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PubMed1995

Immunomodulation of transplant rejection using monoclonal antibodies and soluble receptors.

The main objective of our studies has been to optimize the effects of monoclonal antibodies (MAbs) and other immunosuppressive reagents to enhance organ graft survival. One such agent is OKT3, a MAb that is directed against the CD3 component of the human T-cell receptor (TCR) complex. Treatment of a rejection episode with OKT3 results in a rapid and efficient clearing of circulating T cells and reversal of most rejection episodes. Its wider use in transplantation and in the treatment of immune-mediated disease is limited by adverse reactions that follow the initial dose, the production of neutralizing Abs, and the transient nature of the immunosuppression. We have engineered CDR-grafted "humanized" anti-CD3 MAbs that lack Fc-receptor binding activity through mutagenesis of amino acids in the Fc portion of the MAb. This results in an immunosuppressive anti-CD3 MAb that is less antigenic and one that does not induce the first-dose side effects. In addition, we have pursued a goal of developing a therapy that will induce donor-specific tolerance while maintaining overall recipient immune competency. Because antigen-specific T-cell activation depends not only on TCR-ligand interaction, but also on additional costimulatory signals mediated by accessory molecules such as CD28, blocking the binding of CD28 on T cells to its ligand B7, during TCR engagement, might modulate transplantation responses. Using a soluble fusion protein of human CTLA4, CTLA4-Ig, that binds B7 with high affinity, inhibition of human pancreatic islet rejection that occurs, at least in part, by affecting T-cell recognition of human B7+ antigen-presenting cells has been demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed2006

Differential interactions of phytochrome A (Pr vs. Pfr) with monoclonal antibodies probed by a surface plasmon resonance technique.

Phytochromes are red- and far-red light-reversible photoreceptors for photomorphogenesis in plants. Phytochrome A is a dimeric chromopeptide that mediates very low fluence and high irradiance responses. To analyze the surface properties of phytochrome A (phyA), the epitopes of 21 anti-phyA monoclonal antibodies were determined by variously engineered recombinant phyA proteins and the dissociation constants of seven anti-phyA monoclonal antibodies with phyA were measured using a surface plasmon resonance (SPR)-based resonant mirror biosensor (IAsys). Purified oat phyA was immobilized on the sensor surface using a carboxymethyl dextran cuvette in advance, and the interactions of each chosen monoclonal antibody against phyA in either red light absorbing form (Pr) or far-red light absorbing form (Pfr) at different concentrations were monitored. The binding profiles were analyzed using the FAST Fit program of IAsys. The resultant values of dissociation constants clearly demonstrated the differential affinities between the phyA epitopes and the monoclonal antibodies dependent upon Pr vs. Pfr conformations. Monoclonal antibody mAP20 preferentially recognized the epitope at amino acids 653-731 in the Pr form, whereas mAA02, mAP21 and mAR07/mAR08 displayed preferential affinities for the Pfr's surfaces at epitopes 494-601 (the hinge region between the N- and C-terminal domains), 601-653 (hinge in PASI domain), and 772-1128 (C-terminal domain), respectively. The N-terminal extension (1-74) was not recognized by mAP09 and mAP15, suggesting that the N-terminal extreme is not exposed in the native conformation of phyA. On the other hand, the C-terminal domain becomes apparently exposed on Pr-to-Pfr phototransformation, suggesting an inter-domain cross-talk. The use of surface plasmon resonance spectroscopy offers a new approach to study the surface properties of phytochromes associated with the photoreversible structural changes, as well as for the study of protein-protein interactions of phytochromes with their interacting proteins involved in light signaling events in plants.

Antibodies, Monoclonal↗
PubMed2004

Restricted entry of R5 HIV Type 1 strains into eosinophilic cells.

