Exercise training-induced modification of the gut microbiota persists after microbiota colonization and attenuates the response to chemically-induced colitis in gnotobiotic mice.

Exercise reduces the risk of inflammatory disease by modulating a variety of tissue and cell types, including those within the gastrointestinal tract. Recent data indicates that exercise can also alter the gut microbiota, but little is known as to whether these changes affect host function. Here, we use a germ-free (GF) animal model to test whether exercise-induced modifications in the gut microbiota can directly affect host responses to microbiota colonization and chemically-induced colitis. Donor mice (n = 19) received access to a running wheel (n = 10) or remained without access (n = 9) for a period of six weeks. After euthanasia, cecal contents were pooled by activity treatment and transplanted into two separate cohorts of GF mice. Two experiments were then conducted. First, mice were euthanized five weeks after the microbiota transplant and tissues were collected for analysis. A second cohort of GF mice were colonized by donor microbiotas for four weeks before dextran-sodium-sulfate was administered to induce acute colitis, after which mice were euthanized for tissue analysis. We observed that microbial transplants from donor (exercised or control) mice led to differences in microbiota ß-diversity, metabolite profiles, colon inflammation, and body mass in recipient mice five weeks after colonization. We also demonstrate that colonization of mice with a gut microbiota from exercise-trained mice led to an attenuated response to chemical colitis, evidenced by reduced colon shortening, attenuated mucus depletion and augmented expression of cytokines involved in tissue regeneration. Exercise-induced modifications in the gut microbiota can mediate host-microbial interactions with potentially beneficial outcomes for the host.

Potential risks to freshwater aquatic organisms following a silvicultural application of herbicides in Oregon's Coast Range.

Glyphosate, aminomethylphosphonic acid (AMPA), imazapyr, sulfometuron methyl (SMM), and metsulfuron methyl (MSM) were measured in streamwater collected during and after a routine application of herbicides to a forestry site in Oregon's Coast Range. Samples were collected at 3 stations: HIGH at the fish-no-fish interface in the middle of the harvest and spray unit, MID at the bottom of the unit, and LOW downstream of the unit. All herbicides were applied by helicopter in a single tank mix. AMPA, imazapyr, SMM, and MSM were not detected (ND) in any sample at 15, 600, 500, and 1000 ng/L, respectively. A pulse of glyphosate peaking at approximately equal to 62 ng/L manifested at HIGH during the application. Glyphosate pulses peaking at 115 ng/L (MID) and 42 ng/L (HIGH) were found during the first 2 postapplication storm events 8 and 10 days after treatment (DAT), respectively: glyphosate was less than 20 ng/L (ND) at all stations during all subsequent storm events. All glyphosate pulses were short-lived (4-12 h). Glyphosate in baseflow was approximately equal to 25 ng/L at all stations 3 DAT and was still approximately equal to 25 ng/L at HIGH, but ND at the other stations, 8 DAT: subsequently, glyphosate was ND in baseflow at all stations. Aquatic organisms were subjected to multiple short-duration, low-concentration glyphosate pulses corresponding to a cumulative time-weighted average (TWA) exposure of 6634 ng/L × h. Comparisons to TWA exposures associated with a range of toxicological endpoints for sensitive aquatic organisms suggests a margin of safety exceeding 100 at the experimental site, with the only potential exception resulting from the ability of fish to detect glyphosate via olfaction. For imazapyr, SMM, and MSM the NDs were at concentrations low enough to rule out effects on all organisms other than aquatic plants, and the low concentration and (assumed) pulsed nature of any exposure should mitigate this potential. Integr Environ Assess Manag 2017;13:396-409. © 2016 SETAC.

Regulation and secretion of an extracellular esterase from Streptomyces scabies.

Production of a heat-stable, extracellular esterase by Streptomyces scabies is regulated by zinc ions. The esterase-encoding gene (est) from S. scabies was cloned and expressed in Streptomyces lividans. In S. lividans, expression of the est gene is also regulated by Zn2+, and the esterase is efficiently secreted in this organism. The sequence of the est gene suggests that a 39-amino acid signal peptide is removed during secretion of this protein. Deletion analysis has indicated that the hydrophobic domain of the signal peptide is required for secretion. Gel retardation assays and DNaseI footprinting using an S-30 protein extract from S. scabies have previously identified a specific 23-bp protein-binding site upstream from the est coding sequence. Deletion of this protein-binding sequence significantly decreased expression of the est gene.

