The gas5 gene is disrupted by a frameshift mutation within its longest open reading frame in several inbred mouse strains and maps to murine chromosome 1.
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Animals↗
Biomedical subjects
S Chatterjee
Publications and source records attributed to S Chatterjee.
Comparison of antiviral compounds against human herpesvirus 6 and 7.
Four classes of antiviral compounds were evaluated for inhibitory activity against two variants of human herpesvirus 6 (HHV-6A and -6B) and human herpesvirus 7 (HHV-7). These included: (1) a pyrophosphate analog, phosphonoformic acid (PFA); (2) beta-guanine analogs, 9-(2-hydroxyethoxymethyl)guanine (acyclovir or ACV), 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (ganciclovir or GCV) and 9-(4-hydroxy-3-hydroxy-3-hydroxymethylbutylyl)guanine (penciclovir or PCV); (3) acyclic nucleoside phosphonates, (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine [cidofovir or (S)-HPMPC] and its cyclic derivative (S)-cyclic-HPMPC (cHPMPC), 9-[[2-hydroxy-1-phosphonomethoxy)ethoxy]methyl]guanine (HPMEMG) and 9-[(2-phosphonylmethoxy)ethyl]-2,6-diaminopurine (PMEDAP), and the seven other related compounds; and (4) a series of benzimidazole ribonucleosides, including 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (BDCRB). End-point inhibitory concentration (EPC) and 50% effective inhibitory concentration (EC50) values were determined by a dot-blot antigen detection method in cord blood mononuclear cells infected with HHV-6A, HHV-6B or HHV-7 at a multiplicity of infection of 0.004 CCID50/cell. (S)-HPMPC and cHPMPC had an EC50 value of approximately 0.3 microg/ml for HHV-6A, 1.2 microg/ml for HHV-6B and 3.0 microg/ml for HHV-7. These compounds were the most active of those tested against each virus. The EC50 value of GCV for HHV-6A was 0.65 microg/ml, 1.33 microg/ml for HHV-6B, and >7 microg/ml for HHV-7. The EC50 values of ACV and PCV were approximately 6-8 microg/ml for HHV-6A, 16-24 microg/ml for HHV-6B and 121-128 microg/ml for HHV-7. These drugs were the least active. The sensitivity of HHV-7 to the guanine analogs was different from HHV-6, suggesting a difference in selectivity of specific viral enzymes.
Antiviral Agents↗
Exploration of the importance of the P2-P3-NHCO-moiety in a potent di- or tripeptide inhibitor of calpain I: insights into the development of nonpeptidic inhibitors of calpain I.
Calpain I, an intracellular cysteine protease, has been implicated in the neurodegeneration following an episode of cerebral ischemia. In this paper, we report on a series of peptidomimetic ketomethylene and carbamethylene inhibitors of recombinant human calpain I (rh calpain I). Our study reveals that the -NHCO-moiety (possible hydrogen-bonding site) at the P2-P3 region of a potent tripeptide or a dipeptide inhibitor of calpain I is not a strict requirement for enzyme recognition. Compounds 7d ((R)-2-isobutyl-4-oxo-4-(9-xanthenyl)butanoic acid ((S)-1-formyl-3-methyl)butyl amide), 31 ((R)-2-isobutyl-4-(2-sulfonylnaphthyl)butyric acid ((S)1-formyl-3-methyl)butyl amide) and 34 ((R)-2-isobutyl-4-(2-sulfoxylnaphthyl)butyric acid ((S)-1-formyl-3-methyl)butyl amide) which exhibited good activity in the enzyme assay, also inhibited calpain I in a human cell line.
Calpain↗
Aerobiologic and immunochemical studies on Borassus flabellifer pollen: evidence for a 90-kD allergen.