A cell culture system previously developed by our laboratory demonstrated that T cell-tropic (CXCR4-using) but not macrophage-tropic (CCR5-using) HIV-1 strains productively infected eosinophilic cells. In the current study, an improved model was used to determine the level of this viral restriction by assessing viral entry and coreceptor usage. The model was improved by using AML14.3D10 cells that were engineered to express CCR3 in addition to the major HIV-1 coreceptors, CD4, CXCR4, and CCR5, thus making them more like primary eosinophils. A polymerase chain reaction (PCR) assay was used to detect viral entry. In the PCR assay, primers specific for early reverse transcription products were used to amplify minus strand viral DNA from HIV-1-infected AML14.3D10-CCR3 eosinophilic cells. Coreceptor blocking experiments, using the CXCR4 antagonist AMD3100, were performed to determine coreceptor usage by the CXCR4-using (X4) strain known to productively infect the cells. Virus production was measured by p24 immunoassay. As expected, viral DNA was detected in AML14.3D10-CCR3 cells infected with X4 HIV-1, and cell viability was decreased during maximal viral production. Conversely, viral DNA was not detected in eosinophilic cells exposed to a CCR5-using (R5) HIV-1 strain that is also capable of using CCR3, indicating that R5 HIV-1 is unable to enter eosinophilic cells despite the presence of the appropriate coreceptors. Infection of AML14.3D10-CCR3 cells by HTLV-III(B) was completely inhibited by AMD3100, indicating that X4 HIV-1 enters the AML14.3D10-CCR3 cell line by using the CXCR4 coreceptor exclusively. Since X4 strains predominate during the late stages of HIV-1 infection in many patients, when eosinophil numbers also tend to increase, the ability of these HIV-1 strains to infect eosinophilic cells has important implications for the involvement of eosinophils in the pathogenesis of AIDS.

Cell Line↗
PubMed2006

Context-dependent mutations predominate in an engineered high-affinity single chain antibody fragment.

A mutational analysis of the femtomolar-affinity anti-fluorescein antibody 4M5.3, compared to its wild-type progenitor, 4-4-20, indicates both context-dependent and -independent mutations are responsible for the 1800-fold affinity improvement. 4M5.3 was engineered from 4-4-20 by directed evolution and contains 14 mutations. The seven mutations identified as present in each of 10 final round affinity maturation clones were studied here. Affinities of the 4-4-20 single mutant addition and 4M5.3 single site reversion mutants were compared. These experiments identified four mutations, of these seven, that were context-dependent in their contribution to higher affinity. A simplified mutant containing only these seven mutations was created to analyze complete double mutant cycles of selected sets of mutations. Specific mutational sets studied included the ligand contact mutations, the heavy chain CDR3 mutations, the heavy chain CDR3 mutations plus the neighboring residue at site H108, and the early and late acquired mutations on the directed evolution pathway. The heavy chain CDR3 mutational set and the ligand-contacting mutations were shown to provide -1.4 and -2.0 kcal/mol, respectively, of the total -3.5 kcal/mol change in free energy of binding of the seven-site consensus mutant. The mutations acquired late in the directed evolution rounds provided much of the change in free energy without the earlier acquired mutations (-3.1 kcal/mol of the total -3.5 kcal/mol). Prior structural data and electrostatic calculations presented several hypotheses for the higher affinity contributions, some of which are supported by these mutational data.

Animals↗
PubMed1996

The simian foamy virus type 1 transcriptional transactivator (Tas) binds and activates an enhancer element in the gag gene.

Simian and human foamy viruses (SFV and HFV) encode a transcriptional transactivator, Tas, which governs the levels of viral transcripts initiated by both the promoter in the long terminal repeat (LTR) and the internal promoter (IP) located within the env gene of these viruses. Tas-responsive target elements,(TRE) LTR in the LTR and (TRE) IP in the env gene, are located 5' of the TATA box in both viral promoters and function as orientation- and position-independent enhancers. We have identified a strong Tas-responsive element, designated TRE (GP), near the 3' end of the gag gene and preceding the pol gene of SFV-1. In transient-expression assays with plasmids containing reporter genes, a 59-bp DNA fragment containing TRE (GP) (nucleotides 2224 to 2282) functioned as an enhancer element, dependent on Tas, in several cell types and in the context of a heterologous basal promoter. DNase footprinting revealed that the fusion protein glutathione S-transferase-Tas, purified from genetically engineered bacteria, interacts with about 40 hp (nucleotides 2237 to 2279) in the TRE (GP). A low degree of sequence homology was noted between TRE (GP) and TRE (IP). In virus-infected cells, novel transcripts with 5' ends immediately upstream from the reverse transcriptase translation frame (nucleotides 2611 to 5778) were identified. Upstream of the start site for these transcripts is a TATA box (nucleotides 2575 to 2579), which was required for transcription in transient-expression assays. Although a spliced mRNA initiated in the viral LTR is implicated in the synthesis of the HFV Pol polyprotein which encodes protease, reverse transcriptase, and integrase, it is possible that SFV-1 contains a promoter within the pol gene for initiating a reverse transcriptase transcript. Taken together, these studies define a novel Tas-responsive enhancer element, which binds the viral transactivator, and a potential promoter within the pol gene.

