Antigenic variation of a major surface glycoprotein of Pneumocystis carinii

The mannosylated surface glycoprotein (gp) of Pneumocystis carinii has one known conserved epitope that is recognized by the monoclonal antibody 85-1-5E12. The gp exhibits host species-specific antigenic variation, exhibits host species-specific collagenase sensitivity, and varies in size depending on the host of origin and the method of preparation. These data support the existence of host species-specific serotypes of P. carinii.     

Pneumocystis carinii telomere repeats are composed of TTAGGG and the subtelomeric sequence contains a gene encoding the major surface glycoprotein

We have cloned a telomere and adjacent sequences from rat-derived Pneumocystis carinii using the ability of foreign telomeres to complement a yeast artificial chromosome (YAC) deficient by one telomere in Saccharomyces cerevisiae . Characterization of the cloned DNA in the recombinant YAC demonstrated that it was a chimera of two P. carinii sequences, namely a 13.5 kb fragment of mitochondrial DNA and an 8.3 kb distal portion consisting of subtelomeric DNA. The P. carinii telomere repeat was demonstrated to be TTAGGG, the most common telomere repeat found in organisms from the animal and fungal kingdoms. Karyotype analysis confirmed that this sequence was present on all the P. carinii chromosomes. Sequence adjacent to the telomere repeats was shown by Bal 31 exonuclease digestion to be located at the chromosome ends. Analysis of the subtelomeric fragment revealed homology to the gene encoding the major surface glycoprotein of P. carinii     

Epitope study and cDNA screening of major surface glycoprotein of Pneumocystis carinii.

Use of monoclonal antibodies against the major glycoprotein of Pneumocystis carinii (P115) implicated the sugar moiety as being strongly antigenic. Furthermore, monoclonal antibodies directed against the peptide portion of P115 were generated by using synthetic oligopeptides after amino acid sequencing was done on P115 proteolytic fragments.     

Characterization of the expression site of the major surface glycoprotein of human-derived Pneumocystis carinii

The major surface glycoprotein (MSG) of Pneumocystis carinii, a pathogen responsible for pulmonary infection in AIDS and other immunocompromised patients, is an abundant surface protein that potentially allows the organism to evade host defences by antigenic variation. MSG is encoded by a multicopy gene family; in two specific forms of rat-derived P. carinii, regulation of MSG expression uses a single expression site, termed the upstream conserved sequence (UCS), through two related but distinct mechanisms. In the current study, the UCS of the MSG from human-derived P. carinii was obtained using an RNA ligase-mediated rapid amplification of cDNA ends technique. Southern blot analysis demonstrated that the UCS was present in a single copy per genome, whereas multiple copies of the downstream MSG gene were present. Sequencing and restriction fragment length polymorphism analysis of polymerase chain reaction products amplified from pulmonary samples of patients with P. carinii pneumonia demonstrated that multiple MSG genes were expressed in a given host, and that different patterns of MSG expression were seen among different patients. Tandem repeats present in the single intron occurred with varying frequency in different patient isolates, potentially providing a new method for typing human isolates. Thus, human-derived P. carinii regulates MSG expression in a manner similar to P. carinii f. sp. carinii and, in immunosuppressed patients, in whom immune pressures that probably drive antigenic variation are functioning inadequately, P. carinii can express a broad repertoire of MSG variants.     

Specific T-cell response to a Pneumocystis carinii surface glycoprotein (gp120).

Pneumocystis carinii gp120 can elicit a specific T-cell proliferative response in mice after immunization with a gp120 preparation or with a crude P. carinii homogenate. It can also elicit a proliferative response from SCID mice after recovery from natural infection with P. carinii, implicating this glycoprotein as an important antigen in the host's response to P. carinii infection.     

