Biomedical subjects

J Chen

Publications and source records attributed to J Chen.

At least 757 records · Page 42Linked to original sources

PubMed2000

Melanoma-targeting properties of (99m)technetium-labeled cyclic alpha-melanocyte-stimulating hormone peptide analogues.

Preliminary reports have demonstrated that (99m)technetium (Tc)-labeled cyclic [Cys(3,4,10), D-Phe7]alpha-MSH(3-13) (CCMSH) exhibits high tumor uptake and retention values in a murine melanoma mouse model. In this report, the tumor targeting mechanism of 99mTc-CCMSH was studied and compared with four other radiolabeled alpha-melanocyte stimulating hormone (alpha-MSH) peptide analogues: 125I-(Tyr2)-[Nle4, D-Phe7]alpha-MSH [125I-(Tyr2)-NDP]; 99mTc-CGCG-NDP; 99mTc-Gly11-CCMSH; and 99mTc-Nle11-CCMSH. In vitro receptor binding, internalization, and cellular retention of radiolabeled alpha-MSH analogues in B16/F1 murine cell line demonstrated that >70% of the receptor-bound radiolabeled analogues were internalized together with the receptor. Ninety % of the internalized 125I-(Tyr2)-NDP, whereas only 36% of internalized 99mTc-CCMSH, was released from the cells into the medium during a 4-h incubation at 37 degrees C. Two mouse models, C57 mice and severe combined immunodeficient (Scid) mice, inoculated s.c. with B16/F1 murine and TXM-13 human melanoma cells were used for the in vivo studies. Tumor uptake values of 11.32 and 2.39 [% injected dose (ID)/g] for 99mTc-CCMSH at 4 h after injection, resulted in an uptake ratio of tumor:blood of 39.0 and 11.5 in murine melanoma-C57 and human melanoma-Scid mouse models, respectively. Two strategies for decreasing the nonspecific kidney uptake of 99mTc-CCMSH, substitution of Lys11 in CCMSH with Gly11 or Nle11, and lysine coinjection, were evaluated. The biodistribution data for the modified peptides showed that Lys11 replacement dramatically decreased the kidney uptake, whereas the tumor uptakes of 99mTc-Nle11- and 99mTc-Gly11-CCMSH were significantly lower than that of 99mTc-CCMSH. Lysine coinjection significantly decreased the kidney uptake (e.g., from 14.6% ID/g to 4.5% ID/g at 4 h after injection in murine melanoma-C57 mice) without significantly changing the value of tumor uptake of 99mTc-CCMSH. In conclusion, the compact cyclic structure of 99mTc-CCMSH, its resistance to degradation, and its enhanced intracellular retention are the major contributing factors to the superior in vivo tumor targeting properties of 99mTc-CCMSH. Lys11 residue in 99mTc-CCMSH is critical to the tumor targeting in vivo, and lysine coinjection rather than lysine replacement can significantly decrease the nonspecific renal radioactivity accumulation without impeding the high melanoma-targeting properties of 99Tc-CCMSH. The metal-cyclized CCMSH molecule displays excellent potential for the development of melanoma-specific diagnostic and therapeutic agents.

Amino Acid Sequence↗

PubMed2000

The fourth transmembrane segment of the dopamine D2 receptor: accessibility in the binding-site crevice and position in the transmembrane bundle.

