Ron tyrosine kinase receptor synergises with EGFR to confer adverse features in head and neck squamous cell carcinoma.

BACKGROUND: Although EGFR inhibitors have shown some success in the treatment of head and neck squamous cell carcinomas (HNSCCs), the results are not dramatic. Additional molecular targets are urgently needed. We previously showed that the loss of Ron receptor activity significantly slowed squamous tumour growth and progression in a murine model. Based on these data, we hypothesised that Ron expression confers an aggressive phenotype in HNSCCs. We prospectively collected and evaluated 154 snap-frozen, primary HNSCCs for Ron and EGFR expression/phosphorylation. Biomarker correlation with clinical, pathological and outcome data was performed. The biological responses of HNSCC cell lines to Ron knockdown, its activation and the biochemical interaction between Ron and EGFR were examined. RESULTS: We discovered that 64.3% (99 out of 154) HNSCCs expressed Ron. The carcinomas expressed exclusively mature functional Ron, whereas the adjacent nonmalignant epithelium expressed predominantly nonfunctional Ron precursor. There was no significant association between Ron and sex, tumour differentiation, perineural/vascular invasion or staging. However, patients with Ron+HNSCC were significantly older and more likely to have oropharyngeal tumours. Ron+HNSCC also had significantly higher EGFR expression and correlated strongly with phosphorylated EGFR (pEGFR). Newly diagnosed HNSCC with either Ron/pEGFR or both had lower disease-free survival than those without Ron and pEGFR. Knocking down Ron in SCC9 cells significantly blunted their migratory response to not only the Ron ligand, MSP, but also EGF. Stimulation of Ron in SCC9 cells significantly augmented the growth effect of EGF; the synergistic effect of both growth factors in SCC9 cells was dependent on Ron expression. Activated Ron also interacted with and transactivated EGFR. CONCLUSION: Ron synergises with EGFR to confer certain adverse features in HNSCCs.

An experimentally confirmed statistical model on arm movement.

The purpose of this research work was to develop a methodology to model arm movement in normal subjects and neurologically impaired individuals through the application of a statistical modelling method. Thirteen subjects with Parkinson's disease and 29 normal controls were recruited to participate in an arm motor task. An infrared optoelectronic kinematic movement analysis system was employed to record arm movement at 50 times per second. This study identified the modified extended Freundlich model as one that could be used to describe this task. Results showed that this model fit the data well and that it has a good correspondence between the observed and the predicted data. However, verification of the model showed that the residuals contained a sizeable autocorrelation factor. The Cochrane and Orcutt method was applied to remove this factor, which improved the fit of the model. Results showed that Parkinson's disease subjects had a higher autocorrelation coefficient than the normal subjects for this task. A positive correlation (r(s) = 0.72, p < 0.001) was found between the Langton-Hewer stage and the autocorrelation coefficient of PD subjects. This finding suggests that if autocorrelation is positively correlated with disease progression, clinicians in their clinical practice might use the autocorrelation value as a useful indicator to quantify the progression of a subjects' disease. Significant differences in model parameters were seen between normal and Parkinson's disease subjects. The use of such a model to represent and quantify movement patterns provides an important base for future study.

Conjoint IGF-I and insulin infusion shows diverse interactive effects in diabetic rats.

Studies in diabetic rodents and humans provide evidence that IGF-I may alleviate the diabetic state and insulin resistance to some degree. To assess the efficacy of IGFs as an adjunct treatment with insulin in diabetes, we infused IGF-I or des(1-3)IGF-I for 7 days at 0, 10.7, 26.7, and 66.8 nmol/day to streptozotocin-induced diabetic rats in conjunction with infusions of 0, 2.2, 5.6, or 14 nmol/day insulin. Both insulin and des(1-3)IGF-I increased body weight gain by 7 g/day compared with controls (1.2 g/day), but there was no additive effect. However, for nitrogen retention, the effects of des(1-3)IGF-I were additive with those of 2.2 nmol/day insulin. Des(1-3)IGF-I was two- to threefold more potent than IGF-I. At comparable rates of total nitrogen retention, carcass nitrogen retention was approximately 35% higher with insulin than with IGF treatment, indicating a differential tissue response. IGFs did not alter carcass fat content. Des(1-3)IGF-I increased liver glycogen additively with insulin but reduced glucosuria only when given with 5.6 nmol insulin per day, indicating the possibility of a facilitatory effect, perhaps via increased insulin sensitivity. Insulin was 10- to 25-fold more potent in these glucoregulatory actions. Differential effects of the hormones were also observed for kidney, liver, and thymus weights. We conclude that IGFs and especially the more potent des(1-3)IGF-I may have a role as an adjunct to insulin therapy in diabetic patients.

