Generation of novel radiolabeled opiates through site-selective iodination

Tritiated opioid radioligands have proven valuable in exploring opioid binding sites. However, tritium has many limitations. Its low specific activity and limited counting efficiency makes it difficult to examine low abundant, high affinity sites and its disposal is problematic due to the need to use organic scintillants and its relatively long half-life. To overcome these issues, we have synthesized both unlabeled and carrier-free radioiodinated iodobenzoyl derivatives of 6β-naltrexamine (¹²⁵I-BNtxA, 18), 6β-naloxamine (¹²⁵I-BNalA, 19) and 6β-oxymorphamine (¹²⁵I-BOxyA, 20) with specific activities of 2100 Ci/mmol. To optimize the utility of the radioligand, we designed a synthesis in which the radiolabel is incorporated in the last synthetic step, which required the selective iodination of the benzoyl moiety without incorporation into the phenolic A ring. Competition studies demonstrated high affinity of the unlabelled compounds for opioid receptors in transfected cell lines, as did the direct binding of the ¹²⁵I-ligands to the opioid receptors. The radioligand displayed very high sensitivity, enabling a marked reduction in tissue, as well as excellent signal/noise characteristics. These new ¹²⁵I-radioligands should prove valuable in future studies of opioid binding sites.     

Identification of calmodulin and MlcC as light chains for Dictyostelium myosin-I isozymes

Dictyostelium discoideum express seven single-headed myosin-I isozymes (MyoA-MyoE and MyoK) that drive motile processes at the cell membrane. The light chains for MyoA and MyoE were identified by expressing Flag-tagged constructs consisting of the motor domain and the two IQ motifs in the neck region in Dictyostelium. The MyoA and MyoE constructs both copurified with calmodulin. Isothermal titration calorimetry (ITC) showed that apo-calmodulin bound to peptides corresponding to the MyoA and MyoE IQ motifs with micromolar affinity. In the presence of calcium, calmodulin cross-linked two IQ motif peptides, with one domain binding with nanomolar affinity and the other with micromolar affinity. The IQ motifs were required for the actin-activated MgATPase activity of MyoA but not MyoE; however, neither myosin exhibited calcium-dependent activity. A Flag-tagged construct consisting of the MyoC motor domain and the three IQ motifs in the adjacent neck region bound a novel 8.6 kDa two EF-hand protein named MlcC, for myosin light chain for MyoC. MlcC is most similar to the C-terminal domain of calmodulin but does not bind calcium. ITC studies showed that MlcC binds IQ1 and IQ2 but not IQ3 of MyoC. IQ3 contains a proline residue that may render it nonfunctional. Each long-tailed Dictyostelium myosin-I has now been shown to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD), whereas the short-tailed myosins-I, MyoA and MyoE, have the multifunctional calmodulin as a light chain. The diversity in light chain composition is likely to contribute to the distinct cellular functions of each myosin-I isozyme.     

Inflammatory macrophages induce Nrf2 transcription factor-dependent proteasome activity in colonic NCM460 cells and thereby confer anti-apoptotic protection

Adaptation of epithelial cells to persistent oxidative stress plays an important role in inflammation-associated carcinogenesis. This adaptation process involves activation of Nrf2 (nuclear factor-E2-related factor-2), which has been recently shown to contribute to carcinogenesis through the induction of proteasomal gene expression and proteasome activity. To verify this possible link between inflammation, oxidative stress, and Nrf2-dependent proteasome activation, we explored the impact of inflammatory (M1) macrophages on the human colon epithelial cell line NCM460. Transwell cocultures with macrophages differentiated from granulocyte monocyte-colony-stimulating factor-treated monocytes led to an increased activity of Nrf2 in NCM460 cells along with an elevated proteasome activity. This higher proteasome activity resulted from Nrf2-dependent induction of proteasomal gene expression, as shown for the 19 and 20 S subunit proteins S5a and α5, respectively. These effects of macrophage coculture were preceded by an increase of reactive oxygen species in cocultured NCM460 cells and could be blocked by catalase or by the reactive oxygen species scavenger Tiron, whereas transient treatment of NCM460 cells with H(2)O(2) similarly led to Nrf2-dependent proteasome activation. Through the Nrf2-dependent increase of proteasomal gene expression and proteasome activity, the sensitivity of NCM460 cells to tumor necrosis factor-related apoptosis-inducing ligand- or irinotecan-induced apoptosis declined. These findings indicate that inflammatory conditions such as the presence of M1 macrophages and the resulting oxidative stress are involved in the Nrf2-dependent gain of proteasome activity in epithelial cells, e.g. colonocytes, giving rise of greater resistance to apoptosis. This mechanism might contribute to inflammation-associated carcinogenesis, e.g. of the colon.     

High-resolution melting genotyping of Enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms

We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 "melting types" (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure.     

