Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells

Rapid phylogenetic identification of single microbial cells was achieved with a new staining method. Formaldehyde-fixed, intact cells were hybridized with fluorescently labeled oligodeoxynucleotides complementary to 16S ribosomal RNA (rRNA) and viewed by fluorescence microscopy. Because of the abundance of rRNA in cells, the binding of the fluorescent probes to individual cells is readily visualized. Phylogenetic identification is achieved by the use of oligonucleotides (length 17 to 34 nucleotides) that are complementary to phylogenetic group-specific 16S rRNA sequences. Appropriate probes can be composed of oligonucleotide sequences that distinguish between the primary kingdoms (eukaryotes, eubacteria, archaebacteria) and between closely related organisms. The simultaneous use of multiple probes, labeled with different fluorescent dyes, allows the identification of different cell types in the same microscopic field. Quantitative microfluorimetry shows that the amount of an rRNA-specific probe that binds to Escherichia coli varies with the ribosome content and therefore reflects growth rate.     

Early warnings of regime shifts: a whole-ecosystem experiment

Catastrophic ecological regime shifts may be announced in advance by statistical early warning signals such as slowing return rates from perturbation and rising variance. The theoretical background for these indicators is rich, but real-world tests are rare, especially for whole ecosystems. We tested the hypothesis that these statistics would be early warning signals for an experimentally induced regime shift in an aquatic food web. We gradually added top predators to a lake over 3 years to destabilize its food web. An adjacent lake was monitored simultaneously as a reference ecosystem. Warning signals of a regime shift were evident in the manipulated lake during reorganization of the food web more than a year before the food web transition was complete, corroborating theory for leading indicators of ecological regime shifts.     

Pattern of variation for seed size traits and molecular markers in Italian germplasm of Phaseolus coccineus L.

Variation in Italian germplasm of Phaseolus coccineus L. was assessed for seed traits and molecular markers. A total of 130 seeds and seedlings, five for each of 21 Italian landraces, an Italian commercial cultivar and four Mesoamerican landraces of P. coccineus, were analysed using seven selected PCR markers: three RAPDs, two ISSRs and two ETs. Seed weight of the Mesoamerican landraces was ≤1 g, whereas that of the Italian landraces varied from 1 g to 2.5 g and was related to their origin. Oval shape was more frequent, with round shape observed only in Mesoamerican landraces. Three seed coat colours were observed: white, violet mottled or spotted black and buff spotted brown, also this trait was related to the origin. The level of polymorphism detected by molecular markers was low but with significant discriminant power. ISSRs were the most effective markers prone to unravel molecular polymorphism. The within accession component of variation exceeded that among accessions, as expected for an allogamous species. However correct classification of the individuals was achieved performing either discriminant analysis of the seed phenotypic traits or cluster analysis of seedling similarity measure based on the whole banding patterns obtained by the three marker types. Our data suggest that the Italian farmers, starting with ancestral Mesoamerican runner bean introductions in Europe, bred their own landraces through selection for seed size and seed coat colour, but occasional gene flow maintained variability within landraces bred by different farmers in the same Italian Region. Selection favored molecular and seed trait uniformity within several landraces making them suitable for certification.     

Improvement of seed yield in Vicia faba L. By using experimental mutagenesis. II. Comparison of gamma-radiation and ethyl-methane-sulphonate (EMS) in production of morphological mutants

Summary Gamma-rays and EMS have been used in two dosages to test their effectiveness in inducing mutations in one major (cv. Aguadulce) and one minor (cv. Manfredini) Vicia faba cultivar. The plants of the M₁ and M₂ generations showed reduced emergence, survival, and fertility. EMS was more effective than gammarays in inducing mutations giving a mutation frequency in M₁ and a frequency of M₂ mutants from two to four times higher. The two mutagenic agents do not seem to differ in mutation spectrum. The minor cv. Manfredini was more sensitive to the lower dosage of gamma-rays than the major cv. Aguadulce. Both mutagens proved to be effective in enlarging the morphological variation of both cultivars.Research work supported by C.N.R. special grant I.P.R.A.-Sub-project 1. Paper N. 515.     

