Biomedical subjects

Y Lin

Publications and source records attributed to Y Lin.

At least 73 records · Page 4Linked to original sources

PubMed2001

Transcriptional control of intestinal cytochrome P-4503A by 1alpha,25-dihydroxy vitamin D3.

It was previously shown that CYP3A4 is induced in the human intestinal Caco-2 cell model by treatment with 1alpha,25-dihydroxy vitamin D3 (1,25-D3). We demonstrate the vitamin D analog, 19-nor-1alpha,25-dihydroxy vitamin D2, is also an effective inducer of CYP3A4 in Caco-2 cells, but with half the potency of 1,25-D3. We report that treatment of LS180 cells, a human intestinal cell line, with 1 to 10 nM 1,25-D3 dose dependently increased CYP3A4 protein and CYP3A4 mRNA expression. CYP3A4- and CYP3A23-promoter-Luciferase reporter constructs transiently transfected into LS180 cells were transcriptionally activated in a dose-dependent manner by 1,25-D3, whereas mutation of the nuclear hormone receptor binding motif (ER6) in the CYP3A4 promoter abrogated 1,25-D3 activation of CYP3A4. Although the CYP3A4 ER6 promoter element has been shown to bind the pregnane X receptor (PXR), this receptor does not mediate 1,25-D3 induction of CYP3A4 because a) PXR is not expressed in Caco-2 cells; b) PXR mRNA expression is not induced by 1,25-D3 treatment of LS180 cells; and c) the ligand binding domain of human PXR was not activated by 1,25-D3. 1,25-D3 uses the vitamin D receptor to induce CYP3A4 because a) the vitamin D receptor (VDR)-retinoid X receptor (RXR) heterodimer binds specifically to the CYP3A4 ER6; b) selective mutation of the CYP3A4 ER6 disrupted the binding of VDR-RXR; and c) reporter constructs containing only three copies of the CYP3A4 ER6 linked to a TK-CAT reporter were activated by 1,25-D3 only in cells cotransfected with a human VDR expression plasmid. These data support the hypothesis that 1,25-D3 and VDR induce expression of intestinal CYP3A by binding of the activated VDR-RXR heterodimer to the CYP3A PXR response element and promoting gene transcription.

Animals↗

PubMed2001

The alpha and beta subunits of IkappaB kinase (IKK) mediate TRAF2-dependent IKK recruitment to tumor necrosis factor (TNF) receptor 1 in response to TNF.

The activation of IkappaB kinase (IKK) is a key step in the nuclear translocation of the transcription factor NF-kappaB. IKK is a complex composed of three subunits: IKKalpha, IKKbeta, and IKKgamma (also called NEMO). In response to the proinflammatory cytokine tumor necrosis factor (TNF), IKK is activated after being recruited to the TNF receptor 1 (TNF-R1) complex via TNF receptor-associated factor 2 (TRAF2). We found that the IKKalpha and IKKbeta catalytic subunits are required for IKK-TRAF2 interaction. This interaction occurs through the leucine zipper motif common to IKKalpha, IKKbeta, and the RING finger domain of TRAF2, and either IKKalpha or IKKbeta alone is sufficient for the recruitment of IKK to TNF-R1. Importantly, IKKgamma is not essential for TNF-induced IKK recruitment to TNF-R1, as this occurs efficiently in IKKgamma-deficient cells. Using TRAF2(-/-) cells, we demonstrated that the TNF-induced interaction between IKKgamma and the death domain kinase RIP is TRAF2 dependent and that one possible function of this interaction is to stabilize the IKK complex when it interacts with TRAF2.

Animals↗

PubMed2001

Inhibition of insulin signaling and glycogen synthesis by phorbol dibutyrate in rat skeletal muscle.

