Therapy-induced clearance of HCV core antigen from plasma predicts an end of treatment viral response.

During viral assembly, viral proteins are released into plasma and can be used to infer viral load. The Architect hepatitis C virus (HCV) core antigen (Ag) assay is a potential alternative to HCV RNA quantification for measuring response to therapy and predicting an end of treatment viral response (EOTR). The HCVp22Ag assay was used to infer viral load in 68 window RNA-containing samples and in 284 samples from baseline to week 14 of ribavirin/interferon treatment in 23 patients with EOTR including three who relapsed, 20 not achieving EOTR and 11 controls. HCV Ag and RNA correlated well (r = 0.86) with linear dose responses on dilution. In patients on therapy and control patients, plasma HCV antigen was detected in 51 of 54 with an interpolated LOD cut off between 10(3) and 10(4) RNA IU/mL. Plasma HCV antigenaemia and plasma RNA levels were significantly different in EOTR from non-EOTR patients at 3 days after treatment start and all times thereafter. Positive and negative EOTR predictive values for HCV RNA >2 log drop and HCV Ag loss at 12 weeks were 70% and 74%, 85% and 93% respectively. HCV Ag reactivity has a linear dose response independent of genotype and correlates well with HCV RNA. The failure to clear HCV Ag is as accurate as the failure to clear HCV RNA at twelve weeks into therapy in predicting the likelihood of failure to achieve EOTR. HCV Ag potentially offers a convenient alternative to RNA measurement for defining a futility flag in HCV therapy.

QTL for relative water content in field-grown barley and their stability across Mediterranean environments.

A quantitative genetics approach was developed to identify the genomic regions that control relative water content (RWC) in field-grown barley. The trait was previously demonstrated to be a relevant screening tool of drought-tolerance in cereals, as well as a good indicator of plant water-status. The trait was measured at the heading stage on flag leaves recorded from 167 recombinant inbred lines grown in several Mediterranean sites (Montpellier, France; Meknès, Morocco; Le Kef, Tunisia). The results obtained confirmed that several genomic regions are implicated in the total phenotypic variation of RWC. A total of nine chromosomal regions were identified. One region situated on the long arm of chromosome 6H contains the most-consistent QTL obtained in the present study. This region was previously identified as controlling RWC, as well as leaf osmotic potential under water stress and osmotic adjustment, from an experiment conducted in growth-chamber conditions with the same genetic background. The confirmation of the role of this region in the genetic control of water and turgor status underlined its interest for breeding purposes in the Mediterranean area. In addition, the presence of several dehydrin loci in the same chromosomal area reinforce its interest for genomics analyses to confirm, or not to confirm, the implication of these genes in the variation of RWC.

Normal sexual development of two strains of rat exposed in utero to low doses of bisphenol A.

Pregnant Sprague-Dawley (SD) and Alderley Park (Wistar derived) rats were exposed by gavage during gestation days 6-21 to 20 microg/kg, 100 microg/kg, or 50 mg/kg body weight of BPA with ethinylestradiol (EE; 200 microg/kg) acting as a positive control agent. The sexual development of the derived pups was monitored until termination at postnatal day 90-98. The endpoints evaluated were litter size and weight, anogenital distance at birth, days of vaginal opening, first estrus and prepuce separation, weights of the liver, seminal vesicles, epididimydes, testes, ventral prostate, uterus, vagina, cervix and ovaries, and daily sperm production. Males were terminated at postnatal day 90 and females at postnatal day 98. The only statistically significant effects observed for any dose of BPA were a decrease in daily sperm production and an increase in the age of vaginal opening for the Alderley Park animals at the highest dose evaluated (50 mg/kg). The dose of EE evaluated proved to be maternally toxic in our laboratory, but provided gross evidence of endocrine disruption in the treated dams. These results diverge from those of Chahoud and his colleagues who indicated disturbances to the sexual development of both male and female SD rat pups administered the same 3 doses of BPA. This failure to confirm low dose endocrine effects for BPA is discussed within the context of similar divergent conclusions derived from other assessments of the endocrine toxicity of this agent to rats.

Comparison of the developmental and reproductive toxicity of diethylstilbestrol administered to rats in utero, lactationally, preweaning, or postweaning.

