Changes in the blood level, efficacy, and safety of tacrolimus in pregnancy and the lactation period in patients with systemic lupus erythematosus.

OBJECTIVES: We investigated the efficacy and safety of tacrolimus (TAC) by monitoring its serum concentration for mothers and infants in pregnant patients with systemic lupus erythematosus (SLE). METHODS: We measured trough concentrations of TAC in 25 pregnant patients with SLE to assess influence of TAC on the disease activity. Additionally, we measured the concentrations of TAC in umbilical arterial blood, breast milk, and breastfed infants to investigate the safety of TAC for the mothers and infants. RESULTS: The trough concentrations of TAC in the mothers significantly decreased in the second trimester as compared with those before pregnancy. However, the decrease in the trough concentrations of TAC did not lead to the deterioration of SLE. When examined, the doses of TAC were significantly lower in the second trimester and postpartum in the deteriorating group than those in the non-deteriorating group. There were no adverse events by TAC in mothers and fetuses. The concentrations of TAC in the umbilical cord blood were lower than those in the maternal blood. The relative infant dose in breastfed infants of TAC was < 1%. The level of TAC in infant bloods was below detectable limits. CONCLUSION: These findings suggest that TAC is one of the most effective and safest immunosuppressive drugs for use in pregnant patients with SLE.

Peripartum type B aortic dissection in patients with Marfan syndrome who underwent aortic root replacement: a case series study.

OBJECTIVE: To investigate pregnancy outcomes, especially the risk of pregnancy-related aortic dissection (AD), in patients with Marfan syndrome (MFS) after prophylactic aortic root replacement (ARR). DESIGN: Retrospective case series study. SETTING: Tertiary perinatal care centre at a university hospital. POPULATION: Pregnant women fulfilling the revised Ghent nosology (2010) criteria for MFS who were managed at our institute. METHODS: The pregnancy outcomes of all patients with MFS managed at our institute between 1982 and September 2016 were reviewed retrospectively based on medical records. MAIN OUTCOME MEASURES: Obstetrical management and complication including the incidence of AD throughout the peripartum period. RESULTS: Among 22 patients (28 pregnancies) who had been managed as potential MFS or related disorders, 14 (17 pregnancies) fulfilled the revised Ghent nosology (2010) criteria for MFS and were enrolled in this study. Five patients (five pregnancies) had received ARR before conception: three (60%) developed type B aortic dissection [AD(B)] during the peripartum period, compared with only one of 10 patients (12 pregnancies) without ARR (P < 0.05, Chi-square test). CONCLUSIONS: Our study results suggest that MFS patients after prophylactic ARR are still at high risk of AD(B) during the peripartum period. Careful pre-pregnancy counselling and multidisciplinary care throughout the peripartum period are essential for the management of MFS, even after surgical repair of an ascending aortic aneurysm. TWEETABLE ABSTRACT: MFS patients after prophylactic ARR are still at high risk of type B aortic dissection during the peripartum period.

Primary prophylaxis to prevent obstetric complications in asymptomatic women with antiphospholipid antibodies: a systematic review.

OBJECTIVE: Obstetric complications are common in patients with antiphospholipid syndrome. However, the impact of antiphosholipid antibodies (aPL) in the pregnancy outcomes of asymptomatic aPL carriers is uncertain. The aim of this systematic review is to assess whether primary prophylaxis is beneficial to prevent obstetric complications during pregnancy in asymptomatic women positive for aPL who have no history of recurrent pregnancy loss or intrauterine fetal death. METHODS: Studies evaluating the effect of prophylactic treatment versus no treatment in asymptomatic pregnant aPL carriers were identified in an electronic database search. Design, population and outcome homogeneity of studies was assessed and meta-analysis was performed. The pooled Mantel-Haenszel relative risk of specific pregnancy outcomes was obtained using random effects models. Heterogeneity was measured with the I(2) statistic. All analyses were conducted using Review Manager 5.3. RESULTS: Data from five studies involving 154 pregnancies were included and three studies were meta-analysed. The risk ratio and 95% confidence interval (CI) of live birth rates, preterm birth, low birth weight and overall pregnancy complications in treated and untreated pregnancies were 1.14 (0.18-7.31); 1.71 (0.32-8.98); 0.98 (0.07-13.54) and 2.15 (0.63-7.33),respectively. Results from the meta-analysis revealed that prophylactic treatment with aspirin is not superior to placebo to prevent pregnancy complications in asymptomatic aPL carriers. CONCLUSION: This systematic review did not find evidence of the superiority of prophylactic treatment with aspirin compared to placebo or usual care to prevent unfavourable obstetric outcomes in otherwise healthy women with aPL during the first pregnancy.

