70-kDa Band Research Articles

The Insulin-Like Factor 3 (INSL3)-Receptor (RXFP2) Network Functions as a Germ Cell Survival/Anti-Apoptotic Factor in Boar Testes

Relaxin-like factor, commonly known as insulin-like factor (INSL3), is essential for testis descent during fetal development; however, its function in the adult testis is still being elucidated. The study aimed to identify a relaxin family peptide receptor 2 (RXFP2)-specific antibody suitable for immunological approaches, analyze which testicular germ cell types express RXFP2, and clarify its expression dynamics in the boar testis. In addition, the function of INSL3-RXFP2 signaling on the germ cells was explored by neutralizing INSL3 using long-term active immunization. Samples were collected from Duroc boars, and a commercially available RXFP2-specific antibody directed against the human RXFP2 endodomain was identified by characterizing its specificity in HEK-293 cells expressing mouse RXFP2, and by demonstrating the suitability for analyzing RXFP2 expression in porcine tissues. RXFP2 mRNA and protein were both localized mainly in meiotic and post-meiotic germ cells, but not in Leydig cells. Functional RXFP2, which enables INSL3 to bind, was detected as an ∼85-kDa band, which increased in intensity from the pubertal stage onward. Interestingly, INSL3 immunization significantly reduced testis weight and induced a 4-fold increase in the frequency of apoptotic germ cells, which was associated with the up-regulation of pro-apoptotic caspase-3 (CASP3) and BAX, and the down-regulation of anti-apoptotic XIAP and BCL2, and a substantial reduction in sperm concentration. These results revealed that RXFP2 was expressed in boar meiotic and post-meiotic germ cells, where INSL3 neutralization led to increased germ cell apoptosis and reduced sperm output, suggesting that INSL3 acts as a survival/anti-apoptotic factor in maintaining sperm production.

Proteolytic Processing and Activation of Clostridium perfringens Epsilon Toxin by Caprine Small Intestinal Contents

ABSTRACTEpsilon toxin (ETX), a pore-forming toxin produced by type B and D strains of Clostridium perfringens, mediates severe enterotoxemia in livestock and possibly plays a role in human disease. During enterotoxemia, the nearly inactive ETX prototoxin is produced in the intestines but then must be activated by proteolytic processing. The current study sought to examine ETX prototoxin processing and activation ex vivo using the intestinal contents of a goat, a natural host species for ETX-mediated disease. First, this study showed that the prototoxin has a KEIS N-terminal sequence with a molecular mass of 33,054 Da. When the activation of ETX prototoxin ex vivo by goat small intestinal contents was assessed by SDS-PAGE, the prototoxin was processed in a stepwise fashion into an ~27-kDa band or higher-molecular-mass material that could be toxin oligomers. Purified ETX corresponding to the ~27-kDa band was cytotoxic. When it was biochemically characterized by mass spectrometry, the copresence of three ETX species, each with different C-terminal residues, was identified in the purified ~27-kDa ETX preparation. Cytotoxicity of each of the three ETX species was then demonstrated using recombinant DNA approaches. Serine protease inhibitors blocked the initial proteotoxin processing, while carboxypeptidase inhibitors blocked further processing events. Taken together, this study provides important new insights indicating that, in the intestinal lumen, serine protease (including trypsin and possibly chymotrypsin) initiates the processing of the prototoxin but other proteases, including carboxypeptidases, then process the prototoxin into multiple active and stable species.

Green synthesis of protein capped silver nanoparticles from phytopathogenic fungus Macrophomina phaseolina (Tassi) Goid with antimicrobial properties against multidrug-resistant bacteria

