Genetic diversity of durum wheat as determined by AFLP in fluorescence

The DNA of fifteen Italian cultivars of durum wheat (Triticum turgidum L. ssp. durum) were analyzed by in fluorescence amplified fragment length polymorphism (fAFLP) in order to obtain the characteristic fingerprintings of genotypes and assess their genetic relatedness. Among 64 combinations of fluorescence labelled primers, three different combinations were chosen as producing a total of 6630 AFLP fragments, 2277 (34.3 %) of them being polymorphic. By using this fAFLP methodology a DNA fingerprinting of each durum wheat cultivar was generated for genotype identification. Analysis of the genetic relationships show the low variability among durum wheat cultivars.     

Limb reconstruction by free-tissue transfer combined with the Ilizarov method.

In four complex cases of extremity reconstruction, we have been able to overcome the problems of combined bone and soft-tissue loss and length discrepancy by a combination of free-tissue transfer and the Ilizarov method of slow distraction. It is our observation that gradual distraction of a free tissue is a safe and viable procedure; the free tissue tolerates the pins of the circular external fixator well, and there is an equal degree of distraction and regeneration of the transferred free tissue and the native recipient tissue without evidence of wound dehiscence. Corticotomy through free tissue and in close proximity to vascularized bone is safe, with the subsequent bone regeneration not unlike that of normal bone. Manipulation by slow distraction does not appear to compromise the vasculature of the recipient bed for later microsurgical procedures or endanger the axial flow pattern of the transferred free tissue.     

Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space of Escherichia coli

Summary The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A--lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5pH6.0) and at low temperatures. These conditios were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn²⁺ ions. Offsprint requests to: G. Georgiou     

The peculiar binding properties of 4''-deoxy,4''-iododoxorubicin to isolated DNA and 175 bp nucleosomes.

The thermodynamic parameters governing the interaction of 4'-deoxy,4'-iododoxorubicin (4'-IAM) to double stranded DNA or 175 bp nucleosomes have been evaluated at different ionic strength and temperature conditions by means of fluorescence techniques. The iodo-anthracycline exhibits quite different characteristics from the parent compounds adriamycin (AM) and daunomycin (DM) and other second generation derivatives. In fact, the contribution of electrostatic interactions to the total free energy of binding is rather poor and the changes in enthalpy, usually high and negative, are low and eventually positive. Unlike other members of its family, 4'-IAM exhibits preference for the nucleosomal structure. In addition, its binding to isolated DNA is remarkably cooperative. Circular dichroism studies show changes in the geometry of the intercalation complex when the drug binds to nucleosomes. The possibility for the iodo-sugar moiety to act as an alkylating or free-radical producing species was also considered as an alternative mechanism of action. However, no evidence was obtained to support these hypotheses. Thus the major differences observed in DNA-binding in comparison to parent anthracyclines appear to be mostly related to the lower pKa and higher lipophilicity exhibited by 4'-IAM.     

Starch-Ampicillin Agar for the Quantitative Detection of Aeromonas hydrophila

Interest in Aeromonas hydrophila as a food-borne and human pathogen is increasing. Isolation media from the clinical laboratory were evaluated for food use and either did not give quantitative recovery of A. hydrophila or did not permit ready differentiation of A. hydrophila from the background microflora. A new medium was developed which permitted quantitative recovery of A. hydrophila from foods. The medium consisted of phenol red agar base (Difco Laboratories), soluble starch (10 g/liter), and ampicillin (10 mg/liter). All foods surveyed contained A. hydrophila. Foods sampled included red meats, chicken, raw milk, and seafood (fish, shrimp, scallops, crab, and oysters). The count of A. hydrophila at the time of purchase ranged from 1 × 10²/g (lower limit of detection) to 5 × 10⁵/g. In most instances, the count of A. hydrophila increased during 1 week of storage at 5°C. The starch-ampicillin agar developed permitted rapid quantitative recovery of A. hydrophila from foods in the presence of very large numbers of competing microflora.     

Interaction of calcium ions with peptide hormones of the gastrin family

The interactions of Des-Trp¹-Nle¹²-minigastrin I (Nle¹¹-HG-13) and Nle¹⁵-little gastrin I (Nle¹⁵-HG-17) with calcium ions have been investigated in water and in trifluoroethanol solution using uv and CD absorption techniques. Both hormones strongly interact with Ca²⁺ in the organic medium. In the case of Nle¹¹-HG-13, the near-uv chiroptical properties (dominated by the transitions of the Trp residue in the C-terminal tetrapeptide sequence) indicate that three metal ions per mole of hormone are involved in the binding process. From the different response of near-uv and far-uv CD properties to the addition of calcium and from the change of the CD spectra in the aromatic absorption region, it is concluded that the biologically important C-terminal sequence is directly involved in the interaction with calcium. Elongation of the peptide chain from Nle¹¹-HG-13 to Nle¹⁵-HG-17 (Nle¹⁵-little gastrin I) does not provide any additional binding site for calcium ions. The change of the CD properties in the near- and far-uv indicates that three metal ions per mole of hormone are involved in the binding process. The dichroic absorption in the aromatic region indicates that only one of the two Trp residues of the little gastrin analog is sensitive to the presence of calcium. On the assumption that the variation of the CD properties is proportional to the extent of calcium binding, the binding constants K₁, K₂, and K₃ have been estimated roughly. Two similar sets of binding constants have been found, with K₁ ≥ 10⁶M^?1 and K₃ of the order of 10⁵M^?1, indicating similar binding sites in the two hormones with high affinity for calcium ions.