Boron Supply Enhances Aluminum Tolerance in Root Border Cells of Pea (Pisum sativum) by Interacting with Cell Wall Pectins.

Aluminum (Al) toxicity is the primary factor limiting crop growth in acidic soils. Boron (B) alleviates Al toxicity in plants, which is mainly considered to be due to the formation of Rhamnogalacturonan II-B (RGII-B) complexes, which helps to stabilize the cytoskeleton. It is unclear yet whether this is due to the increasing of net negative charges and/or further mechanisms. Kinetics of Al accumulation and adsorption were investigated using entire cells, cell wall and pectin of root border cells (RBCs) of pea (Pisum sativum), to reveal the mechanism of B in interacting with alkali-soluble and chelator-soluble pectin for an increased Al tolerance in RBCs. The results show that B could rescue RBCs from Al-induced cell death by accumulating more Al in the cell wall, predominately in alkali-soluble pectin. Boron also promotes Al3+ adsorption and inhibits Al3+ desorption from alkali-soluble pectin. Thus, more Al3+ is immobilized within the alkali-soluble pectin fraction and less in the chelator-soluble pectin, rendering Al3+ less mobile. Boron induces an increase of RG-II (KDO,2-keto-3-deoxyoctonic acid) content for forming more borate-RGII complexes, and the decrease of pectin methyl-esterification, thus creates more negative charges to immobilize Al3+ in cell wall pectin. The study provides evidence that abundant B supply enhances the immobilization of Al in alkali-soluble pectin, thus most likely reducing the entry of Al3+ into the symplast from the surroundings.

Alkali-Soluble Pectin Is the Primary Target of Aluminum Immobilization in Root Border Cells of Pea (Pisum sativum).

We investigated the hypothesis that a discrepancy of Al binding in cell wall constituents determines Al mobility in root border cells (RBCs) of pea (Pisum sativum), which provides protection for RBCs and root apices under Al toxicity. Plants of pea (P. sativum L. 'Zhongwan no. 6') were subjected to Al treatments under mist culture. The concentration of Al in RBCs was much higher than that in the root apex. The Al content in RBCs surrounding one root apex (10(4) RBCs) was approximately 24.5% of the total Al in the root apex (0-2.5 mm), indicating a shielding role of RBCs for the root apex under Al toxicity. Cell wall analysis showed that Al accumulated predominantly in alkali-soluble pectin (pectin 2) of RBCs. This could be attributed to a significant increase of uronic acids under Al toxicity, higher capacity of Al adsorption in pectin 2 [5.3-fold higher than that of chelate-soluble pectin (pectin 1)], and lower ratio of Al desorption from pectin 2 (8.5%) compared with pectin 1 (68.5%). These results indicate that pectin 2 is the primary target of Al immobilization in RBCs of pea, which impairs Al access to the intracellular space of RBCs and mobility to root apices, and therefore protects root apices and RBCs from Al toxicity.

Down-regulation of ß3-integrin inhibits bone metastasis of small cell lung cancer.

Bone is one of the most frequent targets of small cell lung cancer (SCLC) metastasis, but the molecular mechanism remains unclear. ß3-integrin plays an important role in invasion of various kinds of tumors. Yet, its role in bone-metastasis of SCLC is still unknown. In this study, we first examined the expression of ß3-integrin in SBC-5 and SBC-3 cells by real-time PCR, western blot and immunofluorescence. We found that, compared to none bone-metastatic SBC-3 cells, ß3-integrin was highly expressed in SBC-5 cells, a specific bone-metastatic SCLC cells line characterized in our previous study. We next constructed ß3-integrin siRNA and transfected SBC-5 cell line, and found that ß3-integrin siRNA significantly down-regulated the ß3-integrin mRNA level and protein expression in SBC-5 cell line. We further found that inhibition of ß3-integrin significantly reduced tumor cell proliferation and induced apoptosis. In addition, the ß3-integrin down-regulated cells presented significant decrease in cell adhesion, migration and invasion activity. Our results suggest the ß3-integrin has an essential effect on tumor cell proliferation and progression, and may be a potential therapeutic target for the prevention of skeletal metastases of lung cancer.

The effect of down regulation of calcineurin Aα by lentiviral vector-mediated RNAi on the biological behavior of small-cell lung cancer and its bone metastasis.

