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We trained volunteers from conservation organizations to collect environmental DNA (eDNA) from 21 ponds with amphibian communities that had a history of Batrachochytrium dendrobatidis (Bd) and ranavirus (Rv) infections. Volunteers were given sampling kits to filter pond water and preserve eDNA on filter paper, as were the principal investigators (PIs), who made independent collections within 48 h of volunteer collections. Using multi-scale occupancy modeling, we found no evidence to suggest the observer who collected the water sample (volunteer or PI) influenced either the probability of capturing eDNA on a filter or the probability of detecting extracted eDNA in a quantitative PCR (qPCR) reaction. The cumulative detection probability of Bd eDNA at a pond decreased from May through July 2017 because there was a decrease in the probability of detecting eDNA in qPCR reactions. In contrast, cumulative detection probability increased from May to July for Rv due to a higher probability of capturing eDNA on filters later in the year. Our models estimate that both pathogens could be detected with 95% confidence in as few as 5 water samples taken in June or July tested with either 4 or 3 qPCR reactions, respectively. Our eDNA protocols appeared to detect pathogens with 95% confidence using considerably fewer samples than protocols which typically recommend sampling ≥30 individual animals. In addition, eDNA sampling could reduce some biosecurity concerns, jurisdictional and institutional permitting, and stress to biota at ponds.

Highlights • A quantitative LAMP method for identification of EEDV has been developed. • Analytical sensitivity of the qLAMP is as low as 78 pg extracted DNA from tissue. • The method is highly specific for EEDV. • The EEDV qLAMP method was evaluated against the qPCR method., Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus-3) causes a serious disease hatchery-reared lake trout (Salvelinus namaycush), threatening restoration efforts of this species in North America. The current inability to replicate EEDV in vitro necessitates the search for a reproducible, sensitive, and specific assay that allows for its detection and quantitation in a time- and cost-effective manner. Herein, we describe a loop-mediated isothermal amplification (LAMP) assay that was developed for the quantitative detection of EEDV in infected fish tissues. The newly developed LAMP reaction was optimized in the presence of calcein, and the best results were produced using 2 mM MgCl2, 1.8 mM dNTPs and at an incubation temperature of 67.1 °C. This method was highly specific to EEDV, as it showed no cross-reactivity with several fish viruses, including Salmonid Herpesvirus-1, -2, -4, and -5, Infectious Pancreatic Necrosis Virus, Spring Viremia of Carp Virus, Infectious Hematopoietic Necrosis Virus, Golden Shiner Reovirus, Fathead Minnow Nidovirus, and Viral Hemorrhagic Septicemia Virus. The analytical sensitivity of the EEDV-LAMP method was estimated to be as low as 16 copies of plasmid per reaction. When infected fish tissue was used, a positive reaction could be obtained when an infected gill tissue sample that contained 430 viral copies/μg was diluted up to five orders of magnitude. The sensitivity and specificity of the newly developed LAMP assay compared to the SYBR Green qPCR assay were 84.3% and 93.3%, respectively. The quantitative LAMP for EEDV had a correlation coefficient (R2 = 0.980), and did not differ significantly from the SYBR Green quantitative PCR assay (p > 0.05). Given its cost- and time-effectiveness, this quantitative LAMP assay is suitable for screening lake trout populations and for the initial diagnosis of clinical cases.

Over the past century, populations of Lake Trout Salvelinus namaycush have declined throughout the Great Lakes basin due to overfishing, habitat destruction, introduction of invasive species, and associated recruitment issues from high thiaminase, as well as emerging infectious diseases. To combat these declines, state and federal fishery management agencies undertook substantial stock enhancement efforts, including more stringent regulation of sport and commercial catch limits and increasing hatchery propagation of Lake Trout stocked into Great Lakes basin waterways. One state fish hatchery involved in these rehabilitation efforts experienced mass mortality events in 2012 and 2017. In 2012, following a period of abnormally heavy rain, hatchery staff observed abnormal behavior followed by increased mortalities in two strains of Lake Trout fingerlings, reaching upwards of 20% mortality and totaling a loss of approximately 100,000 fish. In 2017, following another heavy-rain season, 6-8% of 2-year-old Lake Trout experienced morbidity and mortality similar to that observed in 2012. During the 2012 event, Brook Trout Salvelinus fontinalis and splake (Lake Trout × Brook Trout hybrid) reared in flow-through systems receiving water from diseased Lake Trout remained clinically unaffected. Molecular analyses revealed all lots of affected Lake Trout were infected with the salmonid herpesvirus-3 (epizootic epitheliotropic disease virus [EEDV]), a disease that caused complete depopulation of this hatchery in the late 1980s and until 2012 was never again detected in this hatchery or in Michigan. Further sampling detected EEDV in apparently healthy 5-year-old Lake Trout and in wild Mottled Sculpin Cottus bairdii collected in the hatchery source water. The ability of the virus to replicate in tissues of infected fish was verified by exposing naïve Lake Trout to the filtered tissue homogenates of infected fish resulting in similar disease signs. Despite the virus going undetected for many years, these two EEDV episodes clearly demonstrate the continued presence of this deadly herpesvirus in the Great Lakes basin.

Few studies have documented seasonal variation of Batrachochytrium dendrobatidis (Bd) infection rates in larval amphibians. We identified 4 natural populations of northern green frogs Lithobates clamitans melanota in Pennsylvania (USA) that contained Bd-infected tadpoles during post-wintering collections in May and June, after hibernating tadpoles had overwintered in wetlands. However, we failed to detect infected tadpoles at those wetlands when pre-wintering collections were made in late July through early September. We observed 2 cohorts of tadpoles that appeared to lack Bd-infected individuals in pre-wintering collections, yet contained Bd-infected individuals the following spring. We also observed 4 cohorts of pre-wintering tadpoles that were Bd-free, even though post-wintering tadpoles collected earlier in the year were infected with Bd. Our results suggest that tadpoles either reduce Bd infections during the summer months, and/or infections proliferate sometime prior to (or shortly after) tadpoles emerge from hibernation. It is unlikely that pre-wintering tadpoles were too small to detect Bd zoospores because (1) there was no correlation between Bd zoospore levels and tadpole size or stage, and (2) size was not a significant predictor of infection status. These results suggest that, while sampling larvae can be an effective means of collecting large sample sizes, investigators in our Mid-Atlantic region should conduct sampling by early summer to maximize the chances of detecting Bd. Further research is warranted to determine whether wetland topography and warm, shallow microhabitats within wetlands contribute to a population's ability to drastically reduce Bd prevalence prior to overwintering at ponds.

