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The preparation of Agrobacterium tumefaciens cultures with strains encoding proteins intended for therapeutic or industrial purposes is an important activity prior to treatment of plants for transient expression of valuable protein products. The rising demand for biologic products such as these underscores the expansion of molecular pharming and warrants the need to produce transformed plants at an industrial scale. This requires large quantities of A. tumefaciens culture, which is challenging using traditional growth methods (e.g., shake flask). To overcome this limitation, we investigate the use of bioreactors as an alternative to shake flasks to meet production demands. Here, we observe differences in bacterial growth among the tested parameters and define conditions for consistent bacterial culturing between shake flask and bioreactor. Quantitative proteomic profiling of cultures from each growth condition defines unique growth-specific responses in bacterial protein abundance and highlights the functional roles of these proteins, which may influence bacterial processes important for effective agroinfiltration and transformation. Overall, our study establishes and optimizes comparable growth conditions for shake flask versus bioreactors and provides novel insights into fundamental biological processes of A. tumefaciens influenced by such growth conditions.

The activity of broadly neutralizing antibodies (bNAbs) targeting HIV-1 depends on pleiotropic functions, including viral neutralization and the elimination of HIV-1-infected cells. Several in vivo studies have suggested that passive administration of bNAbs represents a valuable strategy for the prevention or treatment of HIV-1. In addition, different strategies are currently being tested to scale up the production of bNAbs to obtain the large quantities of antibodies required for clinical trials. Production of antibodies in plants permits low-cost and large-scale production of valuable therapeutics; furthermore, pertinent to this work, it also includes an advanced glycoengineering platform. In this study, we used Nicotiana benthamiana to produce different Fc-glycovariants of a potent bNAb, PGT121, with near-homogeneous profiles and evaluated their antiviral activities. Structural analyses identified a close similarity in overall structure and glycosylation patterns of Fc regions for these plant-derived Abs and mammalian cell-derived Abs. When tested for Fc-effector activities, afucosylated PGT121 showed significantly enhanced FcγRIIIa interaction and antibody dependent cellular cytotoxicity (ADCC) against primary HIV-1-infected cells, both in vitro and ex vivo. However, the overall galactosylation profiles of plant PGT121 did not affect ADCC activities against infected primary CD4(+) T cells. Our results suggest that the abrogation of the Fc N-linked glycan fucosylation of PGT121 is a worthwhile strategy to boost its Fc-effector functionality. IMPORTANCE PGT121 is a highly potent bNAb and its antiviral activities for HIV-1 prevention and therapy are currently being evaluated in clinical trials. The importance of its Fc-effector functions in clearing HIV-1-infected cells is also under investigation. Our results highlight enhanced Fc-effector activities of afucosylated PGT121 MAbs that could be important in a therapeutic context to accelerate infected cell clearance and slow disease progression. Future studies to evaluate the potential of plant-produced afucosylated PGT121 in controlling HIV-1 replication in vivo are warranted.

In Hong Kong, many primary teachers of English have to prepare students for examinations which are mainly written assessment. Both teachers and students focus their attention on exam-based literacy such as reading and writing skills. Consequently, oral skills are overlooked. Indeed, public examinations sideline speaking assessment because of its subjective nature; thus English teachers do not provide students with many conversational activities. Without sufficient speaking practice, many students tend to learn English by rote. Conversely, English syllabuses always suggest that students should be given more opportunities to practice speaking. It seems there are inconsistencies between syllabuses and public assessment, but the latter certainly impacts upon classroom practices. In this paper, it is suggested that an Oral Recording System should be used for speaking assessment in public examinations; this system could reduce biases and subjectivity because the recorded data could serve as evidence to justify ...

When Hong Kong was returned to China in 1997, the Hong Kong Special Administrative Region Government implemented mother-tongue education in secondary schools. Only a quarter of the secondary schools were allowed to use English as a medium of instruction (EMI). As a result, more and more parents wanted to send their children to EMI schools. This prompted the local government to ‘fine-tune’ its language policy. The new policy will give schools more flexibility to choose their medium of instruction in September, 2010. However, the root problem still remains: a substantial number of secondary students still have little opportunity to use English in their daily lives. This paper will propose that English teachers should be encouraged to speak English to students outside the classroom in school as a first step. Furthermore, Hong Kong English should be promoted in order to encourage students to speak up. Lastly, the weight given to oral English in public examinations for secondary students should be incr...

