Distinct Protein Bands Research Articles

Glycosylation of aquaporins in the freeze tolerant tree frog, Hyla chrysoscelis

HC‐1, ‐2, and ‐3 are aquaporins expressed in tissues of the freeze‐tolerant treefrog Hyla chrysoscelis. We hypothesize that the aquaporins are involved in osmoregulation during cold acclimation and freezing. Western blots reveal distinct protein bands at the nominal MW of the proteins (29 ‐ 31 kDa), and also at higher MW. We hypothesized that the high MW "smear" represents glycosylated forms of aquaporin involved in proper protein folding and localization to the membrane. The purpose of the present project was to investigate the magnitude of this glycosylation and its importance in quantifying aquaporin expression. We used Peptide N‐glycosidase (PNGase F), an enzyme that cleaves the oligosaccharides attached to extracellular asparagine residue, to examine N‐linked glycosylation of HC‐1, 2, and 3. In all tissues examined (skin, muscle, stomach, liver, blood, kidney, small and large intestine), addition of PNGase F decreased the intensity of signal at high MW and increased the intensity of the 29 ‐ 31 kDa band. We conclude that variation in aquaporin protein expression in treefrogs experiencing cold or freezing is associated with changing patterns of glycosylation. However, even with PNGase F, we continue to observe high MW bands in some tissues and conditions; further studies are exploring whether this represents an alternative form, O‐linked, glycosylation. Supported by NSF IOB‐0517301 and APS UGSRF.

A simple method for purifying the White Spot Syndrome Virus using ultrafiltration

A very simple and efficient method was developed for isolating intact White Spot Syndrome Virus (WSSV) particles from infected Litopenaeus vannamei tissue. No density gradient centrifugation, ultracentrifugation or protease inhibitors were required for the purification of intact WSSV virions using microfilters (100 kDa cut-off) combined with several steps of conventional centrifugation procedures. A mortality assay was run using healthy shrimp to prove that the virions obtained were infective. The concentrated viral preparations were further studied using polyacrylamide gel electrophoresis (PAGE). At least five distinct protein bands were detected when intact purified WSSV virions were found by sodium dodecyl sulphate-PAGE, followed by Coomassie Brilliant R-250 staining. The estimated molecular weights of these proteins were 23, 24, 29, 32 and 42-kDa, which could correspond to viral protein. Using this method, the virus does not lose its ability to infect healthy shrimp.

Chitinase Production by Streptomyces sp. ANU 6277.

Chitinase production by a terrestrial Streptomyces sp. ANU 6277 was studied under sub-merged fermentation. Chitinase production started after 24 h of incubation and reached maximum levels after 60 h of cultivation. A high level of chitinase activity was observed in the culture medium with pH 6 at 35°C. Culture medium amended with 1% chitin was found to be suitable for maximum production of chitinase. An optimum concentration of colloidal chitin for chitinase production was determined. Studies on the influence of additional carbon and nitrogen sources on chitinase production revealed that starch and yeast extract served as good carbon and nitrogen sources to enhance chitinase yield. Chitinase was purified from crude enzyme extract by single step gel filtration by Sephadex G-100. Purified chitinase of the strain exhibited a distinct protein band near 45 kDa by means of SDS-PAGE.

Enzymatic recovery of platinum (IV) from industrial wastewater using a biosulphidogenic hydrogenase

