L-Proline Prevents Endoplasmic Reticulum Stress in Microglial Cells Exposed to L-azetidine-2-carboxylic Acid.

L-Azetidine-2-carboxylic acid (AZE) is a non-protein amino acid that shares structural similarities with its proteogenic L-proline amino acid counterpart. For this reason, AZE can be misincorporated in place of L-proline, contributing to AZE toxicity. In previous work, we have shown that AZE induces both polarization and apoptosis in BV2 microglial cells. However, it is still unknown if these detrimental effects involve endoplasmic reticulum (ER) stress and whether L-proline co-administration prevents AZE-induced damage to microglia. Here, we investigated the gene expression of ER stress markers in BV2 microglial cells treated with AZE alone (1000 µM), or co-treated with L-proline (50 µM), for 6 or 24 h. AZE reduced cell viability, nitric oxide (NO) secretion and caused a robust activation of the unfolded protein response (UPR) genes (ATF4, ATF6, ERN1, PERK, XBP1, DDIT3, GADD34). These results were confirmed by immunofluorescence in BV2 and primary microglial cultures. AZE also altered the expression of microglial M1 phenotypic markers (increased IL-6, decreased CD206 and TREM2 expression). These effects were almost completely prevented upon L-proline co-administration. Finally, triple/quadrupole mass spectrometry demonstrated a robust increase in AZE-bound proteins after AZE treatment, which was reduced by 84% upon L-proline co-supplementation. This study identified ER stress as a pathogenic mechanism for AZE-induced microglial activation and death, which is reversed by co-administration of L-proline.

Limonin alleviates macro- and micro-vascular complications of metabolic syndrome in rats: A comparative study with azelnidipine.

BACKGROUND: Hypertension is a serious component of metabolic syndrome (MetS). HYPOTHESIS: This research investigates the potential protective effect of limonin against MetS-associated hypertension in comparison with azelnidipine, a common calcium channel blocker. STUDY DESIGN: MetS was induced in rats by 10% fructose in water and 3% salt in diet over a 16-week period. Limonin (50 mg/kg) and azelnidipine (5 mg/kg) were administered daily in the last four weeks METHODS: Non-invasive blood pressure (BP) was recorded in conscious animals. Concentration-response curves for phenylephrine (PE) and acetylcholine (ACh) were analysed in thoracic aorta (macrovessels) and kidney microvessels. Blood glucose level, serum insulin level, advanced glycation end products (AGEs), tumor necrosis factor-α (TNF-α), malondialdehyde (MDA) and transforming growth factor-ß1 (TGF-ß1) were determined. RESULTS: Limonin alleviated elevations in systolic and diastolic BP associated with MetS similar to levels associated with azelnidipine. Limonin prevented the MetS induced exaggerated macro- and micro-vascular contractility to PE and the impaired dilatation to ACh. However, in vitro incubation with limonin partially alleviated the deteriorated vascular reactivity of aorta isolated from MetS animals or AGEs injured aorta. Limonin did not have direct relaxant effect on the isolated vessel. On the other hand, limonin reduced the elevated serum levels of AGEs, TNF-α and MDA. Limonin suppressed the vascular fibrosis through reducing the elevated serum level of TGF-ß1 and excessive aortic collagen deposition. Limonin decreased the elevated HOMA-IR in MetS animals. CONCLUSION: Limonin offsets the hypertensive and vascular impairment associated with MetS via attenuation of inflammation and fibrosis. Its impact is comparable to that of azelnidipine.

A novel siderophore system is essential for the growth of Pseudomonas aeruginosa in airway mucus.

Pseudomonas aeruginosa establishes airway infections in Cystic Fibrosis patients. Here, we investigate the molecular interactions between P. aeruginosa and airway mucus secretions (AMS) derived from the primary cultures of normal human tracheal epithelial (NHTE) cells. PAO1, a prototype strain of P. aeruginosa, was capable of proliferating during incubation with AMS, while all other tested bacterial species perished. A PAO1 mutant lacking PA4834 gene became susceptible to AMS treatment. The ΔPA4834 mutant was grown in AMS supplemented with 100 µM ferric iron, suggesting that the PA4834 gene product is involved in iron metabolism. Consistently, intracellular iron content was decreased in the mutant, but not in PAO1 after the AMS treatment. Importantly, a PAO1 mutant unable to produce both pyoverdine and pyochelin remained viable, suggesting that these two major siderophore molecules are dispensable for maintaining viability during incubation with AMS. The ΔPA4834 mutant was regrown in AMS amended with 100 µM nicotianamine, a phytosiderophore whose production is predicted to be mediated by the PA4836 gene. Infectivity of the ΔPA4834 mutant was also significantly compromised in vivo. Together, our results identify a genetic element encoding a novel iron acquisition system that plays a previously undiscovered role in P. aeruginosa airway infection.

