We examined, using physiological and morphological techniques, the distribution of Ca(2+)-gated K+ (gKca) channels relative to the location of Ca2+ channels and transmitter release sites at the frog neuromuscular junction (NM). Charybdotoxin (ChTx) and iberiotoxin, blockers of gKca channels with large conductances, increase transmitter release at the frog NMJ. Intracellular Ca2+ buffers with rapid binding kinetics, dimethyl BAPTA and BAPTA, prevented the effect of ChTx, but EGTA, a Ca2+ buffer with similar affinity for Ca2+ but slower binding kinetics, did not. Dimethyl BAPTA and BAPTA, but not EGTA, caused a temporary increase in transmitter release. Labeling of gKca channels with ChTx-biotin revealed a series of bands located at the sites of Ca2+ channels, but this labeling did not occur in denervated preparations. Cross sections of NMJs revealed that gKca channels are clustered in the presynaptic membrane facing the postsynaptic membrane. We conclude that gKca channels are strategically clustered at the neurotransmitter release sites, where they can be quickly activated by Ca2+ entering the terminal.

Tandem affinity strategies reach exceptional protein purification grades and have considerably improved the outcome of mass spectrometry-based proteomic experiments. However, current tandem affinity tags are incompatible with two-step purification under fully denaturing conditions. Such stringent purification conditions are desirable for mass spectrometric analyses of protein modifications as they result in maximal preservation of posttranslational modifications. Here we describe the histidine-biotin (HB) tag, a new tandem affinity tag for two-step purification under denaturing conditions. The HB tag consists of a hexahistidine tag and a bacterially derived in vivo biotinylation signal peptide that induces efficient biotin attachment to the HB tag in yeast and mammalian cells. HB-tagged proteins can be sequentially purified under fully denaturing conditions, such as 8 m urea, by Ni(2+) chelate chromatography and binding to streptavidin resins. The stringent separation conditions compatible with the HB tag prevent loss of protein modifications, and the high purification grade achieved by the tandem affinity strategy facilitates mass spectrometric analysis of posttranslational modifications. Ubiquitination is a particularly sensitive protein modification that is rapidly lost during purification under native conditions due to ubiquitin hydrolase activity. The HB tag is ideal to study ubiquitination because the denaturing conditions inhibit hydrolase activity, and the tandem affinity strategy greatly reduces nonspecific background. We tested the HB tag in proteome-wide ubiquitin profiling experiments in yeast and identified a number of known ubiquitinated proteins as well as so far unidentified candidate ubiquitination targets. In addition, the stringent purification conditions compatible with the HB tag allow effective mass spectrometric identification of in vivo cross-linked protein complexes, thereby expanding proteomic analyses to the description of weakly or transiently associated protein complexes.

In the past two decades, high-affinity nucleic acid aptamers have been developed for a wide variety of pure molecules and complex systems such as live cells. Conceptually, aptamers are developed by an evolutionary process, whereby, as selection progresses, sequences with a certain conformation capable of binding to the target of interest emerge and dominate the pool. This protocol, cell-SELEX (systematic evolution of ligands by exponential enrichment), is a method that can generate DNA aptamers that can bind specifically to a cell type of interest. Commonly, a cancer cell line is used as the target to generate aptamers that can differentiate that cell type from other cancers or normal cells. A single-stranded DNA (ssDNA) library pool is incubated with the target cells. Nonbinding sequences are washed off and bound sequences are recovered from the cells by heating cell-DNA complexes at 95 degrees C, followed by centrifugation. The recovered pool is incubated with the control cell line to filter out the sequences that bind to common molecules on both the target and the control, leading to the enrichment of specific binders to the target. Binding sequences are amplified by PCR using fluorescein isothiocyanate-labeled sense and biotin-labeled antisense primers. This is followed by removal of antisense strands to generate an ssDNA pool for subsequent rounds of selection. The enrichment of the selected pools is monitored by flow cytometry binding assays, with selected pools having increased fluorescence compared with the unselected DNA library. The procedure, from design of oligonucleotides to enrichment of the selected pools, takes approximately 3 months.

