Calorie Restriction Effect of Heat-Processed Onion Extract (ONI) Using In Vitro and In Vivo Animal Models.

Onion (Allium cepa L.) is widely consumed as food or medicinal plant due to its well-defined health benefits. The antioxidant and antihyperlipidemic effects of onion and its extracts have been reported well. However, very limited information on anti-hyperglycemic effect is available in processed onion extracts. In our previous study, we reported that Amadori rearrangement compounds (ARCs) produced by heat-processing in Korean ginseng can reduce carbohydrate absorption by inhibiting intestinal carbohydrate hydrolyzing enzymes in both in vitro and in vivo animal models. To prove the enhancement of anti-hyperglycemic effect and ARCs content by heat-processing in onion extract, a correlation between the anti-hyperglycemic activity and the total content of ARCs of heat-processed onion extract (ONI) was investigated. ONI has a high content of ARCs and had high rat small intestinal sucrase inhibitory activity (0.34 ± 0.03 mg/mL, IC50) relevant for the potential management of postprandial hyperglycemia. The effect of ONI on the postprandial blood glucose increase was investigated in Sprague Dawley (SD) rats fed on sucrose or starch meals. The maximum blood glucose levels (Cmax) of heat-processed onion extract were significantly decreased by about 8.7% (from 188.60 ± 5.37 to 172.27 ± 3.96, p < 0.001) and 14.2% (from 204.04 ± 8.73 to 175.13 ± 14.09, p < 0.01) in sucrose and starch loading tests, respectively. These results indicate that ARCs in onion extract produced by heat-processing have anti-diabetic effect by suppressing carbohydrate absorption via inhibition of intestinal sucrase, thereby reducing the postprandial increase of blood glucose. Therefore, enhancement of ARCs in onion by heat-processing might be a good strategy for the development of the new product on the management of hyperglycemia.

Effect of supplementation of low-molecular-weight chitosan oligosaccharide, GO2KA1, on postprandial blood glucose levels in healthy individuals following bread consumption.

The effect of chitosan oligosaccharide (GO2KA1) administration on postprandial blood glucose levels of subjects with normal blood glucose levels was evaluated following bread consumption. Postprandial blood glucose levels were determined for 2 h after bread ingestion with or without 500 mg of GO2KA1. GO2KA1 significantly lowered the mean, maximum, and minimum levels of postprandial blood glucose at 30 min after the meal. Postprandial blood glucose levels were decreased by about 25% (from 155.11±13.06 to 138.50±13.59, p<0.01) at 30 min when compared to control. Furthermore, we observed that the area under the concentration-time curve (AUCt) was decreased by about 6% (from 255.46±15.43 to 240.15±14.22, p<0.05) and the peak concentration of blood glucose (C max) was decreased by about 11% (from 157.94±10.90 to 140.61±12.52, p<0.01) when compared to control. However, postprandial the time to reach C max (Tmax) levels were the same as those found in control. Our findings suggest that GO2KA1 limits the increase in postprandial blood glucose levels following bread consumption.

Identification and characterization of a new type of inhibitor against the human immunodeficiency virus type-1 nucleocapsid protein.

BACKGROUND: The human immunodeficiency virus type-1 (HIV-1) nucleocapsid protein (NC) is an essential and multifunctional protein involved in multiple stages of the viral life cycle such as reverse transcription, integration of proviral DNA, and especially genome RNA packaging. For this reason, it has been considered as an attractive target for the development of new anti-HIV drugs. Although a number of inhibitors of NC have been reported thus far, the search for NC-specific and functional inhibitor(s) with a good antiviral activity continues. RESULTS: In this study, we report the identification of A1752, a small molecule with inhibitory action against HIV-1 NC, which shows a strong antiviral efficacy and an IC50 around 1 µM. A1752 binds directly to HIV-1 NC, thereby inhibiting specific chaperone functions of NC including Psi RNA dimerization and complementary trans-activation response element (cTAR) DNA destabilization, and it also disrupts the proper Gag processing. Further analysis of the mechanisms of action of A1752 also showed that it generates noninfectious viral particles with defects in uncoating and reverse transcription in the infected cells. CONCLUSIONS: These results demonstrate that A1752 is a specific and functional inhibitor of NC with a novel mode of action and good antiviral efficacy. Thus, this agent provides a new type of anti-HIV NC inhibitor candidate for further drug development.

The determination of importance of sequences neighboring the Psi sequence in lentiviral vector transduction and packaging efficiency.

