Distinct Protein Bands Research Articles

Antigenic components, isolation and partial characterization of excretion–secretion fraction of Paramphistomum cervi

The immunogenic components of adult Paramphistomum cervi excretion-secretion (ES) fraction were revealed by SDS-PAGE and immunoblotting technique using sera from cattle naturally infected with P. cervi, Fasciola gigantica, strongylids, Trichuris sp., and Strongyloides sp. By SDS-PAGE, it was found that the ES fraction comprised 13 distinct protein bands. Immunoblotting analysis of these proteins exhibited nine prominent antigenic bands which were recognized by paramphistomosis antisera. These antigenic proteins had molecular weights ranging from 10-170 kDa. One antigenic protein band of 40 kDa was found to give a consistent reaction with sera from all infected cattle. Its diagnostic sensitivity, specificity and accuracy using this test were 100%, 98.9% and 99.3%, respectively. The positive and negative predictive values were 98% and 100%, respectively. The 40 kDa antigen was partially purified by gel filtration and ion-exchange chromatography. The antigenicity of 40 kDa protein for diagnosis of P. cervi infection was confirmed by immunoblotting and indirect ELISA (at 1:78,125 dilution) using a pool of sera and individual serum samples from infected cattle. The present findings suggest that the 40 kDa protein may be used as a diagnostic antigen for paramphistomosis.

Structural Characterization of Baxter's Recombinant Human ADAMTS13 Drug Candidate.

Abstract 2236Baxter has developed a human recombinant (r) ADAMTS13 for treatment of patients with hereditary TTP. The protein is produced in a CHO cell-line using serum and protein-free fermentation technology. The purification process does not use immune-affinity chromatography, and includes two viral reduction steps. The final drug product is formulated without proteins of animal or human origin.Here we characterize preclinical and clinical lots of rADAMTS13 with respect to their structural properties. The molecular weight of rADAMTS13 was determined by MALDI TOF as ∼173 kDa. SDS-PAGE showed one band at ∼190 kDa; no additional distinct protein bands corresponding to other proteins or product related degradation products were visible in the gels. Investigation of the primary structure of rADAMTS13 by peptide mapping revealed a sequence coverage of 98% compared to the theoretical amino acid sequence, and no peptides from the signal or propeptide. Peptide mapping was also used to determine posttranslational modifications of rADAMTS13. Seven O-glycosylation sites were identified in addition to the predicted N-glycosylation, O-fucosylation and C-mannosylation sites. The relative monosaccharide and N-linked oligosaccharides composition was consistent in all preclinical and clinical rADAMTS13 batches. The main N-glycan structure found on rADAMTS13 is a biantennary, core fucosylated complex type oligosaccharide with one or two sialic acids. Quantification of sialic acid determination showed consistent amounts of sialic acid on the terminal ends of the glycans in preclinical and clinical rADAMTS13 samples. N-glycolylneuraminic acid, a potentially immunogenic variant of sialic acid, was <1% the amount of total sialic acids.Higher order structure analysis using dynamic light scattering showed a similar hydrodynamic diameter for all preclinical and clinical lots. Finally, FT-IR and CD analysis showed comparable amounts of α-helix and β-sheet in preclinical and clinical lots.Taken together, similarity was shown between the preclinical and clinical lots of rADAMTS13 in all assays. A phase I clinical trial using rADAMTS13 for the treatment of hereditary TTP patients is expected to start soon. Disclosures:Foettinger-Vacha:Baxter Innovations GmbH: Employment. Kaliwoda:Baxter Innovations GmbH: Employment. Matthiessen:Baxter Innovations GmbH: Employment. Hasslacher:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Mitterer:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

