Effect of DMT1 knockdown on iron,cadmium, and lead uptake in Caco-2 cells

DMT1 (divalent metal transporter 1) is ahydrogen-coupled divalent metal transporter with a substrate preferencefor iron, although the protein when expressed in frog oocytestransports a broad range of metals, including the toxic metals cadmiumand lead. Wild-type Caco-2 cells displayed saturable transport of leadand iron that was stimulated by acid. Cadmium and manganese inhibitedtransport of iron, but zinc and lead did not. The involvement of DMT1in the transport of toxic metals was examined by establishing clonalDMT1 knockdown and control Caco-2 cell lines. Knockdown cell linesdisplayed much lower levels of DMT1 mRNA and a smaller V_max for iron uptake compared with control celllines. One clone was further characterized and found to display an~50% reduction in uptake of iron across a pH range from 5.5 to 7.4. Uptake for cadmium also decreased 50% across the same pH range, butuptake for lead did not. These results show that DMT1 is important in iron and cadmium transport in Caco-2 cells but that lead enters thesecells through an independent hydrogen-driven mechanism.

     

Protein structure plays a critical role in peanut allergen stability and may determine immunodominant IgE-binding epitopes

Hypersensitivity to peanuts is a reaction mediated by IgE Abs in response to several peanut protein allergens. Among these allergenic proteins, Ara h 2 is one of the most commonly recognized allergens. Ara h 2 is a 17-kDa protein that has eight cysteine residues that could form up to four disulfide bonds. Circular dichroism studies showed substantial changes in the secondary and tertiary structures of the reduced Ara h 2 as compared with the native protein. Upon treatment with trypsin, chymotrypsin, or pepsin, a number of relatively large fragments are produced that are resistant to further enzymatic digestion. These resistant Ara h 2 peptide fragments contain intact IgE-binding epitopes and several potential enzyme cut sites that are protected from the enzymes by the compact structure of the protein. The enzyme-treated allergen remains essentially intact despite the action of proteases until the fragments are dissociated when the disulfide linkages are reduced. Amino acid sequence analysis of the resistant protein fragments indicates that they contain most of the immunodominant IgE-binding epitopes. These results provide a link between allergen structure and the immunodominant IgE-binding epitopes within a population of food-allergic individuals.     

Further phenotypic delineation of subtelomeric (terminal) 4q deletion with emphasis on intracranial and reproductive anatomy

Hypoplastic left heart syndrome(HLHS) refers to the abnormal development of the left-sided cardiac structures, resulting in obstruction to blood flow from the left ventricular outflow tract. In addition, the syndrome includes underdevelopment of the left ventricle, aorta, and aortic arch, as well as mitral atresia or stenosis. HLHS has been reported to occur in approximately 0.016 to 0.036% of all live births. Newborn infants with the condition generally are born at full term and initially appear healthy. As the arterial duct closes, the systemic perfusion becomes decreased, resulting in hypoxemia, acidosis, and shock. Usually, no heart murmur, or a non-specific heart murmur, may be detected. The second heart sound is loud and single because of aortic atresia. Often the liver is enlarged secondary to congestive heart failure. The embryologic cause of the disease, as in the case of most congenital cardiac defects, is not fully known. The most useful diagnostic modality is the echocardiogram. The syndrome can be diagnosed by fetal echocardiography between 18 and 22 weeks of gestation. Differential diagnosis includes other left-sided obstructive lesions where the systemic circulation is dependent on ductal flow (critical aortic stenosis, coarctation of the aorta, interrupted aortic arch). Children with the syndrome require surgery as neonates, as they have duct-dependent systemic circulation. Currently, there are two major modalities, primary cardiac transplantation or a series of staged functionally univentricular palliations. The treatment chosen is dependent on the preference of the institution, its experience, and also preference. Although survival following initial surgical intervention has improved significantly over the last 20 years, significant mortality and morbidity are present for both surgical strategies. As a result pediatric cardiologists continue to be challenged by discussions with families regarding initial decision relative to treatment, and long-term prognosis as information on long-term survival and quality of life for those born with the syndrome is limited.     

