Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is the causative agent of KS, the second most common AIDS-associated malignancy. KSHV expresses at least 18 different mature microRNAs (miRNAs) during latency. To identify cellular targets of KSHV miRNAs, we have analyzed a previously reported series of microarrays examining changes in cellular gene expression in the presence of KSHV miRNAs. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) receptor (TWEAKR) was among the most consistently and robustly downregulated genes in the presence of KSHV miR-K12-10a (miR-K10a). Results from luciferase assays with reporter plasmids containing the 3' untranslated region (UTR) of TWEAKR suggest a targeting of TWEAKR by miR-K10a. The mutation of two predicted miR-K10a recognition sites within the 3' UTR of TWEAKR completely disrupts inhibition by miR-K10a. The expression of TWEAKR was downregulated in cells transfected with miR-K10a as well as during de novo KSHV infection. In a KS tumor-derived endothelial cell line, the downregulation of TWEAKR by miR-K10a resulted in reduced levels of TWEAK-induced caspase activation. In addition, cells transfected with miR-K10a showed less induction of apoptosis by annexin V staining and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays. Finally, the downregulation of TWEAKR by miR-K10a in primary human endothelial cells resulted in a decrease in levels of expression of the proinflammatory cytokines interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in response to TWEAK. These results identify and validate an important cellular target of KSHV miRNAs. Furthermore, we demonstrate that a viral miRNA protects cells from apoptosis and suppresses a proinflammatory response, which may have significant implications in the complex context of KS lesions.

The present report describes an analysis of two virulence genes of Helicobacter pylori. Parts of the cagA gene, as well as parts from the signal (s) and middle (m) regions of the mosaic vacA gene, were amplified with biotin-labelled PCR primers and the products were subsequently analyzed by a single-step reverse hybridization line probe assay (LiPA). This assay comprises a strip containing multiple specific probes for the vacA s region (sla, slb, and s2 alleles), the vacA m region (ml and m2 alleles), and the cagA gene. A total of 103 H. pylori-positive materials, including cultured isolates, gastric biopsy specimens, and surgical specimens from patients living in Portugal (n = 55) and The Netherlands (n = 48) were tested by the PCR-LiPA. cagA was detected in 84 and 73% of the Portuguese and Dutch patients, respectively. vacA typing results, as determined by reverse hybridization, were completely concordant with those of sequence analysis. Most Portuguese patients (72%) contained type slb, whereas most Dutch patients (61%) contained type sla (P < 0.001). The method is also very effective at detecting the presence of multiple genotypes in a single biopsy specimen. The prevalence of multiple strains in Portuguese patient samples was significantly higher (29%) than that in Dutch patient samples (8%) (P = 0.001). There was a significant association between the presence of ulcers or gastric carcinoma and the presence of vacA type sl (sla or slb; P = 0.008) and cagA (P = 0.003) genes.

Phosphatidylserine (PS) normally localizes to the inner leaflet of cell membranes but becomes exposed in abnormal or apoptotic cells, signaling macrophages to ingest them. Along similar lines, it seemed possible that the removal of red cells from circulation because of normal aging or in hemolytic anemias might be triggered by PS exposure. To investigate the role of PS exposure in normal red cell aging, we used N-hydroxysuccinimide-biotin to tag rabbit red cells in vivo, then used phycoerythrin-streptavidin to label the biotinylated cells, and annexin V-fluorescein isothiocyanate (FITC) to detect the exposed PS. Flow cytometric analysis of these cells drawn at 10-day intervals up to 70 days after biotinylation indicated that older, biotinylated cells expose more PS. Furthermore, our data match a simple model of red cell senescence that assumes both an age-dependent destruction of senescent red cells preceded by several hours of PS exposure and a random destruction of red cells without PS exposure. By using this model, we demonstrated that the exposure of PS parallels the rate at which biotinylated red cells are removed from circulation. On the other hand, using an annexin V-FITC label and flow cytometry demonstrates that exposed PS does not cause the reduced red cell life span of patients with hemolytic anemia, with the possible exception of those with unstable hemoglobins or sickle cell anemia. Thus, in some cases PS exposure on the cell surface may signal the removal of red cells from circulation, but in other cases some other signal must trigger the sequestration of cells.

