mice received potassium iodide in their drinking water 24 hr prior to injection of radiolabeled antibody to minimize the uptake by the thyroid of any releasedradioiodine.Micewereinjectedvia the tail vein with 100 zCi ‘24I-ICR12(10 @iCi/@tg).The mice were anesthetized with pentobarbitone and imaged using the RMH/ICR MUP-PET positron camera (20) at 24, 48 and 120 hr postinjection. Fifty thousand counts per image were collected.

Pi Technology has been working on hybrid propulsion systems for its various customers for several years, including the Ford Fuel Cell Focus and Hybrid Escape. The systems we have worked on cover a wide range of technologies, from fuel cell/battery and engine/battery systems to hydraulic based systems. As a result we are only too aware of the multitude of issues that need to be addressed. Each of the systems when viewed in isolation appears to be completely different to each of the others; however, we have found this not to be the case, in that common architectural challenges have emerged from each system. In this paper we review the issues we encountered on two early hybrid projects and discuss our experimental efforts at creating a unified control strategy framework for combining different components, in a way that provides a consistency of view between the individual subsystems and the system as a whole. We then go on to examine how we applied this experience to a hydraulic hybrid vehicle, and to some of the novel issues that such systems possess.

PRODUCT INNOVATION IN EARLY MODERN LONDON Get access John Styles John Styles Victoria and Albert Museum Search for other works by this author on: Oxford Academic Google Scholar Past & Present, Volume 168, Issue 1, August 2000, Pages 124–169, https://doi.org/10.1093/past/168.1.124 Published: 01 August 2000

Phosphoinositides bind to profilin and regulate actin-based cytoskeletal protein assembly. We report here that profilin is phosphorylated in vitro by protein kinase C (PKC) in the presence of phosphoinositides and micromolar concentrations of calcium. PKC-mediated phosphorylation of profilin was observed only in the presence of phosphoinositides; phosphatidylserine and diacylglycerol (known activators of PKC) and other lipids, including phosphatidic acid and phosphatidylglycerol phosphate, did not activate the phosphorylation. The activation of PKC-mediated phosphorylation of profilin by phosphoinositides was as follows: phosphatidylinositol (PI) 4-phosphate (K(m) = 18 microM) > PI 4,5-bisphosphate (K(m) = 30 microM) > PI (no activation). About 0.5 mol phosphate was incorporated per mol of profilin. Phosphorylation of profilin by PKC was not affected by the presence of various concentrations of actin. Phospho-amino acid analysis showed serine to be the only amino acid phosphorylated. The amino acid sequence of a phosphopeptide from CNBr-digested profilin corresponded to the COOH-terminal peptide of profilin (Ala-Ser-His-Leu-Arg-Ser-Gln-Tyr). Further digestion of this phosphopeptide by trypsin generated two phosphopeptides (Arg-Ser-Gln-Tyr and Ser-Gln-Tyr), thereby confirming that the phosphorylation site was the antepenultimate Ser (Ala-Ser-His-Leu-Arg-Arg-Ser(P)-Gln-Tyr).

A series of rat monoclonal antibodies (MAbs) has been generated against the extracellular domain of the receptor for EGF which block the binding of EGF and TGF alpha to the receptor and inhibit the growth in vitro of a range of carcinoma cell lines that over-express the receptor for EGF. Some of these antibodies were able also to induce the complete regression of xenografts of EGFR-over-expressing tumours when treatment was started, either at the time of tumour inoculation or later when the tumours were established. The most effective of these antibodies was ICR62, which was also able to activate host immune effector functions. We conclude that antibodies which block growth-factor-ligand interaction can have a profound influence on the proliferative capacity of tumour cells in vivo and may have useful clinical application.

