Programmed cell death (PCD) in the form of apoptosis is recognized as one of the central events in the development of the central nervous system. To study the time of onset, extent and distribution of PCD in the human telencephalon, embryos and fetuses from 4.5 to 27 gestational weeks (g.w.) were examined using the TUNEL (TdT-mediated dUTP-biotin nick-end labelling) in situ method. At 4.5 g.w. sparse TUNEL(+) nuclei were observed in the ventricular zone of the neural tube. With the formation of the cortical plate at 7-8 g.w. , TUNEL(+) nuclei were seen in all developmental layers of the cortical anlage, as well as in the subcortical regions such as the ganglionic eminence and the internal capsule. The proliferative zones (the ventricular zone, the subventricular zone and the ganglionic eminence) contained the majority of all apoptotic nuclei observed in each specimen. However, the apoptotic index was highest in the subplate zone and in layer I. Double-labelling experiments suggested that neuronal precursors were the main population of cells undergoing PCD in the first trimester of gestation, whereas glial cells probably start dying around midgestation. The onset of labelling of microglial cells and apoptotic nuclei were synchronous, indicating the involvement of microglia in PCD. In conclusion, two distinct types of PCD were observed during human telencephalic development: embryonic apoptosis, which was synchronous with proliferation and migration of neuronal cells and probably not related to establishment of neuronal circuitry, and fetal apoptosis, which coincided with differentiation and synaptogenesis, and therefore may be related to the development of axonal-target connectivity.

The thermodynamic and structural cooperativity between the Ser45- and D128-biotin hydrogen bonds was measured by calorimetric and X-ray crystallographic studies of the S45A/D128A double mutant of streptavidin. The double mutant exhibits a binding affinity approximately 2x10(7) times lower than that of wild-type streptavidin at 25 degrees C. The corresponding reduction in binding free energy (DeltaDeltaG) of 10.1 kcal/mol was nearly completely due to binding enthalpy losses at this temperature. The loss of binding affinity is 11-fold greater than that predicted by a linear combination of the single-mutant energetic perturbations (8.7 kcal/mol), indicating that these two mutations interact cooperatively. Crystallographic characterization of the double mutant and comparison with the two single mutant structures suggest that structural rearrangements at the S45 position, when the D128 carboxylate is removed, mask the true energetic contribution of the D128-biotin interaction. Taken together, the thermodynamic and structural analyses support the conclusion that the wild-type hydrogen bond between D128-OD and biotin-N2 is thermodynamically stronger than that between S45-OG and biotin-N1.

Formalin is a commonly used fixative for tissue preservation in pathology laboratories. A major adverse effect of this fixative is the concealing of tissue antigens by protein cross-linking. To achieve a universal antigen retrieval method for immunohistochemistry under a constant condition, we developed a new method in which the effects of formalin fixation were reversed with citraconic anhydride (a reversible protein cross-linking agent) plus heating. Formalin-fixed, paraffin-embedded tissues from various organs were examined for immunohistochemical localization of a wide variety of antigens. Deparaffinized tissue sections were placed in an electric kitchen pot containing 0.05% citraconic anhydride solution, pH 7.4, and the pot was set at "keep warm" temperature mode of 98C for 45 min. This mode allowed heating the sections at a constant temperature. The sections were then washed in buffer solution and immunostained using a labeled streptavidin-biotin method using an automated stainer. In general, formalin-fixed tissues demonstrated specific immunostainings comparable to that in fresh frozen tissues and significantly more enhanced than after conventional antigen retrieval methods. In particular, even difficult-to-detect antigens such as CD4, cyclin D1, granzyme beta, bcl-6, CD25, and lambda chain revealed distinct immunostainings. Different classes of antigens such as cellular markers and receptors, as well as cytoplasmic and nuclear proteins, consistently produced enhanced reactions. This method provides efficient antigen retrieval for successful immunostaining of a wide variety of antigens under an optimized condition. It also allows standardization of immunohistochemistry for formalin-fixed tissues in pathology laboratories, eliminating inter-laboratory discrepancies in results for accurate clinical and research studies.

