Privacy and patient-clergy access: perspectives of patients admitted to hospital.

BACKGROUND: For patients admitted to hospital both pastoral care and privacy or confidentiality are important. Rules related to each have come into conflict recently in the US. Federal laws and other rules protect confidentiality in ways that countermand hospitals' methods for facilitating access to pastoral care. This leads to conflicts and poses an unusual type of dilemma-one of conflicting values and rights. As interests are elements necessary for establishing rights, it is important to explore patients' interests in privacy compared with their desire for attention from a cleric. AIM: To assess the willingness of patients to have their names and rooms included on a list by religion, having that information given to clergy without their consent, their sense of privacy violation if that were done and their views about patients' privacy rights. METHODS AND PARTICIPANTS: 179 patients, aged 18-92 years, admitted to hospital in an acute care setting, were interviewed and asked about their preferences for confidentiality and pastoral support. RESULTS: Most (57%) patients did not want to be listed by religion; 58% did not think hospitals should give lists to clergy without their consent and 84% welcomed a visit by their own clergy even if triggered from a hospital list. CONCLUSIONS: Values related to confidentiality or privacy and pastoral care were found to be inconsistent and more complicated than expected. Balancing the right to privacy and the value of religious support continue to present a challenge for hospitals. Patients' preferences support the importance of providing balance in a way that protects rights while offering comprehensive services.

Reduced misreading of asparagine codons by Escherichia coli tRNALys with hypomodified derivatives of 5-methylaminomethyl-2-thiouridine in the wobble position.

It has been suggested that modified nucleosides of the xm5(s2)U(m)34-type restrict the wobble capacity of the base, and that their function is to prevent misreading in the third position of the codon in mixed codon family boxes that encode two different amino acids. In this study in Escherichia coli, the misreading in vivo of asparagine codons in bacteriophage MS2 mRNA by different hypomodified derivatives of tRNALys, normally containing 5-methylaminomethyl-2-thiouridine (mnm5s2U34) in the wobble position, has been analysed. Contrary to what would be predicted from the general hypothesis for the function of mnm5s2U, it was found that the misreading of asparagine codons by tRNALys was greatly reduced in the mnmA (formerly asuE or trmU) and mnmE (formerly trmE) mutants which contain the hypomodified mnm5U34 and s2U34, respectively, instead of the fully modified mnm5s2U34. In addition, it was found that these hypomodified tRNAs were efficiently charged with lysine in vivo, under the growth conditions employed. The latter result is at variance with results obtained in vitro. The results are discussed in relation to the postulated function for modified nucleosides of the xm5s2U type.

Tubercidin analogs from the ascidian Didemnum voeltzkowi.

Two new 5'-deoxypyrrolo[2,3-d]pyrimidine (7-deazapurine) nucleosides, 5'-deoxytubercidin and 5'-deoxy-3-bromotubercidin, were identified from the ascidian Didemnum voeltzkowi. Two known anomers of 5'-deoxy-3-iodotubercidin were also purified from the extract. Assignments were made on the basis of 1H and 13C chemical shifts as well as HPLC-MS experiments.

A novel modified nucleoside found at the first position of the anticodon of methionine tRNA from bovine liver mitochondria.

Methionine tRNA was purified from bovine liver mitochondria, and its nucleotide sequence was determined. The tRNA possesses only three posttranscriptionally modified nucleosides, two pseudouridines in the anticodon and T stems and a previously unknown nucleoside specified by the gene sequence as cytidine, in the first position of the anticodon. Structure analysis of the anticodon nucleoside by mass spectrometry revealed a molecular mass 28 Da greater than that of cytidine, and unmodified ribose, with substitution at C-5 implied by hydrogen-deuterium exchange experiments. Proton NMR of the intact tRNA showed presence of a formyl moiety, thus leading to the candidate structure 5-formylcytidine (f5C), not a previously known compound. The structure assignment was confirmed by chemical synthesis and comparison of data from combined HPLC/mass spectrometry and proton NMR for the natural and synthetic nucleosides. The potential function of f5C in the tRNA(Met) anticodon is discussed with regard to codon-anticodon interactions.

Editing does not exist for mammalian selenocysteine tRNAs.

It has been reported that selenocysteine tRNA from bovine liver is completely edited to two isoacceptor species, called tRNA([Ser]SecNCA) and tRNA([Ser]SecCmCa), which differ from the gene sequence. We used direct tRNA sequencing, mobility shift analyses, primer extension, restriction enzyme digestion and single strand conformational polymorphism (SSCP) analyses of products from reverse transcription coupled with polymerase chain reaction (RT/PCR), sequencing of RT/PCR products and HPLC-coupled mass spectrometry to reproduce this result and show here that editing of these tRNAs does not occur.

A novel method for the determination of post-transcriptional modification in RNA by mass spectrometry.

