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J Sun

Publications and source records attributed to J Sun.

At least 505 records · Page 28Linked to original sources
PubMed1996

Tolerance to rat liver allografts: IV. Acceptance depends on the quantity of donor tissue and on donor leukocytes.

Liver allografts in some rat strains are often spontaneously accepted across a complete major histocompatibility barrier without the requirement for immunosuppression while other nonliver allografts are rejected. In previous studies, we have shown that spontaneous acceptance is dependent on liver passenger leukocytes. Depletion of passenger leukocytes by donor irradiation allows rejection, with DA recipients of irradiated PVG livers having a median survival time (MST) of 16 days. Here we show that, in this model, spontaneous acceptance is reconstituted by intravenous injection of donor leukocytes. Intravenous injection of 3-5x10(7) PVG liver leukocytes significantly prolonged DA survival time (MST=96 days, P=0.026), as did 5x10(7) spleen leukocytes (MST>100 days, P=0.002). Deletion of T cells from the reconstituting inoculum reduced survival time (MST=78 days, P=0.039), whereas deletion of B cells or monocytes/macrophages had no effect on survival time. In contrast, PVG hearts are regularly rejected by DA recipients, and PVG liver or spleen leukocytes, even at doses of greater than 3x10(8) cells/recipient, were unable to induce heart acceptance. To investigate the possibility that acceptance of the irradiated liver but not the heart might be due to the large mass of the liver, two kidneys and two hearts of PVG origin were transplanted to each DA recipient together with 1.5x10(8) PVG leukocytes. These organs survived for greater than 200 days, thereby showing that a large mass of donor tissue, in association with donor leukocytes, leads to acceptance of organs that are rejected if transplanted singly. It appears likely that spontaneous liver transplant tolerance is a high-dose or activation-associated immune phenomenon.

Animals↗
PubMed1996

A cytosolic granzyme B inhibitor related to the viral apoptotic regulator cytokine response modifier A is present in cytotoxic lymphocytes.

Using a polymerase chain reaction strategy we identified a serine proteinase inhibitor (serpin) in human bone marrow that is related to the cellular serpin proteinase inhibitor 6 (PI-6) and the viral serpin cytokine response modifier A (CrmA). This serpin, proteinase inhibitor 9 (PI-9), has an unusual reactive center P1(Glu)-P1'(Cys), which suggests that it inhibits serine proteinases that cleave after acidic residues. The only known serine proteinase with this specificity is granzyme B, a granule cytotoxin produced by cytotoxic lymphocytes. To test the interaction of PI-9 with granzyme B we prepared recombinant hexa-histidine tagged PI-9 in a yeast expression system. Addition of the recombinant protein to native granzyme B resulted in an SDS-resistant complex typical of serpin-serine proteinase interactions. Further analysis showed that complex formation followed bimolecular kinetics with a second order rate constant of 1.7 +/- 0.3 x 10(6) M-1 s-1, which is in the range for a physiologically significant serpin-proteinase interaction. Recombinant PI-9 also completely abrogated granzyme B and perforin-mediated cytotoxicity in vitro. Examination of PI-9 mRNA distribution demonstrated that it is expressed in immune tissue, primarily in lymphocytes. The highest levels of PI-9 mRNA and protein were observed in natural killer cell leukemia cell lines and in interleukin-2 stimulated peripheral blood mononuclear cells, which also produce granzyme B. Like PI-6, PI-9 was shown to be a cytosolic protein that is not secreted. Fractionation of natural killer cells and stimulated peripheral blood mononuclear cells demonstrated that PI-9 is in a separate subcellular compartment to granzyme B. These results suggest that PI-9 serves to inactivate misdirected granzyme B following cytotoxic cell degranulation. This may explain why cytotoxic cells are not damaged by their own granzyme B during destruction of abnormal cells.

Amino Acid Sequence↗
PubMed1996

Cardiovascular effects of an ultra-short-acting nitric oxide-releasing compound, zwitterionic diamine/NO adduct, in dogs.