Excretome of the chitinolytic bacterium Clostridium paraputrificum J4

A strictly anaerobic mesophilic chitinolytic bacterial strain identified as Clostridium paraputrificum J4 was isolated from human feces. In response to various types of growth substrates, the bacterium produced an array of chitinolytic enzymes representing significant components of the J4 strain secretome. The excreted active proteins were characterized by estimating the enzymatic activities of endochitinase, exochitinase, and N-acetylglucosaminidase induced by cultivation in medium M-10 with colloidal chitin. The enzyme activities produced by J4 strain cultivated in medium M-10 with glucose were significantly lower. The spectrum of extracellularly excreted proteins was separated by SDS-PAGE. The chitinase variability was confirmed on zymograms of renatured SDS-PAGE. The enzymes were visualized under ultraviolet light by using 4-methylumbelliferyl derivatives of N-acetyl-β-D: -glucosaminide, N,N´-diacetyl-β-D: -chitobiose, or N,N´,N˝-triacetyl-β-D: -chitotriose for β-N-acetylglucosaminidase, chitobiosidase, or endochitinase activities, respectively. Protein components of the secretome were separated by 2D-PAGE analysis. The distinct protein bands were excised, isolated, and subsequently characterized by using MALDI-TOF/TOF tandem mass spectrometry. The final identification was performed according to sequence homology by database searching.

Multiple Endo-β-1,4-glucanases Present in the Gut Fluid of a Defoliating Beetle, Podontia quatuordecimpunctata (Coleoptera: Chrysomelidae)

Endo-beta-1, 4-glucanase (EC 3.2.1.4) activity was measured in the gut fluid of phytophagous insect Podontia quatuordecimpunctata Linnaeus (Coleoptera: Chrysomelidae) in different days of development. The eight day-old larva showed maximum activity with 1.73 U mg(-1) of protein, which was confirmed by gel zymography. In zymogram, using Carboxymethyl Cellulose (CMC) as substrate, four distinct cellulolytic protein bands were detected in leaf borer gut fluids through out of its development. The optimum temperature and pH were 60 degrees C and 5.0, respectively. This endo-beta-1,4-glucanase showed maximum stability at 20-45 degrees C with approximately 20% remaining activity. Zymography also showed complete loss of endo-beta-1,4 glucanase activity at 55 degrees C. This is the first report that the cellulolytic enzyme is produced in the gut of P. quatuordecimpunctata through the whole developmental stages, from the 1st instar to the adult, except for pupae.

Molecular properties and antioxidant activities of polysaccharide–protein complexes from selected mushrooms by ultrasound-assisted extraction

Ultrasound-assisted extraction (UAE) was evaluated for isolation of polysaccharide–protein (PSP) complexes from three important medicinal mushrooms (Grifola frondosa, Coriolus versicolor and Lentinus edodes). Compared with those of conventional hot-water extraction (HWE), the PSP yield of UAE was similar with G. frondosa, notably higher with L. edodes but lower with C. versicolor, and the extraction rate of UAE was notably higher with G. frondosa and L. edodes but much lower with C. versicolor. The PSPs from all three mushrooms by UAE had higher protein but lower carbohydrate contents than those by HWE, and their molecular weight (MW) profiles exhibited an overall shift to lower MW and a major low-MW peak near 1.0kDa. All PSPs from UAE but none from HWE exhibited 3–4 distinct protein bands between 10 and 130kDa. The antioxidant activities of PSPs extracted by UAE were generally higher than those by HWE.