The amino-terminal and internal sequences of the isolated large subunit of trifunctional β-oxidation complex from pig heart mitochondria were determined by Edman degradation. The results demonstrated that the sequence of this novel β-oxidation enzyme is identical with the sequence recently reported for a porcine gastrin binding protein that serves as the gastrin receptor on parietal cell surfaces. Evidence is provided to show that it is unlikely that the porcine gastrin binding protein has such a sequence. The data lead us to conclude that the mature large subunit of porcine trifunctional β-oxidation complex is composed of 727 amino acid residues with a calculated molecular weight of 79,113, while the precursor of this long-chain fatty acid oxidation enzyme has a mitochondrial presequence consisting of 36 residues and a calculated Mr of 83,099.

In mammalian cells, the transmembrane beta-amyloid peptide precursor (beta-APP) undergoes a complex series of alternative proteolytic processing steps that result in the secretion of varying proportions of its extra-cellular domain (protease nexin II) and beta-amyloid peptide. The protein is also reinternalized and degraded in the endosomal-lysosomal system. The relative efficiencies of these competing processes determine the yield of beta-amyloid peptide. Several proteases have been implicated in this complex processing pathway, although none has been identified to date. The yeast secretory system contains proteases homologous to mammalian pro-hormone convertases and is susceptible to genetic manipulation. We therefore investigated the expression and processing of the beta-amyloid peptide precursors (beta-APP-695 and beta-APP-751) in Saccharomyces cerevisiae transformed with human beta-APP cDNA's. beta-APP (695 or 751) cDNA either with its authentic signal sequence or the yeast-derived prepro-alpha-factor leader, was inserted into a glucose-regulated expression vector and transfected into a protease-deficient yeast strain. In all instances, expression of beta-APP was about 1% of total protein. Protease protection studies indicated that either the natural human signal sequence or the alpha-factor leader sequence targetted beta-APP to the endoplasmic reticulum and inserted it with the amino-terminal domain in the lumen. All of the beta-APP fused to the alpha-factor leader proceeded to the trans-Golgi, where Kex2 endopeptidase removed the leader and released the normal amino-terminus of beta-APP. About one-half of the beta-APP was also cleaved at the "alpha-secretase" site in the middle of the beta-peptide sequence, 12 residues before the membrane-spanning sequence. A fraction of the alpha-secretase-cleaved beta-APP appeared in the culture medium; however, most of it associated with the exterior of the cells. The carboxyl-terminal fragments formed by cleavage at the alpha-secretase site accumulated in the membranes. Other proteolytic processes generated membrane-associated carboxyl-terminal fragments that also resembled those found in mammalian cells. These results indicate that the secretory system of S. cerevisiae possesses proteases with specificities similar to the mammalian enzymes that process beta-APP.

Athymic mice bearing xenografts of human tumours that overexpress the receptor (EGFR) for EGF and TGF alpha have been used to evaluate the therapeutic potential of three new rat monoclonal antibodies (mAbs) directed against two distinct epitopes on the extracellular domain of the human EGFR. The antibodies, ICR16 (IgG2a), ICR62 (IgG2b) and ICR64 (IgG1), have been shown (Modjtahedi et al., 1993) to be potent inhibitors of the growth in vitro of a number of human squamous cell carcinomas because they block receptor-ligand interaction. When given i.p. at 200 micrograms dose, the three antibodies were found to induce complete regression of xenografts of the HN5 tumour if treatment with antibody commenced at the time of tumour implantation (total doses: ICR16, 3.0 mg; ICR62, 1.2 mg; ICR64, 2.2 mg). More importantly when treatment was delayed until the tumours were established (mean diam. 0.5 cm) both ICR16 and ICR62 induced complete or almost complete regression of the tumours. Furthermore, treatment with a total dose of only 0.44 mg of ICR62 was found to induce complete remission of xenografts of the breast carcinoma MDA-MB 468, but ICR16 was less effective at this dose of antibody and only 4/8 tumours regressed completely. ICR16 and ICR62 were poor inhibitors of the growth in vitro of the vulval carcinoma A431, but both induced a substantial delay in the growth of xenografts of this tumour and 4/8 tumours regressed completely in the mice treated with ICR62 (total dose 2.2 mg). Although ICR16 and ICR64 were more effective than ICR62 as growth inhibitors in vitro, ICR62 was found to be substantially better at inducing regression of the tumour xenografts due perhaps to additional activation of host immune effector functions by the IgG2b antibody. We conclude that these antibodies may be useful therapeutic agents that can be used alone without conjugation to other cytotoxic moieties.

