Zinc-induced decrease of the thermal stability and regeneration of rhodopsin

Zinc is present at high concentrations in the photoreceptor cells of the retina where it has been proposed to play a role in the visual phototransduction process. In order to obtain more information about this role, the study of the effect of zinc on several properties of the visual photoreceptor rhodopsin has been investigated. A specific effect of Zn(2+) on the thermal stability of rhodopsin, obtained from bovine retinas and solubilized in dodecyl maltoside detergent, in the dark is reported. The thermal stability of rhodopsin in its ground state (dark state) is clearly reduced with increasing Zn(2+) concentrations (0-50 microm Zn(2+)). The thermal bleaching process is accelerated in the presence of Zn(2+) with k rate constants, at 55 degrees C, of 0.028 +/- 0.002 min(-1) (0 microm Zn(2+)) and 0.056 +/- 0.003 min(-1) (50 microm Zn(2+)), corresponding to t(12) values of 24.4 +/- 1.6 min and 11.8 +/- 0.1 min, respectively. Thermodynamic parameters derived from Arrhenius plots show a significant E(a) increase at 50 microm Zn(2+) for the process, with deltaG++ decrease and increase in deltaH++ and deltaS++ possibly reflecting conformational rearrangements and reordering of water molecules. The stability of the metarhodopsin II intermediate is also decreased and changes in the metarhodopsin II decay pathway are also detected. The extent of rhodopsin regeneration in vitro is also reduced by zinc. These effects, specific for zinc, are also seen for rhodopsin in native disc membranes, and may be relevant to the suggested role of Zn(2+) in normal and pathological retinal function.     

Single strand conformational polymorphism evaluation for the identification of TH3R alleles of the circumsporozoite protein gene of Plasmodium falciparum

Highly polymorphic regions of the circumsporozoite protein (CSP) of Plasmodium falciparum are associated with cellular immune responses. One of these regions, the TH3R polymorfic region of the csp gene codes for known T-cell epitopes. The present study tested the use of SSCP to determine sequence variations of the TH3R regions of four clones of P. falciparum (3D7, HB3, Dd2 and K1) which are known to have different TH3R regions. Single-strand conformation polymorphism (SSCP) technique was performed on amplified products labelled with fluorescent primer (both strands) and electrophoresed in an automated sequencer. Various gel compositions and electrophoresis conditions were tested. Even if some electrophoretogram differences were observed between clones, they could not distinguish between the alleles.     

PCR template preparation for capillary DNA sequencing

Fluorescence-based capillary DNA sequencing has facilitated the early completion of several complex sequencing projects. While capillary systems offer great benefits in terms of ease of use and automation, we find that they are sufficiently different from slab gel separation methodologies, demanding re-examination of the protocols used to generate and use DNA sequencing templates. We have recently initiated a large-scale Human Open Reading Frame EST project involving 30 laboratories feeding 11 MegaBace 1000 capillary sequencers. The group has already produced more than 300,000 valid sequences. The most successful template preparation protocol we have found is described here. We have found that a crucial step is the standardization of the quantity and quality of the templates, which have been achieved by overnight bacterial culture followed by PCR using limiting amounts of primers. Using this protocol, there is no need for post-PCR purification, and the final preparation cost is US $0.09/template. After sequencing 10,848 templates using this protocol, 78% of the reads were accepted (after discarding vectors without inserts and inserts smaller than 100 nucleotides), and 85% of the total number of bases had Phred scores of 15 or above.     

Genome plasticity in Lactococcus lactis

Comparative genome analyses contribute significantly to our understanding of bacterial evolution and indicate that bacterial genomes are constantly evolving structures. The gene content and organisation of chromosomes of lactic acid bacteria probably result from a strong evolutionary pressure toward optimal growth of these microorganisms in milk. The genome plasticity of Lactococcus lactis was evaluated at inter- and intrasubspecies levels by different experimental approaches. Comparative genomics showed that the lactococcal genomes are not highly plastic although large rearrangements (a.o. deletions, inversions) can occur. Experimental genome shuffling using a new genetic strategy based on the Cre-loxP recombination system revealed that two domains are under strong constraints acting to maintain the original chromosome organisation: a large region around the replication origin, and a smaller one around the putative terminus of replication. Future knowledge of the rules leading to an optimal genome organisation could facilitate the definition of new strategies for industrial strain improvement.     

Functional conservation of subfamilies of putative UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases in Drosophila, Caenorhabditis elegans, and mammals. One subfamily composed of l(2)35Aa is essential in Drosophila