The binding site of the dopamine D2 receptor, like that of homologous G-protein-coupled receptors (GPCRs), is contained within a water-accessible crevice formed among its seven transmembrane segments (TMSs). Using the substituted-cysteine-accessibility method (SCAM), we are mapping the residues that contribute to the surface of this binding-site crevice. We have mutated to cysteine, one at a time, 21 consecutive residues in the fourth TMS (TM4). Eleven of these mutants reacted with charged sulfhydryl-specific reagents, and bound antagonist protected nine of these from reaction. For the mutants in which cysteine was substituted for residues in the cytoplasmic half of TM4, treatment with the reagents had no effect on binding, consistent with these residues being inaccessible and with the low-resolution structure of the homologous rhodopsin, in which TM3 and TM5 occlude the cytoplasmic half of TM4. Although hydrophobicity analysis positions the C-terminus of TM4 at 4.64, Pro-Pro and Pro-X-Pro motifs, which are known to disrupt alpha-helices, occur at position 4.59 in a number of homologous GPCRs. The SCAM data were consistent with a C-terminus at 4.58, but it is also possible that the alpha-helix extends one additional turn to 4.62 in the D2 receptor, which has a single Pro at 4.59. In homologous GPCRs, the high degree of sequence variation between 4.59 and 4.68 is more characteristic of a loop domain than a helical segment. This region is shown here to be very conserved within functionally related receptors, suggesting an important functional role for this putative nonhelical domain. This inference is supported by observed ligand-specific effects of mutations in this region and by the predicted spatial proximity of this segment to known ligand binding sites in other TMs.

Amino Acid Sequence↗

PubMed2000

Intracellular delivery of phosphoinositides and inositol phosphates using polyamine carriers.

Phosphoinositide signaling regulates events in endocytosis and exocytosis, vesicular trafficking of proteins, transduction of extracellular signals, remodeling of the actin cytoskeleton, regulation of calcium flux, and apoptosis. Obtaining mechanistic insights in living cells is impeded by the membrane impermeability of these anionic lipids. We describe a carrier system for intracellular delivery of phosphoinositide polyphosphates (PIP(n)s) and fluorescently labeled PIP(n)s into living cells, such that intracellular localization can be directly observed. Preincubation of PIP(n)s or inositol phosphates with carrier polyamines produced complexes that entered mammalian, plant, yeast, bacterial, and protozoal cells in seconds to minutes via a nonendocytic mechanism. Time-dependent transit of both PIP(n)s and the carrier to specific cytosolic and nuclear compartments was readily visualized by fluorescence microscopy. Platelet-derived growth factor treatment of NIH 3T3 fibroblasts containing carrier-delivered phosphatidylinositol 4,5-bisphosphate [PtdIns(4, 5)P(2)]-7-nitrobenz-2-oxa-1,3-diazole resulted in the redistribution of the fluorescent signal, suggesting that fluorescent PtdIns(4, 5)P(2) was a substrate for phospholipase C. We also observed a calcium flux in NIH 3T3 cells when complexes of carrier and PtdIns(4, 5)P(2) or inositol 1,4,5-trisphosphate were added extracellularly. This simple intracellular delivery system allows for the efficient translocation of biologically active PIP(n)s, inositol phosphates, and their fluorescent derivatives into living cells in a physiologically relevant context.

3T3 Cells↗

PubMed2000

Comparison of bisoprolol and carvedilol cardioprotection in a rabbit ischemia and reperfusion model.

Carvedilol, a selective alpha(1) and non-selective beta-adrenoceptor antagonist and antioxidant, has been shown to provide significant cardiac protection in animal models of myocardial ischemia. To further explore the mechanisms contributing to the efficacy of carvedilol cardioprotection, the effects of carvedilol on hemodynamic variables, infarct size and myeloperoxidase activity (an index of neutrophil accumulation) were compared with a beta(1) selective adrenoceptor antagonist, bisoprolol. Carvedilol (1 mg/kg) or bisoprolol (1 mg/kg) was given intravenously 5 min before reperfusion. In vehicle-treated rabbits, ischemia (45 min) and reperfusion (240 min) resulted in significant increases in left ventricular end diastolic pressure, large myocardial infarction (64.7+/-2.6% of area-at-risk) and a marked increase in myeloperoxidase activity (64+/-14 U/g protein in area-at-risk). Carvedilol treatment resulted in sustained reduction of the pressure-rate-index and significantly smaller infarcts (30+/-2.9, P<0.01 vs. vehicle) as well as decreased myeloperoxidase activity (26+/-11 U/g protein in area-at-risk, P<0.01 vs. vehicle). Administration of bisoprolol at 1 mg/kg resulted in a pressure-rate-index comparable to that of carvedilol and also decreased infarct size (48.4+/-2.5%, P<0.001 vs. vehicle, P<0.05 vs. carvedilol), although to a significantly lesser extent than that observed with carvedilol. Treatment with bisoprolol failed to reduce myeloperoxidase activity in the ischemic myocardial tissue. In addition, carvedilol, but not bisoprolol, markedly decreased cardiac membrane lipid peroxidation measured by thiobarbituric acid formation. Taken together, this study suggests that the superior cardioprotection of carvedilol over bisoprolol is possibly the result of carvedilol's antioxidant and anti-neutrophil effects, not its hemodynamic properties.

