In mammalian cells, inhibition of translation interferes with synthesis of the lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2-P-P-dolichol as measured with radioactive sugar precursors. Conflicting hypotheses have been proposed, and the fundamental basis for this regulation has remained elusive. Here, fluorophore-assisted carbohydrate electrophoresis (FACE) was used to measure LLO concentrations directly in cells treated with translation blockers. Further, LLO biosynthetic enzymes were assayed in vitro with endogenous acceptor substrates using either cells gently permeabilized with streptolysin-O (SLO) or microsomes from homogenized cells. In Chinese hamster ovary (CHO)-K1 cells treated with translation blockers, FACE did not detect changes in concentrations of Glc3Man9GlcNAc2-P-P-dolichol or early LLO intermediates. These results do not support earlier proposals for feedback repression of LLO initiation by accumulated Glc3Man9GlcNAc2-P-P-dolichol, or inhibition of a GDP-mannose dependent transferase. With microsomes from cells treated with translation blockers, there was no interference with LLO initiation by GlcNAc-1-P transferase (GPT), mannose-P-dolichol synthase, glucose-P-dolichol synthase, or LLO synthesis in vitro, as reported previously. Surprisingly, inhibition of all of these was detected with the SLO in vitro system. Additional experiments with the SLO system showed that the three transferases shared a limited pool of dolichol-P that was trapped as Glc3Man9GlcNAc2-P-P-dolichol by translation arrest. Overexpression of GPT was unable to reverse the effects of translation arrest on LLO initiation, and experiments with FACE and the SLO system showed that overexpressed GPT was not functional in vivo, although it was highly active in microsomal assays. Thus, the combined use of the SLO in vitro system and FACE showed that LLO biosynthesis depends upon a limited primary pool of dolichol-P. Physical perturbation associated with microsome preparation appears to make available a secondary pool of dolichol-P, masking inhibition by translation arrest, as well as activating a nonfunctional fraction of GPT. The implications of these results for the organization of the LLO pathway are discussed.

The human placenta is an organ for fetus development and abundant reservoir of various bioactive molecules. Interest to human placenta extract (hPE) is growing, and application with trial of hPE is widening in oriental medicine including in liver diseases. However, underlying mechanisms for therapeutic effects are still unclear. Here, we investigated therapeutic effects of hPE in carbon tetrachloride (CCl(4))-injured rat liver model in vivo and in damaged rat hepatic cells exposed to CCl(4) in vitro. In addition, regulation of inflammatory responses by treatment of hPE was investigated. Serum levels of GOT/AST and GPT/ALT were significantly reduced (P<0.05), and uptake/excretion of indocyanine green in serum was significantly induced at 3 weeks after intravenous hPE administration in CCl(4)-injured rat model (P<0.05). Expression of type I collagen (Col I) and α-smooth muscle actin (α-SMA) was decreased, whereas that of matrix metalloproteinase-9 (MMP-9) was increased resulting in improvement of score for fibrotic grade in hPE group. Also, albumin, proliferation activities and molecules associated with liver regeneration (e.g. interleukin-6, gp130, ATP binding cassette transporters, cyclin A) were more increased in hPE administration group than Non-hPE group. hPE administration suppressed activated T-cell proliferation via increasing anti-inflammatory cytokines and decreasing pro-inflammatory cytokines. These results suggest that hPE could be effective for liver disease through reduction of fibrosis, induction of liver regeneration, and regulation of inflammatory responses. These findings are important for understanding the roles of hPE and provide evidences for therapeutic effects of hPE in hepatic diseases which could lead to potential clinical applications.

An iridoid glucoside, aucubin was isolated from Aucuba japonica leaves and its protective activities against CCl4-induced hepatotoxicity were evaluated by measuring the duration of hypnosis induced by hexobarbital after CCl4 challenge (0.2 ml/kg/day, po) and the levels of serum glutamic-oxalacetic (GOT) and serum glutamic-pyruvic transaminase (GPT). The duration of hypnosis for the saline control group, the CCl4 alone treated group and the aucubin plus CCl4 treated group was 24.8 +/- 8.5, 60.5 +/- 9.5 and 28.0 +/- 3.2 min, respectively. Treatment of mice with aucubin also effectively protected against CCl4-induced increased serum GOT and GPT activities. It was found that aucubin inhibited hepatic RNA and protein syntheses in vivo. Such inhibitory effects of aucubin might be responsible for protective activities against CCl4-induced liver damage.

