An optimized protocol for detecting guard cell specific gene expression by in situ RT-PCR in Brassica rapa

It is important to detect specific genes expressed in the guard cells,which control gas exchange and play key roles in response to drought and salt stresses.Due to the genetic transformation of Chinese cabbage(Brassica rapa)has not been well developed,in situ RT-PCR is a valuable option for detecting guard cell specific genes.We reported an optimized protocol of in situ RT-PCR by using an FAMA homologous gene Bra001929 in Brassica rapa.FAMA in Arabidopsis has been verified to be specially expressed in guard cells.We designed specific RT-PCR primers and optimized the protocol in terms of the(a)reverse transcription time,(b)blocking time,(c)antigen-antibody incubation time,and(d)washing temperature.Our approach provides a sensitive and effective in situ RT-PCR method for locating expression in the guard cells in Brassica rapa.Moreover,we proved the guard cell specific expression of Bra001929 in the epidermis indicating its’applicability as a marker gene for guard cells of Brassica rapa.

A STUDY ON EXPRESSION OF ARmRNA IN HUMEN BPH TISSUE WITH IN SITU RT-PCR

Objecive To check and compare the expression levels of human androgen receptor (AR) mRNA in both normal adult prostate (NAP) and benign prostate hyperplasia (BPH) tissues, and to try to find if BPH results from the abnormal transcription of ARmRNA in prostate. Methods Expression of the human ARmRNA in 14 paraffin-embedded prostate tissues (4 cases of NAP and 10 cases of BPH) was studied with the direct in situ reverse transcription-polymerase chain reaction (RT-PCR). Quantitative analysis of the ARmRNA products was performed using the image analysis system. Results ①Specific ARmRNA was detected in both NAP and BPH specimens and in both epithelia and interstitial cells. The positive products were relatively densely localized in the cytoplasm of perinuclear zone. ② The intensity of ARmRNA signals in epithelial cells was significantly stronger than that in interstitial cells (P<0.001). However, there was no statistically significant difference in ARmRNA level between NAP and BPH; ③ The heterogeneity of ARmRNA signal and the androgen-independent cells were observed in prostatic epithelia. Stronger positive signals of ARmRNA were shown in a few basal-cell layer (BCL) cells of BPH tissue, but were not found in that of NAP tissue. Conclusion Results of this study show that there is no significant difference in the ARmRNA expression between NAP and BPH groups in both epithelium and interstitial cells. It may indicate that BPH does not result from the ARmRNA transcription in the prostate.

Comparison of FGFR1 expression on lens epithelial cells between adults and fetuses

AIM: To study the differences of fibroblast growth factor receptor 1 (FGFR1) gene on human lens epithelial cells (HLECs) of adults and fetuses. METHODS: Indirect in situ RT-PCR was adopted for detection of FGFR1 gene. The cDNA of the nnRNA in the paraffin sections of fetus and adult HLEC was synthesized by reverse transcription reaction. After PCR amplification, in situ hybridization test was performed with synthesized oligonucleotide probe and relative quantification was carried out using image analysis. RESULTS: HLECs of adults and fetuses expressed FGFR1 gene, the expression level was higher in fetuses than in adults. The difference between them had significance (P<0.05). CONCLUSION: FGFR1 Exist in HLEC and the expression is age-related, which could be one of causes of the high occurrence of post operational after-cataract in children.

A nuclear localized protein ZCCHC9 is expressed in cerebral cortex and suppresses the MAPK signal pathway

The CCHC-type zinc finger motif has numerous biological activities （such as DNA binding and RNA binding） and can also mediate protein-protein interaction. This article gives a primary report about the human ZCCHC9 gene. Protein ZCCHC9 contains four CCHC motifs and is highly conserved in humans, mice, and rats. The whole eDNA sequence of the ZCCHC9 gene has been amplified by PCR and a number of plasmids have been constructed for further study. The results show that ZCCHC9 is localized in the nucleus, and especially concentrated in the nueleolus. It is highly expressed in the brain and testicles of the mouse. This has been confirmed by real-time reverse transcription polymerase chain reaction （RT-PCR）. In situ hybridization of the mouse brain indicates that ZCCHC9 is mainly expressed in the cerebral cortex. Reporter gene assay shows that ZCCHC9 suppresses the transcription activities of NF-kappa B and SRE, and may play roles in the Mitogen-Aetivated Protein Kinase （MAPK） signaling transduetion pathway.

Polypeptide Daintain as a New Biomarker for Detecting Breast Tumor

Daintain, a novel bioactive peptide produced and secreted by macrophages, was expressed in breast tumor tissues. The spatial distributions of daintain in 66 breast tumor specimens were investigated with immuno-histochemistry method. Reverse transcription polymerase chain reaction （RT-PCR） and in situ hybridization inspection system were also used to detect daintain in 45 cases of malignant breast tumors. The final results show that 93% high positive responses to daintain on breast cancer tumors. RT-PCR demonstrated that, no smear of daintain transcripted in benign tissues was found, and light smear in peri-cancer tissue was observed. Distribution of daintain was distinguishable among benign tissues, hyperplasia tissues, immature hyperplasia and invasive breast cancer, which can be used to mark the progression of the malignant lesion development. We conclude that the expression of daintain is up-regulated in breast cancers, which indicates that the peptide is closely associated with the disease progression. So daintain could be used as the biomarker for detecting breast cancer.

