Cancer and thrombosis: new insights to an old problem.

Venous thromboembolism (VTE) is a common complication in patients with cancer and portends a poor prognosis. Our understanding of the underlying pathophysiology of VTE in cancer has advanced since Trousseau first described hypercoagulability in patients with malignancy and Virchow described his famous triad of thrombosis formation. Malignancy itself induces a thrombophilic state by increasing the risk of venous stasis, endothelial injury and an imbalance of pro and anti-thrombotic factors leading to a hypercoaguable state. Additional insults to this thrombotic balance are introduced by patient-specific, treatment related and tumor-specific factors. The importance of understanding the factors associated with increased thrombosis in cancer is paramount in order to adequately identify patients who will benefit from thromboprophylaxis.

Multi-locus DNA metabarcoding of zooplankton communities and scat reveal trophic interactions of a generalist predator.

To understand the ecosystem dynamics that underpin the year-round presence of a large generalist consumer, the Bryde's whale (Balaenoptera edeni brydei), we use a DNA metabarcoding approach and systematic zooplankton surveys to investigate seasonal and regional changes in zooplankton communities and if whale diet reflects such changes. Twenty-four zooplankton community samples were collected from three regions throughout the Hauraki Gulf, New Zealand, over two temperature regimes (warm and cool seasons), as well as 20 samples of opportunistically collected Bryde's whale scat. Multi-locus DNA barcode libraries were constructed from 18S and COI gene fragments, representing a trade-off between identification and resolution of metazoan taxa. Zooplankton community OTU occurrence and relative read abundance showed regional and seasonal differences based on permutational analyses of variance in both DNA barcodes, with significant changes in biodiversity indices linked to season in COI only. In contrast, we did not find evidence that Bryde's whale diet shows seasonal or regional trends, but instead indicated clear prey preferences for krill-like crustaceans, copepods, salps and ray-finned fishes independent of prey availability. The year-round presence of Bryde's whales in the Hauraki Gulf is likely associated with the patterns of distribution and abundance of these key prey items.

Late boosting phenomenon in TST conversion among health care workers.

BACKGROUND: Available information is insufficient to guide determination of whether tuberculin skin test (TST) conversions of health care workers (HCWs) within 2 years of two-step testing are related to occupational exposures or to other causes, including late boosting. AIMS: To describe the epidemiologic factors of TST conversion in HCWs, comparing early TST conversion (≤2 years after two-step testing) with late conversion to possibly distinguish late boosting phenomenon from occupational TST conversion. METHODS: Retrospective analysis of a database of TSTs of HCWs from 1 January 1998, through 31 May 2014, in the United States Midwest. RESULTS: In total, 40142 HCWs had 197932 tests over the 16 years, with 123 conversions (conversion rate: 0.3%; 95% CI 0.3-0.4%). Among 61 HCWs with a negative two-step TST, 30 (49%) were found to have early TST conversion within 2 years; 31 (51%) had late conversion, with likely occupational exposure but no identifiable community risks. Persons with early conversion were more likely to be born outside the USA (89% versus 57%; P < 0.05), had a higher rate of prior bacille Calmette-Guérin (BCG) vaccination (89% versus 52%; P < 0.05) and had no identifiable risk factors for conversion (63% versus 58%; P < 0.05). CONCLUSIONS: Early conversions among HCWs after negative two-step TST are associated with various nonoccupational factors, including international birth and BCG vaccination history. Therefore, conversion is not a reliable indicator of recent tuberculosis contact in this population, and two-step TST is insufficient to discount a delayed boosting response for HCWs.

Screening for depression in the occupational health setting.

