The presence of F3-F2a1 dimers and F1 oligomers in chromatin.

The oligomeric structure of histones in nuclei and chromatin has been studied by crosslinking nuclei and chromatin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Crosslinked histones were detected as new high molecular weight components on SDS gels, and the protomers of the crosslinked histones were identified by their characteristic ¹²⁵I-fingerprints. The results show that a considerable portion of histones F3 and F2a1 exist in nuclei and chromatin as an F3-F2a1 dimer. Evidence is presented that histone F1 probably exists in chromatin as large oligomers.     

Histone-histone associations within chromatin. Cross-linking studies using tetranitromethane.

Treatment of chromatin with the protein cross-linker tetranitromethane (TNM) results in a product identified as an F2a1-F2b dimer. The same product appears after treatment with TNM of HeLa cells growing in culture. Furthermore acid-extracted histones which have been fractionated into the five separate species can be recombined and mixed with DNA to produce a nucleohistone preparation which is also cross-linked by TNM to give the F2a1-F2b dimer. F1 and F3 can be excluded from the reconstitution mixture without effect on the dimer production. In contrast, the presence of F2a2 is essential to the proper reconstitution of F2a1 and F2b with DNA. The specificity of TNM and the characteristics of the reaction suggest that F2a1 and F2b are cross-linked at their specific binding sites. These results provide evidence that F2a1, F2a2, and F2b interact specifically in chromatin.     

Structural and hormonal changes during follicular maturation in the ovary of the domestic goose

Follicle maturation in the ovary of sexually mature domestic geese in the spring reproductive cycle was investigated by histological methods and steroid-RIA. The single-layer granulosa of primary follicles temporarily transformed in the growing white follicles into several layers or a simple membrana granulosa with nuclei at several different levels in the cell. In the yolky follicles the granulosa represents a cuboidal epithelium (F4-F3) and subsequently a high cylindrical epithelium (F1). The originally connective tissue-like cells of the theca interna show a glandular proliferation in the largest white (F7) and the small yolky follicles (F6-F5). Glandular cell nests in the theca externa are typical in the generation of small white follicles and are absent in the wall of yolky follicles. Progesterone-content in the follicular wall (granulosa + theca) is the highest in the F1-F2 and F6-F5 types and is low in small white follicles (F8, F9 and F10). E2 concentration shows only slight variations between F1-F10. TEST content shows a slight increase between F1 and F3 and is high in medium-sized white follicles (F8-F9). The results suggest that in addition to the granulosa, the theca interna is also capable of an intensive progesterone synthesis.     

Subcellular distribution of [3H]-prostaglandin E2 binding sites in porcine gastric mucosa

The distribution of PGE2 binding sites in four subcellular fractions (F1-F4) from porcine fundic mucosa obtained by gradient centrifugation was examined. Binding of 3HPGE2 to fractions F2-F4 was specific, dissociable, saturable and pH dependent. A significant degree of specific binding was not evident in F1. The Scatchard analysis of binding to F2 and F3 revealed heterogenous populations of binding sites with similar dissociation constants but greater concentrations of binding sites than was evident in the initial 30,000 xg homogenate protein. A single class of low affinity binding sites was evident in F4. The ratio of total: nonspecific binding was approximately equal in F2 and F3. The ratio was considerably smaller in F4. The activity of 5' nucleotidase the marker enzyme for plasma membranes followed this ratio. There was no correlation between the binding ratio and marker enzyme activities for mitrochondrial membranes and endoplasmic reticulum. These data suggest that high affinity PGE2 binding sites occur predominantly on the plasma membrane from gastric mucosal tissue.     

