Muscarinic ACh receptors mediate complex and clinically important effects in the striatum. To better understand the roles of the different muscarinic receptor subtypes (m1-m4), we have determined the cellular and subcellular distribution of the m1-m4 receptor proteins in the rat neostriatum using subtype-specific antibodies and avidin-biotin-peroxidase immunocytochemistry for light and electron microscopy. m1 receptor protein is expressed in 78% of neurons and is enriched in spiny dendrites and at postsynaptic densities. A small number of m1-immunoreactive axon terminals were observed, all forming asymmetrical synapses. About 2.5% of striatal neurons express m2 receptor protein with reaction product evident, by light microscopy in scattered large oval neurons with enfolded nuclei and long aspiny dendrites. By electron microscopy, m2 immunocytochemistry labeled somata, aspiny dendrites, and many axon terminals. Most axon terminals containing m2 make symmetrical synapses with somata, and dendritic shafts and spines. In addition, many m2-immunoreactive axon terminals formed asymmetrical synapses with spines or dendrites. m3 receptor protein was not evident in somata by light microscopy but was present in a distinct population of small-caliber spiny dendrites as well as in axon terminals forming asymmetrical synapses with spines. m4 receptor protein was heterogeneously distributed in the neostriatum and localized to 44% of striatal cells. m4-positive neurons had the ultrastructural features of medium spiny neurons with reaction product particularly concentrated in spines, often at postsynaptic densities. Axon terminals containing m4 form asymmetrical synapses, primarily with spines. These findings indicate that the muscarinic receptor proteins occur in distinct populations of striatal neurons; that the receptor proteins concentrate postsynaptically at synapses, including many considered to be noncholinergic; that m2 is the predominant muscarinic autoreceptor in the striatum; and that each receptor subtype may be a presynaptic heteroceptor in the striatum modulating extrinsic striatal afferents.

We have devised a method for efficiently constructing high-content full-length cDNA libraries based on chemical introduction of a biotin group into the diol residue of the cap structure of eukaryotic mRNA, followed by RNase I treatment to select full-length cDNA. The selection occurs by trapping the biotin residue at the cap sites using streptavidin-coated magnetic beads, thus eliminating incompletely synthesized cDNAs. When this method was used to construct a mouse brain full-length cDNA library, our evaluation showed that more than 95% of the total clones were of full length, and recombinant clones could be produced with high efficiency (1.2 x 10(7)/10 micrograms starting mRNA). The analysis of 120 randomly picked clones indicates an unbiased representation of the starting mRNA population.

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables, such as broccoli and broccoli sprouts. This anticarcinogen was first identified as a potent inducer of Phase 2 detoxification enzymes, but evidence is mounting that SFN also acts through epigenetic mechanisms. SFN has been shown to inhibit histone deacetylase (HDAC) activity in human colon and prostate cancer lines, with an increase in global and local histone acetylation status, such as on the promoter regions of P21 and bax genes. SFN also inhibited the growth of prostate cancer xenografts and spontaneous intestinal polyps in mouse models, with evidence for altered histone acetylation and HDAC activities in vivo. In human subjects, a single ingestion of 68 g broccoli sprouts inhibited HDAC activity in circulating peripheral blood mononuclear cells 3-6 h after consumption, with concomitant induction of histone H3 and H4 acetylation. These findings provide evidence that one mechanism of cancer chemoprevention by SFN is via epigenetic changes associated with inhibition of HDAC activity. Other dietary agents such as butyrate, biotin, lipoic acid, garlic organosulfur compounds, and metabolites of vitamin E have structural features compatible with HDAC inhibition. The ability of dietary compounds to de-repress epigenetically silenced genes in cancer cells, and to activate these genes in normal cells, has important implications for cancer prevention and therapy. In a broader context, there is growing interest in dietary HDAC inhibitors and their impact on epigenetic mechanisms affecting other chronic conditions, such as cardiovascular disease, neurodegeneration and aging.

