Budd-Chiari syndrome associated with coagulation abnormalities in a child with carbohydrate deficient glycoprotein syndrome type Ix.

A 6-year-old male patient presented with Budd-Chiari syndrome and glycoprotein abnormalities associated with carbohydrate deficient glycoprotein syndrome type I with yet unidentified molecular defect (type Ix). Budd-Chiari syndrome most likely developed after hepatic venous thrombosis caused by coagulation abnormalities resulting from hypoglycosylation and functional impairment of anticoagulant proteins.

Modulation of RNA function by aminoglycoside antibiotics.

One of the most important families of antibiotics are the aminoglycosides, including drugs such as neomycin B, paromomycin, gentamicin and streptomycin. With the discovery of the catalytic potential of RNA, these antibiotics became very popular due to their RNA-binding capacity. They serve for the analysis of RNA function as well as for the study of RNA as a potential therapeutic target. Improvements in RNA structure determination recently provided first insights into the decoding site of the ribosome at high resolution and how aminoglycosides might induce misreading of the genetic code. In addition to inhibiting prokaryotic translation, aminoglycosides inhibit several catalytic RNAs such as self-splicing group I introns, RNase P and small ribozymes in vitro. Furthermore, these antibiotics interfere with human immunodeficiency virus (HIV) replication by disrupting essential RNA-protein contacts. Most exciting is the potential of many RNA-binding antibiotics to stimulate RNA activities, conceiving small-molecule partners for the hypothesis of an ancient RNA world. SELEX (systematic evolution of ligands by exponential enrichment) has been used in this evolutionary game leading to small synthetic RNAs, whose NMR structures gave valuable information on how aminoglycosides interact with RNA, which could possibly be used in applied science.

The antibiotic viomycin as a model peptide for the origin of the co-evolution of RNA and proteins.

Viomycin is an RNA-binding peptide antibiotic which inhibits prokaryotic protein synthesis and group I intron self-splicing. This antibiotic enhances the activity of the ribozyme derived from the Neurospora crassa VS RNA, and at sub-inhibitory concentrations it induces the formation of group I intron oligomers. Here, we address the question whether viomycin exerts specificity in the promotion of RNA-RNA interactions. In an in vitro selection experiment we tested the ability of viomycin to specifically select molecules out of an RNA pool. Group I intron RNA was incubated with a pool of random sequence RNA, or with a pool of RNA molecules which had previously been enriched for viomycin-binding RNAs. Viomycin was added in order to select viomycin-binding RNAs and to guide their interaction with the intron RNA resulting in recombinant molecules. Viomycin was indeed capable of specifically selecting RNA molecules which contain viomycin-binding sites promoting recombination. These results suggest that small peptides are able to play the role of selector molecules in a putative 'RNA World' launching the co-evolution of RNA and proteins into an 'RNA-protein World'.

A reverse transcriptase/maturase promotes splicing by binding at its own coding segment in a group II intron RNA.

Group II introns encode reverse transcriptases that promote RNA splicing (maturase activity) and then with the excised intron form a DNA endonuclease that mediates intron mobility by target DNA-primed reverse transcription (TPRT). Here, we show that the primary binding site for the maturase (LtrA) encoded by the Lactococcus lactis Ll.LtrB intron is within a region of intron domain IV that includes the start codon of the LtrA ORF. This binding is enhanced by other elements, particularly domain I and the EBS/IBS interactions, and helps position LtrA to initiate cDNA synthesis in the 3' exon as occurs during TPRT. Our results suggest how the maturase functions in RNA splicing and support the hypothesis that the reverse transcriptase coding region was derived from an independent genetic element that was inserted into a preexisting group II intron.

RNA and protein catalysis in group II intron splicing and mobility reactions using purified components.

Group II introns encode proteins with reverse transcriptase activity. These proteins also promote RNA splicing (maturase activity) and then, with the excised intron, form a site-specific DNA endonuclease that promotes intron mobility by reverse splicing into DNA followed by target DNA-primed reverse transcription. Here, we used an Escherichia coli expression system for the Lactococcus lactis group II intron Ll.LtrB to show that the intron-encoded protein (LtrA) alone is sufficient for maturase activity, and that RNP particles containing only the LtrA protein and excised intron RNA have site-specific DNA endonuclease and target DNA-primed reverse transcriptase activity. Detailed analysis of the splicing reaction indicates that LtrA is an intron-specific splicing factor that binds to unspliced precursor RNA with a K(d) of

Cord blood leucocytes/basophils produce and release sulfidoleucotrienes in response to allergen stimulation.

