Biomedical subjects

C Gu

Publications and source records attributed to C Gu.

At least 37 records · Page 2Linked to original sources

PubMed2001

The role of sexual related Y gene detection in the diagnosis of patients with gonadal dysgenesis.

OBJECTIVE: To clarify the role of sexual related Y (SRY) gene detection in the diagnosis of gonadal dysgenesis. METHODS: Sixteen cases of gonadal dysgenesis were included in this study: 5 with androgen insensitivity syndrome, 1 with 17-alpha-hydroxylase deficiency, 4 with true hermaphrodite, 2 with 45, X/46, XY gonadal dysgenesis, 1 with 45, X gonadal dysgenesis, 1 with XY pure gonadal dysgenesis, 1 with testicular regression, and 1 XY female who gave birth to a normal baby. SRY gene was detected by using polymerase chain reaction (PCR) in blood and gonad samples and by direct sequencing of the SRY motif. RESULTS: Among the 16 cases, 15 were blood SRY positive, among which 13 (86.7%) showed the presence of testicular tissue, and 2 showed ovaries without testicular tissue. One SRY negative case showed the presence of testicular tissue. In 3 cases, SRY detection in gonadal tissue correlated with pathological findings but not with blood karyotype. The correlation between peripheral blood SRY and the pathology of the gonads was 81.25% and the correlation between the presence of peripheral blood Y chromosome and pathology of the gonads was 68.75%. Sequencing of the SRY motif in an XY female who gave birth to a normal baby showed no mutation. CONCLUSIONS: SRY detection is more sensitive and specific than blood karyotype in the prediction of the presence of testicular tissue. Peripheral blood karyotype does not necessarily reflect gonadal type. There may be testicular related factors other than the SRY gene.

DNA↗

PubMed2000

Selective gas-phase cleavage at the peptide bond C-terminal to aspartic acid in fixed-charge derivatives of Asp-containing peptides.

This study focuses on the molecular level interpretation of the selective gas-phase cleavage at aspartic acid residues (Asp) in protonated peptides. A phi3P+CH2C(=O)group (phi = 2,4,6-trimethoxyphenyl) is attached to the N-terminal nitrogen of the selected peptides LDIFSDF and LDIFSDFR, via solid-phase synthesis, to "mimic" the tightly held charge of a protonated arginine (Arg) residue. Collision-induced dissociation in a quadrupole ion trap instrument and surface-induced dissociation in a dual quadrupole instrument were performed for electrospray-generated ions of the fixed-charge peptide derivatives. Selective cleavages at Asp-Xxx are observed for those ions with charge provided only by the fixed charge or for those with a fixed charge and one Arg plus one added proton. This supports a previously proposed mechanism which suggests that the cleavages at Asp-Xxx, initiated by the acidic hydrogen of the Asp residue, become significant when ionizing protons are strongly bound by Arg in the protonated peptides. It is clear that the fixed charge is indeed serving as a "mimic" of protonated Arg and that a protonated Arg side chain is not required to interact with the Asp to induce cleavage at Asp-Xxx. When the number of protons exceeds the number of Arg in a peptide containing Arg and Asp, nonselective cleavages occur. The fragmentation efficiency of the peptides is consistent with the idea that these nonselective cleavages are promoted by a mobile proton. The peptide with a fixed charge and one added proton, [phi3P+CH2C(=O)-LDIFSDF + H]2+, fragments much more efficiently than the corresponding peptide with a fixed charge, an Arg and one added proton, [phi3P+CH2C(=O)-LDIFSDFR + H]2+; both of these fragment more efficiently than the peptide with a fixed charge and no added proton, phi3P+CH2C(=O)-LDIFSDF. MS/MS/MS (i.e., MS3) experimental results for bn ions formed at Asp-Xxx from phi3P+CH2C(=O)-LDIFSDF and its H/D exchange derivative, phi3P+CH2C(=O)-LDIFSDF-d11, are consistent with the bn ions formed at Asp-Xxx having a succinic anhydride cyclic structure. MS/MS experiments were also carried out for phi3P+CH2C(=O)-AAAA, a peptide derivative containing active hydrogens only at amide nitrogens plus the C-terminus, and its active H/D exchange product, phi3P+CH2C(=O)-AAAA-d5. The results show that a hydrogen originally located at an amide nitrogen is transferred away in the formation of a cyclic charge remote b ion.