Numerous studies have shown a correlation between changes in protein kinase C (PKC) distribution and/or activity and insulin resistance in skeletal muscle. To investigate which PKC isoforms might be involved and how they affect insulin action and signaling, studies were carried out in rat soleus muscle incubated with phorbol esters. Muscles preincubated for 1 h with 1 microM phorbol 12,13-dibutyrate (PDBu) showed an impaired ability of insulin to stimulate glucose incorporation into glycogen and a translocation of PKC-alpha, -betaI, -theta, and -epsilon, and probably -betaII, from the cytosol to membranes. Preincubation with 1 microM PDBu decreased activation of the insulin receptor tyrosine kinase by insulin and to an even greater extent the phosphorylation of Akt/protein kinase B and glycogen synthase kinase-3. However, it failed to diminish the activation of phosphatidylinositol 3'-kinase by insulin. Despite these changes in signaling, the stimulation by insulin of glucose transport (2-deoxyglucose uptake) and glucose incorporation into lipid and oxidation to CO2 was unaffected. The results indicate that preincubation of skeletal muscle with phorbol ester leads to a translocation of multiple conventional and novel PKC isoforms and to an impairment of several, but not all, events in the insulin-signaling cascade. They also demonstrate that these changes are associated with an inhibition of insulin-stimulated glycogen synthesis but that, at the concentration of PDBu used here, glucose transport, its incorporation into lipid, and its oxidation to CO2 are unaffected.

Animals↗

PubMed2001

Requirement of protein tyrosine phosphatase SHP2 for NO-stimulated vascular smooth muscle cell motility.

We have previously reported that nitric oxide (NO) increases the motility of differentiated cultured primary aortic smooth muscle cells from adult rats. There is little information on the role of protein tyrosine phosphatases in vascular biology. One such phosphatase, Src homology 2 phosphatase 2 (SHP2), is essential for motility. We tested the hypothesis that NO increases SHP2 levels via a cGMP-mediated mechanism and that this effect is necessary for NO-stimulated cell motility. Here we report that two different NO donors increased SHP2 protein levels and enzyme activity. This effect was mimicked by several cGMP agonists and blocked by an inhibitor of guanylyl cyclase. Specific decrease of SHP2 protein levels via the use of antisense oligodeoxynucleotides (ODNs), but not several control ODNs attenuated the motogenic effect of NO, which indicates the involvement of SHP2 in NO-elicited motogenesis. S-nitroso-N-acetylpenicillamine failed to increase SHP2 protein levels in subcultured aortic smooth muscle cells. This provides a potential explanation for the lack of effect of NO on cell motility in dedifferentiated subcultured cells. These results support the hypothesis that NO-elicited upregulation of SHP2 via a cGMP-mediated pathway is necessary for NO-induced motogenesis in differentiated aortic smooth muscle cells.

Animals↗

PubMed2001

Development and use of a gene promoter-based screen to identify novel inhibitors of cyclooxygenase-2 transcription.

Cyclooxygenase-2 (COX-2) is a recognized target for cancer prevention and possibly treatment. To identify novel inhibitors of COX-2, we developed a high throughput reporter gene assay that utilizes a region of the human COX-2 promoter to drive luciferase expression. A total of 968 extracts from 266 plants were screened. Extracts from 12 plants (4.5%), including Arnebia euchroma, a medicinal plant used in the Far East to treat inflammation, inhibited the stimulation of COX-2 promoter activity. The gene promoter assay then was used to identify shikonin, a compound with known anti-inflammatory and chemopreventive properties, as an active compound in A. euchroma. To complement the gene promoter studies, we determined the effects of a mixture of shikonins on phorbol 12-myristate 13-acetate (PMA)-mediated induction of COX-2 in transformed human mammary epithelial cells. Shikonins inhibited PMA-mediated induction of COX-2 mRNA, protein, and prostaglandin E(2) synthesis. In transient transfections, PMA caused a severalfold increase in COX-2 promoter activity, an effect that was suppressed by shikonins. Shikonins also inhibited PMA-mediated stimulation of extracellular signal-regulated kinase1/2 mitogen-activated protein kinases and activator protein-1 activity. Collectively, these results demonstrate the successful development and use of a high throughput reporter gene assay for the identification of a novel inhibitor of COX-2 expression.