Paraformaldehyde-fixed tissue from mouse cerebellum was hybridized with biotin-labeled satellite DNA for identification of centromeres. By using avidin-peroxidase conjugates, it was possible to define the nuclear position of centromeres at the ultrastructural level. Three-dimensional analysis of well-resolved centromere arrays were aided by computer reconstruction of serial sections. Different cell types displayed distinct, nonrandom centromere locations. In Purkinje neurons, the majority of detected sequences were clustered together around the central nucleolus, whereas in granule neurons, more numerous, dispersed centromere clusters were associated with the nuclear membrane. In Purkinje cells, peroxidase-labeled regions corresponded to dense heterochromatic aggregates were detected in Purkinje cells of several different species. These observations suggest that in these highly differentiated cells, the nuclear position of centromeres is maintained in evolution despite species differences in centromeric DNA sequence. Such defined ordering of centromeres may be integral to specific functional capacities.

An array of aligned carbon nanotubes (CNTs) was incorporated across a polymer film to form a well-ordered nanoporous membrane structure. This membrane structure was confirmed by electron microscopy, anisotropic electrical conductivity, gas flow, and ionic transport studies. The measured nitrogen permeance was consistent with the flux calculated by Knudsen diffusion through nanometer-scale tubes of the observed microstructure. Data on Ru(NH3)6(3+) transport across the membrane in aqueous solution also indicated transport through aligned CNT cores of the observed microstructure. The lengths of the nanotubes within the polymer film were reduced by selective electrochemical oxidation, allowing for tunable pore lengths. Oxidative trimming processes resulted in carboxylate end groups that were readily functionalized at the entrance to each CNT inner core. Membranes with CNT tips that were functionalized with biotin showed a reduction in Ru(NH3)6(3+) flux by a factor of 15 when bound with streptavidin, thereby demonstrating the ability to gate molecular transport through CNT cores for potential applications in chemical separations and sensing.

The purpose of this study is to investigate the efficacy and the mechanism of Hsp90 inhibition of Withaferin A (WA), a steroidal lactone occurring in Withania somnifera, in pancreatic cancer in vitro and in vivo. Withaferin A exhibited potent antiproliferative activity against pancreatic cancer cells in vitro (with IC(50)s of 1.24, 2.93 and 2.78 microM) in pancreatic cancer cell lines Panc-1, MiaPaCa2 and BxPc3, respectively. Annexin V staining showed that WA induced significant apoptosis in Panc-1 cells in a dose-dependent manner. Western blotting demonstrated that WA inhibited Hsp90 chaperone activity to induce degradation of Hsp90 client proteins (Akt, Cdk4 and glucocorticoid receptor), which was reversed by the proteasomal inhibitor, MG132. WA-biotin pull down assay of Hsp90 using Panc-1 cancer cell lysates and purified Hsp90 showed that WA-biotin binds to C-terminus of Hsp90 which was competitively blocked by unlabeled WA. Co-immunoprecipitation exhibited that WA (10 microM) disrupted Hsp90-Cdc37 complexes from 1 to 24h post-treatment, while it neither blocked ATP binding to Hsp90, nor changed Hsp90-P23 association. WA (3, 6mg/kg) inhibited tumor growth in pancreatic Panc-1 xenografts by 30% and 58%, respectively. These data demonstrate that Withaferin A binds Hsp90, inhibits Hsp90 chaperone activity through an ATP-independent mechanism, results in Hsp90 client protein degradation, and exhibits in vivo anticancer activity against pancreatic cancer.

Protein sulfenic acids are reactive intermediates in the catalytic cycles of many enzymes as well as the in formation of other redox states. Sulfenic acid formation is a reversible post-translational modification with potential for protein regulation. Dimedone (5,5-dimethyl-1,3-cyclohexanedione) is commonly used in vitro to study sulfenation of purified proteins, selectively "tagging" them, allowing monitoring by mass spectrometry. However dimedone is of little use in complex protein mixtures because selective monitoring of labeling is not possible. To address this issue, we synthesized a novel biotinylated derivative of dimedone, keeping the dione cassette required for sulfenate reactivity but adding the functionality of a biotin tag. Biotin-amido(5-methyl-5-carboxamidocyclohexane 1,3-dione) tetragol (biotin dimedone) was prepared in six steps, combining 3,5-dimethoxybenzoic acid (Birch reduction, ultimately leading to the dimedone unit with a carboxylate functionality), 1-amino-11-azido-3,6,9-trioxaundecane (a differentially substituted tetragol spacer), and biotin. We loaded biotin dimedone (0.1 mm, 30 min) into rat ventricular myocytes, treated them with H(2)O(2) (0.1-10,000 microm, 5 min), and monitored derivatization on Western blots using streptavidin-horseradish peroxidase. There was a dose-dependent increase in labeling of multiple proteins that was maximal at 0.1 or 1 mm H(2)O(2) and declined sharply below basal with 10 mm treatment. Cell-wide labeling was observed in fixed cells probed with avidin-FITC using a confocal fluorescence microscope. Similar H(2)O(2)-induced labeling was observed in isolated rat hearts. Hearts loaded and subjected to hypoxia showed a striking loss of labeling, which returned when oxygen was resupplied, highlighting the protein sulfenates as oxygen sensors. Cardiac proteins that were sulfenated during oxidative stress were purified with avidin-agarose and identified by separation of tryptic digests by liquid chromatography with on-line analysis by mass spectrometry.

