Cloning and characterization of the Atlantic salmon serum lectin, a long-form C-type lectin expressed in kidney.

We report the cloning of four distinct cDNAs and a genomic sequence encoding a multimeric serum lectin found in the blood of Atlantic salmon (Salmo salar). The sequence variation among the cDNAs as well as genomic Southern blotting analysis revealed a multi-gene family. Expression of the salmon serum lectin (SSL) was specific to kidney, as demonstrated by RT-PCR. Analysis of the 173-amino acid sequence of SSL confirmed that it is a member of the C-type lectin superfamily. Sequence alignments and intron/exon structure of the SSL gene showed it to belong to the type VII C-type lectins, which normally bind to galactose or other ligands, whereas the SSL protein sequence contains the EPN motif of mannose-binding C-type lectins, that bind mannose or related carbohydrates.

Assembly and characterisation of a multi-component cytoskeletal gel from adrenal medulla.

Incubation of bovine adrenal medullary cytoplasmic extracts results in the formation of three-dimensional supramolecular gels. Ultrastructurally, the gels display a network of fibres similar in appearance to the cytoskeleton within intact chromaffin cells. Analysis of the protein composition using both electrophoretic and immunoblotting techniques indicates that the gels are composed exclusively of cytoskeletal elements; microfilaments, microtubules and intermediate filament proteins have been identified as having a number of actin-associated proteins. Among the latter class of components the following polypeptides have been identified: filamin (300 kDa), fodrin (240 kDa), a 235 kDa polypeptide, myosin (200 kDa), caldesmon (70 kDa) and tropomyosins (39 kDa). All of these polypeptides co-sedimented with F-actin when gels were assembled in the absence of Ca2+. When gelation was performed in the presence of 10 microM Ca2+ actin, the 235 kDa polypeptide, 70 kDa caldesmon and tropomyosin were all absent from the gels. These results may suggest that the 235 kDa polypeptide, 70 kDa caldesmon and tropomyosins could act either individually or as a functional regulatory unit in controlling the Ca2+-activated reorganisation of the actin network in the cytoplasmic gels.

Fc receptor-directed phagocytic stimuli induce transient actin assembly at an early stage of phagocytosis in neutrophil leukocytes.

The phagocytic pathway for the uptake of multivalent Fc complexes by neutrophil leukocytes can be partially dissected by using a pulse-chase protocol at different chase temperatures. Utilising a deoxyribonuclease I inhibition assay, we show that an early response to recognition of phagocytic substrate by neutrophils is a rapid (less than 10 sec) polymerization of actin. Net actin assembly is transient and is reversed at higher chase temperatures which appears to allow for further progress of the pulse of phagocytic substrate along the pathway. Assembly of actin monitored in whole cells is paralleled by an increase in total actin retained in the cortical filamentous network of detergent-insoluble ghosts prepared from phagocytosing neutrophils. These data provide direct evidence for the reversible reorganisation of actin filaments in the cortical cytoplasm of phagocytic cells during phagocytosis. Furthermore, this experimental system provides an opportunity to study the characteristics of ligand-induced actin assembly, and subsequent disassembly in situ.

Localization of EGF receptors in frozen tissue sections by antibody and biotinylated EGF-based techniques.

We developed a sensitive EGF receptor detection method for frozen tissue sections using biotinylated EGF as the primary reagent. The method was directly compared with an immunohistochemical technique based on an anti-EGF receptor monoclonal antibody (MAb EGFR1) in normal human and rat tissues and in human tumors. The method was more sensitive than a previously published biotinylated EGF-based technique. In normal human tissues and in 37 of the 50 tumors, the binding pattern mirrored that of positive staining with EGFR1. Five further tumors showed weak immunoreactivity, but in these no binding of biotinylated EGF was detected. The remaining eight tumors were negative by both techniques. The discordant cases may reflect a lower level of sensitivity of the ligand-binding technique or, alternatively, abnormal receptors may have been expressed in these tissues. EGF receptors could be detected in rat liver with biotinylated EGF but not with the antibody, indicating the usefulness of the ligand-based technique in cross-species studies.

Concentrations of epidermal growth factor in mouse milk throughout lactation.

A radioimmunoassay was used to measure the concentration of epidermal growth factor (EGF) in mouse milk throughout lactation. The EGF content of mouse milk increased steadily from birth to a concentration of 427 micrograms/l at day 8 of lactation. These high levels were maintained until the approach of weaning, when values decreased from day 17 to 130 micrograms/l at day 22. Milk samples chromatographed on a Biogel P10 column gave a major peak of immunoreactivity at the point at which pure standard EGF was eluted. The origin of milk EGF is unknown, but the high concentrations of this peptide identified in mouse milk suggest that it must play a role in the neonate.

Epidermal growth factor receptors on isolated human placental syncytiotrophoblast plasma membrane.