Antineoplastic Agents, Phytogenic↗

PubMed2001

Embryonic control of epidermal cell patterning in the root and hypocotyl of Arabidopsis.

A position-dependent pattern of epidermal cell types is produced during the development of the Arabidopsis seedling root and hypocotyl. To understand the origin and regulation of this patterning mechanism, we have examined the embryonic expression of the GLABRA2 (GL2) gene, which encodes a cell-type-specific transcription factor. Using in situ RNA hybridization and a sensitive GL2::GFP reporter, we discovered that a position-dependent pattern of GL2 expression is established within protodermal cells at the heart stage and is maintained throughout the remainder of embryogenesis. In addition, we show that an exceptional GL2 expression character and epidermal cell pattern arises during development of the root-hypocotyl junction, which represents an anatomical transition zone. Furthermore, we find that two of the genes regulating seedling epidermal patterning, TRANSPARENT TESTA GLABRA (TTG) and WEREWOLF (WER), also control the embryonic GL2 pattern, whereas the CAPRICE (CPC) and GL2 genes are not required to establish this pattern. These results indicate that position-dependent patterning of epidermal cell types begins at an early stage of embryogenesis, before formation of the apical meristems and shortly after the cellular anatomy of the protoderm and outer ground tissue layer is established. Thus, epidermal cell specification in the Arabidopsis seedling relies on the embryonic establishment of a patterning mechanism that is perpetuated postembryonically.

Arabidopsis↗

PubMed2001

Induced repatterning of type XVIII collagen expression in ureter bud from kidney to lung type: association with sonic hedgehog and ectopic surfactant protein C.

Epithelial-mesenchymal tissue interactions regulate the formation of signaling centers that play a role in the coordination of organogenesis, but it is not clear how their activity leads to differences in organogenesis. We report that type XVIII collagen, which contains both a frizzled and an endostatin domain, is expressed throughout the respective epithelial bud at the initiation of lung and kidney organogenesis. It becomes localized to the epithelial tips in the lung during the early stages of epithelial branching, while its expression in the kidney is confined to the epithelial stalk region and is lost from the nearly formed ureter tips, thus displaying the reverse pattern to that in the lung. In recombinants, between ureter bud and lung mesenchyme, type XVIII collagen expression pattern in the ureter bud shifts from the kidney to the lung type, accompanied by a shift in sonic hedgehog expression in the epithelium. The lung mesenchyme is also sufficient to induce ectopic lung surfactant protein C expression in the ureter bud. Moreover, the shift in type XVIII collagen expression is associated with changes in ureter development, thus resembling aspects of early lung type epigenesis in the recombinants. Respecification of collagen is necessary for the repatterning process, as type XVIII collagen antibody blocking had no effect on ureter development in the intact kidney, whereas it reduced the number of epithelial tips in the lung and completely blocked ureter development with lung mesenchyme. Type XVIII collagen antibody blocking also led to a notable reduction in the expression of Wnt2, which is expressed in the lung mesenchyme but not in that of the kidney, suggesting a regulatory interaction between this collagen and Wnt2. Respecification also occurred in a chimeric organ containing the ureter bud and both kidney and lung mesenchymes, indicating that the epithelial tips can integrate the morphogenetic signals independently. A glial cell line-derived neurotrophic factor signal induces loss of type XVIII collagen from the ureter tips and renders the ureter bud competent for repatterning by lung mesenchyme-derived signals. Our data suggest that differential organ morphogenesis is regulated by an intra-organ patterning process that involves coordination between inductive signals and matrix molecules, such as type XVIII collagen.

Animals↗

PubMed2001

The influence of chronic antihypertensive treatment on the central pressor response in SHR.