A novel herpesvirus was found by molecular methods in samples of Lake Trout Salvelinus namaycush from Lake Erie, Pennsylvania, and Lake Ontario, Keuka Lake, and Lake Otsego, New York. Based on PCR amplification and partial sequencing of polymerase, terminase, and glycoprotein genes, a number of isolates were identified as a novel virus, which we have named Namaycush herpesvirus (NamHV) salmonid herpesvirus 5 (SalHV5). Phylogenetic analyses of three NamHV genes indicated strong clustering with other members of the genus Salmonivirus, placing these isolates into family Alloherpesviridae. The NamHV isolates were identical in the three partially sequenced genes; however, they varied from other salmonid herpesviruses in nucleotide sequence identity. In all three of the genes sequenced, NamHV shared the highest sequence identity with Atlantic Salmon papillomatosis virus (ASPV; SalHV4) isolated from Atlantic Salmon Salmo salar in northern Europe, including northwestern Russia. These results lead one to believe that NamHV and ASPV have a common ancestor that may have made a relatively recent host jump from Atlantic Salmon to Lake Trout or vice versa. Partial nucleotide sequence comparisons between NamHV and ASPV for the polymerase and glycoprotein genes differ by >5% and >10%, respectively. Additional nucleotide sequence comparisons between NamHV and epizootic epitheliotropic disease virus (EEDV/SalHV3) in the terminase, glycoprotein, and polymerase genes differ by >5%, >20%, and >10%, respectively. Thus, NamHV and EEDV may be occupying discrete ecological niches in Lake Trout. Even though NamHV shared the highest genetic identity with ASPV, each of these viruses has a separate host species, which also implies speciation. Additionally, NamHV has been detected over the last 4 years in four separate water bodies across two states, which suggests that NamHV is a distinct, naturally replicating lineage. This, in combination with a divergence in nucleotide sequence from EEDV, indicates that NamHV is a new species in the genus Salmonivirus. Received April 20, 2015; accepted October 11, 2015.

Epizootic epitheliotropic disease virus (EEDV; salmonid herpesvirus [SalHV3]; family Alloherpesviridae) causes a systemic disease of juvenile and yearling Lake Trout Salvelinus namaycush. No cell lines are currently available for the culture and propagation of EEDV, so primary diagnosis is limited to PCR and electron microscopy. To better understand the pervasiveness of EEDV (carrier or latent state of infection) in domesticated and wild Lake Trout populations, we developed a sensitive TaqMan quantitative PCR (qPCR) assay to detect the presence of the EEDV terminase gene in Lake Trout tissues. This assay was able to detect a linear standard curve over nine logs of plasmid dilution and was sensitive enough to detect single-digit copies of EEDV. The efficiency of the PCR assay was 99.4 ± 0.06% (mean ± SD), with a 95% confidence limit of 0.0296 (R(2) = 0.994). Methods were successfully applied to collect preliminary data from a number of species and water bodies in the states of Pennsylvania, New York, and Vermont, indicating that EEDV is more common in wild fish than previously known. In addition, through the development of this qPCR assay, we detected EEDV in a new salmonid species, the Cisco Coregonus artedi. The qPCR assay was unexpectedly able to detect two additional herpesviruses, the Atlantic Salmon papillomatosis virus (ASPV; SalHV4) and the Namaycush herpesvirus (NamHV; SalHV5), which both share high sequence identity with the EEDV terminase gene. With these unexpected findings, we subsequently designed three primer sets to confirm initial TaqMan qPCR assay positives and to differentiate among EEDV, ASPV, and NamHV by detecting the glycoprotein genes via SYBR Green qPCR. Received April 20, 2015; accepted November 10, 2015.

The tumor necrosis factor superfamily (TNFSF) and the TNF receptor superfamily (TNFRSF) have an ancient evolutionary origin that can be traced back to single copy genes within Arthropods. In humans, 18 TNFSF and 29 TNFRSF genes have been identified. Evolutionary models account for the increase in gene number primarily through multiple whole genome duplication events as well as by lineage and/or species-specific tandem duplication and translocation. The identification and functional analyses of teleost ligands and receptors provide insight into the critical transition between invertebrates and higher vertebrates. Bioinformatic analyses of fish genomes and EST datasets identify 14 distinct ligand groups, some of which are novel to teleosts, while to date, only limited numbers of receptors have been characterized in fish. The most studied ligand is TNF of which teleost species possess between 1 and 3 copies as well as a receptor similar to TNFR1. Functional studies using zebrafish indicate a conserved role of this ligand-receptor system in the regulation of cell survival and resistance to infectious disease. The increasing interest and use of TNFSF and TNFRSF modulators in human and animal medicine underscores the need to understand the evolutionary origins as well as conserved and novel functions of these biologically important molecules.

To detect aquatic animal diseases of national concern, 111 individual amphibians, including wood frogs Rana sylvatica (28), spring peepers Pseudacris crucifer (35), red-spotted newts Notophthalmus viridescens (41), and gray tree frogs Hyla versicolor (7), were sampled at seven different sites in the Delaware Water Gap National Recreation Area (DGNRA), Pennsylvania, from June 14 to July 19, 2007. These samples were screened for Batrachochytrium dendrobatidis and viral pathogens at the U.S. Fish and Wildlife Service's Fish Health Center in Lamar, Pennsylvania. Cell culture revealed cytopathic effect (CPE) in two cell lines (epithelioma papillosum cyprini and fathead minnow) inoculated with liver, kidney, and spleen samples from one sample pool of Notophthalmus viridescens (4 individuals). Polymerase chain reaction was conducted on cell culture supernatant exhibiting CPE. Sequencing revealed the resulting product to be identical to frog virus 3, a ranavirus in the family Iridoviridae. Upon gross examination, two Notophthalmus viridescens were found to exhibit dermal swelling and lethargy. Histological examination of these lesions revealed involvement by an Ichthyophonus sp. In summary, two pathogens of concern were found in amphibians in the DGNRA: a ranavirus with a major capsid protein sequence identical to that of frog virus 3 and a mesomycetozoan, Ichthyophonus sp. Although no epizootic die-offs were observed during this health survey, the results warrant further research into the distribution of these pathogens throughout the DGNRA because they have the potential to cause mass mortalities in amphibians.

Much research has been carried out over the years examining cell wall glucans from Saccharomyces cerevisiae and this study further examines aspects of the binding of (1r4)-α-D-glucan in the yeast cell wall, using a number of isolation techniques as well as monoclonal antibodies able to recognize a mixed (1r4)-α-D-glucan/(1r6)-β-D-glucan. Extraction of purified glucan, from S. cerevisiae cell wall, with 0.1N HCl, at 80°C for 6 h, released into the solution (1r4)-α-D-glucan and (1r6)-β-D-glucan as the major polysaccharides, along with an insoluble pellet highly enriched in (1r3)-β-D-glucan. The released (1r4)-α-D-glucan was composed of a high molecular size >100 kDa fraction (7.2% w/w) and a medium 5–50 kDa polysaccharide (10.2% w/w), with the (1r4)-α-D-glucan covalently bound to the (1r6)-β-D-glucan. The average molar ratio of the α:β glucan was 47: 53 in this mixed polysaccharide. The structure of this polysaccharide was different from the structure of plant starch or animal glycogen as monoclonal antibodies specific to yeast (1r4)-α-D-glucan/(1r6)-β-D-glucan did not recognize the plant starch or animal glycogen standards.

Selective breeding of animals for increased innate resistance offers an attractive strategy to control disease in agriculture. However, this approach is limited by an incomplete knowledge of the heritability, duration, and mechanism(s) of resistance, as well as the impact of selection on the immune response to unrelated pathogens. Herein, as part of a rainbow trout broodstock improvement program, we evaluated factors involved in resistance against a bacterial disease agent, Flavobacterium psychrophilum. In 2005, 71 full-sibling crosses, weighing an average of 2.4 g, were screened, and resistant and susceptible crosses were identified. Naive cohorts were evaluated at 10 and 800 g in size, and most maintained their original relative resistant or susceptible phenotypes, indicating that these traits were stable as size increased >300-fold. During the course of these studies, we observed that the normalized spleen weights of the resistant fish crosses were greater than those of the susceptible fish crosses. To test for direct association, we determined the spleen-somatic index of 103 fish crosses; created high, medium, and low spleen-index groups; and determined survival following challenge with F. psychrophilum or Yersinia ruckeri. Consistent with our previous observations, trout with larger spleen indices were significantly more resistant to F. psychrophilum challenge; however, this result was pathogen-specific, as there was no correlation of spleen size with survival following Y. ruckeri challenge. To our knowledge, this is the first report of a positive association between spleen size and disease resistance in a teleost fish. Further evaluation of spleen index as an indirect measure of disease resistance is warranted.

