70-kDa Band Research Articles

Expression of ferritin-like protein in Listeria monocytogenes after cold and freezing stress

The cold shock protein family consists of the transfer of the foodborne pathogen Listeria monocytogenes from 37 to 4 and −20 °C and was characterized by the sharp induction of a low molecular mass protein. This major cold shock protein ferritin-like protein (Flp) has an important role in regulation of various microbial physiological processes. Flp have a molecular mass of about 18 kDa, as observed on SDS–PAGE. The purification procedure including ammonium sulfate fractionation was used. Monospecific polyclonal antibodies raised in rabbits against the purified new Flp immunostained a single 18-kDa Flp band in extracts from different cytoplasmic proteins blotted onto nitrocellulose. A 411-bp cDNA fragment that corresponds to an internal region of an flp gene was obtained by RT-PCR. Our result indicated a surexpression of major cold shock protein and an important increase in flp mRNA amount after a downshift temperature especially at −20 °C.

Molecular Cloning and Characterization of First Organic Matrix Protein from Sclerites of Red Coral, Corallium rubrum

We report here for the first time the isolation and characterization of a protein from the organic matrix (OM) of the sclerites of the alcyonarian, Corallium rubrum. This protein named scleritin is one of the predominant proteins extracted from the EDTA-soluble fraction of the OM. The entire open reading frame (ORF) was obtained by comparing amino acid sequences from de novo mass spectrometry and Edman degradation with an expressed sequence tag library dataset of C. rubrum. Scleritin is a secreted basic phosphorylated protein which exhibits a short amino acid sequence of 135 amino acids and a signal peptide of 20 amino acids. From specific antibodies raised against peptide sequences of scleritin, we obtained immunolabeling of scleroblasts and OM of the sclerites which provides information on the biomineralization pathway in C. rubrum.

Molecular identification and functional characterisation of uncoupling protein 4 in larva and pupa fat body mitochondria from the beetle Zophobas atratus

Uncoupling protein 4 (UCP4) is a member of the UCP subfamily that mediates mitochondrial uncoupling, and sequence alignment predicts the existence of UCP4 in several insects. The present study demonstrates the first molecular identification of a partial Zophobas atratus UCP4-coding sequence and the functional characterisation of ZaUCP4 in the mitochondria of larval and pupal fat bodies of the beetle. ZaUCP4 shows a high similarity to predicted insect UCP4 isoforms and known mammalian UCP4s, both at the nucleotide and amino acid sequence levels. Bioenergetic studies clearly demonstrate UCP function in mitochondria from larval and pupal fat bodies. In non-phosphorylating mitochondria, ZaUCP activity was stimulated by palmitic acid and inhibited by the purine nucleotide GTP. In phosphorylating mitochondria, ZaUCP4 activity decreased the yield of oxidative phosphorylation. ZaUCP4 was immunodetected with antibodies raised against human UCP4 as a single 36-kDa band. A lower expression of ZaUCP4 at the level of mRNA and protein and a decreased ZaUCP4 activity were observed in the Z. atratus pupal fat body compared with the larval fat body. The different expression patterns and activity of ZaUCP4 during the larval-pupal transformation indicates an important physiological role for UCP4 in insect fat body development and function during insect metamorphosis.

Pre-freezing and post-thawing quality of boar sperm for distinct portions of the ejaculate and as a function of protein bands present in seminal plasma

This study evaluated pre-freezing and post-thawing boar sperm quality in distinct portions of the ejaculates and identified protein bands potentially related to boar sperm tolerance to freezing. The sperm-rich portion of the ejaculate was fractioned in: the first 10mL (P1); and its remaining volume (P2). Those portions were frozen and the bands present in both portions were evaluated by one-dimensional electrophoresis. Inter- and intra-boar ANOVA evaluated how the presence of bands related to pre-freezing and post-thawing sperm motility and membrane integrity and to post-thawing acrosome integrity. Post-thawing sperm motility, membrane integrity and acrosome integrity were greater for P1 than for P2 (P<0.05). The presence of the 19kDa band was associated with improved pre-freezing sperm motility and membrane integrity, but with reduced post-thawing acrosome integrity (P<0.05). The presence of the 44kDa band was associated with reduced post-thawing sperm motility and acrosome integrity (P<0.05). Post-thawing acrosome integrity was enhanced when the 80kDa band were present (P<0.05). Reduced pre-freezing sperm membrane integrity and post-thawing sperm motility were associated to the presence of the 100kDa band (P<0.05). The presence of the 18kDa band in P2 was associated with reduction in both post-thawing sperm motility and membrane integrity (P<0.05), but such an effect was not observed in P1 (P>0.05). Thus, sperm from P1 presented greater tolerance to freezing than sperm from P2 and the 18, 19, 44, 65, 80 and 100kDa bands may be potential markers for boar sperm tolerance to cryopreservation.