UNLABELLED: We examined the influence of chronic antihypertensive treatment on the central pressor response in SHR. Adult male SHR were divided into 5 groups, i.e., those receiving 1) enalapril (Enal: 25 mg/kg/day in drinking water, n=12); 2) losartan (Los: 40 mg/kg/day, n=11); 3) candesartan (Cand: 4 mg/kg/day, n=12); 4) hydralazine+hydrochlorothiazide (H&H: 50+7.5 mg/kg/day, n=9); 5) vehicle ( CONTROL: n=9). At 4 weeks of treatment, hypertonic saline (0.25, 0.5 M) was intracerebroventricularly (i.c.v.) injected into conscious rats. Plasma catecholamines were measured before and after i.c.v. injection. On completion of the experiment, heart weight was measured, and angiotensin-converting enzyme (ACE) activity of the cerebrum was determined. All antihypertensive drugs elicited comparable reductions in systolic blood pressure, while heart rate was significantly higher in the H&H group than in the other groups during treatment. Pressor response to i.c.v. hypertonic saline (0.5 M) was significantly smaller in the Enal (12 +/- 3 mmHg) and Cand (11 +/- 2 mmHg) groups than in the Los (22 +/- 2 mmHg), H&H (16 +/- 2 mmHg), and CONTROL (29 +/- 5 mmHg) groups. Plasma catecholamines did not differ among the groups. Heart weight was lowest in the Enal group, followed by the Los and Cand groups. ACE activity of the cerebrum was significantly decreased in the Enal group. The results suggest that chronic treatment with various antihypertensive drugs differentially alters the central pressor response in SHR, and enalapril and candesartan are effective in attenuating this response.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2001

Enzyme kinetics of cytochrome P450-mediated reactions.

The most common drug-drug interactions may be understood in terms of alterations of metabolism, associated primarily with changes in the activity of cytochrome P450 (CYP) enzymes. Kinetic parameters such as Km, Vmax, Ki and Ka, which describe metabolism-based drug interactions, are usually determined by appropriate kinetic models and may be used to predict the pharmacokinetic consequences of exposure to one or multiple drugs. According to classic Michaelis-Menten (M-M) kinetics, one binding site models can be employed to simply interpret inhibition (pure competitive, non-competitive and uncompetitive) or activation of the enzyme. However, some cytochromes P450, in particular CYP3A4, exhibit unusual kinetic characteristics. In this instance, the changes in apparent kinetic constants in the presence of inhibitor or activator or second substrate do not obey the rules of M-M kinetics, and the resulting kinetics are not straightforward and hamper mechanistic interpretation of the interaction in question. These unusual kinetics include substrate activation (autoactivation), substrate inhibition, partial inhibition, activation, differential kinetics and others. To address this problem, several kinetic models can be proposed, based upon the assumption that multiple substrate binding sites exist at the active site of a particular P450, and the resulting kinetic constants are, therefore, solved to adequately describe the observed interaction between multiple drugs. The following is an overview of some cytochrome P450-mediated classic and atypical enzyme kinetics, and the associated kinetic models. Applications of these kinetic models can provide some new insights into the mechanism of P450-mediated drug-drug interactions.

Algorithms↗

PubMed2001

Plasmablastic lymphoma of the lung: report of a unique case and review of the literature.

Non-Hodgkin lymphomas associated with acquired immunodeficiency syndrome are heterogeneous. Recently, a novel subtype of non-Hodgkin lymphoma occurring mostly in patients with acquired immunodeficiency syndrome has been described and designated as plasmablastic lymphoma. The histomorphologic and immunophenotypic findings of this distinct subtype of non-Hodgkin lymphoma have been characterized previously. Most patients present with oral cavity involvement. We report a case of plasmablastic lymphoma presenting as a lung tumor. To our knowledge, this is the first case report of this unusual subtype of diffuse large B-cell lymphoma in this location.

Acquired Immunodeficiency Syndrome↗

PubMed2001

Substrate inhibition kinetics for cytochrome P450-catalyzed reactions.

