Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins

Abstract:  The identification of tumor-specific proteins located at the plasma membrane is hampered by numerous methodological pitfalls many of which are associated with the post-translational modification of such proteins. Here, we present a new combination of detergent fractionation of cells and of subtractive suppression hybridization (SSH) to gain overexpressed genes coding for membrane-associated or secreted proteins. Fractionation of subcellular components by digitonin allowed sequestering mRNA of the rough Endoplasmatic reticulum and thereby increasing the percentage of sequences coding for membrane-bound proteins. Fractionated mRNAs from the cutaneous T-cell lymphoma (CTCL) cell line HuT78 and from normal peripheral blood monocytes were used for SSH leading to the enrichment of sequences overexpressed in the tumor cells. We identified some 21 overexpressed genes, among them are GPR137B, FAM62A, NOMO1, HSP90, SLIT1, IBP2, CLIF, IRAK and ARC. mRNA expression was tested for selected genes in CTCL cell lines, skin specimens and peripheral blood samples from CTCL patients and healthy donors. Several of the detected sequences are clearly related to cancer, but have not yet been associated with CTCL. qPCR confirmed an enrichment of these mRNAs in the rough endoplasmic reticulum fraction. RT-PCR confirmed the expression of these genes in skin specimens and peripheral blood of CTCL patients. Western blotting verified protein expression of HSP90 and IBP2 in HuT78. GPR137B could be detected by immunohistology in HuT78 and in keratinocytes of dysplastic epidermis, but also in sweat glands of healthy skin. In summary, we developed a new technique, which allows identifying overexpressed genes coding preferentially for membrane-associated proteins.     

Peripheral T-cell lymphoma in childhood. A clinicopathological study of six cases

A review of the pathological material from 42 children with non-Hodgkin's lymphoma seen over a 44 month period revealed 10 large cell tumours. Of these, six were classified as peripheral T-cell lymphoma, an entity rarely reported in childhood. Three patients were boys and three girls (median age 9.5 years), and extranodal presentation was a feature of two patients. Five had high-grade tumours; of these, three were classified as large cell anaplastic, Ki-1 positive and two as pleomorphic large cell. The remaining patient had a low-grade tumour of angioimmunoblastic type. T-cell subsets were examined in three cases and showed the following phenotypes: CD4-, CD8-; CD4+, CD8-; CD4-, CD8+. Three of the patients with high-grade tumours died, with a mean survival of 22 weeks. The remaining patients are alive and clinically disease-free for between 10 and 24 months after treatment.     

Bayesian estimation of a random effects heteroscedastic probit model

Summary Bayesian analysis is given of a random effects binary probit model that allows for heteroscedasticity. Real and simulated examples illustrate the approach and show that ignoring heteroscedasticity when it exists may lead to biased estimates and poor prediction. The computation is carried out by an efficient Markov chain Monte Carlo sampling scheme that generates the parameters in blocks. We use the Bayes factor, cross‐validation of the predictive density, the deviance information criterion and Receiver Operating Characteristic (ROC) curves for model comparison.     

超声引导下犬肝注射醋酸高渗氯化钠混合液对射频消融毁损体积的影响

目的探讨超声引导下活体犬肝注射不同剂量的醋酸高渗氯化钠液(acetic acid hypertonic saline solution,AHS)后行射频消融(RFA),观察犬肝一次性毁损体积的变化。方法使用LDRF-120S多极RFA系统联合AHS对活体犬肝行RFA。健康成年杂种犬30只,随机分为5组(n=6)。A组:注射2ml AHS后立即行RFA;B组:注射2ml AHS后延时5min行RFA;C组:注射4ml AHS后立即行RFA;D组:注射4ml AHS后延时5min行RFA;E组:注射6ml AHS后立即行RFA。结果5组间平均起始阻抗差异无统计学意义(P>0.05);5组间平均消融时间差异有统计学意义(F=83.831,P<0.001),LSD-t检验分析各组间两两比较差异有统计学意义(P<0.001),在观察范围内E组平均消融时间最长;5组间平均毁损直径差异有统计学意义(F=53.488,P<0.001),在观察范围内E组平均毁损直径最大,LSD-t检验分析除D与E组间差异无统计学意义(P>0.05)外,其余各组间两两比较差异有统计学意义(P<0.001);网状纤维染色显示凝固坏死区及其邻近的正常肝组织均可见血管内血栓形成;E组在AHS注射过程中出现不同程度的外溢;A、B、C及D组14d内无动物死亡,E组死亡4只。结论活体犬肝局部注射AHS4ml后延时5min行RFA即可达到较理想的毁损体积。     

