MOP and NOP receptor interaction: Studies with a dual expression system and bivalent peptide ligands.

Opioids targeting mu;µ (MOP) receptors produce analgesia in the peri-operative period and palliative care. They also produce side effects including respiratory depression, tolerance/dependence and addiction. The N/OFQ opioid receptor (NOP) also produces analgesia but is devoid of the major MOP side effects. Evidence exists for MOP-NOP interaction and mixed MOP-NOP ligands produce analgesia with reduced side effects. We have generated a HEKMOP/NOP human expression system and used bivalent MOP-NOP and fluorescent ligands to (i) probe for receptor interaction and (ii) consequences of that interaction. We used HEKMOP/NOP cells and two bivalent ligands; Dermorphin-N/OFQ (MOP agonist-NOP agonist; DeNO) and Dermorphin-UFP101 (MOP agonist-NOP antagonist; De101). We have determined receptor binding profiles, GTPγ[35S] binding, cAMP formation and ERK1/2 activation. We have also probed MOP and NOP receptor interactions in HEK cells and hippocampal neurones using the novel MOP fluorescent ligand, DermorphinATTO488 and the NOP fluorescent ligand N/OFQATTO594. In HEKMOP/NOP MOP ligands displaced NOP binding and NOP ligands displaced MOP binding. Using fluorescent probes in HEKMOP/NOP cells we demonstrated MOP-NOP probe overlap and a FRET signal indicating co-localisation. MOP-NOP were also co-localised in hippocampal tissue. In GTPγ[35S] and cAMP assays NOP stimulation shifted the response to MOP rightwards. At ERK1/2 the response to bivalent ligands generally peaked later. We provide evidence for MOP-NOP interaction in recombinant and native tissue. NOP activation reduces responsiveness of MOP activation; this was shown with conventional and bivalent ligands.

A phase II study (ARCHER 1042) to evaluate prophylactic treatment of dacomitinib-induced dermatologic and gastrointestinal adverse events in advanced non-small-cell lung cancer.

BACKGROUND: ARCHER 1042, a randomized phase II trial, explored the impact of prophylactic treatment on select dermatologic adverse events of interest (SDAEI), diarrhea, and mucositis associated with dacomitinib, an oral irreversible pan-human epidermal growth factor receptor (HER) inhibitor, in development for advanced non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients with advanced NSCLC treated with dacomitinib were enrolled in two cohorts. Cohort I patients were randomized 1:1 to receive oral doxycycline or placebo (4 weeks). Cohort II patients received oral VSL#3 probiotic plus topical alclometasone. Primary end points for Cohorts I and II were incidence of all grade and grade ≥2 SDAEI in the first 8 weeks of treatment and quality of life (QoL) assessed by the Skindex-16 survey. Additional primary end points for Cohort II were incidence of all grade and grade ≥2 diarrhea and mucositis in the first 8 weeks of treatment; QoL regarding diarrhea and mucositis incidence was assessed by the modified-Oral Mucositis Daily Questionnaire. RESULTS: Cohort I randomized 114 evaluable patients: 56 in the doxycycline arm, 58 in the placebo arm. Cohort II enrolled 59 evaluable patients. Doxycycline significantly reduced the incidence of grade ≥2 SDAEI by 50% (P = 0.016) compared with placebo. The incidence of all grade SDAEI was lower with doxycycline than with placebo but did not reach statistical significance. Doxycycline was associated with less deterioration in QoL compared with placebo. Alclometasone was associated with less deterioration in QoL compared with placebo but did not statistically significantly reduce the incidence of all grade or grade ≥2 SDAEI. VSL#3 did not reduce the incidence of all grade or grade ≥2 diarrhea and did not impact mucositis scores. CONCLUSIONS: Doxycycline was effective as a prophylactic treatment for dacomitinib-induced grade ≥2 SDAEI. Both doxycycline and alclometasone reduced the negative impact in patient-reported dermatologic AEs. The probiotic was not effective for preventing diarrhea or mucositis.

Activation of purinergic receptors induces proliferation and neuronal differentiation in Swiss Webster mouse olfactory epithelium.