It has been established that dissolved heavy metals escaping into the environment pose a serious health hazard. As a result, there is an urgent need for controlling metal emissions into the environment. The aims of this study were to purify and biochemically characterise hydrogenase(s) from sulphate reducing consortium (SRB) and investigate the potential of the purified enzyme(s) in the recovery of platinum from wastewaters. A hydrogenase from sulphate reducing consortium was purified by a combination of PEG 20000 concentration, ion exchange (Toyopearl-Super Q 650 S) and size exclusion (Sephacryl S-200) chromatographies. SDS-PAGE analysis revealed a distinct protein band with a molecular mass of 58 kDa. The investigation of enzymatic platinum (IV) reduction in vitro, showed highest hydrogen-dependent platinum (IV) reducing activity in the presence of hydrogenase and its physiological electron carrier, cytochrome c3. When the purified hydrogenase enzyme (with and without cytochrome c3) was used with the industrial effluent, containing 7.9 mg.l-1 platinum, only 10 – 15% recovery was noted pointing to a suppression of enzyme activity due to the low pH (0.38) of the effluent. Bioremediation studies on industrial effluent using resting SRB cells showed a 34% platinum removal from the effluent while growing SRB cells, within a sulphidogenic reactor, gave a platinum removal of 78%, with the pH of the system fluctuating at around 5.6. Evidence of sulphate reduction and sulphide generation were not observed during this treatment process suggesting that platinum sulphide was not formed and supporting the argument that the increased amount (78%) of platinum removal from the industrial wastewater by the growing SRB cells was due to more hydrogenase/cytochrome c3 enzyme complex being available.

Differential subcellular distribution and colocalization of the microsomal and soluble epoxide hydrolases in cultured neonatal rat brain cortical astrocytes

The microsomal epoxide hydrolase (mEH) and soluble epoxide hydrolase (sEH) enzymes exist in a variety of cells and tissues, including liver, kidney, and testis. However, very little is known about brain epoxide hydrolases. Here we report the expression, localization, and subcellular distribution of mEH and sEH in cultured neonatal rat cortical astrocytes by immunocytochemistry, subcellular fractionation, Western blotting, and radiometric enzyme assays. Our results showed a diffuse immunofluorescence pattern for mEH, which colocalized with the astroglial cytoskeletal marker glial fibrillary acidic protein (GFAP). The GFAP-positive cells also expressed sEH, which was localized mainly in the cytoplasm, especially in and around the nucleus. Western blot analyses revealed a distinct protein band with a molecular mass of approximately 50 kDa, the signal intensity of which increased about 1.5-fold in the microsomal fraction over the whole-cell lysate and other subcellular fractions. The polyclonal anti-human sEH rabbit serum recognized a protein band with a molecular mass similar to that of the affinity-purified sEH protein (approximately 62 kDa), the signal intensity of which increased over 1.7-fold in the 105,000g supernatant fraction over the cell lysate. Furthermore, the corresponding enzyme activities measured by using mEH- and sEH-selective substrates generally corroborated the immunocytochemical and Western blotting data. These results suggest that rat brain cortical astrocytes differentially coexpress mEH and sEH enzymes. The differential subcellular localization of mEH and sEH may play a role in the cerebrovascular functions that are known to be affected by brain-derived vasoactive epoxides.

Environmental Exposure to Allergens of Different Dog Breeds and Relevance in Allergological Diagnostics

In our environment, dogs are a relevant source of allergens, but diagnosing dog-related allergies may present difficulties, as in diagnostic tests with commercial dog allergens, some patients show only slight positive or negative results, even though they suffer from dog-related symptoms. Occasionally, allergy tests with extracts of dog hair belonging to patients' dogs or from dogs of the same breed were found to yield more reliable results, possibly due to breed-specific allergen components. The purpose of this study was to determine breed-specific differences or possibly hypo- or hyperallergenic dog breeds. The dog allergen content and protein patterns of different commercial and self-prepared dog allergen extracts were compared. Protein extracts were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with silver. The major allergen Can f 1 was quantified using the commercially available enzyme-linked immunosorbent assay (ELISA) technique. The majority of the bands in the self-prepared extracts of different breeds had a molecular mass lower than 30 kD. Notably, the self-prepared extracts of hair of common breeds showed distinct protein bands with a molecular mass lower than 14 kD, which the commercial extracts did not. With regard to Can f 1 content, a marked variability occurred. Factors related to individual dogs seem to influence the allergenicity more than breed or gender. This is the first report to describe allergens with low molecular mass that are absent in extracts of commercial test kits. Consequently, skin tests with self-prepared dog allergen extracts need to be performed in case of inconsistent test results with commercial extracts.

