In high doses with stem-cell transplantation, melphalan is an effective but toxic therapy for patients with systemic light-chain (AL-) amyloidosis, a protein deposition and monoclonal plasma cell disease. Melphalan can eliminate the indolent clonal plasma cells that cause the disease, an achievement called a complete response. Such a response is usually associated with extended survival, while no response (a less than 50% reduction) is not. Gene-expression studies and a stringently supervised analysis identified calreticulin as having significantly higher expression in the pretreatment plasma cells of patients with systemic AL-amyloidosis who then had a complete response to high-dose melphalan. Calreticulin is a pleiotropic calcium-binding protein found in the endoplasmic reticulum and the nucleus whose overexpression is associated with increased sensitivity to apoptotic stimuli. Real-time PCR and immunohistochemical staining also showed that expression of calreticulin was higher in the plasma cells of those with a complete response. Furthermore, wild-type murine embryonic fibroblasts were significantly more sensitive to melphalan than calreticulin knock-out murine embryonic fibroblasts. These data have important implications for understanding the activity of melphalan in plasma-cell diseases and support further investigation of calreticulin and its modulation in patients with systemic AL-amyloidosis receiving high-dose melphalan.

The cancer-testis antigens (CTA) are highly immunogenic antigens expressed in various tumors but not in normal tissues (except during gametogenesis), making them an attractive target for cancer immunotherapy. Expression of CTAs such as MAGE-A3, MAGE-C1 (CT7), MAGE-C2 (CT10), NY-ESO1 and the SSX antigens has been previously reported in multiple myeloma (MM). To date, however, these reports have included a heterogeneous group of newly diagnosed and relapsed/refractory patients, all in different stages of treatment. Therefore, the extent and prognostic significance of CTA expression, and of de novo immune responses against CTA in newly-diagnosed MM patients are not known. We now report on both CTA expression and antibody responses in MM patients at diagnosis and on their prognostic significance. From 8/00-11/04, we treated 67 newly-diagnosed, symptomatic patients with a thalidomide, doxorubicin, and dexamethasone-based induction regimen. (Brit J Haematol 2006; 132:155). Median age was 58; 54% were ISS stage I, 28% ISS II, and 18% ISS III. Nine of 63 tested (14%) had deletion 13q by FISH, while 24% had soft tissue involvement by MM. Responses to induction therapy included 10 (15%) CR, 16 (24%) VGPR, 26 (39%) PR, 6 (8%) stable or progressive disease, and 9 (13%) inevaluable. Post-induction 54 underwent autoSCT and 9 also underwent alloSCT.. Median overall survival (OS) has not been reached with 61% alive at median follow up of 65 months. Cryopreserved pre-treatment bone marrow plasma cells were used to assess CTA expression by RT-PCR. Pre- and post-treatment sera were used to assess antibody (Ab) responses against CTA proteins by ELISA. Fifty-two patients had sufficient RNA for PCR, and 46 had baseline serum for ELISA. OS of these groups did not differ significantly from the entire cohort. At least 1 CTA was expressed in 77% of cases, including MAGE-A3 (52%), SSX1 (40%), CT7 (29%), CT10 (25%), NY-ESO1 (21%), and SSX5 (17%). Three or more CTA were expressed in 29% of cases. Individually MAGE-A3 or NY-ESO1 expression at diagnosis conferred a poorer prognosis (MAGE-A3: median OS 66 mos. vs. not reached, p=0.02 by log-rank; NY-ESO1: median OS 65 mos. vs. not reached, p=0.09). These poorer outcomes were independent of ISS stage, presence of del 13q, or response to induction therapy. No other CTA was associated with an OS difference, nor was the total number of CTA expressed prognostically significant. Baseline Ab responses, all at titers > 1:1600, were noted to NY-ESO1 in 6/46 (13%) patients, 5 of whom also had Ab to the NY-ESO1 homologue LAGE-1. Ab responses were also noted to CT7 (n=2), CT10 (n=1) and SSX4 (n=1). No Ab responses were noted to MAGE-A3. The effect of induction therapy on antibody titers was inconsistent, with increases, decreases, and no changes seen. Interestingly, 2 of the 6 NY-ESO1 Ab+ patients had no NY-ESO1 expression in bone marrow plasma cells. Both, however, had extensive soft tissue (ST) plasmacytomas, suggesting another source of NY-ESO1 antigen. Presence of NY-ESO1 Ab correlated significantly with baseline ST involvement, with 67% of Ab+ patients having ST disease compared with 20% of Ab− patients (p=0.05). NY-ESO1 Ab+ patients also had significantly poorer OS (med 21 mos. vs. not reached, p=0.009), independent of other prognostic factors. In sum, CTA expression is frequent in newly diagnosed MM patients, and expression of MAGE-A3 or NY-ESO1 is associated with worse long-term survival. Spontaneous antibody responses against NY-ESO1 are seen in untreated patients, and are associated with ST involvement and poorer survival. Further exploration of biologic differences between CTA+ and CTA-MM, as well as immunotherapeutic strategies which target these antigens, are warranted.