OBJECTIVE

The antitumor effect of bee honey against bladder cancer was examined in vitro and in vivo.

METHODS

Three human bladder cancer cell lines (T24, 253J and RT4) and one murine bladder cancer cell line (MBT-2) were used in these experiments. In an in vitro study, the antitumor activity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay, 5-Bromodeoxyuridine (BrdU) labeling index and flowcytometry (FCM). In the in vivo study, cancer cells were implanted subcutaneously in the abdomens of mice, and the effects were assessed by the tumor growth.

RESULTS

In vitro studies revealed significant inhibition of the proliferation of T24 and MBT-2 cell lines by 1-25% honey and of RT4 and 253J cell lines by 6-25% honey. BrdU labeling index was significantly lower. FCM showed lower S-phase fraction, as well as absence of aneuploidy compared with control cells. In the in vivo studies, intralesional injection of 6 and 12% honey as well as oral ingestion of honey significantly inhibited tumor growth.

CONCLUSIONS

Bee honey is an effective agent for inhibiting the growth of T24, RT4, 253J and MBT-2 bladder cancer cell lines in vitro. It is also effective when administered intralesionally or orally in the MBT-2 bladder cancer implantation models. Our results are promising, and further research is needed to clarify the mechanisms of the antitumor activity of honey.

The distribution of the ribosomal genes and their ribosomal RNA (rRNA) products in the different compartments of the nucleolus of HeLa cells was examined on thin sections of Lowicryl embedded material. The ribosomal nucleic acids were visualized after hybridization with a set of biotinylated double-stranded ribosomal DNA (rDNA) probes from different locations along the gene, followed by immunogold labelling of biotin. Ribosomal genes were detected over both the entire fibrillar centres (FCs) and some masses of intranucleolar condensed chromatin. As for the rRNA components, comparison of the signal levels obtained with the different probes provides some information about the compartmentalization of distinct stages of ribosome biogenesis. Thus a probe specific for the 5' external transcribed spacer (5'ETS) portion of pre-rRNA labels almost exclusively the dense fibrillar component (DFC) and the border of the FCs, while the interior of the FCs appears devoid of any kind of rRNA species. By contrast, probes recognizing either 18S or 28S mature rRNA sequences label both the DFC and the granular component (GC). Moreover, mature 18S rRNA sequences are markedly under-represented relative to mature 28S rRNA sequences in the GC, as compared with the other nucleolar compartments. Our observations are consistent with the view that DFCs contain elongating and 47S-45S precursor rRNA molecules whereas the subsequent various rRNA processing intermediates are mainly located within the GC. Since the border of FCs is the only site where both rDNA and newly synthesized pre-rRNA coexist, the transcription of ribosomal genes seems to take place at the periphery of the FCs, and not in the DFC, suggesting that elongating and newly completed transcripts are immediately transferred into the surrounding DFC where they transiently accumulate before undergoing processing reactions and transfer to the GC.

A Phase II study of yttrium-90-tetra-azacyclododecanetetra-acetic acid-biotin (90Y-DOTA-biotin) pretargeted by NR-LU-10 antibody/streptavidin (SA) was performed. The primary objectives of the study were to evaluate the efficacy and safety of this therapy in patients with metastatic colon cancer. Twenty-five patients were treated with a single dose of 110 mCi/m2 (mean administered dose, 106.5 +/- 10.3 mCi/m2) of 90Y-DOTA-biotin. There were three components of the therapy. Patients first received NR-LU-10/SA on day 1. A clearing agent (biotin-galactose-human serum albumin) was administered approximately 48 h after the NR-LU-10/SA to remove residual circulating unbound NR-LU-10/SA. Lastly, 24 h after administration of clearing agent, patients received biotin-DOTA-labeled with 110 mCi/m2 90Y. All three components of the therapy were administered i.v. Both hematological and nonhematological toxicities were observed. Diarrhea was the most frequent grade 4 nonhematological toxicity (16%; with 16% grade 3 diarrhea). Hematological toxicity was less severe with 8% grade 3 and 8% grade 4 neutropenia and 8% grade 3 and 16% grade 4 thrombocytopenia. The overall response rate was 8%. Two partial responders had freedom from progression of 16 weeks. Four patients (16%) had stable disease with freedom from progression of 10-20 weeks. Despite the relatively disappointing results of this study in terms of therapeutic efficacy and toxicity, proof of principle was obtained for the pretargeting approach. In addition, valuable new information was obtained about normal tissue tolerance to low-dose-rate irradiation that will help to provide useful guidelines for future study designs.

