Crystal structure of thermally stable homodimeric cytochrome c'-ß from Thermus thermophilus.

Cytochrome c'-ß is a heme protein that belongs to the cytochrome P460 family and consists of homodimeric subunits with a predominantly antiparallel ß-sheet fold. Here, the crystal structure of cytochrome c'-ß from the thermophilic Thermus thermophilus (TTCP-ß) is reported at 1.74 Šresolution. TTCP-ß has a typical antiparallel ß-sheet fold similar to that of cytochrome c'-ß from the moderately thermophilic Methylococcus capsulatus (MCCP-ß). The phenylalanine cap structure around the distal side of the heme is also similar in TTCP-ß and MCCP-ß, indicating that both proteins similarly bind nitric oxide and carbon monoxide, as observed spectroscopically. Notably, TTCP-ß exhibits a denaturation temperature of 117°C, which is higher than that of MCCP-ß. Mutational analysis reveals that the increased homodimeric interface area of TTCP-ß contributes to its high thermal stability. Furthermore, 14 proline residues, which are mostly located in the TTCP-ß loop regions, possibly contribute to the rigid loop structure compared with MCCP-ß, which has only six proline residues. These findings, together with those from phylogenetic analysis, suggest that the structures of Thermus cytochromes c'-ß, including TTCP-ß, are optimized for function under the high-temperature conditions in which the source organisms live.

Thermal stability tuning without affecting gas-binding function of Thermochromatium tepidum cytochrome c'.

Hydrogenophilus thermoluteolus, Thermochromatium tepidum, and Allochromatium vinosum, which grow optimally at 52, 49, and 25 °C, respectively, have homologous cytochromes c' (PHCP, TTCP, and AVCP, respectively) exhibiting at least 50% amino acid sequence identity. Here, the thermal stability of the recombinant TTCP protein was first confirmed to be between those of PHCP and AVCP. Structure comparison of the 3 proteins and a mutagenesis study on TTCP revealed that hydrogen bonds and hydrophobic interactions between the heme and amino acid residues were responsible for their stability differences. In addition, PHCP, TTCP, and AVCP and their variants with altered stability similarly bound nitric oxide and carbon oxide, but not oxygen. Therefore, the thermal stability of TTCP together with PHCP and AVCP can be tuned through specific interactions around the heme without affecting their gas-binding function. These cytochromes c' will be useful as specific gas sensor proteins exhibiting a wide thermal stability range.

Artificial hydration therapy, laboratory findings, and fluid balance in terminally ill patients with abdominal malignancies.

To explore the association between hydration volume and laboratory findings, and between calculated fluid balance and changes in clinical signs of dehydration and fluid retention in terminally ill cancer patients, a secondary analysis of a large multicenter, prospective, observational study was performed. The study enrolled 125 abdominal cancer patients who received laboratory examinations in the last week before death. Patients were classified into two groups: the hydration group (n = 44), who received 1L or more of artificial hydration per day both 1 and 3 weeks before death, and the nonhydration group (n = 81). The mean albumin level 1 week before death was significantly lower in the hydration group than in the nonhydration group, and the interaction between hydration group and decrease in the albumin level was statistically significant after adjusting multiple covariates (from 2.8 +/- 0.68 mg/dL 3 weeks before death to 2.4 +/- 0.56 mg/dL 24 hours before death in the hydration group vs. a decrease of 2.8 +/- 0.53 to 2.6+ /- 0.45 mg/dL in the nonhydration group, P = 0.015). There was no significant difference between the groups in the mean blood urea nitrogen/creatinine, sodium, or potassium levels 1 week before death. Among 53 patients who had oral fluid intake of less than 500 mL/day throughout the last 3 weeks and completed a fluid balance study, the median of calculated fluid balance was -400 mL/day 3 weeks before death, -521 mL/day 1 week before death, and -421 mL/day 24 hours before death. Calculated fluid balances did not significantly differ between the patients with deterioration of dehydration signs, edema, ascites, and pleural effusion during the final 3 weeks and those without. These data suggest that active artificial hydration might result in hypoalbuminemia, with no clear beneficial effects on normalizing blood urea nitrogen/creatinine, sodium, or potassium levels. Fluid balance did not significantly correlate with changes in dehydration-and fluid retention-signs. Calculated fluid balance is not an appropriate alternative to direct monitoring of patient symptoms. More studies are needed to determine the clinical efficacy of artificial hydration for terminally ill cancer patients.

Incidence and underlying etiologies of bronchial secretion in terminally ill cancer patients: a multicenter, prospective, observational study.

Although bronchial secretion is frequently observed in terminally ill cancer patients and can cause significant distress for both patients and family members, the pathophysiology is unclear. The primary aim of this study was to investigate the incidence and underlying etiologies of bronchial secretion. A multicenter, prospective, observational study was conducted on consecutive terminally ill patients with lung or abdominal malignancies. Primary physicians and nurses prospectively evaluated patients' symptoms. Of 310 patients enrolled, bronchial secretions were observed in 41% in the final 3 weeks, and oral/bronchial suctioning, with considerable distress, was required in 9%; bronchial secretions were severe in 4.5% of all patients. Multiple logistic regression analyses revealed that the determinants of the development of bronchial secretion were primary lung cancer, pneumonia, and dysphagia. There were no statistically significant effects of severity of peripheral edema and pleural effusion on development of bronchial secretions and requirement for oral/bronchial suctioning. Etiology-based classification of bronchial secretion is useful to identify the most suitable palliative treatments and to clarify treatment efficacy in each specific pathophysiology.