The basic treatment of leishmaniasis consists in the administration of pentavalent antimonials. The mechanisms that contribute to pentavalent antimonial toxicity against the intracellular stage of the parasite (i.e., amastigote) are still unknown. In this study, the combined use of several techniques including DNA fragmentation assay and in situ and cytofluorometry terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling methods and YOPRO-1 staining allowed us to demonstrate that potassium antimonyl tartrate, an Sb(III)-containing drug, was able to induce cell death associated with DNA fragmentation in axenic amastigotes of Leishmania infantum at low concentrations (10 microg/ml). This observation was in close correlation with the toxicity of Sb(III) species against axenic amastigotes (50% inhibitory concentration of 4.75 microg/ml). Despite some similarities to apoptosis, nuclease activation was not a consequence of caspase-1, caspase-3, calpain, cysteine protease, or proteasome activation. Altogether, our results demonstrate that the antileishmanial toxicity of Sb(III) antimonials is associated with parasite oligonucleosomal DNA fragmentation, indicative of the occurrence of late events in the overall process of apoptosis. The elucidation of the biochemical pathways leading to cell death could allow the isolation of new therapeutic targets.

Biotin synthase catalyzes the insertion of a sulfur atom between the saturated C6 and C9 carbons of dethiobiotin. Catalysis requires AdoMet and flavodoxin and generates 5'-deoxyadenosine and methionine, suggesting that biotin synthase is an AdoMet-dependent radical enzyme. Biotin synthase (BioB) is aerobically purified as a dimer of 38.4 kDa monomers that contains 1-1.5 [2Fe-2S](2+) clusters per monomer and can be reconstituted with exogenous iron, sulfide, and reductants to contain up to two [4Fe-4S] clusters per monomer. The iron-sulfur clusters may play a dual role in biotin synthase: a reduced iron-sulfur cluster is probably involved in radical generation by mediating the reductive cleavage of AdoMet, while recent in vitro labeling studies suggest that an iron-sulfur cluster also serves as the immediate source of sulfur for the biotin thioether ring. Consistent with this dual role for iron-sulfur clusters in biotin synthase, we have found that the protein is stable, containing one [2Fe-2S](2+) cluster and one [4Fe-4S](2+) cluster per monomer. In the present study, we demonstrate that this mixed cluster state is essential for optimal activity. We follow changes in the Fe and S content and UV/visible and EPR spectra of the enzyme during a single turnover and conclude that during catalysis the [4Fe-4S](2+) cluster is preserved while the [2Fe-2S](2+) cluster is destroyed. We propose a mechanism for incorporation of sulfur into dethiobiotin in which a sulfur atom is oxidatively extracted from the [2Fe-2S](2+) cluster.

BACKGROUND

Multipotent human mesenchymal stromal cells (hMSCs) are considered as promising biological tools for regenerative medicine. Their antibody-based isolation relies on the identification of reliable cell surface markers.

RESULTS

To obtain a comprehensive view of the cell surface proteome of bone marrow-derived hMSCs, we have developed an analytical pipeline relying on cell surface biotinylation of intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin to enrich the plasma membrane proteins and mass spectrometry for identification with extremely high confidence. Among the 888 proteins identified, we found ≈200 bona fide plasma membrane proteins including 33 cell adhesion molecules and 26 signaling receptors. In total 41 CD markers including 5 novel ones (CD97, CD112, CD239, CD276, and CD316) were identified. The CD markers are distributed homogenously within plastic-adherent hMSC populations and their expression is modulated during the process of adipogenesis or osteogenesis. Moreover, our in silico analysis revealed a significant difference between the cell surface proteome of hMSCs and that of human embryonic stem cells reported previously.

CONCLUSIONS

Collectively, our analytical methods not only provide a basis for further studies of mechanisms maintaining the multipotency of hMSCs within their niches and triggering their differentiation after signaling, but also a toolbox for a refined antibody-based identification of hMSC populations from different tissues and their isolation for therapeutic intervention.

