Biomedical subjects
Publications and source records attributed to J Ma.
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The ryanodine receptor/Ca(2+) release channels from skeletal (RyR1) and cardiac (RyR2) muscle cells exhibit different inactivation profiles by cytosolic Ca(2+). D3 is one of the divergent regions between RyR1 (amino acids (aa) 1872-1923) and RyR2 (aa 1852-1890) and may contain putative binding site(s) for Ca(2+)-dependent inactivation of RyR. To test this possibility, we have deleted the D3 region from RyR1 (DeltaD3-RyR1), residues 1038-3355 from RyR2 (Delta(1038-3355)-RyR2) and inserted the skeletal D3 into Delta(1038-3355)-RyR2 to generate sD3-RyR2. The channels formed by DeltaD3-RyR1 and Delta(1038-3355)-RyR2 are resistant to inactivation by mM [Ca(2+)], whereas the chimeric sD3-RyR2 channel exhibits significant inactivation at mM [Ca(2+)]. The DeltaD3-RyR1 channel retains its sensitivity to activation by caffeine, but is resistant to inactivation by Mg(2+). The data suggest that the skeletal D3 region is involved in the Ca(2+)-dependent regulation of the RyR1 channel.
Recent electrostatics calculations on the cytochrome c oxidase from Paracoccus denitrificans revealed an unexpected coupling between the redox state of the heme-copper center and the state of protonation of a glutamic acid (E78II) that is 25 A away in subunit II of the oxidase. Examination of more than 300 sequences of the homologous subunit in other heme-copper oxidases shows that this residue is virtually totally conserved and is in a cluster of very highly conserved residues at the "negative" end (bacterial cytoplasm or mitochondrial matrix) of the second transmembrane helix. The functional importance of several residues in this cluster (E89II, W93II, T94II, and P96II) was examined by site-directed mutagenesis of the corresponding region of the cytochrome bo(3) quinol oxidase from Escherichia coli (where E89II is the equivalent of residue E78II of the P. denitrificans oxidase). Substitution of E89II with either alanine or glutamine resulted in reducing the rate of turnover to about 43 or 10% of the wild-type value, respectively, whereas E89D has only about 60% of the activity of the control oxidase. The quinol oxidase activity of the W93V mutant is also reduced to about 30% of that of the wild-type oxidase. Spectroscopic studies with the purified E89A and E89Q mutants indicate no perturbation of the heme-copper center. The data suggest that E89II (E. coli numbering) is critical for the function of the heme copper oxidases. The proximity to K362 suggests that this glutamic acid residue may regulate proton entry or transit through the K-channel. This hypothesis is supported by the finding that the degree of oxidation of the low-spin heme b is greater in the steady state using hydrogen peroxide as an oxidant in place of dioxygen for the E89Q mutant. Thus, it appears that the inhibition resulting from the E89II mutation is due to a block in the reduction of the heme-copper binuclear center, expected for K-channel mutants.
Extracellular adenosine critically modulates ischemic brain injury, at least in part through activation of the A(1) adenosine receptor. However, the role played by the A(2A) receptor has been obscured by intrinsic limitations of A(2A) adenosinergic agents. To overcome these pharmacological limitations, we explored the consequences of deleting the A(2A) adenosine receptor on brain damage after transient focal ischemia. Cerebral morphology, as well as vascular and physiological measures (before, during, and after ischemia) did not differ between A(2A) receptor knock-out and wild-type littermates. The volume of cerebral infarction, as well as the associated neurological deficit induced by transient filament occlusion of the middle cerebral artery, were significantly attenuated in A(2A) receptor knock-out mice. This neuroprotective phenotype of A(2A) receptor-deficient mice was observed in different genetic backgrounds, confirming A(2A) receptor disruption as its cause. Together with complimentary pharmacological studies, these data suggest that A(2A) receptors play a prominent role in the development of ischemic injury within brain and demonstrate the potential for anatomical and functional neuroprotection against stroke by A(2A) receptor antagonists.
