NEDD8 protein is involved in ubiquitinated inclusion bodies

Proteolysis by the ubiquitin-proteasome system is considered to play a pathological role in several degenerative diseases that involve ubiquitinated inclusion bodies. In recent years, several ubiquitin-like proteins have been isolated, but it is uncertain whether their roles are associated with protein degradation through the ubiquitin-proteasome system. NEDD8 (neural precursor cell-expressed and developmentally down-regulated gene), which consists of 81 amino acid residues, possesses the highest sequence similarity to ubiquitin. Recent studies have indicated that NEDD8 is covalently ligated to cullin family proteins, which are components of certain ubiquitin E3 ligases, by a pathway analogous to that of ubiquitin. Thus, by focusing on the structural and functional association between NEDD8 and ubiquitin, it would be of interest to know whether the NEDD8 system is involved in pathological disorders of the ubiquitin-proteasome system. This study has examined the immunohistochemical distribution of NEDD8 protein by using a highly purified antibody in normal tissues and in tissues known to contain ubiquitinated inclusions. NEDD8 protein expression was widely observed in most types of tissues. Furthermore, accumulation of the NEDD8 protein was commonly observed in ubiquitinated inclusion bodies, including Lewy bodies in Parkinson's disease, Mallory bodies in alcoholic liver disease, and Rosenthal fibres in astrocytoma. Two of ten cases of neurofibrillary tangles and senile plaques from patients with Alzheimer's disease showed intense staining for NEDD8 as well as for ubiquitin. These findings suggest the possibility that the NEDD8 system is involved in the metabolism of these inclusion bodies via the ubiquitin-proteasome system.     

Insertion of non-homologous DNA sequences into a regulatory gene cause a constitutive maltase synthesis in yeast

Summary Two maltase constitutive alleles MAL1-1 ^c and MAL1-2 ^c were obtained as revertants from a defective mall-1 mutant allele not promoting maltose fermentation. Classical genetical analysis showed that the mutations were linked or allelic to the MAL1 locus. Dominance relations were established by testing -glucosidase activities in diploids containing various allele combinations.The maltose regulatory genes belonging to the MAL1, MAL1-1 ^c and MAL1-2 ^c alleles were cloned. Differences in restriction sites were found between the wild type MAL1 and the derived MAL1-constitutive alleles. The MAL1 regulatory gene was located in a 1.15 kb EcoRI fragment (Rodicio and Zimmermann 1985a, b). An EcoRI fragment of this size was found in plasmids containing the MAL1 regulatory wild type allele but was absent from plasmids carrying the constitutive alleles.The genomic organization of the MAL loci in the constitutive mutants was confirmed by Southern analysis. Various fragments containing sequences of the different MAL1 alleles were used to probe genomic digests of MAL1, MAL1-1 ^c and MAL1-2 ^c strains. The results obtained support the conclusion that the constitutive mutations had arisen by a rearrangement between the original mal1-1 mutant allele and sequences with different location in the genome.Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthday     

Context of deletions and insertions in human coding sequences

We studied the dependence of the rate of short deletions and insertions on their contexts using the data on mutations within coding exons at 19 human loci that cause mendelian diseases. We confirm that periodic sequences consisting of three to five or more nucleotides are mutagenic. Mutability of sequences with strongly biased nucleotide composition is also elevated, even when mutations within homonucleotide runs longer than three nucleotides are ignored. In contrast, no elevated mutation rates have been detected for imperfect direct or inverted repeats. Among known candidate contexts, the indel context GTAAGT and regions with purine-pyrimidine imbalance between the two DNA strands are mutagenic in our sample, and many others are not mutagenic. Data on mutation hot spots suggest two novel contexts that increase the deletion rate. Comprehensive analysis of mutability of all possible contexts of lengths four, six, and eight indicates a substantially elevated deletion rate within YYYTG and similar sequences, which is one of the two contexts revealed by the hot spots. Possible contexts that increase the insertion rate (AT(A/C)(A/C)GCC and TACCRC) and decrease deletion (TATCGC) or insertion (GCGG) rates have also been identified. Two-thirds of deletions remove a repeat, and over 80% of insertions create a repeat, i.e., they are duplications.     

