Toward the experimental codon reassignment in vivo: protein building with an expanded amino acid repertoire.

The high precision and fidelity of the genetic message transmission are ensured by numerous proofreading steps, from DNA replication and transcription to protein translation. The key event for translational fidelity is the proper codon assignment for 20 canonical amino acids. An experimental codon reassignment is possible for noncanonical amino acids in vivo using artificially constructed expression hosts under efficient selective pressure. However, such amino acids may interfere with the cellular metabolism and thus do not belong to the 'first' or 'restricted' part of the universal code, but rather to a second or 'relaxed' part, which is limited mainly by the downstream proofreading in the natural translational machinery. Correspondingly, not all possible alpha-amino acids can be introduced into proteins. The aim of this study is to discuss biological and evolutionary constraints on possible candidates for this second coding level of the universal code. Engineering of such a 'second' code is expected to have great academic as well as practical impact, ranging from protein folding studies to biomedicine.

Variability of precordial electrode placement during routine electrocardiography.

Placement of precordial electrodes by 30 experienced technicians during routine electrocardiography was compared with anatomically ascertained sites marked by ink visible only under ultraviolet "black light" in three men and five women. The distance and direction from each marked site to the center of the applied electrode was measured to allow calculation of both the linear magnitude of difference (derived from the square root of the sum of the squares of rise and run) and also the direction of difference (in quadrants) between the anatomically established location and the electrode at each precordial site. The average difference from the applied electrodes to the corresponding marked sites was 1.14 inches, ranging from a mean of 1.31 inches at lead V1 to 0.98 inches at lead V4. Overall, 64% of precordial electrodes were placed within a radius of 1.25 inch, ranging from 56% for lead V6 to 74% for lead V4, and 27% of precordial electrodes were placed within a radius of 0.625 inch. There was superior quadrant displacement of more than 0.625 inch in more than 50% of routine applications of leads V1 and V2, indicating that these electrodes are commonly placed both high and wide of their anatomically defined precordial sites. Similarly, there was an inferior and left-ward displacement of more than 0.625 inch in 30-50% leads of routine applications of V4 through V6, indicating that these lateral precordial electrodes are commonly placed both low and wide of their respective anatomic sites.

Structural and functional properties of adult rat heart myocytes lysed with digitonin.

Low concentrations of digitonin disrupt the sarcolemma of adult rat heart myocytes selectively and completely. When the digitonin lysis is carried out in the presence of 10 mM Mg-ATP, the permeabilized cells retain the rod-cell morphology typical of heart cells in situ and show spontaneous phasic contractions. The rate of contraction is a function of the free Ca2+ concentration from a pCa of 7.2 to 5.2. Higher levels of free Ca2+ result in hypercontracture of the myocytes into round cells with characteristically distorted morphology. The sarcoplasmic reticulum of digitonin-lysed myocytes takes up Ca2+ in an ATP-dependent reaction that is inhibited and reversed by caffeine and strongly enhanced by procaine or ruthenium red. The Ca2+ accumulation has a Km of 0.6 microM Ca2+, depends on Pi (Km of 13 mM), and is strongly inhibited by bicarbonate ion. The hypercontracture of digitonin-lysed myocytes is a function of both the pCa and the Mg-ATP concentration of the suspending medium. Hypercontracture requires ATP. Hypercontracture due to Ca2+ overload occurs at lower Ca2+ concentrations when Mg-ATP is decreased from 10 to 1 mM. However, at low concentrations of Mg-ATP (in the range from 1 to 10 microM), hypercontracture also occurs and is essentially Ca2+-independent. Since hypercontracture of heart myocytes appears analogous to the formation of contraction bands in situ, these observations may be relevant to the phenomena of oxygen paradox and of Ca2+ paradox in intact myocardial tissue.

Mitochondrial enzyme retention by irreversibly damaged rectangular isolated adult rat heart myocytes.

A one hour hypoxic incubation causes the release of a small but significant amount of cytosolic lactic dehydrogenase from glucose-deprived isolated adult rat heart myocytes. However, enzymes associated with the mitochondria are not liberated, and there is no increase in the number of hypercontracted cells. These observations led Piper et al. (Life Sciences 35, 127-134 [1984]) to conclude that reversibly injured myocytes can release cytosolic proteins. This conclusion was based on the supposition that irreversibly hypoxic injury must cause mitochondrial enzyme efflux and hypercontracture. The present study establishes that this supposition is invalid.

Effects of ionic strength and sulfhydryl reagents on the binding of creatine phosphokinase to heart mitochondrial inner membranes.

