To understand the RNA expression in response to acid stress of Helicobacter pylori in genomic scale, a microarray membrane containing 1,534 open reading frames (ORFs) from strain 26695 was used. Total RNAs of H. pylori under growth conditions of pH 7.2 and 5.5 were extracted, reverse transcribed into cDNA, and labeled with biotin. Each microarray membrane was hybridized with cDNA probe from the same strain under two different pH conditions and developed by a catalyzed reporter deposition method. Gene expression of all ORFs was measured by densitometry. Among the 1,534 ORFs, 53 ORFs were highly expressed >> or = 30% of rRNA control in densitometry ratios). There were 445 ORFs which were stably expressed (<30% of rRNA in densitometry) under both pH conditions without significant variation. A total of 80 ORFs had significantly increased expression levels at low pH, while expressions of 4 ORFs were suppressed under acidic condition. The remaining 952 ORFs were not detectable under either pH condition. These data were highly reproducible and comparable to those obtained by the RNA slot blot method. Our results suggest that microarray can be used in monitoring prokaryotic gene expression in genomic scale.

Neurons exhibiting GABA-like immunoreactivity were identified in the monkey amygdala using an avidin-biotin immunohistochemical technique. The pattern of GABA immunoreactivity was very similar in the basolateral and superficial amygdaloid nuclei. In these regions GABA-positive cells were nonpyramidal neurons that were often arranged in clusters or curvilinear rows. These GABA-positive nonpyramidal neurons constituted about 25% of the total neuronal population of the basolateral and superficial amygdaloid nuclei. Numerous GABA-positive puncta resembling axon terminals were observed both in the neuropil and encapsulating the perikarya of GABA-negative pyramidal cells. The pattern of GABA-like immunoreactivity was different in the central and medial amygdaloid nuclei. These regions contained a very dense array of GABA-positive puncta. There were numerous GABA-positive neurons in the lateral subdivision of the central nucleus and fewer cells in the medial nucleus and medial subdivision of the central nucleus. Many immunoreactive puncta were observed contacting the perikarya and dendrites of GABA-positive cells in these regions. The intercalated nuclei consisted of numerous, small, GABA-positive neurons and a few, larger, GABA-negative cells. Both cell types were contacted by GABA-positive puncta. This study indicates that neuronal subpopulations in each of the amygdaloid nuclei of the monkey are GABAergic. The pattern of immunoreactivity varies in different amygdaloid regions and is very similar to that described in the rat. Certain aspects of the functional organization of this rich GABAergic circuitry can be elucidated by correlating the findings of the present investigation with previous anatomical, physiological, and pharmacological studies of the amygdala.

The syntheses are described of two types of linker molecule useful for the specific attachment of non-radioactive labels such as biotin and fluorophores to the 5' terminus of synthetic oligodeoxyribonucleotides. The linkers are designed such that they can be coupled to the oligonucleotide as a final step in solid-phase synthesis using commercial DNA synthesis machines. Increased sensitivity of biotin detection was possible using an anti-biotin hybridoma/peroxidase detection system.

Chicken avidin and bacterial streptavidin, (strept)avidin, are proteins widely utilized in a number of applications in life science, ranging from purification and labeling techniques to diagnostics, and from targeted drug delivery to nanotechnology. (Strept)avidin-biotin technology relies on the extremely tight and specific affinity between (strept)avidin and biotin (dissociation constant, K(d) approximately 10(-14)-10(-16) M). (Strept)avidins are also exceptionally stable proteins. To study their ligand binding and stability characteristics, the two proteins have been extensively modified both chemically and genetically. There are excellent accounts of this technology and chemically modified (strept)avidins, but no comprehensive reviews exist concerning genetically engineered (strept)avidins. To fill this gap, we here go through the genetically engineered (strept)avidins, summarizing how these constructs were designed and how they have improved our understanding of the structural and functional characteristics of these proteins, and the benefits they have provided for (strept)avidin-biotin technology.

OBJECTIVE

To investigate human umbilical cord-derived mesenchymal stem cells (hUCMSCs) for use in the reversal of mouse hepatic injury.

METHODS

Human umbilical cord-derived mesenchymal stem cells, characterized by flow cytometry, were transplanted into carbon tetrachloride (CCl(4))-injured mice, and then followed for determination of localization and differentiation. Reverse transcriptase-polymerase chain reaction for the human 17alpha gene and fluorescence in situ hybridization analysis for the human X chromosome were used to locate exogenous hUCMSCs in mouse livers. Peripheral blood and liver specimens were collected at 7, 14 and 21 days after transplantation. For evaluating the recovery of injured liver tissues, serum aminotransferase was measured, and the pathological state of the hepatocytes was assessed.

