The 5S rRNA gene in sea barley (Hordeum marinum Hudson sensu lato): sequence variation among repeat units and relationship to the X haplome in barley (Hordeum)

We have investigated the molecular diversity of the 5S rDNA units in sea barley, comprising Hordeum marinum and Hordeum geniculatum. Although we were unable to detect "short" units after screening of 639 clones, we found two unit classes, one 602-607 bp long and the other 507-512 bp long. We classify the shortest unit class of the two as belonging to the "long H1" unit class, identified in previous papers. The longest unit class is not similar to any unit class so far identified, and is therefore unique. It was coined by us as the "long X1," to reflect the X haplome. We present a summary of all the unit classes so far described in Hordeum. We carried out a cladistic analysis, based on the "long H1" (orthologous) sequences, that included H. vulgare, H. spontaneum, H. bulbosum, H. marinum, H. geniculatum, and H. bogdanii. As a result, the first three grouped in one clade, and the other three in the other clade, with the latter clade being more isolated. These results reflect current knowledge of relationships based on morphology, cytology, and genome analysis. Furthermore, the sequences from the 5S unit classes may be potentially useful as DNA probes for genomic identification and genetic transfer in the Triticeae.     

Analysis of a kanamycin resistance gene (kmr) from Streptomyces kanamyceticus and a mutant with increased aminoglycoside resistance

The tyrosine phosphatase IA-2 is a molecular target of pancreatic islet autoimmunity in type 1 diabetes. T-cell epitope peptides in autoantigens have potential diagnostic and therapeutic applications, and they may hold clues to environmental agents with similar sequences that could trigger or exacerbate autoimmune disease. We identified 13 epitope peptides in IA-2 by measuring peripheral blood T-cell proliferation to 68 overlapping, synthetic peptides encompassing the intracytoplasmic domain of IA-2 in six at-risk type 1 diabetes relatives selected for HLA susceptibility haplotypes. The dominant epitope, VIVMLTPLVEDGVKQC (aa 805-820), which elicited the highest T-cell responses in all at-risk relatives, has 56% identity and 100% similarity over 9 amino acids (aa) with a sequence in VP7, a major immunogenic protein of human rotavirus. Both peptides bind to HLA-DR4(*0401) and are deduced to present identical aa to the T-cell receptor. The contiguous sequence of VP7 has 75% identity and 92% similarity over 12 aa with a known T-cell epitope in glutamic acid decarboxylase (GAD), another autoantigen in type 1 diabetes. This dominant IA-2 epitope peptide also has 75-45% identity and 88-64% similarity over 8-14 aa to sequences in Dengue, cytomegalovirus, measles, hepatitis C, and canine distemper viruses, and the bacterium Haemophilus influenzae. Three other IA-2 epitope peptides are 71-100% similar over 7-12 aa to herpes, rhino-, hanta- and flaviviruses. Two others are 80-82% similar over 10-11 aa to sequences in milk, wheat, and bean proteins. Further studies should now be carried out to directly test the hypothesis that T-cell activation by rotavirus and possibly other viruses, and dietary proteins, could trigger or exacerbate beta-cell autoimmunity through molecular mimicry with IA-2 and (for rotavirus) GAD.     

Analysis of xylem formation in pine by cDNA sequencing

Secondary xylem (wood) formation is likely to involve some genes expressed rarely or not at all in herbaceous plants. Moreover, environmental and developmental stimuli influence secondary xylem differentiation, producing morphological and chemical changes in wood. To increase our understanding of xylem formation, and to provide material for comparative analysis of gymnosperm and angiosperm sequences, ESTs were obtained from immature xylem of loblolly pine (Pinus taeda L.). A total of 1,097 single-pass sequences were obtained from 5' ends of cDNAs made from gravistimulated tissue from bent trees. Cluster analysis detected 107 groups of similar sequences, ranging in size from 2 to 20 sequences. A total of 361 sequences fell into these groups, whereas 736 sequences were unique. About 55% of the pine EST sequences show similarity to previously described sequences in public databases. About 10% of the recognized genes encode factors involved in cell wall formation. Sequences similar to cell wall proteins, most known lignin biosynthetic enzymes, and several enzymes of carbohydrate metabolism were found. A number of putative regulatory proteins also are represented. Expression patterns of several of these genes were studied in various tissues and organs of pine. Sequencing novel genes expressed during xylem formation will provide a powerful means of identifying mechanisms controlling this important differentiation pathway.     