Cyclooxygenase (COX)-2, an inducible enzyme that catalyzes the formation of prostaglandins and other eicosanoids from arachidonic acid, is constitutively expressed in LNCaP human prostate cancer cell line. To evaluate the potential role of COX-2 in prostate cancer, LNCaP cells were treated with NS398, a selective COX-2 inhibitor, and the effects on cell viability and apoptosis were determined. NS398 treatment induced apoptosis in LNCaP cells in a time- and dose-dependent fashion. Treatment with 100 microM NS398 caused a down-regulation in bcl-2 protein expression, followed by chromatin condensation, chromosomal DNA fragmentation, and changes in nuclear morphology detected by 4,6-diamidino-2-phenylindole staining, DNA fragmentation assay, and terminal deoxynucleotidyl transferase-mediated UTP-biotin nick end-labeling assay. In contrast, NS398 treatment had no effect on either cell viability or nuclear function and morphology in human fetal prostate fibroblasts. These results demonstrate that NS398 induces apoptosis in LNCaP cells but not in human fetal prostate fibroblasts, and that this induction is associated with a decreased level of bcl-2 protein.

We report calculations of free energies of binding, DeltaG(bind), between a diverse set of nine ligands and avidin as well as between a peptide and streptavidin using the recently developed MM/PBSA approach. This method makes use of a molecular dynamics simulation of the ligand-protein complex to generate a thermally averaged ensemble of conformations of the molecules that are involved in the complex formation. Based on this set of structures, a free energy of binding is calculated using molecular mechanical and continuum solvent energies as well as including estimates of the nonpolar solvation free energy and solute entropy. We compare in our simulations different classes of ligands, involving biotin derivatives, the dye 2-(4'-hydroxyazobenzene)benzoic acid (HABA), and a cyclic hexapeptide, which cover a large range of binding free energies from -5 to -20 kcal/mol. Our calculations are able to reproduce experimental DeltaG(bind) values with a very good correlation coefficient of r(2) = 0.92. This agreement is considerably better than the results obtained with an alternate approach, the linear interaction energy approximation, for this system (r(2) = 0.55).

Genomic studies of cell differentiation and function within a whole organism depend on the ability to isolate specific cell types from a tissue, but this is technically difficult. We developed a method called INTACT (isolation of nuclei tagged in specific cell types) that allows affinity-based isolation of nuclei from individual cell types of a tissue, thereby circumventing the problems associated with mechanical purification techniques. In this method nuclei are affinity-labeled through transgenic expression of a biotinylated nuclear envelope protein in the cell type of interest. Total nuclei are isolated from transgenic plants and biotin-labeled nuclei are then purified using streptavidin-coated magnetic beads, without the need for specialized equipment. INTACT gives high yield and purity of nuclei from the desired cell types, which can be used for genome-wide analysis of gene expression and chromatin features. The entire procedure, from nuclei purification through cDNA preparation or chromatin immunoprecipitation (ChIP), can be completed within 2 d. The protocol we present assumes that transgenic lines are already available, and includes procedural details for amplification of cDNA or ChIP DNA prior to microarray or deep sequencing analysis.

Analysis of previously unknown genetic aberrations in solid tumors has become possible through the use of comparative genomic hybridization (CGH), which is based on competitive binding of tumor and control DNA to normal metaphase chromosomes. CGH allows detection of DNA sequence copy number changes (deletions, gains, and amplifications) on a genome-wide scale in a single hybridization. We describe here an improved CGH technique, which enables reliable detection of copy number changes in archival formalin-fixed paraffin-embedded tumor samples. The technique includes a modified DNA extraction protocol, which produces high molecular weight DNA which is necessary for high quality CGH. The DNA extraction includes a 3-day digestion with proteinase K, which remarkably improves the yield of high molecular weight DNA. Labeling of the test DNA with a directly fluorescein-conjugated nucleotide (instead of biotin labeling) improved significantly the quality of hybridization. Using the paraffin-block technique, we could analyze 70 to 90% of paraffin blocks, including very old samples as well as samples taken at autopsy. CGH from paraffin blocks was highly concordant (95%) with analyses done from matched freshly frozen tumor samples (n = 5 sample pairs; kappa coefficient = 0.83). The method described here has wide applicability in tumor pathology, allowing large retrospective prognostic studies of genetic aberrations as well as studies on genetic pathogenesis of solid tumors, inasmuch as premalignant lesions and primary and metastatic tumors can be analyzed by using archival paraffin-embedded samples.