Cloning and sequencing of a secY homolog from Streptomyces scabies.

The complete DNA sequence of the Streptomyces scabies (Ss) secY homolog and partial sequences of adjacent upstream and downstream open reading frames (ORFs) have been determined. The nucleotide sequence of a 2-kb region predicts a polypeptide of 437 amino acids in length with homology to the SecY protein family. The Ss secY homolog lies upstream from a sequence that has homology to the adenylate kinase gene (adk) family. The translational stop codon of the putative SecY ORF overlaps the predicted start codon for the Adk ORF. Another ORF that lies upstream from the secY homolog has sequence similarity to the genes that code for the L15 r-protein. Within the 243-bp intergenic region between the L15 and SecY coding sequences, the presence of a streptomycete-like promoter sequence and an 18-bp inverted repeat suggests that the secY homolog and the adjacent downstream sequences may be transcribed independently of the L15 coding sequence. Transcript analysis indicates that the secY homolog is expressed in both Ss and Streptomyces lividans. The proposed gene and transcript organization of the L15-SecY-Adk coding regions in the Ss clone resembles that of Micrococcus luteus which, like the streptomycetes, has a G+C-rich genome.

Quantitative CT predicts the severity of physiologic dysfunction in patients with lymphangioleiomyomatosis.

PURPOSE: To assess quantitative high-resolution CT (quantitative CT) as a diagnostic and prognostic tool in pulmonary lymphangioleiomyomatosis. METHODS: Spirometry, lung volumes, diffusing capacity, exercise physiology, and expiratory high-resolution CT (HRCT) examinations were performed on a cohort of ten patients with the diagnosis of lymphangioleiomyomatosis (LAM) referred to a tertiary care center. HRCT examinations were also done on ten normal control subjects. A thresholding technique was used to quantitatively assess the amount of abnormal cystic parenchyma present on each of the two images obtained for each subject with LAM and for each normal control subject. This numeric index of cystic parenchyma, the quantitative CT index, was then examined (1) as a diagnostic measure to distinguish the subjects with LAM from the normal control subjects and (2) as a prognostic measure to assess disease severity in the subjects with LAM. Linear regression of the quantitative CT index against physiologic indexes of pulmonary function and exercise performance was analyzed to determine the relationship between this radiologic assessment of disease severity and functional impairment. RESULTS: The quantitative CT index was significantly greater for the LAM patients, 37.2 +/- 6.9 (SEM), compared with the control group, 0.8 +/- 0.2 (p = 0.0001). Linear regression analysis demonstrated significant linear correlation between the quantitative CT index and measures of airflow (FEV1, r = -0.90, p = 0.0005), air trapping (residual volume, r = 0.70, p = 0.02), diffusing capacity (diffusing capacity for carbon monoxide, r = -0.76, p = 0.01), gas exchange (alveolar to arterial oxygen gradient) at rest, r = 0.69, p = 0.007, and at maximum exercise, r = 0.79, p = 0.007) and exercise performance (maximum workload, r = -0.84, p = 0.002), and oxygen utilization (oxygen utilization at maximum exercise, r = -0.76, p = 0.01). CONCLUSION: Quantitative CT techniques can distinguish subjects with LAM from normal controls. Further, the quantitative CT index correlates well with physiologic measurements of airflow, lung volumes, diffusing capacity, and exercise performance and, thus, may provide a useful measure of disease severity.

A study of the long term results achieved by the Gillies Fry procedure.

