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C Stein

Publications and source records attributed to C Stein.

At least 55 records · Page 3Linked to original sources
PubMed2000

Reducing mass degeneracy in SAR by MS by stable isotopic labeling.

Mass spectrometry (MS) promises to be an invaluable tool for functional genomics, by supporting low-cost, high-throughput experiments. However, large-scale MS faces the potential problem of mass degeneracy--indistinguishable masses for multiple biopolymer fragments (e.g. from a limited proteolytic digest). This paper studies the tasks of planning and interpreting MS experiments that use selective isotopic labeling, thereby substantially reducing potential mass degeneracy. Our algorithms support an experimental-computational protocol called Structure-Activity Relation by Mass Spectrometry (SAR by MS), for elucidating the function of protein-DNA and protein-protein complexes. SAR by MS enzymatically cleaves a crosslinked complex and analyzes the resulting mass spectrum for mass peaks of hypothesized fragments. Depending on binding mode, some cleavage sites will be shielded; the absence of anticipated peaks implicates corresponding fragments as either part of the interaction region or inaccessible due to conformational change upon binding. Thus different mass spectra provide evidence for different structure-activity relations. We address combinatorial and algorithmic questions in the areas of data analysis (constraining binding mode based on mass signature) and experiment planning (determining an isotopic labeling strategy to reduce mass degeneracy and aid data analysis). We explore the computational complexity of these problems, obtaining upper and lower bounds. We report experimental results from implementations of our algorithms.

Algorithms↗
PubMed1999

Systemic hyperosmolality improves beta-glucuronidase distribution and pathology in murine MPS VII brain following intraventricular gene transfer.

Mucopolysaccharidosis VII, a classical lysosomal storage disease, is caused by deficiency of the enzyme beta-glucuronidase. Central nervous system (CNS) manifestations are severe with accumulations of storage vacuoles in all cell types. Intraventricular gene transfer can lead to transduction of the ependyma, with production and secretion of beta-glucuronidase into the cerebral spinal fluid and underlying cortex resulting in reversal of disease pathology restricted to the periventricular areas. We tested if systemic hyperosmolality would increase the distribution of beta-glucuronidase in brain parenchyma after intraventricular virus injection. Mice were administered mannitol, intraperitoneally, 20 days after gene transfer and 1 day prior to sacrifice. Mannitol-induced systemic hyperosmolality caused a marked penetration of beta-glucuronidase into the brain parenchyma. If mannitol was administered at the time of the intraventricular injection of virus, there was penetration of vector across the ependymal cell layer, with infection of cells in the subependymal region. This also resulted in increased beta-glucuronidase activity throughout the brain. Sections of brains from beta-glucuronidase-deficient mice showed correction of cellular pathology in the subependymal region plus cortical structures away from the ventricular wall. These data indicate that virus-mediated gene transfer to the brain via the ventricles, coupled with systemic mannitol administration, can lead to extensive CNS distribution of beta-glucuronidase with concomitant correction of the storage defect. Our findings have positive therapeutic implications for the treatment of CNS disorders with gene transfer vectors and recombinant proteins.

Adenoviridae↗
PubMed1999

Dose-dependency of intra-articular morphine analgesia.

We have examined if the analgesic effects of intra-articular morphine are dose-dependent in patients undergoing elective arthroscopic knee surgery. At the end of surgery, patients were allocated randomly to one of four groups to receive intra-articular saline (n = 22), or morphine 1 mg (n = 24), 2 mg (n = 21) or 4 mg (n = 19). After operation, patients remained in hospital overnight and pain intensity was assessed using a visual analogue scale at 1, 2, 3, 6, 9, 12, 18 and 24 h after intra-articular injection. Patients requesting additional analgesia received a loading dose of piritramide 0.1 mg kg-1 i.v. and were connected to a PCA device using the same drug. Increasing doses of intra-articular morphine were associated with greater analgesic effects and less supplementary analgesic requirements.

Adolescent↗
PubMed1999

Activated mouse Notch1 transactivates Epstein-Barr virus nuclear antigen 2-regulated viral promoters.

