Validation of Suitable Carrier Molecules and Target Genes for Antisense Therapy Using Peptide-Coupled Peptide Nucleic Acids (PNAs) in Streptococci.

Antisense peptide nucleic acids (PNAs) targeting genes involved in metabolism or virulence are a possible means to treat infections or to investigate pathogenic bacteria. Potential targets include essential genes, virulence factor genes, or antibiotic resistance genes. For efficient cellular uptake, PNAs can be coupled to cell-penetrating peptides (CPPs). CPPs are peptides that serve as molecular transporters and are characterized by a comparably low cytotoxicity. So far, there is only limited information about CPPs that mediate PNA uptake by Gram-positive bacteria. Here, we describe two methods to identify suitable CPP-antisense PNA conjugates, novel carrier molecules, and efficient target genes for streptococcal species and to evaluate their antimicrobial efficiency.

Cashew Tree Pollen: An Unknown Source of IgE-Reactive Molecules.

Pollinosis is sub-diagnosed and rarely studied in tropical countries. Cashew tree pollen has been reported as an allergen source although the knowledge of its immunoglobulin E (IgE)-reactive molecules is lacking. Therefore, this work aimed to identify IgE-reactive molecules and provide a proteomic profile of this pollen. From the 830 proteins identified by shotgun analysis, 163 were annotated to gene ontology, and a list of 39 proteins filtered for high confidence was submitted to the Allfam database where nine were assigned to allergenic families. Thus, 12 patients from the northeast of Brazil with persistent allergic rhinitis and aggravation of symptoms during cashew flowering season were selected. Using a 2D-based approach, we identified 20 IgE-reactive proteins, four already recognized as allergens, including a homolog of the birch isoflavone-reductase (Bet v 6). IgE-reactivity against the extract in native form was confirmed for five patients in ELISA, with three being positive for Bet v 6. Herein, we present a group of patients with rhinitis exposed to cashew tree pollen with the first description of IgE-binding proteins and a proteomic profile of the whole pollen. Cashew tree pollen is considered an important trigger of rhinitis symptoms in clinical practice in the northeast of Brazil, and the elucidation of its allergenic molecules can improve the diagnostics and treatment for allergic patients.

Melatonin inhibits endoplasmic reticulum stress-associated TXNIP/NLRP3 inflammasome activation in lipopolysaccharide-induced endometritis in mice.

Endometritis, an inflammatory response of the uterus tissue, is characterized by the production of inflammatory cytokines and migration of neutrophil (PMN) into the uterus tissue. Melatonin has been demonstrated to have anti-inflammatory and antioxidant effects. The purpose of this study was to investigate the protective effects of melatonin on lipopolysaccharide (LPS)-induced endometritis in mice. An endometritis model was induced by LPS and melatonin was given 1 h before LPS treatment. The results showed that melatonin inhibited LPS-induced pathologic changes, Myeloperoxidase (MPO) activity, and levels of interleukin-1 beta (IL-1ß). Melatonin also inhibited LPS-induced thioredoxin-interacting protein (TXNIP)/NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome and nuclear factor kappa B (NF-κB) activation, reactive oxygen species (ROS) production, and endoplasmic reticulum (ER) stress. Furthermore, melatonin was found to increase AMPK activity. In conclusion, our results demonstrated that melatonin inhibited ER stress-associated TXNIP/NLRP3 inflammasome activation with a regulation of adenosine monophosphate activated protein kinase (AMPK) in LPS-induced endometritis. Melatonin may serve as a promising nutritional supplement for the treatment of endometritis.

Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules.

