Mechanisms of action of differentiation inducers: detection of inducer binding protein(s) in murine erythroleukemia cells

We have shown previously that murine erythroleukemia (MEL) and human neuroectodermal RD/TE-671 cells are induced to differentiate by ureido derivatives of pyridine (UDPs) and may contain inducer binding protein(s). In the present study, we prepared radiolabeled [3H]UDP [2-(3-ethylureido)-6-[3H]-acetylaminopyridine] as ligand and investigated whether it interacts selectively with novel binding proteins. MEL and RD/TE-671 cells, incubated with the inducer [3H]UDP and subsequently fractionated, yielded a radiolabeled postmitochondrial soluble fraction containing the [3H]UDP-protein complex. We purified the UDP binding protein by using UDP-sepharose affinity chromatography, gel filtration, and SDS-PAGE electrophoresis and analyzed its structure. The data presented here indicate for the first time that the inducer UDP interacts with a 38,333 +/- 30 Da binding protein(s) (p38), of unknown function, in both cell lines. Microsequencing and sequence alignment search revealed that the p38 protein(s) contains at least two homologous domains, one being part of ABC-type transporters and another found in the Wingless-type (Wnt) proteins. Kinetic analysis revealed that the p38 forms a relatively stable protein complex with [3H]UDP that accumulates within the cytosol and nucleus of MEL cells during the precommitment period. This complex, however, decays later on after commitment to erythroid maturation has been initiated. De novo protein and mRNA synthesis is needed for the UDP-p38 complex to form, as shown by the use of metabolic inhibitors. Purified p38 was used to develop an anti-p38 polyclonal serum, and Western blot analysis revealed that the level of p38 was quite similar in both UDP-inducible and -resistant MEL subclones that we developed. Although only a portion of the primary structure of the p38 is known from microsequencing, the mechanism by which the UDP-p38 complex contributes to induction of differentiation in both UDP-responsive mouse MEL and human RD/TE-671 cells is discussed.     

云南省恶性疟原虫多药抗性基因Asn86Tyr多态性调查

目的检测云南恶性疟原虫多药抗性基因(Pfmdr1)Asn86Tyr的多态性,探索其与氯喹敏感性表现型的关系.方法用等位基因特异PCR和限制性酶切片段长度分析技术(AS-PCR/RFLP).结果在86%(19/22)的氯喹抗性分离株中检测到Pfmdr1基因的Asn86Tyr两种多态性,携带编码86Tyr多态变异的占50%,而含编码Asn86多态的也是50%.结论在云南氯喹抗性高度流行区,Pfmdr1基因Asn86Tyr的多态性是普遍存在的.     

Analysis of the inhibition of commitment of murine erythroleukemia (MEL) cells to terminal maturation by N6-methyladenosine.

Treatment of cultured murine erythroleukemia (MEL or Friend) cells with N6-methylated derivatives of adenosine inhibited erythroid cell differentiation induced by various agents. N6-Methyladenosine (N6mAdo) inhibited initiation of commitment to terminal maturation and prevented accumulation of hemoglobin in a concentration-dependent manner. Treatment with N6mAdo slowed cell growth without causing substantial inhibition in the rate of DNA synthesis and a marked decrease in viability and clonogenic potential of MEL cells. Furthermore, N6mAdo decreased the cytoplasmic accumulation of beta(major) globin mRNA and affected its structural integrity in MEL cells. Cells pre-exposed to N6mAdo failed to initiate commitment as early as control cells upon challenge with the inducer dimethyl sulfoxide. N6mAdo-induced inhibition of commitment was not reversed but rather was potentiated by the presence of adenine, L-homocysteine and/or L-methionine, agents involved in the active methylation cycle. To this respect, N6mAdo-induced inhibition of commitment was found to be different from that caused by cordycepin (3'-deoxyadenosine, an inhibitor of RNA methylation and mRNA polyadenylation). The latter inhibition was fully reversed by the addition of L-methionine. These findings indicate that N6-methyladenosine: (a) blocks a central process that is required for initiation of commitment; and (b) decreases accumulation of beta (major) globin mRNA, causes mRNA degradation and prevents hemoglobin synthesis. Due to the differential sensitivity of N6mAdo- and cordycepin-induced blockade of commitment to L-methionine, these agents inhibit commitment by acting via two different mechanisms impinging on the final pathway of MEL erythroid cell maturation.     

Spiral-shaped biliary endoprosthesis: initial study

A new flexible, spiral-shaped biliary endoprosthesis is described. This spiral endoprosthesis can negotiate acute angles, can be cut to any length, and has been specifically designed to deal with the problem of stent migration. The authors report their initial experience with this endoprosthesis in 14 patients.     

