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Protamine, a mixture of positively charged proteins from salmon roe used in diverse pharmaceutical applications, was reacted with the N-hydroxysuccinimide ester of tetramethylrhodamine to yield tetramethylrhodamine-labeled protamines (Pro(Rh)) containing one mole of fluorochrome per mole of protein. The internalization of tetramethylrhodamine-labeled protamine (Pro(Rh)) and the fluorescein-labeled tat peptide (Tat(Fl)) showed a similar dependence on time and concentration. Pro(Rh) and Tat(Fl) showed strong nuclear localizations, evident with both live cells and fixed cells costained with DAPI, a nuclear stain. The loss of fluorescence when cells were loaded with Pro(Rh) or Tat(Fl) was similar, further supporting a strong similarity between these two materials. Finally, when Pro(Ph) was covalently attached to the amino-CLIO nanoparticle, the cellular uptake of the nanoparticle was greatly enhanced. All experiments were performed with HeLa and CaCo-2 cells with similar results. These observations imply that protamine, a protein in regular clinical use, might be used for the design of novel membrane translocating/nuclear localizing pharmaceuticals whose development was initiated with other membrane-translocating peptides. In addition, the fluorescent protamines developed here might be used to further our understanding of this important pharmaceutical.

The key role played by PI3 kinase in cancer, hormone action, and a host of other biological functions suggests that specific inhibitors whose disposition could be ascertained in vivo would be useful in biological research or, potentially, for imaging PI3K in a clinical setting. Wortmannin (Wm, 1) is an inhibitor of PI3 kinase with high specificity for this enzyme. We synthesized three modified Wm probes, a biotinylated Wm (7a), a 4-hydroxy-3-iodophenylated Wm, which was obtained both unlabeled (7b) and labeled with ¹²⁵I (8), and a fluoresceinated Wm (7c), through modification at C-11, and evaluated their inhibitive activity as inhibitors of PI3 kinase. Biotinylated (7a) and 4-hydroxy-3-iodophenylated Wm's (7b) had IC50s for PI3K of 6.11 and 11.02 nM, respectively, compared to an IC50 for Wm of 1.63 nM. Fluoresceinated Wm (7c) lost considerably more activity than the other derivatives, with an IC50 of 64.9 nM. The ¹²⁵I labeled 4-hydroxy-3-iodophenylated Wm (8) could be detected after reaction with an immunoprecipitate of PI3 kinase. The activity of these reporter Wm's is discussed in relationship to earlier findings on the pharmacological activity of Wm derivatives and the ability of inhibitors to fit into the ATP pocket of PI3 kinase.

Multimodal proteins, or proteins labeled with both fluorescent and magnetic reporter groups, can be used in a wide range of applications including FACS or fluorescence microscopy, MRI and or near-infrared based optical imaging, or to fractionate cells by magnetic cell sorting. A problem with multimodal proteins, however, is the need to maximize bioactivity, often achieved by minimizing the number of modification points of the protein, while attaching fluorescent and magnetic labels. Here we describe the synthesis of a magneto/optical form of annexin V, achieved by reacting the amino-CLIO nanoparticle with Cy5.5 and SPDP, to produce a fluorescent, sulfhydryl reactive nanoparticle. A single reactive sulfhydryl group was added to annexin V by reaction with SATA that preserved the protein's ability to bind apoptotic Jurkat T cells. Reacting SATAylated annexin V with an SPDP activated nanoparticle yielded Anx-CLIO−Cy5.5, a magneto/optical form of annexin V. The binding of Anx-CLIO−Cy5.5 was specific for apoptotic Jurkat T cells and had an EC50 of 3.66 nM. This was comparable to the strength of the interaction of unmodified annexin V with apoptotic cells, measured as the displacement of FITC−annexin by annexin V (2.4 nM). Our conjugation strategy preserves the strength of the interaction between annexin V and apoptotic cells, while yielding a probe, Anx-CLIO−Cy5.5, that is readily detectable by standard MR imaging or NIRF optical methods.

