Identification of essential amino acids within the proposed CuA binding site in subunit II of Cytochrome c oxidase

To explore the nature of proposed ligands to the CuA center in cytochrome c oxidase, site-directed mutagenesis has been initiated in subunit II of the enzyme. Mutations were introduced into the mitochondrial gene from the yeast Saccharomyces cerevisiae by high velocity microprojectile bombardment. A variety of single amino acid substitutions at each of the proposed cysteine and histidine ligands (His-161, Cys-196, Cys-200, and His-204 in the bovine numbering scheme), as well as at the conserved Met-207, all result in yeast which fails to grow on ethanol/glycerol medium. Similarly, all possible paired exchange Cys,His and Cys,Met mutants show the same phenotype. Furthermore, protein stability is severely reduced as evidenced by both the absence of an absorbance maximum at 600 nm in the spectra of mutant cells and the underaccumulation of subunit II, as observed by immunolabeling of mitochondrial extracts. In the same area of the protein, a variety of amino acid substitutions at one of the carboxylates previously implicated in binding cytochrome c, Glu-198, allow (reduced) growth on ethanol/glycerol medium, with normal intracellular levels of protein. These results suggest that a precise folding environment of the CuA site within subunit II is essential for assembly or stable accumulation of cytochrome c oxidase in yeast.     

Symptom reduction and sobriety in the male alcoholic

The relationship between subjective symptom reduction and sobriety was studied for 82 male alcoholics who had completed an inpatient Alcoholic Rehabilitation Unit that included six or more biofeedback/relaxation sessions. Specific symptom relief for anxiety was significantly correlated with sobriety. In addition, the reduction of symptoms showed a positive trend with sobriety. The discussion focuses on the relationship of anxiety-related symptoms and alcohol abuse.     

Epitope mapping of prostate-specific antigen with monoclonal antibodies

Prostate-specific antigen (PSA) is a widely used marker for screening and monitoring prostate cancer. We identified and characterized the epitopes of two anti-PSA monoclonal antibodies (mAbs) designated B80 and B87. The epitopes were initially mapped as nonoverlapping by developing a sandwich immunoassay to measure PSA with the two anti-PSA mAbs. The two antibodies do not cross-react with homologous pancreatic kallikrein, but recognize epitopes unique to PSA. B80 and B87 can recognize both free and complexed PSA and hence measure total PSA. Epitope scanning and bacteriophage peptide library affinity selection procedures were used to identify and locate an epitope on PSA. A possible epitope for B80 was identified as being located on or near PSA amino acid residues 50-58 (-GRH-SLFHP-). The epitope for B87 was likely on an exposed nonlinear conformational determinant, unique to PSA, and not masked by the binding of B80 or alpha 1-antichymotrypsin.     

A new technique for placement of nasoenteral feeding tubes using external magnetic guidance

OBJECTIVE: To evaluate a new technique in which a hand-held external magnet is used to maneuver nasoenteral feeding tubes through the pylorus and into the duodenum. DESIGN: Prospective case series. SETTING: Critical care units and medical and surgical wards of a university-affiliated community hospital. PATIENTS: Thirty-five patients were entered into the study after the attending physician requested assistance in tube placement. INTERVENTIONS: A standard 12-Fr, 114-cm flexible nasoenteral feeding tube was modified by inserting a small magnet into the distal tip. The tube was inserted per nares into the stomach, using traditional technique. Next, an external magnet was placed over the right upper abdominal quadrant, at the midclavicular line to attract the tube tip along the lesser curvature of the stomach, through the pyloric sphincter, and into the duodenum. Portable abdominal radiography performed immediately after the procedure confirmed the anatomic location of the tube tip. MEASUREMENTS AND MAIN RESULTS: Forty-two intubations were performed in 35 patients (in seven patients, the tube had to be reinserted due to inadvertent removal or surgery). In 37 (88%) of 42 intubations, the tube was passed through the pyloric sphincter and into the duodenum on the first attempt. The mean procedure time was 15 +/- 9 mins (range 10 to 45). There were no complications related to the procedure during the study period. CONCLUSIONS: This report describes a novel technique of enteral feeding tube placement, using external magnetic guidance. Transpyloric placement was achieved in 88% of cases. This reliable and convenient bedside method for rapid placement of the tube into the duodenum allows prompt and safe initiation of enteral nutrition.     

Raman difference studies of GDP and GTP binding to c-Harvey ras

The vibrational spectra of phosphate modes for GDP and GTP bound to the c-Harvey p21(ras) protein have been determined using 18O isotope edited Raman difference spectroscopy. A number of the phosphate stretch frequencies are changed upon GDP/GTP binding to ras, and the results are analyzed by ab initio calculations and through the use of empirical relationships that relate bond orders and bond lengths to vibrational frequencies. Bound GDP is found to be strongly stabilized by its interactions, mostly electrostatic, with the active site Mg2+. Bound GTP also interacts with the active site Mg2+ via its beta-phosphate group, as expected on the basis of crystallographic studies of bound GppNp. The angle between the nonbridging P&bondDot;O bonds of the gamma-phosphate of bound GTP increase by about 1-2 degrees compared to its solution value, thus bringing about a geometry that is closer to planar for these bonds as expected for the putative pentacoordinated transition state geometry of the phosphotransfer reaction. Modeling of the interactions at the nucleotide binding site suggests that the water molecule in-line with the P-O bond is positioned to bring about the change in bond angle. Moreover, a weak fifth bond (about 0.03 vu) appears to be formed between it and the gamma-phosphorus atom of bound GTP with a concomitant weakening of the O-P bond between the GDP leaving group and the gamma-phosphorus atom. Hence, an important role of the active site structure appears to be the strategic positioning of this in-line water. These structural results are consistent with a reaction pathway for GTP hydrolysis in ras of synchronous bond formation between the gamma-phosphorus of GTP and the attacking nucleophile and bond breaking between the gamma-phosphorus and the GDP leaving group.     

Splenic lymphoid tissue of mice after irradiation with fast carbon ions

Reorganization of cellular content of splenic functional zones was studied morphometrically in mice after single irradiation with fast carbon ions. It was demonstrated that irradiation does not cause significant changes in microtopography of splenic structures but is accompanied with changes in cytoarchitecture and rearrangement of lymphoid cells throughout the experiment. Two arbitrary stages are determined in lymphoid tissue response. The first one (30 hrs-7 d) is characterized with destructive processes intensification and decrease of small lymphocytes number in all functional zones of the organ. Strong plasmocellular reaction was observed in the organ except for the lymphoid nodules mantle zone in this period as well. On the second stage (15-60 d) orientation of plasmocellular reaction changes along with the tendency of small lymphocytes to restore population (up to 57-79% from the control level on d 60 of the experiment. As a result plasma cell content decreases sharply in all the organ structures. Low level of lymphocytopoiesis was demonstrated in the course of the experiment resulting from cell mitotic activity decline following the irradiation. At the distant period (d 60 following the irradiation) another decrease of cell lymphoid population was noted, which indicates that irradiation with fast carbon ions is suppressive to the animal immune system.