Vijittra Leardkamolkarn

Structures of some bioactive phytochemicals in bran extract of the black rice cv. Riceberry that had demonstrated anti-cancer activity in leukemic cell line were investigated. After saponification with potassium hydroxide, separation of the unsaponified fraction by reversed-phase high performance liquid chromatography (HPLC) resulted in four sub-fractions that had a certain degree of anti-proliferation against a mouse leukemic cell line (WEHI-3 cell), this being IC50 at 24 h ranging between 2.80-467.11 μg/mL. Further purification of the bioactive substances contained in these four sub-fractions was performed by normal-phase HPLC. Structural characterization by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) resulted in, overall, the structures of seven phytosterols and four triterpenoids. Four phytosterols, 24-methylene-ergosta-5-en-3β-ol, 24-methylene-ergosta-7-en-3β-ol, fucosterol, and gramisterol, along with three triterpenoids, cycloeucalenol, lupenone, and lupeol, were found in the two sub-fractions that showed strong anti-leukemic cell proliferation (IC50 = 2.80 and 32.89 μg/mL). The other sterols and triterpenoids were campesterol, stigmasterol, β-sitosterol and 24-methylenecycloartanol. Together with the data from in vitro biological analysis, we suggest that gramisterol is a significant anti-cancer lead compound in Riceberry bran extract.

The potential anti-cancer activity of compounds extracted from Riceberry bran was evaluated in human cancer cell lines (Caco-2, MCF-7 and HL-60). Anti-proliferation and BrdU incorporation assays indicated a time–dose dependent effect of dichloromethane (DCM) and methanol (MeOH) extracts, and that HL-60 was the most sensitive cell. DNA fragmentation assay revealed that both extracts could induce different degrees of apoptosis. The apoptotic induction pathway of each extract determined by flow cytometry and immunoblotting assays revealed various phases of cell cycle arrest with alteration of pro-apoptotic p53, caspase-3, and cyclin proteins. The bioactive compounds in each extract were chemically analysed by GC–MS and LC–ESI–MS/MS. Results revealed the presence of two major anthocyanins, cyanidin-3-glucoside and peonidin-3-glucoside, in the MeOH extract, while the DCM extract contained higher content of plant sterols. The latter constituents are considered the major contributors to apoptotic mechanism in the sensitive cell. These bran products are worth developing into medicinal supplements.

Dengue virus (DV) is an important re‐emerging arthropod‐borne virus of global significance. The defining characteristic of DV infection‐associated pathology is haemorrhagic fever, which often leads to a fatal shock‐like syndrome (DHF/DSS) owing to an increase in vascular endothelial permeability. Here, we show, in a viral dose‐dependent manner, that DV‐infected immature dendritic cells overproduce soluble gelatinolytic matrix metalloproteinase (MMP)‐9—and to a lesser extent MMP‐2—which enhances endothelial permeability, but which are reduced by specific inhibitors and a neutralizing anti‐MMP‐9 antibody. This permeability was associated with a loss of expression of the platelet endothelial adhesion molecule 1 (PECAM‐1) and vascular endothelium (VE)‐cadherin cell adhesion molecules and redistribution of F‐actin fibres. These in vitro observations were confirmed in an in vivo vascular‐leakage mouse model. These results provide a molecular basis for DHF/DSS that could be a basis for a general model of haemorrhagic fever‐inducing viruses, and identify a new therapeutic approach for the treatment of viral‐induced vascular leakage by specifically targeting gelatinolytic metalloproteases.

To address origins of glomerular endothelial and mesangial cells in embryonic mammalian kidneys, we established interspecies grafts between rats and mice, in which fetal kidneys were implanted into the anterior eye chamber of adult hosts. After 5-7 days, hosts bearing grafts received intravenous injections with species-specific monoclonal antibodies (MAbs) to matrix components. In all cases, glomerular basement membranes and mesangial matrices labeled solely for donor-derived matrix. Additionally, microvessel extracellular matrices within grafts were usually of donor origin. To examine directly the origin of glomerular endothelial and mesangial cells, we grafted embryonic gestational days 11-12 (E11-12) kidneys from normal mice into anterior eye chambers of host reverse-orientation splice acceptor 26 mice, which are transgenic animals that express beta-galactosidase in every cell. When grafts were developed for beta-galactosidase activity, host cells were seen in peripheral vessels, but the majority of glomerular endothelial cells were of donor, not host, origin. Where host-derived-endothelial cells were found in glomeruli, donor endothelial cells were present as well. Mesangial cells were always of donor origin. When E11 mouse kidneys were labeled with the endothelial cell-specific Bandeiraea simplicifolia isolectin B4, we determined that endothelial cells are present from the inception of metanephrogenesis. Together, the evidence shows that cells of endogenous kidney origin were almost entirely responsible for development of the glomerular microvasculature in oculo. External vessels from the host, although important for graft maintenance, were not major contributors to the glomerulus.