Aspartic Acid↗

PubMed2000

Use of high-performance liquid chromatography-tandem mass spectrometry to distinguish Panax ginseng C. A. Meyer (Asian ginseng) and Panax quinquefolius L. (North American ginseng).

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed to distinguish Asian ginseng (Panax ginseng C. A. Meyer) and North American ginseng (Panax quinquefolius L.). The method is based on the baseline chromatographic separation of ginsenoside Rf and 24(R)-pseudoginsenoside F11, two potential chemical markers present in ginseng root methanolic extracts, and their unambiguous on-line identification using tandem mass spectrometry. Consistent with the literature, 24(R)-pseudoginsenoside F11 was detected in abundance in North American ginseng roots in excess of 0.1% (w/w) of the dried root. In contrast to some reports, 24(R)-pseudoginsenoside F11 was also identified in Asian ginseng roots at trace levels using LC-MS-MS but at less than 0.0001% (w/w). Besides showing identical tandem mass spectra to authentic 24(R)-pseudoginsenoside F11, the corresponding compound in Asian ginseng root coeluted with standard under different HPLC conditions, thus confirming this compound as 24(R)-pseudoginsenoside F11. Another ginsenoside often used to distinguish Asian and North American ginseng, ginsenoside Rf, was found in abundance in Asian ginseng roots at more than 0.021% (w/w). In Asian ginseng roots, the ratio of ginsenoside Rf to 24(R)-pseudoginsenoside F11 exceeded 700:1. The limit of detection of ginsenoside Rf or 24(R)-pseudoginsenoside F11 was 120 pg injected on-column, and the limit of quantification was 240 pg on-column. In summary, LC-MS-MS analysis of ginseng products for the presence and ratio of ginsenoside Rf and 24(R)-pseudoginsenoside F11 may be used for the unambiguous identification of Asian and North American ginsengs.

Asia↗

PubMed2000

Introducing a null mutation in the mouse K6alpha and K6beta genes reveals their essential structural role in the oral mucosa.

Mammalian genomes feature multiple genes encoding highly related keratin 6 (K6) isoforms. These type II keratins show a complex regulation with constitutive and inducible components in several stratified epithelia, including the oral mucosa and skin. Two functional genes, K6alpha and K6beta, exist in a head-to-tail tandem array in mouse genomes. We inactivated these two genes simultaneously via targeting and homologous recombination. K6 null mice are viable and initially indistinguishable from their littermates. Starting at two to three days after birth, they show a growth delay associated with reduced milk intake and the presence of white plaques in the posterior region of dorsal tongue and upper palate. These regions are subjected to greater mechanical stress during suckling. Morphological analyses implicate the filiform papillae as being particularly sensitive to trauma in K6alpha/K6beta null mice, and establish the complete absence of keratin filaments in their anterior compartment. All null mice die about a week after birth. These studies demonstrate an essential structural role for K6 isoforms in the oral mucosa, and implicate filiform papillae as being the major stress bearing structures in dorsal tongue epithelium.

Animals↗

PubMed2000

Apoptotic signaling through the beta -adrenergic receptor. A new Gs effector pathway.

Stimulation of beta-adrenergic receptor normally results in signaling by the heterotrimeric G protein G(s), leading to the activation of adenylyl cyclase, production of cAMP, and activation of cAMP-dependent protein kinase (PKA). Here we report that cell death of thymocytes can be induced after stimulation of beta-adrenergic receptor, or by addition of exogenous cAMP. Apoptotic cell death in both cases was observed with the appearance of terminal deoxynucleotidyl transferase-mediated UTP end labeling reactivity and the activation of caspase-3 in S49 T cells. Using thymocytes deficient in either Galpha(s) or PKA, we find that engagement of beta-adrenergic receptors initiated a Galpha(s)-dependent, PKA-independent pathway leading to apoptosis. This alternative pathway involves Src family tyrosine kinase Lck. Furthermore, we show that Lck protein kinase activity can be directly stimulated by purified Galpha(s). Our data reveal a new signaling pathway for Galpha(s), distinct from the classical PKA pathway, that accounts for the apoptotic action of beta-adrenergic receptors.

