It was recently shown that the human atherosclerotic plaque contains significant amounts of T lymphocytes, and also that smooth muscle cells in these plaques express class II MHC (Ia) antigens. These antigens are not normally present on smooth muscle cells but are inducible by interferon-gamma, a secretory product of activated T cells. Therefore, T cell activation in the plaque was analyzed by immunofluorescent detection of activation markers on T cells isolated from the plaques and in cryostat sections of carotid endarterectomy specimens. Of cells isolated from the plaque, 5% exhibited the E rosettes characteristic of T cells. One third of these cells expressed HLA-DR and VLA-1 (very late activation antigen-1), which in T cells are synthesized only in the activated state. T cells were also identified in sections using immunofluorescent detection of the T cell-specific surface protein, CD3 (Leu-4), with rhodamine labeled second-step antibodies. The frequency of activated T cells was then determined by staining the same, or serial, sections with antibodies to HLA-DR or to the interleukin-2 receptor, followed by biotin-avidin-FITC detection. Of the T cells in the plaque, 34% and 6%, respectively, expressed these cell surface proteins. Taken together, these results indicated that a substantial proportion of the T cells in atherosclerotic plaque are in an activated state. The activation pattern, with a high frequency of HLA-DR and VLA-1 expression and a much lower frequency of interleukin-2 receptor expression, was similar to that reported to occur in chronic inflammatory conditions. Interferon-gamma could be detected in and around some of the lymphocytes, suggesting that paracrine secretion of this lymphokine may occur in the plaque. T cells may be activated locally, presumably by antigen(s) presented in the context of class II MHC expressing smooth muscle cells and/or macrophages, in the atherosclerotic lesion. Such activated T cells may in turn modulate the functions of other cells in the plaque.

Nitric oxide has been shown to be an important signaling messenger in ischemic preconditioning (IPC). Accordingly, we investigated whether protein S-nitrosylation occurs in IPC hearts and whether S-nitrosoglutathione (GSNO) elicits similar effects on S-nitrosylation and cardioprotection. Preceding 20 minutes of no-flow ischemia and reperfusion, hearts from C57BL/6J mice were perfused in the Langendorff mode and subjected to the following conditions: (1) control perfusion; (2) IPC; or (3) 0.1 mmol/L GSNO treatment. Compared with control, IPC and GSNO significantly improved postischemic recovery of left ventricular developed pressure and reduced infarct size. IPC and GSNO both significantly increased S-nitrosothiol contents and S-nitrosylation levels of the L-type Ca2+ channel alpha1 subunit in heart membrane fractions. We identified several candidate S-nitrosylated proteins by proteomic analysis following the biotin switch method, including the cardiac sarcoplasmic reticulum Ca2+-ATPase, alpha-ketoglutarate dehydrogenase, and the mitochondrial F1-ATPase alpha1 subunit. The activities of these enzymes were altered in a concentration-dependent manner by GSNO treatment. We further developed a 2D DyLight fluorescence difference gel electrophoresis proteomic method that used DyLight fluors and a modified biotin switch method to identify S-nitrosylated proteins. IPC and GSNO produced a similar pattern of S-nitrosylation modification and cardiac protection against ischemia/reperfusion injury, suggesting that protein S-nitrosylation may play an important cardioprotective role in heart.

A method of in situ cytohybridization is described for the detection of specific viral genomes in infected cell cultures or paraffin-embedded tissue sections without the use of radioisotopes. Biotin-labeled analogs of TTP are incorporated into viral DNA in vitro by nick translation and the resultant DNA probes hybridized to cytologic samples. Cells containing viral genetic material are then revealed by standard immunofluorescence, immunoperoxidase, or affinity cytochemical techniques that are based on the specific interaction between biotin and antibiotin IgG or avidin. Hybridization probes containing nucleotides that have an 11- or 16-atom spacer arm between the biotin molecule and the pyrimidine ring interact with these detector proteins more efficiently than probes containing biotin-nucleotides with a 4-atom spacer arm. The total procedure can be performed fairly rapidly (24 hr or less) and numerous samples can be processed simultaneously. Although the detection methods employed to date are not as sensitive as autoradiographic procedures with high specific activity probes, more sensitive protein detector complexes are currently being constructed. The speed, specificity, and resolving power of this technique should be of general utility in screening for the presence of infectious agents in cell or tissue samples. Here we report the visualization of parvovirus, polyomavirus, herpes simplex virus, adenovirus, and retrovirus genetic material in infected cell cultures and herpes simplex and adenovirus DNA in paraffin-embedded autopsy tissues.

