Predominant secretion of cellobiohydrolases and endo-ß-1,4-glucanases in nutrient-limited medium by Aspergillus spp. isolated from subtropical field.

Biological degradation of cellulose from dead plants in nature and plant biomass from agricultural and food-industry waste is important for sustainable carbon recirculation. This study aimed at searching diverse cellulose-degrading systems of wild filamentous fungi and obtaining fungal lines useful for cellooligosaccharide production from agro-industrial wastes. Fungal lines with cellulolytic activity were screened and isolated from stacked rice straw and soil in subtropical fields. Among 13 isolated lines, in liquid culture with a nutrition-limited cellulose-containing medium, four lines of Aspergillus spp. secreted 50-60 kDa proteins as markedly dominant components and gave clear activity bands of possible endo-ß-1,4-glucanase in zymography. Mass spectroscopy (MS) analysis of the dominant components identified three endo-ß-1,4-glucanases (GH5, GH7 and GH12) and two cellobiohydrolases (GH6 and GH7). Cellulose degradation by the secreted proteins was analysed by LC-MS-based measurement of derivatized reducing sugars. The enzymes from the four Aspergillus spp. produced cellobiose from crystalline cellulose and cellotriose at a low level compared with cellobiose. Moreover, though smaller than that from crystalline cellulose, the enzymes of two representative lines degraded powdered rice straw and produced cellobiose. These fungal lines and enzymes would be effective for production of cellooligosaccharides as cellulose degradation-intermediates with added value other than glucose.

Proteome of equine oviducal fluid: effects of ovulation and pregnancy.

The equine oviduct plays a pivotal role in providing the optimal microenvironment for early embryonic development, but little is known about the protein composition of the oviducal fluid in the horse. The aim of the present study was to provide a large-scale identification of proteins in equine oviducal fluid and to determine the effects of ovulation and pregnancy. Four days after ovulation, the oviducts ipsilateral and contralateral to the ovulation side were collected from five pregnant and five non-pregnant mares. Identification and relative quantification of proteins in the oviducal fluid of the four groups was achieved by isobaric tags for relative and absolute quantification (iTRAQ) labelling and HPLC-tandem mass spectrometry. The presence of an embryo in the ipsilateral oviducal fluid of pregnant mares induced upregulation of 11 and downregulation of two proteins compared with the contralateral side, and upregulation of 19 proteins compared with the ipsilateral side of non-pregnant mares. Several of these upregulated proteins are related to early pregnancy in other species. The present study represents the first high-throughput identification of proteins in the oviducal fluid of the mare. The results support the hypothesis that the equine embryo interacts with the oviduct, affecting the maternal secretion pattern of proteins involved in pregnancy-related pathways.

The effects of Sarconesiopsis magellanica larvae (Diptera: Calliphoridae) excretions and secretions on fibroblasts.

Sarconesiopsis magellanica is a necrophagous blowfly which is relevant in both forensic and medical sciences. Previous studies regarding this species have led to understanding life-cycle, population and reproduction parameters, as well as identifying and characterising proteolytic enzymes derived from larval excretions and secretions (ES). As other studies have shown that ES proteolytic activity plays a significant role in wound healing and fibroblasts play a relevant role in granulation tissue formation during such healing, the present study was aimed at analysing the biological effect of S. magellanica larval ES on fibroblasts. ES were obtained from third-instar larvae and added to fibroblast cells at three concentrations (10, 5 and 1 µg/mL) to evaluate their behaviour. MTT assays were used for analysing cell proliferation and viability, whilst cell adhesion was measured by optical density with 10% SDS. Fibroblast migration and morphology was recorded by microscopic observation. ES did not affect fibroblast viability and induced an increase in cell proliferation; cell adhesion became reduced, whilst cell migration through extracellular matrix increased. ES also induced a decreased cell surface and morphological alterations. Changes in all the above-mentioned parameters were reduced when ES were incubated at 60 °C, probably due to protease denaturation. These results suggested that the proteases contained in S. magellanica larval ES contributed towards granulation tissue formation, increased cell migration and promoted cell proliferation. All these data support carrying out further experiments aimed at validating S. magellanica usefulness in larval therapy.