Chemokines play important roles in controlling leukocyte trafficking under normal and inflammatory conditions. Sixteen CXC chemokines have been identified in the human and mouse genomes, while considerably fewer teleost fish CXC chemokines have been reported. Here, we describe a novel clade of trout (Onchorynchus mykiss) CXC chemokines, designated Onmy CXCd, and we identify a novel gene, CXCd1, and a putative duplicate, CXCd2. The trout CXCd proteins contain 112 amino acids and the CXCd1 gene is comprised of four exons and three introns. Constitutive CXCd mRNA expression was detected in skin, gill, visceral fat, and posterior kidney tissues, while low transcript levels were present in the anterior kidney and spleen. Spleen CXCd transcript abundance increased 1 day after bath vaccination (fourfold) and subsided to basal levels by 7 days postvaccination. Challenge with viable Yersinia ruckeri induced expression of trout CXCd RNA up to ninefold in the spleen. The number of viable Y. ruckeri were significantly correlated with CXCd gene transcript abundance (P = 0.0051, Spearman correlation 0.497, n = 30 fish), and fish with the highest bacterial loads had the highest CXCd expression. In contrast, pro-inflammatory cytokine IL-1-beta2 mRNA levels were elevated in fish infected with low numbers of Y. ruckeri, while diminishing in heavily infected fish. CXCd mRNA expression was not increased in rainbow trout infected with infectious hematopoietic necrosis virus, suggesting that up-regulation may be pathogen-specific. Taken together, these results indicate that CXCd transcript elevation follows the pro-inflammatory cytokine response to Y. ruckeri and may be a relevant immunological marker of exposure.

After 22 years of negative viral screening results, the viral pathogen infectious pancreatic necrosis virus (IPNV) was isolated from the ovarian fluid of two pooled samples of returning Connecticut River Atlantic salmon Salmo salar during the 2007 spawning season at Richard Cronin National Salmon Station (RCNSS), Hadley, Massachusetts. Cytopathic effect was observed in Chinook salmon embryo (CHSE-214) cells, and IPNV was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR). Sequence analysis conducted by the U.S. Geological Survey's Western Fisheries Research Center determined that the isolate closely resembled the Canada_3 strain, falling into Genogroup 4 rather than Genogroup 1, which is more common in the United States. This allowed us to speculate that the Atlantic salmon were not infected during their freshwater life stage in the Connecticut River watershed but somewhere on their migratory route or feeding grounds in the Northwest Atlantic. On November 20, 2007, the Connecticut River Atlantic Salmon Commission voted to depopulate the infected stock at RCNSS and the entire suspect egg lots held at White River National Fish Hatchery, Vermont. Approximately one and a half months later, the 121 Connecticut River Atlantic salmon were euthanized and sampled for a follow-up investigation to determine the prevalence of infection. Only one kidney-spleen homogenate (male) was confirmed IPNV positive via cell culture and RT-PCR. A total of 2,983 base pairs from segment A of the RNA genome were sequenced from this fish and determined to be from a new strain (Connecticut-1) of IPNV that closely resembles Canada_2 and Canada_3 in Genogroup 4. The new strain is genetically identical to one of the first ovarian fluid isolates over a shared 130-nucleotide region, possibly indicating original transmission from a single source. The absence of IPNV from the Connecticut River's subsequent four returning Atlantic salmon year-classes may indicate that the aggressive corrective action was prudent.

Two previously undescribed species of myxozoan parasites were observed in the gills of bass inhabiting the Potomac and James River basins. They are described using morphological characteristics and small-subunit (SSU) rDNA gene sequences. Both were taxonomically identified as new species of Myxobolus; Myxobolus branchiarum n. sp. was found exclusively in smallmouth bass, and Myxobolus micropterii n. sp. was found in largemouth and smallmouth bass. Small, spherical, white plasmodia of M. branchiarum from smallmouth bass were observed grossly in the gills; these plasmodia had an average length of 320.3 µm and width of 246.1 µm. The development of the plasmodia is intralamellar in the secondary lamellae of the gills. Mature spores were pyriform in shape with a length of 12.8 ± 1.4 (8.1-15.1) µm and width of 6.9 ± 1.1 (4.0-9.0) µm. Analysis of SSU rDNA identified M. branchiarum in a sister-group to 3 species of Henneguya , although morphologically caudal appendages were absent. Myxobolus micropterii observed in the gills of largemouth and smallmouth bass had larger, ovoid, cream-colored plasmodia with an average length of 568.1 µm and width of 148.1 µm. The cysts developed at the distal end of the gill filament within the primary lamellae. The mature spores were ovoid in shape with a length of 10.8 ± 0.7 (9.2-12.2) µm and width of 10.6 ± 0.6 (9.0-11.8) µm. SSU rDNA analysis placed M. micropterii in a sister group with Henneguya lobosa and Myxobolus oliveirai . The highest prevalence of M. branchiarum was observed in the gills of bass collected from the Cowpasture River (50.9%). Prevalence was 44.6% in bass from the Potomac River and only 4.3% in bass collected from the Shenandoah River. A seasonal study of M. branchiarum , which included both infected and uninfected smallmouth bass, determined that a significantly higher intensity was observed in the spring than in the summer (P0.001) or fall (P = 0.004). In an analysis excluding uninfected bass, a higher intensity was observed in the spring than in the summer (P = 0.001) or fall (P = 0.008). Prevalence and seasonal differences were not determined for M. micropterii .

The sarcolemma of the smooth muscle cell displays two alternating structural domains in the electron microscope: densely-staining plaques that correspond to the adherens junctions and intervening uncoated regions which are rich in membrane invaginations, or caveolae. The adherens junctions serve as membrane anchorage sites for the actin cytoskeleton and are typically marked by antibodies to vinculin. We show here by immunofluorescence and immunoelectron microscopy that dystrophin is specifically localized in the caveolae-rich domains of the smooth muscle sarcolemma, together with the caveolae-associated molecule caveolin. Additional labeling experiments revealed that beta 1 integrin and fibronectin are confined to the adherens junctions, as indicated by their codistribution with vinculin and tensin. Laminin, on the other hand, is distributed around the entire cell perimeter. The sarcolemma of the smooth muscle cell is thus divided into two distinct domains, featuring different and mutually exclusive components. This simple bipartite domain organization contrasts with the more complex organization of the skeletal muscle sarcolemma: smooth muscle thus offers itself as a useful system for localizing, among other components, potential interacting partners of dystrophin.