Bone is the third most common site of cancer metastasis. Over 30 to 40% of lung cancers can develop skeletal metastasis and no effective curative therapy exists in clinic cases. Previously we screened the different expression of proteins between SBC-5 cells and SBC-3 cells by proteomic study methods (MALDI-TOF/TOF-MS) and found that calcineurin (hereafter referred as Cn) overexpresses in SBC-5 which has special priority in metastasis to bone in a multiple-organ metastasis mice model. However the roles of Cn in osteotropism of SCLC remain to be elucidated. At present study, we decrease CnAα expression in SBC-5 by lentiviral vector-mediated RNAi and found that down regulation of CnAα gene expression can decrease the proliferation and colony formation rate, impede the cell cycle progression, reduce the cell migration and invasion, and inhibit cells adhering to bone matrix, but not change the apoptosis rate of SBC-5 in vitro. In vivo down or up regulation of CnAα gene expression can only decrease or increase the bone metastasis rate, but not affect the metastasis rate to the visceral organs. Our research reveals that CnAα is closely related to the osteotropism metastasis of SCLC and a candidate tumor promotor gene for developing bone metastases.

[The different structure forms of HBx protein influence their intracellular distribution in hepatocellular carcinoma HepG2 cells].

AIM: Different eukaryotic expression vectors were selected in this research, and then we respectively cloned and constructed recombinant plasmids contained HBX gene or its different fusion forms with GFP. We are aimed to explore the influence of the HBX protein with different structure on its intracellular localization. METHODS: Flag-HBX gene was amplified from pcDNA3.0-HBX plasmid in our laboratory, cloned into pMD-18T vectors, sequenced.and then subcloned into different vectors. After right sequenced, respective recombinant plasmids of pFlag- HBX-IRES2-EGFP, pEGFP-C3-Flag-HBX and pFlag-HBX-EGFP-N3 were transiently transfected into hepatoma HepG-2 cells. The intra-cellular localizations and distributions of HBX protein were examined by indirect immunofluorescence. RESULTS: Three different Flag-HBX eukaryotic expression vectors were successfully constructed. After transfection of them into HepG2 cells respectively, indirect immunofluorescence demonstrated that HBX protein fused with GFP in different forms show distint intra-cellular distribution characteristics. CONCLUSION: We have provided significant experimental evidences for research of the role of HBX protein in the development of hepatocellular carcinoma, and especially the references for explanation of the outcomes in vitro transfection experiments.

Calcineurin promotes proliferation, migration, and invasion of small cell lung cancer.

A novel role for calcineurin (Cn) has been reported recently regarding the oncogenic potential in pancreatic and colorectal cancer. The aim of this study was to investigate the putative causal role calcineurin could play in the development of lung cancer with bone metastases. We found that CnAalpha, an isoform of calcineurin, was significantly overexpressed in lung cancer tissues with bone metastasis as compared to tumors with non-bone metastases as investigated by RT-PCR. Strong nuclear staining of tumor cells was observed in small cell lung cancer tissues with bone metastasis. Conversely, cytoplasmic staining of tumor cells was observed in small cell lung cancer tissues with non-bone metastasis. Western blots of nuclear proteins from lung cancer tissues indicated that CnAalpha was highly expressed in lung cancer tissues with bone metastases, but not in those with non-bone metastases. In vitro, it was demonstrated that the CnAalpha gene obviously promoted cell proliferation and inhibited cell apotosis. The CnAalpha gene affected the cell cycle and promoted G1[Symbol: see text]S transition in SBC-3 cells. Transfection with the CnAalpha gene promoted cell migration and invasion. These results indicated that CnAalpha may affect the biological behavior of the human small cell lung cancer cell line SBC-3 in vitro and may be a candidate tumor promotor gene for developing bone metastases.

Hepatitis B virus X-DNA. A serum marker for early detection of resistance development during lamivudine therapy.

HBV genome replication intermediates blocked at early stages of minus strand synthesis have been identified in a study on circulating DNA and RNA during short-term lamivudine therapy. This suggested that the inhibition of HBV replication processes in the liver are mirrored in the blood. Levels of circulating HBV mRNA remained largely unaffected. Here we followed therapy with two patients (patients 1 and 2) up to stages without apparent replication. As in the earlier study, DNA segments produced successively during replication were used as targets for quantitative PCR: X (early minus strand), C (completed minus strand), and preC (nascent plus strand). Corresponding RNA was quantified by RT/PCR. Polyadenylated viral RNA were assayed as full-length (f) and as truncated (tr) RNAs. Blocked X-region intermediates persisted for about one year. After a period of undetectable HBV DNA viral replication resumed in patient 1 because of the emergence of drug-resistant mutants and in patient 2 because of the discontinuation of therapy. In the former case, X-region intermediates reappeared first, then C- and, finally, preC-region intermediates. Stopping therapy, in contrast, led to a simultaneous reappearance of all three types of intermediates. At low replication levels or its absence, trRNA represented the only polyadenylated viral RNA. Apparently, HBV serum nucleic acid markers allow a study of replication and transcription separately. Specifically, it is concluded (1) that PCR assays for monitoring lamivudine therapy must target the X-gene region and (2) that in the absence of HBV replication, trRNA may constitute a serum marker for HBV expression.