RESULTS

The hUCMSCs were positive for the human MSC-specific markers CD13, CD29, CD44, CD105 and nerve growth factor receptor, but negative for the haematopoietic lineage markers CD31, CD34, CD38, CD45 and HLA-DR. Under conditions favouring differentiation in vivo, the expression of tryptophan 2,3-dioxygenase, human alpha-fetoprotein, cytokeratin 18, fibroblast secretory protein 1 and alpha-smooth-muscle-actin was detectable after hUCMSCs administration to mice subjected to liver injury. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labelling and proliferating cell nuclear antigen staining showed that transplanted hUCMSCs could inhibit hepatocyte apoptosis and facilitate proliferation. Serum aminotransferases were decreased after transplantation of hUCMSCs into the injured mice, and hepatocyte denaturation was reduced.

CONCLUSIONS

Human umbilical cord-derived mesenchymal stem cells can enhance recovery of CCl(4)-injured mouse liver, providing evidence that such therapy could be useful for liver disorders or injury.

Release of mitochondrial cytochrome c into the cytosol is a critical step in apoptosis. We have reported that early release of cytochrome c in vivo occurs after permanent focal cerebral ischemia (FCI) and is mediated by the mitochondrial antioxidant manganese superoxide dismutase (SOD). However, the role of reactive oxygen species produced after ischemia-reperfusion in the mitochondrial apoptosis process is still unknown, although overexpression of copper/zinc-SOD (SOD1), a cytosolic isoenzyme, protects against ischemia-reperfusion. We now hypothesize that the overexpression of SOD1 also prevents apoptosis after FCI. To address this issue, we examined the subcellular distribution of the cytochrome c protein in both wild-type mice and in SOD1 transgenic (Tg) mice after transient FCI. Cytosolic cytochrome c was detected as early as 2 hr after reperfusion, and correspondingly, mitochondrial cytochrome c was significantly reduced after FCI. Cytosolic cytochrome c was significantly lower in the SOD1 Tg mice compared with wild types 2 (p < 0.0001) and 4 (p < 0.05) hr after FCI. Apaf-1, which interacts with cytochrome c and activates caspases, was constitutively expressed in both groups of animals, with no alteration after FCI. Double staining with cytochrome c immunohistochemistry and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed a spatial relationship between cytosolic cytochrome c expression and DNA fragmentation. A significant amount of DNA laddering was detected 24 hr after ischemia and was reduced in SOD1 Tg mice. These data suggest that SOD1 blocks cytosolic release of cytochrome c and could thereby reduce apoptosis after transient FCI.

Connective tissue growth factor (CTGF) is a member of an emerging CCN gene family that is implicated in various diseases associated with fibro-proliferative disorder including scleroderma and atherosclerosis. The function of CTGF in human cancer is largely unknown. We now show that CTGF induces apoptosis in the human breast cancer cell line MCF-7. CTGF mRNA was completely absent in MCF-7 but strongly induced by treatment with transforming growth factor beta (TGF-beta). TGF-beta by itself induced apoptosis in MCF-7, and this effect was reversed by co-treatment with CTGF antisense oligonucleotide. Overexpression of CTGF gene in transiently transfected MCF-7 cells significantly augmented apoptosis. Moreover, recombinant CTGF protein significantly enhanced apoptosis in MCF-7 cells as evaluated by DNA fragmentation, Tdt-mediated dUTP biotin nick end-labeling staining, flow cytometry analysis, and nuclear staining using Hoechst 33258. Finally, recombinant CTGF showed no effect on Bax protein expression but significantly reduced Bcl2 protein expression. Taken together, these results suggest that CTGF is a major inducer of apoptosis in the human breast cancer cell line MCF-7 and that TGF-beta-induced apoptosis in MCF-7 cells is mediated, in part, by CTGF.

