Fabrication of pH-degradable supramacromolecular microgels with tunable size and shape via droplet-based microfluidics.

This study presents a versatile method to synthesize stimuli-responsive microgels with supramolecular cross-links exhibiting tunable size and shape via droplet-based microfluidics. The natural polyphenol tannic acid (TA) is used to cross-link poly(N-vinylcaprolactam) (PVCL) chains in aqueous droplets by the formation of hydrogen bonds and hydrophobic interactions between the phenolic groups of TA and the carbonyl group and the hydrophobic segments of lactam ring of PVCL chains. The obtained microgels exhibit diameters in the range of 130-150µm in swollen state in aqueous solution. Synthesized microgels exhibit pH-responsive behavior: at low pH microgels deswell and shrink due to the protonation of phenolic groups and enhanced hydrophobic interactions; at high pH microgels swell and disintegrate due to the deprotonation of phenolic groups and destruction of hydrogen bonds with PVCL chains. Additionally, we present supramacromolecular microgels in cylindrical shape with different aspect ratios using a new design of microfluidic chip by varying flow rates at high concentration of the prepolymerized precursor combined with rapid pH-triggered on-chip gelation. Furthermore, developed synthesis methodology allows on-chip encapsulation of colloidal objects into large supramacromolecular microgels during the cross-linking step. The complete and fast release of objects by pH-triggered degradation indicates that the pH-responsive supramacromolecular microgels can be used for controlled loading/release of various payloads, like probiotics. Moreover, cell studies of L929 fibroblast clearly show the biocompatibility of the microgels.

Mapping the Secretome of Dental Pulp Stem Cells Under Variable Microenvironmental Conditions.

There is substantial evidence supporting the anti-inflammatory and regenerative potential of dental pulp stem cells (DPSCs) through direct cell transplantation or paracrine action. However, DPSC secretome profile remains inadequately studied. This study provides proteomic profiling of the human DPSC secretome by comparatively analysising cell lysates and respective culture supernatants (i.e. conditioned media-CM) under variable oxygen tension conditions (normoxia-20% O2/CM_Norm vs. hypoxia 2% O2/CM_Hyp) and/or stimulation with Tumor Necrosis Factor alpha (TNF-α). DPSC-CM samples and respective crude lysates (DPSC-CL) were collected and subjected to SDS-PAGE, followed by LC-MS/MS analysis. The identified proteins were analyzed by Gene Ontology, Reactome, and String databases. The anti-inflammatory properties of DPSC-CMs were validated via an in vitro RAW_246.7 murine macrophages model through evaluation of the expression of pro-and anti-inflammatory markers by real-time PCR. Results showed a total of 2413 proteins identified in CM_Norm, 2479 in CM_Norm+TNF-α, 1642 in CM_Hyp, and 2002 in CM_Hyp + TNF-α samples. CM_Norm contained 122 proteins statistically significantly upregulated compared to the CM_Hyp and involved in pathways related to "ECM organization", "cellular response to hypoxia", and "IL signaling". Functional network analysis showed that TGFß1, TIMP1 and TIMP2 were key nodes among proteins significantly upregulated in the CM_Norm compared to the CM_Hyp, interacting with more than 10 proteins, each. DPSC-CM application in the in vitro RAW_246.7 model decreased the expression of pro-inflammatory markers (MMP-3, MMP-9, MMP-13, MCP-1), while increasing anti-inflammatory markers (IL-10). Overall, DPSC-CM collected under normoxic conditions is enriched with anti-inflammatory, tissue repair and regenerative factors, which prompts further investigation on its therapeutic applications.

Tunable clustering of magnetic nanoparticles in microgels: enhanced magnetic relaxivity by modulation of network architecture.

In the present work we used microgels as colloidal containers for the loading of hydrophobic magnetic nanoparticles using the solvent exchange method. We varied systematically two parameters: (i) the crosslinking degree of microgels (1-4.5 mol% crosslinker) and (ii) loading of hydrophobic magnetite nanoparticles (d = 7 nm) in microgels (2-10 wt%). The experimental data show that the interplay between these two parameters provides efficient control over the clustering of magnetic nanoparticles in the microgel structure. Transverse magnetization relaxation measurements indicate that the formation of nanoparticle clusters in microgels induces non-linear enhancement of the relaxivity with the increase of nanoparticle loading in microgels. The results suggest that the modulation of the microgel network architecture can be efficiently applied to trigger self-assembly processes inside microgels and design hybrid colloids with unusual morphologies and properties.

