A Multifunctional Neutralizing Antibody-Conjugated Nanoparticle Inhibits and Inactivates SARS-CoV-2.

The outbreak of 2019 coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global pandemic. Despite intensive research, the current treatment options show limited curative efficacies. Here the authors report a strategy incorporating neutralizing antibodies conjugated to the surface of a photothermal nanoparticle (NP) to capture and inactivate SARS-CoV-2. The NP is comprised of a semiconducting polymer core and a biocompatible polyethylene glycol surface decorated with high-affinity neutralizing antibodies. The multifunctional NP efficiently captures SARS-CoV-2 pseudovirions and completely blocks viral infection to host cells in vitro through the surface neutralizing antibodies. In addition to virus capture and blocking function, the NP also possesses photothermal function to generate heat following irradiation for inactivation of virus. Importantly, the NPs described herein significantly outperform neutralizing antibodies at treating authentic SARS-CoV-2 infection in vivo. This multifunctional NP provides a flexible platform that can be readily adapted to other SARS-CoV-2 antibodies and extended to novel therapeutic proteins, thus it is expected to provide a broad range of protection against original SARS-CoV-2 and its variants.

Microbe-Derived Butyrate and Its Receptor, Free Fatty Acid Receptor 3, But Not Free Fatty Acid Receptor 2, Mitigate Neointimal Hyperplasia Susceptibility After Arterial Injury.

Background Arterial restenosis after vascular surgery is a common cause of midterm restenosis and treatment failure. Herein, we aim to investigate the role of microbe-derived butyrate, FFAR2 (free fatty acid receptor 2), and FFAR3 (free fatty acid receptor 3) in mitigating neointimal hyperplasia development in remodeling murine arteries after injury. Methods and Results C57BL/6 mice treated with oral vancomycin before unilateral femoral wire injury to deplete gut microbiota had significantly diminished serum and stool butyrate and more neointimal hyperplasia development after arterial injury, which was reversed by concomitant butyrate supplementation. Deficiency of FFAR3 but not FFAR2, both receptors for butyrate, exacerbated neointimal hyperplasia development after injury. FFAR3 deficiency was also associated with delayed recovery of the endothelial layer in vivo. FFAR3 gene expression was observed in multiple peripheral arteries, and expression was increased after arterial injury. Treatment of endothelial but not vascular smooth muscle cells with the pharmacologic FFAR3 agonist 1-methylcyclopropane carboxylate stimulated cellular migration and proliferation in scratch assays. Conclusions Our results support a protective role for butyrate and FFAR3 in the development of neointimal hyperplasia after arterial injury and delineate activation of the butyrate-FFAR3 pathway as a valuable strategy for the prevention and treatment of neointimal hyperplasia.

Development, implementation, and effects of a cancer center's exercise-oncology program.

BACKGROUND: National and international bodies acknowledge the benefit of exercise for people with cancer, yet limited accessibility to related programing remains. Given their involvement in managing the disease, cancer centers can play a central role in delivering exercise-oncology services. The authors developed and implemented a clinically integrated exercise-oncology program at a major cancer center and evaluated its effectiveness and participant experience. METHODS: A hospital-based program with prescribed at-home exercise was developed and accepted referrals over a 42-month period (3.5 years). Implementation was conducted in 2 phases: a pilot phase for women with breast cancer and men with genitourinary cancer and a roll-out phase for all patients with cancer. Enrolled patients were assessed and received an exercise prescription as well as a program manual, resistance bands, and a stability ball from a kinesiologist. Program participation and effectiveness were evaluated up to 48 weeks after the baseline assessment using intention-to-treat analyses. Participants in the roll-out phase were asked to complete a program experience questionnaire at the completion of the 48-week follow-up. RESULTS: In total, 112 participants enrolled in the pilot, and 150 enrolled in the roll-out phase. Program attrition to 48 weeks was 48% and 65% in the pilot and roll-out phases, respectively. In participants who consented to research evaluation of their performance, objective and patient-reported measures of functional capacity improved significantly from baseline in both phases. Participants were highly satisfied with the program. CONCLUSIONS: Despite significant drop-out to program endpoints, our cancer-exercise program demonstrated clinically relevant improvement in functional outcomes and was highly appreciated by participants.

Time-, Sex-, and Dose-Dependent Alterations of the Gut Microbiota by Consumption of Dietary Daikenchuto (TU-100).