Effect of oviductal fluid proteins on buffalo sperm characteristics during cryopreservation

The objective was to determine the effects of oviductal proteins on sperm function. Abbatoir-derived buffalo oviducts were flushed with PBS; the fluid recovered (protein concentration, 2.3 mg/mL; average of 3.5 mg protein/oviduct) was centrifuged, dialyzed, and clarified, and the supernatant applied to a Heparin-Sepharose affinity column. Unbound fractions were collected and bound proteins were separately eluted (with elution buffer). Eight distinct protein bands (from 12 to 177 kDa) in the H-unbound fraction and 15 distinct protein bands (from 12 to 165 kDa) in the H-bound fraction were detected in SDS-PAGE. Semen from four buffalo bulls was divided into three parts: Parts 1 and 2 were treated with the heparin binding (H-bound) and non-heparin binding (H-unbound) oviductal proteins, respectively, whereas Part 3 remained as an untreated control. Equilibrated and frozen-thawed semen was assessed for motility, viability, intact acrosome percentage, mucus penetration distance, and hypo-osmotic swelling test. The H-bound oviductal fluid proteins enhanced (P<0.05) the proportion of sperm that were progressively motile, alive, had an intact acrosome and functional plasma membrane (hypo-osmotic swelling test), as well as the distance covered in the cervical mucus sperm penetration test during cryopreservation. Addition of the H-unbound oviductal protein fraction did not increase sperm motility and penetration distance but increased (P<0.05) the proportion of sperm that were live, had an intact acrosome, and functional plasma membrane (hypo-osmotic swelling test). We concluded that the H-bound fraction of buffalo oviductal fluid protein(s) maintained sperm motility, viability and membrane integrity during cryopreservation, whereas the H-unbound proteins maintained sperm viability and membrane integrity.

Discrimination and numerical analysis of human pathogenic Candida albicans strains based on SDSPAGE protein profiles

In the present study, 21 Candida albicans strains were investigated using the commercial kit API 20C AUX system and the numerical analysis of whole-cell protein profiles. The results of the commercial kit confirmed that the all the strains belonged to C. albicans species. However, the research indicated that SDS-PAGE of polypeptides of whole-cell extracts can provide more valuable taxonomic information than conventional yeast test kits at the subspecies level. Despite the fact that C. albicans subtypes isolated from different anatomical sites had similar protein profiles, there were some distinctive protein bands. Numerical analysis of whole-cell protein profiles of all strains revealed 2 major clusters at similarity degrees of between 46.26 and 100%. Moreover, the results of numerical analysis confirmed that each cluster had characteristic and distinctive protein profiles. The research showed that, the morphological examination of yeast isolates remains essential to obtaining a correct identification, both the commercial yeast kit system and the numerical analysis of whole-cell protein patterns can be useful for the more reliable identification of C. albicans strains.

The antigen for Hep Par 1 antibody is the urea cycle enzyme carbamoyl phosphate synthetase 1

Hepatocyte paraffin 1 (Hep Par 1), a murine monoclonal antibody, is widely used in surgical pathology practice to determine the hepatocellular origin of neoplasms. However, identity of the antigen for Hep Par 1 is unknown. The aim of this study was to characterize the Hep Par 1 antigen. To identify the antigen, immunoprecipitation was used to isolate the protein from human liver tissue, and a distinct protein band was detected at approximately 165 kDa. The protein band was also present in small intestinal tissue, but was not present in several other non-liver tissues nor in three human hepatocellular carcinoma cell lines, Huh-7, HepG2, and LH86. The protein was purified and analyzed by mass spectrometry. It was identified as carbamoyl phosphate synthetase 1 (CPS1). CPS1 is a rate-limiting enzyme in urea cycle and is located in mitochondria. We demonstrated that hepatoid tumors (gastric and yolk sac) were immunoreactive with both Hep Par 1 antibody and anti-CPS1 antibody, further confirming the results of mass spectrometric analysis. We found that the three human hepatocellular carcinoma cell lines do not express either CPS1 RNA or protein. We confirmed that the gene was present in these cell lines, suggesting that suppression of CPS1 expression occurs at the transcriptional level. This finding may have relevance to liver carcinogenesis, since poorly differentiated hepatocellular carcinomas exhibit poor to absent immunoreactivity to Hep Par 1. In conclusion, we have identified the antigen for Hep Par 1 antibody as a urea cycle enzyme CPS1. Our results should encourage further investigation of potential role that CPS1 expression plays in liver pathobiology and carcinogenesis.