Influence of electrical stimulation of the tibialis anterior muscle in paraplegic subjects. 2. Morphological and histochemical properties.

In adult paraplegic subjects one tibialis anterior muscle received daily electrical stimulation for 4 weeks at twice the motor threshold to determine changes of morphological and histochemical profiles (this paper) and of contractile properties (preceding paper). Bilateral biopsies, obtained 4 weeks before, and immediately after, electrical stimulation, were studied for fibre type proportions, fibre diameters, oxidative capacity, microvasculature and histopathology. Before stimulation the biopsies showed disuse with increased type 2 fibre proportions and decreased oxidative capacity (succinate dehydrogenase (SDH) activity). The effects of two stimulus patterns were compared. Following stimulation SDH activity increased significantly in all stimulated muscles. Inconsistent changes occurred in fibre type proportions, fibre diameters, capillary density and capillary/fibre ratios. Both stimulus patterns evoked similar results. In five/seven subjects subsarcolemmal vacuolation was observed. Electrical stimulation for 4 weeks at only twice motor threshold improves oxidative capacity, but different stimulus parameters are probably needed for significant fibre type conversion.

Influence of electrical stimulation of the tibialis anterior muscle in paraplegic subjects. 1. Contractile properties.

In adult paraplegic subjects one tibialis anterior muscle received daily electrical stimulation for 4 weeks at twice motor threshold to determine changes of its contractile properties (this paper) and its morphological and histochemical profiles (following paper). Force, speed of contraction and fatigue resistance were assessed by percutaneous electrical stimulation of the muscle with torque measured about the ankle. Comparative contractile tests were performed on 51 normal adult subjects and new parameters for force measurement proposed, particularly where maximum voluntary contraction cannot be obtained. In paraplegic subjects before stimulation the tibialis anterior muscle showed evidence of disuse with decreased force, faster contraction and relaxation, and reduced fatigue resistance. The effects of two stimulus patterns were compared: 10 Hz, and 10 Hz with 100 Hz bursts. After stimulation contraction was slower, fatigue resistance increased and there was a tendency for force to increase. No differences occurred using the different stimulus patterns. Four weeks later fatigue resistance was partially maintained, while speed of contraction reverted to pre-stimulus levels.

Effects of insulin and insulin-like growth factors on protein and energy metabolism in tumour-bearing rats.

The effects of insulin-like growth factor-1 (IGF-I), and a more potent variant LR3-IGF-I, which binds poorly to IGF-binding proteins, were investigated in rats bearing a mammary adenocarcinoma. The effect of insulin, either alone or in combination with LR3-IGF-I, was also investigated. Peptides were infused via osmotic minipumps for 6-7 days after tumour size reached 5% of body weight. Infusion of IGFs alone at either 200 or 500 microgram/day significantly decreased food intakes as well as circulating levels of insulin and glucose, and consequently failed to promote muscle protein accretion in the host. Tumour growth was increased by the IGFs, especially by LR3-IGF-I, even though these peptides did not promote growth of the adenocarcinoma in cell culture. Infusion of LR3-IGF-I, and to a lesser extent IGF-I, led to decreased rates of muscle protein synthesis and increased muscle protein breakdown, but each of these measures was closely related to the final tumour burden (r2 = 0.454 and 0.810 respectively; P < 0.01) and possibly resulted from a decrease in substrate supply to the host tissues. Insulin infusion (100 micrograms/day) increased food consumption by more than 50% and significantly decreased tumour growth. Insulin and LR3-IGF-I had a synergistic effect on host weight, which increased by 19.1 +/- 1.9, -1.1 +/- 4.7 and 37.9 +/- 1.5 g for insulin, LR3-IGF-I and combined treatments respectively. Carcass protein was increased by more than 10% with insulin treatment, due to increased rates of synthesis and decreased rates of muscle protein breakdown, but LR3-IGF-I had no positive effect on carcass protein accretion, either alone or in combination with insulin. Similarly, the amount of carcass fat was increased almost 2-fold by insulin treatment, whereas it was decreased by 30% by LR3-IGF-I. These changes may have arisen either from direct hormone effects on metabolism or from the indirect effects of food intake, or both. Our results suggest that IGF administration may exacerbate an insulin insufficiency associated with the tumour-bearing state and further decrease metabolic substrate supply to the host. This can be overcome by co-infusion of insulin.