Medications or dietary components can affect both the host and the host's gut microbiota. Changes in the microbiota may influence medication efficacy and interactions. Daikenchuto (TU-100), a herbal medication, comprised of ginger, ginseng, and Japanese pepper, is widely used in Japanese traditional Kampo medicine for intestinal motility and postoperative paralytic ileus. We previously showed in mice that consumption of TU-100 for 4 weeks changed the gut microbiota and increased bioavailability of bacterial ginsenoside metabolites. Since TU-100 is prescribed in humans for months to years, we examined the time- and sex-dependent effects of TU-100 on mouse gut microbiota. Oral administration of 1.5% TU-100 for 24 weeks caused more pronounced changes in gut microbiota in female than in male mice. Changes in both sexes largely reverted to baseline upon TU-100 withdrawal. Effects were time and dose dependent. The microbial profiles reverted to baseline within 4 weeks after withdrawal of 0.75% TU-100 but were sustained after withdrawal of 3% TU-100. In summary, dietary TU-100 changed mouse microbiota in a time-, sex-, and dose-dependent manner. These findings may be taken into consideration when determining optimizing dose for conditions of human health and disease with the consideration of differences in composition and response of the human intestinal microbiota.

Dietary antioxidant micronutrients alter mucosal inflammatory risk in a murine model of genetic and microbial susceptibility.

Inflammatory bowel diseases (IBD) are caused by the convergence of microbial, environmental, and genetic factors. Diet significantly alters these interactions by affecting both the host and microbiome. Using a mucosal inflammatory model that resembles the human condition of ileal pouchitis, we investigated the effects of Control (CONT) or Antioxidant (AOX) diet, containing pharmacologically relevant levels of 4 micronutrients, on disease risk in wild-type and IL-10-/- animals following surgical self-filling (SF) ileal blind loop placement. Although no differences were found in body weight change or survival, IL-10-/- CONT animals had significantly larger lymphoid organs compared with IL-10-/- AOX or with WT. SF loops from IL-10-/- CONT loop mucosa demonstrated histological inflammation, characterized by goblet cell depletion, increased mucosal myeloperoxidase (MPO), and elevated IFNγ, TNFα, and IL-17α gene expression, which AOX attenuated. AOX elevated luminal IgA in IL-10-/- animals, but not significantly in WT. In IL-10-/- animals, AOX significantly decreased the percentage of CD4 + T-bet and CD4 + RORγ T-cells compared with CONT, with no changes in CD4 + Foxp3+ Treg cells. 16S rRNA gene sequencing demonstrated AOX increased microbial alpha diversity compared with CONT in both genotypes. Notably, colonizing germ-free IL-10-/- hosts with CONT bacterial communities, but not AOX, recapitulated the inflammatory phenotype. Collectively, these findings highlight that common dietary antioxidant micronutrients reshape the gut microbial community to mitigate intestinal inflammatory profiles in genetically susceptible hosts. Insights into the dietary-immune-microbial nexus may improve understanding for recurrent inflammatory episodes in susceptible patient populations and opportunities for practical therapeutics to restore immune and microbial homeostasis.

Daikenchuto (TU-100) alters murine hepatic and intestinal drug metabolizing enzymes in an in vivo dietary model: effects of gender and withdrawal.

Herbal medicines and natural products used for maintenance of health or treatment of diseases have many biological effects, including altering the pharmacokinetics and metabolism of other medications. Daikenchuto (TU-100), an aqueous extract of ginger, ginseng, and Japanese green pepper fruit, is a commonly prescribed Kampo (Japanese herbal medicine) for postoperative ileus or bloating. The effects of TU-100 on drug metabolism have not been investigated. In this study, we analyzed the effect of TU-100 on expression of key drug-metabolizing enzymes (DMEs) and drug transporters (DTs) in murine liver and gastrointestinal tract using a dietary model. Liver, jejunum, and proximal colon were analyzed for phase I and II DMEs and DT mRNA expression by reverse transcription (RT) first by nonquantitative and followed by quantitative polymerase chain reaction (PCR) and protein expression. Liver, jejunum, and proximal colon expressed some identical but also unique DMEs and DTs. TU-100 increased the greatest changes in cytochrome (Cyp) 2b10 and Cyp3a11 and Mdr1a. Basal and TU-100 stimulated levels of DME and DT expression were gender-dependent, dose-dependent and reversible after cessation of TU-100 supplementation, except for some changes in the intestine. Quantitative Western blot analysis of protein extracts confirmed the quantitative PCR results.