Animals↗

PubMed2000

Ca(2+), Sr(2+), and Ba(2+) identify distinct regulatory sites on adenylyl cyclase (AC) types VI and VIII and consolidate the apposition of capacitative cation entry channels and Ca(2+)-sensitive ACs.

Ca(2+)-sensitive adenylyl cyclases may act as early integrators of the two major second messenger-signaling pathways mediated by Ca(2+) and cAMP. Ca(2+) stimulation of adenylyl cyclase type I (ACI) and adenylyl cyclase type VIII (ACVIII) is mediated by calmodulin and the site on these adenylyl cyclases that interacts with calmodulin has been defined. By contrast, the mechanism whereby Ca(2+) inhibits adenylyl cyclase type V (ACV) and adenylyl cyclase type VI (ACVI) is unknown. In this study, Ca(2+), Sr(2+), and Ba(2+) were compared to probe the involvement of E-F hand-like domains in both Ca(2+) stimulation and inhibition of ACVIII and ACVI, respectively. HEK 293 cells transfected with ACVIII cDNA and C6-2B glioma cells (where the endogenous adenylyl cyclases is predominantly ACVI) were used to compare the effects of these three cations in in vitro and in vivo measurements. The in vitro data identified two Ca(2+) regulatory sites for both ACVIII and ACVI. Strikingly different potency series for these cations at mediating high affinity stimulation and inhibition of ACVIII and ACVI, respectively, effectively rule out the possibility that calmodulin or proteins utilizing similar Ca(2+)-binding motifs mediate inhibition of ACVI. On the other hand, the low affinity inhibition that is common to both ACVIII and ACVI showed virtually identical potency profiles for the IIa cation series, indicating a common site of action. Remarkably, whereas Sr(2+) was rather ineffective at regulating these cyclases (particularly ACVI) in vitro, adequate concentrations accumulated in the vicinity of these enzymes as a consequence of capacitative cation entry to partially regulate both of these activities in vivo. This latter finding consolidates earlier observations that Ca(2+)-sensitive adenylyl cyclases detect and respond to capacitative cation entry rather than global cytosolic cation concentrations.

Adenylyl Cyclase Inhibitors↗

PubMed2000

Transcriptional regulatory effects of lymphoma-associated NFKB2/lyt10 protooncogenes.

C-terminal truncations of the NFKB2 p100 gene product have been observed in a number of cases of human cutaneous T cell lymphomas, as well as human B-cell lymphomas and myelomas. The contribution of these alterations to lymphomagenesis is not understood; however, truncation at amino acid 666 to generate 80 - 85 kD proteins in the HUT78 cell line is associated with addition of a short (serine-alanine-serine) fusion at the 3' end of p80HT, as well as with increased expression of NFKB2 mRNA. We therefore examined the effects of p80HT on the regulation of NFKB2 expression, as well as the properties of a series of other tumor-associated, and site directed mutations of NFKB2. While p80HT had not itself acquired novel transcriptional activation properties with respect to the NFKB2 P1 or P2 promoters or the IL-6 kappaB promoter, p80HT had lost the potent inhibitory (IkappaB-like) activity associated with the wild-type, p100 gene product. Loss of the inhibitory property depended on the SAS residues in the fusion protein, direct truncation at aa666 was fully inhibitory, as was a substitution of three alanines for the SAS residues. The presence of as few as two C-terminal ankyrin motifs was sufficient for inhibition of NF-kappaB-mediated transcriptional activation. Assays of a series of additional lymphoma-associated NF-kappaB-2 truncation suggested that the C-terminal truncation associated with these proteins was also associated with a loss of the IkappaB-like activities of p100 NF-kappaB-2, for at least some NF-kappaB target promoters. Thus, the loss of IkappaB-like activity of lymphoma-associated NFKB2 mutations may play an important role in the genesis of a subset of human lymphomas.