The objective of the study was to determine which period of exposure produces the most marked effects on the reproductive capacity and sexual development of the rat, with particular emphasis on the relative sensitivity of in utero and postnatal exposures. The endocrine active chemical, diethylstilbestrol (DES) was used as an agent known to affect many of the endpoints examined. Hitherto, such comparisons have been made between studies, rather than within a study. Our data will be helpful in the interpretation of future multigenerational assay data. In preliminary studies, DES was shown to be active in the immature rat uterotrophic assay with a lowest detected dose of 0.05 mg DES/kg body weight by sc injection and 10 mg DES/l (1.6 mg DES/kg body weight) by administration in drinking water. A dose of 60 microg DES/l drinking water ( approximately 6.5mg DES/kg body weight/day) was selected for the main study since this represented the midpoint of the drinking water uterotrophic dose response and produced no overt maternal toxicity. The study used 10 groups of concomitantly pregnant animals, including 2 control groups. The first comparison was between the effects of exposure to DES in utero, and exposure from conception to weaning. Another group of animals was exposed to DES in utero and cross-fostered to untreated pregnant females to prevent lactational transfer of DES to pups. Two groups were exposed to DES neonatally, either from birth to postnatal day (PND) 10 (pups thus having only lactational exposure), or from birth until weaning (PND 21; pups thus having both lactational exposure and self-exposure via drinking water). In addition, a dose response study to DES was conducted on animals exposed from weaning to PND 100, when the first phase of the study was terminated. Pups exposed to DES in utero and pups exposed from weaning to PND 100 were bred to assess fertility of the F1 animals and the sexual development of F2 offspring. This last comparison was to determine the extent to which weanling rats could be used in endocrine toxicity studies to assess their potential to show activity in utero. The most sensitive period of exposure for inducing developmental effects in F1 animals was from weaning onwards. The neonatal to weaning period (PND 1-21) was the next most sensitive. Essentially no effects were induced in F1 animals exposed in utero. No effects of any kind were observed in animals only exposed over the early neonatal period of PND 1-10. The mean day of vaginal opening, testes descent, and prepuce separation was only altered in groups where postnatal exposure to DES continued beyond PND 10, or was started at weaning. No changes were observed in anogenital distance or caudal sperm counts. Some changes in organ weights were observed, but the interpretation of these was often confused by concomitant changes in body weight. In general, histopathological examination of tissues yielded no additional information. In breeding studies with animals exposed to DES in utero, or from weaning, reduced litter sizes and marginal advances in the day of vaginal opening were observed in the offspring, together with changes in organ weights. However, no unique sensitivity was noted for exposure in utero. Evaluation of the several exposure periods and the many markers monitored in this study may have individual strengths in individual cases, but when rigorously compared using the reference estrogen DES, many preconceptions regarding their absolute or relative value were not upheld. Further, each of these markers is subject to natural variability, as demonstrated by comparisons made among the 5 separate control groups available in parts of the present study. This variability increases the chance that small changes observed in endocrine toxicity studies employing small group sizes and a single control group, or no concomitant control group, may be artifactual. The most marked effects observed in this study were on the developmental landmarks in the F1 animals induced by exposures after PND 10. Some effects on developmental landmarks and organ weights were observed in F2 animals following exposure either in utero or postweaning. This study therefore does not establish a unique role for exposures in utero or during the early neonatal period.

Replacement of surgical castration by GnRH-inhibition or Leydig cell ablation in the male rat Hershberger antiandrogen assay.

An obstacle to the widespread adoption of the Hershberger antiandrogen assay is the surgical castration procedure required to produce androgen deficiency in the test animals. Here we describe two chemical treatments that produce similar effects to surgical castration. The first is use of ethane dimethane sulphonate (EDS), a specific toxin to the testosterone-producing Leydig cells of the mature testes. The second class of compound is the decapeptide inhibitors of the gonadotrophin-releasing hormone (GnRH), compounds such as Antarelix and Antide. Administration of either EDS or the GnRH inhibitors results in loss of weight of the testes, epididymides, and sex-associated tissues. Co-administration of testosterone to these animals leads to reversal of the induced effects. The basic test protocol for both of these assay modifications is described. Flutamide was used as a representative potent antiandrogen, and DDE as an example of a weakly active antiandrogen. The 5alpha-reductase inhibitor finasteride was used to inhibit the transformation of testosterone to dihydrotestosterone. It is shown that the EDS assay is sensitive to the antiandrogen flutamide, but that it fails to detect the weaker antiandrogen DDE. In contrast, the Antarelix assay performs as well as does the classical castration assay, leading to the detection as antiandrogens of flutamide, DDE, and finasteride. It is concluded that the GnRH inhibition Hershberger assay is more convenient to conduct than the original surgical castration assay, and it involves less stress to the test animals.

