Infection experiments with Aphanomyces invadans in four species of estuarine fish.

Along the eastern seaboard of the US, Atlantic menhaden, Brevoortia tyrannus, develop characteristic ulcerative lesions, a condition termed ulcerative mycosis. These lesions are identical to those seen across Asia in fish affected by epizootic ulcerative syndrome, a condition caused by the fungus-like oomycete Aphanomyces invadans. Young-of-the-year menhaden inhabiting estuarine environments are the primary species affected in the USA and little is known about the factors involved in the initiation of the lesions, or why menhaden are predominantly infected. Atlantic menhaden, hogchoker, Trinectus maculatus, striped killifish, Fundulus majalis, and mummichog, Fundulus heteroclitus, were inoculated with A. invadans (80 zoospores per fish) to explore species differences in infection and lesion development. All four species developed lesions. Killifish developed frank lesions similar to those observed in menhaden but the gross lesions occurred later, approximately 5-10 days after those on menhaden. Hogchoker and mummichog did not develop gross skin ulcers; rather, their lesions appeared as reddened areas under the epidermis. Mummichogs also showed evidence of significant healing with a well-developed granuloma and significant myocyte regeneration. These experiments show that species barriers as well as ecological barriers can explain some of the factors involved in the development of lesions in, and specificity of the water mould for, menhaden.

Expression of functional recombinant human procathepsin B in mammalian cells.

Cathepsin B has been implicated in numerous pathobiological processes. In order to study its interactions with other proteins implicated in these processes, quantities of functional recombinant cathepsin B are needed. Therefore, we expressed recombinant human procathepsin B in mammalian cells (BSC-1 monkey kidney cells and HeLa human cervical carcinoma cells) using a vaccinia virus expression system. The recombinant human procathepsin B appeared to be authentic and expressed in its native conformation as indicated by: (1) N-terminal sequencing; (2) molecular size; (3) processing intracellularly to mature double-chain cathepsin B; (4) in vitro cleavage by pepsin to mature cathepsin B coincident with appearance of activity against a selective synthetic substrate; and (5) substrate/inhibitor profiles. This is the first report of the expression of functional recombinant human procathepsin B in mammalian cells. We also report a single-step immunoaffinity purification procedure for the isolation of electrophoretically pure proenzyme. By the methodologies described, human procathepsin B can now be obtained in high yield. This should facilitate studies of its interactions with protease inhibitors, other proteases, extracellular matrices, cell-surface proteins and biological substrates that may be of relevance to the pathobiological functions of this enzyme.

Cathepsins D, B and L in breast carcinoma and in transformed human breast epithelial cells (HBEC).

An increased expression of lysosomal enzymes, cathepsin (Cat) D, Cat B and Cat L, was observed in various human tumours and after in vitro cell transformation. To establish possible co-ordination in their expression, all three cathepsins were determined in human breast tumours and in transformed human breast epithelial cells (HBEC). In breast carcinoma (n = 120) all three cathepsins, determined immunochemically and by enzymatic activity, were increased compared to normal breast tissues. The activities, correlated with the corresponding protein masses for Cat D (r = 0.77, p < 0.01), but not for Cat B and Cat L. Significant increase in Cat B activity was observed in stage II compared to stage I tumours, and Cat L activity in stage III compared to stage II tumours, but no significant correlation of cathepsin protein with tumour stage (TNM) was established. No significant correlation between Cat D and the cysteine cathepsins B and L was observed. Similarly, Cat D, Cat B and Cat L levels did not correlate in the in vitro system, e.g. in the five transformed HBEC, such as evolved after dimethylbenz(a)anthracene treatment and c-Has-ras oncogene transfection of diploid MCF-10F cell line (Calaf et al., 1993). Transformed cells showed increased anchorage-independent growth and invasive capability (MCF-10 < MCF-10FTras < D3 < D3-1 < D3-1Tras). The intracellular level of Cat D was not related to cell invasiveness, while total cellular Cat B and Cat L increased 13 fold and 4 fold, respectively, in the most invasive cell line, D3-1Tras compared to MCF-10F.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated levels of pro-cathepsin-d in the plasma of breast-cancer patients.

Changes in the expression and processing of cathepsin D (CD) have been shown to be associated with cancer invasion and metastasis. However, the value of CD as a prognostic marker remains controversial. Most studies have used immunological methods to measure the mature form of CD, although it is the precursor (pro-CD) that appears to be abnormally secreted by breast cancer cells. A sandwich-type ELISA has been developed that is specific for pro-CD. The assay employs a monoclonal antibody to mature CD as the capture reagent and a rabbit polyclonal to the pro fragment as the detector. The assay is specific for pro-CD and capable of quantitating this antigen in biological samples. Pro-CD levels were measured in plasma samples from 76 breast cancer patients and compared with 36 samples from normal control individuals. The plasmas of breast cancer patients showed elevated levels of pro-CD; 12% were more than two standard deviations above the mean for the normal samples. Immunoblots of the plasma samples using a CD monoclonal antibody revealed a band at the appropriate size for pro-CD that corresponded in intensity with the ELISA results. Affinity adsorption of breast cancer plasmas with pepstatin agarose followed by immunoblot analysis revealed a single protein band that correlated with pro-CD. Only trace amounts were detected in the normal control plasmas. These results demonstrate that cathepsin D is present in the plasma of breast cancer patients primarily in its precursor form and that it may be a useful prognostic indicator.

The extracellular domain of p185/neu is released from the surface of human breast carcinoma cells, SK-BR-3.