In recent years, green synthesis of nanoparticles, i.e., synthesizing nanoparticles using biological sources like bacteria, algae, fungus, or plant extracts have attracted much attention due to its environment-friendly and economic aspects. The present study demonstrates an eco-friendly and low-cost method of biosynthesis of silver nanoparticles using cell-free filtrate of phytopathogenic fungus Macrophomina phaseolina. UV-visible spectrum showed a peak at 450 nm corresponding to the plasmon absorbance of silver nanoparticles. Scanning electron microscopy (SEM), atomic force microscopy (AFM), and transmission electron microscopy (TEM) revealed the presence of spherical silver nanoparticles of the size range 5 to 40 nm, most of these being 16 to 20 nm in diameter. X-ray diffraction (XRD) spectrum of the nanoparticles exhibited 2θ values corresponding to silver nanoparticles. These nanoparticles were found to be naturally protein coated. SDS-PAGE analysis showed the presence of an 85-kDa protein band responsible for capping and stabilization of the silver nanoparticles. Antimicrobial activities of the silver nanoparticles against human as well as plant pathogenic multidrug-resistant bacteria were assayed. The particles showed inhibitory effect on the growth kinetics of human and plant bacteria. Furthermore, the genotoxic potential of the silver nanoparticles with increasing concentrations was evaluated by DNA fragmentation studies using plasmid DNA.

IRES-incorporated lactococcal bicistronic vector for target gene expression in a eukaryotic system

Plasmid DNAs isolated from lactic acid bacteria (LAB) such as Lactococcus lactis (L. lactis) has been gaining more interests for its positive prospects in genetic engineering-related applications. In this study, the lactococcal plasmid, pNZ8048 was modified so as to be able to express multiple genes in the eukaryotic system. Therefore, a cassette containing an internal ribosome entry site (IRES) was cloned between VP2 gene of a very virulent infectious bursal disease (vvIBDV) UPM 04190 of Malaysian local isolates and the reporter gene, green fluorescent protein (GFP) into pNZ:CA, a newly constructed derivative of pNZ8048 harboring the cytomegalovirus promoter (Pcmv) and polyadenylation signal. The new bicistronic vector, denoted as pNZ:vig was subjected to in vitro transcription/translation system followed by SDS–PAGE and Western blot analysis to rapidly verify its functionality. Immunoblotting profiles showed the presence of 49 and 29kDa bands that corresponds to the sizes of the VP2 and GFP proteins respectively. This preliminary result shows that the newly constructed lactococcal bicistronic vector can co-express multiple genes in a eukaryotic system via the IRES element thus suggesting its feasibility to be used for transfection of in vitro cell cultures and vaccine delivery.

Association between the presence of protein bands in ram seminal plasma and sperm tolerance to freezing

This study evaluated associations between the presence of protein bands in ram seminal plasma and the quality of sperm frozen with distinct extenders. Ejaculates were frozen in a Tris–egg yolk based extender, including either 5% glycerol or 100mM trehalose. Seminal plasma samples were submitted to unidimensional electrophoresis. Pre-freezing and post-thawing sperm quality was similar between extenders (P>0.05). A total of 26 bands were identified in ram seminal plasma. Pre-freezing sperm motility was increased when the 15, 19 and 80kDa bands were present in seminal plasma (P<0.05). The presence of an 11kDa band in seminal plasma was associated with reduced pre-freezing membrane integrity (P<0.05). After thawing, both sperm motility and membrane integrity were reduced when a 24kDa band was present in seminal plasma (P<0.05). Post-thawing acrosome integrity was greater in the presence of a 31kDa band in seminal plasma (P<0.05). Regardless of the cryoprotectant included in the freezing extender, these six bands may be potential markers for ram sperm tolerance to freezing.

Developmental Increase in Ecto-5′-Nucleotidase Activity Overlaps with Appearance of Two Immunologically Distinct Enzyme Isoforms in Rat Hippocampal Synaptic Plasma Membranes