Epstein-Barr virus nuclear antigen 2 (EBNA2) is essential for B-cell immortalization by EBV, most probably by its ability to transactivate a number of cellular and viral genes. EBNA2-responsive elements (EBNA2REs) have been identified in several EBNA2-regulated viral promoters, each of them carrying at least one RBP-Jkappa recognition site. RBP-Jkappa recruits EBNA2 to the EBNA2RE and, once complexed to EBNA2, is converted from a repressor into an activator. An activated form of the cellular receptor Notch also interacts with RBP-Jkappa, providing a link between EBNA2 and Notch signalling. To determine whether activated Notch is able to transactivate EBNA2-responsive viral promoters, we performed cotransfection experiments with activated mouse Notch1 (mNotch1-IC) and luciferase constructs of the BamHI C, LMP1, and LMP2A promoters. We present here evidence that mNotch1-IC transactivates viral promoters known to be regulated by EBNA2. As shown for EBNA2, mutations or deletions of the RBP-Jkappa sites diminish or eliminate mNotch1-IC-mediated transactivation of the promoters, pointing to an essential role for Notch-RBP-Jkappa interaction. In addition to RBP-Jkappa, other cellular factors may bind within the EBNA2REs of viral promoters. While some factors appear to play an important role in both EBNA2- and mNotch1-IC-mediated transactivation, others are only important for the activity of either EBNA2 or mNotch1-IC. We could observe specific mNotch1-IC-responsive regions, thereby throwing more light upon which cofactors interact with EBNA2 and mNotch1-IC, thus enabling them to become functionally transactivators in vivo.

Animals↗
PubMed1999

Peripheral effects of the kappa-opioid agonist EMD 61753 on pain and inflammation in rats and humans.

The objective of the present study was to evaluate the effects of EMD 61753 (asimadoline), a kappa-opioid receptor agonist with restricted access to the central nervous system, on postoperative pain in patients who underwent knee surgery and on nociceptive thresholds and inflammation in rats treated with Freund's complete adjuvant. Patients treated with EMD 61753 (10 mg p.o.) tended to report an increase in pain, as evaluated by a visual analog scale and by the time to the first request for and the total amount of supplemental analgesic medication. The global tolerability of EMD 61753 was assessed as significantly inferior to that of a placebo by the investigator. In rats, the bilateral intraplantar (i.pl.) injection of EMD 61753 (0.1-3.2 mg) resulted in dose-dependent antinociception in both inflamed and noninflamed paws, with a peak at 5 min after injection, as evaluated by the paw pressure method. However, at later time points (1 h-4 days), a significant decrease in the paw pressure threshold was observed, confirming its tendency toward a hyperalgesic action in humans. This was accompanied by an increase in paw volume and paw temperature, with a peak at 6 h after injection. EMD 61753 (1.6 mg)-induced analgesia was blocked by the peripheral opioid receptor antagonist naloxone methiodide (2.5-10 mg/kg s.c.) and by the kappa receptor antagonist nor-binaltorphimine (0.1 mg; i.pl.). In contrast, EMD 61753 (1.6 mg)-induced hyperalgesia and increases in paw volume and paw temperature were blocked neither by naloxone methiodide (10-40 mg/kg s.c.) nor by dizocilpine maleate (0.003-0.009 mg i.pl.), a N-methyl-D-aspartic acid receptor antagonist. These data show differentially mediated peripheral actions of EMD 61753: kappa-opioid receptor-induced analgesia and nonopioid, non-N-methyl-D-aspartic acid hyperalgesic and proinflammatory effects.

Acetamides↗
PubMed1998

Extensive beta-glucuronidase activity in murine central nervous system after adenovirus-mediated gene transfer to brain.