The pathological findings of Alzheimer's disease include amyloid deposition in cerebral blood vessels and in senile plaques. Both deposits are known to include peptides that contain a common sequence. Both forms of amyloid were isolated and their peptide compositions were determined. The peptides were resolved by size-exclusion chromatography in 70% formic acid, and reverse-phase chromatography in 60% formic acid, 0-40% acetonitrile. Senile plaque amyloid cores contain about 25% protein, about 70% of which is composed of peptides containing the beta-amyloid sequence. Amino-terminal sequencing of the core amyloid peptides (CAPs) revealed extensive amino-terminal heterogeneity, with variable amounts of blocked amino termini. Matrix-assisted, laser-desorption-time-of-flight mass spectrometry of the CAP mixture revealed an array of peptides the molecular weights of which corresponded to peptides beginning with each of the first 11 amino acids of the beta-peptide sequence and ending with Ala-42 of that sequence. The carboxyl-terminal residues were identified by tandem mass spectrometry of chymotrypsin digests. CAP possessed a minor degree of carboxyl-terminal heterogeneity. Cerebrovascular amyloid peptides (CVAPs) possessed minor degrees of both amino- and carboxyl-terminal heterogeneity. The major CVAP commenced at Asp-1 and ended at Val-40. Minor components of CAP possessed masses of 8000-9000 Da and the same amino-terminal residues as the major components of CAP. They may be precursors to the smaller CAPs. The differences in amino termini and carboxyl termini of CAPs and CVAPs suggest that the two types of amyloid form by different pathways, on which they encounter different proteases.

The receptor (EGFR) for epidermal growth factor (EGF) and transforming growth alpha (TGFα) is often overexpressed in certain types of human malignancy and high levels of expression of the receptor and/ or coexpression of growth factors. EGF and TGFα have also been correlated with poor prognosis in many patients. We have produced a number of rat monoclonal antibodies (MAbs) against four distinct epitopes on the external domain of the EGF receptor and are currently evaluating their potential for therapeutic use. Nine of these of MAbs were found to inhibit the binding of TGF and EGF to the receptor on tumor cells and these MAbs were able to inhibit the growth in vitro and in vivo of tumor cells that overexpress the EGF receptor. Here, we describe the results of experiments to determine the antitumor activity of combination treatment with two antibodies directed against separate epitopes on the external domain of human EGFR. Our results showed that treatment of human tumor xenografts with a combination of two anti-EGFR MAbs that bind to two distinct epitopes on the external domain of EGF receptor was not as effective as treatment with ICR62 alone, which binds to epitope C on the EGFR and is of IgG2b isotype. A phase I clinical trial with antibody ICR62 is currently underway in Royal Marseden Hospital (UK) in patients with head and neck and lung carcinomas.