Glucosamine-induced alterations of mitochondrial function in pancreatic beta-cells: possible role of protein glycosylation

Chronic exposure of rat pancreatic islets and INS-1 insulinoma cells to glucosamine (GlcN) produced a reduction of glucose-induced (22.2 mM) insulin release that was associated with a reduction of ATP levels and ATP/ADP ratio compared with control groups. To further evaluate mitochondrial function and ATP metabolism, we then studied uncoupling protein-2 (UCP2), F1-F0-ATP-synthase, and mitochondrial membrane potential, a marker of F1-F0-ATP-synthase activity. UCP2 protein levels were unchanged after chronic exposure to GlcN on both pancreatic islets and INS-1 beta-cells. Due to the high number of cells required to measure mitochondrial F1-F0-ATP-synthase protein levels and mitochondrial membrane potential, we used INS-1 cells, and we found that chronic culture with GlcN increased F1-F0-ATP-synthase protein levels but decreased glucose-stimulated changes of mitochondrial membrane potential. Moreover, F1-F0-ATP-synthase was highly glycosylated, as demonstrated by experiments with N-glycosidase F and glycoprotein staining. Tunicamycin (an inhibitor of protein N-glycosylation), when added with GlcN in the culture medium, was able to partially prevent all these negative effects on insulin secretion, adenine nucleotide content, mitochondrial membrane potential, and protein glycosylation. Thus we suggest that GlcN-induced pancreatic beta-cell toxicity might be mediated by reduced cell energy production. An excessive protein N-glycosylation of mitochondrial F1-F0-ATP-synthase might lead to cell damage and secretory alterations in pancreatic beta-cells.     

Dynamics and relative stabilities of parallel- and antiparallel-stranded DNA duplexes.

The dynamics and stability of four DNA duplexes are studied by means of molecular dynamics simulations. The four molecules studied are combinations of 4, 15 bases long, single-stranded oligomers, F1, F2, F3, and F4. The sequence of these single strand oligomers are chosen such that F1-F2 and F3-F4 form parallel (ps) DNA double helices, whereas F1-F4 and F2-F3 form antiparallel-stranded (aps) DNA double helices. Simulations were done at low (100 K) and room (300 K) temperatures. At low temperatures the dynamics are quasi-harmonic and the analysis of the trajectories gives good estimates of the low frequency vibrational modes and density of states. These are used to estimate the linear (harmonic) contribution of local fluctuations to the configurational entropy of the systems. Estimates of the differences in enthalpy between ps and aps duplexes show that aps double helices are more stable than the corresponding ps duplexes, in agreement with experiments. At higher temperatures, the distribution of the fluctuations around the average structures are multimodal and estimates of the configurational entropy cannot be obtained. The multi-basin, nonlinear character of the dynamics at 300 K is established using a novel method which extracts large amplitude nonlinear motions from the molecular dynamics trajectories. Our analysis shows that both ps DNA exhibit much larger fluctuations than the two aps DNA. The large fluctuations of ps DNA are explained in terms of correlated transitions in the beta, epsilon, and zeta backbone dihedral angles.     

Preliminary results of providing various combinations of live foods to grouper (Epinephelus coioides) larvae

Fertilized oyster eggs, S-rotifer (Brachionusrotundiformis) and SS-rotifer (Brachionus sp.),were tested either solely or in combination fortheir suitability as feed for early stage grouper (Epinephelus coioides) larvae. Sizes ofS-rotifers ranged between 143-224 µm lorica lengthwith mean 182±21µm, and SS-rotifersbetween 122-176µmlorica length with mean 154±13µm. Theresults indicated that both fertilized oyster eggs andSS-rotifers were suitable as feed at F1 (first feedingday). However, poor growth was recorded when providing oyster eggs solely for the periodsF1-F3. Although growth of larvae at F6 had nodifference between the sole SS-rotifers and the oystereggs additionally provided for F1-F3, better survivalof larvae at F15 was obtained when providingcombinations of SS-rotifers with oyster eggs for F1-F3.Besides, better growth and survival of larvae at F15was found when providing S-rotifers enriched withKirin yeast for F7-F15. The highest survival andfastest growth of larvae at F15 was found whenproviding oyster eggs for F1-F3, SS-rotifers forF1-F6, S-rotifers for F7-F15, both rotifers enrichedwith Kirin yeast, and Isochrysis for F1-F15.Total fatty acid (TFA), EPA, DHA content, and DHA/EPA(D/E) ratio of larvae changed with their sizes andcorresponded to that of their feeds. The F15 larvaehaving a higher TFA grew faster, having higher DHA orEPA survived better.     