The thermodynamic properties and phase behavior of water in confined regions can vary significantly from that observed in the bulk. This is particularly true for systems in which the confinement is on the molecular-length scale. In this study, we use molecular dynamics simulations and a powerful solvent analysis technique based on inhomogenous solvation theory to investigate the properties of water molecules that solvate the confined regions of protein active sites. Our simulations and analysis indicate that the solvation of protein active sites that are characterized by hydrophobic enclosure and correlated hydrogen bonds induce atypical entropic and enthalpic penalties of hydration. These penalties apparently stabilize the protein-ligand complex with respect to the independently solvated ligand and protein, which leads to enhanced binding affinities. Our analysis elucidates several challenging cases, including the super affinity of the streptavidin-biotin system.

Cortisol 3-(o-carboxymethyl)oxime (C3-CMO) and a commercially available biotin-hydrazide derivative were used to synthesize a C3-CMO-biotin conjugate. C3-CMO was converted into a N-hydroxysuccinimide ester derivative which in a second reaction step was allowed to interact with the hydrazide derivative of biotin. This simple-to-perform synthesis yielded a conjugate suitable for use as a tracer in immunoassays for cortisol measurement. Employing biotin as the primary probe in a competitive solid phase immunoassay allows for variable end point determination by means of commercially available labeled avidin or streptavidin derivatives. Streptavidin-Europium was used in conjunction with the DELFIA-system for time-resolved fluorometric end point measurement (TR-FIA) throughout the study. In addition, colorimetric end point determination (ELISA) using streptavidin-alkaline phosphatase as a secondary probe was established and evaluated. Both forms of this non-isotopic assay showed excellent correlation with a commercially available radioimmunoassay adapted for salivary cortisol measurement. The lower detection limit was 0.43 nM for a 50 microliters salivary sample. The intra-assay coefficient of variation was 6.7, 4.7 and 4.0% at cortisol concentrations of 2.2, 5.5 and 13.2 nM, respectively (n = 37), and the corresponding inter-assay coefficients of variation were 9.0, 8.6 and 7.1% (n = 50). The competitive immunoassay requires 1.5 h incubation time and shows robust and reproducible performance. The C3-CMO-biotin conjugate allows for sensitive and flexible end point determination of salivary cortisol levels in immunoassays.

We describe a method, Hi-C, to comprehensively detect chromatin interactions in the mammalian nucleus. This method is based on Chromosome Conformation Capture, in which chromatin is crosslinked with formaldehyde, then digested, and re-ligated in such a way that only DNA fragments that are covalently linked together form ligation products. The ligation products contain the information of not only where they originated from in the genomic sequence but also where they reside, physically, in the 3D organization of the genome. In Hi-C, a biotin-labeled nucleotide is incorporated at the ligation junction, enabling selective purification of chimeric DNA ligation junctions followed by deep sequencing. The compatibility of Hi-C with next generation sequencing platforms makes it possible to detect chromatin interactions on an unprecedented scale. This advance gives Hi-C the power to both explore the biophysical properties of chromatin as well as the implications of chromatin structure for the biological functions of the nucleus. A massively parallel survey of chromatin interaction provides the previously missing dimension of spatial context to other genomic studies. This spatial context will provide a new perspective to studies of chromatin and its role in genome regulation in normal conditions and in disease.

We have modified the biotin switch assay for protein S-nitrosothiols (SNOs), using resin-assisted capture (SNO-RAC). Compared with existing methodologies, SNO-RAC requires fewer steps, detects high-mass S-nitrosylated proteins more efficiently, and facilitates identification and quantification of S-nitrosylated sites by mass spectrometry. When combined with iTRAQ labeling, SNO-RAC revealed that intracellular proteins may undergo rapid denitrosylation on a global scale. This methodology is readily adapted to analyzing diverse cysteine-based protein modifications, including S-acylation.

HeLa cells in early S phase were encapsulated in agarose microbeads, permeabilized, and incubated with biotin-11-dUTP in a "physiological" buffer. Sites of DNA synthesis were then immunolabeled. As others have found, approximately 150 focal sites of synthesis were visible in each nucleus by light microscopy; they also contained DNA polymerase alpha and proliferating cell nuclear antigen. Electron microscopy of thick resinless sections from which approximately 90% of the chromatin had been removed revealed a similar number of dense, morphologically discrete ovoid bodies strung along a nucleoskeleton. The ovoids remained morphologically and functionally intact despite the removal of most of the chromatin. After 2.5 min of incubation with biotin-11-dUTP, the incorporated analog was associated only with ovoids; after 5 min it began to spread into the adjacent chromatin, which became extensively labeled after 1 hr. This provides visual evidence for polymerization "factories" fixed to a skeleton, with replication occurring as the template moves through them.

