Efficiency Of Primers Research Articles

A spectrum of biophysical interaction modes between T cells and different antigen-presenting cells during priming in 3-D collagen and in vivo

AbstractFor activation T cells engage antigen-presenting cells (APCs) in lymphatic tissues. The contact duration and kinetics (static versus dynamic) vary considerably in different model systems; however, it is unclear whether T cells, APCs, or the environment are responsible for the observed discrepancies. Using 3-D collagen matrices as structural scaffold, we directly compared the kinetics of T-cell engagement and activation by functionally major APC types, ie, dendritic cells (DCs) and resting or activated B cells. Resting B cells engaged T cells in long-lived (several hours), adhesive, and leukocyte function-associated antigen-1 (LFA-1)-dependent conjugates in 3-D collagen as well as in intact lymph nodes in vivo. DCs and preactivated B cells, however, supported predominantly dynamic, short-lived (minutes), and sequential contacts to T cells that were dependent on high cytoskeletal activity of the APCs but could not be inhibited by anti-LFA-1 treatment. Naive T cells were most strongly activated by DCs and activated B cells, whereas resting B cells were 100-fold less efficient to induce T-cell proliferation. Thus, in the same 3-D environment, naive T cells respond with a spectrum of different interaction modes dependent on the type and activation state of the APCs. Thereby, more dynamic interaction kinetics is positively correlated with higher T-cell priming efficiency. (Blood. 2004;104: 2801-2809)

Assessing the benefits of implementing micro-CHP systems in the UK

The feasibility of applying combined heat and power rationale to individual homes is discussed, with particular reference to the transient variations that occur in domestic requirements for heat and power. Predicted energy-cost savings, based on simulations employing 1 min demand profiles, are presented for several prospective micro-CHP systems. The effects upon energy-cost savings of prime mover capacity and efficiency, heat-recovery efficiency, central-heating boiler efficiency, and the unit price for exporting electricity are assessed. For various configurations of a nominally 1 kWe micro-CHP system, reductions of 16-39 per cent in annual energy expenditure are identified. Carbon savings per average home of around one tonne of CO2 per annum are predicted, but actual savings will depend strongly upon transient heat-and-power demand variations, the operating mode, capacity and efficiency of the micro-CHP system, and upon the import/export interaction that the household has with the electricity distribution network. If the carbon-reduction potential of this single technical fix is to be maximized, further research attention needs to be given to define preferred implementation strategies for micro-CHP systems in the UK domestic sector.

Semantic priming in a first and second language: evidence from reaction time variability and event-related brain potentials

We investigated individual differences in second language (L2) proficiency by looking at the efficiency or automaticity of semantic priming using behavioural and event-related brain potential (ERP) measures. In Experiment 1, 37 first language (L1) English speakers varying in second language (L2, French) proficiency made living/non-living judgments to English and French nouns in lists blocked by language. Sixty critical words were each presented twice, once primed by a semantic associate in the preceding trial (e.g. ADULT, CHILD) and once unprimed (e.g. RABBIT, CHILD). Measures of response time (RT) and intra-individual variability in response time (coefficient of variation, CV) were obtained. The CV provided an index of processing efficiency that has been related to automaticity. Participants performed faster and with lower CVs (i.e. with greater efficiency) in L1 than L2, and the more highly proficient bilinguals had lower CVs than the less proficient bilinguals. Experiment 2 replicated these results with 29 participants and provided an electrical brain activity measure of processing efficiency using the N400 ERP. The similar pattern of results obtained between the behavioural and N400 ERP CV measures supported the idea that the CV measure of electrical brain activity can provide useful information about the automaticity or efficiency of cognitive processing.

Efficient Initiation of HIV-1 Reverse Transcriptionin Vitro: REQUIREMENT FOR RNA SEQUENCES DOWNSTREAM OF THE PRIMER BINDING SITE ABROGATED BY NUCLEOCAPSID PROTEIN-DEPENDENT PRIMER-TEMPLATE INTERACTIONS

