Architecture of a transcribing-translating expressome.

DNA transcription is functionally coupled to messenger RNA (mRNA) translation in bacteria, but how this is achieved remains unclear. Here we show that RNA polymerase (RNAP) and the ribosome of Escherichia coli can form a defined transcribing and translating "expressome" complex. The cryo-electron microscopic structure of the expressome reveals continuous protection of ~30 nucleotides of mRNA extending from the RNAP active center to the ribosome decoding center. The RNAP-ribosome interface includes the RNAP subunit α carboxyl-terminal domain, which is required for RNAP-ribosome interaction in vitro and for pronounced cell growth defects upon translation inhibition in vivo, consistent with its function in transcription-translation coupling. The expressome structure can only form during transcription elongation and explains how translation can prevent transcriptional pausing, backtracking, and termination.

Use of a whole-slide imaging system to assess the presence and alteration of lymphatic vessels in joint sections of arthritic mice.

We investigated the presence and alteration of lymphatic vessels in joints of arthritic mice using a whole-slide imaging system. Joints and long bone sections were cut from paraffin blocks of two mouse models of arthritis: meniscal-ligamentous injury (MLI)-induced osteoarthritis (OA) and TNF transgene (TNF-Tg)-induced rheumatoid arthritis (RA). MLI-OA mice were fed a high fat diet to accelerate OA development. TNF-Tg mice were treated with lymphatic growth factor VEGF-C virus to stimulate lymphangiogenesis. Sections were double immunofluorescence stained with anti-podoplanin and alpha-smooth muscle actin antibodies. The area and number of lymphatic capillaries and mature lymphatic vessels were determined using a whole-slide imaging system and its associated software. Lymphatic vessels in joints were distributed in soft tissues mainly around the joint capsule, ligaments, fat pads and muscles. In long bones, enriched lymphatic vessels were present in the periosteal areas adjacent to the blood vessels. Occasionally, lymphatic vessels were observed in the cortical bone. Increased lymphatic capillaries, but decreased mature lymphatic vessels, were detected in both OA and RA joints. VEGF-C treatment increased lymphatic capillary and mature vessel formation in RA joints. Our findings suggest that the lymphatic system may play an important role in arthritis pathogenesis and treatment.

EGF-dependent cell cycle progression is controlled by density-dependent regulation of Akt activation.

The normal human breast epithelial cell line, MCF10A, was used to investigate the mechanism by which high-density inhibits EGF-dependent cell cycle progression. EGF-dependent Akt activation was found to be transient in high-density cells and sustained in low-density cells. High-density cells also showed decreased EGF receptor (EGFR) autophosphorylation, decreased retinoblastoma protein phosphorylation, and increased p27 protein expression. Although EGFR activation was decreased in the high-density cells, the activation was sufficient to stimulate EGFR substrates comparable to low-density cells. EGF-dependent activation of the Erk1/2 pathway and the upstream activators of Akt (Gab1, erbB3, PI3 kinase, and PDK1) showed no density dependency. Antagonists of Akt activity provided further evidence that regulation of Akt activation is the critical signal transduction step controlling EGF-dependent cell cycle progression. Both adenovirus-mediated expression of dominant-negative Akt and inhibition of PI3 kinase-mediated Akt activation with LY294002 blocked cell cycle progression of low-density cells. In summary, we report the novel finding that high-density blocks EGF-dependent cell cycle progression by inhibiting EGF signaling at the level of EGF-dependent Akt activation rather than at the level of EGFR activation.

Suppressors of cytokine signaling-1 and -6 associate with and inhibit the insulin receptor. A potential mechanism for cytokine-mediated insulin resistance.

