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PubMed2003

Effects of antisense misexpression of CFC on downstream flectin protein expression during heart looping.

Dextral looping of the heart is regulated on multiple levels. In humans, mutations of the genes CFC and Pitx2/RIEG result in laterality-associated cardiac anomalies. In animal models, a common read-out after the misexpression of laterality genes is heart looping direction. Missing in these studies is how laterality genes impact on downstream morphogenetic processes to coordinate heart looping. Previously, we showed that Pitx2 indirectly regulates flectin protein by regulating the timing of flectin expression in one heart field versus the other (Linask et al. [2002] Dev. Biol. 246:407-417). To address this question further we used a reported loss-of-function approach to interfere with chick CFC expression (Schlange et al. [2001] Dev. Biol. 234:376-389) and assaying for flectin expression during looping. Antisense CFC treatment results in abnormal heart looping or no looping. Our results show that regardless of the sidedness of downstream Pitx2 expression, it is the sidedness of predominant flectin protein expression in the extracellular matrix of the dorsal mesocardial folds and splanchnic mesoderm apposed to the foregut wall that is associated directly with looping direction. Thus, Pitx2 can be experimentally uncoupled from heart looping. The flectin asymmetry continues to be maintained in the secondary heart field during looping.

Animals↗
PubMed2004

A kinematic view of loop closure.

We consider the problem of loop closure, i.e., of finding the ensemble of possible backbone structures of a chain segment of a protein molecule that is geometrically consistent with preceding and following parts of the chain whose structures are given. We reduce this problem of determining the loop conformations of six torsions to finding the real roots of a 16th degree polynomial in one variable, based on the robotics literature on the kinematics of the equivalent rotator linkage in the most general case of oblique rotators. We provide a simple intuitive view and derivation of the polynomial for the case in which each of the three pair of torsional axes has a common point. Our method generalizes previous work on analytical loop closure in that the torsion angles need not be consecutive, and any rigid intervening segments are allowed between the free torsions. Our approach also allows for a small degree of flexibility in the bond angles and the peptide torsion angles; this substantially enlarges the space of solvable configurations as is demonstrated by an application of the method to the modeling of cyclic pentapeptides. We give further applications to two important problems. First, we show that this analytical loop closure algorithm can be efficiently combined with an existing loop-construction algorithm to sample loops longer than three residues. Second, we show that Monte Carlo minimization is made severalfold more efficient by employing the local moves generated by the loop closure algorithm, when applied to the global minimization of an eight-residue loop. Our loop closure algorithm is freely available at http://dillgroup. ucsf.edu/loop_closure/.

Journal Article↗
PubMed1988

Rabbit model for mucosal immunity in the bowel: I. Establishment of virus-infected ileal loops.

An animal model was designed for use in studies of initial cellular immune responses to virus infection of the intestinal mucosa. The animal chosen was the New Zealand White rabbit and the mucosal site the subterminal ileum, isolated in a Thirty-Vella loop. The antigen used was parainfluenzavirus type 3, which would normally be destroyed by bile salts if ingested. Loops approximately 20 cm in length, each containing at least one Peyer's patch, were exteriorised through left paramedian stomata. Atrophic changes began to appear in the loops by 7 days, but no observable diminution in their associated lymphoid tissues was evident. The genesis of parainfluenzavirus type 3 infection in the loops was monitored by assay of sequential loop washings for infectious virus and in fluorescent antibody studies of cells from infected loop epithelia. Infectious virus was recovered for up to 13 days after inoculation and specific intracytoplasmic immunofluorescence was detected in loop epithelial cells. There was little serological evidence of systemic spread of the virus. A localised cellular immune response against parainfluenzavirus type 3 was mounted in the lymphoid tissues associated with the infected loops by day 14, but was not detected in systemic lymphoid tissues. No reactivity was detected in rabbits given inactivated virus via their loops or in those receiving infectious virus intravenously. This model appears to be capable of generating mucosal cellular responses to infection and may therefore be suitable for further studies in this field.

