Inactive Counterpart Research Articles

Src kinase phosphorylates Caspase-8 on Tyr380: a novel mechanism of apoptosis suppression

We identified Caspase-8 as a new substrate for Src kinase. Phosphorylation occurs on Tyr380, situated in the linker region between the large and the small subunits of human Procaspase-8, and results in downregulation of Caspase-8 proapoptotic function. Src activation triggers Caspase-8 phosphorylation on Tyr380 and impairs Fas-induced apoptosis. Accordingly, Src failed to protect Caspase-8-defective human cells in which a Caspase-8-Y380F mutant is expressed from Fas-induced cell death. Remarkably, Src activation upon EGF-receptor stimulation triggers endogenous Caspase-8 phosphorylation and prevents Fas-induced apoptosis. Tyr380 is phosphorylated also in human colon cancers where Src is aberrantly activated. These data provide the first evidence for a direct role of tyrosine phosphorylation in the control of caspases and reveal a new mechanism through which tyrosine kinases inhibit apoptosis and participate in tumor progression.

Overexpression of both catalytically active and -inactive cathepsin D by cancer cells enhances apoptosis-dependent chemo-sensitivity

The aspartic protease cathepsin D (cath-D) is a key mediator of induced-apoptosis and its proteolytic activity has been generally involved in this event. During apoptosis, cath-D is translocated to the cytosol. Because cath-D is one of the lysosomal enzymes that requires a more acidic pH to be proteolytically active relative to the cysteine lysosomal enzymes such as cath-B and -L, it is therefore open to question whether cytosolic cath-D might be able to cleave substrate(s) implicated in the apoptotic cascade. Here, we have investigated the role of wild-type cath-D and its proteolytically inactive counterpart overexpressed by 3Y1-Ad12 cancer cells during chemotherapeutic-induced cytotoxicity and apoptosis, as well as the relevance of cath-D catalytic function. We demonstrate that wild-type or mutated catalytically inactive cath-D strongly enhances chemo-sensitivity and apoptotic response to etoposide. Both wild-type and mutated inactive cath-D are translocated to the cytosol, increasing the release of cytochrome c, the activation of caspases-9 and -3 and the induction of a caspase-dependent apoptosis. In addition, pretreatment of cells with the aspartic protease inhibitor, pepstatin A, does not prevent apoptosis. Interestingly therefore, the stimulatory effect of cath-D on cell death is independent of its catalytic activity. Overall, our results imply that cytosolic cath-D stimulates apoptotic pathways by interacting with a member of the apoptotic machinery rather than by cleaving specific substrate(s).

Modified allelic replication in lymphocytes of patients with neurofibromatosis type 1

Transcription activity of genes is related to their replication timing, accordingly gene activation is coupled with a shift from late replication to early replication and vice versa. The relationship between replication timing and gene expression is best manifested by monoallelically expressed genes which show an asynchronous pattern of allelic replication, with the active allele replicating earlier than the inactive counterpart. Biallelically expressed genes, which normally replicate highly synchronously, when present in lymphocytes derived from patients with various types of malignancies or premalignancies, replicate highly asynchronously, similar to monoallelically expressed genes. Since neurofibromatosis-type 1 (NF1) patients are at an increased risk to develop malignancies, we used the fluorescence in situ hybridization (FISH) replication assay and evaluated the level of replication synchrony of three cancer-implicated genes ( RB1, AML1, and CMYC) in lymphocytes derived from patients with NF1 without malignancy. Each gene, which normally displayed synchrony in allelic replication, in the patients' cells displayed loss of synchrony. The loss of replication synchrony, of each gene, in the patients' cells was achieved by an advanced replication of a single allele, which replicated remarkably earlier than its normal scheduled timing. In addition, the second allele showed slightly earlier replication timing than that normal for the gene. Thus, it is assumed that the NF1 condition is associated with activation of cancer-implicated genes that may be the cause for increased risk of patients to develop malignancies. As loss of synchrony in allelic replication timing differentiates well between NF1 patients and control subjects, this marker may have a potential use for identification of presymptomatic carriers of NF1 disorders.

