SDZ MRL 953, a lipid A analog as selective cytokine inducer.
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Adjuvants, Immunologic↗
Biomedical subjects
P Mayer
Publications and source records attributed to P Mayer.
Primary structures for a mammalian cellular and serum copper amine oxidase.
The 6-hydroxydopa quinone-containing active site peptide from bovine serum amine oxidase has been found to be highly homologous to a segment of a cloned human kidney amiloride-binding protein (Barbry, P., Champe, M., Chassande, O., Munemitsu, S., Champigny, G., Lingueglia, E., Maes, P., Frelin, C., Tartar, A., Ullrich, A., and Lazdunski, M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7347-7351). Additionally, a second 38-residue tryptic peptide (peptide XI) isolated from bovine serum amine oxidase shows 82% identity with a portion near the carboxyl terminus of the human kidney amiloride-binding protein. When an extended active site peptide was isolated from porcine kidney diamine oxidase (Janes, S. M., Palcic, M. M., Scaman, C. H., Smith, A. J., Brown, D. E., Dooley, D. M., Mure, M., and Klinman, J. P. (1992) Biochemistry 31, 12147-12154), it was found to be fully contained in the human kidney amiloride-binding protein. Examination of amiloride binding to bovine serum amine oxidase and porcine kidney diamine oxidase reveals dissociation constants of 196 and 9.1 microM, respectively. Taken together, these findings indicate that the cDNA isolated for human kidney amiloride-binding protein encodes a human kidney diamine oxidase. Two oligonucleotides, based on the tryptic peptide XI and active-site peptide of bovine serum amine oxidase, were used to amplify a portion of cDNA from a commercial bovine liver cDNA library through the use of the polymerase chain reaction. A full-length clone (2.7 kilobase pairs) for bovine serum amine oxidase was subsequently obtained through screening of the same cDNA library with the amplified 0.7-kilobase pair cDNA. These studies provide the first primary sequences for a mammalian cellular and serum copper amine oxidase. Computer alignment of amine oxidase cDNA-derived protein sequences reveals three conserved histidine residues, which are likely to be ligands to copper.
Amine Oxidase (Copper-Containing)↗
Additional isoforms of multifunctional calcium/calmodulin-dependent protein kinase II in rat heart tissue.
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Amino Acid Sequence↗
Characterization of gamma- and delta-subunits of Ca2+/calmodulin-dependent protein kinase II in rat gastric mucosal cell populations.
We searched for the occurrence of a Ca2+/calmodulin-dependent protein kinase in rat gastric cell types as a likely member in the chain of gastrin- and muscarinic-receptor-mediated signal transmission. A Ca(2+)- and calmodulin-dependent phosphorylation of major 50, 60 and 100 kDa substrates was observed in parietal cell cytosol and a major 60 and 61 kDa protein doublet was found to bind 125I-calmodulin in 125I-calmodulin-gel overlays. A specific substrate of the multifunctional Ca2+/calmodulin-dependent protein kinase II, autocamtide II, was phosphorylated in a calmodulin-dependent manner. The specific inhibitor of this enzyme, KN-62, antagonized protein kinase activity. RNA extracted from gastric mucosal cells was shown to contain sequences of the gamma- and delta- but not alpha- and beta-subunits of the calmodulin-dependent kinase II, and mRNA of both subtypes was demonstrated in highly purified parietal, chief and mucous cells. A calmodulin-dependent kinase II composed of gamma- and delta-subunits is a likely mediator of Ca(2+)-dependent signal transmission in these populations of gastric cells.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Simulation of reduced band broadening during single-stranded DNA pulsed field electrophoresis in polyacrylamide gels.
Using a computer simulation algorithm based on the reptation model, we investigated the effects of pulsed field gel electrophoresis on the separation of single-stranded DNA molecules in denaturing polyacrylamide gels. Pulsed fields that combine two different field intensities were found to affect the orientation of the reptation tube as well as the electrophoretic velocity, the rate of band broadening, the plate height and the molecular length for which the minimum of mobility was found, in agreement with available experimental results. Due to "memory" effects, pulses alternating between the forward and backward directions can reduce the diffusion constant by many orders of magnitude. Pulses of identical polarity but different intensities reduce the diffusion because stretches of less-oriented DNA segments are conserved during the migration. We suggest that, for DNA sequencing, pulsed fields of fixed polarity should be used to reduce band broadening and not to overcome band inversion or suppress molecular orientation. We conclude that, using a low intensity, constant electric field will lead to smaller band widths than any pulsed field regime, in a similar length of experimental time, but with less complications.
Algorithms↗
Prediction of the role of granulocyte-macrophage colony-stimulating factor in animals and man from in vitro results.
