Constitutive expression of rice MADS box gene using seed explants in hot pepper (Capsicum annuum L.).

Two transgenic pepper plants were obtained from 255 seed explants that were infected with Agrobacterium LBA4404 (pGA1209). One of them (PT2) showed morphological change, such as dwarfism and early flowering by the constitutive expression of the rice OsMADS1 gene. The in vitro condition of the plant regeneration has been optimized from hypocotyl explants on a MS medium that was supplemented with zeatin 3 mg/L, IAA 0.3 mg/L for shoot induction. The optimal rooting condition was at NAA 0.3 mg/L. The transformation frequency was 0.8% from the total hypocotyls. DNA and RNA hybridization analyses showed that the introduced gene was integrated and stably expressed in regenerated plants.

Molecular cloning, characterization and expression analysis of a catalase cDNA from hot pepper (Capsicum annuum L.).

We report the cloning of a catalase cDNA from hot pepper (Capsicum annuum L.) and its expression patterns. CaCat1 is consisted of 1837 bp containing one open reading frame (ORF) of 1479 and 45 bp/313 bp of 5'/3'-untranslated region. Deduced amino acid sequence of CaCat1 showed the 95% and 78% identity with that of Nicotiana tabacum Cat1 and Nicotiana plumbaginifolia Cat2, respectively. Northern hybridization shows that CaCat1 transcripts are more abundant in stems than in leaves and roots, and in early stages than in mature stage of fruit development. In roots its transcripts are induced in response to aluminum and NaCl. In addition, its transcription levels under light (12 h)/dark (12 h) cycle and constant light conditions exhibit circadian rhythm, reaching a maximum at late in the dark period or early in the light period. The morning specific circadian regulation of CaCat1 was also observed in NpCat1, but not in NtCat1, suggesting difference in evolutionary rates between coding region and regulatory region of the catalase genes.

CDNA cloning of chloroplast omega-3 fatty acid desaturase from Capsicum annuum and its expression upon wounding.

A clone for a plastid omega-3 fatty acid desaturase (FAD) was isolated from a leaf cDNA library of hot pepper (Capsicum annuum L.). The nucleotide sequence of a 1,317 bp open reading frame in the CachFAD showed 80.9% homology with that of tobacco plant. It codes for a polypeptide of 438 amino acids with molecular mass of 50.5 kDa and a pI of 8.14. The CachFAD had a putative transit peptide for targeting the chloroplast. Genomic Southern hybridization indicated that it exists as small gene family. Northern hybridization revealed that its mRNA was present in leaves, but not in roots. Transcript levels in the leaves upon wounding increased rapidly to reach the first peak between 1-3 h, decreased thereafter and slightly increased at 24 h after wounding. The levels of linolenic acid (18:3) in wounded leaves also reached the first peak at 6 h, decreased thereafter and reached the second peak at 18 h. These results indicated that wounding not only enhanced the accumulation of the CachFAD mRNA but also increased the conversion of linoleic acid (18:2) to linolenic acid (18:3) in leaf lipids of hot pepper.

Molecular cloning of a cDNA encoding ribosome inactivating protein from Amaranthus viridis and its expression in E. coli.

In order to isolate a cDNA clone of ribosome inactivating protein (RIP), a cDNA library was constructed in Uni-ZAP XL vector with poly(A) RNA purified from leaves of Amaranthus viridis. To get the probe for screening the library, PCR of phage DNA was conducted using the vector primer and degenerate primer designed from a conserved putative active site of the RIPs. Twenty-six cDNA clones from about 600,000 plaques were isolated, and one of these clones was fully sequenced. It was 1,047 bp and contained an open reading frame encoding 270 amino acids. The deduced amino acid sequence had a putative signal sequence of 17 amino acids and a putative active site (AIQMVAEAARFFKYIE) conserved in other RIPs. E. coli cells expressing A. viridis RIP cDNA did not grow well as compared to control cells, indicating that recombinant A. viridis RIP presumably inactivated E. coli ribosomes. In addition, recombinant A. viridis RIP cDNA produced by E. coli had translation inhibition activity in vitro.

Rehmannia glutinosa inhibits tumour necrosis factor-alpha and interleukin-1 secretion from mouse astrocytes.

We investigated whether an aqueous extract of Rehmannia glutinosa steamed root (RGAE) inhibits secretion of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) from primary cultures of mouse astrocytes. RGAE dose-dependently inhibited the TNF-alpha secretion by astrocytes stimulated with substance P (SP) and lipopolysaccharide (LPS). IL-1 has been shown to elevate TNF-alpha secretion from LPS-stimulated astrocytes while having no effect on astrocytes in the absence of LPS. We therefore investigated whether IL-1 mediated inhibition of TNF-alpha secretion from astrocytes by RGAE. Treatment of RGAE to astrocytes stimulated with both LPS+SP decreased IL-1 secretion. Moreover, incubation of astrocytes with IL-1 antibody abolished the synergistic cooperative effect of LPS+SP. These results suggest that RGAE may inhibits TNF-alpha secretion by inhibiting IL-1 secretion and that RGAE has an anti-inflammatory activity in the central nervous system curing some pathological disease states.

Isolation and characterization of the catalase gene from Rhizobium sp. SNU003, a root nodule symbiont of Canavalia lineata.