Dihydropiperazine neonicotinoid compounds. Synthesis and insecticidal activity.

Syntheses of various isomeric dihydropiperazines can be approached successfully by taking advantage of the regioselective monothionation of their respective diones. Preparation of the precursor unsymmetrical N-substituted piperazinediones from readily available diamines is key to this selectivity. The dihydropiperazine ring system, as exemplified in 1-[(6-chloropyridin-3-yl)methyl]-4-methyl-3-oxopiperazin-2-ylidenecyanamide (4) and 1-[(2-chloro-1,3-thiazol-5-yl)methyl]-4-methyl-3-oxopiperazin-2-ylidenecyanamide (25), has been shown to be a suitable bioisosteric replacement for the imidazolidine ring system contained in neonicotinoid compounds. However, placement of the cyanoimino electron-withdrawing group further removed from the pyridine ring, as in 4-[(6-chloropyridin-3-yl)methyl]-3-oxopiperazin-2-ylidenecyanamide (3a), or relocation of the carbonyl group, as in 1-[(6-chloropyridin-3-yl)methyl]-4-methyl-5-oxopiperazin-2-ylidenecyanamide (5), results in significantly decreased bioisosterism. The dihydropiperazine ring system of 4 and 25 also lends a degree of rigidity to the molecule that is not offered by the inactive acyclic counterpart 2-[(6-chloropyridin-3-yl)-methyl-(methyl)amino]-2-(cyanoimino)-N,N-dimethylacetamide (6). A pharmacophore model is proposed that qualitatively explains the results on the basis of good overlap of the key pharmacophore elements of 4 and imidacloprid (1); the less active regioisomers of 4 (3a, 5, and 6) feature a smaller degree of overlap.

Immunosuppressants leflunomide and mycophenolic acid inhibit fibroblast IL-6 production by distinct mechanisms.

Mycophenolic acid (MPA) and A77 1726, the active components of the immunosuppressants mycophenolate mophetil and leflunomide, respectively, in a dose-dependent manner inhibited interferon (IFN)-gamma/LPS-induced interleukin (IL)-6 release in confluent cultures of mouse L929 fibrosarcoma cells. In addition, both drugs markedly reduced the production of the free radical gas nitric oxide (NO), without affecting the viability of L929 cells. The inhibitors of NO synthase, aminoguanidine and L-NMMA, but not L-NMMA inactive counterpart D-NMMA, mimicked the effects of A77 1726 and MPA on IL-6 generation in L929 fibroblasts. Furthermore, NO-releasing substance SNP completely reverted IL-6 accumulation in L929 cultures treated with A77 1726, while only partial recovery of IL-6 production was observed in the presence of MPA. MPA, but not A77 1726, significantly suppressed NO-independent IL-6 release triggered by cAMP-elevating agent rolipram. Thus, while A77 1726 effect on IL-6 production was mediated through concomitant reduction of NO synthesis, MPA action was mainly independent of the interference with NO generation. Finally, both agents inhibited IFN-gamma/LPS-triggered IL-6 production in mouse primary fibroblasts, but not in mouse peritoneal macrophages, indicating cell-specificity of this novel anti-inflammatory action of A77 1726 and MPA.

IS DNA METHYLATION UNDERLYING SOMACLONAL VARIATION IN OIL PALM?

The role of DNA methylation in the regulation of gene expression and its implication in somaclonal variation has been extensively investigated. Significant changes in the methylation level of genomic DNA during dedifferentiation and somatic embryogenesis have been reported for higher plants. The occurrence of somaclonal variants (ca 5%) among populations of somatic embryo-derived oil palms (Elaeis guineensis Jacq.) currently hampers the scaling-up of clonal plant production. Variant mantled palms show a feminisation of flower male parts, which can result in complete sterility in the most severe cases. Given the observed features of the abnormality (spatial heterogeneity, temporal instability, non-Mendelian sexual transmission, absence of any detectable defect in DNA organisation as revealed by AFLP/RAPD studies) the hypothesis of an epigenetic alteration of genome expression has been proposed to explain the origin of the variant plants. The relationship between the mantled variation and hypomethylation of genomic DNA has been assessed, using two complementary approaches: HPLC quantification of relative amounts of 5-methyl-deoxycytidine and the Methylase Accepting Assay (MAA), based on the enzymatic saturation of CG sites with methyl groups (Jaligot et al., 2000). RFLP and AFLP studies are currently implemented, using isoschizomeric restriction enzyme pairs showing differential sensitivity to the methylation of dC residues (eg MspI and HpaII). As active chromatin may show reduced methylation rates as compared with its inactive counterpart, we are now comparing methylation rates in DNA corresponding to active chromatin in normal and variant regenerants. (Resume d'auteur)

