Human globin chain separation by isoelectric focusing

Human globin chain separation, a key procedure in the study of hemoglobinopathies, is routinely performed by chromatography on carboxymethylcellulose. This method, though relatively easy and highly reliable, is expensive and time consuming. A new procedure, based on isoelectric focusing, is presented which allows the simultaneous separation of globin chains from multiple samples (at least 20 per gel slab). The method is rapid, inexpensive and can be easily carried out in clinical laboratories, and its high sensitivity allows the identification of radioactive bands even with minute amounts of labelled material. A new phenomenon, called the 'Nonidet P-40 effect', which greatly enhances the separation between gamma and beta chains by binding to these two chains and shifting their pI values in opposite directions, is described.     

Role of drugs in recovery of metabolic function of rat brain following severe hypoglycemia

Severe hypoglycemia with isoelectric EEG induced extensive deterioration of the energy state and gross alteration of amino acid contents on the rat cerebral and cerebellar cortex. During recovery, tissue glucose concentration returned to normal, while both lactate and pyruvate concentrations increased to above normal. In the recovery period, the ATP concentration increased but the adenine nucleotide pool remained reduced, even if the ADP and AMP contents were close to normal. Phosphocreatine was restored to normal concentration with reciprocal changes in creatine content. During recovery there was a rise in glutamate and glutamine concentrations, gamma-aminobutyrate content returning to normal value. Ammonia and aspartate decreased below normal, while alanine increased above normal. The effect of some pharmacological agents on the posthypoglycemic recovery was tested: (a) Ergot alkaloids (dihydroergocristine, dihydroergocriptine, dihydroergocornine); (b) Vinca minor alkaloids (vincamine TPS, (–) eburnamonine); (c) Rauwolfia serpentina alkaloids (reserpine, raubasine); (d) synthetic agent (piracetam). During the posthypoglycemic recovery, these different agents exhibited different, or even contrasting, interferences on glycolytic metabolites, amino acids and energy-rich phosphates. The metabolic alterations in the cerebellar cortex were qualitatively of the same character of those in neocortex. However, the metabolic alterations were less extensive and more sensitive to drug action.     

Detection of DNA in Ancient Bones Using Histochemical Methods

We describe histochemical techniques for detecting DNA within the osteocytic lacunae of ancient bones. The bones examined were fragments of femurs from two human individuals found in the Pompeian C. I. Polybius house and fragments of metacarpals from two horses (Equus sp.) found in the Pompeian “Casti Amanti” house. Both buildings were buried by the 79 A. D. Vesuvius eruption. Fragments of femurs from a modern horse, a modern swine and a modern amphibian also were studied as controls. Some bone sections were stained with two different DNA-specific fluorochromes, 4' -' 6-diamidino-2-phenylindole (DAPI) and chromomycin A3 (CMA), while others were stained by the Feulgen reaction. All of the techniques gave a positive reaction within the osteocytic lacunae. Histological analysis of the undecalcified, ground and unstained sections agreed well with results of bone sections stained with either the fluorochromes or the Feulgen reaction. Bones showing good histology also were positive by our DNA-specific stain. Histochemical and histological analyses correlated well with the success of DNA extraction and amplification. Using conventional DNA-specific histochemical techniques in conjunction with histological analysis can be useful in the study of DNA extracted from ancient bone remains while reducing both the amount of time and cost.     

Engineering of the redox imbalance of Fusarium oxysporum enables anaerobic growth on xylose

Dissimilatory nitrate reduction metabolism, of the natural xylose-fermenting fungus Fusarium oxysporum, was used as a strategy to achieve anaerobic growth and ethanol production from xylose. Beneficial alterations of the redox fluxes and thereby of the xylose metabolism were obtained by taking advantage of the regeneration of the cofactor NAD(+) during the denitrification process. In batch cultivations, nitrate sustained growth under anaerobic conditions (1.21 g L(-1) biomass) and simultaneously a maximum yield of 0.55 moles of ethanol per mole of xylose was achieved, whereas substitution of nitrate with ammonium limited the growth significantly (0.15 g L(-1) biomass). Using nitrate, the maximum acetate yield was 0.21 moles per mole of xylose and no xylitol excretion was observed. Furthermore, the network structure in the central carbon metabolism of F. oxysporum was characterized in steady state. F. oxysporum grew anaerobically on [1-(13)C] labelled glucose and unlabelled xylose in chemostat cultivation with nitrate as nitrogen source. The use of labelled substrate allowed the precise determination of the glucose and xylose contribution to the carbon fluxes in the central metabolism of this poorly described microorganism. It was demonstrated that dissimilatory nitrate reduction allows F. oxysporum to exhibit typical respiratory metabolic behaviour with a highly active TCA cycle and a large demand for NADPH.     

