Screening for 185delAG in the Ashkenazim.

A study was initiated to assess interest, educational effectiveness, and implications of genetic testing for the common BRCA1 mutation, 185delAG, in the Ashkenazim. Of 333 individuals who attended group sessions, 309 (92%) participated in the study. Participants were categorized as having negative family history (67%), positive family history (defined, by a relaxed criterion, as one first-degree relative or two second-degree relatives with breast [premenopausal] or ovarian cancer) (22%), positive personal history (7%), and both positive personal history and positive family history (4%). Group education was effective, as shown by the improvement in participant scores from pre- to posteducation tests. For the 289 individuals (94%) who requested testing, the major reasons included concern for their own risk, concern for the risk of their children, and desire to learn about surveillance options. The most common reason given by participants who declined testing was concern about health insurance. Six participants found to be heterozygous for the 185delAG mutation received results and were offered genetic counseling. Participants had consented for additional testing without receiving results and were screened for the 6174delT mutation in BRCA2, and seven were found to be positive. All identified carriers reported at least one first- or second-degree relative with a history of breast or ovarian cancer, although they did not all meet our study criteria for positive family history. Given these outcomes, we conclude that screening for breast and ovarian cancer susceptibility is most appropriate for individuals with a positive personal or positive family cancer history. We propose a guideline for future studies designed to identify individuals who may benefit from genetic testing for inherited breast and ovarian cancer.     

A neuronal protein (NP185) associated with clathrin-coated vesicles. Characterization of NP185 with monoclonal antibodies

Two monoclonal antibodies (S-8G8 and S-6G7) are characterized that react with an abundant neuronal protein associated with brain clathrin-coated vesicles (CCVs). This 185-kDa polypeptide (NP185) is not a transmembrane cargo molecule and is distinguishable from clathrin by several criteria including neuronal specificity, chymotryptic sensitivity, migration during two-dimensional gel electrophoresis, lack of cross-reactivity of S-8G8 or S-6G7 with purified clathrin, and lack of associated clathrin light chains. When 0.9 M NaCl extracts of CCVs were diluted and immunoprecipitated by either S-8G8 or S-6G7, NP185 precipitated as a complex with a fraction of the CCV assembly polypeptides. Immunofluorescence microscopy of PC12 cells cultured in nerve growth factor (NGF) revealed that NP185 was distributed in a punctate manner throughout the mature neurites. Immunoblot analysis of PC12 cell extracts, taken at various times during NGF-induced differentiation, revealed that steady-state accumulation of NP185 reaches significant levels 3 days after the addition of NGF and returns to undetectable levels when NGF is removed from the cultures. Significantly, the quantity of NP185 detected in differentiated PC12 cells exceeded the quantity of clathrin. These data indicate that while NP185 may be a specialized component of neuronal CCVs, its function in neuronal cells cannot be associated exclusively with these organelles.     

p185, an immunodominant epitope, is an autoantigen mimotope

An immunodominant peptide (p185(378-394)) derived from the c-erbB2 gene product, was recognized by an anti-DNA antibody, B3, and importantly by two classical DNA-binding proteins, Tgo polymerase and Pa-UDG. These reactivities were inhibited by DNA, confirming that the peptide mimicked DNA. BALB/c mice immunized with p185(378-394) developed significant titers of IgG anti-dsDNA antibodies. Screening of 39 human lupus sera revealed that 5% of these sera possessed reactivity toward p185(378-394). Representative mouse and human sera with anti-p185(378-394) reactivity bound intact p185, and this binding was inhibited by dsDNA. This is the first demonstration of a naturally occurring autoantigen mimotope. The present study identifies a potential antigenic stimulus that might trigger systemic lupus erythematosus in a subset of patients.     

An extra cysteine proximal to the transmembrane domain induces differential cross-linking of p185neu and p185neu.

The neu proto-oncogene encodes a receptor tyrosine kinase (p185) that is closely related to the epidermal growth factor receptor. It has been proposed that receptor tyrosine kinases are activated through oligomerization. Because this clustering model predicts that oligomerization of receptors is sufficient to activate them, we determined if p185 can be activated by introducing an extra cysteine proximal to the transmembrane domain. This should induce inter-receptor disulfide bonding and, according to the clustering model, activate the receptor. This amino acid substitution enhanced recovery of both normal and transforming neu proteins as dimers, with normal p185 recovered predominantly as monomers and transforming p185* as dimers. However, the cysteine substitution did not affect the transforming activity of the two proteins.     