The human gastrointestinal tract (GI tract) harbors a complex community of microbes. The microbiota composition varies between different locations in the GI tract, but most studies focus on the fecal microbiota, and that inhabiting the colonic mucosa. Consequently, little is known about the microbiota at other parts of the GI tract, which is especially true for the small intestine because of its limited accessibility. Here we deduce an ecological model of the microbiota composition and function in the small intestine, using complementing culture-independent approaches. Phylogenetic microarray analyses demonstrated that microbiota compositions that are typically found in effluent samples from ileostomists (subjects without a colon) can also be encountered in the small intestine of healthy individuals. Phylogenetic mapping of small intestinal metagenome of three different ileostomy effluent samples from a single individual indicated that Streptococcus sp., Escherichia coli, Clostridium sp. and high G+C organisms are most abundant in the small intestine. The compositions of these populations fluctuated in time and correlated to the short-chain fatty acids profiles that were determined in parallel. Comparative functional analysis with fecal metagenomes identified functions that are overrepresented in the small intestine, including simple carbohydrate transport phosphotransferase systems (PTS), central metabolism and biotin production. Moreover, metatranscriptome analysis supported high level in-situ expression of PTS and carbohydrate metabolic genes, especially those belonging to Streptococcus sp. Overall, our findings suggest that rapid uptake and fermentation of available carbohydrates contribute to maintaining the microbiota in the human small intestine.

Results of transgenetic studies argue that the scrapie isoform of the prion protein (PrPSc) interacts with the substrate cellular PrP (PrPC) during conversion into nascent PrPSc. While PrPSc appears to accumulate primarily in lysosomes, caveolae-like domains (CLDs) have been suggested to be the site where PrPC is converted into PrPSc. We report herein that CLDs isolated from scrapie-infected neuroblastoma (ScN2a) cells contain PrPC and PrPSc. After lysis of ScN2a cells in ice-cold Triton X-100, both PrP isoforms and an N-terminally truncated form of PrPC (PrPC-II) were found concentrated in detergent-insoluble complexes resembling CLDs that were isolated by flotation in sucrose gradients. Similar results were obtained when CLDs were purified from plasma membranes by sonication and gradient centrifugation; with this procedure no detergents are used, which minimizes artifacts that might arise from redistribution of proteins among subcellular fractions. The caveolar markers ganglioside GM1 and H-ras were found concentrated in the CLD fractions. When plasma membrane proteins were labeled with the impermeant reagent sulfo-N-hydroxysuccinimide-biotin, both PrPC and PrPSc were found biotinylated in CLD fractions. Similar results on the colocalization of PrPC and PrPSc were obtained when CLDs were isolated from Syrian hamster brains. Our findings demonstrate that both PrPC and PrPSc are present in CLDs and, thus, support the hypothesis that the PrPSc formation occurs within this subcellular compartment.

EZH2 or EZH1 is the catalytic subunit of the polycomb repressive complex 2 that catalyzes methylation of histone H3 lysine 27 (H3K27). The trimethylation of H3K27 (H3K27me3) is a transcriptionally repressive post-translational modification. Overexpression of EZH2 and hypertrimethylation of H3K27 have been implicated in a number of cancers. Several selective inhibitors of EZH2 have been reported recently. Herein we disclose UNC1999, the first orally bioavailable inhibitor that has high in vitro potency for wild-type and mutant EZH2 as well as EZH1, a closely related H3K27 methyltransferase that shares 96% sequence identity with EZH2 in their respective catalytic domains. UNC1999 was highly selective for EZH2 and EZH1 over a broad range of epigenetic and non-epigenetic targets, competitive with the cofactor SAM and non-competitive with the peptide substrate. This inhibitor potently reduced H3K27me3 levels in cells and selectively killed diffused large B cell lymphoma cell lines harboring the EZH2(Y641N) mutant. Importantly, UNC1999 was orally bioavailable in mice, making this inhibitor a valuable tool for investigating the role of EZH2 and EZH1 in chronic animal studies. We also designed and synthesized UNC2400, a close analogue of UNC1999 with potency >1,000-fold lower than that of UNC1999 as a negative control for cell-based studies. Finally, we created a biotin-tagged UNC1999 (UNC2399), which enriched EZH2 in pull-down studies, and a UNC1999-dye conjugate (UNC2239) for co-localization studies with EZH2 in live cells. Taken together, these compounds represent a set of useful tools for the biomedical community to investigate the role of EZH2 and EZH1 in health and disease.