In patients with psychotic disorders, the effects of psychological post-traumatic stress disorder (PTSD) treatment on symptoms of psychosis, depression and social functioning are largely unknown

In a single-blind randomized controlled trial (RCT) 155 outpatients in treatment for psychosis (61.3% schizophrenic disorder, 29% schizoaffective disorder) were randomized to eight sessions prolonged exposure (PE; n = 53) or eye movement desensitization and reprocessing (EMDR) (n = 55), or a waiting-list condition (WL, n = 47) for treatment of their co-morbid PTSD. Measures were performed on (1) psychosis: severity of delusions (PSYRATS-DRS), paranoid thoughts (GPTS), auditory verbal hallucinations (PSYRATS-AHRS), and remission from psychotic disorder (SCI-SR-PANSS); (2) depression (BDI-II); (3) social functioning (PSP). Outcomes were compared at baseline, post-treatment, 6-month follow-up and over all data points.

Both PE and EMDR were significantly associated with less severe paranoid thoughts post-treatment and at 6-month follow-up, and with more patients remitting from schizophrenia, at post-treatment (PE and EMDR) and over time (PE). Moreover, PE was significantly associated with a greater reduction of depression at post-treatment and at 6-month follow-up. Auditory verbal hallucinations and social functioning remained unchanged.

In patients with chronic psychotic disorders PE and EMDR not only reduced PTSD symptoms, but also paranoid thoughts. Importantly, in PE and EMDR more patients accomplished the status of their psychotic disorder in remission. Clinically, these effects are highly relevant and provide empirical support to the notion that delivering PTSD treatment to patients with psychotic disorders and PTSD deserves increasing recognition and acceptance among clinicians.

The methanolic extract from the flowers of Tilia argentea (linden) was found to show a hepatoprotective effect against D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced liver injury in mice. By bioassay-guided separation using in vitro D-GalN-induced damage to hepatocytes, five flavonol glycosides were isolated as the hepatoprotective constituents of the methanolic extract. Tiliroside, the principal flavonol glycoside, strongly inhibited serum GPT and GOT elevations at doses of 25-100 mg/kg (p.o.) in D-GalN/LPS-treated mice. By comparing the inhibitory effects of tiliroside with those of its components alone, the kaempferol 3-O-beta-D-glucopyranoside moiety was found to be essential for the activity, and its effect was suggested to depend on the inhibition of tumor necrosis factor-alpha (TNF-alpha) production, decreased sensitivity of hepatocytes to TNF-alpha, and on the protection of hepatocytes against D-GalN.

Two frequent protein variants of glutamate pyruvate transminase (GPT) (E.C.2.6.1.2) have been used as genetic markers in humans for more than two decades, although chromosomal mapping of the GPT locus in the 1980s produced conflicting results. To resolve this conflict and develop useful DNA markers for this gene, we isolated and characterized cDNA and genomic clones of GPT. We have definitively mapped human GPT to the terminus of 8q using several methods. First, two cosmids shown to contain the GPT sequence were derived from a chromosome 8-specific library. Second, by fluorescence in situ hybridization, we mapped the cosmid containing the human GPT gene to chromosome band 8q24.3. Third, we mapped the rat gpt cDNA to the syntenic region of rat chromosome 7. Finally, PCR primers specific to human GPT amplify sequences contained within a "half-YAC" from the long arm of chromosome 8, that is, a YAC containing the 8q telomere. The human GPT genomic sequence spans 2,7 kb and consists of 11 exons, ranging in size from 79 to 243 bp. The exonic sequence encodes a protein of 495 amino acids that is nearly identical to the previously reported protein sequence of human GPT-1. The two polymorphic GPT isozymes are the results of a nucleotide substitution in codon 14, coding for a histidine in GPT-1 and an asparagine in GPT-2, which causes a gain or loss of an NlaIII restriction site. In addition, a cosmid containing the GPT sequence also contains a previously unmapped, polymorphic microsatellite sequence, D8S421. The cloned GPT gene and associated polymorphisms will be useful for linkage and physical mapping of disease loci that map to the terminus of 8q, including atypical vitelliform macular dystrophy (VMD1) and epidermolysis bullosa simplex, type Ogna (EBS1). In addition, this will be a useful system for characterizing the telomeric region of 8q. Finally, determination of the molecular basis of the GPT isozyme variants will permit PCR-based detection of this world-wide polymorphism.

