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V Lang

Publications and source records attributed to V Lang.

At least 37 records · Page 2Linked to original sources
PubMed1997

Formation of dimeric Fabs in Escherichia coli: effect of hinge size and isotype, presence of interchain disulphide bond, Fab' expression levels, tail piece sequences and growth conditions.

We have made hinge variants of two human Fab's in order to investigate the factors involved in the formation of dimeric Fab's in the periplasm of E. coli. Hinges containing one or more copies of the IgG1 hinge with various numbers of spacing residues were tested. Fab's with hinges based on the gamma2, gamma3 and gamma4 isotypes were also tested. We find that the IgG1 hinge sequence can form approximately 35% F(ab')2 in vivo in shake flask experiments, but that only (approximately) 5% F(ab')2 can be produced during fermentation. IgM and IgA tail-pieces added to Fab's did not effect their multimerisation. The possible role of growth conditions upon F(ab')2 formation in vivo is discussed.

Amino Acid Sequence↗
PubMed1997

In vivo association of CD5 with tyrosine-phosphorylated ZAP-70 and p21 phospho-zeta molecules in human CD3+ thymocytes.

CD5 is a 67-kDa T cell surface Ag that can be found physically associated with the CD3-TCR molecular complex. In different experimental models it has been shown to act as a costimulatory receptor for T cell activation. Unexpectedly, studies in CD5-deficient mice suggested a negative role for the CD5 Ag in CD3-TCR signaling in the thymus. In this report we assessed the constitutive interactions of CD5 in freshly isolated human thymocytes with signaling elements of the CD3-TCR complex. We determined that the ZAP-70 protein tyrosine kinase was present in CD5 immunoprecipitates. The two molecules were constitutively tyrosine phosphorylated in a complex also associating with unphosphorylated as well as phosphorylated zeta-chains. Although both p21 and p23 tyrosine-phosphorylated forms of zeta as well as phospho-CD3 epsilon molecules were constitutively present in human thymocytes and could be immunoprecipitated with ZAP-70- or CD3 epsilon-specific Abs, the p21 species of zeta was predominant in CD5 immune complexes. The interaction between CD5 and ZAP-70 was not observed in CD3-negative thymocytes, where the constitutive tyrosine phosphorylation of ZAP-70 was very low. We conclude that CD5 may affect in vivo the signaling capacity of TCRs expressed by human thymocytes by altering the phosphorylation state of CD3 and/or by retaining ZAP-70 with the p21 species of zeta.

Biotinylation↗
PubMed1997

The human semaphorin-like leukocyte cell surface molecule CD100 associates with a serine kinase activity.

CD100 is a 150-kDa homodimeric glycoprotein broadly expressed on the surface of human hematopoietic cells. CD100 has been recently identified as the first lymphoid gene that belongs to the semaphorin gene family. Semaphorins function as chemorepellent molecules in the nervous system, but the function of CD100 remains poorly understood. In lymphoid cells, it has been suggested to play a role in homotypic cell adhesion and in T cell activation. We demonstrate that in T cells and natural killer cells a serine kinase activity is immunoprecipitated with CD100. Distinct epitopes of CD100 have been defined with specific monoclonal antibodies, mediating opposite effects at the functional level, especially in T cells. The kinase activity is retained only with an antibody against a particular epitope of CD100. Additionally, a fusion protein containing the cytoplasmic domain of the molecule retains the kinase activity in cellular lysates, and CD100 itself is presumably a favorite substrate of the kinase. These findings suggest that a serine kinase pathway may participate in the different functional effects triggered through the distinct epitopes of CD100 and is likely involved in the biological effects of this semaphorin-like leukocyte cell surface molecule.

Amino Acid Sequence↗
PubMed1997

Identification of native complexes containing the yeast coactivator/repressor proteins NGG1/ADA3 and ADA2.

