Biology and host range of Tecmessa elegans (Lepidoptera: Notodontidae), a leaf-feeding moth evaluated as a potential biological control agent for Schinus terebinthifolius (Sapindales: Anacardiaceae) in the United States

During surveys for natural enemies that could be used as classical biological control agents of Schinus terebinthifolius Raddi (Brazilian pepper), the caterpillar, Tecmessa elegans Schaus (Lepidoptera: Notodontidae), was recorded feeding on the leaves of the shrub in South America. The biology and larval and adult host range of this species were examined to determine the insect's suitability for biological control of this invasive weed in North America and Hawaii. Biological observations indicate that the larvae have five instars. When disturbed, the late instar larvae emit formic acid from a prothoracic gland that may protect larvae from generalist predators. Larval host range tests conducted both in South and North America indicated that this species feeds and completes development primarily on members of the Anacardiaceae within the tribe Rhoeae. Oviposition tests indicated that when given a choice in large cages the adults will select the target weed over Pistacia spp. However, considering the many valued plant species in its host range, especially several North American natives, this species will not be considered further for biological control of S. terebinthifolius in North America.     

Importance of circulating platelet aggregates and haemodynamic changes in ophthalmic artery and progression of visual field loss at pseudoexfoliation glaucoma

The aim of this work is to examine the role of circulating platelet aggregates (CPA) at pseudoexfoliation glaucoma (PXG), haemodynamic changes in the ophthalmic artery by ultrasonic color Doppler, searching for visual field progression. Vascular component at PXG and its role in VF progression dynamics has not been sufficiently explained, as well as CPA influence to ischaemic events related to optic nerve damage and VF progression. The examination included 80 patients, where of 35 (44%) men average age 68.3 +/- 7.0 and 45 (56%) women average age 65.7 +/- 7.0 (t = 1.66; p = 0.101). Forthy of them suffered from primary open angle glaucoma (POAG) as a control group (healthy), and 40 from pseudoexfoliative glaucoma (PXG) as an experimental group. All the examinees underwent complete ophthalmological examination: visual acuity, ocular fundus, intraocular pressure measured, anterior eye segment biomicroscopy with gonioscopy performed. Also VF examination was performed three times at 6 months intervals. Laboratory testing of CPA proportion values was performed by means of Wu an Hoak method and ultrasonic measurement of blood perfusion in the carotid tree, particularly concerning ophthalmic artery by means of color Doppler. Obtained decreased values of CPA proportion resulted in hypercoagulability of blood in PXG group. At PXG were also found increased blood flow resistivity indexes in ophthalmic artery (RI AO) and internal carotid artery (RI ACI), resulting with ischemia and hypoxia and finally progression of the visual filed damage. In conclusion, our study shows that examining CPA and ultrasonic monitoring of vascular parameters in ophthalmic artery with color Doppler may be the way of better understanding the vascular role in PXG prognosis.     

Afferent signaling drives oxytocinergic preautonomic neurons and mediates training-induced plasticity

We showed previously that oxytocinergic (OTergic) projections from the hypothalamic paraventricular nucleus (PVN) to the dorsal brain stem mediate training-induced heart rate (HR) adjustments and that beneficial effects of training are blocked by sinoaortic denervation (SAD; Exp Physiol 94: 630-640; 1103-1113, 2009). We sought now to determine the combined effect of training and SAD on PVN OTergic neurons in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. Rats underwent SAD or sham surgery and were trained (55% of maximal capacity) or kept sedentary for 3 mo. After hemodynamic measurements were taken at rest, rats were deeply anesthetized. Fresh brains were frozen and sliced to isolate the PVN; samples were processed for OT expression (real-time PCR) and fixed brains were processed for OT immunofluorescence. In sham rats, training improved treadmill performance and increased the gain of baroreflex control of HR. Training reduced resting HR (-8%) in both groups, with a fall in blood pressure (-10%) only in SHR rats. These changes were accompanied by marked increases in PVN OT mRNA expression (3.9- and 2.2-fold in WKY and SHR rats, respectively) and peptide density in PVN OTergic neurons (2.6-fold in both groups), with significant correlations between OT content and training-induced resting bradycardia. SAD abolished PVN OT mRNA expression and markedly reduced PVN OT density in WKY and SHR. Training had no effect on HR, PVN OT mRNA, or OT content following SAD. The chronic absence of inputs from baroreceptors and chemoreceptors uncovers the pivotal role of afferent signaling in driving both the plasticity and activity of PVN OTergic neurons, as well as the beneficial effects of training on cardiovascular control.     

