Post-partum thyroiditis in South Brazil presenting as thyrotoxicosis: prevalence and risk factors.

The prevalence of post-partum thyroiditis (PPT) has been reported in several countries (1.9 to 16.7%) but is not known in Brazil. Several factors have been associated to its development, such as a female sex of the newborn, PPT in a previous pregnancy, a family history of thyroid disease and cigarette smoking. To investigate the prevalence of PPT and its risk factors in a southern Brazilian city, a three-cross-sectional observation study was performed. PPT was diagnosed in 14/284 subjects (5.3%) and all cases had thyrotoxicosis (13 sub-clinical and one clinical). Serum total T4 and free T4 were higher and serum TSH was lower in PPT subjects. Anti-thyroid antibodies were positive in 16.7% of PPT subjects and in 4.5% of those with no thyroid dysfunction. Goiter was identified in 14.3% of PPT subjects and in 15% of no PPT subjects. Thyroid was hardened more frequently in PPT subjects (21.4%) than in others (5.2%). Male sex of the newborn was associated to PPT, increasing 11 times the risk of PPT. Cigarette smoking was associated to PPT in group II subjects. There was no clinical sign or symptom able to contribute to this diagnosis, except the presence of hardened thyroid. Based on these findings, PPT, manifesting itself as mild thyrotoxicosis, is a common problem in southern Brazil and is associated to male sex of the newborn.

Real-time transmission of full-motion echocardiography over a high-speed data network: impact of data rate and network quality of service.

UNLABELLED: With high-resolution network transmission required for telemedicine, education, and guided-image acquisition, the impact of errors and transmission rates on image quality needs evaluation. METHODS: We transmitted clinical echocardiograms from 2 National Aeronautics and Space Administration (NASA) research centers with the use of Motion Picture Expert Group-2 (MPEG-2) encoding and asynchronous transmission mode (ATM) network protocol over the NASA Research and Education Network. Data rates and network quality (cell losses [CLR], errors [CER], and delay variability [CVD]) were altered and image quality was judged. RESULTS: At speeds of 3 to 5 megabits per second (Mbps), digital images were superior to those on videotape; at 2 Mbps, images were equivalent. Increasing CLR caused occasional, brief pauses. Extreme CER and CDV increases still yielded high-quality images. CONCLUSIONS: Real-time echocardiographic acquisition, guidance, and transmission is feasible with the use of MPEG-2 and ATM with broadcast quality seen above 3 Mbps, even with severe network quality degradation. These techniques can be applied to telemedicine and used for planned echocardiography aboard the International Space Station.

Activation of JNK3 alpha 1 requires both MKK4 and MKK7: kinetic characterization of in vitro phosphorylated JNK3 alpha 1.

JNK3 alpha 1 is predominantly a neuronal specific MAP kinase that is believed to require, like all MAP kinases, both threonine and tyrosine phosphorylation for maximal enzyme activity. In this study we investigated the in vitro activation of JNK3 alpha 1 by MAP kinase kinase 4 (MKK4), MAP kinase kinase 7 (MKK7), and the combination of MKK4 + MKK7. Mass spectral analysis showed that MKK7 was capable of monophosphorylating JNK3 alpha 1 in vitro, whereas both MKK4 and MKK7 were required for bisphosphorylation and maximal enzyme activity. Measuring catalysis under Vmax conditions showed MKK4 + MKK7-activated JNK3 alpha 1 had Vmax 715-fold greater than nonactivated JNK3 alpha 1 and MKK7-activated JNK3 alpha 1 had Vmax 250-fold greater than nonactivated JNK3 alpha 1. In contrast, MKK4-activated JNK3 alpha 1 had no increase in Vmax compared to nonactivated levels and had no phosphorylation on the basis of mass spectrometry. These data suggest that MKK7 was largely responsible for JNK3 alpha 1 activation and that a single threonine phosphorylation may be all that is needed for JNK3 alpha 1 to be active. The steady-state rate constants kcat, Km(GST-ATF2++), and Km(ATP) for both monophosphorylated and bisphosphorylated JNK3 alpha 1 were within 2-fold between the two enzyme forms, suggesting the addition of tyrosine phosphorylation does not affect the binding of ATF2, ATP, or maximal turnover. Finally, the MAP kinase inhibitor, SB203580, had an IC50 value approximately 4-fold more potent on the monophosphorylated JNK3 alpha 1 compared to the bisphosphorylated JNK3 alpha 1, suggesting only a modest effect of tyrosine phosphorylation on inhibitor binding.