BACKGROUND: This study was conducted to clarify the cardiovascular effects of a new NO-releasing compound, NOC-7, and to compare it with other nitrovasodilators, sodium nitroprusside (SNP) and nitroglycerin, in dogs anesthetized with pentobarbital. METHODS AND RESULTS: A bolus injection of NOC-7 decreased mean aortic blood pressure in a dose-dependent manner. The onset was rapid and the recovery quick. Continuous infusion of NOC-7 decreased mean aortic pressure from 115 +/- 3.9 to 84 +/- 2.9 mm Hg and infusion of SNP, from 118 +/- 3.8 to 87 +/- 3.1 mm Hg. The optimum doses of NOC-7 and SNP were determined to be 2.73 +/- 0.77 and 11.5 +/- 6.1 micrograms.kg-1.min-1, respectively. During infusion of NOC-7, heart rate and cardiac output were increased (P < .05), pulmonary artery pressure was not changed, and systemic and pulmonary vascular resistances were decreased (P < .05). Electromagnetic flowmetry showed that portal venous and internal carotid arterial blood flow were increased (P < .05) and that hepatic and renal arterial blood flows were not changed. These hemodynamic changes during NOC-7 infusion were similar to those with SNP. The plasma level of NO2-/NO3 did not change, but methemoglobin increased slightly (P < .05). Comparison between hypotensive responses before and after a 3.5-hour infusion of NOC-7 or nitroglycerin showed that acute tolerance developed to nitroglycerin but not to NOC-7. CONCLUSIONS: The results indicate that NOC-7 may be useful as an ultra-short-acting nitrovasodilator that has no major adverse effect or tolerance.

Animals↗
PubMed1996

Cysteine-scanning mutagenesis of transmembrane domain XII and the flanking periplasmic loop in the lactose permease of EScherichia coli.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain XII and the periplasmic loop between putative helices XI and XII (loop XI/XII) was replaced individually with Cys. Out of 34 mutants, 31 exhibit 60-100% or more of C-less activity, mutants Gly377-->Cys and Leu385-->Cys exhibit lower rates of transport but accumulate lactose about 60-70% as well as C-less, and mutant Leu400-->Cys exhibits < 20% of C-less activity. Immunoblots reveal that all of the mutant proteins are present in the membrane in amounts comparable to that of C-less with the exception of mutants Gly377-->Cys and Leu385-->Cys which are expressed about 40% as well as C-less and mutant Leu400-->Cys which is hardly detectable. When transferred to the wild-type background, however, mutant Leu400-->Cys is expressed normally and exhibits highly significant transport activity. Finally, each active Cys-replacement mutant was assayed for sensitivity to N-ethylmaleimide, and with three exceptions, the mutants are essentially unaffected by the alkylating agent. Mutants Val367-->Cys, Gly370-->Cys, and Tyr373-->Cys which are predicted to be immediately distal to helix XI in loop XI/XII are significantly inactivated. The periodicity observed suggests that the periplasmic end of transmembrane domain XI may extend to position 373. In the following paper [Voss, J., He, M. M., Hubbell, W. L., & Kaback, H. R. (1996) Biochemistry 35, 12915-12918], site-directed spin labeling of single-Cys mutants at positions 387-402 is used to demonstrate that transmembrane domain XII is in an alpha-helical conformation.

Amino Acid Sequence↗
PubMed1996

Rescue of the horseradish peroxidase His-170-->Ala mutant activity by imidazole: importance of proximal ligand tethering.