Hydrophobic superparamagnetic FePt nanoparticles in hydrophilic poly(N-vinylcaprolactam) microgels: a new multifunctional hybrid system.

We report the synthesis of a new multifunctional colloidal hybrid system consisting of thermoresponsive amphiphilic biocompatible poly(N-vinylcaprolactam) microgels loaded with hydrophobic superparamagnetic FePt nanoparticles (NPs). In the first step, water swellable poly(N-vinylcaprolactam) microgels were mixed with hydrophobically coated sub-10 nm superparamagnetic FePt NPs in a tetrahydrofuran (THF) solution. In the second step, changing the surrounding solvent from THF to water forces the FePt NPs to migrate into the amphiphilic microgels. These new hybrid microgels (i) are colloidally stable in water and their thermo-responsive properties in terms of volume phase transition are retained, (ii) exhibit superparamagnetic characteristics introduced by FePt NPs, (iii) show a drastically reduced cytotoxicity compared to water-soluble FePt NPs of similar size, as known from the literature. This makes the new hybrid microgels suitable e.g. as biocompatible containers for drug delivery or for imaging.

miR-139-5p controls translation in myeloid leukemia through EIF4G2.

MicroRNAs (miRNAs) are crucial components of homeostatic and developmental gene regulation. In turn, dysregulation of miRNA expression is a common feature of different types of cancer, which can be harnessed therapeutically. Here we identify miR-139-5p suppression across several cytogenetically defined acute myeloid leukemia (AML) subgroups. The promoter of mir-139 was transcriptionally silenced and could be reactivated by histone deacetylase inhibitors in a dose-dependent manner. Restoration of mir-139 expression in cell lines representing the major AML subgroups (t[8;21], inv[16], mixed lineage leukemia-rearranged and complex karyotype AML) caused cell cycle arrest and apoptosis in vitro and in xenograft mouse models in vivo. During normal hematopoiesis, mir-139 is exclusively expressed in terminally differentiated neutrophils and macrophages. Ectopic expression of mir-139 repressed proliferation of normal CD34(+)-hematopoietic stem and progenitor cells and perturbed myelomonocytic in vitro differentiation. Mechanistically, mir-139 exerts its effects by repressing the translation initiation factor EIF4G2, thereby reducing overall protein synthesis while specifically inducing the translation of cell cycle inhibitor p27(Kip1). Knockdown of EIF4G2 recapitulated the effects of mir-139, whereas restoring EIF4G2 expression rescued the mir-139 phenotype. Moreover, elevated miR-139-5p expression is associated with a favorable outcome in a cohort of 165 pediatric patients with AML. Thus, mir-139 acts as a global tumor suppressor-miR in AML by controlling protein translation. As AML cells are dependent on high protein synthesis rates controlling the expression of mir-139 constitutes a novel path for the treatment of AML.

Differential expression of miR-17~92 identifies BCL2 as a therapeutic target in BCR-ABL-positive B-lineage acute lymphoblastic leukemia.

Despite advances in allogeneic stem cell transplantation, BCR-ABL-positive acute lymphoblastic leukaemia (ALL) remains a high-risk disease, necessitating the development of novel treatment strategies. As the known oncomir, miR-17~92, is regulated by BCR-ABL fusion in chronic myeloid leukaemia, we investigated its role in BCR-ABL translocated ALL. miR-17~92-encoded miRNAs were significantly less abundant in BCR-ABL-positive as compared to -negative ALL-cells and overexpression of miR-17~19b triggered apoptosis in a BCR-ABL-dependent manner. Stable isotope labelling of amino acids in culture (SILAC) followed by liquid chromatography and mass spectroscopy (LC-MS) identified several apoptosis-related proteins including Bcl2 as potential targets of miR-17~19b. We validated Bcl2 as a direct target of this miRNA cluster in mice and humans, and, similar to miR-17~19b overexpression, Bcl2-specific RNAi strongly induced apoptosis in BCR-ABL-positive cells. Furthermore, BCR-ABL-positive human ALL cell lines were more sensitive to pharmacological BCL2 inhibition than negative ones. Finally, in a xenograft model using patient-derived leukaemic blasts, real-time, in vivo imaging confirmed pharmacological inhibition of BCL2 as a new therapeutic strategy in BCR-ABL-positive ALL. These data demonstrate the role of miR-17~92 in regulation of apoptosis, and identify BCL2 as a therapeutic target of particular relevance in BCR-ABL-positive ALL.