The Transfection of BDNF to Dopamine Neurons Potentiates the Effect of Dopamine D3 Receptor Agonist Recovering the Striatal Innervation,Dendritic Spines and Motor Behavior in an Aged Rat Model of Parkinson’s Disease

The progressive degeneration of the dopamine neurons of the pars compacta of substantia nigra and the consequent loss of the dopamine innervation of the striatum leads to the impairment of motor behavior in Parkinson’s disease. Accordingly, an efficient therapy of the disease should protect and regenerate the dopamine neurons of the substantia nigra and the dopamine innervation of the striatum. Nigral neurons express Brain Derived Neurotropic Factor (BDNF) and dopamine D3 receptors, both of which protect the dopamine neurons. The chronic activation of dopamine D3 receptors by their agonists, in addition, restores, in part, the dopamine innervation of the striatum. Here we explored whether the over-expression of BDNF by dopamine neurons potentiates the effect of the activation of D3 receptors restoring nigrostriatal innervation. Twelve-month old Wistar rats were unilaterally injected with 6-hydroxydopamine into the striatum. Five months later, rats were treated with the D3 agonist 7-hydroxy-N,N-di-n-propy1-2-aminotetralin (7-OH-DPAT) administered i.p. during 4½ months via osmotic pumps and the BDNF gene transfection into nigral cells using the neurotensin-polyplex nanovector (a non-viral transfection) that selectively transfect the dopamine neurons via the high-affinity neurotensin receptor expressed by these neurons. Two months after the withdrawal of 7-OH-DPAT when rats were aged (24 months old), immunohistochemistry assays were made. The over-expression of BDNF in rats receiving the D3 agonist normalized gait and motor coordination; in addition, it eliminated the muscle rigidity produced by the loss of dopamine. The recovery of motor behavior was associated with the recovery of the nigral neurons, the dopamine innervation of the striatum and of the number of dendritic spines of the striatal neurons. Thus, the over-expression of BDNF in dopamine neurons associated with the chronic activation of the D3 receptors appears to be a promising strategy for restoring dopamine neurons in Parkinson’s disease.     

In vitro zygotic embryo culture of wild Musa acuminata ssp.malaccensis and factors affecting germination and seedling growth

In vitro zygotic embryo culture of wild banana significantly increased the germination compared to greenhouse grown seeds. Embryo orientation and BAP concentration significantly affected germination rate. These factors together with gelling agent, dark and light conditions and coconut water, also showed variable effects on the number of roots per plant, root length, shoot length, number of days to root emergence and number of days to shoot emergence.     

Construction and Characterization of a Magnaporthe grisea Bacterial Artificial Chromosome Library

Diaz-Perez, S. V., Crouch, V. W., and Orbach, M. J. 1996. Construction and characterization of a Magnaporthe grisea bacterial artificial chromosome library. Fungal Genet. Biol. 20, 280-288. A bacterial artificial chromosome (BAC) library of Magnaporthe grisea containing 4128 clones with an average insert size of 66-kb has been constructed. This library represents seven genome equivalents of M. grisea and has been demonstrated to be representative of the genome by screening for the presence of several single-copy genes and DNA markers. The utility of the library for use in map-based cloning projects was shown by the spanning of a nine-cosmid, 207-kb DNA contig with only 3 BAC clones. In addition, using a lys1-3 auxotroph, we have shown that BAC clones at least 113 kb can be transformed into M. grisea to screen for complementation of mutations. Thus, BACs isolated in chromosome walks can be rapidly screened for the presence of the sought after gene. The ease of construction of BAC libraries and of isolation and manipulation of BAC clones makes the BAC system an ideal one for physical analyses of fungal genomes.     

