[Spontaneous Consolidation of Exercise-Induced Bilateral Apophyseolysis of Lesser Trochanter in a Young Ice Hockey Player - Long-Term Follow-up by MRI]. / Spontánní konsolidace ponámahové bilaterální apofyzeolýzy trochanter minor femoris u mladého hokejisty ­ dlouhodobé sledování magnetickou rezonancí.

Exercise-induced bilateral apophyseolysis of lesser trochanter with enthesopathy of iliopsoas muscle is a less frequent cause of the groin pain syndrome in children and adolescents. In a 13-year-old boy, an active ice hockey player, spontaneous consolidation of exercise-induced bilateral apophyseolysis of lesser trochanter and regression of enthesopathy of iliopsoas muscle is documented by a long-term follow-up through MRI. The conservative treatment comprises targeted rehabilitation focusing on the hypertonic muscles, but of equal importance is also comprehensive rehabilitation focusing on correction of the posture and coordination of the muscle groups. It is also necessary to include compensatory exercise in the training plan. Key words: groin pain syndrome, children, apophyseolysis of lesser trochanter, enthesopathy of iliopsoas muscle.

An appetite suppressant from Hoodia species.

Studies conducted at the Council for Scientific and Industrial Research (CSIR, South Africa) identified extracts from Hoodia species, in particular Hoodia pilifera and Hoodia gordonii, as possessing appetite suppressing properties. Two pregnane glycosides were isolated by fractionation of the dried stems of H. gordonii. Their structures were determined as 3beta-[beta-D-thevetopyranosyl-(1-->4)-beta-D- cymaropyranosyl-(1-->4)-beta-D-cymaropyranosyloxy]-12beta-tigloyloxy-14beta-hydroxypregn-5-en-20-one (1) and 3beta-[beta-D-cymaropyranosyl-(1-->4)-beta-D-6-thevetopyranosyl-(1-->4)-beta-D-cymaropyranosyl-(1-->4)-beta-D-cymaropyranosyloxy]-12beta-tigloyloxy-14beta-hydroxypregn-5-en-20-one (2) on the basis of spectroscopic studies and conversion to known compounds. Compounds 1 and 2 were also isolated from H. pilifera. Compound 1 was tested for its appetite suppressant properties in rats by oral gavage at 6.25-50 mg/kg and the results showed that all doses resulted in a decrease of food consumption over an eight day period and a body mass decrease when compared to the control sample receiving only the vehicle. In a comparative study against a fenfluramine control sample, compound 1 resulted in a reduction in food intake over the study period, with a concomitant overall decrease in body weight while fenfluramine resulted in a small decrease in food intake, but an increase in body weight (though less than control group) over the same period of time.

Involvement of sigma(S) in starvation-induced transposition of Pseudomonas putida transposon Tn4652.

Transpositional activity of mobile elements can be induced by different environmental stresses. Here, we present evidence that transposition of Tn4652 is elevated in stationary-phase Pseudomonas putida and suppressed in an isogenic sigma(S)-defective strain. We demonstrate that transcription from the Tn4652 transposase promoter is controlled by the stationary-phase-specific sigma factor sigma(S). To our knowledge, this is the first example of direct stationary-phase-specific regulation of a mobile element transposase. Data presented in this report support the idea that activation of transposition under stressful conditions could be an inducible process.

Transcription from fusion promoters generated during transposition of transposon Tn4652 is positively affected by integration host factor in Pseudomonas putida.

We have previously shown that both ends of the Tn3 family transposon Tn4652 contain integration host factor (IHF) binding sites and that IHF positively regulates expression of the Tn4652 transposase gene tnpA in Pseudomonas putida (R. Hõrak, and M. Kivisaar, J. Bacteriol. 180:2822-2829, 1998). Tn4652 can activate silent genes by creating fusion promoters during the transposition. The promoters are created as fusions between the -35 hexamer provided by the terminal inverted repeats of Tn4652 and the -10 hexamers in the target DNA. Two fusion promoters, PRA1 and PLA1, that contain sequences of the right and left termini of Tn4652, respectively, were chosen for the study of mechanisms of transcription activation. Gel mobility shift analysis using crude extracts from P. putida cells allowed us to detect specific binding of P. putida IHF to the ends of the transposon Tn4652. We found that the rate of transcription from the fusion promoter PRA1 is enhanced by IHF. Notably, the positive effect of IHF on transcription from the promoter PRA1 appeared only when cells of P. putida reached the stationary growth phase. We speculate that the intracellular concentration of IHF might be critical for the in vivo effect of IHF on transcription from the fusion promoters in P. putida. In the case of PLA1, the mechanism of transcription modulation by IHF is different than that observed for PRA1. Our results demonstrate that transcription of neighboring genes from outwardly directed promoters at the ends of a mobile DNA element could be influenced by the same factors that control transposition of the element.