Purification and characterisation of monoamine oxidase from Avena sativa

FAD-containing monoamine oxidase (MAO; EC 1.4.3.4) oxidises monoamines to their corresponding aldehydes, H2O2, and NH3. It has been purified to homogeneity in mammals, but to our knowledge, there have been no reports of the enzyme in plants. MAO activity was detected in Avena sativa seedlings during germination using benzylamine as substrate. The enzyme was purified to homogeneity (as assessed by native PAGE) by Sephadex G-25, DEAE Sephacel, hydroxyapatite, Mono Q, and TSK-GEL column chromatographies. The molecular mass estimated by gel filtration using the TSK-GEL column was 220 kDa. SDS-PAGE yielded four distinct protein bands of 78, 58, 55, and 32 kDa molecular masses. The pI value of the enzyme was 6.3. The enzyme showed high substrate specificity for an endogenous amine, phenethylamine, which was oxidised to phenylacetaldehde, but not for ethylamine, propylamine, butylamine, pentylamine, dopamine, serotonin, tryptamine, or tyramine. The K m values for benzylamine and phenethylamine were 2.7 × 10−4 and 7.1 × 10−4 M, respectively. Enzyme activity was not inhibited by pargyline, clorgyline, semicarbazide, or Na-diethyldithiocarbamate. Benzaldehyde, the product of benzylamine oxidation, exhibited strong competitive inhibition of enzyme activity with a Ki of 3 μM. FAD was identified by ODS-column chromatography as an enzyme cofactor. The enzyme contained 2 mol of FAD per 220,000 g of enzyme.

Degradation of Nuclease-Stabilized RNA Oligonucleotides in Mycoplasma-Contaminated Cell Culture Media

Artificial RNA reagents such as small interfering RNAs (siRNAs) and aptamers often must be chemically modified for optimal effectiveness in environments that include ribonucleases. Mycoplasmas are common bacterial contaminants of mammalian cell cultures that are known to produce ribonucleases. Here we describe the rapid degradation of nuclease-stabilized RNA oligonucleotides in a human embryonic kidney 293 (HEK) cell culture contaminated with Mycoplasma fermentans, a common species of mycoplasma. RNA with 2'-fluoro- or 2'-O-methyl- modified pyrimidines was readily degraded in conditioned media from this culture, but was stable in conditioned media from uncontaminated HEK cells. RNA completely modified with 2'-O-methyls was not degraded in the mycoplasma-contaminated media. RNA zymogram analysis of conditioned culture media and material centrifuged from the media revealed several distinct protein bands (ranging from 30 to 68 kDa) capable of degrading RNA with 2'-fluoro- or 2'-O-methyl-modified pyrimidines. Finally, the mycoplasma-associated nuclease was detected in material centrifuged from the contaminated culture supernatants in as little as 15 minutes with an RNA oligo-containing 2'-O-methyl-modified pyrimidines and labeled with a 5'-fluorescein amidite (FAM) and 3'-quencher. These results suggest that mycoplasma contamination may be a critical confounding variable for cell culture experiments involving RNA-based reagents, with particular relevance for applications involving naked RNA (e.g., aptamer-siRNA chimeras).

Detection of eukaryotic translation initiation factor 4E and its clinical significance in hepatocellular carcinoma

To study the expression of eukaryotic translation initiation factor 4E (eIF4E), which is closely correlated with malignant tumors, and its relationship to prognosis in hepatocellular carcinoma. Western blotting was performed to quantify the elF4E protein expression in the normal human liver cell line L02 and the hepatoma cell lines Hep3B, HepG2, and Huh7. Forty-six hepatocellular carcinoma samples with complete clinical data were obtained from Changzheng Hospital during the period of December 2008 to July 2009. The expression of eIF4E in the tumor samples and their adjacent tissues were detected by immunohistochemistry. The relationship between the test results and hepatocellular carcinoma (HCC) prognosis was statistically analysed by using a COX proportional hazard model. Western blotting analysis showed that there were distinct eIF4E protein bands in all three of the hepatoma cell lines. In particular, the HepG2 cell line had the highest level of eIF4E protein expression. The L02 cell group had a low eIF4E expression. Immunohistochemical assay showed that there were 32 cases in which the tumour tissue expression was higher than their adjacent tissues, accounting for 69.57%. There were also 14 cases in which the tumour tissue expression was lower or no significant difference was found, accounting for 30.43%. COX proportional hazards model analysis showed that HCC prognosis was related to the depth of invasion, the overexpression of eIF4E and p53, possibly as independent HCC prognostic predictors. In summary, eIF4E expression is associated with liver cancer, and patients with high eIF4E expression levels have a higher risk of recurrence.