BACKGROUND: The cost of workplace absenteeism and presenteeism due to depression in the USA is substantial. AIMS: To assess the frequency of depression and its impact at the point of care in an occupational health (OH) practice. METHODS: Patients presenting to an OH practice completed a standardized depression screening tool and were compared to an unscreened group in the same clinic. Respondents with a nine-item Patient Health Questionnaire (PHQ-9) score >15 and untreated for depression were referred for further evaluation per usual practice. A comparison group of unscreened patients were selected from the same clinic from 1 year prior and records were reviewed for evidence of prior depression, treatment and outcomes. After 1 year, frequency of depression, PHQ-9 scoring for screened patients, days absent from work, days on restricted duties and permanent restrictions were recorded for both groups. RESULTS: Two hundred and five patients were screened for depression. Screening was associated with increased frequency of a diagnosis of current depression (30 versus 4%; P < 0.05). Screening was associated with similar rates of absenteeism but lower number of days on restricted duties (97 versus 159 days; P < 0.001). After adjusting for age, sex, history of and treatment for depression, screening was associated with lower odds of being on work restrictions [odds ratio (OR) 0.55; 95% confidence interval (CI) 0.38-0.78] or permanent restrictions (OR 0.35; 95% CI 0.23-0.52). CONCLUSIONS: Depression was common in this OH practice. Screening for depression, with appropriate recognition and referral, may reduce time for employed patients on restricted duties and permanent restrictions.

A survey of physicians' perceptions of their health care needs.

BACKGROUND: Physicians may face unique challenges in accessing health care and managing their own health. AIMS: To evaluate physicians' perceptions of their health care needs and desired services. METHODS: A written survey, distributed and collected anonymously among attendees at a large primary care continuing medical education conference. RESULTS: The survey was given to 346 physicians and 141 (41%) responded. The majority of physicians (53%) reported having difficulty accessing health care and reverting to self-diagnosis and treatment (63%). Over 83% reported having or knowing a colleague who had a career-threatening illness and 42% had experienced concern about a colleague's ability to practise safely. CONCLUSIONS: Physicians as an occupational group have challenges in accessing health care, very commonly suffer career-limiting illnesses and revert to self-diagnosis and treatment. Programmes tailored to providing health care to physicians are needed.

Expressed sequence tags and proteomics of antennae from the tortricid moth, Epiphyas postvittana.

Genomic and proteomic analyses of the antennae of the light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae) were undertaken to identify genes and proteins potentially involved in odorant and pheromone binding and turnover. An EST approach yielded 5739 sequences, comprising 808 contigs and 1545 singletons. InterPro and Blast analyses revealed members of families implicated in odorant and pheromone binding (PBPs, GOBPs, ABPXs and CSPs) and turnover (CXEs, GSTs, CYPs). Of the three pheromone binding proteins (PBPs) identified, two were more highly expressed at the RNA and protein levels in adult male antennae (EpPBP1, EpPBP3), while a third was more highly expressed in female antennae (EpPBP2). To identify proteins involved in the detection of sex-specific signals, differential 2D gel electrophoresis (pH 5-8) followed by mass spectrometry was conducted on antennal proteins from males versus females. Identified male-biased proteins included a pheromone binding protein, a porin, a short chain dehydrogenase/reductase, and a member of the takeout family.

Expressed sequence tags from the midgut of Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae).

The midgut is a key tissue in insect science. Physiological roles include digestion and peritrophic membrane function, as well as being an important target for insecticides. We used an expressed sequence tag (EST) approach to identify candidate genes and gene families involved in these processes in the light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae). Two cDNA libraries were constructed from dissected midgut of third to fifth instar larvae. Clustering analysis of 6416 expressed sequence tags produced 1178 tentative unique genes comprising 725 tentative contigs and 453 singletons. The sequences show similar codon usage to sequences from other lepidopterans, a Kozak consensus sequence similar to Drosophila and single nucleotide polymorphisms (SNPs) were detected at a frequency of 1.35/kb. The identity of the most common Interpro families correlates well with major known functions of the midgut. Phylogenetic analysis was conducted on representative sequences from selected multigene families. Gene families include a broad range of digestive proteases, lipases and carbohydrases that appear to have degradative capacity against the major food components found in leaves, the diet of these larvae; and carboxylesterases, glutathione-S-transferases and cytochrome P450 monooxygenases, potentially involved in xenobiotic degradation. Two of the larger multigene families, serine proteases and lipases, expressed a high proportion of genes that are likely to be catalytically inactive.

