Young adults with cystic fibrosis have altered trabecular microstructure by ITS-based morphological analysis.

UNLABELLED: Young adults with cystic fibrosis have compromised plate-like trabecular microstructure, altered axial alignment of trabeculae, and reduced connectivity between trabeculae that may contribute to the reduced bone strength and increased fracture risk observed in this patient population. INTRODUCTION: The risk of fracture is increased in patients with cystic fibrosis (CF). Individual trabecular segmentation (ITS)-based morphological analysis of high-resolution peripheral quantitative computed tomography (HR-pQCT) images segments trabecular bone into individual plates and rods of different alignment and connectivity, which are important determinants of trabecular bone strength. We sought to determine whether alterations in ITS variables are present in patients with CF and may help explain their increased fracture risk. METHODS: Thirty patients with CF ages 18-40 years underwent DXA scans of the hip and spine and HR-pQCT scans of the radius and tibia with further assessment of trabecular microstructure by ITS. These CF patients were compared with 60 healthy controls matched for age (±2 years), race, and gender. RESULTS: Plate volume fraction, thickness, and density as well as plate-plate and plate-rod connectivity were reduced, and axial alignment of trabeculae was lower in subjects with CF at both the radius and the tibia (p < 0.05 for all). At the radius, adjustment for BMI eliminated most of these differences. At the tibia, however, reductions in plate volume fraction and number, axially aligned trabeculae, and plate-plate connectivity remained significant after adjustment for BMI alone and for BMI and aBMD (p < 0.05 for all). CONCLUSIONS: Young adults with CF have compromised plate-like and axially aligned trabecular morphology and reduced connectivity between trabeculae. ITS analysis provides unique information about bone integrity, and these trabecular deficits may help explain the increased fracture risk in adults with CF not accounted for by BMD and/or traditional bone microarchitecture measurements.

Polymerase chain reaction/denaturing gradient gel electrophoresis (PCR/DGGE): sensitivity, band pattern analysis, and methodologic optimization.

Polymerase chain reaction/denaturing gradient gel electrophoresis (PCR/DGGE) has been gaining popularity as a preferred method to determine the clonality of T-cell populations in small or sparsely infiltrated specimens such as skin biopsies. T-cell receptor (TCR)-gamma gene rearrangements are amplified using nested consensus primers in two rounds of PCR and then are separated by DGGE. Sensitivity is better than with conventional Southern blot analysis but not fully defined. In addition to a discrete primary band resulting from a monoclonal TCR-gamma gene rearrangement, there are often weaker secondary bands of unknown origin. Our goals were to define the PCR/DGGE clonal detection threshold, determine the genesis of the multiband pattern, and optimize methods to minimize extraneous bands. Titration studies showed that the sensitivity of PCR/DGGE for detecting clonal T-cell DNA is affected by the polyclonal T-cell content of the background DNA. The detection threshold is 0.001% using keratinocyte DNA as diluent but only 1% with tonsil DNA. Analysis of monoclonal T-cell lines showed that multiple bands can be produced by a single TCR-gamma gene rearrangement. Mixing of inner and outer primer pair PCR products showed that this is an artifact resulting from different sized PCR products produced during the two rounds of nested PCR required for optimal specificity. Repeat DGGE of isolated bands ruled out variable mobility of partially melted PCR products. Reduction of first round PCR product used as second round target from 10 microl to 1 microl, or a decrease of first round primers from 40 pmole to 5 pmole, resulted in diminished secondary bands without compromising primary band intensity. These results show that: 1) PCR/DGGE has a realistic clonal detection threshold of 0.1% to 1%, 2) multiple bands are consistent with a monoclonal T-cell population, and 3) conditions can be optimized to minimize artifactual secondary bands.

High-affinity agonist binding is not sufficient for agonist efficacy at 5-hydroxytryptamine2A receptors: evidence in favor of a modified ternary complex model.

