High Lytic Activity Research Articles

Isolation of activity and partial characterization of large non-proteinaceous lytic allelochemicals produced by the marine dinoflagellate Alexandrium tamarense

Certain strains of the toxigenic dinoflagellate Alexandrium tamarense produce potent allelochemicals with lytic activity against a wide variety of marine microorganisms. Our efforts to characterize these allelochemicals from a lytic strain focused on the less polar components because of their higher lytic activity. Fractionation and partial purification after solid phase extraction (SPE) were achieved via alternative chromatographic methods, namely HPLC separation on C8 and HILIC phases. Through MALDI-TOF mass spectrometry we compared the mass differences in SPE, C8 HPLC, and HILIC fractions between a lytic and non-lytic strain of A. tamarense. Several large species with masses between 7 kDa and 15 kDa were found in the HILIC lytic fraction by MALDI-TOF MS. Tryptic digestion and tryptic digestion-coupled size-exclusion chromatography (SEC) suggested that the lytic compounds are large non-proteinaceous molecules (<23.3 kDa, trypsin). Although there is no direct proof that the large molecules found in the lytic HILIC fraction are responsible for the lytic activity of this fraction, the mass range deduced from SEC strongly supports this hypothesis. Total sugar content analysis showed that the lytic HILIC fraction contained two-fold more sugar than the non-lytic one. Nevertheless, the low percentage of saccharide per dry mass equivalent (0.18 ± 0.01%) indicates that sugar residues are likely not a major component of the lytic compounds. We concluded that at least one group of lytic allelochemicals produced by A. tamarense comprise a suite of large non-proteinaceous and probably non-polysaccharide compounds between 7 kDa and 15 kDa.

Bacteriophage phi297, a new species of Pseudomonas aeruginosa temperate phages with a mosaic genome: Potential use in phage therapy

The genome of halo-forming temperate Pseudomonas aeruginosa phage phi297 and lytic activity of its virulent mutant were studied. A mosaic structure was revealed for phi297 genome by its complete sequencing. The phi297 genome was partly homologous to the genomes of phages D3 and F116. High lytic activity was assumed for temperate P. aeruginosa bacteriophage phi297 on the basis of morphological features of negative colonies. Virulent mutant phi297vir, which was capable of lysing the wild-type phage bacteria, was isolated. Lytic activity was compared for phi297 and the phages from commercial mixtures of two manufacturers (facilities of Nizhnii Novgorod and Perm’). Phage phi297 caused lysis of the mutant PAO1 bacteria that were resistant to the phages from commercial preparations, but the lytic activity spectrum of phi297 was narrower that the spectra of the commercial phages. The use of nonreverting virulent mutants of certain temperate bacteriophages was proposed for the treatment of P. aeruginosa infections.

INTERACTION OF VIRAL AND BACTERIAL LYSOZYMES WITH ERWINIA AMYLOVORA AND THEIR INHIBITION BY A BACTERIAL PROTEIN

Viral lysozymes can interact with Erwinia amylovora, and their genes may control fire blight when expressed in the pathogen. We have investigated lysozymes from E. amylovora phages and from genomes of several Erwinia species. With chloroform-treated cells as substrate, the highest lytic activity was found for viral lysozymes. Fusion proteins with the (phi)Ea1h lysozyme, such as addition of a His-tag reduced lysis efficiency significantly. For large fusions at the N- or C-terminus lytic activity was completely lost. When a protein fused to glutathione S-transferase was cleaved off with a specific protease, the activity of the viral lysozyme was restored. Intact lysozyme from E. amylovora phage ΦEa1h had a similar lytic activity as E. coli phage T4 lysozyme. Viable cells of E. amylovora were not inhibited by external addition of lysozyme from sonicated cell extracts in assays with (phi)Ea1h lysozyme and T4 lysozyme. The cellular expression of a viral lysozyme gene can be toxic for a bacterial host. The gene was therefore expressed in yeast as safe environment for its secretion. Lysozyme-specific antibodies detected the protein in culture supernatants of transgenic yeast. When E. amylovora was transformed with a viral lysozyme gene, the cells were killed after induction by IPTG. Transfer of a lyz-gene into E. amylovora can thus strongly reduce the viability of the pathogen. The (phi)Ea1h lysozyme was inhibited by a cellular protein from E. amylovora related to an inhibitor for vertebrate lysozymes of E. coli. The lytic activity of lysozyme from phage (phi)Ea104 and (phi)Ea116 was not affected by the enzyme inhibitor.

