Protection by zinc against UVA— and UVB-induced cellular and genomic damage in vivo and in vitro

For many years, zinc salts have been used both topically and orally to treat minor burns and abrasions as well as to enhance wound repair in man and animals. In this study we describe the protective effects of zinc against UV-induced genotoxicity in vitro and against sunburn cell formation in mouse skin in vivo. Cultured skin cells from neonatal mice showed a dramatic increase in the number of micronuclei as a result of UVA and UVB irradiation. Inclusion of zinc at 5 μg/mL in the medium significantly reduced the frequency of micronuclei and of micronucleated cells. In hairless mice, topical application of zinc chloride for 5 consecutive days or a single application 2 h prior to UV exposure reduced the number of sunburn cells in the epidermis as did application of zinc 1 h after exposure. Application 2 h after irradiation also tended to have a protective effect, although there was a large variation between animals. It is proposed that an influx of zinc can protect epidermal cells against some of the more delayed effects of UV-induced damage.     

Oxygen evolution and phosphorylation in Scenedesmus as influenced by the inhibitor-β complex from potato and by phloridzin

Summary Scenedesmus cells were tried as a photosynthesizing test system for the inhibitor- complex from potato (var. Majestic). The main effect on oxygen evolution is an inhibition of the second gush during the induction period. The formation of bound phosphate is increased by the inhibitor when the cell suspensions are shaken in the light, whereas no significant trend is observed in similar experiments in the dark. — Phloridzin inhibits the same part of oxygen evolution as inhibitor- from potato when cells pretreated in light are the test object. Concentrations of 1 mM increase the formation of bound phosphate in light, but at 2 mM there is a decrease in this action. Phloridzin and inhibitor- from potato have different R_f values. — The biochemical effects studied precede possible effects on cell division. — The results are discussed in relation to the localization of the site(s) of action.     

β-Methylamino-L-alanine-induced protein aggregation in vitro and protection by L-serine

The cyanobacterial non-protein amino acid α-amino-β-methylaminopropionic acid, more commonly known as BMAA, was first discovered in the seeds of the ancient gymnosperm Cycad circinalis (now Cycas micronesica Hill). BMAA was linked to the high incidence of neurological disorders on the island of Guam first reported in the 1950s. BMAA still attracts interest as a possible causative factor in amyotrophic lateral sclerosis (ALS) following the identification of ALS disease clusters associated with living in proximity to lakes with regular cyanobacterial blooms. Since its discovery, BMAA toxicity has been the subject of many in vivo and in vitro studies. A number of mechanisms of toxicity have been proposed including an agonist effect at glutamate receptors, competition with cysteine for transport system x_c^_ and other mechanisms capable of generating cellular oxidative stress. In addition, a wide range of studies have reported effects related to disturbances in proteostasis including endoplasmic reticulum stress and activation of the unfolded protein response. In the present studies we examine the effects of BMAA on the ubiquitin-proteasome system (UPS) and on chaperone-mediated autophagy (CMA) by measuring levels of ubiquitinated proteins and lamp2a protein levels in a differentiated neuronal cell line exposed to BMAA. The BMAA induced increases in oxidised proteins and the increase in CMA activity reported could be prevented by co-administration of L-serine but not by the two antioxidants examined. These data provide further evidence of a protective role for L-serine against the deleterious effects of BMAA.

     

Visfatin is regulated by rosiglitazone in type 2 diabetes mellitus and influenced by NFκB and JNK in human abdominal subcutaneous adipocytes