Micronuclei formation in liver fibrosis samples from patients infected by hepatitis C virus

Genetic research on fibrosis outset and its progression in chronic hepatitis (CH) by hepatitis C virus (HCV) are limited. The lack of cytogenetic data led us to investigate the presence of micronuclei (MNi), as a sign of genomic damage. Hepatocytes of hepatic parenchyma from 62 cases diagnosed with CH associated with HCV and displaying different degrees of fibrosis (F1-F4) were analyzed. These data were compared to 15 cases without fibrosis (F0). Twelve healthy liver parenchyma samples were included as control. All samples were obtained from paraffin-embedded archival material. Micronucleated hepatocytes (MN-Heps) were analyzed through Feulgen/Fast-green staining. Results showed that the rates of MN-Heps in the F4 group were statistically significant (p < 0.05) and higher than those in the control group. Like results were also obtained on comparing F4 with F0, F1, F2 and F3 cases. Conversely, differences were not significant (p > 0.05) on comparing F0, F1, F2, F3, one against the other, as well as individual versus control. Although chromosomal losses in CH were detected, it was shown that liver parenchyma with fibrosis in the initial stages (F1-F3) cannot be considered cytogenetically abnormal.     

Myoelectric reactions to ultra-low frequency and low-frequency whole body vibration

5 healthy males were exposed to vertical sinusoidal whole body vibration (WBV) at 5 frequencies (F1 = 0.315 Hz, F2 = 0.63 Hz, F3 = 1.25 Hz, F4 = 2.5 Hz, F5 = 5.0 Hz) and 2 intensities (I1 = 1.2 ms-2 rms, F1-F5; I2 = 2.0 ms-2 rms, F2-F5). Erector spinae EMGs were derived at the levels of the first thoracic (T1) and third lumbar (L3) spinous processes, rectified and synchronously averaged, as were the accelerations of the seat and the head. WBV induced vibration-synchronous EMG activity (T1 and L3) which exceeded the activity without WBV during enhanced gravitation and decreased during lowered gravitation from F1 to F3. At F4 and F5, these phase relations changed drastically, thus suggesting a different trigger mechanism. The extreme average EMG-amplitudes remained nearly constant at F1 to F3 and increased at higher frequencies. Maximum EMG activity was higher at I2 than at I1. WBV from F1 to F3 is supposed to cause tonic muscular activity triggered by the otoliths; at higher frequencies, stretch reflexes probably gain additional importance. The results hint at an increasing sensory conflict with decreasing frequency of WBV and are interpreted within the theoretical framework of different modes of motor control. Relations between transmissibility and muscle activity suggest the usefulness of including time-variant spring-characteristics into biomechanical models.     

Bacteriophage Mu-1: A tool to transpose and to localize bacterial genes

In a previous publication (Faelen et al., 1975), it was predicted that the temperate phage Mu-1 would mediate transposition of bacterial genes. Here we show that this is indeed the case. By mating either induced F′ strains (which carry a thermoinducible Mu prophage in the bacterial chromosome), or sensitive F′ infected with Mu, with appropriate recipients, we were able to isolate new F′ episomes which carry various lengths of bacterial DNA. The frequency of transposition of a given marker can be as high as 10^?4. The episomes which carry the transposed DNA always carry Mu as well. When this is coupled with the fact that induction or infection with Mu is necessary for transposition to occur, it is probable that both Mu enzymes and Mu DNA are required by the transposition process. Episomes selected for the presence of a given marker were analyzed for the presence of unselected markers. It was found that: (1) only markers linked to the selected marker can be cotransposed with it; (2) when two markers are simultaneously transposed, all markers lying between them on the chromosome are also transposed; (3) the frequency at which an unselected marker is cotransposed is in some way related to the distance between that marker and the selected marker; (4) the transposition process occurs in both Rec⁺ and Rec^? strains. Mu-mediated transposition offers a new way to isolate F′ episomes and to localize and order bacterial genes as far apart as three minutes.     