Near-infrared fluorescence (NIRF) optical probes have been able to provide a noninvasive assessment of enzyme activity for a number of different enzymes and types of pathology. Here we describe a dual fluorochrome enzyme-activatable probe featuring one NIRF fluorochrome that is activated by protease activity and a second fluorochrome that is protease resistant and serves as an internal standard. The probe was prepared by attaching Cy7 directly to an amino-CLIO, an amine functional cross-linked iron oxide (CLIO) nanoparticle carrier, in a protease resistant manner. Cy5.5 was attached to a protease sensitive polyarginine peptide spacer, also attached to amino-CLIO. In vitro and in vivo the ratio of the Cy5.5 to Cy7 fluorescence was increased by protease, reflecting the increase in Cy5.5 fluorescence by protease in the vicinity of the probe. In vitro and in vivo the absolute values of the Cy5.5 and Cy7 fluorescence reflected lesion size and the distance of lesions from the surface, while the ratio of Cy5.5 to Cy7 fluorescence obtained was constant and independent of lesion size and depth. The dual fluorochrome probe, and related dual wavelength imaging method, represents a novel approach for imaging protease activity in vivo.

Surface modification of superparamagnetic contrast agents with HIV-1 tat peptide has emerged as a promising means for intracellular magnetic labeling and noninvasive tracking of a large number of cell types with MRI. To achieve efficient intracellular delivery of the nanoparticles, we investigated the effect on cellular uptake of superparamagnetic iron oxide particles by varying the number of attached tat peptides. First, we report here a modified P2T method in measuring the numbers of surface attachments per particle through disulfide linkage. The method was shown to have desirable simplicity and reproducibility. With the P2T method as a tool, conjugates with progressively higher ratios of peptide-to-particle were synthesized. We were able to demonstrate that higher numbers of tat peptide facilitate the cellular uptake of iron oxide nanoparticles in a nonlinear fashion. Cells labeled with these optimized preparations were readily detectable by MR imaging. The increase in sensitivity could allow in vivo tracking of 100-fold lower cell concentration than currently described.

A number of quantitative three-dimensional tomographic near-infrared fluorescence imaging techniques have recently been developed and combined with MR imaging to yield highly detailed anatomic and molecular information in living organisms (1, 2). Here we describe magnetic nanoparticle based MR contrast agents that have a near-infrared fluorescence (NIRF) that is activated by certain enzymes. The probes are prepared by conjugation of arginyl peptides to cross-linked iron oxide amine (amino-CLIO), either by a disulfide linkage or a thioether linker, followed by the attachment of the indocyanine dye Cy5.5. The NIRF of disulfide-linked conjugate was activated by DTT, while the NIRF of thioether-linked conjugate was activated by trypsin. Fluorescent quenching of the attached fluorochrome occurs in part due to the interaction with iron oxide, as evident by the activation of fluorescence with DTT when nanoparticles that have less than one dye attached per particle. With a SC injection of the probe, axillary and brachial lymph nodes were darkened on MR images and easily delineated by NIRF imaging. The probes may provide the basis for a new class of so-called smart nanoparticles, capable of pinpointing their position through their magnetic properties, while providing information on their environment by optical imaging techniques.

Covalent conjugates of the cross-linked iron oxide nanoparticles (CLIO) and high-affinity (Kd^app = 8.5 nM) anti-human E-selectin (CD62E) F(ab‘)2 fragments were prepared and tested in vitro to establish feasibility of endothelial proinflammatory marker magnetic resonance (MR) imaging. The conjugates were obtained by using thiol−disulfide exchange reaction between 3-(2-pyridyl)propionyl-CLIO and S-acetylthioacetate-modified F(ab‘)2 fragments. The purified CLIO−F(ab‘)2 conjugates (average hydrodynamic diameter 40.6 nm) were used in experiments with the live human endothelial umbilical vein cells (HUVEC). Cells treated with IL-1β expressed E-selectin and showed a 100−200 times higher binding of CLIO particles (83−104 ng iron/million cells) than control cells. The binding resulted in a high superparamagnetism of HUVEC with the transverse water proton relaxation time (T2) decrease to 30−40 ms in cell precipitates. Cells did not bind/internalize CLIO−F(ab‘)2 conjugates prepared using a control fragment or nonconjugated iron oxide particles before or after treatment with IL-1β. MR imaging of cells showed a highly specific T2-weighted signal darkening associated with cells treated with IL-1β and incubated with anti-E selectin. Demonstration of MR imaging of E-selectin expression justifies further development of MR-targeted agents for monitoring tumor vascular endothelial proliferation, angiogenesis, and atherosclerosis.