Assembly of physalis mottle virus capsid protein in Escherichia coli and the role of amino and carboxy termini in the formation of the icosahedral particles

The coat protein gene of physalis mottle tymovirus (PhMV) was over expressed in Escherichia coli using pET-3d vector. The recombinant protein was found to self assemble into capsids in vivo. The purified recombinant capsids had an apparent s value of 56.5 S and a diameter of 29(+/-2) nm. In order to establish the role of amino and carboxy-terminal regions in capsid assembly, two amino-terminal deletions clones lacking the first 11 and 26 amino acid residues and two carboxy-terminal deletions lacking the last five and ten amino acid residues were constructed and overexpressed. The proteins lacking N-terminal 11 (PhCPN1) and 26 (PhCPN2) amino acid residues self assembled into T=3 capsids in vivo, as evident from electron microscopy, ultracentrifugation and agarose gel electrophoresis. The recombinant, PhCPN1 and PhCPN2 capsids were as stable as the empty capsids formed in vivo and encapsidated a small amount of mRNA. The monoclonal antibody PA3B2, which recognizes the epitope within region 22 to 36, failed to react with PhCPN2 capsids while it recognized the recombinant and PhCPN1 capsids. Disassembly of the capsids upon treatment with urea showed that PhCPN2 capsids were most stable. These results demonstrate that the N-terminal 26 amino acid residues are not essential for T=3 capsid assembly in PhMV. In contrast, both the proteins lacking the C-terminal five and ten amino acid residues were present only in the insoluble fraction and could not assemble into capsids, suggesting that these residues are crucial for folding and assembly of the particles.     

Complicated cataracts in various forms of retinitis pigmentosa. Type and incidence

PURPOSE: To study the incidence and types of cataract in retinitis pigmentosa (RP) and their variations among different forms of RP. PATIENTS AND METHODS: This analysis was based on data from 473 patients with RP (autosomal dominant, n = 87; autosomal recessive, n = 79; x chromosomal recessive, n = 23; simplex RP, n = 215; Usher's syndrome n = 80; M. Refsum and others, n = 9) that were retrieved from the literature and patient charts in our clinic. RESULTS: Posterior subcapsular cataract (PSC) developed with the following frequencies for the different genetic types of RP: autosomal dominant, 45.3%; autosomal recessive, 44.0%; x chromosomal recessive, 40.7%; simplex RP, 46.1%; Usher's syndrome, 52.9%. PSC was the only type of lens opacity in patients with Usher's syndrome and autosomal recessive RP.PSC development correlated with early onset of RP symptoms. Nuclear cataracts showed a statistically significant higher frequency in patients with simplex RP (14.8%) than in other genetic types (0-5.9%) (P < 0.01). In addition, nuclear cataracts developed in simplex RP at a significantly later age (69.6 +/- 12.4 years) than PSC (44.4 +/- 12.3 years) (P < 0.001). Patients with cataracts showed significantly worse visual fields than patients with clear lenses (P = 0.00067). CONCLUSIONS: The typical RP cataract (PSC) was found in similar frequencies among all genetic types of RP.PSC was the only type of lens opacity in patients with Usher's syndrome and autosomal recessive RP. Nuclear cataracts developed on average 20 years later than PSC and had their highest incidence in patients with simplex RP. Patients with cataracts showed significantly worse visual field results, indicating a more pronounced retinal pathology.     

Gradual and rapid weight loss: effects on nutrition and performance in male athletes

We studied seven male wrestlers and three judo athletes (weight 55-93 kg) during two weight reductions. In the "gradual" procedure (GP), a 5.0 +/- 0.4% (mean +/- SEM) weight loss was achieved in 3 weeks by energy restriction. In the "rapid" procedure (RP), 6.0 +/- 0.6% of body weight was lost in 2.4 days by fluid and diet restriction and forced sweating, and followed by a 5-h "loading" (food and drinks ad libitum). The net weight loss after GP and loading was 2.7 +/- 0.5%. Protein intakes (4-d food records) during GP and RP were 71 +/- 16 and RP 56 +/- 17 g.d-1, respectively. Carbohydrate intakes were 239 +/- 56 (GP) and 182 +/- 55 g.d-1 (RP). During GP and RP, mean thiamin, magnesium, and zinc intakes were at or below the respective recommendation. Thiamin, riboflavin, potassium, iron, and zinc status, assessed from blood chemistry, remained stable during both procedures. Changes in vitamin B6 indicator (E-ASTAC) and S-magnesium concentration were different (P < 0.01) between the procedures, suggesting negative trends during GP. Sprint (30-m run) and anaerobic (1-min Wingate test) performance was similar throughout the study. Following GP, vertical jump height with extra load increased by 6-8% (P < 0.01). Jumping results were not affected by RP. Hence, < or = 5% loss in body weight by either method did not impair experienced athletes' performance.     

