Incorporation Of Radioactivity Research Articles

Interaction of apolipoproteins A-I and E in the regulation of DNA, RNA, and protein biosynthesis in cultured rat hepatocytes

Experiments on hepatocyte culture showed that apolipoprotein A-I-tetrahydrocortisol complex increases the rate of DNA, RNA, and protein biosynthesis measured by radioactive label incorporation. Apolipoprotein E acted as competitor of the apolipoprotein A-I-tetrahydrocortisol complex and abolished biological activity of the latter. We hypothesize that this mechanism of regulation plays an important role in processes of intracellular regeneration and proliferation.

High Iron Content and Bioavailability in Humans from Four Species of Marine Algae ,

Searching for economical, nonconventional sources of iron is important in underdeveloped countries to combat iron deficiency and anemia. Our objective was to study iron, vitamin C, and phytic acid composition and also iron bioavailability from 4 species of marine algae included in a rice-based meal. Marine algae (Ulva sp, Sargassum sp, Porphyra sp, and Gracilariopsis sp) were analyzed for monthly variations in iron and for ascorbic acid and phytic acid concentrations. A total of 96 subjects received rice-based meals containing the 4 species of marine algae in different proportions, raw or cooked. All meals contained radioactive iron. Absorption was evaluated by calculating the radioactive iron incorporation in subjects’ blood. Iron concentrations in algae were high and varied widely, depending on the species and time of year. The highest iron concentrations were found in Sargassum (157 mg/100 g) and Gracilariopsis (196 mg/100 g). Phytates were not detected in the algae and ascorbic acid concentration fluctuated between 38 μg/g dry weight (Ulva) and 362 μg/g dry weight (Sargassum). Algae significantly increased iron absorption in rice-based meals. Cooking did not affect iron absorption compared with raw algae. Results indicate that Ulva sp, Sargassum sp, Porphyra sp, and Gracilariopsis sp are good sources of ascorbic acid and bioavailable iron. The percentage of iron absorption was similar among all algae tested, although Sargassum sp resulted in the highest iron intake. Based on these results, and on the high reproduction rates of algae during certain seasons, promoting algae consumption in some countries could help to improve iron nutrition.

Improved synthesis of no-carrier-added p-[ 124I]iodo- l-phenylalanine and p-[ 131I]iodo- l-phenylalanine for nuclear medicine applications in malignant gliomas

This work describes the synthesis and the tumor affinity testing of no-carrier-added (n.c.a.) p-[ 124I]iodo- l-phenyalanine ([ 124I]IPA) and n.c.a. p-[ 131I]iodo- l-phenyalanine ([ 131I]IPA) as radiopharmaceuticals for imaging brain tumors with PET and for radionuclid-based therapy, respectively. Parameters for labeling were optimized with regard to the amount of precursor, temperature and time. Thereafter, n.c.a. [ 124I]IPA and n.c.a. [ 131I]IPA were investigated in rat F98 glioma and in primary human A1207 and HOM-T3868 glioblastoma cells in vitro, followed by an in vivo evaluation in CD1 nu/nu mice engrafted with human glioblastoma. No-carrier-added [ 124I]IPA and n.c.a. [ 131I]IPA were obtained in 90±6% radiochemical yield and >99% radiochemical purity by iododestannylation of N-Boc-4-(tri- n-butylstannyl)- l-phenylalanine methylester in the presence of chloramine-T, followed by hydrolysis of the protecting groups. The total synthesis time, including the HPLC separation and pharmacological formulation, was less than 60 min and compatible with a clinical routine production. Both amino acid tracers accumulated intensively in rat and in human glioma cells. The radioactivity incorporation in tumor cells following a 15-min incubation at 37 °C/pH 7.4 varied from 25% to 42% of the total loaded activity per 10 6 tumor cells (296–540 cpm/1000 cells). Inhibition experiments confirmed that n.c.a. [ 124I]IPA and n.c.a. [ 131I]IPA were taken up into tumor by the sodium-independent L- and ASC-type transporters. Biodistribution and whole-body imaging by a gamma-camera and a PET scanner demonstrated a high targeting level and a prolonged retention of n.c.a. [ 124I]IPA and n.c.a. [ 131I]IPA within the xenotransplanted human glioblastoma and a primarily renal excretion. However, an accurate delineation of the tumors in mice was not possible by our imaging systems. Radioactivity accumulation in the thyroid and in the stomach as a secondary indication of deiodination was less than 1% of the injected dose at 24 h p.i., confirming the high in vivo stability of the radiopharmaceuticals. In conclusion, n.c.a. [ 124I]IPA and n.c.a. [ 131I]IPA are new promising radiopharmaceuticals, which can now be prepared in high radiochemical yields and high purity for widespread clinical applications. The specific and high-level targeting of n.c.a. [ 124I]IPA and n.c.a. [ 131I]IPA to glioma cells in vitro and to glioblastoma engrafts in vivo encourages further in vivo validations to ascertain their clinical potential as agent for imaging and quantitation of gliomas with PET, and for radionuclid-based therapy, respectively.

