Acetophenones with selective antimycobacterial activity.

AIMS: Mycobacteria are a serious cause of infections in humans, with limited treatment options, as no new antibiotics have been developed against mycobacteria since the 1960s. In this study, the antimycobacterial activity of a small library of acetophenone (AP) compounds was analysed. METHODS AND RESULTS: Twenty-three AP derivatives were examined for activity against mycobacteria using a microbroth assay. The compounds were bacteriostatic, with the most effective (cyclohexylacetophenone and piperidinoacetophenone) having minimal inhibitory concentrations of 246 microM. Active compounds tended to be more hydrophobic, and may work by alkylation of as yet undetermined intracellular target protein(s). Cytotoxicity against eukaryotic cells was also determined and appears to be unrelated to the bacteriostatic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: AP may serve as a novel group of useful therapeutics against the mycobacteria.

Hormone binding by protein disulfide isomerase, a high capacity hormone reservoir of the endoplasmic reticulum.

Protein disulfide isomerase (PDI) is a folding assistant of the eukaryotic endoplasmic reticulum, but it also binds the hormones, estradiol, and 3,3',5-triiodo-l-thyronine (T(3)). Hormone binding could be at discrete hormone binding sites, or it could be a nonphysiological consequence of binding site(s) that are involved in the interaction PDI with its peptide and protein substrates. Equilibrium dialysis, fluorescent hydrophobic probe binding (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS)), competition binding, and enzyme activity assays reveal that the hormone binding sites are distinct from the peptide/protein binding sites. PDI has one estradiol binding site with modest affinity (2.1 +/- 0.5 microm). There are two binding sites with comparable affinity for T(3) (4.3 +/- 1.4 microm). One of these overlaps the estradiol site, whereas the other binds the hydrophobic probe, bis-ANS. Neither estradiol nor T(3) inhibit the catalytic or chaperone activity of PDI. Although the affinity of PDI for the hormones estradiol and T(3) is modest, the high local concentration of PDI in the endoplasmic reticulum (>200 microm) would drive hormone binding and result in the association of a substantial fraction (>90%) of the hormones in the cell with PDI. High capacity, low affinity hormone sites may function to buffer hormone concentration in the cell and allow tight, specific binding to the true receptor while preserving a reasonable number of hormone molecules in the very small volume of the cellular environment.

[Callus distraction of the mid-face in severe mid-face hypoplasia]. / Kallusdistraktion des Mittelgesichts bei ausgeprägter Mittelgesichtshypoplasie.

Callus distraction in the treatment of severe midfacial hypoplasia/atrophy may offer new therapeutical possibilities. Between May 1998 and March 1999, six patients underwent a subtotal Le Fort I-II osteotomy with subsequent distraction via a haloborne distractor (RED). Five patients suffered from CLP, four patients were totally or almost edentulous in the upper jaw. Corrections of sagittal discrepancies were possible in all cases (distraction 16-31 mm). No case of increased velopharyngeal insufficiency was seen. In one patient the halo had to be refixed. Three patients suffered from tension-like pain in the soft palate during distraction. Follow-up studies suggest the necessity for overcorrection of 20% in edentulous senior patients. Callus midfacial distraction is a promising new alternative to conventional orthognathic surgery in severe cases.

The stringent response of Mycobacterium tuberculosis is required for long-term survival.

The stringent response utilizes hyperphosphorylated guanine [(p)ppGpp] as a signaling molecule to control bacterial gene expression involved in long-term survival under starvation conditions. In gram-negative bacteria, (p)ppGpp is produced by the activity of the related RelA and SpoT proteins. Mycobacterium tuberculosis contains a single homolog of these proteins (Rel(Mtb)) and responds to nutrient starvation by producing (p)ppGpp. A rel(Mtb) knockout strain was constructed in a virulent strain of M. tuberculosis, H37Rv, by allelic replacement. The rel(Mtb) mutant displayed a significantly slower aerobic growth rate than the wild type in synthetic liquid media, whether rich or minimal. The growth rate of the wild type was equivalent to that of the mutant when citrate or phospholipid was employed as the sole carbon source. These two organisms also showed identical growth rates within a human macrophage-like cell line. These results suggest that the in vivo carbon source does not represent a stressful condition for the bacilli, since it appears to be utilized in a similar Rel(Mtb)-independent manner. In vitro growth in liquid media represents a condition that benefits from Rel(Mtb)-mediated adaptation. Long-term survival of the rel(Mtb) mutant during in vitro starvation or nutrient run out in normal media was significantly impaired compared to that in the wild type. In addition, the mutant was significantly less able to survive extended anaerobic incubation than the wild-type virulent organism. Thus, the Rel(Mtb) protein is required for long-term survival of pathogenic mycobacteria under starvation conditions.

