Long-Term Results of the Treatment of Patients With Chronic Ischemia of the Lower Limbs in Combination With Ischemic Heart Disease.

Aim      To compare long-term outcomes of x-ray endovascular (percutaneous coronary intervention, PCI, and lower limb angioplasty with stent placement, LLA; group 1) and combination treatments (PCI and open LLA surgery; group 2) in patients with chronic lower limb ischemia (CLLI) associated with ischemic heart disease (IHD).Material and methods  This retrospective study has been conducted in the Vishnevsky National Medical Research Center of Surgery since 2019. The study includes 92 patients with stage 2B CLLI associated with IHD who were managed from January 1, 2017 through December 31, 2020. Long-term outcomes were evaluated in 76 (82.6 %) patients. The endpoint was severe cardiovascular complications (CVC), including death, myocardial infarction, and acute cerebrovascular disease (ACVD).Results In group 1 during the long-term period, 1 (2.7%) fatal outcome due to pneumonia was observed. In group 2, 4 (10 %) patients died: 1 (2.5 %) patient due to ACVD, 1 (2.5 %) patient due to progression of oncological process, and 2 2 (5 %) patients due to COVID-19. Also, 2 (5.5 %) and 1 (2.5 %) cases of acute coronary syndrome (ACS) were observed in groups 1 and 2, respectively (p=0.61).Conclusion      In the x-ray endovascular (group1) and the combination (group 2) intervention groups, lethal outcomes due to myocardial infarction were absent. This fact confirms the importance of PCI in patients with CLLI for prevention of possible ACS in the long-term. Both therapeutic tactics in managing CLLI patients with IHD demonstrated high safety and clinical efficacy during the hospital and long-term periods and can be extensively used in routine clinical practice.

[Determination of thermodynamic binding parameters and type of interaction between the А/Bangkok/1/1979 (Н3N2) influenza virus hemagglutinin and phosphatidylcholine liposome]. / Opredelenie termodinamicheskikh parametrov sviazyvaniia i kharakter vzaimodeistviia gemaggliutinina virusa grippa A/Bangkok/1/1979(N3N2) s liposomami iz fosfatidilkholina.

The study of interaction between surface viral proteins and model phospholipids is important for learning more details about the mechanisms of viral penetration into cells during infection. In this context, liposomes represent suitable systems for modeling a cell membrane. The binding of hemagglutinin (HA) of influenza virus with phosphatidylcholine liposomes was studied by equilibrium adsorption. It was interesting elucidate changes occurring in the structure of a protein during its translocation from the surface into the interior part of the membrane. In this work, we have studied characteristics of the protein-lipid interaction during HA complex formation with phospholipids including adsorption of HA on a phospholipid bilayer. Using the Scatchard equation and the Gibbs-Helmholtz equation at pH 4.0 and pH 6.0 thermodynamic parameters were determined. The results concluded the hydrophobic type of interaction between viral protein and liposomes. The additional confirmation of hydrophobic protein-lipid interaction presence was determination of HA distribution constants in two-phase systems: dextran-polyethylene glycol (K1) and dextran-polyethylene glycol esterified with palmitic acid (K2). The presence of hydrophobic interaction between HA and the liposome membrane was also confirmed using the quenching method of intrinsic protein fluorescence by a neutral quencher with acrylamide. At pH 4.0, an increase in the Stern-Volmer quenching constant was observed for the HA+liposome from phosphatidylcholine system, which is caused by structural changes in HA upon incorporation into the liposome bilayer. The fluorescence quenching rate constants calculated using the Stern-Volmer equation indicate a static quenching mechanism in which the quencher interacts with fluophors of a stationary protein molecule. The obtained results are interesting for not only studying virus and cell fusion theoretically, but also have practical applications. Using values of the protein-bilayer binding constant and free energy constant, it is possible to select the optimal phospholipid composition of liposomes or virosomes to obtain a stronger complex with various viral proteins. With two-phase systems, it is possible to determine the presence of hydrophobic sites on the viral protein surface, which can be used for evaluation both protein-lipid and protein-protein interaction.

Increased CD4+CD25-Foxp3+ T cells in Chinese systemic lupus erythematosus: correlate with disease activity and organ involvement.