Paramphistomum cervi: Antigenic profile of adults as recognized by infected cattle sera

An antigenic profile of adult Paramphistomum cervi was revealed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting using sera from cattle naturally infected with P. cervi, Fasciola gigantica and strongylids. SDS–PAGE of whole worm extracts exhibited 26 distinct protein bands. Immunoblotting analysis of these proteins showed five major antigenic bands which were recognized by serum of individual cattle naturally infected with P. cervi. These antigenic proteins had molecular weights ranging from 23 to 116 kDa. One antigenic protein with a molecular weight of 52 kDa exhibited a consistent reaction with sera from all infected cattle. It’s diagnostic sensitivity, specificity and accuracy using this test were 100%, 98% and 98.9%, respectively. The positive and negative predictive values were 97.6% and 100%, respectively. This finding suggests that the 52 kDa protein may be a diagnostic antigen for paramphistomosis.

P2X5 Subunit Assembly Requires Scaffolding by the Second Transmembrane Domain and a Conserved Aspartate

Functional homomeric and heteromeric ATP-gated P2X receptor channels have been shown to display a characteristic trimeric architecture. Of the seven different isoforms (designated P2X(1)-P2X(7)), P2X(5) occurs in humans primarily as a non-functional variant lacking the C-terminal end of the ectodomain and the outer half of the second transmembrane domain. We show that this truncated variant, which results from the splice-skipping of exon 10, is prone to subunit aggregation because the residual transmembrane domain 2 is too short to insert into the membrane. Alleviation of the negative hydrophobic mismatch by the addition of a stretch of moderately hydrophobic residues enabled formation of a second membrane-spanning domain and strictly parallel homotrimerization. Systematic mutagenesis identified only one transmembrane domain 2 residue, Asp(355), which supported homotrimerization in a side chain-specific manner. Our results indicate that transmembrane domain 2 formation contributes 2-fold to hP2X(5) homotrimerization by tethering the end of the ectodomain to the membrane, thereby topologically restricting conformational mobility, and by intramembrane positioning of Asp(355). While transmembrane domain 2 appears to favor assembly by enabling productive subunit interactions in the ectodomain, Asp(355) seems to assist by simultaneously driving intramembrane helix interactions. Overall, these results indicate a complex interplay between topology, helix-helix interactions, and oligomerization to achieve a correctly folded structure.

Enzymatically Active N-Deacetylase/N-Sulfotransferase-2 Is Present in Liver but Does Not Contribute to Heparan Sulfate N-Sulfation