Diabetes is a serious health problem. Searching for alternative natural antioxidants is considered important strategy to manage diabetes. This study evaluated the effect of Riceberry bran oil (RBBO) supplementation on oxidative stress and organ histology in streptozotocin-induced diabetic rats fed a high fat (HF) diet. Adult male Sprague-Dawley rats with hyperglycemia were divided into four groups: DM group fed a HF diet alone; DMRL group fed a HF diet and 5% RBBO; DMRM group fed a HF diet and 7.5% RBBO; DMRH group fed a HF diet and 15% RBBO. Normal rats were used as normal control and were divided into NC and NR group fed a normal diet containing either 5% corn oil or 5% RBBO, respectively. After 12 weeks, RBBO significantly decreased malondialdehyde and restored superoxide dismutase, catalase, glutathione peroxidase, coenzyme Q10 and ORAC levels in diabetic rats. RBBO additionally improved the regenerative changes of the pancreas, kidneys, heart and liver. These findings indicate that pigmented RBBO could provide beneficial effect on diabetes by decreasing oxidative stress and recovering organ histology.

Dark purple riceberry bran contains a higher dietary fiber and antioxidant compounds than unpigmented rice bran. Riceberry supplement (RB) was used to evaluate the effects on biochemical parameters, skeletal muscle glucose transporter 4 (GLUT4), oxidative stress and inflammation in a streptozotocin (STZ)-induced diabetes rat. To elucidate the effects were due to dietary fiber supplementation and/or bioactive components, equivalent amounts of dietary fiber present in RB were also fed to STZ-induced diabetic rats. Diabetes Sprague–Dawley rats (non-FBG ⩾ 16.65 mM) were randomly divided into five groups: DM fed a high fat (HF) diet, DM-RB1 fed 5% RB, DM-RB2 fed 41% RB, DM-F1 fed 0.6% fiber and DM-F2 fed 5% fiber. After 12 weeks, significant improvement of BG, insulin, HbA1C, IPGTT and GLUT4 levels were observed in DM-RB1 and DM-RB2 groups. Hyperlipidemia was significantly improved in DM-RB2 and DM-F2 groups. Oxidative stress (TBARS), antioxidant enzymes (SOD, CAT, and GPx), antioxidant capacity (ORAC), pro-inflammation cytokine (TNF-α and IL-6) were improved in DM-RB1 and DM-RB2 groups. Improvement of pancreas and spleen histology was found in DM-RB1 and DM-RB2 groups. These indicate the potential of RB to improve hyperglycemia and hyperlipidemia conditions as well as alleviate oxidative stress and inflammation.

ABSTRACT The antiviral activities of Houttuynia cordata (H. cordata) aqueous extract against dengue virus serotype 2 (DEN-2), strain 16681, were evaluated in this study. The results showed that pre- and post-incubation of H. cordata extract (10–100 µg/mL) with HepG2 cells significantly reduced intracellular DEN-2 RNA production correlating with the decrease in dengue protein expression. In the direct blocking mode, the extract bound with DEN-2 and strongly inhibited the intracellular viral RNA replication with an effective dose (EC50) of 0.8 µg/mL. Concentrations as low as 10–40 µg/mL of H. cordata extract also exhibited protective effect on virion release from infected LLC-MK2 cells. High-performance liquid chromatography analysis of H. cordata extract indicated that hyperoside was the predominant bioactive compound, and was likely to play a role in this inhibition. The extract was also shown to have no genotoxic effect on human blood cells. PRACTICAL APPLICATIONS Houttuynia cordata is recognized as medicinal vegetable consumed by people in East and Southeast Asia. It has multilateral activities in health promotion and regulation of inflammatory reactions induced by several pathogens. Dengue viral infection is a global health problem since licensed vaccines or specific antiviral drug treatments are not yet available. The current study provides scientific data to support the phytomedicinal properties of H. cordata aqueous extract on anti-dengue virus in three modes of action (prevention, treatment and virucidal). A major constituent in the extract, hyperoside, seems to be the bioactive compound effectively acting against dengue infection. H. cordata aqueous extract was shown to be nontoxic and hyperoside may be considered a lead compound possessing drug potential for further development.