Anabolic effects of insulin-like growth factor-I (IGF-I) and an IGF-I variant in normal female rats.

Administration of IGF-I over a 14-day period to growing female rats via s.c. implanted osmotic pumps led to an increased body weight gain, an improved N retention and a greater food conversion efficiency. The effects were dose-dependent, with the highest daily dose tested, 278 micrograms/day, producing 18-26% increases in these measurements. LR3IGF-I, a variant of human IGF-I that contains an amino terminal extension peptide as well as glutamate-3 replaced by arginine and exhibits very weak binding to IGF-binding proteins, was substantially more potent than the natural growth factor, in the 44 micrograms/day of this peptide produced similar effects to the high IGF-I dose. Organ weight and carcass composition measurements showed that the two IGF peptides generally maintained body proportions at those existing when the experiment began. Muscle protein synthesis and myofibrillar protein breakdown were both slightly increased by IGF treatment, so that the observed improvement in N retention could not be explained through protein accretion rates calculated from these measures. Infusion of human GH at a dose of 213 micrograms/day did not stimulate body growth. This investigation establishes that IGF peptides stimulate the growth of normal growing animals, with IGF-I variants that bind less well to IGF-binding proteins being more active than IGF-I.

Insulin-like growth factor-I and more potent variants restore growth of diabetic rats without inducing all characteristic insulin effects.

The effects of graded doses of insulin-like growth factor-I (IGF-I) and two variants which bind poorly to IGF-binding proteins were investigated in 160 g streptozotocin-induced diabetic rats. The two variants were the truncated form, des(1-3)IGF-I, and another with arginine at residue 3 and an N-terminal extension, termed LR3-IGF-I. The peptides were infused via mini-osmotic pumps. Reference groups received either vehicle or insulin (30 i.u. per day). Treatment led to a marked dose-dependent increase in growth rate and nitrogen balance. The highest dose (695 micrograms/day) of IGF-I increased body weight by 48.1 +/- 1.7 g/7 days, compared with 11.0 +/- 2.8 g/7 days for the vehicle-treated group. The two variants were 2.5-3 times more potent than IGF-I in restoring growth. The insulin-treated group gained more weight (64.5 +/- 1.6 g/7 days), but the added gain was fat (92.5 +/- 4.8 g of fat/kg carcass wet wt., compared with 32.2 +/- 2.1 for all other groups) rather than protein. All peptides increased muscle protein-synthesis rates and RNA levels by up to 50%, with IGF-I the least potent. These high doses of IGFs did not decrease either the glucosuria or the daily excretion rate of N tau-methyl-histidine (N tau-MH). On the other hand, insulin treatment markedly decreased both glucosuria (from 82.7 +/- 5.4 to 4.5 +/- 3.3 mmol/day) and N tau-MH excretion (from 9.3 +/- 0.3 to 7.1 +/- 0.4 mumol/day per kg). This experiment shows that, although IGF-I and variants can restore growth in diabetic rats, other insulin-dependent metabolic processes in liver, muscle and adipose tissue are not restored.

Growth hormone increases whole-body protein turnover in growing pigs.

Ten pigs with an average initial live weight of 65 kg were used to investigate the effects of daily exogenous porcine pituitary growth hormone (pGH; .1 mg.kg-1.d-1) for a 13-d period on N retention and whole-body protein turnover. Feed intake was restricted to both the control (treated with excipient) and pGH-treated groups to ensure that animals in each group consumed equal amounts. Whole-body protein turnover was estimated from the excretion of 15N in urinary urea and ammonia after a single oral dose of [15N]glycine. Nitrogen balance and whole-body N flux were increased by 35 to 40% with pGH treatment (P less than .001). Protein synthesis and breakdown were increased by 56 and 59% (P less than .001), respectively, in pGH-treated pigs relative to controls. These higher rates of protein turnover seemed to lower slightly the efficiency of the metabolic process for protein deposition. However, the absolute increment in protein synthesis rate was greater than that for breakdown, leading to the increased net N retention. Thus, pGH treatment improved the utilization of dietary amino acids for protein deposition.