The concept that creatine phosphokinase is bound to the outer surface of the heart mitochondrial inner membrane originated from observations that the enzyme is retained by water-swollen heart mitochondria and by digitonin-treated heart mitochondria suspended in isotonic sucrose. The present study establishes that digitonin-treated mitochondria release creatine phosphokinase in isotonic KCl, and other investigators have reported an identical response for the water-swollen organelles. These observations suggest that mitochondrial creatine phosphokinase is not bound to the outer surface of the inner membrane at a site adjacent to the adenine nucleotide translocase under physiologic conditions.

Ionic movements and irreversible anoxic damage.

Myocyte hypercontracture can be produced by adding Ca2+ to calcium-intolerant myocytes. A similar morphologic change occurs in Ca2+-free media when anoxic, ATP-depleted myocytes are reoxygenated or when respiring myocytes are lysed with digitonin. Hypercontracture in Ca2+-free media is abolished by rotenone, an inhibitor of NADH-linked respiration. Rotenone-treated, digitonin-permeabilized myocytes were used to examine the effects of MgATP, pCa, and respiration on hypercontracture. In the absence of Ca2+ (pCa 8.5), hypercontracture occurred at low MgATP but not when ATP was increased above 1 mM. At high MgATP (1-10 mM), hypercontracture was Ca2+-dependent. Succinate did not cause hypercontracture in the absence of added MgATP, but it shifted the concentration dependence for Ca2+-independent hypercontracture to lower values by regenerating ATP.

Release of acetylcholine from fragments of human term placenta and effects of the choline acetyltransferase inhibitor (2-benzoylethyl)trimethylammonium iodide.

Intact tissue from human term placenta contained 102 +/- 17 (n = 8) nmoles of acetylcholine (ACh)/g fresh tissue when analyzed by pyrolysis gas chromatography (GC). Free-hand dissection followed by extensive washing led to reductions of tissue ACh content (57 +/- 11 nmoles/g; n = 5) presumably as a result of release into the bathing medium. Presence or absence of physostigmine as an anticholinesterase agent in all solutions used had no significant effect on the ACh content of intact fragments immediately after dissection. Upon incubation at 37 degrees C a new steady state ACh concentration was established within 40-50 min (177 +/- 14 nmoles/g; n = 11) which reflected the content typical for purified villus. ACh was released continuously in amounts linearly related to incubation time up to 80 min, the longest time point examined. When the incubation volume was doubled there was an apparently immediate significant effect on release of ACh. Thereafter liberation of ACh continued at the rate observed prior to the volume perturbation. The stable choline acetyltransferase inhibitor (2-benzoylethyl)trimethylammonium (BETA) inhibited the enzyme rapidly and almost completely in tissue fragments. The drug affected the rate of ACh release significantly 40 min after addition to the bathing solution. At the same time it led to a depletion of the total ACh content that could be extracted from human placenta fragments.

Interlaboratory survey of enzyme analyses IV. Human versus porcine tissue as source of creatine kinase for survey serum.

A special enzyme survey was carried out in 185 laboratories on specimens fortified with either porcine heart or human skeletal muscle creatine kinase (CK). All analytical systems were examined to see if they gave different results for porcine and human CK as compared with analysis of the same specimens on the duPont ACA. Analytical differences were found. However, these differences were small, and, with some exceptions, do not prevent interconversion of units based solely on specimens fortified with porcine CK. Both types of specimens serve equally well in estimating bias and variability of CK analyses. Based on the results for the human material, many laboratories appear to use inappropriate reference ranges for CK.

Choline metabolism in placenta: evidence for the biosynthesis of phosphatidylcholine in microsomes via the methylation pathway.

Microsomes from human, mouse and rat placenta were found to contain enzymatic activity which methylates the phospholipids phosphatidylethanolamine (PE), phosphatidyl-N-monomethylethanolamine (PMME) and phosphatidyl-N,N-dimethylethanolamine (PDME) to form phosphatidylcholine (PCh) with 3H-methyl-S-adenosyl-l-methionine as the methyl donor. The three labelled reaction products were isolated by solvent extraction and separated on thin-layer chromatography (TLC) plates. The endogenous methyltransferase activity was low, indicating that the methylation pathway is quantitatively not important for the synthesis of free choline to meet the fetal needs. The distribution of 3H-methyl among PMME, PDME and PCh revealed fairly even labelling of all products when analysed by TLC. Addition of authentic PE, PMME and PDME to a level of approximately 2.5 mM stimulated the incorporation of 3H-methyl into the total lipid-soluble fraction with all three substrates, but was most pronounced with PMME. Present observations suggest that all three methylation steps were catalysed by one enzyme with a pH optimum of 9.0 in a reaction that does not require Mg++.