携带新城疫病毒DNA疫苗的鼠伤寒沙门氏菌及其安全性与免疫效力

根据GenBank中发表的新城疫病毒(NDV)融合蛋白(F)基因序列,设计一对引物,通过RTPCR扩增出鹅源新城疫病毒分离株JS5F基因,测序确认后,将其克隆入真核表达载体pVAX1,获得重组真核表达质粒pVAX1F。将pVAX1F转化减毒鼠伤寒沙门氏菌SL7207,构建成携带DNA疫苗的重组沙门氏菌SL7207(pVAX1F)。重组菌以不同剂量口服免疫1日龄雏鸡,结果表明,细菌对雏鸡具有良好的安全性,且不影响鸡的增重。将SL7207(pVAX1F)分别以108CFU和109CFU的剂量3次口服免疫1日龄商品代伊莎褐蛋鸡,抗体检测结果显示,在三免后1周,SL7207(pVAX1F)109CFU剂量组的血清抗体效价与空载体组之间存在显著性差异(p<0.05)。重组菌两个剂量组在首免后2周开始出现粘膜抗体,并于二免后2周和3周达到较高水平。免疫保护试验结果显示,SL7207(pVAX1F)109CFU剂量组的保护率为77.27%,与空载体组之间存在显著性差异(p<0.05)。     

Serum MicroRNAs as Potential Biomarkers for Early Diagnosis of Hepatitis C Virus-Related Hepatocellular Carcinoma in Egyptian Patients

Circulating microRNAs are deregulated in liver fibrosis and hepatocellular carcinoma (HCC) and are candidate biomarkers. This study investigated the potential of serum microRNAs; miR-19a, miR-296, miR-130a, miR-195, miR-192, miR-34a, and miR-146a as early diagnostic biomarkers for hepatitis C virus (HCV)-related HCC. As how these microRNAs change during liver fibrosis progression is not clear, we explored their serum levels during fibrosis progression in HCV-associated chronic liver disease (CLD) and if they could serve as non-invasive biomarkers for fibrosis progression to HCC. 112 Egyptian HCV-HCC patients, 125 non-malignant HCV-CLD patients, and 42 healthy controls were included. CLD patients were subdivided according to Metavir fibrosis-scoring. Serum microRNAs were measured by qRT-PCR custom array. Serum microRNAs were deregulated in HCC versus controls, and except miR-130a, they were differentially expressed between HCC and CLD or late fibrosis (F3-F4) subgroup. Serum microRNAs were not significantly different between individual fibrosis-stages or between F1-F2 (early/moderate fibrosis) and F3-F4. Only miR-19a was significantly downregulated from liver fibrosis (F1-F3) to cirrhosis (F4) to HCC. Individual microRNAs discriminated HCC from controls, and except miR-130a, they distinguished HCC from CLD or F3-F4 patients by receiver-operating-characteristic analysis. Multivariate logistic analysis revealed a panel of four microRNAs (miR-19a, miR-195, miR-192, and miR-146a) with high diagnostic accuracy for HCC (AUC = 0.946). The microRNA panel also discriminated HCC from controls (AUC = 0.949), CLD (AUC = 0.945), and F3-F4 (AUC = 0.955). Studied microRNAs were positively correlated in HCC group. miR-19a and miR-34a were correlated with portal vein thrombosis and HCC staging scores, respectively. In conclusion, studied microRNAs, but not miR-130a, could serve as potential early biomarkers for HCC in high-risk groups, with miR-19a as a biomarker for liver fibrosis progression to cirrhosis to HCC. We identified a panel of four serum microRNAs with high accuracy in HCC diagnosis. Additional studies are required to confirm this panel and test its prognostic significance.     

