Prediction structure type for human leukocyte interferon subtype A from circular dichroism.

Vacuum UV circular dichroism studies were carried out on human leukocyte interferon subtype A. The secondary structure analysis for the CD spectrum shows 59% alpha-helix, 16% antiparallel beta-sheet, no parallel beta-sheet, 18% beta-turns and 13% other structures. The analysis of the CD features for the prediction of tertiary structural class reveals that it is an all-alpha type protein.

Structure of rhesus monkey relaxin predicted by analysis of the single-copy rhesus monkey relaxin gene.

The gene encoding rhesus monkey relaxin has been investigated. A cDNA library was prepared using corpus luteal RNA from a pregnant rhesus monkey, cDNA clones encoding relaxin were isolated and the nucleotide sequence was determined. The amino acid sequence of rhesus monkey preprorelaxin, predicted from the cDNA, demonstrates that the sequence has not been strongly conserved when compared with that of man, although features characteristic of the relaxin molecule have been maintained. This structural information will allow production of rhesus monkey relaxin, leading to studies investigating the bioactivity of relaxin in a homologous primate system. Southern blot analysis indicated that there is only one relaxin gene in the rhesus monkey and baboon genomes. In this respect these primate genomes are different from the human genome which contains two relaxin genes.

Immunohistochemical localization of relaxin in human prostate.

We identified relaxin in human male prostate by use of an anti-human relaxin analogue polyclonal antibody and the avidin-biotin-immunoperoxidase method. The antibody was obtained by immunizing a rabbit with a synthetic human relaxin analogue which has 95% sequence homology with native human relaxin. Human prostate tissues incubated with the anti-human relaxin analogue exhibited positive immunostaining up to an antibody dilution of 1:3200. Inhibition of immunostaining with this antibody by excess relaxin analogue demonstrated specificity of the antibody. The exact role of relaxin in human male reproductive physiology remains to be fully elucidated.

An enzyme-linked immunosorbent assay to study human relaxin in human pregnancy and in pregnant rhesus monkeys.

A sensitive and specific double-antibody enzyme-linked immunoassay, using a synthetic analogue of human relaxin for standard and immunogen, was developed for the measurement of human relaxin (hRLX) in serum and plasma. No cross-reactivity was observed for human insulin, human insulin-like growth factor-I, hGH, human chorionic gonadotropin, hFSH, hLH or human prolactin. The assay was used to monitor RLX concentrations in samples from men, non-pregnant and pregnant women, and in pregnant rhesus monkeys infused with hRLX. RLX was not detected in serum from men nor from non-pregnant women, while a concentration of 600 ng/l was measured in pooled sera from two pregnant women (pregnancies achieved by in-vitro fertilization). Immunoreactive RLX (1.1 micrograms/g) was found in human corpora lutea taken from ectopic pregnancies at 7 weeks. In an experiment with a pregnant rhesus monkey infused with human RLX analogue, less than 1.5% of the maternal concentration was measured in the fetal circulation. Even though preliminary, these data suggest a low level of transfer of human analogue relaxin across the placenta in a rhesus monkey. Further studies of the physiology of RLX in human pregnancy will be facilitated by the availability of this immunoassay.

Relaxin in human pregnancy serum measured with an homologous radioimmunoassay.

This study reports serum levels of relaxin in normal and special-interest pregnancies using an homologous radioimmunoassay for human relaxin. The mean levels in uncomplicated antenatal patients were lower than those reported in studies using heterologous assays, but the trend in serum levels was similar. Serum levels peaked at ten weeks' gestation and decreased progressively to term. Relaxin was detectable in all pregnant subjects assessed at the time of the first missed menses. The mean relaxin levels in patients having in vitro fertilization and embryo transfer who subsequently delivered a single infant were significantly higher than those in normal antenatal patients at an equivalent gestational age. Patients with twin pregnancies after in vitro fertilization and embryo transfer generally had higher levels than patients with single pregnancies. Some pregnant patients who aborted after in vitro fertilization and embryo transfer had declining levels of relaxin before 40 days postlaparoscopy.

Relaxin, CA-125, progesterone, estradiol, Schwangerschaft protein, and human chorionic gonadotropin as predictors of outcome in threatened and nonthreatened pregnancies.