BACKGROUND: The pollen grains of Arecaceae have been proved to be a very common aeroallergen in India. Except for the pollen grains of Cocos nucifera, detailed information about the allergenicity of other dominant Indian palm pollen is not available. OBJECTIVES: To explore the aerobiologic and allergenic significance of the pollen of palmyra palm (Borassus flabellifer Linn.), one of the important and common palm trees in India, with a view to isolate and purify its major allergenic components. METHODS: The aerobiologic survey was performed continuously using Burkard volumetric and Rotorod samplers from July, 1994 to June, 1996 in a northern suburb of greater Calcutta. The allergenic potential of the pollen extract and different fractions were studied by skin prick tests (SPT). ELISA and ELISA inhibition experiments were performed with individual and pooled patient sera to detect the specific IgE level. By 2-step ammonium sulphate fractionation and gel filtration study, the allergic fraction Fr.IIA1 was isolated. Its molecular weight was determined by SDS-PAGE and homogeneity was confirmed by rocket and crossed immunoelectrophoresis using rabbit antisera. RESULTS: Pollen grains of B. flabellifer were found to be present in air from February to May and June contributing more than 7% of the total airborne pollen load of the study area. The pollen elicited maximum concentration at 19 to 20 hours in the evening and at a height of 4 m from ground level. Skin prick testing of 455 respiratory allergic patients with whole pollen extract exhibited 31.64% positive response. After 2-step ammonium sulphate fraction and gel filtration, a fraction (Fr.IIA1) having remarkable allergenicity was isolated. In native and SDS-PAGE, it was found as a single protein component of 90 kD and as homogeneous in RIE and CIE. The soluble protein-carbohydrate analysis indicated the probability of the component to be a glycoprotein. CONCLUSION: The aerobiologic, clinical, and immunochemical, studies demonstrated that the pollen of B. flabellifer is a dominant aeroallergen to cause respiratory trouble in the area where these trees grow. The isolated 90-kD component is one of the major allergens present in the pollen extract.
Adult↗
Blocked hepatic-stage parasites and decreased susceptibility to Plasmodium berghei infections in BALB/c mice.
The BALB/c strain of mice is comparatively more resistant to sporozoite infections of Plasmodium berghei than the C57BL6 strain. Infection with live sporozoites results in the formation of small hepatic forms in the BALB/c liver that persist for as long as 6 days. Upon infection with small numbers of sporozoites, some of the parasites are destroyed in the liver whereas the rest persist as blocked forms. When larger numbers of sporozoites are injected the same process occurs but, in addition, a fraction of the liver-stage parasites complete full development and give rise to blood forms. Although blocked liver forms persist until day 6 post-infection they actually develop to only 24 h of maturity. The nature of these persistent forms is similar to those obtained from irradiated sporozoite immunization. There is a stronger cell proliferation to liver-stage antigens by spleen lymphocytes of irradiated sporozoite-immunized BALB/c mice in comparison to that of immunized C57BL6 mice suggesting that a stronger priming to liver-stage antigens, probably due to the presence of blocked hepatic forms in the liver for a longer period of time (as compared to C57BL6), occurs in the BALB/c mice. This could be a reason for the long-lasting protective memory observed in BALB/c mice.
Animals↗
Immunohistochemical localization of constitutive and inducible cyclo-oxygenases in rat uterus during the oestrous cycle and pregnancy.