The chicken egg resists most environmental microbes suggesting that it potentially contains efficient antimicrobial molecules. Considering that some heparin-binding proteins in mammals are antibacterial, we investigated the presence and the antimicrobial activity of heparin-binding proteins from chicken egg white. Mass spectrometry analysis of the proteins recovered after heparin-affinity chromatography, revealed 20 proteins, including known antimicrobial proteins (avidin, lysozyme, TENP, ovalbumin-related protein X and avian bêta-defensin 11). The antibacterial activity of three new egg candidates (vitelline membrane outer layer protein 1, beta-microseminoprotein-like (LOC101750704) and pleiotrophin) was demonstrated against Listeria monocytogenes and/or Salmonella enterica Enteritidis. We showed that all these molecules share the property to inhibit bacterial growth through their heparin-binding domains. However, vitelline membrane outer layer 1 has additional specific structural features that can contribute to its antimicrobial potential. Moreover, we identified potential supplementary effectors of innate immunity including mucin 5B, E-selectin ligand 1, whey acidic protein 3, peptidyl prolyl isomerase B and retinoic acid receptor responder protein 2. These data support the concept of using heparin affinity combined to mass spectrometry to obtain an overview of the various effectors of innate immunity composing biological milieus, and to identify novel antimicrobial candidates of interest in the race for alternatives to antibiotics.

Shotgun proteomic analysis of porcine colostrum and mature milk.

The epitheliochorial nature of the porcine placenta prevents the transfer of maternal immunity. Therefore, ingestion of the colostrum immediately after birth is crucial for neonatal piglets to acquire passive immunity from the sow. We performed a shotgun proteomic analysis of porcine milk to reveal in detail the protein composition of porcine milk. On the basis of the Swiss-Prot database, 113 and 118 proteins were identified in the porcine colostrum and mature milk, respectively, and 50 of these proteins were common to both samples. Some immune-related proteins, including interleukin-18 (IL-18), were unique to the colostrum. The IL-18 concentration in the colostrum and mature milk of four sows was measured to validate the proteomic analysis, and IL-18 was only detected in the colostrum (191.0 ± 53.9 pg/mL) and not in mature milk. In addition, some proteins involved in primary defense, such as azurocidin, which has never been detected in any other mammal's milk, were also identified in the colostrum.

Grape seed extract suppresses MDA-MB231 breast cancer cell migration and invasion.

BACKGROUND AND AIM: Breast cancer remains a leading cause of mortality among women. In metastasis, cascade migration of cancer cells and invasion of extracellular matrix (ECM) represent critical steps. Urokinase-type plasminogen activator (uPA), as well as metalloproteinases MMP-2 and MMP-9, strongly contribute to ECM remodelling, thus becoming associated with tumour migration and invasion. In addition, the high expression of cytoskeletal (CSK) proteins, as fascin, has been correlated with clinically aggressive metastatic tumours, and CSK proteins are thought to affect the migration of cancer cells. Consumption of fruits and vegetables, characterized by high procyanidin content, has been associated to a reduced mortality for breast cancer. Therefore, we investigated the biological effect of grape seed extract (GSE) on the highly metastatic MDA-MB231 breast cancer cell line, focusing on studying GSE ability in inhibiting two main metastatic processes, i.e., cell migration and invasion. METHODS: After MDA-MB231 breast cancer cells stimulated with GSE migration and invasion were evaluated by means of trans-well assays and uPA as well as MMPs activity was detected by gelatin zymography. Fascin, ß-catenin and nuclear factor-κB (NF-κB) expression were determined using western blot technique. ß-Catenin localization was observed by confocal microscopy. RESULTS: We observed that high concentrations of GSE inhibited cell proliferation and apoptosis. Conversely, low GSE concentration decreased cell migration and invasion, likely by hampering ß-catenin expression and localization, fascin and NF-κB expression, as well as by decreasing the activity of uPA, MMP-2 and MMP-9. CONCLUSIONS: These results make GSE a powerful candidate for developing preventive agents against cancer metastasis.

Exocrine pancreatic insufficiency following esophagectomy.