Significant difference in active metabolite levels of ginseng in humans consuming Asian or Western diet: The link with enteric microbiota.

After ingestion of ginseng, the bioavailability of its parent compounds is low and enteric microbiota plays an important role in parent compound biotransformation to their metabolites. Diet type can influence the enteric microbiota profile. When human subjects on different diets ingest ginseng, their different gut microbiota profiles may influence the metabolism of ginseng parent compounds. In this study, the effects of different diet type on gut microbiota metabolism of American ginseng saponins were investigated. We recruited six healthy adults who regularly consumed different diet types. These subjects received 7 days' oral American ginseng, and their biological samples were collected for LC-Q-TOF-MS analysis. We observed significant ginsenoside Rb1 (a major parent compound) and compound K (a major active metabolite) level differences in the samples from the subjects consuming different diets. Subjects on an Asian diet had much higher Rb1 levels but much lower compound K levels compared with those on a Western diet. Since compound K possesses much better cancer chemoprevention potential, our data suggested that consumers on a Western diet should obtain better cancer prevention effects with American ginseng intake compared with those on an Asian diet. Ginseng compound levels could be enhanced or reduced via gut microbiota manipulation for clinical utility.

Daikenchuto (TU-100) Suppresses Tumor Development in the Azoxymethane and APCmin/+ Mouse Models of Experimental Colon Cancer.

Chemopreventative properties of traditional medicines and underlying mechanisms of action are incompletely investigated. This study demonstrates that dietary daikenchuto (TU-100), comprised of ginger, ginseng, and Japanese pepper effectively suppresses intestinal tumor development and progression in the azoxymethane (AOM) and APCmin/+ mouse models. For the AOM model, TU-100 was provided after the first of six biweekly AOM injections. Mice were sacrificed at 30 weeks. APCmin/+ mice were fed diet without or with TU-100 starting at 6 weeks, and sacrificed at 24 weeks. In both models, dietary TU-100 decreased tumor size. In APC min/+ mice, the number of small intestinal tumors was significantly decreased. In the AOM model, both TU-100 and Japanese ginseng decreased colon tumor numbers. Decreased Ki-67 and ß-catenin immunostaining and activation of numerous transduction pathways involved in tumor initiation and progression were observed. EGF receptor expression and stimulation/phosphorylation in vitro were investigated in C2BBe1 cells. TU-100, ginger, and 6-gingerol suppressed EGF receptor induced Akt activation. TU-100 and ginseng and to a lesser extent ginger or 6-gingerol inhibited EGF ERK1/2 activation. TU-100 and some of its components and metabolites of these components inhibit tumor progression in two mouse models of colon cancer by blocking downstream pathways of EGF receptor activation. Copyright © 2016 John Wiley & Sons, Ltd.

Daikenchuto (TU-100) shapes gut microbiota architecture and increases the production of ginsenoside metabolite compound K.

Many pharmaceutical agents not only require microbial metabolism for increased bioavailability and bioactivity, but also have direct effects on gut microbial assemblage and function. We examined the possibility that these actions are not mutually exclusive and may be mutually reinforcing in ways that enhance long-term of these agents. Daikenchuto, TU-100, is a traditional Japanese medicine containing ginseng. Conversion of the ginsenoside Rb1 (Rb1) to bioactive compound K (CK) requires bacterial metabolism. Diet-incorporated TU-100 was administered to mice over a period of several weeks. T-RFLP and 454 pyrosequencing were performed to analyze the time-dependent effects on fecal microbial membership. Fecal microbial capacity to metabolize Rb1 to CK was measured by adding TU-100 or ginseng to stool samples to assess the generation of bioactive metabolites. Levels of metabolized TU-100 components in plasma and in stool samples were measured by LC-MS/MS. Cecal and stool short-chain fatty acids were measured by GC-MS. Dietary administration of TU-100 for 28 days altered the gut microbiota, increasing several bacteria genera including members of Clostridia and Lactococcus lactis. Progressive capacity of microbiota to convert Rb1 to CK was observed over the 28 days administration of dietary TU-100. Concomitantly with these changes, increases in all SCFA were observed in cecal contents and in acetate and butyrate content of the stool. Chronic consumption of dietary TU-100 promotes changes in gut microbiota enhancing metabolic capacity of TU-100 and increased bioavailability. We believe these findings have broad implications in optimizing the efficacy of natural compounds that depend on microbial bioconversion in general.