Most cytochrome P450 (P450 or CYP)-catalyzed reactions are adequately described by classical Michaelis-Menten kinetic parameters (e.g., Km and Vmax), which are usually determined by a saturation profile of velocity of product formation versus substrate concentration. In turn, these parameters may be used to predict pharmacokinetics. However, some P450 enzymes exhibit atypical or non-Michaelis-Menten kinetics, due largely to substrate inhibition at higher concentrations of substrate. Although the mechanism of substrate inhibition is unknown, ignoring it and truncating the data can lead to erroneous estimates of kinetic parameters. In the present study, 13 P450 marker substrates were examined with 10 recombinant P450 proteins, and 6 were found, to varying degrees, to exhibit substrate inhibition. To understand the nature of the inhibition, a kinetic model was proposed (assuming that two binding sites exist on the enzyme) and used to fit the experimental data. The derived data indicated that 1) the K(I) values (substrate inhibition) were approximately 1.2- to 10-fold greater than the respective K(S) values; 2) both K(S) and K(I) values may be affected by the interaction of the two bound substrates within the enzyme, exhibited by a factor alpha (alpha = 5.1-23.3); and 3) enzyme activity was inhibited markedly (39-97%) at excess concentrations of the substrates (beta = 0.03-0.61). These findings suggest that substrates have access to both the inhibitory site and catalytic site simultaneously (K(I) > K(S)). Furthermore, the two sites, in the presence of substrate, can interact with each other. Therefore, the degree of inhibition of the enzyme is dependent on the concentration of the substrate (usually >K(I)) that sufficiently occupies the inhibitory site.

Antineoplastic Agents↗

PubMed2001

[Toxic effects of Cu, Zn and Mn on the inhibition of Chlorella pyrenoidosa's growth].

A standard method of algal bioassay for evaluating the toxicity of toxic chemicals was applied in the study. It was found that safety concentrations of Cu, Zn and Mn for Chlorella pyrenoidosa's growth were 31.8 micrograms/L, 65.0 micrograms/L and 5.5 mg/L, and 96 h-EC50 were 67.3 micrograms/L, 473.0 micrograms/L and 17.0 mg/L, respectively. According to both safety concentration and 96 h-EC50, the toxic sequences of these metals from high to low on the inhibition of C. pyrenoidosa's growth were Cu, Zn and Mn. The different affinities between different metal ions and algal cells are mainly responsible for the different inhibitions of C. pyrenoidosa's growth.

Chlorella↗

PubMed2001

[Treatment of dilute solution anilines-containing with displacement by extraction].

It was found that the biodegradability (indicated by BOD5/CODCr value of water solution containing anilire or chloroaniline declined with the increasing of their concentration. In order to bio-treat such water, decrease of the solute's concentration was necessary. Based on the research of organic solvents' and complexing agent's biodegradability, a series of extraction experiments were conducted for aniline and m-chloroaniline with complexing agent or physical solvent. The factors influencing the distribution ratio were discussed. The BOD5/CODCr value of raffinates showed that the raffinate can be treated by biological degradation without dilution if suitable solvent was choosed. It was proved that displacement by extraction had great potentiality for treating wastewater containing non-biodegradable organic contaminant.

Aniline Compounds↗

PubMed2001

Dendritic growth induced by BMP-7 requires Smad1 and proteasome activity.