Site-directed alkylation of cysteine to test solvent accessibility of membrane proteins

This protocol describes a detailed method to study the static and dynamic features of membrane proteins, as well as solvent accessibility, by utilizing the lactose permease of Escherichia coli (LacY) as a model. The method relies on the use of functional single-Cys mutants, an affinity tag and a PhosphoImager. The membrane-permeant, radioactive thiol reagent N-[ethyl-1-14C]ethylmaleimide ([14C]NEM) is used to detect site-directed alkylation of engineered single-Cys mutants in situ. The solvent accessibility of the Cys residues is also determined by blockage of [14C]NEM labeling with membrane-impermeant thiol reagents such as methanethiosulfonate ethylsulfonate (MTSES). The labeled proteins are purified by mini-scale affinity chromatography and analyzed by gel electrophoresis. Gels are dried and exposed to a PhosphoImager screen for 1-5 d, and incorporation of radioactivity is visualized. Initial results can be obtained in 24 h.

Maternal allergy influences the proliferation of neonatal T cells expressing CCR4, CXCR5 or CD103

Elevated proliferative response to allergen in cord blood mononuclear cells (CBMCs) is related to subsequent allergy development of the neonate and has been suggested as a screening marker for high allergy risk. To characterize the proliferating cells in CBMCs from a neonatal group influenced by maternal allergy compared with a control group without known allergic heredity. CBMCs were stimulated with bovine beta-lactoglobulin (beta-LG) and proliferation was analysed by radioactive thymidine incorporation and expressed both as the traditional stimulation index (SI) and SI corrected by eliminating non-specific proliferation. After beta-LG combined with endotoxin stimulation, cellular expression of IL-4 and IFN-gamma mRNA was determined by quantitative RT-PCR and adhesion as well as chemokine receptors were analysed by three-colour flow cytometry in proliferating T cells (CD3+ Ki-67+). The percentage of CCR4+ cells correlated weakly with concurrent IL-4 expression (r(S)=0.5, P<0.05), while CXCR3 correlated strongly with IFN-gamma expression (r(S)=0.83, P<0.001). In the allergy risk group, the percentage of proliferating T cells expressing CCR4 or integrin alphaE (CD103) was significantly reduced compared with the control group, while CXCR5 and the corrected SI were relatively increased (CCR4: P=0.01; integrin alphaE: P=0.03; CXCR5: P=0.04; SI: P=0.04). Our results implied delayed maturation of immune functions involved in cellular migration, cell-cell interaction and immunoregulatory functions in neonates with hereditary allergy risk. The alterations observed in this subject group suggested that the corrected SI as well as proliferation of CCR4+, CXCR5+ or CD103+ T cells in allergen-stimulated CBMCs might serve as early screening markers for allergy risk.

