Magnetic resonance imaging-guided core needle biopsy and needle localized excision of occult breast lesions.

BACKGROUND: Breast magnetic resonance imaging (MRI) has been reported to be twice as sensitive and three times more specific in detecting breast cancer. We report a series of MRI-guided stereotactic breast biopsies (SCNBB) and needle localized breast biopsies (NLBB) to evaluate MRI as a localization tool. METHODS: Forty-one breast lesions were identified in 39 patients who subsequently had SCNBB or NLBB. Suspicious areas of enhancement were stereotactically biopsied with 16-G core biopsy needles or localized with 22-G wires for excision under laser guidance. RESULTS: Forty-one breast lesions were identified from 1,292 breast MRIs. SCNBB identified three malignancies and two areas of atypia. Two additional cancers were found after NLBB. In patients having NLBB alone, five cancers and two areas of atypia were identified. CONCLUSIONS: In this initial series, breast MRI-guided SCNBB and NLBB were valuable tools in the management of patients with suspicious abnormalities seen only on MRI.

Development of a high throughput scintillation proximity assay for hepatitis C virus NS3 protease that reduces the proportion of competitive inhibitors identified.

A screening assay has been developed for hepatitis C virus (HCV) NS3 protease using the scintillation proximity assay (SPA) technology. The sequence of the peptide substrate used was taken from the site cleaved by the enzyme in the mature nonstructural protein of HCV. The peptide was biotinylated at the N-terminus and tritiated at the C-terminus so that a decrease in signal was detected as a result of enzyme activity. IC(50) values were calculated for the cleaved product, and it was shown that the value obtained was dependent on the substrate concentration used. The effect of substrate concentration on the inhibition of HCV NS3 protease was further highlighted in a mock screening assay, using colored natural product samples, in which the hit rate was altered by a change in substrate concentration. An increase in substrate concentration reduced the proportion of competitive inhibitors identified. This study highlighted the importance of optimizing the components used in SPA assays in order to obtain an assay format valid for high throughput screening.

Effects of topiramate on kainate- and domoate-activated [14C]guanidinium ion flux through GluR6 channels in transfected BHK cells using Cytostar-T scintillating microplates.

PURPOSE: This study was undertaken to test the hypothesis that topiramate (TPM) exerts a negative modulatory effect on some types of alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA)/kainate receptors by binding to the site at which protein kinase A (PKA) phosphorylates the receptor-channel complex. METHODS: The effect of TPM on kainate- or domoate-induced [14C]guanidinium ion flux through iGluR6 channels expressed in baby hamster kidney (BHK) cells was evaluated. Because the hypothesis predicts that TPM will bind only in the dephosphorylated state, a variety of experimental conditions were used to either promote or impede the phosphorylation of the receptor-channel complex. These included the use of dibutyryl cyclic adenosine monophosphate (cAMP) and forskolin to activate PKA, H-9 and H-89 to inhibit PKA, and okadaic acid to inhibit protein phosphatases. RESULTS: Kainate (1 microM) induced a gradual accumulation of [14C]guanidinium into the cells that plateaued approximately 30 min after initiation of the reaction, whereas domoate (0.1 microM) caused a rapid accumulation into the cells that peaked within 5 min; thereafter, the amount of [14C]guanidinium in the cells declined gradually. Topiramate, at 0.1 and 100 microM, did not significantly affect the [14C]guanidinium accumulation under any of the experimental conditions used. CONCLUSIONS: The results of this study are not consistent with the hypothesis tested. However, the results must be interpreted cautiously because iGluR6 receptors expressed in the BHK cells and the functional state of proteins that regulate AMPA/receptors (e.g., PSD-95) may not be sufficiently similar to the receptors and functional state in neurons to serve as a true test of the hypothesis.

Measurement of calcium flux through ionotropic glutamate receptors using Cytostar-T scintillating microplates.

Human embryonic kidney cells (HEK293), expressing the human GluR4 receptor sub-unit of 2-amino-3-hydroxy-methylisoxazol-4-ylpropionic acid (AMPA) type non-NMDA receptors were used, in combination with Cytostar-T scintillating microplates, to develop an assay system for the screening of novel compounds with potential AMPA antagonistic characteristics. Agonist dose responses were measured using the agonists: AMPA; quisqualic acid; L-glutamic acid and kainic acid (KA), and EC50 values of 40, 10, 100 and 100 microM were estimated for each of the agonists, respectively. The AMPA receptor antagonists LY293558 and GYK152466 were tested and shown to inhibit agonist induced [45Ca] influx into the cells. An IC50 value of 600 microM was estimated for the competitive antagonist LY293558 and a value of 100 microM estimated for the non-competitive antagonist GYK152466. The developed assay system is homogeneous, allowing increased assay precision and speed. This allows the potential for automation of the assay and it may be used for screening large numbers of novel compounds.