In the central nervous system (CNS), adenosine 5'-triphosphate (ATP) induces the synthesis and release of neurotrophic factors, cell proliferation, and differentiation. The olfactory system is one site where multipotent progenitor cells continue to proliferate and differentiate into neurons throughout life. We tested the hypothesis that ATP initiates proliferation in olfactory epithelium by measuring 5-bromo-2-deoxyuridine incorporation. Adult mice were pre-treated intraperitoneally (i.p.) or intranasally with saline or purinergic receptor antagonists (pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate+suramin) 30 min prior to nasal instillation of ATP, uridine 5'-triphosphate (UTP), adenosine 5'-(3-thiotriphosphate) (ATP gamma S) or saline (0 h). Mice received three injections of 5-bromo-2-deoxyuridine between 42 and 46 h, and were sacrificed at 2, 9 or 16 days post-ATP instillation. ATP, UTP or ATP gamma S significantly increased 5-bromo-2-deoxyuridine incorporation compared to intranasal saline controls in groups pre-treated with saline. Saline, ATP, UTP or ATP gamma S instillation did not significantly increase 5-bromo-2-deoxyuridine incorporation in groups pre-treated with purinergic receptor antagonists. Similar results were observed in neonates and in a cultured slice preparation. Intranasal instillation of ATP also increased the protein levels of proliferating cell nuclear antigen in adults. Pre-treatment with purinergic receptor antagonists inhibited the ATP-induced increase in proliferating cell nuclear antigen. In adults, a subset of the cells that incorporated 5-bromo-2-deoxyuridine was immunoreactive to neuronal markers mammalian achaete-schute homolog 1, growth-associated protein 43, and olfactory marker protein at 2, 9, and 16 days, respectively. Collectively, these data indicate that purinergic receptor activation induces proliferation and neuronal differentiation in the mouse olfactory epithelium. We propose that extracellular ATP released upon injury could induce proliferation and promote the neuroregeneration of the olfactory epithelium.

China's one-child policy: the economic choices and consequences faced by pregnant women.

This paper addresses the important issue of the effect of China's one-child policy on prenatal and obstetric care utilization. The paper provides the first detailed empirical approach to this question, exploiting a unique high quality household survey. China officially codified a set of rules and regulations in 1979 governing the approved size of Chinese families, commonly known as the one-child policy. The policy imposed economic and social costs on families failing to adhere to the family size limits. In particular, the policy raised the price of obstetric medical services for unapproved pregnancies in comparison to approved pregnancies and imposed fines on families with unapproved births. Using data from an eight-province longitudinal household survey (The China Health and Nutrition Survey), we investigate whether or not the one-child policy's financial penalties were associated with the avoidance of obstetric care by pregnant Chinese women with unapproved pregnancies. The one-child policy variables of particular interest were a dichotomous measure of the approval status of the pregnancy, a continuous measure of the fine imposed upon families with unapproved births, and a continuous measure of the prices of prenatal care and delivery services net of any subsidy available for approved births. The results partially confirm the hypotheses that the one-child policy's economic and social costs caused women to forego seeking modern obstetric care services. The fine was found to be a significant deterrent to the utilization of prenatal care. Additionally, the unapproved-status of a pregnancy was strongly negatively associated with "the use of obstetric care. However, higher prices were not consistently found to be a significant deterrent to the use of obstetric care.

DNA methylation in the promoter region of the p16 (CDKN2/MTS-1/INK4A) gene in human breast tumours.

The p16 (CDKN2/MTS-1/INK4A) gene is one of several tumour-suppressor genes that have been shown to be inactivated by DNA methylation in various human cancers including breast tumours. We have used bisulphite genomic sequencing to examine the detailed sequence specificity of DNA methylation in the CpG island promoter/exon 1 region in the p16 gene in DNA from a series of human breast cancer specimens and normal human breast tissue (from reductive mammaplasty). The p16 region examined was unmethylated in the four normal human breast specimens and in four out of nine breast tumours. In the other five independent breast tumour specimens, a uniform pattern of DNA methylation was observed. Of the nine major sites of DNA methylation in the amplified region from these tumour DNAs, four were in non-CG sequences. This unusual concentration of non-CG methylation sites was not a general phenomenon present throughout the genome of these tumour cells because the methylated CpG island regions of interspersed L1 repeats had a pattern of (almost exclusively) CG methylation similar to that found in normal breast tissue DNA and in DNA from tumours with unmethylated p16 genes. These data suggest that DNA methylation of the p16 gene in some breast tumours could be the result of an active process that generates a discrete methylation pattern and, hence, could ultimately be amenable to therapeutic manipulation.

Asymmetric methylation in the hypermethylated CpG promoter region of the human L1 retrotransposon.