Insulin resistance contributes to a number of metabolic disorders, including type II diabetes, hypertension, and atherosclerosis. Cytokines, such as tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6, and hormones, such as growth hormone, are known to cause insulin resistance, but the mechanisms by which they inhibit the cellular response to insulin have not been elucidated. One mechanism by which these agents could cause insulin resistance is by inducing the expression of cellular proteins that inhibit insulin receptor (IR) signaling. Suppressors of cytokine signaling (SOCS) proteins are negative regulators of cytokine signaling pathways, the expression of which is regulated by certain cytokines. SOCS proteins are therefore attractive candidates as mediators of cytokine-induced insulin resistance. We have found that SOCS-1 and SOCS-6 interact with the IR when expressed in human hepatoma cells (HepG2) or in rat hepatoma cells overexpressing the human IR. In SOCS-1-expressing cells, insulin treatment increases the extent of interaction with the IR, whereas in SOCS-6-expressing cells the association with the IR appears to require insulin treatment. SOCS-1 and SOCS-6 do not inhibit insulin-dependent IR autophosphorylation, but both proteins inhibit insulin-dependent activation of ERK1/2 and protein kinase B in vivo and IR-directed phosphorylation of IRS-1 in vitro. These results suggest that SOCS proteins may be inhibitors of IR signaling and could mediate cytokine-induced insulin resistance and contribute to the pathogenesis of type II diabetes.

Direct localization of a beta-subunit domain on the three-dimensional structure of Escherichia coli RNA polymerase.

To identify the location of a domain of the beta-subunit of Escherichia coli RNA polymerase (RNAP) on the three-dimensional structure, we developed a method to tag a nonessential surface of the multisubunit enzyme with a protein density easily detectable by electron microscopy and image processing. Four repeats of the IgG-binding domain of Staphylococcus aureus protein A were inserted at position 998 of the E. coli RNAP beta-subunit. The mutant RNAP supported E. coli growth and showed no apparent functional defects in vitro. The structure of the mutant RNAP was determined by cryoelectron microscopy and image processing of frozen-hydrated helical crystals. Comparison of the mutant RNAP structure with the previously determined wild-type RNAP structure by Fourier difference analysis at 20-A resolution directly revealed the location of the inserted protein domain, thereby locating the region around position 998 of the beta-subunit within the RNAP three-dimensional structure and refining a model for the subunit locations within the enzyme.

PTP LAR expression compared to prognostic indices in metastatic and non-metastatic breast cancer.

Several prognostic indices in breast cancer, including c-erbB2, epithelial growth factor receptors (EGFR), estrogen and progesterone receptors are signal transduction molecules. Recently, expression of another signal transduction molecule, the protein tyrosine phosphatase LAR, has been suggested to be increased in breast cancer. The objective of the current investigation was to examine the relationship between LAR expression and prognostic parameters in breast cancer. LAR expression was associated with metastatic potential in the well-characterized 13762NF rat mammary adenocarcinoma clones. The metastatic MTLn3 and MTLn2 clones expressed sizable amounts of LAR. The essentially non-metastatic MTC clone had little LAR expression. C-erbB2 had highest expression in the highly metastatic MTLn3 clone, but c-erbB2 levels were sizeable in the weakly metastatic MTLn2 and non-metastatic MTC clone. EGFR expression had the strongest association with a clone's metastatic potential, being very high in MTLn3, weak in MTLn2, and undetectable in MTC. In human breast cancer specimens, LAR expression was strongly positive in 50% of metastatic cases but in only 21% of 'non-metastatic' cases. As with the 13762NF-derived clones, c-erbB2 expression was strongly positive independent of metastatic phenotype. However, 46% (6/13) of cases that were strongly positive for c-erbB2 were strongly positive for LAR. Only 17% (2/11) of negative or weakly c-erbB2 positive samples were strongly positive for LAR. All ER+ positive tumors (n = 15) were positive for LAR and 53% of these tumors were strongly positive for LAR. In ER negative cases, only 1 of 11 was strongly positive for LAR. While the current data indicate a strong association between ER and LAR expression in breast cancer tissue (p = 0.003), additional studies are warranted to further explore the relationship between LAR and prognostic indices of breast cancer progression.

Dissociation of PTPase levels from their modulation of insulin receptor signal transduction.

