Abnormalities of the intracellular metabolism of the hydrophobic surfactant proteins SP-B and SP-C and their precursors may be causally linked to chronic childhood diffuse lung diseases. The profile of these proteins in the alveolar space is unknown in such subjects.We analyzed bronchoalveolar lavage fluid by Western blotting for SP-B, SP-C and their proforms in children with pulmonary alveolar proteinosis , children with no SP-B (n = 6), children with chronic respiratory distress of unknown cause , in comparison to children without lung disease (n = 15) or chronic obstructive bronchitis (n = 19).SFTPB or SPTPC genes were found. Pro-SP-C forms were identified at very low frequency. Their presence was clearly, but not exclusively associated with mutations of the SFTPB and SPTPC genes, impeding their usage as candidates for diagnostic screening.Pro-SP-B of 25–26 kD was commonly abundant in all groups of subjects, suggesting that their presence is not of diagnostic value for processing defects. In contrast, pro-SP-B peptides cleaved off during intracellular processing of SP-B and smaller than 19–21 kD, were exclusively found in PAP and cRD. In 4 of 6 children with no SP-B, mutations of Immuno-analysis of the hydrophobic surfactant proteins and their precursor forms in bronchoalveolar lavage is minimally invasive and can give valuable clues for the involvement of processing abnormalities in pediatric pulmonary disorders. SFTPB) [SFTPC gene on chromosome 8 [SFTPB mutations, aberrant pro-SP-C forms (Mr 6–12 kD) are observed [SFTPB mutations [Pulmonary surfactant is a highly surface active complex of lipids and specific proteins, including surfactant proteins (SP-) A, B, C and D . The maiSFTPB)  and tranSFTPB) -7. SP-C mosome 8 -11 is inmosome 8 ,13. In lobserved . Similarutations .Bronchoalveolar lavage (BAL) is a commonly used first line diagnostic tool to sample the alveolar space content and this technique is much less invasive than open lung biopsy. Thus the profiles of SP-B, SP-C and their propeptide precursors present in the extracellular, intraalveolar space represent a potential diagnostic tool for assessment of neonatal and childhood lung disease.Neonates with respiratory distress of unknown cause are likely candidates for abnormalities of SP-B and SP-C metabolism. Similarly, but much less appreciated, SP-B and SP-C abnormalities might play a role in infants or older children with chronic respiratory distress developing beyond the neonatal period. Pediatric pulmonary alveolar proteinosis (PAP) is a rare abnormality of the surfactant metabolism, characterized by the accumulation of large amounts of surfactant in the alveolar space, leading to gas exchange abnormalities ,16. In cUsing defined pediatric patient populations, Western blotting of BAL identified several distinct banding profiles for the hydrophobic surfactant proteins and their precursors. These data support the feasibility of using immunoanalyses of BAL fluid to evaluate chronic pediatric pulmonary disorders in more detail.The lavage effluents from 15 children without lung disease and 19 children with chronic obstructive bronchitis were used as controls or disease controls, for comparison with the lavage effluents that were available from our previously described cohort of neonatal, pediatric or juvenile patients with respiratory distress of unknown cause. These children were seen in western European medical hospital centers (mainly from France and Germany) and were analyzed for a genetic defect leading to deficiency in SP-B and SP-C ,18.The lavage effluents from the children without lung disease were aliquots obtained previously in a study that assessed inflammation in children with chronic tracheostoma in comparison to these controls . The lavChildren with chronic obstructive bronchitis in whom anomalies of the airways, cystic fibrosis, primary ciliary dyskinesia, gastro-esophageal reflux, immuno deficiencies, allergic asthma and passive smoke exposure were excluded as causes and in whom a lavage was performed during the diagnostic work up, were used as a disease control group. The obstruction was determined by chest auscultation during the course of the disease. Details of these patients are given in table From the cases with SP-B deficiency we initially described, sufficient BAL material for analysis was available from 6 neonates , now labeled no-SP-B 1–6. All these babies had respiratory distress, and alveolar infiltrates with various degrees of interstitial involvement. A congenital heart disease or a lung disease due to mycoplasma, chlamydia, and viruses had also been ruled out. Details on the subjects are given in table From the cases with pulmonary alveolar proteinosis, sufficient BAL material for analysis was available from 15 children , now labeled PAP 1–15. Most of these cases were less severely affected, had dyspnea and progressive cough, sometimes accompanied by cyanosis, finger clubbing, failure to thrive in the younger ones, and asthenia or weight loss in the others. Chest x-ray showed typical alveolar as well as interstitial infiltrates  of the initial 15 patients and from another infant born at 36 wks of gestation, with acute respiratory distress and development of chronic respiratory distress of unknown cause, after exclusion of SP-B, SP-C deficiency, and pulmonary alveolar proteinosis. None of these children was investigated for ABCA3 mutations. The children were labeled cRD 1–7 and their outcomes are given in table Routinely, the fluid recovered from BAL  was pooled and the cells separated before analysis. Alternatively, in very sick neonates, repetitive tracheal aspirates after the instillation of 1 ml 0.9%NaCl/kg b.w. were collected over time periods of several hours up to a week, pooled and used for biochemical analyses.All antisera used were polyclonal and raised in rabbits. The antibodies against SP-B (c329) and SP-C (22/96) were gifts from Dr W. Steinhilber, Altana AG, Konstanz, FRG and were used at a dilution of 1:10,000 . The antTotal protein content of the samples was determined with the Biorad Protein Assay Kit , which is based on the method by Bradford . Ten to Surfactant proteins and their pro-forms were detected on the PVDF membrane by immunoblot using the polyclonal rabbit antisera described in detail above, and the enhanced chemiluminescence assay  with horseradish peroxidase conjugated goat anti-rabbit polyclonal anti-IgG .To verify the specificity of the antibodies used to probe the pro-forms of SP-B and SP-C, a duplicate blot was prepared in each case and probed with an antibody solution containing 1 μM of the peptide, against which the antibody was raised. Antigen specific bands on the blot disappeared under these conditions. The blots were developed by exposure of X-ray film  to the blots.In the group of controls blots were first incubated with antibody against CTERMB and after that with the SP-B antibody respectively first with antibody against SP-C and after that with the pro-SP-C antibody, with and without competing peptide. In the other groups there were separate blots for each incubation with antibodies against SP-B and SP-C and their proforms, with and without competing peptide. Under these conditions the assay could detect about 2.5 ng of SP-B or SP-C per lane. In several experiments, aliquots of a patient with pro-SP-C forms were run in parallel as a positive control for pro-SP-C forms.Immunoblots and silver stained gels were scanned with the Fluor-S MultiImager  gel documentation system, and the resulting images were analyzed with the Software MultiAnalyst .To determine if the proteins that reacted with the CTERMB antibody on the immunoblots were glycosylated, the samples were deglycosylated before applying them on the gel (4). In brief, 1 unit of recombinant N-glycosidase F  was added to 500 μl incubation buffer . The vacuum dried sample was resuspended in 20 μl of this solution and incubated for 15 h at 37°C. The sample was then vacuum dried and analyzed by Western immunoblot.SFTPB mutation screening, first the 121ins2 frame-shift mutation was searched using the restriction enzyme cleavage SfuI endonuclease by PCR. In 121ins2-negative patients, SFTPB exons 1–11 and the promoter region were PCR-amplified and the purified PCR products served as templates in the sequencing reaction using Ready Reaction Dye Terminator Cycle Sequencing Kit With AmpliTaq® DNA Polymerase, FS  with forward and reverse PCR oligonucleotides used as extension primers. Extension products were analyzed using the ABIPRISM™ 310 Genetic analysis System (PEBiosystems), as previously reported in detail [SFTPC exons 1–6 were analysed [For n detail . Similaranalysed .Statistical calculations were performed with the Software GraphPad Prism 4.0 . Differences in nonparametric values were calculated with the Kruskal-Wallis test. For pair wise comparisons of groups we used Dunn's test (2). Differences in frequencies were calculated with the Fisher exact test. Correlation coefficients were determined according to Pearson. Results with a p ≤ 0.05 were considered significant.The children with chronic obstructive bronchitis had a slight increase in neutrophils ) compared to children without lung disease ; p = 0.035) and a somewhat lower viability  and 90%  in children without lung disease; p = 0,035). The other variables did not differ and were within the normal range, i.e. children with chronic obstructive bronchitis: total cell count 150/μl , macrophages 80%  of total cells, lymphocytes 10% (4/14), eosinophils 0%  and recovery was 54%  and the children without lung disease: total cell count 115/μl , macrophages 87%  of total cells, lymphocytes 11.5% , eosinophils 0%  and recovery was 48% .r of 5.0 kDa was present in both controls and children with chronic bronchitis, whereas pro-SP-C forms were never detected in BAL  exclusively in BAL from patients with alveolar proteinosis and chronic respiratory distress. Taken together, the data suggest that immunobiochemical analysis of BAL can detect abnormalities in surfactant biosynthesis and metabolism associated with a variety of parenchymal lung diseases.In this study we defined the presence and characteristics of SP-B, SP-C and their processing forms in bronchoalveolar lavages from children with severe chronic respiratory distress and in comparison groups of normal children and children with chronic obstructive bronchitis Fig. . The majSFTPB  cells; however, complete processing of the precursor to the biologically active, mature peptide occurs only in type II cells. Clara cells merely generate the 25 and 42 kDa precursors [An important finding of this study is the regular detection of certain pro-SP-B peptides in ecursors . Thus, tecursors ,29. HereIn children with pulmonary alveolar proteinosis we discovered increased amounts of a 19–21 kD intermediate which reacted against C-terminal pro-SP-B antisera and with the NFLANK SP-B antibody. This finding of a complex pro-SP-B intermediate containing both the C-terminal propeptide and a vestigial N-terminal propeptide (approximate residues 186–201) extends the work of Brasch and colleagues who also noted the presence of pro-SP-B forms containing C-terminal propeptide epitopes . ConsistChildren with chronic respiratory distress of unknown cause (cRD) exhibited the 40–42 kD proprotein with increased frequency. The N'-terminal peptides liberated from pro-SP-B pre-protein during intracellular processing, i.e. 13–15 kDa peptides or smaller fragments and reacting with NFPROX, were found exclusively in both cRD and PAP . The presence of these relatively rarely observed bands at 11 to 15 kDa was not related to specific clinical features of the subjects, i.e. more pronounced lung injury, high protein to phospholipids ratio or high abundance of SP-B. Both, a 9 kDa intermediate, reactive to NFLANK  and a 9 SFTPC mutation. On the other