Factors secreted from dental pulp stem cells show multifaceted benefits for treating experimental temporomandibular joint osteoarthritis.

OBJECTIVE: Temporomandibular joint osteoarthritis (TMJOA) is a degenerative disease characterized by progressive cartilage degeneration, abnormal bone remodeling, and chronic pain. In this study, we aimed to investigate effective therapies to reverse or suppress TMJOA progression. DESIGN: To this end, we performed intravenous administration of serum free conditioned media from human exfoliated deciduous teeth stem cells (SHED-CM) into a mechanical-stress induced murine TMJOA model. RESULTS: SHED-CM administration markedly suppressed temporal muscle inflammation, and improved bone integrity and surface smoothness of the destroyed condylar cartilage. Moreover, SHED-CM treatment decreased the number of IL-1ß, iNOS, and MMP-13 expressing chondrocytes, whereas it specifically increased PCNA-positive cells in the multipotent polymorphic cell layer. Notably, the numbers of TdT-mediated dUTP nick end labeling (TUNEL)-positive apoptotic chondrocytes in the SHED-CM treated condyles were significantly lower than in those treated with DMEM, whereas the proteoglycan positive area was restored to a level similar to that of the sham treated group, demonstrating that SHED-CM treatment regenerated the mechanical-stress injured condylar cartilage and subchondral bone. Secretome analysis revealed that SHED-CM contained multiple therapeutic factors that act in osteochondral regeneration. CONCLUSIONS: Our data demonstrated that SHED-CM treatment promoted the regeneration and repair of mechanical-stress induced mouse TMJOA. Our observations suggest that SHED-CM has potential to be a potent tissue-regenerating therapeutic agent for patients with severe TMJOA.

Diagnostic role of mean platelet volume in tonsillitis with and without peritonsillar abscess.

OBJECTIVE: To assess the diagnostic role of mean platelet volume in tonsillitis with and without peritonsillar abscess. METHODS: Mean platelet volume and other laboratory data were retrospectively investigated. RESULTS: Mean platelet volume was significantly lower in the tonsillitis group (7.8 per cent ± 0.7 per cent) than in the control group (8.7 per cent ± 0.6 per cent; p < 0.0001), and it was significantly lower in the abscess group (7.5 per cent ± 0.6 per cent) than in the no abscess group (8.0 per cent ± 0.7 per cent; p = 0.0277). White blood cell counts and C-reactive protein levels were not significantly different between patients with an abscess and those without. The mean platelet volume cut-off values for the diagnosis of tonsillitis and peritonsillar abscess were 7.95 fl and 7.75 fl, respectively. CONCLUSION: Our results suggest that a decreased mean platelet volume is associated with the development and severity of tonsillitis. This finding provides useful diagnostic information for physicians treating patients with tonsillitis.

Toxoplasma gondii: cloning, sequencing, expression, and antigenic characterization of ROP2, GRA5 and GRA7.

Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO vector which contains thioredoxin and polyhistidine tags at the C- and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 microg/ml growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.

Toxoplasma gondii: evaluation of an intranasal vaccine using recombinant proteins against brain cyst formation in BALB/c mice.

The purpose of this work was to evaluate protective activity against brain cyst formation in BALB/c mice intranasally vaccinated with recombinant proteins from Toxoplasma gondii. The recombinant proteins rROP2, rGRA5 and rGRA7 were used in vaccine preparation. Thirty-three female mice were divided into three groups, these animals received two doses by intranasal route at days 0 and 21 as follows; group 1 (G1, n=11) received 12.5 microg of each recombinant protein plus 0.5 microg of cholera toxin, group 2 (G2, n=11) received phosphate buffer saline (PBS) plus 0.5 microg of cholera toxin, and group 3 (G3, n=11) received PBS only. At challenge day (day 33) three animals from each group were euthanatized for IgA measure from intestine. Mice were infected orally with 50 cysts from the VEG strain at day 33. At challenge day the G1 animals had high immunoglobulin A levels, however, they only showed IgG antibody titers against rROP2 and rGRA7. Animals from G1 also exhibited strong resistance to cyst formation compared with the control group (G3, P<0.05). However, we did not observe differences in protection against brain cyst formation between G1 and G2 (P>0.1). These results indicate that intranasal immunization in BALB/c mice with recombinant proteins rROP2, rGRA5 and rGRA7 associated with cholera toxin induced partial protection, when compared with G3, against tissue cyst formation after oral infection with tissue cysts from T. gondii.