High concentrations of glucose are considered to be toxic for the pancreatic beta-cell. However, the mechanisms underlying beta-cell dysfunction and resulting cell death are not fully characterized. In the present study we have demonstrated that incubation of pancreatic islets and beta-cells from ob/ob mice and Wistar rats with glucose induced a process of apoptotic beta-cell death, as shown by DNA laddering, TdT-mediated dUTP-biotin nick end-labeling (TUNEL) technique, and by using DNA-staining dye HOECHST 33342. The obtained results show that the percentage of apoptotic cells was dependent on glucose concentration, being minimal at 11 mM glucose. At a concentration of 100 microM, aurintricarboxylic acid, an inhibitor of endonuclease activity, almost completely inhibited apoptosis triggered by 17 mM glucose. We have also shown that long term incubation with 100 microM sulfonylurea, tolbutamide, triggered apoptosis in pancreatic beta-cells. The process of beta-cell death induced by high glucose concentration and tolbutamide were Ca2+-dependent, because introduction to the culture medium of 50 microM D-600 or 200 microM diazoxide, which blocked glucose- and tolbutamide-induced [Ca2+]i increase, inhibited apoptosis. Thus, this study shows for the first time that high glucose concentrations and tolbutamide induce apoptosis in pancreatic beta-cells, and that this process is Ca2+-dependent.

Methanosphaera stadtmaniae is a non-motile, Gram-positive spherical-shaped organism that obtains energy for growth by using hydrogen to reduce methanol to methane. It does not produce methane from hydrogen and carbon dioxide, formate, acetate or methylamines and cannot grow with hydrogen and carbon monoxide, nitrate, fumarate, sulfate or choline. Its pH optimum is 6.5 to 6.9 and its temperature optimum is 36 degrees to 40 degrees C. It is not inhibited by bile salts, inhibitors of the synthesis of folic acid coenzymes, cephalothin or clindamycin but is inhibited by metronidazole, bacitracin, monensin, lasalocid, or bromoethanesulfonate. It requires acetate, carbon dioxide, isoleucine, ammonium, and thiamin for growth and biotin is stimulatory. It does not contain cytochromes and the mol % G + C of its DNA is 25.8. The composition of its cell wall and 16 S rRNA and its immunological fingerprint are consistent with characterization of the organism as a member of a new genus of the family Methanobacteriaceae. The habitat of the type strain is the human large intestine.

The attractive properties of avidin (streptavidin) and biotin, in particular their strong affinities (Kd = 10(-15)M), may be used to advantage in imaging applications. These molecules have been used in this preliminary investigation to improve the targeting of 111In in animals. Antibodies have been conjugated with biotin and administered unlabeled while, at a later time, the radiolabel was administered attached to DTPA-coupled avidin or streptavidin. An alternative procedure was also considered whereby the antibodies were conjugated with avidin and administered before the administration of radiolabeled biotin. Using a model in which the target consisted of conjugated agarose beads deposited in the peritoneum of mice, it has been shown that the target/nontarget radioactivity ratios may be significantly improved with respect to the conventional procedures through the use of this approach.

OBJECTIVE

The receptor for advanced glycation end products (RAGE) binds multiple ligands, including S100 proteins, high mobility group box chromosomal protein 1 (HMGB-1), and AGEs, all of which are present in articular cartilage. Stimulation of RAGE signaling can lead to MAP kinase activation and increased NF-kappaB activity. The objective of the present study was to determine if chondrocytes express functional RAGE.

