MCA/MR syndrome in two female siblings: new entity or variant examples of Coffin-Lowry versus Atkin-Flaitz syndromes?

We report two sisters with mental retardation, coarse facial features, telecanthus, flat malar region, prominent lower lip, kyphoscoliosis, and tapering fingers. Although these patients' phenotypes showed considerable overlap with the Coffin-Lowry and the Atkin-Flaitz syndromes, their overall picture makes these diagnoses controversial.     

Chlamydia trachomatis Infection of Endocervical Epithelial Cells Enhances Early HIV Transmission Events

Chlamydia trachomatis causes a predominantly asymptomatic, but generally inflammatory, genital infection that is associated with an increased risk for HIV acquisition. Endocervical epithelial cells provide the major niche for this obligate intracellular bacterium in women, and the endocervix is also a tissue in which HIV transmission can occur. The mechanism by which CT infection enhances HIV susceptibility at this site, however, is not well understood. Utilizing the A2EN immortalized endocervical epithelial cell line grown on cell culture inserts, we evaluated the direct role that CT-infected epithelial cells play in facilitating HIV transmission events. We determined that CT infection significantly enhanced the apical-to-basolateral migration of cell-associated, but not cell-free, HIV_BaL, a CCR5-tropic strain of virus, across the endocervical epithelial barrier. We also established that basolateral supernatants from CT-infected A2EN cells significantly enhanced HIV replication in peripheral mononuclear cells and a CCR5+ T cell line. These results suggest that CT infection of endocervical epithelial cells could facilitate both HIV crossing the mucosal barrier and subsequent infection or replication in underlying target cells. Our studies provide a mechanism by which this common STI could potentially promote the establishment of founder virus populations and the maintenance of local HIV reservoirs in the endocervix. Development of an HIV/STI co-infection model also provides a tool to further explore the role of other sexually transmitted infections in enhancing HIV acquisition.     

Staff Nurses’ Perceptions and Experiences about Structural Empowerment: A Qualitative Phenomenological Study

The aim of the study reported in this article was to investigate staff nurses’ perceptions and experiences about structural empowerment and perceptions regarding the extent to which structural empowerment supports safe quality patient care. To address the complex needs of patients, staff nurse involvement in clinical and organizational decision-making processes within interdisciplinary care settings is crucial. A qualitative study was conducted using individual semi-structured interviews of 11 staff nurses assigned to medical or surgical units in a 600-bed university hospital in Belgium. During the study period, the hospital was going through an organizational transformation process to move from a classic hierarchical and departmental organizational structure to one that was flat and interdisciplinary. Staff nurses reported experiencing structural empowerment and they were willing to be involved in decision-making processes primarily about patient care within the context of their practice unit. However, participants were not always fully aware of the challenges and the effect of empowerment on their daily practice, the quality of care and patient safety. Ongoing hospital change initiatives supported staff nurses’ involvement in decision-making processes for certain matters but for some decisions, a classic hierarchical and departmental process still remained. Nurses perceived relatively high work demands and at times viewed empowerment as presenting additional. Staff nurses recognized the opportunities structural empowerment provided within their daily practice. Nurse managers and unit climate were seen as crucial for success while lack of time and perceived work demands were viewed as barriers to empowerment.     

Regulation of intracellular pH in LLC-PK1 cells studied using 31P-NMR spectroscopy

