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The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana.

The RXLR cytoplasmic effector AVR3a of Phytophthora infestans confers avirulence on potato plants carrying the R3a gene. Two alleles of Avr3a encode secreted proteins that differ in only three amino acid residues, two of which are in the mature protein. Avirulent isolates carry the Avr3a allele, which encodes AVR3aKI (containing amino acids C19, K80 and I103), whereas virulent isolates express only the virulence allele avr3a, encoding AVR3aEM (S19, E80 and M103). Only the AVR3aKI protein is recognized inside the plant cytoplasm where it triggers R3a-mediated hypersensitivity. Similar to other oomycete avirulence proteins, AVR3aKI carries a signal peptide followed by a conserved motif centered on the consensus RXLR sequence that is functionally similar to a host cell-targeting signal of malaria parasites. The interaction between Avr3a and R3a can be reconstructed by their transient co-expression in Nicotiana benthamiana. We exploited the N. benthamiana experimental system to further characterize the Avr3a-R3a interaction. R3a activation by AVR3aKI is dependent on the ubiquitin ligase-associated protein SGT1 and heat-shock protein HSP90. The AVR3aKI and AVR3aEM proteins are equally stable in planta, suggesting that the difference in R3a-mediated death cannot be attributed to AVR3aEM protein instability. AVR3aKI is able to suppress cell death induced by the elicitin INF1 of P. infestans, suggesting a possible virulence function for this protein. Structure-function experiments indicated that the 75-amino acid C-terminal half of AVR3aKI, which excludes the RXLR region, is sufficient for avirulence and suppression functions, consistent with the view that the N-terminal region of AVR3aKI and other RXLR effectors is involved in secretion and targeting but is not required for effector activity. We also found that both polymorphic amino acids, K80 and I103, of mature AVR3a contribute to the effector functions.

Algal Proteins↗

The epithelium of the human endolymphatic sac: immunohistochemical characterization.

A panel of monoclonal and polyclonal antibodies has been used to study the epithelium of the extraosseous part of the human endolymphatic sac (ES) by immunohistochemistry. The ES epithelium reacted with several epithelial markers such as Lu-5, different anticytokeratins, antiepithelial membrane antigen, and anticarcinoembryonic antigen. Unexpectedly, all epithelial cells also revealed a strong positive reaction for the mesenchymal marker vimentin and for S-100 protein. 'Neuroendocrine', a neurosecretory antigen, and neuron-specific enolase reactivity was detected in a few epithelial cells. The results support the assumption that the ES epithelium is metabolically active and capable of secretion and resorption. These findings are in keeping with results of functional experiments in animals. The demonstration of neurosecretory antigen and neuron-specific enolase in some cells indicate that the epithelium may also have paracrine functions.

Antibodies, Monoclonal↗

Expression of aquaporin-4 in fast-twitch fibers of mammalian skeletal muscle.

In this study we analyzed the expression of aquaporin-4 (AQP4) in mammalian skeletal muscle. Immunohistochemical experiments revealed that affinity-purified AQP4 antibodies stained selectively the sarcolemma of fast-twitch fibers. By immunogold electron microscopy, little or no intracellular labeling was detected. Western blot analysis showed the presence of two immunopositive bands with apparent molecular masses of 30 and 32 kD specifically present in membrane fraction of a fast-twitch rat skeletal muscle (extensor digitorum longus, EDL) and not revealed in a slow-twitch muscle (soleus). PCR Southern blot experiments resulted in a selective amplification in EDL of a 960-bp cDNA fragment encoding for the full-length rat form of AQP4. Functional experiments carried out on isolated skeletal muscle bundle fibers demonstrated that the osmotic response is faster in EDL than in soleus fibers isolated from the same rat. These results provide for the first time evidence for the expression of an aquaporin in skeletal muscle correlated to a specific fiber-type metabolism. Furthermore, we have analyzed AQP4 expression in skeletal muscle of mdx mice in which a decreased density of orthogonal arrays of particles, a typical morphological feature of AQP4, has been reported. Immunofluorescence experiments showed a marked reduction of AQP4 expression suggesting a critical role in the membrane alteration of Duchenne muscular dystrophy.