Epidermal growth factor (EGF) treatment of cells expressing the human EGF receptor (EGFr) results in rapid tyrosine phosphorylation of several cellular proteins including mitogen-activated protein (MAP) kinase. EGF treatment of cells expressing a tyrosine kinase-inactive EGFr failed to induce the tyrosine phosphorylation of endogenous substrates in response to EGF; however, the tyrosine phosphorylation and activation of MAP kinase did occur. This observation indicates that MAP kinase is activated in response to a signal other than the tyrosine kinase activity of the EGFr. Because EGF does not stimulate cells expressing the inactive EGFr to proliferate, phosphorylation of MAP kinase may not be sufficient for the EGF-dependent mitogenesis.

Caveolae have been implicated in the transcytosis of macromolecules across endothelial cells and in the receptor-mediated uptake of 5-methyltetrahydrofolate. Structural studies indicate that caveolae are decorated on their cytoplasmic surface by a unique array of filaments or strands that form striated coatings. To understand how these nonclathrin-coated pits function, we performed structural analysis of the striated coat and searched for the molecular component(s) of the coat material. The coat cannot be removed by washing with high salt; however, exposure of membranes to cholesterol-binding drugs caused invaginated caveolae to flatten and the striated coat to disassemble. Antibodies directed against a 22 kd substrate for v-src tyrosine kinase in virus-transformed chick embryo fibroblasts decorated the filaments, suggesting that this molecule is a component of the coat. We have named the molecule caveolin. Caveolae represent a third type of coated membrane specialization that is involved in molecular transport.

Phosphotyrosine and similar analogs have been used to elicit antibodies that have found widespread use in the study of cellular tyrosine phosphorylation. In order to better understand the anti-phosphotyrosine immune response and to elucidate the details of the specific association between a tyrosine phosphate and an antibody combining site, we have undertaken a detailed comparison of antibody stability, specificity, apparent affinity, and primary structure for eight different anti-phosphotyrosine antibodies derived from immunizations with three different antigens. Two of these, 2G8 and 1G2, were derived from an immunization using azobenzylphosphonate conjugated to carrier, and five others, Py2, Py20, Py42, Py54, and Py69, were the products of an immunization with phosphotyrosine conjugated to carrier. Each of these anti-hapten antibodies was an IgG. One antibody, 129, an IgM, was the result of an immunization with a mixture of tyrosine-phosphorylated proteins which had been purified from growth factor treated cells. We found that antibody binding was significantly inhibited by millimolar levels of divalent cations or high concentrations of monovalent salt, with the exception of the antibody 129 where binding was significantly enhanced by both. Under optimal conditions, the highest apparent affinities for phosphotyrosine were observed for antibodies Py69 and Py20 (10(-6)-10(-7) M) and the lowest for 129 and 1G2 (10(-3)-10(-4). The heavy and light chain variable regions of seven of these antibodies were cloned and sequenced and a predominant anti-phosphotyrosine response was observed. The light chains of these antibodies could be assigned to one of two major VK groups, VK10 and VK19, with sequence identity between the different light chains of each class ranging from 65 to 100% at the amino acid level. Similar sequence identity was found among the heavy chain sequences (89-98% identity at the amino acid level) with the exception of one antibody, 2G8, which was only distantly related to the others (61-64% amino acid identity). These heavy chains belong to the same heavy chain family, J558. Two of the antibodies, Py20 and Py69, were clearly derived from the same progenitor cell since both share a highly unusual apparent V-D-D-JH organization. However, a significant level of somatic mutation has occurred between the two antibodies resulting in subtle changes in their apparent affinity and specificity.

Many receptors for cellular growth factors are known to be protein tyrosine kinases which become activated upon ligand binding at their extracellular domain. We describe here a method to detect the activation state of Epidermal Growth Factor receptor (EGFr) with a monoclonal antibody (mAb74). This antibody was found to preferentially recognize the ligand-activated EGFr as detected by immunoprecipitation, Western blotting and immunocytochemical techniques. mAb74 did not recognize other tyrosine-phosphorylated proteins and was not inhibited by phosphotyrosine, suggesting that it is recognizing an epitope specific for the ligand-activated EGF receptor. The reactivity of mAb74 towards EGFr was closely correlated with the EGF-dependent tyrosine phosphorylation of endogenous substrates. This antibody allows one to detect the activated EGF receptor in vitro or in vivo even in a complex mixture of other tyrosine kinases and substrates.

The TNF superfamily (TNFSF) of proteins are cytokines involved in diverse immunological and developmental pathways. Little is known about their evolution or expression in lower vertebrate species. Bioinformatic searches of Zebrafish, Tetraodon, and Fugu genome and other teleost expressed sequence tag databases identified 44 novel gene sequences containing a TNF homology domain. This work reveals the following: 1) teleosts possess orthologs of BAFF, APRIL, EDA, TWEAK, 4-1BBL, Fas ligand, LIGHT, CD40L, RANKL, and possibly TL1A; 2) the BAFF-APRIL subfamily is enriched by a third member, BALM, unique to fish; 3) orthologs of lymphotoxins α and β were not clearly identified in teleosts and are substituted by a related ligand, TNF-New; 4) as many as four TRAIL-like genes are present in teleosts, as compared with only one in mammals; and 5) T cell activation ligands OX40L, CD27L, CD30L, and GITRL were not identified in any fish species. Finally, we characterize mRNA expression of TNFSF members CD40L, LIGHT, BALM, APRIL, Fas ligand, RANKL, TRAIL-like, and TNF-New in rainbow trout, Oncorhynchus mykiss, immune and nonimmune tissues. In conclusion, we identified a total of 14 distinct TNFSF members in fishes, indicating expansion of this superfamily before the divergence of bony fish and tetrapods, ∼360–450 million years ago. Based on these findings, we extend a model of TNFSF evolution and the coemergence of the vertebrate adaptive immune system.

Particulate antigen uptake by the mucosa of developing channel catfish was determined by immersing larvae and fry [2-day post-hatch (dph), 1-, 2-, 3-, 4-, and 8-week post-hatch (wph)] to two forms of fluorescent microspheres (FMS): blue FMS were carboxylated, and green FMS were coated via conjugation with a crude extract of Edwardsiella ictaluri outer membrane protein (OMP). Phagocytosis, destination, and clearance appeared similar for the two types of FMS used. In the older age classes, primary uptake was observed in epithelial cells of the torso, fins, nares and to a lesser extent the gills. Fluorescent microspheres were less frequently observed within mononuclear phagocytes in the epidermis, dermis and underlying connective tissue of the tissue mentioned above. Limited FMS trafficking was observed from 4- to 24-h post-immersion (hpi). Significantly higher numbers of FMS (blue and green)/mm(3) of tissue were observed in the posterior kidney of the 4- and 8-wph age classes and in the anterior kidney and spleen of the 8-wph age class when compared to younger age classes (p < 0.05). Significantly higher FMS (blue and green)/mm(3) of tissue were observed in the posterior kidney of 4- and 8-wph fish when compared to all other organs (p < 0.05). The present study indicates that FMS uptake increases with age in channel catfish. The younger age classes may possess an increased ability to exclude particulate antigen, or lack the specific mechanisms that needed to take up particulates in the form of FMS.