The ability to image specific molecular targets in vivo would have significant impact in allowing earlier disease detection and in tailoring molecular therapies. One of the rate-limiting steps in the development of novel compounds as reporter probes has been the lack of cell-based, biologically relevant, high throughput screening methods. Here we describe the development and validation of magnetic resonance imaging (MRI) as a technique to rapidly screen compounds that are potential MR reporter agents for their interaction with specific cellular targets. We show that MR imaging can (1) evaluate thousands of samples simultaneously and rapidly, (2) provide exceedingly accurate measurements, and (3) provide receptor binding/internalization data as validated by radioactive assays. The technique allows the screening of libraries of peptide−nanoparticle conjugates against target cells and the identification of conjugates that may be subsequently used as reporter agents in vivo. The technology should greatly accelerate the development of target-specific or cell-specific MR contrast agents.

Recently, it has been demonstrated that magnetic resonance imaging (MRI) utilizing monocrystalline iron oxide nanoparticles (MIONs) targeted to an engineered transferrin receptor enables imaging of gene expression. However, the relatively high doses of iron oxides used indicated the need for improved MR imaging probes to monitor changes in gene expression in vivo. Using alternative conjugation chemistries to link targeting ligands and iron oxide nanoparticles, we present the development and characterization as well as improved receptor binding and MRI detection of a novel imaging probe. Iron oxide nanoparticles with a cross-linked dextran coat were conjugated to transferrin (Tf) through the linker molecule N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) to yield Tf-S-S-CLIO. The characteristics of this conjugate were evaluated in comparison to Tf-MION and Tf-CLIO generated by oxidative activation of the dextran-coat with subsequent reduction of Schiff's base. SPDP conjugation allowed approximately a 4-fold increase in the number of Tf molecules attached per iron oxide nanoparticle and resulted in a more than 10-fold improvement of binding and uptake by cells. This translated into an imaging probe that was 16 times better for imaging gene expression in a cellular MRI assay. This novel probe for MRI may substantially increase the sensitivity for the detection of endogenous or genetically induced transferrin receptor expression in small numbers of cells and may significantly reduce the imaging dose from over 100 mg/kg to doses of iron oxides that are currently used in clinical imaging.

A biocompatible, dextran coated superparamagnetic iron oxide particle was derivatized with a peptide sequence from the HIV-tat protein to improve intracellular magnetic labeling of different target cells. The conjugate had a mean particle size of 41 nm and contained an average of 6.7 tat peptides. Derivatized particles were internalized into lymphocytes over 100-fold more efficiently than nonmodified particles, resulting in up to 12.7 × 10⁶ particles/cell. Internalized particles localized in cytoplasm and nuclear compartments as demonstrated by fluorescence microscopy and immunohistochemistry. Labeled cells were highly magnetic, were detectable by NMR imaging, and could be retained on magnetic separation columns. The described method has potential applications for in vivo tracking of magnetically labeled cells by MR imaging and for recovering intracellularly labeled cells from organs.

Considerable work has been reported on the influence of fertilizer elements, particularly potassium, on yields and lodging of corn (Zea maysL.). Additional information is needed on the influence of other nutrients and different genotypes on stalk quality characteristics and yield. This paper reports the influence of four levels of nitrogen, phosphorus, and potassium on the percent of senescent stalks, crushing strength, rind thickness, stalk lodging, and yield of the six F1hybrids of a four‐parent diallel of inbreds T101, T105, Till, and T115. The data were obtained using three independent experiments; one associated with each fertility element for 3 years. Nitrogen and phosphorus had little or no influence on stalk characteristics. Potassium additions reduced the percent of senescent stalks and stalk lodging and increased crushing strength and rind thickness. The influence of potassium on senescent stalks was dependent on the hybrid involved. Yield increased with additions of nitrogen and potassium, but phosphorus had little influence.

