Production of nickel bio-ore from hyperaccumulator plant biomass: applications in phytomining

An important step in phytomining operations is the recovery of metal from harvested plant material. In this work, a laboratory-scale horizontal tube furnace was used to generate Ni-enriched bio-ore from the dried biomass of Ni hyperaccumulator plants. Prior to furnace treatment, hairy roots of Alyssum bertolonii were exposed to Ni in liquid medium to give biomass Ni concentrations of 1.9% to 7.7% dry weight; whole plants of Berkheya coddii were grown in Ni-containing soil to produce above-ground Ni levels of up to 0.49% dry weight. The concentration of Ca in the Ni-treated B. coddii biomass was about 15 times greater than in A. bertolonii. After furnace treatment at 1200 degrees C under air, Ni-bearing residues with crystalline morphology and containing up to 82% Ni were generated from A. bertolonii. The net weight loss in the furnace and the degree of concentration of Ni were significantly reduced when the furnace was purged with nitrogen, reflecting the importance of oxidative processes in Ni enrichment. Ni in the B. coddii biomass was concentrated by a factor of about 17 to yield a residue containing 8.6% Ni; this bio-ore Ni content is substantially higher than the 1% to 2% Ni typically found in mined ore. However, the B. coddii samples after furnace treatment also contained about 34% Ca, mainly in the form of hydroxyapatite Ca(5)(PO(4))(3)OH. Such high Ca levels may present significant challenges for further metallurgical processing. This work demonstrates the feasibility of furnace treatment for generating Ni-rich bio-ore from hyperaccumulator plants. The results also suggest that minimizing the uptake of Ca and/or reducing the Ca content of the biomass prior to furnace treatment would be a worthwhile strategy for improving the quality of Ni bio-ore produced in phytomining operations.     

Direct measurement of the association constant of HER2/neu antisense oligonucleotide to its target RNA sequence using a molecular beacon

A molecular beacon approach was developed to directly determine the association constant of RNA-DNA hybrid formation. The molecular beacon was composed of a 15-nt loop structure containing the antisense sequence that can hybridize with the AUG translational start site of the HER2/neu gene, which is overexpressed in a significant proportion of breast, ovarian, and lung tumors. The equilibrium association constant (Ka) of DNA binding to the RNA oligonucleotide was 6.4 +/- 0.14 x 10(7) M(-1) in the presence of 150 mM NaCl at 22 degrees C. The free energy change (AG) associated with RNA-DNA hybrid formation was -10.7 kcal/mole. The melting temperature (Tm) of RNA-DNA hybrid was 64.4 degrees C +/- 1 degree C in the presence of 150 mM NaCl. The RNA-DNA hybrid was more stable than the corresponding DNA-DNA duplex in 150 mM NaCl, as judged by both Ka and Tm data. We also determined the Ka, deltaG, and Tm values of RNA-DNA and DNA-DNA duplex formation in the presence of three monovalent cations, Li+, K+, and Cs+. The feasibility of this method was also investigated using a phosphorothioate molecular beacon. The information generated through this new approach for thermodynamic measurements might be useful for the design of oligonucleotides for antisense therapeutics.     

Ectrodactyly/split hand feet malformation

Split-hand/split-foot malformation is a rare limb malformation with median clefts of the hands and feet and aplasia/hypoplasia of the phalanges, metacarpals and metatarsals. When present as an isolated anomaly, it is usually inherited as an autosomal dominant form. We report a case of autosomal recessive inheritance and discuss the antenatal diagnosis, genetic counseling and treatment for the malformation.     