The proximal iron ligand in horseradish peroxidase (HRP) is His-170. The H170A mutant of polyhistidine-tagged HRP (hHRP) has been expressed in a baculovirus system and has been purified and characterized. At pH 7, the Soret maximum of the mutant is at 414 nm rather than 403 nm. Resonance Raman spectra indicate that the protein is primarily 6-coordinate low-spin in the ferric state with a band in the ferrous state at 212 cm-1 indicative of distal histidine coordination to the iron. Exogenous imidazole (Im) binds to the enzyme with Kd = 22 +/- 4 mM. Reaction of H170A hHRP with H2O2 does not give spectroscopically detectable compound I or compound II intermediates but results in gradual degradation of the heme group. Nevertheless, H170A hHRP is catalytically active, and its guaiacol and ABTS peroxidase activities are improved 260- and 125-fold, respectively, in the presence of saturating concentrations of Im. The Km for the stimulatory effect of Im is 24 mM for both guaiacol and ABTS. The pH profile of H170A hHRP differs from that of wild-type hHRP, but the differences are essentially eliminated by Im. The rate of formation of "compound I" for H170A hHRP, determined by steady state kinetic methods, is k1 = 16 M-1 s-1 without Im and k1 = 2.4 x 10(4) M-1 s-1 with Im. The corresponding rate for wild-type hHRP is k1 = 4.4 x 10(6) M-1 s-1. The results indicate that Im binds in the cavity created by the H170A mutation, coordinates to the heme iron atom, and restores a large part of the catalytic activity by rescuing the rate of compound I formation. However, this rescue of the catalytic activity by Im is possibly limited by coordination of the heme to the distal histidine (His-42) in the H170A mutant. Thus, a primary function of the proximal histidine is to tether the iron atom to disfavor sixth ligand binding, particularly coordination of the iron to the distal histidine. In addition, strong hydrogen bonding of the proximal ligand may be critical for facilitating O-O bond cleavage in the formation of compound I.

Baculoviridae↗
PubMed1996

Defining a novel cis-element in the 3'-untranslated region of mammalian ribonucleotide reductase component R2 mRNA. cis-trans-interactions and message stability.

Mammalian ribonucleotide reductase is a highly regulated activity essential for DNA synthesis and repair. The 3'-untranslated region (3'-UTR) of mammalian ribonucleotide reductase R2 mRNA has been implicated in the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate-mediated stabilization of mouse BALB/c 3T3 R2 message. We investigated the possibility that the 3'-UTR contains regulatory information for R2 mRNA turnover. Using 3'-end-labeled RNA in gel shift and UV cross-linking analyses, we detected in the 3'-UTR a novel 9-nucleotide cis-element, 5'-UCGUGUGCU-3', which interacted with a widely distributed cellular cytosolic protease-sensitive factor(s) in a sequence-specific manner to form a 45-kDa R2 binding protein complex. The binding activity was redox-sensitive and down-regulated by 12-O-tetradecanoylphorbol-13-acetate and okadaic acid in a dose-dependent manner. Insertion of a 154-base pair fragment containing the cis-element led to markedly reduced accumulation of chloramphenicol acetyltransferase hybrid mRNA relative to the same insert carrying a series of G --> A mutations within this element that eliminated binding. We suggest that the 9-nucleotide region functions as a destabilizing element. These results provide a model for ribonucleotide reductase gene expression through a novel and specific mRNA cis-trans-interaction involving a phosphorylation signal pathway that leads to changes in the stability of R2 message.

3T3 Cells↗
PubMed1996

Platelet endothelial cell adhesion molecule-1 (PECAM-1) homophilic adhesion is mediated by immunoglobulin-like domains 1 and 2 and depends on the cytoplasmic domain and the level of surface expression.