Biomarker Development for the Clinical Activity of the mTOR Inhibitor Everolimus (RAD001): Processes,Limitations, and Further Proposals

The mTOR inhibitor everolimus (RAD001, Afinitor) is an orally active anticancer agent. Everolimus demonstrates growth-inhibitory activity against a broad range of tumor cell histotypes in vitro and has the capacity to retard tumor growth in preclinical tumor models in vivo through mechanisms directed against both the tumor cell and the solid tumor stroma components. These properties have rendered it to be a clinically active drug, with subsequent registration in renal cell carcinoma (Motzer et al. [2008]. Lancet 372, 449–456) as well as showing strong potential as a combination partner (André F et al. [2008]. J Clin Oncol 26. Abstract 1003). Although everolimus has a high specificity for its molecular target, the ubiquitous nature of mTOR and the multifactorial influence that mTOR signaling has on cell physiology have made studies difficult on the identification and validation of a biomarker set to predict and monitor drug sensitivity for clinical use. In this review, a summary of the preclinical and clinical data relevant to biomarker development for everolimus is presented, and the advantages and problems of current biomarkers are reviewed. In addition, alternative approaches to biomarker development are proposed on the basis of examples of a combination of markers and functional noninvasive imaging. In particular, we show how basal levels of pAKT and pS6 together could, in principle, be used to stratify patients for likely response to an mTOR inhibitor.     

Comparison of the anti-scorbutic activity of L-ascorbic acid and Ester C in the non-ascorbate synthesizing Osteogenic Disorder Shionogi (ODS) rat

The Osteogenic Disorder Shionogi (ODS) rat, Clea Inc., Tokyo, Japan lacks the ability to synthesize L-ascorbic acid (AA). As with man, monkey and the guinea pig, this rat lacks L-gulonolactone oxidase necessary for the synthesis of AA from glucose. This study shows this animal to be an alternative to the guinea pig in AA studies. The anti-scorbutic potency of Ester C (EC), a calcium ascorbate and calcium threonate mixture, was compared with an AA dose of equal ascorbate activity equivalents (AAE) for anti-scorbutic activity in the ODS rat. The minimal anti-scorbutic dose of EC was determined to be 0.44 mg/kg/day (AAE), while an AA dose of 0.51 mg/kg/day (AAE) was not anti-scorbutic in a 24 day study. At 24 days EC rats gained 125% of initial body weight (BW) and the AA rats only 45% BW. Scorbutic signs at 24 days were scored on a 0 (min) to 3 (max) scale. The EC/AA ratio scores were: hemorrhage 0/1.4, behavior change 0/2.0, piloerection 0/2.2, mobility 0.4/2.2, dysbasia 0.6/2.8 and ataxia 0.4/1.0. Pearson's correlation coefficient for BW versus AAE was r = .34 for the AA group and r = .90 for the EC group. The morbidity index for EC was 0/5 and for the AA group 2/5. The AAE dose of AA which was 16% higher/day than the EC AAE dose was not anti-scorbutic, while the EC dose was anti-scorbutic. EC rats had 3.5X greater weight gain, a sensitive indicator of scurvy, than the AA rats. EC rats had 3-4 times less, if any, scorbutic signs than AA rats. The results clearly show that, based on ascorbate activity equivalents, EC has more available ascorbate activity/potency than AA. The mechanism of this increased potency is believed to be due to the facilitated transport of AAE into the cell by the threonate (a normal in vivo metabolite of AA) present in the EC product. In addition, previous studies have shown EC (AAE) to be higher in plasma and excreted less rapidly than the AAE derived from AA administered orally.     

Uric acid is a strong independent predictor of renal dysfunction in patients with rheumatoid arthritis

Introduction  

Recent evidence suggests that uric acid (UA), regardless of crystal deposition, may play a direct pathogenic role in renal disease. We have shown that UA is an independent predictor of hypertension and cardiovascular disease (CVD), and that CVD risk factors associate with renal dysfunction, in patients with rheumatoid arthritis (RA). In this study we investigated whether UA associates with renal dysfunction in patients with RA and whether such an association is independent or mediated through other comorbidities or risk factors for renal impairment.