Colicin Ia, a bacterial protein toxin of 626 amino acid residues, forms voltage-dependent channels in planar lipid bilayer membranes. We have exploited the high affinity binding of streptavidin to biotin to map the topology of the channel-forming domain (roughly 175 residues of the COOH-terminal end) with respect to the membrane. That is, we have determined, for the channel's open and closed states, which parts of this domain are exposed to the aqueous solutions on either side of the membrane and which are inserted into the bilayer. This was done by biotinylating cysteine residues introduced by site-directed mutagenesis, and monitoring by electrophysiological methods the effect of streptavidin addition on channel behavior. We have identified a region of at least 68 residues that flips back and forth across the membrane in association with channel opening and closing. This identification was based on our observations that for mutants biotinylated in this region, streptavidin added to the cis (colicin-containing) compartment interfered with channel opening, and trans streptavidin interfered with channel closing. (If biotin was linked to the colicin by a disulfide bond, the effects of streptavidin on channel closing could be reversed by detaching the streptavidin-biotin complex from the colicin, using a water-soluble reducing agent. This showed that the cysteine sulfur, not just the biotin, is exposed to the trans solution). The upstream and downstream segments flanking the translocated region move into and out of the bilayer during channel opening and closing, forming two transmembrane segments. Surprisingly, if any of several residues near the upstream end of the translocated region is held on the cis side by streptavidin, the colicin still forms voltage-dependent channels, indicating that a part of the protein that normally is fully translocated across the membrane can become the upstream transmembrane segment. Evidently, the identity of the upstream transmembrane segment is not crucial to channel formation, and several open channel structures can exist.

Resistance and susceptibility of macrophages to mycobacteria are under the control of the Bcg/Nramp1 gene, which also controls the NO- production in response to macrophage activators. There is recent evidence indicating that mycobacteria induces apoptosis in infected macrophages. Using murine macrophage lines, congenic at the Bcg/Nramp1 gene, this report shows that B10R are more prone than B10S macrophages to undergo apoptosis after exposure to live virulent Mycobacterium tuberculosis H37Rv (Mtb) or PPD, as determined by cell viability, DNA fragmentation, hypoploidy, and the terminal deoxynucleotide transferase dUTP-biotin nick-end labeling assay. Induction of apoptosis correlated with NO- production. Aminoguanidine and anti-TNF-alpha inhibited NO- production and apoptosis. B10R and B10S macrophages were equally affected by sodium nitroprusside, a donor of NO-, but its effect, mainly in B10R cells, was enhanced by the presence of Mtb. Nonvirulent mycobacteria induced lower levels of NO- and did not cause cell death. Killed Mtb, mannose-capped lipoarabinomannan (ManLAM), and LPS rescued macrophages from apoptosis albeit induce NO-. These findings suggest the existence of opposite pathways: metabolically active mycobacteria promotes apoptosis whereas their structural components inhibit it. Apoptosis may be a critical mechanism by which Nramp1 gene controls the macrophage infection with virulent mycobacteria.