Nicotianamine is a novel angiotensin-converting enzyme 2 inhibitor in soybean.

Angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase which is highly homologous to angiotensin-converting enzyme (ACE). ACE2 produces vasodilator peptides angiotensin 1-7 from angiotensin II. In the present study, we synthesized various internally quenched fluorogenic (IQF) substrates (fluorophore-Xaa-Pro-quencher) based on the cleavage site of angiotensin II introducing N-terminal fluorophore N-methylanthranilic acid (Nma) and C-terminal quencher N(ε)-2,4- dinitrophenyl-lysine [Lys(Dnp)]. The synthesized mixed substrates "Nma-Xaa-Pro-Lys(Dnp)" were hydrolyzed by recombinant human (rh) ACE2. The amount of each product was determined by liquid chromatography mass spectrometry (LC-MS) with fluorescence detection and it was found that Nma-His-Pro-Lys(Dnp) is the most suitable substrate for rhACE2. The K(m), k(cat), and k(cat)/K(m) values of Nma-His-Pro-Lys(Dnp) on rhACE2 were determined to be 23.3 µM, 167 s(-1), and 7.17 µM(-1) s(-1), respectively. Using the rhACE2 and the newly developed IQF substrate, we found rhACE2 inhibitory activity in soybean and isolated the active compound soybean ACE2 inhibitor (ACE2iSB). The physicochemical data on the isolated ACE2iSB were identical to those of nicotianamine. ACE2iSB strongly inhibited rhACE2 activity with an IC50 value of 84 nM. This is the first demonstration of an ACE2 inhibitor from foodstuffs.

Regression of glomerular and tubulointerstitial injuries by dietary salt reduction with combination therapy of angiotensin II receptor blocker and calcium channel blocker in Dahl salt-sensitive rats.

A growing body of evidence indicates that renal tissue injuries are reversible. We investigated whether dietary salt reduction with the combination therapy of angiotensin II type 1 receptor blocker (ARB) plus calcium channel blocker (CCB) reverses renal tissue injury in Dahl salt-sensitive (DSS) hypertensive rats. DSS rats were fed a high-salt diet (HS; 4% NaCl) for 4 weeks. Then, DSS rats were given one of the following for 10 weeks: HS diet; normal-salt diet (NS; 0.5% NaCl), NS + an ARB (olmesartan, 10 mg/kg/day), NS + a CCB (azelnidipine, 3 mg/kg/day), NS + olmesartan + azelnidipine or NS + hydralazine (50 mg/kg/day). Four weeks of treatment with HS diet induced hypertension, proteinuria, glomerular sclerosis and hypertrophy, glomerular podocyte injury, and tubulointerstitial fibrosis in DSS rats. A continued HS diet progressed hypertension, proteinuria and renal tissue injury, which was associated with inflammatory cell infiltration and increased proinflammatory cytokine mRNA levels, NADPH oxidase activity and NADPH oxidase-dependent superoxide production in the kidney. In contrast, switching to NS halted the progression of hypertension, renal glomerular and tubular injuries. Dietary salt reduction with ARB or with CCB treatment further reduced blood pressure and partially reversed renal tissues injury. Furthermore, dietary salt reduction with the combination of ARB plus CCB elicited a strong recovery from HS-induced renal tissue injury including the attenuation of inflammation and oxidative stress. These data support the hypothesis that dietary salt reduction with combination therapy of an ARB plus CCB restores glomerular and tubulointerstitial injury in DSS rats.

Zn and Fe biofortification: the right chemical environment for human bioavailability.