A method is described for the detection, chemical characterization and sequence placement of post-transcriptionally modified nucleotides in RNA. Molecular masses of oligonucleotides produced by RNase T1 hydrolysis can be measured by electrospray mass spectrometry with errors of less than 1 Da, which provides exact base composition, and recognition of modifications resulting from incremental increases in mass. Used in conjunction with combined liquid chromatography-mass spectrometry and gene sequence data, modified residues can be completely characterized at the nucleoside level, and assigned to sequence sites within oligonucleotides defined by selective RNase cleavage. The procedures are demonstrated using E.coli 5S rRNA, in which all RNase T1 fragments predicted from the rDNA sequence are identified solely on the basis of their molecular masses, and using E.coli 16S rRNA for analysis of post-transcriptional modification, including placement of 3-methyluridine at position 1498. The principles described are generally applicable to other covalent structural modifications of RNA which produce a change in mass, such as those resulting from editing, photochemical cross-linking, or xenobiotic modification.

Dietary selenium affects methylation of the wobble nucleoside in the anticodon of selenocysteine tRNA([Ser]Sec).

We reported previously that the presence of selenium in culture media of mammalian cells influences both the steady-state levels and distributions of two tRNA isoacceptors involved in the insertion of selenocysteine into protein in response to certain UGA codons. In this study, we demonstrate an increase in the levels of these isoacceptors in rats fed a selenium-adequate diet compared to animals fed a selenium-deficient diet, as well as a shift in the relative distribution toward the tRNA which elutes later from an RPC-5 column. These effects were found to occur in a tissue-specific manner. Both selenocysteine tRNAs were isolated from rat liver, sequenced, analyzed by mass spectrometry, and shown to differ only by ribose 2'-O-methylation of 5-methylcarboxymethyluridine that occurs in the wobble position of the anticodon. This modified nucleoside has been documented previously only in yeast tRNA while the corresponding 2'-O-methylribose derivative has not been observed. The structure of these nucleosides was established by mass spectrometry and confirmed by chemical synthesis. Although the role of methylation of the wobble nucleotide is not known, the differences in elution properties from RPC-5 columns are consistent with other experimental observations indicating that a change in tRNA conformation accompanies this methylation.

Determination of oligonucleotide composition from mass spectrometrically measured molecular weight.

Extensive calculations for molecular mass versus subunit composition have been made for oligonucleotides from RNA and DNA to determine the extent to which base compositions might be derived from mass spectrometrically determined molecular weights. In the absence of compositional constraints (e.g., any numbers of A, U, G, C), measurement of molecular weight leads to only modest restrictions in allowable number of base compositions; however, if the compositional value for any one residue is known, such as from selective chemical modification or enzymatic cleavage, the number of allowable base compositions becomes unexpectedly low. For example, hydrolysis of RNA by ribonuclease T1 produces oligonucleotides for which G=1, for which all base compositions can be uniquely specified up to the 14-mer level, solely by measurement of mass to within ±0,01%. The effects of methylation, phosphorylation state of nucleotide termini, and knowledge of chain length on the determination of subunit composition are discussed.

Isolation, purification, and characterization of two new chemical decomposition products of methylazoxyprocarbazine.

We have previously reported that the antineoplastic agent, procarbazine, in aqueous solutions was chemically oxidized to its azoxy metabolites (methylazoxy and benzylazoxy). To determine if there was additional metabolism of the most active metabolite, methylazoxyprocarbazine, it was incubated in the presence and absence of CCRF-CEM human leukemia cells. Incubations were extracted, and potential metabolites were detected by HPLC with UV detection and by combined HPLC and thermospray mass spectrometric analysis. The major metabolite identified by HPLC with UV detection of the extracts was N-isopropyl-p-formylbenzamide; this was identified by comparison of its retention time with that of a synthesized standard. This identification was further corroborated by HPLC/thermospray mass spectrometry (LC/MS). Analysis of the extracts by LC/MS also showed the presence of a closely eluting peak that had a protonated molecular ion at m/z 207. This new metabolite was identified as N-isopropyl-(benzene-1,4-bis-carboxamide) by 1H NMR and gas chromatography/ion trap mass spectrometry. This metabolite is postulated to arise from breakage of the N-N bond in the hydrazine portion of the molecule. Reconstructed ion (m/z 236) current profiles from the analysis of the cell extracts indicated that there was only a trace amount of methylazoxyprocarbazine left after a 72-hr incubation. Interestingly, a peak with the same molecular weight as the parent compound (methylazoxyprocarbazine) was observed in the cellular incubations and also in extracts of control incubations in which methylazoxyprocarbazine was incubated in medium without cells. This unknown was silylated and identified as a hydroxyazo compound by an ion trap mass spectrometer operated under both single and multiple-stage mass analysis. Formation of this decomposition product appears to involve a novel intramolecular rearrangement of methylazoxyprocarbazine in solution. This pathway may be responsible for the formation of the ultimate cytotoxic species by chemical decomposition of procarbazine.

Non-enzymatic activation of procarbazine to active cytotoxic species.

