Shi Ke

Recombinant pp60c−src from baculovirus-infected insect cells: purification and characterization

A simple and effective method has been developed to purify the recombinant protein tyrosine kinase pp60c-src from a baculovirus-insect cell expression system. The procedure includes affinity chromatography and HPLC. Milligram quantities of protein have been isolated with an activity of 3.9 mumol/min/mg protein using the substrate poly E4Y. This specific activity is many times higher than any published protocol. The enzyme is stable for months when stored in buffered 10% glycerol at -70 degrees C. This purification technique is compared to the immuno-affinity technique which is widely used for this enzyme. Enzyme kinetics were characterized with respect to substrate specificity, the effect of temperature, ionic strength, pH, and Mg+2 versus Mn+2 ions. Similar to the enzyme expressed in human cells, the recombinant enzyme demonstrated a higher Vmax and substrate specificity for poly E4Y over 5V-Agt-II. An activation energy of 14.2 kcal/mol was determined. Inhibition by increasing ionic strength is mostly due to an increase in Km for the poly E4Y substrate and hence was substrate dependent. The Km(ATP) was pH dependent while the Km(poly E4Y) was pH independent. For the phosphorylation of poly E4Y, free Mg+2 was stimulatory while Mn+2 was inhibitory. In contrast, Mn+2 stimulated the phosphorylation of 5V-Agt-II.

Targeting Gelatinases with a Near-Infrared Fluorescent Cyclic His-Try-Gly-Phe Peptide

PurposeThe purpose of the study is to synthesize and characterize near-infrared (NIR) fluorescence imaging probes targeted to gelatinases.ProceduresA phage display-selected cyclic peptide containing the His-Try-Gly-Phe (HWGF) motif was used as the lead compound. Structure–activity relationship analysis was used to identify stable and potent gelatinase inhibitors suitable for NIR imaging applications.ResultsReplacing the S–S bond in cyclic peptide c(CTTHWGFTLC)NH2 (C1) with an amide bond between the ε-amino group of Lys and the side chain of Asp resulted in a significant increase in stability and a fourfold increase in gelatinase inhibition of the resulting peptide, c(KAHWGFTLD)NH2 (C6). Conjugation of Cy5.5 to C6 led to Cy5.5–C6, which was selectively taken up by MMP-2 expressing human glioma U87 cells. In vivo, selective accumulation of Cy5.5–C6, but not Cy5.5–C1 or a Cy5.5-scrambled peptide conjugate, was visualized in intratibial prostate PC-3 tumors 48xa0h after their intravenous injection. Moreover, Cy5.5–C6 was readily visualized in orthotopically inoculated U87 brain tumors.ConclusionsCy5.5–C6 may be a useful agent for molecular imaging of gelatinases. The approach of producing stable cyclic peptides through side chain amide linkage should be applicable to other peptide-based imaging agents.

Use of synthetic peptides and copolymers to study the substrate specificity and inhibition of the protein tyrosine kinase pp60c-src

The ability of synthetic peptides and polypeptides to act as substrates and/or inhibitors of pp60c-src was examined. The random copolymer, poly(K4Y) had a threefold lower specificity than poly(E4Y). Peptides containing lysine vs. glutamate were also found to have a lower substrate specificity (Vmax:Km ratio). In order to assess the substrate specificity of acidic peptides, an assay protocol using DEAE-membranes was developed. Peptides containing a (YXE)5YXD motif (X = G, A, V, P, or norvaline) were tested as inhibitors and substrates of pp60c-src. The glycine-containing peptide was the best substrate having a specificity 16,000-fold higher than 5Val-angiotensin II, the most commonly used peptide substrate. Most of the peptides, except for the proline containing peptide, had Ki values of 20-100 microM. In a series of (XGE)5XGD peptides, where X = Y or F, tyrosine at position 10 was found to be the preferred site for accepting a phosphate. Analogs in which the glycine was replaced with alanine indicated that loss of flexibility around position 10 was detrimental to substrate specificity. Results suggest that conformational requirements of the peptides tested was important and substrate specificity was a more sensitive parameter than binding as measured by Ki values.

In vivo fluorescent optical imaging of cytotoxic T lymphocyte migration using IRDye800CW near-infrared dye.