Identification and characterization of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from the Gram-positive pathogen Streptococcus pneumoniae.

The UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from a Gram-positive pathogen, Streptococcus pneumoniae, was identified and characterized. The enzyme from S. pneumoniae shows 31% identity with the MurB protein from Escherichia coli, and contains the catalytic residues, substrate-binding residues and FAD-binding motif identified previously in the E. coli protein. The gene was cloned into the pET28a+ expression vector, and the 34.5 kDa protein that it encodes was overexpressed in E. coli strain BL21(DE3) to 30% of total cell protein. The majority of the protein was found to be insoluble. A variety of methods were used to increase the amount of soluble protein to 10%. This was then purified to near homogeneity in a two-step process. The absorption spectrum of the purified protein indicated it to be a flavoprotein, like its E. coli homologue, with a characteristic absorption at 463 nm. The enzyme was shown to be active, reducing UDP-N-acetylglucosamine enolpyruvate with the concomitant oxidation of NADPH, and was characterized kinetically with respect to its two substrates. The enzyme showed properties similar to those of its E. coli counterpart, being activated by univalent cations and being subject to substrate inhibition. The characterization of an important cell wall biosynthesis enzyme from a Gram-positive pathogen provides a good starting point for the discovery of antibacterial agents against MurB.

Two active forms of UDP-N-acetylglucosamine enolpyruvyl transferase in gram-positive bacteria.

Gene sequences encoding the enzymes UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from many bacterial sources were analyzed. It was shown that whereas gram-negative bacteria have only one murA gene, gram-positive bacteria have two distinct genes encoding these enzymes which have possibly arisen from gene duplication. The two murA genes of the gram-positive organism Streptococcus pneumoniae were studied further. Each of the murA genes was individually inactivated by allelic replacement. In each case, the organism was viable despite losing one of its murA genes. However, when attempts were made to construct a double-deletion strain, no mutants were obtained. This indicates that both genes encode active enzymes that can substitute for each other, but that the presence of a MurA function is essential to the organism. The two genes were further cloned and overexpressed, and the enzymes they encode were purified. Both enzymes catalyzed the transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine, confirming they are both active UDP-N-acetylglucosamine enolpyruvyl transferases. The catalytic parameters of the two enzymes were similar, and they were both inhibited by the antibiotic fosfomycin.

A genomic analysis of two-component signal transduction in Streptococcus pneumoniae.

A genomics-based approach was used to identify the entire gene complement of putative two-component signal transduction systems (TCSTSs) in Streptococcus pneumoniae. A total of 14 open reading frames (ORFs) were identified as putative response regulators, 13 of which were adjacent to genes encoding probable histidine kinases. Both the histidine kinase and response regulator proteins were categorized into subfamilies on the basis of phylogeny. Through a systematic programme of mutagenesis, the importance of each novel TCSTS was determined with respect to viability and pathogenicity. One TCSTS was identified that was essential for the growth of S. pneumoniaeThis locus was highly homologous to the yycFG gene pair encoding the essential response regulator/histidine kinase proteins identified in Bacillus subtilis and Staphylococcus aureus. Separate deletions of eight other loci led in each case to a dramatic attenuation of growth in a mouse respiratory tract infection model, suggesting that these signal transduction systems are important for the in vivo adaptation and pathogenesis of S. pneumoniae. The identification of conserved TCSTSs important for both pathogenicity and viability in a Gram-positive pathogen highlights the potential of two-component signal transduction as a multicomponent target for antibacterial drug discovery.

The kinetic mechanism of 5-enolpyruvylshikimate-3-phosphate synthase from a gram-positive pathogen Streptococcus pneumoniae.

