<Emphasis Type="Italic">In vitro</Emphasis> assessment of immunomodulating activity of the two <Emphasis Type="Italic">Lactobacillus</Emphasis> strains isolated from traditional fermented milk

The objective of this study was to evaluate the in vitro immunomodulating capacity of Lactobacillus coryniformis subsp torquens T3L (L. coryniformis T3L) isolated from traditional fermented yak’s milk in Tibet, China, and Lactobacillus paracasei supsp. paracasei M5L (L. paracasei M5L)isolated from kumiss in Sinkiang, China was used as control. The effects of live bacteria, cell wall and genomic DNA of the two Lactobacillus strains on human peripheral blood mononuclear cells (PBMCs) proliferation, production of interleukin-12 (IL-12 p70), interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), and natural killer (NK) cell activity were assessed. The live bacteria, cell wall and genomic DNA of the two lactobacilli exerted proliferative effects on PBMCs. Live bacteria at 1 × 10⁶ c.f.u. ml⁻¹, cell wall at 20 μg protein ml⁻¹ and DNA at 50 μg DNA ml⁻¹ of the strainS induced the secretion of IL-12 (p70), IFN-γ and TNF-α by PBMCs. NK cell activities increased after cultivation of PBMCs with live bacteria, cell wall and DNA of the strains. Overall, these results demonstrate that the live bacteria, cell wall and genomic DNA of the two Lactobacillus strains exhibit immunomodulating activity.     

Immunogenicity of tumor peptides: importance of peptide length and stability of peptide/MHC class II complex

Nonameric P815AB, a cytotoxic-T-lymphocyte-defined minimal core peptide encoded by the murine mastocytoma gene P1A, fails to initiate CD4⁺ cell-dependent reactivity in vivo to class-I-restricted epitopes when mice are administered peptide-pulsed dendritic cells. Effective immunization requires T helper effects, such as those mediated by coimmunization with class-II-restricted (helper) peptides or by the use of recombinant interleukin-12 (rIL-12). Although P815AB does possess class-II-restricted epitopes, they are likely suboptimal, resulting in poor affinity and/or stability of MHC/P815AB complexes and inadequate activation of the antigen-presenting cell function of dendritic cells. The present study has examined a series of longer, P815AB-centered peptides (11–14 amino acids in length, all P1A-encoded) for their ability to initiate CD4⁺ and CD8⁺ cell-mediated responses to the nonamer in vivo, their ability to bind class II MHC in vitro, and their ability to assemble class II molecules stably. By means of a class-I-restricted skin test assay in mice receiving peptide-pulsed dendritic cells, we found that a 12-mer and a 13-mer effectively immunized against the core P815AB peptide, and that this correlated with IL-2 production in vitro by CD4⁺ cells in response to the nonamer. In vitro studies, involving affinity-purified class II molecules, showed that the capacity to assemble class II molecules stably, more than the affinity for class II MHC, correlated with the ability of the different P815AB peptides to prime the host to the core peptide seen by the T cells. Received: 25 February 1999 / Accepted: 14 April 1999     

Antitumor effect of heat-killed Lactobacillus plantarum L-137 through restoration of impaired interleukin-12 production in tumor-bearing mice

 We have previously reported that heat-killed Lactobacillus plantarum L-137 is a potent inducer of interleukin-12 (IL-12) in vivo as well as in vitro in mice. In order to develop effective usage of L. plantarum L-137 for tumor immunotherapy, we examined its antitumor effect against DBA/2 mice inoculated with syngenic P388D1 tumor cells in different treatment schedules. Daily injection of L. plantarum L-137 from the day of tumor inoculation induced a steep increase in plasma IL-12 only after the first injection but not after subsequent injections, and had no effect on tumor growth and survival time. In contrast, daily injection of L. plantarum L-137 from the 7th day after tumor inoculation exerted a marked antitumor effect but such an effect was not evident in mice treated with L. plantarum L-137 twice a week from the 7th day. IL-12 production was considerably impaired at the first injection but steeply increased after the third injection in the mice injected daily with L. plantarum L-137 from the 7th day. Our results suggest that daily administration of L. plantarum L-137 is required to exert an antitumor effect at the late stages of tumor development when IL-12 production is considerably impaired. Received: 15 July 1999 / Accepted: 28 January 2000     