NGG1p/ADA3p and ADA2p are dual function regulators that stimulate or inhibit a set of yeast transcriptional activator proteins. In vitro, NGG1p and ADA2p associate in a complex that also contains GCN5p (Horiuchi, J., Silverman, N., Marcus, G. A., and Guarente, L. (1995) Mol. Cell. Biol. 15, 1203-1209). We have found that NGG1p and ADA2p are coimmunoprecipitated from yeast whole cell extracts. In fact, <2% of cellular ADA2p was not associated with NGG1p. Also in agreement with their association in vivo, the stability of ADA2p and NGG1p depended on the presence of each other. In addition, three NGG1p- and ADA2p-containing peak fractions were resolved by Q-Sepharose Fast Flow ion-exchange chromatography of whole cell extract. The presence of another high molecular mass complex was supported by the separation of one of the NGG1p- and ADA2p-containing peak fractions by gel-filtration chromatography. Together, the combination of ion-exchange and gel-filtration chromatography suggests a total of four complexes, two with sizes of >2 MDa and single complexes of approximately 900 and 200 kDa. At least one of these complexes was found to associate with the TATA-binding protein (TBP) since TBP was present in immunoprecipitates with NGG1p. The association of TBP with the ADA proteins required amino acids 274-307 of NGG1p, a region of NGG1p required for activity. This supports a role for NGG1p in the interaction with TBP and suggests that the interaction with TBP is functionally relevant.

Blotting, Western↗
PubMed1997

A dual participation of ZAP-70 and scr protein tyrosine kinases is required for TCR-induced tyrosine phosphorylation of Sam68 in Jurkat T cells.

Sam68 has been initially described as a substrate of src kinases during mitosis in fibroblasts. Recent evidence suggests that in T lymphocytes Sam68 may act as an adaptor protein and participate in the early biochemical cascade triggered after CD3 stimulation. A direct interaction between Sam68 and the two src kinases involved in T cell activation, p59(fyn) and p56(lck), as well as a partnership of Sam68 with various key downstream signaling molecules, like phospholipase Cgamma-1 and Grb2, has been shown. In this study we analyze the contribution of p56(lck), as well as the role of ZAP-70, the second class of protein tyrosine kinase involved in T cell activation, in Sam68 tyrosine phosphorylation in the human Jurkat T cell line. Using the src inhibitor PP1 [4-amino-5-(4-methylphenyl)7-(t-butyl) pyrazolo [3,4-d] pyrymidine] and cell variants with defective expression of p56(lck) or expressing a dominant negative form of ZAP-70, we demonstrate that, while both p56(lck) and ZAP-70 are dispensable for the low constitutive phosphorylation of Sam68 observed in Jurkat cells, a cooperation between the two kinases is required to increase its rapid phosphorylation observed in vivo after CD3 stimulation. We also show that recombinant forms of both p56(lck) and ZAP-70 phosphorylate Sam68 in vitro. However, using CD2 stimulated cells, we observe that p56(lck) activation by itself does not induce Sam68 tyrosine phosphorylation. We conclude that p59(fyn) and p56(lck) differently participate in regulating the phosphorylation state of Sam68 in T cells and that ZAP-70 may contribute to Sam68 tyrosine phosphorylation and to the specific recruitment of this molecule after CD3 stimulation.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Molecular cloning and expression of a human phosphodiesterase 4C.

A cDNA coding for a human phosphodiesterase 4C (PDE4C2) was isolated from the mRNA prepared from the glioblastoma cell line, U87. The cDNA contained an ORF of 1818 bp corresponding to a 605 amino acid polypeptide. The sequence differed at the 5' end from the human PDE4C previously reported (Engels, P. et al, 1995 FEBs Letters 358, 305-310) indicating that it represents a novel splice variant of the human PDE4C gene. Evidence was also obtained for a third 5' splice variant. The PDE4C2 cDNA was transfected into both COS 1 cells and yeast cells, and shown to direct the expression of an 80 kD polypeptide by Western blotting using a PDE4C specific antiserum. The activity of cell lysates was typical of PDE4 being specific for cAMP and inhibitable by the selective inhibitor, rolipram. However, the Km for cAMP of the enzyme produced in COS cells was 0.6 microM compared to 2.6 microM for the yeast 4C activity. In addition the COS cell PDE4 activity was much more sensitive to R rolipram than the yeast PDE4 enzyme (IC50 of 23 nM compared to 1648 nM). This difference in rolipram sensitivity was associated with the detection of a high affinity [3H] R rolipram binding site on the COS cell 4C enzyme but not on the yeast expressed enzyme. The results indicate that the enzyme can adopt more than one active conformation, which are distinguished by their interaction with rolipram.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

Normal Syk protein level but abnormal tyrosine phosphorylation in B-CLL cells.