Bone morphogenetic proteins (BMPs) induce dendritic growth in cultured sympathetic neurons; however, the signaling pathways that mediate this dendrite-promoting activity have not been previously characterized. Here we report studies of the signaling events that regulate the growth of these afferent processes. We find that Smad1 is expressed in sympathetic neurons and that BMPs rapidly induce its phosphorylation and translocation from the cytoplasm to the nucleus. Furthermore, a dominant negative form of Smad1 inhibits BMP-7-induced dendritic growth, suggesting a requirement for Smad1 activation in this biological activity of BMP-7. A physical interaction between Smad1 and components involved in the proteasome-mediated degradation system was detected with a yeast two-hybrid screen, thereby prompting an examination of the effects of proteasome inhibitors on dendritic growth. Lactacystin and ALLN (N-acetyl-Leu-Leu-norleucinal) selectively blocked BMP-7-induced dendritic growth without adversely affecting either cell viability or axonal growth. Moreover, studies of transfected P19 cells suggest that the proteasome inhibitors directly block the effects of Smad1 on the transcriptional activity of the Tlx-2 promoter. These data indicate that BMP-induced dendritic growth requires Smad1 activation and involves proteasome-mediated degradation events.

Acetylcysteine↗

PubMed2001

Testosterone, 7-benzyloxyquinoline, and 7-benzyloxy-4-trifluoromethyl-coumarin bind to different domains within the active site of cytochrome P450 3A4.

Testosterone, 7-benzyloxyquinoline, and 7-benzyloxy-4-trifluoromethyl-coumarin, marker substrates for cytochrome P450 3A4 are commonly used within the pharmaceutical industry to screen new chemical entities as inhibitors of CYP3A4 in a high-throughput manner to predict the potential for drug-drug interactions. However, it has been observed that inhibition data obtained with a given CYP3A4 probe substrate may not correlate well with results from a different probe. As a consequence, the choice of the probe compound becomes an important consideration in such screens. In the present study, kinetic interactions between either two of the above three substrates were evaluated, and three-dimensional nonlinear regression analysis was performed to understand the kinetic mechanisms of drug interaction. Our results demonstrate that the kinetic interaction between each pair of substrates does not appear to be competitive and that the interactions are characterized by an unchanged or a decrease in both apparent K(m) (a = 0.21-0.72, a change of K(m) in the absence of the effector) and V(max) (alpha and beta = 0.09-0.75, changes of V(max) in the absence of the effector). These data suggest that 1) the three substrates bind to different domains; 2) at least two substrates can coexist in the active site of CYP3A4; and 3) the two bound substrates interact kinetically with each other (e.g., through steric hindrance), thereby leading to a change in both apparent kinetic parameters and partial inhibition. Selection of multiple substrates, which are shown not to be competitive, is necessary to accurately predict CYP3A4 inhibition and the potential for drug-drug interaction.

Baculoviridae↗

PubMed2001

Glucocorticoid receptor and heat shock protein 90 in peripheral blood mononuclear cells from asthmatics.

OBJECTIVE: To investigate the expression of glucocorticoid receptor (GR) and heat shock protein 90 (HSP90) mRNA in peripheral blood mononuclear cells (PBMCs) from steroid-sensitive (SS), steroid-dependent (SD) and steroid-resistant (SR) asthmatics patients, and to evaluate the role of GR and HSP90 in the pathogenesis of SR. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the expressions of GR and HSP90 mRNA in PBMC stimulated with IL-2 and/or IL-4 from 10 normal volunteers, 10 SS, 5 SD and 6 SR patients. RESULTS: The expressions of GR and HSP90 mRNA were the highest in PBMC from SR patients (0.730 +/- 0.171 and 1.122 +/- 0.165, respectively) compared with the normals (P < 0.05). The second was from SS patients (0.359 +/- 0.350 and 0.885 +/- 0.250, respectively). The lowest was from the SD patients (0.017 +/- 0.008 and 0.078 +/- 0.039, respectively). The ratio of HSP90/GR in SR was significantly lower than that in the others, and it suggested that the expression of HSP90 mRNA was not sufficient in this group of patients. When PBMC from SS, SD and SR was incubated with IL-2 or IL-4 alone, no changes in GR and HSP90 mRNA expression were observed. While incubated with combination of IL-2 and IL-4, a significantly higher expression of GR mRNA was observed in all asthmatics, and a significantly higher expression of HSP90 mRNA was observed only in SS and SD patients. CONCLUSION: The lowering of HSP90/GR ratio may be one of the causes of SR. IL-2 and IL-4 may play roles in the imbalance of HSP90/GR.