Insulin-like growth factor-I (IGF-I) and especially IGF-I variants are anabolic in dexamethasone-treated rats.

The administration of insulin-like growth factor-I (IGF-I) via subcutaneously implanted osmotic pumps partially reversed a catabolic state produced by the co-administration of 20 micrograms of dexamethasone/day to 150 g male rats. Marked dose-dependent effects on body weight and nitrogen retention were produced, with the highest IGF-I dose, 695 micrograms/day, giving a 6 g increase in body weight over 7 days, compared with a 19 g loss in the dexamethasone-only group and an 18 g gain in pair-fed controls. Two IGF-I analogues that bind poorly to IGF-binding proteins, the truncated form, des(1-3)IGF-I, and a variant with an N-terminal extension as well as arginine at residue 3, LR3IGF-I, were approx. 2.5-fold more potent than IGF-I. The response with LR3IGF-I was particularly striking because this peptide binds 3-fold less well than IGF-I to the type 1 IGF receptor. The increased potencies of the IGF-I variants may relate to the substantially increased plasma levels of IGF-binding proteins, particularly IGFBP-3, produced by the combined treatment of dexamethasone with IGF-I or the variants. These binding proteins would be expected to decrease the transfer of IGF-I, but not that of the variants, from blood to tissue sites of action. Measurements of muscle protein synthesis at the end of the treatment period and muscle protein breakdown by 3-methylhistidine (3MH) excretion throughout the experiment indicated coordinate anabolic effects of the IGF peptides on both processes. Thus 3MH excretion was decreased at the highest IGF-I dose from 83.5 +/- 4.2 (S.E.M.) mumol/kg per 7 days to 65.1 +/- 2.2, compared with 54.9 +/- 1.2 in the pair-fed controls. Part of this response in 3MH excretion may have reflected a decrease in gut protein breakdown, because IGF-I and especially the IGF analogues increased the gut weight by up to 45%. Notwithstanding the effects on protein synthesis and breakdown, the fractional carcass weights remained low in the IGF-treated groups, although the increase in total carcass weight reflected nitrogen rather than fat gain. The dexamethasone-induced changes in liver, spleen and heart weight were restored towards normal by the IGF treatment. The experiment demonstrates the potential of IGF-I treatment of catabolic states and especially the value of modified forms of growth factors that bind weakly to IGF-binding proteins.

Increased weight gain, nitrogen retention and muscle protein synthesis following treatment of diabetic rats with insulin-like growth factor (IGF)-I and des(1-3)IGF-I.

We have examined the effects of infusing recombinant human growth hormone (hGH), insulin-like growth factor-I (IGF-I), the truncated IGF-I analogue, des(1-3)IGF-I, and insulin over a 7-day period in streptozotocin-induced diabetic rats. IGF-I at a dose of 1.05 or 1.08 mg/kg per day in two experiments increased body weight and nitrogen retention above those of vehicle-infused controls to about 30% of the improvement achieved with 25 or 30 units of insulin/kg per day, but only in the second experiment were the differences statistically significant (P less than 0.05). A 2.5-fold higher IGF-I dose, or des(1-3)IGF-I at 1.08 mg/kg per day, gave effects that were approx. 70% of those obtained with insulin. hGH at 1.38 mg/kg per day was not effective. The IGF peptides, unlike insulin, did not ameliorate the diabetic glucosuria. The improvements in nitrogen balance could be accounted for in part by increases in muscle protein synthesis. Muscle protein breakdown, as assessed by 3-methylhistidine excretion, was inhibited by insulin, but not by the IGF peptides. Carcass fat increased substantially following insulin administration. This did not occur with the IGF peptides, suggesting that IGF predominantly stimulates the growth of lean tissue. IGF-I concentrations and IGF-I-binding proteins in plasma were increased by IGF-I, especially at the higher dose, whereas hGH produced only a transient increase in IGF-I. Des(1-3)IGF-I induced binding proteins, but had only a slight effect on measured IGF-I concentrations. We conclude that IGF peptides stimulate muscle protein synthesis and improve nitrogen balance in diabetes without obviously influencing the abnormal carbohydrate metabolism. Moreover, des(1-3)IGF-I is at least as potent as the full-length IGF-I.