Despite the well-established tropism of the Epstein-Barr virus (EBV) for human B lymphocytes, the cell type within the oropharynx capable of allowing EBV replication has never been conclusively identified. Using in situ cytohybridization, we demonstrated EBV DNA in oropharyngeal epithelial cells from 10 of 12 patients with infectious mononucleosis. In duplicates of specimens found to contain cell-associated EBV DNA, we detected EBV RNA in two of four samples, using a biotin-labeled EBV DNA probe, thereby confirming the intracellular location of the viral genome. In 20 of 28 throat washings analyzed, cytohybridization results and assays for cell-free infectious virus were in agreement. In seven of the eight remaining specimens, cytohybridization identified intracellular EBV DNA in the absence of detectable extracellular virus. We conclude that the oropharyngeal epithelial cell may be the target cell type that is productively infected in infectious mononucleosis.

To gain insight into the regulation of expression of peroxisome proliferator-activated receptor (PPAR) isoforms, we have determined the structural organization of the mouse PPAR gamma (mPPAR gamma) gene. This gene extends>> 105 kb and gives rise to two mRNAs (mPPAR gamma 1 and mPPAR gamma 2) that differ at their 5' ends. The mPPAR gamma 2 cDNA encodes an additional 30 amino acids N-terminal to the first ATG codon of mPPAR gamma 1 and reveals a different 5' untranslated sequence. We show that mPPAR gamma 1 mRNA is encoded by eight exons, whereas the mPPAR gamma 2 mRNA is encoded by seven exons. Most of the 5' untranslated sequence of mPPAR gamma 1 mRNA is encoded by two exons, whereas the 5' untranslated sequence and the extra 30 N-terminal amino acids of mPPAR gamma 2 are encoded by one exon, which is located between the second and third exons coding for mPPAR gamma 1. The last six exons of mPPAR gamma gene code for identical sequences in mPPAR gamma 1 and mPPAR gamma 2 isoforms. The mPPAR gamma 1 and mPPAR gamma 2 isoforms are transcribed from different promoters. The mPPAR gamma gene has been mapped to chromosome 6 E3-F1 by in situ hybridization using a biotin-labeled probe. These results establish that at least one of the PPAR genes yields more than one protein product, similar to that encountered with retinoid X receptor and retinoic acid receptor genes. The existence of multiple PPAR isoforms transcribed from different promoters could increase the diversity of ligand and tissue-specific transcriptional responses.

BACKGROUND

Selective protein import into the cell nucleus occurs in two steps: binding to the nuclear envelope, followed by energy-dependent transit through the nuclear pore complex. A 60 kD protein, importin, is essential for the first nuclear import step, and the small G protein Ran/TC4 is essential for the second. We have previously purified the 60kD importin protein (importin 60) as a single polypeptide.

RESULTS

We have identified importin 90, a 90 kD second subunit that dissociates from importin 60 during affinity chromatography on nickel (II)-nitrolotriacetic acid-Sepharose, a technique that was originally used to purify importin 60. Partial amino-acid sequencing of Xenopus importin 90 allowed us to clone and sequence its human homologue; the amino-acid sequence of importin 90 is strikingly conserved between the two species. We have also identified a homologous budding yeast sequence from a database entry. Importin 90 potentiates the effects of importin 60 on nuclear protein import, indicating that the importin complex is the physiological unit responsible for import. To assess whether nuclear localization sequences are recognized by cytosolic receptor proteins, a biotin-tagged conjugate of nuclear localization signals linked to bovine serum albumin was allowed to form complexes with cytosolic proteins in Xenopus egg extracts; the complexes were then retrieved with streptavidin-agarose. The pattern of bound proteins was surprisingly simple and showed only two predominant bands: those of the importin complex. We also expressed the human homologue of importin 60, Rch1p, and found that it was able to replace its Xenopus counterpart in a functional assay. We discuss the relationship of importin 60 and importin 90 to other nuclear import factors.

CONCLUSIONS

Importin consists of a 60 and a 90 kD subunit. Together, they constitute a cytosolic receptor for nuclear localization signals that enables import substrates to bind to the nuclear envelope.

