Identification and characterization of novel antagonists of the CCR3 receptor

Eotaxin, an inducer of eosinophil migration and activation, exerts its activity by binding to CCR3, the C-C chemokine receptor 3. An inhibitor of the eotaxin-CCR3 binding interaction may have potential as an anti-inflammatory drug for treatment of asthma, parasitic infections, and allergic disorders. A radioligand binding assay was developed using HEK cells transfected with CCR3, with (125)I eotaxin as the ligand. Whole cells grown on polylysine-coated plates were used as the receptor source for the screen. Screening of more than 200,000 compounds with this assay yielded a number of screening hits, and of these, 2 active novel antagonists were identified. These compounds showed inhibitory effects on eosinophil chemotaxis in both in vitro and in vivo assays.     

Thermoregulatory and metabolic changes during fever in young and old rats

We injected old and young rats with lipopolysaccharide (LPS; 50 microg/kg ip) at two ambient temperatures (Ta; 21 and 31 degrees C). Young rats mounted equivalent fevers at both Tas [peak body temperatures (Tb) of 38.3 and 38.7 degrees C, respectively]. The Tb of old rats was not different from baseline (37.3 degrees C) after LPS at Ta 21 degrees C, whereas, at 31 degrees C, their Tb rose to a mean peak of 38.4 degrees C. We also measured the associated thermoregulatory responses by use of calorimetry. At 21 degrees C, young rats developed a fever by increasing both O2 consumption and heat conservation. Old rats did not become febrile, and O2 consumption fell by 15%. Heat loss was the same in old and young rats. At 31 degrees C, young and old rats developed similar fevers with similar increases in heat production and conservation. Our results suggest that the lack of LPS fever in old rats at 21 degrees C is due mainly to the lowered metabolic rate.     

Importance of extra- and intracellular domains of TLR1 and TLR2 in NFkappa B signaling

Recognition of ligands by toll-like receptor (TLR) 2 requires interactions with other TLRs. TLRs form a combinatorial repertoire to discriminate between the diverse microbial ligands. Diversity results from extracellular and intracellular interactions of different TLRs. This paper demonstrates that TLR1 and TLR2 are required for ara-lipoarabinomannan- and tripalmitoyl cysteinyl lipopeptide-stimulated cytokine secretion from mononuclear cells. Confocal microscopy revealed that TLR1 and TLR2 cotranslationally form heterodimeric complexes on the cell surface and in the cytosol. Simultaneous cross-linking of both receptors resulted in ligand-independent signal transduction. Using chimeric TLRs, we found that expression of the extracellular domains along with simultaneous expression of the intracellular domains of both TLRs was necessary to achieve functional signaling. The domains from each receptor did not need to be contained within a single contiguous protein. Chimeric TLR analysis further defined the toll/IL-1R domains as the area of crucial intracellular TLR1-TLR2 interaction.     

Synchronized prostate cancer cells for studying androgen regulated events in cell cycle progression from G1 into S phase

Androgen-ablation is a most commonly prescribed treatment for metastatic prostate cancer but it is not curative. Development of new strategies for treatment of prostate cancer is limited partly by a lack of full understanding of the mechanism by which androgen regulates prostate cancer cell proliferation. This is due, mainly, to the limitations in currently available experimental models to distinguish androgen/androgen receptor (AR)-induced events specific to proliferation from those that are required for cell viability. We have, therefore, developed an experimental model system in which both androgen-sensitive (LNCaP) and androgen-independent (DU145) prostate cancer cells can be reversibly blocked in G(0)/G(1) phase of cell cycle by isoleucine deprivation without affecting their viability. Pulse-labeling studies with (3)H-thymidine indicated that isoleucine-deprivation caused LNCaP and DU145 cells to arrest at a point in G(1) phase which is 12-15 and 6-8 h, respectively, before the start of S phase and that their progression into S phase was dependent on serum factors. Furthermore, LNCaP, but not DU145, cells required AR activity for progression from G(1) into S phase. Western blot analysis of the cell extracts prepared at regular intervals following release from isoleucine-block revealed remarkable differences in the expression of cyclin E, p21(Cip1), p27(Kip1), and Rb at the protein level between LNCaP and DU145 cells during progression from G(1) into S phase. However, in both cell types Cdk-2 activity associated with cyclin E and cyclin A showed an increase only when the cells transited from G(1) into S phase. These observations were further corroborated by studies using exponentially growing cells that were enriched in specific phases of the cell cycle by centrifugal elutriation. These studies demonstrate usefulness of the isoleucine-deprivation method for synchronization of androgen-sensitive and androgen-independent prostate cancer cells, and for examining the role of androgen and AR in progression of androgen-sensitive prostate cancer cells from G(1) into S phase.     

