Maternal plasma soluble neuropilin-1 is downregulated in fetal growth restriction complicated by abnormal umbilical artery Doppler: a pilot study.

OBJECTIVES: Placental expression of neuropilin-1 (NRP1), a proangiogenic member of the vascular endothelial growth factor receptor family involved in sprouting angiogenesis, was recently discovered to be downregulated in pregnancies with fetal growth restriction (FGR) and abnormal umbilical artery (UA) Doppler. Soluble NRP1 (sNRP1) is an antagonist to NRP1; however, little is known about its role in normal and FGR pregnancies. This study tested the hypotheses that, first, sNRP1 would be detectable in maternal circulation and, second, its concentration would be upregulated in FGR pregnancies compared to those with normal fetal growth and this would correlate with the severity of the disease as assessed by UA Doppler. METHODS: This was a prospective case-control pilot study of 40 singleton pregnancies (20 FGR cases and 20 uncomplicated controls) between 24 + 0 and 40 + 0 weeks' gestation followed in an academic perinatal center from January 2015 to May 2017. FGR was defined as an ultrasound-estimated fetal weight < 10th percentile for gestational age. The control group was matched to the FGR group for maternal age and gestational age at assessment. Fetal ultrasound biometry and UA Doppler were performed using standard protocols. Maternal plasma sNRP1 measurements were performed using a commercially available ELISA. RESULTS: Contrary to the study hypothesis, maternal plasma sNRP1 levels were significantly decreased in FGR pregnancies as compared to those with normal fetal growth (137.4 ± 44.8 pg/mL vs 166.7 ± 36.9 pg/mL; P = 0.03). However, there was no significant difference in sNRP1 concentration between the control group and FGR pregnancies that had normal UA Doppler. Plasma sNRP1 was downregulated in FGR pregnancies with elevated UA systolic/diastolic ratio (P = 0.023) and those with UA absent or reversed end-diastolic flow (P = 0.005) in comparison to FGR pregnancies with normal UA Doppler. This suggests that biometrically small fetuses without hemodynamic compromise are small-for-gestational age rather than FGR. CONCLUSIONS: This study demonstrated a significant decrease in maternal plasma sNRP1 concentration in growth-restricted pregnancies with fetoplacental circulatory compromise. These findings suggest a possible role of sNRP1 in modulating fetal growth and its potential as a biomarker for FGR. © 2021 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Drugs affecting microtubule dynamics increase alpha-tubulin mRNA accumulation via transcription in Tetrahymena thermophila.

In cultured mammalian cells, an increase in the amount of tubulin monomer due to treatment with a microtubule-depolymerizing agent results in a rapid decline in tubulin synthesis. This autoregulatory response is mediated through a posttranscriptional mechanism which decreases the stability of tubulin message with no change in transcriptional activity of tubulin genes. Conversely, treatment with a microtubule-polymerizing drug, such as taxol, results in a slight increase in the synthesis of tubulin. Surprisingly, we find that two microtubule-depolymerizing agents, colchicine and oryzalin, actually cause an increase in alpha-tubulin synthesis and alpha-tubulin message in starved Tetrahymena thermophila. This increase is paralleled by an increase in transcription of alpha-tubulin sequences measured by run-on transcription, while the half-life of tubulin message measured by decay in the presence of actinomycin D does not change appreciably. Treatment of starved cells with taxol also produces an increase in alpha-tubulin synthesis via an increase in message abundance due to an increase in transcription of the alpha-tubulin gene. These results indicate that tubulin synthesis in T. thermophila is regulated very differently than in cultured mammalian cells.

Nicotinamide Phosphoribosyltransferase Attenuates Methotrexate Response in Juvenile Idiopathic Arthritis and In Vitro.