Animals↗
PubMed1995

Simultaneous modeling of multiple loops in proteins.

The most reliable methods for predicting protein structure are by way of homologous extension, using structural information from a closely related protein, or by "threading" through a set of predefined protein folds ("inverse folding"). Both sets of methods provide a model for the core of the protein--the structurally conserved secondary structures. Due to the large variability both in sequence and size of the loops that connect these secondary structures, they generally cannot be modeled using these techniques. Loop-closure algorithms are aimed at predicting loop structures, given their end-to-end distance. Various such algorithms have been described, and all have been tested by predicting the structure of a single loop in a known protein. In this paper we propose a method, which is based on the bond-scaling-relaxation loop-closure algorithm, for simultaneously predicting the structures of multiple loops, and demonstrate that, for two spatially close loops, simultaneous closure invariably leads to more accurate predictions than sequential closure. The accuracy of the predictions obtained for pairs of loops in the size range of 5-7 residues each is comparable to that obtained by other methods, when predicting the structures of single loops: the RMS deviations from the native conformations of various test cases modeled are approximately 0.6-1.7 A for backbone atoms and 1.1-3.3 A for all-atoms.

Algorithms↗
PubMed1997

Analysis of the structure and stability of omega loop A replacements in yeast iso-1-cytochrome c.

Omega (omega)-loop A, residues 18-32 in wild-type yeast iso-1-cytochrome c, has been deleted and replaced with loop sequences from three other cytochromes c and one from esterase. Yeast expressing a partial loop deletion do not contain perceptible amounts of holoprotein as measured by low-temperature spectroscopy and cannot grow on nonfermentable media. Strains expressing loop replacement mutations accumulate holoprotein in vivo, but the protein function varies depending on the sequence and length of the replacement loop; in vivo expression levels do not correlate with their thermal denaturation temperatures. In vitro spectroscopic studies of the loop replacement proteins indicate that all fold into a native-like cytochrome c conformation, but are less stable than the wild-type protein. Decreases in thermal stability are caused by perturbation of loop C backbone in one case and a slight reorganization of the protein hydrophobic core in another case, rather than rearrangement of the loop A backbone. A single-site mutation in one of the replacement mutants designed to relieve inefficient hydrophobic core packing caused by the new loop recovers some, but not all, of the lost stability.

Amino Acid Sequence↗
PubMed2002

Probing reactive center loop insertion in serpins: a simple method for ovalbumin.

Insertion of the reactive center loop in beta-sheet A in serpins has been typically inferred from the increased stability of the cleaved form to thermal- and urea-induced denaturation. We describe a convenient and rapid fluorescence-based method that differentiates the loop-inserted form from the loop-exposed form in ovalbumin, a prototypic noninhibitory serpin. Recombinant wild-type and R345A ovalbumins in the intact form bind ANS with equilibrium dissociation constants of 116 and 125 microM and a maximal fluorescence increase of 200 and 264%, respectively, in pH 6.8 buffer. Cleavage of the two proteins with porcine pancreatic elastase results in a 1.6- and 2.6-fold increase in the ANS-binding affinity. While cleavage of the reactive center loop in rR345A ovalbumin results in a approximately 200% increase in the ANS fluorescence, the rWT protein exhibits a approximately 50% decrease. Similar experiments with alpha(1)-proteinase inhibitor and antithrombin, two inhibitory serpins that exhibit reactive center loop insertion, show a decrease in ANS fluorescence on cleavage with porcine pancreatic elastase and thrombin, respectively. Denaturation studies in guanidinium hydrochloride indicate that the reactive center loop is inserted in the main body of the serpin in the cleaved form of rR345A mutant, while it is exposed in the cleaved form of rWT ovalbumin. These results demonstrate that ANS fluorescence change is an indicator of the loop-inserted or loop-exposed form in these recombinant ovalbumins, and thus could be advantageously used for probing reactive center loop insertion in ovalbumins. The major increase in fluorescence for the rR345A mutant on cleavage primarily arises from a change in ANS binding rather than from the generation of an additional ANS-binding site.