Fluorescent microspheres (FMS) were injected intraperitoneally into channel catfish fry at 2 days post hatch (dph), 1, 2, 3, 4 and 8 weeks post hatch (wph). The FMS were observed in the vasculature almost immediately after injection in all age groups except 2 dph. Fluorescent microspheres were observed within mononuclear phagocytes in the vasculature after 0.16 dph in all age groups. Fluorescent microspheres were first phagocytized in the coelomic cavity immediately after injection, while the majority of coelomic FMS were phagocytized between 0.16 and 1 dph for all ages. Enzyme cytochemical staining indicated that both polymorphonuclear (neutrophilic granulocytes) and mononuclear phagocytes had phagocytized FMS in the coelomic cavity and organs, with a predominance of FMS found in mononuclear phagocytic cells in all age groups across all sample periods. The predominant organs associated with the observed cellular responses were the posterior kidney, spleen, and anterior kidney. Splenic organization and melanomacrophage development and activity were more pronounced as the fish aged from 2 wph on. Particulate clearance rates were faster in the 2 dph and 1 wph fish than the older ages of fish. These results suggest that to facilitate particulate retention, channel catfish should be vaccinated at 4 wph or older.

Three types of seed (eggs, sac-fry, and fry) production for Rocky Mountain White® hybrid tilapia, (O. niloticus x O. aureus), were compared under green water conditions over a six-month period in an environmentally-controlled greenhouse at the Virginia Tech Aquaculture Center. Rectangular tanks were stocked with broodstock (mean wt. 680 g), at a sex ratio of 3 females to 1 male. Nine tanks were stocked at one of three densities (1, 2, and 4 females/m2), and seed was collected from the females' mouths weekly. Three additional tanks were stocked at a density of 2 females/m2, and fry were collected from the edges of the tanks daily. Average number of viable fry produced by the clutch removal method at 1 female/m2 was significantly higher than the combined average production of densities at 2 and 4 females/m2 (p< 0.02). Even though there was no significant difference between viable fry production per meter sq. (p>0.05), the highest density consistently produced more fry/m2. No significant difference was observed in viable fry production between the clutch removal method and the natural mouth-brooding method (p>0.05). The mean monthly hatchery seed survival was 65.7 ± 2.3%, which varied largely depending on initial seed developmental stage. The effects of stocking density on growth and survival were evaluated by stocking 14-16 day old artificially incubated fry (25.5 ± 0.32 mg, 12.1 ± 0.04 mm), into 150-liter troughs at three densities (3, 6, and 12 fry/ liter) under green water conditions for 30 days. Significant differences were observed between mean weight, length, survival, and feed conversion ratios among the various fry stocking densities (p

Bacterially expressed Fv regions of the anti-phosphotyrosine antibody Py20 have been shown to have an affinity similar to the parent IgG. Previous studies revealed a requirement for both heavy and light chain variable regions, VH and VK joining amino acids and the heavy chain D region for high affinity binding. In order to identify amino acids which might play a role in complexing phosphotyrosine by this antibody, a molecular model for the Py20 Fv was generated using the 3-D crystal structure coordinates of a closely related IgG (R19.9) and the molecular modeling programs CHARMm and CONGEN. The resulting model was tested by mutational analysis. Twelve amino acid residues in both the Py20 heavy and light chain variable regions were altered and the effects of these mutations on phosphotyrosine recognition were determined by affinity chromatography, a competitive ELISA and an antigen overlay assay. The results from this analysis revealed that light chain tryptophan 96 and heavy chain arginines 99 and 59, both of two tyrosines 105 and 106 and a histidine 35 were necessary for high affinity binding. Surprisingly, changing the heavy chain tyrosines 105 and 106 individually resulted in an FV that bound phosphotyrosine with an apparent affinity 10-fold greater than the wild type molecule. Even though the 105 and 106 FVs are monovalent, they recognize tyrosine-phosphorylated proteins displayed on a Western blot as well as the intact IgG. Five other amino acid changes had no obvious effect on antigen binding in any of the assays used.

Caveolin is a protein present in membrane specializations termed caveolae where it may play a structural role. Previous studies on the cDNA sequence of chicken caveolin demonstrated that it is an integral membrane protein without significant homology to any known protein. The cDNA sequence of human lung caveolin is presented here. A striking sequence homology is observed with the chicken protein, as well as a very recently reported protein termed VIP21 [(1992) J. Cell Biol. 118, 1003–1014], a putative vesicle transport protein isolated from MDCK cells. Genomic Southern blots suggest that caveolin is present in a single copy, and Northern blots confirm that the caveolin mRNA is elevated in muscle.

Tyrosine phosphorylation of cytoskeletal proteins at adhesive junctions has been speculated to play a role in the regulation of cell signaling at these sites. Previously, monoclonal antibodies were generated against phosphotyrosine-containing proteins from Rous sarcoma virus-transformed chick embryo fibroblasts, resulting in two antibodies which recognized antigens of 76 and 215 kDa that localized to focal contacts. We have now localized the 215-kDa antigen to a number of adhesive junctions in vivo, including the zonula adherens, intercalated discs, and myotendinous and neuromuscular junctions. In sections of skeletal muscle and in isolated myofibrils, the 215-kDa protein was localized to the I-band. By immunoprecipitation and immunoblot analysis, we determined that the 215-kDa antigen cross-reacts with a polyclonal anti-tensin antibody.

Caveolae are flask-shaped plasma membrane invaginations abundant in endothelium and muscle but may be present in all cells. They contain a filamentous coat material thought to be important in their structure and function. Recent studies have demonstrated that a 22-kDa protein (caveolin) phosphorylated on tyrosine in Rous sarcoma virus-transformed chicken fibroblasts is a component of the caveolae coat on the inner aspect of the membrane. We now report the deduced protein sequence of chicken caveolin derived from cDNA PCR products and genomic DNA clones. Caveolin is a unique protein of 178 amino acids and displays little sequence similarity to other proteins in the GenBank data base. Hydrophobicity predictions indicate an unusual 40-amino acid hydrophobic region near the C terminus that may be used to anchor the protein to the membrane. When chicken caveolin was expressed in mouse 3T3 cells and detected by immunofluorescence microscopy, the typical caveolae pattern was observed. This includes brightly fluorescent membrane patches in many cases concentrated at the margin of cells and in arrays. Caveolae may be distinct from other membrane domains due at least in part to caveolin.

Treatment of normal human fibroblasts with epidermal growth factor (EGF) results in the rapid (0.5 min) and simultaneous tyrosine phosphorylation of the EGF receptor (EGFr) and several other proteins. An exception to this tyrosine phosphorylation wave was a protein (42 kDa) that became phosphorylated on tyrosine only after a short lag time (5 min). We identified this p42 kDa substrate as the microtubule-associated protein (MAP) kinase using a monoclonal antibody to a peptide corresponding to the C-terminus of the predicted protein (Science 249, 64-67, 1990). EGF treatment of human fibroblasts at 37 degrees C for 5 min resulted in the tyrosine phosphorylation of 60-70% of MAP kinase as determined by the percent that was immunoprecipitated with antiphosphotyrosine antibodies. Like other tyrosine kinase growth factor receptors, the EGFr is activated and phosphorylated at 4 degrees C but is not internalized. Whereas most other substrates were readily tyrosine phosphorylated at 4 degrees C, MAP kinase was not. When cells were first stimulated with EGF at 4 degrees C and then warmed to 37 degrees C without EGF, tyrosine phosphorylation of MAP kinase was again observed. Treatment of cells with the protein kinase C activator phorbol myristate acetate (PMA) also resulted in the tyrosine phosphorylation of MAP kinase, and again only at 37 degrees C. Tryptic phosphopeptide maps demonstrated that EGF and PMA both induced the phosphorylation of the same peptide on tyrosine and threonine. This temperature and PMA sensitivity distinguishes MAP kinase from most other tyrosine kinase substrates in activated human fibroblasts.