Advances in quantitative trait analysis in yeast

Understanding the genetic mechanisms underlying complex traits is one of the next frontiers in biology. The budding yeast Saccharomyces cerevisiae has become an important model for elucidating the mechanisms that govern natural genetic and phenotypic variation. This success is partially due to its intrinsic biological features, such as the short sexual generation time, high meiotic recombination rate, and small genome size. Precise reverse genetics technologies allow the high throughput manipulation of genetic information with exquisite precision, offering the unique opportunity to experimentally measure the phenotypic effect of genetic variants. Population genomic and phenomic studies have revealed widespread variation between diverged populations, characteristic of man-made environments, as well as geographic clusters of wild strains along with naturally occurring recombinant strains (mosaics). Here, we review these recent studies and provide a perspective on how these previously unappreciated levels of variation can help to bridge our understanding of the genotype-phenotype gap, keeping budding yeast at the forefront of genetic studies. Not only are quantitative trait loci (QTL) being mapped with high resolution down to the nucleotide, for the first time QTLs of modest effect and complex interactions between these QTLs and between QTLs and the environment are being determined experimentally at unprecedented levels using next generation techniques of deep sequencing selected pools of individuals as well as multi-generational crosses.     

Loss of soluble N-ethylmaleimide-sensitive factor attachment protein α (αSNAP) induces epithelial cell apoptosis via down-regulation of Bcl-2 expression and disruption of the Golgi

Intracellular trafficking represents a key mechanism that regulates cell fate by participating in either prodeath or prosurvival signaling. Soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein α (αSNAP) is a well known component of vesicle trafficking machinery that mediates intermembrane fusion. αSNAP increases cell resistance to cytotoxic stimuli, although mechanisms of its prosurvival function are poorly understood. In this study, we found that either siRNA-mediated knockdown of αSNAP or expression of its dominant negative mutant induced epithelial cell apoptosis. Apoptosis was not caused by activation of the major prodeath regulators Bax and p53 and was independent of a key αSNAP binding partner, NSF. Instead, death of αSNAP-depleted cells was accompanied by down-regulation of the antiapoptotic Bcl-2 protein; it was mimicked by inhibition and attenuated by overexpression of Bcl-2. Knockdown of αSNAP resulted in impairment of Golgi to endoplasmic reticulum (ER) trafficking and fragmentation of the Golgi. Moreover, pharmacological disruption of ER-Golgi transport by brefeldin A and eeyarestatin 1 or siRNA-mediated depletion of an ER/Golgi-associated p97 ATPase recapitulated the effects of αSNAP inhibition by decreasing Bcl-2 level and triggering apoptosis. These results reveal a novel role for αSNAP in promoting epithelial cell survival by unique mechanisms involving regulation of Bcl-2 expression and Golgi biogenesis.     

Immunoglobulin superantigen protein L induces IL-4 and IL-13 secretion from human Fc epsilon RI+ cells through interaction with the kappa light chains of IgE

Peptostreptococcus magnus protein L is a multidomain bacterial surface protein that correlates with virulence. It consists of up to five homologous Ig-binding domains (B1-B5) that interact with the variable domain of Ig kappa L chains. Intact protein L stimulates the synthesis and the release of IL-4 and IL-13 from human basophils in vitro. A protein L fragment covering the Ig-binding domains B1-B4 also induced IL-4 and IL-13 release from basophils. There was an excellent correlation (r(s) = 0.82; p < 0.001) between the maximal percent IL-4 release induced by protein L and that induced by anti-IgE and between intact protein L and the B1-B4 fragment (r(s) = 0.90; p < 0.01). Removal of IgE bound to basophils markedly reduced the IL-4 release induced by anti-IgE, protein L, and B1-B4. Preincubation of basophils with protein L or anti-IgE caused complete cross-desensitization to subsequent challenge with the heterologous stimulus. IgE purified from myeloma patients PS and PP (lambda chains) blocked anti-IgE-induced IL-4 release, but not the releasing activity of protein L. In contrast, IgE purified from myeloma patient ADZ (kappa chains) blocked both anti-IgE- and protein L-induced secretion. Cyclosporin A, but not cyclosporin H, inhibited protein L-induced release of IL-4 and IL-13 from basophils. Thus, protein L acts as a bacterial Ig superantigen to induce the synthesis and release of IL-4 and IL-13 from basophils by interacting with kappa L chains of the IgE isotype.     