31P-NMR spectroscopy was used to monitor intracellular pH (pHi) in a suspension of LLC-PK1 cells, a renal epithelial cell line. The regulation of intracellular pH (pHi) was studied during intracellular acidification with 20% CO2 or intracellular alkalinization with 30 mM NH4Cl. The steady-state pHi in bicarbonate-containing Ringer's solution (pHo 7.40) was 7.14 +/- 0.04 and in bicarbonate-free Ringer's solution (pHo 7.40) 7.24 +/- 0.04. When pHo was altered in nominally HCO3(-)-free Ringer's, the intracellular pHi changed to only a small extent between pHo 6.6 and pHo 7.6; beyond this range pHi was linearly related to pHo. Below pHo 6.6 the cell was capable of maintaining a delta pH of 0.2 pH unit (inside more alkaline), above pH 7.6 a delta pH of 0.4 unit could be generated (inside more acid). During exposure to 20% CO2 in HCO3(-)-free Ringer's solution, pHi dropped initially to 6.9 +/- 0.05, the rate of realkalinisation was found to be 0.071 pH unit X min-1. After removal of CO2 the pHi increased by 0.65 and the rate of reacidification was 0.056 pH unit X min-1. Exposure to 30 mM NH4Cl caused a raise of pHi by 0.48 pH unit and an initial rate of re-acidification of 0.063 pH unit X min-1, after removal of NH4Cl the pHi fell by 0.58 pH unit below the steady-state pHi, followed by a subsequent re-alkalinization of 0.083 pH unit X min-1. Under both experimental conditions, the pHi recovery after an intracellular acidification, introduced by exposure to 20% CO2 and by removal of NH4+, was found to be inhibited by 53% and 63%, respectively, in the absence of sodium and 60% and 72%, respectively, by 1 mM amiloride. These studies indicate that 31P-NMR can be used to monitor steady-state intracellular pH as well a pHi transients in suspensions of epithelial cells. The results support the view that LLC-PK1 cells use an Na+-H+ exchange system to readjust their internal pH after acid loading of the cell.     

13C-n.m.r. study of citrate metabolism in rabbit renal proximal-tubule cells.

U.v.-visible-absorption and e.p.r. spectroscopy were used to study the type 2 and type 3 copper centres in the mercury derivative of laccase. After treatment with peroxide the mercury derivative of laccase exhibits a fully developed absorption band at 330 nm (delta epsilon = 2900 +/- 100 M-1.cm-1, which is characteristic of type 3 copper in the oxidized state. In addition, there is a weak ligand-field absorption at 740 nm (epsilon = 380 +/- 30 M-1.cm-1), which can be assigned to the type 3 pair. Because the e.p.r. spectrum of the type 2 copper is well resolved in the case of the mercury derivative of laccase, for the first time we have been able to observe spectroscopic evidence for a pH-dependent structural transition that has been invoked to explain the kinetics of enzyme reduction [Andréasson & Reinhammar (1979) Biochim. Biophys. Acta 568, 145-156]. According to the e.p.r. data the pKa lies in the range 6-7, and comparisons with a model compound show that the spectral changes can plausibly be interpreted in terms of the deprotonation of a water molecule in the co-ordination sphere of the type 2 copper.     

The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen.

We have previously demonstrated [Rihs, H.-P. and Peters, R. (1989) EMBO J., 8, 1479-1484] that the nuclear transport of recombinant proteins in which short fragments of the SV40 T-antigen are fused to the amino terminus of Escherichia coli beta-galactosidase is dependent on both the nuclear localization sequence (NLS, T-antigen residues 126-132) and a phosphorylation-site-containing sequence (T-antigen residues 111-125). While the NLS determines the specificity, the rate of transport is controlled by the phosphorylation-site-containing sequence. The present study furthers this observation and examines the role of the various phosphorylation sites. Purified, fluorescently labeled recombinant proteins were injected into the cytoplasm of Vero or hepatoma (HTC) cells and the kinetics of nuclear transport measured by laser microfluorimetry. By replacing serine and threonine residues known to be phosphorylated in vivo, we identified the casein kinase II (CK-II) site S111/S112 to be the determining factor in the enhancement of the transport. Either of the residues 111 or 112 was sufficient to elicit the maximum transport enhancement. The other phosphorylation sites (S120, S123, T124) had no influence on the transport rate. Examination of the literature suggested that many proteins harboring a nuclear localization sequence also contain putative CK-II sites at a distance of approximately 10-30 amino acid residues from the NLS. CK-II has been previously implicated in the transmission of growth signals to the nucleus. Our results suggest that CK-II may exert this role by controlling the rate of nuclear protein transport.     

Pathways for organic osmolyte synthesis in rabbit renal papillary tissue, a metabolic study using 13C-labeled substrates

Renal papillary collecting duct cells have been postulated to adapt their intracellular osmolality to the large changes in interstitial osmolality by changing their content of 'non-perturbing' organic osmolytes such as sorbitol and myo-inositol. 13C-NMR was used in this study to elucidate the metabolic pathways leading to a synthesis of those compounds. Incubation of rabbit renal papillary tissue with [1-13C]glucose showed label scrambling mainly into sorbitol (C-1) and lactate (C-3). This result confirms activity of aldose reductase and glycolytic enzymes in renal papillary cells. Using [3-13C]alanine or [2-13C]pyruvate as carbon source, 13C-labeling of sorbitol and myo-inositol was observed, indicating that renal papillary tissue possesses, in addition, gluconeogenic activity. The latter assumption is supported by the result that in enzyme assays rabbit kidney papilla and isolated rat kidney papillary collecting duct cells show significant fructose-1,6-bisphosphatase activity.     