The retina is sensitive to light stimuli varying over more than 12 log units in intensity. It accomplishes this, in part, by switching between rod-dominated circuits designed for maximum utilization of scarce photons and cone circuits designed for greater acuity. Rod signals are integrated into the cone pathways through AII amacrine cells, which are connected by gap junctions both to other AII amacrine cells and to cone bipolar cells. To determine the relative permeabilities of the two junctional pathways, we have measured the distribution of biotinylated tracers across this heterologous cell assembly after injecting a single AII amacrine cell. We found that neurobiotin (relative molecular mass, 286) passed easily through both types of gap junctions, but that biotin-X cadaverine (relative molecular mass, 442) passed through AII/bipolar cell gap junctions poorly compared to AII/AII gap junctions. Thus, the AII/bipolar cell channel has a lower permeability to large molecules than does the AII/AII amacrine cell channel. The two pathways are also regulated differently. Dopamine and cyclic AMP agonists, known to diminish AII-AII coupling, did not change the relative labelling intensity of AII to bipolar cells. However, nitric oxide and cGMP agonists selectively reduced labelling in bipolar cells relative to AII amacrine cells, perhaps by acting at the bipolar side of this gap junction. This suggests that increased cGMP controls the network switching between rod and cone pathways associated with light adaptation.

Nanoparticles have emerged as key materials for biomedical applications because of their unique and tunable physical properties, multivalent targeting capability, and high cargo capacity. Motivated by these properties and by current clinical needs, numerous diagnostic and therapeutic nanomaterials have recently emerged. Here we describe a novel nanoparticle targeting platform that uses a rapid, catalyst-free cycloaddition as the coupling mechanism. Antibodies against biomarkers of interest were modified with trans-cyclooctene and used as scaffolds to couple tetrazine-modified nanoparticles onto live cells. We show that the technique is fast, chemoselective, adaptable to metal nanomaterials, and scalable for biomedical use. This method also supports amplification of biomarker signals, making it superior to alternative targeting techniques including avidin/biotin.

The temporal pattern of apoptosis in the adult rat brain after lateral fluid-percussion (FP) brain injury was characterized using terminal deoxynucleotidyl-transferase-mediated biotin-dUTP nick end labeling (TUNEL) histochemistry and agarose gel electrophoresis. Male Sprague Dawley rats were subjected to brain injury and killed for histological analysis at intervals from 12 hr to 2 months after injury (n = 3/time point). Sham (uninjured) controls were subjected to anesthesia with (n = 3) or without (n = 3) surgery. Apoptotic TUNEL-positive cells were defined using stringent morphological criteria including nuclear shrinkage and fragmentation and condensation of chromatin and cytoplasm. Double-labeled immunocytochemistry was performed to identify TUNEL-positive neurons (anti-neurofilament monoclonal antibody RM044), astrocytes (anti-glial fibrillary acidic protein polyclonal antibody), and oligodendrocytes (anti-cyclic nucleotide phosphohydrolase polyclonal antibody). Compared with that seen with sham controls, in the injured cortex, significant apoptosis occurred at 24 hr (65 +/- 19 cells; p < 0.05) with a second, more pronounced response at 1 week after injury (91 +/- 24 cells; p < 0.05). The number of apoptotic cells in the white matter was increased as early as 12 hr after injury and peaked by 1 week (33 +/- 6 cells; p < 0.05). An increase in apoptotic cells was observed in the hippocampus at 48 hr (13 +/- 8), whereas in the thalamus, the apoptotic response was delayed, peaking at 2 weeks after injury (151 +/- 71 cells; p < 0.05). By 2 months, the number of apoptotic cells in most regions had returned to uninjured levels. At 24 hr after injury, TUNEL-labeled neurons and oligodendrocytes were localized primarily to injured cortex. By 1 week after injury, populations of TUNEL-labeled astrocytes and oligodendrocytes were present in the injured cortex, while double-labeled neurons were present predominantly in injured cortex and thalamus, with a few scattered in the hippocampus. DNA agarose gels confirmed morphological identification of apoptosis. These data suggest that the apoptotic response to trauma is regionally distinct and may be involved in both acute and delayed patterns of cell death.