BACKGROUND: The Physicians' Health Study was a randomized, double-blind, placebo-controlled trial using a 2x2 factorial design including supplementation with beta-carotene (50 mg every other day) in the primary prevention of cancer among 22,071 U.S. male physicians ages 40-84 years at randomization. Before randomization, the authors collected baseline blood specimens to determine whether any benefit was greater among or confined to those with low baseline levels of beta-carotene. METHODS: Baseline blood samples were collected from 14,916 participants. These samples were assayed, according to a nested case-control design, from 1439 men subsequently diagnosed with cancer over 12 years of follow-up (631 with prostate carcinoma) and 2204 controls matched by age and smoking habits. RESULTS: Men in the lowest quartile for plasma beta-carotene at baseline had a marginally significant (P = 0.07) increased risk of cancer compared with those in the highest quartile (relative risk [RR] = 1.30, 95% confidence interval [CI], 0.98-1.74). Men in the lowest quartile assigned at random to beta-carotene supplementation had a possible but nonsignificant decrease in overall cancer risk (RR = 0.83, 95% CI, 0.63-1.09) compared with those assigned to placebo. This was primarily due to a significant reduction in the risk of prostate carcinoma (RR = 0.68, 95% CI, 0. 46-0.99) in this group. After the first 2 years of follow-up were excluded, the results were virtually unchanged. CONCLUSIONS: These prespecified subgroup analyses appeared to support the idea that beta-carotene supplementation may reduce risk of prostate carcinoma among those with low baseline levels. Further follow-up of this population will help determine whether these findings are valid.
Several N-substituted 11-azaartemisinins were prepared from 11-azaartemisinin in high yield by the DMAP catalyzed addition of terminal acetylenes conjugated with electron-withdrawing groups. Their antimalarial activities against two drug-resistant strains of Plasmodium falciparum were determined.
To test the role of nucleotide-binding fold (NBF) 2 and its interaction with the regulatory (R) domain in the function of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, we used three deletion mutants of CFTR: DeltaR(708-835), DeltaNBF2(1185-1349) and DeltaR-DeltaNBF2. In lipid bilayers, DeltaNBF2 channel activity is ATP- and cAMP-dependent protein kinase (PKA)-dependent, but unlike wild-type (wt) CFTR, it displays a reduced activity and insensitivity to 5'-adenylylimidodiphosphate (AMP-PNP). Both DeltaR and DeltaR-DeltaNBF2 channels are PKA-independent, but DeltaR activity is reduced whereas DeltaR-DeltaNBF2 activity is increased. Deletion of NBF2 from CFTR affects protein trafficking and channel gating kinetics. The data suggest that NBF2 could have inhibitory and stimulatory roles in CFTR activity by interaction with NBF1 directly or indirectly via the R domain.
Apoptosis is the final pathway of many forms of retinal degeneration. E1A-NR3 is an immortalized retinal cell line that manifests specific phenotypes of retinal neurons. The present study induced apoptosis in these cells by two ischemic assaults, serum deprivation and hypoxia. The results demonstrated that both the assaults decreased viable cell numbers significantly by inducing apoptosis, as revealed by viable cell count, DNA fragmentation analysis, and in situ labeling of apoptotic cells by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method. l-Deprenyl is known to be a monoamine oxidase inhibitor, and it was found recently to have neurotrophic activities. We set out to determine the protective effect of l-deprenyl on retinal cells and delineate its mechanism independent of monoamine oxidase inhibition. At concentrations as low as 0.0001 and 0.001 microM, l-deprenyl significantly increased the numbers of surviving cells under serum-free and hypoxic conditions, respectively. This effect appeared to be dependent upon the l-deprenyl concentration within the range of 0.001 to 10 microM. The neurotrophic activity was via blocking apoptosis, as l-deprenyl decreased the fragmented DNA and the numbers of positively stained apoptotic cells under serum-free or hypoxic conditions. Using mRNA differential display, nine mRNAs were identified and confirmed by northern blot analysis to have altered expression levels at 8 hr of exposure to hypoxia. Five of them do not match any existing sequences in GenBank, whereas the other four represent known genes including c-jun, heat-shock protein hsp70, phosphoglycerate kinase (PGK), and calpactin I heavy chain. All of the four mRNAs were increased significantly by hypoxia. The c-jun, PGK, and calpactin mRNAs, but not hsp70, also were increased by serum withdrawal. l-Deprenyl partially reversed the increase in c-jun and hsp70 mRNA levels, but not in PGK and calpactin. These results suggest that l-deprenyl blocks apoptosis induced by hypoxia as well as by growth factor withdrawal and regulates the expression of apoptosis-related genes.