To test our hypothesis that substitution of domain III of Bacillus thuringiensis delta-endotoxin (Cry) proteins might improve toxicity to pest insects, e.g., Spodoptera exigua, in vivo recombination was used to produce a number of cryIA(b)-cryIC hybrid genes. A rapid screening assay was subsequently exploited to select hybrid genes encoding soluble protoxins. Screening of 120 recombinants yielded two different hybrid genes encoding soluble proteins with domains I and II of CryIA(b) and domain III of CryIC. These proteins differed by only one amino acid residue. Both hybrid protoxins gave a protease-resistant toxin upon in vitro activation by trypsin. Bioassays showed that one of these CryIA(b)-CryIC hybrid proteins (H04) was highly toxic to S. exigua compared with the parental CryIA(b) protein and significantly more toxic than CryIC. In semiquantitative binding studies with biotin-labelled toxins and intact brush border membrane vesicles of S. exigua, this domain III substitution appeared not to affect binding-site specificity. However, binding to a 200-kDa protein by CryIA(b) in preparations of solubilized and blotted brush border membrane vesicle proteins was completely abolished by the domain III substitution. A reciprocal hybrid containing domains I and II of CryIC and domain III of CryIA(b) did bind to the 200-kDa protein, confirming that domain III of CryIA(b) was essential for this reaction. These results show that domain III of CryIC protein plays an important role in the level of toxicity to S. exigua, that substitution of domain III may be a powerful tool to increase the repertoire of available active toxins for pest insects, and that domain III is involved in binding to gut epithelium membrane proteins of S. exigua.

We describe a method to postlabel, in vivo, biotinylated monoclonal antibodies pretargeted onto tumor deposits when most of the non-tumor-bound antibodies have already been cleared as avidin-bound complexes. The application of this principle to tumor detection by immunoscintigraphy was tested in 20 patients with histologically documented cancer and increased circulating carcinoembryonic antigen levels. One mg of biotinylated anti-carcinoembryonic antigen monoclonal antibody (FO23C5) was administered i.v. (first step). After 3 days, 4-6 mg of cold avidin were injected i.v. (second step), followed 48 h later by 0.2-0.3 mg of a biotin derivative labeled with 111In (2-3 mCi) (third step). No evidence of toxicity was observed. Whole body radioactivity distribution was measured in five patients at various intervals postinjection by the conjugate counting technique. Tumors and metastases were detected in 18 of 19 patients (the remaining patient was a true negative) within 3 h after administration of 111In-biotin by planar or single photon emission tomography imaging. At the time of imaging, tumor/blood pool ratio was 5.5 +/- 3.2, and tumor/liver ratio was 6.7 +/- 3.9. Blood clearance of 111In-biotin was multiexponential, with the fast component having a t1/2 of 5 +/- 3 min. Urinary excretion of radioactivity over 3 h was 63.5 +/- 4.9% of the injected dose. Radioactivity at 3 h was 6.5 +/- 1.8% in blood, 1.6 +/- 0.3% in the kidney, and 2.4 +/- 0.6% in the liver. This approach represents an improvement in immunoscintigraphic techniques for tumor localization. The potential use for radioimmunotherapy is discussed.

We demonstrate by immunofluorescence that a 70-kD protein (P70) purified from Xenopus egg extracts is associated with subnuclear foci (about 200) which we propose to be an assembly of DNA pre-replication centers (preRCs). A cDNA encoding this protein reveals that P70 is the Xenopus homologue of replication protein A (RPA also called RF-A). RPA is know to be a cellular, three-subunit single-stranded DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. The punctated preRCs exist transiently; they form post-mitotically during the period of nuclear membrane breakdown and disappear during ongoing DNA replication. P70 is homogeneously associated with chromatin at the later stages of the S-phase and is displaced from chromatin post replication, so that P70 cannot be detected on mitotic chromosomes. Double-immunofluorescence studies using biotin-dUTP demonstrate that initiation of DNA synthesis is confined to preRCs, resulting in the punctated replication pattern observed previously by others. PreRCs form efficiently on decondensed chromatin in membrane-free egg extracts if ATP and divalent cations are present. Our results suggest that preRCs are composed of an assembly of a large number of pre-initiation replication complexes poised for initiation at discreet subnuclear regions prior to nuclear reconstruction and initiation of DNA synthesis.