β‐amyloid (25–35) enhances lipid metabolism and protein ubiquitination in cultured neurons

We investigated the effect of beta-amyloid (Abeta) (25-35), a cytotoxic fragment of Abeta peptide, on lipid metabolism and protein ubiquitination in cultured rat hippocampal neurons. After treatment with Abeta under conditions leading to apoptotis, as assessed by caspase activity assay, the total cell mass of lipids changed following a biphasic behavior, with an increase that reached a maximum after 16 hr of treatment, followed by a decrease. The increase at 16 hr was 15.3% in the case of phospholipids and 103.0% in the case of gangliosides and was due to enhanced biosynthesis as confirmed by increase of radioactivity incorporation (phospholipids +52.0%, gangliosides +193.1%) in cells fed with tritiated palmitic acid. No change with respect to cholesterol was observed. Strikingly, under these conditions, the ubiquitination state of cell proteins strongly increased. These effects were not observed with the (35-25) reverse sequence peptide. Similarly to Abeta, lactacystin treatment increased lipid synthesis and protein ubiquitination; only lactacystin, and not Abeta, induced a strong decrease of proteasome chimotrypsin activity. These results suggest that Abeta enhances protein ubiquitination, without inhibiting proteasomal activity, and lipid synthesis. These results may shed new light on the mechanisms of Abeta toxicity.

Synapsin Phosphorylation by Src Tyrosine Kinase Enhances Src Activity in Synaptic Vesicles

Synapsins are synaptic vesicle-associated phosphoproteins implicated in the regulation of neurotransmitter release. Synapsin I is the major binding protein for the SH3 domain of the kinase c-Src in synaptic vesicles. Its binding leads to stimulation of synaptic vesicle-associated c-Src activity. We investigated the mechanism and role of Src activation by synapsins on synaptic vesicles. We found that synapsin is tyrosine phosphorylated by c-Src in vitro and on intact synaptic vesicles independently of its phosphorylation state on serine. Mass spectrometry revealed a single major phosphorylation site at Tyr(301), which is highly conserved in all synapsin isoforms and orthologues. Synapsin tyrosine phosphorylation triggered its binding to the SH2 domains of Src or Fyn. However, synapsin selectively activated and was phosphorylated by Src, consistent with the specific enrichment of c-Src in synaptic vesicles over Fyn or n-Src. The activity of Src on synaptic vesicles was controlled by the amount of vesicle-associated synapsin, which is in turn dependent on synapsin serine phosphorylation. Synaptic vesicles depleted of synapsin in vitro or derived from synapsin null mice exhibited greatly reduced Src activity and tyrosine phosphorylation of other synaptic vesicle proteins. Disruption of the Src-synapsin interaction by internalization of either the Src SH3 or SH2 domains into synaptosomes decreased synapsin tyrosine phosphorylation and concomitantly increased neurotransmitter release in response to Ca(2+)-ionophores. We conclude that synapsin is an endogenous substrate and activator of synaptic vesicle-associated c-Src and that regulation of Src activity on synaptic vesicles participates in the regulation of neurotransmitter release by synapsin.

Assessment and Continued Validation of the Malaria SYBR Green I-Based Fluorescence Assay for Use in Malaria Drug Screening

Several new fluorescence malaria in vitro drug susceptibility microtiter plate assays that detect the presence of malarial DNA in infected erythrocytes have recently been reported, in contrast to traditional isotopic screens that involve radioactive substrate incorporation to measure in vitro malaria growth inhibition. We have assessed and further characterized the malaria SYBR Green I-based fluorescence (MSF) assay for its ability to monitor drug resistance. In order to use the MSF assay as a drug screen, all assay conditions must be thoroughly examined. In this study we expanded upon the capabilities of this assay by including antibiotics and antifolates in the drug panel and testing folic acid-free growth conditions. To do this, we evaluated a more expansive panel of antimalarials in combination with various drug assay culture conditions commonly used in drug sensitivity screening for their activity against Plasmodium falciparum strains D6 and W2. The detection and quantitation limits of the MSF assay were 0.04 to 0.08% and approximately 0.5% parasitemia, respectively. The MSF assay quality was significantly robust, displaying a Z' range of 0.73 to 0.95. The 50% inhibitory concentrations for each drug and culture condition combination were determined by using the MSF assay. Compared to the standard [(3)H]hypoxanthine assay, the MSF assay displayed the expected parasite drug resistance patterns with a high degree of global and phenotypic correlation (r(2) >/= 0.9238), regardless of which culture condition combination was used. In conclusion, the MSF assay allows for reliable one-plate high-throughput, automated malaria in vitro susceptibility testing without the expense, time consumption, and hazard of other screening assays.