The completed fruit fly genome was found to contain up to 15 putative UDP-N-acetyl-alpha-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) genes. Phylogenetic analysis of the putative catalytic domains of the large GalNAc-transferase enzyme families of Drosophila melanogaster (13 available), Caenorhabditis elegans (9 genes), and mammals (12 genes) indicated that distinct subfamilies of orthologous genes are conserved in each species. In support of this hypothesis, we provide evidence that distinctive functional properties of Drosophila and human GalNAc-transferase isoforms were exhibited by evolutionarily conserved members of two subfamilies (dGalNAc-T1 (l(2)35Aa) and GalNAc-T11; dGalNAc-T2 (CG6394) and GalNAc-T7). dGalNAc-T1 and novel human GalNAc-T11 were shown to encode functional GalNAc-transferases with the same polypeptide acceptor substrate specificity, and dGalNAc-T2 was shown to encode a GalNAc-transferase with similar GalNAc glycopeptide substrate specificity as GalNAc-T7. Previous data suggested that the putative GalNAc-transferase encoded by l(2)35Aa had a lethal phenotype (Flores, C., and Engels, W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2964-2969), and this was substantiated by sequencing of three lethal alleles l(2)35Aa(HG8), l(2)35Aa(SF12), and l(2)35Aa(SF32). The finding that subfamilies of GalNAc-transferases with distinct catalytic functions are evolutionarily conserved stresses that GalNAc-transferase isoforms may serve unique biological functions rather than providing functional redundancy, and this is further supported by the lethal phenotype of l(2)35Aa.     

Characterization of pregnancy-associated glycoproteins extracted from zebu (Bos indicus) placentas removed at different gestational periods

In the present work, two biochemical approaches were used to characterize PAGs isolated from Bos indicus fetal cotyledons removed at different gestational ages. The first procedure included acidic and ammonium sulfate precipitations, anion and cation exchange chromatographies and the second included pepstatin-agarose affinity chromatography. A bovine PAG radioimmunoassay was used to monitor the immunoreactivity throughout the isolation procedures. The most immunoreactive fractions issued from cation exchange and affinity chromatographies were analyzed by SDS-PAGE and Western blotting, before transfer to a polyvinylidene difluoride (PVDF) membrane for NH2-microsequence determination. Use SDS-PAGE and Western blotting, different isoforms of PAG with apparent molecular masses of 51 to 69 kDa and isoelectric points varying from 4.4 to 6.7 were identified in the placentas from different gestational ages. N-terminal microsequencing (10 to 25 aa long) indicates the expression of one single terminal amino acid sequence in the Bos indicus placenta, which is 100% identical to the bovine PAG-1.     

Gene sequence and the 1.8 A crystal structure of the tungsten-containing formate dehydrogenase from Desulfovibrio gigas

Desulfovibrio gigas formate dehydrogenase is the first representative of a tungsten-containing enzyme from a mesophile that has been structurally characterized. It is a heterodimer of 110 and 24 kDa subunits. The large subunit, homologous to E. coli FDH-H and to D. desulfuricans nitrate reductase, harbors the W site and one [4Fe-4S] center. No small subunit ortholog containing three [4Fe-4S] clusters has been reported. The structural homology with E. coli FDH-H shows that the essential residues (SeCys158, His159, and Arg407) at the active site are conserved. The active site is accessible via a positively charged tunnel, while product release may be facilitated, for H(+) by buried waters and protonable amino acids and for CO(2) through a hydrophobic channel.     

Process development of a recombinant antibody/interleukin-2 fusion protein expressed in protein-free medium by BHK cells

The production, purification and stability of quality (in terms of integrity and glycosylation) of an antibody/interleukin-2 fusion protein with potential application in tumour-targeted therapy expressed in BHK21 cells are described. Consistency of the product throughout time was determined by analysis of glycosylation of the fusion protein using MALDI-TOF mass spectroscopy and HPAEC-PAD combined with product integrity studies by SDS-PAGE and Western blotting. These investigations showed consistent expression in terms of integrity and of three major oligosaccharide structures of the fusion protein after 62 generations. The data obtained at this stage indicated the suitability of the cell line for production purposes. Different approaches for the production of this protein were subsequently carried out. The relative productivity of the recombinant fusion protein and general performance of the cells in two different protein-free medium (PFM) culture systems, continuous chemostat and continuous perfusion using a Centritech centrifuge as a cell retention device, were studied. The results indicate that the chemostat culture resulted in more stable and controllable nutrient environment, which could indicate better product consistency, in accordance with what has been observed under serum-containing conditions, in relation to the perfusion culture. Finally, product obtained from the chemostat culture was analysed and purified. The purification process was optimised with an increase in the overall yield from 38 to 70% being obtained, a significant improvement with important consequences for the implementation of an industrial-scale culture system. In conclusion, it was possible to produce and purify the recombinant antibody/interleukin-2 fusion protein assuring the quality and stability of the product in terms of integrity and glycosylation. Therefore, a candidate production process was established.     

The attachment of Clinostomum sp. (Digenea,Clinostomidae) to the oesophagus of the bird Ardea cocoi (Aves,Ardeidae)

The attachment of Clinostomum sp. to the oesophagus of the bird definitive host Ardea cocoi is reported. The parasite attaches to the mucous layer of the oesophagus by the acetabulum, oral sucker, and oral collar. The mucous layer of the oesophagus engulfed by the oral sucker presents an intense liquefactive necrosis, and the engulfment is not likely to contribute significantly to the attachment of the parasite. The oral collar of the parasite, surrounding the oral sucker, is juxtaposed to the oesophagus mucous layer, showing a perfect fitting of both surfaces. It is concluded that the main attachment mechanism of the parasites to the oesophagus of the host is provided by the acetabulum and by the juxtaposition of the oral collar of the parasites to the oesophagus host surface.