PECAM-1/CD31 is vascular cell adhesion and signaling molecule of the Ig superfamily that plays a role in neutrophil recruitment at inflammatory sites and may be involved the release of leukocytes from the bone marrow and in cardiovascular development. The interactions of PECAM-1 with its ligands are complex in that it is able to bind both with itself (homophilic adhesion) or with non-PECAM-1 ligands (heterophilic adhesion). Although the factors that regulate ligand binding are not fully understood, these interactions are regulated in part by its large cytoplasmic domain, a region of 118 amino acids encoded by 8 exons of its gene (exons 9-16). The purpose of this work was to better define the mechanisms of PECAM-1-dependent homophilic adhesion by analyzing the binding interactions of L-cells expressing full-length and selectively mutated forms of human, murine, and human/murine chimeric PECAM-1 molecules in an established aggregation assay. These studies demonstrate that 1) the minimal length of the cytoplasmic domain required for cellular aggregation is represented within the sequences encoded by exons 9 and 10, 2) removal or addition of the sequences encoded by exon 14 from the cytoplasmic domain can determine whether the mechanism of aggregation is a heterophilic calcium-dependent process or a homophilic calcium-independent process, 3) high levels of surface expression of PECAM-1 on the cell surface change the mechanism of aggregation from heterophilic to homophilic, and 4) PECAM-1-dependent homophilic binding appears to involve the direct interaction of only the first two extracellular Ig-like domains. These data suggest that PECAM-1-ligand interactions can be regulated through multiple pathways including alterations of the cytoplasmic domain and the level of surface expression.

Animals↗
PubMed1996

A non-parametric test for interval-censored failure time data with application to AIDS studies.

Interest often centres on the comparison of failure time distributions based on interval-censored failure time data such as in the work by Finkelstein, in which she proposed a score test under continuous proportional hazards model. In this article, we consider a different situation in which the underlying failure time is a discrete variable or the observed times correspond with a discrete scale. To compare failure time distributions in these situations, we propose a non-parametric test, a generalization of the usual logrank test for right-censored failure time data. Simulation results indicate that the test performs satisfactorily.

Cohort Studies↗
PubMed1996

Characterization of manganese(II) binding site mutants of manganese peroxidase.

A series of site-directed mutants, E35Q, E39Q, and E35Q-D179N, in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium, was created by overlap extension, using the polymerase chain reaction. The mutant genes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The mutant manganese peroxidases (MnPs) were purified and characterized. The molecular masses of the mutant proteins, as well as UV-vis spectral features of their oxidized states, were very similar to those of the wild-type enzyme. Resonance Raman spectral results indicated that the heme environment of the mutant MnP proteins also was similar to that of the wild-type protein. Steady-state kinetic analyses of the E35Q and E39Q mutant MnPs yielded K(m) values for the substrate MnII that were approximately 50-fold greater than the corresponding K(m) value for the wild-type enzyme. Likewise, the kcat values for MnII oxidation were approximately 300-fold lower than that for wild-type MnP. With the E35Q-D179N double mutant, the K(m) value for MnII was approximately 120-fold greater, and the kcat value was approximately 1000-fold less than that for the wild-type MnP1. Transient-state kinetic analysis of the reduction of MnP compound II by MnII allowed the determination of the equilibrium dissociation constants (KD) and first- order rate constants for the mutant proteins. The KD values were approximately 100-fold higher for the single mutants and approximately 200-fold higher for the double mutant, as compared with the wild-type enzyme. The first-order rate constants for the single and double mutants were approximately 200-fold and approximately 4000-fold less, respectively, than that of the wild-type enzyme. In contrast, the K(m) values for H2O2 and the rates of compound I formation were similar for the mutant and wild-type MnPs. The second-order rate constants for p-cresol and ferrocyanide reduction of the mutant compounds II also were similar to those of the wild-type enzyme.

Aspartic Acid↗
PubMed1996

Tolerance to rat liver allografts. III. Donor cell migration and tolerance-associated cytokine production in peripheral lymphoid tissues.