Gene Expression Regulation, Neoplastic↗

PubMed2000

Characterization of atherosclerosis in LDL receptor knockout mice: macrophage accumulation correlates with rapid and sustained expression of aortic MCP-1/JE.

Atherosclerosis and the expression of monocyte chemoattractant protein-1 (MCP-1) were quantified in low density lipoprotein receptor knockout (LDLR KO) mice fed 1.25% cholesterol (study #1) or 0.2% cholesterol (study #2). In study #1 plasma total cholesterols leveled-off at 1800 mg/dl whereas plasma triglycerides remained low. In en face specimens of the aortic root and arch, intimal foam cells plus extracellular lipid particles accumulated and by 8 weeks the fatty streak surface area had rapidly expanded at both sites. In study #2, total cholesterols averaged 400 mg/dl and fatty streaks were 2-3-fold smaller compared to those in study #1. In study #3, LDLR KO mice were fed chow or 1.25% cholesterol, and immunostaining demonstrated a few Mac-2-positive intimal macrophages in mice fed chow, and during the first 10 weeks of hypercholesterolemia the number of intimal macrophages increased continuously. In chow-fed mice (0 weeks) there was little MCP-1 in the aorta. After 2 days of hypercholesterolemia intimal macrophages stained for MCP-1, and during the next 10 weeks recently recruited arterial macrophages also expressed MCP-1. Macrophage accumulation was highly correlated with MCP-1 expression. In study #4, feeding LDLR KO mice 1.25% cholesterol for 6 months produced atherosclerotic plaques at both sites and they contained a fibrous cap of smooth muscle cells, macrophage-foam cells, connective tissue and cholesterol crystals. In summary, LDLR KO mice fed cholesterol develop fatty streaks that transform into fibrous plaques. Hypercholesterolemia rapidly triggers MCP-1 expression in resident intimal macrophages, which is followed by the accumulation of more macrophages that also express MCP-1, suggesting that this chemokine may both initiate and amplify monocyte recruitment to the artery wall during early atherogenesis.

Animals↗

PubMed2000

Genomic structure and promoter analysis of the mouse EphA8 receptor tyrosine kinase gene.

The gene encoding the mouse EphA8 receptor tyrosine kinase has been isolated from a mouse genomic library, and its complete genomic structure has been determined. This gene spans approximately 28 kb and consists of 17 exons. This gene structure is similar to the structure of the chick EphB2 (Cek5) gene, except for one intron present between the first two exons encoding the EphA8 kinase domain. This difference may reflect an evolutionary divergence of the catalytic domain between EphA and EphB subgroup receptors. The site for transcription initiation has been mapped to the 19th nucleotide upstream from the translation start codon ATG. A feature of this gene is an unmethylated CpG island spanning exon 1 and the flanking sequence. The putative promoter of the EphA8 gene lacks a TATA box and contains multiple copies of the sequence GGGCGG, the core sequence of the putative Sp1-binding site. The 3.5-kb upstream genomic region containing part of the first exon showed strong promoter activity in NG108-15 neuroblastoma cells but much less in 293T cells, suggesting that this fragment is sufficient for neural cell-directed promoter activity. By deleting the genomic region containing the five GC boxes, it was shown that the minimal promoter region is primarily comprised of five copies of the Sp1-binding site located upstream from the transcription initiation site. Finally, in situ RNA hybridization studies revealed a very specific pattern of EphA8 gene expression restricted to the rostral region of midbrain tectum during embryonic development. Isolation of a functional promoter for the EphA8 gene is a first step in understanding how expression of this gene is controlled at the molecular level.

Amino Acid Sequence↗

PubMed2000

[Reevaluation of the nomenclature and diagnostic criteria in 477 patients with severe hepatitis].