Design and synthesis of potent, selective, and orally bioavailable tetrasubstituted imidazole inhibitors of p38 mitogen-activated protein kinase.

Novel potent and selective diarylimidazole inhibitors of p38 MAP (mitogen-activated protein) kinase are described which have activity in both cell-based assays of tumor necrosis factor-alpha (TNF-alpha) release and an animal model of rheumatoid arthritis. The SAR leading to the development of selectivity against c-Raf and JNK2alpha1 kinases is presented, with key features being substitution of the 4-aryl ring with m-trifluoromethyl and substitution of the 5-heteroaryl ring with a 2-amino substituent. Cell-based activity was significantly enhanced by incorporation of a 4-piperidinyl moiety at the 2-position of the imidazole which also enhanced aqueous solubility. In general, oral bioavailability of this class of compounds was found to be poor unless the imidazole was methylated on nitrogen. This work led to identification of 48, a potent (p38 MAP kinase inhibition IC50 0.24 nM) and selective p38 MAP kinase inhibitor which inhibits lipopolysaccharide-stimulated release of TNF-alpha from human blood with an IC50 2.2 nM, shows good oral bioavailability in rat and rhesus monkey, and demonstrates significant improvement in measures of disease progression in a rat adjuvant-induced arthritis model.

Molecular basis for p38 protein kinase inhibitor specificity.

p38 is a member of the mitogen-activated protein (MAP) kinase family and is a critical enzyme in the proinflammatory cytokine pathway. Other MAP kinase group members that share both structural and functional homology to p38 include the c-Jun NH2-terminal kinases (JNKs or SAPKs) and the extracellular-regulated protein kinases (ERKs). In this study, we determined the molecular basis for p38alpha inhibitor specificity exhibited by five compounds in the diarylimidazole, triarylimidazole, and triarylpyrrole classes of protein kinase inhibitors. These compounds are significantly more potent inhibitors of p38 compared to the JNKs and ERKs. Three active site ATP-binding domain residues in p38, T106, M109, and A157, selected based on primary sequence alignment, molecular modeling, and X-ray crystal structure data, were mutated to assess their role in inhibitor binding and enzymatic catalysis. All mutants, with the exception of T106M, had kinase activity within 3-fold of wild-type p38. Mutation of T106 to glutamine, the residue present at the corresponding position in ERK-2, or methionine, the corresponding residue in p38gamma, p38delta, and the JNKs, rendered all five inhibitors ineffective. The diarylimidazoles had approximately a 6-fold decrease in potency toward M109A p38. For the mutant A157V, all diarylimidazoles and triarylimidazoles tested were 5-10-fold more potent compared with wild-type p38. In contrast, two triarylpyrroles were 15-40-fold less potent versus A157V p38. These results showed that the molecular basis for the specificity of the p38 inhibitors was attributed largely to threonine 106 in p38 and that methionine 109 contributes to increased binding affinity for imidazole based inhibitors.

The activation state of p38 mitogen-activated protein kinase determines the efficiency of ATP competition for pyridinylimidazole inhibitor binding.

The serine/threonine kinase p38 is a ubiquitous, highly conserved, stress responsive, signal-transducing enzyme. It regulates the production of proinflammatory mediators and is the target of the cytokine synthesis inhibitory pyridinylimidazoles. We have expressed human p38 in Drosophila S2 cells and characterized preparations of mixed unphosphorylated/monophosphorylated (inactive) and homogeneously diphosphorylated (active) forms of the enzyme. We observed that only the active preparation of the enzyme has significant kinase activity when assayed using an ATF2-GST fusion protein as the substrate. We determined that the value of KM[ATP] in this reaction is 25 microM and that the pyridinylimidazole inhibitor of p38 kinase activity, SB203580, competes with ATP. We have found that a tritiated pyridinylimidazole, SB202190, has an equal affinity for both the active and inactive forms of the enzyme and that SB203580 competes with it equally well for binding to either form of the enzyme. However, ATP can compete with the tritiated inhibitor for binding to only the active form of the enzyme. Further, we demonstrate in vivo that at concentrations consistent with its IC50 as a cytokine inhibitor, SB203580 can inhibit stimulus-induced phosphorylation of p38 at the Thr-Gly-Tyr activation motif. Our observations suggest that pyridinylimidazoles may block the biological activity of p38 kinase by binding to the inactive form of p38 and reducing its rate of activation. Under these conditions, ATP would not effectively compete with the inhibitors in vivo.