Synthesis of HIV-1 (-) strong-stop DNA is initiated following annealing of the 3' 18 nucleotides (nt) of tRNA(3)(Lys) to the primer binding site (PBS) near the 5' terminus of viral RNA. Here, we have investigated whether sequences downstream of the PBS play a role in promoting efficient (-) strong-stop DNA synthesis. Our findings demonstrate a template requirement for at least 24 bases downstream of the PBS when tRNA(3)(Lys) or an 18-nt RNA complementary to the PBS (R18), but not an 18-nt DNA primer, are used. Additional assays using 18-nt DNA-RNA chimeric primers, as well as melting studies and circular dichroism spectra of 18-nt primer:PBS duplexes, suggest that priming efficiency is correlated with duplex conformation and stability. Interestingly, in the presence of nucleocapsid protein (NC), the 24 downstream bases are dispensable for synthesis primed by tRNA(3)(Lys) but not by R18. We present data supporting the conclusion that NC promotes extended interactions between the anticodon stem and variable loop of tRNA(3)(Lys) and a sequence upstream of the A-rich loop in the template. Taken together, this study leads to new insights into the initiation of HIV-1 reverse transcription and the functional role of NC-facilitated tRNA-template interactions in this process.

DNTP versus NTP discrimination by phenylalanine 451 in duck hepatitis B virus P protein indicates a common structure of the dNTP-binding pocket with other reverse transcriptases.

Hepatitis B viruses, or hepadnaviruses, are small DNA-containing viruses that replicate through reverse transcription. Their prototype, HBV, causes severe liver disease in humans. The hepadnaviral P protein is an unusual reverse transcriptase (RT) that initiates DNA synthesis by host-factor-dependent protein priming on a specific RNA stem-loop template, epsilon, yielding a short DNA oligonucleotide covalently attached to the RT. This priming reaction can be reconstituted with in vitro-translated duck hepatitis B virus (DHBV) P protein. No direct structural data are available for any P protein. However, P proteins share a number of conserved motifs with other polymerases. Box A contains an invariant bulky residue recently shown to be crucial for dNTP versus NTP discrimination in RTs and some DNA polymerases; its equivalent in DHBV P protein would be phenylalanine 451 (F451). Four mutants, containing glycine (F451G), alanine (F451A), valine (F451V) and aspartate (F451D), were therefore analyzed for their ability to utilize dNTPs and NTPs in in vitro priming. Priming efficiencies with dNTPs decreased with decreasing side chain size but GTP utilization increased; the wild-type enzyme was inactive with GTP. In the context of complete DHBV genomes, all mutant proteins were competent for RNA encapsidation, indicating the absence of global structural alterations. Because the function of the discriminatory residue depends on its specific spatial disposition this strongly suggests a similar architecture for the P protein dNTP-binding pocket as in other RTs.

Human T lymphocyte priming in vitro by haptenated autologous dendritic cells.

Dendritic cells (DC), generated from adherent peripheral blood mononuclear cells (PBMC) by culturing with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4, were used to study in vitro sensitization of naive, hapten-specific T cells and to analyse cross-reactivities to related compounds. DC were hapten-derivatized with nickel sulphate (Ni) or 2-hydroxyethyl-methacrylate (HEMA), followed by tumour necrosis factor-alpha (TNF-alpha)-induced maturation, before autologous T cells and a cytokine cocktail of IL-1beta, IL-2 and IL-7 were added. After T cell priming for 7 days, wells were split and challenged for another 7 days with Ni or HEMA, and potentially cross-reactive haptens. Hapten-specificity of in vitro priming was demonstrated by proliferative responses to the haptens used for priming but not to the unrelated haptens. Highest priming efficiencies were obtained when both IL-4 and IL-12 were added to the cytokine supplement. Marked interferon-gamma (IFN-gamma) release (up to 4 ng/ml) was found when IL-12 was included in the cultures, whereas IL-5 release (up to 500 pg/ml) was observed after addition of IL-4 alone, or in combination with IL-12. Nickel-primed T cells showed frequent cross-reactivities with other metals closely positioned in the periodic table, i.e. palladium and copper, whereas HEMA-primed T cells showed distinct cross-reactivities with selected methacrylate congeners. Similar cross-reactivities are known to occur in allergic patients. Thus, in vitro T cell priming provides a promising tool for studying factors regulating cytokine synthesis, and cross-reactivity patterns of hapten-specific T cells.

Designing Peptide Vaccines for Cellular Cross-Presentation

Synthetic peptides are safe and relatively cheap vaccine components. However, the efficiency of peptide vaccines is limited by peptide interaction with non-professional antigen-presenting cells, which may hamper induction of productive T-cell responses. This paper argues that peptide vaccines should be modified for exclusive uptake by cells with the capacity to prime T-cell responses. Moreover, design of peptide vaccines should take intracellular antigen processing into account and exploit cellular mechanisms of proteolysis, transport and HLA class I assembly of antigenic peptides to enhance efficiency of T-cell priming and stimulation.