Toxoplasma gondii: cloning, sequencing, expression, and antigenic characterization of ROP2, GRA5 and GRA7

Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO® vector which contains thioredoxin and polyhistidine tags at the C- and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 μg/ml growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.

Cloning, sequencing, expression, and antigenic characterization of rMSP4 from Anaplasma marginale isolated from Paraná State, Brazil.

Anaplasmosis is a bovine intraerythrocytic disease caused by the bacterium Anaplasma marginale; it causes significant economic losses in tropical and subtropical regions, worldwide. The msp4 gene of an A. marginale strain isolated in Paran , Brazil, was amplified by PCR and sequenced; its cloning into the pET102/D-TOPO vector produced an msp4-6xHis-V5-HP thioredoxin fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21(DE-3); the expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in the cell lysate. The inclusion bodies were solubilized with urea and the recombinant protein was purified by Ni-NTA column and dialyzed. This method produced a relatively high yield of rMSP4, which was used to immunize rabbits. The deduced amino acid sequence encoded by MSP4 showed 99% homology to A. marginale isolates from Florida, USA, and from Minas Gerais, Brazil. Both rMSP4 and native MSP4 were recognized by post-immunization rabbit serum, showing that rMSP4 has conserved epitopes. As antigenicity was preserved, rMSP4 might be useful for the development of vaccine against anaplasmosis.

Cloning, sequencing, expression, and antigenic characterization of rMSP4 from Anaplasma marginale isolated from Paraná State, Brazil

Anaplasmosis is a bovine intraerythrocytic disease caused by the bacterium Anaplasma marginale; it causes significant economic losses in tropical and subtropical regions, worldwide. The msp4 gene of an A. marginale strain isolated in Paraná, Brazil, was amplified by PCR and sequenced; its cloning into the pET102/D-TOPO® vector produced an msp4-6xHis-V5-HP thioredoxin fusion gene construct. This recombinantclone was over-expressed in Escherichia coli BL21(DE-3); the expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in the cell lysate. The inclusion bodies were solubilized with urea and the recombinant protein was purified by Ni-NTA column and dialyzed. This method produced a relatively high yield of rMSP4, which was used to immunize rabbits. The deduced amino acid sequence encoded by MSP4 showed 99% homology to A. marginale isolates from Florida, USA, and from Minas Gerais, Brazil. Both rMSP4 and native MSP4 were recognized by post- immunization rabbit serum, showing that rMSP4 has conserved epitopes. As antigenicity was preserved, rMSP4 might be useful for the development of vaccine against anaplasmosis.

Antigenic characterization of Anaplasma marginale isolates from different regions of Brazil.

Antigenic characterization of A. marginale isolates has contributed to identifying the presence of common and restricts epitopes of major surface proteins (MSPs). The data may improve vaccine development to protect against A. marginale isolates from different regions. Brazilian A. marginale isolates were characterized antigenically by Western blot with monoclonal antibodies (MAbs) against MSPs and rabbit anti-MSP-4 from Florida strain. Six A. marginale isolates from MS, MG (AUFV1), SP, PR-L1, PR-HV, RS and Florida strain were tested with ANA22B1 to MSP-1a, AMR36A6 to MSP-1b, ANAF19E2 to MSP-2, AMG75C1 and AMG76B2 to MSP-3 and ANAF16C1 to MSP-5. ANA22B1 recognized MSP-1a epitope in all A. marginale isolates, and reacted with polypeptides of different size ranging 46-105kDa. MSP2 was not detected in MS and SP isolates by ANAF19E2, and only PR-L1 and MG (AUFV1) isolates reacted with MAbs which recognize MSP3 epitope. MSP4 and MSP5 were detected in all A. marginale isolates analyzed. The results revealed conservation of MSP-1a and MSP-5 epitopes among all Brazilian isolates, and showed antigenic variability to MSP-1b, MSP-2 and MSP-3 proteins, agreeing with recent data about the genetic diversity found in the polimorphic multigene family responsible for these proteins.

