Neurotoxicity of 2,4-dichlorophenoxyacetic butyl ester in chick embryos

Fertilized hens' eggs were externally treated, before starting incubation, with a single dose of 2,4-Dichlorophenoxyacetic butyl ester (2,4-D b.e., 3.1 mg/egg). Chicks at different developmental stages were examined, extending from 10 day embryos to one day after hatching. Previously, we demonstrated that 2,4-D b.e. produces hypomyelination in chicks born from treated eggs. In search of the causes of this hypomyelination, myelin markers such as sulphatides, cerebrosides and 2 3-cyclic nucleotide 3-phosphohydrolase (CNPase) activity, as well as protein and nucleic acid contents were determined in the embryonic brains. We have shown in the present study that the chemical alterations occurred even before the period of active myelination, since myelin appears in chicken brain stem and cerebrum approximately after 17 days of incubation, and most of the chemical parameters studied are diminished before that time. The DNA content in brain of treated group is increased from the 14^th embryonic day (with a transient diminution at 12^th day) to the first hatching day, when compared to the control, suggesting a proliferation of glial cells, possibly oligodendrocytes.     

Processing of transgenic corn seed and its effect on the recovery of recombinant beta-glucuronidase

The tools of plant biotechnology that have been developed to improve agronomic traits are now being applied to generate recombinant protein products for the food, feed, and pharmaceutical industry. This study addresses several processing and protein recovery issues that are relevant to utilizing transgenic corn as a protein production system. The gus gene coding for beta-glucuronidase (rGUS) was stably integrated and expressed over four generations. The accumulation level of rGUS reached 0.4% of total extractable protein. Within the kernel, rGUS was preferentially accumulated in the germ even though a constitutive ubiquitin promoter was used to direct gus expression. Fourth-generation transgenic seed was used to investigate the effect of seed processing on the activity and the recovery of rGUS. Transgenic seed containing rGUS could be stored at an ambient temperature for up to two weeks and for at least three months at 10 degrees C without a significant loss of enzyme activity. rGUS exposed to dry heat was more stable in ground than in whole kernels. The enzyme stability was correlated with the moisture loss of the samples during the heating. Transgenic seed was dry-milled, fractionated, and hexane extracted to produce full-fat and defatted germ fractions. The results of the aqueous extraction of rGUS from ground kernels, full-fat germ, and defatted-germ samples revealed that approximately 10 times more rGUS per gram of solids could be extracted from the ground full-fat germ and defatted-germ than from the kernel samples. The extraction of corn oil from ground germ with hot hexane (60 degrees C) did not affect the extractable rGUS activity. rGUS was purified from ground kernels and full-fat germ extracts by ion exchange, hydrophobic interaction, and size exclusion chromatography. Similar purity and yield of rGUS were obtained from both extracts. Biochemical properties of rGUS purified from transgenic corn seed were similar to those of E. coli GUS.     

Specific recognition of human pi glutathione transferase by an antipeptide antibody

Antibodies were raised in a rabbit by using a 12-residue synthetic peptide, corresponding to fragment 2-13 of rat placental glutathione S-transferase, as the immunogen. The antiserum appeared to react with the fragment as well as with the corresponding human enzyme (GST-pi), which shares with the rat transferase a 92% sequence homology at the N terminus. In addition, the binding of the antibody to the protein was completely inhibited by small amounts of peptide. The enzymatic activity of glutathione transferase was not affected by the antibody. This might indicate that the N-terminal fragment is not involved in the catalytic activity of the enzyme. This antibody of predetermined specificity might thus find a useful application for the detection and approximate quantitation of this marker in human preneoplastic lesions.     

Preparation and utilization of fluorescent synthetic peptides

In this study, several methods for controlled labelling of synthetic peptides by the use of fluorescent compounds (fluorescein isothiocyanate and dimethylaminonaphthalene sulfonyl chloride) were investigated. The first reagent yielded monofluoresceinated, active compounds only when the peptides lacked lysine residues. Monolabelling of peptides in solution with dimethylaminonaphthalenesulphonyl chloride was hindered by the broad reactivity of the reagent, but was achieved by reacting the fluorochrome on protected resin-bound peptides in solid-phase synthesis. The remarkable stability of the linkage allowed the cleavage of the peptide from the resin and deprotection of side-chain functions without hydrolysis of the labelled group. The binding of antipeptide antibodies to the labelled fragments was then estimated using different techniques.     

