Mycobacterium Phlei Cell Research Articles

Evaluation of Locally Injected Mycobacterium Cell Wall Fraction in Horses with Sarcoids

A reformulation of Mycobacterium cell wall fraction immunotherapeutic can be used to successfully treat sarcoids in horses. Sarcoids are reported to be the most common equine skin tumors with tumor type and location influencing the choice of treatment. Wide surgical excision is curative for many tumors, but may not always be feasible. Previous studies have reported sarcoid regression after injection with mycobacterial cell wall immunotherapeutics. A new formulation of the Mycobacterium phlei cell wall fraction immunostimulant (Immunocidin Equine) was used to treat cutaneous tumors in horses. Equids with skin tumors diagnosed as sarcoids were enrolled in the study. Sarcoids were injected at the initial visit with Immunocidin Equine and subsequently at approximately 2-week intervals. Of 17 cases, nine cases were completely resolved at the end of the study period evaluation or at the time of final follow-up (52.9%). Three cases were reported as improved (smaller), but not resolved (17.6%). Three cases were discontinued from the study as the respective masses were growing larger or not resolving (17.6%). One case (5.8%) with two masses had resolution of one mass, whereas the other tumor had a small regrowth 5 months after the last treatment. One case (5.8%) was lost to follow-up. All cases had mild to moderate swelling of the injection site, and some cases had discharge after the second, third, or fourth injections. No serious systemic side effects or complications were encountered during the study.

Systematic Review of the Therapeutic Efficacy of Bladder-preserving Treatments for Non–muscle-invasive Bladder Cancer Following Intravesical Bacillus Calmette-Guérin

ContextThere is a critical need for effective bladder-sparing therapies for bacillus Calmette-Guérin (BCG)-unresponsive non–muscle-invasive bladder cancer (NMIBC). Owing to the current lack of effective agents that can be used as a control, the US Food and Drug Administration began to accept single-arm trials for patients with carcinoma in situ (CIS), using complete response rate (CRR) and duration of response as the primary endpoints to support marketing applications. Despite the ensuing growth of clinical trials in this space, no consensus exists on a clinically relevant benchmark for CRR. ObjectiveTo elucidate the CRR and recurrence-free rate (RFR) using bladder-sparing agents after BCG failure in order to provide a frame of reference for future clinical trial results. Evidence acquisitionWe performed a systematic review of clinical trials utilizing bladder-sparing therapeutics for NMIBC recurring after intravesical BCG (PROSPERO CRD42019130553). The search was performed in MEDLINE, EMBASE, and Cochrane Library. Relevant studies identified from bibliography search and conference abstracts were searched to complement the systematic review. A total of 42 studies utilizing 24 treatment options and consisting of 2254 patients were included for final analysis. Evidence synthesisMedian CRRs in the treatment of CIS-containing tumors were 26% at 6 mo, 17% at 12 mo, and 8% at 24 mo after treatment. In comparison, median RFRs in the papillary-only studies were 67% at 6 mo, 44% at 12 mo, and 10% at 24 mo. Specifically in the BCG-unresponsive population, 6- and 12-mo CRRs in CIS-containing patients treated with Mycobacterium phlei cell wall–nucleic acid complex were 45% and 27%, respectively, and the median 6-, 12-, and 24-mo disease-free rates in the other studies were 43%, 35%, and 18%, respectively. The median progression-free rate was 91%: 95% in the CIS-containing studies and 89% in studies restricted to papillary-only recurrences. Toxicities of intravesical agents were generally mild, with very few dose limiting toxicities. ConclusionsWe demonstrate that, to date, bladder-sparing therapies achieved modest efficacy in patients with NMIBC after BCG. Results from the current study will serve as a frame of reference for emerging trial results in the BCG-unresponsive space. Patient summaryIn this study, we found that bladder-sparing therapies achieved modest efficacy in patients with non–muscle-invasive bladder cancer after bacillus Calmette-Guérin (BCG). These results will serve to inform future clinical trial results for salvage agents used to treat BCG-unresponsive bladder cancer.