Genotoxicity of 2-(3-Chlorobenzyloxy)-6-(piperazinyl)pyrazine, a Novel 5-Hydroxytryptamine2c Receptor Agonist for the Treatment of Obesity: Role of Metabolic Activation

2-(3-Chlorobenzyloxy)-6-(piperazin-1-yl)pyrazine (3) is a potent and selective 5-HT(2C) agonist that exhibits dose-dependent inhibition of food intake and reduction in body weight in rats, making it an attractive candidate for treatment of obesity. However, examination of the genotoxicity potential of 3 in the Salmonella Ames assay using tester strains TA98, TA100, TA1535, and TA1537 revealed a metabolism (rat S9/NADPH)- and dose-dependent increase of reverse mutations in strains TA100 and TA1537. The increase in reverse mutations was attenuated upon coincubation with methoxylamine and glutathione. The irreversible and concentration-dependent incorporation of radioactivity in calf thymus DNA after incubations with [14C]3 in the presence of rat S9/NADPH suggested that 3 was bioactivated to a reactive intermediate that covalently bound DNA. In vitro metabolism studies on 3 with rat S9/NADPH in the presence of methoxylamine and cyanide led to the detection of amine and cyano conjugates of 3. The mass spectrum of the amine conjugate was consistent with condensation of amine with an aldehyde metabolite derived from hydroxylation of the secondary piperazine nitrogen-alpha-carbon bond. The mass spectrum of the cyano conjugate suggested a bioactivation pathway involving N-hydroxylation of the secondary piperazine nitrogen followed by two-electron oxidation to generate an electrophilic nitrone, which reacted with cyanide. The 3-chlorobenzyl motif in 3 was also bioactivated via initial aromatic ring hydroxylation followed by elimination to a quinone-methide species that reacted with glutathione or with the secondary piperazine ring nitrogen in 3 and its monohydroxylated metabolite(s). The metabolism studies described herein provide a mechanistic basis for the mutagenicity of 3.

Killed but Metabolically ActiveSalmonella typhimurium:Application of a New Technology to an Old Vector

Previous studies have shown that attenuated salmonellae utilized as vaccine vectors engender strong immune responses; however, balancing immunogenicity with reactogenicity remains problematic. Recent work in other bacteria has shown that photochemical treatment of DNA excision repair mutants ( Delta uvrAB) renders organisms "killed but metabolically active" (KBMA). Here, we extend this concept to Salmonella typhimurium. A strain of attenuated S. typhimurium previously evaluated in human volunteers was further deleted for uvrAB genes and designated CKS362. Photochemical treatment of CKS362 resulted in significant inactivation. These KBMA organisms were metabolically active as shown by radioactive methionine incorporation and lactate dehydrogenase activity. In mice inoculated intraperitoneally, KBMA CKS362 was markedly less reactogenic and stimulated a humoral immune equivalent to its live counterpart. Because the parental strain has previously been found to elicit strong immune responses to Salmonella antigens, we propose CKS362 as a prototype strain to test the immunogenicity of KBMA organisms in humans.

Inhibition of Gαi2 Activation by Gαi3 in CXCR3-mediated Signaling

G protein-coupled receptors (GPCRs) convey extracellular stimulation into dynamic intracellular action, leading to the regulation of cell migration and differentiation. T lymphocytes express G alpha(i2) and G alpha(i3), two members of the G alpha(i/o) protein family, but whether these two G alpha(i) proteins have distinguishable roles guiding T cell migration remains largely unknown because of a lack of member-specific inhibitors. This study details distinct G alpha(i2) and G alpha(i3) effects on chemokine receptor CXCR3-mediated signaling. Our data showed that G alpha(i2) was indispensable for T cell responses to three CXCR3 ligands, CXCL9, CXCL10, and CXCL11, as the lack of G alpha(i2) abolished CXCR3-stimulated migration and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) incorporation. In sharp contrast, T cells isolated from G alpha(i3) knock-out mice displayed a significant increase in both GTPgammaS incorporation and migration as compared with wild type T cells when stimulated with CXCR3 agonists. The increased GTPgammaS incorporation was blocked by G alpha(i3) protein in a dose-dependent manner. G alpha(i3)-mediated blockade of G alpha(i2) activation did not result from G alpha(i3) activation, but instead resulted from competition or steric hindrance of G alpha(i2) interaction with the CXCR3 receptor via the N terminus of the second intracellular loop. A mutation in this domain abrogated not only G alpha(i2) activation induced by a CXCR3 agonist but also the interaction of G alpha(i3) to the CXCR3 receptor. These findings reveal for the first time an interplay of G alpha(i) proteins in transmitting G protein-coupled receptor signals. This interplay has heretofore been masked by the use of pertussis toxin, a broad inhibitor of the G alpha(i/o) protein family.