Lactate dehydrogenase and caspase activity in nasopharyngeal secretions are predictors of bronchiolitis severity.

BACKGROUND: Bronchiolitis is the leading cause of hospitalization in infants. Biomarkers of disease severity might help in clinical management. OBJECTIVE: To determine the clinical predictiveness of NW-LDH, NW-caspase 3/7, and NW-LDH/NW-caspase 3/7 ratio in bronchiolitis. METHODS: Previously healthy children less than 24 months of age with bronchiolitis were recruited from the Texas Children's emergency room and intensive care unit from October 2010 to April 2011. Demographic, clinical information, and NW samples were obtained at enrollment. NW samples were analyzed for respiratory viruses, caspase 3/7, and LDH. RESULTS: A viral pathogen was detected in 91·6% of 131 children, with the most common being respiratory syncytial virus and human rhinovirus. A single infection was found in 61·8% of subjects and co-infection in 29·8%. Children admitted to ICU had significantly higher NW-LDH than children sent home from the ER or admitted to the general floor (P = 0·02). Children infected with RSV had the highest NW-LDH concentration (P = 0·03) compared with other viral infections. NW-LDH and NW-caspase were significantly correlated (r = 0·77, P < 0·0001). The univariate models showed NW-LDH and NW-LDH/NW- caspase 3/7 ratio were directly associated with hospitalization. Mutivariate regression analyses suggested a complex interaction between the biomarkers, demographics, and disposition. CONCLUSIONS: NW-LDH, NW-caspase 3/7 and NW-LDH/NW-caspase 3/7 ratio and their interactions with demographic factors are predictive of bronchiolitis severity and can help distinguish children requiring ICU-level care from those admitted to the general floor, or discharged home from the emergency center.

Alkaline phosphatase in nasal secretion of cattle: biochemical and molecular characterisation.

BACKGROUND: Nasal secretion (NS) was investigated as a source of information regarding the mucosal and systemic immune status of cattle challenged by respiratory disease. A method for the collection of substantial volumes (~12 ml) of NS from cattle was developed to establish a reference range of analytes that are present in the NS of healthy cattle. Biochemical profiles of NS from a group of 38 healthy Holstein-Friesian cows revealed high alkaline phosphatase (AP) activity of up to 2392 IU/L. The character and source of the high activity of AP in bovine NS was investigated. RESULTS: Histochemical analysis confirmed the localization of the AP enzyme activity to epithelial cells and serous glands of the nasal respiratory mucosa. Analysis of mRNA levels from nasal mucosa by end point RT-PCR and PCR product sequencing confirmed that the AP was locally produced and is identical at the nucleotide level to the non-specific AP splice variant found in bovine liver, bone and kidney. Analysis by isoelectric focussing confirmed that AP was produced locally at a high level in nasal epithelium demonstrating that AP from nasal secretion and nasal mucosa had similar pI bands, though differing from those of the liver, kidney, bone and intestine, suggesting different post-translational modification (PTM) of AP in these tissues. CONCLUSIONS: A nasal isozyme of AP has been identified that is present at a high activity in NS, resulting from local production and showing distinctive PTM and may be active in NS as an anti-endotoxin mediator.

Proteolytic activity regarding Sarconesiopsis magellanica (Diptera: Calliphoridae) larval excretions and secretions.