The association of baseline fatty acid composition in plasma cholesterol esters with 6-year incidence of hypertension was examined in middle-aged Minneapolis participants of the Atherosclerosis Risk in Communities (ARIC) Study (1987-1995). Compared with those who were never hypertensive (n = 1,975), incident hypertensives (n = 413) had statistically significantly higher baseline levels of palmitic (16:0) and palmitoleic (16:1n7) acids but lower levels of linoleic (18:2n6) acid and the polyunsaturated/saturated fatty acids ratio (P/S ratio). Among polyunsaturated fatty acids, levels of dihomo-gamma-linolenic (20:3n6) and arachidonic (20:4n6) acids were statistically significantly higher in incident hypertensives, compared with normotensives. After adjustment for age, sex, body mass index, waist/hip ratio, smoking status, ethanol intake, education level, physical activity, and baseline systolic blood pressure in separate models, the odds ratio estimates of incident hypertension for an interquartile increment of a fatty acid in cholesterol esters were 1.26 (95% confidence interval (CI): 1.05, 1.51) for 16:0, 1.11 (95% CI: 0.96, 1.28) for 16:1n7, 1.01 (95% CI: 0.85, 1.21) for 20:3n6, 1.14 (95% CI: 1.03, 1.27) for 20:5n3, 0.81 (95% CI: 0.68, 0.96) for 18:2n6, and 0.83 (95% CI: 0.70, 0.99) for the P/S ratio. The authors conclude that reduced levels of linoleic acid and the P/S ratio and elevated levels of palmitic and arachidonic acids are associated with a higher risk of hypertension.
WD repeat proteins are components of multiprotein complexes that are involved in a wide spectrum of cellular activities, such as cell cycle progression, signal transduction, apoptosis, and gene regulation. These proteins are characterized by repeat units bracketed by Gly-His and Trp-Asp (GH-WD). We report here the isolation of a new member of the WD repeat gene family, WDR3, which encodes a putative 943-amino-acid nuclear protein consisting of 10 WD repeat modules. WDR3 is widely expressed in hematopoietic cell lines and in nonhematopoietic tissues. Fluorescence in situ hybridization mapped WDR3 to human chromosome 1p12-p13, a region that is affected by chromosomal rearrangements in a number of hematologic malignancies and solid tumors.
Hippocampal EEG and behavior of freely moving rats were studied before and after a hippocampal afterdischarge (AD), with or without reversible inactivation of the medial septum (MS) by muscimol. Muscimol suppressed the normal hippocampal EEG, including theta (5-10 Hz) and gamma (30-70 Hz) waves. After a hippocampal AD, hippocampal gamma waves were decreased for about 2 min and then increased at 3-10 min, while power of EEG of <30 Hz was decreased at </=3 min and showed no increase after an AD. Muscimol injection in the MS before the AD blocked the post-ictal increase of the gamma waves. In rats injected with vehicle in the MS, post-ictal increase in gamma waves was accompanied by an increase in horizontal locomotion, rearings, face washes, and wet dog shakes. In rats injected with muscimol in the MS before the AD, both the post-ictal increase in gamma waves and behaviors were completely suppressed. Muscimol injection in the MS after the AD significantly suppressed both gamma waves and post-ictal behaviors, as compared to vehicle-injected rats, but to a lesser degree than rats injected with muscimol in the MS before the AD. Hippocampal AD duration was not significantly affected by muscimol injection in the MS. We conclude that the MS is responsible for the maintenance of the normal and the post-ictal gamma waves. We suggest that the post-ictal gamma waves in the hippocampus may drive post-ictal behaviors, and in previous studies, we showed that this may occur through the nucleus accumbens.
