Differential extractability of influenza virus hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts

Biochemical changes in the influenza virus hemagglutinin during intracellular transport to the apical plasma membrane of epithelial cells were investigated in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells stably transfected with a hemagglutinin gene. After pulse-labeling a substantial fraction of hemagglutinin was observed to become insoluble in isotonic solutions of Triton X-100. Insolubility of hemagglutinin was detected late in the transport pathway after addition of complex sugars in the Golgi complex but before insertion of the protein in the plasma membrane. Insolubility was not dependent on oligosaccharide modification since deoxymannojirimycin (dMM), which inhibits mannose trimming, failed to prevent its onset. Insolubility was not due to assembly of virus particles at the plasma membrane because insoluble hemagglutinin was also observed in transfected cells. Hemagglutinin insolubility was also seen in MDCK cells cultured in suspension and in chick embryo fibroblasts, indicating that insolubility and plasma membrane polarity are not simply correlated. In addition to insolubility, an apparent transport-dependent reduction of the disulfide bond linking HA1 and HA2 in hemagglutinin was detected. Because of the timing of both insolubility and the loss of the disulfide bond, these modifications may be important in the delivery of the hemagglutinin to the cell surface.     

Characterization of the secondary structure of calmodulin in complex with a calmodulin-binding domain peptide.

The interaction between calcium-saturated chicken calmodulin and a peptide corresponding to the calmodulin-binding domain of the chicken smooth muscle myosin light chain kinase has been studied by multinuclear and multidimensional nuclear magnetic resonance methods. Extensive 1H and 15N resonance assignments of calmodulin in the complex have been obtained from the analysis of two- and three-dimensional nuclear magnetic resonance spectra. The assignment of calmodulin in the complex was facilitated by the use of selective labeling of the protein with alpha-15N-labeled valine, alanine, lysine, leucine, and glycine. These provided reference points during the main-chain-directed analysis of three-dimensional spectra of complexes prepared with uniformly 15N-labeled calmodulin. The pattern of nuclear Overhauser effects (NOE) seen among main-chain amide NH, C alpha H, and C beta H hydrogens indicates that the secondary structure of the globular domains of calmodulin in the complex closely corresponds to that observed in the calcium-saturated state of the protein in the absence of bound peptide. However, the backbone conformation of residues 76-84 adopts an extended chain conformation upon binding of the peptide in contrast to its helical conformation in the absence of peptide. A sufficient number of NOEs between the globular domains of calmodulin and the bound peptide have been found to indicate that the N- and C-terminal regions of the peptide interact with the C- and N-terminal domains of calmodulin, respectively. The significance of these results are discussed in terms of recently proposed models for the structure of calmodulin-peptide complexes.     

The incidence of Listeria spp. in soft cheeses, butter and raw milk in the province of Bologna

Samples of soft cheese, butter and raw milk were examined for Listeria species. Listeria monocytogenes (serotype 1, haemolytic and virulent for mice) and L. innocua (the only other Listeria sp. isolated) were each found in 2/21 (1.6%) of soft cheese samples. Five per cent of butter samples were contaminated with L. innocua . No Listeria spp. were detected in 40 raw milk samples. The results were compared with similar studies in Italy and abroad. and accepted 14 June 1989     

Cloning and characterization of a 70 kd heat shock cognate (hsc70) gene from the zebrafish (Danio rerio)

The heat shock 70 family of proteins is one of the most highly conserved among all species. The genes encoding these proteins have been cloned and sequenced from bacterial species to humans with a high degree of homology preserved throughout evolution. Here we describe the cloning and characterization of a cDNA encoding a 70 kd heat shock cognate (hsc70) gene from the zebrafish (Danio rerio). A high degree of conservation is observed among hsc70 genes of other species as shown by phylogenetic analysis. The characterization of a hsc70 gene in the zebrafish provides a marker for studying the role of a constitutively expressed member of the hsp70 family in an important developmental and evolutionary model system.     