Renewed interest in delayed closure for cleft palate subjects revealed that no evaluation had been undertaken of patients who underwent the original Gillies Fry procedure. In the present investigation, 10 subjects operated upon prior to 1948 according to the Gillies Fry recommendations were evaluated for facial development, occlusal relationship, speech performance and velopharyngeal seal. Comparison of the cephalometric findings with accepted norms and examination of study casts gave encouraging results for facial and occlusal development. Speech quality covered a wide range of attainment, the best being rated highly. Endoscopic examination demonstrated regular velopharyngeal seal during swallowing but only one subject who regularly achieved velopharyngeal seal in speech. It was concluded that, although some claims for the procedure may have been a little enthusiastic, advantages accrued from it which were not accorded by other surgical approaches of the time, particularly so far as facial development and occlusion were concerned.

The interconversion of prednisone and prednisolone in human and rabbit organs in vitro is a function of tissue integrity.

The interconversion and elimination of prednisone (PO) and prednisolone (POH) were examined in human and rabbit liver, kidney, lung and skeletal muscle preparations to determine the effect of organ disruption on in vitro metabolism within multiple organs of two species. Results from microsomes, homogenates and minces were compared to determine the effect of various stages of tissue disruption on the reversible and irreversible metabolism of the glucocorticoids. Oxidation of POH to PO was enhanced by homogenization of all the organs examined; further disruption by subcellular fractionation to microsomes reduced oxidation relative to homogenates but yielded values greater than the original minces. The reverse reaction, reduction of PO to POH, was also enhanced by homogenization, but microsomal preparations were less active than the minces. The irreversible elimination of the glucocorticoids was progressively diminished by disruption of normal architecture in all organs. Results of in vitro studies of glucocorticoid metabolism have provided discrepant results, as a function of the laboratory, the species, the organ and the organ preparation examined. These results provide insights into the source of discrepant results regarding steroid metabolism, as it appears that results of experimentation with glucocorticoid conversion and/or elimination may be a function of the method used. Additionally, the data support the hypothesis that the enzyme system involved in the interconversion of the glucocorticoids may not be a simple single protein which acts bidirectionally.

Mutational analysis of the Streptomyces scabies esterase signal peptide.

Ten site-directed mutations affecting the predicted 39-amino-acid signal peptide of the Streptomyces scabies esterase were used to examine start-codon usage and esterase secretion in S. lividans. The first of two in-frame AUG codons was preferred for translation initiation. Removal of 2 of the 4 positively charged amino acids at the amino terminus of the signal peptide reduced esterase expression more than 100-fold; however, deletion of all 4 charged residues reduced expression by only 2- to 5-fold. Deletion of 4 or 8 amino acids from the hydrophobic core of the signal peptide reduced esterase production more than 200-fold, and a signal peptide processing site deletion completely disrupted esterase expression. For all constructs in which a mutation in the signal sequence decreased esterase production, esterase mRNA levels were also reduced, suggesting that a defect in secretion or processing affected esterase transcript abundance.

Prednisolone and prednisone exhibit linear extraction in the perfused rabbit liver.

The rabbit and human both display nonlinear pharmacokinetics of prednisone (PO) and prednisolone (POH). The in vivo apparent clearances of these compounds increase with dose. To determine whether one source of the kinetic nonlinearity is due to hepatic metabolism, the isolated liver of the rabbit was perfused with widely varying concentrations of both PO and POH in the absence of corticosteroid binding globulin. Both compounds exhibited constant extraction ratios over a wide concentration range, even at concentrations exceeding 100 times those expected in portal venous blood during absorption of orally administered drug. Hepatic extraction of POH averaged 0.49 and that of PO was nearly complete at 0.96. The apparent hepatic blood clearance of POH was about 16 ml/min and that of PO approximated liver blood flow, at 30 ml/min. Furthermore, the liver was predominantly reductive toward these compounds: upon PO perfusion, POH was recovered as about half of the dose of PO administered. POH perfusion yielded no detectable PO in the exiting perfusate. The liver is believed to be the most significant organ involved in glucocorticoid biotransformation. Its capacity to eliminate PO and POH does not appear to be saturable, and greatly favors the pharmacologically active, reduced compound, POH. Hypotheses that attribute the nonlinear pharmacokinetics of PO and POH as partially due to saturable hepatic metabolism may be incorrect. It has been previously assumed that all metabolic organs produce both interconversion products. Since the liver apparently yields no PO (oxidized form), another organ must be responsible for the in vivo oxidation.