Efficacy of mycobacterium phlei cell wall-nucleic acid complex (MCNA) in BCG-unresponsive patients.

342 Background: Previously, we reported the results of a single-armed, multi-institutional study on the efficacy of MCNA in patients who failed intravesical BCG treatment. Since that publication, a new standardized definition for BCG-unresponsiveness has been established. We re-analyzed the oncologic outcomes following intravesical MCNA in patients classified as BCG-unresponsive according to the new definition. Methods: Patients with recurring high grade NMIBC after intravesical BCG were enrolled. For this analysis, we focused on the patients satisfying the minimum criterion for adequate BCG treatment (5/6 induction and 2/3 maintenance). Treatment course included 6 weekly intravesical instillations of 8mg MCNA followed by 3 weekly instillations at months 3, 6, 12, 18, and 24. Followup assessments included cystoscopy, urine cytology and biopsy. Patients absent of high grade disease confirmed by central review of biopsy were deemed disease-free. Results: Of the 129 patients enrolled, 94 (68 CIS with/without papillary tumors, 26 papillary only tumors) fit the criteria for the new BCG-unresponsive definition. In the group with papillary tumors, DFS measured at months 6, 12, and 24 were 61.2%(38.2-77.8%), 61.2(38.2-77.8%), and 50.1%(27.5-69%). In the CIS-containing group, the corresponding DFS were 44.8%(32.3-56.4%), 26.5%(16.3-37.9%), and 16.6%(8.6-26.9%), respectively. Conclusions: Intravesical MCNA therapy has the potential to offer 26.5% of patients with CIS and 61.2% of patients with papillary tumors a chance to safely preserve their bladder for at least 1 year. The efficacy in BCG-unresponsive patients was higher than that reported with all-comers, suggesting that adequate priming with BCG might be required for further immune-based response.

Efficacy of Mycobacterium Phlei Cell Wall-Nucleic Acid Complex (MCNA) inBCG-Unresponsive Patients.

Background: We have previously reported the results of a prospective multi-institutional study on the efficacy of MCNA in patients who recurred after intravesical BCG treatment [1]. Since that publication, a new standardized definition for BCG-unresponsiveness has been established [2].Objective: We re-analyzed the oncologic outcomes following intravesical MCNA in patients classified as BCG-unresponsive according to the new definition.Methods: For this analysis, we focused on the enrolled patients who satisfied the requirements for BCG Unresponsiveness: i.e. adequate BCG treatment (at least 5/6 induction and 2/3 maintenance instillations) and high grade tumor within 6 months of prior BCG. Treatment course included 6 weekly intravesical instillations of 8 mg MCNA followed by 3 weekly instillations at months 3, 6, 12, 18, and 24. Followup assessments included cystoscopy, urine cytology and biopsy. Patients absent of high grade disease confirmed by central review of biopsy were deemed disease-free.Results: Of the 129 patients enrolled, 94 (68 CIS with/without papillary tumors, 26 papillary only tumors) fit the criteria for the new BCG Unresponsive definition. Overall, disease free survival (DFS) for all BCG unresponsive patients was 48.9% (95% CI 38.0–59.0%) at 6 months, 34.8% (95% CI 24.7–45%) at 1 year and 28.3% (15.7–34.3%) at 2 years post induction. In the group with papillary tumors, DFS measured at months 6, 12, and 24 were: 61.2% (38.2–77.8%), 61.2(38.2–77.8%), and 50.1% (27.5–69%). In the CIS-containing group, the corresponding DFS were: 44.8% (32.3–56.4%), 26.5% (16.3–37.9%), and 16.6% (8.6–26.9%), respectively.Conclusions: For patients who are BCG Unresponsive, MCNA has the potential to render 26.5% of patients with CIS and 61.2% of patients with papillary tumors disease-free for at least 1 year with an intact bladder. The higher efficacy noted in the true BCG-unresponsive cohort than was previously reported with all-comers emphasizes the importance of having clearly defined criteria for clinical trials investigating new intravesical therapies after BCG failure.