Ecto-5'-nucleotidase (e-5NT), a glycosylphosphatidylinositol-linked membrane protein, catalyzes a conversion of AMP to adenosine, which influences nearly every aspect of brain physiology, including embryonic and postnatal brain development. The present study aimed to investigate a pattern of expression, activity and kinetic properties of e-5NT in the hippocampal formation and synaptic plasma membrane (SPM) preparations in rats at postnatal days (PDs) 7, 15, 20, 30 and 90. By combining gene expression analysis and enzyme histochemistry, we observed that e-5NT mRNA reached the adult level at PD20, while the enzyme activity continued to increase beyond this age. Further analysis revealed that hippocampal layers rich in synapses expressed the highest levels of e-5NT activity, while in layers populated with neuronal cell bodies, the enzyme activity was weak or absent. Therefore, activity and expression of e-5NT were analyzed in SPM preparations isolated from rats at different ages. The presence of two protein bands of about 65 and 68 kDa was determined by immunoblot analysis. The 65-kDa band was present at all ages, and its abundance increased from PD7 to PD20. The 68-kDa band appeared at PD15 and increased until PD30, coinciding with the increase of e-5NT activity, substrate affinity and enzymatic efficiency. Since distinct e-5NT isoforms may derive from different patterns of the enzyme protein N-glycosylation, we speculate that long-term regulation of e-5NT activity in adulthood may be effectuated at posttranslational level and without overall change in the gene and protein expression.

Detection of grass carp reovirus (GCRV) with monoclonal antibodies

Grass carp reovirus (GCRV) is a pathogen that causes hemorrhagic disease of grass carp. It is the most serious infectious disease of carp and causes serious losses of fingerlings of grass carp and black carp. In this study, a recombinant VP4, one of the viral core proteins, was constructed with a histidine tag and expressed at a high level in E. coli, and the expressed protein was mainly found in the form of inclusion bodies. The expressed VP4 protein was recognized by an anti-His-tag monoclonal antibody and goat anti-GCRV serum. Four monoclonal antibodies (16B7, 39E12, 13C3 and 14D1) against the recombinant VP4 protein were produced. These MAbs did not react with any of the tested viruses or fish cells lines in the ELISA tests except GCRV. In western blotting analysis, a protein band was observed when the recombinant VP4 protein of GCRV was used as an antigen, but a 68-kDa band was observed when natural capsid proteins of GCRV were used as antigens. Furthermore, a sandwich ELISA was developed for detection of GCRV. The detection limit of the test was 105 TCID50 of GCRV per mL.

Uterine fluid proteins and egg quality characteristics for 2 commercial and 2 heritage laying hen lines in response to manipulation of dietary calcium and vitamin D3

The aim of this study was to evaluate the quality of eggs from 2 selected commercial strains of laying hens and 2 unselected lines of chickens fed diets with different combinations of Ca and vitamin D and relate it to the profile of uterine proteins and ultrastructure of the shell. A group of 4 chickens was housed in each of 24 cages. The group consisted of one representative from each of the following breeds: Lohmann LSL- Lite, Lohmann Classic-Brown, Fayoumi, and Light Sussex. Six dietary combinations of Ca and vitamin D3 (3.35%, 2,500 IU; 4.10%, 2,500 IU (control); 4.85%, 2,500 IU; 3.35%, 200 IU; 4.10%, 200 IU; and 4.85%, 200 IU) were randomly assigned to 4 replicate cages for 2 treatment periods (26–29 and 56–59 wk of age). Data were analyzed as a split-plot design with cage as the main plot and hen as the subplot. Egg quality traits were different (P < 0.0001) between commercial and heritage breeds. Lohmann Brown had stronger shells with higher specific gravity compared with other breeds. Both commercial and heritage birds responded to a drop in vitamin D3 level by marked reduction in shell thickness. The SDS-PAGE profiles of uterine fluid samples revealed a decrease (P < 0.05) in 200-, 150-, 116-, and ≤6.5-kDa proteins, whereas proteins with molecular weight (MW) of 80, 55, 52, 45, 42, and 28 kDa increased with bird age. A 36- and 52-kDa protein band was most intense for Fayoumi compared with other breeds. Ultrastructural characteristics showed flattened and deeply etched mammillary caps for Lohmann Brown and the presence of type A and type B bodies between mammillary cones in eggshells from Fayoumi and Lohmann Lite. The negative correlation between ultrastructural characteristics, which decrease with bird age, and the 116-kDa uterine protein band could provide insight into reduced eggshell quality as hens age.