Effects of full-length and truncated insulin-like growth factor-I on nitrogen balance and muscle protein metabolism in nitrogen-restricted rats.

The ability of insulin-like growth factor-I (IGF-I) to protect against losses of body protein during periods of dietary nitrogen restriction has been evaluated in young rats. Recombinant human IGF-I was administered by osmotic pumps at dose rates of 0, 1.2 or 2.9 mg/kg per day over a 7-day period beginning with the transfer of animals from an 18% to a 4% protein diet. A fourth group received the potent truncated IGF-I analogue, des(1-3)IGF-I, at a dose of 1.2 mg/kg per day over a comparable 7-day period. Plasma IGF-I levels were reduced by 60% following nitrogen restriction, a reduction that was partly prevented by IGF-I administration, especially at the higher dose, but not measurably by des(1-3)IGF-I. The major IGF-binding protein circulating in blood, IGFBP-3, demonstrated a similar pattern of change. A significant (P less than 0.05) protection of body weight was achieved in the low dose IGF-I and des(1-3)IGF-I groups, but only after differences in food intake had been eliminated by analysis of covariance. Nitrogen balances were not significantly different unless analysis of covariance was used to adjust for the nitrogen intakes, whereupon all treatment groups showed improved balance, especially the animals treated with the low IGF-I dose and des(1-3)IGF-I (both P less than 0.01). The rate of muscle protein breakdown calculated from the urinary excretion of 3-methyl-histidine was not significantly altered by the treatments, but fell progressively throughout the 7 days. The fractional rate of muscle protein synthesis measured on the final day was increased by 31,26 and 21% respectively by the low and high doses of IGF-I and by des(1-3)IGF-I. Organ weights (g/kg body weight) showed no effects of IGF-I treatment except for 16% increases in the weight of kidneys in the high dose IGF-I and the des(1-3)IGF-I groups. Carcass analyses demonstrated higher water and lower fat contents (all P less than 0.01) in the same groups. These results suggest that exogenous IGF-I and especially des(1-3)IGF-I can partly protect body protein reserves during nitrogen restriction.

Regulation of the breakdown rates of biotin-containing proteins in Swiss 3T3-L1 cells.

1. Degradation rate constants for individual biotin-labelled proteins were measured in Swiss 3T3-L1 adipocytes that had been incubated with inhibitors of autophagy or of lysosomal proteolysis. 2. Inhibitory effects produced by 10 mM-3-methyladenine and a combination of 5 mM-NH4Cl and leupeptin (50 micrograms/ml) were approximately equal. The inclusion of NH4Cl did not significantly enhance the responses to 3-methyladenine, suggesting that autophagy was already maximally inhibited. 3. The extent of inhibition by 3-methyladenine or by the NH4Cl/leupeptin mixture was similar for the cytosolic enzyme acetyl-CoA carboxylase and for the three mitochondrial carboxylases. This inhibition averaged 50%. The breakdown rate of a more-stable 38 kDa biotin-containing mitochondrial protein was more responsive to the inhibitory agents. These results are best explained by mitochondrial proteolysis occurring via a combination of the degradation of whole mitochondria within autophagic vacuoles, supplemented by the selective intramitochondrial breakdown of more labile proteins. 4. A number of intermediate products in the degradation of biotin-containing proteins were detected. Differences in the patterns of radioactivity between these peptides after incubation of cells in the presence of inhibitors of the breakdown process provided evidence that some peptides were produced before autophagy, others as a result of intralysosomal inhibition, while at least one was associated with intramitochondrial proteolysis.

Multiple biotin-containing proteins in 3T3-L1 cells.

Extracts of 3T3-L1 cells prepared after labelling the monolayer cultures with [3H]biotin contained numerous protein bands that were detected by fluorography of dried SDS/polyacrylamide electrophoresis gels. All labelled proteins in the extracts could be removed by avidin affinity chromatography. The biotin-containing subunits of acetyl-CoA carboxylase, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase, with molecular masses of approx. 220, 120, 75 and 72 kDa respectively, were detected together with minor bands at 100, 85 and 37 kDa that did not appear to be partial degradation products. Additional labelled bands increased in amount during incubation of cell extracts or did not occur in extracts prepared with trichloroacetic acid, 9.5 M-urea or proteolytic inhibitors, and were tentatively classified as partial degradation products. The unknown bands were not removed by incubation of cell monolayers for 24 h, a treatment that gave degradation rate constants of 0.47 day-1 for acetyl-CoA carboxylase and 0.28 day-1 for pyruvate carboxylase. Upon two-dimensional electrophoresis, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase had isoelectric points of 6.4, 7.2 and 6.4 respectively. Several additional discrete spots with isoelectric points below 6.2 were also present. All the unknown biotin-containing proteins banded with intact mitochondria during density-gradient centrifugation. We conclude that several unknown biotin-containing proteins are present in the mitochondria of 3T3-L1 cells, whereas others are partial breakdown products of mitochondrial proteolysis.

