Determination of the total concentration and speciation of Al(III) in tea infusions

The aluminium species in different tea infusions were investigated, by determining their stability constants and concentration. This was done for some particular samples using a simple experimental method based on the sorption of aluminium on the strongly sorbing resin Chelex 100, by a batch procedure. From the thermodynamic information obtained it is possible to calculate the concentration of the different species, and in particular that of the free metal ion, which is very important for evaluating the adsorption of aluminium on biological membranes. It was found that aluminium in the tea infusions here considered is present at high total concentration, approximately 0.1 mM, but mainly linked to strong complexes, for instance with side reaction coefficient higher than 10(5.11) at pH 3.95 in one case (tea 1). This could be the reason for the low toxicity of aluminium in tea. These strong complexes were not dissociated even in the presence of Chelex 100. In this case only a limiting value of the reaction coefficient could be evaluated. The presence of the very strong complexes was found in all the tea sample here considered. In two of the considered samples (one black and one green tea) a part of Al(III) was linked to less strong complexes, for example with a reaction coefficient 10(4.14) (tea 2, pH 4.20). The presence in the considered tea infusions of other substances able to complex aluminium was also detected, by the well known ligand titration procedure, at concentration ranging from 0.65 to 3.37 mM in three tea infusions, and at somewhat higher concentration in the case of the ready drink, which was also considered for comparison.     

Ectopic expression of maize polyamine oxidase and pea copper amine oxidase in the cell wall of tobacco plants

To test the feasibility of altering polyamine levels by influencing their catabolic pathway, we obtained transgenic tobacco (Nicotiana tabacum) plants constitutively expressing either maize (Zea mays) polyamine oxidase (MPAO) or pea (Pisum sativum) copper amine oxidase (PCuAO), two extracellular and H(2)O(2)-producing enzymes. Despite the high expression levels of the transgenes in the extracellular space, the amount of free polyamines in the homozygous transgenic plants was similar to that in the wild-type ones, suggesting either a tight regulation of polyamine levels or a different compartmentalization of the two recombinant proteins and the bulk amount of endogenous polyamines. Furthermore, no change in lignification levels and plant morphology was observed in the transgenic plants compared to untransformed plants, while a small but significant change in reactive oxygen species-scavenging capacity was verified. Both the MPAO and the PCuAO tobacco transgenic plants produced high amounts of H(2)O(2) only in the presence of exogenously added enzyme substrates. These observations provided evidence for the limiting amount of freely available polyamines in the extracellular space in tobacco plants under physiological conditions, which was further confirmed for untransformed maize and pea plants. The amount of H(2)O(2) produced by exogenously added polyamines in cell suspensions from the MPAO transgenic plants was sufficient to induce programmed cell death, which was sensitive to catalase treatment and required gene expression and caspase-like activity. The MPAO and PCuAO transgenic plants represent excellent tools to study polyamine secretion and conjugation in the extracellular space, as well as to determine when and how polyamine catabolism actually intervenes both in cell wall development and in response to stress.     

Stk33 is required for spermatid differentiation and male fertility in mice

Spermiogenesis is the final phase during sperm cell development in which round spermatids undergo dramatic morphological changes to generate spermatozoa. Here we report that the serine/threonine kinase Stk33 is essential for the differentiation of round spermatids into functional sperm cells and male fertility. Constitutive Stk33 deletion in mice results in severely malformed and immotile spermatozoa that are particularly characterized by disordered structural tail elements. Stk33 expression first appears in primary spermatocytes, and targeted deletion of Stk33 in these cells recapitulates the defects observed in constitutive knockout mice, confirming a germ cell-intrinsic function. Stk33 protein resides in the cytoplasm and partially co-localizes with the caudal end of the manchette, a transient structure that guides tail elongation, in elongating spermatids, and loss of Stk33 leads to the appearance of a tight, straight and elongated manchette. Together, these results identify Stk33 as an essential regulator of spermatid differentiation and male fertility.     