High-intensity focused ultrasound has been discovered to be able to locally and reversibly increase the permeability of the blood-brain barrier (BBB), which can be detected using magnetic resonance imaging (MRI). However, side effects such as microhemorrhage, erythrocyte extravasations or even extensive hemorrhage may also occur. Although current contrast-enhanced T1-weighted MRI can be used to detect the changes in BBB permeability, its efficacy in detecting tissue hemorrhage after focused-ultrasound sonication remains limited. The purpose of this study is to investigate the feasibility of magnetic resonance susceptibility-weighted imaging (MR-SWI) for identifying possible tissue hemorrhage associated with disruption of the BBB induced by focused ultrasound in a rat model. The brains of 42 Sprague-Dawley rats were subjected to 107 sonications, either unilaterally or bilaterally. Localized BBB opening was achieved by delivering burst-mode focused ultrasound energy into brain tissue in the presence of microbubbles. Rats were studied by T2-weighted and contrast-enhanced T1-weighted MRI techniques, as well as by SWI. Tissue changes were analyzed histologically and the extent of apoptosis was investigated with the terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling method. The results demonstrated that SWI is more sensitive than standard T2-weighted and contrast-enhanced T1-weighted MRI techniques in detecting hemorrhages after brain sonication. Longitudinal study showed that SWI is sensitive to the recovery process of the damage and, therefore, could provide important and complementary information to the conventional MR images. Potential applications such as drug delivery in the brain might be benefited.

The dioecious plant Rumex acetosa has a multiple sex chromosome system: XX in female and XY(1)Y(2) in male. Both types of Y chromosome were isolated from chromosome spreads of males by manual microdissection, and their chromosomal DNA was amplified using degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). When the biotin-labeled DOP-PCR product was hybridized with competitor DNA in situ, the fluorescent signal painted the Y chromosomes. A library of Y chromosome DNA was constructed from the DOP-PCR product and screened for DNA sequences specific to the Y chromosome. One Y chromosome-specific DNA sequence was identified and designated RAYSI (R. acetosa Y chromosome-specific sequence I). RAYSI is a tandemly arranged repetitive DNA sequence that maps to the 4',6-diamidino-2-phenylindole bands of both Y chromosomes.

OBJECTIVE

This study was designed to document the association of endocardial fibroelastosis (EFE) and maternal autoantibodies.

BACKGROUND

Neonatal lupus erythematosus is associated with the transplacental passage of maternal anti-Ro and anti-La antibodies, leading to complete atrioventricular block (CAVB). In some cases, CAVB is associated with EFE. Isolated EFE may be independently related to maternal anti-Ro and anti-La antibodies.

METHODS

We identified three cases (one fetus and two infants, all female) of isolated EFE in infants born to autoantibody-positive mothers in the absence of CAVB. Demographics, echocardiograms, and pathology were reviewed. Immunohistochemical analyses for immunoglobulin (Ig)G, IgM, IgA, T-cell, B-cell, and terminal deoxynucleoleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) (test for cell apoptosis) staining were performed on multiple sections of the heart in each case and compared with negative controls.

RESULTS

Two cases died and one received a cardiac transplant. All three cases had histologically confirmed EFE. All cases demonstrated significant diffuse IgG infiltration. To a lesser extent, myocardial tissue was also positive for IgM, CD43, and Granzyme B. None of the specimens were TUNEL positive.

CONCLUSIONS

These are the first reported cases of isolated EFE associated with maternal anti-Ro and anti-La antibodies in the absence of CAVB. The diffuse deposition of IgG and the presence of a T-cell infiltrate throughout the myocardium suggest that the transplacental passage of maternal autoantibodies induces an immune reaction within the myocardium, leading to isolated EFE. Autoantibody-mediated EFE may be an etiologic factor in cases of fetal and neonatal "idiopathic" dilated cardiomyopathy.