Visfatin has been proposed as an insulin-mimicking adipocytokine, predominantly secreted from adipose tissue and correlated with obesity. However, recent studies suggest visfatin may act as a proinflammatory cytokine. Our studies sought to determine the significance of this adipocytokine and its potential role in the pathogenesis of T2DM. Firstly, we examined the effects of diabetic status on circulating visfatin levels, and several other adipocytokines, demonstrating that diabetic status increased visfatin*, TNF-α*** and IL-6*** compared with non-diabetic subjects (*p<0.05, **p<0.01, ***p<0.001, respectively). We then assessed the effects of an insulin sensitizer, rosiglitazone (RSG), in treatment naïve T2DM subjects, on circulating visfatin levels. Our findings showed that visfatin was reduced post-RSG treatment [vs. pre-treatment (*p<0.05)] accompanied by a reduction in HOMA-IR**, thus implicating a role for insulin in visfatin regulation. Further studies addressed the intracellular mechanisms by which visfatin may be regulated, and may exert pro-inflammatory effects, in human abdominal subcutaneous (Abd Sc) adipocytes. Following insulin (Ins) and RSG treatment, our in vitro findings highlighted that insulin (100 nM), alone, upregulated visfatin protein expression whereas, in combination with RSG (10 nM), it reduced visfatin*, IKKβ** and p-JNK1/2*. Furthermore, inhibition of JNK protein exacted a significant reduction in visfatin expression (**p<0.01), whilst NF-κB blockade increased visfatin (*p<0.05), thus identifying JNK as the more influential factor in visfatin regulation. Additional in vitro analysis on adipokines regulating visfatin showed that only Abd Sc adipocytes treated with recombinant human (rh)IL-6 increased visfatin protein (*p<0.05), whilst rh visfatin treatment, itself, had no influence on TNF-α, IL-6 or resistin secretion from Sc adipocytes. These data highlight visfatin''s regulation by insulin and RSG, potentially acting through NF-κB and JNK mechanisms, with only rh IL-6 modestly affecting visfatin regulation. Taken together, these findings suggest that visfatin may represent a pro-inflammatory cytokine that is influenced by insulin/insulin sensitivity via the NF-κB and JNK pathways.     

Localization by immunofluorescence and by light- and electron-microscopic immunoperoxidase techniques of Tamm–Horsfall glycoprotein in adult hamster kidney

1. Tamm-Horsfall glycoprotein was isolated from hamster urine and antiserum against it was produced in rabbits. Immunoglobulin G was isolated from the antiserum. 2. Indirect methods of immunofluorescence staining were applied to kidney sections previously fixed by both perfusion and immersion methods. Tamm-Horsfall glycoprotein was identified associated with only the cells of the ascending limb of the loop of Henle and the distal convoluted tubule. Maculae densae were free of the glycoprotein. 3. Indirect immunoperoxidase procedures with light microscopy were applied to kidney sections. The results extended those found by immunofluorescence by showing that the glycoprotein is largely associated with the plasma membrane of the cells. Macula densa cells were shown to be free of the glycoprotein, although the luminal surface of the remaining cells in the transverse section of the nephron at that region was shown to contain it. 4. A variety of immuno-electron-microscopic techniques were applied to sections previously fixed in a number of ways. Providing periodate/lysine/paraformaldehyde was used as the fixative, the glycoprotein was often seen to be present not only on the luminal surface of the cells of the thick ascending limb of the loop of Henle and of the distal convoluted tubule, but also on the basal plasma membrane, including the infoldings. 5. It is generally accepted that the hyperosmolarity in the medulla of the kidney results from passage of Cl(-) ions with their accompanying Na(+) ions across the single cell layer of the lumen of the thick ascending limb of the loop of Henle, a region of the nephron with relatively high impermeability to water. We suggest that Tamm-Horsfall glycoprotein operates as a barrier to decrease the passage of water molecules by trapping the latter at the membrane of the cells. Our hypothesis requires the glycoprotein on the basal plasma membrane also.     

Regulation of chemokine and chemokine receptor expression by PPARγ in adipocytes and macrophages

Background

PPARγ plays a key role in adipocyte biology, and Rosiglitazone (Rosi), a thiazolidinedione (TZD)/PPARγ agonist, is a potent insulin-sensitizing agent. Recent evidences demonstrate that adipose tissue inflammation links obesity with insulin resistance and that the insulin-sensitizing effects of TZDs result, in part, from their anti-inflammatory properties. However the underlying mechanisms are unclear.

