Podophyllum peltatum possesses a beta-glucosidase with high substrate specificity for the aryltetralin lignan podophyllotoxin

A beta-glucosidase with high specificity for podophyllotoxin-4-O-beta-D-glucopyranoside was purified from the leaves of Podophyllum peltatum. The 65-kDa polypeptide had optimum activity at pH 5.0 and was essentially inactive at pH 6.5 or above. Maximum catalytic activity of this glucosidase was obtained at 45 degrees C, but the enzyme was not heat stable. This beta-glucosidase displayed higher substrate specificity for podophyllotoxin-4-O-beta-D-glucopyranoside than for the other lignans tested, and for the (1-->3) linkage of laminaribiose than for other glucosidic linkages.     

Hybrid sterility,Haldane's rule and speciation in Heliconius cydno and H. melpomene

Most genetic studies of Haldane's rule, in which hybrid sterility or inviability affects the heterogametic sex preferentially, have focused on Drosophila. It therefore remains unclear to what extent the conclusions of that work apply more generally, particularly in female-heterogametic taxa such as birds and Lepidoptera. Here we present a genetic analysis of Haldane's rule in Heliconius butterflies. Female F(1) hybrids between Heliconius melpomene and H. cydno are completely sterile, while males have normal to mildly reduced fertility. In backcrosses of male F(1) hybrids, female offspring range from completely sterile to fully fertile. Linkage analysis using the Z-linked triose-phosphate isomerase locus demonstrates a "large X" (Z) effect on sterility. Expression of female sterility varies among crosses in this and a previous study of Heliconius. Sterility may result from the production of normal but infertile eggs, production of small infertile eggs, or from a complete failure to develop ovarioles, which suggests multiple routes to the evolution of hybrid sterility in these Heliconius species. These results conform to the expectations of the "dominance" rather than "faster male" theories of Haldane's rule and suggest that relatively few loci are responsible. The two species are broadly sympatric and hybridize in the wild, so that female hybrid sterility forms one of several strong but incomplete barriers to gene flow in nature. The effect of female sterility is comparable to that of selection against non-mimetic hybrids, while mate choice forms a much stronger barrier to gene transfer.     

Nitric Oxide Synthase Inhibition Prevents Acute Quinolinate-Induced Striatal Neurotoxicity

Quinolinic acid (QUIN) is an endogenous excitotoxin acting on N-methyl-D-aspartate (NMDA) receptors, that leads to neurotoxic damage resembling the alterations observed in Huntington's disease. Two major end-points of QUIN induced neurotoxicity are both circling behavior (CB) and lipid peroxidation (LP). Recently, nitric oxide (NO) has been implicated as a mediator of cell injury in some neurological disorders, thus, NO as a free radical might be involved in QUIN-induced neurotoxicity and oxidative stress. In the present study we evaluated the possible role of NO on QUIN-induced neurotoxicity, by measuring nitric oxide synthase activity (NOS), before and after QUIN-induced damage and by evaluating the effect of NOS inhibition on acute QUIN-induced CB and LP. Rats were striatally microinjected with QUIN (240 nmol/1l). QUIN administration increased NOS activity by 327% as compared to control values and this enhancement was inhibited by i.v. pretreatment with a NOS inhibitor the N^G-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg). QUIN-induced CB was also attenuated by pretreatment of rats with 1, 5, 10 and 15 mg/kg of L-NAME by –37, –55, –62 and –74% vs QUIN respectively. Similarly, L-NAME also reduced by 32% the QUIN-induced LP. These findings suggest that enhanced NOS activity may participate in QUIN-induced neurotoxicity and oxidative stress.     