Pp-GalNAc-T13 induces high metastatic potential of murine Lewis lung cancer by generating trimeric Tn antigen

In order to analyze the mechanisms for cancer metastasis, high metastatic sublines (H7-A, H7-Lu, H7-O, C4-sc, and C4-ly) were obtained by repeated injection of mouse Lewis lung cancer sublines H7 and C4 into C57BL/6 mice. These sublines exhibited increased proliferation and invasion activity in vitro. Ganglioside profiles exhibited lower expression of GM1 in high metastatic sublines than the parent lines. Then, we established GM1-Si-1 and GM1-Si-2 by stable silencing of GM1 synthase in H7 cells. These GM1-knockdown clones exhibited increased proliferation and invasion. Then, we explored genes that markedly altered in the expression levels by DNA microarray in the combination of C4 vs. C4-ly or H7 vs. H7 (GM1-Si). Consequently, pp-GalNAc-T13 gene was identified as up-regulated genes in the high metastatic sublines. Stable transfection of pp-GalNAc-T13 cDNA into C4 (T13-TF) resulted in increased invasion and motility. Then, immunoblotting and flow cytometry using various antibodies and lectins were performed. Only anti-trimeric Tn antibody (mAb MLS128), showed increased expression levels of trimeric Tn antigen in T13-TF clones. Moreover, immunoprecipitation/immunoblotting was performed by mAb MLS128, leading to the identification of an 80kDa band carrying trimeric Tn antigen, i.e. Syndecan-1. Stable silencing of endogenous pp-GalNAc-T13 in C4-sc (T13-KD) revealed that primary tumors generated by subcutaneous injection of T13-KD clones showed lower coalescence to fascia and peritoneum, and significantly reduced lung metastasis than control clones. These data suggested that high expression of pp-GalNAc-T13 gene generated trimeric Tn antigen on Syndecan-1, leading to the enhanced metastasis.

Role of Pro-Ala-Rich Extension of Troponin in Insect Flight Muscle as Examined by X-Ray Diffraction

The mechanism of stretch activation is essential for the asynchronous action of insect flight muscle (IFM). Our previous study has shown that the 1,1 row-line spot on the 1st layer line reflection from bumblebee IFM is the first one to respond to the stretch signal in a calcium-dependent manner, and the model calculation has suggested that it may come from the structural change of troponin. This makes IFM troponin a candidate for the stretch sensor. Then the question is what structure transmits stretch information to troponin. IFM troponin-I (troponin-H) is known to have a long Pro-Ala-rich extension, and this may extend to the thick filament and pick up the stretch information. To test this possibility, the extension was severed by a specific endoproteinase (Igase) in bumblebee IFM, and its mechanical properties and X-ray diffraction patterns were examined. In honeybee troponin-H, the susceptible sequence is located at the base of the extension. Unexpectedly, the ability of stretch activation was not affected. The severing had a dramatic effect on diffraction patterns: In IFMs in general, the equatorial 2,0 reflection is much more intense than the 1,1 reflection, but after Igase treatment, the intensities of these reflections became almost equal. If this change is caused by the reduced mass of the thin filament, its mass should be reduced to 1/4 while the extension accounts for only 10% of the thin filament mass. In the SDS gel pattern, the 80-KDa band for troponin H disappeared from the fiber and several protein bands appeared in the solution after treatment. A possibility is that each extension binds several GST molecules (Clayton et al., 1998) to process reactive oxygen generated by the high mitochondrial activities in IFM cells.