OBJECTIVE: To explore the possibility of establishing more reasonable nomenclatures and diagnostic criteria for patients with severe hepatitis (SH) through analyzing clinical data of 477 cases of SH. METHODS: The clinical characteristics and outcomes of SH were analyzed according to different criteria. RESULTS: Chronic severe hepatitis (CSH) made up 88.5% of total cases of SH. The survival rate in the patients with hepatic encephalopathy was much lower than that without hepatic encephalopathy. About 1/5 of cases of subacute severe hepatitis (SSH) and CSH had neither ascites nor hepatic encephalopathy. When the period of 2 weeks was used in replace of 10 days for the diagnosis for acute severe hepatitis (ASH), the newly added cases were consistent with the characteristics of ASH. CONCLUSION: We suggest dividing SH into 2 types: encephalopathy and non-encephalopathy by using the nomenclature of fulminant hepatitis and severe type hepatitis, respectively. The late-onset form should be added besides of acute form and subacute form. It seems to use the period of 2 weeks as the new definition of onset time for ASH. The criteria of dividing SH into 3 forms, i.e. ascites, encephalopathy and ascites plus encephalopathy, and the nomenclature recommended by the International Association for the Study of the Liver Subcommittee are not satisfactory when used in clinical cases. The typing of CSH remains to be clarified.

Adolescent↗

PubMed2000

[The role of intercellular adhesion molecule-1/lymphocyte function-associated antigen-1 in the pathogenesis of viral hepatitis B].

OBJECTIVE: To study the role of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) in the pathogenesis of viral hepatitis B. METHODS: ICAM-1, LFA-1 and CD(8) expression in the livers of 11 healthy persons and 70 patients with HBV infection were studied by using immunohistochemical techniques. Hepatic coexpression of ICAM-1 and LFA-1 were studied using double staining immunohistochemical techniques. RESULTS: In the areas of inflammation and necrosis, intensive infiltration of LFA-1 positive lymphocytes were often observed around hepatocytes with enhanced ICAM-1 expression, with some of these lymphocytes in close contact with hepatocytes expressing ICAM-1. In addition, the debris of necrotic cells and injured hepatocytes were found at the surrounding of LFA-1 positive lymphocytes. CONCLUSION: The interaction of ICAM-1 and LFA-1 is involved in mediating the adhesion of lymphocytes and hepatocytes, and it plays an important part in the immunopathogenesis of chronic viral hepatitis B and severe viral hepatitis B.

Adult↗

PubMed2000

[Loss and inactivation of PTEN/MMAC1/TEP1 gene in lung cancer].

OBJECTIVE: The recently identified PTEN/MMAC1/TEP1 gene is the first tumor suppressor encoding a phosphatase, which mutated in multiple tumors. Our research aims to detect loss of heterozygosity and inactivation of PTEN in lung cancer. METHODS: 24 lung cancer fresh samples paired with normal tissue, 18 archival paraffin-embedded sections of small cell lung cancer (SCLC) were included. For 24 fresh samples, PCR-LOH was used to detect Loss of heterozygosity (LOH) in or near PTEN loci. In situ hybridization, Western blot and or immunohistochemistry were proceeded for mRNA and protein expression of PTEN in lung cancer samples. Southern, Northern and Western blot were done to exam PTEN abnormality in one NSCLC and three SCLC cell lines. RESULTS: Among these 24 fresh samples PTEN LOH was detected in 11 out of 24 cases (45.8%) and 6 cases had no PTEN mRNA and protein expression. Results in situ hybridization were in conformity with that of immunohistochemistry/Western blot in our research. No protein expression was detected in 8/18 (44%) archival paraffin cases of SCLC. Southern, Northern and Western blot confirmed homozygous deletion in one SCLC cell line. CONCLUSION: Lung cancers had LOH and inactivation of PTEN.

Adenocarcinoma↗

PubMed2000

[HPV16 E6E7 fragments transform immortalized human bronchial epithelial cells into neoplastic cells].

