CD14 C-159T and early infection with Pseudomonas aeruginosa in children with cystic fibrosis

Early acquisition of Pseudomonas aeruginosa is associated with a poorer prognosis in patients with cystic fibrosis. We investigated whether polymorphisms in CD14, the lipopolysaccharide receptor, increase the risk of early infection. Forty-five children with cystic fibrosis were investigated with annual bronchoalveolar lavage (BAL) and plasma sCD14 levels. Plasma sCD14 levels were significantly lower in children from whom P.aeruginosa was subsequently isolated (492.75 μg/ml vs. 1339.43 μg/ml, p = 0.018). Those with the CD14 -159CC genotype had a significantly increased risk of early infection with P.aeruginosa suggesting that CD14 C-159T plays a role in determining the risk of early infection with P.aeruginosa.     

Fluoride Exposure Aggravates the Testicular Damage and Sperm Quality in Diabetic Mice: Protective Role of Ginseng and Banaba

The current study was conducted to investigate the effects of dietary zinc oxide (ZnO) on the antioxidant capacity, small intestine development, and jejunal gene expression in weaned piglets. Ninety-six 21-day-old piglets were randomly assigned to three dietary treatments. Each treatment had eight replicates with four piglets per replicate. The piglets were fed either control diet (control) or control diet supplemented with in-feed antibiotics (300 mg/kg chlortetracycline and 60 mg/kg colistin sulfate) or pharmacological doses of ZnO (3000 mg/kg). The experiment lasted 4 weeks. Blood samples were collected at days 14 and 28, while intestinal samples were harvested at day 28 of the experiment. Dietary high doses of ZnO supplementation significantly increased the body weight (BW) at day 14 and average daily gain (ADG) of days 1 to 14 in weaned piglets, when compared to control group (P < 0.05). The incidence of diarrhea of piglets fed ZnO-supplemented diets, at either days 1 to 14, days 14 to 28, or the overall experimental period, was significantly decreased in comparison with those in other groups (P < 0.05). Supplementation with ZnO increased the villus height of the duodenum and ileum in weaned piglets and decreased the crypt depth of the duodenum, when compared to the other groups (P < 0.05). Dietary ZnO supplementation decreased the malondialdehyde (MDA) concentration at either day 14 or day 28, but increased total superoxide dismutase (T-SOD) at day 14, when compared to that in the control (P < 0.05). ZnO supplementation upregulated the messenger RNA (mRNA) expression of zonula occludens-1 (ZO-1) and occludin in the jejunum mucosa of weaned piglets, compared to those in the control (P < 0.05). The pro-inflammatory cytokine interleukin-lβ (IL-1β) mRNA expression in the jejunum mucosa was downregulated in the ZnO-supplemented group, compared with the control (P < 0.05). Both in-feed antibiotics and ZnO supplementation decreased the mRNA expression of interferon-γ (IFN-γ), but increased the mRNA expression of transforming growth factor-β (TGF-β), in the jejunum mucosa of piglets, when compared to those in the control (P < 0.05). In summary, supplemental ZnO was effective on the prevention of post-weaning diarrhea (PWD) in weaned piglets and showed comparative growth-promoting effect on in-feed antibiotics, probably by the mechanism of improvement of the antioxidant capacity, restoration of intestinal barrier function and development, and modulation of immune functions.     

Molecular phylogenetics reveal multiple tertiary vicariance origins of the African rain forest trees

Background  

Tropical rain forests are the most diverse terrestrial ecosystems on the planet. How this diversity evolved remains largely unexplained. In Africa, rain forests are situated in two geographically isolated regions: the West-Central Guineo-Congolian region and the coastal and montane regions of East Africa. These regions have strong floristic affinities with each other, suggesting a former connection via an Eocene pan-African rain forest. High levels of endemism observed in both regions have been hypothesized to be the result of either 1) a single break-up followed by a long isolation or 2) multiple fragmentation and reconnection since the Oligocene. To test these hypotheses the evolutionary history of endemic taxa within a rain forest restricted African lineage of the plant family Annonaceae was studied. Molecular phylogenies and divergence dates were estimated using a Bayesian relaxed uncorrelated molecular clock assumption accounting for both calibration and phylogenetic uncertainties.     