One characteristic of B cells that accumulate during chronic lymphocytic leukemia (CLL) is their highly heterogeneous functional responses to B cell receptor (BCR) stimulation. Leukemic B cells with very poor responses have defective rapid tyrosine phosphorylation of numerous substrates, especially phospholipase C (PLC)gamma, as well as a defective calcium elevation on BCR stimulation. This points to a defect in BCR-associated protein tyrosine kinase (PTK). We investigated whether a defect in Syk, a PTK that is pivotal in coupling BCR to downstream signaling events, could account for these alterations. Syk tyrosine phosphorylation triggered by BCR ligation was severely impaired in B-CLL cells with low calcium responses to anti-mu stimulation. Syk associations were also defective, as concomitant tyrosine phosphorylation of a Syk-associated 145 kDa protein comigrating with PLCgamma-2 was only detected in responding B-CLL cells. By contrast, we found similar expression of the kinase regardless of B-CLL cell responsiveness. These results are consistent with the possibility that very proximal BCR signaling elements in some B-CLL cells are unable to connect with downstream biochemical events dominated by tyrosine phosphorylation and the potential docking function of Syk PTK.

Calcium↗
PubMed1996

Tyrosine phosphorylation and recruitment of ZAP-70 to the CD3-TCR complex are defective after CD2 stimulation.

CD2 has been described as an alternative transduction pathway sharing the same biochemical cascade as CD3. The T cell-specific protein tyrosine kinase (PTK) ZAP-70 is believed to play a key role in early tyrosine phosphorylations of cellular proteins induced by CD3 stimulation. We show in the present report that ZAP-70 is insignificantly tyrosine phosphorylated and recruited to CD3 after CD2 stimulation in Jurkat T cells. The same result is obtained for p72syk, a PTK structurally and functionally related to ZAP-70. Several studies have suggested a model in which p56lck would be responsible after CD3 triggering for the phosphorylation of particular tyrosine residues in the immunoreceptor tyrosine-based activation motifs of the CD3 chains, inducing the recruitment and the tyrosine phosphorylation of ZAP-70. In Jurkat cells, p56lck is required for CD3- or CD2-induced tyrosine phosphorylations and clearly activated after CD2 cross-linking. However, we find that the CD3 complex, and especially its zeta-chain, are faintly tyrosine phosphorylated after CD2 triggering. By contrast, CD2 induces PLCgamma-1 tyrosine phosphorylation as efficiently as CD3, with a correlated inositol phosphate production and intracellular calcium increase, and even a higher production of IL-2. Interestingly, the SH2 domains of PLCgamma-1 associate with ZAP-70 upon CD3 stimulation while they bind, in CD2-activated cells, to a heavily tyrosine-phosphorylated 62-kDa protein. Altogether, these findings suggest that CD2 could bypass the PTK ZAP-70 for PLCgamma-1 activation and involves a preferential cascade comprising p56lck and a p62 protein, possibly acting as an anchor molecule.

CD2 Antigens↗
PubMed1995

The highly acidic C-terminal region of the yeast initiation factor subunit 2 alpha (eIF-2 alpha) contains casein kinase phosphorylation sites and is essential for maintaining normal regulation of GCN4.

Regulation of the effective activity of eukaryotic initiation factor 2 (eIF-2) in protein synthesis is known to involve phosphorylation of its alpha subunit. Two mammalian enzymes, the haem-controlled repressor (HCR) and the double-stranded RNA-activated inhibitor (dsI), phosphorylate Ser-51 of the alpha subunit, thereby inhibiting the exchange of bound nucleotides on, and thus the recycling of, eIF-2. In Saccharomyces cerevisiae, the equivalent serine seems to be phosphorylated by the GCN2 protein kinase, which is activated by amino acid starvation. However, in the present paper we show that this is not the only site of phosphorylation in yeast eIF-2 alpha. We report the preparation of recombinant yeast eIF-2 alpha from Escherichia coli and its use in in vitro phosphorylation studies. Mammalian HCR and dsI are shown to phosphorylate specifically Ser-51 of yeast eIF-2 alpha, whereas extracts from yeast cells do not. Instead, at least one of three serine residue in the acidic C-terminal region of this protein is phosphorylated by fractions of yeast possessing casein kinase activities 1 and 2. A triple Ser-->Ala mutant form of yeast eIF-2 alpha was found to be no longer phosphorylated by either of the yeast (or mammalian) casein kinase activities in vitro. Isoelectric focusing of yeast extracts confirmed that the mutated sites normally act as sites of phosphorylation in vivo. The same mutant was used to show that the three sites have no essential function under normal physiological conditions in yeast. In contrast, deletion of the 13 amino acid long C-terminal region of eIF-2 alpha, including the three phosphorylation sites, led to derepression of GCN4 in vivo. Thus removal of the short, highly acidic C-terminal region of eIF-2 alpha has the same regulatory effect on translational (re)initiation as phosphorylation of the Ser-51 residue of the wild-type protein. This result provides new insight into the role of eIF-2 alpha activity in the regulation of translational (re-) initiation.