The Streptococcus pneumoniae 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is a potential novel antibacterial target. The enzyme catalyzes a reversible transfer of an enolpyruvyl group from phospho(enol)pyruvate (PEP) to shikimate 3-phosphate (S3P) to give EPSP with the release of inorganic phosphate (Pi). Understanding the kinetic mechanism of this enzyme is crucial to the design of novel inhibitors of this enzyme that may have potential as antibacterial agents. Steady-state kinetic studies of product inhibition and inhibition by glyphosate (GLP) have demonstrated diverse inhibition patterns of the enzyme. In the forward reaction, GLP is a competitive inhibitor with respect to PEP, but an uncompetitive inhibitor relative to S3P. Product inhibition shows that EPSP is a competitive inhibitor versus both PEP and S3P, suggesting that the forward reaction follows a random sequential mechanism. In the reverse reaction, GLP is an uncompetitive inhibitor versus EPSP, but a noncompetitive inhibitor versus Pi. This indicates that a non-productive quaternary complex might be formed between the enzyme, EPSP, GLP and Pi. Product inhibition in the reverse reaction has also been investigated. The inhibition patterns of the S. pneumoniae EPSP synthase are not entirely consistent with those of EPSP synthases from other species, indicating that EPSP synthases from different organisms may adopt unique mechanisms to catalyze the same reactions.

Characterization of Streptococcus pneumoniae 5-enolpyruvylshikimate 3-phosphate synthase and its activation by univalent cations.

The aroA gene (Escherichia coli nomenclature) encoding 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the gram-positive pathogen Streptococcus pneumoniae has been identified, cloned and overexpressed in E. coli, and the enzyme purified to homogeneity. It was shown to catalyze a reversible conversion of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to EPSP and inorganic phosphate. Activation by univalent cations was observed in the forward reaction, with NH+4, Rb+ and K+ exerting the greatest effects. Km(PEP) was lowered by increasing [NH+4] and [K+], whereas Km(S3P) rose with increasing [K+], but fell with increasing [NH+4]. Increasing [NH+4] and [K+] resulted in an overall increase in kcat. Glyphosate (GLP) was found to be a competitive inhibitor with PEP, but the potency of inhibition was profoundly affected by [NH+4] and [K+]. For example, increasing [NH+4] and [K+] reduced Ki(GLP versus PEP) up to 600-fold. In the reverse reaction, the enzyme catalysis was less sensitive to univalent cations. Our analysis included univalent cation concentrations comparable with those found in bacterial cells. Therefore, the observed effects of these metal ions are more likely to reflect the physiological behavior of EPSP synthase and also add to our understanding of how to inhibit this enzyme in the host organism. As there is a much evidence to suggest that EPSP synthase is essential for bacterial survival, its discovery in the serious gram-positive pathogen S. pneumoniae and its inhibition by GLP indicate its potential as a broad-spectrum antibacterial target.

The impact of genomics on novel antibacterial targets.

Antibiotic discovery has remained primarily focused on improving versions of existing classes of antibiotics which work on a limited set of bacterial targets. In addition, the characterization of these targets has focused almost entirely on Escherichia coli homologs. The advancing problems associated with resistant pathogens has driven a critical need for the discovery of new classes of antibiotics which will target novel bacterial functions required for viability and pathogenicity. Recent advances in DNA sequencing technology have now made it possible to elucidate the entire genomes of pathogenic bacteria. Comparative analysis of these genome sequences, driven by advancements in the availability of bioinformatic tools, is dramatically increasing our ability to interrogate the spectrum and selectivity of novel antibacterial target areas. In this review, we present an update on the antibiotic target areas of tRNA synthetases, two-component signal transduction systems, peptidoglycan biosynthesis, fatty acid biosynthesis and chorismate biosynthesis. We illustrate how the availability of genomes from a range of clinically important pathogens has enabled valid considerations of the limitations and advantages of particular targets based on their predicted spectrum and selectivity. Furthermore, we demonstrate how genomics is facilitating the characterization of targets from relevant pathogens and how these data, coupled with genomic-based technologies, provide new approaches for drug discovery.

Gastrointestinal tolerability of meloxicam and piroxicam: a double-blind placebo-controlled study.

