Interphase FISH as a new tool in tumor pathology

Interphase FISH (IFISH) analysis is an intriguing molecular cytogenetic approach to study chromosome abnormalities in cancer. IFISH is a high sensitivity technique because of its ability to identify aberrations on a cell-to-cell level. The possibility to perform IFISH on different types of nuclei obtained both from fresh and archived samples, makes this technique an advantageous method to identify specific chromosome aberrations in cancer and correlate them to prognosis and therapy. The aim of this review is to outline the technical aspects, the sensitivity and specificity and the current strategies for employment of IFISH in tumor pathology, and to discuss the enormous range of novel applications.     

两种少量小鼠外周血间期核FISH技术的比较

目的:探讨低渗法与红细胞裂解法应用于小鼠少量外周血间期核荧光原位杂交(fluorescence in situ hybridization,FISH)检测的优缺点,以便于根据实验条件选取最合适的方法。方法:小鼠剪尾巴法获取外周血,采用低渗法或红细胞裂解法破坏红细胞,涂片前先用免疫组化笔在玻片上划好预计涂片区域,重悬沉淀后稍等片刻再吸中间部分用于涂片,涂完后在相差显微镜下观察密度,行常规FISH,比较两种方法处理后的FISH结果。结果:低渗法和红细胞裂解法处理后的片子背景都较干净,密度适中,信号较强。结论:在外周血间期核荧光原位杂交技术中,低渗法和红细胞裂解法都可以获得较满意的FISH结果。低渗法的特点在于试剂便宜且易于获取,但耗时稍长;而红细胞裂解法的特点在于可节省实验时间,但需专用试剂。     

Recurrence risks for down syndrome

Summary The recurrence risks for Down syndrome due to an inherited translocation are estimated from empirical data in the literature for two maternal age groups: mothers under 30 and mothers 30 and over. These risks were found to be approximately 0.3% and 0.05%, respectively. The probability for two Down syndrome sibs both having an inherited translocation was estimated as about 18.2% for the former age group and 2.7% for the latter. The relative effectiveness in preventing Down syndrome births by karyotyping affected children is discussed.     

Chemotherapy-induced hemolytic uremic syndrome: Description of a potential animal model

Hemolytic uremic syndrome (HUS) is an uncommon complication of chemotherapy that contributes to the morbidity of oncology and bone marrow transplant patients. The pathogenesis is not well understood and no established clinical animal model exists. We studied four rhesus monkeys (RM) that developed fatal HUS following high-dose chemotherapy. Microangiopathic hemolytic anemia (pre-Hct 40% and day 5–8 Hct 31 % (P <.05), increased BUN (168 mg/dl), creatinine (8.2 mg/dl), and lactate dehydrogenase (1458 IU/L) (mean day 5–8 measurements) were observed. Platelets counts decreased to 39±15 × 10⁹/l from a mean of 397±31 × 10⁹/L (P < .0001). vWF, AT_III, thrombin:anti-thrombin complex (T:AT) and prothrombin fragment F1.2 levels were not different from a control group (N = 2). The data presented describe chemotherapy-induced HUS with typical clinical and laboratory features which may provide an animal model for the study of this important syndrome.     

Characterization of PTZ-induced seizure susceptibility in a down syndrome mouse model that overexpresses CSTB

Down syndrome (DS) is a complex genetic syndrome characterized by intellectual disability, dysmorphism and variable additional physiological traits. Current research progress has begun to decipher the neural mechanisms underlying cognitive impairment, leading to new therapeutic perspectives. Pentylenetetrazol (PTZ) has recently been found to have positive effects on learning and memory capacities of a DS mouse model and is foreseen to treat DS patients. But PTZ is also known to be a convulsant drug at higher dose and DS persons are more prone to epileptic seizures than the general population. This raises concerns over what long-term effects of treatment might be in the DS population. The cause of increased propensity for epilepsy in the DS population and which Hsa21 gene(s) are implicated remain unknown. Among Hsa21 candidate genes in epilepsy, CSTB, coding for the cystein protease inhibitor cystatin B, is involved in progressive myoclonus epilepsy and ataxia in both mice and human. Thus we aim to evaluate the effect of an increase in Cstb gene dosage on spontaneous epileptic activity and susceptibility to PTZ-induced seizure. To this end we generated a new mouse model trisomic for Cstb by homologous recombination. We verified that increasing copy number of Cstb from Trisomy (Ts) to Tetrasomy (Tt) was driving overexpression of the gene in the brain, we checked transgenic animals for presence of locomotor activity and electroencephalogram (EEG) abnormalities characteristic of myoclonic epilepsy and we tested if those animals were prone to PTZ-induced seizure. Overall, the results of the analysis shows that an increase in Cstb does not induce any spontaneous epileptic activity and neither increase or decrease the propensity of Ts and Tt mice to myoclonic seizures suggesting that Ctsb dosage should not interfere with PTZ-treatment.     

Membrane mutants of animal cells: rapid identification of those with a primary defect in glycosylation.