Animals↗
PubMed2001

Effects of ANG II on bradykinin receptor gene expression in cardiomyocytes and vascular smooth muscle cells.

Bradykinin has vasodilatory and tissue-protective effects exerted via its B(2) type receptor, whereas the B(1) receptor is constitutively absent but inducible by inflammation and toxins. In previous studies, we found that B(2) receptor gene knockout mice exhibit overexpression of the B(1) receptor, which assumes a vasodilatory function and is further upgraded in renovascular hypertension. The present study was designed to explore the effects of excess angiotensin II (ANG II) on B(1) receptor and B(2) receptor gene expression in mouse cardiomyocytes and rat vascular smooth muscle cells (VSMC) in vivo (after a 3-day infusion of 30 ng/min ANG II in 11 wild-type and in 13 genetically engineered mice with deleted B(2) receptor gene) and in vitro (ANG II added in rat VSMC culture in the presence or absence of AT(1) or AT(2) receptor antagonist). Expression of B(1) and B(2) receptor mRNA was assessed by reverse transcriptase-polymerase chain reaction. ANG II infusion caused upregulation by 30% of the already significantly overexpressed B(1) receptors in cardiomyocytes of the B(2) receptor gene knockout mice, but in the wild-type mice it upregulated only the B(2) receptor mRNA by 47%. The addition of ANG II in VSMC culture produced a time-dependent induction of B(1) and upregulation of B(2) receptor gene expression, maximal at 3 h (by fivefold), declining almost to baseline by 24 h. The addition of losartan completely blocked this effect, whereas the AT(2) blocker PD-123319 made no difference, indicating that this is an AT(1)-mediated effect of ANG II. The data indicate that excess ANG II in subpressor doses in vivo upregulates expression of the B(2) receptor, but in its absence, the already overexpressed B(1) receptor is further upregulated, evidently assuming a counterregulatory response; in vitro, it transiently upregulates both bradykinin receptors.

Angiotensin II↗
PubMed1998

[Refolding of recombinant proteins in vitro].

Refolding of recombinant proteins is an important event in the downstream process of genetic engineering. On the basis of the mechanism of protein folding in vitro, general strategy of recombinant protein refolding is postulated, and major developments in recent years on this field are reviewed, including: molecular chaperone mediated refolding, detergent-assisted refolding, protein refolding in reverse micelle, addition of folding enhancer and methods for the formation of disulfide bond during protein refolding.

Animals↗
PubMed2003

[Cloning and sequence analysis of active fragments of human thrombospondin-1].

OBJECTIVE: To lay a foundation for researches on the expression of gene engineering anti-tumor drugs. METHODS: On the basis of mRNA sequence of human thrombospondin-1(TSP-1), two pairs of primers were designed and synthesized. Two active fragments TSP-1, called TSP-1-I and TSP-1-III, were obtained by using reverse transcription-polymerase chain reaction(RT-PCR). The sizes of those two fragments were found to be 728 bp and 257 bp respectively. They were purified and then cloned into pMDT vector after transformation into Escherichia coli with recombined DNAs. RESULTS: Plasmid DNAs were isolated from transformants and digested with restriction enzyme. The results from agarose gel electrophorasis showed that these two fragments had been inserted into vector and their sizes were in accord with the expected values. Sequence analysis demonstrated that TSP-1-I is 728 bp in size and encodes a peptide containing 212 amino acid residues with relative molecular mass of 23.6 x 10(3) and the TSP-I III is 257 bp in size and encodes a peptide containing 86 amino acid residues with relative molecular mass of 9.5 x 10(3). CONCLUSION: The results from homology analysis indicate that the sequence of these two fragments is identified with that of TSP-1 of Genbank.