The possibility of predicting the clinical effects of cytokines from in vitro data is discussed, using GM-CSF as an example. GM-CSF incubated with bone marrow cells has been shown to induce proliferation and colony formation, predominantly of the colony-forming unit granulocyte and granulocyte-macrophage types. Daily treatment of normal monkeys with GM-CSF resulted in transient neutropenia followed by neutrophilia. After withdrawal of GM-CSF the neutrophil levels returned to baseline. Predictably, GM-CSF administration results in accelerated neutrophil recovery in patients with chemotherapy-induced neutropenia. GM-CSF has also been shown to induce microbial killing by neutrophils and monocytes in vitro. This activity translated into a dose-related protection of GM-CSF-pretreated mice infected with lethal doses of micro-organisms. Interleukin-3 (IL-3) increases the cellularity of the bone marrow and GM-CSF can induce mobilization of bone marrow cells into the peripheral blood. Therefore, it was predicted and subsequently proved that a combination of these cytokines is synergistic, increasing the yields of peripheral blood progenitor cells which could be collected and then retransplanted into patients undergoing myeloablative chemotherapy. Monkeys injected with recombinant human IL-3 and GM-CSF had increased antibody titres to human IL-3 compared with monkeys given IL-3 alone, suggesting a potential use of GM-CSF which was not predicted from its in vitro results, that of vaccine adjuvancy.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Chemotactic activity of substances derived from antibody-loaded tumor cells on granulocytes.
Chemotactic activity of granulocytes attracted by tumor cells loaded either with anti-ganglioside monoclonal antibodies (mAb) or with antibody-glucose oxidase conjugates (mAb-GO) was investigated. The melanoma cell line SK-Mel-28 which expresses the ganglioside GD3 at high density as well as the neuroectodermal cell line SK-N-LO which expresses GD2 were used for the experiments. In the presence of 50% human AB-serum, antibody-loaded tumor cells induced chemotactic activity on granulocytes, probably due to the generation of C3a/C5a which could be detected in serum incubated with anti-GD3 loaded SK-Mel-28 cells. Both compounds could also be detected in vivo in the plasma of patients suffering from neuroblastoma during therapy with anti-GD2 antibodies. In another set of experiments mAb-GO conjugates generating high amounts of H2O2 in the presence of glucose were bound to these tumor cells. A significant lipid peroxidation could be observed in the simultaneous presence of iron and ascorbate. The lipid peroxidation products were measured as thiobarbituric acid-reactive substances (TBARS) and were also shown to induce chemotactic effects on granulocytes.
ABO Blood-Group System↗
Okadaic acid indicates a major function for protein phosphatases in stimulus-response coupling of RINm5F rat insulinoma cells.
Stimulus-induced insulin secretion involves the activation of several protein kinases within the beta cell. Most prominent are protein kinase A, protein kinase C and calcium/calmodulin-dependent protein kinases. Protein kinase action is functionally antagonized by protein phosphatases. The four ubiquious serine/threonine protein phosphatases are termed PP-1, PP-2A, -2B and -2C. PP-1 and PP-2A are in vivo parts of major protein complexes. These complexes presumably regulate the phosphatase activity and direct the enzyme to its site of action. Therefore, PP-1 and -2A could play an important role in controlling intracellular signal transmission. Two different toxins, okadaic acid and calyculin A, both from marine invertebrates, were recently discovered and identified as potent and highly specific inhibitors of PP-1 and PP-2A. Both compounds emerged as very useful tools for studying intracellular phosphorylation events. We took advantage of these substances to investigate the significance of protein phosphatase action in stimulus-induced insulin secretion. To avoid major complexity, we confined our study to the cAMP and the phosphoinositide signal pathway. Okadaic acid alone evoked virtually no secretory response. cAMP-dependent secretion was markedly enhanced by 1 microM okadaic acid. The stimulatory effect of okadaic acid was strongly dependent on the concentration of cAMP analoga. In contrast, insulin release caused by the cholinergic agonist carbachol was not influenced by okadaic acid. Calyculin A (10 nM) slightly increased cAMP-induced secretion, but its high toxicity prohibited accurate interpretation of the data. Our findings support the idea that serine/threonine phosphatases act as important regulators in stimulus response coupling.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Multiple recurrent benign schwannomas of deep and superficial nerves of the upper extremity: a new variant of segmental neurofibromatosis.
Benign schwannomas of the brachial plexus are uncommon tumors, first described in the late 19th century. These lesions, which are histologically benign, can generally be excised without sacrifice of neural elements. We present the first known case of multiple concurrent and recurrent benign schwannomas of the upper extremity in an individual who demonstrated no other evidence of neurofibromatosis, and we suggest that this case may represent a new subtype of type V neurofibromatosis.
Adult↗
Activation of cellular cytotoxicity and complement-mediated lysis of melanoma and neuroblastoma cells in vitro by murine antiganglioside antibodies MB 3.6 and 14.G2a.
