Identification of structural elements involved in the interaction of simian virus 40 small tumor antigen with protein phosphatase 2A

SV40 small tumor antigen (small-t) was used as a model to identify structural elements involved in the interactions between regulatory proteins and protein phosphatase 2A (PP2A). Using mutant proteins and synthetic peptides, we identified a small domain within small-t that is a major site for interaction with the dimeric form of PP2A. A series of small-t truncation mutants identified a region surrounding the first of two conserved cysteine clusters that was critical for interaction with PP2A. These mutants also identified additional regions of small-t that contribute to high affinity interaction. Deletion of residues 110-119, which encompass the first cysteine cluster, resulted in a protein that failed to bind to PP2A. Synthetic peptides that contained residues 105-122 of small-t blocked binding of small-t to PP2A. These peptides also inhibited the phosphatase activity of PP2A in a manner analogous to full-length small-t. The active small-t peptides adopt a beta-strand structure that was essential for high affinity interaction with the PP2A dimer. Based on circular dichroism measurements, the same cysteine cluster-containing peptides that bind to PP2A also interact with zinc. Interaction with zinc required the conserved cysteines but was not required for interaction with PP2A.     

Altered modulation of the in vitro antibody synthesis by placental factors from the CBA/J x DBA/2 abortion-prone mating combination

BACKGROUND: We determined the accuracy of intraductal ultrasonography (IDUS) in distinguishing between bile duct cancer and benign bile duct disease. METHODS: Patients (n=42) who required bile duct biopsy using percutaneous transhepatic cholangioscopy (PTCS) to evaluate bile duct strictures or filling defects were studied. A thin-caliber ultrasonic probe (2.0 mm diameter and 20 MHz frequency) was inserted into the bile duct, and its images were prospectively reviewed before PTCS. RESULTS: Disruption of the bile duct wall structure, seen on IDUS, was associated with malignancy in 25 of 26 patients. When IDUS demonstrated a lesion with normal bile duct structure, six of nine patients were found to have no malignancy. IDUS demonstrated no intraductal lesion in seven patients, and bile duct biopsy also did not indicate cancer in any of these patients. The accuracy, sensitivity, and specificity of IDUS for diagnosing bile duct cancer were 76%, 89%, and 50%, respectively. When used in tandem with IDUS, the sensitivity of bile cytology (64%) and PTCS (93%) improved to 96% and 100%, respectively. CONCLUSIONS: The accuracy of IDUS for diagnosing bile duct cancer was less than that of PTCS (95%). However, the sensitivity for bile cytology, or bile duct biopsy improved when performed in combination with IDUS.     

Differential interaction of the human cholesteryl ester transfer protein with plasma high density lipoproteins (HDLs) from humans, control mice, and transgenic mice to human HDL apolipoproteins. Lack of lipid transfer inhibitory activity in transgenic mice expressing human apoA-I

Plasma high density lipoproteins (HDLs) from humans, from transgenic mice to human apolipoprotein A-I (HuAITg mice), from transgenic mice to human apolipoprotein A-II (HuAIITg mice), from transgenic mice to human apolipoproteins A-I and A-II (HuAIAIITg mice), and from C57BL/6 control mice were isolated, and their ability to interact with the human cholesteryl ester transfer protein (CETP) was studied. Whereas cholesteryl ester transfer rates were gradually enhanced by the addition of moderate amounts of HDL from the different sources, striking differences appeared when HDL levels kept increasing beyond a maximal transfer value. Indeed, while a plateau value corresponding to maximal CETP activity was maintained when raising the concentration of HuAITg HDL and HuAIAIITg HDL, inhibitions could be observed with the highest levels of human, control mouse, and HuAIITg mouse HDL. The concentration-dependent inhibition of CETP activity could be reproduced by the addition of delipidated HDL apolipoproteins from control mice, but it was abolished by a 1-h preheating treatment at 56 degrees C. In contrast, no significant inhibition of CETP activity was observed with the delipidated protein moiety of HuAITg HDL, and cholesteryl ester transfer rates remained unchanged before and after a 1-h, 56 degrees C preheating step. Finally, the CETP-mediated transfer of radiolabeled cholesteryl esters from human low density lipoprotein to human HDL was significantly higher in the presence of lipoprotein-deficient plasma from HuAITg mice than in the presence of lipoprotein-deficient plasma from control mice. Interestingly, cholesteryl ester transfer rates measured with both control and HuAITg lipoprotein-deficient plasmas became remarkably similar following a 1-h, 56 degrees C preheating treatment. It is concluded that human, control mouse, and HuAIITg mouse HDL contain a heat-labile lipid transfer inhibitory activity that is absent from HDL of HuAITg and HuAIAIITg mice. Alterations in CETP-lipoprotein binding did not account for differential lipid transfer inhibitory activities.     