hand, not all infants with SFTPB (496delC) or SFTPC (R167Q) mutations had pro-SP-C in their lavages. Thus the presence of pro-SP-C in lavages may give strong, but surely not definitive, diagnostic evidence for SP-B and SP-C processing defects.Pro-SP-C peptides were never detected in the control groups. This is in agreement with an earlier observation on a limited number of samples . HoweverSFTPB mutation clearly differed in molecular weights from those detected in SP-B deficiency, suggesting that several processing defects may result in aberrant pro-SP-C in the alveolar space.The aberrant pro-SP-C species observed in patients with SP-B deficiency carrying the 121ins2 mutation consists of a N-terminal extension of SP-C by the N-flanking 12 aminoacids of pro-SP-C . The proHere we defined the presence and characteristics of SP-B, SP-C and their processing forms in bronchoalveolar lavage fluids from children with severe chronic respiratory distress and in comparison groups of normal children and children with chronic obstructive bronchitis. Pro-SP-B of 25–26 kD was commonly detected in all groups, suggesting that this form currently does not appear to be of great diagnostic value for processing defects. In contrast, pro-SP-B of 19–21 kD was increased in children with alveolar proteinosis while the cleaved flanking propeptides liberated during intracellular processing of pro-SP-B were exclusively found in these children and in chronic respirator distress of unknown cause. Furthermore, although identified at low frequency, pro-SP-C forms when present in the BAL suggest the presence of one of the parenchymal diseases studied in this report. Though often associated with mutations in SFTPB and SFTPC genes, this was not an exclusive finding limiting the usage of pro-SP-C as a surrogate for SFTP/SFTPC diagnostic screening procedures. Taken together, our results demonstrate that significant perturbations in the metabolism of these hydrophobic surfactant proteins occur in a variety of chronic lung diseases.The author(s) declare that they have no competing interests.MG designed the study, categorized and organized the subjects, wrote initial drafts of the manuscript, SS performed the blots, MT and MB determined the genotype of the patients, MG, SS, MS, AB, MT and MB collected the case histories, reviewed the subjects data and clinical courses, SG and MFB participated in the design for the methods to blot for the surfactant proteins, helped to organize the data and the results, and to prepare the manuscript. All authors read and approved the final manuscript.


We examined protein kinase C (PKC) activity in the cytosolic and particulate fractions of homogenates obtained from 25 colorectal adenomas and adjacent normal mucosa in patients with colorectal carcinoma. The total PKC activity of colorectal adenomas was significantly reduced compared with that of normal mucosa in all cases (122 +/- 45.8 vs 174 +/- 50.5 pmol min-1 mg-1) (means +/- s.d.) (P less than 0.001). The particulate fraction PKC activity of adenomas was also significantly lower than in normal mucosa (71.4 +/- 31.3 vs 115 +/- 39.6 pmol min-1 mg-1) (P less than 0.001). Adenomas were classified by size, histological type and degree of dysplasia. The average particulate PKC activity ratio  of tubulovillous adenomas or those with severe dysplasia was significantly reduced compared with that of tubular adenomas or tumours with mild and moderate dysplasia (both P less than 0.001), while there were no significant differences in the cytosolic PKC activity ratio. The particulate PKC activity ratio decreased significantly with increasing adenoma size (P less than 0.001), while the cytosolic ratio again showed no difference. These findings suggested that the particulate PKC activity ratio had a possible correlation with the malignant potential of colorectal adenomas and that this ratio may be a useful biological indicator of colorectal carcinogenesis.


Pulmonary surfactant protein (SP)-D modulates immune and inflammatory responses in the lung. We have shown previously that SP-D plays a protective role in a mouse model of allergic airway inflammation. Here we studied the role and regulation of SP-D in O3-induced inflammatory changes in the lung.Ozone (O3 exposure in mouse strains with genetically different expression levels of SP-D we exposed Balb/c, C57BL/6 and SP-D knockout mice to O3 or air. BAL cellular and cytokine content and SP-D levels were evaluated and compared between the different strains. The kinetics of SP-D production and inflammatory parameters were studied at 0, 2, 6, 12, 24, 48, and 72 hrs after O3 exposure. The effect of IL-6, an O3-inducible cytokine, on the expression of SP-D was investigated in vitro using a primary alveolar type II cell culture.To evaluate the effects of O3 exposure. IL-6, which was highly up-regulated in O3 exposed mice, was capable of inducing the expression of SP-D in vitro in a dose dependent manner.Ozone-exposed Balb/c mice demonstrated significantly enhanced acute inflammatory changes including recruitment of inflammatory cells and release of KC and IL-12p70 when compared with age- and sex-matched C57BL/6 mice. On the other hand, C57BL/6 mice had significantly higher levels of SP-D and released more IL-10 and IL-6. Increase in SP-D production coincided with the resolution of inflammatory changes. Mice deficient in SP-D had significantly higher numbers of inflammatory cells when compared to controls supporting the notion that SP-D has an anti-inflammatory function in our model of O3 exposure and elevation of SP-D in the lung is associated with the resolution of inflammation. Absence or low levels of SP-D predispose to enhanced inflammatory changes following acute oxidative stress.Our data suggest that IL-6 contributes to the up-regulation of SP-D after acute O O3 is associated with an immediate impairment of lung function and contributes to increased morbidity in patients with asthma and chronic obstructive pulmonary disease (COPD) [3 increases levels of the vascular adhesion molecules P-selectin and ICAM-1 in airway lavages and bronchial tissue and induces influx of neutrophils and mast cells [3-induced acute pathological changes are characterized by an influx of neutrophils and airway hyperresponsiveness (AHR). Long-term or chronic exposure to O3, however, attenuates inflammatory responses, a phenomenon referred to as adaptation [3 exposure) is largely IL-6 dependent but the late adaptive response  involves mobilization of pulmonary antioxidants and leads to hypertrophy and metaplasia of epithelial cells in the upper as well as in the lower respiratory tract [Ozone (Oe (COPD) ,2. Even st cells . The O3-aptation . The earry tract -8. The m3 but also express different levels of SP-D, we hypothesized that there is a causal relationship between these two characteristics. To test our hypothesis and to better characterize the role SP-D may play in O3-induced inflammation, we used mice with reportedly different SP-D levels [3-inducible cytokine IL-6 selectively induced the expression of SP-D in vitro.Surfactant protein-D (SP-D), a pattern-recognition molecule of the pulmonary innate immune system, enhances the phagocytosis and clearance of various inhaled pathogens, allergens, and apoptotic cells in the lung and serves as a potent immuno-modulator -11. SP-DD levels  and miceD levels  We foundad libitum. The protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania and GlaxoSmithKline.All experimental animals used in this study were housed under pathogen-free conditions. Experiments were performed between 8 and 12 weeks of age. Animals received water and food 3 exposure in mouse strains with different SP-D levels, we used Balb/c and C57BL/6 mice and exposed them to 3.0 ppm O3 for a 2 hrs period. BAL SP-D levels, cellular and cytokine content were evaluated 6 hrs later. To define the kinetics of the O3-induced inflammation and SP-D production in more detail, C57BL/6 mice  were exposed to 3.0 ppm O3 or air for a 2 hrs period and studied 2, 6, 12, 24, 48, and 72 hrs later. Finally, to study the effects of a complete lack of SP-D, SP-D knockout mice [3 for 2 hrs or to 0.5 ppm O3 for 24 hrs. BAL was performed 12 hrs (2 hrs exposure) and 24 and 48 hrs (24 hrs exposure) later. In all experiments age- and strain-matched controls were exposed to room air concurrently. The levels and exposure times were based on a previous pilot study (unpublished) and were chosen to accommodate all three different mouse strains and to allow us to study and compare the temporal inflammatory changes. After exposure, groups of mice (n = 6) were euthanized and BAL was performed.To evaluate the effects of Oout mice  were expg and the pellet was resuspended in 1 ml of PBS. Total cell counts were determined from an aliquot of the cell suspension. Differential cell counts were done on cytocentrifuge preparations  stained with Kwik™Diff , and 200 – 500 cells were counted from each individual.BAL was performed as previously described . Briefly®100™ assay System  and Endogen®SearchLight™ Mouse Cytokine and Chemokine arrays , respectively, and was performed according to the manufacturer's instructions.Cytokine and chemokine levels in the cell-free BAL were determined as part of a LuminexTotal protein from cell free supernatant of the BAL fluids was assessed using the Bradford Assay . Western blots for SP-D levels in cell-free BAL fluid were performed as previously described . BrieflySP-D protein recovered from BAL was quantified by ELISA using an in-house rabbit polyclonal anti-SP-D antibody . AliquotLung alveolar type II cells were isolated from neonatal Sprague-Dawley rats  as previously described ,18. Our t-test was used for two-group comparisons. Data are expressed as mean ± SEM, p < 0.05 was considered statistically significant.Statistical analysis was performed with Prism4 software . Multiple comparisons were performed by one-way-ANOVA followed by Barlett's test or Post test for linear trend. Student 3. In these experiments BAL SP-D levels in the different moue strains were normalized to 100%, i.e. their mean baseline level. We previously published results of a direct comparison between naïve Balb/c and naïve C57BL/6 mice in which Western blot analysis demonstrated that C57BL/6 mice had approximately twice as much SP-D as Balb/c mice [3-exposure caused a significant drop in SP-D levels in Balb/c (but not in C57BL/6) in comparison with air exposed controls (p = 0.0249). Six hours after O3 exposure, the amount of SP-D recovered from the BAL  was significantly lower in Balb/c mice compared with C57BL/6 mice (p = 0.0027).We have previously reported that SP-D levels in Balb/c and C57BL/6 mice differ under normal conditions as well as upon allergen sensitization and challenge . Since Sb/c mice . As show3 challenge. In contrast, C57BL/6 mice released more IL-10 and IL-6  compared to C57BL/6 mice  and followed the onset and resolution of the inflammation at 0, 2, 6, 12, 24, 48, and 72 hrs after O3 exposure. ELISA for SP-D and SP-A recovered from the BAL fluid of O3-exposed mice showed significant elevation of SP-D levels with approximately 50 % increase from baseline 12 hrs post-exposure. SP-D continued to increase until the last time point of the experiment (72 hrs) when SP-D levels were about 150 % above control levels . KC levels were back to baseline by 24 hrs. Interestingly, IL-6 was highly induced, with a peak at the 6 hrs time point and a return to baseline levels 48 hrs post-exposure , a sub-acute exposure, this finding was confirmed, since SP-D -/- mice had increased numbers of inflammatory cells both 24 and 48 hrs after cessation of O3 exposure  on mouse chromosome (MMU) 14 contains one or more relevant genes. It is noteworthy, that the gene coding for SP-D is located on MMU 14 in the area of 37.2 Mb. Additionally, two recent reports identified sequence polymorphisms in the human SP-D gene that lead to differences in constitutive serum levels and influence the multimeric assembly and function of SP-D protein [The drop in SP-D levels seen in Balb/c mice 6 hrs after Oal cells . It is lhrs Fig.  [13,18,2ammation ,28. Thisresponse , no hostresponse . Their i protein ,30.3 exposure in C57BL/6 mice. However, whether or not SP-D has a direct regulatory effect on KC remains to be determined and is the current focus of our studies.Neutrophils play a vital role in the pulmonary host defense. However, due to their release of large amounts of histo-toxic and pro-inflammatory agents, these cells can cause significant tissue damage. Hence, a stringent control of neutrophil priming, recruitment, activation, apoptosis and clearance is crucial to confine tissue damage . Mice ge3 induced a significant rise in BAL SP-D suggesting that there are other mechanisms to promote SP-D expression during a non-allergen induced inflammation. Indeed, our in vitro studies using rat alveolar type II cells show that IL-6 is capable of inducing SP-D production. In a different model of airway inflammation, elicited by allergic sensitization and challenge, we have previously shown that a rapid release of pro-inflammatory cytokines is followed by a relatively slow, gradual elevation of SP-D protein levels in the airways, with a peak 48 hours after allergen challenge [3[et al. and Ward et al. also provide indirect support of our findings that IL-6 is important in launching a protective pulmonary response. Our results showed that the neutrophil chemoattractant KC and the immunosuppressive cytokine, IL-10 were also elevated in the BAL fluid after O3 exposure. While the possibility was raised that these mediators could contribute to the up-regulation of SP-D, we found no evidence that lung epithelial cells would express receptor or show any functional response to exogenous IL-10 or KC [Ozone exposure does not induce the release of classical Th2 type cytokines such IL-4 or IL-13 which have been shown to stimulate the expression of SP-D . Howeverhallenge . In accohallenge . Whetherllenge [3. On the llenge [3. These f10 or KC .3-induced injury and during the resolution of the inflammation is confirmed in SP-D knockout mice, as the absence of SP-D resulted in enhancement of the inflammation in these animals. We propose that IL-6 may contribute to the up-regulation of SP-D expression, which in turn inhibits pro-inflammatory changes and promotes resolution of the inflammation following O3 exposure.Strain dependent differences in SP-D production are associated with differences in the severity of the inflammation. The importance of SP-D in the protection against the initial OThe author(s) declare that they have no competing interests.SK participated in the animal experiments, BAL cell counts, Western blots, data analysis and prepared the manuscript.FRP supervised the animal experiments using SP-D deficient mice and advised on data analysis.YC participated in most of the animal experiments and developed the ELISA for SP-D.MG performed the time course study in C57BL/6 mice.RM performed the animals experiments using SP-D -/- mice.GK developed a template for statistical data analysis and participated in the preparation of the manuscript.MFB gave helpful advice for data analysis and preparation of the manuscript.MS participated in the design of the experiment, took part in the time course study and gave helpful advice for the preparation of the manuscript.RAPJr. gave helpful advice for the preparation of the manuscript.AH designed the study, coordinated the experiments, and helped to draft the manuscript.


Molecular Pain , describes its potential role in feed-forward inhibition in lamina V of the dorsal horn of the spinal cord. This inhibition seems to function through a direct excitation of GABAergic interneurons by substance P released from primary afferent fibers and has a distinct temporal phase of action from the well-described glutamate-dependent feed-forward inhibition. It is believed that through this inhibition, substance P can balance nociceptive output from the spinal cord.Substance P (SP) is a neuropeptide well known for its contribution to pain transmission in the spinal cord, however, less is known about the possible modulatory effects of SP. A new study by Gu and colleagues, published in  Upon noxious stimulation, particularly intense stimulation, tachykinins such as SP and neurokinin A (NKA) are released from primary afferent fibers and excite dorsal horn neurons via activation of the neurokinin-1 and neurokinin-2 receptors (NK1R and NK2R), respectively [The spinal cord dorsal horn is one of the relay stations for nociceptive information transmitted by peripheral sensory afferents. Some of these sensory afferents are substance P (SP) and glutamate-containing Aectively ,4. Mice ectively , both diectively . One posectively . Similarectively . Other sectively .Molecular Pain, Gu and colleagues used a combination of electrophysiological, pharmacological, genetic and behavioral techniques in rats and found that SP can modulate inhibitory transmission in lamina V of the spinal cord dorsal horn, thereby exerting an analgesic effect on nociceptive sensory processing [Although many studies have highlighted the importance of SP in pain transmission, the synaptic mechanisms underlying the antinociceptive effect of SP remain unclear. In a recent study published in ocessing . ThroughThe neuronal network in the spinal dorsal horn is extremely complex. Within this circuit, inhibitory interneurons containing GABA and/or glycine play important roles in controlling network excitability. Activation of these neurons can initiate feedback inhibition and feed-forward inhibition, which are believed to be critical for the fine tuning of sensory information at the spinal level . SP and Gu and colleagues stimulated the dorsal root of the spinal cord, which contains primary afferent fibers, with high frequency stimulation to cause the release of both SP and glutamate in the presence of N-methyl-D-aspartate (NMDA) receptors and non-NMDA receptors antagonists. They found a robust and long-lasting increase in both the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs). Similar results were obtained with chemical stimulation using capsaicin, which also excites primary afferent fibers to release both glutamate and SP . This in-/-) mice. Taken together, these results demonstrate that increases of SP can drive inhibitory activity in the dorsal horn of spinal cord.Could SP be responsible for glutamate-independent feed-forward inhibition? First, among different neuropeptides such as galanin, neuropeptide Y, somatostatin, calcitonin gene-related peptide and SP tested, only SP enhanced the frequency and amplitude of sIPSCs. These results demonstrate that the effect of SP is selective. Second, application of NK1R antagonists blocked both electrical and chemical stimulation-induced increases of sIPSCs. Third, exogenously applied SP- or stimulation-induced increases of sIPSCs were abolished in NK1R knockout (NK1R2+. Since SP had no effect on both miniature inhibitory postsynaptic currents (mIPSC) and evoked inhibitory postsynaptic currents (eIPSC), SP-driven feed-forward inhibition is more likely due to the direct excitation of GABAergic interneurons by SP releasing primary afferent fibers than to action potential-independent modulation of activity within the synapse.To further explore the neuronal mechanisms underlying SP-driven feed-forward inhibition, pharmacological tools were used to show that pertussis toxin-sensitive G proteins such as Gi and Go are involved in SP-driven feed-forward inhibition. Moreover, GIN mice, a strain of transgenic mice that express enhanced green fluorescent protein in GABAergic neurons, were used to show that SP can directly excite some GABAergic neurons by inducing prolonged depolarization, firing of action potentials, and increases of intracellular Caδ- and C- fibers, induces the release of both glutamate and SP, mainly in lamina I and V. In lamina I, SP release activates projection neurons that relay pain-related information to higher centers in the nervous system to process the different qualities of the stimulus. In lamina V, SP binds pertussis toxin-sensitive Gi/Go-coupled NK1R on inhibitory interneurons, which leads to a cascade of downstream events that may result in changes in the activity of non-selective cation channels or inwardly rectifying potassium channels [SP-driven feed-forward inhibition is summarized in Figure channels  ultimateEven though SP coexists with glutamate in primary nociceptive afferent synaptic terminals, their roles in various pain conditions may be distinct considering that higher frequencies of primary afferent stimulation are required to evoke the release of SP compared to glutamate . Consist-/- mice. However, electrical stimulation-induced immediate eIPSCs, which could be blocked by glutamate receptor antagonists and are believed to be glutamate-driven feed-forward inhibition, were similar between wild-type and NK1R-/- mice. Taken together, these results provide evidence for a long-lasting, glutamate-independent SP-driven feed-forward inhibition that is distinct from the immediate pulse-by-pulse glutamate-driven feed-forward inhibition.To address this issue, Gu and colleagues first studied SP-driven feed-forward inhibition in the absence of glutamate receptor antagonists . Their rAfter confirming the existence of the SP dependent feed-forward inhibition as well as delineating its distinct temporal phase, Gu and colleagues searched for the functional significance of the SP-driven feed-forward inhibition in the spinal cord dorsal horn. They hypothesized that the SP-driven feed-forward inhibition may serve to balance neuronal activity by counteracting SP-mediated excitatory nociceptive responses. This hypothesis challenges the traditional role of SP as a purely "pro-pain" substance in favor of a more homeostatic role.A clever set of experiments were devised to address this question. First, SP-mediated nociceptive transmission in the superficial dorsal horn was blocked with a selective lesion of NK1R-expressing neurons using an intrathecal injection of SP-conjugated saporin (SP-SAP) ,18. It ivs lamina V) and an the type of neuron it synapses with (projection neurons vs inhibitory neurons).Behavior responses to nociceptive heat stimuli were studied in SP-SAP treated animals after capsaicin treatment and intrathecal application of NK1R antagonists, resulting in two major findings . First, The elegant study by Gu and colleagues proposes a novel cellular mechanism for the neurokinin system in pain transmission and modulation. Dissecting its intricacies will not only contribute to a better understanding of how somatosensory inputs, including pain information, are coded within the spinal dorsal horn, but may also foster the development of more efficacious treatments for pain control.i/Go-coupled NK1R is reported to activate phospholipase A2 and mobilize arachidonic acid [Due to the important implications of SP-driven feed-forward inhibition in nociceptive transmission, some limitations of these findings need to be addressed. (1) SP can be released from at least three different sources: primary afferent fibers, descending projections fibers and local neurons. The current study uses dorsal root stimulation so it is likely that the primary afferents are the cause of the SP release ,21. Sincnic acid . The finAlthough the complexity of nociceptive transmission and modulation in the spinal cord make it difficult to address all questions in a single model, we believe the description of a SP-driven feed-forward inhibition to be a crucial finding towards the understanding of the role of SP in sensory transmission, modulation and plasticity.