Reconstitution of brefeldin A-induced golgi tubulation and fusion with the endoplasmic reticulum in semi-intact chinese hamster ovary cells.

The fungal metabolite brefeldin A (BFA) induces the disassembly of the Golgi complex in mammalian cells. The drug seems to accentuate tubule formation and causes the subsequent fusion with the endoplasmic reticulum (ER). To investigate the biochemical requirements and kinetics of BFA-induced Golgi disassembly, we have reconstituted the process of green fluorescent protein-tagged Golgi complex disassembly in streptolysin O-permeabilized semi-intact Chinese hamster ovary cells. For quantitative analysis of the morphological changes to the Golgi complex in semi-intact cells, we developed a novel morphometric analysis. Based on this analysis, we have dissected the BFA-induced Golgi disassembly process biochemically into two processes, Golgi tubule formation and fusion with the ER, and found that the formation is induced by only ATP and the residual factors in the cells and that the subsequent fusion is mediated in an N-ethylmaleimide-sensitive factor-dependent manner via Golgi tubules. Tubulation occurs by two pathways that depend on either microtubule integrity or exogenously added cytosol. In the presence of GTPgammaS, coat protein I inhibited the Golgi tubule fusion with the ER but showed no apparent effect on tubulation. Additionally, we analyzed the kinetics of tubulation and fusion independently in nocodazole-treated and -untreated semi-intact cells and found that tubulation is a rate-limiting step of the Golgi disassembly.

Reconstitution of the Golgi reassembly process in semi-intact MDCK cells.

The Golgi apparatus, which consists of stacks of cisternae during interphase, is fragmented or dispersed throughout the cytoplasm at the onset of mitosis. A sea sponge metabolite, ilimaquinone (IQ), causes Golgi membranes to vesiculate. And after its removal, the vesiculated membranes reassemble into stacks of cisternae in the perinuclear region. To study the mechanism of Golgi membrane dynamics during mitosis, we have reconstituted the reassembly process of IQ-induced vesiculated Golgi membranes in streptolysin O-permeabilized Mardin-Darby canine kidney (MDCK) cells. Monitoring the dynamics of Golgi membranes labeled with a green fluorescence protein (GFP)-tagged protein, we dissected the process into two elementary components: the reassembly of vesiculated Golgi membranes into punctate structures; and the subsequent reformation of these structures into stacks of cisternae near the nucleus. Using morphometric analysis, we studied the kinetics and biochemical requirements for the process, and revealed that an NEM-sensitive factor, cytoplasmic dynein, and GTP binding protein were involved in the Golgi reassembly.

MEK and Cdc2 kinase are sequentially required for Golgi disassembly in MDCK cells by the mitotic Xenopus extracts.