Translesion DNA synthesis (TLS) is an important pathway that avoids genotoxicity induced by endogenous and exogenous agents. DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in TLS but its protective roles against DNA damage in vivo are still unclear. To better understand these roles, we have established knock-in mice that express catalytically-inactive Polk and crossbred them with gpt delta mice, which possess reporter genes for mutations. The resulting mice (inactivated Polk KI mice) were exposed to mitomycin C (MMC), and the frequency of point mutations, micronucleus formation in peripheral erythrocytes, and γH2AX induction in the bone marrow was determined. The inactivated Polk KI mice exhibited significantly higher frequency of mutations at CpG and GpG sites, micronucleated cells, and γH2AX foci-positive cells than did the Polk wild-type (Polk(+)) mice. Recovery from MMC-induced DNA damage, which was evaluated by γH2AX induction, was retarded in embryonic fibroblasts from the knock-in mice when compared to those from the Polk(+) mice. These results suggest that Polk mediates TLS, which suppresses point mutations and DNA double-strand breaks caused by intra- and interstrand cross-links induced by MMC treatment. The established knock-in mice are extremely useful to elucidate the in vivo roles of the catalytic activity of Polk in suppressing DNA damage that was induced by a variety of genotoxic stresses.

OBJECTIVE

To investigate baseline factors and neurologic function tests (NFTs) that may predict the development of grade 2 or higher peripheral neuropathy (PN) after treatment with ixabepilone, an epothilone microtubule-stabilizing agent with antitumor activity.

METHODS

Advanced breast cancer patients were treated with ixabepilone (6 mg/m2) for 5 consecutive days every 3 weeks in a phase II clinical trial. Physical examinations, questionnaires, nerve conduction studies, and NFTs, including the Jebsen Test of Hand Function (JTH) and the Grooved Pegboard Test (GPT), were performed at baseline and during subsequent cycles.

RESULTS

Forty-seven patients assessable for PN received a median of five cycles of therapy (range, one to 22 cycles). Nine of these patients developed grade 2 PN, and two developed grade 3 PN, with a median time to onset of 144 days (range, 6 to 189 days). Among these 11 patients, PN resolved in eight patients, with a median of 15 days (range, 6 to 346 days) after onset, but PN did not resolve in three patients during follow-ups at 76, 361, and 746 days after onset. GPT and changes of JTH scores at onset of PN were significantly different between patients with and without PN at comparable follow-up times (P = .006 and P = .002, respectively). Changes in GPT and JTH scores over the first two cycles were often associated with the development of PN by exploratory actuarial analysis.

CONCLUSIONS

Serious ixabepilone-induced neuropathy was relatively rare on the treatment schedule used. NFTs, such as JTH and GPT, may have utility for predicting PN, but further testing is needed.

Monolayer cultures of diploid human fibroblasts and suspensions of S. typhimurium TA100 cells were treated with [3H]-labelled enantiomeric forms of benzo[a]pyrene anti and syn 7,8-dihydrodiol 9,10-epoxides. In both cell types, all of the enantiomers induced the formation of mutant 6-thioguanine (human) or 8-azaguanine-(bacterial)resistant cells. Diol-epoxide-modified nucleosides from human and from bacterial DNA hydrolysates were characterized by HPLC and showed essentially the same adduct species for human and bacterial cells treated with the same enantiomers. There were substantial differences, however, in the efficiency with which structurally-different adduct species were converted to mutant genotypes. In human cells, the mutagenic efficiency (mutation frequency/unit modified DNA) of the respective adduct species (+ anti much greater than -anti = +/- syn) at the hprt locus was exactly the opposite of that seen at a similar gene locus (gpt) in TA100 (-anti = +/- syn greater than + anti). The results suggest that the structural configuration of adducts in genomic DNA is important in determining whether a mutant genotype will result, and likewise, that there are differences in specificity between the human and bacterial systems which process these adduct lesions.