Sarconesiopsis magellanica (Diptera: Calliphoridae) is a medically important necrophagous fly which is used for establishing the post-mortem interval. Diptera maggots release proteolytic enzymes contained in larval excretion and secretion (ES) products playing a key role in digestion. Special interest in proteolytic enzymes has also been aroused regarding understanding their role in wound healing since they degrade necrotic tissue during larval therapy. This study was thus aimed at identifying and characterising S. magellanica proteolytic enzyme ES products for the first time. These products were obtained from first-, second- and third-instar larvae taken from a previously-established colony. ES proteins were separated by SDS-PAGE and their proteolytic activity was characterised by zymograms and inhibition assays involving BAPNA (Nα-benzoyl-dl-Arg-p-nitroanilide) and SAPNA substrates, using synthetic inhibitors. The protein profile ranged from ∼69kDa to ∼23kDa; several of them coincided with the Lucilia sericata ES protein profile. Serine-protease hydrolysis activity (measured by zymogram) was confirmed when a ∼25kDa band disappeared upon ES incubation with PMSF inhibitor at pH 7.8. Analysis of larval ES proteolytic activity on BAPNA and SAPNA substrates (determined by using TLCK and TPCK specific inhibitors) suggested a greater amount of trypsin-like protease. These results support the need for further experiments aimed at validating S. magellanica use in larval therapy.

Role of host xanthine oxidase in infection due to enteropathogenic and Shiga-toxigenic Escherichia coli.

Xanthine oxidase (XO), also known as xanthine oxidoreductase, has long been considered an important host defense molecule in the intestine and in breastfed infants. Here, we present evidence that XO is released from and active in intestinal tissues and fluids in response to infection with enteropathogenic Escherichia coli (EPEC) and Shiga-toxigenic E. coli (STEC), also known as enterohemorrhagic E. coli (EHEC). XO is released into intestinal fluids in EPEC and STEC infection in a rabbit animal model. XO activity results in the generation of surprisingly high concentrations of uric acid in both cultured cell and animal models of infection. Hydrogen peroxide (H(2)O(2)) generated by XO activity triggered a chloride secretory response in intestinal cell monolayers within minutes but decreased transepithelial electrical resistance at 6 to 22 h. H(2)O(2) generated by XO activity was effective at killing laboratory strains of E. coli, commensal microbiotas, and anaerobes, but wild-type EPEC and STEC strains were 100 to 1,000 times more resistant to killing or growth inhibition by this pathway. Instead of killing pathogenic bacteria, physiologic concentrations of XO increased virulence by inducing the production of Shiga toxins from STEC strains. In vivo, exogenous XO plus the substrate hypoxanthine did not protect and instead worsened the outcome of STEC infection in the rabbit ligated intestinal loop model of infection. XO released during EPEC and STEC infection may serve as a virulence-inducing signal to the pathogen and not solely as a protective host defense.

Vitamin A deficiency induces fluid hyposecretion from the airway submucosal glands of mice.

Vitamin A deficiency (VAD) alters the phenotype of airway epithelium and attenuates the epithelial defense system, and many studies have reported the association of VAD with respiratory disease. In this study, we investigated changes in submucosal glands (SMG) in a mouse model of VAD. C57BL/6 mice were fed a vitamin A-devoid diet and the others were fed a control diet (1.2 mg retinol/kg). The areas of serous and mucous cells of SMG were measured in 4-, 8-, and 20-wk-old male mice. The volume and lysozyme concentration of glandular secretions were also measured. The 2 groups did not differ in body weight or general morbidity at 3-10 wk of age, although serum retinol concentrations were greater in the control mice than in the VAD mice after 4 wk. Upon histological evaluation, we found that the areal ratio of serous cells:total SMG cells was significantly lower after 8 wk in the VAD mice compared with the control mice, although the total area of SMG did not differ between groups throughout the 20-wk experiment. The number of secretory bubbles did not differ between the groups, but total secretion volume was reduced by 35% in 8-wk-old VAD mice compared with controls. Furthermore, the concentration of lysozyme in secretions from 8-wk-old VAD mice was also less than in controls, compounding the effect of diminished secretion volume. In this study, we found serous cell hypotrophy/hypoplasia and dysfunction in VAD mice, which may contribute to the susceptibility to airway infection linked to VAD.