Dengue virus (DV) is an important re-emerging arthropod-borne virus of global significance. The defining characteristic of DV infection-associated pathology is haemorrhagic fever, which often leads to a fatal shock-like syndrome (DHF/DSS) owing to an increase in vascular endothelial permeability. Here, we show, in a viral dose-dependent manner, that DV-infected immature dendritic cells overproduce soluble gelatinolytic matrix metalloproteinase (MMP)-9-and to a lesser extent MMP-2-which enhances endothelial permeability, but which are reduced by specific inhibitors and a neutralizing anti-MMP-9 antibody. This permeability was associated with a loss of expression of the platelet endothelial adhesion molecule 1 (PECAM-1) and vascular endothelium (VE)-cadherin cell adhesion molecules and redistribution of F-actin fibres. These in vitro observations were confirmed in an in vivo vascular-leakage mouse model. These results provide a molecular basis for DHF/DSS that could be a basis for a general model of haemorrhagic fever-inducing viruses, and identify a new therapeutic approach for the treatment of viral-induced vascular leakage by specifically targeting gelatinolytic metalloproteases.

Although some progress has been made in recent years, there are truly large gaps in our basic knowledge on how the TBM is assembled during development. Some of the new evidence presented here indicates that both the tubular epithelium and interstitial fibroblasts participate in TBM protein biosynthesis during nephrogenesis. In addition, newly assembled segments of TBM are spliced or inserted into existing TBM during tubule expansion and elongation. A similar splicing mechanism has been described previously in the GBM, endocrine organs, and intestinal villi, and this mechanism therefore probably represents a fundamental process of basement membrane formation. A major unresolved question at present, however, is how this mechanism operates at the molecular level. Does the newly formed basement membrane contain identical components as that already present? Since an enzymatic process is likely occurring in the insertion of new matrix into old, which enzymes are involved? What is the cellular origin of these enzymes and which matrix component(s) is their substrate? Even more fundamental yet unanswered questions have to do with the mechanisms of epithelial induction, basement membrane gene activation, and tubular morphogenesis. Once the basement membrane is fully formed at the completion of nephrogenesis, what controls basement membrane turnover and how does this operate? Clearly, much additional research is necessary to address these questions. This work is needed, however, before we can fully understand the important roles basement membranes play in normal development as well as in disease.

Acute myelogenous leukemia (AML) is a cancer of the blood that most commonly affects human adults. The specific cause of AML is unclear, but it induces abnormality of white blood cells that grow rapidly and accumulate in bone marrow interfering with the production and functions of the normal blood cells. AML patients face poor prognosis and low quality of life during chemotherapy or transplantation of hematopoietic stem cells due to the progressive impairment of their immune system. The goal of this study is to find natural products that have the potential to delay growth or eliminate the abnormal leukemic cells but cause less harmful effect to the body's immune system.The unsaponified fraction of Riceberry rice bran (RBDS) and the main pure compound, gramisterol, were studied for cytotoxicity and biological activities in WEHI-3 cells and in the leukemic mouse model induced by transplantation of WEHI-3 cells intraperitoneally. In the in vitro assay, RBDS and gramisterol exerted sub-G1 phase cell cycle arrest with a potent induction of apoptosis. Both of them effectively decreased cell cycle controlling proteins (cyclin D1 and cyclin E), suppressed cellular DNA synthesis and mitotic division, and reduced anti-apoptosis Bcl-2 protein, but increased apoptotic proteins (p53 and Bax) and activated caspase-3 enzyme in the intrinsic cell death stimulation pathway. In leukemic mice, daily feeding of RBDS significantly increased the amount of immune function-related cells including CD3+, CD19+, and CD11b+, and elevated the serum levels of IFN-γ, TNF-α, IL-2, and IL-12β cytokines, but suppressed IL-10 level. At the tumor sites, CD11b+ cells were polarized and became active phagocytotic cells. Treatment of mice normal immune cells with gramisterol alone or a combination of gramisterol with cytokines released from RBDS-treated leukemic mice splenocytes culture synergistically increased pSTAT1 transcriptional factor that up-regulated the genes controlling cell survival and function. Phosphorylation of STAT1 was absent in WEHI-3. Instead, similar treatments significantly decreased pSTAT3 signaling that regulates transcription of genes controlling tumor growth and proliferation.Rice bran gramisterol possesses a promising anti-cancer effect against a tumor of white blood cells and induces the production of anti-cancer immune-related cytokines. Gramisterol induces cell cycle arrest and apoptosis via suppression of pSTAT3 signaling control of tumor cells' growth and progression. Gramisterol increased IFN-γ production and prevented the dysfunctional immune system of leukemic mice by enhancing pSTAT1 transcription signal controlling proliferation and functions of hematopoietic cells in the spleen. Together with IFN-γ, gramisterol efficiently facilitates leukemic mice immune system modulation leading to improvement of the AML condition. Administration of RBDS containing gramisterol potentiates immune recovery of leukemic mice and extends their survival. This finding encourages the medicinal application of rice bran gramisterol as a palliative treatment or an alternative agent for future drug development against AML.