A considerable fraction of global disease burden and child mortality is attributed to Fe and Zn deficiencies. Biofortification, i.e. the development of plants with more bioavailable Zn and Fe, is widely seen as the most sustainable solution, provided suitable crops can be generated. In a cereal-dominated diet availability of Fe and Zn for absorption by the human gut is generally low and influenced by a highly complex chemistry. This complexity has mostly been attributed to the inhibitory effect of Fe and Zn binding by phytate, the principal phosphorus storage compound in cereal and legume seeds. However, phytate is only part of the answer to the multifaceted bioavailability question, albeit an important one. Recent analyses addressing elemental distribution and micronutrient speciation in seeds strongly suggest the existence of different Fe and Zn pools. Exploration of natural variation in maize showed partial separation of phytate levels and Fe bioavailability. Observations made with transgenic plants engineered for biofortification lend further support to this view. From a series of studies the metal chelator nicotianamine is emerging as a key molecule. Importantly, nicotianamine levels have been found to not only increase the loading of Fe and Zn into grains. Bioavailability assays indicate a strong activity of nicotianamine also as an enhancer of intestinal Fe and Zn absorption.

Sex differences in response to angiotensin II receptor blocker-based therapy in elderly, high-risk, hypertensive Japanese patients: a subanalysis of the OSCAR study.

The OlmeSartan Calcium Antagonists Randomized (OSCAR) study is a multicenter, prospective, randomized, open-label, blinded, end point study of elderly hypertensive Japanese patients that compared the efficacy of a high-dose angiotensin II receptor blocker (ARB) treatment to an ARB plus calcium channel blocker (CCB) combination. In this pre-specified subgroup analysis, we compared the response to such therapy according to sex. A total of 1164 patients (515 (44%) men and 649 (56%) women) were included, and each gender was split into two nearly equal treatment groups. The primary end point was a composite of cardiovascular events and non-cardiovascular death. The baseline characteristics between the two treatment groups in each sex were similar, except for some variables. Male patients had lower systolic and higher diastolic blood pressure than female patients (156.8/85.7 vs. 158.5/84.2 mm Hg). At the end of the study, the mean systolic pressure was higher in the ARB group (134.4 mm Hg) than in the ARB plus CCB group (131.5 mm Hg; P=0.03) for men but not for women (135.4 vs. 133.4 mm Hg; P=0.12). For men, the primary outcome events tended to be higher in the ARB group than in the ARB plus CCB group (hazard ratio (HR)=1.66; P=0.055) but not for women (HR=0.97; P=0.92). This difference in men was due to cardiovascular events (HR=1.86; P=0.03). The interaction between sex and treatment group was not significant (P=0.17). These findings suggest that, in addition to blood pressure control, appropriate patient risk assessment is important for the treatment of hypertension, especially in male patients, as opposed to possible sex differences in treatment effects.

Recycling of methylthioadenosine is essential for normal vascular development and reproduction in Arabidopsis.

5'-Methylthioadenosine (MTA) is the common by-product of polyamine (PA), nicotianamine (NA), and ethylene biosynthesis in Arabidopsis (Arabidopsis thaliana). The methylthiol moiety of MTA is salvaged by 5'-methylthioadenosine nucleosidase (MTN) in a reaction producing methylthioribose (MTR) and adenine. The MTN double mutant, mtn1-1mtn2-1, retains approximately 14% of the MTN enzyme activity present in the wild type and displays a pleiotropic phenotype that includes altered vasculature and impaired fertility. These abnormal traits were associated with increased MTA levels, altered PA profiles, and reduced NA content. Exogenous feeding of PAs partially recovered fertility, whereas NA supplementation improved fertility and also reversed interveinal chlorosis. The analysis of PA synthase crystal structures containing bound MTA suggests that the corresponding enzyme activities are sensitive to available MTA. Mutant plants that expressed either MTN or human methylthioadenosine phosphorylase (which metabolizes MTA without producing MTR) appeared wild type, proving that the abnormal traits of the mutant are due to MTA accumulation rather than reduced MTR. Based on our results, we propose that the key targets affected by increased MTA content are thermospermine synthase activity and spermidine-dependent posttranslational modification of eukaryotic initiation factor 5A.

Functional compounds in fermented buckwheat sprouts.

Fermented buckwheat sprouts (FBS) are used as multifunctional foods. Their production process includes fermentation with lactic acid bacteria. The major strains were found to include Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus pentosus, Lactococcus lactis subsp. lactis, and Pediococcus pentosaceus in an investigation of the lactic acid bacteria. We searched for the functional components, and nicotianamine (NA) and 2″-hydroxynicotianamine (HNA) were identified as angiotensin I-converting enzyme (ACE) inhibitors. NA and HNA increased during fermentation. Indole-3-ethanol was identified as an antioxidant (a SOD active substance), and may have been generated from tryptophan during fermentation because it was not contained in green buckwheat juice. A safety test demonstrated that FBS contained were safe functional food components, showing negative results in buckwheat allergy tests. Any buckwheat allergy substances might have been degraded during the fermentation process.