Circulating hepatitis B virus RNA in newborns from carrier mothers.

OBJECTIVE: Despite intermittent flares, progression of chronic infection with hepatitis B virus has generally been related to a decrease in replication levels. In our laboratory, this decrease has been found to be associated with a decline in circulating full-length transcripts (fRNA) and to a shift to truncated transcripts (trRNA). Monitoring fRNA and trRNA as markers allows a more detailed analysis of high or low or non-replicative HBV infection stages. METHODS: Here, we determined circulating HBV RNA in newborns from a total of 69 HBsAg-positive carrier mothers with and without immunotherapy administered during the last 3 months of pregnancy. RESULTS: In line with previous observations, serum HBV DNA in newborns was only found when their mothers had not been treated (7/23). HBV RNA measured as trRNA was detected both in the presence and in apparent absence of serum HBV DNA. fRNA (coexisting with HBV DNA and HBeAg) was detected only in newborns from untreated mothers (8/23) and was always combined with trRNA. In contrast, trRNA was found in newborns from both untreated (20/23) and treated mothers (26/46). CONCLUSIONS: Our data strongly suggest a direct intrauterine transmission of a low-replicative (unapparent) HBV infection stage. However, a feto-maternal transfer of trRNA (transfer of a marker) rather than transmission of the infectious virus cannot be excluded.

Skewed X chromosome inactivation of blood cells is associated with early development of lung cancer in females.

The skewed X chromosome inactivation (SXCI) was found mainly in adult females. It has been linked to development of ovarian and breast cancers. The present study aimed to describe the relationship between SXCI and development of lung cancer in females. DNA was isolated from blood cells from patients with primary lung cancer (n=148) and reference subjects (n=289). The androgen receptor (AR) gene exon 1 was amplified, with its products from different alleles resolved on denaturing polyacrylamide gels and visualized by silver staining. The corrected ratio (CR) between products from AR alleles after and before HpaII pretreatment was calculated. Occurrence of SXCI was detected in both the patients and reference subjects at similar frequency. However, the phenomenon was more frequent in the patients below 40 years compared to the corresponding reference group, either taking CR >/=3 (25 and 5.8%, respectively; P=0.048) or CR >/=10 as the criterion of SXCI (16.7 and 0.8%, respectively; P=0.022). A higher frequency of SXCI was also found in the patients below 50 years compared to that for the corresponding reference group when CR >/=10 adopted as the criterion (7.9 and 1.2%, respectively; P=0.046). The cancer patients with SXCI were more than 10 years younger in average age than those without SXCI. SXCI of blood cells is associated with early development of lung cancer in females. The X chromosomal inactivation assay, therefore, may be used to screen for females predisposed to malignancies including lung cancer.

Liver cell adenoma: a case report with clonal analysis and literature review.

We report a case of liver cell adenoma (LCA) in a 33-year-old female patient with special respect to its clonality status, pathogenic factors and differential diagnosis. The case was examined by histopathology, immunohistochemistry and a clonality assay based on X-chromosomal inactivation mosaicism in female somatic tissues and polymorphism at androgen receptor focus. The clinicopathological features of the reported cases from China and other countries were compared. The lesion was spherical, sizing 2 cm in its maximal dimension. Histologically, it was composed of cells arranged in cords, most of which were two-cell-thick and separated by sinusoids. Focal fatty change and excessive glycogen storage were observed. The tumor cells were round or polygonal in shape, resembling the surrounding parenchymal cells. Mitosis was not found. No portal tract, central vein or ductule was found within the lesion. The tumor tissue showed a positive reaction for cytokeratin (CK) 18, but not for CK19, vimentin, estrogen and progesterone receptors. Monoclonality was demonstrated for the lesion, confirming the diagnosis of an LCA. Clonality analysis is helpful for its distinction from focal nodular hyperplasia.