The role of anxiety on sexual arousal.

Sex therapists have long argued that anxiety is the primary psychological mechanism that underlies the interference with sexual arousal that is responsible for such conditions as erectile failure. However, laboratory research on anxiety's effects has yielded mixed results. Studies have found that sexual arousal may be unaffected, disrupted, or even facilitated by anxiety. Fifty-four men, ages 21-46, viewed a series of erotic videotape segments after having received one of three types of information; (i) neutral feedback, (ii) feedback indicating that they would be receiving a painful electric shock, and (iii) feedback indicating that their level of sexual arousal during baseline measurement was subnormal. Subsequent plethysmographic measurement of sexual arousal while viewing the erotic material revealed that leading subjects to worry either about their physical well-being (shock threat) or their psychosexual well-being (negative feedback) were both effective strategies for interfering substantially with sexual arousal. The hypothesis that attention/memory would mediate the effects of anxiety on arousal received only partial support. Theoretical and clinical implications of these findings are explored.

Heterologous expression and secretion of a Streptomyces scabies esterase in Streptomyces lividans and Escherichia coli.

The esterase gene from Streptomyces scabies FL1 was cloned and expressed in Streptomyces lividans on plasmids pIJ486 and pIJ702. In S. lividans, the esterase gene was expressed during later stages of growth and was regulated by zinc, as is seen with S. scabies. The 36-kDa secreted form of the esterase was purified from S. lividans. N-terminal amino acid sequencing indicated that the processing site utilized in S. lividans for the removal of the signal sequence was the same as that recognized for processing in S. scabies. Western blots (immunoblots) revealed the presence of a 40-kDa precursor form of the esterase in cytoplasmic extracts. A 23-amino-acid deletion was introduced into the putative signal sequence for the esterase. When this deleted form of the esterase was expressed in S. lividans, a cytoplasmic 38-kDa precursor protein was produced but no secreted esterase was detected, suggesting the importance of the deleted sequence for efficient processing and secretion. The esterase gene was also cloned into the pUC119 plasmid in Escherichia coli. By using the lac promoter sequence, the esterase gene was expressed, and the majority of the esterase was localized to the periplasmic space.

Predictability of the clinical potency of NSAIDs from the preclinical pharmacodynamics in rats.

OBJECTIVE AND DESIGN: Relevance of the preclinical pharmacodynamic, toxicity and pharmacokinetic parameters predicting the clinical potency of nonsteroidal antiinflammatory drugs (NSAIDs) was evaluated. MATERIAL: Data for oral potencies of 24 NSAIDs in rats were collected from the literature and from New Drug Applications with respect to the following parameters: antiinflammatory, analgesic, antipyretic, acute ulcerogenic activities, acute toxicity, in vitro inhibition of prostaglandin synthesis, acid dissociation constant (pKa), octanol-water partition coefficient and elimination half-life. TREATMENT: Data for most of the in vivo parameters in rats were collected following single dose administration with the exception of adjuvant arthritis. Single and daily clinical doses were considered. All of these NSAIDs have been approved for marketing although not all have been sold in the USA. METHODS: The preclinical data were compared to human dose (unit or daily doses) using single and multiple stepwise regression analyses. RESULTS: Analyses suggest that NSAIDs are effective in all models of preclinical tests for fever, pain and inflammation, however, carrageenin-induced rat paw edema model is clearly the best predictor of human dose. Rank order of preclinical models for predicting human dose is carrageenin > yeast induced fever > pressure induced pain = adjuvant arthritis in rats. The analysis suggested that the pain and adjuvant arthritis models in rats may also involve a prostaglandin independent mechanism. Of the two physicochemical factors tested, pKa contributed best to the carrageenin model towards predicting the clinical potency of NSAIDs. Mathematical relationships between human dose, carrageenin ED50 and pKa were established that may assist in the future clinical development of NSAIDs. CONCLUSIONS: Carrageenin-induced paw edema model in rats is the most robust predictor of the clinical potency of NSAIDs. Acid dissociation constant (pKa) appears to be a secondary contributor to the potency of NSAIDs. The relevance of the data analyses for developing cyclooxygenase-2 (COX-2) selective NSAIDs is discussed.