Protein tyrosine phosphatases have been implicated in the regulation of receptor tyrosine kinase signalling pathways, including that of the insulin receptor. Here, cell density-dependent changes in PTPase expression have been exploited to investigate the relationship between cellular PTPase levels and the insulin receptor signal transduction pathway. Increasing cell density (20%, 50%, and >90%) in the rat McA-RH7777 hepatoma cell line resulted in increased protein expression of the receptor-like PTPase LAR (14-fold), and the nonreceptor PTPases PTP1B (11-fold) and SHP2 (10-fold). Each of these PTPases has previously been implicated in regulating insulin receptor signal transduction. Despite these marked increases, maximum insulin receptor autophosphorylation as well as receptor expression actually increased 2-fold. MAP kinase also increased approximately 2-fold as a function of cell density and paralleled increases in expression levels. Neither sensitivity nor maximum responsiveness to insulin were decreased at increasing cell densities as assessed by activation of PI 3-kinase. Duration of response was also unimpaired. These results suggest that expression levels of relevant PTPases are not the primary determinant in their modulation of insulin receptor kinase activity. Restricted accessibility at the molecular level or involvement of accessory proteins may be more critical parameters.

Maternal serum analyte levels in pregnancies with fetal Down syndrome resulting from translocations.

OBJECTIVE: Our purpose was to determine whether pregnancies affected by fetal Down syndrome resulting from Robertsonian translocations are associated with second-trimester maternal serum analyte levels different from those resulting from fetal trisomy 21. STUDY DESIGN: Pregnancies with Down syndrome caused by Robertsonian translocations were identified through the cytogenetics laboratories at the participating institutions. Those with maternal serum screening values between 15 and 20 weeks were evaluated. RESULTS: Eleven cases of fetal Down syndrome caused by Robertsonian translocations were identified. The median alpha-fetoprotein, unconjugated estriol, and human chorionic gonadotropin levels were 0.68, 0.67, and 2.83 multiples of the median, respectively. These analyte levels are similar to those for fetal trisomy 21. CONCLUSIONS: These data suggest that Down syndrome resulting from either Robertsonian translocations or trisomy 21 will be detected in a similar percentage of cases because the second-trimester maternal serum analyte levels are similar.

Suppression of the protein tyrosine phosphatase LAR reduces apolipoprotein B secretion by McA-RH7777 rat hepatoma cells.

Apolipoprotein B (apo B) secretion is reduced by insulin in rat hepatocytes. To evaluate possible mechanisms by which insulin action leads to inhibition of apo B secretion, we evaluated the effect of suppression of the protein-tyrosine phosphatase LAR on apo B secretion by McA-RH7777 (McA) rat hepatoma cells. A reduction in cellular LAR levels was accomplished by stable transfection of McA cells with LAR antisense cDNA. Previous studies indicate that LAR-antisense transfectants demonstrate increased insulin receptor signaling. In current studies, reduced LAR expression results in a 60% to 70% reduction in apo B secretion compared with null vector control. The reduction in apo B secretion correlated with a significant decrease in cellular apo B mRNA levels. Results suggests there is a relationship of protein tyrosine phosphorylation with regulation of apo B mRNA abundance in McA cells.

The protein tyrosine phosphatase LAR has a major impact on insulin receptor dephosphorylation.

Transmembrane protein tyrosine phosphatases (PTPases) may act as regulators of the insulin receptor. Supporting this hypothesis, antisense suppression of the PTPase LAR in McA-RH7777 hepatoma cells increased insulin receptor signaling (Kulas et. al., J. Biol. Chem. (1996) 271, 748-754). The effects of decreased LAR expression may be mediated by decreased dephosphorylation of the insulin receptor. The rate of insulin receptor dephosphorylation was examined in situ, following elution of surface bound insulin at pH 4.0. In LAR antisense cells, dephosphorylation was prolonged by 2.6-fold with a t(1/2) of 87+/-11 sec compared to a t(1/2) of 34+/-6 sec in control cells. EGF receptor dephosphorylation was also prolonged in LAR antisense cells. These results are further evidence that LAR is a physiological regulator of the insulin receptor and is consistent with its direct interaction with the tyrosine phosphorylated insulin receptor.

The transmembrane protein-tyrosine phosphatase LAR modulates signaling by multiple receptor tyrosine kinases.