Adult↗

PubMed2000

Delineation of the roles of amino acids involved in the catalytic functions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase.

The roles of particular amino acids in substrate and coenzyme binding and catalysis of glucose-6-phosphate dehydrogenase of Leuconostoc mesenteroides have been investigated by site-directed mutagenesis, kinetic analysis, and determination of binding constants. The enzyme from this species has functional dual NADP(+)/NAD(+) specificity. Previous investigations in our laboratories determined the three-dimensional structure. Kinetic studies showed an ordered mechanism for the NADP-linked reaction while the NAD-linked reaction is random. His-240 was identified as the catalytic base, and Arg-46 was identified as important for NADP(+) but not NAD(+) binding. Mutations have been selected on the basis of the three-dimensional structure. Kinetic studies of 14 mutant enzymes are reported and kinetic mechanisms are reported for 5 mutant enzymes. Fourteen substrate or coenzyme dissociation constants have been measured for 11 mutant enzymes. Roles of particular residues are inferred from k(cat), K(m), k(cat)/K(m), K(d), and changes in kinetic mechanism. Results for enzymes K182R, K182Q, K343R, and K343Q establish Lys-182 and Lys-343 as important in binding substrate both to free enzyme and during catalysis. Studies of mutant enzymes Y415F and Y179F showed no significant contribution for Tyr-415 to substrate binding and only a small contribution for Tyr-179. Changes in kinetics for T14A, Q47E, and R46A enzymes implicate these residues, to differing extents, in coenzyme binding and discrimination between NADP(+) and NAD(+). By the same measure, Lys-343 is also involved in defining coenzyme specificity. Decrease in k(cat) and k(cat)/K(m) for the D374Q mutant enzyme defines the way Asp-374, unique to L. mesenteroides G6PD, modulates stabilization of the enzyme during catalysis by its interaction with Lys-182. The greatly reduced k(cat) values of enzymes P149V and P149G indicate the importance of the cis conformation of Pro-149 in accessing the correct transition state.

Arginine↗

PubMed2000

A tetravalent single-chain antibody-streptavidin fusion protein for pretargeted lymphoma therapy.

Single-chain Fv antibody fragments from the CD20-specific murine monoclonal antibody B9E9 were genetically engineered as streptavidin fusions [single-chain Fv-streptavidin (scFvSA) fusion protein] for use in pretargeted radioimmunotherapy. The scFvSA constructs were expressed as soluble, tetrameric species in the periplasm of Escherichia coli. Expression levels were affected by the order of the variable regions and the length and composition of the single-chain Fv linker. The best expressor was obtained with the variable regions in the heavy chain-light chain configuration separated by a 25-mer Gly4Ser linker. This construct produced 250-300 mg of soluble, tetrameric fusion protein per liter of fermentor culture. The fusion protein (Mr 173,600) was purified from crude lysates by iminobiotin affinity chromatography with an overall yield of about 50% and was analyzed for functionality both in vitro and in vivo. Immunoreactivity of the scFvSA fusion protein and its nanomolar affinity to CD20-positive Ramos cells were comparable with the B9E9 monoclonal antibody. The fusion protein had a biotin dissociation rate identical to recombinant streptavidin and bound an average of 3.6 biotins/molecule of a possible 4 biotins/molecule. Labeled fusion protein cleared from the blood of BALB/c mice with a P half-life of about 16 h. In nude mice bearing Ramos xenografts, the fusion protein demonstrated sufficient tumor localization of functional streptavidin to enable efficient, tumor-specific targeting of a subsequently administered radionuclide-chelate/biotin molecule. These results suggest that large quantities of functional scFvSA can be produced for clinical testing as a therapy for non-Hodgkin's lymphoma.

Amino Acid Sequence↗