Characterization of a <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Strain Able to Produce Tyramine and Partial Cloning of a Putative Tyrosine Decarboxylase Gene

The aim of this article was to analyze the ability of wine Lactobacillus plantarum strains to form tyramine. Preliminary identification of L. plantarum strains was performed by amplification of the recA gene. Primers pREV and PlanF, ParaF and PentF were used respectively as reverse and forward primers in the polymerase chain reaction tests as previously reported. Furthermore, the gene encoding for the tyrosine decarboxylase (TDC) was partially cloned from one strain identified as L. plantarum. The strain was further analyzed by 16S rDNA sequence and confirmed as belonging to L. plantarum species. The tyrosine decarboxylase activity was investigated and tyramine was determined by the high-performance liquid chromatography method. Moreover, a negative effect of sugars such as glucose and fructose and L-malic acid on tyrosine decarboxylase activity was observed. The results suggest that, occasionally, L. plantarum is able to produce tyramine in wine and this ability is apparently confined only to L. plantarum strains harboring the tdc gene.     

Ribonucleic Acid Polymerase in a Thermosensitive Sporulation Mutant (ts-4) of Bacillus subtilis

Partially synchronized cultures of a Bacillus subtilis thermosensitive sporulation mutant (ts-4) and the 168 try⁻try⁻ (168tt) parental strain were infected with the virulent phage e at various times during their growth cycle at 30 and 42 C (permissive and restrictive temperatures, respectively). It was shown that at the restrictive temperature the burst size in the parental strain was two- to threefold lower than in the ts-4 mutant. No such difference was observed at the permissive temperature. However, the time at which this difference was observed excludes a correlation between the burst size and initiation of the sporulation process. It was further found that the capacity to transcribe in vitro phage e deoxyribonucleic acid by partially purified ribonucleic acid (RNA) polymerase from both strains decreased sharply if the source of enzyme was sporulating cells instead of vegetative ones. However, a similar decrease, although to a lesser extent, was observed with the RNA polymerase isolated from stationary-phase cells of the ts-4 mutant grown at the nonpermissive temperature, or with the enzyme derived from several other zero-stage sporulation mutants. At no time was a structural modification in the β subunits of the RNA polymerase observed during growth of the sporulating bacteria. We have also shown that, in addition to the relatively low specific activity of the RNA polymerase, the level of the intracellular protease activity is about 15-fold lower in the ts-4 mutant grown at the restrictive temperature than that of the parental strain grown at the same temperature. At the permissive temperature no such difference was observed between these two strains. However, the present data do not allow us to establish a correlation among the low content of intracellular protease, the weak specific activity of the RNA polymerase, and the loss of the sporulation capacity in the ts-4 mutant grown at the restrictive temperature.     

Zooplankton (Cladocera) species turnover and long-term decline of Daphnia in two high mountain lakes in the Austrian Alps

We investigated long-term succession of sedimentary cladoceran assemblages in two morphologically divergent mountain lakes by utilizing sediment traps and previously available sediment data. We aimed to detect lake-specific changes in pelagic communities potentially attributable to climate warming under the presumption that lakes and biotic communities may respond individually to the same regional climatic forcing. Both lakes showed a similar community turnover, as Daphnia was first replaced by Chydorus cf. sphaericus and during the twentieth century by the latest colonizer Bosmina. The community succession was similar among the lakes and consistent with the regional temperature increase, although the timing of community shift, the magnitude of change, and taxa in question differed. Decline of Daphnia mismatched with historical fish stockings, but the eventual extirpation of Daphnia in one of the lakes corresponded to the start of fish introductions. We propose that the observed shifts were mainly governed by increasing temperatures and its limnoecological consequences (e.g., deeper mixing). We suggest that Bosmina distribution may be extending to lakes at higher altitudes as a response to climate warming, and that it may replace Daphnia as the key component of pelagic alpine food webs by coping in interspecific resource competition under changed limnological regimes.     