Administration of Mycobacterium phlei cell wall-nucleic acid complex in the immediate postoperative period for the treatment of non-muscle-invasive bladder cancer.

This review sought to investigate the safety of intravesical administration of Mycobacterium phlei cell wall-nucleic acid (MCNA) in the immediate postoperative period after biopsy/resection for non-muscle-invasive bladder cancer (NMIBC). Patients with NMIBC who failed bacillus Calmette-Guérin (BCG) therapy and at high risk of recurrence and progression participated in this study. Treatment involved an induction phase of six weeks and maintenance of three weekly instillations every six months for two years. Biopsies were mandatory at six months and resections/biopsies as indicated. Of the 129 patients enrolled, 18 (14%) received one or more instillations of MCNA within 24 hours of an endoscopic procedure for a total of 32 instillations. Fourteen patients (78%) received MCNA in the immediate postoperative period. Two (11%) received treatment the day after surgery, but a second treatment immediately after a transurethral resection of the bladder tumour (TURBT). The remaining two patients received an instillation each the day after surgery. Adverse events (AEs) occurred in 31.3% of those treated immediately after the procedure; they were mild, limited to the lower urinary tract, and not drug-related. Only one patient experienced systemic symptoms of moderate severity. None of the AEs resulted in postponement of treatment. There were no AEs among those receiving MCNA the day after surgery. The dual mechanism of action of MCNA suggests that early treatment would take advantage of its chemotherapeutic (pro-apoptotic) activity. Concerns about early administration due to the presence of live bacteria are circumvented with this sterile preparation. These preliminary results warrant further investigation to confirm the safety of perioperative administration of MCNA.

Mycobacterium phlei cell wall-nucleic acid complex in the treatment of nonmuscle invasive bladder cancer unresponsive to bacillus Calmette-Guerin

ABSTRACTIntroduction Current guidelines recommend cystectomy in patients with high risk NMIBC who fail to respond to BCG. However due to the significant morbidity and mortality of the procedure, many are not candidates or refuse it. No new treatments for this indication have been approved by the US FDA since 1998.Areas covered A cell wall-nucleic acid complex (MCNA) from M. phlei has been investigated for possible application in patients with BCG refractory NMIBC. The development of this biological from the original studies is reviewed, together with the clinical trials leading to a submission to the FDA. Its efficacy and safety are presented together with comparative analysis of alternative treatments, most of which are used off-label. In addition, new combinations of standard therapies are described as well as single agents exhibiting activity against these tumors.Expert opinion MCNA has shown activity against high risk BCG refractory bladder cancer and offers an alternative to current treatments. The clinical experience remains limited and the optimal therapeutic regimen (dose, frequency) have not been firmly established. Patients and clinicians would welcome the introduction of a compound that may delay or prevent the risks and negative impact in quality of life of cystectomy and urinary diversion.

Intravesical immunotherapy in nonmuscle invasive bladder cancer.