Currently, serum prostate-specific antigen (PSA) is used for the early detection of prostate cancer despite its low specificity in the range of 4-10 ng/mL. Because aberrant glycosylation is a fundamental characteristic of tumor genesis, the objective of this study was to investigate whether changes in PSA glycosylation may be used to improve the cancer specificity of PSA. We developed five lectin immunosorbant assays to analyze the glycosylation patterns of PSA in serum. Each assay sandwiches serum PSA between a PSA monoclonal antibody and a biotinylated lectin and then tags the biotin complex using a streptavidin SULFO TAG for electrochemiluminescence detection. Low limits of detection (0.04-1.35 ng/mL), good reproducibility (%CVs < 10%), and direct analysis of PSA glycosylation in sera suggest these assays may have a potential role in improving PSA's cancer specificity. Clinical performance was evaluated in 52 human subjects (26 cancer and 26 noncancer). ROC analysis showed that the total SNA assay (AUC = 0.71) appeared to perform better than percent free PSA (AUC = 0.54) in its diagnostic gray zone between 10 and 20% in a subset of 21 subjects. A separate study of 16 additional subjects showed similar findings.

The serine-threonine kinase, Akt, is involved in the survival signaling pathways in many cell systems. The present study examined phosphorylation of Akt at serine-473 and DNA fragmentation after traumatic brain injury (TBI) in mice. Immunohistochemistry showed phospho-Akt was decreased in the injured cortex 1 h after TBI, whereas it was temporally increased at 4 h in the perifocal damaged cortex. In the CA1 region of the hippocampus, phospho-Akt was increased after TBI. Western blot analysis showed that Akt was significantly decreased as early as 1 h after trauma; however, the phosphorylation was accelerated at 4 h. Double staining with phospho-Akt and phospho-BAD or phospho-GSK-3beta revealed the colocalization of phospho-Akt and downstream elements. Double staining with phospho-Akt and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling showed different cellular distributions after TBI. The present study implicates Akt phosphorylation in the signaling pathways that are involved in cell survival after TBI.

A nonradioactive method is described that detects 10 to 100 legionellae in 1 ml of bronchoalveolar lavage fluid. DNA is purified by a proteinase K-phenol protocol or with a commercial DNA preparation kit and amplified by PCR with amplimers specific for the 16S rRNA gene of Legionella pneumophila. The upstream primer is 5' biotinylated. The amplification product is immobilized on streptavidin-coated microtiter plates. Because of the high binding capacity, no removal of nonincorporated biotin from the PCR product is required. After alkaline denaturation, the single-stranded PCR product is hybridized with a 5' digoxigenin-labeled probing oligomer. The amplification product is then detected by using peroxidase-labeled anti-digoxigenin antibodies in a luminescence or colorimetric reaction. The assay detects as few as 10 legionellae in 1-ml bronchoalveolar lavage fluid specimens. It is specific for medically relevant Legionella species, including Legionella pneumophila, L. bozemanii, and L. longbeachae. Of over 250 clinical specimens examined, 8 were positive for legionellae by both culture and the PCR assay. Six further specimens were culture negative but PCR positive for legionellae; of these, five specimens were from patients receiving high-dose erythromycin therapy for suspected or previously diagnosed legionella pneumonia. None of the remaining 240 specimens that were culture negative for legionellae yielded a positive PCR test, although a total of over 30 different bacterial species were cultured from these specimens. The PCR assay therefore appears to exhibit high sensitivity and specificity and thus could prove suitable for use in the routine microbiological diagnostic laboratory.

In recent years, polymeric nanoparticles have appeared as a most viable and versatile delivery system for targeted cancer therapy. Various in vivo studies have demonstrated that virus-sized stealth particles are able to circulate for a prolonged time and preferentially accumulate in the tumor site via the enhanced permeability and retention (EPR) effect (so-called "passive tumor-targeting"). The surface decoration of stealth nanoparticles by a specific tumor-homing ligand, such as antibody, antibody fragment, peptide, aptamer, polysaccharide, saccharide, folic acid, and so on, might further lead to increased retention and accumulation of nanoparticles in the tumor vasculature as well as selective and efficient internalization by target tumor cells (termed as "active tumor-targeting"). Notably, these active targeting nanoparticulate drug formulations have shown improved, though to varying degrees, therapeutic performances in different tumor models as compared to their passive targeting counterparts. In addition to type of ligands, several other factors such as in vivo stability of nanoparticles, particle shape and size, and ligand density also play an important role in targeted cancer chemotherapy. In this review, concept and recent development of polymeric nanoparticles conjugated with specific targeting ligands, ranging from proteins (e.g., antibodies, antibody fragments, growth factors, and transferrin), peptides (e.g., cyclic RGD, octreotide, AP peptide, and tLyp-1 peptide), aptamers (e.g., A10 and AS1411), polysaccharides (e.g., hyaluronic acid), to small biomolecules (e.g., folic acid, galactose, bisphosphonates, and biotin), for active tumor-targeting drug delivery in vitro and in vivo are highlighted and discussed. With promise to maximize therapeutic efficacy while minimizing systemic side effects, ligand-mediated active tumor-targeting treatment modality has become an emerging and indispensable platform for safe and efficient cancer therapy.