Adenocarcinoma↗
PubMed2003

Studies on osteogenic differentiation of rat bone marrow stromal cells cultured in type I collagen gel by RT-PCR analysis.

The purpose of this investigation was to examine by reverse-transcriptase polymerase chain reaction analysis the osteogenic differentiation of twice-passaged Sprague-Dawley rat bone marrow stromal cells in type I collagen gel cultured for 3 weeks. Two culture media were used here, namely Dulbecco's modified Eagle (DME) medium supplemented with vitamin C [Dex (-)] and those with vitamin C, dexamethasone and beta-glycerophosphate [Dex (+)]. Culture with Dex (-) medium in collagen gel for 3 weeks brought about the well-developed cell network and middle-stage osteogenic phenotype expression characterized by mRNA for alkaline phosphatase, osteonectin and osteopontin while those for bone sialo protein and osteocalcin were not detected. On the contrary, culture with Dex (+) medium in collagen gel for 3 weeks lead to necrosis of the cells. These results indicate that culture in collagen gel with Dex (-) DME medium containing vitamin C was useful for three-dimensional culture and middle-stage osteogenic differentiation of twice-passaged bone marrow stromal cells. This study might contribute to tissue engineering therapy to fix bone and periodontal defects in the future.

Animals↗
PubMed1996

[Transformation of genetically engineered oxytocinoyllysine into oxytocin].

Various methods for obtaining oxytocin from its recombinant precursor oxytocinoyllysine were studied. For splitting off the C-terminal lysine residue in oxytocinoyllysine, the carboxypeptidase B and an analogous carboxypeptidase from king crab hepatopancreas were used. Ammonolysis of oxytocinic acid methyl ester proved to be the most efficient method for the last stage of the oxytocin preparation. Reversed-phase HPLC was used for the product analysis at each stage of the recombinant oxytocinoyllysine conversion into oxytocin.

Amino Acid Sequence↗
PubMedDate not supplied

[The antidiphtheria activity of the pre-EGF fragment].

The DNA fragment, coding a part of the protein molecule--the precursor of the epidermal growth factor (pre-EGF76-208)--and containing the sequence of 133 N-end amino acid residues, was obtained with the use of gene engineering and molecular biological techniques. For this purpose a fraction of poly(A+) = RNA was isolated from the kidney of a newborn infant; on this fraction the "library" of cDNA fragments whose coding capacity corresponded to the required sequence of mRNA in the pre-EGF gene was obtained in the reaction of reverse transcription, conjugated with the polymerase chain reaction (PCR). After the determination of the nucleotide sequences in 11 fragments only 1 fragment which contained practically no mutations appearing in PCR as the result of amplifications was chosen. This fragment was used for the construction of a hybrid plasmid controlling the synthesis of hybrid fusion protein with the sequence of pre-EGF76-208. Fusion protein was synthesized with very low effectiveness, but the use of metallo-affinity chromatography permitted to isolate and purify it, its purity reaching 98%. Then its antitoxic activity was determined in the skin test on guinea pigs and found to be not less then 10(6) I.U./mg of protein.

Animals↗
PubMed2005

Hysteresis in a synthetic mammalian gene network.

Bistable and hysteretic switches, enabling cells to adopt multiple internal expression states in response to a single external input signal, have a pivotal impact on biological systems, ranging from cell-fate decisions to cell-cycle control. We have designed a synthetic hysteretic mammalian transcription network. A positive feedback loop, consisting of a transgene and transactivator (TA) cotranscribed by TA's cognate promoter, is repressed by constitutive expression of a macrolide-dependent transcriptional silencer, whose activity is modulated by the macrolide antibiotic erythromycin. The antibiotic concentration, at which a quasi-discontinuous switch of transgene expression occurs, depends on the history of the synthetic transcription circuitry. If the network components are imbalanced, a graded rather than a quasi-discontinuous signal integration takes place. These findings are consistent with a mathematical model. Synthetic gene networks, which are able to emulate natural gene expression behavior, may foster progress in future gene therapy and tissue engineering initiatives.