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The natural history of de novo cardiac amyloidosis is poorly described, a limitation that makes clinical decision-making difficult given the growing number of therapies for light-chain (AL)-amyloidosis (AL). We identified all patients presenting to our center from 3/99 to 8/07 at, or within 4 months of, diagnosis with symptomatic cardiac amyloidosis, and analyzed the baseline and post-treatment factors influencing survival for those with AL. During that period, 34.5% (157/455) of amyloid patients were diagnosed with de novo cardiac amyloidosis: 112 men and 45 women with a median age of 62 (31–83). Heart biopsies were obtained and were positive in 39% of men (44/112) and 31% of women (14/45). AL was diagnosed in 86% of patients (n=135) and hereditary (n=7) and senile cardiac (n=15) in the rest. Eight patients underwent heart transplant for hereditary (2 men) and AL (5 men, 1 woman). AL was diagnosed in 81% of men and 98% of women (p=0.005, chi square). We analyzed survival from diagnosis based on intention-to-treat. Ninety percent of patients with AL (n=122) received chemotherapy. Hematologic responses were scored as complete (CR), partial (PR, > 50% reduction) or non. Patients received IV melphalan with stem cell transplant (SCT, n=45), oral melphalan and dexamethasone (MDex, n=42) or other regimens (n = 35). Selection for SCT was based on age and extent of organ disease. Thirty-eight patients (28%) subsequently received second- and third-line therapies. The median survival for all patients was 10 months (range, 1 to 94). Baseline predictors of survival included age, gender and troponin I. Patients ≤ 60 years old had a median survival of 22 and those > 60 of 8 months (p=0.007). Women had a median survival of 24 and men of 8 months (p=0.02). Patients with troponin I ≤ 0.10 lived a median of 21 and those with levels > 0.10 of 9 months (p=0.01). Median ages at diagnosis of the 91 men and 44 women with AL were 59 (31–83) and 63 (38–82) respectively (p=0.19), and there were no differences in baseline albumin, alkaline phosphatase, CRP, brain natriuretic peptide, troponin I or clonal free light chains. There was a significant difference in serum creatinine (medians of 1.35 and 1.00 in men and women, p < 0.01). Overall, 60% of patients responded to chemotherapy: 55% of men and 74% of women (p=0.04, chi-square). Responders lived a median of 40 and non-responders 7 months (p<0.0001), and there was no difference in median survival based on gender. With SCT, mobilization-related and 100-day mortality was 6.7% (3/45, all men) and deaths due to progression of disease after mobilization pre-SCT were 11% (5/45, 4 men, 1 women). With SCT the median survival is not yet reached, median follow up of survivors is 39 mos (range 12–94), and there is no difference in median survival by gender. With MDex, the median survival is 14 months and for men is 9 and for women 20 months (p=0.08). With other therapies the median survival is 7 months with no difference by gender. In patients who received second- and third-line therapies median survival is 37 months. In sum, in patients with de novo cardiac AL, factors influencing survival include age, gender, troponin I and response to therapy. Achievement of a prompt hematologic response should be a primary goal of initial therapy and should guide adjuvant and second-line treatments. The gender-related difference in overall survival with cardiac AL merits further study.