OBJECTIVE

Glaucoma is a leading cause of irreversible visual impairment and blindness in the world. A major risk factor for glaucoma is elevated intraocular pressure due to increased resistance of aqueous humor outflow through the trabecular meshwork (TM). In the glaucomatous TM, there is increased accumulation of extracellular matrix (ECM) material due to a disruption of the normal balance between ECM deposition and degradation. Tissue transglutaminase (TGM2) belongs to a family of calcium-dependent enzymes that catalyze the posttranslational modification of the ECM by cross-linking proteins, thus making these proteins resistant to enzymatic and physical degradation. It is possible that the increase in ECM proteins in the glaucomatous TM is due to increased cross-linking activity of TGM2. The purpose of this study was to determine whether there are differences in expression and activity of TGM2 between normal and glaucoma TM cells and tissues.

METHODS

Normal (n = 3 NTM) and glaucomatous (n = 3 GTM) human TM cell lines were grown until confluent. Western immunoblot analysis of cell lysates was used to compare TGM2 protein levels in NTM and GTM cells. TGM2 enzyme activity between NTM and GTM cells was studied by using a biotin cadaverine assay. In addition, immunohistochemistry of three normal and three glaucomatous TM tissues was used to evaluate the in vivo expression of TGM2, fibronectin (FN) and epsilon-(gamma-glutamyl) lysine (GGEL) proteins.

RESULTS

Western blot analysis and immunohistochemistry demonstrated the presence of TGM2 protein in both NTM and GTM cells. There was an increase in TGM2 protein in GTM cells compared with NTM cells, and GTM cells also had increased in TGM2 enzyme activity compared with NTM cells. Immunohistochemical results demonstrated increased expression of TGM2 and FN in GTM tissues. FN and GGEL proteins were colocalized in GTM tissues, indicating significant cross-linking of FN by TGM2.

CONCLUSIONS

This study demonstrated that both NTM and GTM cells express TGM2. In addition, TGM2 protein levels and enzyme activities were elevated in GTM cells. There was also an increase in colocalization of FN and GGEL protein in GTM tissues. These results indicate that TGM2 may play an important role in the pathogenesis of glaucoma by cross-linking ECM proteins such as FN and thus making the ECM more resistant to degradation.

We have previously demonstrated that when cardiac mitochondria were challenged with H2O2, NADH production and oxidative phosphorylation declined. Upon consumption of H2O2, mitochondrial function was restored. These alterations were due, in large part, to reversible glutathionylation and inhibition of the Krebs cycle enzyme alpha-ketoglutarate dehydrogenase. The current study was undertaken to identify the site and consequences of alpha-ketoglutarate dehydrogenase glutathionylation. Mitochondria were treated with H2O2 for varying periods of time. Protein sulfhydryls that had undergone H2O2 mediated glutathionylation were specifically derivatized with N-ethylmaleimide-biotin. Subsequent purification of biotin labeled (glutathionylated) protein and Western blot analysis revealed that the E2 subunit of alpha-ketoglutarate dehydrogenase was reversibly glutathionylated. Further analysis revealed that lipoic acid, a required cofactor covalently attached to the E2 subunit, was the site of glutathionylation. The relative level of glutathionylated lipoic acid closely paralleled the degree of enzyme inhibition and reactivation. Glutathionylation of alpha-ketoglutarate dehydrogenase protected lipoic acid from modification by the electrophilic lipid peroxidation product 4-hydroxy-2-nonenal. Glutathionylation of alpha-ketoglutarate dehydrogenase can therefore be viewed as an antioxidant response protecting the enzyme from oxidative damage.

BACKGROUND

Serotonin (5-hydroxytryptamine, 5-HT) was named for its source (sero-) and ability to modify smooth muscle tone (tonin). The biological effects of 5-HT are believed to be carried out by stimulation of serotonin receptors at the plasma membrane. Serotonin has recently been shown to be synthesized in vascular smooth muscle and taken up from external sources, placing 5-HT inside the cell. The enzyme transglutaminase uses primary amines such as 5-HT to covalently modify proteins on glutamine residues. We tested the hypothesis that 5-HT is a substrate for transglutaminase in arterial vascular smooth muscle, with protein serotonylation having physiological function.