QCD and strongly coupled gauge theories: challenges and perspectives.

We highlight the progress, current status, and open challenges of QCD-driven physics, in theory and in experiment. We discuss how the strong interaction is intimately connected to a broad sweep of physical problems, in settings ranging from astrophysics and cosmology to strongly coupled, complex systems in particle and condensed-matter physics, as well as to searches for physics beyond the Standard Model. We also discuss how success in describing the strong interaction impacts other fields, and, in turn, how such subjects can impact studies of the strong interaction. In the course of the work we offer a perspective on the many research streams which flow into and out of QCD, as well as a vision for future developments.

JAK2V617F activating mutation is associated with the myeloproliferative type of chronic myelomonocytic leukaemia.

BACKGROUND: Chronic myelomonocytic leukaemia (CMML) is a haematopoietic malignancy with heterogeneous clinical and morphological features. It is classified in the World Health Organization myeloproliferative-myelodysplastic overlap category. JAK2(V617F) mutation can be found in a large percentage of patients with myeloproliferative neoplasms. AIMS: To investigate the association between JAK2(V617F) mutation and clinical, haematological and bone marrow histological features in CMML and to verify whether the mutation is associated with the myeloproliferative type of the disease. METHODS: 78 consecutive patients with newly diagnosed CMML from 2004 to 2008 were included in the study. JAK2(V617F) mutation was assessed using direct sequencing of exon 14 or by allele-specific PCR from total peripheral blood or bone marrow samples. RESULTS: JAK2(V617F) mutation was identified in eight cases (10.2%). All patients with the mutation presented with splenomegaly and had a significantly higher haemoglobin level and neutrophil count than patients without the mutation. All bone marrow biopsies of JAK2(V617F)-mutated CMML showed increased erythropoiesis, a marked myeloid and megakaryocytic hyperplasia with occasionally clustered megakaryocytes, and a mild or moderate (grade 1 or 2) fibrosis; six cases showed an increased number of dilated sinusoids and reactive lymphoid nodules. CONCLUSIONS: The results indicate that JAK2(V617F) mutation is associated with clinical and morphological features of the myeloproliferative type of CMML. Therefore, JAK2 mutation analysis together with bone marrow morphology could help in a more appropriate classification of the disease.

Identification of biomarkers indicating cellular changes after treatment of neuronal cells with the C3 exoenzyme from Clostridium botulinum using the iTRAQ protocol and LC-MS/MS analysis.

Proteomic approaches are used to identify biomarkers, to monitor pathological changes inside of cells and for a better diseases diagnosis. Comparable changes in protein homeostasis also occur in differentiating cells and proteomic techniques should be suitable to identify biomarkers that indicate different steps of cellular development. The C3 exoenzyme from Clostridium botulinum (C3bot) inactivates Rho GTPases and induces morphological cellular changes like cell rounding and neurite outgrowth [G. Ahnert-Hilger, M. Höltje, G. Grosse, G. Pickert, C. Mucke, B. Nixdorf-Bergweiler, P. Boquet, F. Hofmann, I. Just, J. Neurochem. 90 (2004) 9]. To investigate these observations further a comparative proteomic approach has been chosen to elucidate C3bot effects in the neuroblastoma cell line model SH-SY5Y. The screening method applied for biomarker detection was based on the stable isotope approach isobaric tagging for relative and absolute quantification (iTRAQ). Proteins of C3bot-treated and untreated cells were digested and peptides were labeled by the iTRAQ reagent, combined, and separated by means of a two-dimensional nano-HPLC system. Peptide analysis was performed in a MALDI-TOF/TOF mass spectrometer. Identification and quantification of peptides and their corresponding proteins were accomplished by MS/MS spectra analysis. Overall, five replicate measurements identified 355 different proteins of which 235 were accessible for quantification. C3bot altered the concentration of 55 proteins (at least 1.3-fold) and several proteins were identified as possible biomarker candidates that indicate C3bot-induced cellular changes.

The impact of liquid-based oral cytology on the diagnosis of oral squamous dysplasia and carcinoma.