Immunostimulatory properties of phosphorothioate CpG DNA containing both 3'-5'- and 2'-5'-internucleotide linkages

Synthetic oligodeoxyribonucleotides containing CpG-dinucleotides (CpG DNA) in specific sequence contexts activate the vertebrate immune system. We have examined the effect of 3′-deoxy-2′–5′-ribonucleoside (3′-deoxynucleoside) incorporation into CpG DNA on the immunostimulatory activity. Incorporation of 3′-deoxynucleosides results in the formation of 2′–5′-internucleotide linkages in an otherwise 3′–5′-linked CpG DNA. In studies, both in vitro and in vivo, CpG DNA containing unnatural 3′-deoxynucleoside either within the CpG-dinucleotide or adjacent to the CpG-dinucleotide failed to induce immunostimulatory activity, suggesting that the modification was not recognized by the receptors. Incorporation of the same modification distal to the CpG-dinucleotide in the 5′-flanking sequence potentiated the immunostimulatory activity of the CpG DNA. The same modification when incorporated in the 3′-flanking sequence had an insignificant effect on immunostimulatory activity of CpG DNA. Interestingly, substitution of a 3′-deoxynucleoside in the 5′-flanking sequence distal to the CpG-dinucleotide resulted in increased IL-6 and IL-10 secretion with similar levels of IL-12 compared with parent CpG DNA. The incorporation of the same modification in the 3′-flanking sequence resulted in lower IL-6 and IL-10 secretion with similar levels of IL-12 compared with parent CpG DNA. These results suggest that site-specific incorporation of 3′-deoxynucleotides in CpG DNA modulates immunostimulatory properties.     

Isolation and characterisation of cell wall polysaccharides from cocoa (Theobroma cacao L.) beans

 Cell wall material (CWM) was prepared from sun-dried cocoa (Theobroma cacao L.) bean cotyledons before and after fermentation. The monosaccharide composition of the CWM was identical for unfermented and fermented beans. Polysaccharides of the CWM were solubilised by sequential extraction with 0.05 M trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), 0.05 M Na₂CO₃, and 1 M, 4 M and 8 M KOH. The non-cellulosic sugar composition for each fraction was similar for unfermented and fermented samples, indicating that fermentation caused no significant modification of the structural features of individual cell wall polysaccharides. Pectic polysaccharides accounted for 60% of the cell wall polysaccharides but only small amounts could be solubilised in solutions of CDTA, Na₂CO₃, and 1 M and 4 M KOH. The bulk of the pectic polysaccharides were solubilised in 8 M KOH and were characterised by a rhamnogalacturonan backbone heavily substituted with side-chains of 5-linked arabinose and 4-linked galactose. Linkage analysis indicated the presence of additional acidic polysaccharides, including a xylogalacturonan and a glucuronoxylan. Cellulose, xyloglucan and a galactoglucomannan accounted for 28%, 8% and 3% of the cell wall polysaccharides, respectively. It is concluded that the types and structural features of cell wall polysaccharides in cocoa beans resemble those found in the parenchymatous tissue of many fruits and vegetables rather than those reported for many seed storage polysaccharides. Received: 29 May 1999 / Accepted: 19 October 1999     

A polysaccharide carrier to effectively deliver native phosphodiester CpG DNA to antigen-presenting cells

Oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNAs) activate the vertebrate innate immune system via toll-like receptor 9 (TLR-9). Although CpG DNA is a promising immunotherapeutic agent, its short circulation time in biological fluids due to nuclease is the major drawback. This paper proposes that a natural polysaccharide called schizophyllan (SPG) can be used as an effective CpG DNA carrier because SPG can complex with CpG DNA and the resultant complex shows the nuclease resistance of the bound DNA. In order to increase cellular uptake in vitro, we chemically attached spermine, cholesterol, arginine octamer, or RGD peptide to SPG. The complexes made of the chemically modified SPG and CpG DNA having a phosphorothioate (PS) or phosphodiester (PO) backbone led to increased secretion of cytokines of about 4- to 15-fold, compared with the uncomplexed dose. Furthermore, when PO CpG DNA was complexed with unmodified SPG, the IL-12 level increased by almost 3- to 11-fold compared with the naked dose. The PO CpG DNA/unmodified SPG complex data suggested that unmodified SPG might effectively deliver PO in vivo due to the electrically neutral nature of unmodified SPG. When the complexed CpG DNAs were injected intraperitoneally, a large amount of IL-12 production was observed compared with the uncomplexed material. Both in vivo and vitro assays indicated that the SPG complex may be of use for CpG DNA therapy.     

Synergistic antitumor effects of interleukin-12 gene transfer and systemic administration of interleukin-18 in a mouse bladder cancer model

We introduced the interleukin-12 (IL-12) gene into the mouse bladder cancer cell line (MBT2) to establish sublines that secrete bioactive IL-12. IL-12-secreting MBT2 (MBT2/IL-12) sublines were completely rejected when subcutaneously implanted into immunocompetent syngeneic C3H mice. Although this antitumor effect did not change when IL-12-secreting cells were injected into immunodeficient mice whose CD8⁺ T or CD4⁺ T cells had been depleted by the corresponding antibody, it was abrogated when natural killer cells were depleted by anti-asialoGM1 antibody. In addition, when parental MBT2 cells mixed with MBT2/IL-12 cells were subcutaneously injected into mice, admixed MBT2/IL-12 inhibited the growth of the parental tumor. Furthermore, this antitumor effect was enhanced by systemic IL-18 administration. This synergism was abrogated when the mice were treated with interferon-γ-neutralizing antibody in vivo. In conclusion, local secretion of IL-12 led to effective antitumor activity that was enhanced by systemic administration of IL-18. Interferon-γ plays an important role in the synergism of IL-12 gene transduction and systemic administration of IL-18. Received: 7 May 1998 / Accepted: 27 May 1999     

Haplotypes of IL12B promoter polymorphisms condition susceptibility to severe malaria and functional changes in cytokine levels in Thai adults

Polymorphic variability in immune response genes, such as IL12B, encoding the IL-12p40 subunit is associated with susceptibility to severe malaria in African populations. Since the role of genetic variation in conditioning severe malaria in Thai adults is largely unexplored, the functional association between IL12B polymorphisms [i.e. IL12Bpro (rs17860508) and IL12B 3′ UTR T/G (rs3212227)], severe malaria and cytokine production was examined in patients with Plasmodium falciparum infections (n = 355) recruited from malaria endemic areas along the Thai–Myanmar border in northwest Thailand. Circulating IL-12p40 (p = 0.049) and IFN-γ (p = 0.051) were elevated in patients with severe malaria, while only IL-12p40 was significantly higher in severe malaria patients with hyperparasitaemia (p = 0.046). Carriage of the IL12Bpro1.1 genotype was associated with enhanced severity of malaria (OR, 2.34; 95% CI, 0.94–5.81; p = 0.066) and hyperparasitaemia (OR, 3.42; 95% CI, 1.17–9.87; p = 0.025) relative to the IL12Bpro2.2 genotype (wild type). Individuals with the IL12Bpro1.1 genotype also had the lowest IL-12p40 (p = 0.002) and the highest IFN-γ (p = 0.004) levels. Construction of haplotypes revealed that carriage of the IL12Bpro-2/3′ UTR-T haplotype was associated with protection against severe malaria (OR, 0.51; 95% CI, 0.29–0.90; p = 0.020) and reduced circulating IFN-γ (p = 0.06). Thus, genotypic and haplotypic variation at IL12Bpro and IL12B 3′ UTR in this population influences susceptibility to severe malaria and functional changes in circulating IL-12p40 and IFN-γ levels. Results presented here suggest that protection against severe malaria in Thai adults is associated with genotypic variants that condition enhanced IL-12p40 and reduced IFN-γ levels.     