Sensitivity of Treponema denticola isolated from infected periodontal pockets to some mouth rinses and common antibiotics

Aims:
Treponema denticola is one of the most important periodontal pathogens because of its high lytic enzymatic activity leading to tooth lose and its ability to invade tissues and spread via blood stream causing systemic infections, this study coming to be the first local study concerning with this anaerobic fastidious bacterium and looking at the possibility of rely on the culturing methods in determining the most preferred chemotherapeutic drugs for limiting of bacterial activity and spread, as well as investigating the range of the antimicrobial activity of some mouth rinses and number of antibiotics.
Materials and Methods: 139 samples were collected from periodontal pockets with a depth of
3 mm under supervision of specialist dentitist, then placed in a reduced transport medium. The activity and effective spectrum of different concentrations for three kinds of widely used mouth rinses in the treatment of periodontal infections including Biofresh K, Biofresh F and Zak towards this bacterium was studied using sensitivity test methods, furthermore, the minimum inhibitory cocentration of antibiotics amoxillin, ciprofloxacin, clindamycin, azithromycin and mitronidazole was determined using antibiotic discs diffusion method. Results: The results showed that Biofresh K is the best in the term of bacterial killing as its inhibition activity continued to 1:16 dilution and when the sensitivity of T.denticola isolates to some antibiotics was tested, it was appeared that ciprofloxacin is the best causing growth inhibition with the lowest minimum inhibitory cocentration (0.0001mg), and when the synergistic effect of the tested antibiotics was studied it is becoming clear that the lowest antibiotic concentrations can cause growth inhibition when the two antibiotics (mitronidazole + ciprofloxacin) or (mitronidazole + amoxillin) are used in combination. Conclusions: It is possible to depend on culturing methods for determining the sensitivity of the bacterium T.denticola to chemotherapeutic drugs. Biofresh K is the best among the rinses under study and the antibiotic CIP is the best one with the lowest MIC and the lowest concentration of the antibiotics resulting in growth inhibition can be achieved when they are used in combination.

Influence of carbon dioxide on the activity of chicken egg white lysozyme

Rapid cooling of shell eggs by using liquid CO2 has shown increased bactericidal effects along with saturation of the egg albumen with CO2. Lysozyme is a bactericidal enzyme present in chicken eggs, and it lyses gram-positive bacteria. Newly laid chicken eggs have an initial pH of 7.6 to 8.5 and are saturated with CO2. During storage, the pH gradually increases to 9.7, accompanied by a loss of CO2. It is hypothesized that the lysozyme activity is influenced by either CO2 concentration or pH changes resulting from CO2 loss. The objective of this study was to determine the lytic activity of purified lysozyme and chicken egg white (unpurified lysozyme) under varying conditions of temperature, pH, and CO2 gas concentration. Lytic activity was determined by a standard microbial assay using lyophilized Micrococcus lysodeikticus. A 2 × 4 × 2 × 2 × 3 factorial design consisting of 2 temperatures (5 and 22°C), 4 pH (4.5, 6.5, 8.0, and 9.5), 2 treatments (with and without CO2), 2 types of lysozyme (purified and unpurified egg white), and 3 replicates was used. The highest lytic activity was found at pH 6.5 and 22°C. At pH 4.5 and 8.0, the addition of CO2 increased lytic activity by more than 50% at both temperatures. At pH 6.5, lytic activity was maintained with CO2 addition at both temperatures. At pH 9.5, lytic activity without CO2 addition was high; however, adding CO2 reduced lytic activity to zero. In conclusion, both pH and CO2 treatment influence lysozyme activity.

Combination of DTIC and peptide-vaccination elicit a renewal of high-avidity late-differentiated T-cell clones in melanoma patients (156.6)

Abstract Combination of chemo/immunotherapy to increase the effectiveness of the antitumor immune responses associated with a clinical efficacy is an attractive antitumor strategy. We have recently reported that the administration of dacarbazine (DTIC) one day before peptide (Melan-A and gp100)-vaccination in disease-free melanoma patients increases the number of peptide-specific effector-memory CD8+ lymphocytes. Notably, patients treated with DTIC before vaccination, showing a longer survival, display a progressive enhancement of TCR repertoire diversity of Melan-A+ CD8+ clones, accompanied by the maintenance of high-avidity, whereas patients treated with vaccine alone showed a reduction of TCR repertoire diversity and a decline of their tumor lytic activity. To study the phenotype and the functional lytic activity of CD8+ T-cell clones in association with their renewal rate we have analyzed the expression of CD28 and CD27 co-stimulatory molecules, in both Melan-A- and gp100-specific T-cell clones, isolated before and at different times after the treatments. We found newly recruited clones with high-lytic activity and highly-differentiated phenotype only after the combined therapy. Studies are in progress to define the role of replicative senescence in the continuous renewal induced by the combined therapy, by studying activity and regulation of telomerase in endogenous-induced or vaccine-driven clones, in correlation with their different phenotype and anti-tumor lytic capability.