Cartographic study: Breakpoints in 1574 families carrying human reciprocal translocations

Reciprocal translocations (rcp) are among the most common constitutional chromosomal aberrations in man. Using a European database of 1574 families carrying autosomal rcp, a cartographic study was done on the breakpoints involved. The breakpoints are non-randomly distributed along the different chromosomes, indicating “hot spots”. Breakpoints of rcp that result in descendants that are unbalanced chromosomally at birth are more frequent in a distal position on chromosomal arms, and 65% of them are localised in R-bands. Among the R-bands, bands rich in GC islands and poor in Alu repetitive sequences are more frequently the site of breakpoints, as well as bands that include a fragile site. This result suggests that the variation in degree of methylation in GC islands could be involved in chromosomal breakage and hence in chromosomal rearrangements. Received: 10 April 1995 / Revised: 1 July 1995     

Effect of oocyte-sperm co-incubation on acrosome reaction in the goat

The induction of acrosome reaction of goat spermatozoa was investigated. The acrosomal status of spermatozoa was determined by a triple-staining technique. The effect of the presence of goat oocytes on the proportion of acrosome-reacted spermatozoa was also determined. Ovulated oocytes were obtained from superstimulated adult goats. Other sources of oocytes were adult and prepubertal goats; oocytes from both sources were maturated in vitro. There was an increase in the percentage of acrosome-reacted spermatozoa from 4% +/- 0.98 to 9% +/- 1.41 when oocytes from adult females were used. Similar induction rates were measured with prepubertal and adult oocytes maturated in vitro (10.4% +/- 2.06 and 8.75% +/- 1.06, respectively). The influence of several qualities of cumulus oophorus as well as the presence of zona pellucida was also investigated. No significant differences were obtained with any cumulus oophorus or zona pellucida oocyte complexes. Although oocyte quality is important for high fertilization rates, it does not seem to be crucial for the induction of acrosome reaction.     

Thermodynamics elucidation of the structural stability of a thermophilic protein

The structural stability of the protein, phycocyanin isolated from two strains of cyanophyta, Synechococcus lividus (thermophile) and Phormidium luridum (mesophile), are investigated by comparative thermal and denaturant unfolding, using differential scanning calorimetry, visible absorption spectrophotometry, and circular dichroism. The thermophilic protein exhibits a much higher temperature and enthalpy of unfolding from the native to the denatured state. The concentration of urea at half-completion of thermal unfolding is essentially the same between the thermophilic and mesophilic proteins; in contrast, the corresponding temperature and the enthalpy of thermal unfolding are much higher for the thermophilic protein. In addition, the concentration of urea at which the non-thermal (denaturant) unfolding of protein is half-completed, as detected by either circular dichroism or absorption spectroscopy, is significantly higher in the thermophilic protein, while the apparent free energy of unfolding only shows a moderate difference between the two proteins. The distinct differences in the enthalpy of thermal unfolding and the free energy of denaturant unfolding are interpreted in terms of a significant entropy change associated with the unfolding of these proteins. This entropy contribution is much higher in the thermophilic protein, and may be derived from its more rigid overall structure that possesses higher internal hydrophobicity and stronger internal packing.     

Atypical Chordoid Structures in the Aristotle's Lantern of Regular Echinoids

Regular sea–urchins, as a rule, prepare and process their food as pellets in their buccal cavity before ingesting them. This is obtained through the synergistic interaction of the five teeth with five fleshy protuberances (paradental tongues) which extend into the oral cavity and work like tongues. Each of these structures consists of two anatomically distinct parts: the outer component is a deep pouch of the terminal tract of the pharynx and shows a histological organization similar to that usually shown by the pharyngeal wall itself. The deeper component (paradental axis) is represented by a supporting rod and shows a peculiar histological structure, which consists of glycogen containing vesiculate cells mixed to slim muscle fibres and held together by a thin fibrillar stroma. The paradental axes are very problematic structures in terms of comparative anatomy and phylogenetic aspects. Their microscopic and submicroscopic organization is unusual for an echinoderm and recalls that of different types of chordoid organs commonly found in other invertebrates (Protostomata and Deuterostomata).     

