Cuticular proteins from the shrimp, Pandalus borealis.

1. The percentages of mineral salts, chitin, and urea-extractable and non-extractable proteins were determined in pieces of cuticle from selected body regions of the shrimp, Pandalus borealis. 2. Two-dimensional gel-electrophoresis of the urea-extractable proteins shows that a large number of different proteins are present. Identical protein patterns are obtained from the various cuticular regions. 3. A fractionation scheme is presented, which is suitable for obtaining the major proteins in quantities sufficient for further characterization. The amino acid compositions are reported for several of the proteins.     

Primary structure of an analog of crustacean pigment-dispersing hormone from the lubber grasshopper Romalea microptera

An octadecapeptide capable of inducing pigment dispersion in the chromatophores of the fiddler crab Uca pugilator has been isolated from lyophilized heads of the lubber grasshopper Romalea microptera. This pigment-dispersing factor (PDF) was purified by gel filtration, ion-exchange chromatography, partition chromatography, and reversed-phase high performance liquid chromatography. Automated gas-phase sequencing, followed by the identification of the carboxyl-terminal amide, established the primary structure of this PDF as Asn-Ser-Glu-Ile-Ile-Asn-Ser-Leu-Leu-Gly-Leu-Pro-Lys-Leu-Leu-Asn-Asp-Ala- NH2. This structure was confirmed by chemical synthesis and by demonstrating that the synthetic and native PDF displayed identical chromatographic behavior and biological activity. The Romalea PDF is structurally related to the crustacean pigment-dispersing hormones (PDHs), which are also octadecapeptides. The sequence of grasshopper PDF shows 78% homology with beta-PDH (from the crabs U. pugilator and Cancer magister) and 50% homology with alpha-PDH (from the prawn Pandalus borealis). This study provides the first direct chemical evidence for the structural relatedness of insect PDF to the crustacean PDHs, thus identifying them as an authentic family of arthropod peptides.     

Molecular targets for detection and immunotherapy in Cryptosporidium parvum

Cryptosporidium parvum is an obligate protozoan parasite responsible for the diarrheal illness cryptosporidiosis in humans and animals. Although C. parvum is particularly pathogenic in immunocompromised hosts, the molecular mechanisms by which C. parvum invades the host epithelial cells are not well understood. Characterization of molecular-based antigenic targets of C. parvum is required to improve the specificity of detection, viability assessments, and immunotherapy (treatment). A number of zoite surface (glyco)proteins are known to be expressed during, and believed to be involved in, invasion and infection of host epithelial cells. In the absence of protective treatments for this illness, antibodies targeted against these zoite surface (glyco)proteins offers a rational approach to therapy. Monoclonal, polyclonal and recombinant antibodies represent useful immunotherapeutic means of combating infection, especially when highly immunogenic C. parvum antigens are utilized as targets. Interruption of life cycle stages of this parasite via antibodies that target critical surface-exposed proteins can potentially decrease the severity of disease symptoms and subsequent re-infection of host tissues. In addition, development of vaccines to this parasite based on the same antigens may be a valuable means of preventing infection. This paper describes many of the zoite surface glycoproteins potentially involved in infection, as well as summarizes many of the immunotherapeutic studies completed to date. The identification and characterization of antibodies that bind to C. parvum-specific cell surface antigens of the oocyst and sporozoite will allow researchers to fully realize the potential of molecular-based immunotherapy to this parasite.     

Mass spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab, Cancer borealis

The crustacean stomatogastric ganglion (STG) is modulated by both locally released neuroactive compounds and circulating hormones. This study presents mass spectrometric characterization of the complement of peptide hormones present in one of the major neurosecretory structures, the pericardial organs (POs), and the detection of neurohormones released from the POs. Direct peptide profiling of Cancer borealis PO tissues using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) revealed many previously identified peptides, including proctolin, red pigment concentrating hormone (RPCH), crustacean cardioactive peptide (CCAP), several orcokinins, and SDRNFLRFamide. This technique also detected corazonin, a well-known insect hormone, in the POs for the first time. However, most mass spectral peaks did not correspond to previously known peptides. To characterize and identify these novel peptides, we performed MALDI postsource decay (PSD) and electrospray ionization (ESI) MS/MS de novo sequencing of peptides fractionated from PO extracts. We characterized a truncated form of previously identified TNRNFLRFamide, NRNFLRFamide. In addition, we sequenced five other novel peptides sharing a common C-terminus of RYamide from the PO tissue extracts. High K+ depolarization of isolated POs released many peptides present in this tissue, including several of the novel peptides sequenced in the current study.     