The recognition mechanisms and dissociation pathways of the avidin-biotin complex and of actin monomers in actin filaments were investigated. The unbinding forces of discrete complexes of avidin or streptavidin with biotin analogs are proportional to the enthalpy change of the complex formation but independent of changes in the free energy. This result indicates that the unbinding process is adiabatic and that entropic changes occur after unbinding. On the basis of the measured forces and binding energies, an effective rupture length of 9.5 +/- 1 angstroms was calculated for all biotin-avidin pairs and approximately 1 to 3 angstroms for the actin monomer-monomer interaction. A model for the correlation among binding forces, intermolecular potential, and molecular function is proposed.

Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional factor. Under normal oxygen tension, HIF-1 activity is usually suppressed due to the rapid, oxygen-dependent degradation of one of its two subunits, HIF-1alpha. Here we report that normoxic HIF-1 activity can be upregulated through NO-mediated S-nitrosylation and stabilization of HIF-1alpha. In murine tumors, exposure to ionizing radiation stimulated the generation of NO in tumor-associated macrophages. As a result, the HIF-1alpha protein is S-nitrosylated at Cys533 (through "biotin switch" assay) in the oxygen-dependent degradation domain, which prevents its destruction. Importantly, this mechanism appears to be independent of the prolylhydroxylase-based pathway that is involved in oxygen-dependent regulation of HIF-1alpha. Selective disruption of this S-nitrosylation significantly attenuated both radiation-induced and macrophage-induced activation of HIF-1alpha. This interaction between NO and HIF-1 sheds new light on their involvement in tumor response to treatment as well as mammalian inflammation process in general.

We explore the question of what are the best ligands for macromolecular targets. A survey of experimental data on a large number of the strongest-binding ligands indicates that the free energy of binding increases with the number of nonhydrogen atoms with an initial slope of approximately -1.5 kcal/mol (1 cal = 4.18 J) per atom. For ligands that contain more than 15 nonhydrogen atoms, the free energy of binding increases very little with relative molecular mass. This nonlinearity is largely ascribed to nonthermodynamic factors. An analysis of the dominant interactions suggests that van der Waals interactions and hydrophobic effects provide a reasonable basis for understanding binding affinities across the entire set of ligands. Interesting outliers that bind unusually strongly on a per atom basis include metal ions, covalently attached ligands, and a few well known complexes such as biotin-avidin.

Although nitric oxide (NO) has grown into a key signaling molecule in plants during the last few years, less is known about how NO regulates different events in plants. Analyses of NO-dependent processes in animal systems have demonstrated protein S-nitrosylation of cysteine (Cys) residues to be one of the dominant regulation mechanisms for many animal proteins. For plants, the principle of S-nitrosylation remained to be elucidated. We generated S-nitrosothiols by treating extracts from Arabidopsis (Arabidopsis thaliana) cell suspension cultures with the NO-donor S-nitrosoglutathione. Furthermore, Arabidopsis plants were treated with gaseous NO to analyze whether S-nitrosylation can occur in the specific redox environment of a plant cell in vivo. S-Nitrosylated proteins were detected by a biotin switch method, converting S-nitrosylated Cys to biotinylated Cys. Biotin-labeled proteins were purified and analyzed using nano liquid chromatography in combination with mass spectrometry. We identified 63 proteins from cell cultures and 52 proteins from leaves that represent candidates for S-nitrosylation, including stress-related, redox-related, signaling/regulating, cytoskeleton, and metabolic proteins. Strikingly, many of these proteins have been identified previously as targets of S-nitrosylation in animals. At the enzymatic level, a case study demonstrated NO-dependent reversible inhibition of plant glyceraldehyde-3-phosphate dehydrogenase, suggesting that this enzyme could be affected by S-nitrosylation. The results of this work are the starting point for further investigation to get insight into signaling pathways and other cellular processes regulated by protein S-nitrosylation in plants.

An unmet need in cell engineering is the availability of a single transgene encoded, functionally inert, human polypeptide that can serve multiple purposes, including ex vivo cell selection, in vivo cell tracking, and as a target for in vivo cell ablation. Here we describe a truncated human EGFR polypeptide (huEGFRt) that is devoid of extracellular N-terminal ligand binding domains and intracellular receptor tyrosine kinase activity but retains the native amino acid sequence, type I transmembrane cell surface localization, and a conformationally intact binding epitope for pharmaceutical-grade anti-EGFR monoclonal antibody, cetuximab (Erbitux). After lentiviral transduction of human T cells with vectors that coordinately express tumor-specific chimeric antigen receptors and huEGFRt, we show that huEGFRt serves as a highly efficient selection epitope for chimeric antigen receptor(+) T cells using biotinylated cetuximab in conjunction with current good manufacturing practices (cGMP)-grade anti-biotin immunomagnetic microbeads. Moreover, huEGFRt provides a cell surface marker for in vivo tracking of adoptively transferred T cells using both flow cytometry and immunohistochemistry, and a target for cetuximab-mediated antibody-dependent cellular cytotoxicity and in vivo elimination. The versatility of huEGFRt and the availability of pharmaceutical-grade reagents for its clinical application denote huEGFRt as a significant new tool for cellular engineering.