The role of counterions in the membrane-disruptive properties of pH-sensitive lysine-based surfactants

Surfactants are among the most versatile and widely used excipients in pharmaceuticals. This versatility, together with their pH-responsive membrane-disruptive activity and low toxicity, could also enable their potential application in drug delivery systems. Five anionic lysine-based surfactants which differ in the nature of their counterion were studied. Their capacity to disrupt the cell membrane was examined under a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model for endosomal membranes. The surfactants showed pH-sensitive hemolytic activity and improved kinetics at the endosomal pH range. Low concentrations resulted in negligible hemolysis at physiological pH and high membrane lytic activity at pH 5.4, which is in the range characteristic of late endosomes. With increasing concentration, the surfactants showed an enhanced capacity to lyse cell membranes, and also caused significant membrane disruption at physiological pH. This observation indicates that, at high concentrations, surfactant behavior is independent of pH. The mechanism of surfactant-mediated membrane destabilization was addressed, and scanning electron microscopy studies were also performed to evaluate the effects of the compounds on erythrocyte morphology as a function of pH. The in vitro cytotoxicity of the surfactants was assessed by MTT and NRU assays with the 3T3 cell line. The influence of different types of counterion on hemolytic activity and the potential applications of these surfactants in drug delivery are discussed. The possibility of using pH-sensitive surfactants for endosome disruption could hold great promise for intracellular drug delivery systems in future therapeutic applications.

A novel analog of antimicrobial peptide Polybia-MPI, with thioamide bond substitution, exhibits increased therapeutic efficacy against cancer and diminished toxicity in mice

Polybia-MPI (MPI), a short cationic α-helical antimicrobial peptide, exhibited excellent anticancer activity and selectivity in vitro in our previous studies. To improve its in vivo application, we synthesized an analog (MPI-1) of MPI by replacing the C terminal amide –[CO–NH 2] with thioamide –ψ[CS–NH 2]. Although there is just one atom difference, the MPI-1 exhibited some surprising properties. In vitro studies revealed that MPI-1 exhibited relatively high lytic activity over MPI, whereas its stability to enzymatic degradation in serum was improved remarkably. Despite the enhanced toxicity in vitro, MPI-1 exhibited significantly lower mortality to mice than MPI at 75 mg/kg. Importantly, in vivo anticancer activity study indicated that MPI-1 could remarkably suppress the growth of sarcoma xenograft tumors more efficiently than MPI. Therefore, the significantly improved anticancer activity and predominantly lower in vivo toxicity might allow MPI-1 to be a good candidate for future anticancer treatment.

Lytic activity against primary AML cells is stimulated in vitro by an autologous whole cell vaccine expressing IL-2 and CD80.

Despite being of the myeloid lineage, acute myeloid leukaemia (AML) blasts are of low immunogenicity, probably because they lack the costimulatory molecule CD80 and secrete immunosuppressive factors. We have previously shown that in vitro stimulation of autologous peripheral blood mononuclear cells (PBMCs) with primary AML cells modified to express CD80 and IL-2 promotes proliferation, secretion of Th1 cytokines and expansion of activated CD8(+) T cells. In this study, we show that allogeneic effector cells (from a healthy donor or AML patients) when stimulated with IL-2/CD80 modified AML blasts were able to induce the lysis of unmodified AML blasts. Effector cells stimulated with IL-2/CD80AML blasts had higher lytic activity than cells stimulated with AML cells expressing CD80 or IL-2 alone. Similarly, AML patient PBMCs primed with autologous IL-2/CD80 AML cells had a higher frequency of IFN-gamma secreting cells and show cytotoxicity against autologous, unmodified blasts. Crucially, the response appears to be leukaemia specific, since stimulated patient PBMCs show higher frequencies of IFN-gamma secreting effector cells in response to AML blasts than to remission bone marrow cells from the same patients. Although studied in a small number of heterogeneous patient samples, the data are encouraging and support the continuing development of vaccination for poor prognosis AML patients with autologous cells genetically modified to express IL-2/CD80.

