New mutations in acetylcholine receptor subunit genes reveal heterogeneity in the slow-channel congenital myasthenic syndrome

Mutations in genes encoding the epsilon, delta, beta and alpha subunits of the end plate acetylcholine (ACh) receptor (AChR) are described and functionally characterized in three slow-channel congenital myasthenic syndrome patients. All three had prolonged end plate currents and AChR channel opening episodes and an end plate myopathy with loss of AChR from degenerating junctional folds. Genetic analysis revealed heterozygous mutations: epsilon L269F and delta Q267E in Patient 1, beta V266M in Patient 2, and alpha N217K in Patient 3 that were not detected in 100 normal controls. Patients 1 and 2 have no similarly affected relatives; in Patient 3, the mutation cosegregates with the disease in three generations. epsilon L269F, delta Q267E and beta V266M occur in the second and alpha N217K in the first transmembrane domain of AChR subunits; all have been postulated to contribute to the lining of the upper half of the channel lumen and all but delta Q267E are positioned toward the channel lumen, and introduce an enlarged side chain. Expression studies in HEK cells indicate that all of the mutations express normal amounts of AChR. epsilon L269F, beta V266M, and alpha N217K slow the rate of channel closure in the presence of ACh and increase apparent affinity for ACh; epsilon L269F and alpha N217K enhance desensitization, and epsilon L269F and beta V266M cause pathologic channel openings in the absence of ACh, rendering the channel leaky, delta Q267E has none of these effects and is therefore a rare polymorphism or a benign mutation. The end plate myopathy stems from cationic overloading of the postsynaptic region. The safety margin of neuromuscular transmission is compromised by AChR loss from the junctional folds and by a depolarization block owing to temporal summation of prolonged end plate potentials at physiologic rates of stimulation.      

Identification of three mutations and associated haplotypes in the protoporphyrinogen oxidase gene in South African families with variegate porphyria

Mutation analysis of genomic DNA samples obtained from 17 unrelated South African patients with variegate porphyria (VP) revealed three novel missense mutations in the protoporphyrinogen oxidase (PPOX) gene. A common C to T transition at nucleotide position 452 (R59W) was identified in 15 of the patients analysed, while base changes at positions 336 (H20P) and 779 (R168C) were identified in the remaining two patients. Using protein analysis software we were able to predict that all three mutations have a similar biophysical effect on the protein, being the disturbance of amphiphatic regions within the protein, which might result in misfolding of the protein. Mutation R59W, identified in the majority of South African VP families, was shown to create a Styl restriction site, while mutation R168C would abolish a Dsal restriction site in genomic DNA of affected individuals. As 100% of the index patients analysed were molecularly characterized, the combined use of restriction enzyme and single-strand conformation polymorphism (SSCP) analysis now allows a rapid and accurate diagnosis of VP in South Africa. Mutation R59W was furthermore shown to be in association with one of four potential haplotypes defined by two newly described polymorphisms in exon 1 of the PPOX gene. Our molecular data thus strongly support the founder hypothesis for VP in South Africa.      

Insuffisance rénale aiguë et infarctus cérébral au décours d’un traitement par immunoglobulines intraveineuses à fortes doses

Introduction. — Side-effects of immunoglobulins administered via the intravenous route are usually minor. However, acute renal failure and more rarely thrombotic events, including ischemic stroke, have been reported in association with immunoglobulin infusion. To our knowledge, no case of both acute renal failure and stroke following immunoglobulin has been described until now.Exegesis. — Two days after immunoglobulin infusion (2 g/kg), a patient who presented with autoimmune thrombocytopenia suffered severe acute renal failure associated with an ischemic stroke in the right anterior choroid artery territory. Moreover, the stroke worsened immediately following a second infusion (1 g/kg). Clinical and neuroradiological examinations were conducted.Conclusion. — Results indicate the need for both close monitoring of serum creatinine and diuresis before starting immunoglobulin therapy, and limiting the total dose to prevent thrombotic events such as stroke.     

Synergistic factors for stem cell proliferation: further studies of the target stem cells and the mechanism of stimulation by interleukin-1, interleukin-6, and granulocyte colony-stimulating factor

Serial observations of blast cell colony development from spleen cells of mice treated with 5-fluorouracil (5-FU) four days earlier revealed that either form of human interleukin-1 (IL-1 alpha or IL-1 beta) hastens the emergence of interleukin-3 (IL-3)-dependent blast cell colonies. This activity was essentially indistinguishable from the effect of interleukin-6 (IL-6) or granulocyte colony-stimulating factor (G-CSF) in the same system, an effect that we have ascribed previously to a shortening of the G0 period of the dormant stem cells. We also analyzed the time courses of colony formation from cultures of day-2 post-5-FU marrow cells supported by IL-1 alpha, IL-6, or G-CSF alone or in combination with IL-3. In the presence of IL-3, G-CSF and IL-6 but not IL-1 alpha hastened the development of colonies and increased the numbers of multilineage colonies relative to cultures of IL-3 alone. This observation, together with our previous data from the human system, suggests that the synergistic effect of IL-1 is likely due to induction of secondary growth factors, including IL-6 and G-CSF, by accessory cells in culture. The effect of IL-6 on G0 was confirmed by analysis of the cycling status of progenitor cells in short-term culture. While neither IL-3 nor IL-6 alone had any effect on the cycling status, the combination of factors resulted in a rapid recruitment of quiescent cells into cell cycle (within 48 hours) as represented by a twofold increase in the numbers of multipotential progenitors and a significant increase in the sensitivity of these cells to 3H-thymidine with high specific activity. Combinational testing of all of these synergistic factors revealed that the target cell populations for the IL-1, IL-6, and G-CSF overlap considerably, suggesting that they all may act through a common mechanism. This is further supported by our finding that cells from blast cell colonies grown in the presence of a combination of any one of the synergistic factors with IL-3 replate with higher efficiency and yield more multilineage secondary colonies than those from colonies grown in IL-3 alone. These findings provide further evidence that IL-1, IL-6, and G- CSF serve to integrate the immediate host responses to infection through augmentation of effector cells and antibody production as well as the longer term host responses by recruitment of dormant hemopoietic stem cells into active cell cycling.     