Prostate cancer is one of the high incidences and the most invasive cancer that is also highly resistant to chemotherapy.Currently, several natural products have been considering using as the supplements for anti-cancer therapy.This study aims to identify the potential active anti-cancer ingredients in the bran extracts of the native Thai rice (Luempua cultivar).Rice bran fraction enriched in anthocyanins was successively isolated and processed until the major purified compound obtained.The sub-fractions and the purified, rice bran, cyanidin 3-glucoside (RBC3G), were studied for biological effects (cell viability, migration, and invasion assays) on human prostatic cancer (PC3) cells using immunohistochemicalstaining and immuno-blotting approaches.The sub-fractions and the purified RBC3G inhibited epithelial mesenchymal transition (EMT) characteristics of PC3 cells by blocking the expression of several cytoskeletal associate proteins in a concentration dependent manner, leading to decreasing of the cancer cell motility.RBC3G reduced the expression of Smad/Snail signaling molecules but enhanced the expression of cell surface protein, E-cadherin, leading to a delay tumor transformation.The RBC3G also inhibited matrix metalloproteinase-9 and nuclear factor-kappa B expression levels and the enzymes activity in PC3 cells, leading to a slow cell migration/invasion process.The results suggested that RBC3G blunt and/ or delay the progressive cancer cell behaviors by inhibit EMT through Smad signaling pathway(s) mediating Snail/E-cadherin expression.

Abstract We studied the immunohistochemical and ultrastrural distribution of laminin in ovaries of immature and mature rats. When sections from 1–8‐week‐old rat ovaries were labeled directly with conjugates of affinity purified anti‐laminin IgG‐horseradish peroxidase (HRP), the antibodies bound to all ovarian basement membranes including those surrounding follicles in different stages of maturation. In addition, intracellular labeling was seen in granulosa and theca cells of follicles undergoing rapid development (preantral and antral stages) and in basement membrane‐like structures of the Call‐Exner bodies. Intracellular laminin was generally not detected, however, in any cells of primordial or atretic follicles. Tissue processed for immunoelectron microscopy 1 hour after the intravenous injection of anti‐laminin IgG‐HRP showed binding of antibody in linear patterns along endothelial and follicular epithelial basement membranes. Discontinuous strands of laminin‐positive, extracellular matrices were also seen between theca cells of all follicles. In addition, injected anti‐laminin IgG labeled perisinusoidal basement membranes located within corpora luteae and patches of basement membranes material between granulosa lutein cells. When ovaries were examined 5 d after the intravenous injections of anti‐laminin IgG‐HRP, uneven or segmented labeling was found in subepithelial basement membranes surrounding developing follicles. Our results therefore indicate that granulosa and theca cells participate directly in basement membrane laminin biosynthesis and suggest that this new laminin is spliced into existing basement membranes during follicular growth. © 1992 Wiley‐Liss, Inc.

This study aimed to generate a stable cell line harboring subgenomic dengue virus replicon and a green fluorescent gene (DENV/GFP) for a cell-based model to screen anti-DENV compounds. The gene-encoding envelope protein of DENV-2 was deleted and then replaced with fragments of the GFP gene and a foot-and-mouth-disease virus 2A-derived cleavage site. The human cytomegalovirus immediate early and antisense hepatitis delta virus ribozyme sequences were added at the 5'- and 3'-ends. An internal ribosome entry site and neomycin resistance genes were placed upstream and next to the NS1 gene. The recombinant plasmids were propagated in a mammalian cell line. A stable cell line with the brightest green fluorescent protein and the highest viral protein and RNA expression was selected from six clones. The clone was then examined for effectiveness in an antiviral drug screening assay with compounds isolated from the local plants using two known antiviral agents as controls. Two novel flavones, PMF and TMF, were discovered having DENV-inhibitory properties. The data were validated by a conventional plaque titration assay. The results indicate that this newly developed cell line is efficient for use as a cell-based model for primary screening of anti-DENV compounds.