A tumor-homing peptide, F3, selectively binds to endothelial cells in tumor blood vessels and to tumor cells. Here, we show that the cell surface molecule recognized by F3 is nucleolin. Nucleolin specifically bound to an F3 peptide affinity matrix from extracts of cultured breast carcinoma cells. Antibodies and cell surface biotin labeling revealed nucleolin at the surface of actively growing cells, and these cells bound and internalized fluorescein-conjugated F3 peptide, transporting it into the nucleus. In contrast, nucleolin was exclusively nuclear in serum-starved cells, and F3 did not bind to these cells. The binding and subsequent internalization of F3 were blocked by an antinucleolin antibody. Like the F3 peptide, intravenously injected antinucleolin antibodies selectively accumulated in tumor vessels and in angiogenic vessels of implanted "matrigel" plugs. These results show that cell surface nucleolin is a specific marker of angiogenic endothelial cells within the vasculature. It may be a useful target molecule for diagnostic tests and drug delivery applications.

Primary cultures of human fetal brain cells were transfected with plasmid DNA pMK16, containing an origin-defective mutant of simian virus 40 (SV40). Several weeks after DNA treatment, proliferation of glial cells was evident in the culture, allowing passage of the cells at low split ratios. Initially, only 10% of the cells demonstrated nuclear fluorescence staining using a hamster tumor antibody to the SV40 T protein. By the sixth passage, however, 100% of the cells reacted positively to the same antibody. During these early passages, the cells designated SVG began growing very rapidly and acquired a homogeneous morphology. Cell division required only low serum concentrations, was not contact-inhibited, and remained anchorage dependent. These characteristics of the SVG cells have been stable through 25 passages or approximately equal to 80 cell generations. The SV40 T protein is continuously produced in the cells and can direct the replication of DNA inserts in the pSV2 vector, determined by in situ hybridization using biotin-labeled DNA probes, which contains the SV40 replication origin. More importantly, SVG cells support the multiplication of the human papovavirus JCV at levels comparable to primary cultures of human fetal glial cells, producing infectious virus as early as 1 week after viral adsorption. Their brain-cell derivation has been established as astroglial, based on their reactivity with a monoclonal antibody to glial fibrillary acid protein and lack of activity with an anti-galactocerebroside antibody, which identifies oligodendroglial cells. The SVG cells represent a unique line of continuous rapidly growing human fetal astroglial cells that synthesizes a replication-proficient SV40 T protein. Their susceptibility to JC virus (JCV) infection obviates a host restriction barrier that limited JCV studies to primary cultures of human fetal brain and thus should allow for more detailed molecular studies of human brain cells and JCV that infects them.

Best vitelliform macular dystrophy is a dominantly inherited, early onset, macular degenerative disease that exhibits some histopathologic similarities to age-related macular degeneration. Although the vitelliform lesion is common in the fundus of individuals with Best disease, diagnosis is based on a reduced ratio of the light peak to dark trough in the electrooculogram. Recently, the VMD2 gene on chromosome 11q13, encoding the protein bestrophin, was identified. The function of bestrophin is unknown. To facilitate studies of bestrophin, we produced both rabbit polyclonal and mouse monoclonal antibodies that proved useful for Western blotting, immunoprecipitation, and immunocytochemistry. To characterize bestrophin, we initially probed the retinal pigment epithelium (RPE)-derived cell lines ARPE-19, D407, and RPE-J. All of the cell lines expressed bestrophin mRNA by reverse transcription-PCR, but not on Western blots. Bestrophin in human RPE partitioned in the detergent phase during Triton X-114 extraction and could be modified by biotin in intact cells, indicative of a plasma membrane localization. Immunocytochemical staining of macaque and porcine eyes indicated that bestrophin is localized at the basolateral plasma membrane of RPE cells. When expressed in RPE-J cells by adenovirus-mediated gene transfer, bestrophin again was determined by confocal microscopy and cell surface biotinylation to be a basolateral plasma membrane protein. The basolateral plasma membrane localization of bestrophin suggests the possibility that bestrophin plays a role in generating the altered electrooculogram of individuals with Best disease.