Regulation of hematopoiesis-IV: The role of interleukin-3 and bryostatin 1 in the growth of erythropoietic progenitors from normal and anemic W/Wv mice

We and others have established a role for T lymphocytes and their products in the regulation of erythropoiesis. Interleukin-3 (IL-3) is a multipotential lymphokine with burst-promoting activity that is produced by activated T lymphocytes. In the anemic, stem cell-defective W/Wv mouse we have described the absence of a functionally active thymocyte population that in normal animals enhances erythroid progenitor growth and stem cell self-renewal. In studies reported here we find that W/Wv mouse marrow responds to exogenous IL-3 by increased erythroid progenitor cell growth. The BFU-E and CFU-E from anemic donors are more sensitive to IL-3 than are those in +/+ marrow. We have recently observed a stimulatory effect of bryostatin 1 (a macrocyclic lactone derived from a marine invertebrate) on normal erythropoiesis in human bone marrow progenitor assays. To test the effects of this molecule on murine normal and anemic W/Wv cells we grew these cells in the presence of increasing doses of bryostatin 1. Bryostatin mimics the stimulatory action of IL-3 on W/Wv bone marrow. Polyclonal antibody directed against murine IL-3 blocks the stimulatory effect of bryostatin on erythropoiesis. Otherwise inactive thymocytes from W/Wv mice in coculture with W/Wv bone marrow showed stimulation of erythropoiesis in the presence of bryostatin. We believe that bryostatin may in part act by stimulating T lymphocytes to release physiologic concentrations of lymphokines.     

Erythropoietin induces association of the JAK2 protein tyrosine kinase with the erythropoietin receptor in vivo

Protein tyrosine phosphorylation has been hypothesized to play a key role in the growth signaling induced by erythropoietin (Epo), although the Epo receptor (EpoR), a member of the cytokine receptor superfamily, lacks a tyrosine kinase domain. Recently, the JAK2 tyrosine kinase was shown to be activated on Epo stimulation and to bind to the cytoplasmic domain of EpoR in vitro. To further explore the mechanisms of activation of JAK2 in EpoR-mediated signal transduction, we assessed the conditions for association of JAK2 with EpoR in vivo. Epo stimulation rapidly induced association of JAK2 with the EpoR in an interleukin 3 (IL-3)-dependent cell line transfected with the wild-type EpoR. On Epo stimulation JAK2 also associated with a truncated mutant EpoR (H-mutant), which is mitogenetically active but not tyrosine phosphorylated, indicating that association does not require receptor phosphorylation and occurs in the membrane proximal region. However, association was not detected with mutant receptors inactivated by an internal deletion or a point mutation, Trp282 to Arg, in a membrane- proximal cytoplasmic region (PB or PM4 mutant, respectively). Immune complex kinase assays of anti-EpoR immunoprecipitates also revealed that activated JAK2 associates with the EpoR in Epo-stimulated cells. By this approach, association also occurred with the mitogenically active H mutant but not with the mitogenically inactive PB or PM4 mutants. In the immune complex kinases assays, EpoR, JAK2, and a 150-kD protein were phosphorylated on tyrosine. Taken together, the results further support the hypothesis that, on Epo stimulation, JAK2 associates with the membrane-proximal cytoplasmic region of the EpoR to be activated and induces tyrosine phosphorylation of cellular substrates, including the EpoR, to transduce a growth signal.     

兔膝关节内注射透明质酸钠后胶原纤维及关节外相关韧带组织的变化

目的:在兔固定的膝关节内定期注入透明质酸钠,观察关节内外组织的变化。方法:实验于2003-10/2004-04在大连医科大学完成。实验分组:新西兰兔24只,随机分为透明质酸钠组、生理盐水组、单纯对照组,每组8只。实验干预:用树脂绷带将兔右膝关节固定于伸直位,膝关节注射部位开窗,左膝自由活动(自身对照侧)。透明质酸钠组膝关节腔内注入透明质酸钠0.1mL,生理盐水组注射同等剂量的生理盐水,单纯对照组不向关节内注射任何药物,每周1次,共5次。实验评估:5周后麻醉下处死动物,去除外固定,采用改良Clarke-Weeknesser关节伸屈范围检查标准测量右膝关节的活动范围;采用改良的Rydell-Balazes肉眼粘连评分标准评估膝关节内纤维粘连情况;光镜下观察股间肌、股直肌、髌内外侧支持带的纤维变性情况。结果:动物饲养过程中死亡4只,进入结果分析透明质酸钠组7只,生理盐水组6只,单纯对照组7只。①右膝关节的活动范围:透明质酸钠组大于生理盐水组和单纯对照组[(37.86±2.94)°,(15.67±2.23)°,(14.29±1.96)°,P<0.01]。②膝关节内纤维粘连评分:透明质酸钠组小于生理盐水组和单纯对照组(1.44±0.49,3.33±0.44,3.44±0.57,P<0.01)。③光镜下透明质酸钠组股间肌、股直肌的纤维变性较生理盐水组和单纯对照组减轻,髌内外侧支持带胶原纤维成分的变化也减轻。结论:在固定的兔膝关节内注射透明质酸钠不但可以明显抑制关节内的纤维性粘连;还可以抑制关节外股间肌、股直肌、髌内外侧支持带的组织变性。