Laparoscopic repair of a perforated marginal ulcer 2 years after gastric bypass

The authors present the case of a 43-year-old women who underwent a laparoscopic gastric bypass in 2003 for morbid obesity. They report that 2 years later, she had maintained significant weight loss, but had developed acute abdominal pain, followed by nausea and emesis. In the emergency room, she had diffuse tenderness, tachycardia, and leukocytosis. After initial resuscitation, a computed tomography was performed, which showed free air above the liver and thickened small bowel loops. She was brought emergently to the operating room for laparoscopy. At surgery, turbid fluid and inflamed small bowel loops were seen. A perforated marginal ulcer was discovered in the Roux limb, approximately 2 cm distal to the gastrojejunal anastomosis. The perforation was oversewn primarily and patched with omentum. The repair was tested by intraoperative endoscopy. A gastrostomy tube also was placed within the gastric remnant for enteral access. The patient did extremely well postoperatively, and had an uneventful postoperative course. She was discharged on postoperative day 4. The gastrostomy tube was removed at 1 month, and at this writing, she remains well since surgery. An upper endoscopy at 2 months was completely normal, and the Helicobacter pylori test results were negative. The gastric pouch had not significantly enlarged since initial surgery, as indicated by both endoscopy and barium study. Marginal ulcer is reported to be 0.6% to 16% after laparoscopic gastric bypass [1]. Etiologies include gastrogastric fistula, excessively large gastric pouch containing antral mucosa, H. pylori infection, nonsteroidal antiinflammatory use, and smoking [2]. Unfortunately, none of these applied to the reported patient. Because her exact etiology remains unknown, she at this writing continues to receive proton pump inhibitor therapy. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Hypophyseal Non-Hodgkin's Lymphoma presenting with clinical panhypopituitarism successfully treated with chemotherapy

Summary A patient is described with a testicular Non-Hodgkin's Lymphoma (NHL) presenting with panhypopituitarism caused by a hypophyseal localization. A⁶⁷Gallium scintigraphy showed avid uptake in the hypophyseal region. Obviously⁶⁷Gallium could reach the tumor, by the intravenous route, which was the reason to treat the patient with intravenous chemotherapy. A complete remission was induced, which seems to be lasting (+ 25 months). As far as we know this is the first report of panhypopituitarism caused by a hypophyseal NHL in the hypophysis and successfully treated by intravenous chemotherapy.     

Effects of specific antibodies on the ultrastructure of mouse oocytes

The effects of antiovarian antiserum and monoclonal antibodies to the oolemma antigens on the ultrastructure of mouse oocytes and their microenvironment are studied. The antioolemma monoclonal antibodies cause more pronounced degenerative changes in the oocyte that in its microenvironment. Antiovarian antiserum induces greater changes in the microenvironment than in the oocyte. Changes induced in the oocyte by the antiserum are secondary relative to changes occurring in the microenvironment, while changes observed in the oocyte treated with monoclonal antibodies are primary. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 123, No. 1, pp. 115–119, January, 1997     

Immunoglobulin heavy chain Diversity genes rearrangement pattern indicates that MALT-type gastric lymphoma B cells have undergone an antigen selection process

Gastric MALT lymphoma usually develops from chronic gastritis, the vast majority of which (>90%) is associated with Helicobacter pylori infection. We sequenced the third complementarity determining region (CDR3) of immunoglobulin heavy chain genes in 19 gastric MALT lymphoma clones to determine the pattern of variable (V), diversity (D) and joining (J) gene utilization during immunoglobulin gene rearrangement.
DNA was extracted from paraffin-embedded sections and the rearranged CDR3 regions were amplified using a semi-nested polymerase chain reaction (with primers complementary to the conserved framework-three segment of the variable region [FR3A] and J regions). The DNA used for cloning and sequencing was obtained after purification of monoclonal bands excised from polyacrylamide gels. The N-D-N region specific to each clone was compared with known germline D sequences.
Similarly to that observed in normal and leukaemic B cells, our series of gastric MALT lymphomas showed apparent preferential utilization of genes from the DXP family. In two cases no similarity between the CDR3 nucleotide sequences of the neoplastic clones and the known germline D sequences could be found. In 10/19 analysed alleles the lymphoma B-cell clones appeared to contain two D gene segments (D-D recombination), a rare occurrence in normal individuals but one which has been described as a significant event in the determination of idiotype expression and antigen-binding affinity. Remarkably, despite the use of different D and J segments, the resultant amino acid sequences matched in two patients, suggesting the presence of a common selecting antigen.
The observed pattern of D gene rearrangement suggests that MALT lymphoma B-cell clones have undergone antigen selection, which seems to indicate that the antigen stimulation plays a pivotal role in the development of the lymphoma.     

Hemophagocytic syndrome and T-cell lymphoma after kidney transplantation: a case report

Lymphoma in immunocompromised transplant patients is a feared cause of morbidity and mortality. Superimposed on the lymphoma and the transplantation immunosuppression is a rare condition: hemophagocytic syndrome (HS). HS is characterized by fever, hepatosplenomegaly and lymphadenopathy, skin rashes, jaundice, coagulopathy, and phagocytosis of blood elements with pancytopenia. Here we describe a rare but fatal case of a kidney transplant patient who developed T-cell lymphoma and HS, without evidence of EBV replication. A short review of the diagnosis, treatment, and prognosis of HS is given. Received: 4 March 1997 Received after revision: 6 June 1997 Accepted: 30 June 1997     

43例鼻咽,鼻腔恶性淋巴瘤的临床病理及免疫表型研究

本文对遵义地区４３例鼻咽、鼻腔恶淋巴瘤的临床病理及免疫学表型进行了研究，结果显示：全部病例均为弥漫型，无１例滤泡型，组织学类型以多表细胞性淋巴瘤为主，共３３例（包括小细胞型１３例，中多形１５例，大多形５例），占７６．７％。用ＵＣＨＬ－１、ＣＤ２０和Ｍａｃ３８７等多单克隆抗体进行免疫表型研究。显示Ｔ细胞淋巴瘤３７例（８６％），Ｂ细胞淋巴瘤４例（９．３％），无１例组织细胞性淋巴瘤。鼻咽部Ｔ．Ｂ淋巴瘤比