I describe a technique for screening peptide libraries of over 10(9) independent clones for substrates of peptide-modifying enzymes. The peptides, linked to their genetic material by the lac repressor, are exposed to the enzyme and then screened by affinity purification on a receptor specific for the modified product. The enzyme characterized, E. coli biotin holoenzyme synthetase, normally adds biotin to a specific lysine residue in complex protein domains. The 13 residue substrate identified by this library screening approach is much smaller than the 75 amino acid required sequence of the natural substrate, and can function at either end of a fusion protein. The sequence is quite distinct at some positions from that region of the natural substrates, presumably because the peptides have to mimic the folded structure formed by the natural substrate. This technique should be useful for mapping the substrate specificity of a variety of peptide-modifying enzymes. In addition, small peptide substrates that are enzymatically biotinylated at a single site should be useful for a variety of purposes in labeling, purification, detection, and immobilization of proteins.

To investigate the behavioral mechanism of chemotaxis in Caenorhabditis elegans, we recorded the instantaneous position, speed, and turning rate of single worms as a function of time during chemotaxis in gradients of the attractants ammonium chloride or biotin. Analysis of turning rate showed that each worm track could be divided into periods of smooth swimming (runs) and periods of frequent turning (pirouettes). The initiation of pirouettes was correlated with the rate of change of concentration (dC/dt) but not with absolute concentration. Pirouettes were most likely to occur when a worm was heading down the gradient (dC/dt < 0) and least likely to occur when a worm was heading up the gradient (dC/dt>> 0). Further analysis revealed that the average direction of movement after a pirouette was up the gradient. These observations suggest that chemotaxis is produced by a series of pirouettes that reorient the animal to the gradient. We tested this idea by imposing the correlation between pirouettes and dC/dt on a stochastic point model of worm motion. The model exhibited chemotaxis behavior in a radial gradient and also in a novel planar gradient. Thus, the pirouette model of C. elegans chemotaxis is sufficient and general.

The present study investigates the induction of neurogenesis, reduction of apoptosis, and promotion of basic fibroblast growth factor (bFGF) expression as possible mechanisms by which treatment of stroke with bone marrow stromal cells (MSCs) improves neurological functional recovery. Additionally, for the first time, we treated cerebral ischemia in female rats with intraveneous administration of MSCs. Female rats were subjected to 2 hr of middle cerebral artery occlusion (MCAo), followed by an injection of 3 x 10(6) male (for Y chromosome labeling) rat MSCs or phosphate-buffered saline (PBS) into the tail vein 24 hr after MCAo. All animals received daily injection of bromodeoxyuridine (BrdU; 50 mg/kg, i.p.) for 13 days after treatment for identification of newly synthesized DNA. Animals were sacrificed at 14 days after MCAo. Behavioral tests (rotarod and adhesive-removal tests) were performed. In situ hybridization, immunohistochemistry, and terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) were performed to identify transplanted MSCs (Y chromosome), BrdU, bFGF, and apoptotic cells in the brain. Significant recovery of behavior was found in MSC-treated rats at 7 days in the somatosensory test and at 14 days in the motor test after MCAo compared with control, PBS-treated animals (P<.05). MSCs were found to survive and preferentially localize to the ipsilateral ischemic hemisphere. Significantly more BrdU-positive cells were located in the subventricular zone (P<.05), and significantly fewer apoptotic cells and more bFGF immunoreactive cell were found in the ischemic boundary area (P<.05) of MSC-treated rats than in PBS-treated animals. Here we demonstrate that intravenously administered male MSCs increase bFGF expression, reduce apoptosis, promote endogenous cellular proliferation, and improve functional recovery after stroke in female rats.