The impact of various growth conditions on the expression of toxins and other proteins by Clostridium difficile VPI 10463 was studied. During non-starved conditions, the rate of toxin synthesis paralleled that of total protein during both exponential growth and stationary phase, and in both defined and complex media. Biotin limitation reduced growth rate and bulk protein synthesis, whereas toxin expression continued, leading to a 50- to 200-fold increase in intracellular toxin levels. Concomitantly, several 22 kDa proteins were up-regulated as revealed by two-dimensional PAGE analysis. The toxin yield was 30-fold higher in peptone yeast extract (PY) than in PY containing glucose (PYG). By contrast, glucose limitation reduced toxin yields by 20- to 100-fold in defined media. By elevating the buffering capacity and bicarbonate concentration, toxin yields were increased by 10-fold in PY and PYG. The high toxin production by C. difficile during growth in PY was lowered 100-fold by adding a blend of nine amino acids and several 60-100 kDa proteins were concomitantly down-regulated. It was concluded that toxin expression in C. difficile VPI 10463 was not affected by growth rate, growth phase, catabolite repression or the stringent response. Instead the co-expression of toxins and a few specific additional proteins appeared to be influenced by metabolic pathways involving CO2 assimilation, carboxylation reactions and metabolism of certain amino acids.

Radioimmunotherapy with anti-CD20 monoclonal antibodies is a promising new treatment approach for patients with relapsed B-cell lymphomas. However, the majority of patients treated with conventional radiolabeled anti-CD20 antibodies eventually have a relapse because the low tumor-to-blood and tumor-to-normal organ ratios of absorbed radioactivity limit the dose that can be safely administered without hematopoietic stem cell support. This study assessed the ability of a streptavidin-biotin "pretargeting" approach to improve the biodistribution of radioactivity in mice bearing Ramos lymphoma xenografts. A pretargeted streptavidin-conjugated anti-CD20 1F5 antibody was infused, followed 24 hours later by a biotinylated N-acetylgalactosamine-containing "clearing agent" and finally 3 hours later by (111)In-labeled DOTA-biotin. Tumor-to-blood ratios were 3:1 or more with pretargeting, compared with 0.5:1 or less with conventional (111)In-1F5. Tumor-to-normal organ ratios of absorbed radioactivity up to 56:1 were observed with pretargeting, but were 6:1 or less with conventional (111)In-1F5. Therapy experiments demonstrated that 400 microCi (14.8 MBq) or more of conventional (90)Y-1F5 was required to obtain major tumor responses, but this dose was associated with lethal toxicity in 100% of mice. In marked contrast, up to 800 microCi (29.6 MBq) (90)Y-DOTA-biotin could be safely administered by the pretargeting approach with only minor toxicity, and 89% of the mice were cured. These data suggest that anti-CD20 pretargeting shows great promise for improving current therapeutic options for B-cell lymphomas and warrants further preclinical and clinical testing.

The detection of biological molecules and their interactions is a significant component of modern biomedical research. In current biosensor technologies, simultaneous detection is limited to a small number of analytes by the spectral overlap of their signals. We have developed an NMR-based xenon biosensor that capitalizes on the enhanced signal-to-noise, spectral simplicity, and chemical-shift sensitivity of laser-polarized xenon to detect specific biomolecules at the level of tens of nanomoles. We present results using xenon "functionalized" by a biotin-modified supramolecular cage to detect biotin-avidin binding. This biosensor methodology can be extended to a multiplexing assay for multiple analytes.

The flagellins of Campylobacter spp. differ antigenically. In variants of C. coli strain VC167, two antigenic flagellin types determined by sero-specific antibodies have been described (termed T1 and T2). Post-translational modification has been suggested to be responsible for T1 and T2 epitopes, and, using mild periodate treatment and biotin hydrazide labelling, flagellin from both VC167-T1 and T2 were shown to be glycosylated. Glycosylation was also shown to be present on other Campylobacter flagellins. The ability to label all Campylobacter flagellins examined with the lectin LFA demonstrated the presence of a terminal sialic acid moiety. Furthermore, mild periodate treatment of the flagellins of VC167 eliminated reactivity with T1 and T2 specific antibodies LAH1 and LAH2, respectively, and LFA could also compete with LAH1 and LAH2 antibodies for binding to their respective flagellins. These data implicate terminal sialic acid as part of the LAH strain-specific epitopes. However, using mutants in genes affecting LAH serorecognition of flagellin it was demonstrated that sialic acid alone is not the LAH epitope. Rather, the epitope(s) is complex, probably involving multiple glycosyl and/or amino acid residues.