Chaperone Properties of Mammalian Mitochondrial Translation Elongation Factor Tu

The main function of the prokaryotic translation elongation factor Tu (EF-Tu) and its eukaryotic counterpart eEF1A is to deliver aminoacyl-tRNA to the A-site on the ribosome. In addition to this primary function, it has been reported that EF-Tu from various sources has chaperone activity. At present, little information is available about the chaperone activity of mitochondrial EF-Tu. In the present study, we have examined the chaperone function of mammalian mitochondrial EF-Tu (EF-Tumt). We demonstrate that recombinant EF-Tumt prevents thermal aggregation of proteins and enhances protein refolding in vitro and that this EF-Tumt chaperone activity proceeds in a GTP-independent manner. We also demonstrate that, under heat stress, the newly synthesized peptides from the mitochondrial ribosome specifically co-immunoprecipitate with EF-Tumt and are destabilized in EF-Tumt-overexpressing cells. We show that most of the EF-Tumt localizes on the mitochondrial inner membrane where most mitochondrial ribosomes are found. We discuss the possible role of EF-Tumt chaperone activity in protein quality control in mitochondria, with regard to the recently reported in vivo chaperone function of eEF1A.

Disease-associated Mutations at Copper Ligand Histidine Residues of Superoxide Dismutase 1 Diminish the Binding of Copper and Compromise Dimer Stability

A subset of superoxide dismutase 1 (Cu/Zn-SOD1) mutants that cause familial amyotrophic lateral sclerosis (FALS) have heightened reactivity with (-)ONOO and H(2)O(2) in vitro. This reactivity requires a copper ion bound in the active site and is a suggested mechanism of motor neuron injury. However, we have found that transgenic mice that express SOD1-H46R/H48Q, which combines natural FALS mutations at ligands for copper and which is inactive, develop motor neuron disease. Using a direct radioactive copper incorporation assay in transfected cells and the established tools of single crystal x-ray diffraction, we now demonstrate that this variant does not stably bind copper. We find that single mutations at copper ligands, including H46R, H48Q, and a quadruple mutant H46R/H48Q/H63G/H120G, also diminish the binding of radioactive copper. Further, using native polyacrylamide gel electrophoresis and a yeast two-hybrid assay, the binding of copper was found to be related to the formation of the stable dimeric enzyme. Collectively, our data demonstrate a relationship between copper and assembly of SOD1 into stable dimers and also define disease-causing SOD1 mutants that are unlikely to robustly produce toxic radicals via copper-mediated chemistry.

High-Throughput Plasmodium falciparum Growth Assay for Malaria Drug Discovery

New therapeutic agents for the treatment of malaria, the world's most deadly parasitic disease, are urgently needed. Malaria afflicts 300 to 500 million people and results in 1 to 2 million deaths annually, and more than 85% of all malaria-related mortality involves young children and pregnant women in sub-Saharan Africa. The emergence of multidrug-resistant parasites, especially in Plasmodium falciparum, has eroded the efficacy of almost all currently available therapeutic agents. The discovery of new drugs, including drugs with novel cellular targets, could be accelerated with a whole-organism high-throughput screen (HTS) of structurally diverse small-molecule libraries. The standard whole-organism screen is based on incorporation of [3H]hypoxanthine and has liabilities, such as limited throughput, high cost, multiple labor-intensive steps, and disposal of radioactive waste. Recently, screens have been reported that do not use radioactive incorporation, but their reporter signal is not robust enough for HTS. We report a P. falciparum growth assay that is technically simple, robust, and compatible with the automation necessary for HTS. The assay monitors DNA content by addition of the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI) as a reporter of blood-stage parasite growth. This DAPI P. falciparum growth assay was used to measure the 50% inhibitory concentrations (IC50s) of a diverse set of known antimalarials. The resultant IC50s compared favorably with those obtained in the [3H]hypoxanthine incorporation assay. Over 79,000 small molecules have been tested for antiplasmodial activity using the DAPI P. falciparum growth assay, and 181 small molecules were identified as highly active against multidrug-resistant parasites.