Here we describe a new type of biosensor element for detecting proteins in solution at nanomolar concentrations. We tethered a 3.4 kDa polyethylene glycol chain at a defined site within the lumen of the transmembrane protein pore formed by staphylococcal alpha-hemolysin. The free end of the polymer was covalently attached to a biotin molecule. On incorporation of the modified pore into a lipid bilayer, the biotinyl group moves from one side of the membrane to the other, and is detected by reversible capture with a mutant streptavidin. The capture events are observed as changes in ionic current passing through single pores in planar bilayers. Accordingly, the modified pore allows detection of a protein analyte at the single-molecule level, facilitating both quantification and identification through a distinctive current signature. The approach has higher time resolution compared with other kinetic measurements, such as those obtained by surface plasmon resonance.

The polymeric immunoglobulin receptor (pIgR) on mucosal epithelial cells binds dimeric IgA (dIgA) on the basolateral surface and mediates transport of dIgA to the apical surface. Using Madin-Darby canine kidney epithelial cells stably transfected with pIgR cDNA, we found that soluble immune complexes (ICs) of 125I-labeled rat monoclonal antidinitrophenyl (DNP) dIgA (125I-dIgA) and DNP/biotin-bovine serum albumin were transported from the basolateral to the apical surface and then released. Monomeric IgA ICs were not transported, consistent with the specificity of pIgR for polymeric immunoglobulins. Essentially all the 125I-dIgA in apical culture supernatants was streptavidin precipitable, indicating that dIgA remained bound to antigen during transcytosis. While both dIgA and dIgA ICs bound pIgR with equal affinity (Kd approximately 8 nM), the number of high-affinity binding sites per cell was 2- to 3-fold greater for dIgA than for dIgA ICs. The extent of endocytosis of dIgA and dIgA ICs was correlated with the number of high-affinity binding sites. SDS/PAGE analysis of intracellular dIgA and dIgA ICs demonstrated that in both cases IgA remained undegraded during transport. The results suggest that the pathways of epithelial transcytosis of free dIgA and dIgA ICs are the same. Given the high population density of mucosal IgA plasma cells and the enormous surface area of pIgR-expressing mucosal epithelium, it is likely that significant local transcytosis of IgA ICs occurs in vivo. Such a process would allow direct elimination of IgA ICs at the mucosal sites where they are likely to form, thus providing an important defense function for IgA.

