Three-dimensional structure of the histidine-containing phosphocarrier protein (HPr) from Enterococcus faecalis in solution.

The histidine-containing phosphocarrier protein (HPr) transfers a phosphate group between components of the prokaryotic phosphoenolpyruvate-dependent phosphotransferase system (PTS), which is finally used to phosphorylate the carbohydrate transported by the PTS through the cell membrane. Recently it has also been found to act as an intermediate in the signaling cascade that regulates transcription of genes related to the carbohydrate-response system. Both functions involve phosphorylation/dephosphorylation reactions, but at different sites. Using multidimensional (1)H-NMR spectroscopy and angular space simulated annealing calculations, we determined the structure of HPr from Enterococcus faecalis in aqueous solution using 1469 distance and 44 angle constraints derived from homonuclear NMR data. It has a similar overall fold to that found in HPrs from other organisms. Four beta strands, A, B, C, D, encompassing residues 2-7, 32-37, 40-42 and 60-66, form an antiparallel beta sheet lying opposite the two antiparallel alpha helices, a and c (residues 16-26 and 70-83). A short alpha helix, b, from residues 47-53 is also observed. The pairwise root mean square displacement for the backbone heavy atoms of the mean of the 16 NMR structures to the crystal structure is 0.164 nm. In contrast with the crystalline state, in which a torsion angle strain in the active-center loop has been described [Jia, Z., Vandonselaar, M., Quail, J.W. & Delbaere, L.T.J. (1993) Nature (London) 361, 94-97], in the solution structure, the active-site His15 rests on top of helix a, and the phosphorylation site N(delta 1) of the histidine ring is oriented towards the surface, making it easily accessible to the solvent. Back calculation of the 2D NOESY NMR spectra from both the NMR and X-ray structures shows that the active-center structure derived by X-ray crystallography is not compatible with experimental data recorded in solution. The observed torsional strain must either be a crystallization artefact or represents a conformational state that exists only to a small extent in solution.

Pressure-induced local unfolding of the Ras binding domain of RalGDS.

The reliable prediction of the precise three-dimensional structure of proteins from their amino acid sequence is a major, still unresolved problem in biochemistry. Pressure is a parameter that controls folding/unfolding transitions of proteins through the volume change DeltaV of the protein-solvent system. By varying the pressure from 30 to 2,000 bar we detected using 15N/ 1H 2D NMR spectroscopy a unique equilibrium unfolding intermediate I in the Ras binding domain of the Ral guanine nucleotide dissociation stimulator (Ral GDS). It is characterized by a local melting of specific structural elements near hydrophobic cavities while the overall folded structure is maintained.

[Short-term hypofractionated radiotherapy followed by total mesorectal excision]. / Kleinvolumige hypofraktionierte Radiotherapie vor totaler mesorektaler Exzision.

PURPOSE: Is preoperative short-term radiotherapy of operable rectal carcinoma feasible with regard to early side effects and perioperative complications? PATIENTS AND METHODS: As of December 1996 to March 1999, 34 patients with locally advanced rectal cancer have been irradiated preoperatively with 5 times 5 Gy. After CT-planning, radiotherapy was administered using a 3-field or 4-field box technique with 2 anterior-posterior fields or a posterior field of 9 +/- 2 cm x 11.5 +/- 2.4 cm and 2 opposed bilateral fields of 9 +/- 1.5 cm x 11.5 +/- 2 cm with 6- to 25-MV photons. Surgery was performed 14 +/- 6 days after irradiation in 33/34 patients (82% anterior resection with total mesorectal excision, 18% abdomino-perineal resection). Patients with a positive lymph node status or pT3/4 lesions underwent adjuvant chemotherapy with 5-Fluorouracil (5-FU). The median follow-up period is 189 days (range: 15 to 548 days). RESULTS: The following early side reactions were registered: increased bowel movements (4/34), fatigue (2/34), pain in the groins (1/34), nausea and perianal smart (1/34), vertigo (1/34), temporary urinary obstruction (1/34). One patient with heart failure NYHA Grade III died of a heart attack after 21 days. Preoperative T and N categories showed a distribution of 3, 29 and 2 for T4, T3 and unknown and 20, 11 and 3 for N+, N- and unknown; postoperative T and N categories showed a distribution of 3, 19 and 11 for T4, T3 and T2 and 19 and 14 for N+ and N-. In 32 of 33 patients tumor-free margins were achieved. One patient with peritoneal metastases had a R1 resection. In 3 patients metastases were detected intraoperatively. Perioperative complications were: 2 cases of leaking anastomosis and postoperative bowel atonia, 1 case with bowel obstruction, delayed wound healing, wound dehiscence and temporary renal dysfunction. CONCLUSION: Preoperative radiotherapy is feasible with moderate toxicity and is able to induce down staging despite the short time interval between radiotherapy and surgery.

Determination of mean and standard deviation of dihedral angles.

Backbone torsional angles are a characteristic and useful parameter for the description and characterisation of protein structures determined by x-ray crystallography or NMR spectroscopy. For the comparison of an ensemble of three-dimensional structures the calculation of the statistical parameters mean and standard deviation would be very useful. However, they are not defined unambiguously for periodic quantities such as the dihedral angles. In this paper a plausible and unique definition of these parameters is introduced and a straightforward method for their calculation is given.

[Interdisciplinary therapy of anal carcinoma]. / Interdisziplinäre Therapie des Analkarzinoms.