Variability in response to methotrexate (MTX) in the treatment of juvenile idiopathic arthritis (JIA) remains unpredictable and poorly understood. Based on previous studies implicating an interaction between nicotinamide phosphoribosyltransferase (NAMPT) expression and MTX therapy in inflammatory arthritis, we hypothesized that increased NAMPT expression would be associated with reduced therapeutic response to MTX in patients with JIA. A significant association was found between increased plasma concentrations of NAMPT and reduced therapeutic response in patients with JIA treated with MTX. Inhibition of NAMPT in cell culture by either siRNA-based gene silencing or pharmacological inhibition with FK-866 was found to result in a fourfold increase in the pharmacological activity of MTX. Collectively, these findings provide evidence that NAMPT inhibits the pharmacological activity of MTX and may represent a predictive biomarker of response, as well as a therapeutic target, in the treatment of JIA with MTX.

Cytokine-mediated stimulation of laminin expression and cell-growth arrest in a human submandibular gland duct-cell line (HSG).

Increased expression of laminin and various cytokines, including interferon-y (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) has been demonstrated in minor salivary glands from patients with Sjögren's syndrome. Previous reports state that exposure of a human salivary-gland cell line (HSG) to IFN-gamma results in cellular changes similar to those in vivo Sjögren's syndrome. To begin studies of the cause of increased laminin expression in salivary glands in Sjögren's syndrome and laminin's role in the pathological process, the effects of IFN-gamma on laminin expression and growth of HSG cells were examined here. Subconfluent cultures of HSG cells were treated or not with IFN-gamma (1000 units/ml) for 1, 3 or 6 days. Immunoprecipitation showed that the expression of cell-associated laminin was significantly greater in IFN-gamma-treated cells at 3 or 6 days than in untreated cells, while no significant differences in laminin counts precipitated from the media were evident among any of the IFN-gamma-treated or untreated samples. Western blot analysis strongly suggested that this immunoprecipitated product is a dimer of the beta- and gamma-chains of laminin. Intracellular laminin was demonstrated immunocytochemically in a distinct, perinuclear pattern in both cytokine-treated and untreated cells. However, only faint staining for type IV collagen, and no staining for fibronectin were evident in untreated and cytokine-treated cells. An RNase protection assay showed only slight upregulation of the laminin beta-chain mRNA at 3 days, but no significant difference at 6 days of treatment. Taken together, these data suggest enhanced accumulation of a dimer of laminin beta- and gamma-chains in the cytoplasm of cytokine-treated HSG cells. However, mRNA for glyceraldehyde 3-phosphate dehydrogenase was significantly reduced at 6 days of treatment, suggestive of cytokine-mediated metabolic abnormalities. IFN-gamma treatment also resulted in significant reductions in cell numbers over time, in agreement with previous reports. Treatment of HSG cells for 3 days with IFN-gamma (1000 U/ml) and TNF-alpha (20 U/ml) resulted in no significant changes in cell proliferation or laminin protein and/or mRNA species compared to cells treated with IFN-gamma alone. Karyotype analysis of HSG cells revealed human chromosomes with triploid chromosome numbers and rearrangements, characteristic of transformed cells. These data demonstrate that IFN-gamma increases the amount of intracellular laminin beta-gamma dimers while decreasing cell growth. Further studies are required to define an interaction between laminin expression and the growth and viability of HSG cells.

Immunoglobulins that bind to uncoated ELISA plate surfaces: appearance in mice during infection with lactate-dehydrogenase-elevating virus and in human anti-nuclear antibody-positive sera.