Pyrroles and other heterocycles as inhibitors of p38 kinase.

Investigation of furans, pyrroles and pyrazolones identified 3-pyridyl-2,5-diaryl-pyrroles as potent, orally bioavailable inhibitors of p38 kinase. 3-(4-pyridyl-2-(4-fluoro-phenyl)-5-(4-methylsulfinylphenyl)-pyrrol e (L-167307) reduces secondary paw swelling in the rat adjuvant arthritis model: ID50 = 7.4 mg/kg/b.i.d.

Kinetic mechanism for p38 MAP kinase.

p38 has been shown to be a critical enzyme in the pro-inflammatory cytokine pathway and is a member of the mitogen-activated protein (MAP) kinase family. While the details for p38 activation and subsequent signal transduction have begun to be elucidated, little is known about the kinetic mechanism for p38. In this study, we have determined the kinetic mechanism for p38 MAP kinase. Data from initial velocity patterns in the presence and absence of a dead-end inhibitor and two triarylimidazole p38 inhibitors were consistent with an ordered sequential mechanism for p38 with protein substrate, glutathione S-transferase-activating transcription factor 2 (GST-ATF2), binding before ATP. The ATP analog, adenylyl methylenediphosphonate (AMP-PCP), and two triarylimidazoles were competitive inhibitors versus ATP and uncompetitive inhibitors versus GST-ATF2. Equilibrium binding studies utilizing a tritiated ATP-competitive inhibitor were also consistent with this mechanism and suggest an inability of ATP to bind to p38 in the absence of protein substrate. Moreover, the Michaelis constant for GST-ATF2 was 12-fold greater than the dissociation constant, indicating that the binding of ATP affected the binding of GST-ATF2. An ordered sequential mechanism with protein substrate binding first is unique to p38 compared to cyclic AMP-dependent protein kinase (cAPK) and most tyrosine kinases and helps to explain the interaction between enzyme, substrates, and inhibitors.

Regulation of IkB alpha phosphorylation by PKC- and Ca(2+)-dependent signal transduction pathways.

The Ca(2+)-dependent phosphatase calcineurin, a target of FK506 and CsA, synergizes with PKC-induced activation of nuclear factor (NF)-kappa B in T cell lines. We have investigated whether this synergy is present in other cell types and the mechanism(s) by which these two pathways lead to NF-kappa B activation. While this synergy is present in other cell types, in the monocytic cell line U937 calcineurin is also sufficient to activate NF-kappa B. Having previously shown that Ca(2+)- and PKC-dependent pathways synergize by accelerating the degradation of IkB alpha, we focused on the regulation of IkB alpha phosphorylation. While PKC-dependent pathways sequentially result in the phosphorylation and in an incomplete degradation of IkB alpha in T cell lines, co-activation of Ca(2+)-dependent pathways accelerates the rate of IkB alpha phosphorylation and results in its complete degradation. Activation of Ca(2+)-dependent pathways alone do not result in the phosphorylation and/or degradation of IkB alpha in Jurkat T or in U937 cells. Treatment of T cells with the selective PKC inhibitor GF109203X abrogates the PMA-induced IkB alpha phosphorylation/degradation irrespective of activation of Ca(2+)-dependent pathways, but not the phosphorylation and degradation of IkB alpha induced by TNF-alpha, a PKC-independent stimulus. Contrary to the interaction with PKC, Ca(2+)-dependent pathways synergize with TNF-alpha not at the level of IkB alpha phosphorylation, but at the level of its degradation. These results indicate that Ca(2+)-dependent pathways, including the phosphatase calcineurin, participate in the regulation of NF-kappa B in a cell specific fashion and synergize with PKC-dependent and -independent pathways at the level of IkB alpha phosphorylation and degradation.