Complementation between uncF alleles affecting assembly of the F1F0-ATPase complex of Escherichia coli.

A mutant affected in the b subunit (coded by the uncF gene) of the F1F0-ATPase in Escherichia coli was isolated by a localized mutagenesis procedure in which a plasmid carrying the unc genes was mutagenized in vivo. The biochemical properties of cells carrying the uncF515 allele were examined in a strain carrying the allele on a multicopy plasmid and a mutator-induced polar unc mutation on the chromosome. The strain carrying the mutant unc allele was uncoupled with respect to oxidative phosphorylation. Membrane-bound ATPase activity was very low or absent, and membranes were somewhat proton permeable. It was concluded that the F0 sector was assembled. Determination of the DNA sequence of the uncF515 allele showed it differed from wild type in that a G----A substitution occurred at position 392, resulting in glycine being replaced by aspartate at position 131. Genetic complementation tests indicated that the uncF515 allele complemented the uncF476 allele (Gly 9----Asp). Two-dimensional gel electrophoresis of membrane preparations indicated that the uncF515 and uncF476 alleles interrupted assembly of the F1F0-ATPase at different stages.     

Mutations in the uncE gene affecting assembly of the c-subunit of the adenosine triphosphatase of Escherichia coli.

The amino acid substitutions in the mutant c-subunits of Escherichia coli F1F0-ATPase coded for by the uncE429, uncE408 and uncE463 alleles affect the incorporation of these proteins into the cell membrane. The DNA sequence of the uncE429 allele differed from normal in that a G leads to A base change occurred at nucleotide 68 of the uncE gene, resulting in glycine being replaced by aspartic acid at position 23 in the c-subunit. The uncE408 and uncE463 mutant DNA sequences were identical and differed from normal in that a C leads to T base change occurred at nucleotide 91 of the uncE gene, resulting in leucine being replaced by phenylalanine at position 31 in the c-subunit. An increased gene dosage of the uncE408 or uncE463 alleles resulted in the incorporation into the membranes of the mutant c-subunits. The results are discussed in terms of the 'Helical Hairpin Hypothesis' of Engelman & Steitz [(1981) Cell 23,411-422].     

N-glycosylation plays a role in biosynthesis and internalization of the adenylate cyclase stimulating vasopressin V2-receptor of LLC-PK1 renal epithelial cells: an effect of concanavalin A on binding and expression.

The role of N-glycosylation in the function and biosynthesis of the vasopressin V2-receptor in LLC-PK1 renal epithelial cells was examined using various lectins and inhibitors operating at different steps of the glycosidic pathway. Tunicamycin, which blocks all N-glycosylation, and castanospermine, which inhibits glycosidase I and hence blocks formation of high-mannose-type N-glycosylated intermediates, resembled one another in affecting V2-receptor biosynthesis and internalization in a concentration-dependent manner. In contrast, swainsonine, an inhibitor of mannosidase II and hence of complex-type oligosaccharide formation, had no effect. Interestingly, the alpha-D-mannose/alpha-D-glucose-specific lectin concanavalin A, (Con A), in contrast to the beta-D-galactose-specific lectin ricin, had a marked effect on the V2-receptor in LLC-PK1 cells, increasing both receptor numbers up to twofold in vivo and specific [3H]AVP binding up to 50% in vitro in a concentration-dependent manner. The concentrations inducing half-maximal response were about 0.2 and 20 micrograms/ml for the in vivo and in vitro responses, respectively, implying distinct effects on V2-expression and ligand binding. That the in vitro effect on binding was due to a direct effect on the V2-receptor could be shown by the lack of a Con A effect on [3H]AVP binding in membranes prepared from LLC-PK1 cells down-regulated for the V2-receptor or from cells of the LLC-PK1 V2-receptor deficient mutant M18. All results were consistent with a functional role for N-glycosylation of the V2-receptor in LLC-PK1 cells.