Noise is the most common hazardous agent at workplaces. Noise induced hearing loss (NIHL) has been known since the industrial revolution. Although NIHL is permanent, irreversible and frequent, it is preventable. The economic costs of NIHL have been estimated to be about billions of dollars. Besides, cigarette smoking is a common habit worldwide, and according to some recent studies smoking and noise may act in common causal pathways for hearing loss.A cross-sectional study was designed to study the effect of smoking on NIHL in 206 male smoker workers and 206 male non-smoker workers in a large food-producing factory, in which workers were exposed to noise levels exceeding 85dBA. To determine noise exposure level, we used sound level measurements reported by industrial hygienists.A qualified audiologist assessed hearing acuity by using standardized audiometric procedures assuring at least 14 h of noise avoidance.We observed that the percentage of workers with hearing threshold differences of greater than or equal to 30 dB between 4000 Hz and 1000 Hz in both ears were 49.5% and 11.2% in smoker and non smoker groups, respectively , and the percentage of workers with a hearing threshold of greater than 25dB at 4000 Hz in the better ear were 63.6% and 18.4% in smoker and non smoker groups, respectively. This difference was statistically significant after adjustment for age and exposure duration.It can be concluded that smoking can accelerate noise induced hearing loss, but more research is needed to understand the underlying mechanisms. Accurate follow up of smoker workers who are exposed to noise levels exceeding 85 dBA is suggested. Smokers should periodically attend educational courses on "smoking cessation", especially in noisy workplaces. Noise is the most pervasive hazardous agent at workplaces ,2. ApproGenerally, NIHL (Noise- Induced Hearing loss) is a sensorineural hearing deficit which begins at higher frequencies 3,000 to 6,000 Hz) and develops gradually as a result of chronic exposure to excessive sound levels ,000 Hz a,8. EarlyAccording to NIOSH's  estimate, 14% of the working population in the USA is employed in environments where the A-weighted sound exposure level exceeds 90 decibels. In some manufacturing plants such as those producing textile mill products, lumber and wood products and food and kindred products this ratio exceeds 25% ,13. LikeThis cross-sectional study was conducted in a large food-producing factory situated in the capital city of Iran. There were no other known occupational hazards affecting hearing acuity, but noise. Noisy work-places were selected based on industrial hygienists' reports on sound level measurements; these measurements were performed annually, and saved as "noise monitoring records". In this study, the last 3 records were used and all 6 parts of the factory in which the noise levels exceeded 85 dBA in all 3 records, were selected as noisy workplaces. At different stations of these 6 parts the lowest and highest noise levels were 87dB and 94dB, respectively. Almost all non-noisy parts were offices. The turnover of workers among noisy parts was so fast that it was impossible to estimate the exact level of exposure of each worker, but the turnover of worker between noisy and non-noisy parts was slow and exposure duration could be calculated almost exactly. Our study population were all the male smoker workers of these noisy parts (n = 253). Female workers were not enrolled in this study because of the low noise exposure in this group . After filling in the questionnaire, all workers with a history of ototoxic drug use, diabetes mellitus, hyperlipidemia, hypothyroidism, severe or frequent ear infection, ear surgery, exposure to non-occupational noise , noise exposure in previous job/jobs, unilateral or conductive hearing loss or any kind of hearing loss with a known etiology except noise exposure were excluded (n = 47). 206 male smoker workers who worked in noisy workplaces and participated in periodic medical examinations became the objective population. All the subjects participated voluntarily in this study and an inform consent was filled in by each (written consent in Persian was obtained).Smoking was ascertained based on the following questions: 'Have you ever smoked?' and 'Do you smoke?' Duration of smoking, working in noisy workplaces and age were recorded in years. A qualified audiologist assessed hearing acuity using standardized audiometric procedures assuring at least 14 hours of noise avoidance. The exams were done in an isolated acoustic room meeting the ANSI S3.1–1991 standards with a diagnostic audiometer . The pure-tone hearing thresholds at 500, 1000, 2000, 3000, 4000, and 6000 Hz were measured for air and bone conduction, in both ears.2 test was applied to examine differences between the study groups for categorical parameters. Association of hearing loss with smoking habit was examined by binary logistic regression. The modeling strategy followed Hosmer and Lemeshow [The hearing threshold differences of greater than or equal to 30 dB between 4000 Hz and 1000 Hz in both ears, hearing threshold level of greater than 25dB at 4000 Hz in the better ear, and hearing threshold at 4000 Hz in the better ear were used as the indicators of hearing loss. After the initial review of the results of our study we decided to complete our study by choosing non-smoker workers as a reference group. There were 508 male non-smoker workers in these noisy parts. So 206 non-smoker male workers, who worked in the same parts were randomly selected as the reference group (42 workers were excluded by applying the exclusion criteria). Inclusion and exclusion criteria for this group were the same as the smokers group. Data collection method was also the same. Thus the only difference between the two groups was cigarette smoking. This study was approved by the Ethics Committee of Tehran University of Medical Sciences. Data were analyzed using SPSS version 11.5. The following statistical tests were applied in analysis of the data presented in this article. The two-sample t test was applied for testing differences between the study groups for quantitative parameters. Pearson χLemeshow . All varThe percentage of workers with hearing threshold differences of greater than or equal to 30 dB between 4000 Hz and 1000 Hz in both ears were 49.5% in smokers and 11.2 % in non-smokers , and the percentage of hearing threshold level of greater than 25dB at 4000 Hz in the better ear were 63.6% and 18.4% in smoker and non-smoker groups, respectively . Descriptive statistics of all 412 workers are shown in Table As shown in Table Age-related hearing loss (presbycusis) is one of the most common causes of high frequency hearing loss and its effects begin around the age of 40. 57.3% of non-smokers and 68.4% of smokers were older than 40, and this difference was statistically significant. To adjust the effect of age, we divided all workers into 2 groups; aged 40 or less (n = 153) and older than 40 (n = 259) and then each group was divided into 2 subgroups; smokers and non-smokers. Then we compared the hearing threshold difference between 4000 Hz and 1000 Hz of greater than or equal to 30 dB in both ears, hearing threshold of greater than 25dB at 4000 Hz in the better ear by chi square and means of hearing threshold at 4000 Hz by t-test between the two subgroups of smoker and non-smoker workers. This was done separately for the two age groups of 40 or less and older than 40  is one of the most common causes of high frequency hearing loss and its effects begin around the age of 40 ,33. The It can be concluded that smoking may accelerate noise induced hearing loss. The cross- sectional design of this study does not permit causal inference from the observed associations. But the observed relationships give valuable evidence for further researches. If prospective studies confirm this idea, smoking cessation or modification of smoking habits may delay the appearance of hearing loss. Therefore it is recommended that in order to detect hearing loss at its early stages, smoker workers exposed to noise levels exceeding 85 dBA, should be followed carefully. It is also recommended that they should attend educational courses on smoking cessation periodically, especially in noisy workplacesEvidences have accumulated in recent years on the adverse effects of smoking on hearing among the working population, such as studies conducted by Nakanishi N, et al in Ozaka university, Japan ; Barone A possible explanation for the underlying pathogenic mechanism may be the well-known vascular changes and the consequent cochlear hypoxia related to both smoking and long-term intense noise exposure ,22,38-40Ultimately it can be mentioned that there are few studies about the combined effect of occupational exposure to noise and smoking on hearing loss. But the results of most of them (including our study) demonstrate that smoking can accelerate hearing loss with various degrees. More research is needed to understand the underlying mechanisms.NIHL: noise induced hearing lossThe author(s) declare that they have no competing interests.Dr. Pouryaghoub has worked as a designer and leader of study, Dr. Mehrdad has worked specially in analyzing data, and Dr. Mohammadi has worked specially in gathering data as his residential thesis. All authors have been involved in drafting the manuscript.The pre-publication history for this paper can be accessed here:


A series of 24 paired samples of colorectal carcinoma and the respective normal colorectal mucosa were analysed for Epidermal Growth Factor Receptor (EGFR) content by means of a standardised ligand binding assay. We, for the first time, found that EGFR levels are statistically significantly higher in normal colorectal mucosa biopsy samples than they are in colorectal carcinoma biopsy samples, the median EGFR levels being 77.5 fmol mg-1 of membrane protein (range 35-239), against 46 fmol mg-1 of membrane protein (range 22-81), respectively, P less than 0.001. In addition, we found that there are significant regional differences in EGFR expression in the normal human colon mucosa. The EGFR levels were significantly higher in samples from the proximal part of the colon than they were in samples from the distal part, the median EGFR levels being 124 fmol mg-1 of membrane protein (range 70-239) vs 55 fmol mg-1 membrane protein (range 35-156), P less than 0.05. The EGFR levels of the colorectal carcinoma samples did not show any regional variation.


