The matrix protein Tiggrin regulates plasmatocyte maturation in Drosophila larva.

The lymph gland (LG) is a major source of hematopoiesis during Drosophila development. In this tissue, prohemocytes differentiate into multiple lineages, including macrophage-like plasmatocytes, which comprise the vast majority of mature hemocytes. Previous studies have uncovered genetic pathways that regulate prohemocyte maintenance and some cell fate choices between hemocyte lineages. However, less is known about how the plasmatocyte pool of the LG is established and matures. Here, we report that Tiggrin, a matrix protein expressed in the LG, is a specific regulator of plasmatocyte maturation. Tiggrin mutants exhibit precocious maturation of plasmatocytes, whereas Tiggrin overexpression blocks this process, resulting in a buildup of intermediate progenitors (IPs) expressing prohemocyte and hemocyte markers. These IPs likely represent a transitory state in prohemocyte to plasmatocyte differentiation. We also found that overexpression of Wee1 kinase, which slows G2/M progression, results in a phenotype similar to Tiggrin overexpression, whereas String/Cdc25 expression phenocopies Tiggrin mutants. Further analysis revealed that Wee1 inhibits plasmatocyte maturation through upregulation of Tiggrin transcription. Our results elucidate connections between the extracellular matrix and cell cycle regulators in the regulation of hematopoiesis.

Wnt-mediated repression via bipartite DNA recognition by TCF in the Drosophila hematopoietic system.

The Wnt/ß-catenin signaling pathway plays many important roles in animal development, tissue homeostasis and human disease. Transcription factors of the TCF family mediate many Wnt transcriptional responses, promoting signal-dependent activation or repression of target gene expression. The mechanism of this specificity is poorly understood. Previously, we demonstrated that for activated targets in Drosophila, TCF/Pangolin (the fly TCF) recognizes regulatory DNA through two DNA binding domains, with the High Mobility Group (HMG) domain binding HMG sites and the adjacent C-clamp domain binding Helper sites. Here, we report that TCF/Pangolin utilizes a similar bipartite mechanism to recognize and regulate several Wnt-repressed targets, but through HMG and Helper sites whose sequences are distinct from those found in activated targets. The type of HMG and Helper sites is sufficient to direct activation or repression of Wnt regulated cis-regulatory modules, and protease digestion studies suggest that TCF/Pangolin adopts distinct conformations when bound to either HMG-Helper site pair. This repressive mechanism occurs in the fly lymph gland, the larval hematopoietic organ, where Wnt/ß-catenin signaling controls prohemocytic differentiation. Our study provides a paradigm for direct repression of target gene expression by Wnt/ß-catenin signaling and allosteric regulation of a transcription factor by DNA.

Do we have a workable clinical protocol for differentiating lympho-hematopoietic stem cells from the source of embryonic stem cells and induced pluripotent stem cells in culture?

In recent years, many researchers are focusing on deriving lympho-hematopoietic stem cells (L-HSC) from human embryonic stem cells (ESC) and/or induced pluripotent stem cells (iPSC) in culture as alternative sources for transplantation. Two protocols are available for research purposes: mouse stroma cell line coculture system and embryoid bodies (EBs) suspension culture system. However, due to the lack of human stroma cell line, which could support the derivation of L-HSC in culture, the generation of therapeutic lympho-hematopoietic cells for clinical purpose can only be achieved using EBs suspension culture system. In this short communication/review, the results of EBs suspension culture system using mouse and human ESC/iPSC are summarized and the potential clinical application is discussed.

Some properties and applications of cell lines and clones established from tet-responsive-SV40 tag mice and mES cell lines.

In this article, 11 cell lines established from transgenic mice and mouse embryonic stem cells (mESC) expressing SV40Tag under control of tetracycline/doxycycline (tet-off, tet-on) are described. Several cell lines were further transfected with a plasmid vector containing genes coding for a cytokine/protein under tet-regulation to obtain tet-co-regulated expression of cytokine/protein. A total of 29 clones and 234 subclones have been established so far. Partial characterization of these tet-responsive cell lines, clones and subclones was performed. Questions related to the rare frequency of establishing permanent cycling cell lines from this source, the unusual expression pattern of SV40Tag protein in the subcellular compartment and the phenotype of 'stemness' of several such cell lines are raised. Some future applications of these cells, related to immunology and transplantation, are discussed.