Species identification of the Northern shrimp (Pandalus borealis) by polymerase chain reaction-restriction fragment length polymorphism and proteomic analysis

Genomic and proteomic techniques for species identification of meat and seafood products are being widely used. In this study, a genomic approach was used to differentiate Pandalus borealis (the Northern shrimp), which belongs to the superfamily Pandaloidea, from 30 crustaceans consisting of 19 commercially relevant prawns/shrimps species that belong to the superfamily Penaeoidea, which include the families Penaeidae and Solenoceridae, and 11 other crustacean species, including prawns, shrimps, lobsters, and crabs. For this purpose, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was designed based on the amplification of the 16S rRNA/tRNA(Val)/12S rRNA mitochondrial regions using the primers 16S-CruF and 16S-CruR. The 966-bp PCR products were produced and cleaved with the restriction enzymes AluI, TaqI, and HinfI, which provided species-specific restriction patterns. In addition, a proteomic approach, based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization-ion trap (ESI-IT) mass spectrometry, was used to identify and characterize new P. borealis-specific peptides that could be useful as potential markers of this species in protein-based detection methods. To our knowledge, this is the first time a molecular method has been successfully applied to identify a wide range of prawn and shrimp species, including P. borealis, for either whole individuals or processed products. However, validation of the methods proposed here is required by applying them to a larger sample of individuals from different populations and geographic origins in order to avoid mainly false-negative results.     

Immunohistochemical Double Staining of Microwave Enhanced and Nonenhanced Nuclear and Cytoplasmic Antigens

Many of the antigens commonly investigated in histopathology can be enhanced by microwave pretreatment (MWPT) of formalin fixed, paraffin embedded tissue sections. We developed a double labeling method using microwave heating to detect otherwise undetectable nuclear antigens combined.with immunohisto-chemistry (IHC) of cytoplasmic or membranous antigens that do not benefit from MWPT. We used the same primary antibody solutions used in single antibody IHC. The staining technique is based on the alkaline phosphatase anti-alkaline phosphatase (APAAP) and the labeled avidin-biotin (LSAB) methods. Four different protocols were tested, each modifying the sequence of MWPT, APAAP and LSAB staining. In this study Ki67, estrogen receptor, progesterone receptor, c-neu, CD68 and desmin primary antibodies were used in routinely formalin fixed, paraffin embedded tissues of 50 tumor specimens. MWPT followed by LSAB for microwave enhanced antigens and APAAP for antigens that cannot be enhanced by MWPT gave the best double staining results. This method improves characterization of tumor cell features from paraffin embedded tissue and should aid analysis of tumor differentiation, receptor status and nuclear proteins in the single cells in archival tissues.     

Immunological relationship between oocyte nuclear proteins of Xenopus laevis and X. borealis

Monoclonal antibodies (mABs) have been raised against oocyte nuclear proteins of Xenopus laevis and X. borealis and have been screened for species specificity. Although about 40% of all germinal vesicle polypeptides differ between the two species as judged by two-dimensional gel analysis, most mABs react with polypeptides of both species. Biochemical analysis of the antigens by immunoblotting revealed that a homologue of each antigen of one species could be detected in the other species, despite frequent differences in molecular structure. Nevertheless, five strictly species-specific mABs have been identified. All five are directed against the same acidic polypeptide B3 of X. borealis, which is a structurally altered homologue of the protein N1, previously described in X. laevis germinal vesicles. In oocyte nuclei of hybrids between X. laevis females and X. borealis males, polypeptide of both species appear to be accumulated equivalently. Exceptions to this rule are most easily explained by differences between individuals and by the loss of certain alleles resulting from the cross.     