Therapeutic Effects of a NEDD8-Activating Enzyme Inhibitor,Pevonedistat, on Sclerodermatous Graft-versus-Host Disease in Mice

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the sole treatment option for highly malignant hematologic disease; however, the major complication—graft-versus-host disease (GVHD)—still hinders its clinical application. In addition, chronic GVHD remains the major cause of long-term morbidity and mortality after allo-HSCT. Previously we showed that bortezomib, a proteasome inhibitor, can ameliorate the sclerodermatous GVHD response while maintaining graft-versus-tumor (GVT) effects. Here we report that pevonedistat (MLN4924), an inhibitor of the Nedd8-activating enzyme, which functions upstream of the proteasome in the ubiquitin-proteasome pathway, can also show similar protective effects. Recipient mice treated with pevonedistat demonstrated inhibitory effects on sclerodermatous GVHD pathogenesis. The beneficial effect of pevonedistat was observed to be temporally dependent. Whereas treatment given at the time of allo-HSCT administration or before the onset of symptoms worsened the scleroderma response, therapeutic administration starting at 20 days post-transplantation ameliorated the sclerodermatous GVHD. Flow cytometry analysis revealed differential effects on immune subsets, with inhibition of only antigen-presenting cells and not of donor T cells. Finally, pevonedistat preserved GVT effects in a sclerodermatous murine model of B cell lymphoma. Taken together, these data suggest that inhibition of neddylation with pevonedistat can serve as an alternative approach for the treatment of GVHD while maintaining GVT effects in a murine model of sclerodermatous GVHD.     

基于显微图像序列的细胞形态变化分析

目的细胞的形态变化与细胞的生理特性密切相关,其定量的描述和分析对探究生命的生理或病理状态过程有重要意义。本文基于显微图像序列提取细胞形态的动态变化信息,以实现对细胞不同形态变化的定量描述及分类。方法采用运动历史图像和局部二值模式分别提取细胞轮廓和内部运动信息,并使用一系列不同尺度的时间窗口将上述特征映射为多时间尺度的特征矢量,再采用支持向量机对细胞的不同形态变化进行分类。通过对4组不同形态变化等级的小鼠淋巴细胞图像序列进行分类实验,以验证本方法的分类效果。结果对形态变化由缓慢到剧烈的4组淋巴细胞视频,分类精确度达到75%,能有效区分不同程度的细胞形态变化。结论与径向距离、Zernike矩、傅里叶描述子等常用的形状描述方法相比,本文方法更加全面地描述了细胞形态变化的动态信息,对细胞的多样性运动具有更好的适应性和稳定性。对细胞形态变化的分类,可用于异常细胞形态变化的检测,为疾病的早期诊断提供了客观依据。     

A probabilistic model for detecting coding regions in DNA sequences

A probabilistic model is presented to predict whether or notan anonymous sequence of DNA contains exons. The method is shownto be at least as reliable as Grail, a well-known neural networksolution to the problem, and to be significantly more amenableto customization for specific prediction problems.     

Insertion in the coding region of the movement protein improves stability of the plasmid encoding a tomato mosaic virus-based expression vector

A major obstacle in the genetic manipulation of tomato mosaic virus (ToMV) is the instability of the plasmid containing the infectious full-length cDNA of the ToMV vector, which often prevents the subcloning of a foreign gene of interest into the vector. We found that an insertion of a 0.3-1.6-kbp DNA fragment in the movement protein (MP) coding region effectively attenuated bacterial toxicity of the plasmid and greatly increased plasmid yield. Accumulation of a modified ToMV containing a 0.3-kb insertion in the MP coding region was comparable to that of a modified ToMV without an insertion in tobacco BY-2 protoplasts, while an insertion more than 0.6 kb significantly reduced accumulation of the viral RNA. The modified ToMV vector containing a 0.3-kb insertion was easily manipulated to introduce a coding sequence for human interferon-gamma (HuIFN-gamma) and successfully utilized to produce HuIFN-gamma in both BY-2 protoplasts and transgenic BY-2 cells.     