We describe a method to measure in vivo migration of human T cells by using the near-infrared (NIR) dye IRDye800CW. Labeling of Epstein-Barr virus-specific T cells with IRDye800CW did not affect viability, proliferation, or T cell function. Following tail vein injection into mice bearing subcutaneous tumors, the NIR signal could be measured in vivo at the tumor site. Analysis of tumors revealed T cell infiltration and an increased NIR signal, confirming T cell migration. To test specific migration with IRDye800CW, tumors were modified to express CCL5 to measure site-specific migration. The NIR signal was increased at CCL5-secreting tumors compared with control tumors. Together, these data suggest that IRDye800CW may be used to study the trafficking of T cells in a small animal model and may have potential as a short-term reporter molecule for human immunotherapy studies.

Dual-modality imaging in vivo with an NIR and gamma emitter using an intensified CCD camera and a conventional gamma camera

Fluorescence-enhanced optical imaging measurements and conventional gamma camera images on human M21 melanoma xenografts were acquired for a dual-modality molecular imaging study. The avb3 integrin cell surface receptors were imaged using a cyclic peptide, cyclopentapeptide cyclo(lys-Arg-Gly-Asp-phe) [c(KRGDf)] probe which is known to target the membrane receptor. The probe, dual-labeled with a radiotracer, 111Indium, for gamma scintigraphy as well as with a near-infrared dye, IRDye800, was injected into six nude mice at a dose equivalent to 90mCi of 111In and 5 nanomoles of near-infrared (NIR) dye. A 15 min gamma scan and 800 millisecond NIR-sensitive ICCD optical photograph were collected 24 hours after injection of the dual-labeled probe. The image quality between the nuclear and optical data was investigated with the results showing similar target-to-background ratios (TBR) based on the origin of fluorescence and gamma emissions at the targeted tumor site. Furthermore, an analysis of SNR versus contrast showed greater sensitivity of optical over nuclear imaging for the subcutaneous tumor targets measured by surface regions of interest.

The influence of improved interference filter performance for molecular imaging using frequency domain photon migration measurements

Several phantom and in vivo small animal imaging studies have been performed to detect the re-emitted fluorescence signal arising from micro to pico molar concentrations of fluorophore by employing band-pass and band-rejection filters. However, elimination of the back-reflected excitation light still remains a major challenge for further reducing the noise floor in fluorescence imaging. Furthermore, despite the well-known deterioration of interference filter performance as the angle of incidence deviates from zero degrees, most studies do not employ collimated light optical design required for efficient excitation light rejection using interference filters. In this study, we measured quantities in frequency domain data for the combination of three-cavity interference and holographic super notch filters. To assess excitation leakage, the “out-of-band (S (λx ) )” to “in-band (S (λm ) - S (λx ) )” signal ratio, AC amplitude (IAC ), and phase delay (δ-δ*) measured from a gain modulated, intensified CCD imaging system with and without collimating optics was evaluated. The addition of collimating optics resulted in a reduction of 82% to 91% of the out-of-band to in-band ratio for the phantom studies and an increase of 1.4 to 3.7 times of the target-to-background ratio (T:B) for small animal studies.

In vivo pharmacokinetic analysis for fluorescently labeled RGD peptide targeted to the α vβ 3 integrin in Kaposi's sarcoma

The dose dependence of near-infrared (NIR) fluorescent labeled RGD peptide targeted to the αvβ3 integrin was assessed from xenografts bearing a subcutaneous human Kaposi’s sarcoma (KS1767) with dynamic NIR fluorescence optical imaging. The three-compartment pharmacokinetic (PK) model was used to determine PK parameters from fluorescence images acquired with an intensified charge-coupled device (ICCD) system. Dynamic imaging of Kaposi’s sarcoma bearing animals was conducted with i.v. administration of Cy5.5-c(KRGDf) at doses of 0.75 to 6 nmol/animal and at the doses of 300 or 600 nmol of c(KRGDf) administered 1 hour before the injection of 3 nmol dose of the conjugate. The results show early and rapid uptake of Cy5.5-c(KRGDf), which was mediated by the administration of c(KRGDf) 1 hour before administration at the conjugate agent. From the results we found a linear increase in PK uptake rates at doses of 0.75 to 1.5 nmol, reflecting unsaturated binding to the integrin receptor. However, the results show the dose independence at large dose amounts from 3 to 6 nmol per animal. The effects of cancer treatments as well as diagnostics may be evaluated by in vivo PK analysis with NIR fluorescence optical imaging.

Dynamic fluorescence imaging for evaluating molecular targets of disease

Dynamic fluorescent intensity measurements of targeting exogenous dyes within small animal models of disease can be used in conjunction with a pharmacokinetic model to evaluate the expression of molecular markers of disease.