A helix-turn-helix structure unit in human centromere protein B (CENP-B)

CENP-B has been suggested to organize arrays of centromere satellite DNA into a higher order structure which then directs centromere formation and kinetochore assembly in mammalian chromosomes. The N-terminal portion of CENP-B is a 15 kDa DNA binding domain (DBD) consisting of two repeating units, RP1 and RP2. The DBD specifically binds to the CENP-B box sequence (17 bp) in centromere DNA. We determined the solution structure of human CENP-B DBD RP1 by multi-dimensional 1H, 13C and 15N NMR methods. The CENP-B DBD RP1 structure consists of four helices and has a helix-turn-helix structure. The overall folding is similar to those of some other eukaryotic DBDs, although significant sequence homology with these proteins was not found. The DBD of yeast RAP1, a telomere binding protein, is most similar to CENP-B DBD RP1. We studied the interaction between CENP-B DBD RP1 and the CENP-B box by the use of NMR chemical shift perturbation. The results suggest that CENP-B DBD RP1 interacts with one of the essential regions of the CENP-B box DNA, mainly at the N-terminal basic region, the N-terminal portion of helix 2 and helix 3.     

Group counseling versus individualized relapse prevention aftercare following intensive outpatient treatment for cocaine dependence: Initial results.

Ninety-eight male cocaine-dependent patients who completed an intensive outpatient program (IOP) were randomly assigned to either standard group counseling (STND) or individualized relapse prevention (RP) aftercare. Heavier cocaine and alcohol use during IOP and low self-efficacy predicted more cocaine use during the treatment phase of the study, whereas lifetime diagnoses of alcohol dependence, major depression, and any anxiety disorder predicted less cocaine use. Rates of complete abstinence during the 6-month study period were higher in STND than RP, whereas RP was more effective in limiting the extent of cocaine use in those who used during Months 1-3. Matching analyses indicated patients who failed to achieve remission from cocaine dependence during IOP and those with a commitment to absolute abstinence did better in RP than in STND, whereas patients with other abstinence goals did better in STND than RP. Several differences in experiences before cocaine use and "near-miss" episodes were also identified. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Secretory response to cholera toxin in the porcine jejunum under different types of general anaesthesia

Both classical pancreatic lipase (DPL) and pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by dog exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with dog PLRP1 on any of the substrates tested: di- and tri-glycerides, phospholipids, etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.10 A. Its structure is similar to that of the classical PL structures in the absence of any inhibitors or micelles. The lid domain that controls the access to the active site was found to have a closed conformation. An amino-acid substitution (Ala 178 Val) in the DPLRP1 may result in a steric clash with one of the acyl chains observed in the structures of a C11 alkyl phosphonate inhibitor, a transition state analogue, bound to the classical PL. This substitution was suspected of being responsible for the absence of DPLRP1 activity. The presence of Val and Ala residues in positions 178 and 180, respectively, are characteristic of all the known PLRP1, whereas Ala and Pro residues are always present in the same positions in all the other members of the PL gene family. Introducing the double mutation Val 178 Ala and Ala 180 Pro into the human pancreatic RP1 (HPLRP1) gene yielded a well expressed and folded enzyme in insect cells. This enzyme is kinetically active on triglycerides. Our findings on DPLRP1 and HPLRP1 are therefore likely to apply to all the RP1 lipases.     