Weight loss following esophagectomy is a management challenge for all patients. It is multifactorial with contributing factors including loss of gastric reservoir, rapid small bowel transit, malabsorption, and adjuvant chemotherapy. The development of a postoperative malabsorption syndrome, as a result of exocrine pancreatic insufficiency (EPI), is recognized in a subgroup of patients following gastrectomy. This has not previously been documented following esophageal resection. EPI can result in symptoms of flatulence, diarrhea, steatorrhea, vitamin deficiencies, and weight loss. It therefore has the potential to pose a significant level of morbidity in postoperative patients. There is some evidence that patients with proven EPI (fecal elastase-1 < 200 µg/g) may benefit from a trial of pancreatic enzyme replacement therapy (PERT). We observed symptoms compatible with EPI in a subgroup of patients following esophagectomy. We hypothesized that this was contributing to malabsorption and malnutrition in these patients. To investigate this, fecal elastase-1 was measured in postoperative patients, and in those with proven EPI, a trial of PERT was commenced in combination with specialist dietary education. At routine postoperative follow-up, which included assessment by a specialist dietitian, those patients with symptoms suggestive of malabsorption were given the opportunity to have their fecal elastase-1 measured. PERT was then offered to patients with fecal elastase-1 less than 200 µg/g (EPI) as well as those in the 200-500 µg/g range (mild EPI) with more severe symptoms. Fecal elastase-1 was measured in 63 patients between June 2009 and January 2011 at a median of 4 months (range 1-42) following surgery. Ten patients had fecal elastase-1 less than 200 µg/g, and all had failed to maintain preoperative weight. All accepted a trial of PERT. Nine (90%) had symptomatic improvement, and seven (70%) increased their weight. Thirty-nine patients had a fecal elastase-1 in the 200-500 µg/g range. Twelve were given a trial of PERT based on level of symptoms, five (42%) reported an improvement in symptoms, but only two (17%) gained weight. Our early results support the observation that EPI is a factor contributing to postoperative morbidity in patients recovering from esophagectomy and that these patients can benefit from a trial of PERT. Our study has limitations, and a formal trial is required to evaluate the impact of EPI and PERT following esophagectomy. Currently, our practice is to measure fecal elastase-1 in any patient with unexplained weight loss or symptoms of malabsorption. In patients with proven EPI or those who are symptomatic with mild EPI, a trial of PERT should be offered and symptoms reassessed.

Low AZGP1 expression predicts for recurrence in margin-positive, localized prostate cancer.

BACKGROUND: Men with positive margins after radical prostatectomy (RP) for localized prostate cancer (PC) have a 40-50% biochemical relapse rate at 5 years. Adjuvant radiotherapy improves biochemical progression-free and overall survival in men with positive margins, but is associated with increased toxicity. There is an urgent need to identify new prognostic markers to define the group of patients who would benefit from multimodality therapy. METHODS: Nuclear ß-catenin, membranous secreted frizzled-related protein 4 (sFRP4), zinc-alpha 2-glycoprotein (AZGP1), and macrophage inhibitory cytokine-1 (MIC-1) have previously been identified as molecular markers of outcome in localized PC. From these published studies, we identified a subset of patients with positive margins. The aim of this study was to assess the association between these four molecular markers and outcome in men with margin-positive, localized PC. RESULTS: We identified 186 men with positive margins from 330 men with localized PC; 53% had preoperative PSA >10 ng/ml, 72% extraprostatic extension (EPE), 24% seminal vesicles involvement (SVI), and 57% RP Gleason score ≥ 7. AZGP1 (P = 0.009), membranous sFRP4 (P = 0.03) and MIC-1 (P = 0.04) expression predicted for biochemical relapse on univariate analysis. Only absent/low AZGP1 expression (P = 0.01) was an independent predictor of recurrence in margin-positive, localized PC when modeled with preoperative PSA (P = 0.2), EPE (P = 0.2), SVI (P = 0.4), Gleason score ≥ 7 (P = 0.5) and adjuvant treatment (P = 0.4). Furthermore, there was an association between absent/low AZGP1 expression and clinical recurrence (P = 0.007). CONCLUSIONS: AZGP1 is a potential molecular marker for biochemical relapse in men with margin-positive, localized PC. Routine assessment of this biomarker may lead to better selection of patients who will benefit from post-RP radiotherapy.