[Callus distraction of the midface. Requirements for diagnostic imaging]. / Kallusdistraktion des Mittelgesichts. Anforderungen an die bildgebende Diagnostik.

Callus distraction of the midface is a recently developed surgical method for treatment of atrophic or hypoplastic maxillae. The treatment planning is based on individual shaped models, being constructed with the help of computed tomographic data. Our experience is based on 10 patients (3 male/7 female; age: 11-55 years). The demands for imaging are reported. The following imaging procedure is recommended: 1. preoperative: lateral x-ray, orthopantomogram, spiral CT, 2. during the time of callus distraction with normaly takes 4 to 5 weeks: lateral x-ray (every 10 days), and 3. for follow up: lateral x-ray and spiral CT. The Radiation exposure caused by the repetitive images is calculated in detail.

The soft tissue cover of the mandibular condyle. Differentiation in histological forms and age-related changes of aggrecan- and versican-like proteoglycans.

Age-related changes of the composition of the extracellular matrix of the soft tissue cover of the mandibular condyle (STC), especially of the large proteoglycans, have been investigated. Proteoglycans were extracted from the STC of neonatal, juvenile and adult domestic pigs, fractionated by density gradient centrifugation and analyzed by electrophoresis/Western blotting. Experiments revealed firstly that a large CS/KS proteoglycan (aggrecan) is an essential constituent of the STC at all ages. This proteoglycan is required for nutrition of avascular tissues, and age-related changes in its average size and substitution with KS (keratan sulfate) may be a response to altered functional loading and tissue architecture of the STC. Secondly it was shown that a large CS/DS (chondroitin sulfate/dermatan sulfate) proteoglycan characterized by a doublet of core proteins at 200 and 250 kDa, thereby resembling perlecan, is present in the tissue of adults, but not of neonates and juveniles. Thirdly a large CS/DS proteoglycan characterized by core proteins at 350, 450 and 550 kDa, thereby resembling versican, was present in juveniles. It was detectable only weakly in neonates and not in adults. Results of core protein analysis were confirmed by results of agarose gel electrophoresis/Western blotting of the undigested proteoglycans isolated directly from the tissue extracts. Versican is believed to destabilize cell-matrix interactions required for cell proliferation and differentiation. In this context, presence of versican-like proteoglycans in the STC of growing individuals and its disappearance in adults appears to be related to the growth potential of the mandibular condyle.

A 21-kDa C-terminal fragment of protein-disulfide isomerase has isomerase, chaperone, and anti-chaperone activities.

A catalyst of disulfide formation and isomerization during protein folding, protein-disulfide isomerase (PDI) has two catalytic sites housed in two domains homologous to thioredoxin, one near the N terminus and the other near the C terminus. The thioredoxin domains, by themselves, can catalyze disulfide formation, but they are unable to catalyze disulfide isomerizations (Darby, N. J. and Creighton, T. E. (1995) Biochemistry 34, 11725-11735). A 21-kDa, C-terminal fragment of PDI (amino acids 308-491), termed weePDI, comprises the C-terminal third of the molecule. The kcat for ribonuclease oxidative folding by weePDI is 0.26 +/- 0.02 min-1, 3-fold lower than the wild-type enzyme but indistinguishable from the activity of a full-length mutant of PDI in which both active site cysteines of the N-terminal thioredoxin domain have been mutated to serine. Eliminating the ability of weePDI to escape easily from covalent complexes with substrate by mutating the active site cysteine nearer the C terminus to serine has a large effect on the isomerase activity of weePDI compared with its effect on the full-length enzyme. weePDI also displays chaperone and anti-chaperone activity characteristic of the full-length molecule. As isolated, weePDI is a disulfide-linked dimer in which the single cysteine (Cys-326) outside active site cross-links two weePDI monomers. The presence of the intermolecular disulfide decreases the activity by more than 2-fold. The results imply that the functions of the core thioredoxin domains of PDI and other members of the thioredoxin superfamily might be modified quite easily by the addition of relatively small accessory domains.