Adrenergic beta-Agonists↗

PubMed2000

A [4 + 4] 2-pyridone approach to taxol. 3. Stereocontrol during elaboration of the cyclooctane.

Intramolecular photocycloaddition of 2-pyridones connected through a four-carbon tether (6-[4-(1,2-dihydro-1-methyl-2-oxo-3-pyridinyl)-4-[[(1,1-dimethylethyl)++ +dimethylsilyl]oxy]butyl]-4-methoxy-1,3-dimethyl-2(1H)-pyridinone) yields a single tetracyclic product with four new stereogenic centers. The diastereoselectivity of this [4 + 4] reaction is fully controlled by a stereogenic carbon of the tether. Treatment of the photoproduct with osmium tetraoxide transforms the alkene to a diol and the enol ether to an alpha-hydroxy ketone, with stereocontrol dictated by nearby lactams that block one face of each alkene. Allylmagnesium bromide addition to the ketone also yields a single diastereomer, but unexpectedly this product results from approach of the nucleophile to the most-hindered face of the ketone. Study of this reaction in a model system has found the allylic nucleophile to be unique, with nonallylic reagents approaching along the expected, least-hindered path. This contrasteric addition likely results from coordination of the allylic nucleophile to the nearby amide. The amide can therefore act either as a steric shield or as a directing group. The three steps of photocycloaddition, cis-hydroxylation, and nucleophilic addition constructs both quaternary carbons of the cyclooctane and four of the five stereogenic centers found in the eight-membered ring of Taxol.

Antineoplastic Agents, Phytogenic↗

PubMed2000

Feasibility of electrokinetic soil remediation in horizontal Lasagna cells.

An integrated soil remediation technology called Lasagna has been developed that combines electrokinetics with treatment zones for use in low permeability soils where the rates of hydraulic and electrokinetic transport are too low to be useful for remediation of contaminants. The technology was developed by two groups, one involving industrial partners and the DOE and another involving US EPA and the University of Cincinnati, who pursued different electrode geometries. The Industry/DOE group has demonstrated the technology using electrodes and treatment zones installed vertically from the soil surface. We have demonstrated the feasibility of installing horizontal electrodes and treatment zones in subsurface soils by hydraulic fracturing, a process that we adapted from petroleum industry practices. When horizontal electrodes were connected to a dc power supply, uniform electrical potential gradients of 10-40 V/m were created in soil between the electrodes, inducing electroosmotic flow that facilitated movement of water and contaminants into treatment zones between the electrodes.

Electrochemistry↗

PubMed2000

A quantitative model for the dependence of solute permeability on peptide and cholesterol content in biomembranes.