Identification of two new polar tube proteins related to polar tube protein 2 in the microsporidianAntonospora locustae

Microsporidia are obligate intracellular eukaryotic parasites with a broad host spectrum characterized by a unique and highly sophisticated invasion apparatus, the polar tube (PT). In a previous study, two PT proteins, named AlPTP1 (50 kDa) and AlPTP2 (35 kDa), were identified in Antonospora locustae, an orthoptera parasite that is used as a biological control agent against locusts. Antibodies raised against AlPTP2 cross-reacted with a band migrating at ~70 kDa, suggesting that this 70-kDa antigen is closely related to AlPTP2. A blastp search against the A. locustae genome database allowed the identification of two further PTP2-like proteins named AlPTP2b (568 aa) and AlPTP2c (599 aa). Both proteins are characterized by a specific serine- and glycine-rich N-terminal extension with elastomeric structural features and share a common C-terminal end conserved with AlPTP2 (~88% identity for the last 250 aa). MS analysis of the 70-kDa band revealed the presence of AlPTP2b. Specific anti-AlPTP2b antibodies labelled the extruded PTs of the A. locustae spores, confirming that this antigen is a PT component. Finally, we showed that several PTP2-like proteins are also present in other phylogenetically related insect microsporidia, including Anncaliia algerae and Paranosema grylli.

Dot immunogold filtration assay (DIGFA) for the rapid detection of specific antibodies against the rat lungwormAngiostrongylus cantonensis(Nematoda: Metastrongyloidea) using purified 31-kDa antigen

A rapid dot immunogold filtration assay (DIGFA) was adopted for specific immunodiagnosis of human cerebral angiostrongyliasis, using purified 31-kDa glycoprotein specific to Angiostrongylus cantonensis as diagnostic antigen and protein A colloidal gold conjugate as antigen-antibody detector. A total of 59 serum samples were assayed - 11 samples from clinically diagnosed patients with detectable A. cantonensis-specific antibody in immunoblotting; 23 samples from patients with other related parasitic diseases, i.e. gnathostomiasis (n=8), cysticercosis (n=5), toxocariasis (n=2), filariasis (n=4), paragonimiasis (n=2) and malaria (n=2); and 25 samples from normal healthy subjects. The sensitivity and specificity of DIGFA to detect anti-A. cantonensis specific antibodies in serologically confirmed angiostrongyliasis cases, were both 100%. No positive DIGFA was observed in cases with other parasitic diseases, and the healthy control subjects. The 3-min DIGFA is as sensitive and specific as the 3-h immunoblot test in angiostrongyliasis confirmed cases that revealed a 31-kDa reactive band. The gold-based DIGFA is more rapid and easier to perform than the traditional enzyme-linked immunosorbent assay (ELISA). The test utilizing purified A. cantonensis antigen is reliable and reproducible for specific immunodiagnosis of human infection with A. cantonensis - thus can be applied as an additional routine test for clinical diagnostic support. Large-scale sero-epidemiological studies in endemic communities in north-east Thailand are under way to evaluate its usefulness under field conditions.

Clonaje y expresión de un fragmento recombinante del gen cagA de Helicobacter pylori y su evaluación preliminar en el serodiagnóstico

Helicobacter pylori strains expressing cytotoxic CagA protein are more commonly associated with peptic ulceration, atrophic gastritis and gastric adenocarcinoma than those lacking CagA. Determination of anti-CagA antibodies, therefore, acquires a relevant clinical significance in the serological detection of H. pylori infection and disease risk prediction. However, the CagA-serology has been questioned due to the differences found in their performance evaluations in different populations. To obtain a recombinant CagA fragment useful for serodiagnosis of H. pylori infection A fragment of the cagA gene was cloned into a prokaryotic T7 RNA polymerase expression vector. A recombinant C-terminal His 6 -tagged CagA was expressed, subsequently solubilized with urea and purified by immobilized metal affinity chromatography. The performance of the recombinant protein was evaluated using 180 human serum samples with an in-house Western blot assay compared to the Helicoblot 2.1 reference test. The expressed His 6 -tagged CagA showed an immunoreactive 80kDa band as was revealed by SDS-PAGE and Western blot analysis using two different specific anti-CagA polyclonal antibodies. The recombinant protein was successfully purified obtaining a 93% of purity. The performance analysis of the purified recombinant antigen showed good immunoreactivity and exhibited values of sensitivity, specificity and accuracy of 88.1%, 100% and 92.7%, respectively. The CagA fragment of the study may constitute a useful tool for serological diagnosis of CagA-positive H. pylori infection.