Lack of effector cell function and altered tetramer binding of tumor-infiltrating lymphocytes

Tumor-specific CD8 T cell responses to MCA102 fibrosarcoma cells expressing the cytotoxic T cell epitope gp33 from lymphocytic choriomeningitis virus were studied. MCA102(gp33) tumors grew progressively in C57BL/6 mice, despite induction of peripheral gp33-tetramer(+) T cells that were capable of mediating antiviral protection, specific cell rejection, and concomitant tumor immunity. MCA102(gp33) tumors were infiltrated with a high number ( approximately 20%) of CD11b(+)CD11c(-) macrophage-phenotype cells that were able to cross-present the gp33 epitope to T cells. Tumor-infiltrating CD8 T cells exhibited a highly activated phenotype but lacked effector cell function. Strikingly, a significant portion of tumor-infiltrating lymphocytes expressed TCRs specific for gp33 but bound MHC tetramers only after cell purification and a 24-h resting period in vitro. The phenomenon of "tetramer-negative T cells" was not restricted to tumor-infiltrating lymphocytes from MCA102(gp33) tumors, but was also observed when Ag-specific T cells derived from an environment with high Ag load were analyzed ex vivo. Thus, using a novel tumor model, allowing us to trace tumor-specific T cells at the single cell level in vivo, we demonstrate that the tumor microenvironment is able to alter the functional activity of T cells infiltrating the tumor mass.     

Short-term changes in phosphorus storage in an oligotrophic Everglades wetland ecosystem receiving experimental nutrient enrichment

Natural, unenriched Evergladeswetlands are known to be limited by phosphorus(P) and responsive to P enrichment. However,whole-ecosystem evaluations of experimental Padditions are rare in Everglades or otherwetlands. We tested the response of theEverglades wetland ecosystem to continuous,low-level additions of P (0, 5, 15, and30 g L^–1 above ambient) in replicate,100 m flow-through flumes located in unenrichedEverglades National Park. After the first sixmonths of dosing, the concentration andstanding stock of phosphorus increased in thesurface water, periphyton, and flocculentdetrital layer, but not in the soil or macrophytes. Of the ecosystem components measured, total P concentration increased the most in the floating periphyton mat (30 g L^–1: mean = 1916 g P g^–1, control: mean =149 g P g^–1), while the flocculentdetrital layer stored most of the accumulated P(30 g L^–1: mean = 1.732 g P m^–2,control: mean = 0.769 g P m^–2). Significant short-term responsesof P concentration and standing stock wereobserved primarily in the high dose (30 gL^–1 above ambient) treatment. Inaddition, the biomass and estimated P standingstock of aquatic consumers increased in the 30and 5 g L^–1 treatments. Alterationsin P concentration and standing stock occurredonly at the upstream ends of the flumes nearestto the point source of added nutrient. Thetotal amount of P stored by the ecosystemwithin the flume increased with P dosing,although the ecosystem in the flumes retainedonly a small proportion of the P added over thefirst six months. These results indicate thatoligotrophic Everglades wetlands respondrapidly to short-term, low-level P enrichment,and the initial response is most noticeable inthe periphyton and flocculent detrital layer.     