Studies on the mechanism of antihypertensive action by nicotianamine.

Nicotianamine (NA), which is obtained from vegetables, lowers blood pressure through the renin-angiotensin system, and we clarified that NA preferentially inhibits the activity of angiotensin I-converting enzyme (ACE)-a zinc-containing enzyme. In this study, we elucidated the mechanism of antihypertensive action of NA through the Magnus method by using rat aortic blood vessels. Angiotensin I-induced contractions were inhibited by NA in a concentration-dependant manner. Because NA did not inhibit angiotensin II-induced contractions, it was believed that NA inhibited ACE activity in vascular smooth muscles. NA did not affect KCl-induced contractions, but it affected norepinephrine-induced contractions to a small extent. NA exerted similar effects on endothelium-denuded and endothelium-intact blood vessels. Therefore, the antihypertensive action of NA did not play a role in the opening of voltage-dependent calcium channels, but this effect influenced vasoconstriction by the activation of α-adrenergic receptors. These results suggest that after absorption from the intestinal tract, NA may exert antihypertensive effects via 2 mechanisms: direct inhibition of ACE in vascular smooth muscle and activation of α-adrenergic receptors.

Cardiovascular effects of azelnidipine in comparison with those of amlodipine assessed in the halothane-anaesthetized dog.

Azelnidipine is a new dihydropyridine Ca(2+) channel blocker with long plasma half-life. To understand the in vivo cardiovascular profile of azelnidipine, it was assessed in the halothane-anaesthetized, closed-chest canine model and compared with the effect of amlodipine. We administered azelnidipine in doses of 10, 20 and 70 microg/kg, i.v. or amlodipine in doses of 30, 70 and 200 microg/kg, i.v. cumulatively to the animals. The hypotensive effects of azelnidipine and amlodipine were slow in onset and long-lasted, while their extents of dose-related hypotensive effects were similar. Azelnidipine hardly affected the heart rate or plasma noradrenaline concentration at any doses, whereas the high dose of amlodipine increased these parameters. Azelnidipine as well as amlodipine tended to increase the ventricular contraction, which did not achieve statistical significance. During autonomic receptor blockade with atropine and propranolol, neither drug affected the heart rate, ventricular contraction or plasma noradrenaline concentration, although a more significant hypotensive action was observed. These results indicate that azelnidipine and amlodipine do not directly affect cardiac function. Amlodipine may induce sinus tachycardia via reflex-mediated increase in sympathetic tone. Such lack of reflex tachycardia with azelnidipine will provide potential therapeutic strategy for treatment of patients with cardiovascular diseases, being more beneficial than amlodipine.

In vivo imaging of renal redox status during azelnidipine treatment.

The effect of the calcium channel blocker azelnidipine on the redox status of a murine hypertension model was analyzed and imaged using in vivo low frequency electron paramagnetic resonance (EPR). A murine two kidney-one clip (2K1C) hypertension model was produced by a clipping of the right renal artery. The resulting hypertensive mice were treated with low-dose azelnidipine (1 mg/kg/d), with high-dose azelnidipine (3 mg/kg/d) or without azelnidipine (HT group). An EPR system equipped with a loop-gap resonator and an imaging system was employed. Redox status was evaluated as organ reducing activity measured by means of the decay rate (half-lives) of the spin probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (Carbamoyl-PROXYL). Four weeks after clipping the mice demonstrated hypertension as expected. After the additional 2 weeks of azelnidipine treatments, the Carbamoyl-PROXYL half-lives of the Low and High azelnidipine groups measured in the upper abdominal area were significantly shorter than those of the HT group, suggesting improvements in the reducing activity. The blood pressures of the three groups showed no significant differences at this time, and there was no correlation between the renal reducing activity and either blood pressure or serum creatinine values. EPR imaging studies revealed that the improvement in abdominal reducing activity was mainly recognized in the kidney but not in the liver. These results indicate that azelnidipine ameliorates renal redox status through an improvement in reducing activity independent of blood pressure control.

Azelnidipine attenuates cardiovascular and sympathetic responses to air-jet stress in genetically hypertensive rats.

