Survival and initial chromatid breakage in normal and tumour cells exposed in vitro to gamma rays and carbon ions at the HIRFL

Human hepatoma and normal liver cells were irradiated with C-12(6+) ion beams (linear energy transfer (LET) = 96 keV mu m(-1)) and gamma-rays at the Heavy Ion Research Facility in Lanzhou (HIRFL). The numbers and types of chromatid breaks were detected using the premature chromosome condensation technique. Irradiation with C-12(6+) ions produced a majority of isochromatid break types, while chromatid breaks were dominant for irradiation with gamma-rays. Experimental results showed that the initial level of chromatid breaks is clearly related to the absorbed dose from C-12(6+), ions and gamma-rays. The (12)C(6+)ions are relatively more effective at inducing initial chromatid breaks when compared with the gamma-rays. A relative biological effectiveness (RBE) of about 2.5 resulted for the induction of initial chromatid breaks by C-12(6+) ions relative to gamma-rays in both cell lines.

Effects of ion beam irradiation on adventitious shoot regeneration from in vitro leaf explants of Saintpaulia ionahta

The effects of 960 MeV carbon ion beam and 8 MeV X-ray irradiation on adventitious shoots from in vitro leaf explants of two different Saintpaulia ionahta (Mauve and Indikon) cultivars were studied with regard to tissue increase, shoots differentiation and morphology changes in the shoots. The experimental results showed that the survival fraction of shoot formation for the Mauve and Indikon irradiated with the carbon ion beam at 20 Gy were 0.715 and 0.600, respectively, while those for both the cultivars exposed to the Xray irradiation at the same dose were 1.000. Relative biological effectiveness (RBE) of Mauve with respect to X-ray was about two. Secondly, the percentage of regenerating explants with malformed shoots in all Mauve regenerating explants irradiated with carbon ion beam at 20 Gy accounted for 49.6%, while that irradiated with the same dose of X-ray irradiation was only 4.7%; as for Saintpatdia ionahta Indikon irradiated with 20 Gy carbon ion beam, the percentage was 43.3%, which was higher than that of X-ray irradiation. Last, many chlorophyll deficient and other varieties of mutants were obtained in this study. Based on the results above, it can be concluded that the effect of mutation induction by carbon ion beam irradiation on the leaf explants of Saintpaulia ionahta is better than that by X-ray irradiation; and the optimal mutagenic dose varies from 20 Gy to 25 Gy for carbon ion beam irradiation.

Optimizing the formation of in vitro sponge primmorphs from the Chinese sponge Stylotella agminata (Ridley)

The establishment and optimization of in vitro primmorph formation from a Chinese sponge, Stylotella agminata (Ridley), collected from the South China Sea, were investigated. Our aims were to identify the key factors affecting primmorph formation in this species and to optimize the technique for developing an in vitro primmorph culture system. The size of dissociated cells from S. agminata is relatively small, in the range between 5 and 10 mum. Round-shaped primmorphs of less than 100 gm were formed 3 days after transferring the dissociated cells into seawater containing Ca2+ and Mg2+. The effect of various cell dissociation conditions, inoculum. cell density, concentration of antibiotics, pH, and temperature was further investigated upon the formation of primmorphs. The time required for primmorph formation, primmorph size distribution, and the proliferating capability were microscopically documented. Healthy sponge S. agminata, inoculum. cell density and culture temperature play a critical role for the successful formation of primmorphs and that the microbial contamination will have to be controlled. (C) 2002 Elsevier Science B.V. All rights reserved.

Ultrafine PEG-PLA fibers loaded with both paclitaxel and doxorubicin hydrochloride and their in vitro cytotoxicity

By means of "emulsion-electrospinning", both hydrophobic and hydrophilic drugs, paclitaxel (PTX) and doxorubicin hydrochloride (DOX), were successfully loaded into PEG-PLA nanofiber mats to realize multi-drug delivery. The release behaviors of both the drugs from the same fiber mats were ascribed to their solubility properties and distribution status in the fibers. Due to its high hydrophilicity, DOX was easy to diffuse out from the fibers, and its release rate was always faster than that of hydrophobic PTX. Moreover, the release rate of PTX was accelerated by DOX's release from the same drug-loaded fibers. In vitro cytotoxicity against rat Glioma C6 cells indicated that the dual drug combination showed a higher inhibition and apoptosis against C6 cells than a single drug-loaded system, which suggests the promise for multi-drug delivery on combination therapy.