Sorting Nexin 27 Protein Regulates Trafficking of a p21-activated Kinase (PAK) Interacting Exchange Factor (β-Pix)-G Protein-coupled Receptor Kinase Interacting Protein (GIT) Complex via a PDZ Domain Interaction

Sorting nexin 27 (SNX27) is a 62-kDa protein localized to early endosomes and known to regulate the intracellular trafficking of ion channels and receptors. In addition to a PX domain, SNX27 is the only sorting family member that contains a PDZ domain. To identify novel SNX27-PDZ binding partners, we performed a proteomic screen in mouse principal kidney cortical collecting duct cells using a GST-SNX27 fusion construct as bait. We found that β-Pix (p21-activated kinase-interactive exchange factor), a guanine nucleotide exchange factor for the Rho family of small GTPases known to regulate cell motility directly interacted with SNX27. The association of β-Pix and SNX27 is specific for β-Pix isoforms terminating in the type-1 PDZ binding motif (ETNL). In the same screen we also identified Git1/2 as a potential SNX27 interacting protein. The interaction between SNX27 and Git1/2 is indirect and mediated by β-Pix. Furthermore, we show recruitment of the β-Pix·Git complex to endosomal sites in a SNX27-dependent manner. Finally, migration assays revealed that depletion of SNX27 from HeLa and mouse principal kidney cortical collecting duct cells significantly decreases cell motility. We propose a model by which SNX27 regulates trafficking of β-Pix to focal adhesions and thereby influences cell motility.

Specificity of immunoblotting analyses in eosinophilic meningitis

Angiostrongylus cantonensis and Gnathostoma spinigerum are the two most common causative parasites of eosinophilic meningitis (EOM). Serological tests are helpful tools for confirming the identity of the pathogen. Recent reports determined the specificity of such tests by using normal healthy controls. There have been limited studies done to rule out the cross-reactivity between these two causative parasites of EOM. This study aims to assess the specificity of the serological test in EOM by using each condition as a control for the other. Thirty-three patients with a diagnosis of EOM were enrolled. Sera from 22 patients with a positive 29-kDa antigenic diagnostic band of A. cantonensis were tested for the 21 and 24-kDa antigenic bands of G. spinigerum. Similarly, sera of 11 gnathostomiasis patients were tested for the 29-kDa diagnostic band for A. cantonensis. Only one patient in the angiostrongyliasis group had a positive result for the 21 and 24-kDa antigenic bands of G. spinigerum, while no gnathostomiasis patients showed a positive result for the 29-kDa antigenic band of A. cantonensis. The specificity of the 21 and 24-kDa antigenic bands for gnathostomiasis and the 29-kDa antigenic band for A. cantonensis was 95.5% and 100%, respectively. The antigenic bands for the diagnosis of gnathostomiasis and angiostrongyliasis in EOM were highly specific.

Tyrosine kinase B protein expression is reduced in the cerebellum of patients with bipolar disorder

The role of the cerebellum in coordinating mental activity is supported by its connections with cerebral regions involved in cognitive/affective functioning, with decreased activities on functional neuroimaging observed in the cerebellum of schizophrenia patients performing mental tasks. Brain-derived neurotrophic factor (BDNF)-induced activation of tyrosine kinase B (TrkB) is essential to synaptic plasticity. We hypothesized that alterations in BDNF and TrkB expression in the cerebellum were associated with schizophrenia and affective disorders. We employed immunohistochemistry and immunoblotting to quantify protein expression of BDNF and TrkB in the cerebellum of patients with schizophrenia, bipolar disorder, and major depression compared to controls (n=15 each). While TrkB immunoreactivity in each of the molecular and granule-cell layers was reduced in all 3 disease groups (12-34%) compared to the control (P=0.018 and 0.038, respectively, ANOVA), only the reduction in bipolar disorder remained statistically significant upon Tukey-Kramer post hoc analyses (P=0.019 and 0.021, respectively). Apparent decreases in BDNF immunoreactivity in all 3 disease groups (12-30%) compared to the control were not statistically significant. TrkB immunoreactivity was not significantly associated with any of the demographic, clinical, and postmortem variables. Immunoblotting displayed an 85-kDa TrkB-immunoreactive band, consistent with a truncated isoform, in all 60 cases. On immunoblotting, apparent decreases in 85-kDa-TrkB levels in all 3 disease groups compared to the control were not statistically significant. Our finding of reduced TrkB expression in bipolar disorder suggests that dysregulation of TrkB-mediated neurotrophin signaling in the cerebellum may play a role in the pathophysiology of this disease.