Mucopolysaccharidosis type VII (MPS VII), caused by beta-glucuronidase deficiency, is a classic lysosomal storage disease. In the central nervous system (CNS), there is widespread pathology with distention of vacuoles in neurons and glia. An approach to therapy for MPS VII would require extensive delivery of enzyme to the CNS and subsequent uptake by the affected cells. In this study we show that intrastriatal injection of recombinant adenovirus encoding beta-glucuronidase (Ad betagluc) to MPS VII or wild-type mice results in focal, intense beta-glucuronidase mRNA expression near the injection site. Further, histochemical staining for enzyme activity showed that beta-glucuronidase activity extended well beyond transduced cells. Activity was detected throughout the ipsilateral striatum as well as in the corpus callosum, ventricles, and bilateral neocortex. Similarly, after injection into the right lateral ventricle or cisterna magna, enzyme activity was present in the ependymal cells of the ventricles, in the subarachnoid spaces, and also in the underlying cortex (150-500 microm from ependyma). The distribution of enzyme was most extensive 21 days after gene transfer to normal mouse brain, with more than 50% of the hemisphere positive for beta-glucuronidase activity. Eighty-four days after adenovirus injection a substantial level of enzyme expression remained (>40% of hemisphere positive for beta-glucuronidase activity). Histological sections from striatum of beta-glucuronidase-deficient mice injected with Ad betagluc showed a marked reduction in the number of distended vacuoles in both neurons and glia, as compared with uninjected striatum. Importantly, correction was noted in both hemispheres. Our finding that a relatively small number of transduced cells produce enzyme that reaches a large proportion of the CNS has favorable implications in developing direct gene transfer therapies for lysosomal storage disorders.

Adenoviridae↗
PubMed1998

Peripheral morphine analgesia in dental surgery.

The recent identification of opioid receptors on peripheral nerve endings of primary afferent neurons and the expression of their mRNA in dorsal root ganglia support earlier experimental data about peripheral analgesic effects of locally applied opioids. These effects are most prominent under localized inflammatory conditions. The clinical use of such peripheral analgesic effects of opioids was soon investigated in numerous controlled clinical trials. The majority of these have tested the local, intraarticular administration of morphine in knee surgery and have demonstrated potent and long-lasting postoperative analgesia. As the direct application of morphine into the pain-generating site of injury and inflammation appears most promising, we examined direct morphine infiltration of the surgical site in a unique clinical model of inflammatory tooth pain. Forty-four patients undergoing dental surgery entered into this prospective, randomized, double-blind study. Before surgery they received, together with a standard local anesthetic solution (articaine plus epinephrine) a submucous injection of either 1 mg of morphine (group A) or saline (group B). Postoperative pain intensity was assessed using the visual analog scale (VAS) and numeric rating scale (NRS) at 2, 4, 6, 8, 10, 12, 16, 20 and 24 h after surgery. In addition, patients recorded the occurrence of side effects and the supplemental consumption of diclofenac tablets. Results of 27 patients were analyzed (group A: n=14, group B: n=13). Pain scores which were moderate to severe preoperatively were reduced to a similar extent in both groups up to 8 h postoperatively. Thereafter, pain scores in group A were significantly lower than those in group B for up to 24 h, demonstrating the analgesic efficacy of additional morphine. The time to first analgesic intake and the total amount of supplemental diclofenac were less in group A than in group B. No serious side effects were reported. Our results show that 1 mg of morphine added to a local anesthetic for dental surgery results in significant improvement of postoperative analgesia. Since the majority of dental surgeries is accompanied with an inflammatory reaction, supplemental morphine may be of benefit for the relief of postoperative dental pain.

Adult↗
PubMed1998

Cholecystokinin inhibits peripheral opioid analgesia in inflamed tissue.