This study provides a critical examination of protein labeling with Cy3, Cy5, and other Cy dyes. Two alternate situations were tested. (i) Antibodies were covalently labeled with Cy dye succinimidyl ester at various fluorophore/protein ratios and the fluorescence of the labeled antibodies was compared to that of free Cy dye. (ii) Fluorescent <em>biotin</em> derivatives were synthesized by derivatizing ethylenediamine with one <em>biotin</em> and one Cy3 (or Cy5) residue. The fluorescence properties of these <em>biotin</em>-Cy dye conjugates were examined at all ligand/(strept)avidin ratios (0 </= n </= 4). The results showed an astounding discrepancy between Cy3 and Cy5: Cy3-labeled antibodies fluoresced very well, even at high Cy3/protein ratios, and the same applied to (strept)avidin with up to four bound <em>biotin</em>-Cy3 conjugates. In contrast, antibodies with six covalently bound Cy5 labels (obtained with the recommended procedure) were almost nonfluorescent, only at 2-3 Cy5 labels/IgG some moderate fluorescence was obtained. By analogy, the <em>biotin</em>-Cy3 conjugate fluoresced intensely, even at high ligand/avidin ratio, in contrast to the weakly fluorescing <em>biotin</em>-Cy5 conjugate. Three mechanisms are responsible for the discrepancy between Cy3 and Cy5. (i) Attachment of Cy3 to a protein's surface causes an anomalous enhancement in fluorescence (by 2-3-fold) while no enhancement occurs with Cy5. (ii) Mutual quenching of IgG-bound Cy dyes by resonance energy transfer is much more pronounced for Cy5 labels than for Cy3. (iii) In IgG with six bound Cy5 labels, about one-third of the labels adopt a nonfluorescent state which is characterized by a large UV-vis absorption maximum at 600 nm instead of at 650 nm. Cy3.5 was found to mimick the properties of Cy3, while Cy7, and to some extent also Cy5.5, were similar to Cy5. In conclusion the Cy dye series is divided into two groups: Antibodies with multiple Cy3 or Cy3.5 labels yield bright fluorescence while extensive quenching occurs in antibodies labeled with Cy5 and Cy7.

Recently, it has become increasingly evident that fracture healing involves a complex interaction of many local and systemic regulatory factors. The roles of some of these growth factors have been described; however, little is understood about the presence of the bone morphogenetic proteins in fracture repair, despite the fact that they are the most potent osteoinductive proteins known. This study defines and characterizes the physiologic presence, localization, and chronology of the bone morphogenetic proteins in fracture healing with an established rat fracture healing model. With use of a recently developed monoclonal antibody against bone morphogenetic proteins 2 and 4 developed with standard avidin-biotin complex/immunoperoxidase protocols, frozen undecalcified fracture calluses were analyzed semiquantitatively for the percentage of various types of fracture cells staining positively. During the early stages of fracture healing, only a minimum number of primitive cells stained positively in the fracture callus. As the process of endochondral ossification proceeded, the presence of bone morphogenetic proteins 2 and 4 increased dramatically, especially in the primitive mesenchymal and chondrocytic cells. While the cartilaginous component of the callus matured with a concomitant decrease in the number of primitive cells, there was a concomitant decrease in both the intensity and the number of positively staining cells. As osteoblasts started to lay down woven bone on the chondroid matrix, these osteoblastic cells exhibited strong positive staining. The intensity of this staining decreased, however, as lamellar bone replaced the primitive woven bone. A similar observation was noted for the areas of the callus undergoing intramembranous ossification. Initially, within several days after the fracture, periosteal cells and osteoblasts exhibited intense staining for bone morphogenetic proteins 2 and 4. As the woven bone was replaced with mature lamellar bone, this staining decreased. These data, and the awareness of the strong osteoinductive capacities of bone morphogenetic protein, suggest that bone morphogenetic proteins 2 and 4 are important regulators of cell differentiation during fracture repair.