F2-dihomo-isoprostanes as potential early biomarkers of lipid oxidative damage in Rett syndrome

Oxidative damage has been reported in Rett syndrome (RTT), a pervasive developmental disorder caused in up to 95% of cases by mutations in the X-linked methyl-CpG binding protein 2 gene. Herein, we have synthesized F(2)-dihomo-isoprostanes (F(2)-dihomo-IsoPs), peroxidation products from adrenic acid (22:4 n-6), a known component of myelin, and tested the potential value of F(2)-dihomo-IsoPs as a novel disease marker and its relationship with clinical presentation and disease progression. F(2)-dihomo-IsoPs were determined by gas chromatography/negative-ion chemical ionization tandem mass spectrometry. Newly synthesized F(2)-dihomo-IsoP isomers [ent-7(RS)-F(2t)-dihomo-IsoP and 17-F(2t)-dihomo-IsoP] were used as reference standards. The measured ions were the product ions at m/z 327 derived from the [M-181](-) precursor ions (m/z 597) produced from both the derivatized ent-7(RS)-F(2t)-dihomo-IsoP and 17-F(2t)-dihomo-IsoP. Average plasma F(2)-dihomo-IsoP levels in RTT were about one order of magnitude higher than those in healthy controls, being higher in typical RTT as compared with RTT variants, with a remarkable increase of about two orders of magnitude in patients at the earliest stage of the disease followed by a steady decrease during the natural clinical progression. hese data indicate for the first time that quantification of F(2)-dihomo-IsoPs in plasma represents an early marker of the disease and may provide a better understanding of the pathogenic mechanisms behind the neurological regression in patients with RTT.     

Sequence specific cleavage of African green monkey alpha-satellite DNA by micrococcal nuclease

The sequence specificity of micrococcal nuclease complicates its use in experiments addressed to the still controversial issue of nucleosome phasing. In the case of alpha-satellite DNA containing chromatin from African green monkey (AGM) cells cleavage by micrococcal nuclease in the nucleus was reported to occur predominantly at only one location around position 126 of the satellite repeat unit (Musich et al. (1982) Proc. Natl. Acad. Sci. USA 79, 118-122). DNA control experiments conducted in the same study indicated the presence of many preferential cleavage sites for micrococcal nuclease on the 172 bp long alpha-satellite repeat unit. This difference was taken as evidence for a direct and simple phase relationship between the alpha-satellite DNA sequence and the position of the nucleosomes on the DNA. We have quantitatively analyzed the digestion products of the protein-free satellite monomer with micrococcal nuclease and found that 50% of all cuts occur at positions 123 and 132, 5% at position 79, and to a level of 1-3% at about 20 other positions. We also digested high molecular weight alpha-satellite DNA from AGM nuclei with micrococcal nuclease. Again cleavage occurred mostly at positions 123 and 132 of the satellite repeat unit. Thus digestion of free DNA yields results very similar to those reported by Musich et al. for the digestion of chromatin. Therefore no conclusions on a possible phase relationship can be drawn from the chromatin digestion experiments.     

The effect of valproic acid on hepatic and plasma levels of 15-F2t-isoprostane in rats