Analysis of latent forms of renin using antibodies raised against the propart segment of human prorenin: validation with representative samples of ovarian cyst and follicular fluids.

Antisera were raised against synthetic peptides from the prosegment of human prorenin. The use of each of these for detection of the appropriate prosegment region of prorenin was validated by development of an ELISA protocol standardised with recombinant prorenin present in culture medium conditioned by myeloma cells transfected with a prorenin expression plasmid. Detection of the respective epitopes in the prosegment required prior exposure of the prorenin in the medium to acid pH in order to partially unfold the prorenin molecule by dislodging the prosegment from the main body of the protein. By these ELISA protocols, the form of latent renin present in representative samples from ovarian cyst and follicular fluids was analysed; one follicular cyst fluid was found to contain full-length prorenin whereas the fluid from a benign cyst and ovarian follicular fluid samples contained the precursor in truncated form.

The V3 loops of the HIV-1 and HIV-2 surface glycoproteins contain proteolytic cleavage sites: a possible function in viral fusion?

Located close to the crown of the V3 type-specific neutralization loop of the human immunodeficiency virus type 1 (HIV-1) (IIIB) SU glycoprotein gp120, are several potential sites that should be susceptible to proteolytic cleavage by enzymes of trypsinlike or chymotrypsinlike specificity, or by aspartic proteinases. The linkages potentially sensitive to chymotryptic/aspartic proteinase cleavage are retained also within the equivalent domain of HIV-2 (ROD) gp105. We show that thrombin and tryptase cleave HIV-1 gp120 specifically at the tryptic site (GPGR decreases AFVT), and that cathepsin E, an endosomal aspartic proteinase, cleaves at the chymotrypsinlike site (GPGRAF decreases VT). HIV-2 gp105 is also cut by cathepsin E at a site (QIML decreases MSGH) in its V3 loop. Cleavage of HIV-1 gp120 by thrombin is enhanced by sCD4 binding, but is prevented by transient exposure of gp120 to nonionic detergent. Thrombin treatment of HIV-1 gp120 destroys the binding sites for some neutralizing monoclonal antibodies (MAbs) on the V3 loop, but does not affect the affinity of gp120 for sCD4. Conversely, binding of neutralizing MAbs to the HIV-1 V3 loop prior to addition of thrombin or cathepsin E blocks the cleavage reactions, and the binding of some HIV-positive sera to gp120 blocks thrombin cleavage. Analysis of published sequences suggests that all HIV-1, HIV-2, and simian immunovirus (SIV) isolates contain potential proteolytic cleavage sites at similar positions in their V3 loops or equivalent domains. We suggest that cleavage of the V3 loop by a cell surface or endosomal proteinase occurs during the HIV-cell fusion reaction, and that neutralizing antibodies directed against the V3 loop might act by inhibition of this reaction.

The control of CTP:choline-phosphate cytidylyltransferase activity in pea (Pisum sativum L.).

Several possible control mechanisms for CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) activity in pea (Pisum sativum L.) stems were investigated. Indol-3-ylacetic acid (IAA) treatment of the pea stems decreased total cytidylyltransferase activity but did not affect its subcellular distribution. Oleate (2 mM) caused some stimulation of enzyme activity by release of activity from the microsomal fraction into the cytosol, but neither phosphatidylglycerol nor monoacyl phosphatidylethanolamine had an effect on activity or subcellular distribution. A decrease in soluble cytidylyltransferase protein concentrations was found in IAA-treated pea stems, but this was not sufficient to account for all of the decrease in cytidylyltransferase activity. A 50% inhibition of enzyme activity could be obtained with 0.2 mM-CMP, which indicated possible allosteric regulation. Similar inhibition was obtained with 1.5 mM-ATP, but other nucleotides had no effect. The cytidylyltransferase enzyme protein was not directly phosphorylated, and the inhibition with 1.5 mM-ATP occurred with the purified enzyme, thus excluding an obligatory mediation via a modulator protein. The results indicate that the cytosolic form of cytidylyltransferase is the most important in pea stem tissue and that the decrease in cytidylyltransferase activity in IAA-treated material appears to be brought about by several methods.

Hormonal regulation of phosphatidylcholine synthesis in plants. The inhibition of cytidylyltransferase activity by indol-3-ylacetic acid.

Indol-3-ylacetic acid stimulated stem elongation within 1 h of treatment of Pisum sativum L. cv. Feltham First stem sections. This elongation was accompanied by an increase in the endogenous level of phosphocholine and a decrease in that of CDP-choline. Measurements in vitro of the CDP-base pathway enzymes showed an increase in choline phosphotransferase and a decrease in cytidylyltransferase activity on hormone treatment. These results indicate that the decrease in phosphatidylcholine labelling from [14C]choline that is observed on indol-3-ylacetic acid treatment of pea stem sections is caused by the decrease in cytidylyltransferase activity.