The biosynthesis of heparin and heparan sulfate involves a series of polymer-modification reactions that is initiated by N-deacetylation and subsequent N-sulfation of N-acetylglucosamine residues. These reactions are catalysed by a combined N-deacetylase/N-sulfotransferase. Proteins expressing both activities have previously been purified from mouse mastocytoma, which generates heparin, and from rat liver, which produces heparan sulfate. In the present study, the mouse mastocytoma enzyme has been expressed in the human kidney cell line, 293, to investigate whether it could promote modification of the endogenous heparan sulfate precursor polysaccharide into a heparan-like molecule. The N-deacetylase activity of the stably transfected cell clones as approximately 8-fold higher, on a cell-protein basis, than that of control cells, while the N-sulfotransferase activity was increased approximately 2.5 fold. The amounts of glycosaminoglycans synthesized were the same in control and transfected cells, measured as incorporation of [3H]-glucosamine, whereas 35S-labeled glycosaminoglycans were approximately 50% increased in transfected cells, with an increased relative content of heparin sulfate. Structural analysis demonstrated the the glucosamine units of the "heparan sulfate" from transfected cells were almost exclusively N-sulfated, as expected for heparin, whereas more than half of the glucosamine units of the control polysaccharide remained N-acetylated. Notably, the increased N-sulfation was not accompanied by increased O-sulfation, not by C-5 epimerization of D-glucuronic to L-iduronic acid units. The implications of these findings are discussed with regard to the regulation of the biosynthetic process.

To study the structural requirements for factor IXa binding to platelets, we have carried out equilibrium binding studies with human factor IXa after replacing the second epidermal growth factor (EGF) domain by the corresponding polypeptide region of factor X. The chimeric protein, factor IX(Xegf2), and the wild-type, factor IXwt, produced in embryonic kidney cells 293 were radiolabelled with 125I and activated with factor XIa. Direct binding studies with thrombin-activated platelets showed normal stoichiometry and affinity of binding of factor IXawt in the presence of factor VIIIa (2 units/ml) and factor X (1.5 microM). However, under similar experimental conditions, factor IXa(Xegf2) was bound to a smaller number of sites (396 sites/platelet) with decreased affinity, i.e. a dissociation constant (Kd) of 1.4 nM, compared with normal factor IXa, factor IXaN (558 sites/platelet; Kd 0.67 nM), or factor IXawt (590 sites/platelet; Kd 0.61 nM). The concentrations of factor IXaN and factor IXawt required for half-maximal rates of factor-X activation were 0.63 nM and 0.7 nM, indicating a close correspondence of the Kd, app. for binding of factor IXawt to the factor-X activating complex on activated platelets to the Kd obtained in equilibrium binding studies. In contrast, kinetic parameters for factor-X activation by factor IXa(Xegf2) showed a decreased affinity (Kd 1.5 nM), in agreement with results of binding studies. These studies with factor IX(Xegf2) suggest that the EGF-2 domain may be important for specific high-affinity factor IXa binding to platelets in the presence of factor VIIIa and factor X.

To identify the amino acids in the Gla domain that mediate factor IXa binding to human platelets, we have used chimeric molecules and point mutations in the Gla domain of recombinant factor IX, based on molecular modeling using the coordinates of the Gla domain of bovine prothrombin, which reveals two surface structures whose sequences differ among factor IX, factor X, and factor VII. Binding to thrombin-activated platelets of factor IXa in the presence of factor VIIIa (2 units/mL) and factor X (1.5 microM) revealed a stoichiometry of approximately 550 sites per platelet with a Kd of approximately 0.65 nM compared with a Kd of approximately 2.5 nM in the absence of factor VIIIa and factor X. In contrast, mutations of factor IX to factor X residues at positions 4 and 5 or at positions 9, 10, and 11 results in decreases in the number of sites and affinity of factor IXa binding in the presence or absence of factor VIIIa and factor X. A chimera consisting of the Gla domain of factor VII with factor IX residues at positions 33, 34, 35, 39, and 40 displayed abnormal factor IXa binding and a decreased Vmax and a normal Km for factor X activation, and the replacement of amino acid residues 3-10 with those of factor IX restored normal binding and factor X activation kinetics to this chimeric protein.(ABSTRACT TRUNCATED AT 250 WORDS)