Insertion of green fluorescent protein (GFP) encoding-gene into virus genes has provided a valuable tool for flavivirus research. This study aimed to develop dengue virus (DENV) replicons expressing GFP reporter that would provide a fast in vitro system to analyze functional roles of specific DENV sequences in viral replication. Two classes of recombinant replicon constructs were generated; one was a RNA-launched replicon with a GFP gene directly inserted into a full-length DENV genome (FL-DENV/GFP), and the other consisted of 4 types of DNA-launched DENV subgenomic replicons with GFP replacement at various structural genes (Δ-DENV/GFP). The FL-DENV/GFP resulted in GFP expression in transfected cells with no viable DENV being recovered from the transfection. The Δ-DENV/GFP constructs with partial structural gene deletion (ΔC-, ΔCprM/M-, ΔprM/M-, or ΔE-) expressed bright and long lasting GFP. The GFP expression intensity in living cells correlated well with the level of RNA replication. Various mutations in the 5'noncoding region of DENV-2 previously shown to be important genetic determinants for virus replication and mouse virulence were incorporated into the 5 different replicon constructs. Characterizations of 29 mutants demonstrated that these replicons can provide a useful platform for a quick and powerful in vitro system to analyze genetic determinants of DENV replication. These constructs can also be useful for development of vectors expressing foreign genes for various researches.

A C57U nucleotide mutation in a predicted RNA stem structure (nt 11-16/56-61) of the 5' non-coding region (5'NCR) of dengue 2 (DEN-2) 16681 virus is partially attenuating, but unstable during serial passage of certain candidate DEN-2 PDK-53-based vaccine viruses containing this mutation. Here, 11 different mutations (one or more point substitution and/or deletion) between nt 54 and 70 in the 5'NCR of the pD2/IC-30P-A (16681) infectious clone are described. Four mutants were infectious. Three mutants with single point substitutions replicated well in cell culture and exhibited variable neurovirulence in mice. Constructs containing multiple substitutions or any deletions failed to produce infectious viruses. Unexpectedly, a double C57U+G58C mutant replicated as efficiently as D2/IC-30P-A virus, and was more neurovirulent for newborn ICR mice. Thus, despite its predicted additional disruption of the RNA stem structure, the engineered contiguous secondary G58C mutation caused reversion of the partially attenuated phenotype caused by the 5'NCR-C57U mutation.

The distribution and synthetic rate of glomerular basement membrane components was examined in the Passive Heymann Nephritis model of experimental membranous nephropathy. The extensive tissue injury that developed included subepithelial electron-dense deposits, podocyte foot process effacement, and expansion of the glomerular basement membrane. Levels of mRNA for type IV collagen, laminin, and fibronectin from isolated glomeruli was quantitated by slot-blot analysis and showed no change in experimental animals as compared to controls at either 1 week, 3 weeks, or 3 months after disease induction. Immunoelectron microscopy with gold-labeled anti-laminin IgG revealed no difference in the number of particles bound to the glomerular basement membrane of experimental animals and controls. Immunofluorescence with both type IV collagen antisera and anti-laminin antibody showed no difference in the intensity or pattern of staining. Despite extensive glomerular damage and glomerular basement membrane thickening, no evidence was found for either an increase in the synthetic rate of type IV collagen, laminin, or fibronectin or for an accumulation of basement membrane laminin within the damaged glomeruli. Alternate processes, such as diminished density of matrix components or accumulation of other unmeasured matrix constituents, presumably account for the expansion of the glomerular basement membrane seen in experimental membranous nephropathy.

Variants of wild-type dengue serotype 2 (DEN-2) virus containing nucleotide substitutions at positions 14, 15, or 57 in the<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msup><mml:mn>5</mml:mn><mml:mo>′</mml:mo></mml:msup></mml:math>NCR were constructed by PCR-mediated site-directed mutagenesis. All three viruses containing a single point substitution demonstrated attenuation phenotype as evidenced by decreases replication and plaque size in cell culture assay. All three variants were less neurovirulent in newborn mice compared to the wild type. The mutants were immunogenic in adult mice immunogenicity and maintained stable replication characteristics following passage in mice. The variant viruses were competent for replication in Aedes aegypi mosquito vector, albeit at lower levels of infection and dissemination in the mosquito than the wild-type Den-2 16681 virus. Although all of the viruses, including the wild type, were found transmissible in mosquito life cycles, they were found subsequentially decreased in efficiency of infection, transmission, and dissemination rates along the mosquito generations and all of them remained genetically stable.