RESULTS

The model was the rat aorta and cultured aortic smooth muscle cells. Western analysis demonstrated that transglutaminase II was present in vascular tissue, and transglutaminase activity was observed as a cystamine-inhibitable incorporation of the free amine pentylamine-biotin into arterial proteins. Serotonin-biotin was incorporated into alpha-actin, beta-actin, gamma-actin, myosin heavy chain and filamin A as shown through tandem mass spectrometry. Using antibodies directed against biotin or 5-HT, immunoprecipitation and immunocytochemistry confirmed serotonylation of smooth muscle alpha-actin. Importantly, the alpha-actin-dependent process of arterial isometric contraction to 5-HT was reduced by cystamine.

CONCLUSIONS

5-HT covalently modifies proteins integral to contractility and the cytoskeleton. These findings suggest new mechanisms of action for 5-HT in vascular smooth muscle and consideration for intracellular effects of primary amines.

The hypothesis was tested that hyperbaric oxygen therapy (HBO) reduced brain infarction by preventing apoptotic death in ischemic cortex in a rat model of focal cerebral ischemia. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) and subsequently were exposed to HBO (2.5 atmospheres absolute) for 2 h, at 6 h after reperfusion. Rats were killed and brain samples were collected at 24, 48, 72 h, and 7 days after reperfusion. Neurologic deficits, infarction area, and apoptotic changes were evaluated by clinical scores, 2,3,7-triphenyltetrazolium chloride staining, caspase-3 expression, DNA fragmentation assay, and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL)-hematoxylin and eosin (H&E) costaining. In MCAO/R without HBO treatment animals, DNA fragmentation was observed in injured cortex at 24, 48, and 72 h but not in samples at 7 days after reperfusion. Double labeling of brain slides with NeuN and caspase-3 demonstrated neurons in the injured cortex labeled with caspase-3. TUNEL+H&E costaining revealed morphologic apoptotic changes at 24, 48, and 72 h after reperfusion. Hyperbaric oxygen therapy abolished DNA fragmentation and reduced the number of TUNEL-positive cells. Hyperbaric oxygen therapy reduced infarct area and improved neurologic scores at 7 days after reperfusion. One of the molecular mechanisms of HBO-induced brain protection is to prevent apoptosis, and this effect of HBO might preserve more brain tissues and promote neurologic functional recovery.

The inner membrane complex (IMC) of Toxoplasma gondii is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell invasion. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. In this study, we have adapted a technique named proximity-dependent biotin identification (BioID) for use in T. gondii to identify novel components of the IMC. Using IMC proteins in both the alveoli and the cytoskeletal network as bait, we have uncovered a total of 19 new IMC proteins in both of these suborganellar compartments, two of which we functionally evaluate by gene knockout. Importantly, labeling of IMC proteins using this approach has revealed a group of proteins that localize to the sutures of the alveolar sacs that have been seen in their entirety in Toxoplasma species only by freeze fracture electron microscopy. Collectively, our study greatly expands the repertoire of known proteins in the IMC and experimentally validates BioID as a strategy for discovering novel constituents of specific cellular compartments of T. gondii.

OBJECTIVE

The identification of binding partners is critical for determining protein function within cellular compartments. However, discovery of protein-protein interactions within membrane or cytoskeletal compartments is challenging, particularly for transient or unstable interactions that are often disrupted by experimental manipulation of these compartments. To circumvent these problems, we adapted an in vivo biotinylation technique called BioID for Toxoplasma species to identify binding partners and proximal proteins within native cellular environments. We used BioID to identify 19 novel proteins in the parasite IMC, an organelle consisting of fused membrane sacs and an underlying cytoskeleton, whose protein composition is largely unknown. We also demonstrate the power of BioID for targeted discovery of proteins within specific compartments, such as the IMC cytoskeleton. In addition, we uncovered a new group of proteins localizing to the alveolar sutures of the IMC. BioID promises to reveal new insights on protein constituents and interactions within cellular compartments of Toxoplasma.

OBJECTIVE

Alpha-fetoprotein (AFP)-producing gastric cancer has been associated with a poor prognosis. In the present study, the cell proliferation, apoptosis, and angiogenesis of this cancer were studied histochemically to determine its malignant potential.