There is abundant evidence that opioid receptors are present on peripheral terminals of primary afferent neurons. Experimental and clinical studies have shown that activation of these peripheral opioid receptors produces potent analgesia. In addition to peripheral opioid receptors, cholecystokinin receptors are present in sensory neurons. We examined the hypothesis that cholecystokinin receptors may be present on the same primary afferent neuron and that either exogenous or endogenous cholecystokinin may modulate peripheral antinociceptive effects of mu-opioid receptor agonists. Administration of cholecystokinin into inflamed paws, of the rat, but not intravenously attenuated peripheral antinociceptive effects induced by two mu-opioid receptor agonists, [D-Ala2,N-methyl-Phe4,Gly-ol5]-enkephalin and fentanyl. Only the desulphated form of cholecystokinin produced significant and dose-dependent attenuation. Cholecystokinin alone did not alter nociceptive baseline values in inflamed or non-inflamed paws. The anti-opioid effect of cholecystokinin was dose-dependently antagonized by the cholecystokininB receptor-selective antagonist L-365260, but not by the cholecystokininA receptor-selective antagonist L-364718. Local pretreatment with the protein kinase C specific inhibitor calphostin C abolished cholecystokinin's effect. Peripheral antinociceptive effects of [D-Ala2,N-methyl-Phe4,Gly-ol5]-enkephalin and fentanyl were not altered by intraplantar L-365260 alone. These results indicate that activation of peripheral cholecystokininB but not cholecystokininA receptors attenuates the local antinociceptive effects of mu-opioid receptor agonists in inflamed tissue. This anti-opioid effect may be mediated by protein kinase C in sensory nerve terminals. Endogenous cholecystokinin does not seem to influence the efficacy of peripheral opioids under both normal and inflammatory conditions.

Analgesics, Opioid↗
PubMed1998

Effects of neurotoxins and hindpaw inflammation on opioid receptor immunoreactivities in dorsal root ganglia.

Three types of opioid receptors mediate peripheral opioid antinociception in inflammation. Recently, antisera that recognize unique epitopes of the cloned mu-, delta-, and kappa-opioid receptors have been developed. Using these antisera we examined the regulation of opioid receptors in rat dorsal root ganglia after hindpaw inflammation and the influence of neurotoxins for primary afferent neurons and sympathetic neurons thereon. Peripheral tissue inflammation was produced by injection of complete Freund's adjuvant into the right hindpaw. Capsaicin was injected subcutaneously once a day for three days using a total dose of 150 mg/kg. 6-hydroxydopamine was injected intraperitoneally 75 mg/kg/day for three days. Freund's adjuvant induced a marked increase in the percentage of mu-, but a decrease in delta- and kappa-opioid receptor-positive neurons. Capsaicin significantly decreased mu-, delta- and kappa-opioid receptor immunoreactivity in both Freund's adjuvant treated and non-treated rats. No significant changes on the mu-, delta- and kappa-opioid receptor immunoreactivities were observed after 6-hydroxydopamine treatment in either Freund's adjuvant-treated or non-treated rats. Our studies indicate: (1) Peripheral inflammation can differentially regulate the expression of the three opioid receptors in dorsal root ganglia neurons with an up-regulation of mu- and down-regulation of delta- and kappa-opioid receptors. 2) A significant portion of mu-, delta- and kappa-opioid receptors are located on capsaicin-sensitive neurons in dorsal root ganglia of both non-inflamed and inflamed hindlimbs. 3) The expression of opioid receptors in dorsal root ganglia of either inflamed or non-inflamed hindlimbs is not influenced by sympathetic postganglionic neurons.

Animals↗
PubMed1998

Pain control in inflammation governed by selectins.

Opioid-containing immune cells migrate preferentially to inflamed sites, where they release beta-endorphin which activates peripheral opioid receptors to inhibit pain. Immunocyte recruitment is a multistep, sequential engagement of various adhesion molecules located on immune cells and vascular endothelium. Selectins mediate the initial phase of immunoctye extravasation into inflamed sites. Here we show that anti-selectin treatment abolishes peripheral opioid analgesia elicited either endogenously (by stress) or by corticotropin-releasing factor. This results from a blockade of the infiltration of immunocytes containing beta-endorphin and the consequent decrease of the beta-endorphin content in the inflamed tissue. These findings indicate that the immune system uses mechanisms of cell migration not only to fight pathogens but also to control pain in injured tissue. Thus, pain is exacerbated by measures inhibiting the immigration of opioid-producing cells or, conversely, analgesia might be conveyed by adhesive interactions that recruit those cells to injured tissue.

Analgesia↗
PubMed1998

Detection of CBFbeta/MYH11 fusion transcripts in patients with inv(16) acute myeloid leukemia after allogeneic bone marrow or peripheral blood progenitor cell transplantation.