Alkaline Phosphatase↗
PubMed2006

Hepatic differentiation of mouse ES cells into BE cells in vitro.

To date, the hepatic differentiation of embryonic stem (ES) cells into biliary epithelial (BE) cells has only been identified in hepatocytes. In this study, an attempt was made to induce the differentiation of ES cells to BE cells. In order to induce hepatic, and then BE cell differentiation, growth factors such as TGF, FGF, HGF and EGF were added to the culture medium supplied to embryonic bodies (EBs) that were derived from ES cells. The marker genes and corresponding proteins of hepatocytes and BE cells such as AFP, ALB, CK8, CK18, CK7, CK19 and GGT, etc. were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), immunocytochemistry (ICC) and enzymatic histochemistry. Lastly, the ratio of BE-like cells to all EBs cells was analyzed and determined by flow cytometry (FCM). Hepatocyte and BE cell marker genes and proteins were found to be expressed in the cytoplasm of differentiated cells. On day 10 of differentiation, many round structures appeared in the EBs culture system and the marker proteins of BE cells were found to be expressed in these structures. The BE cell differentiation ratio continually increased from its initial value of 1.7% on day 13 to 7.4% on day 21. ES cells were found to be able to differentiate into BE cells when cultured using medium with appropriate cell growth factors. These BE cells may be a novel source of differentiated cell types for liver engineering.

Animals↗
PubMed1995

Effects of beta 1-integrin antisense phosphorothioate-modified oligonucleotide on myoblast behaviour in vitro.

Myoblasts gene-engineered in vitro and then injected in vivo are safe, efficient options for gene therapy. While isolation of satellite cells is routinely achieved, their proliferation potential in vitro remains a limiting factor for cell transplantation under clinical conditions. We have studied the role of reversible inhibition of gene expression by antisense oligonucleotides on the proliferation of the myogenic cells. Addition of antisense oligonucleotides to myoblast cultures has been used to inhibit specifically the expression of the beta 1-integrin subunit gene. Here we show that the effects of multiple pulses of a phosphorothioate oligodeoxinucleotide antisense on the attachment to substrata and on the proliferation of myoblasts are dose-dependent. The addition of antisense to rat myoblasts caused rounding up of the cells and most of the cells became detached after several days in culture. A single pulse did not show any consistent effect, while in the presence of continuously administered antisense, the relative numbers of myoblasts in the treated muscle culture increased. We have no evidence of inhibition of myoblast fusion under these conditions. On the other hand, [3H]-TdR incorporation, total DNA and total number of cells decreased in antisense-treated cultures thus demonstrating an inhibitory effect of the phosphorothioate oligonucleotides on DNA synthesis. These side-effects could be overcome by substituting the phosphorothioate by unmodified oligonucleotides, so decreasing the half-life of the antisense, but also its toxicity. The overall results suggest a potential role of integrin antisense strategy in modulating the potential of myoblasts to proliferate.

Animals↗
PubMed2006

Expression of Vitreoscilla hemoglobin enhances growth and levels of alpha-amylase in Schwanniomyces occidentalis.