Selection for lower ear placement in corn (Zea maysL.) was done to obtain synthetics with a high plant to ear height ratio, to maintain maturities at or near the original materials, and to obtain additional information on whether selection for lower ear placement would reduce the number and length of internodes below the ear. An early and a late flowering synthetic was selected for lower ear placement by sib‐mating over a 10‐generation period; then the 10 cycles were compared in performance trials. Ear height was reduced from 67 to 41 cm in 10 cycles of selection in the early synthetic, or an average decrease of 4.3% per cycle. In the late synthetic, the reduction was from 69 to 51 cm, or an average decrease of 2.9% per cycle. Plant heights remained fairly constant in both synthetics. Plant to ear height ratios increased from 2.7 to 4.2 in the early synthetic and from 2.7 to 3.7 in the late synthetic. In both synthetics one less leaf was produced below the ear and one leaf added above the ear, while the total number of leaves remained fairly constant. Average internode lengths below the ear were reduced from 10.1 to 7.3 cm in the early synthetic and from 9.0 to 7.7 cm in the late synthetic after 10 generations. The effect of selection for lower ears was therefore shorter internodes and the ears being borne at a lower node. There was no change in number of days to silking in the early synthetic but there was a 2‐day delay in the late synthetic. Slightly higher grain moistures were obtained in the later cycles of both synthetics. Grain yield decreased in successive cycles of selection with correlations of 0.97 and .87 (both significant at the 0.01 level) between ear height and yield in the early and late synthetics, respectively. Stalk lodging was generally reduced as ear height was lowered but correlations were not significant. Uses of the synthetics in breeding programs are discussed.

The study examined the relationship of Aspergillus flavusLink ex Fr. in insects collected from developing ears and aflatoxin contamination of the kernels of corn (Zea maysL.). In 1976 two hybrids adapted for growth in the South and two hybrids adapted to the Corn Belt were grown at 11 diverse locations in the USA. Planting and harvest dates were staggered to examine maturity factors. Of 1621 insect larvae collected, A. flavuswas identified on 54 specimens (3.3%). The distribution of A. flavuson the insects was relatively uniform between test locations and across maturity groups and hybrids. However, common occurrence of aflatoxin was restricted to corn grown in the South, particularly in hybrids not adapted to the region. A detailed study of Georgia‐grown corn demonstrated that aflatoxin was not present in kernels 20 days post‐flowering, but the toxin was routinely found in samples collected 40 days after flowering. The results show a broad geographical occurrence of A. flavuson insects feeding on developing corn but a distinct interregional difference in the contamination of the grain by aflatoxin.

Three phenylalanine injections (1 mg/g body wt) at 4-hour intervals resulted in increases in phenylalanine and tyrosine levels which were age- (4–42 days) and tissue (plasma, brain, liver)-dependent. Incorporation of [1-14C]-acetate into lipid was constantly reduced by 20–40% in brain but not liver of 7-, 11-, 14- and 21-day-old phenylalanine-injected rats, while in adult brain incorporation was near normal. A similar distribution of label between lipid classes of differing brain regions in injected rats, compared to controls at all ages, indicates a lack of a selective effect on myelin lipid synthesis, suggesting that reduced myelination in hyperphenylalaninemia is not related to high phenylalanine levels per se.