Phage Lysin LysK Can Be Truncated to Its CHAP Domain and Retain Lytic Activity against Live Antibiotic-Resistant Staphylococci

A truncated derivative of the phage endolysin LysK containing only the CHAP (cysteine- and histidine-dependent amidohydrolase/peptidase) domain exhibited lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus. This is the first known report of a truncated phage lysin which retains high lytic activity against live staphylococcal cells.

The effects of lipids on the structure of the eukaryotic cytolysin equinatoxin II: A synchrotron radiation circular dichroism spectroscopic study

Synchrotron radiation circular dichroism (SRCD) spectroscopy studies of the eukaryotic pore-forming protein equinatoxin II (EqtII) were carried out in solution and in the presence of micelles or small unilamellar vesicles (SUV) of different lipid composition. The SRCD structural data was correlated with calcein leakage from SUV and with partitioning of EqtII to liposomes, and micelles, according to haemolysis assays. The structure of EqtII in water and dodecylphosphocholine micelles as determined by SRCD was similar to the values calculated from crystal and solution structures of the protein, and no changes were observed with the addition of sphingomyelin (SM). SM is required to trigger pore formation in biological and model membranes, but our results suggest that SM alone is not sufficient to trigger dissociation of the N-terminal helix and further structural rearrangements required to produce a pore. Significant changes in conformation of EqtII were detected with unsaturated phospholipid (DOPC) vesicles when SM was added, but not with saturated phospholipids (DMPC), which suggests that not only is membrane curvature important, but also the fluidity of the bilayer. The SRCD data indicated that the EqtII structure in the presence of DOPC:SM SUV represents the ‘bound’ state and the ‘free’ state is represented by spectra for DOPC or DOPC:Chol vesicles, which correlates with the high lytic activity for SUV of DOPC:SM. The SRCD results provide insight into the lipid requirements for structural rearrangements associated with EqtII toxicity and lysis.

A cold-active salmon goose-type lysozyme with high heat tolerance.

The Atlantic salmon (Salmo salar) goose-type lysozyme gene was isolated and revealed alternative splicing within exon 2 affecting the signal peptide-encoding region. The lysozyme was produced in Escherichia coli, and the recombinant enzyme showed a high specific lytic activity that was stimulated by low or moderate concentrations of mono- or divalent cations. Relative lytic activities of 70 and 100% were measured at 4 degrees C and 22 degrees C, respectively, and there was no detectable activity at 60 degrees C. However, 30% activity was retained after heating the enzyme for 3 h at 90 degrees C. This unique combination of thermal properties was surprising since the salmon goose-type lysozyme contains no cysteines for protein structure stabilization through disulphide bond formation. The results point to a rapid reversal of inactivation, probably due to instant protein refolding.

Forceps size and immune function in the earwig Forficula auricularia L.

Females of many species select their mates on the basis of the size or intensity of sexual ornaments, and it has been suggested that these provide reliable signals of a male's ability to resist parasites and pathogens. European earwigs, Forficula auricularia, are sexually dimorphic in forceps shape and length. Male forceps are used as weapons in male contests for access to females, but recent findings suggest that females also choose males on the basis of their forceps length. In the present study, we tested the hypotheses that in the European earwig, F. auricularia, the size of forceps is correlated with immune function and that immune function differs between the sexes. We found that encapsulation rate was not correlated with the length of forceps in either sex, but was negatively correlated with body size. By contrast, lytic activity of the haemolymph increased with overall body size in both sexes but, in females, lytic activity increased with relative forceps length whereas, in males, it decreased with relative forceps length. After accounting for effects of body size, there was no remaining significant correlation in females but the negative correlation in males remained. Furthermore, we found that males had higher encapsulation rate and higher lytic activity than females, suggesting that males have stronger immune defence. The results of the study indicate that the size of forceps in male earwigs does not reliably reflect male immune defence.