Proteomic analysis of sericin in Bombyx mori cocoons

Cocoon sericin plays an important role in the reeling of silk and serves as a valuable biomaterial in the field of biomedicine, skincare, and food industries; however, knowledge about cocoon sericin proteins has been limited. For a comprehensive study on sericin, cocoons of eight varieties of silkworm of different geographic origin and with varied cocoon color were analyzed utilizing proteomics and bioinformatics approaches. The electrophoresis pattern demonstrated some common protein bands for all silkworm varieties and distinctive protein bands for some of those examined in the present study. The Ser2 protein, a new Ser3 protein, and four other novel sericin proteins were identified in cocoons for the first time. Products of both Ser1 and Ser3 genes appear to be ubiquitous in the cocoon shell of Bombyx mori. In addition, cocoons with especially high-reelability produced by the mutant strain B84 had an unique protein product of the Ser2 gene, indicating that the protein may play an important role in cocoon reelability. A series of sequence conflicts and post-translational modifications (PTMs) were also revealed in sericin proteins. Lipid modifications of sericin proteins, which promote waterproofing of the cocoon shell, were observed. Further, hydroxylation was identified, which provided evidence for intermolecular bonds among neighboring molecules of sericin as found in collagens. The sericin proteome data obtained from this study illuminated the molecular complexity of cocoon sericin and contributed to our understanding of the properties of sericin in filature and biomaterials.

First identification of tannin‐binding proteins in saliva of Papio hamadryas using MS/MS mass spectrometry

Hamadryas baboons possess salivary proline-rich proteins (PRP), as indicated by the presence of pink-staining protein bands using 1D SDS gel electrophoresis and Coomassie R250 staining. The ability of these protein bands to interact with tannic acid was further examined. In a tannin-binding assay using 5 µg tannic acid mixed with hamadryas whole saliva, we recently found four distinct protein bands of apparently 72, 55, 20, and 15 kDa that were precipitated during the experiments. In this work, we were able to identify these protein bands in a follow-up analysis using MS/MS mass spectrometry after excising such bands out of air-dried gels. Albumin and α-amylase were present in the tannic acid-protein complexes, with albumin already known to nonspecifically interact with a great diversity of chemical compounds. More interesting, we also identified a basic PRP and a cystatin precursor protein. This was the first successful attempt to identify a PRP from precipitated tannin-protein complexes in hamadryas baboons using MS/MS mass spectrometry. On the other hand, the role of cystatins in tannin binding is not yet well understood. However, there are recent reports on cystatin expression in saliva of rats responding to astringent dietary compounds. In conclusion, the follow-up data on tannin-binding proteins present in salivary secretions from hamadryas baboons adds important knowledge to primate physiology and feeding ecology, in order to shed light on the establishment and development of food adaptations in primates. It also demonstrates that tannin binding is characteristic for PRP, but might not be restricted to this particular group of proteins in primate species.

Beneficial effects of diatomite on the growth, the biochemical contents and polymorphic DNA in Lupinus albus plants grown under water stress

Water is a major limiting factor in world agriculture and most crop plants are highly sensitive to even a mild water stress.The beneficial effects of diatomite is characterized by helping plants to overcome various biotic and abiotic stresses. Thus , this experiment was performed to study the ameliorative effect of diatomite (10g/kg) on each of growth, productivity, gas exchange , biochemical contents and polymorphic DNA of Lupinus albus L. plants grown in either well watered or two different soil water regimes (1 and 4 days withdrawal). Results showed that diatomite was able to alleviate the deteriorative effect of water stress on almost all measured criteria specially in plants grown under mild water stress (1+D).Soil fertilization by diatomite significantly increased all measured growth parameters expressed as shoot and root length, fresh and dry weight of shoot, root and pods and number of pods/plant above both untreated and water stress -free diatomite treated plants. Diatomite application improved the rate of photosynthesis, stomatal conductance, the contents of each of total soluble sugars (TSS), total soluble protein (TSP) , total phenols, auxins, gibberellins, cytokinins and ABA and the activity of four antioxidant enzymes namely, superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and polyphenol oxidase (PPO) as compared to both control and stressed plants maintained at both levels of water regime. On the contrary, diatomite treatment markedly reduced transpiration rate and lipid peroxidation level to a minimum . Moreover, diatomite application either alone or combined with stress induced a distinctive protein electrophoratic band (92KD) which was absent in either the control or stressed plants. Three distinctive protein bands were also induced in stressed white lupin, two (65 and 25 KD) under one day drought and one (143K) under four day drought. Finally, diatomite induced several distinguished polymorphic DNA fragments using PCRRAPD analysis.