Methodology and Principal Findings

In this study, we establish a link between free fatty acids (FFAs) and PPARγ in the context of obesity-associated inflammation. We show that treatment of adipocytes with FFAs, in particular Arachidonic Acid (ARA), downregulates PPARγ protein and mRNA levels. Furthermore, we demonstrate that the downregulation of PPARγ by ARA requires the activation the of Endoplamsic Reticulum (ER) stress by the TLR4 pathway. Knockdown of adipocyte PPARγ resulted in upregulation of MCP1 gene expression and secretion, leading to enhanced macrophage chemotaxis. Rosi inhibited these effects. In a high fat feeding mouse model, we show that Rosi treatment decreases recruitment of proinflammatory macrophages to epididymal fat. This correlates with decreased chemokine and decreased chemokine receptor expression in adipocytes and macrophages, respectively.

Conclusions and Significance

In summary, we describe a novel link between FAs, the TLR4/ER stress pathway and PPARγ, and adipocyte-driven recruitment of macrophages. We thus both describe an additional potential mechanism for the anti-inflammatory and insulin-sensitizing actions of TZDs and an additional detrimental property associated with the activation of the TLR4 pathway by FA.     

Interleukin-23 is constitutively expressed in the human annulus in vivo and in vitro,and is up-regulated in vitro by TNF-α

ABSTRACT

Interleukin-23 (IL-23, IL-23p19) is a proinflammatory cytokine in the IL-12-related family. Although inflammatory cells in herniated discs have been shown to contain IL-23, little is known about the presence and role of IL-23 in human disc cells. We analyzed disc specimens for IL-23 localization using immunohistochemistry in control, herniated and non-herniated discs from which annulus fibrosus (annulus) cells were isolated and cultured to identify IL-23 gene expression and production. Microarray analysis was used to assess the expression of IL-23 in disc tissue and in cells exposed to two proinflammatory cytokines, IL-1ß and TNF-α. IL-23 was present in annulus cells at the protein level and its expression was up-regulated significantly in herniated compared to control disc tissue. Direct measurement of medium components confirmed production of IL-23 and its receptor, IL-23R, by annulus cells in vitro. Annulus cells in three-dimensional culture exposed to TNF-α, but not IL-1ß, resulted in significant up-regulation of IL-23 expression compared to control cells. Our findings are evidence for the constitutive presence of IL-23 in the human disc and that its expression in vitro is modified by exposure to TNF-α.     

Brothers in arms? Common and contrasting themes in pathogen perception by plant NB-LRR and animal NACHT-LRR proteins

Both plant and animal genomes encode proteins with nucleotide binding domains fused to leucine-rich repeat domains that are involved in responses to pathogens. While these domain structures are probably an example of convergent evolution, there are a number of similarities in the core mechanisms by which these proteins are regulated.     

Prolactin secretion in cattle as affected by haloperidol and α-Methyl-p-Tyrosine

Prolactin (PRL) secretion in response to i.v. injection of different doses of α-Methyl-p-Tyrosine (αMT) and haloperidol (HAL) was studied in one cow and three bulls. HAL was tested at doses of 0.033, 0.1, and 0.33 mg/kg body weight (BW), and αMT was tested at doses of 0.1, 1.0, 10, and 30 mg/kg BW. Blood was collected via an indwelling catheter into the external jugular vein, and plasma PRL was analysed by radioimmunoassay. Dose-related increases in plasma PRL concentrations were observed after administration of both αMT and HAL. Peak PRL concentrations after injection of HAL at a low, medium, and high dose were 22, 45, and 70 ng/ml, respectively. Peak PRL concentrations after injection of increasing doses of αMT were 3.0, 10, 40 and 70 ng/ml. HAL (0.1 mg/kg BW) and αMT (10 mg/kg BW) were administered intravenously to four heifers during summer and winter. Response to both drugs, as measured by changes in PRL secretion, was greater in summer than winter. Peak plasma PRL levels after HAL injection were 327 ± 58 ng/ml in June and 149 ± 27 ng/ml in January, whereas peak levels after αMT were 166±29 and 60±9 ng/ml, respectively. Basal PRL secretion was also greater in summer than winter. The results of this study demonstrate that PRL in peripheral plasma of cattle is increased in response to administration of antidopaminergic drugs and that this increase is greater during the summer than the winter.     

Chemiluminescence in neutrophils and Lettré cells induced by myxoviruses.