Feasibility of biodegradable PLGA common bile duct stents: An in vitro and in vivo study

The current study investigates the feasibility of using a biodegradable polymeric stent in common bile duct (CBD) repair and reconstruction. Here, poly(l-lactide-co-glycolide) (PLGA, molar ratio LA/GA = 80/20) was processed into a circular tube- and dumbbell-shaped specimens to determine the in vitro degradation behavior in bile. The morphology, weight loss, and molecular weight changes were then investigated in conjunction with evaluations of the mechanical properties of the specimen. Circular tube-shaped PLGA stents with X-ray opacity were subsequently used in common bile duct exploration (CBDE) and primary suturing in canine models. Next, X-ray images of CBD stents in vivo were compared and levels of serum liver enzymes and a histological analysis were conducted after stent transplantation. The results showed that the PLGA stents exhibited the required biomedical properties and spontaneously disappeared from CBDs in 4-5 weeks. The degradation period and function match the requirements in repair and reconstruction of CBDs to support the duct, guide bile drainage, and reduce T-tube-related complications.

Mannose-Escherichia coli interaction in the presence of metal cations studied in vitro by colorimetric polydiacetylene/glycolipid liposomes

Supramolecular assemblies of liposomes (vesicles) made of diacetylenic lipids and synthetic mannoside derivative glycolipid receptors were successfully used to mimic the molecular recognition occurring between mannose and Escherichia coli. This specific molecular recognition was translated into visible blue-to-red color transition (biochromism) of the polymerized liposomes, readily quantified by UV-visible spectroscopy. Some transition metal cations (Cd2+, Ag+, Cu2+, Fe3+, Zn2+ and Ni2+) and alkali earth metal cations (Ca2+, Mg2+ and Ba2+) were introduced into the system to analyze their effects on specific biochromism. Results showed that the presence of Cd2+, Ag+, Ca2+, Mg2+ and Ba2+ enhanced biochromisin. A possible enhancement mechanism was proposed in the process of bacterial adhesion to host cells. However, Cu2+, Fe3+, Zn2+ and Ni2+ exhibited inhibitory effects that cooperated with diacetylene lipid with a carboxylic group and increased the rigidity of the liposomal outer leaflet, blocking changes in the side chain conformation and electrical structure of polydiacetylene polymer during biochromism.

BCNU-loaded PEG-PLLA ultrafine fibers and their in vitro antitumor activity against glioma C6 cells

The purpose of the present study was to develop implantable BCNU-toaded poly(ethylene glycol)poly(L-lactic acid) (PEG-PLLA) diblock copolymer fibers for the controlled release of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU was well incorporated and dispersed uniformly in biodegradable PEG-PLLA fibers by using electrospinning method. Environmental Scanning Electron Microscope (ESEM) images indicated that the BCNU-loaded PEG-PLLA fibers looked uniform and their surfaces were reasonably smooth. Their average diameters were below 1500 nm. The release rate of BCNU from the fiber mats increased with the increase of BCNU loading amount. In vitro cytotoxicity assay showed that the PEG-PLLA fibers themselves did not affect the growth of rat Glioma C6 cells. Antitumor activity of the BCNU-loaded fibers against the cells was kept over the whole experiment process, while that of pristine BCNU disappeared within 48 h. These results strongly suggest that the BCNU/PEG-PLLA fibers have an effect of controlled release of BCNU and are suitable for postoperative chemotherapy of cancers.

In vitro study on the binding of neutral red to bovine serum albumin by molecular spectroscopy

In this paper, the binding of neutral red (NR) to bovine serum albumin (BSA) under physiological conditions has been studied by spectroscopy method including fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. The Stern-Volmer fluorescence quenching constant (K-SV), binding constant (K-b) and the number of binding sites (It) were measured by fluorescence quenching method. Fluorescence experiments were also performed at different ionic strengths. It was found K-SV was ionic strength dependent, which indicated the electrostatic interactions were part of the binding forces. The distance r between donor (BSA) and acceptor (NR) was obtained according to Foster's non-radiative energy transfer theory. CD spectroscopy and FT-IR spectroscopy were used to investigate the structural information of BSA molecules on the binding of NR, and the results showed no change of BSA conformation in our experimental conditions.

Studies on the transport of lanthanides ions through the membrane of human erythrocyte in vitro by ICP-MS

A method was developed for the determination of lanthanum in the cytoplasm of human erythrocytes after they were incubated in lanthanum nitrate or citrate solutions. The lanthanum concentration in the cytoplasm of incubated erythrocytes is much higher than that in normal erythrocytes. It is suggested lanthanum can transport through the membrane of erythrocyte in vitro. Solutions containing chelator are unsuitable to be washing buffer in the investigation.

In vitro antioxidative activities of extract and semi-purified fractions of the marine red alga, Rhodomela confervoides (Rhodomelaceae)

The methanol-chloroform extract of the marine red alga, Rhodomela confervoides, was measured for antioxidant activity, using the alpha,alpha-diphenyl-beta-picrylhydrazyl radical-scavenging assay and the beta-carotene-linoleate bleaching assay systems, and compared with those of the positive Controls of butylated hydroxytoluene, gallic acid and ascorbic acid, The active extract was further purified by liquid-liquid partition to afford four fractions, of which the ethyl acetate-soluble (EA) fraction exhibited the strongest antioxidant activity in both assay systems. This fraction was further divided into seven subfractions, designated as EA1-EA7, by silica gel vacuum liquid chromatography. in most cases, EA1 and EM Were found to possess the strongest activity. The total phenolic contents and reducing powers of the extract, fractions, and subfractions were also determined. Significant associations between the antioxidant potency and the total phenolic content, as well as between the antioxidant potency and the reducing power, were found for the tested fractions and subfractions. (c) 2008 Elsevier Ltd. All rights reserved.

