[DEMONSTRATION OF AN INHIBITORY EFFECT OF POLYPHOSPHATES ON ACCEPTOR ACTIVITY OF YEAST SOLUBLE RIBONUCLEIC ACID].
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We can now have considerable confidence that the broad features of protein synthesis are understood. The involvement of RNA is very much more complicated than was imagined in 1953. There is not one functional RNA. Instead, protein synthesis demands the ordered interaction of three classes of RNA-ribosomal, soluble, and messenger. Many important questions, however, remain unanswered. For instance, there is no theoretical framework for the ribosomal subunits, nor for that matter, do we understand the functional significance of ribosomal RNA. Most satisfying is the realization that all the steps in protein replication will be shown to involve well-understood chemical forces. As yet we do not know all the details. For example, are the DNA base pairs involved in messenger RNA selection of the corresponding amino-acyl-sRNA? With luck, this will soon be known. We can thus have every expectation that future progress in understanding selective protein synthesis (and its consequences for embryology) will have a similarly well-defined and, when understood, easy-to-comprehend chemical basis (62).
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OBJECTIVE: To study the progression of HIV infection in relation to immunological and virological variables with emphasis on the role of CD8+ lymphocytes. DESIGN: Prospective follow-up from October 1991 of patients observed for at least 18 months allowing nucleoside analogue monotherapy. Peripheral CD4+ and CD8+ lymphocyte counts, HIV RNA, and soluble CD8 were analysed by statistics allowing the evaluation of serial data, avoiding time points with concurrent infections. SETTING: Tertiary university clinic. PATIENTS: Forty-nine patients were followed for 52.6 months, baseline CD4+ count of 300 x 10(6)/l, sample interval of 5.9 months (medians). MAIN OUTCOME MEASURES: AIDS, death, and CDC groups B- or C-related events. RESULTS: AIDS developed in 28% of patients. Baseline CD8+ counts above the median were significantly associated with AIDS development; the best Cox model included CD8+ cells and the log10RNA/CD4 ratio. A decline in CD8+ counts relative to baseline most significantly predicted AIDS, along with higher baseline RNA and actual CD4+ counts of less than 200 x 10(6)/l. Levels of soluble CD8 in the blood relative to total CD8+ cells significantly increased in patients developing AIDS. Death occurred in 16% of the patients, and was only predicted by high CD8+ cell counts at baseline. CDC B- and C-related events occurred in 35% of the patients and were best predicted by high baseline CD8+ counts and high RNA levels. CONCLUSIONS: The serial quantitation of CD8+ lymphocytes gave highly significant predictive information on the natural progression of HIV infection in patients with moderate to severe immune deficiency. Our data suggest that the hyperactivation of CD8+ lymphocytes is an important factor leading to a numerical decrease of CD8+ lymphocytes in progressive HIV infection.
Salmonella enteritidis starved in Fernbach flasks used acid-alcohol-soluble material and RNA as endogenous reserves during starvation. Organisms starved in a fermentor system consumed acid-alcohol-soluble material, RNA and protein to maintain viability. Half-life survival times were 132 h and 118 h for the Fernbach and fermentor-starved cells, respectively. The acid-alcohol-soluble fraction of the cell consisted mainly of peptides or protein. This fraction accounted for most of the loss of label from 14C-labelled cells during the first 5 days of starvation and presumably contains the primary endogenous reserve. Although the residue fraction of fermentor-starved cells provided 35% of the total loss of 14C from the cells by the 5th day, a small net increase in 14C activity of the residue fraction of Fernbach-starved cells was observed. Differences observed appeared to be due to the method of starvation.
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In a model of secondary liver cancer in Wistar rats, the incorporation of tracer doses of pyrimidines and 5-fluoropyrimidines into the acid-soluble fraction, RNA and DNA of several normal tissues and of an experimental adenocarcinoma of the colon transplanted to the liver of rat was determined 90 min after infusion of the substances via the gastroduodenal artery. There was a higher incorporation after injection of FUra than after uracil into tumor RNA. There was very little labeling of DNA by FdUrd but a greater labeling of RNA following injection of this substance than following deoxyuridine in all tissues including tumor except in liver, where it was of the same magnitude. All fluoro compounds gave high labeling of the acid-soluble fraction of the kidney and liver. The experiments will be continued with therapeutic doses of the fluoro compounds.
Soluble forms of the interleukin (IL)-2, IL-4 and IL-7 receptors which lack the transmembrane domain have been described. IL-6 is a growth factor important in the final differentiation of B-cells into plasma cells and in the pathogenesis of multiple myeloma. To determine whether the receptor for IL-6 may exist as a soluble molecule, RNA was analysed from the transformed B-cell lines U266, CESS and Daudi, from bone marrow from two myeloma patients, and from normal leukocytes. Using polymerase chain reaction, oligonucleotide primers which flank the transmembrane domain were selected to generate a 339 bp fragment. All samples produced equivalent amounts of the expected 339 bp fragment plus a smaller 245 bp fragment except Daudi which exhibited virtual absence of both. Sequence analysis of the smaller fragments from each of the five samples demonstrated the deletion of the entire transmembrane region from codons 356 (G-TG) to 387 (AG-G). The boundaries of this deletion were identical in all cases. Partial sequence analysis of the ligand-binding domain for U266 demonstrated identical sequences for the membrane-bound and soluble forms of the IL-6 receptor cDNAs. In summary, an mRNA which encodes a soluble form of the IL-6 receptor is expressed in both normal and myeloma cells.
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The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine 'tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.
The RNA-dependent DNA polymerase(s) in murine leukemia, murine mammary tumor, and avian myeloblastosis viruses require maganese for optimal activity. The transcription of added synthetic polyribonucleotides is greatly enhanced when manganese is used in place of magnesium. A soluble RNA-dependent DNA polymerase activity has been released from murine leukemia particles in the presence of manganese and high detergent concentrations. Two RNA viruses, visna virus of sheep and a primate syncytial virus, not known to have tumor-producing ability, also contain the polymerase.