Restriction endonuclease fragment length variations (RFLV) were detected by use of the cDNA probe Hox-3.1 for the homeo box-3.1 gene and also the c-myc oncogene probe for exon 2. RFLV of Hox-3.1 were found in HindIII restriction patterns, and RFLV of the Myc-1.2 gene in EcoRV patterns. From the RFLV, the Hox-3.1 and Myc-1.2 genes were mapped on chromosome 15. Three-point cross test data showed that the frequency of recombination is 26.4% between Myc-1.2 and Gpt-1, 30.2% between Gpt-1 and Gdc-1, and 9.4% between Gdc-1 and Hox-3.1. The following order of these genes is proposed, Myc-1.2--Gpt-1--Gdc-1--Hox-3.1. All laboratory strains carry the Hox-3.1a and Myc-1.2a alleles. Among strains of wild origin, domesticus strains carry only the Hox-3.1a and Myc-1.2a alleles, as do the laboratory strains. One strain of brevirostris carries the Hox-3.1a and Myc-1.2b alleles. Other wild subspecies from Europe and Asia, M. m. musculus, M. m. castaneus, M. m. molossinus, Chinese mice of wild origin, and M. m. yamashinai carry the Hox-3.1b and Myc-1.2b alleles.

AIM:To evaluate the safety and efficacy of the bioartificial liver support system in canines with acute liver failure (ALF).METHODS:Nine canines with acute liver failure by acetaminophen-induced received TECA-I bioartificial liver support system (BALSS) from Hong Kong TECA LTD Co. Blood was perfused through a hollow fiber tube containing (1-2)X10(10) the porcine hepatocytes.In contrast, another 10 canines with acute liver failure by Acetaminophen received drugs. Each treatment lasted 6 hours.RESULTS:BALSS treatment resulted in beneficial effects for acetaminophen-induced ALF canines with survival and with the recovery of the liver functions and tissues, and plasma ammonia decreased from 135.9&mgr;mol/L plus minus 17.5&mgr;mol/L to 65.7&mgr;mol/L plus minus 22.0&mgr;mol/L, 32.5&mgr;mol/L plus minus 8.8&mgr;mol/L, GPT from 97.8U/L plus minus 8.7U/L to 64.8U/L plus minus 11.9U/L, 19.0U/L plus minus 6.3U/L, GOT from 103.0U/L plus minus 16.7U/L to 75.7U/L plus minus 19.6U/L, 26.5U/L plus minus 5.0U/L, and AKP from 158.3U/L plus minus 12.1U/L to 114.5U/L plus minus 19.8U/L, 43.8U/L plus minus 5.6U/L during and after the treatment. In contrast, 10 ALF canines in both the drug and control groups died 1 or 2 days after treatment.CONCLUSION: TECA-1 artificial liver support system is safe and efficacious for canines with acute liver failure.

The extent of the indirect DNA damage generated in mammalian cells by visible light because of the presence of endogenous photosensitizers was studied by means of repair endonucleases. In immortalized human keratinocytes (HaCaT cells) exposed to low doses of natural sunlight, the yield of oxidative DNA base modifications sensitive to the repair endonuclease formamidopyrimidine-DNA glycosylase (Fpg protein) generated by this indirect mechanism was 10% of that of pyrimidine dimers (generated by direct DNA excitation). A similar yield of Fpg-sensitive modifications, which include 8-hydroxyguanine, was observed in primary keratinocytes. The relative yield of oxidative base modifications decreased at higher light doses, probably as a result of photodecomposition of the endogenous chromophore involved. For the three cell lines tested, viz. HaCaT cells, L1210 mouse leukemia cells and AS52 Chinese hamster cells, the yield of oxidative base modifications generated by a low dose of visible light appeared to be correlated with the basal concentrations of porphyrins in the cells. Induction of cellular porphyrin synthesis by pretreatment with 5-aminolaevulinic acid increased the light-induced oxidative damage in L1210 cells several-fold. In both induced and uninduced cells, the damage was inhibited by more than 50% in the presence of ascorbic acid (100 microM), while alpha-tocopherol and the iron chelator alpha-phenanthroline had no effect and beta-carotene even increased the damage. Even high doses of visible light did not significantly increase the numbers of micronuclei in L1210 cells or of gpt mutations in AS52 cells. The negative outcome can be fully explained by the photobleaching of the endogenous photosensitizers, which prevents the generation of sufficiently high levels of oxidative DNA damage. Therefore, the mutagenic risk arising from the indirectly generated oxidative DNA modifications induced by sunlight may be underestimated when results obtained at high doses are extrapolated to low doses or low dose rates.