Prenatal exposure of testosterone prevents SDN-POA neurons of postnatal male rats from apoptosis through NMDA receptor.

The role of N-methyl-D-aspartate (NMDA) receptor in mediating the effect of testosterone exposure prenatally on neuronal apoptosis in the sexual dimorphic nucleus of the preoptic area (SDN-POA) of rats was studied. The endogenous testosterone was diminished by prenatal stress (PNS) or simulated by testosterone exposure (TE) to understand the effect of testosterone on NR(1) (a functional subunit protein of NMDA receptor) expression and neuronal apoptosis. To further study whether the testosterone, after being converted into estradiol, modulates NR(1) expression, 4-androstein-4-ol-3,17-dione (ATD; an aromatase inhibitor) was used to block the conversion of estradiol from testosterone. The expressions of the NR(1) mRNA and NR(1) subunit protein were quantified by RT-PCR and western blotting analysis, respectively. In addition, a noncompetitive antagonist of NMDA receptor, MK-801, was used to find out whether blockage of NMDA receptor affects the naturally occurring apoptosis in SDN-POA. The results showed the following. 1) Expression of perinatal NR(1) subunit protein in the central part of the medial preoptic area of male rats was significantly higher than that of females, especially on postnatal days 1 and 3. 2) The testosterone level of male fetuses on embryonic day 18 was significantly higher than that of females, while the testosterone level of TE females or PNS males was similar to that of intact males or intact females, respectively. 3) The apoptotic incidence of intact male rats was significantly less than that of females, and the apoptosis was stimulated by PNS in male or inhibited by TE in female. 4) The expression of NR(1) subunit protein could be inhibited by PNS or ATD-treatment in male, while stimulated by TE in female. 5) NR(1) mRNA showed no significant difference among intact male, PNS male, ATD-treated male, TE female and intact female rats. 6) The low apoptotic incidence of male rats was significantly increased when NMDA receptor was blocked by MK-801. These results suggest that testosterone, after being converted to estradiol, may prevent the SDN-POA neurons of male rats from apoptosis through enhancing the expression of NR(1) at the posttranscriptional level.

Sit-to-stand at different periods of pregnancy.

OBJECTIVE: This study was performed to determine the biomechanics of chair rising by pregnant women. DESIGN: Relative body joint position and ground reaction forces were measured by a motion analysis system and one force plate. BACKGROUND: Physiological and psychological changes during pregnancy impose postural demands and limit the performance of daily living activities such as rising from sitting to standing position. METHODS: Twenty-four pregnant women, divided into three groups, were studied performing sit-to-stand transition from an armless and adjustable chair. By kinematic and kinetic analysis, the angles and moments of hip, knee and ankle joints were investigated. RESULTS: The chair height has great influence on knee joint and hip joint moments, but less on ankle joints. In the third trimester for all chair heights, because of a marked increase in abdominal depth, the maximum hip moment is significantly less than that in first trimester, while the maximum knee moment is significantly larger. Pregnant women in third trimester produced larger knee moment during sit-to-stand transition from lower chair height. CONCLUSIONS: The mechanism of sit-to-stand is affected by the physical changes of pregnant women at different periods of pregnancy, e.g. increased loading of knee joint and decreased hip joint moment, especially in the last trimester period of pregnancy.

Cell-based delivery of cytokines allows for the differentiation of a doxycycline inducible oligodendrocyte precursor cell line in vitro.