The role of anxiety in premature ejaculation: a psychophysiological model.

Despite the prevalence of premature ejaculation and the success in its treatment, researchers have yet to offer a compelling, empirically based theory regarding its nature or etiology. This study explored a model that argues that anxiety may not be necessary for the existence of this dysfunction. Fifteen premature ejaculators (PEs) and 17 nonpremature ejaculators (NPEs) engaged in self-stimulation to orgasm both in the laboratory and at home. The following specific hypotheses were tested: Compared to NPEs, PEs would demonstrate (i) shorter orgasmic latencies, both in the lab and at home, and (ii) equally accurate estimates of these latencies. Results offered strong support for both hypotheses. These findings, and those derived from a questionnaire completed by subjects, were seen as consistent with a psychophysiologic model of premature ejaculation. According to this model, the role of anxiety is seen as variable, interacting with the somatic vulnerability of the individual to determine orgasmic latency.

Disposition of prednisone and prednisolone in the perfused rabbit liver: modeling hepatic metabolic processes.

The livers of 15 rabbits were perfused in situ with prednisone (PO) or prednisolone (POH) over a wide range of steady state concentrations, resulting in multiple experimental measurements per organ. Linearity of extraction, an apparent lack of oxidative conversion, and marked preference for the reduction of PO to POH was observed. Predictions of hepatic tissue concentrations were made using both the well-stirred and parallel-tube model approximations. Glucocorticoid disposition across the liver was described by a series of differential equations. Discrimination between the two models was accomplished by examining the effects of changes in flow rate upon the availability of the highly extracted drug PO. The well-stirred model very closely predicted the observed changes in availability of PO, whereas the parallel-tube model provided poor predictions. The intrinsic clearances of interconversion and elimination of PO and POH were subsequently calculated by population analysis using NONMEM. This method assumed the well-stirred model and resulted in intrinsic clearance estimates of 26 ml/min for the elimination of POH, 157 ml/min for reductive conversion of PO to POH, and 205 ml/min for the irreversible elimination of PO. A mechanism of intrahepatic disposition of these glucocorticoids was proposed using well-stirred model predictions of hepatic drug concentrations, the perfusion rate limitation to drug transport, and the assumption of no oxidative interconversion of POH to PO. In this case, the capacity for reduction of PO to POH approaches the elimination clearance of PO and the elimination of PO is about 13 times greater than the elimination clearance of POH.

Analysis of neurotrophin/receptor interactions with a gD-flag-modified quantitative kinase receptor activation (gD.KIRA) enzyme-linked immunosorbent assay.

A rapid, sensitive, and high-capacity assay has been developed to quantify ligand-induced receptor tyrosine kinase activation in terms of receptor phosphorylation. The assay, termed a "kinase receptor activation" or KIRA-ELISA, utilizes two separate microtiter plates, one for cell culture and ligand stimulation, and the other for receptor capture and phosphotyrosine ELISA. The assay was developed for analysis of neurotrophin-induced trkA, trkB, or trkC activation. It utilizes a trkA, trkB, or trkC receptor fused with a 26-amino-acid polypeptide flag derived from HSV glycoprotein D (gD.trkA, B, or C, respectively) on the amino-terminus, stably transfected into CHO cells. Stimulated receptors were solubilized with Triton X-100 buffer and then captured in ELISA wells coated with gD-specific mAb. The degree of receptor autophosphorylation was quantified by anti-phosphotyrosine ELISA. Reproducible standard curves were generated with an EC50 of approximately 16 ng/ml NGF for gD.trkA KIRA, 11 ng/ml for NT4/5 and 20 ng/ml for BDNF in gD.trkB KIRA, and 9.4 ng/ml for NT3 in gD.trkC KIRA. When the gD.trkA KIRA assay was used to quantify serum NGF or NT3 following administration to rats, the assay agreed well with currently existing ELISA assays. When the gD.trkA KIRA assay was used to test several NGF variants, as well as NGF stability samples, the capacity of the assay to quantify ligand bioactivity compared well with the more widely used radioreceptor binding and PC 12 cell survival assays. The gD.trk KIRA assays show great potential as rapid bioassays, capable of quantitative, consistent, and stability indicating analyses.