Atomic force microscopy examination of conformations of polynucleotides in response to platinum isomers: significance of GC content at broken ends.

Atomic force microscopy is a technique that enables visualization of macromolecular conformations of polynucleotides at nanometer resolution. We investigated the results of interactions of cisplatin, a DNA binding anticancer drug, and its inactive counterpart, transplatin isomer, on the molecular conformation of polynucleotides: poly d(G-C). poly d(G-C) (polyGC) and poly d(A-T). poly d(A-T) (polyAT). We observed that polyAT exhibited an increased number of enlarged ends of molecules, which we attribute to unwound and/or collapsed regions of polyAT. PolyGC molecules did not show such ends unless cisplatin was added to the PolyGC polymers. Transplatin had the apparent effect of causing overlapping or stacking of the polymer molecules. Addition of exonuclease-III to these polymers removed the visible enlarged ends. The effects of cisplatin as compared to transplatin on the polyGC duplex polymers provide support for the presence of intrastrand covalent linkages, consistent with known N7 guanine interaction of the cis isomer on molecular conformation. Furthermore, our results indicate that the mechanism of interactions of DNA with cisplatin may be dependent on the GC content of the molecules. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 68-72, 2000.

An analysis of the role of active site protic residues of cytochrome P-450s: mechanistic and mutational studies on 17alpha-hydroxylase-17,20-lyase (P-45017alpha also CYP17).

Certain cytochrome P-450s involved in the transformation of steroids catalyse not only the hydroxylation process associated with the group of enzymes, but also an acyl-carbon cleavage reaction. The hydroxylation occurs using an iron-monooxygen species while the acyl-carbon cleavage has been suggested to be promoted by an iron peroxide. In this paper we have studied the role of active site protic residues, Glu305 and Thr306, in modulating the two activities. For this purpose, the kinetic parameters for the hydroxylation reaction (pregnenolone-->17alpha-hydroxypregnenolone) and two different versions of acyl-carbon cleavage (17alpha-hydroxypregnenolone-->dehydroepiandrosterone and 3beta-hydroxyandrost-5-ene-17beta-carbaldehyde-->3beta-hydroxya ndrost -5,16-diene+androst-5-ene-3beta,17alpha-diol) were determined using the wild-type human CYP17 and its eight different single and double mutants. In addition the propensity of the proteins to undergo a subtle rearrangement converting the 450 nm active-form into an inactive counterpart absorbing at 420 nm, was monitored by measuring the t12 of the P-450-->P-420 conversion. The results are interpreted to draw the following conclusions. The functional groups of Glu305 and Thr306 do not directly participate in the two proton delivery steps required for hydroxylation but may be important participants for the provision of a net work of hydrogen bonds for 'activating' water that then acts as a proton donor. The loss of any one of these residues is, therefore, only partially debilitating. That the mutation of Thr306 impairs the hydroxylation reaction more than it does the acyl-carbon cleavage is consistent with the detailed mechanistic scheme considered in this paper. Furthermore attention is drawn to the fact that the mutation of Glu305 and Thr306 subtly perturbed the architecture of the active site, which affects the geometry of this region of the protein and therefore its catalytic properties.