Heat shock protein 70 family: multiple sequence comparisons, function, and evolution

The heat shock protein 70 kDa sequences (HSP70) are of great importance as molecular chaperones in protein folding and transport. They are abundant under conditions of cellular stress. They are highly conserved in all domains of life: Archaea, eubacteria, eukaryotes, and organelles (mitochondria, chloroplasts). A multiple alignment of a large collection of these sequences was obtained employing our symmetric-iterative ITERALIGN program (Brocchieri and Karlin 1998). Assessments of conservation are interpreted in evolutionary terms and with respect to functional implications. Many archaeal sequences (methanogens and halophiles) tend to align best with the Gram-positive sequences. These two groups also miss a signature segment [about 25 amino acids (aa) long] present in all other HSP70 species (Gupta and Golding 1993). We observed a second signature sequence of about 4 aa absent from all eukaryotic homologues, significantly aligned in all prokaryotic sequences. Consensus sequences were developed for eight groups [Archaea, Gram-positive, proteobacterial Gram-negative, singular bacteria, mitochondria, plastids, eukaryotic endoplasmic reticulum (ER) isoforms, eukaryotic cytoplasmic isoforms]. All group consensus comparisons tend to summarize better the alignments than do the individual sequence comparisons. The global individual consensus "matches" 87% with the consensus of consensuses sequence. A functional analysis of the global consensus identifies a (new) highly significant mixed charge cluster proximal to the carboxyl terminus of the sequence highlighting the hypercharge run EEDKKRRER (one-letter aa code used). The individual Archaea and Gram-positive sequences contain a corresponding significant mixed charge cluster in the location of the charge cluster of the consensus sequence. In contrast, the four Gram-negative proteobacterial sequences of the alignment do not have a charge cluster (even at the 5% significance level). All eukaryotic HSP70 sequences have the analogous charge cluster. Strikingly, several of the eukaryotic isoforms show multiple mixed charged clusters. These clusters were interpreted with supporting data related to HSP70 activity in facilitating chaperone, transport, and secretion function. We observed that the consensus contains only a single tryptophan residue and a single conserved cysteine. This is interpreted with respect to the target rule for disaggregating misfolded proteins. The mitochondrial HSP70 connections to bacterial HSP70 are analyzed, suggesting a polyphyletic split of Trypanosoma and Leishmania protist mitochondrial (Mt) homologues separated from Mt-animal/fungal/plant homologues. Moreover, the HSP70 sequences from the amitochondrial Entamoeba histolytica and Trichomonas vaginalis species were analyzed. The E. histolytica HSP70 is most similar to the higher eukaryotic cytoplasmic sequences, with significantly weaker alignments to ER sequences and much diminished matching to all eubacterial, mitochondrial, and chloroplast sequences. This appears to be at variance with the hypothesis that E. histolytica rather recently lost its mitochondrial organelle. T. vaginalis contains two HSP70 sequences, one Mt-like and the second similar to eukaryotic cytoplasmic sequences suggesting two diverse origins.     

Human extracellular proteins display a different pattern of local sequence similarity with the four classes of human T-cell receptor V-regions than foreign proteins and human intracellular proteins: a preliminary report

A pool of 110 randomly selected/generated amino acids sequences was used to perform specific local sequence similarity alignment analysis with the pool of 279 reported sequences of human T-cell receptor (TCR) V-regions. The 110 analyzed sequences were divided, according to their origin and nature, into six protein groups, as: human intracellular (hi), extracellular/transmembrane (he) and extracellular adhesive matrix (ha) proteins, 'average' human proteins (hum), proteins of non-human origin (nhum) and randomly generated quasi-protein sequences (r). These sequences were decomposed into all their overlapping 11-mer segments, generating a total of 56,836 derived peptides (at least 8000 per group). Each derived peptide was aligned with the 279 human TCR V-regions and assigned to the category (alpha-like, beta-like, gamma-like or delta-like) corresponding to the class (V alpha, Vbeta, Vgamma or Vdelta) of the V-region encompassing the most similar segment, as determined by the performed similarity-search. The six protein groups were found to differ significantly in their distribution of derived peptides among the four categories. According to the binomial tests results, human proteins from the extracellular compartment (he, ha) comprise a higher proportion of delta-like segments (P = 2.3 x 10(-2) and P < 10(-8), respectively) than the 'average' human proteins (hum). In addition, and in accordance with this finding, proteins that are normally not found in that topological compartment comprise a lower proportion of delta-like peptides (P = 1.4 x 10(-5) and P < 10(-8) for groups nhum and hi, respectively) than the 'average' human proteins (hum). In contrast, these proteins comprise a higher proportion of gamma-like segments (P = 8.3 x 10(-3), P = 1.4 x 10(-3) and P = 1.7 x 10(-4), for groups r, nhum and hi, respectively) than the 'average' human proteins (hum). These findings indicate significant differences between proteins encountered in the extracellular compartment--that are normally immunologically tolerated--and those the presence of which is usually non-tolerated. The results suggest that the discrimination and the reaction of the human immune network to proteins found in the extracellular compartment correlate with the proteins' pattern of preferential local sequence similarity with the Vgamma and Vdelta classes of human TCR V-regions, implying a specific and an important role of gammadelta-cells in the maintenance of the immune homeostasis. Whether this implication represents a rule associated with self-tolerance, will be investigated by future analyses.     