Introduction:Nonmuscle invasive urothelial cell carcinoma is the most frequent malignancy of the urinary bladder. The high recurrence rate (up to 80%) and risk of progression (up to 30%) reflect the need for long-term follow-up and sometimes multiple interventions. To reduce the rate of recurrences and tumor progression, intravesical immunotherapy, especially the use of Bacille Calmette-Guerin (BCG), represents the gold standard adjuvant treatment of high-risk nonmuscle invasive bladder cancer (NMIBC). This article reviews the role of BCG therapy and several promising new immunotherapeutic approaches such as mycobacterium phlei cell wall-nucleic acid complex, interleukin-10 (IL-10) antibody, vaccine-based therapy, alpha-emitter therapy, and photodynamic therapy checkpoint inhibitors.Methods:A systematic literature review was performed using the terms (immunotherapy, NMIBC, BCG, and intravesical) using PubMed and Cochrane databases.Results:BCG represents the most common intravesical immunotherapeutic agent for the adjuvant treatment of high-risk NMIBC. Its use is associated with a significant reduction of recurrence and progression. Patients with NMIBC of intermediate and high-risk benefit the most from BCG therapy. To achieve maximal efficacy, an induction therapy followed by a maintenance schedule should be used. Full-dose BCG is recommended to obtain ideal antitumoral activity and there is no evidence of a reduction of side effects in patients treated with a reduced dose. There are multiple new approaches and agents in immunotherapy with potential and promising antineoplastic effects.Conclusions:The beneficial effect of BCG is well documented and established. To reduce the tumor specific mortality, it is essential to follow guideline-based treatment. In patients with BCG-failure, there are new promising alternatives other than BCG but BCG remains the gold standard at this stage.

Sequestration of Reactive Light-Yellow K-4G by Free <i>Mycobacterium phlei</i> Cells and its Extracellular Polysaccharide

Production of extracellular polysaccharide by the culture of Mycobacterium phlei were examined, using molasses wastewater to replace glucose as carbon source and energy source in the culture medium. Results showed that the COD concentration in molasses wastewater favorable for the growth of the Mycobacterium phlei cell was 5000 mg•L-1, and inoculum size of 5%(v/v), 28°C, initial pH 7.0 and shaking speed of 150 r•min-1, under the optimal culture conditions, the highest flocculating activity achieved for Kaolin suspension was 95.5% and 5.0 g cell/L broth was obtained. The Mycobacterium phlei cell was effective in flocculating some soluble reactive dye in aqueous solution, reactive Light-Yellow K-4G with a decolorization efficiency of 93.5%, respectively, using 20 mL of the flocculant in 500 mL of 100 mg•L-1 dye solution.

Therapeutic effects of Mycobacterium phlei cell wall extracts

To investigate the cytokine-stimulating activity of several Mycobacterium phlei cell wall extracts. The bacillus Calmette-Guérin application is the gold standard treatment for some types of superficial bladder carcinoma. However, its usage is limited due to its harmful side effects. Intensive research has been carried out to find less toxic and more potent therapeutic agents for treatment. Researchers focus on M. phlei for this purpose. The cell wall extract of M. phlei is sufficient for antitumoral activity. Materials and methods: In this study, M. phlei cells were cultured and several different cell wall extracts were prepared by physicochemical procedures. Human monocytic cell lines were incubated with these extracts, and tumor necrosis factor-\alpha and interleukin-12 were determined in the samples. Results: Various cell extracts showed tumor necrosis factor-\alpha and interleukin-12 stimulating activity. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) profiles of the samples were also obtained. The MALDI-MS spectra of the samples showed that peptides of 3808, 9207, 11,533, 12,460, and 21,587 Da may be involved in the immunostimulation activity. Conclusion: This study showed that peptides obtained from mycobacteria cell wall extracts may be used to prepare new antitumoral chemicals for the treatment of superficial bladder carcinoma.

Selective adsorption of Mycobacterium Phlei on pyrite and sphalerite

The adsorption of Mycobacterium Phlei cells on the surfaces of pyrite and sphalerite was studied as functions of time and pH. The results indicated that a higher amount of cells adsorbing onto pyrite compared with that onto sphalerite under neutral and alkaline conditions, and it was also observed from photographs of scanning electron micrograph. To gain a better insight into the mechanisms of differential adsorption, the functional groups on cell surfaces and the chemical states of each element on mineral surfaces before and after interaction with bacterial cells were investigated. The results showed that many groups presented on cells surface, such as C–O–H, C–O–C, CO, C–N, N–H and PO. The change in state of each element on pyrite and sphalerite surfaces after interaction with bacterial cells revealed that there were chemical reactions between metal ions and S on mineral surface and atoms like N, O, P, etc. on cell surface, and the shifts in binding energy of each element on pyrite surface is larger than that of sphalerite. Possible mechanisms for selective adsorption of bacterial cells onto pyrite and sphalerite were discussed in the latter part of this paper.