Salivary secretions from the honeybee mite,Varroa destructor:effects on insect haemocytes and preliminary biochemical characterization

The ectoparasitic honey bee mite Varroa destructor feeds on the haemolymph of the honey bee, Apis mellifera, through a single puncture wound that does not heal but remains open for several days. It was hypothesized that factors in the varroa saliva are responsible for this aberrant wound healing. An in vitro procedure was developed for collecting salivary gland secretions from V. destructor. Mites were incubated on balls of cotton wool soaked in a tissue culture medium (TC-100), and then induced to spit by topical application of an ethanolic pilocarpine solution. Elution of secretions from balls of cotton wool, followed by electrophoretic analysis by SDS-PAGE and electroblotting indicated the presence of at least 15 distinct protein bands, with molecular weights ranging from 130 kDa to <17 kDa. Serial titration of V. destructor salivary secretions in TC-100 followed by an 18-h incubation with haemocytes from the caterpillar, Lacanobia oleracea, indicated that the secretions damage the haemocytes and suppresses their ability to extend pseudopods and form aggregates. We suggest that these secretions facilitate the ability of V. destructor to feed repeatedly off their bee hosts by suppressing haemocyte-mediated wound healing and plugging responses in the host.

Characterization of cellulolytic activity from digestive fluids of Dissosteira carolina (Orthoptera: Acrididae)

Previous screening of head-derived and gut fluid extracts of Carolina grasshoppers, Dissosteira carolina (L.) revealed relatively high activity against cellulase substrates when compared to other insect groups. In this work we report on the characterization and identification of enzymes involved in cellulolytic activity in digestive fluids of D. carolina. In zymograms using carboxymethylcellulose (CMC) as substrate, we detected four distinct cellulolytic protein bands in D. carolina gut fluids, common to all developmental stages. These cellulolytic enzymes were localized to foregut and midgut regions of the D. carolina digestive tract. Cellulases were purified from D. carolina head and gut fluid extracts by liquid chromatography to obtain N-terminal amino acid sequence tags. Database searches with sequence tags from head fluids indicated high similarity with invertebrate, bacterial and plant beta1,4-endoglucanases, while no homologues were identified for the gut-derived protein. Our data demonstrate the presence of cellulolytic activity in the digestive system of D. carolina and suggest that cellulases of endogenous origin are present in this organism. Considering that this grasshopper species is a pest of grasses, including switchgrass that has been suggested bioethanol feedstock, characterization of insect cellulolytic systems may aid in developing applications for plant biomass biodegradation for biofuel production.