Azelnidipine is a new dihydropyridine calcium channel blocker that causes minimal stimulation of the sympathetic nervous system despite its significant depressor effect. In the present study, we examined the effects of oral or intravenous administration of azelnidipine on cardiovascular and renal sympathetic nerve activity (RSNA) responses to air-jet stress in conscious, unrestrained stroke-prone spontaneously hypertensive rats. Oral administration of high-dose azelnidipine (10 mg/kg per day) or nicardipine (150 mg/kg per day) for 10 days caused a significant and comparable decrease in blood pressure, but low-dose azelnidipine (3 mg/kg per day) did not. Air-jet stress increased mean arterial pressure (MAP), heart rate (HR) and RSNA. High-dose azelnidipine significantly attenuated the increases in MAP, HR and RSNA in response to air-jet stress while nicardipine did not. Low-dose azelnidipine significantly attenuated the pressor response with a trend of decrease in RSNA. Intravenous injection of azelnidipine induced a slowly developing depressor effect. To obtain a similar time course of decrease in MAP by azelnidipine, nicardipine was continuously infused at adjusted doses. Both drugs increased HR and RSNA significantly, while the change in RSNA was smaller in the azelnidipine group. In addition, intravenous administration of azelnidipine attenuated the responses of MAP, HR, and RSNA to air-jet stress; by comparison, the inhibitory actions of nicardipine were weak. In conclusion, oral or intravenous administration of azelnidipine inhibited cardiovascular and sympathetic responses to air-jet stress. This action of azelnidipine may be mediated at least in part by the inhibition of the sympathetic nervous system.

A third-generation, long-acting, dihydropyridine calcium antagonist, azelnidipine, attenuates stent-associated neointimal formation in non-human primates.

BACKGROUND: Calcium antagonists have been shown to reduce atherogenesis and improve clinical outcomes in atherosclerotic vascular disease. No study has so far, however, addressed the effects of calcium antagonists on stent-associated neointimal formation. We therefore investigated whether a third-generation calcium antagonist, azelnidipine, attenuates in-stent neointimal formation in non-human primates. METHOD: Male cynomolgus monkeys were fed a high cholesterol diet for 4 weeks, and were randomly assigned to three groups: a vehicle group and two other groups treated with azelnidipine at 3 and 10 mg/kg per day for an additional 24 weeks (n = 12 each). Multi-link stents were then implanted in the iliac artery. RESULTS: Azelnidipine at the high dose reduced neointimal thickness (0.25 +/- 0.02 versus 0.19 +/- 0.02 mm; P < 0.05). Azelnidipine also reduced local oxidative stress and monocyte chemoattractant protein 1 (MCP-1) expression. No difference was found between the three groups in the degrees of injury score, inflammation score, plaque neovascularization, or plasma lipid levels. Azelnidipine also reduced MCP-1-induced proliferation/migration of vascular smooth muscle cells in vitro. CONCLUSIONS: This study demonstrated for the first time that azelnidipine attenuates in-stent neointimal formation associated with the reduced expression of MCP-1 and smooth muscle proliferation/migration in the neointima. These data in non-human primates suggest potential clinical benefits of azelnidipine as a 'vasculoprotective calcium antagonist' in patients undergoing vascular interventions.

An angiotensin-I converting enzyme inhibitor from buckwheat (Fagopyrum esculentum Moench) flour.

A compound that inhibited angiotensin-I converting enzyme (ACE) activity was isolated from buckwheat powder. This compound is thought to be the hydroxy derivative of nicotianamine and its chemical structure is 2''-hydroxynicotianamine. This compound showed a very high inhibitory activity toward ACE, and the IC(50) was 0.08 microM. Only this hydroxy analog was found in buckwheat powder, at about 30 mg/100g, and no nicotianamine was detected. However, nicotianamine was detected in the buckwheat plant body. 2''-hydroxynicotianamine was also found in other polygonaceous plants.

Effects of ascorbic acid and ascorbic acid 2-phosphate, a long-acting vitamin C derivative, on the proliferation and differentiation of human osteoblast-like cells.