We report the functional characterization of site-directed biotin binding-site mutants of recombinant core streptavidin. The mutagenesis studies were aimed at characterizing the contributions of Trp residues known to contact biotin that have been postulated to control the exceptional binding affinity observed in this system. The functional properties of single site-directed mutants replacing Trp residues with Phe or Ala at positions 79, 108, and 120 were investigated by quantitating the EC50 binding parameters of these mutants to biotin and 2-iminobiotin in an ELISA format. The biotin EC50 for all mutants was the same as wild-type streptavidin, demonstrating that their delta Ka values relative to wild type were < 10(6). The conservative W79F and W108F mutants displayed only a 2- to 3-fold increase in EC50 for 2-iminobiotin, corresponding to an estimated delta Ka < 10, while the W120F mutant displayed a much greater alteration in 2-iminobiotin EC50, corresponding to an estimated delta Ka of 10(2). These delta Ka values are likely to reflect similar changes for biotin. The 2-iminobiotin EC50 values for the Ala mutants fell outside the accessible concentration range of the ELISA assay, demonstrating that these mutations lowered the Ka by a factor of 10(4) to 10(6). Direct estimation of biotin Ka values for W79A, W120A, and W120F in an ultrafiltration binding assay yielded Ka values of 4.3 x 10(7) M-1, 8.6 x 10(6) M-1, and>> 5 x 10(9) M-1, respectively, in excellent agreement with the ELISA estimates of delta Ka with 2-iminobiotin as a reporter ligand. The results of these preliminary functional studies suggest that these aromatic side chains contribute significantly to the streptavidin-biotin binding free energy.

The ability of estrogen to prevent glucocorticoid-induced apoptosis in osteoblasts was studied both in vitro and in vivo. Glucocorticoid treatment for 72 h produced a dose-dependent increase in the number of apoptotic cells, determined by acridine orange/ethidium bromide staining, with a maximal response of 31+/-2% and 26+/-3% with 100 nM corticosterone in primary rat and mouse osteoblasts, respectively. Simultaneous administration of varying concentrations of 17beta-estradiol and 100 nM corticosterone decreased apoptotic osteoblasts in a dose-dependent manner, with a maximal decrease of 70% with 0.01 nM 17beta-estradiol. Terminal deoxynucleotidyltransferase-mediated deoxy-UTP-biotin nick end labeling also demonstrated glucocorticoid-induced DNA fragmentation that was inhibited by estrogen. Estrogen was shown to inhibit apoptosis induced by lipopolysaccharide treatment. As early as 6 h, Western blots demonstrated a dose-dependent decrease in the Bcl-2/Bax ratio, which reached a minimum of 0.18 in osteoblasts treated with 1000 nM corticosterone for 72 h. This reduction in Bcl-2/Bax was abolished by treating osteoblasts simultaneously with 17beta-estradiol, but not with 17alpha-estradiol. In 7-day-old mice, administration of varying concentrations of dexamethasone for 72 h resulted in a dose-dependent increase in the number of apoptotic osteoblasts as demonstrated by in situ terminal deoxynucleotidyltransferase-mediated deoxy-UTP-biotin nick end labeling staining of calvaria. A maximum of 22+/-1% apoptotic osteoblasts on the bone surface was found with 1 mg/kg BW dexamethasone compared with 2+/-1% in vehicle-treated mice. Injection of varying concentrations of 17beta-estradiol (0.5-5 mg/kg BW), but not 17alpha-estradiol, with 1 mg/kg dexamethasone produced a dose-dependent decrease in the number of apoptotic osteoblasts to 5+/-1% with 5 mg/kg 17beta-estradiol. Thus, glucocorticoid-induced apoptosis of osteoblasts may be prevented at least in part by 17beta-estradiol.