The uterus is a rich source of eicosanoids synthesized from arachidonic acid metabolism through the cyclo-oxygenase pathway. Two isoforms of cyclo-oxygenase, constitutive (COX-I) and inducible (COX-II) enzyme, have been reported. In the present study, we have immunohistochemically mapped the distribution of both COX-I and COX-II during various physiological states of the rat uterus. Uterine tissue was collected from female rats (a) during different stages of the oestrous cycle, (b) on days 1, 4, 8 and 18 of gestation, (c) after spontaneous delivery and (d) post partum, and fixed in Bouin's fixative. After paraffin wax embedding, 5-microm-thick sections were immunohistochemically stained by the ABC technique. Observation of the stained sections under the light microscope revealed that, in non-pregnant rat uterus, both COX-I and COX-II were abundantly expressed in the endometrium, with minimal staining observed in the myometrium. Staining was more prominent in epithelial cells than in stromal cells. The intensity of staining in epithelial cells was highest at pro-oestrus and oestrus and lowest at dioestrus. In pregnant rats, although the expression of both COX-I and COX-II was localized primarily to the endometrium with very little staining in the myometrium on day 1 of gestation, both of these enzymes were also apparent in myometrial cells by day 4 of gestation. The staining intensity of endometrial and myometrial cells increased further with the progression of gestation, being maximal at the time of spontaneous delivery. During the post-partum period, however, the staining intensity for both of the enzymes in endometrium and myometrium was decreased. Thus, our studies show that the expression of cyclo-oxygenases in various uterine cells vary with the oestrous cycle and with pregnancy. Furthermore, prominent increases in the expression of cyclo-oxygenases in the myometrium during pregnancy and parturition imply that the cyclo-oxygenase system in the myometrium may play a major role in modulating uterine contractility during pregnancy and labour.
Animals↗
Identification, partial purification, and localization of a neutral sphingomyelinase in rabbit skeletal muscle: neutral sphingomyelinase in skeletal muscle.
We have investigated the presence of neutral sphingomyelinases present in rabbit skeletal muscle fractions. Neutral sphingomyelinase activity measurements and immunoblot analysis of various skeletal muscle fractions indicated that most of the neutral sphingomyelinase was associated with the junctional transverse tubules. Activity gel analysis of the detergent solubilized transverse tubule fraction revealed two distinct bands corresponding to molecular weight on the order of approximately 92 and 53 kDa. Moreover, monospecific antibody raised against pure neutral sphingomyelinase recognized both the 53 and the 92 kDa protein. Peptide mapping studies revealed that both neutral sphingomyelinase isoforms were similar. Moreover, both the enzymes catalyzed the hydrolysis of sphingomyelin to phosphocholine and ceramide. Lithium stimulated and Cu2+ inhibited the activity of both of the enzyme isoforms. However, the 53 kDa isoform was insensitive to activation by Mg2+, and thus differed from the 92 kDa isoform of neutral sphingomyelinase. The localization of neutral sphingomyelinase in skeletal muscle transverse tubule membrane is consistent with transverse tubule production of the sphingomyelin-derived second messenger, sphingosine. Since sphingosine has been shown to modulate calcium release from sarcoplasmic reticulum membranes (Sabbadini et al. (1992) J Biol Chem 207: 15473-15684), our work suggests that neutral sphingomyelinase/sphingosine signaling system may be a physiologically relevant regulator of calcium levels in skeletal muscle.
Animals↗
Ordered cloned DNA map of the genome of Vibrio cholerae 569B and localization of genetic markers.
By using a low-resolution macrorestriction map as the foundation (R. Majumder et al., J. Bacteriol. 176:1105-1112, 1996), an ordered cloned DNA map of the 3.2-Mb chromosome of the hypertoxinogenic strain 569B of Vibrio cholerae has been constructed. A cosmid library the size of about 4,000 clones containing more than 120 Mb of V. cholerae genomic DNA (40-genome equivalent) was generated. By combining landmark analysis and chromosome walking, the cosmid clones were assembled into 13 contigs covering about 90% of the V. cholerae genome. A total of 92 cosmid clones were assigned to the genome and to regions defined by NotI, SfiI, and CeuI macrorestriction maps. Twenty-seven cloned genes, 9 rrn operons, and 10 copies of a repetitive DNA sequence (IS1004) have been positioned on the ordered cloned DNA map.
Cholera Toxin↗
Sphingolipids in atherosclerosis and vascular biology.