AIMS: The aim of the study was to compare the effects of meloxicam and piroxicam on the gastroduodenal mucosa in healthy adults. METHODS: Forty-four healthy volunteers were given a 28 day course of either meloxicam 15 mg, piroxicam 20 mg or placebo. Damage to the oesophageal, gastric and duodenal mucosa was assessed, mucosal blood flow (MBF) measured at endoscopy and biopsies taken for prostaglandin content and microscopic assessment of damage before NSAID administration and during days 1, 7 and 28 of continued intake. RESULTS: Maximal macroscopic gastric mucosal damage (median grade+IQR) occurred within 24 h of piroxicam administration, the damage score increasing from 0 to 2.5 (0-3) (P=0.02) at day 1 before falling to 2.0 (0-2) at day 7 and 0 (0-1) at day 28 with resolution of damage observed in six out of the seven subjects who sustained acute injury. No significant macroscopic gastric damage occurred in either of the two other groups although some minor damage was observed in seven subjects taking placebo and five taking meloxicam. There was a trend towards piroxicam causing more acute gastric damage than meloxicam (P=0.06). Baseline antral, body and duodenal MBF were similar in all three groups. No significant changes occurred in any of the groups on any of the visits. There were also no changes in gastric mucosal prostaglandin content in any group. CONCLUSIONS: These observations suggest that meloxicam causes little acute damage to the upper gastrointestinal tract and piroxicam causes some acute gastric injury but such damage resolves in most subjects by 28 days.

Adenomyoepithelioma of the skin: a case report with immunohistochemical and ultrastructural observations.

AIMS: The histological, immunohistochemical and electron microscopic features of a primary adenomyoepithelioma of skin, a rare sweat gland tumour, are reported. METHODS AND RESULTS: The tumour occurred on the back of a 92-year-old woman. It was composed of well-formed tubules lined by epithelial cells surrounded by clear or spindled myoepithelial cells. Immunohistochemically, the epithelial cells exhibited strong cytokeratin (CAM5.2) and weak carcinoembryonic antigen positivity. The myoepithelial cells showed diffuse positivity for smooth muscle actin and focal positivity for S100 protein. Ultrastructurally, the myoepithelial cells contained myofilaments with focal densities and hemidesmosomes. They were limited by well-formed basal lamina. The tumour was associated with a small eccrine spiradenoma. CONCLUSION: We predict that the tumour will behave in a benign fashion. There is no evidence of recurrence or metastasis 28 months later.

Granular cell tumour of the spinal cord: case report.

A granular cell tumour presenting within the spinal cord of a 17-year-old woman is described. This distinctive tumour has a widespread distribution, but has been reported only rarely in the central nervous system. The literature is reviewed.

Recognition of a surface loop of the lipoyl domain underlies substrate channelling in the pyruvate dehydrogenase multienzyme complex.

In the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus, the interaction between the pyruvate decarboxylase (E1p) component and the lipoyl domain of the dihydrolipoyl acetyltransferase (E2) component was investigated using a combination of site-directed mutagenesis and NMR spectroscopy. Residues 11 to 15 (EGIHE) of the lipoyl domain, part of a surface loop close in space to the beta-turn containing the lipoyl-lysine residue (position 42), were deleted or replaced. The mutant domains all retained their three-dimensional structures and ability to become lipoylated, but in the absence of the loop the lipoyl-lysine residue could no longer be reductively acetylated by E1p. A mutation (N40A) in the N- terminal part of the lipoyl-lysine hairpin showed that it is involved in recognition of the domain by E1p but other mutations in the loop (E15A) and close to the lipoyl-lysine hairpin (V44S, V45S and E46A) were without effect. The heteronuclear multiple quantum coherence NMR spectra of 15N-labelled lipoyl domain in the presence and absence of B. stearothermophilus E1p were recorded. Of the 85 amino acid residues in the lipoyl domain, 13 exhibited significant differences in chemical shift. These differences, most of which were associated with residues in the surface loop between positions 8 and 15 and in, or close to, the lipoyl-lysine hairpin, indicate that E1p makes contact with the lipoyl domain in these areas. The combined results of directed mutagenesis and NMR spectroscopy point to the surface loop as a major determinant of the interaction of lipoyl domain with E1p. The specificity of this essential interaction provides the molecular basis of substrate channelling in this, the first committed, step of the enzyme reaction mechanism.

Influence of Helicobacter pylori on gastric mucosal adaptation to naproxen in man.