Nascent transcripts in permeabilized HeLa cells were elongated by approximately 30-2,000 nucleotides in Br-UTP or biotin-14-CTP, before incorporation sites were immunolabelled either pre- or post-embedding, and visualized by light or electron microscopy. Analogues were concentrated in approximately 2,100 (range 2,000-2,700) discrete sites attached to a nucleoskeleton and surrounded by chromatin. A typical site contained a cluster (diameter 71 nm) of at least 4, and probably about 20, engaged polymerases, plus associated transcripts that partially overlapped a zone of RNA polymerase II, ribonucleoproteins, and proteins rich in thiols and acidic groups. As each site probably contains many transcription units, these results suggest that active polymerases are confined to these sites, which we call transcription 'factories'. Results are consistent with transcription occurring as templates slide past attached polymerases, as nascent RNA is extruded into the factories.

Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant K(d) = 48 fM and slower dissociation kinetics (half-time>> 5 days) than those for the streptavidin-biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 10(5)-10(7) yeast surface-displayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wild-type complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.

Escherichia coli biotin ligase site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (AP) sequence. We show that mammalian cell surface proteins tagged with AP can be biotinylated by biotin ligase added to the medium, while endogenous proteins remain unmodified. The biotin group then serves as a handle for targeting streptavidin-conjugated quantum dots (QDs). This labeling method helps to address the two major deficiencies of antibody-based labeling, which is currently the most common method for targeting QDs to cells: the size of the QD conjugate after antibody attachment and the instability of many antibody-antigen interactions. To demonstrate the versatility of our method, we targeted QDs to cell surface cyan fluorescent protein and epidermal growth factor receptor in HeLa cells and to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in neurons. Labeling requires only 2 min, is extremely specific for the AP-tagged protein, and is highly sensitive. We performed time-lapse imaging of single QDs bound to AMPA receptors in neurons, and we compared the trafficking of different AMPA receptor subunits by using two-color pulse-chase labeling.

Strategies for cardiac repair include injection of cells, but these approaches have been hampered by poor cell engraftment, survival, and differentiation. To address these shortcomings for the purpose of improving cardiac function after injury, we designed self-assembling peptide nanofibers for prolonged delivery of insulin-like growth factor 1 (IGF-1), a cardiomyocyte growth and differentiation factor, to the myocardium, using a "biotin sandwich" approach. Biotinylated IGF-1 was complexed with tetravalent streptavidin and then bound to biotinylated self-assembling peptides. This biotin sandwich strategy allowed binding of IGF-1 but did not prevent self-assembly of the peptides into nanofibers within the myocardium. IGF-1 that was bound to peptide nanofibers activated Akt, decreased activation of caspase-3, and increased expression of cardiac troponin I in cardiomyocytes. After injection into rat myocardium, biotinylated nanofibers provided sustained IGF-1 delivery for 28 days, and targeted delivery of IGF-1 in vivo increased activation of Akt in the myocardium. When combined with transplanted cardiomyocytes, IGF-1 delivery by biotinylated nanofibers decreased caspase-3 cleavage by 28% and increased the myocyte cross-sectional area by 25% compared with cells embedded within nanofibers alone or with untethered IGF-1. Finally, cell therapy with IGF-1 delivery by biotinylated nanofibers improved systolic function after experimental myocardial infarction, demonstrating how engineering the local cellular microenvironment can improve cell therapy.

This proteomic protocol purifies and identifies palmitoylated proteins (i.e., S-acylated proteins) from complex protein extracts. The method relies on an acyl-biotinyl exchange chemistry in which biotin moieties are substituted for the thioester-linked protein acyl-modifications through a sequence of three in vitro chemical steps: (i) blockade of free thiols with N-ethylmaleimide; (ii) cleavage of the Cys-palmitoyl thioester linkages with hydroxylamine; and (iii) labeling of thiols, newly exposed by the hydroxylamine, with biotin-HPDP (Biotin-HPDP-N-[6-(Biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide. The biotinylated proteins are then affinity-purified using streptavidin-agarose and identified by multi-dimensional protein identification technology (MuDPIT), a high-throughput, tandem mass spectrometry (MS/MS)-based proteomic technology. MuDPIT also affords a semi-quantitative analysis that may be used to assess the gross changes induced to the global palmitoylation profile by mutation or drugs. Typically, 2-3 weeks are required for this analysis.