DNA nanoparticles and development of DNA delivery vehicles for gene therapy

DNA transport through the cell membrane is an essential requirement for gene therapy, which utilizes oligonucleotides and plasmid DNA. However, membrane transport of DNA is an inefficient process, and the mechanism(s) by which this process occurs is not clear. Although viral vectors are effective in gene therapy, the immune response elicited by viral proteins poses a major problem. Therefore, several laboratories are involved in the development of nonviral DNA delivery vehicles. These vehicles include polyamines, polycationic lipids, and neutral polymers, capable of condensing DNA to nanoparticles with radii of 20-100 nm. Although the structural and energetic forces involved in DNA condensation have been studied by physical biochemists for the past 25 years, this area has experienced a resurgence of interest in recent years because of the influx of biotechnologists involved in developing gene therapy protocols to combat a variety of human diseases. Despite an intense effort to study the mechanism(s) of DNA condensation using a variety of microscopic, light scattering, fluorescence, and calorimetric techniques, the precise details of the energetics of DNA nanoparticle formation and their packing assembly are not known at present. Future studies aimed at defining the mechanism(s) of DNA compaction and structural features of DNA nanoparticles might aid in the development of novel gene delivery vehicles.     

Podophyllotoxin and nocodazole counter the effect of IKP104 on tubulin decay

Tubulin, the subunit protein of microtubules, undergoes a time-dependent loss of functional properties known as decay. We have previously shown that the drug 2-(4-fluorophenyl)-1-(2-chloro-3,5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone (IKP104) accelerates decay, but that in the presence of colchicine, IKP104 becomes a stabilizer of tubulin. To see if this is due to conformational effects specific to colchicine or simply to occupancy at the colchicine site, we examined the effects of nocodazole and podophyllotoxin, two well-known competitive inhibitors of colchicine for binding to tubulin, on IKP104’s acceleration of decay. We found that podophyllotoxin abolished IKP104’s accelerating effect and, like colchicine, turned it into a stabilizer of tubulin. Nocodazole’s effects were similar to those of podophyllotoxin and colchicine, in that it abolished IKP104-induced enhancement of decay; however, in the presence of nocodazole, IKP104 caused little or no stabilization of tubulin. Since colchicine, nocodazole, and podophyllotoxin have very different interactions with tubulin, but all inhibit the IKP104-induced enhancement of decay, our findings suggest that this inhibition arises from occupancy of the colchicine site rather than from a direct conformational effect of these two drugs.     

Gastropathy and defense mechanisms in common bile duct ligated portal hypertensive rats

Portal hypertensive gastropathy is associated with a broad spectrum of gastric mucosal damage inspite of decreased gastric acid secretion, suggestive of compromised endogenous protective mechanisms. To determine the mechanisms of damage in portal hypertensive gastropathy we measured lipid peroxidation, glutathione, antioxidant and lysosomal enzymes in gastric mucosal homogenates from male Wistar rats with elevated intrasplenic pulp pressure, eighteen days after common bile duct ligation. Thiobarbituric acid-reactive substances and lysosomal enzymes (-glucuronidase and acid phosphatase) were increased in the common bile duct ligated group as compared to the sham-operated group. The levels of antioxidant defense enzymes, superoxide dismutase, glutathione peroxidase, catalase and glutathione were decreased as compared to the sham-operated controls. Pre-operative vitamin E administration decreased mucosal lipid peroxidation increased the levels of antioxidant defense enzymes and lowered the lysosomal enzymes. The plasma vitamin E levels in this group were lower when compared to animals receiving it post-operatively. In conclusion, free radical and lysosomal enzyme mediated damage may play a role in portal hypertensive gastropathy.     

Sequestration of pRb by cyclin D3 causes intranuclear reorganization of lamin A/C during muscle cell differentiation

The A-type lamins that localize in nuclear domains termed lamin speckles are reorganized and antigenically masked specifically during myoblast differentiation. This rearrangement was observed to be linked to the myogenic program as lamin speckles, stained with monoclonal antibody (mAb) LA-2H10, were reorganized in MyoD-transfected fibroblasts induced to transdifferentiate to muscle cells. In C2C12 myoblasts, speckles were reorganized early during differentiation in cyclin D3-expressing cells. Ectopic cyclin D3 induced lamin reorganization in C2C12 myoblasts but not in other cell types. Experiments with adenovirus E1A protein that can bind to and segregate the retinoblastoma protein (pRb) indicated that pRb was essential for the cyclin D3-mediated reorganization of lamin speckles. Cyclin D3-expressing myoblasts displayed site-specific reduction of pRb phosphorylation. Furthermore, disruption of lamin structures by overexpression of lamins inhibited expression of the muscle regulatory factor myogenin. Our results suggest that the reorganization of internal lamins in muscle cells is mediated by key regulators of the muscle differentiation program.     