OBJECTIVE: Even though diagnostic oral exfoliative cytology is a useful, economical and practical tool in the diagnosis of oral dysplasia and carcinoma, it is not yet extensively used. The results of conventional exfoliative and liquid-based diagnostic cytology in oral potentially malignant lesions (PML) are herein reported and compared with the histological diagnosis. METHODS: Either conventional (89) or liquid-based (384) exfoliative cytology was used for the diagnosis of oral dysplasia/carcinoma in 473 subjects and the results were compared with scalpel biopsy histology. Cells were collected using a Cytobrush device for conventional smears and with a dermatological curette for the liquid-based cytology. The 'curette technique' also allowed for the collection of 'accidental' tissue fragments, utilized as microbiopsies. RESULTS: Histological diagnosis was squamous carcinoma in 96 of 473 cases, high-grade dysplasia (oral intraepithelial neoplasia two to three) in 24 and other lesions in 353 cases. The smears in the conventional cytology group were inadequate in 12.4%, with an 85.7% sensitivity and a 95.9% specificity. There were 8.8% of inadequate specimens in the liquid-based cytology group; sensitivity was 95.1% and specificity was 99.0%. CONCLUSIONS: Although conventional cytology is useful when diagnosing oral PML (better sensitivity and predictive positive value if compared with the cervical smear test with similar specificity) and can improve the accuracy of histological diagnosis, liquid-based cytology gives better results, as it not only enhances both sensitivity and specificity, but also provides material for further investigation (AgNORs, DNA, microbiopsies, etc.).

Redispergation of TiO2 particles in aqueous solutions.

The colloidal stability of TiO2 dispersions in aqueous solutions was studied. Aqueous solutions of ATLAS G-3300 (1.57 x 10(-3) mol/l), TRITON X-100 (5 x 10(-5) mol/l), and PMAA (4 x 10(-6) and 5.81 x 10(-3) mol/l) have been used as medium for redispergation of TiO2 particles. Stability of dispersions was investigated at different pH values by two different methods. By using analytical centrifuge the sedimentation velocity of TiO2 particles was directly measured and by means of light scattering the particle size of dispersed particles has been monitored. Combination of these two methods allowed determination of the aggregation degree of TiO2 particles as well as structure of the aggregates formed in aqueous phase. It has been found that redispergation process does not provide complete separation of virgin TiO2 particles. Even in the case of stable dispersions some aggregates were found, which consisted of 2-4 virgin TiO2 particles. With increasing colloidal stability of dispersions aggregates appear to be spherically shaped. In the system where TRITON X-100 was used, formation of secondary aggregates by fusion of primary ones was observed.

Prognostic relevance of cell proliferation in head and neck tumors.

Cell proliferative activity has been extensively investigated in head and neck tumors. Ki67/MIB-1 immunostaining, tritiated thymidine or bromodeoxyuridine labeling indices, DNA S-phase fraction, proliferating cell nuclear antigen expression, potential doubling time and analysis of the nucleolar organizer region associated proteins (AgNORs) have shown significant correlation with prognosis in 4806 cases of tumors of the oral cavity, salivary glands, pharynx and larynx. However, this was not observed in 2968 other reported cases. Discrepancies may depend on various factors: the heterogeneity of the series, which include tumors from various anatomic sites and patients treated with different therapy, and the lack of standardization of methods for assessing cell proliferation. Furthermore, none of the methods currently applied can by themselves define the actual proliferative activity, as it depends both on the proportion of cells committed to the cycle (growth fraction) and the speed of the cell cycle. Indeed, the actual proliferative activity of a tumor could well be measured by the equation [PA = Ki67 or MIB-1 scores x AgNORs], as we did in pharyngeal carcinoma. Provided that large and homogeneous series are evaluated by standardized methods, cell proliferative activity can still be regarded as an inexpensive and reliable prognostic factor in head and neck tumors.

[Usefulness of oral exfoliative cytology for the diagnosis of oral squamous dysplasia and carcinoma]. / Utilità della citologia esfoliativa orale per la diagnosi di displasia e carcinoma squamoso orale.

