Asbestos,chromosomal deletions,and tumor suppressor gene alterations in human malignant mesothelioma

Exposure to the carcinogen asbestos is considered to be a major factor contributing to the development of most malignant mesotheliomas (MMs). We highlight the role of asbestos in MM and summarize cytogenetic and molecular genetic findings in this malignancy. The accumulation of numerous clonal chromosomal deletions in most MMs suggests a multistep process of tumorigenesis, characterized by the loss and/or inactivation of multiple tumor suppressor genes (TSGs). Cytogenetic and loss of heterozygosity (LOH) analyses of MMs have demonstrated frequent deletions of specific sites within chromosome arms 1p, 3p, 6q, 9p, 13q, 15q, and 22q. Furthermore, TSGs within two of these regions, i.e., p16/CDKN2A-p14^ARF at 9p21 and NF2 at 22q12, are frequently altered in MMs. Homozygous deletion appears to be the major mechanism affecting p16/CDKN2A-p14^ARF, whereas inactivating mutations coupled with allelic loss occur at the NF2 locus. Finally, recent studies have demonstrated the presence and expression of simian virus 40 (SV40) in many MMs. SV40 large T antigen has been shown to inactivate the TSG products Rb and p53, suggesting the possibility that asbestos and SV40 could act as cocarcinogens in MM. The frequent occurrence of homozygous deletions of p16/CDKN2A-p14^ARF and the ability of SV40 Tag to bind TSG products suggest that perturbations of both Rb- and p53-dependent growth-regulatory pathways are critically involved in the pathogenesis of MM. J. Cell. Physiol. 180:150–157, 1999. © 1999 Wiley-Liss, Inc.     

Isolation and characterization of the dehydration stress-inducible GhRDL1 promoter from the cultivated upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>)

Cotton fiber is the basic raw material used in the textile industry. The fiber yield is severely affected by a number of biotic and abiotic factors, such as insects, viruses, drought and salinity. Drought is a major factor that negatively impacts the yields and quality of cotton fiber. Promoters that respond to stress conditions and up-regulate transgenes are of great significance in crop improvement using genetic engineering approach. Although dehydration-responsive gene promoters, such as RD22 and RD29 from Arabidopsis, have been characterized, not much information is available regarding stress-responsive promoters from Gossypium hirsutum, which accounts for approximately 90 % of cultivated cotton. In this study, we isolated and characterized the promoter of a dehydration-responsive gene (GhRDL1) from G. hirsutum using Agrobacterium-mediated transformation in tobacco and cotton. Transgenic tobacco plants expressing uidA under the GhRDL1 promoter showed GUS activity in the trichomes. Also, GUS expression was observed to some extent in leaf, stem and floral tissues. Similar results were observed when GhRDL1 promoter was tested in transgenic cotton. Most importantly, our study showed that the GhRDL1 promoter is up-regulated in the presence of polyethylene glycol that creates water stress under invitro conditions. Thus, the GhRDL1 promoter may find its usefulness in the development of stress-tolerant cotton and other crop species in the near future.     

Using homology modeling to interrogate binding affinity in neutralization of ricin toxin by a family of single domain antibodies

In this report we investigated, within a group of closely related single domain camelid antibodies (V_HHs), the relationship between binding affinity and neutralizing activity as it pertains to ricin, a fast‐acting toxin and biothreat agent. The V1C7‐like V_HHs (V1C7, V2B9, V2E8, and V5C1) are similar in amino acid sequence, but differ in their binding affinities and toxin‐neutralizing activities. Using the X‐ray crystal structure of V1C7 in complex with ricin's enzymatic subunit (RTA) as a template, Rosetta‐based homology modeling coupled with energetic decomposition led us to predict that a single pairwise interaction between Arg29 on V5C1 and Glu67 on RTA was responsible for the difference in ricin toxin binding affinity between V1C7, a weak neutralizer, and V5C1, a moderate neutralizer. This prediction was borne out experimentally: substitution of Arg for Gly at position 29 enhanced V1C7's binding affinity for ricin, whereas the reverse (ie, Gly for Arg at position 29) diminished V5C1's binding affinity by >10 fold. As expected, the V5C1_R29G mutant was largely devoid of toxin‐neutralizing activity (TNA). However, the TNA of the V1C7_G29R mutant was not correspondingly improved, indicating that in the V1C7 family binding affinity alone does not account for differences in antibody function. V1C7 and V5C1, as well as their respective point mutants, recognized indistinguishable epitopes on RTA, at least at the level of sensitivity afforded by hydrogen‐deuterium mass spectrometry. The results of this study have implications for engineering therapeutic antibodies because they demonstrate that even subtle differences in epitope specificity can account for important differences in antibody function.     

