Isolation and characterization of neurokinin A, neurokinin A(3-10) and neurokinin A(4-10) from a neutral water extract of a metastatic ileal carcinoid tumour

A metastasis to the right liver lobe of an argyrophil/argentaffin midgut carcinoid tumour in a patient with the classical carcinoid syndrome was examined for the presence of tachykinins other than substance P, using a specific antiserum. The extract was initially purified using SepPak cartridges, and subsequently subjected to cation-exchange chromatography on SP Sephadex C-25 which separated the immunoreactive material into two main components (components I and II). Both were further purified by anion-exchange chromatography on DEAE-Sephadex A-25, and by reverse-phase fast protein liquid chromatography. Component II was identified as neurokinin A by its immunochemical and chromatographic properties and amino acid sequence analysis. Component I consisted of two molecular forms which were identified as neurokinin A(3-10) and neurokinin A(4-10) by amino acid sequence analysis. The tumour tissue contained only small amounts of the eledoisin-like peptide that has earlier been demonstrated in mammalian tissues. Although this component behaved like the nonmammalian peptide eledoisin on reverse-phase HPLC and on reverse-phase ion-pair chromatography, eledoisin-specific antiserum E2 indicated that eledoisin-like peptide is not identical to eledoisin. Neurokinin A in carcinoid tumours has an N-terminal heterogeneity; this multiplicity constitutes a further support for the hypothesis that carcinoid tumours produce a number of tachykinins which may be present in different relative amounts in individual patients and may contribute to the individual differences in symptomatology.     

EGF-induced PGE2 release is synergistically enhanced in retinoic acid treated fetal rat lung cells

Retinoic acid has been shown to induce a 2.5-fold increase in 125I-EGF binding capacity through increased EGF receptor synthesis in a fetal rat lung (FRL) cell line (1). In FRL cells, incubation with either EGF or retinoic acid induces a modest increase in PGE2 secretion (80% or 40%, respectively). However, in the presence of both EGF and retinoic acid, FRL cells exhibit a 6.4-fold increase in PGE2 secretion. Retinoic acid and EGF dose-response curves demonstrate that the effect on PGE2 secretion correlates with the retinoic acid induced increase in EGF receptors. These data suggest a relationship between increased EGF receptor expression and increased EGF responsiveness. Furthermore, these data indicate a potential mechanism by which EGF and retinoic acid may interact in lung physiology.     

Antisera raised against eledoisin and kassinin detect elevated levels of immunoreactive material in plasma and tumor tissues from patients with carcinoid tumors

Radioimmunoassays based on antisera raised against the tachykinins eledoisin (antiserum E7) and kassinin (antiserum K12) were used to measure the concentration of tachykinin-like immunoreactivity (TKLI) in plasma from 52 healthy subjects. 65 patients with carcinoid tumors (of which 46 had symptoms of both flushing and diarrhoea), and 6 patients with endocrine pancreatic tumors. The antisera did not crossreact with substance P (SP). Elevated concentrations of TKLI, as compared with healthy subjects, were found in 75% of the carcinoid patients, but in none of the patients with pancreatic tumors. Tumor metastases from 8 of the carcinoid patients all contained TKLI. Ion-exchange chromatography of plasma samples and tumor tissue extracts indicated the presence of several immunoreactive molecular forms. The elution patterns of the immunoreactivity detected by antisera E7 and K12 were similar, indicating that the same molecular species are measured by these antisera. None of the components coeluted with synthetic SP. One of the immunoreactive components in carcinoid tumor extracts coeluted with synthetic NKA. The major immunoreactive components in plasma from the patients eluted in a position different from that of all currently known mammalian tachykinins. Tachykinin immunoreactive material detected in tumor tissue and plasma of patients with carcinoid tumor may play a role in the symptomatology of the carcinoid syndrome.     

High-yield purification of HIV-1 proteinase expressed by a synthetic gene in Escherichia coli.

A rapid and simple purification procedure for human immunodeficiency virus type 1 (HIV-1) proteinase from a synthetic gene expressed in Escherichia coli has been developed. The synthetic gene was constructed from oligonucleotides containing several restriction enzyme sites in order to allow simple construction of homologous genes. The protein was translated as a precursor which was autocatalytically processed into the mature protein as shown by N-terminal sequence analysis of the purified protein. Immunoblot analysis was used to verify the nature of the expression product and it was found that 2 of 10 anti-peptide antibodies, covering the whole proteinase sequence, were able to react with the enzyme in crude bacterial lysates. These two anti-peptide antibodies represent a continuous sequence partially overlapping the active site. The purification involves two initial precipitation steps followed by cation-exchange and size-exclusion chromatography. A high yield and a high specific activity were achieved.     

In vivo and in vitro synthesis of adenovirus type 2 early proteins.