AIM: It is well known that diagnostic oral exfoliative cytology, even if a useful, economical and practical tool in the diagnosis of oral dysplasia and carcinoma, is not yet used so extensively as is cervico-vaginal cytology. METHODS: Exfoliative cytology was used for the diagnosis of oral dysplasia and carcinoma, and the results compared to the histological examination. Cytological smears were taken from 89 patients with oral lesions suspicious for neoplasia (in particular erythro- and leukoplakia and lichen). All patients were also subjected to oral biopsy and histological examination. RESULTS: Out of 89 cases studied, histology showed the presence of an invasive squamous carcinoma in 32, dysplasia in 17, phlogosis in 15 and other types of lesions (2 of which malignant non-epithelial tumours) in 25. The cytological smear was inadequate for diagnosis in 11/89 cases (12.4%). In cytologically adequate and histologically positive cases, cytology confirmed the histological diagnosis of dysplasia and/or carcinoma in 38/45 cases (sensitivity 86.5%, accuracy 89.6%). Moreover, 1 case which was histologically negative at the onset, proved positive at cytology. There were 2 false-positive cytology results (specificity 94.3%, predictive positive value 95.7%). CONCLUSIONS: Despite the small number of cases in the cohort, oral cytology can improve the accuracy of histology, and may be a useful screening tool for the diagnosis of oral neoplasia/dysplasia.

Isospin violation in epsilon'.

On the basis of a next-to-leading-order calculation in chiral perturbation theory, the first complete analysis of isospin breaking for direct CP violation in K0-->2 pi decays is performed. We find a destructive interference between three different sources of isospin violation in the CP violation parameter epsilon'. Within the uncertainties of large-N(c) estimates for the low-energy constants, the isospin violating correction for epsilon' is below 15%.

[Significance of the AgNOR in tumor pathology]. / Significato delle AgNOR in patologia tumorale.

Analysis of silver-stained nucleolar organizer regions (AgNORs), originally regarded as a diagnostic tool, is now considered mainly as a prognostic parameter. Indeed, the expression of AgNOR proteins is associated with several biological properties of neoplastic cells: metabolic activity, DNA content, histological grade of differentiation and, especially, the rapidity of cellular proliferation. Thus, a high AgNOR quantity is a marker of aggressive tumour phenotype, and a large number of papers have shown the independent prognostic value of AgNOR analysis in several human neoplasias. Moreover, the method can be applied to small biopsies, can identify neoplastic clones with different proliferative activities and may stratify patients into different risk groups. The standardized method for AgNOR quantification offers objective and reproducible results. The evaluation of AgNOR quantity in cycling cells, either by immunohistochemistry or by a novel flow cytometry technique, may represent the future of AgNOR analysis.

[LAT (linker for activation of T cells): a useful marker for megakaryocyte evaluation on bone marrow biopsies]. / LAT (linker for activation of T cells): un utile marcatore per l'identificazione di megacariociti in biopsie osteomidollari.

Detection of atypical megakaryocytes in bone marrow biopsies, especially in cases of myelodysplastic syndromes (MDS), chronic myeloproliferative disorders (CMPD) and acute leukemias, is facilitated by staining for markers such as Ulex europaeus agglutinin (UEA)-J, CD31, CD61 and von Willebrand factor (VWF), the latter being considered the most sensitive. Recently, LAT (linker for activation of T cells), a molecule involved in T-cell activation and platelet aggregation, was found to be expressed by megakaryocytes and platelets in tissue sections. We compared VWF and LAT immunoreactivity on megakaryocytes in 64 bone marrow biopsies from 12 normal controls (NC), and from patients with MDS (n=18), CMPD (n=21) and acute megakaryocytic leukemia (AML-M7, n=13). Immunostaining was performed on paraffin sections with polyclonal antibodies against VWF and LAT. Immunoreactivity was evaluated by counting positive megakaryocytes in 10 high-power fields, and values were compared using Student's t test for paired data. Both VWF and LAT predominantly stained the cytoplasm of megakaryocytes, although LAT was also recognizable on the cell membrane. In most biopsies, the immunoreactivity of the two antibodies was quite similar. No significant differences were noticed between the mean values of VWF+ and LAT+ megakaryocytes. However, in 22 cases (5 NC; 5 MDS; 6 CMPD; 6 AML-M7), the number of LAT+ megakaryocytes was at least 30% higher than VWF+cells, while in 3 cases opposite findings were found. In 3 AML-M7 cases, anti-LAT antibodies stained numerous megakaryocytes, but anti-VWF staining was practically negative; in another 5 AML-M7 cases, anti-LAT labeling was much stronger than anti-VWF staining. LAT represents a useful immunohistochemical marker for megakaryocytes in normal and pathological conditions. It seems to be expressed by megakaryocytes more than VWF in most cases and, particularly, in conditions associated with poorly differentiated megakaryocytes, such as acute megakaryocytic leukemias. The use of LAT staining should be recommended in association with other megakaryocyte markers in the study of bone marrow biopsies in cases of hematopoietic disorders.

