Gestational regulation of granulocyte-colony stimulating factor receptor expression in the human placenta

A number of cytokines and their receptors are abundantly expressed at the materno-fetal interface and are thought to have a function in the regulation of placentation. Granulocyte-colony stimulating factor (G-CSF) is expressed by stromal cells in both placental tissue and maternal decidua throughout placentation. In this study, we examined the expression of placental G-CSF receptor (G-CSFR) mRNA and protein throughout gestation by ribonuclease protection assays, Western blotting, and immunohistochemistry. The major placental form of G-CSFR mRNA, corresponding to a membrane-bound form of the protein, was present in first-trimester placental tissues; levels decreased in second- and were highest in third-trimester placental tissues. Two placental G-CSFR molecules, 120 kDa and 150 kDa, were detected in first- and third-, but not second-, trimester tissues. The level of the 150-kDa G-CSFR was greater in the third- than in first-trimester samples. These differences were irrespective of whether or not the patients had received prostaglandin E1 analogues, prostaglandin E1 analogues and oxytocin, oxytocin alone, or mifepristone before labor. We demonstrated by immunohistochemistry that interstitial cytotrophoblast in first- and second-trimester decidual tissue and cytotrophoblast in term fetal membranes express G-CSFR. These data demonstrate that the expression of specific forms of placental G-CSFR is strictly cell type- and developmental stage-specific, and they suggest that G-CSFR may be important in decidual invasion of cytotrophoblast and in trophoblast function during placentation.     

The cellulose-binding domains from Cellulomonas fimi beta-1, 4-glucanase CenC bind nitroxide spin-labeled cellooligosaccharides in multiple orientations

The N-terminal cellulose-binding domains CBDN1 and CBDN2 from Cellulomonas fimi cellulase CenC each adopt a jelly-roll beta-sandwich structure with a cleft into which amorphous cellulose and soluble cellooligosaccharides bind. To determine the orientation of the sugar chain within these binding clefts, the association of TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl) spin-labeled derivatives of cellotriose and cellotetraose with isolated CBDN1 and CBDN2 was studied using heteronuclear 1H-15N NMR spectroscopy. Quantitative binding measurements indicate that the TEMPO moiety does not significantly perturb the affinity of the cellooligo-saccharide derivatives for the CBDs. The paramagnetic enhancements of the amide 1HN longitudinal (DeltaR1) and transverse (DeltaR2) relaxation rates were measured by comparing the effects of TEMPO-cellotetraose in its nitroxide (oxidized) and hydroxylamine (reduced) forms on the two CBDs. The bound spin-label affects most significantly the relaxation rates of amides located at both ends of the sugar-binding cleft of each CBD. Similar results are observed with TEMPO-cellotriose bound to CBDN1. This demonstrates that the TEMPO-labeled cellooligosaccharides, and by inference strands of amorphous cellulose, can associate with CBDN1 and CBDN2 in either orientation across their beta-sheet binding clefts. The ratio of the association constants for binding in each of these two orientations is estimated to be within a factor of five to tenfold. This finding is consistent with the approximate symmetry of the hydrogen-bonding groups on both the cellooligosaccharides and the residues forming the binding clefts of the CenC CBDs.     

The genera Ceratomyxa Thélohan, 1892, Leptotheca Thélohan, 1895 and Sphaeromyxa Thélohan, 1892 (Myxosporea: Bivalvulida) in gadid fish of the Northeast Atlantic

The gall-bladders of four species of gadid fish from the North Sea and Norwegian waters were examined for myxosporeans. The host species were cod Gadus morhua L. (350 examined), haddock Melanogrammus aeglefinus (L.) (592 examined), saithe Pollachius virens (L.) (205 examined) and whiting Merlangius merlangus (L.) (368 examined). Four species of myxosporeans are redescribed from these fish. Ceratomyxa arcuata Thélohan, 1892 was the most common species and was found in whiting (42.8%) and cod (0.6%). Leptotheca informis Auerbach, 1910 was found only in whiting (6.5%). L. longipes Auerbach, 1910 was found only in haddock (6.2%). Sphaeromyxa hellandi Auerbach, 1909 was found in haddock (9.1%) and whiting (0.3%). None of the saithe examined, and no cod or haddock from Norwegian waters, was infected with these myxosporeans. All four species appear to have distributions limited to the Northeast Atlantic, with S. hellandi having a more northern distribution than the other three. The validity of reports of C. arcuata, L. informis and L. longipes from outside this area is discussed.     