The purpose of this study was to design a method by which immunoperoxidase staining can be applied to formalin-fixed, paraffin-embedded tissue sections to demonstrate amyloid deposits in cerebral and systemic amyloidotic tissues. We used anti-prion protein, anti-beta-protein, anti-amyloid A, and anti-prealbumin antisera. The tissue sections were first treated with 100% formic acid for 5, 20, or 60 minutes and the unlabeled immunoperoxidase method (biotin-streptavidin system reagents) was used. This formic acid pretreatment enhanced immunoreactivity of the amyloid deposits which reacted positively with specific antiserum. The specificity of the immunostainings was well preserved. This method can also be used to demonstrate interspecies cross-reactivity, by using anti-human amyloid A and anti-scrapie hamster prion protein antisera, which stained negatively or faintly with amyloid deposits of heterogenous species. The technique is expected to reveal the buried epitopes of amyloid deposits in tissue sections.

Three metallic ions, NiCl2, CoCl2, and CuSO4, were found to modify the color of the normally brown diaminobenzidine (DAB) reaction. The colors ranged from purplish blue (NiCl2), dark blue/bluish black (CoCl2), to greyish blue (CuSO4). We have found that the CoCl2 + DAB is the ion of choice because: 1) it yields a distinct dark blue color that is easily distinguishable from brown DAB; 2) the blue reaction product is very stable throughout the entire staining procedure; and 3) background staining is minimal. These findings can be applied to the double staining technique of two different antigens in the same section. Among three staining procedures discussed, the avidin-biotin peroxidase complex (Co-DAB)-peroxidase-antiperoxidase (DAB) technique produced the best results because: 1) no antibody elution was needed following the avidin-biotin-peroxidase complex procedure when the CoCl2-DAB modification was used; and 2) no background staining occurred.

We have microinjected biotinylated tubulin into mitotic fibroblast cells to identify the sites in the spindle at which new subunits are incorporated into microtubules (MTs). Labeled subunits were visualized in the electron microscope using an antibody to biotin followed by a secondary antibody coupled to colloidal gold. Astral MTs incorporate labeled subunits very rapidly by elongation of existing MTs and by new nucleation from the centrosome. At a slower rate, kinetochore MTs incorporate subunits at the kinetochore progressively during metaphase, suggesting a slow poleward flux of subunits in the kinetochore fiber. When cells injected in metaphase were examined in anaphase, a significant fraction of kinetochore MTs was unlabeled, suggesting that depolymerization had occurred at the kinetochore concomitant with chromosome to pole movement. The existence of opposite fluxes at the kinetochore during metaphase and anaphase suggests that two separate forces are responsible for chromosome congression and anaphase movement.

Libraries of random peptide sequences were constructed and screened to identify peptides that specifically bind to proteins. In one of these about 2 X 10(7) different 15-residue peptide sequences were expressed on the surface of the coliphage M13. Each phage encoded a single random sequence and expressed it as a fusion complex with pIII, a minor coat protein present at five molecules per phage. Phage encoding nine different streptavidin-binding peptide sequences were isolated from this library. The core consensus sequence was His-Pro-Gln and binding of these phage to streptavidin was inhibited by biotin. This type of library makes it possible to identify peptides that bind to proteins (or other macromolecules) that have no previously known affinity for peptides.

We examined the expression, activation, and cellular localization of caspase-3 (CPP32) using immunohistochemistry, immunoblots, and cleavage of the fluorogenic substrate N-benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (zDEVD-afc) in adult mouse brain after temporary (2 hr) middle cerebral artery occlusion produced by filament insertion into the carotid artery. Immunoreactive caspase-3p32 but not its cleavage product caspase-3p20 was constitutively expressed in neurons throughout brain and was most prominent in neuronal perikarya within piriform cortex. Caspase-like enzyme activity was elevated in brain homogenate 0-3 hr after reperfusion and reached a peak within 30 to 60 min. Caspase-3p20 immunoreactivity became prominent in neuronal perikarya within the middle cerebral artery territory at the time of reperfusion and on immunoblots 1-12 hr later. DNA laddering (agarose gels) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL)-stained cells were detected 6-24 hr after reperfusion. At 12-24 hr, immunoreactive p20 was visualized in TUNEL-positive cells, a finding also observed in apoptotic mouse cerebellar granule cells on postnatal day 5. Together, these observations suggest the existence of a time-dependent evolution of ischemic injury characterized by the close correspondence between caspase-like enzyme activation and an associated increase in immunoreactive product (caspase-3p20) beginning at or before reperfusion and followed several hours later by morphological and biochemical features of apoptosis.