Sphingolipids and their metabolic products are now known to have second-messenger functions in a variety of cellular signaling pathways. Lactosylceramide (LacCer), a glycosphingolipid (GSL) present in vascular cells such as endothelial cells, smooth muscle cells, macrophages, neutrophils, platelets, and monocytes, contributes to atherosclerosis. Large amounts of LacCer accumulate in fatty streaks, intimal plaque, and calcified intimal plaque, along with oxidized low density lipoproteins (Ox-LDLs), growth factors, and proinflammatory cytokines. A possible role for LacCer in vascular cell biology was suggested when this GSL was found to stimulate the proliferation in vitro of aortic smooth muscle cells (ASMCs). A further link of LacCer in atherosclerosis was uncovered by the finding that Ox-LDLs stimulated specifically the biosynthesis of LacCer. Ox-LDL-stimulated endogenous synthesis of LacCer by activation of UDP-Gal:GlcCer,beta1-4galtransferase (GalT-2) is an early step in this signaling pathway. In turn, LacCer serves as a lipid second messenger that orchestrates a signal transduction pathway, ultimately leading to cell proliferation. This signaling pathway includes LacCer-mediated activation of NADPH oxidase that produces superoxide. Such superoxide molecules stimulate the GTP loading of p21(ras). Subsequently, the kinase cascade (Raf-1, Mek2, and p44MAPK [mitogen-activated protein kinase]) is activated. The phosphorylated form of p44MAPK translocates from the cytoplasm to the nucleus and engages in c-fos expression, proliferating cell nuclear antigen (PCNA) such as cyclin activation, and cell proliferation takes place. Interestingly, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of GalT-2, can abrogate the Ox-LDL-mediated activation of GalT-2, the signal kinase cascade noted above, as well as cell proliferation. Additional studies have revealed that LacCer mediates the tumor necrosis factor-alpha (TNF-alpha)-induced nuclear factor-kappaB expression and intercellular adhesion molecule (ICAM-1) expression in vascular endothelial cells via the redox-dependent transcriptional pathway. LacCer also stimulates the expression of CD11/CD8, or Mac-1, on the surface of human neutrophils. Collectively, this phenomenon may contribute to the adhesion of neutrophils or monocytes to the endothelial cell surface and thus initiate the process of atherosclerosis. In addition, the LacCer-mediated proliferation of ASMCs may contribute to the progression of atherosclerosis. On the other hand, programmed cell death (apoptosis) by proinflammatory cytokines such as TNF-alpha, interleukin-1, and high concentrations of Ox-LDL occur via activation of a cell membrane-associated neutral sphingomyelinase (N-SMase). N-SMase hydrolyzes sphingomyelin into ceramide and phosphocholine. In turn, ceramide or a homologue serves as an important stress-signaling molecule. Interestingly, an antibody against N-SMase can abrogate Ox-LDL- and TNF-alpha-induced apoptosis and therefore may be useful for in vivo studies of apoptosis in experimental animals. Because plaque stability is an integral aspect of atherosclerosis management, activation of N-SMase and subsequent apoptosis may be vital events in the onset of plaque rupture, stroke, or heart failure. Interestingly, in human liver cells, N-SMase action mediates the TNF-alpha-induced maturation of the sterol regulatory-element binding protein. Moreover, a cell-permeable ceramide can reconstitute the phenomenon above in a sterol-independent fashion. Such findings may provide new avenues for therapy for patients with atherosclerosis. The findings described here indicate an important role for sphingolipids in vascular biology and provide an exciting opportunity for further research in vascular disease and atherosclerosis.
Animals↗
Limitations of specific reverse-transcriptase polymerase chain reaction markers in the detection of metastases in the lymph nodes and blood of breast cancer patients.