At the onset of mitosis, the Golgi apparatus, which consists of several cisternae, disperses throughout the cell to be partitioned into daughter cells. The molecular mechanisms of this process are now beginning to be understood. To investigate the biochemical requirements and kinetics of mitotic Golgi membrane dynamics in polarized cells, we have reconstituted the disassembly of the Golgi apparatus by introducing Xenopus egg extracts into permeabilized Mardin-Darby canine kidney (MDCK) cells. We used green fluorescence protein (GFP)-tagged galactosyltransferase-expressing MDCK cells to analyze the morphological changes of the Golgi membrane in the semi-intact system. Analyses by fluorescence and electron microscopies showed that the Golgi disassembly can be dissected into two elementary processes morphologically. In the first process, the perinuclear Golgi stacks break into punctate structures, intermediates, which are comprised of mini-stacks of cisternae associating with apical microtubule networks. In the second process, the structures fragment more thoroughly or substantially relocate to the ER. Our analyses further showed that cdc2 kinase and mitogen-activated protein kinase kinase (MAPKK = MEK) are differently involved in these two processes: the first process is mainly regulated by MEK and the second mainly by cdc2.

Structure-activity relationship of the affinity of 5-substituted uracil nucleoside analogues for varicella-zoster virus thymidine kinase and their activity against varicella-zoster virus.

We investigated structure-activity relationships of 5-substituted uracil nucleoside analogues for their selective antiviral activity against varicella-zoster virus (VZV) and affinity for VZV thymidine kinase (TK). Anti-proliferative activity of the compounds was measured using human lymphoblastoid cells. Most 2'-deoxyribofuranosyluracil, arabinofuranosyluracil (araU) and 2'-deoxy-2'-fluoro-arabinofuranosyluracil derivatives showed selective anti-VZV activity as well as activity against herpes simplex virus types 1 and 2. 2'-Deoxyuridine derivatives showed higher affinity than the corresponding araU analogues. A correlation was seen between the 50% effective doses for VZV and the Ki values for VZV TK, except for 5-ethyl-2'-deoxyuridine and 5-ethyl araU that showed relatively high affinity for VZV TK without showing any activity against VZV. 5-Halogenovinyluracil nucleosides showed the highest affinity and the most potent and selective anti-VZV activity. 2'-Deoxy-2'-fluoro-arabinofuranosyluracil derivatives exhibited high anti-VZV potency though they showed relatively low affinity for VZV TK. Some 3'-deoxythymidine analogues having anti-human immunodeficiency virus activity were inactive against herpesviruses.

Deglycosylation of antiherpesviral 5-substituted arabinosyluracil derivatives by rat liver extract and enterobacteria cells.

A number of antiherpesviral 5-substituted derivatives of 1-beta-D-arabinofuranosyluracil (araU) were significantly resistant to phosphorolysis by rat liver extract (S-9), but were gradually deglycosylated in a 2% enterobacteria cell suspension. The relative order of the resistance conferred by the different C-5 substituents was: 5-propynyl > 5-(E)-2-bromovinyl > 5-(E)-2-chlorovinyl > 5-methyl > 5-iodo. The 2'-fluoro derivatives of araU were completely resistant to phosphorolysis by both liver extract and enterobacteria, whereas the corresponding ribofuranosyl and 2'-deoxyribofuranosyl nucleosides were easily phosphorolysed by S-9, and were immediately cleaved in a 1% enterobacteria cell suspension. These findings suggest that antiherpesviral 5-substituted araU analogues can be relatively stable in vivo, when injected intravenously, and that degradation of 1-beta-D-arabinofuranosyl-5-(E-2-bromovinyl)uracil (sorivudine) following oral administration is due primarily to the action of enterobacteria.

Comparison of the selectivity of anti-varicella-zoster virus nucleoside analogues.