Conjugated linoleic acid (CLA) is a naturally occurring group of dienoic derivatives of linoleic acid found in beef and dairy products. CLA has been reported to reduce body fat. To examine the mechanism(s) of CLA reduction of fat mass, female C57BL/6J mice were fed standard semipurified diets (10% fat of total energy) with or without CLA (1% wt/wt). Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick endlabeling (TUNEL) and DNA fragmentation analysis revealed that fat-mass decrease by CLA was mainly due to apoptosis. Tumor necrosis factor (TNF)-alpha and uncoupling protein (UCP)-2 mRNA levels increased 12- and 6-fold, respectively, in isolated adipocytes from CLA-fed mice compared with control mice. Because it is known that TNF-alpha induces apoptosis of adipocytes and upregulates UCP2 mRNA, a marked increase of TNF-alpha mRNA with an increase of UCP2 in adipocytes caused CLA-induced apoptosis. However, with a decrease of fat mass, CLA supplementation resulted in a state resembling lipoatrophic diabetes: ablation of brown adipose tissue, a marked reduction of white adipose tissue, marked hepatomegaly, and marked insulin resistance. CLA supplementation decreased blood leptin levels, but continuous leptin infusion reversed hyperinsulinemia, indicating that leptin depletion contributes to the development of insulin resistance. These results demonstrate that intake of CLA reduces adipose tissue by apoptosis and results in lipodystrophy, but hyperinsulinemia by CLA can be normalized by leptin administration.

OBJECTIVE

The hepatocyte growth factor/c-MET axis is implicated in tumor cell proliferation, survival, and angiogenesis. ARQ 197 is an oral, selective, non-adenosine triphosphate competitive c-MET inhibitor. A phase I trial of ARQ 197 was conducted to assess safety, tolerability, and target inhibition, including intratumoral c-MET signaling, apoptosis, and angiogenesis.

METHODS

Patients with solid tumors amenable to pharmacokinetic and pharmacodynamic studies using serial biopsies, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), and circulating endothelial cell (CEC) and circulating tumor cell (CTC) enumeration were enrolled.

RESULTS

Fifty-one patients received ARQ 197 at 100 to 400 mg twice per day. ARQ 197 was well tolerated, with the most common toxicities being grade 1 to 2 fatigue, nausea, and vomiting. Dose-limiting toxicities included grade 3 fatigue (200 mg twice per day; n = 1); grade 3 mucositis, palmar-plantar erythrodysesthesia, and hypokalemia (400 mg twice per day; n = 1); and grade 3 to 4 febrile neutropenia (400 mg twice per day, n = 2; 360 mg twice per day, n = 1). The recommended phase II dose was 360 mg twice per day. ARQ 197 systemic exposure was dose dependent and supported twice per day oral dosing. ARQ 197 decreased phosphorylated c-MET, total c-MET, and phosphorylated focal adhesion kinase and increased terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) staining in tumor biopsies (n = 15). CECs decreased in 25 (58.1%) of 43 patients, but no significant changes in DCE-MRI parameters were observed after ARQ 197 treatment. Of 15 patients with detectable CTCs, eight (53.3%) had ≥ 30% decline in CTCs after treatment. Stable disease, as defined by Response Evaluation Criteria in Solid Tumors (RECIST), ≥ 4 months was observed in 14 patients, with minor regressions in gastric and Merkel cell cancers.

CONCLUSIONS

ARQ 197 safely inhibited intratumoral c-MET signaling. Further clinical evaluation focusing on combination approaches, including an erlotinib combination in non-small-cell lung cancer, is ongoing.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity (2-6). Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible (7-10). We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments. Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