KARAKTERISASI INDIVIDU WERENG HIJAU NEPHOTETTIX VIRESCENS DISTANT PENULAR AKTIF VIRUS TUNGRO PADI

Characterization of active transmitter among population of the leafhopper, Nephotettix virescens Distant in relation to the transmission of the rice tungro virus. The objective of this research was to identify the characteristics of Nephotettix virescens as the active transmitter of the rice tungro virus. The specimens of N. virescens active transmitter were determined by differing in their ability to transmit the tungro virus to healthy plants. These specimens were used as samples to identify their morphological and molecular characters. Five external morphological characters, namely length of head to abdomen, hind femur, head, fore wing, and length of stylet were measured to determined their character. The protein banding patterns of the active transmitter were identified by protein separating technique on SDS-Page. Based on the morphological characters, especially the length of head to abdomen, hind femur, head, fore wing, and length of stylet, it showed that N. virescens active transmitter and non transmitter were similar. There were no specific morphological characters for N. virescens active transmitter. However, N. virescens active transmitter showed different protein banding pattern. Three distinct protein bands, estimated as 173, 134, and 68 kDa were observed in the specimens of active transmitter. These three protein bands, were absent in the non transmitter.

Identification and characterization of venom proteins of two solitary wasps, Eumenes pomiformis and Orancistrocerus drewseni

Secretory proteins were identified in the venoms of two solitary hunting wasps, Eumenes pomiformis and Orancistrocerus drewseni, by SDS-PAGE in conjunction with mass analysis. More than 30 protein bands (2–300 kDa) were detected from the crude venom of each wasp. With the aid of the previously constructed venom gland/sac-specific EST libraries, a total of 31 and 20 proteins were identified from 18 to 20 distinctive protein bands of E. pomiformis and O. drewseni venoms, respectively. Arginine kinase was the most predominant protein in both wasp venoms. Along with the full-length arginine kinase, a truncated form, which was known to have paralytic activity on a spider, was a common predominant protein in the two wasp venoms. Insulin/insulin-like peptide-binding protein was abundantly found only in E. pomiformis venom, which might be due to its unique behaviors of oviposition and provision. The presence of various immune response-related proteins and antioxidants suggested that wasps might use their venom to maintain prey fresh while feeding wasp larvae by protecting the prey from microbial invasion and physiological stresses. It seemed that some venom proteins are secreted into venom fluid from venom gland cells via exosomes, not by signal sequence-mediated transport processes.

Oligoclonal bands and immunoglobulin isotype switch during monitoring of patients with multiple myeloma and autologous hematopoietic cell transplantation: a 16-year experience

Over the last 20 years, high dose therapy followed by hemopoietic stem cell transplantation has been employed in patients with multiple myeloma (MM). During 16 years of follow-up, the degree of tumor response and survival in 238 patients with autologous stem cell transplantation (ASCT) and changes in the serum protein electrophoretic pattern were analyzed. Agarose gel electrophoresis with densitometric analysis and immunofixation were performed to evaluate serum monoclonal protein. IgM, IgA, IgG and beta(2)-microglobulin (beta2M) were quantitated. Urine protein electrophoresis with IF was performed on cellulose acetate gel using colloidal silver staining without concentrating. After 34 months of follow-up (range 1-160 months), eight patients (3.4%) showed a distinct monoclonal protein band that was different from their original isotype switch. This was observed to be a transient phenomenon (22.2 months). Thirty-seven patients (15.5%) developed oligoclonal bands (OB) between the first and the twentieth month after ASCT (mean 4.4 months), which persisted for 7.9 months (1-36 months). The mean overall survival time was statistically different (p<0.05) between the group with OB and the group without them. Mean values of serum albumin, beta2M, and non-involved immunoglobulins did not show statistical differences. The occurrence of OB could be a potential favorable prognostic marker after transplantation due to the prolonged survival observed. Close follow-up of anomalous protein bands, either in serum or urine, is essential due to the additional difficulty in interpretation when the therapeutic response and evolution are evaluated.