The Major Autolysin of Streptococcus gordonii Is Subject to Complex Regulation and Modulates Stress Tolerance, Biofilm Formation, and Extracellular-DNA Release

A gene, designated atlS, encoding a major autolysin from Streptococcus gordonii, was identified and characterized. The predicted AtlS protein is 1,160 amino acids and 127 kDa and has a conserved β1,4-N-acetylmuramidase domain. Zymographic analysis of wild-type S. gordonii revealed peptidoglycan hydrolase activities with molecular masses of 130 and 90 kDa that were absent in an atlS deletion mutant. Western blotting revealed that the 90-kDa band was derived from the 130-kDa protein. Inactivation of atlS resulted in formation of long chains by the cells, markedly decreased autolytic capacity, poor biofilm formation, diminished tolerance of acid and oxidative stress, and decreased production of extracellular DNA (eDNA). The biofilm-forming capacity of the atlS mutant could be almost completely restored to that of the wild-type strain by adding purified recombinant AtlA autolysin of S. mutans but was only partially restored by addition of eDNA. Autolysis, eDNA release, and atlS expression increased sharply when cells entered stationary phase and were greatly enhanced in cells growing with aeration. The LytST and VicRK two-component systems were both required for the induction of atlS by aeration, and purified LytT was able to bind to the promoter region of atlS in vitro. Thus, AtlS and its associated regulatory cascade dominantly control phenotypes of S. gordonii that are critical to colonization, persistence, and competition with other commensal and pathogenic oral bacteria in response to the redox environment and growth domain.

Anti-Borrelia burgdorferi Antibody Profile in Post-Lyme Disease Syndrome

Patients with post-Lyme disease syndrome (PLDS) report persistent symptoms of pain, fatigue, and/or concentration and memory disturbances despite antibiotic treatment for Lyme borreliosis. The etiopathogenesis of these symptoms remains unknown and no effective therapies have been identified. We sought to examine the antiborrelia antibody profile in affected patients with the aim of finding clues to the mechanism of the syndrome and its relationship to the original spirochetal infection. Serum specimens from 54 borrelia-seropositive PLDS patients were examined for antibodies to Borrelia burgdorferi proteins p18, p25, p28, p30, p31, p34, p39, p41, p45, p58, p66, p93, and VlsE by automated immunoblotting and software-assisted band analysis. The presence of serum antibodies to the 31-kDa band was further investigated by examination of reactivity against purified recombinant OspA protein. Control specimens included sera from 14 borrelia-seropositive individuals with a history of early localized or disseminated Lyme disease who were symptom free (post-Lyme healthy group), as well as 20 healthy individuals without serologic evidence or history of Lyme disease. In comparison to the post-Lyme healthy group, higher frequencies of antibodies to p28 (P < 0.05), p30 (P < 0.05), p31 (P < 0.0001), and p34 (P < 0.05) proteins were found in the PLDS group. Assessment of antibody reactivity to recombinant OspA confirmed the presence of elevated levels in PLDS patients (P < 0.005). The described antiborrelia antibody profile in PLDS offers clues about the course of the antecedent infection in affected patients, which may be useful for understanding the pathogenic mechanism of the disease.