We reconstituted bilayer nuclear membranes, multilayer membranes, and organelles from mixtures of Xenopus laevis egg extracts and demembranated Xenopus sperm nuclei. Varying proportions of the cytosolic and vesicular fractions from the eggs were used in the reconstitution mixtures. A cytosol:vesicle ratio of 10:1 promoted reassembly of the normal bilayer nuclear membrane with inserted nuclear pore complexes around the decondensed Xenopus sperm chromatin. A cytosol:vesicle ratio of 5:1 caused decondensed and dispersed sperm chromatin to be either surrounded by or divided by unusual multilayer membrane structures with inlaid pore complexes. A cytosol:vesicle ratio of 2.5:1 promoted reconstitution of mitochondria, endoplasmic reticulum networks, and Golgi apparatus. During reassembly of the endoplasmic reticulum and Golgi apparatus, vesicular fragments of the corresponding organelles fused together and changed their shape to form flattened cisternae, which were then stacked one on top of another.

Nuclear envelope is a dynamic structure in the cell cycle. At the beginning of mitosis, nuclear envelope breaks down and its components disperse into the cytoplasm. At the end of mitosis, nuclear envelope reassembles using the dispersed components. Searching for the mechanisms of the nuclear disassembly and reassembly has for a long time been one of the key projects for cell biologists. In this report we show that microtubules take a role in the nuclear envelope assembly around the sperm chromatin inXenopus egg extracts. Microtubule cytoskeleton has been demonstrated to take roles in the transport of intracellular membranes such as Golgi and ER vesicles. We found that the nuclear envelope assembly needs functional microtubules. At the beginning of the nuclear assembly, microtubules nucleated to form a microtubule aster around the centrosome at the base of the sperm head. Using the microtubule drug colchicine to disrupt the microtubule nucleation, nuclear envelope reassembly was seriously inhibited. If the microtubules were stabilized by taxol, another microtubule drug, the nuclear envelope reassembly was also interfered, although a significantly large aster formed around the chromatin. Based on these observations, we propose that microtubules play an important role in the nuclear envelope reassembly maybe by transporting the nuclear envelope precursors to the chromatin surfaces.

报道了由微管组成的星体在爪蟾卵细胞提取物中的组装过程、动态变化及其在核膜装配过程中的作用.用精子染色质在爪蟾卵非细胞体系中诱导细胞核重建.在初期阶段, 总是在精子染色质基部的中心体周围通过微管装配而形成星体.随着星体的形成, 具有DNA复制功能的细胞核逐步装配.当完整的细胞核形成后, 星体开始去组装直至消失.在实验体系中加入微管装配特异抑制剂秋水仙素, 可以有效地抑制星体的组装, 核膜的组装同时受到严重的影响.高浓度的秋水仙素还抑制核膜膜泡的融合.若加入微管稳定剂紫杉醇, 微管可以装配, 并在染色质周围形成大而稳定的星体.在此情况下, 核膜的装配也受到严重影响.观察发现, 在此被稳定的星体中, 有转运受阻的膜泡存在.基于以上实验结果, 认为由微管组成的星体在核膜重建中具有重要的作用, 其机制可能是微管参与核膜前体物质向染色质表面的输运.

用扫描近场光学显微镜对凋亡的HeLa细胞进行了近场光学成像,得到优于光学衍射极限的光学分辨率.由于近场光学显微镜同时测量细胞表面形貌信息和透射光强信息,可以将细胞表面及其内部的物质结构特性和光学特性与空间位置相结合,获得凋亡细胞的结构信息与光学细节信息的对应关系.运用近场光谱方法在不同波长进行透射光谱成像时,不同波长的光在细胞内的传播与吸收所造成的光强分布存在显著差别,这种特性可以用于对细胞内不同组分的超高空间分辨率成像.结合近场光学成像和光谱成像结果,表明凋亡HeLa细胞内部为非均匀结构,并且其物质分布也极不均匀.研究表明,运用近场光学显微镜和近场光谱成像技术,不但提供优于衍射极限的高分辨本领,还可以提供生物细胞精细结构的更深层次的光学信息.

动物爪蟾(Xenopus laevis)卵提取物诱导植物诸葛菜(Orychophragmus violaceus)去膜精子实现非细胞体系核重建. 诸葛菜去膜精子在爪蟾卵提取物中温育30 min左右开始膨大, 随着温育时间的延长, 膨大的精子染色质逐渐去凝集. 电子显微镜观察和荧光显微镜观察均表明重建核有核膜的装配, 膜泡在去凝集的染色质周围逐渐融合形成双层核膜. 用细胞分级抽提整装电子显微镜技术观察到重建核中有核纤层和核骨架结构的装配.