Amino Acid Substitution↗
PubMed1999

R-Loop in the replication origin of human mitochondrial DNA is resolved by RecG, a Holliday junction-specific helicase.

Stable RNA-DNA hybrids (R-loops) prime the initiation of replication in Escherichia coli cells. The R-loops are resolved by Escherichia coli RecG protein, a Holliday junction specific helicase. A stable RNA-DNA hybrid formation in the mitochondrial D-loop region is also implicated in priming the replication of mitochondrial DNA. Consistent with this hypothesis, the 3' ends of the mitochondrial R-loop formed by in vitro transcription are located close to the initiation sites of the mitochondrial DNA replication. This mitochondrial R-loop is resolved by RecG in a dose-dependent manner. Since the resolution by RecG requires ATP, the resolution is dependent on the helicase activity of RecG. A linear RNA-DNA heteroduplex is not resolved by RecG, suggesting that RecG specifically recognizes the higher structure of the mitochondrial R-loop. This is the first example that R-loops of an eukaryotic origin is sensitive to a junction-specific helicase. The resolution of the mitochondrial R-loop by RecG suggests that the replication-priming R-loops have a common structural feature recognized by RecG.

Bacterial Proteins↗
PubMed2002

Loops within the CD11c I domain critical for specific recognition of fibrinogen.

The I domains of CD11 are responsible for the binding of ligands and have a unique structure with 6-7 alpha helices and 6 beta sheets with interconnecting loops. To determine loops recognizing fibrinogen in CD11c I domain, five oligopeptides corresponding to CD11c loops were used to prevent fibrinogen binding to isolated CD11c I domain. The results of the inhibition experiment indicated that all of the loops except the one between E-beta sheet and 6-alpha helix were involved in the binding to fibrinogen. The peptide beta D alpha 5 and alpha 3 alpha 4 showed higher inhibitory activity than others, and the combination of four peptides blocked fibrinogen binding to the I domain completely. These peptides (beta A alpha 1, alpha 3 alpha 4, beta D alpha 5 and beta F alpha 7) could block THP-1 cell binding to fibrinogen coated surface as well. Alanine substitution of amino acids on the I domain such as Y249A and Q201A (which are on the loops beta D-alpha 5 and alpha 3-alpha 4 respectively) abolished fibrinogen binding, while mutation on the loop beta E-alpha 6 (Q273A) had no effect on fibrinogen binding. Taken together, the results from this study suggest that the loops on the top of CD11c I domain such as loop beta A-alpha 1, alpha 3-alpha 4, beta D-alpha 5 and beta F-alpha 7 are involved in fibrinogen binding, and two loops (alpha 3-alpha 4 and beta D-alpha 5) are more important than others for the recognition of fibrinogen.

Amino Acid Sequence↗
PubMed1996

Structure of a hexanucleotide RNA hairpin loop conserved in ribosomal RNAs.

The structure of 5'-pppGGAC(GUAAUA)GUCC has been deduced from NMR data. The six-nucleotide hairpin loop is highly conserved in large subunit ribosomal RNA and confers unusual stability on RNA hairpins. Standard assignment methods, including through-bond strategies for backbone protons, were used to assign all 31P resonances and all but two of the non-exchangeable protons. The model calculated by restrained molecular dynamics shows that the loop adopts a specific structure stabilized by five non-canonical hydrogen bonds. An edge-to-edge G-A pair (hydrogen bonds G5 N3-A10 NH6, G5 H2-A1O N7 and A10 NH6-G5 O2') closes the hairpin loop. A trans 5' P-O bond at A7 reverses the direction of the backbone and is stabilized by two base-backbone hydrogen bonds (U6 NH3-U9 OP and U6 O2'-A8 N7). The only unstructured part of the loop is U9, which is completely unstacked and also the only phylogenetically variable position. The U6-A7-A8 "U-turn" reproduces hydrogen bonds and backbone torsion seen in the tRNA anticodon loop and TTpsiC loop, and in an internal loop of the hammerhead ribozyme. G-A is a common closing mismatch in ribosomal RNA hairpin loops. The U-turn and G-A pair found in this hexaloop structure may therefore be common structural units of RNA hairpin loops.