Amino Acid Sequence↗
PubMed1994

Initiation factor eIF-4E of Saccharomyces cerevisiae. Distribution within the cell, binding to mRNA, and consequences of its overproduction.

The eukaryotic translational initiation factor 4E (eIF-4E) is an essential protein that binds the 5' cap structure with high specificity and affinity. Yeast eIF-4E is homologous to eIF-4E of higher eukaryotes, but interacts with a different set of cap-binding complex proteins. In the present study the distribution of yeast eIF-4E in Saccharomyces cerevisiae was found to be similar to that observed in higher cells, whereby the yeast factor was more concentrated in the nucleus than in the cytoplasm. Overexpression of yeast eIF-4E in S. cerevisiae exerted at most a minimal effect on growth in liquid minimal medium and was not found to influence the translation of reporter gene mRNAs bearing secondary structure in their leader regions. In a new method to study mRNA-protein interactions, biotinylated mRNAs were synthesized in vitro for use in studies of the binding of eIF-4E in yeast extracts. Streptavidin was used to adsorb the biotinylated mRNAs plus bound initiation factors. Stem-loop structures in the leader region did not influence the binding of eIF-4E or, in comparative experiments, of eIF-4A. Thus yeast eIF-4E shows both similarities and differences with respect to the distribution and function of its counterparts in higher eukaryotes.

Base Sequence↗
PubMed1992

Polychlorinated biphenyls in the environment.

This review surveys the problems arising from the release of PCBs into the environment from the point of view of the analytical chemist. These problems are very complex and interdependent and so it is essential to recognize their mutual links rather than to separate one problem from another (sources of contamination, fate in the environment, toxic properties and particular capabilities, limitations and purposes of analytical methods). Prominent attention should be paid in the future to congener-specific analyses of "toxic" congeners using high-resolution gas chromatography and to toxicity-assessing biological methods.

Animals↗
PubMed1992

Cortical DC potential shifts accompanying auditory and visual short-term memory.

Negative DC potential shifts appeared over the scalp during the performance of verbal and non-verbal short-term memory tasks. Three items were successively presented (presentation of memory items) and then had to be retained in memory for 3 sec (memory retention) before being compared to a probe which was either a member (in set) or not a member (out of set) of the memory set. Verbal items (the digits "1" through "9") were tested in the auditory and visual modality and non-verbal items (musical notes) were tested in the auditory modality. Stimulus modality had a significant effect on DC potential shifts during both presentation of memory items and memory retention. There was a sustained negative shift during these periods which was larger over frontal regions with auditory than with visual material whereas the negative shift was larger over posterior temporal regions with visual than with auditory material. Out of 21 subjects who participated in the study, 9 reported the use of visual images in the auditory task, 5 used subvocal auditory rehearsal in the visual task and 7 used imagery concordant with the stimulus modality being memorized. These different strategies had a significant effect on the amplitudes and distribution of the DC potential shifts. The speed of response affected the amplitude of the DC potential shifts in the frontal regions, being larger with fast RTs than with slow RTs but only when verbal items were being processed. These results indicate that stimulus modality, modality of mental imagery, and speed of scanning of the memory store affect DC potential shifts during a 3 sec period of memory retention.

Acoustic Stimulation↗
PubMed1990

[Comparative study on the biological properties of plastics for temporary crowns and bridges].

Six different plastic materials for temporary crowns and bridges were tested comparatively concerning their biological properties. To this the temperature in the pulp cavity was measured in vitro while making temporary crowns. Also the cytotoxicity was determined by the hemolysis-test. The results obtained showed that there is no thermal irritation of the pulp if the temporary plastic material is applied in an alginate impression as usual in practice. Cytotoxicity of the hardened plastics was not provable. Nevertheless tissue irritation can not be justified when using polymethacrylates and epimine-resin.

Alginates↗