METHODS

The presence of chondrocyte RAGE was analyzed by immunohistochemistry using normal and osteoarthritic (OA) cartilage from young and old monkeys and humans, immunoblotting of chondrocyte lysates and human cartilage extracts, and reverse transcription-polymerase chain reaction (RT-PCR) analysis of RNA from chondrocytes treated with interleukin-1 (IL-1) and fibronectin fragments. RAGE signaling was evaluated by stimulating chondrocytes with S100B and HMGB-1 and analyzing for activation of the ERK MAP kinase and NF-kappaB. The ability of S100B and HMGB-1 to stimulate matrix metalloproteinase 13 (MMP-13) production was also assessed. A pull-down assay using biotin-labeled S100B was used to demonstrate binding to RAGE.

RESULTS

RAGE was detected in sections of monkey knee cartilage and human knee and ankle cartilage. Increased immunostaining for RAGE was noted in cartilage from older adult monkeys and humans and was further increased in OA tissue. RAGE was also detected by immunoblotting and by RT-PCR, where IL-1beta and fibronectin fragments were found to stimulate RAGE expression. Stimulation of chondrocytes with S100B or HMGB-1 increased phosphorylation of the ERK MAP kinase and the p65 subunit of NF-kappaB and increased the production of MMP-13. This signaling was inhibited in cells pretreated with soluble RAGE, and S100B was shown to bind to chondrocyte RAGE.

CONCLUSIONS

Articular chondrocytes express functional RAGE. The increase in RAGE noted in OA cartilage and the ability of RAGE ligands to stimulate chondrocyte MAP kinase and NF-kappaB activity and to stimulate MMP-13 production suggests that chondrocyte RAGE signaling could play a role in OA.

The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and protecting the brain from toxic substances. Breakdown of this barrier results in severe brain pathologies, whereas impermeability of the BBB is a major obstacle for drug delivery to the brain. Despite its importance, our understanding of the maturation and modulation of the BBB is limited. Zebrafish (Danio rerio) has emerged as a useful model organism for studying vertebrate development and disease mechanisms, as well as for preclinical drug screening. However, the nature of the BBB has not yet been examined in teleost fish. In this paper, we report that with the exception of the circumventricular organs, the cerebral microvessels in zebrafish are impermeable to sulfo-NHS-biotin and horseradish peroxidase (HRP). Brain endothelial cells show immunoreactivity to Claudin-5 and Zonula Occludens-1 (ZO-1), implying the presence of tight junctions in these cells. The expression of Claudin-5 and ZO-1 was detected in cerebral microvessels from 3 days post-fertilization (dpf), concomitant with maturation of the BBB, as determined by restricted permeability to HRP and various fluorescent tracers. Real-time analysis of fluorescent tracer leakage in embryonic zebrafish suggests that they may be used as an in vivo model for BBB breakdown. Taken together, our results show that the endothelial tight junction-based BBB of zebrafish is similar to that of higher vertebrates and thus, zebrafish may be an excellent genetic and experimental model organism for studying development and maintenance of the BBB.

Apurinic/apyrimidinic (AP) sites are common DNA lesions that arise from spontaneous depurination or by base excision repair (BER) of modified bases. A biotin-containing aldehyde-reactive probe (ARP) [Kubo, K., Ide, H., Wallace, S. S. & Kow, Y. W. (1992) Biochemistry 31, 3703-3708] is used to measure AP sites in living cells. ARP penetrates the plasma membrane of cells and reacts with AP sites in DNA to form a stable ARP-DNA adduct. The DNA is isolated and treated with avidin-horseradish peroxidase (HRP), forming a DNA-HRP complex at each biotin residue, which is rapidly separated from free avidin-HRP by selective precipitation with a DNA precipitating dye (DAPER). The number of AP sites is estimated by HRP activity toward chromogenic substrate in an ELISA assay. The assay integrates the AP sites formed by the different glycosylases of BER during a 1-h incubation and eliminates artifactual depurination or loss of AP sites during DNA isolation. The assay was applied to living cells and nuclei. The number of AP sites after a 1-h incubation in old IMR90 cells was about two to three times higher than that in young cells, and the number in human leukocytes from old donors was about seven times that in young donors. The repair of AP sites was slower in senescent compared with young IMR90 cells. An age-dependent decline is shown in the activity of the glycosylase that removes methylated bases in IMR90 cells and in human leukocytes. The decline in excision of methylated bases from DNA suggests an age-dependent decline in 3-methyladenine DNA glycosylase, a BER enzyme responsible for removing alkylated bases.