The cellular cytotoxicity of procarbazine is thought to result from bioactivation of the parent compound through reactive intermediates to an ultimate alkylating species. Procarbazine is converted initially to azoprocarbazine, which is then N-oxidized through a cytochrome P-450-mediated process to a mixture of the positional isomers, benzylazoxyprocarbazine and methylazoxyprocarbazine. In order to define the bioactivation events that lead to the cytotoxic species, the in vitro cytotoxicities of the purified azoxy isomers as well as of the parent compound, procarbazine, were evaluated with the human leukemia cell line, CCRF-CEM. The methylazoxy isomer was found to be the most active species. Procarbazine inhibited the growth of CCRF-CEM cells but at a concentration much higher than that required for the methylazoxy isomer. Since procarbazine must be metabolized to form the cytotoxic species, we sought to determine if the active metabolite, methylazoxyprocarbazine, was being formed in the incubations. Solutions of procarbazine incubated with and without cells at 37 degrees C were analyzed by combined liquid chromatography-mass spectrometry with a thermospray interface. The azoxy metabolites of procarbazine appeared rapidly in cellular incubations and in the aqueous solutions without cells. More of the methylazoxy isomer was formed initially, but by 72 hr the benzylazoxy isomer was the predominant species. Thus, in these studies it appears that procarbazine was benzylazoxy isomer was the predominant species. Thus, in these studies it appears that procarbazine was non-enzymatically oxidized to the two azoxyprocarbazine isomers and that the methylazoxy compound was the most cytotoxic to CCRF-CEM cells.

The mechanism of adenosine to inosine conversion by the double-stranded RNA unwinding/modifying activity: a high-performance liquid chromatography-mass spectrometry analysis.

We have used directly combined high-performance liquid chromatography-mass spectrometry (LC/MS) to examine the mechanism of the reaction catalyzed by the double-stranded RNA unwinding/modifying activity [Bass & Weintraub (1988) Cell 55, 1089-1098]. A double-stranded RNA substrate in which all adenosines were uniformly labeled with 13C was synthesized. An LC/MS analysis of the nucleoside products from the modified, labeled substrate confirmed that adenosine is modified to inosine during the unwinding/modifying reaction. Most importantly, we found that no carbons are exchanged during the reaction. By including H2(18)O in the reaction, we showed that water serves efficiently as the oxygen donor in vitro. These results are consistent with a hydrolytic deamination mechanism and rule out a base replacement mechanism. Although the double-stranded RNA unwinding/modifying activity appears to utilize a catalytic mechanism similar to that of adenosine deaminase, coformycin, a transition-state analogue, will not inhibit the unwinding/modifying activity.

Posttranscriptional modification of tRNA in thermophilic archaea (Archaebacteria).

Nucleoside modification has been studied in unfractionated tRNA from 11 thermophilic archaea (archaebacteria), including phylogenetically diverse representatives of thermophilic methanogens and sulfur-metabolizing hyperthermophiles which grow optimally in the temperature range of 56 (Thermoplasma acidophilum) to 105 degrees C (Pyrodictium occultum), and for comparison from the most thermophilic bacterium (eubacterium) known, Thermotoga maritima (80 degrees C). Nine nucleosides are found to be unique to the archaea, six of which are structurally novel in being modified both in the base and by methylation in ribose and occur primarily in tRNA from the extreme thermophiles in the Crenarchaeota of the archaeal phylogenetic tree. 2-Thiothymine occurs in tRNA from Thermococcus sp., and constitutes the only known occurrence of the thymine moiety in archaeal RNA, in contrast to its near-ubiquitous presence in tRNA from bacteria and eukarya. A total of 33 modified nucleosides are rigorously characterized in archaeal tRNA in the present study, demonstrating that the structural range of posttranscriptional modifications in archaeal tRNA is more extensive than previously known. From a phylogenetic standpoint, certain tRNA modifications occur in the archaea which are otherwise unique to either the bacterial or eukaryal domain, although the overall patterns of modification are more typical of eukaryotes than bacteria.

Detection of ribose-methylated nucleotides in enzymatic hydrolysates of RNA by thermospray liquid chromatography-mass spectrometry.

A procedure has been developed for the detection and characterization of ribose-methylated dinucleotides of the type NmpN' in enzymatic digests of RNA. Differences in reaction products from hydrolysis using RNase T2, which will not cleave NmpN', and hydrolysis by nuclease P1 are analyzed by thermospray liquid chromatography-mass spectrometry. The method is applicable to dinucleotides present in nanogram range quantities and is suited for the characterization of new or unexpected ribose-methylated nucleotides for which chromatographic mobilities are not known.

Sex differences in the relative importance of self-esteem, physical self-satisfaction, and identity in predicting adolescent satisfaction.

Theoretical discussions of adolescence tend to emphasize the importance of physical development, self-views, and a transitional aspect of adolescence. Few research studies examine these variables in combination. The purpose of the present research was to examine the interrelationships of physical self-satisfaction, self-esteem, and identity in addition to their ability to predict satisfaction with an individual's social milieu. Multiple regression analyses were conducted for six groups consisting of eighth-, tenth-, and twelfth-grade males and females. While the prediction patterns yielded no differences for grade level, differences between males and females were found. Self-esteem was the best predictor for males, while identity and physical self-satisfaction were the best predictors for females.