S-nitrosylation both in vitro and by endogenous, pulmonary derived nitric oxide (NO) within a rodent acute lung injury model. S-nitrosylation is becoming increasingly recognized as an important post-translational modification with signaling consequences. The formation of S-nitrosothiol (SNO)-SP-D both in vivo and in vitro results in a disruption of SP-D multimers such that trimers become evident. SNO-SP-D but not SP-D, either dodecameric or trimeric, is chemoattractive for macrophages and induces p38 MAPK phosphorylation. The signaling capacity of SNO-SP-D appears to be mediated by binding to calreticulin/CD91. We propose that NO controls the dichotomous nature of this pulmonary collectin and that posttranslational modification by S-nitrosylation causes quaternary structural alterations in SP-D, causing it to switch its inflammatory signaling role. This represents new insight into both the regulation of protein function by S-nitrosylation and NO's role in innate immunity.The pulmonary collectins, surfactant proteins A and D (SP-A and SP-D) have been implicated in the regulation of the innate immune system within the lung. In particular, SP-D appears to have both pro- and anti-inflammatory signaling functions. At present, the molecular mechanisms involved in switching between these functions remain unclear. SP-D differs in its quaternary structure from SP-A and the other members of the collectin family, such as C1q, in that it forms large multimers held together by the N-terminal domain, rather than aligning the triple helix domains in the traditional “bunch of flowers” arrangement. There are two cysteine residues within the hydrophobic N terminus of SP-D that are critical for multimer assembly and have been proposed to be involved in stabilizing disulfide bonds. Here we show that these cysteines exist within the reduced state in dodecameric SP-D and form a specific target for  S-nitrosothiols. In this multimeric state, the tail domains are buried, limiting the ability of SP-D to activate inflammation. S-nitrosylation causes the multimers to fall apart into trimers, exposing the tail domain. S-nitrosylated SP-D induces inflammatory cell activation as determined by chemotaxis, calcium influx, and phosphorylation state. This activity is dependent upon both the S-nitrosothiol and the disruption of SP-D's multimeric structure. These modifications are observed in an in vivo model of inflammation and form a critical part of the process. A model is proposed in which nitric oxide operates as a molecular switch for SP-D.Cells of the lung lining secrete a microbe-binding molecule called surfactant protein D (SP-D) that helps activate the inflammatory system against invading pathogens. In the absence of infection, SP-D is important in limiting inflammation, demonstrated by the fact that mice lacking the SP-D gene have chronic inflammation and emphysema. SP-D has two structural features—a lectin-like head domain and a collagenous tail domain—that, respectively, inhibit and stimulate inflammation. Here we define a mechanism for generating the active “inflammatory” version of SP-D. SP-D is held together in its multimeric state by interacting cysteine residues, which are susceptible to modification by the gaseous second messenger, nitric oxide, to form  S-nitrosylation.Nitric oxide is shown to control the pro- and anti-inflammatory functions of surfactant protein D by altering its quaternary structure via Nitric oxide (NO) has long presented a curious dichotomy within biologic systems, namely that it is both an important physiological regulator and the mediator of many pathologies –3. NowheS-nitrosylation is emerging as an important regulator of protein function -1-(2-pyridyldithio) propionamide (Pierce). Biotinylated proteins were precipitated with Streptavidin-agarose beads and Western blot analysis was performed to detect the amount of SP-D remaining in the samples.h method . BAL . Proteins were electrophoresed using MEPS-SDS buffer at 200 V for 35–45 min per the manufacturer's instructions (Invitrogen). Native gel electrophoresis was performed using a Novex 4% tris-glycine gel (Invitrogen). Samples were mixed with a cold native gel sample buffer (Invitrogen) before loading. Electrophoresis was run at room temperature at a constant voltage of 80 V for 5 h. Proteins were then transferred from the gel to a nitrocellulose membrane using the XCell II Mini-Cell and sandwich blot module (Invitrogen) in Bicine-10% methanol-0.01% SDS transfer buffer (Invitrogen) at 30 V for 1 h. Blots were blocked for 1 h at room temperature with 10% nonfat milk and then incubated with primary SP-D antibody  overnight at 4 °C. Blots were then incubated with 1:5,000 goat anti-rabbit IgG-horseradish peroxidase for 2 h at room temperature. Signal was detected using the enhanced chemiluminescence kit (Amersham), and blots were exposed to Kodak Biomax MS film.All animals for this study were housed in the Animal Care Facility of The Children's Hospital of Philadelphia under standard conditions with free access to food and water. All animal experimental protocols were reviewed and approved by the Animal Care and Use Committees of both the Children's Hospital of Philadelphia and The University of Pennsylvania. Either human clinical-grade, sterile, and lipopolysaccharide (LPS)-negative saline or 8.0 U/kg of bleomycin sulfate (Bristol Myers Squibb) in 25 μl of saline was administered intratracheally to 6-wk-old (200–250 g) male Sprague-Dawley rat littermates (Charles River Breeding Laboratories) as previously described . Similar6 cells/ml in DMEM were placed in the upper wells of a 48-well microchemotaxis chamber (Neuro Probe). The lower chambers contained 41 μl of test solution, consisting of DMEM and either nothing (control); various concentrations of SP-D, SNO-SP-D, BAL, or SNO-BAL. All test solutions were used in triplicate in each assay. A polyvinylpyrrolidone-free polycarbonate filter was placed between the wells along with the rubber gasket of the assembly. The filters used for macrophage chemotaxis had 5-μm pores (Neuro Probe). The chamber was incubated at 37 °C with 5% CO2 for 3 h, and then disassembled. Nonmigrating cells were scraped from the upper surface, and the migrating cells were stained with the Hemacolor differential blood stain. The filter was placed on a glass coverslip and mounted with immersion oil onto a glass slide. Cells that migrated through the filter were counted in ten randomly selected oil-immersion fields in each well at 1,000× magnifications. Data were expressed as cells per oil- immersion field for the three wells used for each solution.Directed migration (chemotaxis) of cells was performed as previously described  Briefly,6) were plated on glass coverslips (Fisher Scientific) sized to fit a homeothermic perfusion chamber platform of an inverted Nikon microscope. The cells were loaded with 5μM fura-2 acetoxymethylester (Molecular Probes) and 0.2 mg/ml pluronic F-127 (Molecular Probes) in 2 ml HBSS supplemented with 1% FBS and 1.25 mM CaCl2 for 30 min at 37 °C. The cells were stimulated with HBSS at 37 °C containing BAL or SNO-BAL and excitation was performed at 334 and 380 nm with two narrow-bandpass filters. The emitted fluorescence was filtered (520 nm), captured with a Hamamatso CCD video camera (512 × 480-pixel resolution), digitized (256 gray levels), and analyzed with SimplePCI (Version 3.7.9) software. The amount of Ca2+ was calculated by comparing the ratio of fluorescence at each pixel to an in vitro 2-point calibration curve. The Ca2+ concentration presented was obtained by averaging the values of all pixels over a cell body. The data points were collected at intervals of 5 s.RAW cells (2 × 106 cells./ml) were cultured overnight, then incudbated with Bal or SNO-BAL (100 μg/ml) for 10 min. If anti-caltericulin was used in the experiments, the antibody (2 μg/ml) was added 30 min before the stimulation. Cells were lysed in lysis buffer  containing protease inhibitors , and resolved in 4–10% SDS-PAGE, and blotted to a PVDF membrane as outlined above. The membranes were probed with a phosphospecific antibody to p38. To confirm the equal loading, the membranes were stripped and reprobed for p38.RAW cells (1 × 10