A metabolic engineering approach was exploited to improve growth and protein secretion in the non-conventional yeast, Schwanniomyces occidentalis. Vitreoscilla hemoglobin (VHb) gene was expressed in S. occidentalis under the control of the native alpha-amylase (AMY1) promoter. Expression of VHb was confirmed by reverse transcriptase polymerase chain reaction and Western blot hybridization analysis. Effect of VHb on growth and protein secretion was studied in synthetic medium under both limiting and non-limiting dissolved oxygen conditions. Under both conditions, VHb-expressing cells exhibited higher oxygen uptake and higher specific growth rates. Levels of extracellular alpha-amylase were also elevated in the VHb-transformed strain relative to the control strain. In amylase production medium, VHb-expressing cells showed 3-fold elevated levels of alpha-amylase and a 31% increase in the total secreted protein under oxygen-limiting environment. VHb was found to localize in the mitochondria in addition to its cytoplasmic location. Inhibition of respiration by antimycin A resulted in the loss of the growth-enhancing effects of VHb. A 2.5-fold increase in the cytochrome c oxidase (COX) activity was observed in VHb-expressing cells relative to the control. In addition to this, exogenously added VHb in the assay mixture augmented COX activity.

Antifungal Agents↗
PubMed2000

Genetic therapy for pain management.

Two approaches to genetic therapy for the management of chronic pain have recently been investigated in animal models of pain. First, transgene-mediated delivery of antinociceptive molecules to the cerebrospinal fluid has been performed with engineered cell lines transplanted to the subarachnoid space and with recombinant adenoviruses that transduce pia mater cells. Second, the phenotype of nociceptive neurons has been altered by recombinant herpes viruses overexpressing antinociceptive peptides or reducing expression of endogenous nociceptive molecules. Both approaches attenuate or reverse persistent nociceptive states, suggesting use in the development of genetic therapy for pain management in humans.

Genetic Therapy↗
PubMed2026

Current landscape of Cys-OxiPTMs in plants: from hormone signaling to phenotypic control and their potential in sustainable agriculture.

The integration of environmental and developmental cues into coherent physiological responses is fundamental to plant survival. Reactive oxygen, nitrogen, and sulfur species (ROS/RNS/RSS) are now recognized as essential signaling molecules, not merely cytotoxic byproducts. Their specificity is largely achieved through reversible, site-specific cysteine oxidative post-translational modifications (Cys-OxiPTMs), which constitute a dynamic and sophisticated "redox code". This review provides a systematic synthesis of the current landscape of Cys-OxiPTMs in plants, bridging chemistry, hormone biology, agronomy, detection, and engineering. The chemical and enzymatic basis of major Cys-OxiPTMs is detailed, along with a discussion of how their spatiotemporal interplay orchestrates signaling specificity. A critical examination is then presented on how these modifications decode and integrate plant hormone signaling networks to regulate key agronomic traits. Cutting-edge proteomic technologies that have revolutionized the identification of redox-sensitive cysteines are also evaluated. Finally, forward-looking strategies to "write" the redox code are explored. By moving the field from descriptive cataloging to predictive "redox breeding", this review establishes a foundational framework for manipulating Cys-OxiPTMs to develop climate-resilient, high-yielding crops for sustainable agriculture.

Agronomic traits↗
PubMed1999

Pressure versus heat-induced unfolding of ribonuclease A: the case of hydrophobic interactions within a chain-folding initiation site.

To investigate the characteristics of the postulated carboxy terminal chain-folding initiation site in bovine pancreatic ribonuclease A (RNase A) (residues 106-118), important in the early stages of the folding pathway, we have engineered by site-directed mutagenesis a set of 14 predominantly conservative hydrophobic variants of the protein. The stability of each variant has been compared by pressure and temperature-induced unfolding, monitored by fourth derivative UV absorbance spectroscopy. Apparently simple two-state, reversible unfolding transitions are observed, suggesting that the disruption of tertiary structure of each protein at high pressure or temperature is strongly cooperative. Within the limits of the technique, we are unable to detect significant differences between the two processes of denaturation. Both steady-state kinetic parameters for the enzyme reaction and UV CD spectra of each RNase A variant indicate that truncation of hydrophobic side chains in this region has, in general, little or no effect on the native structure and function of the enzyme. Furthermore, the decreases in free energy of unfolding upon pressure and thermal denaturation of all the variants, particularly those modified at residues 106 and 108, suggest that the hydrophobic residues and side chain packing interactions of this region play an important role in maintaining the conformational stability of RNase A. We also demonstrate the potential of Tyr115 replacement by Trp as a non-destabilizing fluorescence probe of conformational changes local to the region.

Animals↗