Correlation of CT findings with clinical evaluations in 261 patients with symptomatic bronchiectasis.

OBJECTIVE: In a multicenter study, we evaluated the relationships between the extent and severity of bronchiectasis on CT and clinical symptoms, spirometric abnormality, and sputum characteristics. SUBJECTS AND METHODS: The study population included 261 patients with symptomatic, physiologically significant bronchiectasis, who were enrolled in another study evaluating the clinical efficacy of deoxyribonudease in treatment of bronchiectasis. Patients with cystic fibrosis, allergic bronchopulmonary aspergillosis, and fungal or mycobacterial infection were excluded. In addition to high-resolution CT scanning, all patients underwent clinical evaluation, spirometry, and sputum culture. CT features scored by consensus of two observers included the extent of bronchiectasis, type of bronchiectasis (cylindric, varicose, or cystic), extent of mucoid impaction, and degree of bronchial wall thickening. RESULTS: Scores for the severity and extent of bronchiectasis correlated with the forced expiratory volume in 1 sec (FEV1) (r = -.362, p < .0001) and with the forced vital capacity (FVC) (r = -.362, p < .0001). Scores for bronchial wall thickening correlated with the FEV1 (r = -.367, p < .0001) and FVC (r = -.239, p < .001). Patients with cystic bronchiectasis were significantly more likely to grow Pseudomonas from their sputa and to have purulent sputa than were patients with cylindric or varicose bronchiectasis. Patients with cystic bronchiectasis had significantly lower FEV1 and FVC values than did patients with cylindric or varicose bronchiectasis. CONCLUSION: In this patient population, we found weak but significant correlations between the degree of morphologic abnormality on CT and the extent of physiologic impairment. Cystic bronchiectasis was associated with sputum purulence and with the growth of Pseudomonas. CT classification of the type of bronchiectasis may be useful as an index of severity of disease.

Lipoid pneumonia: CT findings.

OBJECTIVE: To assess the CT findings of lipoid pneumonia. METHODS: Chest radiography and CT performed in six patients with proven lipoid pneumonia were reviewed by two observers. Diagnosis was confirmed by biopsy (five cases) or bronchoalveolar lavage (one case). The clinical history of taking oily substance could be obtained retrospectively in all patients. RESULTS: Chest radiography showed bilateral air space consolidation in three cases, irregular mass-like lesions in two, and a reticulonodular pattern in one case. Computed tomography demonstrated diffuse parenchymal consolidation in three cases, localized areas of consolidation in two, and subpleural pulmonary fibrosis in one case. In two cases, fat with localized areas of consolidation could be seen on CT. In three cases with diffuse consolidation the attenuation was decreased but higher than that of subcutaneous fat. In one case with subpleural fibrosis no areas of low attenuation could be seen on CT. CONCLUSION: We conclude that in patients with lipoid pneumonia CT may demonstrate areas with low attenuation diagnostic of fat or areas with nonspecific low attenuation or soft tissue density.

Tissue disposition and pharmacokinetics of recombinant human nerve growth factor after acute and chronic subcutaneous administration in monkeys.