Activating SRC mutation in a subset of advanced human colon cancers.

The discovery of Rous sarcoma virus (RSV) led to the identification of cellular Src (c-Src), a non-receptor tyrosine kinase, which has since been implicated in the development of numerous human cancers. c-Src has been found to be highly activated in colon cancers, particularly in those metastatic to the liver. Studies of the mechanism of c-Src regulation have suggested that c-Src kinase activity is downregulated by phosphorylation of a critical carboxy-terminal tyrosine (Tyr 530 in human c-Src, equivalent to Tyr 527 in chicken Src) and have implied the existence of activating mutations in this C-terminal regulatory region. We report here the identification of a truncating mutation in SRC at codon 531 in 12% of cases of advanced human colon cancer tested and demonstrate that the mutation is activating, transforming, tumorigenic and promotes metastasis. These results provide, for the first time, genetic evidence that activating SRC mutations may have a role in the malignant progression of human colon cancer.

Cardiac hamartoma in a young squirrel monkey who died suddenly.

A case of cardiac hamartoma in a 2-month-old squirrel monkey is reported. The monkey showed a loss of appetite and died suddenly. Microscopically, an encapsulated nodular lesion was found at the right atrial wall. The lesion consisted of irregularly shaped, slender myocytes intermingled with a few fibroblasts and collagen fibers. Neither nuclear atypia nor inflammatory cell infiltrate was seen. The constituting cells had stratified striations in the cytoplasm and reacted immunohistochemically for desmin, indicating the nature of myocytes. Based on the above findings, a diagnosis of cardiac hamartoma was made. This is the first case of cardiac hamartoma in this species.

Prostaglandin F(2alpha) negatively regulates bone resorption in murine osteoclast development.

Prostaglandin (PG) E(2) promotes osteoclastic cell differentiation, but the physiological function of PGF(2alpha) remains unclear. We examined the physiological effects of PGF(2alpha) on osteoclast differentiation using a murine cell line, RAW264, and the column-purified murine bone marrow cells, both of which are differentiable into osteoclast-like multi-nuclear cells. Although PGF(2alpha) did not affect the number of differentiated osteoclasts, PGF(2alpha) reduced the bone resorption activity of osteoclasts developed from both cell types in a pit formation assay. Thus, PGF(2alpha) inhibits bone resorption without affecting the number of osteoclasts, providing a novel molecular mechanism underlying bone metabolism.

SUMO modification of Sam68 enhances its ability to repress cyclin D1 expression and inhibits its ability to induce apoptosis.

Sam68 (Src associated in mitosis; 68 kDa) is an RNA-binding protein and substrate of Src family kinases. It is thought to play a role in cell cycle progression. Overexpression of Sam68 in fibroblasts was reported to have two separable functions dependent on its ability to bind RNA--cell cycle arrest or the induction of apoptosis. Post-translational modification with SUMO (small ubiquitin-like modifier) is common to many transcription factors and can regulate protein localization, stability and function. Here we show Sam68 to be modified by SUMO, and demonstrate that the SUMO E3 ligase (PIAS1) (protein inhibitor of activated STAT1) can enhance Sam68 sumoylation. Lysine 96, the first lysine in the amino-terminal region of Sam68, was found to be the major SUMO acceptor site. Mutation of the SUMO acceptor lysine to arginine enhanced the ability of Sam68 to induce apoptosis but inhibited its ability to act as a transcriptional inhibitor of cyclin D1 expression. A SUMO-1 Sam68 fusion protein, on the other hand, inhibited the ability of Sam68 to induce apoptosis but was a strong repressor of cyclin D1 expression. Thus, SUMO may be an important regulator of Sam68 function in cell cycle progression.

Expression of a molecularly cloned human c-src oncogene by using a replication-competent retroviral vector.