BACKGROUND: Leucocyte-derived sulfidoleucotrienes (SLT) from children and adults can be detected in vitro in response to specific allergen stimulation, a mechanism thought to require the presence of allergen-specific immunoglobulin (Ig)E antibodies on the surface of basophils. It is unknown whether this mechanism is functional in cord blood basophils. OBJECTIVE: We studied the in vitro SLT-release of leucocytes in response to allergen and anti-IgE stimulation in term newborns and children with allergic diseases. METHODS: Cord blood from randomly selected term newborns were analysed for total IgE-antibodies and in vitro SLT-release in response to allergen and anti-IgE stimulation. Children from an allergy outpatient clinic were used as the control group. The Cellular Allergen Stimulation Test (CAST) was used as read-out system. Allergen stimulation was performed with an allergen-mix containing 21 nutritive and inhalant allergens. RESULTS: Peripheral blood leucocytes/basophils derived from allergic children (n = 56; median SLT release 1049 pg/mL) were more responsive to anti-IgE stimulation as cord blood leucocytes/basophils (n = 104; median 419 pg/mL P < 0.0001). In response to stimulation with an allergen-mix, the two groups did not differ significantly from each other. Only SLT-releasability in response to anti-IgE showed a correlation with cord blood IgE. CONCLUSIONS: Sulfidoleucotriene-release of cord blood basophils is functional in response to allergens. It appears possible that cord blood basophils are armed with allergen-specific IgE-antibodies though not detectable in serum. Therefore, cord blood SLT-release may indirectly reflect prenatal priming with allergens with subsequent production of allergen-specific IgE.

Clinical significance of lupus anticoagulants in children.

OBJECTIVES: To determine the spectrum of associated clinical manifestations and time course of lupus anticoagulants (LA) in children. STUDY DESIGN: Retrospective study of 95 consecutive children (46 boys and 47 girls), with a median age of 5.3 years (range, 1.7 to 17.1 years), diagnosed with presence of LA at a hemostasis referral center; 83 were followed up over a median of 2.9 years (range, 6 weeks to 21.6 years). RESULTS: At diagnosis, 80 of 95 (84%) children were free of symptoms, and presence of LA was found incidentally. Nine children (10%) had bleeding symptoms, 5 (5%) had thrombotic events, and 1 had systemic lupus erythematosus. Among the patients with bleeding, 5 had transient severe hypoprothrombinemia after adenovirus infections, and 3 had thrombocytopenia. None of the children who were initially free of symptoms had bleeding, thrombotic complications, or autoimmune disease subsequently. At follow-up, 48 of 83 (58%) patients had normal activated partial thromboplastin time values after 1.9 years (5 weeks to 19.1 years). Thirty-two (38%) still had activated partial thromboplastin time elevations but did not fulfill all criteria for presence of LA after 3.2 years (7.4 months to 9.3 years). Three (4%) patients, who had presented with thrombosis, had persistent positive LA, anti-cardiolipin, and antinuclear antibodies after 1.4, 2.8, and 7.5 years, respectively. One of these had recurrent thrombosis. CONCLUSIONS: In most children the presence of LA did not lead to clinical complications and was transient. Bleeding occurred with additional hypoprothrombinemia or thrombocytopenia. Thrombosis was rare and strongly associated with persistently positive LA.

A bacterial group II intron encoding reverse transcriptase, maturase, and DNA endonuclease activities: biochemical demonstration of maturase activity and insertion of new genetic information within the intron.

The Lactococcus lactis group II intron Ll.ltrB is similar to mobile yeast mtDNA group II introns, which encode reverse transcriptase, RNA maturase, and DNA endonuclease activities for site-specific DNA insertion. Here, we show that the Lactococcal intron can be expressed and spliced efficiently in Escherichia coli. The intron-encoded protein LtrA has reverse transcriptase and RNA maturase activities, with the latter activity shown both in vivo and in vitro, a first for any group II intron-encoded protein. As for the yeast mtDNA introns, the DNA endonuclease activity of the Lactococcal intron is associated with RNP particles containing both the intron-encoded protein and the excised intron RNA. Also, the intron RNA cleaves the sense-strand of the recipient DNA by a reverse splicing reaction, whereas the intron-encoded protein cleaves the antisense strand. The Lactococcal intron endonuclease can be obtained in large quantities by coexpression of the LtrA protein with the intron RNA in E. coli or reconstituted in vitro by incubating the expressed LtrA protein with in vitro-synthesized intron RNA. Furthermore, the specificity of the endonuclease and reverse splicing reactions can be changed predictably by modifying the RNA component. Expression in E. coli facilitates the use of group II introns for the targeting of specific foreign sequences to a desired site in DNA.