A novel method for preparation of HAMLET-like protein complexes

Some natural proteins induce tumor-selective apoptosis. α-Lactalbumin (α-LA), a milk calcium-binding protein, is converted into an antitumor form, called HAMLET/BAMLET, via partial unfolding and association with oleic acid (OA). Besides triggering multiple cell death mechanisms in tumor cells, HAMLET exhibits bactericidal activity against Streptococcus pneumoniae. The existing methods for preparation of active complexes of α-LA with OA employ neutral pH solutions, which greatly limit water solubility of OA. Therefore these methods suffer from low scalability and/or heterogeneity of the resulting α-LA - OA samples. In this study we present a novel method for preparation of α-LA - OA complexes using alkaline conditions that favor aqueous solubility of OA. The unbound OA is removed by precipitation under acidic conditions. The resulting sample, bLA-OA-45, bears 11 OA molecules and exhibits physico-chemical properties similar to those of BAMLET. Cytotoxic activities of bLA-OA-45 against human epidermoid larynx carcinoma and S. pneumoniae D39 cells are close to those of HAMLET. Treatment of S. pneumoniae with bLA-OA-45 or HAMLET induces depolarization and rupture of the membrane. The cells are markedly rescued from death upon pretreatment with an inhibitor of Ca²⁺ transport. Hence, the activation mechanisms of S. pneumoniae death are analogous for these two complexes. The developed express method for preparation of active α-LA - OA complex is high-throughput and suited for development of other protein complexes with low-molecular-weight amphiphilic substances possessing valuable cytotoxic properties.     

Inhibition of metastasis development by daily administration of ultralow doses of RNase A and DNase I

Recent data on the involvement of miRNA and circulating tumor-derived DNA in regulation of tumorigenesis showed a great prospect for these molecules as a novel class of therapeutic targets and gave a new start for the study of enzymes cleaving nucleic acids as potential antitumor and antimetastatic agents. In the present paper using two murine tumor models with pulmonary or liver metastases we studied the antimetastatic potential of RNase A and DNase I and performed a search for possible molecular targets of the enzymes. Herein, we show for the first time that daily administration of ultralow doses of RNase A (0.5-50 μg/kg) and DNase I (0.02-2.3 mg/kg) inhibits the development of metastasis to 60-90% and RNase A exerts 30% retardation of tumor growth. Remarkably, the increase in RNase A dose from 50 μg/kg to 10 mg/kg leads to a disappearance of antitumor and antimetastatic effects. Simultaneous treatment of tumor-bearing animals with RNase A and DNase I leads to an additive effect and results in almost total absence of metastases. The use of RNase A as an adjuvant in conjunction with conventional cytostatic cyclophosphamide results in a reliable enhancement of antitumor and antimetastatic effect of the therapy compared with the use of these agents individually. The search for possible molecular mechanism of antimetastatic effect of nucleases showed that daily administration of the enzymes reduced the pathologically increased level of extracellular nucleic acids and increased nuclease activity of the blood plasma of tumor-bearing mice back to the level of healthy animals. Thus, we unequivocally show that the proposed protocol of treatment of tumor-bearing animals with RNase A and DNase I has a general systemic and immunomodulatory effect, leads to a drastic suppression of metastasis development, and in perspective may become an effective component of intensive complex therapy of cancer.     

Improved production of monoclonal antibodies through oxygen-limited cultivation of glycoengineered yeast

Glycoengineering technology can elucidate and exploit glycan related structure-function relationships for therapeutic proteins. Glycoengineered yeast has been established as a safe, robust, scalable, and economically viable expression platform. It has been found that specific productivity of antibodies in glycoengineered Pichia pastoris is a non-linear function of specific growth rate that is dictated by a limited methanol feed rate. The optimal carbon-limited cultivation requires an exponential methanol feed rate with an increasing biomass concentration and more significantly an increase in heat and mass transfer requirements that often become the limiting factor in scale-up. Both heat and mass transfer are stoichiometrically linked to the oxygen uptake rate. Consequently an oxygen-limited cultivation approach was evaluated to limit the oxygen uptake rate and ensure robust and reliable scale-up. The oxygen-limited process not only limited the maximum oxygen uptake rate (and consequently the required heat removal rate) in mut+ P. pastoris strains but also enabled extension of the induction phase leading to an increased antibody concentration (1.9 g L⁻¹ vs. 1.2 g L⁻¹), improved N-glycan composition and galactosylation, and reduced antibody fragmentation. Furthermore, the oxygen-limited process was successfully scaled to manufacturing pilot scale and thus presents a promising process option for the glycoengineered yeast protein expression platform.     