Antisense-mediated suppression of the transmembrane protein-tyrosine phosphatase (PTPase) LAR has been shown previously to increase insulin-dependent phosphatidylinositol 3-kinase (PI 3-kinase) activation by greater than 300% in the rat hepatoma cell line McA-RH7777. Here, insulin-dependent insulin receptor tyrosine kinase activation was examined with recombinant insulin receptor substrate 1 (IRS-1) as the substrate and shown to be 3-fold greater in cells with suppressed LAR levels. Consistent with a receptor level effect, in vivo insulin-dependent tyrosine phosphorylation of both IRS-1 and Shc was increased by a similar 3-fold with LAR suppression. These increases in IRS-1 and Shc phosphorylation were paralleled by increases in insulin-dependent PI 3-kinase association with IRS-1 and activation of the MAP kinase pathway. Reduced LAR levels also resulted in increases of over 300% and 250% in epidermal growth factor (EGF)- and hepatocyte growth factor (HGF)-dependent receptor autophosphorylation, respectively, as well as a severalfold increase in substrate tyrosine phosphorylation. In a post-receptor response, EGF- and HGF-dependent MAP kinase activation was increased by 300% and 350%, respectively, with LAR suppression. Similarly, growth factor-dependent PI 3-kinase activation was increased in LAR antisense expressing cells when compared to null vector expressing cells. These results demonstrate that the transmembrane PTPase LAR modulates ligand-dependent activation of at least three receptor tyrosine kinases.

The transmembrane protein-tyrosine phosphatase CD45 is associated with decreased insulin receptor signaling.

Overexpression of the transmembrane protein-tyrosine phosphatase (PTPase) CD45 in nonhematopoietic cells results in decreased signaling through growth factor receptor tyrosine kinases. Consistent with these data, insulin receptor signaling is increased when the CD45-related PTPase LAR is reduced by antisense suppression in a rat hepatoma cell line. To test whether the hematopoietic cell-specific PTPase CD45 functions in a manner similar to LAR by negatively modulating insulin receptor signaling in hematopoietic cells, the insulin-responsive human multiple myeloma cell line U266 was isolated into two subpopulations that differed in CD45 expression. In CD45 nonexpressing (CD45-) cells, insulin receptor autophosphorylation was increased by 3-fold after insulin treatment when compared to CD45 expressing (CD45+) cells. This increase in receptor autophosphorylation was associated with similar increases in insulin-dependent tyrosine kinase activation. These receptor level effects were paralleled by postreceptor responses. Insulin-dependent tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) and Shc was 3-fold greater in CD45- cells. In addition, insulin-dependent IRS-1/phosphatidylinositol 3-kinase association and MAP kinase activation in CD45- cells were also 3-fold larger. While expression of CD45 was associated with a decrease in the responsiveness of early insulin receptor signaling, interleukin 6-dependent activation of mitogen-activated protein kinase kinase and mitogen-activated protein kinase was equivalent between CD45- and CD45+ cells. These observations indicate that CD45 can function as a negative modulator of growth factor receptor tyrosine kinases in addition to its well-established role as an activator of src family tyrosine kinases.

Decreased maternal serum hCG levels with increasing gravidity and parity.

OBJECTIVE: To investigate the preliminary observation that primigravid women have higher hCG multiples of the median (MoM) than multigravid women. METHODS: An analysis of the effect of gravidity and parity on maternal serum alpha-fetoprotein (MSAFP) and hCG was performed using data from 20,009 consecutive singleton pregnancies of 15-20 weeks' gestation in a maternal serum screening program. RESULTS: The human chorionic gonadotropin MoM for primigravid women was 0.1 MoM higher than for multigravid women. As parity or gravidity increased, maternal serum hCG decreased. The median hCG MoM for nulliparous women was 1.05, compared with 0.94 MoM for para 3 women. The decrease in hCG was similar at each gestational week from 15-20. In contrast, MSAFP and MSAFP MoM were unaffected by parity. Maternal age and race were potential contributing factors to the effect of parity. However, the decrease in hCG MoM with parity was observed within each 5-year increment of maternal age. Similarly, both black and non-black populations displayed decreases in hCG with parity, although black women had a consistently higher MoM in all matched sets. The decrease in hCG MoM with parity was also observed in 50 Down syndrome cases. Correcting patient data for parity resulted in the hCG MoM changing only 2.7% on average. The detection rate for the 50 Down syndrome cases would not have changed. CONCLUSION: The decrease in maternal serum hCG with increasing parity demonstrates that pregnancy history influences the level of maternal serum hCG. Further studies are needed to define the contributing factors, but the impact of parity on Down syndrome screening appears to be small.

Insulin receptor signaling is augmented by antisense inhibition of the protein tyrosine phosphatase LAR.

