Chromosomal aberrations induced in human cultured cells by liposome-encapsulated deoxyribonuclease I

Experiments of incorporation of a nucleolytic enzyme into human cells cultured in vitro have been carried out with the aim of inducing structural chromosome variations. Human heteroploid cells, either as asynchronous populations or enriched in mitoses, and PHA-stimulated lymphocytes were used as recipients. We found that all these cells when exposed to pancreatic DNAase I encapsulated in liposomes, either of multilamellar (MLV) or of small unilamellar (SUV) type, show an incidence of chromosome damage higher than that induced by the enzyme free in the incubation buffer. Our results indicate that liposomes are suitable vehicles for the transfer of an exogenous nuclease into human cultured cells. The enzyme remains functionally active and interacts with nuclear DNA, giving rise to chromosome lesions.     

Preparation of asymmetric,cerebroside sulfate-containing phospholipid vesicles

A procedure is described which inserts asymmetrically cerebroside sulfate (‘sulfatide’) into the outer leaflet of bilayered phospholipid vesicles. Cerebroside sulfate is adsorbed onto a cellulose, filter-paper support and, when incubated with phosphatidylcholine vesicles is transferred to and inserted into the outer leaflet of these vesicles. This transfer occurs at, or above the transition temperature of the phospholipid and follows a similar pattern with small or larger (‘fused’) unilamellar vesicles. The transfer is linear with time for 1–2 h and is maximal after about 6 h, when the sulfatide content reaches about 6 mol% of the total quantity of phospholipid, corresponding to about 10 mol% of the phospholipids present in the outer layer. Initial rates of sulfatide transfer were somewhat increased when the vesicles contained a positively charged lipid (e.g. stearylamine) and decreased when this lipid was negatively charged (e.g. dicetyl phosphate) or hydrophobic (e.g. cholesterol). Divalent ions markedly inhibited sulfatide transfer and monovalent ions did so to a lesser degree. Once incorporated into the outer leaflet of the vesicle, the sulfatide could not be removed by washing with buffer, 1 M NaCl or 1 M urea.     

A single-chain antibody fragment is functionally expressed in the cytoplasm of both Escherichia coli and transgenic plants.

Despite the well-known crucial role of intradomain disulfide bridges for immunoglobulin folding and stability, the single-chain variable fragment of the anti-viral antibody F8 is functionally expressed when targeted to the reducing environment of the plant cytoplasm. We show here that this antibody fragment is also functionally expressed in the cytoplasm of Escherichia coli. A gel shift assay revealed that the single-chain variable fragment (scFv) accumulating in the plant and bacterial cytoplasm bears free sulfhydryl groups. Guanidinium chloride denaturation/renaturation studies indicated that refolding occurs even in a reducing environment, producing a functional molecule with the same spectral properties of the native scFv(F8). Taken together, these results suggest that folding and functionality of this antibody fragment are not prevented in a reducing environment. This antibody fragment could therefore represent a suitable framework for engineering recombinant antibodies to be targeted to the cytoplasm.     

Nuove ricerche sulla coltura in vitro di Aucuba japonica Thunb

Abstract

New researches on the culture « in vitro » of Aucuba japonica Thunb, – Caulinar fragments grown in vitro in nutrient media solidified with agar are compared with fragments grown on a filter paper support on unshaken liquid media. Tissue cultures of Aucuba are established putting down caulinar fragments just taken from the plant with isolated callus; subcultures are obtained more easily in July. We have observed that during the months of March or April, caulinar fragments, taken from the plant at various months of the year, of different ages and cultivated on various media, unexpectedly produce many roots.     

Environmental DNA metabarcoding as an effective and rapid tool for fish monitoring in canals

We focus on a case study along an English canal comparing environmental DNA (eDNA) metabarcoding with two types of electrofishing techniques (wade-and-reach and boom-boat). In addition to corroborating data obtained by electrofishing, eDNA provided a wider snapshot of fish assemblages. Given the semi-lotic nature of canals, we encourage the use of eDNA as a fast and cost-effective tool to detect and monitor whole fish communities.     

Chimeric plant virus particles as immunogens for inducing murine and human immune responses against human immunodeficiency virus type 1

The high-yield expression of a neutralizing epitope from human immunodeficiency virus type 1 (HIV-1) on the surface of a plant virus and its immunogenicity are presented. The highly conserved ELDKWA epitope from glycoprotein (gp) 41 was expressed as an N-terminal translational fusion with the potato virus X (PVX) coat protein. The resulting chimeric virus particles (CVPs), purified and used to immunize mice intraperitoneally or intranasally, were able to elicit high levels of HIV-1-specific immunoglobulin G (IgG) and IgA antibodies. Furthermore, the human immune response to CVPs was studied with severe combined immunodeficient mice reconstituted with human peripheral blood lymphocytes (hu-PBL-SCID). hu-PBL-SCID mice immunized with CVP-pulsed autologous dendritic cells were able to mount a specific human primary antibody response against the gp41-derived epitope. Notably, sera from both normal and hu-PBL-SCID mice showed an anti-HIV-1-neutralizing activity. Thus, PVX-based CVPs carrying neutralizing epitopes can offer novel perspectives for the development of effective vaccines against HIV and, more generally, for the design of new vaccination strategies in humans.     