BACKGROUND: Stem cells, having the property of self renewal, offer the promise of lifelong repair of damaged tissue. However, somatic tissue-committed primary stem cells are rare and difficult to expand in vitro. Genetically modified stem-like cells with the ability to expand conditionally provide a valuable tool with which to study stem cell biology, especially the cellular events of proliferation and differentiation. In addition, stem cells may be appropriate candidates for therapeutic applications. METHODS: Double transgenic mice possesing SV40 T antigen (Tag) under the control of the reverse tetracycline-transactivator (rtTA) were used to establish cell lines. One brain cell line was partially characterized by DNA sequencing, morphology, antigen expression using flow cytometry, confocal microscopy, and electrophysiology using the patch clamp technique. Cell cycle analysis was performed using propidium iodide staining; cell viability and H3-thymidine incorporation assays. The ability of this cell line to differentiate was assessed by confocal microscopy following co-culture with stem cells secreting cytokines. RESULTS: We report here the establishment and partial characterization of a cell line derived from the brain tissue of rtTA-SV40 Tag transgenic mice. Analysis of the morphology and antigen markers has shown that this cell line mimics some aspects of primary glial precursors. The results of electrophysiology are consistent with this and suggest that the cell line is derived from O2A glial precursor cells. Cell cycle progression of this cell line is doxycycline-dependent. In the absence of doxycycline, cells become apoptotic. Differentiation into mature type 2 astrocytes and (precursor) oligodendrocytes can be induced upon withdrawal of doxycycline and addition of epithelial stem cells secreting cytokine, such as hIL3 (human Interleukine 3) or hIL6 to the culture. In contrast, co-culturing with hCNTF (human Ciliary NeuroTrophic Factor)-secreting epithelial stem cells did not induce them to mature into progeny cell types. CONCLUSION: The differentiation of this O2A glial precursor line does not occur automatically in culture. Additional external help is required from the cell-based delivery of appropriate transgenic cytokines. Withdrawal of doxycycline from the culture medium removes the proliferation signals and induces a fatal outcome.

p21Cip1/Waf1 disrupts the recruitment of human Fen1 by proliferating-cell nuclear antigen into the DNA replication complex.

Fen1 or maturation factor 1 is a 5'-3' exonuclease essential for the degradation of the RNA primer-DNA junctions at the 5' ends of immature Okazaki fragments prior to their ligation into a continuous DNA strand. The gene is also necessary for repair of damaged DNA in yeast. We report that human proliferating-cell nuclear antigen (PCNA) associates with human Fen1 with a Kd of 60 nM and an apparent stoichiometry of three Fen1 molecules per PCNA trimer. The Fen1-PCNA association is seen in cell extracts without overexpression of either partner and is mediated by a basic region at the C terminus of Fen1. Therefore, the polymerase delta-PCNA-Fen1 complex has all the activities associated with prokaryotic DNA polymerases involved in replication: 5'-3' polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. Although p21, a regulatory protein induced by p53 in response to DNA damage, interacts with PCNA with a comparable Kd (10 nM) and a stoichiometry of three molecules of p21 per PCNA trimer, a p21-PCNA-Fen1 complex is not formed. This mutually exclusive interaction suggests that the conformation of a PCNA trimer switches such that it can either bind p21 or Fen1. Furthermore, overexpression of p21 can disrupt Fen1-PCNA interaction in vivo. Therefore, besides interfering with the processivity of polymerase delta-PCNA, p21 also uncouples Fen1 from the PCNA scaffold.

Comparison of myocardial ischaemia in post-infarction patients with and without angina: assessment by dipyridamole 201T1 imaging.

Ninety patients with old myocardial infarction were studied to evaluate the efficacy of intravenous dipyridamole 201Tl imaging for the detection of myocardial ischaemia in post-infarction patients, and to compare the prevalence of ischaemia in 63 patients with post-infarction angina (group I) and 27 patients without angina (group II). Thirty-four of the patients in group I and 15 of the patients in group II received coronary arteriography (CAG) for comparison; these were labelled groups IA and IIA, respectively. On 201Tl imaging, the incidence of scar with ischaemia in the infarct zone and scar with ischaemia at a distance were 72% and 42% in all patients, 89% and 52% in group I vs 33% and 19% in group II (P < 0.001 and P < 0.01, respectively). On CAG testing, the rates of infarct-related recanalization vessel and multi-vessel disease were 76% and 68% in group IA vs 40% and 40% in group IIA (P < 0.05 and P > 0.05, respectively). Thus, dipyridamole 201Tl imaging is a useful modality and post-infarction angina a proper indicator in the detection of myocardial ischaemia in post-infarction patients

Technetium-99m albumin scintigraphy in the diagnosis of protein-losing enteropathy: a case report.