Primary radiation therapy is a safe sphincter sparing treatment for anal carcinomas less than 4 cm. In larger tumors results have improved dramatically in recent years by use of chemoradiation. Simultaneous radiochemotherapy of anal carcinoma leads to a 5-year survival rate of 80% with only 3 to 8% severe side effects, a local control of 75% and a conservation of sphincter function in 80%. If the tumor is smaller than 4 cm, radiotherapy alone is sufficient in patients with contraindications for chemotherapy; however, the risk of severe late side effects increases to 10%. A colostomy is indicated in patients with severe or complete stenosis of the anal canal with incontinence or obstruction by the tumor, in case of tumor nonresponding to radiotherapy or inoperable with painful defecation. Abdominoperineal resection should be limited to residual tumors increasing two months after radiotherapy, to salvage after relapse, to fistulas and necroses developing as complications of radiotherapy, to ulcers and fecal incontinence and to cases of a extended primary tumor (T4). Clinically suspicious lymph nodes should be biopsied. Histologically positive inguinal nodes should be treated with radiochemotherapy without groin dissection. A monthly follow-up is necessary in cases with residual tumor. If the size of the tumor increases, a biopsy is indicated. In case of relapse a second radiochemotherapy should be considered. Otherwise an abdominoperineal resection is indicated.

[The prognostic factors following the simultaneous radiochemotherapy of anal canal carcinoma in a multicenter series of 139 patients]. / Prognostische Faktoren nach simultaner Radiochemotherapie des Analkanalkarzinoms in einer multizentrischen Serie von 139 Patienten.

PURPOSE: Evaluation of prognostic variables, results and toxicity after chemo-radiation (CRT) of anal canal carcinoma (ACC). MATERIAL AND METHODS: Between 1982 and 1992, 139 patients with epidermoid carcinoma of the anal canal were treated by radiation and chemotherapy with 5-fluorouracil (5-Fu) and mitomycin C (MMC). 99 patients belonged to a prospectively designed trial (50 Gy, 2 courses of chemotherapy) and 40 to a historical control group (40 Gy, 1 course of chemotherapy). The female/male ratio was 116/23. Median age was 62 years. Staging (UICC 1987): T1: 16.5%; T2: 49%; T3: 23%; T4: 9.4%; unknown: 2.1%. Abnormal regional nodes were present in 15% of the patients. HISTOLOGY: Squamous cell carcinoma: 68%; cloacogenic carcinoma: 31%; unknown: 1%. External beam radiation (ERT) was given to the primary tumour including perirectal, inguinal and iliac nodes by a 3 to 4 field box technique (50 patients) or parallel opposed fields (89 patients). Median single fraction and total dose were 1.8 Gy and 50 Gy. An additional boost to the involved sites was delivered by ERT (32 cases; median dose 16 Gy) or interstitial brachytherapy (BT) in 28 cases with a median dose 14 Gy. 84 patients (60%) received 2 or more cycles, 50 patients (36%) 1 cycle, 5 patients (4%) no chemotherapy. RESULTS: The survival rate, NED-survival rate and local tumour control rate were 78%, 64% and 69% at 5 years. Anorectal function was retained in 94 of 139 patients (68%). Multivariate analysis indicated that T-stage (p = 0.037) and belonging to the historical control group (p = 0.003) were significant variables for local tumour control. T-stage was a marginally significant factor (p = 0.09) for NED-survival. Acute toxicity of grade 3 and 4 (WHO) was observed in 36%, severe late toxicity (grade 3 Eschwege) in 3% of the patients. CONCLUSIONS: CRT with 2 courses 5-FU, MMC and ERT to a total dose of 45 to 50 Gy is a safe and effective treatment for ACC. Intensification of treatment is recommended in advanced stages T3/4.

[The value of postoperative radiotherapy in colonic carcinomas]. / Wertigkeit der postoperativen Strahlentherapie bei Kolonkarzinomen.

From 1979 to 1988 63 patients with cancer of the colon received postoperative adjuvant radiotherapy. The mean age was 62 years and the median follow-up period was 71 months. Pathologic staging showed a B1-tumour in nine cases, B2 in 25, C1 in three and a C2-tumour in 26 cases according to the Astler and Coller modification of the Duke's classification. 53 patients received doses between 41 and 50 Gy. 47 patients were treated with a 60Co megavoltage unit and 16 cases with 8 MV photons. The overall five years survival rate is 65%. There was no significant difference between the survival rates for all stages. Only four patients with primary positive lymph nodes experienced a local relapse. 16 developed metastatic disease. In twelve cases (19%) liver metastases appeared: four patients (6%) developed intraperitoneal metastases, one patient had a metastasis in the abdominal wall and another showed pulmonary carcinosis. Acute and transient side effects were seen in 35% of our patients. Three patients underwent a second operation for radiation induced bowel stenosis. Compared with the results of surgery adjuvant radiotherapy yields improved five-year survival data in patients with C2 and G4 tumours.

Maintenance of D-alanine ester substitution of lipoteichoic acid by reesterification in Staphylococcus aureus.

Toluene-treated Staphylococcus aureus cells did not synthesize teichoic acid and lipoteichoic acid under the conditions used. The organism displayed, however, a high capacity of incorporating D-[14C]alanine into previously formed polymers. The reaction was dependent on ATP and enhanced by magnesium ions. The incorporation rate into lipoteichoic acid correlated with the rate of loss of alanine ester which occurred through transfer to teichoic acid and base-catalyzed hydrolysis. At pH 6.5 the loss (20% within 4 h) was completely compensated for by reesterification. At pH 7.5 the loss was 60%, but by accelerated incorporation it was reduced to 10%. Incorporation was also enhanced when the original substitution of lipoteichoic acid was lowered by previous growth of S. aureus at high salt concentration. The newly added alanine was randomly distributed along the poly(glycerophosphate) chain. The decreased alanine substitution of lipoteichoic acid after growth at high salt concentration was shown to result from a direct inhibition of alanine incorporation.