OBJECTIVE: To study the effects of HPV16 E6E7 fragments on the biological behavior of immortalized human bronchial epithelial cells TR. METHODS: E6E7 fragments were constructed into retrovirus expression vector and then transfected into TR cells to observe changes in growth character and tumorigenesis in nude mice. IP-Western blots were done to analyze function of p27 protein as well as expression and phosphorylation of FAK and paxillin. RESULTS: Southern, Northern, Western blot confirmed the stable expression of E6E7 fragments in puromycin-resistant cell strain TR/E6E7. TR/E6E7 proliferated faster, showed stronger growth ability in soft agar and eventually formed tumor after being inoculated into nude mice. IP-Western blot also showed sequestering of p27 protein from cyclin E/CDK2 complex, which implied increased activity of cyclin E/CDK2 and promoting G1 to S cell cycle. CONCLUSION: TR/E6E7 cells formed tumor in nude mice indicating that HPV infection played a role in pulmonary carcinogenesis. Sequestering of p27 protein from cyclin E/CDK2 complex may be responsible for increased proliferation in cells transfected by HPV16 E6E7.

Animals↗

PubMed2000

[Effects of cytokine gene therapy on prolonging survival time of allografted skin after scalding in a murine model].

OBJECTIVE: To explore the role of cytokine gene therapy on prolonging survival time of allografted skin after scalding in a murine model. METHODS: Interleukin 10 (IL-10) gene was employed as therapeutic objective gene and fibroblast was as a carrier cell. Gene transcription technique was adopted to establish an experimental murine model in which fibroblast-mediated gene therapy was used to prolong allografted skin survival time after scalding. IL-10 was transferred into fibroblastocyte (NIH3T3) by reverse transcriptive virus vector. The mice were grafted with alloskin after scalding. In addition, collagen capsulized NIH3T-3-IL-10 cells were implanted intraperitoneally in the mice so as to observe its influence on allografted skin survival time and on the changes of their main internal organs. RESULTS: Cytokine gene therapy could obviously prolong the survival time of allografted skin (P < 0.01) without any evident detrimental effect on the internal organs. CONCLUSION: These results demonstrated that skin allograft rejection could be inhibited and the survival time be prolonged with the implantation of the fibroblastocyte transferred IL-10 gene.

Animals↗

PubMed2000

[Gastro-pyloro-duodenal motor cooperation in patients with dyspepsia].

OBJECTIVE: To investigate the role of the midgastric transverse band(MTB) and gastro-pyloro-duodenal motility in functional dyspepsia (FD) and diabetic dyspepsia (DD). METHODS: Healthy subjects, patients with FD and DD participated in the study. Of them, 65 subjects received radionuclide gastric emptying, 48 complex ultrasonograph, and 35 gastro-pyloro-duodenal manometry. RESULTS: (1) MTB was (7.01 +/- 1.31), (3.39 +/- 0.21) cm in FD and DD, which was significantly wider than that in HS group [(2.42 +/- 0.16) cm, P < 0.01, P < 0.001]. (2) The half gastric emptying time (t 1/2) in FD and DD were prolonged, gastric remained rate at 120 min increased, the gastric emptying velocity reduced when compared with HS. (3) The gastric occlusive and non-occlusive peristaltic contractions could be clearly shown under ultrasonograph. The percentage of emptying circle > 10 seconds was reduced in FD and DD. (4) There was decreased number of migrating motor complex phase III in FD and DD, when compared with HS. CONCLUSIONS: MTB is an area with physiological function, which affects intra-gastric food distribution. Patients with FD and DD showed delayed gastric emptying associated with decreased gastric motor activity and antro-pyloro-duodenal in-cooperative function. There may be different pathogenesis in FD and DD. Further study needs to be performed.

Adult↗

PubMed2000

[Electrogastograph during interdigestive and digestive stages in patients with functional dyspepsia].