Regulation of the transposase of Tn4652 by the transposon-encoded protein TnpC.

Transposition is a DNA reorganization reaction potentially deleterious for the host. The frequency of transposition is limited by the amount of transposase. Therefore, strict regulation of a transposase is required to keep control over the destructive multiplication of the mobile element. We have shown previously that the expression of the transposase (tnpA) of the Pseudomonas putida PaW85 transposon Tn4652 is positively affected by integration host factor. Here, we present evidence that the amount of the transposase of Tn4652 in P. putida cells is controlled by the transposon-encoded protein (TnpC). Sequence analysis of the 120-amino-acid-long TnpC, coded just downstream of the tnpA gene, showed that it has remarkable similarity to the putative polypeptide encoded by the mercury resistance transposon Tn5041. As determined by quantitative Western blot analysis, the abundance of TnpA was reduced up to 10-fold in the intact tnpC background. In vivo experiments using transcriptional and translational fusions of the tnpA gene and the reporter gene gusA indicated that TnpC operates in the regulation of the transposase of Tn4652 at the post-transcriptional level.

Identification and characterization of IS1411, a new insertion sequence which causes transcriptional activation of the phenol degradation genes in Pseudomonas putida.

A new insertion sequence (IS element), IS1411, was identified downstream of the phenol degradation genes pheBA that originated from plasmid DNA of Pseudomonas sp. strain EST1001. According to sequence analysis, IS1411 belongs to a new family of IS elements that has recently been named the ISL3 family (J. Mahillon and M. Chandler, Microbiol. Mol. Biol. Rev. 62:725-774, 1998). IS1411 generates 8-bp duplication of the target DNA and carries 24-bp inverted repeats (IRs), highly homologous to the IRs of other IS elements belonging to this family. IS1411 was discovered as a result of insertional activation of promoterless pheBA genes in Pseudomonas putida due to the presence of outward-directed promoters at the left end of IS1411. Both promoters located on the IS element have sequences that are similar to the consensus sequence of Escherichia coli sigma70. IS1411 can produce IS circles, and the circle formation is enhanced when two copies of the element are present in the same plasmid.

Expression of the transposase gene tnpA of Tn4652 is positively affected by integration host factor.

Tn4652 is a derivative of the toluene degradation transposon Tn4651 that belongs to the Tn3 family of transposons (M. Tsuda and T. Iino, Mol. Gen. Genet. 210:270-276, 1987). We have sequenced the transposase gene tnpA of transposon Tn4652 and mapped its promoter to the right end of the element. The deduced amino acid sequence of tnpA revealed 96.2% identity with the putative transposase of Tn5041. Homology with other Tn3 family transposases was only moderate (about 20 to 24% identity), suggesting that Tn4652 and Tn5041 are distantly related members of the Tn3 family. Functional analysis of the tnpA promoter revealed that it is active in Pseudomonas putida but silent in Escherichia coli, indicating that some P. putida-specific factor is required for the transcription from this promoter. Additionally, tnpA promoter activity was shown to be modulated by integration host factor (IHF). The presence of an IHF-binding site upstream of the tnpA promoter enhanced the promoter activity. The positive role of IHF was also confirmed by the finding that the enhancing effect of IHF was not detected in the P. putida ihfA-deficient strain A8759. Moreover, the Tn4652 terminal sequences had a negative effect on transcription from the tnpA promoter in the ihfA-defective strain. This finding suggests that IHF not only enhances transcription from the tnpA promoter but also alleviates the negative effect of terminal sequences of Tn4652 on the promoter activity. Also, an in vitro binding assay demonstrated that both ends of Tn4652 bind IHF from a cell lysate of E. coli.

Promoter-creating mutations in Pseudomonas putida: a model system for the study of mutation in starving bacteria.