Expression of Syntaxin‐2 in Bovine Sperm

Syntaxins are membrane integrated Q‐SNARE proteins known to participate in exocytosis. Vesicle docking involves the binding of two plasma membrane proteins, syntaxin and SNAP‐25, to the vesicle membrane protein VAMP to form a stable trimeric core complex; synaptophysin is thought to regulate the formation of this complex. Although the members of Q‐SNARE proteins are characterized in somatic cells, it is not known whether related proteins function in the sperm acrosome reaction. The objective of the present study is to identify and localize syntaxin and synaptophysin in bovine epididymal spermatozoa. Western blots of caput and cauda sperm lysates and plasma membrane fractions stained with anti‐syntaxin and anti‐synaptophysin showed the presence of a 31kDa and 38kDa band in both sperm lysates and plasma membrane fractions, respectively. Indirect immunofluorescence localized syntaxin to the anterior but not equatorial regions of the acrosomal segment. Syntaxin was recovered in the anti‐syntaxin immunoprecipitation pellet. Coimmunoprecipitation analysis will be utilized to identify the syntaxin‐associated protein(s) complex during capacitation and acrosome reaction of bovine spermatozoa. It is assumed that syntaxin‐2 and synaptophysin may be involved in triggering the acrosome reaction through a ligand‐receptor mediated signal transduction pathway. NIH 1SC3GM096875 and FSU‐RISE ‐ STEM Grants.

Z-band Alternatively Spliced PDZ Motif Protein (ZASP) Is the Major O-Linked β-N-Acetylglucosamine-substituted Protein in Human Heart Myofibrils

We studied O-linked β-N-acetylglucosamine (O-GlcNAc) modification of contractile proteins in human heart using SDS-PAGE and three detection methods: specific enzymatic conjugation of O-GlcNAc with UDP-N-azidoacetylgalactosamine (UDP-GalNAz) that is then linked to a tetramethylrhodamine fluorescent tag and CTD110.6 and RL2 monoclonal antibodies to O-GlcNAc. All three methods showed that O-GlcNAc modification was predominantly in a group of bands ~90 kDa that did not correspond to any of the major myofibrillar proteins. MALDI-MS/MS identified the 90-kDa band as the protein ZASP (Z-band alternatively spliced PDZ motif protein), a minor component of the Z-disc (about 1 per 400 α-actinin) important for myofibrillar development and mechanotransduction. This was confirmed by the co-localization of O-GlcNAc and ZASP in Western blotting and by immunofluorescence microscopy. O-GlcNAcylation of ZASP increased in diseased heart, being 49 ± 5% of all O-GlcNAc in donor, 68 ± 9% in end-stage failing heart, and 76 ± 6% in myectomy muscle samples (donor versus myectomy p < 0.05). ZASP is only 22% of all O-GlcNAcylated proteins in mouse heart myofibrils.