The aim of this work was to investigate the mechanism of spontaneous rat liver allograft tolerance. Liver allografts from a LEW donor into DA recipient (LEW-->DA) or of PVG-->DA were spontaneously tolerated (TOL) across a complete MHC mismatch. In contrast, DA-->LEW or PVG-->LEW liver allografts were rejected in 10 to 15 days (REJ). We examined whether donor cell migration to recipient lymphoid tissues might be associated with TOL. Many donor cells were observed in draining (celiac) lymph nodes (LN) and spleen, reaching a peak on day 1 and then decreasing rapidly thereafter. Irradiation of liver donors, which we have previously shown to delete tolerance, significantly reduced the number of donor leukocytes in recipient lymphoid tissues. While this suggested an association between donor cell migration and tolerance, the number, distribution, and type of donor cells in recipient lymphoid tissues of REJ was similar to those of TOL. Expression of cytokine mRNA in LN and spleen showed an early increase in the expression of IL-2 and IFN-gamma mRNA on day 1 and then a rapid decrease to constitutive levels. Spleen and LN levels of IL-6, IL-10, TNF-alpha, or TGF-beta mRNA showed much less up-regulation than IL-2 or IFN-gamma. Paradoxically, there was greater expression of IL-2 and IFN-gamma mRNA in TOL lymphoid tissues than in REJ, and this superinduction was partially prevented by donor irradiation. Superinduction of IL-2 and IFN-gamma was, therefore, more closely associated with TOL than was donor cell migration. This was confirmed by treatment of TOL recipients with a short course of methylprednisolone, which reduced survival of subsequent donor strain skin grafts. This finding has implications for treatment of human liver transplants and is evidence for a novel pathway of transplant tolerance.

Animals↗
PubMed1996

Magnesium activated adenosine formation in intact perfused heart: predominance of ecto 5'-nucleotidase during hypermagnesemia.

Magnesium ion is an allosteric effector of 5'-nucleotidase and thus activates adenosine production from AMP. Two distinct 5'-nucleotidase systems, the membrane-bound ecto and the soluble cytosolic isoforms, exist in mammalian myocardium. The aim of this study was to delineate the contributions of the ecto vs. cytosolic isoforms to Mg2+-stimulated cardiac purine nucleoside formation and release. Isolated guinea pig hearts were retrogradely perfused at their physiological aortic pressure with Krebs-Henseleit bicarbonate buffer fortified with 10 mM glucose. AMP and the adenylate degradatives adenosine and inosine were measured in coronary venous effluent and in epicardial transudate, which was sampled to estimate concentrations of adenylate degradatives in the interstitium. When perfusate Mg2+ was increased from 0.6 to 6 mM, coronary vascular resistance and spontaneous heart rate fell, and steady-state coronary venous release of adenosine + inosine rose severalfold. Cytosolic free magnesium, as estimated by 31P-NMR after 15 min of perfusion with 6 mM Mg2+ or from chemically measured indicator metabolites after 30 min, rose 60 and 144% respectively (P < 0.05). Excess Mg2+ stimulated purine nucleoside release nearly threefold in coronary venous effluent and four- to sevenfold in epicardial transudate. 50 microM, alpha,beta-methylene adenosine 5'-diphosphate (AOPCP), a selective inhibitor of ecto 5'-nucleotidase, elevated interstitial AMP concentration tenfold, did not attenuate basal nucleoside release, but completely inhibited Mg2+-stimulated coronary venous purine nucleoside release and blunted Mg2+-stimulated interstitial purine nucleoside formation by 69%. During perfusion with exogenous 1 microM [8-14C]AMP, excess perfusate MgCl2 increased [14C]adenosine release by 63% in coronary effluent and 133% in epicardial transudate. AOPCP decreased baseline [14C]adenosine release in coronary effluent and epicardial transudate by 85-90%, caused equilibration of arterial and epicardial AMP, and attenuated MgCl2 activation of p[14C]adenosine formation by approx. 75%, in both the vascular and interstitial compartments. Intramyocytic concentrations of allosteric regulators of the cytosolic 5'-nucleotidases were evaluated in stop-frozen myocardium. Excess magnesium did not appreciably alter intracellular pH and ATP concentration, but lowered free cytosolic ADP and AMP concentrations by 50 and 70%, respectively. A simplified model of compartmentalized adenosine metabolism is proposed in which magnesium ion-activated cardiac purine release originates predominantly from the ecto 5'-nucleotidase; magnesium ion stimulation of metabolic flux through the cytosolic isoforms was constrained by concomitant reductions in intracellular AMP substrate and allosteric activator ADP. Magnesium ion-enhanced adenosine formation by 5'-nucleotidase could contribute to the known cardioprotective effects of this clinically used cation.