The methods used above allow one to generate the tools to study individual phosphotyrosine-containing proteins. While all of the substrates we have identified in this way contain phosphotyrosine, this cannot be assumed. Some polypeptides may bind to the anti-phosphotyrosine column because of association with other phosphotyrosine-containing proteins or by nonspecific binding to the anti-phosphotyrosine affinity column. The antibodies generated to these substrates allow one to assess potential questions directly. At a minimum, phosphoamino acid analysis must be performed on the candidate substrate labeled in intact cells with 32PO4 and precipitated with the antibody to the substrate. Methods for this can be found in Cooper and Hunter.3

Publisher Summary Calpactins are Ca2+-binding proteins, which interact with phospholipids and the cytoskeletal proteins actin and spectrin. Calpactins I and II (also termed p34, p36, p39, or lipocortin or annexin) are substrates of protein tyrosine kinases in vivo and are found in cells as either a 38-kDa monomer or an M 90,000 tetramer of heavy (38 kDa) and fight (11 kDa) chain subunits. This chapter outlines the method for the isolation of calpactins I and II from readily available tissue sources (lung and placenta) in quantities (20–100 mg) sufficient for most biochemical studies. This method relies on the CA2+-dependent precipitation of calpactin together with cytoskeletal or membrane fragments, followed by solubilization with EGTA to achieve a high level of purity prior to column chromatography. For the higher yield of calpactin II, a phosphatidylserine affinity column, developed for the purification of protein kinase C, is found to work well. Since the two calpactins described here represent members of a much larger family of proteins with similar structure and function, it may be important in isolating putative homologs from other tissue or cell sources to prove their identity.

The 68-kD protein (paxillin) is a cytoskeletal component that localizes to the focal adhesions at the ends of actin stress fibers in chicken embryo fibroblasts. It is also present in the focal adhesions of Madin-Darby bovine kidney (MDBK) epithelial cells but is absent, like talin, from the cell-cell adherens junctions of these cells. Paxillin purified from chicken gizzard smooth muscle migrates as a diffuse band on SDS-PAGE gels with a molecular mass of 65-70 kD. It is a protein of multiple isoforms with pIs ranging from 6.31 to 6.85. Using purified paxillin, we have demonstrated a specific interaction in vitro with another focal adhesion protein, vinculin. Cleavage of vinculin with Staphylococcus aureus V8 protease results in the generation of two fragments of approximately 85 and 27 kD. Unlike talin, which binds to the large vinculin fragment, paxillin was found to bind to the small vinculin fragment, which represents the rod domain of the molecule. Together with the previous observation that paxillin is a major substrate of pp60src in Rous sarcoma virus-transformed cells (Glenney, J. R., and L. Zokas. 1989. J. Cell Biol. 108:2401-2408), this interaction with vinculin suggests paxillin may be a key component in the control of focal adhesion organization.

Previous studies have shown that molecules related to erythrocyte spectrin are present in the cortical cytoplasm of nonerythroid cells. We report here the localization by immunoelectron microscopy of one such molecule, TW-260/240, in the brush border of intestinal epithelial cells. Using highly specific antibodies against TW-260 and TW-240 as well as antibodies against fodrin, another spectrinlike molecule, we have found that the TW-260/240 molecules are displayed between rootlets at all levels of the terminal web. Occasionally, extended structures appear labeled suggestive of the fine filaments known to cross-link actin bundles. These results are in line with previous in vitro studies showing that TW-260/240 binds to, and cross-links, actin filaments. The results are discussed in terms of a model in which rootlets are immobilized in the terminal web in a matrix of TW-260/240.

The intestinal epithelial cell and specifically the cytoskeleton of the brush border are thought to be controlled by micromolar levels of free calcium. Calcium-binding proteins of this system include intestinal calcium binding protein (CaBP), calmodulin (CaM), villin, and a 36,000-mol-wt protein substrate of tyrosine kinases. To assess the sequence of events as the intracellular Ca++ level rises, we determined the amount of CaM and CaBP in the intestinal epithelium by western blotting and tested the Ca++ binding of CaM and CaBP by equilibrium dialysis. The Ca++-dependent actin severing activity of villin was analyzed in the presence of physiological CaM levels and increasing calcium concentrations. In addition, we analyzed the Ca++ levels required for interaction between CaM and the microvillus 110,000-mol-wt protein as well as fodrin and the interaction between a polypeptide of 36,000 mol wt (P-36) and actin. The results suggest that CaBP serves as the predominant Ca++ buffer in the cell, but CaM can effectively buffer ionic calcium in the microvillus and thus protect actin from the severing activity of villin. CaM binds to its cytoskeletal receptors, 110,000-mol-wt protein and fodrin differently, governed by the free Ca++ and pH. The interaction between P-36 and actin, however, appears to require an unphysiologically high calcium concentration (10(-4) to 10(-3) M) to be meaningful. The results provide a coherent picture of the different Ca++ regulated events occurring when the free calcium rises into the micromolar level in this unique system. This study would suggest that as the Ca++ rises in the intestinal epithelial cell an ordered sequence of Ca++ saturation of intracellular receptors occurs with the order from the lowest to highest Ca++ requirements being CaBP less than CaM less than villin less than P-36.

A protein of 110,000 MW connects actin filaments to the plasma membrane in microvilli of intestinal epithelial cells. In the present study four independent lines of evidence suggest that the 110K protein is directly bound to the lipid bilayer. The solubilization of the 110K protein requires detergents and removal of detergent after solubilization results in aggregation. The 110K protein partitions into the detergent phase in Triton X-114 solutions. It is selectively incorporated into liposomes. It is specifically labeled with the hydrophobic probe 14C-phenylisothiocyanate. In addition we present a purification scheme for the 110K protein in milligram amounts. This represents the simplest system of membrane to filament attachment, in which an integral membrane protein is also a cytoskeletal protein.

Spectrin and related proteins are made up of a common calmodulin-binding subunit tightly associated with a variant subunit. We have analyzed the distribution of the variant subunits in various cell types using subunit-specific antibodies in immunofluorescence as well as western blotting and in some cases have compared the subunits by two-dimensional peptide mapping. We have found that in the majority of cell types (lymphocytes, hepatocytes, neurons, fibroblasts) fodrin 235 K is present in the absence of the other two variant subunits, spectrin 220 K and TW260. Two cell types were found (skeletal muscle and erythrocytes) which contained only the spectrin variant. Two cell types display two distinct variant subunits. Both fodrin 235 K and spectrin 220 K are detected in cardiac muscle whereas TW260 is present in addition to fodrin 235 K in intestinal epithelial cells. During the early stages of embryonic development of the chicken intestine, fodrin 235 K is expressed in the epithelial cells whereas TW260 and spectrin are not detectable. TW260 is expressed relatively late in development (15-16 days) and is inserted only in the apical (brush border) membrane compartment whereas fodrin 235 K is present in these same cells and underlies the entire plasma membrane. These results suggest that fodrin provides the general linkage system between microfilaments and the membrane in nonerythroid and nonmuscle cells.