A longitudinal study of free leptin index in pre-eclamptic pregnancies

The ratio between circulating levels of leptin and soluble leptin receptor (sOB-R), the free leptin index (FLI), is used as a marker of leptin resistance. Therefore, the aim of our study was to investigate the FLI in mild pre-eclamptic pregnancies in a nested case–control study within a prospective observational study. Circulating levels of leptin and sOB-R levels rise significantly during pregnancy in healthy (p < 0.05) (n = 46) and pre-eclamptic pregnancies (p < 0.05) (n = 20). Serum levels of leptin were significantly higher in pre-eclamptic compared to healthy pregnancies at second and third trimesters of pregnancy (p < 0.05). Additionally, serum levels of sOB-R were significantly lower in pre-eclamptic pregnancies during the second and third trimesters of pregnancy compared to healthy pregnancies (p < 0.05). Moreover, we found that FLI did not vary significantly during pregnancy in healthy women (p > 0.05), while it increases in pre-eclamptic pregnancies (p < 0.05). Indeed, FLI was significantly higher at second and third trimesters of pregnancy in pre-eclamptic compared to healthy pregnancies (p < 0.05). In addition, FLI was significantly higher in the luteal phase compared with the follicular phase of the menstrual cycle in eumenorrheic women (p < 0.05). Receiver operating characteristic (ROC) curve analysis revealed the ability of leptin (AUC = 0.72) and FLI (AUC = 0.67) as a reliable predictor for mild pre-eclampsia during the second trimester of pregnancy. In conclusion, our findings show that FLI were significantly increased in mild pre-eclamptic pregnancies and allowed us to hypothesize that this rise might alter leptin bioavailability and bioactivity which might lead to the sympathetic hyperactivity and the hypertensive disorders during pregnancy.     

Identifying and characterising the Plasmodium falciparum merozoite surface protein 10 Plasmodium vivax homologue

Plasmodium vivax malaria is one of the most prevalent parasitic diseases in Asia and Latin-America. The difficulty of maintaining this parasite culture in vitro has hampered identifying and characterising proteins implied in merozoite invasion of red blood cells. We have been able to identify an open reading frame in P. vivax encoding the Plasmodium falciparum merozoite surface protein 10 homologous protein using the partial sequences from this parasite's genome reported during 2004. This new protein contains 479 amino-acids, two epidermal growth factor-like domains, hydrophobic regions at the N- and C-termini, being compatible with a signal peptide and a glycosylphosphatidylinositol anchor site, respectively. The protein is expressed during the parasite's asexual stage and is recognised by polyclonal sera in parasite lysate using Western blot. P. vivax-infected patients' sera highly recognised recombinant protein by ELISA.     

Production of macrophage-activated killer cells for targeting of glioblastoma cells with bispecific antibody to FcγRI and the epidermal growth factor receptor

Objective: The aim was to determine the ability of macrophage-activated killer cells (MAK cells) obtained from peripheral blood of normal volunteers to kill glioblastoma multiforme (GBM) cell lines. Another goal was to investigate whether a bispecific antibody (bsAb) MDX-447, recognizing the high-affinity Fc receptor for IgG (FcγRI) and epidermal growth factor receptor (EGFR), would enhance MAK cell tumoricidal activity. Methods: Monocytes, from leukapheresis product, were isolated by countercurrent elutriation and differentiated into MAK cells by culture with granulocyte/macrophage-colony-stimulating factor, vitamin D₃ and interferon γ. Cells were checked for sterility, endotoxin and phenotypic markers. MAK cell functional activity was measured by a flow-cytometric phagocytosis assay. Target cells, a carcinoma cell line and two glioma cell lines expressing EGFR, were stained with PKH-26. MAK cells were labeled with fluorescein-conjugated anti-CD14. Combined effectors, targets and bsAb were incubated and the percentage of MAK cells with phagocytosed targets was determined by flow cytometry. Conclusion: We demonstrate that a large number of highly purified monocytes, isolated from peripheral blood, can be differentiated into MAK cells for use as an adjuvant for cancer treatment. After culture these cells are sterile, endotoxin-free and comprise more than 95% MAK cells. Increased amounts of CD14, CD64 and HLA-DR, which are characteristics of macrophage activation, were expressed. MAK cells were extremely phagocytic in comparison to monocytes, even in the absence of bsAb. Moreover, bsAb enhanced the tumoricidal activity of elutriated MAK cells targeted against GBM cell lines. Therefore, intracavity MAK cells armed with MDX-447 could be an effective adoptive immunotherapy for EGFR-positive GBM. Received: 5 April 2000 / Accepted: 12 July 2000     