An artificial primosome: design, function, and applications.

Double-stranded (ds) DNA is capable of the sequence-specific accommodation of an additional oligodeoxyribonucleotide strand by the peptide nucleic acid(PNA)-assisted formation of a so-called PD-loop. We demonstrate here that the PD-loop may function as an artificial primosome within linear, nonsupercoiled DNA duplexes. DNA polymerase with its strand displacement activity uses this construct to initiate the primer extension reaction at a designated dsDNA site. The primer is extended by several hundred nucleotides. The efficiency of dsDNA priming by the artificial primosome assembly is comparable to the single-stranded DNA priming used in various assays. The ability of the PD-loop structure to perform like an artificial primosome on linear dsDNA may find applications in biochemistry, molecular biology, and molecular biotechnology, as well as for DNA diagnostics. In particular, multiple labels can be incorporated into a chosen dsDNA site resulting in ultrasensitive direct quantification of specific sequences. Furthermore, nondenaturing dsDNA sequencing proceeds from the PD-loop. This approach opens the way to direct isothermal reading of the DNA sequence against a background of unrelated DNA, thereby eliminating the need for purification of the target DNA.

Influence of DNA Target Melting Behavior on Real-Time PCR Quantification

Quantitative real-time PCR is increasingly used to quantify copy numbers of nucleic acids for clinical applications. We observed that the measurements of allele imbalances of the tumor suppressor gene p16 and the oncogene ErbB-2 yielded results with variable precision under certain experimental conditions. We used the LightCycler(TM) real-time PCR system to quantify different genomic target sequences using hybridization probes or SYBR Green for detection. With two primer/template systems (p16 and ErbB-2), we observed sinusoidal scattering of the threshold cycle values depending on the capillary position in the thermostated reaction chamber. This scattering depended on the denaturation temperature only when complete genomic DNA was used as template and did not occur when PCR product or restricted or boiled genomic DNA was used or the denaturation temperature in the first cycles was increased (and other targets, such as p53, HBB, IGF-1, GAPDH, and PBGD, did not show this behavior). Before a primer system is used for precise quantitative real-time PCR, the dependence of the quantification results on the positions of reaction tubes in the thermocycler should be tested. Our data indicate that amplification efficiencies, especially in the first cycles, depend not only on the priming efficiencies of the primers and the melting temperature of the amplicon, but also on the melting behavior of the amplicon's genomic vicinity. Complete denaturation of genomic DNA is necessary to maximize precision of quantitative PCR. Higher denaturation temperatures in the initial cycles or boiling of DNA before the PCR can improve the accuracy of quantification in some cases.

Sugarbeet seed priming: effects of priming conditions on germination, solubilization of 11-S globulin and accumulation of LEA proteins

AbstractThe relationship between initial mobilization of 11-S globulin storage protein (leading to solubilization of its B-subunit) and germination performance after priming was studied in sugarbeet (Beta vulgarisL.) seeds. Priming was conducted for 2 d either in water (hydropriming) or in –2.0 MPa polyethylene glycol-8000 (osmopriming), at various temperatures ranging from 5 to 40°C and oxygen concentrations in the atmosphere ranging from 0 to 21%. For both types of pre-treatments, the range of temperatures and the concentrations of oxygen which were effective in priming were very similar to those which allowed solubilization of the B-subunit of 11-S globulin, supporting the robustness of this protein marker for optimization of sugarbeet seed priming. Furthermore, the temperature and oxygen dependence of priming efficiency closely paralleled that for germination of the untreated seeds, reinforcing the finding that the beneficial effect of priming corresponded to the advancement of germinationsensu stricto(i.e. phase II of the germination process). For priming times longer than 2 d, particularly for osmopriming, there was a very dramatic decrease in germination of the treated seeds. For instance, following a 14 d osmopriming at 25°C as much as 60% of the pre-treated seed population failed to germinate when transferred to water. This loss in germination performance quite closely paralleled degradation of LEA (late embryogenesis abundant) proteins, notably a heat-stable seed-specific protein of about 60 kDa and a seed-specific biotinylated LEA protein.

