Expanding the Genetic Code of a Photoautotrophic Organism.

The photoautotrophic freshwater cyanobacterium Synechococcus elongatus is widely used as a chassis for biotechnological applications as well as a photosynthetic bacterial model. In this study, a method for expanding the genetic code of this cyanobacterium has been established, thereby allowing the incorporation of unnatural amino acids into proteins. This was achieved through UAG stop codon suppression, using an archaeal pyrrolysyl orthogonal translation system. We demonstrate incorporation of unnatural amino acids into green fluorescent protein with 20 ± 3.5% suppression efficiency. The introduced components were shown to be orthogonal to the host translational machinery. In addition, we observed that no significant growth impairment resulted from the integration of the system. To interpret the observations, we modeled and investigated the competition over the UAG codon between release factor 1 and pyl-tRNACUA. On the basis of the model results, and the fact that 39.6% of the stop codons in the S. elongatus genome are UAG stop codons, the suppression efficiency in S. elongatus is unexpectedly high. The reason for this unexpected suppression efficiency has yet to be determined.

Cardiac index and incidence of heart failure cells.

Inverse correlations were established between the percentage of iron-laden macrophages (heart-failure cells), found in the sputum of patients with various types of heart ailment, and cardiac index. Less than 2% iron-laden macrophages were seen when cardiac index was in the region of 3 liter/min/sq mm of body surface area (BSA). This fraction increased to approximately 75% when the cardiac index attained 1 liter/min/sq mm of BSA. Since there was noticeable heterogeneity of data when the cardiac index was below 3 liters/min/sq mm of BSA and above 1 liter/min/sq mm of BSA, it was concluded that a second factor was probably at play: this was the degree of activity of the reticuloendothelial system of the subjects concerned.

Bronchial cytologic changes during cardiopulmonary bypass: iron transport by histiocytes in low flow states.

We previously reported (1) that tracheobronchial secretions of patients in hemorrhagic shock contained significantly more iron laden histiocytes than secretions of matched controls and demonstrated by histiologic studies in rats bled to hemorrhagic shock that these animals' lungs contained significantly more iron laden histiocytes than those of a control group. Two explanations appeared plausible: 1) abnormal hemoglobin breakdown products of systemic hypoperfusion during shock (2, 3, 5) may have accumulated in the pulmonary circulation where they were phagocytozed by pulmonary histiocytes, or 2) circulating iron laden histiocytes may have been arrested in the lung, which is well known to act as a filter during low-flow states (4). The present study reports on assay of bronchial secretions of 12 open-heart surgery patients under cardiopulmonary bypass from specimens obtained 1) immediately after onset of anesthesia; 2) during; and 3) after termination of cardiopulmonary bypass. The percentage of iron laden histiocytes rose from 15.6+/-6.0 after intubation, to 49.6+/-6.8 after 1 hour on bypass with occluded pulmonary arteries (p less than 0.0025), remaining close to the latter value after pulmonary circulation was restored. Hypoperfusion, accumulation of metabolites, or falling pH at the tissue level are probable explanations for these findings.

Tracheobronchial cytologic changes and abnormal serum heme pigments in hemorrhagic shock.

Significantly elevated numbers of iron laden histiocytes have been shown to appear in the tracheobronchial secretions of subjects in hemorrhagic shock and in patients undergoing open heart-surgery during cardiopulmonary bypass (3,4). Abnormal heme pigments have also been demonstrated in the serum of dogs bled to hemorrhagic shock (6) and have been felt to be toxic. Because the ingestion of abnormal heme pigments by histiocytes may be part of a defense mechanism, we have attempted to correlate the percentage of iron laden histiocytes found in the tracheobronchial secretions of patients in hemorrhagic shock (Prussian blue method) with the presence of degradation of products of hemoglobin found in their serum by scanning spectrophotometry. There were, generally speaking, few iron laden histiocytes when hemoglobin degradation was advanced, and always numerous iron laden histiocytes when abnormal heme pigments were absent or spectrophotometric findings revealed minor degrees of degradation. Our findings probably reflect the process in the reticuloendothelial systems which eliminates toxic products accumulating in the circulation in low-flow states.

Abnormal heme-protein patterns in hemorrhagic shock.

Allegedly toxic heme pigments have been described in the serum of animals bled to hemorrhagic shock (5,7). In addition, Sears et al. (9), and Braun et al. (1) have shown that the products derived from the degradation of hemoglobin following intravascular hemolysis were toxic (heme carried by hemopexin and albumin). The accumulation of metabolites, caused by the impeded circulation, degrades free hemoglobin into heme pigments and their concentration then reaches a level which exceeds the normal heme-carrying capacity of serum proteins. We have already demonstrated the presence of abnormal heme pigments in clinical cases of shock using scanning spectrophotometry (3). We have endeavored to identify these pigments by serum electrophoresis, and to relate the appearance of some of these compounds to mortality rates. There were no deaths associated with the presence of haptoglobin-hemoglobin alone in serum. As shock deepened and mortality rose, hemopexin-heme and methemalbumin appeared. The highest mortality rate (4 out of 5 cases) was found when both hemopexin-heme and methemalbumin were present. It appears, therefore, that the administration of serum proteins in shock may reduce the toxicity of the products of degradation formed in low-flow states.