Neurospheres Enriched in Cancer Stem–Like Cells Are Highly Effective in Eliciting a Dendritic Cell–Mediated Immune Response against Malignant Gliomas

Cancer stem-like cells (CSC) could be a novel target for cancer therapy, including dendritic cell (DC) immunotherapy. To address this, we developed experiments aimed at DC targeting of neurospheres (NS) from GL261 glioma cells because neurospheres can be enriched in CSC. We obtained murine neurospheres by growing GL261 cells in epidermal growth factor/basic fibroblast growth factor without serum. GL261-NS recapitulated important features of glioblastoma CSC and expressed higher levels of radial glia stem cell markers than GL261 cells growing under standard conditions (GL261 adherent cells, GL261-AC), as assessed by DNA microarray and real-time PCR. GL261-NS brain gliomas were highly infiltrating and more rapidly lethal than GL261-AC, as evidenced by survival analysis (P < 0.0001), magnetic resonance imaging and histology. DC from the bone marrow of syngeneic mice were then used for immunotherapy of GL261-NS and GL261-AC tumors. Strikingly, DC loaded with GL261-NS (DC-NS) cured 80% and 60% of GL261-AC and GL261-NS tumors, respectively (P < 0.0001), whereas DC-AC cured only 50% of GL261-AC tumors (P = 0.0022) and none of the GL261-NS tumors. GL261-NS expressed higher levels of MHC and costimulatory molecules (CD80 and CD86) than GL261-AC; the JAM assay indicated that DC-NS splenocytes had higher lytic activity than DC-AC splenocytes on both GL261-NS and GL261-AC, and immunohistochemistry showed that DC-NS vaccination was associated with robust tumor infiltration by CD8+ and CD4+ T lymphocytes. These findings suggest that DC targeting of CSC provides a higher level of protection against GL261 gliomas, a finding with potential implications for the design of clinical trials based on DC vaccination.

Melittin analogs with high lytic activity at endosomal pH enhance transfection with purified targeted PEI polyplexes

Melittin–polyethylenimine (PEI) conjugates have been shown to enhance gene transfer efficiency of polyplexes due to their membrane-destabilizing properties. Inherent lytic activity at neutral pH however also provokes high cytotoxicity due to plasma membrane damage. In order to shift the lytic activity towards the endosomal membrane, several melittin analogs were designed. Acidic modification of melittin by replacing neutral glutamines (Gln-25 and Gln-26) with glutamic acid residues greatly improved the lytic activity of C-terminally linked PEI conjugates at the endosomal pH of 5. This activity correlated well with the gene transfer efficiency of polyplexes in four different cell lines. Melittin–PEI conjugates with high lytic activities at endosomal pH were then incorporated into EGF receptor-targeted and polyethylene glycol-shielded polyplexes. The resulting particles had virus-like dimension (150 nm) with a neutral surface charge and were subsequently purified by size exclusion chromatography to remove unbound toxic PEI conjugate. These purified polyplexes mediated EGF-receptor-specific gene transfer with up to 70-fold higher activity compared to the corresponding PEI polyplexes without melittin.

Improvement of the Lytic Properties of a β-1,3-Glucanase by Directed Evolution

BGLII is a bacterial endoglucanase that hydrolyzes the beta-1,3-glucan present in yeast cell walls, resulting in lysis of Saccharomyces cerevisiae. As a result of this property, BGLII is considered a potential tool for downstream processing and recovery of biotechnological products produced in yeast. Here we describe the improvement of the yeast lytic activity of BGLII, achieved by a directed evolution approach involving random mutagenesis and screening for variants with improved catalytic activity, combined with site-directed mutagenesis. A BGLII variant having three times the wild-type hydrolytic activity on laminarin was identified. The purified enzyme also exhibited higher lytic activity on yeast cells. Mutations causing the improvements are located very close to each other in the amino acid sequence, suggesting that the region should be considered as a target for further improvements of the glucanase activity. These results demonstrate the feasibility of molecular evolution methods for the improvement of the BGLII hydrolytic activity, and open a window for further improvement of this or other properties in glycosyl hydrolases in general.

Coexistence of a Two-States Organization for a Cell-Penetrating Peptide in Lipid Bilayer

Primary amphipathic cell-penetrating peptides transport cargoes across cell membranes with high efficiency and low lytic activity. These primary amphipathic peptides were previously shown to form aggregates or supramolecular structures in mixed lipid-peptide monolayers, but their behavior in lipid bilayers remains to be characterized. Using atomic force microscopy, we have examined the interactions of P ( α) , a primary amphipathic cell-penetrating peptide which remains α-helical whatever the environment, with dipalmitoylphosphatidylcholine (DPPC) bilayers. Addition of P ( α) at concentrations up to 5 mol % markedly modified the supported bilayers topography. Long and thin filaments lying flat at the membrane surface coexisted with deeply embedded peptides which induced a local thinning of the bilayer. On the other hand, addition of P ( α) only exerted very limited effects on the corresponding liposome’s bilayer physical state, as estimated from differential scanning calorimetry and diphenylhexatriene fluorescence anisotropy experiments. The use of a gel-fluid phase separated supported bilayers made of a dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine mixture confirmed both the existence of long filaments, which at low peptide concentration were preferentially localized in the fluid phase domains and the membrane disorganizing effects of 5 mol % P ( α) . The simultaneous two-states organization of P ( α) , at the membrane surface and deeply embedded in the bilayer, may be involved in the transmembrane carrier function of this primary amphipathic peptide.