Female-biased expression of odourant receptor genes in the adult antennae of the silkworm, Bombyx mori.

Olfaction plays an important role in the life history of insects, including key behaviours such as host selection, oviposition and mate recognition. Odour perception by insects is primarily mediated by the large diverse family of odourant receptors (Ors) that are expressed on the dendrites of olfactory neurones housed within chemosensilla. However, few Or sequences have been identified from the Lepidoptera, an insect order that includes some of the most important pest species worldwide. We have identified 41 Or gene sequences from the silkworm (Bombyx mori) genome, more than double the number of published Or sequences from the Lepidoptera. Many silkworm Ors appear to be orthologs of the 17 published tobacco budworm (Heliothis virescens) Ors indicating that many Or lineages may be conserved within the Lepidoptera. The majority of the Or genes are expressed in adult female and male antennae (determined by quantitative real-time PCR analysis), supporting their probable roles in adult olfaction. Several Or genes are expressed at high levels in both male and female antennae, suggesting they mediate the perception of common host or conspecific volatiles important to both sexes. BmOrs 45-47 group together in the same phylogenetic branch and all three are expressed at moderate female-biased ratios, six to eight times higher in female compared to male moth antennae. Interestingly, BmOrs19 and 30 appear to be expressed predominantly in female antennae, opposite to that of the published silkworm pheromone receptors BmOrs 1 and 3 that are specific to male antennae. These results suggest that BmOr19 and 30 may detect odours critical to female behaviour, such as oviposition cues or male-produced courtship pheromones.

RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-stranded RNA feeding.

RNA interference (RNAi) or gene silencing is typically induced in insects by the injection of double-stranded RNAs (dsRNAs), short interfering RNAs, or through the use of hairpin constructs in transgenic insects. Here we demonstrate in the horticultural pest, Epiphyas postvittana (Lepidoptera: Tortricidae), that RNAi can be triggered by oral delivery of dsRNA to larvae. Transcript levels of a larval gut carboxylesterase gene (EposCXE1) were reduced to less than half that of controls within 2 days of being fed EposCXE1 dsRNA. Transcript levels of the pheromone binding protein gene (EposPBP1) were reduced in adult antennae by feeding larvae EposPBP1 dsRNA. Knockdown of EposPBP1 transcripts was observed for the first 2 days after adult eclosion but recovered to wild-type levels at 4 days posteclosion. The potential mechanisms involved in the initiation, movement and amplification of the silencing signal are discussed.

Amplification of DNA from preserved specimens shows blowflies were preadapted for the rapid evolution of insecticide resistance.

Mutations of esterase 3 confer two forms of organophosphate resistance on contemporary Australasian Lucilia cuprina. One form, called diazinon resistance, is slightly more effective against commonly used insecticides and is now more prevalent than the other form, called malathion resistance. We report here that the single amino acid replacement associated with diazinon resistance and two replacements associated with malathion resistance also occur in esterase 3 in the sibling species Lucilia sericata, suggesting convergent evolution around a finite set of resistance options. We also find parallels between the species in the geographic distributions of the polymorphisms: In both cases, the diazinon-resistance change is absent or rare outside Australasia where insecticide pressure is lower, whereas the changes associated with malathion resistance are widespread. Furthermore, PCR analysis of pinned specimens of Australasian L. cuprina collected before the release of organophosphate insecticides reveals no cases of the diazinon-resistance change but several cases of those associated with malathion resistance. Thus, the early outbreak of resistance in this species can be explained by the preexistence of mutant alleles encoding malathion resistance. The pinned specimen analysis also shows much higher genetic diversity at the locus before organophosphate use, suggesting that the subsequent sweep of diazinon resistance in Australasia has compromised the scope for the locus to respond further to the ongoing challenge of the insecticides.