Evaluation of bovine cutaneous delayed-type hypersensitivity (DTH) to various test antigens and a mitogen using several adjuvants

The Bacillus Calmette Guerin (BCG)-induced/purified protein derivative (PPD)-elicited tuberculin skin test is a reliable measure of cell-mediated immune response (CMIR), specifically delayed-type hypersensitivity (DTH); however, its use in livestock may confound diagnosis of Mycobacterium tuberculosis. Therefore, various alternative antigen/adjuvant combinations were evaluated as inducers of DTH that were compared to the BCG/PPD test system with the purpose of finding a skin DTH protocol that does not cross-react with the tuberculin test and allows identification of high and low CMIR responder phenotypes. Specifically, 30 non-lactating cows (five/treatment) were sensitized on day 0 with mycobacteria [BCG, M. tuberculosis or Mycobacterium phlei cell wall extract (MCWE)], and ovalbumin (OVA) emulsified in Freund's complete adjuvant (FCA), non-ulcerative Freund's adjuvant (NUFA), complete NUFA or MCWE. On day 21, cows were injected intradermally with various test antigens including PPD tuberculin, phlein, and OVA. Phosphate buffered saline was included as the negative control and the T-cell mitogen phytohemagglutinin (PHA) was also administered. Double skin-fold thickness was evaluated before and at 6, 24, and 48 h post-injection. Skin biopsies were taken at 24 and 48 h to assess oedema, necrosis, and inflammatory cell infiltration. BCG/PPD and M. phlei/phlein treatments when given with a Freund's adjuvant induced equivalent DTH with peak reactions at 24-48 h after antigen injection. Cows receiving NUFA had fewer injection site granulomas than FCA or CNUFA treatments. The change in skin thickness response to PHA peaked at 6 h. Only cows receiving mycobacteria in NUFA had skin response to OVA, which peaked 6-24 h post-injection. Only sites tested with PPD or phlein had significantly higher lymphocyte infiltration than control, whereas neutrophils were significantly higher at PHA test sites and eosinophils predominated at the PHA test sites. Macrophages were significantly more numerous at the PPD and/or phlein test sites in treatment groups that received killed mycobacteria in a Freund's adjuvant and/or with BCG, and at the PHA test sites in all treatment groups. It was concluded that the M. phlei/phlein system did induce DTH and was similar to the DTH induced by the BCG/PPD system when MCWE was administered with a Freund's adjuvant. Therefore, this protocol is suitable for detecting high/low CMIR responders in research herds. However, cross-reaction to PPD was evident following induction of DTH using M. phlei. Hence, this protocol does not alleviate the problem of artificial induction of DTH cross-reactivity and would not be suitable for commercial herds where tuberculin testing is required.

282: Mycobacterium Phlei Cell Wall Extract for the Treatment of Superficial Bladder Cancer: Final Results of a Phase 2 Trial

282: Mycobacterium Phlei Cell Wall Extract for the Treatment of Superficial Bladder Cancer: Final Results of a Phase 2 Trial

Mycobacterium phlei cell wall complex directly induces apoptosis in human bladder cancer cells.