We have investigated the effect of treatment with epidermal growth factor (EGF) and/or monoclonal antibodies (Mabs) to the epidermal growth factor receptor (EGFR) on the growth in vitro of a number of human tumour cell lines. Mabs ICR10, ICR11, and ICR16 that prevent the binding of both I-125-EGF and I-125-TGFalpha to the EGF receptor were found to inhibit the growth of human tumour cell lines overexpressing the EGF receptor. At high concentrations (5 nM), EGF was also found. to inhibit the growth of HN5, A431 and MDA MB-468 cells whereas the proliferation of SKBR3 and HN6 cells was stimulated. While some of the cell lines (e.g. HN5 and HN6) were very susceptible to growth inhibition by antibodies to the EGFR others, known to secrete autocrine growth factors (e.g. A431 and MDA MB-468), were less affected by these antibodies. Indeed, growth of the latter cell lines was inhibited more effectively by the addition of 5 nM of exogenous EGF than by treatment with 156 nM of the anti-EGFR Mabs ICR16, ICR11, and ICR10. In addition, we show that the growth of HN5 cells, which express very large numbers of EGF receptors (1.4x10(7)/cell), was stimulated at picomolar concentrations of EGF but inhibited at concentrations of EGF in the nanomolar range. Maximal stimulation (25% above control) was observed with the addition of 78 pM EGF to the cultures. The mitogenic effect of low concentrations of EGF and the growth inhibitory effect of nanomolar concentrations of EGF on HN5 cells could be reversed by the addition of 156 nM of the anti-EGFR antibodies. We conclude that growth inhibition can follow from either inhibition of signal transduction by blocking ligand binding to the EGFR or from excess binding of ligand. With tumours that produce significant quantities of autocrine growth factors the latter will compete with the Mabs for binding to the receptor and reduce their effectiveness.

The product of the c-erbB-2 protooncogene (p185) is a member of the EGF receptor family of transmembrane tyrosine kinases. Amplification of this gene and overexpression of the product has been observed in adenocarcinomas and has been correlated with poor prognosis in patients with breast and ovarian cancer. The very low levels of expression of p185 by normal adult tissues makes the receptor an almost tumor-specific target. We have prepared rat monoclonal antibodies against five distinct epitopes on the external domain of the c-erbB-2 product overexpressed by the breast cancer line BT474. The antibodies bind to the protein core of p185 and stain specifically the membranes in frozen sections of tumors overexpressing the c-erbB-2 product. Three of the antibodies, ICR12 (epitope A), ICR54, and ICR55 (epitope E), also stain the cell membrane in formalin-fixed, paraffin-embedded sections and bind to p185 in Western blots. An investigation of the stability of the antigen-antibody complexes indicates that the majority are not readily internalized by SKOV3 cells growing in vitro. Antibodies ICR12 (IgG2a) and ICR55 (IgG2a), which are directed against separate epitopes on the c-erbB-2 p185, are both of high affinity and immunoreactivity (> 75%) and localize specifically and stably to xenografted breast and ovarian carcinomas growing in athymic mice when labeled with 125I (up to 13% injected dose/g, ICR12 and ICR55) or a range of other radionuclides (up to 20% id/g, ICR12). We conclude that these antibodies may be useful as therapeutic vehicles for targeting radionuclides (imaging and therapy) or enzymes for activating prodrugs (ADEPT).

Lymph node status is still the single most important prognostic factor in breast cancer. Axillary surgery remains the only reliable means of providing this information. This pilot study evaluates using a highly specific radiolabelled monoclonal antibody to provide equivalent information by a non-invasive technique.After optimisation of labelling conditions, our first antibody, ICR12 (against the gene product of c-erbB-2) was evaluated in a mouse model system. Twenty-four hours post i.v. injection the mice were killed and their organs, blood and tumours harvested for counting. Tumour localisation was four times greater than that into normal tissues, reaching 20% injected dose per gram of tumour.Eight patients have had this Tc99m-ICR12. Patient selection was by immunocytochemical staining of fine needle aspirates from the patient's own breast cancer. After intravenous administration of the immunoconjugate, tomographic images were obtained at 24 h. These results were compared to the subsequent histopathological examinations. Three patients acted as normal controls, one patient was negative due to inappropriate sampling, and two patients had strong membrane staining and provided excellent tumour localisation to both breast primary and regional node metastases. A further two patients only had moderate antigen expression on staining and did not localise well.The good performance of this radiolabelled antibody with patients that strongly stain for the antigen encourages the development of this system as both a method of staging breast cancer and a potential means of immunotherapy in this subgroup of patients.