Cloning of human brevican cDNA and expression of its mRNA in human glioma

Objective: To clone the cDNA of human brevican secreting isoform and to investigate its mRNA expression in human glioma. Methods: The full-length cDNA of human brevican secreted isoform was cloned from a human anaplastic astrocytoma by RT-PCR, and the expression of human brevican mRNA in 22 cases of human glioma and 13 cases of non-glial brain tumors were investigated by in situ hybridization. Results: The cDNA which including the whole open reading frame of human brevican secreted isoform was obtained. In situ hybridization showed that brevican positive cells were present in all of the 22 cases of gliomas (100%), whereas none were found in the 13 cases of non-glial and metastasis brain tumors examined. Conclusion: The results suggest that brevican mRNA is highly and specifically expressed in human glioma.

The expression of TRPA1 mRNA in the rat brain

Objective： To investigate the distribution of TRPA1 （one kind of the TRP-like ion channel family） channel in the hippocampus and cerebral cortex of rat. Methods： RT-PCR was used to amplify the fragment of TRPA1 in the DRG （dorsal root ganglion）, hippocampus and cerebral cortex of adult SD rat. In situ hybridization staining was used to show the distribution of TRPA1 mRNA in the hippocampus and cerebral cortex of adult rat brain. Results： Both RT-PCR and in situ hybridization staining showed that TRPA1 mRNA was expressed in hippocampus and cerebral cortex of the adult rat brain. Conclusion： Our results suggest that there is expression of TRPA1 mRNA both in the hippocampus and cerebral cortex of the adult rat brain.

Expression of Vascular Endothelial Growth Factor C and Its Clinical Significance in Human Esophageal Squamous Cell Carcinoma

OBJECTIVE To examine the expression of vascular endothelial growth factor C （VEGF-C） in human esophageal squamous cell carcinoma （ESCC）, and to clarify its role in lymphatic metastasis in ESCC patients.METHODS Esophageal carcinoma EC9706 cells and samples from 49 patients with primary ESCC were investigated by using S-P immunohistochemistry （IHC）, the semi-quantitative reverse transcriptase-polymerase chain reaction （RT-PCR） and in situ hybridization （ISH） methods for VEGF-C expression. RESULTS VEGF-C positive expression was found in EC9706 cells through IHC, ISH and RT-PCR. Positive IHC for VEGF-C was observed in 36 of 49 cases of ESCC. There was a significant difference between the expression of VEGF-C in a lymph-node-positive group compared to a node-negative group （χ^2=4.7, P〈0.05）. Positive ISH for VEGF-C mRNA was observed in 23 of 49 cases of ESCC. There was a significant difference between the expression of VEGF-C in the lymph-node-positive group and node-negative group （χ^2=31.3, P〈0.01）. The expression of VEGF-C was significantly higher in the lymph-node-positive group compared to the node-negative group. Of 49 ESCC tissues, RT-PCR for VEGF-C mRNA was observed positively in 29 cases. There was a significant difference between the expression of VEGF-C in the lymph-node-positive group and node-negative group （χ^2=23.3, P〈0.01）. The expression of VEGF-C was significantly higher in the lymphnode-positive group compared to the node-negative group. Expressions of VEGF-C were not significantly associated with age, gender, and pathological grade. There was a relationship between VEGF-C mRNA expressions by RT-PCR and ISH （χ^2=18.5, P〈0.01） in ESCC cases, but with no significant difference between the two methods. CONCLUSION VEGF-C expression may induce lymphangiogenesis in human ESCC. There was a close correlation between VEGF-C expression and lymph node metastasis. VEGF-C can serve as a useful prognostic factor for ESCC patients.

A novel cytogenetic abnormality r(7)(::p11.2->q36.3::) in a Philadelphia-positive chronic myeloid leukemia case

The so-calledPhiladelphia(Ph) chromosome is present in more than 90% of chronic myeloid leukemia (CML) cases. It results in juxtaposition of the 5' part of the BCR gene on chromosome 22 and the 3' part of the ABL1 gene on chromosome 9. An additional acquired monosomy 7 or deletion of 7q is associated with poor prognosis in a variety of myeloid disorders. Here we report a novel Ph chromosome positive CML case with a ring chromosome 7 [r(7)]. Immunophenotyping was compatible with CML, although 4.5% of total leucocytes appeared like acute myelogeneous leukemia (AML) subtype M2. The r(7) was characterized in detail by array-proven multicolor banding (aMCB), the latter being of enormous significance to characterize breakpoint regions in detail. Underlying mechanisms and prognostic are discussed, as ring chromosomes are rare cytogenetic abnormalities in hematopoietic malignancies.