Losartan decreases glomerular filtration rate in isolated perfused porcine slaughterhouse kidneys

We investigated whether Losartan, an angiotensin II (Ang II) AT1 receptor antagonist, decreases renal vascular resistance (RVR) and increases glomerular filtration rate (GFR) in isolated perfused porcine slaughterhouse kidneys (11 control experiments and 11 Losartan experiments with 7.5mg Losartan in the preservation solution and 100(g/minute Losartan infused during perfusion). With perfusion, plasma renin activity (PRA) increased markedly from 3 +/- 1 to 90 +/- 17 ng Ang I/ml/h (control), and from 4 +/- 1 to 70 +/- 8 ng Ang I/ml/h (Losartan), plasma Ang II increased from 86 +/- 63 to 482 +/- 111 pg/ml (control), and from 73 +/- 42 to 410 +/- 91 pg/ml (Losartan). The GFR was decreased in Losartan experiments as compared with control experiments (5 +/- 1 versus 10 +/- 2 ml/min/100g kidney wt; p < 0.05). The RVR was the same in both groups (0.2 +/- 0.01 mm Hg/100g kidney wt/min/ml). Tubular sodium reabsorption was decreased in Losartan experiments as compared with control experiments (0.7 +/- 0.1 versus 1.4 +/- 0.3 mmol/min/100g kidney wt). Overall, Losartan accentuated pathophysiological signs of acute renal failure. Although other drugs have to be investigated, these results suggest that porcine slaughterhouse kidneys could be useful as a tool for research in areas such as transplantation and intensive-care medicine.     

Stress-induced hyperthermia depends on both time of day and light condition

Rats placed in an environment other than their home cage increase their body temperature (Tb) by more than 1 degree C. This stress-induced hyperthermia is considered to be a fever, in the sense that the Tb rise seems to reflect an upward shift in the level of regulated Tb (set point). The circadian rhythm of Tb also reflects changes in set point. One might therefore expect to see differences in response to such stress during various phases of the light-dark (LD) cycle as Tb fluctuates between L and D. To test this, 3- to 6-month-old male Long-Evans rats were taken from their home cages (12:12 LD) and placed individually in a Plexiglas container for 30 min. Tb and activity were measured via telemetry. In the first experiment, rats were placed in the container during day (from 1 to 3 h after lights on) and night (from 1 to 3 h after lights off), with light on or off during the test. There was a significant Tb rise in response to placement in the container at all times except when the rats were tested during the night with light on in the container; in that condition there was no Tb rise. In the second experiment, the authors determined that 30 min of light in the home cage before the test did not affect Tb: If the light was on in the test situation, hyperthermia was inhibited, and if it was off, hyperthermia was as high as control levels. In the third experiment, to determine whether this effect was time dependent, the test was performed at 4-h intervals, with light on or off during the test. The strongest inhibiting effect of light was in early night. In the fourth experiment, the authors turned the lights on during early night while the rats were in their home cages. This reduced their Tb significantly by less than 0.3 degrees C. The authors conclude that both clock time and light condition during testing are factors affecting the Tb rise in response to stress.     

The effects of an exercise physiology program on physical fitness variables, body satisfaction, and physiology knowledge

This study was performed to determine the effects of an exercise physiology program on physical fitness, body satisfaction, and knowledge. A total of 161 students (mean age = 16.5 +/- 0.89 years) of the Coral Gables Senior High School served as the experimental group volunteers, whereas 33 students enrolled in a standard biology course served as the control group (mean age = 15.61 +/- 0.84 years). The experimental group received exercise physiology theory coupled with active aerobic and resistance exercise. Age (p = 0.0001) and lean body mass (p = 0.023) were the only physical characteristics significantly greater in the experimental group at pretesting. An analysis of covariance controlling for pretest values showed better results in the experimental compared with the control group for sit and reach (p = 0.008), step test, recovery heart rate (p = 0.0002), overhead press (p = 0.002), bench press (p = 0.017), leg press (p = 0.012), sit-ups (p = 0.001), body satisfaction (p = 0.0009), and physiology knowledge (p = 0.0001) at posttesting. Findings indicated that a biology curriculum integrated with exercise physiology theory and exercise activities may result in significant improvements in physical fitness, body size satisfaction, and physiology knowledge in high school adolescents.