Base Sequence↗
PubMed1996

Hairpin loops consisting of single adenine residues closed by sheared A.A and G.G pairs formed by the DNA triplets AAA and GAG: solution structure of the d(GTACAAAGTAC) hairpin.

The DNA undecamers GTACAAAGTAC (AAA 11-mer) and GTACGAGGTAC (GAG 11-mer) have been studied in solution by high-resolution NMR spectroscopy. Both duplexes form stable hairpins containing single deoxyadenosine loops and stems containing five base-pairs that are closed at the loop end by sheared AxA and GxC pairs, respectively. These molecules thus contain new AAA and GAG loop turn motifs. All protons, including the chiral H5'/H5" protons of the loop residues, were assigned using NOESY, DQF-COSY and heteronuclear 1H-31P COSY experiments. The backbone torsion angles were constrained using experimental data from NOE crosspeaks, three-bond 1H-1H coupling constants and four-bond 1H-31P coupling constants and four-bond 1H-31P coupling constants. The AAA and GAG 11-mers form similar structures in solution. The detailed structure of the AAA 11-mer was determined by the combined use of NMR, distance geometry and energy minimization methods. This structure exhibits good stacking of the loop adenosine base on the closing 5Ax7A sheared pair, with the 6A base stacking on the 5A base and the 6A deoxyribose stacking with the 7A base. All sugars in the AAA 11-mer hairpin adopt the typical DNA C2'-endo conformation and a sharp backbone turn occurs between residues 6A and 7A. This loop turn is brought about mainly by a change in the backbone phosphate torsion angles from zeta(g-) alpha(g-) to zeta(g+) alphat(g+) at the turn. The gamma torsion angle of residue 7A in the closing sheared pair also changes from gauche+ to trans. In Pu1NPu2 loop turns of the GCA, AAA and GAG types, the chemical shift of the H4' proton of the loop deoxyribose depends on the nature of Pu2; this reflects the stacking of the loop sugar on the Pu2 base and the different ring current effects of A or G in this position.

Adenine↗
PubMed1999

Evidence for an RNA pseudoknot loop-helix interaction essential for efficient -1 ribosomal frameshifting.

RNA pseudoknots are structural elements that participate in a variety of biological processes. At -1 ribosomal frameshifting sites, several types of pseudoknot have been identified which differ in their organisation and functionality. The pseudoknot found in infectious bronchitis virus (IBV) is typical of those that possess a long stem 1 of 11-12 bp and a long loop 2 (30-164 nt). A second group of pseudoknots are distinguishable that contain stems of only 5 to 7 bp and shorter loops. The NMR structure of one such pseudoknot, that of mouse mammary tumor virus (MMTV), has revealed that it is kinked at the stem 1-stem 2 junction, and that this kinked conformation is essential for efficient frameshifting. We recently investigated the effect on frameshifting of modulating stem 1 length and stability in IBV-based pseudoknots, and found that a stem 1 with at least 11 bp was needed for efficient frameshifting. Here, we describe the sequence manipulations that are necessary to bypass the requirement for an 11 bp stem 1 and to convert a short non-functional IBV-derived pseudoknot into a highly efficient, kinked frameshifter pseudoknot. Simple insertion of an adenine residue at the stem 1-stem 2 junction (an essential feature of a kinked pseudoknot) was not sufficient to create a functional pseudoknot. An additional change was needed: efficient frameshifting was recovered only when the last nucleotide of loop 2 was changed from a G to an A. The requirement for an A at the end of loop 2 is consistent with a loop-helix contact similar to those described in other RNA tertiary structures. A mutational analysis of both partners of the proposed interaction, the loop 2 terminal adenine residue and two G.C pairs near the top of stem 1, revealed that the interaction was essential for efficient frameshifting. The specific requirement for a 3'-terminal A residue was lost when loop 2 was increased from 8 to 14 nt, suggesting that the loop-helix contact may be required only in those pseudoknots with a short loop 2.