We studied the expression of a molecularly cloned human c-src gene, c-src-1, localized on chromosome 20, whose coding region consists of 11 exons and spans a 19.5-kilobase (kb) distance. Using a replication-competent retroviral vector derived from molecularly cloned Rous sarcoma virus DNA (pSRA-2), we obtained two constructs: one (pSR-CS) carrying the unmodified human c-src coding sequence and another (pSR-CVS) with a chimeric gene formed between the human c-src gene and the carboxy-terminal 12-amino acid v-src-specific coding sequence. From chicken embryo fibroblasts transfected with these DNA constructs, infectious viruses designated as WO CS and WO CVS, respectively, were recovered. WO CS virus did not cause cell transformation, whereas WO CVS induced cell transformation. Analyses of the proviral DNAs indicated that all introns were spliced out such that the 19-kb inserts were converted to 1.7-kb cDNA forms. Analyses of src proteins in infected cells, using monoclonal antibody MAb327 against v-src protein, showed the following results. The CVS and CS src proteins were about 60 and 61 kilodaltons in size, respectively; the specific protein kinase activity assayed in vitro of the CVS src protein was about 20-fold higher than that of the CS src protein and comparable to that of the v-src protein; the transforming CVS src protein reacted to an antibody against a v-src-specific peptide, whereas the CS src protein did not. These results indicate that the human c-src gene has a potential transforming ability and suggest that the v-src-specific sequence played an important role in the generation of Rous sarcoma virus.

The SH3 domain of p56lck is involved in binding to phosphatidylinositol 3'-kinase from T lymphocytes.

Many of the Src-like tyrosine kinases are thought to participate in multiprotein complexes that modulate transmembrane signalling through tyrosine phosphorylation. We have used in vitro binding studies employing bacterially expressed glutathione S-transferase-p56lck fusion proteins and cell extracts to map regions on p56lck that are involved in binding to phosphatidylinositol 3'-kinase (PI3K). Deletions within the SH3 domain of p56lck abolished binding of PI3K activity from T-cell lysates, whereas deletion of the SH2 domain caused only a slight reduction in the level of PI3K activity bound to p56lck sequences. The binding of PI3K from T-cell extracts to p56lck was not blocked by antiphosphotyrosine antibodies, but p56lck-bound PI3K activity was sensitive to phosphatase treatment. The SH3 domain of p56lck also bound the majority of PI3K activity from uninfected chicken embryo fibroblasts. However, a drastically different binding specificity was observed with use of extracts of Rous sarcoma virus v-src-transformed cells, in which the majority of PI3K activity bound to the SH2 domain of p56lck in a phosphotyrosine-dependent manner. These results suggest that are two modes of PI3K binding to p56lck, and presumably to other Src-like tyrosine kinases. In one mode, PI3K from T cells or uninfected chicken embryo fibroblasts binds predominantly to the SH3 domain of p56lck. In the other mode, involving PI3K from Rous sarcoma virus-transformed cells, binding is largely phosphotyrosine dependent and requires the SH2 domain of p56lck.

Association of type I phosphatidylinositol kinase activity with mutationally activated forms of human pp60c-src.

Chicken embryo fibroblast cells overexpressing activated mutant forms of human pp60c-src, but not those overexpressing normal human pp60c-src, exhibited high levels of type I phosphatidylinositol (PI) kinase activity associated with pp60c-src. Levels of PI kinase activity were positively correlated with src tyrosine protein kinase activity and not with absolute levels of pp60c-src. Our results suggest that a linkage exists between certain forms of pp60c-src and the PI signal transduction pathway.

DNA sequence encoding the amino-terminal region of the human c-src protein: implications of sequence divergence among src-type kinase oncogenes.

We sequenced the 5'-coding region of the human c-src gene, exons 2 through 5, corresponding to one-third of the human c-src protein consisting of 536 amino acids. Sequence analysis of the src type of protein kinases revealed that the amino-terminal region encoded by exon 2 contains sequences specific for the src proteins and raised the possibility that this region is involved in the recognition of a src-specific substrate(s) or receptor(s).

Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxy-terminal two-thirds of pp60c-src.

The nucleotide sequence of the 3' two-thirds of a highly conserved, molecularly cloned human cellular src gene (c-src) has been determined. This region of the c-src gene encodes the tyrosine kinase domain of the cellular src protein (pp60c-src) and corresponds to exons 6 through 12 of the chicken c-src gene, as well as nucleotides 545 to 1542 of the Rous sarcoma virus src gene (v-src). The human c-src sequence is very strongly conserved with respect to both the chicken c-src and the Rous sarcoma virus v-src genes, with nearly 90% nucleotide homology observed in this region. Amino acid sequence conservation in this region is even greater; 98% of the amino acids are conserved between human and chicken c-src. Furthermore, the exon sizes and the locations of the exon-intron boundaries are identical in the human and chicken c-src genes. However, sequences within the introns have not been conserved, and the introns within the human c-src gene are significantly larger than the corresponding introns within the chicken c-src gene. The strong amino acid conservation between the carboxy-terminal two-thirds of pp60c-src of species as divergent as humans and chickens suggests that this portion of the pp60c-src protein specifies one or more functional domains that are of great importance to some aspect of normal cellular growth or differentiation.