Facilitated protein aggregation. Effects of calcium on the chaperone and anti-chaperone activity of protein disulfide-isomerase.

Protein disulfide-isomerase (PDI) catalyzes the formation and isomerization of disulfides during oxidative protein folding in the eukaryotic endoplasmic reticulum. At high concentrations, it also serves as a chaperone and inhibits aggregation. However, at lower concentrations, PDI can display the unusual ability to facilitate aggregation, termed anti-chaperone activity (Puig, A., and Gilbert, H. F. (1994) J. Biol. Chem. 269, 7764-7771). Under reducing conditions (10 mM dithiothreitol) and at a low concentration (0.1-0. 3 microM) relative to the unfolded protein substrate, PDI facilitates aggregation of alcohol dehydrogenase (11 microM) that has been denatured thermally or chemically. But at higher concentrations (>0.8 microM), PDI inhibits aggregation under the same conditions. With denatured citrate synthase, PDI does not facilitate aggregation, but higher concentrations do inhibit aggregation. Anti-chaperone behavior is associated with the appearance of both PDI and substrate proteins in insoluble complexes, while chaperone behavior results in the formation of large (>500 kDa) but soluble complexes that contain both proteins. Physiological concentrations of calcium and magnesium specifically increase the apparent rate of PDI-dependent aggregation and shift the chaperone activity to higher PDI concentrations. However, calcium has no effect on the Km or Vmax for PDI-catalyzed oxidative folding, suggesting that the interactions that lead to chaperone/anti-chaperone behavior are distinct from those required for catalytic activity. To account for this unusual behavior of a folding catalyst, a model with analogy to classic immunoprecipitation is proposed; multivalent interactions between PDI and a partially aggregated protein stimulate further aggregate formation by noncovalently cross-linking smaller aggregates. However, at high ratios of PDI to substrate, cross-linking may be inhibited by saturation of the sites with PDI. The effects of PDI concentration on substrate aggregation and the modulation of the behavior by physiological levels of calcium may have implications for the involvement of PDI in protein folding, aggregation, and retention in the endoplasmic reticulum.

Mycobacterium tuberculosis 16-kDa antigen (Hsp16.3) functions as an oligomeric structure in vitro to suppress thermal aggregation.

Tuberculosis continues to be a major disease threatening millions of lives worldwide. Several antigens of Mycobacterium tuberculosis, identified by monoclonal antibodies, have been cloned and are being exploited in the development of improved vaccines and diagnostic reagents. We have expressed and purified the 16-kDa antigen, an immunodominant antigen with serodiagnostic value, which has been previously cloned and shown to share low sequence homology with the alpha-crystallin-related small heat shock protein family. Sedimentation equilibrium analytical ultracentrifugation and dynamic light scattering demonstrate the formation of a specific oligomer, 149 +/- 8 kDa, consisting of approximately nine monomers. In 4 M urea, a smaller oligomer of 47 +/- 6 kDa (or trimer) is produced. Analysis by electron cryomicroscopy reveals a triangular shaped oligomeric structure arising from the presence of three subparticles or globules. Taken together, the data suggest an antigen complex structure of a trimer of trimers. This antigen, independent of ATP addition, effectively suppresses the thermal aggregation of citrate synthase at 40 degrees C, indicating that it can function as a molecular chaperone in vitro. A complex between the antigen and heat-denatured citrate synthase can be detected and isolated using high performance liquid chromatography. We propose to rename the 16-kDa antigen Hsp16.3 to be consistent with other members of the small heat shock protein family.