In vitro selection of a viomycin-binding RNA pseudoknot.

BACKGROUND: The peptide antibiotic viomycin inhibits ribosomal protein synthesis, group I intron self-splicing and self-cleavage of the human hepatitis delta virus ribozyme. To understand the molecular basis of RNA binding and recognition by viomycin, we isolated a variety of novel viomycin-binding RNA molecules using in vitro selection. RESULTS: More than 90% of the selected RNA molecules shared one continuous highly conserved region of 14 nucleotides. Mutational analyses, structural probing, together with footprinting experiments by chemical modification, and Pb2+-induced cleavage showed that this conserved sequence harbours the antibiotic-binding site and forms a stem-loop structure. Moreover, the loop is engaged in a long-range interaction forming a pseudoknot. CONCLUSIONS: A comparison between the novel viomycin-binding motif and the natural RNA target sites for viomycin showed that all these segments form a pseudoknot at the antibiotic-binding site. We therefore conclude that this peptide antibiotic has a strong selectivity for particular RNA pseudoknots.

Antibiotic-induced oligomerisation of group I intron RNA.

Antibiotics act as inhibitors of various biological processes. Here we demonstrate that some tuberactinomycins, hitherto known as inhibitors of prokaryotic protein synthesis and of group I intron self-splicing, have a modulatory effect on group I intron RNAs. The linear intron, which is excised during the self-splicing process, is still an active molecular capable of performing an intramolecular transesterification resulting in a circular molecule. However, in the presence of sub-inhibitory concentrations of tuberactinomycins, the intron reacts intermolecularly leading to the formation of linear head-to-tail intron-oligomers. The antibiotic stimulates the intron to react in trans instead of in cis. The phage T4-derived td intron uses the same sites for oligomerisation as for circularisation. Gel- retardation experiments demonstrate that the intron RNA forms non-covalent complexes in the presence of the antibiotic. It might be envisaged that the role of these peptide antibiotics is to bridge RNA molecules mediating RNA-RNA interactions and thus enabling their reaction. The tuberactinomycins are further able to induce the interaction of heterologous introns. The ligation of the T4 phage-derived td intron with the Tetrahymena rRNA intron is very efficient, resulting in molecules composed of two introns derived from different species. The td intron attacks the Tetrahymena intron at various sites, which are located within double-stranded regions. These observations suggest that small molecules like these basic peptide antibiotics could have mediated RNA-RNA interactions in a pre-protein era.

Polymerase chain reaction for detection of Chlamydia pneumoniae in gargled-water specimens of children.

The objective of the present study was to establish the occurrence of Chlamydia pneumoniae by direct detection in gargled-water specimens obtained from 193 children suffering from acute or chronic respiratory infections. Specimens were analyzed by an indirect immunofluorescence test (IIF), a genus-specific antigen enzyme immunosorbent assay (EIA) and the polymerase chain reaction (PCR). The pathogen was detected in three children by PCR only. As underlying disease, chronic obstructive bronchitis resistant to therapy was reported. In two of the children, the presence of pneumonia could be verified by X-ray. With a detection threshold of target DNA obtained from two inclusion forming units (IFU), the PCR proved clearly more sensitive than EIA becoming positive at levels of 100 IFU and above. No interpretable results could be obtained for the IIF.

Peptide antibiotics of the tuberactinomycin family as inhibitors of group I intron RNA splicing.

The tuberactinomycins are a group of cyclic peptide antibiotics, which are potent inhibitors of prokaryotic protein synthesis. We report the inhibitory effect of viomycin, di-beta-lysyl-capreomycin IIA and tuberactinomycin A on group I intron self-splicing. They compete with the guanosine cofactor for the G-binding site located in the conserved core of the intron. They are 100-fold more active than all other competitive inhibitors described so far (dGTP, arginine or streptomycin), inhibiting splicing at concentrations between 10 and 50 microM. Mutation of the G-binding site leads to partial resistance, and the inhibitory effect of these drugs is dependent on Mg2+ concentration. This suggests that the tuberactinomycins have more than one contact site with the intron RNA: via the G-binding site and via additional contacts with the RNA backbone. Positioning the tuberactinomycins in the three-dimensional model of the td intron core suggests that the charged lysyl side-chain (R1) is in contact with the backbone of the P1 helix. Structure/function analyses with various tuberactinomycin analogues with different activities confirm the involvement of this sidechain in inhibition of group I self-splicing. The demonstration of a new class of splicing inhibitors, the peptide antibiotics, illustrates how antibiotics may interact with catalytic RNA.