A dual inhibitor of platelet-derived growth factor beta-receptor and Src kinase activity potently interferes with motogenic and mitogenic responses to PDGF in vascular smooth muscle cells. A novel candidate for prevention of vascular remodeling.

PP1 has previously been described as an inhibitor of the Src-family kinases p56(Lck) and FynT. We have therefore decided to use PP1 to determine the functional role of Src in platelet-derived growth factor (PDGF)-induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed, and the inhibitory activity of PP1/AGL1872 against Src was examined. PP1/AGL1872 potently inhibited recombinant p60(c-src) in vitro and Src-dependent tyrosine phosphorylation in p60(c-srcF572)-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs, as determined in the modified Boyden chamber, as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly, in addition to inhibition of Src kinase, PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells, including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR, PP1/AGL1872 had only a weak effect on vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases, the activities of the epidermal growth factor receptor, fibroblast growth factor receptor-1, and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872, whereas PDGF alpha-receptor was susceptible, albeit to a lesser extent than PDGF beta-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for the Src family of tyrosine kinases. It is also a potent inhibitor of the PDGF beta-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events, creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity, such as vascular remodeling and restenosis.

Organization and analysis of the promoter region and 5' non-coding exons of the human c-src proto-oncogene.

In order to help clarify the cellular mechanisms that regulate expression of the human c-src proto-oncogene, we have isolated a series of overlapping genomic clones that contain the c-src promoter region, as well as three previously uncharacterized exons. These exons encode the 350-bp 5' untranslated region of the c-src mRNA and span 35 kb of genomic DNA, extending the human c-src locus to approximately 60 kb. Subcloning and sequence analysis of the 5' flanking region of the gene revealed a high GC content and several consensus Sp1 and AP2 binding sites. However, TATA or CAAT boxes were not present, a characteristic shared by other GC-rich promoters. Promoter-CAT constructs demonstrated that the promoter was functional in transfection assays and that its activity was dependent on correct orientation. CAT-promoter deletion constructs were used to define the 5' boundary for maximal promoter activity and to reveal the presence of both positive and negative regulatory elements. S1 analyses of human c-src mRNA from cell lines indicated that multiple transcription start sites were utilized.

Selected glimpses into the activation and function of Src kinase.

Since the discovery of the v-src and c-src genes and their products, much progress has been made in the elucidation of the structure, regulation, localization, and function of the Src protein. Src is a non-receptor protein tyrosine kinase that transduces signals that are involved in the control of a variety of cellular processes such as proliferation, differentiation, motility, and adhesion. Src is normally maintained in an inactive state, but can be activated transiently during cellular events such as mitosis, or constitutively by abnormal events such as mutation (i.e. v-Src and some human cancers). Activation of Src occurs as a result of disruption of the negative regulatory processes that normally suppress Src activity, and understanding the various mechanisms behind Src activation has been a target of intense study. Src associates with cellular membranes, in particular the plasma membrane, and endosomal membranes. Studies indicate that the different subcellular localizations of Src could be important for the regulation of specific cellular processes such as mitogenesis, cytoskeletal organization, and/or membrane trafficking. This review will discuss the history behind the discovery and initial characterization of Src and the regulatory mechanisms of Src activation, in particular, regulation by modification of the carboxy-terminal regulatory tyrosine by phosphatases and kinases. Its focus will then turn to the different subcellular localizations of Src and the possible roles of nuclear and perinuclear targets of Src. Finally, a brief section will review some of our present knowledge regarding Src involvement in human cancers.

Activation of Src in human breast tumor cell lines: elevated levels of phosphotyrosine phosphatase activity that preferentially recognizes the Src carboxy terminal negative regulatory tyrosine 530.

