Efficient Semiparametric Marginal Estimation for the Partially Linear Additive Model for Longitudinal/Clustered Data

We consider the efficient estimation of a regression parameter in a partially linear additive nonparametric regression model from repeated measures data when the covariates are multivariate. To date, while there is some literature in the scalar covariate case, the problem has not been addressed in the multivariate additive model case. Ours represents a first contribution in this direction. As part of this work, we first describe the behavior of nonparametric estimators for additive models with repeated measures when the underlying model is not additive. These results are critical when one considers variants of the basic additive model. We apply them to the partially linear additive repeated-measures model, deriving an explicit consistent estimator of the parametric component; if the errors are in addition Gaussian, the estimator is semiparametric efficient. We also apply our basic methods to a unique testing problem that arises in genetic epidemiology; in combination with a projection argument we develop an efficient and easily computed testing scheme. Simulations and an empirical example from nutritional epidemiology illustrate our methods.     

Identification of salivary components that induce transition of hyphae to yeast in Candida albicans

Candida albicans , the major human fungal pathogen, undergoes a reversible morphological transition from single yeast cells to pseudohyphae and hyphae filaments. The hyphae form is considered the most invasive form of the fungus. The purpose of this study is to investigate the effect of saliva on hyphae growth of C. albicans. Candida albicans hyphae were inoculated in Roswell Park Memorial Institute medium with whole saliva, parotid saliva or buffer mimicking the saliva ion composition, and cultured for 18 h at 37 °C under aerobic conditions with 5% CO₂. Whole saliva and parotid saliva induced transition to yeast growth, whereas the culture with buffer remained in the hyphae form. Parotid saliva was fractionated on a reverse-phase C8 column and each fraction was tested for inducing transition to yeast growth. By immunoblotting, the salivary component in the active fraction was identified as statherin, a phosphoprotein of 43 amino acids that has been implicated in remineralization of the teeth. Synthetically made statherin induced transition of hyphae to yeast. By deletion of five amino acids at the negatively charged N-terminal site (DpSpSEE), yeast-inducing activity and binding to C. albicans were increased. In conclusion, statherin induces transition to yeast of C. albicans hyphae and may thus contribute to the oral defense against candidiasis.     

Routes of epithelial water flow: aquaporins versus cotransporters

The routes water takes through membrane barriers is still a matter of debate. Although aquaporins only allow transmembrane water movement along an osmotic gradient, cotransporters are believed to be capable of water transport against the osmotic gradient. Here we show that the renal potassium-chloride-cotransporter (KCC1) does not pump a fixed amount of water molecules per movement of one K⁺ and one Cl⁻, as was reported for the analogous transporter in the choroid plexus. We monitored water and potassium fluxes through monolayers of primary cultured renal epithelial cells by detecting tiny solute concentration changes in the immediate vicinity of the monolayer. KCC1 extruded K⁺ ions in the presence of a transepithelial K⁺ gradient, but did not transport water. KCC1 inhibition reduced epithelial osmotic water permeability P_f by roughly one-third, i.e., the effect of inhibitors was small in resting cells and substantial in hormonal stimulated cells that contained high concentrations of aquaporin-2 in their apical membranes. The furosemide or DIOA (dihydroindenyl-oxy-alkanoic acid)-sensitive water flux was much larger than expected when water passively followed the KCC1-mediated ion flow. The inhibitory effect of these drugs on water flux was reversed by the K⁺-H⁺ exchanger nigericin, indicating that KCC1 affects water transport solely by K⁺ extrusion. Intracellular K⁺ retention conceivably leads to cell swelling, followed by an increased rate of endocytic AQP2 retrieval from the apical membrane.     

Protective endogenous cyclic adenosine 5'-monophosphate signaling triggered by pemphigus autoantibodies

Pemphigus vulgaris (PV) is an autoimmune skin disease mediated by autoantibodies directed against the cadherin-type cell adhesion molecules desmoglein (Dsg) 3 and Dsg1 and is characterized by loss of keratinocyte cohesion and epidermal blistering. Several intracellular signaling pathways, such as p38MAPK activation and RhoA inhibition, have been demonstrated to be altered following autoantibody binding and to be causally involved in loss of keratinocyte cohesion. In this paper, we demonstrate that cAMP-mediated signaling completely prevented blister formation in a neonatal pemphigus mouse model. Furthermore, elevation of cellular cAMP levels by forskolin/rolipram or β receptor agonist isoproterenol blocked loss of intercellular adhesion, depletion of cellular Dsg3, and morphologic changes induced by Ab fractions of PV patients (PV-IgG) in cultured keratinocytes. Incubation with PV-IgG alone increased cAMP levels, indicating that cAMP elevation may be a cellular response pathway to strengthen intercellular adhesion. Our data furthermore demonstrate that this protective pathway may involve protein kinase A signaling because protein kinase A inhibition attenuated recovery from PV-IgG-induced cell dissociation. Finally, cAMP increase interfered with PV-IgG-induced signaling by preventing p38MAPK activation both in vitro and in vivo. Taken together, our data provide insights into the cellular response mechanisms following pemphigus autoantibody binding and point to a possible novel and more specific therapeutic approach in pemphigus.     