The catalytic subunit of cAMP-dependent protein kinase (cAPK) is susceptible to inactivation by a number of thiol-modifying reagents. Inactivation occurs through modification of cysteine 199, which is located near the active site. Because cysteine 199 is reactive at physiological pH, and modification of this site inhibits activity, we hypothesized that cAPK is a likely target for regulation by an oxidative mechanism, specifically glutathionylation. In vitro studies demonstrated the susceptibility of kinase activity to the sulfhydryl oxidant diamide, which inhibited by promoting an intramolecular disulfide bond between cysteines 199 and 343. In the presence of a low concentration of diamide and reduced glutathione, the kinase was rapidly and reversibly inhibited by glutathionylation. Mutant kinase containing an alanine to cysteine mutation at position 199 was resistant to inhibition by both diamide and glutathionylation, thus implicating this as the oxidation-sensitive site. Mouse fibroblast cells treated with diamide showed a reversible decrease in cAPK activity. Inhibition was dramatically enhanced when cells were first treated with cAPK activators. Using biotin-cysteine as means for detecting and purifying thiolated cAPK from cells, we were able to show that, under conditions in which cAPK is inactivated by diamide, it is also readily thiolated.

BACKGROUND

MicroRNAs (miRNAs) bind to mRNAs and target them for translational inhibition or transcriptional degradation. It is thought that most miRNA-mRNA interactions involve the seed region at the 5' end of the miRNA. The importance of seed sites is supported by experimental evidence, although there is growing interest in interactions mediated by the central region of the miRNA, termed centered sites. To investigate the prevalence of these interactions, we apply a biotin pull-down method to determine the direct targets of ten human miRNAs, including four isomiRs that share centered sites, but not seeds, with their canonical partner miRNAs.

RESULTS

We confirm that miRNAs and their isomiRs can interact with hundreds of mRNAs, and that imperfect centered sites are common mediators of miRNA-mRNA interactions. We experimentally demonstrate that these sites can repress mRNA activity, typically through translational repression, and are enriched in regions of the transcriptome bound by AGO. Finally, we show that the identification of imperfect centered sites is unlikely to be an artifact of our protocol caused by the biotinylation of the miRNA. However, the fact that there was a slight bias against seed sites in our protocol may have inflated the apparent prevalence of centered site-mediated interactions.

CONCLUSIONS

Our results suggest that centered site-mediated interactions are much more frequent than previously thought. This may explain the evolutionary conservation of the central region of miRNAs, and has significant implications for decoding miRNA-regulated genetic networks, and for predicting the functional effect of variants that do not alter protein sequence.

The gene product of an open reading frame of the chloroplast genome, accD, that has sequence similarity with a subunit of acetyl-CoA carboxylase from Escherichia coli was detected immunochemically in pea chloroplasts. The apparent molecular mass of the accD protein was 87 kDa on SDS-polyacrylamide gel electrophoresis. The protein was acidic and had less mobility than the calculated value, 67,116. Acetyl-CoA carboxylase activity solubilized from pea chloroplasts was inhibited by antibodies against recombinant accD protein. The antibodies precipitated a polypeptide of 35 kDa containing biotin and a polypeptide of 91 kDa together with the 87-kDa-accD protein. The accD protein formed a complex with the molecular mass of about 700 kDa, probably with the 35- and 91-kDa proteins. These results indicate that the chloroplast-encoded polypeptide, accD protein, is a component of a functional acetyl-CoA carboxylase in chloroplasts and this enzyme is a multi-subunit complex, like that from E. coli. The synthesis of accD protein was not induced by light.

BACKGROUND

Propionibacterium freudenreichii is essential as a ripening culture in Swiss-type cheeses and is also considered for its probiotic use. This species exhibits slow growth, low nutritional requirements, and hardiness in many habitats. It belongs to the taxonomic group of dairy propionibacteria, in contrast to the cutaneous species P. acnes. The genome of the type strain, P. freudenreichii subsp. shermanii CIRM-BIA1 (CIP 103027(T)), was sequenced with an 11-fold coverage.