Calcineurin acts in synergy with PMA to inactivate I kappa B/MAD3, an inhibitor of NF-kappa B.

The interleukin-2 (IL-2) promoter consists of several independent T cell receptor (TcR) responsive elements. The induction of promoters dependent on these elements is inhibitable by the immunosuppressants cyclosporin A (CsA) and tacrolimus (FK-506). Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is the FK-506- and CsA-sensitive enzyme required for TcR mediated activation of the IL-2 promoter. We report that a constitutively active form of calcineurin partially substitutes for the Ca2+ co-stimulus required to activate the IL-2 promoter elements IL-2A (which binds the factors OAP and Oct-1) and IL-2E (which binds NF-AT), and completely substitutes for the Ca2+ co-stimulus required to stimulate an NF-kappa B-dependent element. Calcineurin stimulates the NF-kappa B element by enhancing inactivation of I kappa B/MAD3, an inhibitor of NF-kappa B, thereby increasing the amount of nuclear NF-kappa B DNA binding activity. These data provide the first demonstration in vivo that activation of a protein phosphatase can inactivate I kappa B, and suggest one possible explanation for mechanism-based toxicities associated with FK-506 and CsA by demonstrating that these drugs can inhibit the calcineurin-dependent activation of a virtually ubiquitous transcription factor.

Gene sequence, overproduction, purification and determination of the wild-type level of the Escherichia coli flagellar switch protein FliG.

The flagellar motor switch in Escherichia coli and Salmonella typhimurium controls swimming behavior by regulating the direction of flagellar rotation. The switch is a complex apparatus composed of at least three proteins--FliG, FliM and FliN. During chemotactic behavior, the switch responds to signals transduced by the chemotaxis sensory signaling system. CheY, the chemotaxis response regulator, is thought to act directly on the switch to induce tumbles in the swimming pattern, but physical interaction of CheY and switch proteins has not been shown. We have undertaken this work to develop the molecular tools to investigate CheY binding to switch proteins, as well as to understand more about the structure and function of the switch. We present here the sequences of the fliG gene and its protein product, the engineering and amplification of fliG by the polymerase chain reaction (PCR) and its subcloning, and the overproduction, purification and determination of the wild-type (wt) level of the FliG protein. The sequence data revealed a 91.8% amino acid (aa) identity between E. coli and S. typhimurium FliG. Engineering and amplifying fliG by PCR allowed convenient cloning into an efficient expression vector. FliG was successfully overproduced and purified to > 98% purity. Polyclonal antibodies (Ab) were generated against purified FliG and used in quantitative Western blots to determine that the wt expression level of fliG results in about 3700 FliG copies per cell. Purified FliG and anti-FliG Ab will be useful for direct biochemical analyses of CheY-switch protein interaction.

Comparison between mechanical cleaning and an antimicrobial rinse for the treatment and prevention of interdental gingivitis.

This study compared the efficacy of an antimicrobial mouthrinse (0.12% chlorhexidine gluconate) plus toothbrushing (mouthrinse group), mechanical interdental cleaning plus toothbrushing (mechanical group), and toothbrushing alone (control group), at reducing and preventing interdental gingival inflammation. 92 male subjects were examined for interdental inflammation using the Eastman interdental bleeding index at baseline, then monthly for 3 months after using one of the above oral hygiene regimens. The mechanical cleaning group had significant reductions in bleeding sites compared to baseline at 1 month (56.90% versus 13.17%) that persisted throughout the study (2 months = 6.65%, 3 months = 5.70%). The other regimens showed no significant bleeding reduction at any time point in the study. The mechanical interdental cleaning group showed improvement over baseline at 1 month with the full benefit apparent after 2 months. The effect of location in the mouth on bleeding reduction was also assessed. The % of posterior sites which bled was always higher than anterior sites. Analysis of maxillary versus mandibular, and buccal versus lingual sites showed no significant differences. Additional observations of the data demonstrated that sites which bled at baseline were more likely to stop bleeding in the mechanical cleaning group. Also, sites which did not bleed at baseline were unlikely to bleed subsequently when mechanical cleaning was used. Neither of these observations were true for the other cleaning regimens. These data show that only mechanical interdental plaque removal combined with toothbrushing is effective at reducing or preventing interdental inflammation. This underscores the importance of instituting mechanical interdental cleaning to eliminate interdental inflammation.

