Reversal of depressed miduterine arterial flow during hyperthermia-induced respiratory alkalosis

The influence of ambient and arterial PCO2 on miduterine arterial flow of pregnant sheep acutely exposed to hot environments was investigated. Five mixed-breed ewes between 120 and 130 days of gestation were subjected to hot environments (increasing from thermoneutral 23 to 40 degrees C), and arterial blood pH, PCO2, and PO2 were determined at 5-min intervals. Respiratory rate, heart rate, rectal temperature, blood pressure, and miduterine arterial flow were continuously monitored prior to and during elevation of ambient air temperature. When miduterine arterial flow had decreased to 50% of thermoneutral control levels, ambient air CO2 was increased to 2.5%. Elevated ambient inspired CO2 caused a reversal in arterial pH and PCO2 to near thermoneutral levels. Miduterine arterial flow increased to 77% of the control levels following the elevated ambient PCO2 period. Respiratory rate also decreased when ambient CO2 was increased but remained 136% greater than the thermoneutral control level. All other parameters remained near their heat stress (40 degrees C) level during the elevation of ambient CO2. These data indicate that heat-stress-induced depression of miduterine arterial flow is vasoactively regulated, and cause-effect related to both arterial pH and PCO2, and thermoregulatory shunting of blood to heat-dissipating surfaces.     

Energy coupling to nitrite respiration in the sulfate-reducing bacterium Desulfovibrio gigas.

By use of a membrane fraction prepared from Desulfovibrio gigas grown in a lactate-sulfate medium, synthesis of ATP was demonstrated to be coupled to the oxidation of molecular hydrogen and reduction of either nitrite or hydroxylamine. This phosphorylation was uncoupled from electron transport by pentachlorophenol, methyl viologen, and gramicidin, but not by oligomycin. The extrusion of protons from the cells was shown to be coupled to the hydrogen-nitrite respiratory system, and, assuming the localization of nitrite reductase on the outer side of the plasma membrane, H+/2e- values of 2.0 +/- 0.3 were obtained. Energy coupling observed with this system appears to be due to electron transfer-coupled proton translocation rather than vectorial electron transfer associated with hydrogen oxidation.     

Localization of the elongation factor Tu binding site on Escherichia coli ribosomes

Fluorescent techniques were used to study binding of peptide elongation factor Tu (EF-Tu) to Escherichia coli ribosomes and to determine the distances of the bound factor to points on the ribosome. Thermus thermophilus EF-Tu was labeled with 3-(4-maleimidylphenyl)-4-methyl-7-(diethyl-amino)coumarin (CPM) without loss of activity. In the presence of Phe-tRNA and a nonhydrolyzable analogue of GTP, 70S ribosomes bind the CPM-EF-Tu [Kb = (3 +/- 1.2) X 10(6) M-1] causing a decrease of CPM fluorescence. Binding of CPM-EF-Tu to 50S subunits was at least 1 order of magnitude lower than with 70S ribosomes, and binding to 30S subunits could not be detected. Reconstituted 70S ribosomes containing either S1 labeled with fluoresceinmaleimide or ribosomal RNAs labeled at their 3' ends with fluorescein thiosemicarbazide were used for energy transfer from CPM-EF-Tu. The distances between CPM-EF-Tu bound to the ribosomes and the 3' ends of 16S RNA, 5S RNA, 23S RNA, and the closest sulfhydryl group of S1 were calculated to be 82, 70, 73, and 62-68 A, respectively.     

Evidence for RNA in the peptidyl transferase center of Escherichia coli ribosomes as indicated by fluorescence.