Clone 33D1 is a mouse-rat hybridoma that secretes a specific anti-dendritic cell (DC) monoclonal antibody (14). Because the antibody kills DC in the presence of rabbit complement, it can be used to study the functional consequences of selective DC depletion. Previous data on the cell specificity of 33D1 were first extended. By cytotoxicity (rabbit complement) and indirect immunofluorescence (biotin-avidin technique), 33D1 reacted with DC but not with macrophages nor other splenocytes. In contrast, the monoclonal antibody, F4/80 (15), reacted with macrophages but not DC. The functional assay evaluated in this paper was stimulation of the primary mixed leukocyte reaction (MLR). 33D1 antibody itself did not inhibit stimulation by enriched populations of DC. In the presence of complement, 33D1 killed DC and ablated stimulatory function. The effect of 33D1 and complement on MLR stimulation by heterogenous cell mixtures was then evaluated. Removal of DC from unfractionated spleen suspensions reduced stimulatory capacity 75-90 percent, comparable to that produced with specific anti-Ia antibody and complement. Stimulation of both proliferative and cytotoxic responses was reduced. DC depletion had similar effects on MLR generated across full strain differences, or across selected subregions (H2I, H-2K/D) of the major histocompatibility complex. To further compare the functional properties of spleen DC and macrophages, MLR stimulation by adherent and nonadherent fractions of spleen were tested separately. 62 +/- 8 percent of the total stimulatory capacity of spleen was in the plastic adherent population. Activity was ablated greater than 90 percent after elimination of DC. MLR stimulation by 24-h cultures of spleen adherent cells, which contained a three- to sixfold excess of Ia(+) macrophages, was also ablated when DC were removed. Stimulation by nonadherent spleen was more resistant, but was reduced 50-75 percent by 33D1 and complement. The function of spleen cells treated with 33D1 or anti-Ia antibody and complement was restored with a small inoculum of purified DC. The latter corresponded to 0.5 percent of total stimulator cells and were enriched by previously described techniques that did not require the 33D1 antibody. We conclude that the DC, a trace component of mouse spleen, is the principal cell type required for stimulation of the primary MLR. Because other cells are not immunogenic, but do express Ia and H-2 alloantigens, DC likely represent the critical accessory cell required for the induction of lymphocyte responses.

Synapses relay information through the release of neurotransmitters stored in presynaptic vesicles. The identity, kinetics and location of the vesicle pools that are mobilized by neuronal activity have been studied using a variety of techniques. We created a genetically encoded probe, biosyn, which consists of a biotinylated VAMP2 expressed at presynaptic terminals. We exploited the high-affinity interaction between streptavidin and biotin to label biosyn with fluorescent streptavidin during vesicle fusion. This approach allowed us to tag vesicles sequentially to visualize and establish the identity of presynaptic pools. Using this technique, we were able to distinguish between two different pools of vesicles in rat hippocampal neurons: one that was released in response to presynaptic activity and another, distinct vesicle pool that spontaneously fused with the plasma membrane. We found that the spontaneous vesicles belonged to a 'resting pool' that is normally not mobilized by neuronal activity and whose function was previously unknown.

The analysis of gene expression data in clinical medicine has been plagued by the lack of a critical evaluation of accepted methodologies for the collection, processing, and labeling of RNA. In the present report, the reliability of two commonly used techniques to isolate RNA from whole blood or its leukocyte compartment was compared by examining their reproducibility, variance, and signal-to-noise ratios. Whole blood was obtained from healthy subjects and was either untreated or stimulated ex vivo with Staphylococcus enterotoxin B (SEB). Blood samples were also obtained from trauma patients but were not stimulated with SEB ex vivo. Total RNA was isolated from whole blood with the PAXgene proprietary blood collection system or from isolated leukocytes. Biotin-labeled cRNA was hybridized to Affymetrix GeneChips. The Pearson correlation coefficient for gene expression measurements in replicates from healthy subjects with both techniques was excellent, exceeding 0.985. Unsupervised analyses, including hierarchical cluster analysis, however, revealed that the RNA isolation method resulted in greater differences in gene expression than stimulation with SEB or among different trauma patients. The intraclass correlation, a measure of signal-to-noise ratio, of the difference between SEB-stimulated and unstimulated blood from healthy subjects was significantly higher in leukocyte-derived samples than in whole blood: 0.75 vs. 0.46 (P = 0.002). At the P < 0.001 level of significance, twice as many probe sets discriminated between SEB-stimulated and unstimulated blood with leukocyte isolation than with PAXgene. The findings suggest that the method of RNA isolation from whole blood is a critical variable in the design of clinical studies using microarray analyses.