Receptor sites for mEGF that are specific, saturable and reversible have been demonstrated on isolated syncytiotrophoblast microvillous plasma membrane from term human placentae. Analysis of the binding data indicated the presence of two receptor populations. The high affinity population had 1.7 X 10(13) sites per mg membrane protein with an affinity constant (Ka) of 5.4 X 10(9)/M. The receptors of lower affinity had a Ka of 4.1 X 10(8)/M and 3.1 X 10(13) sites per mg membrane protein.

Desmoid tumors and mesenteric fibromatosis in Gardner's syndrome: report of kindred 109.

Kindred 109, from which Gardner's syndrome was first described, now has 224 members, 28 of whom have inherited the syndrome. Among the group of 28, desmoid tumors or mesenteric fibromatosis or both have developed in eight (29%). In four patients, the fibrous dysplasia appeared to originate in postsurgical abdominal incisional scars; in two of these cases, the mesenteric involvement resulted in death. In one patient, the process was present in the mesentery at the original operation. In three patients, the fibrous dysplastic lesions were extraincisional, with no known trauma associated with their occurrence.

Splenectomy for immune thrombocytopenic purpura.

Of 481 splenectomies performed at the University of Utah, Salt Lake City, 78 (16.2%) were for immune (idiopathic) thrombocytopenic purpura (ITP). The mean platelet count prior to therapy was 13,800/cu mm (range, 500 to 80,000/cu mm). All but two patients were initially treated with corticosteroids, and 58.2% responded with an increase in platelets (mean, 77,900/cu mm). The indications for splenectomy included (1) failure to respond to steroids (33.8%); (2) inability to taper steroids (52.1%); (3) recurrent ITP (5.6%); and (4) miscellaneous (8.5%). There was one death following splenectomy, and the postoperative morbidity was 14.1%. Complete remission occurred in 77.3%, and 84.8% were judged to have benefited from the procedure. The remission rate following splenectomy in patients who responded to preoperative steroids was 93.2%, whereas improvement fell to 68.3% in patients who failed to respond to steroids. This study confirms the benefit of splenectomy for ITP and demonstrates a predictive correlation with response to preoperative preparation with corticosteroids in these patients.

Epidermal growth factor (urogastrone) in human seminal plasma from fertile and infertile males.

Epidermal growth factor/urogastrone (EGF/URO) was measured in the seminal plasma of 214 untreated patients attending an infertility clinic. Patients were assigned groups according to sperm density and progressive motility and in the case of azoospermia into an obstructive etiology or germinal failure. There was no significant variation in mean EGF/URO levels between groups, suggesting that this peptide plays no role in the density or motility of sperm associated with fertility. Other functions this peptide may fulfill in the female reproductive tract, including that of immunosuppression, are discussed.

The current role of thymectomy for myasthenia gravis.

Myasthenia gravis is an autoimmune disease characterized by muscle weakness and fatigability due to a reduction in available acetylcholine receptors at the neuromuscular junction. Treatment with anticholinesterase drugs and corticosteroids has improved the prognosis for patients with this disease. However, controversy continues concerning the indications for thymectomy. During a 9 year period, 27 patients who underwent thymectomy by median sternotomy were reviewed. Eighty-one percent of these patients benefited from the procedure. Clinical improvement did not correlate with age, sex, duration of symptoms, severity of disease or thymic histology, This suggests that the indications for thymectomy should be liberalized to include most patients with generalized myasthenia who fail to respond readily to conventional medical therapy. The importance of immunosuppression nd plasmapheresis in the therapy of myasthenia gravis awaits further delineation of the immune defect associated with the disease.

Cloning of glycerol-3-phosphate dehydrogenase cDNAs from two fish species and effect of temperature on enzyme expression in rainbow smelt (Osmerus mordax).

Rainbow smelt (Osmerus mordax) can accumulate extreme levels of glycerol in their blood during winter. Low temperatures are required for glycerol accumulation in smelt blood and the enzyme glycerol-3-phosphate dehydrogenase (GPDH) has been suggested to play a role in glycerol production/concentration in this species. In the present study, cDNA sequences encoding glycerol-3-phosphate dehydrogenase (GPDH) from rainbow smelt and Atlantic salmon (Salmo salar) were cloned. The encoded GPDH protein sequences were very similar to one another (88% identity). Using RT-PCR, GPDH mRNA was detected in skin, gill, heart, head kidney, brain and liver from both salmon and smelt obtained in December. However, GPDH was not detected in salmon intestine and spleen or in smelt intestine. Examination of GPDH expression in smelt liver during February by Northern blotting revealed temperature regulation. Elevation of the temperature resulted in a significant decrease in liver GPDH transcript level. Serum glycerol levels decreased concomitantly. These findings suggest a role for GPDH in the accumulation of glycerol in smelt at low temperatures.