Procoagulant activity of blood cells in pestivirus infection

An increased level of procoagulant activity (PCA) in leukocytes of pigs with acute classical swine fever (CSF) was observed on day 4 postinfection; PCA level normalized during the moribund state. CSF vaccine strain either did not induce an increase of PCA level or induced an increase that persisted for at least 11 days. Time course of PCA changes in the leukocytes from sheep infected with borderline sheep disease was similar to the time course of PCA in acute CSF. In vitro each of the pestiviruses induced an increase in PCA in homologous and heterologous leukocytes.     

Evolutionary variants of mouse adenovirus containing cellular DNA sequences

This study was initiated to determine the amount of mouse adenovirus strain FL that is available for substitution. Evolutionary variants were isolated following serial passage at high input multiplicities. Virions containing altered genomes were detected by altered size of their DNA in agarose gels. These variants demonstrated a selective growth advantage and became the predominant species as passages were continued. Both deletions and substitutions were found; they ranged in size from a small percentage to over 90% of the AdFL genome. The ends of the viral DNA appeared to be conserved. No common DNA sequences were deleted in all variants. The DNA from one mutant was shown to contain a continuous insert of about 16 million daltons of cellular DNA that included two regions of reiterated cellular sequences.     

Identification of a novel virus in pigs--Bungowannah virus: a possible new species of pestivirus

In 2003 an outbreak of sudden deaths occurred in 3-4-week-old piglets on a farm in New South Wales, Australia. There was a marked increase in the birth of stillborn foetuses. Pathological changes consisted of a multifocal non-suppurative myocarditis. A viral infection was suspected but a wide range of known agents were excluded. A modified sequence independent single primer amplification (SISPA) method was used to identify a novel virus associated with this outbreak. Conserved 5'UTR motifs, the presence of a putative N(pro) coding region and limited antigenic cross-reactivity with other members of the Pestivirus genus, support the placement of this virus in the Pestivirus genus. Phylogenetic analysis of the 5'UTR, N(pro) and E2 coding regions showed this virus to be the most divergent pestivirus identified to date.     

Analysis of integrated hepatitis B virus DNA and flanking cellular sequences in a childhood hepatocellular carcinoma

Hepatitis C virus (HCV) is transmitted mainly by the parenteral route after percutaneous exposure to virus-infected products or body fluids. Thus, HCV shares with hepatitis B and D (HBV, HDV) viruses this common transmission route. The prevalence of antibody against HCV (anti-HCV) was studied in 1155 serum samples from individuals at risk of infection by bloodborne or sexually transmitted agents, as well as from others lacking such risk factors, from the city of Maracaibo, Venezuela. Anti-HCV and serological markers of infection by HBV and HDV were also studied in further 550 samples taken from Bari Indians living in different communities in the Perija mountains. State of Zulia, Venezuela. The results obtained showed that recipients of blood or blood products are at increased risk of HCV infection in Maracaibo, whereas sexual transmission plays only a minor role if any. Both HBV and HDV infections were highly prevalent among Bari Indians (64.4% positive for anti-HBc; 11.1% of HBsAg carriers; 15.3% positive for anti-HDV among HBsAg carriers). No anti-HCV positive samples were, however, detected among them, thus suggesting either that HCV has not still reached this population or that HBV and HDV are transmitted by routes unshared by HCV. Anti-HCV was also absent among samples from mentally retarded patients from Maracaibo, thus confirming similar findings from other countries and supporting the existence of specific transmission mechanisms for HBV and HDV which are not working for HCV. © 1994 Wiley-Liss, Inc.     