Base Sequence↗
PubMed2001

Structure and dimerization of HIV-1 kissing loop aptamers.

Dimerization of two homologous strands of genomic RNA is an essential feature of the retroviral replication cycle. In HIV-1, genomic RNA dimerization is facilitated by a conserved stem-loop structure located near the 5' end of the viral RNA called the dimerization initiation site (DIS). The DIS loop is comprised of nine nucleotides, six of which define an autocomplementary sequence flanked by three conserved purine residues. Base- pairing between the loop sequences of two copies of genomic RNA is necessary for efficient dimerization. We previously used in vitro evolution to investigate a possible structural basis for the marked sequence conservation of the DIS loop. In this study, chemical structure probing, measurements of the apparent dissociation constants, and computer structure analysis of dimerization-competent aptamers were used to analyze the dimers' structure and binding. The selected aptamers were variants of the naturally occurring A and B subtypes. The data suggest that a sheared base-pair closing the loop of the DIS is important for dimerization in both subtypes. On the other hand, the open or closed state of the last base-pair in the stem differed in the two subtypes. This base-pair appeared closed in the subtype A DIS dimer and open in subtype B. Finally, evidence for a cross-talk between nucleotides 2, 5, and 6 was found in some, but not all, loop contexts, indicating some structural plasticity depending on loop sequence. Discriminating between the general rules governing dimer formation and the particular characteristics of individual DIS aptamers helps to explain the affinity and specificity of loop-loop interactions and could provide the basis for development of drugs targeted against the dimerization step during retroviral replication.

Base Pairing↗
PubMed2002

Stiffness of the distal loop restricts the structural heterogeneity of the transition state ensemble in SH3 domains.

Protein engineering experiments and Phi(F)-value analysis of SH3 domains reveal that their transition state ensemble (TSE) is conformationally restricted, i.e. the fluctuations in the transition state (TS) structures are small. In the TS of src SH3 and alpha-spectrin SH3 the distal loop and the associated hairpin are fully structured, while the rest of the protein is relatively disordered. If native structure predominantly determines the folding mechanism, the findings for SH3 folds raise the question: What are the features of the native topology that determine the nature of the TSE? We propose that the presence of stiff loops in the native state that connect local structural elements (such as the distal hairpin in SH3 domains) conformationally restricts TSE. We validate this hypothesis using the simulations of a "control" system (16 residue beta-hairpin forming C-terminal fragment of the GBl protein) and its variants. In these fragments the role of bending rigidity in determining the nature of the TSE can be directly examined without complications arising from interactions with the rest of the protein. The TSE structures in the beta-hairpins are determined computationally using cluster analysis and limited Phi(F)-value analysis. Both techniques prove that the conformational heterogeneity decreases as the bending rigidity of the loop increases. To extend this finding to SH3 domains a measure of bending rigidity based on loop curvature, which utilizes native structures in the Protein Data Bank (PDB), is introduced. Using this measure we show that, with few exceptions, the ordering of stiffness of the distal, n-src, and RT loops in the 29 PDB structures of SH3 domains is conserved. Combining the simulation results for beta-hairpins and the analysis of PDB structures for SH3 domains, we propose that the stiff distal loop restricts the conformational fluctuations in the TSE. We also predict that constraining the distal loop to be preformed in the denatured ensemble should not alter the nature of TSE. On the other hand, if the amino and carboxy terminals are cross-linked to form a circular polypeptide chain, the pathways and TSs are altered. These contrasting scenarios are illustrated using simulations of cross-linked WT beta-hairpin fragments. Computations of bending rigidities for immunoglobulin-like domain proteins reveal no clear separation in the stiffness of their loops. In the beta-sandwich proteins, which have large fractions of non-local native contacts, the nature of the TSE cannot be apparently determined using purely local structural characteristics. Nevertheless, the measure of loop stiffness still provides qualitative predictions of the ordered regions in the TSE of Ig27 and TenFn3.