A coumarin derivative was covalently attached to either the amino acid or the 5' end of phenylalanine-specific transfer RNA (tRNA(phe)). Its fluorescence was quenched by methyl viologen when the tRNA was free in solution or bound to Escherichia coli ribosomes. Methyl viologen as a cation in solution has a strong affinity for the ionized phosphates of a nucleic acid and so can be used to qualitatively measure the presence of RNA in the immediate vicinity of the tRNA-linked coumarins upon binding to ribosomes. Fluorescence lifetime measurements indicate that the increase in fluorescence quenching observed when the tRNAs are bound into the peptidyl site of ribosomes is due to static quenching by methyl viologen bound to RNA in the immediate vicinity of the fluorophore. The data lead to the conclusion that the ribosome peptidyl transferase center is rich in ribosomal RNA. Movement of the fluorophore at the N-terminus of the nascent peptide as it is extended or movement of the tRNA acceptor stem away from the peptidyl transferase center during peptide bond formation appears to result in movement of the probe into a region containing less rRNA.     

Fluorescence characterization of the environment encountered by nascent polyalanine and polyserine as they exit Escherichia coli ribosomes during translation.

The fate of the amino termini of nascent polyalanine, polyserine, and polylysine was monitored by fluorescence techniques as each was translated on Escherichia coli ribosomes. A coumarin probe was placed at the alpha-amino group of a synthetic elongator alanyl-tRNA or a synthetic initiator alanyl-tRNA or at the epsilon-amino group of natural lysyl-tRNA, and each was used to nonenzymatically initiate peptide synthesis. The fluorescent alanyl-tRNAs containing an AAA anticodon were used to initiate polyserine (with a synthetic tRNA(Ser] or polyalanine synthesis from a poly(uridylic acid) template. The fluorescent lysyl-tRNA was used to initiate polylysine synthesis from poly(adenylic acid). Changes in the fluorescence of the amino-terminal coumarin were examined to characterize the environment of the probe as the nascent peptides were extended. Protection from proteolysis and the binding of anti-coumarin antibodies or Fab fragments suggest that the amino terminus of each polypeptide is protected from interaction with proteins (Mr greater than 28,000) until the peptides are extended to an average length of 40-50 residues; however, the fluorescence from the amino terminus of shorter nascent polyalanine and polyserine peptides was readily quenched by methyl viologen (Mr = 257), indicating ribosomes do not shield the nascent peptide from molecules of this size. The data appear to indicate that polyalanine, polyserine, and polylysine are extended from the peptidyl transferase into a protected region of the ribosome such as a groove or tunnel but that this region is readily accessible to small molecules.     

Cytoskeleton-amyloplast interactions in sweet clover

The distribution of organelles within columella cells of sweet clover was examined by transmission electron microscopy following growth under static or clinorotating conditions. A developmentally conditioned polarity was observed, with a proximal location of the nucleus and a distal accumulation of the endoplasmic reticulum. This polarity was insensitive to clinorotation. In contrast, clinorotation altered the location of amyloplasts. Application of cytoskeletal poisons (colchicine, cytochalasin D, taxol, and phalloidin), especially during clinorotation, had interesting effects on the maintenance of columella cell polarity, with a profound effect on the extent, location, and structure of the endoplasmic reticulum. The site of cytoskeletal interactions with sedimenting amyloplasts is thought to be the amyloplast envelope. An envelope fraction, having over 17 polypeptides, was isolated using immobilized antibody technology, and will provide a means of assessing the role of specific peptides in cytoskeleton/amyloplast interactions.     

The distance between S1, S21, and the 3' end of 16S RNA in 30S ribosomal subunits. The effect of poly(uridylic acid) and 50S subunits on these distances

The apparent distances between probes covalently attached to the cysteine thiols of S1 or S21 and the 3' end of 16S RNA in Escherichia coli 30S ribosomal subunits were determined by non-radiative energy transfer to be: S21-16S RNA, 5.1 nm; S21-S1, 6.9 nm; S1-16S RNA, 6.8 nm. Binding of poly(uridylic acid) to 30S subunits causes the apparent distances between S1 and 16S RNA or S21 and 16S RNA to increase by more than 1.2 nm and 0.5 nm, respectively, but has little or no effect on the S1-S21 distance. Binding of 50S subunits causes an apparent increase in the S21-16S RNA and S21-S1 distances by 1.0 nm and 0.8 nm, respectively, but has little or no effect on the S1-16S-RNA distance.