Understanding the Direct Synaptic Effects of Estradiol

More than 35 years ago, Kelly and coworkers (1) demonstrated that iontophoretic application of an estradiol ester altered the firing rates of a subset of hypothalamic neurons in a matter of milliseconds, an effect that appeared to be stereospecific in that responses were observed to esters of 17 -estradiol, but not its 17 isomer. These observations accompanied the almost simultaneous demonstration by Pietras and Szego (2) that endometrial cells had estradiol recognition sites on their plasma membranes. Kelly et al (1) proposed that neurons might also express stereospecific membrane receptor sites, capable of mediating rapid electrophysiological responses to changes in ovarian estradiol secretion. Progress in characterizing these putative membrane receptors was initially slow, because most work continued to focus on estrogen receptor (ER) actions in the cell nucleus and the regulation of transcriptional response mechanisms. This situation changed dramatically in the early 1990s with the demonstration of rapid effects of estradiol on a number of intracellular signaling pathways (3, 4) as well as direct evidence of ER-like immunoreactivity in dendrites, axon terminals, and postsynaptic densities in the brain (5–7) (reviewed in Blaustein, Ref. 8). Over the ensuing 20 years, multiple potential receptor systems for estradiol have been identified in the central nervous system, signaling via a number of different intracellular pathways (reviewed in Micevych and Christensen, Ref. 9). Studies in cells transfected with vectors expressing ER or ER have shown that whereas newly synthesized receptors concentrate in the cell nucleus, they also incorporate into the plasma membrane (10) where they can initiate estradiol-dependent changes in ERK phosphorylation (11), suggesting that plasma membrane and nuclear ER or ER may be derived from a common source. The demonstration that ER immunoreactivity is present in multiple neuronal cellular compartments raises the question of how the distribution of extranuclear ER is controlled. Are ERs associated with specific cellular organelles or do synaptic and dendritic ERs represent receptors en passant to the plasma membrane? Studies in nonneuronal systems have demonstrated that that ER is trafficked to the plasma membrane via a caveolin-dependent process (12) where it can be anchored in place by palmitoylation (13). Is the same true for ER in neurons? Is the density of ER in the plasma membrane regulated independently of nuclear receptor concentrations? Is ER distributed evenly throughout neuronal cell membranes or is it concentrated specifically in target sites where it can both respond to locally synthesized steroids and exert rapid effects on neurotransmission? The paper by Tabatadze et al (14) in this issue of Endocrinology begins to answer some of these questions. Using tissue fractionationtechniques, theauthorsdemonstrate that ER is concentrated in synaptosomes, particularly in association with synaptic vesicles and neuropeptide-containing large dense-core vesicles. Immunoisolation experiments showthatER is associatedwithbothgammaaminobutryic acid (GABA)and glutamate-containing vesicles. On Western blots, synaptosomal ER immunoreactivity migrates as two bands at approximately 66 and 70 kDa, the ratio of the higher to lower molecular mass forms being greatly decreased by phosphatase treatment, suggesting that the 70kDa band represents a phosphorylated form of the receptor. Finally, treatment of hippocampal slices in vitro was found to decrease the quantity of ER associated with synaptosomal membranes, via a depalmitoylation-dependent mechanism. Overall, the results suggest that ER associates with synaptosomal membranes via a palmitoyla-

A Lectin with Highly Potent Inhibitory Activity toward Breast Cancer Cells from Edible Tubers of Dioscorea opposita cv. Nagaimo

A 70-kDa galactose-specific lectin was purified from the tubers of Dioscorea opposita cv. nagaimo. The purification involved three chromatographic steps: anion exchange chromatography on a Q-Sepharose column, FPLC-anion exchange chromatography on a Mono Q column, and FPLC-gel filtration on a Superdex 75 column. The purified nagaimo lectin presented as a single 35-kDa band in reducing SDS-PAGE while it exhibited a 70-kDa single band in non-reducing SDS-PAGE suggesting its dimeric nature. Nagaimo lectin displayed moderate thermostability, retaining full hemagglutinating activity after heating up to 62°C for 30 minutes. It also manifested stability over a wide pH range from pH 2 to 13. Nagaimo lectin was a galactose-specific lectin, as evidenced by binding with galactose and galactose-containing sugars such as lactose and raffinose. The minimum concentration of galactose, lactose and raffinose required to exert an inhibitory effect on hemagglutinating activity of nagaimo lectin was 20 mM, 5 mM and 40 mM, respectively. Nagaimo lectin inhibited the growth of some cancer cell lines including breast cancer MCF7 cells, hepatoma HepG2 cells and nasopharyngeal carcinoma CNE2 cells, with IC50 values of 3.71 µM, 7.12 µM and 19.79 µM, respectively, after 24 hour treatment with nagaimo lectin. The induction of phosphatidylserine externalization and mitochondrial depolarization indicated that nagaimo lectin evoked apoptosis in MCF7 cells. However, the anti-proliferative activity of nagaimo lectin was not blocked by application of galactose, signifying that the activity was not related to the carbohydrate binding specificity of the lectin.