Amino Acid Sequence↗
PubMed1998

An alternative way of thinking about stem-loops in DNA. A case study of the human G0S2 gene.

Single strands extruded from duplex DNA have the potential to form stem-loop structures, which may be involved in the homology search preceding recombination. The total stem-loop potential in a sequence window can be analysed in terms of the relative contributions of base composition and base order. There are at least 10 base composition-determined parameters of relevance to the energetics of stem-loop formation. These are the quantities of the four bases themselves, and six derived parameters: ATmin, CGmin, Chargaff differences for the W and S bases, and two base products. The quantities of the least represented base of a Watson-Crick base pair (ATmin, CGmin) might provide an index of the total stem potential of a widow. The degrees to which one base of a Watson-Crick pair exceeds the other (the Chargaff differences for the W bases and for the S bases) might provide an index of the total loop potential of a window. Base products (A x T, C x G) might provide an index both of stem and of loop potentials. Multiple regression analysis of the relationship of the 10 parameters to the energetics of stem-loop formation in the G0S2 gene reveals major roles of S bases, and of base products. While base composition may primarily serve genome or genome sector "strategies", it becomes of local relevance in the case of CpG islands. Base order serves many local "strategies", whose demands may conflict. Base order serves the encoding of protein or of recognition motifs for regulatory factors. On the other hand there appear to be circumstances under which base order synergizes with, or antagonises, base composition in determining total stem-loop potential. Antagonism is evident when the base composition-dependent component of the stem-loop potential of a region is greater than the total stem-loop potential of that region.

DNA↗
PubMed1990

Transcription of a satellite DNA on two Y chromosome loops of Drosophila melanogaster.

Primary spermatocyte nuclei of Drosophila melanogaster exhibit three giant lampbrush-like loops formed by the kl-5, kl-3 and ks-1 Y chromosome fertility factors. Detailed mapping of satellite DNA sequences along the Y chromosome has recently shown that AA-GAC satellite repeats are a significant component of the kl-5 and ks-1 loop-forming regions. To determine whether these simple repeated sequences are transcribed on the loop structures we performed a series of DNA-RNA in situ hybridization experiments to fixed loop preparations using as a probe cloned AAGAC repeats. These experiments showed that the probe hybridizes with homologous transcripts specifically associated with the kl-5 and ks-1 loops. These transcripts are detected at all stages of development of these two loops, do not appear to migrate to the cytoplasm and are degraded when loops disintegrate during the first meiotic prophase. Moreover, an examination of the testes revealed that the transcription of the AAGAC sequences is restricted to the loops of primary spermatocytes; the other cell types of D. melanogaster spermatogenesis do not exhibit nuclear or cytoplasmic labeling. These experiments were confirmed by RNA blotting analysis which showed that transcription of the AAGAC sequences occurs in wild-type testes but not in X/O testes. The patterns of hybridization to the RNA blots indicated that the transcripts are highly heterogeneous in size, from large (migration at limiting mobility) to less than 1 kb. We discuss the possible function of the AAGAC satellite transcripts, in the light of the available information on the Y chromosome loops of D. melanogaster.

Animals↗
PubMed1995

Analysis of the loop-helix interaction in bundle motif protein structures.