Human cationic trypsinogen is sulfated on Tyr154

The crystal structure of human pancreatic cationic trypsin showed the chemical modification of Tyr154, which was originally described as phosphorylation [Gaboriaud C, Serre L, Guy-Crotte O, Forest E & Fontecilla-Camps JC (1996) J Mol Biol259, 995-1010]. Here we report that Tyr154 is sulfated, not phosphorylated. Cationic and anionic trypsinogens were purified from human pancreatic juice and subjected to alkaline hydrolysis. Modified tyrosine amino acids were separated on a Dowex cation-exchange column and analyzed by thin layer chromatography. Both human cationic and anionic trypsinogens contained tyrosine sulfate, but no tyrosine phosphate, whereas bovine trypsinogen contained neither. Furthermore, incorporation of [(35)S]SO(4) into human cationic trypsinogen transiently expressed by human embryonic kidney 239T cells was demonstrated. Mutation of Tyr154 to Phe abolished radioactive sulfate incorporation, confirming that Tyr154 is the site of sulfation in cationic trypsinogen. Sulfated pancreatic cationic trypsinogen exhibited faster autoactivation than a nonsulfated recombinant form, suggesting that tyrosine sulfation of trypsinogens might enhance intestinal digestive zymogen activation in humans. Finally, sequence alignment revealed that the sulfation motif is only conserved in primate trypsinogens, suggesting that typsinogen sulfation is absent in other vertebrates.

Role of adenosine salvage in wound-induced adenylate biosynthesis in potato tuber slices

Levels of ATP and other nucleotides increased in wounded potato tuber slices, maintained on moist paper for 24 h after preparation. The relative expression intensity of genes encoding adenosine kinase ( AK) and adenine phosphoribosyltransferase ( APRT) in wounded slices was greater than the intensity of genes of the de novo pathway, glycineamide ribonucleotide formyltransferase ( GART) and 5-aminoimidazole ribonucleotide synthetase ( AIRS). In vitro activities of adenosine kinase (ATP:adenosine 5′-phosphotransferase; EC 2.7.1.20) and adenine phosphoribosyltransferase (AMP:pyrophosphate phospho- d-ribosyltransferase; EC 2.4.2.7) increased during wounding. Adenosine nucleosidase (adenosine ribohydrolase; EC 3.2.2.7) activity was negligible in freshly prepared slices, but its activity is dramatically enhanced in wounded slices. In situ adenosine salvage activity, estimated from the incorporation of radioactivity from exogenously supplied [8- 14C]adenosine into nucleotides and RNA, increased more than five times in the wounded slices. These results strongly suggest that greater expression of the genes encoding enzymes of adenosine salvage during wounding is closely related to the increased supply of adenine nucleotides in the wounded slices.

Preparation of tritiated oostatic peptides for study of radioactivity incorporation in flesh fly Neobellieria bullata

A series of insect oostatic peptides containing 3,4-dehydroproline in the C-terminal part or inside of the peptide chain was synthesized and tritiated by addition of (3)H2 to double bond of 3,4-dehydroproline residue. (3)H-label was introduced also into tyrosine residue of oostatic tetra- and pentapeptides by isotopic exchange of benzyl beta-hydrogens. In this way, three types of tritiated peptides were prepared, different in the radiolabeled amino acid position: [(3)H] Tyr-Asp-Pro-Ala-OH, H-Tyr-Asp-[(3)H] Pro-Ala-OH, [(3)H] Tyr-Asp-Pro-Ala-Pro-OH, H-Tyr-Asp-[(3)H] Pro-Ala-Pro-OH, H-Tyr-Asp-Pro-Ala-[(3)H] Pro-OH, H-Tyr-Asp-Pro-Ala-Pro(5)-[(3)H] Pro-OH and H-Asp-[(3)H] Pro-OH. These peptides made possible a highly sensitive comparative study on radioactivity incorporation into head and ovaries of the flesh fly Neobellieria bullata, which revealed this process to proceed differently. The reasons of the found differences are discussed.