Time course of the antibody response in humans compared with rats experimentally infected with hepatic alveolar echinococcosis

Human alveolar echinococcosis (AE) is caused by the accidental ingestion of Echinococcus multilocularis eggs. Early detection is essential as surgical resection is the only treatment for a complete cure. However, details are unclear about changes in the antibody response during the initial stages of infection, yet such information is useful for early serodiagnosis. Therefore, a long-term investigation was performed into the time course of the antibody response before 'positive' detection. Patient sera were used for enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) analysis using crude antigens extracted from E. multilocularis protoscoleces. Rats were experimentally infected with AE and similarly analysed by ELISA and WB. Among the markers for diagnoses, the 18kDa band (main marker) appeared last in the preoperative stages and disappeared first after operation in a WB test. Although the 18kDa antigen can be useful as a marker for AE diagnosis, it cannot contribute to the detection of some patients before the 18kDa band appearance. To avoid misdiagnosis, different diagnostic antigens such as the 26-28 and 7-8kDa bands should also be considered. These bands tend to appear earlier than the 18kDa band and thus offer the potential for early detection of AE. We first observed changes in the antibody response in a relatively early stage after infection in human AE cases. Notably, changes in the antibody response of two intermediate species were similar. These findings provide valuable information for the early detection of human AE cases in the future.

Molybdophyllysin, a toxic metalloendopeptidase from the tropical toadstool, Chlorophyllum molybdites

A toxic protein, dubbed molybdophyllysin, was isolated from the tropical toadstool Chlorophyllum molybdites by following its lethal effect in mice. Analysis of the protein using SDS–PAGE revealed a single 23-kDa band. Sequence analysis of molybdophyllysin tryptic fragments showed that this protein is highly homologous to metalloendopeptidases (MEPs) obtained from edible mushrooms, such as Grifola frondosa, Pleurotus ostreatus, and Armillaria mellea. These proteins include a HEXXH+D zinc-binding motif known as aspzincin. Accordingly, molybdophyllysin is a member of the deuterolysin family of zinc proteases. Molybdophyllysin retained its proteolytic activity at temperatures up to 60°C with an optimum pH of 7.0. The activity was inhibited by both 1,10-phenanthroline and N-bromosuccinimide, but molybdophyllysin exhibited strong resistance to SDS.

The 31-kDa Antigen of Angiostrongylus cantonensis Comprises Distinct Antigenic Glycoproteins

Human angiostrongyliasis results from accidental infection with Angiostrongylus, an intra-arterial nematode. Angiostrongylus cantonensis infections result in eosinophilic meningitis, and A. costaricensis infections cause eosinophilic enteritis. Immunological methodologies are critical to the diagnosis of both infections, since these parasites cannot be isolated from fecal matter and are rarely found in cerebrospinal fluid samples. A. costaricensis and A. cantonensis share common antigenic epitopes which elicit antibodies that recognize proteins present in either species. Detection of antibodies to a 31-kDa A. cantonensis protein present in crude adult worm extracts is a sensitive and specific method for immunodiagnosis of cerebral angiostrongyliasis. The objective of the present work was to isolate and characterize the 31-kDa proteins using soluble protein extracts derived from adult female worms using both one- (1DE) and two-dimensional (2DE) gel electrophoresis. Separated proteins were blotted onto nitrocellulose and probed using sera from infected and non-infected controls. The 31-kDa band present in 1DE gels and the 4 spots identified in 2DE gels were excised and analyzed by electrospray ionization mass spectrometry. Using the highest scores obtained following Mascot analysis, amino acid sequences were obtained that matched four unique proteins: tropomyosin, the 14-3-3 phosphoserine-binding protein, a protein containing a nascent polypeptide-associated complex domain, and the putative epsilon subunit of coatomer protein complex isoform 2. Oxidative cleavage of diols using sodium m-periodate demonstrated that carbohydrate moieties are essential for the antigenicity of all four spots of the 31-kDa antigen. In this article we describe the identification of the 31-kDa antigen, and provide DNA sequencing of the targets. In conclusion, these data suggest that reactivity to the 31-kDa proteins may represent antibody recognition of more than one protein, and recombinant protein-based assays for cerebral angiostrongyliasis diagnosis may require eukaryotic expression systems to maintain antigenicity.