Animals↗

Different types of receptor interaction of peptide and nonpeptide angiotensin II antagonists revealed by receptor binding and functional studies.

The pharmacological effects of angiotensin II (AII) are potently inhibited by several peptide and recently synthesized nonpeptide AII receptor antagonists. The interaction of sarcosine1, isoleucine8-AII (sarile), sarcosine1,O-methyltyrosine4-AII (sarmesin), and the nonpeptide AII antagonists 2-n-butyl-4-chloro-5- hydroxymethyl-1-[(2'-(1H-tetrazole-5-yl)biphenyl-4-yl)- methyl]imidazole (DuP 753, Losartan potassium) and its metabolite 2-n-butyl-4-chloro-1-[(2'-(1H-tetrazole-5-yl)biphenyl-4-yl)methyl]imidaz ole - 5-carboxylic acid (EXP3174) with AII binding sites was investigated in radioligand binding and functional studies. Sarile, sarmesin, DuP 753, and EXP3174 inhibited 125I-AII binding to rat lung tissue, with Ki values of 3.5, 16.1, 23.7, and 10.4 nM, respectively. The Hill coefficients of all displacement curves, except for sarile (nH, 1.45), were not significantly different from unity. In functional experiments using rabbit aorta, sarmesin and DuP 753 competitively inhibited the contractile response to AII, with pA2 values of 6.75 and 8.01, respectively. Sarile, in contrast, revealed noncompetitive antagonism, i.e., the maximum contractile force and the slope of the concentration-contractile force curve were significantly and concentration-dependently depressed. The concentration-contractile response curve for AII was shifted to the right in a parallel fashion in the presence of EXP3174 (3 nM to 1 microM); however, the maximum contractile force was significantly decreased, by 24%. The marked noncompetitive antagonism of sarile (3 nM) was reversed in the presence of increasing concentrations of sarmesin (30 nM to 30 microM) or DuP 753 (10 nM to 1 microM), whereas in the presence of increasing concentrations of EXP3174 (3-300 nM) a 25% depression in maximum contractile force persisted. Moreover, the reduction of the maximum contractile force by EXP3174 (10 nM) was concentration-dependently restored in the presence of increasing concentrations of DuP 753 (10 nM to 1 microM), indicating interaction with the same binding site. Whereas sarile (0.3-10 nM) did not affect the 125I-AII binding capacity in radioligand saturation experiments, a 54% reduction of Bmax was observed in the presence of 100 nM EXP3174. The data provide evidence that all antagonists inhibit the functional response to AII by interacting with a common binding site at the receptor. The noncompetitive behavior of sarile seems to be due to slow dissociation from this receptor site. An additional mechanism must be postulated for EXP3174. An allosteric interaction with the receptor, as suggested by the reduction in Bmax, may be, at least in part, responsible for the nonclassical antagonism of this compound.

Angiotensin II↗

Functional expression and characterization of G-protein-gated inwardly rectifying K+ channels containing GIRK3.

The G-protein-gated inwardly rectifying K+(GIRK) family of ion channels form functional Gbetagamma-sensitive channels as heteromultimers of GIRK1 and either the GIRK2 or GIRK4 subunits. However, the homologous mouse brain GIRK3 clone failed to express in the earliest reported functional experiments in Xenopus oocytes. We recloned the GIRK3 subunit from mouse brain and found that the new clone differed significantly from that originally reported. The functional aspects of GIRK3 were reinvestigated by expression in CHO cells. The single channel properties of GIRK1/GIRK3 were characterized and compared to those of the GIRK1/GIRK2 and GIRK1/GIRK4 channels. All three GIRK1/GIRKx combinations produced channels with nearly indistinguishable conductances and kinetics. The response of GIRK1/GIRK3 to Gbetagamma in the 1-47 nm range was examined and found to be indistinguishable from that of GIRK1/GIRK4 channels. We conclude that GIRK1, with either GIRK2, 3, or 4, gives rise to heteromultimeric channels with virtually identical conductances, kinetics, and Gbetagamma sensitivities.

Amino Acid Sequence↗

The orthotopic T pouch ileal neobladder: experience with 209 patients.