In this study, we have characterized the metabolism, tissue disposition, excretion routes, and plasma pharmacokinetics of recombinant human nerve growth factor after single and multiple s.c. administration in male cynomolgus monkeys. Unlabeled nerve growth factor (NGF; 2 mg/kg) was administered three times a week for 4 weeks and a full pharmacokinetic profile was obtained for doses 1 and 12. For the tissue distribution studies, 0.8 microg/kg of trace (125)I-labeled recombinant human nerve growth factor was dosed. Histological analysis of emulsion-microautoradiography indicated that specific (125)I-NGF labeling was confined to sections of nerves most frequently localized adjacent to large vessels in sections of kidney, spleen, liver, and salivary gland. A small percentage of large neurons within the sympathetic ganglia were intensely labeled, as well as large neurons within the dorsal root ganglia. We found an increased disposition of (125)I-NGF in parts of the peripheral nervous system (including sympathetic ganglia) from 8 to 24 h postdose. In contrast, radioactivity in most non-neuronal tissues declined. This suggests specific uptake in these target tissues known to express specific receptors for NGF. We also identified changes in pharmacokinetic parameters after single versus chronic s. c. administration. These studies demonstrated that s.c. administration of NGF at 0.8 microg/kg doses in monkeys is capable of accessing and localizing in the target tissues.

Biosynthesis of ansatrienin (mycotrienin) and naphthomycin. Identification and analysis of two separate biosynthetic gene clusters in Streptomyces collinus Tü 1892.

The polyketide chains of the two ansamycin antibiotics, ansatrienin (mycotrienin) and naphthomycin produced by Streptomyces collinus are assembled using 3-amino-5-hydroxybenzoic acid (AHBA) as a starter unit. The gene encoding AHBA synthase, an enzyme which catalyzes the final step of AHBA biosynthesis in the recently discovered aminoshikimate pathway, has been used to identify two separate antibiotic biosynthetic gene clusters in S. collinus. In one of these clusters, analysis of approximately 20 kb of contiguous sequence has revealed both a cluster of six genes presumed to play a role in the AHBA pathway and the beginning of a polyketide synthase (PKS) gene containing an acyl ACP ligase domain. This domain is likely responsible for loading AHBA onto the PKS. This gene cluster also contains chcA, encoding the enzyme 1-cyclohexenylcarbonyl CoA reductase, which is essential for the biosynthesis of the cyclohexanecarboxylic acid moiety of ansatrienin from shikimic acid, and a peptide synthetase. This gene cluster thus seems to control the biosynthesis of ansatrienin, which contains a side chain of N-cyclohexanecarbonyl-d-alanine esterified to the macrocyclic lactam backbone. In the putative naphthomycin biosynthetic gene cluster approximately 13 kb of contiguous sequence has revealed a second set of the genes required for AHBA biosynthesis. In addition the end of a polyketide synthase and a gene putatively involved in termination of the chain extension process, formation of an intramolecular amide bond between the AHBA nitrogen and the carboxyl group of the fully extended polyketide chain, have been identified. Thus, despite commonality in biosynthesis, the ansatrienin and naphthomycin biosynthetic gene clusters show clear organizational differences and carry separate sets of genes for AHBA biosynthesis.

Obliterative bronchiolitis: varying presentations and clinicopathological correlation.

In obliterative bronchiolitis, inflammation and fibrosis lead to narrowing or occlusion of bronchiolar lumina. To determine how bronchiolar structural alterations relate to lung physiology, 19 patients with a pathological diagnosis of obliterative bronchiolitis were studied. The bronchiolar inflammatory and fibrotic features were correlated to the clinical presentation, and lung function tests. Eleven patients demonstrated airflow limitation, one had a restrictive pattern and one had a mixed pattern, two had isolated gas trapping, but four had normal spirometry. Mild-to-moderate bronchiolar inflammation was invariably present. It involved 60% of bronchioles subepithelially and 54% in the adventitia. Subepithelial fibrosis was observed in 15 patients and adventitial in 12. Adventitial bronchiolar inflammation correlated with forced expiratory volume in one second and forced vital capacity and inversely correlated with residual volume. Subepithelial fibrosis inversely correlated with subepithelial and adventitial inflammation. High-resolution computed tomography in 10 patients revealed inspiratory (five out of 10) and expiratory air trapping (five out of five), ground glass opacities (seven out of 10), bronchial wall thickening (five out of 10), bronchiectasis (two out of 10) and centrilobular nodules (two out of 10). The present study suggests that inflammation and fibrosis occurs in bronchioles at different time points in the disease process, or that there is no transition between these types of pathology in the same patient. No correlation was observed between the degree of bronchiolar fibrosis and the degree of airflow limitation.