Five highly resistant (R) and five highly susceptible (S) inbred lines of corn (Zea maysL.) were evaluated in a diallel set of 45 single crosses to study the inheritance of resistance to the corn virus disease complex. The entries were evaluated under conditions of mechanical inoculation at Knoxville and natural infection at Waverly, Tenn. Evaluations were made prior to flowering and again on fully grown plants. Highly significant differences for mean severity ratings among genotypes were obtained in all experiments. Correlations between ratings in the two locations and for both times of rating were highly significant for both the inbreds and the hybrids. R ✕ R crosses were highly resistant in all evaluations while S ✕ S crosses were highly susceptible. Mean ratings for R ✕ S crosses were significantly lower than the mean of R ✕ R and S ✕ S crosses and the mean of all crosses indicating partial dominance of resistance. General combining ability (gca) and specific combining ability (sea) mean squares were highly significant in all evaluations but gca mean squares were substantially larger than the sea mean squares. Inbred T232 had the largest negative gca effect with both times of rating, followed in order by Tx601, Ky226, Ga209, and Mo18W. By contrast, inbred CI872 had the largest positive gca effect on susceptibility while inbred T105 had the least at both times of rating. Vr, Wr graphs and components of variance analyses of the data provided similar results to those with combining ability analysis. Among the resistant inbreds, T232 indicated the most dominant genes for resistance when tested in both locations and with both times of rating while Mo18W indicated the least number. Inbreds Tx601, Ga209, and Ky226 varied in the two locations and time of rating. As many as four major genes for resistance were indicated to be present in the parental inbreds. Hybrids having high resistance are obtainable from crosses involving these resistant inbreds. Since some degree of dominance was indicated, a backcrossing procedure to transfer the resistance factors into elite lines is suggested. Some form of recurrent selection in populations having a degree of resistance also is suggested. Host reaction and the nature of the genetic variation governing resistance showed that the inbreds used in this study reacted in a similar manner to the virus complex under all conditions of testing. The data also indicate that evaluations for virus resistance can be made prior to flowering and that breeders can make pollinations concurrent with evaluation.

Twelve corn (Zea maysL.) hybrids, including one reciprocal, with differences in trypsin inhibitor levels and insect resistance were grown during 1978 at 12 locations in the United States. The study examined the effect of planting date, inoculation with Aspergillus flavusLink ex Fr., and mechanical damage of developing kernels on aflatoxin accumulation in kernels before harvest. Although a broad occurrence of toxin was found in kernels of untreated ears from locations outside the Corn Belt, addition of A. flavusspores to silks markedly increased toxin accumulation. To a lesser extent, mechanical damage was also associated with elevated aflatoxin concert. trations. At specific locations, toxin levels varied among hybrids, but no consistent interlocation pattern of absolute toxin resistance was detected. Visual insect damage on mature ears was not linked quantitatively to toxin levels. More than 90% of the Georgia, Mississippi, Missouri, North Carolina, South Carolina, and Tennessee samples had detectable aflatoxin, but less than 30% of the samples from Illinois, Iowa, and Ohio were toxin positive. In general, bright greenish‐yellow (BGY) fluores. cence in kernels and high levels of toxin were observed, but a number of samples with low levels of toxin did not exhibit the fluorescence.

Two six‐parent sets of crosses, each consisting of three resistant inbreds and three susceptible inbreds, were grown under heavy natural infections of maize dwarf mosaic virus (MDMV) in two locations in Tennessee in 1968. Highly significant differences among genotypes were obtained in all experiments for reaction to the virus. White inbred Ga209 and yellow inbred T232 were the most resistant both as inbreds and in F1crosses. Mean MDMV ratings for R ✕ R, R ― S, and S ✕ S crosses were 1.09, 1.78, and 3.41, respectively. The data showed that resistance to MDMV was largely dominant and was controlled by only a few major genes. Indications were that minor genes also were necessary for a high degree of resistance. General combining ability effects for reaction to MDMV were highly significant and were more important than specific effects. Significant specific effects were associated with crosses involving highly susceptible inbreds. Hybrids having high tolerance are obtainable from crosses involving these resistant inbreds. The data indicate that a backcrossing procedure can be used to transfer the resistance factors into elite lines. Alternating generations of backcrossing with selfing or sib‐pollination of resistant plants will facilitate incorporation of any minor genes necessary for a high degree of resistance. Some form of recurrent selection in populations having a degree of resistance also is suggested.