The effect of fluoride exposure during gestation and post gestation periods were studied to check the status of oxidant, antioxidant and macromolecular changes in CNS and ameliorative role of antioxidants. The pregnant Wistar albino rats were exposed to 50 and 150 ppm fluoride in drinking water and the pups born to them were used for experimentation. After postpartum, the pups were administered daily selected antioxidants through oral gavage. On 21st postnatal day pups were sacrificed and biochemical parameters were assessed. Fluoride exposure substantially increased the activity/levels of fluoride, LPO, protein oxidation, MAO-B, GOT, GPT and decreased protein thiols, RNA and total proteins in discrete regions of CNS. The findings evidenced fluoride induced dyshomeostasis caused on antioxidants, enzymes, macromolecules and governed the pathophysiological events leading to functional loss in a dose dependent manner. The administration of antioxidants remedied the disquiet caused by high fluoride exposure at extreme vulnerable periods of life.

We have analyzed the specificity of mutations induced by ethyl methanesulfonate (EtMes) in mouse cells carrying a selectable bacterial gene. The target gene was the Escherichia coli gpt gene contained within a retroviral shuttle vector integrated into mouse chromosomal DNA. Following mutagenesis by EtMes, cells with mutations in the gpt gene were selected as resistant to 6-thioguanine. Shuttle vector sequences were recovered from the mutant cell lines following fusion with monkey COS cells and introduced into bacteria as part of a bacterial plasmid. The DNA base sequences of the mutant genes were directly determined from plasmid DNA. All of the EtMes-induced mutations involving single base changes were found to be G:C to A:T transitions.

7,8-dihydro-8-oxo-guanine (8-oxoG) is a mutagenic DNA lesion that is induced by ultraviolet A (UVA) radiation. 8-oxoG results in increased frequency of GC->>TA transversion mutations. UVA-induced mutant frequency was measured in the guanine phosphoribosyl transferase (gpt) gene of Chinese hamster ovary cells (AS52) that were stably transfected to overexpress the hOGG1 protein, the human DNA repair glycosylase for 8-oxoG. This mutant frequency was compared with ultraviolet A-induced mutant frequency in AS52 cells stably transfected with the same vector without the hOGG1 gene. The mutant frequency was significantly decreased in the hOGG1 overexpressing cells irradiated with 300 and 400 kJ/m2 ultraviolet A radiation, corresponding to 25% and 10% cell survival, respectively. The hOGG1 overexpressing cells repaired oxidative DNA lesions three times faster than the vector only cells as measured by a semi-automated alkaline elution assay using FPG enzyme, the bacterial OGG1 analogue, to cut DNA at oxidative base modifications. Thus, the lower mutation frequency in UVA-induced mutations of the hOGG1 overexpressing cells may be related to the increased repair of 8-oxoG. No GC->>TA mutations were detected in the UVA-irradiated hOGG1 overexpressing cells. The results suggest a link between the 8-oxoG lesion and UVA-induced mutagenesis. We propose that hOGG1 has a role in maintaining genomic stability in mammalian cells after oxidative stress.

A pSV2gpt-transformed Chinese hamster ovary (CHO) cell line has been used to study mutation at the molecular level. This cell line, designated AS52, was constructed from a hypoxanthine-guanine phosphoribosyl transferase (HPRT)-deficient CHO cell line, and has been previously shown to contain a single, functional copy of the E. coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) stably integrated into the Chinese hamster genome. In this study, conditions for its use in the study of mammalian cell mutagenesis have been stringently defined. The spontaneous mutation rate (2 X 10(-6)/cell division) and phenotypic expression time (7 days) of the gpt locus compare favorably with those of the hprt locus in wild-type CHO-K1-BH4 cells. While both cell lines exhibit similar cytotoxic responses to ethyl methanesulfonate (EMSO and ICR 191, significant differences in mutation induction were observed. Ratios of XPRT to HPRT mutants induced per unit dose of EMS and ICR 191 are 0.70 and 1.6, respectively. Southern blot hybridization analyses revealed that most XPRT mutant cell lines which arose following treatment with EMS (20/22) or ICR 191 (20/24) exhibited no alterations of the gpt locus detectable by this technique. Similar observations were made for the hprt locus in EMS-(21/21) and ICR 191-induced (22/22) HPRT mutants. In contrast, most spontaneous gpt mutants (14/23) contained deletions, while most spontaneous hprt mutants (18/23) exhibited no detectable alterations. Results of this study indicate that the AS52 cell line promises to be useful for future study of mutation in mammalian cells at the DNA sequence level.