Guided tissue regeneration in interproximal defects in the monkey.

The potential for guided tissue regeneration was evaluated in one-walled interproximal sites in Macaca fascicularis. Histologic differences were evaluated at 1 and 3 months. Within the experimental (barrier) group, 100% of the root surfaces with potential for regeneration were covered with new cementum, whereas the control specimens had 20% or less new cementum. The amount of regeneration was determined by the position of the barrier membrane; the more coronal the barrier, the greater the regeneration. Observations indicated that the optimal time for barrier removal is between 1 and 3 months.

DNA distortion accompanies transcriptional activation by the metal-responsive gene-regulatory protein MerR.

Transcriptional regulation of the bacterial mercuric ion resistance operon (mer) in response to nanomolar concentrations of mercuric ion is achieved by the allosterically modulated transcriptional activator protein MerR. We now show that mercuric ion modification of MerR activates transcription, facilitating the conversion of an RNA polymerase complex with the mer promoter from the closed conformation to the strand-separated, transcriptionally competent open complex. An Hg-MerR-induced structural alteration at the center of the promoter has been detected in the presence or absence of RNA polymerase by use of chemical nucleases sensitive to variations in DNA secondary structure. This hypersensitivity correlates directly with transcriptional activation, lending further support to a previous proposal that a protein-induced distortion in local DNA structure can be the key step in an allosterically modulated transcription activation mechanism.

Characterization and nucleotide sequence determination of a repeat element isolated from a 2,4,5-T degrading strain of Pseudomonas cepacia.

Pseudomonas cepacia strain AC1100, capable of growth on 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), was mutated to the 2,4,5-T- strain PT88 by a ColE1::Tn5 chromosomal insertion. Using cloned DNA from the region flanking the insertion, a 1477-bp sequence (designated RS1100) was identified which was repeated several times on the wild-type chromosome and was also present on AC1100 plasmid DNA. Various chromosomal fragments containing this sequence were cloned and their nucleotide sequence was determined. Examination of RS1100 revealed the presence of 38-39-bp terminal inverted repeats immediately flanked by 8-bp direct repeats. The translated sequence of the single large open reading frame of RS1100 showed structural similarity to the phage Mu transposase and other DNA-binding proteins. Thus the AC1100 repeated sequence has several structural features in common with insertion sequence elements. Three copies of RS1100 were mapped near 2,4,5-t genes encoding degradation of 5-chloro-1,2,4-trihydroxybenzene, an intermediate in 2,4,5-T degradation. Neither RS1100 nor the 2,4,5-t genes hybridized to DNA isolated from Pseudomonas strains, including P. cepacia, suggesting that both gene fragments may be of foreign origin recruited in strain AC1100. The origin of these two DNA segments as well as the role played by RS1100 in the recruitment of 2,4,5-t genes in AC1100 are presently under investigation.

The MerR heavy metal receptor mediates positive activation in a topologically novel transcription complex.

Several physical and chemical signals from the extracellular environment are known to be transduced into changes in gene expression through multiple step pathways; however, mechanisms for triggering cellular responses to heavy metal stress have yet to be elucidated. We demonstrate here one such mechanism that employs a single heavy metal receptor protein, MerR, to directly activate transcription of the bacterial mercuric ion resistance operon. The mercuric ion-MerR complex and E. coli RNA polymerase holoenzyme synergistically bind to the metal responsive promoter in an unprecedented spatial relationship to form transcriptionally competent complexes. The activator binds adjacent to and overlaps with the polymerase molecule between the consensus -35 and -10 promoter regions. Our results support a model for transcriptional activation that includes both effector-induced protein-protein interactions and activator-induced alteration in DNA structure.