A 34-year-old female presenting with bilateral lower leg edema and distended abdomen was admitted to our hospital. The serum albumin was 1.42g/dl. Renal function and hepatic function were normal. Urinalysis did not show proteinuria. Tc-99m albumin scintigraphy was arranged for this patient to rule out protein-losing enteropathy. The results demonstrated loss of albumin into the intestines. We conclude that Tc-99m albumin scintigraphy of the abdomen is a valuable adjunct in the diagnosis of protein-losing enteropathy.

Development of mouse embryonic stem (ES) cells: IV. Differentiation to mature T and B lymphocytes after implantation of embryoid bodies into nude mice.

Mouse embryonic stem (ES) cells in culture can differentiate into late stages of many lineage-committed precursor cells. Under appropriate organ-culture conditions, ES cells differentiate into lymphoidlike cells at a stage equivalent to lymphoid cells found in fetal liver. These hematopoietic precursors are located in cup-shaped structures found in some embryoid bodies; we called such embryoid bodies "ES fetuses." In this study, we have followed the maturation of hematopoietic cells after implantation of ES fetuses into nude mice for 3 weeks. ES-cell-derived lymphoid cells-pre-B cells, mature B cells, and mature T cells were found in all lymphoid organs. Interestingly, there was also an increase of T cells of host origin. Because native nude mouse lack thymus, these T cells might be educated by thymuslike epithelium generated from ES fetuses. Practical applications of this combined in vitro and in vivo system are discussed.

Comparison of intravenous dipyridamole and adenosine Tc-99m MIBI myocardial imaging for detection of coronary artery disease.

A total of 44 patients referred for Tc-99m MIBI myocardial imaging for detection of coronary artery disease (CAD) were studied to compare the differences in heart beat, blood pressure, electrocardiographic changes and side effects during intravenous infusion of dipyridamole (ID) and adenosine (IA) and also to determine the degree of concordance between ID and IA Tc-99m MIBI imaging. These patients were divided into two groups: 20 suspected CAD patients constituted group I and 24 proven CAD patients formed group II. All patients received ID 0.56 mg/kg for 4 min and within about 10 days IA 0.14 microgram/kg/min for 6 min with Tc-99m MIBI imaging. The results revealed that maximal heart beat increased and maximal systolic blood pressure decreased in both IA and ID patients with no statistically significant differences. Transient second-degree AV block occurred with IA in 3 patients. Side effects, such as, chest pain, headache, dizziness and shortness of breath occurred more often and were in general more intense in IA patients, but they were typically mild and resolved spontaneously within 1 or 2 min of discontinuation of IA. Both IA and ID Tc-99m MIBI imaging were normal in 18 of 20 group I patients and were concordant for the presence of perfusion defects in the other 2 patients. Of 24 group II patients, all had myocardial perfusion defects on both tests and were concordant for the severity of the perfusion abnormalities. However, in other 2 patients. Of 24 group II patients, all had myocardial perfusion defects on both tests and were concordant for the severity of the perfusion abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)

One Herbert double-threaded compression screw fixation of displaced type II odontoid fractures.

Surgical treatment of type II odontoid fractures (OFs) has usually entailed C1-2 arthrodesis rather than fracture fixation. An alternative treatment of direct screw fixation is used to treat the fractures for preservation of atlantoaxial rotation. Type II OFs that cannot be completely reduced by close means are generally believed to be a contraindication for anterior screw fixation. Seven patients (group I) with displaced type II OFs that could be completely reduced were treated with fracture fixation by one 4.5-mm double-threaded compression screw and five patients (group II) with displaced type II OFs that could only be partially reduced were treated with fracture fixation by one 3.0-mm double-threaded compression screw. All patients had a minimum of 1-year follow-up. No major complications occurred. No loss of reduction occurred in group I patients. Group II patients had an average loss of reduction of 0.8 mm anterior displacement and 5 degrees anterior angulation. The overall rate of fracture union was 100%, and fracture resolution averaged 4.1 months. Ten patients had a normal range of cervical rotation, and there was no difference in preservation of cervical rotation between the two groups. Our results suggest that close reduction and compressive osteosynthesis by one double-threaded compression screw is an optimal method of treatment for displaced type II OFs that can be completely reduced and for some cases that can only be partially reduced. A 100% rate of fracture union and preservation of cervical rotation are the major advantages of this method. However, significant complications have been reported by other investigators. (ABSTRACT TRUNCATED AT 250 WORDS)

The role of ultrasound in scrotal trauma.