Overexpression of tumor necrosis factor-alpha produces an increase in lung volumes and pulmonary hypertension

Tumor necrosis factor (TNF)-alpha is a key proinflammatory cytokine that is thought to be important in the development of pulmonary fibrosis, whereas its role in pulmonary emphysema has not been as thoroughly documented. In the present study, TNF-alpha was overexpressed in alveolar type II cells under the control of the human surfactant protein C promoter. In this report, we further characterized the pulmonary abnormalities and provided a physiological assessment of these mice. Histopathology of the lungs revealed chronic inflammation, severe alveolar air space enlargement and septal destruction, and bronchiolitis. However, pulmonary fibrosis was very limited and only seen in the subpleural, peribronchiolar, and perivascular regions. Physiological assessment showed an increase in lung volumes and a decrease in elastic recoil characteristic of emphysema; there was no evidence of restrictive lung disease characteristic of pulmonary fibrosis. In addition, the mice raised in ambient conditions in Denver developed pulmonary hypertension. Gelatinase activity was increased in the lavage fluid from these lungs. These results suggest that in these mice TNF-alpha contributed to the development of pulmonary emphysema through chronic lung inflammation and activation of the elastolytic enzymes but by itself was unable to produce significant pulmonary fibrosis.     

Dynamic interaction of cAMP with the Rap guanine-nucleotide exchange factor Epac1

Epac1 is a Rap-specific guanine-nucleotide exchange factor (GEF) which is activated by the binding of cAMP to a cyclic nucleotide monophosphate (cNMP)-binding domain. We investigated the equilibrium and dynamics of the interaction of cAMP and Epac1 using a newly designed fluorescence analogue of cAMP, 8-MABA-cAMP. We observed that the interaction of cAMP, measured by competition with 8-MABA-cAMP, with an isolated cNMP binding domain of Epac1 has an overall equilibrium constant (Kd) of 4 microM and that the kinetics of the interaction are highly dynamic. The binding properties of cAMP are apparently not affected when the catalytic domain is present, despite the fact that binding of cAMP results in activation of Epac1. This indicates that for the activation process, no appreciable binding energy is required. However, when bound to Rap1b, the apparent Kd of Epac to cAMP was about fivefold lower, suggesting that substrate interaction stabilizes cAMP binding. Since the fluorescent analogues used here were either less able or unable to induce activation of Epac1, we concluded that the binding of nucleotide to Epac and the activation of GEF activity are uncoupled processes and that thus appropriate cAMP analogues can be used as inhibitors of the Epac1-mediated signal transduction pathway of Rap.     

Development and maintenance of a B220- memory B cell compartment

We have recently demonstrated that a novel somatically mutated B220(-) memory B cell subset rapidly dominates the secondary immune response to (4-hydroxy-3-nitrophenyl) acetyl (NP). Upon adoptive transfer with Ag, B220(+)NP(+) memory B cells produce large numbers of B220(-)NP(+) B cells that can rapidly differentiate into plasma cells. Therefore, it is not clear whether the novel B220(-) memory compartment is a consequence of secondary Ag challenge or whether it develops as a stable memory subset after initial Ag challenge. In this study, we demonstrate the gradual emergence of B220(-)NP(+) B cells in the spleen to maximal numbers 3 wk after initial Ag exposure. Like their B220(+) counterparts, the B220(-) B cells initially appear unmutated at days 5-7; however, the majority rapidly accumulate affinity increasing mutations by days 9-14 of the primary immune response. More extensive cell surface phenotype (GL7(-)BLA-1(-)CD24(-)CD43(+)) argues strongly against germinal center localization and direct analysis in situ places a cohort of B220(-)CD11b(+)NP(+) B cells in the red pulp of the spleen and not in the MZs. These data provide direct evidence for the development of B220(-) memory B cells as a unique cellular consequence of primary Ag exposure. The cellular dynamics and molecular attributes of these unique memory B cells suggest they are distinct cellular products of the germinal center reaction in the primary response and are maintained long-term in the spleen and bone marrow.     