Heparan sulfate (HS) proteoglycans influence embryonic development through interactions with growth factors and morphogens. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. NDST (glucosaminyl N-deacetylase/N-sulfotransferase), responsible for HS N-sulfation, is a key enzyme directing further modifications including O-sulfation. To elucidate the roles of the different NDST isoforms in HS biosynthesis, we took advantage of mice with targeted mutations in NDST1 and NDST2 and used liver as our model organ. Of the four NDST isoforms, only NDST1 and NDST2 transcripts were shown to be expressed in control liver. The absence of NDST1 or NDST2 in the knock-out mice did not affect transcript levels of other NDST isoforms or other HS modification enzymes. Although the sulfation level of HS synthesized in NDST1-/- mice was drastically lowered, liver HS from wild-type mice, from NDST1+/-, NDST2-/-, and NDST1+/- / NDST2-/- mice all had the same structure despite greatly reduced NDST enzyme activity (30% of control levels in NDST1+/- / NDST2-/- embryonic day 18.5 embryos). Enzymatically active NDST2 was shown to be present in similar amounts in wild-type, NDST1-/-, and NDST1+/- embryonic day 18.5 liver. Despite the substantial contribution of NDST2 to total NDST enzyme activity in embryonic day 18.5 liver (approximately 40%), its presence did not appear to affect HS structure as long as NDST1 was also present. In NDST1-/- embryonic day 18.5 liver, in contrast, NDST2 was responsible for N-sulfation of the low sulfated HS. A tentative model to explain these results is presented.

Binding and Transport of Metal Ions at the Dimer Interface of the Escherichia coli Metal Transporter YiiP

YiiP is a representative member of the cation diffusion facilitator (CDF) family, a class of ubiquitous metal transporters that play an essential role in metal homeostasis. Recently, a pair of Zn2+/Cd2+-selective binding sites has been localized to two highly conserved aspartyl residues (Asp157), each in a 2-fold-symmetry-related transmembrane segment 5 (TM5) of a YiiP homodimer. Here we report the functional and structural interactions between Asp157 and yet another highly conserved Asp49 in the TM2. Calorimetric binding analysis indicated that Asp49 and Asp157 contribute to a common Cd2+ binding site in each subunit. Copper phenanthroline oxidation of YiiP(D49C), YiiP(D157C), and YiiP(D49C/D157C) yielded inter- and intra-subunit cross-links among Cys49 and Cys157, consistent with the spatial proximity of two (Asp49-Asp157) sites at the dimer interface. Hg2+ binding to YiiP(D49C) or YiiP(D49C/D157C) also yielded a Cys49-Hg2+-Cys49 biscysteinate complex across the dimer interface, further establishing the interfacial location of a (Asp49-Asp157)2 bimetal binding center. Two bound Cd2+ ions were found transported cooperatively with a sigmoidal dependence on the Cd2+ concentration (n = 1.4). The binding affinity, transport cooperativity, and rate were modestly reduced by either a D49C or D157C mutation, but greatly diminished when all the bidentate aspartate O-ligands in (Asp49-Asp157)2 were replaced by the monodentate cysteine S-ligands. The functional significance of these findings is discussed based on the unique coordination chemistry of aspartyl residues and a model for the translocation pathway of metal ions at the YiiP dimer interface.

Quantitative Proteomic Analysis of Post-translational Modifications of Human Histones

Histone proteins are subject to a range of post-transcriptional modifications in living cells. The combinatorial nature of these modifications constitutes the "histone code" that dictates chromatin structure and function during development, growth, differentiation, and homeostasis of cells. Deciphering of the histone code is hampered by the lack of analytical methods for monitoring the combinatorial complexity of reversible multisite modifications of histones, including acetylation and methylation. To address this problem, we used LC-MSMS technology and Virtual Expert Mass Spectrometrist software for qualitative and quantitative proteomic analysis of histones extracted from human small cell lung cancer cells. A total of 32 acetylations, methylations, and ubiquitinations were located in the human histones H2A, H2B, H3, and H4, including seven novel modifications. An LC-MSMS-based method was applied in a quantitative proteomic study of the dose-response effect of the histone deacetylase inhibitor (HDACi) PXD101 on histone acetylation in human cell cultures. Triplicate LC-MSMS runs at six different HDACi concentrations demonstrated that PXD101 affects acetylation of histones H2A, H2B, H3, and H4 in a site-specific and dose-dependent manner. This unbiased analysis revealed that a relative increase in acetylated peptide from the histone variants H2A, H2B, and H4 was accompanied by a relative decrease of dimethylated Lys(57) from histone H2B. The dose-response results obtained by quantitative proteomics of histones from HDACi-treated cells were consistent with Western blot analysis of histone acetylation, cytotoxicity, and dose-dependent expression profiles of p21 and cyclin A2. This demonstrates that mass spectrometry-based quantitative proteomic analysis of post-translational modifications is a viable approach for functional analysis of candidate drugs, such as HDAC inhibitors.