We have used chimeras and point mutations of recombinant coagulation factor IX to examine factor IX's specific interaction with bovine endothelial cells. Previously (Toomey, J. R., Smith, K. J., Roberts, H. R., and Stafford, D. W. (1992) Biochemistry 31, 1806-1808), we restricted the region of factor IX responsible for binding to endothelial cells to its Gla domain. Molecular modeling of the Gla domain of factor IX using the coordinates of the Gla domain of bovine prothrombin-(1-145) (Soriano-Garcia, M., Padmanabhan, K., deVos, A. M., and Tulinsky, A. (1992) Biochemistry 31, 2554-2566) reveals two major surface determinants whose sequences differ among factors IX, X, and VII. A chimeric protein comprised of the Gla domain of factor VII with the remainder of the molecule of factor IX did not bind to the endothelial cell binding site. We changed residues 33, 34, 35, 39, and 40 to those of factor IX without restoring endothelial cell binding. Replacement of amino acid residues 3-10 with those of factor IX restored normal binding. With the knowledge that specific binding was localized to the first 11 amino acids, point mutations were made at residues predicted to be on the surface in this region of the factor IX molecule. Changing lysine 5 to alanine (K5A) or valine 10 to lysine (V10K) resulted in loss of binding with total retention of in vitro clotting activity. The lysine 5 to arginine (K5R) mutation also was fully active in vitro but displayed 3-fold tighter binding. In addition to defining the sequence of factor IX necessary for binding to endothelial cells, these results suggest that the binding site is not phospholipid but instead is specific, and in all likelihood, protein.

Irreparable degradation of articular cartilage is a characteristic feature of arthritic diseases and much of this degradation occurs as a result of increased levels of matrix metalloproteinases (MMPs) such as collagenase and stromelysin [1-6]. The elevated levels of degradative enzymes are due to increased synthesis by cells in joint tissue such as chondrocytes and synoviocytes, as well as invading cells, such as macrophages and neutrophils [7-9]. Thus, depending on the type of joint disease, several cell types may be involved in promoting cartilage destruction. In osteoarthritic cartilage, there is a marked depletion of matrix around the chondrocyte suggesting that this cell plays a key role in cartilage degradation. In inflammatory arthritis, both resident cells, synoviocytes and chondrocytes, as well as invading inflammatory cells are involved.

The cDNA for human gamma-glutamyl carboxylase, which accomplishes the post-translational modification required for the activity of all of the vitamin K-dependent proteins, was cloned. The enzyme is a 758-residue integral membrane protein and appears to have three transmembrane domains near its amino terminus. The hydrophilic COOH-terminal half of the carboxylase has 19.3 percent identity with soybean seed lipoxygenase. Expression of the cloned cDNA resulted in an increase in carboxylase activity in microsomes of transfected cells compared to mock-transfected cells.

The binding of Ca2+ induces a conformational change in factor IX which can be monitored with conformation specific antibodies. Anti-FIX:Mg(II) antibodies recognize a conformational epitope (FIX') that can be induced by several metal ions such as Ca2+, Mg2+, Mn2+ and Ba2+, while anti-FIX:Ca(II) antibodies recognize a conformational epitope (FIX*) that can be only induced by Ca2+ and Sr2+ ions (Liebman et al., J. Biol. Chem., vol. 262 (1987) pp. 7605-7612). The latter conformation is essential for the function of factor IX. In this study we tried to identify residues in the Gla-domain of factor IX which are involved in binding to anti-factor IX:Mg(II) and anti-factor IX:Ca(II) antibodies. For this we substituted residues in recombinant human factor IX for those of factor X or factor VII. The substitution of residues 1-40 of factor IX by those of factor VII eliminated binding to both types of antibodies. Re-introduction of factor IX specific residues increased the binding to conformation specific anti-factor IX antibodies, but reduced the binding to conformation specific anti-factor VII antibodies, indicating that the structural integrity of the Gla-domain was not seriously affected by the mutations. We provide evidence that residues 33, 39 and 40 of human factor IX are important for binding to anti-factor IX:Mg(II) antibodies, while residues 1-11 are important for binding to anti-factor IX:Ca(II) antibodies.

