[Clinical significance of syndecan-1 and syndecan-2 expression in gallbladder squamous cell/adenosquamous carcinoma and adenocarcinoma].

Objective: To investigate the expression of syndecan-1 and syndecan-2 and their clinicopathological significance in patients with gallbladder squamous cell (SC)/adenosquamous carcinoma (ASC) and adenocarcinoma (AC). Methods: A total of 126 patients with SC/ASC (n=46) and AC (n=80) were included in this study. The expression levels of syndecan-1 and syndecan-2 were detected by Envison™ immunohistochemistry assay. The clinical and prognostic significance of syndecan-1 and syndecan-2 were analyzed. Results: In the 46 SC/ASC samples, syndecan-1 and syndecan-2 were positively expressed in 29 (63.0%) and 28 (60.9%) tumor tissues, respectively. (Positive expression was defined based on the staining in the component of squamous cell carcinoma. That is to say, the tissue which adenocarcinoma part was positively stained, but squamous cell carcinoma part was negatively stained is also regarded as negative.) In the 80 AC samples, 47 (58.8%) cases showed syndecan-1 positive expression, and 51 (63.8%) showed syndecan-2 positive expression. There was no significant difference in the positive rates of syndecan-1 and syndecan-2 between SC/ASC and AC groups (P>0.05 for all). The levels of syndecan-1 and syndecan-2 were associated with tumor size, TNM staging, lymph node metastasis, invasion of adjacent tissue, and surgical procedures in SC/ASC patients (P<0.05 for all). However, their expression was associated with tumor differentiation, tumor size, TNM staging, lymph node metastasis, invasion of adjacent tissue, and surgical procedures in AC patients (P<0.05 for all). The Kaplan-Meier survival analysis of SC/ASC and AC patients revealed that the average survival time for patients with positive syndecan-1 and syndecan-2 expression was significantly shorter than that of those with negative expression (P<0.01 for all). Cox multivariate analysis indicated that syndecan-1 and syndecan-2 expression were independent unfavorable prognostic factors for SC/ASC and AC patients (P<0.05 for all). Conclusion: The syndecan-1 and syndecan-2 expression are associated with the tumor progression and poor prognosis in patients with gallbladder SC/ASC and AC.

Comparative study of clinicopathological significance, BIRC7, and STC2 expression between squamous cell/adenosquamous carcinomas and adenocarcinoma of gallbladder.

Gallbladder cancers (GBCs) are uncommon, but highly aggressive cancers. The majority of GBCs are adenocarcinomas (ACs), but rare subtypes of GBCs such as squamous cell carcinoma (SC) and adenosquamous carcinoma (ASC) are observed as well. The clinicopathological characteristics of SC/ASC have not been well documented. Expressions of BIRC7 and STC2 were observed in some tumors. However, BIRC7 and STC2 expressions and clinical significances in gallbladder cancer have not been reported.In this study, the protein expressions of BIRC7 and STC2 in 46 SCs/ASCs and 80 ACs were measured using immunohistochemistry. We demonstrated that positive BIRC7 and STC2 expressions were significantly associated with large tumor mass (>3cm), high TNM stage and lymph node metastasis in SC/ASC and AC (p<0.05). Positive expression of BIRC7 was significantly associated with invasion of around tissues and organs in both SC/ASC and AC. Additionally, negative BIRC7 and STC2 expressions were significantly associated with surgical curability in AC. Univariate Kaplan-Meier analysis showed that BIRC7 and STC2 expressions, differentiation, tumor size, TNM stages, invasion, lymph node metastasis, and surgical curability were significantly associated with post-operative survival in both SC/ASC and AC patients(p < 0.001). Multivariate Cox regression analysis showed that positive BIRC7 and STC2 expressions are independent poor-prognostic factors in both SC/ASC and AC patients. Our study suggested that positive BIRC7 and STC2 expressions are closely correlated with clinical, pathological, and biological behaviors as well as poor-prognosis of gallbladder cancer.

Mouse Zp1 encodes a zona pellucida protein homologous to egg envelope proteins in mammals and fish.

Zp1 encodes one of the three major glycoproteins of the zona pellucida, an extracellular matrix that surrounds growing oocytes, ovulated eggs, and preimplantation embryos. The mouse gene is composed of 12 exons ranging in size from 82 to 364 base pairs and spans 6.5 kilobase pairs on chromosome 19 (2.13 +/- 1.5 centimorgans distal to D19Bir1). The Zp1 exon map is similar to ZPB, a human orthologue, and an E-box (CANNTG), implicated in oocyte-specific gene expression of mouse Zp2 and Zp3, is similarly located upstream of the transcription start site. The single copy Zp1 gene encodes a 623-amino acid protein, the carboxyl-terminal half of which is significantly similar to a corresponding region of mouse ZP2. The conservation of this same region in a fish egg envelope protein suggests that not only has this protein domain been duplicated in mammals but that it has been conserved and used as an egg envelope protein in species that diverged 650 million years ago.