Recently, molecules highly related to erythrocyte spectrin have been identified in nonerythroid cells. Here we summarize our current understanding of these molecules and suggest a model for their organization. Significant differences exist between this family of proteins isolated from mammalian cells and avian cells, and this may explain the variability in antibody preparations as well as differences in peptide maps of these subunits which have been reported. We have prepared antibodies specific for the variant subunits of the spectrinlike proteins fodrin, spectrin, and TW260/240 and analyzed the distribution of these variant subunits in different chicken cell types as well as their developmental distribution in the intestine. The results suggest that fodrin is the general member of this family of proteins and can even coexist with other spectrinlike proteins in the same cells.

Spectrin, fodrin, and TW-260/240 form a group of structurally and functionally similar but not identical high molecular weight actin-binding proteins from chicken erythrocytes, brain tissue, or intestinal epithelial brush borders. Immunological data and one-dimensional peptide maps of the separated subunits suggest that a common (Mr 240,000) and a variant (Mr 220,000, 235,000, or 260,000) subunit account for the three different heterodimers. These results are in line with the related but distinct morphology of the three proteins observed in micrographs of rotary-shadowed molecules and the finding that the common (Mr 240,000) subunit seems to account for the calcium-dependent calmodulin-binding activity displayed by the three proteins. The possible functions of spectrin-like molecules in nonerythroid cells are discussed.

Fodrin is an axonally transported F-actin crosslinking rod-shaped multidomain protein having a length of 200 nm. It is thought to be related to erythroid spectrin (220,000 and 240,000 M r ) as it is built as a tetramer derived from a heterodimer comprised of two large subunits (235,000 and 240,000 M r ). A panel of 24 monoclonal antibodies has been used to probe the fodrin structure by direct microscopical decoration and identification of large tryptic fragments retaining antigenicity. The combined results unambiguously define the fodrin organization and show it to be strongly related to erythrocyte spectrin. They demonstrate the heterodimer organization, the head-to-head association of dimers, the proteaseresistant domain structure of each subunit and indicate that different termini of the polypeptides are localized at the free F-actin binding ends and the heterodimer association sites. They also show that within the same mammalian species even the highly related 240,000 M r subunits of fodrin and spectrin are immunologically distinct, most likely reflecting different gene products.

The microvillus core-filament bundle from intestinal epithelial cells is a highly ordered structure containing actin and four major associated proteins. Two of these, villin and calmodulin, bind calcium ions (Kd approximately 10(-6) M) in the physiologically important range. Because ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid is present throughout the purification and the isolated cores contain levels of calcium substoichiometric to calmodulin, the protein is bound in the structure without calcium saturation. 10-[3-(4-Methyl-1-piperazinyl)propyl]-2-trifluoromethylphenothiazine, a calmodulin-specific drug, removes the protein from the cores without visibly affecting their ultrastructure. Calmodulin-depleted cores rebind exogenously supplied brain calmodulin. Although the core filaments are stable when the calcium level is less than 10(-7) M, they dissassemble when it is greater than 10(-6) M. This appears to be due to the calcium-sensitive allosteric transition of villin from an F-actin bundling protein to an F-actin severing protein. The actions of the two calcium-binding proteins, villin and calmodulin, are discussed in terms of the calcium sensitivity of the filament bundle. We suggest that villin may act as a calcium-sensitive factor regulating microfilament assembly and disassembly and that calmodulin serves as a buffer modulating the free calcium concentration. This hypothesis may explain some aspects of the physiological process of calcium uptake in the intestine and of the effects of calcium fluxes on the submembranous organization of microfilaments in other cells and tissues.

Spectrin is composed of two nonidentical subunits, with the 240-kDa subunit of nonerythroid spectrin (fodrin) able to bind calmodulin (CaM) Ca2+-dependently. It was found that in the presence of chaotropic salts this binding site was still expressed, although the subunits of fodrin were dissociated. This has been exploited for separating the fodrin subunits rapidly and quantitatively by affinity chromatography on calmodulin-Sepharose. When bovine fodrin was dissolved in 2 m KI + 1 m m Ca2+ and applied to CaM-Sepharose the β subunit (235-kDa) passed through unretarded whereas the α subunit (240-kDa) bound and could be eluted with ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid. These subunits would reform the intact molecule when mixed and dialyzed.

p36 is a major substrate of both viral and growth factor-receptor-associated tyrosine protein kinases. p36 can be isolated as a complex consisting of a subunit of Mr 36,000 (p36) and a subunit of Mr 10,000 (p10), and it represents an abundant cellular protein. We have isolated the p36-p10 complex from bovine intestinal epithelium and analyzed the amino terminus of both subunits. Sequence analysis of the first 56 amino acids of p10 demonstrates a striking sequence homology (48% identically placed residues) with the Mr 10,000 calcium-binding proteins from bovine brain, termed S-100. Intestinal p36 could be effectively labeled on a single tyrosine in vitro with immunoprecipitated pp60v-src and [gamma-32P]ATP. Mild proteolysis of p36 with chymotrypsin resulted in the cleavage into large (Mr, 33,000) and small domains (Mr, 3000), with the latter representing the phosphorylated amino terminus. Although the amino terminus is apparently blocked, sequence analysis of a secondary tryptic peptide of the Mr 3000 fragment as well as the amino-terminal sequence of the Mr 33,000 domain and overlapping peptides clearly established the site of tyrosine phosphorylation.

Calpactins I and II are related 39-kilodalton (kDa) proteins that interact with phospholipids and actin in a calcium-dependent manner and are substrates of tyrosine protein kinases. They contain a short amino-terminal tail attached to a 36-kDa core domain. Monoclonal antibodies (Mabs) were raised to bovine calpactin II and used as site-specific probes of its structure and function. All of the antibodies reacted with native calpactin II and gave rise to a single band of 39 kDa among total cell protein displayed on Western blots. Most of the antibodies (9/14) reacted with determinants on the tail as shown by Western blots and competition with a synthetic tail peptide. Four antibodies reacted with determinants on the core and a 10-kDa tryptic fragment. Antibody-calpactin II complexes were tested for their ability to interact with lipid, actin, and Ca2+ and to serve as substrates of the epidermal growth factor (EGF) receptor tyrosine protein kinase. Whereas none of the antibodies had a detectable effect on actin binding, two anticore antibodies reduced calpactin's affinity for phospholipid. Ca2+-binding sites are known to reside within the core region, yet most antitail antibodies markedly increased the affinity of calpactin II for Ca2+, with four Ca2+-binding sites observed. Antitail antibodies either (i) abolished or (ii) greatly stimulated (10-fold) the phosphorylation of calpactin II by the EGF receptor. These results suggest that the interactions between calpactin II and Ca2+, phospholipid, or the EGF receptor are more complex than previously thought and can be modulated by interactions occurring in the tail.

A low molecular mass protein which we term S100L was isolated from bovine lung. S100L possesses many of the properties of brain S100 such as self association, Ca++-binding (2 sites per subunit) with moderate affinity, and exposure of a hydrophobic site upon Ca++-saturation. Antibodies to brain S100 proteins, however, do not cross react with S100L. Tryptic peptides derived from S100L were sequenced revealing similarity to other members of the S100 family. Oligonucleotide probes based on these sequences were used to screen a cDNA library derived from a bovine kidney cell line (MDBK). A 562-nucleotide cDNA was sequenced and found to contain the complete coding region of S100L. The predicted amino acid sequence displays striking similarity, yet is clearly distinct from other members of the S100 protein family. Polyclonal and monoclonal antibodies were raised against S100L and used to determine the tissue and subcellular distribution of this molecule. The S100L protein is expressed at high levels in bovine kidney and lung tissue, low levels in brain and intestine, with intermediate levels in muscle. The MDBK cell line was found to contain both S100L and the calpactin light chain, another member of this protein family. S100L was not found associated with a higher molecular mass subunit in MDBK cells while the calpactin light chain was tightly bound to the calpactin heavy chain. Double label immunofluorescence microscopy confirmed the observation that the calpactin light chain and S100L have a different distribution in these cells.

