The AXH module: an independently folded domain common to ataxin-1 and HBP1.

Ataxin-1 (ATX1), a human protein responsible for spinocerebellar ataxia type 1 in humans, shares a region of homology, named AXH module, with the apparently unrelated transcription factor HBP1. Here, we describe the first characterisation of the AXH module in terms of its structural properties and stability. By producing protein constructs spanning the AXH modules of ATX1 and HBP1 and by comparing their properties, we have identified the minimal region sufficient for forming independently folded units (domains). Knowledge of the AXH domain boundaries allows us to map many of the interactions of ATX1 with other molecules onto the AXH module. We further show that the AXH of ATX1 is a dimerisation domain and is able to recognise RNA with the same nucleotide preference previously described for the full-length protein. AXH is therefore a novel protein-protein and RNA binding motif.

PcF protein from Phytophthora cactorum and its recombinant homologue elicit phenylalanine ammonia lyase activation in tomato.

The phytotoxic protein PcF (Phytophthora cactorum-Fragaria) is a 5.6-kDa cysteine-rich, hydroxyproline-containing protein that is secreted in limited amounts by P. cactorum, an oomycete pathogen of tomato, strawberry and other relevant crop plants. Although we have shown that pure PcF triggers plant reactivity, its mechanism of action is not yet understood. Here we show that PcF, like other known fungal protein elicitors involved in pathogen-plant interaction, stimulates the activity of the defense enzyme phenylalanine ammonia lyase (EC 4.3.1.5) in tomato seedlings. Recognizing that a key step in understanding the mechanism of action of PcF at a molecular level is knowledge of its three-dimensional structure, we overexpressed this protein extracellularly in Pichia pastoris. The preliminary structural and functional characterization of a recombinant PcF homologue, N4-rPcF, is reported. Interestingly, although N4-rPcF is devoid of proline hydroxylation and has four additional amino acid residues attached to its N terminus, its secondary structure and biological activity are indistinguishable from wild-type PcF.

NMR solution structure of a novel thioredoxin from Bacillus acidocaldarius possible determinants of protein stability.

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx), an eubacterium growing optimally at 333 K, is the first Trx described to date from a moderate thermophilic source. To understand the molecular basis of its thermostability, the three-dimensional structure in the oxidized form was determined by NMR methods. A total of 2276 1H-NMR derived distance constraints along with 23 hydrogen-bonds, 72 phi and 27 chi1 torsion angle restraints, were used in a protocol employing simulated annealing followed by restrained molecular dynamics and restrained energy minimization. BacTrx consists of a well-defined core region of five strands of beta-sheet, surrounded by four exposed alpha-helices, features shared by other members of the thioredoxin family. The BacTrx 3D structure was compared with the Escherichia coli Trx (EcTrx) determined by X-ray crystallographic diffraction, and a number of structural differences were observed that may contribute to its thermostabilty. The results of structural analysis indicated that protein stability is due to cumulative effects, the main factor being an increased number of ionic interactions cross-linking different secondary structural elements and clamping the C-terminal alpha-helix to the core of the protein.

Radiation late effects in children treated for orbital rhabdomyosarcoma.

BACKGROUND AND PURPOSE: The experience resulting from large cooperative studies shows that correct radiation therapy at doses adequate to the tumor bulk are crucial for local control of rhabdomyosarcoma. The aim of the present study was to document the correlation between modalities and doses of radiotherapy and radiation side effects. PATIENTS AND METHODS: Between 1980 and 1997, 19 patients affected by primary orbital rhabdomyosarcoma have been followed at the University Federico II of Naples. All but three patients, who received 45, 54 and 55 Gy respectively, have been treated by immediate radiation at the dose of 60 Gy, delivered in 2 Gy fractions, five times per week, by cobalt 60 megavoltage equipment. Combined chemotherapy using vincristine and vincristine plus dactinomycin on alternate weeks was also administered as part of induction therapy. RESULTS: An overall survival rate of 94.7% was registered. In our patients the majority of radiation late effects were paid by orbit and ocular adnexa. Side effects to lens and ocular structures were fewer and of low grade. CONCLUSIONS: Radiation therapy is still essential for local control of orbital rhabdomyosarcoma, however radiation side effects have to be carefully considered together with the therapeutic goal to be obtained.

Thioredoxin from Bacillus acidocaldarius: characterization, high-level expression in Escherichia coli and molecular modelling.

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx) was purified to homogeneity by anion-exchange chromatography and gel-filtration chromatography, based on its ability to catalyse the dithiothreitol-dependent reduction of bovine insulin disulphides. The protein has a molecular mass of 11577 Da, determined by electrospray mass spectrometry, a pI of 4.2, and its primary structure was obtained by automated Edman degradation after cleavage with trypsin and cyanogen bromide. The sequences of known bacterial Trxs were aligned at the active site: BacTrx has an identity ranging from 45 to 53% with all sequences except that of the unusual Anabaena strain 7120 Trx (37% identity). The gene coding for BacTrx was isolated by a strategy based on PCR gene amplification and cloned in a plasmid downstream of a lac-derived promoter sequence; the recombinant clone was used as the expression vector for Escherichia coli. The expression was optimized by varying both the time of cell growth and the time of exposure to the inducer isopropyl beta-d-thiogalactoside; expressed BacTrx represents approx. 5% of the total cytosolic protein. CD spectra and differential scanning calorimetry measurements demonstrated that BacTrx is endowed with a higher conformational heat stability than the Trx from E. coli. Nanogravimetry experiments showed a lower content of bound water in BacTrx than in E. coli Trx, and a transition temperature approx. 10 degrees C higher for BacTrx. The three-dimensional model of the oxidized form of BacTrx was constructed by a comparative molecular modelling technique, using E. coli Trx and Anabaena strain 7120 Trx as reference proteins. Increased networks of ion-pairs and shorter loops emerged as major features of the BacTrx structure compared with those of the template proteins. The findings are discussed in the light of the current knowledge about molecular determinants of protein stability.

Primary Ewing's sarcoma of the maxilla, a rare and curable localization: report of two new cases, successfully treated by radiotherapy and systemic chemotherapy.

Primary maxillary localization of Ewing's sarcoma is unusual. Involvement of facial bones is characterized by clinical and radiological features distinct from those commonly observed in other sites. Because of the above peculiarities a delay in diagnosis and thus in starting treatment is very probable in such cases. We report here two new cases of Ewing's sarcoma localized to facial bones, successfully treated by local high dosage radiotherapy and systemic chemotherapy. Our experience suggests that, especially for particular sites not suitable to radical surgery, radiation therapy can represent an effective tool to achieve local control of the tumor.

A novel chromosomal abnormality detected in a case of acute myelomonocytic leukemia.

A not yet recorded translocation, t(3:17)(q21;q23), detected in a case of acute myelomonocytic leukemia, is reported. In spite of an aggressive cytotoxic chemotherapy, the disease showed a rapid fatal course, confirming the dismal prognostic significance of structural, 'primary' chromosomal abnormalities in acute leukemias.

Familial erythrophagocytic lymphohistiocytosis: adverse prognostic significance of delayed diagnosis.

Familial erythrophagocytic lymphohistiocytosis (FEL) is a rare disorder of the monocyte-macrophage system, for which an autosomal recessive mode of inheritance has been postulated. It is characterized by a dismal prognosis and is peculiar of early infancy. Three new cases of infants affected by FEL are reported. All three patients were diagnosed about three months after the onset of symptoms, and all three died shortly after diagnosis. The need for early diagnosis and prompt, intensive cytotoxic chemotherapy is emphasized.