Monoclonal antibodies to murine thrombospondin-1 and thrombospondin-2 reveal differential expression patterns in cancer and low antigen expression in normal tissues

There is a considerable interest for the discovery and characterization of tumor-associated antigens, which may facilitate antibody-based pharmacodelivery strategies. Thrombospondin-1 and thrombospondin-2 are homologous secreted proteins, which have previously been reported to be overexpressed during remodeling typical for wound healing and tumor progression and to possibly play a functional role in cell proliferation, migration and apoptosis. To our knowledge, a complete immunohistochemical characterization of thrombospondins levels in normal rodent tissues has not been reported so far. Using antibody phage technology, we have generated and characterized monoclonal antibodies specific to murine thrombospondin-1 and thrombospondin-2, two antigens which share 62% aminoacid identity. An immunofluorescence analysis revealed that both antigens are virtually undetectable in normal mouse tissues, except for a weak staining of heart tissue by antibodies specific to thrombospondin-1. The analysis also showed that thrombospondin-1 was strongly expressed in 5/7 human tumors xenografted in nude mice, while it was only barely detectable in 3/8 murine tumors grafted in immunocompetent mice. By contrast, a high-affinity antibody to thrombospondin-2 revealed a much lower level of expression of this antigen in cancer specimens. Our analysis resolves ambiguities related to conflicting reports on thrombosponding expression in health and disease. Based on our findings, thrombospondin-1 (and not thrombospondin-2) may be considered as a target for antibody-based pharmacodelivery strategies, in consideration of its low expression in normal tissues and its upregulation in cancer.     

Isolation and characterization of the novel lipophilic protein, Pb CP-12.7, from the shell of the pink shrimp, Pandalus borealis.

Past research on diapause-inducing substances of the silkworm has isolated an extremely lipophilic peptide and demonstrated its unique characteristics. In the present work, similar lipophilic proteins were searched for in the shell of the shrimp, Pandalus borealis, and one novel protein, Pb CP-12.7, was isolated. Its structure comprising 126 amino acids was revealed by a combination of a sequence analysis and the enzymic fragmentation technique. Pb CP-12.7 is unique in that it was insoluble in neutral-slightly basic water, but highly soluble in some organic solvents. It contained an abundance of hydrophobic amino acids and repeating sequences. In addition, it was adsorbed to chitin, a major component of the shell of the shrimp.     

Antibacterial activity in four marine crustacean decapods

A search for antibacterial activity in different body-parts of Pandalus borealis (northern shrimp), Pagurus bernhardus (hermit crab), Hyas araneus (spider crab) and Paralithodes camtschatica (king crab) was conducted. Dried samples were extracted with 60% (v/v) acetonitrile, containing 0.1% (v/v) trifluoroacetic acid, and further extracted and concentrated on C18 cartridges. Eluates from the solid phase extraction were tested for antibacterial, lysozyme and haemolytic activity. Antibacterial activity against Escherichia coli, Vibrio anguillarum, Corynebacterium glutamicum and Staphylococcus aureus was detected in extracts from several tissues in all species tested, but mainly in the haemolymph and haemocyte extracts. V. anguillarum and C. glutamicum were generally the most sensitive micro-organisms. In P. borealis and P. bernhardus most of the active fractions were not affected by proteinase K treatment, while in H. araneus and P. camtschatica most fractions were sensitive to proteinase K treatment, indicating antibacterial factors of proteinaceous nature. In P. bernhardus the active fractions were generally heat labile, whereas in H. araneus the activities were resistant to heat. Differences between active extracts regarding hydrophobicity and sensitivity for heat and proteinase K treatment indicate that several compounds are responsible for the antibacterial activities detected. Lysozyme-like activity could be detected in some fractions and haemolytic activity against human red blood cells could be detected in haemolymph/haemocyte and exoskeleton extracts from all species tested.     

Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS)

A simple method for antigen retrieval in tissue sections and cell cultures is described. Because many antibodies recognize denatured proteins on western blots, but are poorly reactive by immunocytochemistry, the effect of applying sodium dodecyl sulfate (SDS) to cryostat sections of tissues and to cell cultures prior to immunostaining was examined. In many cases, a 5-min pretreatment with 1% SDS produced a dramatic increase in staining intensity by indirect immunofluorescence. Among the antibodies tested that showed a positive effect of SDS were an anti-Na/K-ATPase monoclonal antibody, an anti-AE1/2 anion exchanger polyclonal antipeptide antibody, a monoclonal anti-caveolin antibody, and an anti-rab4 monoclonal antibody. In other cases, including antibodies against gp330, aquaporin 1, and aquaporin 2, no effect of SDS was detected. The results show that SDS treatment can be used as a simple method of antigen retrieval in cryostat sections and on cultured cells. In some cases, antigens were not detectable without pretreatment with SDS.     

Monoclonal antibodies against Aleutian disease virus distinguish virus strains and differentiate sites of virus replication from sites of viral antigen sequestration.

Monoclonal antibodies (mAbs) were used to study antigenic differences among strains of Aleutian disease virus (ADV) and to characterize viral proteins in vitro and in vivo. A number of ADV field strains could be discriminated, and highly virulent Utah I ADV was clearly delineated from the tissue culture-adapted avirulent ADV-G strain. This specificity could be demonstrated by indirect immunofluorescence against infected cultures of Crandell feline kidney cells or against tissues of Utah I ADV-infected mink. Viral antigens were demonstrated in both the nuclei and the cytoplasm of infected tissue culture cells. However, in mink mesenteric lymph node, spleen, and liver, viral antigen was observed only in the cytoplasm. Absence of nuclear fluorescence suggested that the detected antigen represented phagocytized viral antigens rather than replicating virus. This conclusion was supported by the finding that mAbs reactive only against low-molecular-weight polypeptides derived from intact viral proteins gave the same pattern of in vivo fluorescence as mAbs with broad reactivity for large or small (or both) viral polypeptides. The distribution of infected cells was the same as that described for macrophages in these tissues and suggested that cells of the reticuloendothelial system had sequestered viral antigens.     

The secreted antigens of Mycobacterium tuberculosis and their relationship to those recognized by the available antibodies

Proteins secreted by strains of Mycobacterium tuberculosis during short-term, zinc-sufficient batch culture were identified in order to define antigens likely to be relevant to the pathogenesis of human disease. [35S]Methionine-labelled proteins in supernatants of 4-7 d cultures were separated by PAGE under both denaturing and non-denaturing conditions, and the position of labelled material was determined. Secreted protein patterns of M. tuberculosis were quite similar to those of Bacillus Calmette-Guérin (BCG) but differed by the absence of the 46 kDa dimeric protein specific to BCG and by the presence in large amounts of a 23 kDa protein which, when denatured, gave 13 kDa subunits. This 13 kDa subunit protein constituted up to 20% of secreted proteins in classical strains of M. tuberculosis of phage type B but was not detected in phage type I strains from South India. This may be relevant to the different pathogenicity of these strains. Western blot analysis showed that antigens defined in supernatants of short-term (3 d) cultures of M. tuberculosis constituted a small subset of those seen in supernatants of organisms cultured for longer periods. One of the secreted proteins has the interesting property of binding to fibronectin. The available monoclonal antibodies and antisera have been used to identify lines on immunoblots corresponding to the secreted/released antigens of M. tuberculosis. The present findings suggest that there are major secreted antigens to which antibodies do not yet appear to have been produced experimentally.     