OBJECTIVE: To investigate the relationship between dynamic change of electrogastrogram (EGG) with motility index of antrum during migrating motor complex (MMC) cycle in healthy subjects and functional dyspepsia, and to verify whether a 100% coupling of electrical and mechanical events existed after meal. METHODS: Eleven healthy subjects (HS) and 14 patients with functional dyspepsia (FD) were studied. An 8-channel Dent-sleeve catheter was positioned under fluoroscopic guidance across antropyloroduodenal region and bipolar electrodes were attached to the skin corresponding to the location of antrum. Antropyloroduodenal pressure waves and electrical signals were transferred simultaneously to PC Polygraph (CTD) and recorded for 3 hours preprandially and 2 hours after ingestion of 40 g solid meal (336 kJ). The power of EGG and motility index (MI) of antral contractions were calculated as the area under curve (AUC) by a build-in computer program. RESULTS: The power increases of EGG increased significantly in phase II (P < 0.01), phase III (P < 0.01) and after meal (P < 0.01) compared with phase I in HS, however, this dynamic increase was not found in FD. The power of EGG was positively correlated with the motility index of antrum both in HS (r = 0.87, P < 0.01) and in FD (r = 0.79, P < 0.01) during the interdigestive period. A significant increase of EGG power after meal was found in HS, which corresponded to the degree of phase III and kept stable for the whole postprandial period. In contrast, power increase was not found in FD except during 30 min after test meal. CONCLUSIONS: Prolonged cutaneous EGG recording can reflect the periodic variation of MMC cycle. EGG response in HS but not in FD after meal is characterized as persistent increase which is corresponded to phase III.

Adult↗

PubMed1999

Oligodendrocyte apoptosis mediated by caspase activation.

Treatment with NGF causes long-term cultures of oligodendrocytes to die via a yet undefined mechanism mediated by the p75 neurotrophin receptor. The p75 receptor belongs to the TNF receptor superfamily of molecules, which includes Fas and p55 TNF receptors. The Fas and TNF receptors use adaptor molecules to recruit and activate caspase-8 to the receptor. Using a combination of immunohistochemical and Western blotting assays, we have examined caspase activity during NGF-induced apoptosis. Interestingly, although caspase-1 [interleukin-1beta-converting enzyme (ICE)], caspase-2, caspase-3, and caspase-8 were expressed in oligodendrocytes, only caspase-1, -2, and -3 were activated after NGF treatment, whereas caspase-8 was not. These data suggest that the mechanism of apoptosis by NGF through the p75 receptor is different from TNF and Fas-mediated killing. gamma Radiation of oligodendrocytes also activated a similar subset of caspases as NGF, indicating that NGF-induced oligodendrocyte apoptosis uses a similar cell death execution mechanism as injury models. This consolidates a potential role of the p75 neurotrophin receptor during stress and inflammatory conditions.

Animals↗

PubMed1999

Calmodulin-binding sites on adenylyl cyclase type VIII.

Ca2+ stimulation of adenylyl cyclase type VIII (ACVIII) occurs through loosely bound calmodulin. However, where calmodulin binds in ACVIII and how the binding activates this cyclase have not yet been investigated. We have located two putative calmodulin-binding sites in ACVIII. One site is located at the N terminus as revealed by overlay assays; the other is located at the C terminus, as indicated by mutagenesis studies. Both of these calmodulin-binding sites were confirmed by synthetic peptide studies. The N-terminal site has the typical motif of a Ca2+-dependent calmodulin-binding domain, which is defined by a characteristic pattern of hydrophobic amino acids, basic and aromatic amino acids, and a tendency to form amphipathic alpha-helix structures. Functional, mutagenesis studies suggest that this binding makes a minor contribution to the Ca2+ stimulation of ACVIII activity, although it might be involved in calmodulin trapping by ACVIII. The primary structure of the C-terminal site resembles another calmodulin-binding motif, the so-called IQ motif, which is commonly Ca2+-independent. Mutagenesis and functional assays indicate that this latter site is a calcium-dependent calmodulin-binding site, which is largely responsible for the Ca2+ stimulation of ACVIII. Removal of this latter calmodulin-binding region from ACVIII results in a hyperactivated enzyme state and a loss of Ca2+ sensitivity. Thus, Ca2+/calmodulin regulation of ACVIII may be through a disinhibitory mechanism, as is the case for a number of other targets of Ca2+/calmodulin.

Adenylyl Cyclases↗