PURPOSE: This study was performed to evaluate the potential of specific mRNA markers to detect micrometastases by reverse-transcriptase polymerase chain reaction (RT-PCR) and Southern blot analysis of sentinel lymph nodes (SNs) and blood from patients with breast cancer. PATIENTS AND METHODS: We assessed the specificity of carcinoembryonic antigen (CEA), cytokeratin-19 (CK-19), CK-20, gastrointestinal tumor-associated antigen-733.2 (GA733.2), and mucin-1 (MUC-1) in the blood of healthy donors (n = 13) and lymph nodes from patients without cancer (n = 3) by RT-PCR assay. The sensitivity of the RT-PCR assay for the target mRNA markers was assessed in breast cancer cell lines (n = 4), primary breast tumors (n = 8), and the frozen sections of SNs (n = 22) from 22 patients with American Joint Committee on Cancer (AJCC) stages I to IIIA breast cancer. RESULTS: CK-20 was the only mRNA marker not detected in lymph nodes or blood from patients without cancer. Both the blood and lymph nodes from patients without cancer expressed CEA, CK-19, GA733.2, and MUC-1 mRNA. All four breast cancer cell lines and six of eight primary breast tumors expressed all five mRNA markers. Expression of mRNA by the RT-PCR assay in the frozen-section SNs (n = 12) without metastases by conventional histopathology ranged from 8% (CK-20) to 92% (GA733.2). Detection of RT-PCR cDNA products in frozen-section SNs was increased with Southern blot analysis compared with ethidium bromide gel electrophoresis (EtBr) for all mRNA markers except CK-19. CONCLUSION: CEA, CK-19, GA733.2, and MUC-1 show no diagnostic value as mRNA markers for the detection of micrometastases by the RT-PCR assay because they are expressed in the blood and lymph nodes of patients without cancer. Further studies are needed to assess the sensitivity of CK-20 to detect micrometastases by the RT-PCR assay in the blood and frozen-section SNs of patients with breast cancer.
Antigens, Neoplasm↗
Diffusion of proteins across the nuclear envelope of HeLa cells.
We describe an experimental system to study nucleocytoplasmic diffusion of proteins in living HeLa cells. To localize proteins to the nucleus, substrates were created that contain a nuclear localization sequence fused to Aequorea victoria green fluorescent protein (GFP). Transiently and stably transfected HeLa cells were used for these assays. A protein of 29-kDa molecular mass that harbors GFP and the bipartite Xenopus nucleoplasmin nuclear localization sequence (NLS) accumulates efficiently in nuclei of HeLa cells. However, in the absence of active facilitated nuclear import, the reporter protein exits the nucleus and equilibrates between nucleus and cytoplasm. We define different conditions that promote the diffusion of small nuclear proteins across the nuclear envelope of mammalian culture cells. Our results set the stage to analyze the competence of nuclear pore complexes for nucleocytoplasmic diffusion of macromolecules in living cells.
Animals↗
Structure-activity relationships of new aranorosin analogs to antifungal activity.
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Antifungal Agents↗
Cortical binocularity and monocular optokinetic asymmetry in early-onset esotropia.
PURPOSE: To investigate the correlation between directional asymmetry in ocular responses to monocularly viewed optokinetic stimuli (monocular optokinetic nystagmus, MOKN) and sensory fusion in infants and toddlers with early-onset esotropia. METHODS: Subjects were 14 infants and toddlers with early-onset esotropia (7-26 months old; median, 10 months), and 16 with no esotropia (6-22 months; median, 11 months) who provided control data. Monocular optokinetic nystagmus in response to a 30 degrees/sec square-wave grating (0.25 cycles/degree) was measured by electro-oculogram. Sensory fusion was assessed with visual evoked potentials (VEPs) to random-dot correlograms after correction of the strabismus angle with Fresnel prisms. RESULTS: All subjects with early-onset esotropia had MOKN with a faster slow-phase component for temporal-to-nasalward (TN) than nasal-to-temporalward (NT) motion. Ninety-three percent of subjects had MOKN asymmetry higher than the 95th percentile of the control group. Of subjects who cooperated with VEP fusion testing, 5 subjects with early-onset esotropia (45%) and 11 control subjects (92%) showed evidence of sensory fusion. CONCLUSIONS: Symmetrical MOKN did not develop in infants and toddlers with early-onset esotropia. This deficit existed in most infants who showed sensory- cortical fusion. These results are consistent with the belief that optokinetic nystagmus asymmetry may not be associated with a deficit in the cortical fusion facility, but rather with deficits in binocular pathways projecting to MOKN control centers. These deficits may be associated with abnormal processing subsequent to sensory fusion or with abnormal processing in motion pathways, which run parallel to sensory fusion pathways.