Metabolic products of microorganisms. 185. The anthraquinones of the Aspergillus glaucus group. I. Occurrence,isolation, identification and antimicrobial activity

The occurrence of emodin, erythroglaucin, physcion, physcion-9-anthrone, questin, catenarin, and catenarin-8-methyl ether in different species of the Aspergillus glaucus group (genus Eurotium) was investigated. So far catenarin-8-methyl ether (1, 4, 6-trihydroxy-8-methoxy-3-methylanthraquinone) has not been described as a natural product; it was therefore given the name rubrocristin. The chemical and physical properties of rubrocristin are reported. In addition a new violet pigment (C₁₆H₁₂O₅) was isolated and characterized by its MS-, IR- and UV-spectra.The antimicrobial properties of all substances were examined in the agar diffusion assay. Gram-positive bacteria were the most sensitive organisms and catenarin was the most active naturally occurring substance. Synthetically obtained 1, 4, 6, 8-tetrahydroxy-anthraquinone was slightly more active than catenarin, whereas rubrocristin showed no antibacterial activity.Abbreviations MIC Minimal inhibitory concentration - TLC Thin layer chromatography - PTLC Preparative thin layer chromatography Metabolic products of microorganisms. 184. H. Anke: On the mode of action of cladosporin. J. Antibiotics 32, 952–958 (1979)     

Catalytic mechanism of S-adenosylhomocysteine hydrolase. Site-directed mutagenesis of Asp-130, Lys-185, Asp-189, and Asn-190

S-Adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of S-adenosylhomocysteine to form adenosine and homocysteine. On the bases of crystal structures of the wild type enzyme and the D244E mutated enzyme complexed with 3'-keto-adenosine (D244E.Ado*), we have identified the important amino acid residues, Asp-130, Lys-185, Asp-189, and Asn-190, for the catalytic reaction and have proposed a catalytic mechanism (Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (2000) J. Biol. Chem. 275, 32147-32156). To confirm the proposed catalytic mechanism, we have made the D130N, K185N, D189N, and N190S mutated enzymes and measured the catalytic activities. The catalytic rates (k(cat)) of D130N, K185N, D189N, and N190S mutated enzymes are reduced to 0.7%, 0.5%, 0.1%, and 0.5%, respectively, in comparison with the wild type enzyme, indicating that Asp-130, Lys-185, Asp-189, and Asn-190 are involved in the catalytic reaction. K(m) values of the mutated enzymes are increased significantly, except for the N190S mutation, suggesting that Asp-130, Lys-185, and Asp-189 participate in the substrate binding. To interpret the kinetic data, the oxidation states of the bound NAD molecules of the wild type and mutated enzymes were measured during the catalytic reaction by monitoring the absorbance at 340 nm. The crystal structures of the WT and D244E.Ado*, containing four subunits in the crystallographic asymmetric unit, were re-refined to have the same subunit structures. A detailed catalytic mechanism of AdoHcyase has been revealed based on the oxidation states of the bound NAD and the re-refined crystal structures of WT and D244E.Ado*. Lys-185 and Asp-130 abstract hydrogen atoms from 3'-OH and 4'-CH, respectively. Asp-189 removes a proton from Lys-185 and produces the neutral N zeta (-NH(2)), and Asn-190 facilitates formation of the neutral Lys-185. His-54 and His-300 hold and polarize a water molecule, which nucleophilically attacks the C5'- of 3'-keto-4',5'-dehydroadenosine to produce 3'-keto-Ado.     

185 Phytate Utilization by Tetrahymena

Until recently the commercial collection of beach-cast seaweeds in New Zealand was prohibited but the legislation has recently been amended to allow permitting of this activity. This review collates existing information on the role of beach-cast seaweed in coastal ecosystems to describe the nature and extent of the effects that commercial removals of beach cast seaweed may have on the marine environment. It outlines the amount of beach-cast seaweed available for harvest in New Zealand and the fate of seaweed when not collected; reviewing current information on the importance of beach-cast seaweeds and its inhabitants on: feeding and nesting shorebirds, and nutritional contribution of seaweed inhabitants to nearshore coastal ecosystems when seaweed is washed back into the sea It also identifies key research gaps related to any environmental impacts that the removal of beach cast seaweed might have.     

p185, a product of the neu proto-oncogene, is a receptorlike protein associated with tyrosine kinase activity.

The neu oncogene was originally identified in cell lines derived from rat neuroectodermal tumors. neu is related to but distinct from the c-erbB gene, which encodes the epidermal growth factor (EGF) receptor. neu encodes a protein, designated p185, that is serologically related to the EGF receptor. Identification of the normal homolog of p185 encoded by the neu proto-oncogene enabled us to compare the product of the neu proto-oncogene with the mutated version specified by the neu oncogene and with the EGF receptor. The normal form of p185 was structurally similar to its transforming counterpart, indicating that activation of the neu oncogene did not cause major structural alterations in the gene product. Both normal and transforming forms of p185 were associated with tyrosine kinase activity, supporting the idea that normal p185 functions as a growth factor receptor. p185 differed both structurally and functionally from the EGF receptor. p185 and the EGF receptor had distinct electrophoretic mobilities when synthesized under normal culture conditions or in the presence of tunicamycin. EGF did not stimulate increased turnover of p185 and did not bind quantitatively to p185. A number of other growth factors failed to stimulate degradation of p185 or tyrosine phosphorylation of p185 and are therefore unlikely to be ligands for p185.     

p185HER2 signal transduction in breast cancer cells.