The mechanism by which valproic acid (VPA) induces liver injury remains unknown, but it is hypothesized to involve the generation of toxic metabolites and/or reactive oxygen species. This study's objectives were to determine the effect of VPA on plasma and hepatic levels of the F(2)-isoprostane, 15-F(2t)-IsoP, a marker for oxidative stress, and to investigate the influence of cytochrome P450- (P450-) mediated VPA biotransformation on 15-F(2t)-IsoP levels in rats. In rats treated with VPA (500 mg/kg), plasma 15-F(2t)-IsoP was increased 2.5-fold at t(max) = 0.5 h. Phenobarbital pretreatment (80 mg/kg/d for 4 d) in VPA-treated rats increased plasma and liver levels of free 15-F(2t)-IsoP by 5-fold and 3-fold, respectively, when compared to control groups. This was accompanied by an elevation in plasma and liver levels of P450-mediated VPA metabolites. Pretreatment with SKF-525A (80 mg/kg) or 1-aminobenzotriazole (100 mg/kg), which inhibited P450-mediated VPA metabolism, did not attenuate the increased levels of plasma 15-F(2t)-IsoP in VPA-treated groups. Plasma and hepatic levels of 15-F(2t)-IsoP were further elevated after 14 d of VPA treatment compared to single-dose treatment. Our data indicate that VPA increases plasma and hepatic levels of 15-F(2t)-IsoP and this effect can be enhanced by phenobarbital by a mechanism not involving P450-catalyzed VPA biotransformation.     

Antimicrobial HPA3NT3 peptide analogs: Placement of aromatic rings and positive charges are key determinants for cell selectivity and mechanism of action

In an earlier study, we determined that HP(2‐20) (residues 2‐20 of parental HP derived from the N-terminus of the Helicobacter pylori ribosomal protein L1) and its analog, HPA3NT3, had potent antimicrobial effects. However, HPA3NT3 also showed undesirable cytotoxicity against HaCaT cells. In the present study, we designed peptide analogs including HPA3NT3-F1A (‐F1A), HPA3NT3-F8A (‐F8A), HPA3NT3-F1AF8A (‐F1AF8A), HPA3NT3-A1 (‐A1) and HPA3NT3-A2 (‐A2) in an effort to investigate the effects of amino acid substitutions in reducing their hydrophobicity or increasing their cationicity, and any resulting effects on their selectivity in their interactions with human cells and pathogens, as well as their mechanism of antimicrobial action. With the exception of HPA3NT3-A1, all of these peptides showed potent antimicrobial activity. Moreover, substitution of Ala for Phe at positions 1 and/or 8 of the HPA3NT3 peptides (‐F1A, -F8A and -F1AF8A) dramatically reduced their cytotoxicity. Thus the cytotoxicity of HPA3NT3 appears to be related to its Phe residues (positions 1 and 8), which strongly interact with sphingomyelin in the mammalian cell membrane. HPA3NT3 exerted its bactericidal effects through membrane permeabilization mediated by pore formation. In contrast, fluorescent dye leakage and nucleic acid gel retardation assays showed that ‐A2 acted by penetrating into the cytoplasm, where it bound to nucleic acids and inhibited protein synthesis. Notably, Staphylococcus aureus did not develop resistance to -A2 as it did with rifampin. These results suggest that the -A2 peptide could potentially serve as an effective antibiotic agent against multidrug-resistant bacterial strains.     

Isomer-specific contractile effects of a series of synthetic f2-isoprostanes on retinal and cerebral microvasculature

F2-isoprostanes (F2-IsoP's) are biologically active prostanoids formed by free radical-mediated peroxidation of arachidonic acid. Four different F2-IsoP regioisomers (5-, 8-, 12-, and 15-series), each comprising eight racemic diastereomers, total 64 compounds. Information regarding the biological activity of IsoP's is largely limited to 15-F2t-IsoP (8-iso-PGF2alpha). We recently demonstrated that 15-F2t-IsoP and its metabolite, 2,3-dinor-5,6-dihydro-15-F2t-IsoP, evoked vasoconstriction and TXA2 generation in retina and brain microvasculature. We have now examined and compared the biological activities of a series of recently synthesized new 5-, 12-, and 15-series F2-IsoP isomers in pig retinal and brain microvasculature. We hereby show that other 15-series F2-IsoP isomers, 15-epi-15-F2t-IsoP, ent-15-F2t-IsoP, and ent-15-epi-15-F2t-IsoP, are also potent vasoconstrictors. The 12-series isomers tested, 12-F2t-IsoP and 12-epi-12-F2t-IsoP, also caused marked vasoconstriction. Of the 5-series isomers tested, 5-F2t-IsoP and 5-epi-5-F2t-IsoP possessed no vasomotor properties, whereas ent-5-F2t-IsoP caused modest vasoconstriction. The vasoconstriction of ent-5-F2t-IsoP, 12-F2t-IsoP, and 12-epi-12-F2t-IsoP was abolished by removal of the endothelium, by TXA2 synthase and receptor inhibitor (CGS12970, L670,596), and by receptor-mediated Ca2+ channel blockade (SK & F96365); correspondingly, these isomers increased TXB2 formation by activating Ca2+ influx (detected with fura 2-AM) through non-voltage-dependent receptor-mediated Ca2+ entry (SK & F96365 sensitive) in endothelial cells. In conclusion, as seen with 15-F2t-IsoP, ent-5-F2t-IsoP, 12-F2t-IsoP, and 12-epi-12-F2t-IsoP constricted both retinal and brain microvessels by inducing endothelium-dependent TXA2 synthesis. These new findings broaden the scope of our understanding regarding the potential involvement of F2-IsoP's as mediators of oxidant injury.     