We hypothesize that the efficacy of doxorubicin (DOX) can be maximized and dose-limiting cardiotoxicity minimized by controlled release from PEGylated nanoparticles. To test this hypothesis, a unique surface modification technique was used to create PEGylated poly(lactic-co-glycolic acid) (PLGA) nanoparticles encapsulating DOX. An avidin-biotin coupling system was used to control poly(ethylene glycol) conjugation to the surface of PLGA nanoparticles, of diameter approximately 130 nm, loaded with DOX to 5% (wt/wt). Encapsulation in nanoparticles did not compromise the efficacy of DOX; drug-loaded nanoparticles were found to be at least as potent as free DOX against A20 murine B-cell lymphoma cells in culture and of comparable efficacy against subcutaneously implanted tumors. Cardiotoxicity in mice as measured by echocardiography, serum creatine phosphokinase (CPK), and histopathology was reduced for DOX-loaded nanoparticles as compared with free DOX. Administration of 18 mg/kg of free DOX induced a sevenfold increase in CPK levels and significant decreases in left ventricular fractional shortening over control animals, whereas nanoparticle-encapsulated DOX produced none of these pathological changes.

UNASSIGNED

The efficacy of doxorubicin (DOX) may be maximized and dose-limiting cardiotoxicity minimized by controlled release from PEGylated nanoparticles. Administration of 18 mg/kg of free DOX induced a sevenfold increase in CPK levels and significant decreases in left ventricular fractional shortening in mice, whereas nanoparticle-encapsulated DOX produced none of these pathological changes.

Protein interaction networks and protein compartmentalization underlie all signaling and regulatory processes in cells. Enzyme-catalyzed proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in living cells. However, current PL methods require over 18 h of labeling time or utilize chemicals with limited cell permeability or high toxicity. We used yeast display-based directed evolution to engineer two promiscuous mutants of biotin ligase, TurboID and miniTurbo, which catalyze PL with much greater efficiency than BioID or BioID2, and enable 10-min PL in cells with non-toxic and easily deliverable biotin. Furthermore, TurboID extends biotin-based PL to flies and worms.

We have evaluated the utility of the hepatoma-derived hybrid cell line, WIF-B, for in vitro studies of polarized hepatocyte functions. The majority >> 70%) of cells in confluent culture formed closed spaces with adjacent cells. These bile canalicular-like spaces (BC) accumulated fluorescein, a property of bile canaliculi in vivo. By indirect immunofluorescence, six plasma membrane (PM) proteins showed polarized distributions similar to rat hepatocytes in situ. Four apical PM proteins were concentrated in the BC membrane of WIF-B cells. Microtubules radiated from the BC (apical) membrane, and actin and foci of gamma-tubulin were concentrated in this region. The tight junction-associated protein ZO-1 was present in belts marking the boundary between apical and basolateral PM domains. We explored the functional properties of this boundary in living cells using fluorescent membrane lipid analogs and soluble tracers. When cells were incubated at 4 degrees C with a fluorescent analog of sphingomyelin, only the basolateral PM was labeled. In contrast, when both PM domains were labeled by de novo synthesis of fluorescent sphingomyelin from ceramide, fluorescent lipid could only be removed from the basolateral domain. These data demonstrate the presence of a barrier to the lateral diffusion of lipids between the PM domains. However, small soluble FITC-dextrans (4,400 mol wt) were able to diffuse into BC, while larger FITC-dextrans were restricted to various degrees depending on their size and incubation temperature. At 4 degrees C, the surface labeling reagent sNHS-LC-biotin (557 mol wt) had access to the entire PM, but streptavidin (60,000 mol wt), which binds to biotinylated molecules, was restricted to only the basolateral domain. Such differential accessibility of well-characterized probes can be used to mark each membrane domain separately. These results show that WIF-B cells are a suitable model to study membrane trafficking and targeting in hepatocytes in vitro.