The interactions between various functionalized carbon nanotubes and several types of human cancer cells are explored. We have prepared modified nanotubes and have shown that these can be derivatized in a way that enables attachment of small molecules and of proteins, the latter through a novel noncovalent association. The functionalized carbon nanotubes enter nonadherent human cancer cells as well as adherent cell lines (CHO and 3T3) and by themselves are not toxic. While the fluoresceinated protein streptavidin (MW approximately 60 kD) by itself does not enter cells, it readily enters cells when complexed to a nanotube-biotin transporter and exhibits dose-dependent cytotoxicity. The uptake pathway is consistent with adsorption-mediated endocytosis. The use of carbon nanotubes as molecular transporters could be exploited for various cargos. The biocompatibility and unique physical, electrical, optical, and mechanical properties of nanotubes provide the basis for new classes of materials for drug, protein, and gene delivery applications.

OBJECTIVE

To establish the presence of inflammation in and cytokine production by synovial membranes from patients with various stages of early osteoarthritis (OA), with knee pain, normal knee radiographs, and arthroscopic evidence of chondral damage.

METHODS

Synovial membrane samples were obtained from the knees of 63 patients at the time of arthroscopy for unexplained knee pain or at the time of joint replacement surgery. Evaluations of synovial membrane variables including thickness of lining layer, vascularity, and inflammatory cell infiltrate were by a blinded observer. In a subset of 20 patients, production of interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), and IL-1 receptor antagonist (IL-1ra) at the mRNA and protein levels was determined using in situ hybridization with biotin labeled ribo-probes and immunohistochemistry.

RESULTS

There was evidence of thickening of the lining layer, increased vascularity, and inflammatory cell infiltration in synovial membranes from patients with all grades of OA, with the most marked changes seen in synovial tissue from patients with advanced grades of OA. Similarly, production of IL-1 alpha, IL-1 beta, and TNF-alpha was present in synovial membranes from all patients with OA, irrespective of the degree of articular cartilage damage. There was a trend to decreased levels of IL-1ra in synovial membranes from patients with OA that did not attain statistical significance. Similarly, there was a decrease in the ratio of IL-1ra to IL-1 alpha and beta with increasing grades of OA.

CONCLUSIONS

Chronic inflammatory changes with production of proinflammatory cytokines are a feature of synovial membranes from patients with early OA, with the most severe changes seen in patients at the time of joint replacement surgery resembling those seen in rheumatoid arthritis. This low grade synovitis results in the production of cytokines that may contribute to the pathogenesis of OA.

Triangular silver nanoparticles ( approximately 100 nm wide and 50 nm high) have remarkable optical properties. In particular, the peak extinction wavelength, lambda(max) of their localized surface plasmon resonance (LSPR) spectrum is unexpectedly sensitive to nanoparticle size, shape, and local ( approximately 10-30 nm) external dielectric environment. This sensitivity of the LSPR lambda(max) to the nanoenvironment has allowed us to develop a new class of nanoscale affinity biosensors. The essential characteristics and operational principles of these LSPR nanobiosensors will be illustrated using the well-studied biotin-streptavidin system. Exposure of biotin-functionalized Ag nanotriangles to 100 nM streptavidin (SA) caused a 27.0 nm red-shift in the LSPR lambda(max). The LSPR lambda(max) shift, DeltaR/DeltaR(max), versus [SA] response curve was measured over the concentration range 10(-)(15) M < [SA] < 10(-)(6) M. Comparison of the data with the theoretical normalized response expected for 1:1 binding of a ligand to a multivalent receptor with different sites but invariant affinities yielded approximate values for the saturation response, DeltaR(max) = 26.5 nm, and the surface-confined thermodynamic binding constant K(a,surf) = 10(11) M(-)(1). At present, the limit of detection (LOD) for the LSPR nanobiosensor is found to be in the low-picomolar to high-femtomolar region. A strategy to amplify the response of the LSPR nanobiosensor using biotinylated Au colloids and thereby further improve the LOD is demonstrated. Several control experiments were performed to define the LSPR nanobiosensor's response to nonspecific binding as well as to demonstrate its response to the specific binding of another protein. These include the following: (1) electrostatic binding of SA to a nonbiotinylated surface, (2) nonspecific interactions of prebiotinylated SA to a biotinylated surface, (3) nonspecific interactions of bovine serum albumin to a biotinylated surface, and (4) specific binding of anti-biotin to a biotinylated surface. The LSPR nanobiosensor provides a pathway to ultrasensitive biodetection experiments with extremely simple, small, light, robust, low-cost instrumentation that will greatly facilitate field-portable environmental or point-of-service medical diagnostic applications.