A cDNA encoding a new cytochrome P450 was isolated from a mouse liver library. Sequence analysis reveals that this 1,886-base pair cDNA encodes a 501-amino acid polypeptide that is 69-74% identical to CYP2J subfamily P450s and is designated CYP2J5. Recombinant CYP2J5 was co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 cells using a baculovirus system. Microsomal fractions of CYP2J5/NADPH-cytochrome P450 oxidoreductase-transfected cells metabolize arachidonic acid to 14,15-, 11,12-, and 8, 9-epoxyeicosatrienoic acids and 11- and 15-hydroxyeicosatetraenoic acids (catalytic turnover, 4.5 nmol of product/nmol of cytochrome P450/min at 37 degrees C); thus CYP2J5 is enzymologically distinct. Northern analysis reveals that CYP2J5 transcripts are most abundant in mouse kidney and present at lower levels in liver. Immunoblotting using a polyclonal antibody against a CYP2J5-specific peptide detects a protein with the same electrophoretic mobility as recombinant CYP2J5 most abundantly in mouse kidney microsomes. CYP2J5 is regulated during development in a tissue-specific fashion. In the kidney, CYP2J5 is present before birth and reaches maximal levels at 2-4 weeks of age. In the liver, CYP2J5 is absent prenatally and during the early postnatal period, first appears at 1 week, and then remains relatively constant. Immunohistochemical staining of kidney sections with anti-human CYP2J2 IgG reveals that CYP2J protein(s) are present primarily in the proximal tubules and collecting ducts, sites where the epoxyeicosatrienoic acids are known to modulate fluid/electrolyte transport and mediate hormonal action. In situ hybridization confirms abundant CYP2J5 mRNA within tubules of the renal cortex and outer medulla. Epoxyeicosatrienoic acids are endogenous constituents of mouse kidney thus providing direct evidence for the in vivo metabolism of arachidonic acid by the mouse renal epoxygenase(s). Based on these data, we conclude that CYP2J5 is an enzymologically distinct, developmentally regulated, protein that is localized to specific nephron segments and contributes to the oxidation of endogenous renal arachidonic acid pools. In light of the well documented effects of epoxyeicosatrienoic acids in modulating renal tubular transport processes, we postulate that CYP2J5 products play important functional roles in the kidney.
BACKGROUND: Insulin-like growth factor-I (IGF-I) is a potent mitogen for normal and neoplastic cells, whereas IGF-binding protein-3 (IGFBP-3) inhibits cell growth in many experimental systems. Acromegalics, who have abnormally high levels of growth hormone and IGF-I, have higher rates of colorectal cancer. We therefore examined associations of plasma levels of IGF-I and IGFBP-3 with the risk of colorectal cancer in a prospective case-control study nested in the Physicians' Health Study. METHODS: Plasma samples were collected at baseline from 14916 men without diagnosed cancer. IGF-I, IGF-II, and IGFBP-3 were assayed among 193 men later diagnosed with colorectal cancer during 14 years of follow-up and among 318 age- and smoking-matched control subjects. All P values are two-sided. RESULTS: IGFBP-3 levels correlated with IGF-I levels (r=.64) and with IGF-II levels (r=.90). After controlling for IGFBP-3, age, smoking, body mass index (weight in kg/[height in m]2), and alcohol intake, men in the highest quintile for IGF-I had an increased risk of colorectal cancer compared with men in the lowest quintile (relative risk [RR]=2.51; 95% confidence interval [CI]=1.15-5.46; P for trend = .02). After controlling for IGF-I and other covariates, men with higher IGFBP-3 had a lower risk (RR=0.28; 95% CI=0.12-0.66; P for trend = .005, comparing extreme quintiles). The associations were consistent during the first and the second 7-year follow-up intervals and among younger and older men. IGF-II was not associated with risk. CONCLUSIONS: Our findings suggest that circulating IGF-I and IGFBP-3 are related to future risk of colorectal cancer.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase (PKA)- and ATP-regulated chloride channel, whose gating process involves intra- or intermolecular interactions among the cytosolic domains of the CFTR protein. Tandem linkage of two CFTR molecules produces a functional chloride channel with properties that are similar to those of the native CFTR channel, including trafficking to the plasma membrane, ATP- and PKA-dependent gating, and a unitary conductance of 8 picosiemens (pS). A heterodimer, consisting of a wild type and a mutant CFTR, also forms an 8-pS chloride channel with mixed gating properties of the wild type and mutant CFTR channels. The data suggest that two CFTR molecules interact together to form a single conductance pore for chloride ions.