In mammalian tissues, circadian gene expression can be driven by local oscillators or systemic signals controlled by the master pacemaker in the suprachiasmatic nucleus. We show that simulated body temperature cycles, but not peripheral oscillators, controlled the rhythmic expression of cold-inducible RNA-binding protein (CIRP) in cultured fibroblasts. In turn, loss-of-function experiments indicated that CIRP was required for high-amplitude circadian gene expression. The transcriptome-wide identification of CIRP-bound RNAs by a biotin-streptavidin-based cross-linking and immunoprecipitation (CLIP) procedure revealed several transcripts encoding circadian oscillator proteins, including CLOCK. Moreover, CLOCK accumulation was strongly reduced in CIRP-depleted fibroblasts. Because ectopic expression of CLOCK improved circadian gene expression in these cells, we surmise that CIRP confers robustness to circadian oscillators through regulation of CLOCK expression.

The objective of this study was to determine the incidence of DNA fragmentation in human sperm, and to correlate any detected DNA damage with semen analysis parameters and fertilization rates in in vitro fertilization (IVF). A total of 298 semen samples were collected from men in the infertility program at The Toronto Hospital. For each sample, the percentage of sperm with DNA fragmentation was determined using the method of terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) and fluorescence-activated cell sorting. The percentage of sperm with fragmented DNA was less than 4% in the majority of samples but ranged from 5% to 40% in approximately 27% of the samples. A negative correlation was found between the percentage of DNA fragmentation and the motility, morphology, and concentration of the ejaculated sperm. In 143 IVF samples, a significant negative association was also found between the percentage of sperm with DNA fragmentation and fertilization rate (p = 0.008) and embryo cleavage rate (p = 0.01). In addition, 35 men who smoked demonstrated an increased percentage of sperm with fragmented DNA (4.7 +/- 1.2%) as compared to 78 nonsmokers (1.1 +/- 0.2%; p = 0.01). These results demonstrate a negative association between semen analysis parameters and sperm with fragmented DNA. Since extremely poor semen samples are the indication for intracytoplasmic sperm injection, there is a high likelihood that sperm with fragmented DNA may be selected by chance and used for oocyte injection, resulting in poor fertilization and/or cleavage rates.

RNA affinity tags would be very useful for the study of RNAs and ribonucleoproteins (RNPs) as a means for rapid detection, immobilization, and purification. To develop a new affinity tag, streptavidin-binding RNA ligands, termed "aptamers," were identified from a random RNA library using in vitro selection. Individual aptamers were classified into two groups based on common sequences, and representative members of the groups had sufficiently low dissociation constants to suggest they would be useful affinity tools. Binding of the aptamers to streptavidin was blocked by presaturation of the streptavidin with biotin, and biotin could be used to dissociate RNA/streptavidin complexes. To investigate the practicality of using the aptamer as an affinity tag, one of the higher affinity aptamers was inserted into RPR1 RNA, the large RNA subunit of RNase P. The aptamer-tagged RNase P could be specifically isolated using commercially available streptavidin-agarose and recovered in a catalytically active form when biotin was used as an eluting agent under mild conditions. The aptamer tag was also used to demonstrate that RNase P exists in a monomeric form, and is not tightly associated with RNase MRP, a closely related ribonucleoprotein enzyme. These results show that the streptavidin aptamers are potentially powerful tools for the study of RNAs or RNPs.

We report an advanced chemoenzymatic strategy for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc-modified proteins. O-GlcNAc residues are selectively labeled with fluorescent or biotin tags using an engineered galactosyltransferase enzyme and [3 + 2] azide-alkyne cycloaddition chemistry. We demonstrate that this approach can be used for direct in-gel detection and mass spectrometric identification of O-GlcNAc proteins, identifying 146 novel glycoproteins from the mammalian brain. Furthermore, we show that the method can be exploited to quantify dynamic changes in cellular O-GlcNAc levels and to image O-GlcNAc-glycosylated proteins within cells. As such, this strategy enables studies of O-GlcNAc glycosylation that were previously inaccessible and provides a new tool for uncovering the physiological functions of O-GlcNAc.