Progesterone (P), estradiol (E2), relaxin, CA-125, Schwangerschaft protein, and human chorionic gonadotropin (hCG) were measured in 221 pregnancies (less than or equal to 77 days gestation). The cohort was divided into asymptomatic subjects (group I, n = 117) and those with threatening symptoms (group II, n = 104). Outcome was ascertained as viable (normal at 14 weeks, n = 131), spontaneous abortion (n = 58), or ectopic gestation (n = 32). Statistical analysis revealed no differences in the mean maternal or gestational ages among the viable pregnancies, abortions, and ectopics in group I and group II. In group I, significant differences in the means were noted for P, hCG, relaxin, and CA-125 among those destined to abort, compared with those who were not. In group II, differences were noted in P, hCG, relaxin, and E2 when viable and nonviable pregnancies were compared. Within group II, there were significant differences between the means of E2 and CA-125 when the aborters were contrasted with ectopics. Receiver operating characteristic curve analysis revealed that P was the single most reliable predictor and was most effective in threatened pregnancies. Stepwise logistic regression of the six markers in group II provided an equation of possible clinical utility in differentiating abortion versus ectopic pregnancy in threatened gestations based on CA-125 and E2 levels.

Relaxin levels in antenatal patients following in vitro fertilization.

The aim of this study was to investigate the relationship between serum relaxin and pregnancy outcome in a group of patients pregnant after in vitro fertilization (IVF). Patients who delivered a single live infant at term after IVF had mean serum levels of relaxin more than double the mean levels in normal antenatal patients. The high relaxin levels were compatible with delivery at term. However, because of the proposed role of relaxin in the process of cervical ripening, the high serum levels may help explain the high rate of preterm labor observed among IVF patients.

A study of worker dose distributions with respect to ICRP dose limitation recommendations.

This paper describes an investigation into the distribution of radiation doses to workers, undertaken at the request of ICRP Committee 4. Three main questions are considered. Firstly, the distribution of annual doses to workers is described. Doses are generally well below the ICRP dose limits and most are below 15 mSv y-1. However, doses are not distributed randomly from year to year. The second part of the paper examines recording levels in use and the implications of using the recording level recommended by the ICRP. The data presented here show that little dosimetric information would be lost and many fewer doses would need to be stored if the ICRP-recommended recording level were used. The last part of the paper considers the distribution of doses in individual monitoring periods. It is shown that the distribution of doses to all individuals in a particular monitoring period is not necessarily the same as the distribution of doses to a particular individual in all monitoring periods. This places limitations on the extent to which missing doses may be estimated from doses to the workforce as a whole.

An NMR study of the exchange rates for protons involved in the secondary and tertiary structure of yeast tRNA Phe.

Solvent exchange rates of all the protons of yeast tRNAphe resonating in the lowfield NMR region (-11 to-15 ppm from DSS) have been measured by saturation-recovery long-pulse Fourier transform NMR. All these protons in yeast tRNAphe are in the fast exchange limit with H2O relative to their intrinsic longitudinal relaxation processes. Most rates show very little temperature dependence; however, tertiary base pair protons are preferentially destabilized in the absence of Mg++ at higher temperatures. The measured exchange rates are between 2 and 125 sec-1 for a temperature range from 10 degrees C to 45 degrees C and MgCl2 concentrations between 0 and 15 mM.

Nuclear magnetic resonance and nuclear Overhauser effect study of yeast phenylalanine transfer ribonucleic acid imino protons.

Results directed primarily toward spectral assignment and nuclear spin dynamics are described for yeast tRNAPhe in 0.1 M NaCl, pH 7. Magnesium titrations were performed. Changes in the spectrum occur for Mg2+/tRNA ratios of about 2 and above 10. Difference spectroscopy between 43 and 29 degrees C in zero Mg2+ concentration, together with prior identification of the GU4 acceptor stem base pair, indicates early acceptor melting and is used to identify acceptor resonances. Transport of spin energy (spin diffusion) is described in tRNA together with a summary of relevant experiments. A survey of nuclear Overhauser effects (NOE's) between imino and aromatic and amino protons is included, together with some recent conclusions based on methyl NOE's and experiments with tRNAs deuterated at the purine C8 position. Assignment of the imino NMR spectrum on the basis of these and previous data is reviewed and discussed in detail. Preliminary distance estimates based on the NOE for AU and GU4 base pairs are in reasonable agreement with the expected distances.

Study of transfer ribonucleic acid unfolding by dynamic nuclear magnetic resonance.