5'-Nucleotidase↗
PubMed1996

A phospholipid acts as a chaperone in assembly of a membrane transport protein.

A mutant of Escherichia coli lacking phosphatidylethanolamine (PE) and a monoclonal antibody (mAb 4B1) directed against a conformationally sensitive epitope (4B1) of lactose permease were used to establish a novel role for a phospholipid in the assembly of a membrane protein. Epitope 4B1 is readily detectable in spheroplasts and right-side-out membrane vesicles from PE-containing but not from PE-deficient cells expressing lactose permease. Lactose permease from PE-containing membranes, but not from PE-deficient membranes, subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western blot analysis is also recognized by mAb 4B1. If total E. coli phospholipids or PE (but not phosphatidylcholine, phosphatidylglycerol, or cardiolipin) are blotted on nitrocellulose sheets (Eastern blot) prior to transfer of proteins from SDS-polyacrylamide gels, the permease from PE-deficient cells regains its recognition by mAb 4B1. Therefore, PE is required during assembly to form epitope 4B1, but, once formed, sufficient "conformational memory" is retained in the permease to either retain or reform this epitope in the absence of PE. Lactose permease lacking epitope 4B1 can be induced to form the epitope if partially denatured and then renatured in the presence of PE specifically. These results establish for the first time a role for PE as a molecular chaperone in the assembly of the lactose permease.

Antibodies, Monoclonal↗
PubMed1996

CWR22 xenograft as an ex vivo human tumor model for prostate cancer gene therapy.

BACKGROUND: Lack of well-defined relevant in vivo or in vitro tumor models is one of the major limitations in assessing candidate therapeutic regimens, especially gene therapy, for prostate cancer. Since gene therapy is emerging as a potentially powerful therapeutic modality, it is desirable to evaluate this approach for the treatment of human prostate cancer. PURPOSE: We sought to establish a relevant ex vivo tumor model for gene therapy studies of human prostate cancer. METHODS: We constructed and established a transgenic human tumor model consisting of three major components: 1) human primary prostate cancer cells, CWR22, reactivated for growth after storage in liquid nitrogen; 2) a collagen gel ex vivo tissue culture system useful for short-term maintenance and manipulation of CWR22 cells under in vitro experimental conditions; and 3) a high-velocity, particle-mediated gene transfer system that is highly efficient in the ex vivo transfection of target cells. Prostate-specific antigen (PSA) levels in the cell culture media were monitored after transfecting CWR22 cells with candidate therapeutic genes, including the cytokines human interleukin 2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF), both as complementary DNAs [cDNAs]). CWR22 cells, transfected with firefly luciferase cDNA as a reporter gene, served as control cells for cytokine gene expression. CWR22 cells, transfected with the bacterial beta-galactosidase cDNA as a reporter gene, were used to assess the efficiency of gene transfer. Transcription of each of the cDNAs was driven by the cytomegalovirus (CMV) early gene promoter. RESULTS: The three-dimensional organization of tumor cells and functional characteristics of human prostate cancers were maintained in this ex vivo model of prostate cancer. Candidate therapeutic genes, CMV-IL-2 and CMV-GM-CSF, were expressed at peak levels of up to 38 ng of protein per 10(6) cells every 24 hours. IL-2 and GM-CSF secretion was sustained at approximately 40%-50% of peak levels during the entire experimental period (9-10 days in culture). At 7 days after gene delivery, a more than twofold reduction in the secretion of PSA was detected in the IL-2 (3.8 +/- 1.3 ng/10(4) cells every 24 hours [mean +/- standard deviation]) or GM-CSF (4.0 +/- 1.7 ng/10(4) cells every 24 hours) cDNA transfected cells as compared with the control cells transfected with luciferase cDNA (9.3 +/- 1.0 ng/10(4) cells every 24 hours). Up to 10% of the cells transfected with beta-galactosidase cDNA expressed measurable beta-galactosidase activity. CONCLUSION: This study demonstrated an efficient, rapid, and reliable system for gene transfer and expression in primary human prostatic carcinoma cells maintained in a collagen gel culture system. IMPLICATIONS: Our findings suggest a broad application of this CWR22 xenograft primary culture system as an ex vivo tumor model for the evaluation and characterization of various candidate therapeutic genes for human prostate cancer gene therapy, including a cytokine gene-modified tumor vaccine strategy.