We reviewed the sonograms of 16 cases with scrotal trauma and documented the spectrum of abnormalities that may be present in sonography after a scrotal injury. The sonographic findings included scrotal hematoceles, post-traumatic epididymitis, epididymal hematoma, testicular hematoma and/or infarction, testicular rupture, testicular swelling and hyperemia. Ultrasound is able to distinguish intratesticular and extratesticular lesions. The integrity of the testis could be evaluated as well. It should be the imaging modality of choice for the evaluation of scrotal trauma.

Differentiation of mouse embryonic stem cells in vitro: III. Morphological evaluation of tissues developed after implantation of differentiated mouse embryoid bodies.

Mouse embryonic stem cells (ES) were allowed to differentiate in a liquid culture system. After 2-3 weeks, complex cystic embryoid bodies developed. These bodies were composed of several structures identified as cardiac muscle and yolk sac blood islands as well as cup-shape compartments containing a mixed population of hematopoietic stem cells. When these cystic embryoid bodies were implanted into adult mice, either subcutaneously or under the kidney capsule, they developed into various tissues. These included bone, blood vessels, cardiac muscle, nerves, and skin with hair follicles. In addition, highly differentiated, complicated tissues resembling intestinal epithelium with mucus glands or salivary glandular tissue were derived. The ES tissues from these in vitro developed embryoid bodies developed quickly within 2 to 3 weeks of implantation. This is in contrast to a minimal of 6 weeks for teratocarcinomas derived from embryonic carcinoma cells and/or the direct implantation of undifferentiated embryonic stem cells. Moreover, we found that there are different types of tissue developed upon different sites of implantation. The data suggest a local environment and/or growth factors are influential for ES tissue development. This system provides a possible means to purify and identify stem cells that give rise to specific tissues, and to study the factors regulating the commitment of these stem cells.

Application of BrdU/Hoechst-ethidium bromide two parameter flow cytometry in studying synchronous and non-synchronous mouse cells.

BrdU/Hoechst-EB bivariate flow cytometry has a wide application in the study of factors controlling cell cycle for asynchronous cells such as embryonic stem cells (ES), and for synchronous cells such as stimulated resting B cells (Bo). The technique allows one to calculate the average cell cycle duration time. ES cells are found to cycle every 8-10 h, and most B cells are 11-12 h, but there is a small subset of B cells with a cycle time of only 6-7 h. Using this technique, we also study the roles of different T lymphocytes on B cell activation when B cells are stimulated with anti-IgM antibodies (commonly used, anti-mu). Exposure to anti-mu recruits small B cells into the cell cycle, but arrests them in the G1 phase of the second cycle. Interleukin (IL) 4 is a costimulator of anti-mu. In addition, IL-4 is an S-phase progression factor. Contrary to that seen when B cells are stimulated by other mitogens, very few cells are in the G2 compartments after anti-mu plus IL-4 stimulation. This phenomenon is reminiscent of embryonic cells. Our findings provide strong evidence to propose that there are two restriction points for B cell activation: at the transition from G0 to G1 and at the transition from G1 to S phase.

Establishment and characterization of lymphoid and myeloid mixed-cell populations from mouse late embryoid bodies, "embryonic-stem-cell fetuses".

Mouse embryonic stem (ES) cells have the potential to differentiate into embryoid bodies in vitro and mimic normal embryonic development. The "ES fetus" is a specific development at a late stage seen under our culture conditions. We have established several mixed populations from ES fetuses by using combinations of retroviruses carrying different oncogenes (v-abl, v-raf, c-myc), interleukins 2 and 3, and Con A. Six groups of mixed populations were characterized by immunophenotyping. For some groups, transfer of cells into sublethally irradiated mice resulted in the development of macrophages, mature T and B lymphocytes, and plasma cells of donor origin. Thus, these mixed populations may contain immortalized precursors of hematopoietic lineages. These mixed populations should be valuable for defining hematopoietic stem cells and their committed progenitors.