Tissue-specific expression and functional role of dehydrins in heat tolerance of sugarcane (Saccharum officinarum)

Studies on the functional roles of dehydrins (DHNs) in heat tolerance of plants are scarce. This study was conducted to immunohistolocalize DHNs in leaves of heat-tolerant (CP-4333) and heat-sensitive (HSF-240) sugarcane (Saccharum officinarum L.) clones at three phenological stages in order to elucidate their putative roles under heat stress. CP-4333 indicated greater amounts of heat-stable proteins than HSF-240 under heat stress. Western blotting revealed the expression of three DHNs in CP-4333 (13- and 15-kDa peptides at 48h and an additional 18-kDa band at 72h) and two (13 and 15kDa at 48h) in HSF-240 at formative stage; two DHNs in CP-4333 (20 and 25kDa) and one in HSF-240 (20kDa) at grand growth stage, while two DHNs in CP-4333 (20 and 22kDa) and one in HSF-240 (20kDa) at maturity stage. Tissue-specific immunohistolocalization showed that DHNs were expressed in stele particularly the phloem and the cells intervening bundle sheath and vascular bundles. Furthermore, DHNs were also found scattered along the epidermal and parenchymatous cells. Recovery of sugarcane from heat stress manifested a gradual disappearance of DHNs in both the clones, being quicker in sensitive clone (HSF-240). Results suggested specific implications for DHNs synthesis. Their synthesis in epidermis appears to protect the mesophyll tissues from heat injury. When associated to vascular tissue, they tend to ensure the normal photoassimilate loading into the sieve element-companion cell complex. DHNs diminution during recovery suggested that their expression was transitory. However, prolonged retention of DHNs by tolerant clone appears to be an adaptive advantage of sugarcane to withstand heat stress.

Osteopetrosis Mutation R444L Causes Endoplasmic Reticulum Retention and Misprocessing of Vacuolar H+-ATPase a3 Subunit

Osteopetrosis is a genetic bone disease characterized by increased bone density and fragility. The R444L missense mutation in the human V-ATPase a3 subunit (TCIRG1) is one of several known mutations in a3 and other proteins that can cause this disease. The autosomal recessive R444L mutation results in a particularly malignant form of infantile osteopetrosis that is lethal in infancy, or early childhood. We have studied this mutation using the pMSCV retroviral vector system to integrate the cDNA construct for green fluorescent protein (GFP)-fused a3(R445L) mutant protein into the RAW 264.7 mouse osteoclast differentiation model. In comparison with wild-type a3, the mutant glycoprotein localized to the ER instead of lysosomes and its oligosaccharide moiety was misprocessed, suggesting inability of the core-glycosylated glycoprotein to traffic to the Golgi. Reduced steady-state expression of the mutant protein, in comparison with wild type, suggested that the former was being degraded, likely through the endoplasmic reticulum-associated degradation pathway. In differentiated osteoclasts, a3(R445L) was found to degrade at an increased rate over the course of osteoclastogenesis. Limited proteolysis studies suggested that the R445L mutation alters mouse a3 protein conformation. Together, these data suggest that Arg-445 plays a role in protein folding, or stability, and that infantile malignant osteopetrosis caused by the R444L mutation in the human V-ATPase a3 subunit is another member of the growing class of protein folding diseases. This may have implications for early-intervention treatment, using protein rescue strategies.