The T/B cell interaction involved in induction of the mouse IgG2ab suppression is restricted by major histocompatibility complex class I, but not class II molecules

To determine the major histocompatibility complex (MHC) restriction of the T/ B cell interaction involved in a negative regulation of Ig production, we used mouse model of T cell-induced IgG2ab suppression in vivo. Normal or specifically triggered T splenocytes from mice of the Igha haplotype, when neonatally transferred into histocompatible Igha/b heterozygotes, are able to induce a specific and total suppression of the IgG2ab allotype. Nevertheless, only transfer of IgG2ab-primed Igha T splenocytes induces this suppression in Ighb/b homozygous congenic mice in which the whole IgG2a isotype production is inhibited. This suppression is chronically maintained by CD8+ T cells, but can be experimentally reversed. We have established that the suppression induction required a CD4+CD8+ T cell cooperation and operated via the recognition by the involved TCR of C gamma 2ab-derived peptides presented by the target B cells in an MHC haplotype-restricted manner. Here, by using Ighb mice genetically deficient for MHC class I (beta 2-microglobulin%, or beta 2m%) or class II (I-A beta%) molecules, we demonstrate functionally that the suppression induction implicates an MHC class I-, but not class II-restricted interaction. Indeed, the anti-IgG2ab T cells transferred into Ighb H-2b I-A beta% mice carry out the suppression process normally, while in Ighb H-2b beta 2m% recipients, their suppression induction capacity is significantly inhibited. Moreover, the C gamma 2ab 103-118 peptide, identified as the sole C gamma 2ab-derived peptide able to amplify the anti-IgG2ab T cell reactivity in Igha H-2b mice, is also able to stabilize the H-2Db, but not the H-2Kb class I molecules at the surface of RMA-S (TAP2-, H-2b) cells. These results indicate that, despite the CD4+/CD8+ T cell cooperation during the induction phase of suppression only MHC class I molecule expression is required at the surface of IgG2ab+ B cells for suppression establishment.     

Lipoprotein lipase induces catabolism of normal triglyceride-rich lipoproteins via the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor in vitro. A process facilitated by cell-surface proteoglycans

Bovine milk lipoprotein lipase (LPL) induced binding, uptake, and degradation of 125I-labeled normal human triglyceride-rich lipoproteins by cultured mutant fibroblasts lacking LDL receptors. The induction was dose-dependent and occurred whether LPL and 125I-lipoproteins were added to incubation media simultaneously or LPL was allowed to bind to cell surfaces, and unbound LPL was removed by washing prior to the assay. Lipolytic modification of lipoproteins did not appear to be necessary for increased catabolism because the effect of LPL was not prevented by inhibitors of LPL's enzymatic activity, p-nitrophenyl N-dodecylcarbamate or phenylmethylsulfonyl fluoride. However, the effect was abolished by boiling LPL prior to the assay suggesting that major structural features of LPL were required. Also, LPL-induced binding to cells was blocked by an anti-LPL monoclonal antibody but not by antibodies that are known to block apolipoprotein E- or B-100-mediated binding to low density lipoprotein (LDL) receptors. This indicates that LPL itself mediated 125I-lipoprotein binding to cells. Cellular degradation of 125I-lipoproteins was partially or completely blocked by two previously described ligands for the LDL receptor-related protein/alpha 2-macroglobulin receptor (LRP): activated alpha 2-macroglobulin (alpha 2M*), and the 39-kDa receptor-associated protein. These data implicated LRP as mediating LPL-induced lipoprotein degradation and were confirmed by showing that LPL's effects were prevented by an immunoaffinity-isolated polyclonal antibody against LRP. Furthermore, LPL promoted binding of 125I-lipoproteins to highly purified LRP in a solid-phase assay. Heparin or heparinase treatment of cells markedly decreased LPL-induced binding, uptake, and degradation of lipoproteins, but had no effect on catabolism of alpha 2M*. Thus, cell-surface proteoglycans were obligatory participants in the effects of LPL but were not required for LRP-mediated catabolism of alpha 2M*. Taken together, these in vitro findings establish that through interaction with cell-surface proteoglycans, LPL induces catabolism of normal human triglyceride-rich lipoproteins via LRP.     

Reductions in the activation of ERK and JNK are associated with decreased IL-2 production in T cells from elderly humans stimulated by the TCR/CD3 complex and costimulatory signals

T cells from elderly humans often display impaired IL-2 production, but the mechanisms are unknown. Because the activities of extracellular signal-regulated kinases (ERK) and c-Jun NH2-terminal kinases (JNK) are important for IL-2 production, the current study evaluated if aberrancies in the expression and activation of ERK2 or JNK might underlie decreased IL-2 production by human T cells during aging. The present results show that diminished ERK2 and JNK catalytic activities were commonly detected in T cells from elderly humans stimulated with anti-CD3 mAb OKT3 plus PMA. These reductions did not represent temporal shifts in activation or altered expression of ERK2 or JNK. In addition, the reductions of ERK2 activation in stimulated T cells from elderly individuals were accompanied by decreased Raf-1 kinase activation and could be observed without coexisting impairments in JNK activation. Stimulation of ERK2 activation in elderly T cells correlated with IL-2 production and decreased ERK2 activation was consistently associated with reduced IL-2 production. Although the age-related decreases in JNK activation were accompanied by reduced IL-2 production, substantial impairments of JNK activation were observed with diminished ERK2 activation. Moreover, anti-CD3/PMA-stimulated T cells from elderly individuals that displayed normal JNK activation and impaired ERK2 activation continued to demonstrate reduced IL-2 production. These findings show that impairments in the activation of ERK2 and JNK can accompany decreased IL-2 production by T cells from elderly humans and further suggest that aberrancies in TCR/CD3-dependent activation of the Raf-1/MEK/ERK2 cascade may be rate-limiting for the full induction of IL-2.     