The influence of varying concentrations of a transmembrane peptide, gramicidin A (gA), and cholesterol (Chol) on the passive permeation of p-methylhippuric acid (MHA) and alpha-carbamoyl-p-methylhippuric acid (CMHA) across egg-lecithin membranes (EPC) has been investigated in vesicle efflux experiments. Incorporation of 0.25 volume fraction of gA in its nonchannel conformation increased the permeability coefficient (Pm) for CMHA by a factor of 6.0 +/- 1.8 but did not alter Pm for MHA, a more lipophilic permeant. In contrast, incorporation of 0.26 volume fraction Chol with no added protein decreased the Pm values for both CMHA and MHA by similar factors of 4.2 +/- 1.1 and 3.5 +/- 1.2, respectively. A quantitative structure-transport model has been developed to account for the dependence of Pm on the membrane concentrations of gA and Chol in terms of induced changes in both membrane chain ordering and hydrophobicity. Chain ordering is assumed to affect Pm for both permeants similarly since they are comparable in molecular size, while changes in Pm ratios in the presence of gA or Chol are attributed to alterations in membrane hydrophobicity. Changes in lipid chain ordering were detected by monitoring membrane fluidity using fluorescence anisotropy of 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene incorporated into the membranes. The influence of additives on membrane hydrophobicity, which governs Pm ratios through effects on solute partitioning into the barrier domain, were rationalized within the framework of regular solution theory using solubility parameters as a measure of membrane hydrophobicity. Fits of the Pm ratios using the theoretical model yielded solubility parameters for gA and Chol in EPC membranes of 13.2 and 7.7 (cal/ml)(1/2), respectively, suggesting that gA decreases the barrier domain hydrophobicity while Chol has a minimal effect on barrier hydrophobicity. After correcting for barrier domain hydrophobicity, permeability decrements due to membrane ordering induced by gA or Chol were found to exhibit a strong correlation with membrane order as predicted by free-surface-area theory, regardless of whether gA or Chol is used as the ordering agent.

Cholesterol↗

PubMed2000

Autocrine action and its underlying mechanism of nitric oxide on intracellular Ca2+ homeostasis in vascular endothelial cells.

The rise in cytosolic Ca(2+) concentration (Ca(2+)(i)) in vascular endothelial cells (ECs) activates the production and release of nitric oxide (NO). NO modifies Ca(2+)(i) homeostasis in many types of nonendothelial cells. However, its effect on endothelial Ca(2+)(i) homeostasis at basal and excited states remains unclear. In the present study, to elucidate the effect of NO on basal Ca(2+)(i), inositol 1,4,5-trisphosphate-induced Ca(2+)(i) release (IICR) was blocked by expressing an antisense against type-1 inositol 1,4,5-trisphosphate receptors or by microinjecting heparin to individual ECs, and the effects of NO that was released by and diffused from adjacent IICR-intact ECs were recorded. After ATP or bradykinin stimulation, IICR-inhibited ECs showed a marked reduction of basal Ca(2+)(i), which was abolished by N(G)-monomethyl-l-arginine monoacetate pretreatment. The reduction disappeared in sparsely seeded ECs. Exogenous NO gas mimicked the effect of ATP or bradykinin to reduce basal Ca(2+)(i). Blocking plasma membrane Ca(2+)-ATPase (PMCA), but not Na(+)-Ca(2+) exchange or sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase, suppressed the reduction, indicating that the reduction resulted from a NO-dependent potentiation of PMCA. To elucidate the effect of NO on elevated Ca(2+)(i), ATP-, bradykinin-, or thapsigargin-evoked Ca(2+)(i) response in the presence and absence of NO production was compared in adjacent IICR-intact ECs. NO was found to potentiate PMCA, which, in turn, greatly attenuated agonist-evoked Ca(2+)(i) elevation. NO also potentiated Ca(2+) influx, which markedly increased the sustained phase of Ca(2+)(i) elevation and possibly NO production. NO did not affect other Ca(2+)(i)-elevating and Ca(2+)(i)-sequestrating components. Thus, NO-dependent potentiation of PMCA is crucial for Ca(2+)(i) homeostasis over a wide Ca(2+)(i) range.

Adenosine Triphosphate↗

PubMed2000

Ataxia telangiectasia-related protein is involved in the phosphorylation of BRCA1 following deoxyribonucleic acid damage.

The breast/ovarian cancer susceptibility gene BRCA1 exerts its tumor suppressor function, at least in part, by participating in DNA repair and/or DNA damage-responsive pathways. BRCA1 protein is hyperphosphorylated following various DNA-damaging events. Here, we report that the ataxia telangiectasia mutated protein-related protein kinase (ATR) is involved in the phosphorylation of BRCA1 following gamma radiation and hydroxyurea treatment. We have shown that ATR can phosphorylate several BRCA1 fragments in vitro and that a kinase-inactive mutant of ATR interacts with BRCA1 in vivo. Taken together, these results suggest that ATR directly phosphorylates BRCA1 following DNA damage.