Considerable evidence has shown that most physiologic responses to insulin require activation of the intrinsic tyrosine kinase of the insulin receptor. Biochemical studies have also supported the hypothesis that receptor kinase activity can be modulated by cellular protein tyrosine phosphatases (PTPases), which have not yet been identified. To test the hypothesis that the transmembrane PTPase LAR can modulate insulin receptor signaling in vivo, antisense RNA expression was used to specifically suppress LAR protein levels by 63% in the rat hepatoma cell line, McA-RH7777. Hormone-dependent autophosphorylation of the insulin receptor was increased by approximately 150% in the antisense-expressing cells at all insulin concentrations tested. This increase in autophosphorylation was paralleled by a 35% increase in insulin receptor tyrosine kinase activity. Reduced LAR levels did not alter non-hormone-dependent tyrosine phosphorylation nor basal insulin receptor tyrosine phosphorylation and kinase activity. Most significantly, reduced LAR levels resulted in a 350% increase in insulin-dependent phosphatidylinositol 3-kinase activity. These studies provide unique in vivo evidence that LAR is involved in the modulation of insulin receptor signaling in intact cells.

The PI3-kinase serine kinase phosphorylates its p85 subunit and IRS-1 in PI3-kinase/IRS-1 complexes.

Insulin receptor tyrosine kinase activity accounts for tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), but the serine kinase(s) responsible for serine phosphorylation of IRS-1 is(are) unknown. In vitro kinase assays performed on PI3-kinase and IRS-1 immunoprecipitates demonstrated insulin-dependent serine phosphorylation of IRS-1. IRS-1 was associated with both insulin-dependent and independent serine kinases. Only the insulin-dependent serine kinase preferred Mn2+ over Mg2+ and was recovered from cell lysates containing dithiothreitol. In complexes of tyrosine phosphorylated recombinant IRS-1 and PI3-kinase, phosphorylation of IRS-1 was associated with decreased phosphorylation of the p85 subunit of PI3-kinase. These results are consistent with PI3-kinase being responsible for insulin-dependent serine phosphorylation of IRS-1 and suggest that this phosphorylation reaction may affect functions of both IRS-1 and the PI3-kinase.

Differential effects of phosphotyrosine phosphatase expression on hormone-dependent and independent pp60c-src activity.

pp60c-src kinase activity can be increased by phosphotyrosine dephosphorylation or growth factor-dependent phosphorylation reactions. Expression of the transmembrane phosphotyrosine phosphatase (PTPase) CD45 has been shown to inhibit growth factor receptor signal transduction (Mooney, RA, Freund, GG, Way, BA and Bordwell, KL (1992) J Biol Chem 267, 23443-23446). Here it is shown that PTPase expression decreased platelet-derived growth factor (PDGF)-dependent activation of pp60c-src but failed to increase hormone independent (basal) pp60c-src activity. PDGF-dependent tyrosine phosphorylation of its receptor was reduced by approximately 60% in cells expressing the PTPase. In contrast, a change in phosphotyrosine content of pp60c-src was not detected in response to PDGF or in PTPase+ cells. PDGF increased the intrinsic tyrosine kinase activity of pp60c-src in both control and PTPase+ cells, but the effect was smaller in PTPase+ cells. In an in vitro assay, hormone-stimulated pp60c-src autophosphorylation from PTPase+ cells was decreased 64 +/- 22%, and substrate phosphorylation by pp60c-src was reduced 54 +/- 16% compared to controls. Hormone-independent pp60c-src kinase activity was unchanged by expression of the PTPase. pp60c-src was, however, an in vitro substrate for CD45, being dephosphorylated at both the regulatory (Tyr527) and kinase domain (Tyr416) residues. In addition, in vitro dephosphorylation by CD45 increased pp60c-src activity. These findings suggest that the PDGF receptor was an in vivo substrate of CD45 but pp60c-src was not. The lack of activation of pp60c-src in the presence of expressed PTPase may demonstrate the importance of compartmentalization and/or accessory proteins to PTPase-substrate interactions.

Effectiveness of combining maternal serum alpha-fetoprotein and hCG in a second-trimester screening program for Down syndrome.