Abstract The relationships between urinary enzyme levels and changes in blood urea nitrogen (BUN) and plasma creatinine levels, along with simultaneous ultrastructural changes of the kidney, were studied in rats treated with stevioside. BUN levels increased at 3 h onward after subcutaneous injection (s.c.) with stevioside (1.5 g/kg BW). The maximum increases in BUN and creatinine were approximately 180% and 132% at 9 h after stevioside injection, respectively. At this time, stevioside also caused significant increases in glucosuria, alkaline phosphatase (AP) and γ‐glutamyl transpeptidase (γ‐GTP) but no significant changes in proteinuria, N‐acetyl‐β‐D‐glucuronidase (NAG) or glutathione‐S‐transferase (GSH‐S‐TF). Histopathological examination of the kidney induced by stevioside revealed degeneration of the proximal convoluted tubule cells but no relation to lipid peroxide formation was detected. These results suggest that stevioside induced nephrotoxicity at the proximal convoluted tubules rather than at the glomeruli and other tubules presumably by a defect of cell volume regulation due to depletion of intracellular ATP and disruption of microvilli, and nuclear dysfunction.

Infection by trematode parasites generally affects life history traits of their intermediate hosts. Reduction in life expectancy and reproductive capacity have previously been documented in Fasciola gigantica-infected Lymnaea ollula, but the influence of the endocrine system on this specific host–parasite interaction has not been previously examined. In the present study, we observed survival, growth pattern, and reproductive output of L. ollula following exposure to F. gigantica. Both the survival and the growth pattern of infected snails were similar to those of the non-infected control group. However, a significant difference was apparent in fecundity, as infected snails consistently showed lower levels of egg and embryo production throughout a 7-wk observation period. Semi-quantitative RT-PCR also revealed down-regulation of estrogen receptor expression in infected snails during the first 4 wk of infection. Nonetheless, the inhibition of estrogen signaling was transient, as they regained expression in the later phase of infection. It is, therefore, suggested that other hormones of the complex endocrine system may be involved in the reduced fecundity of L. ollula following F. gigantica infection.

We used antibodies against mouse Englebreth-Holm-Swarm (EHS) tumor laminin to screen a newborn rat kidney lambda gt11 expression library and isolated three overlapping cDNA clones, termed 2b-11 (401 bp), 10-b7 (779 bp), and 2a (2,095 bp). DNA sequence analysis identified these cDNAs as encoding much of the carboxy terminal domain I/II of laminin gamma 1 chain (formerly referred to as B2e), and 1436 bp of the 3' untranslated region. In situ hybridization of fetal (E15) rat sections localized laminin gamma 1 chain mRNA primarily to meninges of the brain, auditory and peripheral nerve fibers, gastrointestinal system, and developing lung airway epithelium. Intense hybridization was also found in early nephric structures and glomeruli of fetal kidneys. In kidneys of three-day-old rats, hybridization persisted over early nephric figures, developing glomeruli, and collecting ducts, but considerably less hybridization was seen over tubules. On Northern blots of neonatal kidney RNA, the three cDNA clones hybridized to two species of 7.5 and 5.5 kb, suggesting that developing rat kidney laminin gamma 1 mRNAs are processed using two different polyadenylation signals.

Many earlier studies have shown that the intravenous injection into rats of sheep antibodies against rat glomerular basement membrane (GBM) induces a rapid influx of neutrophils and proteinuria (nephrotoxic nephritis or NTN). The GBM antigens recognized by nephrotoxic antibodies (NTAbs) have not been identified conclusively. Our experiments presented here, however, showed that NTAbs did not significantly reduce binding of anti-laminin IgGs to laminin-coated enzyme-linked immunosorbent assay (ELISA) plates or to the GBM in vivo, indicating little cross-reactivity between the NTAbs and laminin. To evaluate possible changes in GBM architecture during acute stages of NTN, the ultrastructural distribution of laminin was determined by postfixation, postembedding immunogold labeling, and compared between normal and nephritic rats. The density of immunoreactive GBM laminin was significantly reduced in rats with acute NTN. In addition, conjugates of anti-laminin IgG and horseradish peroxidase were intravenously injected into rats that then received injections of NTAbs. Anti-laminin peroxidase conjugates were also injected after administering NTAbs. In both cases, an overall decrease in anti-laminin peroxidase reaction product was observed as compared to normal controls. The densest labeling was seen in the lamina rara interna, especially in areas of endothelial cell detachment. Some immunoperoxidase reaction product was also bound to basal surfaces of detaching endothelial cells, demonstrating the removal of at least some laminin from the GBM. A decrease in GBM binding of intravenously injected anti-laminin IgG, both before and after injection of rats with NTAbs, was also confirmed by postembedding immunogold labeling. Furthermore, morphometry showed that the GBM was significantly wider in nephritic rats than in controls, indicating a redistribution of laminin over a greatly increased area. These immunoultrastructural findings show, therefore, that GBM architecture is altered in the early phase of NTN.