Immunoglobulins present in the blood plasma of mice infected with lactate-dehydrogenase-elevating virus (LDV) were found to bind strongly in the presence of 0.05% Tween 20 to the uncoated surfaces of wells of certain ELISA plates with previously recognized high protein-binding capacity. The binding was readily distinguishable from non-specific background binding of immunoglobulins present in normal mouse plasma. The binding components absorbed to protein A and had molecular weights in the 150-300 kDa range. Binding of the purified IgG fraction was progressively inhibited by increasing the concentration of Tween 20 in the diluent and by preincubation of the fraction at pH 3-4 for 10 min. The appearance of plate-binding IgM and IgG during LDV infection corresponded approximately with previously reported time courses of appearance of IgM- and IgG-containing circulating immune complexes and of specific IgM and IgG anti-LDV antibodies in LDV-infected mice. We conclude that complexes of IgG and IgM with LDV antigens have a much higher affinity for ELISA plates with high protein-binding capacity than uncomplexed immunoglobulins. Immune complexes did not significantly bind to ELISA plates with low protein-binding capacity, which, therefore, are suitable for measuring specific antiviral antibodies. Preliminary experiments with human anti-nuclear antibody-positive serum samples demonstrated markedly elevated non-specific binding of immunoglobulins to high-binding-capacity ELISA plates.

Influence of protein tyrosine phosphorylation on the expression of the c-myc oncogene in cancer of the large bowel.

We tested the potential impact of tyrosine phosphorylation on the expression of the c-myc gene in two colon cancer cell lines, HCT8 and SW837. We found that the protein tyrosine kinase inhibitor genistein causes a decrease in the abundance of c-myc RNA and an inhibition of proliferation with a similar dose response. Geldanamycin, a mechanistically different tyrosine kinase inhibitor, also causes a decrease in both the expression of c-myc RNA and proliferation. Genistein has also been found to inhibit topoisomerase II, but the topoisomerase II inhibitor novobiocin did not lower the expression of c-myc. The most likely interpretation is that inhibition of protein tyrosine kinase activity caused a decrease in c-myc expression in these cells. The impact of tyrosine phosphorylation on the expression of the c-myc gene is further supported by the finding that inhibition of phosphotyrosine phosphatase using orthovanadate causes an increase in the level of c-myc RNA. The effect of genistein on HCT8 cells is not dependent on the synthesis of new protein and does not involve an alteration in the stability of the message. Analysis of transcription in the c-myc gene reveals a more complicated picture with a decrease in initiation and an increase in elongation but no net change in transcription. We speculate that the genistein induced reduction in myc expression is the result of a posttranscriptional intranuclear event(s).

Characterization of genetic determinants for R body synthesis and assembly in Caedibacter taeniospiralis 47 and 116.

Caedibacter taeniospiralis, an obligate bacterial endosymbiont of Paramecium tetraurelia, confers a killing trait upon its host paramecium. Type 51 R bodies (refractile inclusion bodies) are synthesized by these endosymbionts and are required for expression of the killing trait. The nucleotide sequence of the genetic determinants for type 51 R body synthesis and assembly was determined for C. taeniospiralis 47 and 116. Three independently transcribed genes (rebA, rebB, and rebC) were characterized. To date these are the only genes from C. taeniospiralis to be sequenced and characterized. DNA regulatory regions are recognized by Escherichia coli, and codon usage appears similar to that in E. coli. A fourth open reading frame with appropriate regulatory sequences was found within the reb locus, but no evidence was obtained to suggest that this putative gene is expressed in E. coli. The R body-encoding sequences from both strains are identical. Two-dimensional gel electrophoresis of deletion derivatives shows that two polymerization events are involved in R body assembly. One polymerization event requires only RebB and RebC; the other requires all three proteins. Expression of RebC is necessary for the posttranslational modification of RebA and RebB into species with three and two different molecular weights, respectively. In the presence of RebC, each species of RebB with a different molecular weight has six different isoelectric points.

Sodium butyrate causes an increase in the block to transcriptional elongation in the c-myc gene in SW837 rectal carcinoma cells.