Androstadienes↗

PubMed2000

Spinal cord injury in rat: treatment with bone marrow stromal cell transplantation.

We tested the hypothesis that transplantation of bone marrow stromal cells (MSCs) into the spinal cord after a contusion injury promotes functional outcome. Rats (n = 31) were subjected to a weight driven implant injury. MSCs or phosphate buffered saline was injected into the spinal cord 1 week after injury. Sections of tissue were analyzed by double-labeled immunohistochemistry for MSC identification. Functional outcome measurements using the Basso-Beattie-Bresnehan score were performed weekly to 5 weeks post-injury. The data indicate significant improvement in functional outcome in animals treated with MSC transplantation compared to control animals. Scattered cells derived from MSCs expressed neural protein markers. These data suggest that transplantation of MSCs may have a therapeutic role after spinal cord injury.

Animals↗

PubMed2000

The nuclear matrix protein CDP represses hepatic transcription of the human cholesterol-7alpha hydroxylase gene.

To date, the molecular mechanisms that govern hepatic-specific transcription of the human cholesterol 7alpha-hydroxylase (CYP7A1) gene are poorly understood. We recently reported that the region extending from -1888 to +46, which includes the promoter, is not capable of conferring expression to human CYP7A1 promoter lacZ transgenes in the livers of mice, but that expression is observed with transgenes containing the entire structural gene. To locate liver-specific elements in other segments of the human gene, DNase I hypersensitivity studies were performed with transcriptionally active, liver-derived HepG2 cells and with transcriptionally inactive HeLa cells. Three DNase I hypersensitivity sites were detected within the first intron of the human CYP7A1 gene, but only in HepG2 cells. Transient transfection experiments with HepG2 cells revealed a transcriptional repressor within intron 1. Five binding sites for the CAAT displacement protein (CDP) were detected within intron 1. Since CDP is a nuclear matrix protein, two methods were employed to localize nuclear matrix attachment sites within intron 1 of the human CYP7A1 gene. A matrix attachment site was found throughout the entirety of intron 1. Gel retardation experiments and cell transfection studies provided evidence for the repression mechanism. Repression is achieved by displacement by CDP of two hepatic activators, namely HNF-1alpha and C/EBPalpha, that bind to three different sites within intron 1. Additionally, CDP represses transactivation mediated by these two activators.

Animals↗

PubMed2000

Homeostasis-stimulated proliferation drives naive T cells to differentiate directly into memory T cells.

The developmental requirements for immunological memory, a central feature of adaptive immune responses, is largely obscure. We show that as naive CD8 T cells undergo homeostasis-driven proliferation in lymphopenic mice in the absence of overt antigenic stimulation, they progressively acquire phenotypic and functional characteristics of antigen-induced memory CD8 T cells. Thus, the homeostasis-induced memory CD8 T cells express typical memory cell markers, lyse target cells directly in vitro and in vivo, respond to lower doses of antigen than naive cells, and secrete interferon gamma faster upon restimulation. Like antigen-induced memory T cell differentiation, the homeostasis-driven process requires T cell proliferation and, initially, the presence of appropriate restricting major histocompatibility complexes, but it differs by occurring without effector cell formation and without requiring interleukin 2 or costimulation via CD28. These findings define repetitive cell division plus T cell receptor ligation as the basic requirements for naive to memory T cell differentiation.

Adoptive Transfer↗

PubMed2000

Purification, cDNA cloning and function assessment of BmK abT, a unique component from the Old World scorpion species.