We have previously reported the production of monoclonal antibodies directed against phosphotyrosine, which is the modification induced by many oncogene products and growth factor receptors. One of these antiphosphotyrosine antibodies (py20) was used in affinity chromatography to isolate phosphotyrosine (PY)-containing proteins from Rous sarcoma virus-transformed chick embryo fibroblasts (RSV-CEFs). Mice were immunized with these PY-proteins for the production of monoclonal antibodies to individual substrates. Fifteen antibodies were generated in this way to antigens with molecular masses of 215, 76, 60, and 22 kD. Antibodies to individual substrates were used to analyze the subcellular location in normal and RSV-CEFs. Antibodies to the 215- and 76-kD antigen stained focal contacts when used in immunofluorescence microscopy while anti-22-kD protein antibodies resulted in punctate staining concentrated in the margins of cells and in parallel arrays. Both distributions were altered in transformed cells. When cells were extracted with nonionic detergent under conditions that stabilize the cytoskeleton, 50% of the 76-kD protein and greater than 90% of the 22-kD protein fractionated with the cytoskeleton. This study offers a new approach to both the identification of membrane skeletal proteins in fibroblasts and changes that occur upon transformation by an activated tyrosine kinase.

Villin, one of the calcium regulated modulator proteins of F-actin organization, restricts F-actin to short filaments in the presence of calcium and bundles F-actin in the absence of calcium. Limited in vitro proteolysis of villin generates, in addition to a large core fragment (apparent Mr = 90,000) previously described, a small headpiece (Mr = 8,500). The finding that the F-actin nucleation and severing activity of villin, but not its bundling activity, is retained by the core suggested that the headpiece may be directly involved in bundling. Headpiece has now been purified and characterized. It shows strong F-actin binding both in the presence and absence of calcium, leading to a final stoichiometry of 1 headpiece to 1 F-actin monomer. Headpiece also inhibits villin-induced F-actin bundling. Thus villin expresses at least two distinct actin-binding sites localized on separate functional domains. Protein sequence analysis documents that the core comprises the NH2-terminal portion of intact villin, whereas the headpiece covers the COOH-terminal 76 amino acids. We provide the amino acid sequence of the headpiece, which is currently the smallest F-actin binding peptide.

Previous drug studies have suggested that concanavalin A (ConA)-induced cytoagglutination may be influenced by a system of contractile microfilaments. The present study was undertaken to determine the effects of microfilament-active drugs (cytochalasins B and D) (CB and CD) and inhibitors of ATP formation (NaN 3 and 2,4-dinitrophenol (DNP)) on ConA-induced agglutination of Novikoff cells and to investigate the mechanism(s) whereby these agents alter the surface properties of cells. The study described herein demonstrated that 1. 1. CB or CD inhibit cytoagglutination at concentrations above 1 or 0.1 μg/ml, respectively. 2. 2. NaN 3 and DNP both inhibit cytoagglutination, but DNP is more effective and specific. 3. 3. Combinations of metabolic inhibitors (NaN 3 or DNP) with CB lead to a greater reduction of cytoagglutination than with either agent alone. 4. 4. Maximal (>95%) cytoagglutination is still achieved in the presence of CB, CD, NaN 3 , DNP, or combinations of these drugs. 5. 5. None of the drugs tested induced or allowed the redistribution of ConA receptors as measured by ferritin-ConA labeling. 6. 6. Both CB and CD induced the appearance of numerous blebs (zeioses) at the cell periphery, whereas metabolic inhibitors did not. 7. 7. The concentration of either CB or CD required to alter cell-surface morphology paralleled the concentration necessary to inhibit cytoagglutination. These results suggest that the cytochalasins inhibit ConA-induced agglutination of Novikoff cells by their effect on cell-surface morphology, rather than by their effects on the topographical distribution of cell-surface lectin receptors, and that metabolic inhibitors reduce agglutinability by a different mechanism than the cytochalasins.

p36 is a major substrate of tyrosine kinases that co-localizes with spectrin in non-erythroid cells. Recent studies by Gerke & Weber [14] have shown that p36 can be isolated from intestine by selective extraction with the Ca2+-chelating agent EGTA. We now show that p36 can be re-precipitated by adding free Ca2+ to 1 mM with the co-precipitation of a high molecular weight (MW) factor and a polypeptide of 73K. The 73K protein was purified to apparent homogeneity, rabbit antibodies were raised to it and used in Western blots and immunofluorescence microscopy. The 73K protein is found in a wide range of tissues and is particularly concentrated in fibroblasts, where its distribution partially overlaps that of non-erythroid spectrin.

Villin is a major F-actin-bundling protein present in the microfilament bundle underlying the plasma membrane of the microvilli present on intestinal epithelial cells. Mild in vitro proteolysis converts villin (Mr, 95,000) into a large fragment, the villin core (apparent Mr, 90,000). Villin core has lost the F-actin-bundling activity expressed by villin in the absence of calcium but retains the micromolar Kd calcium-binding site and the calcium-dependent restriction of actin filament length (F-actin severing) of intact villin. This finding suggests a common structural and functional relatedness between the known calcium-dependent F-actin-severing proteins from different cell types, even though not all of them reveal F-actin-bundling activity.

India ink can be used to stain proteins bound to nitrocellulose. Subsequently, specific proteins can be identified with antibodies and 125I-protein A. In most cases, india ink did not significantly inhibit detection with antibody, nor was the ink washed off during the antibody incubation steps. Using this method, a direct comparison of antibody-reactive protein and total protein can be made with the same replica.

Calpactins are a family of related Ca++-regulated cytoskeletal proteins. To analyze the expression and cytoskeletal association of calpactins we raised monoclonal antibodies with specificity for the heavy or light chains of calpactin I or to calpactin II. Comparison of the tissue distribution of calpactin I heavy and light chains by Western blots revealed that these subunits are coordinately expressed. Both soluble and cytoskeletal forms of the heavy chain of calpactin I were detected in human fibroblasts whereas only a soluble pool of calpactin II was found. These two forms of the calpactin I heavy chain differed both in their state of association with the light chain and in their rate of turnover. Both the soluble pool of the calpactin I heavy chain and calpactin II turned over three to four times faster than the cytoskeletal pool of heavy and light chains. Immunofluorescence microscopy revealed that the calpactin I light chain was present exclusively in the cytoskeleton whereas the calpactin I heavy chain distribution was more diffuse. No difference in the amount of light chain or the cytoskeletal attachment of phosphorylated calpactin I heavy chain was found in Rous sarcoma virus-transformed chick embryo fibroblasts compared with their normal counterpart. The antibody to the light chain of calpactin I was microinjected into cultured fibroblasts and kidney epithelial cells. In many cases antibody clustering was observed with the concomitant aggregation of the associated calpactin I heavy chain. The distribution of fodrin and calpactin II in injected cells remained unchanged. These results are consistent with the existence of two functionally distinct pools of calpactin I which differ in their association with the cytoskeleton.