Evolution of secondary metabolite production: potential roles for antibiotics as prebiotic effectors of catalytic RNA reactions.

It has been proposed that organic molecules related to known secondary metabolites have existed since the beginning of biochemical evolution and were present in primordial soups. Under primitive earth conditions certain of these molecules may have played roles as effectors in prebiotic reactions, especially those involving catalytic RNA (ribozymes). We demonstrate that a number of antibiotic-related secondary metabolites bind to group I introns and either inhibit splicing reactions or promote the formation of intron oligomers. This is consistent with the functional co-evolution of catalytic RNA and secondary metabolites as antibiotic inhibitors of translation, and supports the notion of an evolutionary relationship between group I introns and ribosomal RNA.

Altered condylar morphology associated with disc displacement in TMJ dysfunction: observations by MRI.

Magnetic resonance images of 276 TM joints in 138 symptomatic patients were analyzed in a retrospective study to determine the condylar shape and size and to correlate it with internal derangement. Internal derangement by MRI was noted in 66% of the TM joints. Our study demonstrates that the regressive condylar changes in TM joints with internal derangement were more common (61%) than proliferative bony changes (39%). On the converse, none of the TM joints with regressive condyles revealed normal disc. The altered bony morphology also correlated with the severity of internal derangement, i.e. bony changes in TM joints with anterior closed lock were noted in 64% compared to 45% with reducible disc. The cause and effect relationship of the regressive condylar remodeling and disc abnormality is not clear and needs further study.

TMJ arthroscopy--a preoperative and postoperative rehabilitation protocol.

The purpose of this paper is to familiarize the specialist with surgical arthroscopy of the temporomandibular joint and to present a preoperative and postoperative rehabilitation protocol for patients undergoing this increasingly popular procedure. Clinical uses of arthroscopy are listed, and the criteria for arthroscopic intervention are described. The rationale for a comprehensive rehabilitation program is discussed in view of anatomic, neurologic, and functional relationships between the craniomandibular and craniocervical regions. Next, a preoperative and postoperative rehabilitation protocol is presented along with therapeutic goals for each phase of care. The timing and sequence of the protocol is intended to be a general guide, since modifications will be necessary depending on the extent of disk and supportive ligament damage as well as individual responses to rehabilitation.

Hepatitis B vaccination and immune response in children with malignant diseases.

Fifty children with malignant diseases were vaccinated against hepatitis B. Twenty-nine children suffered from leukaemia or non-Hodgkin's lymphoma; 14 of these were on intensive chemotherapy (group I) and 15 were without intensive therapy (group II). The other 21 children had various forms of solid tumours, 14 of them were on intensive therapy (group III) and 7 were without intensive therapy (group IV). To evaluate the immune response, we determined antibody titres over a period of more than 14 weeks after the first vaccination. As 22 out of 50 patients had received passive immunisation together with either the first or the first and second vaccination, antibody titres at the 14th and 18th week (i.e. more than 10 weeks after passive immunisation) were used to evaluate the vaccination results. An antibody titre of greater than or equal to 10 mIU/ml was considered to be a positive response. All patients of group IV, but only 4 out of 14 in group III, 4 out of 15 in group II, and 0 out of 14 in group I produced antibody titres higher than 50 mIU/ml. In contrast to the full response in group IV, two-thirds of all other patients had no immune response (less than 10 mIU/ml). Based on our experience we recommend vaccinating patients suffering from solid tumours and receiving no intensive therapy (group IV) against hepatitis B and protecting all the other children with malignant diseases by passive immunisation, if necessary.

A quantitative measurement of bacteremia and its relationship to plaque control.

Despite the improvement in the patients' oral hygiene, there was no significant decrease in the frequency of bacteremia. There was no significant difference in bacteremia between brushing, flossing, or deplaquing either before or after initial periodontal preparation and plaque control in 21 healthy subjects. Utilizing more sophisticated bacteriologic techniques for the cultivation of obligate anaerobes, a relatively high frequency of anaerobic bacteremia was found.