Reduction of allogeneic blood transfusions after open heart operations by lowering cardiopulmonary bypass prime volume

BACKGROUND: Despite recent advances in blood conservation techniques, up to 30% to 80% of patients undergoing open heart operations require allogeneic blood transfusions. A prospective, randomized study was performed to test the effect of lowering cardiopulmonary bypass prime volume (as an additional component of an integrated blood conservation strategy) on clinical outcome and allogeneic blood transfusion. METHODS: One hundred fourteen patients undergoing open heart operations were randomized to either full prime (FP) volume (1,400 mL of Plasmalyte solution) or reduced prime (RP) volume (600 to 800 mL). The reduction of prime volume was achieved by slowly draining the cardiopulmonary bypass circuit into a cell-saving device before the initiation of bypass. Firm transfusion thresholds were observed. RESULTS: There were no significant differences between the groups with respect to baseline characteristics, body surface area, type and urgency of the procedures, perfusion technique, and hematologic profile. Mortality (FP, 1.7%; RP, 0%; p approximately 1.0) and overall morbidity (FP, 28.1%; RP, 22.8%; p = 0.53) were similar. However, transfusion requirements were significantly lower in the RP group: total donor exposure, 3.8 +/- 10.1 versus 1.0 +/- 2.4 units (p = 0.044); percentage of patients transfused, 54% (n = 31) versus 35% (n = 20) (p = 0.036). Twenty-four-hour chest tube drainage was similar: 455 +/- 223 mL for FP versus 472 +/- 173 mL for RP (p = 0.66). The lowest hematocrit on bypass was significantly higher in the RP group: 29.3% +/- 4% versus 26.3% +/- 5.3% (p = 0.009). CONCLUSIONS: Lowering cardiopulmonary bypass prime volume resulted in a significant decrease in allogeneic blood product use. Because postoperative 24-hour chest tube drainage was similar in both groups, and hematocrit during bypass was higher in the RP group, the reduction in allogeneic blood transfusions appears to be related to a decrease in prime-induced hemodilution. This technique is effective, simple, and safe. It therefore should be strongly considered for patients undergoing operations using normothermic or near-normothermic cardiopulmonary bypass who are at high risk for allogeneic blood transfusion.     

Designing amino acid sequences to fold with good hydrophobic cores

We present two methods for designing amino acid sequences of proteins that will fold to have good hydrophobic cores. Given the coordinates of the desired target protein or polymer structure, the methods generate sequences of hydrophobic (H) and polar (P) monomers that are intended to fold to these structures. One method designs hydrophobic inside, polar outside; the other minimizes an energy function in a sequence evolution process. The sequences generated by these methods agree at the level of 60-80% of the sequence positions in 20 proteins in the Protein Data Bank. A major challenge in protein design is to create sequences that can fold uniquely, i.e. to a single conformation rather than to many. While an earlier lattice-based sequence evolution method was shown not to design unique folders, our method generates unique folders in lattice model tests. These methods may also be useful in designing other types of foldable polymer not based on amino acids.     

Mental effort required for walking: effects of retinitis pigmentosa

PURPOSE: To determine whether retinitis pigmentosa (RP) increases the mental effort required for walking. METHODS: A dual-task methodology was used; walking speed and reaction time (RT) to randomly emitted tones were measured in 13 persons with RP and 29 persons with normal vision. Measures of RT were used to estimate the mental effort required for walking. In a second experiment, 15 persons with RP and 17 persons with normal vision navigated a "simple" and a "complex" route. RESULTS: The RP subjects had longer RT compared with the normal-vision subjects when walking the complex but not the simple route. Normalized to each person's baseline, RT while walking the complex route was significantly correlated with log contrast sensitivity (r = -0.63) and log retinal area (r = -0.64) in the RP group. The amount of mental effort required for walking was shown to also depend on familiarity with the route. CONCLUSIONS: Route complexity is critical in determining whether walking requires more mental effort for persons with RP than for persons with normal vision. The magnitude of mental effort required for mobility covaries with the visual impairment measures which correlate with mobility performance in RP.     