Th1 and th2 cytokine expression in nasopharyngeal secretions during acute bronchiolitis in children younger than two years old

Objectives: We characterised the T helper cytokine profiles on the surface of nasal mucosa of children with acute bronchiolitis caused by Respiratory Syncytial Virus, Parainfluenza Virus, Influenza Virus, Adenovirus, or without any viral identification, in order to examine whether these viral types modified cytokine responses. As an additional objective we sought to determine if T helper polarisation was associated with other demographic and environmental factors. Methods: A prospective study of children with acute bronchiolitis was performed. Patients were recruited from the emergency department of a central hospital in Lisbon, Portugal. Demographical, epidemiological and clinical data were gathered from a questionnaire. Nasal swabs were collected for viral studies (immunofluorescence) and T cell cytokine responses (detection of expression of interleukins 4, 13, 12 and interferon-ã by real-time polymerase chain reaction assays). Results: Respiratory Syncytial Virus elicited lower levels of interleukin 4, when compared with samples without virus identification. A similar tendency to Th1 polarisation was found in older children, in those who attended day-care centres, and in breastfed individuals. Exposure to tobacco smoke was associated with a Th2 bias in this population.ConclusionsThese findings show that Respiratory Syncytial Virus infection contributes to Th1 polarisation in immune response of respiratory mucosa, an effect that is similar to other environmental factors. Further studies are needed to assess immune response to other infectious causes of acute bronchiolitis (AU)

Rapid diagnosis of bacterial sinusitis in patients using a simple test of nasal secretions.

Upper respiratory symptoms and sinusitis constitute a major reason for patient visits to their physician. The diagnosis of sinusitis is often made based on history and physical exam, but the accuracy of such diagnosis is questioned. Sinus films or CT scans are expensive. Obtaining pus from the middle meatus is impractical. We studied whether analysis of four easily measured substances (protein, pH, leukocyte esterase and nitrite) in nasal secretions could predict the presence or absence of bacterial sinusitis, as diagnosed by history combined with sinus x ray or CT. We enrolled 217 consecutive patients, aged 4-61 years, with clinically suspected bacterial sinusitis (duration of symptoms, 7-26 days), who had radiographic studies. All had their nasal secretions assayed using a simple rapid test. A clinical scoring system was developed to allow for a simple interpretation of test results of four assays in a single clinical score. All 52 patients with scores of 0 or 1 were CT or x-ray negative for bacterial sinusitis. All 144 with scores of > or =4 were imaging study positive. Of the 21 patients (10%) with scores of 2 or 3, 14 were imaging study negative and 7 were positive. We concluded that combining these four separate assays on nasal secretion into one number, it is possible to rule in or rule out bacterial sinusitis in 90% of patients. This inexpensive, simple test can decrease the cost and help increase the accuracy of the diagnosis, thus improving the care of patients with bacterial sinusitis.

Matrix metalloprotease-9 dysregulation in lower airway secretions of cystic fibrosis patients.

Matrix metalloproteases (MMPs) are proteolytic enzymes that regulate extracellular matrix turnover and aid in restoring tissue architecture following injury. There is an emerging role for extracellular matrix destruction in the pathogenesis of chronic neutrophilic lung diseases. In this study, we examined the expression and activity profiles of MMPs in lower airway secretions from cystic fibrosis (CF) patients, patients with acute respiratory failure (ARF), and normal controls. A discrete repertoire of MMP isoforms was found in the CF samples, with robust MMP-9 expression compared with normal controls and ARF. CF samples possessed increased levels of active MMP-9, as well as decreased amounts of tissue inhibitor of metalloprotease-1 (TIMP-1), a natural inhibitor of MMP-9. The CF inpatient samples demonstrated fully active MMP-9 activity compared with CF outpatients, ARF, and normal controls. CF samples also demonstrated increased human neutrophil elastase (HNE) levels compared with ARF and normal controls. To examine potential mechanisms for the protease dysregulation seen in the CF clinical samples, in vitro studies demonstrated that HNE could activate pro-MMP-9 and also degrade TIMP-1; this HNE-based activation, however, was not seen with MMP-8. A strong correlation was seen between HNE and MMP-9 activity in CF inpatient samples. Finally, the dysregulated MMP-9 activity seen in CF inpatient sputum samples could be significantly reduced by the use of MMP-9 inhibitors. Collectively, these findings further emphasize the proposed protease/antiprotease imbalance in chronic neutrophilic lung disease, providing a potential mechanism contributing to this proteolytic dysregulation.