Electroporation was used to introduce a mixture of two plasmid-cloned genes into Chinese hamster ovary (CHO) cells, and the location of the two genes was subsequently determined by fluorescence in situ hybridization (FISH). The 25 kb Chinese hamster gene for dihydrofolate reductase (dhfr) in the form of a cosmid-derived 40 kb BglI fragment and the SV40 promoter-driven E. coli gene for guanine phosphoribosyltransferase (gpt) were co-electroporated and gpt + transfectants selected. Clones that had also integrated a single copy of the dhfr gene were studied by 2-color fluorescence in situ hybridization (FISH) to localize the integration site(s) of the exogenous DNA in metaphase chromosomes. All 9 clones examined showed co-localization of the two transgenes. The chromosomal site of integration was different in each clone. Co-integration was confirmed by co-amplification experiments. We conclude that, even when provided at low concentrations, separate soluble DNA molecules become linked upon gene transfer by electroporation, either by intracellular ligation prior to integration, or by co-integration at a common site in a given recipient cell.

We have developed a system to study mutations which affect expression of the E. coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) in hypoxanthine-guanine phosphoribosyl transferase-deficient (HPRT-) Chinese hamster ovary (CHO) cells that have been transformed by the plasmid pSV2gpt. Several gpt-transformed cell lines have been isolated and characterized with respect to integrated pSV2gpt sequences, expression of the gpt gene, and cytotoxic and mutagenic responses to UV light. While the gpt-transformed CHO and wild-type CHO-K1-BH4 cell lines have similar cytotoxic responses to UV light, the gpt-transformed cell lines respond differently from the parental CHO-K1-BH4 cell line in terms of mutation induction. As with CHO-K1-BH4 HPRT mutants, spontaneous or induced XPRT mutants derived from the gpt+ cell lines can be selected for 6-thioguanine resistance (TGr). Analysis of cell-free extracts from a number of these TGr clones indicates that the mutant phenotype is due to the absence of XPRT activity. One transformant, designated AS52, has previously been described in limited detail. Here we describe additional characteristics of this cell line, as well as several related transformants.

Groups of Brahman-cross steers maintained on two nutritional planes were infected intravenously with a stabilate of Anaplasma marginale. In general, animals on the higher plane of nutrition were more severely affected. Fever was the first clinical sign of anaplasmosis but, like anaemia, was absent in the mildest cases. When present anaemia appeared two to three weeks after infection. There was a corresponding increase in erythrocyte sedimentation rates when read after 24 h but not at 1 h. The haemolytic nature of the anaemia was indicated by a significant increase in unconjugated bilirubin during the acute phase. Some visceral damage was suggested by a significant increase of serum aspartate amino-transferase (GOT) especially in severely affected animals of the 'high' nutrition group but no significant change occurred in levels of alanine amino-transferase (GPT).

From a series of 1,3,4-thiadiazole-2-arylhydrazone derivatives of megazol screened in vitro against Trypanosoma cruzi, eight (S1 to S8) were selected for in vivo screening by single-dose oral administration (200 mg/kg of body weight) to infected mice at 5 days postinfection (dpi). Based on significant decreases in both parasitemia levels and mortality rates, S2 and S3 were selected for further assays. Despite having no in vivo effect, S1 was included since it was 2-fold more potent against trypomastigotes than megazol in vitro. Trypomastigotes treated with S1, S2, or S3 showed alterations of the flagellar structure and of the nuclear envelope. When assayed on intracellular amastigotes, the selectivity index (SI) for macrophages was in the range of >27 to >63 and for cardiac cells was >32 for S1 and >48 for megazol. In noninfected mice, S1 did not alter the levels of glutamic oxalacetic transaminase (GOT), glutamate pyruvate transaminase (GPT), or urea. S2 led to an increase in GOT, S3 to increases in GOT and GPT, and megazol to an increase in GOT. Infected mice were treated with each derivative at 50 and 100 mg/kg from dpi 6 to 15: S1 did not interfere with the course of infection or reduce the number of inflammatory foci in the cardiac tissue, S2 led to a significant decrease of parasitemia, and S3 decreased mortality. There was no direct correlation between the in vitro effect on trypomastigotes and amastigotes and the results of the treatment in experimental models, as S1 showed a high potency in vitro while, in two different schemes of in vivo treatment, no decrease of parasitemia or mortality was observed.