PURPOSE To evaluate the prognostic importance of immunocytochemically determined c-erbB-2 overexpression in the primary tumors of patients with breast cancer. PATIENTS AND METHODS Primary tumors from 1,506 breast cancer patients (760 node-negative and 746 node-positive) who were treated in the International (Ludwig) Breast Cancer Study Group Trial V were studied. Node-negative patients were allocated randomly to either a single cycle of perioperative chemotherapy (PeCT) or no adjuvant treatment, and node-positive patients received either a prolonged chemotherapy (with tamoxifen for postmenopausal patients) or a single perioperative cycle. RESULTS Tumors from 16% of the node-negative patients and 19% of the node-positive patients were found to be c-erbB-2-positive. In both groups c-erbB-2 positivity correlated with negative progesterone receptors (PR), negative estrogen receptors (ER), and high tumor grade. Lobular carcinomas were all negative, and, thus support the view that such tumors represent a defined subtype of breast carcinoma. The expression of c-erbB-2 was prognostically significant for node-positive but not for node-negative patients. However, in both subgroups, the prognostic significance was greater for patients who had received more adjuvant therapy. For node-positive patients, the effect of prolonged-duration therapy on disease-free survival (DFS) was greater for patients without c-erbB-2 overexpression (hazards ratio [HR], = 0.57; 95% confidence interval [CI], 0.46 to 0.72) than for those with c-erbB-2 overexpression (HR, 0.77; 95% CI, 0.51 to 1.16). Similarly, for node-negative patients, the effect of PeCT on DFS was greater for those without c-erbB-2 overexpression (HR, 0.82; 95% CI, 0.61 to 1.09) than for those with c-erbB-2 overexpression (HR, 1.22; 95% CI, 0.66 to 2.25). CONCLUSION We conclude that tumors with overexpression of the c-erbB-2 oncogene are less responsive to cyclophosphamide, methotrexate, and fluorouracil (CMF)-containing adjuvant therapy regimens than those with a normal amount of gene product.

The epithelial membrane antigen (EMA) is expressed by the majority of colorectal cancers but has not previously been investigated as a target for radiation-labeled monoclonal antibodies (MoAb) in the imaging of patients with colorectal cancer. A rat IgG2a MoAb that recognizes EMA, ICR2, was labeled with Indium-111 (100 megabecquerel per milligram [MBq/mg]MoAb) using the bicyclic anhydride of the chelating agent diethylene triamine pentacetic acid (ccDTPA) and was administered intravenously to 22 patients known to have or thought to have colorectal cancer. Daily gamma camera imaging was performed for 3 days during the time between the administration of the radiation-labeled antibody and surgical procedure. At operation, the biopsies were done of the tumors and the normal colon, and the uptake of radiation-labeled MoAb was measured in a gamma well-counter. Immunocytochemistry for EMA expression also was done on resected tumors. Independent unblinded and blinded reporting was done on all scans. The sensitivity of 111In-ICR2 for detecting cancers preoperatively was 80% and 60%, respectively, on unblinded and blinded reporting, and the corresponding specificity 20% and 60%. The low unblinded specificity was attributable to a false-positive localization in severely dysplastic benign tumors (n = 2) and inflammatory tissue (n = 2). Liver metastases present in three patients were cold relative to normal liver. Lymph node metastases were localized in 1 of 6 patients preoperatively. The mean absolute uptake of 111In-ICR2 in tumor tissue was 7.75 +/- 3.77 x 10(-3) percent of injected dose per gram, and the ratio to normal colon was 2.10 +/- 0.92:1. On immunohistochemistry, EMA was expressed by 16 of the 17 primary cancers, both dysplastic adenomas, and all nodal metastatic deposits. EMA-negative tumors (1 cancer + 1 colonic lipoma) had negative antibody scans, and patients whose tumor was negative or only focally positive for EMA expression had lower tumor/normal colon ratios of radioactivity (1.30 +/- 0.26 versus 2.45 +/- 0.65, P = 0.005) on gamma well-counting of excised specimens. These results suggest a possible role for 111In-ICR2 in the detection of colorectal cancer and metastases but not its liver deposits.