We report a new chemoenzymatic strategy for the rapid and sensitive detection of O-GlcNAc posttranslational modifications. The approach exploits the ability of an engineered mutant of beta-1,4-galactosyltransferase to selectively transfer an unnatural ketone functionality onto O-GlcNAc glycosylated proteins. Once transferred, the ketone moiety serves as a versatile handle for the attachment of biotin, thereby enabling chemiluminescent detection of the modified protein. Importantly, this approach permits the rapid visualization of proteins that are at the limits of detection using traditional methods. Moreover, it bypasses the need for radioactive precursors and captures the glycosylated species without perturbing metabolic pathways. We anticipate that this general chemoenzymatic strategy will have broad application to the study of posttranslational modifications.

Nitric oxide is implicated in a variety of signaling pathways in different systems, notably in endothelial cells. Some of its effects can be exerted through covalent modifications of proteins and, among these modifications, increasing attention is being paid to S-nitrosylation as a signaling mechanism. In this work, we show by a variety of methods (ozone chemiluminescence, biotin switch, and mass spectrometry) that the molecular chaperone Hsp90 is a target of S-nitrosylation and identify a susceptible cysteine residue in the region of the C-terminal domain that interacts with endothelial nitric oxide synthase (eNOS). We also show that the modification occurs in endothelial cells when they are treated with S-nitroso-l-cysteine and when they are exposed to eNOS activators. Hsp90 ATPase activity and its positive effect on eNOS activity are both inhibited by S-nitrosylation. Together, these data suggest that S-nitrosylation may functionally regulate the general activities of Hsp90 and provide a feedback mechanism for limiting eNOS activation.

HIV-1 Gag is the only protein required for retroviral particle assembly. There is evidence suggesting that phosphatidylinositol phosphate and nucleic acid are essential for viruslike particle assembly. To elucidate structural foundations of interactions of HIV-1 Gag with the assembly cofactors PI(4,5)P2 and RNA, we employed mass spectrometric protein footprinting. In particular, the NHS-biotin modification approach was used to identify the lysine residues that are exposed to the solvent in free Gag and are protected from biotinylation by direct protein-ligand or protein-protein contacts in Gag complexes with PI(4,5)P2 and/or RNA. Of 21 surface lysines readily modified in free Gag, only K30 and K32, located in the matrix domain, were strongly protected in the Gag-PI(4,5)P2 complex. Nucleic acid also protected these lysines, but only at significantly higher concentrations. In contrast, nucleic acids and not PI(4,5)P2 exhibited strong protection of two nucleocapsid domain residues: K391 and K424. In addition, K314, located in the capsid domain, was specifically protected only in the presence of both PI(4,5)P2 and nucleic acid. We suggest that concerted binding of PI(4,5)P2 and nucleic acid to the matrix and nucleocapsid domains, respectively, promotes protein-protein interactions involving capsid domains. These protein-protein interactions must be involved in virus particle assembly.