Agarases are the enzymes which catalyze the hydrolysis of agar. They are classified into α-agarase (E.C. 3.2.1.158) and β-agarase (E.C. 3.2.1.81) according to the cleavage pattern. Several agarases have been isolated from different genera of bacteria found in seawater and marine sediments, as well as engineered microorganisms. Agarases have wide applications in food industry, cosmetics, and medical fields because they produce oligosaccharides with remarkable activities. They are also used as a tool enzyme for biological, physiological, and cytological studies. The paper reviews the category, source, purification method, major characteristics, and application fields of these native and gene cloned agarases in the past, present, and future. JAMB-A33 and Alteromonas agarlyticus GJ1B belong to the glycoside hydrolase (GH) family 96 (http://www.cazy.org/fam/acc_GH.html). The amino acid sequences of the two α-agarases have been identified, with GeneBank accession number of BAF44076.1 (JAMB-A33) and AAF26838.1 (GJ1B), respectively. However, there is no publication available on the catalytic domain in α-agarase. The two α-agarases feature a type-VI cellulose-binding domain by the alignment of amino acid sequence in the NCBI protein database (http://www.ncbi.nlm.nih.gov/protein/6724084?report=genpept).Two α-agarases produced by i.e., GH-16, GH-50, and GH-86 (http://www.cazy.org/fam/acc_GH.html). GH-16 family has most abundant members, including agarase, carrageenase, glucanase, galactosidase, laminarinase, etc., while agarase is the only member of GH-50 and GH-86 families. On the other hand, most β-agarases belong to GH-16 family, while only a few β-agarases belong to GH-50 and GH-86 families  is reported to be the first agarase classified in the GH-86 family , howeverc module ,35. Thouc system .Lugol’s iodine solution has been used to visualize agarase activity both for screening the agarase production by microorganism on a culture plate and for identification of protein band of agarase activity after electrophoresis . It staiin situ detection of agarase is performed on one gel, and Coomassie Brilliant Blue staining is performed on the other gel [2 and 10% KI in distilled water). A clear zone is formed around the agarase band. To determine the molecular mass by electrophoresis, the other gel is stained by Coomassie Brilliant Blue after electrophoresis. The molecular mass of the agarase can be determined by comparing the two gels.After electrophoresis, ther gel . SDS in d-galactose as a standard. Enzyme activity (U/mL) was defined as the amount of enzyme required to liberate 1 μmol d-galactose per min.Agarases degrade agar in α-1,3 or β-1,4 glycosidic bonds and from reducing ends. The agarase activity is commonly quantified by spectrophotometric determination of the increase in the concentration of reducing sugars by Nelson method  or DNS mI values lower than 7.The purification procedure of ammonium sulfate fractionation followed by anion exchange chromatography and gel filtration chromatography has often been used in purification of several agarases ,22,27,31Thalassomonas sp. JAMB-A33 has been purified by hydroxyapatite, followed by anion exchange chromatography, column purification by hydroxyapatite, anion exchange chromatography, and gel filtration chromatography [Vibrio sp. PO303 [Hydroxyapatite has been successfully used for purification of a few agarases ,35,36. Ttography . Column p. PO303 .2+ Sepharose which is commonly used to trap the 6-His tagged recombinant protein. It is used for one-step purifications of rAgaD cloned from Vibrio sp. PO-303 [S. degradans 2-40 [Agarivorans sp. LQ48 [Agarivorans sp. YKW-34 [Agarivorans sp. JA-1 [Three kinds of affinity chromatography media have been used in agarase purification up to date. The first kind of medium is the cross-linked agarose such as agarose CL-6B, which results in a marked increase of agarase specific activity based on the specific affinity of enzyme and substrate. This kind of affinity chromatography has been used cooperating with anion exchange chromatography ,39 or ge. PO-303 , five reans 2-40 , rAgaA fsp. LQ48 , and rAg. YKW-34 . The thisp. JA-1 .Zobellia galactanivorans have been highly purified [Most native and cloned agarases have been purified to high purity with a satisfactory yield. Two agarases AgaA and AgaB from the marine bacterium purified , and thepurified .Vibrio sp. AP-2 from marine algae with a Mr of 20 kDa [Alteromonas agarlyticus GJ1B from seawater with a Mr of 360 kDa [Alteromonas agarlyticus GJ1B. Using SDS-PAGE, this purified agarase has been detected as a single band with a molecular mass of 180 kDa. After the affinity-chromatography step, however, the native molecular mass was approximately 360 kDa, suggesting that the native enzyme is a dimer [Various agarases from marine microorganisms derived from seawater, marine sediments, marine algae, and marine mollusks have been purified and characterized. Their important properties are listed in f 20 kDa , while tVibrio have lower specific activities, which are 7.54 and 20.8 U/mg from strain PO303 [Alteromonas and Pseudoalteromonas exhibit high specific activities, which are 83.5 U/mg from Alteromonas sp. SY37-12 [Alteromonas sp. C-1 [Pseudoalteromonas antarctica N-1 [As shown in in PO303  and 6.3 in PO303 . Agarase SY37-12 , 234 U/m sp. C-1 , and 292tica N-1 .i.e., Vibrio sp. JT0107 and Alteromonas sp. C-1, possess optimal activity at temperature of 30 °C [Alteromonas sp. SY37-12 and Agarivorans YKW-34 have been reported to be stable up to temperature of 50 °C, others are stable up to 45 °C [The optimal temperatures for the activity of agarases are similar. The gelling temperature of agar is around 38 °C, and most of reported agarases show optimal activity at temperature above this level . Except of 30 °C ,16, whicto 45 °C , 40 °C [to 45 °C , 30 °C [to 45 °C ,23, and to 45 °C .Alteromonas sp. C-1 and Vibrio sp. AP-2 has been reported to possess maximum activity at pH 6.5 [Most agarases have been reported to show maximum activity at a neutral –23 or a t pH 6.5  and pH 5t pH 6.5 , respecti.e., Alteromonas agarlyticus GJ1B and Thalassomonas sp. JAMB-A33, which produce agarotetraose as the main product. Other β-agarases have been reported to produce NA2 [Vibrio sp. JT0107 has been reported to produce the mixture products of NA2 and NA4, however, NA2 is not detected when NA4 is used as a substrate [i.e., agarase-a, agarase-b, and agarase-c, secrete by a marine bacterium, Vibrio sp. strain PO-303, have been reported to exhibit different action patterns against agarose. Agarase-a hydrolyzed agarose into NA4 and NA6, agarase-b hydrolyzed agarose into NA2, and agarase-c produced NA6 and NA8 from agarose [The products are totally different between different categories of the agarase. Two α-agarases derived from marine environment have been reported, duce NA2 –25, NA4 duce NA2 ,22 and Nduce NA2  as the pubstrate . Three k agarose .Cytophaga flevensis is 26 kDa [Acinetobacter sp. AGLSL-1 [Bacillus sp. MK03 [Alteromonas sp. E-l [Acinetobacter sp. AGLSL-1 show a specific activity of 397 U/mg [Alteromonas sp. E-l [Acinetobacter sp. AGLSL-1 [Most agarases are from marine environment, except four agarases have been reported deriving from other environments . The mol AGLSL-1 , 113 kDasp. MK03  and 180 397 U/mg , which i AGLSL-1  have beeThalassomonas sp. JAMB-A33 [Bacillus subtilis as a host with an extraordinary production of 6950 U/L [rAgaA33 has been cloned from a deep sea sediment bacterium JAMB-A33 . Two natJAMB-A33 ,19, whil6950 U/L , whereas6950 U/L . Thus thEscherichia coli is commonly used as a host for recombinant production of agarases  by using three kinds of enzymes, i.e., β-1,4-mannanase, β-1,3-xylanase, and agarase prepared from three marine bacteria, i.e., Vibrio sp. MA-138, Alcaligenes sp. XY-234, and Vibrio sp. PO-303 [Vibrio sp. PO303 [O-linked β-d-galactopyranose and 4-O-linked 3,6-anhydro-α-l-galactose. It is contained in the cell wall of Porphyra which is an important edible red seaweed. Hence, by combination with β-1,4-mannanase and β-1,3-xylanase, these agarases might be a useful tool for isolation of protoplasts from Porphyra.Agarases have potential application in degrade the cell wall of red algae for extraction of labile substances with biological activities such as unsaturated fatty acids, vitamins, carotenoids from algae. Yaphe has used the agarase from atlantic  in the iatlantic . Agaraseatlantic . Araki h. PO-303 , respect. PO-303 . The rAgp. PO303 ,40,47 haFrom the collective information on agarases, we conclude that agarases are important enzymes naturally purified and cloned from variety of microorganisms. They are characterized into two categories with more than four families which can produce various oligosaccharides with different DP values. A few agarases have a remarkable production and possess excellent properties such as high specific activity, excellent temperature and pH stability, which enlighten their widely potential applications.