In this study, the relationship between high-affinity agonist binding and second messenger production was examined at native and mutant 5-hydroxytryptamine2A receptors. At native 5-hydroxytryptamine2A receptors all agonists, with the exception of quipazine, discriminated between high- and low-affinity states of the receptor, as determined by analysis of competition binding assays. There was no correlation between the ability of selected agonists to label the high-affinity agonist state and to augment phosphoinositide hydrolysis. Quipazine, which did not discriminate between the affinity states of the receptor, behaved as a full agonist. Similar results were obtained when a point mutation (F340L) of a highly conserved phenylalanine located in transmembrane domain VI was examined. With the F340L mutant, most of the agonists tested labeled significantly fewer high-affinity sites, compared with the native receptor. There was no significant relationship between high-affinity agonist binding and second messenger production. Bufotenine and 4-iodo-3,5-dimethoxyphenylisopropylamine labeled similar percentages of high-affinity agonist binding sites (22% vs. 26%), but 4-iodo-3,5-dimethoxyphenylisopropylamine behaved as a full agonist, whereas bufotenine was devoid of detectable agonist activity. The inability of selected agonists to activate phosphoinositide hydrolysis was not due solely to lower agonist affinity for the mutant receptor, because the binding affinity of quipazine was unchanged by the F340L mutation but quipazine had no detectable agonist activity at the mutant receptor. Our results demonstrate that the ability of an agonist to promote the high-affinity state of the 5-hydroxytryptamine2A receptor is not correlated with its ability to augment second messenger production. These results are consistent with recent models of G protein-receptor functioning (e.g., modified ternary complex model) that predict that additional transition states of the receptor-ligand complex are essential for agonist efficacy.

5-Hydroxytryptamine2A (5-HT2A) receptor desensitization can occur without down-regulation.

The connection between agonist-induced desensitization and down-regulation of 5-hydroxytryptamine2A (5-HT2A) receptors was examined in a clonal cell line that stably expresses the 5-HT2A receptor. Brief (2-hr) and prolonged (24-hr) exposure to the agonist quipazine or the agonist 4-iodo-(2,5-dimethoxy)- phenylisopropylamine (DOI) diminished 5-HT2A receptor-mediated phosphoinositide hydrolysis; no change in 5-HT2A receptor number or affinity was measured after 24 hr of exposure to DOI or quipazine. Immunohistochemical studies demonstrated that a 24-hr exposure to DOI did not alter surface 5-HT2A receptor immunoreactivity. Western blot analysis with G alpha q- and G alpha 11-selective antibodies indicate that a 24-hr agonist exposure did not alter the levels of phospholipase C-dependent G proteins. These results suggest that desensitization after prolonged DOI exposure can occur via a process independent of the levels of phospholipase C-coupled G proteins. Studies with a mutant 5-HT2A receptor (F340L) indicated that binding per se is not sufficient for desensitization. Down-regulation of the protein kinase C isozymes alpha and epsilon by overnight exposure to phorbol-12,13-dibutyrate attenuated the intermediate phase (i.e., after 2-6 hr of agonist exposure) of DOI- and quipazine-induced desensitization. These results indicate that the intermediate phase of DOI-induced desensitization is mediated by the alpha- and/or epsilon-protein kinase C isozymes but that neither is involved in the later phase (i.e., after 24 hr of agonist exposure) of desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphomatoid papulosis and associated cutaneous lymphoproliferative disorders exhibit a common clonal origin.

Determination of the clonal relationship among multiple lymphoproliferative disorders occurring in individual patients has been hampered by dependence on molecular biologic techniques that require analysis of advanced lesions containing high tumor clone densities to isolate dominant, clonal antigen-receptor gene rearrangements. Polymerase chain reaction/denaturing gradient gel electrophoresis (PCR/DGGE) involves the amplification of T-cell receptor (TCR)-gamma gene rearrangements followed by their electrophoresis in denaturing gradient gels. This method detects dominant TCR-gamma gene rearrangements at tumor clone densities as low as 0.1%, making this assay suitable for analysis of early as well as late lesions. Using this approach, we analyzed skin lesions of lymphomatoid papulosis and either CD30+ large-cell lymphoma or early patch/plaque mycosis fungoides that developed in three patients. In each case, the dual specimens exhibited an identical band pattern by PCR/DGGE analysis, suggesting a common clonal origin. To confirm this clonal relationship, the dominant TCR-gamma gene rearrangements were eluted, amplified, cloned, and sequenced. In each case, they showed identical junctional sequences. These findings are significant for several reasons: 1) they demonstrate the common clonal origin of lymphomatoid papulosis and CD30+ large-cell lymphoma or mycosis fungoides occurring in individual patients; 2) they confirm that co-migrating PCR/DGGE bands exhibit identical nucleotide sequences; and 3) they provide a method for determining the sequence of a tumor-derived TCR-gamma gene rearrangement in early lesions containing a low tumor clone density. This latter feature should allow the prospective molecular staging of early cutaneous lymphoproliferative disorders.