PURPOSE: A serous lined extramural ileal flap valve technique called the T limb was developed to prevent reflux of urine in an orthotopic bladder substitute called the T pouch. We evaluate our intermediate clinical and functional experience with the orthotopic T pouch ileal neobladder. MATERIALS AND METHODS: From November 1996 through May 2000, 209 patients (169 men [80%], 40 women), with a mean age of 69 years (range 33 to 93) underwent construction of an orthotopic T pouch ileal neobladder after cystectomy. The indication for cystectomy included bladder cancer in 198 patients (95%). Median followup for the entire cohort was 33 months (range 0 to 69). Data were analyzed according to perioperative mortality, early (within 3 months) and late diversion related and diversion unrelated complications, radiographic evaluation of the upper urinary tract and urinary reservoir, and determination of renal function. RESULTS: Three patients (1.4%) died perioperatively. A total of 63 (30%) early complications occurred, 53 (25%) diversion unrelated and 10 (5%) diversion related. The most common early diversion unrelated complication was dehydration (10 patients). The most common early diversion related complication was urine leak in 6 patients. There were no early complications directly related to the antirefluxing T limb. Late complications occurred in 68 (32%) patients including 30 (14%) diversion unrelated and 38 (18%) diversion related. The most common late diversion unrelated complication was incisional hernia in 16 patients. Of the 38 late diversion related complications the most common were pouch calculi in 17 and ureteroileal obstruction in 9 patients. The only late complication directly related to the T limb was stenosis in 4 patients, 3 of whom received adjuvant pelvic radiation. A total of 181 patients had radiographic evaluation of the upper urinary tract including 162 (90%) with a normal radiographic study or evidence of postoperative decompression. An abnormal upper tract study was seen in 18 patients (10%) including 9 with ureteroileal obstruction and 4 with afferent T limb stenosis. Gravity cystography of the neobladder was normal in 143 of 158 (90%) evaluable patients. Reflux was seen in 15 patients (10%). Renal function as determined by serum creatinine was stable or improved in 96% of patients. Good daytime and nighttime continence was reported in 87% and 72% of evaluable patients, respectively. Overall 75% of patients complete void while 25% required some form of intermittent catheterization to empty the neobladder completely including 20% of men and 43% of women. CONCLUSIONS: With intermediate followup the functional results of the T pouch ileal neobladder are acceptable. The antirefluxing T limb provides unobstructed urinary flow in 95% and reflux prevention in 90% of patients. Although these results are encouraging, further followup is required to assess the long-term results of the T pouch ileal neobladder.

Adult↗

Quantitative nature of the Prolamin-box, ACGT and AACA motifs in a rice glutelin gene promoter: minimal cis-element requirements for endosperm-specific gene expression.

The -197 bp promoter of the rice seed storage protein gene, GluB-1, is capable of conferring endosperm-specific gene expression. This proximal 5' flanking region contains four motifs, GCN4, AACA, ACGT and Prolamin-box, which are conserved in many seed storage protein genes. We previously showed that multiple copies of GCN4 conferred endosperm expression pattern when fused to the -46 core promoter of CaMV 35S. In this paper we demonstrate, using a similar approach, that tandem repeated copies of any of the other three motifs are unable to direct expression in seeds as well as other tissues of transgenic rice plants. Mutational analysis of individual motifs in the -197 bp promoter resulted in remarkable reductions in promoter activity. These results indicate that the GCN4 motif acts as an essential element determining endosperm-specific expression and that the AACA, ACGT and Prolamin-box are involved in quantitative regulation of the GluB-1 gene. A set of gain-of-function experiments using transgenic rice showed that either the Prolamin-box or AACA, although often coupled with GCN4 in many genes, is insufficient to form a functional promoter unit with GCN4, whereas a combination of GCN4, AACA and ACGT motifs was found sufficient to confer a detectable level of endosperm expression. Taken together, our results provide direct insight into the importance of combinatorial interplay between cis-elements in regulating the expression of seed storage protein genes.

Base Sequence↗

A novel role for lbx1 in Xenopus hypaxial myogenesis.