OBJECTIVE: The increment of CD4+CD25-Foxp3+T cells has been reported in systemic lupus erythematosus (SLE) patients. However, the exact identity of this T cell subset is still unclear. Thus, we analyzed CD4+CD25-Foxp3+T cells and Treg cells (CD4+CD25+Foxp3+ T cells) in a large sample of Chinese SLE patients in different disease states. METHODS: A total of 280 SLE patients and 38 healthy volunteers were enrolled, which included 21 patients with untreated new-onset lupus (UNOL), 13 patients with drug withdrawal more than 6 months and 246 patients with treatments. Phenotypic and functional analysis of peripheral blood CD4+CD25-Foxp3+ T cells and Treg cells were performed by flow cytometry. The correlation of CD4+CD25-Foxp3+T cells and Treg cells with disease activity, clinical indicators and organ involvement were analyzed. RESULTS: CD4+CD25-Foxp3+ T cells and Treg cells were significantly increased in SLE patients and showed significantly positive correlations with disease activity. CD4+CD25-Foxp3+ T cells were significantly increased in patients with skin and hematologic involvement as well as arthritis. Diverse changes between CD4+CD25-Foxp3+ T cells and Treg cells when faced with different medications, especially HCQ and MMF. CD4+CD25-Foxp3+ T cells expressed more IFN-γ and less CTLA-4 than CD4+CD25+Foxp3+ T cells, which were similar to CD4+CD25+Foxp3- T cells, and expressed similar IL-17, ICOS and Helios to CD4+CD25+Foxp3+ T cells. The synthesis capacity of IL-10 of CD4+CD25-Foxp3+ T cells and the expression of GITR on CD4+CD25-Foxp3+ T cells were between CD4+CD25+Foxp3+ and CD4+CD25+Foxp3- T cells. CONCLUSIONS: Our results indicate that increased CD4+CD25-Foxp3+ T cells in lupus patients, which combined the features of suppression and pro-inflammatory, may serve as a biomarker for disease activity and organ involvement in SLE.

Highly Dispersible Buckled Nanospring Carbon Nanotubes for Polymer Nano Composites.

We propose the unique structure of highly dispersible single-walled carbon nanotubes (SWCNTs) in various solvents and polymers using the ZnO nano particle template. Buckled nanospring-shaped carbon nanotubes (NS-CNTs) were synthesized by a chemical reaction of ZnO nanoparticles with acid-treated SWCNTs and then dissolving ZnO through chemical etching. The unique structure of distorted hexagonal NS-CNTs encircled around ZnO nanoparticles was formed by the bending of SWCNTs caused by the agglomeration of chemically adsorbed Zn(OH)2, which is further crystallized as the polycrystalline ZnO inner core. The highly dispersible NS-CNTs could be incorporated in the poly[(vinylidenefluoride-co-trifluoroethylene] [P(VDF-TrFE)] copolymer, one of widely studied ferro- and piezo-electric polymer, up to the value of 15 wt% as nanofillers. The relative dielectric constant (K) of polymer nanocomposite, at 1 kHz, was greatly enhanced from 12.7 to the value of 62.5 at 11 wt% of NS-CNTs, corresponding to a 492% increase compared to that of pristine P(VDF-TrFE) with only a small dielectric loss tangent (D) of 0.1.

[Concepts of "urinary bladder" and "vesicles (bao)" , "jin ye" (fluid and humor) and "urine" and other associated issues].