Effects of the ErbB1/ErbB2 kinase inhibitor GW2974 on androgen-independent prostate cancer PC-3 cell line growth and NSE, chromogranin A and osteopontin content.

Prostate cancer is one of the most frequently diagnosed cancer in men. Treatment by radical prostatectomy, radiotherapy and anti-androgen drugs is successful in patients with localized cancer. However, prolonged androgen deprivation often leads to hormone refractory condition, associated with disease relapse. ErbB1 and ErbB2 activity has been correlated with androgen-independence. We determined the effects of GW2974, a dual inhibitor of ErbB-1 and ErbB-2 tyrosine kinase activity, on growth, NSE, chromogranin A and osteopontin cytosol content in the androgen-independent prostate cancer cell line PC-3. We found that PC-3 cell growth was inhibited by GW2974, whereas NSE and chromogranin A cell contents were stimulated and osteopontin cytosol level was not affected. The present data may have clinical implications for the treatment of advanced prostate cancer.

Time-dependant protective effects of mangenese(III) tetrakis (1-methyl-4-pyridyl) porphyrin on mitochondrial function following renal ischemia-reperfusion injury.

This study examined the time-dependent effects of a cell permeable SOD mimetic, MnTMPyP, on mitochondrial function in renal ischemia-reperfusion injury (IRI). Male SD rats were subject to either sham operation or bilateral renal ischemia for 45 min followed by reperfusion for 1, 4 or 24 h. A sub-set of animals was treated with either saline vehicle or 5 mg/Kg of MnTMPyP (i.p.). EPR measurements showed that at 1-h reperfusion MnTMPyP prevented a decrease in aconitase activity (p < 0.05) and attenuated the increase in the high spin heme at g = 6 and oxidation of 4Fe4S to 3Fe4S signal at g = 2.015 (p < 0.01). MnTMPyP was effective in preventing loss of mitochondrial complexes and prevented the loss of cytochrome c and Smac/Diablo from mitochondria early in reperfusion. Following 24 h of reperfusion MnTMPyP was effective in attenuating caspase-3 and blocking apoptosis (p < 0.05). In conclusion, MnTMPyP has biphasic effects in renal IRI, inhibiting mitochondrial dysfunction at the early phases of reperfusion and prevention of apoptosis following longer durations of reperfusion.

Relationship between dietary and supplemental intake of folate, methionine, vitamin B6 and folate receptor alpha expression in ovarian tumors.

Because folate receptor alpha (FRalpha) is frequently over-expressed in epithelial ovarian tumors, we hypothesized that its association with folate may differ by FRalpha expression or by the timing of intake. We examined the association between folate and other cofactors in 152 ovarian cancers evaluated for FRalpha expression from the Nurses' Health Study. Multivariate odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using unconditional logistic regression. FRalpha expression was higher in serous invasive and advanced stage ovarian tumors. Recent dietary folate intake > or = 300 microg/day compared to < 300 microg/day was associated with decreased risk of developing ovarian cancer (OR = 0.62; 95%CI 0.40-0.96). There was suggestion of an increased risk with total folate (dietary and supplemental) (OR=1.42; 95%CI 0.94-2.14 for past and OR = 1.53; 95%CI 0.99-2.37 for recent intake). These results did not vary by FRalpha status of the tumor. Methyl group score, a marker of high dietary folate and methionine intake but low alcohol consumption, was inversely associated with risk of ovarian cancer (OR = 0.44; 95%CI 0.23-0.84 for past and OR=0.46; 95%CI 0.24-0.88 for recent intake). There were no clear individual associations between methionine, vitamin B(6), or multivitamin use and ovarian cancer risk overall or by FRalpha tumor status. Our data do not support the hypothesis that the relationship between factors involved in one-carbon metabolism and ovarian cancer risk differs by FRalpha status of the tumor.

Tomato allergy: detection of IgE-binding lipid transfer proteins in tomato derivatives and in fresh tomato peel, pulp, and seeds.