We have examined lbx1 expression in early X. laevis tadpoles. In contrast to amniotes, lbx1 is expressed in all of the myoblasts that contribute to the body wall musculature, as well as in a group of cells that migrate into the head. Despite this different expression, the function of lbx1 appears to be conserved. Morpholino (MO) knockdown of lbx1 causes a specific reduction of body wall muscles and hypoglossal muscles originating from the somites. Although myoblast migratory defects are observed in antisense MO injected tadpoles targeting lbx1, this results at least in part from a lack of myoblast proliferation in the hypaxial muscle domain. Conversely, overexpression of lbx1 mRNA results in enlarged somites, an increase in cell proliferation, but a lack of differentiated muscle. The control of cell proliferation is linked to a strong downregulation of myoD expression in gain-of-function experiments. Co-injection of myoD mRNA with lbx1 mRNA eliminates the overproliferation phenotype observed when lbx1 is injected alone. The results indicate that a primary function of lbx1 in hypaxial muscle development is to repress myoD, allowing myoblasts to proliferate before the eventual onset of terminal differentiation.

Animals↗

The role of beta(3)-adrenoceptors in mediating relaxation of porcine detrusor muscle.

1. beta-adrenoceptors mediate relaxation of bladder detrusor smooth muscle. This study investigates the contribution of beta(3)-adrenoceptors to relaxation of the pig urinary bladder. 2. Cell membranes were prepared from detrusor muscle of the pig bladder dome and competition experiments with [(3)H]-dihydroalprenolol (DHA), a non-selective beta-adrenoceptor antagonist was used as a specific radioligand to determine the presence of beta-adrenoceptor subtypes. In functional experiments, isolated detrusor muscle strips were used to determine the potency of agonists and the affinity of antagonists. 3. In competition binding experiments, CGP20712A (beta(1)-adrenoceptor selective) displaced [(3)H]-DHA from a single binding site with a low affinity. In contrast, displacement data for ICI 118551 (beta(2)-adrenoceptor antagonist) and SR59230A (beta(3)-adrenoceptor antagonist) best fitted a two-site model suggesting a predominant (70%) population of beta(3)-adrenoceptors. 4. In functional studies, isoprenaline and salbutamol (beta(2)-adrenoceptor agonist) relaxed KCl precontracted muscle strips with high potency (pEC(50) 7.7 and 7.2, respectively), whilst CGP12177 and BRL37344 (beta(3)-adrenoceptor agonists) had low potency and were partial agonists. CGP20712A and atenolol (beta(1)-adrenoceptor antagonists) antagonised responses with a low affinity. ICI118551 antagonized responses to isoprenaline and salbutamol with a high affinity (pK(B)=7.8 and 8.7, respectively), but the Schild slopes were low suggesting that responses were mediated by more than one beta-adrenoceptor. The Schild plot for SR59230A was biphasic, apparent pK(B) values for 3 - 10 nM SR59230A being 8.6 and those for 30 nM - 1 microM being 7.7. 5. These data suggest that beta(3)-adrenoceptors are the predominant beta-adrenoceptor subtype present in the pig bladder and that beta-adrenoceptor mediated responses of this tissue are mediated via both the beta(2)- and beta(3)-adrenoceptor subtypes.

Adrenergic beta-Agonists↗

Biochemical evidence of functional interaction between mu- and delta-opioid receptors in SK-N-BE neuroblastoma cell line.

Radioligand binding assays and functional experiments revealed that the SK-N-BE neuroblastoma cell line expresses a similar ratio of mu- and delta-opioid receptors, both negatively coupled to adenylyl cyclase through pertussis toxin-sensitive G proteins. Our findings also indicate that some functional interaction occurred between the two opioid subtypes; in fact, long-term exposure to [D-Ala2-N-methyl-Phe4-Gly-ol5]enkephalin (DAMGO), a mu-selective agonist, sensitized the functional response of the delta-selective agonist but not vice versa. It is interesting that in acute interaction experiments, we observed a shift to the right of the concentration-effect curve of either DAMGO or [D-Pen2,5]enkephalin (DPDPE), a delta-selective agonist, as a result of DPDPE or DAMGO administration, respectively. In addition, low doses of naloxone, an antagonist selective for mu receptors, increased the inhibitory effect [D-Ala2-D-Met5]enkephalinamide (DAME), a mixed mu/delta agonist, on adenylyl cyclase activity. Taken overall, these data support the hypothesis of the existence of a cross talk between mu and delta receptors in the SK-N-BE cell line.