In the Huang di nei jing(Huangdi's Internal Classic), jin ye (fluid and humor) is described in two senses, broad and narrow, though not so strictly.Sometimes, jin ye is explained ambiguously as "sweat" and "urine" , as in the phrase "the bladder, being a house of jin ye" , here "jin ye" refers to the urine. In the Qi jue lun pian of Su wen (Chapter on Qi-Syncope of Plain Questions) , the "bao" in the sentence "heat of bao moved to bladder" refersto the uterus. In the Shi cong rong lun pian (Chapter of Readily Inspecting) of Plain Questions, the "bladder" in the phrase "gallbladder, stomach, large intestine, small intestine, spleen, bao and bladder" , which, being an annotation of "bao" originally, is mistakenly incorporated into the text of the Classic. In the Wu wei lun of Ling shu (On Five Tastes in Miraculous Pivot) , the "bao" in "bao of bladder" refers to the external hou (external manifestation) of the bladder, that is the scrotum. In the Bei ji qian jin yao fang (Essential Prescriptions Worth a Thousand Gold for Emergencies) , the short sentence "pang guang zou bao" is an error in itself. In the sentence of "settled in the bao and zhi causing to dream of defecation and urination" in the Yin xie fa meng (Dreams due to Evils) of Miraculous Pivot, "bao" refers to uterus, and "zhi" to anus. In Bi lun pian(Chapter on Impediment) of Plain Questions, "the man suffered bao bimight feel internal pain when the lesser abdomen and bladder are pressed" , here, "bao" refers to the bladder. In the Wu yin wu wei(Chapter on Five Sound and Five Tastes) of Miraculous Pivot, the "bao" in the sentence "thoroughfare vessel and conception vessel all starts from bao" , again, "bao" here refers to the bladder, rather than to the uterus. From the above descriptions of "bladder" and "bao" in the Huangdi's Internal Classic, the "bladder" in ancient medical books refers to the substantial bladder, an anatomical organ, and "bao" refers to cystiform organs, including the bladder, uterus, scrotum etc.

Evolution and heterogeneity of non-hereditary colorectal cancer revealed by single-cell exome sequencing.

Recently single-cell whole-exome sequencing (scWES) has deeply expanded and sharpened our knowledge of cancer evolution and subclonality. Herein, with scWES and matched bulk whole-exome sequencing (bulk WES) on two colorectal cancer (CRC) patients with normal or adenomatous polyps, we found that both the adenoma and cancer were of monoclonal origin, and both shared partial mutations in the same signaling pathways, but each showed a specific spectrum of heterogeneous somatic mutations. In addition, the adenoma and cancer further developed intratumor heterogeneity with the accumulation of nonrandom somatic mutations specifically in GPCR, PI3K-Akt and FGFR signaling pathways. We identified novel driver mutations that developed during adenoma and cancer evolution, particularly in OR1B1 (GPCR signaling pathway) for adenoma evolution, and LAMA1 (PI3K-Akt signaling pathway) and ADCY3 (FGFR signaling pathway) for CRC evolution. In summary, we demonstrated that both colorectal adenoma and CRC are monoclonal in origin, and the CRCs further diversified into different subclones with heterogeneous mutation profiles accumulating in GPCR, PI3K-Akt and FGFR signaling pathways. ScWES provides evidence for the importance of mutations in certain pathways that would not be as apparent from bulk sequencing of tumors, and can potentially establish whether specific mutations are mutually exclusive or occur sequentially in the same subclone of cells.

Bone morphogenetic protein-2 provokes interleukin-18-induced human intervertebral disc degeneration.

OBJECTIVES: Interleukin 18 (IL-18) is a regulatory cytokine that degrades the disc matrix. Bone morphogenetic protein-2 (BMP-2) stimulates synthesis of the disc extracellular matrix. However, the combined effects of BMP-2 and IL-18 on human intervertebral disc degeneration have not previously been reported. The aim of this study was to investigate the effects of the anabolic cytokine BMP-2 and the catabolic cytokine IL-18 on human nucleus pulposus (NP) and annulus fibrosus (AF) cells and, therefore, to identify potential therapeutic and clinical benefits of recombinant human (rh)BMP-2 in intervertebral disc degeneration. METHODS: Levels of IL-18 were measured in the blood of patients with intervertebral disc degenerative disease and in control patients. Human NP and AF cells were cultured in a NP cell medium and treated with IL-18 or IL-18 plus BMP-2. mRNA levels of target genes were measured by real-time polymerase chain reaction, and protein levels of aggrecan, type II collagen, SOX6, and matrix metalloproteinase 13 (MMP13) were assessed by western blot analysis. RESULTS: The serum level of patients (IL-18) increased significantly with the grade of IVD degeneration. There was a dramatic alteration in IL-18 level between the advanced degeneration (Grade III to V) group and the normal group (p = 0.008) Furthermore, IL-18 induced upregulation of the catabolic regulator MMP13 and downregulation of the anabolic regulators aggrecan, type II collagen, and SOX6 at 24 hours, contributing to degradation of disc matrix enzymes. However, BMP-2 antagonised the IL-18 induced upregulation of aggrecan, type II collagen, and SOX6, resulting in reversal of IL-18 mediated disc degeneration. CONCLUSIONS: BMP-2 is anti-catabolic in human NP and AF cells, and its effects are partially mediated through provocation of the catabolic effect of IL-18. These findings indicate that BMP-2 may be a unique therapeutic option for prevention and reversal of disc degeneration.Cite this article: S. Ye, B. Ju, H. Wang, K-B. Lee. Bone morphogenetic protein-2 provokes interleukin-18-induced human intervertebral disc degeneration. Bone Joint Res 2016;5:412-418. DOI: 10.1302/2046-3758.59.BJR-2016-0032.R1.