The human breast carcinoma cell line SK-BR-3, expresses the neu oncogene product, p185, which is a receptor tyrosine kinase. Using a double monoclonal antibody capture enzyme-linked immunosorbent assay for p185, activity was detected in conditioned media from cultures of SK-BR-3 cells. Two monoclonal antibodies specific for the extracellular domain of p185/neu immunoprecipitated a protein with a molecular mass of approximately 105 kDa. p105 was further shown to compete with p185 for binding to monoclonal antibodies and pulse-chase experiments indicate that it was generated by post-translational processing. Peptide maps showed that p105 and p185 are related polypeptides. Since p105 is close to the predicted size for the extracellular domain of p185/neu, we propose that SK-BR-3 cells specifically process and release this portion of the receptor into the medium. The release of the extracellular domain may have implications in oncogenesis and its detection could prove useful as a cancer diagnostic.

Type I macrophage scavenger receptor contains alpha-helical and collagen-like coiled coils.

The macrophage scavenger receptor is a trimeric membrane glycoprotein with unusual ligand-binding properties which has been implicated in the development of atherosclerosis. The trimeric structure of the bovine type I scavenger receptor, deduced by complementary DNA cloning, contains three extracellular C-terminal cysteine-rich domains connected to the transmembrane domain by a long fibrous stalk. This stalk structure, composed of an alpha-helical coiled coil and a collagen-like triple helix, has not previously been observed in an integral membrane protein.

The role of lipids in the function of the acetylcholine receptor.

The composition of membranes containing acetylcholine receptor was altered in order to examine the possible role of lipids in receptor function. Polyethylene glycol was used to fuse AcChR-rich membranes with an excess of lipid vesicles of defined composition. By this procedure, the cholesterol composition was reduced to as little as 20% of that found in native membranes. Using a T1+ flux assay it was possible to measure receptor function in such altered membrane environments. The apparent Kd for carbamylcholine was found to decrease as the cholesterol content was reduced. This result was confirmed by measuring the agonist-induced fluorescence change of the covalently attached probe, 4-[N-(iodoacetoxy)-ethyl-N-methyl]amino-7-nitrobenz-2-oxa-1,3-diaz ole. When the phospholipid composition was manipulated by membrane fusion, ion flux was found to be optimal when the lipid composition resembled that of native receptor membranes. These results indicate that membrane lipids potentially play a role in the regulation of acetylcholine receptor function.

Localization of the ATP binding site on alpha-tubulin.

The binding site for ATP to tubulin was established by use of the photoaffinity label [gamma-32P]N3ATP. Photolysis of the analog in the presence of tubulin resulted in covalent modification of the protein as revealed by autoradiography of electropherograms. Scanning the autoradiograms showed that the ATP analog was bound mainly to the alpha subunit of the tubulin dimer; the alpha subunit was two to three times more radioactive than was the beta subunit. The location of a particular site on the alpha subunit was further defined by peptide maps. The alpha and beta subunits from affinity-labeled tubulin were separated and digested with Staphylococcus protease. Radioactivity was found predominantly in one peptide band from the alpha subunit. The location of the [gamma-32P]N3ATP binding site on the alpha subunit distinguishes it from the previously known exchangeable GTP binding site which is on the beta subunit. Moreover, excess GTP did not compete with [gamma-32P]N3ATP binding. The ATP binding site is distinct from the nonexchangeable GTP binding site. The GTP content of tubulin was the same after dialysis in 0.5 mM ATP as it was following dialysis against ATP-free buffer. Proof that the binding site for [gamma-32P]N3ATP is the same as that for ATP was obtained by competition experiments. In the presence of ATP, photolysis of the affinity analog did not label the alpha subunit preferentially.

Effect of ATP on the kinetics of microtubule assembly.

We investigated the role of ATP in the assembly of microtubules. Tubulin, prepared by chromatography on DEAE-cellulose, was nearly devoid of nucleoside diphosphokinase activity. ATP induced assembly in such preparations for a single assembly/disassembly cycle; then further assembly could not be induced by ATP unless the system was supplemented with additional GTP. This suggests that the E-site must contain GTP for polymerization and ATP interacts at a different site on tubulin. Although tubulin can be assembled into microtubules in 1.0 mM GTP, the inclusion of 0.2 mM ATP along with the GTP increases the rate and extent of assembly. The enhancement increased with increasing ATP concentrations. The inclusion of 0.2 mM ATP reduced the critical concentration for tubulin assembly from 1.5 to 0.9 mg/ml. Analysis of assembly rate versus protein concentration suggested that ATP also affects nucleation. Aggregates of tubulin rings formed by warming tubulin in the presence of 1.0 mM ATP and 5.0 mM Mg2+ were capable of initiating assembly in a solution of tubulin which was not able to polymerize. Furthermore, the extent of microtubule formation was dependent on the concentration of aggregated rings added to the solution. We propose that ATP interacts with tubulin at a binding site that is distinct from the N- and E-sites that bind GTP. A function of ATP binding is to stimulate the formation of tubulin rings as nucleation centers for polymerization.

ATP-induced aggregates of tubulin rings.

Aggregates of double-walled rings (46 nm diameter) were formed upon warming (37 degrees C) ion exchange purified bovine brain tubulin (1.5 to 2.0 mg/ml) in the presence of 1.0 mM ATP and 5.0 mM Mg2+. The formation of aggregated rings was blocked by GDP (1.0 mM), but when GTP (1.0 mM) was added subsequently, the block was overcome. Warming in the presence of ATP and GTP simultaneously produced a mixture of microtubules and aggregated rings. The tubulin preparations used were demonstrated to be devoid of transphosphorylase, and it is therefore clear that the action of ATP in the induction of aggregated rings was not mediated by transphosphorylation. These results are consistent with an interaction of nucleotide with tubulin at a site distinct from the previously described N- and E-sites.