Effects of dietary addition of heat-killed Mycobacterium phlei on growth performance,immune status and anti-oxidative capacity in early weaned piglets

The contradiction between high susceptibility of early weaned piglets to enteric pathogens and rigid restriction of antibiotic use in the diet is still prominent in the livestock production industry. To address this issue, the study was designed to replace dietary antibiotics partly or completely by an immunostimulant, namely heat-killed Mycobacterium phlei (M. phlei). Piglets (n = 192) were randomly assigned to one of the four groups: (1) basal diet (Group A), (2) basal diet + a mixture of antibiotics (80 mg/kg diet, Group B), (3) basal diet + a mixture of antibiotics (same as in Group B, but 40 mg/kg diet) + heat-killed M. phlei (1.5 g/kg diet) (Group C) and (4) basal diet + heat-killed M. phlei (3 g/kg diet) (Group D). All piglets received the respective diets from days 21 to 51 of age and were weaned at the age of 28 d. Compared with the Control (Group A), in all other groups the average daily gain, average daily feed intake, small intestinal villus height:crypt depth ratio and protein levels of occludin and ZO-1 in the jejunal mucosa were increased. A decreased incidence of diarrhoea in conjunction with an increased sIgA concentration in the intestinal mucosa and serum IL-12 and IFN-γ concentrations was found in groups supplemented with heat-killed M. phlei (Groups C and D), but not in Group B. Groups C and D also showed decreased IL-2 concentrations in the intestinal mucosa with lower TLR4 and phosphor-IκB protein levels. The antioxidant capacity was reinforced in Groups C and D, as evidenced by the reduction in malondialdehyde and enhanced activities of antioxidant enzymes in serum. These data indicate that heat-killed M. phlei is a promising alternative to antibiotic use for early weaned piglets via induction of protective immune responses.     

Mycobacteria activate γδ T-cell anti-tumour responses via cytokines from type 1 myeloid dendritic cells: a mechanism of action for cancer immunotherapy

Attenuated and heat-killed mycobacteria display demonstrable activity against cancer in the clinic; however, the induced immune response is poorly characterised and potential biomarkers of response ill-defined. We investigated whether three mycobacterial preparations currently used in the clinic (BCG and heat-killed Mycobacterium vaccae and Mycobacterium obuense) can stimulate anti-tumour effector responses in human γδ T-cells. γδ T-cell responses were characterised by measuring cytokine production, expression of granzyme B and cytotoxicity against tumour target cells. Results show that γδ T-cells are activated by these mycobacterial preparations, as indicated by upregulation of activation marker expression and proliferation. Activated γδ T-cells display enhanced effector responses, as shown by upregulated granzyme B expression, production of the T_H1 cytokines IFN-γ and TNF-α, and enhanced degranulation in response to susceptible and zoledronic acid-treated resistant tumour cells. Moreover, γδ T-cell activation is induced by IL-12, IL-1β and TNF-α from circulating type 1 myeloid dendritic cells (DCs), but not from type 2 myeloid DCs or plasmacytoid DCs. Taken together, we show that BCG, M. vaccae and M. obuense induce γδ T-cell anti-tumour effector responses indirectly via a specific subset of circulating DCs and suggest a mechanism for the potential immunotherapeutic effects of BCG, M. vaccae and M. obuense in cancer.     