BACKGROUND

Prostate specific membrane antigen (PSM) is a membrane-bound antigen that is highly specific for benign and malignant prostate epithelial cells. Its expression in high grade prostatic intraepithelial neoplasia (PIN) has not been compared with that in prostate carcinoma.

METHODS

The authors performed an immunohistochemical study of representative sections from 184 radical prostatectomies from previously untreated patients with pathologic stage T2N0M0 adenocarcinoma treated at the Mayo Clinic between 1987 and 1991. Affinity-purified monoclonal antibody 7E11-5.3 directed against PSM was employed at a concentration of 20 microg/mL overnight. For comparison, serial sections in each case were stained with prostate specific antigen (PSA). Staining for all antibodies was performed using the streptavidin-biotin method. For each case, the percentage of immunoreactive cells in benign epithelium, PIN, and adenocarcinoma was estimated in increments of 10%. Cox proportional hazards models were used to identify the risk of carcinoma recurrence according to the number of immunoreactive PIN or cancer cells for PSM and PSA; the date of radical prostatectomy was used as the starting time, and serum PSA (biochemical) failure or clinical failure was the event. PSA biochemical failure was defined as serum PSA>> 0.2 ng/mL at least 30 days after surgery.

RESULTS

Intense cytoplasmic immunoreactivity for PSM was observed in the benign and neoplastic epithelial cells in all cases (100% of cases staining). The number of cells staining was lower in benign epithelium and PIN than in adenocarcinoma (69.5+/-17.3% [range, 20-90%] vs. 77.9+/-13.2% [range, 30-100%] vs. 80.2+/-13.7% [range, 30-100%], respectively). With rare exceptions, basal cells were negative, and there was no immunoreactivity of the prostate stroma, urothelium, or vasculature. Adenocarcinoma gave the most intense and extensive staining, and the highest grades of adenocarcinoma (Gleason primary patterns 4 and 5) showed staining in virtually every cell; there was greater heterogeneity of staining in lower grades of adenocarcinoma. By contrast, PSA immunoreactivity was more intense and extensive in benign epithelium than in PIN and adenocarcinoma. The number of immunoreactive PIN or cancer cells for PSM and PSA was not predictive of PSA biochemical or clinical failure as defined in this study.

CONCLUSIONS

PSM was expressed in all cases of prostate adenocarcinoma, with the greatest extent and intensity observed in the highest grades. The expression increased incrementally from benign epithelium to high grade PIN or adenocarcinoma. Conversely, PSA showed the greatest staining in benign epithelium, with decreased expression incrementally from benign epithelium to high grade PIN or adenocarcinoma. Expression of PSM is clinically useful for the identification of prostate epithelium, particularly PIN or adenocarcinoma, and its expression is regulated independent of PSA. The number of PSM immunoreactive cells was not predictive of recurrence, most likely because of the presence of abundant immunoreactivity in most cases, or because of differential expression in primary and metastatic disease.