RNA Degradation and Primer Selection by Moloney Murine Leukemia Virus Reverse Transcriptase Contribute to the Accuracy of Plus Strand Initiation

During reverse transcription, plus strand DNA synthesis is initiated at a purine-rich RNA primer generated by the RNase H activity of reverse transcriptase (RT). Specific initiation of plus strand synthesis from this polypurine tract (PPT) RNA is essential for the subsequent integration of the linear viral DNA product. Based on current models, it is predicted that priming from sites upstream of the PPT may be tolerated by the virus, whereas efficient extension from RNA primers located downstream from the PPT is predicted to generate dead-end products. By using hybrid duplex substrates derived from the Moloney murine leukemia virus long terminal repeat, we investigated the extent to which RNase H degrades the viral RNA during time course cleavage assays, and we tested the capacity of the polymerase activity of RT to use the resulting cleavage products as primers. We find that the majority of the RNA fragments generated by RNase H are 2-25 nucleotides in length, and only following extensive degradation are most fragments reduced to 10 nucleotides or smaller. Although extensive RNA degradation by RNase H likely eliminates many potential RNA primers, based on thermostability predictions it appears that some RNA fragments remain stably annealed to the DNA template. RNA primers generated by RNase H within the long terminal repeat sequence are found to have the capacity to initiate DNA synthesis by RT; however, the priming efficiency is significantly less than that observed with the PPT primer. We find that Moloney murine leukemia virus nucleocapsid protein reduces RNase H degradation and slightly alters the cleavage specificity of RT; however, nucleocapsid protein does not appear to enhance PPT primer utilization or suppress extension from non-PPT RNA primers.

Molecular nature of RAPD markers from Haemophilus influenzae Rd genome

Despite the widespread application of the random amplified polymorphic DNA (RAPD) technique, there is no experimental evidence of the molecular mechanism of random amplification starting from a complex template. To investigate this mechanism, we cloned and sequenced 23 selected RAPD bands amplified from Haemophilus influenzae Rd genomic DNA using eight decamer primers different in GC content and/or nucleotide sequence. As the whole genome sequence of H. influenzae Rd has been reported, the exact nucleotide sequence of each primer-template annealing site was identified. Results showed that, on an average, a homology of eight base pairs was involved in priming events and that the number of nonhomologous base pairings declined exponentially from the 5' end of the primer to its 3' end. The interaction between the primer and the template DNA was stabilized by the formation of secondary structures, and a perfect match of the 3' terminal region of the primer was not necessary for successful amplification. The complexity of the annealing process suggested that, in the studied reaction conditions, many primer-template annealing sites were extended in the first cycles and that differences in the efficiency of priming and replication processes led to amplification of RAPD fragments. Moreover, the distribution of the amplified regions on the H. influenzae chromosome was analyzed.

Cell cycle activity during seed priming is not essential for germination advancement in tomato

Seed priming is a technique of controlled hydration and drying that results in more rapid germination when the seeds are reimbibed. Advancement of radicle meristem cells into the S and G 2 phases of the cell cycle, increasing the percentage of nuclei having a 4C DNA content, has been reported to occur during priming. It has been suggested that the efficiency of priming is related to the accumulation of 4C nuclei in the radicle meristem, but the extent of cell cycle activity varied among different treatments and seed lots. A wide range of priming treatments across temperatures, water potentials and durations can be compared on a common basis using the hydrothermal priming time model. Flow cytometry was used to monitor cell cycle activity in a number of tomato (Lycopersicon esculentum Mill.) seed lots during priming in relation to the accumulation of hydrothermal priming time and the subsequent germination rate response. In some seed lots, the percentage of 4C nuclei in the radicle meristems prior to emergence increased in proportion to accumulated hydrothermal priming time, while in other lots, no increase in nuclear DNA content was detected. All lots, however, demonstrated rapid radicle emergence following priming. Thus, replicative DNA synthesis in radicle meristem nuclei often occurred during seed priming, but an increase in the percentage of 4C nuclei was not essential for germination advancement.

Interdependence between DNA template secondary structure and priming efficiencies of short primers.

Here we analyze the effect of DNA folding on the performance of short primers and describe a simple technique for assessing hitherto uncertain values of thermodynamic parameters that determine the folding of single-stranded DNA into secondary structure. An 8mer with two degenerate positions is extended simultaneously at several complementary sites on a known template (M13mp18) using one, two or three (but never all four) of the possible dNTPs. The length of the extension is site specific because it is limited by the first occurrence in the downstream template sequence of a base whose complementary dNTP is not present. The relative priming efficiencies of different sites are then ranked by comparing their band brightnesses on a gel. The priming efficiency of a short primer (unlike conventional long primers) depends dramatically on the secondary structure of the template at and around the priming site. We calculated the secondary structure and its effect on priming using a simple model with relatively few parameters which were then optimized to achieve the best match between the predictions and the actual rankings of the sites in terms of priming efficiency. This work introduces an efficient and conceptually novel approach that in the future can make use of more data to optimize a larger set of DNA folding parameters in a more refined model. The model we used, however crude it may be, significantly improved the prediction of priming efficiencies of 8mer primers and appreciably raised the success rate of our DNA sequencing technique (from 67 to 91% with a significance of P < 7 x 10(-5)), which uses such primers.