Nitric Oxide Inhibits Neuronal Activity in the Supraoptic Nucleus of the Rat Hypothalamic Slices

The presence of abundant nitric oxide synthase (NOS) in magnocellular neurons of the rat hypothalamus suggests that nitric oxide (NO) may be involved in controlling the release of oxytocin and vasopressin. To test this possibility, we examined the effect of NO-related drugs on extracellular discharges of 124 supraoptic nucleus (SON) neurons from slices of rat hypothalamus in vitro. Twenty-three (43%) of 53 neurons were inhibited by sodium nitroprusside (SNP), a spontaneous releaser of NO, at 1–3 mM. This inhibition was prevented by preincubation of the slices with 1 μM hemoglobin, an inactivator of NO (n = 14), whereas hemoglobin alone enhanced neuronal activity in seven (35%) of 20 neurons. l-Arginine (1 mM), a precursor of NO, inhibited neuronal activity in five (36%) of 14 neurons, while d-arginine (1 mM), the inactive counterpart of l-arginine, was ineffective (n = 12). N-ω-nitro-l-arginine methyl ester (l-NAME, 10 μM), an inhibitor of NOS, also enhanced neuronal activity in five (29%) of 17 neurons, while N-ω-nitro-d-arginine methyl ester (DNAME, 10 μM), the inactive enantiomer of l-NAME, was without effect (n = 11). Together, our data show that NO exerts predominantly an inhibitory effect on SON neurons and may serve as a negative feedback loop in controlling release of oxytocin and vasopressin.

Effect of synthetic and natural protein tyrosine kinase inhibitors on auxin efflux in zucchini (Cucurbita pepo) hypocotyls

Basipetal auxin transport along the zucchini hypocotyl requires the cell‐to‐cell translocation of this hormone. Several authors have demonstrated that naphthylphthalamic acid (NPA) blocks auxin efflux. It is believed that NPA binds to a regulatory protein that modulates the activity of the carrier rather than to the carrier itself. A few regulatory mechanisms have been suggested but all lack strong supporting experimental evidence. Four classes of protein tyrosine kinase inhibitors (PTKIs) have been used to investigate the NPA perception pathway. First, six synthetic tyrphostins, inhibitors of the epidermal growth factor receptor (EGF‐R), were shown to displace NPA on plasmalemma fractions. The most potent compound was (3,4‐dihydroxybenzylidene)‐thiocyanoacetamide (tyrphostin A47). The inactive counterpart of tyrphostin A47, (4‐methoxybenzylidene)‐malononitrile (tyrphostin A1), was unable to displace NPA. Tyrphostins did not inhibit auxin efflux but were antagonists of the inhibition of auxin efflux by NPA. Again, tyrphostin A47 was the most effective and tyrphostin A1 was inactive. Second, the flavonoid genistein, also an EGF‐R inhibitor, showed an effect on NPA binding and NPA antagonism similar to the one for the tyrphostins. Daidzein, the inactive counterpart of genistein, was ineffective in displacing NPA and in antagonizing its inhibition of auxin efflux. Two other PTKIs, curcumin and lavendustin A, displayed similar characteristics. Calmodulin antagonists, protein serine/threonine kinase inhibitors, and phosphatase inhibitors were inactive. These results suggest that the NPA binding protein may be related to mammalian tyrosine kinases and may regulate the auxin efflux carrier by phosphorylation.

Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5.

Phosphatidylinositol (PI) 3-kinases have been implicated in several aspects of intracellular membrane trafficking, although a detailed mechanism is yet to be established. In this study we demonstrated that wortmannin, a specific inhibitor of PI 3-kinases, inhibited constitutive endocytosis of horseradish peroxidase and transferrin in BHK-21 and TRVb-1 cells. The IC50 was approximately 40 ng/ml (93 nM). In addition, wortmannin blocked the stimulation of horseradish peroxidase uptake by the small GTPase Rab5 but not the stimulation by the GTPase-defective, constitutively activated Rab5 Gln79-->Leu mutant (Rab5:Q79L), providing further evidence that PI 3-kinase activity is essential for the early endocytic process. To further investigate the mechanism, we examined the effect of wortmannin on early endosome fusion in vitro. Wortmannin decreased endosome fusion by 80% with an IC50 value similar to that in intact cells. Addition of Rab5:Q79L but not wild-type Rab5 reversed the inhibitory effect of wortmannin. Furthermore, addition of a constitutively activated PI 3-kinase but not its inactive counterpart stimulated early endosome fusion in vitro. These results strongly indicate that PI 3-kinase plays an important role in regulation of early endosome fusion, probably via activation of Rab5.