A catalase gene from Rhizobium sp. SNU003, a root nodule symbiont of Canavalia lineata, was cloned and its nucleotide sequence was determined. The Rhizobium DNA of about 280 bp was amplified using two PCR primers synthesized from the conserved sequences of the type I catalase gene. The nucleotide sequence of the amplified fragment revealed three regions that were conserved in the catalase, showing it as being part of the catalase gene. A genomic Southern hybridization using this fragment as a probe showed that the 5.5 kb PstI, 1.8 kb EcoRI, and 0.7 kb StyI fragments hybridized strongly with the probe. The Rhizobium genomic library constructed into the EMBL3 vector was screened, and one catalase clone was selected. The nucleotide sequence of the 5.5 kb PstI fragment from the clone revealed an open reading frame of 1455 bp, encoding a polypeptide of 485 amino acids with a molecular mass of 54,958 Da and a pI of 6.54. The predicted amino acid sequence of the catalase is 66.3% identical to that of Bacteroides fragilis, but was only 53.3% identical to the Rhizobium meliloti catalase.

Isolation and characterization of mitochondrial manganese superoxide dismutase (MnSOD) from Capsicum annuum L.

A cDNA clone for a mitochondrial manganese superoxide dismutase (MnSOD) was isolated and characterized from red pepper (Capsicum annuum L.). The clone consisted of 941 bp containing one open reading frame (ORF) of 687 bp, 34 bp/220 bp of 5'/3'-untranslated region. Amino acid sequence of the ORF showed the highest homology (86%) with that of Nicotiana plumbaginifolia. It encodes for a polypeptide of 228 amino acids with a molecular mass of 25.5 kDa and a pI value of 8.39. Genomic Southern hybridization suggested that more than one copy are present. Northern hybridization showed that the MnSOD transcript was more abundant in stems than in leaves and roots. When seedlings were treated with arsenate (0.1-10 mM), the MnSOD transcript level increased slightly at 0.1 mM and then dropped, while the Cu/ZnSOD transcript level increased at 1 mM, and also dropped at higher concentrations.

Construction of retroviral vectors with improved safety, gene expression, and versatility.

Murine leukemia virus (MLV)-based retroviral vectors are the most frequently used gene delivery vehicles. However, the current vectors are still not fully optimized for gene expression and viral titer, and many genetic and biochemical features of MLV-based vectors are poorly understood. We have previously reported that the retroviral vector MFG, where the gene of interest is expressed as a spliced mRNA, is superior in the level of gene expression with respect to other vectors compared in the study. As one approach to developing improved retroviral vectors, we have systematically performed mutational analysis of the MFG retroviral vector. We demonstrated that the entire gag coding sequence, together with the immediate upstream region, could be deleted without significantly affecting viral packaging or gene expression. To our knowledge, this region is included in all currently available retroviral vectors. In addition, almost the entire U3 region could be replaced with the heterologous human cytomegalovirus immediately-early promoter without deleterious effects. We could also insert internal ribosome entry sites (IRES) and multicloning sites into MFG without adverse effects. Based on these observations, we have constructed a series of new, improved retroviral constructs. These vectors produced viral titers comparable to MFG, expressed high levels of gene expression, and stably transferred genes to the target cells. Our vectors are more convenient to use because of the presence of multicloning sites and IRESs, and they are also more versatile because they can be readily converted to various applications. Our results have general implications regarding the design and development of improved retroviral vectors for gene therapy.

Isolation and characterization of cytosolic copper/zinc superoxide dismutase from Capsicum annuum L.

A cDNA clone for a cytosolic Cu/Zn superoxide dismutase (Cu/ZnSOD) was isolated and characterized from red pepper (Capsicum annuum L.). The clone consisted of 735 bp containing one open reading frame (ORF) of 459 bp, 46 bp of 5'- and 230 bp of 3'-untranslated region. The nucleotide sequence of the ORF showed 93% homology with that of Nicotiana plumbaginifolia and tomato. It encodes a polypeptide of 154 amino acids with a molecular weight of 15,300. Genomic Southern hybridization suggested that only one copy is present. During cold treatment at 4 degrees C, its expression was maintained at a similar level regardless of illumination.

The application of image analysis and neural network technology to the study of large-cell liver-cell dysplasia and hepatocellular carcinoma.

Liver cell dysplasia (LCD) is considered a preneoplastic lesion, whose characterization and differentiation from hepatocellular carcinoma (HCC) and from the reactive changes seen in cirrhosis has been controversial. We studied 12 cases of LCD (large cell type) with image analysis techniques (IA) and compared the findings with those of HCC (n = 40), and a spectrum of non-neoplastic hepatic lesions including normal liver and cirrhosis (n = 49). A minimum of 200 Feulgen-stained nuclei were measured from each lesion with the CAS 200 image analysis system. The data were collected with the aid of CellSheet software. Thirty-four variables were measured, including geometric, textural, and photometric nuclear features and DNA ploidy. The data were analyzed with multivariate statistics and a backpropagation neural network (NN). Stepwise statistical analysis selected 22 variables that were statistically significant in the three groups with P values <.05. Various NN architectures were developed using these variables. The best NN architecture included a sigmoidal transfer function, 14 input, 16 hidden, and 3 output neurons. It trained to completion after 8,887 runs using 90% of the lesions. This NN yielded a 100% cross-validation rate for unknown cases. These data support the concept of LCD (large cell type) as a lesion that can be objectively distinguished from HCC and non-neoplastic liver. Our study also demonstrates the potential usefulness of IA for the evaluation of difficult histopathological problems.

A ribosome-inactivating protein from Amaranthus viridis.

An antiviral protein purified from the leaves of Amaranthus viridis was named amaranthin. The in vivo antiviral activity of amaranthin was confirmed in tobacco mosaic virus (TMV) infection test on Nicotiana glutinosa leaves. The molecular mass of the amaranthin was estimated about 30 kDa by SDS-PAGE and the pI was measured as 9.8 by isoelectric focusing (IEF) analysis. Cytotoxicity of the amaranthin using in vitro translation inhibition assay was similar to that of pokeweed antiviral protein (PAP) with IC50 of 25 pM. Depurination activity (N-glycosidase activity) against animal rRNA was also confirmed.