Mouse monoclonal antibodies against tumour-associated gangliosides GD2 (14.G2a) and GD3 (MB 3.6) were tested to mediate antibody-dependent cellular cytotoxicity (ADCC) with various effector cells or complement-dependent cytolysis (CDC). We also evaluated the immunomodulating potential of interferons in combination with cellular cytotoxicity. Using effector:target (E/T) ratios of 40:1, ADCC with effector cells such as granulocytes or mononuclear blood cells was not detectable against melanoma cell lines GR, SK-MEL-28 and G-361 which preferentially express GD3 and bind antibody MB 3.6. Neuroblastoma cell line SK-N-LO, which was used for comparative purposes, mainly expressed GD2 and the tumour cells were killed effectively after labelling with antibody 14.G2a. Granulocytes did not show significant killing of melanoma cells by ADCC, but neuroblastoma cells were killed very efficiently. Peripheral blood mononuclear cells (PBMC) also failed to kill melanoma cells. Interferon-beta slightly stimulated PBMC and increased killing of neuroblastoma cells, but no additive effects with ADCC were detectable. Incubation of target cells with interferons produced no significant differences in susceptibility of the target cells to interferon-activated PBMC cytotoxicity. Despite the lack of effectiveness in mediating cellular cytotoxicity, GD3 antibody MB 3.6 showed strong complement-dependent cytolysis in the presence of human plasma. There were remarkable differences in individual activity and different susceptibility of the melanoma cell lines. We assume that CDC may have more activity against melanoma cells than cytotoxicity associated with various effector cells.
Animals↗
Inhibition of human immunodeficiency virus type 1 replication by SDZ NIM 811, a nonimmunosuppressive cyclosporine analog.
(Me-Ile-4)cyclosporin (SDZ NIM 811) is a 4-substituted cyclosporin which is devoid of immunosuppressive activity but retains full capacity for binding to cyclophilin and exhibits potent anti-human immunodeficiency virus type 1 (HIV-1) activity. SDZ NIM 811 selectively inhibits HIV-1 replication in T4 lymphocyte cell lines, in a monocytic cell line, and in HeLa T4 cells. Furthermore, its antiviral activity against laboratory strains and against clinical isolates from geographically distinct regions in primary T4 lymphocytes and in primary monocytes (50% inhibitory concentration = 0.011 to 0.057 micrograms/ml) was demonstrated. SDZ NIM 811 does not inhibit proviral gene expression or virus-specific enzyme functions, either free or bound to cyclophilin. The compound does not influence CD4 expression or inhibit fusion between virus-infected and uninfected cells. SDZ NIM 811 was, however, found to block formation of infectious particles from chronically infected cells. Oral administration to mice, rats, dogs, and monkeys resulted in levels in blood considerably exceeding the drug concentration, which completely blocked virus replication in primary cells. SDZ NIM 811 caused changes of toxicity parameters in rats to a smaller degree than cyclosporine (formerly cyclosporin A). Thus, the potent and selective anti-HIV-1 activity of SDZ NIM 811 and its favorable pharmacokinetic behavior together with its lower nephrotoxicity than that of cyclosporine make this compound a promising candidate for development as an anti-HIV drug.
Amino Acid Isomerases↗
[Effects of aclarubicin on growth, differentiation and apoptosis of tumor cells in vitro].
Aclarubicine induces various effects after several days of incubation with human leukemic cells HL60: cell growth inhibition, inductions of differentiation, necrosis and apoptosis. Several methods of detection of differentiated and apoptotic cells were studied. The methods utilizing optical microscopy were used as references. Flow cytometry (FCM) appeared to have a higher sensitivity, but it yielded somewhat different results. Aclarubicine and doxorubicin have dose-dependent effects, except for the differentiation induction by aclarubicine, which had a maximum effect and then decreased; this probably means that the inductions of differentiation and apoptosis could be independent. Different kinetic studies indicated that differentiation increased quickly after 24 to 48 hours and reached a plateau toward 72 hours, where approximately 80% of the cells were differentiated. Necrosis increased at the same time, but less, with a 1 hour incubation with 75 nmol/l of aclarubicine. Apoptosis appeared in an irregular and non-reproducible manner. The kinetic study also indicated a certain independence between the differentiation and apoptosis inductions.
Aclarubicin↗
Anti-proliferative activity of natural interferon-alpha, isotretinoin and their combination varies in different human melanoma cell lines.