A partially agonistic monoclonal antibody, 4D5, known to bind to the extracellular domain of p185HER2 and shown to inhibit long term growth of p185HER2-overexpressing breast cancer cells, was used to study signal transduction and phosphotyrosyl protein substrates associated with this receptor. Normal breast epithelial cells and breast carcinoma cells expressing low levels of p185HER2 were not affected by 4D5. HER2/neu-overexpressing breast cancer cells (BT-474 and SK-Br-3) exposed to 4D5 exhibited rapid phosphorylation of both p185HER2 and an associated 56-kDa phosphotyrosyl protein (ptyr56). Paralleling the 4D5- stimulated phosphorylation of p185HER2 and ptyr56 was a 5-10-fold induction of c-fos mRNA and phosphatidylinositol 4-kinase activity and a 2-fold induction of inositol 1,4,5-trisphosphate 3'-kinase activity. The increased phosphatidylinositol 4-kinase activity immunoprecipitated with p185HER2 and also co-eluted with ptyr56 from an antiphosphotyrosine immunoaffinity column. These results indicate that short term (less than 6 h) 4D5 activation of p185HER2 in overexpressing breast cancer cells produces agonistic-like signaling typical of homologous tyrosine kinase growth factor receptors such as epidermal growth factor receptor. The data also suggest that ptyr56 represents a novel phosphorylated substrate associated with 4D5-stimulated p185HER2.     

GM3 content modulates the EGF-activated p185c-neu levels, but not those of the constitutively activated oncoprotein p185neu

The functional relationship between ganglioside GM(3) and two tyrosine-kinase receptors, the normal protein p185(c-neu) and the mutant oncogenic protein p185(neu), was examined in HC11 cells and in MG1361 cells, respectively. In the former, p185(c-neu) expression and activation are controlled by EGF addition to the culture medium and by epidermal growth factor receptor (EGFR) activity, whereas the latter express unchangingly high levels of constitutively activated p185(neu). Studies were carried out using (+/-)-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP), which inhibits ganglioside biosynthesis resulting in ganglioside depletion, and addition of exogenous GM(3) to the culture medium. In HC11 cells treated with only [D]-PDMP, p185(c-neu) levels remain similar to control cells, whereas levels of tyrosine-phosphorylated p185(c-neu) increase after treatment with [D]-PDMP in combination with EGF. When exogenous GM(3) is added in combination with [D]-PDMP and EGF, the enhanced phosphorylated-p185(c-neu) returns to control levels. Interestingly, EGFR levels also vary and, analogously to phosphorylated-p185(c-neu), the increase of EGFR content consequent to the [D]-PDMP and EGF addition is reversed by exogenous GM(3). In contrast, the addition of neither [D]-PDMP nor exogenous GM(3) modifies expression and tyrosine-phosphorylation levels of p185(neu) in MG1361 cells. These findings indicate that changes in GM(3) content modulate the tyrosine-phosphorylated p185(c-neu) levels in a reversible manner, but this is not specific for p185(c-neu) because EGFR levels are also modified. Furthermore, these data suggest that GM(3) may play a functional role by affecting the internalisation pathway of p185(c-neu)/EGFR heterodimers, but not of p185(neu) homodimers.     

Oncogenic activation of p185neu stimulates tyrosine phosphorylation in vivo.

p185, the product of the neu/erbB2 proto-oncogene, is oncogenically activated by a point mutation that substitutes glutamic acid for valine in the transmembrane domain of the protein. We have found that the transforming form of p185 differs from its normal counterpart in inducing increased tyrosine phosphorylation of other proteins in vivo and in having a much shorter half-life. These results support the model that the transforming p185 resembles a ligand-activated receptor.     

185W-labelled nitrate reductase from Chlorella

By adding ¹⁸⁵W-tungstate to a Chlorella culture, it has beenpossible to incorporate this metal into the nitrate reductasecomplex. The W-labelled enzyme was completely inactive as nitratereductase, but maintained unaffected its diaphorase activity.In vivo incorporation of tungsten into the enzyme was competitivelyhindered by molybdenum. ¹ This work was supported by a grant from the Instituto de EstudiosNucleares, J.E.N., Spain. (Received July 6, 1971; )