Luminol-mediated chemiluminescence in neutrophils is stimulated by Sendai virus and by influenza virus; Lettré cells also exhibit chemiluminescence (less than 10% of that of neutrophils), which is stimulated by Sendai virus and by influenza virus. Virally induced permeability changes are not responsible for chemiluminescence, since (i) extracellular Ca2+ inhibits permeability changes but stimulates chemiluminescence, and (ii) influenza virus, which induces permeability changes at pH 5.3 but not at pH 7.4, induces chemiluminescence at either pH. Other agents [zymosan, N-formyl-L-methionyl-L-leucyl-L-phenylalanine, 4-phorbol 12-myristate 13-acetate (phorbol ester), A23187] likewise induce chemiluminescence in the absence of permeability changes.     

α-Amylase production in batch and continuous cultures by Bacillus caldolyticus

Summary The concentration and productivity of -amylase increased remarkably, 15- and 11-fold respectively, in a continuous culture of Bacillus caldolyticus DSM 405 compared with batch culture, provided starch was used as the sugar source in a casitone medium. In the casitone medium with or without glucose hardly any improvement of enzyme production was observed in continuous culture. The addition of a small amount of starch to the glucose-casitone medium had a marked effect in stimulating amylase formation in continuous culture but no effect in batch culture.It was suggested that the higher production of -amylase in the continuous culture using starch as the inducer was partly related to the predominance of some conditional non-sporulating variants with a higher amylase forming activity and to derepression of the enzyme at a low glucose concentration.     

Mutation induction by γ and X-ray irradiation in tissue cultured lotus

Mutations of tissue cultured lotus were induced by treating plantlets with either acute -rays at doses of 0, 2, 3, 4, 5 or 6 krad or X-rays at doses of 0, 1, 2, 3, 4 or 5 krad. The 2-krad dose of either - or X-ray treatments resulted in a 50% survival rate. The use of - and X-rays to induce mutation in lotus resulted in 21 altered characteristics. Mutants from 1- and 2-krad of either or X-rays had long secondary roots and numerous adventitious roots. These mutants also exhibited good shoot growth and healthy rhizome development. Most plants treated with 3–5 krad of either - or X-rays exhibited abnormal characteristics including vitrification, chlorosis, deformed petioles and in addition had inhibited growth of lateral buds, secondary roots and rhizomes. All plants treated with 6 krad of -rays died within 4 weeks. Control plants had stoma lengths of 2.56 m and cytological analysis of the root tips confirmed the diploid chromosome number of 16. Two groups of aneuploid cells were achieved using irradiation at doses of 3 and 4 krad of either - or X-ray. Chromosome numbers were 2n=18 and 20 with associated stoma lengths of 3.43 and 4.34 m, respectively. Abnormal stomata (cyclocytic and deformity) were observed in plants treated with 4 krad of -ray.     

Sustained by Snakes? Seasonal Livelihood Strategies and Resource Conservation by Tonle Sap Fishers in Cambodia

This paper situates concerns for conservation of aquatic snakes and livelihood sustainability in Cambodia within a social–ecological systems context and thereby presents a challenge to conventional species-based conservation programmes. Fishing for low-value water snakes has become a widespread activity within the floating communities of Tonle Sap Lake in the last 20 years in response to new market opportunities, provided primarily by a crocodile farming industry. The scale and intensity of this new form of exploitation and reports of declines in catch per fisher have highlighted this activity as a conservation concern, yet its role within local livelihood strategies was previously unknown. We show that it is of increasing importance to the less well-off, and is linked to higher incomes within this group, where it potentially reduces their vulnerability to fluctuations and declines in fish catches. It is particularly important as a means to smooth seasonality of incomes in this flood pulse-driven social–ecological system. We argue that shifts between snake-hunting and fishing, as a market-driven adaptive livelihood strategy by the poor, may be more compatible with wider ecosystem conservation and development goals than alternatives such as increased fishing effort or converting floodplain habitats for seasonal agriculture.