A novel experimental system to study mutation in starving bacteria was designed, relying on the activation of a promoterless phenol degradation operon of Pseudomonas putida. The Phe+ (phenol-utilizing) mutants accumulated in the starving culture of P. putida in the presence of phenol but not in the absence of it. We ruled out the possibility that the absence of phenol eliminates Phe+ mutants from the starving population. Sequence analysis of the Phe+ mutants revealed that base substitutions, deletions, and insertion of Tn4652 can result in creation of a sequence similar to the sigma70-specific promoter consensus. One particular C --> A transversion was predominant in the Phe+ mutants that arose in the starving population under selection for phenol use. In contrast, various deletions were the most frequent Phe+ mutants occurring in a culture growing without selection. The accumulation rate of the Phe+ mutants on selective plates was found to be higher for bacteria plated from stationary-phase culture than that from exponentially growing cells. This suggests that some specific processes, occurring predominantly in stationary-phase cells, facilitate generation and/or fixation of such mutations.

Regulation of the catechol 1,2-dioxygenase- and phenol monooxygenase-encoding pheBA operon in Pseudomonas putida PaW85.

In Pseudomonas putida PaW85, the ortho-cleavage pathway is used for catechol degradation. The 11.4-kb XhoI fragment cloned from phenol degradation plasmid pEST1226 into pKT240 (recombinant plasmid pAT1140) contains the inducible pheBA operon that encodes catechol 1,2-dioxygenase (gene pheB) and phenol monooxygenase (gene pheA), the first two enzymes for the phenol degradation pathway. The promoter of the pheBA operon is mapped 1.5 kb upstream of the pheB gene. The plasmid pAT1140, when introduced into P. putida PaW85, enables the bacteria to use the hybrid plasmid-chromosome-encoded pathway for phenol degradation. The synthesis of the plasmid-encoded phenol monooxygenase and catechol 1,2-dioxygenase is induced by cis,cis-muconate. The expression studies of the deletion subclones derived from pAT1140 revealed that the transcription of the pheBA operon is positively controlled by a regulatory protein that is chromosomally encoded in P. putida. cis,cis-Muconate in cooperation with positive transcription factor CatR activates the transcription of the chromosomal ortho-pathway genes catA and catBC in P. putida (R. K. Rothmel, T. L. Aldrich, J. E. Houghton, W. M. Coco, L. N. Ornston, and A. M. Chakrabarty, J. Bacteriol. 172:922-931, 1990). The inability to express the pheBA operon in a P. putida CatR- background and activation of transcription of the pheBA operon in Escherichia coli in the presence of the catR-expressing plasmid demonstrated that the transcription of the pheBA operon in P. putida PaW85 carrying pEST1226 is controlled by the chromosomally encoded CatR.

In-vivo-generated fusion promoters in Pseudomonas putida.

Plasmid pEST1463 carrying the promoterless pheBA operon was cloned into Pseudomonas putida PaW85, and phenol-utilizing colonies were isolated on minimal plates containing phenol as the only carbon and energy source. In these clones, chromosomally located Tn4652 was transposed upstream from the coding sequencing of pheA (encoding phenol monooxygenase). Sequence analysis together with mapping of the transcription start point of the pheBA operon in the recombinant plasmids revealed that fusions of the -10 sequences present in the pheBA operon and -35 sequence located in the terminal inverted repeats of Tn4652 had generated functional promoters under selective pressure in P. putida cells. These promoter sequences show similarity to the Escherichia coli RNA polymerase sigma 70 promoter consensus sequence. In three of the six fusion promoters studied, the generation combined two distinct events: transposition of Tn4652 into DNA containing potential -10 sequences and point mutations in these sequences. These mutations made the -10 sequences more like the sigma 70 promoter consensus sequences.

Selection of independent plasmids determining phenol degradation in Pseudomonas putida and the cloning and expression of genes encoding phenol monooxygenase and catechol 1,2-dioxygenase.

Long-term cultivation of the Pseudomonas putida multiplasmid strain EST1020 on phenol resulted in the formation of individual PHE plasmids determining phenol degradation. Four types of PHE plasmids, pEST1024, pEST1026, pEST1028, and pEST1029, are characterized. They all contain a transferrable replicon similar to pWWO-8 with a partly duplicated DNA sequence of the 17-kb transposable element of this plasmid and include various amounts of DNA that carry genes encoding phenol degradation (phe genes). We cloned the genes determining phenol monooxygenase and catechol 1,2-dioxygenase from the Pseudomonas sp. parent strain plasmid DNA into the broad host range vector pAYC32 and studied the expression of the cloned DNA. The formation of a new hybrid metabolic plasmid, pEST1354, was demonstrated in P. putida PaW85 as the result of transposition of the 17-kb genetic element from the chromosome of PaW85 into the plasmid carrying cloned phe genes. The target site for the 17-kb transposon was localized in the vector DNA, just near the cloning site. In subcloning experiments we found two regions in the 17-kb DNA stretch that are involved in the expression of the cloned phe genes.