Three-step purification of lipopolysaccharide-free, polyhistidine-tagged recombinant antigens of Mycobacterium tuberculosis

Previous work has shown that the study of host immune responses against Mycobacterium tuberculosis, the causative agent of tuberculosis, requires the availability of multiple mycobacterial antigens. Since purification of protein from M. tuberculosis cells is extremely cumbersome, we developed a protocol for purifying milligram amounts of ten recombinant antigens of M. tuberculosis from E. coli cells. Purified proteins were immunologically active and free of contaminants that confound interpretation of cell-based immunological assays. The method utilizes a three-step purification protocol consisting of immobilized metal-chelate affinity chromatography, size exclusion chromatography and anion-exchange chromatography. The first two chromatographic steps yielded recombinant protein free of protein contaminants, while the third step (anion-exchange chromatography) efficiently removed E. coli lipopolysaccharide, a potent polyclonal activator of lymphoid cells. The recombinant proteins were immunologically indistinguishable from their native (i.e., purified from M. tuberculosis) counterparts. Thus the method provides a way to utilize recombinant proteins for immunological analyses that require highly purified antigens.     

Invasion genetics of the Eurasian spiny waterflea: evidence for bottlenecks and gene flow using microsatellites

The Eurasian spiny waterflea (Bythotrephes longimanus) is a predacious zooplankter that has increased its range in Europe and is rapidly invading inland water-bodies throughout North America's Great Lakes region. To examine the genetics of these invasions, we isolated five microsatellite DNA loci with between 5 and 19 alleles per locus. We sampled three populations where B. longimanus has been historically present (Switzerland, Italy, and Finland) as well as an introduced European population (the Netherlands) and three North American populations (Lakes Erie, Superior, Shebandowan). Consistent with a bottleneck during colonization (i.e. founder effect), average heterozygosities of the four European populations ranged from 0.310 to 0.599, and were higher than that of three North American populations (0.151-0.220). Pairwise F(ST) estimates among North American populations (0.002-0.063) were not significantly different from zero and were much lower than among European populations (0.208-0.474). This is consistent with a scenario of high gene flow among North American populations relative to that of European ones. Contrary to an invasion bottleneck, however, Erie and Superior populations contained similar numbers of rare alleles as European populations. Assignment tests identified several migrant genotypes in all introduced populations (the Netherlands, Erie, Superior, Shebandowan), but rarely in native ones (Switzerland, Italy and Finland). A large number of genotypes from North America were assigned to our Italian population suggesting a second, previously unidentified, invasion source somewhere in the region of northern Italy. Together, our results support an invasion bottleneck for North American populations that has been largely offset by gene flow from multiple native sources, as well as gene flow among introduced populations.     

Mitochondrial Capture Misleads about Ecological Speciation in the Daphnia pulex Complex

The North American ecological species Daphnia pulicaria and Daphnia pulex are thought to have diverged from a common ancestor by adaptation to sympatric but ecologically distinct lake and pond habitats respectively. Based on mtDNA relationships, European D . pulicaria is considered a different species only distantly related to its North American counterpart, but both species share a lactate dehydrogenase (Ldh) allele F supposedly involved in lake adaptation in North America, and the same allele is also carried by the related Holarctic Daphnia tenebrosa . The correct inference of the species’ ancestral relationships is therefore critical for understanding the origin of their adaptive divergence. Our species tree inferred from unlinked nuclear loci for D . pulicaria and D . pulex resolved the European and North American D . pulicaria as sister clades, and we argue that the discordant mtDNA gene tree is best explained by capture of D . pulex mtDNA by D . pulicaria in North America. The Ldh gene tree shows that F-class alleles in D . pulicaria and D . tenebrosa are due to common descent (as opposed to introgression), with D . tenebrosa alleles paraphyletic with respect to D . pulicaria alleles. That D . tenebrosa still segregates the ancestral and derived amino acids at the two sites distinguishing the pond and lake alleles suggests that D . pulicaria inherited the derived states from the D . tenebrosa ancestry. Our results suggest that some adaptations restricting the gene flow between D . pulicaria and D . pulex might have evolved in response to selection in ancestral environments rather than in the species’ current sympatric habitats. The Arctic ( D . tenebrosa ) populations are likely to provide important clues about these issues.     