Pseudanthocotyloides heterocotyle (van Beneden, 1871) Euzet & Prost, 1969 (Monogenea: Polyopisthocotylea: Mazocraeidae), a parasite of herring Clupea harengus L. and sprat Sprattus sprattus L. (Teleostei: Clupeidae)

A total of 690 herring Clupea harengus L. and 88 sprat Sprattus sprattus L. caught off the west coast of Sweden, in the North Sea and off the west and south coasts of the United Kingdom, were examined for gill parasites. The monogenean Pseudanthocotyloides heterocotyle (van Beneden, 1871) Euzet & Prost, 1969 was found in 38 (5.5%) herring and one (1.1%) sprat. The parasite was significantly (P>0.05) more common off the west coast of Sweden than elsewhere and most specimens (62.5%) were found on the pseudobranchs. Only the smaller herring were infected. P. heterocotyle is redescribed and its taxonomy discussed, together with the possibility of host and parasite misidentification in previous reports.     

The MAP kinase ERK2 inhibits the cyclic AMP-specific phosphodiesterase HSPDE4D3 by phosphorylating it at Ser579

The extracellular receptor stimulated kinase ERK2 (p42(MAPK))-phosphorylated human cAMP-specific phosphodiesterase PDE4D3 at Ser579 and profoundly reduced ( approximately 75%) its activity. These effects could be reversed by the action of protein phosphatase PP1. The inhibitory state of PDE4D3, engendered by ERK2 phosphorylation, was mimicked by the Ser579-->Asp mutant form of PDE4D3. In COS1 cells transfected to express PDE4D3, challenge with epidermal growth factor (EGF) caused the phosphorylation and inhibition of PDE4D3. This effect was blocked by the MEK inhibitor PD98059 and was not apparent using the Ser579-->Ala mutant form of PDE4D3. Challenge of HEK293 and F442A cells with EGF led to the PD98059-ablatable inhibition of endogenous PDE4D3 and PDE4D5 activities. EGF challenge of COS1 cells transfected to express PDE4D3 increased cAMP levels through a process ablated by PD98059. The activity of the Ser579-->Asp mutant form of PDE4D3 was increased by PKA phosphorylation. The transient form of the EGF-induced inhibition of PDE4D3 is thus suggested to be due to feedback regulation by PKA causing the ablation of the ERK2-induced inhibition of PDE4D3. We identify a novel means of cross-talk between the cAMP and ERK signalling pathways whereby cell stimuli that lead to ERK2 activation may modulate cAMP signalling.     

Long-term physical impairment and functional outcomes after complex facial fractures