Salmonella recD mutations increase recombination in a short sequence transduction assay.

We have identified recD mutants of Salmonella typhimurium by their ability to support growth of phage P22 abc (anti-RecBCD) mutants, whose growth is prevented by normal host RecBCD function. As in Escherichia coli, the recD gene of S. typhimurium lies between the recB and argA genes at min 61 of the genetic map. Plasmids carrying the Salmonella recBCD+ genes restore ATP-dependent exonuclease V activity to an E. coli recBCD deletion mutant. The new Salmonella recD mutations (placed on this plasmid) eliminate the exonuclease activity and enable the plasmid-bearing E. coli deletion mutant to support growth of phage T4 gene 2 mutants. The Salmonella recD mutations caused a 3- to 61-fold increase in the ability of a recipient strain to inherit (by transduction) a large inserted element (MudA prophage; 38 kb). In this cross, recombination events must occur in the short (3-kb) sequences that flank the element in the 44-kb transduced fragment. The effect of the recD mutation depends on the nature of the flanking sequences and is likely to be greatest when those sequences lack a Chi site. The recD mutation appears to minimize fragment degradation and/or cause RecBC-dependent recombination events to occur closer to the ends of the transduced fragment. The effect of a recipient recD mutation was eliminated if the donor P22 phage expressed its Abc (anti-RecBC) function. We hypothesize that in standard (high multiplicity of infection) P22-mediated transduction crosses, recombination is stimulated both by Chi sequences (when present in the transduced fragment) and by the phage-encoded Abc protein which inhibits the host RecBCD exonuclease.     

Redox-active bis-cysteinyl peptides. II. Comparative study on the sequence-dependent tendency for disulfide loop formation

Bis (cysteinyl) octapeptides related to the active sites of the oxidoreductases protein disulfide isomerase (PDI), thioredoxin reductase (trr), glutaredoxin (grx), and thioredoxin (trx) were analyzed for their propensity to form the intramolecular 14-membered disulfide ring in oxidation experiments. The rank order of percentage of cyclic monomer formed in aqueous buffer (pH 7.0) at 10^?3 M concentration was found to be very similar, but opposite to that of the K_ox and, correspondingly, of the redox potentials of the native enzymes. Attempts to induce intrinsic conformational preferences of the peptides by addition of trifluoroethanol led to enhancements of β-turn structures as reflected by the CD and Fourier transform ir spectra. The induced secondary structure, instead of aligning the tendencies of the excised fragments for loop formation with those of the intact proteins, was found to suppress the differences by significantly increasing the preference for cyclic monomers (≈ 90%). Similarly, operating under denaturing conditions, i.e., in 6M guanidinium hydrochloride, only for the trx peptide was the statistical product distribution obtained. For the remaining peptides, again a strong increase of cyclic monomer contents was observed that could not be correlated with dissolution of β-sheet type aggregates. The CD spectra are more consistent with the presence of ordered structure to some extent, possibly resulting from an hydrophobic collapse of the sparingly soluble peptides. The results of the oxidation experiments further support previous findings from thiol disulfide interchange equilibria, which clearly revealed a decisive role of the characteristic thioredoxin structural motif in dictating the redox properties of the enzymes. Point mutations in the active sites of the oxidoreductases allowed us to affect their redox potentials strongly, but apparently only in the constraint form of the three-dimensional structure as similar exchanges in the excised fragments did not produce the expected effect. This observation contrasts with numerous reports that the conformation of short disulfide loops is mainly dictated by the amino acid sequence. © 1994 John Wiley & Sons, Inc. © 1994 John Wiley & Sons, Inc.