There is an increasing consumption of tomatoes worldwide: fresh in salads, cooked in household sauces, or industrially processed. Although many tomato allergens have been identified, there is no information in the literature on the allergenic components found in commercial tomato products. The primary aim of the study was to evaluate the allergenic profile of commercial tomato products by skin prick tests (SPTs) and IgE/immunoblotting in tomato-allergic subjects. The secondary end point was the study of the IgE-binding profile of tomato peel, pulp, and seeds. Forty tomato-allergic patients, reporting oral allergy syndrome (OAS) at different grades of severity for fresh and, in some cases, also for cooked tomato, were selected on the basis of positive tomato allergy history or open food challenge (OFC). They were evaluated by SPTs with different experimental tomato extracts. SDS-PAGE/immunoblotting was performed to detect tomato allergens, which were then identified by Edman degradation. Twenty-three patients (57.5%) presented first-grade OAS at the OFC, whereas 17 (42.5%) reported severe symptoms. Ten of these 17 patients (25%) reported allergic reactions to cooked tomatoes; in immunoblotting tests, their sera reacted only to lipid transfer protein (LTP). In commercial products, LTP was the only detectable allergen. In contrast to other LTP-containing fruits, in tomato, an IgE-binding LTP was identified not only in the peel but also in the pulp and seeds. This study demonstrates that, in fresh tomato, different LTP isoforms are present and allergenic. Industrial tomato derivatives still contain LTP, thus presenting a problem for LTP-allergic patients.

Short communication: evidence for the presence of a putative odorant-binding protein in bovine colostrum.

Using a combination of PAGE and mass spectrometry for protein identification, we obtained evidence that a putative odorant-binding protein, designated hypothetical protein LOC517854, occurs in bovine colostrum. This protein, termed as a putative bovine colostral odorant-binding protein (bcOBP), consists of 172 AA residues, including a putative 16-AA signal peptide. The theoretical isoelectric point value and molecular mass of the full-length sequence of bcOBP were calculated to be 4.57 and 19604.18, respectively. The highest sequence similarity (83%) was observed with a potential pheromone transporter, Allergen Bos d 2. An odorant-binding protein derived from bovine nasal mucosa showed relatively low sequence similarity (52%) against bcOBP. Its biological function is unclear, but pheromone transport could be considered. This is the first report of a putative odorant-binding protein in bovine colostrum.

Taurine supplementation increases skeletal muscle force production and protects muscle function during and after high-frequency in vitro stimulation.

Recent studies report that depletion and repletion of muscle taurine (Tau) to endogenous levels affects skeletal muscle contractility in vitro. In this study, muscle Tau content was raised above endogenous levels by supplementing male Sprague-Dawley rats with 2.5% (wt/vol) Tau in drinking water for 2 wk, after which extensor digitorum longus (EDL) muscles were examined for in vitro contractile properties, fatigue resistance, and recovery from fatigue after two different high-frequency stimulation bouts. Tau supplementation increased muscle Tau content by approximately 40% and isometric twitch force by 19%, shifted the force-frequency relationship upward and to the left, increased specific force by 4.2%, and increased muscle calsequestrin protein content by 49%. Force at the end of a 10-s (100 Hz) continuous tetanic stimulation was 6% greater than controls, while force at the end of the 3-min intermittent high-frequency stimulation bout was significantly higher than controls, with a 12% greater area under the force curve. For 1 h after the 10-s continuous stimulation, tetanic force in Tau-supplemented muscles remained relatively stable while control muscle force gradually deteriorated. After the 3-min intermittent bout, tetanic force continued to slowly recover over the next 1 h, while control muscle force again began to decline. Tau supplementation attenuated F(2)-isoprostane production (a sensitive indicator of reactive oxygen species-induced lipid peroxidation) during the 3-min intermittent stimulation bout. Finally, Tau transporter protein expression was not altered by the Tau supplementation. Our results demonstrate that raising Tau content above endogenous levels increases twitch and subtetanic and specific force in rat fast-twitch skeletal muscle. Also, we demonstrate that raising Tau protects muscle function during high-frequency in vitro stimulation and the ensuing recovery period and helps reduce oxidative stress during prolonged stimulation.