A Permanent Automated Real-Time Passive Acoustic Monitoring System for Bottlenose Dolphin Conservation in the Mediterranean Sea

Within the framework of the EU Life+ project named LIFE09 NAT/IT/000190 ARION, a permanent automated real-time passive acoustic monitoring system for the improvement of the conservation status of the transient and resident population of bottlenose dolphin (Tursiops truncatus) has been implemented and installed in the Portofino Marine Protected Area (MPA), Ligurian Sea. The system is able to detect the simultaneous presence of dolphins and boats in the area and to give their position in real time. This information is used to prevent collisions by diffusing warning messages to all the categories involved (tourists, professional fishermen and so on). The system consists of two gps-synchronized acoustic units, based on a particular type of marine buoy (elastic beacon), deployed about 1 km off the Portofino headland. Each one is equipped with a four-hydrophone array and an onboard acquisition system which can record the typical social communication whistles emitted by the dolphins and the sound emitted by boat engines. Signals are pre-filtered, digitized and then broadcast to the ground station via wi-fi. The raw data are elaborated to get the direction of the acoustic target to each unit, and hence the position of dolphins and boats in real time by triangulation.     

New insights into the Lpt machinery for lipopolysaccharide transport to the cell surface: LptA-LptC interaction and LptA stability as sensors of a properly assembled transenvelope complex

Lipopolysaccharide (LPS) is a major glycolipid present in the outer membrane (OM) of Gram-negative bacteria. The peculiar permeability barrier of the OM is due to the presence of LPS at the outer leaflet of this membrane that prevents many toxic compounds from entering the cell. In Escherichia coli LPS synthesized inside the cell is first translocated over the inner membrane (IM) by the essential MsbA flippase; then, seven essential Lpt proteins located in the IM (LptBCDF), in the periplasm (LptA), and in the OM (LptDE) are responsible for LPS transport across the periplasmic space and its assembly at the cell surface. The Lpt proteins constitute a transenvelope complex spanning IM and OM that appears to operate as a single device. We show here that in vivo LptA and LptC physically interact, forming a stable complex and, based on the analysis of loss-of-function mutations in LptC, we suggest that the C-terminal region of LptC is implicated in LptA binding. Moreover, we show that defects in Lpt components of either IM or OM result in LptA degradation; thus, LptA abundance in the cell appears to be a marker of properly bridged IM and OM. Collectively, our data support the recently proposed transenvelope model for LPS transport.     

Characterization of conjugative transposon Tn5251 of Streptococcus pneumoniae

Abstract Tn5251 belongs to the Tn916-Tn1545 family of conjugative transposons (CT) and was found integrated into CT Tn5252 , to form the composite element Tn5253 of Streptococcus pneumoniae . We show that Tn5251 is identical in structure and size to Tn916 . DNA sequence analysis of a 4,419-bp segment containing the tet(M) gene showed that only 73 nucleotides out of 4,419 were different in the the two CT. Essentially all differences (66 / 73) were clustered in a 688-bp segment of tet(M) , which was 90% identical to Tn916 and 100% identical to the tet(M) genes of Tn1545 from S. pneumoniae and pOZ101 from Neisseria gonorrhoeae . DNA sequence analysis of the Tn5251/Tn5252 junction fragments allowed us (i) to determine Tn5251 termini, (ii) to define the 6-bp coupling sequences flanking the CT, and (iii) to infer the structure of the integration site ( attB ) of Tn5251 into Tn5252 . Conjugal transfer of Tn5251 independent from Tn5253 could not be detected, even if we could show excision and formation of Tn5251 circular intermediates at a level of 5.4 copies per 10⁶ chromosomes.