Membrane mutants of animal cells have been isolated by several laboratories, using a variety of selection protocols. The majority are lectin receptor mutants arising from altered glycosylation of membrane molecules. They have been obtained by selection for resistance to cytotoxic plant lectins or by alternative protocols designed, in many cases, to isolate different classes of receptor mutants. The identification of most membrane mutants expressing altered surface carbohydrates is rapidly achieved by determining their resistance to several lectins of different carbohydrate-binding specificities. For Chinese hamster ovary mutants, genetic novelty may subsequently be determined by complementation analysis with selected members of 10 recessive, glycosylation-defective complementation groups defined by this laboratory. In an attempt to identify new complementation groups, 11 Chinese hamster ovary membrane mutants independently isolated in different laboratories have been investigated for their lectin resistance and complementation properties. Only one new complementation group was defined by these studies. The remaining 10 mutants fell into complementation group 1, 2, 3, or 8. Although no evidence for intragenic complementation was observed, indirect evidence for different mutations within some genes was obtained. Seven of the independent isolates fell into complementation group 1, reflecting the high probability of isolating the Lec1 phenotype from Chinese hamster ovary populations. The results emphasize the importance of performing a genetic analysis before biochemical characterization of putative new membrane mutants.     

Utility of Interphase FISH Panels for Routine Clinical Cytogenetic Evaluation of Chronic Lymphocytic Leukemia and Multiple Myeloma

Specific genetic abnormalities are of prognostic significance for patients with chronic lymphocytic leukemia (CLL) and multiple myeloma (MM); however, routine cytogenetic analysis usually provides normal results. We utilized two probe panels for interphase fluorescence in situ hybridization (FISH) studies to enhance the ability to detect genetic abnormalities in samples that were referred for routine cytogenetic studies for possible diagnoses of CLL or MM. The CLL panel consisted of probes for 11q22.3 (ATM gene), 13q14 (D13S319), the centromere of chromosome 12 (D12Z3) and 17p13.1 (P53 gene). The MM panel included probes for 14q32 (IgH gene) and/or t(11:14)(q13;q32) (BCL1/IgH), 13q14 (D13S319) and 17p13.1 (P53 gene). FISH detected clonal aberrations not identified by conventional cytogenetics in an additional 8 of 23 (35%) samples referred for possible CLL and 7 of 42 (17%) samples with possible MM. The prognostic significance of the aberrations identified ranged from favorable, to intermediate, to poor. Our studies indicate that many samples referred for routine cytogenetics testing for CLL and MM yield normal results for both conventional and FISH testing, likely due to lack of definitive diagnosis in a percentage of cases. However, FISH is more sensitive for the detection of clinically significant chromosome abnormalities and should be the testing methodology of choice for these disorders.     

Proteomic analysis of maternal serum in down syndrome: identification of novel protein biomarkers

Down syndrome (DS) is the most prevalent chromosomal disorder, accounting for significant morbidity and mortality. Definitive diagnosis requires invasive amniocentesis, and current maternal serum-based testing requires a false-positive rate of about 5% to detect 85% of affected pregnancies. We have performed a comprehensive proteomic analysis to identify potential serum biomarkers to detect DS. First- and second-trimester maternal serum samples of DS and gestational age-matched controls were analyzed using multiple, complementary proteomic approaches, including fluorescence 2-dimensional gel electrophoresis (2D-DIGE), 2-dimensional liquid chromatography-chromatofocusing (2D-CF), multidimensional protein identification technology (MudPIT; LC/LC-MS/MS), and MALDI-TOF-MS peptide profiling. In total, 28 and 26 proteins were differentially present in first- and second-trimester samples, respectively. Of these, 19 were specific for the first trimester and 16 for the second trimester, and 10 were differentially present in both trimesters. Analysis of MALDI-TOF-MS peptide profiles with pattern-recognition software also discriminated between DS and controls in both trimesters, with an average recognition capability approaching 96%. A majority of the biomarkers identified are serum glycoproteins that may play a role in cellular differentiation and growth of fetus. Further characterization and quantification of these markers in a larger cohort of subjects may provide the basis for new tests for improved DS screening.     

Riboprints as a tool for rapid preliminary identification of sphingomonads

Fourtythree strains of the genus Sphingomonas and close relatives were subjected to riboprint analyses generated after digestion of genomic DNA with the restriction enzyme EcoRI and hybridization with E. coli rrnB operon. The majority of strains were characterized by a complex banding pattern in the riboprints. High degrees of similarities in the riboprints were only observed among strains of the same species such as S. yanoikuyae, S. aromaticivorans, S. subarctica and S. chlorophenolica. Strains of different species including close phylogenetic relatives such as S. asaccharolytica, S. mali and S. pruni were easily distinguished by the differences in the riboprints even after visual evaluation. Thus, our data demonstrate that riboprint analysis is useful for preliminary identification of new sphingomonad isolates at the species level.     