Mucorales species are deadly opportunistic fungi with a rapidly invasive nature. A rare disease, mucormycosis is most commonly reported in patients with diabetes mellitus, because the favorable carbohydrate-rich environment allows the Mucorales fungi to flourish, especially in the setting of ketoacidosis. However, case reports over the past 20 years show that a growing number of cases of mucormycosis are occurring during treatment following bone marrow transplants (BMT) and hematological malignancies (HM) such as leukemia and lymphoma. This is due to the prolonged treatment of these patients with steroids and immunosuppressive agents. Liposomal amphotericin B treatment and posaconazole are two pharmacologic agents that seem to be effective against mucormycosis, but the inherently rapid onset and course of the disease, in conjunction with the difficulty in correctly identifying it, hinder prompt institution of appropriate antifungal therapy. This review of the literature discusses the clinical presentation, diagnosis, and treatment of mucormycosis among the BMT and HM populations. Mucormycosis is a rare and rapidly progressive opportunistic fungal infection that most often presents among patients with diabetes mellitus, immunodeficiency, neutropenia, iron overload, and severe burns. There has been a reported rise in mucormycosis among patients with hematological malignancies (HM), e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), and in bone marrow transplant (BMT) recipients due to the neutropenia and immunodeficiency associated with chemotherapy and post-transplant steroid treatment.–3 Due toRhizopus, Rhizomucor, Mucor, and Absidia.[Though a relatively uncommon fungal infection in comparison with candidiasis and aspergillosis, mucormycosis cases have been on the rise over the past decade. One stud Absidia.7 MucormyCunninghamella infections from the Cunninghamellaceae family are also being reported. Mucorales, first described in 1885 by Paltauf,[Mucorales fungi are characterized by fast-growing fibrous mycelium and thin-walled aseptate or hyposeptate hyphae that measure 10–20 µm in width. Right-angled branching is also seen as it rapidly grows within the host tissue. As the hyphae grow they invade the blood vessels and result in tissue infarction.9 This ul7Although it is an uncommon infection in the general population, exposure to Mucorales is common. Most species are saprotrophic, obtaining their nutrients from dead organic matter, and grow in soil, fruit, bread, and plants. Some are characterized as parasites of animals, plants, and other fungi. Mucormycosis infection in humans is usually acquired through airborne fungal spores.10 The nu9/l. The reference range is 1.8 × 109/l to 7 × 109/l.For optimal growth in humans, Mucorales fungi require the host to have decreased levels of neutrophils. Neutrophils are the key defense against the fungi in the host tissue. In healthy humans with intact immune systems, neutrophils phagocytize these pathogens. However, in neutropenic individuals, the unregulated pathogens proliferate in the host tissue and infection ensues. Neutropenia is often a side effect of chemotherapy in cancer patients or of the malignancy itself and is also seen in immunocompromised individuals. The vast majority of patients with mucormycosis are neutropenic, with neutrophil counts usually below 0.5 × 10et al., based on their study of 5589 transplant patients at the Fred Hutchinson Cancer Center, report that the number of cases of mucormycosis have more than doubled from 1985–1989 to 1995–1999.[Rhizopus are the most commonly isolated species among these patients. Less common, but increasingly frequent in the general population, is the Cunninghamella bertholletiae species.[The literature reports an increase in mucormycosis, as compared to invasive aspergillosis, among immunocompromised patients over the last 2 decades. Marr et  species.16 No stuAccording to one study, the annual incidence of mucormycosis in the general population is 1.7 cases for every 1 million individuals, which amounts to approximately 500 affected Americans each year. However,10223et al. in a 5-year (2001–2005) retrospective study of 20 HM patients admitted to the Medical Oncology Division at the Cukurova University Hospital in Turkey[in Turkey; the autin Turkeyet al. of 87 pulmonary mucormycosis patients indicated that those with HM comprised 32% of the cases; the majority (56%) of the cases were those with diabetes mellitus.[Mucormycosis commonly presents in five forms: it may affect the pulmonary, rhinocerebral, gastrointestinal, cutaneous, or the central nervous systems. Disseminmajority 6% of theThe most common symptom in pulmonary mucormycosis is a high-grade fever (> 39°C) that does not subside despite the administration of antibiotics.51624323642–473236Cases of rhinocerebral mucormycosis present with facial pain, periorbital cellulitis, proptosis, visual deficiencies, black necrotic lesions and discharge from the nasal and palatal mucosa, and fever.4849 Biop48Gastrointestinal mucormycosis presents with features similar to that seen in bowel obstruction. Tenderness on palpation, with or without the presence of a mass, and hepatomegaly or splenomegaly have been reported.34 This fA corymbifera. The site of the lesion may be where catheters are placed.[3033Cutaneous involvement by mucormycosis has been reported as presenting with black necrotic lesions, most often on the face and extremities. Many of the cases of cutaneous mucormycosis reported in BMT and HM patients seem to have been caused by e placed. Reports e placed.303340et al. have reported a CML patient in whom mucormycosis spread through the ear to invade cerebral tissue; the patient died 15 days post-BMT.[Brain mucormycosis is not common by itself; CNS involvement is usually a result of dissemination of the fungal infection from pulmonary or rhinocerebral sources. Patersonpost-BMT.Mucormycosis can be a fatal infection and early diagnosis and treatment are of extreme importance for successful eradication of the infection and for patient survival. Differential diagnoses may be varied due to the different clinical presentations and organ systems involved. The most commonly employed technique for differentiating between mucormycosis and other diseases is a histopathological study. However,For a definitive diagnosis of mucormycosis, the histopathological specimen need not be of the isolated fungus, but may be a sample of the necrotic tissue from the site in question. The Grocott-Gomori methenamine silver (GMS) stain is most effective for identifying fungi. However, hematoxylin and eosin, periodic acid-Schiff (PAS), or Calcofluor White stains may also be used. InvasionCulture studies often fail to grow Mucorales in mucormycosis patients. Blood cultures are especially known to produce false negative results. The ideal culturing technique is to receive the infarcted tissue and submit it for processing by mincing the tissue with a sterile scalpel and not by grinding it. The syncytial cells of the Mucorales fail to grow in culture if submitted to a grinding procedure, which will produce small fragments devoid of any enclosing septal cell walls. Mucorales grow well on Sabouraud dextrose, brain-heart infusion, or potato dextrose agar incubated at 25–30°C. Identification of saprophytic mold does not conclusively indicate that an infection with that organism has occurred; therefore culture is not usually used alone for diagnosis.et al. recently conducted a study showing the usefulness of panfungal PCR on serial serum samples for diagnosing pulmonary mucormycosis caused by Cunninghamella bertholletiae.[et al. reported the use of a combination of two nested PCR assays to differentiate between Aspergillus and Zygomycetes  and successfully identified the Zygomycetes to the species or genus level.[A recent Mayo clinic study using the MicroSeq D2 large-subunit ribosomal DNA sequencing kit to identify filamentous fungi concluded that nucleic acid sequencing using this technique holds much promise, with accurate identification possible in under 24 h. However,olletiae. Rickertsus level. They werus level.54At present, serological testing is not recommended for diagnosis due to its investigational status. Only a fAspergillus utilizes fluorescence-based microplate assays. This method may come into use for Zygomycetes in the future but has not yet been comprehensively evaluated. A recent study by Antachopoulos showed that clinically important Zygomycetes show significant metabolic activity in XTT assays even before visual or spectrophotometric evidence of activity becomes apparent.[Susceptibility testing is used to determine a drug's potential effectiveness for eradicating infection and inhibiting growth of the pathogen. One susceptibility testing method commonly used for apparent. XTT is tapparent. These XTMortality rates among patients with HM who have mucormycosis remain high despite antifungal therapy. Surgical intervention and debridement is the gold standard of treatment for eradicating the infection.758 Howev7There are many similarities in the clinical presentations of aspergillosis and mucormycosis. The ineffectiveness of the drugs used in aspergillosis for the treatment of mucormycosis makes it especially important to establish the diagnoses as early as possible in the course of disease. For leukemia and BMT patients who receive prophylactic antifungal drugs as part of their treatment, it is crucial to distinguish between the different fungal infections as soon as possible after symptoms appear in order to administer the correct antifungal therapy. Early correction of predisposing factors such as neutropenia and immunodeficiency are also important in controlling the progression of the infection.Rhizopus species.[The most effective treatments for mucormycosis in BMT and leukemia patients are various formulations of amphotericin B combined with posaconazole. Itracona species.59 there  species.61in vitro and in vivo.[46596364Aspergillus infection, only to find that they develop mucormycosis later. These cases showed breakthrough mucormycosis at 9–12 days after voriconazole administration was started as an empiric antifungal treatment in leukemia. Marty et al. also reports breakthrough mucormycosis in stem cell transplant recipients who received voriconazole as prophylactic or empiric therapy.[Aspergillus, Candida, Scedosporium, and Fusarium.[Voriconazole, an effective drug in aspergillosis, is not r in vivo.465963–65vivo.4659636466 Se therapy. VoriconaFusarium.68 The poet al., administration of CAmB to treat mucormycosis may not be favorable and may even be toxic due to the high dosage levels and prolonged treatment required.[Amphotericin B has been the drug of choice against mucormycosis for over 50 years due to its superior effectiveness compared to other therapies. Treatment duration is usually between 3 and 6 weeks and the total dose that needs to be administered ranges from 2.0–4.0 g, depending on the specific case. Among threquired. Another et al. reported the case of a leukemia patient in whom disseminated mucormycosis was successfully treated with the use of a combination of LAmB and posaconazole without surgical intervention. Other authors have also reported similar success.[Posaconazole is a relatively new drug that appears to be a promising alternative or adjunctive to LAmB administration.7071 Rick70 success.72 Many eAnother alternative or adjuvant therapy is hyperbaric oxygen (HBO). A study published in 2004 by Garcia-Covarrubias showed 60% survival rate using HBO as an adjuvant therapy to amphotericin B and surgical debridement. A 1994 sThe high mortality rate among mucormycosis patients calls for an increased index of suspicion and aggressive attempts to diagnose and treat the disease at an early stage. Because of its rarity, mucormycosis is often not included in the differential diagnosis until widespread Mucorales invasion has already taken place in the patient. Its commonplace presence in the environment emphasizes the importance of keeping the wards and hospital instruments free of these opportunistic fungi. Increasing the implementation of promising new diagnostic methods such as PCR nested assays and ITS2 sequencing may help increase the rate of successful Mucorales identification in the early stages of infection. However, the new assays await FDA approval for implementation in hospital-based clinical microbiology laboratories. LAmB and posaconazole treatment have helped to treat many cases of mucormycosis. However, despite these advances, the incidence of the infection has risen in recent years, in part due to the use of prolonged chemotherapy and immunosuppressive treatment in HM and BMT patients. Given the increase in cases of breakthrough mucormycosis after general antifungal prophylaxis and the overall increase in cases in this population, anti-Mucorales prophylaxis studies are warranted and should be given due importance, alongside the search for better methods of diagnosis and treatment.