Adenylyl Cyclases↗

Neurosurgical intensive care unit organization and function: an American experience.

This article describes the organization and function of a university-based neurosurgical intensive care unit. The unit's success has been based in part on its physical structure and in larger part on its organization. Patients are comanaged by a team of neurosurgeons and anesthesiologist intensive care specialists, who receive twice-weekly input from all other participating patient care specialists, including nurses, physical therapists, social workers, and others. This type of approach promotes teamwork and fosters mutual respect among the team. It also improves patient care and, frequently, outcome.

Critical Care↗

Out-of-body experience and personality functioning.

Reporters of out-of-body experiences (n = 21) and nonreporters (n = 159) showed no significant differences on the Spheres of Control Scale, Self-efficacy Scale, and Purpose in Life Scale. However, reporters of out-of-body experiences showed significantly greater belief in Psi, Spiritualism, and Extraordinary Life Forms than nonreporters.

Adult↗

Integrative Genomic and Functional Investigation of the Multi-Layered Genetic Architecture Between Anorexia Nervosa and Bone Loss.

OBJECTIVE: Bone loss is a severe and often irreversible complication of anorexia nervosa (AN), yet the genetic mechanisms underlying this comorbidity remain underexplored. This study focuses on constructing a comprehensive genetic architecture between AN and estimated calcaneal bone mineral density (eBMD). METHOD: We applied an integrative framework incorporating genetic correlation, pleiotropic association, and causal inference across single-variant, multi-variant, and gene expression levels. Functional validation was conducted in vitro to investigate the biological role of the key candidate gene. RESULTS: Local genetic correlation analysis identified significant signals at 8p21.2 and 10q26.3, despite the lack of significant global correlation. Mendelian randomization analysis pointed to a suggestive negative causal effect of genetically predisposed AN on eBMD. Extensive pleiotropic signals were detected, particularly at 3p21.31 and 10q26.3, loci enriched with genes associated with both traits. Notably, we identified a novel pleiotropic signal near NCAM1 at 11q23.2, which was supported by multi-layered genetic evidence and confirmed through in vitro functional experiments. NCAM1, a well-established neural-associated gene, promoted osteoclastic differentiation and bone resorption when overexpressed in osteoclast precursor cells, indicating that NCAM1 possesses distinct functional roles in both neural and skeletal tissues. DISCUSSION: This study constructs a comprehensive genetic architecture underlying AN and eBMD and highlights NCAM1 as a key pleiotropic gene.

anorexia nervosa↗

Brain perivascular macrophages regulate endothelial cell function via a cMAF-dependent transcriptional program in mouse and human.

Brain perivascular macrophages maintain brain physiology, yet their transcriptional regulators and functions in health and disease remain unclear. Using single-cell multi-omics and functional experiments, we identify cellular musculoaponeurotic fibrosarcoma oncogene (cMAF) as a key transcription factor for brain perivascular macrophages, and conditional deletion of cMAF disrupts their phenotype in vivo. Functionally, cMAF drives insulin-like growth factor-1 (IGF1) expression in perivascular macrophages, enabling communication with endothelial cells. Consistently, cMAF deletion in perivascular macrophages causes transcriptional alterations in cerebral arteries, affecting vascular functions. Notably, cMAF emerges as the main transcription factor for human perivascular macrophages, suggesting conservation of this transcriptional module. During Alzheimer's disease (AD), human perivascular macrophages upregulate cMAF and IGF1 to enhance communication with vascular cells, and this response is abrogated in APOE4 carriers. Lastly, we explore an uncharacterized polymorphism in cMAF, providing evidence that the cMAF program is protective against AD. Targeting cMAF in perivascular macrophages may offer new therapeutic strategies for neurodegenerative and cerebrovascular diseases.

APOE4↗

Vax1, a novel homeobox-containing gene, directs development of the basal forebrain and visual system.