Acetylcholine in human term placenta: tissue levels in intact fragments after inhibition in vitro of choline acetyltransferase and relationship to [14C]alpha-aminoisobutyric acid uptake.

Choline acetyltransferase (ChAc), the enzyme catalysing the biosynthesis of acetylcholine (ACh) in the non-innervated human placenta, was rapidly and persistently inhibited by (2-benzoylethyl)trimethylammonium (BETA) when the drug was applied to intact tissue fragments. This inhibition (50 per cent at congruent to 0.4 mmol/1 BETA) was coupled to a concomitant reduction in the active uptake against a concentration gradient of the nonmetabolizable amino acid alpha-aminoisobutyric acid (AIB). The reduction of AIB accumulation (50 per cent at congruent to 0.1 mmol/1 BETA) was temporally related to inhibition of ChAc. These effects suggest that AIB uptake by the human placenta and ACh biosynthesis catalysed by ChAc are related. Measurements of total ACh content in tissue samples treated in parallel with those destined for ChAc and AIB uptake determinations revealed that BETA (3 mmol/1) significantly reduced the ACh levels by 35 to 50 per cent. This drug concentration caused almost complete inhibition of ChAc and blockade of AIB accumulation.

Lactose and D-galactose metabolism in Staphylococcus aureus. II. Isomerization of D-galactose 6-phosphate to D-tagatose 6-phosphate by a specific D-galactose-6-phosphate isomerase.

The inducible D-galactose-6-phosphate isomerase that functions in the metabolism of lactose and D-galactose in Staphylococcus aureus was partially purified from extracts of D-galactose-grown cells. It was shown to catalyze specifically the reversible isomerization of D-galactose 6-phosphate to D-tagatose 6-phosphate, the apparent Km values being 9.6 mM and 1.9 mM, respectively. At equilibrium, the ratio of D-galactose 6-phosphate to D-tagatose 6-phosphate was 9.0. The enzyme was not simulated by mono- or divalent cations and was not inhibited by EDTA, but it was inactivated reversibly by the thiol reagent N-ethylmaleimide. Its molecular weight was estimated to be about 100,000 both by gel filtration and by sedimentation in a sucrose density gradient. Data on stability, pH optimum, and inducibility of the enzyme are also presented. An improved procedure for the chemical synthesis of D-tagatose 6-phosphate is described, and resolution of the anomers of D-tagatose 6-phosphate by gas-liquid chromatography is reported.

Acetylcholine in human placenta. Identification by pyrolysis gas chromatography/mass spectrometry and tissue levels following different modes of delivery.

Extracts from human term placentae were analyzed by pyrolysis gas chromatography (GC) combined with mass spectrometry. The only choline ester of carbonic acid whose presence could be unequivocally established was acetylcholine (ACh). The mass spectrum of its demethylated tertiary derivative dimethylaminoethyl acetate (nor-ACh) agreed entirely with that of authentic pyrolyzed ACh. The concentrations of ACh were determined in placentae obtained after vaginal or Caesarean delivery to investigate the claim that the latter resulted in tissue with higher concentrations of ACh-like activity presumably because ACh was not expended during labor and delivery. No differences inACh content were found with GC analysis. The ACh concentrations were 102 +/- 17 (n=8) nmoles/g fresh tissue (vaginal delivery) vs. 105 +/- 16 (n=6) nmoles/g (Caesarean section). The fetal membranes (amnion and chorion) and the umbilical cord contained no ACh under the analytical conditions with a limit of sensitivity of 200 pmoles ACh.

Reliability and stability of the Comrey Personality Scales in a clinical setting.

Examined split-half reliabilities, retest reliabilities, and stability of the Comrey Personality Scales (CPS) (Comrey, 1970) for a Navy sample of 200 young drug abusers tested at the beginning and end of rehabilitation in a no-feedback, compulsory participation setting. The pre-rehabilitation split-half reliabilities for the eight personality scales ranged from .73 to .94, with an average of .85, and ranged from .74 to .91, with an average of .83, on the post-rehabilitation administration. Retest reliabilities for the eight scales were between .39 and .64, with an average of .52. Post-rehabilitation means were significantly higher on four of the eight scales, and one scale mean was significantly lower. It is concluded that the CPS is appropriate in a clinical setting in which test participation is compulsory and test feedback is not made available to the respondents.