Coordinate expression of the three zona pellucida genes during mouse oogenesis.

The mammalian zona pellucida is an extracellular matrix that surrounds growing oocytes, ovulated eggs and early embryos. The mouse zona is composed of three sulfated glycoproteins: ZP1, ZP2 and ZP3. Each is critically involved in fertilization, the postfertilization block to polyspermy and protection of the preimplantation embryo. We have previously isolated cDNAs encoding mouse ZP2 and ZP3 and now report the isolation of a full-length cDNA encoding ZP1. Mouse ZP1 is composed of a 623 amino acid polypeptide chain with a signal peptide and a carboxyl terminal transmembrane domain, typical of all zona proteins. Sequence comparison demonstrate that mouse ZP1 is an orthologue of a rabbit zona protein, R55. The expression of R55 has been reported previously in both oocytes and granulosa cells. However, by northern analysis and in situ hybridization with 33P-labelled antisense probes to each of the three mouse zona mRNAs, we have determined that the expression of each mouse zona gene is restricted to the oocyte. ZP2 transcripts, but not ZP1 or ZP3, are detected in resting (15 microns diameter) oocytes, and all three zona transcripts coordinately accumulate as oocytes begin to grow. Together they represent approximately 1.5% of the total poly(A)+ RNA in 50-60 microns oocytes. In the latter stages of oogenesis, their abundance declines and each zona transcript is present in ovulated eggs at less than 5% of its maximal level. No zona transcripts were detected above background signal in granulosa cells. We conclude that, in mice, the three zona pellucida genes are expressed in a coordinate, oocyte-specific manner during the growth phase of oogenesis. Our data support the hypothesis that the transcription of the zona genes is controlled, in part, by shared regulatory element(s).

POM-ZP3, a bipartite transcript derived from human ZP3 and a POM121 homologue.

Human POM-ZP3 is a novel bipartite RNA transcript that is derived from a gene homologous to rat POM121 (a nuclear pore membrane protein) and ZP3 (a sperm receptor ligand in the zona pellucida). The 5' region is 77% identical to the 5' end of the coding region of rat POM121 and appears to represent a partial duplication of a gene encoding a human homologue of this rodent gene. The 3' end of the POM-ZP3 transcript is 99% identical to ZP3 and appears to have arisen from a duplication of the last four exons (exons 5-8) of ZP3. Using Northern blots and RT-PCR, POM-ZP3 transcripts were detected in human ovaries, testes, spleen, thymus, lymphocytes, prostate, and intestines. The longest open reading frame encodes a conceptual protein of 210 amino acids, the first 76 of which are 83% identical to residues 241-315 of rat POM121. The next 125 amino acids are 98% identical to residues 239-363 of the 424-amino-acid human ZP3 protein. By fluorescence in situ hybridization, genomic fragments of ZP3 and a human homologue of POM121 were localized to chromosome 7q11.23. Taken together, these data suggest that partial duplications of human ZP3 and a POM121-like gene have resulted in a fusion transcript, POM-ZP3, that is expressed in multiple human tissues.

Conservation of mammalian secondary sperm receptor genes enables the promoter of the human gene to function in mouse oocytes.

The human zona pellucida is an extracellular sheath composed of three major proteins (ZP1, ZP2, and ZP3) which surround the ovulated egg and mediate the initial interactions with sperm. Although fertilization is relatively species-specific and human sperm will not bind to mouse zona, there is a high degree of conservation between the coding regions of human ZP3 and mouse Zp-3 (the primary sperm receptor) genes. We now report the characterization of the human ZP2 gene and demonstrate that the sequences of its coding regions are 70% identical with those of the mouse Zp-2 (the secondary sperm receptor) gene. In addition, the first 300 bp of the 5' flanking regions of human ZP2 and mouse Zp-2 are highly conserved. This region of 5' flanking DNA contains a previously described 12-bp DNA sequence (element IV) that forms an oocyte-specific DNA-protein complex important for mouse Zp-2 and Zp-3 promoter activity. Human element IV forms a DNA-protein complex in gel mobility shift assays when incubated with human or mouse ovarian extracts. The formation of this complex is inhibited with molar excess of either human or mouse element IV sequences and is not present in extracts of testes, uterus, spleen, lung, or kidney. The human promoter region (0.3 kbp), coupled to a luciferase reporter gene and microinjected into the nuclei of 50-microns-diameter mouse oocytes, results in reporter gene activity at a level comparable to that of the homologous mouse promoter. Clustered point mutations in element IV in either the mouse or the human sequence dramatically decrease reporter gene activity. These results indicate that the similarity between mouse Zp-2 and human ZP2 genes enables the human promoter to utilize the heterologous transcription machinery in mouse oocytes. The observed transcription may involve the recognition of promoter sequences in element IV by conserved transcription factor(s).

Oocyte-specific factors bind a conserved upstream sequence required for mouse zona pellucida promoter activity.