Nonthermal plasma (NTP) is generated by ionization of neutral gas molecules, which results in a mixture of energy particles including electrons and ions. Recent progress in the understanding of NTP has led to its application in the treatment of various diseases, including cancer. However, the molecular mechanisms of NTP-induced cell death are unclear. The purpose of this study was to evaluate the molecular mechanism of NTP in the induction of apoptosis of head and neck cancer (HNC) cells. The effects of NTP on apoptosis were investigated using MTT, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling, Annexin V assays, and western blot analysis. The cells were examined for production of reactive oxygen species (ROS) using DCFCA or MitoSOX staining, intracellular signaling, and an animal model. NTP reduced HNC cell viability in a dose-dependent manner and induced apoptosis. NTP resulted in alteration of mitochondrial membrane potential and accumulation of intracellular ROS generated from the mitochondria in HNC cells. Blockade of ROS production by N-acetyl-L-cysteine inhibited NTP-induced apoptosis. NTP led to the phosphorylation of c-JUN N-terminal kinase (JNK) and p38, but not extracellular-regulated kinase. Treatment with JNK and p38 inhibitors alleviated NTP-induced apoptosis via ROS generation. Taken together, these results show that NTP induced apoptosis of HNC cells by a mechanism involving MAPK-dependent mitochondrial ROS. NTP inhibited the growth of pre-established FaDu tumors in a nude mouse xenograft model and resulted in accumulation of intracellular ROS. In conclusion, NTP induced apoptosis in HNC cells through a novel mechanism involving MAPK-mediated mitochondrial ROS. These findings show the therapeutic potential of NTP in HNC.

The physiological activity of hyaluronic acid (HA) polymers and oligomers makes it a promising material for a variety of applications. The development of HA-hydrogel scaffolds with improved mechanical stability against degradation and biochemical functionality may enhance their application to tissue engineering. In this report, a crosslinking strategy targeting the alcohol groups via a poly(ethylene glycol) diepoxide crosslinker was investigated for the generation of degradable HA hydrogels. To provide support for cell adhesion in vitro, collagen was incorporated into the HA solution prior to the crosslinking process. The hydrogels have a continuous exterior and a porous interior, with pore diameters ranging from 6 to 9 microm. HA and HA-collagen hydrogels degrade in the presence of hyaluronidase and collagenase enzymes, indicating that the chemical modification does not prevent biodegradation. Complete degradation of the hydrogels occurred within 14 days in hyaluronidase (100 U/ml) and 3 days in collagenase (66 U/ml). Pattern transfer was employed to introduce a surface topography onto the hydrogel, which was able to orient cell growth. Furthermore, the hydrogels could be functionalized with the biomolecule neutravidin by incorporation of biotin along the HA backbone. This biotinylation approach may allow attachment of bioactive molecules that are conjugated to avidin.

Identifying the interaction partners of noncoding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA crosslinking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-crosslinking and Argonaute 2 immunopurification followed by streptavidin affinity purification of probe-linked RNAs provided selectivity in the capture of targets, which were identified by deep sequencing. miR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. miR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long noncoding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation, levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a 'sponge' for these miRNAs.

A saccharolytic spirochete that associated and interacted with cellulolytic bacteria was isolated from bovine rumen fluid. Isolation was accomplished by means of a procedure involving serial dilution of a sample of rumen fluid into a cellulose-containing agar medium. Clear zones appeared within the medium as a result of cellulose hydrolysis by rumen bacteria. The saccharolytic spirochete and a cellulolytic bacterium later identified as a strain of Bacteroides succinogenes were isolated from the clear zones. The spirochete did not utilize cellulose, but grew in coculture with the cellulotytic bacterium in cellulose-containing media. When cocultured in these media the spirochete used, as fermentable substrates, soluble sugars released from cellulose by the cellulolytic bacterium. In cellulose-containing agar medium the spirochete enhanced cellulose breakdown by the B. succinogenes strain. Electron microscopy showed that the helical spirochete cells possessed an outer sheath, a protoplasmic cylinder, and two periplasmic fibrils. Under a CO2 atmosphere, in a reduced medium containing inorganic salts, rumen fluid, glucose, and NaHCO3, the spirochete grew to a final density of 1.9 X 10(9) cells/ml. Succinate, acetate, and formate were products of the fermentation of glucose by growing cells. CO2 (HCO3-), branched short-chain fatty acids, folic acid, biotin, niacinamide, thiamine, pyridoxal, and a carbohydrate were required for growth of the spirochete. The results of this study indicated that the rumen spirochete represents a new species of Treponema. It is proposed that the new species be named Treponema bryantii.