We compared the selectivity of six anti-varicella-zoster virus (VZV) drugs, which are clinically available or of which clinical efficacy for the treatment of VZV infections has been reported. Sorivudine (BV-araU) had the most potent anti-VZV effect in the plaque inhibition assay, followed by brivudine (BVDU) and 5-propynyl-arabinofuranosyluracil (Pry-araU). All test compounds, except vidarabine (AraA), had only a very weak effect on human embryonic lung cell growth. The selectivity indexes (ID50 for cell growth/ED50 for VZV plaque inhibition) of BV-araU, BVDU, and Pry-araU were > 1,000,000, 20,000, and > 10,000, respectively, while those of acyclovir and penciclovir ranged from 600 to 800. AraA was much less selective than any of the other drugs tested. We measured the amount of [3H] thymidine incorporated into the acid-insoluble fraction of VZV-infected cells to determine the ability of these drugs to selectively inhibit viral DNA synthesis. [3H]Thymidine incorporation was markedly inhibited by all anti-VZV compounds, except BVDU. Treatment of infected cells with drugs from 32 to 38 hr after infection inhibited the DNA synthesis to the same extent as VZV plaque formation, except that AraA inhibited the DNA synthesis at a lower dose than for VZV plaque formation. DNA synthesis in non-infected growing cells was inhibited to the same extent as cell growth. A particularly high selectivity index for the inhibition of DNA synthesis was noted for BV-araU, which was defined as the ratio of inhibitions of DNA synthesis in VZV-infected and non-infected. The highest selectivity indexes were recorded for BV-araU > Pry-araU > acyclovir > or = penciclovir > AraA.

In vitro and in vivo anti-herpes viral activities and biological properties of CV-araU.

We compared the in vitro and in vivo antiviral effects against herpes simplex virus type 1 (HSV-1) and other biological properties of 1-beta-D-arabinofuranosyl-5-[(E)-2-chlorovinyl]uracil (CV-araU) and 1-beta-D-arabinofuranosyl-5-[(E)-2-bromovinyl]uracil (BV-araU, sorivudine). Both CV-araU and BV-araU exhibited antiviral activities against HSV-1 in the cell culture derived from mouse, though the activities were lower than those seen in human cells. For intraperitoneal and intracerebral infections in mice with HSV-1 strain WT-51, both compounds, administered twice daily, were effective in increase in the survival rate at doses of 15 mg/kg and 30 mg/kg, respectively. In pharmacokinetic analysis, both drugs were absorbed well in the rat gastrointestinal tract following oral administration. There was no difference between the metabolism of orally administered CV-araU and BV-araU in rats. High levels of the corresponding base were found in plasma after oral administration of CV-araU and BV-araU, but much lower base levels were seen after intravenous doses. Both drugs were resistant to degradation by rat liver enzymes.

Novel uracil derivatives: newly synthesized centrally acting agents.

A series of 1-amino-5-substituted uracils and their 4-thio or 2,4-dithio substituted analogues were synthesized and assayed for anti-conflict activity in rats and anesthetic activity in mice. 1-Amino-5-halogenouracils 3b-e, 1-amino-4-thiouracil (9a), and 1-amino-5-halogeno-4-thiouracils 9c, d showed both anti-conflict and anesthetic activities. The most active compound was 1-amino-5-chloro-4-thiouracil (9d) which showed anxiolytic activity at 2 mg/kg of oral administration (p.o.) on a modified Geller-Seifter conflict schedule. Its minimum effective dose (MED) was lower than that of diazepam. The 50 percent effective dose (ED50) for anesthetic activity in mice of the compound (9d) was 32.9 mg/kg, p.o.

Effect of surface ionization of dimyristoylphosphatidic acid vesicle membranes on the main phase-transition enthalpy and temperature.

The main phase transition of phospholipid bilayers is a property expressed by the order-disorder conformational change of the lipid tails. Nevertheless, with ionizable phospholipids, changes in the surface charge have large effects on the membrane properties. The free energy of a charged phospholipid membrane depends on the degree of ionization, area per phospholipid molecule, and the temperature. Here, the effect of surface electrostatic charges on the temperature and the enthalpy of the main phase transition of dimyristoylphosphatidic acid vesicle membranes is analyzed. A simple equation is presented that describes the relationship among the surface charge density, the phase-transition temperature, the surface area ratio between solid and liquid membranes, and the excess enthalpy. The theory indicated that the pH-induced shift in the excess enthalpy is attributable to the change in the surface area ratio between the solid and liquid membranes.