The influence of bisnaphthalimidopropyl polyamines on DNA instability and repair in Caco-2 colon epithelial cells

Bisnaphthalimido compounds bis-intercalate to DNA via the major groove and are potentially potent cancer therapeutics. Previously, we incorporated natural polyamines as linkers connecting the two naphthalimido ring moieties to create a series of soluble bisnaphthalimidopropyl polyamines (BNIPPs). Here, extending earlier work on bisnaphthalimidopropylspermidine (BNIPSpd)-induced apoptosis in colon adenocarcinoma Caco-2 cells, we compare the cytotoxicity and genotoxicity of BNIPSpd relative to the spermine and oxaspermine derivatives, bisnaphthalimidopropylspermine (BNIPSpm) and bisnaphthalimidopropyloxaspermine (BNIPOSpm). The order of cytotoxicity after 24 h was BNIPSpd (IC₅₀ = 0.47 μM) > BNIPSpm (IC₅₀ = 10.04 μM) > BNIPOSpm (IC₅₀ >50 μM). After a 72-h BNIPOSpm exposure, an IC₅₀ = 10.25 μM was achieved. With 4-h exposure to BNIPSpd or BNIPSpm or 12-h exposure to BNIPOSpm, concentrations ≥1 μM induced a significant dose-dependent increase in DNA damage as measured by alkaline single-cell gel electrophoresis. The longer incubation times required for BNIPOSpm to induce DNA strand breaks reflect a slower rate of BNIPOSpm cellular distribution as monitored via BNIPP fluorescence within the cells. Moreover, exposure to a non-genotoxic concentration of BNIPSpd, BNIPSpm (0.1 μM for 4 h) or BNIPOSpm (0.1 μM for 12 h) induced a significant decrease in repair of oxidative DNA damage induced by hydrogen peroxide. In conclusion, BNIPP exposure in Caco-2 cells is associated with significant induction of DNA damage and inhibition of DNA repair at non-genotoxic concentrations. The latter is a novel consequence of BNIPP–cell interactions which adds to the spectrum of therapeutically relevant activities that may be exploited for the design and development of naphthalimide-based therapeutics.     

Tumor-derived Fas ligand induces toxicity in lymphoid organs and plays an important role in successful chemotherapy

 Recent studies have suggested that Fas ligand (FasL⁺) tumor cells can induce apoptosis in Fas⁺ T cells. However, the effect of growth of FasL⁺ tumors in vivo, on lymphoid tissues of the host is not clear and therefore was the subject of this investigation. Injection of FasL⁺ LSA tumor caused a significant decrease in cellularity of the thymus and spleen, resulting from marked apoptosis, in syngeneic C57BL/6+/+ (wild-type) but not C57BL/6-lpr/lpr (Fas-deficient) mice. The tumor-induced toxicity resulted from tumor-derived rather than host-derived FasL, inasmuch as LSA tumor growth in C57BL/6-gld/gld (FasL-defective) mice, induced marked apoptosis and toxicity in the thymus and spleen. The LSA tumor growth induced a significant decrease in the percentage of CD4⁺CD8⁺ T cells in the thymus of C57BL/6+/+ mice and an increase in the percentage of CD4⁺, CD8⁺ and CD4⁻CD8⁻ T cells. Of the four subpopulations tested, the CD4⁺CD8⁺ T cells showed maximum apoptosis. The LSA (FasL⁺) but not P815(FasL⁻) tumor cell lysates and culture supernatants induced marked apoptosis in Fas⁺ thymocytes, when tested both in vitro and in vivo. The LSA-tumor-induced apoptosis in vitro was inhibited by antibodies against FasL or by caspase and other inhibitors of apoptosis. Chemotherapy of LSA-tumor-bearing C57BL/6+/+ mice at advanced stages of tumor growth failed to cure the mice, whereas, more than 80% of LSA-tumor-bearing C57BL/6-lpr/lpr mice, similarly treated, survived. Together, the current study demonstrates that FasL produced by LSA tumor cells is functional in vivo and can cause severe toxicity in lymphoid organs of the host. Also, Fas/FasL interactions may play an important role in the successful chemotherapy of FasL-bearing tumor. Received: 31 August 1999 / Accepted: 12 November 1999     