Natural interferon alpha (nIFN-alpha) isotretinoin and their combination were tested for their capacity to modulate the proliferation of different human melanoma cell lines. Modulation of cell growth was measured using the MTT-assay. Isotretinoin and nIFN-alpha as single agents inhibited the proliferation in a dose dependent manner in the three cell lines: SK-Mel-30, SK-Mel-28 and MaRi. The combination of isotretinoin and nIFN-alpha led to a marked enhancement of the antiproliferative effect compared with either nIFN-alpha or isotretinoin alone in SK-Mel-30 cells. In contrast, there were no additive effects on growth inhibition of melanoma cell lines SK-Mel-28 and MaRi when nIFN-alpha and isotretinoin was combined. In one melanoma cell line (MaRi) proliferation was actually enhanced by isotretinoin in combination with nIFN-alpha. These results demonstrate the heterogeneous response of human melanoma cell lines to noncytotoxic concentrations of isotretinoin and nIFN-alpha alone and in combination.
Cell Division↗
The biased reptation model of DNA gel electrophoresis: a user guide for constant field mobilities.
The biased reptation model of DNA gel electrophoresis is simple enough to allow one to obtain detailed analytical and numerical predictions for experimentally relevant situations. Although it is not always applicable for explaining experimental results, the biased reptation model is usually a good starting point for data analysis. Unfortunately, the model is often reported as being incapable of explaining experimental data because the users have not analyzed the data properly or because they attempted to use the model outside its expected range of applicability. This article presents a detailed practical guide to the model and its limitations, as well as a complete description of its predictions regarding the analysis of constant field mobilities.
DNA↗
New isoforms of multifunctional calcium/calmodulin-dependent protein kinase II.
Calcium/calmodulin dependent protein kinase II (CaM kinase II) seems to act as an important regulator of intracellular signal transmission. Four subtypes, termed alpha to delta, have been cloned; some of them can exist as different splicing variants. All these isoforms share a great overall homology, and they contain 3 areas of low homology. We have identified 5 new variants of subtype delta so that the total number of different isoforms now adds up to 12. These variants are probably a result of different splicing and show several deletions in regard to subtype delta. The deletion sites do exactly match regions of low homology between the subtypes. This suggests a functional division of the CaM kinase II molecule into homologous and variable domains. The homologous domains are highly conserved. Therefore, it might be the case that the constitution of the variable domains is more significant for a certain isoform than its belonging to one of the 4 subtypes alpha to delta.
Alternative Splicing↗
Recombinant human leukemia inhibitory factor induces acute phase proteins and raises the blood platelet counts in nonhuman primates.
Recombinant human leukemia inhibitory factor (rhLIF) produced by Escherichia coli was administered subcutaneously (sc) to rhesus monkeys at doses of 2, 10, and 50 micrograms/kg body weight/d for 14 days to assess its biologic activities in vivo. Serum levels of positively regulated acute phase proteins (APP) (C-reactive protein, alpha 1-antitrypsin, haptoglobin, and ceruloplasmin) were increased, whereas the negatively regulated APP prealbumin decreased in response to rhLIF treatment. During the second week of treatment, blood platelet counts began to increase, resulting in a maximum of a twofold increase above normal levels a week after termination of the rhLIF treatment. No changes were seen in total and differential white blood cell counts in blood progenitor levels and in red blood cell numbers. The low- and medium-dose rhLIF treatments were tolerated without significant side effects. The animals treated with the high dose showed a reduction in body weight of approximately 10%. In conclusion, rhLIF was shown to stimulate APP and to increase the number of platelets in circulation in nonhuman primates.
Acute-Phase Proteins↗
On the limits of near-equilibrium DNA gel electrophoretic sequencing.
High field intensities deeply affect the migration mechanisms of DNA in gels. The corresponding large electric forces lead to far-from-equilibrium transport properties such as the limiting mobility, increased diffusion and strong pulsed field responses. We present a theoretical study of the conditions required to retain a near-equilibrium reptation transport behaviour. We then examine the possibility of increasing both the field intensity and the gel concentration to sequence large single-stranded DNA molecules rapidly and efficiently. Finally, we discuss possible experimental approaches based on our theoretical predictions.
Base Sequence↗
Dynamics of DNA under pulsed-field gel electrophoresis as analyzed from birefringence rise and decay.
The orientation of DNA induced by electrophoretic transport in agarose gel has been studied by optical birefringence. From its field-free decay, it is clearly demonstrated that the degree of orientation results from two processes: alignment along the electric field (stretching of the end-to-end vector) and elongation of the primitive path in the gel (overstretching). Separation of the two contributions allows the experimental determination of the effective charge per base pair, the gel pore size seen by the reptating molecule, the reptation time, the degree of overstretching and the mean relaxation time of the overstretching. Their field and DNA length dependences compare well with theoretical predictions. Similarly, the time at which overstretching presents an overshoot in the rise of the orientation follows closely the predictions of a model based on the evolution of J-shaped conformations. The recovery of such conformations is studied by a sequence of two pulses with variable delay time. The use of directly measured or extrapolated characteristic times and fields in the design of efficient pulse schemes for pulsed-field gel electrophoresis is emphasized.
Birefringence↗