Intact mycobacteria and mycobacterial cell wall extracts have been shown to inhibit the growth of human and murine bladder cancer. Their mechanism of action is, however, poorly understood. Mycobacterium phlei mycobacterial cell complex (MCC) is a cell wall preparation that has mycobacterial DNA in the form of short oligonucleotides complexed on the cell wall surface. In this study, we have investigated the possibility that MCC has anti-cancer activity that is mediated by two different mechanisms – a direct effect on cancer cell proliferation and viability and an indirect effect mediated by the production of interleukin 12 (IL-12), a cytokine known to possess anti-cancer activity. We have found that, although MCC is a potent inducer of IL-12 and IL-6 synthesis in monocytes and macrophages either in vitro or in vivo, it is unable to induce the synthesis of either IL-12, IL-6 or granulocyte–macrophage colony-stimulating factor (GM-CSF) by the human transitional bladder cancer cell lines HT-1197 and HT-1376. MCC is not directly cytotoxic towards these cancer cells, but induces apoptosis as determined by nuclear DNA fragmentation and by the release of nuclear mitotic apparatus protein. Mycobacterium phlei DNA associated with MCC is responsible for the induction of apoptosis. Our results indicate that MCC directly effects bladder cancer cells by inhibiting cellular proliferation through the induction of apoptosis, and has the potential for an indirect anti-cancer activity by stimulating cancer-infiltrating monocytes/macrophages to synthesize IL-12. © 1999 Cancer Research Campaign

Effective adjuvants for the induction of antigen-specific delayed-type hypersensitivity

Vaccines utilizing poorly immunogenic subunit antigens are dependent upon adjuvants to drive the appropriate T cell responses. In an effort to determine the ability of several adjuvants to promote cell-mediated immunity (CMI), we assessed delayed-type hypersensitivity (DTH) in mice inoculated with heat-killed Listeria monocytogenes (HKLM) vaccines. The vaccines were formulated as oil-in-water emulsions containing one or more of the following bacterial-derived immunostimulators: MPL ® immunostimulant, a monophosphoryl lipid A preparation, synthetic trehalose dicorynomycolate (TDCM) and Mycobacterium phlei cell wall skeleton (CWS). Oil-in-water emulsions containing HKLM without adjuvants did not induce DTH responsiveness in mice. The incorporation of TDCM, or MPL ® plus TDCM and/or CWS to the formulation enabled the HKLM vaccine to stimulate CMI characterized by DTH responsiveness. Following antigen challenge the resulting increases in footpad thickness ranged from 15–20% and were comparable to the DTH driven by complete Freund's adjuvant. Adjuvants composed of MPL/TDCM and MPL/TDCM/CWS induced responses equivalent to those measured in mice immunized with viable L. monocytogenes, and the responses remained at these levels for at least 2 months. Furthermore, in vivo of CD4+ T cells, but not CD8+ T cells, abrogated the induction and expression of DTH, indicating that the response is mediated by CD4+ T cells.

Immunotherapy of Guinea Pigs with a Transplanted Hepatoma: Comparison of Intralesionally Injected Emulsions Containing Heat-Killed <i>Nocardia rubra</i>, <i>Mycobacterium bovis</i> (BCG) and <i>Mycobacterium phlei</i>

Heat-killed cells of Mycobacterium bovis (BCG), Mycobacterium phlei and Nocardia rubra were each tested in emulsified form for their ability to cause regression of established dermal transplants and lymph node metastases of a syngeneic hepatocarcinoma in guinea pigs. On a weight basis, BCG was superior to N. rubra in causing tumor regression. Under the conditions tested N. rubra was inferior to M. phlei in its antitumor activity. M. phlei and BCG were approximately the same in their therapeutic potency. In BCG-sensitized guinea pigs, N. rubra provoked a weaker delayed cutaneous hypersensitivity (DCH) reaction than did BCG. In N. rubra-sensitized guinea pigs, BCG provoked a weaker DCH reaction than did N. rubra. Purified protein derivative of M. tuberculosis was more active in eliciting DCH in BCG-sensitized guinea pigs than in animals sensitized with N. rubra.