Animals↗
PubMed1996

Purification and functional characterization of the C-terminal half of the lactose permease of Escherichia coli.

The lactose permease has been expressed in contiguous, non-overlapping polypeptide fragments containing the N-terminal (N6) and C-terminal (C6) transmembrane domains of the protein [Bibi, E., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4325; Zen, K., et al. (1994) Biochemistry 33, 8198]. When expressed individually, N6 and C6 are unstable and do not catalyze active transport. However, when expressed simultaneously, the polypeptides stabilize each other and form a complex that catalyzes active lactose transport. Moreover, a deletion construct containing the first transmembrane domain and the six C-terminal transmembrane domains mediates downhill lactose translocation [Bibi et al. (1991) proc. Natl. Acad. Sci. U.S.A. 88, 7271]. Here we report that C6 can be expressed independently in a relatively stable form that binds monoclonal antibodies 4B1 and 4B11, which interact with conformationally dependent epitopes on the periplasmic and cytoplasmic surfaces of the membrane, respectively. In addition, C6 retains the ability to catalyze lactose translocation down a concentration gradient in a specific manner. Finally, as observed with full-length Val331Cys permease, beta-D-galactopyranosyl 10thio-beta-D-galactopyranoside quenches the fluorescence of 2-(4'-maleimidylanilino)naphthalene- 6-sulfonic acid (MIANS)- labeled C6 with a single-Cys residue in place of Val331, exhibiting as apparent Kd of 0.2 mM. Unlike full-length Val331Cys permease, however, ligand does not induce a chance in the position of the emission maximum of MIANS-labeled C6(Val331Cys) permease not in the reactivity of C6 (Val331Cys) permease with MINAS. the results indicate that C6 retains a conformation similar to that on the native permease and that most of the structure required of high-affinity binding and substrate translocation is located in the C-terminal half of the molecule.

Amino Acid Sequence↗
PubMed1996

Resonance Raman spectroscopic identification of a histidine ligand of b595 and the nature of the ligation of chlorin d in the fully reduced Escherichia coli cytochrome bd oxidase.