Our objective was to determine whether H. pylori influences gastric mucosal injury and adaptation caused by naproxen. Twenty-four healthy volunteers, 12 H. pylori-positive and 12 H. pylori-negative, were given a 28-day course of naproxen 500 mg twice a day. They were each gastroscoped to assess gastric mucosal damage and mucosal blood flow before and at 1, 7, and 28 days during treatment. Maximal gastric mucosal damage (median grade + IQR) occurred during the first 24 hr in both groups and was of similar magnitude (H. pylori-positive: 2.5, 2.0-3.0 P < 0.01; H. pylori-negative: 2.0, 1.0-3.0 P < 0.01). This damage was associated with a fall in antral but not corpus mucosal blood flow. With continued NSAID administration, gastric damage resolved confirming adaptation (H. pylori-positive 1.0, 0-2.0, H. pylori-negative: 1.0, 0-1.0) and antral mucosal blood flow returned to baseline in both groups by day 28. These observations suggest that initial gastric mucosal injury is not influenced by H. pylori colonization and adaptation occurs regardless of its presence.

Gastric mucosal adaptation to etodolac and naproxen.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) frequently cause damage to the gastroduodenal mucosa, principally by suppressing mucosal prostaglandin synthesis. However, such acute mucosal injury usually resolves, despite continued NSAID administration, by a process known as adaptation. Newer NSAIDs, such as etodolac, have been developed to minimize effects on prostaglandin synthesis. AIM: To determine whether etodolac causes less acute damage than naproxen, and whether the damage produced resolves with continued NSAID administration. METHODS: Twenty-four healthy volunteers were given a 28-day course of either etodolac 300 mg b.d. or naproxen 500 mg b.d. Gastroduodenal damage was assessed using a modified Lanza scoring system and mucosal blood flow with laser doppler flowmetry at endoscopy before NSAID administration and during days 1, 7 and 28 of continued intake. RESULTS: Maximum gastric damage (median grade and interquartile range, IQR) occurred during the first 24 h of administration, being greater with naproxen (2.0, IQR 1.0-3.0) than etodolac (1.0, IQR 1.0-1.5; P = 0.03). Such damage was associated with a fall in antral blood flow in the naproxen group (mean +/- S.E.M.) from 54.5 +/- 3.4 to 43.8 +/- 3.4 arbitrary units (P = 0.07) and a slight increase in mucosal blood flow in the etodolac group from 43.5 +/- 2.24 to 49.5 +/- 3.6 arbitrary units. With continued intake this damage resolved in all subjects taking etodolac and in eight of 14 subjects on naproxen. Resolution in the naproxen group was associated with a return to normal of antral blood flow. CONCLUSIONS: These observations suggest that etodolac causes less mucosal damage than naproxen and that adaptation occurs to both.

Heteronuclear NMR studies of the interactions of 15N-labeled methionine-specific transfer RNAs with methionyl-tRNA transformylase.

In Escherichia coli the methionylated initiator methionyl-tRNA (tRNAfMet) is formylated on the aminoacyl moiety by the enzyme methionyl-tRNA transformylase. The methionylated elongator methionyl-tRNA (tRNAmMet) is not modified in this way. In order to gain structural information about this specific recognition, solution NMR studies were carried out. To be able to identify changes that were occurring in the tRNA molecule on interaction with the methionyl-tRNA transformylase, the imino protons involved in secondary and tertiary base pairing in the tRNAfMet and tRNAmMet molecules first had to be assigned to specific resonances in the NMR spectra. A combination of 2D NOESY, 2D HMQC, and 3D NOESY--HMQC spectra were used on uniformly 15N-labeled samples. After assignment of the base pairs of the tRNA, the two forms of tRNA were separately mixed with transformylase in a 1:1 molar ratio. The HMQC spectra of both the tRNAmMet and the tRNAfMet showed general broadening, but in the tRNAfMet HMQC spectra a decrease in the intensity of several resonances was also observed. These resonances had been assigned to the acceptor stem of the tRNA, confirming site-directed mutagenesis experiments that it is the acceptor stem of the tRNA which is important in conferring the specificity for the transformylase. The loss of intensity of the acceptor stem resonances suggests that this part of tRNAfMet melts upon binding to the enzyme.