To develop an understanding of the expected functional outcomes after facial trauma, a retrospective cohort study of patients with complex facial fractures was conducted. A cohort of adults aged 18 to 55 years who were admitted to the R. Adams Cowley Shock Trauma Center between July of 1986 and July of 1994 for treatment of a Le Fort midface fracture (resulting from blunt force) was retrospectively identified.Outcomes of interest included measures of general health status and psychosocial well being in addition to self-reported somatic symptoms. General health status was ascertained using the Medical Outcomes Study 36-Item Short Form Health Survey (SF-36). The Body Satisfaction Scale was used to define patient concerns about altered body image and shape. To determine whether complex maxillofacial trauma and facial fractures contributed to altered social interactions, the Social Avoidance and Distress scale was used. In addition, information about a patient, his or her injury, and its treatment were ascertained from the medical records.Using the methods described above, 265 patients with Le Fort fractures were identified. These individuals were matched to a similar group of 242 general injury patients. A total of 190 of the Le Fort patients (72 percent of those eligible for the study) and 144 (60 percent) general injury patients were successfully located, and long-term interview data were acquired.Le Fort fracture patients as a group had similar health status outcomes when compared with the group of general injury patients. However, when outcomes were examined by the complexity of the Le Fort fracture, the authors found that study subjects with severe, comminuted Le Fort injuries (group D) had significantly lower SF-36 scores (worse outcomes) for the two dimensions related to role limitations: role limitations due to physical problems and role limitations due to emotional problems (p < 0.05). SF-36 scores for all other dimensions except physical function were also lower for comminuted versus less complex Le Fort fractures, although differences were not statistically significant.Specifically, there was a direct relationship between severity of facial injury and patients reporting work disability. Of group C and D Le Fort patients (severely comminuted fractures) only 55 and 58 percent, respectively, had returned to work at the time of follow-up interview. These figures are significantly lower than the back-to-work percentage of patients with less severe facial injury (70 percent).When study participants were asked if they were experiencing specific somatic symptoms at the time of the interview that they had not experienced before the injury, a significantly larger percent of the Le Fort fracture patients (compared with the general injury patients) responded in the affirmative. Differences between the Le Fort fracture and general injury groups were statistically significant (p < 0.05) for all 11 symptoms.The percentage of patients reporting complaints increased with increasing complexity of facial fracture in the areas of visual problems, alterations in smell, difficulty with mastication, difficulty with breathing, and epiphora, and these differences reached statistical significance.Patients sustaining comminuted Le Fort facial fractures report poorer health outcomes than patients with less severe facial injury and substantially worse outcomes than population norms. It is also this severely injured population that reports the greatest percentage of injury-related disability, preventing employment at long-term follow-up. The long-term goal of centralized tertiary trauma treatment centers must be to return the patient to a productive, active lifestyle.     

Residues involved in the mechanism of the bifunctional methylenetetrahydrofolate dehydrogenase-cyclohydrolase: the roles of glutamine 100 and aspartate 125

The human bifunctional dehydrogenase-cyclohydrolase domain catalyzes the interconversion of 5,10-methylene-H(4)folate and 10-formyl-H(4)folate. Although previous structure and mutagenesis studies indicated the importance of lysine 56 in cyclohydrolase catalysis, the role of several surrounding residues had not been explored. In addition to further defining the role of lysine 56, the work presented in this study explores the functions of glutamine 100 and aspartate 125 through the use of site-directed mutagenesis and chemical modification. Mutants at position 100 are inactive with respect to cyclohydrolase activity while preserving significant dehydrogenase levels. We succeeded in producing a K56Q/Q100K double mutant, which has no cyclohydrolase yet retains more than two-thirds of wild type dehydrogenase activity. Neither activity is detectable in aspartate 125 mutants with the exception of D125E. The results indicate that the function of glutamine 100 is to activate lysine 56 for cyclohydrolase catalysis and that aspartate 125 is involved in the binding of the H(4)folate substrates. In highlighting the importance of these residues, catalytic mechanisms are proposed for both activities as well as an explanation for the differences in channeling efficiency in the forward and reverse directions.     

TAPAS-1, a novel microdomain within the unique N-terminal region of the PDE4A1 cAMP-specific phosphodiesterase that allows rapid, Ca2+-triggered membrane association with selectivity for interaction with phosphatidic acid

Here we identify an 11-residue helical module in the unique N-terminal region of the cyclic AMP-specific phosphodiesterase PDE4A1 that determines association with phospholipid bilayers and shows a profound selectivity for interaction with phosphatidic acid (PA). This module contains a core bilayer insertion unit that is formed by two tryptophan residues, Trp(19) and Trp(20), whose orientation is optimized for bilayer insertion by the Leu(16):Val(17) pairing. Ca(2+), at submicromolar levels, interacts with Asp(21) in this module and serves to gate bilayer insertion, which is completed within 10 ms. Selectivity for interaction with PA is suggested to be achieved primarily through the formation of a charge network of the form (Asp(21-):Ca(2+):PA(2-):Lys(24+)) with overall neutrality at the bilayer surface. This novel phospholipid-binding domain, which we call TAPAS-1 (tryptophan anchoring phosphatidic acid selective-binding domain 1), is here identified as being responsible for membrane association of the PDE4A1 cAMP-specific phosphodiesterase. TAPAS-1 may not only serve as a paradigm for other PA-binding domains but also aid in detecting related phospholipid-binding domains and in generating simple chimeras for conferring membrane association and intracellular targeting on defined proteins.