In vitro assembly of R-phycoerythrin from marine red alga Polysiphonia urceolata

Scanning tunneling microscope was used to investigate the in vitro assembly of R-phycoerythrin (R-PE) from the marine red alga Polysiphonia urceolata. The results showed that R-PE molecules assembled together by disc-to-disc while absorbing on HOPG surface, which just looked like the rods in the phycobilisomes. When the water-soluble R-PE was dissolved in 2% ethanol/water spreading solution, they could form monolayer film at the air/water interface. Similar disc-to-disc array of R-PE was constituted in the two-dimensional Langmuir-Blodgett film by the external force. It could be concluded that, apart from the key role of time linker polypeptides, the in vivo assembly of phycobiliproteins into phycobilisomes is also dependent on the endogenous properties of phycobiliprotein themselves.

N-acetylchitooligosaccharide is a potent angiogenic inhibitor both in vivo and in vitro

N-Acetylchitooligosaccharide (N-acetyl-COs) was prepared by N-acetylation of chitooligosaccharide (COs). In vitro study using human umbilical vein endothelial cells (HUVECs) revealed that both N-acetyl-COs and COs inhibited the proliferation of HUVECs by inducing apoptosis. Treatment of HUVECs by N-acetyl-COs resulted in a significant reduction of density of the migration cells and repressed tubulogenesis process. The antiangiogenic effects of the oligosaccharides were further evaluated using in vivo zebrafish angiogenesis model, and the results showed that both oligosaccharides inhibited the growth of subintestinal vessels (SIV) of zebrafish embryos in a dose-dependent manner, as observed by endogenous alkaline phosphatase (EAP) staining assay. In contrast, no cytotoxicity was found when treating the NIH3T3 and several other cancer cells with the oligosaccharides. Our results also confirmed the antiangiogenic activity of N-acetyl-COs was significantly stronger than the parent oligosaccharide, COs. (c) 2007 Published by Elsevier Inc.

In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (>10(13) different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents.

Zebrafish thymidylate synthase binds to its own mRNA in vitro and in vivo

Thymidylate synthase (TS), an essential enzyme for DNA de novo synthesis, is a critical therapeutic target in cancer therapy. Previous study has shown that TS was able to bind to its own mRNA in human and E.coli, resulting in translational repression. Zebrafish is the best animal model for vertebrate study. In order to study the regulatory mechanism of zebrafish TS, the enzyme were expressed in E. coli BL21 (DE3) and it was purified to homogeneity. Electrophoretic mobility shift assay (EMSA) was used to detect the interaction of zebrafish TS protein and its own TS transcript in vitro and the results showed that zebrafish TS could bound with its own mRNA specifically. Further study revealed that zebrafish TS was able to interact with its own mRNA in vivo using immunoprecipitation : RT-PCR technique. The results provide evidence that zebrafish may be developed as an useful model for studying the anti-metabolism agents.

In vitro iron enrichment experiments in the Prydz Bay, the Southern Ocean: A test of the iron hypothesis

In "high nitrate, low chlorophyll" (HNLC) ocean regions, iron has been typically regarded as the limiting factor for phytoplankton production. This "iron hypothesis" needs to be tested in various oceanic environments to understand the role of iron in marine biological and biogeochemical processes. In this paper, three in vitro iron enrichment experiments were performed in Prydz Bay and at the Polar Front north of the Ross Sea, to study the role of iron on phytoplankton production. At the Polar Front of Ross Sea, iron addition significantly (P < 0.05, Student's t-test) stimulated phytoplankton growth. In Prydz Bay, however, both the iron treatments and the controls showed rapid phytoplankton growth, and no significant effect (P > 0.05, Student's t-test) as a consequence of iron addition was observed. These results confirmed the limiting role of iron in the Ross Sea and indicated that iron was not the primary factor limiting phytoplankton growth in Prydz Bay. Because the light environment for phytoplankton was enhanced in experimental bottles, light was assumed to be responsible for the rapid growth of phytoplankton in all treatments and to be the limiting factor controlling field phytoplankton growth in Prydz Bay. During the incubation experiments, nutrient consumption ratios also changed with the physiological status and the growth phases of phytoplankton cells. When phytoplankton growth was stimulated by iron addition, N was the first and Si was the last nutrient which absorption enhanced. The Si/N and Si/P consumption ratios of phytoplankton in the stationary and decay phases were significantly higher than those of rapidly growing phytoplankton. These findings were helpful for studies of the marine ecosystem and biogeochemistry in Prydz Bay, and were also valuable for biogeochemical studies of carbon and nutrients in various marine environments.