We have investigated the function and sequence specificity of DNA methylation in the hypermethylated CpG island promoter region of the endogenous human LINE-1 (L1) retrotransposon family. In nontransformed human embryonic fibroblasts, inhibition of DNA methylation with 5-azadeoxycytidine induced a greater than 4-fold increase in transcription from potentially functional L1 elements without increasing the transcription level of the majority of degenerate elements, implicating hypermethylation in the repression of L1 activity. Using bisulfite genomic sequencing to assess the pattern of methylation in a subset of nondegenerate L1 elements, we found 29 sites within a 460-base pair region of the noncoding (top) DNA strand of the L1 promoter in which cytosine methylation was maintained with high efficiency. Of these, 25 were at CG dinucleotides and four were in non-CG sites. When the methylation sites were analyzed for the complementary (bottom) strand, the only highly conserved sites of methylation were in CG dinucleotides. Several of these sites of CG methylation in the bottom (coding) strand were at positions where top (noncoding) strand-derived sequences were unmethylated, suggesting that these sites might be maintained in a hemi-methylated state. Hence, there is a subset of human L1 elements in which methylation is efficiently maintained in asymmetric non-CG sites and further that this non-CG methylation may be part of a wider phenomenon involving hemi-methylation at CG dinucleotides. Maintenance of asymmetric methylation at non-CG sites (and possibly at hemi-methylated CG dinucleotides) could be through a novel DNA methyltransferase activity. Alternatively, the promoter region of L1 elements may be induced by factor binding to form some type of secondary structure that presents as a highly efficient substrate for de novo methylation.

A sensitive RNase protection assay to detect transcripts from potentially functional human endogenous L1 retrotransposons.

A high background of read-through transcripts from degenerate human L1 retrotransposons is present in almost all human cell types. This prevents the detection of RNA transcripts from potentially functional elements. To overcome this, we have developed an RNase protection assay based on the reconstructed consensus sequence for the 5' end of the major L1 family. In the human Ntera2D1 teratocarcinoma cell line, this assay readily detected L1 transcripts that were located primarily in the cytoplasm and where 20% were in filterable particles. By this assay, potentially functional L1 elements are also transcriptionally active in lymphocytes from some but not all normal individuals. Together with the full length protection product, there were three other discrete L1 RNAs, two of which (305 and 275 bases) were transcribed from the 5' end of the L1 element. These smaller L1 RNAs do not appear to be derived from transcripts from divergent L1 families but are either discrete shorter transcripts or specifically processed products from longer initial transcripts.

Modified-cytosine restriction-system-induced recombinant cloning artefacts in Escherichia coli.

We have tested whether, and to what extent, recombinant clones from DNA segments with 5-methylation of cytosines recovered in methylation-restrictive (mcr+) hosts contain mutations. We constructed a model system in which the tetracycline-resistance-encoding gene (tet) from pBR322 was cloned into the plasmid pGEM3Zf+. The central region of tet was removed from the construct, methylated in vitro and then religated back into the unmethylated remainder of the construct. The central region of tet was either (1) methylated with a combination of four bacterial methyltransferases (M.AluI, M.HaeIII, M.HpaII plus M.HhaI) or (2) methylated with M.SssI which methylates at all CpG dinucleotides. These two protocols generated theoretical levels of DNA methylation in the central fragment of 10.5% and 33%, respectively. The construct was transformed into a series of isogenic (recA+) bacterial strains that were mcrA+ mcrB+C+, mcrA+ mcrB-C+, mcrA- mcrB+C+, mcrA- mcrB-C+ or mcrA- delta mcrBC, and also into a set of isogenic recA- derivatives of these strains. With the two methylation protocols, there was an average 48- and 141-fold reduction, respectively, in the number of transformants recovered from the recA+ mcr+ hosts compared with a methylation-tolerant host (mcr-). Of the clones recovered in recA+mcr+ hosts, > 20% of clones had an inactivating mutation in tet. The majority of such mutant clones contained deletions that frequently extended into the unmethylated portion of tet and even into the plasmid sequences beyond the end of the polylinker. With the recA- mcr+ hosts, effective restriction was much more stringent, rendering the plasmid containing the methylated segment effectively unclonable.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli host strains SURE and SRB fail to preserve a palindrome cloned in lambda phage: improved alternate host strains.