Spike bursts generated by the thermosensitive (cold) neuron from the antennal campaniform sensilla of the ground beetle Platynus assimilis

Responses of the antennal thermosensitive neuron of the ground beetle Platynus assimilis to warming from 20 to 50 °C were measured and analysed. During warming, neurons switched from regular spiking to bursting. ISI analysis showed that the number of spikes in the burst and spike frequency within the burst were temperature dependent and may precisely encode unfavourably or dangerously high temperatures in a graded manner. In contrast, regular spikes of the neuron encode moderate temperatures at 20-30 °C. The threshold temperature of spike bursting varied in different neurons from 25 to 47 °C. As a result, the number of bursting neurons increased with temperature increase. Therefore, in addition to the burst characteristics, the total number of bursting neurons may also contain useful information on external temperature. A relationship between the spike bursts and locomotor activity of the beetles was found which may have importance in behavioural thermoregulation of the species. At 44.4 ± 0.6 °C, first indications of partial paralysis (of the hind legs) were observed. We emphasize, that in contrast to various sensory systems studied, the thermoreceptor neuron of P. assimilis has a stable and continuous burst train, no temporal information is encoded in the timing of the bursts.     

Protein-bound dinitrosyl-iron complexes appearing in blood of rabbit added with a low-molecular dinitrosyl-iron complex: EPR studies.

The formation of protein-bound dinitrosyl-iron complexes (DNIC) in blood plasma and packed red cell fraction has been demonstrated by the EPR method in the experiments on rabbits which were i/v injected with the low-molecular DNIC with thiosulphate. This formation was ensured by transfer of Fe(+)(NO(+))(2) moieties from low-molecular DNIC onto serum albumin or hemoglobin molecules. Protein-bound DNICs appeared immediately after low-molecular DNIC injection followed with gradually decreasing their amounts. The complexes could be detected by EPR technique during more than two days. The addition of water-soluble NO scavenger, the iron complex with N-methyl-d-glucamine dithiocarbamate (MGD) resulted in decomposition of a part of protein-bound DNICs and in effective excretion of secondary products (mainly mononitrosyl-iron complexes with MGD) from the blood flow.     

Consequences of cysteine mutations in calcium-binding epidermal growth factor modules of fibrillin-1

Mutations in fibrillin-1 lead to Marfan syndrome and some related genetic disorders. Many of the more than 600 mutations currently known in fibrillin-1 eliminate or introduce cysteine residues in epidermal growth factor-like modules. Here we report structural and functional consequences of three selected cysteine mutations (R627C, C750G, and C926R) in fibrillin-1. The mutations have been analyzed by means of recombinant polypeptides produced in mammalian expression systems. The mRNA levels for the mutation constructs were similar to wild-type levels. All three mutated polypeptides were secreted by embryonic kidney cells (293) into the culture medium. Purification was readily feasible for mutants R627C and C750G, but not for C926R, which restricted the availability of this mutant polypeptide to selected analyses. The overall folds of the mutant polypeptides were indistinguishable from the wild-type as judged by the ultrastructural shape, CD analysis, and reactivity with a specific antibody sensitive for intact disulfide bonds. Subtle structural changes caused by R627C and C750G, however, were monitored by proteolysis and heat denaturation experiments. These changes occurred in the vicinity of the mutations either as short range effects (R627C) or both short and long range effects (C750G). Enhanced proteolytic susceptibility was observed for R627C and C750G to a variety of proteases. These results expand and further strengthen the concept that proteolytic degradation of mutated fibrillin-1 might be an important potential mechanism in the pathogenesis of Marfan syndrome and other disorders caused by mutations in fibrillin-1.     

Exportin 6: a novel nuclear export receptor that is specific for profilin.actin complexes

Active macromolecular transport between the nucleus and cytoplasm proceeds through nuclear pore complexes and is mostly mediated by transport receptors of the importin beta-superfamily. Here we identify exportin 6 (Exp6) as a novel family member from higher eukaryotes and show that it mediates nuclear export of profilin.actin complexes. Exp6 appears to contact primarily actin, but the interaction is greatly enhanced by the presence of profilin. Profilin thus functions not only as the nucleotide exchange factor for actin, but can also be regarded as a cofactor of actin export and hence as a suppressor of actin polymerization in the nucleus. Even though human and Drosophila Exp6 share only approximately 20% identical amino acid residues, their function in profilin.actin export is conserved. A knock-down of Drosophila Exp6 by RNA interference abolishes nuclear exclusion of actin and results in the appearance of nuclear actin paracrystals. In contrast to a previous report, we found no indications of a major and direct role for CRM1 in actin export from mammalian or insect nuclei.