Genomic organization and chromosomal localization of a new repeat MS7, specific for heterochromatin of sex chromosomes in Microtus species voles of the arvalis group

The tandemly arranged MS4 repeat with monomeric units of 4.1 kb is species-specifically distributed in heterochromatin of sex chromosomes of four common vole species of genus Microtus, group arvalis [1, 2]. In this work, we studied the genomic organization of the MS4 homolog in euchromatin of the X chromosome of M. arvalis. It has been shown by analyzing the phage genomic clones that one MS4 copy makes a part of a monomeric unit exceeding 8.5 kb that also includes a new MS7 repeat and, possibly, LINE fragments. MS7 is located together with MS4 in heterochromatin of common vole sex chromosomes, but in a substantially lesser amount. Probably, as a result of an evolutionary transition of an original repeat from euchromatin of the X chromosome to heterochromatin of the Y chromosome, MS4 underwent multiple amplification, and MS7 spread throughout heterochromatin, being surrounded by the MS4 tandem arrays.     

Discriminatory residues in Ras and Rap for guanine nucleotide exchange factor recognition

The inability of the S17N mutant of Rap1A to sequester the catalytic domain of the Rap guanine nucleotide exchange factor C3G (van den Berghe, N., Cool, R. H., Horn, G., and Wittinghofer, A. (1997) Oncogene 15, 845-850) prompted us to study possible fundamental differences in the way Rap1 interacts with C3G compared with the interaction of Ras with the catalytic domain of the mouse Ras guanine nucleotide exchange factor Cdc25(Mm). A variety of mutants in both Ras and Rap1A were designed, and both the C3G and Cdc25(Mm) catalyzed release of guanine nucleotide from these mutants was studied. In addition, we could identify regions in Rap2A that are responsible for the lack of recognition by C3G and induce high C3G activity by replacement of these residues with the corresponding Rap1A residues. The different Ras and Rap mutants showed that many residues were equally important for both C3G and Cdc25(Mm), suggesting that they interact similarly with their substrates. However, several residues were also identified to be important for the exchange reaction with only C3G (Leu70) or only Cdc25(Mm) (Gln61 and Tyr40). These results are discussed in the light of the structure of the Ras-Sos complex and suggest that some important differences in the interaction of Rap1 with C3G and Ras with Cdc25(Mm) indeed exist and that marker residues have been identified for the different structural requirements.     

Extraction of oxytocin and arginine-vasopressin from serum and plasma for radioimmunoassay and surface-enhanced laser desorption-ionization time-of-flight mass spectrometry

Oxytocin and arginine-vasopressin (AVP) are secreted into the blood in low concentrations. To analyze these peptides, we investigated two common extraction procedures, acetone-ether precipitation and C(18)-SepPak columns. Recovery from both procedures approached 70-80% of the spiked amount, though the SepPak columns were more efficient. C(18)-SepPak columns were used to sequentially separate oxytocin from AVP by eluting oxytocin first with 98% acetone followed by elution of AVP with 80% acetonitrile. Surface-enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF MS) was used to analyze oxytocin and AVP extracted with C(18)-SepPak columns from an autistic patient's plasma sample. We conclude that C(18)-SepPaks provide more consistent and efficient peptide extraction from serum or plasma that augments both quantitative and qualitative analysis by radioimmunoassay and SELDI-TOF MS.     

Fibroblast growth factor receptor 4 (FGFR4) expression in newborn murine calvaria and primary osteoblast cultures

Fibroblast growth factor receptor (FGFR) signalling is important in the initiation and regulation of osteogenesis. Although mutations in FGFR1, 2, and 3 genes are known to cause skeletal deformities, the expression of FGFR4 in bony tissue remains unclear. We have investigated the expression pattern of FGFR4 in the neonatal mouse calvaria and compared it to the expression pattern in cultures of primary osteoblasts. Immunohistochemistry demonstrated that FGFR4 was highly expressed in rudimentary membranous bone and strictly localised to the cellular components (osteoblasts) between the periosteal and endosteal layers. Cells in close proximity to the newly formed osteoid (preosteoblasts) also expressed FGFR4 on both the endosteal and periosteal surfaces. Immunocytochemical analysis of primary osteoblast cultures taken from the same cranial region also revealed high levels of FGFR4 expression, suggesting a similar pattern of cellular expression in vivo and in vitro. RT-PCR and Western blotting for FGFR4 confirmed its presence in primary osteoblast cultures. These results suggest that FGFR4 may be an important regulator of osteogenesis with involvement in preosteoblast proliferation and differentiation as well as osteoblast functioning during intramembranous ossification. The consistent expression of FGFR4 in vivo and in vitro supports the use of primary osteoblast cultures for elucidating the role of FGFR4 during osteogenesis.