Mimicry between the hepatitis B virus DNA polymerase and the antigenic targets of nuclear and smooth muscle antibodies in chronic hepatitis B virus infection

Autoantibodies to nuclear and smooth muscle are common in hepatitis B virus (HBV) infection. To understand their origin, we scanned protein databases and found that HBV-DNA polymerase (HBV-pol) shares 7-9 amino acid sequences with nuclear (MHC II trans-activator, nuclear pore core protein, nuclear mitotic apparatus, and polymyositis sclerosis Ag) and smooth muscle proteins (caldesmon and myosin). Twenty-mer peptides with relevant homologues and an irrelevant control peptide were constructed and ELISAs were established. Sixty-five children with chronic HBV infection, 104 patients with other chronic liver diseases (CLD), 36 patients with extrahepatic autoimmune diseases, and 24 healthy controls were investigated. Double reactivity to HBV-pol peptides and corresponding self homologues was observed in 40% of HBV-positive patients as compared with four (4%) with other chronic liver diseases, two (6%) with extrahepatic autoimmune diseases, and in none of the healthy controls (p < 0.001 for all). Double reactivity to myosin or caldesmon peptides and their HBV-pol homologues was associated with anti-smooth muscle Ab positivity by immunofluorescence (p < 0.05 for both). HBV-positive sera double reactive for myosin or caldesmon and their homologous HBV-pol peptides also reacted with the native proteins on immunoblot. Fifty to ninety percent Ab inhibition to individual HBV-pol and HBV-pol99-118 peptides was noted by preincubation with individual HBV-pol/self homologue peptide and native proteins, respectively, but not with control peptide. Our results show that cross-reactive immunity targeting homologous sequences of viral and self proteins may partly account for autoantibody production in HBV infection.     

Effects of RP 73401, a novel, potent and selective phosphodiesterase type 4 inhibitor, on contractility of human, isolated bronchial muscle

1. The aim of this study was to investigate the smooth muscle relaxant effects of the novel, selective phosphodiesterase (PDE) type 4 inhibitor, RP 73401 in comparison with the classical PDE 4 inhibitor, rolipram, the non-selective PDE inhibitor, theophylline and the beta-adrenoceptor agonist, isoprenaline on the human, isolated bronchus. 2. At resting tone, the rank order of potency (pD2) for the relaxants was RP 73401 > or = rolipram > or = isoprenaline > theophylline. In terms of maximum relaxation produced (Emax) the PDE 4-selective inhibitors were similar, but the maximal effects (70-75% of theophylline, 3 mM) were lower than that observed with isoprenaline (98% of theophylline, 3 mM) or theophylline itself (100%). 3. On the human isolated bronchus pre-contracted with acetylcholine (ACh, 0.1 or 1.0 mM), the rank order of potency remained the same. The maximal responses to RP 73401 and rolipram were however markedly reduced (Emax 39.9-46.6%) compared with isoprenaline (Emax 79-85%). 4. In tissues pre-contracted with ACh (0.1 mM), RP 73401 and rolipram (10(-9)-10(-7) M) significantly and concentration-dependently increased tissue sensitivity to isoprenaline. RP 73401 and rolipram were similar in potency. Both selective PDE 4 inhibitors also significantly increased the maximal relaxant effects of isoprenaline. These effects were not observed with the PDE 3 inhibitor, siguazodan. 5. In terms of retention by tissues (an index of duration of action), the onset of action of RP 73401 (2.11 +/- 0.53 min) and rolipram (1.70 +/- 0.45 min) was significantly slower than that of isoprenaline (0.33 +/- 0.06 min) or theophylline (1.17 +/- 0.25 min). The retention of RP 73401 (89.0 +/- 21.9 min) on the human isolated bronchial tissues after washing was however dramatically longer than that of rolipram (18.3 +/- 4.5 min), theophylline (3.43 +/- 0.58 min) or isoprenaline (2.81 +/- 0.31 min). 6. These data indicate that RP 73401 is a potent and long acting relaxant of human bronchial muscle in vitro. RP 73401 is more potent than the classical PDE 4-selective inhibitor rolipram and the non-selective PDE inhibitor theophylline and is retained in bronchial tissue for a much longer period of time.