A New Real-Time PCR for the Detection of Plasmodium ovale wallikeri

It has been proposed that ovale malaria in humans is caused by two closely related but distinct species of malaria parasites: P. ovale curtisi and P. ovale wallikeri. We have extended and optimized a Real-time PCR assay targeting the parasite’s small subunit ribosomal RNA (ssrRNA) gene to detect both these species. When the assay was applied to 31 archival blood samples from patients diagnosed with P. ovale, it was found that the infection in 20 was due to P. ovale curtisi and in the remaining 11 to P. ovale wallikeri. Thus, this assay provides a useful tool that can be applied to epidemiological investigations of the two newly recognized distinct P. ovale species, that might reveal if these species also differ in their clinical manifestation, drugs susceptibility and relapse periodicity. The results presented confirm that P. ovale wallikeri is not confined to Southeast Asia, since the majority of the patients analyzed in this study had acquired their P. ovale infection in African countries, mostly situated in West Africa.     

Pectolytic clostridia isolated from sugar beet pulp silages in Italy

Twenty-eight strains of pectolytic clostridia were isolated from sugar beet pulp silages. Seventeen non-pigmented strains were presumed to be Clostridium acetobutylicum ; the remaining 11 pigmented strains were similar to Cl. felsineum. The addition of molasses to sugar beet pulps favoured the growth of other bacteria, particularly lactic acid organisms, whereas pectolytic clostridia were only occasionally found. The pectolytic clostridia promoted the structure loss of simulated silages. The use of molasses in sugar beet pulp ensiling was suggested to prevent texture loss of the ensiled mass.     

Chromosome-specific subsets of human alphoid DNA identified by a chromosome 2-derived clone

We have cloned an alphoid DNA fragment, pBS4D, from the DNA of a human-hamster hybrid cell line containing chromosome 2 as its only cytologically detectable human component. Under high stringency conditions, pBS4D hybridized in situ mostly to chromosome 2 and to a lesser extent to chromosomes 18 and 20. Restriction analysis using the DNA from selected somatic hybrid cell lines revealed that the genomic organization of this alphoid DNA differs on each of these three chromosomes.     

Importance of some factors on the dimorphism of Candida albicans

The passage between the yeast and mycelial forms of Candida albicans B 311-10 was studied by using the minimal syntehtic medium of Shepherd et al. [19] modified without biotin and with low glucose concentrations. It was observed that biotin, aminoacids and particularly pH are not important factors in the dimorphism of C. albicans. The only factor of notable importance in the passage of yeast form to mycelial form in C. albicans was glucose concentration.     

Development of tool use in a macaque and a gorilla

The development of the capacity to use a stick as a tool was tested in a macaque (Macaca fuscata) and a gorilla (Gorilla gorilla gorilla) infants that had previously shown to be able to use strings and supports as dragging tools. Subjects were tested between 15 and 38 months of age. Different levels of competence between the subjects emerged over testing. The macaque developed a stereotyped strategy to cope with the problem, only getting random successes, whilst the gorilla developed a flexible strategy and revealed to be able to mentally represent the solution of the problem. In fact, when not successful using the stick, the gorilla thought out an alternative strategy, choosing and adapting a new object to use it as a tool.     

Multiple variants in subtelomeric regions of normal karyotypes

We describe a human genomic cosmid clone, 56.1.1, that contains subtelomeric sequences present on multiple human chromosomes. In particular, using fluorescence in situ hybridization, we have identified 16 sites of hybridization on 12 chromosomes. In a sample of 8 unrelated individuals, 10 of these sites showed interindividual variation. Co-hybridization with other polymorphic probes allowed us to demonstrate cytologically heterozygosity at three sites in six individuals. The chromosomal distribution of hybridization sites in a family strongly suggests that these variants are inherited in a Mendelian fashion. These data show that subtelomeric repeats are a rich source of genetic variability. Possible mechanisms of generation of such variants are discussed.     

Mischarging Escherichia coli tRNAPhe with L-4'-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenylalanine, a photoactivatable analogue of phenylalanine

The Boc-protected derivative of a photoactivatable, carbene-generating analogue of phenylalanine, L-4'-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenylalanine [(Tmd)Phe], was used to acylate 5'-O-phosphorylcytidylyl(3'-5')adenosine (pCpA). A diacyl species was isolated which upon successive treatments with trifluoroacetic acid and 0.01 M HCl yielded a 1:1 mixture of 2'(3')-O-(Tmd)phenylalanyl-pCpA and of its 2'-5'-phosphodiester isomeric form. Adapting a procedure introduced by Hecht's group [Heckler, T.G., Chang, L.H., Zama, Y., Naka, T., Chorghade, M.S., & Hecht, S.M. (1984) Biochemistry 23, 1468-1473], brief incubation of a 15 molar excess of this material with Escherichia coli tRNAPhe, missing at the acceptor stem the last two nucleotides (pCpA), in the presence of T4 RNA ligase and ATP afforded "chemically misaminoacylated" tRNAPhe in approximately 50% yield. Following chromatographic purification on DEAE-Sephadex A-25, benzoylated DEAE-cellulose, and Bio-Gel P-6, the misaminoacylated tRNAPhe was characterized by (i) urea-polyacrylamide gel electrophoresis, (ii) enzymatic reaminoacylation under homologous conditions following chemical deacylation, and (iii) its ability to stimulate protein synthesis in an in vitro translation system which, through the addition of the phenylalanyl-tRNA synthetase inhibitor phenylalaninyl-AMP, was unable to charge its endogenous tRNAPhe. The data demonstrate that we have prepared a biologically active misaminoacylated tRNAPhe.     

Glycerol stress and platelet integrity

Platelet response to glycerol gradient was studied using a few in vitro parameters. These were platelet count, mean platelet size, platelet response to hypotonic stress (PHRS), and collagen-induced platelet aggregation. An equal volume of 1-10% (w/v) glycerol in plasma was added at once to the platelet concentrate resulting in 0.5-5% (w/v) final glycerol concentration. The concentrate was kept at 22 degrees C for 60 min. Platelets were then separated by one centrifugation and resuspended in glycerol-free plasma. A loss in platelet count was observed when the gradient of glycerol was more than 3%. This was associated with an increase in mean cell size and a reduction in aggregability. With 5% glycerol stress, a loss of 30% in cell count, an increase in 18% in cell size, and a 78% loss in aggregability was observed. Declining of PRHS was shown already with a 1% glycerol gradient and 69% of this function was suppressed by 5% glycerol stress. In other experiments, 5% glycerol was first added, them removed in 5 steps with a gradient of 1% each. When time interval between each step was less than 0.5 min, platelet loss and PRHS reduction were 17 and 47% respectively. These values were gradually improved to 4% and 11-20%, respectively, as increasing time interval up to 15 min. It was concluded that a gradient of 1% glycerol and a 15-min interval for each step minimizes the detrimental osmotic stress on platelets while glycerol is added or removed. Our findings may lead up to devising an improved protocol for platelet cryopreservation with glycerol.