Insertion/deletion coding polymorphisms in hHAVcr-1 are not associated with atopic asthma in the Japanese population

Hepatitis A virus receptor (HAVcr-1) and T-cell immunoglobulin- and mucin-domain-containing molecule (TIM)-3 were recently implicated as asthma susceptibility genes in the study of congenic mice. In a genome-wide screen, we found strong evidence for linkage of atopic asthma with marker D5S820, located approximately 0.5 Mb from hHAVcr-1 and human TIM3. We screened for mutations in human HAVcr-1 (hHAVcr-1) and in TIM3 and found seven, including two insertion/deletion polymorphisms, in hHAVcr-1 and two in TIM3. We conducted transmission disequilibrium tests (TDTs) in families identified through children with atopic asthma. None of the hHAVcr-1 allele were transmitted preferentially to asthma-affected children (P>0.1). In quantitative TDT analysis, no association was observed between the log[total IgE] and either allele of the hHAVcr-1 polymorphism (P>0.1). The two TIM3 mutations were rare in the Japanese population, occurring in only one of 48 unrelated asthmatic subjects. Our results indicate that hHAVcr-1 polymorphisms are not likely to be associated with the development of atopy-related phenotypes in the Japanese population.     

Evolution of instability at coding and non-coding repeat sequences in human MSI-H colorectal cancers

A number of human genes containing coding mononucleotide repeat sequences are particularly prone to mutations in tumors with defects in mismatch repair (MMR) genes (MSI-H cancers). In a large series of MSI-H colorectal tumors, we looked for mutations in 25 coding repeats contained in eight genes already known to be mutated in these cancers or in 17 other genes with an expected role in carcinogenesis. Mutations were found in 19 of the 25 candidate genes. Using a maximum likelihood statistical method, they were separated into two different groups that differed significantly in their mutation frequencies, and were likely to represent mutations that do or do not provide selective pressures during MSI-H tumoral progression, respectively. Three new target genes were found (GRB-14, RHAMM, RAD50). Our results provide evidence that MSI-H tumoral progression involves the cumulative mutations of a large number of genes. For each MSI-H tumor we calculated indexes representing the number of mutations found in genes of these groups. We also evaluated a shortening index at both the Bat-25 and Bat-26 non-coding mononucleotide tracts that are known to be almost always unstable in MSI-H cancers. A significant correlation was observed between instability at both coding and non-coding repeats, suggesting that Bat-25 and Bat-26 could be used as simple phenotypical markers of the tumoral evolution. A preferential order of mutations was deduced. During this process, hMSH3 alterations, a target gene encoding for a MMR protein, was found to play an important role by increasing the instability phenomenon characterizing these cancers.     

Complete coding sequences and phylogenetic analysis of Human Bocavirus (HBoV)

Human Bocavirus (HBoV) is a novel virus which can cause respiratory tract disease in infants or children. Recently, the prevalence of this virus has been studied worldwide. In this study, 18 of 252 (7.14%) nasopharyngeal suctions from infants or children between 1 month and 9 years of age with respiratory tract illness were HBoV-positive by PCR. Three positive samples were selected for sequencing the entire coding sequences using a new conserved set of primers. The results showed that the most conserved regions of HBoV are the NS1 and NP1 genes, whereas VP1 and VP2 showed frequent variations. However, the complete coding sequences showed that the variations did not depend on the origin of virus found. The complete coding sequences determined in this study can resolve the problem of an HBoV detection method, which can be advantageously implemented in laboratory detection.     

Absence of HHV-8 DNA sequences in malignant mesothelioma.

Human herpesvirus 8 (HHV-8) associated primary effusion lymphomas arise and grow in the body cavities as effusions, but it is not known whether the lining of body cavities and mesothelium derived malignancies are potential targets of HHV-8 infection. We examined a series of 13 diffuse malignant mesotheliomas and four mesothelial cell rich effusion samples of non-neoplastic aetiology from non-immunodepressed patients using the polymerase chain reaction to detect HHV-8 specific sequences. HHV-8 amplification products were absent in diffuse malignant mesotheliomas and in non-neoplastic effusions samples. These results suggest that HHV-8 has a selective tropism among body cavity based tumours, being confined to primary effusion lymphomas.