The cell-free extracts from animal Xenopus laevis egg could induce chromatin decondensation and pronuclear formation from demembranated plant (Orychophragmus violaceus) sperm. The demembranated Orychophragmus violaceus sperm began to swell in 30 min incubation, and then were gradually decondensed. The reassembly of nuclear envelope in the reconstituted nuclei had been visualized by means of electron microscopy and fluorescent microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nucleus, with a double membrane, was similar to those nuclei after fertilization. Transmission electron microscope micrograph of the whole mount prepared nuclear matrix-lamina showed the reconstituted nucleus to be filled with a dense network.

Nicotiana tabaccum ovule extracts induced nuclear reconstitution of demembranated Xenopus leavis sperm in a cell-free system. Demembranated Xenopus sperm began to swell after 15 min of incubation with Nicotiana ovule extracts. Accompanying the process of incubation, Xenopus sperm decondensed and their shapes changed gradually from long and ellipse to round. The completely decondensed chromatin was surrounded with membrane structure, which was a mixture envelope of a double membrane and a single membrane. Nucleosome assembly was verified by means of micrococcal nuclease digestion to re-constituted nuclei and DNA electrophoresis. Nicotiana ovule extracts supplied one more experimental model and system. The new system could promote powerfully the research on mechanisms of cell division and cell cycle regulation.

The cell-free preparation derived from Nicotiana tabaccum ovules induced chromatin decondensation and pronuclear formation from demembranated Xenopus laevis sperm nuclei. Fluorescent microscope and phase-contrast microscope visualization of assembly intermediates indicated that 95.6% of X. leavis sperm changed their tadpole-like shape to circular shape or elliptical shape after over 1.5 h of incubation. Transmission electron microscope visualization showed that nuclear membrane was assembled around the periphery of the dispersed chromatin. Nuclear envelope of most reassembled nuclei was composed of a double membrane inlaid with a little single membrane. Nucleosome assembly was verified by means of micrococcal nuclease digestion. After 2 to 5 h of incubation, digestion of the product of nuclear assembly with micrococcal nuclease produced at least six nucleosome fragments of about 250 bp each.

非洲爪蟾(Xenopus laevis)去膜精子在茄科植物烟草(Nicotiana tabaccum)胚珠提取物中实现非细胞体系核重建.爪蟾去膜精子在烟草胚珠提取物中温育15 min左右即开始膨大;继续温育,精子染色质去凝集,爪蟾精子形状由细长形经由长形、半月形、椭圆形等逐渐变圆.在去凝集的染色质周围有膜状结构出现,这种膜状结构由双层膜和单层膜组成.重建核经微球菌核酸酶酶切,DNA电泳结果表明爪蟾精子染色质在烟草胚珠提取物中装配形成核小体结构.植物烟草胚珠提取物用于非细胞体系核重建,为离体核重建研究提供了又一模式和实验体系,可促进细胞分裂机制和细胞周期调控机制的研究.

目的：研究稀土元素对药用植物细胞组织培养的作用和意义。方法：以药用植物黄连为材料，研究不同浓度的稀土化合物对愈伤组织鲜重增加和生物碱含量的影响。结果：０．１ｍｇ·Ｌ－１Ｙｂ３＋和１ｍｇ·Ｌ－１Ｅｕ３＋促进愈伤组织的生长，高浓度稀土化合物抑制生长。在不含稀土化合物的培养基上的继代培养中，来源于稀土化合物处理的愈伤组织鲜重增加值均比对照低。稀土化合物影响愈伤组织小檗碱、药根碱和巴马汀含量。结论：低浓度的稀土化合物对黄连愈伤组织的生长具有促进作用，低浓度和高浓度的Ｙｂ３＋和Ｅｕ３＋对黄连愈伤组织后续的继代培养均有不利的影响。Ｙｂ３＋抑制愈伤组织中三种生物碱的积累，０．１ｍｇ·Ｌ－１Ｅｕ３＋有利于药根碱和巴马汀的累积