Analysis of Polypurine Tract-associated DNA Plus-strand Priming in Vivo Utilizing a Plant Pararetroviral Vector Carrying Redundant Ectopic Priming Elements

Initiation of DNA plus-strand synthesis in most reverse-transcribing elements requires primer generation by reverse transcriptase-associated RNase H at one or more template polypurine tracts (PPTs). We have exploited infectious clones of the plant pararetrovirus cauliflower mosaic virus carrying redundant ectopic plus-strand priming elements to study priming in vivo. Ectopic priming generated an additional discontinuity in progeny virion DNA during infection of plants. We found that altering the length of the 13-base pair PPT by +/-25% significantly reduced priming efficiency. A short pyrimidine tract 5' to the PPT, highly conserved among diverse reverse-transcribing elements, was shown to play an important role in PPT recognition in vivo. The predominant DNA plus-strand 5' end remained 3 nucleotides from the PPT 3' end in mutant primers that were longer or shorter than the wild-type primer. Use of an ectopic redundant primer to study replication-dependent priming was validated by demonstrating that it could rescue infectivity following destruction of the wild-type priming elements. We propose a model for plant pararetroviral plus-strand priming in which pyrimidines enhance PPT recognition during polymerase-dependent RNase H cleavages, and suggest that fidelity of primer maturation during polymerase-independent cleavages involves PPT length measurement and 3' end recognition by RNase H.

Identification of putative sequence specific PCR primers for detection of the toxigenic fungal speciesStachybotrys chartarum

The nucleotide sequence of ac936 bp segment of the nuclear rRNA gene operon was determined for the toxigenic fungal speciesStachybotrys chartarumand for other species ofStachybotrysand the related genusMemnoniella. This information was used to infer the phylogenetic relationships of these organisms and to search for sequence specific polymerase chain reaction (PCR) primers forS. chartarumin the internal transcribed spacer (ITS) regions. Searches for candidate primers were performed both by computer using the commercially available Oligo® v5.0 primer analysis software package and by manual inspection of the aligned sequences. Primers identified in both types of searches were evaluated for their specificities using a priming efficiency analysis algorithm available in the Oligo® 5.0 software. The automated computer searches were unsuccessful in findingS. chartarum-specific primers but did identify a group-specific reverse primer (designated as StacR4) for a phylogenetically related cluster of species that includedS. chartarum. Manual searches led to the identification of a reverse primer (designated as StacR3) that was predicted to be specific for onlyS. chartarumand one other species ofStachybotrys. Experimental PCR analyses using these primers in conjunction with a universal forward primer indicated that the computer-generated amplification efficiency predictions were correct in most instances. A notable exception was the finding that StacR3 was specific only forS. chartarum. The relative merits of different PCR strategies for the detection ofS. chartarumemploying either one or both of the primers identified in this study are discussed.

Enhancement of MHC class I-restricted peptide-specific T cell induction by a DNA prime/MVA boost vaccination regime

Human immunodeficiency virus (HIV) vaccine candidates were previously constructed as a string of cytotoxic T lymphocyte (CTL) epitopes delivered and expressed using DNA and modified virus Ankara (MVA; an attenuated vaccinia virus) vectors. These vaccines were shown to induce interferon (IFN)-γ-producing and cytolytic CD8 + T cells after a single vaccine administration. In the course of this work, immunization protocols were sought which would improve the levels of induced HIV-specific T cells. It was found that previous immunological exposure to MVA reduced the efficiency of subsequent priming and boosting using the same vaccine vehicle. However, a combined regime whereby the animals were first primed with the DNA vaccine and then boosted with MVA was the most potent protocol for the induction of both interferon-γ-producing and cytolytic T cells against two CTL epitopes simultaneously. The general applicability of this novel vaccination method for induction of major histocompatibility complex class I-restricted T cells is discussed.

Differential regulation of T cell activation for primary versus secondary proliferative responses.