Nuclear magnetic resonance (NMR) measurements of proton exchange were performed on yeast tRNAPhe, and in much less detail on Escherichia coli tRNAfMet, over a range of Mg2+ concentrations and temperatures, at neutral pH and 0.1 M NaCl. The resonances studied were those of ring nitrogen protons, resonating between 10 and 15 ppm downfield from sodium 3-(trimethylsilyl)-1-propanesulfonate, which partake in hydrogen bonding between bases of secondary and tertiary pairs. Methods include saturation--recovery, line width, and real-time observation after a change to deuterated solvent. The relevant theory is briefly reviewed. We believe that most of the higher temperature rates reflect major unfolding of the molecule. For E. coli tRNAfMet, the temperature dependence of the rate for the U8--A14 resonance maps well onto previous optical T-jump studies for a transition assigned to tertiary melting. For yeast tRNAPhe, exchange rates of several resolved protons could be studied from 30 to 45 degrees C in zero Mg2+ concentration and had activation energies on the order of 40 kcal/mol. Initially, the tertiary structure melts, followed shortly by the acceptor stem. At high Mg2+ concentration, relatively few exchange rates are measurable below the general cooperative melt at about 60 degrees C; these are attributed to tertiary changes. Real-time observations suggest a change in the exchange mechanism at room temperature with a lower activation energy. The results are compared with those obtained by other methods directed toward assaying ribonucleic acid dynamics.

Pulsed FT-NMR double resonance studies of yeast tRNAPhe: specific nuclear Overhauser effects and reinterpretation of low temperature relaxation data.

Cross-relaxation effects are demonstrated between the imino protons and other protons in yeast tRNAPhe and H2O. A detailed examination has been made of the observed relaxation rate of the proton resonance at 11.8 ppm from DSS as a function of the D2O content in the solvent. This result, as well as the size and number of observed nuclear Overhauser effects, suggests that dipolar magnetization transfer between solvent H2O, amino, imino, and other tRNA protons may dominate the relaxation processes of the imino protons at low temperature. At higher temperatures the observed relaxation rate is dominated by chemical exchange. The selective nuclear Overhauser effects are shown to be an important aid in resonance assignments. By these means we were able to identify tow protons from the wobble base pair GU4 at 11.8 ppm and 10.4 ppm.

Real-time solvent exchange studies of the imino and amino protons of yeast phenylalanine transfer RNA by Fourier transform NMR.

Real-time solvent exchange measurements using Fourier transform NMR at 270 MHz are presented. By means of the fast gel filtration column techniques originally developed for tritium exchange experiments, we were able to replace the solvent of a tRNA sample from an 1H2O to an 2H2O buffer and obtain a useful spectrum in 2-5 min. At 15 degrees C, there are 5 +/- 1 lowfield (-11 to -15 ppm relative to 2,2-dimethyl-2-silapentane-5-sulfonate) imino protons with exchange half times of minutes to hours. In addition, the m7G-46 C(8) proton and several amino protons are observed to exchange with similar rates. Analogous studies on unfractionated yeast tRNA suggest that such a class of slowly exchanging imino protons is present in several tRNAs, and that the activation energy for exchange is small [[approximatley 5 kcal/mol (21 kJ/mol)]. We speculate that these imino resonances arise from D-stem protons and that their slow exchange reflects stabilization by the numerous tertiary interactions involving this stem and the Mg2+ bound at the P-10 bend.

Roles of the 29-138 disulfide bond of subtype A of human alpha interferon in its antiviral activity and conformational stability.

Human alpha (leukocyte) interferons contain two disulfide bonds between Cys-1 and Cys-98 and between Cys-29 and Cys-138. Reduction of interferon under native conditions leads to irreversible loss of antiviral activity; reduction in denaturant, followed by oxidation in native conditions, leads to restoration of activity. This behavior, unusual for disulfide-containing proteins, was studied by using a thiosulfonate derivative of subtype A of human alpha interferon (IFN-alpha A). The disulfide-free thiosulfonate formed at 25 degrees C has essentially no antiviral activity, while maintaining a conformation related to that of native IFN-alpha A. This species can regain activity after regeneration of its 29-138 disulfide, by thiol-disulfide interchange in native buffer. Incubation of the disulfide-free thiosulfonate under nonreducing conditions at 37 degrees C generates a monomeric species that has lost its native conformation as well as its ability to regain antiviral activity after thiol-disulfide interchange. These results explain the difficulty in obtaining, under native conditions, a reduced species that regains activity upon oxidation; complete reduction of IFN-alpha A in 100 mM 2-mercaptoethanol requires 37 degrees C, a temperature that promotes conformational decay of the disulfide-free form.

Nuclear Overhauser effect in specifically deuterated macromolecules: NMR assay for unusual base pairing in transfer RNA.

