. There were no significant differences in 5α-DHT serum levels in the male offspring among the treatments. However in non-injected control female offspring displayed significantly lower 5α-DHT levels than all the other groups. Mothers treated prenatally with equol displayed significantly higher circulating equol levels compared to controls values. Rats injected with 63.0 mg/kg of equol gained the least weight during pregnancy. Their offspring also had the lowest body weights at birth. Male and female offspring displayed similar behaviors in the Porsolt forced-swim test among the treatment groups. The low and high equol groups displayed the least depressive-like behaviors. The offspring from mothers treated with the medium and high equol doses both gained the most weight from birth to day 29. Treating pregnant rats with equol during the last week of gestation does not appear to have any affect on morphological genital development of the offspring.

The Liver Kinase B1 (LKB1)/AMP-Activated Protein Kinase (AMPK) signaling pathway is a major regulator of skeletal muscle metabolic processes. During exercise, LKB1-mediated phosphorylation of AMPK leads to its activation, promoting mitochondrial biogenesis and glucose transport, among other effects. The roles of LKB1 and AMPK have not been fully characterized in the diaphragm. Two methods of AMPK activation were used to characterize LKB1/AMPK signaling in diaphragms from muscle-specific LKB1 knockout (KO) and littermate control (C) mice: (1) acute injection of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and (2) 5-min direct electrical stimulation (ES) of the diaphragm. Diaphragms were excised 60 minutes post-AICAR injection and immediately after ES. AMPK phosphorylation increased with AICAR and ES in C but not KO mice. Acetyl CoA carboxylase (ACC) phosphorylation increased with AICAR in C but not KO mice, but increased in both genotypes with ES. While the majority of mitochondrial enzyme levels were lower in KO diaphragms, uncoupling protein 3 (UCP-3) levels were not different between genotypes. A IIx to IIb fiber type switch was observed in KO diaphragms. While in vitro peak force generation was similar between genotypes, KO diaphragms fatigued more quickly and had an impaired ability to recover. In conclusion, LKB1 regulates AMPK phosphorylation, mitochondrial enzyme expression, fiber type distribution, as well as recovery of the diaphragm from fatigue.