Biocytin is a biotin-lysine complex of low molecular weight containing about 65% biotin, which retains a high affinity for avidin. Since the latter molecule has been conjugated to several histochemical markers, the use of biocytin as an intracellular marker was investigated. Electrodes were filled with a solution of 4-6% biocytin dissolved in 0.5 M KCl and 0.05 M Tris buffer, pH 7-7.6. Neurons were recorded intracellularly in the supraoptic nucleus of an explant preparation of the rat supraoptico-neurohypophysial system and injected for 1-20 min with either hyperpolarizing or depolarizing current. Following variable recovery times, the explants were fixed in either 10% formalin or 4% paraformaldehyde overnight, sectioned on a vibratome, and incubated with the avidin-biotin complex (ABC) or avidin which had been conjugated to fluorescein, rhodamine, Texas Red or horseradish peroxidase and containing 1% Triton-X 100. A high percentage of injected neurons were recovered using each of the labels with about equal success. Both negative or positive current injection could be used with little electrode clogging. Labeling with fluorescent conjugates was qualitatively similar to that of Lucifer Yellow, whereas labeling with avidin coupled to horseradish peroxidase or with ABC was qualitatively similar to filling neurons directly with horseradish peroxidase. The advantages of this technique are the ease of injection of biocytin and the versatility in allowing the investigator to choose among light-emitting and light-absorbing images.

Cannabinoid type 1 receptors and transient receptor potential vanilloid type 1 channels have been proposed to act as metabotropic and ionotropic receptors, respectively, for two classes of endogenous polyunsaturated fatty acid amides, the acylethanolamides and the acyldopamides. Furthermore, we and others have shown that functional crosstalk occurs between these two receptors when they are expressed in the same cell. Although demonstrated in sensory neurons of the dorsal root ganglia, spinal cord and myenteric neurons, co-expression of cannabinoid type 1 and transient receptor potential vanilloid type 1 has not yet been studied in the brain. In the present study, we addressed this issue by using commercially available specific antibodies whose specificity was confirmed by data obtained with brains from cannabinoid type 1(-/-) and transient receptor potential vanilloid type 1(-/-) mice. Double cannabinoid type 1/transient receptor potential vanilloid type 1 immunofluorescence and single cannabinoid type 1 or transient receptor potential vanilloid type 1 avidin-biotin complex immunohistochemistry techniques were performed and both methods used point to the same results. Cannabinoid type 1/transient receptor potential vanilloid type 1 expression was observed in the hippocampus, basal ganglia, thalamus, hypothalamus, cerebral peduncle, pontine nuclei, periaqueductal gray matter, cerebellar cortex and dentate cerebellar nucleus. In particular, in the hippocampus, cannabinoid type 1/transient receptor potential vanilloid type 1 expression was detected on cell bodies of many pyramidal neurons throughout the CA1-CA3 subfields and in the molecular layer of dentate gyrus. In the cerebellar cortex, expression of cannabinoid type 1/transient receptor potential vanilloid type 1 receptors was found surrounding soma and axons of the vast majority of Purkinje cell bodies, whose cytoplasm was found unstained for both receptors. Cannabinoid type 1 and transient receptor potential vanilloid type 1 immunoreactivity was also detected in: a) the globus pallidus and substantia nigra, in which some intensely transient receptor potential vanilloid type 1 immunopositive cell bodies were found in dense and fine cannabinoid type 1/transient receptor potential vanilloid type 1 positive and cannabinoid type 1 positive nerve fiber meshworks, respectively; b) the cytoplasm of thalamic and hypothalamic neurons; and c) some neurons of the ventral periaqueductal gray. These data support the hypothesis of a functional relationship between the two receptor types in the CNS.

AAV is unique among eukaryotic viruses in the ability of its DNA to integrate preferentially into a specific region of the human genome. Understanding AAV integration may aid in developing gene therapy systems with predictable integration sites. Using a gel mobility-shift assay, we have identified a DNA sequence within the AAV integration locus on human chromosome 19 which is specifically bound by the AAV Rep78 and Rep68 proteins. This Rep recognition sequence is a GCTC repeating motif very similar to sequences within the inverted terminal repeats of the AAV genome which are also bound by Rep78 and Rep68. Cloned oligonucleotides containing the recognition sequence can direct specific binding by Rep proteins. Binding assays with mutant Rep proteins show that the amino-terminal portion of Rep78 and Rep68 can direct binding to either the AAV terminal repeat hairpin DNA or chromosome 19. This human genomic DNA can be complexed with AAV DNA by Rep proteins as demonstrated by a dual-label (32P/biotin) assay. These results suggest a role for Rep in targeting viral integration.