miR-145 regulates chemoresistance in hepatocellular carcinoma via epithelial mesenchymal transition.

Resistance to chemotherapeutic drugs is a major obstacle in hepatocellular carcinoma (HCC) therapy. MicroRNA—145 (miR—145) has been shown to be down—regulated in several cancers and may be involved in the process of carcinogenesis. The present study aimed to evaluate the effects of miR—145 in adriamycin (ADM)—resistant human HCC cells. We found that miR—145 was significantly reduced in HepG2/ADM and HuH7/ADM cells compared with the chemosensitive parental cells. Up—regulation of miR—145 increased the ADM cytotoxicity in chemoresistant tumor cells. In addition, Smad3 was identified as the target of miR—145 and miR—145 overexpression inhibited Smad3 expression both at the mRNA and protein levels. The luciferase reporter assay confirmed that Smad3 was a direct target of miR—145. Moreover, up—regulation of miR—145 suppressed Smad3 related EMT features as shown by increased expression of E—cadherin and reduced vimentin level in HepG2/ADM and HuH7/ADM cells. Our study demonstrated that miR—145 modulated both chemoresistance and EMT in HCC cells, and up—regulation of miR—145 might be a potential therapeutic strategy for treatment of chemoresistant HCC.

[EFFECTIVENESS OF URETERAL STENTS WITH NANOSTRUCTURED COATING IN RENAL TRANSPLANTATION (PRELIMINARY RESULTS)].

From a surgeon's perspective, intraureteral jj-stent is an optimal tool to ensure upper urinary tract drainage. This paper presents preliminary results of our study investigating the use of ureteral stents with nanostructured coating in renal transplant recipients. The use of nanostructured coating based on amorphous carbon and silver nanocrystallites eliminated bacteriuria by week 4 after stenting in the treatment group with significant decrease of urine sediments while in the control group bacteriuria was found in 83,3% cases. Symptoms of bladder irritability depended on stent construction rather than presence of coating.

Orthotopic models of pediatric brain tumors in zebrafish.

High-throughput screens (HTS) of compound toxicity against cancer cells can identify thousands of potential new drug-leads. But only limited numbers of these compounds can progress to expensive and labor-intensive efficacy studies in mice, creating a 'bottle neck' in the drug development pipeline. Approaches that triage drug-leads for further study are greatly needed. Here we provide an intermediary platform between HTS and mice by adapting mouse models of pediatric brain tumors to grow as orthotopic xenografts in the brains of zebrafish. Freshly isolated mouse ependymoma, glioma and choroid plexus carcinoma cells expressing red fluorescence protein were conditioned to grow at 34 °C. Conditioned tumor cells were then transplanted orthotopically into the brains of zebrafish acclimatized to ambient temperatures of 34 °C. Live in vivo fluorescence imaging identified robust, quantifiable and reproducible brain tumor growth as well as spinal metastasis in zebrafish. All tumor xenografts in zebrafish retained the histological characteristics of the corresponding parent mouse tumor and efficiently recruited fish endothelial cells to form a tumor vasculature. Finally, by treating zebrafish harboring ERBB2-driven gliomas with an appropriate cytotoxic chemotherapy (5-fluorouracil) or tyrosine kinase inhibitor (erlotinib), we show that these models can effectively assess drug efficacy. Our data demonstrate, for the first time, that mouse brain tumors can grow orthotopically in fish and serve as a platform to study drug efficacy. As large cohorts of brain tumor-bearing zebrafish can be generated rapidly and inexpensively, these models may serve as a powerful tool to triage drug-leads from HTS for formal efficacy testing in mice.

Activation of Sonic hedgehog signaling in neural progenitor cells promotes glioma development in the zebrafish optic pathway.