SNP/haplotype associations in cytokine and cytokine receptor genes and immunity to rubella vaccine

An effective immune response to vaccination is, in part, a complex interaction of alleles of multiple genes regulating cytokine networks. We conducted a genotyping study of Th1/Th2/inflammatory cytokines/cytokine receptors in healthy children (n = 738, 11–19 years) to determine associations between individual single-nucleotide polymorphisms (SNPs)/haplotypes and immune outcomes after two doses of rubella vaccine. SNPs (n = 501) were selected using the ldSelect-approach and genotyped using Illumina GoldenGate™ and TaqMan assays. Rubella-IgG levels were measured by immunoassay and secreted cytokines by ELISA. Linear regression and post hoc haplotype analyses were used to determine associations between single SNPs/haplotypes and immune outcomes. Increased carriage of minor alleles for the promoter SNPs (rs2844482 and rs2857708) of the TNFA gene were associated with dose-related increases in rubella antibodies. IL-6 secretion was co-directionally associated (p ≤ 0.01) with five intronic SNPs in the TNFRSF1B gene in an allele dose-related manner, while five promoter/intronic SNPs in the IL12B gene were associated with variations in IL-6 secretion. TNFA haplotype AAACGGGGC (t-statistic = 3.32) and IL12B promoter haplotype TAG (t-statistic = 2.66) were associated with higher levels of (p ≤ 0.01) rubella-IgG and IL-6 secretion, respectively. We identified individual SNPs/haplotypes in TNFA/TNFRSF1B and IL12B genes that appear to modulate immunity to rubella vaccination. Identification of such “genetic fingerprints” may predict the outcome of vaccine response and inform new vaccine strategies.     

CK2 phosphorylates TNFAIP1 to affect its subcellular localization and interaction with PCNA

TNFAIP1 is a protein which can be induced by tumor necrosis factorα (TNFα) and interleukin-6 (IL-6), it may play roles in DNA synthesis, DNA repair, cell apoptosis and human diseases. However, very little has been known about how TNFAIP1 acts in these physiological processes. In this paper, CK2β was identified as a partner of TNFAIP1 by screening the HeLa cDNA library in yeast two-hybrid system with TNFAIP1 as a bait. Furthermore, it was demonstrated that CK2 could phosphorylate TNFAIP1 in vitro and in vivo, which facilitated the distribution of TNFAIP1 in nucleus and enhanced its interaction with PCNA. It is suggested that the phosphorylation of TNFAIP1 may be required for its functions.     

3D-QSAR studies on caspase-mediated apoptosis activity of phenolic analogues

Phenols and its analogues are known to induce caspase-mediated apoptosis activity and cytotoxicity on various cancer cell lines. In the current work, two types of molecular field analysis techniques were used to perform the three dimension quantitative structure activity relationship (3D-QSAR) modeling between structural characters and anticancer activity of two sets of phenolic compounds, which are comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). Then two 3D-QSAR models for two sets of phenolic analogues were obtained with good results. The first QSAR model, which was derived from CoMFA for phenols with caspase-mediated apoptosis activity against L1210 cells, had good predictability (q ² = 0.874, r ² = 0.930), and the other one was derived from CoMSIA for electron-attracting phenols with cytotoxicity in L1210 cell (q ² = 0.836, r ² = 0.950). In addition, the CoMFA and CoMSIA contour maps provide valuable guidance for designing highly active phenolic compounds.     

Inhibition of phosphatidylcholine synthesis is associated with excitotoxic cell death in cerebellar granule cell cultures