Determination of American ginseng saponins and their metabolites in human plasma, urine and feces samples by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

American ginseng is a commonly consumed herbal medicine in the United States and other countries. Ginseng saponins are considered to be its active constituents. We have previously demonstrated in an in vitro experiment that human enteric microbiota metabolize ginseng parent compounds into their metabolites. In this study, we analyzed American ginseng saponins and their metabolites in human plasma, urine and feces samples by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). Six healthy male volunteers ingested 1 g of American ginseng twice a day for 7 days. On day 7, biological samples were obtained and pretreated with solid phase extraction. The ginseng constituents and their metabolites were characterized, including 5 ginseng metabolites in plasma, 10 in urine, and 26 in feces. For the plasma, urine and feces samples, the levels of ginsenoside Rb1 (a major parent compound) were 8.6, 56.8 and 57.7 ng/mL, respectively, and the levels of compound K (a major metabolite) were 58.4 ng/mL, 109.8 ng/mL and 10.06 µg/mL, respectively. It suggested that compound K had a remarkably high level in all three samples. Moreover, in human feces, ginsenoside Rk1 and Rg5, Rk3 and Rh4, Rg6 and F4 were detected as the products of dehydration. Further studies are needed to evaluate the pharmacological activities of the identified ginseng metabolites.

TU-100 (Daikenchuto) and ginger ameliorate anti-CD3 antibody induced T cell-mediated murine enteritis: microbe-independent effects involving Akt and NF-κB suppression.

The Japanese traditional medicine daikenchuto (TU-100) has anti-inflammatory activities, but the mechanisms remain incompletely understood. TU-100 includes ginger, ginseng, and Japanese pepper, each component possessing bioactive properties. The effects of TU-100 and individual components were investigated in a model of intestinal T lymphocyte activation using anti-CD3 antibody. To determine contribution of intestinal bacteria, specific pathogen free (SPF) and germ free (GF) mice were used. TU-100 or its components were delivered by diet or by gavage. Anti-CD3 antibody increased jejunal accumulation of fluid, increased TNFα, and induced intestinal epithelial apoptosis in both SPF and GF mice, which was blocked by either TU-100 or ginger, but not by ginseng or Japanese pepper. TU-100 and ginger also blocked anti-CD3-stimulated Akt and NF-κB activation. A co-culture system of colonic Caco2BBE and Jurkat-1 cells was used to examine T-lymphocyte/epithelial cells interactions. Jurkat-1 cells were stimulated with anti-CD3 to produce TNFα that activates epithelial cell NF-κB. TU-100 and ginger blocked anti-CD3 antibody activation of Akt in Jurkat cells, decreasing their TNFα production. Additionally, TU-100 and ginger alone blocked direct TNFα stimulation of Caco2BBE cells and decreased activation of caspase-3 and polyADP ribose. The present studies demonstrate a new anti-inflammatory action of TU-100 that is microbe-independent and due to its ginger component.

Composition of dietary fat source shapes gut microbiota architecture and alters host inflammatory mediators in mouse adipose tissue.

BACKGROUND: Growing evidence shows that dietary factors can dramatically alter the gut microbiome in ways that contribute to metabolic disturbance and progression of obesity. In this regard, mesenteric adipose tissue has been implicated in mediating these processes through the elaboration of proinflammatory adipokines. In this study, we examined the relationship of these events by determining the effects of dietary fat content and source on gut microbiota, as well as the effects on adipokine profiles of mesenteric and peripheral adipocytes. METHODS: Adult male C57Bl/6 mice were fed milk fat-based, lard-based (saturated fatty acid sources), or safflower oil (polyunsaturated fatty acid)-based high-fat diets for 4 weeks. Body mass and food consumption were measured. Stool 16S ribosomal RNA (rRNA) was isolated and analyzed via terminal restriction fragment length polymorphism as well as variable V3-4 sequence tags via next-generation sequencing. Mesenteric and gonadal adipose samples were analyzed for both lipogenic and inflammatory mediators via quantitative real-time polymerase chain reaction. RESULTS: High-fat feedings caused more weight gain with concomitant increases in caloric consumption relative to low-fat diets. In addition, each of the high-fat diets induced dramatic and specific 16S rRNA phylogenic profiles that were associated with different inflammatory and lipogenic mediator profiles of mesenteric and gonadal fat depots. CONCLUSIONS: Our findings support the notion that dietary fat composition can both reshape the gut microbiota and alter host adipose tissue inflammatory/lipogenic profiles. They also demonstrate the interdependency of dietary fat source, commensal gut microbiota, and inflammatory profile of mesenteric fat that can collectively affect the host metabolic state.

Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10-/- mice.

The composite human microbiome of Western populations has probably changed over the past century, brought on by new environmental triggers that often have a negative impact on human health. Here we show that consumption of a diet high in saturated (milk-derived) fat, but not polyunsaturated (safflower oil) fat, changes the conditions for microbial assemblage and promotes the expansion of a low-abundance, sulphite-reducing pathobiont, Bilophila wadsworthia. This was associated with a pro-inflammatory T helper type 1 (T(H)1) immune response and increased incidence of colitis in genetically susceptible Il10(−/−), but not wild-type mice. These effects are mediated by milk-derived-fat-promoted taurine conjugation of hepatic bile acids, which increases the availability of organic sulphur used by sulphite-reducing microorganisms like B. wadsworthia. When mice were fed a low-fat diet supplemented with taurocholic acid, but not with glycocholic acid, for example, a bloom of B. wadsworthia and development of colitis were observed in Il10(−/−) mice. Together these data show that dietary fats, by promoting changes in host bile acid composition, can markedly alter conditions for gut microbial assemblage, resulting in dysbiosis that can perturb immune homeostasis. The data provide a plausible mechanistic basis by which Western-type diets high in certain saturated fats might increase the prevalence of complex immune-mediated diseases like inflammatory bowel disease in genetically susceptible hosts.

Ultra-performance liquid chromatography and time-of-flight mass spectrometry analysis of ginsenoside metabolites in human plasma.

American ginseng is a commonly used herbal medicine in the United States. When ginseng is taken orally, its active components, ginsenosides, are reportedly biotransformed by intestinal microbiota. Previous pharmacokinetic evaluations of ginseng in humans have focused on its parent constituents. However, the metabolites, especially those transformed by intestinal microbiota, have not been carefully studied. We used an ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOF-MS) method to determine 15 ginsenosides and/or metabolites and their bioavailability in humans. Six healthy human subjects received a single oral dose of 10 g of American ginseng root powder, after which samples of their blood were collected at 0, 2, 4, 7, 9 and 12 h for measurement of ginsenoside/metabolite levels in plasma. Ginsenosides Rb1, Rd, Rg2 and compound K (C-K) were detected in human plasma samples at different time points. The Rb1 concentration peak was 19.90 ± 5.43 ng/ml at 4 h. C-K was detected from 7 h to 12 h with 7.32 ± 1.35 ng/ml at 12 h. Since the last time point was at 12 h, C-K peak level was not observed. The areas under the concentration curves (AUC) from 0 to 12 h were 155.0 ± 19.5 ng⋅h/ml for Rb1 and 26.4 ± 6.4 ng⋅h/ml for C-K, respectively. The gradual decrease of Rb1 levels and the delayed increase in levels of C-K observed in human subjects supported previous reports that enteric microbiota played a key role in transforming Rb1 to C-K.

American ginseng suppresses Western diet-promoted tumorigenesis in model of inflammation-associated colon cancer: role of EGFR.