METHODS

Tissue samples were taken from four patients with AFP-producing gastric cancer and 26 patients with AFP-negative gastric cancer. Cell proliferation was evaluated by Ki-67 immunostaining, and the Ki-67 labeling index (LI) was determined. Apoptosis was studied by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling method, and the apoptotic index was determined. Angiogenesis was evaluated by measuring the microvessel density using factor VIII immunostaining, and immunostainings for vascular endothelial growth factor and thymidine phosphorylase were performed.

RESULTS

The Ki-67 LI of the AFP-producing gastric cancers was significantly higher than that of the AFP-negative gastric cancers (p < 0.01). The apoptotic index of the AFP-producing gastric cancers was significantly lower than that of the AFP-negative gastric cancers (p < 0.01). The microvessel density of the AFP-producing gastric cancers was significantly higher than that of the AFP-negative gastric cancers (p < 0.01). Vascular endothelial growth factor expression was observed in all four of the AFP-producing gastric cancers, whereas thymidine phosphorylase was not expressed in any of the AFP-producing gastric cancers.

CONCLUSIONS

These results suggest that AFP-producing gastric cancers have high malignant potential (high proliferative activity, weak apoptosis, and rich neovascularization) compared with that of AFP-negative gastric cancers. These biological characteristics of AFP-producing gastric cancer reflect the aggressive behavior and the poor prognosis of patients with this type of cancer.

Adhesion molecules are thought to play a vital role in the induction and maintenance of tissue differentiation and their loss or down-regulation has been implicated in the neoplastic process. Recent studies have shown that the morphoregulatory activities are a consequence of interactive processes between several cell adhesion molecules rather than the function of a single molecule. Therefore, we have investigated a panel of adhesion molecules including members of the integrin, cadherin and immunoglobin superfamily in colorectal cancer. Twenty-eight consecutive colorectal adenocarcinomas were stained using an avidin-biotin indirect immunoperoxidase technique. Our results showed a consistent loss of the alpha 2 and beta 1 integrin subunits (21/28 = 75% and 22/28 = 78.6% respectively) and a decrease in expression of E-cadherin in 5/5 poorly differentiated adenocarcinomas. Carcinoembryonic antigen expression was preserved but with basolateral accentuation seen in tumours. There was no statistical correlation with Dukes' stage. These results provide further evidence that in colorectal cancer there is a widespread deregulated expression of cell-cell and cell-matrix adhesion molecules. Changes in the expression and function of adhesion molecules which regulate growth and differentiation may play a role in the behaviour of colorectal cancer.

A simple and inexpensive method of condensing and linking plasmid DNA to carrier adenovirus particles is described. The synthetic polycation polyethylenimine is used to condense plasmid DNA into positively charged 100 nm complexes. These PEI-DNA complexes are then bound to adenovirus particles through charge interactions with negative domains on the viral hexon. The resulting transfection complexes deliver plasmid DNA to cells by the adenovirus infectious route without interference from virus gene expression because psoralen-inactivated virus is employed. The PEI-DNA-adenovirus complexes display DNA delivery comparable to more sophisticated DNA virus complexes employing streptavidin/biotin linkage, but require no special reagents and are much easier to prepare.

Short peptide affinity tags have become indispensable in protein research. They cannot only be used for affinity purification but also downstream for detection and assay of an arbitrary fused recombinant protein without the need for any prior knowledge of its biochemical properties. Strep-tag®II is particularly popular for providing recombinant proteins at high purity and functionality by using physiological conditions within a rapid one-step protocol. The affinity receptor for Strep-tag®II is affinity engineered streptavidin, named Strep-Tactin®. Strep-tag®II binds to the biotin binding pocket enabling mild competitive elution with biotin derivatives, preferably desthiobiotin, for repeated use of the Strep-Tactin® affinity resins. Fast binding and dissociation kinetics allow comparatively high flow rates throughout column chromatography including elution. Fast dissociation kinetics may be, however, limiting for using Strep-tag®II for direct purification of target proteins from large volumes of diluted extracts like mammalian cell culture supernatants or in assay formats requiring extended washing like ELISA. For this reason, binding characteristics were improved by development of the Twin-Strep-tag® consisting of two Strep-tag®II moieties connected by a short linker. The resulting avidity effect, i.e., the combined synergistic binding of two Strep-tag®II moieties to tetrameric Strep-Tactin®, reduces the off-rate for more steady binding under non-competitive conditions. The addition of a competitor, however, reverses the synergistic avidity effect and, hence, efficient elution capability is preserved. In fact, the Twin-Strep-tag® features all beneficial properties of Strep-tag®II, including efficient elution under gentle competitive conditions, but, due to its higher affinity, additionally enables a more universal use in applications requiring stable binding.