Elevated levels of Src kinase activity have been reported in a number of human cancers, including colon and breast cancer. We have analysed four human breast tumor cell lines that exhibit high levels of Src kinase activity, and have determined that these cell lines also exhibit a high level of a phosphotyrosine phosphatase activity that recognizes the Src carboxy-terminal P-Tyr530 negative regulatory site. Total Src kinase activity in these cell lines is elevated as much as 30-fold over activity in normal control cells and specific activity is elevated as much as 5.6-fold. When the breast tumor cells were grown in the presence of the tyrosine phosphatase inhibitor vanadate, Src kinase activity was reduced in all four breast tumor cell lines, suggesting that Src was being activated by a phosphatase which could recognize the Tyr530 negative regulatory site. In fractionated cell extracts from the breast tumor cells, we found elevated levels of a membrane associated tyrosine phosphatase activity that preferentially dephosphorylated a Src family carboxy-terminal phosphopeptide containing the regulatory tyrosine 530 site. Src was hypophosphorylated in vivo at tyrosine 530 in at least two of the tumor cell lines, further suggesting that Src was being activated by a phosphatase in these cells. In preliminary immunoprecipitation and antibody depletion experiments, we were unable to correlate the major portion of this phosphatase activity with several known phosphatases.

Coordinate alteration of pp60c-src abundance and c-src RNA expression in human neuroblastoma variants.

We examined the expression, abundance, and protein kinase activity of pp60c-src in two different pairs of genetically indistinguishable cloned human neuroblastoma cell variants which display altered phenotypes as the result of conversion from a neuronal to a non-neuronal phenotype. The results demonstrate that cells which exhibit the neuroblastic (N-type) phenotype possess high levels of pp60c-src protein and that one of the two N-type cell lines is capable of expressing the neuronal-specific isoenzyme of pp60c-src. In contrast, cells which display the substrate-adherent (S-type) phenotype have low levels of pp60c-src protein and express exclusively the non-neuronal isoenzyme of pp60c-src. In all cells examined the abundance of pp60c-src was found to be proportional to the steady-state level of c-src RNA. In each case the protein kinase activity of pp60c-src was found to be proportional to the abundance of the protein and independent of the ratio of neuronal to non-neuronal species of pp60c-src.

An aberrant lck mRNA in two human T-cell lines.

Low stringency screening of a human T-cell cDNA library with a c-src probe resulted in the isolation of several cDNAs of approx. 800 base pairs. Sequence analysis revealed that these clones encoded partial cDNAs for the src-family tyrosine kinase p56lck. A novel feature of this partial cDNA was that it contained a 93 bp intron insertion that was not observed in other full length or partial lck cDNA. This aberrant lck RNA was also detected in the mRNA pool by Northern blotting and PCR amplification of poly(A)+ RNA isolated from two human leukemic T-cell lines.

Influence of antifouling paint on freshwater invertebrates (Mytilidae, Chironomidae and Naididae): density, richness and composition.

We conducted a study about invertebrates on artificial substrates with different antifouling paints in order to answer the following questions 1) is there lower accumulation of organic matter on substrates with antifouling paints, 2) is invertebrate colonization influenced by the release of biocides from antifouling paints, 3) is the colonization of aquatic invertebrates positively influenced by the material accumulated upon the substrate surface and 4) is the assemblage composition of invertebrates similar among the different antifouling paints? To answer these questions, four structures were installed in the Baía River in February 1st, 2007. Each structure was composed of 7 wood boards: 5 boards painted with each type of antifouling paints (T1, T2, T3, T4 and T5), one painted only with the primer (Pr) and the other without any paint (Cn). After 365 days, we observed a greater accumulation of organic matter in the substrates with T2 and T3 paint coatings. Limnoperna fortunei was recorded in all tested paints, with higher densities in the control, primer, T2 and T3. The colonization of Chironomidae and Naididae on the substrate was positively influenced by L. fortunei density. The non-metric multidimensional scaling (NMDS) of the invertebrate community provided evidence of the clear distinction of invertebrate assemblages among the paints. Paints T2 and T3 were the most similar to the control and primer. Our results suggest that antifouling paints applied on substrates hinder invertebrate colonization by decreasing the density and richness of invertebrates.

Effect of heparin on phosphorylation site specificity of neuronal Cdc2-like kinase.

Neuronal Cdc2-like kinase (Nclk) can be stimulated by heparin in a substrate-dependent manner. While phosphorylation of tau is markedly enhanced by heparin, phosphorylation of histone H1 by Nclk is essentially unaffected. A histone H1 peptide, HS(9-18): PKTPKKAKKL, and its substitution analogues were used to examine the basis of the differential heparin effect. In the presence of heparin, the phosphorylation site specificity of Nclk is altered and only proline immediately following the phosphorylation site is still an essential substrate determinant. This change in the site specificity may adequately account for the differential heparin effect on histone H1 and tau phosphorylation.