Isolation and Characterisation of Two cDNAs Encoding the Neuromedin U Receptor from Goldfish Brain

Intracerebroventricular administration of neuromedin U (NMU) exerts an anorexigenic effect in a goldfish model. However, little is known about the NMU receptor and its signalling system in fish. In the present study, we isolated and cloned two cDNAs encoding different proteins comprising 429 and 388 amino acid residues from the goldfish brain based on the nucleotide sequences of human NMU receptor 1 (NMU-R1) and receptor 2 (NMU-R2). Hydropathy and phylogenetic analyses suggested that these two proteins were orthologues of NMU-R1 and -R2 of goldfish. We established two human embryonic kidney 293 cell lines stably expressing putative NMU-R1 and -R2, respectively, and showed that NMU induced an increase in intracellular calcium concentration ([Ca(2+)](i)) in these cells. We examined the presence of NMU-R1 and -R2 in the goldfish brain by western blotting analysis using affinity-purified antisera raised against peptide fragments derived from these receptors. NMU-R1-specific and NMU-R2-specific antisera detected a 49-kDa and 45-kDa immunopositive bands, respectively, in the brain extract. The mass of each band corresponded to that of the deduced respective primary structures. Reverse transcriptase-polymerase chain reaction analysis showed that NMU-R1 and -R2 transcripts were detected in several tissues. In particular, both mRNAs were strongly expressed in the goldfish brain. By contrast, NMU-R2 mRNA was also expressed in the gut. These results indicate for the first time that NMU-R orthologues exist in goldfish, and suggest physiological roles of NMU and its receptor system in fish.

Burkholderia pseudomallei Antibodies in Individuals Living in Endemic Regions in Northeastern Brazil

A seroepidemiological investigation was conducted among the population of two municipalities in Northeastern Brazil. Immunoglobulin M (IgM) and IgG antibodies to Burkholderia pseudomallei were positive in 51.27% (161 in 317 samples) and 58.49% (186), respectively. IgM titers were higher in children than in adults. On the contrary, IgG increased progressively with age. We observed a significant association between agricultural occupation and raised IgM titers (P < 0.005) and IgG titers (P < 0.001), and between construction workers and raised IgG titers (P = 0.005). Antibody IgG avidities did not correlate with age. The highest titers of antibodies (1/800) showed the highest antibody avidity indexes (P < 0.01). Most of the serum samples recognized 45-kDa and 200-kDa bands by IgG1 and IgG2 subclasses. Our study showed a high seropositivity among individuals living in endemic regions of the state of Ceará, and highlights the need for further surveillance close to water courses such as dams and rivers in Northeastern Brazil.

Relationships among hemocytes, tunic cells, germ cells, and accessory cells in the colonial ascidian Botryllus schlosseri

Monoclonal antibodies were raised against hemocytes of the colonial ascidian Botryllus schlosseri as possible tools to study hemocyte differentiation. In this species, blood cells are involved in various biological functions, such as immunosurveillance, encapsulation of foreign bodies, metal accumulation, and allorecognition. The latter process drives the fusion or rejection of contacting colonies, according to whether they do or do not share at least one allele at the fusibility/histocompatibility (Fu/HC) locus. Hemocytes take part in the rejection reaction, which suggests that they express molecules, coded by the Fu/HC locus, on their surface. A homozygous colony at the Fu/HC locus was used to produce the antibodies, which were screened by immunocytochemistry on hemocyte monolayers, immunohistochemistry on colony paraffin sections, and immunoblotting on colony homogenates. Here, we report on one of the obtained antibodies (1D8), which recognized a surface epitope on hemocytes of the donor colony and other colonies, apparently in a manner specific to the Fu/HC genotype. It also labeled a single 80-kDa band in colony homogenates. In addition, it specifically recognized tunic cells, germ cells, and their accessory cells. These results strengthen the assumption of a close relationship among these types of cells and blood cells, and suggest a close relationship among the above cells, probably deriving from undifferentiated blood cells.

Isolation and Characterization of Vitreous Insulin-like Growth Factor Binding Proteins