Identification of neuropeptides from the decapod crustacean sinus glands using nanoscale liquid chromatography tandem mass spectrometry

Neurosecretory systems are known to synthesize and secrete a diverse class of peptide hormones which regulate many physiological processes. The crustacean sinus gland (SG) is a well-defined neuroendocrine site that produces numerous hemolymph-borne agents including the most complex class of endocrine signaling molecules--neuropeptides. As an ongoing effort to define the peptidome of the crustacean SG, we determine the neuropeptide complements of the SG of the Jonah crab, Cancer borealis, and the Maine lobster, Homarus americanus, using nanoflow liquid chromatography electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS. Numerous neuropeptides were identified, including orcokinins, orcomyotropin, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptides (CPRPs), red pigment concentrating hormone (RPCH), beta-pigment dispersing hormone (beta-PDH), proctolin and HL/IGSL/IYRamide. Among them, two novel orcokinins were de novo sequenced from the SG of H. americanus. Three CPRPs including a novel isoform were sequenced in H. americanus. Four new CPRPs were sequenced from the SG of C. borealis. Our results show that structural polymorphisms in CPRPs (and thus the CHH precursors) are common in Dendrobranchiata as well as in Pleocyemata. The evolutionary relationship between the CPRPs is also discussed.     

Biochemistry of the erythrocyte Rh polypeptides: a review

The clinically important Rh blood group system is complex, consisting of multiple distinct antigens. Despite clinical recognition for over 50 years, the Rh blood group antigens have remained poorly understood on a molecular level until the recent identification and characterization of the "Rh polypeptides," the core structural proteins of the Rh antigens. This group of erythrocyte membrane proteins of molecular weight 30,000-35,000 daltons was first recognized by employing Rh-specific antibodies to immunoprecipitate radiolabeled components of erythrocyte membranes. By using antibodies specific for the Rh D, c, and E antigens, a series of highly related non-identical proteins were immunoprecipitated, indicating that the Rh antigens are composed of multiple related proteins. The Rh polypeptides have been purified and characterized, and they were found to have several unusual biochemical characteristics. The Rh polypeptides penetrate the membrane bilayer; they are linked to the underlying membrane skeleton; they are covalently fatty acid acylated with palmitate. While the Rh antigenic reactivity is unique to human erythrocytes, the Rh polypeptides have been isolated from erythrocytes of diverse species and are thought to be fundamental components of all mammalian erythrocyte membranes. The functional role of the Rh polypeptides remains undefined, but a role in the organization of membrane phospholipid is suspected.     

The Organization of the lamina ganglionaris of the prawn,Pandalus borealis (Kröyer)

The Lamina ganglionaris (first optic neuropile) of the decapod crustacean Pandalus borealis has its optic cartridges (synaptic compartments) arranged in horizontal rows. Each optic cartridge contains seven receptor axon terminals and the branching axis fibres of five monopolar second order neurons. Four types of monopolar neurons are classified. Their cell bodies are arranged in two layers. The inner layer contains the cell bodies of exclusively one of these types, and each cartridge is invaded by two neurons of this neuron type (type M 1:a and M 1:b). The outer layer contains the cell bodies of the remaining three types (M 2, M3 and M4). One gives rise to a large radially branched axis fibre in the centre of the cartridge. The other two have wide branches which may make inter-cartridge contacts, one proximally and the other distally in the plexiform layer, which is clearly bistratified. The receptor axons terminate in two levels corresponding to these strata. Two sets of tangenital fibres form networks in the proximal and the mid-portion of the lamina. Both networks have fibres with primary branches in the vertical plane and secondary branches in the horizontal plane. The fibres of the networks are derived from axons that pass from the second optic neuropile, the medulla externa.     

Reduction of High Background Staining by Heating Unfixed Mouse Skeletal Muscle Tissue Sections Allows for Detection of Thermostable Antigens With Murine Monoclonal Antibodies

Antigen detection with indirect immunohistochemical methods is hampered by high background staining if the primary antibody is from the same species as the examined tissue. This high background can be eliminated in unfixed cryostat sections of mouse skeletal muscle by boiling sections in PBS, and several proteins including even the low abundant dystrophin protein can then be easily detected with murine monoclonal antibodies. However, not all antigens withstand the boiling procedure. Immunoreactivity of some of these antigens can be restored by subsequent washing in Triton X-100, whereas immunoreactivity of other proteins is not restored by this detergent treatment. When such thermolabile proteins are labeled with polyclonal primary antibodies followed by dichlorotriazinylaminofluorescein–conjugated secondary antibodies and boiled, the fluorescence signal persists, and sections can then be processed with a monoclonal antibody for double immunostaining of a protein unaffected by boiling. This stability of certain fluorochromes on heating can also be exploited for double immunofluorescence labeling of two different thermostable proteins with murine monoclonal antibodies as well as for combination with Y-chromosome fluorescence in situ hybridization. Our method should extend the range of monoclonal antibodies applicable to tissues derived from the same species as the monoclonal antibodies. (J Histochem Cytochem 56:969–975, 2008)     

Epstein-Barr virus-transformed lymphocytes produce monoclonal autoantibodies that react with antigens in multiple organs.