Differential ergoline and ergopeptine binding to 5-hydroxytryptamine2A receptors: ergolines require an aromatic residue at position 340 for high affinity binding.

In this paper we show that a highly conserved aromatic residue, phenylalanine at the 340-position, is essential for ergoline binding to 5-hydroxytryptamine2A receptors. We hypothesized that F340 was essential for a specific aromatic-aromatic interaction (e.g., pi-pi or hydrophobic) between the phenyl moiety of F340 and the aromatic ring of the ergoline nucleus. To test this hypothesis, eight point mutations of adjacent (F340 and F339) and nonadjacent (F125) phenylanaines were made, using conservative (phenylalanine to tyrosine) and nonconservative (phenylalanine to leucine, alanine, or serine) substitutions. The binding affinities of all of the tested simple ergolines were greatly reduced by specific mutations of F340 in which aromatic-aromatic interactions (e.g., F340A and F340L) were abolished, but they were unaffected when the replacement residue was aromatic (e.g., F340Y). In contrast, the binding affinities of four ergopeptines (bromocryptine, ergocryptine, ergocornine, and ergotamine) were relatively unaffected by the F340L substitution. Neither ergoline nor ergopeptine affinities were consistently altered by F339 mutations. These results support the notion that aromatic-aromatic interactions (either pi-pi of hydrophobic) with F340 are essential for the binding of simple ergolines but not ergopeptines to 5-hydroxytryptamine2A receptors. Our findings support models of ergoline and ergopeptine binding to serotonin receptors that suggest that the nature of the substituent at the 8-position of the ergoline nucleus may give rise to different modes of binding for the two classes of agents, particularly with respect to the phenyl ring of F340.

Binding of typical and atypical antipsychotic agents to 5-hydroxytryptamine-6 and 5-hydroxytryptamine-7 receptors.

The authors examined the affinities of 36 typical and atypical antipsychotic agents for the cloned rat 5-hydroxytryptamine-6 (5-HT6) and rat 5-hydroxytryptamine-7 (5-HT7) receptors in transiently expressed COS-7 cells (5-HT7) or stably transfected HEK-293 cells (5-HT6 receptors). Clozapine and several related atypical antipsychotic agents (rilapine, olanzepine, tiospirone, fluperlapine, clorotepine and zotepine) had high affinities for the newly discovered 5-HT6 receptor (Kis < 20 nM). The 5-HT7 receptor bound clozapine, rilapine, fluperlapine, clorotepine, zotepine and risperidone but not tiospirone and olanzepine, with affinities less than 15 nM. In addition, several typical antipsychotic agents (chloroprothixene, chlorpromazine, clothiapine and fluphenazine) had high affinities for both the 5-HT6 and 5-HT7 receptors. Pimozide, a diphenylbutylpiperidine, had the highest affinity of all the typical antipsychotic agents tested for the 5-HT7 receptor (Ki = 0.5 nM). Three putative atypical antipsychotic agents melperone, amperozide and MDL 100907 did not bind with high affinities to either the 5-HT6 or 5-HT7 receptors (Kis > 50 nM). Several dopamine-selective antipsychotic agents (raclopride, rimcazole and penfluridol) had essentially no affinity for either the 5-HT6 or 5-HT7 receptors (Ki values > 5000 nM).(ABSTRACT TRUNCATED AT 250 WORDS)