Previous studies from this laboratory revealed that vitreous insulin-like growth factor (IGF) biological activity increases in proliferative diabetic retinopathy and that this activity is normally attenuated by IGFBPs. The goal of this study was to identify and characterize the species involved. Human and porcine vitreous, plasma, recombinant IGFBP-2, and IGFBP-3 were separated by gel electrophoresis. Functional IGFBPs were detected in Western ligand blots with biotinylated IGF-II. IGFBPs were identified using IGFBP-specific antibodies. Western ligand blots of normal vitreous and plasma detected two major proteins at ∼35 kDa and ∼29 kDa. Western blot analysis of human and porcine vitreous and plasma confirmed the identity of the ∼35-kDa band as IGFBP-2 and the ∼29-kDa band as a fragment of IGFBP-3. Western blot and Western ligand blot analyses of vitreous and plasma proteins separated by two-dimensional gel electrophoresis revealed that the IGFBP-3 fragments in vitreous and plasma have virtually identical profiles. Lyase digestion revealed that the ∼29-kDa IGFBP-3 fragment is a glycoprotein with a peptide core of ∼25 kDa. N-terminal sequence data obtained from vitreous IGFBP-3 revealed that the protein is proteolytically truncated at the C terminus. CONCLUSIONS. Normal human and porcine vitreous contain two major IGFBPs, IGFBP-2 and an ∼29-kDa fragment of IGFBP-3. Both IGFBPs retain biological activity, and IGFBP-3 has one or more glycosylation sites with a protein core of ∼25 kDa. Systematic comparisons indicate that the vitreous IGFBP-3 is similar to and perhaps identical with a previously described IGFBP-3 fragment in plasma with reduced growth factor affinities.

Purification and biochemical properties of a cytochrome bc complex from the aerobic hyperthermophilic archaeon Aeropyrum pernix

BackgroundThe bioenergetics of Archaea with respect to the evolution of electron transfer systems is very interesting. In contrast to terminal oxidases, a canonical bc1 complex has not yet been isolated from Archaea. In particular, c-type cytochromes have been reported only for a limited number of species.ResultsHere, we isolated a c-type cytochrome-containing enzyme complex from the membranes of the hyperthermophilic archaeon, Aeropyrum pernix, grown aerobically. The redox spectrum of the isolated c-type cytochrome showed a characteristic α-band peak at 553 nm corresponding to heme C. The pyridine hemochrome spectrum also revealed the presence of heme B. In non-denaturing polyacrylamide gel electrophoresis, the cytochrome migrated as a single band with an apparent molecular mass of 80 kDa, and successive SDS-PAGE separated the 80-kDa band into 3 polypeptides with apparent molecular masses of 40, 30, and 25 kDa. The results of mass spectrometry indicated that the 25-kDa band corresponded to the hypothetical cytochrome c subunit encoded by the ORF APE_1719.1. In addition, the c-type cytochrome-containing polypeptide complex exhibited menaquinone: yeast cytochrome c oxidoreductase activities.ConclusionIn conclusion, we showed that A. pernix, a hyperthemophilic archaeon, has a "full" bc complex that includes a c-type cytochrome, and to the best of our knowledge, A. pernix is the first archaea from which such a bc complex has been identified. However, an electron donor candidates for cytochrome c oxidase, such as a blue copper protein, have not yet been identified in the whole genome data of this archaeon. We are currently trying to identify an authentic substrate between a bc complex and terminal oxidase.

Overexpression of nerve growth factor in peritoneal fluid from women with endometriosis may promote neurite outgrowth in endometriotic lesions

To investigate the role of the nerve growth factor (NGF) in the endometriosis-associated innervation in the development of endometriosis-associated symptoms, 41 peritoneal fluid samples (PF) from patients with surgically and histologically proven endometriosis and 20 PF from patients with other gynecologic conditions were analyzed with Western blot and a novel in vitro model using dorsal root ganglia (DRG) to show neuronal outgrowth; endometrial cells also were analyzed. The results suggest that the PF of endometriosis patients and endometriotic lesions have neurotropic properties, because the Western blot analysis and the cell culture stainings showed NGF expression, and the neurite outgrowth of DRG treated with PF of patients with endometriosis was significantly higher than when treated with PF of patients without endometriosis. Furthermore, blocking NGF with both anti-NGF and K252a leads to a significant decrease in neurite outgrowth.