Peripheral blood lymphocytes from normal individuals and patients with autoimmune abnormalities such as insulin-dependent diabetes mellitus and thyroiditis were infected with Epstein-Barr virus, and the culture supernatants were tested for autoantibodies that reacted with normal tissues. Between 58 and 86% of Epstein-Barr virus-transformed cultures produced immunoglobulin M antibodies, and between 9 and 24% of the transformed cultures produced immunoglobulin G antibodies that reacted with normal tissues. Ten Epstein-Barr virus-transformed clones secreting human immunoglobulin M monoclonal autoantibodies were isolated. Four of these monoclonal autoantibodies were studied in depth and found to react with antigens in multiple organs, including thyroid, pancreas, stomach, smooth muscle, and nerves. It is concluded that Epstein-Barr virus can trigger the production of autoantibodies without infecting the target cells to which the autoantibodies are directed.     

Modelling gastric evacuation in gadoids feeding on crustaceans

A mechanistic, prey surface‐dependent model was expanded to describe the course and rate of gastric evacuation in predatory fishes feeding on crustacean prey with robust exoskeletons. This was accomplished by adding a layer of higher resistance to the digestive processes outside the inner softer parts of a prey cylinder abstraction and splitting up the prey evacuation into two stages: an initial stage where the exoskeleton is cracked and a second where the prey remains are digested and evacuated. The model was parameterized for crustaceans with different levels of armour fed to Atlantic cod Gadus morhua or whiting Merlangius merlangus and recovered from the stomachs at different post‐prandial times. The prey species were krill Meganyctiphanes norvegica; shrimps and prawns Crangon crangon, Pandalus borealis, Pandalus montagui and Eualus macilentus; crabs Liocarcinus depurator and Chionoecetes opilio. In accordance with the apparent intraspecific isometric relationship between exoskeleton mass and total body mass, the model described stage duration and rate of evacuation of the crustacean prey independently of meal and prey sizes. The duration of the first stage increased (0–33 h) and the evacuation rate of both stages decreased (by a half) with increasing level of the crustacean armament in terms of chitin and ash. A common, interspecific parameterization of the model within each of the categories krill, shrimp and crab can probably be used if the contents of chitin and ash are similar among prey species per prey category. The model offers a simple way for estimating evacuation rates from stomach content data in order to obtain food consumption rates of wild fishes, provided that information about digestion stage of crustacean prey is available.     

Determination of membrane antigens by a covalent crosslinking method with monoclonal antibodies

Monoclonal antibodies that recognize cell surface proteins may serve as very useful tools for the study of the biological functions of membrane proteins. However, solubilization of the antigens with detergents may lead to major conformational changes of the protein, making their determination with monoclonal antibodies by immune blot or ordinary immunoprecipitation methods difficult. This is especially evident when the monoclonal antibodies recognize tertiary structures of the proteins in the membrane. We have generated two monoclonal antibodies which are specific for the cell surface antigens of multidrug-resistant human cell lines. However, the antigens of both monoclonal antibodies were difficult to detect by either immune blot or ordinary immunoprecipitation methods. We used a cleavable crosslinking reagent dithiobis(succinimidyl propionate) to covalently link the monoclonal antibody with its antigenic determinant in the membrane of intact cells. By this method, we were able to detect the antigens for these two monoclonal antibodies following solubilization, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This method should have wide applicability in determination of membrane antigens recognized by monoclonal antibodies when immune blot or ordinary immunoprecipitation methods are not successful.