In order to investigate the effect of ascorbic acid (AsA) and ascorbic acid 2-phosphate (Asc 2-P), a long-acting vitamin C derivative, on the growth and differentiation of human osteoblast-like cells, we supplemented the culture medium of MG-63 cells with various concentrations (0.25 to 1 mM) of these factors. Asc 2-P significantly stimulated nascent cell growth at all concentrations in the presence of fetal bovine serum (FBS). On the other hand, AsA showed a growth repressive effect depending on its concentration, and that of FBS. Asc 2-P also increased expression of osteoblast differentiation markers, such as collagen synthesis and alkaline phosphatase (ALP) activity. These stimulative activities of Asc 2-P were attenuated by inhibitors of collagen synthesis, indicating that these effects were dependent on collagen synthesis. Electron micrographs of the cells showed the formation of a three-dimensional tissue-like structure endowed with a mature extracellular matrix in the presence of Asc 2-P.

Novel function of ascorbic acid as an angiostatic factor.

Endothelial permeability is increased by vascular endothelial cell growth factor and decreased by antioxidants. Whether or not l-ascorbic acid (Asc), which decreases endothelial permeability by stimulating the endothelial barrier function, is anti-angiogenic (angiostatic) remains unknown. We examined the role of Asc on angiogenesis using two assay systems. At first, the potential role of Asc on four steps of angiogenesis was investigated in cultured bovine microvascular endothelial cells. Asc inhibited the formation of vessel-like tubular structures of endothelial cells cultured on Matrigel; however, it did not decrease the activity of plasminogen activator (PA), which creates the space into which vascular vessels extend. Furthermore, even at high concentrations, Asc did not inhibit either the proliferation or migration of endothelial cell cultures. Secondly, whether Asc inhibited in vivo angiogenesis or not was studied on chick chorioallantoic membrane (CAM) during the 4-6 days of embryogenesis when neovascularization is rapid. It also revealed that angiogenesis was dose-dependently inhibited by Asc from 0.5 micro mol/CAM with half-maximal inhibition at 2.5 micro mol/CAM. Because it was previously reported that the endothelial barrier function decreases permeability via the stimulation of collagen synthesis induced by Asc, we treated CAM with the inhibitor of collagen synthesis, l-azetidine 2-carboxylic acid (AzC). This compound partially attenuated the angiostatic function of Asc on CAM. To understand the involvement of an antioxidant activity in the angiostatic function of Asc, we further examined the effect of glutathione (GSH), which is an endogenous antioxidant, on angiogenesis in CAM and endothelial cells. GSH inhibited CAM angiogenesis, as well as the formation of vessel-like tubular structures of endothelial cell cultures on Matrigel. Both Asc and GSH inhibited hydrogen peroxide (H(2)O(2)) induced tubular morphogenesis. These findings suggest that Asc affects angiogenesis through both its antioxidant properties and the stimulation of collagen synthesis. As the angiostatic activity of Asc may be one of the many effects involved in host resistance to the growth or invasiveness of solid cancer, it may be useful as a supplementary therapy in various angiogenic diseases.

An internal survival standard for Salmonella typhimurium forward mutation assays.

A novel S. typhimurium forward mutation assay which avoids plating density artifacts is described. The new method uses a pair of multiply drug-resistant substrains of TM677, a bacterial strain used in previous forward mutation studies. This technique permits the measurement of cell survival following mutagen treatment by plating the culture on specially supplemented plates at the same cell concentration used to measure mutant yield. Thus this method is both technically easier and theoretically superior to our previous method.

Effect of L-azetidine 2-carboxylic acid on growth and proline metabolism in Escherichia coli.

The effects of L-azetidine 2-carboxylic acid on growth and proline metabolism in a proline-requiring auxotroph of Escherichia coli are described. The homologue inhibited growth of the wild type and it, alone, did not substitute effectively for proline as a growth supplement for the mutant. In medium containing 0.05 mM proline, the addition of increasing amounts of homologue progressively inhibited growth of the wild type but stimulated growth of the mutant at homologue: proline ratios of 10 : 1 and 50 : 1. This suggested that the homologue exerted a "sparing effect" on proline in the mutant. The incorporation of L-[U-14C]proline and L-[3H]azetidine 2-carboxylic acid into hot trichloroacetic acid-insoluble material in the mutant was measured. Amino acid analysis of the insoluble material from cells incubated with radiolabeled proline alone revealed that proline was partially degraded and metabolized to other amino acids prior to incorporation into protein. The addition of unlabeled homologue to the incubation medium significantly reduced proline catabolism, suggesting that the homologue exerted a sparing effect on proline in this mutant. In medium containing unlabeled proline and radiolabeled L-azetidine 2-carboxylic acid, the homologue was incorporated both intact and partially degraded prior to incorporation into protein. Alanine was the major L-azetidine 2-carboxylic acid catabolite.