Disease severities following dengue virus (DV) infection are the result of increased vascular permeability leading to hypovolemic shock. Matrix metalloproteinases (MMPs) are believed to play a key role in promoting such severities. A previous study reported that supernatants of DV-infected dendritic cells (DCs), which contained high levels of MMP-2 and MMP-9, induced vascular leakage in a mouse model. In the present study, we investigated whether hepatocytes (HepG2) and monocytes (U937) could be additional sources of MMPs during DV infection. HepG2 and U937 cells were exposed to DV serotype 2 strain 16681. The secretion of MMP-2 and MMP-9 was detected using gelatin zymography. We found that DV infection in the HepG2 cells promoted MMP-2 production while that in the U937 cells promoted MMP-9 production. Semi-quantitative RT-PCR results also confirmed that DV infection in the HepG2 cells up-regulated the expression of MMP-2 mRNA, whereas that in the U937 cells enhanced the expression of MMP-9 mRNA. We monitored the expression of endogenous TIMP-1 and TIMP-2. DV infection induced TIMP-1 expression in the U937 cells. However, lower expression of TIMP-2 was observed in the infected HepG2 cells. We believed that following DV infection, monocytes and hepatocytes can act as MMP-9 and MMP-2 producers, respectively. Their responses could be attributed to the disturbance of TIMP expression by DV in different cell types.

Cleistanthin A (CleinA) and cleistanthoside A (CleisA) isolated from plant Phyllanthus taxodiifolius Beille have previously shown potent anticancer effects. To promote their medicinal benefits, CleisA was modified to cleistanthoside A tetraacetate (CleisTA) and evaluated for genotoxic and anti-mutagenic properties in comparison with CleinA. Both compounds showed no significant mutagenic activity to S. typhimulium bacteria and no cytotoxic effect to normal mammalian cells. The non genotoxic effect of CleinA was further confirmed by un-alteration of cytokinesis-block proliferation index (CBPI) and micronucleus (MN) frequency assays in Chinese hamster lung fibroblast (V79) cells, and of CleisTA was confirmed by un-changes of human peripheral blood lymphocytes (HPBL) chromosomal structure assay. Moreover, the metabolic form of CleinA efficiently demonstrated cytostasis effect to V79 cell and prevented mutagen induced Salmonella TA98 and TA100 reversion, whereas both metabolic and non-metabolic forms of CleisTA reduced HPBL mitotic index (%M.I) in a concentration-dependent relationship. The results support CleinA and CleisTA as the new lead compounds for anti-cancer drug development.

Structural chromosome aberrations, especially de novo translocations and marker chromosomes, often remain uncharacterized in clinical cytogenetic analysis. Due to the limitations of conventional banding analysis in precisely identifying these rearrangements and marker chromosomes, genetic counselling is exceedingly difficult. Microdissection combined with fluorescence in situ hybridization (micro-FISH) has become a powerful tool in clinical genetics for the characterization of cytogenetically unclassifiable aberrations. Micro-FISH was used to elucidate the chromosomal origin of two different de novo structural chromosome abnormalities. Ten copies of aberrant chromosomes were collected with microneedles from patient's metaphases, transferred to a collecting drop and amplified by means of degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR). The PCR products were labeled by nick-translation with digoxigenin-11-dUTP and used as FISH probes. They were hybridized to metaphase spreads from patients to confirm the specificity of the probe and normal metaphases to determine the origin of the aberrant chromosomes. With this strategy, a de novo marker chromosome and a de novo isodicentric chromosome in peripheral blood samples were successfully identified in two cases. One marker of a small ring chromosome appeared to be derived from the pericentromeric region on the short arm and long arm (Xp11.1-q12) of the X chromosome and the second aberrant was identified as an isodicentric X chromosome (idic(X)(q28)). Based on the analysis of both G-banding and micro-FISH, the karyotypes for the patients were defined as mos 46,X,r(X)(p11.1q12)(64)/45,X(26)/46,XX(10) for the first patient and mos 46,X,idic(X)(q28)(86)/ 45,X(12)/46,XX(2) for the second case, respectively.