A new neurotoxic component named BmK abT was purified from the venom of Chinese scorpion Buthus martensi Karsch. The molecular weight of BmK abT was determined to be 7212 Da on a mass spectrum. The minimum lethal dose of BmK abT was tested to be about 1.5 microg per mouse by intracerebroventricular injection, and the dose induced significant paralysis effect on cockroach was about 5 microg by i.p. injection. The partial amino acid sequence indicated that it was a distinctive polypeptide in the scorpion neurotoxin family. Thereafter, the whole amino acid sequence of mature BmK abT was deduced from cDNA sequence by 5'- and 3'-rapid amplification of cDNA ends. Finally, it was defined to be composed of 63 residues with amidation at the C-terminal residue. By sequence comparison, BmK abT was found to be most similar to Ts VII, a beta-toxin from the New World scorpion. The patch-clamp recording on DRG neurons, unexpectedly, showed this toxin could prolong the action potential and increase the amplitude of the peak Na+ currents, which are the typical characters of alpha-toxin. These results suggested that BmK abT was a new toxic component found in the Old World scorpion species structurally similar to beta-toxins, but functionally similar to alpha-toxins.

Action Potentials↗

PubMed2000

Genetic diversity and disease control in rice.

Crop heterogeneity is a possible solution to the vulnerability of monocultured crops to disease. Both theory and observation indicate that genetic heterogeneity provides greater disease suppression when used over large areas, though experimental data are lacking. Here we report a unique cooperation among farmers, researchers and extension personnel in Yunnan Province, China--genetically diversified rice crops were planted in all the rice fields in five townships in 1998 and ten townships in 1999. Control plots of monocultured crops allowed us to calculate the effect of diversity on the severity of rice blast, the major disease of rice. Disease-susceptible rice varieties planted in mixtures with resistant varieties had 89% greater yield and blast was 94% less severe than when they were grown in monoculture. The experiment was so successful that fungicidal sprays were no longer applied by the end of the two-year programme. Our results support the view that intraspecific crop diversification provides an ecological approach to disease control that can be highly effective over a large area and contribute to the sustainability of crop production.

Agriculture↗

PubMed2000

SMAR1, a novel, alternatively spliced gene product, binds the Scaffold/Matrix-associated region at the T cell receptor beta locus.

Rearrangement and expression of the T cell receptor beta gene are critical events for early T lymphocyte development. To characterize cis-regulatory elements and their associated trans-factors that mediate these events, we have previously identified a nuclear matrix/scaffold-associated region, referred to as MARbeta, 400 bp upstream of the Ebeta enhancer. Electrophoretic mobility shift assay showed that two known MAR-binding proteins, SATB1 and Cux, bind MARbeta. In this article, we report the identification of a novel MAR-binding protein, named SMAR1, that also binds MARbeta. SMAR1 shares homology with SATB1 and Cux in the MAR-binding domain/Cut repeat and also with the tetramerization domain of a B cell-specific MAR-binding protein, Bright. The binding of GST-SMAR1 fusion protein to MARbeta is inhibited by the presence of an excess amount of MAR-containing DNA from the immunoglobulin kappa locus. Smar1 transcripts are most abundant in the thymus and are alternatively spliced. The smar1 gene maps to the distal portion of mouse chromosome 8 at a distance of 111.8 cM.

Alternative Splicing↗

PubMed2000

Complexes of the G protein subunit gbeta 5 with the regulators of G protein signaling RGS7 and RGS9. Characterization in native tissues and in transfected cells.

A novel protein class, termed regulators of G protein signaling (RGS), negatively regulates G protein pathways through a direct interaction with Galpha subunits and stimulation of GTP hydrolysis. An RGS subfamily including RGS6, -7, -9, and -11, which contain a characteristic Ggamma -like domain, also has the unique ability to interact with the G protein beta subunit Gbeta(5). Here, we examined the behavior of Gbeta(5), RGS7, RGS9, and Galpha in tissue extracts using immunoprecipitation and conventional chromatography. Native Gbeta(5) and RGS7 from brain, as well as photoreceptor-specific Gbeta(5)L and RGS9, always co-purified as tightly associated dimers, and neither RGS-free Gbeta(5) nor Gbeta(5)-free RGS could be detected. Co-expression in COS-7 cells of Gbeta(5) dramatically increased the protein level of RGS7 and vice versa, indicating that cells maintain Gbeta(5):RGS stoichiometry in a manner similar to Gbetagamma complexes. This mechanism is non-transcriptional and is based on increased protein stability upon dimerization. Thus, analysis of native Gbeta(5)-RGS and their coupled expression argue that in vivo, Gbeta(5) and Ggamma-like domain-containing RGSs only exist as heterodimers. Native Gbeta(5)-RGS7 did not co-precipitate or co-purify with Galpha(o) or Galpha(q); nor did Gbeta(5)L-RGS9 with Galpha(t). However, in transfected cells, RGS7 and Gbeta(5)-RGS7 inhibited Galpha(q)-mediated Ca(2+) response to muscarinic M3 receptor activation. Thus, Gbeta(5)-RGS dimers differ from other RGS proteins in that they do not bind to Galpha with high affinity, but they can still inhibit G protein signaling.