Involvement of mitogen-activated protein kinase in 2-hydroxyestradiol-17β-induced oocyte maturation in the catfish Heteropneustes fossilis and a note on possible interaction with protein phosphatases

Mitogen-activated protein kinase (MAPK) was demonstrated in the postvitellogenic follicles (theca-granulosa and oocyte) of catfish by Western blotting using a polyclonal anti-rabbit serum, which recognized both ERK1 and ERK2. Two distinct protein bands resolved in the 46–48 kDa range of 12% SDS–PAGE were immunoblotted. Incubation of the follicles with 5 μM 2-OHE 2 elicited GVBD significantly in a duration-dependent manner with a concomitant increase in the expression of MAPK (ERK1 and ERK2). Densitometric analysis of the immunoblots showed significant variations in the intensity of staining. The ERK1 expression increased significantly from 6 h onwards but the changes were less pronounced. On the other hand, ERK2 registered a sharp significant increase after 3 h, which paralleled the GVBD response. The MEK inhibitor PD098059 alone did not induce GVBD. Co-incubation of the follicles with 2-OHE 2 and PD098059 significantly inhibited the steroid-induced GVBD at all concentrations. Immunoblot analysis showed that PD098059 inhibited MAPK activity significantly compared to the 2-OHE 2 group. The addition of okadaic acid (OA) in the incubation medium containing both 2-OHE 2 and PD098059 reversed the inhibitory effect of the latter and GVBD was elevated significantly over that of the 2-OHE 2 group but significantly lower than that of the 2-OHE 2 + OA group. The results suggest an involvement of MAPK in meiotic maturation but the site(s) of action: oocyte, follicular envelope or both needs further investigation.

Rapid and selective isolation of β‐xylosidase through an activity‐based chemical approach

beta-Xylosidase is a key enzyme in the xylanolytic system with a great potential in many biotechnological applications, especially in the food as well as the pulp and paper industries. We have developed a chemical approach for the rapid screening and isolation of beta-xylosidase. Activity probe LCL-6X targeting beta-xylosidase was utilized in this study. It carries a beta-xylopyranosyl recognition head, a latent trapping device consisting of a 2-fluoromethylphenoxyl group, and a biotin reporter group. The biotin reporter group serves both as a readout device and as a tool for enriching the labeled proteins. LCL-6X could selectively label a model beta-xylosidase from Trichoderma koningii. All other bystander proteins used in this study, including phosphorylase b, BSA, ovalbumin, carbonic anhydrase, and trypsin inhibitor, gave negligible cross-labeling effect. With the assistance of streptavidin agarose beads and mass spectrophotometry for the recovery and identification of the biotinylated proteins, we demonstrated that LCL-6X could be successfully applied to identify a bi-functional enzyme with alpha-L-arabinofuranosidase/beta-xylosidase activity from the total protein extract of a Pichia expressing system and a prospective beta-xylosidase in the culture medium of Aspergillus fumigatus. The beta-xylosidase activities from numerous microbes were also screened using the LCL-6X probe. Preliminary results showed significant differences among these microbial sources and some distinct protein bands were observed. Thus, we have successfully developed a novel chemical probe that has potential applications in xylan-related research.

Effect of nutritional parameters on laccase production by the culinary and medicinal mushroom, Grifola frondosa

Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 (M Cu over the basal level (1.6 mM Cu) and peak levels observed at 300 mM Cu were ∼ ∼7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu2+. SDS-PAGE combined with activity staining revealed the presence of a single protein band (Mr ∼ ∼70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct second laccase protein band (Mr␣∼ ∼45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. The optimal temperature and pH values for laccase activity were 65 °C and pH 2.2 (using 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) {ABTS} as substrate), respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development.