[X-chromosomal inactivation skewing in blood cells is associated with early development of lung].

OBJECTIVE: To observe the relationship between skewed X-chromosomal inactivation (SXCI) and development of lung cancer in females. METHODS: DNA was isolated from peripheral blood cells from patients with primary lung cancer (n = 148) and control subjects (n =289). Exon 1 of androgen receptor ( AR) gene was amplified, with its products from different alleles resolved on denaturing polyacrylamide gels and visualized by silver staining. The corrected ratio (CR) between products from different AR alleles before and after Hpa II pretreatment was calculated. All statistical tests were two-sided. RESULTS: With CR> or = 10 adopted as the criterion, SXCI was found more frequently in the younger patients ( C50 years; 7. 9%) than in the controls of the same age group (1. 2% ; P = 0. 046). The SXCI frequency, however, were not significantly different between the old patients ( > 50 years; 4. 5% ) and the controls of the same age group (5. 4% ; P =0. 488). Whether taking CR> or =3 or CR> or =10 as the criteria, the average ages of the patients with SXCI were more than 10 years younger than those without SXCI (P < 0. 05). CONCLUSION: SXCI in blood cells is associated with early development of lung cancer in females.

Establishment and characterization of human hepatocellular carcinoma cell line FHCC-98.

AIM: To establish a novel human hepatocellular carcinoma (HCC) cell line FHCC-98 from HCC tissue and to provide a suitable model for studying HCC occurrence, progress and metastasis. METHODS: Serially passaged cells were cultured and their morphologies were observed under light and electron microscope. Cytogenetic study was conducted by using flow cytometry and chromosome analysis. Expressions of tumor markers such as alpha-fetoprotein (AFP), cytokeratin (CK) and hepatoma metastasis-associated factor HAb18G/CD147 on the FHCC-98 cells were detected by immunocytochemistry or Western blotting. Lactic dehydrogenase (LDH) isoenzymes were detected by polyacrylamide gel electrophoresis (PAGE). Xenograft was performed by inoculating FHCC-98 cells into the flanks of nude mice. RESULTS: Morphology of FHCC-98 cells was the same as that of other malignant cells. The expressions of the cells were positive for HAb18G/CD147 and CK, and negative for AFP. Its population doubling time was 21.4 h. The cell DNA was tetraploid and the major chromosomes were triploid by cytogenetics analysis. The tumorigenicity in nude mice was 100%. PAGE showed four bands representing LDH2, LDH3, LDH4 and LDH5. CONCLUSION: FHCC-98 is a novel HCC cell line and an ideal cell model for further exploring the mechanism of hepatocellular carcinoma invasion and metastasis.

[X-chromosome inactivation patterns of different tumor nodules in multiple leiomyomas of uterus].

OBJECTIVE: To describe the relationship among different tumor nodules in multiple leiomyomas of uterus. METHODS: Genomic DNA was extracted from fresh tissue samples, digested through incubation with methylation-sensitive Hha I or Hpa II, and amplified via PCR for androgen receptor (AR) and phosphoglycerate kinase (PGK) genes. The length polymorphism on AR gene was demonstrated by denaturing polyacrylamide gel electrophoresis and silver staining, the PGK gene products were treated with Bst XI and resolved on agarose gels. RESULTS: 112 cases of leiomyomas and one case of leiomyosarcoma were examined, 89% showing the length polymorphism for AR gene and 30% carrying the polymorphic Bst XI site at PGK locus. Loss of X-chromosome inactivation mosaicism was observed in all the 321 tumor nodules examined from 77 cases, reflecting their clonal cellular composition. The relationship between different nodules was evaluated by their X-chromosome inactivation patterns in the 295 tumor nodules taken from 57 multiple leiomyomas. Similar inactivated alleles were found in all nodules in 30, in most nodules in 7 cases, similar to a multi-nodular leiomyosarcoma, while 20 cases showed near-random distribution of the inactivated alleles in different nodules, indicating their multicentric origins. No relevance was found between this difference and any histopathological parameters including number of mitotic figures and occurrence of bizarre nuclei and degeneration. In addition, an identical mutation and loss of heterozygosity were found at the AR locus in two apparently discrete tumor nodules in one case, providing further evidence for the unicellular origin of these lesions. CONCLUSIONS: The multi-nodular leiomyomas may be classified into multicentric, unicentric types, as well as a mixed type. It remains to be clarified whether different nodules in the unicentric cases originate from a parent tumor by migration or by spreading.