Isolation and properties of naphthoate synthetase from Mycobacterium phlei

Cell-free extracts obtained by sonication of Mycobacterium phlei cells contain an important enzyme of the menaquinone (= vitamin K 2) biosynthetic pathway. This enzyme, naphthoate synthetase (1,4-dihydroxy-2-naphthoate synthetase), was partially purified by chromatography on Sepharose 6BCL. Conversion of o-succinylbenzoate to 1,4-dihydroxy-2-naphthoate was followed by a radioactivity assay using o-[2,3- 14C 2]succinylbenzoate, or by a spectrophotofluorometric assay. o-[1- 13C]Succinylbenzoate was converted intact by the extracts to dihydroxynaphthoate containing 13C only in the carboxyl carbon atom. For maximum activity, the enzyme requires ATP, Mg 2+, and coenzyme A. The pH optimum is 6.9 and the molecular weight approximately 44,000. In the presence of farnesyl pyrophosphate, the extracts convert o-[2,3- 14C 2]succinylbenzoate to 14C-containing menaquinone.

Purification and properties of a triacylglycerol lipase from Mycobacterium phlei

In order to study the metabolism of triacylglycerol in mycobacteria, an intracellular particulate triacylglycerol lipase (EC 3.1.1.3) was purified 800-fold from stationary phase cells of Mycobacterium phlei. Extraction of whole cell suspensions with 5% Triton X-100, followed by ion-exchange chromatography of the extract on two successive DEAE-cellulose columns produced a preparation which was nearly homogeneous by the criterion of analytical isoelectric focusing in acrylamide gels (one band, pI. 3.8) and by polyacrylamide gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the preparation into six protein bands. Lipase activity stable to electrophoresis in sodium dodecyl sulfate was extracted from the 40 000 molecular weight region of the gels. With phosphate or maleate buffer the enzyme exhibits a broad pH optimum around 6.0 with sigmoid saturation kinetics (Hill number 2), and an apparent K m of 8.8 mM for tripalmitoylglycerol. Citrate and other carboxylic acids increase the apparent V up to 3-fold with the Hill number approaching 1.0. In a series of p-nitrophenyl esters tested ( C 2– C 18), p-nitrophenylmyristate was hydrolyzed most rapidly. The saturation curve for p-nitrophenylmyristate was sigmoid and unaffected by citrate. The role of this activity in the metabolism of triacylglycerols by Mycobacteria is discussed.

Different binding sites for entry and exit of amino acids in whole cells of Mycobacterium phlei

On the basis of mutual inhibition of uptake with different amino acids in whole cells of Mycobacterium phlei, it was demonstrated that the binding site of proline was different from those of all other amino acids studied. Other groups of amino acids share a common binding site: lysine, histidine, and arginine; valine, leucine, and isoleucine; tryptophan, tyrosine, and phenylalanine; glutamic acid and aspartic acid. The exit and entry processes were studied for proline, glutamine, and glutamic acid. It was observed that in each case the entry and exit processes were mediated by different membrane sites.

Different mechanisms of energy coupling for transport of various amino acids in cells of Mycobacterium phlei.

Whole cells of Mycobacterium phlei were shown to actively accumulate proline, leucine, lysine, tryptophan, histidine, glutamine, and glutamic acid to different steady state levels. The transport of proline, in contrast to that of other amino acids, was found to be insensitive to various respiratory inhibitors, e.g. cyanide, arsenate, azide, and sulfhydryl reagents. However, oxygen was an obligatory requirement for the uptake of proline, as well as for the other amino acids. The results indicate that the energy requirements for proline uptake are different from those of other amino acids. In contrast to the system from Escherichia coli, the mode of energy transduction for the uptake of proline, glutamine, and glutamic acid is different even though these amino acids are shock resistant in the M. phlei system.

Delayed-type skin reactivity of enzyme proteins from mycobacteria.

Five highly purified enzymes (aminoacylases, lactate oxygenase, and fumarases), isolated from the cells of Mycobacterium phlei or Mycobacterium smegmatis, were tested for their delayed-type skin re...