Breath sulfides and pulmonary function in cystic fibrosis.

We have determined the concentrations of carbonyl sulfide (OCS), dimethylsulfide, and carbon disulfide (CS(2)) in the breath of a group of cystic fibrosis (CF) patients and one of healthy controls. At the detection sensitivity in these experiments, room air always contained measurable quantities of these three gases. For each subject the inhaled room concentrations were subtracted from the time-coincident concentrations in exhaled breath air. The most significant differences between the CF and control cohorts in these breath-minus-room values were found for OCS. The control group demonstrated a net uptake of 250 +/- 20 parts-per-trillion-by-volume (pptv), whereas the CF cohort had a net uptake of 110 +/- 60 pptv (P = 0.00003). Three CF patients exhaled more OCS than they inhaled from the room. The OCS concentrations in the CF cohort were strongly correlated with pulmonary function. The dimethylsulfide concentrations in breath were greatly enhanced over ambient, but no significant difference was observed between the CF and healthy control groups. The net (breath minus room) CS(2) concentrations for individuals ranged between +180 and -100 pptv. They were slightly greater in the CF cohort (+26 +/- 38 pptv) vs. the control group (-17 +/- 15 pptv; P = 0.04). Lung disease in CF is accompanied by the subsistence of chronic bacterial infections. Sulfides are known to be produced by bacteria in various systems and were therefore the special target for this investigation. Our results suggest that breath sulfide content deserves attention as a noninvasive marker of respiratory colonization.

Pheromone binding proteins of Epiphyas postvittana (Lepidoptera: Tortricidae) are encoded at a single locus.

The light brown apple moth, Epiphyas postvittana (Tortricidae: Lepidoptera) uses a blend of (E)-11-tetradecenyl acetate and (E,E)-9,11-tetradecadienyl acetate as its sex pheromone. Odorant binding proteins, abundant in the antennae of male and female E. postvittana, were separated by native PAGE to reveal four major proteins with distinct mobilities. Microsequencing of their N-terminal residues showed that two were general odorant binding proteins (GOBPs) while two were pheromone binding proteins (PBPs). Full length cDNAs encoding these proteins were amplified using a combination of PCR and RACE-PCR. Sequence of the GOBPs revealed two genes (EposGOBP1, EposGOBP2), similar to orthologues in other species of Lepidoptera. Eleven cDNAs of the PBP gene were amplified, cloned and sequenced revealing two major phylogenetic clusters of PBP sequences differing by six amino acid substitutions. The position of the six amino acid differences on the protein was predicted by mapping onto the three-dimensional structure of PBP of Bombyx mori. All six substitutions were predicted to fall on the outside of the protein away from the inner pheromone binding pocket. One substitution does fall close to the putative dimerisation region of the protein (Ser63Thr). Expression of three of the cDNAs in a baculovirus expression system revealed that one class encodes an electrophoretically slow form (EposPBP1-12) while the other encodes a fast form (EposPBP1-2, EposPBP1-3). A native Western of these expressed proteins compared with antennal protein extracts demonstrated that PBP is also expressed in female antennae and that PBP may be present as a dimer as well as a monomer in E. postvittana. The fast and slow forms of EposPBP1 are allelic. Westerns on single antennal pair protein extracts and allele-specific PCR from genomic DNA both show a segregating pattern of inheritance in laboratory and wild populations. Radio labelled (E)-11-tetradecenyl acetate binds to both fast and slow PBP forms in gel assays. Taken together, the genetic and biochemical data do not support the hypothesis that these PBPs are specific for each component of the E. postvittana pheromone. However, duplication of this PBP locus in the future might allow such diversification to evolve, as observed in the other species.

Mechanisms of synergistic cytokine-induced nitric oxide production in human alveolar epithelial cells.