Androgens are essential for the maintenance of normal spermatogenesis in the rat. We assessed the sites, developmental pattern, and hormonal control of androgen receptors (AR) in the rat testis. Adult male rats were studied after 1) no treatment; 2) ethane dimethane sulfonate (EDS), which eradicates Leydig cells and endogenous testosterone (T); 3) EDS plus T replacement beginning at the time of EDS administration; or 4) methoxyacetic acid, which leads to the loss of specific germ cell types. Testes were also obtained from normal immature rats (aged 5, 14, 16, 21, 28, 31, 35, 38, and 45 days). After microwave antigen retrieval, immunohistochemistry was performed using a rabbit polyclonal antibody (Novocastra) raised against a peptide unique to the N-terminal region of the AR and detection with biotinylated swine antirabbit immunoglobulin G, avidin-biotin complex/alkaline phosphatase, and nitroblue tetrazolium salt (NBT)/5 bromo-4-chloro-3-indolylphosphate (BCIP) substrate. In adults, nuclear immunostaining of Sertoli cells (SC) increased progressively in intensity from stages II through VII of the spermatogenic cycle, and then declined precipitously during stage VIII to become barely detectable in stages IX-XIII. Prominent AR immunostaining was also evident in peritubular myoid cells, arterioles, and interstitial cells; staining in these cells did not vary with the stage of the cycle of the adjacent tubules. EDS caused a severe loss of AR immunostaining in all cell types. Replacement of T in EDS-treated animals resulted in a pattern of AR immunostaining comparable to that in controls, although staining intensity was reduced. Methoxyacetic acid administration did not affect the pattern of AR staining. In immature rats, peritubular myoid cell immunostaining was prominent from day 5; SC staining was detectable on day 5, increased in intensity with age, and became stage dependent between days 21-35. The following conclusions were reached. 1) Immunohistochemically detectable AR expression in SC occurs predominantly in stages II-VII of the spermatogenic cycle, with highest levels at stage VII. 2) AR immunostaining is also prominent in peritubular myoid cells, arterioles, and Leydig cells (but not in germ cells), but is unrelated to the stage of adjacent tubules. 3) Endogenous T and/or its metabolites control the expression of AR in the testis. 4) AR immunostaining is detectable by day 5 of age and becomes stage specific in SC between days 21-35.

Despite their high DNA identity and a proposal to group classical Brucella species as biovars of Brucella melitensis, the commonly recognized Brucella species can be distinguished by distinct biochemical and fatty acid characters, as well as by a marked host range (e.g., Brucella suis for swine, B. melitensis for sheep and goats, and Brucella abortus for cattle). Here we present the genome of B. abortus 2308, the virulent prototype biovar 1 strain, and its comparison to the two other human pathogenic Brucella species and to B. abortus field isolate 9-941. The global distribution of pseudogenes, deletions, and insertions supports previous indications that B. abortus and B. melitensis share a common ancestor that diverged from B. suis. With the exception of a dozen genes, the genetic complements of both B. abortus strains are identical, whereas the three species differ in gene content and pseudogenes. The pattern of species-specific gene inactivations affecting transcriptional regulators and outer membrane proteins suggests that these inactivations may play an important role in the establishment of host specificity and may have been a primary driver of speciation in the genus Brucella. Despite being nonmotile, the brucellae contain flagellum gene clusters and display species-specific flagellar gene inactivations, which lead to the putative generation of different versions of flagellum-derived structures and may contribute to differences in host specificity and virulence. Metabolic changes such as the lack of complete metabolic pathways for the synthesis of numerous compounds (e.g., glycogen, biotin, NAD, and choline) are consistent with adaptation of brucellae to an intracellular life-style.

IgA1 proteins from sera of patients with IgA nephropathy (IgAN) are galactosylated to a lesser degree than those from healthy controls. The increased reactivity of intact or de-sialylated serum IgA1 with N-acetylgalactosamine (GalNAc)-specific lectins, Helix aspersa (HAA) and Caragana arborescens (CAA) and de-sialylated IgA1 with Helix pomatia (HPA) and Bauhinia purpurea (BPA) indicated that the Gal deficiency is in glycans located in the hinge region of IgA1 molecules. De-sialylated IgA from sera of 81 IgAN patients bound biotin-labeled lectin HAA more effectively than did de-sialylated IgA from 56 healthy controls (P < 0.0001). Similar results were observed for 67 IgAN patients and 52 controls with second lectin, CAA (P < 0.001). The binding patterns for 9 patients with mesangial proliferative glomerulonephritis of non-IgA origin were similar to those for controls. Incompletely galactosylated IgA1 capable of binding GalNAc-specific lectins was detected in complexes with IgG as demonstrated by ELISA, size-exclusion chromatography and sucrose gradient ultracentrifugation. The formation of IgA1-IgG complexes may affect the serum level of IgA1 by reducing the rate of its elimination and catabolic degradation by the liver.