Picrorhiza kurroa (Pk), a known hepatoprotective plant, was studied in experimental and clinical situtations. The standardization of active principles--Picroside 1 and 2 was done with High Performance Liquid Chromatography. Picroside 1 ranged from 2.72 to 2.88 mg/capsule and picroside 2 from 5.50 to 6.00 mg/capsule. In the galactosamine-induced liver injury in rats, Pk at a dose of 200 mg/kg p.o. showed a significant reduction (p < 0.05) in liver lipid content, GOT and GPT. In a randomised, double-blind placebo controlled trial in patients diagnosed to have acute viral hepatitis (HBsAg negative), Pk root powder 375 mg three times a day was given for 2 weeks (n = 15) or a matching placebo (n = 18) was given. Difference in values of bilirubin, SGOT and SGPT was significant between placebo and Pk groups. The time in days required for total serum bilirubin to drop to average value of 2.5 mg% was 75.9 days in placebo as against 27.44 days in Pk group. The present study has shown a biological plausability of efficacy of Pk as supported by clinical trial in viral hepatitis, hepatoprotection in animal model and an approach for standardizing extracts based on picroside content.

The composition of a Brazilian green propolis ethanolic extract (Et-Bra) and its effect on Trypanosoma cruzi trypomastigotes and other pathogenic microorganisms have already been reported. Here, we further investigated Et-Bra targets in T. cruzi and its effect on experimental infection of mice. The IC(50)/4 days for inhibition of amastigote proliferation was 8.5 ± 1.8 μg mL(-1), with no damage to the host cells. In epimastigotes Et-Bra induced alterations in reservosomes, Golgi complex and mitochondrion. These effects were confirmed by flow cytometry analysis. In trypomastigotes, Et-Bra led to the loss of plasma membrane integrity. The in vitro studies indicate that Et-Bra interferes in the functionality of the plasma membrane in trypomastigotes and of reservosomes and mitochondrion in epimastigotes. Acutely infected mice were treated orally with Et-Bra and the parasitemia, mortality and GPT, GOT, CK and urea levels were monitored. The extract (25-300 mg kg(-1) body weight/day for 10 days) reduced the parasitemia, although not at significant levels; increased the survival of the animals and did not induce any hepatic, muscular lesion or renal toxicity. Since Et-Bra was not toxic to the animals, it could be assayed in combination with other drugs. Et-Bra could be a potential metacyclogenesis blocker, considering its effect on reservosomes, which are an important energy source during parasite differentiation.

Tissue engineering therapies require biocompatible and bioactive biomaterials that are capable of encouraging an angiogenic response for effective tissue regeneration. In this study, a SVVYGLR peptide, which functions as a potent angiogenic factor, was conjugated into injectable gelatin-poly(ethylene glycol)-tyramine (GPT) hydrogels in situ to enhance endothelial cell activities and neo-vascularization. SVVYGLRGGY (SV-Y) conjugated GPT (SV-GPT) hydrogels were formed in situ via enzyme-mediated reaction using horseradish peroxidase (HRP) and hydrogen peroxide (H(2)O(2)). The physico-chemical properties were characterized and could be controlled depending on the feed peptide and H(2)O(2) concentration. The concentration of conjugated peptide ranged from 0.37 to 0.81 μmol/mL, and the elastic moduli (G') of the hydrogels were 600-4900 Pa. In vitro cell studies using human umbilical vein endothelial cells (HUVECs) and in vivo subcutaneous injection studies were performed to confirm the effect of the SVVYGLR peptide on HUVEC activity and neo-vascularization. Obtained results demonstrated that the in situ conjugation of SVVYGLR sequences into phenol residues of GPT hydrogels enhanced the activity of HUVECs in vitro and stimulated the formation of new blood vessels in the hydrogel matrices in vivo. From the results, we suggest that in situ conjugation of SV-Y to GPT hydrogels via the enzymatic reaction may be an efficient tool to prepare injectable bioactive hydrogels that can enhance endothelial cell activities and promoting angiogenesis for tissue regeneration.