The Isolation and Characterization of Glycosylated Phosphoproteins from Herring Fish Bones

Past studies of bone extracellular matrix phosphoproteins such as osteopontin and bone sialoprotein have yielded important biological information regarding their role in calcification and the regulation of cellular activity. Most of these studies have been limited to proteins extracted from mammalian and avian vertebrates and nonvertebrates. The present work describes the isolation and purification of two major highly glycosylated and phosphorylated extracellular matrix proteins of 70 and 22 kDa from herring fish bones. The 70-kDa phosphoprotein has some characteristics of osteopontin with respect to amino acid composition and susceptibility to thrombin cleavage. Unlike osteopontin, however, it was found to contain high levels of sialic acid similar to bone sialoprotein. The 22-kDa protein has very different properties such as very high content of phosphoserine (∼270 Ser(P) residues/1000 amino acid residues), Ala, and Asx residues. The N-terminal amino acid sequence analysis of both the 70-kDa (NPIMA(M)ETTS(M)DSKVNPLL) and the 22-kDa (NQDMAMEASSDPEAA) fish phosphoproteins indicate that these unique amino acid sequences are unlike any published in protein databases. An enzyme-linked immunosorbent assay revealed that the 70-kDa phosphoprotein was present principally in bone and in calcified scales, whereas the 22-kDa phosphoprotein was detected only in bone. Immunohistological analysis revealed diffusely positive immunostaining for both the 70- and 22-kDa phosphoproteins throughout the matrix of the bone. Overall, this work adds additional support to the concept that the mechanism of biological calcification has common evolutionary and fundamental bases throughout vertebrate species.

Purification of the Major Group 1 Allergen from Bahia Grass Pollen, Pas n 1

Background: Group 1 grass pollen allergens are glycoproteins of the β-expansin family. They are a predominant component of pollen and are potent allergens with a high frequency of serum IgE reactivity in grass pollen-allergic patients. Bahia grass is distinct from temperate grasses and has a prolonged pollination period and wide distribution in warmer climates. Here we describe the purification of the group 1 pollen allergen, Pas n 1, from Bahia grass (Paspalum notatum), an important subtropical aeroallergen source. Methods: Pas n 1 was purified from an aqueous Bahia grass pollen extract by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography, and assessed by one- and two-dimensional gel electrophoresis, immunoblotting and ELISA. Results: Pas n 1 was purified to a single 29-kDa protein band containing two dominant isoforms detected by an allergen-specific monoclonal antibody and serum IgE of a Bahia grass pollen-allergic donor. The frequency of serum IgE reactivity with purified Pas n 1 in 51 Bahia grass pollen-allergic patients was 90.6%. Serum IgE reactivity with purified Pas n 1 was highly correlated with serum IgE reactivity with Bahia grass pollen extract and recombinant Pas n 1 (r = 0.821 and 0.913, respectively). Conclusions: Pas n 1 is a major allergen reactive at high frequency with serum IgE of Bahia grass pollen-allergic patients. Purified natural Pas n 1 has utility for improved specific diagnosis and immunotherapy for Bahia grass pollen allergy.

The Cystic Fibrosis Transmembrane Conductance Regulator Impedes Proteolytic Stimulation of the Epithelial Na+ Channel

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) that prevent its proper folding and trafficking to the apical membrane of epithelial cells. Absence of cAMP-mediated Cl(-) secretion in CF airways causes poorly hydrated airway surfaces in CF patients, and this condition is exacerbated by excessive Na(+) absorption. The mechanistic link between missing CFTR and increased Na(+) absorption in airway epithelia has remained elusive, although substantial evidence implicates hyperactivity of the epithelial Na(+) channel (ENaC). ENaC is known to be activated by selective endoproteolysis of the extracellular domains of its α- and γ-subunits, and it was recently reported that ENaC and CFTR physically associate in mammalian cells. We confirmed this interaction in oocytes by co-immunoprecipitation and found that ENaC associated with wild-type CFTR was protected from proteolytic cleavage and stimulation of open probability. In contrast, ΔF508 CFTR, the most common mutant protein in CF patients, failed to protect ENaC from proteolytic cleavage and stimulation. In normal airway epithelial cells, ENaC was contained in the anti-CFTR immunoprecipitate. In CF airway epithelial cultures, the proportion of full-length to total α-ENaC protein signal was consistently reduced compared with normal cultures. Our results identify limiting proteolytic cleavage of ENaC as a mechanism by which CFTR down-regulates Na(+) absorption.