Dysregulation of Sonic hedgehog (Shh) signaling has been implicated in glioma pathogenesis. Yet, the role of this pathway in gliomagenesis remains controversial because of the lack of relevant animal models. Using the cytokeratin 5 promoter, we ectopically expressed a constitutively active zebrafish Smoothened (Smoa1) in neural progenitor cells and analyzed tumorigenic capacity of activated Shh signaling in both transient and stable transgenic fish. Transient transgenic fish overexpressing Smoa1 developed retinal and brain tumors, suggesting smoa1 is oncogenic in the zebrafish central nervous system (CNS). We further established stable transgenic lines that simultaneously developed optic pathway glioma (OPG) and various retinal tumors. In one of these lines, up to 80% of F1 and F2 fish developed tumors within 1 year of age. Microarray analysis of tumor samples showed upregulated expression of genes involved in the cell cycle, cancer signaling and Shh downstream targets ptc1, gli1 and gli2a. Tumors also exhibited specific gene signatures characteristic of radial glia and progenitor cells as transcriptions of radial glia genes cyp19a1b, s100ß, blbp, gfap and the stem/progenitor genes nestin and sox2 were significantly upregulated. Overexpression of GFAP, S100ß, BLBP and Sox2 was confirmed by immunofluorescence. We also detected overexpression of Mdm2 throughout the optic pathway in fish with OPG, therefore implicating the Mdm2-Tp53 pathway in glioma pathogenesis. In conclusion, we demonstrate that activated Shh signaling initiates tumorigenesis in the zebrafish CNS and provide the first OPG model not associated with neurofibromatosis 1.

Suitable calcineurin inhibitor concentrations for liver transplant recipients in the Chinese population.

AIM: The aim was to deduce suitable calcineurin inhibitor concentrations for the Chinese liver transplantation population. METHODS: We retrospectively studied 97 liver transplant recipients who displayed stable liver and renal function. No grafts were obtained from prisoners, procurements were performed with donor consent conforming to international ethics regulations. At 3, 6, and 12 months, we increased the concentrations and doses of calcineurin inhibitors as well as the values of alanine transaminase and serum creatinine. RESULTS: Twenty-eight recipients received cyclosporine and 69 tacrolimus. The mean cyclosporine daily dosages were 203 ± 62 mg at 3, 188 ± 55 mg at 6, and 173 ± 52 mg at 12 months, the tacrolimus daily dosages were 3.08 ± 0.98, 2.82 ± 0.98, and 2.58 ± 0.93 mg, respectively. The corresponding mean cyclosporine peak concentrations (C(2)) were 806 ± 322 ng/mL, 681 ± 206 ng/mL, and 644 ± 190 ng/mL and the mean tacrolimus trought concentrations (C(0)) 6.61 ± 3.02 ng/mL, 5.85 ± 2.44 ng/mL, and 5.22 ± 2.33 ng/mL, respectively. In both groups, transaminases and serum creatinine were stable over time. CONCLUSIONS: An individualized immunosuppressive regimen for the local population is necessary. We delayed calcineurin inhibitors with subsequent low-dose mycophenolate mofetil plus minimized calcineurin inhibitors, which seemed to be nephroprotective and safe for Chinese liver transplantation patients.

Droplet-based magnetically activated cell separation.

In this study, we developed a method that target cells in suspension can be separated by combining magnetic force and gravitation force. Since the newly developed method involves a separating process of a droplet containing nontarget cells in suspension by applying magnetic force to separate target cells, we called it droplet-based magnetic activated cell sorting (dMACS). To demonstrate the efficiency of the dMACS system, Ter119 (+) cells from mouse bone marrow cells were separated by both conventional MACS and our dMACS systems. Effects of three parameters on separation efficiency were examined in the dMACS system. As a result, both volume of droplet of cell suspension, and magnetic force did not affect the efficiency of cell separation markedly. However, the time for cell settlement in the droplet showed a critical role in the efficiency of cell separation according to increasing time. Therefore, we tried to verify that the saturation time affected increase of its efficiency and that flow rate injected to get rid of the negative cell resulted in the decrease of its efficiency. Using this dMACS system, we were able to pinpoint that the flow rate of cell suspension injected into a magnetic platform results in disturbance in the droplet, leading to turbulence in the cell suspension.