Reversible addition of NO to Cys-sulfur in proteins, a modification termed S-nitrosylation, has emerged as a ubiquitous signaling mechanism for regulating diverse cellular processes. A key first-step toward elucidating the mechanism by which S-nitrosylation modulates a protein's function is specification of the targeted Cys (SNO-Cys) residue. To date, S-nitrosylation site specification has been laboriously tackled on a protein-by-protein basis. Here we describe a high-throughput proteomic approach that enables simultaneous identification of SNO-Cys sites and their cognate proteins in complex biological mixtures. The approach, termed SNOSID (SNO Site Identification), is a modification of the biotin-swap technique [Jaffrey, S. R., Erdjument-Bromage, H., Ferris, C. D., Tempst, P. & Snyder, S. H. (2001) Nat. Cell. Biol. 3, 193-197], comprising biotinylation of protein SNO-Cys residues, trypsinolysis, affinity purification of biotinylated-peptides, and amino acid sequencing by liquid chromatography tandem MS. With this approach, 68 SNO-Cys sites were specified on 56 distinct proteins in S-nitrosoglutathione-treated (2-10 microM) rat cerebellum lysates. In addition to enumerating these S-nitrosylation sites, the method revealed endogenous SNO-Cys modification sites on cerebellum proteins, including alpha-tubulin, beta-tubulin, GAPDH, and dihydropyrimidinase-related protein-2. Whereas these endogenous SNO proteins were previously recognized, we extend prior knowledge by specifying the SNO-Cys modification sites. Considering all 68 SNO-Cys sites identified, a machine learning approach failed to reveal a linear Cys-flanking motif that predicts stable transnitrosation by S-nitrosoglutathione under test conditions, suggesting that undefined 3D structural features determine S-nitrosylation specificity. SNOSID provides the first effective tool for unbiased elucidation of the SNO proteome, identifying Cys residues that undergo reversible S-nitrosylation.

Cell surface oligosaccharides can be engineered to display unusual functional groups for the selective chemical remodeling of cell surfaces. An unnatural derivative of N-acetyl-mannosamine, which has a ketone group, was converted to the corresponding sialic acid and incorporated into cell surface oligosaccharides metabolically, resulting in the cell surface display of ketone groups. The ketone group on the cell surface can then be covalently ligated under physiological conditions with molecules carrying a complementary reactive functional group such as the hydrazide. Cell surface reactions of this kind should prove useful in the introduction of new recognition epitopes, such as peptides, oligosaccharides, or small organic molecules, onto cell surfaces and in the subsequent modulation of cell-cell or cell-small molecule binding events. The versatility of this technology was demonstrated by an example of selective drug delivery. Cells were decorated with biotin through selective conjugation to ketone groups, and selectively killed in the presence of a ricin A chain-avidin conjugate.

Cell kinetic information is an important adjunct to histologically-based tumor classifications. Presently, cell kinetic data can be obtained from slide-based material only with monoclonal antibodies such as Ki-67, which require the use of frozen sections and cannot be applied to archival, paraffin-embedded material. Monoclonal antibodies have recently been generated to PCNA/cyclin, a 36 kd, S-phase-associated nuclear protein. The authors investigated whether monoclonal antibody 19A2 could be used to identify proliferating cells within fixed, embedded tissue sections. Deparaffinized sections of 41 methacarn-fixed human tumors were immunostained with 19A2 using a streptavidin biotin immunoperoxidase system. A semiquantitative scoring system was used to evaluate the fraction of cells that were PCNA/cyclin-positive, and this score was compared with cell kinetic data obtained from parallel flow cytometric S-phase analysis that had been performed on fresh samples of the same tumors. While there was general agreement between the slide-based, antibody-derived and the flow cytometrically-derived cell kinetic information, some discrepancies were observed. Some of the latter represented cases in which the anti-PCNA/cyclin antibody preparations demonstrated significant heterogeneity in the numbers of proliferating cells in different regions of the tumor. In other cases, a significant fraction of the positive cells corresponded to nontumor stromal and/or inflammatory cells. In these cases, the slide-based method provided more information about the tumor cell population than did the flow cytometry data. It is concluded that semiquantitative immunocytochemical analysis with anti-PCNA/cyclin antibodies may represent a simple, reproducible, yet powerful technique for the routine analysis of cell kinetic data in alcohol-fixed, paraffin-embedded tissue by the surgical pathologist.