Reduced drug accumulation due to overexpression of individual members of the ATP binding cassette (ABC) superfamily of membrane transporters has been investigated as a cause of multidrug resistance and treatment failure in oncology. This study was designed to develop an immunohistochemical assay to determine the expression and localization of the 72kDa ABC half-transporter ABCG2 in normal tissues. Formalin-fixed, paraffin embedded archival tissue from 31 distinct normal tissues with an average of eight separate tissue samples of each were immunostained with rabbit-anti-ABCG2 antibody 405 using a modified avidin-biotin procedure. As a negative control, each sample was also stained with antibody pre-adsorbed with peptide to assess background staining. As a means of verification, selected tissues were also stained with the commercially available monoclonal antibody 5D3. ABCG2 positivity was consistently found in alveolar pneumocytes, sebaceous glands, transitional epithelium of bladder, interstitial cells of testes, prostate epithelium, endocervical cells of uterus, squamous epithelium of cervix, small and large intestinal mucosa/epithelial cells, islet and acinar cells of pancreas, zona reticularis layer of adrenal gland, kidney cortical tubules and hepatocytes. Placental syncytiotrophoblasts showed both cytoplasmic and surface staining. Our results support a hypothesis concluding that ABCG2 plays a role in the protection of organs from cytotoxins. However, many of the cell types expressing ABCG2 have a significant secretory function. These data suggest a dual function for ABCG2 in some tissues: the excretion of toxins and xenobiotics including anti-cancer agents and a potential, as-yet undefined role in the secretion of endogenous substrates.

The ATP-grasp enzymes consist of a superfamily of 21 proteins that contain an atypical ATP-binding site, called the ATP-grasp fold. The ATP-grasp fold is comprised of two α+β domains that "grasp" a molecule of ATP between them and members of the family typically have an overall structural design containing three common conserved focal domains. The founding members of the family consist of biotin carboxylase, d-ala-d-ala ligase and glutathione synthetase, all of which catalyze the ATP-assisted reaction of a carboxylic acid with a nucleophile via the formation of an acylphosphate intermediate. While most members of the superfamily follow this mechanistic pathway, studies have demonstrated that two enzymes catalyze only the phosphoryl transfer step and thus are kinases instead of ligases. Members of the ATP-grasp superfamily are found in several metabolic pathways including de novo purine biosynthesis, gluconeogenesis, and fatty acid synthesis. Given the critical nature of these enzymes, researchers have actively sought the development of potent inhibitors of several members of the superfamily as antibacterial and anti-obseity agents. In this review, we will discuss the structure, function, mechanism, and inhibition of the ATP-grasp enzymes.

OBJECTIVE

To determine the cellular and molecular forms of MMP-8 (collagenase-2) and MMP-13 (collagenase-3) associated with chronic adult periodontitis by examining the species present in gingival crevicular fluid (GCF) and enzyme distribution in gingival tissue.

METHODS

30-s GCF samples were collected directly from the periodontal pockets of 12 untreated patients using filter paper strips. After elution into buffer, the samples were examined by Western immunoblotting with polyclonal antibodies for MMP-8 and MMP-13 and quantification by scanning image analysis. Individual band intensities were expressed as a percentage of total sample absorbance and mean patient values were calculated. Gingival tissue from 6 patients was fixed in formalin and embedded in paraffin wax. MMP-8 and MMP-13 were localised using the same antibodies and an avidin-biotin-peroxidase detecting system. Double staining was performed with a contrasting substrate reaction.