Child, Preschool↗
Hypersensitivity to DNA cross-linking agents associated with up-regulation of glucose-regulated stress protein GRP78.
We have shown previously that NAD/poly(ADP-ribose) polymerase-deficient cells that overexpress Mr 78,000 glucose-regulated stress protein (GRP78) are resistant to topoisomerase II inhibitors, such as etoposide, m-amsacrine, and doxorubicin. However, these cells have been found to be hypersensitive to DNA cross-linking agents, including melphalan, cisplatin, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). These observations prompted us to examine whether overexpression of GRP78 is associated with modulation of cytotoxicity of clinically useful DNA-cross-linking agents such as melphalan, BCNU, and cisplatin. We up-regulated GRP78 in V79 Chinese hamster cells by 2-5-fold using two independent approaches that include exposure to 6-aminonicotinamide, or 2-deoxyglucose. Subsequently, these GRP78-overexpressing cells were trypsinized, plated in regular medium without GRP78-inducing agents, and allowed a 5-h attachment time before being treated with melphalan, BCNU, or cisplatin for 1 h to determine clonogenic survivals. In addition, repair of DNA cross-links induced by those agents were determined by alkaline elution assay. Our results show that the GRP78-overexpressing V79 cells are hypersensitive to DNA cross-linking agents compared to the control V79 cells. Furthermore, repair of drug-induced DNA cross-links appears to be considerably slower in these cells relative to that found in control V79 cells. Thus, our results suggest that (a) up-regulation of GRP78 is associated with an impairment of DNA cross-link repair, (b) up-regulation of GRP78 is associated with potentiation of cytotoxicity induced by alkylating and platinating agents, and (c) up-regulation of GRP78 can be considered as a potentially useful tool to modulate the cytotoxicity of clinically useful alkylating and platinating agents.
6-Aminonicotinamide↗
Synthesis and biological activity of a series of potent fluoromethyl ketone inhibitors of recombinant human calpain I.
Calpain I, an intracellular cysteine protease, has been implicated in the neurodegeneration following an episode of stroke. In this paper, we report on a series of potent dipeptide fluoromethyl ketone inhibitors of recombinant human calpain I (rh calpain I). SAR studies revealed that while calpain I tolerates a variety of hydrophobic groups at the P1 site, Leu at P2 is preferred. However, the nature of the N-terminal capping group has a significant effect on the inhibitory activity of this series of compounds. Compound 4e [(1,2,3,4-tetrahydroisoquinolin-2-yl)carbonyl-Leu-D,L-Phe-CH2F+ ++], having a tetrahydroisoquinoline containing urea as the N-terminal capping group, is the most potent dipeptide fluoromethyl ketone inhibitor of calpain I (with a second-order rate constant for inactivation of 276,000 M-1 s-1) yet reported; tripeptide 4k (Cbz-Leu-Leu-D,L-Phe-CH2F) is equipotent. A number of compounds presented in this study displayed excellent selectivity for calpain I over cathepsins B and L, two related cysteine proteases. Compounds which exhibited good inhibitory activity in the assay against isolated rh calpain I also inhibited intracellular calpain I in a human cell line. Thus, in an intact cell assay, compounds 4e and 4k inhibited calpain I with IC50 values of 0.2 and 0.1 microM, respectively. Finally, we also disclose the first example of fluorination of a dipeptide enol silyl ether to generate the corresponding dipeptide fluoromethyl ketone.
Calpain↗
In vivo analysis of nuclear protein traffic in mammalian cells.
We have developed an experimental system to study nucleocytoplasmic traffic of proteins in living mammalian cells. Toward this goal, substrates were generated that contain several copies of Aequorea victoria green fluorescent protein (GFP). To follow facilitated transport across the nuclear envelope we created reporter proteins that carry different nuclear localization sequences (NLSs). The expression of reporter genes was controlled by an inducible promoter. Transiently transfected HeLa cells were employed to follow the sorting of fluorescent reporter proteins. When NLS-GFP fusions were located in HeLa cells, we found that direct fusion of the NLS derived from SV40 T-antigen to GFP prevented nuclear accumulation of the protein. However, insertion between NLS and GFP of different linkers encoding small amino acid residues produced reporter proteins that were competent for nuclear import. Taken together, we have generated unique tools for the characterization of nuclear protein import in dividing mammalian cells.