Comparative genomic in situ hybridization (CGH) provides a new possibility for searching genomes for imbalanced genetic material. Labeled genomic test DNA, prepared from clinical or tumor specimens, is mixed with differently labeled control DNA prepared from cells with normal chromosome complements. The mixed probe is used for chromosomal in situ suppression (CISS) hybridization to normal metaphase spreads (CGH-metaphase spreads). Hybridized test and control DNA sequences are detected via different fluorochromes, e.g., fluorescein isothiocyanate (FITC) and tetraethylrhodamine isothiocyanate (TRITC). The ratios of FITC/TRITC fluorescence intensities for each chromosome or chromosome segment should then reflect its relative copy number in the test genome compared with the control genome, e.g., 0.5 for monosomies, 1 for disomies, 1.5 for trisomies, etc. Initially, model experiments were designed to test the accuracy of fluorescence ratio measurements on single chromosomes. DNAs from up to five human chromosome-specific plasmid libraries were labeled with biotin and digoxigenin in different hapten proportions. Probe mixtures were used for CISS hybridization to normal human metaphase spreads and detected with FITC and TRITC. An epifluorescence microscope equipped with a cooled charge coupled device (CCD) camera was used for image acquisition. Procedures for fluorescence ratio measurements were developed on the basis of commercial image analysis software. For hapten ratios 4/1, 1/1 and 1/4, fluorescence ratio values measured for individual chromosomes could be used as a single reliable parameter for chromosome identification. Our findings indicate (1) a tight correlation of fluorescence ratio values with hapten ratios, and (2) the potential of fluorescence ratio measurements for multiple color chromosome painting. Subsequently, genomic test DNAs, prepared from a patient with Down syndrome, from blood of a patient with T-cell prolymphocytic leukemia, and from cultured cells of a renal papillary carcinoma cell line, were applied in CGH experiments. As expected, significant differences in the fluorescence ratios could be measured for chromosome types present in different copy numbers in these test genomes, including a trisomy of chromosome 21, the smallest autosome of the human complement. In addition, chromosome material involved in partial gains and losses of the different tumors could be mapped to their normal chromosome counterparts in CGH-metaphase spreads. An alternative and simpler evaluation procedure based on visual inspection of CCD images of CGH-metaphase spreads also yielded consistent results from several independent observers. Pitfalls, methodological improvements, and potential applications of CGH analyses are discussed.

OC125, a murine monoclonal antibody, recognizes an antigenic determinant (CA125) that is associated with greater than 80% of epithelial ovarian neoplasms of serous, endometrioid, clear cell, and undifferentiated types. In the present report, a sensitive biotin-avidin immunoperoxidase technique was used to determine reactivity of OC125 with normal adult and fetal tissues, as well as with neoplasms of nonovarian origin. In fetal tissues, the antibody reacted with amnion and with derivatives of the coelomic epithelium, i.e., the müllerian epithelium and the lining cells of the peritoneum, pleura, and pericardium. Among adult tissues, OC125 reacted with the epithelium of fallopian tubes, endometrium, and endocervix. The CA125 determinant was also detected on mesothelial cells in the adult pleura, pericardium, and peritoneum, particularly in areas of inflammation and adhesion. Curiously, the surface epithelium of normal fetal and adult ovaries, thought to be derived from coelomic epithelium, did not express the determinant, except in inclusion cysts, areas of metaplasia, and papillary excrescences. Of neoplastic tissues of nonovarian origin, OC125 reacted consistently only with adenocarcinomas of the endocervix, endometrium, and fallopian tube, and with mesotheliomas. Only seven of 64 nongynecological tumors tested reacted with OC125. Thus, OC125 detects a differentiation antigen shared by fetal coelomic epithelium and its derivatives in the fetus and the adult. Apparently, this antigen disappears early in the course of formation of the ovarian epithelium and is reexpressed in certain reactive and neoplastic lesions, a process that could be termed "molecular metaplasia."

We have probed the relationship between tubulin posttranslational modification and microtubule stability, using a variation of the antibody-blocking technique. In human retinoblastoma cells we find that acetylated and detyrosinated microtubules represent congruent subsets of the cells' total microtubules. We also find that stable microtubules defined as those that had not undergone polymerization within 1 h after injection of biotin-tubulin were all posttranslationally modified; furthermore dynamic microtubules were all unmodified. We therefore conclude that in these cells the stable, acetylated, and detyrosinated microtubules represent the same subset of the cells' total network. Posttranslational modification, however, is not a prerequisite for microtubule stability and vice versa. Potorous tridactylis kidney cells have no detectable acetylated microtubules but do have a sizable subset of stable ones, and chick embryo fibroblast cells are extensively modified but have few stable microtubules. We conclude that different cell types can create specific microtubule subsets by modulating the relative rates of posttranslational modification and microtubule turnover.