Distribution and degradation of biotin-containing carboxylases in human cell lines.

Incubation of cultured cells with [3H]biotin leads to the labelling of acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase and methylcrotonyl-CoA carboxylase. The biotin-containing subunits of the last two enzymes from rat cell lines are not separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, but adequate separation is achieved with the enzymes from human cells. Since incorporated biotin is only released upon complete protein breakdown, the loss of radioactivity from gel slices coinciding with fluorograph bands was used to quantify degradation rates for each protein. In HE(39)L diploid human fibroblasts, the degradation rate constants are 0.55, 0.40, 0.31 and 0.19 day-1 for acetyl-CoA carboxylase, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase respectively. A similar series of rate constants is found for AG2804 transformed fibroblasts. The degradation rate constants are decreased by 31-67% in the presence of 50 micrograms of leupeptin/ml plus 5 mM-NH4Cl. Although the largest percentage effect was noted with the most stable enzyme, propionyl-CoA carboxylase, the absolute change in rate constant produced by the lysosomotropic inhibitors was similar for the three mitochondrial carboxylases, but greater for the cytosolic enzyme acetyl-CoA carboxylase. The heterogeneity in degradation rate constants for the mitochondrial carboxylases indicates that only part of their catabolism can occur via the autophagy-mediated unit destruction of mitochondria. Calculations showed that the autophagy-linked process had degradation rate constants of 0.084 and 0.102 day-1 respectively in HE(39)L and AG2804 cells. It accounted for two-thirds of the catabolic rate of propionyl-CoA carboxylase and a lesser proportion for the other enzymes.

Inhibition of pyruvate carboxylase degradation and total protein breakdown by lysosomotropic agents in 3T3-L1 cells.

1. Exposure to [3H]biotin during the differentiation of 3T3-L1 cells to adipocytes selectively labelled pyruvate carboxylase (EC 6.4.1.1). A subsequent incubation of labelled cells permitted the measurement of the degradation rate constant of this mitochondrial enzyme. 2. In medium without serum, pyruvate carboxylase was degraded with a half-life of 64 h, considerably longer than that found for average cell protein. The long half-life is commensurate with the enzyme being catabolized when whole mitochondria are destroyed. 3. The breakdown of pyruvate carboxylase was inhibited to a greater extent than the breakdown of total cell protein by insulin, NH4Cl and inhibitors of lysosomal proteinases, suggesting that the enzyme is degraded by the autophagic lysosomal system of the cell. 4. The above evidence implies that whole mitochondria are degraded in lysosomes, a conclusion that agrees with earlier electron-microscopic evidence showing mitochondria within autophagic vacuoles. 5. A second degradative pathway must be invoked to account for the breakdown of mitochondrial proteins of short half-life.

Accelerated breakdown of reticulocyte protein formed under conditions of amino acid depletion.

1. Labile protein is formed when rat or rabbit reticulocytes are incubated in medium deficient in individual amino acids, especially histidine, valine or alanine. The fraction of unstable protein is increased to about 35% of the total protein synthesized when the histidinyl-tRNA-charging inhibitor, histidinol, is added to histidine-deficient media. 2. The molecular weights of the labile proteins measured by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of urea are less than haemoglobin and probably represent prematurely terminated haemoglobin chains. 3. Although protein synthesis is always lower under conditions that produce labile protein, inhibition of protein synthesis by fluoride or cycloheximide does not give an effect similar to amino acid depletion. 4. The synthesis of protein in deficient medium does not alter the degradation rate of pre-existing protein in reticulocytes and is thus unrelated to the stringent response in bacteria. 5. We propose that amino acid-deficient medium leads to a decreased charging of the appropriate tRNA, a concomitant decrease in protein synthesis and the degradation of nascent peptides.