RESULTS

The majority of MMP-8 staining in pre-treatment GCF was present in 80, 75 and 60 kD bands corresponding to prepro-, pro- and active forms of PMN-type enzyme. 43 and 38 kD bands evidently represented active, fibroblast-type MMP-8. Immunoreactivities at >100 kD and < or =30 kD were probably enzyme-inhibitor complex and degraded fragments, respectively. MMP-13 was seen mainly as 60 kD proenzyme with some 40 kD active enzyme and a small proportion of >100 kD complex. The percentages of MMP-8 PMN-type enzyme and MMP-13 proenzyme bands correlated significantly with gingival and bleeding indices (p<0.05). Immunohistochemistry demonstrated MMP-8 in PMNs, sulcular epithelial and also plasma cells in inflamed gingival connective tissue. MMP-13 immunoreactivity was detected in the sulcular epithelium and in macrophage-like cells.

CONCLUSIONS

Multiple species and elevated levels of both MMP-8 and MMP-13 from many rather than single cellular sources in the diseased periodontium are identified in untreated periodontitis GCF and active forms contribute to GCF collagenase activity.

Recent studies from this laboratory have demonstrated the presence of thyroid hormone response elements (TREs) in the 5'-flanking region of the rat alpha and TSH beta subunit genes. Using an avidin-biotin complex DNA binding assay, we have shown that these TREs bind the thyroid hormone (T3) receptor present in nuclear extracts of GH3 cells, as well as the in vitro synthesized Hc-erbA beta, which has been identified as a member of the family of T3 receptors. The binding of Hc-erbA beta to the alpha subunit TRE can be enhanced 3-4-fold by including GH3 nuclear extract in the binding assay. Binding to the TRE present in the TSH beta gene or the rat growth hormone gene was similarly enhanced, although to a lesser degree. The enhanced binding activity is trypsin-sensitive and heat labile, and is not reproduced by the addition of histones, bovine serum albumin, or cytosol instead of nuclear extract. Gel exclusion chromatography suggests a molecular size of approximately 65,000 Da. This protein, which is present in several different cell types, is also able to complement binding of the rat erbA alpha-1 and the pituitary-specific erbA beta-2 forms of the receptor. These data suggest that the binding of the T3 receptor to a TRE is augmented by another nuclear protein, which may be involved in the mechanism of action of thyroid hormone.

The authors have studied the expression of keratin 19 in normal oral mucosa and in oral lesions exhibiting a range of histopathologic changes that are thought to precede squamous cell carcinoma. Formalin-fixed, paraffin-embedded sections were pretreated with pronase and stained with a K19-specific antibody by the avidin-biotin immunoperoxidase method. In nonkeratinized mucosa, whether normal or benign hyperplastic, K19 was detectable in the basal cell layer. In keratinized mucosa, whether normal or benign hyperplastic, there was no detectable K19. All lesions from any oral site that exhibited atypia diagnosed from hematoxylin and eosin stained sections as moderate-to-severe dysplasia or carcinoma in situ, whether hyperkeratotic or not, stained strongly for K19 in the basal and suprabasal cell layers. The number of cell layers that were K19-positive correlated with the level in the epithelium to which dysplasia persisted. Suprabasal K19 staining tended to occur in regions of the epithelium in which expression of the terminal differentiation protein involucrin was delayed or absent. Thus, K19 expression may be linked to the retention of stem cell character or a state otherwise uncommitted to terminal squamous differentiation. Suprabasal K19 staining is clearly correlated with premalignant change in oral epithelium and therefore promises to be a useful tool in oral histopathologic diagnosis.