Signaling pathways involved in isoprostane-mediated fibrogenic effects in rat hepatic stellate cells

Despite evidence supporting a potential role for F2-isoprostanes (F2-IsoP's) in liver fibrosis, their signaling mechanisms are poorly understood. We have previously provided evidence that F2-IsoP's stimulate hepatic stellate cell (HSC) proliferation and collagen hyperproduction by activation of a modified form of isoprostane receptor homologous to the classic thromboxane receptor (TP). In this paper, we examined which signal transduction pathways are set into motion by F2-IsoP's to exert their fibrogenic effects. HSCs were isolated from rat liver, cultured to their activated myofibroblast-like phenotype, and then treated with the isoprostane 15-F2t-isoprostane (15-F2t-IsoP). Inositol trisphosphate (IP₃) and adenosine 3′,5′-cyclic monophosphate (cAMP) levels were determined using commercial kits. Mitogen-activated protein kinase (MAPK) and cyclin D1 expression was assessed by Western blotting. Cell proliferation and collagen synthesis were determined by measuring [³H]thymidine and [³H]proline incorporation, respectively. 15-F2t-IsoP elicited an activation of extracellular-signal-regulated kinase (ERK), p38 MAPK, and c-Jun NH₂-terminal kinase (JNK), which are known to be also regulated by G-protein-coupled receptors. Preincubation with specific ERK (PD98059), p38 (SB203580), or JNK (SP600125) inhibitors prevented 15-F2t-IsoP-induced cell proliferation and collagen synthesis. 15-F2t-IsoP decreased cAMP levels within 30 min, suggesting binding to the TPβ isoform and activation of Giα protein. Also, 15-F2t-IsoP increased IP₃ levels within a few minutes, suggesting that the Gq protein pathway is also involved. In conclusion, the fibrogenic effects of F2-IsoP's in HSCs are mediated by downstream activation of MAPKs, through TP binding that couples via both Gqα and Giα proteins. Targeting TP receptor, or its downstream pathways, may contribute to preventing oxidative damage in liver fibrosis.     

In vitro anti-oxidant activity, fluorescence quenching study and structural features of carbohydrate polymers from Phyllanthus emblica

The water-extracted carbohydrate polymers (WE) of Phyllanthus emblica are analyzed using chemical, chromatographic, and spectroscopic methods. Anion-exchange-chromatography of WE yielded four fractions (F1-F4) with different chemical compositions and all of them contain phenolics. The major fraction F4 possesses 50% polysaccharide and 26% phenol, and is a glycoconjugate. The antioxidant capacities of WE and F4 are comparable to standard anti-oxidants. Notably, activities of F1-F4 correlate with their phenol content. Evidence for the complexation of F4 with bovine serum albumin is presented by fluorescence quenching measurement. The results also indicate conformational change of protein at high carbohydrate polymer concentration.