Effect of imported fire ant extract on the degradation of mountain cedar pollen allergen

Dust mite, cockroach, and mold extracts have been shown to contain proteases capable of degrading the proteins in other extracts. Loss of potency of allergens has been reported in mixtures containing cockroach and fungal extracts. Fire ant venoms consist of 90% to 95% n-alkyl and n-alkenyl piperidine alkaloids, which are not allergenic. No studies are available addressing the mixture of imported fire ant (IFA) whole-body extract with other allergens or the presence of proteolytic activity in the venom extract. To evaluate the stability of mountain cedar pollen extract mixed with IFA whole-body extract and to qualitatively analyze the extract mixture for degradation of mountain cedar protein. One milliliter each of mountain cedar and IFA whole-body extracts at a concentration of 500 microg/mL were combined and stored at 4 degrees C for 1, 3, 6, 15, 30, 60, 90, and 180 days. Separate mixtures of 1 mL of mountain cedar and IFA with 1 mL of human serum albumin were used as controls. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed, and protein bands were qualitatively analyzed for degradation. We detected 3 distinct IFA protein bands and 1 mountain cedar protein band. With respect to these bands, no protein degradation was observed during 6 months of study in the extract mixture compared with the controls. Imported fire ant whole-body extract does not seem to degrade mountain cedar protein. Mixtures of allergenic extracts may be able to include IFA whole-body extract.

ERp29 Triggers a Conformational Change in Polyomavirus to Stimulate Membrane Binding

Membrane penetration of nonenveloped viruses is a poorly understood process. We have investigated early stages of this process by studying the conformational change experienced by polyomavirus (Py) in the lumen of the endoplasmic reticulum (ER), a step that precedes its transport into the cytosol. We show that a PDI-like protein, ERp29, exposes the C-terminal arm of Py's VP1 protein, leading to formation of a hydrophobic particle that binds to a lipid bilayer; this reaction likely mimics initiation of Py penetration across the ER membrane. Expression of a dominant-negative ERp29 decreases Py infection, indicating ERp29 facilitates viral infection. Interestingly, cholera toxin, another toxic agent that crosses the ER membrane into the cytosol, is unfolded by PDI in the ER. Our data thus identify an ER factor that mediates membrane penetration of a nonenveloped virus and suggest that PDI family members are generally involved in ER remodeling reactions.

Allergenic and mutagenic characterization of 14 Penicillium species

Extracts of 14 Penicillium species, P. aurantiogriseum, P. brevicompactum, P. citrinum, P. chrysogenum, P. expansum, P. glabrum, P. hirsutum, P. italicum, P. janthinellum, P. melini, P. oxalicum, P. purpurescens, P. simplicissimum, and P. viridicatum were investigated by total protein, specific enzyme determinations, isoelectric focusing (IEF), sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using pooled human atopic IgE. Considerable variation was observed between the Penicillium species with respect to protein yield and the number of distinct protein bands resolved in IEF. Using the Api-Zym system, the most common activities observed among the extracts included acid and alkaline phosphatase, phosphodiamidase and β-glucosaminidase. The number of discrete atopic IgE-reactive bands in immunoblots of Penicillium extracts ranged from 1 (P. chrysogenum) to 9 (P. viridicatum). Certain allergens showed potential for cross-reactivity between species, including 52 and 54 kDa proteins in P. citrinum, P. purpurescens, P. viridicatum and 40 kDa proteins in several species. The extracts were also nonmutagenic when tested with the Ames assay using Salmonella strains TA98 and TA100. The results tend to indicate that P. viridicatum, P. janthinellum, P. oxalicum, P. brevicompactum and P. italicum, which are highly immunogenic as well as allergenic, could possibly be good candidates for allergen cloning studies through the construction of cDNA libraries. The extracts were non-mutagenic and can be used safely for skin testing.