Elevated expression of the c-myc oncogene is a frequent finding in tumors and cell lines derived from carcinomas of the colon and rectum. In a previous study we demonstrated that the differentiation agent sodium butyrate causes a rapid reduction in the expression of c-myc RNA in the rectal carcinoma cell line SW837. This effect was blocked by inhibitors of protein synthesis, suggesting that butyrate causes the induction of an activity that has a negative effect on c-myc expression. In the present work we demonstrate that the rapid decrease in the level of c-myc RNA, upon treatment of SW837 cells with 2 mM butyrate, is followed by a slower decrease in the level of p53 RNA and an increase in the RNA levels for fibronectin and a placental type alkaline phosphatase. Using in vitro elongation of nascent transcripts to measure transcription and actinomycin D chase experiments to measure RNA stability, we show that the reduction in expression of c-myc RNA is due to an increase in the block to transcriptional elongation, rather than a decrease in transcriptional initiation or an increase in degradation of the RNA. We conclude that sodium butyrate induces an activity that increases the transcriptional block in SW837 cells, and that regulation of transcriptional elongation is an important mechanism for regulating c-myc expression in this cell type. A shift in relative usage of the two major promoters in the c-myc gene accompanies the reduction in expression. The potential significance of this finding with respect to transcriptional elongation is discussed. Mutations in the exon 1/intron 1 boundary region of the c-myc gene cause an increase in transcriptional elongation in Burkitt lymphoma. We sequenced this region in a series of cell lines derived from colorectal carcinomas, all of which had an elevated level of c-myc expression, to determine if a similar mutational mechanism is at work in this disease. All of the lines examined had a normal c-myc DNA sequence, suggesting that the deregulation of c-myc expression in colon cancer is not due to a cis mutation in this region.

Regulation and evolution of the single alpha-tubulin gene of the ciliate Tetrahymena thermophila.

The single alpha-tubulin gene of Tetrahymena thermophila was isolated from a genomic library and shown to encode a single protein. Comparisons of the rates of evolution of this gene with other alpha-tubulin sequences revealed that it belongs to a group of more evolutionarily constrained alpha-tubulin proteins in animals, plants, and protozoans versus the group of more rapidly evolving fungal and variant animal alpha-tubulins. The single alpha-tubulin of Tetrahymena must be used in a variety of microtubule structures, and we suggest that equivalently conserved alpha-tubulins in other organisms are evolutionarily constrained because they, too, are multifunctional. Reduced constraints on fungal tubulins are consistent with their simpler microtubule systems. The animal variant alpha-tubulins may also have diverged because of fewer functional requirements or they could be examples of specialized tubulins. To analyze the role of tubulin gene expression in regulation of the complex microtubule system of Tetrahymena, alpha-tubulin mRNA amounts were examined in a number of cell states. Message levels increased in growing versus starved cells and also during early stages of conjugation. These changes were correlated with increases in transcription rates. Additionally, alpha-tubulin mRNA levels oscillate in a cell cycle dependent fashion caused by changes in both transcription and decay rates. Therefore, as in other organisms, Tetrahymena adjusts alpha-tubulin message amounts via message decay. However the complex control of alpha-tubulin mRNA during the Tetrahymena life cycle involves regulation of both decay and transcription rates.

A deletion polymorphism due to Alu-Alu recombination in intron 2 of the retinoblastoma gene: association with human gliomas.

The retinoblastoma gene (RB) encodes a tumor suppressor that is inactivated in a number of different types of cancer. We searched for gross alterations of this gene in tumors of the central nervous system by using Southern blot hybridization. A common alteration was found in several tumors and was mapped to the region around exon 2. Nucleotide sequencing showed that the alteration was caused by a 799-bp deletion in intron 2 of the RB gene and was probably due to homologous recombination between two Alu repeats. Deletions of this type have not been found previously in the RB gene. The deletion turned out to be a polymorphism with an allele frequency estimated at 2.2% in 185 patients without cancer. The deletion was found in five of 48 patients with brain tumors (allele frequency of 5.2%). This difference is not statistically significant (P = 0.149, Fisher's exact test). Confining the analysis only to glioma brain tumors revealed a statistically significant difference compared with the cancer-free patient controls (P = 0.027, Fisher's exact test). Further study is needed to determine if the deletion is a weak brain cancer-predisposing mutation or a harmless polymorphism. Finding this mutation in a tumor and the germline DNA of a retinoblastoma patient could lead to incorrect estimation of the heritability of a tumor.