Plant regeneration from mesophyll protoplasts in commercial potato cultivars (Primura,Kennebec, Spunta,Desirée)

In view of an evaluation of the relative efficiency of different in vitro systems for mutant induction and isolation in potato, a procedure of plant regeneration from protoplasts of some potato (Solanum tuberosum L.) cultivars was developed. Four cultivars (Primura, Kennebec, Spunta, Desirée) were used for isolation, culture and regeneration of leaf mesophyll protoplasts.These lines were chosen because of their economic importance in Italy and in the case of cv. Desirée for the presence of markers useful in the morphological characterization of regenerants.Abbreviations MS Murashige and Skoog's medium - BAP 6-benzylaminopurine - 2,4 D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - GA ₃ gibberellic acid - ZT zeatin     

cDNA cloning of artichoke mottled crinkle virus RNA and localization and sequencing of the coat protein gene

We report the cDNA cloning of the genomic RNA of artichoke mottled crinkle virus (AMCV), which is a member of Tombusvirus group. AMCV has a monopartite positive sense RNA genome, which is not polyadenylated at the 3 end. The genome size is 4.8 kb.We have localized and sequenced the open reading frame (ORF) encoding the coat protein. Unlike most monopartite positive-strand RNA plant viruses, the ORF is not located near the 3 end, but like other members of the Tombusvirus group, CyRSV (cymbidium ringspot virus), TBSV-cherry (tomato bushy stunt virus cherry strain) and CNV (cucumber necrosis virus) it starts ca. 2.7 kb downstream of the 5 end and stops ca. 1 kb upstream of the 3 end. This ORF predicts a polypeptide chain of 387 amino acids.Comparison of the coat proteins of AMCV, TBSV-BS3, TBSV-cherry and CNV confirms that, within the Tombusvirus group, there exists a high degree of similarity among coat proteins but that this similarity is not uniformly distributed among domains. In particular, the N-terminal region, thought to make contact with the phosphate groups of the viral RNA, and the C-terminal region, considered the most immunogenic portion of the capsid, are found to be the least homologous.     

‘Phytoantibodies’: a general vector for the expression of immunoglobulin domains in transgenic plants

Sequences encoding the immunoglobulin heavy-chain variable (VH) domains were engineered in a new general purpose vector to transform plants via Agrobacterium. The expression of an isolated VH domain (IVD) after introduction into the plant genome has been monitored by northern, western and immuno-histochemical analysis. Immunoblotting showed that the polypeptide was stably expressed and accounted for up to 1% of the soluble protein fraction. It is therefore proposed that single immunoglobulin domains of suitable specificity expressed in plants may constitute an effective system to inhibit the activity of molecules involved in plant pathology or plant development.     

Molecular screening and fetal diagnosis of β-thalassemia in the Italian population

Summary This paper reports our experience of molecular screening and fetal diagnosis of -thalassemia in 457 at risk couples of Italian descent. Molecular screening was carried out by dot blot analysis on amplified DNA with oligonucleotide probes complementary to the eight most common mutations in Italians [39 (CT); 6 (-A); ⁺-87 (CG); ⁺ IVSI nt 110 (GA); IVSI nt 1 (GA); ⁺ IVSI nt 6 (TC); IVSII nt 1 (GA); ⁺ IVSII nt 745 (CG)]. By using this approach, we have been able to define the mutation in 92.8% of cases. The rest (all but four) were defined by direct sequencing and this led to the detection of nine rare mutations [76 (-C); ⁺ IVSI nt 5 (GA); ⁺ IVSI nt 5 (GC); ⁺ IVSI -1 (cod 30) (GC); ⁺-87 (CT), -290 bp del.; ⁺-101 (CT)], and to the characterization of a novel mutation consisting of the deletion of the G at the invariant AG of the IVSII splice acceptor site of the -globin gene ( IVSII nt 850-1 bp). In the remaining four cases, the -globin gene showed entirely normal sequences and the -globin gene cluster was intact, as indicated by Southern blot analysis. Fetal diagnosis was carried out by dot blot analysis with the oligonucleotide probes defined in the parents. The procedure is simple and reliable, and the results can be obtained within 1 week of sampling. No misdiagnosis has so far occurred. The results indicate that fetal diagnosis of -thalassemia by DNA analysis may be obtained in practically all cases (even in a population showing marked heterogeneity of -thalassemia) by the combination of dot blot analysis for detecting common mutations, and direct sequencing for defining those that are uncommon.