Structure of major surface determinants and DNA diagnosis of Pneumocystis carinii

Pneumocystis carinii is a pathogen which causes fatal pneumonia in patients with the acquired immune deficiency syndrome (AIDS). To facilitate the basic study of P. carinii, we have analyzed its major surface proteins by both immunochemical and biochemical methods. The major protein components of both cysts and trophozoites are a group of proteins called "P115" with apparent masses of 105-120 kd. It includes 6 isoelectric variants. A monoclonal antibody raised against cysts recognizes all 6 variants and reacts with epitopes located in the cell wall indicating that P115 is an immunoreactive surface component. The isoelectric variants contain identical or closely related protein components and they are mannose-rich glycoproteins. The isoelectric variation may be due primarily to differences in glycosylation. The majority of sera from humans with diagnosed pneumocystosis that were tested reacted strongly with the P115 proteins. To develop probes for DNA diagnosis and to facilitate molecular studies, a genomic DNA library of P. carinii has been constructed. Some of these clones were used for DNA hybridization analysis of rat and human lungs.     

Analysis of a surface antigen of Pneumocystis carinii

The 115 kd band in polyacrylamide gels is a major antigen of Pneumocystis carinii. Data obtained from treatment with enzymes, binding to lectins, and labelling the surface with biotin suggest that this moiety is a glycoprotein containing mannosyl/glucosyl and N-acetylglucosamine residues, and that it is located on the cell wall of the organism. Other rat and human P. carinii antigens also are glycoproteins but differ in specific protein or carbohydrate residues or location on the organism.     

Detection of different developmental stages of malaria parasites by non-radioactive DNAin situ hybridization

Journal of Molecular Histology - A highly sensitive non-radioactive DNAin situ hybridization procedure is described that enables detection and unequivocal identification of various developmental...     

原位杂交检测microRNA-205在乳腺癌中的表达

目的探讨microRNA-205表达与乳腺恶性病变的关系。方法乳腺疾病及癌组织芯片原位杂交分析microRNA-205的表达;实时定量RT-PCR方法检测正常乳腺细胞株、恶性程度不同的乳腺癌细胞株中microRNA-205的表达。结果原位杂交分析显示,36例正常与良性乳腺病变中,33例(91.67%)表达阳性;36例乳腺癌中,23例(63.89%)表达阳性。microRNA-205的表达在乳腺正常与良性病变中的表达较恶性病变中高且有统计学差异(P=0.011),但与乳腺癌TNM分期、临床分期无关(P0.05)。实时定量RT-PCR结果显示,四个高度恶性乳腺癌细胞株(MDA-MB-231、HS578T、BT549和SUM159PT)中microRNA-205的表达较永生化正常乳腺上皮细胞株MCF10A和四个低度恶性细胞株(MDA-MB-468、T-47D、ZR-75-1和SKBR3)中为低(P0.05)。结论原位杂交适用于microRNA-205的表达分析;组织芯片标本原位杂交与乳腺细胞株实时定量RT-PCR分析结果提示,microRNA-205可能参与了乳腺癌的发生、发展,并随着乳腺癌的演进呈下调趋势。     

Structure and identity of a late-replicating and transcriptionally active gene

Eukaryotic genes are usually replicated early during S-phase in the cell lineages in which they are expressed. Using partially characterized cDNA probes, we recently established two exceptions to this rule in the slime mold Physarum polycephalum. In this paper, we analyzed the structure and the identity of one of these two genes. By genomic cloning and Southern analysis we demonstrate that it is a single-copy gene and decipher the structure of the two alleles by taking advantage of a restriction fragment length polymorphism. By cDNA cloning and sequencing, we deduced the amino acid coding capacity of the mRNA. Finally, we confirmed the late replication of this abundantly expressed gene by "gene dosage" analysis, an experiment that did not require any drug treatment of the cell. Our results provide for the characterization and the structure of the first developmentally regulated gene known to be replicated late in S-phase and abundantly expressed within a eukaryotic cell.     

Inhibition of In Vitro Splicing of a Group I Intron of Pneumocystis carinii

Unlike its mammalian hosts, the opportunistic fungal pathogen Pneumocystis carinii harbors group I self-splicing introns in its chromosomal genes encoding rRNA. This difference between pathogen and host suggests that intron splicing is a promising target for chemotherapy. We have found that intron splicing in vitro is inhibited by the anti- Pneumocystis agent pentamidine and by a series of pentamidine analogues, as well as by some aminoglycosides, tetracycline, L-arginine and ethidium bromide. Further studies will be needed to determine if this is the mechanism of action of pentamidine against P. carinii .