Control of G2/M transition in Xenopus by a member of the p21-activated kinase (PAK) family: a link between protein kinase A and PAK signaling pathways?

X-PAKs are involved in negative control of the process of oocyte maturation in Xenopus (). In the present study, we define more precisely the events targetted by the kinase in the inhibition of the G2/M transition. We show that microinjection of recombinant X-PAK1-Cter active kinase into progesterone-treated oocytes prevents c-Mos accumulation and activation of both MAPK and maturation-promoting factor (MPF). In conditions permissive for MAPK activation, MPF activation still fails. We demonstrate that a constitutive truncated version of X-PAK1 (X-PAK1-Cter) does not prevent the association of cyclin B with p34(cdc2) but rather prevents the activation of the inactive complexes present in the oocyte. Proteins participating in the MPF amplification loop, including the Cdc25-activating Polo-like kinase are all blocked. Indeed, using active MPF, the amplification loop is not turned on in the presence of X-PAK1. Our results indicate that X-PAK and protein kinase A targets in the control of oocyte maturation are similar and furthermore that this negative regulation is not restricted to meiosis, because we demonstrate that G2/M progression is also prevented in Xenopus cycling extracts in the presence of active X-PAK1.     

One hundred seventy-fold increase in excretion of an FV fragment-tumor necrosis factor alpha fusion protein (sFV/TNF-alpha) from Escherichia coli caused by the synergistic effects of glycine and triton X-100

To target tumor necrosis factor alpha (TNF-alpha) to tumor cells, recombinant DNA techniques were used to construct and express the fused gene VKLVH-TNF-alpha, which encodes the secreted form of single-chain fusion protein sFV/TNF-alpha in Escherichia coli. sFV/TNF-alpha was secreted into the culture medium and purified by affinity chromatography. The production of the fusion protein in the culture medium under the optimal conditions of 30 degrees C and 37 micromol of isopropyl-beta-D-thiogalactopyranoside (IPTG) per liter was 16- and 5-fold higher than that under the standard conditions of 37 degrees C and 1 mmol of IPTG per liter. Fusion protein excretion into culture medium with 2% glycine, 1% Triton X-100, or both of these two chemicals was either 14-, 38-, or 170-fold higher, respectively than that without the two chemicals. The final yield of sFV/TNF-alpha was estimated to be 50 mg/liter. The loss of integrity of the cellular membrane may be a potential mechanism for enhancement of fusion protein production and excretion by treatment with glycine and Triton X-100. This study thus provides a practical, large-scale method for more efficient production of the heterologous fusion protein sFV/TNF-alpha in E. coli by using glycine and Triton X-100.     

The Ala/Val98 polymorphism of the hepatocyte nuclear factor-1alpha gene contributes to the interindividual variation in serum C-peptide response during an oral glucose tolerance test: evidence from studies of 231 glucose-tolerant first degree relatives of type 2 diabetic probands

The third form of maturity-onset diabetes of the young is caused by mutations in the hepatocyte nuclear factor-1alpha gene. Recently, we demonstrated an association between a prevalent polymorphism at codon 98, Ala/Val98, of this gene and a 20% decreased insulin release during an oral glucose tolerance test (OGTT) in middle-aged glucose-tolerant Danish Caucasian subjects. The major objective of the present study was to replicate this finding among glucose-tolerant first degree relatives of type 2 diabetic patients of the same ethnic origin. All participants, 231 glucose-tolerant offspring of 62 type 2 diabetic probands, underwent an OGTT with measurements of plasma glucose, serum insulin, and serum C peptide during the test. Thirty-three heterozygous carriers of the Ala/Val variant were identified, whereas no subjects had the variant in its homozygous form. Ala/Val carriers had a 20% reduction in serum C peptide at 30 min during the OGTT (1225+/-636 vs. 1507+/-624 pmol/L; P=0.02) compared to wild-type carriers. No significant differences in serum insulin levels during the OGTT were observed between carriers of the variant and Ala/Ala homozygotes. In conclusion, among Danish glucose-tolerant first degree relatives of type 2 diabetic patients the Ala/Val98 polymorphism of the hepatocyte nuclear factor-1alpha gene is associated with a decreased serum C-peptide secretion during an OGTT. This finding confirms our previously reported observation of the functional importance of the variant to insulin secretion during an OGTT among middle-aged healthy subjects.