Summary.  Glucose deprivation (GD) enhances the sensitivity of cerebellar granule cells to die by excitotoxicity. Neither 70 min of GD, a treatment that depletes cell energy resources, nor exposure to 20 μM glutamate (GLU) for 30 min, induce significant cell death in cultures of cerebellar granule cells. However, the combined treatment with GLU and GD induces choline (Cho) release before excitotoxic cell death. We investigated whether the neurotoxic effect of this treatment is related with inhibition of phosphatidylcholine (PC) synthesis. We found that exposure to GLU for 30 min, to GD for 70 min, and to the combination of both, inhibited PC synthesis at the end of treament by 71%, 92% and 91%, respectively. The inhibition of PC synthesis was accompanied by a decrease in the incorporation of [³H]Cho into phosphocholine and by an increase of the intracellular content of free [³H]Cho, indicating that these treatments inhibit the synthesis of PC by inhibiting choline kinase activity. However, only the combined treatment with GLU and GD induced a prolonged inhibition of PC synthesis that extented after the end of treatment. These results show that excitotoxic death is associated with sustained inhibition of PC synthesis and suggest that this effect of the combined treatment with GLU and GD on PC synthesis is produced by an action on an enzymatic step downstream of choline kinase activity. Received June 29, 2001 Accepted August 6, 2001 Published online June 3, 2002     

Identification of a cation-specific channel (TipA) in the cell wall of the gram-positive mycolata Tsukamurella inchonensis: the gene of the channel-forming protein is identical to mspA of Mycobacterium smegmatis and mppA of Mycobacterium phlei

Detergent extracts of whole cells of the Gram-positive bacterium Tsukamurella inchonensis ATCC 700082, which belongs to the mycolata, were studied for the presence of ion-permeable channels using lipid bilayer experiments. One channel with a conductance of about 4.5 nS in 1 M KCl was identified in the extracts. The channel-forming protein was purified to homogeneity by preparative SDS-PAGE. The protein responsible for channel-forming activity had an apparent molecular mass of about 33 kDa as judged by SDS-PAGE. Interestingly, the protein showed cross-reactivity with polyclonal antibodies raised against a polypeptide derived from MspA of Mycobacterium smegmatis similarly as the cell wall channel of Mycobacterium phlei. Primers derived from mspA were used to clone and sequence the gene of the cell wall channels of T. inchonensis (named tipA for T. inchonensis porin A) and M. phlei (named mppA for M. phlei porin A). Surprisingly, both genes, tipA and mppA, were found to be identical to mspA of M. smegmatis, indicating that the genomes of T. inchonensis, M. phlei and M. smegmatis contain the same genes for the major cell wall channel. RT-PCR revealed that tipA is transcribed in T. inchonensis and mppA in M. phlei. The results suggest that despite a certain distance between the three organisms, their genomes contain the same gene coding for the major cell wall channel, with a molecular mass of 22 kDa for the monomer.     

Physicochemical Characterization of Tween 80-Hydrolyzing Esterases Produced by Rapidly Growing Mycobacteria

Tween 80-hydrolyzing esterases produced by various species of rapidly growing mycobacteria were partially purified from sonicated cell lysates by diethylaminoethyl (DEAE) cellulose and subsequent Sephadex G-150 column chromatographies. The amount of the esterase produced per gram of bacterial cells varied markedly with each species. Mycobacterium smegmatis, M. chelonei, and M. phlei were high producers and M. chitae and M. diernhoferi were low producers of Tween-hydrolyzing esterase. The resistance of each mycobacterial strain to oleic acid correlated well with their esterase-producing ability. All the esterases studied were adsorbed on DEAE cellulose in 50 mM Tris-HCl buffer (pH 7.5), indicating that they are acidic proteins. Esterases of M. smegmatis, M. chitae, M. fortuitum, and M. phlei were eluted from DEAE at high concentrations (0.11–0.18 m) of ammonium sulfate, while those of M. parafortuitum and M. diernhoferi were eluted at lower concentrations (0.05–0.08 m). With Sephadex G-150 gel filtration, all esterases were shown to have similar molecular weights (36,000 to 58,000). On the basis of heat-stability and trypsin- or chymotrypsin-sensitivity, these esterases were divided into three groups: one was heat-stable and protease-sensitive (M. smegmatis and M. fotuitum), another was heat-labile and protease-resistant (M. chelonei), and the other was the intermediate of the above two groups (M. diernhoferi).