ProNGF\NGF imbalance triggers learning and memory deficits,neurodegeneration and spontaneous epileptic-like discharges in transgenic mice

ProNGF, the precursor of mature nerve growth factor (NGF), is the most abundant form of NGF in the brain. ProNGF and mature NGF differ significantly in their receptor interaction properties and in their bioactivity. ProNGF increases markedly in the cortex of Alzheimer''s disease (AD) brains and proNGF\NGF imbalance has been postulated to play a role in neurodegeneration. However, a direct proof for a causal link between increased proNGF and AD neurodegeneration is lacking. In order to evaluate the consequences of increased levels of proNGF in the postnatal brain, transgenic mice expressing a furin cleavage-resistant form of proNGF, under the control of the neuron-specific mouse Thy1.2 promoter, were derived and characterized. Different transgenic lines displayed a phenotypic gradient of neurodegenerative severity features. We focused the analysis on the two lines TgproNGF#3 and TgproNGF#72, which shared learning and memory impairments in behavioral tests, cholinergic deficit and increased Aβ-peptide immunoreactivity. In addition, TgproNGF#3 mice developed Aβ oligomer immunoreactivity, as well as late diffuse astrocytosis. Both TgproNGF lines also display electrophysiological alterations related to spontaneous epileptic-like events. The results provide direct evidence that alterations in the proNGF/NGF balance in the adult brain can be an upstream driver of neurodegeneration, contributing to a circular loop linking alterations of proNGF/NGF equilibrium to excitatory/inhibitory synaptic imbalance and amyloid precursor protein (APP) dysmetabolism.     

Reticulate evolution of the Daphnia pulex complex as revealed by nuclear markers

The study of species complexes is of particular interest to understand how evolutionary young species maintain genomic integrity. The Daphnia pulex complex has been intensively studied as it includes species that dominate freshwater environments in the Northern hemisphere and as it is the sole North American complex that shows transitions to obligate parthenogenesis. Past studies using mitochondrial markers have revealed the presence of 10 distinct lineages in the complex. This study is the first to examine genetic relationships among seven species of the complex at nuclear markers (nine microsatellite loci and one protein-coding gene). Clones belonging to the seven species of the Daphnia pulex complex were characterized at the mitochondrial NADH dehydrogenase (ND5) gene and at the Lactate dehydrogenase (LDH) locus. K-means, principal coordinate analyses and phylogenetic network analyses on the microsatellite data all separated European D. pulicaria, D. tenebrosa, North American D. pulex, D. pulicaria and their hybrids into distinct clusters. The hybrid cluster was composed of diploid and polyploid hybrids with D. pulex mitochondria and some clones with D. pulicaria mitochondria. By contrast, the phylogeny of the D. pulex complex using Rab4 was not well resolved but still showed clusters consisting mostly of D. pulex alleles and others of D. pulicaria alleles. Incomplete lineage sorting and hybridization may obscure genetic relationships at this locus. This study shows that hybridization and introgression have played an important role in the evolution of this complex.     

Arginine metabolism inLactobacillus leichmannii

Lactobacillus leichmannii ATCC 4797 metabolizes arginine via the arginine dihydrolase pathway producing ornithine, ammonia, carbon dioxide, and ATP. The specific activities of arginine deiminase and ornithine transcarbamylase were two-or threefold lower (stationary growth phase) in galactose-grown cells. The addition of arginine increased the specific activities of these two enzymes with all growth sugars. When glucose was virtually exhausted from the medium, maximum activities of both enzymes were achieved. The formation of two first enzymes of the arginine dihydrolase pathway inL. leichmannii ATCC 4797 seems to be under the control of two processes: induction by arginine and repression of the induced synthesis by glucose.Dedicated to Dr. Luis F. Leloir on the occasion of his 80th birthday, 6 September 1986.     

Functional and computer modelling studies of haemoglobin from horse. The haemoglobin system of the Sardinian wild dwarf horse.

A study was made of the haemoglobin (Hb) system from the Sardinian dwarf horse (Equus caballus jara), one of the last surviving wild horse species in Europe. The oxygen binding properties of the whole haemolysate and of the four different horse Hbs, separated by ion-exchange chromatography, were studied with special regard to the effect of chloride, 2,3-diphosphoglycerate and lactate. Results indicate that no significant functional differences exist between the four Hb components of horse haemolysate. Moreover, the molecular basis of the intrinsically low oxygen affinity and of the weak interaction of horse Hb with 2,3-diphosphoglycerate is discussed in the light of the primary structure of the molecule and of the results of a computer modelling approach. On these bases, it is suggested that the A1 (Thr-->Ser) and A2 (Pro-->Gly) substitutions observed in the beta chains from horse Hb may be responsible for the displacement of the A helix that is known to be a key structural feature of those Hbs that display an altered interaction with 2,3-diphosphoglycerate as compared with human Hb.