We have attempted to produce Escherichia coli strains with the optimal combination of host mutations required for the construction of genomic libraries in lambda and cosmid vectors. For lambda vectors, we defined this as a strain that combined high efficiency of phage plating with optimal tolerance to DNA methylation and the ability to propagate recombinants containing regions of potential secondary structure. To optimize this latter property, we have tested a series of strains for the ability to propagate a lambda phage containing a palindromic sequence. These included an mcr- derivative of a strain shown by Ishiura et al. [J. Bacteriol. 171 (1989) 1068-1074] to allow optimal stability of inserts in cosmid clones. All the sbcC strains allowed plaque formation of the palindrome-containing lambda phage. However, while the palindrome-containing phage plated with reasonable efficiency on SURE (recB sbcC recJ umuC uvrC) and SRB (sbcC recJ umuC uvrC), the majority of phage recovered from these strains no longer required an sbcC host for subsequent plating. These two strains also gave poorer titres with a low-yielding phage clone from the human Prader-Willi chromosome region. Optimal phage hosts appear to be those that are mcrA delta(mcrBC-hsd-mrr) combined with mutations in sbcC plus recBC or recD and without mutations in additional recombination functions such as recJ or recJ umuC uvrC (all of our E. coli strains are available on request).

A sizing artifact of DNA restriction fragments with unusually long single-stranded termini.

The restriction endonucleases (ENases) BstNI (CCATGG) and EcoRII (CCATGG) both cleave DNA at the same time sequences, but only EcoRII produces 5-nucleotide (nt) cohesive ends and is inhibited by 5-methylation of the inner cytosine. The low-Mr fragments in digests of mouse DNA made with these two ENases exhibit different mobilities during agarose-gel electrophoresis. The difference in the mobilities of the BstNI and EcoRII fragments from mouse DNA was not due to closely spaced, differentially methylated sites, or to alternate mechanisms such as circularization of the long cohesive ends of the EcoRII fragments, or to residual bound protein. Rather, it was due to the unusually long 5-nt single-stranded (ss) ends of fragments produced by EcoRII digestion, since the slower mobility of the EcoRII fragments was abolished by treatment with ss-specific nuclease. Similar mobility differences between BstNI and EcoRII fragments which could be removed by ss nuclease were also observed in digests of simian virus 40 DNA.

Revised genomic consensus for the hypermethylated CpG island region of the human L1 transposon and integration sites of full length L1 elements from recombinant clones made using methylation-tolerant host strains.

Efficient recovery of clones from the 5' end of the human L1 dispersed repetitive elements necessitates the use of deletion mcr- host strains since this region contains a CpG island which is hypermethylated in vivo. Clones recovered with conventional mcr+ hosts seem to have been derived preferentially from L1 members which have accumulated mutations that have removed sites of methylation. We present a revised consensus from the 5' presumptive control region of these elements. This revised consensus contains a consensus RNA polymerase III promoter which would permit the synthesis of transcripts from the 5' end of full length L1 elements. Such potential transcripts are likely to exhibit a high degree of secondary structure. In addition, we have determined the flanking sequences for 6 full length L1 elements. The majority of full length L1 clones show no convincing evidence for target site duplication in the insertion site as commonly observed with truncated L1 elements. These data would be consistent with two mechanisms of integration of transposing L1 elements with different mechanisms predominating for full length and truncated elements.

Effects of mcr restriction of methylated CpG islands of the L1 transposons during packaging and plating stages of mammalian genomic library construction.

The use of optimally methylation-tolerant mcrA- mcrB- strains has been shown to produce an over tenfold increase in the plating efficiencies of mammalian genomic libraries, compared to a superior conventional phage host strain LE392 which is mcrB+. However, there is an even more significant effect of mcr restriction. Amongst the recombinants recovered with an mcrB+ host, we have found that there is an additional 30-fold reduction in the frequencies of clones containing the heavily methylated 5'-CpG island sequences of both the human and rat L1 repetitive elements. The mcrA product was also found to restrict clones of these methylated genomic segments, but not as strongly as mcrB. However, the use of packaging extracts made from mcrA+ lysogens did not result in convincing reductions in the recoveries of these dispersed methylated elements. The magnitude of mcr restriction during plating due to methylated dispersed elements is sufficient to make a significant proportion of mammalian genomes unclonable from genomic libraries constructed previously using conventional mcr+ hosts.

Expression of two conserved leptospiral antigens in Escherichia coli.

The genes encoding two protein antigens of Leptospira interrogans serovar pomona were cloned and expressed in Escherichia coli. Rabbit antisera raised against the cloned proteins, designated p12 and p20, were used to identify the antigens in Western blots of disrupted leptospiral cells. The proteins p12 and p20 were conserved within the genus Leptospira and were not detected in Leptonema illini. Although both proteins were present in leptospiral outer envelope preparations they did not elicit the production of agglutinating or opsonising antibodies.

AIDS: one psychosocial response.

In 1982 only 20 cases of AIDS were recognized in the Chicago area. Today 383 cases are known. The Howard Brown Memorial Clinic provides the 120 surviving individuals and their loved ones with comprehensive and coordinated services ranging from financial assistance to significant other support groups.