Stepwise expansion of the bacteriophage ϕ6 procapsid: possible packaging intermediates

The initial assembly product of bacteriophage ?6, the procapsid, undergoes major structural transformation during the sequential packaging of its three segments of single-stranded RNA. The procapsid, a compact icosahedrally symmetric particle with deeply recessed vertices, expands to the spherical mature capsid, increasing the volume available to accommodate the genome by 2.5-fold. It has been proposed that expansion and packaging are linked, with each stage in expansion presenting a binding site for a particular RNA segment. To investigate procapsid transformability, we induced expansion by acidification, heating, and elevated salt concentration. Cryo-electron microscopy reconstructions after all three treatments yielded the same partially expanded particle. Analysis by cryo-electron tomography showed that all vertices of a given capsid were either in a compact or an expanded state, indicating a highly cooperative transition. To benchmark the mature capsid, we analyzed filled (in vivo packaged) capsids. When these particles were induced to release their RNA, they reverted to the same intermediate state as expanded procapsids (intermediate 1) or to a second, further expanded state (intermediate 2). This partial reversibility of expansion suggests that the mature spherical capsid conformation is obtained only when sufficient outward pressure is exerted by packaged RNA. The observation of two intermediates is consistent with the proposed three-step packaging process. The model is further supported by the observation that a mutant capable of packaging the second RNA segment without previously packaging the first segment has enhanced susceptibility for switching spontaneously from the procapsid to the first intermediate state.     

Glucocorticosteroids modify Langerhans cells to produce TGF-β and expand regulatory T cells

Although glucocorticosteroids (GCSs) have been used for many decades in transplantation and (auto)inflammatory diseases, the exact mechanisms responsible for their immunosuppressive properties are not fully understood. The purpose of this study was to characterize the effects of oral GCSs on the cutaneous immune response. We analyzed, by immunofluorescence staining and quantitative RT-PCR, residual skin biopsy material from a clinical study in which we had used oral GCS as positive control for determining the effects of candidate anti-inflammatory compounds on epicutaneous patch tests of Ni-allergic patients. Expectedly, oral GCS treatment led to a reduction of clinical symptoms and infiltrating leukocytes. Notably, we observed increased numbers of dermal FOXP3(+)CD25(+) T cells and epidermal Langerhans cells (LCs) that were associated with upregulated mRNA expression of TGF-β in lesions of GCS-treated Ni-allergic patients. To investigate this phenomenon further, we exposed purified LCs to GCS. They exhibited, in contrast to GCS-nonexposed LCs, 1) a more immature phenotype, 2) higher intracellular amounts of TGF-β, and 3) increased receptor activator for NF-κB expression, conditions that reportedly favor the expansion of regulatory T cells (Tregs). Indeed, we observed an enhancement of functionally suppressive FOXP3(+) T cells when CD3(+) cells were incubated with GCS-pretreated LCs. The expansion of Tregs was inhibited by TGF-β blockage alone, and their suppressive activity was neutralized by a combination of anti-TGF-β and anti-IL-10 Abs. Our data show that systemically applied GCSs endow LCs with Treg-promoting properties and thus shed new light on the mechanisms of GCS-mediated immunosuppression.     

The Heat-Resistant Agglutinin Family Includes a Novel Adhesin from Enteroaggregative Escherichia coli Strain 60A

Heat-resistant agglutinin 1 (Hra1) is an accessory colonization factor of enteroaggregative Escherichia coli (EAEC) strain 042. Tia, a close homolog of Hra1, is an invasin and adhesin that has been described in enterotoxigenic E. coli. We devised a PCR-restriction fragment length polymorphism screen for the associated genes and found that they occur among 55 (36.7%) of the enteroaggregative E. coli isolates screened, as well as lower proportions of enterotoxigenic, enteropathogenic, enterohemorrhagic, and commensal E. coli isolates. Overall, 25%, 8%, and 3% of 150 EAEC strains harbored hra1 alone, tia alone, or both genes, respectively. One EAEC isolate, 60A, produced an amplicon with a unique restriction profile, distinct from those of hra1 and tia. We cloned and sequenced the full-length agglutinin gene from strain 60A and have designated it hra2. The hra2 gene was not detected in any of 257 diarrheagenic E. coli isolates in our collection but is present in the genome of Salmonella enterica serovar Heidelberg strain SL476. The cloned hra2 gene from strain 60A, which encodes a predicted amino acid sequence that is 64% identical to that of Hra1 and 68% identical to that of Tia, was sufficient to confer adherence on E. coli K-12. We constructed an hra2 deletion mutant of EAEC strain 60A. The mutant was deficient in adherence but not autoaggregation or invasion, pointing to a functional distinction from the autoagglutinin Hra1 and the Tia invasin. Hra1, Tia, and the novel accessory adhesin Hra2 are members of a family of integral outer membrane proteins that confer different colonization-associated phenotypes.     