Effects of weight loss on erythrocyte membrane composition and fluidity in overweight and moderately obese women

A previous study showed chemical and physical impairment of the erythrocyte membrane of overweight and moderately obese women. The present study investigated the effects of a low-calorie diet (800 kcal/day deficit for 8 weeks) on erythrocyte membrane properties in 70 overweight and moderately obese (body mass index, 25-33 kg/m²) normotensive, nondiabetic women. At the end of dietary intervention, 24.3% of women dropped out, 45.7% lost less than 5% of their initial weight (Group I) and only 30% of patients lost at least 5% of their initial body weight (Group II). Group I showed no significant changes in erythrocyte membrane composition and function. The erythrocyte membranes of Group II showed significant reductions in malondialdehyde, lipofuscin, cholesterol, sphingomyelin, palmitic acid and nervonic acid and an increase in di-homo-γ-linolenic acid, arachidonic acid and membrane fluidity. Moreover, Group II showed an improvement in total cholesterol, low-density lipoprotein cholesterol, glycemia and insulin resistance. These changes in erythrocyte membrane composition could reflect a virtuous cycle resulting from the reduction in insulin resistance associated with increased membrane fluidity that, in turn, results in a sequence of metabolic events that concur to further improve membrane fluidity.     

Development of a gas chromatography silicon-based microsystem in clinical diagnostics

The accurate determination of biological parameters by means of rapid, on-line measurements at low-concentrations is an important task within the fields of pharmaceutical screening and medical diagnostic. Nevertheless, in biological samples, the analytes of interest are present as minor components in complex mixtures and with interfering species. Biosensors are the best candidates for these applications providing a direct solution to this need of accuracy, but their intrinsic selectivity often excludes all the other components in the sample. A separation step introduced prior to the sensing component could allow both the increase of selectivity with respect the interfering species and the identification of a large spectrum of molecular components in the sample. This work reports the development of a silicon-based integrated separation microsystem for gas chromatography aimed to biomedical applications, with particular emphasis to monitor the homovanillic acid (HVA) and vanillylmandelic acid (VMA) ratios in mass population screening for neuroblastoma diagnosis and prognosis. The miniaturised system consists of two main modules: (i) a metal oxide semiconductor detector and (ii) a micromachined separation capillary column. As first step, the metal oxide semiconductor capability to detect HVA and VMA has been demonstrated. Then, a technology for a silicon separation capillary microcolumn including the on-chip gas sensor housing has been proposed and a first prototype has been developed. The proposed microsystem is an analytical device with biosensing capabilities for diagnostic and biomedical applications, which yield an electronic signal proportional to the concentration of a specific analyte or group of analytes.     

Concentrative uptake of cyclic ADP-ribose generated by BST-1+ stroma stimulates proliferation of human hematopoietic progenitors

Cyclic ADP-ribose (cADPR) is an intracellular calcium mobilizer generated from NAD(+) by the ADP-ribosyl cyclases CD38 and BST-1. cADPR, both exogenously added and paracrinally produced by a CD38(+) feeder layer, has recently been demonstrated to stimulate the in vitro proliferation of human hemopoietic progenitors (HP) and also the in vivo expansion of hemopoietic stem cells. The low density of BST-1 expression on bone marrow (BM) stromal cells and the low specific activity of the enzyme made it unclear whether cADPR generation by a BST-1(+) stroma could stimulate HP proliferation in the BM microenvironment. We developed and characterized two BST-1(+) stromal cell lines, expressing an ectocellular cyclase activity similar to that of BST-1(+) human mesenchymal stem cells, the precursors of BM stromal cells. Long term co-culture of cord blood-derived HP over these BST-1(+) feeders determined their expansion. Influx of paracrinally generated cADPR into clonogenic HP was mediated by a concentrative, nitrobenzylthioinosine- and dipyridamole-inhibitable nucleoside transporter, this providing a possible explanation to the effectiveness of the hormone-like concentrations of the cyclic nucleotide measured in the medium conditioned by BST-1(+) feeders. These results suggest that the BST-1-catalyzed generation of extracellular cADPR, followed by the concentrative uptake of the cyclic nucleotide by HP, may be physiologically relevant in normal hemopoiesis.     

Optimisation of the purification process of a tumour-targeting antibody produced in <Emphasis Type="Italic">N. benthamiana</Emphasis> using vacuum-agroinfiltration

It was previously demonstrated that the tumour-targeting antibody mAb H10 can be transiently expressed and purified at high levels in Nicotiana benthamiana by using a vacuum-agroinfiltration system boosted by the use of a virus silencing suppressor protein. Scope of this work was to analyse different steps of protein extraction from agroinfiltrated leaves to optimise the purification process of the secretory mAb H10 providing new insights in the field of large-scale plant production. Two different extraction procedures (mechanical shearing/homogenisation and recovery of intercellular fluids -IFs-) were evaluated and compared in terms of purified antibody yields, antibody degradation and total phenolic compounds content. Mechanical grinding from fresh leaf tissues gave the highest purification yield (75 mg/kg Fresh Weight -75% intact tetrameric IgG-) and total phenolics concentration in the range of 420 μg/g FW. The second extraction procedure, based on the recovery of IFs, gave purification yields of 15–20 mg/kg FW (corresponding to 27% of total soluble protein) in which about 40% of purified protein is constituted by fully assembled IgG with a total phenolic compounds content reduced by one order of magnitude (21 μg/g FW). Despite a higher antibody degradation, purification from intercellular fluids demonstrated to be very promising since extraction procedures resulted extremely fast and amenable to scaling-up. Overall data highlight that different extraction procedures can dramatically affect the proteolytic degradation and quality of antibody purified from agroinfiltrated N. benthamiana leaves. Based on these results, we optimised a pilot-scale purification protocol using a two-step purification procedure from batches of fresh agroinfiltrated leaves (250 g) allowing purification of milligram quantities (average yield 40 mg/kg FW) of fully assembled and functional IgG with a 99.4% purity, free of phenolic and alkaloid compounds with low endotoxin levels (<1 EU/ml).