Molecular dynamics simulations and energy analysis have been carried out to study the structural mobility and stability of the four alpha-helix bundle motifs. The simulation results as well as the X-ray data show that the atomic RMS fluctuation is larger at the loop region for four representative proteins investigated: methemerythrin, cytochrome b-562, cytochrome c', and bovine somatotropin. The loop-loop, helix-helix, and loop-helix interactions are computed for the unfolded and folded proteins. In the folded and solvated protein structures the loop-helix interaction is stronger than the helix-helix interaction, especially in the electrostatic component. But the stabilization energies of both the loop-helix and the helix-helix interactions relative to those of an unfolded structure are of the same order of magnitude. The stabilization due to protein-solvent interaction is greater in the helix region than in the loop region. The percentage of hydrophilic solvent accessible area for the four proteins studied was calculated with the method of Eisenberg and McLachlan. The percentage of the hydrophilic area is greater in the loops than in the helices. A Poisson-Boltzmann calculation shows that the potential from the loops acting on a helix is generally more negative than that from other helices.

Animals↗
PubMed2001

Novel technique to average breathing loops for infant respiratory function testing.

Breathing loops can be obtained by plotting two respiratory signals on an x-y diagram: the resulting loops represent a non-parametric description of the respiratory system. In infancy, loops are commonly measured during tidal breathing and their interpretation is hampered by high within-subject variability. Therefore a two-dimensional averaging technique for loops has been developed. The algorithm is based on segmentation of the loops and required two steps. First, the total length of the loop of every breathing cycle was divided into a specified number of equidistant intervals and the co-ordinates calculated by stepwise linear approximation of the curve. Second, averaged loops were calculated using the arithmetical mean (or the median if there were artifacts) of the x and y co-ordinates of the loops for all calculated points. To compare the new technique with averaging in the time domain a simulation study was performed using respiratory signals with a coefficient of variation (CV) of 5%, 10%, 15% and 20%. In contrast to the new technique, with increasing CV, averaging in the time domain led to increasing contortions in the averaged flow-volume loops. Mean errors of peak tidal expiratory flow were -3.3%, -13.9%, -21.3% and -34.2%, whereas errors with the new technique were considerably lower (0.5%, 0.4%, 0.5% and -0.1%) and independent of the level of CV.

Algorithms↗
PubMed1998

Loop diuretics in patients aged 75 years or older: general practitioners' assessment of indications and possibilities for withdrawal.

OBJECTIVE: To examine the indications for prescription and possibilities for withdrawal of loop diuretics in community-dwelling patients aged 75 years or older, as reported by their general practitioners (GPs). METHODS: Analysis of dispensary data to identify patients aged 75 years or older using loop diuretics on 1 February 1996. Questionnaires were sent to the GPs of these patients to inquire about the indications for loop diuretic prescription and the necessity for continuation. We subsequently determined loop diuretic prescription rates in the survey population over the next 9 months. SETTING: A panel of nine Dutch community pharmacies. RESULTS: Questionnaires were returned for 338 out of 667 patients (50.7%) using loop diuretics on 1 February 1996. Reported indications for loop diuretic use were heart failure in 223 patients (66.0%), hypertension in 35 patients (10.4%), and a combination of both in 23 patients (6.8%). Loop diuretics were used for ankle edema in 27 patients (8.0%) and for unknown reasons in another 27 patients (8.0%). Continuation of treatment was considered unnecessary by GPs in 66 out of 338 patients (19.5%). However, prescription rates for these 66 patients in the following months were no different from rates for the remaining 272 patients. Loop diuretics were still prescribed to 47 of 66 patients (71.2%) after 12 weeks, and to 26 patients (39.4%) after 36 weeks. CONCLUSIONS: GPs reported substantial opportunities for withdrawal of loop diuretics in patients over 75 years of age, but this did not influence actual prescription rates in these patients. Future studies should explore means of facilitating withdrawal of these medications in this population.

Age Factors↗