Birdsong and human vocal communication are both complex behaviours which show striking similarities mainly thought to be present in the area of development and learning. Recent studies, however, suggest that there are also parallels in vocal production mechanisms. While it has been long thought that vocal tract filtering, as it occurs in human speech, only plays a minor role in birdsong there is an increasing number of studies indicating the presence of sound filtering mechanisms in bird vocalizations as well.Taeniopygia guttata) to song structures we identified beak gape and the expansion of the oropharyngeal-esophageal cavity (OEC) as potential articulators. We subsequently manipulated both structures in an experiment in which we played sound through the vocal tract of dead birds. Comparing acoustic input with acoustic output showed that OEC expansion causes an energy shift towards lower frequencies and an amplitude increase whereas a wide beak gape emphasizes frequencies around 5 kilohertz and above.Correlating high-speed X-ray cinematographic imaging of singing zebra finches (These findings confirm that birds can modulate their song by using vocal tract filtering and demonstrate how OEC and beak gape contribute to this modulation. Birdsong is a complex vocal behaviour often considered to show striking developmental and structural similarities with human speech In humans, voiced speech is produced by vibrations of the vocal folds which are subsequently filtered in order to produce different speech sounds that form an important part of our phonetic repertoire In contrast to this source-filter theory of human speech Zonotrichia albicollis), swamp sparrows (Melospiza georgiana) Taeniopygia guttata) Serinus canaria) Some of the first evidence derives from experiments showing that both songbirds and non-songbirds singing in heliox exhibit deviating vocal characteristics Streptopelia risoria) Cardinalis cardinalis) Myiopsitta monachus) also seem to have a filtering effect on the sound produced Other studies indicate that expanding the oropharyngeal-esophageal cavity (OEC) plays a role in vocal tract filtering as well by tuning it to the fundamental frequency of the vocalizations in doves .We used five male and two female zebra finches for the X-ray cinematographic imaging and five male zebra finches for the experiment in which we replaced the syrinx by a mini-loudspeaker. The female birds only served as stimulus birds in the X-ray setting to stimulate the males to sing. During X-ray recordings male birds were individually transferred into a small cage (30 cm wide ×20 cm high ×10 cm deep) built from wood with plexi glass on both long sides. The small size of the cage allowed to optically focus on the birds, but still allowed the typical dancing movements during singing −1, shutter speed 1/500 s by replacing the original camera of the X-ray apparatus by the Kodak system. The images which had a resolution of 512×240 pixels were loaded into the camera's onboard memory. The maximum recording time of the Kodak Motion Corder which was triggered manually is 8.7 s at 500 fields s−1, making it necessary to save the video sequences immediately on digital video for permanent storage −1 we played back the video sequences from the Kodak system with 25 frames s−1 to prevent data loss while re-recording. We continuously applied an X-ray dose of 56 kV, 60 mA. The videos were captured either from the Mini DV tapes or from the DVDs using Adobe Premiere Pro software version 7.0 for Windows. Due to a distinct tone produced by the X-ray apparatus only while the shutter was open it was possible to align sound and video using the frame-matching features of Adobe Premiere with an accuracy of 2 milliseconds. In order to accurately follow and quantify the movements of certain articulators we also glued several lead markers (ca. 0.5 mm3) on head and beak of the birds using tissue adhesive . In two birds we implanted additional lead markers into the tongue and larynx. These procedures were conducted under anesthesia using isoflurane .A Philips Optimus M 200 X-ray apparatus was combined with a Kodak Motion Corder Analyzer SR- 500 s that records at 500 field sTo quantify beak gape and OEC expansion we measured the distance between the tips of mandible and maxilla and the distance between the most ventral point of the OEC and the midpoint of the neck of the bird for several note types per song and always at the temporal midpoint of those notes as identified on song spectrograms. These measurements were taken from still images using AviDigitiser (© Peter Ch. Snelderwaard) which provides the coordinates of manually selected points within each video frame and from which distances can be calculated. Only X-ray images in which the birds kept their head in a perfect lateral position towards the camera were used, so that in the end we obtained measurements from at least 8 songs for every measured note type. Although we recorded with a high frame rate, the stereotyped dancing movements of zebra finches allowed sampling of only a few notes per song per bird, namely those in which the bird's head was perfectly lateral to the camera. This method is well suited for comparing beak gape and OEC expansion for various elements within a song, as well as for qualitative comparison between birds, but not for quantitative comparisons between birds. Furthermore, due to the small size of zebra finches it was not possible to identify structures such as single vertebrae, but the overall shape of OEC and neck was used to obtain measurements.Afterwards we carried out a direct discriminant function analysis using beak gape and OEC expansion as predictors for determining which note types were produced. Since zebra finches produce several different and complex note types we did not relate our articulation measurements to signal analytic features such as fundamental or peak frequency as has been done in other studies www.praat.org) Prior to the X-ray cinematography we recorded the songs of each bird in a sound-attenuating chamber (ca. 1.80 m ×1.20 m ×2.00 m) that was lined with acoustic foam  to reduce sonic reflections from the walls. From these recordings we later took amplitude measurements using the software PRAAT  in the pectoral muscle. Afterwards a small incision was made posterior from the lower jaw to expose the urohyal bone We obtained sufficient video data from four male birds and measured beak gape and OEC expansion of different note types within each song per individual. For every bird separately a direct discriminant function analysis was carried out –3 with In all four birds beak gape is weighted heavier in the first discriminant function whereas OEC expansion is weighted more in the second, as shown by the standardized coefficients and the correlation between each variable and any discriminant function .The classification results  show thaWhile it is difficult to compare note types between individuals since the four males sing different note types, it is possible to detect some similarities between birds that produce comparable note types. Bird 498 for instance produces a frequency modulated note (note 4) with an upwards sweep in the second half of the note which is comparable to note 5 in bird 705. In both cases these notes show a relatively high OEC expansion and a similar beak gape. Another comparison can be drawn in individuals 499 and 498 since they produce a rather dense harmonic stack  and in both birds these elements are produced with a relatively large beak gape, although OEC expansion varies remarkably . This agAnother factor that might be influenced by beak gape and OEC expansion is amplitude and indeed three out of four birds produce the loudest note measured with the largest OEC expansion and in two cases also with a wide beak gape ; Table 4Generally speaking the results indicate that beak gape as well as OEC expansion might act as vocal tract articulators to generate different note types within each zebra finch song. However, no clear picture concerning the specific effects of these articulators on sound modulation is emerging yet and the speaker experiment was carried out to directly address the role of beak gape and OEC expansion.The results of this experiment are displayed in The overall frequency and amplitude modulation pattern is rather similar in all five individuals indicating a fairly specific effect of the mentioned articulators on the sound signal. The clearest and most consistent effect is given by the expansion of the OEC leading to an amplitude increase especially in the frequency region around 5 kHz which is particularly obvious at a wide beak gape, but also clearly visible when the beak is only slightly open. The total amplitude increases on average 10.9 dB when the beak is only slightly open and 8 dB when the beak is wide open. With a closed beak this effect is not so clear since amplitude first increases with OEC expansion but at a certain point drops again; in three birds even below the intensity level that was gained at position 0. Also at the other two beak gapes amplitude first increases rapidly, but drops again at a certain point although it clearly remains above the intensity level at the beginning of a series. Furthermore OEC expansion causes an energy shift towards relatively lower frequencies which again is especially obvious with a wide beak gape, but also visible in the two other conditions.Beak gape has a strong effect on amplitude too. The wider the beak opens the louder the sound becomes with an average amplitude increase of 5.8 dB compared between a closed and a wide beak gape.Regarding frequency range it seems that a closed beak filters frequencies above 6 kHz whereas a wide beak gape emphasizes high frequencies above 5 kHz and over a broader frequency range.All of the nine subplots show a low prominent formant around 1.5 kHz probably mostly influenced by the resonating trachea, the dimensions of which do not change under the different articulator configurations tested in this experiment.In the current study we combined observational and correlational data on song production in zebra finches with findings derived from experimental manipulations of beak gape and OEC expansion to provide insight into the mechanisms of vocal tract filtering in this species. Based on our results it seems clear that zebra finches can use both beak gape and OEC expansion as vocal articulators to filter the sound produced by the syrinx. However, while some of our results support conclusions drawn by other studies and are in line with some of the hypotheses formulated earlier, not all our findings confirm what has been discovered regarding vocal tract filtering in other bird species.Our X-ray cinematographic imaging of singing zebra finches revealed that the expansion of the OEC is caused by a cyclical posterior-ventral movement of the hyoid skeleton which is comparable to northern cardinals Melospiza melodia) Branta leucopsis) Beak gape has been shown to essentially influence frequency patterns of bird vocalizations although different studies arrive at different conclusions. The general picture emerging from the literature is that wider beak gapes correlate with higher frequencies whereas smaller beak gapes correlate with lower frequencies. This has been shown in several songbird species such as the white-throated sparrow, the swamp sparrow Pipilo erythrophthalmus) the hypothesis that beak gape articulation causes significant modulation of the vocal tract filtering function. In this species frequencies between 4 and 7.5 kHz are attenuated when beak gape width is reduced. Furthermore the authors propose that towhees in particular and songbirds in general might vary beak gape as a mechanism to exclude or concentrate energy in distinct frequency bands which often results in the production of narrow-band or pure-tone sounds.Another study Based on the results of our speaker experiment it seems that on the one hand large beak gapes indeed sustain high frequencies  and therAnother observation made in song sparrows is that coordinated beak movements develop rather late during song learning and appear to correspond with improved tonal quality of the sound produced whereas they are not necessary for producing the acoustic fine structure of notes A different study found a strong positive correlation between beak gape and fundamental frequency as well as peak frequency in zebra finches in most of the individuals tested although the relationship between beak gape and fundamental frequency did not apply to harmonic stacks Phonygammus keraudrenii) exhibit elongated tracheas which are assumed to lower the pitch of the vocalizations Other structures that might be involved in vocal tract filtering include the trachea itself and glottal opening. Whereas the role of glottal opening has not been examined yet there are indications that zebra finches actively shorten the trachea at the beginning of a song bout In any case it has become clear that vocal tract filtering in birds can enhance vocal complexity and serve to code biologically relevant information such as size In summary we have shown that zebra finches can use both beak gape and OEC expansion to modulate their vocalizations to a substantial degree. However, the wide variety of different note types that these birds produce does not seem to be solely based on the interaction of these articulators but is likely to be affected also by other factors related to the sound source.Video S1X-ray video of a singing zebra finch(5.69 MB WMV)Click here for additional data file.