The zona pellucida of mouse oocytes, composed of three major glycoproteins (ZP1, ZP2, and ZP3), performs crucial functions at fertilization and in early development. The transcripts encoding mouse ZP2 and ZP3 are coordinately expressed and accumulate in oocytes during a 2-week growth phase prior to ovulation. The 5'-flanking regions of mouse Zp-2 and Zp-3 genes and their human homologs contain five short DNA sequences (4 to 12 bp) that are 60 to 100% identical and are approximately equidistant upstream of the TATAA box in the four genes. Mutation of these five elements (I, IIA, IIB, III, and IV) in Zp-luciferase constructs demonstrates that the 12-bp element IV, positioned approximately 200 bp upstream from the TATAA box, is necessary for high-level expression from the mouse Zp-2 and Zp-3 promoters after microinjection into the nuclei of 50-microns-diameter oocytes. Injection of minimal Zp-3 promoter constructs containing element IV in either orientation also resulted in high levels of reporter gene activity, suggesting that the element is not only necessary but also sufficient for expression from zona pellucida promoters. Oligonucleotides containing the conserved element from either Zp-2 or Zp-3 form DNA-protein complexes of identical mobility in gel retardation assays using extracts of oocytes but not other tissues. These data are consistent with the hypothesis that common factors binding to conserved element IV are involved in coordinate expression of the oocyte-specific Zp-2 and Zp-3 zona pellucida genes.

Oocyte-specific expression of mouse Zp-2: developmental regulation of the zona pellucida genes.

The zona pellucida surrounds all mammalian oocytes and plays a vital role at fertilization and in early development. The genes that code for two of the mouse zona proteins (ZP2 and ZP3) represent a developmentally regulated set of genes whose expression serves as markers of mouse oocyte growth and differentiation. We previously characterized the single-copy Zp-3 gene and showed that its expression is oocyte specific and restricted to a narrow window of oocyte development. We now define the Zp-2 gene transcript and show that it is coordinately expressed with Zp-3 only during the 2-week growth phase of oogenesis that occurs prior to ovulation. Like Zp-3, the expression of Zp-2 is restricted to oocytes, and, although not detectable in resting oocytes, both ZP2 and ZP3 transcripts accumulate to become very abundant messengers in 50-microns-diameter oocytes. Ovulated eggs contain ZP2 and ZP3 transcripts which are 200 nucleotides shorter than those found in growing oocytes and have an abundance of less than 5% of the peak levels. In an attempt to understand the molecular details associated with the developmentally regulated, tissue-specific gene expression of the zona genes, the Zp-2 genetic locus has been characterized and its 5' flanking sequences have been compared with those of Zp-3. Both genes contain three short (8- to 12-base-pair) DNA sequences of 80 to 88% identity located within 250 base pairs of their transcription start sites.

Genomic mapping of murine Zp-2 and Zp-3, two oocyte-specific loci encoding zona pellucida proteins.

The zona pellucida is a unique, oocyte-specific matrix that coats the surface of all mammalian eggs. Composed of three sulfated glycoproteins in the mouse (ZP1, ZP2, and ZP3), the zona pellucida facilitates early events in fertilization and protects the embryo during preimplantation development. Using DNA isolated from hamster-mouse somatic cell hybrids and from C57BL/6J X Mus spretus interspecific backcross progeny, Zp-2 was located on chromosome 7, 11.3 +/- 3.2 cM distal to Tyr, and Zp-3 was located on chromosome 5, 9.2 +/- 2.9 cM distal to Gus.

In vitro synthesis of large RNAs by an unusual defective interfering particle of vesicular stomatitis virus.

We analyzed an unusual defective interfering (DI) particle of the New Jersey serotype of vesicular stomatitis virus. In vitro transcription experiments demonstrated that this DI particle was unusual in that it transcribed large RNAs in addition to the standard DI leader RNA. S1 nuclease mapping assays indicated that the large transcripts probably resulted from polymerase reinitiation downstream of the DI leader termination site. A sequence related to the Indiana serotype putative polymerase promoter sequence was identified which may regulate the synthesis of the large RNA transcripts.

Exogenous interferon administration in experimental leishmaniasis: in vivo and in vitro studies.

The potential feasibility of using exogenously administered human interferon for the treatment of selected cases of leishmaniasis prompted us to study the effects of murine interferon on the course of Leishmania tropica infection in C57Bl/6 mice. L cell-derived mouse interferon was administered daily by intraperitoneal (1,000 and 10,000 U) or intralesional (100 U) injection in mice inoculated into footpads with L. tropica amastigotes. Footpad swelling and tissue parasite density were assessed over the course of infection. Interferon treatment did not significantly affect these clinical and parasitological parameters. Furthermore, addition of interferon (100--100,000 U) to cultures of amastigote-infected mouse peritoneal macrophages or to axenic cultures of promastigotes did not affect replication. We conclude that interferon lacks intrinsic antileishmanial activity and does not significantly enhance host defense against Leishmania.