Phenotypic and functional analysis of lymphocytes infiltrating osteolytic tumors: use as a possible therapeutic approach of osteosarcoma.

BackgroundOsteosarcoma is the most common type of primary bone tumor. The use of aggressive chemotherapy has drastically improved the prognosis of the patients with non-metastatic osteosarcomas, however the prognosis of the patients with metastasis is still very poor. Then, new and more effective treatments for curing osteosarcoma, such as immunotherapy are needed. Tumor-infiltrating lymphocytes (TIL) have been involved in the control of tumor development and already assessed with success for the treatment of several cancers including melanoma. While TIL represent a fascinating therapeutic approach in numerous malignant pathologies, there is few report concerning adult bone-associated tumors including osteosarcoma.MethodsHuman TIL were isolated and characterized (phenotype, lytic activity) from twenty-seven patients with bone-associated tumors (osteosarcoma, Ewing's sarcoma, giant cell tumor, chondrosarcoma, plasmocytoma and bone metastases). Similar experiments were performed using rat osteosarcoma model.ResultsWhile TIL with a main CD4+ profile were easily isolated from most of the tumor samples, only TIL extracted from osteosarcoma were cytotoxic against allogeneic tumor cells. In all cases, TIL lytic activity was significantly higher compared to autologous peripheral blood leukocytes. Similar data were observed in rat osteosarcoma model where TIL were characterized by a main CD4+ profile and high lytic activity against allogeneic and autologous tumor cells. Moreover, rat TIL expansion was not accompanied by refractoriness to further activation stimulus mainly by tumor antigens.ConclusionThese results demonstrated that TIL therapy could be a very efficient strategy for the treatment of adult osteosarcoma.

Rituximab mediates in vitro antileukemic activity in pediatric patients after allogeneic transplantation

Relapse is a major problem after allogeneic transplantation in children with acute B-lineage lymphoblastic leukemias (ALL) and lymphomas and additional therapeutic strategies are needed to increase graft versus leukemia effects without inducing graft versus host disease (GvHD). Several studies have shown the efficacy of a humanized CD20 antibody (rituximab) for treatment of CD20+ malignancies together with conventional chemotherapy but less is known about its post transplant usefulness. We studied the ability of rituximab to mediate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) with effector cells and complement from patients who were transplanted with T-cell-depleted grafts from unrelated or mismatched related donors. Highest lytic activity (ADCC) was observed against leukemia-derived MHH4 cells and Burkitt's lymphoma-derived Raji cells in the first months after transplantation, corresponding to the high percentage of regenerating CD56+ CD16+ cells. Moreover, primary cryopreserved ALL-blasts from a pediatric patient were also efficiently lysed. Increased lysis was obtained after stimulation with interleukin-2. Combination of ADCC and CDC had additive effects. These findings encourage clinical trials on the use of rituximab for improving minimal residual disease control and relapse prevention after allogeneic high-risk transplantation in the small group of pediatric patients with CD20+ leukemias/lymphomas.

Lytic enzyme, labiase for a broad range of Gram-positive bacteria and its application to analyze functional DNA/RNA

The lytic activity of labiase and achromopeptidase for bacterial DNA/RNA extraction were compared. Rapid lysis of many bacterial strains was observed with labiase followed by SDS treatment. Both labiase and achromopeptidase showed high lytic activity against bacterial strains with the A1α chemotype (e.g., Aerococcus viridans) and the A3α chemotype (e.g., Staphylococcus epidermidis) for cell wall peptidoglycan structures. The lytic activity of labiase was higher than that of achromopeptidase against strains with the A1γ chemotype (e.g., Bacillus subtilis). The activity of labiase was not detrimentally affected with increasing NaCl concentration. Labiase lysates were successfully used for rapid extraction of DNA and RNA, whereas achromopeptidase lysates degraded RNA. The DNA and RNA obtained were successfully used for 16 S rRNA amplification and real-time RT-PCR detection. It is concluded that labiase is useful for rapid lysis of a wide variety of Gram-positive bacteria and can be used for DNA/RNA isolation protocols.