Towards the identification of type II secretion signals in a nonacylated variant of pullulanase from Klebsiella oxytoca.

Pullulanase (PulA) from the gram-negative bacterium Klebsiella oxytoca is a 116-kDa surface-anchored lipoprotein of the isoamylase family that allows growth on branched maltodextrin polymers. PulA is specifically secreted via a type II secretion system. PelBsp-PulA, a nonacylated variant of PulA made by replacing the lipoprotein signal peptide (sp) with the signal peptide of pectate lyase PelB from Erwinia chrysanthemi, was efficiently secreted into the medium. Two 80-amino-acid regions of PulA, designated A and B, were previously shown to promote secretion of beta-lactamase (BlaM) and endoglucanase CelZ fused to the C terminus. We show that A and B fused to the PelB signal peptide can also promote secretion of BlaM and CelZ but not that of nuclease NucB or several other reporter proteins. However, the deletion of most of region A or all of region B, either individually or together, had only a minor effect on PelBsp-PulA secretion. Four independent linker insertions between amino acids 234 and 324 in PelBsp-PulA abolished secretion. This part of PulA, region C, could contain part of the PulA secretion signal or be important for its correct presentation. Deletion of region C abolished PelBsp-PulA secretion without dramatically affecting its stability. PelBsp-PulA-NucB chimeras were secreted only if the PulA-NucB fusion point was located downstream from region C. The data show that at least three regions of PulA contain information that influences its secretion, depending on their context, and that some reporter proteins might contribute to the secretion of chimeras of which they are a part.

Specific cleavage of beta-amyloid peptides by a metallopeptidase from Xenopus laevis skin secretions.

Dactylysin (EC 3.5.24.60) is a metalloendopeptidase first isolated from the skin granular gland secretions of Xenopus laevis. This peptidase hydrolyzes bonds on the amino-terminus of singlets and between doublets of hydrophobic amino acids and was considered to play a role in the in vivo inactivation of biologically active regulatory peptides. Here, we show that dactylysin has also the ability to cleave human beta[1-40]-amyloid peptide and related peptides. Cleavage of the wild type beta[1-40]-amyloid peptide form, and to a lesser extent Flemish and Dutch mutants, occurred predominantly at the His14-Glu15 bond. We demonstrate that frog skin exudate contains a full-length amyloid protein precursor detected by immunochemical cross-reactivity with monoclonal antibody against C-terminal human amyloid protein precursor. The possibility that dactylysin, might be involved in normal catabolism of beta amyloid peptide of Xenopus laevis is discussed.

The synthesis of phosphate-repressible alkaline phosphatase do not appear to be regulated by ambient pH in the filamentous mould Neurospora crassa

In order to investigate further the adaptive response of moulds to ambient pH, we have measured by ELISA the pho-2-encoded Pi-repressible alkaline phosphatase synthesised by Neurospora crassa. We showed that the 74A and pho-2A strains of this mould secrete similar amounts of the pho-2-encoded enzyme irrespective of ambient pH, when both the preg and pgov genes are not functional, i.e., in strains nuc-2+ growing under Pi-starvation. This suggests that pho-2, which is responsive to Pistarvation via the action of genes nuc-2, preg, pgov and nuc-1, is not a gene responsive to ambient pH and that the differential glycosylation observed for the Pi-repressible alkaline phosphatase retained by the mycelium at pH 5.6 or secreted into the growth medium at pH 8.0 is the genetic response to ambient pH sensing in N. crassa.