We investigated the functional role of gamma-carboxyglutamic acid (Gla) residue 21 of human factor IX, using site-directed mutagenesis to change the glutamic acid residue to aspartic acid (FIX21D). FIX21D had reduced activity in an activated partial thromboplastin time (aPTT) assay and was activated by factor XIa more slowly than wild-type factor IX (FIXwt). FIX21D underwent normal, two-stage calcium-dependent intrinsic fluorescence quenching, indicating that a folding event similar to that seen in FIXwt occurred upon the addition of calcium ions. Antibody A-7, which recognizes factor IX-specific residues at positions 33-40, bound FIX21D as well as FIXwt; however, the calcium-specific monoclonal antibody, JK-IX-2, whose epitope includes residues 1 and 22, did not recognize FIX21D. FIX21D bound phosphatidylserine/phosphatidylcholine (PS/PC) vesicles with Kd approximately 10-fold greater than FIXwt, as measured by a fluorescence light scattering assay. Finally, although FIXwt binds endothelial cells with a Kd of 2.8 nM, FIX21D did not bind endothelial cells. Molecular modeling simulations of FIXwt and FIX21D indicate that mutating Gla 21 to Asp causes structural changes in residues 3-5 and 8-10, as well as in two exposed calcium ions, consistent with the reduced function of FIX21D. Immunological and intrinsic fluorescence quenching assays and the molecular dynamics simulations suggest normal folding in the C-terminal region of the Gla domain. Thus we hypothesize the FIX21D has reduced JK-IX-2 and phospholipid and endothelial cell binding due to localized structural changes in residues 3-10 and the exposed calcium ions. Our study suggests that the Gla 21 to Asp mutation disrupts function in the N-terminal region of the Gla domain without affecting structure in the C-terminal Gla domain region.

We have used site-directed mutagenesis to define the epitope of calcium-dependent monoclonal antibodies to human factor IX. We demonstrate that the calcium-specific epitope includes residues 1–11 of factor IX, with apparent contributions from other regions of the protein. Antibodies JK.IX-1, -3, and -4 had critical portions of their epitopes in the first elevent amino acids of the Gla domain. These three antibodies could bind to a chimera containing the factor VII Gla domain, in which surface residues 3,4,5,9,10, and 11 were modified to those of factor IX. In contrast, the epitope of JK.IX-2 was unaffected by mutations in residues 3–11 of factor IX, but was dependent on the amino terminal tyrosine residue. Furthermore, the calcium-dependent monoclonal antibodies, JK.IX-1, -3, and -4, whose epitope include residues 3 through 11, inhibit factor IX's binding to endothelial cells, for which the binding site on factor IX has been localized to this region. Our results, together with previous studies, confirm the existence of discrete calcium and metal-dependent epitopes within the Gla domain of factor IX and show that the calcium-specific epitope lies near the amino terminus region of this domain.

We previously demonstrated that the primary region of factor IX and IXa responsible for saturable specific binding to bovine aortic endothelial cells resides in residues 3-11 at the amino terminus of factor IX. We also demonstrated that mutations of lysine to alanine at residue 5, factor IX K5A, or valine to lysine at residue 10, factor IX V10K, resulted in a molecule unable to bind to endothelial cells. Moreover, a mutation with lysine to arginine at residue 5, factor IX K5R, resulted in a factor IX molecule with increased affinity for the endothelial cell binding site. In this paper we report that collagen IV is a strong candidate for the factor IX binding site on endothelial cells. Factor IX and factor IX K5R compete with 125I-labeled factor IX for binding to tetrameric collagen IV immobilized on microtiter plates, while factor X, factor VII, and factor IX K5A or V10K fail to compete. The Kd for wild-type factor IX binding to collagen IV in the presence of heparin was 6.8 +/- 2 nM, and the Kd for factor IX K5R was 1.1 +/- 0.2 nM, which agrees well with our previously published Kd values of 7.4 and 2.4 nM for binding of the same proteins to endothelial cells. Our working assumption is that we have identified the endothelial cell binding site and that it is collagen IV. Its physiological relevance remains to be determined.

Metal binding sites within the Gla domain of vitamin K-dependent coagulation factors have been divided into nonspecific metal sites and calcium-specific sites. We demonstrate here that five residues within the Gla domain of factor IX are responsible for the reactivity with the metal-dependent factor IX monoclonal antibody, A-7. First we demonstrate that modifying any one of three residues within this site in factor IX abolishes the binding of A-7. To confirm the specificity of the antibody, the Gla domain of factor VII was changed at residues 32, 33, 34, 38 and 39 to the homologous residues of human factor IX. These changes were sufficient to generate a factor VII Gla domain with an A-7 binding site of the same affinity as that in factor IX. The site identified is one of the two major surfaces of the Gla domain and may represent the metal-dependent binding site.

The epitope of a calcium-dependent murine monoclonal antibody specific for human factor VII (Thim et.al., Biochemistry (1988) 27, 7785-7793) has been determined. Site-directed mutagenesis of residues 3 through 10 of factor VII eliminates the binding of this antibody but does not disturb the binding of a second antibody which binds nearby.