BACKGROUND: Western diets increase colon cancer risk. Epidemiological evidence and experimental studies suggest that ginseng can inhibit colon cancer development. In this study we asked if ginseng could inhibit Western diet (20% fat) promoted colonic tumorigenesis and if compound K, a microbial metabolite of ginseng could suppress colon cancer xenograft growth. METHODS: Mice were initiated with azoxymethane (AOM) and, two weeks later fed a Western diet (WD, 20% fat) alone, or WD supplemented with 250-ppm ginseng. After 1 wk, mice received 2.5% dextran sulfate sodium (DSS) for 5 days and were sacrificed 12 wks after AOM. Tumors were harvested and cell proliferation measured by Ki67 staining and apoptosis by TUNEL assay. Levels of EGF-related signaling molecules and apoptosis regulators were determined by Western blotting. Anti-tumor effects of intraperitoneal compound K were examined using a tumor xenograft model and compound K absorption measured following oral ginseng gavage by UPLC-mass spectrometry. Effects of dietary ginseng on microbial diversity were measured by analysis of bacterial 16S rRNA. RESULTS: Ginseng significantly inhibited colonic inflammation and tumorigenesis and concomitantly reduced proliferation and increased apoptosis. The EGFR cascade was up-regulated in colonic tumors and ginseng significantly reduced EGFR and ErbB2 activation and Cox-2 expression. Dietary ginseng altered colonic microbial diversity, and bacterial suppression with metronidazole reduced serum compound K following ginseng gavage. Furthermore, compound K significantly inhibited tumor xenograft growth. CONCLUSIONS: Ginseng inhibited colonic inflammation and tumorigenesis promoted by Western diet. We speculate that the ginseng metabolite compound K contributes to the chemopreventive effects of this agent in colonic tumorigenesis.

Polyamines mediate glutamine-dependent induction of the intestinal epithelial heat shock response.

Heat shock proteins (Hsps) are highly conserved proteins that play a role in cytoprotection and maintaining intestinal homeostasis. Glutamine is essential for the optimal induction of intestinal epithelial Hsp expression, but its mechanisms of action are incompletely understood. Glutamine is a substrate for polyamine synthesis and stimulates the activity of ornithine decarboxylase (ODC), a key enzyme for polyamine synthesis, in intestinal epithelial cells. Thus we investigated whether polyamines (putrescine, spermidine, or spermine) and their precursor ornithine mediate the induction of Hsp expression in IEC-18 rat intestinal epithelial cells. As previously observed, glutamine was required for heat stress induction of Hsp70 and Hsp25, although it had little effect under basal conditions. Under conditions of glutamine depletion, supplementation of ornithine or polyamines restored the heat-induced expression of Hsp70 and Hsp25. When ODC was inhibited by α-difluoromethylornithine (DFMO), an irreversible ODC inhibitor, the heat stress induction of Hsp70 and Hsp25 was decreased significantly, even in the presence of glutamine. Ornithine, polyamines, and DFMO did not modify the nuclear localization of heat shock transcription factor 1 (HSF-1). However, DFMO dramatically reduced glutamine-dependent HSF-1 binding to an oligonucleotide with heat shock elements (HSE), which was increased by glutamine. In addition, exogenous polyamines recovered the DNA-binding activity. These results indicate that polyamines play a critical role in the glutamine-dependent induction of the intestinal epithelial heat shock response through facilitation of HSF-1 binding to HSE.

Heat induction of heat shock protein 25 requires cellular glutamine in intestinal epithelial cells.

Glutamine is considered a nonessential amino acid; however, it becomes conditionally essential during critical illness when consumption exceeds production. Glutamine may modulate the heat shock/stress response, an important adaptive cellular response for survival. Glutamine increases heat induction of heat shock protein (Hsp) 25 in both intestinal epithelial cells (IEC-18) and mesenchymal NIH/3T3 cells, an effect that is neither glucose nor serum dependent. Neither arginine, histidine, proline, leucine, asparagine, nor tyrosine acts as physiological substitutes for glutamine for heat induction of Hsp25. The lack of effect of these amino acids was not caused by deficient transport, although some amino acids, including glutamate (a major direct metabolite of glutamine), were transported poorly by IEC-18 cells. Glutamate uptake could be augmented in a concentration- and time-dependent manner by increasing either media concentration and/or duration of exposure. Under these conditions, glutamate promoted heat induction of Hsp25, albeit not as efficiently as glutamine. Further evidence for the role of glutamine conversion to glutamate was obtained with the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine (DON), which inhibited the effect of glutamine on heat-induced Hsp25. DON inhibited phosphate-dependent glutaminase by 75% after 3 h, decreasing cell glutamate. Increased glutamine/glutamate conversion to glutathione was not involved, since the glutathione synthesis inhibitor, buthionine sulfoximine, did not block glutamine's effect on heat induction of Hsp25. A large drop in ATP levels did not appear to account for the diminished Hsp25 induction during glutamine deficiency. In summary, glutamine is an important amino acid, and its requirement for heat-induced Hsp25 supports a role for glutamine supplementation to optimize cellular responses to pathophysiological stress.