Pancreatic secretory trypsin inhibitor is a major motogenic and protective factor in human breast milk.

Colostrum is the first milk produced after birth and is rich in immunoglobulins and bioactive molecules. We examined whether human colostrum and milk contained pancreatic secretory trypsin inhibitor (PSTI), a peptide of potential relevance for mucosal defense and, using in vitro and in vivo models, determined whether its presence influenced gut integrity and repair. Human milk was collected from individuals over various times from parturition and PSTI concentrations determined with the use of immunoassay. Human milk samples were analyzed for proliferation and promigratory activity (wounded monolayers) and antiapoptotic activity (caspase-3 activity) with the use of intestinal HT29 cells with or without neutralizing antibodies to PSTI and epidermal growth factor (EGF). Rats were restrained and given indomethacin to induce gastric injury. Effect of gavage with human breast milk with or without neutralizing antibodies on amount of injury were compared with animals receiving a commercial formula feed. PSTI is secreted into human milk, with colostrum containing a much higher concentration of PSTI than human milk obtained later. Human milk stimulated migration and proliferation about threefold and reduced indomethacin-induced apoptosis by about 70-80%. Sixty-five percent of the migratory effect of human milk could be removed by immunoneutralization of PSTI. PSTI worked synergistically with EGF in mediating these effects. Gastric damage in rats was reduced by about 75% in the presence of human milk and was more efficacious than the formula feed (P<0.001). Protective effects of the milk were reduced by about 60% by PSTI immunoneutralization. We concluded that PSTI is secreted into human milk at concentrations that have probable pathophysiological relevance.

A fluorescence polarization assay to quantify biotin and biotin-binding proteins in whole plant extracts using Alexa-Fluor 594 biocytin.

A high-throughput fluorescence polarization assay has been developed for the detection of biotin and biotin-binding proteins in whole leaf extracts. Various groups are investigating the insecticidal properties of avidin and other biotin-binding proteins expressed in leaves of transgenic plants. The methods commonly used to quantify biotin and avidin in leaf extracts are enzyme-linked immunosorbent assay (ELISA) and Western blotting. Here we describe a homogeneous fluorescence polarization (FP) method that quantifies transgenic avidin in whole leaf extract by the simple addition of the fluorescent avidin ligand Alexa-Fluor 594 biocytin (AFB). The FP assay exploits the fact that AFB excites and emits in regions of the spectrum that are relatively free of background fluorescence in leaf extract. Transgenic leaf avidin can be quantified within 1-2 h by the FP method, in comparison with 1-2 days for ELISA and Western blotting. The FP method can also measure the amount of biotin in control leaves, not expressing avidin. Functional avidin levels of 1.54 microM (26.1 microg/g leaf tissue) were detected in tobacco leaves expressing vacuole-targeted avidin. Control leaves had biotin levels of around 0.74 microM (approximately 0.18 microg/g leaf tissue). Reagent costs are minimal: typically AFB is used at concentrations of 1-10 nM, avidin is used at 1-100 nM, and sample volumes are 20 microL in 384-well microplates.

Vitamin D(2) supplementation induces the development of aortic stenosis in rabbits: interactions with endothelial function and thioredoxin-interacting protein.