We demonstrate a fairly general method for identification of NMR absorption lines of macromolecues extracted from microorganisms, based on nuclear Overhauser effects (NOE). Several NOE in tRNA are observable between resolved imino proton resonances and ring carbon resonances that are either C(2) protons of adenine or C(8) protons of adenine or guanine. Yeast tRNAPhe was deuterated at the purine C(8) positins by heating in 2H2O and also biosynthetically. NOE between imino protons and adenine C(2) protons of standard A . U base pairs would not be affected by such a label, but some other NOE that might be otherwise similar, such as those of reverse Hoogsteen base pairs, should disappear. Six NOE were shown to be from standard A . U pairs by their nondisappearance. Four NOE from methyl resonances to aromatic proton resonances did disappear. The results disagree with previous assignments based on ring-current theories of imino proton NMR shifts.

Directly iodinated rat relaxin as a tracer for use in radioimmunoassays.

A newly developed, specific, sensitive and reliable RIA for rat relaxin is described. Radioiodinated rat relaxin, with a specific activity of 22 to 37 kBq/micrograms, was prepared from purified authentic ovarian rat relaxin using the chloramine T method. Polyclonal rat relaxin antiserum was raised in rabbits. Assay sensitivity was 30 pg/tube. Intra- and interassay coefficients of variation were 8% and 13%, respectively. Initial experiments demonstrated rising concentrations of rat relaxin in pregnant rats. Serum of adult male or non-pregnant female rats contained undetectable levels of rat relaxin. The assay is suitable for measurements of rat relaxin in biological fluids, thus making the assay a valuable tool for further studies on the physiology of relaxin.

Increase in cyclic AMP levels by relaxin in newborn rhesus monkey uterus cell culture.

A novel relaxin sensitive cell line of apparent smooth muscle origin has been established from a newborn rhesus monkey uterus (NRMU). NRMU cells respond to relaxin, in the presence of 1 microM forskolin, by producing intracellular adenosine 3', 5'-cyclic monophosphate (cAMP). The increase in cAMP levels is dose, time and cell density dependent, reaching peak levels at 10 min when cells are seeded at 1 X 10(5) cells/well. Specificity was demonstrated by neutralization of the relaxin activity with anti-relaxin monoclonal and polyclonal antibodies, degradation of cAMP in the presence of phosphodiesterase, and confirmation of the absence of cGMP. Three synthetic analogs of human relaxin generated a dose-related cAMP response as did synthetic native human relaxin. Natural relaxin purified from human corpora lutea tissue also generated a response similar to synthetic human relaxin. Porcine and rat relaxins also increased levels of cAMP. Insulin, but not IGF I or IGF II, was capable of increasing cAMP levels in NRMU cells, however, 200 ng/mL were required to achieve cAMP levels comparable to 6.25 ng/ml relaxin. Combinations of relaxin with insulin, IGF I or IGF II did not increase cAMP levels above levels obtained with relaxin alone. The effect on NRMU cells of other hormones, growth factors and drugs potentially present in cell culture systems or serum samples was evaluated. In combination with relaxin, oxytocin significantly decreased the cAMP production below the levels induced by relaxin alone, whereas progesterone and prostaglandin E2 resulted in additive increases in cAMP. These data suggest that the NRMU cell line is an appropriate target tissue for studying relaxin-mediated biological responses in vitro as well as functioning as the primary component of a relaxin in vitro bioassay.

Antenatal serum levels of relaxin in patients having preterm labour.

This study is the first report of antenatal levels of relaxin measured by homologous radioimmunoassay in peripheral serum from patients who subsequently had a preterm delivery. Serial blood samples were collected antenatally from a group of subjects known to be at increased risk of preterm labour because of a past history of shortened pregnancy. Serum relaxin was measured using an homologous radioimmunoassay based on a synthetic bioactive analogue of the native hormone. In women whose pregnancies ended preterm most measurements were within the range of values previously established in normal antenatal patients although some measurements early in pregnancy were above the normal range. These findings suggest that low serum levels of relaxin are not causatively related to the onset of labour before term.

Radioimmunoassay of relaxin in pregnancy with an analogue of human relaxin.

A radioimmunoassay for relaxin was developed in which a synthetic analogue of human relaxin was used as standard, tracer, and immunogen. Relaxin could not be measured in sera from men or non-pregnant women, but was measurable in pregnant women from the tenth week of gestation until term. Concentrations ranged from 0.19-1.18 ng/ml, with highest levels measured in the first trimester.