Temporal regulation of a paired-like homeodomain repressor/TLE corepressor complex and a related activator is required for pituitary organogenesis.

Understanding the functional significance of the coordinate expression of specific corepressors and DNA-binding transcription factors remains a critical question in mammalian development. During the development of the pituitary gland, two highly related paired-like homeodomain factors, a repressor, Hesx1/Rpx and an activator, Prop-1, are expressed in sequential, overlapping temporal patterns. Here we show that while the repressive actions of Hesx1/Rpx may be required for initial pituitary organ commitment, progression beyond the appearance of the first pituitary (POMC) lineage requires both loss of Hesx1 expression and the actions of Prop-1. Although Hesx1 recruits both the Groucho-related corepressor TLE1 and the N-CoR/Sin3/HDAC complex on distinct domains, the repressor functions of Hesx1 in vivo prove to require the specific recruitment of TLE1, which exhibits a spatial and temporal pattern of coexpression during pituitary organogenesis. Furthermore, Hesx1-mediated repression coordinates a negative feedback loop with FGF8/FGF10 signaling in the ventral diencephalon, required to prevent induction of multiple pituitary glands from oral ectoderm. Our data suggest that the opposing actions of two structurally-related DNA-binding paired-like homeodomain transcription factors, binding to similar cognate elements, coordinate pituitary organogenesis by reciprocally repressing and activating target genes in a temporally specific fashion, on the basis of the actions of a critical, coexpressed TLE corepressor.

Fertile and diploid nuclear transplants derived from embryonic cells of a small laboratory fish, medaka (Oryzias latipes).

Fertile and diploid nuclear transplants were successfully generated by using embryonic cells as donors in a small laboratory fish, medaka (Oryzias latipes). Embryonic cell nuclei from transgenic fish carrying the green fluorescent protein (GFP) gene were transplanted into unfertilized eggs enucleated by x-ray irradiation. In this study, 1 out of 588 eggs transplanted in the first experiment and 5 out of 298 eggs transplanted in the second experiment reached the adult stage. All of these nuclear transplants were fertile and diploid, and the natural and GFP markers of the donor nuclei were transmitted to the F(1) and F(2) offspring in a Mendelian fashion. This systematic study proves the feasibility of generating nuclear transplants by using embryonic cells from fish as donors, and it is supported by convincing evidence.

Green fluorescent protein (GFP) transgenic fish and their applications.

The coupling of the GFP reporter system with the optical clarity of embryogenesis in model fish such as zebrafish and medaka is beginning to change the picture of transgenic fish study. Since the advent of first GFP transgenic fish in 1995, GFP transgenic fish technology have been quickly employed in many areas such as analyses of gene expression patterns and tissue/organ development, dissection of promoters/enhancers, cell lineage and axonal pathfinding, cellular localization of protein products, chimeric embryo and nuclear transplantation, cell sorting, etc. The GFP transgenic fish also have the potentials in analysis of upstream regulatory factors, mutagenesis screening and characterization, and promoter/enhancer trap. Our own studies indicate that GFP transgenic fish may become a new source of novel variety of ornamental fish. Efforts are also being made in our laboratory to turn GFP transgenic fish into biomonitoring organisms for surveillance of environmental pollution.

Effect of all-trans retinoic acid and arsenic trioxide on tissue factor expression in acute promyelocytic leukemia cells.

OBJECTIVE: To study the effect of all-trans retinoic acid (ATRA) and arsenic troxide (As2O3) on tissue factor (TF) expression and procoagulant activity (PCA) of acute promyelocytic leukemia (APL) cells in vivo and in vitro. METHODS: PCA from freshly isolated APL blasts from APL patients treated with ATRA or As2O3 was detected using a one-stage clotting assay. TF antigen was detected by ELISA and TF mRNA by RT-PCR. The maturation sensitive (NB4) or resistant subclones (NB4-R1) of the promyelocytic NB4 cell line, as well as U937 cells infected with pMSCV-PML-RARa treated with or without ATRA or As2O3, were also examined. RESULTS: Both ATRA and As2O3 can down-regulate the TF antigen, its mRNA transcription and membrane PCA of APL cells in vivo and in vitro, in a time-dependent manner. The TF antigen level in PML-RARa + U937 cells was significantly higher than that in U937 cells infected with retrovirus vector. Both ATRA and As2O3 can also down-regulate the TF antigen in U937 cells transfected with or without PML-RARa. CONCLUSION: Tissue factor expression and PCA in APL cells may be down-regulated by ATRA and As2O3. By down-regulating TF expression, As2O3 might also be used to improve the DIC-related hemorrhage in APL. Our data indicate that elevated TF antigen in PML-RARa + U937 may be related to the fusion protein PML-RARa. The down-regulating effect of ATRA and As2O3 on TF expression in U937 cells might not involve this fusion protein.