Nitric oxide (NO) derived from inducible NO synthase (iNOS) at sites of inflammation is closely related to host defense against infection and airway inflammation. Cytokines are known to stimulate NO production in human alveolar epithelial cells in a synergistic (nonlinear or nonadditive) manner. The mechanism of this synergy is not known. We measured the activation of the transcription factor NF-kappaB, the iNOS protein, and NO production in A549 monolayers (human alveolar epithelial cell line) in response to different combinations of IL-1beta, INF-gamma, and TNF-alpha (100 ng/ml), and the cofactors FMN, FAD, and BH4. We found that both IL-1beta and TNF-alpha could independently activate cytosolic NF-kappaB, direct its translocation into the nucleus, and induce iNOS monomer synthesis. In addition, different combinations of cytokines produced synergistic amounts of iNOS monomers. Exogenous BH4 (0.1 microM) had no impact on NO production induced by cytokine combinations that included IL-1beta, but significantly enhanced NO production in the presence of INF-gamma and TNF-alpha, and allowed TNF-alpha independently to produce NO. We conclude that there are at least three mechanisms of synergistic cytokine-induced NO production: (1) the biosynthesis of iNOS monomer due to nonlinear interactions by transcription factors, (2) synergistic cytosolic activation of NF-kappaB, and (3) parallel biosynthesis of BH4 in the presence of cytokine combinations that include IL-1beta.

Distribution of somatic H1 subtypes is non-random on active vs. inactive chromatin II: distribution in human adult fibroblasts.

For nearly twenty years researchers have observed changes in the histone H1 subtype content of tissues as an organism develops into an adult. To better understand the consequences of such changes, immunofractionation of chromatin using previously characterized antibodies specific for human H1 subtypes was employed in the analysis of a fibroblast cell strain derived from a 37-year-old individual. DNAs isolated from immunoprecipitates were probed for the existence of a variety of DNA sequences. The results presented lend further support to a previously-proposed model (Parseghian et al. [2000] Chromosome Res 8:405-424) in which transcription of a sequence is accompanied by the selective depletion of subtypes. The data also suggest that there is more total H1 on actively transcribed sequences in these cells as compared to fetal fibroblasts and that there is less difference in the subtype compositions of active genes vs. inactive sequences in this strain. Specifically, the consequences of these changes appear to correlate with the attenuation of the heat shock response in aging fibroblasts. In a broader context, these results could explain why there are reductions in transcription in cells from mature tissue that approach senescence.

Profiles of glutamate and GABA efflux in core versus peripheral zones of focal cerebral ischemia in mice.

Efflux of glutamate during cerebral ischemia is known to contribute to brain cell death via processes of excitotoxicity. However, gamma-aminobutyric acid (GABA) is also released during ischemia, and may be protective. In this study, we used in vivo microdialysis to map the efflux of glutamate and GABA from central core and peripheral zones of focal ischemia in mouse brain. We show that the temporal profiles of glutamate and GABA efflux are significantly different in core versus peripheral zones. Calculation of glutamate/GABA ratios demonstrate that, in the core, there is a significant increase above baseline ratios during the first 30 mm of ischemia, which then rapidly renormalizes. In contrast, no significant changes in glutamate/GABA ratios were seen in the ischemic periphery. These data suggest that imbalances in glutamate versus GABA efflux may be an initial trigger of excitotoxic brain damage in the core but not the peripheral zones of focal cerebral ischemia.

Fitness, training, and the growth hormone-->insulin-like growth factor I axis in prepubertal girls.