Global Translational Responses to Oxidative Stress Impact upon Multiple Levels of Protein Synthesis

Global inhibition of protein synthesis is a common response to stress conditions. We have analyzed the regulation of protein synthesis in response to oxidative stress induced by exposure to H(2)O(2) in the yeast Saccharomyces cerevisiae. Our data show that H(2)O(2) causes an inhibition of translation initiation dependent on the Gcn2 protein kinase, which phosphorylates the alpha-subunit of eukaryotic initiation factor-2. Additionally, our data indicate that translation is regulated in a Gcn2-independent manner because protein synthesis was still inhibited in response to H(2)O(2) in a gcn2 mutant. Polysome analysis indicated that H(2)O(2) causes a slower rate of ribosomal runoff, consistent with an inhibitory effect on translation elongation or termination. Furthermore, analysis of ribosomal transit times indicated that oxidative stress increases the average mRNA transit time, confirming a post-initiation inhibition of translation. Using microarray analysis of polysome- and monosome-associated mRNA pools, we demonstrate that certain mRNAs, including mRNAs encoding stress protective molecules, increase in association with ribosomes following H(2)O(2) stress. For some candidate mRNAs, we show that a low concentration of H(2)O(2) results in increased protein production. In contrast, a high concentration of H(2)O(2) promotes polyribosome association but does not necessarily lead to increased protein production. We suggest that these mRNAs may represent an mRNA store that could become rapidly activated following relief of the stress condition. In summary, oxidative stress elicits complex translational reprogramming that is fundamental for adaptation to the stress.

Bone Morphogenetic Protein-4, a Novel Modulator of Melanogenesis

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta family, signal in many cells including neural precursors. Two receptors, types 1 and 2, coordinately mediate BMP signaling, and type 1 receptor has two forms: A and B. Using RT-PCR we found that neural crest-derived human melanocytes express BMP receptor-1A, -1B, and -2. Furthermore, melanocytes and the surrounding keratinocytes express BMP-4, suggesting both autocrine and paracrine effects of this molecule. Moreover, BMP-4 supplementation of cultured human melanocytes decreases melanin synthesis, tyrosinase mRNA, and protein. The mechanism of this BMP-4 effect on tyrosinase and ultimately on melanogenesis involves modest decreases of tyrosinase transcription rate and mRNA stability. Moreover, ultraviolet irradiation, the best recognized environmental stimulator of melanogenesis, down-regulated the mRNA of BMP receptor-1B in melanocytes. Our data provide evidence of a novel regulatory pathway for melanogenesis in human skin.

Low‐intensity ultrasound stimulates the viability and matrix gene expression of human articular chondrocytes in alginate bead culture

We investigated the effects of low-intensity ultrasound (LIUS) on the activity of human articular chondrocytes isolated from osteoarthritis patients and cultured in the three-dimensional alginate beads. LIUS was treated at 0, 100, 200, and 300 mW/cm(2) for 10 min everyday for 2, 7, or 15 days. LIUS induced the viability of cells only at day 15 but not until day 7 after treatment, when examined by trypan blue exclusion and LIVE/DEAD(R) assay kit. When examined at day 7, the proliferation of cells was not changed by LIUS in the (3)H-thymine incorporation. The expression of matrix producing proteins (type II collagen and proteoglycan) was clearly induced by 200-300 mW/cm(2) LIUS in the incorporation of radioactivity and Northern blot analysis. Although the expression of MMP-1, a matrix degrading protein, was decreased, that of TIMP-1, an inhibitor of MMPs, was not affected by LIUS. Histological analysis revealed an increase in the number and size of glycosaminoglycan-positive lacunae and cellular organelles, appearing as rough endoplasmic reticulum and mitochondria by LIUS. These results showed that the viability and metabolism of human articular chondrocytes in alginate culture was induced by LIUS treatment, suggesting that they could be a promising autologous source for cartilage tissue engineering.