Variation in durum wheat populations from three geographical origins. I. Material and spike characteristics

Summary Sixteen durum wheat landraces. i.e. 6 from Algeria, 5 from Ethiopia and 5 from Italy were analysed in order to ascertain the amount of variation for all 11 spike characters. For each population the differences among spike progenies were significant for all the observed characters. Principal component analysis showed that 4 factors accounted for 80% of the total variation. Component 1 accounted for 32% of the total variation and was associated with spike width, spike density, number of kernels per spikelet and kernel weight. Component 2 accounted for 24% of the total variation and was associated with number of fertile spikelets, total number of spikelets and spike length. In a scatter diagram of all 1127 spike progenies for the first two principal components, the material from Ethiopia and Italy clustered at two opposite sides, while the material from Algeria had the largest swarm and overlapped the other two groups of material. Differences among populations from the same origin were found. The ecological and anthropological causes that may have played a role in the creation of the observed variation in the examined populations, are discussed.     

Proximate composition and mineral content of selected Tanzanian vegetables and the effect of traditional processing on the retention of ascorbic acid,riboflavin and thiamine

Proximate composition, mineral content and the effect of traditional processing practices on the retention of ascorbic acid, riboflavin and thiamine were studied using amaranth, cowpea, peanut, pumpkin and sweetpotato leaves. Results of this study indicated that, crude protein, crude fat, carbohydrate and ash contents were in the range of 20.64–46.56 percent, 2.57–4.34 percent, 35.43–63.50 percent and 8.92–15.69 percent respectively. The mineral content per 100 g of fresh vegetables was in the range of 83.64–229.34 mg, 145.97–780.19 mg, 11.56–21.31 mg, 43.02–110.30 mg, 0.96–5.90 mg and 0.40–2.24 mg for Ca, K, Na, Mg, Fe and Zn respectively. For ascorbic acid, riboflavin and thiamine, concentrations in 100 g of fresh vegetables were in the range of 43.78–89.00 mg, 0.62–1.71 mg and 0.09–0.30 mg respectively. The traditional processing practices of sun/shade drying and storing in ventilated containers resulted in a significant (P<0.05) decrease in ascorbic acid, riboflavin and thiamine for all vegetables. Conventional blanching and cooking for up to 15 minutes resulted in a significant (P<0.05) increase in riboflavin content in cowpea, peanut and pumpkin greens while in amaranth and sweetpotato leaves, thermal processing resulted in a significant (P<0.05) decrease in the vitamin. Based on the results of this study, the vegetables were good dietary sources of minerals, carbohydrate and protein.     

Gene targeting in stem cells from individuals with osteogenesis imperfecta

Adult stem cells offer the potential to treat many diseases through a combination of ex vivo genetic manipulation and autologous transplantation. Mesenchymal stem cells (MSCs, also referred to as marrow stromal cells) are adult stem cells that can be isolated as proliferating, adherent cells from bones. MSCs can differentiate into multiple cell types present in several tissues, including bone, fat, cartilage, and muscle, making them ideal candidates for a variety of cell-based therapies. Here, we have used adeno-associated virus vectors to disrupt dominant-negative mutant COL1A1 collagen genes in MSCs from individuals with the brittle bone disorder osteogenesis imperfecta, demonstrating successful gene targeting in adult human stem cells.     

Identification of Ruscus steroidal saponins by HPLC-MS analysis

A novel HPLC-UV method has been developed for the fingerprint analysis of the steroidal saponins in the rhizomes of three Ruscus species (Ruscus aculeatus, Ruscus hypoglossum and Ruscus colchicus). Saponins were identified by HPLC-ESI-MS. During the study a new major saponin was detected in the rhizomes of R. hypoglossum and R. colchicus. The structure of the new compound was defined as 1-O-[alpha-L-rhamnopyranosyl-(1-->2)-6-O-acetyl-beta-D-galactopyranosyl]-1beta,3beta,22xi,26-tetrahydroxy-furost-5(6)-en-26-O-beta-D-glucopyranoside (8) by spectral analysis.