RESULTS

The circular chromosome of 2.7 Mb of the CIRM-BIA1 strain has a GC-content of 67% and contains 22 different insertion sequences (3.5% of the genome in base pairs). Using a proteomic approach, 490 of the 2439 predicted proteins were confirmed. The annotation revealed the genetic basis for the hardiness of P. freudenreichii, as the bacterium possesses a complete enzymatic arsenal for de novo biosynthesis of aminoacids and vitamins (except panthotenate and biotin) as well as sequences involved in metabolism of various carbon sources, immunity against phages, duplicated chaperone genes and, interestingly, genes involved in the management of polyphosphate, glycogen and trehalose storage. The complete biosynthesis pathway for a bifidogenic compound is described, as well as a high number of surface proteins involved in interactions with the host and present in other probiotic bacteria. By comparative genomics, no pathogenicity factors found in P. acnes or in other pathogenic microbial species were identified in P. freudenreichii, which is consistent with the Generally Recognized As Safe and Qualified Presumption of Safety status of P. freudenreichii. Various pathways for formation of cheese flavor compounds were identified: the Wood-Werkman cycle for propionic acid formation, amino acid degradation pathways resulting in the formation of volatile branched chain fatty acids, and esterases involved in the formation of free fatty acids and esters.

CONCLUSIONS

With the exception of its ability to degrade lactose, P. freudenreichii seems poorly adapted to dairy niches. This genome annotation opens up new prospects for the understanding of the P. freudenreichii probiotic activity.

Stimuli-responsive polymers exhibit reversible phase changes in response to changes in environmental factors such as pH or temperature. Conjugating such polymers to antibodies and proteins provides molecular systems for applications such as affinity separations, immunoassays and enzyme recovery and recycling. Here we show that conjugating a temperature-sensitive polymer to a genetically engineered site on a protein allows the protein's ligand binding affinity to be controlled. We synthesized a mutant of the protein streptavidin to enable site-specific conjugation of the responsive polymer near the protein's binding site. Normal binding of biotin to the modified protein occurs below 32 degrees C, whereas above this temperature the polymer collapses and blocks binding. The collapse of the polymer and thus the enabling and disabling of binding, is reversible. Such environmentally triggered control of binding may find many applications in biotechnology and biomedicine, such as the control of enzyme reaction rates and of biosensor activity, and the controlled release of drugs.

Lipid peroxidation (LPO) is induced by a variety of abiotic and biotic stresses. Although LPO is involved in diverse signaling processes, little is known about the oxidation mechanisms and major lipid targets. A systematic lipidomics analysis of LPO in the interaction of Arabidopsis (Arabidopsis thaliana) with Pseudomonas syringae revealed that LPO is predominantly confined to plastid lipids comprising galactolipid and triacylglyceride species and precedes programmed cell death. Singlet oxygen was identified as the major cause of lipid oxidation under basal conditions, while a 13-lipoxygenase (LOX2) and free radical-catalyzed lipid oxidation substantially contribute to the increase upon pathogen infection. Analysis of lox2 mutants revealed that LOX2 is essential for enzymatic membrane peroxidation but not for the pathogen-induced free jasmonate production. Despite massive oxidative modification of plastid lipids, levels of nonoxidized lipids dramatically increased after infection. Pathogen infection also induced an accumulation of fragmented lipids. Analysis of mutants defective in 9-lipoxygenases and LOX2 showed that galactolipid fragmentation is independent of LOXs. We provide strong in vivo evidence for a free radical-catalyzed galactolipid fragmentation mechanism responsible for the formation of the essential biotin precursor pimelic acid as well as of azelaic acid, which was previously postulated to prime the immune response of Arabidopsis. Our results suggest that azelaic acid is a general marker for LPO rather than a general immune signal. The proposed fragmentation mechanism rationalizes the pathogen-induced radical amplification and formation of electrophile signals such as phytoprostanes, malondialdehyde, and hexenal in plastids.