A number of lentiviral vector systems have been developed for gene delivery and therapy by eliminating and/or modifying viral genetic elements. However, all lentiviral vector systems derived from HIV-1 must have a viral packaging signal sequence, Psi (Ψ), which is placed downstream of 5' long terminal repeat in a transgene plasmid to effectively package and deliver transgene mRNA. In this study, we examined feasible regions or sequences around Psi that could be manipulated to further modify the packaging sequence. Surprisingly, we found that the sequences immediately upstream of the Psi are highly refractory to any modification and resulted in transgene vectors with very poor gene transduction efficiency. Analysis around the Psi region revealed that there are a few sites that can be used for manipulation of the Psi sequence without disturbing the virus production as well as the efficiency of transgene RNA packaging and gene transduction. By exploiting this new vector system, we investigated the requirement of each of four individual stem-loops of the Psi sequence by deletion mapping analysis and found that all stem-loops, including the SL4 region, are needed for efficient transgene RNA packaging and gene delivery. These results suggest a possible frame of the lentiviral vector that might be useful for further modifying the region/sequence around the packaging sequence as well as directly on the Psi sequence without destroying transduction efficiency.

Identification of in vivo interaction between Hepatitis C Virus core protein and 5' and 3' UTR RNA.

Here, we investigated the ability of the Hepatitis C Virus (HCV) core protein to interact specifically with the 5' and 3' untranslated regions (UTRs) of HCV using an in vivo cell-based translation inhibition assay. HCV core protein interacts weakly but specifically with the SLIII stem loop in the 5' UTR in which the SLIIIb subdomain is the major determinant and the SL2 loop in the X region of the 3' UTR. These results revealed for the first time in vivo interaction of the core protein with 5' and 3' UTRs involved in the viral life cycle. This system provides a useful tool for further investigating interactions between the HCV core protein and 5' and 3' UTRs.

Investigation of requirements for efficient gene delivery using the HIV-1 based lentiviral transduction system.

The specific recognition and selection of the HIV-1 packaging signal psi (Psi) sequence which is mediated by Gag protein is believed to be pivotal for selective viral genomic RNA packaging and has been a basis for the development of HIV-based transgene delivery systems. However, the requirement of the psi sequence has been questioned recently by a report postulating that the psi element is not absolutely required for transgene transduction. Here, we used a four-plasmid transgene delivery system and analyzed the results by HIV p24 antigen assay, MT4 infection assay, HT1080 colony assay, and reverse transcription-PCR (RT-PCR). The results clearly demonstrate that the psi sequence must be present for efficient transgene encapsidation and transduction.

Examination of specific binding activity of aptamer RNAs to the HIV-NC by using a cell-based in vivo assay for protein-RNA interaction.

The nucleocapsid (NC) protein of the Human Immunodeficiency Virus-1 plays a key role in viral genomic packaging by specifically recognizing the Psi(Psi) RNA sequence within the HIV-1 genome RNA. Recently, a novel cell-based assay was developed to probe the specific interactions in vivo between the NC and Psi-RNA using E.coli cells (J. Virol. 81: 6151-55, 2007). In order to examine the extendibility of this cell-based assay to RNAs other than Psi-RNA, this study tested the RNA aptamers isolated in vitro using the SELEX method, but whose specific binding ability to NC in a living cellular environment has not been established. The results demonstrate for the first time that each of those aptamer RNAs can bind specifically to NC in a NC zinc finger motif dependent manner within the cell. This confirms that the cell-based assay developed for NC-Psi interaction can be further extended and applied to NC-binding RNAs other than Psi-RNA.

Expression profiles and pathway analysis in HEK 293 T cells overexpressing HIV-1 Tat and nucleocapsid using cDNA microarray.

Human immunodeficiency virus type 1 (HIV-1) infections are responsible for a substantial number of deaths annually and represent a significant threat to public health. According to the latest study, the Tat (Transactivator of transcription) protein is essential in transcription and replication of viral genes, and is among the early expression genes involved in the life cycle of HIV. The virion NC (nucleocapsid) plays an important role in early mRNA expression and contributes to the rapid viral replication that occurs during HIV-1 infection. Therefore, we attempted to elucidate the relationship between the Tat protein and nucleocapsid protein. In a comparison of two independently prepared and hybridized samples, flag NC overexpressed HEK 293T cells and pTat overexpressed HEK 293T cells, and hybridization showed the differences in expression in each case. Among the microarray results confirmed with real-time reverse transcriptase assay, twelve genes were identified to be involved according to their gene expression profiles. Of approximately 8,208 human genes that were analyzed, we monitored candidate genes that might have been related to NC and Tat genes from gene expression profiles. Additionally, the pathways could be viewed and analyzed through the use of PathwayStudio software. The pathways from the gene list were built and paths were found among the molecules/cell objects/processes by the curation method.

Development of a cell-based assay probing the specific interaction between the human immunodeficiency virus type 1 nucleocapsid and psi RNA in vivo.

Here, we describe a cell-based in vivo assay that probes the specific interaction between nucleocapsid (NC) protein and Psi (Psi) RNA, the human immunodeficiency virus (HIV) packaging signal. The results demonstrate for the first time a specific NC-Psi interaction within living cells. The specificity and applicability of the assay were confirmed by mutational studies of NC and deletion-mapping analyses of Psi-RNA as well as by testing the in vivo NC-binding effects of NC-aptamer RNAs identified previously in vitro. This assay system would facilitate further detailed studies of the NC-Psi interaction in vivo and the screening of various anti-HIV molecules targeting NC and the specific interaction.