Management of unstable angina

One-hundred and seven patients with unstable angina were treated between 1981 and 1987. Platelet dysfunction and elevation of fibrinogen was observed. The efficacy of aspirin, dipyridamole and heparin on coagulation profile is compared. Left main disease was seen in 11.4 per cent. Left anterior descending was occluded in 90.7 per cent. Aortocoronary bypass graft using saphenous vein graft was carried out on all patients. On an average, each patients received 3.5 grafts. Emergency surgery was done in nineteen. Associated ventricular aneurysm was resected in nine. Thirteen patients died. Operative mortality decreased to nil in 1986–1987. Perioperative infarction was seen in five patients. Clinical improvement was seen in 80.3 per cent and 48.9 per cent were asymptomatic. Actuarial survival was 92 per cent after six years. Postoperative coronary angiography in 28 patients showed patency of grafts in 18, partial patency in six and occlusion in four. Aspirin was found useful after surgery to relieve angina. Spiroergometry and Thallium²⁰¹ scintigraphy was useful to assess medical and surgical treatment.     

Anti-glomerular basement membrane disease and dual positivity for antineutrophil cytoplasmic antibody in a patient with membranous nephropathy

We present the case of a 50-year-old man who underwent kidney biopsy for nephrotic syndrome. In addition to a membranous pattern, anti-glomerular basement membrane (anti-GBM) staining was noted before manifestations of anti-GBM disease. Hematuria and renal failure ensued 2 weeks later. In addition, he had simultaneous circulating levels of anti-GBM antibody and both perinuclear (P-) and cytoplasmic (C-) antineutrophil cytoplasmic antibody (ANCA).     

Chemotherapeutic Delivery from a Self-Assembling Peptide Nanofiber Hydrogel for the Management of Glioblastoma

Purpose

Localized chemotherapy has gained significant impetus for the management of malignant brain tumors. In the present study, we appraised the versatility of an in-situ gel forming self-assembling peptide, ac-(RADA)₄-CONH₂, as a biocompatible delivery depot of the chemotherapeutic drug doxorubicin (DOX) and the anticancer agent curcumin (CUR), respectively.

Methods

The morphology and mechanical properties of ac-(RADA)₄-CONH₂ were assessed with scanning electron microscopy (SEM) and rheological studies. The in vitro drug release from ac-(RADA)₄-CONH₂ was monitored in phosphate-buffered saline pH 7.4. Distribution of the fluorescent actives within the peptide matrix was visualized with confocal laser scanning microscopy (CLSM). The in vitro biological performance of the ac-(RADA)₄-CONH₂-DOX and ac-(RADA)₄-CONH₂-CUR was evaluated on the human glioblastoma U-87 MG cell line.

Results

SEM studies revealed that the ac-(RADA)₄-CONH₂ hydrogel contains an entangled nanofiber network. Rheology studies showed that the more hydrophobic CUR resulted in a stiffer hydrogel compared with ac-(RADA)₄-CONH₂ and ac-(RADA)₄-CONH₂-DOX, due to the interaction of CUR with the hydrophobic domains of the peptide nanofibers as confirmed by CLSM. In vitro release studies showed a complete DOX release from ac-(RADA)₄-CONH₂ within 4 days and a prolonged release for ac-(RADA)₄-CONH₂-CUR over 20 days. An increased cellular uptake and a higher cytotoxic effect were observed for ac-(RADA)₄-CONH₂-DOX, compared with DOX solution. Higher levels of early apoptosis were observed for the cells treated with the ac-(RADA)₄-CONH₂-CUR, compared to CUR solution.

Conclusions

The current findings highlight the potential utility of the in-situ depot forming ac-(RADA)₄-CONH₂ hydrogel for the local delivery of both water soluble and insoluble chemotherapeutic drugs.

     

Management of malignant colon polyps: Current status and controversies

Colon cancer remains a significant clinical problem worldwide and in the United States it is the third most common cancer diagnosed in men and women.It is generally accepted that most malignant neoplasms of the colon arise from precursor adenomatous polyps.This stepwise progression of normal epithelium to carcinoma,often with intervening dysplasia,occurs as a result of multiple sequential,genetic mutations-some are inherited while others are acquired.Malignant polyps are defined by the presence of cancer cells invading through the muscularis mucosa into the underlying submucosa(T1).They can appear benign endoscopically but the presence of malignant invasion histologically poses a difficult and often controversial clinical scenario.Emphasis should be initially focused on the endoscopic assessment of these lesions.Suitable polyps should be resected en-bloc,if possible,to facilitate thorough evaluation by pathology.In these cases,proper attention must be given to the risks of residual cancer in the bowel wall or in the surrounding lymph nodes.If resection is not feasible endoscopically,thenthese patients should be referred for surgical resection.This review will discuss the important prognostic features of malignant polyps that will most profoundly affect this risk profile.Additionally,we will discuss effective strategies for their overall management.