Microbiological and chemical-sensory characteristics of three coffee varieties processed by wet fermentation

This work evaluated the bacterial diversity during coffee wet fermentation of the three coffee varieties—Mundo Novo (MN), Ouro Amarelo (OA), and Catuaí Vermelho (CV). Isolates were identified by polyphasic techniques: biochemical tests, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and DNA sequencing. Chemical compositions were determined by high (HPLC) and gas chromatography-mass spectrometry (GC-MS) and the roasted beans were sensorial evaluated using the cupping test. Thirty-six mesophilic bacteria and six lactic acid bacteria were identified. Lactobacillus plantarum and Leuconostoc mesenteroides were often found in all varieties. Citric acid was the acid detected in higher concentrations. The volatile profile of the green coffee beans changed during the fermentation in the tank, but more significantly, during the roasting process. These volatiles belonged to the classes of acids, alcohols, aldehydes, and hydrocarbons. Temporal dominance of sensations analysis showed sensorial sensations of acidity (OA and CV), bitterness, chocolate, nuts (MN), and sweetness (CV). The characteristics of each coffee variety were distinct, mainly in relation to total bacteria population, volatile compounds, and sensorial profile. In conclusion polyphasic methodology was efficiently done for bacteria identification; the dominant bacteria might be used for starter cultures and the chemical and sensory analyses helped to understand the changes in coffee fermentation. Our findings are relevant to future select starter bacteria for coffee processing to improve quality and standardization of quality.     

Population dynamics and feeding behavior of Triatoma brasiliensis and Triatoma pseudomaculata, main vectors of chagas disease in Northeastern Brazil

Biological parameters of Triatoma brasiliensis and T. pseudomaculata that could influence the epidemiological importance of these insects as vectors of Trypanosoma cruzi were compared. The parameters studied were incubation period, interval between hatching or moulting and first feeding, number of blood meals, development time, mortality, net reproductive rate, instantaneous daily reproductive rate, time-lapse before starting feeding, duration of feeding, blood ingestion capacity, occurrence of defecation and blood ingestion velocity. Most aspects of feeding were similar for the two species, although T. pseudomaculata had a longer life cycle than T. brasiliensis producing one and two generations per year, respectively. The two species had similar instantaneous daily rates of population growth.     

Homogeneous Cell- and Bead-Based Assays for High Throughput Screening Using Fluorometric Microvolume Assay Technology

High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds has resulted in a requirement for assays and instrumentation that are amenable to nonradioactive formats and that can be miniaturized. Homogeneous assays that minimize upstream automation of the individual assays are also preferable. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based platform for the development of nonradioactive cell- and bead-based assays for HTS. This technology is plate format-independent, and while it was designed specifically for homogeneous ligand binding and immunological assays, it is amenable to any assay utilizing a fluorescent cell or bead. The instrument fits on a standard laboratory bench and consists of a laser scanner that generates a 1 mm(2) digitized image of a 100-μmm deep section of the bottom of a microwell plate. The instrument is directly compatible with a Zymark Twistertrade mark (Zymark Corp., Hopkinton, MA) for robotic loading of the scanner and unattended operation in HTS mode. Fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using data processing. Unbound flurophore comprising the background signal is ignored, allowing for the development of a wide variety of homogeneous assays. The use of FMAT for peptide ligand binding assays, immunofluorescence, apoptosis and cytotoxicity, and bead-based immunocapture assays is described here, along with a general overview of the instrument and software.     

A proteomic approach to the investigation of early events involved in the activation of vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) are mature cells that maintain great plasticity. This distinctive quality is the basis of the migration and proliferation of VSMC in cardiovascular diseases. We have investigated, via a proteomic approach, the molecular changes that promote VSMC switching from a quiescent to an activated-proliferating phenotype. In particular, we focus on the modulation in tyrosine phosphorylation that occurs in cell activation by serum or by single growth factors, such as insulin-like growth factor 1 (IGF-1) or platelet-derived growth factor (PDGF-BB). A comparison of profiles from two-dimensional polyacrylamide gel electrophoresis analysis of quiescent and activated-proliferating VSMC has revealed a number of differences in protein expression. Several differentially expressed proteins have been identified by mass spectrometry, and their changes during the time course of tyrosine phosphorylation have been documented from time zero up to 48 h after stimulus. The tyrosine-phosphorylation level generally decreases within a few minutes of stimulation, followed by a rapid dramatic recovery of some chaperones and redox enzymes, but no significant recovery for glucose metabolism enzymes. With respect to cytoskeleton components, no remarkable fluctuations have been detected at the earliest time points, except for those relating to α-actin, which displays an impressive decrease. A comparison of the early stages of cell stimulation after serum or after single growth factor administration has revealed important differences in the phosphorylation of chaperones, thereby suggesting their crucial role in VSMC activation. This work was partially supported by two FIRB 2001 project grants to Dr. G. Rainaldi and to Prof. G. Camici.