Understanding of the pathophysiology of aortic valve stenosis (AVS) and finding potentially effective treatments are impeded by the lack of suitable AVS animal models. A previous study demonstrated the development of AVS in rabbits with vitamin D(2) and cholesterol supplementation without any hemodynamic changes in the cholesterol supplemented group alone. The current study aimed to determine whether AVS develops in an animal model with vitamin D(2) supplementation alone, and to explore pathophysiological mechanisms underlying this process. The effects of 8 weeks' treatment with vitamin D(2) alone (n=8) at 25,000 IU/4 days weekly on aortic valve structure and function were examined in male New Zealand white rabbits. Echocardiographic aortic valve backscatter (AV(BS)), transvalvular velocity, and transvalvular pressure gradient were utilized to quantitate changes in valve structure and function. Valvular histology/immunochemistry and function were examined after 8 weeks. Changes in valves were compared with those in endothelial function and in valvular measurement of thioredoxin-interacting protein (TXNIP), a marker/mediator of reactive oxygen species-induced oxidative stress. Vitamin D(2) treated rabbits developed AVS with increased AV(BS) (17.6+/-1.4 dB vs 6.7+/-0.8 dB, P<0.0001), increased transvalvular velocity and transvalvular pressure gradient (both P<0.01 via 2-way ANOVA) compared to the control group. There was associated valve calcification, lipid deposition and macrophage infiltration. Endothelial function was markedly impaired, and intravalvular TXNIP concentration increased. In this model, vitamin D(2) induces the development of AVS with histological features similar to those of early AVS in humans and associated endothelial dysfunction/redox stress. AVS development may result from the loss of nitric oxide suppression of TXNIP expression.

Preclinical radioimmunotargeting of folate receptor alpha using the monoclonal antibody conjugate DOTA-MORAb-003.

INTRODUCTION: The in vitro and in vivo behavior of the radiolabeled monoclonal antibody MORAb-003 was investigated as a prelude to a clinical trial. METHODS: The cellular retention of 111In- and 131I-labeled MORAb-003 was investigated using IGROV1 and SW620 cells. Biodistribution studies in tumor-bearing mice were performed with the more favorable agent. RESULTS: Five 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA) molecules were conjugated to MORAb-003 with no apparent loss of immunoreactivity. Radiolabeled MORAb-003 had a high affinity for the folate receptor alpha (FRA) expressed by both IGROV1 and SW620 cells and was found to bind to around 8 x 10(5) and 7 x 10(5) sites/cell, respectively. Both cancer cell lines were found to internalize both 131I- and 111In-labeled MORAb-003, but 111In was retained and 131I was released as iodide. In athymic mice, 111In-DOTA-MORAb-003 was cleared from the blood with a single exponential biological clearance rate of 110 h. The uptake in SW620 tumors was 32+/-5%ID/g after 4 days. The clearance rate of activity from normal organs such as liver, kidney and spleen was similar to the blood clearance and was 5.36%ID/g, 4.03%ID/g and 4.36%ID/g at 1 day postinjection and 2.14%ID/g, 1.65%ID/g and 3.74%ID/g after 8 days, respectively. In a pilot clinical study, the biodistribution and tumor targeting of 111In-MORAb-003 was assessed in three patients undergoing treatment with cold MORAb-003. CONCLUSION: MORAb-003 is an attractive antibody for radioimmunoscintigraphy and possibly radioimmunotherapy of FRA-expressing cancers in addition to its potential direct therapeutic effects.

Cardioprotective effects of Astragali Radix against isoproterenol-induced myocardial injury in rats and its possible mechanism.

The purpose of the present study was to investigate the effects of the Chinese medical herb Astragali Radix on myocardial injury in vivo and its possible mechanisms. Myocardial injury in rats was induced by the subcutaneous injection of a high dose of isoproterenol for 10 days, and the therapeutic effects of Astragali Radix were observed. Cardiac hemodynamics, heart coefficient and marker enzymes in serum showed that Astragali Radix prevented isoproterenol-induced myocardial damage. Astragali Radix also improved the antioxidant status by decreasing the lipid peroxidative product malondialdehyde and increasing the activity of the antioxidant enzyme superoxide dismutase. The observed depressions in sarcoplasmic reticulum Ca2+-ATPase mRNA and protein expression as well as Ser(16)-phosphorylated phospholamban protein expression in isoproterenol-treated rats were attenuated by Astragali Radix treatment. Moreover, treatment with Astragali Radix showed higher myocardial cAMP content compared with the isoproterenol-alone group. These results suggest that the antioxidant property and partial prevention of changes in protein and gene expression of cardiac sarcoplasmic reticulum Ca2+ regulatory proteins which may be mediated through the cAMP pathway could help to explain the beneficial effects of Astragali Radix on myocardial injury in vivo.