Autophagy is a known mechanism of cells under internal stress that regulates cellular function via internal protein recycling and the cleaning up of debris, leading to healthy live cells. However, the stimulation of autophagy by external factors such as chemical compounds or viral infection mostly tends to induce apoptosis/cell death. This study hypothesizes that manipulation of the autophagy mechanism to the pro-cell survival and/or decreased pro-viral niche can be a strategy for effective antiviral and anticancer treatment. Cells susceptible to viral infection, namely LLC-MK2, normal monkey epithelium, and K562, human immune-related lymphocyte, which is also a cancer cell line, were treated with fludarabine nucleoside analog (Fdb), interferon alpha (IFN-α), and a combination of Fdb and IFN-α, and then were evaluated for signs of adaptive autophagy and STAT1 antiviral signaling by Western blotting and immunolabeling assays. The results showed that the low concentration of Fdb was able to activate an autophagy response in both cell types, as demonstrated by the intense immunostaining of LC3B foci in the autophagosomes of living cells. Treatment with IFN-α (10 U/mL) showed no alteration in the initiator of mTOR autophagy but dramatically increased the intracellular STAT1 signaling molecules in both cell types. Although in the combined Fdb and IFN-α treatment, both LLC-MK2 and K562 cells showed only slight changes in the autophagy-responsive proteins p-mTOR and LC3B, an adaptive autophagy event was clearly shown in the autophagosome of the LLC-MK2 cell, suggesting the survival phase of the normal cell. The combined effect of Fdb and IFN-α treatment on the antiviral response was identified by the level of activation of the STAT1 antiviral marker. Significantly, the adaptive autophagy mediated by Fdb was able to suppress the IFN-α-mediated pSTAT1 signaling in both cell types to a level that is appropriate for cellular function. It is concluded that the administration of an appropriate dose of Fdb and IFN-α in combination is beneficial for the treatment of some types of cancer and viral infection.

Structural chromosome aberrations, especially de novo translocations and marker chromosomes, often remain uncharacterized in clinical cytogenetic analysis.Due to the limitations of conventional banding analysis in precisely identifying these rearrangements and marker chromosomes, genetic counselling is exceedingly difficult.Microdissection combined with fluorescence in situ hybridization (micro-FISH) has become a powerful tool in clinical genetics for the characterization of cytogenetically unclassifiable aberrations.Micro-FISH was used to elucidate the chromosomal origin of two different de novo structural chromosome abnormalities.Ten copies of aberrant chromosomes were collected with microneedles from patient' s metaphases, transferred to a collecting drop and amplified by means of degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR).The PCR products were labeled by nick-translation with digoxigenin-11-dUTP and used as FISH probes.They were hybridized to metaphase spreads from patients to confirm the specificity of the probe and normal metaphases to determine the origin of the aberrant chromosomes.With this strategy, a de novo marker chromosome and a de novo isodicentric chromosome in peripheral blood samples were successfully identified in two cases.One marker of a small ring chromosome appeared to be derived from the pericentromeric region on the short arm and long arm (Xp11.1-q12) of the X chromosome and the second aberrant was identified as an isodicentric X chromosome (idic(X)(q28)).Based on the analysis of both G-banding and micro-FISH, the karyotypes for the patients were defined as mos 46,X,r(X)(p11.1q12)[64]/45,X[26]/46,XX[10] for the first patient and mos 46,X,idic(X)(q28)[86]/ 45,X[12]/46,XX[2] for the second case, respectively.

Female Fisher rats aged 35 days were injected intraperitoneally with 50 μg and 100 μg per day of bovine thyrotropin(TSH) for 44 days. GH content and concentration in the TSH treated groups were significantly lower than that of the control group. Wet weight of anterior pituitary and thyroid gland were not altered by TSH treatment, while the doses of 100 μg TSH significantly lowered adrenal, ovarian and uterine weights. The formation of antibodies to bovine TSH after chronic administration, which can cross react to rat endogenous TSH, may lead to thyroid hypofunction which in turn lower pituitary GH synthesis. The bovine TSH antiserum may also cross react with rat FSH and LH and lead to adrenal and ovarian hypofunction.