T cell activation in vivo results in proliferation and generation of effector cytokine-secreting cells, as also in development of memory cells that mount enhanced responses upon restimulation. However, differences in the signals promoting generation of effector vs memory T cells are not yet characterized. In this study, using various strategies to modulate an allorecognition system for priming human T cells in vitro, we show that there are indeed differences between the signaling requirements for a first proliferative response and those for priming T cells for enhanced recall proliferative responses. Using APCs fixed with varying concentrations of paraformaldehyde, we show that the loss of ability of these APCs to generate a first response is not matched by a similar loss in their ability to prime responder T cells for recall responses. Prevention of DNA replication during T cell priming with aphidicolin, a DNA polymerase inhibitor, is not inimical to successful T cell priming. Thus, clonal expansion during priming is less crucial than the primed activation status of T cells for the enhanced recall response. We also show that pentoxifylline, a phosphodiesterase inhibitor, inhibits the primary proliferative response, but its presence during priming enhances the recall response capabilities of T cells. On the other hand, the presence of the calcineurin inhibitor cyclosporin A during priming reduces the efficiency of priming, but at low concentrations it induces, like pentoxifylline, enhancement in recall response capability. These findings have significant implications in designing immunosuppressive therapy and in the analysis of signals for T cell memory commitment.

Synthesis of the Mammalian Telomere Lagging Strand in Vitro

Using a synthetic telomere DNA template and whole cell extracts, we have identified proteins capable of synthesizing the telomere complementary strand. Synthesis of the complementary strand required a DNA template consisting of 10 repeats of the human telomeric sequence d(TTAGGG) and deoxy- and ribonucleosidetriphosphates and was inhibited by neutralizing antibodies to DNA polymerase alpha. No evidence for RNA-independent synthesis of the lagging strand was observed, suggesting that a stable DNA secondary structure capable of priming the lagging strand is unlikely. Purified DNA polymerase alpha/primase was capable of catalyzing synthesis of the lagging strand with the same requirements as those observed in crude cell extracts. A ladder of products was observed with an interval of six bases, suggesting a unique RNA priming site and site-specific pausing or dissociation of polymerase alpha on the d(TTAGGG)10 template. Removal of the RNA primers was observed upon the addition of purified RNase HI. By varying the input rNTP, the RNA priming site was determined to be opposite the 3' thymidine nucleotide generating a five-base RNA primer with the sequence 5'-AACCC. The addition of UTP did not increase the efficiency of priming and extension, suggesting that the five-base RNA primer is sufficient for extension with dNTPs by DNA polymerase alpha. This represents the first experimental evidence for RNA priming and DNA extension as the mechanism of mammalian telomeric lagging strand replication.

Clonidine potentiates the growth hormone response to a growth hormone releasing hormone challenge in hypothalamic growth hormone releasing hormone deficient rats.

This study was designed to further investigate our postulate regarding the inhibitory role played by central alpha 2-adrenergic pathways on hypothalamic somatostatin (SS) release in rats. The growth hormone (GH) responses to exogenous GH-releasing factor (GRF; 3 micrograms/kg i.v.) or clonidine (CLO; 100 micrograms/kg i.v.), either given alone or in combination, were tested in 3-month-old male rats made GH-releasing hormone (GH-RH) deficient neonatally by administration of monosodium glutamate (MSG; 4 mg/g body weight s.c.). To prevent the presumable decrease in the pituitary GH content in these animals from leading to an erroneous interpretation of the results obtained, half of these rats were given GRF (MSG-GRF rats; 30 micrograms/kg s.c.) for 3 days immediately prior to GH testing. The other half of MSG-treated and non MSG-treated rats received saline during these days (MSG-S and controls, respectively). To establish the efficiency of GRF priming, the pituitary GH content was measured in other MSG-GRF, MSG-S, and control animals. The mean (+/- SEM) GH peaks in response to GRF challenge were significantly higher in controls than in MSG-GRF rats (125.2 +/- 28.5 vs. 67.5 +/- 19.4 micrograms/l; p < 0.05), while no significant GRF-induced GH release was observed in the MSG-S group. Most likely these results are related to the different pituitary GH content, significantly (p < 0.01) higher in controls than in MSG-GRF rats, and in the latter higher than in MSG-S animals (p < 0.05). CLO administration did not evoke a significant GH release in MSG rats, whether primed with GRF or not.(ABSTRACT TRUNCATED AT 250 WORDS)