Evidence for an inhibitory effect of nitric oxides on neuropeptide secretion from isolated neural lobe of the rat pituitary gland

The present study aims at investigating the effect of pharmacological manipulation of nitric oxides (NOs) formation in the rat neurohypophysis on the secretion of vasopressin (AVP). We found that the NO synthase antagonist l-NAME and free-ferrous hemoglobin (an NO inactivator) produced a transient and significant enhancement of basal secretion of AVP from incubated glands. Conversely, the NO precursor L-arginine (but not its inactive counterpart d-arginine) antagonized the stimulatory influence of l-NAME on both AVP and oxytocin (OT) output. Elevation of NOs formation triggered by means of the NO donor SIN-1 likewise dampened spontaneous, as well as stimulated, AVP release. It is concluded that NOs molecules show up as potent regulators of neuropeptide secretion at the level of nerve terminals in the neurohypophysis.

Active catalytic fragment of Ca2+/calmodulin-dependent protein kinase II. Purification, characterization, and structural analysis

We report the purification and characterization of an active catalytic fragment of Ca2+/calmodulin-dependent protein kinase II, derived from autophosphorylation and subsequent limited chymotryptic digestion of the purified rat forebrain soluble kinase. The purified fragment was completely Ca2+/calmodulin-independent, existed as a monomer, and phosphorylated synapsin I at the same sites as does the native form of Ca2+/calmodulin-dependent protein kinase II. Kinetic studies with the purified fragment revealed a more than 10-fold increase in Vmax and a 50% decrease in Km for synthetic peptide substrates, compared with native Ca2+/calmodulin-dependent protein kinase II. No 32P-labeled autophosphorylated residues were detected in the purified active fragment, indicating that the autophosphorylation sites were not contained within this fragment. Comparative studies of this active fragment (30 kDa) and its inactive counterpart (32-kDa fragment) revealed certain structural details of both fragments. Calmodulin-overlay study, immunoblot analysis, and direct amino acid sequencing suggest that both fragments contain the entire NH2-terminal catalytic domain and were generated by distinct cleavage within the regulatory domain. The putative cleavage sites for both fragments are discussed.

Hog Intrinsic Factor: II. SOME PHYSICOCHEMICAL PROPERTIES OF VITAMIN B12-BINDING FRACTIONS FROM HOG PYLORUS

Abstract Two vitamin B12-binding glycoproteins, isolated from hog pyloric mucosa as their vitamin B12 complexes and in noncomplexed form, differ greatly in intrinsic factor activity but are similar in chemical and physical properties. However, intrinsic factor, either as its vitamin B12 complex or in noncomplexed form, contains more galactose and less galactosamine and glucosamine than its inactive counterpart. About one-third of the weight of these preparations is carbohydrate, resulting in a low partial specific volume of 0.68 ml per g. The weight average molecular weight of the noncomplexed preparations was about 50,000, and the sedimentation coefficient was 3.7 S. The vitamin B12 complexes had weight average molecular weights of about 100,000 and sedimentation coefficients of 4.6 S. Viscosity measurements with a noncomplexed preparation gave an intrinsic viscosity of 0.06 dl per g, indicating a nearly spherical molecule. Incubation of a noncomplexed preparation with vitamin B12 at 37° slowly produced the 100,000 molecular weight form. Conversely, treatment of the 100,000 molecular weight form with 5 m guanidinium chloride slowly gave rise to the 50,000 molecular weight form. Prolonged treatment with guanidinium chloride did not reduce the molecular weight below 50,000. These data are interpreted as showing that following vitamin B12 binding, intrinsic factor undergoes a slow dimerization which is reversible by guanidinium treatment. Based on vitamin B12-binding capacity and molecular weight, a model of the dimerization process is proposed in which the dimer may contain either 1 or 2 molecules of vitamin B12.