We recently demonstrated that a brief endurance type training program led to increases in thigh muscle mass and peak oxygen uptake (VO(2)) in prepubertal girls. In this study, we examined the effect of training on the GH-->insulin-like growth factor I (GH-->IGF-I) axis, a system known to be involved both in the process of growth and development and in the response to exercise. Healthy girls (mean age 9.17 +/- 0.10 yr old) volunteered for the study and were randomized to control (n = 20) and training groups (n = 19) for 5 weeks. Peak VO(2), thigh muscle volume, and blood samples [for IGF-I, IGF-binding proteins (IGFBP)-1 to -6, and GHBP] were measured. At baseline, IGF-I was significantly correlated with both peak VO(2) (r = 0.44, P < 0.02) and muscle volume (r = 0.58, P < 0.004). IGFBP-1 was negatively correlated with muscle volume (r = -0.71, P < 0.0001), as was IGFBP-2. IGFBP-4 and -5 were significantly correlated with muscle volume. We found a threshold value of body mass index percentile (by age) of about 71, above which systematic changes in GHBP, IGFBP-1, and peak VO(2) per kilogram were noted, suggesting decreases in the following: 1) GH function, 2) insulin sensitivity, and 3) fitness. Following the training intervention, IGF-I increased in control (19.4 +/- 9.6%, P < 0.05) but not trained subjects, and both IGFBP-3 and GHBP decreased in the training group (-4.2 +/- 3.1% and -9.9 +/- 3.8%, respectively, P < 0.05). Fitness in prepubertal girls is associated with an activated GH-->IGF-I axis, but, paradoxically, early in a training program, children first pass through what appears to be a neuroendocrine state more consistent with catabolism.

The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina.

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphorous (OP) and carbamate insecticides. Ace mutations have been identified in OP resistants strains of Drosophila melanogaster. However, in the Australian sheep blowfly, Lucilia cuprina, resistance in field and laboratory generated strains is determined by point mutations in the Rop-1 gene, which encodes a carboxylesterase, E3. To investigate the apparent bias for the Rop-1/E3 mechanism in the evolution of OP resistance in L. cuprina, we have cloned the Ace gene from this species and characterized its product. Southern hybridization indicates the existence of a single Ace gene in L. cuprina. The amino acid sequence of L. cuprina AChE shares 85.3% identity with D. melanogaster and 92.4% with Musca domestica AChE. Five point mutations in Ace associated with reduced sensitivity to OP insecticides have been previously detected in resistant strains of D. melanogaster. These residues are identical in susceptible strains of D. melanogaster and L. cuprina, although different codons are used. Each of the amino acid substitutions that confer OP resistance in D. melanogaster could also occur in L. cuprina by a single non-synonymous substitution. These data suggest that the resistance mechanism used in L. cuprina is determined by factors other than codon bias. The same point mutations, singly and in combination, were introduced into the Ace gene of L. cuprina by site-directed mutagenesis and the resulting AChE enzymes expressed using a baculovirus system to characterise their kinetic properties and interactions with OP insecticides. The K(m) of wild type AChE for acetylthiocholine (ASCh) is 23.13 microM and the point mutations change the affinity to the substrate. The turnover number of Lucilia AChE for ASCh was estimated to be 1.27x10(3) min(-1), similar to Drosophila or housefly AChE. The single amino acid replacements reduce the affinities of the AChE for OPs and give up to 8.7-fold OP insensitivity, while combined mutations give up to 35-fold insensitivity. However, other published studies indicate these same mutations yield higher levels of OP insensitivity in D. melanogaster and A. aegypti. The inhibition data indicate that the wild type form of AChE of L. cuprina is 12.4-fold less sensitive to OP inhibition than the susceptible form of E3, suggesting that the carboxylesterases may have a role in the protection of AChE via a sequestration mechanism. This provides a possible explanation for the bias towards the evolution of resistance via the Rop-1/E3 mechanism in L. cuprina.

Pernin: a novel, self-aggregating haemolymph protein from the New Zealand green-lipped mussel, Perna canaliculus (Bivalvia: Mytilidae).