Phytochemicals and protein–polyamine conjugates by transglutaminase as chemopreventive and chemotherapeutic tools in cancer

Identifying novel chemopreventive and chemotherapeutic agents and targeting them to patients at high risk of developing cancer or following curative treatment may go some way towards improving prognosis. This review examines current knowledge regarding the chemopreventive and chemotherapeutic potential of phytochemicals in cancer. Both in vitro and animal studies demonstrate that several phytochemicals increase the activity of intracellular transglutaminases, a family of enzymes involved in cell differentiation, through the covalent conjugation of polyamine to cellular protein, with promising anti-neoplastic properties. The substantial data available on certain plant secondary metabolites makes a strong case for integrating these safe and well-tolerated agents into clinical practice.     

Tricholoma matsutake– an assessment of in situ and in vitro infection by observing cleared and stained whole roots

 Structures present within field-collected Tricholoma matsutake/Pinus densiflora ectomycorrhizas and in vitro infections of P. densiflora roots by T. matsutake were observed by clearing, bleaching and staining whole lateral roots and mycorrhizas. Field mycorrhizas were characterized by a lack of root hairs, by the presence of a sparse discontinuous mantle composed of irregularly darkly staining hyphae over the root surface, primarily behind the root cap, and by the presence of Hartig net mycelium within the root cortex. Hartig net 'palmettis' were classified into three basic structures, each with distinctive morphologies. Aerial hyphae, bearing terminal swellings, were observed emanating from the mantle. Cleared, bleached and stained in vitro-infected roots possessed multibranched hyphal structures within the host root cortex and aerial hyphae bearing terminal swellings were observed arising from the mycelium colonizing the root surface. T. matsutake on P. densiflora conforms to the accepted morphology of an ectomycorrhiza. This staining protocol is particularly suited to the study of Matsutake mycorrhizal roots and gives rapid, clear, high-contrast images using standard light microscopy while conserving spatial relationships between hyphal elements and host tissues. Accepted: 26 August 1999     

Destabilization of microRNAs in human cells by 3′ deadenylation mediated by PARN and CUGBP1

MicroRNA-122 (miR-122), which is expressed at high levels in hepatocytes, is selectively stabilized by 3′-adenylation mediated by the cytoplasmic poly(A) polymerase GLD-2. Here, we report that poly(A)-specific ribonuclease (PARN) is responsible for the deadenylation and destabilization of miR-122. The 3′-oligoadenylated variant of miR-122 was detected in Huh7 cells when PARN was down-regulated. In addition, both the steady-state level and stability of miR-122 were increased in PARN knockdown cells. We also demonstrate that CUG-binding protein 1 (CUGBP1) specifically interacts with miR-122 and other UG-rich miRNAs, and promotes their destabilization. Overexpression of CUGBP1 or PARN in Huh7 cells reduced the steady-state levels of these miRNAs. Because CUGBP1 interacts directly with PARN, we hypothesized that it specifically recruits PARN to miR-122. In fact, CUGBP1 enhanced PARN-mediated deadenylation and degradation of miR-122 in a dose-dependent manner in vitro. These results indicate that the cellular level of miR-122 is determined by the balance between the opposing effects of GLD-2 and PARN/CUGBP1 on the metabolism of its 3′-terminus.     

Acquisition of an Agrobacterium Ri Plasmid and Pathogenicity by Other α-Proteobacteria in Cucumber and Tomato Crops Affected by Root Mat

Root mat of cucumbers and tomatoes has previously been shown to be caused by Agrobacterium radiobacter strains harboring a root-inducing Ri plasmid (pRi). Nine other pRi-harboring α-Proteobacteria have subsequently been isolated from root mat-infected crops. Fatty acid profiling and partial 16S rRNA sequence analysis identified three of these strains as being in the genus Ochrobactrum, five as being in the genus Rhizobium, and one as being in the genus Sinorhizobium. An in vitro pathogenicity test involving inoculation of cucumber cotyledons was developed. All pRi-harboring α-Proteobacteria induced typical root mat symptoms from the cotyledons. Average transformation rates for rhizogenic Ochrobactrum (46%) and Rhizobium (44%) strains were lower than those observed for rhizogenic A. radiobacter strains (64%). However, individual strains from these three genera all had transformation rates comparable to those observed from cotyledons inoculated with a rhizogenic Sinorhizobium strain (75%).