Cytochrome bd oxidase is a bacterial terminal oxidase that contains three cofactors: a low-spin heme (b558), a high-spin heme (b595), and a chlorin d. The center of dioxygen reduction has been proposed to be a binuclear b595/d site, whereas b558 is mainly involved in transferring electrons from ubiquinol to the oxidase. Information on the nature of the axial ligands of the three heme centers has come from site-directed mutagenesis and spectroscopy, which have implicated a His/Met coordination for b558 (Spinner, F., Cheesman, M. R., Thomson, A. J., Kaysser, T., Gennis, R. B., Peng, Q., & Peterson, J. (1995) Biochem. J. 308, 641-644; Kaysser, T. M., Ghaim, J. B., Georgiou, C., & Gennis, R. B. (1995) Biochemistry 34, 13491-13501), but the ligands to b595 and d are not known with certainty. In this work, the three heme chromophores of the fully reduced cytochrome bd oxidase are studied individually by selective enhancement of their resonance Raman (rR) spectra at particular excitation wavelengths. The rR spectrum obtained with 413.1-nm excitation is dominated by the bands of the 5cHS b595(2+) cofactor. Excitation close to 560 nm yields a rR spectrum dominated by the 6cLS b558(2+) heme. Wavelengths between these values enhance contributions from both b595(2+) and b558(2+) chromophores. The rR bands of the ferrous chlorin become the major features with red laser excitation (595-650 nm). The rR data indicate that d2+ is a 5cHS system whose axial ligand is either a weakly coordinating protein donor or a water molecule. In the low-frequency region of the 441.6-nm spectrum, we assign a rR band at 225 cm-1 to the (b595)Fe(II)-N(His) stretching vibration, based on its 1.2-cm(-1) upshift in the 54Fe-labeled enzyme. This observation provides the first physical evidence that the proximal ligand of b595 is a histidine. Site-directed mutagenesis had suggested that His 19 is associated with either b595 or d (Fang, H., Lin, R. -J., & Gennis, R. B. (1989) J. Biol. Chem. 264, 8026-8032). On the basis of the present study, we propose that the proximal ligand of b595 is His 19. We have also studied the reaction of cyanide with the fully reduced cytochrome bd oxidase. In approximately 700-fold excess cyanide (approximately 35 mM), the 629-nm UV/vis band of d2+ is blue-shifted to 625 nm and diminished in intensity. However, the rR spectra at each of three different gamma(0) (413.1, 514.5, and 647.1 nm) are identical with or without cyanide, thus indicating that both b595 and d remain as 5cHS species in the presence of CN-. This observation leads to the proposal that a native ligand of ferrous chlorin d is replaced by CN- to form the 5cHS d2+ cyano adduct. These findings corroborate our companion study of the "as-isolated" enzyme in which we proposed a 5cHS d3+ cyano adduct (Sun, J., Osborne, J. P., Kahlow, M. A., Kaysser, T. M., Hill, J. J., Gennis, R. B., & Loehr, T. M. (1995) Biochemistry 34, 12144-12151). To further characterize the unusual and unexpected nature of these proposed high-spin cyanide adducts, we have obtained EPR spectral evidence that binding of cyanide to fully oxidized cytochrome bd oxidase perturbs a spin-state equilibrium in the chlorin d3+ to yield entirely the high-spin form of the cofactor.

Cytochrome a Group↗
PubMed1996

Efficient extraction and partial purification of the polyribosome-associated stem-loop binding protein bound to the 3' end of histone mRNA.

Replication-dependent histone mRNAs end in a highly conserved stem-loop sequence rather than a polyA sequence. A 45-kDa stem-loop binding protein (SLBP), which specifically binds the stem-loop of histone mRNA, is present in both polyribosomes and nuclei. An identical 45-kDa protein, as determined by partial protease digestion, is cross-linked to a 30 nt RNA containing the 3' stem-loop from both nuclei and polyribosomes. The SLBP can also be detected by a Northwestern blot procedure using the 30 nt RNA as a probe. As judged from the Northwestern assay, more than 90% of the SLBP in the cell is found in the polyribosomes with the remaining SLBP localized to the nucleus. Only 5-10% of the SLBP could be extracted from the polyribosomes with salt. Treatment of the polyribosomes with micrococcal nuclease prior to salt extraction solubilized 5-10 times more SLBP as an RNA-protein complex. The SLBP could be subsequently partially purified from this complex.

Animals↗
PubMed1996

Transformation of NIH 3T3 cells by HER3 or HER4 receptors requires the presence of HER1 or HER2.

Members of the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases have been implicated in the pathogenesis of various malignancies. The ability of one EGFR subfamily member to influence, or function synergistically with, another is likely to be a general feature of these receptors. To assess the role of receptor heterodimerization, we analyzed the ability of Neu differentiation factor (NDF) to induce cell growth and transformation of NIH 3T3 cells transfected with different combinations of the EGFR subfamily of receptors. NDF induced mitogenesis, but not transformation, of cells expressing either HER3 or HER4 alone. However, NDF-induced cell transformation was observed when either HER1 or HER2 was coexpressed with HER3 or HER4. In analogous receptor phosphorylation experiments, NDF-induced transphosphorylation appears to be correlated with synergistic transformation of NIH 3T3 cells. Interestingly, transphosphorylation between HER1 and HER4 can be stimulated by either EGF or NDF.

3T3 Cells↗