A protein, designated pernin, found in the New Zealand green-lipped mussel, comprises almost all of the protein in cell-free haemolymph. It occurs as large, aggregate structures of several hundred units resembling small virus-like particles. Pernin is a non-pigmented, glycosylated protein, composed of 497 amino acids, which has an estimated molecular mass of 60 kDa. It is exceptionally rich in histidine (13.7%) and aspartic acid (12.3%), amino acids both known to participate in the binding of divalent metal cations. In addition, pernin has serine protease inhibitor activity, likely due to a sequence of eight N-terminal amino acid residues, separated from the remainder of the protein via a histidine-aspartate spacer. The pernin monomer comprises three regions of obvious sequence duplication. These make up approximately 95% of the pernin molecule and have sequences clearly homologous to the active-site domain of Cu-Zn SODs (superoxide dismutases). Despite several of the metal ion co-ordinating histidine residues being retained, pernin contains no Cu or Zn. It is, however, associated with Fe with an apparent stoichiometry of 1 atom of Fe to 6 molecules of pernin. Since pernin has no demonstrable SOD activity, these SOD-derived sequences presumably have been modified for another function.

Muscle size and cardiorespiratory response to exercise in cystic fibrosis.

The mechanism responsible for diminished exercise performance in cystic fibrosis (CF) is not clear. We hypothesized that reduced muscle size, rather than an intrinsic muscle defect, was the primary factor in such diminished exercise performance. Twenty-two subjects with CF (14 females and eight males, aged 6.5 to 17.7 yr, with FEV(1) of 46% to 111% predicted) participated in a study of this hypothesis, and were compared with healthy children tested in the same laboratory. Muscle size was estimated from midthigh muscle cross-sectional area (CSA) obtained by magnetic resonance imaging, and fitness was determined by progressive cycle ergometer exercise testing with breath-by-breath measurements of gas exchange. Peak oxygen consumption (V O(2)) was reduced in CF subjects (956 +/- 81 [mean +/- SEM] ml/min, as compared with 1,473 +/- 54 ml/min in controls; p < 0.00001). Surprisingly, CF subjects had a lower peak V O(2) per CSA (mean for CF subjects 70 +/- 3% predicted, p < 0.0001) than did controls, whereas muscle CSA in CF subjects was not significantly smaller than in controls. The scaling parameters of peak V O(2) and muscle CSA did not differ significantly between healthy controls (0.80 +/- 0.16) and CF subjects (1.03 +/- 0.12). Indexes of aerobic function that are less effort-dependent than peak V O(2) were also lower in the CF subjects (e.g., the slope of V O(2) versus work rate [WR] (DeltaV O(2)/DeltaWR) was 68 +/- 2% predicted; p < 0.005). The study data did not support the initial hypothesis, and suggest a muscle-related abnormality in oxygen metabolism in patients with CF.

Binding of Bacillus thuringiensis delta-endotoxins Cry1Ac and Cry1Ba to a 120-kDa aminopeptidase-N of Epiphyas postvittana purified from both brush border membrane vesicles and baculovirus-infected Sf9 cells.

A 120-kDa protein was purified from brush border membrane vesicles of the tortricid moth Epiphyas postvittana (Walker) based both on its activity as an aminopeptidase and the ability to bind the Bacillus thuringiensis delta-endotoxin Cry1Ac. The purified enzyme had a pI of 5.6 and was a leucine aminopeptidase, with some isoleucine, phenylalanine and tryptophan aminopeptidase activity. Further characterisation showed that the protein was also able to bind Cry1Ba. During purification, the molecular weight of the protein decreased from 120 to 115 kDa due to the loss of a glycophosphatidinyl anchor. The protein was N-terminally sequenced and, using this information and conserved regions within other insect aminopeptidase-N (APN) sequences, redundant primers were designed to amplify the aminopeptidase coding sequence from E. postvittana midgut cDNA. The predicted protein sequence from the full-length cDNA was most closely related to the APN protein sequence from Heliothis virescens (61% identity) and shared other features of insect APNs including a Zn(2+) binding site motif and four conserved cysteines. The E. postvittana was expressed in Sf9 cells using baculovirus, yielding a protein of molecular weight 130 kDa, but with unchanged N-terminal sequence. Purified recombinant protein bound both Cry1Ac and Cry1Ba by ligand blot assays. However, despite the protein being expressed on the external surface of the Sf9 cells, it bound neither Cry1Ac nor Cry1Ba in vivo.