Protein prenylation is a widespread phenomenon in eukaryotic cells that affects many important signaling molecules. We describe the structure-guided design of engineered protein prenyltransferases and their universal synthetic substrate, biotin-geranylpyrophosphate. These new tools allowed us to detect femtomolar amounts of prenylatable proteins in cells and organs and to identify their cognate protein prenyltransferases. Using this approach, we analyzed the in vivo effects of protein prenyltransferase inhibitors. Whereas some of the inhibitors displayed the expected activities, others lacked in vivo activity or targeted a broader spectrum of prenyltransferases than previously believed. To quantitate the in vivo effect of the prenylation inhibitors, we profiled biotin-geranyl-tagged RabGTPases across the proteome by mass spectrometry. We also demonstrate that sites of active vesicular transport carry most of the RabGTPases. This approach enables a quantitative proteome-wide analysis of the regulation of protein prenylation and its modulation by therapeutic agents.

Mammalian transglutaminase (TGase) catalyzes covalent cross-linking of peptide-bound lysine residues or incorporation of primary amines to limited glutamine residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive glutamine residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII (Factor XIIIa). We identified several amino acid sequences that were preferred as glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxPphiD(P), QxPphi, and QxxphiDP; Factor XIIIa, QxxphixWP (where x and phi represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa.

In some cell types the paramyxovirus simian virus 5 (SV5) causes little cytopathic effect (CPE) and infection continues productively for long periods of time; e.g., SV5 can be produced from MDBK cells for up to 40 days with little CPE. SV5 differs from most paramyxoviruses in that it encodes a small (44-amino-acid) hydrophobic integral membrane protein (SH). When MDBK cells were infected with a recombinant SV5 containing a deletion of the SH gene (rSV5DeltaSH), the MDBK cells exhibited an increase in CPE compared to cells infected with wild-type SV5 (recovered from cDNA; rSV5). The increased CPE correlated with an increase in apoptosis in rSV5DeltaSH-infected cells over mock-infected and rSV5-infected cells when assayed for annexin V binding, DNA content (propidium iodide staining), and DNA fragmentation (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay). In rSV5DeltaSH-infected MDBK cells an increase in caspase-2 and caspase-3 activities was observed. By using peptide inhibitors of individual caspases it was found that caspase-2 and caspase-3 were activated separately in rSV5DeltaSH-infected cells. Expression of caspase-2 and -3 in rSV5DeltaSH-infected MDBK cells appeared not to require STAT1 protein, as STAT1 protein could not be detected in SV5-infected MDBK cells. When mutant mice homologous for a targeted disruption of STAT1 were used as a model animal system and infected with the viruses it was found that rSV5DeltaSH caused less mortality than wild-type rSV5, consistent with the notion of clearance of apoptotic cells in a host species.

Several gammaherpesviruses contain open reading frames encoding proteins homologous to mammalian D-type cyclins. In this study, we analyzed the expression and function of the murine gammaherpesvirus 68 (gammaHV68) viral cyclin (v-cyclin). The gammaHV68 v-cyclin gene was expressed in lytically infected fibroblasts as a leaky-late mRNA of approximately 0.9 kb encoding a protein of approximately 25 kDa. To evaluate the effect of the gammaHV68 v-cyclin on cell cycle progression in primary lymphocytes and to determine if the gammaHV68 v-cyclin gene is an oncogene, we generated transgenic mice by using the lck proximal promoter to express the gammaHV68 v-cyclin in early T cells. Expression of the gammaHV68 v-cyclin significantly increased the number of thymocytes in cell culture, as determined by measuring both DNA content and incorporation of 5-bromo-2-deoxyuridine following in vivo pulse-labeling. Expression of the gammaHV68 v-cyclin interfered with normal thymocyte maturation, as shown by increased numbers of CD4(+) CD8(+) double-positive thymocytes and decreased numbers of CD4(+) or CD8(+) single-positive and T-cell-receptor-bright thymocytes and splenocytes in transgenic mice. Despite increased numbers of cycling thymocytes, gammaHV68-v-cyclin-transgenic mice did not have proportionately increased thymocyte numbers, and staining by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling demonstrated increased apoptosis in the thymi of v-cyclin-transgenic mice. Fifteen of 38 gammaHV68-v-cyclin-transgenic mice developed high-grade lymphoblastic lymphoma between 3 and 12 months of age. We conclude that (i) the gammaHV68 v-cyclin is expressed as a leaky-late gene in lytically infected cells, (ii) expression of the gammaHV68 v-cyclin in thymocytes promotes cell cycle progression and inhibits normal T-cell differentiation, and (iii) the gammaHV68 v-cyclin gene is an oncogene.