Animals↗

PubMed2000

Phenotypic screening of mutations in Pmr1, the yeast secretory pathway Ca2+/Mn2+-ATPase, reveals residues critical for ion selectivity and transport.

Thirty-five mutations were generated in the yeast secretory pathway/Golgi ion pump, Pmr1, targeting oxygen-containing side chains within the predicted transmembrane segments M4, M5, M6, M7, and M8, likely to be involved in coordination of Ca(2+) and Mn(2+) ions. Mutants were expressed in low copy number in a yeast strain devoid of endogenous Ca(2+) pumps and screened for loss of Ca(2+) and Mn(2+) transport on the basis of hypersensitivity to 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and Mn(2+) toxicity, respectively. Three classes of mutants were found: mutants indistinguishable from wild type (Class 1), mutants indistinguishable from the pmr1 null strain (Class 2), and mutants with differential sensitivity to BAPTA and Mn(2+) toxicity (Class 3). We show that Class 1 mutants retain normal/near normal properties, including (45)Ca transport, Golgi localization, and polypeptide conformation. In contrast, Class 2 mutants lacked any detectable (45)Ca transport; of these, a subset also showed defects in trafficking and protein folding, indicative of structural problems. Two residues identified as Class 2 mutants in this screen, Asn(774) and Asp(778) in M6, also play critical roles in related ion pumps and are therefore likely to be common architectural components of the cation-binding site. Class 3 mutants appear to have altered selectivity for Ca(2+) and Mn(2+) ions, as exemplified by mutant Q783A in M6. These results demonstrate the utility of phenotypic screening in the identification of residues critical for ion transport and selectivity in cation pumps.

ATP-Binding Cassette Transporters↗

PubMed2000

Recovery of Penaeus monodon from functional anaemia after exposure to sublethal concentration of nitrite at different pH levels.

Tiger shrimp Penaeus monodon (14.79+/-0.19 g) which had been exposed individually to 0.72 mM nitrite at pH 6.8, 8.2 and 9.8 after 3, 6, 12, 24 and 48 h were examined for the water nitrite concentration, hemolymph nitrite, oxyhemocyanin, protein and osmolality levels. Same parameters were examined for shrimp depurated in nitrite-free water after 3, 6, 12 and 24 h following 48 h exposure to nitrite. In the nitrite-exposed test, nitrite influx, hemolymph nitrite and osmotic differential (medium osmolality - hemolymph osmolality) increased with exposure time, and were higher at pH 6.8, whereas water nitrite concentration, oxyhemocyanin, protein, ratio of oxyhemocyanin to protein and hemolymph osmolality decreased with exposure time and were lower at pH 6.8. In the depuration test, water nitrite concentration increased with depuration time, and was higher at pH 6.8, whereas, hemolymph oxyhemocyanin, ratio of oxyhemocyanin to protein and hemolymph osmolality increased with depuration time and were higher at pH 9.8. Hemolymph nitrite decreased with depuration time, and was 2.52, 2.19 and 0.24 µmol ml(-1) after 6 h at pH 6.8, 8.2 and 9.8, respectively. Nitrite entry is considered to occur mainly via the form of nitrous acid (HNO(2)) in water uptake. The fact that the hemolymph nitrite decreased to non-detectable level with 72-88% recovery of oxyhemocyanin after 24 h depuration suggests an occurrence of reductase for nitrite-exposed shrimps.

Journal Article↗