Human papillomavirus (HPV) is frequently associated with cervical carcinoma and derived cell lines. In primary tissues of the carcinoma, the viral genome may be present in episomal or integrated configuration. In cell lines, however, only integrated HPV sequences have been reported. In this article, we describe the presence of episomal type 16 HPV (HPV16), demonstrated by electron microscopy and two-dimensional agarose gel electrophoresis, in a cervical carcinoma cell line, CC7T/VGH, established in 1980 in Taiwan. In CC7T/VGH, the HPV16 sequences are transcriptionally active, and at least three major HPV16 RNA species were detected in Northern blots. Results from restriction enzyme and S1 nuclease analysis suggest a composition of oligomeric HPV16 molecules in dimeric repeats. In addition, the HPV16 oligomers exist as catenated molecules of interlocking rings instead of concatemers. A monomeric copy of the HPV16 episome was cloned from a Hirt supernatant of CC7T/VGH by using a plasmid vector. Mapping and partial sequencing studies revealed an internal deletion of 163 base pairs within the L1 open reading frame. However, insertion of an A.C nucleotide pair at the deletion junction restored the otherwise frame-shifted L1 open reading frame. Two base transitions were also found within the E7 and the E1 open reading frames. Our findings suggest the need for closer examination for HPV episomal catenation in other cervical carcinoma cell lines as well as in primary carcinoma tissues of the uterine cervix and the anogenital tract. With CC7T/VGH, a way is now available for studies of many important aspects of the biology of HPV such as replication and gene expression of the extrachromosomal viral genome.

Integrated human papillomavirus type 16 (HPV16) sequences were cloned from a cervical carcinoma and analyzed by restriction mapping and nucleotide sequencing. The viral integration sites were mapped within the E1 and E2 open reading frames (ORFs). The E4 and E5 ORFs were entirely deleted. An internal deletion of 376 base pairs (bp) was found disrupting the L1 and L2 ORFs. Sequencing analysis showed that an AGATGT/ACATCT inverted repeat marked the deletion junction with two flanking direct repeats 14 and 8 bp in length. A 1,330-bp sequence duplication containing the long control region (LCR) and the E6 and E7 ORFs was also found. The duplication junction was formed by two 24-bp direct repeats with 79% (19 of 24) homology located within the LCR and the E2 ORF of the prototype viral genome, respectively. This observation leads us to propose that the initial viral integration involved an HPV16 dimer in which the direct repeats in tandem units recombined, resulting in reiteration of only a portion of the original duplication. A guanosine insertion between nucleotides 1137 and 1138 created a continuous E1 ORF which was previously shown to be disrupted. Results from this study indicate that sequence reiteration and internal deletion in the integrated, and possibly in the episomal, HPV16 genome are influenced by specific nucleotide sequences in the viral genome. Moreover, reiteration of the LCR/E6/E7 sequences further supports the hypothesis that the E6/E7 ORFs may code for oncogenic proteins and that regulatory signals in the LCR may play a role in cellular transformation.

Tripeptidyl-peptidase II (TPP II) is an exopeptidase with a remarkably high native Mr (> 106). Recently, an alternatively spliced, murine cDNA variant was identified which contains an additional 39 bp, encoding 13 aniino acids in the C- terminal end of the protein. The two enzyme variants were expressed in human kidney 293 cells. Both types of subunit were found to form the active oligomers. In addition, subunits containing the extra 13 amino acids formed an even larger complex eluting in the void volume of a Sepharose CL-4B column. Thus, it appears that this sequence is important for aggregation of subunits. © 1997 Federation of European Biochemical Societies.

We used recombinant DNA techniques to map a panel of six monoclonal antibodies (MoAbs) to regions of the human factor IX molecule. A-2 maps to 17 amino acids at the amino terminus of the heavy chain of IXa; 2D5, an inhibitor of clotting, is defined to 36 amino acids of the first EGF- like domain of human factor IX. A-4, A-5, C10D, and FXC008 all map to a region of the heavy chain containing amino acids 180 through 310, suggesting an immunodominant site. FXC008 has been reported to interfere with binding of factor IXa to factor VIII:Ca.

To determine the function and specificity in factor IX of the first epidermal growth factor (EGF)-like domain and the eight-amino acid hydrophobic stack encoded by exon C (residues 39-46), these domains were replaced by the corresponding polypeptide regions of factor X and chimeric proteins were produced in human embryo kidney cells. Both chimeras were activated by factor XIa at a rate similar to plasma factor IX and exhibited calcium-dependent fluorescence quenching similar to plasma factor IX. Both chimeras competed equally for binding to the endothelial cell receptor. Our findings make it unlikely that the first EGF-like domain or the hydrophobic stack of factor IX are responsible for the specific binding of factor IX to its endothelial cell receptor.