ST6Gal-I (alpha2,6-sialyltransferase) is expressed as two isoforms, STTyr and STCys, which exhibit differences in catalytic activity, trafficking through the secretory pathway, and proteolytic processing and secretion. We have found that the ST6Gal-I isoforms are phosphorylated on luminal Ser and Thr residues. Immunoprecipitation of 35S- and 32P-labeled proteins expressed in COS-1 cells suggests that the STTyr isoform is phosphorylated to a greater extent than the STCys isoform. Analysis of domain deletion mutants revealed that STTyr is phosphorylated on stem and catalytic domain amino acids, whereas STCys is phosphorylated on catalytic domain amino acids. An endoplasmic reticulum retained/retrieved chimeric Iip33-ST protein demonstrates drastically lower phosphorylation than does the wild type STTyr isoform. This suggests that the bulk of the ST6Gal-I phosphorylation is occurring in the Golgi. Treatment of cells with the ionophore monensin does not significantly block phosphorylation of the STTyr isoform, suggesting that phosphorylation is occurring in the cis-medial Golgi prior to the monensin block. This study demonstrates the presence of kinase activities in the cis-medial Golgi and the substantial phosphorylation of the luminal sequences of a glycosyltransferase.
Dietary consumption of the carotenoid lycopene (mostly from tomato products) has been associated with a lower risk of prostate cancer. Evidence relating other carotenoids, tocopherols, and retinol to prostate cancer risk has been equivocal. This prospective study was designed to examine the relationship between plasma concentrations of several major antioxidants and risk of prostate cancer. We conducted a nested case-control study using plasma samples obtained in 1982 from healthy men enrolled in the Physicians' Health Study, a randomized, placebo-controlled trial of aspirin and beta-carotene. Subjects included 578 men who developed prostate cancer within 13 years of follow-up and 1294 age- and smoking status-matched controls. We quantified the five major plasma carotenoid peaks (alpha- and beta-carotene, beta-cryptoxanthin, lutein, and lycopene) plus alpha- and gamma-tocopherol and retinol using high-performance liquid chromatography. Results for plasma beta-carotene are reported separately. Odds ratios (ORs), 95% confidence intervals (Cls), and Ps for trend were calculated for each quintile of plasma antioxidant using logistic regression models that allowed for adjustment of potential confounders and estimation of effect modification by assignment to either active beta-carotene or placebo in the trial. Lycopene was the only antioxidant found at significantly lower mean levels in cases than in matched controls (P = 0.04 for all cases). The ORs for all prostate cancers declined slightly with increasing quintile of plasma lycopene (5th quintile OR = 0.75, 95% CI = 0.54-1.06; P, trend = 0.12); there was a stronger inverse association for aggressive prostate cancers (5th quintile OR = 0.56, 95% CI = 0.34-0.91; P, trend = 0.05). In the placebo group, plasma lycopene was very strongly related to lower prostate cancer risk (5th quintile OR = 0.40; P, trend = 0.006 for aggressive cancer), whereas there was no evidence for a trend among those assigned to beta-carotene supplements. However, in the beta-carotene group, prostate cancer risk was reduced in each lycopene quintile relative to men with low lycopene and placebo. The only other notable association was a reduced risk of aggressive cancer with higher alpha-tocopherol levels that was not statistically significant. None of the associations for lycopene were confounded by age, smoking, body mass index, exercise, alcohol, multivitamin use, or plasma total cholesterol level. These results concur with a recent prospective dietary analysis, which identified lycopene as the carotenoid with the clearest inverse relation to the development of prostate cancer. The inverse association was particularly apparent for aggressive cancer and for men not consuming beta-carotene supplements. For men with low lycopene, beta-carotene supplements were associated with risk reductions comparable to those observed with high lycopene. These data provide further evidence that increased consumption of tomato products and other lycopene-containing foods might reduce the occurrence or progression of prostate cancer.
Bicoid is a Drosophila morphogenetic protein required for the development of anterior structures in the embryo. To gain a better understanding of how Bicoid works as a transcriptional activator, we systematically analysed various functions of Bicoid required for gene activation. We provide evidence suggesting that Bicoid is an intrinsically weak activator. First, our biochemical experiments demonstrate that the Bicoid-DNA complexes are very unstable, suggesting a weak DNA-binding function of Bicoid. This idea is further supported by our experiments demonstrating that the same number of LexA-Bicoid fusion molecules can activate transcription more effectively from LexA sites than from Bicoid sites. Secondly, we demonstrate that transcriptional activation by the weak activator Bicoid is readily influenced by the local enhancer environment. These influences are decreased when the Bicoid function is enforced by attaching to it either a known dimerization domain or the strong activation domain VP16. VP16 can also compensate for the loss of some Bicoid sites in an enhancer element. Our experiments demonstrate that the outcome of transcriptional activation by Bicoid is determined by multiple weak functions that are interconnected, a finding that can further help us to understand how this morphogenetic protein achieves its molecular functions.