We evaluated the occurrence of the CBFbeta/MYH11 fusion transcripts by PCR analysis in 10 patients with inv(16)(p13;q22) acute myeloid leukemia (AML) who underwent allogeneic bone marrow transplantation (BMT) (n = 5), peripheral blood progenitor cell transplantation (PBPCT) (n = 3), or autologous transplantation (n = 2). In addition to the analysis of minimal residual disease (MRD), the chimerism status of patients after allogeneic transplant was studied by PCR. The CBFbeta/MYH11 fusion transcript was not detectable in six of seven patients who remained in remission after allogeneic BMT or PBPCT. Two of these patients in remission were monitored for 50 months and 64 months post-BMT. One patient in remission was PCR-positive for CBFbeta 3 months post-BMT in a single BM sample, but not in a simultaneously examined blood sample, suggesting that analyses from BM samples are more sensitive than those from blood samples. Sequential PCR assays performed 6 and 12 months post-BMT obtained from the same patient were negative. Another patient with a positive PCR assay 3 months post-allogeneic PBPCT, remained PCR positive for the CBFbeta/MYH11 fusion transcript when tested 6 months post-PBPCT. A chimerism analysis by PCR revealed a mixed chimerism status in this patient. He relapsed 7 months post-transplant. Before transplant, in all nine patients who were in complete remission of AML (eight patients in 1CR, one patient in 2CR), the CBFbeta/MYH11 transcript was detectable. In one patient in relapse, the fusion transcript was not only detectable in blood and bone marrow, but also in a cerebrospinal fluid sample prior to transplant. Two patients who received autologous BMT were monitored for CBFbeta/MYH11 transcripts 3 months after BMT. The CBFbeta/MYH11 was detected in these patients. Both patients subsequently relapsed 3 months and 23 months post-autologous BMT. The results study show that analysis of the CBFbeta/MYH11 fusion transcript by PCR seems to be a suitable method for monitoring minimal residual disease in AML patients with inv (16).

Adolescent↗
PubMed1998

Contribution of opioid receptors on primary afferent versus sympathetic neurons to peripheral opioid analgesia.

Opioid receptors are synthesized in dorsal root ganglia and transported into peripheral terminals of primary afferent neurons. Activation of such receptors results in antinociceptive effects that are most prominent in inflammation. In addition, opioid receptors located on sympathetic postganglionic neuron terminals may be involved in these effects. This study investigates the peripheral analgesic efficacy of the mu, delta and kappa receptor agonists [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin, [D-Pen2,5]-enkephalin and trans-(+/-)3, 4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamid e, the effective number of peripheral mu, delta and kappa receptors in relation to the development of inflammation and the contribution of sympathetic vs. sensory neurons by use of capsaicin and 6-hydroxydopamine, respectively. In Wistar rats with Freund's adjuvant-induced hindpaw inflammation, antinociceptive effects of intraplantar [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (1.0-32 microg), [D-Pen2,5]-enkephalin (10-100 microg) and trans-(+/-)3, 4-Dichloro-N-methyl-N-[2-(l-pyrrolidiny)-cyclohexyl]-benzeneace tam ide (10-100 microg) were evaluated by paw pressure test. These effects increased linearly between 6 and 24 hr, but did not change between 24 and 96 hr of inflammation, whereas the doses of the irreversible antagonists beta-funaltrexamine, [D-Ala2,Leu5,Cys6]enkephalin or (+/-)-(5beta,7a,8beta)-3, 4-dichloro-N-[3-methylene-2-oxo-8-(1-pyrrolidinyl)-1-oxaspir[4, 5]dec-7-yl]benzeneacetamide required to abolish the respective agonist effects increased between 12 and 96 hr. Pretreatment with capsaicin (30, 50, 70 mg/kg s.c. over 3 days) but not with 6-hydroxydopamine (75 mg/kg i.p. over 3 days) reversed the hyperalgesia in inflamed paws and almost abolished antinociceptive effects of all three agonists. These results suggest that the increased opioid agonist efficacy is due to an increased number of peripheral opioid receptors at later stages of inflammation and that peripheral opioid antinociceptive effects are primarily mediated by mu, delta and kappa opioid receptors on primary afferent neurons.

Analgesics, Opioid↗