Studies of the cryptic allylic pyrophosphate isomerase activity of trichodiene synthase using the anomalous substrate 6,7-dihydrofarnesyl pyrophosphate.

Two enantiomeric analogues of farnesyl pyrophosphate (1) were tested as inhibitors and anomalous substrates of trichodiene synthase, which catalyzes the cyclization of trans,trans-farnesyl pyrophosphate (1) to the sesquiterpene hydrocarbon trichodiene (2). The reaction has been shown to involve preliminary isomerization of 1 to the tertiary allylic isomer nerolidyl pyrophosphate (3) which is cyclized without detectable release of the intermediate from the active site of the cyclase. Both (7S)-trans-6,7-dihydrofarnesyl pyrophosphate (7a) and (7R)-trans-6,7-dihydrofarnesyl pyrophosphate (7b), prepared from (3R)- and (3S)- citronellol (9a and 9b), respectively, proved to be modest competitive inhibitors of trichodiene synthase. The values of Ki(7a), 395 nM, and Ki(7b), 220 nM, were 10-15 times the observed Km for 1 and half the Ki of inorganic pyrophosphate alone. Incubation of either 7a or 7b with trichodiene synthase resulted in formation of a mixture of products which by radio/gas-liquid chromatographic and GC/selected ion mass spectrometric analysis was shown to be composed of 80-85% isomeric trienes 19-21 and 15-20% allylic alcohols 12 and 18. Examination of the water-soluble products resulting from incubation of 7a also revealed the generation of 24% of the isomeric cis-6,7-dihydrofarnesyl pyrophosphate (26). The combined rate of formation of anomalous alcoholic and olefinic products was 10% the Vmax determined for the conversion of 1 to 2. The results can be explained by initial enzyme-catalyzed isomerization of dihydrofarnesyl pyrophosphate (7) to the corresponding tertiary allylic isomer dihydronerolidyl pyrophosphate (8). Since the latter intermediate is unable to cyclize due to the absence of the 6,7-double bond, ionization of 8 and quenching of the resulting ion pair by deprotonation, capture of water, or collapse to the isomeric primary pyrophosphate esters will generate the observed spectrum of anomalous products.

Large-scale purification of the mycotoxins aflatoxin B1, B2 and G1.

The isolation and purification of gram quantities of the important mycotoxins aflatoxin B1, B2 and G1 are described. The method involves final purification on a Waters Prep LC-500 instrument, loaded with silica cartridges, and elution with chloroform.

Fumonisins--novel mycotoxins with cancer-promoting activity produced by Fusarium moniliforme.

Cultures on corn of Fusarium moniliforme MRC 826 are known to cause leukoencephalomalacia in horses and to be toxic and hepatocarcinogenic in rats. Culture material of this F. moniliforme isolate has also been shown to exhibit cancer-promoting activity in a short-term cancer initiation-promotion bioassay with diethylnitrosamine-initiated rats and the induction of gamma-glutamyl-transpeptidase-positive (GGT+) foci as an endpoint after 4 weeks of promotion. This bioassay was used as a monitoring system to isolate cancer-promoting compounds from cultures of F. moniliforme MRC 826. Culture material was successively extracted with ethyl acetate and CH3OH-H2O (3:1). Most of the cancer-promoting activity was recovered in the CH3OH-H2O extract and remained in the aqueous phase following partitioning of this extract between CH3OH-H2O (1:3) and CHCl3. The CH3OH-H2O fraction was chromatographed on an Amberlite XAD-2 column, and the active fraction was eluted with CH3OH. This fraction was chromatographed on a silica gel column with CHCl3-CH3OH-CH3COOH (6:3:1) as eluent and further purified on a C18 reverse-phase column. Two pure compounds were isolated, and these have been chemically characterized and given the trivial names fumonisin B1 and B2. At least 2 g of the major compound fumonisin B1 was purified from 1 kg of culture material. Fumonisin B1 in the diet (0.1%) significantly (P less than 0.001) induced the formation of GGT+ foci in the livers of initiated as well as noninitiated rats.(ABSTRACT TRUNCATED AT 250 WORDS)