The novel homeobox-containing gene Vax1, a member of the Emx/Not gene family, is specifically expressed in the developing basal forebrain and optic nerve. Here, we show that Vax1 is essential for normal development of these structures. Mice carrying a targeted mutation of Vax1 show dysgenesis of the optic nerve, coloboma, defects in the basal telencephalon, and lobar holoprosencephaly. With the help of molecular markers we determined that in the developing visual system, the absence of Vax1 results in a proximal expansion of the activity of Pax6 and Rx. This observation suggests that Vax1 may interfere negatively with the expression of Pax6 and Rx. In reciprocal gain-of-function experiments, injection of Xvax1 mRNA or Shh into Xenopus embryos primarily affects the brain at the level of the eye primordium. Consistent with the loss-of-function results, the injection of Xvax1 results in a down-regulation of Rx. Similarly, Shh injection expands the Vax1 and Pax2 territory at the expense of the Pax6 and Rx region. On the basis of these results, we propose a model for a molecular cascade involved in the establishment of structures of the visual system.

Abnormalities, Multiple↗

Identification of the ligand binding site in Fas (CD95) and analysis of Fas-ligand interactions.

Fas (CD95), a member of the tumor necrosis factor receptor superfamily, and its ligand (FasL), a tumor necrosis factor-like protein, are intensely studied because their interaction on the cell surface is critical for the induction of programmed cell death (apoptosis) and the regulation of immune responses. The structure and specificity of the extracellular binding domains of Fas and its ligand were studied, in different laboratories, by combining molecular modeling, mutagenesis, and a variety of binding and functional experiments. Residues critical for the receptor-ligand interaction were identified and, in the absence of experimentally determined structures, binding sites and details of the Fas-ligand interactions were predicted. These studies provide an instructive example for the close combination of prediction and experiment and illustrate how insights into the structure and binding characteristics of Fas and its ligand were gradually refined. Discussed methodological aspects are representative of structure-function studies on extracellular domains of other single-path transmembrane proteins.

Amino Acid Sequence↗

In vivo treatment with anti-I-A antibodies: differential effects on Ia antigens and antigen-presenting cell function of spleen cells and epidermal Langerhans cells.

The in vivo activation of T cells by a variety of antigens can be inhibited by the administration of anti-I-A antibodies (Ab) at the time of antigen priming. This inhibition can partially be explained by the temporary loss of Ia molecules from Ia-bearing antigen-presenting cells (APC) in the spleen. In this study, the effects of i.p. injected monoclonal Ab specific for I-A glycoproteins of different H-2 haplotypes on Ia antigen expression and APC function of spleen cells and epidermal Langerhans cells were compared. It was found that anti-I-A Ab quickly bound to both spleen cell and Langerhans cell Ia antigens. Although spleen cell Ia antigens were modulated and thus temporarily disappeared, Ia antigen expression by epidermal Langerhans cells was not modulated. In functional studies, the capacity of spleen cells and epidermal cells from anti-I-A Ab treated vs control animals to function as APC for antigen-specific, I-A- or I-E-restricted T cell clones was tested. A single injection of anti-I-A Ab completely abolished the APC function of spleen cells as shown in several inbred mouse strains, F1 animals, and with the use of several different Ab and T cell clones. In contrast, Langerhans cell-dependent APC function of epidermal cells remained completely unaltered. Even multiple injections of high doses of Ab never caused any inhibition of Langerhans cell function. Experiments with anti-I-Ak or anti-I-Ad Ab in an (H-2k X H-2d)F1 animal showed abrogation of APC function of spleen cells, but again not of Langerhans cells. Thus in vivo anti-I-A Ab administration appears to differentially affect Ia antigen expression and APC function from spleen and epidermis: Ia antigens are modulated from spleen cells but not from epidermis, and APC function disappears in the spleen but not in the epidermis. The abrogation of splenic but not of Langerhans cell APC function with anti-I-A Ab will facilitate the dissection of the relative contributions of Langerhans cells as compared with other APC in the generation of cutaneous immune responses.

Animals↗

Psychosocial functioning and subjective experience in schizophrenia: a reanalysis.

Data collected by Brekke et al. (1993) on the symptomatology, psychosocial functioning, and subjective experience of schizophrenia outpatients were reanalyzed using LISREL to elucidate a causal model that would depict the functional relationships between the variables. The model that best fit the data parallels another model tested previously on cardiac patients and shows that subjective experience is much more influenced by symptomatology than by social functioning. This confirms Brekke et al.'s main finding. The implications of these results for intervention and for future research are considered.

Activities of Daily Living↗