We have developed methods that allow detection, quantitation, purification, and identification of cardiac proteins S-thiolated during ischemia and reperfusion. Cysteine was biotinylated and loaded into isolated rat hearts. During oxidative stress, biotin-cysteine forms a disulfide bond with reactive protein cysteines, and these can be detected by probing Western blots with streptavidin-horseradish peroxidase. S-Thiolated proteins were purified using streptavidin-agarose. Thus, we demonstrated that reperfusion and diamide treatment increased S-thiolation of a number of cardiac proteins by 3- and 10-fold, respectively. Dithiothreitol treatment of homogenates fully abolished the signals detected. Fractionation studies indicated that the modified proteins are located within the cytosol, membrane, and myofilament/cytoskeletal compartments of the cardiac cells. This shows that biotin-cysteine gains rapid and efficient intracellular access and acts as a probe for reactive protein cysteines in all cellular locations. Using Western blotting of affinity-purified proteins we identified actin, glyceraldehyde-3-phosphate dehydrogenase, HSP27, protein-tyrosine phosphatase 1B, protein kinase Calpha, and the small G-protein ras as substrates for S-thiolation during reperfusion of the ischemic rat heart. MALDI-TOF mass fingerprint analysis of tryptic peptides independently confirmed actin and glyceraldehyde-3-phosphate dehydrogenase S-thiolation during reperfusion. This approach has also shown that triosephosphate isomerase, aconitate hydratase, M-protein, nucleoside diphosphate kinase B, and myoglobin are S-thiolated during post-ischemic reperfusion.

Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis (HGA), one of the major tick-borne zoonoses in the United States. The surface of A. phagocytophilum plays a crucial role in subverting the hostile host cell environment. However, except for the P44/Msp2 outer membrane protein family, the surface components of A. phagocytophilum are largely unknown. To identify the major surface proteins of A. phagocytophilum, a membrane-impermeable, cleavable biotin reagent, sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-Biotin), was used to label intact bacteria. The biotinylated bacterial surface proteins were isolated by streptavidin agarose affinity purification and then separated by electrophoresis, followed by capillary liquid chromatography-nanospray tandem mass spectrometry analysis. Among the major proteins captured by affinity purification were five A. phagocytophilum proteins, Omp85, hypothetical proteins APH_0404 (designated Asp62) and APH_0405 (designated Asp55), P44 family proteins, and Omp-1A. The surface exposure of Asp62 and Asp55 was verified by immunofluorescence microscopy. Recombinant Asp62 and Asp55 proteins were recognized by an HGA patient serum. Anti-Asp62 and anti-Asp55 peptide sera partially neutralized A. phagocytophilum infection of HL-60 cells in vitro. We found that the Asp62 and Asp55 genes were cotranscribed and conserved among members of the family Anaplasmataceae. With the exception of P44-18, all of the proteins were newly revealed major surface-exposed proteins whose study should facilitate understanding the interaction between A. phagocytophilum and the host. These proteins may serve as targets for development of chemotherapy, diagnostics, and vaccines.

Bovine herpesvirus 1 (BHV-1) is an important pathogen of cattle and infection is usually initiated via the ocular or nasal cavity. After acute infection, the primary site for BHV-1 latency is sensory neurons in the trigeminal ganglia (TG). Reactivation from latency occurs sporadically, resulting in virus shedding and transmission to uninfected cattle. The only abundant viral transcript expressed during latency is the latency-related (LR) RNA. An LR mutant was constructed by inserting three stop codons near the beginning of the LR RNA. This mutant grows to wild-type (wt) efficiency in bovine kidney cells and in the nasal cavity of acutely infected calves. However, shedding of infectious virus from the eye and TG was dramatically reduced in calves infected with the LR mutant. Calves latently infected with the LR mutant do not reactivate after dexamethasone treatment. In contrast, all calves latently infected with wt BHV-1 or the LR rescued mutant reactivate from latency after dexamethasone treatment. In the present study, we compared the frequency of apoptosis in calves infected with the LR mutant to calves infected with wt BHV-1 because LR gene products inhibit apoptosis in transiently transfected cells. A sensitive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay and an antibody that detects cleaved caspase-3 were used to identify apoptotic cells in TG. Both assays demonstrated that calves infected with the LR mutant for 14 days had higher levels of apoptosis in TG compared to calves infected with wt BHV-1 or to mock-infected calves. Viral gene expression, except for the LR gene, is extinguished by 14 days after infection, and thus this time frame is operationally defined as the establishment of latency. Real-time PCR analysis indicated that lower levels of viral DNA were present in the TG of calves infected with the LR mutant throughout acute infection. Taken together, these results suggest that the antiapoptotic properties of the LR gene play an important role during the establishment of latency.