Renal Fanconi syndrome: developmental basis for a new animal model with relevance to human disease

Using succinylacetone (SA), a metabolite of tyrosine excreted in excess by infants and children with hereditary tyrosinemia and the renal Fanconi syndrome (FS), we have investigated developmentally-related membrane transport events leading to emergence of the generalized renal tubular dysfunction seen in human FS. SA was found to impair sugar and amino acid uptake by both newborn renal tubules and 7-day renal brush-border membrane vesicles (BBMV). This impairment by SA was due in part to a slowing of substrate cotransport rate of 22Na+-entry into BBMV. Concentration-dependent uptake studies indicated SA inhibited the newborn high-affinity transport systems for sugars and amino acids. SA also caused an increase in membrane fluidity and a shift in the thermotropic transition temperature. The demonstrated dual nature of SA's effect on membrane fluidity and O2 consumption, together with the relative contribution of each component to SA-induced transport impairment helps to provide a basis for an understanding of the age-related increases in glucosuria, aminoaciduria and natriuria seen in infants with FS.     

Spectral karyotyping, a 24-colour FISH technique for the identification of chromosomal rearrangements

 Spectral karyotyping (SKY) is a new fluorescence in situ hybridisation (FISH) technique that refers to the molecular cytogenetic analysis of metaphase preparations by means of spectral microscopy. For SKY of human metaphase chromosomes, 24 chromosome-specific painting probes are used in just one FISH experiment. The probes are labelled by degenerate oligonucleotide-primed PCR using three fluorochromes and two haptens. Each probe is differentially labelled with one, two, three or four fluorescent dyes, resulting in a unique spectral signature for every chromosome. After in situ hybridisation and immunodetection, a spectral image is acquired using a conventional fluorescence light microscope equipped with a custom-designed triple-bandpass filter and the SpectraCube, which is able to retrieve spectral information for every pixel in a digital CCD image. The 24-colour display and chromosome classification are based on the unique emission spectra of the chromosomes. Together with chromosome banding information from an inverted DAPI or a G-banded metaphase, a comprehensive overview of chromosomal aberrations is presented. Accepted: 3 July 1997     

Towards a small animal model for hepatitis C

Hepatitis C virus (HCV) causes chronic liver disease and affects an estimated 3% of the world's population. Options for the prevention or therapy of HCV infection are limited; there is no vaccine and the nonspecific, interferon‐based treatments now in use are frequently ineffective and have significant side effects. A small‐animal model for HCV infection would significantly expedite antiviral compound development and preclinical testing, as well as open new avenues to decipher the mechanisms that underlie viral pathogenesis. The natural species tropism of HCV is, however, limited to humans and chimpanzees. Here, we discuss the prospects of developing a mouse model for HCV infection, taking into consideration recent results on HCV entry and replication, and new prospects in xenotransplantation biology. We highlight three independent, but possibly complementary, approaches towards overcoming current species barriers and generating a small‐animal model for HCV pathogenesis.     

Annotation of a serum N-glycan library for rapid identification of structures

Glycosylation is one of the most common post-translational modifications of proteins and has been shown to change with various pathological states including cancer. Global glycan profiling of human serum based on mass spectrometry has already led to several promising markers for diseases. The changes in glycan structure can result in altered monosaccharide composition as well as in the linkages between the monosaccharides. High-throughput glycan structural elucidation is not possible because of the lack of a glycan template to expedite identification. In an effort toward rapid profiling and identification of glycans, we have constructed a library of structures for the serum glycome to aid in the rapid identification of serum glycans. N-Glycans from human serum glycoproteins are used as a standard and compiled into a library with exact structure (composition and linkage), liquid chromatography retention time, and accurate mass. Development of the library relies on highly reproducible nanoLC-MS retention times. Tandem MS and exoglycosidase digestions were used for structural elucidation. The library currently contains over 300 entries with 50 structures completely elucidated and over 60 partially elucidated structures. This database is steadily growing and will be used to rapidly identify glycans in unknown biological samples.     

一种鹿科动物标本的分子鉴定

中国是世界上鹿科动物资源最丰富的国家之一,共有18种,分属獐亚科、麂亚科、鹿亚科和美洲鹿亚科,占全球鹿种的41.7％(盛和林等,1992).在皖南分布的鹿科动物有梅花鹿华南亚种(Cervus nippon pseudaxis)、黑麂(Muntiacus crinifrons)、黄麂(Muntiacus reevesi)和毛冠鹿(Elaphadus cephalophus)(王岐山,1990).2001年11月从泾县获得一具标本(图1-A,标本现保存于安徽师范大学标本馆),体形及大小似梅花鹿,身体背部毛棕褐色,无明显白斑,尾背面黑色、腹面纯白,但鹿角主干表面光滑,无眉叉及其它分叉.鹿角是鹿科动物所特有的结构,是区别于其它有蹄类的标志之一.     

Development and evaluation of a rapid identification test for candida albicans

Summary A new technique for the rapid identification ofC. albicans has been developed and evaluated. This yeast can be identified in one hour by the formation of germ tubes after inoculation in 1/2 ml of human or animal plasma, and commercial plasma substitutes.C. albicans also forms germ tubes within 2 to 4 hours after inoculation in human serum and incubation at 37° C.Filamentation ofC. albicans in these blood derivatives is a reliable method for the identification of this yeast. It is more rapid than the assimilation and fermentation sugar tests and chlamydospore formation.Assimilation and fermentation sugar tests are used to identify those isolates ofCandida that fail to produce filaments in plasma or serum.