Biologic differences between noninvasive papillary urothelial neoplasms of low malignant potential and low-grade (grade 1) papillary carcinomas of the bladder.

We investigated the expression of oncogenes p53, c-erbB-2, and bcl-2 and cell proliferative activity in 62 newly diagnosed superficial pTa papillary bladder tumors. Based on the 1998 World Health Organization/International Society of Urological Pathology (WHO/ISUP) and 1999 WHO classifications, 19 were urothelial neoplasias of low malignant potential (LMP) and 43 low-grade (grade 1) papillary carcinomas. All the patients underwent transurethral resection and were followed up to 97 months; 42 had recurrences. Initial biopsies were tested for p53, c-erbB-2, and bcl-2 proteins using DO7, CB11, and bcl-2 124 monoclonal antibodies. Cell proliferation was assessed by MIB-1 mAb and mitotic count. LMP had significantly lower MIB-1 (p = 0.002) and p53 immunopositivity (p = 0.03), mitotic count (p = 0.006), and recurrence rates (p = 0.04) than did grade 1 cases, whereas no difference was observed for c-erbB-2 and bcl-2 expression. The median disease-free survival for LMP was 76 months but only 15 months for grade 1 cases (p = 0.002). Although the cohort is small, the results indicate that the distinction between LMP and low-grade (grade 1) papillary urothelial neoplasias, as proposed by the 1998 WHO/ISUP and 1999 WHO classifications, reflects different biologic activity and clinical behavior; however, a long-term follow-up is advisable also for patients with LMP.

Fe homeostasis in plant cells: does nicotianamine play multiple roles in the regulation of cytoplasmic Fe concentration?

The cellular and intracellular localization of the non-proteogenic amino acid nicotianamine (NA) in leaves and root elongation zones was immunochemically investigated in pea (Pisum sativum L.) and tomato (Lycopersicon esculentum Mill.) plants grown under various iron regimes and in three mutants defective in the regulation of iron uptake. Strongest immunostaining was observed in the over-accumulating pea mutants brz and dgl, and in iron-loaded wild-type plants. Fe concentration and NA level paralleled staining intensity, indicating that NA synthesis is induced by high iron availability. While label was mainly present in the cytoplasm under normal (10 microM) Fe supply and under Fe deprivation, most of the labeling was present in the vacuole in iron-loaded plants. This pattern resembled the distribution of NA in Fe over-accumulating mutants, indicating the possible importance of vacuolar sequestration in the detoxification of excess Fe. Based on the dependence of the cellular distribution of NA on the iron nutritional status of the plant, a possible role of NA in buffering free Fe in root and leaf cells was inferred. We show here for the first time that the NA concentration is increased in response to iron overload, indicating that, besides other classes of intracellular metal-binding ligands, NA may play an essential role in iron tolerance.

In vitro processing of the proproteins GrdE of protein B of glycine reductase and PrdA of D-proline reductase from Clostridium sticklandii: formation of a pyruvoyl group from a cysteine residue.

GrdE and PrdA of Clostridium sticklandii are subunits of glycine reductase and D-proline reductase, respectively, that are processed post-translationally to form a catalytic active pyruvoyl group. The cleavage occurred on the N-terminal side of a cysteine residue, which is thus the precursor of a pyruvoyl moiety. Both proproteins could be over-expressed in Escherichia coli and conditions were developed for in vitro processing. GrdE could be expressed as full-size protein, whereas PrdA had to be truncated N-terminally to achieve successful over-expression. Both proproteins were cleaved at the in vivo observed cleavage site after addition of 200 mM NaBH4 in Tris buffer (pH 7.6) at room temperature as analysed by SDS/PAGE and MS. Cleavage of GrdE was observed with a half-time of approximately 30 min. Cys242, as the precursor of the pyruvoyl group in GrdE, was changed to alanine, serine, or threonine by site-directed mutagenesis. The Cys242-->Ser and Cys242-->Thr mutant proteins were also cleaved under similar conditions with extended half-times. However, the Cys242-->Ala mutant protein was not cleaved indicating a pivotal role of the thiol group of cysteine or hydroxyl group of serine and threonine during the processing of pyruvoyl group-dependent reductases.