Animals↗
Redox-regulated signaling by lactosylceramide in the proliferation of human aortic smooth muscle cells.
Previously, our laboratory reported that lactosylceramide (LacCer) stimulated human aortic smooth muscle cell proliferation via specific activation of p44 mitogen-activated protein kinase (MAPK) in the p21(ras)/Raf-1/MEK2 pathway and induced expression of the transcription factor c-fos downstream to the p44 MAPK signaling cascade (Bhunia A. K., Han, H., Snowden, A., and Chatterjee S. (1996) J. Biol. Chem. 271, 10660-10666). In the present study, we explored the role of free oxygen radicals in LacCer-mediated induction of cell proliferation. Superoxide levels were measured by the lucigenin chemiluminescence method, MAPK activity was measured by immunocomplex kinase assays, and Western blot analysis and c-fos expression were measured by Northern blot assay. We found that LacCer (10 microM) stimulates endogenous superoxide production (7-fold compared with control) in human aortic smooth muscle cells specifically by activating membrane-associated NADPH oxidase, but not NADH or xanthine oxidase. This process was inhibited by an inhibitor of NADPH oxidase, diphenylene iodonium (DPI), and by antioxidants, N-acetyl-L-cysteine (NAC) or pyrrolidine dithiocarbamate. NAC and DPI both abrogated individual steps in the signaling pathway leading to cell proliferation. For example, the p21(ras).GTP loading, p44 MAPK activity, and induction of transcription factor c-fos all were inhibited by NAC and DPI as well as an antioxidant pyrrolidine dithiocarbamate or reduced glutathione (GSH). In contrast, depletion of GSH by L-buthionine (S, R)-sulfoximine up-regulated the above described signaling cascade. In sum, LacCer, by virtue of activating NADPH oxidase, produces superoxide (a redox stress signaling molecule), which mediates cell proliferation via activation of the kinase cascade. Our findings may explain the potential role of LacCer in the pathogenesis of atherosclerosis involving the proliferation of aortic smooth muscle cells.
Acetylcysteine↗
Interaction of Gal repressor with inducer and operator: induction of gal transcription from repressor-bound DNA.
Gal repressor inhibits transcription from the gal promoter (P1) when it binds to the cognate operator (O(E)). The repression is relieved by the presence of the inducer D-galactose. Compared with its interaction with free repressor, D-galactose binds to the repressor-operator complex with 10-fold reduced affinity as determined by fluorescence enhancement measurements. Thermodynamic analysis and fluorescence anisotropy showed that the stability of the repressor-operator complex is reduced by only 7-fold by the presence of the inducer in the complex. The formation of the inducer-repressor-operator ternary complex has been confirmed by CD spectral analysis. Fluorescence spectroscopy and energy transfer experiments suggest that individual allosteric effects of the two ligands, inducer and operator, on Gal repressor are responsible for the slightly weakened stability of the ternary complex compared with the stability of the inducer-repressor and repressor-operator complexes. In vitro transcription results demonstrated full derepression of transcription of the P1 promoter under conditions in which the concentrations of the inducer-repressor binary complex are severalfold higher than the dissociation constant of the inducer-repressor-operator ternary complex into inducer-repressor and free DNA. These results strongly suggest that the inducer binding to the repressor-operator complex does not lead to dissociation of the repressor from the operator during transcription induction. Because Gal repressor inhibits transcription by modulating the alpha subunit of the P1-bound RNA polymerase, we conclude that the inducer binding to the operator-bound repressor only allosterically relieves the inhibitory effect of repressor on RNA polymerase without dissociating the repressor from DNA.
Circular Dichroism↗