The 2-Cys Peroxiredoxin Alkyl Hydroperoxide Reductase C Binds Heme and Participates in Its Intracellular Availability in Streptococcus agalactiae

Heme is a redox-reactive molecule with vital and complex roles in bacterial metabolism, survival, and virulence. However, few intracellular heme partners were identified to date and are not well conserved in bacteria. The opportunistic pathogen Streptococcus agalactiae (group B Streptococcus) is a heme auxotroph, which acquires exogenous heme to activate an aerobic respiratory chain. We identified the alkyl hydroperoxide reductase AhpC, a member of the highly conserved thiol-dependent 2-Cys peroxiredoxins, as a heme-binding protein. AhpC binds hemin with a K(d) of 0.5 microm and a 1:1 stoichiometry. Mutagenesis of cysteines revealed that hemin binding is dissociable from catalytic activity and multimerization. AhpC reductase activity was unchanged upon interaction with heme in vitro and in vivo. A group B Streptococcus ahpC mutant displayed attenuation of two heme-dependent functions, respiration and activity of a heterologous catalase, suggesting a role for AhpC in heme intracellular fate. In support of this hypothesis, AhpC-bound hemin was protected from chemical degradation in vitro. Our results reveal for the first time a role for AhpC as a heme-binding protein.

Novel virus-associated proteins encoded by UL112–113 of human cytomegalovirus

Evidence suggests that the products of the human cytomegalovirus (HCMV) UL112-113 genes are involved in viral DNA replication during lytic infection. A polyclonal antibody was raised against the UL112 open reading frame (ORF) to characterize its function in detail. Immunoblots utilizing the UL112 antibody identified seven distinct protein bands (p20, p26, p28, p34, p43, p50 and p84) expressed during the HCMV infectious cycle. After screening a cDNA library constructed from cells 72 h after infection with HCMV, only four different cDNA protein-producing constructs were obtained, and their ORFs corresponded to p34, p43, p50 and p84. The proteins p20, p26 and p28 were further shown to be selectively included within mature HCMV particles, virions, non-infectious enveloped particles and dense bodies. Immunoaffinity protein purification was used to prepare the samples for liquid chromatography coupled to tandem mass spectrometry. This analysis revealed that p20, p26 and p28 were derived from the UL112 ORF, most likely through post-translational proteolytic cleavage.

Characterisation and expression of SPLUNC2, the human orthologue of rodent parotid secretory protein

We recently described the Palate Lung Nasal Clone (PLUNC) family of proteins as an extended group of proteins expressed in the upper airways, nose and mouth. Little is known about these proteins, but they are secreted into the airway and nasal lining fluids and saliva where, due to their structural similarity with lipopolysaccharide-binding protein and bactericidal/permeability-increasing protein, they may play a role in the innate immune defence. We now describe the generation and characterisation of novel affinity-purified antibodies to SPLUNC2, and use them to determine the expression of this, the major salivary gland PLUNC. Western blotting showed that the antibodies identified a number of distinct protein bands in saliva, whilst immunohistochemical analysis demonstrated protein expression in serous cells of the major salivary glands and in the ductal lumens as well as in cells of minor mucosal glands. Antibodies directed against distinct epitopes of the protein yielded different staining patterns in both minor and major salivary glands. Using RT-PCR of tissues from the oral cavity, coupled with EST analysis, we showed that the gene undergoes alternative splicing using two 5' non-coding exons, suggesting that the gene is regulated by alternative promoters. Comprehensive RACE analysis using salivary gland RNA as template failed to identify any additional exons. Analysis of saliva showed that SPLUNC2 is subject to N-glycosylation. Thus, our study shows that multiple SPLUNC2 isoforms are found in the oral cavity and suggest that these proteins may be differentially regulated in distinct tissues where they may function in the innate immune response.

Micro disc gradient gel electrophoresis of seminal plasma proteins before and after vasectomy and in different split ejaculate fractions.

The seminal plasma proteins of patients before and after vasectomy as well as in different split ejaculate fractions were investigated using gradient polyacrylamide gel electrophoresis. Considerable interindividual variations of the protein patterns were observed. A distinct high molecular weight proteins band was found to disappear after vasectomy. This protein band could b4 attributed to the sperm-rich fraction of split ejaculates. It seems to be of testicular-epididymal origin and may be of interest for functional characterization of the testicular-epididymal fluid and for diagnosis of an occlusion of the excretory duct system.