The synthesis of adenovirus type 2 (Ad2)-induced early polypeptides was examined in vivo and in vitro by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis alone and specific immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of total [35S]methionine-labeled polypeptides synthesized in vivo at 3 h postinfection allowed us to detect in infected cells at lease 13 distinct polypeptides that are either absent or less conspicuous in extracts from mock-infected cells. These Ad2-induced early polypeptides have molecular weights ranging from 72 x 10(3) to 10.5 x 10(3) and have accordingly been designated as E72K to E10.5K. Nine of the in vivo synthesized early polypeptides can be precipitated specifically from infected cell extracts by antisera with specificity against early adenovirus proteins. In vitro translation of mRNA extracted from mock-infected cells and from Ad2-infected cells was carried out in preincubated Ehrlich ascites cell extracts. All the early Ad2-induced polypeptides identified in the extracts from infected cells labeled in vivo were also detected among the polypeptides immunoprecipitated specifically from the in vitro reaction mixtures programmed by RNA extracted at 4 h postinfection from Ad2-infected cells.     

Purification of adenovirus messenger ribonucleic acid by an aqueous polymer two-phase system.

An aqueous polymer phase system containing 6.3% (w/w) dextran and 3.5% (w/w) poly(ethylene glycol) in 10 mM phosphate buffer (pH 8.0) was developed to select RNA-DNA hybrids from unhybridized RNA. The top phase of this phase system, which contains DNA and the RNA-DNA hybrids, can be used to purify adenovirus messenger RNA both early and late in the infectious cycle. The hybrids can be melted by heat in the top phase and the messenger RNA selected by oligo(dT)cellulose chromatography whereupon the polymers and the DNA percolate and the polyadenylated messenger RNA absorb to the column. The isolated messenger RNA appears to be almost quantitatively recovered at a purity from 70 to 90% depending on the concentration of the specific messenger RNA in the starting material. Early and late viral messenger RNA were selected on the complementary strands of adenovirus DNA according to this procedure.     

Saturable binding to cell membranes of the presynanptic neurotoxin, β-Bungarotoxin

Brief exposure to the protein neurotoxin, β-bungarotoxin, is known to disrupt neuromuscular transmission irreversibly by blocking the release of transmitter from the nerve terminal. This neurotoxin also has a phospholipase A2 activity, although phospholipases in general are not very toxic. To determine if the toxicity of this molecule might result from specific binding to neural tissue, we have looked for high affinity, saturable binding using ¹²⁵I-labeled toxin. At low membrane protein concentration ¹²⁵I-labeled toxin binding was directly proportional to the amount of membrane; at fixed membrane concentration ¹²⁵I-labeled toxin showed saturable binding. It was unlikely that iodination markedly changed the toxin's properties since the iodinated toxin had a comparable binding affinity to that of native toxin as judged by competition experiments. Comparison of toxin binding to brain, liver and red blood cell membranes showed that all had high affinity binding sites with dissociation constants between one and two nanomolar. This is comparable to the concentrations previously shown to inhibit mitochondrial function. However, the density of these sites showed marked variation such that the density of sites was 13.0 pmol/mg protein for a brain membrane preparation, 2.4 pmol/mg for liver and 0.25 pmol/mg for red blood cell membranes.In earlier work we had shown that calcium uptake by brain mitochondria is inhibited at much lower toxin concentrations than is liver mitochondrial uptake. Both liver and brain mitochondria bind toxin specifically, but the density of ¹²⁵I-labeled toxin binding sites on brain mitochondrial prepartions ($\text{3.3 ± 0.3}\text{pmol/mg}$) exceeded by a factor of ten the density on liver mitochondrial preparations ($\text{0.3 ± 0.05}\text{pmol/mg}$). It is also shown that the labeled toxin does not cross synaptosomal membranes, suggesting that mitochondria may not be the site of action of the toxin in vivo. We conclude the β-bungarotoxin is an enzyme which can bind specifically with high affinity to cell membranes.     

Electromyographic changes in work-related myalgia of the trapezius muscle.

In 11 patients, all women, 21-55 years of age, with unilateral work-related myalgia of the trapezius muscle, the right and left trapezius muscles were examined simultaneously for electromyogram (EMG) signs of localized muscle fatigue. All patients were tested with 0-kg hand load for 5 min, holding the arms straight at 90 degrees of elevation in the scapular plane. Only 4 of the patients tolerated exposure to higher load levels. They were tested with 1 kg hand load for 3 min and 2 kg hand load for 2 min, with a period of rest of 30 min between the trials. The EMG mean power frequency (MPF) and root mean square (rms) were calculated. Data were normalized with the initial value as a reference and regression analyses were performed. On both sides a decrease of MPF and an increase of rms were found with increasing time and load, i.e. classical EMG signs of localized muscle fatigue. Compared with the nonaffected side smaller changes were found on the affected side, possibly due to pain inhibition, impaired microcirculation and biochemical changes along the muscle fibres. At 0-kg hand load we found no change of MPF on either side despite subjective feelings of fatigue and pain. We interpreted these findings as an indication of reduced capacity of the affected trapezius muscle to sustain static load with early development of pain-associated local fatigue.     

Characterizing the normal proteome of human ciliary body

Background

The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.

Results

In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.

Conclusions

More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.