Flow cytometric analysis of glyoxalase-1 expression in human leukocytes

Altered glyoxalase‐1 (GLO‐1) activity and expression is associated with the development of late diabetic complications, malignancy and oxidative stress‐ and aging‐related diseases. In the present study, we developed a flow cytometry method for GLO‐1 detection in human leukocytes isolated from peripheral blood samples to investigate GLO‐1 expression in leukocyte subsets from type 1 and 2 diabetes mellitus patients (n = 11) and healthy subjects (n = 8). The flow cytometry analysis of GLO‐1 in leukocytes showed that expression index of GLO‐1‐positive cells was slightly increased in mononuclear leukocytes from diabetic patients. This result correlated with the increase in GLO‐1 activity in the whole blood samples of type 2 diabetes patients. In conclusion, the present study demonstrates that flow cytometry is suitable for the detection of the GLO‐1 enzyme in human leukocytes and that this method could be used to investigate the fast adaptation of the glyoxalase system related to the pathogenesis of late complications of diabetes mellitus and other glycation stress‐related disorders. Copyright © 2011 John Wiley & Sons, Ltd.     

Oldies (but goldies!): extracting DNA from cryopreserved allozyme homogenates

In this work, we describe a method that allows the extraction of plant genomic DNA from frozen enzyme homogenates. To test if reliable DNA was recovered from allozyme extracts, we compared these results with that obtained from fresh frozen and dried material. Sequencing and microsatellite fragments were analyzed for samples from 5 plant families in order to check for chloroplast and nuclear DNA quality. High-molecular weight DNA was recovered from frozen homogenates, and it was found that the quality was independent of storage time, which varied from 1 to 9 years. High-quality PCR products along with sequences and microsatellite fragments were obtained for nearly all analyzed samples. Frozen protein homogenates from previous population studies could thus be used for further phylogeographical and phylogenetic analyses using DNA molecular tools. This simple method provides an opportunity to take advantage of stored cryopreserved samples.     

Passive immunotherapies protect WRvFire and IHD-J-Luc vaccinia virus-infected mice from lethality by reducing viral loads in the upper respiratory tract and internal organs

Whole-body bioimaging was employed to study the effects of passive immunotherapies on lethality and viral dissemination in BALB/c mice challenged with recombinant vaccinia viruses expressing luciferase. WRvFire and IHD-J-Luc vaccinia viruses induced lethality with similar times to death following intranasal infection, but WRvFire replicated at higher levels than IHD-J-Luc in the upper and lower respiratory tracts. Three types of therapies were tested: licensed human anti-vaccinia virus immunoglobulin intravenous (VIGIV); recombinant anti-vaccinia virus immunoglobulin (rVIG; Symphogen, Denmark), an investigational product containing a mixture of 26 human monoclonal antibodies (HuMAbs) against mature virion (MV) and enveloped virion (EV); and HuMAb compositions targeting subsets of MV or EV proteins. Bioluminescence recorded daily showed that pretreatment with VIGIV (30 mg) or with rVIG (100 μg) on day -2 protected mice from death but did not prevent viral replication at the site of inoculation and dissemination to internal organs. Compositions containing HuMAbs against MV or EV proteins were protective in both infection models at 100 μg per animal, but at 30 μg, only anti-EV antibodies conferred protection. Importantly, the t statistic of the mean total fluxes revealed that viral loads in surviving mice were significantly reduced in at least 3 sites for 3 consecutive days (days 3 to 5) postchallenge, while significant reduction for 1 or 2 days in any individual site did not confer protection. Our data suggest that reduction of viral replication at multiple sites, including respiratory tract, spleen, and liver, as monitored by whole-body bioluminescence can be used to predict the effectiveness of passive immunotherapies in mouse models.