Cyclosporin-A Inhibits Constitutive Nitric Oxide Synthase Activity and Neuronal and Endothelial Nitric Oxide Synthase Expressions after Spinal Cord Injury in Rats

Nitric oxide (NO) plays a role in the pathophysiology of spinal cord injury (SCI). NO is produced by three types of nitric oxide synthase (NOS) enzymes: The constitutive Ca²⁺/calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms, and the inducible calcium-independent isoform (iNOS). During the early stages of SCI, nNOS and eNOS produce significant amounts of NO, therefore, the regulation of their activity and expression may participate in the damage after SCI. In the present study, we used Cyclosporin-A (CsA) to further substantiate the role of Ca-dependent NOS in neural responses associated to SCI. Female Wistar rats were subjected to SCI by contusion, and killed 4 h after lesion. Results showed an increase in the activity of constitutive NOS (cNOS) after lesion, inhibited by CsA (2.5 mg/kg i.p.). Western blot assays showed an increased expression of both nNOS and eNOS after trauma, also antagonized by CsA administration.     

Effect of nicotine on lung S-adenosylmethionine and development of Pneumocystis pneumonia

Because S-adenosylmethionine (AdoMet) is required by Pneumocystis carinii in vitro, Pneumocystis infection depletes plasma AdoMet of rats and humans, nicotine reduces AdoMet of guinea pig lungs, and smoking correlates with reduced episodes of Pneumocystis pneumonia (PCP) in AIDS patients, we tested the effect of nicotine treatment on PCP using a rat model. Intraperitoneal infusion of 400 microg of R-(+) nicotine kg(-1) h(-1) intraperitoneal for 21 days caused a 15-fold reduction in lung AdoMet although neither plasma nor liver were changed. Infusion of 4 and 400 microg kg(-1) h(-1) into immunosuppressed rats, beginning when rats were inoculated with P. carinii, caused 85 and 99.88% reductions, respectively, in P. carinii cysts at sacrifice 21 days later; P. carinii nuclei were reduced by 91.2 and >99.99%, respectively. This effect was reversed by concomitant administration of AdoMet with nicotine. Treatment with AdoMet alone increased infection intensity. We conclude that AdoMet is a critical and limiting nutrient for Pneumocystis thus can serve as a therapeutic target for PCP. Regarding the mechanism, nicotine treatment caused no change in rat lung activity of AdoMet synthesizing methionine ATP transferase activity nor was there any evidence of increased AdoMet utilization for methylation reactions. Except of a doubling of putrescine, nicotine treatment also did not change lung polyamine content. However, key polyamine anabolic and catabolic enzymes were upregulated, and there were corresponding changes in polyamine metabolic intermediates. We conclude that chronic nicotine treatment increases lung polyamine catabolic/anabolic cycling and/or excretion leading to increased AdoMet-consuming polyamine biosynthesis and depletion of lung AdoMet.     

High-affinity near-infrared fluorescent small-molecule contrast agents for in vivo imaging of prostate-specific membrane antigen

Surgical resection remains a definitive treatment for prostate cancer. Yet, prostate cancer surgery is performed without image guidance for tumor margin, extension beyond the capsule and lymph node positivity, and without verification of other occult metastases in the surgical field. Recently, several imaging systems have been described that exploit near-infrared (NIR) fluorescent light for sensitive, real-time detection of disease pathology intraoperatively. In this study, we describe a high-affinity (9 nM), single nucleophile-containing, small molecule specific for the active site of the enzyme PSMA. We demonstrate production of a tetra-sulfonated heptamethine indocyanine NIR fluorescent derivative of this molecule using a high-yield LC/MS purification strategy. Interestingly, NIR fluorophore conjugation improves affinity over 20-fold, and we provide mechanistic insight into this observation. We describe the preparative production of enzymatically active PSMA using a baculovirus expression system and an adenovirus that co-expresses PSMA and GFP. We demonstrate sensitive and specific in vitro imaging of endogenous and ectopically expressed PSMA in human cells and in vivo imaging of xenograft tumors. We also discuss chemical strategies for improving performance even further. Taken together, this study describes nearly complete preclinical development of an optically based small-molecule contrast agent for image-guided surgery.