N-Acetyl-L-glutamate kinase (NAGK), a member of the amino acid kinase family, catalyzes the second and frequently controlling step of arginine synthesis. The Escherichia coli NAGK crystal structure to 1.5 A resolution reveals a 258-residue subunit homodimer nucleated by a central 16-stranded molecular open beta sheet sandwiched between alpha helices. In each subunit, AMPPNP, as an alphabetagamma-phosphate-Mg2+ complex, binds along the sheet C edge, and N-acetyl-L-glutamate binds near the dyadic axis with its gamma-COO- aligned at short distance from the gamma-phosphoryl, indicating associative phosphoryl transfer assisted by: (1) Mg2+ complexation; (2) the positive charges on Lys8, Lys217, and on two helix dipoles; and (3) by hydrogen bonding with the y-phosphate. The structural resemblance with carbamate kinase and the alignment of the sequences suggest that NAGK is a structural and functional prototype for the amino acid kinase family, which differs from other acylphosphate-making devices represented by phosphoglycerate kinase, acetate kinase, and biotin carboxylase.

Sulfenic acids, formed as transient intermediates during the reaction of cysteine residues with peroxides, play significant roles in enzyme catalysis and regulation, and are also involved in the redox regulation of transcription factors and other signaling proteins. Therefore, interest in the identification of protein sulfenic acids has grown substantially in the past few years. Dimedone, which specifically traps sulfenic acids, has provided the basis for the synthesis of a novel group of compounds that derivatize 1,3-cyclohexadione, a dimedone analogue, with reporter tags such as biotin for affinity capture and fluorescent labels for visual detection. These reagents allow identification of the cysteine sites and proteins that are sensitive to oxidation and permit identification of the cellular conditions under which such oxidations occur. We have shown that these compounds are reactive and specific toward sulfenic acids and that the labeled proteins can be detected at high sensitivity using gel analysis or mass spectrometry. Here, we further characterize these reagents, showing that the DCP-Bio1 incorporation rates into three sulfenic acid containing proteins, papaya papain, Escherichia coli fRMsr, and the Salmonella typhimurium peroxiredoxin AhpC, are significantly different and, in the case of fRMsr, are unaffected by changes in buffer pH from 5.5 and 8.0. We also provide protocols to label protein sulfenic acids in cellular proteins, either by in situ labeling of intact cells or by labeling at the time of lysis. We show that the addition of alkylating reagents and catalase to the lysis buffer is critical in preventing the formation of sulfenic acid subsequent to cell lysis. Data presented herein also indicate that the need to standardize, as much as possible, the protein and reagent concentrations during labeling. Finally, we introduce several new test or control proteins that can be used to evaluate labeling procedures and efficiencies.

The effects of soluble Nef protein on CD4(+) T cells were examined. CD4(+)-T-cell cultures exposed to soluble Nef were analyzed for apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and hallmarks of apoptosis including cytoplasmic shrinkage, nuclear fragmentation, DNA laddering, and caspase activation. We observed dose- and time-dependent inductions of apoptosis. DNA laddering and activated caspase 3 were also evident. Cells treated with Nef/protein kinase inhibitor complexes were protected from Nef-induced apoptosis, suggesting possible roles for protein kinases in the apoptosis pathway. Similarly, cells treated with Nef/anti-Nef antibody complexes were protected from Nef-induced apoptosis. The cellular receptor responsible for Nef-induced apoptosis was identified through antibody- and ligand-blocking experiments as a receptor commonly involved in viral entry. CXCR4 antibodies, as well as the endogenous ligand SDF-1alpha, were effective in blocking Nef-induced apoptosis, while CCR5 and CD4 antibodies were ineffective. Moreover, a CXCR4-deficient cell line, MDA-MB-468, which was resistant to Nef-induced apoptosis, became sensitive upon transfection with a CXCR4-expressing vector. This study suggests that extracellular Nef protein could contribute to the decline of CD4 counts prior to and during the onset of AIDS in patients with human immunodeficiency virus type 1 infections.