Radioprotective effect of <Emphasis Type="SmallCaps">dl</Emphasis>-α-lipoic acid on mice skin fibroblasts

During the course of cancer radiation treatment, normal skin invariably suffers from the cytotoxic effects of γ-radiation and reactive oxygen species (ROS), which are generated from the interaction between radiation and the water molecules in cells. The present study was designed to investigate the radioprotective role of α-lipoic acid (LA), an antioxidant on murine skin fibroblasts exposed to a single dose of 2, 4, 6, or 8Gy γ-radiation. Irradiation of fibroblasts significantly increased ROS, nitric oxide, and lipid peroxidation (P < 0.001); all of these factors substantially decreased with 100 μM LA treatment. Hydroxyl radical (OH^⋅) production from 8Gy irradiated fibroblasts was measured directly by electron spin resonance using spin-trapping techniques. LA was found to inhibit OH^⋅ production at 100-μM concentrations. Dose-dependent depletion of antioxidants, such as catalase and glutathione reductase, was observed in irradiated fibroblasts (P < 0.001), along with increased superoxide dismutase (P < 0.001). LA treatment restored antioxidant levels. Concentration of the pro-inflammatory cytokine IL-1β was significantly reduced in irradiated fibroblasts when treated with LA. MTT and lactate dehydrogenase assays demonstrated that LA treatment reduced cell injury and protected cells against irradiation-induced cytotoxicity. Thus, we conclude that results are encouraging and need further experiments to demonstrate a possible benefit in cancer patients and the reduction of harmful effects of radiation therapy.     

Structural prediction and analysis of VIH-related peptides from selected crustacean species

The tentative elucidation of the 3D-structure of vitellogenesis inhibiting hormone (VIH) peptides is conversely underprivileged by difficulties in gaining enough peptide or protein, diffracting crystals, and numerous extra technical aspects. As a result, no structural information is available for VIH peptide sequences registered in the Genbank. In this situation, it is not surprising that predictive methods have achieved great interest. Here, in this study the molt-inhibiting hormone (MIH) of the kuruma prawn (Marsupenaeus japonicus) is used, to predict the structure of four VIHrelated peptides in the crustacean species. The high similarity of the 3D-structures and the calculated physiochemical characteristics of these peptides suggest a common fold for the entire family.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and micropropagation of <Emphasis Type="Italic">Givotia rottleriformis</Emphasis> Griff.

The propagation of Givotia rottleriformis Griff. is difficult as a result of long seed dormancy associated with poor seed germination. The present study was undertaken to develop a protocol to overcome seed dormancy by culture of zygotic embryo axes and then develop an efficient method for micropropagation of Givotia. Best germination frequency (78.3%) was achieved from mature zygotic embryo axes isolated from acid-scarified fresh seeds when cultured on Murashige and Skoog (MS) medium (half-strength major salts) with 28.9 μM gibberellic acid (GA₃). Efficient plant conversion was achieved by transfer of 10-d-old germinated embryos to MS medium (half-strength major salts) supplemented with 1.2 μM kinetin (KN) and 0.5 μM indole-3-butyric acid (IBA). However, acid scarification of 1-yr-old seeds decreased the germination frequency of zygotic embryo axes in comparison to those obtained from non-acid-scarified seeds which germinated (96.2%) and converted into plants (80.3%) on MS basal (half-strength major salts) medium. Multiple shoot bud induction was achieved by culture of shoot tips derived from in vitro germinated seedlings on MS medium with 0.5 μM thidiazuron for 4 wk, and the shoots elongated after transfer to a secondary medium with 1.2 μM KN. A maximum number of 7.8 shoots per explant with an average shoot length of 3.2 cm was achieved after two subcultures on this medium. The in vitro regenerated shoots rooted (41.5%) on half-strength MS medium with 0.5 μM IBA. The in vitro generated seedlings and micropropagated plants were established in soil with a survival frequency of 70% and 60%, respectively.     

Endoplasmic reticulum stress response in an INS-1 pancreatic β-cell line with inducible expression of a folding-deficient proinsulin

Background  

Cells respond to endoplasmic reticulum stress (ER) stress by activating the unfolded protein response. To study the ER stress response in pancreatic β-cells we developed a model system that allows for pathophysiological ER stress based on the Akita mouse. This mouse strain expresses a mutant insulin 2 gene (C96Y), which prevents normal proinsulin folding causing ER stress and eventual β-cell apoptosis. A double-stable pancreatic β-cell line (pTet-ON INS-1) with inducible expression of insulin 2 (C96Y) fused to EGFP was generated to study the ER stress response.