OBJECTIVE: To evaluate the efficacy of combining hCG and alpha-fetoprotein (AFP) with maternal age in a two-analyte maternal serum screening program for Down syndrome. METHODS: A prospective study involved the screening of 12,170 maternal sera from patients at 14-25 weeks of gestation. The risk for Down syndrome at term was calculated from maternal serum hCG and AFP, and maternal age. For women 36 years of age and younger, a risk of 1:307 or greater was considered screen-positive. For women over 36, a risk greater than that a priori was considered screen-positive. False-positive rates and detection rates were compared with those resulting from a screening protocol using only AFP and age. RESULTS: Seven hundred eighty-two sera were initially screen-positive (6.4%). Subsequent sonography decreased this total to 687 (5.6%), and 467 (3.8%) of these patients accepted amniocentesis. Ten cases of Down syndrome and seven other chromosomal abnormalities were detected. Follow-up investigations revealed eight additional Down syndrome cases that were missed by screening. The identification of 18 Down syndrome cases in 12,170 pregnancies corresponds closely with the prediction of 14.1 Down syndrome births (18.2 second-trimester fetuses) in this population calculated from age-dependent risks. The detection rate for Down syndrome was 56% (ten of 18 expected cases). Only five of 18 (28%) would have been detected by AFP and age alone. CONCLUSION: These results support the mathematical model that hCG is the major contributor to the increased sensitivity of multi-analyte screening and demonstrate that screening programs can attain substantial improvement in detection of second-trimester Down syndrome by adding hCG to AFP and age.

Functional insulin and insulin-like growth factor-1 receptors are preferentially expressed in multiple myeloma cell lines as compared to B-lymphoblastoid cell lines.

While IGF-1 plays a role in early B-cell development, little is known of insulin and insulin-like growth factor-1 (IGF-1) action in post-marrow B-cells. Recently, our laboratory demonstrated that mouse and human multiple myeloma (MM) cell lines possess functional insulin receptors (IRs) and IGF-1 receptors (IGF-1Rs). In this study, we show that responsiveness to insulin and IGF-1 is more developed in human MM cell lines than in human B-lymphoblastoid cell lines. Two human MM cell lines (U266 and RPMI 8226) were compared to three B-lymphoblastoid cell lines [Epstein-Barr virus immortalized B-cells (EBV), a Burkitt lymphoma cell line (Ramos), and a non-EBV lymphoblastoid cell line (HS Sultan)]. Surface IR and IGF-1R expression, measured by flow cytometry, demonstrated that the MM cell lines expressed more IRs and IGF-1Rs than did the EBV, Ramos, or HS Sultan cell lines. In vitro receptor kinase activity of affinity-purified receptors showed that the MM cells had more phosphorylated receptors than did the EBV, Ramos, or HS Sultan cells. Intracellular receptor signaling was also markedly different between the two cell groups. Whole cell phosphorylation studies showed that MM cells possessed not only hormone-dependent receptor autophosphorylation (M(r) 97,000) but also substrate phosphorylation (M(r) 185,000; 60,000). The lymphoblastoid cells, while demonstrating receptor autophosphorylation (IR autophosphorylation in the EBV cell line at 200 nM hormone was similar to MM receptor phosphorylation at 2 nM), lacked hormone-responsive substrates. The MM cell lines contained significantly more hormone-stimulated phosphatidylinositol 3-kinase (PI 3-kinase) activity than the B-lymphoblastoid cell lines. In the MM cells, PI 3-kinase was activated by at least 10-fold, but, in the B-lymphoblastoid cell lines, it was activated by no more than 2-fold. Hormone-responsive glucose metabolism was also greater in the MM cell lines. In the U266 cells, insulin increased lactate production 62 +/- 9 and 101 +/- 12% (mean +/- SE) at concentrations of 2 nM and 200 nM, respectively. IGF-1 produced 72 +/- 9 and 99 +/- 13% increases at similar concentrations. In the 8226 cells, insulin increased lactate production 4 +/- 4 and 36 +/- 15% at 2 and 200 nM, respectively. IGF-1 produced a 13 +/- 6 and 70 +/- 18% increase. In the EBV and Ramos cells, neither hormone increased lactate production by more than 10 +/- 3%.(ABSTRACT TRUNCATED AT 400 WORDS)