Aerial Dispersal and Epiphytic Survival of Pseudomonas syringae during a Pretest for the Release of Genetically Engineered Strains into the Environment

Prospective experimental field evaluation of genetically engineered microorganisms, such as microbial pest control agents, raises issues of how to properly ascertain their fate and survival in the environment. Field trials with recombinant organisms must reflect requirements for sampling and monitoring. Field trials were conducted at Tulelake, Calif., to monitor the numbers of viable cells of a nonrecombinant strain of Pseudomonas syringae that entered the atmosphere and landed on plants and soil during and after an aerosol spray application. An exponential decrease in numbers of viable cells deposited at increasing distances from three sprayed plots was observed. The relative rate of survival of cells sprayed directly on plants was more than 10 times higher than that of cells dispersed through the air to similar adjacent plants. Results are being used to gain experience with the characteristics of a release site that influence containment or dispersal and to develop appropriate sampling methodologies for evaluating survival and dispersal characteristics of genetically engineered bacteria released into the environment. The ability to make predictions about microbial dispersal and survival will reduce the uncertainties associated with environmental releases of recombinant organisms.     

The EGFR/ErbB3 Pathway Acts as a Compensatory Survival Mechanism upon c-Met Inhibition in Human c-Met+ Hepatocellular Carcinoma

Backgroundc-Met, a high-affinity receptor for Hepatocyte Growth Factor (HGF), plays a critical role in tumor growth, invasion, and metastasis. Hepatocellular carcinoma (HCC) patients with activated HGF/c-Met signaling have a significantly worse prognosis. Targeted therapies using c-Met tyrosine kinase inhibitors are currently in clinical trials for HCC, although receptor tyrosine kinase inhibition in other cancers has demonstrated early success. Unfortunately, therapeutic effect is frequently not durable due to acquired resistance.MethodsWe utilized the human MHCC97-H c-Met positive (c-Met⁺) HCC cell line to explore the compensatory survival mechanisms that are acquired after c-Met inhibition. MHCC97-H cells with stable c-Met knockdown (MHCC97-H c-Met KD cells) were generated using a c-Met shRNA vector with puromycin selection and stably transfected scrambled shRNA as a control. Gene expression profiling was conducted, and protein expression was analyzed to characterize MHCC97-H cells after blockade of the c-Met oncogene. A high-throughput siRNA screen was performed to find putative compensatory survival proteins, which could drive HCC growth in the absence of c-Met. Findings from this screen were validated through subsequent analyses.ResultsWe have previously demonstrated that treatment of MHCC97-H cells with a c-Met inhibitor, PHA665752, results in stasis of tumor growth in vivo. MHCC97-H c-Met KD cells demonstrate slower growth kinetics, similar to c-Met inhibitor treated tumors. Using gene expression profiling and siRNA screening against 873 kinases and phosphatases, we identified ErbB3 and TGF-α as compensatory survival factors that are upregulated after c-Met inhibition. Suppressing these factors in c-Met KD MHCC97-H cells suppresses tumor growth in vitro. In addition, we found that the PI3K/Akt signaling pathway serves as a negative feedback signal responsible for the ErbB3 upregulation after c-Met inhibition. Furthermore, in vitro studies demonstrate that combination therapy with PHA665752 and Gefitinib (an EGFR inhibitor) significantly reduced cell viability and increased apoptosis compared with either PHA665752 or Gefitinib treatment alone.Conclusionc-Met inhibition monotherapy is not sufficient to eliminate c-Met⁺ HCC tumor growth. Inhibition of both c-Met and EGFR oncogenic pathways provides superior suppression of HCC tumor growth. Thus, combination of c-Met and EGFR inhibition may represent a superior therapeutic regimen for c-Met⁺ HCC.     

A novel endo‐β‐N‐acetylglucosaminidase releases specific N‐glycans depending on different reaction conditions

Milk glycoproteins are involved in different functions and contribute to different cellular processes, including adhesion and signaling, and shape the development of the infant microbiome. Methods have been developed to study the complexities of milk protein glycosylation and understand the role of N‐glycans in protein functionality. Endo‐β‐N‐acetylglucosaminidase (EndoBI‐1) isolated from Bifidobacterium longum subsp. infantis ATCC 15697 is a recently isolated heat‐stable enzyme that cleaves the N‐N′‐diacetyl chitobiose moiety found in the N‐glycan core. The effects of different processing conditions (pH, temperature, reaction time, and enzyme/protein ratio) were evaluated for their ability to change EndoBI‐1 activity on bovine colostrum whey glycoproteins using advanced mass spectrometry. This study shows that EndoBI‐1 is able to cleave a high diversity of N‐glycan structures. Nano‐LC‐Chip–Q‐TOF MS data also revealed that different reaction conditions resulted in different N‐glycan compositions released, thus modifying the relative abundance of N‐glycan types. In general, more sialylated N‐glycans were released at lower temperatures and pH values. These results demonstrated that EndoBI‐1 is able to release a wide variety of N‐glycans, whose compositions can be selectively manipulated using different processing conditions. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1323–1330, 2015