The role of molecular markers in predicting response to therapy in patients with colorectal cancer

Advances in systemic therapy for colorectal cancer have dramatically improved prognosis. While disease stage has traditionally been the main determinant of disease course, several molecular characteristics of tumor specimens have recently been shown to have prognostic significance. Although to date no molecular characteristics have emerged as consistent predictors of response to therapy, retrospective studies have investigated the role of a variety of biomarkers, including microsatellite instability, loss of heterozygosity of 18q, type II transforming growth factor beta receptor, thymidylate synthase, epidermal growth factor receptor, and Kirsten-ras (KRAS). This paper reviews the current literature, ongoing prospective studies evaluating the role of these markers, and novel techniques such as gene profiling, which may help to uncover the more complex molecular interactions that will predict response to chemotherapy in patients with colorectal cancer.     

Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

Senescence of alveolar type 2 (ATII) cells, progenitors of the alveolar epithelium, is implicated in the pathogeneses of idiopathic pulmonary fibrosis (IPF), an aging‐related progressive fatal lung disorder with unknown etiology. The mechanism underlying ATII cell senescence in fibrotic lung diseases, however, remains poorly understood. In this study, we report that ATII cells in IPF lungs express higher levels of serpine 1, also known as plasminogen activator inhibitor 1 (PAI‐1), and cell senescence markers p21 and p16, compared to ATII cells in control lungs. Silencing PAI‐1 or inhibition of PAI‐1 activity in cultured rat ATII (L2) cells leads to decreases in p53 serine 18 phosphorylation (p53^S18P), p53 and p21 protein expressions; an increase in retinoblastoma protein phosphorylation (ppRb); and a reduction in the sensitivity to bleomycin‐ and doxorubicin‐induced senescence. Silencing p53, on the other hand, abrogates PAI‐1 protein‐stimulated p21 expression and cell senescence. In vivo studies, using ATII cell‐specific PAI‐1 conditional knockout mouse model generated recently in this laboratory, further support the role of PAI‐1 in the activation of p53‐p21‐Rb cell cycle repression pathway, ATII cell senescence, and lung fibrosis induced by bleomycin. This study reveals a novel function of PAI‐1 in regulation of cell cycle and suggests that elevation of PAI‐1 contributes importantly to ATII cell senescence in fibrotic lung diseases.     

Acetylcholinesterase inhibitors neostigmine and physostigmine inhibit induction of alpha-amylase activity during seed germination in barley, Hordeum vulgare var. Jyoti

Acetylcholine (ACh) is an important neurotransmitter whose non-neuronal biological roles are being widely accepted. ACh and components of its metabolism are present in plants. ACh and some inhibitors of acetylcholinesterase (AChE) share structural similarity (quaternary ammonium group) with some inhibitors of biosynthesis of a plant hormone, gibberellic acid (GA); e.g., 2-Isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine carboxylate methyl chloride (AMO-1618) inhibits GA biosynthesis as well as AChE. The present study explores the possibility that ACh and antiAChE may inhibit GA biosynthesis. Seeds of barley var. Jyoti were germinated in the presence of ACh, its breakdown products - choline and acetate, and two antiAChE - neostigmine and physostigmine (all 10(-5) M). Alpha amylase activity in germinating seeds was measured as a reliable indicator of the level of GA biosynthesis. Alpha amylase activity in barley seeds was significantly reduced after 72 h of treatment with antiChE but not by ACh or its breakdown products. Since germinating barley seeds contain AChE, much of the ACh may have been broken down before its uptake. Quaternary ammonium antiChE neostigmine was more effective (50% inhibition at 10(-5) M) as compared to tertiary ammonium physostigmine (15% inhibition at 10(-5) M). ACh, choline, acetate, neostigmine and physostigmine (all 10(-5) M) did not affect formation of starch-iodine complex or activity of alpha-amylase per se. Our results indicate that quaternary ammonium inhibitors of AChE may inhibit GA biosynthesis.