Cloning of a cDNA encoding cathepsin D from salamander, Hynobius leechii, and its expression in the limb regenerates.

Cathepsin D is a major lysosomal aspartic proteinase participating in the degradation or modification of intra- and extracellular matrix molecules, and its activity is known to increase in the process of tissue reorganization during the early phase of salamander limb regeneration. Here, we report the cloning of a salamander cathepsin D cDNA from Hynobius leechii and its expression profile in normal and retinoic acid (RA) treated limb regenerates. The gene expression of cathepsin D increased notably during the dedifferentiation stage and decreased gradually thereafter. Furthermore, RA that enhances dedifferentiation of regenerating salamander limb caused the elevation of cathepsin D expression both in terms of level and duration. These results suggest that cathepsin D plays important role(s) in the dedifferentiation process, and enhancement of cathepsin D expression might be closely related to RA-evoked pattern duplication.

Fringe forms a complex with Notch.

The Fringe protein of Drosophila and its vertebrate homologues function in boundary determination during pattern formation. Fringe has been proposed to inhibit Serrate-Notch signalling but to potentiate Delta-Notch signalling. Here we show that Fringe and Notch form a complex through both the Lin-Notch repeats and the epidermal growth factor repeats 22-36 (EGF22-36) of Notch when they are co-expressed. The Abruptex59b (Ax59b) and AxM1 mutations, which are caused by missense mutations in EGF repeats 24 and 25, respectively, abolish the Fringe-Notch interaction through EGF22-36, whereas the l(1)N(B) mutation in the third Lin-Notch repeat of Notch abolishes the interaction through Lin-Notch repeats. Ax mutations also greatly affect the Notch response to ectopic Fringe in vivo. Results from in vitro protein mixing experiments and subcellular colocalization experiments indicate that the Fringe-Notch complex may form before their secretion. These findings explain how Fringe acts cell-autonomously to modulate the ligand preference of Notch and why the Fringe-Notch relationship is conserved between phyla and in the development of very diverse structures.

Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters.

Although the zebrafish has become a popular model organism for vertebrate developmental and genetic analyses, its use in transgenic studies still suffers from the scarcity of homologous gene promoters. In the present study, three different zebrafish cDNA clones were isolated and sequenced completely, and their expression patterns were characterized by whole-mount in situ hybridization as well as by Northern blot hybridization. The first clone encodes a type II cytokeratin (CK), which is specifically expressed in skin epithelia in early embryos and prominently expressed in the adult skin tissue. The second clone is muscle specific and encodes a muscle creatine kinase (MCK). The third clone, expressed ubiquitously in all tissues, is derived from an acidic ribosomal phosphoprotein P0 (arp) gene. In order to test the fidelity of zebrafish embryos in transgenic expression, the promoters of the three genes were isolated using a rapid linker-mediated PCR approach and subsequently ligated to a modified green fluorescent protein (gfp) reporter gene. When the three hybrid GFP constructs were introduced into zebrafish embryos by microinjection, the three promoters were activated faithfully in developing zebrafish embryos. The 2.2-kb ck promoter was sufficient to direct GFP expression in skin epithelia, although a weak expression in muscle was also observed in a few embryos. This pattern of transgenic expression is consistent with the expression pattern of the endogenous cytokeratin gene. The 1.5-kb mck promoter/gfp was expressed exclusively in skeletal muscles and not elsewhere. By contrast, the 0.8-kb ubiquitous promoter plus the first intron of the arp gene were capable of expressing GFP in a variety of tissues, including the skin, muscle, lens, neurons, notochord, and circulating blood cells. Our experiments, therefore, further demonstrated that zebrafish embryos can faithfully express exogenously introduced genes under the control of zebrafish promoters.